Transcript Development of a new generation of anti

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
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.
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
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.
Development of a new generation of anti-HIV
preparations
O
Outcome:
HOOC
O
HOOC
COO
COO
HOOC
O
HOOC
COO
COO
HOOC
O
HOOC
COO
COO
HOOC
HOOC
COO
CO
CO
NH
NH
(CH2)m
(CH2)n
COO
Na+n
Peptide imitators coreceptors HIV-1/2
(CCR5 or CxCR4)
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)
Evaluation of candidate microbicides
1. Microbicide
(pretreat 1 hour)
2.Virus
(1 hour exposure)
Permission cell culture
(MT-4 or PBMC)
5.Analysis cell viability, IFA,
and p24 HIV-1 production
3. Cell culture washed x3 with PBS
4.Cells incubated for 4 days at 37 oC, 5% CO2
Development of a new generation of anti-HIV
preparations
14
12
С, мкг/мл
10
8
6
4
2
0
As337 As337 As525 As347 As347 As348 As488 As488
evk
Z
evk
evk
Z
Z
evk
Z
IС50
IС90
Development of a new generation of anti-HIV
preparations
100.00
ЕС, %
80.00
60.00
40.00
20.00
0.00
0.1
1
10
100
C, мкг/мл
As643 (М-P3)
As470+P3
пептид P3
Development of a new generation of anti-HIV
preparations
14
12
C, мкг/мл
10
8
6
4
2
0
As337
As504
IC 50
As639
ВИЧ-1 Z
As641
ВИЧ-1 evk
As643
As645
Development of a new generation of anti-HIV
preparations
PAA
M-Nb
.
PVP
M-Nb/PAA/PVP
Formation of complex M-Nb/PAA/PVP
Development of a new generation of anti-HIV
preparations
1.8
CL 113
As 646
1.6
As 646-CL 113
As 646-CL 113-LVG
1.4
LVG
As 646-CL 113-LVGg
Adsorpbance
1.2
1
0.8
0.6
0.4
0.2
0
190
290
390
490
590
W avelength, nm
Formation of complex As646(M-Nb-P4)/CL113/LVG
690
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
Comparison of anti-HIV activity different components
microbicide candidates in vitro
Cells
viability,
%
62,5
Protection
cells infections,
%
Concentration
р24, ng/ml
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
91,0
90,6
106,66
97,4
94,1
93,6
137,64
96,7
Compounds
PAA – PVP
M-Nb (10 mkg/ml)
AZT (0,1 mkg/ml)
Inhibition
p24 HIV-1, %
Comparison of anti-HIV activity different
components microbicide candidates in vitro
Compounds
Concentration,
mkg/ml
Cells
viability,
%
Index of
cell proliferation
Protection cells
from HIV
infections, %
Inhibition
р24, %
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
93.0
4.8
100.0
97
M-Nb - CL213-LVG-PAA-PVP
26
26
88.8
4.6
91.7
95
M-P, рН,5,0
26
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 vitro
Strains
Microorganism
(normoflora)
Quantity colonies at the presence of different
compounds (average for 5 measurements)
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
No significant differences between original compounds and control in terms of:
1. Genital saprophyte microorganisms
2. Rectal saprophyte microorganisms
3. Transistors microorganisms
Structure and morphology genital mucous (mice) with
microbicide compounds at local introduction
polymeric matrix
(3mg/ml)
interpolymeric complex (IPC)
Peptide-imitator with
(15mg/ml)
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
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)
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
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 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.
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