ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS Timofeyev I.V.,...
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Transcript of ANTI-HIV MICROBICIDES WITH INCLUSION OF PHARMACOPHORE MODIFIERS AND PSEUDO-LIGANDS Timofeyev I.V.,...
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)
Development of a new generation of anti-HIV preparations
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
Development of a new generation of anti-HIV preparations
0
2
4
6
8
10
12
14
As337evk
As337Z
As525evk
As347evk
As347Z
As348Z
As488evk
As488Z
С, м
кг/м
л
IС50
IС90
Development of a new generation of anti-HIV preparations
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
Development of a new generation of anti-HIV preparations
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
Development of a new generation of anti-HIV preparations
. Formation of complex M-Nb/PAA/PVP
M-NbPAA
M-Nb/PAA/PVPPVP
Development of a new generation of anti-HIV preparations
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
Development of a new generation of anti-HIV preparations
Interpolymeric complex (IPC) is stability at рН 4,5-6,0
Development of a new generation of anti-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
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.