Hidden Secret - Blocking A Treatment for HIV and Cancer

8/14/2019 Hidden Secret - Blocking A Treatment for HIV and Cancer

1/124


2/124


3/124


4/124


5/124


6/124


7/124


8/124


9/124


10/124


11/124


12/124


13/124


14/124


15/124


16/124


17/124


18/124


19/124


20/124


21/124


22/124


23/124


24/124


25/124


26/124


27/124


28/124


29/124


30/124


31/124


32/124


33/124


34/124


35/124


36/124


37/124


38/124


39/124


40/124


41/124


42/124


43/124


44/124


45/124


46/124


47/124


48/124


49/124


50/124


51/124


52/124


53/124


54/124


55/124


56/124


57/124


58/124


59/124


60/124


61/124


62/124


63/124


64/124

I[

rE

L

cL

(ii).

(ii ).

The peroral medication has antimetastatic activity in animals withspontaneous pulmonary metastases. The greatest reduction of thenumber and volume of lung metastases was achieved when thetreatment began in the early phases of tumor dissemination or aftersurgical resection of th e primary tumor (fig. 7). The antimetastatic

activity of Factor-R increases host defence against tumors, .affectingAIDS patients .

The combined therapy with Factor-R and cyclophosphamide led tomore effective inhibition of the metastatic growth in comparison withchemotherapy alone . This combined therapy could avoid the severeside effects associated with high doses of cyclophosphamide . Theseresults suggested that the application of Factor -R could be successful inthe therapy of secondary pulmonary micrometastases . The success ofimmunotherapy of tumor metastases depends on the capacity of

Factor-R to activate immune mechanisms in the target organ whichcontains metastatic foci. The prooved effectivity of combined Factor-Rand cytostatic therapy ma y reflect in reduction of the dosage ofcytostatic agents in patients with AIDS, leading to the increase of theirtoxic effects

(iv). The cytolytic activity of AM recovered after peroral therapy with Factor-R for tendays was higher than that of the control AM. The increase of cytolitic activity ofphagocytes may result in stimulation of host defence against tumors and certainintracellular microorganisms .

(v). It was proven that there was a correlation between the capacity of Factor-R toinduce the accumulation of tumoricidal AM in the lower respiratory path ways andlungs and the substances anti metastatic properties.

In conclusion we can say that the activation of AM is one of the basic means ofinhibition of the growth of neoplasms.

82


65/124

I .

rr

C.

TOXICITY STUDY

1. ACUTE TOXICITY

1. 1 Acute toxicity on albino mice

As experimental animals 60 albino mice of the strain ICR were used, weighting18-20 g each. Twenty of them were fed per os through a probe with Factor-R - 50 mgin capacity of 0,5 ml physiological solution 5 days. The control group of 10 mice werefed in the same way with physiological solution only. The dose of 50 mg Factor-Radministered to mice is equivalent to about 5 000 daily human doses . All mice werecontrolled regarding their general state, feeding and weight, and blood probes weretaken for determination of the main hematological indices (erythrocytes, leukocytes,hemoglobin and differential counting).

The mice were sectioned under light ether narcosis on the 2nd day after the lastfeeding and a macroscopic view was made too. Material was collected for histologicand electron-microscopic examination of the main parenchymal organs (lung, liver,spleen and brain).

No changes in general state, weight and behaviour, of the fed with Factor-Rcompared with those from the control group were observed. The hematologicalindices were in the limits of norm in both groups. The histological and electronmicroscopic study did not establish pathological changes in the examined organs.

1.2 Acute toxicity on rabbits

As experimental animals 15 rabbits of the "Chinchilla" race were used, weighing1.500-1.600 g each . Ten of them were fed through probe with Factor-R - 2 g, incapacity of 2 ml physiological solution 5 days. This dose of Factor-R is equivalent toabout 2,000 daily human doses. Five rabbits were fed with 2 ml physiological

solution on the same scheme. All rabbits were controlled daily regarding their generalstate, feeding and weight, and blood probes were taken for determination of theirmain hematological indices (erythrocytes, leukocytes, hemoglobin and differentialcounting).

The rabbits were sectioned after treatment with 300 mg hexobarbital sodiumintracutaneously on the second day after the last feeding. A macroscopic view wasmade of the sectioned animals and material was collected fo r histological andelectronmicroscopic examination of their parenchymal organs (lung, liver, spleen and

brain).

83


66/124

rE

L

Compared with those of the control group, no changes in the general state, weightand behaviour, of the animals fed with Factor-R were observed. The hematologicalindices were in the limits of norm in both groups. No pathological changes wereobserved in the organs examined through histological and electron-microscopicstudies.

2. CHRONIC TOXICITY

Chronic Toxicity in Rats

30 rats of the Wistar race, weighing 180 - 200 g each were used as experimentalanimals. 20 of them were fed per os through probe with 0.5 g of Factor-R in capacityof 1 ml physiological solution daily in the course of 4 months. One dose per rat isequivalent to 5 000 daily human doses. The remaining 10 rats were fed with 1 ml

physiological solution on the same scheme.

The rats were controlled daily with regard to their general state, weight, feeding,morbidity and mortality. Their hematological indices were examined every monthduring the 4-month period. The animals were sectioned under light ether narcosis 7

days after the chronical trials had been completed. A macroscopic view was madeand material for hematological and electron-microscopic examination of the mainparenchymal organs (lung, liver, spleen and brain) was collected.

As a result from th e studies no changes in the general state (weight, feeding,

morbidity and mortality) of the animals fed with Factor-R was observed compared tothose of the control group. The hematological indices were within the limits of normin both groups. No pathological changes were observed in the examined organsthrough histological and electron-microscopic studies.

84


67/124

I

I

rF

r.

Name of drug: SUMMARY TABLE Processing numberFACTOR-R ref. to

III. A. . I ........

Generic name Page Number

Report date/Number:Study period IV. 1987

SINGLE DOSE TOXICITY Referring to documentationVolume: Page: toAddendum No. :

Species/Strain: White mice/JCR

Administration route: Number of animals: 60per os

Treatment of controls: Dose : max. non lethal: 5000 human doses

10 white mice strain JCR min. lethal:were treated per os with 0.5 ml

Observation period: (Appl. day = Day 1)saline and observed for 7 days

Study group (1) Contr. (2) (3) (4) (5) (6)

Dosage 0 1000 h. 5000 hdoses doses

Sex (m/t) m f m f m f m f m f m f

Animals per dosage 10 20 10 25 5

within 6 hours - - - - -til

.c 7 - 24 hours - - - - --a day 2-7 - - - - -DC day 8 - end of observ.I - - - - -

Summary of salient findings: All mice were under daily control regarding their generalstate, feeding and weight and blood testing was made fo r determination of the main hemato-logical indices (erythrocytes, leukocytes, hemoglobin and differential counting). On the secondda y after the last feeding the animals were sectioned under light ether narcosis and macro-scopically viewed. Samples were taken from the main parenchymal organs (lung, liver, spleen andbrain) fo r histological and electron-microscopical testing . No changes were observed in thegeneral state (weight and behaviour) of th e animals fed with Factor-R in comparison withha t of the control group. Th e hematological indices in both groups were within th e normalimits. No pathological changes in the examined organs were observed during the histological andelectron-microscopical studies.

Study conducted by the applicant: yes < > no < X >If "no", indicate the name and address of the insti tute that conducted the study:National Center of Infectious and Parasitic Diseases, Sofia, BulgariaStudy in compliance with GLP: yes < X> no

Page

85


68/124

I :

.

[

.r

L

L

L

Name of drug: SUMMARY TABLE Processing numberFACTOR-R ref. to

III. B .... 1.....Generic name Page Number

REPEATED DOSE TOXICITY Report date/Number:Study period IV. 1987Referring to documentation

1. Subacute toxicity Volume: Page: to(up to 3 months) Addendum No.:

Species/Strain: Rabbits/Chinchilla

Number of animals: 15 Duration of treatment: 5 days

Observation period after the end of dosing: 3 days

Administration route: per as

Treatment of controls: 5 rabbits Age: at studywere treated per os with 2 ml Body weight: 1500 - 1600 g initiationsaline for 5 days

Treatment da ys Der week: 5 consecutive da vsStudy group (1) Contr. (2) (3) (4)

Dosage 0 2000 humandoses

Sex (m/t) m f m f m f m f

Number of test animals 5 - 10 5 ,-

Number of animals died - - - -or sacrificed in extremis

Clinical observations: yes < X > no< > Clin. chemis try: yes < > no< > 1rinalysis: yes no < > > no< >I

Water consumption: yes < X > no< > Organ weights: yes < X > no < > IBody weights: yes < X > no < > Necropsy: yes < X > no < >

Haematology: yes < X > no < > Histology: yes < X > no < >

Additional examination: electron microscopy of the main parenchymal organs IAdditional information:

Histology performed according to EEC Note fo r Guidance: yes < > no< >Study conducted by the applicant: yes < > no < X >If "no", indicate the name and address of the insti tute that conducted the study:National Center of Infectious and Parasitic Diseases, Sofia, BulgariaStudy in compliance with GLP: yes < X > no < > Page

86


69/124

l[[

[[

[

ll

Name of drug: Processing numberFACTOR-R

Generic name

REPEATED DOSE TOXICITYSUPPLEMENTAY SHEET

to III. b

Page from

Important findiings

referring toIII.bSUPPLEMENTARY SHEET

Report date/Number:

Study period (years): -Referring to documentationVolume: Page:Addendum No. :

Group 2M F

Group 3M F

Explanations: 0 = decrease 1= increase p= permanent t= transitoryns = not significant * = p


70/124

I

I

:

~

r

L

L

Name of drug:FACTOR-R

Generic name

SUPPLEMENTARY SHEETTO SUMMARY TABLE .III.to register importantfindingsPage from

referring toIIISUPPLEMENTARY SHEET

Report date/Number:

Processing number

Study period (years): -Referring to documentationVolume: Page:Addendum No.:

to

The rats were under a daily control regarding their general state (weight, feeding,morbidity and mortality). the hematological indices of both groups were investigated everymonth during a 4-month period . Seven days after the chronic trial was completed the animalswere sectioned under a light ether narcosis . They were macroscopically viewed and

samples were taken for histological and electron-microscopical examination of the mainparenchymal organs (lung, liver , spleen and brain) . No changes in the general state (weightand behaviour) of the animals fed with Factor-R were observed in comparison with that ofthe control group of rats. In both groups hematological indices were within the normallimits. No pathological changes in the examined organs were observed during the histologicaland electron-microscopical studies.

Page

89


71/124

I~

.

LL

L

RESULTS OF CLINICAL TRIALS


72/124

I

I.

Ir.

~

CLINICAL TRIALS

A. Description of subject population

The stimulating effect of Factor-R on the immune system was confirmed in extensive trials onlarge contingents of subjects with various diseases.

B. Eligibility criteria

Trials were conducted in subjects with the following diseases:

- non-specific respiratory diseases,

- syphilis,

- lung cancer,

- HI V infection.

Although ou r investigations were conducted in subjects with different nosologic entities, thecommon eligibility criterion was the presence of immunodeficiency in all of the remaining groups.

C. Study design

Subjects were treated with Factor-R perorally at the following doses: 25 mg, 40 g, 50 mg, 60 mg,

and 80 mg daily.

D. Objective parameters for both laboratory and clinical improvement

The clinical monitoring of subjects included: case history, physical examination and objectivecondition, results of laboratory tests and some immunologic indices.

E. Toxicity

No adverse effects of toxicity induced by Factor-R were observed in any of the subjects.

F. Concomitant therapies allowed and utilized

Different groups of subjects received the following therapeutic agents in combination with FactorR, depending of the specificity of disease:

Antibiotics and chemotherapeutic drugs in subjects with syphilis and 8 AIDS subjectsou t of 17;

Immunosuppressive therapy in subjects with lung cancer;

9 subjects with HIV-infection - antiretroviral drugs;

All subjects with nonspecific diseases and 9 HIV-infected subjects ou t of 17 receivedno etiologic therapy to the date of clinical trials.

91


73/124

c

L

L

LL

G. Subject status at the conclusion of trial

The following effects were observed in subjects with chronic nonspecific pulmonary diseasesfollowing Factor-R treatment:

beneficial effect on the clinical characteristics of the disease (severity, course andoutcome);

improvement of spirometric indices;

shortening of treatment duration;

favourable anti recurrent effect in long-term treated subjects;

immunologic indices showed an increase of non-specific defence mechanisms

(phagocytosis and stimulation of local and systemic humoral immune response).

The conducted study shows that treatment with Factor-R has a stimulating effect on haemopoesis.This reflects on the haemoglobin level and may lead to the improvement of the anaemic syndromein HI V infected persons.

The increase in the level of neutrophil granulocites and monocytes contribute to the activation ofthe intracellular killing of phagocyted infectious agents. It also leads to the restoration of theparticipation ofmonocytes and neutrophil granulocytes in the induction of humoral and cell-mediatedimmune responces. As a result of the stimulation of the immune system against secondary

opportunistic infections, the applicationof

Factor-R will lead to significant reduction in the useof

antibiotics and chimiotherapeutics for the treatment of AIDS-subjects.

There is a demonstrabale increase in the level of total protein is especially beneficial in AIDSsubjects, due to their multiple reasons to develop deep hypoproteinemia.

These diverse effects of Factor-R have led to the improvement in the quality of life for HIV -infectedpeople and AIDS sufferers. The lack of stimulating effect on CD4 Iymphocites when accompaniedby the activation of the CDB Iymphocites, that produce IL-4 and IL- l0 could lead to an importantreduction in viral replication in host cells.

Negative side effects were no t observed in any of the subjects both during the appllication of F actorR as monotherapy and during its combined application with antiretroviral drugs. The foregotinglogically leads to the suggestion that future wider research of the effect of Factor-R in combinationwith antiretroviral agents could produce promising results. Although the research to date can beregard as preliminary, we can suggest that the multiple effect of Factor-R on the protectivemecanisms may optimize the treatment of HIV infected and AIDS sufferes .

92


74/124

._ ...

I.

r

L

L

I. Examination of the effect of Factor-R on non-specificrespiratory diseases in humans.

1. Broad base clinical application of Factor-R for prophylaxis of broncho

pulmonary infections in childrenA basic problem of the medical practice in cases of chronic and recurrent bronchopulmonary infections is prophylaxis. Modern research has entirely rejected thepreventive antibiotic treatment because of possible allergies or secondary activationof pathogenic flora. Yet, HIV and AIDS sufferers receive such treatment as standartcourse.

This necessitates the application of bacterial vaccines which through activation of theimmunocompetent system lead to the creation of short-term or long-term, local or

systemic specific or non-specific immunity. Factor-R is intended for strengthening ofthe body's defensive mechanisms against acute and chronic diseases includingfrequently occurring pulminary diseases in children.

The similarity between this condition and the frequent occurence of pulmonarydisorders in HIV/AIDS sufferers makes the subject of this examination vital.

Clinical Experiment:

50 children at the age of 4 to 12 registered as frequently suffering from acute

broncho-pulmonary infections were observed for three months.

The immunostimulant was administered according to the following dosage scheme:1 tablet of 25 mg daily in the morning before breakfast for 30 days, then followed a30-day period of rest, afterwards according to the above dosage scheme the first 10days in 3 consecutive months. Side effects of the preparation were not observed.

The children were divided into two groups:

(1) A group of 30 children treated with Factor-R

(2) Placebo group of 20 children

For proper observation of the clinical effect of the application of Factor-R the

following indices were examined:

(1) The rate of secretory IgA in saliva

(2) The bactericidal activity of the serum

93


75/124

I

[

I

I

c

L

Serum and saliva were examined before the course of treatment, on the tenth,

thirtieth and ninetieth day after its beginning.

Results:

The results before the course of treatment and those from the tenth day werecompared with a placebo group of children whose serum and saliva were observed.

The results of the clinical effect of the administration of Factor-R are shown on table 1.

Table 1

Diseases, antibiotic treatment and number of days of stay in hospital of the examined

children for a 3-month period of the previous year and the period of observation -

controlled study.

Studied patients

Inflammatory disease

(total number)

Days of antibiotic

treatment (total number)

Number of days of

stay in hospital

n - number of observations

For the previous

year

Factor-R placebo

95 65

505 285

138 127

Studied patients: with Factor-R - 30, with placebo - 20

For the period of

observation

Factor-R placebo

37 61

123 261

33 137

It is evident that there is a significant decrease in the number of diseases and areduction of the period of antibiotic treatment which is a marker of the seriousness ofthe diseases.

In support of th e results of the clinical effect are the results of th e dynamicexamination of several immunologic parameters (see Table 2).

94


76/124

I

[

rL

Table 2

Dynamics of the level of IgA in the saliva of the children treated with F-R and those

from the control (placebo) group.

Before 10th day 30th day 60th day 90th day

treatment

Children trea ted 9 .64 14 .58 15 .36 14 .2 13 .95

with Factor-R

Control group 8 .92 10.18 10 .18 10.18 10.18

Conclusions:

(i) .

(ii).

(ii ) .

The application of Factor-R fo r prophylaxis of broncho- pulmonaryinfections in frequently ailing children led to a significant decrease inthe number of diseases.

The administration of the immunostimulant led to a considerable

increase of the level of IgA in the saliva of the children for the wholeperiod of observation.

We observed an increase in phagocytal and bactericydal activitytowards the bacterial strains contained in the vaccine.

1.2 Prophylaxis of broncho-pulmonary infections with Factor-R in adults

IgA has a great importance fo r the protection against bacterial infections of therespiratory system. The immunological strength of a given immunostimulant can beestimated by the increase of the IgA concentration in the serum and bronchial secretion.In case of peroral administration of Factor-R a protection of the respiratory tract isrealized as a result of considerable increase of secretory IgA, e.g. Factor-R inducesresistance in patients suffering from chronic bronchitis.

Clinical Experiment:

34 patients with chronic bronchitis (bronchial form of chronic obstructive pulmonarydisease) at the age of 19 to 65 were treated with Factor-R. Factor-R was administered

during the period of clinical stabilization of the patients.

95


77/124

I

I

r

L

During the first month the patients were given 1 tablet daily in the morning beforebreakfast. Then followed a 30-day period of rest and in th e end 1 tablet wasadministered during the first 10 days of the third, forth, fifth months out of a totaltreatment period of six months. Every month the following indices were examined:from the anamnesis - cough, expectoration: from a physical study of the lung -evidence of bronchial obstruction, functional study of the breathing - vital capacity,forced expiratory volume pe r second and Tiffno index; laboratory studies -quantitative determination of IgA, IgG, IgM in the blood serum, of IgA, IgG, of thesecretory component SC and of albumin in the bronchial secretion using Mancini'smethod.

The changes in the characteristic of the passing of the chronic bronchitis weredetermined by the frequency and duration of the fits as well as by the duration of thetemporary incapacity for work compared with the same period of the previous year .

With each observation the tolerabil ity and the possible side effects of the preparationwere taken into consideration.

Results:

After the treatment with Factor-R the clinical picture changed. In the beginning all thepatients had a cough . At the end of the first month it stopped in 20 of the patients(58 .8%) and at the end of the sixth month only 4 of them had a cough (1,1 %).Similar changes were observed in the character of th e expectoration . At th ebeginning of the treatment course all patients had mucous expectoration (up to 20

cm 3 - 22 and above 20 cm 3 - 12 patients). At the end of the first month only 2patients (0 .6%) remained with more abundant expectoration (above 20 cm 3). Theexpectoration was scarce in 20 (58 .8 %) and completely disappeared in 12 patients(35.3%).

In the beginning while a physical study of the lungs was carried out 17 patients hadsymptoms of bronchial obstruction . At the end of the first month obstruction wasestablished only in 4 patients (23 .5 %) and at the end of the treatment in none ofthem.

Important changes in the basic functional indices of breathing were observed. Thevital capacity (VC) was increased from 2,500 up to 3,500 cm3 . Similar were thechanges in the forced expiratory volume (FEV1) and Tiffno index. They were a resultof an influence of the inf lammatory component on the bronchial obstruction.

The changes in serum immunoglobulins can be seen on Fig. 1. A certain increase inIgA and IgM was observed. The stimulating effect was slow and long. The decrease inIgG probably reflected a tendency toward stabilization of the inflammatory process.

96


78/124

Ir

rrF

2.01 __ , , ~6--. - - - - : > - -I

200 -r

150 -- - - - -lOa t

.0

~ . - - - - - l g M- - 0 - - IgA I

l a o o ; _ _ ~,

1 ' 0 0 -

1200 -

1000 -

800 -

600 -

400 -

200 -

0 ~ 1 - - - - - - - - - - - - - - -- - - - - - ----------- --- -30 60 90 12 0 30 eo " 020D.,.. D.,..

Fig . 1 Levels of the serum immunoglobulins(IgCI IgA and IgM) during the 1 t 2nd,3rd and 4th months

1s t month 2 nd month. _ _ - - . ,

O lg A IgG Al b ~ SC i. - - - - - . - - ~

. : . - - - - . - - - ~ ,,, ,- - - - . - - - -

i

3rd month

Fig. 2 Levels of the immunoglobulins (lgA, Ige), of the albumin (Alb) and of he freesecretory component (5C) in the bronchial secretion during the 1st 2nd and 3rdmonths.

The changes in the local immunity had a crucial importance for the passing of the

chronic bronchitis. Fig. 2 shows the changes which occurred in some of the basiccomponents of the bronchial secretion. The quantity of albumin and of IgGdecreased. This reflected a decrease of the degree of inflammation of the bronchialmucouse membrane. The stimulation of local immunity is presented with anincreased level of IgA no matter that a certain decrease of the secretory componentcould be observed. This is probably a result of the general stabilization of the localinflammatory changes. A certain stimulation of cellular immunity could be observedduring the treatment period.

9 7


79/124

I

r

[

C

LL

L

Significant changes were established in the passing ofthe chronic bronchitis. Numberof fits as compared to those observed fo r the same period of the previous yeardecreased from 3.1 to 1.3 and their duration - from 14.6 to 6.8 days. The temporaryincapacity for work for the two compared years decreased from 45.5 to 22.7 days.Side effects as a result of the administration of the preparation were not observed.

Conclusions:

The clinical treatment that was carried ou t gave reason for the following conclusions:

(i). In the treatment of patients with chronic bronchitis with Factor-R it wasestablished that the main clinical indices changed for the better . Thebronchial obstruction was influenced significantly which was a resultof changes in its inflammatory component.

(ii).

(iii).

(iv).

Changes in the levels of the serum immunoglobulins were observed(stimulation of the humoral immunity). Some components of thebronchial secretion which reflects the condition of the local immunitychanged significantly. Mainly the production of IgA was beingstimulated. A certain stimulation of the cellular immunity could beobserved during the treatment period .

As a result of the treatment the number and duration of the bronchialfits decreased. The temporary incapacity for work dropped with 50%.

The preparation has a very good tolerability.

98


80/124

\0\0

ToPagePart

1

r - -

Investigation:Investigator,CoordinatingOrganization

2

K. Kisiova,Zl. Zlatanov,M. Peneva,K. Yankov,An. Tzonev,T. Mavrodiev.

Estimation of SomeImmunos t imulan tsApplied in PulmonaryPractice.

Department of InternalDiseases,Clinic of Pneumology,Medical University,Varna, Bulgaria.

I I I I

Project

3

Th e immunostimulatingactivity of Factor-R wascompared in patients withrecurrent non-specificdiseases with data ofimmune deficiency

Participants,number,age, sex

4

161 patients (73female and 88male) aged 18 to 60

L J L J r -l

Diagnosisan d

criteria foradmittance

5

Periodof

treatment

6

4 months

r- :J [ -_-I

Test product.Dosage and

administration

7

Factor-R: 50 mg daily inth e morning beforebreakfast for 30 days,afterwards according tothe above dosage scheme10 days in 3 consecutivemonth

I I l T l ~

Criterion of evaluation

8Patients with data of immundeficiency suffering fromreccurent chronic non-specificpulmonary diseases wereselected. Before the treatmentand every following month animmunological study was madeincluding: phagocytic index,complement, serum IgG, IgA,IgM, circulating immunecomplexes, intradermal skin testswith kits of bacterial and mouldantigens for the determination ofcell immunity state, of E-RFCtotal and active, the bronchialsecretion obtained byfibrobronchoscopy was tetstedfo r alveolar macrophages,protease inhibitors, lysozymes,immunoglobulin. Th e clinicaleffect was estimated taking into

account the less frequency of theexudates, the shortening oftreatment periods an d thereduction of treatment in anacute phase.

~ ~ [ J

Results

9

F a c t o r - R

provoked anantirecurrenteffect in 66.2%of the treatedpatients, in82.25% theperiod oftreatment wasshortened andin 30.6% it ledto reduction ofthe treatment. Itwas establishedthat some of heimmunol;ogicali n d i c e s ,

especially thep h a g o c y t i cindex and therate ofIgA wereinfluenced.

Sideeffects

10

Notobserved


81/124

......

oo

ToPage

Part

1

~

Investiga tion:Investigator,

CoordinatingOrganization

2

K. Kissiova,D. Petkova,J. Radkov,A. Tzonev,M. Peneva.

Factor-R in ClinicalPractice - Present andFuture Prospects.

Department of InternalDiseases,Clinic of Pneumology,Medical University,Varna, Bulgaria

I l I l

Project

3

The clinical effectiveness ofFactor-R in patients withrecurrent chronic nonspecific pulmonary diseaseswas evaluated.

r-------'J

Participants,number,

age, sex

4

93 patients (42female and 51male) aged 47 !o65, divided into 4groups

L J ........---, r J

Diagnosisand

criteria foradmittance

5

Periodof

treatment

6

Chronic I 4 monthsobstructivepulmonarydiseases,pneumosclerosis,infectiousbronchialasthma, morbusbronchoectaticus.

r J r l


administration

7

Factor-R : 50 mgdaily in the morningbefore breakfast for30 days, afterwardsaccording to th eabove dosage scheme10 days in 3consecutive month.

1 I f T I : - - - l

Criterion of evaluation

8The patients observed weredivided into 4 groups: withchronic obstructive pulmonarydisease, chronic bronchitis withrecurrences, infectious bronchialasthma, morbus bronchoectaticus. All patients had dataof suppression of the immunesystem determined by phagocyticindex, titer of complement, rateof serum IgA, IgM, IgG,circulating immune complexes,E-RFC and cell immunity usingkits of bacterial and mouldantigens injected intradermally.After the completion oftreatment these immunologicalindices were observed monthlyfor a period of 6 months. Theclinical indices were alsoinvestigated: shortening of theexudates, red uction of thetreatment, shortening of thetreatment periods. The patientstreated with Factor-R werecompared with those undergoingconventional therapy.

~ 1 I 1 I

Results

9

Sideeffects

10

In 81.9% of the I otpatientsthe period observedof antibacterialtreatment wasshortened and agood antirecur-ren t ef fect wasobtained. Only in30.1 % of thepatients the basictreatment withbronchodilatingand inspectorat-ing agents wasreduced. Fromthe immunolo-gical indices theincrease of phago-cytic activity, rateof complementand serum IgAtiter was statist-ically reliable.


82/124

L J . - - - ,..

. ---- , 1 IJ r : J r T l ~l ~ I I

To Investigation: Project Participants, Diagnosis Period Test product. Criterion of Results SidePage Investigator, number, and of Dosage and evaluation effects

Part Coordinating age, sex criteria for treatment administrationOrganization admittance

1 2 3 4 5 6 7 8 9 10

......

o......

I. Iliev, Clinical immunological 37 ailing and 10 Non-specific 4 months Factor-R: 25 mg daily The treated children with Application of NotV. Radanova, criteria for i mm un o- c li ni ca l healthy r e c u r r e n t in the morning before immunodeficiency data were Factor-R leads to an observedP. Georgieva, stimulating treatment with children aged 3 p u l m o n a r y breakfast for 30 days, selected among 153 increase in th eP. Petrova Factor-R of various to 7 tuberculos is afterwards according examined ones. They were number of T-

infections of the respiratory accompanied by to the above dosage divided into 3 groups: 1st - lymphocytes, IgG,

Clinical system in children had to be recurrent non- scheme 10 days in 325 children with non-specific IgA rate and of

Immunological found specific viral consecutive months.recurrent pulmonary phagocytic activity in

Results of the and bacterialprocesses, 2nd - 12 children the treated children

Application of infections.with primary pulmonary from both groups. Atuberculosis, 3rd - control

Factor-R to Children group of 10 healthy children. generalclinical

With Various Before the treatment and on estimation showed, in

Pulmonary Diseases the 14th and 30th day after statistical values of

its completion the clinical s i g n i f i c a n c e

Clinic of Pediatry, state was evaluated. The rate compared with those

Medical Academy of IgG, IgA, IgM in serum,before the treatment,

Sofia, Bulgaria the number of T- and B-u n d o u b t e d l y

limphocytes, the immune favourable effect oncomplexes, the phagocytic the characteristics of

index and the state of cellthe disease process

immunity by intradermal (heaviness, coursetests with kits of bacterial and and outcome) inmould antigens were also more than 50% of thedetermined. children.


83/124

otv

r - - r--o

ToPage

Part

1

r-- ~

Investigation:Investigator,


2K. Kissiova,D. Kovachev,B. Petrunov,P. Nenkov,D. Petkova,M. Pencheva.

Optimisationof Factor-RTherapeutical Schemesby Determinationof the Titer of SpecificAntibodies to ItsBacterial Components.

Department ofInternal Diseases,Clinic of Allergy and

Clinical Immunology,Medical University,Varna , Bulgaria.

~ . r - - l ~ I J

Project Participants,number,

age, sex

3 4

An impartial assessment of 64 patients (30the effect of polybacterial female and 34 male)immunostimulant Factor- aged in 18 to 65,R applied to patients with taking Factor-Rrecurrent chronic non - perorally and 14specific pulmonary patients, aged 25 todiseases was made by 40, taking Factor- Rdetermination of the titer through inhalations.of specific antibodies to itscomponents in order to fIXthe right dosage scheme .

r - J r - - r - l I " j L " l I I I T J ~ ---""]

Diagnosis Period Test product. Criterion Results Sidean d of Dosage and of evaluation effects

criteria for treatment administrationadmittance

5 6 7 8 9 10R e c u r r e n t 4 months Factor -R : 50 mg daily , The patient s were The favourable clinical Notchronic nOn - in the morning before observed before effect of Factor-R was observedtreatment, the first and proved. It was founds p e c i f i c breakfast for 30 days, u ~ to the 3rd month that the ti ters ofp u l m o n a r y afterwards according to a ter the treatment 's specific antibodies ofdiseases. th e above dosage completion, re gardin8 IgG, IgA, IgM classesscheme 10 days for 3 the clinical indices an to bacterial compo-

consecutive months . paying attention to the nents significantlyantirecurrent effect of increased at the end offactor-R as well as to the the 30day course andtiters of complement decreases, through notand C3, pha!ocytic to the initial low values,index, rate 0 serum at the end of the 3-ISG, Ig.A , ISM and month supportingcirculatIng Immune course. The obtainedcomplexes, and the titer results led to theof specific antibodies to creation of an im-bacterial components of r o v e d scheme ofFactor-R were actor-R administra-determined by ELISA. tion. It was concludedFactor-R was applied that inhalatory treat -through inhalations to a ment could be usedg r o u ~of 14 patient s and when an urgent effecton t e 7th, 14th, 21st is needed in patientsand 28th day the above without bronchialmentioned results werefixed.

hyperreactivity.


84/124

ow

ToPage

Part

1

Investiga tion:Investigator,


2

Y. Petrovska,L. Tzvetkova,O. Petrova

ClinicalImmunologicalStudies of PatientsWith InfectiousBronchial AsthmaTreated With thePolybacterialImmunostimulantFactor-R.

Clinic of PulmonaryDiseases,Research Institute ofResort, Health andPhysiotherapy,Medical Academy,Sofia, Bulgaria.

[ ~ L J L . l I I

Project Participants, Diagnosis Periodnumber, and of

age, sex criteria for treatmentadmittance

3 4 5 6

The clinical effectiveness 30 patients aged 29 Infectious 4 monthsof Factor -R and changes to 57. bronchialof some functional and asthma.imm uno ogical indiceswere studied in patientswith infectious bronchialasthma.

: - -J L-:l 1 1 ~ ~ ~

Test product. Criterion of Results SideDosage and evaluation effects

administra tion

7 8 9 10

Factor-R: 50 mg daily in In the beginning featients The results of the study Notthe morning before with ~ r o v e din ectious showed a considerable observedbreakfast for 30 days, bronc ial asthma were improvement of theexamined on the 45th, clinical paramete rs evenafterwards according to 120th and 240th da f of as early as the 45th daythe above dosage the treatment. he from the beginning ofscheme 10 days for 3 following clinical symp- treatment (in more thanconsecutive months. toms were under obser- 70% of the patients) but

vation: intensity and the most remarkablefrequency of asthma results were observed onattacks, cough, rhinitis, the 240th (in more thanexpectoration, weakness, 80% ofthe patients). Thephysical overtaxing, Ftre- s ~ i r o m e t r l cindicessence of rales. The un- s owed a considerablectional examination of improvement on the 240the lung durin 3 he same th day in more than 50%periods inclu ed deter- of the treated patients.mination of YC, FEY, The immunologicalFEY3, EMD, EMD50, studies showed aEMD75, MEMD 25-75 decrease of bronchialand the immunological mucose sensitivity toexamination included inflammatory agents anddetermination of serum an imlirovement of non-IgG, IgM, IgA in saliva - ~ p e c iic indices of theIgA, IgG, and albumin, Immune system,complement and C3, cell including th e cellimmunity level. immunity.


85/124


86/124

oVI

ToPage

Part

1



2

S. Garov.

Clinical Resultsof the Administrationof the New BulgarianImmunostimulantFactor-Rin ComplexTreatmentof GingivitisCatarrhalis Chronica .

StomatologicalFaculty,Medical University,Plovdiv, Bulgari a .

r ~ ,

Project

3

A study was made of theclinical effectiveness ofFactor-R in the treatment ofgingivitis catarrhalischronica in patients withestablished immunodeficiency .

r J

Participants,number,

age, sex

4

24 patients (12females and 12males) aged to 12to 24 .

L J l . J L I

Diagnosis Periodan d of

criteria for treatmentadmittance5 6

G i n g i v i t i s 4 monthsc a t a r r h a l ischronica .

I J r - l


administration

7

Factor-R: 50 mg daily ,in the morning beforebreakfast for 30 days ,afterwards accordingthe above dosagescheme 10 days for 3consecutive months .

[ J ----rI

Criterion ofevaluation

8

The treated patientswere divided into 2 groups- 1st with Factor-Rapplied simultaneouslywith the undergoing localand general treatmentand 2nd - withoutFactor-R. Both groupsconsisted of 12 patients .They were followed upmonthly for a periodof 2 years from thebeginning of thetreatment. The effect wasreviewed under the fol-lowing criteria: clinical -local state, antirecurrenteffect and shortening of

the treatment period;immunological - phagocytic index , rate ofsera IgG, IgA , IgM.

~ ~ ~ ~

Results I Sideeffects

9 I 10

Factor-R application I otgave very good clinical Observedresults. On the 30th dayfrom the beginning of thetreatment 100% ofthosetaking it came throughthe illness . From thoseundergoing a convent-ional treatment only 75%got better. The favour-able antirecurrent effectof Facto r-R was stronglyexpressed when theresults of both groups,which were 92% and25% two years after thebeginning of the treat-ment compared. Dataabout the stimulation ofnon-specific immuno-

logical indices - phago-cytic activity and rate ofserum IgA, wereestablished.


87/124

o0 \

ToPage

Part

1

r - - ~



2

M. Vassileva .

Immunoprophyla xisand Immunotherapywith Factor-Rof Children up to3 years of Age .

Departmentof Pediatrics ,Medical University,Varna, Bulgaria .

- -

I : : ! I I

Project

3

The possibility of Factor-Rapplication to children intheir early age as well asits effectiveness in cases ofrecurrent infections of therespiratory tract wasstudied.

r-:=J r - - ' L J ~ ~

Participants, Diagnosis Periodnumber, and of

age, sex criteria for treatmentadmittance

4 5 6

158 children at 10 R e c u r r e n t 4 monthsmonths to 3 years non-specificof age (83 boys and infections of75 girls) . the respiratory

tract, otitis,rhinitis .

~ ,...----, ,..------.. - - r - - ' - - - . - - - - ,

Test product. Criterion of evaluation Results SideDosage and effects

administration

7 8 9 10

Factor-R : 25 mg for The study included children at 10 Th e clinical Notchildren above 13 kg months to 3 years of age with observations oIRrvOO.and of 12 .5 mg for recurrent infections of the and the physic-children under 13 kg, respiratory tract, recurrent al examinationsdaily, in the morning bronchitis with obstructive showed that inbefore breakfast for 30 syndrome, otitis and rhinitis . They 80% of thedays , afterwards were under observation for 6 children a veryaccording to above months after the treatment 's good clinicaldosage scheme 10 days complection . The effectiveness of effect wasin 3 consecutive Factor-R was evaluated by clinical achieved andmonths . indices. no side effects

were observed .It is proposedthat Factor-Rcan be used inimmunotherapyand immuno-prophylaxis of

children in theirearly age .


88/124

I

Ir

r

~

Immunoreactivity in cases of lung cancer,treated with cytostatics and Factor-R.

(preliminary report)

K. Kassabov 1, D . Damianov 2, A. Mihailova 3, I. Stoichkov 1

Introduction

Currently the leading oncological disease among .males in Bulgaria is lung cancer. The averagerate of contraction among Bulgarian males is 60

%0, equal to 2600 ne w cases registered each

year. Nearly 80 % of these cases constitute the non-small cell lung cancer clinical group. Only20 % to 27 % of these cases are detected in clinical stages I or II. Consequently, more than 70%of the patients with lung cancer cannot be subjected to radical operat ive programs. The treatmentof these patients, especially of those in stages III B and IV, is a challenge to clinical oncology. Theambition to improve the patients' quality of life in the long run stimulates the search fo r ne wmethods and preparations to optimise the complex treatment of non-small cell lung cancer.

Some lung cancer patients demonstrate digressions and disturbances in cell mediated and humoralimmune response (9)*. Radiation therapy and/or combined chemotherapy also lead to changesin immune status, mostly in a negative direc tion over different periods of time (1, 10). Consequently,the complex treatment of lung cancer should include a simultaneous stimulation of the immunesystem, aimed at restoration or reinforcement of its normal functions. This practice would improvethe final results of the treatment (2, 11).

The aim of this study is to determine some essential parameters which characterise the immunestatus of patients with locally advanced and/or metastasid non-small cell lung cancer after acombined treatment with cytostatics and Factor-R immunotherapy.

This clinical study is based on previous experimental data on the inhibition of the growth of thelung metastasis in patients with Lewis's carcinoma, caused by treatment with the Respivaxpreparation (4, 5, 6, 8) .

Material and methods

Patients.

Seven patients, 6 men and 1 woman aged from 44 to 71, are included in the clinical study so far.This number to increase to 20. In all cases the diagnosis has been morphologically confirmed -flat-cell lung cancer. The apportionment of the cases in terms of clinical stage of the disease is asfollows:

Tw o patients in stage II (inoperable accord ing to functional parameters or refusal of operativetreatment),4 patients in stage III, and 1 patient in stage IV. The combined chemotherapy appliesa quadruple combination designed by the Clinic of Medicinal Oncology, National OncologicalCenter. The combination includes Vinblastin, Endoxan, Etoposide and Cisplatin (chart 1).

1 Tumor immunology and therapy division, Department of clinical immunology and laboratory studies, Medical Academy, Sofia2 Clinic of Medicinal Onkotherapy, National Oncological Institute, Sofia .3 Division ofTransplantation Immunology, Department of clinical immunology and laboratory research, Medical Academy, Sofia* See section reference of this chapter

107


89/124

r

rI

l

L

E

LLL

....... , . . .... -.... .,. ..... ~ - . . .. ....... ~ - - ~... ' , ..

On the Bth day of treatment a simultaneous peroral immunotherapy with Factor-R (during aperiod of 2B days, 40 gm per day in two applications) is introduced. After that a combined chemoimmunotherapy is applied according to the same scheme. The above-mentioned schemeprovided fo r a blood examination of the patients one day before and after the combinedchemotherapy, as well as after the immunotherapy with Factor-R. In most of the cases the combi nedchemotherapy lead to a salient leucopenia which prevented us from isolating the number of

blood cells necessary to carry outall planned tests. For this reason we interrupted b ~ o o dexaminationimmediately after the combined chemotherapy.

Scheme of administration

1 course CT Factor-R (IT) 2 course CT Factor-R (IT) 3 course CT

Chart 1. Scheme of treatment and examination of patients suffering from non-small cell lung cancer undergoinga combined type o f chemical therapy (Cl) and a immunotherapy with Factor-R (IT)

Isolation of peripheral mononuclear cells (PMC).

PMCs were isolated from heparinized venous blood according to the traditional method of gradientcentrofuge separation on Ficoll - Hypaque (Lymphocyte separation medium, ICN Flow). Thecells were resuspended in a nutritive environment RPMI1640, enhanced with 2rnM L -glutamin,100 U penicill in/ml, 100 microg streptomycin /ml, and 10% heat inactivated fetal calf serum (ICNFlow). The isolated cells were counted and used to determine the values of the followingimmunological parameters:

Cytokines Secretion in vitro.

We tested the spontaneous and the inducted (with PHA and LPS) secretion of interleucine-2 (lL-2) and the secretion of the tumor-necrocizing factor-alpha (TNF-alpha). The isolated PMC,concentrated to 2x10 6 /ml, were distributed among several 24-hole plates (Limbro, Flow-Laboratories), 1 mllhole. The cells were cultivated for 24 hours with or without inductive agents ina thermostat set on 37C in a 5 % CO 2 environment. The resulting cell super-natants were frozenand conserved at -20C. The IL-2 and TNF-alpha levels were determined according to aninnunoenzymic method (ELISA)with standard kits (IntertestTM-2x IL-Elisa Kit, PredictaTM TFNalpha Elisa Kit, Genzyme).

Activity of he natural killer cells (NK).

Determined according to the NK-sensitive erythroleucemic cell line K562. The target tumor cells

were counted, then concentrated to 105 cells/ml and spread on a 96-hole rounded-bottom plate(Limro, Flow Laboratories), 100 microllhole. The effector cells were added to the target cells in E: T proFortions of 50:1,25:1, 12:1, and 6:1. Enhanced RPMI1640 envi ronment was added to thecontro tumor cells at a rate of 100 microl. After a 20-hour treatment in a thermostat at 37C and5 % C02, the living tumor cells in the control (C) and test (T) holes were counted after the additionof a 0.5 % solution of a tripan blau colouring agent (Flow Laboratories). The cytotoxicity wasdetermined according to the following formula: % cytotoxicity = K - O/ K x 100; K - number of theliving tumor cells in the control holes, T - number of the living tumor cells in the test cells.

Surface leukocyte markers.

Determined with flowcytometer (FACStar, Becton Dickinson). We used a set of monoclonal

antibodies (Monoclonal Antibodies Laboratory, National Institute of Contagious and ParasiticDiseases), which allowed us to determine the general T lymphocytes (CD3 +), helper/inducer(CD4 + ) and suppressor/cytotoxic (COB + ) T lymphocytes, NK-similar (COB + CDS7 + ) andactivated (HLA-DR + ) T lymphocytes, as well as the general (CDS7 + ) and the typical (CDBCD57 + ) NK cells.

108


90/124

r

r

Results and Discussion

Activity of he NK cells.

As indicated in Table 1, the examined patients show a decreased NK activity before the treatment

(as compared to healthy persons who volunteered for the tests). .

Table 1

Patient Time for E:T (% )

N! determination 50:1 25 :1 12 : 1 6 :1

1 at entering clinic 70.2 56.2 31.8 20.1after 1 course of CT 28.4 16.9 21.2after 1 course of IT 64.3 34.6 9 .6after 2 course of CT 13.7 6.6 14.3after 2 course of IT 26.2 9.7 6.9

2 at entering clinic 88 .9 63 .5 36.5 7.9after 1 course of IT 32.6 18.6 4.7 2.3

3 at entering clinic 71 .6 50 .2 31.4 18.5after 1 course of IT 49.6 31 5.8 1 .8after 2 course of IT 48.4 54.6 59.3 28.1

4 at entering clinic 63 .2 32.4 20.1 9.4after 1 course of CT 16.2 8.3 3after 1 course of IT 21.7 16.4 9.9 1.8after 2 course of IT 49.3 52 29.1 21.6

5 at entering clinic 82.1 57.3 20.4 6.7after 1 course of CT 30.1 38.4 14.9 7.2after 1 course of IT 73.7 67.1 51.3 28.9

6 at entering clinic 63.8 47 .9 19.1 9after 1 course of IT 64 68.7 65.6 48.4

7 at entering clinic 45.8 30.2 19.7 8 .3after 1 course of IT 31.2 31.2 35 .9 17.1

X SD at entering clinic 69.4 12.9 48.2 11.7 25.6 6.8 11.6 5.3after 1 course of cr 30 .1 27.7 9.1 13.4 3.7 6 .6 2.7after 1 course of IT 45.5 18.7 42.5 21 .6 29.7 22.1 15.716 .3after 2 course of IT 43.1 8.2 44.3 12.8 32.7 20.4 18.9 8.9

Healty volunteers 74 .5 14.1 55 .3 9.7 34.1 8.6 20.1 7.7

(n=6)

Table 1. Effects of the chemical therapy (eT) and immunotherapy with Factor-R (IT) on the NK activity of diseased suffering fromnon-small cell lung cancer

Immediately after the cytostatic therapy most of the patients developed salient leucopenia,which allowed us to determine the NK activity in only three of the cases. In all threecases a decrease in the cytotoxicity of the NK cells in vitro was observed. After completionof the 1 st period of chemo-immunotherapy the NK activity was fully (4 cases) or partly (3cases) restored. After completion of the 2nd period of treatment fully or partly restoredactivity was observed in tw o of the cases. In lower concentrations of effector cells thevalues came close to or even exceeded the initial values.

Four of the patients examined after immunotherapy (two of them after the 1 st period oftreatment, and tw o - after the 2nd) showed a relatively higher NK cytotoxicity in lessereffector cell concentrations. One of the possible explanations of this phenomenon is

stimulation of the monocytes in the peripheral blood. Some scholars affirm that monocytesexert an inhibiting effect on the NK cells (1, 3) (especially higher concentrations ofmonocytes). This statement might be verified by determining the activity of the tw o effectorpopulations together and separately.

109


91/124

[

fr

L

'. . - . . ~ ....... ----- .... - .. - - - ~ .

Cytokines secretion in vitro.

Table 2 describes the spontaneous and the induced (with PHA and LPS respectively) secretion ofinterleukine-2 (lL-2) and tumor-necrotizing factor-alpha (TNF-alpha).

TABLE 2

PATIENT TIME FOR IL-2 [pglml THF-a [pglmll

N! Determination Spontaneous Induced Spontaneous Induced

1 at entering clinic 527 .1 653 .2 380 .5after 1 course CT 607.7 968 .9 509.4after 1 course IT 709.7 >4000 166.7 306.3after 2 course CT 13.7 6.6 14 .3after 2 course IT 116.2 294.6 160 .8 328.8

2 at entering clinic 495 .8 333.7 > 1200 >1200after 1 course CT 239.4 560.4 600.7after 1 course IT 706 .9 1976.1 455.2 500.7

3 at entering clinic 578 505.7 103 .5 142.7

after 1 courseCT

824.4 517.1 116.7 163.2after 1 course IT 712 .8 745 .1 246 .5 260 .1

4 at entering clinic 1314.6 2794.9 270.5 345.1after 1 course CT 306 689 .9 48.6 100.7after 1 course IT 1167.3 1265 97.4 269.4

5 at ente ring clinic 862.7 1804.7 347 .2 434.4after 1 course CT 802 .1 1003.2 590 .1 > 1200after 1 course IT 463.5 612 537 .8 669.4

6 at entering clinic 1221.1 1616.3 470 .5 549.3

7 at enteri ng clinic 675.7 1357 .1 447 .9 567.7

X SO at ente ring clinic 811 311 936 592 460 322 540 328after 1 course CT 556 244 795 201 365 233 516439after 1 course IT 752 229 17201237 301 169 401 160

Healthy volunteers(N=6)514 242 854 662 93 77 203 108

Table 2. Effect of chemical therapy (CT) and the immunotherapy with Factor-R (IT) on the secretion of interleukin-2 (IL-2) andtumor-necrotising factor-alpha (TNF-a) from peripheral mononuclear cells

In most of cases there was an increased secretion of tested cytokines (compared to the healthyvolunteers). After the first period of chemotherapy the average values indicated decreasedsecretion of the two cytokines (especially IL-2). After a one-month of peroral immunotherapywith Factor-R the IL-2 secretion increased (especially the one induced with PHA). There was nosignificant change in the TNF-alpha secretion.

Both in tested patients and healthy volunteers the levels of secretion showed a large range ofvariation. The different patients responded in a rather individual way to the treatment whichaccounts for the different models of secretion. For example, patients with an initially increasedlevel of secretion often reacted to the cytostatic therapy by an increase in the IL-2 and TNFalpha secretion. Patients with initially decreased level of secretion reacted Similarly after thecompletion of the 1 st period of chemotherapy. The overall analysis of the measured parametersmight help us give a satisfactory explanation of the observed phenomena.

Surface Leukocyte Markers

Table 3 describes the analysis of the percentage content of several group determinants (CD),typical for the T- and NK- cell populations. The indicated surface markers were measured beforethe beginning of the treatment and after the complet ion of the 1 st and the 2nd period of chemoimmunotherapy.

110


92/124

r

r~

L

LL

. ~ - . _ . . . . .". . . . ,.... .. . . .a . .

TABLE 3

Patient Time for CD3+ CD4+ CD8+ CD8+ HLA- CD4/ General CD8-~ distribution


93/124


94/124

I

r

G

L

L

L

Clinical and serological investigations of Factor-Raction on different clinical forms of Syphilis

G. Spirov, O. Niagolova(Preliminary report)

Nonspecific therapy of syphilis was a well known procedure prior to the discovery of Spirochaetapallida(1905) and the diagnostic serological reactions (1906). Historically different therapies wereused: malaria therapy, di fferent pyrogenic substance (vaccine , 2 % sulfur emulsion, pyrogenic,etc.), vitamins, biogenic stimulators (placenta, aloe, fibs), n.v. radiation, corticosteroids,balneotherapy etc.

For nearly a century, when discussing the problems of immunity in syphilis the fact that in theevolution of the disease there are periods without any symptom (latent syphilis), could be explainedthrough relative immunity .

Even after treatment serological reactions for syphilis remain positive in a very high percentage oftreated patients - a disturbing fact for the doctors, the patients and their relatives . It is due to thealready accepted fact in syphilisology that alive, but non active spirochaetes in the lymphaticnodes, produce of penicillinase, L-forms capable of reversion etc. According to Ljuben Popov(1947) nonspecific treatment of syphilis is necessary because "without the participation of theorganism in the treatment all the antisyphilitics means remain problematic" .

The foregoing provided a motive to investigate the action of a modern immunostimulant in th etreatment of syphilis together with specific Penicillin therapy (Factor-R).

MATERIALS AND METHODS

Two groups of patients with contagious forms of syphilis were under observation, one of themwas treated with crystalline Penicillin G (4x2 000 000 IU) per day - the other with crystallinepenicillin and Factor-R 50 mg daily. The duration of the treatment with Penicillin was 15 to 30days according to the clinical form of the disease, and Factor-R with the first application on thethird day of Penicillin therapy for three month. Factor-R was appl ied by th e following scheme:30 days 50 mg in the morning before breakfast, 20 days rest, 10 days 50 mg, 20 days rest and again10 days rest.

The efficacy of applied treatment was valued with the help of the following criteria:

a) clinical observation of persisting skin and mucose changes (in days);

b)

c)

term of negativisation of the serological reaction for syphilis -microchlorholinictest (VORL)and ReF "WASSERMANN" - type "KOLMER" (in days).

immunological tracing

The observed patients are randomly chosen . The group treated only with Penicillin consist of 95patients (38 female and 57 male with average age 22.6 years) divided in clinical forms : 30 syphilisprimaria seropositiva (4 female and 26 male), 35 syphilis secundaria recens (18 female and 17male), 30 - syphilis secundaria recidiva (16 female and 14 male).

113


95/124

I

G

The group treated with Penicillin G and Factor-R consist of 66 patients (25 female and 41 male)average age 20.8 years divided by clinical forms in : syphilis primaria seropositiva 31 (4 femaleand 27 male), syphilis secundaria recens 22 (12 female and 27 male), syphilis secundaria recidiva- 13 (9 female and 4 male).

RESULTS

I. Clinical observation (persistence of th e skin and mucose changes)

Data fo r the duration of the persistence of the different skin and mucose changes (erosive orulcerous shancar, papulas, condylomata lata, changes on the palms and feet, angina syphilitica,laryngitis catarrhalis, papulosa syphilitica, deflovium syphiliticum, alopecia syphilitica etc. werecalculated separately and after that for the different clinical forms were processed by the methodWilkokson and White.

The results fo r the different forms are given in the tables 1,2, 3 . They are approximately similar inthe two methods and the differences are statistically doubtful. These results show, that the clinicalobservations conserning the persistence of skin and mucosa changes in contagious syphilis, treatedby the tw o methods, is no t enough to give an impartial assessment fo r th e effectiveness of thetreatment.

Table 1

STATISTICAL IMPORTANCEOF DIFERENCE IN THE PERSISTENCE OF THE SKIN EFFLORESCENCE

IN SYPHILIS PRIMARIA SEROPOSITIVA

Method of treatment:

N ~

1

2

1. Crystalline Penicillin "G" (4 x 2 mil E pro die)

2. Crystalline Penicillin "Gil (4 x 2 mil E pro die) +Factor - R (50 mg pr o die)

Method ofTreatment

Penicillin "G"

Penicillin "G"

+ FactQr - R

Criteria P < 0.05

114

-Number of X (days)

patients X M1N - X M AX

9.1030

(6 - 13)

9.1631

(4 - 15)


96/124

I

I

t

ELL

Table 2

STATISTICAL IMPORTANCEOF DIFFERENCE IN THE PERSISTENCE OF THE SKIN EFFLORESCENCE

IN SYPHILIS SfCUNDARIA RfCfNS


N!!

1

2


2. Crystalline Penicillin "G" (4 x 2 mil E pro die) +Factor - R (50 mg pro die)

Method ofTreatment

Penicillin "G"

Penicillin "G"

+ Factor - R

Criteria P < 0.05

Table 3

Number ofpatients

35

22

STATISTICAL IMPORTANCE

X (days)

XM1N - xMAX

10.23(6 - 16)

10.10

(6 - 17)

OF DIFFERENCE IN THE PERSISTENCE OF THE SKIN EFflORESCENCEIN SYPHILIS SfCUNDARIA RfClDlVA



2. Crystalline Penicillin "G" (4 x 2 mi l E pro die) +Factor - R (50 mg pro die)

N2 Method ofTreatment

1 Penicillin "G"

Penicillin "G"2 + Factor - R

Criteria P < 0.05

115

-Number of X (days)

patients XM1N - XMAX

12.2330

(7 - 18)

11.4813

(6 - 18)


97/124


98/124

l

I

Investigations on later forms of syphil is: syphilis latens recent and syph His latens tarda show si milarresults at the end, but the necessity of one year and longer tracing and the small number ofpatients currently prevent interpretation.

It was observed however that in two female patients with late latent syphilis (4 year period) andseveral treatments with Penicillin G, cardiolipoid reactions were neutralized within 6 monthsafter adding Factor-R to the treatment. We are cont inuing the immune tracing of those subjects.

In conclusion, although the authors do not make definite conclusions, it is strongly suggestedthat Factor-R in combination with classical antisyphilitic treatment is more effective for thetreatment of contagious forms of syphilis and has in test subjects led to greater percentage ofrecovered patients.

117


99/124

I

r

l

r

TRIALS ON HIV/AIDS SUBJECTS

The following information is the chronological subjective and objective evaluation of our clinicalexperience in the therapeutic application of Factor-R in 133 seropositive subjects. All participantsin these trials were volunteers.

Introduction

While there is a wealth of information on the effects of Factor-R, results, prior to thecommencement of these studies, were derived primarily from numerous laboratory experimentswith animals . Even though immune deficiency was induced in these early studies animals havetypically failed to provide adequate research profiles to satisfy HIV investigation. The substantialclinical application of the substance in non-HIV/AIDS subjects, all wi th a demonstrated therapeutiCbenefit, also fails to provide the needed substantiation. Because of this history, the Investigatorsencountered significant resistance from clinicians and researchers due to a lack of demonstrableresults in seropositive and AIDS subjects. The lack of these studies in HIV/AIDS sufferers providedsupport for critics of the proposed therapeutic procedure. It also allowed pur ists to challenge thetheoretical hypothesis put forth by the investigators.

In order to meet these challenges and to demonstrate both subjectively and objectively thetherapeutic benefit of this form of immuno-modulation, the investigators accepted the applicationsof two subjects in highly advanced stages of AIDS to participate in an early study. Since thesubstance, Factor-R, has not at any time demonstrated any toxic effects on human subjects, theinvestigators elected to proceed .

First applicat ion of Factor-R in HIV-positive subjects begun in 1993. The product was initiallygiven to two men:

Subject 1.

A 39-year-old, male, who was diagnosed HIV-positive on or about 1990 has suffered from fungalinfection (seborrhea dermatitis) and shingles during the past three years. In the preceeding yearhe suffered from recurrent sinusitis, ocular herpes, weight loss, fatigue and diarrhea. In the twomonths prior to commenCing the study he had salmonella sepsis. He has previously been treatedwith AZT, antibiotics and antifungals. His very poor health condit ion reqUired anexsangvinotransfusion some days before the commencement of Factor-R treatment. As a resultbase statistics are skewed.

The subject took Factor-R (80 mglday) beginning October 15, 1993. From the laboratory datapresented in Fig. 1 - 10 it is evident that Factor-R stimulated leucopoiesis (fig . 1, 2) . Lymphocytesincreased significantly and then stabilized (fig. 4). This resulted from a rise in CD8 cells (fig. 9).

Factor-R did not influence the number of CD4 lymphocytes and they stayed at very low levelsduring observation (fig. 8). Although some laboratory data such as WB C (fig. 1), platelets(fig. 6) and hemoglobin (fig. 5) remained below the normal, they did increase in number after twomonths of treatment with Factor-R and to the end of the investigation the subjects general healthimproved significantly. The subject had no further sinusitis or opportunistic infections, or chronicdiarrhea. The ocular herpes subsided . The subject demonstrated an unusually rapid recoveryfrom Salmonella sepsis.

Subject 2.

A 34-year-old, male, HIV-positive since 1987 had for almost two years persistent diarrhea andweight loss. As well as Factor R (80 mglday) the Subject also took antiretroviral medication andvitamins. From the laboratory data presented in Fig. 1,5 and 6 it is obvious that factor R stimulatedleucopoiesis, erythropoiesis and thrombocytopoiesis. It is very -important to note that after theinitial lo w values of WBC (fig. 1), neutrophils (fig. 2), thrombocytes (fig. 6) and hemoglobin (fig. 5)these populations, after treatment, entered the normal range.

118


100/124

I "',' .

r[

rrE

Factor-R did not influence the total number of lymphocytes (fig. 4), including CD4's (fig. 8). At thesame time the number of CD8 lymphocytes (fig. 9) and monocytes (fig. 3)increased considerablyand constantly during the 6-month period of 06servation. A clearly expressed rise of protein levelwas also observed (fig. 7). As a result the Subject reported an increased sense of well-being. Ofparticular interest, and as demonstrated in Subject 1, an unexpected effect was that after almosttwo years of persistent diarrhea and the loss of 45 pounds, his weight stabilized at 150-155 lb. andthe diarrhea in fact stoped.

woe

Range 4 8 10 8 k/cumm5.5

- 5p.'

~ Q----/ ~-

L 5

4.5

4

/_? c/' / _ ~ 1

) ( -_2J -3.5

3

2.5--"'------ Starting Factor R 90th da y 180th day

- Subject 1- )(- Subject 2

Figure 1

WBC in tw o subjects increased after treatment with F-R. The initial WBC values in bothwere below referent ranges. In Subject 2 WBC reached normal range (5,5 k!cumm). Theincrease in Subject 1 was also considerable (from 2,0 to 3,1 k!cumm).

60

55

50

45

40

35

30

25

20

15

ii----- .----

/\V

Starting Factor-R

-.-)( - '

NEUTROPHILS

Range: 40-74 % 11

.-'

1

54-.--.-J(-

/ /

90th da y

Subject 1Subject 2

.--

J i

_12.--

180th da y

Figure 2

Neutrophils increased significantly in both subjects. In. Subject 1 cell populations rosefrom 46 to 56 % and in Subject 2 the initial lo w count (16 %) reached the normal range(41 %).

119


101/124

[

rIt

L

L

11

10

9

8

7

6

5

4

3

MONOCYfES

Range: 3-11 %

II

------- ---------0

------

) tStarting Factor-R

-I I- )(

..1,_ - - -- "~ -

Subject 1Subject 2

90th day

-

-- 10-

...- "

180th da y

Figure 3

Observations varied slightly within the limits of the normal. In Subject 2, wh o demonstratedthe lowest count, experianced a consistent elevation, from 3,4 to 5 %, during a 6 monthperiod of treatment.

36

32

28

24

20

16

12

8

4

LYMPHOCYfES

Range: 19-48 %

~ ,-....------- --.-.-

..-'

--'/ -

,--i - ./

8 a n- -Starting Factor R

Subject 1Subject 2

90th day

~ l : ;

1- - - --

7

180th da y

Figure 4

Varieties of lymphocyte levels differed in the tw o subjects. In Subject 1 a significant increasewas evident after 90 days oftreatment and remained high (35 %) on 180th day_ The lo winitial level of lymphocytes (8%) in Subject 2 was no t substantially effected throughoutobservation .

120


102/124

[

rr

L

- - '. - ' - ~ ' - " -- - - - ' - ~ - -

16

15

14

13

12

11

/1 ~V

HEMOGLOBIN

Range: 14-18 g/d/

/ ~-----------

/

t ! ~ _ _ _ _ _ _ _ _

- - - - - . _~ L _.----10

9Starting Factor R

---- )( -90th day

Subject 1Subject 2

~ o

9.9

--- ---180th da y

FigureS

A significant increase in the level of hemoglobin level was observed in Subject 2 - from13,3 to 15,6 gldl on the 90th day. A slight Decrease occurred on the 180th day (14,6 gldl)but within normal limits as compared to the initial value. In Subject 1 the hemoglobin didnot change Significantly although some increase was shown at the end of the investigation.It should- be noted that Subject 1 had, only days before the initial draw, undergoneexsanguinotransfusion. All values in Subject 1 remained below normal ones.

180

160

140

120

100

80

60

40

0404 ..~qL. ~

PlATELET COUNT

Range: 130-400 klcumm

/ / ~

0 4 " ". /

i(7

-

............... - ..............

~ ~ ~

Starting Factor R

' ...

, ~

-90th da y

Subject 1Subject 2

-

179~ ./ "

. / '"

~ - -- ! j >

180th day

Figure 6

Both Subjects entered the study with a platelet count under the normal. A significantincrease of platelet count within the l imit of the referent ranges (114 to 134 klcumm), wasevident after a 90-day course of treatment in Subject 2. This tendency remained stableand Subject 2 demonstrated a platelet count of 179 klcumm platelets. In Subject 1 a

decrease from97

to59

and 66 klcumm on day 90th and 120th respectively was observed.In Subject 1 the platelet count and hemoglobin level were most likely influenced by theexsanguinotransfusion made immediatly before the begining of treatment. It is quiteprobable that Subject 1 had an effectivly lower count prior to tranfusion and that thevalues reflected on the 90th and 180th days may reflect an actual improvement.

121


103/124

r~

8.2

8

7.8

7.6

7,; ____.--

7.4

7.2

7l _- .-

6.8 Starting Factor R

---)( --

PROTEIN TOTAL

Range: 6-8 gldl

---->-:::.- _. -...

Subject 1Subject 2

/7/

.-'

--..... -J.. -- .. --90th day

8 )

///

/

7';;'

180th day

Figure 7

Protein levels were found to be normal at all times during observation in both Subjects.There was a demonstratable tendency for protein levels to increase (from 7 to 7,2 and 8,2gldl) in both Subjects, most significantly in Subject 2.

28

24

20

16

12

8

4

T Helper/Inducer (CD4)Range: 370-1482 percumm

ir- - - - - - -~ -

.,.~ -

Starting Factor-R

----J ( - .

-.,.) ( - - _ .-

90th day

Subject 1Subject 2

,.,.,.

-l*

."

~,...

180th day

Figure 8

Factor R treatment did not influence the number of CD4 cells in either subject. Theirvalues were very low and characteristic for advanced stage subjects .

122


104/124

I

r

T suppressor/cyto (CD8)

Range: 162-682 percumm

560

-_~ 4

-~ : : - - - --4 ~ 8__

/ ---- ----. ~ ~ 1V

520

480

440

400 / /

360 ./

3 ~'-20

Starting Factor-R 90th day 180th day

Subject 1)( Subject 2 Figure 9

CD8 cell values were found to be at normal levels at all times during observation. Aconsiderable increase however was found in Subject 1. A slight decrease from 496 to 441cells/cumm on day 180 after initial rise was found in Subject 2.

IN CONCLUSION Factor-R prOVided demonstrable efficacy in Subjects 1 and 2. Both demonstrateda reduction in the number of and recovery period from existing and later infections (herpes,sinusitis, fungal infection etc.) . Subject 1 recovered rapidly from salmonella sepsis. As well bothsubjects were relieved of long term diarrhea. An obvious weight gain in both subjects was anadditionally encouraging sign of improved overall health. As a result of the stimulation ofhemopoiesis in both subjects WBC and hemoglobin increased, and in all subjects platelet countincreased by the 3rd draw. The increase of both neutrophils and monocytes (in Subject 2) isindicative of a heightened capability for phagocytosis against extra- and intracellular parasitesand is a prerequisite forthe development of cell-mediated immunity against infections and tumors.According to recent findings the elevation in CD8 is very important for conducting antiviral, cellmediated immunity against HIV. The subjective results in combination with improved laboratoryparameters provided positive early evidence that Factor-R treatment in advanced cases of AIDSaltered measurable parameters. It must be remembered that these subjects were extremely ill atthe time they commenced treatment. They had attempted and later abandoned all other acceptedtreatment modalities. The overall improvement in quality of life in such advanced cases was inand of itself an encouraging sign. This motivated investigators to consider further extendedstudies in an attempt to reproduce and verify the reported results.

EXTENDED STUDY 133 SUBJECTS

Here we provide a profile of Subject populations. The distribution of all subjects is compared tothose for which Investigators currently have results and are used as an early sampling. Subjects inthe sampling are almost evenly divided according to applied therapy (anti-retrovirals, antibiotics,

etc.).

The sampling population is representative of the general population as far as sex, period ofseropositivity, age and secondary infections/malignancies.

123


105/124

[

I

-:

L

LL

EVALUATION OF EFFECTSOF FACTOR-R ON PATIENTS WITH HIV-INFECTION

(Preliminary report)

Current results in the treatment of HIV-infected patients with antiretroviral drugs has, regretfully,but finally, been recognized as limited in effect and not the sole solution to AIDS. While there issome evidence that antiviral treatment may prolong the lives of some patients it has proven to betoo toxic for prolonged use. The profound depletion of a variety of defensive capabilities of theorganism, reflected in secondary immunodeficiency, imposes the need to apply preparations,that restore HI V damaged defensive mechanisms and stimulate others unaffected by the virus.

The objective of our study was to examine the effect of a polybacterial immunomodulatingpreparation, Factor-R, on some biological parameters in a statistically significant number ofHIV-infected patients.

MATERIALSAND METHODS

Ou r study included 133 subjects with HIV-infection. To the moment there are results after one90-day course of treatment for 40 % of them. Patient age varies between 24 and 64 years. Durationof seropositivity is between 1 and 11 years. 59,1 % of the participants in the study are undergoingtreatment with antiretroviral drugs, which is no w combined with Factor-R, 8 of the patients hadnever taken antiretrovirals.

Subjects were divided in several groups according to the following criteria;

- Group 1, including patients with CD4 from 0 to 200 lymphocytes;- Group 2, including patients with CD4 from 201 to 499 lymphocytes;- Group 3, including patients with CD4 more then 500 lymphocytes.

The following parameters were initially studied in all subjects:

- CBC with manual different.ial;- platelet count;- total protein;- CD4 absolute number;- CD8 absolute number.

At the time of this report investigators had complete data on only 17 subjects. Partial data for 44subjects was obtained on the number of CD4 and CDB.

64.66%

% Subjects Recieving Antiretrovira l Therapy(AZT, DOC, 001)

GeneraL population Sampling

50.00% - - ~ - - - 5 0 . 0 0 %

Treated with antiretrovirals Not trea ted with antiretroviral

124


106/124

rI

L

r

21.31

% Subjects Recieving Antibiotics And Other Therapies,Used for secondary opportunistic infections in patients with AIDS:

GeneraL popuLation Sampling

41.6

78.69%

With ethiological therapy Without ethiological therapy

GeneraL population

AgeFrom 24 to 64 years old

Mean range 37 ,5 years old

Sampling

58.33%

40.98% 0.82%10.66%

16.67%

From 24 to 34 years oldFrom 46 to 56 years old

47.22%

From 35 to 45 years old57 and more years old

Sex of Subjects

GeneraL popuLation Sampling

91.80%

Female Male125

36.11 %

88.89%


107/124

I

r

51.64%

Duration of Sero Positivity:(as reported by subjects)

From 0.5 to 11 yearsMean range: 4.4 years

GeneraL population

43.44%

From 0.5 to 5 yearsMore than 10 years

61.11%

SampLing

33.33%

From 6 to 9 years

The percentage of the more than 10 year survivors within the subject population and samplingis representative of the same group in the general seropositive population.

47.54%

AIDS related diseases

Secondary infections(as repor ted by subjects)

GeneraL popuLation

27.78%

Sampling

72.22 %

With secondary infect ions Without secondary infections

L Malignancies

17.50%

GeneraL population Sampling

With

82.50%

13.89%

126

Without

86.11 %


108/124

r

RESULTS AND DISCUSSIONS

5.4 ...-- -----,-- -- 1After a 90-day course of treatment with

Factor-R; in Group 1 the average values ofwhi te blood cell (WBC) count increased by35 %. An increase in the absolute values ofWBC count was observed in 75 % of thesubjects in this group. In Group 2 theaverage values decreased by 4.25 %; theincrease in absolute values was observedhowever in 55.9 % of the subjects in thisgroup.

5 . 2 ~ - - - - - - - : - - - -' "

51- - - - - - - - - - ' - - I - - - - - - - i

4.8-1 - - - - - - - - t - 1- - - - -4.6-1- - - - - - ~ ....... ::--- -4.4 -f- - - -

4.2 - -

4 1- - - - -11.._ -

3.8 L - _ _ _ _ _ _ _ _ _ _ _ _ _ _

Before treatment the average values of WBCin subjects of Group I were below thereferent values (4 .8 - 10.8 klcumm) . AfterFactor-R treatment WBC increased andreached normal levels, Fig. 1 .

Starting Factor R 90th day

_ _ _ Gr

Documents

Hidden Secret - Blocking A Treatment for HIV and Cancer