1

THE RESULTS OF BIOCHEMICAL RESEARCH AND THE RESULTS OF BIOCHEMICAL RESEARCH AND DEVELOPMENT OF NITROGEN-CONTAINING DEVELOPMENT OF NITROGEN-CONTAINING

COMPOUNDS OF NATURAL ORIGIN: COMPOUNDS OF NATURAL ORIGIN: methodology of exploitation of biological methodology of exploitation of biological

properties as universal natural regulators of properties as universal natural regulators of metabolism and drugsmetabolism and drugs

Leonid NefyodovLeonid Nefyodov

Doktor of Medical Science, Professor,Doktor of Medical Science, Professor,

Head of the Research Laboratory ofHead of the Research Laboratory of

biochemistry of bioactive substances,biochemistry of bioactive substances,

Professor of Biochemistry Department,Professor of Biochemistry Department,

Yanka Kupala State University of GrodnoYanka Kupala State University of Grodnowww.nil.grsu.bywww.nil.grsu.by

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BIOCHEMISTRY OF AMINO ACIDS AND BIOCHEMISTRY OF AMINO ACIDS AND DEVELOPMENT OF METHODS OF THEIR DEVELOPMENT OF METHODS OF THEIR

PRACTICAL USE AS UNIVERSAL ENDOGENOUS PRACTICAL USE AS UNIVERSAL ENDOGENOUS REGULATORS OF METABOLISM, MEDICAL AND REGULATORS OF METABOLISM, MEDICAL AND PROPHYLACTIC AGENTS AND COMPONENTS OF PROPHYLACTIC AGENTS AND COMPONENTS OF

HUMAN NUTRITION HUMAN NUTRITION

Grodno "amino acid" scientific school represented by Grodno "amino acid" scientific school represented by 20 scientists, including 3 scientists of the second 20 scientists, including 3 scientists of the second

generation; successfully were defended 12 generation; successfully were defended 12 candidate theses and 3 doctoral theses, one more is candidate theses and 3 doctoral theses, one more is

being prepared by a post-graduate.being prepared by a post-graduate.

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ACCREDITATION OF THE RL “BIOCHEMISTRY OF ACCREDITATION OF THE RL “BIOCHEMISTRY OF BIOACTIVE SUBSTANCES” ON ITS CONFORMANCE BIOACTIVE SUBSTANCES” ON ITS CONFORMANCE

TO THE REQUIREMENTS OF ISO/IEC 17025TO THE REQUIREMENTS OF ISO/IEC 17025

Material and technical base of the RL was Material and technical base of the RL was created using directed funds of the created using directed funds of the Council of Ministers and the Ministry of Council of Ministers and the Ministry of Education of the Republic of Belarus, Education of the Republic of Belarus, international contracts (Austria, United international contracts (Austria, United Kingdom) and the Central Fund of the Kingdom) and the Central Fund of the University.University.

Due to this the laboratory acquired the Due to this the laboratory acquired the opportunities to develop and implement opportunities to develop and implement modern methods of biochemical modern methods of biochemical research, perform researches in research, perform researches in accordance with the international accordance with the international standards of GLP at a competitive level.standards of GLP at a competitive level.

The Laboratory is a part of Grodno The Laboratory is a part of Grodno Regional Clinical Hospital, and is Regional Clinical Hospital, and is integrated into its Department of Clinical integrated into its Department of Clinical and Laboratory Diagnosis, what and Laboratory Diagnosis, what contributes to the efficiency of basic and contributes to the efficiency of basic and applied research.applied research.

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DEVELOPMENT OF DEMANDED, NEW AND ADAPTATION DEVELOPMENT OF DEMANDED, NEW AND ADAPTATION OF KNOWN HIGHLY ANALYTICAL METHODS OF OF KNOWN HIGHLY ANALYTICAL METHODS OF

DETECTION OF DIFFERENT NATURAL COMPOUNDS’ IN DETECTION OF DIFFERENT NATURAL COMPOUNDS’ IN BIOLOGICAL MATERIALBIOLOGICAL MATERIAL

Years of experience and qualification of the Years of experience and qualification of the staff enable to set and achieve goals not staff enable to set and achieve goals not only with the help of well-known and only with the help of well-known and standard laboratory methods but also by standard laboratory methods but also by the development or adaptation of new the development or adaptation of new methods, which often meet unexpected and methods, which often meet unexpected and changing requirements of customers (for changing requirements of customers (for example - combination of HPLC with example - combination of HPLC with modern physical-chemical methods of NMR, modern physical-chemical methods of NMR, mass spectrometry, differential mass spectrometry, differential centrifugation, spectrofluorometry), what centrifugation, spectrofluorometry), what allows to obtain evidential results, allows to obtain evidential results, comparing them in independent researches.comparing them in independent researches.

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HPLC of alkaloids Chelidonium majus L., HPLC of alkaloids Chelidonium majus L., free amino acids, their derivatives, free amino acids, their derivatives,

precursors and metabolitesprecursors and metabolites

One of the best used in One of the best used in practice, evidence, high-practice, evidence, high-sensitive, universal and, sensitive, universal and, simultaneously, specific simultaneously, specific methods is high-efficient methods is high-efficient liquid chromatography liquid chromatography (HPLC).(HPLC). min0 10 20 30 40 50 60 70 80

LU

0

5

10

15

20

25

30

35

40

45

FLD1 A, Ex=231, Em=445, TT (AACID\AA000020.D)

64

.95

0 -

T

rp

17

.28

0 -

H

is

19

.06

6 -

M

His

3

32

.29

1 -

Ta

u

25

.31

8 -

Arg

3.1

70

57

.06

3 -

M

et

66

.88

7 -

Ile

6.3

45

-

Glu

18

.57

8 -

G

ly

42

.65

9 -

T

yr

3.6

01

- A

sp

48

.92

9 -

E

A

24

.38

5 -

C

tr

15

.62

8 -

G

ln

9.8

34

- A

sn

67

.41

7

63

.24

5

60

.70

9 -

C

tn

55

.78

2 -

V

al

44

.58

4 -

a

AB

A

29

.32

4 -

Ala

21

.09

5 -

T

hr

62

.83

3

38

.04

7 -

G

AB

A

14

.08

4

33

.12

2

69

.21

8 -

L

eu

13

.67

7

50

.34

2 -

d

AV

A

30

.45

8

11

.75

7

61

.62

7

53

.96

1

20

.49

6

71

.82

9

7.2

91

64

.34

1

74

.73

5 -

O

rn

58

.92

2

77

.10

9

47

.09

0

FLD1 B, Ex=231, Em=313, TT (AACID\AA000020.D)

2.4

79

26

.57

7

4.0

97

min0 5 10 15 20 25

mAU

0

2

4

6

8

DAD1 E, Sig=285,16 Ref=450,100 (D:\DATA\UKR00008.D)

proto

pine

allok

ryptop

ine ch

elido

nine

copti

sine

sang

uinari

ne stylop

ine be

rberin

e

chele

rythri

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6

Methods of HPLC /mass-spectrometry, Methods of HPLC /mass-spectrometry, NMR and flow cytometryNMR and flow cytometry

400 500 600 700 800 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Inten.(x10,000)

582.35

817.65

719.00616.75534.00

392.75 652.95564.90877.15

746.00 829.75892.80

7

Largely thanks to this, agreements Largely thanks to this, agreements with the next organizations were with the next organizations were

implemented:implemented:

- - Agreement with the Center for collective use of devices, Agreement with the Center for collective use of devices, Federation of European Biochemical Communities Federation of European Biochemical Communities (FEBS SARS, London, UK) on free technical assistance;(FEBS SARS, London, UK) on free technical assistance;

- Agreement with Anticancer Institute in Vienna (Austria) - Agreement with Anticancer Institute in Vienna (Austria) on the early diagnosis of malignant disease on the on the early diagnosis of malignant disease on the basis of identification of specific compounds, which basis of identification of specific compounds, which appear in the range of the second and third lines of the appear in the range of the second and third lines of the anticancer defense;anticancer defense;

- Agreements with Institutions of the Ministry of Health of - Agreements with Institutions of the Ministry of Health of the Republic of Belarus on new domestic medicines’ the Republic of Belarus on new domestic medicines’ test, in particular – on piracetam, produced by Grodno test, in particular – on piracetam, produced by Grodno (Skidel) factory.(Skidel) factory.

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- - Agreement with Grodno executive committee to Agreement with Grodno executive committee to implement the tasks of Regional Scientific and implement the tasks of Regional Scientific and Technical Programme (RSTP) and develop new Technical Programme (RSTP) and develop new products of functional food on the basis of domestic products of functional food on the basis of domestic herbal;herbal;

- Agreements with Belarusian Republican Fund for - Agreements with Belarusian Republican Fund for Fundamental Research (BRFFR) and the Ministry of Fundamental Research (BRFFR) and the Ministry of Education to study mechanisms of therapeutic action Education to study mechanisms of therapeutic action of natural nitrogen-containing compounds;of natural nitrogen-containing compounds;

- Agreements with enterprises of meat and dairy - Agreements with enterprises of meat and dairy industry in the region to provide an advisory service industry in the region to provide an advisory service in the questions of safety and quality of food;in the questions of safety and quality of food;

- There was made an independent examination of RSTP - There was made an independent examination of RSTP and SSTP projects related to research and and SSTP projects related to research and development in the field of biochemistry of development in the field of biochemistry of biologically active substances.biologically active substances.

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STUDY OF BIOLOGICAL ACTIVITY AND EFECT STUDY OF BIOLOGICAL ACTIVITY AND EFECT MECHANISMS, INCLUDING ANTICANCER EFFECT MECHANISMS, INCLUDING ANTICANCER EFFECT

OF NATURAL NITROGEN-CONTAINING OF NATURAL NITROGEN-CONTAINING COMPOUNDS AND THEIR DERIVATIVESCOMPOUNDS AND THEIR DERIVATIVES

Priority methodology of the RL based on the Priority methodology of the RL based on the exploitation of regulatory action of natural exploitation of regulatory action of natural or close to natural (endogenous) or close to natural (endogenous) concentrations of nitrogen-containing concentrations of nitrogen-containing natural compounds in the first place - amino natural compounds in the first place - amino acids and related substances, which allows acids and related substances, which allows to fulfill biochemical and clinical sections of to fulfill biochemical and clinical sections of the fourteenth tasks of VI section of SSTP the fourteenth tasks of VI section of SSTP "Drugs: highly purified amino acids" and to "Drugs: highly purified amino acids" and to develop medicines manufactured by Grodno develop medicines manufactured by Grodno (Skidel) factory (pills "taurine", "lejcine" (Skidel) factory (pills "taurine", "lejcine" capsules "tavamin", "tryptophan").capsules "tavamin", "tryptophan").

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L. NefyodovL. Nefyodov, , M. VelichloM. Velichlo, , A. KaravayA. Karavay, , and othersand others. . Medication containing Medication containing derivative of L-glutamine, which possesses the anti-cancer activity derivative of L-glutamine, which possesses the anti-cancer activity /Inventor’s application/Inventor’s application 19990937 19990937 d/dd/d 15.10. 1999 15.10. 1999

L. Nefyodov L. Nefyodov , , M. VelichkoM. Velichko, , A. KaravayA. Karavay, , I. Lednyova, E. Doroshenko, D. I. Lednyova, E. Doroshenko, D. Maslakov New counter-drugs agent based on L-triptophaneMaslakov New counter-drugs agent based on L-triptophane / Inventor’s / Inventor’s applicationapplication 19991021 19991021 d/dd/d 17.11. 1999. 17.11. 1999.

V. SoldatovV. Soldatov, , L. NefyodovL. Nefyodov, , N. KurbatN. Kurbat MedicamentMedicament “ “taurinetaurine”” / Registration / Registration certificate certificate МЗ РБ N 08-1-6/856.МЗ РБ N 08-1-6/856.

V. SoldatovV. Soldatov, , L. NefyodovL. Nefyodov, , N. KurbatN. Kurbat MedicamentMedicament “ “leucineleucine”” / / Registration Registration certificate certificate МЗ РБ N 12-1-6. МЗ РБ N 12-1-6.

L. NefyodovL. Nefyodov, , E. DoroshenkoE. Doroshenko, , V. Smirnov Medication withV. Smirnov Medication with hepatoprotective, antioxidant and antiradiation effect hepatoprotective, antioxidant and antiradiation effect ““tavaminetavamine““ / / Inventor’s applicationInventor’s application 19991021 19991021 d/dd/d 17.09.2001. МПК А 61 К 31/195 17.09.2001. МПК А 61 К 31/195

L. NefyodovL. Nefyodov, , M. VelichkoM. Velichko, , A. KaravayA. Karavay, , I. Lednyova, T. Valyuk Medication I. Lednyova, T. Valyuk Medication containing derivative of L-glutamine, which possesses the anti-cancer containing derivative of L-glutamine, which possesses the anti-cancer activity / Official bulletin of the Center for Intellectual Property of activity / Official bulletin of the Center for Intellectual Property of BelarusBelarus - - 2004.-N1.- 2004.-N1.- pp.103. .103. PatentPatent BY N 5882 CI. BY N 5882 CI.

Zh. MotylevichZh. Motylevich, , L. NefyodovL. Nefyodov, , E. SudnikovichE. Sudnikovich, , T. RudyakT. Rudyak, , S. ZabrodskayaS. Zabrodskaya Medication which is a magnesium salt hydrate of Medication which is a magnesium salt hydrate of -- toluydine acid with toluydine acid with and has an anti-cancer effect / Official bulletin of the Center for and has an anti-cancer effect / Official bulletin of the Center for Intellectual Property of BelarusIntellectual Property of Belarus - - 2005.-N.- 2005.-N.- PatentPatent BY N 5882 CI. BY N 5882 CI.

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We were the first to demonstrate that endogenous levels We were the first to demonstrate that endogenous levels of free amino acids’ of men’s fluids and tissues are the of free amino acids’ of men’s fluids and tissues are the most important integral indicators and regulators of most important integral indicators and regulators of metabolism.metabolism.

This enables to prove the use of individual amino acids This enables to prove the use of individual amino acids or their combinations for guided correction of or their combinations for guided correction of metabolism with specific human diseases and metabolism with specific human diseases and significantly expand the area of practical application of significantly expand the area of practical application of these compounds.these compounds.

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Under Regulator effect of amino acids is Under Regulator effect of amino acids is understood their effect on the biochemical understood their effect on the biochemical processes and vital functions, which is processes and vital functions, which is manifested in natural (endogenous) or close manifested in natural (endogenous) or close to natural concentrations of these to natural concentrations of these compounds.compounds.

A regulatory effect after introduction of amino A regulatory effect after introduction of amino acids can be achieved by using either acids can be achieved by using either individual amino acids, or a combination of individual amino acids, or a combination of their small sets.their small sets.

The structure of these compositions in specific The structure of these compositions in specific ratios may include almost any amino acids, ratios may include almost any amino acids, their structural analogs or derivatives with a their structural analogs or derivatives with a known mechanism of action.known mechanism of action.

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The trend we develop is notable for the The trend we develop is notable for the fund of amino acids in biological fluids fund of amino acids in biological fluids and tissues is assessed as a single and tissues is assessed as a single information unit, and while analyzing it, information unit, and while analyzing it, it is possible to estimate the segments it is possible to estimate the segments which disrupt the metabolic balance which disrupt the metabolic balance (changes in the metabolic flows ratio (changes in the metabolic flows ratio and their balance shifts).and their balance shifts).

Thus, the studied characteristics Thus, the studied characteristics represent a kind of "chemical represent a kind of "chemical projection" of the genome that is being projection" of the genome that is being undertaken through the proteome.undertaken through the proteome.

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Thus, field of our studies and Thus, field of our studies and developments includes new, developments includes new, postgenome technologies which are on postgenome technologies which are on the list of EU scientific priorities, but the list of EU scientific priorities, but not so developed in the Republic of not so developed in the Republic of Belarus Belarus

These research solve not only These research solve not only fundamental tasks but also enable us fundamental tasks but also enable us to solve key problems of biotechnology, to solve key problems of biotechnology, medicine and pharmacology, in this medicine and pharmacology, in this connection most countries devote great connection most countries devote great funds to perform such researches.funds to perform such researches.

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The results of such researches enable to The results of such researches enable to distinguish "normal" metabolism from distinguish "normal" metabolism from a “modified“ one and since the year a “modified“ one and since the year 2002 has being successfully applied in 2002 has being successfully applied in USA, Britain, Japan and EU countries in USA, Britain, Japan and EU countries in the development of new medicines and the development of new medicines and methods of molecular diagnostics. They methods of molecular diagnostics. They accelerate the development of new accelerate the development of new medicines and increase the chances for medicines and increase the chances for their safe and effective application.their safe and effective application.

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It is believed that research in this It is believed that research in this area will have more significant area will have more significant impact on medicine than the impact on medicine than the project “Genome Project”, as they project “Genome Project”, as they reflect more accurately reflect more accurately physiological and biochemical physiological and biochemical processes in the body, what processes in the body, what enables to use them not only for enables to use them not only for early diagnosis of human early diagnosis of human diseases, but also to assess the diseases, but also to assess the aptitude to them.aptitude to them.

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Performance of tasks of RSTP Performance of tasks of RSTP "Sustainable development: science, "Sustainable development: science,

innovation and technology in Grodno innovation and technology in Grodno region," grants of BRFFR and the region," grants of BRFFR and the

Ministry of Education, scientific and Ministry of Education, scientific and methodical support of clinical trials of methodical support of clinical trials of

new home made drugsnew home made drugsThe methodology of the regulatory effect of amino The methodology of the regulatory effect of amino

acids and their derivatives was used by us for:acids and their derivatives was used by us for: realization of the project on the development of new realization of the project on the development of new

domestic sorts of functional food based on vegetable domestic sorts of functional food based on vegetable supplement “Lofantus anisatis” together with JSC supplement “Lofantus anisatis” together with JSC “Oshmyany meat-packing factory” and farm “Gury”.“Oshmyany meat-packing factory” and farm “Gury”.

development of alternative, applied and used as development of alternative, applied and used as evidence methods for assessing nutritional quality evidence methods for assessing nutritional quality and safety of functional food, and addition of and safety of functional food, and addition of vegetarian supplements with unique chemistry and vegetarian supplements with unique chemistry and similarity in the basic qualitative and quantitative similarity in the basic qualitative and quantitative characteristics with the amino acid Fund of human characteristics with the amino acid Fund of human plasma;plasma;

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Percentage of free amino acids in Percentage of free amino acids in anise giant hyssopanise giant hyssop

Trp0,5%

Ile2,3%

Tau0,2%

GABA5,9%

bABA0,0%

Ala3,7%bAla

0,5%Arg2,5%

1MHis0,0%

Ctr0,1%

Thr7,1%

Gly1,0%

3MHis0,2%

PEA0,2%

His2,2%

aAAA0,4%

Asn20,0%

Glu8,4%

Hcy0,2%

Val2,2%

aABA0,0%

Tyr2,2%

Pro17,1%

Ser1,5%

Gln1,3%

Asp7,6%

CA0,8%

GSH1,7%

CysGly0,2%

Cys1,5%

Lys0,3%

Orn1,2%

HPro0,0%

Leu2,4%

Phe1,6%

CysCys0,8%

Ctn0,5%

EA1,3%

Met0,0%

20

Profiles of free amino acids and their derivativesProfiles of free amino acids and their derivativesanise giant hyssop and human plasma anise giant hyssop and human plasma

0

5

10

15

20

25

30

35

40

CA

Ta

u

As

p

Th

r

Se

r

Glu

Gln

Pro

Gly

Ala

Va

l

Cy

s

Ile

Le

u

Ty

r

Ph

e

bA

la

EA

Orn

Ly

s

His

%,

от с

ум

.

плазма крови человека

Лофант анисовый

21

Content of free amino acids and their derivatives in Content of free amino acids and their derivatives in food production before and after addition of anise food production before and after addition of anise

giant hyssop’s extractgiant hyssop’s extract

0

100

200

300

400

500

600

700

800

CA

Asp

Glu

Asn

Ser

aA

AA

Gln

His

Gly

3-M

His

PE

A

Th

r

1-M

His

Ctr

Arg

bA

la

Ala

Tau

bA

BA

GA

BA

Tyr

aA

BA

EA

Val

Met

Ctn

Trp

Ph

e

Ile

Leu

HP

ro

Orn

Lys

Pro

Cys

Hcy

CysG

ly

GS

H

С, м

г/кг

пищевая продукция с лофантом

пищевая продукция без лофанта

22

Implementation of grants of BRFFR and the Implementation of grants of BRFFR and the Ministry of Education of the Republic of Belarus, Ministry of Education of the Republic of Belarus, which result not only in development and which result not only in development and optimization of highly selective HPLC, NMR, mass optimization of highly selective HPLC, NMR, mass and flow cytometric methods of nitrogen-and flow cytometric methods of nitrogen-containing biologically active substances’ containing biologically active substances’ determination (free α-amino acids and their determination (free α-amino acids and their metabolites, as well as the alkaloids of celandine), metabolites, as well as the alkaloids of celandine), which allow to identify them in one biological which allow to identify them in one biological sample, but in vitro a specific selective binding of sample, but in vitro a specific selective binding of these compounds with components of plasma and these compounds with components of plasma and formed blood elements of cancer patients were formed blood elements of cancer patients were determined .determined .

High sensitivity (91.7%), specificity (83.3%) and High sensitivity (91.7%), specificity (83.3%) and efficiency (88.9%) of the proposed method of efficiency (88.9%) of the proposed method of malignant tumors’ diagnosis enable us to develop malignant tumors’ diagnosis enable us to develop a new test-system for diagnosis and monitoring a new test-system for diagnosis and monitoring the treatment of cancer patients.the treatment of cancer patients.

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Plane projection of two main Plane projection of two main components and groups’ clustering components and groups’ clustering according to the levels of specific according to the levels of specific

compounds in blood plasma of healthy compounds in blood plasma of healthy people and patients with malignant or people and patients with malignant or

benign tumorsbenign tumors

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Improving the quality of education and Improving the quality of education and upbringing, work with talented student upbringing, work with talented student

youth, youth, and training of higher qualified and training of higher qualified

personnelpersonnelWe pay particular attention to We pay particular attention to education and training of talented education and training of talented students: search and find students students: search and find students with fresh thinking, ability to with fresh thinking, ability to analyze and solve tasks with the analyze and solve tasks with the help of not just well-known but help of not just well-known but original ways. In reality, as shows original ways. In reality, as shows the experience, according to the the experience, according to the normal distribution law, the normal distribution law, the percentage of researchers among percentage of researchers among population doesn’t exceed 5%. population doesn’t exceed 5%. When invited to the Laboratory When invited to the Laboratory undergraduates work together with undergraduates work together with the lab’s personnel in the normal the lab’s personnel in the normal working conditions, perform their working conditions, perform their daily work, get practice and learn. daily work, get practice and learn. Few of them remain till the 5Few of them remain till the 5thth year. Diligence, active research year. Diligence, active research work and guidance bring results. In work and guidance bring results. In such a way, the Laboratory such a way, the Laboratory prepared Anton Glazov, who prepared Anton Glazov, who became an Associate Professor became an Associate Professor (new representative of "amino (new representative of "amino acid" school), a JRS of the acid" school), a JRS of the Laboratory, a lecturer at the Laboratory, a lecturer at the Faculty of Biology and Ecology and Faculty of Biology and Ecology and a talented biochemist. The others a talented biochemist. The others are coming soon.are coming soon.

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Today the Laboratory:Today the Laboratory: - - Submitted all the documentation for new RSTP Submitted all the documentation for new RSTP

tasks - 2010;tasks - 2010; - Completed the preparation of two SSTP projects for - Completed the preparation of two SSTP projects for

2011 - 2015. ("Metabolism" and "Development of 2011 - 2015. ("Metabolism" and "Development of new effective domestic drugs and blood substitutes new effective domestic drugs and blood substitutes with directional effect");with directional effect");

- Accumulated experience in development and - Accumulated experience in development and production of new domestic products of functional production of new domestic products of functional food, delivery of consultative help to companies of food, delivery of consultative help to companies of meat and dairy industry in questions concerning meat and dairy industry in questions concerning quality assurance and food safety, food staples, quality assurance and food safety, food staples, premixes and feed additives, as production of the premixes and feed additives, as production of the indicated enterprises is marketing in both the indicated enterprises is marketing in both the Republic of Belarus, and in Russia and EU countries;Republic of Belarus, and in Russia and EU countries;

- Negotiates for participation in establishment of a - Negotiates for participation in establishment of a cross-border inter-agency center for promotion of cross-border inter-agency center for promotion of food quality assurance and safety in accordance with food quality assurance and safety in accordance with EU regulations.EU regulations.

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The publications of Laboratory:The publications of Laboratory: 1. Nefyodov L.I. Taurine, Grodno, 1999, 140 p.1. Nefyodov L.I. Taurine, Grodno, 1999, 140 p.

2. Uglyanitsa K.N., Nefyodov L.I., Bzosko W. Ukrain® (biological activity & medical 2. Uglyanitsa K.N., Nefyodov L.I., Bzosko W. Ukrain® (biological activity & medical applications), Grodno, 2000, 315 p.applications), Grodno, 2000, 315 p.3. Ledneva I.O., Velichko M.G., Nefyodov L.I. Effects of deryvativs of L- glutamin and 3. Ledneva I.O., Velichko M.G., Nefyodov L.I. Effects of deryvativs of L- glutamin and L- phenilalanin in the tumorbraiding, Grodno, GSU, 2003, 108 p.L- phenilalanin in the tumorbraiding, Grodno, GSU, 2003, 108 p.4. Amino Acids And Their Derivatives//Proc of Internat. Symp/Ed. V. Soldatov, L. 4. Amino Acids And Their Derivatives//Proc of Internat. Symp/Ed. V. Soldatov, L. Nefydov, 1996, Grodno, 125 pNefydov, 1996, Grodno, 125 p5. Nefyodov L.I., Uglynitsa K.N., Godysh Yu. et al. Biochemical mechanisms of rea 5. Nefyodov L.I., Uglynitsa K.N., Godysh Yu. et al. Biochemical mechanisms of rea lization of Ukrain antitumor activity // European Journal of Cancer, 1997, V. 33, lization of Ukrain antitumor activity // European Journal of Cancer, 1997, V. 33, Supp.5, S27. Supp.5, S27. 6. Nefyodov L.I., Mechanisms of regulatory effects and strategy of using amino acids 6. Nefyodov L.I., Mechanisms of regulatory effects and strategy of using amino acids and their derivatives as effective agents for metabolic therapy and medicinal and their derivatives as effective agents for metabolic therapy and medicinal preparations Proc of Internat. Symp. Biological activity and transport of drugs. /Ed. L. preparations Proc of Internat. Symp. Biological activity and transport of drugs. /Ed. L. Nefydov, Grodno, 1999, 189p.Nefydov, Grodno, 1999, 189p.7. Nefyodov L.I., Smirnov V.Y. Brzosko W. et al. Mechanisms of the anticancer effect 7. Nefyodov L.I., Smirnov V.Y. Brzosko W. et al. Mechanisms of the anticancer effect of Ukrain in the treatment of cancer of the urinary bladder // Drugs under Exptl. of Ukrain in the treatment of cancer of the urinary bladder // Drugs under Exptl. Clin. Clin. Res., 2000, V.XXVI, N 5/6, p. 334 -344 Res., 2000, V.XXVI, N 5/6, p. 334 -344 88. Nefyodov L.I., Planowa regulacja równowagi metabolizmu aminokwasami i . Nefyodov L.I., Planowa regulacja równowagi metabolizmu aminokwasami i strategia ich zastosowania w jakości nowych efektywnych preparatów leczniczych // strategia ich zastosowania w jakości nowych efektywnych preparatów leczniczych // XXXVII Zjazd Polskiego towarzystwa biochemicznego. XXXVII Zjazd Polskiego towarzystwa biochemicznego. Torun, 10-14 IX 2001. s.327Torun, 10-14 IX 2001. s.3279. Biologically active compounds in control of metabolic homeostasis //Proc of 9. Biologically active compounds in control of metabolic homeostasis //Proc of Internat. Symp /Ed. L. Nefyodov, 2000, Grodno, 325 pInternat. Symp /Ed. L. Nefyodov, 2000, Grodno, 325 p10. VI Ordinary General Assembly Society of Biochemistry of Belarus /Ed. L. 10. VI Ordinary General Assembly Society of Biochemistry of Belarus /Ed. L. Nefyodov, 2000, Grodno, 225 p Nefyodov, 2000, Grodno, 225 p 11. Amino Acids And Their Derivatives // Proc Internat. Symp. /Ed. L. Nefyodov, 2001 11. Amino Acids And Their Derivatives // Proc Internat. Symp. /Ed. L. Nefyodov, 2001 Grodno, 124 p. Grodno, 124 p. 12. Nefyodov LI., Doroshenko E.M., Smirnov V. et al. Metabolic control and treatment 12. Nefyodov LI., Doroshenko E.M., Smirnov V. et al. Metabolic control and treatment of malignant growth: tumor development, patterns of amino acid imbalance and of malignant growth: tumor development, patterns of amino acid imbalance and Ukrain // Int. J. Immunotherapy, 2003,V.XIX (2-4), p.109-114.Ukrain // Int. J. Immunotherapy, 2003,V.XIX (2-4), p.109-114.

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13. Nefyodov LI., Doroshenko E.M., Smirnov V. Yu. at al. Metabolic 13. Nefyodov LI., Doroshenko E.M., Smirnov V. Yu. at al. Metabolic control and treatment of malignant growth: tumor development, control and treatment of malignant growth: tumor development, patterns of amino acid imbalance and Ukrain/ FEBS Journal", 2005, patterns of amino acid imbalance and Ukrain/ FEBS Journal", 2005, V 272, N1 (Suppl), p.558, // Abst. 30th FEBS Congress & 9th IUBMB V 272, N1 (Suppl), p.558, // Abst. 30th FEBS Congress & 9th IUBMB Conference 'The Protein World Proteins and Peptides: Structure, Conference 'The Protein World Proteins and Peptides: Structure, Function & organization", 3-7 July 2005, V1-067P BudapestFunction & organization", 3-7 July 2005, V1-067P Budapest14. Nefyodov L., Nowicky J., Glazev A., Karavay A. Anticancer drug 14. Nefyodov L., Nowicky J., Glazev A., Karavay A. Anticancer drug Ukrain®: mechanisms of anticancer effects and perspectives of its Ukrain®: mechanisms of anticancer effects and perspectives of its application in the cancer diagnostics // Abstracts of the 41st application in the cancer diagnostics // Abstracts of the 41st Meeting of the Polish Biochemical Society, September 12-15, 2006, Meeting of the Polish Biochemical Society, September 12-15, 2006, Bialystok, Poland. – 2006. – P. 92.Bialystok, Poland. – 2006. – P. 92.15. Glazev A, L. Nefyodov Changes in amino acid patterns of blood 15. Glazev A, L. Nefyodov Changes in amino acid patterns of blood plasma in tumor patients with anticancer drug NSC-631570: plasma in tumor patients with anticancer drug NSC-631570: possible approaches to cancer diagnostics// Biochemistry (Moscow). possible approaches to cancer diagnostics// Biochemistry (Moscow). Suppl. series B. Biomedical chemistry, 2008, V. 2, № 3, p. 318–324.Suppl. series B. Biomedical chemistry, 2008, V. 2, № 3, p. 318–324.16. Glazev, A. Nefyodov L., Nowicky W.HPLC–method for detection 16. Glazev, A. Nefyodov L., Nowicky W.HPLC–method for detection of alkaloids and its derivatives in anticancer drug Ukrain® for its of alkaloids and its derivatives in anticancer drug Ukrain® for its using in the cancer detection, effective treatment and investigating using in the cancer detection, effective treatment and investigating the mechanisms of carcinogenesis // Abstracts of the 41st Meeting the mechanisms of carcinogenesis // Abstracts of the 41st Meeting of the Polish Biochemical Society, September 12-15, 2006, of the Polish Biochemical Society, September 12-15, 2006, Bialystok, Poland. – 2006. – P. 88.Bialystok, Poland. – 2006. – P. 88.17. W. Nowicky, L. Nefyodov, A. Glazev, [et al] The effect of NSC 17. W. Nowicky, L. Nefyodov, A. Glazev, [et al] The effect of NSC 631570 on amino acid profile as a possible assay for early cancer 631570 on amino acid profile as a possible assay for early cancer diagnostics // Int. Sypm. Clinical Biomarкer Summit, March 29-31, diagnostics // Int. Sypm. Clinical Biomarкer Summit, March 29-31, 2006, Coronado, California, USA. – 2006. – P. 25.2006, Coronado, California, USA. – 2006. – P. 25.

2424. . L. NefyodovL. Nefyodov, , E. DoroshenkoE. Doroshenko, , V. Smirnov Medication withV. Smirnov Medication with hepatoprotective, antioxidant and antiradiation effect hepatoprotective, antioxidant and antiradiation effect ““tavaminetavamine““ / / Inventor’s applicationInventor’s application 19991021 19991021 d/dd/d 17.09.2001. 17.09.2001. МПК А 61 К 31/195МПК А 61 К 31/195

25. 25. L. NefyodovL. Nefyodov, , M. VelichkoM. Velichko, , A. KaravayA. Karavay, , I. Lednyova, T. Valyuk I. Lednyova, T. Valyuk Medication containing derivative of L-glutamine, which possesses Medication containing derivative of L-glutamine, which possesses the anti-cancer activity / Official bulletin of the Center for the anti-cancer activity / Official bulletin of the Center for Intellectual Property of BelarusIntellectual Property of Belarus - - 2004.-N1.- 2004.-N1.- pp.103. .103. PatentPatent BY N BY N 5882 CI.5882 CI.

26. 26. Zh. MotylevichZh. Motylevich, , L. NefyodovL. Nefyodov, , E. SudnikovichE. Sudnikovich, , T. RudyakT. Rudyak, , S. S. ZabrodskayaZabrodskaya Medication which is a magnesium salt hydrate of Medication which is a magnesium salt hydrate of -- toluydine acid with and has an anti-cancer effect / Official bulletin toluydine acid with and has an anti-cancer effect / Official bulletin of the Center for Intellectual Property of Belarusof the Center for Intellectual Property of Belarus - - 2005.-N.- 2005.-N.- PatentPatent BY N 5882 CI. BY N 5882 CI.