ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS Timofeyev I.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Karpyshev

ANTI-HIV MICROBICIDES WITH ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE INCLUSION OF PHARMACOPHORE

MODIFIERS AND PSEUDO-LIGANDS MODIFIERS AND PSEUDO-LIGANDS

Timofeyev I.V.,Timofeyev I.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Karpyshev N.N., Ryabicheva T.G., Varaksin N.A., Larionov P.M., Karpyshev N.N., Ryabicheva T.G., Varaksin N.A., Larionov P.M.,

Kiseleva Y.Y., Nekludov V.V., Perminova N.G.Kiseleva Y.Y., Nekludov V.V., Perminova N.G.

SRC Virology & Biotechnology “Vector”, Koltsovo, Novosibirsk SRC Virology & Biotechnology “Vector”, Koltsovo, Novosibirsk region; *region; *HRDF, Moscow, HRDF, Moscow, RussiaRussia

IntroductionIntroduction

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.

AimsAims

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

Development of a new generation of anti-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.

Outcome:

Optimal topical microbicides formula:

we included efficient membrane-acting anti-HIV compounds we included efficient membrane-acting anti-HIV compounds into the pH-dependent interpolymeric complex (IPC) into the pH-dependent interpolymeric complex (IPC)

• PAA – PVP (polyacrylic acid, polyvinylpyrrolidone)

• CL213-LVG (hydrolyzed chitosan, sodium alginate)


O

HOOC COO

COOHOOC

O

HOOC COO

COOHOOC

O

HOOC COO

COOHOOC

O

HOOC COO

COOHOOCNa+

n

CO

NH

(CH2)m

CO

NH

(CH2)n

Peptide imitators co-receptors HIV-1/2(CCR5 or CxCR4)

Evaluation of candidate microbicidesEvaluation of candidate microbicides

1. Microbicide (pretreat 1 hour)

2.Virus(1 hour exposure)

3. Cell culture washed x3 with PBS 4.Cells incubated for 4 days at 37 oC, 5% CO2

Permission cell culture (MT-4 or PBMC)

5.Analysis cell viability, IFA, and p24 HIV-1 production


0

2

4

6

8

10

12

14

As337evk

As337Z

As525evk

As347evk

As347Z

As348Z

As488evk

As488Z

С, м

кг/м

л

IС50

IС90


0.00

20.00

40.00

60.00

80.00

100.00

0.1 1 10 100C, мкг/мл

ЕС

, %

As643 (М-P3) As470+P3 пептид P3


IC50

0

2

4

6

8

10

12

14

As337 As504 As639 As641 As643 As645

C, м

кг/

мл

ВИЧ-1 Z ВИЧ-1 evk


. Formation of complex M-Nb/PAA/PVP

M-NbPAA

M-Nb/PAA/PVPPVP


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

190 290 390 490 590 690

W avelength, nm

Ad

sorp

ban

ce

CL 113

As 646

As 646-CL 113

As 646-CL 113-LVG

LVG

As 646-CL 113-LVGg

Formation of complex As646(M-Nb-P4)/CL113/LVG


Interpolymeric complex (IPC) is stability at рН 4,5-6,0


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

Compounds Cellsviability,

%

Protection cells infections, %

Concentration р24, 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

Comparison of anti-HIV activity different components microbicide candidates in vitro

Compounds Concen-tration, mkg/ml

Cellsviability,

%

Index of cell proli-feration

Protection cells from HIV

infections, %

Inhi-bitionр24, %

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 2626

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

Comparison of anti-bacterial activity in vitroComparison of anti-bacterial activity in vitro

No significant differences between original compounds and control in terms of:1. Genital saprophyte microorganisms2. Rectal saprophyte microorganisms3. Transistors microorganisms

Strains Microorganism (normoflora) As 504 As 641 Control

Lactobacillus Ded. 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

Quantity colonies at the presence of different compounds (average for 5 measurements)

Structure and morphology genital mucous (mice) with Structure and morphology genital mucous (mice) with microbicide compomicrobicide compounds at local introductionunds at local introduction

polymeric matrix (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

Peptide-imitator with

pharmacophore (3mg/ml)

interpolymeric complex (IPC) (15mg/ml)

Structure and morphology of the mouse organs with Structure and morphology of the mouse organs with microbicide compoundsmicrobicide compounds at parenteral introduction 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)

Summary 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 candidateshad of anti-HIV potent activity ≥ 160 g/ml

Peptide imitator candidate & pharmacophores microbicide candidates had of anti-HIV potent activity ≥ 1g/ml

Summary Summary

Using test in vivo, topical microbicides:

We have developed a new non toxic microbicides which is suitable for the preclinical and clinical assessment.

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 candidates did not show toxicity both at local (genital, 3mg/ml), and at parenteral introduction doses to 150 mg/kg at mouse

*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 Process Development (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.

AcknowledgementsAcknowledgements

SRC VB “Vector”, Koltsovo, Russia:

Igor TimofeyevOlga PlyasunovaDenis TimofeyevNikolay Karpyshev Tatyana Ryabicheva Nikolay Varaksin Peter LarionovYana KiselevaVitaliy Nekludov Nataliya Perminova

This work was supported by:BTEP021/ISTC-2175p project as part of the Microbicide Development Programme

HRDF, MoscowRussia:

Alexandr Serbin

Poster presentations at Int. Conference “Development of International Collaboration in Infectious Disease Research” - Novosibirsk 2004

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.

Documents

ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS Timofeyev I.V., Plyasunova O.A., Timofeyev D.I., *Serbin A.V., Karpyshev