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THE RESULTS OF BIOCHEMICAL RESEARCH AND DEVELOPMENT OF NITROGEN-CONTAINING COMPOUNDS OF NATURAL ORIGIN: methodology of exploitation of biological properties as universal natural regulators of metabolism and drugs Leonid Nefyodov Doktor of Medical Science, Professor, Head of the Research Laboratory of biochemistry of bioactive substances, Professor of Biochemistry Department, Yanka Kupala State University of Grodno www.nil.grsu.by 1 BIOCHEMISTRY OF AMINO ACIDS AND DEVELOPMENT OF METHODS OF THEIR PRACTICAL USE AS UNIVERSAL ENDOGENOUS REGULATORS OF METABOLISM, MEDICAL AND PROPHYLACTIC AGENTS AND COMPONENTS OF HUMAN NUTRITION Grodno "amino acid" scientific school represented by 20 scientists, including 3 scientists of the second generation; successfully were defended 12 candidate theses and 3 doctoral theses, one more is being prepared by a postgraduate. 2 ACCREDITATION OF THE RL “BIOCHEMISTRY OF BIOACTIVE SUBSTANCES” ON ITS CONFORMANCE TO THE REQUIREMENTS OF ISO/IEC 17025 Material and technical base of the RL was created using directed funds of the Council of Ministers and the Ministry of Education of the Republic of Belarus, international contracts (Austria, United Kingdom) and the Central Fund of the University. Due to this the laboratory acquired the opportunities to develop and implement modern methods of biochemical research, perform researches in accordance with the international standards of GLP at a competitive level. The Laboratory is a part of Grodno Regional Clinical Hospital, and is integrated into its Department of Clinical and Laboratory Diagnosis, what contributes to the efficiency of basic and applied research. 3 DEVELOPMENT OF DEMANDED, NEW AND ADAPTATION OF KNOWN HIGHLY ANALYTICAL METHODS OF DETECTION OF DIFFERENT NATURAL COMPOUNDS’ IN BIOLOGICAL MATERIAL Years of experience and qualification of the staff enable to set and achieve goals not only with the help of well-known and standard laboratory methods but also by the development or adaptation of new methods, which often meet unexpected and changing requirements of customers (for example - combination of HPLC with modern physical-chemical methods of NMR, mass spectrometry, differential centrifugation, spectrofluorometry), what allows to obtain evidential results, comparing them in independent researches. 4 4 protopine allokryptopine chelidonine coptisine 6 0 5 chelerythrine stylopine berberine 2 sanguinarine One of the best used in practice, evidence, high-sensitive, universal and, simultaneously, specific methods is high-efficient liquid chromatography (HPLC). 10 15 20 25 min 5 0 10 20 30 40 50 60 70 mAU DAD1 E, Sig=285,16 Ref=450,100 (D:\DATA\UKR00008.D) 8 0 5 77.109 74.735 - Orn 63.245 60.709 - Ctn 55.782 - Val 57.063 - Met 32.291 - Tau 25.318 - Arg 66.887 - Ile 48.929 - EA 67.417 69.218 - Leu 62.833 50.342 - dAVA 44.584 - aABA 42.659 - Tyr 38.047 - GABA 33.122 29.324 - Ala 24.385 - Ctr 64.950 - Trp LU 71.829 64.341 61.627 58.922 53.961 47.090 30.458 26.577 20 21.095 - Thr 35 17.280 - His 19.066 - MHis3 40 18.578 - Gly 15.628 - Gln 45 20.496 10 13.677 14.084 9.834 - Asn 6.345 - Glu 3.601 - Asp 3.170 25 11.757 15 2.479 30 7.291 4.097 HPLC of alkaloids Chelidonium majus L., free amino acids, their derivatives, precursors and metabolites FLD1 A, Ex=231, Em=445, TT (A ACID\A A000020.D) FLD1 B, Ex=231, Em=313, TT (A ACID\A A000020.D) 0 80 min Methods of HPLC /mass-spectrometry, NMR and flow cytometry Inten.(x10,000) 582.35 1.75 1.50 1.25 1.00 817.65 0.75 534.00 0.50 392.75 564.90 0.25 719.00 616.75 877.15 652.95 746.00 892.80 829.75 0.00 400 500 600 700 800 m/z 6 Largely thanks to this, agreements with the next organizations were implemented: - Agreement with the Center for collective use of devices, Federation of European Biochemical Communities (FEBS SARS, London, UK) on free technical assistance; - Agreement with Anticancer Institute in Vienna (Austria) on the early diagnosis of malignant disease on the basis of identification of specific compounds, which appear in the range of the second and third lines of the anticancer defense; - Agreements with Institutions of the Ministry of Health of the Republic of Belarus on new domestic medicines’ test, in particular – on piracetam, produced by Grodno (Skidel) factory. 7 - Agreement with Grodno executive committee to implement the tasks of Regional Scientific and Technical Programme (RSTP) and develop new products of functional food on the basis of domestic herbal; - Agreements with Belarusian Republican Fund for Fundamental Research (BRFFR) and the Ministry of Education to study mechanisms of therapeutic action of natural nitrogen-containing compounds; - Agreements with enterprises of meat and dairy industry in the region to provide an advisory service in the questions of safety and quality of food; - There was made an independent examination of RSTP and SSTP projects related to research and development in the field of biochemistry of biologically active substances. 8 STUDY OF BIOLOGICAL ACTIVITY AND EFECT MECHANISMS, INCLUDING ANTICANCER EFFECT OF NATURAL NITROGEN-CONTAINING COMPOUNDS AND THEIR DERIVATIVES Priority methodology of the RL based on the exploitation of regulatory action of natural or close to natural (endogenous) concentrations of nitrogen-containing natural compounds in the first place - amino acids and related substances, which allows to fulfill biochemical and clinical sections of the fourteenth tasks of VI section of SSTP "Drugs: highly purified amino acids" and to develop medicines manufactured by Grodno (Skidel) factory (pills "taurine", "lejcine" capsules "tavamin", "tryptophan"). 9 10 L. Nefyodov, M. Velichlo, A. Karavay, and others. Medication containing derivative of L-glutamine, which possesses the anti-cancer activity /Inventor’s application 19990937 d/d 15.10. 1999 L. Nefyodov , M. Velichko, A. Karavay, I. Lednyova, E. Doroshenko, D. Maslakov New counter-drugs agent based on L-triptophane / Inventor’s application 19991021 d/d 17.11. 1999. V. Soldatov, L. Nefyodov, N. Kurbat Medicament “taurine” / Registration certificate МЗ РБ N 08-1-6/856. V. Soldatov, L. Nefyodov, N. Kurbat Medicament “leucine” / Registration certificate МЗ РБ N 12-1-6. L. Nefyodov, E. Doroshenko, V. Smirnov Medication with hepatoprotective, antioxidant and antiradiation effect “tavamine“ / Inventor’s application 19991021 d/d 17.09.2001. МПК А 61 К 31/195 L. Nefyodov, M. Velichko, A. Karavay, I. Lednyova, T. Valyuk Medication containing derivative of L-glutamine, which possesses the anti-cancer activity / Official bulletin of the Center for Intellectual Property of Belarus - 2004.-N1.p.103. Patent BY N 5882 CI. Zh. Motylevich, L. Nefyodov, E. Sudnikovich, T. Rudyak, S. Zabrodskaya Medication which is a magnesium salt hydrate of - toluydine acid with and has an anti-cancer effect / Official bulletin of the Center for Intellectual Property of Belarus - 2005.-N.- Patent BY N 5882 CI. 11 We were the first to demonstrate that endogenous levels of free amino acids’ of men’s fluids and tissues are the most important integral indicators and regulators of metabolism. This enables to prove the use of individual amino acids or their combinations for guided correction of metabolism with specific human diseases and significantly expand the area of practical application of these compounds. 12 Under Regulator effect of amino acids is understood their effect on the biochemical processes and vital functions, which is manifested in natural (endogenous) or close to natural concentrations of these compounds. A regulatory effect after introduction of amino acids can be achieved by using either individual amino acids, or a combination of their small sets. The structure of these compositions in specific ratios may include almost any amino acids, their structural analogs or derivatives with a known mechanism of action. 13 The trend we develop is notable for the fund of amino acids in biological fluids and tissues is assessed as a single information unit, and while analyzing it, it is possible to estimate the segments which disrupt the metabolic balance (changes in the metabolic flows ratio and their balance shifts). Thus, the studied characteristics represent a kind of "chemical projection" of the genome that is being undertaken through the proteome. 14 Thus, field of our studies and developments includes new, postgenome technologies which are on the list of EU scientific priorities, but not so developed in the Republic of Belarus These research solve not only fundamental tasks but also enable us to solve key problems of biotechnology, medicine and pharmacology, in this connection most countries devote great funds to perform such researches. 15 The results of such researches enable to distinguish "normal" metabolism from a “modified“ one and since the year 2002 has being successfully applied in USA, Britain, Japan and EU countries in the development of new medicines and methods of molecular diagnostics. They accelerate the development of new medicines and increase the chances for their safe and effective application. 16 It is believed that research in this area will have more significant impact on medicine than the project “Genome Project”, as they reflect more accurately physiological and biochemical processes in the body, what enables to use them not only for early diagnosis of human diseases, but also to assess the aptitude to them. 17 Performance of tasks of RSTP "Sustainable development: science, innovation and technology in Grodno region," grants of BRFFR and the Ministry of Education, scientific and methodical support of clinical trials of new home made drugs The methodology of the regulatory effect of amino acids and their derivatives was used by us for: realization of the project on the development of new domestic sorts of functional food based on vegetable supplement “Lofantus anisatis” together with JSC “Oshmyany meat-packing factory” and farm “Gury”. development of alternative, applied and used as evidence methods for assessing nutritional quality and safety of functional food, and addition of vegetarian supplements with unique chemistry and similarity in the basic qualitative and quantitative characteristics with the amino acid Fund of human plasma; 18 Percentage of free amino acids in anise giant hyssop Cys 1,5% Hcy 0,2% CysGly 0,2% GSH 1,7% CA 0,8% Asp 7,6% Pro 17,1% Glu 8,4% Lys 0,3% Orn 1,2% HPro 0,0% Leu 2,4% Asn 20,0% Ile 2,3% Phe 1,6% Trp 0,5% CysCys 0,8% Ctn 0,5% Met 0,0% Ser 1,5% Val 2,2% EA 1,3% aABA 0,0% Tyr 2,2% GABA 5,9% bABA 0,0% Ala 3,7%bAla Arg Tau 0,5% 2,5% 0,2% 1MHis 0,0% PEA Thr 0,2% Ctr 7,1% 0,1% Gly 1,0% His 2,2% aAAA 0,4% Gln 1,3% 3MHis 0,2% 19 Profiles of free amino acids and their derivatives anise giant hyssop and human plasma 40 35 25 20 15 10 5 His Lys Orn EA bAla Phe Tyr Leu Ile Cys Val Ala Gly Pro Gln Glu Ser Thr Asp Tau 0 CA %, от сум. 30 плазма крови человека Лофант анисовый 20 Content of free amino acids and their derivatives in food production before and after addition of anise giant hyssop’s extract 800 700 600 400 300 200 100 0 CA Asp Glu Asn Ser aAAA Gln His Gly 3-MHis PEA Thr 1-MHis Ctr Arg bAla Ala Tau bABA GABA Tyr aABA EA Val Met Ctn Trp Phe Ile Leu HPro Orn Lys Pro Cys Hcy CysGly GSH С, мг/кг 500 пищевая продукция с лофантом пищевая продукция без лофанта 21 Implementation of grants of BRFFR and the Ministry of Education of the Republic of Belarus, which result not only in development and optimization of highly selective HPLC, NMR, mass and flow cytometric methods of nitrogen-containing biologically active substances’ determination (free α-amino acids and their metabolites, as well as the alkaloids of celandine), which allow to identify them in one biological sample, but in vitro a specific selective binding of these compounds with components of plasma and formed blood elements of cancer patients were determined . High sensitivity (91.7%), specificity (83.3%) and efficiency (88.9%) of the proposed method of malignant tumors’ diagnosis enable us to develop a new testsystem for diagnosis and monitoring the treatment of cancer patients. 22 Plane projection of two main components and groups’ clustering according to the levels of specific compounds in blood plasma of healthy people and patients with malignant or benign tumors 23 Improving the quality of education and upbringing, work with talented student youth, and training of higher qualified personnel We pay particular attention to education and training of talented students: search and find students with fresh thinking, ability to analyze and solve tasks with the help of not just wellknown but original ways. In reality, as shows the experience, according to the normal distribution law, the percentage of researchers among population doesn’t exceed 5%. When invited to the Laboratory undergraduates work together with the lab’s personnel in the normal working conditions, perform their daily work, get practice and learn. Few of them remain till the 5th year. Diligence, active research work and guidance bring results. In such a way, the Laboratory prepared Anton Glazov, who became an Associate Professor (new representative of "amino acid" school), a JRS of the Laboratory, a lecturer at the Faculty of Biology and Ecology and a talented biochemist. The others are coming soon. 24 Today the Laboratory: - Submitted all the documentation for new RSTP tasks 2010; - Completed the preparation of two SSTP projects for 2011 2015. ("Metabolism" and "Development of new effective domestic drugs and blood substitutes with directional effect"); - Accumulated experience in development and production of new domestic products of functional food, delivery of consultative help to companies of meat and dairy industry in questions concerning quality assurance and food safety, food staples, premixes and feed additives, as production of the indicated enterprises is marketing in both the Republic of Belarus, and in Russia and EU countries; - Negotiates for participation in establishment of a crossborder inter-agency center for promotion of food quality assurance and safety in accordance with EU regulations. 25 The publications of Laboratory: 1. Nefyodov L.I. Taurine, Grodno, 1999, 140 p. 2. Uglyanitsa K.N., Nefyodov L.I., Bzosko W. Ukrain® (biological activity & medical applications), Grodno, 2000, 315 p. 3. Ledneva I.O., Velichko M.G., Nefyodov L.I. Effects of deryvativs of L- glutamin and Lphenilalanin in the tumorbraiding, Grodno, GSU, 2003, 108 p. 4. Amino Acids And Their Derivatives//Proc of Internat. Symp/Ed. V. Soldatov, L. Nefydov, 1996, Grodno, 125 p 5. Nefyodov L.I., Uglynitsa K.N., Godysh Yu. et al. Biochemical mechanisms of realization of Ukrain antitumor activity // European Journal of Cancer, 1997, V. 33, Supp.5, S27. 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 preparations Proc of Internat. Symp. Biological activity and transport of drugs. /Ed. L. Nefydov, Grodno, 1999, 189p. 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. Clin. Res., 2000, V.XXVI, N 5/6, p. 334 -344 8. Nefyodov L.I., Planowa regulacja równowagi metabolizmu aminokwasami i strategia ich zastosowania w jakości nowych efektywnych preparatów leczniczych // XXXVII Zjazd Polskiego towarzystwa biochemicznego. Torun, 10-14 IX 2001. s.327 9. Biologically active compounds in control of metabolic homeostasis //Proc of Internat. Symp /Ed. L. Nefyodov, 2000, Grodno, 325 p 10. VI Ordinary General Assembly Society of Biochemistry of Belarus /Ed. L. Nefyodov, 2000, Grodno, 225 p 11. Amino Acids And Their Derivatives // Proc Internat. Symp. /Ed. L. Nefyodov, 2001 Grodno, 124 p. 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 Ukrain // Int. J. Immunotherapy, 2003,V.XIX (2-4), p.109-114. 26 13. Nefyodov LI., Doroshenko E.M., Smirnov V. Yu. at al. Metabolic control and treatment of malignant growth: tumor development, patterns of amino acid imbalance and Ukrain/ FEBS Journal", 2005, V 272, N1 (Suppl), p.558, // Abst. 30th FEBS Congress & 9th IUBMB Conference 'The Protein World Proteins and Peptides: Structure, Function & organization", 3-7 July 2005, V1-067P Budapest 14. Nefyodov L., Nowicky J., Glazev A., Karavay A. Anticancer drug Ukrain®: mechanisms of anticancer effects and perspectives of its application in the cancer diagnostics // Abstracts of the 41st Meeting of the Polish Biochemical Society, September 12-15, 2006, Bialystok, Poland. – 2006. – P. 92. 15. Glazev A, L. Nefyodov Changes in amino acid patterns of blood plasma in tumor patients with anticancer drug NSC-631570: possible approaches to cancer diagnostics// Biochemistry (Moscow). Suppl. series B. Biomedical chemistry, 2008, V. 2, № 3, p. 318–324. 16. Glazev, A. Nefyodov L., Nowicky W.HPLC–method for detection of alkaloids and its derivatives in anticancer drug Ukrain® for its using in the cancer detection, effective treatment and investigating the mechanisms of carcinogenesis // Abstracts of the 41st Meeting of the Polish Biochemical Society, September 12-15, 2006, Bialystok, Poland. – 2006. – P. 88. 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 diagnostics // Int. Sypm. Clinical Biomarкer Summit, March 29-31, 2006, Coronado, California, USA. – 2006. – P. 25. 27 24. L. Nefyodov, E. Doroshenko, V. Smirnov Medication with hepatoprotective, antioxidant and antiradiation effect “tavamine“ / Inventor’s application 19991021 d/d 17.09.2001. МПК А 61 К 31/195 25. L. Nefyodov, M. Velichko, A. Karavay, I. Lednyova, T. Valyuk Medication containing derivative of L-glutamine, which possesses the anticancer activity / Official bulletin of the Center for Intellectual Property of Belarus 2004.-N1.p.103. Patent BY N 5882 CI. 26. Zh. Motylevich, L. Nefyodov, E. Sudnikovich, T. Rudyak, S. Zabrodskaya Medication which is a magnesium salt hydrate of - toluydine acid with and has an anti-cancer effect / Official bulletin of the Center for Intellectual Property of Belarus - 2005.-N.- Patent BY N 5882 CI.