Ultralow doses (history of one research)

1

“All flesh is coupled by the wave ofcircular rotational similarity”

B. L. Pasternak

2

Ultralow doses

3

MoscowRAMS Publishing House

2009

RUSSIAN ACADEMY OF MEDICAL SCIENCES

O. I. EPSTEIN

ULTRALOW DOSES(HISTORY OF ONE RESEARCH)

This edition was recommended and approved for publicationby the Editorial Advisory Board of the Presidium

of the Russian Academy of Medical Sciences

4

Ultralow doses

Reviewers:Seredenin Sergey Borisovich,Academician of the Russian Academy of Medical SciencesChereshnev Valeriy Akeksandrovich,Academician of the Russian Academy of Sciences

Scien c e editor:

Sergeeva Svetlana Aleksandrovna,Doctor of Biological Sciences, Professor

Epstein O. I. Ultralow doses (history of one research). Moscow:Publishing Office of the Russian Academy of Medical Sciences,2008. 336 pages.

ISBN 978�5�7901�0101�4

This monograph is devoted to a systemic study of the effects ofpotentiated (activated) preparations that contain an active substance inultralow concentrations. The activated preparations were shown to havepreviously unknown modifying properties. Taking into account thesedata, a new class of medical products (antibodies in ultralow doses) wasdeveloped. A new direction of bipathic pharmacotherapy was proposed.The first part of this monograph not only illustrates the results of ourstudies, but also describes the physiological and physical aspects andworld outlook of ultralow doses. The final chapters are devoted toexperimental and clinical pharmacology of antibodies in ultralow doses.The monograph is addressed to physicians of various specialties.

© RAMS Publishing House, 2009

UDC 615.015.32UDC 615.015.32UDC 615.015.32UDC 615.015.32UDC 615.015.32

LBC 52.81LBC 52.81LBC 52.81LBC 52.81LBC 52.81

E736E736E736E736E736

ISBN 978�5�7901�0101�4

E736

5

Table of contents

Preface ............................................................................................ 7

List of abbreviations ............................................................................ 10

Introduction ......................................................................................... 13

Chapter 1. Analysis of the experience of homeopathy ....................... 15

Chapter 2. Three types of effects of ultralow doses ...........................24

Chapter 3. Dual organization of vital activity ....................................45

Chapter 4. Holographic control of vital activityby the immune system ......................................................62

Chapter 5. Principle of maintenance of the initial integrity ..............77

Chapter 6. On the way to pharmacology of ultralow doses ............... 91

Chapter 7. Experimental pharmacology of productsfrom ultralow doses of antibodies .................................. 111

7.1. Experimental study of antibodies to S�100 proteinin ultralow doses ................................................................ 111

7.2. Preclinical study of Impaza .................................................. 152

7.3. Preclinical study of Anaferon and Anaferon for children ........... 160

7.4. Preclinical study of Artrofoon ............................................... 173

7.5. Preclinical study of Epigam .................................................. 180

7.6. Preclinical study of Afala ..................................................... 190

7.7. Preclinical study of Kardos ................................................... 198

7.8. Study for antidiabetic activity of a new productfrom ultralow doses of antibodies on the modelof streptozotocin�induced diabetes in rats .............................. 204

Chapter 8. Clinical pharmacology of productsfrom ultralow doses of antibodies .................................. 210

Devoted to my teachers

6

Ultralow doses

8.1. Use of medical products from antibodies to S�100 proteinin the therapy for alcoholism and anxiety disorders ................. 210

8.2. Use of Impaza in monotherapy and combinedtreatment for erectile dysfunction ......................................... 223

8.3. Clinical effectiveness and mechanismsfor action of Anaferon ......................................................... 241

8.4. Artrofoon as a promising drug for pathogenetictherapy of chronic arthropathies ........................................... 252

8.5. Epigam in the therapy for gastric ulcerand duodenal ulcer ............................................................. 259

8.6. Afala in the therapy for benign prostate hyperplasia ................ 262

8.7. Clinical pharmacology of Kardos .......................................... 265

Conclusion ........................................................................................ 275

References ........................................................................................277

7

Preface

This book tells you about the history of a 10�year study, which was per�formed at the interface between immunology, pharmacology, pathophysiology,and problem of ultralow doses and resulted in the development of a new classof medical products.

Ultralow doses were used in alternative academic medicine (homeopathy)over two centuries. Hence, this problem received little attention of scientists. Atthe beginning of the 1980s, advanced�technology studies in Russia and othercountries showed that ultralow doses have biological activity. The scientists sho�wed a cautious attitude toward these results, although they were not associated withhomeopathic doctrine. Moreover, such studies did not attract much attention ofthose investigators who was interested in them (primarily, of pharmacologists).

Due to certain reasons, a systemic pharmacological study of ultralowdoses was first performed in Russia. The author of this book, O. I. Epstein, wasan initiator of such investigations in 1995. Now O. I. Epstein is the Doctor ofMedical Sciences and Professor. He was awarded the prize of the Governmentof the Russian Federation in the field of science and technology. During thatperiod, a young physician was enthusiastic in homeopathy. He was the head ofa small pharmaceutical company, who searched for a specific direction of ac�tivity. Famous Russian specialists (investigators and clinicians) became interestedin the idea of O. I. Epstein, which determined the success of a scientific search.The scientific way of O. I. Epstein extends from orthodox homeopathy to phar�macology (i.e., immunopharmacology). Success attended him, since the direc�tion of many researches was selected intuitively. Initially, the goal of his studywas to develop new indications for the use of homeopathic remedies. If someGod of Science protected O. I. Epstein, it was the God of Immunology.

The study may be divided into three stages. Stage I may be designated as“non�homeopathy”. The author assumed that ultralow doses and homeopathyare not identical to each other. It was proposed that the phenomenon of ho�meopathy is associated with hypersensitivity of the organism to ultralow doses.Hence, the effects of ultralow doses were explained by immunological mecha�nisms.

8

Ultralow doses

In stage II, an unusual application was found for ultralow doses. It wasshown that ultralow dose of a certain medical product modifies activity of theoriginal substance. Therefore, ultralow doses of modern pharmaceutical prod�ucts may be used to potentiate their effects and to reduce toxicity. Combinedtreatment with the medical product in normal dose and ultralow dose receivedthe name “bipathy” (O. I. Epstein). Bipathy holds much promise for pharmaco�logy. The researches could focus their attention on study of bipathy.

However, the direction of investigations sharply changed. In stage III,these researches were in close contact with immunology. To confirm the phe�nomenon of bipathy, O. I. Epstein in collaboration with M. B. Shtark (Acade�mician of the Russian Academy of Medical Sciences) and high colleagues stud�ied the effects of antibodies in ultralow doses on neurobiological models. Theyshowed that antibodies in ultralow and normal doses have various effects. An�tibodies in ultralow doses did not inhibit, but modified the activity of a specif�ic antigenic molecule. The discovery of “pro�antigenic” effects of antibodies inultralow doses resulted in the development of new effective and safe drugs forthe therapy of various diseases.

As an immunologist, I know that very low doses (or sometimes nano�quantities) of immunogenic proteins, peptides, and polysaccharides may inducea strong physiological and pathophysiological effect. It mainly concerns antibod�ies, allergens, and other immunologically active molecules.

This monograph illustrates the results of advanced�technology experi�ments and clinical studies. However, the data that extremely low concentrationsof medical products (from the viewpoint of molecular biology) exhibit the ac�tivity seem to be unexpected and paradoxical. At the modern of stage of sciencedevelopment, the mechanism for action of ultralow doses can be described hy�pothetically. The author gives his opinion on this problem. Sometimes, the un�usual effects of ultralow doses are explained by uncommon events. As a pioneerin this field, O. I. Epstein can do it.

Numerous preclinical and clinical trials were performed in leading insti�tutions of the Russian Academy of Medical Sciences and Russian Ministry ofHealth and Social Development. The results of these studies indicate that ul�tralow doses have a reproducible effect, which may be evaluated and used inevidence�based medicine. However, the author notes that the exception is ho�meopathic therapy. “An individual (similar) prescription of medical products issimilar to art. The methodology of homeopathy is not associated with generalpathophysiological approaches in pharmacology”. It is really true.

O. I. Epstein is so infatuated with his hypotheses that sometimes he passesfrom a strongly scientific presentation to the emotional, speculative, or evenphilosophical conclusions. It is not necessarily that strong evidence exists forthese conclusions. O. I. Epstein believes that there are no two medicines (allo�

9

pathic medicine and homeopathic medicine). At a particular fine�structure level,the effects of normal and homeopathic doses in an organism are mediated bysimilar mechanisms. This level includes the distant intermolecular relationships,which are unique for each individual. O. I. Epstein assumes that the preserva�tion of individuality is an evolutionary purpose of vital activity of the organism.This theory is close to the principles of immunology. M. Bernet, one of thefounders of modern immunology, believed that a major role of the immune sys�tem is regulation of genetic integrity in an organism.

This monograph integrates the author’s notion of distant interactions inan organism with general principles of physiology. A lot of surprising and, some�times, doubtful facts will be of interest to the reader. The monograph is writ�ten in a vigorous and interesting style, which facilitates the understanding ofcomplex biological problems. O. I. Epstein not only tells us about new medi�cal products, which have high therapeutic effectiveness and hold promise for thetreatment of various diseases. He wants the reader to form an opinion of newdrugs in ultralow doses (particularly of those from antibodies).

R. M. KhaitovAcademician of the

Russian Academy of Medical Sciencesand Russian Academy of Sciences

Preface

10

Ultralow doses

List of abbreviationsAWS — alcohol withdrawal syndromeBP — blood pressureAID50 — aerogenic infective doseAnti�S100 — antiserum to brain�specific protein S�100AFC — antibody�forming cellsAPC — antigen�presenting cellsACE — angiotensin�converting enzymeARA — American Rheumatology AssociationAT — angiotensinAT1 — type 1 angiotensin II receptorAB�IRβ — antibodies to insulin receptor beta�subunitATP — adenosine triphosphateATPase — adenosine triphosphataseADC — analog�to�digital converterEPSP — evoked postsynaptic potentialGABA — gamma�aminobutyric acidDTHR — delayed�type hypersensitivity reactionGCD — glucocorticoid drugsGCSF — granulocyte colony�stimulating factorGMP — guanosine 3,5�monophosphateGAD — generalized anxiety disorderBPH — benign prostate hyperplasiaCI 95% — 95% confidence intervalDNA — deoxyribonucleic acidLPTP — long�term posttetanic potentiationNK — natural killer cellsGIT — gastrointestinal tractCHD — coronary heart diseaseIL — interleukinEIA — enzyme immunoassayIFN — interferonCIA — collagen�induced arthritisLD — lethal dose

11

List of abbreviations

LV — left ventricleLC — latencyLPS — lipopolysaccharideIIEF — International index of erectile functionICD — International classification of diseasesMTD — maximum tolerable doseISIAH — inherited stress�induced arterial hypertensionNAID — nonsteroid antiinflammatory drugsAE — adverse eventOA — osteoarthritisARVI — acute respiratory viral infectionsHTP — hydroxytryptophanAP — action potentialEPM — elevated plus�mazeRP — resting potentialCGM — complete growth mediumPSA — prostate�specific antigenRA — rheumatoid arthritisLBTR — lymphocyte blast transformation reactionRNA — ribonucleic acidRSV — respiratory syncytial virusSBP — systolic blood pressureDM — diabetes mellitusDBPM — daily (24�h) blood pressure monitoringULD — ultralow dosesULDH — ultralow doses of haloperidolULDP — ultralow doses of phenazepamALS — average lifespanMADD — mixed anxiety and depression disorderTRUSE — transrectal ultrasound examinationTS — testosteroneUA — urogenic reactive arthritisUSE� ultrasound examinationCAAR — conditioned active avoidance reflexCPAR — conditioned passive avoidance reflexLVEF — left ventricular ejection fractionPHA — phytohemagglutininPDE�5 — type 5 phosphodiesterasePI — phagocytic indexTNF�β — tumor necrosis factor�βPBS — phosphate�buffered salinePN — phagocytic number

12

Ultralow doses

CHF — chronic heart failurecAMP — cyclic adenosine 3',5'�monophosphatecGMP — cyclic guanosine 3',5'�monophosphateCNS — central nervous systemCP — cyclophosphaneHR — heart rateSE — sheep erythrocytesED — erectile dysfunctionECG — electrocardiogram5�HT — serotonin receptorsACR20 — 20% improvement by American College of Rheumatology criteriaARA — American Rheumatology AssociationAS�100 — antiserum to brain�specific protein S�100AUC — trapezoid method for estimation of the areaunder the concentration�time curveC — centesimal dilutionD — decimal dilutionEGF — epidermal growth factoreNOS — endothelial NO synthaseFDA — USA Food and Drug AdministrationHAMA — Hamilton anxiety scaleIg — immunoglobulinIPSS — International questionnaire for symptomsof prostate diseases (International Prostate Symptom Score)ITT — analysis of the results for patients included in the trial(intention to treat analysis)MHC — major histocompatibility complexNMMA — NG�monomethyl�L�arginineNOS — NO synthaseNYHA — New York Heart AssociationQoL — quality�of�life index (IPSS questionnaire)STAI — Spielberger scale (State�Trait Anxiety Inventory)STAI�S and STAI�T — state and trait anxiety by the Spielberger scaleVEGF — vascular endothelial growth factorWOMAC — index for the severity of osteoarthritis(Western Ontario and Mc Master Universities Osteoarthritis Index)

13

The author of this book, as well as his colleagues, succeeded in an intriguingscientific path from homeopathy to immunopharmacology, and from

traditional homeopathic remedies to high�technology, safe, and effective medicalproducts. These drugs were developed on the basis of a newly discoveredphenomenon of ultralow doses of antibodies.

Several preparations from ultralow doses of antibodies, including Ana�feron, Impaza, and Proproten�100, are well known. However, many physiciansand specialists do not have enough information on the mechanism for actionof these products. The phenomenon of antibodies in ultralow doses was openedat the boundary of the following three medical disciplines: pharmacology,immunology, and homeopathy. Therefore, this monograph includes some dataon homeopathy and immunology.

Experimental and clinical trials allowed us to obtain new data, which arenot consistent with the common notions about vital functions of the organism.The interpretation of these facts requires other approaches and new knowledge.The pragmatic purpose of this monograph is to develop the notion of a newpharmacological direction.

During the preparation of this manuscript, the author should becomefamiliar with previously unknown areas of knowledge. It was necessary for himto learn the notions and terminology that exist in each field of science. Medicalscience consists of several special directions. A fruitful professional dialog doesnot necessarily occur between experimenters and clinicians. We would like thisbook to be available not only for representatives of theoretical medicine(pharmacologists, physiologists, and immunologists), but also for physicians. Tosimplify the understanding of some facts that require special biologicalknowledge, they are given in a popular scientific form (history of one research).Fundamentally, the monograph is divided into two sections. The first sixchapters are devoted to the general problem of ultralow doses. The final twochapters show some data on experimental and clinical effectiveness of newproducts. It may be of interest to various specialists, including neurologists(Tenoten), infectious disease physicians and podiatrists (Tenoten for children

IntroductionIntroductionIntroductionIntroductionIntroduction

14

Ultralow doses

and Anaferon for children), urologists (Afala and Impaza), rheumatologists(Artrofoon), narcologists (Proproten�100 and Anar), and cardiologists (Kardos).

The author is grateful to Professor S. A. Sergeeva, Yu. L. Dugina, I. A.Kheifets, and all colleagues from the Science Department of the “MateriaMedica Holding” Research�and�Production Company for their help in thepreparation of this monograph.

15

The author of this monograph, a recent graduate of the Medical Institute,received the book “Homeopathy” (G. Kohler) as a gift from his father in

1989. It was a rare book in the period of commodity deficit. A period of 1 yearwas required to overcome a skeptical attitude of the Soviet physician toward this“superficial” discipline. The book was opened and read. This moment may beconsidered as the first successful step in a study described here. As differentiatedfrom various manuals on homeopathy, the general part in the book of G. Kohlerwas written in down�to�earth language. Otherwise, the familiarity and furtherfascination with homeopathy could not occur.

After ten years of neglect of homeopathy in our country, thousands ofphysicians were able to learn this discipline by visiting a variety of quasi�legaltraining courses. However, only some of them became the practitioners. It wasvery difficult to learn homeopathy without assistance of a teacher. The authorof this monograph was successful. A twist of fate introduced him to a famousphysician T. D. Popova, who headed the Kiev school of homeopathy. She wasa bright person. O. I. Epstein had the possibility to collaborate with Tat’yanaDem’yanovna and to look for the reception of patients. It helped him to learnthe basic principles of homeopathy.

Homeopathy is inseparably linked with the name of S. F. Hahnemann. In1976, he published the manuscript on a new therapeutic direction. He describedthe method for preparation of medical products in ultralow doses, principles ofprescription, and results of clinical trials. S. Hahnemann is one of the pioneers

C h a p t e r 1

Analysis of theexperience of homeopathy

16

Ultralow doses

in clinical trials with medical products. There are ambiguous data on pharma�ceutical studies in old time. The first comparative analyses of therapeutic agentswere performed only in the 18th century. However, R. Virchow believed that the“farther of experimental pharmacology” is S. Hahnemann. Before Hahnemann,there was no integral and intelligent approach to study of medical products. Hereceived a fine education and learned the ancient languages. In previousmanuscripts, Hahnemann could learn the “like cures like” principle. The deve�lopment of a new rational scientific method for medicinal treatment of variousdiseases was associated with activity of this unique man. Hahnemann decidedto test the effect of cinchona bark with himself to confirm the reliability ofpublished data. This substance caused fever, which was typical of malaria. Thescientist concluded that a medicine for the therapy of some disease can inducea similar state in healthy individuals.

S. Hahnemann tested the effects of various medical products (mainly ofherbs and minerals) on healthy volunteers. Clearly the trial did not meet mo�dern requirements and was performed with a small number of people (relatives,friends, pupils, and colleagues). The results confirmed the hypothesis of Hah�nemann. The reactions induced by some substance in healthy volunteers maybe considered as an indication for therapy of similar disorders with the sameagent. The Hahnemann’s Law of Similars was formulated for the first time inthe journal of Hufeland. “One should apply in the disease to be healed, parti�cularly if chronic, that remedy which is able to stimulate another artificially pro�duced disease, as similar as possible; and the former will be healed – similiasimilibus*”.

It should be emphasized that the Law of Similars was discovered by S. Hah�nemann with normal doses**. Some patients were characterized by severe drug�induced exacerbation. Hence, Hahnemann decided to reduce the dose of re�medies. Due to pedantry, the scientist developed a method for dilution of theoriginal substance. He empirically showed that it is necessary to combine(exactly to combine!) a repeated dilution of the initial solution and tenfoldmechanical shaking. Hahnemann proposed to use the centesimal (C) scale ofdilution. At each stage of preparation, the initial amount of medical product isdiluted by 100 times. In the follow�up period, homeopaths introduced the so�called “decimal dilution” (D, successive tenfold dilution of the original substan�ce). For example, dilution C30 means that the original substance was diluted30 times. Moreover, the concentration of this substance was reduced by 100times at each dilution. D6 means that the concentration of the original

* Similia similibus (lat.) � likes with likes.** The term “homeopathy” originates from the Greek word “homo” (similar). It mainly

designates the principle of “remedy prescription”, but not the dose.

17

substance decreased by 10 times at each of six dilutions (Fig. 1.1). In the SIsystem, high dilutions by the method of Hahnemann correspond to 10—n M and100—n M. Formally, the dilutions of more than 10—24 M (D24) and 100—12 M(C12) are submolar. They do not contain molecules of the original substance.This fact did not disturb the founder of homeopathy. Hahnemann did not knowabout the unit of “mole”, which was proposed by the physician AmedeoAvogadro (1776�1856). Hence, Hahnemann operated with submolar doses.

In the 18th century, drug manufacturers were in conflict with Hahne�mann. It was related to commercial reasons, but not to the absence of moleculesof the original substance in homeopathic remedies. Hahnemann prepared thesehomeopathic remedies by himself and, therefore, deprived the manufacturers ofearnings.

S. Hahnemann and his followers studied the effects of various prepara�tions on healthy volunteers. They showed that treatment with these agents insubmolar concentrations is followed by drug�induced exacerbation. Even at highdilution of one or another substance, there were two or three individuals(respondents) with hypersensitivity to the prescribed remedy.

It should be emphasized that the respondents had similar personalcharacteristics, appearance, behavioral habits, and dietary predilections. Theywere predisposed to certain diseases, including inherited disorders. For example,the subjects reacting to ultralow doses of arsenic mainly appeared as lean, fine�boned, and light�skinned individuals. They usually had a geographic tongue,drank a considerable amount of fluid in small sips, and felt comfortable in warmclimate. These subjects differed in pedantry, anxious mood, and predisposition

Fig. 1.1.Fig. 1.1.Fig. 1.1.Fig. 1.1.Fig. 1.1. Scheme for the preparation of homeopathic remedies (V. G. Zilov et al.,2000).

Chapter 1. Chapter 1. Chapter 1. Chapter 1. Chapter 1. Analysis of the experience of homeopathy

(Н2О) (Н2О) (Н2О) (Н2О) (Н2О)

Molecular 1:1 1:102 1:104 1:106 1:108 1:1010

dilution

Homeopathic Basic 1С 2С 3С 4С 5Сpotency solution

Increase in the potency of remedies

Alcoholsolution

⏐ ↓ 1/100

⏐ ↓ 1/100

⏐ ↓ 1/100

⏐ ↓ 1/100

⏐ ↓ 1/100

18

Ultralow doses

to psoriasis, bronchial asthma, and other diseases with common clinical symp�toms. A complex description of the effects of any substance in ultralow dose andcharacteristics of hypersensitive patients received the name “pathogenesis ofhomeopathic remedy” (in this case, arsenic pathogenesis). It includes the phar�macodynamic properties of a substance and markers of individual sensitivity,which should be taken into account in the prescription of this remedy*. S.Hahnemann demonstrated that the maximum individualization of therapy isnecessary for clinical practice. The major therapeutic principle appeared asfollows: “cure only with a similar drug”. The similarity was considered not onlyas the prescription of a medical product for certain symptoms (indications), butalso as the use of a specific remedy in patients with high individual sensitivity.

Hundreds of medical products are extensively used in homeopathy. Thereare 2000 homeopathic remedies. Homeopathy was born 200 years ago. There�fore, homeopathic remedies are prepared from available raw materials (plants,minerals and, more rarely, biological substances). The major advantage ofhomeopathic remedies is that their effects were studied in details. Undoubtedly,the arsenal of homeopathy will always include well�known “ancient” prepara�tions. Some modern pharmaceutical (allopathic) products, including aspirin,nitroglycerine, insulin, and prednisolone, are sometimes given in ultralow doses.The quintessence of homoeopathy is the evaluation of sensitivity of each patientto one of the homeopathic remedies. In this case, the treatment will producea therapeutic effect. Hahnemann developed the integral therapeutic disciplineof homeopathy, which differs in all�sufficiency. Potentially, a skilled homeopathmay affect any disease state. The success of therapy strongly depends on the“appropriateness” of medical treatment, but not on the nosological form orseverity of disease. A skilled homeopath is a general practitioner and good spe�cialist in propaedeutic. The prescriptions of a homeopath are based on historytaking, thorough examination, power of observation, and evaluation of patientcharacteristics.

By the 20th century, homeopathy was widely distributed in Europe, Northand South America, India, and Russia. Pharmaceutical chemistry, experimentalpharmacology, and molecular biology became the specific areas of knowledgeat a later period. They determine the type of modern medicine. Due to severalreasons, homeopathy was separated from modern science. Symbiotic relation�ships between academic medicine and homeopathy developed in the follow�upperiod. Traditionally, a European patient knows the problems that require him

* The term “drug pathogenesis” is very close to the modern notion of constitution inpsychiatry and pediatrics. Hahnemann did not use the term “constitution” or“constitutional type”. They were introduced into the homeopathic lexicon by followersof Hahnemann.

19

to consult with a homeopath. Homeopathic therapy also develops in Russia.After long�term neglect, this method was officially approved in Russia in 1995.

Let’s consider the main principles of the Hahnemann theory.Principle of similarity. First of all, it should be remembered that the prin�

ciple of similarity is not a prerogative of ultralow doses. The first prescriptions byHahnemann were made in “normal” doses. This principle suggests that the remedyshould be prescribed for a “similar” clinical manifestation. For example, arseniccauses fever in healthy volunteers. By contrast, arsenic in homeopathic doses maybe used to cure fever in patients. However, arsenic�induced fever has several specificfeatures. Fever is observed in a certain time (midnight) and accompanied bytypical chill, which spreads in an upward direction of the back. Arsenic inhomeopathic doses will be therapeutically effective in patients with this type offever. Moreover, the effect will occur only in patients that are susceptible toultralow concentrations of arsenic (primarily in lean and fine�boned pedants).

These features of symptoms and constitutional characteristics serve as amarker for individual sensitivity. Modern pharmacology also postulates thenecessity of individual pharmacotherapy. Much attention is paid to the searchfor genetic and phenotypic criteria of individual sensitivity.

It should be noted that each patient may have various markers. These dataindicate that individual sensitivity (at least, phenotypic sensitivity) to a substanceis determined by several markers, but not by one marker. The experience ofhomeopathy shows that one sign (e.g., geographic tongue) may serve as one ofthe markers for a large group of medical products. Only a specific combinationof various markers is a reliable criterion for individual sensitivity to the remedy*.

Trial with healthy volunteers. The trial with healthy volunteers was of con�siderable significance. This approach allowed S. Hahnemann and his followersto perform a simple, rapid, and safe evaluation of indications for a large numberof medical products. The following two facts are of importance for our study.

First, medical products in ultralow concentrations cause exacerbation ina small number of patients. Hence, this state is associated with the reaction ofhypersensitivity. Several types of immediate and delayed�type hypersensitivitywere studied and described in immunology. However, little is known about theirmechanisms. All these reactions are nonspecific. For example, anaphylactic shockmay be induced by a variety of substances. By contrast, the symptoms of drug�induced exacerbation in homeopathy are always specific for a certain substance.

And second, arsenic in toxic doses causes hyperthermia in all subjects.Low doses of arsenic may produce the same clinical symptoms of fever inindividual volunteers, which is related to hypersensitivity. Let’s consider fever as


* In the future, this observation allowed the author to develop a new concept of seman�tically organized constellations.

20

Ultralow doses

a protective response. In this case, the substance in high doses serves as a“pathogen” for all individuals. By contrast, the same substance in low doses hasa pathogenic effect only on several subjects. A similar protective response shouldbe mediated by similar mechanisms. However, the substance in high dosetriggers these mechanisms in all subjects. It remains unclear why the substancein low dose affects only sensitive individuals*.

Preparation of ultralow doses. It is known that highly diluted solutions donot have biological activity. Otherwise, biological activity of these solutions isextremely unstable. Moreover, the Hahnemann’s method of mechanical shakingmay be substituted for another external influence (electromagnetic or ultrasoundexposure). However, ultra�diluted solutions do not exhibit activity without suc�cessive dilutions of the original substance in combination with external treat�ment. It is surprising that Hahnemann could combine various procedures intoa common system: preparation of medical products, study of pharmacologicalactivity, and principle of prescription (similarity).

The physical mechanisms for memory retention of the original substancein highly diluted solutions remain unknown. Several scientists have cast doubton the use of ultralow doses and effectiveness of homeopathy. Modern studieswere performed 200 years after the discovery of homeopathy. They illustratedthat ultra�diluted solutions (method of Hahnemann) have fine biological activity.

The technological process of S. Hahnemann received the name “poten�tiation”. The prepared remedies were designated as potentiated or dynamicsubstances. Hahnemann proposed that these agents are characterized by therelease of an “active basis” or “vital force”. A clinical effect of potentiated pro�ducts was not observed without maximum individualization. Hahnemann sug�gested that potentiated products are ineffective without the principle of similarity(all�or�none law). Various properties of potentiated agents were revealed inmodern molecular and cellular studies. Hahnemann had no technicalpossibilities to determine the general properties of ultralow doses, but proposedan approach to their use (homeopathy).

The primacy of the principle of similarity took root in the mind of Hah�nemann followers. Until the present time, homeopaths know little about other(non�homeopathic) variants for the use of medical products in ultralow doses.The term “homeopathic dose” has been commonly accepted. It is morepreferable to tell about the individual (homeopathic) prescription of a medicalproduct in ultralow or low dose, but not about the dose. In our opinion, theterm “homeopathic remedy” means that the indications for treatment with a

* The mechanisms of individual sensitivity are a major problem, which underlies thehypothesis on dual (holographic) organization of vital functions in an organism. Thisproblem is discussed in the next chapters.

21

certain drug were evaluated in trials with healthy volunteers. However, theindications for modern pharmaceutics are estimated by other methods.

The terms “low” and “ultralow” dose are also indefinite. Some authorsbelieve that the doses of up to 10—12 M original substance are low (molar) do�ses. The doses of not more than 10—24 M are ultralow doses. However, activityof the potentiated agent depends little on the presence or absence of several mo�lecules of the original substance (see below). By contrast, this activity is deter�mined by the technology of preparation. The method of potentiation suppliesbiological activity to ultra�diluted solutions. Hence, these solutions can be usedaccording to the doctrine of homeopathy. It is more appropriate to use the term“potentiated” or “activated” substance, but not the homeopathic, low, or ultra�low dose. The scale of preparation (C or D) and number of dilution cycles(usually 6, 12, 30, 200, or 1000) should be designated.

The term “activated” preparation seems to be preferable. First, this term in�dicates than biological activity of the ultra�diluted solution is related to externaltreatment. Second, the term “potentiation” has another meaning in modernpharmacology. And third, we showed that medical products in ultralow doseshave a potentiating effect under certain conditions. These features may introduceterminological difficulties. However, the preparation of ultralow doses shouldretain its historical name of “potentiation” or “dynamization” (S. Hahnemann).

Besides a rational analysis of homeopathy experience, it is necessaryto consider the subjective feelings of homeopathic physicians. At a certain stageof professional activity, any homeopath notices that nearly all (even rarelyobserved) physiological signs are manifested in the description or pathogenesisof homeopathic remedies. For example, you see that one of the guests asks youto prepare tea. Then he gulps down boiling water. Another guest asks you toclose the window since he cannot hear a noise (the yard�keeper cleans asphaltwith a scraper). Under these conditions a homeopath will remember thepathogenesis of Lycopodium (club moss) and Asarum (snakeroot), respectively.

When studying the temperamental characteristics of patients sensitive toone or another homeopathic remedy, you can see that they have a commonfeature (“stem” or “algorithm”). It is difficult to explain this algorithm. How�ever, a homeopath cannot select the effective medical product without under�standing this algorithm.

Many homeopaths know that the psychological type of patients sensitive tosome medical product is progressively transformed into the psychological type ofanother remedy. This feature contributes to the mosaic pattern, which resemblesa change in the properties of chemical elements in the periodic table of D. I.Mendeleev. Psychological portraits of sensitive patients are similar in thepathogenesis of several preparations (mineral potassium sulfate and Pulsatilla of thefamily Ranunculaceae; calcium carbonate and belladonna; etc.). The remedies with


22

Ultralow doses

similar “psychological characteristics” have the same therapeutic effect. These datasuggest the systemic nature of homeopathy, which is difficult to verbalize.

This impression becomes stronger in learning the other basic principles ofhomeopathy. Using the terminology of the 18th century, homeopaths classify allpathological processes into the following three groups (depending on general char�acteristics): syphilis (destructive processes), sycosis (productive processes, includingcough), and psora (subacute areactive states). Each type of pathological processesis characterized by a certain group of preferable homeopathic remedies. Otherprinciples of homeopathy are also unusual. For example, some activatedpreparations are tropic for the right�sided or left�sided disease. Caustic (lime) andLachesis (bushmaster snake venom) are prescribed for the therapy of right�sidedand left�sided hemiplegia, respectively. The rules of C. Hering also seemuncommon. C. Hering (1800�1880) is the farther of American homeopathy. Hedescribed the spatial and temporal response of patients to homeopathic remedies.

C. Hering revealed (1998) that “the patient will be cured and the symp�toms will permanently disappear when they develop in the following direction:from within outwards, from above downwards, and from later symptoms toearlier symptoms (i.e. in the reverse order of their coming)”. The use of homeo�pathic remedies is rarely followed by drug�induced exacerbation. When thedynamics of drug�induced exacerbation is consistent with the rules of C. Hering,it may be considered as a prognostically favorable process. Under these condi�tions, the prescribed remedy is not withdrawn by a homeopath.

Homeopathic practitioners know that each patient is sensitive to severalhomeopathic remedies (hierarchy of individual sensitivity). The higher is thesensitivity to the remedy, the greater is the effectiveness of this remedy.

Summarizing the above we conclude that vital activity of the organism isbased not only on well�known physiological and chemical processes, but alsoon spatial and temporal laws of harmony. They are closely related to individualcharacteristics of each patient. Even though the homeopathic knowledge seemsarchaic, it is worthy of notice. This knowledge was obtained in a directedclinical study of medical products. Hence, these data are objective. Moreover,separate observations illustrate the existence of various clinical, phenotypic,psychological, and topic criteria for individual sensitivity of an organism.

Most important in ChaptMost important in ChaptMost important in ChaptMost important in ChaptMost important in Chapter 1er 1er 1er 1er 1

Homeopathy is a drug therapy based on the principle of similarity.

The principle of similarity suggests maximum individualization in the prescrip�tion of medical products, which involves a propaedeutic approach of S. Hah�nemann.

23

Homeopathy suggests the use of activated agents that are prepared by the meth�od of potentiation (successive dilutions of the original substance and rhythmicmechanical shaking).

Activity of ultra�diluted solutions depends on the technology of poten�tiation, but not on the concentration of the original substance. This fact isproved, but does not have a physical explanation.

Ideally low doses in homeopathy are associated with the method of preparation.

Over two centuries, potentiated (activated) preparations were believed to produceonly the clinical effect. It was postulated that these drugs are ineffective with�out individualization of therapy.

Recent experiments showed that ultralow doses produce a fine molecular�and�cellular effect, which is not directly related to the principle of similarity. Acti�vated preparations have a small effect that is insufficient for therapeutic activi�ty. Taking into account these data, we assumed that homeopathic therapy isbased on increasing the strength of ultralow doses by the mechanism of hyper�ergia.

After studying the ultralow doses of remedies with healthy volunteers, S.Hahnemann revealed the existence of individual sensitivity to certain medicalproducts (complex of clinical, psychological, psychological, and topic criteria).


24

Ultralow doses

The first professional experience of homeopathy showed that it is notnecessary to follow the principle of similarity in achieving a therapeutic

effect of remedies in ultralow doses. For example, a group of preparations existsthat are a priori tropic for the liver (Lycopodium, Chelidonium, and Carduusmarianus). Even without individualization of therapy, these medical productsimprove the state of most patients with hepatobiliary disorders. Moreover, travelsickness (acute paroxysmal state) may be treated with several homeopathicremedies that reduce the symptoms of autonomic disorders. Individualization oftherapy is not required under these conditions.

The “Materia Medica Holding” company was established in 1992. In theinitial period, this company manufactured the so�called “complex homeopathicremedies”. The pharmaceutical formulation consisted of three or four homeo�pathic drugs (granules and tablets). Other Russian and foreign complexons ofwell�known homeopathic components from plants and minerals, as well asorganotropic preparations of animal (embryonic) tissues or organs, appeared onthe pharmaceutical market. The effects of potentiated remedies and tissuepreparations are related to their tropism for a certain pathological condition andorgan, respectively.

A complex preparation Agri (homeopathic Antigrippin) was the brand ofthe “Materia Medica Holding” company in the 1990s. Surprisingly, this drughad a preventive effect on influenza and chill. These facts seem to contradictthe principle of similarity.

C h a p t e r 2

Three types of effectsof ultralow doses

25

Such “discrepancies” with the homeopathic doctrine did not alter ourrelation to the basic principles of homeopathy. These data stimulated us tosearch for new indications for ultralow doses.

Experimental and clinical trials of potentiated products wereconducted at the “Materia Medica Holding” company beginning from 1995.Several discoveries were made. The first steps of a large�scale scientific researchwere devoted to study the effect of combined treatment with a medical productin normal dose and activated form of the same remedy. The phenomenon ofisopathy was well known. It suggests that the symptoms of poisoning with acertain substance are treated by the potentiated form of this substance. Due tohigh risk of chemical attacks of the German army during World War II, Englishvolunteers were exposed to skin burns with mustard gas. These burns were thentreated with potentiated mustard gas. Complications of corticosteroid therapy,including Itsenko�Cushing syndrome, are homeopathically treated with Cortex(activated prednisolone). However, isopathy was not subjected to a complexclinical study. Moreover, there are no experimental data on this problem.

Similarly to the preventive effect of Agri, we decided to test ultralow dosesof a certain substance for protective activity during intoxication that was inducedby this substance in high (toxic or subtoxic) doses. As differentiated from theisopathic method, ultralow dose (“antidote”) was administered in combination,but not after treatment with the same substance in toxic doses (poison). Webelieved that this approach does not contradict the homeopathic doctrine.Administration of the substance in toxic doses served as a model to induce thesymptoms typical of treatment with the activated form.

The principle of our experiments was quite paradoxical. We tried tointroduce a “drop” (activated substance) into the “sea” (toxic dose of thesubstance). Combined treatment with ultralow doses was performed in variousregimens. Ultralow doses were administered simultaneously or before treatmentwith the toxic dose (one�to�ten minutes). Various routes of treatment with ultra�low dose were also analyzed. For example, the potentiated preparation wasmixed with a toxic dose of the same substance. The mixture was administeredperorally through a probe. Otherwise, the original substance was administeredparenterally, while the activated preparation was given perorally.

Combined treatment with the substance in ultralow (homeopathic) andnormal (therapeutic or toxic) doses was performed simultaneously or in a smallinterval. This approach received the name bipathic* treatment (O. I. Epstein,1996, 1997). Prednisolone was the first medical product for combined treatment.It was the first step from homeopathy to immunopharmacology. Therefore, thisstudy should be described in details.

Chapter 2. Chapter 2. Chapter 2. Chapter 2. Chapter 2. Three types of effects of ultralow doses

* Bipathic administration, allopathy + homeopathy.

26

Ultralow doses

Bipathic administration of prednisolone was performed at the Laboratory ofBiophysics (Kiev Institute of Otorhinolaryngologist) headed by A.F. Karas’. The animalssimultaneously received prednisolone in “normal” and potentiated doses (dilution C30). Inthe latter case, the conventional concentration of prednisolone was 10—60 wt % (10—60 M).

Series I was performed on 30 rats. The effect of bipathic treatment with pred�nisolone was studied on animals with experimental acute inflammation. This state wasinduced by injection of 0.05 mg 1% carrageenan into the hindlimb of rats. Activatedprednisolone was administered through a probe to group 1 animals with experimentalinflammation. Group 2 animals received prednisolone in a total dose of 20 mg per rat.Group 3 animals were subjected to combined treatment with both drugs. The standard andpotentiated forms of prednisolone were administered twice (1 h before and 2 h aftercarrageenan injection). The control groups consisted of intact animals and untreated ratswith carrageenan�induced inflammation.

We showed that administration of prednisolone alone or in combination withactivated prednisolone is followed by the reduction of inflammatory edema of the paw.Potentiated prednisolone had no antiinflammatory activity and did not potentiate theantiinflammatory effect. However, bipathic administration of prednisolone wasaccompanied by several positive effects.

Migration of peritoneal macrophages significantly decreased during inflammation.Normal doses of prednisolone did not improve macrophage migration. However,macrophage migration rapidly returned to the control level after combined treatment withstudy drugs.

An electron microscopic and morphological study showed that administration of 20mg prednisolone is followed by moderate destructive changes in the liver and thymus ofanimals. However, the structure of these organs was not impaired after combinedadministration of prednisolone and activated prednisolone in the same doses.

Carrageenan�induced inflammation is accompanied by the increase in energyconsumption due to activation of the protective response. Prednisolone has no effect onblood ATP concentration. Bipathic administration of prednisolone is followed by a sharpdecrease in ATP content, which reduces energy supply to the inflammatory process.Combined administration of prednisolone in the toxic and ultralow dose improves enzymeactivity in blood cells, activates alkaline phosphatase in neutrophils, and has a normalizingeffect on the activities of ATPase, 5�nucleotidase, and lactate dehydrogenase. Thesechanges illustrate the restoration of energy supply to cells.

As compared to “standard” prednisolone, bipathic administration was followed bya greater increase in biosynthetic activity of rat peripheral blood lymphocytes. Thisconclusion was derived from the increase in RNA level.

Series II was designed to evaluate whether potentiated prednisolone (10—60 wt %)may prevent the side effect of chronic treatment with prednisolone in normal doses for 2weeks. Prednisolone in a daily dose of 50 mg/kg body weight was administered through aprobe. This dose of prednisolone produced a strong antiinflammatory effect on the modelof carrageenin�induced inflammation. The potentiated substance had a variety of protectiveeffects under these conditions (Table 2.1; V.G. Zilov et al., 2000).

These data indicate that the activated agent prevents metabolic disordersin lymphocytes and neutrophils and, probably, has a normalizing effect onmembrane processes after treatment with normal doses of prednisolone.

A potentiated form of prednisolone also prevented the development of de�structive changes in the liver, adrenal glands, and lymph nodes and gastric

27


mucosal erosion, which is typical of long�term treatment with this drug.Activated prednisolone had a normalizing effect on synthetic activity of lym�phocytes, which was suppressed after long�term administration of prednisolone.It was manifested in an increase in the amount of chromatin protein�uncoupledDNA. Hence, functional activity of lymphocytes returned to normal under theinfluence of activated prednisolone.

We conclude that during combined (bipathic) treatment, potentiatedprednisolone has protective (adaptive) activity and abolishes the effect ofprednisolone in the toxic dose*.

The next series of experiments was performed in collaboration withProfessor Tamara Mikhailovna Vorob’eva (Head of the Laboratory of Neuro�physiology and Immunology, Ukrainian Institute of Neurology andPsychiatry). When the protective effect of activated prednisolone wasestablished, the author of this monograph (professional psychiatrist) decidedto develop a new drug for the therapy of alcohol abuse and opium addiction.We asked Tamara Mikhailovna to perform an experimental study with po�tentiated ethanol and morphine. The animals were subjected to chronicintoxication with these substances. These experiments involved the standardneurophysiological behavioral tests, biochemical assays, and immunological

* In some experiments, a potentiated form of prednisolone tended to increase the anti�inflammatory activity of prednisolone. Initially, this fact received little attention.

Table 2.1.Table 2.1.Table 2.1.Table 2.1.Table 2.1. Protective effects of potentiated prednisolone

Significant increase in the number Slight increase in the number of stabof stab granulocytes granulocytes; significant decrease

in the absolute number of monocytes

Decrease in leukocyte count compared Leukocyte count does not differto the control from the control

No effect on peroxide chemiluminescence Activation of peroxidation in blood plasma

Two�fold decrease in adenosine triphosphate Increase in blood ATP concentration(ATP) content in the blood above normal

Two�fold decrease in alkaline phosphatase Slight decrease in alkaline phosphataselevel in neutrophilic leukocytes level in neutrophils

Increase in lactate dehydrogenase activity Lactate dehydrogenase activity in neutrophilsin neutrophils practically does not differ from normal

Decrease in ATPase activity in lymphocytes No changes in lymphocyte ATPase(nearly by 2 times) activity

Prednisolone Prednisolone +potentiated form

28

Ultralow doses

methods. Electrical activity of the brain and self�stimulation of the “pleasurecenter” in the lateral hypothalamus were studied*.

I would like to briefly describe the results of “bipathic” treatment withethanol. It was shown that ultralow dose of test substance modifies the effectof the same substance in normal dose. Activated ethanol had a strong protective(adaptive) effect against alcohol in the toxic dose. It was manifested in the “re�gulation” of animal behavior (e.g., conditioned responses) and normalization ofseveral parameters (electrical activity of the brain, structure of sleep,neurotransmitter balance, and blood alcohol level). It should be emphasized thatthe animals could stimulate the “pleasure center” via a stereotaxic electrode bypressing the lever. However, they refused to perform self�stimulation. T. M. Vo�rob’eva believed that this unusual effect reflects a well�balanced emotional state.Hence, pathological alcohol addiction was reduced in these animals. Furtherclinical trials with ultralow doses of alcohol showed that they have anti�absti�nence properties. An antialcohol drug Anti�E (activated alcohol) was approved bythe Russian Ministry of Health. This drug was manufactured by the “MateriaMedica Holding” Research�and�Production Company beginning from 1998.

Experiments of T. M. Vorob’eva and further clinical trials showed thatpotentiated morphine has a wide range of protective activity during intoxicationwith morphine or opium surrogates. However, this drug was not introduced intoclinical practice.

Our studies revealed the phenomenon of bipathy in 1996 (200�yearanniversary of homeopathy). Followers of Hahnemann performed the effective,but extremely conservative studies for 2 centuries. Homeopaths believed that theprinciple of similarity is absolutely essential for the effectiveness of ultralowdoses. Initially, we shared this opinion. The results of experimental studies in themid�1990s showed that activated preparations have biological activity. However,this fact received insufficient attention of homeopaths. There was a greatdiscrepancy between homeopathic physicians and experimental biologists. Onthe one hand, homeopaths could not ignore the homeopathic doctrine. On theother hand, biologists knew a little about homeopathy and, therefore, could notintroduce the phenomenon of homeopathy into the area of rational scientificknowledge. At the same time, studies of low and ultralow doses were conductedfor a long time. At the beginning of the 1920s, a famous Russian pharmacologistN. P. Kravkov (1924) showed that blood flow variations in the rabbit ear can beinduced by vasoconstricting and vasodilating agents in low concentration (up to10—32 wt %). Further experiments of A. N. Kudrin (1991) revealed that

* The results of this experiment and further studies are described in the monograph“Informational and ontological models of adaptation” (O. I. Epstein et al., 1997) andjoint articles.

29

administration of epinephrine in a concentration of 10—16 wt % has a similareffect on blood flow in frog mesenteric vessels. The effectiveness of phosphatasein a concentration of 10—16 wt % was demonstrated by A. M. Kuzin in 1947.The results of these experiments were published only in 1997. At the beginningof the 1950s, A. Gay and J. Boiron showed that sodium chloride at dilution C27modifies the dielectric constant of water. On the basis of these data, one vesselwith potentiated sodium chloride in the submolar concentration was correctlyselected from 100 vessels (99 vessels with placebo). In 1941, W. Boyddemonstrated that activated mercury chloride in a concentration 10—6 wt %accelerates enzymatic hydrolysis of starch.

The effects of substances in ultralow concentrations on biological objected(primarily on plants) were described by various authors, including G. N.Shangin�Berezovskii (1982, 1986) and L. Kolisko (1953).

In the 1980s, high�technology studies with ultra�diluted solutions were de�voted to the evaluation of their biological activity. A group of investigators underthe direction of Professor E. B. Burlakova (N. E. Emanuel’ Institute of Bio�chemical Physics, 1986) showed that antioxidants at submolar dilution (10—15

M) have a stronger effect on electrical activity of the isolated snail neuron thanthose in the physiological concentration (10—3 M). Further experiments ofE. B. Burlakova et al. (1986, 1990) revealed that ultralow doses have variousbiological effects.

The results of a well�known study by E. Danevas and J. Benveniste (1988)were published in Nature. They showed that high and low doses have a similareffect. Until the present time, this experiment is one of the most “academic”researches in the field of ultralow doses. The authors revealed that degranulationof basophils with surface immunoglobulin E (IgE) may be induced by anti�IgEat concentrations of 10—2�10—120 M. Treatment with the substance in theseconcentrations was followed by successive peaks of degranulation in 40�60%basophils. Molecules of anti�IgE were absent in several dilutions, whichexceeded the Avogadro constant. However, the authors hypothesized that thismethod for preparation of homeopathic dilutions (thorough shaking of thesolution) provides transmission of biological information due to the arrangementof water molecules. Basophil degranulation was also observed in the presenceof other substances at high and low dilutions, including the specific allergen(basophils from allergic patients) and peroxidase (basophils from peroxidase�immunized rabbits). J. Benveniste et al. evaluated the degree of basophildegranulation in the presence of phospholipase A2 from bee venom or pigpancreas, sodium ionophore monensin (up to 90% degranulation at anequivalent concentration of 10—30 M), and calcium ionophores A23187 andionomycin (10—38 M). The specific effect of high dilutions was confirmed byexperiments with ionophores. Degranulation of basophils decreased after the


30

Ultralow doses

removal of the corresponding ion from the extracellular medium (E. Danevaset al., 1988).

These results were reproduced in six laboratories of four universities (ParisSouth University, Toronto University, Jewish University, and Milan University)and published in Nature. However, an Editorial Article (J. Maddox, 1988) hascast doubt on the reliability of these data. In the follow�up period, a similarexperiment was repeated under strict conditions. The results of this study werepublished in the Journal of the French Academy of Sciences (J. Benveniste, 1991).

To bridge the gap between modern biology, medicine, and homeopathy, itwas necessary to follow simple steps. The phenomenological (narrow) view ofultralow doses should be changed to a detailed systemic evaluation of their activity.Except for several researches in the 1980s and mid�1990s, a rational study ofultralow doses was terra incognita. Until recent times, homeopathic remedies shouldmeet simple requirements of Medical Regulatory Authorities in various countries.Hence, even worldwide leaders in the production of homeopathic remedies donot have the experience of high�level experimental studies with ultralow doses.

In discussing the results of studies with bipathic (combined) admi�nistration of ethanol and morphine, T. M. Vorob’eva supposed that the pheno�menon of bipathy is related to biological properties of ultralow doses. Theseproperties should not be associated with the homeopathic doctrine. Sheproposed to perform a detailed study of ultralow doses. Neurophysiologicalstudies of morphine in ultralow doses were performed in 1996.

A large�scale study showed that the systemic effect of activated morphineis qualitatively similar to that of normal�dose morphine. Similarly to morphinein normal doses, potentiated morphine decreased the pain threshold, facilitatedthe acquisition of conditioned reactions in animals, and modulated theemotional state (model of self�stimulation). However, morphine in ultralowdoses did not cause euphoria and addiction. These data could break the tabooof homeopathy, which was associated with the principle of similarity. Morphinein ultralow doses had a strong effect on experimental animals withoutindividualization of treatment.

Tamara Mikhailovna Vorob’eva showed that ultralow doses have a specificbiological activity, which does not depend on individual treatment. It becameclear that this activity is a general property of ultralow doses. Similarly tohomeopathy, bipathy should be considered as a particular approach to the useof potentiated drugs. Morphine and other substances in ultralow doses have littleeffect, which limits the therapeutic use of these drugs. Hence, a particularapplication (homeopathy and bipathy) is of greater importance than a generalapplication. The indication for use, but not the dose, serves as a “watershed”between allopathy and homeopathy. Trials of homeopathic remedies areperformed on healthy volunteers. Homeopathic remedies can cause allergic

31

reactions in some of these individuals. They serve as a clinical criterion for theuse of this remedy. Modern pharmaceutical products are tested on patients. Thehyperergic reaction to these pharmaceutics is considered as a side effect. Theindications for use of “standard” pharmaceutics are based on their general(physiological) properties.

Previous studies of cyclophosphane, phenazepam, and haloperidol showedthat combined treatment with a medical product and potentiated substanceholds much promise. Combined administration of cyclophosphane and ultralowdose of this drug to experimental animals with melanoma, lung cancer, andcarcinosarcoma was followed by an increase in antitumor activity of thecytostatic. The antimetastatic effect of cyclophosphane increased mostsignificantly after bipathic administration (E. N. Amosova, 2003).

To understand the mechanisms of bipathy we evaluated whether thisphenomenon is the prerogative of a living organism, or ultralow dose maymodify the effect of “high” dose in simple physicochemical systems. In vitroexperiments were performed to answer this question. The first study was con�ducted under the direction of Professor A. V. Zakharov and Senior ResearcherV. G. Shtyrlin (Candidate of Chemical Sciences) at the Kazan State University.

A nuclear magnetic resonance study was performed to estimate the effectof potentiation on the kinetics of ATP hydrolysis with citrate buffer atphysiological pH. The rate of hydrolysis was measured after addition ofpotentiated ATP or potentiated buffer (Table 2.2).

The rate of hydrolysis decreased slightly after addition of any componentin a bipathic form.

Our further studies were performed in collaboration with Professor S. I.Petrov (Institute of Oil and Gas). Potentiated preparations of lithium chlorideand mercury nitrate were shown to modulate electroconductivity of a simple


Table 2.2.Table 2.2.Table 2.2.Table 2.2.Table 2.2. Effect of potentiated substances on hydrolysis

1 Reference (3.35+0.07)×10—5

2 Reference (3.45±0.12)×10—5

3 Bipathic (3.02±0.05)×10—5

(with potentiated ATP solution)

4 Bipathic (2.60±007)×10—5

(with potentiated buffer solution)

Note.Note.Note.Note.Note. (1, 2) Reactions with two various reference preparations; (3) addition of 10 vol % potentiatedhomeopathic solution of ATP at dilution C30; (4) addition of 10 vol % potentiated homeopathic solutionof citrate buffer at dilution C30. K, hydrolysis rate constant for ATP in aqueous solutions of the referenceand potentiated sample with citrate buffer at pH 6.7 and T�378K.

№ Hydrolysis rate, K (sec–1)Type of sample

32

Ultralow doses

electrochemical system, which contained these electrolytes in normalconcentrations (S. I. Petrov et al., 2003). Professor M. A. Myagkova et al.(2003) revealed that activated antibodies in ultralow doses have a modulatoryeffect on the antibody�antigen binding constant in EIA. Similar results wereobtained in further experiments of E. A. Dukhanina.

The effect of potentiated antibodies on “standard” antibodies was studiedby means of EIA (E. A. Dukhanina).

A reaction mixture consisted of antigen (sorbed in plate wells), standard antibodies(diluted in phosphate�buffered saline, PBS), and potentiated dilutions. The control systemswere composed of potentiated water and PBS instead of potentiated dilutions and standardantibodies, respectively. The optical density in wells with potentiated dilutions was muchhigher than that in wells with standard antibodies and water. The average optical densityAst+dilut was estimated in four independent experiments with 10�12 samples of potentiateddilutions. This parameter varied from 0.276±0 to 0.643±0.024. The average optical densityAst+water varied from 0.210±0.046 to 0.531±0.026. These values were not the sum of opticaldensities for the reference substance and potentiated dilutions. For a quantitative study ofthe effect, the relative optical density was calculated as follows: (Ast+dilutOAst+water)×100%/Ast+water. The average value was 23.2±7.2%. These data suggest that potentiated dilutionshave a direct effect on the antigen�antibody interaction. Standard antibodies and sorbedantigen in various concentrations were used for a detailed study of this phenomenon. Theconcentration of standard antibodies varied from 0.3 to 100 ng/ml. The content of standardantibodies varied from 5.7±3.0 (at 0.3 ng/ml) to 22.7±3.9% (at 100 ng/ml). Hence, decreasingthe concentration of standard antibodies was accompanied by the reduction of effect.

We discovered the phenomenon of bipathy. During combined (bipathic)administration of ultralow dose and normal dose, the activated preparationalways modifies the effect of the original substance. A potentiated form in vivoand in vitro modifies the effect of the original substance. It should beemphasized that the potentiated preparation modifies not only biological, butalso fine physicochemical properties of the original substance.

As regards the toxic and subtoxic dose* of a medical product, itspotentiated form has a strong protective (adaptive) effect. Sometimes thepotentiated preparation “strengthens” a therapeutic effect of the originalsubstance (e.g., antimetastatic activity of cyclophosphane). Under otherconditions the potentiated preparation has no effect on pharmacological activityof the original substance (e.g., antiinflammatory effect of prednisolone).

Bipathy could become a major direction of activity in the “MateriaMedica Holding” Research�and�Production Company. Moreover, potentiationof well�known pharmaceutics and correction of their toxicity are the urgentproblems. These approaches are developed by the world’s leading pharma�

* The potentiated substance can produce a complex polymodal effect on toxic doses (seeChapter 5).

33

ceutical companies. Unexpectedly, our study gained a new direction. We met afamous scientist and one of the leading specialists in neuroimmunology M. B.Shtark (Academician of the Russian Academy of Medical Sciences). MarkBorisovich thoroughly examined the results of our experiments. He proposedfurther studies on simple biological models to formally confirm the presence ofbipathy. In the opinion of M. B. Shtark, the so�called long�term posttetanicpotentiation (LTPTP) in surviving brain slices serves as an adequate model.Ultrathin sections of the animal brain can retain functional activity for a longtime in a special nutrient medium. LTPTP is a well�known electrophysiologicalphenomenon. This phenomenon has been extensively used in neurobiology formany years. Neurotropic activity of various drugs may be evaluated from theireffect on LTPTP. The phenomenon of bipathy was studied on the model ofLTPTP with antibodies to S�100 protein from nervous tissue*.

In immunology, any molecule that causes the formation of complemen�tary antibodies is designated as an antigen. Hence, we shall use the term“antibodies to S�100 antigen”.

The description of our experiment may seem complicated to the readerof this book (i.e., general practitioner). Let’s consider only the main results.Antibodies to S�100 antigen (anti�S100) in normal doses have an inhibitoryeffect on LTPTP. Under certain technical conditions, the potentiated substancecompletely abolished a physiological effect of anti�S100 in “normal” doses. Amajor advantage of our experiment is the uniqueness of results. Anti�S100 innormal doses inhibited the electrophysiological reaction (LTPTP—). However,this reaction returned to normal after combined treatment with potentiated anti�S100 (LTPTP+; O. I. Epstein et al., 1999).

This experiment was performed by M. A. Starostina, N. A. Borisov, andN. S. Sorokina under the supervision of M. B. Shtark (Novosibirsk Institute ofMolecular Biology and Biophysics, Siberian Division of the Russian Academyof Medical Sciences). These data are of particular importance for our scientificresearch. A complete description of this experiment is presented below (ac�cording to the monograph of V. G. Zilov et al., 2000).

The study was performed on surviving slices with an artificial medium, which retainsphysiological activity for a long time. It was based on a well�known electrophysiologicalphenomenon of LTPTP. During tetanic electrical stimulation in one of the regions of rathippocampal slice, the evoked postsynaptic potential is recorded in another region with aspecial electrode. This potential persists for a long time (from 40 min to several hours) andhas specific characteristics. Synaptic effectiveness (transmission) is evaluated by recordingof the potential.


* An unusual name of this protein is associated with the procedure of isolation. In oneof the stages, this protein is dissolved in 100% saturated solution of ammonium sulfate.

34

Ultralow doses

Fig. 2.1.Fig. 2.1.Fig. 2.1.Fig. 2.1.Fig. 2.1. Scheme of an experimental chamber to study LTPTP in survivinghippocampal slices.

The incubation medium is delivered from reservoir 1, passes successively through polyvinyl tubes 2,tap 3 (regulation and maintenance of the flow rate at 250 ml/min), and dropper 4 (prevention of airbubble formation in the system), and enters compartment 6 (heat�capacity fluid). The constanttemperature of this fluid was maintained using thermostat 5. The incubation medium was deliveredthrough tube spirals in compartment 6, heated to a certain temperature, and entered chamber 7 (5�10 ml in volume). The medium outflowed from chamber 7 through water�jet pump 8. Incubationchamber 7 was closed with cap 10. The fluid in chamber 7 was heated and aerated with carbogen,which contributed to water evaporation and carbon dioxide exchange between the medium and air.Cap 10 had the only hole above chamber 9 for the release of water vapor and CO2 that moistenedthe above�chamber space and prevented a change in pH.

1

2

5

4

3

5

6

79

10

8

The development of LTPTP is a calcium�dependent process. S�100 protein is acalcium�binding protein, which has an important role in synaptic processes. The antiserumto S�100 protein inhibits these processes, including LTPTP.

However, the synaptic effect was abolished after bipathic (combined) administrationof antiserum in normal dose and potentiated form C6 (10—12 wt %). A homeopathic doseof the substance (i.e., immunological preparation) blocked the action of an effective dose.

It is important that this experiment involved an electrophysiological method, whichallowed us to repeat the measurements under standard conditions.

This series was performed to compare the effects of antibodies toneurospecific protein S�100 and potentiated sample of the same antibodies. Theinfluence of combined (“bipathic”) treatment with these antibodies was studiedon the model of LTPTP in the hippocampus of animals.

Experiments were performed on hippocampal slices from Wistar ratsweighing 180�299 g. Transverse hippocampal sections (400 m in width) wereplaced in a temperature�controlled chamber at 35�37oC (Fig. 2.1). FlowYamamoto medium (Fig. 2.2) was aerated by carbogen (95% O2 and 5% CO2).

35

Evoked postsynaptic potentials (EPSP) were recorded after 40�60�minincubation. A stimulatory electrolytically sharpened bipolar wolfram electrodewas introduced into the zone of mossy fibers. A reference glass electrode (tipthickness 3�4 m, resistance 2�5 mO) was filled with 2.5 M NaCl and placed inCA3 region (initial segments of apical dendrites; Fig. 2.3).

Testing was performed with single rectangular pulses (duration 200 msec)delivered at intervals of not less than 5 min. The amplitude of test stimuli varied

Fig. 2.2.Fig. 2.2.Fig. 2.2.Fig. 2.2.Fig. 2.2. General view of an experimental device to study LTPTP in survivinghippocampal slices: chamber with a flow system, micromanipulators that carrycathode followers, preamplifiers, and stimulating and reference electrodes. Positionof electrodes is shown in Fig. 2.3.

Fig. 2.3.Fig. 2.3.Fig. 2.3.Fig. 2.3.Fig. 2.3. Scheme for the position of a reference (1) and stimulating electrode (2)to study the dynamics of LTPTP in surviving hippocampal slices. CA1�4, fields ofthe Ammon’s horn (hippocampus); DF, dentate fascia.


СА1

СА2

СА3

21

СА4DF

!!!!!!!!!!!!!!!!!!!

!

36

Ultralow doses

from 10 to 30 V. EPSP were recorded on a 12�digit analog�to�digital converter(Digidata, Axon Instruments Inc.). The results were analyzed on a computerwith pClamp�6 (Axon Instruments Inc.) and Microcal Origin softwares.

To induce LTPTP, the amplitude of a test stimulus was selected so thatthe response corresponded to 50% of the maximum value. Tetanization wasproduced by three consecutive series of stimulation at 200 Hz. The length ofeach series was 1 sec. Stimulation was applied at 2�sec intervals. The procedureof tetanization was repeated after 10 min. EPSP were recorded for at least 40min after the first tetanization, which allowed us to make a conclusion aboutthe induction or absence of LTPTP. A significant increase in the amplitude ofEPSP (by 1.5�2 times), which persisted for at least 20 min after the secondtetanization, served as a criterion for the induction of potentiation.

The effect of antibodies to S�100 protein was studied as follows.Tetanization was induced in one or two slices of each series. Furtherexperiments with slices of this series were performed only after the induction ofLTPTP. All slices were maintained in the incubation medium after addition ofa specified amount of antibodies or reference solutions. The initial latency ofeffect was considered to be 20 min (according to D. Levis and T. Teyler). Theeffect of antiserum to neurospecific protein S�100 on the induction of LTPTPin rat hippocampal slices was described previously (D. Levis et al., 1986). Thenthe period of incubation was selected experimentally. After study of eachdilution, the chamber was repeatedly washed with distilled water and ethylalcohol and completely dried with compressed air.

The indication of LTPTP is characterized by a significant increase in theamplitude of EPSP in synapses of mossy fibers in the hippocampal dentatefascia in response to the test stimulus after tetanization (Fig. 2.4).

Twenty�minute incubation with antiserum to neurospecific protein S�100(anti�S100, final dilution 1:50) completely inhibited the induction of LTPTP

Fig. 2.4.Fig. 2.4.Fig. 2.4.Fig. 2.4.Fig. 2.4. Dynamics of EPSP in CA3 region of the Ammon’s horn during extracellularrecording. Amplitude of the test stimulus is 20 V. (1) Before and (2) 10 min aftertetanization.

0

1

2

0

5

5 10 15 20

mV

Time, μsec

37

(Fig. 2.5), which is consistent with the results of previous experiments. Highdilution of antiserum in our experiments is related to differences in the titer ofantibodies at various laboratories.

Nonimmune rabbit antiserum at the same dilution had no effect onLTPTP induction in rat hippocampal slices (Fig. 2.6). Incubation of slices withethanol at a concentration present in potentiated preparations (similar dilution)did not impair the induction of LTPTP in slices (Fig. 2.7).

To study the combined effect of test samples, anti�S100 and itspotentiated form were added simultaneously to the incubation medium. Thistreatment completely blocked the induction of LTPTP in slices. It should beemphasized that combined administration of these substances did not abolish

Fig. 2.5.Fig. 2.5.Fig. 2.5.Fig. 2.5.Fig. 2.5. Effect of anti�S100 on the induction of LTPTP in hippocampal slices.Ordinate, amplitude of EPSP. (1) Before treatment with anti�S100; (2�5) over 20min after treatment with anti�S100 (5�7�min interval); and (6, 7) 7 and 12 min afterthe second tetanization, respectively. Amplitude of the test stimulus is 30 V.

10

0.1

0.2

0.3

0.4

0.5

32 4 6 75

mV0.6

Fig. 2.6.Fig. 2.6.Fig. 2.6.Fig. 2.6.Fig. 2.6. Induction of LTPTP in the presence of nonimmune rabbit serum. Dilution1:50. Ordinate: average amplitude of EPSP. (1) After 10�min incubation in non�immune serum; (2, 3) 5 and 10 min the first tetanization, respectively; and (4�6)over 20 min after the second tetanization. Amplitude of the test stimulus is 12 V.

10.0

0.2

0.4

0.6

0.8

1.0

32 4 65


38

Ultralow doses

Fig. 2.7.Fig. 2.7.Fig. 2.7.Fig. 2.7.Fig. 2.7. Induction of LTPTP after addition of 40 ml 40% ethanol. Volume of anexperimental chamber is 10 m. Ordinate: average amplitude of EPSP. (1, 2) 20�minincubation in Yamamoto medium after addition of ethanol; (3) 10 min after thefirst tetanization; and (4, 5) 10 and 30 min after the second tetanization,respectively. Amplitude of the test stimulus is 15 V.

10.0

0.2

0.4

0.6

0.5

0.3

0.1

32 4 5

the effect of anti�S100. Similar results were obtained after 10�min preincubationof the slice with potentiated anti�S100, further addition of anti�S100, and 20�min incubation in a solution of both substances.

The effect of anti�S100 was abolished after 20�min preincubation of slicesin a solution of potentiated anti�S100 (concentration 10�12) and 20�minincubation in a solution of native and potentiated antiserum. Hence, theinduction of LTPTP in slices was similar to that in control samples not exposedto antibodies (Figs. 2.8 and 2.9).

It could be suggested that the effect of native anti�S100 is abolished due to long�term incubation of the slice with an ethanol�containing solution of the potentiated form,which results in modulation of the membrane state and/or impairment of antibody�antigenbinding. The next series was performed to test this hypothesis. Preincubation was performedin an ethanol solution, whose concentration did not differ from that in potentiated anti�S100. Other manipulations were similar to those in the previous series. Under theseconditions, anti�S100 retained the ability to block the induction of LTPTP.

These data show that nonimmune serum and 40% ethanol did not prevent theinduction of LTPTP. The inhibition of LTPTP was observed only in anti�S100 solutions.Preincubation with potentiated anti�S100 for 20 min abolished the inhibition of LTPTP byanti�S100. This procedure did not prevent a normal reaction of the hippocampal CA3region, which had a potentiating effect on synaptic effectiveness.

A study of the model of LTPTP provides strong evidence for thephenomenon of bipathy. The experiment had unexpected consequences. Indiscussing the results of this research, we hypothesized that activated antibodiesexhibit inexplicable “pro�antigenic” activity. T. M. Vorob’eva and M. B. Shtarkdid not exclude this possibility. However, it was necessary to confirm ourhypothesis. Studies with potentiated antibodies to various neurotropic

39


Fig. 2.8.Fig. 2.8.Fig. 2.8.Fig. 2.8.Fig. 2.8. “Bipathic effect”.

(a) Induction of LTPTP in thepresence of anti�S100 (final dilution1:50): (1�3) incubation in Yamamotomedium with anti�S100 for 20 min,interstimulus interval 5�7 min; (4�6)10 min after the first tetanization, 3�4 min intervals; and (7, 8) 10 and 25min after the second tetanization,respectively. Amplitude of the teststimulus is 12 V.

(b) Induction of LTPTP in thepresence of potentiated anti�S100 ata concentration of 10–12 (40 mmol):(1�3) incubation in Yamamotomedium with potentiated anti�S100(10�12) for 20 min, interstimulusinterval 5�7 min; (4�6) over 10 minafter the first tetanization, 3�4 minintervals; and (7�11) over 30 minafter the second tetanization, 5�7�min intervals. Amplitude of the teststimulus is 20 V.

(c) Induction of LTPTP in the pre�sence of anti�S100 at a concentra�tion of 10—12 (40 mmol) and dilution1:50: (1�3) incubation in Yamamotomedium with potentiated anti�S100at a concentration of 10–12 for 20min, interstimulus interval 10 min;(4�6) incubation with potentiatedanti�S100 for 20 min, 5�7�minintervals; (7�10) over 10 min afterfirst tetanization, 2�3�min intervals;and (11�21) over 40 min after thesecond tetanization, 3�5�minintervals. Amplitude of the teststimulus is 10 V.

10.0

0.2

0.4

0.6

0.8

1.0mV

32 4 7 865

a

10.0

0.2

0.4

0.6

0.8

1.0

1.2

mV

32 4 7 8 9 10 1165

1.4b

0.0

0.5

1.0

1.5

2.0

mV

5 15 2010

c

substances, including morphine, delta sleep�inducing peptide, histamine, andserotonin, were performed in Kharkov and Novosibirsk. These investigationssupported our hypothesis. Under various conditions, potentiated antibodies andantigen had “codirectional” activity. It became obvious that this is a newimmunological phenomenon. In standard immunological reactions, binding ofantibodies to the complementary antigen is followed by the inhibition of its

40

Ultralow doses

Fig. 2.9.Fig. 2.9.Fig. 2.9.Fig. 2.9.Fig. 2.9. “Bipathic effect: induction of LTPTP under “bipathic” conditions: (1, 2)20�min preincubation with potentiated anti�S100 (10–12); (3, 4) incubation withnative anti�S100; (5) after the first tetanization; and (6, 7) 5 and 10 min after thesecond tetanization, respectively.

10.0

0.2

0.4

0.6

32 4 6 75

V, mV

0.7

activity. By contrast, antibodies in ultralow doses modify the activity of thisantigen.

We asked T. M. Vorob’eva to perform the next series of experiments.Several pairs of potentiated antibodies and antigen (S�100 protein andantibodies to S�100 protein; morphine and antibodies to morphine; delta sleep�inducing peptide and antibodies to delta sleep�inducing peptide; etc.) werestudied on the model of behavior and brain self�stimulation. It was shown thatpsychotropic activity of antibodies in ultralow doses is higher than that ofantigen in ultralow doses.

It should be emphasized that during this period the author of thismonograph was not familiar with immunology. Similarly to the phenomenonof bipathy, we assumed that there is a mediator between activated antibodiesand antigens (endogenous molecules, i.e., S�100 protein) in the organism. I.P. Ashmarin and I. S. Freidlin hypothesized that the so�called naturalantibodies have regulatory functions (I. P. Ashmarin et al., 1989). Thishypothesis developed the theory of a famous immunologist Pierre Grabarabout the physiological role of autoantibodies (P. N. Grabar, 1969). Undernormal conditions, nearly all molecules in the organism have “predetermined”antibodies in very low physiological concentrations. These antibodies exhibitaffinity for the corresponding antigens and stabilize, but not inhibit theiractivity. We suggested that natural antibodies serve as a target for activatedantibodies in the organism. The effect of antibodies in ultralow doses inmediated by a change in physiological functions of predetermined antibodies(“regulation of regulator”).

Scientific collaboration between the Institute of Molecular Biology andBiophysics (Siberian Division of the Russian Academy of Medical Sciences),

41

Ukrainian Institute of Neurology and Psychiatry, and “Materia MedicaHolding” Research�and�Production Company resulted in the development ofthree medical products with activated antibodies of a new class (Proprotein�100,Anar, and Tenoten). An antialcohol drug Proprotein�100 contains antibodies toS�100 at dilution C1000. This is the first drug of a new pharmacological class.Tenoten and Tenoten for children were synthesized from activated antibodies toS�100 protein in other doses. They have a wide range of pharmacologicalactivity. Anar is a drug for the therapy of opium withdrawal syndrome, whichcontains ultralow doses of antibodies to morphine.

The discovery of a new immunological phenomenon in 1998 wasaccidental. However, this discovery was associated with the results of previousstudies with ultralow doses. The phenomenon of bipathy was historicallypreceded by isopathy. However, it was impossible to foresee new properties ofpotentiated antibodies. Before 1998, there was only one historic “junction”between ultralow doses and immunology. J. Benveniste showed that anti�IgEantiserum causes degranulation of basophils (E. Danevas et al., 1099). It couldbe suggested that treatment with anti�IgE in ultralow and normal dose causesthe same physiological phenomenon. However, new properties of activatedantibodies were not revealed due to technical reasons. These studies should beperformed on another experimental model (i.e., monospecific antibodies).Moreover, the phenomenon of bipathy should form the basis for a newideology.

After “accidental contact” with immunology, we studied the basicprinciples of this relatively young and rapidly developing area of biology andmedicine. It was unexpected that homeopathy and immunology have commonroots. Biologically, homeopathy is based on individual sensitivity. The immunesystem maintains the individual and genetically determined integrity in anorganism. The knowledge of homeopathy and results of studying the biologicalactivity of ultralow doses provide new insight into physiological functions of theimmune system (see Chapter 4).

It became evident that preparations of antibodies should bedeveloped only by qualified pharmacologists. Rational scientists express skep�ticism about ultralow doses. We are grateful to E. D. Gol’dberg (Academicianof the Russian Academy of Medical Sciences) and A. M. Dygai (Academicianof the Russian Academy of Medical Sciences) from the Institute of Phar�macology (Tomsk Institute of Pharmacology, Siberian Division of the RussianAcademy of Medical Sciences). Despite dogmatic statements, our collaborativestudies of ultralow doses were initiated 9 years ago. The Tomsk Institute ofPharmacology is equipped with specialized laboratories for a variety of pharma�cological investigations. The collaborative study yielded high�value results. Agroup of medical products from activated antibodies was developed.


42

Ultralow doses

Besides the study of potentiated substances, Tomsk specialists made acontribution to the introduction and popularization of a new class of medicalproducts. Some remedies with ultralow doses were developed in collaboration withthe Tomsk Institute of Pharmacology and extensively used in clinical practice. Theyinclude antibodies to the following endogenous molecules (antigens): IFN�γ(Anaferon and Anaferon for children), tumor necrosis factor�β (TNF�β, Artrofoon),endothelial NO synthase (Impaza), prostate�specific antigen (Afala), etc. During ourcollaboration with Tomsk scientists, the basic mechanisms for action of antibodiesin homeopathic doses were evaluated. For example, our experiments showed thatpotentiated antibodies to the endogenous regulator modify its expression. Moreover,potentiated antibodies have a modulatory effect on functional and metabolicprocesses that are associated with this regulator (O. I. Epstein et al., 2004).

Collaborative studies with Tomsk scientists confirmed the fact that that thesystem of natural antibodies has a role in the effect of antibodies in ultralowdoses. A clinical trial of potentiated antibodies to S�100 protein and morphinein patients with alcohol and heroin intoxication, respectively, was performed atthe Institute of Mental Health (Tomsk Research Center, Siberian Division ofthe Russian Academy of Medical Sciences). Administration of drugs withultralow doses of antibodies improved the somatopsychic state of these patientsand had a normalizing effect on natural (predetermined) antibodies to S�100protein and opiates, respectively (T. P. Vetlugina, 2003; N. A. Bokhan et al., 2003).Further studies showed that activated antibodies to IFN�γ have a normalizingeffect on natural antibodies to this endogenous regulator. For example,chickenpox patients have the increased level of natural antibodies (A. Caruso etal., 1997). The amount of natural antibodies in these patients rapidly returnedto normal after treatment with antibodies to IFN�γ in ultralow doses (ascompared to the control group).

Then clinical trials of new drugs had a major role in applied studies.During clinical trials, the scientists had to overcome skepticism about substancesin ultralow doses. We thank V. I. Petrov (Academician of the Russian Academyof Medical Sciences) and specialists from the Volgograd Medical University forthe first systemic study with a new class of medical products in clinicalpharmacology. Our collaboration with Volgograd colleagues resulted in thedevelopment of a new drug Kardos for the therapy of chronic heart failure. Thispreparation of antibodies to the angiotensin II receptor in ultralow doses wasinitially considered as a hypotensive drug.

The major contribution to a detailed clinical study of potentiated drugswas made by famous Moscow scientists E. B. Mazo (Corresponding Memberof the Russian Academy of Medical Sciences), V. F. Uchaikin (Academician ofthe Russian Academy of Medical Sciences), and V. N. Yarygin (Academicianof the Russian Academy of Medical Sciences).

43

In recent years, advanced clinical and experimental studies were headedby Professor S. A. Sergeeva (Scientific Director of the “Materia Medica Hol�ding” Research�and�Production Company). Svetlana Aleksandrovna built acreative team of young scientists. They summarized and systematized the dataon pharmacology of ultralow doses.

Collaborative studies of several research institutes and “Materia MedicaHolding” Research�and�Production Company to develop a new class of medicalproducts from ultralow doses of antibodies were awarded with the Prize of theGovernment of the Russian Federation in 2005. Pupils of S. A. Sergeevareceived the same prize for a detailed investigation of Tenoten in 2006 (category“Young Scientists”). These experiments were performed in collaboration withthe Institute of Neurology (Russian Academy of Medical Sciences).

A famous scientist Professor T. A. Voronina also made a contribution tostudy of the mechanisms for action of activated products. Before ourcollaboration, a qualified psychopharmacologist Tat’yana Aleksandrovna had theexperience of working with ultralow doses.

The author thanks K. V. Sudakov (Academician of the Russian Academy ofMedical Sciences) for attention to our theoretical problems. In 1997, KonstantinViktorovich supported the idea of “horizontal” and “vertical” regulation*. Heproposed a collaborative approach to ultralow doses. We were greatly influenced bythe theory of K. V. Sudakov about the holographic principle of functional systemsand quantization of vital activity. This theory is close to our notion of structuralorganization of the organism. Our collaboration with K. V. Sudakov and V. G.Zilov (Academician of the Russian Academy of Medical Sciences) yielded themonograph “Elements of information biology and medicine” (2000).

A new class of medical products was developed during a difficult periodof transition in Russia. Despite difficulties, the collaboration between allparticipants of this scientific research opened a new chapter in the history ofpharmacology.

Most important in Chapter 2

Three types of effects of ultralow doses were evaluated.

“Direct” fine molecular�and�cellular activity (confirmed by modern evidentialstudies by E. B. Bulakova, J. Benveniste, and other scientists in the 1980s and1990s).

The phenomenon of homeopathy was discovered by S. Hahnemann in study�ing the effect of ultralow doses on healthy volunteers (1976). According to


* This problem is discussed in Chapter 3.

44

Ultralow doses

modern notions, this phenomenon can be considered as a hyperergic reactionof the organism to ultralow doses.

Bipathy (i.e., combined treatment with a medical product in normal dose andits activated form) suggests that ultralow dose modifies the effect of normal dose.The phenomenon of bipathy was demonstrated on biological and physicochem�ical systems. Clinical and experimental studies showed that potentiated antibod�ies modify, but do not inhibit the activity of a specific antigen (as differentiat�ed from antibodies in normal doses). This is the phenomenon of antibodies inultralow doses.

45

In the mid�1990s, we knew the following three types of effects of ultralowdoses: direct activity and phenomena of homeopathy and bipathy.

Homeopathy and bipathy are particular cases of the activity of ultralow doses(Fig. 3.1). S. Hahnemann studied the particular (i.e., hyperergic reactions toultralow doses). It was technically impossible to reveal and evaluate the generalproperties of potentiated products. The latter fact emphasizes the role of thisscientist, who developed a new integral clinical�and�therapeutic discipline. Inthe epoch of bloodletting and leeching (200 years ago), homeopathy was themost effective therapeutic method.

Potentiated products exhibit activity with no concern for the homeopathicdoctrine. However, this fact did not facilitate an understanding of the nature ofultralow doses. By contrast, this situation was similar to the mathematicalsystem of equations with unknowns. The greater was the number of questionsabout ultralow doses, the stronger was the desire to evaluate the mechanism oftheir action. It was at least necessary to systematize the data and to develop anew approach to the understanding of these facts.

The direct activity of ultralow doses should be evaluated to under�stand the mechanisms of their action. What do we know about ultralow doses?Experiments of J. Benveniste and E. B. Burlakova demonstrated that highlydiluted solutions exhibit physiological activity. This activity has the so�calledpeaks or extremes. E. B. Burlakova revealed that ultralow dose retains the “split”(reduced) activity of a substance. Hence, this substance may produce only some

C h a p t e r 3

Dual organizationof vital activity

46

Ultralow doses

of the effects. However, the opposite results were not revealed in ourexperiments and published data. Ultralow dose does not have the specific targetsthat are untypical of the initial dose. Hence, the effects of ultralow and initial doseare qualitatively similar. This conclusion was made by E. B. Burlakova and J. Ben�veniste. Experiments of T. M. Vorob’eva produced similar results for potentiatedethanol and morphine (O. I. Epstein et al., 1996b; V. G. Zilov et al., 2000).

The effects of activated products are smaller than those of the initial dose.Experiments with the isolated neuron from Helix pomatia showed that the effectof activated substances cannot compete with the influence of drugs in normaltherapeutic doses (O. I. Epstein et al., 1999b). The results of this study aredescribed below.

Experiments were performed on the identified spontaneously active neurons ofsubesophageal ganglia V2�V6, PPa1, and PPa2 from 28 edible snails. The snails were in anactive state for at least 2 weeks before study. The following parameters were measured:resting potential (RP), action potential amplitude (Vo), time derivative of the actionpotential (AP), maximum rate of rise of AP (Vmax), spike discharge frequency, andcurrent�voltage and inactivation characteristics of ion channels for inward and outwardcurrents. In some measurements with isolated neurons, calcium currents were recorded bythe voltage clamp technique. A simulating electrode was connected to the output of afixation block. The value of current to hold the membrane potential at a certain level wasmeasured with an amplifier. The signal from this amplifier was delivered to the secondinput of an oscillograph.

Under control conditions, the substitution of physiological saline for nonimmuneserum or antiserum to sheep erythrocytes had no effect on electrical properties of themembrane (Fig. 3.2).

Fig. 3.1.Fig. 3.1.Fig. 3.1.Fig. 3.1.Fig. 3.1. Three types of effects of ultralow doses.

Technologies of potentiation

Ultrahigh dilutions

General propertiesHomeopathy Bipathy

• hyperergic reactions • reduced activity;• polymodal dose�dependence

• modifying properties

Ultralow doses of antibodies

• normalizing effect on the sys�tem of natural antibodies

↓

↓

→ ←

↓

47

Vmax decreased sharply 20 min after application of antiserum to S�100 protein (AS�100) at antibody dilutions of 0.2 and 2% (by 22�28 and 37�45%, respectively). Vmaxdecreased by 60�80% over the first 10�15 min after treatment with AS�100 at dilutions of6 and 12% (Fig. 3.2).

Similar results were obtained in experiments with potentiated AS�100 (pAS�100) inconcentrations of 10�2 and 10�400 wt %. Vmax decreased by 14�8% over 30�35 min (Figs.3.2�3.4).

Independently on the concentration of antibodies, the action of AS�100 on inwardcurrent channels is followed by a decrease in the strength of current and increase in thesteady�state inactivation at zero conditioning pulse. Vmax and inactivation curves wereshifted toward negative values of the membrane potential (Fig. 3.4).

The pAS�100�induced reduction of inward current was mainly associated with adecrease in the maximum conductance of inward current channels, but not with anincrease in the steady�state inactivation. These changes probably contribute to an AS�100�induced decrease in the amplitude of AP. Similar results were obtained inexperiments with pAS�100 at various dilutions. Outward current remained unchangedunder these conditions (Fig. 3.5).

Potentiated AS�100 decreased the amplitude of AP and inward current, but had littleeffect on leakage current. These data indicate that AS�100 at normal dilutions andpotentiated form has a depolarizing effect (Fig. 3.6).

Studying the influence of antiserum to S�100 on functional activity of isolatedneurons showed that all dilutions of this substance (e.g., potentiated dilutions atconcentrations of 10�2 and 10�400 wt %) have a similar, but quantitatively different effect.This is manifested in membrane depolarization, decrease in the amplitude of AP, increasein the maximum rate of rise of AP, decrease in the maximum conductance, andinactivation of channels. A change in electrical properties of the membrane becomes lesssignificant with a decrease in the dilution of antiserum to S�100 (V. G. Zilov et al., 2000).

Fig. 3.2.Fig. 3.2.Fig. 3.2.Fig. 3.2.Fig. 3.2. Maximum rate of rise of the action potential in giant neurons of snailsubesophageal ganglion after application of antibodies to S�100 antigen at variousdilutions.

50

20

40

60

80

100%

10 15 20 25 30 35

10-400

10-12

0.2%

2%

6%

12%

Time, min

Control

Chapter 3. Chapter 3. Chapter 3. Chapter 3. Chapter 3. Dual organization of vital activity

48

Ultralow doses

Fig. 3.3.Fig. 3.3.Fig. 3.3.Fig. 3.3.Fig. 3.3. Amplitude of the action potential after application of potentiated antiserumto S�100 protein. (1) Nonimmune serum, dilution 1:5; (2) potentiated AS�100 in aconcentration of 10�400 wt %; (3) 0.2% dilution of AS�100; (4) 2% dilution of AS�10;and (5) 6% dilution of AS�10.

05

1

2

3

4

5

10 15 20 25 30 35 40 45

Время, мин

80

85

90

95

100

%105

50

Fig. 3.4.Fig. 3.4.Fig. 3.4.Fig. 3.4.Fig. 3.4. Resting potential of giant neurons from H. pomatia after application ofpotentiated and non�potentiated antiserum to S�100 protein. (1) Nonimmuneserum; (2) potentiated AS�100 in a concentration of 10�400 wt %; (3) potentiatedAS�100 in a concentration of 10�2 wt %; (4) 2% AS�100; and (5) 6% AS�100.

05 10 15 20 25 30 35

Time, min

80

85

90

95

100

%

1

2

3

4

5

105

40

49

Fig. 3.5.Fig. 3.5.Fig. 3.5.Fig. 3.5.Fig. 3.5. Current�voltage characteristics of inward current channels in themembrane of giant neurons from snail subesophageal ganglion after applicationof physiological saline (1), antibodies to S�100 antigen at 12% dilution (2), andpotentiated antiserum to S�100 protein in a concentration of 10�2 wt % (3). Initialmembrane potential 43 mV.

�50 �40 �30 �20 �10

10

2

1

2

3

�1

�6

�5

�4

�3

�2

4

6

8

10

12

14

16

20 30 40 50 60 70

5x10�8 A

mV

Fig. 3.6.Fig. 3.6.Fig. 3.6.Fig. 3.6.Fig. 3.6. Action potential generation in snail neurons under the influence of pAS�100 in concentrations of 10�60 (C30) and 10�2 wt % (C6). PS, physiological salinefor H. pomatia; NS, nonimmune serum.

10 mV

15 msec

О

PS PS

PS С6

NS С30

10 mV

40 msec

О О О


50

Ultralow doses

The activity of ultralow doses depends on the method of preparation(scale of dilution, number of dilutions, and conditions of dilution), but not onthe content of the original substance. Hence, the preparation of ultrahighdilutions is standardized. Our experiments were not directed toward studying thedose dependence of ultra�diluted solutions. However, it became obvious thatsuch dependence exists. This fact is of considerable importance. It seemed thatthe dose dependence has a polymodal (cyclic) nature. We used the standardhomeopathic dilutions C12, C30, C200, C1000 and, more rarely, C6 and C50.The effects were more pronounced at low (C16, C12, and C30) or highdilutions (C200 and C1000). In some experiments, ultrahigh dilutions did notdiffer in activity. For example, the effectiveness of dilutions appeared as follows:C1000>C30=C200.

Neurobiological studies confirmed a well�known homeopathic fact thatthat the maximum neurotropism is typical of high dilutions C1000 and C200.These investigations showed that a mixture (chords) of various dilutions causesthe cumulative effect. For example, experiments with ultralow doses of anti�S100 revealed that the mixture of dilutions C12+C30+C200 has a strongerantianxiety effect that C1000. The original substance and its ultralow dosesalways produce a unidirectional systemic effect. Sometimes, the direction of localeffects of potentiated substances depended on the dose. In experiments of T. M.Vorob’eva, activated ethanol was given to animals after 2�week consumption ofalcohol in high doses. Blood alcohol concentration decreased after administra�tion of potentiated alcohol in dose C30, but increased in treatment with doseC200. Dose C30 decreased, while dose C200 increased the contents ofdopamine and serotonin in the brain. It should be emphasized that potentiatedalcohol in doses C30 and C200 had a protective effect during chronic alcoholintoxication (A. M. Titkova et al., 2002).

Submolar dilutions produce the dose�dependent effect. Hence, the proper�ties of ultra�diluted solutions are determined by the technology of preparation.The effects of “molar” dilutions C6 and C12 with molecules of the original sub�stance are similar to those of “submolar” dilutions C30, C50, and C200 (O. I.Epstein et al., 2004). These data indicate that the activity of ultra�diluted solu�tions depends on the method of preparation (potentiation), but not on theconcentration of the original substance. A slight increase in the dose of medicalproduct (by several micrograms) has little effect on its properties. Duringcombined (bipathic) administration of the medical product and its activatedform, the latter compound in vitro and in vivo modifies the effect of the originalsubstance. Therefore, the phenomenon of bipathy illustrates that the activity ofultralow doses is determined by the process of preparation.

The technology of potentiation allows us to retain some basic pro�perties of the original substance in ultra�diluted solutions. Homeopathic studies

51

showed that the ability of ultra�diluted solutions to produce a therapeutic effectis observed for a long time (several years). The data show that potentiationprovides not only activity, but also stability of these solutions. After preparationby the method of Hahnemann, potentiated solutions gain the unique modifyingproperties. Despite the fact that activated products were used in medical practicefor 200 years, the mechanism of potentiation remains unclear. It is poorlyunderstood how a potentiated solution “remembers” the properties of theoriginal substance. Why these properties are transmitted to a solid pharmaceuti�cal form (carrier)? It remains unknown which mechanism is responsible foractivity of these preparations in the organism.

During potentiation, molecules of the solvent (water, alcohol, or water�alcohol mixtures) are probably arranged in a certain cluster order around thedissolved substance. Physical properties of this substance are “fixed” in the spa�tial organization of clusters. They retain this information even when dilutedbeyond Avogadro’s number (10�24 M, no molecules of the original substance).

Some authors believe that the formation of these clusters results fromspatial reconstruction of an aqueous medium. For example, 912 water moleculesare arranged in the cubic structure and interact with each other by the principleof charge complementarity (S. Zenin, 1999). Otherwise, these clusters consistof fluid subsystems (e.g., proton subsystem; F. R. Chernikov, 1998). Indepen�dently on the basis of solution “memory” (water molecules or submolecularparticles), the potentiated solution is structured according to a fine organizationof the original (dissolved) substance. Solvent molecules are in constant motion.Therefore, the potentiated solution is characterized by a dynamic structure. Thisis a stable dynamic constellation of solvent particles (organized spatial structure).

The structure of any substance in normal doses determines its physico�chemical and biological properties. Surprisingly, this structure may be accuratelyperceived and stored in the solution. Even without the molecule, its image andproperties are retained in dynamic constellations of molecules in the solution.The structure of this “missed” molecule is a semantic content of constellations.This fact is of considerable importance for the theory of ultralow doses. Weproposed the term “semantically organized molecular constellations”* (O. I. Ep�stein, 2002). The term “molecular constellations” was introduced by A. G. Gur�witsch. He proposed the concept of biological field (A. G. Gurwitsch, 1944).The field is considered as an integral factor, which determines the direction andorder of biological processes. According to A. G. Gurwitsch, the constellationsconsist of molecular ensembles that are combined by a common suprasystemicfactor. However, A. G. Gurwitsch did not consider the physical nature of re�lationships in constellations.


* Constellation (Latin name).

52

Ultralow doses

During potentiation, the memory of various substances is retained in thesolvent. It may be suggested that specific physicochemical properties of thedissolved substance are stored in the structure (relationships), but not in thenature of associations in constellations. Despite constant motion of moleculesin a solution, memory of the original substance is retained in constellations(even during strong heating and cooling of potentiated solutions).

The process of potentiation is a surprising empirical discovery of Hah�nemann, which comprises the following two factors: successive dilution andexternal influence. This process requires fundamental physical investigations.

Studies of the bipathic phenomenon on simple models showed that acti�vated solutions and, therefore, process of potentiation may be analyzed by stan�dard physicochemical methods*. We hope that this problem will be of interestfor physicists. However, several conclusions were made about the process ofpotentiation.

The external influence** (10�sec mechanical shaking by Hahnemann)probably provides an additional energy source for structural reconstruction ofthe solution. Repeated treatment has proper oscillation characteristics.Hypothetically, the mechanisms of resonance contribute to the selection andstorage of a typical vibration spectrum in the solution. A well�known study byJ. Benveniste (see Chapter 2) showed that 10 sec of shaking (according toHahnemann) is the minimum time required for activation of a potentiatedsolution***.

Repeated removal and further transfer of only one drop from the solutioninto the intact solution probably contribute to the preservation of other “basic”oscillation parameters in the previous dilution of solvent. Repeated dilution maybe considered as a filtration of “information noise”. This fact probably deter�mines an increase in the effect with increasing the number of dilutions (e.g., inexperiments with neurotropic agents).

* There is some controversy as to whether the potentiated solution has particular physicalproperties. We can remember a humorous poem of S. Ya. Marshak:

The matrix of a song and its essenceCannot be made out of thin air by the author.Even God could not create anythingIn the absence of raw material.

** Little is known about other types of external influences (electromagnetic and ultrasoundgenerators).

*** Moreover, some technological principles were demonstrated by J. Benveniste et al.They illustrate an important role of potentiation in the activation of ultra�dilutedsolutions. These authors showed that potentiation can occur in water, ethanol, andpropanol, but not in dimethylsulfoxide. Various substances in usual doses differ inthermal resistance. However, the activity of potentiated products decreases in the sametemperature range (from 70 to 80oC).

53

Oscillation parameters of the electromagnetic spectrum and acoustic rangeare typical of any molecule. Theoretically, they may be stored in newly formedconstellations and determine the properties of these constellations. Thesemantically organized constellations comprise not only solvent molecules, butalso atomic and subatomic particles and specific wave characteristics. The“wave” constituent of constellations probably serves as a basis for distant transferof information about the original (dissolved) substance in a structured solution.These data indicate that constellations may be considered as a wave structure.

The term “semantically organized constellations” suggests the essential orontological interpretation of the notion “information”. Information about the dis�solved substance is an essence or substantive content of constellations. This es�sence combines the relationships between solution molecules into a singlewhole, “couples” the specific wave characteristics, and serves as a complexspatial structure. The notion of semantically organized constellations is ofparticular importance for physics and philosophy. From the standpoint of phy�sics, we postulate the existence of distant intermolecular (information) rela�tionships of unknown nature. Philosophically, semantic constellations are aparticular case when the “spirit” organizes the matter.

The question arises: whether the “coupled” oscillation parameters ofmolecules may form a basis for distant transfer of information about the originalsubstance in constellations? Let’s look at some published data.

It is a well�known fact that all biological objects have weak electromag�netic radiation. Theoretically, weak electromagnetic field may transfer informa�tion about some physiological parameters of the object and affect other biolo�gical structures. Some authors believe that the effect of electromagnetic field onan organism is realized via membrane�bound acceptors with a specific frequencyof coherent oscillations. Due to resonance with these structures, electromagneticimpulses provide synchronization of oscillations in membrane acceptors. Thesereceptors mediate the influence of electromagnetic impulses.

A. G. Gurwitsch (1944), V. P. Kaznacheev (1981), Yu. V. Tszyankan’�chzhen’ (1993) and other investigators attempted to confirm the possibility fordistant transfer of information between biological objects. These studies have twofeatures. First, not all information about the biological object, but only somegeneral (modal) characteristics are distantly transferred under various conditions.And second, the biological objects serve as an inductor of information whentheir vital functions are strongly suppressed or activated under the influence ofsome factors. In experiments of A. G. Gurwitsch and Yu. V. Tszyankan’chzhen’these factors were presented by degradation mitogenetic radiation and stronggrowth of young plants, respectively. In studies of V. P. Kaznacheev, the tissueculture was suppressed by viruses, mercuric chloride, and radiation. This culturewas then connected to the intact culture through an optical channel. A cyto�


54

Ultralow doses

pathic effect was sometimes observed in the intact culture (suppression of vitalactivity). The inhibition or activation of vital functions in the early period afterexposure to a strong external factor was probably followed by synchronization ofphysiological processes (e.g., mitotic activity). Synchronous rhythms of vitalfunctions were accompanied by synchronization of weak electromagnetic radiationfrom biological objects. Electromagnetic field was characterized by modulation andserved as a carrier of some general properties of the biological system in space.

These data suggest that electromagnetic field and, probably, other fieldscontribute to a distant effect on the biological system. It should be emphasizedthat the potentiated product causes a specific response, which differs from othertypes of distant interaction. Our study and other researches showed that ultralowdoses have a reproducible effect, which may be used in practice (asdifferentiated from the majority of distant interactions).

The quintessence of our study is the notion of semanticallyorganized constellation as a unique spatial structure.

It became clear that the principle of similarity is not an exclusiveprinciple. Ultralow doses can produce at least two types of effects. These resultswere absolutely unexpected. It was very difficult to explain a variety of propertiesof potentiated products. The most surprising discovery was that ultralow dosesproduce a specific physiological response in each individual. This response maybe investigated by modern molecular and cellular methods. It was unclear whythe substance in ultralow dose causes “drug exacerbation” only in sensitivepatients. Which mechanisms are triggered by the essence of ultralow dose toinduce a hyperergic reaction? To answer this question, we made the followingsuggestion: not only memory units of the potentiated solution are a semanticallyorganized spatial structure. The whole organism can be also considered as acomplex spatial structure, which consists of molecular constellations. Asdifferentiated from inanimate nature, the spatial structure of biological systemshas dual individual�and�species organization. Due to spatial duality of theorganism, ultralow doses sometimes cause a hyperergic reaction.

However, all facts should be considered one after another. We studied theprocess of potentiation and activity of potentiated products. Potentiation wasdiscovered 200 years ago, but received little attention for a long time. It was aunique chance to investigate a simple physical process at the dawn of the 21stcentury. In natural sciences, there were no data on the phenomenology of thisprocess. We hypothesized that potentiation may be considered as dematerializa�tion, since this process results in “extraction” of the original specific informationstructure from any substance. It could be suggested that the reverse process alsooccurs during evolution (materialization of information). Taking into accountthis hypothesis, we proposed an evolutionary approach to explain the principleof dual organization in constellations.

55

There is no general evolutionary theory. We hold to the idea of Logos thatlife originated from one common source due to self�development. The principleof the original matter is postulated in the Big Bang theory, which suggestssaltatory evolution of the World. We also share the idea of V. I. Vernadsky thatbiotic and non�biotic processes occurred simultaneously, and biosphere forma�tion was a one�time�only process (V. I. Vernadsky, 1988). Whatever the causeand mechanism of evolution, we should emphasize that the original discretenon�vacuum matter (“preform of material existence”) appeared in the hypo�thetical absolute vacuum at a certain primary stage of life development. Self�complication and self�development of the original matter were followed by theappearance of the World. The original form of material existence should havethe specific geometric properties and semantic content. In our opinion, it wasan essential information structure. Self�complication of the original informationstructure resulted in the appearance of complex information structures. Thesestructures were spatially congruent (identical).

The evolutionary process was probably associated with competitionbetween at least two original substances for the spatial and temporal organizationof a vacuum (according to principles of each original substance). The logicalconsequence is a tendency toward self�complication of the original matter andappearance of complex spatial structures. Only these structures provide transferof life principles (“jurisdiction” of the original matter) to all hypostases of theWorld. The evolution of a spatially simple vacuum resulted in the formation ofa three�dimensional space. The “plane” information structures were “materia�lized” or hypostatized* into complex spatial structures (e.g., biological systems).

Complex information structures were formed from simple structures(elements) during evolution. The information structures of similar complexitywere characterized by various combinations of constituent elements, whichprovided them with individual architectonics. Despite such individuality, eachinformation structure is similar to the original matter.

Materialization of complex information structures into biological systems(if it occurred) has several important consequences**. First, the structure ofbiological systems should have a “regular” hierarchy. This hierarchy is similarto the original matter and reflects prebiotic self�complication of life. Andsecond, any biological system should have a dual individual�and�species spatial


* Hypostatization is the ascription of material existence to any abstract notion, property,or idea.

** The idea that each biological species develops from an information precursor hassomething in common with the concept of nomogenesis (L. S. Berg and A. A.Lyubishchev). This seems contrary to the theory of C. Darwin. However, this conceptis consistent with the notion of natural selection. Natural selection occurs in animatenature, but does not serve as an “engine” of evolution.

56

Ultralow doses

organization to retain the initial predetermined integrity of functions (seeChapter 5). It is provided by specific principles of regulation.

These assumptions contribute to the revision of common notions aboutthe meaning of life. Evolutionarily, each living organism should reproduce itselfin the next generation and have a “normal” function. A hypothetical structuredvacuum with information structures is the ether in a three�dimensional space.In biological systems, the relationship exists between physiological processes andvacuum. A “normal” function contributes to a “correct” reflection and memo�rization of the environment. After biological death of the individual, its expe�rience as a “regular” information structure remains in a vacuum. This “cell” ofthe universe is similar to the original matter.

Primary information structures are characterized by a certain hierarchicrelationship between constituent elements (architectonics). The initialarchitectonics is preserved during materialization into objects of a three�dimensional space (e.g., biological objects). This transition without an increasein complexity of new three�dimensional systems received the name horizontaltransition (similarly to the relationships between constituent elements ormolecules in biological systems that determine the general species properties).

The species similarity does not abolish the individual properties. Hence,there are some individual relationships between elements in each biologicalsystem.

Spatial relationships between constituent elements of each biologicalsystem have a specific species organization (horizontal relationships). However,some individual relations between elements of the system also exist within thelimits of general species relationships. They received the name verticalrelationships or vertical plane of biological systems (O. I. Epstein, 1996).

To facilitate the understanding of horizontal and vertical relation�ships, we proposed the “theory” of four billiard balls. Any plane passes throughthree points. Let’s assume that these three points are the billiard balls. The ballsare adjacent to each other and form a triangle on the playing field (Fig. 3.7).

A shot with ball 4 is followed by scattering of these balls into differentdirections (Fig. 3.8).

* The theory of physical vacuum was very popular. However, this theory was rejectedwhen A. Michelson failed to confirm the existence of a vacuum by astronomicalmethods.

Fig. 3.7.Fig. 3.7.Fig. 3.7.Fig. 3.7.Fig. 3.7. “Theory” of four billiard balls: step I.

57

The greater is the power of the shot, the longer is the distance betweenballs 1, 2, and 3 after scattering. Moreover, the quality of the shot with ball 4may be estimated from the type of scattering (regular or rotational, smooth orsharp, and slow or fast). An experienced player can evaluate the quality of thebreak shot. Conventionally, the movement of balls 1, 2, and 3 may be con�sidered as a semantically organized constellation. It reflects the quality and vari�ous parameters of the shot with ball 4 (triangle 1'�2'�3 in Fig. 3.8, outlined witha dotted line).

Let’s assume that we have four, but not three balls. These are the air balls,but not the billiard balls. They scatter in the air after playing a shot with thefifth ball (Fig. 3.9).

According to the simplest geometrical laws, three balls will lie in the sameplane. In the majority of cases, the fourth ball with be beyond this plane. There�fore, such constellation of four air balls will appear like a pyramid (Fig. 3.10).

The “species” relations are preserved in the base and each face of thispyramid. The face is formed by three balls (planes 1'�2'�3', 2'�3'�4', 1'�3'�4', and1'�2'�4'). Any three balls are within certain spatial limits. The fourth ball maybe situated in an individual, but predetermined place relative to the center of

Fig. 3.8. Fig. 3.8. Fig. 3.8. Fig. 3.8. Fig. 3.8. “Theory” of four billiard balls: step II (plane constellations).

Fig. 3.9.Fig. 3.9.Fig. 3.9.Fig. 3.9.Fig. 3.9. Constellations of “air balls”.


Shot

Shot

Fig. 3.10.Fig. 3.10.Fig. 3.10.Fig. 3.10.Fig. 3.10. Principle of the spatial organization of constellations.

58

Ultralow doses

each plane. In any case, this position of the fourth ball will contribute to a “re�gular” species organization of pyramid faces. Hence, the “individual” will bealways incorporated into the “species”.

To achieve the maximum stability, each electron occupies a certain orbitduring rotation around the nucleus. Similarly to the electron, each element hasa specific position in the biological structure. This feature provides the irrever�sibility of physiological processes. Various biological theorists, including I.Prigozhin (1986, 2006) and E. Bauer (1935), attempted to substantiate theirreversibility of processes on the basis of thermodynamic principles. Theysuggested that the organism is an open thermodynamic system with the arrangedand directed energy flow, which provides the function of biological systemsagainst the chaos (anti�entropic vital activity). We believe that the spatiallynonlinear individual�and�species organization of biological systems contributesto the prevention of stochastic (random) processes.

Ideally, molecules of the organism may be conceived as balls (points).Moreover, biological systems can be imagined as thermodynamic systems*. Theresponse to external influences will appear as molecular constellations. They areassociated with the semantic structure of a certain external factor. First of all,we are interested in the local and systemic response to medical products. Theexperience of pharmacology indicates that any medical product has a specificactivity. The specificity of response is determined by new relationships betweenmetabolic or functional processes, but not by the involvement of one molecule.Any substance causes a species�specific response, which does not exclude thepossibility of individual (hyperergic) reactions.

What is the cause of hypersensitivity? This state may appear as follows. Anymedical product triggers a series of genetically determined molecular (ther�modynamic) events in the organism. These events are in certain relations witheach other (constellations). Similarly to constellations in the potentiated solution,these events are spatially “coupled” with each other by a fine structure of themedical product. Such events reflect a three�dimensional image of this structure.

“Reflection” of the medical product reaches maximum at the “peak” ofa pharmacological response. In this period, the biological system is significantlydeviated from the previous state of thermodynamic equilibrium. A negativefeedback mechanism induces the secondary reconstruction of this system, whichachieves another equilibrium state**. The achievement of equilibrium in this

* Thermodynamics is a science that deals with the inner state of macroscopic bodies inequilibrium. According to another definition, thermodynamics is a science that dealswith the laws of interconversion and transfer of energy.

** The equilibrium state of biological systems will be designated as an integrative state(Chapter 5).

59

system serves as a biological adaptation. Pharmacologically, this process reflectsa therapeutic effect of the medical product. The experience of immunologyindicates that before responding to an external stimulus, the biological systemshould evaluate its major characteristics. Semantically (i.e., spatially), this factorcan be “self” or “non�self” for the system.

The organism is considered as a combination of considerable amounts ofmolecular constellations. Any organism has the conditional horizontal and ver�tical planes. A paradoxical situation may occur. The semantic structure (archi�tectonics) of an external stimulus may be similar to the individual structure ofa biological system. Hence, the newly formed constellations in an individual(vertical) plane of the organism do not reflect (!) a fine structure of the medicalproduct. Spatial perception is impossible under these conditions, which ne�cessitates the construction of large constellations. The local response is trans�formed into the systemic response.

The studies to understand a major problem of homeopathy (nature ofhyperergic reactions to ultralow doses) yielded the evolutionary postulate andnotions on a dual individual�and�species spatial organization of vital functionsin biological systems.

The notion of semantic constellations allows us to illustrate schema�tically not only the mechanisms of hyperergic reactions to ultralow doses (biologicalbasis of homeopathy), but also the protective effect of activated products. Eachmedical product is biotransformed in the organism and induces a specific metabolicand physiological response. It results in the formation of local and systemicconstellations that are coupled by semantic parameters of the medical product.

Potentiation is accompanied by the formation of semantically organizedconstellations of solvent molecules in a solution. When molecular constellationsin the organism and potentiated solution consist of the same medical product,their spatial structures are similar (congruent).

Let’s return to the abstraction with three billiard balls. Biotransformationin an organism and potentiation in a tube are accompanied by three stages ofbiotransformation or potentiation of substance A. They are shown in Fig. 3.11.

Constellations 1'�2'�3', 1'’�2'’�3'’, and 1'’’�2'’’�3'’’ are the successive stagesof reflection (“reading”) of the image (structure) of substance A at variousperiods. If the specific response to an external stimulus is considered as afunction, the constellations appear as derivatives of this function.

After combined administration of substance A and its activated form (A’)during stage 1'’’�2'’’�3'’’ (Fig. 3.12), molecular constellations of the activatedsolution initiate an adaptive response to external treatment (negative feedbackmechanism) in the earlier period than the intact non�biotransformed molecule.

The potentiated substance is characterized by greater degree of “reading”in time. It initiates an adaptive response of the organism, which occurs before


60

Ultralow doses

biotransformation of the same substance in normal dose. During combinedtreatment with medical product A and its activated form, the latter “prepares”the organism to an external influence. This process determines the protectiveeffect of ultralow dose.

The next chapters will show that the phenomenon of bipathy is mediatedby more complex mechanisms. Activated forms of medical products have amodulatory effect not only on specific targets in the organism, but also on finecharacteristics of the substance in normal doses.

Most important in ChaptMost important in ChaptMost important in ChaptMost important in ChaptMost important in Chapter 3er 3er 3er 3er 3

There is a large body of evidence that ultralow doses have physiologicalproperties (polymodal dose�dependence, “splitting” of the effect, unidirection�

Fig. 3.11.Fig. 3.11.Fig. 3.11.Fig. 3.11.Fig. 3.11. Scheme for the phase states of constellations.

Fig. 3.12.Fig. 3.12.Fig. 3.12.Fig. 3.12.Fig. 3.12. Scheme for the protective effect of activated substances.

Substance A

Biological feedback and induction of adaptive mechanisms

↓

Advanced (predetermined) “reading” of substance A

↓

Activated substance A’↓

61


al systemic effect of the activated and original substance, and effectiveness ofpotentiated products in molar and submolar concentrations). The physical prop�erties of ultralow doses remain unknown. However, it is obvious that the activityof ultra�diluted substances results is associated with their preparation (poten�tiation).

The effects of ultralow doses are qualitatively similar to those of the orig�inal substance. These data suggest that the potentiated solution retains a finestructure of the dissolved substance. The memory unit of an ultra�diluted struc�tured solution is presented by stable relationships between solvent molecules.These semantically organized constellations are “coupled” by parameters of theoriginal substance, which is absent in the solution.

Molecules, submolecular particles of the solvent, and field processes(acoustic and electromagnetic fields) are integrated into the common spatialstructure of semantically organized constellations. Semantically organized con�stellations in potentiated solutions may appear as the wave structures that arecapable of distant interactions.

To explain the basic principle of homeopathic therapy (hyperergic reac�tions), biological systems are considered as the integral spatial structures. Theyconsist of molecular constellations and have a dual individual�and�species or�ganization. The dual structure of living organisms is related to evolution of bi�ological objects from the common material (essential) information source.

Semantically organized constellations have a dynamic spatial structure,which is characterized by specific phase�and�temporal parameters. These dataallow us to explain the phenomenon of bipathy.

62

Ultralow doses

In the previous chapter, we attempted to explain the mechanism of hyperegicreactions to ultralow doses. It was hypothesized that they are related to a dual

(individual�and�species) spatial structure of the organism. This approach is veryclose to the notion of holography. The term “holography” is formed by twoGreek words: “holos”, whole; and “graphein”, to write. This method to obtaina three�dimensional image of the object was proposed by Dennis Gabor in 1948.To develop the holographic image, a photographic film is exposed to the lightbeam. This beam passes through a prism and is split into two beams. Whereasthe reference beam is projected directly onto a holographic film, the objectbeam first reflects off the object before reaching this film. According to the lawof wave interference, these beams are combined in the plane of a holographicfilm to produce the picture of dark and light bands. The hologram is trans�formed into a three�dimensional image by means of a laser technique and othermethods. The following consequences of the holographic theory are importantfor our research:

1) a holographic image is stable; and2) each point and elementary unit of the hologram include the whole

image.The basic principle of any hologram is spatial “coupling” of constituent

elements by the general suprasystemic factor (light waves in physical holo�graphy). Before separation by a prism, optical beams are spatially coupled in the

C h a p t e r 4

Holographic controlof vital activityby the immune system

63

whole beam. These relationships are preserved after separation. Hence, thedirect (reference) and reflected (object) waves can be combined into a three�dimensional image.

In essence, semantically organized constellations of molecules are similarto holographic structures. They are combined into a single whole byphysicochemical properties of the dissolved substance*. As differentiated froma simple process of dissolution, potentiation allows the highly diluted solutionsto retain activity and stability. Molecules of water and other solvents are linkedby bonds (hydrogen, Van der Waals, and other bonds). These bonds exist for ashort time and easily dissociate in heating. During the manufacture of homeo�pathic remedies, it was concluded that ultra�diluted solutions are resistant toheating. These data serve as indirect evidence that constellations in the solutionare linked by other distant bonds. Conventionally, they may be designated asinformation bonds.

There is no consensus on the physical nature of information (torsionfields by G. I. Shipov, 1993; P�waves by N. D. Kolpakov, 1997; etc.). I do notwant to be an amateur. This serious physical problem should be solved byspecialists. For medicine and biology, it is important that the molecules andmolecular processes can be coupled by information parameters.

In the 1950s, a number of interesting facts were coming to light. Memoryis distributed over the whole brain, but not stored in a certain local “cell”. Someauthors believe that these data illustrate a holographic distribution ofinformation in the brain. Physical holography postulates that each elementaryunit reflects the whole image. According to P. J. Beurle (1956), damage to oneor several regions in the brain does not impair an integral perception. A famousscientist K. Pribram (1975) assumed that any brain area includes the wholeimage (according to the principle of holography). In experiments of N. Yu.Belenkov, several regions of the cortex in animals were “switched off” by coldexposure. The animals were trained in the follow�up period. Then the damagedareas were returned to a normal state (N. Yu. Belenkov, 1980). It was shown thatthe interference of “trained” and “non�trained” regions in the brain is followedby the disappearance of acquired skills in animals. These data can be explainedfrom the viewpoint of holography.

K. V. Sudakov is one of the authors of the theory of functional systems.He proposed that the principle of holographic interference forms the basis for“coupling” between signaling and satisfaction of the demand. This processdetermines the activity of any functional system (K. V. Sudakov, 1984, 1990,1996a,b).

* The horizontal and vertical planes of an organism may be conventionally designated asanalogues of the reference and object waves, respectively.

Chapter 4. Chapter 4. Chapter 4. Chapter 4. Chapter 4. Holographic control of vital activity by the immune system

64

Ultralow doses

It is very attractive to explain the effect of ultralow doses by holographicprinciples. For the extrapolation of these data to vital activity, it is necessary toevaluate the analogues of potentiation in an organism.

Potentiation consists of the following two components: repeated dilutionand shaking. Dilution is probably followed by separation of essential informationabout the properties of dissolved molecules from “information noise” in thesolution (see above). The process of dilution does not occur in an organism. Theanalogues of dilution (network processes) will be discussed below. However,autooscillations (analogue of shaking) are typical of many structures in biolo�gical objects. It remains unclear whether all these processes are accompanied bystable structuralization of liquid media in the organism (similarly to poten�tiation). We believe that this property is typical only of complex molecules witha three�dimensional or four�dimensional spatial structure (polypeptides andproteins). Autooscillations in the spatially complex molecule of DNA areprobably followed by structuralization of liquid media in an organism, whichresults in the dual spatial organization.

Russian scientists proposed that hereditary information has a wave(filed) nature (A. G. Gurwitsch, 1944; P. P. Garyaev, 1997). It is difficult toimagine that inherited characteristics have a certain “linear” arrangement inDNA (nucleotide sequence). Obviously, hereditary “memory” of DNA isstored in a more complex form (constellations of coding and noncodingregions in this supermolecule*). These data suggest that genetic informationis encoded in oscillation parameters of DNA regions. It remains unclearwhether this information is transmitted directly or indirectly (thought genomicproducts) into the cell. It should be emphasized that genomic products(polypeptides and proteins) have a more complex spatial structure than thematrix of DNA. Proteins and polypeptides probably regulate the spatialstructure of vital functions. The mechanism of these events is similar topotentiation in the manufacture of ultra�diluted solutions. Duringpotentiation, a holographic image (i.e., constellations of solvent molecules) is“extracted” from the molecule of the original substance. A three�dimensionaland four�dimensional structure of regulatory molecules is probably organizedby the holographic principle. Even in the absence of successive dilution (oneof the stages in potentiation), autooscillations of spatially complex moleculescan result in stable structuralization of liquid media in the organism. Theminimal liquid locus is structured around the genomic product of any location(in the cell or intercellular space). This process is related to autooscillationsand results in the “regular” spatial organization of molecular or ionic events.

* Recent studies showed that the genes are united into constellations or gene networks(V. A. Ratner et al., 1985; N. A. Kolchanov et al., 2004).

65

It contributes to the “regular” spatial realization of “wavy” inherited characte�ristics. Hence, DNA plays a dual role. First, the individual species hereditaryinformation is stored in constellations of DNA regions. And second, DNAserves as a matrix for the synthesis of proteins and/or polypeptides that havea specific “vertical” structure and regulate the realization of hereditary infor�mation in an organism.

The primary control is genetically mediated and involves proteins andpolypeptides. Beginning from a certain evolutionary level of multicellularity, theimmune mechanisms became involved in the regulation of vital activity. Itresulted in the appearance of secondary genetic control, or immunity. Theimmune regulation in an organism is directed toward epitopes that serve as themajor molecular regions of proteins, polypeptides, and polysaccharides. Epitopesare the smallest structures that can be “recognized” by the immune system.They are spatially distributed within the molecule. The molecule serves as askeleton to combine these structures.

The notions of semantic and holographic constellations are closely relatedto each other. The notion “semantic constellations” is more inclusive and em�phasizes the “determination” of constellations by information parameters. Thenotion “holographic constellations” refers to the spatial coupling betweenmolecular and submolecular elements in constellations.

Following the apologists of holography in biology, we believe that one ofthe holographic properties can be extrapolated to semantic constellations. Itsuggests that properties of the whole are reflected in each element of thehologram. It becomes clear that the immune system regulates a part of themolecule (epitope) and, therefore, has a modulatory effect on spatial integrityof the whole molecule (O. I. Epstein, 2002a). Holographic regulation of themolecular structure contributes to the evaluation of its “normal” function. Theimmune system has a great regulatory capacity. However, the principle ofimmune function is very simple. The immune system “supervises” the whole(function of the organism) through the small (epitope).

Epitopes are spatially distributed within the molecule. They are thesmallest structures, which can be recognized by the immune system. We believethat epitopes are submolecular semantic constellations, which retain the spatialand temporal properties of the whole molecule.

Immunology is a relatively young science. This science was taught ininstitutes of higher education for a short time. Let’s briefly describe a physio�logical role of the immune system.

Antibodies are a major factor of humoral immunity. They were discoveredby Emil von Behring and Kitazato in 1890. These scientists revealed thatimmunization of mice with tetanus toxin is followed by the appearance of serumantitoxin (protein “bodies”) in the plasma. A famous German scientist Paul


66

Ultralow doses

Ehrlich designated the protein substances in blood plasma from animals withbacterial infection as antibodies.

Until the middle of the 20th century, immunologists were mainly engagedin the development of new vaccines and sera. Much attention was paid toparticular problems of antiinfective immunity. A famous Australian scientist M.F. Bernet (1963) radically altered the role of the immune system. He proposedthat the immune response is directed to differentiation of “self” and “non�self”.Bernet believed that a major function of immunity is the maintenance of geneticintegrity during individual (ontogenetic) development of the organism. The im�mune mechanisms should be particularly specific. They can distinguish, recog�nize, and meet the foreign agent (antigen). A simplified scheme of immuno�logical specificity appears as follows: one antigen — one antibody and oneclone* of lymphocytes.

Antigens are structurally foreign substances (molecules) that can cause animmune response in the individual organism. Usually, the immune response isinduced by high�molecular�weight molecules (proteins, polypeptides, andpolysaccharides). The immune response can be triggered by a small molecule(haptene), which is conjugated with protein (Fig. 4.1).

The immune response is induced by a small spatial region (epitope), butnot by the whole molecule. The epitope of a protein molecule usually consistsof 6�12 amino acids. The epitopes interacting with T cells and B cells are de�signated as the T cell epitope and B cell epitope, respectively. The B cell directlyinteracts with epitopes via the surface receptor. Immunoglobulin M is locatedon the cell membrane and serves as a surface receptor during the first inter�action. By contrast, the T cell cannot directly interact with the epitope. T cellsrecognize the protein molecule only in a complex with histocompatibility mole�cules. This is one of the most important features, which will be discussed below.

In 1937, an electrophoretic study showed that antibodies belong to theγ�globulin fraction of blood plasma. These antibodies are now designated asimmunoglobulins (Ig). There are the following five classes of immunoglobulins:IgM, IgG, IgA, IgE, and IgD. They have a specific domain structure (Fig. 4.2)and consist of two easy chains and two heavy chains. Each polypeptide chaincontains one variable domain (V), which contributes to specific binding ofantibodies to the corresponding antigen. There are also three or four constantdomains (C) in the polypeptide chain. Besides binding to the antigen, antibodiescan interact with the complement or special receptors on various cells due tothe presence of constant domains. Hence, structural dualism of antibodiesdetermines their binding to the specific (original) antigen and involvement incommon reactions (through the C domain).

* Clone is the progeny of cells from one precursor.

67

Fig. 4.1.Fig. 4.1.Fig. 4.1.Fig. 4.1.Fig. 4.1. Structure of myoglobin in cetacean sperm (X�ray structure analysis).

Shaded areas, sequences of amino acid residues that play a role of B cell epitopes. Numerals, ordernumbers of amino acid residues in polypeptide (V. G. Galaktionov, 1998).

GemCOOH

NH2

15�21 (22)

(145) 146�151

113�119

56�62


Antigen�recognizing receptors on the surface of B cells and T cells arestructurally similar. The active antigen�recognizing site of these receptors isformed due to the interaction between variable domains (V domains).

Similarly to all protein molecules, antigen�recognizing receptors areencoded by specific genes. The following fact is of considerable importance: asdifferentiated from other somatic cells, T lymphocytes and B lymphocytes arecharacterized by recombination of gene fragments that encode the light andheavy chain of immunoglobulins. This process is mediated by a specificmechanism. A certain degree of variability contributes to the formation of upto 240 billion types of various antibodies, which bind at least the same numberof types of various antigens.

Antibodies may directly bind the antigen. Before the interaction with Tcell receptors, antigens of foreign viruses and bacteria are exposed to theintracellular preparation with antigen�presenting cells (APC). Thy bind to majorhistocompatibility complex (MHC) class I and II molecules and are transformedto the surface of APC (Fig. 4.3). This complex binds to the T cell receptor. Thenext important fact suggests that antigens are squeezed in specific antigen�binding clefts of MHC molecules. Therefore, they gain another spatialconfiguration before the interaction with T cell receptors (Fig. 4.4).

The immune response�inducing foreign antigens may exist in liquid mediaof an organism, as well as on the surface of cells. In the first case, the foreignagent is neutralized by humoral effector antibody molecules (humoral immuneresponse). In the second case, foreign antigens are killed by cytotoxic Tlymphocytes (direct mechanism) or inflammatory T cells and T helper cells(indirect mechanism). They induce a series of molecular and cellular events

68

Ultralow doses

(cellular immunity). The cellular and humoral immune responses mainly involveT lymphocytes and B lymphocytes, respectively.

T lymphocytes received their name from maturation in the thymus. Tlymphocyte precursors migrate from the bone marrow in the thymus. B cellsoriginate from the bone marrow and become mature in peripheral lymphoidstructures. Historically, the name B cells is derived from the bursa of Fabriciusin birds (one of the peripheral lymphoid organs).

The development of lymphocytes has two distinctive features.

Fig. 4.2.Fig. 4.2.Fig. 4.2.Fig. 4.2.Fig. 4.2. Scheme (a) and molecule (b) of immunoglobulin (V. G. Galaktionov, 1988;R. M. Khaitov, 2005).

L, light chains; H, heavy chains; V, variable domain; C, constant domain. N�Terminal regions of L chainsand H chains (V domain) form two antigen�binding sites. The Fab fragment and Fc fragment of themolecule interact with a specific membrane receptor on various cells, including macrophages,neutrophils, and mast cells.

V Н

С Н1

C L

V L

H�cha

inL�

chain

Domain

Domain

a

CH2

CL

CL

CH2

CH2

VL

VL

VH

VH

CH3

CH3

CH4

CH4

CH2

Hingeregion

Antigen�bindingsite

Fc fragment Fab fragment

S�S

S�SS�S

S�S

b

69

First, during differentiation T cells interact closely with the stroma of thethymus. They are selected for the ability to recognize MHC class I and IImolecules (positive selection) and to interact with body’s own molecules(negative selection for autoimmune reactions). The cells that cannot interactwith MHC molecules or react with autoantigens are removed from thepopulation of T cells (thymocytes).

And second, the immune response is accompanied by the followinginterrelated processes:

1) shift in antibody synthesis from one type (isotype) to another (prev�alence of IgM and IgG in the initial and late stages of an immuneresponse, respectively); and


Fig. 4.3.Fig. 4.3.Fig. 4.3.Fig. 4.3.Fig. 4.3. Scheme for the recognition of a complex of antigenic peptide and MHCclass I and II molecules by the receptor and coreceptor on T lymphocyte (a).Interaction between T helper cells and antigen�presenting cells (b).

TCR, T cell receptor; CD4� and CD8, coreceptors, MHC I and MHC II, histocompatibility complex classI and II antigens; and AG, antigen (by R. M. Khaitov, 2005).

CD8CD4

α β

α β

α β

α β

peptide

MHC II MHC I

T helper cell

APC

T killer cell

TCR TCR

Target cell

а

CD80/86

MHC II TCR

CD28

CD4

CD154CD40

AG

Т helper cellAPC

reception

costimulation

costimulation

b

70

Ultralow doses

α1

α2

N

b

Antigen�bindingcleft

Fig. 4.4.Fig. 4.4.Fig. 4.4.Fig. 4.4.Fig. 4.4. Spatial structure of MCH�A2 class I antigen (X�ray structure analysis). Sideview (a) and top view (b). Arrows, regions of the antiparallel β�structure; spirals,α�spiral fragments (R. M. Khaitov, 2005).

α1

α3β

2—m

α2

N

CC

N

71

2) increase in the affinity of antibodies for an immune response�indu�cing antigen.

The properties of T cells and B cells are determined before theirinteraction with a foreign molecule (pre�antigenic stage of development). Theinteraction of T cells and B cells with the antigen results in cell proliferationand differentiation into mature effector cells, which are capable of neutralizingand killing this antigen. The degree of lymphocyte “maturity” determines theirfunctional capacity and direction of the effect. Special molecules (markers) ordifferentiation clusters appear on the cell surface of certain function. Core�ceptors also play a role in cell�to�cell interactions during the immuneresponse. They improve the interaction between lymphocyte receptors andantigen. This process also involves costimulators that are located on thesurface of APC.

Each event of the immune response is regulated by cytokines. Cytokinesare synthesized by various cells, including lymphocytes. The majority of cyto�kines, immunoglobulins, lymphocyte receptors, MHC molecules, coreceptors,and several adhesion molecules belong to a superfamily of immunoglobulins.They have a common evolutionary precursor and similar domain structure.

The most extensively studied phenomenology of an antiinfective immuneresponse is the large�scale cascade molecular�and�cellular events that involvelymphocytes, accessory cells, and various classes of biologically active substan�ces. However, some problems of immunology are poorly understood. The “loca�lization” of immunological memory, fine mechanisms of tolerance and hyper�sensitivity, and progression of autoimmune reactions remain unknown.

The Nobel Prize winner M. Bernet (1963) believed that the main role ofthe immune system is regulation of genetic integrity in an organism duringontogeny. This function is based on the ability of the immune system to dis�tinguish “self” from “non�self”. Let’s compare the commonly accepted notionsof Bernet with our “spatial” postulates.

At first, it is necessary to consider the notions of “self” and “non�self”.In our opinion the individuality of each organism is determined by its uniquespatial structure, which combines the individual and species properties. Hence,the maintenance of “self” integrity means the preservation of an individualspatial structure that is organized by holographic principles.

Lymphocytes are the only somatic cells in an organism. The lymphocytegenes encoding T cell receptors and B cell receptors of the immunoglobulinsuperfamily are characterized by allowed recombination. According to the germ�line theory of L. Hood, a whole set of V genes and C genes is included in thegenome and transferred between generations with no changes. The majority ofauthors believe that variability of immunoglobulins is associated with randomrecombination of V segments and C segments.


72

Ultralow doses

However, this problem appears to be more complex. First, allrecombinations are predetermined and depend on oscillation parameters ofDNA. Second, the combination of individual gene fragments is directed towardthe construction of an encoding region with unique spatial conformation* (in�corporation of “individual” into “general”). Hence, the encoded receptors onT cells and B cells are characterized by a “regular” individual�and�species orga�nization. This property contributes to the holographic recognition of antigen.And third, recombinant processes are followed not only by the appearance ofT cell receptors or specific antibodies, but also by the synthesis of immunoglo�bulin molecules of certain spatial complexity. Hence, IgM and T cell receptorcan bind several antigens of the same spatial structure.

Let us digress briefly to be more specific. In the periodic table of D. I.Mendeleev, an increase in the weight of elements is accompanied by a changein their chemical properties. Probably, a similar process occurs in the nature.The spatial structure of protein (polypeptide) molecules became more complexin evolution, which resulted in an increase of the semantic content andappearance of “semantic series”. The semantic series may be imagined as a shelfwith books of the same genre (one detective story with another detective story;and one novel with another novel). IgM and T cell receptor recognize theantigenic epitope consisting of a small number of amino acid residues (6�12).However, the structure of each epitope holographically reflects the overallstructure of an antigenic molecule. Hence, the spatial (semantic) complexity ofthis epitope should not correlate with the molecular weight. The immune systemis prepared to analyze a spatial structure of any complexity, but not onemolecule. The T cell receptor or immunoglobulin is polyreactive and recognizesa group of endogenous or exogenous molecules with the same degree of spatialcomplexity (but not a specific antigen).

Polyvalence (hypervariability) suggests the recognition of up to 240 billiongroups of epitopes. This is only one of the unique properties of immune system.Functional activity is also regulated by the opposite mechanism (unification ofrecognition), which involves MHC. MHC is a complex of linked genes. Thiscomplex was discovered in studying the mechanisms of tissue incompatibility inthe 1920s. The main properties of MHC (polygeny and polymorphism)determine the individuality of MHC antigens in specimens of the same species.The MHC�encoded antigens (molecules) belong to three classes. Class I and IImolecules are particularly important for immunology. They share structuralsimilarity and consist of four domains, which form an antigen�binding site

* Such approach suggests that the individual�and�species spatial hierarchy (specificity) ofan organism is determined during the prenatal period. This problem will be discussedbelow.

73

(cleft). The antigen�binding clefts in MHC class I and II molecules have asimilar spatial structure (Fig. 4.4).

Due to the peculiar structure of class I and II MHC, they may beconsidered as the standard of individuality. The antigen�binding cleft has a roleof the Procrustean bed. All antigens undergo proteolysis and degradation in thecytoplasm of APC. They bind to MHC class I and II molecules. A complex ofantigens and molecules is translocated to the cell surface. In the cleft of MHCclass I and II molecules, an antigenic peptide is squeezed between specific an�chor sites and forms the convolution of different shape. This convolutioninteracts with the T cell receptor (Fig. 4.5). The T cell receptor does not recog�nize “self” and “non�self”, but evaluates the degree of spatial differences between“self” and “non�self”. The greater is the differences, the stronger is the immuneresponse*.

Hence, the immune system follows even small conformational changes inthe “self”. Spatial characteristics of “non�self” are compared with those of“self” (MHC class I and II molecules).

The elimination of foreign agents probably serves as an “emergency” or“immediate” response of the immune system (antiinfective immunity). Undernormal physiological conditions, the regulatory capacity of the immune systemis directed to routine regulation of vital activity. This capacity depends on thepredetermined clone of native lymphocytes and natural autoantibodies.Physiological functions of autoantibodies were extensively studied.

As mentioned above, the hypothesis of a regulatory role of naturalantibodies was proposed by famous Russian scientists I. P. Ashmarin and I. S.Freidlin at the end of the 1980s (I. P. Ashmarin et al., 1989).

The notion of natural antibodies is currently undergoing revision. Recentstudies showed that natural antibodies are involved in multifactor regulation of

* Previous experiments showed that the immune response depends on one autosomaldominant gene. MHC class I and II molecules are the phenotypic products of this gene.


Fig. 4.5.Fig. 4.5.Fig. 4.5.Fig. 4.5.Fig. 4.5. Specific interaction of antigenic peptides with MHC class I molecules (V.G. Galaktionov, 1998).

74

Ultralow doses

natural functions (I. P. Ashmarin, 1989, 1997; M. A. Myagkova, 2001; Y.Shoenfeld et al., 1993). The activity of these antibodies does not correlate withautoimmune disorders. The set of natural antibodies reflects the molecularspecificity of each adult individual (I. P. Ashmarin, 1997). There are naturalantibodies to some low�molecular�weight and high�molecular�weightsubstances (peptides), surface membrane structures, and intranuclearstructures. The majority of circulating natural antibodies are presented by IgG(up to 50%).

Autoantibodies to endogenous regulatory peptides play a role in theiractive transport to membrane receptors. They prevent peptide molecules frompreterm proteolysis. It was reported that natural antibodies have catalytic(enzyme�like or abzyme) activity. These antibodies modulate cell proliferation,myelinization, activation of membrane ion channels, etc. There is a growingbody of evidence on directed transport of natural antibodies through membranestructures and blood�tissue barriers. A combination study of natural antibodiesto endogenous regulators is used for diagnostics of various diseases (A. B.Poletaev et al., 2002).

Physicians should consider the immune system as a global regulatorysystem. Regulatory activity of the immune system is highly competitive with thatof CNS. The immune system may be imagined as a brain with dispersedlydistributed lymphocytes. Similarly to the nervous system, the immune systemis involved in the regulation of functional and metabolic processes in anorganism. This system has a role in the development of any pathologicalsyndrome (e.g., infectious or noninfectious syndrome). Antiinfective protectionis only a small part of physiological activity of the immune system. The mainfunctions of the immune system are the maintenance of homeostasis andregulation of normal physiological functions. These functions are provided byregulating the spatial structure of own molecules (antigens). The immune systemis responsible for local and distant regulation. During local regulation,endogenous antigenic molecules interact with detectors of immune individuality(MHC molecules, T cell receptors, and B cell receptors). Under normal con�ditions this interaction is not followed by antigen elimination, but initiates aseries of fine co�adjustment reactions.

Natural antibodies have a distant effect. The antigenic pattern of avariable region in immunoglobulins received the name “idiotype” (Fig. 4.6). Thisregion may induce an immune response. Antibody 1 always induces theproduction of anti�idiopathic antibody 2. This process is continuous. The sameantibody 1 is induced by antibody 2. The Nobel Prize winner H. Hjдrnedesignated this phenomenon as “idiotypic/anti�idiotypic network”. By thenetwork principle of organization, natural antibodies resemble the nervoussystem. The system of natural antibodies attracted our attention. Our studies

75

confirmed the fact that this system has a role in the effect of antibodies inultralow doses (see Chapter 2).

Natural antibodies may be considered as a “top point” of humoralregulation. First, the synthesis of anti�idiotypes provides a continuous patternof the antibody network (independently on the half�life of immunoglobulins).And second, the continuous “mirror�like” synthesis of antibodies providesgenomic regulation of the spatial structure of antibody idiotypes. Autoantibodiesare characterized by low affinity. Moreover, the concentration of autoantibodiesin the blood and liquid media of an organism is low. These data suggest thateven a single contact with the molecule (antigen) is sufficient for regulatoryactivity of natural antibodies. The information about antigen is stored inconstellations of antibodies and provides distant regulation (relative to antigen).The whole network of antibodies may be considered as a large constellation,which reflects the parameters of all antigens in an organism. Due to the dualfunction of antibodies, such holographic network follows the “regular”individuality of an organism through V domains. It contributes to the regulationof main functions through C domains. Natural autoantibodies may be involved inantibody�antigen interactions for antigen elimination, which is mediated by a shiftin the synthesis of idiotypes and increase in affinity. These reactions appear to beautoimmune in relation to own molecules. It is necessary to distinguish normalautoimmune processes that occur in each organism under normal conditions from

Fig. 4.6.Fig. 4.6.Fig. 4.6.Fig. 4.6.Fig. 4.6. Idiotypes, anti�idiotypes, and their networks (J. Pleifer, 1999). Part of theidiotypic/anti�idiotypic network (a); antigen�induced disturbance of the network (b).

VH

CH3 CH2CH1

VHVL

CLVL

IdiotypeImmunoglobin molecule

«Internal image»

Antigen

a b


76

Ultralow doses

autoimmune diseases. Besides this, the term “autoantibody” should not beassociated with a pathological condition in medical practice.

The cause of autoimmune disease is poorly understood. These disorders maybe related not only to immune deficiency or exhaustion, but also to structuralchanges in endogenous molecules. According to holographic principles, themolecule that has a major role in certain pathological conditions may undergodeformation and change in the fine “regular” structure. This molecule becomes“foreign” for the organism and induces immune aggression (beyond the scope ofnormal autoimmune regulation). However, physiological co�adjustment functionsof antibodies are not taken into account in clinical practice.

While assuming that the system of natural antibodies serves as a target foractivated antibodies in the organism, it is necessary to understand which fineregulatory mechanisms are affected. It can be said that we “regulate theregulator”.


The semantically organized molecular constellations are holographicstructures.

Autooscillations of molecules with a complex spatial structure (similarlyto potentiation) can determine the appearance of structured liquid loci.

Hereditary information is stored in constellations of DNA (RNA) molecules.This information is translated to liquid media through autooscillations of DNAregions. Autooscillations of genomic products (proteins and polypeptides) pro�vide the regulation of vital activity in the structured locus (primary genetic con�trol).

The interaction with epitopes (individual regions of “heavy” molecules, includ�ing proteins, polypeptides, and sugars) contributes to secondary genetic controlof vital functions by the immune system.

A major function of the immune system is the maintenance of individu�al�and�species spatial integrity in an organism (homeostasis).

After genetic reconstruction, immune detectors (T cell receptors, B cell receptors,and MHC molecules) gain a specific individual structure and provide the integralspatial (holographic) evaluation of own molecules (antigens).

The immune system is as potent as the nervous system for the overallregulation of functional and metabolic processes.

Natural autoantibodies are the components of the immune system that play arole in humoral regulation of vital activity. Previous studies suggest that natu�ral autoantibodies serve as a target for medical products from potentiated anti�bodies.

77

Much progress in molecular biology, biochemistry, and genetics wasachieved during the second half of the 20th century. The double�stranded

nature of DNA and new classes of regulatory molecules were discovered. Thegenome was “decoded”. Advances in biology and biotechnology provided thedevelopment of new potent drugs. The effect of medical products was explainedschematically from their influence on the chain of “gene–protein–sign”. Mo�dern notions of vital functions are based on the linear continuum (continuity)of vital activity. This hypothesis suggests successive transformation of molecular(biochemical) processes to other processes.

Our study of ultralow doses showed for the first time that drugs have adistant effect on the organism. The notion of essential information was used toretain a scientific materialistic position. An informational approach does notabandon the general physiological principles. In older times, the laws of classicalmechanics appeared to be insufficient to explain some physical processes. Itresulted in the appearance of quantum mechanics and new notions of the spaceand time. The data on ultralow doses illustrate some new principles of vitalactivity. The pharmacotherapeutic effects (e.g., adaptive activity) of ultralowdoses should be analyzed using a coordinate system.

In the previous chapter, we hypothesized that inherited characte�ristics are encoded in constellation�and�oscillation parameters of DNA. Auto�oscillations of DNA and genomic products (peptides and proteins) result instructuralization of the liquid medium in an organism. All processes in this me�

C h a p t e r 5

Principle of maintenanceof the initial integrity

78

Ultralow doses

dium develop in the genetically determined spatial and temporal limits. In�herited characteristics (i.e., “wave” parameters of genetic information in struc�tured loci) appear as a semantic factor, which “integrates” the molecular pro�cesses into constellations. Figuratively, functions of a structured medium havethe semantic content and holographic shape.

Based on the experience of homeopathy, we emphasized that the specificmarker of individual sensitivity serves as an indication for treatment with severalhomeopathic remedies. It depends on the association of this marker with othermarkers. Analogously, we believe that each endogenous molecule enters aconsiderable number of constellations and becomes involved in a variety ofdistant constellation relationships. These relationships extend beyond the cell orsynapse, which forms the basis for cell�to�cell interactions and systemic regu�lation (M. A. Pal’tsev et al., 1995). “Network” constellation processes in thenervous, immune, and endocrine systems are particularly important for systemicregulation. Billions of cells and subcellular factors are involved in reciprocalrelationships between the nervous and immune systems. By the degree ofcomplexity, these relationships are comparable with the Cosmos*. However,local physiological processes are also based on the constellation principle. Anexample is the cascade of metabolic events during intracellular transduction,which involves secondary messengers, calcium, adenylate cyclase, phospho�inositide cycle, and protein kinases.

The existence of molecular constellations that combine cellular and inter�cellular processes eliminates the distinction between such notions as “substrate”and “function”. More than 60 years ago the author of the theory of biologicalfield (A. G. Gurwitsch, 1944) assumed the existence of structured processes, butnot of the substrate or function. We shall use this good term. The formation ofconstellations from hereditary information endows them with the property ofmemory (i.e., holographic memory of normal function in certain molecularensembles). Hence, constellations are the unity of structure, function, andregulations (holographic memory) of functioning.

The molecular ensembles maintain this trinity due to constant motionand permanent dynamic changes in constellations. Each element of con�stellations should constantly deviate from the mean value. Functions are regu�lated by the “accuracy” of this deviation (i.e., on the basis of constellation me�mory). Hence, the most important information is stored in particular networkstructures (neural networks in CNS, anti�idiotypic network of natural anti�bodies, etc.).

Due to interference, all molecular and submolecular constellationsof an organism are combined into the holographic sphere. The dual individual�

* The term “constellations” is most appropriate for these mega�ensembles.

79

and�species organization of distant intermolecular relationships in this structurecontributes to the existence of horizontal (general�species) and vertical (indi�vidual) planes. The notion of “holographic sphere” amplifies the term “homeo�stasis” by a new biophysical (spatial) content. The evolutionary principle pos�tulates that structure of any biological system should be similar to the originalmatter. All manifestations of vital activity should be characterized by thespecific, “regular”, and evolutionarily determined spatial and temporal relation�ships between elements of the biological system. This is achieved by couplingbetween the horizontal and vertical plane of an organism’s holographic sphere.Vital functions are stable, structured, and homeostatic only within the “regular”spatial limits. To emphasize this idea, we introduced one new term into ourhypothesis. This term (initial integrity) designates the ability of biological systemsto retain a unique individual�and�spatial structure according to evolutionary(original) principles of the spatial and temporal organization of vital activity*.

The integration of all constellations into a common holographic sphereprovides the specific spatial architectonics of an organism. Spatial�and�hierarchic relationships are not typical of individual molecules. They are char�acteristic of intermolecular distant (information) relations that serve as a primaryelement of the holographic sphere. The hierarchy of a biological system is basedon the interrelated dual individual�and�species relationships, which develop inthe prebiotic stage of evolution.

The unique architectonics of an organism is formed during embryo�genesis. The integration (interference) of maternal and paternal “wave” infor�mation is probably a long�term process, which determines embryogenesis andterminates only during the postnatal development. Before this period, anyforeign molecule introduced into the embryo has a specific place in thedeveloping architectonics. New relationships of this molecule serve as aconstituent element of the whole holographic memory in an organism (acquiredtolerance). The immune system becomes mature in the postnatal period, whichis related to genetic reconstruction of lymphocytes. This system is capable ofevaluating the structure of any endogenous or exogenous molecule. The immunesystem estimates the identity of this molecule to the whole holographic structureof an organism (principle of “self” or “non�self”).

On the basis of spatial�and�holographic notions, we believe that “non�self” factors are hazardous for the integrity of an organism. Exogenous substan�ces also have the property of integrity, which may be “imposed” upon the orga�nism. A structurally “non�self” molecule forms the extraneous spatial constel�lation relationships and causes disintegration in the organism. Toxic doses of a

* By convention the compliance of organism functioning with basic principles isdesignated the “correct function”.

Chapter 5.Chapter 5.Chapter 5.Chapter 5.Chapter 5. Principle of maintenance of the initial integrity

80

Ultralow doses

* Let’s remember semantic series (see Chapter 4).

slightly foreign substance also induce the formation of “incorrect” relationshipsin an organism. Under these conditions, the existent information relationshipswill be impaired due to a strong response to the toxic dose.

The maintenance of initial integrity is a normal physiological property ofthe organism. This is similar not only to the idea of homeostasis (W. Cannon,1915), but also to the notions of genetic integrity (M. Bernet, 1963) and anti�entropic activity (I. Prigozhin, 1986, 2006; E. Bauer, 1935). A systemic or localdisturbance in the initial integrity is always accompanied by disease. Oncologicaldiseases are the most prominent example. Spatial and temporal laws for theregulation of atypical cells differ from those of normal cells.

Before the specific response to an exogenous molecule, the organismevaluates the degree of its difference from the initial integrity. This moleculeshould interact with conformational regions of endogenous protein moleculesthat have a complex spatial structure (hormones, enzymes, cell receptors, andantibodies). During the interaction of exogenous molecules with the surface ofsomatic cells, these molecules are recognized by well�known cell receptors.During the interaction with conformational sites of microsomal enzymes, low�molecular�weight xenobiotics undergo oxidation�reduction and/or conjugation.An exogenous substance is not only neutralized, but also induces the formationof constellations to “read” its structure. Antibodies and lymphocyte receptorsare responsible for the primary evaluation of integrity during the first interactionof antigen with immune system. This is typical of natural and synthetic antigenswith any degree of spatial complexity. The recognizing site of such antibodiesand receptors is specific for this antigen*. Large molecules are hydrolyzed in thecell. Low�molecular�weight fragments are also evaluated by the immune system.Under these conditions, a low�molecular�weight exogenous molecule or itsfragment interacts with the conformational site of a higher molecular weightendogenous molecule. The weight of endogenous molecules probably “amor�tizes” autooscillations of xenobiotics, which prevents the imposition of “foreign”integrity. Enzymes, receptors, and antibodies not only transform the signal ofan exogenous molecule, but also maintain the initial integrity. This conceptexplains the domain structure of molecules that belong to a superfamily ofimmunoglobulins and originate from a common gene. Non�species�specificdomains of complex structure and high molecular weight provide the integraland safe evaluation of antigen by the immune system.

As mentioned above, the medical product and its potentiated formhave a qualitatively similar effect. It is not surprising since the potentiatedsubstance consists of constellations of solvent molecules. After the first stage ofbiotransformation, this drug in normal doses causes the formation of

81

constellations in an organism. Drug�induced constellations have the same spatialstructure in vivo and in vitro. They reflect spatially a fine structure of this drug.A systemic effect of medical product will depend on the cascade of semanticallyorganized molecular�and�constellation events, but on the linear process(independently on drug dose).

If both forms of the drug are constellation�holographic structures, whatis their target? Probably, this target is also a holographic structure with specificwave properties. Both forms of the drug (original and potentiated substance) caninteract with this target by the resonance�like mechanism. A therapeutic effectof any drug is related to the direct action on pathological syndrome (i.e., holo�graphic structure), but not on a specific molecular target (concept of “magicbullet”, P. Ehrlich).

Any pathological syndrome is a defense response. Chronic diseases areusually characterized by hereditary (constitutional) predisposition. These dataillustrate the relationship between pathological syndrome and individual�and�species structure of an organism. The question arises: what mechanisms do pro�tect the organism in pathological syndrome (abnormal structure)? First of all,these mechanisms should maintain the initial integrity. Our evolutionary pos�tulate suggests that the hypothetic information vacuum (structured vacuum) ischaracterized by a constant fight between at least two original substances for theorganization of information processes in accordance with their structure. Thetendency toward a “regular” spatial and temporal organization of normal func�tions is restricted at the information level. Hence, the inertia is typical of phy�siological processes (similarly to mechanical processes). The strength of in�dividual�and�species regulation probably decreases with time, which contributesto the accumulation of stochastic events and natural aging of an organism.

Due to the inertia of vital functions, sooner or later the organism ischaracterized by a tendency to the systemic or local formation of extraneousspatial and temporal relationships. The increased formation of “regular”relationships in “weak points” probably has a compensatory role. The stage offunctional disorders (pre�disease) is followed by deformation of the holographicsphere in an organism. Such deformation is pathological syndrome, whichprobably contributes to the maintenance of “self�properties” (initial integrity) ina “non�self” form. There are no “pathological” molecules in the organism. Theinduced pathological distant relationships between these molecules determine aconventionally abnormal state. Pathological syndrome should be diagnosed by theappearance of new relationships (correlations) between clinical and laboratoryparameters, but not by a change in one biochemical or functional marker.

Holographic notions of pathological syndrome give rise to a newunderstanding of the therapeutic effect of pharmaceutical products. Probably,the remedy will be effective if it has the properties of tropism for pathological


82

Ultralow doses

syndrome and integrity. Tropic activity is associated with the resonanceinteraction between drug�induced constellations and pathological syndrome. Theinfluence should be integrative to produce a therapeutic effect. Integrity ismainly determined by the weight of medical product. This property may beconsidered as the ability of substance to retain specific physicochemical activity(integrity) during the interaction with an organism. The relationship betweenmedical product and pathological syndrome depends on the integrity. It shouldbe sufficient to disturb the dynamic equilibrium of pathological syndrome.

The notion of pathological syndrome as a spatial structure does notcontradict the physiological hypothesis of disease pathogenesis. When one oranother endogenous molecule regulates a certain physiological process undernormal conditions, it will be involved in the development of pathological syn�drome to compensate disturbances in this process. These events are related tothe formation of “pathological” relationships.

Pathological syndrome has a specific hierarchic structure. The moleculesinvolved in this pathological syndrome are subordinate to each other. Withoutpharmacological screening, it is difficult to estimate a priori which molecule hasa “key role” in the spatial structure of pathological syndrome. This approachis also essential to evaluate the medical product, which will exhibit the highesttropism for one or another type of pathological condition. Some potent phar�maceutics (aspirin, phenobarbital, and Viagra) were developed empirically.Knowing a physiological role of these drugs, it was difficult to anticipate the in�dications for use. Pharmacological screening is a long�term and expensive process.A new notion of tropism will allow us to develop some methods to search forpharmaceutical products (e.g., resonance frequency analysis). Such studies can beperformed with potentiated substances, but not with medical products.

The mechanism of tropism is resonance. Therefore, tropism as a propertyof substance is related to its semantic analysis in the organism. A “correct”reflection of environmental events is the major evolutionary goal of eachorganism. Any physical and chemical factors have a modulatory effect onorganism function. The first exposure to “neutral” factors is followed by onlyone response of the organism (reaction to novelty; T. M. Vorob’eva, 1962). Allfactors that may disturb the integrity always induce an organism’s response.

Each external factor has a specific semantic (information) structure,which is manifested in the structure of induced molecular constellations. Forexample, some information is perceived through the second signaling system andtransformed into plastic memory of the brain. These engrams areholographically distributed in the brain. Metabolism of chemical factors (e.g.,medical products) is followed by the appearance of constellations. Distantrelationships between all elements of the external factor and constituents of abiological system have the same physical (information) nature. In the

83

coincidence (resonance) of oscillation parameters for relationships betweendiscrete (final and simplest) elements of any external factor and distant inter�molecular relations in an organism (information acceptors), the image of thisexternal factor is distributed over the holographic structure of an organism (“re�cording”). External information is refracted through the holographic sphere(prism) and gains an individual nature. This information is stored in compliancewith a unique structure of the organism, which constitutes its evolutionary purpose.

By convention the “portion” of external information may be comparedwith the grammatical sentence, which is linguistically divided into “words” and“syllables”. On the basis of interference, these “syllables” are combined intonew words and new “phrases”. The relationship between new phrases and ori�ginal sentence is not always obvious.

A famous linguist and Nobel Prize winner Noam Chomsky proposed theterm “generative grammar”. According to Chomsky, each notion causes the for�mation of brain associations that reflect its structure. The greater is the numberof words to describe any notion, the more exact and clear is the definition ofits meaning (N. Chomsky, 1999). A semantic analysis of medical products isbased on the same principles.

The question arises: which is the way to perform a semanticanalysis of pharmaceutical products in various doses?

A study of super�diluted solutions showed that they reproduce activity ofthe original substance in a reduced form (E. B. Burlakova et al., 1990). It maybe suggested that due to little effect of the activated substance, its structure isonly partially evaluated in an organism. The activated substance causes a smallphysiological response, which is mediated by the resonance mechanism andsufficient for the image analysis of ultralow doses. Clinically, the exposure to anactivated product is followed by little activating effect.

Other results are obtained when a homeopath prescribes the potentiatedremedy according to the principle of “similarity” (i.e., individual sensitivity ofpatient). The image of a potentiated substance is also “read” and “recorded”in the holographic sphere of an organism. However, the whole spatial image ofthis exposure cannot be formed in an organism. The structure of an activatedsubstance coincides with the individual vertical plane of a holographic sphereand, therefore, is not reflected. To perform a complete evaluation of thepotentiated substance, drug�induced constellations are combined to largeconstructions in the organism. These changes contribute to the hyperergicreaction. A specific response to ultralow dose increases due to hyperergia. In thecase of tropism for pathological syndrome, the activated product has atherapeutic effect.

The constellations induced by a therapeutic dose also resound withpathological syndrome. Under ideal conditions, a holographic sphere of the


84

Ultralow doses

initial integrity is restored in this locus. These changes are followed by recoveryof the patient.

It is well known that the drug has individual toxicity. A prescribed medicalproduct in the therapeutic dose becomes toxic when drug�induced “wave”constellations directly interact (resound) with pathological syndrome. However,these constellations are so great that pathological syndrome cannot “absorb”them. Treatment with the toxic dose is a priori dangerous for the initial integrityof an organism. With respect to the initial integrity, such nonspecific event asunreactivity to high dose of any substance appears to be substantiated. Theorganism does not interact with this substance to retain its initial integrity.

The toxic dose is similar to stress exposure. Hence, stress may be con�sidered as a process to maintain the initial integrity (refusal to interact spe�cifically with a strong pathogenic factor). A similar evolutionary appropriatenessis typical of the opposite event (class reactions of hyperergia). It suggests therefusal to interact specifically with a substance whose structure is dangerous forthe initial integrity. The experience of homeopathy shows that hyperergicreactions retain the specific nature only after treatment with ultralow doses.

Let’s consider the interaction between ultralow dose and normal dose inthe context of the phenomenon of bipathy. Our experiments showed thatsometimes this phenomenon is reproduced during combined treatment withboth doses. Under several conditions, ultralow dose should be administeredbefore (several tens of minutes) treatment with “high dose”. These data indicatethat modifying activity of the potentiated substance is associated not only witha direct effect of ultralow dose on “high” dose, but also with the readiness ofan organism to treatment with the standard drug.

It will be remembered that during bipathic treatment with prednisolone,ethanol, morphine, and cyclophosphane, ultralow dose serves as a protectivefactor against the toxic dose. The specific preparation of an organism to thisexposure probably contributes to a variety of positive protective effects that wereobserved under experimental conditions.

We showed that the ultra�diluted substance sometimes has a potentiatingeffect on the same medical product. For example, antimetastatic activity ofcyclophosphane increased after bipathic treatment. The potentiating effect ofultralow dose is probably related not only to its direct influence on normal dose,but also to the preparation of common oscillatory target acceptors in an organism.The drug in normal dose is biotransformed in an organism, which results in the“reading” of its structure. The potentiated product is a ready constellation form.An analysis of the potentiated product occurs more rapidly than that of the normaldose, which determines the preparation of an organism to “high” dose.

Studying the effect of anti�S100 in ultralow doses on the isolatedneuronal membrane showed that their basic property is sensitizing activity. It

85

was manifested in subthreshold depolarization (with no generation of the actionpotential), increase in the maximum amplitude of inward current, and moderatefunctional modulation of ion channels. The systemic sensitizing effect ofactivated anti�S100 is manifested in variations of synaptic plasticity. Thisconclusion was made in experiments on the model of LTPTP. The alteredsynaptic plasticity determines a wide range of properties of neuropsychotropicdrugs in ultralow doses.

Sensitization is an increase in the individual sensitivity to a certain externalstimulus under the influence of another external factor. This phenomenon wasdemonstrated in some bipathic experiments. On the one hand, we revealed thatultralow doses of a drug specifically prepare the organism to normal dose of thesame drug. On the other hand, studies of American scientists showed thatpretreatment of neurons in the tissue culture with activated glutamate protectsthem from damage by glutamate in toxic doses (W. Jonas et al., 2001).Moreover, the protective effect against glutamate in toxic doses was alsoachieved after administration of cycloheximide in ultralow doses. However, otherneurotropic drugs in ultralow doses did not have a protective effect (D. Marottaet al., 2002). Hence, sensitization with ultralow doses is a specific process. Thisconclusion was confirmed by the results of our studies with morphine (T. A.Zapora et al., 1999), caffeine and cyclosporine A (O. I. Epstein et al., 2004),theophylline and morphine (O. I. Epstein et al., 2003), and 5F5�B6 antigen(N. A. Beregovoi et al., 1999).

Long�term observations of antibodies in ultralow doses showed that theyhave an adaptive effect. It is not surprising. Neurophysiological studies revealedthat weak and subthreshold factors have a sensitizing effect, which increases theadaptive capacity of an organism. More than 30 years ago, I. A. Arshavskiidemonstrated the existence of adaptogenic remedies. They include extracts ofEleutherococcus, Schizandra, ginseng, Rhodiola, etc. (I. A. Arshavskii, 1976).

Adaptive activity of these remedies is related to the nonspecific activatingeffect. This activity is short�lasting and mild. Therefore, adaptogens should notbe identified with the adaptive effect of antibodies in ultralow doses. Antibodiesin ultralow doses have a strong, rapid, and specific effect. The main advantagesof activated antibodies are sensitization of natural antibodies and mobilizationof predetermined normal functions (memory). Hence, activated antibodiesproduce a specific effect on the corresponding antigen. It should be emphasizedthat treatment with these antibodies (i.e., mild exposure) has a sparing effect.

Adaptation is of the most complicated problems in physiology.Adaptation to a certain environmental factor is based on the structural trace ofmemory (F. Z. Meerson, 1993). By the principle of dominant, this processcontributes to the integration of topographically different nervous centers intoone constellation (A. A. Ukhtomskii, 1950, 1952). The final stage of adaptation


86

Ultralow doses

(model) is encoded in this constellation. It was named “a model of the desiredfuture” (N. A. Bernstein, 1990) or “useful adaptive result of activity” (P. K.Anokhin, 1975).

According to P. K. Anokhin, parameters of the useful result integratevarious elements of a biological system into dynamic, self�organizing, and self�regulating functional systems. In other words, the goal (adaptation) combinesvarious physiological processes into a single whole. The integration is based onacquired “adaptable” plastic relationships and “rigid” inherited relationships (N.P. Bekhtereva, 1977). Functional systems are structured by the holographicprinciple (K. V. Sudakov, 1984, 1990, 1996a,b). Our notions about semantic�and�holographic functional principles of biological systems are close to theclassical concept of adaptation. During a semantic analysis, any external factorinduces a specific response of the organism. The image of this factor integratessome physiological processes into dynamic constructions of memory (long�termmemory). All constellations in the organism are “coupled” by wave hereditaryinformation. During interference of genetic and acquired information, the latterbecomes individual for this organism. The formation of an adaptive responseresults from individual experience, which is “superimposed” on general�speciesphysiological processes (genetic memory).

Our notions of adaption contradict the general theory. Approximately 10years ago, the notions of evolutionary purposes of biological systems were revisedafter experiments of Professor B. I. Lyubimov. The animals (mice) were treatedsimultaneously with toxic doses of morphine and potentiated morphine C200 (10�

400 wt %). Control animals received morphine in the same doses (Table 5.1).The following results were obtained for females: 257.476(244.302:271.361)LD50= 260.8298(238.9089:284.7621) mg/kg,

198.854(194.241:195.496)LD16= 152,3292(148.764:155,9799) mg/kg,

340.223(339.159:341.297)LD84= 446,6136(436,1606:457,317) mg/kg;

The following results were obtained for males:

257.476(244.302:271.361)LD50= 275.4606(192.2007:394.7883) mg/kg,

198.854(194.241:195.496)LD16= 138.0255(73.4526:259.3652) mg/kg,

87

Table 5.1.Table 5.1.Table 5.1.Table 5.1.Table 5.1. Toxicity of morphine after individual treatment and bipathic administration in combination with potentiated morphine (O. I.Esptein, 1999)

150 72 72 0 7 72 70 0 7

175 72 72 10 24 72 70 10 29

200 72 72 12 24 72 70 12 26

250 72 72 36 32 72 70 36 30

300 72 72 48 42 72 70 48 35

Dose, mg/kg number of died animals

bipathicadministration

individualadministration

number of animals per group

Males



number of died animals



number of animals per group

Females



Ch

ap

ter 5

.C

ha

pte

r 5.

Ch

ap

ter 5

.C

ha

pte

r 5.

Ch

ap

ter 5

. Prin

ciple

of m

ain

ten

an

ce o

f the

initia

l inte

grity

88

Ultralow doses

340.223(339.159:341.297)LD84= 549.7437(292.5553:1033.029) mg/kg.

The standard (individual) and bipathic methods of morphine treatmentare shown in the numerator and denominator, respectively.

The experiment yielded ambiguous results. On the one hand, thepotentiated product had a protective effect against morphine in toxic doses(LD84). On the other hand, toxicity of morphine in safe doses (LD16) was elevatedafter administration of the potentiated product. A polymodal adaptive response istypical of ultralow doses (E. B. Burlakova, 1986, 1990) and normal doses (well�known experiments of L. Kh. Garkavi et al., 1998). The question arise: what is thebiological significance of a polymodal response to substance in any dose?

We believe that the main role of adaptive capacity is not so much to pro�vide environmental adaptation as to maintain the initial integrity in an orga�nism. The tendency to integrity may be considered as a major factor, whichcombines biological systems into a single whole. Biophysically, it is manifestedin an attempt of the organism to achieve a harmonic “regular” holographicstate. The analogy to electron was drawn above. Each electron occupies a certainorbit during rotation around the nucleus. Similarly to this electron, the holographicsphere of an organism has steady integrative multiparametric functional states(orbits). Any medical product induces “wave” constellations in an organism.Phase characteristics of these constellations are determined by drug dose.Depending on the dose, this direct will “direct” all physicochemical processesin an organism at the closest functional orbit (relative to phase characteristics).

Probably, multiparametric characteristics of a certain steady sate of theorganism’s holographic sphere are the useful result of activity (according to P.K. Anokhin). In other words, a biological system always tends to achieve thepredetermined integrative state. These data introduce new definitions into theclassical notions of adaption in medicine and biology. Integrity appears to be ata higher evolutionary level than health. Therefore, adaptation may be achievedthrough disease. From this standpoint, any pathological condition should beconsidered as the lowest level of adaptation. The organism continues to functionso long as it maintains the initial integrity and fulfils a main evolutionarypurpose (correct reflection of reality). Functional inertia contributes to thedissociation of individual and general�species relationships in molecularensembles of one or another specific locus in the organism’s holographic sphere.To compensate this dissociation, the organism integrates molecular processeswithin the framework of pathological syndrome. During drug treatment forpathological syndrome, we propose another program to achieve the organism’sintegrity. In regard to allopathic drugs, this is related to integrative properties ofthe dose (weight). As for ultralow doses of antibodies, the specified condition

89


is associated with particular properties of these agents (i.e., type of naturalantibodies).

First, the amount of natural antibodies in an organism is extremely lowto perform the role of endogenous regulators. Hence, the “degree” ofconstellations induced by potentiated antibodies is sufficient for the interactionwith natural antibodies. The smaller is the target, the weaker is the weapon to“strike” this target. Despite low physiological concentration of natural anti�bodies in the organism, their constellations include the regulations for functionof all antigens. Hence, global problems (regulation of functions) can be solvedby a “small force”.

Second, natural antibodies have a unique morphofunctional dualism thatis manifested in the existence of V domains and C domains. This propertyallows natural antibodies to “trigger” co�adjustment regulatory (general�species)reactions in an organism. It is realized through C domains during the inter�action of one or another molecule (antigen) with V domains.

Third, antibodies are combined into the anti�idiopathic network. Adisturbance from the interaction of antigen with one or several molecules ofantibodies can spread over the whole network.

And fourth, a continuous transfer of information in network structures(e.g., anti�idiopathic network of antibodies) seems to be analogues to the processof potentiation. During continuous circulation, the same spatial images(constellations) are superimposed on their traces. Hence, each image is dividedinto various temporal (phase) constituents. The anti�idiopathic network not onlyincludes the memory of normal function of antigens, but also encodes a step�by�step program of using this memory (program for result of action, P. K.Anokhin)*.

Activated compounds have an “accelerating” effect on reflection of theoriginal substance in an organism (advanced programming of properties of theuseful adaptive result, P. K. Anokhin) and reduce the latency of an adaptiveresponse. This has been demonstrated with the phenomenon of bipathy. Thesedata suggest that the effect of products from ultralow doses of antibodies isrealized via advanced regulation of functions by natural antibodies.


The cell of vital activity consists of semantically organized molecular andsubmolecular ensembles (constellations) that appear as a trinity of structure,functions, and hereditary principles (memory) of functioning.

* Antibodies at various dilutions can affect various stages of this program. Hence, theeffect of antibodies in ultralow doses depends on their potency.

90

Ultralow doses

Any organism as a sum of molecular constellations is the integral holo�graphic structure (sphere) with a unique dual (individual�and�species) architec�tonics.

The maintenance of a unique specific structure in accordance with evo�lutionary (basic) principles of the spatial and temporal organization of vital ac�tivity is a main evolutionary purpose of any biological system.

All physiological reactions of the organism (including an adaptive re�sponse) are informationally predetermined and directed to maintain the initialintegrity.

Adaptive pathological syndrome is a holographic structure, which may beconsidered as an effort to retain the initial integrity (“self”) in another structuralform (“non�self”).

The medical product in any dose can interact directly with pathologicalsyndrome and has a therapeutic effect under certain conditions (combinationof tropism for a certain pathological condition and sufficient integrity).

The main physiological property of remedies with ultralow doses is a sen�sitizing effect on the certain structured processes.

A specific nature of antibodies indicates that they can be used as mo�dern high�effectiveness remedies in ultralow doses. As differentiated from ho�meopathy, this approach dies not require individualization of therapy. Potenti�ated antibodies have a sensitizing effect on the system of natural antibodiesand, therefore, increase their regulatory activity (“regulation of regulator”).

91

Experiments with ultralow doses showed that the division of medicine intohomeopathic and allopathic medicine is arbitrary. Medicine is a unified

science. The organism specifically reacts to a substance independently on itsdose. This simple fact emphasizes that biological systems are characterized bya fine “informational�and�essential” level of organization. At this level, theeffect of medical products in “allopathic” and “homeopathic” doses is mediatedby similar mechanisms. The effects of ultralow doses are reproducible, may beevaluated by standard methods and, therefore, hold promise for evidence�basedmedicine. The exception is homeopathic therapy. The individual (“similar”)prescription of remedies is like art. The methodology of homeopathy is notassociated with a pathophysiological approach of modern pharmacology.

However, the experience of homeopathy is worthy of attention.Physicians can adopt a wise (holistic) attitude of homeopaths to the patient.Moreover, the drugs should be prescribed with caution. Skilled homeopaths takean individual approach. They usually prescribe only one remedy, follow carefullythe patient’s reaction, and do not tend to cure the disease “at any price”. Ahomeopathic approach to drug�induced exacerbation is of interest to clinicians.This state is associated with drug treatment when the observed symptomsconstitute the so�called “pathogenesis” (see Chapter 1). There is no cause�and�effect relationship between the remaining symptoms and drug treatment. A

C h a p t e r 6

On the way topharmacologyof ultralow doses

92

Ultralow doses

famous physician D. V. Popov (founder of the Kiev homeopathic school)proposed the following three groups of symptoms in exacerbation: 1) existing ina patient and increasing during drug treatment; 2) present in the anamnesis; and3) observed at any time in immediate relatives. According to the rules of C. He�ring, the homeopath can predict a favorable or poor prognosis of exacerbation.The drug is not withdrawn in a favorable prognosis. However, this state requiresa decrease in the frequency of drug treatment or short�term interruption oftherapy. Therapy is interrupted only when drug�induced exacerbation concernsvital organs. When clinical symptoms “migrate” from vital organs to otherorgans (e.g., skin) during treatment with the potentiated product, this trans�formation is considered as a positive reaction (“minimal” harm) and therapycontinues.

The historical experience of homeopathy (i.e., study of medical productsin ultralow doses with healthy volunteers, S. Hahnemann) is of particularimportance for modern clinical pharmacology. This is a paradigmatic exampleof simplicity and greatness.

Generally, up to one third of patients may exhibit an atypical responseto any pharmaceutical product. The incidence of side effects, including severecomplications, is high in the group of patients with atypical reactions.Genotyping of individual drug sensitivity is not introduced into clinicalpractice. We believe that standard clinical trials of medical products shouldbe supplemented by the introduction of a group of healthy volunteersreceiving the activated form of drug. Such approach will allow us to evaluatethe hyperergic reaction to study drug in sensitive respondents. Therefore, allpossible complications will be rapidly and safely revealed at the initial stageof therapy. The remaining side effects of study drug are associated with itscumulative action and may be evaluated only in normal�dose trials. Besidesthe evaluation of adverse events, a study of activated products with healthyvolunteers before the start of standard clinical trials will demonstrate thephenotypic and genetic markers (correlates) for individual sensitivity ofrespondents to a certain drug.

Moreover, the activated forms of pharmaceutical agents should be testedalso in patients. Before the start of clinical trials, one or another drug in ultralowdose may be given to a patient (one�to�two times). In the case of a stronghyperergic reaction, the respondents should be excluded from clinical trials orreceive this drug in the reduced dose.

Besides the experience of homeopathy, combined (bipathic) admi�nistration of the drug and its activated form is of particular interest. Bipathy asa whole may be introduced into modern medicine. After the discovery of thisphenomenon, there was a review on the protective effect of heavy metal saltsin ultralow doses against the toxic concentration. Administration of the activated

93

substance before and after treatment with heavy metals was followed by theincreased elimination of a toxic agent (A. Delbancut et al., 1997). There wereno data on the general modifying activity (except for protective properties) ofultralow doses, which occurs after combined (bipathic) administration of thesubstance and its activated form. Unfortunately, beginning from 1999 thedevelopment and introduction of products from ultralow doses of antibodiesdrew us away from further studies of bipathy. However, the results of previousexperiments are sufficient to understand the importance of this phenomenon.The activated remedy potentiates* a pharmacological effect of the originalsubstance, which is of particular significance for medical practice.

Experiments at the laboratory of T. A. Voronina showed that combinedtreatment with the normal and ultralow dose of phenazepam (ULDP, dilutionC12+30+200) is followed by a significant increase in anxiolytic activity of thisdrug (O. I. Epstein et al., 2007). Activated phenazepam was given simultane�ously or 10 min before administration of the original substance. These studieswere conducted on the model of punished drinking. Electric current was appliedto the floor of cages and spout of drinking bowls. The animals attempted tosatisfy their natural demands for water (drinking from bowls). However, theywere punished by a mild electric current. Antianxiety drugs allowed the animalsto adapt and satisfy their biological demands for water (in spite of punishment).The stronger was the anxiolytic (antianxiety) effect of study drug, the greater wasthe number of punished drinking episodes (Table 6.1).

Another experiment of T. A. Voronina was designed to study the effect ofbipathic treatment with phenazepam on animals with corazol�induced seizures

* For this reason, we proposed the term “activated” form instead of “potentiated” form.

Chapter 6. Chapter 6. Chapter 6. Chapter 6. Chapter 6. On the way to pharmacology of ultralow doses

Table 6.1.Table 6.1.Table 6.1.Table 6.1.Table 6.1. Effect of individual or combined treatment with phenazepam in the thera�peutic and ultralow dose on the number of punished drinking episodes inrats (conflict situation)

Control 20 177.75±43.02

Phenazepam 1 ml/kg 20 415.67±113.96*

ULDP 2.5 ml/kg 20 260.67±38.21

ULDP 2.5 ml/kg 30 358.33±60.75*

ULDP+phenazepam(simultaneously) 2.5 ml/kg + 1 mg/kg 20 1279.33±82.28**

ULDP before phenazepam 2.5 ml/kg+1 mg/kg 20 1022.00±46.36**

Note.Note.Note.Note.Note. *p<0.05 and **p<0.01 compared to the control.

GroupNumber of punisheddrinking episodes at

0.25�mA current

Period betweendrug treatment and

recording of the effectDose

94

Ultralow doses

Table 6.2.Table 6.2.Table 6.2.Table 6.2.Table 6.2. Effect of phenazepam in the therapeutic and ultralow dose on outbredalbino rats with corazol�induced seizures

Parameter

Latency of clonic seizures 0:11:20± 0:22:20± 0:09:40± 0:37:20± 0:32:00±0:03:13 0:05:42* 0:00:35 0:06:48*+ 0:10:26*

Latency of tonic seizures 0:16:40± — 0:13:20± — 0:33:00±0:04:56 0:00:34 0:09:24*

Tonic seizures, % 100 0 100 0 40

Mortality, % 80 0 100 0 20

Note.Note.Note.Note.Note. p<0.05: *compared to corazol; +compared to phenazepam in normal dose.

Corazol+ULDP+

phenazepam(simultaneously)

Corazol+ULDPbefore

phenazepam

Corazol+ULDP

Corazol+phena�zepam

Corazol

(O. I. Epstein et al., 2007c). Ultralow doses were administered simultaneouslyor 10 min before treatment with phenazepam in normal dose. Activatedphenazepam had a potentiating effect not only on anxiolytic activity, but alsoon the antianxiety properties of this drug. It was manifested in an increase inthe latency of seizures, decrease in the ratio of animals with seizures, andreduction of the mortality rate. The side effects of phenazepam were notrevealed under these conditions (sedative and myorelaxant activity; Table 6.2).

Further studies at the laboratory of T. A. Voronina showed that psycho�tropic properties of a well�known neuroleptic drug haloperidol are not observedafter bipathic treatment of experimental animals.

Combined treatment with normal dose and ultralow dose of haloperidol(ULDH) was followed by a decrease in the cataleptogenic effect of this drug.The degree of catalepsy decreased by 24, 46, and 33% by the 60th, 120th, and190th minute after administration of ULDH, respectively (compared to animalsof the haloperidol group).

Administration of Cyclodol (6 mg/kg) in combination with haloperidolhad a strong antagonistic effect on cataleptogenic activity of haloperidol.Catalepsy was completely abolished after 60, 120, and 180 min.

The cataleptogenic effect was less pronounced after combined administra�tion of Cyclodol and ULDH. It should be emphasized that the cataleptogeniceffect was lower compared to that of Cyclodol, but higher than the activity ofpotentiated haloperidol (Table 6.3).

It will be remembered that the anti�blastoma effect of cyclophosphane in�creases after bipathic administration (E. N. Amosova et al., 2003). Experimentalstudies (O. I. Epstein et al., 1997; V. G. Zilov et al., 2000; O. I. Epstein et al.,2002b; A. M. Titkova et al., 2002; O. G. Berchenko et al., 2003; I. F. Pavlovet al., 2003) and some clinical observations (N. V. Aleksandrova et al., 2003)

95

revealed the protective effect of prednisolone, morphine, and ethanol duringcombined administration of the original substance and its activated form. Severalexperiments were performed with detoxification of heavy metals. The results ofthese researches suggest that the phenomenon of bipathy holds much promisenot only for the potentiation of pharmacological activity, but also for thecorrection of toxic properties of pharmaceutical products. Unfortunately, noneof the “bipathic” remedies is used in clinical practice (as differentiated frommedical products from ultralow doses of antibodies). It should be noted thatpatents for bipathy have been granted in some countries.

Bipathy suggests a specific modifying effect of activated drugs on theactivity of original substances. Previous studies on various experimental modelsshowed that ultralow doses of cadmium prevent the toxic effect of cadmium.Mercury in ultralow doses had a protective effect against toxic activity ofmercury salts (A. Delbancut et al., 1997). There are some data on the protectiveactivity of one substance against the toxic dose of another substance. Asmentioned above, activated cycloheximide protects neurons from glutamate intoxic doses (D. Marotta et al., 2002). Moreover, ultralow doses of somesubstances potentiate the effect of an antitumor antibiotic adriamycin (N. P.Konovalova et al., 2002; N. P. Pal’mina et al., 2002).

We proposed that ultralow doses of antibodies can be used in clinicalpractice. Activated products have a wide range of experimental and clinicaleffects. Therefore, this approach may be considered as a precursor ofpharmacology of ultralow doses. At the present time, potentiated antibodies arethe most studied substances of ultralow doses.

Activated antibodies are a new class of medical products that meetthe requirements of evidence�based medicine. Due to technical reasons, they


Table 6.3.Table 6.3.Table 6.3.Table 6.3.Table 6.3. Effect of ULDH and Cyclodol on the degree of haloperidol�induced catalepsyin rats (Morpurgo method)

Haloperidol, 0.7 mg/kg 1.7±0.2 2.6±0.3 2.4±0.3

Haloperidol (0.7 mg/kg) +Cyclodol (6.0 mg/kg), simultaneously 0.0±0.0* 0.0±0.0* 0.1±0.1*

Haloperidol (0.7 mg/kg) +ULDH (2.5 mg/kg), simultaneously 1.3±0.1* 1.4±0.2* 1.6±0.2*

Haloperidol (0.7 mg/kg) +Cyclodol (6.0 mg/kg) +ULDH (2.5 mg/kg), simultaneously 0.5±0.2* 0.5±0.2* 1.0±0.2*

Note.Note.Note.Note.Note. *p<0.05 compared to animals receiving haloperidol in a dose of 0.7 mg/kg

Group, doseafter 180 minafter 120 minafter 60 min

Average score per group

96

Ultralow doses

are formally designated as homeopathic remedies. Probably, this classificationwill change after the solution of normative questions. Principally, all productsof antibodies in ultralow doses are developed according to the general principlesof modern pharmacology. They include an experimental evaluation of specificpharmacological activity, conduction of required toxicology studies, use of adouble�blind placebo�controlled method in clinical trials, comparison of newagents with modern pharmaceutical products, etc.

The results of experimental and clinical studies with products fromactivated antibodies are presented in the final chapters of this monograph. Theyshow that ultralow doses of antibodies exhibit high effectiveness, which isparticularly important for modern pharmacology. The effectiveness of mostproducts was highly competitive with or surpassed that of reference drugs. Eventhough the effectiveness of antibody�containing agents in ultralow doses is lowerthan that of reference drugs (Impaza and sildenafil; and Artrofoon andindomethacin), the integral effectiveness/safety criterion of study preparationscompares well with pharmaceutical products. Sometimes the effectiveness ofantibodies in ultralow doses was unexpectedly high (even for developers). Forexample, a hypoglycemic effect of peroral treatment with activated antibodiesto the insulin receptor β�subunit surpassed that of parenteral insulin. Besidesthis, ultralow doses of antibodies to interferon�γ (IFN�γ) were effective on themodel of avian influenza. Antiinflammatory activity of Artrofoon allowed thepatients to avoid the use of nonsteroid antiinflammatory drugs.

The estimated safety of new products was expected. Ultralow doses couldnot be toxic a priori. All trials for acute and chronic toxicity, reproductivetoxicity, embryotoxicity, immunogenicity, immunotoxicity, etc. confirmed thesafety of activated antibodies. Moreover, ultralow doses of antibodies to IFN�γwere shown to have antimutagenic activity.

The effect of antibodies in ultralow doses can develop in the early (e.g.,several tens of minutes for Anaferon�induced hypothermia) or late period aftertreatment (chronic diseases). It may be said that activated antibodies had theimmediate and delayed therapeutic effects in acute and chronic diseases,respectively.

A general tendency was revealed during the therapy of chronic diseases(rheumatoid arthritis, osteoarthritis, prostate adenoma, and cardiovascularfailure) with ultralow doses of antibodies. The improvement was observed by theend of 1�month treatment with study drugs, progressively increased in thefollow�up period, and reached maximum after 3 or 6 months of therapy. Theeffect of “allopathic” reference drugs usually occurred after 2 weeks of therapyand was most pronounced by the 4th week. However, many patients were char�acterized by exacerbation of the disease and development of side effects. Mildexacerbation of the underlying disease was rarely observed after treatment with

97

ultralow doses of drugs. The adverse events were not revealed under theseconditions. Otherwise, the relationship between treatment with antibody�containing products and development of undesirable effects was ambiguous.During combination therapy with standard drugs and products from antibodiesin ultralow doses, activated antibodies had a potentiating therapeutic effect.Sometimes the incidence of side effects of “allopathic” drugs tended to decreaseunder these conditions.

The effectiveness and safety are associated with a particular effect ofantibodies in ultralow doses on pathological syndrome. It will be rememberedthat pathological syndrome is considered as an adaptive response to a changein the individual�and�species integrity of an organism. Chronic diseases oftendevelop in response to the uncoupling of physiological reactions due to theirinertia, which increases with age. Pathological syndrome is a spatial�and�holographic structure, which allows the organism to maintain its initial integrity.

According to the general laws of matter, any pharmaceutical agent mayexhibit tropism for one or another structured process in an organism (includingthe pathological process). After a “semantic” analysis, the drug�inducedmolecular constellations can directly interact with pathological syndrome. Thisinteraction is probably mediated by the mechanism of resonance. The structureof medical product is reflected in constellations. Because of the integrity, thisdrug becomes “incorporated” into the structure of pathological syndrome. Theobserved changes result in a change in spatial configuration of pathologicalsyndrome. Until this moment, the pathological syndrome is not “noticed” byholographic memory of an organism. This is associated with the absence ofnovelty, which serves as a major prerequisite for memory stimulation. Anyexternal agent or internal event of unknown integrity serves as a novel factor forthe organism. A drug�induced disturbance in the dynamic equilibrium ofpathological syndrome is followed by structural changes and loss of an individualinitial integrity. The pathological syndrome becomes an object of regard withholographic memory*. After the loss of integrity, this organism programs newmultiparametric characteristics of function to achieve the initial integrity.Clinically, the transformation of pathological syndrome may be followed notonly by its complete or partial reduction, but also by the development ofundesired events.


* Some data of experimental immunology are analogous to this suggestion.Administration of an antigenic molecule to newborn or adult animals (under specifictechnological conditions) may inhibit the immune response to a specific antigenicmolecule (tolerance). As mentioned above, the immune system does not respond tosmall molecules or haptens (see Chapter 4). After conjugation of this “non�reactive”antigen with hapten, the immune system “notices” a modified molecule and producesspecific antibodies.

98

Ultralow doses

A distinctive property of products from ultralow doses of antibodies is thatthey modulate a unique regulatory system of natural antibodies. We emphasizedthat antibodies have a specific individual structure and are combined into theanti�idiopathic network. This network includes the principles of function(memory) for all antigens (molecules) in an organism. Potentiated antibodieshave a sensitizing effect on the anti�idiopathic network, which results in theactualization of holographic memory without structural reconstruction ofpathological syndrome. Hence, they produce a sparing effect. Our studiesshowed that the therapeutic effect is achieved only after modification (throughactivated antibodies) of the molecule or antigen, which has a key role in thepathogenesis of a pathological condition. Under these conditions, the therapywith ultralow doses of antibodies may be considered as pathogenetic. Someevidence exists for our assumption, which is unusual for pathophysiology.Artrofoon, which consists of antibodies to TNF�a in ultralow doses, has a strongdisease�modifying effect after long�term therapy for rheumatoid arthritis andosteoarthritis (2 years).

A pathogenetic effect of antibodies in ultralow doses is also confirmed bythe persistence of drug�induced changes after cessation of therapy. As distinctfrom one of the benzodiazepine drugs, the antianxiety effect of Tenoten(ultralow doses of antibodies to S�100) is observed for at least 4 weeks (N. P.Vanchakova et al., 2007).

The following clinical observation requires special attention: thecumulative effect of long�term therapy with ultralow doses of antibodies is notaccompanied by an increase in the dose of prescribed drug.

The experience of homeopathy indicates that the effect of ultralow dosesmay depend on their dilution (potency). Further studies revealed the unusualtypes of selectivity of ultralow doses. For example, mollusk neurons of differentfunctions exhibit a response to various dilutions of anti�S100. Otherwise, anti�S100 of different potency have a modulatory effect on various systems ofintracellular kinases. Clinical trials of Artrofoon in patients with rheumatoidarthritis yielded a surprising result. The antiinflammatory and analgetic effectswere more significant after treatment with Artrofoon in a dose of 2 tablets 4times a day (but not 1 tablet 4 times a day). It was unexpected that the effectdepends on the volume (number) of dilutions of activated substances. Furtherstudies of anti�S100 were performed at the laboratory of T. A. Voronina and“Porsolt & Partners Pharmacology” research company. The effect of theseantibodies in animals was characterized by an inverted U�shaped dependenceon the volume of dilutions of activated antibodies to S�100 protein (V. Castagniet al., 2007; Fig. 6.1).

The observed dependence is important to understand the mechanism foreffect of antibodies in ultralow doses. These data will be discussed later. It

99

should be emphasized that the notion “dose” is also applicable to activatedproducts. Cumulation of the therapeutic effect without increasing the dose is aspecific feature of activated products. Moreover, clinical trials showed thatpatients with rheumatoid arthritis and osteochondrosis may be treated withlower (maintaining) doses when a therapeutic effect of the antibody�containingproduct reaches a plateau.

Tolerance of an organism to medical products results from the inertia,which is typical of physiological processes. The stage of functional�and�metabolic “reading” of any pharmaceutical product is followed by the formationof molecular constellations. This stage is also inert. During protracted treatment,the therapeutic effect of prescribed drug may be maintained only underconditions of “novelty” (i.e., increase in the dose). However, this procedure canresult in the development of drug dependence. Activated solutions are the readyconstellation product. Therefore, various drugs from ultralow doses of antibodiesshould not overcome the inertia during formation of constellations. Drugtolerance does not occur under these conditions.

The inertia is typical not only of physiological processes, but also ofconventionally pathological processes. This inertia does not allow the organismto “recover” naturally from one or another chronic disease. Not only thestructure, but also the dynamics of pathological syndrome is encoded in thegenetic memory. This information appears as the successive and pathologicallyrelated functional�and�metabolic stages. The program of each stage is encodedin the previous stage. The next stage does not start before the end of theprevious stage. Increasing the dose of a therapeutic drug does not necessarilyallow us to overcome the inertia of pathological process. The therapy withultralow doses of antibodies has great advantages in torpid diseases. As


Fig. 6.1.Fig. 6.1.Fig. 6.1.Fig. 6.1.Fig. 6.1. Dependence of the anxiolytic effect of Tenoten on its dose (elevated plus�maze test): “Porsolt & Partners Pharmacology”. *p<0.05 and **p<0.001 comparedto the control.

0

�20

�402.5 5.0

Tenoten dose, ml/kg

7.5 15

20

40

60

80

*

*

**

100

Ultralow doses

mentioned earlier, potentiated antibodies have the specific phase�and�dynamiccharacteristics. They serve as “time derivatives” of physiological functions ofnatural antibodies in the organism. Under conditions of pathological syndrome,these antibodies are coupled with an ensemble of other endogenous molecules.They “freeze” at the unrealized stage of pathological process.

Ultralow doses of antibodies affect natural antibodies and transform them(in advance) into another phasic functional state. These changes also concernthe antibody�regulated antigens and result in an “imbalance” of pathologicalsyndrome.

Sometimes ultralow doses of antibodies directly improve the dynamics ofpathological process (up to complete recovery or remission). These antibodiescan increase the sensitivity to previous therapy, which allows reducing the doseof standard pharmacological agents. The course of treatment with ultralow dosesof antibodies to TNF�α contributes to a twofold decrease in the dose ofnonsteroid antiinflammatory drugs and cessation of therapy in patients withrheumatoid arthritis and osteoarthritis, respectively. The following observationsserve as an example of improved tolerance after administration of potentiatedantibodies.

Ultralow doses of antibodies to the insulin receptor beta�subunitsignificantly increase tolerance to glucose on the model of streptozotocin�induced diabetes. These antibodies are more potent than glybenclamide, but lesseffective than insulin*.

T. M. Vorob’eva studied an antialcohol drug Proproten on the model oflateral hypothalamic self�stimulation. The results of these experiments weresurprising for experimental narcology**. All animals refused the ability to self�stimulate a “pleasure center”, which is untypical of allopathic psychotropicdrugs. As differentiated from a variety of psychopharmacological agents, thesame phenomenon was observed without increasing the dose of study drug.

The ability to maintain a specific effect without increasing the doseis important for long�term therapy (particularly during treatment for nearlyincurable diseases, which result from low level of adaptation). This is related tohigh toxicity and risk of undesired transformation of the underlying disease.

The term “level of adaptation” is extensively used in physiology. Clini�cians also know that the lower are the adaptive capacities of an organism, thehigher is the predisposition to severe destructive diseases. The most prominentclinical example of multistage adaptation is the development of mentaldisorders due to external influences (e.g., craniocerebral injury). These

* See Chapter 7.** Ultralow doses of ethanol had a similar, but less pronounced effect on this experimental

model.

101

disturbances are transitory (Wick’s symptoms), alternate in a certain sequence,and usually result in asthenia. In this case, asthenia serves as a generaladaptive response.

Hence, clinical transformation of pathological syndrome is a normal signof adaptive capacities in the organism. The pathomorphosis of disease may bespontaneous or induced by drug therapy. A physician must evaluate whether theprescribed drug therapy and subsequent transformation are the symptoms of afavorable prognosis (i.e., transfer of the disease to a higher level of adaptation).The transformation of symptoms serves as an unfavorable prognostic factor andmay be accompanied by adverse events in severe chronic diseases (low level ofadaptation). According to FDA, 100000 people in the USA annually die fromcomplications of pharmacotherapy. The mortality of most patients is associatedwith undesirable transformation of destructive chronic diseases. There is needto minimize “harm” under these conditions. Incredible as it may seem, a“severe” pathological symptom should not be “cured”. It is more important toprevent the transformation of this symptom. Sparing therapy with potentiatedantibodies is most appropriate for such patients.

Therapy with ultralow doses of antibodies complies with the followingprinciple: “First, do not harm”. By the mechanism, this therapy serves as anadaptive exposure of high effectiveness and safety. We would like a physicianto known the mechanism for action of new�generation drugs. The safety ofpotentiated products is not related to the presence of low doses of the originalsubstance. These drugs are an activated form of the original substance, whichhas specific biological properties and involves other mechanisms ofadaptation.

Thirteen preparations from ultralow doses of antibodies wereapproved in the Russian Federation. However, the number of widely useddrugs of this type is twofold lower. In recent years, antibodies in normal doseswere used for the therapy of noninfectious diseases and manufactured by thelargest international pharmaceutical companies (Table 4.6). The majority ofthem are monoclonal humanized antibodies. They appear to be identical tohuman antibodies and, therefore, are not perceived as a foreign agent afterparenteral treatment. These drugs are mainly used in the therapy for severedisorders, including oncological diseases (N. I. Olovnikova et al., 2007).Previous studies showed that these drugs are effective, but not harmless. Thedevelopment of these drugs is based on published data that antibodies directlyblock one or another molecular target. We believe that the primary effect isassociated with complementary binding of antibodies to antigens. Thesystemic effect of antibodies in normal and low doses may be realized viaactivation of natural antibodies. This assumption is confirmed by the fact thatRemikeid and Artrofoon (preparations from normal and potentiated


102

Ultralow doses

antibodies to TNF�α) have a similar effect. Long�term administration of bothdrugs is followed by a decrease in the level of TNF�α (cytokine withproinflammatory activity).

As regards the molecular targets (antigens), all products from ultralowdoses of antibodies are classified into the following five groups:

• antibodies to cytokines and growth factors (Anaferon, Artrofoon, andPoetam for IFN�γ, TNF�α, and erythropoietin, respectively);

• antibodies to brain�specific protein S�100 (Proproten, Tenoten, andTenoten for children);

• antibodies to enzymes (Impaza, endothelial NO synthase; and Afa�la, trypsin�like protease or prostate�specific antigen);

• antibodies to receptors (Kardos, angiotensin II AT1 receptor; andexperimental product Bation, insulin receptor b�subunit); and

Table 6.4.Table 6.4.Table 6.4.Table 6.4.Table 6.4. Antibody�containing products in the world

Rituxan (Genetech) CD20 (B lymphocytes) Non�Hodgkin’s B cell lymphoma

Herceptin (Genetech) HER2 antigen Breast cancer

Avastin (Genetech) VEGF (vascular Large intestine cancerendothelial growth factor)

Erbitux (Merck) Oncological diseasesCampath (Bayer)Zevalin (Genetech)MyloTarg (Wyeth Ayerst)Bexxar (GlaxoSmithKline)

Remicade (Centocor) TNF�α Psoriasis, Crohn’s disease,ankylosing spondyloarthritis, rheu�

matoid arthritis, and ulcerative colitis

Humira (Abbott) Autoimmune inflammatoryRaptiva (Genetech) diseasesSimulect (Novartis)Zenapax (Hoffmann�La Roche)OrthoClone OKT3(Qrtho Biotech)

ReoPro (Eli Lilly) Platelet glycoprotein Prevention of thrombusIIb/IIIa receptor formation in angioplasty

(surgical treatment for CHD)

Synagis (Medimmune) Respiratory syncytial Therapy of children with respiratoryvirus protein syncytial virus infection

Xolair Immunoglobulin E Atopic diseases(Genetech/Novartis)

Product (company) Indications for useMolecular target

103

• antibodies to low�molecular�weight compounds (histamine, Prohistamand Epigam; cholecystokinin, Cholestam; and morphine*, Anar).

Published data show that the effectiveness of medical product fromultralow doses of antibodies does not depend on morphofunctionalcharacteristics of antigen, but is determined by “tropism” for one or anotherpathological condition. The effects of activated antibodies are determined by therange of physiological activity of a particular antigen. For example, brain�specific protein S�100 does not have a narrow range of physiological activity.This protein is responsible for some basic functions of the nervous systems,including the generation and conduction of nerve impulses, synaptic plasticity,etc. (M. B. Shtark, 1985). It is not surprising that ultralow doses of anti�S100have a wide range of neurophysiological properties (from sensitization of theneuronal membrane to early gene expression) and psychopharmacologicalactivity**. By contrast, activated antibodies to histamine exhibit an expectedhigh specificity for ulcer disease and allergy (as expected).

The possibility to predict pharmacological activity of antibody�containingproducts from the knowledge of pharmacological properties of a specific antigensignificantly facilitates the screening for drugs. It should be emphasized thatpotentiation allows us to modulate the previously “inaccessible” molecules andextends the number of therapeutic targets in pharmacology.

For example, the mechanisms of action for Impaza (therapy of erectiledysfunction) differ from those for drugs consisting of phosphodiesterase type 5inhibitors (Viagra, Levitra, and Sialis). The physiological mechanism of erectionsuggests NO release in the cavernous bodies during sexual stimulation. Thisprocess contributes to the elevation of cGMP concentration, relaxation ofsmooth muscles, and increase in blood supply to the penis. Type 5 phos�phodiesterase is responsible for the consumption of cGMP. The inhibition ofthis enzyme is followed by an increase in cGMP concentration. Impaza has anormalizing effect on the reduced activity of endothelial NO synthase, increasesNO level, activates guanylate cyclase, and elevates cGMP concentration in thecavernous bodies. Hence, endothelial NO synthase is a new pharmacologicaltarget for ultralow doses of antibodies. Moreover, ultralow doses of antibodieshave a more physiological effect on this enzyme (modification, but notinhibition). Impaza has a mild effect, holds promise for the therapy of patientswith hypertonia and CHD, and may be used in combination with nitrates. Thetechnology of potentiation allowed us to reveal a variety of new pharmacological


* Morphine is the only endogenous antigen used in our studies. However, antibodies tomorphine can interact with some of the opiate�like low�molecular�weight endogenousmolecules.

** See Chapter 7.

104

Ultralow doses

targets (besides endothelial NO synthase), including S�100 protein, prostate�specific antigen, and insulin receptor. It is most important that ultralow dosesof antibodies have a specific effect.

In the previous chapters, we described various aspects of the effectof ultralow doses. In conclusion, we would like to briefly summarize the generalprinciples that concern the mechanisms of action and specific effects ofactivated antibodies.

Similarly to other drugs in ultralow doses, ultralow doses of antibodies havebiological activity that is related to the technology of potentiation. Potentiatedsolutions of antibodies have a similar systemic effect, which does not depend onthe presence of molecules of the original substance (O. I. Epstein et al., 2004).

The technology of potentiation provides a new property of antibodies inultralow doses. Subthreshold sensitization contributes to the modifying effect ofthese antibodies. The physical nature of ultralow doses remains unknown.Hence, the term “sensitization” is secondary in relation to activated products.This term does not reflect the effect of ultralow doses, but suggests the re�storation of sensitivity (reactivity) of structured processes. Recovery of reactivityis associated with the influence of activated antibodies on “wave” geneticmemory of an organism.

We revealed two types of the dose�effect relationship for activatedproducts. The effect of these substances is determined by dilution (potency) andvolume of the activated solution. The potentiated product (liquid or solidsubstance) may be considered as an oscillatory circuit, whose characteristics aredetermined by the method of preparation (number of dilutions) and size(volume of the activated solution). The activated product has a selective dose�dependent effect on various phases of the same physiological process withdifferent frequency�oscillation characteristics, which is mediated by themechanism of resonance. Maximum therapeutic effect is not induced by thehighest volume of potentiated solution, but occurs when the amount of thissolution is optimal for the resonance interaction with a target. Our observationsare consistent with the general notion of U�shaped dependence (E. J. Calabreseet al., 2001). Complex mathematical models showed that an inverted U�shapedcurve for the dose�effect relationship is not typical of substances with thestandard receptor mechanism of action (as differentiated from a monotonicdose�effect relationship). This curve is appropriate for exogenous substances thatactivate nuclear receptors (similarly to natural endogenous ligands). A U�shapeddose�effect curve does not depend on receptor affinity for the exogenous sub�stance (M. C. Kohn et al., 2002). This approach correlates with the assump�tion of T. A. Voronina that Tenoten equilibrates the systems induced byexogenous and endogenous substances (A. V. Martyushev�Poklad et al., 2003,2004). Previous studies of various antagonists on the model of LTPTP and

105

behavioral tests showed that the GABA�A and GABA�B systems are involvedin the effect of Tenoten. However, the effects of activated antibodies to S�100were not completely abolished under these conditions. These data serve asindirect evidence that antibodies in ultralow doses have a wide rage ofmodulatory (normalizing) activity.

Experiments with combined (bipathic) administration of themedical product and its ultralow dose showed that the activated substance notonly prepares one or another structured process to the interaction with studydrug, but also potentiates the effect of this drug. The nature does not like com�plexity. Obviously, both phenomena are mediated by the same mechanism. Itmay be suggested that the potentiated substance affects fine conformational pro�perties of the original molecule. Any endogenous molecule has the specific con�formation at a certain moment of time. Hypothetically, the number of mole�cular conformations is infinite or sufficiently great. The association of variousmolecules in physiological or pathological processes is probably followed by syn�chronization of their conformational and oscillation parameters. Exposure of theoriginal molecule to its activated form is followed not only by a change in themolecular structure, but also by modulation of distant functional relationships.

There is indirect evidence that functional activity of natural anti�bodies may be modified. The use of activated antibodies in EIA is followed bya change in affinity of natural antibodies for the antigen (O. I. Epstein et al.,2000). Clinical trials revealed that the content of natural antibodies returns tonormal after the course of treatment with potentiated antibodies. The reducedlevel of natural antibodies to IFN�γ in patients with viral infections increaseson day 1 after administration of anti�IFN�γ antibodies in ultralow doses (A.Caruso et al., 1997). The content of natural antibodies to S�100 protein in bloodplasma of alcoholic patients returned to normal by the end of long�termtreatment with ultralow doses of antibodies to S�100 protein. A change in theactivity of natural antibodies is probably followed by programming of theadaptive multiparametric state. Within the framework of this program, thesystemic adaptive effect of activated antibodies is mediated by a variety ofnegative feedback biological reactions. The main effects are illustrated by theexample of antibodies to IFN�γ in ultralow doses.

First of all, activated antibodies have a specific effect on expressionof the corresponding antigen. We showed that the course of treatment withpotentiated antibodies to IFN�γ is followed by the increased production ofendogenous IFN�γ in experimental animals. It should be emphasized thatactivated antibodies to other cytokines had little effect on IFN�γ expression*.

Besides the increase in IFN�γ expression, ultralow doses of


* See Chapter 7.

106

Ultralow doses

antibodies to IFN�γ had an indirect modulatory effect on the production offunctionally related IL�2, IL�4, IL�10, and IFN�α/β. These propertiescontribute to a wide range of antiviral and immunomodulatory activity ofAnaferon. It is most important that Anaferon optimizes the interferon status*.The preventive effect of Anaferon in ARVI is probably related to a normalizingaction of IFN�γ on various components of immunity.

The sensitizing activity of antibodies in ultralow doses serves as aphysiological basis for their use in the prevention of various diseases.

A specific effect of activated antibodies is illustrated by the exampleof anti�IFN�γ antibodies in ultralow doses. As shown above, potentiatedantibodies to IFN�γ significantly increase the production of IFN�γ, have a mildactivating effect on the expression of functionally related TNF�α, and do notmodulate erythropoietin secretion (O. I. Epstein et al., 2004). Moreover,potentiated antibodies have a species�specific effect. Ultralow doses of antibodiesto chicken IFN�γ improved the state of chickens infected with avian influenza.However, activated antibodies to human IFN�γ were ineffective under theseconditions. High specificity of ultralow doses was also observed in studying thephenomenon of bipathy. This specificity indicates that the activity of ultralow dosesis strongly determined. It remains unclear why potentiated species�specificpolyclonal antibodies (e.g., rabbit antibodies) modify the corresponding naturalantibodies of a certain patient under clinical conditions. It will be recalled that anti�bodies of any organism are individualized by the V domain due to recombinationsin the genome of lymphocytes. Hence, potentiated antibodies have a primaryeffect on non�species�specific regions of the C domain in natural antibodies.

The specific activity of antibodies in ultralow doses is associatedwith a certain direction of effect, which depends on the initial functional state.For example, ultralow doses of antibodies to erythropoietin and GCSF increaseerythropoietic and granulocyte�macrophage activity, respectively, underconditions of cytostatic myelosuppression. By contrast, these antibodies have aninhibitory effect on test parameters during immobilization stress (A. M. Dygaiet al., 2003, 2004). After administration of anti�S in ultralow doses thefrequency of action potential generation decreases in neurons with high basallevel of spontaneous activity, but increases in neurons with spontaneous activityof low frequency (V. V. Andrianov et al., 2003). The effect of antibodies inultralow doses depends on the initial state of target organs, which illustrates theadaptive nature of antibody�induced changes.

The remedies with ultralow doses of antibodies are manufactured at the“Materia Medica Holding” Research�and�Production Company. They differ inthe implementation phase. Some products, including Impaza, Proproten�100,

* See Chapters 7 and 8.

107

Anaferon, and Anaferon for children, are extensively used in medical practice.Tenoten, Afala, and Artrofoon have recently appeared in pharmacies. Kardosand Epigam will soon be ready for manufacture. Experimental and clinical trialswith ultralow doses of antibodies will be described in Chapters 7 and 8. Wewould like to show the prospects of treatment with these drugs.

Tenoten and Proprotein�100 include ultralow doses of antibodies to brain�specific protein S�100. The dose of anti�S100 in Proprotein is C1000. Tenotenis a mixture of activated dilutions C12+30+200. Previous experiments showedthat antianxiety activity of the mixture is higher than that of dilution C1000.Tenoten was approved for use as an anxiolytic. Tenoten is a typical daytimeanxiolytic, since it combines the incompatible properties (antianxiety andactivating effects). As differentiated from benzodiazepine drugs and plantpreparations, ultralow doses of antibodies to S�100 have no sedative andmyorelaxant properties and do not cause drug dependence. The patientsreported that Tenoten has a “gentle”, mild, and progressive effect (“sudden andimperceptible improvement of the state”). The drug provides a feeling of light�ness and naturalness. After treatment with Tenoten the capacity for work returnsto normal (but not increases). Tenoten may be prescribed in psychologicaltension (driving, examination, managing, and operating activity) and stressconditions. Tenoten�induced “lightness” was not perceived as euphoria oroverexcitation. Clinical trials revealed that Tenoten does not cause drug addic�tion. Tenoten may be extensively used as a drug of the “normal state”, whichprevents internal stress and anxiety. Tenoten has a normalizing effect on moodand general activity. It should be emphasized that Tenoten is effective not onlyin patients with borderline mental disorders, but also in practically healthypeople with a variety of situational neurotic states. Tenoten has an antiastheniceffect and improves cognitive function, which is particularly important forpractically healthy subjects.

In the future, we plan to introduce Tenoten into psychiatry. It will be usedmainly for the therapy of patients with depressive disorders. A unique effect ofTenoten on synaptic plasticity should be taken into account in combinedtreatment with antidepressant and antipsychotic drugs. Combined administrationof Tenoten and psychotropic drugs will allow us to reduce the volume ofpharmacotherapy and to prevent side effects of various pharmaceutical products,including neuroleptics (J. L. Dugina et al., 2006).

Previous experiments revealed that Tenoten has a neurotrophic and protec�tive effect on the model of ischemic and hemorrhagic stroke. Hence, Tenoten holdsmuch promise for combination therapy of these diseases. In several trials, Tenotenwas effective for Parkinsonism, Alzheimer’s disease, and disseminated sclerosis.

Tenoten for children contains a mixture of dilutions C12+C30+C50. Thisproduct is studied at the “Materia Medica Holding” Research�and�Production


108

Ultralow doses

* In collaboration with E. N. Krylov, we studied the possibility of treatment with anti�bodies to morphine in ultralow doses (activated neuropsychotropic drugs) as a non�nar�cotic replacement therapy in additions (E. E. Krylov, 2003a; N. N. Ivanets et al., 2002).

Company. Tenoten for children holds promise for the therapy of children withnervousness and memory deficit.

The composition of Proproten�100 is similar to that of Tenoten.Proproten�100 was manufactured from 1999. Proproten�100 is widely used asan antialcohol drug in Russia. The effect of Proproten is associated with theability of activated anti�S100 to “saturate” a system of positive emotionalreinforcement, which results in the decrease in alcohol abuse. Proproten doesnot cause euphoria and addiction. It can be said that Proproten has apseudoeuphoric effect*. The patients reported that Proproten produces a “mild”and “specific”, but not intoxicant effect. T. M. Vorob’eva studied the effect ofProproten on the model of lateral hypothalamic self�stimulation. These ex�periments showed that Proproten induces the so�called “emotional equili�brium”, which is difficult to describe in words. In discussing the emotiotropiceffect of Proproten, it is more appropriate to say about “normalizing activity”.

Proproten decreases alcohol addiction and has the antidepressant,antianxiety, and neurotrophic�and�protective effects. Therefore, Proproten maybe used to for the prevention of alcoholism and recurrences of this disease. Theeffectiveness of drug treatment for many years (e.g., in combination withpsychotherapy) will be evaluated in future studies.

Activated antibodies to IFN�γ, including Anaferon for children (mixtureof dilutions C12+30+50) and Anaferon (C12+C30+C200), were manufacturedby the “Materia Medica Holding” Research�and�Production Company for 5years. Ultralow doses of antibodies to IFN�γ were shown to be effective in viralinfections, including influenza, chickenpox, infectious mononucleosis, genitalherpes, ophthalmic herpes, adenovirus infection, respiratory syncytial virus in�fection, rotavirus infection, coronavirus infection, calicivirus infection, tick�boneencephalitis, etc. Due to high effectiveness and safety, activated antibodies toIFN�γ are extensively for the therapy and prevention of influenza and ARVI inRussia.

Taking into account a wide range of immunomodulatory properties ofAnaferon, we believe that the future lies with administration of this preparationin combination with other antiviral and/or antiinflammatory drugs, vaccines,and sera. For example, clinical trials showed that the effectiveness of acyclovirsignificantly increases after combined administration of this drug and Anaferonin patients with herpes infection (A. E. Shul’zhenko et al., 2005). After over�coming the distrust of ultralow doses, Anaferon may be used in combinationtherapy for infectious diseases that are resistant to any treatment (e.g., AIDS,

109

tuberculosis, and avian influenza). Promising results were obtained in study ofavian influenza. Activated antibodies to chicken IFN�γ improve the survival rateof chickens with avian influenza. We assume that Anaferon may be used in thetherapy of noninfectious diseases, whose pathogenesis involves interferon(disseminated sclerosis, some mental disorders, etc.). The main indication forAnaferon therapy will be preventive treatment (particularly, in children ofdecreed groups).

Besides Anaferon, the preparation from ultralow doses of antibodies tocytokines (Artrofoon) was developed at the “Materia Medica Holding” Re�search�and�Production Company. Artrofoon consists of activated antibodies toTNF�α. This drug has a strong antiinflammatory effect. At the present time,Artrofoon is mainly used in rheumatology. It should be emphasized thatTNF�α has a wide range of physiological activity. Probably, ultralow doses ofantibodies to this cytokine may be used as an antiinflammatory agent inpulmonology, surgery, and gastroenterology. Strong evidence exists thatArtrofoon is effective in Crohn’s disease. Our experiments showed thatultralow doses of antibodies to TNF�α have antitumor activity, which isparticularly important for oncology.

Impaza has been extensively used in recent years. This product ofantibodies to NO synthase in ultralow doses is used in the therapy for erectiledysfunction. Impaza is a drug of choice for elderly patients. Impaza may begiven to nitrate�receiving patients with coronary heart disease. Impaza has amild hypotensive effect. This drug may be used for a long time to prevent thedevelopment of erectile disorders. Impaza modulates the production of NO andhas a multifactor regulatory effect on intracellular processes. Therefore, Impazaholds much promise for the therapy of endothelial dysfunction.

The other two products, Afala (activated antibodies to prostate�specificantigen) and Kardos* (ultralow doses of antibodies to the type 1 angiotensin IIreceptor), were introduced into clinical practice in recent years. Afala is usedin the therapy for adenoma and inflammation of the prostate gland. Kardos isused in the therapy for chronic cardiovascular failure. Both drugs have hightherapeutic effectiveness. In our opinion they hold promise for the preventivetherapy. The effectiveness of drug treatment for many years will be evaluated infurther studies.

The products from ultralow doses of antibodies to histamine, Epigam andProhistam, were also developed at the “Materia Medica Holding” Research�and�Production Company. They may be used in the therapy for gastric ulcer andallergic diseases, respectively. These drugs hold much promise for secondaryprevention of diseases.


* Beginning from 2008, the drug will be designated “Kardosten”.

110

Ultralow doses


There are the following three directions to the use of potentiated prod�ucts: homeopathy, bipathy, and pharmacology of antibodies in ultralow doses.

The methodology of individual prescription of ultralow doses makes itdifficult to use the experience of homeopathy by untrained physicians. The bi�pathic method and treatment with potentiated antibodies do not contradict theparadigm of modern medicine and may be widely used in the therapy for vari�ous diseases.

The major advantages of products from ultralow doses of antibodies area combination of effectiveness and safety, mild (adaptive) effect, cumulativeaction in long�term therapy, and absence of drug tolerance.

111

7.1. Experimental study of antibodiesto S�100 protein in ultralow doses

An experimental study showed that ultralow doses (ULD) of antibodies toS�100 protein (ULD anti�S100; Proproten�100, Tenoten, and Tenoten for

children) have pharmacological activity. They exhibited the anxiolytic, antiasthenic(activating), antidepressant, GABA�mimetic, antiaggressive, stress�protective,antihypoxic, antiischemic, neuroprotective, and nootropic properties (Fig. 7.1).

ULD anti�S100 did not cause side effects (sedative and myorelaxanteffects) that are typical of benzodiazepine anxiolytics. The action of ULD anti�S100 was shown to be realized via the GABAergic and serotoninergic systems.ULD anti�S100. ULD anti�S100 had a modulatory effect on neuronal plasticityand c�fos gene expression.

We studied the consequences of combined treatment with ULD anti�S100and haloperidol. ULD anti�S100 did not modulate the specific psychotropicactivity, but abolished the cataleptic effect of a neuroleptic drug. The anxiolytic,antidepressant, and nootropic properties of ULD anti�S100 were also revealedin experiments on young rats. ULD anti�S100 were effective in the therapy ofmemory deficit and hyperactivity. ULD anti�S100 had a good safety profile ina toxicology study.

C h a p t e r 7

Experimental pharmacologyof products from ultralowdoses of antibodies

112

Ultralow doses

Anxiolytic activity of ULD anti�S100

Anxiolytic activity of ULD anti�S100 was studied in the Vogel conflict test(T. A. Voronina et al., 2000; G. M. Molodavkin et al., 1995; J. R. Vogel et al.,1971; D. Triet et al., 1985; D. J. Sanger et al., 1991), elevated plus�maze (T. A.Voronina et al., 2000; S. Pellow et al., 1986; G. R. Dawson et al., 1995; S. E.File, 1995), and open�field test (T. A. Voronina et al., 2000). The benzodia�zepine anxiolytic diazepam was used as a reference drug.

Anxiolytic activity of ULD anti�S100 in the Vogel test of “conflict situa�tion”. A conflict situation (G. M. Molodavkin et al., 1995; T. A. Voronina et al.,2000; J. R. Vogel et al., 1971; D. Triet et al., 1985; D. J. Sanger et al., 1991)resulted from the opposition of drinking and defense motivations. Each episodeof drinking was punished by pain electrostimulation. The number of punisheddrinking episodes was recorded for 10 min. The anxiolytic effect of study drugwas estimated from a significant increase in the number of punished drinkingepisodes.

Fig. 7.1.Fig. 7.1.Fig. 7.1.Fig. 7.1.Fig. 7.1. Pharmacological properties of ULD anti�S100.

NEUROPROTECTIVE

ANXIOLYTIC

ANTIAGGRESSIVE

STRESS�PROTECTIVE

ANTIHYPOXIC

ANTIISCHEMIC

ACTIVATING AND ANTIASTHENIC

ANTIDEPRESSANT

NOOTROPIC

Diazepam

Diazepam

Mexidol

Cavinton and

piracetam

Amitriptyline

Piracetam

Nimodipine

ULD anti�S100

113

Experiments were performed on adult outbred albino rats weighing 230�250 g. The animals of groups 1 (control), 2, and 3 received intragastrically 2.5ml/kg distilled water, 2.5 ml/kg ULD anti�S100, and equivalent volume of2 mg/kg diazepam, respectively.

During single and repeated administration (5�day course), the anxiolyticeffect of ULD anti�S100 compared well with that of diazepam (Fig. 7.2; T. A.Voronina et al., 2003a; T. A. Voronina et al., 2006).

ULD anti�S100 had a greater anxiolytic effect on low�activity animalsthat were predisposed to anxiety and depressive behavior (S. B. Seredenin et al.,1994). In high�activity animals, the anxiolytic activity of ULD anti�S100compared well with that of diazepam (Fig. 7.3). These data indicate that ULDanti�S100 not only produces the anxiolytic effect, but also exhibits theantiasthenic or activating properties (as differentiated from diazepam withanxiolytic and sedative activity).

Anxiolytic activity of ULD anti�S100 in the elevated plus�maze test. Theelevated plus�maze (EPM) consisted of four arms (length 0.5 m, width 10 cm)

Chapter 7. Chapter 7. Chapter 7. Chapter 7. Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies

Fig. 7.2.Fig. 7.2.Fig. 7.2.Fig. 7.2.Fig. 7.2. Anxiolytic effect of single administration (a) and 5�day course of treatmentwith ULD anti�S100 (b) in the Vogel conflict test. Control (1), ULD anti�S100 (2),and diazepam (3). *p<0.05 compared to the control.

0

50

100

% of the control

150

200

1 2 3

! !

0

50

100

150

200

250

!

!

% of the control

1 2 3

a b

Fig. 7.3.Fig. 7.3.Fig. 7.3.Fig. 7.3.Fig. 7.3. Effect of ULD anti�S100 on high�activity (a) and low�activity rats (b) inthe Vogel conflict test. *p<0.05 compared to diazepam�receiving animals.

0

100

200

300

400

ULD anti�S100

Diazepam

% of the control

ULD anti�S100

Diazepam

!

a b

114

Ultralow doses

Fig. 7.4.Fig. 7.4.Fig. 7.4.Fig. 7.4.Fig. 7.4. Anxiolytic effect of ULD anti�S100 and diazepam in the elevated plus�maze. Number of entries into the open arms (a), time spent in the open arms (b),and number of overhangs (c). *p<0.05 compared to the control.

0

200

400

600

800

1000

1200% of the control

control

ULD anti�S100

diazepam

!

!!

!

!

!

a b c

that were perpendicular to each other. Two opposite arms were surrounded bya 40�cm wall from three sides. The other two arms were open. This maze wasplaced at a distance of 0.5 m from the floor level. The area of EPM was il�luminated by two luminescent lamps (20 W, 60 cm from the level of arms). Thismethod is based on the fear of open space or fall from a height (T. A. Voronina,et al., 2000; S. Pellow et al., 1986; G. R. Dawson et al., 1995; S. E. File, 1995).

Experimental animals were placed in the central area of EPM. The fol�lowing behavioral parameters were recorded for 3 min: latency (LC) of the firstentry into the open arms, number of complete and incomplete entries, and timespent in the open arms. Emotionality of rats was estimated from the urinationrate and number of fecal boluses in EPM.

Experiments were performed on adult outbred albino rats weighing 230�250 g. The animals of groups 1 (control), 2, and 3 were treated with 2.5 ml/kgdistilled water, 2.5 ml/kg ULD anti�S100, and 2 mg/kg diazepam (2.5 ml/kg,reference drug), respectively. Test substances were administered intragastrically30 min before the study.

ULD anti�S100 had a strong anxiolytic effect. This conclusion was deri�ved from significant changes in the number of entries into the open arms, timespent in the open arms, number of overhangs, and rates of defecation andurination (Fig. 7.4; T. A. Voronina et al., 2003a; T. A. Voronina, 2006). Theeffect of ULD anti�S100 was similar to that of diazepam. Both drugs increasedthe number of entries into the open arms (by 2.1 and 2.4 times, respectively,p<0.05), time spent in the open arms (by 5.6 and 7 times, respectively, p<0.05),and number of overhangs (by 5 and 9 times, respectively, p<0.05). Nodifferences were found in the effects of ULD anti�S100 and diazepam (T. A.Voronina et al., 2003a; T. A. Voronina, 2006).

Anxiolytic activity of ULD anti�S100 in the open�field test. The open�fieldtest is used for a study of rat behavior (locomotor activity and emotionality;

115

T. A. Voronina et al., 2000). The box had an area of 1×1 m and was surroundedby walls (0.5 m in height). The floor was divided into squares (10×10 cm). Theholes (4 cm in diameter) were made at each corner of these squares. The openfield was illuminated by two fluorescent lamps (20 W, 0.5 m above the floorlevel).

The animals were placed in the corner of this box. Horizontal activity wasstudied for 3 min. The following parameters were recorded: number of crossedsquares, vertical activity (number of rearing postures), number of entries into thecenter of the open field, exploratory behavior (exploration of holes in the floor),and frequency of grooming or scratching episodes. This study was performed toevaluate the anxiolytic and sedative effects of drugs (Bruhwyler et al., 1995).

Experiments were performed on adult outbred albino rats weighing 230�250 g. The animals of groups 1 (control), 2, and 3 were treated with 2.5 ml/kgdistilled water and equivalent volumes of ULD anti�S100 and 2 mg/kg diazepam(reference drug), respectively. Test substances were administered intragastrically30 min before the study.

ULD anti�S100 and diazepam had a strong antianxiety effect, which wasmanifested in a significant increase in the number of entries into the center ofthe open field (by 2.5 and 1.8 times, respectively; Fig. 7.5). As differentiatedfrom diazepam, ULD anti�S100 had no sedative activity. Horizontal activity ofanimals did not decrease after treatment with ULD anti�S100 (Fig. 7.6; T. A.Voronina et al., 2003a; T. A. Voronina et al., 2006).

Antidepressant activity of ULD anti�S100

Antidepressant activity of ULD anti�S100 was studied in the Porsolt’sforced swimming test (R. D. Porsolt et al., 1978), as well as in a water tank with


Fig. 7.5.Fig. 7.5.Fig. 7.5.Fig. 7.5.Fig. 7.5. Anxiolytic effect of ULD anti�S100 and diazepam in the open�field test.*p<0.05 compared to the control.

0

0.5

1.0

1.5

0

2.0

2.5

3.0

3.5Number of entries into the center of the open field

Control ULD anti�S100 Diazepam

!

!

116

Ultralow doses

wheels (G. M. Molodavkin et al., 1994; S. Nomura et al., 1982). A standardtricyclic antidepressant amitriptyline was used as the reference drug.

Antidepressant activity of ULD anti�S100 in the Porsolt’s forced swimmingtest. This method is based on recording of the immobility time in rats (R. D.Porsolt et al., 1978). The animals were placed in a water tank (diameter 40 cm,depth 60 cm). Under these conditions, locomotor activity of rats was directedto the avoidance of an aversive (unpleasant) situation. However, these animalsbecame “suspended” in water during the follow�up period. They remainedimmobile or made slight movements to maintain the head above water. Theduration of immobility served as a criterion of depressiveness.

Experiments were performed on male outbred rats weighing 250�300 g.The animals received 2.5 ml/kg distilled water (control group), 2.5 ml/kg ULDanti�S100 (treatment group 1), and equivalent volume of 10 mg/kg amitriptyline(reference drug, treatment group 2). Test substances were administeredintragastrically 30 min before the study.

Single and repeated treatment (5�day course) with ULD anti�S100 andamitriptyline had an antidepressant effect, which was manifested in a significantdecrease in the immobility time. The activity of ULD anti�S100 compared wellwith that of a tricyclic antidepressant (Fig. 7.7; O. I. Epstein et al., 2003c; T. A.Voronina et al. 2006).

Antidepressant activity of ULD anti�S100 in the Nomura’s forcedswimming test in a water tank with wheels. This method is extensively used tostudy the effect of antidepressant drugs (G. M. Molodavkin et al., 1994; S.Nomura et al., 1982). The rats were placed in a water tank. The reservoir wasdivided into four compartments with wheels. The animal’s snout was oppositeto a wheel in each compartment. The number of wheel revolutions was recordedfor 10 min.

Experiments were performed on male outbred rats weighing 250�300 g.The animals received 2.5 ml/kg distilled water (control group), 2.5 ml/kg ULDanti�S100 (treatment group 1), and 10 mg/kg amitriptyline in an equivalent

Fig. 7.6.Fig. 7.6.Fig. 7.6.Fig. 7.6.Fig. 7.6. Effect of ULD anti�S100 and diazepam on horizontal activity of rats inthe open�field test. *p<0.05 compared to the control.

0

5

10

15

20

25


!

Number of entries into the center of the open field

117

volume of the solvent (treatment group 2). Test substances were administeredintragastrically 30 min before the study.

ULD anti�S100 and amitriptyline significantly reduced the signs of adepressive state. The number of wheel revolutions increased by 1.8 times aftertreatment with both drugs (Fig. 7.8; O. I. Epstein et al., 2003c; T. A. Voroninaet al., 2006).

Studying the possible side effects of ULD anti�S100

Previous experiments showed that the anxiolytic activity of ULD anti�S100 is similar to that of a benzodiazepine anxiolytic diazepam. However, therange of pharmacological properties of benzodiazepine drugs includes thesedative and myorelaxant effects. Further investigations were conducted to studythese effects.

Fig. 7.7.Fig. 7.7.Fig. 7.7.Fig. 7.7.Fig. 7.7. Antidepressant activity of ULD anti�S100 and amitriptyline in the Porsolt’sforced swimming test. Single administration (a) and 5�day course of treatment (b).Ordinate: immobility time. *p<0.05 compared to the control.

0

20

40

60

80

100

120

140

% of the control

Control ULD anti�S100 Amitriptyline

!

!

0

20

40

60

80

100

120

140

% of the control


!

!

a b


Fig. 7.8.Fig. 7.8.Fig. 7.8.Fig. 7.8.Fig. 7.8. Antidepressant activity of ULD anti�S100 and amitriptyline (singletreatment) in the Nomura’s forced swimming test in a water tank with wheels.*p<0.05 compared to the control.

0

50

100

150

200

250% of the control


!!

118

Ultralow doses

Fig. 7.9.Fig. 7.9.Fig. 7.9.Fig. 7.9.Fig. 7.9. Effect of ULD anti�S100 and diazepam on muscle tone of rats in therotarod test. Ordinate: percentage of animals (in group). Remaining animals (a) andfalling animals (b). *p<0.05 compared to the control.

0

20

40

60

80

100

120%

Control ULD anti�S100

Diazepam, 2 mg/kg

Diazepam, 4 mg/kg

!

!

0

40

20

60

80%


Diazepam, 2 mg/kg

Diazepam, 4 mg/kg

!

!

a b

The possible myorelaxant and sedative effects of ULD anti�S100 wereevaluated in the rotarod test (T. A. Voronina et al., 2000; N. W. Dunham et al.,1957) and open�field test (T. A. Voronina et al., 2000).

Studying the possible myorelaxant effect of ULD anti�S100 in the rotarodtest. The rats were placed on a rotating rod (diameter 4 cm, rotation speed 10rpm). The number of falling animals and latency�to�fall from the rod wererecorded for 2 min ((T. A. Voronina et al., 2006; N. W. Dunham et al., 1957).

Experiments were performed on male outbred rats weighing 250�300 g.The animals were divided into groups. The rats received 2.5 ml/kg distilled water(control group), 2.5 ml/kg ULD anti�S100, or equivalent volume of diazepamin doses of 1, 2, and 4 mg/kg. Test substances were administered intragastrically30 min before the study.

ULD anti�S100 had no myorelaxant activity (Fig. 7.9; T. A. Voronina etal., 2003). Diazepam produced a dose�dependent myorelaxant effect. The per�centage of animals falling from a rotating rod after administration of diazepamin doses of 2 and 4 mg/kg was 40 and 70%, respectively.

Studying the possible sedative effect of ULD anti�S100 in the open�fieldtest. The sedative or stimulating effect of ULD anti�S100 was evaluated in theopen�field test (increase or decrease in locomotor activity and exploratory be�havior; T. A. Voronina et al., 2000a).

Experiments were performed on adult outbred albino rats weighing 250�300 g. They were divided into the control group and two treatment groups. Theanimals received 2.5 ml/kg distilled water, 2.5 ml/kg ULD anti�S100, or 2 mg/kgdiazepam in an equivalent volume of the solvent (reference drug). Test sub�stances were administered intragastrically 30 min before the study.

ULD anti�S100 had little effect on horizontal and vertical activity andexploratory behavior of animals (exploration of holes). Administration of

119

diazepam in a dose of 2 mg/kg was followed by a significant decrease in horizontalactivity of rats, which reflects the sedative effect of this drug (Table 7.1).

These data indicate that ULD anti�S100 do not cause a sedative effect (asdifferentiated from diazepam; T. A. Voronina et al., 2003a).

Mechanisms for the effect of ULD anti�S100

Role of the GABAergic system in the anxiolytic effect of ULD anti�S100.Anxiolytic activity was studied in the Vogel conflict test (T. A. Voronina et al.,2000; J. R. Vogel et al., 1971). Experiments were performed on male outbredrats weighing 250�300 g. The animals received intragastrically distilled water (2.5ml/kg), ULD anti�S100 (2.5 ml/kg), or reference drug diazepam (2 mg/kg, 2.5ml/kg).

GABA�A receptor blockade was induced by intraperitoneal injection of areceptor antagonist bicuculline in a dose of 1 mg/kg (ICN Biomedicals Inc.; R.W. Olsen et al., 1984). Picrotoxin in a dose of 1 mg/kg (ICN Biomedicals Inc.;M. G. Corda et al., 1984) was injected intraperitoneally to cause Cl� channelblockade in the GABA�benzodiazepine receptor complex.

The anticonflict effects of ULD anti�S100 and diazepam weresignificantly reduced under conditions of bicuculline�induced GABA�A receptorblockade and picrotoxin�induced Cl� channel blockade. Hence, these subunitsof the GABA�benzodiazepine receptor�chloride ionophore complex are involvedin the anxiolytic effect of test substances (Fig. 7.10; T. A. Voronina et al.,2003b).

Role of the GABA(B)�ergic system in the anxiolytic and antidepressanteffects of ULD anti�S100. The role of the GABA(B)�ergic system in theanxiolytic and antidepressant effects of ULD anti�S100 was studied in the Vogelconflict test and forced swimming test (water tank with freely rotating wheels),respectively.


Table 7.1.Table 7.1.Table 7.1.Table 7.1.Table 7.1. Studying the sedative effect of ULD anti�S100 in the open�field test (M±m)

Horizontal activity 18.2±2.4 15.8±2.1 12.5±1.8*

Vertical activity 8.2±3.3 5.8±2.6 6.2±1.4

Exploratory activity 11.2±3.1 8.9±1.6 8.7±1.5

Number of entries into the centerof the open field 0 2.4±0.7* 1.8±0.9*

Note.Note.Note.Note.Note. *p<0.05 compared to the control.

ParameterDiazepam(2 mg/kg)

ULD anti�S100(2.5 ml/kg)Control

120

Ultralow doses

This study was performed with the following substances (equivalentvolume of distilled water as the control):

1) ULD anti�S100 (2.5 ml/kg intragastrically, 40 min before the experi�ment);

2) diazepam (Polfa; 2 mg/kg intragastrically, 40 min before the ex�periment);

3) amitriptyline (Spofa; 10 mg/kg intragastrically, 40 min before the ex�periment);

4) selective GABA�B receptor agonist baclofen (ICN; 1 mg/kg intraperi�toneally; L. Gilbo et al., 2000), 30 min before the experiment and 10min before administration of ULD anti�S100, diazepam, or amitrip�tyline; and

5) GABA�B receptor antagonist faclofen (ICN; 10 mg/kg intraperito�neally; L. Gilbo, 2000), 30 min before the experiment and 10 min be�fore administration of ULD anti�S100, diazepam, or amitriptyline.

The number of punished drinking episodes in rats of the ULD anti�S100group (507.33±120.59) was comparable to that in diazepam�receiving animals(Table 7.2).

A GABA�B receptor agonist baclofen (1 mg/kg) diminished the anxiolyticeffect of ULD anti�S100. The number of punished drinking episodes decreasedto 231.67±79.66 (Table 7.2). By contrast, a GABA�B receptor antagonistfaclofen (10 mg/kg) potentiated the anticonflict effect of ULD anti�S100. It wasmanifested in an increase in the number of punished drinking episodes.

Fig. 7.10.Fig. 7.10.Fig. 7.10.Fig. 7.10.Fig. 7.10. Effect of GABA�benzodiazepine receptor antagonists on anxiolytic activityof ULD anti�S100 and diazepam. Ordinate: number of punished drinking episodes.Control (1); ULD anti�S100 (2); diazepam (2 mg/kg, 3); bicuculline (1 mg/kg, 4);ULD anti�S100 + bicuculline (5); diazepam + bicuculline (6); picrotoxin (1 mg/kg,7); ULD anti�S100 + picrotoxin (8); and diazepam + picrotoxin (9). p<0.05: *com�pared to the control; +compared to the “diazepam + bicuculline” and “diazepam +picrotoxin” groups; xcompared to the “ULD anti�S100 + bicuculline” and “ULD anti�S100 + picrotoxin” groups.

01 2 3 4 5 6 7 8 9

50

100

150

% of thecontrol

!!

+x

121

However, baclofen did not modulate the anxiolytic effect of diazepam (Table7.2; T. A. Voronina et al., 2006b; T. A. Voronina et al., 2003).

Rats of the control group tried to escape from a water tank with wheels.The average number of wheel revolutions was 61.70±28.66. These attemptsfailed due to free rotation of wheels. Sometimes the activity was resumed, butremained unsuccessful.

After administration of ULD anti�S100, the number of wheel revolutionswas 116.1±30.5. The effect of ULD anti�S100 was similar to that of a standardantidepressant amitriptyline. Amitriptyline in a dose of 15 mg/kg increased thenumber of wheel revolutions to 112.40±24.86 (Table 7.2).

GABA(B)�ergic substances baclofen and faclofen were equally potent indiminishing the effect of ULD anti�S100 (decrease in the number of wheelrevolutions).

Our results indicate that administration of ULD anti�S100 in combinationwith GABA(B)�ergic substances is followed by the interaction between thesecompounds. A GABA�B receptor agonist baclofen diminishes, while faclofenpotentiates the anticonflict effect of ULD anti�S100.

Role of the serotoninergic system in the anxiolytic and antidepressanteffects of ULD anti�S100. The serotoninergic system is involved in the genesisof anxiety and depression. Serotonin uptake inhibitors are extensively used forthe therapy of depression. Much attention is paid to the search for new methodsto modulate the serotoninergic system, including serotonin receptors (D. N.Middlemiss et al., 2002). 5�HT1A receptor agonists (buspirone, gepirone,ipsapirone, flesinoxan, etc.) and 5�HT3 receptor antagonists (ondansetron,tropisetron, bemesetron, granisetron, etc.) have the anxiolytic effect.

Control (distilled water) 288.6±39.59 61.70±28.66

ULD anti�S100 (2.5 ml/kg) 507.33±52.59* 116.1±23.5*

ULD anti�S100 (2.5 ml/kg) and baclofen (1 ml/kg) 231.67±39.66+ 78.20±12.51+

ULD anti�S100 (2.5 ml/kg) and baclofen(10 ml/kg) 705.00±81.47*+ 66.60±21.64+

Diazepam (2 mg/kg) 623.33±115.65* Not measured

Diazepam (2 mg/kg) and baclofen (1 ml/kg) 567.00±76.67* Not measured

Amitriptyline (10 mg/kg) Not measured 112.40±24.86*

Amitriptyline (10 mg/kg) and baclofen (1 ml/kg) Not measured 122.10±18.88*

Note.Note.Note.Note.Note. p<0.05: *compared to the control; +compared to the ULD anti�S100 group.

Table 7.2.Table 7.2.Table 7.2.Table 7.2.Table 7.2. Incidence of punished drinking episodes (conflict situation) and number ofwheel revolutions (forced swimming test) in rats after administration of ULDanti�S100 and GABA(B)�ergic substances (M±m)

GroupNumber of wheel

revolutionsNumber of punished

drinking episodes


122

Ultralow doses

Under experimental conditions, ULD anti�S100 exhibit the anxiolytic andantidepressant activity. Hence, a 5�HT2/5�HT1C receptor antagonist ketanserinwas selected for a pharmacological study of the serotoninergic system. Thesereceptors have a role in the development of anxiety and depression. Ketanserinis not approved as a pharmaceutical product in the Russian Federation. Thissubstance is used as an analyzer. Depression is accompanied by serotonindeficiency. Therefore, a serotonin precursor 5�hydroxytryptophan (5�HTP) wasalso used to analyze the effect of ULD anti�S100.

These experiments were designed to evaluate a possible role of theserotoninergic system in the anxiolytic and antidepressant effects of ULD anti�S100. The study was conducted with a selective 5�HT2/5�HT1C receptorantagonist ketanserin and serotonin precursor 5�HTP.

Experiments were performed on male outbred albino rats weighing 200�250 g. Anxiolytic activity of ULD anti�S100 was studied in the Vogel conflicttest. The antidepressant effect of test substances was analyzed in the Nomura’sforced swimming test (water tank with wheels).

The following substances were analyzed:1) ULD anti�S100 (single intragastric dose 2.5 ml/kg, 30 min before the

experiment; distilled water as the control);2) diazepam (Polfa; single intragastric dose 2 mg/kg, 30 min before the

experiment);3) ketanserin (ICN; 1 mg/kg intraperitoneally, 40 min before the experi�

ment and 10 min before administration of ULD anti�S100); and4) 5�HTP (ICN; 50 mg/kg intraperitoneally, 30 min before the experi�

ment and 10 min before administration of ULD anti�S100).In a conflict situation, the number of punished drinking episodes for

control animals was 102.20±25.06. ULD anti�S100 had a strong anxiolyticeffect, which was manifested in an increase in the number of punished drinkingepisodes to 324.09±40.61. The effect of ULD anti�S100 was comparable to thatof diazepam in a dose of 2 mg/kg (Table 7.3).

Ketanserin exhibited anxiolytic activity and increased the number ofpunished drinking episodes to 224.5±31.7. A serotonin precursor 5�HTP alsoincreased the number of punished drinking episodes (statistically insignificant).The anticonflict effect of ULD anti�S100 became less pronounced aftertreatment in combination with ketanserin or 5�HTP (Table 7.3).

In the forced swimming test, control rats tried to escape with freelyrotating wheels. However, their attempts failed. Sometimes the activity wasresumed. The number of wheel revolutions for control animals was73.00±25.19.

After administration of ULD anti�S100, the number of wheel revolu�tions was 159.50±29.77. These data reflect the antidepressant effect of ULD

123

anti�S100. Antidepressant activity of ULD anti�S100 compared well with thatof a standard antidepressant amitriptyline (119.13±19.16 wheel revolutions,Table 7.3).

Individual administration of ketanserin or 5�HTP was followed by anincrease in the number of wheel revolutions, which reflects the antidepressanteffect of drugs. However, the antidepressant effect of study drugs was reducedafter administration in combination with ULD anti�S100. A decrease in theantidepressant effect was most significant after combined treatment withketanserin and ULD anti�S100 (Table 7.3).

Our results illustrate the interaction between ULD anti�S100 andserotoninergic substances. A selective 5�HT2/5�HT1C receptor antagonistketanserin had the anticonflict and antidepressant properties. The anxiolytic andantidepressant effects became less pronounced after combined administration ofULD anti�S100 and ketanserin.

A serotonin precursor 5�HTP induces a significant increase in brainserotonin content. 5�HTP had a strong antidepressant effect and mild anxiolyticeffect. Combined administration of ULD anti�S100 and 5�HTP was alsoaccompanied by a decrease in the anxiolytic and antidepressant properties ofULD anti�S100 (statistically insignificant).

We conclude that the anxiolytic and antidepressant effects of ULD anti�S100 are modified after administration of this product in combination withketanserin and 5�HTP. It may be suggested that the serotoninergic system hasa role in the realization of these effects (T. A. Voronina et al., 2006a; J. L. Du�gina et al., 2005c).

Table 7.3.Table 7.3.Table 7.3.Table 7.3.Table 7.3. Results of the conflict situation test and forced swimming test in rats afteradministration of ULD anti�S100 and serotoninergic substances (M±m)

Control 102.20±15.06 73.00±25.19

ULD anti�S100 (2.5 ml/kg) 324.90±40.61* 159.50±29.77*

Diazepam (2 mg/kg) 372.60±45.18* Not measured

5�HTP (50 mg/kg) 135.00±33.15 146.60±30.16*

Ketanserin (1 mg/kg) 224.5±31.7* 117.10±24.87*

ULD anti�S100 (2.5 ml/kg) and5�HTP (50 mg/kg) 259.2±48.4* 102.90±44.36

ULD anti�S100 (2.5 ml/kg) and 168.70±40.12+ 80.50±18.14+

ketanserin (1 mg/kg)

Amitriptyline (15 mg/kg) Not measured 119.10± 19.16*

Note.Note.Note.Note.Note. p<0.05: *compared to the control; +compared to ULD anti�S100.

GroupNumber of wheel

revolutions (forcedswimming test)

Number of punisheddrinking episodes(conflict situation)


124

Ultralow doses

Effect of ULD anti�S100 on early gene c�fos expression in the hypotha�lamic paraventricular nucleus. An increased expression of the early gene c�fos inthe hypothalamic paraventricular nucleus serves as a criterion for the stressresponse of an organism (L. W. Swanson et al., 1980; T. R. Tolle et al., 1995;Z. Tan et al., 2002).

Experiments were performed on male Wistar rats weighing 250�280 g. Theanimals were divided into groups of high�activity and low�activity specimens inthe open�field test (E. V. Koplik et al., 1995; E. V. Koplik et al., 2001). The ratsreceived intragastrically distilled water (2.5 ml/kg), ULD anti�S100 (2.5 ml/kg),or imipramine (12 mg/kg). Test substances were administered once or severaltimes (20�day course). Each group consisted of active (n=5) and passive rats(n=5). Thirty minutes after acute treatment or last administration of substances,the animals were exposed to 1�h immobilization and electrocutaneous stimu�lation (4�6 V, frequency 50 Hz, pulse duration 1 msec, 16�18 stimulations per1 h). The rats were killed 90 min after stress. The brain was removed and frozenin liquid nitrogen. The slices were prepared and stained immunohistochemicallywith antibodies to c�Fos protein. The number of fos�positive cells in thehypothalamic paraventricular nucleus and intensity of c�fos gene expression wereestimated in five intact (nonstressed) active and passive rats.

Stress was followed by a significant increase in c�fos gene expression inactive and passive animals (by 20�25 times). These changes were particularlypronounced in passive specimens.

The course of treatment with imipramine was accompanied by a decreasein the number of immunoreactive cells in active and passive animals (by 1.2 and1.5 times, respectively). The observed changes were statistically significant inpassive specimens (Fig. 7.11).

Administration of ULD anti�S100 for 20 days was followed by a 1.3�folddecrease in the number of fos�positive cells in passive animals (p<0.0, Fig. 7.11).Imipramine and ULD anti�S100 were equally effective in this respect.

Effect of ULD anti�S100 on long�term posttetanic potentiation in survivinghippocampal slices. The model of long�term posttetanic potentiation (LTPTP)in surviving hippocampal slices is extensively used to study the molecularmechanisms of synaptic plasticity and effect of substances on synaptictransmission. The induction of LTPTP is a Ca2+�dependent process. Thisprocess involves not only Ca2+�regulatory proteins, but also Ca2+�bindingprotein S�100. The induction of LTPTP in hippocampal slices is accompaniedby an increase in the content of membrane�bound protein. Application ofantiserum to S�100 protein inhibits the induction of LTPTP in hippocampalslices (T. Lewis et al., 1986).

Experiments were performed on hippocampal slices from adult Wistar ratsweighing 180�200 g. Transverse sections (400 m in width) were placed in a

125

temperature�controlled chamber at 35�37oC. Flow Yamamoto medium wasaerated by carbogen (95% O2 and 5% CO2). Evoked potentials were recordedafter 40�60�min incubation. A stimulatory electrolytically sharpened bipolarwolfram electrode was introduced into the zone of mossy fibers. A referenceglass electrode (tip thickness 3�5 m, resistance 2�5 mO) was filled with 2.5 MNaCl and placed in CA3 region (initial segments of apical dendrites).

Testing was performed with single rectangular pulses (duration 200 msec)delivered at an interval of not less than 5�7 min.

The amplitude of test stimuli usually varies from 10 to 30 V. Evokedpotentials were recorded on a 12�digit analog�to�digital converter (Digidata,Axon Instruments Inc.). The results were analyzed with pClamp�6 (AxonInstru-ments Inc.) and Microcal Origin softwares.

To induce LTPTP, the amplitude of test stimulus was selected toproduce a half�maximal response. Tetanization was induced by three conse�cutive series of stimulation at 200 Hz. The length of each series was 1 sec. Sti�mulation was applied at 2�sec intervals. The procedure of tetanization was re�peated after 10 min.

The internal potentials were recorded for at least 40 min after the firsttetanization, which reflected the induction or absence of LTPTP. A significantincrease in the amplitude of EPSP (by 1.5�2 times), which persisted for 20 minafter the second tetanization, served as a criterion for the induction ofpotentiation.

After study of each dilution, the chamber was repeatedly washed withdistilled water and ethyl alcohol and completely dried with compressed air.

Experiments were performed with monospecific rabbit antiserum toneurospecific protein S�100 and ULD anti�S100. Nonimmune rabbit antiserumserved as the control.

Fig. 7.11.Fig. 7.11.Fig. 7.11.Fig. 7.11.Fig. 7.11. Effect of single administration (a) and 20�day course of treatment withULD anti�S100 and imipramine (b) on c�fos gene expression in rats afterimmobilization stress and electrocutaneous stimulation. Ordinate: number of Fos�positive cells. p<0.05: *compared to the control; +compared to imipramine�receivinganimals.

0

5

10

15

20

25

Intact Control ULD anti�S100Imipramine

!!

+

0

5

10

15

20

25

Intact Control ULD anti�S100Imipramine

active passive

a b


126

Ultralow doses

The induction of LTPTP was characterized by a significant increase in theamplitude of evoked potentials in response to the test stimulus after tetanization.Twenty�minute incubation with antibodies to S�100 protein (final dilution 1:50)completely inhibited the induction of LTPTP, which is consistent with publisheddata (T. Lewis et al., 1986). Nonimmune rabbit antiserum at the same dilutionhad no effect on LTPTP induction in rat hippocampal slices.

The average amplitude of EPSP after 20�min incubation in Yamamotomedium with antiserum to S�100 (concentration 10�12) at the interstimulusintervals of 10 and 5�7 min was 0.9�1.1 and 0.8�0.9 mV. Over 10 min after firsttetanization at 2�3�min intervals, this parameter was 1.1 mV.

The effect of anti�S100 was completely abolished after preincubation ofslices with ULD anti�S100 (concentration 10�12, 20 min) and subsequentincubation in a solution of native and potentiated antisera (20 min). Hence, theinduction of LTPTP in slices was similar to that in control samples (notreatment with antibodies). Over 40 min after the second tetanization at 3�5�min intervals, the average amplitude of EPSP was 1.7�2.0 mV.

These data show that preincubation with ULD anti�S100 modifies the in�hibitory effect of antiserum to S�100 protein. The inhibition of LTPTP is notobserved under these conditions. This phenomenon is probably related to a di�rect modulatory effect of ULD anti�S100 on the corresponding endogenous li�gand (O. I. Epstein et al., 1999a; M. B. Shtark, 2001).

Effect of ULD anti�S100 on electrical properties of neuronal membranes.Antibodies to S�100 protein cause reversible changes in the passive and activeproperties of neuronal membranes from snail subesophageal ganglia (Kh. L.Gainutdinov et al., 1999).

Moreover, ULD anti�S100 have a strong effect on the membrane of giantneurons from H. pomatia (O. I. Epstein et al., 1996b).

The snails were in an active state for at least 2 weeks before study.Experiments were performed on the identified spontaneously active neurons ofsubesophageal ganglia V2�V6, PPa1, and PPa2. The resting potential, actionpotential amplitude, time derivative of the action potential, maximum rate ofrise of the action potential (Vmax), spike discharge frequency, and current�voltage and inactivation characteristics of ion channels for inward and outwardcurrent were measured on a Hitachi device. In some experiments with isolatedneurons, calcium currents were recorded by the voltage clamp technique. Thestudy was performed with glass microelectrodes (internal resistance 7�20 mΩ).The neuronal membranes were polarized with depolarizing and hyperpolarizingpulses through a reference electrode (up to 30 V).

The dilution of antibodies to S�100 protein was 0.2, 2.6, and 12%. Theconcentration of ULD anti�S100 was 10�12 and 10�400 wt %. Nonimmune andimmune sera of sheep erythrocytes served as the control.

127

Under control conditions, the substitution of physiological saline fornonimmune serum or antiserum to sheep erythrocytes had little effect onelectrical properties of the membrane.

Vmax decreased sharply 20 min after application of antibodies to S�100protein at dilutions of 0.2 and 2% (by 22�28 and 37�45%, respectively). Vmaxdecreased by 60�80% over the first 10�15 min after treatment with antibodiesto S�100 protein at dilutions of 6 and 12%. Similar results were obtained inexperiments with ULD anti�S100. Vmax decreased by 14�8% over 35 min.

Treatment with various dilutions of antibodies to S�100 protein and poten�tiated form of these antibodies was followed by a decrease in the strength of inwardcurrent and increase in the steady�state inactivation at zero conditioning pulse.Current�voltage characteristics and inactivation curves were shifted toward negativevalues of the membrane potential. The antibody�induced reduction of inwardcurrent was mainly associated with a decrease in the maximum conductance ofinward current channels, but not with an increase in the steady�state inactivation.

These data show that ULD anti�S100 have a strong effect on membraneelectrical properties of the isolated neuron. The effect of ULD anti�S100 isqualitatively similar to that of native antiserum in concentrations of 2, 6, and12%. This is manifested in membrane depolarization, decrease in actionpotential amplitude, increase in the maximum rate of rise of the actionpotential, decrease in the maximum conductance, and inactivation of channels(O. I. Epstein et al., 1999b).

Other pharmacological effects of ULD anti�S100

Antiaggressive activity of ULD anti�S100. Antiaggressive activity of ULDanti�S100 was studied under conditions of motivated (Yu. V. Burov et al., 1976)and unmotivated aggression (T. A. Voronina et al., 2000).

Antiaggressive activity of ULD anti�S100 in the test of unmotivatedaggression. The test of unmotivated aggression is based on studying the thresholdof aggressive response for two rats on an electrode floor at increasing thestrength of stimulating current (T. A. Voronina et al., 2000). Pairs of rats wereplaced on the electrode floor of a Plexiglas chamber (27.5×27.5×40 cm).Alternating voltage of increasing amplitude (beginning from 15 V) was appliedto the chamber floor using a special stimulator. The duration of stimulation was3 sec. The interstimulus interval was 1 sec. If three stimulations of similarintensity did not induce the aggressive response, voltage was increased by 1 V.Stimulation was continued until the aggressive response to at least three pulsesof similar strength. This voltage was considered as a threshold. During theaggressive response, both rats stand up on their hindlimbs “face to face” andpaw or bite each other.


128

Ultralow doses

Fig. 7.12.Fig. 7.12.Fig. 7.12.Fig. 7.12.Fig. 7.12. Antiaggressive effect of ULD anti�S100 and diazepam in the test ofunmotivated aggression. Single administration (light bars) and course of treatment(dark bars). *p<0.05 compared to the control.

0

80

40

120

160


Threshold of aggression, % of the control

! !

!!

Experiments were performed on 120 male outbred albino rats weighing250�300 g. Distilled water (2.5 ml/kg) was given to control animals. Theremaining rats received 2.5 ml/kg ULD anti�S100 (treatment group 1) orequivalent volume of 5 mg/kg diazepam (treatment group 2). Test substanceswere administered intragastrically 40 min before the study.

Single administration and, particularly, repeated treatment with ULDanti�S100 and diazepam caused an increase in the aggressive threshold (Fig.7.12). The activity of ULD anti�S100 compared well with that of diazepam (S.A. Sergeeva et al., 2004).

Antiaggressive activity of ULD anti�S100 in the test of motivated aggression.A study of motivated aggression is based on the analysis of aggressive behaviorin two rats that try to avoid punishment on a crowded safe bench (Yu. V. Burovet al., 1976).

Experiments were performed on 120 male outbred rats weighing 250�300 g.Study was conducted in two stages. A conditioned response to avoid nociceptivestimulation of the limbs was elicited on day 1. A safe bench was put in thecenter of the chamber. The animals were placed in this chamber. Pulses ofalternating voltage (35 V, duration 3 sec, interpulse interval 1 sec) were appliedto the electrode floor after 5�min adaptation. After successful avoidance, the ratwas permitted to remain on the bench for 30 sec. Then the rat was placed inthe home cage. The same procedure was repeated after 2 min. The response wasconsidered to be elicited when avoidance LC did not exceed 9 sec (100% trials).

On the next day, pairs of rats were placed in the chamber. The behaviorof animals was studied for 2 min. Control animals fought for a safe bench,although it was sufficient for two specimens. Antiaggressive substances allowedthe rats to avoid nociceptive stimulation by getting to the bench. The efficacyof test substances was estimated from the time of avoidance for both rats.

129

The animals were divided into three groups. They received distilled water(2.5 ml/kg, control group 1), ULD anti�S100 (2.5 ml/kg, group 2), or diazepam(5 mg/kg in an equivalent volume, group 3). Test substances were administeredintragastrically 40 min before the study.

Single administration and course of treatment with ULD anti�S100 anddiazepam had a strong antiaggressive effect (Fig. 7.13). These drugs caused anincrease in the time of avoidance by 3.8 and 3.3 times, respectively (S. A.Sergeeva et al., 2004).

Antihypoxic activity of ULD anti�S100. Antihypoxic activity of ULD anti�S100 was studied under conditions of acute hypobaric hypoxia. Acute hypobarichypoxia in mice was produced in a flow�exhaust attitude chamber (M. V.Korablev et al., 1976). Pressure was measured with an altimeter. The rate ofclimb was measured with a variometer. The animals were “elevated” to a heightof 11,000 m (20 m/sec). The time of exposure was 10 min. The mice werereturned to a normal environment over the next 5 min. Alkali (30�35%) was putinto the chamber to prevent hypercapnia. The lifespan and survival rate ofanimals were calculated relative to the control (100%). The control and treatedmice were simultaneously placed in the chamber to study hypoxia under thesame conditions.

Experiments were performed on male outbred albino mice weighing 18�24 g. ULD anti�S100 were administered on days 1 (2.5 ml/kg twice a day at10.00 and 17.00) and 2 of study (2.5 ml/kg at 10.00; and 5 ml/kg at 15.00). Treat�ment was conducted 30 min before the study. An equivalent volume of mexidol(100 mg/kg) was given similarly. Control animals received physiological saline.

Repeated treatment with ULD anti�S100 was followed by an increase inthe lifespan of mice during hypobaric hypoxia (Fig. 7.14). Antihypoxic activityof ULD anti�S100 was highly competitive with that of mexidol.


Fig. 7.13.Fig. 7.13.Fig. 7.13.Fig. 7.13.Fig. 7.13. Antiaggressive effect of ULD anti�S100 and diazepam in the test ofmotivated aggression. Single administration (light bars) and course of treatment(dark bars). *p<0.05 compared to the control.

0

100

200

300

400

500


Time of collective avoidance, % of the control

!! !

!

130

Ultralow doses

Fig. 7.14.Fig. 7.14.Fig. 7.14.Fig. 7.14.Fig. 7.14. Effect on ULD anti�S100 on the lifespan of mice after acute hypobarichypoxia. *p<0.05 compared to the control.

0

50

100

150

200

250

300

Control Mexidol ULD anti�S100

% of the control

!

!

Neuroprotective activity of ULD anti�S100. Neuroprotective activity of ULDanti�S100 on the model of ischemic stroke. The antiischemic and antiamnesicproperties of ULD anti�S100 were studied on the model of irreversible focalischemia due to bilateral focal photothrombosis of the prefrontal cortex in rats(G. A. Romanova et al., 1998; B. D. Watson et al., 1985, 1998). Bilateral focalischemia of the prefrontal cortex in rats was induced by photochemicalthrombosis. This method was developed by B. D. Watson et al. and modified byI. V. Viktorov. The surgery to induce photothrombosis was performed in animalsunder chloral hydrate anesthesia (300 mg/kg intraperitoneally). The head ofsham�operated and treated rats was fixed using a stereotaxic device.

Amnesia is a prominent symptom of integrative dysfunction in CNSduring photothrombosis of the prefrontal cortex in rats. Integrative dysfunctionof the brain was estimated from the impairment of acquisition and performanceof a conditioned passive avoidance response (CPAR; T. A. Voronona et al.,2000).

CPAR was elicited by the standard method. LC of transition from thedark compartment to the light compartment was measured. On day 1 oftraining, the rat was placed in the light compartment. The animal explored thisarea and moved to the dark compartment after several seconds. The door of thiscompartment was closed, and the rat remained in darkness for 300 sec. Theprocedure was repeated after 1 h. In this trial, the rat was immediately removedfrom the dark compartment. On the next day, this procedure was repeated twotimes at a 1�h interval. When the rat entered the dark compartment, the doorwas closed. Electric current was delivered through a metal grid floor. CPAR wasconsidered to be elicited at LC of 300 sec (preoperative period). The animalswith a shorter period of LC were excluded from further observations.

Before the start of CPAR training, locomotor activity of rats was studiedin the automated open�field test. The number of crossed squares was measuredfor 300 sec.

131

A standard nootropic agent piracetam (200 mg/kg) and cavinton (4 mg/kg)were used as the reference drugs.

The rats were divided into six groups. Group 1 animals (control, n=20)were trained in CPAR and received 0.9% NaCl. Group 2 specimens (n=20)were sham�operated and treated with ULD anti�S100. The rats of groups 3�6were subjected to photothrombosis and received 0.9% NaCl (group 3, n=12),ULD anti�S100 (group 4, n=12), piracetam (group 5, n=12), and cavinton(group 6, n=7).

In groups 3�6, test substances (2.5 ml/kg) were administered orally 1 hafter photothrombosis and then daily for 9 days. In the last day, these drugs weregiven 40 min before testing. Control rats and sham�operated animals receivedphysiological saline or ULD�anti�S100 according to the same regimen.

Locomotor activity of all rats was studied in an automated open field onday 9 after photothrombosis of the prefrontal cortex. A pathomorphologicalstudy was performed immediately after CPAR testing.

The natural extinction of CPAR in intact animals was observed 9 daysafter acquisition. LC decreased by 23% (G. A. Romanova et al., 2003). Irrever�sible focal ischemia of the prefrontal cortex caused amnesia, which was mani�fested in the impairment of response performance and 1.8�fold decrease in thelatency of CPAR.

ULD anti�S100 prevented the natural extinction of CPAR in sham�ope�rated animals. Amnesia due to ischemic stroke was not observed after admini�stration of ULD anti�S100. LC in treated rats did not differ from that in intactanimals. Piracetam and cavinton had little antiamnesic effect (Fig. 7.15).

Administration of ULD anti�S100 was followed by a significant decreasein the area of ischemic injury (by 40%), which illustrates the neuroprotective(antiischemic) effect of this product (Fig. 7.16).


Fig. 7.15.Fig. 7.15.Fig. 7.15.Fig. 7.15.Fig. 7.15. Effect of ULD anti�S100 on CPAR performance in rats on day 9 afterischemic stroke. Intact animals (baseline, 1); control (2); sham operation + ULDanti�S100 (3); photothrombosis (4); photothrombosis + ULD anti�S100 (5);photothrombosis + piracetam (6); and photothrombosis + cavinton (7). *p<0.05compared to untreated rats with stroke.

0

20

40

60

80

100

120

1 2 3 4 5 6 7

CPAR LC, % of the control

!

132

Ultralow doses

Fig. 7.16.Fig. 7.16.Fig. 7.16.Fig. 7.16.Fig. 7.16. Effect of ULD anti�S100 on focal ischemic injury in the prefrontal cortexdue to photothrombosis: control (a) and treated rats (b).

a b

Neuroprotective activity of ULD anti�S100 in the model of hemorrhagicstroke. Hemorrhagic stroke was induced in adult male outbred rats weighing200�250 g (A. N. Makarenko et al., 1990; A. Jackowski et al., 1990). Soft tissuesand periosteum in the parietal and central regions of the cranium were removedunder nembutal anesthesia (40 mg/kg intramuscularly). A hole (diameter 1 mm)was made in the left hemisphere of the cranial bone (1.5�1.88 mm posterior tothe bregma, 2.5�3.0 mm lateral to the sagittal suture). A special device (mandrelknife) was inserted into the hole at a depth of 4 mm. The brain tissue (internalcapsule) was destructed in a stereotaxic device. The blood (0.02�0.03 ml) wastaken from the sublingual region and introduced into the area of injury after 2�3min. A morphological study showed that this treatment causes bilateral focalstroke in the internal capsule (diameter 2 mm, depth 3 mm). The upperstructures of the brain and neocortex remain intact under these conditions.

The dynamics of intracerebral hemorrhagic trauma was monitored for 14days. The state and behavior of animals were examined on days 1, 3, 7, and 14(T. A. Voronina et al., 2006b,c).

ULD anti�S100 (2.5 ml/kg) or nimodipine (0.1 mg/kg) was administered in�tragastrically using a special probe. The probe was equipped with a thickened olive.Test substances were administered once a day. The first treatment was performed4 h after surgery. The animals were tested on days 1, 3, 7, and 14 after surgery.

Sham�operated animals of the control group were subjected to scalpingin the parietal and central regions of the cranium and skull drilling with nodamage to brain structures.

133

We studied the effect of substances on the survival rate of animals,neurological deficit (I. V. Gannushkina, 1996) and cognitive dysfunction (T. A.Voronina et al., 2000) due to hemorrhagic stroke, emotional state of rats (T. A.Voronina et al., 2000), and change in muscle tone (T. A. Voronina et al., 2000;N. W. Dunham et al., 1957).

Blood infusion into the internal capsule (model of hemorrhagic stroke)caused death of some animals (50% mortality on day 14). This treatment wasalso followed by neurological deficit (60% survived animals), reduction ofmuscle tone (50% rats), and amnesia (2�fold decrease in CPAR LC; Figs. 7.17�7.21). High anxiety of animals was manifested in a decrease in the time spentin the open arms (A. N. Makarenko et al., 2004; S. A. Sergeeva et al., 2005).

ULD anti�S100 increased the survival rate of rats to 100%, significantlydecreased the percentage of animals with neurological deficit and reducedmuscle tone, and produced an antiamnesic effect in the CPAR test (nodifferences between treated and intact animals). Administration of ULD anti�S100 also had an anxiolytic effect on day 1 after stroke. It was manifested inan increase in the time spent in the open arms (by 20 times) and elevation oflocomotor activity (by 2 times).

A reference drug nimodipine had little effect on the survival rate of rats.The action of nimodipine on neurological deficit and muscle tone was similarto that of ULD anti�S100. Nimodipine was less potent than ULD anti�S100 inproducing an antiamnesic effect in the CPAR test. The anxiolytic effect ofnimodipine was less pronounced that that of ULD anti�S100.

Nootropic activity of ULD anti�S100. Nootropic activity of ULD anti�S100 was evaluated from CPAR performance by intact animals and specimens


Fig. 7.17.Fig. 7.17.Fig. 7.17.Fig. 7.17.Fig. 7.17. Effect of ULD anti�S100 on the survival rate of rats with hemorrhagicstroke. Control animals (1); sham operation (2); ULD anti�S100 (3); and nimodipine(4). *p<0.05 compared to the control.

1 3 7

2

3

4

1

14Time, days

50

40

60

70

80

% of the control

90

100

110

!

134

Ultralow doses

Fig. 7.19.Fig. 7.19.Fig. 7.19.Fig. 7.19.Fig. 7.19. Effect of ULD anti�S100 on muscle tone in rats with hemorrhagic stroke.Ordinate: animals with reduced muscle tone. Control animals (1); sham operation(2); ULD anti�S100 (3); and nimodipine (4). *p<0.05 compared to the control.

1 3Time, days

7

4

1

3

2

14

20

10

0

30

40

50

60

70

80%

!!

! !

Fig. 7.18.Fig. 7.18.Fig. 7.18.Fig. 7.18.Fig. 7.18. Effect of ULD anti�S100 on neurological deficit in rats with hemorrhagicstroke. Ordinate: animals with neurological deficit. Control animals (1); shamoperation (2); ULD anti�S100 (3); and nimodipine (4). *p<0.05 compared to thecontrol.

1 3 7Time, days

1

34

2

14

20

0

%

40

60

80

100

120

! ! !

!

with scopolamine�induced amnesia or Alzheimer’s disease (Yu. V. Burov et al.,1991; T. A. Voronina et al., 2006b).

Effect of ULD anti�S100 on CPAR performance. The test of CPAR inanimals is based on passive avoidance of aversive events with no activemovements (T. A. Voronina et al., 2006b). CPAR was studied in a special device(Lafaette Instruments Co.). This device consisted of the illuminated platform,which was placed at a height and connected with the dark compartment.Nociceptive stimulation was applied to the floor of this compartment. A holebetween the platform and compartment was closed by a movable partition wall.After appearing on the illuminated platform, all rats were characterized byinstinctive behavior to enter the dark compartment. The animals explored this

135

compartment for 3 min, but sometimes visited the open area. In the follow�upperiod, a partition wall between the platform and dark compartment was closed.Electric current was applied to the floor of this compartment for 10 sec. Hence,the dark compartment became dangerous. CPAR acquisition was tested on thenext day. The rats were placed on the illuminated platform. LC of the first entryto the dark compartment, as well as the total time spent in this compartmentwas recorded for 3 min.

ULD anti�S100 (2.5 ml/kg) or piracetam (400 mg/kg) was administeredimmediately before training (1 h before study), immediately after training, and24 h after training (1 h before performance). Diazepam in a single dose of 5


Fig. 7.20.Fig. 7.20.Fig. 7.20.Fig. 7.20.Fig. 7.20. Effect of ULD anti�S100 on CPAR performance in rats with hemorrhagicstroke (T. A. Voronina et al., 2006b). Intact animals (1); control (hemorrhagic strokeand distilled water, 2); sham operation (3); ULD anti�S100 (4); and nimodipine (5).*p<0.05 compared to the control.

1 7 14Time, days

40

20

0

1

5

2

3 4

60

80

100

120

140

160

180sec

!

!

Fig. 7.21.Fig. 7.21.Fig. 7.21.Fig. 7.21.Fig. 7.21. Anxiolytic effect of ULD anti�S100 on rats with hemorrhagic stroke.Anxiety in the EPM test. Time spent in the open arms (sec, a) and number of entriesinto the arms (b). Intact animals (1); sham operation (2); control (stroke, 3); stroke+ ULD anti�S100 (4); and stroke + nimodipine (5). p<0.05: *compared to animalsof the stroke group; +compared to intact and sham�operated animals.

0

50

100

150

200

250% of intact animals

1 2 4 53 1 2 4 53

!

! !

!

++

a b

136

Ultralow doses

Fig. 7.22.Fig. 7.22.Fig. 7.22.Fig. 7.22.Fig. 7.22. Effect of single treatment with ULD anti�S100 on CPAR performance inrats. Control (1); ULD anti�S100 (2); piracetam (3); and diazepam (4). Control, 100%(post�training level). *p<0.05 compared to the control.

0

20

40

60

80

100

120

1 2 3 4

% of trained animals

!

mg/kg was given 40 min before training. Control animals did not receive anydrug.

As differentiated from ULD anti�S100 and piracetam, diazepam had anadverse effect on the acquisition and performance of CPAR (Fig. 7.22). Thesedata indicate that ULD anti�S100 do not cause one of the side effects, whichis typical of a standard anxiolytic agent diazepam (impairment of cognitivefunction; T. A. Voronina et al., 2006c).

Antiamnesic activity of ULD anti�S100 under conditions of scopolamine�induced amnesia. Antiamnesic activity of ULD anti�S100 was estimated fromCPAR performance by animals with scopolamine�induced amnesia (T. A.Voronina et al., 2006b; R. Ader et al., 1972). A muscarinic receptor antagonistscopolamine (single dose 2 mg/kg) was injected intraperitoneally 15 min beforeCPAR training to induce amnesia in rats.

ULD anti�S100 in a dose of 2.5 ml/kg were administered intragastricallybefore training (1 h before study), immediately after training, or 24 h aftertraining (1 h before performance).

The reference drug piracetam (Nootropil) in a dose of 400 mg/kg wasadministered intragastrically (similarly to ULD anti�S100). Control animalsreceived an equivalent volume of physiological saline.

ULD anti�S100 reduced the symptoms of scopolamine�induced amnesia(increase in learning ability for CPAR; Fig. 7.23). Nootropic activity of ULDanti�S100 compared well with that of piracetam (T. A. Voronina et al., 2006c).

Efficacy of ULD anti�S100 in experimental Alzheimer’s disease.Experimental Alzheimer’s disease in rats was induced by the method of Yu. V.Burov et al. (1991) and T. A. Voronina et al. (2006b). This method is based onthe induction of cholinergic deficit, which occurs during aging and serves as amajor pathogenetic mechanism of Alzheimer’s disease. Experiments wereperformed on male outbred albino rats.

137

The rats were randomized into three groups of control (group 1) and trea�ted specimens (groups 2 and 3). A muscarinic receptor antagonist scopolaminein a daily dose of 1 mg/kg was injected intraperitoneally for 20 days to inducecholinergic deficit in treated rats. Control animals received distilled water.

On days 21�30, the rats of groups 1 (passive control) and 2 (active control)received orally distilled water. ULD anti�S100 were given to group 3 rats (2.5 ml/kg twice a day at 10.00 and 16.00). Cognitive functions were estimated from thedynamics of CAAR acquisition after scopolamine administration (number ofconditioned responses, percent of the total number of presentations; and ratio ofrats reaching the learning criterion). Neurological status was assessed by the McGrow scale. Muscle tone was measured in the rotarod test. Movement coordinationwas studied in the hanging wire test. Anxiety was determined in the EPM test.

Chronic administration of scopolamine was followed by delayed learningof CAAR, weakness, “floppy” movements, unilateral partial ptosis or ptosis(50% rats), and hypotonia (impaired coordination of movements; Figs. 7.24�

Fig. 7.23.Fig. 7.23.Fig. 7.23.Fig. 7.23.Fig. 7.23. CPAR performance in rats with scopolamine�induced amnesia after singleadministration of ULD anti�S100. Control (1); scopolamine (2); ULD anti�S100 + sco�polamine (3); and piracetam + scopolamine (4). *p<0.05 compared to the control.

0

20

40

60

80

100

120

1 2

% of trained animals

4

!

3

!

Fig. 7.24.Fig. 7.24.Fig. 7.24.Fig. 7.24.Fig. 7.24. Effect of ULD anti�S100 on the dynamics of CAAR performance duringexperimental Alzheimer’s disease. Control (1); scopolamine (2); and ULD anti�S100+ scopolamine (3). *p<0.05 compared to the control.

1 2 3 4Training, days

5

1

3

2

6 7

20

0

40

60

80

Performance of conditioned responses, %

!! !


138

Ultralow doses

Fig. 7.26.Fig. 7.26.Fig. 7.26.Fig. 7.26.Fig. 7.26. Effect of ULD anti�S100 on amnesia during experimental Alzheimer’sdisease: no adequate response. Days 1 (a) and 2 (b). Control (1); scopolamine (2);and ULD anti�S100 + scopolamine (3). *p<0.05 compared to the control.

0

5

10

15

20

25

30

1 2 3 1 2 3

% а б

!!

7.26). Anxiety of animals was manifested in a decrease in the time spent in theopen arms of EPM. The rats were also characterized by a decrease in locomotoractivity (T. A. Voronina et al., 2004).

The course of treatment with ULD anti�S100 and piracetam resulted inthe improvement of learning (similarly to intact animals) and decrease in thepercentage of animals with hypotonia (1.5�2�fold decrease in muscle tone) andpartial ptosis or ptosis.

As differentiated from piracetam, ULD anti�S100 had an anxiolytic effect(5�fold increase in the time spent in the open arms of EPM). Moreover,locomotor activity of animals increased after administration of ULD anti�S100.

ULD anti�S100 improved the CAAR learning ability of rats with cognitivedysfunction due to subchronic blockade of muscarinic receptors and furtherdepletion of the cholinergic system. The efficacy of ULD anti�S100 was similarto that of piracetam.

Fig. 7.25.Fig. 7.25.Fig. 7.25.Fig. 7.25.Fig. 7.25. Effect of ULD anti�S100 on amnesia during experimental Alzheimer’sdisease: percentage of animals with the elicited response (5th day of training).Control (1); scopolamine (2); and ULD anti�S100 + scopolamine (3). *p<0.05compared to the control.

0

15

30

45

60

%

1 2 3

!

139

Effects of combined treatmentwith ULD anti�S100 and haloperidol

The modulatory effect of ULD anti�S100 on activity of haloperidol wasstudied in the test of apomorphine verticalization (K. S. Raevskii et al., 2000)and under conditions of catalepsy induced by high dose of haloperidol.

Combined administration of ULD anti�S100 and haloperidol. Influence ofULD anti�S100 on the effect of haloperidol in the test of apomorphineverticalization. Experiments were performed on male outbred albino miceweighing 25�32 g. The animals were randomly divided into four groups of 12specimens each. Apomorphine in a dose of 2.5 mg/kg was injectedsubcutaneously to group 1 mice (control). Group 2 mice were treated withapomorphine 10 min after intraperitoneal injection of haloperidol in a dose of0.1 mg/kg (Gedeon Richter). Group 3 mice were subjected to successivetreatment with ULD anti�S100 (2.5 ml/kg intragastrically), haloperidol (5 minafter ULD anti�S100 administration), and apomorphine (10 min afterhaloperidol injection). Group 4 mice received ULD anti�S100 (2.5 ml/kgintragastrically for 7 days), haloperidol (5 min after the last treatment with ULDanti�S100), and apomorphine (10 min after haloperidol injection).

Immediately after apomorphine injection, the animals were placed incylindrical wire chambers (diameter 13 cm, height 16 cm). Vertical activitywas measured 10 min after injection of apomorphine. The measurements wereperformed for 1 h at 2�min intervals. The degree of verticalization wasdetermined by a 4�point scale (number of limbs to hold on to a vertical wall;M. Vasse et al., 1985). The following parameters were calculated: total scoreof verticalization for each animal over the whole period of study; averagedegree of verticalization for each group; and vertical activity (relative to thecontrol).

Apomorphine injection was followed by a strong stimulatory effect onvertical activity of mice. The average score of vertical activity was 63.7±15.9points over 60 min. A typical neuroleptic haloperidol completely abolished thestimulatory effect of apomorphine on vertical activity of animals (Fig. 7.27).Single administration of ULD anti�S100 did not modulate the influence ofhaloperidol on apomorphine verticalization.

These data indicate that single and repeated administration of ULD anti�S100 has little effect on the specific psychotropic action of haloperidol in thetherapeutic dose on apomorphine verticalization (T. A. Voronina, 2005).

Influence of ULD anti�S100 on the degree of catalepsy induced by high doseof haloperidol. Experiments were performed on male outbred albino ratsweighing 250�280 g. Catalepsy was induced by intraperitoneal injection ofhaloperidol in a dose of 1.0 mg/kg (ampoule solution, Gedeon Richter) 60 min


140

Ultralow doses

before the study (T. A. Voronina et al., 2000a). ULD anti�S100 in a dose of 2.5ml/kg were injected intraperitoneally 40 min after haloperidol administration (20min before the study). Each group consisted of 10 animals.

The degree of catalepsy (ability of animals to maintain a fixed bodyposture for some time) was estimated 60, 120, and 180 min after haloperidolinjection. The placement of paws on the step (7.5 and 12.5 cm in height) wasstudied in the staircase test. We evaluated the ability of rats to hold the paw onthe step for 10 sec.

The degree of catalepsy was determined by a 6�point scale: 1 point, onlyone of the forelimbs remains on the bottom step; 2 points, both forelimbs andone hindlimb remain on the bottom step; 4 points, both hindlimbs remain onthe bottom step; 5 points, only one of the forelimbs remains on the tope step;and 6 points, both forelimbs remain on the top step.

Treatment with high dose of a neuroleptic drug haloperidol causedcatalepsy in rats (maintenance of a given posture for a long time). The degreeof catalepsy in control animals was maximum (6�point scale) over the wholeperiod of study (180 min, Table 7.4).

The degree of catalepsy in haloperidol�receiving animals significantlydecreased 60 min after administration of ULD anti�S100 (by 1.33 times, Table7.4). The effect of ULD anti�S100 was less pronounced after 120 and 180 min.However, the average score of catalepsy in these rats was much lower than incontrol animals (by 1.22 and 1.07 times, respectively).

After administration of ULD anti�S100, the highest degree of catalepsy(6 points) was not observed in 60% animals.

Hence, single treatment with ULD anti�S100 was followed by asignificant decrease in cataleptogenic activity of haloperidol in a dose of 1 mg/kg (T. A. Voronina, 2005).

Fig. 7.27.Fig. 7.27.Fig. 7.27.Fig. 7.27.Fig. 7.27. Effect of haloperidol and ULD anti�S100 on vertical activity ofapomorphine�receiving mice. Apomorphine (1); haloperidol + apomorphine (2); ULDanti�S100 (single administration) + haloperidol + apomorphine (3); and ULD anti�S100 (course of treatment) + haloperidol + apomorphine (4). *p<0.05 comparedto the control.

0

40

20

60

80

Points

4

!

2

!

1 3

!

141

Range of pharmacological propertiesof ULD anti�S100 in young rats

Experiments were performed on 767 outbred rats (384 males and 383females) aging 30�35 days. We studied the anxiolytic, antidepressant, andnootropic properties of ULD anti�S100, efficacy of ULD anti�S100 underexperimental conditions of memory deficit and hyperactivity, and influence ofULD anti�S100 on locomotor and behavioral activity of high�activity and low�activity rat pups.

Anxiolytic activity of ULD anti�S100 in young rats. Anxiolytic activity ofULD anti�S100 was studied in a conflict situation (T. A. Voronina et al., 2000c;G. M. Molodavkin et al., 1995; J. R. Vogel, 1971; D. Triet, 1985; D. J. Sanger,1991) and EPM test (T. A. Voronina et al., 2000c; S. Pellow et al., 1986; G. R.Dawson et al., 1995; S. E. File, 1995). The benzodiazepine anxiolytic diazepamwas used as a reference drug.

Anxiolytic effect of ULD anti�S100 on young rats in the Vogel test. Experimentswere performed as described elsewhere (T. A. Voronina et al., 2000c; J. R. Vogel,1971). The animals (n=36, 30�35 days of life) were divided into three groups. Therats received 2.5 ml/kg distilled water (control group 1, n=12), 1.5 mg/kg diazepam(group 2, n=12), or 2.5 ml/kg ULD anti�S100 (group 3, n=12). Test substanceswere administered intragastrically 30 min before the study.

The behavior of control rat pups was characterized by high anxiety in aconflict situation. This conclusion was derived from a small number ofapproaches to the drinking bowl and low incidence of punished drinkingepisodes.

Anxiety of animals in a conflict situation was significantly reduced afteradministration of diazepam. The number of punished drinking episodes indiazepam�receiving rats was 2.7�fold higher than in control animals (Fig. 7.28).

The anxiolytic effect of ULD anti�S100 in a conflict situation was similarto that of diazepam. It was manifested in a significant increase in the numberof punished drinking episodes compared to the control (Fig. 7.28).

Table 7.4.Table 7.4.Table 7.4.Table 7.4.Table 7.4. Effect of ULD anti�S100 on the degree of haloperidol�induced catalepsy

Haloperidol 6 6 6 0 0 0

Haloperidol +ULD anti�S100 4.50±1.58* 4.90±1.29* 5.60±0.84 60 60 20

Note.Note.Note.Note.Note. *p<0.05 compared to the haloperidol group.

Groupafter

180 minafter

120 minafter

60 min

Animals without the highestdegree of catalepsy (6 points), %

Average score

after180 min

after120 min

after60 min


142

Ultralow doses

Therefore, ULD anti�S100 in a dose of 2.5 ml/kg had the anxiolytic effecton young rats in a conflict situation. Under these conditions, anxiolytic activityof ULD anti�S100 compared well with that of diazepam in a dose of 1.5 mg/kg.

Anxiolytic and antistress effect of ULD anti�S100 on young rats in the EPMtest. Experiments on young animals were performed as described for adultspecimens (T. A. Voronina et al., 2000c; S. Pellow et al., 1986; S. E. File, 1995).The animals (n=36, 30�35 days of life) were divided into the control andtreatment groups of 12 specimens each. The rats received 2.5 ml/kg distilledwater (control group), 1.5 mg/kg diazepam (treatment group 1), and 2.5 ml/kgULD anti�S100 (treatment group 2). Distilled water and test substances wereadministered intragastrically 20 min before the study.

ULD anti�S100 had a normalizing effect on the EPM behavior ofanimals. Administration of this product was followed by a significant increasein the number of entries into the open arms and central area of EPM (by 5.6and 2.1 times, respectively; Fig. 7.29).

Diazepam also had an anxiolytic effect. The number of entries into theopen arms and central area of EPM increased by 8.3 and 3.6 times, respectively,after diazepam treatment (p<0.05, Fig. 7.29).

No significant differences were found in the anxiolytic effect of ULDanti�S100 and diazepam on young rats in EPM.

Antidepressant effect of ULD anti�S100 on young rats. The antidepressanteffect of ULD anti�S100 on young rats was studied in the Porsolt’s forcedswimming test (R. D. Porsolt et al., 1978), as well as in a water tank with wheels(G. M. Molodavkin et al., 1994; S. Nomura et al., 1982).

Antidepressant effect of ULD anti�S100 on young rats: Porsolt’s forcedswimming test. Experiments were performed as described elsewhere (R. D.Porsolt et al., 1978). The animals (n=40) were divided into three groups. They

Fig. 7.28.Fig. 7.28.Fig. 7.28.Fig. 7.28.Fig. 7.28. Anxiolytic effect of ULD anti�S100 on young rats in the Vogel conflicttest. *p<0.05 compared to the control.

0

100

200

300

Number of punished drinking episodes


!

Diazepam

!

143

Fig. 7.29.Fig. 7.29.Fig. 7.29.Fig. 7.29.Fig. 7.29. Anxiolytic effect of ULD anti�S100 on young rats in the EPM test. Lightbars, time spent in the central area; dark bars, time spent in the open arms. p<0.05:*compared to the control; +compared to diazepam.

0

40

20

60

80sec


!!

+ +!

!

received 2.5 ml/kg distilled water (n=20, control group), 10 mg/kg amitriptylinein 2.5 ml/kg distilled water (n=10, treatment group 1), or 2.5 ml/kg ULD anti�S100 (n=10, treatment group 2). Test substances were administeredintragastrically 30 min before the study.

ULD anti�S100 and amitriptyline had an antidepressant effect in thePorsolt’s test of behavioral despair. The immobility time in treated rats wasmuch lower than in control animals (by 1.3 and 1.4 times, respectively; Fig.7.30). Therefore, the antidepressant activity of ULD anti�S100 compared wellwith that of amitriptyline.

Antidepressant effect of ULD anti�S100 on young rats in the Nomura’s forcedswimming test with rotating wheels. Experiments on young rats were performed asdescribed for adult specimens (G. M. Molodavkin et al., 1994; S. Nomura et al.,1982). The animals (n=40, 30�35 days of life) were divided into three groups. Therats received 2.5 ml/kg distilled water (n=20, control group), 10 mg/kg amitriptylinein 2.5 ml/kg distilled water (n=10, group 2), or 2.5 ml/kg ULD anti�S100 (n=10,group 3). Test substances were administered intragastrically 30 min before the study.

The number of wheel revolutions in the Nomura’s forced swimming testwith freely rotating wheels increased by 1.8 and 1.7 times after administration


Fig. 7.30.Fig. 7.30.Fig. 7.30.Fig. 7.30.Fig. 7.30. Antidepressant effect of ULD anti�S100 on young rats in the Porsolt’sforced swimming test. *p<0.05 compared to the control.

0

100

200

300

400

Control Amitriptyline

Immobility time, sec

ULD anti�S100

!

!

144

Ultralow doses

of ULD anti�S100 and amitriptyline, respectively (p<0.05). Drug treatment wasalso followed by an increase in the correlation coefficient between the numbers ofwheel revolutions during the first and second 5�min periods of study (Fig. 7.31).

These data indicate that test substances have a strong antidepressanteffect.

Nootropic effect of ULD anti�S100 on young rats. Nootropic activity ofULD anti�S100 was studied in the CPAR test under conditions of scopolamine�induced amnesia (T. A. Voronina et al., 2000b). The product was also tested inrat pups with poor learning ability. Piracetam was used as a reference drug.

Nootropic effect of ULD anti�S100 (single administration and course oftreatment) during scopolamine�induced amnesia. The device and design ofexperiments were similar to those in studies with adult animals (T. A. Voroninaet al., 2000b). The parameter of DLC was calculated as follows:

ΔLC=LC2�LC1,

where LC2 and LC1 are LC of the first entry into a compartment with theelectrode floor during CPAR acquisition and testing (24 h after training),respectively. The optimal conditions of CPAR training for young rats wereselected in previous experiments (eight series of electrostimulation with 0.6 mA,pulse duration 1 sec, interpulse interval 2 sec).

CPAR amnesia was induced by the standard method. A cholinoceptorantagonist scopolamine in a dose of 1.4 mg/kg was injected subcutaneously15 min before training (according to the Manual on Experimental (Preclinical)Study of New Pharmacological Substances, 2005).

Rat pups were divided into the following groups:• group 1 (passive control, n=27): distilled water, 2.5 ml/kg intragastri�

cally, single or repeated administration (10 days, last treatment 40 minbefore training); and distilled water, 2 ml/kg subcutaneously, 15 minbefore training;

Fig. 7.31.Fig. 7.31.Fig. 7.31.Fig. 7.31.Fig. 7.31. Antidepressant effect of ULD anti�S100 on young rats in the Nomura’sforced swimming test. *p<0.05 compared to the control.

0

40

80

120

160

Control Amitriptyline

Number of wheel revolutions

ULD anti�S100

!

!

145

• group 2 (active control, n=27): distilled water, 2.5 ml/kg intragastri�cally, single or repeated administration (10 days, last treatment 40 minbefore training); and scopolamine, 1.4 mg/kg subcutaneously, 15 minbefore training;

• group 3 (n=15): 400 mg/kg piracetam in 2.5 ml/kg distilled water,intragastrically for 10 days (last treatment 40 min before training); andscopolamine, 1.4 mg/kg subcutaneously, 15 min before training;

• group 4 (n=12): 400 mg/kg piracetam in 2.5 ml/kg distilled water,intragastrically, 40 min before training; and scopolamine, 1.4 mg/kgsubcutaneously, 15 min before training;

• group 5 (n=15): ULD anti�S100, 2.5 ml/kg intragastrically, 40 minbefore training; and scopolamine, 1.4 mg/kg subcutaneously, 15 minbefore training; and

• group 6 (n=12): ULD anti�S100, 2.5 ml/kg intragastrically for 10 days(last treatment 40 min before training); and scopolamine, 1.4 mg/kgsubcutaneously, 15 min before training.

Memory processes were retained in rat pups of the passive control group.These animals remained in the dangerous dark compartment for a longer periodthan during training (by 10 times, p<0.05). Scopolamine�induced amnesia wasmanifested in a decrease in LC of entry into the dark compartment (by 7.6times, p<0.05) and reduction of DLC (by 18.2 times compared to the passivecontrol, p<0.05; Table 7.5).

The amnesic effect of a cholinoceptor antagonist scopolamine was lesspronounced after single administration of piracetam. This conclusion wasderived from an increase in LC of entry into the dangerous dark compartment(by 3.4 times, p<0.05) and DLC (by 7.4 times compared to the active control,p<0.05; Table 7.5).

Single administration of ULD anti�S100 had a similar effect underconditions of scopolamine�induced amnesia. LC of entry into the darkcompartment and DLC increased by 2.2 and 4.6 times, respectively, comparedto the active control (Table 7.5).

Treatment with ULD anti�S100 and piracetam was followed by an in�crease in the number of rat pups that reached the learning criterion (Fig. 7.32).

These data show that single administration of ULD anti�S100 andpiracetam has a strong antiamnesic effect on the model of scopolamine�inducedamnesia. The antiamnesic effect of single treatment with ULD anti�S100 is lesspronounced than that of piracetam. Due to low effectiveness of ULD anti�S100after single treatment, further experiments were performed with repeatedadministration of ULD anti�S100 and piracetam (10�day course).

A 10�day course of treatment with ULD anti�S100 and piracetam had agreater antiamnesic effect. The effect of ULD anti�S100 was most pronounced


146

Ultralow doses

Fig. 7.32.Fig. 7.32.Fig. 7.32.Fig. 7.32.Fig. 7.32. Effect of ULD anti�S100 on CPAR performance during scopolamine�induced amnesia. Control (1); scopolamine (2); piracetam + scopolamine (3); ULDanti�S100 (single administration) + scopolamine (4); and ULD anti�S100 (courseof treatment) + scopolamine (5). p<0.05: *compared to the control; +comparedto scopolamine.

0

20

40

+

+

+60

80

100

120

1 2 3

Trained animals, %

5

!

4

under these conditions. It was manifested in an increase in LC of entry into thedark compartment (by 3.4 times compared to the active control, p<0.05) andDLC (by 8.6 times, p<0.05; Table 7.6). In animals of the piracetam group theseparameters increased by 3.8 and 8.9 times, respectively (p<0.05). It can beconcluded that the antiamnesic effect of repeated treatment with ULD anti�S100 compares well with that of piracetam.

Hence, ULD anti�S100 and piracetam have a strong antiamnesic effectin the test for scopolamine�induced amnesia. During single administration, theantiamnesic effect of ULD anti�S100 was smaller than that of piracetam. Afterthe course of treatment, ULD anti�S100 and piracetam had a similar effect.

Effect of repeated treatment with ULD anti�S100 on training of young ratswith poor learning ability. Experiments were performed by the standard method(T. A. Voronina et al., 2006b). DLC for each animal was calculated as follows:

Table 7.5.Table 7.5.Table 7.5.Table 7.5.Table 7.5. Antiamnesic effect of single treatment with ULD anti�S100 and piracetamin the test for scopolamine�induced amnesia of CPAR (M±m, n=15)

Passive control 10.13±3.81 100.47±21.27 90.27±19.86

Active control(amnesia + scopolamine) 8.3±3.1 13.33±8.08+ 4.97±3.30+

ULD anti�S100 + scopolamine 6.77±2.0 29.80±9.84* 23.03±9.90*

Piracetam + scopolamine 8.07±2.32 44.67±10.58* 36.6±10.9*

Note.Note.Note.Note.Note. p<0.05: *compared to the active control; +compared to the passive control.

Substance

ΔLC=LC2–LC

1LC

2LC

1

24 h after trainingBefore training

147

Table 7.6.Table 7.6.Table 7.6.Table 7.6.Table 7.6. Antiamnesic effects of repeated treatment with ULD anti�S100 andpiracetam (10�day course): CPAR of 30�35�day�old rat pups in the test forscopolamine�induced amnesia (M±m, n=12)

Passive control(distilled water) 8.83±3.15 109.25±25.03 98.25±23.87

Active control (scopolamine) 9.08±2.28 14.83±3.12+ 5.08±1.95+

ULD anti�S100 + scopolamine 7.17±2.25 50.83±17.43* 43.50±16.88*

Piracetam + scopolamine 10.17±1.82 55.58±14.42* 45.42±14.02*

Note.Note.Note.Note.Note. p<0.05: *compared to the active control; +compared to the passive control.

SubstanceΔLC=LC

2–LC

1LC

2LC

1

24 h after trainingTraining

ΔLC=LC2�LC1,

where LC2 and LC1 are LC of the first entry into a compartment with theelectrode floor during CPAR acquisition and testing (24 h after training),respectively.

DLC was low under specified conditions of training (five series of electro�stimulation with 0.45 mA, pulse duration 1 sec, interpulse interval 2 sec). Theanimals were characterized by poor learning ability, which met the requirementsof this series. Previous experiments with scopolamine�induced amnesia showedthat the efficacy of drugs is much higher after the course of treatment. In thepresent study, test substances were administration for 10 days before training.

Rat pups were randomized into three groups of 15 specimens each. Theanimals received intragastrically distilled water (2.5 ml/kg, group 1), ULD anti�S100 (2.5 ml/kg, group 2), or piracetam (400 mg/kg, group 3) for 10 days (lasttreatment 40 min before training).

LC of entry into the dark compartment tended to increase after piracetamadministration (no statistically significant differences from the control, Table 7.7).

LC of entry into the dark compartment and DLC significantly increasedafter treatment with ULD anti�S100 (by 1.8 and 4 times, respectively; Table 7.7).

The results indicate that a 10�day course of treatment with ULD anti�S100 improves memory processes in rat pups with poor learning ability. ULDanti�S100 were more potent than piracetam in modulating the learning abilityof these animals.

Effect of ULD anti�S100 on cognitive function, motor activity, and anxietyof high�activity young rats with inadequate behavior: experimental model ofattention deficiency and hyperactivity. High�activity animals with impulsive(inadequate) behavior in the open�field test and provoking stimulation wereselected from the population of outbred rat pups (scale of Broudy and Nauta).


148

Ultralow doses

The animals were divided into three groups. Rat pups received intragastrically2.5 ml/kg distilled water (group 1, n=49), 125 mg/kg phenibut (group 2, n=20),or 2.5 ml/kg ULD anti�S100 (group 3, n=36) for 7 days.

Each group was divided into two subgroups. Subgroup 1 rats were trainedin CPAR 15 min after the last administration of test substances. EPM anxietyof subgroup 2 animals was studied 20 min after the last treatment.

Administration of phenibut was followed by a 2.8�fold increase in LC ofentry into the dark compartment (p<0.05 compared to the control). Phenibutalso increased the number of animals not entering the dark compartment.Under these conditions the number of transitions between the illuminatedplatform and dark compartment decreased by 3 times (Table 7.8, Fig. 7.33).

Treatment with ULD anti�S100 was followed by an increase in LC ofentry into the dark compartment (by 3.4 times, p<0.05), time spent on theilluminated platform (by 2 times, p<0.05), and number of animals not enteringthe dark compartment. Under these conditions the number of transitionsbetween the illuminated platform and dark compartment decreased by 3 times(Table 7.8, Fig. 7.31).

These data indicate that ULD anti�S100 improve CPAR learning anddecrease the hyperactivity of high�activity young rats with inadequate behavioralreactions. ULD anti�S100 had a modulatory effect on learning ability duringCPAR performance in the dark compartment (i.e., measurement of LC). Thiseffect was manifested in an increase in the time spent on the illuminatedplatform. The number of animals not entering the dark compartment increasedunder these conditions. Moreover, hyperactivity of high�activity rat pups wasshown to decrease after administration of ULD anti�S100. This conclusion wasderived from a decrease in the number of transitions between the illuminatedplatform and dark compartment. The effect of ULD anti�S100 on learning ofhigh�activity rat pups was more pronounced than that of phenibut.

The EPM test showed that the behavior of selected rat pups ischaracterized by high motor activity. The animals exhibited multiple entries into

Table 7.7.Table 7.7.Table 7.7.Table 7.7.Table 7.7. Effects of ULD anti�S100 and piracetam on CPAR acquisition in young ratswith poor learning ability (M±m, n=15)

Control 10.3±1.1 15.1±2.3 4.8±1.9

ULD anti�S100 7.2±1.1 26.4±4.6* 19.2±3.9*

Piracetam 9.2±2.1 19.2±4.8 10.3±5.7


SubstanceΔLCLC

2LC

1

24 h after trainingBefore training

149

the closed arms, active movement in the closed arms, and anxiety (rare visitsto the open arms and central area, little time spent in the open arms and centralarea, considerable number of fecal boluses, and high incidence of groomingepisodes).

ULD anti�S100 had a normalizing effect on the EPM behavior of high�activity animals. ULD anti�S100 decreased the number of entries into theclosed arms (by 3.5 times, p<0.05), but increased the number entries into theopen arms (by 5.2 times, p<0.05) and central area (by 2 times, p>0.05). Thenumber of movements in the open and closed arms was similar afteradministration of ULD anti�S100. The total number of these movements foranimals of the ULD anti�S100 group was lower compared to the control (4.6and 6.1, respectively).

Treatment with ULD anti�S100 was followed by an increase in the timespent in the central area and open arms (by 2.7 and 1.8 times, respectively,p<0.05). Moreover, the number of fecal boluses decreased by 3.1 times (p<0.05).The observed changes reflect a decrease in the degree of anxiety (Table 7.9).

Table 7.8.Table 7.8.Table 7.8.Table 7.8.Table 7.8. Effects of ULD anti�S100 and phenibut on CPAR acquisition in high�activityyoung rats with inadequate behavior (M±m)

Latency of entry into the dark 26.61±11.26 73.40±36.56* 91.33±34.14*compartment, sec

Time spent on the illuminatedplatform, sec 51.99±20.85 73.40±36.56 102.56±32.58*

Time spent in the darkcompartment, sec 128.5±20.8 106.60±36.56 77.72±32.56

Number of transitions 2.40±0.67 0.80±0.26* 0.78±0.34*


Parameter,24 h after training

ULD anti�S100,2.5 ml/kg (n=18)

Phenibut, 125mg/kg (n=10)

Control(n=27)


Fig. 7.33.Fig. 7.33.Fig. 7.33.Fig. 7.33.Fig. 7.33. Effect of ULD anti�S100 on CPAR performance in high�activity youngrats with inadequate behavior. *p<0.05 compared to the control.

0

10

20

30

40

Control ULD anti�S100 Phenibut

Trained animals, %!

150

Ultralow doses

Number of entries

into the closed arms 4.98±0.85 2.50±0.53* 1.44±0.33*

into the open arms 0.33±0.39 1.2±1.0 1.72±0.89*

to the central area 0.75±0.53 2.50±0.73* 1.50±0.66

Time, sec

on the central area 3.71±3.03 20.0±5.3* 10.06±5.21*

in the open arms 3.69±4.79 8.5±7.31 6.56±3.32

Animals entering the open arms, % 25 40 50

Grooming 2.48±0.73 0.80±0.64* 2.50±0.51

Number of boluses 1.88±0.69 0.30±0.42* 0.61±0.39*


Table 7.9.Table 7.9.Table 7.9.Table 7.9.Table 7.9. Effects of ULD anti�S100 and phenibut on the behavior of high�activity ratpups in the elevated plus�maze (M±m)

Phenibut, 125mg/kg (n=10)

ULD anti�S100,2.5 ml/kg (n=18)

Control(n=22)

Parameter

These data indicate that ULD anti�S100 decrease the motor activity andhave an anxiolytic effect on high�anxiety rat pups.

Phenibut also had a normalizing effect on the EPM behavior of high�activity animals. This substance decreased the number of entries into the closedarms (by 2 times, p<0.05), but increased the number of entries into the openarms (by 3.6 times) and central area (by 3.3 times, p<0.05).

However, phenibut was less potent than ULD anti�S100 in decreasing thenumber of entries into the closed arms (by 3.6 and 2 times, respectively; Table7.9). Therefore, phenibut had a smaller effect on the increased motor activityof high�activity animals.

Phenibut also had a strong anxiolytic effect. This substance increased notonly the number of entries into the open arms and central area, but also thetime spent in the illuminated area. The incidence of grooming episodes andnumber of fecal boluses decreased under these conditions (Table 7.9). Phenibuthad a greater anxiolytic effect on high�activity rat pups than ULD anti�S100.

These data indicate that ULD anti�S100 and phenibut have a normalizingeffect on the EPM behavior of high�activity animals. Test substances decreasedthe number of entries into the closed arms, but increased the number of entriesand time spent in the open arms and central area. The number of fecal bolusesdecreased under these conditions. Hence, ULD anti�S100 and phenibutproduce an anxiolytic effect on high�activity rat pups.

The anxiolytic effect of phenibut was greater than that of ULD anti�S100.However, ULD anti�S100 were more potent than phenibut in normalizing theincreased motor activity of high�activity animals.

151

Safety of ULD anti�S100

A complete toxicology study was performed to evaluate the safety profile,possible side effects, target organs, and safe dosage range of ULD anti�S100.These experiments were conducted in accordance with the recommendationsgiven in the Manual on Experimental (Preclinical) Study of NewPharmacological Substances and approved by the Pharmacological Committeeof the Russian Ministry of Health in 2000. The purpose of studies with ULDanti�S100 was to determine the acute toxicity (experiments on mice and rats),chronic toxicity (6�month treatment of rats and rabbits), reproductive andallergic toxicity (experiments on rats), immunotoxicity, mutagenicity (chromo�somal aberration assay in mouse bone marrow cells), and genotoxicity (testsystem for somatic mosaicism in wing cells of Drosophila melanogaster).

Our experiments demonstrated a good safety profile of ULD anti�S100.An acute toxicity study showed that this substance in the maximum permissibledose does not cause death of animals. Drug�related death of animals was notobserved after 6�month treatment with ULD anti�S100 in the highest dose. Theproduct had no toxic effect on organs of experimental animals. Apathomorphological study did not reveal damage to the internal organs or localirritation of the gastric mucosa after drug administration. ULD anti�S100 didnot cause reproductive disorders in male and female rats. The embryotoxic effectof ULD anti�S100 was not detected. ULD anti�S100 had no mutagenic,allergenic, and immunotoxic properties.

Acute toxicity of ULD anti�S100 was also studied on young rats. Due tothe absence of mortality, it was difficult to evaluate LD50 of ULD anti�S100 foryoung animals (similarly to adult specimens). Conventionally, the dose of ULDanti�S100 exceeding the maximum permissible dose (by volume, according tothe route of administration) was considered as LD50.

* * *Experimental studies for pharmacological activity of ULD anti�S100

allowed us to make the following conclusions.Experiments on adult and young animals showed that the product of

ULD anti�S100 has a wide range of psychotropic and neurotropic pharmaco�logical properties, including the anxiolytic, antidepressant, antistress, nootropic,neuroprotective, antiischemic, and antihypoxic effects. ULD anti�S100 differfrom modern neuropsychotropic drugs in a variety of properties, absence of sideeffects (typical of high�efficacy reference drugs), and mechanism of action.

A wide range of pharmacological properties of ULD anti�S100 is related tothe improvement of general adaptive processes in CNS (Fig. 7.34). The impairmentof these processes is followed by a deficiency of endogenous stress�limiting systems


152

Ultralow doses

Fig. 7.34.Fig. 7.34.Fig. 7.34.Fig. 7.34.Fig. 7.34. Possible pathways and mechanisms for pharmacological activity of ULDanti�S100.

Modification of functional activity of endogenous protein S�100

and its ligands

GABA(A) receptor complex

Intracellular calcium,

Ca2+�dependent processes

Membrane adenylate cyclase

Other ligands and regulatory targets

for S�100 protein

GABA�mimetic effect,functional recovery

of the GABAergic system

Restoration of intracellular

Ca2+ homeostasis

Regulation of cAMP�dependent

processes

• Nuclear factor NFkB• Antiapoptotic

factor Bcl�2• Cytoskeletal proteins

(Tau�proteins etc.)

Regulation of the cell cycle, regeneration of neurons and glia,

and cytoskeletal integrity; antiapoptotic effect

• Signal transduction from G protein�coupled receptors

• Regulation of functional plasticity of neurons:� through protein kinase A� through protein kinase G� functional regulation of glutamate

receptors and GABA receptors

• Stabilization of neuronal impulse activity• Stress�protecting effect on cells• Regulation of functional plasticity

of neurons:� through the activity of calmodulin,

protein kinase C, and other Ca2+�dependent kinases

� regulation of enzyme activity for energy metabolism and plastic metabolism (through dephosphorylation or phosphorylation)

� functional regulation of glutamate receptors and GABA receptors Regulation of enzyme expression

for energy metabolism and plastic metabolism

Recovery/increase in neuronal plasticity (natural stress�protective mechanism in CNS)

Recovery/increase in the activity of endogenous stress�limiting systems

• Activation of inhibitory processes in CNS

• Stress�limiting activity• Neuromodulatory effect::

� neuronal plasticity� growth factor for

serotoninergic neurons� reduced production of

hypothalamic corticotropin�releasing hormone

• Functional regulation of other neurotransmitter systems

(primarily of the GABAergic system) and chronic dysregulation of neuronal plas�ticity. Functional recovery is accompanied by a variety of pharmacological effectsof ULD anti�S100. The prevalence of any mechanism for dysregulation determinesthe site of action and effects of this product under various pathological conditions.

The proposed pathways and mechanisms for pharmacological activity ofULD anti�S100 are consistent with the results of experimental and clinicalstudies. Moreover, they fit naturally into the existing neurobiological paradigm.

ULD anti�S100 have a normalizing effect and increase the activity ofendogenous stress�limiting systems. The product regulates neuronal plasticity.These properties determine the neuroprotective and trophic effect of ULD anti�S100. Hence, ULD anti�S100 hold much promise for the pathogenetic therapyof various neurological and mental disorders.

7.2. Preclinical study of Impaza

Impaze consists of affinity purified rabbit polyclonal antibodies toendothelial NO synthase (eNOS) in ULD (mixture of homeopathic dilutionsC12, C30, and C200).

153

Previous experiments showed that Impaza improves sexual behavior anderectile function in male rats with decreased sexual activity. Moreover, Impazahad a positive effect on sexual function in females (Fig. 7.35). Impaza activateseNOS and increases the production of cGMP, nitrates, and nitrites in the ca�vernous bodies of male rats. The drug has no effect on hemodynamics in nor�motensive animals, but decreases blood pressure (BP) in hypertensive rats. Thesedata indicate that Impaza may be used in the therapy of cardiovascular disorders.

A toxicology study was performed in accordance with the recommenda�tions of the Pharmacological Committee of the Russian Ministry of Health andSocial Development. This study showed that Impaza exhibits a good safety pro�file. Combined treatment with Impaza and nitroglycerine was not accompaniedby a further decrease in BP. The data indicate that Impaza may be prescribedfor patients with CHD.

Effect of Impaza on sexual behavior of rats

Effect of Impaza on sexual behavior of male Wistar rats. Although thedevelopment of erectile dysfunction (ED) is associated with a variety of factors, themajor pathogenetic types of this disorder have a common mechanism. It suggestsfunctional insufficiency of the NO synthase — NO guanylate cyclase — cGMPcascade and, primarily, inadequate production of NO (K. E. Andersson, 2001; M.Ushiyama et al., 2004). Previous experiments were designed to study the effectof Impaza on sexual behavior and activity of this regulatory pathway.

The effect of Impaza on sexual behavior of Wistar rats was studied on twomodels of decreased sexual activity (seasonal and age�related suppression).


Fig. 7.35.Fig. 7.35.Fig. 7.35.Fig. 7.35.Fig. 7.35. Preclinical study of Impaza.

IMPAZA

Effect on sexual behavior Mechanism of action Effect on hemodynamics

Males Females Effect on eNOS activity and production of cGMP,

nitrates, and nitrites

Normotensive rats

Seasonal suppression

Age�related suppression

Hypertensive rats

154

Ultralow doses

Fig. 7.36.Fig. 7.36.Fig. 7.36.Fig. 7.36.Fig. 7.36. Effect of Impaza on sexual behavior of male rats with a seasonaldecrease in sexual activity. Number of mountings (a) and matings (b). *p<0.05compared to the baseline value.

Series I was performed on 4�month�old animals (400�450 g) in the winterperiod. Series II was performed on old males (16 months old, 600�700 g).Impaza was administered intragastrically in a daily dose of 1.5 ml for 5 days(treatment group, n=10). Control rats (n=10) received an equivalent volume ofdistilled water. Sexual behavior in the open field was studied before and aftertherapy (J. Bures et al., 1983).

The males were mated with 3�4�month�old females (300�400 g). Thestage of estrus in females was induced by 4�fold injection of 0.05% folliculin ina daily dose of 0.02 mg/kg. The profile of sexual activity was estimated from LCof mounting, total number of mountings, and number of matings (T. G. Bo�rovskaya, et al., 2002).

The course of treatment with Impaza in males with seasonal suppressionof sexual activity was followed by an increase in the total number of mountingsand matings by 2 and 3 times, respectively. These parameters in control animalsincreased by 25 and 35%, respectively (Fig. 7.36).

Therefore, the course of treatment with Impaza improves sexual functionand motivation (T. G. Borovskaya, et al., 2002).

Experiments on old rats showed that Impaza is effective only in animalswith preserved sexual behavior (55% specimens). The number of matings(ejaculations) is one of the major parameters, which determines sexual moti�vation and sexual function. Under basal conditions, this parameter was low in16�month�old males of the control and treatment groups. The number ofmatings (ejaculations) increased by 3.3 times after administration of Impaza for5 days (p<0.05). By contrast, this parameter decreased in control rats. LC ofmating serves as the criterion of sexual motivation. Administration of distilledwater and Impaza had no effect on this parameter in rats. The number ofmountings reflects not only sexual function, but also sexual motivation. The

0

5

10

15

20

25

30

a b

Baseline After treatment


!

!

controlImpaza

155

number of mountings decreased in the control group, but increased after a 5�day course of treatment with Impaza (by 1.8 times, p<0.05 compared to thebaseline value; Fig. 7.37).

The data indicate that a 5�day course of Impaza improves sexual functionin male rats with a seasonal decrease in sexual function and age�related erectiledysfunction (T. G. Borovskaya et al., 2001; T. G. Borovskaya et al., 2002).

The effect of Impaza on the basal activity of the NOS � NO � cGMPcascade in the cavernous bodies was studied on male Wistar rats aging 4 months.The animals received intragastrically Impaza (1.5 ml, single administration or5�day course of treatment), distilled water, or reference drug sildenafil citrate(single dose 10 mg/kg; Pfizer). They were killed 3 h after the last treatment. Thecavernous tissue was isolated. cGMP content was estimated by a direct enzymeimmunoassay with Amersham kits. The concentration of NO derivates wasmeasured colorimetrically with CN Biosciences kits. NOS activity was measuredcolorimetrically (Oxford Biomedical Research).

Impaza improved copulative (erectile) function and had a stimulatoryeffect on sexual behavior under conditions of seasonal or age�related suppressionof reproductive function.

A biochemical study showed that the improvement of erectile functionafter treatment with Impaza is accompanied by significant changes in the basalactivity of NOS, production of NO, and content of cGMP in the cavernoustissue of male rats. Single oral administration of Impaza was followed by anincrease in the activity of endothelial NO synthase and concentration of NOderivates in the cavernous bodies of male rats (by 2 and 1.4 times, respectively;p<0.05). A 5�day course of treatment of Impaza also produced a 4�fold increasein cGMP content in the penile cavernous tissue (Yu. P. Bel’skii et al., 2003; A.V. Martyushev�Poklad et al., 2003). A reference drug sildenafil increased onlythe content of cGMP (Fig. 7.38).

Fig. 7.37.Fig. 7.37.Fig. 7.37.Fig. 7.37.Fig. 7.37. Effect of Impaza on sexual behavior of old male rats with decreasedsexual activity. Number of mountings (a) and matings (b). *p<0.05 compared tothe baseline value.

0

1

2

3

4

5

7a b

6



!

!

controlImpaza


156

Ultralow doses

Fig. 7.38.Fig. 7.38.Fig. 7.38.Fig. 7.38.Fig. 7.38. Major mechanism for the effectof Impaza: content of cGMP (a), concentra�tion of NO derivatives (b), and activity ofeNOS (c) in the cavernous tissue of malerats. *p<0.05 compared to the control (dis�tilled water).

0

100

200

300

400

500

700

600

Single treatment

а

Course of treatment

!

!

!

!

control sildenafil Impaza

% of the baseline

50

7060

8090

100110

140130120

Single treatment Course of treatment

!!


% of the baselineb

0

50

100

150

200

250

300

Single treatment

% of the baseline

Course of treatment

!

!


c

Hence, the most probable peripheral mechanism for Impaza action is anincrease in endothelial NO synthase activity and recovery of NO production(i.e., normalization of endothelial function).

Effect of Impaza on sexual function in females. In addition to studying theeffect of Impaza on sexual behavior of male rats, the influence of this drug onsexual behavior of estrous females was evaluated (T. G. Borovskaya et al., 2002).

Sexual receptivity of females (readiness for mating) is determined by thereduced aggressiveness to males and appearance of the lordosis posture. Impazahad no effect on the number of lordosis postures in females, but increased thelordosis/mounting ratio (by 1.9 times, p<0.05). Moreover, Impaza increased thenumber of active females (exhibiting the lordosis posture) and decreased thenumber of aggressive females.

Effect of Impaza on the cardiovascular system

The vascular endothelium is a neuroendocrine organ, which maintains therelationship between blood and tissues (G. A. Chumakova et al., 2006).Endothelial dysfunction causes a variety of diseases, including hypertension,

157

CHD, and ED (D. V. Nibieridze, 2005; O. D. Ostroumova et al., 2005).Previous experiments showed that Impaza improves endothelial function (NOsynthase — NO guanylate cyclase — cGMP cascade). Further investigationswere performed to evaluate the effect of this drug on the cardiovascular system.

The effects of Impaza on the cardiovascular system were studied in twoseries with normotensive Wistar rats (series I) and hypertensive ISIAH rats(series II).

Effect of Impaza on hemodynamic parameters in normotensive Wistar rats.We studied the effect of test substances on the following parameters of systemichemodynamics: cardiac output (CO), stroke volume, mean BP (MBP), centralvenous pressure, total peripheral resistance, and heart rate (HR).

The drugs to modulate sexual potency (sildenafil, vardenafil, and tadalafil;according to the Instructions for Use) should not be administered in combi�nation with nitrates (nitrites) and hypotensive pharmaceuticals, which limitstheir use in patients with CHD and arterial hypertension. It was interesting toevaluate the effect of combined treatment with nitroglycerine and Impaza.

Experiments were performed on 100 male Wistar rats weighing 200�250 g.The animals were divided into five groups as follows: group 1, single intragastricadministration of distilled water (2 ml); group 2, single intragastric administra�tion of 10 mg/kg sildenafil (2 ml, Pfizer); group 3, single intragastric admini�stration of Impaza (2 ml); group 4, 5�day course of intragastric treatment withImpaza (2 ml); and group 5, 5�day course of intragastric treatment with distilledwater (2 ml).

Single and repeated administration of Impaza had no effect on a short�term decrease in MBP induced by nitroglycerine (5 mg/kg intravenously). Thetime�to�recovery of MBP remained unchanged under these conditions.Moreover, various routes of treatment with Impaza did not affect the systemichemodynamics in healthy rats.

Effect of Impaza on BP in ISIAH rats with inherited hypertension. ISIAHrats were bred at the Institute of Cytology and Genetics of the SiberianDivision of the Russian Academy of Sciences (Novosibirsk). They arecharacterized by inherited stress�induced arterial hypertension. Experimentswere performed on 30 male ISIAH rats aging 5�6 months. The animals weredivided into three groups of 10 specimens each. Group 1 rats (control) receivedorally 0.5 ml distilled water for 10 days (through a glass pipette). Group 2animals were subjected to a 10�day course of treatment with Impaza in a doseof 0.5 ml. Losartan in a dose of 10 mg/kg was administered to group 3 ratsfor 5 days. BP was measured 2 h after the 5th and 10th treatment, as well as7 days after drug withdrawal (day 17 after the start of therapy). Indirectmeasurements were performed with a special tail cuff. During this procedure,the animals were placed in a plastic chamber (B. N. Van Vliet et al., 2000).


158

Ultralow doses

Fig. 7.39.Fig. 7.39.Fig. 7.39.Fig. 7.39.Fig. 7.39. Effect of the course of treatment with Impaza on systolic BP in ISIAHrats with inherited stress�induced arterial hypertension. Effect of Impaza on day5 of treatment (1); effect of Impaza on day 10 of treatment (2); and effect of losartanon day 5 of treatment (3).

0

2

4

6

8

10

14

12

1 2 3

Decrease in MBP, % of the control

Impaza significantly decreased BP in hypertensive rats (A. L. Markel’ et al.,2002). The hypotensive effect of Impaza developed progressively. On the 10thday of treatment, BP in Impaza�receiving rats significantly differed from thecontrol (Figs. 7.39 and 7.40).

The results suggest that Impaza contributes to activation of endothelialNO synthase and recovery or increase in the production of endothelial NO(improvement of endothelial function). A possible mechanism for the effect ofImpaza is shown in Fig. 7.41. Probably, eNOS activity reaches the individualphysiological optimum after long�term administration of Impaza.

Safety profile of Impaza. A complete toxicology study was performed toevaluate the safety profile, possible side effects, target organs, and safe dosagerange of Impaza. These experiments were conducted in accordance with therecommendations given in the Manual on Experimental (Preclinical) Study ofNew Pharmacological Substances and approved by the PharmacologicalCommittee of the Russian Ministry of Health and Social Development in 2000.The purpose of studies with Impaza was to determine the acute toxicity(experiments on mice and rats), chronic toxicity (6�month treatment of rats andrabbits), reproductive and allergic toxicity (experiments on rats),immunotoxicity, mutagenicity (chromosomal aberration assay in mouse bonemarrow cells), and genotoxicity (test system for somatic mosaicism in wing cellsof Drosophila melanogaster).

Impaza had a good safety profile. An acute toxicity study showed thatImpaza in the maximum permissible dose does not cause death of animals.Drug�related death of animals was not observed after 6�month treatment withImpaza in the highest dose. The product had no toxic effect on organs andsystems of organs in experimental animals. A pathomorphological study did not

159

reveal damage to the internal organs or local irritation of the gastric mucosaafter drug administration. Impaza did not produce a damaging or embryotoxiceffect on the reproductive system in male and female rats. Impaza had nomutagenic, allergenic, and immunotoxic properties.

The results of preclinical studies show that Impaza improves copulative(erectile) function, increases sexual motivation, and stimulates sexual behavior

165

170

175

180

185

190

195

Baseline After 2 h

After 5 days

After 10 days

Seven days after

withdrawal

!

MBP, mm Hg

Time after the start of Impaza treatment

Fig. 7.40.Fig. 7.40.Fig. 7.40.Fig. 7.40.Fig. 7.40. Dynamics of systolic BP in ISIAH rats during treatment with Impaza.*p<0.05 compared to the baseline value.

Fig. 7.41.Fig. 7.41.Fig. 7.41.Fig. 7.41.Fig. 7.41. Possible mechanism for the effect of Impaza. NO, nitric oxide; eNO,endothelial NO synthase; GTP, guanosine triphosphate; cGMP, cyclic guanosinemonophosphate.

Impaza

eNOS eNOS

NO

NOGTP Phosphate cGMP

Cell relaxation

Activation of guanylate cyclase

Smooth muscle cell

+ + +

Vascular endothelium


160

Ultralow doses

Fig. 7.42.Fig. 7.42.Fig. 7.42.Fig. 7.42.Fig. 7.42. Preclinical study of specific pharmacological properties of Anaferon andAnaferon for children.

ANAFERON

Antiviral effect Immunomodulatory effect

Models of viral infections In vivo: In vitro:

� humoral response (antibody production)� cellular response (delayed�type hypersensitivity response)� effect on phagocytosis

� T lymphocytes� B lymphocytes� NK cells

� influenza� herpes� genital herpes

Mechanisms of effect (ex vivo)

� effect on the cytokine status (functional activity of type 1 and 2 T helper cells)

� specific effect on IFN�γ induction

of rats. These effects are realized via the NOS – NO guanylate cyclase – cGMPcascade. Impaza had a moderate hypotensive effect on animals with stress�induced arterial hypertension, which was observed after the course of long�termtreatment with this drug. Impaza had no general toxic properties and did notproduce an adverse effect on reproductive function, fertility, and developmentof the offspring. Moreover, Impaza did not possess mutagenic and allergenicproperties.

7.3. Preclinical study of Anaferonand Anaferon for children

The active substances of Anaferon and Anaferon for children are affinitypurified polyclonal antibodies to human interferon�γ (IFN�γ) in ULD.

Experimental studies showed that the course of treatment with oral anti�bodies to IFN�γ in ULD has an antiviral effect on the model of various infec�tions. This effect is related to their ability to stimulate the production of endo�genous IFN�γ and functionally related cytokines. This property also contributesto a wide range of immunomodulatory effects of the product (Fig. 7.42).

161

Antiviral effect of Anaferon

To study antiviral activity of Anaferon, the infectious process was inducedby aerogenic (aerosol) administration of influenza virus strain A/Aichi/2/68(H3N2) in a dose of 50�100 AID50 (aerogenic infectious dose). Anaferon in adaily dose of 0.2 ml was administered intragastrically (through a catheter) tooutbred albino mice. Anaferon was given for 5 days before infection and 5 daysafter infection to study the preventive and therapeutic effects of this drug,respectively. Control mice of the reference group received distilled water. Thepreventive and therapeutic effects of Anaferon were estimated from theconcentration of influenza viruses in the lungs of infected mice (treatment groupand control group) on days 2, 3, 4, and 5 after infection. The presence ofinfluenza viruses in clarified homogenates of the lungs was determined bytitration on 10�day�old developing chick embryos.

The preventive and therapeutic treatment with Anaferon had an antiviraleffect. This conclusion was derived from a decrease in the concentration ofinfluenza viruses in the lungs of animals (by 2.7 times on day 2 of infection; andby 4.6 times on day 4 of infection, respectively) compared to the control group(p<0.05, Fig. 7.43; A. N. Sergeev et al., 2004).

To induce herpes virus infection, outbred albino mice received intraperi�toneal injection of herpes simplex virus type 2 (HSV2; strain MS, ATCC) in adose of 5 LD50.

An aqueous solution of Anaferon was administered intragastrically for 5days before infection (daily treatment). The animals were examined for 14 days.The survival of mice and virus concentration in the brain were estimated on days


Fig. 7.43.Fig. 7.43.Fig. 7.43.Fig. 7.43.Fig. 7.43. Efficacy of Anaferon in mice with experimental influenza: preventive (a)and therapeutic treatment (b). *p<0.05 compared to the control.

3

4

5

6

7

2 3 4 5

!

control Anaferon

4

5

6

7

Time after infection, days

Virus concentration, lg (EID)

Virus concentration, lg (EID)

2

*

*

3 4 5

!

ba

162

Ultralow doses

Fig. 7.44.Fig. 7.44.Fig. 7.44.Fig. 7.44.Fig. 7.44. Efficacy of Anaferon as a prophylactic drug in systemic herpes virusinfection. Survival of mice on day 14 after infection with HSV2 (5 LD

50, a); and HSV2

virus concentration in the brain of mice (b). *p<0.05 compared to the control.

0

*

*

*

20

40

80

60

Survived animals, %

Control Anaferon1

10

100

1000

10000

100000

1000000

2 4 6 9

BFU/mla b

Time after infection, days

controlAnaferon

6 and 9 after infection. Virus concentration in animals of the treatment groupwas much lower than in control specimens. The mortality rate of control andtreated mice was 69.2 and 28.6%, respectively. The average lifespan of died animalsfrom the treatment group was much greater than that of control specimens (by3.3 days). The data indicate that preventive administration of Anaferon has astrong protective effect on mice (p<0.05). This treatment was followed by adecrease in herpes virus concentration in the brain (by 10 times) and increasein the average lifespan of animals (Fig. 7.44; M. A. Susloparov et al., 2004).

The therapeutic efficacy of Anaferon was studied in guinea pigs withexperimental genital herpes infection. The animals were infected with HSV2virus (strain EC). Beginning from the first day after infection, the animalsreceived intragastrically distilled water (5 ml/kg, control), acyclovir (100 mg/kg),or Anaferon (5 ml/kg) twice a day for 15 days. Group 4 guinea pigs were treatedwith Anaferon for 5 days before infection and 15 days after infection. Thegeneral state of animals, local symptoms, and viral titer in vaginal smears wereestimated over 2 months.

The therapeutic and, particularly, therapeutic�and�preventive treatmentwith Anaferon was followed by a significant decrease in the severity and durationof general and local symptoms of herpes infection. Virus elimination alsodecreased under these conditions (Fig. 7.45).

The antiviral effect of ULD anti�IFN�γ on chickens with avian influenzavirus (H5N1, strain A/Chicken/Suzdalka/Nov�11/2005) was studied at the“Vektor” State Research Center for Virology and Biotechnology (Novosibirsk)in 2006�2007.

ULD anti�IFN�γ significantly increased the survival of chickens afterinfection with avian influenza virus in LD75 (p<0.05, Table 7.10). Administra�

163

tion of this product was followed by a significant increase in the survival timeof chickens infected with avian influenza virus in LD90. However, the effect ofULD anti�IFN�γ was less pronounced than that of a reference drug Tamiflu(Fig. 7.46).

These data indicate that the therapeutic, preventive, and therapeutic�and�preventive treatment with Anaferon has an antiviral effect. Hence, the action ofthis product is related to immunomodulatory activity and modulation of the keymechanisms for antiviral protection.

Experimental models for drug efficacy in viral infections reflect functionalactivity of all systems that determine the antiviral resistance of an organism


Fig. 7.45.Fig. 7.45.Fig. 7.45.Fig. 7.45.Fig. 7.45. Efficacy of Anaferon on themodel of experimental genital herpes.Duration of mucosal damage in genitalherpes (a), total score of symptoms (b),and titer of genital herpes virus (c).Control (1); acyclovir (2); Anaferon (3);and preventive and therapeutic treatmentwith Anaferon.

01 2 3 4

1

2

3Days

*

*

01 2 3 4

200

400

600

800

1000

1200Points

*

01 2 3 4

20

40

60

80

100

120% of the control

*

*

а

b

c

Fig. 7.46.Fig. 7.46.Fig. 7.46.Fig. 7.46.Fig. 7.46. Effect of test substances on the survival rate of chickens on day 4 afterinfection with avian influenza virus in LD

90. *p=0.03 and **p=0.01 compared to

the control.

0

*

**

20

40

80% of the baseline

60

Control ULD of antibodies Tamiflu

164

Ultralow doses

Table 7.10.Table 7.10.Table 7.10.Table 7.10.Table 7.10. Effects of ULD anti�IFN�γ on the average lifespan of chickens after infectionwith avian influenza virus in LD

75

Survived chickens, % 25 55*

Average lifespan, days (n=20) 4.5±0.3 4.9±0.3

Note.Note.Note.Note.Note. *p=0.0528 compared to the control.

Parameter ULD anti�IFN�γControl(distilled water)

(interferon system, NK cells, and specific cellular and humoral immunity). Theeffects of Anaferon on these systems were subjected to detailed analysis instudying the immunomodulatory properties.

Studying the immunomodulatory activity of Anaferon

Immunotropic properties of Anaferon and Anaferon for children werestudied in accordance with the Manual on Experimental (Preclinical) Study ofNew Pharmacological Substances (2000).

Experiments were performed on 372 CBA/CaLac mice (318 males and 54females), 25 male F1(CBAґC57Bl/6) mice, and 36 male C57Bl/6 mice (2�2.5months old, 18�20 g), and 120 male and female outbred albino rats (20�23 g).Experiments on females were conducted in studying the delayed type hyper�sensitivity (DTH) reaction and phagocytosis of neutrophils. Other experimentswere conducted on male animals. In vitro experiments were performed with thesuspension of peripheral blood mononuclear cells from 10 healthy donors (22�36 years of age).

To study the immunotropic properties, Anaferon (0.2 ml) was administeredorally for 5 or 10 days. The reference of group of mice was treated with distilledwater (solvent). The control group consisted of intact animals of the same sex.

To study the effect of Anaferon on the humoral immune response, themice were intraperitoneally immunized with sheep erythrocytes (SE) in theminimum dose (5×106/ml). Single immunization was performed at thebeginning of a 5�day course of treatment. Experiments were conducted onhealthy and immunosuppressive CBA mice. To induce immunosuppression,cyclophosphane (CP, Biokhimik) in a single dose of 125 mg/kg (1/2 maximumtolerated dose, MTD) was injected intraperitoneally at the beginning ofAnaferon treatment. The total number of splenocytes (total cellularity of thespleen [TCS], ґ106; E. D. Gol’dberg et al., 1992), relative (%) and absolutenumber (×106) of antibody�producing cells (APC) in the spleen (A. I. Cunning�ham, 1965), and antibody (AB) titer (standard hemagglutination reaction, HAR)were measured on day 5 after immunization.

165

To study the effect of Anaferon on the cellular immune response (DTHreaction), the mice were sensitized with SE (R. V. Petrov et al., 1984). A chal�lenge dose of SE was administered into the hindlimb pad (single subcutaneousinjection) after a 10�day course of Anaferon treatment (5th day after sensiti�zation). An equivalent volume of physiological saline was injected to the controllimb. In a special series with the DTH reaction, some animals of the treatmentand control groups received intraperitoneal injection of a specific NO synthaseinhibitor NG�monomethyl�L�arginine (NMMA) on days 4 and 5 after treatmentwith the sensitizing dose of SE. The reaction index was estimated in eachanimal (relative weight of the SE�injected to control limb, %).

The effect of Anaferon on phagocytic activity of neutrophils andmacrophages in the peritoneal exudate was analyzed after a 10�day course oftreatment with this drug or distilled water. The ability of these cells to phago�cytize a 1�day�old culture of St. aueus was estimated. The following parameterswere evaluated: percent of microbe�engulfing neutrophils or macrophages (pha�gocytic index, PI); and average number of phagocytized staphylococci per cell(phagocytic number, PN).

In in vitro experiments, the aqueous solution of Anaferon was added toa complete nutrient medium (CNM, 50 ml/ml). The in vitro effect of Anaferonon proliferative activity of T lymphocytes and B lymphocytes was studied in thereaction of spontaneous or mitogen�induced blast transformation (LBTR).Induced LBTR was conducted with the T cell mitogen (PHA) or B cell mitogen(pokeweed mitogen) in the suboptimal concentration. Anaferon�free CNMserved as the control. The results were analyzed by the number of pulses perminute and stimulation index (ratio of radioactivity levels in the presence andabsence of mitogen stimulation).

The in vitro effect of Anaferon on production of interleukin�1 (IL�1) orIL�2 was estimated from functional activity of IL�1 and IL�2 in supernatantsof 1�day�old mononuclear cell cultures after incubation with the test substanceand lipopolysaccharide (LPS, induction of IL�1 production) or PHA (inductionof IL�2 production). Control supernatants of 1�day�old mononuclear cellcultures were incubated with LPS, PHA, or test substance. Otherwise, incuba�tion of control samples was performed in CNM. IL�1 activity was determinedfrom comitogenic activity of IL�1 (S. B. Mizel, 1980). The activity of IL�2 wasevaluated from its ability to stimulate proliferation of lymphoblast cells (R. V.Petrov et al., 1984).

The suspension of peripheral blood mononuclear cells served as a sourceof NK cells to study the in vitro effect of Anaferon on cell function. Functionalactivity of NK cells (i.e., ability to lyse tumor cells without presensitization) wasestimated from lysis of myeloblastic K�562 cells in the cytotoxic reaction (B. B.Fuchs radiometric assay; R. V. Petrov et al., 1984).


166

Ultralow doses

Fig. 7.47.Fig. 7.47.Fig. 7.47.Fig. 7.47.Fig. 7.47. Effect of the course of oral Anaferon on the humoral immune responseto SE: production of immune antibodies. Intact animals (1); control (SE, 2); SE +CP (3); SE + Anaferon (4); and SE + CP + Anaferon (4). p<0.05: *compared to thecontrol; +compared to the SE + CP group.

2

0

4

6

10

8*

+

1 2 3 4 5

Antibody titer to SE, log2

The effect of Anaferon on production of IFN�γ, IL�2, IL�4, and IL�10by splenic lymphocytes was studied in mice after a 10�day course of drugtreatment. Lymphocytes were incubated in the absence (spontaneous reaction)or presence of PHA (induced reaction) for 24 h. The content of IFN�γ, IL�2,IL�4, and IL�10 in culture supernatants was measured by enzyme immunoassaywith commercial kits.

Anaferon had a strong immunomodulatory effect. This drug in vivo in�creased the humoral and cellular immune response (course of oral administra�tion) and had a direct stimulatory effect on functional activity of immune cells(A. V. Martyushev�Poklad, 2003).

A 5�day course of treatment with Anaferon contributed to an increase inthe humoral immune response to SE (immunization with the minimum doseof antigen in combination with the first administration of Anaferon). It wasmanifested in an increase in the percentage of APC in the spleen (by 1.7 times)and elevation of hemagglutinin titer in blood plasma (by 1.6 times, p<0.05compared to the control; Fig. 7.47).

The course of Anaferon treatment significantly activated the humoralimmune response to SE in mice with CP�induced immunosuppression (1/2MTD). These mice were characterized by a significant increase in the relativenumber of APC in the spleen (by 2 times) and elevation of specifichemagglutinins in blood plasma (by 3 times) compared to control animals (SEimmunization in cytostatic disease with no drug treatment).

These data show that Anaferon increases the humoral immune responseto a complex corpuscular antigen (e.g., during immunization with the minimumdose of antigen under conditions of cytostatic�induced immunosuppression).Therefore, Anaferon has a positive effect on the regulatory (activation of type

167

2 T helper cells) and/or effector components of the humoral immune response(antibody production).

Studying the DTH reaction to sensitization with SE showed that thecourse of treatment with Anaferon significantly activates the cellular immuneresponse (Fig. 7.48).

In control and Anaferon�receiving specimens, the effector component ofthis reaction was mainly realized via NO�dependent mechanisms. The activationwas abolished after in vivo suppression of NO production by a NO synthaseinhibitor NMMA. The DTH index decreased to 110.5±3.1 and 110.8±3.7%, re�spectively (p<0.05).

The course of treatment with oral Anaferon was accompanied by stimu�lation of the phagocytic immune response in intact mice. The ratio of staphy�lococcus�engulfing neutrophils (phagocytic index) increased from 21.3±0.8 to29.2±3.2% (p<0.05 compared to the control; Fig. 7.49). Phagocytic activity ofperitoneal macrophages significantly increased after administration of Anaferon.The phagocytic index increased from 12.2±0.9 to 19.7±1.1% (p<0.05). However,the phagocytic number remained unchanged under these conditions.

Fig. 7.48.Fig. 7.48.Fig. 7.48.Fig. 7.48.Fig. 7.48. Effect of the course of oral Anaferon on the cellular immune responseto sheep erythrocytes: DTH reaction. *p<0.05 compared to the control.

100

110

120

140

*130

Reaction index, %

Control Anaferon

Fig. 7.49.Fig. 7.49.Fig. 7.49.Fig. 7.49.Fig. 7.49. Effect of the course of Anaferon treatment on phagocytic activity(phagocytic index) of neutrophils (a) and macrophages (b). *p<0.05 compared tothe control.

0

5

10

15

20

25

30

Control Control AnaferonAnaferon

% a b!

!


168

Ultralow doses

Fig. 7.50.Fig. 7.50.Fig. 7.50.Fig. 7.50.Fig. 7.50. Direct in vitro effect of Anaferon on functional activity of NK cells.*p<0.05 compared to the control.

50

55

60

65

70

75

Cytotoxicity index

Control Anaferon

!

These data indicate that the course of oral Anaferon stimulates the pha�gocytic activity of neutrophils and macrophages (another component of the im�mune system), which is related to an increase in the ratio of active phagocytes.

These changes reflect the in vivo effect of Anaferon, which is probablymediated by various regulatory systems of the organism. The next series showedthat Anaferon has a direct effect on immune cells under in vitro conditions.

Addition of Anaferon in combination with the T cell mitogen or B cellmitogen to cultured MNC (induced blast transformation) was followed by anincrease in the stimulation index for T lymphocytes (from 52.9±9.3 to88.6±10.5) and B lymphocytes (from 74.7±32.6 to 120.7±39.7). The observedchanges were statistically insignificant due to a wide scatter of data. However,the drug had no effect on spontaneous blast transformation of lymphocytes.Therefore, addition of Anaferon to the culture of MNC produces a moderatecomitogenic effect on T lymphocytes and B lymphocytes.

IL�1 production in the culture of MNC was much higher after additionof Anaferon and LPS (compared to cell culturing with LPS). The stimulationindex was 20.9±3.1 and 11.5±3.2, respectively. One�day incubation of MNCwith Anaferon also stimulated the production of IL�1 (vs. IL�1 concentrationin supernatants of cell cultures in CNM). However, the effect of Anaferon wasless pronounced than that of the mitogen. During culturing in the presence ofmitogen, the stimulation index increased from 2.7±0.8 to 4.6±1.5.

Anaferon had little effect on in vitro production of IL�2 in the culture ofperipheral blood MNC.

Addition of Anaferon to the culture of MNC was followed by a significantincrease in functional activity of NK cells. It was manifested in an increase inthe cytotoxicity index at a target/effector cell ratio of 1:25 (from 63.6±2.7 to71.7±1.9, p<0.05; Fig. 7.50). Hence, Anaferon increases functional activity ofNK cells that play an important role in the protection from intracellularparasites and tumor cell growth.

169

IFN�γ is one of the major inductors of NO production by effector cellsof the immune system (U. Boehm et al., 1997).

A quantitative study (enzyme immunoassay) was performed to evaluate exvivo production of IFN�γ by splenic lymphocytes from Anaferon�receiving ani�mals. Spontaneous production of IFN�γ by lymphocytes significantly increasedon days 1 and 3�7 after treatment with Anaferon. The observed changes weremost pronounced 3 days after Anaferon administration. IFN�γ production inthis period increased to 116.34±23.71 pg/ml (p<0.001; Fig. 7.51, a, b), whichexceeded the baseline (14.03±1.31 pg/ml, more than 8 times) and control level(17.93±1.75 pg/ml, by 6.5 times).

The increase in PHA�stimulated production of IFN�γ by lymphocytes wasless pronounced. The maximum level was achieved on day 7 after treatment andexceeded the control by 9.5% (2636.22±63.41 and 2407.32±104.59 pg/ml,respectively; p<0.05).

These data show that Anaferon significantly increases spontaneous andmitogen�stimulated ex vivo production of functionally active IFN�γ (keycytokine of type 1 T helper cells) by T lymphocytes from intact animals. IFN�γinduces and regulates the cellular immune response and serves as a componentof the interferon system (autonomic system for antiviral resistance). Oraladministration of Anaferon has a modulatory (inducing) effect on the systemicproduction of endogenous IFN�γ, which confirms the notion that ULD ofantibodies to endogenous regulators have modifying properties.

Spontaneous ex vivo production of IL�2 by lymphocytes from treated miceincreased on days 2�7 after administration of Anaferon. However, statisticallysignificant differences were found only on days 3 and 7. This parameter increasedfrom 42.13±2.92 to 61.07±7.65 pg/ml and from 38.71±2.76 to 56.46±5.70 pg/ml,respectively (p<0.05). The increase in PHA�stimulated production of IFN�2 bylymphocytes was less pronounced under these conditions. The maximum level wasobserved on days 2 (increase from 589.04±33.91 to 689.41±18.34 pg/ml, by 17%),5 (increase from 532.68±22.27 to 684.37±29.89 pg/ml, by 28.6%), and 7 (increasefrom 502.78±41.10 to 649.47±31.97 pg/ml, by 29.3%, p<0.05; Fig. 7.51, c).

Spontaneous production of IL�4 by lymphocytes from experimental animalssignificantly increased on days 7 (from 5.37±0.23 to 16.06±4.10 pg/ml, by 198%)and 10 after administration of Anaferon (from 5.21±0.18 to 7.38±0.80 pg/ml, by41.7%, p<0.05). Anaferon produced the opposite effect on PHA�stimulatedproduction of IL�4 in various periods. The product had a stimulatory effect on days1 (increase from 86.18±2.99 to 121.87±13.92 pg/ml, by 41%), 2 (increase from80.48±0.76 to 111.02±8.49 pg/ml, by 38%), and 4 (increase from 84.27±3.62 to100.67±7.73 pg/ml, by 19%; p<0.05). By contrast, PHA�stimulated production ofIL�4 decreased on days 6 (from 62.74±5.75 to 52.13±5.49 pg/ml, by 17%) and 10(from 52.63±5.77 to 38.50±3.75 pg/ml, by 26.8%; Fig. 7.51, d).


170

Ultralow doses

0

1

2

3

4

5

6

aTreatment/control bTreatment/control

cTreatment/control

eTreatment/control

dTreatment/control

1 2

*

*

*

*

*

* *

*

*

*

*

* **

*

*

*

*

*

3 4 5 6 7 100

0.02

0.04

0.06

0.08

0.10

0.14

0.12

1 2 3 4 5 6 7 10

�0.1

0

0.1

0.2

0.3

0.4

0.5

�0.2

0

�0.4

0.2

0.4

0.6

0.8

1.0

Anaferon administration, days

�0.5

0

0.5

1.0

1.5

2.0

2.5

1 2 3 4 5 6 7 10

1 2 3 4 5 6 7 10

1 2 3 4 5 6 7 10

Fig. 7.51.Fig. 7.51.Fig. 7.51.Fig. 7.51.Fig. 7.51. Effect of the course of oralAnaferon on spontaneous (light bars)and PHA�stimulated ex vivo production(dark bars) of IFN�γ (a, b), IL�2 (c), IL�4(d), and IL�10 by lymphocytes (e). Increa�se, compared to the control.

The course of treatment with Anaferon had no effect on spontaneousproduction of IL�10 by splenic lymphocytes, but caused a significant increasein PHA�stimulated production of this compound on days 1�5 of study (by 40.6�84.5%, p<0.05 compared to the control; Fig. 7.51, e).

171

We conclude that the course of treatment with oral Anaferon has amodulatory effect on the production of not only IFN�γ, but also of functionallyrelated cytokines in type 1 T helper cells (IL�2) and type 2 T helper cells (IL�4and IL�10). These properties contribute to the ability of Anaferon to stimulatethe cellular and humoral immune response.

It should be emphasized that an increase in spontaneous cytokine productionmainly reflects the basal activity of producing cells. The reserve capacity oflymphocytes to produce cellular factors is evaluated from the intensity of sti�mulated cytokine production. Type 1 T helper cells exhibited a spontaneous re�sponse to the test substance. By contrast, the reaction of type 2 T helper cells wasrevealed under conditions of mitogenic stimulation (Fig. 7.52). These differencesillustrate a change in the reserve capacity, but not in the basal activity of cells.

These data indicate that Anaferon possesses the immunotropic properties,stimulates cellular and humoral immunity, has a modulatory effect on the balancebetween regulatory components of the immune system (type 1 and 2 T helpercells), and activates the specific (antibody production and cellular cytotoxicity) andnonspecific immune mechanisms (phagocytosis, NK cells, and interferon system).A strong effect of Anaferon on the IFN system during therapy of acuterespiratory viral infections was confirmed in further clinical studies of this drug.

Studying the toxicity of ULD anti�IFN�γ

A preclinical safety study of Anaferon and Anaferon for children wasperformed in accordance with the recommendations given in the Manual onExperimental (Preclinical) Study of New Pharmacological Substances in 2000.

Fig. 7.52.Fig. 7.52.Fig. 7.52.Fig. 7.52.Fig. 7.52. Effect of the course of oral Anaferon on an increase in spontaneouscytokine production (a) by type 1 T helper cells (light bars) and PHA�stimulatedcytokine production (b) by type 2 T helper cells (dark bars).

0

0.2

0.4

0.6

0.8

1.0

1.4

1.2

�0.2

1 2 3 4 5 7 106�0.4

Anaferon administration, daysIFN+IL�2 IL�4+IL�10

0

1

2

3

a b

4

5

7

6

�1

Treatment/control Treatment/control

1 2 3 4 5 7 106


172

Ultralow doses

The study was designed to evaluate acute toxicity, general chronic toxicity,allergenic properties, genotoxicity, embryotoxicity, reproductive toxicity, andteratogenicity.

Test products were classified to a group of low�hazard substances. Theyhad no general toxic and allergic effects (systemic or local) and did not causereproductive disorders. Antimutagenic properties of ULD anti�IFN�γ were re�vealed in the test for somatic mosaicism in wing cells of Drosophila melanogaster(O. L. Voronova et al., 2002).

Some common effects of ULD antibodiesto endogenous regulators by the exampleof antibodies to IFN�γ in ULD

This experiment was designed to solve two problems. First, we studied thespecificity of the pharmacological effect produced by ULD antibodies to acertain endogenous regulator. And second, we evaluated whether the effect ofantibodies depends on their dose.

The induction of endogenous IFN�γ after oral administration of ULDanti�IFN�γ was estimated ex vivo with the supernatant of splenocytes (O. I.Epstein et al., 2004).

Experiments were performed on 342 CBA/CaLac mice weighing 18�20 g.The study was conducted with goat polyclonal antibodies to human IFN�γ (IgGfraction) and rabbit polyclonal antibodies to erythropoietin (EP) and humantumor necrosis factor�α (TNF�α). ULD antibodies were obtained by thestandard homeopathic method of potentiation. ULD anti�IFN�γ were admini�stered in the molar (dilution C3; equivalent concentration 10�6 wt %; 10�12 M)or submolar dose (mixture of dilutions C12+C30+C50; equivalent concentrationsof 10�24, 10�60, and 10�100 wt %; 10�30 M). ULD antibodies to EP and TNF�αwere used in a mixture of dilutions equivalent to concentrations of 10�24, 10�60,and 10�100 wt % (10�30 M). Test substances (0.2 ml) were given orally for 10days. Control mice received an equivalent volume of the solvent (distilled water).The reference group consisted of intact animals.

The ability of substances to modulate IFN�γ production by lymphocytesfrom experimental animals was studied as described above. IFN�γ concentrationin culture supernatants was measured by enzyme immunoassay with AmershamPharmacia Biotech kits.

Antibodies to an endogenous regulator IFN�γ (oral administration) werehighly potent in inducing the production of this substance (Fig. 7.53). Anti�bodies to IFN�γ in molar and submolar doses had the same effect.

Preclinical studies showed that Anaferon has the immunomodulatory andantiviral properties that are related to the induction of IFN�γ and activation of

173

the key immune mechanisms. The product demonstrated a good safety profile.Moreover, ULD antibodies to IFN�γ produced a specific effect.

7.4. Preclinical study of Artrofoon

The active substances of Artrofoon are antibodies to human TNF�α inULD. Pharmacological activity of Artrofoon was estimated in a large�scale expe�rimental study (Fig. 7.54). Artrofoon not only had a strong antiinflammatory effecton the model of experimental arthritis, but also possessed the analgetic properties.Studying the mechanisms for action of Artrofoon revealed that this product affectsthe system of proinflammatory and antiinflammatory cytokines. A good safety


Fig. 7.53.Fig. 7.53.Fig. 7.53.Fig. 7.53.Fig. 7.53. Specificity and dose dependence of the pharmacological effect of ULDantibodies to an endogenous regulator (antibodies to IFN�γ): spontaneousproduction of IFN�γ by splenocytes after administration of ULD anti�IFN�γ (molarand submolar dilutions, 1�2) and ULD antibodies to erythropoietin (3) and TNF�α(4). *p<0.05 compared to the control.

100

01 2 3 4

200

**

300

500

400

IFN�γ production, % of the control

Fig. 7.54.Fig. 7.54.Fig. 7.54.Fig. 7.54.Fig. 7.54. Preclinical study for the range of pharmacological activity of Artrofoon.

Antiinflammatory activity

Modulation of cytokine production

Analgetic activity

Acetic acid�induced writhing test

Hot�plate test

Adjuvant�induced arthritis (immune

inflammation)

Collagen�induced arthritis (immune

inflammation)

ARTROFOON

174

Ultralow doses

profile of Artrofoon was demonstrated in a toxicology study. The effect of Artrofoonon tumor growth and dissemination was evaluated on various tumor models.

Antiinflammatory activity of Artrofoon

Experiments were performed on the following two models of immuneinflammation: adjuvant�induced arthritis and collagen�induced arthritis (CIA).

Antiinflammatory activity of Artrofoon on the model of adjuvant�inducedarthritis. Experimental adjuvant arthritis is extensively used to study theantiinflammatory effect of pharmaceutical substances in Russia (Manual onExperimental (Preclinical) Study of New Pharmacological Substances, 2000,2005) and other countries (A. Bendele, 1999; F. A. J. Van de Loo, 2004). In thepresent study, immune inflammation was induced by subplantar injection ofcomplete Freund’s adjuvant (ICN) in a single dose of 0.1 ml. Experiments wereperformed on male outbred albino rats weighing 180�200 g. Artrofoon (2.5ml/kg) and reference drug indomethacin (5 mg/kg) were administered 1 daybefore injection of complete Freund’s adjuvant and over the whole period ofinflammation. The severity of edema was evaluated for 21 days at 2�day inter�vals. The measurements were performed using a plethysmograph (evaluation ofthe volume of fluid displaced by the submerged limb).

Antiinflammatory activity of Artrofoon compared well with that ofindomethacin (O. I. Epstein et al., 2001a). The effect was most pronounced onday 10 after drug treatment. The severity of edema decreased by 43.5 and 55%,respectively, compared to the control (p<0.05). No significant differences werefound in the effect of Artrofoon and indomethacin.

Antiinflammatory activity of Artrofoon on the model of collagen�inducedarthritis. CIA serves as an experimental model of rheumatoid arthritis (RA), whichis extensively used to study the effect of antirheumatic drugs (F. Kato et al., 1996;A. Bendele et al., 1999; A. C. Tellander et al., 2000). CIA is progressive auto�immune inflammation of the joints. CIA is a suitable model to study new antirheu�matic drugs for the early stage of disease. In the present work, CIA was studied onmale Wistar rats (D. E. Trentham et al., 1977). CIA was induced by twofold sub�cutaneous injection of rat collagen type II (100 mg) in 100 ml incomplete Freund’sadjuvant. This solution was injected into the tail at a 7�day interval. Experimentswere performed in accordance with the manual “Successful Induction of Collagen�Induced Arthritis in Rats” (Chondrex Company). Forty�five animals of the treat�ment group received intragastrically Artrofoon (0.5 ml) for 90 days beginning fromthe 8th day of study (i.e., next day after repeated injection of collagen). Distilledwater (solvent) was given to 45 control rats according to the same regimen.

The inflammatory response in rats was evaluated three times a week ondays 8�97 of study (i.e., days 1�90 of treatment with Artrofoon or distilled

175

water). The following parameters were studied: onset of arthritis; number ofanimals with arthritis; and count of arthritic joints in each rat. The degree ofdamage was expressed in points (0�4 points for one limb; and 0�16 points,overall severity for four limbs).

Artrofoon had a strong antiinflammatory effect during type II collagen�induced immune inflammation (similarly to the previous series). The course oftreatment with Artrofoon was followed by the reduction of joint inflammationin rats with CIA. It was expressed in a decrease in the severity of joint injury(Fig. 7.55), number of animals with arthritis, and count of arthritic joints.

Analgetic activity of Artrofoon

Nonsteroid antiinflammatory drugs (NAID), including Artrofoon,produce the antiinflammatory and analgetic effects. Analgetic activity ofArtrofoon was studied in the acetic acid�induced writhing test and hot�plate test.

Analgetic activity of Artrofoon in the acetic acid�induced writhing test.Acetic acid�induced writhing was studied on male outbred mice weighing22�25 g. The animals received intraperitoneal injection of 0.75% acetic acid(0.1 ml/10 g). Experimental mice were divided into three groups of 10specimens each. Distilled water (2.5 ml/kg), Artrofoon (2.5 ml/kg), or indo�methacin (5 mk/kg) was administered intragastrically through a probe for5 days (last treatment 1 h before injection of acetic acid). The number ofwrithing episodes and LC of writhing were estimated for each animal over15 min after acetic acid injection.

The analgetic effect of Artrofoon was manifested in a significant decreasein the incidence of writhing episodes compared to the control (by 29%). Theefficacy of Artrofoon was comparable with that of indomethacin. Indomethacindecreased the number of writhing episodes by 44.2% compared to the control(p<0.05, Fig. 7.56; O. I. Epstein, 2001a).

Fig. 7.55.Fig. 7.55.Fig. 7.55.Fig. 7.55.Fig. 7.55. Effect of Artrofoon on the severity of inflammation in rats with collagen�induced arthritis. *p<0.05 compared to the control.

0

0.5

1.01.5

2.0

2.5

3.0

1 3 6 8 10 13 15 17 20 22 24 27 29 31 34 36 38 41 43 45 48 50 52 55 57 59 62 64 66 69 71 73 76 78 80 83 85 87 90

Time after the start of therapy, days

Points

control

Artrofoon

! !

!

!!

!!

!


176

Ultralow doses

Fig. 7.57.Fig. 7.57.Fig. 7.57.Fig. 7.57.Fig. 7.57. Analgetic activity of Artrofoon in the hot�plate test. *p<0.05 comparedto the control.

Analgetic activity of Artrofoon in the hot�plate test. The hot�plate test wasperformed on male outbred rats weighing 180�200 g. The animals were dividedinto three groups of 10 specimens each. The rats received intragastrically 2.5 ml/kg distilled water (group 1), 2.5 ml/kg Artrofoon (group 2), or 5 mg/kgindomethacin (reference drug, group 3) for 5 days (daily treatment). In�flammation of the right hindlimb was induced by subplantar injection of 0.1 mlcomplete Freund’s adjuvant on day 2 of treatment with test substances. Test sub�stances were administered 2 h after the induction of inflammation. Each animalwas placed on a hot plate (64oC) 3 h and 1 or 3 days after injection of Freund’sadjuvant. The analgetic effect was evaluated from the time of staying on a hotplate. LC of paw licking was recorded after the placement of rats to a hot plate.

The nociceptive threshold decreased by 3.7 and 3 times on days 1 and 3after administration of Artrofoon (p<0.05 compared to the control). The effectof Artrofoon persisted for a longer time compared to that of indomethacin.Indomethacin significantly decreased the nociceptive threshold in rats only onday 1 after treatment (Fig. 7.57; O. I. Epstein, 2001a).

Fig. 7.56.Fig. 7.56.Fig. 7.56.Fig. 7.56.Fig. 7.56. Analgetic activity of Artrofoon on the model of acetic�acid�inducedwrithing. *p<0.05 compared to the control.

0

10

20

30

40

50

60

Control Indomethacin Artrofoon

Number of writhing episodes

!

!

10

0

20

30

50

40

3 hTime after injection of complete Freund’s adjuvant

1 day 3 days

LC, seccontrolindomethacinArtrofoon

!

!

!

177

These data show that Artrofoon and typical NAID indomethacin have asimilar effect on two models for studying the analgetic properties of pharma�ceutical substances.

Immunotropic effect of Artrofoon

Immune inflammatory diseases, including RA and systemic lupuserythematosus, are associated with immune dysregulation (C. D. Hamilton,2005). RA is characterized by the prevalence of type 1 T helper cells and over�production of proinflammatory cytokines IL�1 and TNF�α. The antiinflam�matory and analgetic properties of some drugs (glucocorticosteroids, GCS; andmonoclonal antibodies to TNF�α, Infliximab) are related to their influence onthe production of proinflammatory and antiinflammatory cytokines (e.g.,inhibition of TNF�α and IL�1; C. D. Hamilton, 2005). TNF�α can induce theproduction of IL�1, IL�6, and IL�8. Moreover, TNF�α modulates the secretionof IFN�γ, IL�4, IL�10, and other cytokines. Hence, studying the effect ofArtrofoon (ULD antibodies to TNF�α as an active substance) on the cytokineprofile of animals with immune inflammation was necessary to evaluate themechanism for action of this product. Prednisolone that belongs to a group ofGCS was used as a reference drug.

Experiments were performed on 200 male CBA/CalAc mice with CIA(18�20 g). Experimental animals were divided into three groups. The micereceived orally 0.2 ml distilled water (14�day course, from the day before CIAinduction; control group 1), 0.2 ml Artrofoon (14�day course, from the daybefore CIA induction; group 2), or 53 mg/kg prednisolone (Nikomed; 11�daycourse, from the day before CIA induction; group 3).

CIA was induced by subplantar injection of type II collagen (single dose100 mg) in 50 ml complete Freund’s adjuvant. This solution was injected intothe right hindlimb of mice. The concentrations of proinflammatory andantiinflammatory cytokines (TNF�α, IL�1, IL�6, IFN�γ, IL�4, and IL�10) inblood plasma and supernatants of peritoneal macrophages (TNF�α, IL�1, andIL�6) and lymphocytes (IFN�γ, IL�4, and IL�10) were measured after 3 h and1, 3, 5, 9, 13, 17, and 21 days. The overall and mean production of cytokineswas estimated by calculating the area under the concentration�time curve.

Artrofoon had the antiinflammatory and immunomodulatory effect onmice with CIA and immune inflammation. The activity of Artrofoon comparedwell with that of a reference drug prednisolone. The inflammatory index inArtrofoon�receiving animals decreased by 10�27% on days 1�13 of inflammation(p<0.05 compared to the control). Administration of prednisolone was followedby a 36�58% decrease in the inflammatory index on days 1�9 of inflammation(p<0.05 compared to the control).


178

Ultralow doses

Prednisolone and Artrofoon produced the same changes in systemicproduction of cytokines (blood cytokine level; Fig. 7.58).

These data show that Atrtrofoon has the antiinflammatory properties.Similarly to prednisolone, the effect of Artrofoon is related to the inhibition ofproinflammatory cytokine production.

Safety profile of Artrofoon

The antiinflammatory and analgetic effects of Artrofoon compared wellwith those of typical nonsteroid antiinflammatory drugs (indomethacin) andglucocorticoids (prednisolone). Artrofoon reduces the symptoms of experimentalimmune inflammation. Similarly to prednisolone, Artrofoon has a modulatoryeffect on cytokine production. However, NAID and GCS cause some sideeffects. NAID contribute to gastropathy, allergy, and elevation of BP. Treatmentwith GCS is followed by gastropathy, hyperglycemia, and other disorders.Modern pharmaceuticals that affect TNF�α (biological inhibitors of TNF�α,including Infliximab, Etanercept, and Adalimumab) are potent in the therapyof autoimmune diseases, but cause serious side effects. Drug�induced immunesuppression increases the risk of tuberculosis, meningitis, fungal diseases,histoplasmosis, and sepsis (C. D. Hamilton, 2005).

A complete toxicology study was performed to evaluate the safety profile,possible side effects, target organs, and safe dosage range of Artrofoon. Thepurpose of studies with Artrofoon was to determine the acute toxicity

Fig. 7.58.Fig. 7.58.Fig. 7.58.Fig. 7.58.Fig. 7.58. Effect of Artrofoon of systemic cytokine production on days 1�21 ofexperimental arthritis. Ordinate: average concentration of cytokines. *p<0.05compared to the control.

0

20

40

60

80

100

120

140

160

180

200

TNF�α IL�1 IL�6 IFN�γ IL�4 IL�10

% of the control

control Artrofoon prednisolone

!!

!

!

!

!

!

!

!!

!

179

(experiments on mice and rats), chronic toxicity (6�month treatment of rats andrabbits), reproductive and allergic toxicity (experiments on rats), immunoto�xicity, mutagenicity (chromosomal aberration assay in mouse bone marrowcells), and genotoxicity (test system for somatic mosaicism in wing cells ofDrosophila melanogaster).

Artrofoon appears to have a good safety profile. An acute toxicity studyshowed that this substance in the maximum permissible dose does not causedeath of animals. Drug�related death of animals was not observed after 6�monthtreatment with Artrofoon in the highest dose. The product had no toxic effecton organs and functional systems of experimental animals. A pathomorphologi�cal study did not reveal damage to the internal organs or local irritation of thegastric mucosa after drug administration. Artrofoon did not cause reproductivedisorders in male and female rats. The embryotoxic effect of Artrofoon was notobserved. Artrofoon had no mutagenic, allergenic, and immunotoxic properties.

Despite good results of a toxicology study, it was important to evaluate theeffect of Artrofoon on the cytokine system (e.g., TNF�α). TNF�α holds muchpromise for oncology. This cytokine possesses antiblastic activity and has a directcytotoxic effect on malignant cells. This effect is associated with hemorrhagicnecrosis of the tumor and activation of the immune system (S. M. Navashin etal., 1989; S. G. Zubkova et al., 2001; R. Horssen et al., 2006). Multicenterclinical trials confirmed the efficacy of TNF�α infusion for therapy of sometumors (R. Horssen, 2006). However, TNF�α may serve as a tumor�inducingagent. The increased activity of TNF�α provides favorable conditions for tumorcell dissemination, including stimulation of angiogenesis (N. Ahmaazadeh et al.,1990; D. Bertolini et al., 1986; F. Brennan et al., 1989; F. Brennan et al., 1997;P. Cunha et al., 1992; J. Dayer et al., 1985; C.A. Dinareilo et al., 1986;M. Feldman et al., 1986; C. Hawonh et al., 1991; E. Lupia et al., 1996;K. Macnaul et al., 1992; M. Shingu et al., 1993; G. Tilz et al., 1997). A TNF�αinhibitor Infliximab increases the risk of tumor development (P. W. Szlosarek etal., 2006; L. Biancone et al., 2005).

A special series was performed to determine the antitumor activity ofArtrofoon. It was interesting to evaluate whether Artrofoon may contribute totumor progression.

Experiments on tumor models (Lewis lung carcinoma and B�17 mela�noma) showed that Artrofoon has no antitumor activity. The drug did notstimulate primary tumor growth. Moreover, Artrofon had no stimulatory effecton the development and growth of metastases. By contrast, Artrofoon exhibitedthe antitumor and antimetastatic properties under conditions of tumortransplantation with a small number of cells (E. N. Amosova et al., 2001).

This series showed that Artrofoon does not have an adverse effect ontumor growth, but exhibits the antitumor activity.


180

Ultralow doses

Preclinical studies showed that the antiinflammatory and analgetic effectsof Artrofoon compare well with those of GCS and NAID. These properties ofArtrofoon are related to its influence on the cytokine system. Toxicity was notobserved after long�term treatment with Artrofoon in doses that exceed therecommended human dose by more than 1000 times. Artrofoon does not havethe mutagenic, allergenic, immunotoxic, or tumorigenic properties. The resultsof experimental studies were confirmed by clinical observations.

7.5. Preclinical study of Epigam

Epigam consists of affinity purified polyclonal antibodies to histamine inULD.

Studying the antiulcer activity of pharmaceutical products requires a stageof investigations on animals with experimental ulcers. The existence of variousetiopathogenetic factors for ulcer disease and no general agreement concerningthe cause, pathogenetic mechanism, and course of this disorder make it difficultto develop a general model for all manifestations of the pathological processes.Antiulcer activity of drugs is usually studied on several models of ulcer diseasethat differ in etiology and specific characteristics. The antiulcer effect of Epigamwas evaluated on several models of acute and chronic ulcers. Studying thespecific activity of an antiulcer drug suggests the evaluation of its effect onsecretory and motor�evacuation function of the gastrointestinal tract (GIT).Moreover, these experiments were designed to determine the analgetic,spasmolytic, anti�edematous, and antitumor effects of Epigam (Fig. 7.59).

Experiments were performed on 215 outbred albino rats (males, 300�350g; and females, 220�240 g) and 390 outbred mice (females, 22�24 g). Theanimals were obtained from the Laboratory of Experimental BiologicalModeling (Institute of Pharmacology, Tomsk Research Center, Siberian Divisionof the Russian Academy of Medical Sciences).

Antiulcer activity of Epigam

Antiulcer activity of Epigam on the model of acute ulcers. The effect ofEpigam on acute ulcerative lesion of the gastric mucosa was studied on animalmodels for various etiologic factors of ulcer disease, including stress (neurogeniculcer), NAID�induced gastric mucosal injury (indomethacin, acetylsalicylicacid, butadiene), alcohol consumption, and acid�pepsin factor (Shay ulcer).

On the model of acute ulcers, Epigam was administered intragastricallyto mice (0.3 ml) and rats (0.5 ml). The product was given once or several timesfor 5�8 days (last treatment 1 h before the induction of ulcerative lesions).

181

Control animals received distilled water (solvent) according to the sameregimen.

The animals were killed by cervical dislocation under ether anesthesia orether overdose. The number and area of gastric mucosal injury were estimatedmacroscopically. They were differentiated into petechia (less than 1 mm), largeulcers (more than 1 mm), and linear lesions. The average number of ulcerativelesions in one animal and percentage of mice with ulcers were evaluated asdescribed elsewhere (Ya. I. Khadzhai, 1962). The Pauls index was determinedas an integral criterion for the number of lesions (F. Pauls et al., 1947). Antiulceractivity of Epigam was estimated as the control/treatment ratio of the Paulsindex (G. V. Obolentseva et al., 1974).

Neurogenic ulcer. Stress is one of the major etiologic factors for GITdiseases. Immobilization is a strong stress factor, which causes ulceration in GIT.Stress�induced ulceration during immobilization is associated with nervous andhumoral changes (I. S. Zavodskaya et al., 1981). The animals were suspendedby application of dressing forceps to the skinfold of the neck. Partial immo�bilization for 22 h was followed by ulceration of the gastric mucosa (Yu. I.Dobryakov, 1978). The bedding and food were removed from mouse cages 1 day

Fig. 7.59.Fig. 7.59.Fig. 7.59.Fig. 7.59.Fig. 7.59. Studying the pharmacological properties of Epigam

Antiinflammatory activity

Antiulcer activity

Anti�edematous

Analgetic activity

Antiproliferative

Acute ulcers

Chronic ulcer

Effect on functional activity of the gastrointestinal tract

Spasmolytic Secretory Motor�evacuation

EPIGAM


182

Ultralow doses

before stress. The antistress effect of test substances was estimated from thenumber of gastric ulcers and weights of the adrenal glands, thymus, and spleen.

Preventive treatment with Epigam for 6 days had a strong gastroprotectiveeffect under conditions of neurogenic ulceration. The antiulcer activity ofEpigam was 3.52 U. Epigam decreased the number of petechia and large ulcersby 3.5 and 5.5 times, respectively, compared to the control (p<0.05). The aver�age number of ulcers in animals of the Epigam group was 3.8�fold lower thanin the control (p<0.05, Fig. 7.60; J. L. Dugina et al., 2002; 2003a,b,c; S. G.Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

It should be emphasized that single administration of Epigam preventedthe stress�induced involution of the spleen and thymus and had a normalizingeffect on the weight of the adrenal glands (no differences from the intactcontrol).

Indomethacin�induced ulcer. Mucosal damage in GIT is one of the mostcommon side effects of NAID (e.g., indomethacin). Ulcerogenic activity ofthese drugs is associated with barrier dysfunction of the mucous membrane,inhibition of glycosaminoglycan synthesis, blood flow disorders, impaired abilityof the mucosa for reepithelization, and increased secretion of acid and pepsin(F. Bates, 1989). The gastroprotective effect of Epigam on the model of indo�methacin�induced mucosal injury was studied with two species of animals. Indo�methacin�induced damage to the gastric mucosa in mice was produced byintragastric administration of indomethacin in a dose of 20 mg/kg twice dailyat a 4�h interval. The number and severity of destructive changes were estimatedafter 18 h (O. I. Epstein et al., 1001b). Indomethacin�induced damage to thegastric mucosa in rats was produced by intragastric administration ofindomethacin in 1 ml physiological saline (single dose 60 mg/kg; F. Bates et al.,1989). The number of ulcers was evaluated 6 h after the last treatment withindomethacin.

Fig. 7.60.Fig. 7.60.Fig. 7.60.Fig. 7.60.Fig. 7.60. Antiulcer activity of Epigam after prophylactic intragastric administrationto female mice. Neurogenic ulcer (0.3 ml × 1 day, 1); neurogenic ulcer (0.3 ml × 6days, 2); and indomethacin�induced damage (0.3 ml × 6 days, 3). *p<0.05 and**p<0.01 compared to the control.

0

2

4

6

**

8

10Average number of ulcers per mouse

1 2 3

control Epigam

183

After preventive treatment with Epigam for 6 days, 25% mice did not havegastric ulcers (p<0.01). The average number of ulcers in treated mice decreasedby 2.4 times (p<0.05; Fig. 7.61). Epigam also prevented the formation of largeulcers and petechia. The average number of these lesions in mice of the Epigamgroup decreased by 5.2 and 2.0 times, respectively (p<0.05). Moreover, prophy�lactic administration of Epigam completely abolished the formation of linearulcers. Under these conditions, the antiulcer activity of Epigam was 3.22 U.

Prophylactic administration of Epigam decreased the average number ofulcers in rats with indomethacin�induced damage to the gastric mucosa (by 1.33times, p<0.01; Fig. 7.60). The length of petechia, linear lesions, and large ulcersdecreased by 1.4, 1.5, and 1.7 times (p<0.05), respectively, compared to the con�trol. Fig. 7.62 illustrates that treatment with Epigam was followed by a 1.5�folddecrease in the severity of gastric mucosal injury (p<0.05; J. L. Dugina et al., 2002,2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

Fig. 7.61.Fig. 7.61.Fig. 7.61.Fig. 7.61.Fig. 7.61. Antiulcer activity of Epigam after prophylactic intragastric administrationto rats. Indomethacin�induced damage (0.5 ml × 5 days, 1); acetylsalicylic acid�induced damage (0.5 ml × 7 days, 2); butadiene�induced damage (0.5 ml × 7 days,3); ethanol�induced damage (0.5 ml × 8 days, 4); and Shay ulcer (0.5 ml × 7 days,5). *p<0.05 and **p<0.01 compared to the control.

0

5

10

15

20

25

Average number of ulcers per rat

1 2 3 4 5

control Epigam

**

*

**

**


Fig. 7.62.Fig. 7.62.Fig. 7.62.Fig. 7.62.Fig. 7.62. Severity of gastric mucosal injury in female rats after prophylactictreatment with Epigam. Indomethacin�induced damage (0.5 ml × 5 days, 1); buta�diene�induced damage (0.5 ml × 7 days, 2); and ethanol�induced damage (0.5 ml× 8 days, 3). *p<0.05 and **p<0.01 compared to the control.

0

15

30

45

60

75

mm

1 2 3

control treatment

*

**

184

Ultralow doses

Acetylsalicylic acid�induced ulcer. Gastric ulcer in rats was induced by two�fold intragastric administration of acetylsalicylic acid in a dose of 150 mg/kg ata 4�h interval. The animals were examined after 24 h. A direct effect ofacetylsalicylic acid on the gastric mucosa probably results in the loss of pro�tective and barrier properties, desquamation of the epithelium, and formationof a large area of erosions and ulcers.

Petechia, linear ulcers, and large ulcers were revealed in all rats of thecontrol group 24 h after ulcerogenic treatment with acetylsalicylic acid. Theaverage number of ulcers was 15.31±1.89. Prophylactic treatment with Epigamwas followed by a decrease in the number of petechia (by 1.5 times, p<0.05) andaverage number of gastric lesions per rat (by 1.7 times, p<0.05 compared to thecontrol; Fig. 7.61). The gastroprotective effect of Epigam was manifested in adecrease in the incidence of linear ulcers (by 4.5 times, p<0.05) and number ofanimals with this type of gastric lesions. Under these conditions, the antiulceractivity of Epigam was 1.84 U (J. L. Dugina et al., 2002, 2003a,b,c; S. G.Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

Ethanol�induced ulcer. Ethanol�induced gastric mucosal injury in rats wasproduced by single intragastric administration of 1 ml 96o ethanol per 200 gbody weight (C. F. Bou�Abbound et al., 1988). The antiulcer effect was eva�luated 1 h after ethanol administration.

Gastric lesions were found in 100% animals of the control group afterethanol administration. The average number of ulcers was 14.00±0.95. Epigamsignificantly decreased the area and severity of gastric mucosal injury. Thenumber of petechia, linear ulcers, and large ulcers in Epigam�receiving animalsdecreased by 40.6, 72.1, and 39%, respectively, compared to control specimens.The average number of ulcers in rats of the Epigam group was 1.9�fold lower thanin control animals (p<0.01). The antiulcer activity of Epigam was 2.01 U (Fig.7.61). The total length of mucosal lesions per rat decreased by 5.8 times (p<0.05).The severity of damage decreased by 82.5% (p<0.05, Fig. 7.60; J. L. Dugina et al.,2002, 2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

Butadiene�induced ulcer. The rats received intramuscular injection ofbutadiene in a single dose of 300 mg/kg (15% suspension in acetone). Theseverity of ulceration was estimated after 24 h.

Butadiene�induced ulceration of the gastric mucosa was found in controland treated animals. Epigam had an antiulcer effect on the model of butadiene�induced ulcer. The number of large ulcers and average number of ulcers in ratsof the Epigam group decreased by 1.8 and 1.4 times, respectively, compared tothe control (p<0.05). The severity of gastric mucosal injury in Epigam�receivinganimals decreased by 1.2 times (Fig. 7.62).

Shay ulcer. Laparotomy was performed along the white line of the anteriorabdominal wall under light ether anesthesia. The pylorus was ligated (H. Shay

185

et al., 1945). The tissues were sutured in layers. The animals were killed by etheroverdose after 16 h. The forceps were applied to the esophagus. The stomachwas removed. The gastric mucosa was examined under a microscope.

Administration of Epigam was followed by a significant decrease in thenumber of animals with ulcers and average number of ulcers per rat. Theaverage number of large ulcers in 75% control rats was 2.1±0.64. After treatmentwith Epigam only 57% animals had large ulcers. The average number of largeulcers in Epigam�receiving rats decreased to 1.29±0.61 (p<0.05). The numberof rats with petechia decreased from 63 to 14% (p<0.05). Moreover, linear ulcerswere not detected after administration of Epigam. The average number of ulcersin rats of the Epigam group decreased by 2.4 times (p<0.05; Fig. 7.61). Epigamwas potent in reducing the severity of gastric mucosal injury. The area of largeulcers and petechia decreased by 2.3 and 7.3 times, respectively (p<0.05). Theaverage area of gastric lesions decreased by 2.5 times (p<0.05). Hence, theseverity of injury in treated animals decreased by 59.3% (p<0.05). The antiulceractivity of Epigam was 3.31 U.

These data show that Epigam has a strong antiulcer effect on various mo�del of acute ulcerative lesion, which does not depend on the etiology of pa�thological process. This effect is manifested the prevention of ulceration and sig�nificant decrease in the severity of gastric mucosal injury (J. L. Dugina et al., 2002,2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

Antiulcer activity of Epigam on the model of chronic ulcer. Acid acetic�induced chronic ulcer was produced in rats (S. I. Budantseva, 1973; A. A.Kalinichenko, 1973). Laparotomy was performed along the white line of theanterior abdominal wall under light ether anesthesia. A solution of acetic acid(0.05 ml, 5%) was administered into the subserous layer of the anterior wall ofthe stomach. Epigam in a daily dose of 0.5 ml was administered intragastricallyfor 21 days (immediately after the induction of ulceration). Control rats receiveddistilled water (solvent) according to the same regimen. The animals were killedby ether overdose on days 7, 14, and 21. The antiulcer effect of Epigam wasestimated from the area of ulcerative lesions. For a morphological study, thestomach was fixed in formalin and embedded into paraffin. Connective tissuein deparaffinized sections (5 m in width) was stained with hematoxylin andeosin by the Van Gieson technique. Acid glycosaminoglycans were stained bySchiff reagent. RNA was stained by the method of Brachet with methyl greenpyronin (G. A. Merkulov, 1969). During examination of Brachet�stainedsamples, tissue basophils were counted in the ulcer margin (per 1 mm2 section).By the number of granules, they were differentiated into strongly granulated,partially degranulated, and strongly degranulated cells.

The effect of Epigam on rats with acetic acid�induced chronic gastriculcers was evaluated on days 7, 14, and 21. Mucosal ulcers of the stomach were


186

Ultralow doses

Fig. 7.63.Fig. 7.63.Fig. 7.63.Fig. 7.63.Fig. 7.63. Effect of Epigam on healing of acetic acid�induced chronic gastric ulcerin male rats. Area of ulcer�and�swelling (a) and bottom of the ulcer (b). *p<0.05and **p<0.01 compared to the control.

0

6

12

18

24

30mm2

7 14 210

a b

20

40

60

80

120mm2

7 14 21

Time, days

100

control Epigam

** *

*

detected macroscopically in all animals of the control group on day 7 afterulcerogenic treatment. The depth of ulcers was 1�2 mm. The area of the bottomwas 23.52±3.38 mm2. A swollen area around the ulcer was associated withinflammatory infiltration and edema of the mucous membrane (Fig. 7.63). Theaverage size of ulcer�and�swelling in control animals was 98.04±15.04 mm2.Ulcer size in control specimens remained practically unchanged on day 14. Theareas of ulcer�and�swelling and bottom decreased by 21.8 and 20.4%,respectively. Healing of ulcerative lesions in control animals was observed onlyon the 21st day. In this period the area of ulcer�and�swelling and ulcer bottomdecreased by 44.4 and 75.7%, respectively (compared to day 7). The size ofulcerative lesions in Epigam�receiving rats was much lower than in control animals(Fig. 7.63). On day 7 the area of ulcer�and�swelling and ulcer bottom decreasedby 14.6 and 19.9%, respectively, compared to the control. It should be emphasizedthat ulcer healing in Epigam�receiving rats was revealed on day 14 after the induc�tion of ulceration. The area of ulcer�and�swelling and ulcer bottom decreasedby 61.2 (p<0.05) and 75.4% (p<0.01), respectively, compared to the control.

The degree of gastric ulcer healing in Epigam�receiving rats was muchhigher than in the control (J. L. Dugina, 2002, 2003a,b,c; S. G. Krylova, 2002a;O. I. Epstein, 2001b; J. L. Dugina, 2002). An ulcerative lesion was filled withgranulation tissue. The tissue had a greater degree of maturity, included asmaller number of cells, and consisted of thick collagen fibers. The content ofRNA and glycosaminoglycans in the cytoplasm of epithelial cells was highercompared to the control. Chief cells of the fundal glands had a normalstructure. RNA content in the cytoplasm of these cells was higher than in thecontrol. Hence, Epigam accelerates healing of experimental ulcers andcontributes to the formation of glycosaminoglycans in the stomach wall.

187

Healing of chronic gastric ulcer was probably associated with an Epigam�induced increase in the number of mast cells in the zone of ulcerative lesion onday 7 of study (by 1.4 times compared to the control, p<0.05). Similar resultswere obtained on the 14th day. The count of mast cells in the gastric mucosaincreased by 1.5 times after administration of Epigam (p<0.05). These changesshould be considered as a positive event, which accelerates healing of themucous membrane. Previous studies showed that mast cells regulate trophicprocesses in the gastric mucosa and play a role in ulcer healing (M. Barczyk etal., 1995; S. Nakajima et al., 1996).

The model of chronic ulcers is most adequate to study the pathogenesisof peptic ulcer disease. These experiments revealed a strong antiulcer effect ofEpigam.

Effect of Epigam on the functional state of GIT

Evaluation of secretory function of the stomach. The effect of Epigam ongastric secretion in rats was studied on the model of H. Shay ulcer. Surgery wasperformed under light ether anesthesia. The pylorus was ligated. The forcepswere applied to the esophagus. The stomach was removed after 16 h. Thestomach contents were placed in tubes and centrifuged at 1500 rpm for 10 min.The following parameters were measured: volume and pH of gastric juice; totalacidity; and discharge of hydrochloric acid and pepsin (method of V. N.Tugolukov, 1965).

Prophylactic administration of the test substance in a daily dose of 0.5 mlfor 5 days was followed by the decrease in gastric juice secretion over 1 h. Thesechanges reflect a decrease in the strain of gastric secretion. The increase ingastric juice pH (p<0.05) was associated with a significant decrease in acidity.Studying the proteolytic activity of gastric juice showed that Epigam significantlydecreases the discharge of free hydrochloric acid (by 9 times, p<0.01 comparedto the control). Basal H+ concentration was 3.7�fold lower compared to thecontrol (p<0.05). These data show that preventive treatment with Epigam beforethe exposure to an aggressive factor (pylorus ligation) is followed by a decrease inacid�pepsin aggression of gastric juice in rats (Table 7.11; J. L. Dugina et al., 2002,2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).

Evaluation of excretory function of the intestine. Excretory function of theintestine was studied by the method of G. V. Obolentseva (1984). The micereceived Epigam in 0.2 ml suspension of activated charcoal (10 mg/ml). Theappearance of black�colored feces was considered as a positive result. The effectin each animal was estimated after 3, 6, and 24 h and expressed in points.

Fourfold treatment with Epigam had a moderate laxative effect. Thelaxative effect was most pronounced 3 and 6 h after the last administration of


188

Ultralow doses

Control 0.87±0.14 2.03±0.20 17.21±6.80 3.902×10�4±2.16×10�4

Epigam 0.80±0.17 2.97±0.33* 4.71±3.70* 4.35×10�3±0.35×10�4**


Table 7.11.Table 7.11.Table 7.11.Table 7.11.Table 7.11. Effect of prophylactic treatment with Epigam (daily dose 0.5 ml, 5 days) ongastric secretion in rats with ligated pylorus (M±m)

Gastric juicesecretion, ml/h

Group рН

Dischargeof free

hydrochloric acid,mmol/liter/h

H+ concentration,mmol/liter

Epigam (O. I. Epstein, 2001b; J. L. Dugina et al., 2002, 2003a,b,c; S. G.Krylova, 2002a; J. L. Dugina, 2002).

Evaluation of motor�evacuation function of GIT. Motor�evacuation func�tion of the stomach and intestine was studied by the method of “labels” (G. P.Coopman et al., 1977). Activated charcoal suspension of (0.5 ml, 10%) in 2%potato starch served as a label and was administered into the digestive tract ofmice. The animals were killed 10 min after treatment. The effect of testsubstance was evaluated.

Due to BaCl2�induced spastic contraction of pyloric smooth muscles, asuspension of activated charcoal remained in the stomach of 50% animals overthe first 10 min after injection of BaCl2. The degree of charcoal distribution inthe intestine of control animals was 31.43%. Administration of Epigam wasfollowed by an increase in evacuation function of the small intestine. Charcoaltransit in the intestine of Epigam�receiving mice was 52.3% (p<0.05) greaterthan in control animals (O. I. Epstein, 2001b; J. L. Dugina et al., 2002,2003a,b,c; S. G. Krylova, 2002a; J. L. Dugina, 2002).

Evaluation of spasmolytic activity. Spasmolytic activity was studied by themethod of J. Setnicar (1959). Experiments were performed on healthy mice andanimals with indomethacin�induced ulcer. The animals received intraperitoneal�ly 0.2 ml 0.1% BaCl2. Activated charcoal suspension of (0.5 ml, 10%) in 2%potato starch was administered intragastrically. The animals were killed after10 min. Spasmolytic activity of the product was evaluated.

Epigam was potent in increasing the motor activity of GIT. In controlmice, the suspension of charcoal was shown to pass 73.1% intestinal length over10 min. This parameter increased to 98.9% in Epigam�receiving animals(p<0.01). A positive effect of Epigam on animals with indomethacin�inducedulcer was manifested in the increase in motor activity of GIT (by 25.8%, p<0.05compared to the control; O. I. Epstein, 2001b; J. L. Dugina et al., 2002,2003a,b,c; S. G. Krylova, 2002a; J. L. Dugina, 2002).

189

Analgetic activity of Epigam

Analgetic activity of Epigam was studied on outbred mice with aceticacid�induced writhing. The nociceptive response was induced by intraperitonealinjection of acetic acid (0.2 ml, 3% solution; C. Cashik et al., 1977). Thereaction of animals to nociceptive stimulation was estimated from the numberof writhing episodes (20�min period after acetic acid injection) and writhing LC.

Administration of Epigam in a dose of 0.3 ml was followed by a signifi�cant decrease in the number of writhing episodes (by 71.29%, p<0.05). WrithingLC was shown to increase under these conditions. The analgetic effect of areference drug indomethacin was much smaller (Table 7.12; O. I. Epstein, 2001b;J. L. Dugina et al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).

Antiinflammatory activity of Epigam

Anti�edematous effect of Epigam. The anti�edematous effect of Epigamwas studied on the model of agar�induced edema, which results from theinduction of prostaglandin biosynthesis (S. K. Bhattacharya et al., 1989). Theacute inflammatory response (edema) was induced by subplantar injection of 1%agar solution (50 ml) into the hindlimb pad. The animals were killed after 5 h.The paws were cut off to the level of the knee joint and weighted on a torsionbalance. The antiinflammatory effect was evaluated from a change in the volumeof edema and expressed in percent of the control.

At the peak of agar�induced inflammation (5 h after agar injection), singleadministration of Epigam caused a significant decrease in the degree of edema(by 33.6%). We conclude that Epigam has a moderate antiinflammatory effecton mice with agar�induced inflammation (Table 7.13; O. I. Epstein, 2001b;J. L. Dugina et al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).

Antiinflammatory (antiproliferative) activity of Epigam. The course ofchronic proliferative inflammation was studies in experimental rats. A sterile


Group

Table 7.12.Table 7.12.Table 7.12.Table 7.12.Table 7.12. Effect of Epiogam on pain sensitivity of female outbred mice on the modelof acetic acid�induced writhing (M±m)

Control 24.00±3.67 – 3.75±0.25

Indomethacin(10 mg/kg, 7 days) 12.83±2.06* 46.54 4.00±0.68

Epigam (0.3 ml, 7 days) 6.89±1.71** 71.29 4.5±0.8


Time to onsetof writhing, min

Decrease in painsensitivity, %

Number of writhingepisodes over 20 min

190

Ultralow doses

Parameter

Table 7.13.Table 7.13.Table 7.13.Table 7.13.Table 7.13. Antiinflammatory (model of agar�induced edema) and antiproliferativeeffects (model of cotton pellet granuloma) of Epigam in various doses (%)

Reduction of edema at the peakof inflammation 33.6 —

Inhibition of proliferation — 24.0*

Inhibition of exudation — 30.0

Note.Note.Note.Note.Note. *p<0.05 compared to the control

0.5 ml, 8�day course0.3 ml, single treatment

Epigam dose

cotton pellet (13 mg in weight) was implanted subcutaneously on the back ofanimals (R. Meier et al., 1950). Epigam in a daily dose of 0.5 ml wasadministered intragastrically for 8 days. The animals were killed on day 8.Cotton pellets and surrounding granulation tissue were removed, weighted ona torsion balance, and dried to a constant weight at 37oC. The proliferativeresponse was estimated from the difference in the weights of dried granulomaand initial weight of a cotton pellet. The exudative response was evaluated fromthe difference in wet granuloma weight and dry granuloma weight.

The cotton pellet granuloma test showed that Epigam decreases the initialand dry weight of granulation�and�fibrous tissue. It should be emphasized thatEpigam had an inhibitory effect on proliferation in the early proliferative phaseof inflammation (p<0.05, Table 7.13). The degree of exudation tended todecrease in Epigam�receiving rats. The weight of exudate in these animalsdecreased by 1.5 times compared to the control. These data show that Epigamproduces a moderate antiproliferative effect (O. I. Epstein, 2001b; J. L. Duginaet al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).

The data suggest that Epigam affects the histamine�dependent activationof histamine H1, H2, and H3 receptors (Fig. 7.64). Epigam decreases acid�pepsin aggression of gastric juice, has a normalizing effect on motor�evacuationfunction of GIT, and possesses the antiinflammatory and analgetic properties.Therefore, the antiulcer effect of Epigam is realized via central (H3 receptors)and peripheral regulatory mechanisms (H1 and H2 receptors) of histamine�mediated functions.

7.6. Preclinical study of Afala

Antibodies to human prostate�specific antigen (PSA) in ULD constitute theactive substance of Afala. PSA is a promising molecular target in benign prostatichyperplasia (BPH; E. P. Diamandis, 2000; S. P. Balk et al., 2003). Expression of

ГГГГГлава 7.лава 7.лава 7.лава 7.лава 7. Экспериментальная фармакология препаратов сверхмалых доз антител

191

this serine protease is regulated by androgens. PSA has antiangiogenic activity (A.H. Fortier et al., 1999), plays a role in the regulation of stromal cell growth in theprostate (D. M. Sutkowski et al., 1999), and modulates the expression of genesfor tumor growth in prostate tissue (B. Bindukumar et al., 2005).

Experimental preclinical studies on the models of acute and chronicaseptic inflammation and hormone�induced inflammation of the prostaterevealed that Afala exhibits the prostatotropic properties, reduces the severity ofedema and inflammation, has a normalizing effect on prostate function, andprevents prostate sclerosis (Fig. 7.65). A toxicology study showed that Afala hasa good safety profile.

Antiinflammatory effect of Afala

Model of acute aseptic inflammation of the prostate. Experiments wereperformed on 45 male outbred albino rats aging 3 months. The animals were

Fig. 7.64.Fig. 7.64.Fig. 7.64.Fig. 7.64.Fig. 7.64. Mechanisms for action of Epigam. Effect of Epigam on acid�pepsin ag�gression of gastric juice (1); antiinflammatory effect (2); effect of Epigam on mo�tor�evacuation function of GIT (3); and analgetic effect of Epigam (4). His, histamine;H

1, H

2, and H

3, histamine receptors; P, parietal cells; ECL, enterochromaffin cells;

BP, basophils; SM cells, smooth muscle cells; NK, natural killer cells; Ts, T sup�pressor cells.

22

4

2

H+

3

1

1

4

P

Histamine

H2

H3H1

H. p

ylor

i

ECL

TS

NK

Vessel

SMC

BP


192

Ultralow doses

Fig. 7.65.Fig. 7.65.Fig. 7.65.Fig. 7.65.Fig. 7.65. Studying the pharmacological activity of Afala.

Antiinflammatory effect

Prostatotropic effect

ГHormone�induce inflammation

Young rats

Young gonadectomized rats

Model of acute prostatitis

Model of chronic prostatitis

AFALA

divided into three groups. Group 1 rats (n=20) received Afala in a dose of 1.5ml for 10 days. Distilled water in a dose of 1.5 ml was administered to 20animals of group 2 (10�day course). Group 3 consisted of 5 intact specimens.On day 3 after administration of test substances, the prostate was sutured witha silk thread to induce acute inflammation (B. A. Vertapetov, 1970). The animalswere killed 7 days after surgery. The internal organs were examined visually. Ahistological study was performed with the prostate (A. P. Milovanov, 1986; G.G. Avtandilov, 1990). Zn2+ concentration in prostate tissue was measured bymeans of emission spectral analysis (G. V. Kashkan, 1988).

Administration of Afala prevented the formation of adhesions between theprostate and surrounding tissues, had a normalizing effect on the density of theprostate gland, and decreased the severity of edema and hyperemia (Fig. 7.66).Zn2+ concentration in the ventral prostate of Afala�receiving rats was fourfoldhigher than in control specimens (Fig. 7.67). Hence, Afala improves functionof the prostate in rats.

These data show that Afala has an antiinflammatory effect during acuteaseptic inflammation of the prostate.

Model of chronic prostatitis. Experiments were performed on 50 maleoutbred rats weighing 250 g and aging 2.5 months. Chronic prostatitis wasinduced by suturing of the prostate with a silk thread (B. A. Vertapetov, 1970).The treatment group consisted of 20 animals. Afala therapy in these rats wasstarted 1 month after surgery. Afala in a daily dose of 1.5 ml was administeredintragastrically for 1 and 1.5 months. Control animals (n=20) were treated with

193

distilled water in a daily dose of 1.5 ml for 1 and 1.5 months after surgery. Tenanimals remained intact. The treated (n=10), control (n=10), and intact rats(n=5) were killed 2 and 2.5 months after surgery (1 and 1.5 months afteradministration of the test substance). The internal organs were examinedvisually. The weight, volume, weight ratio, and density of the ventral prostatewere measured. The prostate was subjected to a histological study as describedby A. P. Milovanov (1986) and G. G. Avtandilov (1990). Zn2+ concentration inprostate tissue was measured in 5 animals of each group (G. V. Kashkan, 1988).Previous studies showed that chronic prostatitis is accompanied by a decreasein sexual activity. In the present work, sexual activity of animals was estimatedby the method of Ya. Buresh et al. (1991).

Administration of Afala for 1 and 1.5 months contributed to a threefoldincrease in Zn2+ concentration in the ventral prostate (p<0.05 compared to thecontrol, Table 7.14). Atrophy of prostate tissue due to chronic inflammation(decrease in the relative area of the secretory epithelium) was less pronouncedin rats receiving Afala for 1.5 months (as compared to control specimens).

Fig. 7.66.Fig. 7.66.Fig. 7.66.Fig. 7.66.Fig. 7.66. Effect of Afala on the ratio of structural elements and severity of edemain rats with acute aseptic inflammation of the prostate. *p<0.05 compared to thecontrol.

0

20

40

60

80

100

% of the control

Vessels Edema

!

!

!


Fig. 7.67.Fig. 7.67.Fig. 7.67.Fig. 7.67.Fig. 7.67. Effect of Afala on Zn2+ concentration in the ventral prostate of rats withacute inflammation. *p<0.05 compared to the control.

0

0.5

1.0

1.5

2.0

2.5mg%

Control Afala

194

Ultralow doses

Sexual activity of animals with chronic prostatitis was much higher afteradministration of Afala for 1.5 months (compared to the control group).

These data show that Afala has an antiinflammatory effect during chronicprostatitis, reduces the degree of atrophy, and improves function of the prostate(T. G. Borovskaya et al., 2001). Afala had a normalizing effect on sexualbehavior of animals, which was suppressed due to chronic prostatitis.

Prostatotropic effect of Afala

Model of gonadectomized infantile male rats with testosterone propionate�induced androgen deficiency. Experiments were performed on gonadectomizedinfantile male rats aging 23�25 days. Test substances were administered oncedaily for 7 days. The animals were divided into the following groups:

1) intact specimens;2) gonadectomized specimens, olive oil subcutaneously;3) gonadectomized specimens, testosterone propionate (0.5 mg/kg sub�

cutaneously) and distilled water (intragastrically); and4) gonadectomized specimens, testosterone propionate (0.5 mg/kg sub�

cutaneously) and Afala (intragastrically).The animals were killed after the last treatment with test substances. The

internal organs were examined. The weight and weight ratio of target organswere estimated (ventral prostate and seminal vesicles). The weight indexes werecalculated as the ratio of the weight of the organ to body weight.

The weight ratios of the prostate and seminal vesicles in gonadectomizedanimals were lower (by 42.8% for the ventral prostate; and by 33.3% for theseminal vesicles) than in intact specimens (Fig. 7.68). Testosterone propionatecontributed to an increase in the weight of androgen�sensitive organs ingonadectomized male rats. The weight ratios of the prostate and seminal vesiclesin animals of the “distilled water + testosterone” group were 188.8 and 362.5%

Intact 23.1±1.55

Control (distilled water) 19.2±2.80 16.60±0.96* 6.0±1.0 5.6±0.6

Treatment (Afala) 21.80±2.07 19.20±0.48*+ 19.9±2.7+ 20.9±2.3+

Note.Note.Note.Note.Note. p<0.05: *compared to intact animals; +compared to the control.

Table 7.14.Table 7.14.Table 7.14.Table 7.14.Table 7.14. Effect of Afala on Zn2+ concentration in the ventral prostate and area ofthe epithelium in the distal region of the prostate in rats with chronic pro�statitis (M±m)

after 45 daysafter 30 daysafter 45 daysafter 30 days

Zn2+ concentration, mg/literArea of the epithelium in the

distal region, %Parameter

195

of those in rats of the “olive oil” group, respectively (p<0.05, Fig. 7.69). Theweight ratios of the ventral prostate and seminal vesicles in gonadectomizedanimals of the “Afala + testosterone propionate” group were 135.2 and 103%of those in rats of the “testosterone propionate” group, respectively (p=0.05).

These data show that Afala potentiates the androgenic effect oftestosterone on the prostate under conditions of androgen deficiency. However,Afala does not modulate the androgenic effect of this drug on the seminalvesicles (T. G. Borovskaya et al., 2001).

Prostatotropic effect of Afala in young animals. Experiments wereperformed on 28 infantile male rats aging 23 days. The control and treatmentgroups consisted of 14 animals each. Afala (treatment) or distilled water(control) in a dose of 5 ml/kg was administered intragastrically for 10 days. Theanimals were killed on day 11. The testes, ventral prostate, and seminal vesicleswere weighted. The weight ratios were calculated.

The weight ratios of the testes and seminal vesicles did not differ in animalsof the treatment and control groups. The weight ratio of the ventral prostate intreated rats was 43.5% higher than in control animals (p<0.05, Fig. 7.69).

Therefore, Afala has a selective effect on the prostate in young animals(prostatotropic action). The weight ratio of the prostate gland increases, whilethe weight ratios of the testes and seminal vesicles remain unchanged underthese conditions (T. G. Borovskaya et al., 2001).

Efficacy of Afala in benign prostatic hyperplasia

BPH was induced by intraperitoneal injection of sulpiride (Eglonyl,Sintelabo Grupp) in a dose of 40 mg/kg (F. Van Coppenolle, 2001) for 60 days.This treatment resulted in the development of hyperprolactinemia and,therefore, prostatic hyperplasia. Afala in a dose of 50 ml/kg was administered

Fig. 7.68.Fig. 7.68.Fig. 7.68.Fig. 7.68.Fig. 7.68. Effect of Afala on the weight ratio of the ventral prostate (a) and seminalvesicles (b) in gonadectomized animals. 100%, intact animals. Treatment of ratswith olive oil (1), distilled water and testosterone (2), and Afala and testosterone(3). p<0.05: *compared to intact animals; +compared to the olive oil group;xcompared to animals receiving distilled water and testosterone propionate.

0

а b

100

200

300%

2 31 2 31

+х

+

!!


196

Ultralow doses

intragastrically for 60 days. Permixon (Serenoa Repens extract, Pierre FabreMedicament Production) in a dose of 50 mg/kg served as the positive control.Distilled water (5 ml/kg intragastrically, 60 days) served as the negative control.

Sulpiride cause hyperprolactinemia. Plasma prolactin concentration in�creased on days 30 and 60 after sulpiride injection (by 2 and 1.6 times, re�spectively, compared to intact specimens; p<0.05). Hyperplasia of the lateralprostate was observed on day 60 after sulpiride injection. The weight and weightratio of the lateral prostate in treated animals were twofold higher than in intactspecimens (p<0.05). A histological study revealed the signs of lateral prostate hyper�plasia on day 60 after sulpiride injection. They included a significant increase inthe relative area of the glandular epithelium, decrease in the lumen of distalportions of the gland, and thickening of connective tissue layer between the acini.

Afala and Permixon had no effect on plasma prolactin concentration inrats on days 30 and 60 of study. Both substances prevented an increase in theweight ratio of the lateral prostate by the 60th day after sulpiride injection. Theefficacy of Afala was 1.7�fold higher than that of Permixon. The weight ratiosof the lateral prostate in treated rats were 0.07±0.01 and 0.12±0.01 mg/g,respectively (p<0.01; vs. 0.18±0.02 mg/g in the control group; Fig. 7.70). Ahistological study of the prostate from animals of the Afala and Permixon groupsrevealed a decrease in the degree of structural changes due tohyperprolactinemia (compared to control animals). Administration of Afala andPermixon was followed by a decrease in the relative area of epithelial structuresand increase in the relative area of the lumen in distal portions of the gland. Thethickness of connective tissue layer remained unchanged under these conditions.

These data indicate that Afala prevents the development of prolactin�dependent prostatic hyperplasia in rats of late reproductive age (8�10 months;J. L. Dugina et al., 2006). The efficacy of Afala is similar to or higher than thatof Permixon (J. L. Dugina et al., 2006).

0

0.1

0.2

0.3

0.4

mg/g

Control Afala

*

Fig. 7.69.Fig. 7.69.Fig. 7.69.Fig. 7.69.Fig. 7.69. Effect of Afala on the weight ratio of the ventral prostate in young rats.*p<0.05 compared to the control.

197

Safety profile of Afala

A complete toxicology study was performed to evaluate the safety profile,possible side effects, target organs, and safe dosage range of Afala. Theseexperiments were conducted in accordance with the recommendations given inthe Manual on Experimental (Preclinical) Study of New PharmacologicalSubstances (2000). The purpose of studies with Afala was to determine the acutetoxicity (experiments on mice and rats), chronic toxicity (6�month treatment ofrats and rabbits), reproductive and allergic toxicity (experiments on rats),immunotoxicity, mutagenicity (chromosomal aberration assay in mouse bonemarrow cells), and genotoxicity (test system for somatic mosaicism in wing cellsof Drosophila melanogaster).

Our experiments demonstrated a good safety profile of Afala. An acutetoxicity study showed that Afala in the maximum permissible dose does notcause death of animals. Drug�related death of animals was not observed after6�month treatment with Afala in the highest dose. The product had no toxiceffect on organs and systems of experimental animals. A pathomorphologicalstudy did not reveal damage to the internal organs or local irritation of thegastric mucosa after drug administration. Afala did not cause reproductivedisorders in male and female rats. The embryotoxic effect of Afala was notdetected. Afala had no mutagenic, allergenic, and immunotoxic properties.

Some authors reported that chronic inflammation and hyperplasia arerelated to an imbalance between growth factors in prostate tissue (C. L. Eaton,2003). Moreover, BPH is accompanied by structural changes in PSA and de�velopment of the autoimmune response to PSA (A. Zisman et al., 1995, 1999).

Experimental studies revealed that the course of treatment with antibodiesto PSA in ULD modifies the effect of androgens on proliferative activity of prostate

Fig. 7.70.Fig. 7.70.Fig. 7.70.Fig. 7.70.Fig. 7.70. Effect of Afala on the weight ratios of various lobes of the prostate inrats after 60�day treatment with sulpiride. Intact animals (1); distilled water +sulpiride (2); Permixon + sulpiride (3); and Afala + sulpiride (4). p<0.05: *comparedto intact animals; +compared to the control (distilled water and sulpiride); xcomparedto Permixon and sulpiride.

0

0.1

0.2

0.3

0.4mg/g

Anterior lobe Median lobe Posterior lobe

1 432

+

! !

! !

х

х х х

х!


198

Ultralow doses

tissue. Moreover, they have a normalizing effect on the morphofunctional state ofprostate tissue during chronic inflammation. Taking into account the pathogenesisof BPH and possible regulatory role of PSA in proliferative activity of prostatecells, it may be suggested that the pharmacological effect of Afala underspecified conditions is associated with functional modulation of endogenousPSA. Our hypothesis is consistent with modern notions of the generalmechanism for action of products from ULD of antibodies to endogenousregulators (i.e., modification of functional activity; O. I. Epstein et al., 2005).

7.7. Preclinical study of Kardos

ULD of antibodies to the C�terminal fragment of human angiotensin IIAT1 receptor constitute the active substance of Afala. Angiotensin II receptortype 1 (AT1) mediates the key effects of angiotensin II, which has a role in thepathogenesis of arterial hypertension, complications of this disorders, andchronic heart failure (M. de Casparo et al., 2000; R. M. Touyz et al., 2000).

Previous studies showed that Kardos exhibits the hypotensive propertiesunder conditions of arterial hypertension (two strains of rats with inheritedarterial hypertension) and has a normalizing effect on cardiac function duringexperimental chronic heart failure (Fig. 7.71).

Antihypertensive activity of Kardos

Hypotensive activity of Kados in ISIAH rats. Pharmacological activity ofKardos was studied on adult (5�6 months) and young rats (28 days) withinherited stress�induced arterial hypertension (ISIAH). The animals receivedorally (through a pipette) 0.5 ml aqueous solution of Kardos, solvent (distilledwater, control), or reference drug losartan. Losartan was given in a dose of 10mg/kg, which corresponds to the human daily dose (taking into account atenfold correction for interspecies metabolic differences).

The hypotensive effect of single administration and repeated treatmentwith the test substance for 5 days or 4 weeks was studied in adult rats (mea�surement of systolic BP by the cuff method). ECG and behavioral parametersof animals (open�field test and elevated plus�maze test) were recorded after a4�week course of treatment. The effect of Kardos (2�week course) on thehypertensive state was studied in young rats with inherited arterial hypertension.

The hypotensive effect of Kardos compared well with that of anangiotensin II AT1 receptor antagonist losartan in a dose of 10 mg/kg (A. L.Markel’ et al., 2002; O. I. Epstein et al., 2002c). However, the effect of losartandeveloped more rapidly that that of Kardos (Fig. 7.72).

199

Administration of Kardos for 2 weeks prevented the hypertensive state in4�week�old ISIAH rats (treatment from the 4th to the 6th week of life; Fig.7.73).

A 4�week course of treatment with Kardos was accompanied by thefollowing significant changes in ECG: shortening of the QT interval; decreasein the QT/R ratio; and increase in RR. These results are consisted withpublished data on the effect of antihypertensive drugs (ACE inhibitors) on ECG.The observed changes reflect a positive effect of Kardos on the cardiac cycle.

Kardos had no negative (depriming) effect on locomotor, exploratory, andother types of behavioral activity of hypertensive animals.

Fig. 7.71.Fig. 7.71.Fig. 7.71.Fig. 7.71.Fig. 7.71. Experimental study for pharmacological activity of Kardos.

ISIAH rats SHR rats Izadrin model

Antihypertensive activityEfficacy on the model of chronic heart failure

Blood pressure measurement with a cuff

Direct measurement of blood pressureYoung rats

Adult rats

KARDOS


Fig. 7.72.Fig. 7.72.Fig. 7.72.Fig. 7.72.Fig. 7.72. Hypotensive effect of losartan (light bars) and Kardos (dark bars) onadult male ISIAH rats. Single treatment (a); 5�day course (b); 7 days after withdrawal(c); repeated 5�day course (d); and 4�week course (e).

20

�10

15

25

�5

0

5

10

a b c d e

mm Hg

200

Ultralow doses

Fig. 7.73.Fig. 7.73.Fig. 7.73.Fig. 7.73.Fig. 7.73. Systolic BP in ISIAH rats receiving Kardos from the 4th to the 6th weekof life. Control (distilled water, 1); and Kardos (2). *p<0.05 compared to the control.

100

1

2

120

140

160

180

220

200

4 6 8 10 24Age of animals, weeks

mm Hg

!

Hypotensive activity of Kardos in SHR rats. Experiments were performedon the widely used model of inherited arterial hypertension (SHR rats). Anaqueous solution of Kardos in a dose of 2.5 ml/kg was administeredintragastrically. Control animals received the solvent (distilled water). Losartanin a dose of 10 mg/kg was given to specimens of the reference group. Directmeasurements of BP and HR in the abdominal aorta were performed on day28 of treatment. A catheter was introduced through the femoral artery undersodium ethaminal anesthesia.

After 4�week treatment of SHR rats, the hypotensive effect of Kardos wassimilar to that of losartan. As compared to the placebo group, Kardos andlosartan decreased the mean BP (by 14.8 and 17.7%, respectively; Fig. 7.72),systolic BP (by 16.5 and 20%, respectively), and diastolic BP (by 15.6 and17.1%, respectively; N. A. Medvedeva et al., 2006a,b). As differentiated fromlosartan, Kardos significantly decreased HR in SHR rats with hypertension andtachycardia. HR decreased by 9.3 and 1.9% after administration of Kardos andlosartan, respectively (Fig. 7.74; N. A. Medvedeva et al., 2006a,b).

Efficacy of Kardos during experimentalchronic heart failure

The therapeutic efficacy of Kardos was studied in rats with CHF. CHFwas induced by twofold subcutaneous injection of isoproterenol (Izadrin) in adose of 80 mg/kg at a 24�h interval (A. M. Chernukh et al., 1977; M. Ishizawaet al., 2006; C. J. Trivedi et al., 2006). Izadrin�induced hyperactivation of b�adrenoceptors is followed by necrotic damage to the myocardium, myocardialdysfunction, and fibrosis and hypertrophy of the left ventricle (C. J. Friddle etal., 2000; R. Bos et al., 2005). It is accompanied by the impairment ofexcitation/inhibition in cardiomyocytes, actin�myosin interaction, and response

201

to b�adrenergic stimulation (R. M. Saraiva, 2003). This model for the evaluationof existing and potential cardiotonic drugs has several advantages. First, theexperiment involves standard laboratory animals (rats) with high rate ofreproduction (as differentiated from the model of epinephrine myocarditis).Second, the results of this experiment are highly reproducible. And third, theanimals are not exposed to other factors and live under “standard”environmental conditions during Izadrin�induced intoxication and in the follow�up period. Experimental heart failure develops progressively, which is similar tomedical practice (Manual on Experimental (Preclinical) Study of NewPharmacological Substances, 2005).

Experiments were performed on 80 Wistar rats (40 males and 40 females)weighing 220�250 g and aging 4�5 months.

Studying the tolerance of animals to physical exercise (swimming timewith a load of 15% body weight), recording of ECG (standard lead II), andrheography (evaluation of the stroke volume, cardiac output, and otherhemodynamic parameters) were performed in the initial state, 7 days after thesecond injection of Izadrin, and on the 14th and 28th days after administrationof test substances.

After the second test, all rats with signs of CHF (decrease in the swimmingtime by not less than 20% of the initial level) were randomized into three groups.Each group consisted of ten males and ten females. The animals were subjectedto daily intragastric treatment with Kardos (2.5 ml/kg), distilled water, or losartan(10 mg/kg, 2.5 ml/kg aqueous solution; Cozaar, Merck Sharp & Dohme).

On day 28 of therapy, central hemodynamics and myocardial reserve in50% rats were evaluated by the invasive method with an intraventricular catheter.These experiments involved the volume loading test (intravenous infusion ofphysiological saline, 0.3 ml per 100 g), intravenous injection of epinephrine, and

Fig. 7.74.Fig. 7.74.Fig. 7.74.Fig. 7.74.Fig. 7.74. Systolic BP (direct measurement, a) and HR (b) in hypertensive SHR rats:comparative efficacy of Kardos and losartan (28�day course). Control (1); losartan(2); and Kardos (3). *p<0.05 compared to the control.

240

260

250

230

270

280

290

300HR, bpm

!

40

01

a b

2 3 1 2 3

80

120

200

160

mm Hg

!

!


202

Ultralow doses

Fig. 7.75.Fig. 7.75.Fig. 7.75.Fig. 7.75.Fig. 7.75. Tolerance to physical exercise in CHF rats after a 4�week course oftreatment with Kardos and losartan. Ordinate: Izadrine group. *p<0.05 comparedto the control.

0

10

20

40

30

Females Males

%

distilled water losartan Kardos

!

maximum isometric tension (30�sec occlusion of the ascending aortic arch). Themaximum isometric tension in the left ventricular myocardium was calculatedas follows:

P×HR/M,

where P is left ventricular pressure; and M is left ventricular mass. Thisparameter was expressed in mm Hg×g�1×min�1.

The tolerance to physical exercise in females and males decreased by 39and 25.4%, respectively, on day 7 after the last injection of Izadrin. The signsof CHF were more pronounced in females than in males.

Kardos was less potent than losartan in the ability to normalize physicaltolerance. The swimming time of CHF females increased by 30.2±7.7% after4�week administration of Kardos (placebo, by 9.9±6.8%; losartan, by 37.1±5.7%). Fig. 7.75 shows that in males with CHF, the efficacy of test substanceswas similar to that of placebo (Kardos, 7.9%; losartan, 11.9%; placebo, 5.1%).

Izadrin caused sinus tachycardia in females, which was manifested in a16% increase in average HR. The degree of tachycardia decreased mostsignificantly after administration of Kardos (by 8%) as compared to the placebo(by 7%) and losartan group (by 6%). Other parameters of ECG remainedpractically unchanged under these conditions.

A rheographic study with males and females revealed a significant de�crease in the stoke volume, cardiac output (by more than 30%), and strokeindex and increase in total peripheral vascular resistance on day 7 after Izadrininjection. A 4�week course of treatment with Kardos and losartan was accom�panied by the improvement of these parameters.

Figs. 7.76 and 7.77 illustrate that statistically significant differences incentral hemodynamics (invasive functional tests) between the animals receiving

203

losartan or Kardos and specimens of the placebo group are most typical offemales with severe CHF.

The volume loading test showed that losartan and, particularly, Kardosincrease the rate of myocardial contraction and relaxation in rats with CHF (ascompared to the placebo group, distilled water). Volume loading was followedby a greater increase in left ventricular pressure in animals of the Kardos andlosartan groups.

The epinephrine test revealed that animals of the Kardos and losartangroups differ from placebo�receiving rats in a smaller increase in the rate of leftventricular myocardial contraction. Therefore, administration of Kardos andlosartan is accompanied by a slight decrease in myocardial adrenoreactivityduring CHF.

Occlusion of the ascending aorta is used to evaluate the reserve capacityof the left ventricular myocardium. Therapy of CHF rats with Kardos and lo�

Fig. 7.76.Fig. 7.76.Fig. 7.76.Fig. 7.76.Fig. 7.76. Rates of contraction (a) and relaxation (b) of the left ventricular myo�cardium in CHF females after a 4�week course of treatment with Kardos andlosartan. Distilled water (1); losartan (2); and Kardos (3). *p<0.05 compared toanimals receiving distilled water.

0

2000

4000

6000

8000

10 000

12 000

1 2 3

mm Hg/sec

!

0

2000

4000

6000

8000

10 000

12 000

1 2

а b

3

mm Hg/sec

+!

!

+!


Fig. 7.77.Fig. 7.77.Fig. 7.77.Fig. 7.77.Fig. 7.77. Left ventricular developed pressure in CHF rats after a 4�week courseof treatment with Kardos and losartan. *p<0.05 compared to animals receivingdistilled water.

100

96

*

104

108

116

112

Distilled water Losartan Kardos

mm Hg

204

Ultralow doses

sartan was followed by a significant increase in left ventricular developed pres�sure and average maximum value of isometric tension in the left ventricularmyocardium (by 7 and 9.6%, respectively, compared to placebo). The observedchanges were particularly pronounced in females (19 and 20% for Kardos andlosartan, respectively).

These data show that Kardos significantly differs from placebo during a4�week course of intragastric administration to rats with Izadrin�induced CHF.Kardos was as good as losartan for the increase in physical tolerance, impro�vement of systemic hemodynamics, and elevation of myocardial reserve in theleft ventricle (S. Sergeeva et al., 2006; I. N. Tyurenkov et al., 2007).

Kardos safety

A complete toxicology study was performed to evaluate the safety profile,possible side effects, target organs, and safe dosage range of Kardos. The pur�pose of studies with Kardos was to determine the acute toxicity (experiments onmice and rats), chronic toxicity (6�month treatment of rats and rabbits), repro�ductive and allergic toxicity (experiments on rats), immunotoxicity, mutagenicity(chromosomal aberration assay in mouse bone marrow cells), and genotoxicity(test system for somatic mosaicism in wing cells of Drosophila melanogaster).

The experiments demonstrated a good safety profile of Kardos. An acutetoxicity study showed that this substance in the maximum permissible dose doesnot cause death of animals. Drug�related death of animals was not observedafter 6�month treatment with Kardos in the highest dose. The product had notoxic effect on organs and systems of experimental animals. A pathomorpho�logical study did not reveal damage to the internal organs or local irritation ofthe gastric mucosa after drug administration. HR in male and female ratsdecreased by 14�16% after 6�month treatment with Kardos (p<0.01 comparedto the control). Kardos did not cause reproductive disorders in male and femalerats. The embryotoxic effect of Kardos was not observed. Kardos had nomutagenic, allergenic, and immunotoxic properties.

7.8. Study for antidiabetic activityof a new product from ultralowdoses of antibodies on the modelof streptozotocin�induced diabetes in rats

According to the World Health Organization more than 180 millionpeople worldwide suffer from diabetes mellitus (DM). The number of these

205

patients is expected to increase by more than 2 times in 2030 (M. B. Antsiferovet al., 2000). The main criteria of diabetes are an increase in fasting bloodglucose level (above 6.7 mmol/liter) and impaired glucose tolerance (ExpertCommittee, 2003).

The common type of pharmacotherapy for type 1 DM is insulin replace�ment therapy. Blood sugar�lowering drugs (sulfonylurea derivatives, biguanides,meglitinides, etc.) are used in the therapy of type 2 DM. Despite the efficacyof standard pharmacotherapy for DM, antidiabetic drugs cannot compensatecompletely the associated disorders. Moreover, they cause some side effects(M. B. Antsiferov et al., 2000; M. I. Balabolkin et al., 2005; R. K. Bidasee etal., 2003). The search for new hypoglycemic drugs is an urgent problem (M. I.Balabolkon, 1998).

Streptozotocin�induced diabetes is an extensively used experimentalmodel of DM (D. A. Rees et al., 2005). Administration of streptozotocin is fol�lowed by progressive dysfunction of pancreatic β�cells, impairment of glucosetolerance, and development of associated disorders.

This study was designed to study the antidiabetic properties of a newproduct, which belongs to a class of ULD antibodies (O. I. Epstein et al., 2004;A. A. Spasov et al., 2007) and contains antibodies to the insulin receptorβ�subunit (ULD AB�IRb) in ULD for oral administration.

Experiments were performed on 130 male outbred albino rats weighing 250�300 g. DM was induced by intravenous injection of streptozotocin in a single doseof 50 mg/kg. Blood glucose level was measured after 72 h. Blood glucose levelin DM rats was not less than 15 mmol/liter. These animals were divided intogroups and treated in the follow�up period (50 days). The rats received distilledwater (2.5 ml/kg intragastrically once daily; control, n=60); insulin (Aktrapid,daily dose 12 U/kg, subcutaneously twice daily; n=20); glybenclamide (Berlin�Chemie; daily dose 8 m/kg, intragastrically twice daily, n=20); or ULD AB�IRβ(“Materia Medica Holding” Research�and�Production Company, 2.5 ml/kgintragastrically once daily, n=20). The intact group consisted of ten rats.

Body weight, fasting blood glucose (glucose oxidase method withGlyukoza FKD kits), and water consumption were estimated on days 3, 7, 14,21, 28, 35, 42, and 50 of therapy. The glucose tolerance test (1 g/kg glucoseorally) was performed on days 14, 28, and 50 of therapy. The area under theconcentration�time curve (AUC) was calculated by the method of trapezoids.

Streptozotocin caused hyperglycemia in rats. Blood glucose concentrationin control rats was 4�6.5 times higher than that in intact animals. By the endof study, blood glucose level in control specimens reached 17.9±0.06 mmol/liter(Fig. 7.78). The severity of DM was determined from the morality rate ofcontrol rats. Only 15% animals of this group survived by the end of study (Table7.15). On day 50 of observations, body weight loss in control specimens was


206

Ultralow doses

Fig. 7.78.Fig. 7.78.Fig. 7.78.Fig. 7.78.Fig. 7.78. Effect of ULD AB�IRβ, insulin, and glybenclamide on blood glucose levelin rats with streptozotocin�induced diabetes. Intact animals (1); control (2); dia�betes+insulin (3); diabetes+glybenclamide (4); and diabetes+ULD AB�IRβ (5).p<0.05: *compared to the control; +compared to the diabetes+insulin group anddiabetes+glybenclamide group.

0

5

2

43

5

1

10

15

20

25

3

Time, days

mmol/liter

7 14 21 28 35 42 50

+++

++

!

!

!

!!

!

!

!

!

!!

47%. By contrast, body weight of intact animals increased by 20%. Waterconsumption in streptozotocin�receiving rats was 2.7�fold higher than in controlspecimens (Table 7.15).

Insulin injection was followed by a significant decrease in blood glucoselevel (8.96±0.05 mmol/liter on day 50, p<0.001; Fig. 7.78), but had no effecton the degree of polydipsia in rats with experimental diabetes. The survival rateof these animals was 20% (Table 7.15). Body weight remained unchanged in ratsof the insulin group.

Glybenclamide also decreased the degree of hyperglycemia (10.01±0.03mmol/liter on day 50, p<0.001; Fig. 7.78), which probably explains a slightincrease in the survival rate of animals (up to 20%; Table 7.15).

Blood glucose level in DM rats returned to normal on day 7 of treatmentwith ULD AB�IRβ (Fig. 7.78). In the follow�up period, blood glucoseconcentration in animals of this group did not differ from that in intactspecimens. Moreover, blood glucose level in rats receiving ULD AB�IRβ wasmuch lower than in animals of the insulin and glybenclamide groups.Administration of ULD AB�IRβ was also followed by a significant increase inthe survival rate of rats (up to 30%) compared to animals of other groups. Theexternal appearance, behavior, and body weight gain in rats of the ULD AB�IRβ group did not differ from those in intact animals. This product decreasedthe volume of water consumption by 22.5% compared to the control (p<0.05).

The oral glucose loading test showed that streptozotocin injection isfollowed by a 3�6�fold decrease in glucose tolerance (Fig. 7.79). Glucosetolerance increased by 2.5�3 and 2�2.5 times after intragastric administration ofreference drugs (insulin and glybenclamide, respectively; p<0.05 compared tothe control). Administration of ULD AB�IRβ was also followed by the increase

207

in glucose tolerance in diabetic rats (by 2.4�3.7 times compared to the control,p<0.05). It should be emphasized that the effect of ULD AB�IRβ compared wellwith that of reference drugs.

These data indicate that ULD AB�IRβ have a strong antidiabetic effecton the model of streptozotocin�induced DM in rats. The product had anormalizing effect on blood glucose level, glucose tolerance (oral glucoseloading), and body weight gain. The survival rate of rats significantly increasedafter administration of ULD AB�IRβ. The effect of ULD AB�IRβ on glucosetolerance was similar to that of standard drugs for the therapy of type 1 and 2DM (insulin, 12 U/kg; and glybenclamide, 8 mg/kg). Moreover, ULD AB�IRβhad a greater hypoglycemic effect than reference drugs. We conclude that ULDAB�IRβ have high antidiabetic activity.

Group

Table 7.15.Table 7.15.Table 7.15.Table 7.15.Table 7.15. Survival rate, body weight gain, and consumption of food and water in ratswith streptozotocin�induced diabetes on day 50 of study

Intact animals 10 10 +20 57.32±4.63

Control (diabetes) 60 9 �47 151.78±10.5

Diabetes and insulin 20 4 0* 189.23±21.92

Diabetes andglybenclamide 20 4 �30.5 164.05±15.76

Diabetes andULD AB�IRβ 20 6 +12.5* 117.57±8.38*


Water consumption,1 ml per rat

Body weightgain, %

Survivedanimals

Total numberof animals


Fig. 7.79.Fig. 7.79.Fig. 7.79.Fig. 7.79.Fig. 7.79. Glucose tolerance (glucose AUC, oral glucose loading) in rats withstreptozotocin�induced diabetes after administration of test substances. Intactanimals (1); control (diabetes, 2); diabetes+insulin (3); diabetes+glybenclamide(4); and diabetes+ULD AB�IRβ (5). *p<0.05 compared to the control.

0

500

1000

1500

2000

3000AUC, mmolхmin/liter

14 28Duration of therapy, days

50

!!

!

!

!

!

!

!!

2500

� 1 � 4� 3� 2 � 5

208

Ultralow doses

* * *

Chapter 7 was devoted to the results of experimental studies with 11products that contain ULD of antibodies to the following agents: S�100 protein(Proproten�100, Tenoten, and Tenoten for children), NO synthase (Impaza),IFN�γ (Anaferon and Anaferon for children), TNF�α (Artrofoon), histamine(Epigam), PSA (Afala), C�terminal fragment of the angiotensin II AT1 receptor(Kardos), and insulin receptor β�subunit.

These studies were designed to evaluate the range of pharmacologicalactivity of products from antibodies in ULD. ULD anti�S100 have theanxiolytic, antiasthenic, activating, antidepressant, antiaggressive, nootropic,stress�protective, antihypoxic, antiischemic, and neuroprotective properties.Antibodies to NO synthase in ULD improve endothelial function (e.g., erectilefunction) and decrease the increased BP. ULD of antibodies to IFN�γ have theimmunomodulatory and antiviral effects. Antibodies to TNF�α in ULD possessthe antiinflammatory, analgetic, and antitumor properties on some models ofexperimental tumors. ULD of anti�histamine antibodies have the antiulcer effectand improve motor activity of GIT. Antibodies to PSA in ULD exhibit theprostatotropic activity (efficacy during experimental inflammation of the pro�state and BPH). ULD of antibodies to the C�terminal fragment of the angio�tensin II AT1 receptor produce the hypotensive and cardiotropic effects (reco�very of myocardial function and morphology on the model of CHF). ULD ofantibodies to the insulin receptor β�subunit have a strong hypoglycemic andantidiabetic effect.

Each product was subjected to a complete toxicology study in accordancewith the recommendations given in the Manual on Experimental (Preclinical)Study of New Pharmacological Substances (2000, 2005). The purpose of studieswith these products was to determine the acute toxicity (experiments on miceand rats), chronic toxicity (6�month treatment of rats and rabbits), reproductiveand allergic toxicity (experiments on rats), immunotoxicity, mutagenicity(chromosomal aberration assay in mouse bone marrow cells), and genotoxicity(test system for somatic mosaicism in wing cells of Drosophila melanogaster orAmes test).

Antibodies in ULD demonstrated a good safety profile. An acute toxicitystudy showed that intragastric and intraperitoneal administration of products inthe maximum permissible dose does not cause death of animals. Hence, ULDof antibodies were classified to a group of low�hazard substances (GOST12.1.007�76). Drug�related death of animals was not observed after 6�monthtreatment with ULD of antibodies in the highest dose. These products had notoxic effect on organs and systems of experimental animals. A pathomorpholo�gical study did not reveal damage to the internal organs or local irritation of the

209

gastric mucosa after drug treatment. Antibodies in ULD did not causereproductive disorders in male and female rats. The embryotoxic effect was notobserved. Products of antibodies in ULD had no mutagenic, allergenic, andimmunotoxic properties. A toxicology study revealed some specific effects ofproducts from ULD of antibodies.

Hence, preclinical studies demonstrated the efficacy of antibodies inULD. The activity of antibodies in ULD compares well with that of referencedrugs. Moreover, these products have a good safety profile. The results ofpreclinical studies were confirmed by further clinical observations.


210

Ultralow doses

8.1. Use of medical products from antibodiesto S�100 protein in the therapy for alcoholismand anxiety disorders

Pharmacological activity of products from antibodies to S�100 protein(Proproten�100, Tenoten, and Tenoten for children) is associated with

their ability to modify functional activity of the endogenous protein S�100.This property of antibodies in ULD was discovered in vitro on the model oflong�term posttetanic potentiation (Epstein et al., 2003a; O. I. Epstein et al.,2003).

S�100 protein has a wide range of biological functions, which formed atheoretical basis for the development of medical products from antibodies to S�100 protein (new molecular target for drug treatment). This protein is involvedin neuronal plasticity, regulation of GABAergic neurotransmission, intracellularcalcium homeostasis, and neurotrophic processes (A. V. Martyushev�Poklad etal., 2004). Experimental studies showed that the products of antibodies to S�100protein in ULD have a wide range of pharmacological properties, which reflectsa variety of biological functions of this protein (O. I. Epstein et al., 2005). Theefficacy and safety of new medical products from antibodies to S�100 proteinwere confirmed in controlled clinical trials.

C h a p t e r 8

Clinical pharmacologyof products from ultralowdoses of antibodies

211

Proproten�100 in the therapy of alcoholism

Proproten�100 was approved for the use in medical practice in 1999. Thisdrug was shown to be effective in the therapy of alcoholism (O. I. Epstein etal., 2001c; A. G. Gofman et al., 2002; G. A. Livanov et al., 2003; N. A. Bokhanet al., 2003; A. T. Davydov et al., 2004).

The efficacy and safety of Proproten�100 in the therapy for alcoholwithdrawal syndrome (AWS) were studied in a double�blind, randomized,placebo�controlled trial with parallel groups (Proproten�100 monotherapy andplacebo+detoxification; A. G. Gofman et al., 2003). An open comparative studyof Proproten�100 vs. amitriptyline (75 mg daily + detoxification) and phena�zepam (2 mg daily + detoxification) was performed by E. N. Krylov (2003b).Proproten�100 monotherapy was much more effective than placebo treatmentand detoxification. The symptoms of mild and moderate AWS were reduced onday 1 of treatment with Proproten�100 (Fig. 8.1).

Anxiety, depression, alcohol craving, and somatovegetative symptoms ofAWS (tremor, hyperhidrosis, and tachycardia) were relieved in a greater numberof patients on days 1�3 of Proproten�100 therapy. Dysphoria, asthenia, anddyssomnia were not observed in a greater number of patients on days 2�3 oftreatment. Proproten�100 had a strong anxiolytic, antidepressant, antiasthenic,and vegetostabilizing effect, prevented the development of affective disorders,and caused the reduction of alcohol craving in the acute period of alcoholabstinence (Fig. 8.2; A. G. Gofman et al., 2003).

As differentiated from amitriptyline and phenazepam, Proproten�100significantly decreased the time to relief of AWS symptoms. It should beemphasized that Proproten�100 did not cause side effects. Amitriptyline wasmore potent than Proproten�100 in reducing alcohol craving. The severity ofanxiety and sleep disorders decreased most significantly after phenazepamtherapy (Fig. 8.3; E. N. Krylov, 2003b).

Fig. 8.1.Fig. 8.1.Fig. 8.1.Fig. 8.1.Fig. 8.1. Effect of Proproten�100 on the overall severity of AWS. Placebo anddetoxification (1); and Proproten�100 (2).

0

5

10

15

20

25

1 2 3 4Duration of therapy, days

Points

1

2

Chapter 8. Chapter 8. Chapter 8. Chapter 8. Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies

212

Ultralow doses

The efficacy and safety of Proproten�100 in alcoholic patients with post�withdrawal disorders were studied in an open randomized controlled trial. Afterthe relief of AWS (N. A. Bokhan et al., 2003), hospital patients of the controlgroup received Proproten�100 or individually prescribed antidepressants(amitriptyline, up to 100 mg daily), drugs for behavioral disturbances (Neuleptil,up to 30 mg daily; Sonapax, up to 50 mg daily), drugs for sleep disorders(chlorprothixene, up to 100 mg daily), nootropic agents (piracetam, up to 800mg daily), and vegetostabilizing drugs (Pyrroxan, up to 60 mg daily; Grandaxin,up to 100 mg daily; N. A. Bokhan et al., 2003). The degree of affective

0

20

40

60

80

100

!

!

0

20

40

60

80

100

a

c

%

%

b

%

Placebo +detoxification

Proproten Placebo +detoxification

ProprotenProprotenPlacebo +detoxification

!

!

!

!

!

!

1st day 3rd day2nd day

DEPRESSIONANXIETY

d

% INSOMNIADYSSOMNIA

0

20

40

60

80

100


Proproten ProprotenPlacebo +detoxification

!

!

!

!

0

20

40

60

80

100


ProprotenProprotenProproten Placebo +detoxification


!

!

!

!

!!

!

!!TREMOR HYPERHIDROSIS TACHYCARDIA

Fig. 8.2.Fig. 8.2.Fig. 8.2.Fig. 8.2.Fig. 8.2. Effect of Proproten�100 on thesymptoms of AWS. Anxiolytic and antide�pressant effect (a); antiasthenic effect (b);effect on somatovegetative symptoms ofAWS (c); and effect on dyssomnia (d).Ordinate: responding patients. *p<0.05compared to the placebo group.

213

disorders, dyssomnia, neurovegetative disturbances, and alcohol craving wasevaluated daily.

Proproten�100 monotherapy was as good as combination therapy for thereduction of AWS symptoms during the subacute period. The degree of anxietyand depression (Hamilton’s scale) in patients receiving Proproten�100 was re�duced more rapidly than in the control group. Proproten�100 had a strong an�xiolytic and antidepressant effect during the subacute period of AWS. The effi�cacy of Proproten�100 was higher compared to that of reference drugs (Fig. 8.4).

Studying the efficacy, safety profile, and activity showed that Proproten�100 monotherapy has some advantages over other psychopharmacological drugs

Fig. 8.3.Fig. 8.3.Fig. 8.3.Fig. 8.3.Fig. 8.3. Comparative therapeutic efficacy of Proproten�100 in AWS patients.Placebo and detoxification (1); Proproten�100 (2); amitriptyline (75 mg daily) anddetoxification (3); and phenazepam (2 mg daily) and detoxification (4).


0

1

2

3

4

5

6Relief time, days

Alcohol craving Somatovegetative disordersAnxiety Depression Sleep disorders

1 3 42

Fig. 8.4.Fig. 8.4.Fig. 8.4.Fig. 8.4.Fig. 8.4. Anxiolytic (a) and antidepressant effects (b) of Proproten�100 in thetherapy of alcoholic patients with post�withdrawal disorders. *p<0.05 comparedto the reference group.

0

40

80

160

120

Baseline level 7 days 14 days 21 days

Degree of anxiety (Hamilton’s scale), % of the baseline level

!

!!

0

a b

40

80

160

120

Baseline level 7 days

Time, days

14 days 21 days

Degree of anxiety (Hamilton’s scale), % of the baseline level

!

!!

reference group Proproten�100

214

Ultralow doses

during therapy of alcoholic patients with mild or moderate AWS and post�withdrawal disorders.

Previous experiments showed that the product of antibodies to S�100protein had an anxiolytic effect under standard conditions. Moreover, theactivity of Proproten�100 compared well with that of diazepam. As differentiatedfrom diazepam, Proproten�100 did not cause side effects (sedative, myorelaxant,and amnesia�inducing effects). Experimental studies revealed that Proproten�100has a wide range of pharmacological activity, including the antidepressant,antiasthenic, stress�protective, antihypoxic, antiischemic, neuroprotective, andnootropic (antiamnesic) properties. Therapeutic activity of the product fromantibodies to S�100 protein is partly related to the GABA�mimetic effect (O.I. Epsyein et al., 2005). A favorable combination of the anxiolytic and activatingproperties provides a basis for studying the clinical efficacy of Tenoten in anxietydisorders.

Tenoten in the therapy of anxiety disorders

The therapy of anxiety disorders that constitute the most common typeof mental disturbances is an urgent problem of modern medicine. A deficiencyof GABAergic transmission in CNS is one of the major pathogeneticmechanisms for anxiety disorders. The action of well�known anxiolytic drugs(e.g., benzodiazepines) and newly developed products is directed to compensatefor this deficiency. However, the most effective and rapidly acting drugs causea variety of side effects. This disadvantage limits the long�term use of standardpharmaceuticals. Prolonged administration of some antidepressants withanxiolytic activity may be accompanied by side effects, which limits their usein clinical practice.

The efficacy and safety of Tenoten (product of antibodies to S100 protein,ULD for oral administration) in the therapy of anxiety disorders were confirmedin randomized controlled clinical trials at the Institute of Neurology (RussianAcademy of Medical Sciences), V. P. Serbskii State Research Center forForensic and Social Psychiatry (Moscow), V. M. Bekhterev PsychoneurologicalResearch Institute, Military Medical Academy (St. Petersburg) and otherinstitutions.

An open�label randomized trial was designed to compare the efficacy andtolerability of 4�week monotherapy with Tenoten and diazepam (15 mg daily)in patients with borderline disorders. The majority of patients had anxietydisorders, including neurasthenia, adaptation disorder or mixed anxiety�depression, generalized anxiety disorder, and mixed anxiety�depression disorder.

The anxiolytic effect of Tenoten was studied in patients of the treatmentgroup with borderline anxiety (primarily with generalized anxiety disorder).

215

The Hamilton anxiety scale (HAMA) is one of the most common andvalid methods to evaluate the severity of anxiety disorders. There are severalapproaches to study the efficacy of anxiolytic drugs by HAMA (criteria for afavorable response to treatment, remission criteria, etc.; A. Doyle et al., 2003).This study also included a validated test with the Spielberger scale (State�TraitAnxiety Inventory, STAI). As differentiated from HAMA, the patients wereasked to answer the STAI questions.

A multicenter, randomized, parallel�group, comparative trial involved ambu�latory patients (men and women, 18�65 years of age) who met the criteria of neura�sthenia (F48 by ICD�10), adaptation disorders (mixed anxiety�depression, F43.22),generalized anxiety disorder (F41.1), and mixed anxiety�depression disorder (F41.2)and had moderate or severe anxiety (HAMA total score not less than 20).

The exclusion criteria were internal diseases, epilepsy, decompensatedpersonality disorders, comorbid neurological and somatic diseases (difficulties inthe evaluation of anxiety disorder), alcoholism, abuse of psychoactive drugs,pregnancy and breast�feeding, and participation in another clinical trial withinthe past 30 days.

The patients who met the inclusion criteria were selected during the firstvisit. They were informed about the purpose of this trial. The informed consentwas obtained from each patient.

The patients were randomized into groups (1:1 ratio) at each medical center(Institute of Neurology of the Russian Academy of Medical Sciences, V. P. SerbskiiState Research Center for Forensic and Social Psychiatry, V. M. BekhterevPsychoneurological Research Institute, Military Medical Academy, etc.). Therapywas prescribed during the second visit (after entry into the trial). The time of entryinto the trial depended on the half�life of withdrawn drugs (usually 7 days).

The state of patients and adverse events were evaluated in each of the nextfour visits at 7�day intervals. A study of vital parameters (BP and HR), routineblood test, and urine test were performed at the beginning (baseline visit) andby the end of the trial (28 days). All data were recorded in the case report form.The total duration of therapy was 28 days.

The study drug (Tenoten, 1�2 lozenges; 6�12 lozenges daily) or diazepam(5 mg orally, three times daily) was given as monotherapy for 28 days.Medication taking was not associated with food intake.

In patients of the Tenoten group, the frequency of drug treatment couldbe increased to 10�12 times daily (at low effectiveness) or decreased to 2�4 timesdaily. The majority of patients required a higher dose of Tenoten. By the endof study, the average daily dose of Tenoten was 10 tablets.

Psychotropic drugs, psychoactive compounds, and alcoholic beveragescould affect the results of therapy and, therefore, were strictly forbidden duringthe trial period. The patients were allowed to take vitamins and to receive


216

Ultralow doses

physiotherapy or physiotherapy. Hypnotic drugs with a short half�life (Zolpidemand Zopiclon) were prescribed in severe insomnia. The patient’s use of addi�tional drugs was recorded in the case report form during each visit.

The efficacy and safety of study drug were evaluated after 1, 2, and 4weeks of therapy. A decrease in the severity of anxiety (HAMA total score) andsymptoms of anxiety (STAI scale) was considered as the primary efficacyendpoint. The secondary endpoints were a favorable response to treatment(decrease in the HAMA total score by at least 50%) and achievement of partialremission (decrease in the HAMA total score to 10 points or less).

The safety profile was determined after 1, 2, and 4 weeks of therapy usinga structured scale for adverse events (AE). Vital functions (BP and HR) and keylaboratory parameters were measured.

This study was designed to test the hypothesis that Tenoten and diazepamare equally potent in reducing the overall degree of anxiety (HAMA and STAI)and providing a 50% decrease in the anxiety score (HAMA).

The data from all patients who met the inclusion criteria and receiveddrug therapy were subjected to statistical analysis (intent�to�treat analysis). Themean values were compared with the baseline and diazepam group (Student’st test). The data of patients from both groups who demonstrated a favorableresponse to treatment and signs of remission were compared by x2 test forhomogeneity of proportions. All statistical tests were two�sided. The hypotheseswere tested at a significance level of 5%. The number of patients in each groupwas selected to achieve a statistical power of 80%.

Among 300 patients enrolled in the trial, 272 patients were randomizedinto the groups of diazepam (130 subjects) and Tenoten (142 subjects). The finalstage was performed with 247 patients. Six patients of the diazepam group andfifteen patients of the Tenoten group were excluded from the trial due toprotocol non�compliance. Drug safety was evaluated in 272 patients.

Demographic data and key clinical parameters for all patients enrolled inthe trial and further analysis are shown in Table 8.1.

The efficacy of Tenoten and diazepam according to HAMA (primaryendpoints) is illustrated in Fig. 8.5.

Small differences in the HAMA total score were found between patientsof various groups in the basal state. Significant between�group differences wererevealed in the final total score (HAMA) and average decrease in the HAMAtotal score. However, these differences were clinically irrelevant. In clinicalpractice, a statistically significant difference may be characterized by variationsin effect size. Two drugs are considered to be clinically equivalent when thedeviation of effect size does not exceed 0.5. Effect size is calculated as the ratioof the difference between mean values in groups to the standard deviation (SD)of the combined sample. A difference in the efficacy (decrease in the HAMA

217

total score) of diazepam and Tenoten is 2.3 points, which corresponds to theeffect of 0.33. SD of the combined sample is 6.96. Hence, a statisticallysignificant advantage of diazepam over Tenoten is clinically irrelevant. Thesedata indicate that diazepam and Tenoten have similar efficacy.

Small between�group differences were found in the baseline level ofsituational and trait anxiety (STAI). Diazepam and Tenoten caused a similardecrease in situational and trait anxiety. The patients reported that these drugsare equally potent in decreasing the degree of anxiety.

Table 8.1.Table 8.1.Table 8.1.Table 8.1.Table 8.1. Baseline characteristics of patients with anxiety disorders enrolled in thetrial (M±m)

Number of patients in group 120 127

Number of women in group 73 (60.8%) 81 (63.8%)

Average age, years 39.0±1,0 38.6±1.0

Mean duration of disease, months 34.0±3.8 27.7±3.5

Mean total score by HAMA 28.6±0.5 27.4±0.5*

Number of patients with the diagnosis

GAD (F41.1) 40 (33.3%) 40 (31.5%)

MADD (F41.2) 26 (21.7%) 31 (24.4%)

adaptation disorder (F43.22) 16 (13.3%) 17 (13.4%)

neurasthenia (F48.0) 38 (31.7%) 39 (30.7%)

Note.Note.Note.Note.Note. *p<0.05 compared to another group.

ParameterTenotendiazepam

Group

Fig. 8.5.Fig. 8.5.Fig. 8.5.Fig. 8.5.Fig. 8.5. Efficacy of 4�week treatment with diazepam (light bars) and Tenoten (darkbars) in patients with anxiety disorders. (a) Decrease in the total score by theHamilton’s scale; and (b) reduction of situational (STAI�S) and trait anxiety (STAI�T)by the Spielberger scale. *p<0.05 compared to the diazepam group.

20

10

15

25

5

0

STAI�S STAI�Т

!

5

15

10

0

a b

20

25

30

35Points Points

Baseline After 4 weeks

!

!


218

Ultralow doses

Fig. 8.6 illustrates the secondary efficacy endpoints. The number ofpatients who achieved a good response to treatment was insignificantly higherin the diazepam group. The incidence of partial remission was similar in patientsreceiving diazepam and Tenoten.

Diazepam demonstrated a slight advantage over Tenoten in inducing therapid response to treatment (50% decrease in the total score by the Hamiltonanxiety scale and achievement of partial remission; Fig. 8.7). However, nobetween�group differences were found in the number of patients with remissionand favorable response to treatment after 4 weeks of therapy. The “delayedresponse” of Tenoten�receiving patients could be related to selecting the optimaldose of this drug at the beginning of treatment. At the start of our study, theoptimal dose of Tenoten was at least 10 tablets daily. In patients receiving lessthan 10 tablets daily over the 1st week of therapy (n=52), partial remission orfavorable response to treatment was not observed after 2 weeks. The patientsreceiving 10�12 tablets daily from the start of the study (n=75) demonstrated afavorable response (22.7% subjects) and partial remission (9.3% subjects) after2 weeks. By the 4th week of therapy, small differences were found betweensubgroups of patients receiving various doses of Tenoten. The exception was theachievement of remission. Among 82 patients receiving 12 tablets daily,remission was achieved in 17.1% subjects. After treatment with a lower dose ofTenoten (36 patients), remission was observed only in 2.8% subjects. These dataindicate that the optimal dose of Tenoten is 10�12 tablets daily. By the 14th dayof therapy, the HAMA total score in these subjects decreased more significantlythan in patients receiving less than 10 tablets daily (10.1 and 7.1 points,respectively).

Fig. 8.8 illustrates the baseline, final level, and average decrease in thedegree of psychic (psychic anxiety factor; items 1�6 and 14) and somatic anxiety

Fig. 8.6.Fig. 8.6.Fig. 8.6.Fig. 8.6.Fig. 8.6. Efficacy of 4�week treatment with diazepam (light bars) and Tenoten (darkbars) in patients with anxiety disorders: percentage of patients with a favorableresponse to treatment (a) and partial remission (b). *p<0.05 compared to thediazepam group.

20

0

40

60

100% ba

80

!

219

(somatic anxiety factor; items 7�13; Hamilton’s scale) in patients receivingdiazepam and Tenoten. Before the start of therapy, psychic anxiety in thediazepam group was more pronounced than in the Tenoten group. After a 4�week course of therapy, diazepam was more potent that Tenoten in reducing thepsychic and somatic symptoms of anxiety. Similarly to the HAMA total score,diazepam had a slight advantage over Tenoten in effect size.

The primary efficacy endpoints are shown in Fig. 8.9.The efficacy of diazepam and Tenoten practically did not depend on

patient’s sex. Among patients of the Tenoten group, a favorable response totreatment was most typical of women. Similar results were obtained for patientsof the diazepam group (statistically insignificant).

In subgroups of patients below 45 years of age, baseline anxiety, decreasein the HAMA total score, and ratio of responding subjects were slightly higherafter therapy with diazepam. The advantage was clinically irrelevant. However,

Fig. 8.7.Fig. 8.7.Fig. 8.7.Fig. 8.7.Fig. 8.7. Partial response to diazepam (light bars) and Tenoten (dark bars) duringa 4�week course of therapy for anxiety disorders. Patients with a favorable responseto treatment (decrease in the HAMA score by at least 50%) and partial remission(decrease below 11 points). RPT, response to treatment; PR, partial remission.

0

20

40

60

80

PR RPT PR RPT PR RPT

100 1 week 2 weeks 4 weeks

Fig. 8.8.Fig. 8.8.Fig. 8.8.Fig. 8.8.Fig. 8.8. Efficacy of Tenoten (4�week course of treatment) in patients with anxietydisorders. HAMA psychic anxiety (a) and somatic anxiety (b). Light bars, diazepam;dark bars, Tenoten. *p<0.05 compared to the diazepam group.

02468

10121416


!

02468

1012141618

a


!

b


220

Ultralow doses

these drugs did not differ in the post�treatment level of anxiety and ratio ofpatients with partial remission.

The efficacy of study drugs was much lower in patients older than 45years of age (compared to younger subjects). It is probably related to the greaterinertia of pathological processes that require a longer time for regression. Inpatients of this group, Tenoten was as good as diazepam for the reduction ofanxiety. Moreover, the percentage of patients with a favorable response totreatment and achievement of remission did not differ after therapy withTenoten and diazepam.

One�third of patients in both groups had severe anxiety disorder (HAMAtotal score > 30). The effects of diazepam and Tenoten (decrease in the HAMAtotal score; and percentage of patients with a goof response to treatment) wereparticularly pronounced in subgroups of patients with severe anxiety. Diazepam

Fig. 8.9.Fig. 8.9.Fig. 8.9.Fig. 8.9.Fig. 8.9. Therapeutic efficacy of Tenoten in patients with anxiety disorders. Analysisof subgroups. (a) Diagnosis. (b) Baseline characteristics of patients: (1) allparameters; (2�3) sex (women and men, respectively); (4�5) age (not older than45 years of age, older than 45 years of age); and (6�7) baseline anxiety score (lessthan 30 points by HAMA, not more than 30 points by HAMA). Ordinate: percentageof patients with a favorable response. Light bars, diazepam; dark bars, Tenoten.GAD, generalized anxiety disorder; MADD, mixed anxiety�depression disorder.*p<0.05 compared to the diazepam group.

30

0

40

60

100

%

80!

1 2 3 4 5 6 7

30

0

40

60

100

% a

b

80

!

All diagnoses GAD MADD Adaption disorder Neurasthenia

221

was more effective than Tenoten in patients with moderate anxiety. However, theefficacy of Tenoten compared well with that of diazepam in subgroups ofpatients with high level of anxiety.

The size of subgroups with various diagnoses (GAD, MADD, adaptationdisorder, and neurasthenia) was similar in the diazepam and Tenoten groups. Astatistically significant advantage of diazepam over Tenoten was revealed inpatients with MADD and neurasthenia (decrease in the total score). This scoredecreased most significantly in subgroups of MADD patients, which wasprobably related to high level of baseline anxiety. Other criteria for the efficacyof Tenoten and diazepam were similar in all nosological groups. A statisticallysignificant advantage of Tenoten over diazepam (percentage of patients withpartial remission) was revealed in the group of patients with adaptation disorder.

A statistically significant advantage of diazepam over Tenoten indecreasing the HAMA total score was observed not only in main groups, butalso in subgroups (except for patients with severe anxiety, GAD, and adaptationdisorder). However, the percentage of patients with a favorable response totreatment and partial remission did not differ in subgroups of patients receivingTenoten and diazepam.

The safety evaluation was performed with all patients who received studydrugs (n=272).

Significant between�group differences were found in the incidence of AE.The percentage of patients reporting AE was sevenfold lower in the Tenotengroup than in the diazepam group. The total number of AE (per 100 patients)was 15�fold lower in Tenoten�receiving patients (Fig. 8.10).

The majority of AE in diazepam�receiving patients were the typical sideeffects of this drug. The relationship between AE and drug therapy was probableor possible. The most common AE were observed in more than 10% patientsand included daytime sleepiness, muscle weakness, orthostatic disorders, vertigo,and dry mouth.


Fig. 8.10.Fig. 8.10.Fig. 8.10.Fig. 8.10.Fig. 8.10. Reporting of adverse events during a 4�week course of therapy withdiazepam and Tenoten. (a) Percentage of patients; and (b) number of AE per 100patients. *p<0.05 compared to diazepam.

100

0

200

300

!

100

0

200

300a b

!

222

Ultralow doses

Mild AE were revealed in the majority of Tenoten�receiving patients. Acause�and�effect relationship between AE and drug treatment was ambiguous orabsent. Daytime sleepiness and tympanites were observed most frequently (1.4%patients). One patient of the Tenoten group and six patients of the diazepamgroup were excluded from the trial due to side effects.

The results of laboratory tests and vital parameters remained practicallyunchanged during this trial.

A randomized controlled clinical trial showed that 4�week monotherapywith Tenoten (average daily dose 10 tablets) is less potent than diazepam (15 mgdaily) in reducing anxiety in patients with anxiety disorders. The severity ofanxiety was evaluated by a physician (HAMA scale) and patient (STAI scale,effect size 0.33). The efficacy of Tenoten compared well with that of diazepamin patients with severe anxiety (more than 30 points by the HAMA scale).

The effect of Tenoten developed more slowly than that of diazepam. Bythe 4th week of therapy, Tenoten and diazepam were equally potent inproducing a favorable response (at least twofold decrease in anxiety) and partialremission (HAMA total score < 11). The effect of drug is determined by aninitial daily dose. The optimal dose of Tenoten is not less than 10 tablets daily.

Tenoten (average daily dose 10 tablets) had a better safety profile than thereference drug diazepam (15 mg daily) during 4�week monotherapy of patientswith anxiety disorders (18�65 years of age). Serious AE were not observed inpatients of both groups. In the majority of Tenoten�receiving patients, therelationship between AE and drug treatment was ambiguous or absent. Thedesign of this trial suggested an open comparative study of Tenoten anddiazepam. However, the possibility of system errors due to the placebo effectcannot be excluded. Moreover, the duration of this trial could not exceed themaximum length of diazepam treatment (4 weeks).

The trial allowed us to make several conclusions (A. V. Martyushev�Poklad et al., 2005a). During short�term treatment of patients with anxietydisorders (4 weeks), the product of antibodies to S�100 protein (Tenoten) showsa better efficacy�safety ratio (benefit�effect) than diazepam in a daily dose of 15mg. The efficacy of Tenoten in patients with GAD and severe anxiety wassimilar to that of diazepam. Due to the absence of sedative and myorelaxantproperties, Tenoten may be considered as a first�line daytime tranquilizer.

A clinical trial at the Institute of Neurology (Russian Academy of MedicalSciences, Moscow) included the patients who had not only anxiety disorders, butalso chronic cerebrovascular diseases and Parkinson’s disease. Tenoten was welltolerated and exhibited the high antianxiety activity (above the placebo effect).Therefore, Tenoten can be used for the therapy of patients with serious diseases.

Controlled clinical trials showed that the original products of antibodiesto S�100 protein demonstrate high efficacy and good safety profile in the

223

therapy for alcoholism (Proproten�100) and anxiety disorders (Tenoten). By therange of pharmacological properties and benefit/risk ratio, both products haveadvantage over standard pharmaceuticals. Hence, Proproten�100 and Tenotenhold much promise for the use in clinical practice. These products should besubjected to large�scale clinical trials.

8.2. Use of Impaza in monotherapy andcombined treatment for erectile dysfunction

Although the development of erectile dysfunction (ED) is associated witha variety of factors, the major pathogenetic types of this disorder have a com�mon mechanism. It suggests functional insufficiency of the peripheral me�chanism for erection (signal transduction cascade of NO synthase — NO gua�nylate cyclase — cGMP) and, primarily, inadequate production of NO (T. F.Lue, 2000; K. E. Andersson, 2001). Type 5 phosphodiesterase (PDE�5) inhi�bitors are most effective in the therapy for ED. They provide temporal controlover a deficiency in the cavernous tissue, which is related to a modulatory effecton the final stage of this cascade. However, the therapy for ED of various etio�logies should be directed to an increase or recovery of normal NO production.

Impaza consists of antibodies to endothelial NO synthase (ULD for oraladministration). This medical product was approved for the therapy of ED in2001. Preclinical studies showed that Impaza improves copulative function ofmale rats (T. G. Borovskaya et al., 2001, 2002; I. V. Smolenov et al., 2002) anddoes not exhibit the general or reproductive toxicity. The peripheral effects ofImpaza in ED are related to its influence on the signal pathway of NO synthase— NO guanylate cyclase — cyclic GMP in the cavernous tissue (Fig. 8.11). Thecourse of treatment with Impaza increased the activity of NO synthase,production of NO, and concentration of cGMP in the cavernous tissue of malerats.

The efficacy and safety of Impaza in monotherapy and combinationtherapy for ED were studied in controlled clinical trials (A. Martyushev�Pokladet al., 2005c).

A randomized placebo�controlled trial of the efficacy and safety ofImpaza during ED was performed in 2001�2003 (common protocol). This trialinvolved the following five clinical centers in Russia: Department of ClinicalPharmacology, Volgograd State Medical University; Institute of Pharmacology,Volgograd State Medical University; Department of Urology and SurgicalNephrology, Russian State Medical University (Moscow); R. M. FronshteinUrology Clinic, I. M. Sechenov Moscow Medical Academy; and S. P. FedorovSt. Petersburg Urology Society.


224

Ultralow doses

After the primary clinical and laboratory examination, the trial enrolledambulatory patients (18�70 years of age) in heterosexual relationships that com�plained of decreased erection. The diagnosis of ED was made by the Inter�national Index of Erectile Function (IIEF). The integral index of “erectile func�tion” varies from 7 to 25 points. The patients were asked to sign informed con�sent to participate in a clinical trial.

Before enrollment in the trial, all patients were examined for case history,history of sexual activity, and IIEF questionnaire. Laboratory examinationincluded the routine blood test, urine test, measurement of plasma glucose andcreatinine, and study of hormonal status and lipid profile.

The exclusion criteria were alcoholism, drug abuse, anatomical de�formation of the penis, endocrine causes of ED, uncontrolled or decompensatedsomatic disease, therapy with other pharmaceuticals for ED, treatment withmedical products that may cause ED, and participation in other clinical trials.

Impaza and placebo lozenges were prescribed to take in the evening time(1 tablet) every other day. The patents could also receive 1 tablet of Impaza1�3 h before the proposed sexual activity.

The efficacy was evaluated after therapy for 4 and 12 weeks. Patients wereinvited to complete the IIEF questionnaire and to evaluate the overall efficacy

Fig. 8.11.Fig. 8.11.Fig. 8.11.Fig. 8.11.Fig. 8.11. Effect of Impaza on the regulatory cascade of NO synthase � NO � cyclicAMP (experimental data). NO, nitric oxide; eNO, endothelial NO synthase; GTP,guanosine triphosphate; cGMP, cyclic guanosine triphosphate.

IMPAZA

eNOS eNOS

NO

NOGTO Phosphate cGMP

Cell relaxation

Guanylate cyclase activation

Smooth muscle cell

+ + +

Vascular endothelium

225

of therapy. The primary efficacy endpoints were a 3�point increase in theintegral index of IIEF for “erectile function” (percentage of patients with a fa�vorable response), achievement of a normal criterion for “erectile function”(more than 25 points), and patient’s evaluation of the overall efficacy (per�centage of patients who evaluated drug efficacy as “excellent” and “good”).

Three months after the main trial, some patients receiving Impaza (n=18,Volgograd Medical University) were enrolled in the prospective evaluation ofdrug efficacy. The degree of ED (according to IIEF) and patient’s subjectiveevaluation of ED over 3 months after completion of the main trial wereanalyzed in the follow�up visit.

The safety profile was evaluated by monitoring of drug�related adverseevents (side effects) and drug interactions. Clinical examination of patients andstudy of vital indexes were performed by the 4th and 12th weeks.

The patients who refused to participate in a clinical trial or demonstratedserious drug�related adverse events were excluded from the research.

During the first examination in the Department of Urology and SurgicalNephrology (Russian State Medical University, Moscow), all patients weresubjected to intracavernous pharmacological testing, Doppler ultrasonography ofpenile vessels before and after artificial erection, and penile electromyography.

In the Department of Urology and Surgical Nephrology (Russian StateMedical University) and Urology Clinic (I. M. Sechenov Moscow MedicalAcademy), the hormonal status of patients (morning blood test for thyrotropichormone, triiodothyronine, thyroxin, total testosterone, and serum prolactin)was assayed before enrollment in the trial and after the 4th and 12th weeks oftherapy.

One hundred and sixty�nine patients met the inclusion criteria. They wererandomized to the placebo group (30 patients) and Impaza group (139 patients).The trial involved 19�60 patients in each center. After 12 weeks, 23 patients ofthe placebo group (ineffectiveness of therapy) and 1 patient of the Impaza group(irrationality of further treatment due to the recovery of erectile function)discontinued participation in the trial. Six of seven patients from the placebogroup had ED of psychogenic origin.

Study groups were comparable by the major indexes (except for EDetiology; Table 8.2). The placebo group mainly consisted of patients withpsychogenic ED. These patients could be expected to exhibit the maximumfavorable response to treatment. Therefore, a possible system error of the trialmight be related to overestimation of placebo efficacy.

Impaza was much more potent than placebo in the ability to improveerectile function (Table 8.3, Fig. 8.12). As differentiated from placebo, Impazaefficacy significantly increased with an increase in the duration of therapy from4 to 12 weeks.


226

Ultralow doses

Table 8.2.Table 8.2.Table 8.2.Table 8.2.Table 8.2. Data on patients enrolled in the trial (M±m)

Average age, years 47.8±0.98 (19�69) 47.5±1.8 (33�67)

Mean duration of ED, years 3.6±0.32 4.1±0.64

Patients with ED of primarilypsychogenic origin, % 51.1 73.3

Average score of “erectile function”according to IIEF (30 points maximum) 17.70±0.35 16.3±0.7

ED patients, %

severe (EF < 11 points) 7.2 10

moderate (EF = 11�16 points) 29.5 33.3

mild (EF = 17�25 points) 63.3 56.7

Note.Note.Note.Note.Note. EF, erectile function.

Parameterplacebo (n=30)Impaza (n=139)

Group

Impaza had a stronger effect on various components of erectile functioncompared to placebo (IIEF items 1�5 and 15; Table 8.4).

Table 8.4 shows the intention�to�treat analysis of groups without regardto 77% placebo patients who discontinued the trial. The per�protocol analysisrevealed a greater difference between patients of the Impaza and placebo groupson the 12th week of study.

It may be concluded that the efficacy of Impaza is much higher than theplacebo effect (Tables 8.2�8.4). Impaza therapy was followed by significantimprovement of the integral index for “erectile function”. Impaza had a positiveeffect not only on erectile function, but also on other aspects of sexual activityin patients with ED (Table 8.5).

Fig. 8.12.Fig. 8.12.Fig. 8.12.Fig. 8.12.Fig. 8.12. Impaza efficacy in a placebo�controlled trial: patients who achieved thekey efficacy endpoints. *p<0.05 compared to the control.

30

0

40

60

80!

!

!

!

!

Favorable responseto treatment

Recovery of EF

Patient’s evaluation as “excellent”/”good”

Impaza, 4 weeks Placebo, 4 weeks

Impaza, 12 weeks Placebo, 12 weeks

Percentage of patients, %

227

In the Impaza group, three patients suffered from headache (2%) and onepatient had nausea (0.7%) during the 1st week of treatment. These events werebelieved to be associated with drug therapy. Headache was reported by onepatient of the placebo group (3.3%). Serious drug�related adverse events werenot revealed in the Impaza and placebo groups. Adverse drug interactions withImpaza were not found in patients who received medical products for theunderlying disease (e.g., nitrates in CHD).

These data illustrate the efficacy and safety of Impaza in patients withED. The effect was particularly pronounced after long�term treatment withImpaza (12 weeks or more).


Table 8.3.Table 8.3.Table 8.3.Table 8.3.Table 8.3. Therapy efficacy in the Impaza and placebo groups (key endpoints, %)

Patients with improvederectile function(EF, increase by 13 points) 68.3* 30 76.9** 3.3

Patients who achieved a normallevel of EF (> 25 points) 19.4* 6.6 33.8** 0

Patients who evaluatedthe efficacy of therapyas “excellent” or “good” 62.6* 23.3 69.6** 3.3

Note.Note.Note.Note.Note. *p<0.05 and **p<0.01 compared to the placebo group.

4

placebo(n=7)

Impaza(n=138)

12

placebo(n=30)

Impaza(n=138)

Time of observation, weeks

Parameter

Frequency of erection 3.1±0.1 3.8±0.1*+ 4.2±0.1*+ 3.2±0.2 3.0±0.3

Success in insertionof the penis 3.0±0.1 3.7±0.1*+ 4.0±0.1*+ 2.9±0.2 2.7±0.3

Ability to achieve erection 3.1±0.1 3.8±0.1*+ 4.1±0.1*+ 2.9±0.15 3.2±0.3

Ability to maintain erection 2.9±0.1 3.7±0.1*+ 3.9±0.1* 3.0±0.15 3.4±0.3

Ability to completesexual intercourse 2.8±0.1 3.5±0.1*+ 3.8±0.1*+ 3.1±0.15* 2.8±0.2

Erection confidence 2.5±0.1 3.5±0.1*+ 3.8±0.1*+ 2.7±0.2 2.6±0.3

Erectile function 17.4±0.4 22.1±0.3*+ 24.0±0.3*+ 17.8±0.8* 17.9±0.7

Note.Note.Note.Note.Note. p<0.05: *compared to the baseline; +compared to the placebo group.

Parameter

Table 8.4.Table 8.4.Table 8.4.Table 8.4.Table 8.4. Effect of Impaza and placebo on various components of erectile function(completion of the IIEF questionnaire, M±m)

12 weeks(n=7)

4 weeks(n=30)

Placebo

12 weeks4 weeks

Impaza

Baseline

228

Ultralow doses

Fig. 8.13.Fig. 8.13.Fig. 8.13.Fig. 8.13.Fig. 8.13. Efficacy of Impaza in patients of various age groups: achievement ofthe key efficacy endpoints.

Satisfaction withsexual intercourse 8.3±0.2 2.0±0.2**+ 3.0±0.2**+ 0.6±0.2* 0.7±0.9

Orgasm 6.9±0.2 1.0±0.1**+ 1.2±0.2**+ 0.3±0.1* 0.14±0.4

Libido 6.1±0.2 1.0±0.1**+ 1.5±0.2**+ 0.4±0.1** 0.3±0.2

Overall satisfaction 5.1±0.2 1.6±0.2**+ 2.3±0.2**+ 0.4±0.2* 0.1±0.4

Note.Note.Note.Note.Note. *p<0.05 and **p<0.01 compared to the baseline; +p<0.01 compared to placebo.

Parameter

Table 8.5.Table 8.5.Table 8.5.Table 8.5.Table 8.5. Effect of Impaza and placebo on the integral criteria of IIEF (averageincrease; M±m)

12 weeks(n=7)

4 weeks(n=30)

Placebo

12 weeks4 weeks

Impaza

Baseline

A detailed study revealed the specific drug effects in patients of variousage groups (Fig. 8.13). A favorable response to treatment was observed inpatients of the following age groups: younger than 40 years (n=31), 90%patients; 40�49 years of age (n=39), 77% patients; 50�59 years of age (n=48),73% patients; and 60�69 years of age (n=24), 70.8% patients. After Impazatherapy, normal erectile function was achieved in 71, 28, 25, and 8.3% patients,respectively. A favorable response was observed only in 3.3% patients of theplacebo group.

Therefore, Impaza was effective in the majority of ED patients from allage groups. Impaza had a normalizing effect on erectile function in the majorityof patients younger than 40 years (primarily psychogenic ED). Impaza waseffective in more than two�thirds of ED patients from the older age group

0

20

40

60

80

100

120


Impaza, total (n=139)

50�59 years(n=45)

60�69 years(n=24)

Placebo (n=30)

Younger than 40 years (n=31)

40�49 years (n=30)

response to treatment recovery of EF patient’s positive evaluation

229

(primarily organic ED). A complete recovery of erectile function did not occurin the majority of these patients, which was probably related to progressiveorganic changes during ED.

The efficacy of oral pharmacotherapy differed in patients with variouspathogenetic variants of ED. Hence, a detailed analysis of Impaza effect wasperformed by the results of a placebo�controlled trial. Table 8.6 shows thecharacteristics of patients from various groups.

Impaza was most effective in patients with primarily psychogenic ED(Table 8.7; Figs. 8.14 and 8.15). The effect of Impaza in these patientsdeveloped more rapidly compared to subjects of other groups. Moreover, thiseffect remained practically unchanged with an increase in the duration oftherapy from 4 to 12 weeks. The efficacy of Impaza in patients with psychogenicED is probably related to the influence on central and peripheral componentsof erectile function.

Study drug had a progressive effect on patients with organic ED. Thepercentage of responding patients was shown to increase by 10�15% with anincrease in the duration of therapy to 12 weeks. The efficacy of Impaza washighest in patients with the prevalence of arterial factors for ED, but lowest in


Table 8.6.Table 8.6.Table 8.6.Table 8.6.Table 8.6. Subgroups of patients with various pathogenetic factors for ED in a placebo�controlled trial of Imapza

Patients with venous factors 11 51.5±2.4

Patients with neurogenic factors (except for venous factors) 15 53.6±0.8

Patients with arterial factors (except for venous and neurogenic factors) 38 54.4±1.0

Patients with all organic factors 68 53.6±0.8

Patients with primarily psychogenic factors of ED 71 42.3±1.5

Note.Note.Note.Note.Note. n, number of patients.

AgenGroup

Fig. 8.14.Fig. 8.14.Fig. 8.14.Fig. 8.14.Fig. 8.14. Effect of pathogenetic factors for ED on the efficacy of 12�week therapywith Impaza: average increase in the index of “erectile function”.

0

2

4

6

8

10Points

Venous Neurogenic Arterial All organic factors

Psychogenic ED

230

Ultralow doses

Fig. 8.15.Fig. 8.15.Fig. 8.15.Fig. 8.15.Fig. 8.15. Effect of pathogenetic factors for ED on the efficacy of 12�week therapywith Impaza: favorable response to treatment (percentage of responding patients).

vascular venous ED. These results are consistent with published data on theperipheral mechanism of drug effect (recovery of endothelial function).

Particular attention was paid to the efficacy of Impaza in patients withcardiovascular diseases (arterial hypertension and atherosclerosis). A favorableresponse to therapy for ED in 56 and 69% patients of this group (n=48) wasobserved by the 4th and 12th weeks of treatment, respectively. An averageincrease in the index of “erectile function” was 3.1±0.4 (4 weeks) and4.3±0.5 (12 weeks). Normal erectile function was achieved in 16.7 and 18.8%patients by the 4th and 12th weeks of Impaza therapy, respectively. Thepatients with cardiovascular diseases did not report side effects or adverse druginteractions between Impaza and medical products for the underlying disease(e.g., nitrates).

A clinical trial of Impaza efficacy in ED patients with chronic CHD(Institute of Cardiology, Russian Ministry of Health, Saratov) showed that thecourse of drug treatment not only improves erectile function, but also decreasesthe incidence of anginal attacks (K. S. Umetskii et al., 2005, 2006).

Venous (group 1) 27.3 45.5 45.5 45.5 2.9±0.9

Neurogenic (group 2) 46.7 60 60 66.7 3.4±0.7

Arterial (group 3) 63.2 76.3 47.4 55.3 4.7±0.5

All organic factors 53 67.6 50 57.4 4.2±0.4

Prevalence of psychogenic factors 83.1 85.9 74.6 80.3 8.6±0.6

Table 8.7.Table 8.7.Table 8.7.Table 8.7.Table 8.7. Effect of pathogenetic factors for ED on the efficacy of Impaza therapy(percentage of patients who achieved the efficacy endpoints)

Averageincrease in EF

(12 weeks,points)

Favorableresponse totreatment, %

Patient’s eva�luation as “good”or “excellent”, %

4weeks

12weeks

Pathogenetic factor

4weeks

12weeks

0

20

40

60

80

100Patients, %

4 weeks 12 weeks

Venous Neurogenic Arterial All organic factors

Psychogenic ED

231

An initiative controlled clinical study of Impaza efficacy in patients withCHD was performed at the Altai State Medical University (A. I. Neimark et al.,2006a,b). Impaza not only improves erectile function, but also has a positiveeffect on clinical manifestations of angina pectoris, microcirculation, andendothelial function in these patients. The product and standard drugs for CHDtherapy are a good combination. These data indicate that Impaza holds muchpromise for the therapy of CHD.

All patients were asked to complete the IIEF questionnaire 3 months aftercompletion of the trial.

The course of drug treatment was followed by the relief of ED andwithdrawal of medical products to improve erectile function in 44.4% patients.The subjective evaluation was confirmed by the results of IIEF testing. Thisgroup consisted of patients with primarily psychogenic factors of ED.

Drug withdrawal was accompanied by the decrease in erectile function in33.3% patients. Therapy should be continued in these patients. The effect ofImpaza was preserved after reinstitution of therapy. This group mainly consistedof patients with arterial factors of ED (arterial hypertension). The remainingpatients (22.3%) were not satisfied with Impaza therapy and refused to use thisdrug in the follow�up period.

Drug�related adverse events were not reported over 3 months aftercompletion of the main trial.

Serum testosterone (TS) concentration was measured in 60 patients.Impaza therapy for 12 weeks was followed by improvement of erectile functionin 67% patients. Table 8.8 shows that 10% variations in total serum TS weretypical of 72% patients (increase in 52% patients, decrease in 20% patients).

TS concentration significantly increased in 50% responding patients(group 1). The mean values did not differ between patients of this subgroup andentire group (except for an average increase in erectile function).

TS concentration increased by more than 10% in group 2 patients notresponding to Impaza. The average increase in TS level and improvement oflibido in these subjects were twofold lower than in group 1 patients. Thesegroups differed in the etiological structure. The percentage of ED patients withvenous and/or neurogenic factors in group 2 was twofold higher than in group1 (55 and 22.5%, respectively). By contrast, the percentage of patients withprimarily psychogenic ED in group 2 was three times lower than in group 1 (5and 15%, respectively).

The reduced baseline level of TS (group 3) was associated with low indexof erectile function. These patients exhibited an intermediate frequency ofresponse to treatment and improvement of erectile function. By contrast, theincrease in TS concentration was most pronounced in group 3 patients (by morethan 25%). Orgasmic function and libido improved in these subjects.


232

Ultralow doses

Impaza therapy was followed by the decrease in total TS concentrationin group 5 patients, which demonstrated high baseline level of TS and relativelylow index of erectile function. The percentage of group 5 patients respondingto treatment was higher than the average. These patients were characterized byan intermediate increase in erectile function. The increase in orgasmic function,libido, and overall satisfaction was most pronounced in group 5 patients.

Impaza had no effect on TS level in group 6 patients with the higher in�tegral index of orgasmic function, libido, and overall satisfaction. TS concen�tration in these patients was above the average. The incidence of psychogenic/psychological factors for ED in group 6 patients was twofold lower than theaverage. The response to treatment was rarely observed in these patients.

TS concentration increased by more than 10% in six of seven patientswith primarily psychogenic ED (85% vs. 52% on average). These data suggestthat Impaza has a modulatory effect on the central mechanisms of erectilefunction and central regulation of androgen status.

Hence, long�term treatment with Impaza is accompanied by the increasein total serum TS concentration in 50% patients. This effect is particularlypronounced in patients with low baseline level of TS.

Among patients with baseline total serum TS > 14 nM, this parameterremained unchanged in 50% subjects, increased in 20% subjects, and decreasedin 30% subjects. The decrease in TS level was associated with a greater in�cidence of response to treatment and significant increase in orgasmic function,

Table 8.8.Table 8.8.Table 8.8.Table 8.8.Table 8.8. Effect of 12�week treatment with Impaza on serum total TS in ED patients

Improved erectile function 67 53.5±0.9 100 14.9±0.8 14.1±4.5*

No improvementof erectile function 33 53.7±0.9 0 15.1±1.3 7.2±4.1

Baseline TS < 14 nM 42 55.6±1.2 64 9.9±0.4 27.1±5.5*

Increase in TS after 12weeks, by more than 10% 52 55.2±1.1 68 12.7±0.7 30.0±3.8*

Decrease in TS after 12weeks, by more than 10% 20 51.8±1.7 75 18.3±1.5 �20.2±2.6*

No changes in TS level 28 52.3±0.6 59 16.3±1.3 2.4±1.4

Total 100 53.7±0.7 67 14.8±0.7 12.1±3.3*(45�69)

Note.Note.Note.Note.Note. *p<0.05 compared to the baseline value.

Subgroup increase,% of thebaseline

value

baseline, nMTS

Responding to treatment, %

Average age,years

Numberof patients,

% of theentire group

233

libido, and overall satisfaction (above the average). A cause�and�effect rela�tionship between Impaza treatment and decrease in TS concentration should beevaluated in further studies.

The effect of drug therapy for ED depends on the relative contributionof central and peripheral components in the impairment of erectile function ineach patient. A change in androgen status seems to be secondary to the correc�tion of central components of erectile function. It may be suggested that theeffect of Impaza on androgen status is mediated by the central regulation ofandrogen biosynthesis.

The second clinical trial extended the notion of a possible role of Impazain clinical practice (E. B. Mazo et al., 2004a,b). This trial was performed at theDepartment of Urology (Russian State Medical University, Moscow) in 2003�2004.

A prospective, open�label, randomized, parallel�group study was designed toevaluate the efficacy and safety of various schemes of pharmacotherapy for ED.

After clinical and laboratory examination, ambulatory patients (20�75years of age) with complaints of reduced potency were enrolled in the trial.

All patients were examined as follows: case history; history of sexual ac�tivity; IIEF questionnaire; physical examination; Viagra test; intracavernous phar�macological testing with prostaglandin E1; Doppler pharmaco�ultrasonography ofpenile vessels with audiovisual sexual stimulation before and after artificialerection; penile electromyography; blood hormone test; and standard laboratorytests (routine blood test, urine test, blood glucose, creatinine, and lipid profile).

All patients were divided into three groups of comparable size, age,possible etiology, pathogenesis, and severity of ED.

The duration of therapy was 6 months. Group 1 patients (n=81) receivedViagra (sildenafil, Pfizer) in an individual dose. The initial dose of Viagra was100 mg. Viagra dose could be reduced, which depended on the effect,tolerability, and degree of undesired reactions. Viagra was given two�three timesa week. Group 2 patients (n=64) received Sialis (tadalafil, Eli Lilly) in a doseof 20 mg two�three times a week. Group 3 patients (n=73) received Impaza (1sublingual lozenge) every other day.

The effects of PDE�5 inhibitors (Viagra and Sialis) and Impaza aremediated by various mechanisms. These drugs were combined in patients thatdid not respond to monotherapy and had serious side effects.

An additional group consisted of patients who did not respond to Viagraor Sialis in the previous time, received intracavernous injections of vasoactivedrugs (n=22), or were untreated (n=32). The patients received Levitra(vardenafil, Bayer) at an initial dose of 20 mg. This drug was given 20�40 minbefore sexual activity. The dose of Levitra was then selected individually, whichdepended on drug effect and tolerability (reducton to 10 ml and 5 mg; or nochanges, 20 mg). The course of Levitra therapy was 3 months.


234

Ultralow doses

The efficacy of therapy in all groups was evaluated from an increase inthe index of “erectile function” by at least 3 points (IIEF questionnaire,favorable response to treatment) or up to 26 points (recovery of EF).

The percentage of subjects who responded to treatment, achieved normalEF, and rated the efficacy as “good” or “excellent” was evaluated in each groupof patients enrolled in the trial and receiving therapy (ITT). All statistical testswere two�sided. The hypotheses were tested at a significance level of 5%. Thenumber of patients in each group was selected to achieve a statistical power of80%. The analysis of proportions was performed by x2 test for homogeneity ofproportions. Parametric variables were analyzed by Student’s t test for dependent(difference from the baseline) and independent variables (difference fromplacebo or reference drugs).

The main part of this trial (groups 1�3) involved 218 patients with ED(21�73 years of age, average age 58.1±13.2 years): younger than 35 years, 58patients; 35�55 years of age, 69 patients; and older than 55 years of age, 91patients. The possible etiologic factors for ED in 174 patients were essentialarterial hypertension (n=81), diabetes mellitus (n=27), CHD (n=15),lumbosacral osteochondrosis (n=23), chronic pelvic pain syndrome (n=21), andpostoperative period after radical surgery (organs of the pelvis minor, n=7).Eight patients had organic disorders of unknown etiology. Psychogenic ED wasdiagnosed in 36 patients. Mild (18�25 points), moderate (11�17 points), andsevere ED (not more than 10 points) was found in 74 patients (33.9%), 91patients (41.7%), and 53 patients (24.4%), respectively. According to the resultsof a complex andrological examination, these patients had ED of primarilypsychogenic (n=36, 16.5%), arteriogenic (n=87, 39.9%), venoocclusive (n=54,24.8%), and neurogenic origin (n=41, 18.8%).

The groups were comparable by size, age of patients, possible etiology,pathogenesis, and severity of ED (Table 8.9).

The overall efficacy of therapy with Viagra, Sialis, and Impaza was 77.8,81.3, and 56.2%, respectively. The dependence of drug efficacy on the age ofpatients, pathogenesis of ED, and severity of ED is shown in Table 8.10.

The efficacy of Viagra, Sialis, and Impaza was similar in patients youngerthan 35 years. Sialis was much more potent than other drugs in patients of theolder age group (55 years of age or older). The advantage of Sialis over Viagrais probably associated not only with physical capacities of these patients, butalso with the role of prelude to sexual activity and main attributes of sex (e.g.,romantic situation).

Both inhibitors of PDE�5 were pathogenetically effective in all groups ofpatients. The efficacy of Sialis was higher only in patients with venoocclusiveED. These differences are probably related to the pharmacodynamic andpharmacokinetic properties of Sialis (e.g., prolonged circulation in blood

235

plasma). Impaza was most effective in patients with compensated andsubcompensated arteriogenic ED.

The efficacy of study drugs decreased with an increase in the severity of ED.The efficacy of Impaza progressively increased from 33.2% (1 month of

therapy) to 56.2% (6 months of therapy). Significant changes were observed 19patients (26.2%) by the 4th month of therapy (Fig. 8.16). These patients initiallyreported the improvement and recovery of spontaneous erection, penile swelling,and increase in the size of the penis (state of continuous partial tumescence).The recovery of erections was observed after 3�4 months. These changesprobably serve as the early signs for therapeutic action of Impaza. They reflectthe cumulative effect and physiological activity of Impaza. Hence, Impazashould be taken for at least 3�4 months to achieve a stable therapeutic effect.

Age

younger than 35 years (n=58) 22 27.2 17 26.5 19 26.0

35�55 years (n=69) 26 32.1 20 31.3 23 31.5

older than 55 years (n=91) 33 40.7 27 42.2 31 42.5

Etiology

psychogenic ED (n=36) 13 16.1 11 17.2 12 16.4

essential hypertension (n=81) 30 37.0 24 37.5 27 37.0

diabetes mellitus (n=27) 10 12.3 8 12.5 9 12.3

osteochondrosis (n=23) 8 9.9 7 10.9 8 11.0

chronic pelvic pain syndrome (n=21) 8 9.9 6 9.4 7 9.7

CHD (n=15) 6 7.4 3 4.7 6 8.2

after radical surgeries (n=7) 2 2.5 3 4.7 2 2.7

unknown etiology (n=8) 4 4.9 2 3.1 2 2.7

Pathogenesis

psychogenic (n=36) 13 16.1 11 17.2 12 16.4

arteriogenic (n=87) 33 40.7 25 39.0 29 39.7

venoocclusive (n=54) 20 24.7 16 25.0 18 24.7

neurogenic (n=41) 15 18.5 12 18.8 14 19.2

Degree

mild (n=74) 27 33.3 21 32.8 26 35.6

moderate (n=91) 34 42.0 27 42.2 30 41.1

severe (n=53) 20 24.7 16 25.0 17 23.3

Total (n=218) 81 100 64 100 73 100

Table 8.9.Table 8.9.Table 8.9.Table 8.9.Table 8.9. Characteristics of ED patients enrolled in a comparative efficacy study ofmonotherapy with PDE�5 inhibitors and Impaza

Parameter%abs.

ImpazaSialisViagra

%abs.%abs.


236

Ultralow doses

Examination of an additional group showed that Levitra is effective in 44patients (81.5%), including 28 of 32 primary patients (87.5%) and 16 of 22patients not responding to Viagra or Sialis (72.7%). A favorable response toLevitra (10 mg) was observed in 19 primary patients (59.4%). A positive effectof Levitra (20 mg) was also revealed in the majority of patients not respondingto Viagra or Sialis.

Side effects of Viagra therapy (100 mg) were mainly found in fastingpatients. They included headache (11 patients, 13.6%), reddening of the skinand neck (7 patients, 8.6%), dyspepsia (5 patients, 6.2%), and change in colorperception (3 patients, 3.7%). These symptoms persisted for up to 4 h.

AE of Sialis therapy included headache (eight patients, 12.5%), dyspepsia(six patients, 9.4%), reddening of the skin and neck (four patients, 6.3%), andback pain (two patients, 3.1%). The duration of side effects varied from severalhours to several days. In some patients, side effects persisted for the period ofdrug action (36 h or more; up to 3 days).

Levitra was well tolerated. The efficacy and side effects of Levitra did notdepend on food intake and alcohol consumption. Side effects were mostpronounced 50�90 min after the use of Levitra in a dose of 20 mg, whichcorresponded to the maximum concentration of this product in the blood. They

Age

younger than 35 years (n=58) 21 95.5 15 88.2 16 84.2

35�55 years (n=69) 22 84.6 17 85.0 12 52.2

older than 55 years (n=91) 20 60.6 20 74.1 13 41.9

Pathogenesis

psychogenic (n=36) 11 84.6 10 90.9 9 75.0

arteriogenic (n=87) 25 75.6 19 76.0 18 62.1

venoocclusive (n=54) 14 70.0 13 81.3 6 33.3

neurogenic (n=41) 13 86.7 10 83.3 8 57.1

Degree

mild (n=74) 25 92.6 19 90.5 20 76.9

moderate (n=91) 26 76.5 22 81.5 15 50.0

severe (n=53) 12 60.0 11 68.8 6 35.3

Total (n=218) 63 77.8 52 81.3 41 56.2

Table 8.10.Table 8.10.Table 8.10.Table 8.10.Table 8.10. Efficacy of PDE�5 inhibitors and Impaza as monotherapy for ED

Parameter

%abs.

Impaza(n=73)

Sialis(n=64)

Viagra(n=81)

%abs.%abs.

Number of patientswith a favorable response

237

included headache (seven patients, 12.9%), hot flashes to the face or neck (sixpatients, 11.1%), stuffiness of the nose (six patients, 11.1%), and dyspepsia (twopatients, 3.7%). The events were of mild severity, persisted for up to 1.5 h, anddid not require treatment with additional pharmaceuticals or drug withdrawal.

AE were not revealed during Impaza therapy. Impaza had no adverseeffect on the course of underlying diseases. Therapy correction was not required.

The design of study and results of combination therapy are shown inFig. 8.17.

Viagra monotherapy was ineffective in 18 patients. The therapy wassupplemented by Impaza (1 tablet every other day). Combination therapy hada positive effect in eight patients. Ten patients not responding to combinedtreatment were prescribed to take Sialis. Sialis was effective in two of thesepatients. The remaining eight patients were prescribed to receive intracavernousinjections. Combination therapy with Impaza and Viagra was given to 12patients with serious side effects (severe headache), which resulted from the useof Viagra in a dose of 100 mg. The dose of Viagra was reduced to 50 mg in eightof these patients (previously ineffective dose). This therapy was effective and didnot cause side effects. The dose of Viagra could not be reduced in four patients.These patients were successfully treated with Sialis, which did not cause side effects.

Combined treatment with Impaza was followed by a positive effect in 6 of12 patients from the Sialis group. The remaining six patients demonstrated a goodresponse to Viagra in a dose of 100 mg. Five patients received therapy withintracavernous injections and entered the Levitra group in the follow�up period.

Patients not responding to Impaza (n=32) were divided into two equalgroups (group 1, Viagra; group 2, Sialis). Viagra and Sialis had a positive effectin 11 patients (68.7%) and 12 patients (75%). The remaining nine patientsreceived intracavernous injections.

These data show that combination therapy with two inhibitors of PDE�5 and Impaza increases the overall efficacy of oral therapy for ED up to 90%.

Fig. 8.16.Fig. 8.16.Fig. 8.16.Fig. 8.16.Fig. 8.16. Efficacy of Impaza therapy: percentage of patients with a favorableresponse.

0

10

20

30

40

60%

50

1 2 3

Duration of therapy, months

4 5


238

Ultralow doses

Four primary patients did not respond to Levitra monotherapy (Fig. 8.18).None of the PDE�5 inhibitors had a positive effect in six patients. After threeunsuccessful attempts of sexual intercourse during Levitra treatment, tenpatients were prescribed to receive combination therapy with Impaza (1sublingual lozenge every other day). This scheme of treatment had a positiveeffect in seven patients (3 months of therapy). Therefore, combination therapywith Impaza and Levitra was followed by an increase in the efficacy of therapyfrom 81.5 to 94.4%.

The efficacy and safety of Impaza for ED of various etiologies wereconfirmed in accordance with the principles of evidence�based medicine.

The course of prolonged treatment with Impaza was most effective.Impaza differs from other products for pharmacotherapy of ED in the

mechanism of action, profile of therapeutic activity, safety profile, and influenceon androgen status.

Preclinical and clinical studies showed that Impaza has a pathogeneticeffect during ED, including the recovery (increase) of nitric oxide productionin the endothelium (key process in erectile function).

Fig. 8.17.Fig. 8.17.Fig. 8.17.Fig. 8.17.Fig. 8.17. Efficacy of PDE�5 inhibitors and Impaza in combination therapy for ED.“+”, presence of effect; “�”, no effect.

218 patients

64 patients 73 patients81 patients

+Impaza +Impaza

Viagra Sialis Impaza

63 18 52 12 41 32

161666108

8 4 2 8 1 5 12 4 11 5

12

100 mg

Sialis

Sialis50 mg

Viagra

Other forms of therapy

Sialis Viagra+Impaza

+ �

+ �+ �

+ � + �+ �+ �+ �

+ � + �81,3% 56,2%

68,7%

87,7%92,2%90,1%

75%

77,8%

Side effects

239

The pathogenetic mechanism for action of Impaza explains the fact thata clinical effect of this product develops more slowly compared to other oraldrugs for ED.

An individual approach is required to develop the scheme of drugtreatment for each patient. It is necessary to take into account all factors thatmodulate the clinical effect of study drug.

High efficacy of Impaza in patients with primarily psychogenic ED andvascular arterial ED, as well as a good combination of Impaza and nitrates inCHD patients indicate that Impaza holds promise as a first�line drug for EDin patients of these groups.

Taking into account the mechanisms for action of Impaza and oralinhibitors of PDE�5, combination therapy is required to increase the clinicalefficacy of these drugs.

The therapeutic algorithm for ED patients was developed from the resultsof combined treatment (Fig. 8.19).

If a young patient has psychogenic, isolated neurogenic (osteochondrosis),compensated and subcompensated arteriogenic ED of mild or moderate severity,and CHD (nitrate therapy), treatment can be started from Impaza. PDE�5


Fig. 8.18.Fig. 8.18.Fig. 8.18.Fig. 8.18.Fig. 8.18. Efficacy of combination therapy with Impaza and Levitra.

2816 6

7 3

10

4

Levitra

+Impaza

Levitra+ � + �

+ �

72,7%

94,4%

87,5%

22 patients: ineffectiveness

of Viagra and Sialis

32 primary patients with ED

54 patients

240

Ultralow doses

inhibitors are prescribed for patients with Impaza inefficacy, as well as forpatients with severe or moderate venooclusive ED.

Pharmacotherapy with PDE�5 inhibitors should not be withdrawn whenone of these drugs appears to be ineffective. Another inhibitor of PDE�5 canbe prescribed under these conditions. At low efficacy of PDE�5 inhibitors, theyshould be combined with Impaza. This scheme of treatment will allow us toreduce side effects of monotherapy due to a decrease in the dose of PDE�5inhibitors. The efficacy of therapy will remain unchanged.

When therapy is chosen, a physician should perform dynamic monitoringand evaluation of the patient’s state.

From the first days of treatment, Impaza should be given in combinationwith PDE�5 inhibitors. Besides the above mentioned advantages, thiscombination allows us to increase the interval between drug intakes. Thesepatients maintain the ability to complete successful sexual intercourse.

Prolonged therapy with Impaza and PDE�5 inhibitors is accompanied by therecovery of adequate and spontaneous erections. This conclusion is derived fromthe patient’s report, increase in cavernous blood flow (Doppler ultrasonographyof the penis with audiovisual sexual stimulation), and elevation of cavernous

Fig. 8.19.Fig. 8.19.Fig. 8.19.Fig. 8.19.Fig. 8.19. Algorithm of differential combined pharmacotherapy with Impaza forerectile dysfunction (E. B. Mazo et al., 2004).

Impaza Arteriogenic and neurogenic ED

Another pathogenesis of ED

•sexual activity more than twice a week•sexual intercourse in the morning•necessity of spontaneous intercourse

•combination of sex with food intake or alcohol consumption

•necessity of the rapid effect•severe ED

Combination of Impaza and PDE�5 inhibitors

Other forms of therapy

Sialis

Viagra

Levitra

Six months of therapy

Mild Moderate

Complex andrological examination

Severe

+ �

+ �

+ �

+ �

“not” for everything

“not” for everything

no effect

“yes”

“yes”

241

electrical activity (penile electromyography). It is possible to reduce the mini�mum effective dose and to withdraw PDE�5 inhibitors. The patient is transferredto Impaza monotherapy. This drug may be withdrawn in the follow�up period.

The proposed scheme of combined pharmacotherapy with Impaza has apositive effect in more than 90% ED patients, increases the safety of treatment,and significantly decreases the cost of therapy.

8.3. Clinical effectiveness and mechanismsfor action of Anaferon

Among a variety of products for therapy of acute respiratory viralinfections (ARVI), much attention is given to pathogenetic drugs that affect thenatural mechanisms of antiviral resistance (primarily the IFN system). Despitemuch progress in vaccine prevention of influenza, the antigenic variability of thisvirus and high incidence of mixed infections in clinical practice require thedevelopment of universal remedies for ARVI prophylaxis. Modulation of theendogenous IFN system as a natural mechanism of antiviral resistance holdsmuch promise in this respect.

The adequate induction of endogenous IFN provides a mild or abortivecourse of viral infections. Therefore, IFN�inducing agents have a particularplace in the therapy for ARVI. Moreover, viral infections often cause oraccompany secondary immunodeficiency. The strategy of immunorehabilitationwith immunomodulatory drugs should be used for the therapy and preventionof ARVI, particularly in children. IFN inductors are effective in the therapy ofviral infections. A variety of side effects limit the prolonged use of these drugsfor prevention of viral infections, particularly in children.

Experimental and clinical studies showed that Anaferon, a newimmunomodulator with antiviral activity (Russia), holds promise for therapy andprevention of ARVI. Anaferon contains antibodies to human IFN�γ. The drugformulation for children was designated as “Anaferon for children” (V. F.Uchaikin et al., 2003). Anaferon induces the production of endogenous IFN�γand IFN�α/β and affects the expression of functionally related cytokines,including IL�2, IL�4, and IL�10 (A. V. Martyushev�Poklad, 2003).

Previous experiments showed that endogenous IFN�γ�sensitive immunecells serve as a target for Anaferon (Fig. 8.20; A. V. Martyushev�Poklad, 2003).

The most probable mechanisms for therapeutic activity of Anaferon inARVI are shown in Figs. 8.21 and 8.22 (according to the results of experimentaland clinical studies).

The early and frequent use of Anaferon from the onset of viral infectionprovides the induction of IFN�α, IFN�β, and IFN�γ and activation of NK cells.


242

Ultralow doses

IFN suppresses viral replication and prevents infection of other cells. NK cellslyse infected cells. These processes determine the rapid antiviral effect of Anaferon.

The induction of endogenous IFN�γ as a key immunoregulatory cytokinetriggers the cascade of other events in the immune system. They include theinduction of regulatory components for the immune response (type 1 and 2 Thelper cells) and activation of macrophages. Activation of effector componentsof the cellular and humoral response is realized via cytotoxic lymphocytes andantibodies and contributes to lysis of infected cells, binding of viral particles, andelimination of the virus.

Sufficient activation of macrophages and antibody production prevent thedevelopment of bacterial complications after viral infection.

Randomized controlled clinical studies of the efficacy and safety of Anaferonfor children in children with influenza and other ARVI were performed at the In�stitute of Influenza (Russian Academy of Medical Sciences, St. Petersburg), Rus�sian State Medical University (Moscow), and Volgograd State Medical University.The trial involved 390 children (from 1 month to 14 years of age). The childrenreceived inpatient or outpatient therapy for influenza (verified by an immunofluor�

Fig. 20.Fig. 20.Fig. 20.Fig. 20.Fig. 20. Natural regulation of the immune response and targets for Anaferon. Th, Thelper cells; AG, antagonists; AB, antibodies; MP, macrophages; NK, natural killer cells.

NK

NK

TcTh 1

Th 0

Th 2

ANAFERON

TNF�βIFN�γIL�2

IL�4

B

IL�12

IL�12

PL

AB

MP

IL�2IL�10IL�13

IL�10IL�6

IL�12TNF�αIFN�γIFN�γ

IFN�γ

Cellular immune response

Humoral immune response

Th 1 cytokines

Th 2 cytokines

AG

AG

Monocyte

Monocyte

Basophil

ANAFERON

243

Fig. 8.22.Fig. 8.22.Fig. 8.22.Fig. 8.22.Fig. 8.22. Mechanisms for the immunomodulatory effect of Anaferon.

Fig. 8.21.Fig. 8.21.Fig. 8.21.Fig. 8.21.Fig. 8.21. “Rapid” mechanisms for the antiviral effect of Anaferon.

IFN�α/β

Suppression of viral

replication Lysis of infected

cells

Activation of NK cells

Induced resistance

of uninfectedcells

ANTIVIRAL EFFECT

IFN�γ

ANAFERON

Activation of NK

Induction of regulatory

components for the immune

response

IFN�γ

ANAFERON

Activation of macrophages

Antibody production

Lysis of infected

cells

Binding of viral

particles

B lymphocytes (proliferation

and differentiation)

Cytotoxic T lymphocytes (proliferation, differentiation, and activation)

ELIMINATION OF VIRUSES

Type 2 T helper cells

Type 1 T helper cells

IgM, IgG,IgA


244

Ultralow doses

Fig. 8.23.Fig. 8.23.Fig. 8.23.Fig. 8.23.Fig. 8.23. Effect of Anaferon for children on the duration of main clinical mani�festations of influenza. *p<0.05 compared to placebo.

0

1

2

3

4

5

7Days

6

Fever (>37°С)

Intoxication Rhinitis Cough

placebo Anaferon

!

!

!!

escence study) and other ARVI, including those complicated by laryngotracheo�bronchitis with stenosis or bacterial infection. Anaferon lozenges were given three�seven times a day, which depended on the stage of disease. The product was dis�solved in water for treatment of infants. Patients of the reference groups receivedplacebo, Immunal, or Arbidol in combination with symptomatic and/or antibac�terial therapy (when prescribed). The main symptoms of disease (fever, intoxica�tion, and catarrhal symptoms) and possible side effects were evaluated. The in�terferon system and immune status (subpopulations of peripheral blood lympho�cytes, serum IgE, and sIgA in nasal lavage fluid) were studied in some children.

Anaferon therapy was followed by a significant decrease in the severity andduration of main clinical manifestations of ARVI and complications in children(p<0.05 compared to the placebo group). The duration of fever, intoxication,rhinitis, and cough decreased by 35�40, 40�50, 20, and 30%, respectively.

Fig. 8.23 illustrates the main results of a placebo�controlled trial of theefficacy of Anaferon for children during influenza (Institute of Influenza,Russian Academy of Medical Sciences). The diagnosis of influenza was verifiedby laboratory tests.

The trial involved 105 patients (1�10 years of age, average age 5.40±0.02years). Besides the presence of influenza A and/or B virus, 59% patients wereshown to have the adenovirus, respiratory syncytial virus, parainfluenza virus,coronavirus, or mycoplasma as etiologic factors of the disease.

In addition to the reduction of disease duration, patients of the Anaferonfor children group were characterized by a significant decrease in the incidenceof purulent rhinitis as a major complication of influenza (p<0.05; Fig. 8.24).

The use of Anaferon was followed by a decrease in the period of inpatienttreatment and volume of antibacterial and symptomatic therapy. Drug�relatedadverse events were not observed in patients of the Anaferon group.

Anaferon for children improved the IFN status of patients. This remedynot only promoted the induction of endogenous IFN on days 2�3 of therapy,

245

Fig. 8.24.Fig. 8.24.Fig. 8.24.Fig. 8.24.Fig. 8.24. Effect of Anaferon for children on the incidence of purulent rhinitis afterinfluenza. *p<0.05 compared to placebo.

but also prevented a decrease in IFN production by lymphocytes at the stageof reconvalescence (Fig. 8.25).

The ability of Anaferon to induce not only endogenous IFN�γ (as shownin a preclinical study), but also IFN�α in patients with viral infection is relatedto the existence of a close functional relationship between IFN�γ and IFN�α.Published data show that IFN�γ potentiates the induction of IFN�α during viralinfection (A. P. Costa�Pereira et al., 2002). These data are of particular import�ance since one of the major “strategies” of influenza virus in overcoming themechanisms of natural resistance includes the inhibition of early interferon in�duction (X. Wang et al., 2000).

The content of various subpopulations of peripheral blood lymphocytes(CD3+, CD4+, CD8+, CD20+, and CD16+) returned to normal after drug ther�apy (Fig. 8.26). These changes were accompanied by a decrease in IgE level andincrease in sIgA level (Fig. 8.27).

0

5

10

15

20

30

25


Placebo Anaferon

!


Fig. 8.25.Fig. 8.25.Fig. 8.25.Fig. 8.25.Fig. 8.25. Effect of Anaferon for children on the IFN status of influenza patients:stimulated production of IFN�γ (a) and IFN�α by peripheral blood leukocytes (b).Days 1�2 of disease (1); days 2�3 of therapy (2); and convalescence (3). *p<0.05compared to placebo.

01

a b

2 3 1 2 3

100

400pg/ml pg/ml

200

300

placebo Anaferon

0

10

20

30

40

60

50

!

!

!

!

246

Ultralow doses

Fig. 8.27.Fig. 8.27.Fig. 8.27.Fig. 8.27.Fig. 8.27. Parameters of humoral immunity during therapy of influenza patientswith Anaferon for children: secretory IgA in nasal lavage fluid (a); and IgE in bloodserum (b). *p<0.05 compared to the baseline value.

0

40

160

% of the baseline

Control Anaferon Control Anaferon

80

120

0

40

160

80

120

before therapy after therapy

a b

!

!

!

!

% of the baseline

Hence, the therapeutic use of Anaferon has an immunomodulatory effecton influenza patients. These data are consistent with the results of preclinicalstudies and confirm the fact that Anaferon modulates the mechanisms ofantiviral resistance (innate and adaptive cellular and humoral responses; L. V.Osidak et al., 2003).

Fig. 8.26.Fig. 8.26.Fig. 8.26.Fig. 8.26.Fig. 8.26. Effect of Anaferon for children on subpopulations of peripheral bloodlymphocytes in influenza patients. *p<0.05 compared to placebo.

0

10

20

30

40

50

60

70

80

90

100

CD3+ CD4+ CD8+ CD20+

Lymphocyte subpopulationCD16+ CD3+/CD8+

Children with normal content of lymphocytes, %

before therapy (control) after therapy (control)

before therapy (Anaferon) after therapy (Anaferon)

!!!

!

!

!

247

Among a variety of ARVI, respiratory syncytial virus (RSV) infections havea particular place. These infections most often cause complications (disorders ofthe lower respiratory tract) and require hospitalization. A randomized placebo�controlled study of the efficacy of Anaferon for children in patients with RSVinfection was performed at the Institute of Influenza (Russian Academy ofMedical Sciences). The patients with confirmed RSV infection (1�10 years ofage) received Anaferon (n=40) or placebo (n=36) in combination withsymptomatic drugs for 7�10 days (from the 1st or 2nd day of disease). Anaferontherapy was followed by a decrease in the mean duration of hyperthermia (by1.2 days), period of intoxication (by 1 day) and catarrhal syndrome (by 2.2days), and total length of disease (by 2.3 days, p<0.05). Similarly to influenzapatients, Anaferon induced the production of endogenous IFN�α and IFN�γ byperipheral blood leukocytes (days 2�3 of therapy) and prevented a change in theIFN status (period of reconvalescence; E. G. Golovacheva et al., 2003).

A randomized placebo�controlled study of the efficacy of Anaferon forchildren in ARVI prevention was performed at the Institute of Influenza (Rus�sian Academy of Medical Sciences, St. Petersburg) and Russian State MedicalUniversity (Moscow). The trial involved 400 children (from 1 month to 4 yearsof age, 70% patients with poor health). The drug or placebo (lozenges) wasgiven daily for 3 months. The product was dissolved in water for treatment ofinfants. ARVI patients were prescribed to take Anafaron as a therapeutic drugin combination with symptomatic therapy. The morbidity rate, severity of ARVI,incidence of complications, and drug tolerability were evaluated during Anaferontherapy. The IFN status in some patients was studied before and after the startof preventive treatment. Latent infection with various viruses was determined byan immunofluorescent study of nasal lavage fluid.

Both trials in various clinical institutions yielded the same results (Fig.8.28). The average number of ARVI in one patient decreased by 1.5�1.8 times.This drug had a stronger effect on the incidence of severe (febrile) ARVI, whichwas reduced by 2.1�2.4 times.

The prophylactic and therapeutic�and�prophylactic effects of this drug werestudied in details at the Institute of Influenza. During this trial, the patients wereexamined daily. The preventive and therapeutic use of Anaferon for children wasfollowed by a decrease in the incidence and severity of ARVI and complications.The duration of all symptoms (fever, intoxication, and/or catarrhal symptoms)decreased by 2.2 times. The duration of fever and purulent rhinitis decreased by3.8 and 3 times, respectively (Fig. 8.29). The incidence of ARVI complicationsdue to purulent rhinitis and otitis decreased by 2.4 and 2.7 times, respectively.

The decrease in ARVI morbidity was most pronounced in children withpoor health. The percentage of children not suffering from ARVI over a3�month trial increased from 3 to 24.7%.


248

Ultralow doses

Fig. 8.29.Fig. 8.29.Fig. 8.29.Fig. 8.29.Fig. 8.29. Effect of therapeutic and prophylactic treatment with Anaferon forchildren (3�month course) on the total duration of ARVI symptoms (a) and incidenceof ARVI complications in children (b). *p<0.05 compared to placebo.

Purulent rhinitis

Otitis

0

10

20

30

50

60

40!

!

Incidence per 100 patients per monthNumber of days

Any symptom of ARVI/

complication

Subfebrile/febrile state

Purulent rhinitis

placebo Anaferon

0

5

10

15

20

35

a b

30

25

!

!

!

Anaferon did not cause AE. An immunofluorescent study of nasal lavagefluid showed that latent infection of children (occurrence of typical agents forARVI) decreased from 48.1 to 22.9%. Latent infection with herpes simplex virusdecreased from 13.5 to 4.4%. After the course of treatment with Anaferon forchildren, the ability of peripheral blood lymphocytes to produce IFN�α/β uponstimulation increased by 2.2 times.

The efficacy and safety of Anaferon for children as a drug for the therapyand prevention of influenza and other ARVI in children were confirmed inrandomized clinical trials (in accordance with the principles of evidence�basedmedicine). This remedy not only decreases the duration and severity of clinicalmanifestations of ARVI and reduces the morbidity rate from viral infections, but

Fig. 8.28.Fig. 8.28.Fig. 8.28.Fig. 8.28.Fig. 8.28. Effect of prophylactic treatment with Anaferon for children (3�monthcourse) on the overall incidence of ARVI (a) and incidence of febrile ARVI (t>37.5oC,b). Results of two placebo�controlled trials: Institute of Influenza (I) and RussianState Medical University (II). *p<0.05 compared to placebo.

I II0

10

20

30

50

60

40

!

!

ARVI morbidity per 100 patients per month

I II0

1

3

а b

2

placebo Anaferon

!

!

ARVI morbidity per one patient

249

also contributes to immunorehabilitation of patients with clinical and laboratorysigns of secondary immunodeficiency (L. V. Osidak et al., 2003; A. V. Martyu�shev�Poklad et al., 2004a,b; S. A. Sergeeva et al., 2004).

A deficiency of immune resistance and inadequacy of the immuneresponse play an important role in the pathogenesis of viral infections (includingherpes virus infections, enterovirus infections, and viral hepatitides). A combina�tion of immunomodulatory and antiviral properties of Anaferon is related to theinduction of endogenous IFN and provides a significant clinical effect of thedrug during viral infections.

Studying the prophylactic efficacy of Anaferon showed that this remedypromotes the elimination of herpes simplex virus under conditions of latent virusinfection (Institute of Influenza, Russian Academy of Medical Sciences). Theseresults were confirmed by further observations. The family of herpes viruses alsoincludes the agents that cause infectious mononucleosis (Epstein�Barr virus) andchickenpox (Herpes zoster).

The therapeutic efficacy and safety of Anferon for children in patientswith infectious mononucleosis (Epstein�Barr virus) were evaluated in a double�blind placebo�controlled trial at the Siberian State Medical University (Tomsk).The efficacy of Anaferon in various therapeutic schemes for infectious mono�nucleosis was also studied at the Ural State Medical Academy (Ekaterinburg).The trials involved 140 children (3�14 years of age) with moderate�to�severe andsevere infectious mononucleosis. The efficacy of Anaferon for children,acyclovir, cycloferon, combination of Anaferon and acyclovir, and placebo wasevaluated. The patients also received antibacterial and symptomatic therapy(when prescribed). The therapeutic efficacy was estimated from main clinicalmanifestations, including the duration of fever and acute tonsillitis. The trial inTomsk showed that Anaferon significantly decreases the duration of clinicalsymptoms (as compared to placebo). The duration of acute tonsillitis, hepato�megaly, and lymphadenopathy decreased by more than 1, 3, and 4 days, respec�tively. Body temperature rapidly returned to normal after Anaferon therapy.Studying the efficacy of Anaferon in various therapeutic schemes for infectiousmononucleosis was performed in Ekaterinburg. A combination of Anaferon andacyclovir was most effective under these conditions. The mean duration of feverand acute tonsillitis in patients of various groups appeared as follows: 2.4 and 4.3days, respectively, in the Anaferon group; 2.2 and 2.8 days, respectively, in theAnaferon+acyclovir group; 5.1 and 4.1 days, respectively, in the acyclovir group;5.4 and 4.8 days, respectively, in the cycloferon group; and 8.7 and 6.5 days,respectively, in the placebo group. Therefore, Anaferon and other inductors of in�terferon hold much promise for the therapy of infectious mononucleosis. IFN�in�ducing agents should be used in combination with antiviral drugs (L. A. Zhu�ravleva et al., 2003a,b; K. I. Chuikova et al., 2004; V. V. Fomin et al., 2004).


250

Ultralow doses

Fig. 8.30.Fig. 8.30.Fig. 8.30.Fig. 8.30.Fig. 8.30. Efficacy of Anaferon for children in chickenpox: effect on the durationof main clinical symptoms (a) and temperature curve (b). Placebo (1) and Anaferon(2). (b) Evening temperature, e. *p<0.05 compared to placebo.

36,536,736,937,1

37,5

37,9

°С

1 1e 2 2e 3

Period of therapy, daysлечения, сут

3e 4 4e 5

37,3

37,7

38,0

0123

5

8

Duration, days

Fever New spots Itching

7

4

6

placebo Anaferon

!!

!

a b

2

1

A double�blind, randomized, placebo�controlled trial of the efficacy andsafety of Anaferon for children in chickenpox was performed at the Vol’skChildren’s Hospital (Saratov oblast). The trial involved 236 children (1�15 yearsof age). The duration of disease did not exceed 48 h. Anaferon for children(n=136) or placebo (n=100) was given therapeutically in combination withsymptomatic drugs (treatment of rash with 2% solution of brilliant green).

The time to normalization of body temperature, new crops of skin rash,relief of itching, and incidence and severity of complications were evaluated.

Anaferon significantly decreased the duration and severity of disease (Fig.8.30). Body temperature in Anaferon�receiving children returned to normal bythe 3rd day of therapy (vs. 6th day in the reference group). A correlation wasfound between the occurrence of skin rash and body temperature, which isconsistent with published data. Anaferon prevented the appearance of new spotsin an earlier period (by 3 days). The duration of itching decreased by 4 days inthe Anaferon group (as compared to placebo). The percentage of children withbacterial infection�associated pustules was sevenfold higher in the referencegroup compared to the Anaferon group. These data illustrate the efficacy andsafety of Anaferon for children in the therapy of chickenpox (M. V. Kudin,2005a,b; A. V. Martyushev�Poklad et al., 2005b).

The development of acute intestinal infections is one of the urgentproblems in treatment of children’s infections. According to the internationalstatistics, 70�80% of intestinal infections are caused by viruses. Rotaviruses,caliciviruses, and coronaviruses are the major etiologic agents for these diseases.

The efficacy and safety of Anaferon for children in acute intestinalinfections of viral etiology were confirmed by several clinical studies.

A double�blind randomized clinical trial was performed at the Institute ofInfluenza (St. Petersburg). The trial involved 79 patients (from 6 months to 10

251

years of age). The diagnosis of coronavirus infection was confirmed bylaboratory tests. Patients of the main (n=51) and control groups (n=28) receivedAnaferon and placebo, respectively, in combination with standard therapy. Theclinical state at admission was moderate�to�severe. The main symptoms ofcoronavirus infection were rapidly relieved in Anaferon�receiving patients. Thesepatients were characterized by a significant decrease (p<0.05 compared to theplacebo group) in the duration of gastrointestinal symptoms (from 4.4±0.5 to2.5±0.2 days), fever (from 3.4±0.3 to 2.0±0.1 days), intoxication (from 5.1±0.4to 2.9±0.1 days), and catarrhal symptoms in the nasopharynx (from 7.0±0.4 to4.3±0.2 days; E. V. Obraztsova et al., 2006).

Two�day treatment of children with coronavirus infection of the main andcontrol groups was followed by a significant decrease in the number of patientswith fever (by 75 and 44%, respectively).

A double�blind, placebo�controlled clinical trial of the efficacy and safetyof Anaferon for children in calicivirus infection was performed at the Instituteof Children’s Infections (St. Petersburg). The trial involved 60 children (from6 months to 15 years of age) that were hospitalized with symptoms of acuteintestinal infection. The diagnosis of calicivirus infection was verified by meansof scanning electron microscopy. The patients were divided into two groups ofAnaferon for children (n=30) and placebo (n=30). The groups were represen�tative of age, sex, and severity of disease. The use of Anaferon for 5�7 days wasfollowed by a significant decrease in the duration of symptoms of calicivirusinfection. The duration of fever, vomiting, and diarrhea in patients of the maingroup was 1.3±0.2, 1.5±0.2, and 1.2±0.1 days, respectively. Fever, vomiting, andwatery diarrhea in patients of the control group persisted for 2.9±0.3, 2.4±0.2,and 2.0±0.2 days, respectively (p<0.05). Anaferon had a strong effect on theduration of virus elimination. By the end of treatment, caliciviruses weredetected in the feces from 1 patient of the main group (3%) and 16 patients ofthe control group (53%; I. V. Razdyakonova et al., 2005).

A pilot study of the efficacy of Anaferon for children in rotavirusgastroenteritides was performed at the Rostov State Medical University (Rostov�on�Don). The trial involved 27 children (1.5�5.5 years of age). The diagnosis ofrotavirus gastroenteritis was confirmed by laboratory tests. Thirteen patientsreceived Anaferon (therapeutic treatment) and standard therapy. Fourteenpatients received only standard drugs. The introduction of study drug intocombination therapy was followed by a significant decrease in the duration ofmain symptoms of this disease (p<0.001). The duration of fever and diarrheadecreased by 1.5 and 2.3 days, respectively (E. N. Simovan’yan et al., 2004).

Clinical studies demonstrated the efficacy and safety of Anaferon forchildren in treatment of acute intestinal infection of viral etiology. Anaferonsignificantly decreased the time to virus elimination from an organism, which


252

Ultralow doses

reflects the antiviral effect of this remedy. The product was well tolerated anddid not cause AE. These data indicate that Anaferon for children should be usedin combination therapy of children with acute viral infections. Anaferon had apositive effect in patients with tick�borne encephalitis (A. V. Skripchenko et al.,2005), hemorrhagic fever with renal syndrome (E. B. Egorov et al., 2004),pseudotuberculosis (V. N. Timchenko et al., 2004), tubulointerstitial nephritis (I.F. Vladimirtseva et al., 2005), and enteroviral and meningococcal meningitis(Yu. B. Khamanova et al., 2005). Anaferon was also used in emergency pro�phylactic therapy of ARVI in children with bronchial asthma (E. I. Kondrat’evaet al., 2004, 2005, 2006), as well as in the treatment of ARVI in children withheart diseases (L. V. Yakovleva et al., 2005).

Controlled clinical trials in leading medical institutions of the RussianFederation showed that Anaferon holds much promise for the prevention andtherapy of influenza, viral infections of the respiratory tract, and herpesinfection (including chickenpox and infectious mononucleosis). Anaferon canbe used in combination therapy for acute intestinal infections of viral etiology,prevention and treatment of complications of viral infections, therapy ofsecondary immunodeficiency of various etiologies, and combination therapy forbacterial infections.

8.4. Artrofoon as a promising drugfor pathogenetic therapyof chronic arthropathies

The introduction of biological antirheumatic drugs into clinical practicein various countries has opened new perspectives in the therapy of RA, osteo�arthritis (OA), and other autoimmune inflammatory diseases. Unfortunately,these drugs are of limited use in Russia due to the high cost. Artrofoon (ULDof antibodies to TNF�α, Russia) is a new drug for the therapy of RA. Six�month clinical studies were designed to evaluate the efficacy and safety of Artro�foon in RA and OA. The drug was also used in inflammatory and degenerativediseases of the joints.

The clinical efficacy of Artrofoon during RA was evaluated in an openrandomized study at the Volgograd State Medical University (V. I. Petrov et al.,2003, 2005; A. V. Martyushev�Poklad et al., 2003; V. I. Petrov et al., 2003; J.L. Dugina et al., 2005a,b). Diclofenac was used as a reference drug. The trialinvolved 81 patients with RA (according to the criteria of the AmericanRheumatology Association, ARA). Thirty�one patients (average age 54.0±1.1years, mean duration of disease 11.6±1.2 years) received diclofenac in a dailydose of 100 mg. Fifty patients (average age 51.2±1.5 years, mean duration of

253

disease 8.9±1.0 years) were treated with Artofoon (4 tablets twice daily) for 6months. The average number of damaged joints in patients of the diclofenac andArtrofoon groups was 23.8±1.3 and 22.7±1.3, respectively. Seronegative RA wasdiagnosed in 48.4 and 42% patients of the diclofenac and Artrofoon groups,respectively. Among patients enrolled in the trial, 12.9% patients of thediclofenac group and 25% patients of the Artrofoon group previously receivedstandard therapy (methotrexate, 7.5 mg weekly). This treatment was ineffectivein 45 and 26% patients of the diclofenac and Artrofoon groups, respectively.During the trial, standard therapy was continued in 16.1% patients of thediclofenac group and in 18% patients of the Artrofoon group.

A 6�month course of treatment with diclofenac was followed by adecrease in the number of painful joints (from 19.2±1.3 to 17.4±1.2, by 1.9±0.5or 9.4%) and swollen joint (from 8.3±1.0 to 5.7±0.7, by 2.6±0.6 or 27.5%) andreduction of the duration of morning stiffness (from 131±13 to 102±9 min, by29±7 min or 17.3%). The improvement of clinical symptoms was morepronounced in patients of the Artrofoon group. Artrofoon therapy was followedby a decrease in the average number of painful joints (from 17.4±1.1 to13.6±0.9, by 3.8±0.6 or 20.5%) and swollen joint (from 6.2±0.7 to 3.8±0.4, by2.4±0.5 or 33.6%) and reduction of the duration of morning stiffness (from136±10 to 87±6 min, by 49±5 min or 33.2%).

After 6 months of therapy with diclofenac, 25.8% patients reached theACR20 criterion (20% improvement of clinical symptoms, CI 95% = 13.7�43.3%). The number of Artrofoon�receiving patients who reached the ACR20criterion was twofold higher than in the diclofenac group (58% patients, CI95% = 44.2�70.6; Fig. 8.31).

The symptoms of RA in patients were reduced after 1 month of therapywith diclofenac. In the follow�up period, the effect of diclofenac increased lesssignificantly than that of Artrofoon. Artrofoon had a positive effect on local andgeneral symptoms of inflammation in RA patients. A significant improvement


Fig. 8.31.Fig. 8.31.Fig. 8.31.Fig. 8.31.Fig. 8.31. Patients with rheumatoid arthritis who reached the ACR20

improvementcriterion after 6 months of therapy.

%

Artrofoon Diclofenac (100 mg daily)

0

10

20

30

40

60

50

254

Ultralow doses

Fig. 8.32.Fig. 8.32.Fig. 8.32.Fig. 8.32.Fig. 8.32. Effect of 6�month therapy with Artrofoon (light bars) and diclofenac (darkbars) on the severity of rheumatoid arthritis. Mean improvement of the RA activity(a); and percentage of patients with an improvement by at least 20% (b). CRP, C�reactive protein. *p<0.05 compared to the diclofenac group.

01020

4050

70

90

Number of painful

joints

Severity of pain

Duration of morning

stiffness

CRP level

80

60

30

0

a b

10

20

30

40

50

60

Number of painful

joints

Severity of pain

Duration of morning

stiffness

CRP level

% of the baseline % of the baseline

Artrofoon diclofenac, 100 mg daily

!

!

!

!

of the patient’s state was observed 3 months after the start of therapy. The effectof Artrofoon developed progressively and reached maximum by the 6th monthof therapy (Fig. 8.32).

The severity of pain in patients of the diclofenac and Artorfoon groupsdecreased by 21.0 and 34.7%, respectively. Roentgenologically, progression of thedisease was not found in patients of both groups. The effect of study drug wasrated as “good” by 54% patients of the Artrofoon group and 32.3% patients ofthe diclofenac group.

All patients who were enrolled in the trial completed a 6�month courseof therapy. Artrofoon had a better safety profile than diclofenac. Serious adverseevents were not reported in the Artrofoon group. AE in 22.6% patients of thediclofenac group were associated with drug effect on GIT (pain of discomfortin the epigastric region, eructation, and nausea). These events required theprescription of antacid drugs and/or antiemetic agents.

Eleven patients of the Artrofoon group continued to take the drug after6 months of therapy (five patients for 12 months; one patient for 11 months;one patient for 10 months; one patient for 9 months; and three patients for 8months). By the end of treatment, all patients reached the ACR20 criterion.

The relief of clinical symptoms for joint disease in Artrofoon�receivingpatients was accompanied by a significant improvement in laboratory signs ofinflammation, including the concentration of proinflammatory cytokines. Insome patients the concentration of proinflammatory cytokines significantlydecreased (>25%) after 6 months of therapy with Artrofoon. They demonstrateda decrease in the concentrations of plasma TNF�α (50% patients), IL�1 (70%

255

patients), and IL�6 (50% patients). These changes were not found in patientsof the control group.

These data suggest that antiinflammatory activity of Artrofoon is relatedto the regulation of systemic production of proinflammatory cytokines.

An open comparative clinical trial of the efficacy and safety of Artrofoonvs. diclofenac in OA patients was performed at the Volgograd State University(I. V. Kostryukova et al., 2005; I. V. Kostryukova et al., 2006). The trial involved120 patients with the diagnosis of OA (112 women, 93.33%; and 8 men, 6.66%;average age 63.84±0.88 years). The majority of patients were older than 60 yearsof age (66.6% disabled persons). They had polyarticular (101 patients, 84.16%),oligoarticular (11 patients, 9.16%), monoarticular OA (8 patients, 6.66%). Themajority of OA patients (n=72, 60%) suffered from severe synovitis, which wasmanifested in pain at rest, swelling, hyperthermia, and functional disorders.Subclinical synovitis in 48 patients (40%) was manifested in spontaneous nighttimepain, morning stiffness, and local tenderness in palpation. Pain sensation in allpatients was revealed during joint movement and palpation of the joints andperiarticular tissue. Damage was most often observed in the knee joints (90.83%),ankle joints (50.83%), hip joints (20.83%), and small joints of the hand (31.66%).

All patients with OA were randomized into the following three groups:group 1 (60 patients), Artrofoon (4 tablets twice daily); group 2 (30 patients),reference drug diclofenac (100 mg daily); and group 3 (30 patients), com�bination therapy with Artrofoon (8 tablets daily) and diclofenac (100 mg daily).

Drug efficacy was evaluated 1, 3, and 6 months after the start of therapy.The following clinical parameters were evaluated: WOMAC index (WesternOntario and McMaster Universities Osteoarthritis Index); overall pain; articularindex; Ritchie index; swelling index; and Leken’s indexes for gonarthrosis and co�xarthrosis. Artrofoon safety was determined from the incidence of AE (Table 8.11).

The WOMAC total score in Artrofoon�receiving patients was 49.92±1.75(vs. 52.00±0.72 and 51.5±1.97 in the diclofenac and diclofenac+Artrofoongroups, respectively). The WOMAC total score in patients of these groupsdecreased by 10.94, 10.23, and 22.03 points, respectively, 1 month after the startof therapy. The WOMAC total score in these patients decreased by 24.22, 20.6,and 32 points, respectively, 3 months after the start of therapy. Six months afterthe start of therapy, the WOMAC total score in Artrofoon�receiving patients was18.72±1.81 points (decrease by 31.2 points, p<0.001 compared to the diclofenacgroup). This index also decreased in patients receiving diclofenac (by 25 points,27.53±2.52 points) and combination therapy (by 40.4 points, 11.10±1.12 points;p<0.001 and p<0.01 compared to the diclofenac and Artrofoon groups, respec�tively; Fig. 8.33).

Similar changes were found in clinical manifestations of OA (WOMACsubscales of pain, stiffness, and function). Six months after the start of therapy,


25

6

Ultra

low

do

ses

Parameter

Severity of pain 2.17±0.10 1.12±0.10** 0.58±0.10** 2.27±0.14 1.53±0.12** 1.57±0.12** 2.43±0.18 1.40±0.24** 0.57±0.10**

Durationof morning stiffness 19.37±1.77 8.72±1.13* 3.72±0.81* 18.83±2.71 12.17±1.57* 13.43±1.46 17.83±1.06 2.83±0.92* 1.00±0.56*

Ritchie index 6.45±0,40 3.73±0.31* 2.43±0.18** 7.23±1.01 5.47±0.87 4.87±0.87 5.70±0.38 2.77±0.31* 1.90±0.22**

Articular index 5.03±0.31 3.23±0.26* 2.30±0.21** 6.33±1.05 4.60±0.64 3.97±0.52* 4.67±0.25 2.27±0.25* 1.90±0.20**

Lee index 8.53±0.41 5.75±0.41* 4.00±0.30** 9.87±0.79 8.07±0.67 7.83±0.59* 9.03±0.33 5.20±0.27* 4.33±0.25*

Leken’s index 14.95±0.49 10.97±0.58* 8.28±0.54* 16.07±0.59 13.27±0.51* 13.00±0.53* 14.70±0.42 9.93±0.44* 8.00±0.32*

WOMAC index 49.92±1.75 25.70±1.89* 18.72±1.81* 52.00±1.72 31.40±2.49* 27.53±2.52* 51.5±1.97 19.50±1.23* 11.10±1.12*

ESR 17.90±1.16 12.92±0.73* 11.3±0.69* 20.77±1.96 14.83±0.78* 14.13±0.83* 18.13±1.20 11.87±0.88* 10.50±0.62*

C�reactive protein 9.30±0.58 7.20±0.37** 6.80±0.27** 10.20±0.87 7.80±0.77* 7.60±0.64* 10.20±0.96 7.40±0.55* 7.00±0.42*

Note.Note.Note.Note.Note. *p<0.05 and **p<0.01 compared to the baseline value.

Table 8.11.Table 8.11.Table 8.11.Table 8.11.Table 8.11. Main clinical and laboratory parameters of osteoarthritis during therapy with Artrofoon and diclofenac

Artrofoon + diclofenacDiclofenac (100 mg daily)Artrofoon (2 tablets, 4 times daily)

6 months3 months

afterbaseline

6 months3 months

afterbaseline

6 months3 months

afterbaseline

257

a decrease in the severity of pain, stiffness, and dysfunction was most significantin the combined treatment group and least pronounced in the diclofenac group.

After 6 months of monotherapy for OA, Artrofoon (4 tablets twice daily)was more effective than diclofenac (100 mg daily) for the relief of pain andimprovement of functional state and quality of life. The effect of Artrofoondeveloped more slowly than that of diclofenac. One month after the start oftherapy, the clinical efficacy of diclofenac was higher than that of Artrofoon.However, Artrofoon and diclofenac were equally potent by the 3rd month oftherapy. After 6 months of therapy, Artrofoon had a stronger effect thandiclofenac. The improvement of clinical symptoms in patients of the diclofenacgroup was not observed from the 3rd to the 6th month of treatment. The clinicaleffect was most pronounced in patients receiving combination therapy withArtrofoon and diclofenac. A significant clinical effect in these patients wasdetected after 1 month of therapy. By the end of therapy, the efficacy of combinedtreatment did not differ from that of Artrofoon monotherapy. Diclofenac therapywas followed by a variety of gastrointestinal complications in 80% patients. Bycontrast, Artrofoon had an excellent safety profile and did not cause AE.

As differentiated from diclofenac, Artrofoon had a long�term antiinflamma�tory effect and demonstrated an excellent safety profile. Therefore, Artrofoon holdsmuch promise for the therapy of OA. These properties are related to the action ofArtrofoon on pathogenetic mechanisms of degenerative and atrophic processes inthe osteoarthritic joint. During the 1st month of therapy, Artrofoon should beused in combination with diclofenac to produce a rapid effect. Artrofoonmonotherapy can be given from the 2nd month to reduce the incidence of AE.

Similar results were obtained by Prof. B. A. Alikhanov at the MoscowState Medical Stomatological University (B. A. Alikhanov, 2004, 2006). Theclinical efficacy of Artrofoon in various doses was studied in OA patients afterlong�term therapy with this drug (from 6 months to 2 years). The trial involved

Fig. 8.33.Fig. 8.33.Fig. 8.33.Fig. 8.33.Fig. 8.33. WOMAC total score in osteoarthritic patients after 6�month treatmentwith Artrofoon and diclofenac. p<0.05: *compared to the baseline value; +comparedto the diclofenac group.


0

10

20

30

40

50

60

Baseline After 3 months After 6 months

WOMAC total scoreArtrofoon

diclofenac, 100 mg

Artrofoon+diclofenac

!

!

! !

!

!

++

258

Ultralow doses

90 OA patients (stage II�III gonarthrosis) receiving various doses of Artrofoonas monotherapy or in combination with NAID for 6�24 months. The efficacyof therapy was evaluated from the severity of joint syndrome, laboratory andclinical parameters, and ultrasound examination of the joints. Artrofoon had asignificant clinical effect, which was observed on days 20�30 of treatment andreached maximum after 3 months of therapy. After 3 months, the overall effectof therapy was most pronounced in patients receiving Artrofoon as monotherapy(8 tablets daily) or combination with NAID. This effect persisted for up to 6months during therapy with Artrofoon at the specified dose or half dose. Thepositive effect was maintained for 2 years of treatment with Artrofoon in a doseof 2�4 tablets daily. NAID monotherapy had a smaller effect. Long�termtherapy with Artrofoon (for up to 2 years) did not cause AE. Combinationtherapy with Artrofoon and NAID was followed by the reduction of NAID�produced adverse reactions. The efficacy and safety of Artrofoon in OApatients were confirmed by Prof. N. A. Shostak at the Russian State MedicalUniversity (N. A. Shostak et al., 2005).

The effect of Artrofoon on production of cytokines and growth factors inRA patients was studied by Prof. V. I. Mazurov in St. Petersburg (V. I. Mazurovet al., 2007). Besides the high clinical efficacy, Artrofoon decreased theproduction of proinflammatory cytokines IL�1β and TNF�α, stimulated thesecretion of antiinflammatory cytokines IL�4 and IL�10, and had a normalizingeffect on the concentrations of epidermal growth factor (EGF) and vascularendothelial growth factor (VEGF).

The efficacy and safety of Artrofoon in patients with urogenic RA (UA)were evaluated in a pilot study at the Volgograd State Medical University (V. I.Petrov et al., 2005).

An open randomized comparative study of Artrofoon efficacy was per�formed with UA patients (diagnostic criteria of E. R. Agababova). The diagnosesof ureaplasma infection and chlamydial infection were made in 66.7 and 33.3%patients, respectively.

A clinical�and�anatomical type of UA was presented by OA with primarydamage to the joints of the lower extremities. The patients received Artrofoon(8 tablets daily) or diclofenac (100 mg daily) for 3 months in combination withantibacterial therapy (depending on the infectious agent). Clinical and labora�tory parameters were monitored for 1 month at 2�week intervals. The patientswere examined monthly in the follow�up period. Artrofoon therapy was followedby a decrease in the severity of pain (83.3% patients), Ritchie index, andswelling (66.6% patients). The severity of pain, Ritchie index, and swelling werereduced in 66.6% patients of the diclofenac group. Drug�related adverse eventswere not observed in patients of the Artrofoon group. The efficacy of Artrofoonin UA patients should be confirmed in large�scale clinical trials.

259

Artrofoon was effective in the therapy of patients with other diseases ofthe joints and musculoskeletal system, including psoriatic and gouty arthritis,ankylosing spondyloarthritis, osteochondrosis, and periarthritis of the shoulderjoint (V. V. Badokin et al., 2005; O. V. Inamova et al., 2005; I. V. Kudryavtsevaet al., 2005; O. I. Epstein et al., 2005; N. A. Khitrov, 2006).

The therapeutic efficacy of Artrofoon in patients with nonspecific ulcerativecolitis was demonstrated at the Samara Military Medical Institute. TNF�α has animportant role in the pathogenesis of this disease (M. A. Osadchuk et al., 2005).

The results of randomized controlled clinical studies of Artrofoon allowus to make the following conclusions. Pharmacodynamically, Artrofoon holdspromise as a disease�modifying drug with the long�term clinical effect in RApatients. Artrofoon may be used in combined treatment and monotherapy ofthese patients. Artrofoon has a complex effect on the cytokine cascade duringsystemic autoimmune inflammation, which differs from the action of TNF�αantagonists (e.g., Infliximab). The antiinflammatory effect of Artrofoon on RApatients is probably associated with a decrease in the systemic production ofTNF�α. The clinical efficacy of Artrofoon should be evaluated in patients withTNF�α�related diseases.

8.5. Epigam in the therapy for gastric ulcerand duodenal ulcer

An open, randomized, comparative pilot study of the clinical efficacy andsafety of Epigam (ULD to histamine for oral administration, 28�day tripletherapy) during gastric ulcer and duodenal ulcer was performed at the VolgogradState Medical University in 2002�2003 (P. A. Bakumov et al., 2003; J. L.Dugina, 2003a,b; O. I. Epstein et al., 2003b; J. L. Dugina et al., 2002).

The trial involved stationary patients 18�50 years of age. Ulcerative lesionsof the gastric or duodenal mucosa (20 mm in diameter) were revealed duringendoscopic examination. The patients were confirmed to have Helicobacter pyloriinfection.

The patients were divided into two groups of ten subjects each. Group 1patients received Epigam (1 tablet, six times daily for 28 days), amoxicillin (500mg, three times daily for 14 days), and metronidazole (500 mg, twice daily for14 days). Ranitidine (150 mg, twice daily for 28 days), amoxicillin (500 mg,three times daily for 14 days), and metronidazole (500 mg, twice daily for 14days) were given to group 2 patients. During the study, all patients could receivesymptomatic antacid therapy. The main symptoms of peptic ulcer were evaluatedin the basal state and after 1, 2, 3, and 4 weeks of therapy. The overall severityof symptoms was determined during a pretrial period of 1 week. Endoscopic


260

Ultralow doses

Fig. 8.34.Fig. 8.34.Fig. 8.34.Fig. 8.34.Fig. 8.34. Mean time to relief of peptic ulcer symptoms in a controlled clinicalstudy of Epigam.

examination with an Olympus�GIF�E gastrofiberscope was performed beforeand after 4�week therapy. The number and area of ulcerative lesions, endoscopicand histological signs of gastroduodenal mucosal inflammation, and secretorystatus (Congo red test) were evaluated. The benign nature of mediogastric ulcerswas confirmed histologically. Helicobacter pylori was detected by invasive(histological, molecular genetic, and rapid urease test) and noninvasive methodsfor infection diagnostics (indirect solid�phase enzyme immunoassay). The rapidurease test (KhELPIL test, St. Petersburg) was used for primary rapid diag�nostics of H. pylori. This test is based on the measurement of urease activity inbiopsy specimens of the gastric mucosa after endoscopic examination.

The average age of patients in both groups (n=20) was 32.2 years. Twelvemen (60%) and eight women (40%) were enrolled in the trial. The majority ofpatients (n=19, 95%) had a history of peptic ulcer. In one patient (5%), thediagnosis was made for the first time. The mean duration of disease was 3.2 years.

The time to relief of pain syndrome and dyspepsia did not differ inpatients receiving Epigam and ranitidine. Epigastric pain in group 1 patients wasreduced after therapy for 3 (two patients, 20%), 10 (eight patients, 80%), or 14days (all patients). These changes were accompanied by the disappearance ofdyspeptic disorders. Epigastric pain in group 2 patients was reduced after therapyfor 2 (five patients, 50%), 10 (eight patients, 80%), or 14 days (all patients). Themean time to pain relief was 7.50±0.81 days (Fig. 8.34).

The severity of epigastric pain syndrome (including nighttime pain),heartburn, and nausea and volume of symptomatic therapy in patients of theEpigam group decreased significantly after 4�week treatment (p<0.01, Table8.12). The mean time to pain relief was 10.6±1.2 days (6�7 days in the majorityof patients). Pain syndrome and dyspeptic disorders were reduced in the earlyperiod after combination therapy with ranitidine, amoxicillin, and metronidazole(p<0.05). Epigastric and pyloroduodenal pain and dyspeptic disorders wererelieved over 3�11 and 2�14 days, respectively. Pain syndrome in 53.8% patients

0

3

6

9

12

15

Pain Heartburn Eructation Nausea

Duration of symptom, days

Epigam ranitidine

261

was not observed after drug therapy for 5 days. The mean time to relief ofepigastric pain and dyspepsia (mainly of nausea) was 7.5±0.8 and 4.9±0.8 days,respectively.

The duration and severity of symptoms did not depend on basal gastricacid secretion and length of the disease.

The area of gastroduodenal ulcers decreased significantly after 4�weektriple therapy with Epigam (p<0.001). Ulcer cicatrization in nine of ten patients(90%) was observed by the 4th week treatment. The area of ulcerative lesion inone patient decreased by 75�80%. Epigam significantly decreased the severity oferosive changes. In six patients (60%) ulcer cicatrization was accompanied byepithelization of gastric and/or duodenal erosions after 4�week therapy (p<0.05,Table 8.13).

Four�week combination therapy with ranitidine, amoxicillin, and metro�nidazole was also followed by a decrease in the area of mucosal erosions andulcers and reduction of macroscopic (endoscopic) signs for gastritis. Erosivegastroduodenitis was not found in treated patients. Ulcer healing was observedin 100% patients (Table 8.14).


Table 8.12.Table 8.12.Table 8.12.Table 8.12.Table 8.12. Reduction of peptic ulcer symptoms in patients receiving Epigam incombination with amoxicillin and metronidazole (points, M±m)

Epigastric pain 2.8+0.4 0.8+0.04* 2.4±0.5 0.70±0.03*

Nighttime pain 1.8+0.2 0.1+0.01* 1.6±0.1 0.20±0.01*

Heartburn 1.3+0.2 0.5+0.01* 1.6±0.1 0.30±0.01*

Eructation 1.1+0.1 0.70+0.03 0.90±0.04 0.80±0.01

Nausea 0.9+0.1 0.60+0.02 1.20±0.06 0.40±0.02*

Palpatory tenderness 2.9+0.2 0.70+0.03* 2.7±0.2 0.50±0.06*


Symptom

after 4 weeksbaseline

Ranitidine+amoxicillin+metronidazole

Epigam+amoxicillin+metronidazole


Table 8.13.Table 8.13.Table 8.13.Table 8.13.Table 8.13. Clinical efficacy of 4�week combination therapy with Epigam in patients withpeptic ulcer of the stomach and duodenum (n=10)

Incidence of ulcer cicatrization 9 90 10 100

Incidence of epithelizationof erosions 6 60 8 80

Parameter

%abs.



%abs.

262

Ultralow doses

The clinical improvement was accompanied by a decrease in the volumeof symptomatic therapy in patients receiving Epigam (from 3.21±0.16 to0.53±0.09 tablets/spoons daily, p<0.001) and ranitidine (from 2.96±0.11 to0.22±0.04 tablets/spoons daily, p<0.001).

A pilot clinical study confirmed the efficacy of combination therapy withEpigam (6 tablets daily for 28 days) and standard drugs (amoxicillin and me�tronidazole) in patients with H. pylori�associated peptic ulcer of the stomachand duodenum. The efficacy of Epigam compared well with that of the refer�ence drug ranitidine (300 mg daily).

8.6. Afala in the therapy for benignprostatic hyperplasia

The drugs for BPH have two main molecular targets, 5�α�reductase andα�adrenoceptors in the lower urinary tract. 5�α�Reductase inhibitors areeffective in severe hyperplasia of the prostate gland, but do not affect clinicalsymptoms of BPH during the first 3�4 months of therapy. However, these drugsimprove the long�term prognosis in BPH patients. α�Adrenoceptor antagonistsdo not influence prostate tissue, but have a strong effect on the dynamiccomponent of obstruction and rapidly relive the symptoms of BPH. Clinicalsymptoms (total IPSS, International Prostate Symptom Score) and urodynamicsimprove by 20�50 and 20�30%, respectively (S. Madersbacher et al., 2004).

Among a variety of physiotherapeutic products, much attention was paidto Serenoa repens extract. The efficacy of Serenoa repens extract compares wellwith that of finasteride (T. Wilt et al., 2004).

Table 8.14.Table 8.14.Table 8.14.Table 8.14.Table 8.14. Endoscopic examination for reparative and inflammatory processes in thegastric and duodenal mucosa of peptic ulcer patients receiving combinationtherapy with Epigam (points, M±m)

Area of ulcers 1.21±0.07 0* 0.96±0.02 0*

Number of ulcers 1.06±0.04 0* 1.45±0.04 0.07±0.01*

Erosions 0.90±0.03 0.46±0.02* 0.73±0.01 0.24±0.01*

Gastritis 1.29±0.06 1.05±0.01 1.17±0.06 1.03±0.03

Duodenitis 1.35±0.04 1.30±0.05 1.08±0.02 1.01±0.02

Duodenogastric reflux 0.18±0.01 1.17±0.04 0.35±0.01 0.27±0.01


Parameter





263

The target for S. repens was not identified. This extract has the pleiotropicproperties (modulation of cholesterol metabolism; antiestrogenic, antiandro�genic, and antiinflammatory effects; etc.).

PSA is one of the molecular targets during BPH (E. P. Diamandis, 2000;S. P. Balk et al., 2003). The expression of this serine protease is regulated byandrogens. PSA has antiangiogenic activity (A. H. Fortier et al., 1999) and playsa role in the regulation of stromal cell growth in the prostate gland (D. M.Sutkowski et al., 1999).

BPH is accompanied by a strong immune response to PSA, which servesas one of the pathogenetic factors and reflects the progression of otherpathological processes (A. Zisman et al., 1995, 1999).

The pathogenetic role of PSA in BPH requires further investigations.Afala was developed at the “Materia Medica Holding” Research�and�

Production Company. The active components of this product are rabbit poly�clonal antibodies to PSA (ULD for oral administration). Preclinical studiesshowed that Afala decreases the severity of acute and chronic aseptic inflam�mation of the prostate gland (T. G. Borovskaya, 2002). Moreover, this productreduces the degree of prostatic hyperplasia under conditions of sulpiride�induced hyperprolactinemia (K. V. Savel’eva et al., 2007). The effect of Afalais probably related to functional modification of endogenous PSA, which isimpaired during BPH.

Here we describe the results of two randomized, controlled, parallel�groupstudies to evaluate the efficacy and safety of Afala in BPH (V. N. Pavlov et al.,2005a,b; A. A. Martyushev�Poklad et al., 2005; Z. A. Yurmazov et al., 2005;V. I. Petrov et al., 2006; A. Martyushev�Poklad et al., 2005d).

The trials of similar design were performed at the Volgograd State MedicalUniversity, Bashkirian State Medical University (Ufa), Institute of Pharmaco�logy (Tomsk Research Center of the Siberian Division of the Russian Academyof Medical Sciences), and Central Military Medical Hospital No. 32 (Moscow).The patients were randomized into the Afala group, placebo group, and opencontrol group. After 4 weeks of a placebo�controlled phase, the Afala group wasopened. An open randomized comparative study was performed with the activereference drug. The total duration of therapy was 16 weeks.

The study involved patients (40�70 years of age, average age 63.50±0.49years) with moderate symptoms of chronic prostate disease. The diagnosis ofstage I�II BPH (total IPSS 9�25) was confirmed by transrectal ultrasound(TRUS) examination. The volume of the prostate gland was more than 25 cm3.The maximum urine flow rate (Qmax) was 5�15 ml/sec. The informed consentwas obtained from each patient. The exclusion criteria were a history of surgicaltreatment for diseases of the prostate or urinary bladder, residual urine volume> 150 ml, suspected prostate cancer, serum PSA > 4 ng/ml, etc.


264

Ultralow doses

The patients received Afala (16 weeks) or placebo (4 weeks) in a dailydose of two lozenges. The parallel open control group was treated with an activeproduct of Serenoa repens extract (Prostamol Uno, 320 mg daily).

The trial involved 241 patients (stage I�II BPH) receiving Afala (n=132),Prostamol Uno (n=54), or placebo (n=55, control group).

The severity of disease symptoms (total IPSS), quality of life (integralcriterion, IPSS), urodynamics (uroflowmetry), state of the prostate gland(TRUS), and serum PSA level were evaluated during follow�up visits. AE wererecorded during each visit. Routine blood test, urine test, and measurement ofserum glucose or creatinine were performed before and after the study.

The efficacy endpoints were the relief of symptoms, improvement ofurodynamic parameters, and increase in patients’ quality of life.

After 4 weeks of therapy, the efficacy of Afala significantly differed from thatof placebo (according to clinical manifestations and urodynamics). IPSS decreasedby 0.9±0.4 points, while Qmax remained unchanged in the placebo group. IPSSwas 1.9±0.3 points, and Qmax increased by 13.3±2.4% in the Afala group.

Fig. 8.35 illustrates the results of a 16�week open comparative study. Drugtreatment was followed by a significant decrease in the total IPSS (by more than40% after 16 weeks). In 50% patients, IPSS decreased below 8 points.Pharmacotherapy was not required under these conditions. The improvement ofurodynamics (Qmax) in patients of the Afala and Prostamol groups was 45 and36%, respectively. The quality of life (quality�of�life score in IPSS) improvedmore significantly in the Afala group.

TRUS examination revealed a significant decrease in residual urinevolume (from 31.0±3.2 to 12.9±1.7 ml, p<0.001) and size of the prostate gland(from 44.6±1.3 to 41.9±1.3 cm3, p<0.01) after Afala therapy.

Afala (8 tablets daily for 16 weeks) had a good safety profile in patientswith stage I�II BPH. Drug�related AE were not reported. The results of routineblood test, urine test, and biochemical blood analysis did not differ from normalbefore and after therapy.

The concentrations of total and free PSA in blood serum from Afala�receiving patients not only remained within normal limits (less than 4 ng/ml),but even decreased (by 17.6 and 21.7%, respectively). The PSAfree/PSAtotal ratiodid not change after Afala therapy (within normal limits). Serum PSA levelremained unchanged in the Prostamol group.

These data indicate that Afala is an effective and safety drug for thetherapy of stage I�II BPH. The product improves clinical symptoms of thisdisease (particularly those associated with urination disorders), parameters ofurodynamics, size of the prostate, and residual urine volume. A modulatoryeffect of Afala on serum PSA level indirectly reflects the pathogenetic action ofthis product.

265

8.7. Clinical pharmacology of Kardos

Chronic heart failure is an urgent problem of public health. The incidenceof CHF in the European population reaches 0.4�2.0%. The disease is mostcommon in elderly people. The average age of CHF patients is 74 years. Thepercentage of patients with systolic dysfunction not accompanied by CHF issimilar to that of subjects with CHF symptoms.

Nearly half of patients with the diagnosis of CHF die over 4 years. Thesurvival time of 50% patients with severe CHF does not exceed 1 year. CHFpatients have a poor long�term prognosis.

CHF is most often associated with CHD. The left ventricular ejectionfraction in many patients decreases below 45�50%. However, there is no directrelationship between symptoms and degree of cardiac dysfunction (as shown byinstrumental methods). Hence, the severity of CHF is mainly evaluated fromfunctional parameters.

Fig. 8.35.Fig. 8.35.Fig. 8.35.Fig. 8.35.Fig. 8.35. Course of BPH during therapy with Afala (light bars) and Prostamol (darkbars). Q

max (a); average flow rate of urine (b); reduction of disease symptoms (total

IPSS, c); and quality of life (QoL index, d). *p<0.05 compared to the other group.

0

2

3

4

Points

1

Baseline 4 8 12 16

8

12

16

Points

4

Baseline 4 8 12 16

0

3

b

a

d

c

6

Qaver

, ml/sec

Time, weeks

9

Afala Prostamol (320 mg daily)

Baseline 4 8 12 16

0 0

8

12

16

Qmax

, ml/sec

4

Baseline 4 8 12 16


266

Ultralow doses

There are a variety of clinical, electrophysiological, and other signs ofpoor prognosis of CHF. The major signs are a persistent decrease in BP, NYHAfunctional class III or IV CHF (clinical parameter), reduced level of maximumoxygen consumption (functional parameter), and low left ventricular ejectionfraction (LVEF, central hemodynamic parameter).

Therapy for CHF is directed to the prevention of disease progression,maintenance or improvement of the quality of life, decrease in the incidence ofhospitalization for CHF, and increase in the lifespan of patients.

The drugs affecting the renin�angiotensin�aldosterone system are widelyused to decrease the mortality rate of patients and incidence of hospitalization.They include angiotensin�converting enzyme (ACE) inhibitors, angiotensin IIreceptor antagonists (AT II; mainly in combination therapy), aldosteroneantagonists, and β�adrenoreceptor antagonists (Guidelines for the Diagnosis andTreatment of Chronic Heart Failure, 2005).

The angiotensin II receptor type 1 (AT1) mediates the key effects of ATII, which plays a role in the pathogenesis of arterial hypertension, complicationsof this disease, and CHF.

Various fragments of the receptor have different functions. The extracellu�lar fragment is responsible for binding of peptide agonists. The transmembranefragment has a role in binding of peptide agonists, signal transduction to Gproteins (rapid effects of AT), and binding of nonpeptide antagonists (includingthose used in clinical practice). The C�terminal intracellular fragment is in�volved in signal transduction (phosphorylation and, therefore, long�term effectof angiotensin) and receptor internalization.

The long�term effects of AT II include long�term regulation of vasculartone and remodeling of the vascular wall. Internalization is a key event of thereceptor cycle, which serves as one of the regulatory mechanisms for AT IIsignal transduction. Arterial hypertension is accompanied by abnormalities inreceptor internalization (impairment of signal transduction from the receptor)and overphosphorylation (M. de Gasparo et al., 2000; R. M. Touyz et al., 2000).

The C�terminal fragment of the AT II AT1 receptor serves as a moleculartarget for Kardos. Antibodies to this fragment are the active component ofKardos (ULD for oral administration; Fig. 8.36).

Previous experiments showed that Kardos has a hypotensive effect onanimals with inherited arterial hypertension and exhibits the cardioprotectiveproperties on the model of CHF. Toxicology studies revealed that this producthas a good safety profile.

Clinical trials at the Volgograd State Medical University were performed toevaluate Kardos efficacy in patients with arterial hypertension and CHF (P. A. Ba�kumov et al., 2005; V. I. Petrov et al., 2005, 2006; V. V. Ivanenko et al., 2005;S. A. Sergeeva et al., 2006).

267

Clinical studies of Kardos efficacy in patients with arterial hypertension. Anopen clinical study at the Volgograd State Medical University was designed toevaluate the safety profile and effect of Kardos in various doses on BP innormotensive subjects (healthy volunteers).

During the first stage of the trial, Kardos pharmacodynamics was studiedin 48 healthy volunteers (21�48 years of age). These volunteers were divided intothe following four groups (according to the dosage regimen of Kardos): group1, 1 tablet three times daily; group 2, 2 tablets three times daily; group 3, 2tablets five times daily; and group 4, 2 tablets six times daily. Kardos was given2 h after the start of BP monitoring. The 24�h profile of BP and incidence ofAE were evaluated.

According to the results of 24�h BP monitoring, Kardos did not cause thedecrease of BP in all four groups (Fig. 8.37). AE were not reported.

These data indicate that Kardos has a good safety profile and does not causefirst�dose hypotension in normotensive patients (E. A. Zernyukova et al., 2005).

Kardos safety was demonstrated in CHD patients (no first�dose hypoten�sion; N. A. Davydova et al., 2005).

Pharmacodynamics and efficacy of Kardos during therapy of patients witharterial hypertension. An open randomized clinical study at the Volgograd StateMedical University was designed to evaluate the antihypertensive properties,optimal dosage regimen, and safety profile of Kardos during therapy of patientswith arterial hypertension.


Fig. 8.36.Fig. 8.36.Fig. 8.36.Fig. 8.36.Fig. 8.36. Functional fragments of the angiotensin II AT1 receptor. Targets for

peptide agonists and nonpeptide antagonists.

NH2

COOH

С

7474

108108

102102167167

198198 199199256256

252252

301301 300300

295295

292292294294

163163111111

С

P

P

LN

Y

W

YRD

PW

P L

NNY

H

K

VN

D

KTR

A

S

F Y

N

Agonists

Antagonists

Kardos

268

Ultralow doses

Fig. 8.37.Fig. 8.37.Fig. 8.37.Fig. 8.37.Fig. 8.37. 24�h BP monitoring in healthy volunteers. Acute pharmacological testwith Kardos, 2 tablets six times daily (maximum dose). Systolic BP (1) and diastolicBP (2).

0

20

4060

80

100

120

140

mm Hg

0 1 2 3 4 5 6 7 8 9 10 11 12

Time, h

13 14 15 16 17 18 19 20

1

2

21 22 23 24

The trial involved 40 patients (18�70 years of age) with mild arterialhypertension. The patients did not receive hypotensive drugs in the pretrialperiod (2 weeks before the start of study). BP was 140/160 mmHg (systolic)and/or 90/100 mmHg (diastolic).

The patients were divided into four groups. Kardos was given orally(lozenges) in the following four regimens: 1 tablet three times daily (group 1);2 tablets three times daily (group 2); 2 tablets six times daily (group 3); and 1tablet six times daily (group 4).

The duration of therapy was 3 months (12 weeks). The state of patientswas estimated at 4�week intervals. The BP profile was estimated from the resultsof 24�h BP monitoring. BP was measured manually.

In group 1, the hypotensive effect of Kardos became more pronouncedwith an increase in the duration of therapy (significant decrease in diastolic BPduring the nighttime).

The hypotensive effect of Kardos in group 2 patients was observed after1 month of therapy and remained unchanged over the next 2 months. SystolicBP decreased by 10.7% compared to the baseline value (manual measurement;5.6% according to the results of 24�h BP monitoring). A decrease in systolic anddiastolic BP was particularly pronounced in the nighttime (by 18.7 and 27.2%,respectively).

The hypotensive effect of Kardos on systolic and diastolic BP in group 3patients was significant after 2�week therapy (decrease by 7.3 and 7.7%,respectively; p<0.05; manual measurement). After 3 months of therapy, systolicBP decreased by 8.4 (manual measurement) or 11.4% (24�h monitoring). Thesechanges were accompanied by a 20.3% decrease in nighttime systolic BP (24�h monitoring). Nighttime diastolic BP decreased by 26.6%.

The hypotensive effect of Kardos in group 4 patients was similar to thatin group 3 patients.

269

Drug�related AE were not reported by patients of all groups.In various regimens of 3 month�treatment, Kardos had a moderate

hypotensive effect on patients with “mild” arterial hypertension. A stablehypotensive effect of Kardos was observed after 2 months of therapy. Increasingthe dose of Kardos was accompanied by a more rapid development of the effect.This product was most potent in modulating the nighttime level of BP.Therefore, Kardos had a normalizing effect on the 24�h profile of BP. Theproduct was well tolerated in various dosage regimens.

A randomized, controlled, parallel�group study of the efficacy of 4�weektherapy with various pharmaceutical formulations of Kardos (monotherapy andcombination therapy for stage I�II arterial hypertension) was performed at theVolgograd State Medical University. A placebo�controlled study was performedwith the drop formulation of Kardos. Patients of two open control groupsreceived an ACE inhibitor and AT II receptor antagonist. All patients enrolledin the trial were divided into six groups of 15 subjects each: group 1, placebo(6�12 drops, three times daily); group 2, Kardos (6�12 drops, three times daily);group 3, Kardos (1�2 tablets, three times daily); group 4, Kardos (1�2 tablets,three times daily) and enalapril (5 mg twice daily); group 5, lisinopril (10�20 mgonce daily); and group 6, losartan (50�10 mg three times daily). The durationof therapy was 4 weeks. The results of this study are shown in Table 8.15 andFig. 8.38.

Biochemical parameters remained practically unchanged during thecourse of monotherapy or combination therapy with Kardos.


Fig. 8.38.Fig. 8.38.Fig. 8.38.Fig. 8.38.Fig. 8.38. Decrease in BP in patients with stage I�II arterial hypertension receivingantihypertensive therapy for 4 weeks (24�h monitoring). SBP

24h, 24�h systolic BP;

DBP24h

, 24�h diastolic BP; BPm

, mean BP. *p<0.01 compared to group 1.

�2

0

2

4

6

8

10

12

14

1 6543

Group

2

% of the baseline

SBP24h DBP24h BPm

27

0

Ultra

low

do

ses

Table 8.15.Table 8.15.Table 8.15.Table 8.15.Table 8.15. Results of 24�h BP monitoring in patients with stage I�II arterial hypertension during 4�week hypotensive therapy (mmHg,M±m)

24�h systolic BP

baseline 149.5±2.7 149.7±2.1 149.2±2.8 151.2±5.5 150.8±5.4 151.0±2.8

after therapy 150.6±2.9 139.9±2.4* 139.3±2.1 136.0±4.8 134.2±4.6 135.9±2.2

24�h diastolic BP

baseline 90.3±1.9 90.5±1.1 91.4±1.6 91.6±3.2 91.7±3.1 94.4±1.8

after therapy 91.2±2.3 84.9±0.9* 85.3±1.7 80,7±3.4 80.7±3.2 86.3±1.5

Mean BP

baseline 118.9±3.8 113.1±1.9 113.3±1.0 115.4±2.9 114.7±2.8 115.2±3.6

after therapy 120.4±2.7 105.8±1.2* 105.7±0.9 102.8±1.7 101.5±1.9 104.3±2.8

Note.Note.Note.Note.Note. *p<0.05 compared to group 1.

Parameter

654

Group

1 2 3

271

Side effects were not observed during Kardos therapy (including dry coughand first�dose hypotension).

We conclude that 4�week treatment with the drop formulation of Kardoshas a strong hypotensive effect (as differentiated from placebo). Variouspharmaceutical formulations of the product (drops and tablets, 4�week therapy)produce the same hypotensive effect. Combination therapy with Kardos andACE inhibitor potentiates the hypotensive effect of treatment. The hypotensiveeffect of lisinopril and losartan was greater than that of Kardos.

Therapeutic efficacy of Kardos in patients with chronic heart failure. Ablind, randomized, placebo�controlled study was performed at the Departmentof Cardiology and Functional Diagnostics (Faculty of Postgraduate MedicalEducation, Volgograd State Medical University). The trial involved ambulatorypatients (20�80 years of age) with a stable course of functional class II�IVCHF. LVEF did not exceed 35%. The patients received standard therapy forCHF (ACE inhibitors, β�adrenoceptor antagonists, and diuretics). The infor�med consent was obtained from each patient. The patients were randomizedinto groups to receive 6�month therapy with Kardos (1 tablet three timesdaily; 30 patients, including 27 male subjects) or placebo (30 patients, inclu�ding 24 male subjects).

The efficacy and safety of therapy were evaluated during outpatient visitsafter 4, 12, and 24 weeks of treatment.

The percentage of patients with improved functional class of CHF wasconsidered as a primary efficacy endpoint. The secondary efficacy endpointswere an increase in physical tolerance (6�min walking distance), variations inLVEF (echocardiography), and maximum oxygen consumption (treadmill test).

The safety profile was determined by recording of AE.The statistical analysis was performed with patients who met the inclusion

criteria and received therapy. The mean values were compared by Student’s t testfor independent variables. The percentage of patients was compared by χ2 testfor homogeneity of proportions.

Kardos significantly surpassed placebo in the primary efficacy endpoint orone of three secondary efficacy endpoints. No between�group differences wereobserved in the safety profile.

The average age of patients enrolled in the study and receiving placeboand Kardos was 53.9±1.0 and 57.3±1.6 years, respectively. CHF was associatedwith CHD in 28 patients of the placebo group and in 30 patients of the Kardosgroup. Arterial hypertension served as an etiologic factor of CHF in two patientsof the placebo group and in three patients of the Kardos group. The baselineseverity of disease did not differ in patients of both groups. Patients of theplacebo and Kardos groups had FC III (15 and 19 patients, respectively) or FCIV (3 and 1 patients, respectively; Fig. 8.39).


272

Ultralow doses

Baseline 24th week20

24

36

28

32

placebo Kardos

!

LVEF, %Percentage of patients, %

Placebo Kardos

0

20

40

60

80

a b

Fig. 8.40.Fig. 8.40.Fig. 8.40.Fig. 8.40.Fig. 8.40. Kardos efficacy during 6�month treatment of CHF patients. Percentageof patients with improved FC of CHF (CI 95%, a); and systolic function of the leftventricle during therapy (b). *p<0.05 compared to placebo.

Placebo Kardos

40% 34%

50% 63%

10% 3%

FC II FC III FC IV

Fig. 8.39.Fig. 8.39.Fig. 8.39.Fig. 8.39.Fig. 8.39. Severity of CHF in patients enrolled in the study of Kardos efficacy for CHF.

After 6 months of therapy, FC of CHF was reduced in 8 patients of theplacebo group (26.7%; CI 95% = 14.2�44.5) and in 16 patients of the Kardosgroup (53.3%; CI 95% = 36.1�69.8). Among patients with baseline FC III�IV,the improvement was observed in 8 subjects of the placebo group (44.4%; CI95% = 24.6�66.3) and in 14 subjects of the Kardos group (70%; CI 95% = 48.1�85.5; Fig. 8.40).

Six�month therapy was followed by an increase in LVEF in patients of theplacebo group (from 26.4±1.1 to 28.0±1.4%) and Kardos group (from 27.1±0.9to 33.6±1.5%; p<0.01 compared to the baseline value and placebo group; Fig.8.41). Effect size was 1.01. The standard deviation of baseline LVEF for thecombined sample was 5.52.

The 6�min walking distance increased in patients receiving placebo (from390.5±11.9 to 409.1±11.5 m; p=0.12 compared to the baseline value; average

273

increase 6.7±3.7%) and Kardos (from 378.7±12.4 to 419.6±13.7 m; p<0.05compared to the baseline value; average increase 11.9±2.9%).

After 6 months of therapy, maximum oxygen consumption in thetreadmill test increased in patients of the placebo group (from 16.2±1.4 to17.8±1.3 ml/kg/min, statistically insignificant) and Kardos group (from17.4±1.2 to 19.5±1.3 ml/kg/min; p<0.05).

After 6 months of therapy, the total score of the Minnesota questionnairefor assessing quality of life was reduced in patients of the placebo group (from48.3±3.7 to 42.4±3.4 points) and Kardos group (from 47.5±2.8 to 39.1±3.0points; p<0.05).

Patients of the Kardos group were characterized by a significantimprovement of myocardial remodeling (size and volume of the left ventricle),heart rate variability, and endothelial function (pulse wave propagation andendothelium�dependent vasodilation; V. V. Ivanenko et al., 2004a,b,c). Fataloutcome was not observed during 6 months of therapy. None of the patientswithdrew from the trial. One AE was reported in the placebo group. Cardiacfibrillation was followed by decompensation of CHF, which requiredhospitalization of the patient.

Combined treatment with Kardos (3 tablets daily for 6 months) andstandard drugs was much more effective than standard therapy for CHF.Kardos significantly improved the clinical state, morphofunctional parametersof the heart, and tolerance to physical exercise. We conclude that Kardos isan effective drug, which has a good safety profile and may be used incombination with standard therapy for CHF. Kardos improves the prognosisof this disease.


Fig. 8.41.Fig. 8.41.Fig. 8.41.Fig. 8.41.Fig. 8.41. Efficacy of 6�month therapy with Kardos in CHF patients. Influence onphysical tolerance (6�min walking distance, a) and maximum oxygen consumption(treadmill test, b). *p<0.05 compared to the baseline value.

Placebo Kardos10

12

14

16

20

22

18

baseline 24th week

VO2max, ml/kg/min

Placebo Kardos320

340

360

380

420

440

a bm

400

!!

274

Ultralow doses

***

Chapter 8 reviews the results of clinical studies with ULD of antibodies.All clinical trials were performed in accordance with the Manual on ClinicalStudies of New Pharmaceutical Substances in Russia (2005) and InternationalPrinciples of Good Clinical Practice (GCP).

Placebo�controlled clinical trials confirmed the results of preclinicalstudies. It was concluded that products of antibodies in ULD demonstrate theefficacy and good safety profile. Proproten�100 was effective and had a goodsafety profile in the therapy of alcoholism and reduction of alcohol withdrawalsyndrome. Tenoten was successfully used in the therapy of anxiety disorders.Anaferon and Anaferon for children were effective in the therapy of ARVI(influenza, adenovirus infection, respiratory syncytial virus infection, etc.),herpes virus infections (chickenpox, infectious mononucleosis, and genitalherpes), and acute intestinal infections (rotavirus infection, coronavirusinfection, and calicivirus infection). The efficacy and safety of Impaza wererevealed in treatment for ED of various etiologies. Impaza was used asmonotherapy or in combination with other drugs to improve potency (Viagraand Sialis). The efficacy and safety of Artrofoon were demonstrated in patientswith RA, OA, and other autoimmune diseases. Some trials were performed withEpigam (therapy for gastric ulcer and duodenal ulcer), Afala (treatment ofBPH), and Kardos (CHF and arterial hypertension).

It should be emphasized that the possibility of using antibodies in ULDis not limited to the described nosological forms. ULD of anti�TNF�α (Art�rofoon), anti�NOS (Impaza), and anti�IFN�γ (Anaferon and Anaferon forchildren) hold much promise for therapy of autoimmune disease (e.g.., non�specific ulcerative colitis), endothelial dysfunction, and tick�borne encephalitis,respectively.

275

CONCLUSION

The method for potentiation (activation) of ultra�diluted solutions wasempirically discovered by S. Hahnemann at the end of the 18th century.

This approach was used to prepare medical products that have a good safetyprofile and do not cause drug�related adverse events. Treatment with activated(potentiated) substances in ultralow doses was a part of the therapeutic methodof S. Hahnemann (homeopathy). Hence, these products received the name“homeopathic substances”.

A famous botanist K. Negeli (19th century) and other researches (20thcentury) showed that ultra�diluted solutions of various substances exhibit thebiological activity. There was no direct relationship between these properties andhomeopathic doctrine. Experimental studies of ultralow doses by J. Benvenisteand E. B. Burlakova were of high methodological quality and gained generalacceptance in the scientific community.

Homeopathy is based on a particular property of ultralow doses. Theycause a hyperergic reaction, which increases the effect of potentiated products.Homeopathy is an effective, but casuistic approach. The method requiresindividualization of therapy, which limits the use of homeopathic remedies.Potentiated products have low biological activity. Therefore, ultralowconcentrations cannot substitute for standard (therapeutic) doses of medicalproducts.

Our studies revealed that the activated form of a substance, which isprepared by the method of Hahnemann, can modify the effect of this substance(phenomenon of bipathy). The phenomenon of bipathy was demonstrated onbiological and simple physicochemical models. Hence, ultralow doses are notthe prerogative of biology. By contrast, they are the subject matter of varioussciences. Ultralow doses may be used not only in medicine, but also in otherfields of science and technics.

The modifying (bipathic) activity of ultralow doses holds much promisefor modern pharmacology. This property may be used to potentiate the actionof pharmaceutical products, to reduce the adverse effects, and to prevent thedevelopment of drug resistance.

276

Ultralow doses

We showed for the first time that activated antibodies do not inhibit, butmodify the activity of the corresponding antigen. The modifying effect is avariant of the phenomenon of bipathy, which contributes to the development ofa new class of medical products with ultralow doses of antibodies.

The results of experimental and clinical observations are considered as astep to pharmacology of ultralow doses.

The resolution power of modern scientific methods does not allow us toevaluate a physical basis for the activity of ultra�diluted potentiated solutions.At the present stage of science, the mechanisms for action of ultralow doses arehypothetically explained. Our hypothesis of dual spatiotemporal organization ofvital activity provides an explanation for the pleiotropic effects of activatedsubstances.

The reproducibility is a distinctive feature of the phenomenon of bipathy.New medical products developed on the basis of modifying activity of antibodiesin ultralow doses meet the requirements of evidence�based medicine. In recentyears, the number of approved pharmaceuticals decreased sharply throughoutthe world. The efficacy of newly synthesized compounds does not surpass thatof less selective analogues. There is reason to hope that further studies ofactivated products will extend the possibilities of modern pharmacology, whichrequires the development of new approaches.

277

References

Ader R., Weijnen W., Moleman P. Retention of a passive avoidance response as a function ofthe intensity and duration of electric shock // Psychon. Sci. 1972. Vol. 26. P. 125�128.

Ahmaazadeh N., Shingu M., Nobunaga M. The effect of recombinant tumor necrosis factor�a on superoxide and metalloproteinase production by synovial cells and chondro�cytes // Clin. Exp. Rheumalol. 1990. Vol. 8. P. 387�391.

Aivaro�Gracia J., Zvaifler N., Firestein G. Cytokines in chronic inflammatory arthritis. V.Mutual antagonism between interferon�gamma and tumor necrosis factor�alpha onHLA�DR expression, proliferation, collagenase production, and granulocyte macrophagecolony�stimulating factor production by rheumatoid arthritis synoviocytes // J. Clin.Invest. 1980. Vol. 86. P. 1790�1798.

Aleksandrova N.V., Gofman A.G., Krylov E.N., Epstein O.I. Use of potentiated productsfor the relief of alcohol withdrawal syndrome // Byull. Eksp. Biol. Med. 2003. Sup�pl. 1. P. 82�85.

Alikhanov B.A. Artrofoon in the therapy of osteoarthritis // Klinich. Gerontol. 2006.Vol. 12, No. 2. P. 51�54.

Alikhanov B.A. Artrofoon in the therapy of osteoarthritis // Klinich. Gerontol. 2004.Vol. 10, No. 12. P. 63�66.

Amosova E.N., Zueva E.P., Razina T.G., et al. Experimental study of the effect of poten�tiated cyclophosphane on tumor growth and efficacy of cytostatic therapy // Byull.Eksp. Biol. Med. 2003. Suppl. 1. P. 16�19.

Amosova E.N., Zueva E.P., Razina T.G., et al. Perspectives in the use of cytostatics inultralow doses for chemotherapy of malignant neoplasms // Byull. Eksp. Biol. Med.2001. Suppl. 3. P. 54�56.

Andersson K.E. Pharmacology of penile erection // Pharmacol Rev. 2001. Vol. 53, No. 3.P. 417�450.

Andrianov V.V., Gainutdinov Kh.L., Gainutdinova T.Kh., et al. Membranotropic effectsof antibodies to S100 protein in ultralow doses // Byull. Eksp. Biol. Med. 2003.Suppl. 1. P. 24�27.

Anokhin P.K. Cybernetics of functional systems [in Russian]. Moscow, 1998.

Anokhin P.K. Essays on physiology of functional systems [in Russian]. Moscow, 1975.

Anokhin P.K. Selected works. Philosophical aspects of the theory of functional systems[in Russian]. Moscow, 1978.

Antsiferov M.B., Dedov I.I. UKPDS and main principles of oral therapy for type 2 diabe�tes mellitus: obesity, metabolic syndrome, and type 2 diabetes mellitus [in Russian].Moscow, 2000.

278

Ultralow doses

Arshavskii I.A. Biological and medical aspects of adaptation and stress from the stand�point of ontogenetic physiology // Urgent problems of modern physiology [in Rus�sian]. Moscow, 1976. P. 144�191.

Ashmarin I.P. Autoantibodies as regulators of biochemical and physiological processesin a healthy organism. Place in phylogenesis and among other endogenous regula�tors // Zh. Evolyuts. Biokhim. Fiziol. 1997. Vol. 33, No. 2. P. 228�233.

Ashmarin I.P., Freidlin I.S. Hypothesis of antibodies as the newest regulators of physi�ological functions in evolution // Zh. Evolyuts. Biokhim. Fiziol. 1989. Vol. XXV, No.2. P. 176�182.

Ashmarin I.P., Lelekova T.V., Sanzhieva L.Ts. Efficacy of ultralow doses and concentrationsof biologically active compounds // Izv. Ros. Akad. Nauk. 1992. No. 4. P. 531�536.

Avtandilov G.G. Medical morphometry [in Russian]. Moscow, 1990.

Badokin V.V., Kudryavtseva I.V., Korsakova Yu.L. Efficacy and tolerability of Artrofoonduring seronegative spondyloarthritis // 5th Northwestern Conference on Rheuma�tology. St. Petersburg, 2005. Abstracts of Papers. P. 23�24.

Bakumov P.A., Shchepotkin I.V., Dugina J.L., et al. Clinical efficacy of Epigam in pa�tients with exacerbation of gastric ulcer and duodenal ulcer // Byull. Eksp. Biol.Med. 2003. Suppl. 1. P. 77�81.

Bakumov P.A., Zernyukova E.A. Efficacy of anti�P�angiotensin II (G) C12+C30+C200 asmonotherapy and in combination with enalapril and hypothiazide during arterialhypertension // 12th Russian National Congress “Man and Drug”. Moscow, 2005.Abstracts of Papers. P. 638.

Balabolkin M.I. Endocrinology [in Russian]. Moscow, 1998.

Balabolkin M.I., Klebanova E.M., Kreminskaya V.M. Therapy of diabetes mellitus and itscomplications [in Russian]. Moscow, 2005.

Balk S.P., Ko Y.J., Bubley G.J. Biology of prostate�specific antigen // J. Clin. Oncol.2003. Vol. 21, No. 2. P. 383�391.

Barczyk M., Debek W., Chyczewski L. Mast cells in the gastrointestinal tract // Rocz.Akad. Med. Bialymst. 1995. Vol. 40, No. 1. P. 36�57.

Barger S.W., Van Eldik L.J. S100 beta stimulates calcium fluxes in glial and neuronalcells // J. Biol. Chem. 1992. Vol. 15. P. 9689�9694.

Bates F., Buckley G., Strettle R. The action of salmon calcitonin on indomethacin�in�duced gastric ulceration in rat // Br. J. Pharmacology. 1979. Vol. 67, N3. P. 483P�484P.

Bauer E. Theoretical biology [in Russian]. Moscow, 1935. P. 140�144.

Bazzoni F., Beutler B. The tumor necrosis factor ligand and receptor families // N. Engl.J. Med. 1996. Vol. 334. P. 1717�1725.

Bekhtereva N.P. Brain codes of mental activity [in Russian], Leningrad, 1977.

Belenkov N.Yu. Principle of integrity in brain activity [in Russian]. Moscow, 1980.

Bel’skii Yu.P., Agafonov V.I., Martyushev A.V., et al. Mechanisms for the peripheral ef�fect of Impaza // 10th Russian National Congress “Man and Drug”. Moscow, 2003.Abstracts of Papers. P. 581.

Bendele A., McComb J., Gould T., et al. Animal models of arthritis: relevance to humandisease // Toxicol. Pathol. 1999. Vol. 27, No. 1. P. 134�142.

Benveniste J., Davenas E., Ducot B., et al. L’agitation de solutions hautement dilu. Esn’induit pas d’activite biologique spеcifique (Agitation of highly dilute solutions does

279

not induce specific biological activity) // C. R. Acad. Scie. Paris. 1991. Vol. 312,Suppl. II. P. 461�466.

Berchenko O.G., Vorob’eva T.M., Geiko V.V. Morphine and its potentiated form: com�parative study of the effect on pain sensitivity in rats // Byull. Eksp. Biol. Med.2003. Suppl. 1. P. 13�15.

Beregovoi N.A., Pankova T.M., Sorokina N.S., et al. Effect of monoclonal antibodies 5F5�B6 in various dilutions on the induction of long�term posttetanic potentiation insurviving hippocampal slices // Byull. Sib. Otd. Ros. Akad. Med. Nauk. 1999. Vol.91, No. 1. P. 88�91.

Bernet M. Integrity of the organism and immunological reactions [in Russian]. Moscow,1963.

Bernshtein N.A. Physiology of movements and activity [in Russian]. Moscow, 1990. P.373�392.

Bertolini D., Nedwin G., Bringman T. Stimulation of bone resorption and inhibition ofbone formation in vitro by human tumor necrosis factors // Nature. 1986. No. 319.P. 516�518.

Beurle P.J. Properties of mass of cell capable of regenerating pulses // Phil. Trans. Roy.Soc. Sec. B. 1956. Vol. 240, No. 1. P. 55.

Bhattacharya S.K., Rao P.J., Das Gupta G. Effect of centrally administered prostaglandinD2 and some prostaglandin synthesis inhibitors on carrageenan�induced paw edemain rats // J. Pharm. Pharmacol. 1989. Vol. 41, No. 8. P. 569�571.

Biancone L., Orlando A., Kohn A., et al. Infliximab and newly diagnosed neoplasia inCrohn’s disease: a multicentre matched pair study // Gut. 2006. Vol. 55, No. 2.P. 228�233.

Bidasee R.K., Nallani K., Yongqi Y., et al. Chronic diabetes increases advanced glycationend products on cardiac ryanodine receptors // Diabetes. 2003. No. 52. P. 1825�1836.

Bindukumar B., Schwartz S.A., Nair M.P., et al. Prostate�specific antigen modulates theexpression of genes involved in prostate tumor growth // Neoplasia. 2005. Vol. 7,No. 3. P. 241�252.

Boehm U., Klamp T., Groot M., Howard J.C. Cellular responses to interferon�gamma //Annu Rev. Immunol. 1997. Vol. 15. P. 749�795.

Bokhan N.A., Abolonin A.F., Krylov E.N., et al. Comparative efficacy of Proproten�100 inthe therapy of alcoholic patients at the stage of therapeutic remission // Byull. Eksp.Biol. Med. 2003. Suppl. 1. P. 91�96.

Bondarenko L.A., Breslovskii A.S., Vertapetov B.A., Gladkova A.I. Secretion of testicularandrogens during experimental chronic inflammation of the prostate glands // Probl.Endokrinol. 1977. Vol. 23. P. 111�114.

Borovskaya T.G., Fomina T.I., Loskutova O.P., et al. Antibodies to prostate�specific an�tigen in ultralow doses: effect on morphological and functional state of the prostatein rats // Byull. Eksp. Biol. Med. 2002. Suppl. 4. P. 104�106.

Borovskaya T.G., Fomina T.I., Loskutova O.P., et al. Effect of antibodies to prostate�specific antigen in ultralow doses on the prostate in rats // Byull. Eksp. Biol. Med.2001. Suppl. 3. P. 49�51.

Bos R., Mougenot N., Findji L., et al. Inhibition of catecholamine�induced cardiac fibro�sis by an aldosterone antagonist // J. Cardiovasc. Pharmacol. 2005. Vol. 45, No. 1.P. 8�13.

References

280

Ultralow doses

Bou�Abbound Ch.F., Wayland H., Pauisen G., Guth P.H. // Dig. Dis. Sci. 1988. Vol. 33,No. 7. P. 872�882.

Boyd W.E. Biochemical and biological evidence of the activity of high potencies // Br.Hom. J. Vol. 44, No. 1. P. 7�44.

Boyd W.E. The action of microdoses of mercuric chloride on diastase // Br. Hom. J.1941. Vol. 31. P. 1�28.

Brady J.V., Nauta W.J. Subcortical mechanisms in emotional behavioral affective chang�es following septal forebrain lesions in the albino rat // J. Compar. Physiol. Psychol.1953. Vol. 46, No. 3. P. 339�341.

Brennan F., Browne K., Green P., et al. Reduction of serum matrix metalloproteinase1 and matrix metalloproteinase 3 in rheumatoid arthritis patients following anti�tumor necrosis factor (cA2) therapy // Br. J. Rheumatol. 1997. No. 36. P. 643�650.

Brennan F., Chantry D., Turner M., et al. Inhibitory effects of TNF�alpha antibodies onsynovial cell interleukin�1 production in rheumatoid arthritis // Lancet. 1989. No.2. P. 244�247.

Bruhwyler J., Liegeois J.F., Lejeune C., et al. New dibenzazepine derivatives with disin�hibitory and/or antidepressant potential: neurochemical and behavioural study inthe open�field and forced swimming tests // Behav. Pharmacol. 1995. Vol. 6, No. 8.P. 830�838.

Budantseva S.I. Comparative study of the therapeutic efficacy of some drugs in experi�mental chronic nonhealing ulcers of the stomach [in Russian]. Abstract of Cand. Med.Sci. Dissertation. Ryazan, 1973.

Bures J., Buresova O., Huston J.P. Techniques and basic experiments for the study ofbrain and behavior [in Russian]. Moscow, 1991. P. 146�149.

Bures J., Buresova O., Huston J.P. Techniques and basic experiments for the study ofbrain and behavior. Amsterdam�N.Y., 1983.

Burlakova E.B., Konradov A.A., Khudyakov I.B. Effect of chemical agents in ultralowdoses on biological objects // Izv. Akad. Nauk SSSR. 1990, No. 2. P. 184�194.

Burlakova E.B., Terekhova S.F., Grechenko T.N., Sokolov E.N. Effect of inhibitors of radicalreactions of lipid oxidation on electrical activity of the isolated snail neuron // Biofiz�ika. 1986. Vol. 31, No. 5. P. 921.

Burnet F. M. Evolution of the immune process in vertebrates // Nature. 1968. Vol. 218.P. 426�430.

Burov Yu.V., Robakidze T.N., Kadysheva A.E. Study of antiamnesic activity of amiridineon the model of amnesic syndrome // Byull. Eksp. Biol. Med. 1991. Vol. 111, No. 6.P. 614�617.

Burov Yu.V., Salimov P.M. Effect of pharmaceutical substances on intrastrain aggression// Byull. Eksp. Biol. Med. 1976. No. 5. P. 64�67.

Calabrese E.J., Baldwin L.A. The frequency of U�shaped dose responses in the toxicolog�ical literature // Toxicol. Sci. 2001. Vol. 62. P. 330�338.

Cannon W. B. Bodily changes in pain, hunger, fear, and rage. N.Y., 1915.

Caschik C., Dawson W., Ritchen E. Nonsteroid antiinflammatory agents // J. Pharm.Pharmakol. 1977. No. 28. P. 330�336.

Castagni V., Lemaire M., Kheyfets I., et al. Antibodies to S100 proteins have anxiolytic�like activity at ultra�low doses in the adult rat // J. Pharm. Pharmacol. In press.

281

Chernikov F.R., Sorokin V.N. Study of homeopathic remedies in centesimal dilutionsprepared by the method of S. Hahnemann // Homeopathic yearbook [in Russian].Moscow, 1998. P. 93�104.

Chernukh A.M., Kopteva L.A. Myocardial metabolism [in Russian]. Moscow, 1977.P. 329�338.

Chernysheva G.A., Martyushev A., Aliev O.I., et al. Comparison of systemic hemodynam�ic effects of Impaza and sildenafil // 2nd Congress of the Russian Scientific Societyof Pharmacology “Fundamental Problems of Pharmacology”. Moscow, 2003. Abstractsof Papers. Part II. P. 277.

Chuikova K.I., Zhuravleva L.A., Shvab I.A., et al. Antiviral therapy of children with infec�tious mononucleosis // 4th International Conference “Clinical Studies of Drugs”. Mos�cow, 2004. Abstracts of Papers. P. 269�270.

Chumakova G.A., Kiseleva E.V., Ivanov S.V., et al. Endothelial dysfunction as a pathoge�netic mechanism for cardiovascular diseases. Possibilities of diagnostics and correc�tion. Methodical guidelines [in Russian]. Barnaul, 2006.

Chung K.W., Kim S., Chan W.Y., Rennert O.M. Androgen receptors in ventral prostateglands of zinc deficient rats // Life Sci. 1986. Vol. 38, No. 4. P. 351�356.

Coopman G.P., Kennis H.M. Two methods to assess the gastrointestinal transit�time inmice // Z. Versuchstierk. 1977. Vol. 19, No. 5. P. 298�303.

Corda M.G., Orlandi M., Lecca D., et al. Pentylenetetrazole�induced kindling in rats:effect of GABA function inhibitors // Pharmacol. Biochem. Behav. 1991. Vol. 40,No. 2. P. 329�333.

Costa�Pereira A.P., Williams T.M., Strobl B., et al. The antiviral response to gamma�interferon // J. Virol. 2002. Vol. 76, No. 18. P. 9060�9068.

Cunha P., Poole S., Lorenzetii B., Fereira S. The pivotal role of tumor necrosis factor�alpha in the development of inflammatory hyperalgesia // Br. J. Pharmacol. 1992.No. 107. P. 660�669.

Cunningham A.J. A method of increased sensitivity for detecting single antibody�form�ing cells // Nature. 1965. Vol. 207, No. 1. P. 1106�1107.

Danevas E., Beauvais F., Amara J., Benveniste J. Human basophil degranulation trig�gered by very dilute antiserum against IgG // Nature. 1988. Vol. 333. P. 816�818.

Davydov A.T., Bazilenko I.B., Shabanov P.D. Proproten�100 in the therapy of alcoholabuse patients with post�withdrawal disorders // Psikhofarmakol. Biologich. Narkol.2004. No. 2, 3. P. 656�662.

Davydova N.A., Ivanenko V.V., Anisimova L.P., et al. Studying the effect of antibodies toangiotensin II AT

1 receptors in ultralow doses (Kardos) on vascular remodeling in

patients with chronic heart failure // 5th International Conference “Clinical Studiesof Drugs”. Moscow, 2005a. Abstracts of Papers. P. 60�61.

Davydova N.A., Ivanenko V.V., Chepurina N.G., et al. Study of first�dose hypotension inpatients with chronic heart failure after treatment with antibodies to angiotensin IIAT

1 receptors (Kardos) // 5th International Conference “Clinical Studies of Drugs”.

Moscow, 2005d. Abstracts of Papers. P. 65.

Davydova N.A., Ivanenko V.V., Prom A.K., et al. Effect of antibodies to angiotensin IIAT

1 receptors in ultralow doses (Kardos) on physical exercise tolerance in patients

with chronic heart failure // 5th International Conference “Clinical Studies of Drugs”.Moscow, 2005b. Abstracts of Papers. P. 62�63.

References

282

Ultralow doses

Davydova N.A., Ivanenko V.V., Ryazantseva N.V., et al. Effect of antibodies to angio�tensin II AT

1 receptors in ultralow doses (Kardos) on cardiac remodeling in patients

with chronic heart failure // 5th International Conference “Clinical Studies of Drugs”.Moscow, 2005c. Abstracts of Papers. P. 63�64.

Dawson G.R., Tricklebank M.D., Use of the elevated plus�maze in the search for novelanxiolytic agents // Trends Pharmacol. Sci. 1995. Vol. 16, No. 2. P. 33�36.

Dayer J., Beutler B., Cerami A. Cachectin/tumor necrosis factor stimulate collagenaseand prostaglandin E production by human synovial cells and dermal fibroblast // J.Exp. Med. 1985. No. 162. P. 2163�2168.

de Gasparo M., Catt K.J., Inagami T., et al. International union of pharmacology. XXIII.The angiotensin II receptors // Pharmacol. Rev. 2000. Vol. 52, No. 3. P. 415�72.

Delbancut A., Barouillet M.P., Cambar J. Evidence and mechanistic approach of theprotective effects of heavy metal high dilutions in Rodent and renal cell cultures /Ed. M. Bastide. Dordrecht, 1997. P. 71�83.

Devyatkov N.D., Galant M.B., Tager A.S. Role of synchronization in the effect of weakelectromagnetic signals of the millimeter range on living organisms // Biofizika. 1983.Vol. 16, No. 5. P. 895�896.

Diabetes. World Health Organization. Informational Bulletin, No. 312. 2006.

Diamandis E.P. Prostate�specific antigen: a cancer fighter and a valuable messenger? //Clin. Chem. 2000. Vol. 46, No. 7. P. 896�900.

Dinareilo C.A., Cannon J.G., Wolff S.M., et al. Tumor necrosis factor is an endogenouspyrogen and induces production of interleukin 1 // J. Exp. Med. 1986. No. 163. P.1433�1450.

Dobryakov Yu.I. Screening method for studying the antistress effect of drugs // Stressand adaptation [in Russian]. Kishinev, 1978. P. 172.

Donato R. S100: a multigenic family of calcium�modulated proteins of the EF�hand typewith intracellular and extracellular functional roles // Int. J. Biochem. Cell. Biol.2001. Vol. 33. P. 637�668.

Dreen L.C., Wagner D.A., Glogowski J., et al. Analysis of nitrate, nitrite and (15N) ni�trate in biological fluids // Anal. Biochem. 1982. Vol. 126. P. 131�143.

Dugina J.L. Antiulcer properties of antibodies to histamine in ultralow doses [in Rus�sian]. Abstract of Cand. Med. Sci.Dissertation. Tomsk, 2003a. P. 174.

Dugina J.L., Bakumov P.A., Schepotkin I.V., et al. Gastroprotective effects of ultra lowdoses of antibodies to histamine. Hepato�Gastroenterology. Istanbul, Turkey, 2002.Vol. 49, Suppl. II. P. XIII.

Dugina J.L., Bakumov P.A., Shchepotkin I.V., et al. Perspectives in the use of Epigam fortreatment of gastric ulcer and duodenal ulcer // Ros. Zh. Gastroenterol. Gepatol.Koloproktol. 2003c. Vol. XIII, No. 5. Suppl. 21. P. 28.

Dugina J.L., Bakumov P.A., Shchepotkin I.V., Sergeeva S.A. Epigam as a new potentantiulcer agent // Gastroenterologiya Sankt�Peterburga (Nauchno�Prakticheskii Zh.).2003b. No. 2�3. P. 54.

Dugina J.L., Kheifets I.A., Borovskaya T.G., et al. Experimental study of the efficacy ofAfala during hormone�induced inflammation of the prostate gland // 13th RussianNational Congress “Man and Drug”. Moscow, 2006. Abstracts of Papers. P. 127�128.

Dugina J.L., Kheifez I.A., Martyushev�Poklad A.V., et al. Anti�S100 antibodies amelio�rate haloperidol�induced catalepsy and do not change the activity of haloperidol in

283

apomorphine�induced stereotypy // Eur. Neuropsychopharmacol. 2006. Vol. 16,Suppl. 4. S. 436�S.437.

Dugina J.L., Petrov V.I., Babayeva A.R., et al. A randomized, open�label, comparative, 6�month trial of oral ultra�low doses of antibodies to tumor necrosis factor�alpha anddiclofenac in rheumatoid arthritis. // Int. J. Tissue React. 2005a. Vol. 27, No. l.P. 15�21.

Dugina J.L., Petrov V.I., Babayeva A.R., et al. Artorfoon oral ultra�low doses of antibod�ies to TNF�alpha, is effective both in rheumatoid arthritis and osteoarthritis // Ba�sic. Clin. Pharmacol. Toxicol. 2005b. Vol. 97, Suppl. 1. P. 95.

Dugina J.L., Zueva E.P., Amosova E.N., et al. Ultralow doses of antibodies to histamineas a new potent antiulcer agent // Sib. Zh. Gastroenterol. Gepatol. 2002. No. 14�15.P. 79�80.

Dugina J.L., Zueva E.P., Krylova S.G., et al. Experimental study of Epigam as a newantiulcer agent // 10th Russian National Congress “Man and Drug”. Moscow, 2003c.Abstracts of Papers. P. 605.

Dugina Y.L., Voronina T.A., Martyushev�Poklad A.V., et al. The anticonflict and antide�pressant – like effects of proproten are serotonin dependent // Eur. Neuropsychop�harmacol. 2005b. Vol. 15, Suppl. 3. P. 538�539.

Dunham N.W., Miya T.S. A note on a simple apparatus for detecting neurological deficitin rats // J. Amer. Pharm. Ass. 1957. Vol. 46. P. 208�209.

Dygai A.M., Zhdanov V.V., Epstein O.I., et al. Role of humoral factors in the regulationof hematopoiesis during immobilization stress // Byull. Eksp. Biol. Med. 2004.Vol. 137, No. 3. P. 224�248.

Dygai A.M., Zhdanov V.V., Simanina E.V., et al. Role of humoral factors in the regula�tion of erythropoiesis during cytostatic�induced myelosuppression // Byull. Eksp. Biol.Med. 2003. Vol. 125, No. 6. P. 642�646.

Eaton C.L. Aetiology and pathogenesis of benign prostatic hyperplasia // Curr. Opin.Urol. 2003. Vol. 13, No. 1. Р. 7�10.

Effects of biologically active substances in ultralow doses // Ros. Khim. Zh. 1999.Vol. XLIII, N. 5.

Egorov V.B., Pakhomov D.V. Clinical efficacy of Anaferon for children in the therapy ofchildren with hemorrhagic fever and renal syndrome // 4th International Confer�ence “Clinical Studies of Drugs”. Moscow, 2004. Abstracts of Papers. P. 74�75.

Epstein O.I. Conceptual model for the evolution of system adaptation // Depositedmanuscript. Registered in the Russian Authors Society. 1996. No. 1686.

Epstein O.I. From homeopathy to immune regulation of functions // Proceedings of theinterdepartmental scientific council on experimental and applied physiology [inRussian]. 2002d. Vol. 11. P. 123�131.

Epstein O.I. Possible mechanisms for the effect of potentiated remedies and some prob�lems of functions of biological systems // Byull. Sib. Otd. Ros. Akad. Med. Nauk.1999c. No. 1. P. 132�149.

Epstein O.I. Regulatory properties of ultralow doses // Byull. Eksp. Biol. Med. 2002a.Suppl. 4. P. 8�13.

Epstein O.I., Beregovoi N.A., Pankova T.M., et al. Ultralow doses of antibodies: specificin vitro effect of bipathic treatment on the model of long�term posttetanic potentia�tion // Byull. Eksp. Biol. Med. 2003a. Suppl. 1. P. 20�23.

References

284

Ultralow doses

Epstein O.I., Beregovoi N.A., Sorokina N.S., et al. Effect of potentiated antibodies tobrain�specific protein S�100 in various dilutions on the dynamics of posttetanic po�tentiation in surviving hippocampal slices // Byull. Eksp. Biol. Med. 1999a. Vol. 127,No. 3. P. 317�320.

Epstein O.I., Beregovoy N.A., Sorokina N.S., et al. Membrane and synaptic effects ofanti�S�100 are prevented by the same antibodies in low concentrations // Front.Biosci. 2003. Vol. 8. P. A79�A84.

Epstein O.I., Chernova O.V., Kolyadko T.M., Shtark M.B. Method for the correction ofimpaired immune homeostasis and medical product. RF Inventor’s CertificateNo. 2168335, priority of 19.03.1998a.

Epstein O.I., Dugina J.L., Bakumov P.A., et al. Clinical efficacy of Epigam in patientswith gastric ulcer and duodenal ulcer: comparative study with ranitidine // 3rdInternational Conference “Clinical Studies of Drugs”. Moscow, 2003b. Abstracts ofPapers. P. 128�130.

Epstein O.I., Gainutdinov Kh.L., Shtark M.B. Effect of antibodies to S�100 antigen inhomeopathic doses on electrical properties of neuronal membranes // Byull. Eksp.Biol. Med. 1999b. Vol. 127, No. 4. P. 466�467.

Epstein O.I., Komarov E.V., Martyushev A.V., Sergeeva S.A. Proproten�100 as a new�generation drug for the therapy of alcoholism and addictive disorders // Interna�tional Scientific Conference “New Pharmaceutical Products: Synthesis, Technology,Pharmacology, and Clinics”. Minsk, 2001c. Abstracts of Papers. P. 167.

Epstein O.I., Krylova S.G., Zueva E.P., et al. Experimental study of the gastroprotectiveeffect of antibodies to histamine in ultralow doses // Byull. Eksp. Biol. Med. 2001b.Suppl. 3. P. 43�45.

Epstein O.I., Martyushev A.V., Kudryashova D.R., et al. Potentiated antibodies to angio�tensin II and AT

1 receptors: study of the hypotensive effect // Byull. Eksp. Biol. Med.

2002c. Suppl. 4. P. 69�72.

Epstein O.I., Molodavkin G.M., Voronina T.A., Sergeeva S.A. Antidepressant propertiesof Proproten and amitriptyline: comparative experimental study // Byull. Eksp. Biol.Med. 2003c. Suppl. 1. P. 34�36.

Epstein O.I., Myagkova M.A. Method for a qualitative study of the homeopathic remedyor potentiated substance. RF Inventor’s Certificate No. 2195648, priority of03.11.2000.

Epstein O.I., Pashinskii V.G., Zelenskaya K.L., et al. Antiinflammatory and analgetic ef�fects of the homeopathic remedy from antibodies to tumor necrosis factor�a // Byull.Eksp. Biol. Med. 2001a. Suppl. 3. P. 57�59.

Epstein O.I., Pavlov I.F., Shtark M.B. Improvement of memory by means of ultra�lowdoses of antibodies to S�100B antigen // Evidence�based Complementary and Alter�native Medicine. 2006. Vol. 3, No. 4. P. 541�545.

Epstein O.I., Sherstoboev E.Yu., Martyushev�Poklad A.V., et al. Dose dependence andspecificity of the effect of antibodies to endogenous regulators in ultralow doses //Byull. Eksp. Biol. Med. 2004. Vol. 137, No. 5. P. 527�529.

Epstein O.I., Shtark M.B., Dygai A.M., et al. Pharmacology of ultralow doses of antibod�ies to endogenous regulators of functions [in Russian]. Moscow, 2005.

Epstein O.I., Shtark M.B., Simonova O.G., Ratushnyak A.S. Studying the neuronal re�sponse to alkaloid agonists of opiate receptors during modulation of the phosphod�iesterase system // Byull. Eksp. Biol. Med. 2002b. Suppl. 4. P. 18�21.

285

Epstein O.I., Shtark M.B., Timoshenko A.Kh., et al. Protective effect of antibodies toS�100 protein in low doses on the induction of long�term sensitization in ediblesnail // Byull. Eksp. Biol. Med. 2007a. Vol. 143, No. 5. P. 490�493.

Epstein O.I., Vorob’eva O.V., Grinkevich L.N., et al. A psychotropic drug Tenoten activatesthe mitogen�activated MAP/ERK kinase regulatory cascade, which regulates the neu�roprotective effects // Byull. Eksp. Biol. Med. 2007b. Vol. 144, No. 9. P. 293�296.

Epstein O.I., Vorob’eva T.M. Some new notions of the phenomenon of system adapta�tion (architecture of functional regulation). Biological regulation�3: theory and practice[in Russian]. Novosibirsk, 1998b.

Epstein O.I., Vorob’eva T.M., Berchenko O.G., et al. Informational and ontological mo�dels of adaptation [in Russian] / Ed. O. I. Epstein. Moscow, 1997.

Epstein O.I., Voronina T.A., Molodavkin G.M., et al. Studying the bipathic effect ofphenazepam // Byull. Eksp. Biol. Med. 2007c. Vol. 144, No. 10. P. 417�419.

Epstein O.I., Zaparova T., Simonova O.G., et al. Plasticity of neuronal responses inducedby low concentration of exogenous ligands affecting cellular calcium stores // Front.Biosci. 2004. Vol. 9. P. 809�815.

Expert Committee on the Diagnosis and Classification of Diabetes Mellitus // DiabetesCare. 2003. Vol. 26, Suppl. 1. P. 5�20.

Fano G., Biocca S., Fulle S., et al. The S�100: a protein family in search of a function //Prog. Neurobiol. 1995. Vol. 46, No. 1. P. 71�82.

Feldman M., Brennan P., Maini R. Role of cytokines in rheumatoid arthritis // Annu.Rev. Immunol. 1996. No. 14. P. 397�440.

Fermin C.D., Martin D.S. Expression of S100 beta in sensory and secretory cells of thevertebrate inner ear // Cell. Mol. Biol. 1995. Vol. 41, No. 2. P. 213�225.

File S.E. Animal models of different anxiety states // GABAa receptors and anxiety:from neurobiology to treatment. N.Y., 1995. P. 93�113.

Fomin V.V., Tunkina E.E., Saburova E.B., et al. Clinical and immunological study ofAnaferon efficacy in combination therapy of children with infectious mononucleosis// (United Immunological Forum. Ekaterinburg, 2004. Abstracts of Papers). Rus. J.Immunol. 2004. Vol. 9, Suppl. 1. C. 185.

Fortier A.H., Nelson B.J., Grella D.K., Holaday J.W. Antiangiogenic activity of prostate�specific antigen // J. Natl. Cancer Inst. 1999. Vol. 6, No. 91. P. 1635�1640.

Friddle C.J., Koga T., Rubin E.M. Expression profiling reveals distinct sets of genes al�tered during induction and regression of cardiac hypertrophy // Proc. Natl. Acad.Sci. USA. 2000. Vol. 7. P. 6745�6750.

Gannushkina I.V. Pathophysiological mechanisms for disturbances in cerebral blood flowand new directions of their prevention and therapy // Zh. Nevrol. Psikhiatr. 1996.No. 1. P. 14�18.

Garkavi L.Kh., Kvakina I.B., Kuz’menko T.S. Antistress reactions and activation therapy.Reaction of activation as a way to health through the processes of self�organization[in Russian]. Moscow, 1998.

Garyaev P.P. Wave genetic code [in Russian]. Moscow, 1997.

Gay A., Boiron J. Demonstration Phisique de l’Existance Reelle du Remede Homoeo�pathique / Lyon: Editions des Laboratoires P.H.R., 1953.

Gering K. Main symptoms of our Materia Medica [in Russian] / Eds. S. P. Pesonina andYu. V. Vasil’eva. In Ten Volumes. St. Petersburg, 1998.

References

286

Ultralow doses

Gilboi L. M., Adair R.S.S., Malheiros A., et al. Assessment of mechanisms involved inantinociception caused by sesquiterpene polygodial // Pharmacol. Exp. Ther. Vol. 292,Issue 1. P. 164�172.

Gofman A.G., Krylov E.N., Grazhenskii A.V., et al. Efficacy of Proproten�100 in the reliefof alcohol withdrawal syndrome / New medical products (express information) [inRussian]. Moscow, 2002. P. 53�58.

Gofman A.G., Pyatnitskaya I.N., Valentik Yu.V., et al. Proproten�100 in the relief of al�cohol withdrawal syndrome // Byull. Eksp. Biol. Med. 2003. Suppl. 1. P. 86�90.

Gol’dberg E.D., Dygai A.M., Shakhov V.P. Tissue culture methods in hematology [inRussian]. Tomsk, 1992.

Golovacheva E.G. Clinical and laboratory substantiation of immunocorrection therapyfor respiratory syncytial virus infection in children [in Russian]. Abstract of Cand.Med. Sci. Dissertation. St. Petersburg, 2003.

Gubler E.V. Computational methods for the analysis and identification of pathologicalprocesses [in Russian]. Leningrad, 1978. P. 193.

Guidelines for the diagnosis and treatment of chronic heart failure: full text (update2005). Eur. Heart J. The task force for the diagnosis and treatment of CHF of theEuropean Society of Cardiology.

Gulyaev Yu.V., Godik E.E. Physical fields of biological objects // Vestn. Akad. Nauk SSSR.1983. No. 8. P. 73�88.

Gurvich A.G. Theory of biological field [in Russian]. Moscow, 1944.

Gus’kova T.A. Toxicology of medical products [in Russian]. Moscow, 2003.

Hahnemann S. Materia Medica. New Delhi, 1983.

Hahnemann S. Organon of the medical art [in Russian]. Moscow. 1992.

Hamilton C.D. Immunosuppression related to collagen�vascular disease or its treat�ment // Proc. Am. Thorac. Soc. 2005. Vol. 2, No. 5. P. 456�460.

Hawonh C., Brennan D., Chantry D., et al. Expression of granulocyte�macrophage colo�ny�stimulating factor in rheumatoid arthritis, regulation by tumor necrosis factor�α// Eur. J. Immunol. 1991. No. 21. P. 2575�2579.

Heidtmann H.H., Nettelbeck D., Mingels A., et al. Generation of angiostatin�like frag�ments from plasminogen by prostate�specific antigen // Br. J. Cancer. 1999. Vol. 81,No. 8. P. 1269�1273.

Hering C. Condensed Materia Medica. Jain Publishing Co, 1983.

Horssen R., Hagen T.L.M., Eggermont A.M.M. TNF�alpha in cancer treatment: molecularinsights, antitumor effects and clinical utility // Oncologist. 2006. Vol. 11, No. 4.P. 397�408.

Hyden H., Cupello A., Palm A. Effect of anti�S�100 serum on 36Cl� ion permeabilityacross the Deiters’ neuron plasma membrane // Cell. Mol. Neurobiol. 1987. Vol. 7,No. 4. P. 439�445.

Inamova O.V., Suleimanova L.D. Experience in the use of Artrofoon for outpatient treat�ment of various musculoskeletal diseases // 5th Northwestern Conference on Rheu�matology. St. Petersburg, 2005. Abstracts of Papers. P. 48�49.

Ishizawa M., Mizushige K., Noma T., et al. An antioxidant treatment potentially protectsmyocardial energy metabolism by regulating uncoupling protein 2 expression in achronic beta�adrenergic stimulation rat model // Life Sci. 2006. Vol. 78, No. 25.P. 2974�2982.

287

Ivanenko V.V., Davidova N.A., Sergeeva S.A., et al. Ultra�low doses of antibodies to c�ending fragment of the angiotensin II type 1 receptor in patients with congestiveheart failure: first clinical experience // Eur. Heart J. 2005. Vol. 26 (Abstract Sup�plement). P. 412.

Ivanenko V.V., Davydova N.A., Lopatin Yu.M. Endothelial dysfunction in patients withchronic heart failure: modifying effect of ultralow doses of antibodies to angiotensinII AT1 receptors // 1st All�Russian Congress, 5th Annual Conference of Heart Fail�ure Society “Heart Failure 2004”. Moscow, 2004b. Abstracts of Papers. P. 6.

Ivanenko V.V., Davydova N.A., Lopatin Yu.M. Studying the effect of ultralow doses ofantibodies to angiotensin II AT

1 receptors on heart rate variability in patients with

chronic heart failure // 1st All�Russian Congress, 5th Annual Conference of HeartFailure Society “Heart Failure 2004”. Moscow, 2004a. Abstracts of Papers. P. 5�6.

Ivanenko V.V., Davydova N.A., Ryazantseva N.V., et al. First experience in the use ofultralow doses of antibodies to angiotensin II AT

1 receptors in patients with chronic

heart failure // 1st All�Russian Congress, 5th Annual Conference of Heart FailureSociety “Heart Failure 2004”. Moscow, 2004c. Abstracts of Papers. P. 5.

Ivanets N.N., Anokhina I.P., Borisova E.V., Drobysheva V.Ya. Results of scientific studiesin narcology in 2001 // Vopr. Narkol. 2002. N. 3 P. 3�12.

Jackowski A., Crockard A., Burnstock G., et al. The time course of intracranial patho�physiological changes following experimental subarachnoid hemorrhage in the rat// J. Cerebral Blood Flow Metabol. 1990. Vol. 10. P. 835�849.

Jerne N.K. The generative grammar of the immune system. Nobel Lecture, December1984 // Biosci. Reprod. 1985. Vol. 5. P. 439�451.

Jerne N.K. Towards network theory of the immune system // Ann. Immunol. (Inst Pas�teur) 1974. Vol. 125. P. 373�389.

Jonas W., Lin Y., Tortella F. Neuroprotection from glutamate toxicity with ultra�low doseglutamate // Neuroreport. 2001. Vol. 12, No. 2. P. 335�339.

Jonsson M., Linse S., Frohm B., et al. Semenogelins I and II bind zinc and regulate theactivity of prostate�specific antigen // Biochem. J. 2005. Vol. 15, No. 387, Pt. 2.P. 447�453.

Kalinichenko A.A. Experimental and clinical substantiation for using the biological productSpedian�M in treatment of patients with peptic ulcer disease [in Russian]. Abstractof Cand. Med. Sci. Dissertation. Moscow, 1973.

Kashkan G.V. Atomic emission detection of microelements in biological fluids and tis�sues // 1st Regional Scientific Conference “Major Developments of Medical Tech�niques in Institutions and Factories of Tomsk”. Tomsk, 1988. Abstracts of Papers.P. 8�9.

Kato F., Nomura M., Nakamura K. // Ann Rheum Dis. 1996. Vol. 55, No. 8. P. 535�539.

Kaznacheev V.P., Mikhailova L.P. Ultra�low emissions in cell�to�cell interactions [inRussian]. Novosibirsk, 1981.

Keller G. Homeopathy [in Russian]. Moscow, 1989.

Khamanova Yu.B., Tunkina E.E., Shmakova E.A., et al. Clinical and immunological studyof the efficacy of Anaferon for children during infectious mononucleosis and enteroviraland meningococcal meningitis // 12th Russian National Congress “Man and Drug”.Moscow, 2005. Abstracts of Papers. P. 577.

Khitrov N.A. Use of Anaferon in pathogenetic therapy for various forms of shoulderperiarthritis // 7th City Scientific and Practical Conference Devoted to the 40th

References

288

Ultralow doses

Anniversary of Municipal Hospital No. 10 “Medical Rehabilitation of Patients withDiseases of the Musculoskeletal and Nervous Systems”. December 20, 2006. Mos�cow, 2006. Abstracts of Papers. P. 174�176.

Khomskii N. Language and thinking. Language and problems of knowledge [in Russian].Blagoveshchensk, 1999.

Khomskii N. On the nature and language [in Russian]. Moscow, 2005.

Kohn M.C., Melnick R.L. Biochemical origins of the non�monotonic receptor�mediateddose�response // J. Mol. Endocrinol. 2002. Vol. 29. P. 113�123.

Kolchanov N.A., Suslov V.V., Gunbin K.V. Modeling of biological evolution: regulatorygenetic systems and coding of the complexity of biological organization // Vestn.VOGiS. 2004. Vol. 8, No. 2. P. 86�99.

Kolisko L. Capillary dynamolysis; a specific method to study the formative forces ininorganic and organic substances; application in medicine, agriculture, and dietetics// Hippokrates. 1953. Vol. 24, No. 5. P. 130�135.

Kolpakov N.D. Polarization waves // Radio engineering. All�Ukrainian interdepartmen�tal scientific and technical collection [in Russian]. Kharkov, 1997. No. 101.

Kondrat’eva E.I., Shemyakina T.A., Koroleva E.V. Anaferon for children as a drug fornonspecific prevention of ARVI in children with bronchial asthma // Vopr. Sovr.Pediatr. (10th Congress of Pediatricians of Russia “Pathways to Increase the Efficacyof Children’s Medical Care”. Moscow, 2005. Abstracts of Papers) 2005. Vol. 4,Suppl. 1. P. 245.

Kondrat’eva E.I., Shemyakina T.A., Koroleva E.V., Bezlyak V.V. Use of Anaferon for chil�dren in preschool�age children with bronchial asthma // Vopr. Sovr. Pediatr. (10thCongress of Pediatricians of Russia “Urgent Problems of Pediatrics”. Moscow. Febru�ary 6�9, 2006. Abstracts of Papers) 2006. Vol. 5. No. 1.

Kondrat’eva E.I., Shemyakina T.A., Koroleva E.V., et al. Experience in the use of Anaf�eron for children to prevent ARVI and exacerbation of bronchial asthma in preschool�age children // 3rd Congress of Pediatric Infectious Disease Physicians of Russia“Urgent Problems of Infectious Diseases in Children. Infection and Immunity”. Mos�cow, 2004. Abstracts of Papers. P. 111.

Konovalova N.P. Ultralow doses of medical products in chemotherapy for experimentaltumors // Byull. Eksp. Biol. Med. 2002. Suppl. 4. P. 49�51.

Koplik E.V., Gryzunov Yu.A., Kopaeva L.V., et al. Concentration of nonesterified fattyacids in the albumin fraction of blood plasma from rats with different locomotoractivity in the open field // Dokl. Ros. Akad. Nauk. 2001. Vol. 378, No. 1. P. 1�3.

Koplik E.V., Salieva R.M., Gorbunova A.V. Open�field test as a prognostic criterion forthe resistance of Wistar rats to emotional stress // Zh. Vyssh. Nervn. Deyat. 1995.No. 45. P. 775�781.

Korablev M.V., Lukienko P.I. Antihypoxic drugs [in Russian]. Minsk, 1976. P. 28�37.

Kostryukova I.V. Clinical and laboratory evaluation of the efficacy of Artrofoon therapyin patients with osteoarthritis [in Russian]. Abstract of Cand. Med. Sci. Dissertation.Volgograd, 2006. P. 29.

Kostryukova I.V., Babaeva A.R., Petrov V.I., et al. Comparative study of the efficacy andtolerability of Artrofoon in patients with osteoarthritis (4th Congress of Rheumatol�ogists of Russia. Kazan. May 23�26, 2005. Abstracts of Papers) // Nauch.�Praktich.Revmatol. 2005. No. 3. P. 67.

289

Kravkov N.P. Sensitivity limits of living protoplasm // Uspekhi Eksper. Biol. 1924. No. 3�4.P. 147�172.

Krylov E.N. Clinical study of nonnarcotic substitutive effects of Anar in the therapy ofpatients with heroin abuse // Byull. Eksp. Biol. Med. 2003a. Suppl. 1. P. 105�111.

Krylov E.N. Psychotropic activity of Proproten�100 // Byull. Eksp. Biol. Med. 2003b.Suppl. 1. P. 97�101.

Krylova S.G., Razina T.G., Zueva E.P., et al. Analgetic and antiinflammatory activity ofantibodies to histamine under experimental conditions // Byull. Eksp. Biol. Med.2002b. Suppl. 4. P. 95�97.

Krylova S.G., Razina T.G., Zueva E.P., et al. Antiulcer properties of antibodies to hista�mine in ultralow doses under experimental conditions // Byull. Eksp. Biol. Med.2002a. Suppl. 4. P. 92�94.

Kudin M.V. Anaferon for children in the therapy of children with chickenpox // 5thInternational Conference “Clinical Studies of Drugs”. Moscow, 2005a. Abstracts ofPapers. P. 101�103.

Kudin M.V., Maiorov R.N., Sergeeva S.A. Clinical course of chickenpox in children dur�ing treatment with Anaferon for children (10th Congress of Pediatricians of Russia“Pathways to Increase the Efficacy of Children’s Medical Care”. Moscow, 2005. Ab�stracts of Papers) // Vopr. Sovr. Pediatr. 2005b. Vol. 4, Suppl. 1. P. 320.

Kudrin A.N. Lecture on the symposium of the Academy of Sciences “Mechanisms for theEffect of Ultralow Doses” [in Russian]. Moscow, 1991.

Kudryavtseva I.V., Ukolova L.A., Chizhov N.N., et al. Experience in the use of potentia�ted antibodies to tumor necrosis factor�α (Artrofoon) for therapy of ankylosing spondy�loarthritis // Poliklinika. 2005. No. 2. P. 45�49.

Kuzin A.M. Secondary biogenic emissions as rays of life [in Russian] / Puschino, 1997.

Lee K.L., Peehl D.M. Molecular and cellular pathogenesis of benign prostatic hyperplasia// J. Urol. 2004. Vol. 172, No. 5, Pt. 1. P. 1784�1791.

Lewis T., Teyler D.J. Anti�S�100 serum blocks long�term potentiation in the hippocam�pal slice // Brain Res. 1986. Vol. 24, No. 383 (1�2). P. 159�164.

Li X.M., Zhang L., Li J., et al. Measurement of serum zinc improves prostate cancerdetection efficiency in patients with PSA levels between 4 ng/mL and 10 ng/ml //Asian J. Androl. 2005. Vol. 7, No. 3. P. 323�328.

Livanov G.A., Kalmanson M.L., Tikhomirov S.M., Safronov A.G. Standards of addictionhelp. Guidelines for physicians [in Russian]. St. Petersburg, 2003.

Locksley R.M., Killen N., Lenardo M.J. The TNF and TNF receptor superfamilies: inte�grating mammalian biology // Cell. 2001. No. 104. P. 487�501.

Lue TF. Erectile dysfunction // N. Engl. J. Med. 2000. Vol. 342. P. 1802�1813.Lupia E., Montrucchio G., Battaglia E., et al. Role of tumor necrosis factor�α and plate�

let�activating factor in neoangiogenesis induced by synovial fluids of patients withrheumatoid arthritis // Eur. J. Immunol. 1996. No. 26. P. 1690�1694.

Macnaul K., Chartrain N., Lark M., et al. Differential effects of IL�1 and TNF alpha onthe expression of stromelisin, collagenase and their inhibitor, TIMP, in rheumatoidhuman synovial fibroblasts // Matrix Suppl. 1992. No. 1. P. 198�199.

Maddox J. Waves caused by extreme dilution // Nature. 1988. Vol. 335, No. 6193.P. 760�763.

Madersbacher S., Alivizatos G., Nordling J., et al. EAU 2004 guidelines on assessment,therapy and follow�up of men with lower urinary tract symptoms suggestive of be�

References

290

Ultralow doses

nign prostatic obstruction (BPH guidelines) // Eur. Urol. 2004. Vol. 46, No. 5.P. 547�554.

Makarenko A.N., Kositsyn N.S., Karpenko S.V., Mishina V.A. Author’s certificate No.1767518 of 03.11.1990.

Makarenko A.N., Sergeeva S.A., Dugina J.L., et al. Neuroprotective activity of Proprotenon the model of hemorrhagic stroke // 11th Russian National Congress “Man andDrug”. Moscow, 2004. Abstracts of Papers. P. 808�809.

Malm J., Hellman J., Hogg P., Lilja H. Enzymatic action of prostate�specific antigen (PSAor hK3): substrate specificity and regulation by Zn2+, a tight�binding inhibitor //Prostate. 2000. Vol. 1, No. 45. P. 132�139.

Manji H.K., Duman R.S. Impairments of neuroplasticity and cellular resilience in severemood disorders: implications for the development of novel therapeutics // Psychop�harmacol. Bull. 2001. Vol. 35, No. 2. P. 5�49.

Manual on experimental (preclinical) study of new pharmacological substances [in Rus�sian] / Ed. V. P. Fisenko. Moscow, 2000.

Manual on experimental (preclinical) study of new pharmacological substances [in Rus�sian] / Ed. R. U. Khabriev. Moscow, 2000.

Markel’ A.L., Kudryashova D.R., Martyushev A.V., et al. Ultralow doses of antibodies tovascular tone�regulating factors: results of screening for hypotensive activity // Byull.Eksp. Biol. Med. 2002. Suppl. 4. P. 64�66.

Marotta D., Marini A., Banaudha K., et al. Non�linear effects of cycloheximide in glutamate�treated cultured rat cerebellar neurons // Neurotoxicology. 2002. Vol. 23, No. 3.P. 307�312.

Marotta D., Marini A., Banaudha K., et al. Nonlinear effects of glutamate and KCl onglutamate toxicity in cultured rat cerebellar neurons // Int. J. Neurosci. 2003.Vol. 113, No. 4. P. 491�502.

Martyushev�Poklad A., Dugina J., Epstein O., Sergeeva S. A novel antibody anxiolytic:results of short controlled clinical trials // XIII World Congress of Psychiatry. Cairo,Egypt, 10�15 September, 2005f.

Martyushev�Poklad A., Mazo E., Gamidov S., et al. Impaza, an oral antibody therapeuticfor erectile dysfunction: benefits from mono� and combined therapy // Proceedingsof the VIIIth International Congress of Andrology / Eds. J. A. Grootegoed, H. E.Chemes. Medimond, Bologna, Italy. 2005a. P. 49�54.

Martyushev�Poklad A., Petrov V., Dugina J., et al. A novel oral antibody thera�peutic for treatment of BPH: primary findings of a controlled clinical trial //Proceedings of the VIIIth International Congress of Andrology / Eds. Sae�ChulKim, J. A. Grootegoed, H. E. Chemes. Medimond, Bologna, Italy. 2005c. P. 183�188.

Martyushev�Poklad A.V. Mechanisms for the antiviral and immunomodulatory effects ofantibodies to interferon�γ in ultralow doses [in Russian]. Abstract of Cand. Med. Sci.Dissertation. Tomsk, 2003.

Martyushev�Poklad A.V., Babaeva A.R., Tcherevkova E.V., et al. Artrofoon (oral ultra�low doses of anti�TNFα) in rheumatoid arthritis: primary clinical findings // Inflamm.Res. Suppl. 2003. Vol. 2. P. 159.

Martyushev�Poklad A.V., Epstein O.I. Impaza as a new drug for the therapy of erectiledysfunction // Poliklinika. 2003. No. 1. P. 8�9.

291

Martyushev�Poklad A.V., Kotelnikova M.P., Uchaikin V.F., et al. A novel antiviral basedon oral antibodies: clinical benefits in pediatric upper respiratory infections // Clin.Microbiol. Infect. 2004. Vol. 10, Suppl. 3. P. 125�126.

Martyushev�Poklad A.V., Kudin M.V., Dyldina N.V., et al. A novel therapeutic for treat�ment of varicella: peroral anti�interferon gamma antibodies // 4th World Congressof the World Society for Pediatric Infectious Diseases. ESPID, Warsaw, Poland, Sep�tember 1�4, 2005b. Book of abstracts. P. 144.

Martyushev�Poklad A.V., Petrov V.I., Pavlov V.N., et al. New product of oral antibodiesfor the therapy of BPH in Russia: first results of a controlled clinical trial // 2nd All�Russian Conference “Man’s Health”. Moscow, 2005. Abstracts of Papers. P. 290�291.

Martyushev�Poklad A.V., Uchaikin V.F., Dugina I.L., et al. Anaferon – a novel antiviralagent: controlled trials of efficacy in pediatric upper respiratory infections // 22ndAnnual Meeting of the European Society for Pediatric Infectious Diseases. Tampere,Finland. 2004b. May 26�28. P. 11.

Martyushev�Poklad A.V., Voronina T.A. S100B protein as a probable target for neuro�modulation // Vestn. Biologich. Psikhiatr. 2004. No. 3. P. 3�9.

Martyushev�Poklad A.V., Voronina T.A. Stress�limiting systems and neuronal plasticityin the pathogenesis of mental and neurological disorders // Obzory Klinich. Farma�kol. Lekarstv. Ter. 2003. Vol. 2, No. 4. P. 15�25.

Mazo E. B., Gamidov S.I., Ovchinnikov R.I. Oral monotherapy and combination therapyof erectile dysfunction with phosphodiesterase�5 inhibitor and Impaza (new induc�tor of NO synthase in Russia) // Rus. Med. Zh. 2004b. Vol. 12, No. 8. P. 552�559.

Mazo E.B., Gamidov S.I., Ovchinnikov R.I. Viagra, Sialis, and Impaza: what, to whom,when, and how? // Urologiya. 2004a. No. 5. P. 42�48.

Mazo E.B., Petrov V.I. Use of Impaza in the therapy of men with erectile dysfunction.Guidelines for physicians [in Russian]. Moscow, 2004.

Mazurov V.I., Rassokhin V.V. Artrofoon in the therapy of rheumatoid arthritis and os�teoarthritis. Methodical recommendations [in Russian]. St. Petersburg, 2007.

Medvedeva N.A., Artem’eva M.M., Borodavkina M.V., et al. Studying the effect of Kar�dos on baroreflex sensitivity of the cardiovascular system in hypertensive SHR rats// 13th Russian National Congress “Man and Drug”. Moscow, 2006a. Abstracts ofPapers. P. 210.

Medvedeva N.A., Artem’eva M.M., Borodavkina M.V., et al. Studying the effect of Kar�dos on systemic blood pressure and heart rate in hypertensive SHR rats // 13thRussian National Congress “Man and Drug”. Moscow, 2006b. Abstracts of Papers.P. 210�211.

Meerson F.Z. Adaptation medicine: mechanisms and protective effects of adaptation [inRussian]. Moscow, 1993.

Meier R., Schuler W., Dessulles P. // Experimentia (Basel). 1950. No. 6. P. 469.

Merkulov G.A. Course of pathohistological technique [in Russian]. Leningrad, 1969.

Methodical recommendations to study the toxicity of pharmaceutical substances // Far�makologicheskii Gosudarstvennyi Komitet. 1998. No. 1. P. 13�20.

Middlemiss D.N., Price G. W., Watson J.M. // Curr. Opin. Pharmacol. 2002. Vol. 2.P. 18�22.

Migliorini P., Corradin G., Corradin S.B. Macrophage NO2�production as a sensitive andrapid assay for the quantitation of murine IFN�g // J. Immunol. Metods. 1991.Vol. 139. P. 107�114.

References

292

Ultralow doses

Milovanov A.P., Brusilovskii A.I. Standardization of methods for morphometry of hu�man placenta // Arkh. Anat. 1986, No. 8. P. 72�78.

Mizel S.B. Studies on the purification and structure�function relationships of murinelymphocyte activating factor (Interleukin 1) // Mol. Immunol. 1980. Vol. 17.P. 571�577.

Molodavkin G.M., Voronina T.A. Computer software for the recording of results of be�havioral studies // Eksp. Klin. Farmakol. 2005. Vol. 68, No. 6. P. 55�58.

Molodavkin G.M., Voronina T.A. Multichannel device for the search of tranquilizingcompounds and studying the mechanisms of their effect in a conflict situation test// Eksp. Klin. Farmakol. 1995. Vol. 58, No. 2. P. 54�56.

Molodavkin G.M., Voronina T.A., Mdzinarishvili A.L. Specific effects of pharmaceuticalsubstances of various classes on forced swimming in rats // Eksp. Klin. Farmakol.1994. Vol. 57, No. 1. P. 3�5.

Myagkova M.A. Natural antibodies to low�molecular�weight compounds. Monograph [inRussian]. Moscow, 2001.

Myagkova M.A., Abramenko T.V., Panchenko O.N., Epstein O.I. Ultralow doses of anti�bodies to delta sleep�inducing peptide: enzyme immunoassay of the effect // Byull.Eksp. Biol. Med. 2003. Suppl. 1. P. 10�12.

Nakajima S., Arizono N., Hattori T., Bamba T. Increase in mucosal and connective tis�sue�type mast cells in the stomach with acetic acid�induced ulcer in rat // APMIS.1996. Vol. 104, No. 1. P. 19�29.

Navashin S.M., Vyadro M.M. Modifiers of biological reactions in the therapy for malig�nant neoplasms // Itogi Nauki Tekhniki. VINITI. Ser. Onkol. 1989. Vol. 21.

Neimark A.I., Aliev R.T., Muzalevskaya N.I., et al. Interdisciplinary pathogenetic approachto the therapy of endothelial dysfunction // 3rd All�Russian Conference “Man’sHealth”. Moscow, 2006b. Abstracts of Papers. P. 254�255.

Neimark A.I., Aliev R.T., Muzalevskaya N.I., et al. Rational therapy for erectile dysfunc�tion in cardiology patients // 5th Regional Scientific and Practical Conference ofUrologists of Siberia “Urgent Problems of Children’s and Adult Urology. Tomsk, 2006a.Abstracts of Papers. P. 149�151.

Nibieridze D.V. Clinical significance of endothelial dysfunction during arterial hyperten�sion // Consilium Medicum. 2005. No. 1. P. 31�38.

Nomura S., Shimizu J., Kinjo M., et al. A new behavioral test for antidepressant drugs// Eur. J. Pharmacol. 1982. Vol. 83, No. 3�4. P. 171�175.

Note for guidance for non�clinical local tolerance testing of medicinal products (CPMP/CWP/2145/00)

Note for guidance on single dose toxicity (Eudralex vol. 3B)

Obolentseva G.V. Pharmacological study of the effect of some natural and modifiedpolysaccharides on digestive function [in Russian]. Abstract of Cand. Med. Sci. Dis�sertation. Moscow, 1984.

Obolentseva G.V., Khadzhai Ya.I., Vidyukova A.I. Effect of some natural substance onacetylsalicylic acid�induced gastric ulcers in rats // Byull. Eksp. Biol. Med. 1974.No. 3. P. 39�41.

Obraztsova E.V., Osidak L.V., Muradyan A.Ya., et al. Antiviral therapy of children withcoronavirus infection // 13th Russian National Congress “Man and Drug”. Moscow,2006. Abstracts of Papers. P. 676.

293

Ohta T., Hasebe N., Tsuji S., et al. Unequal effects of renin�angiotensin system inhibi�tors in acute cardiac dysfunction induced by isoproterenol // Am. J. Physiol. HeartCirc. Physiol. 2004. Vol. 287, No. 6. P. 2914�2921.

Olovnikova N.I., Miterev G.Yu. Therapeutic monoclonal antibodies and mechanisms oftheir effect // Ter. Arkh. 2007. No. 7. P. 87�96.

Olsen R.W., Snowhill E.W., Wamsley J.K. Autoradiographic localization of low affinityGABA receptors with [3H]bicuculline methochloride // Eur. J. Pharmacol. 1984.Vol. 99. P. 247�248.

Osadchuk M.A., Osadchuk A.M. Comparative clinical study of the therapeutic efficacy ofArtrofoon and Salofalk for mild nonspecific ulcerative colitis // 5th International Con�ference “Clinical Studies of Drugs”. Moscow, 2005. Abstracts of Papers. P. 125�126.

Osidak L.V., Afanas’eva O.I., Drinevskii V.P., et al. Anaferon for children as a new drugfor the therapy and prevention of influenza and other ARVI in children. Methodicalguidelines for physicians [in Russian]. St. Petersburg, Moscow, 2003. P. 23.

Ostroumova O.D., Dubinskaya R.E. Endothelial dysfunction in cardiovascular diseases// Kardiologiya. 2005. No. 2. P. 59�62.

Pal’mina N.P., Gaintseva V.D., Burlakova E.B. Modification of the chemotherapeutic ef�fect of adriablastine with low doses of a synthetic antioxidant // Byull. Eksp. Biol.Med. 2002. Suppl. 4. P. 58�63.

Pal’tsev M.A., Ivanov A.A. Cell�to�cell interactions [in Russian]. Moscow, 1995.

Pauls F., Wick A.M, Mac Key E.M. An assay method for anti�ulcer substances // Gastro�enterology. 1947. No. 8. P. 774�782.

Pavlov I.F., Epstein O.I. Ultralow doses of morphine and antibodies to m�opiate receptors:effect on oxygen consumption // Byull. Eksp. Biol. Med. 2003. Suppl. 1. P. 51�53.

Pavlov V.N., Kazikhinurov R.A., Pushkarev A.M. Studying the clinical efficacy and safetyof Afala as a therapeutic drug for patients with benign prostatic hyperplasia // 12thRussian National Congress “Man and Drug”. Moscow, 2005a. Abstracts of Papers.P. 503.

Pavlov V.N., Martyushev�Poklad A.V., Kazikhinurov R.A., Sergeeva S.A. Clinical efficacyof Afala as a new drug for the therapy of BPH: results of a comparative study //12th Russian National Congress “Man and Drug”. Moscow, 2005b. Abstracts of Pa�pers. P. 126�127.

Pellow S., File S.E. Anxiolytic and anxiogenic drugs effects on exploratory activity in anelevated plus�maze. A novel test of anxiety in the rat // Pharmacol. Biochem. Be�hav. 1986. Vol. 24. P. 525�529.

Petrov R.V., Khaitov R.M., Man’ko V.M., et al. Methodical materials on an experimental(pharmacological) and clinical study of the immunomodulatory effect of pharmaceu�tical substances // Pharmacological Committee [in Russian]. Moscow, 1984.

Petrov S.I., Epstein O.I. Potentiated solutions: effect on mercury (II) signal in inversionvoltammetry // Byull. Eksp. Biol. Med. 2003. Suppl. 1. P. 6�9.

Petrov V.I., Babaeva A.R., Cherevkova E.V., et al. Efficacy of a new antirheumatic drug Art�rofoon in the therapy for inflammatory diseases of the joints // 4th International Con�ference “Clinical Studies of Drugs”. Moscow, 2004. Abstracts of Papers. P. 174�175.

Petrov V.I., Babaeva A.R., Cherevkova E.V., et al. Ultralow doses of antibodies to tumornecrosis factor�α (Artrofoon): efficacy in the therapy of patients with rheumatoidarthritis // Byull. Eksp. Biol. Med. 2003. Suppl. 1. P. 72�76.

References

294

Ultralow doses

Petrov V.I., Babaeva A.R., Tcherevkova E.V., et al. Artrofoon in treatment of rheumatoidarthritis patients: a pilot randomized clinical study. // European collaboration: To�wards drug development and rational drug therapy / Eds. F. C. Tulunay, M. Orme.2003. P. 99.

Petrov V.I., Cherevkova E.V., Babaeva A.R., et al. Anticytokine therapy with Artrofoonfor rheumatoid arthritis and urogenic arthritis // 12th Russian National Congress“Man and Drug”. Moscow, 2005. Abstracts of Papers. P. 507.

Petrov V.I., Davydova N.A., Ivanenko V.V., et al. Effect of ultralow doses of antibodiesto angiotensin II AT

1 receptors on the course of chronic heart failure // 6th Annual

Conference of Heart Failure Society “Heart Failure 2005”. Moscow, 2005. Abstractsof Papers. P. 54�55.

Petrov V.I., Davydova N.A., Ivanenko V.V., et al. Kardos in the therapy of patients withchronic heart failure // 13th Russian National Congress “Man and Drug”. Moscow,2006. Abstracts of Papers. P. 247.

Petrov V.I., Pavlov V.N., Savel’eva K.V., et al. Comparative study of Afala efficacy in thetherapy for benign prostatic hyperplasia // 13th Russian National Congress “Manand Drug”. Moscow, 2006. Abstracts of Papers. P. 437.

Poletaev A.B., Morozov S.G., Kovalev I.E. Regulatory metasystem. Immunoneuroendo�crine regulation of homeostasis [in Russian]. Moscow (2002).

Popova T.D. Essays on homeopathy [in Russian]. Kiev, 2006.

Porsolt R.D., Anton G., Blavet N., Jalfre M. Behavioral despair in rats: a new modelsensitive to antidepressant treatment // Eur. J. Pharmacol. 1978. Vol. 47. P. 379�391.

Portnoi A.S., Grodzovskaya F.L. Cancer and adenoma of the prostate gland [in Russian].Leningrad, 1984. P. 12, 20.

Pribram K. Languages of the brain [in Russian]. Moscow, 1975.

Prigozhin I. From the existing to the developing. Time and complexity in physical sci�ences [in Russian]. Moscow, 2006.

Prigozhin I., Stengers I. Order from chaos: new dialogue of a human with nature [in Russian]/ Eds. V. I. Arshinov, Yu. L. Klimontovich, and Yu. V. Sachkov. Moscow, 1986.

Proproten�100. Ultralow doses of affinity purified antibodies to S�100 protein. Collec�tion of articles [in Russian]. Moscow, 2002.

Pshennikova M.G. Stress: regulatory systems and resistant to stress�induced damages //Dysregulation pathology [in Russian] / Ed. G. N. Kryzhanovskii. Moscow, 2002.P. 307�328.

Raevskii K.S., Narkevich V.B. Manual on experimental (preclinical) study of new phar�macological substances [in Russian] / Ed. V. P. Fisenko. Moscow, 2000. P. 114�120.

Ratner V.A., Zharkikh A.A., Kolchanov N.A. Problems of the theory of molecular evolu�tion [in Russian]. Novosibirsk, 1985.

Razd”yakonova I.V., Tikhomirova O.V., Sirotkin A.K. Clinical course and possibilities fortherapy of children with calicivirus infection // 5th International Conference “Clini�cal Studies of Drugs”. Moscow, 2005. Abstracts of Papers. P. 135�136.

Rees D.A., Alcolado J.C. Animal models of diabetes mellitus // Diabet Med. 2005. Vol. 22,No. 4. P. 359�370.

Regulations of laboratory practice in the Russian Federation. Order of the Russian Mini�stry of Health No. 267 of 19.06.2003.

295

Rizzuto R. Intracellular Ca2+ pools in neuronal signaling // Curr. Opin. Neurobiol. 2001.Vol. 11, No. 3. P. 306�311.

Romanova G.A., Barskov I.V., Ostrovskaya R.U., et al. Behavioral and morphologicaldisorders in rats after bilateral photoinduced thrombosis of cerebral vessels in thefrontal cortex // Patol. Fiziol. Eksper. Ter. 1998. No. 2. P. 8�10.

Romanova G.A., Voronina T.A., Sergeeva S.A., et al. Studying the antiischemic and neu�roprotective effect of Proproten // Sib. Vestn. Psikhiatr. Narkol. 2003. Vol. 27, No. 1.P. 123�125.

Sanger D.J., Perrault G., Morel E., et al., Animal models and recent developments in thesearch for novel anxiolytics // Animal models in psychopharmacology / Eds. B. Oliver,J. L. Slangen. Birkhauser Verlag Basel, 1991. P. 3�14.

Sanotskii I.V. Methods to evaluate the toxicity and safety of chemical substances (toxi�cometry) [in Russian]. Moscow, 1970.

Saraiva R.M., Chedid N.G., Quintero H., et al. Impaired beta�adrenergic response anddecreased L�type calcium current of hypertrophied left ventricular myocytes in postin�farction heart failure // Braz. J. Med. Biol. Res. 2003. Vol. 36, No. 5. P. 635�648.

Seredinin S.B., Voronina T.A., Burlakova E.B., et al. Specific anticonflict effect ofphenazepam on rats selected in the forced swimming test // Zh. Vyssh. Nervn. Deyat.Im. I. P. Pavlova. 1994. Vol. 44, No. 4�5. P. 834�839.

Sergeev A.N., P’yankov O.V., Shishkina L.N., et al. Antiviral activity of ultralow doses ofantibodies to interferon�g during oral administration: experimental study of influen�za infection in mice // Antibiot. Khimioter. 2004. Vol. 49, No. 11. P. 7�11.

Sergeeva S., Martyushev�Poklad A., Lopatin Y., et al. Oral antibodies to AT1 angiotensinII receptor – a novel option in the treatment of chronic heart failure // Acta Phar�macol. Sin. 2006. Vol. 27, Suppl. 1. P. 137.

Sergeeva S.A., Dugina J.L., Martyushev�Poklad A.V., Epstein O.I. Neuroprotective activ�ity of proproten in experimental model of cerebral hemorrhagic stroke // Abstractsof the 8th ECNP Regional Meeting. Moscow, Russia, 2005. Vol. 15. P. 241�242.

Sergeeva S.A., Dugina J.L., Voronina T.A., Epstein O.I. Antiaggressive activity of Propro�ten // 11th Russian National Congress “Man and Drug”. Moscow, 2004. Abstracts ofPapers. P. 832�833.

Sergeeva S.A., Martyushev�Poklad A.V., Petrov V.I., et al. Anaferon, oral anti�interferon�gamma antibodies for treatment and prophylaxis of viral infections // Clin. Exper.Pharmacol. Psychol. 2004. Vol. 31, Suppl. 1. A. 114.

Setnicar J., Da Re P. 3�methyl�6(N�diethyl�amino�methyl)�Flavone � a new smooth�mus�cle relaxant // Arzneimittel � Forsch. 1959. No. 9. P. 653�697.

Shangin�Berezovskii G.N., Adamov V.Ya., Rykhletskaya O.S., Moloskin S.A. Systemicstimulatory effect of supermutagens in ultralow doses // Improvement of cultivatedplants and chemical mutagenesis [in Russian]. Moscow, 1982. P. 65�75.

Shangin�Berezovskii G.N., Moloskin S.A., Rykhletskaya O.S. Paradoxical effect of micro�doses of NDMU and PABA in dependence on the sensitivity of test samples // Chem�ical mutagenesis in the development of varieties with new properties [in Russian].Moscow, 1986. P. 243�248.

Shay M., Komarov S.A., Fels D., et al. A simple method for the uniform production ofgastric ulceration in the rat // Gastroenterology. 1945. No. 5. P. 43�61.

Shingu M., Nagai Y., Isayama T., et al. The effects of cytokines on metalloproteinaseinhibitors (TIMP) and collagenase production by human chondrocytes and TIMP

References

296

Ultralow doses

production by synovial cells and endothelial cells // Clin. Exp. Immunol. 1993. No. 94.P. 145�149.

Shipov G.I. Theory of physical vacuum [in Russian]. Moscow, 1993.

Shoenfeld Y., Isenberg D.A. Natural autoantibodies: their physiological role and regula�tory. CRC Press Inc., 1993.

Shostak N.A., Pavlenko A.Yu., Khomenko V.V., et al. Studying the efficacy and safety ofArtrofoon therapy for pain syndrome in patients with knee osteoarthritis // Vestn.Ros. Gos. Med. Univer. M. 2005. Vol. 47, No. 8. P. 45�48.

Shtark M.B. Brain�specific proteins (antigens) and functions of the neuron [in Russian].Moscow, 1985.

Shtark M.B., Epstein O.I., Komarov E.V. Ultralow doses of antibodies to neurospecificsubstances as a new class of psychotropic drugs // Arkh. Psikhiatr. 2001. Vol. 27,No. 4. P. 166�168.

Shul’zhenko A.E., Zuikova I.N., Shcherbenko V.E., et al. Clinical study of Anaferon foradults in the therapy of chronic recurrent genital herpes // 5th International Con�ference “Clinical Studies of Drugs”. Moscow, 2005. Abstracts of Papers. P. 165�166.

Sil’kis I.G. General postsynaptic mechanism of plasticity in the striatum, neocortex, hip�pocampus, and cerebellum // Ros. Fiziol. Zh. Im. I. M. Sechenova. 2000. Vol. 86,No. 5. P. 519�531.

Simovan’yan E.N., Kolodyazhnaya N.M. Efficacy of Anaferon for children in the therapyof children with rotavirus infection // 3rd Congress of Pediatric Infectious DiseasePhysicians of Russia “Urgent Problems of Infectious Diseases in Children. Infectionand Immunity”. Moscow, 2004. Abstracts of Papers. P. 208�209.

Sit’ko S.P., Chizhko V.V. On the microwave coherent field of an organism and nature ofChinese meridians // Dokl. Akad. Nauk SSSR. Ser. Geol. Khim. Biol. Nauki. 1989.No. 8. P. 77�80.

Skripchenko N.V., Ivanova G.P., Morgatskii N.V., et al. Tick�borne encephalitis in chil�dren (pathogenesis, clinics, diagnostics, and therapy) // Guidelines for physicians[in Russian] / Ed. N. V. Skripchenko. St. Petersburg, 2005.

Smolenov I.V., Dygai A.M., Epshtein O.I., et al. Ultralow doses of antibodies to endothe�lial NOS are effective in erectile dysfunction // Pharmacologist. 2002. Vol. 44, Suppl1. A167.

Sof’ina Z.P. Modeling in chemotherapy // Itogi Nauki Tekhniki. VINITI. Onkol. 1987.P. 64�105.

Spasov A.A., Nazvanova A.N., Chepurina N.G., et al. Studying the antidiabetic activityof a new product from ultralow doses of antibodies on the model of streptozotocin�induced diabetes // Byull. Eksp. Biol. Med. 2007. Vol. 144, No. 7. P. 50�53.

Steiner G., Djavan B., Kramer G., et al. The picture of the prostatic lymphokine networkis becoming increasingly complex. MedReviews: LLC, 2002.

Stiles J. Neural plasticity and cognitive development // Dev. Neuropsychol. 2000. Vol.18, No. 2. P. 237�272.

Sudakov K.V. General theory of functional systems [in Russian]. Moscow, 1984.

Sudakov K.V. Physiology of motivations [in Russian]. Moscow, 1990.

Sudakov K.V. Reflex and functional system [in Russian]. Novgorod, 1997.

Sudakov K.V. System process of reinforcement // Zh. Vyssh. Nerv. Deyat. 1996b. Vol. 46,No. 4. P. 643�655.

297

Sudakov K.V. Theory of functional systems [in Russian]. Moscow, 1996a.

Sudakov K.V., Umriukhin P.E., Koplik E.V., Anokhin K.V. Delta�sleep inducing peptide(DSIP) and ACTH (4�10) analogue influence Fos�induction in the limbic structuresof the rat brain under emotional stress // Stress. 2001. Vol. 4, No. 2. P. 143�153.

Susloparov M.A., Makhova N.M., Noskova N.V., et al. Studying the efficacy of therapeu�tic and prophylactic treatment with ultralow doses of antibodies to interferon�g onthe experimental mouse model of herpes virus infection // Antibiot. Khimioter. 2004.Vol. 49, No. 10. P. 3�6.

Sutkowski D.M., Goode R.L., Baniel J., et al. Growth regulation of prostatic stromal cellsby prostate�specific antigen // J. Natl. Cancer Inst. 1999. Vol. 6, No. 91 (19).P. 1663�1669.

Swanson L.W., Sawchenko P.E. Paraventricular nucleus: a site for the integration ofneuroendocrine and autonomic mechanisms // Neuroendocrinology. 1980. Vol. 31.P. 410�417.

Szlosarek P.W., Grimshaw M.J., Kulbe H., et al. Expression and regulation of tumornecrosis factor alpha in normal and malignant ovarian epithelium // Mol. Cancer.Ther. 2006. Vol. 5, No. 2. P. 382�390.

Tan Z., Nagata S. PVN c�fos expression, HPA axis response and immune cell distributionduring restraint stress // J. UOEH. 2002. Vol. 24, No. 2. P. 131�149.

Tellander A.C., Michalsson E., Brunmark C., Andersson M. J. Potent adjuvant effect byanti�CD40 in collagen�induced arthritis. Enhanced disease is accompanied by increasedproduction of collagen type�II reactive IgG2a and IFN�gamma // Autoimmun. 2000.Vol. 14, No. 4. P. 295�302.

Tilz G., Diez�Ruiz A., Baier�Bitterlich G., et al. Soluble receptor for tumor necrosis fac�tor and neopterin as parameters of cell�mediated immune activation // AC1 Interna�tional. 1997. No. 9. P. 110�118.

Timchenko V.N., Kalinina N.M., Khmylova M.E., et al. Clinical and immunological char�acteristics of pseudotuberculosis in children. Therapy with Anaferon for children //3rd Congress of Pediatric Infectious Disease Physicians of Russia “Urgent Problemsof Infectious Diseases in Children. Infection and Immunity”. Moscow, 2004. Abstractsof Papers.

Titkova A.M., Epstein O.I. Effect of products from potentiated ethanol on biogenic aminecontent and ethanol metabolism in rat tissues during alcoholization // Byull. Eksp.Biol. Med. 2002. Suppl. 4. P. 40�42.

Tolle T.R., Schadrack J., Zieglgensberger W. Immediate early genes in the central ner�vous system. Munich, 1995.

Touyz R.M., Schiffrin E.L. Signal transduction mechanisms mediating the physiologicaland pathophysiological actions of angiotensin II in vascular smooth muscle cells //Pharmacol Rev. 2000. Vol. 52, No. 4. P. 639�672.

Trentham D.E., Townes A.S., Kang A.H. Autoimmunity to type II collagen an experimen�tal model of arthritis // J. Exp. Med. 1977. Vol. 146. P. 857�868.

Triet D. Animal models for the study of anti�anxiety agents, a review // Neurosci. Biobe�hav. Reviews. 1985. Vol. 9. P. 203�222.

Trivedi C.J., Balaraman R., Majithiya J.B., Bothara S.B. Effect of atorvastatin treatmenton isoproterenol�induced myocardial infarction in rats // Pharmacology. 2006.Vol. 77, No. 1. P. 25�32.

References

298

Ultralow doses

Tszyankan’chzhen’ Yu.V. Description of the invention (to patent SU 1828665 A3), 1993.

Tyurenkov I.N., Nazvanova A.N., Chepurina N.G., et al. Comparative study of the car�dioprotective effect of Kardos and losartan on rats with experimental chronic heartfailure // Byull. Eksp. Biol. Med. 2007. Vol. 143, No. 4. P. 411�413.

Uchaikin V.F., Epstein O.I., Sergeeva S.A., et al. Anaferon for children as a Russianimmunocorrecting agent with antiviral properties // Guidelines for pediatricians andinfectious disease physicians [in Russian] / Ed. V. F. Uchaikin (Academician of theRussian Academy of Medical Sciences). Moscow, 2003. P. 31.

Ukhtomskii A.A. Collected works [in Russian]. Leningrad, 1950. Vol. 1.

Ukhtomskii A.A. Collected works [in Russian]. Leningrad, 1950. Vol. 2.

Ulanova I.P., Avilova G.G., Tutarikova V.A. Methods to study the toxicity and hazard ofchemical substances [in Russian]. Moscow, 1970. P. 189�199.

Umetskii K.S., Dovgalevskii P.Ya., Tverdokhleb S.A. Use of Impaza in the therapy forerectile dysfunction in patients with functional class I�II angina pectoris // 13thRussian National Congress “Man and Drug”. Moscow, 2006. Abstracts of Papers.P. 309.

Umetskii K.S., Sergeeva S.A., Tarasov S.A., et al. Efficacy and safety of Impaza in thetherapy for erectile dysfunction in patients with CHD and FC I�II angina pectoris //5th International Conference “Clinical Studies of Drugs”. Moscow, 2005. Abstractsof Papers. P. 154�155.

Ushiyama M., Morita T., Kuramochi T., et al. Erectile dysfunction in hypertensive ratsresults from impairment of the relaxation evoked by neurogenic carbon monoxideand nitric oxide // Hypertens. Res. 2004. Vol. 27, No. 4. P. 253�261.

Uspenskii V.M., Grinevich V.B. Trophic role of mast cells in the gastric mucosa duringsecretion // Ter. Arkh. 1979. No. 3. P. 29�32.

Van Coppenolle F., Slomianny C., Carpentier F., et al. Effects of hyperprolactinemia onrat prostate growth: evidence of androgeno�dependence // Am. J. Physiol. Endo�crinol. Metab. 2001. Vol. 280, No. 1. P. 120�129.

Van de Loo F.A.J., Smeets R.L., van den Berg W.B. Gene therapy in animal models ofrheumatoid arthritis: are we ready for the patients? // Arthritis Res. Ther. 2004.No. 6. P. 183�196.

Van Vliet B.N., Chafe L.L., Antic V., et al. Direct and indirect methods used to study arterialblood pressure// J. Pharmacol. Toxicol. Methods. 2000. Vol. 44, No. 2. P. 361�373.

Vanchakova N.P., Popova A.P. Anxiety disorders in patients with hypertension and cor�onary heart disease. Tenoten in the correction of these disorders // Poliklinika. 2007.No. 2. P. 56�60.

Vasse M., Protais P., Costentin J., Schwartz J.C. Unexpected potentiation by discrimi�nant benzamide derivates of stereotyped behaviors elicited by dopamine agonists inmice // Naunyn�Schmiedeberg’s Arch. Pharmacol. 1985. Vol. 329. P. 108�116.

Vernadskii V.I. Philosophical ideas of a naturalist [in Russian]. Moscow, 1988.

Vertapetov B.A., Nikalaichuk S.P., Sherstyuk P.M. Gonadotropic hormones, prostate gland,and experimental model of its deficiency // Probl. Endokrinol. 1970. Vol. 16, No. 6.P. 80�85.

Vetlugina T.P., Ivanova S.A., Bokhan N.A., Epstein O.I. Immune changes in patients withopium abuse during therapy with Anar // Byull. Eksp. Biol. Med. 2003. Suppl. 1.P. 112�115.

299

Villoutreix B.O., Getzoff E.D., Griffin J.H. A structural model for the prostate diseasemarker, human prostate�specific antigen // Protein. Sci. 1994. Vol. 3, No. 11.P. 2033�2044.

Vladimirtseva I.F., Pusakova N.V., Kurkina A.V. Specific features of outpatient treatmentof children with tubulointerstitial nephritis // Vopr. Sovrem. Pediatr. 2005. Vol. 4,Suppl. 1. P. 92.

Vogel J.R., Beer B., Clody D.E. A simple and reliable conflict procedure for testing an�tianxiety agents // Psychopharmacologia. 1971. Vol. 21. P. 1�7.

Vorob’eva T.M. Novelty as an original stimulus during administration of pharmaceuticalproduct into the organism [in Russian]. Abstract of Cand. Med. Sci. Dissertation.Kharkov, 1962.

Voronina T.A., Borodavkina M.V., Kheifets I.A., et al. Experimental study of nootropicand antiamnesic properties of Tenoten in the test for a conditioned passive avoid�ance response of rats // 2nd National Congress on Social Psychiatry “Social Trans�formations and Mental Health”. Moscow, 2006c. Abstracts of Papers. P. 171.

Voronina T.A., Kheifets I.A., Dugina J.L., et al. Experimental study of combined treat�ment with haloperidol and Proproten // 1st International Interdisciplinary Congress“Progress in Neuroscience for Modern Medicine and Psychology”. Sudak, 2005. Ab�stracts of Papers. P. 58�59.

Voronina T.A., Kheifets I.A., Dugina J.L., et al. Role of the GABA�B system in the effectof antibodies to S�100 protein in ultralow doses // 13th Russian National Congress“Man and Drug”. Moscow, 2006b. Abstracts of Papers. P. 95.

Voronina T.A., Kheifets I.A., Dugina J.L., et al. Role of the serotoninergic system in theeffect of antibodies to S�100 protein in ultralow doses // 13th Russian NationalCongress “Man and Drug”. Moscow, 2006a. Abstracts of Papers. P. 94�95.

Voronina T.A., Molodavkin G.M., Dugina J.L., et al. Anxiolytic activity of proproten:involvement of GABA in this effect // Int. J. Psych. Clin. Pract. 2003. Vol. 7. P. 319.

Voronina T.A., Molodavkin G.M., Sergeeva S.A., Epstein O.I. Anxiolytic effect of Propro�ten under conditions of punished and non�punished behavior // Byull. Eksp. Biol.Med. 2003a. Suppl. 1. P. 31�33.

Voronina T.A., Molodavkin G.M., Sergeeva S.A., Epstein O.I. GABAergic system in theanxiolytic effect of Proproten: experimental study // Byull. Eksp. Biol. Med. 2003b.Suppl. 1. P. 37�39.

Voronina T.A., Ostrovskaya R.U. Methodical recommendations on studying the nootro�pic activity of pharmaceutical substances // Manual on experimental (preclinical)study of new pharmacological substances [in Russian] / Ed. V. P. Fisenko. Moscow,2000. P. 153�158.

Voronina T.A., Seredinin S.B. Methodical recommendations on studying the tranquiliz�ing (anxiolytic) activity of pharmaceutical substances // Manual on experimental(preclinical) study of new pharmacological substances [in Russian] / Ed. V. P. Fisenko.Moscow, 2000. P. 126�130.

Voronina T.A., Sergeeva S.A., Dugina J.L., Epstein O.I. Comparative study of Proprotenand piracetam in rats with experimental Alzheimer’s disease // Neiroimmunologiya.2004. Vol. 2, No. 1. P. 21.

Voronina T.A., Sergeeva S.A., Martyushev�Poklad A.V., et al. Antibodies to S�100 pro�tein in anxiety�depressive disorders in experimental and clinical conditions // Ani�mal models in biological psychiatry / Ed. A. V. Kalueff. N.Y., 2006. P. 137�152.

References

300

Ultralow doses

Voronina T.A., Val’dman E.A., Nerobkova L.N. Methodical recommendations on study�ing the antiparkinson activity of pharmaceutical substances // Manual on experi�mental (preclinical) study of new pharmacological substances [in Russian] / Ed. V.P.Fisenko. Moscow, 2000. P. 147�152.

Voronova O.L., Rogozina O.P., Martyushev A.V., et al. Ultralow doses of antibodies tointerferon�g: study of the cytogenetic effects // Byull. Eksp. Biol. Med. 2002. Suppl. 4.P. 73�75.

Wang X., Li M., Zheng H., et al. Influenza A virus NS1 protein prevents activation ofNF�kappaB and induction of alpha/beta interferon // J. Virol. 2000. Vol. 74, No. 24.P. 1566�1573.

Watson B.D. Animal models of photochemically induced brain ischemia and stroke //Pathophysiology, diagnosis and management. Cerebrovascular disease / Eds. M. D.Ginsberg, J. Bogousslavsky. 1998. Vol. 1. P. 52�73.

Watson B.D., Dietrich W.D., Busto R., et al. Induction of reproducible brain infarctionby photochemically initiated thrombosis // Ann. Neurol. 1985. Vol. 17. P. 497�504.

Wilt T., Ishani A., Mac Donald R. Serenoa repens for benign prostatic hyperplasia //Cochrane Database Syst Rev. 2002. No. 3. CD001423.

Yakovleva L.V., Rayanova R.R., Melititskaya A.V., et al. Experience in the use of Anafer�on for children during therapy of heart disease children with acute respiratory dis�orders // 4th Conference of Ural Immunologists. Ufa, 2005. Abstracts of Papers.Vol. 4, No. 1. P. 161.

Yurmazov Z.A., Savel’eva K.V., Dugina J.L., et al. Afala as a new drug for the therapy ofbenign prostatic hyperplasia // 5th International Conference “Clinical Studies ofDrugs”. Moscow, 2005. Abstracts of Papers. P. 166�168.

Zapara T.A., Simonova O.G., Epstein O.I. Effect of potentiated morphine on electricalparameters of isolated neurons // Byull. Sib. Otd. Ros. Akad. Med. Nauk. 1999.Vol. 91, No. 1. P. 91�92.

Zavodskaya I.S., Moreva E.V. Pharmacology study of the mechanisms and consequencesof stress [in Russian]. Leningrad, 1981.

Zenin S.V. Structured state of water as a basis for the regulation of behavior and safetyof living systems [in Russian]. Abstract of Doct. Chem. Sci. Dissertation. Moscow,1999.

Zernyukova E.A. Comparative efficacy and pharmacodynamics of the product from ul�tralow doses of antibodies to the C�terminal fragment of the angiotensin II AT

1 re�

ceptor, ACE inhibitors, and angiotensin II receptor antagonists in patients with es�sential hypertension [in Russian]. Abstract of Cand. Med. Sci. Dissertation. Volgo�grad, 2005.

Zernyukova E.A., Bakumov P.A., Chepurina N.G., Magnitskaya O.V. Pharmacodynamicsof the product from ultralow doses of antibodies to the C�terminal fragment of theangiotensin II AT

1 receptor (Kardos) in patients with stage 1�2 arterial hypertension

// 5th International Conference “Clinical Studies of Drugs”. Moscow, 2005. Abstractsof Papers. P. 80�81.

Zhuravleva L.A., Chuikova K.I., Galaktionova O.I., et al. Experience in the use of Anaf�eron for children in combination therapy of children with infectious mononucleosis// 2nd National Scientific Medical Congress “Human Health”. Erevan, 2003a. Ab�stracts of Papers. P. 147�148.

301

Zhuravleva L.A., Chuikova K.I., Pan I.R. First experience in the therapy of infectiousmononucleosis with Anaferon for children // 2nd Congress of Pediatric InfectiousDisease Physicians of Russia “Urgent Problems of Infectious Diseases in Children”.Moscow, 2003b. Abstracts of Papers. P. 62.

Zilov V.G., Sudakov K.V., Epstein O.I. Elements of informational biology and medicine[in Russian]. Moscow, 2000.

Zisman A., Lindner A., Zisman E., et al. Prostate�specific antigen induces proliferationof peripheral blood lymphocytes and cytokine secretion in benign prostate hypertro�phy patients // Eur. Urol. 1999. Vol. 36, No. 3. P. 258�265.

Zisman A., Zisman E., Lindner A., et al. Autoantibodies to prostate specific antigen inpatients with benign prostatic hyperplasia // J. Urol. 1995. Vol. 154, No. 3. P. 1052�1055.

Zubova S.G., Okulov V.B. Molecular mechanisms for the effect of tumor necrosis factor�a and transforming growth factor�β during the macrophage response to activation// Immunologiya. 2001. No. 5. P. 18�22.

References

302

Ultralow doses

Scientific edition

Epstein Oleg Il’ich

ULTRALOW DOSES(HISTORY OF ONE RESEARCH)

Head of the Editorial Board, K. V. MovsesyanEditor, T. N. Kuznetsova

Design, A. V. Ustinov

Publishing House of the Russian Academy of Medical Sciences14, Solyanka St., Moscow, 109240 Russia

Tel: +7 (495) 698�57�78, 698�59�89E�mail: [email protected], [email protected]

Internet address: www.iramn.ru