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ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS

ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS. Timofeyev I.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Karpyshev N.N., Ryabicheva T.G., Varaksin N.A., Larionov P.M., Kiseleva Y.Y., Nekludov V.V., Perminova N.G.

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ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS

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  1. ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS Timofeyev I.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Karpyshev N.N., Ryabicheva T.G., Varaksin N.A., Larionov P.M., Kiseleva Y.Y., Nekludov V.V., Perminova N.G. SRC Virology & Biotechnology “Vector”, Koltsovo, Novosibirsk region; *HRDF, Moscow, Russia

  2. Introduction There is a desperate need for microbicides for protection from HIV infection At present, the whole humanity can be considered as a risk group for AIDS. Radically efficient medicines, which could allow curing HIV infection, have not been created yet; available preparations only somewhat slow down the development of AIDS. It is necessary creation of new highly effective preparations for preventive maintenance and treatment HIV-1/2 infection.

  3. Aims To construction of microbicides on basis of membrane-acting anti-HIV preparations of new generation to prevent transmission of HIV-1 across vaginal mucosa 1. To design and to develop optimum topical microbicide compounds, which could protect from transfer and distribution HIV infection 2. Evaluate candidate microbicide compounds for their ability to prevent HIV-1 infection

  4. Development of a new generation ofanti-HIV preparations On the basis: • co-polymers of divinyl ether with maleic anhydride • polymeric matrixes modified with hydrophobic pharmacophores • peptide imitators (pseudo-ligands of HIV-1/2) anti-HIV compounds have been synthesized.

  5. Development of a new generation ofanti-HIV preparations O O O O • Outcome: • Optimal topical microbicides formula: • we included efficient membrane-acting anti-HIV compounds into the pH-dependent interpolymeric complex (IPC) • PAA – PVP (polyacrylic acid, polyvinylpyrrolidone) • CL213-LVG(hydrolyzed chitosan, sodium alginate) H O O C C O O H O O C C O O H O O C C O O H O O C C O O H O O C C O O H O O C C O O H O O C C O O H O O C C O O + N a n C O C O N H N H ( C H ) n ( C H ) m 2 2 Peptide imitatorsco-receptors HIV-1/2 (CCR5 or CxCR4)

  6. 1.Microbicide (pretreat 1 hour) 2.Virus (1 hour exposure) Evaluation of candidate microbicides Permission cell culture (MT-4 or PBMC) 5.Analysis cell viability, IFA, and p24 HIV-1 production 4.Cells incubated for 4 days at 37 oC, 5% CO2 3. Cell culture washed x3 with PBS

  7. Development of a new generation ofanti-HIV preparations

  8. Development of a new generation ofanti-HIV preparations

  9. Development of a new generation ofanti-HIV preparations

  10. Development of a new generation ofanti-HIV preparations PVP PAA M-Nb M-Nb/PAA/PVP . Formation of complex M-Nb/PAA/PVP

  11. Development of a new generation ofanti-HIV preparations Formation of complex As646(M-Nb-P4)/CL113/LVG

  12. Development of a new generation ofanti-HIV preparations Interpolymeric complex (IPC) is stability at рН 4,5-6,0

  13. Development of a new generation ofanti-HIV preparations Destruction of IPC occurs with increasing рН (>6,5) with the release of “ballast” polymer and strengthening of preparation’s bond with HIV-1/2 ligands

  14. Compounds Cells viability, % Protection cells infections, % Concentrationр24,ng/ml Inhibition p24 HIV-1, % PAA – PVP 62,5 31,0 1740,56 58,1 PAA – PVP 70,8 39,4 1639,91 60,5 Carbopol 72,1 40,7 1435,72 65,5 M-Nb– PAA – PVP 91,4 90,9 70,59 98,3 M-Nb– PAA – PVP 93,6 93,1 201,72 95,2 M-Nb – Carbopol –PVP 93,6 93,1 616,8 85,3 M-Nb – PAA –PVP 87,0 86,5 375,50 91,0 M-Nb – PAA – PVP 94,4 93,9 101,90 97,6 M-Nb (10 mkg/ml) 91,0 90,6 106,66 97,4 AZT (0,1 mkg/ml) 94,1 93,6 137,64 96,7 Comparison of anti-HIV activity different components microbicide candidates in vitro

  15. Compounds Concen- tration,mkg/ml Cells viability, % Index of cell proli-feration Protection cells from HIV infections, % Inhi-bition р24,% Comparison of anti-HIV activity different components microbicide candidates in vitro M-Nb, рН,5,0 26 66.7 5.9 58.7 31 M-Nb - CL213-LVG 26 69.2 4.1 60.2 67 M-Nb – PAA-PVP 26 26 93.0 4.8 100.0 97 M-Nb -CL213-LVG-PAA-PVP 26 88.8 4.6 91.7 95 M-P, рН,5,0 43.5 3.9 20.9 44 M-P -CL213-LVG 26 78.2 3.4 77.5 72 M-P – PAA-PVP 26 92.4 5.3 100.0 87 M-P - CL213-LVG-PAA-PVP 26 93.4 5.4 100.0 90 M-Nb-P, рН,5,0 26 85.2 4.0 88.9 92 M-Nb-P - CL213-LVG 26 86.4 3.3 90.7 90 M-Nb-P – PAA-PVP 26 93.7 5.7 100.0 99 M-Nb-P -CL213-LVG-PAA-PVP 26 95.0 5.4 100.0 98

  16. Strains Microorganism (normoflora) As 504 As 641 Control LactobacillusDed. 13±3 15±4 14±3 S.lactis 19±4 21±4 17±4 E.coli 15±4 12±3 16±4 Str. faecalis 23±3 25±5 23±4 Staph. albus 27±5 25±4 23±5 Bac. subtilis 26±4 29±5 24±3 Comparison of anti-bacterial activity in vitro Quantity colonies at the presence of different compounds (average for 5 measurements) • No significant differences between original compounds and control in terms of: • Genital saprophyte microorganisms • Rectal saprophyte microorganisms • Transistors microorganisms

  17. Structure and morphology genital mucous (mice) with microbicide compounds at local introduction polymeric matrix (3mg/ml) interpolymeric complex (IPC) (15mg/ml) Peptide-imitator with pharmacophore(3mg/ml) • Tissue structure remains stable does not change the morphology sign • of inflammation or dystrophic changes in during 10 days of • processing genital mucous

  18. Structure and morphology of the mouse organs with microbicide compounds at parenteral introduction Liver, without pathological changes, no sign of inflammation or dystrophic changes (7 day injection M-Nb-P, 150mg/kg) Spleen without pathological changes, no sign of inflammation or dystrophic changes(7 day injection injection M-Nb-P, 150mg/kg)

  19. Summary We have developed a new anti-HIV-1/2 microbicides which is suitable for the preclinical and clinical assessment. Using test in vitro, topical microbicides: Polymeric matrixes components microbicide candidates had of anti-HIV potent activity ≥ 13 g/ml Interpolymeric complex (IPC) components microbicide candidates had of anti-HIV potent activity ≥ 160 g/ml Peptide imitator candidate & pharmacophores microbicide candidates had of anti-HIV potent activity ≥ 1g/ml

  20. Summary We have developed a new non toxic microbicides which is suitable for the preclinical and clinical assessment. Using test in vivo, topical microbicides: Polymeric matrixes components microbicide candidates did not show toxicity both at local (genital, 3mg/ml), and at parenteral introduction to 150 mg/kg at mouse Interpolymeric complex (IPC) components microbicide candidates did not show toxicity at local (genital, 15mg/ml) introduction doses to 750 mg/kg at mouse Peptide imitator candidate & pharmacophores microbicide candidatesdid not show toxicity both at local (genital, 3mg/ml), and at parenteral introduction doses to 150 mg/kg at mouse

  21. *Microbicides Development *Basic Research *Active Pharmaceutical Ingredients (API) *Manufacturing Process *Pilot scale; *Development API *Good Manufacturing Practice (GMP) Production API *scale-up; validation batches *Formulation Development *Preformulation, *physico-chemical *Analytical, * stability *Manufacturing ProcessDevelopment (formulation) *cGMP Production (formulated product) *Preclinical and Clinical Research * - stages and processes that have been completed by the time being, *- stages and processes that we propose to accomplish through innovations or partner project in collaboration.

  22. Acknowledgements SRC VB “Vector”, Koltsovo, Russia: Igor Timofeyev Olga Plyasunova Denis Timofeyev Nikolay Karpyshev Tatyana Ryabicheva Nikolay Varaksin Peter Larionov Yana Kiseleva Vitaliy Nekludov Nataliya Perminova HRDF, Moscow Russia: Alexandr Serbin The authors express gratitude Dr. L. Margolis (NIH, USA) for the given variants (R5 and X4) strains HIV-1 (SF-162 and LAV.04) and opportunity to execute a part of experiments in his laboratory. This work was supported by: BTEP021/ISTC-2175p project as part of the Microbicide Development Programme Poster presentations at Int. Conference “Development of International Collaboration in Infectious Disease Research” - Novosibirsk 2004

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