Combinations of a caspase inhibitor and an antiviral agent

Abstract

A compound is provided which has a structure I: A-B-C and a method for administering the compound is also provided for use in the prophylaxis and/or treatment of a viral infection, and in particular for preventing and/or inhibiting viral replication, in which A is a quinoline or quinoline-like group, B is a sole amino acid or a peptide or polypeptide having a given amino acid sequence, and C is an O-phenoxy group. According to one embodiment, the compound is a protease inhibitor such as a caspase inhibitor, and the inhibitor can be Q-VD-OPh (N-(2-(quinolyl)valylaspartyl-(2,6-difluorophenoxyl)methyl ketone), optionally in an O-methylated form. Antiviral compositions and kits are also provided.

Claims

1. A combination comprising: a) a caspase inhibitor having the structure: ##STR00004## wherein: B is a single amino acid or a peptide or polypeptide having a given amino acid sequence, R1 and R2 are selected from a hydrogen, an alkyl, an alkoxy, a fluoro, a chloro, a carboxy, a carbonyl, an arylcarbonyl, and an amino, and R3 and R4 are selected from hydrogen, an alkyl, an alkoxy, a fluoro, a chloro, a carboxy, a carbonyl, an arylcarbonyl, and an amino; and b) an anti-HIV agent comprising at least one HIV reverse transcriptase inhibitor or HIV protease inhibitor.

2. The combination according to claim 1, wherein B is a peptide or polypeptide.

3. The combination according to claim 1, wherein the single amino acid is an aspartic acid (D) or the peptide or polypeptide comprises at least one aspartic acid.

4. The combination according to claim 3, wherein the peptide or polypeptide comprises an aspartic acid (D) and a valine (V).

5. The combination according to claim 3, wherein the amino acid sequence of the peptide is valine-aspartic acid (VD) or the sequence valine-alanine-aspartic acid.

6. The combination according to claim 1, wherein the single amino acid or at least one of the amino acids of said peptide or polypeptide is O-methylated.

7. The combination according to claim 1, wherein said caspase inhibitor is O-methylated on the single aspartic acid or on at least one of the aspartic acids of said peptide or polypeptide.

8. The combination according to claim 1, wherein the single amino acid or the amino acid sequence of said peptide or polypeptide is not O-methylated.

9. The combination according to claim 1, wherein R1 or R2 is a hydrogen.

10. The combination according to claim 1, wherein R1 and R2 are a hydrogen.

11. The combination according to claim 1, wherein R3 or R4 is a fluoro.

12. The combination according to claim 1, wherein R3 and R4 are a fluoro.

13. The combination according to claim 1, wherein the caspase inhibitor is N-(2-quinolyl)valyl-O-methyl-aspartyl-(2,6-difluorophenoxy)methyl ketone or N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone.

14. The combination according to claim 13, wherein the caspase inhibitor is N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone.

15. The combination according to claim 1, wherein the anti-HIV agent comprises at least one HIV reverse transcriptase inhibitor.

16. The combination according to claim 15, wherein the HIV reverse transcriptase inhibitor is selected from the group consisting of zidovudine or azidothymidine (AZT), didanosine or ddl, zalcitabine or ddC, stavudine or d4T, lamivudine or 3TC, abacavir or ABC, emtricitabine or FTC, nevirapine, efavirenz, delavirdine and tenofovir or bis-POC-PMPA.

17. The combination according to claim 16, wherein the HIV reverse transcriptase inhibitor is AZT.

18. The combination according to claim 1, wherein the anti-HIV agent comprises at least one HIV protease inhibitor.

19. The combination according to claim 18, wherein the HIV protease inhibitor is selected from the group constituted by the following peptidomimetic molecules: Indinavir or IDV, Nelfinavir or NLFN, Saquinavir or SQN, Ritonavir or RTN, Amprenavir, Lopinavir.

20. The combination according to claim 19, wherein the HIV protease inhibitor is Indinavir.

21. The combination according to claim 13, wherein the anti-HIV agent comprises at least one HIV reverse transcriptase inhibitor.

22. The combination according to claim 21, wherein the HIV reverse transcriptase inhibitor is selected from the group consisting of zidovudine or azidothymidine (AZT), didanosine or ddl, zalcitabine or ddC, stavudine or d4T, lamivudine or 3TC, abacavir or ABC, emtricitabine or FTC, nevirapine, efavirenz, delavirdine and tenofovir or bis-POC-PMPA.

23. The combination according to claim 22, wherein the HIV reverse transcriptase inhibitor is AZT.

24. The combination according to claim 13, wherein the anti-HIV agent comprises at least one HIV protease inhibitor.

25. The combination according to claim 24, wherein the HIV protease inhibitor is selected from the group constituted by the following peptidomimetic molecules: Indinavir or IDV, Nelfinavir or NLFN, Saquinavir or SQN, Ritonavir or RTN, Amprenavir, Lopinavir.

26. The combination according to claim 25, wherein the HIV protease inhibitor is Indinavir.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

(1) FIG. 1: Immunoblot (Western blot) showing the effectiveness of Q-VD-OPh in inhibiting activation of caspase-3 and -8. CD4.sup.+ T lymphocytes were or were not infected by the HIV-1 virus (incubation: 2 hours) and then stimulated by Concanavalin A and IL-2 (incubation: 2 hours). After stimulation, the inhibitor Q-VD-OPh was added to the cell culture at a final concentration of 10 μM and then also added 36 hours after infection (d3) and 96 hours after infection (d4). The immunoblot was carried out on the 6th day post-infection. Caspase-3 and -8 were detected using the anticaspase antibodies indicated above. NI: control cells (CD4.sup.+ T cells not infected by HIV-1 and not treated with Q-VD-OPh); HIV: CD4.sup.+ T cells infected by HIV-1 but not treated with Q-VD-OPh; HIV+QVD: CD4.sup.+ T cells infected by HIV-1 and then cultivated in the presence of 10 μM of Q-VD-OPh. Actin serves as the control for the protein load of the gel. The molecular weight marker RPN 800 (Amersham) was used.

(2) FIG. 2: Analysis of the CD4+ T lymphocytes by flow cytometry on the 5th day after infection by HIV-1 and after stimulation by Concanavalin A and IL-2. A. Analysis of the size and granulometry of the CD4+ T lymphocytes. B. Determination of the percentage of CD4+ cells having a mitochondrial impairment (ΔΨm). C. Determination of the percentage of CD4+ cells infected by HIV after intracellular labelling of the p24 viral antigen. NI: uninfected CD4+ T lymphocytes; HIV: CD4+ T lymphocytes infected by HIV-1; HIV Q-VD-OPh: CD4+ T lymphocytes infected by HIV-1 (incubation: 2 hours), activated by ConA and IL2 (incubation 2 hours) and then incubated with Q-VD-OPh (10 μM final); Q-VD-OPh is added 36 hours (day 3) and 96 hours (day 4) after the start of the infection.

(3) FIG. 3: Immunoblot (Western blot) showing the effect of Q-VD-OPh on mitochondrial damage resulting from viral replication. Primary CD4+ T cells were infected with HIV-Lai and stimulated with a ConA/IL-2 cocktail and then treated or not treated with 10 μM of Q-VD-OPh (QVD) and/or 10 μM of pepstatin A (PA) immediately after stimulation and then 36 hours (day 3) and 96 hours (day 4) after infection. On the 5th day post-infection, the cells were fractionated into two parts: the membrane fraction and the cytosol fraction. The relocalization of the apoptogenic factors Cytochrome c (Cyt c), Smac/Diablo and Endonuclease G (EndoG) was then evaluated by immunotransfer (Western blot). The proteins Cytochrome c, Smac/Diablo and EndoG were detected using the antibodies indicated above. The proteins Cox IV and actin detected by means of the anti-subunit IV antibodies (clone 1068 Molecular Probe) and anti-actin antibodies (SIGMA), respectively, were used as the control for the fractionation procedure and as the control for the protein load of the gel, respectively.

(4) FIG. 4: Graphic representation showing the effect of Q-VD-OPh on replication of the HIV-1 virus. A. Determination of the percentage of CD4+ T lymphocytes infected by HIV-1 by flow cytometry, after intracellular labelling of the p24 viral antigen. B. ELISA assay of the p24 viral antigen in the culture supernatants. All the cells were stimulated by Con A and IL2 within 24 hours of infection. The parameters were measured on the 6th day post-infection. NI: uninfected CD4+ T lymphocytes; HIV: CD4+ T lymphocytes infected by HIV-1; HIV+QVD and HIV+PA+QVD: CD4+ T lymphocytes infected by HIV-1, to which there were administered, on the 3rd day post-infection and then every day, Q-VD-OPh (QVD) or Q-VD-OPh (QVD) and pepstatin A (PA), respectively.

(5) FIG. 5: The time at which Q-VD-OPh is administered to the CD4+ T lymphocytes influences the inhibition of cell death and the inhibition of viral replication. CD4+ T lymphocytes were or were not infected with HIV-Lai and stimulated with a ConA/IL2 cocktail and then treated or not treated with 10 μM of Q-VD-OPh and/or 10 μM of pepstatin A. There were determined, by flow cytometry, the percentage of CD4+ T lymphocytes infected by HIV after intracellular labelling of the p24 antigen (A) and the percentage of dead CD4+ T lymphocytes (B) on the 5th day after infection by HIV-1. The parameters were measured on the 5th day post-infection. NI: CD4+ T lymphocytes not infected by HIV-1; HIV: CD4+ T lymphocytes infected by HIV-1; Q-VD: CD4+ T lymphocytes infected by HIV-1, to which there were administered Q-VD-OPh and pepstatin A (PA); in the first case (Before), Q-VD-OPh and PA were administered between 1 and 2 hours before infection by HIV-1 and stimulation by Concanavalin A and IL-2 and then on d3 and d4 post-infection, at a final concentration of 10 μM. In the second case (After), Q-VD-OPh and PA were administered immediately after infection by HIV-1 and on d3 and d4 post-infection at different final concentrations (0.5; 1; 2; 5; 10 and 20 μM).

(6) FIG. 6: Immunoblot (Western blot) showing the inhibitory effect of Q-VD-OPh on the expression of a group of HIV-1 proteins in primary CD4+ cells. Protein extracts prepared on the 6th day of culturing from uninfected CD4+ T lymphocytes (NI) or CD4+ T lymphocytes infected by the HIV-1 virus and then cultivated in the absence of Q-VD-OPh (HIV) or in the presence of Q-VD-OPh (HIV+Q-VD-OPh) were fractionated into three parts: the cytosol fractions (cytosol), the soluble membrane fractions (soluble) and the insoluble membrane fractions (insoluble). Q-VD-OPh was added at a final concentration of 10 μM, immediately after infection and then 36 hours (d3) and 96 hours (d4) post-infection. The proteins specific to HIV were detected using a mixture of sera from HIV+ patients. They are indicated on the right of the immunoblot. The molecular weight marker RPN 800 (Amersham) was used.

(7) FIG. 7: Graphic representation showing the effect of Q-VD-OPh on primary HIV-1 strains. In all cases (A, B and C), the CD4+ T lymphocytes were stimulated with concanavalin A and IL2 (stimulation being carried out before or during administration of Q-VD-OPh), and Q-VD-OPh was added at a final concentration of 10 μM, immediately after infection and then 36 hours (d3) and 96 hours (d4) post-infection. A. Analysis by flow cytometry of viral replication in CD4+ T lymphocytes infected with the HIV-1Lai virus or with the serum of a chronic HIV+ patient (strain with X4/R5 dual tropism) and then cultivated in the absence or presence (+Q-VD) of Q-VD-OPh. The percentage of CD4+ cells infected by HIV was determined after intracellular labelling of the p24 viral antigen on the 5th day post-infection in the case of the HIV-Lai virus and on the 6th day post-infection in the case of the serum. PE: phycoerythrin, fluorochrome (emission at 578 nm) coupled to the anti-p24 antibody. B. Quantification of viral replication (on the left) and of cell death by measurement of mitochondrial depolarization (on the right) in CD4+ T lymphocytes infected with the serum of the chronic HIV+ patient. C. Analysis of the replication of five viral isolates from HIV+ patients, with R5 tropism, in the absence (−) or in the presence (+) of Q-VD-OPh or of a final concentration of 1 μM of didanosine (ddl, SIGMA), a reverse transcriptase inhibitor also called Vivex EC® (Bristol-Myers Squibb).

(8) FIG. 8: Graphic representation showing the inhibitory effect of Q-VD-OPh on replication of the SIVmac251 virus and the formation of syncitia. The cell line CEMx174 was infected with 200 AID50 (infectious dose necessary for 50% of the animals to be infected) of the strain SIVmac251. Two hours after infection, the cell line was treated with different concentrations of inhibitor Q-VD-OPh (final concentration of 20, 10 or 2.5 μM) or was not treated (0 μM). On the 4th and 5th days post-infection, viral production in the culture supernatant was evaluated by RT-PCR (Taqman) and the number of syncytia was evaluated using an optical microscope.

(9) FIG. 9: Graphic representation showing a synergistic effect of Q-VD-OPh with AZT and Indinavir. Primary CD4+ T cells infected by the HIV-Lai virus and then stimulated with concanavalin A and IL-2 were treated 96 hours after infection with different concentrations of Q-VD-OPh (0, 0.1, 1 and 10 μM) in the absence or presence of azidothymidine (AZT; 0.1 μM) or Indinavir (IND; 1 μM). Inhibition of cell death was quantified by flow cytometry on the 5th day post-infection. It is expressed as follows:
(% cell death induced by HIV in the absence of treatment−% cell death induced by HIV in the presence of treatment)/(% cell death induced by HIV in the absence of treatment−% cell death in the control)×100.

(10) FIG. 10. Analysis by flow cytometry of the fall in the mitochondrial transmembrane potential (% Δ-φm low) starting from Jurkat cells incubated in the absence or presence of different concentrations of anti-CD95 (A) and in the absence or presence of 0.25 μg/ml of anti-CD95 and of different caspase inhibitors (B). C. Analysis by immunoblot of Jurkat cell extracts treated in the presence or absence of anti-CD95 and of different caspase inhibitors.

(11) FIG. 11. Analysis by flow cytometry of the internal viral protein p24 (A) and of the fall in the mitochondrial transmembrane potential (% Δφm low; B) on the 5th (d5) and 7th (d7) day post-infection in primary CD4+ T cells infected by the strain HIV-Lai, after stimulation by Concanavalin A and IL-2 and in the presence or absence of 10 μM of different caspase inhibitors.

(12) FIG. 12. Analysis by immunoblot of the quantity of HIV viral proteins produced from extracts of primary CD4 T cells infected by the strain HIV-Lai in the presence or absence of different caspase inhibitors.

(13) FIG. 13. Analysis by flow cytometry of lymphocyte proliferation after 4 and 5 days' stimulation by 1 μg/ml of anti-CD-3, in the presence or absence of HIV antiproteases or of QVD-OPH.

(14) FIG. 14. Analysis of the fall in the mitochondrial transmembrane potential (% Δφm low) by flow cytometry starting from cells stimulated by 1 μg/ml of anti-CD-3 in the presence or absence of different HIV antiproteases and of QVD-OPH.

(15) FIG. 15. Analysis of the fall in the mitochondrial transmembrane potential (% Δφm low) by flow cytometry in monocytes and lymphocytes in the presence of different HIV antiproteases or of QVD-OPH.

EXAMPLES

Example 1: Analysis of the Properties of Q-VD-OPH

A. Material and Methods

(16) Antibodies

(17) For the immunoblots (Western blot): anti-Smac/Diablo rabbit polyclonal antibodies (ΨProSci), anti-endonuclease-G (ΨProSci), anticaspase-3 (Stressgen), antiactin (Sigma), anticaspase-8 monoclonal antibodies (Cell Signaling), anti-Cytochrome c clone 7H78.2C12 (BD Pharmingen), anti-Cox IV, subunit IV, clone 10G8 (Molecular Probes).

(18) For cytofluorometry: anti-p24 monoclonal antibody, clone KC57-RD1 (Beckman coulter).

(19) Synthetic Inhibitors

(20) Cathepsin D inhibitor: pepstatin A (Sigma).

(21) Broad-spectrum caspase inhibitor: Q-VD-OPh in non-O-methylated form (N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxyl)methyl ketone); Enzyme System Products, MP Biomedicals.

(22) Reverse transcriptase inhibitor: didanosine (or ddl) or Videx EC® (Bristol-Myers Squibb).

(23) Isolation of CD4.sup.+ Cells and Culture Conditions

(24) Peripheral blood mononuclear cells obtained from healthy volunteers (Etablissement Français du sang) were isolated on Ficoll strains (Petit et al., 2002). The majority of the adherent cells were eliminated by incubation in plastics culture dishes. The circulating CD4.sup.+ cells were selected negatively using a CD4.sup.+ cell isolation kit, in accordance with the supplier's instructions (MACS, CD4 T cell isolation kit II; Miltenyi Biotech, Paris, France). The purity of the isolated CD4.sup.+ population, determined by flow cytometry, was ≧96%. Monocytes recovered from the adherent dishes were added to the purified CD4.sup.+ cells in a final percentage of 6%. The composition of the culture medium used is as follows: RPMI 1640, 10% foetal calf serum, 2 mM glutamine, 1 mM pyruvate, 50 units/ml of penicillin and 50 μg/ml of streptomycin.

(25) Measurement of Viral Replication

(26) The CD4.sup.+ cells were incubated for 2 hours at 37° C. in the presence of 10 ng/ml of HIV-1 virus of strain Lai or 50 ng/ml of primary strains of the HIV-1 virus. After 2 washings, the cells were resuspended in a complete medium in the presence of 5 μg/ml of concanavalin A (Con A) and 10 mg/ml of interleukin 2 (IL 2). The HIV p24 antigen, the control for the viral load, was measured in the cell culture supernatants by an ELISA test (Abbott). The intracellular p24 antigen was determined by flow cytometry with the aid of a specific antibody (KC57, Coulter Corp) and after permeabilization of the cells using the permeabilization reagent Intraprep (Coulter Corp.).

(27) Measurement of Cell Death and Analysis by Flow Cytometry

(28) In order to evaluate the change in the transmembrane potential of the inner mitochondrial membrane (Δφm), the CD4.sup.+ cells were labelled for 15 minutes at 37° C. with 40 nM of DIOC.sub.6 (3-3′-diethyloxacarbocyanine). The dead cells exhibit a reduction in labelling intensity. The size and morphometry were also determined. The apoptotic/dead cells were counted by white-light microscopy, on the basis of abnormal cell morphology and/or the absorption of trypan blue.

(29) Immunoblot (Western Blot)

(30) 20 μg extracts of each of the cytosol and mitochondrial fractions were boiled for 5 minutes in Laemmli buffer containing 2% SDS and 10% 2-β-mercaptoethanol and then migrated onto 10-20% polyacrylamide gradient gels (Bio-Rad). After transfer of the proteins to a polyvinylidene difluoride membrane (Bio-Rad), the immunoblots were incubated with the primary and post-secondary antibodies coupled to horseradish peroxidase, obtained from Amersham Biosciences (Orsay, France). They were subsequently developed and revealed by chemiluminescence (ECL from Amersham or West Femto from Pierce) using a CCD camera (Fuji LAS-1000plus) and L process software from Science Lab (Isochem, Paris, France).

(31) Subcellular Fractionation

(32) The cytosol and mitochondrial fractions were obtained by the subcellular fractionation technique based on selective permeabilization by digitonin according to Foghsgaard et al. {Foghsgaard, 2001}. Briefly, 10.sup.7 cells were washed twice in PBS and incubated for 5 minutes on ice with 100 μl of extraction buffer (35 μg/ml digitonin, 250 mM sucrose, 137 mM NaCl, 70 mM KCl, 4.3 mM Na.sub.2HPO.sub.4, 1.4 mM KH.sub.2PO.sub.4, 2 mM EDTA, pH 7.2) supplemented with a protease inhibitor cocktail (“Complete” from Roche Applied Science, Penzberg, Germany). The extracts were centrifuged at 300 g for 5 minutes, and the resulting supernatant was recentrifuged again at 10,000 g for 10 minutes at 4° C. in order to remove the debris. The final supernatant, called the cytosol fraction, was stored at −80° C. The pellet was dissolved in 100 μl of mitochondrial lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 0.5% NP40, 0.2% Triton X100) supplemented with “Complete” protease inhibitor cocktail from Roche, for 30 minutes on ice at 4° C., followed by centrifugation at 10,000 g for 30 minutes at 4° C. in order to obtain the so-called “soluble” mitochondrial fraction. The protein concentration of the cytosol and mitochondrial fractions was determined by the bicinchoninic acid (BCA) method (Bio-Rad).

B. Results

(33) B-1. Q-VD-OPh Inhibits Activation of Caspases-3 and -8.

(34) The effectiveness of Q-VD-OPh in inhibiting activation of caspase-3 and -8 was analyzed by immunoblot (Western blot). CD4.sup.+ T lymphocytes in culture were infected by the HIV-1 virus and then stimulated, 2 hours after infection, by Concanavalin A and IL-2. Following stimulation, 10 μM of inhibitor Q-VD-OPh were added to the cell culture. Q-VD-OPh was added again 36 hours (3 days) and then 96 hours (4 days) after infection, still at a final concentration of 10 μM. Protein extraction and immunoblot were carried out on the 6th day post-infection.

(35) The results obtained (see FIG. 1) show that, in the absence of viral infection, caspase-3 and -8 are substantially in forms p32 and p55, respectively, which correspond to the inactive forms (proforms) of caspase-3 and -8. The intermediate forms p20 for caspase-3 and p44, p26 and p20 for caspase-8 are also found (Alam et al., 1999; Petit et al., 2002).

(36) When the CD4+ cells were infected by HIV-1 and then cultivated in the absence of inhibitor Q-VD-OPh, it is found, on the 6th day post-infection, that the proforms p32 and p55 are detected only weakly, while the intermediate forms are present in a larger amount. Moreover, the presence of forms p17 and p18, which correspond to the active forms (apoptogenic forms) of caspase-3 and -8, respectively, is detected. This reflects the activation of caspase-3 and -8 by proteolysis following the viral infection.

(37) When the CD4+ cells were infected by the HIV-1 virus and cultivated in the presence of the inhibitor Q-VD-OPh, inhibition of the proteolytic degradation of caspase-3 and -8 is observed; the active forms p17 and p18 are not detected, while the intermediate forms and the proforms p32 and p55 are detected much more strongly than when no inhibitor is administered to the cells infected by HIV. Consequently, the use of the inhibitor Q-VD-OPh has the effect of inhibiting the proteolytic degradation of the proforms of caspase-3 and -8 and accordingly of blocking the activation of caspase-3 and -8.

(38) B-2. Q-VD-OPh Inhibits Cell Death Caused by HIV-1 Infection.

(39) The inventors evaluated the effect of the compound Q-VD-OPh on cell death caused by HIV-1 infection and on viral replication by analyzing CD4+ T cells infected by the HIV-1 virus and then stimulated by Concanavalin A and IL-2 and incubated or not incubated in the presence of Q-VD-OPh for 5 days (FIG. 2). Analysis of the size and granulometry of the CD4+ T cells (FIG. 2A) as well as the percentage of CD4+ cells exhibiting mitochondrial depolarization, which is characteristic of cell death (FIG. 2B), shows that HIV-1 infection is accompanied by a pronounced increase in cell death of the CD4+ T cells (63.1% compared with 13.3% for the untreated cells). On the other hand, when the CD4+ T cells were incubated with the inhibitor Q-VD-OPh after having been infected by HIV-1, only a slight increase in cell death of the CD4+ T cells is observed (19.8% as compared with 13.3% for the untreated cells). These results show that Q-VD-OPh is a potent inhibitor of cell death resulting from HIV-1 infection, unlike other broad-spectrum caspase inhibitors, such as zVAD-kmk, which prevents the apoptotic phenotype (condensation and fragmentation of the nuclear chromatin) in cells infected by HIV but does not prevent either mitochondrial depolarization or cell death (Petit et al., 2002).

(40) B-3. Q-VD-OPh Inhibits Apoptogenic Mitochondrial Damage Caused by Viral Replication.

(41) The inventors analyzed, by immunotransfer (Western blot), the effect of Q-VD-OPh on the release of apoptogenic mitochondrial factors caused by viral replication. Primary CD4+ T cells were infected with the HIV-Lai virus and then stimulated with a concanavalin A/IL-2 cocktail before being treated with Q-VD-OPh (10 μM) and/or pepstatin A (10 μM). On day 5 after infection, the cells were fractionated into two parts: (i) the mitochondrial membrane fraction and (ii) the cytosol fraction, which may contain mitochondrial factors released following permeabilization of the mitochondrial membrane caused by the viral infection.

(42) Analysis of the location of the apoptogenic factors Cytochrome C, Smac/Diablo and Endonuclease G (EndoG) (FIG. 3) shows the presence of these apoptogenic factors in the cytosol fraction of the cells infected by HIV-Lai, whereas in the uninfected cells they are located in the mitochondrial membrane fraction, which is the indicator of mitochondrial damage. This suggests that the massive death of the CD4+ T cells observed following an HIV infection is the result of a mechanism of cell death which passes through a loss of permeability of the mitochondrial membrane and a spreading of apoptogenic factors into the cytosol.

(43) In addition, while the use of pepstatin A appears to have no effect after 5 days on the mitochondrial damage caused by the viral infection, the use of Q-VD-OPh enables the presence of the apoptogenic factors Cytochrome C, Smac/Diablo and EndoG in the cytosol fraction to be reduced very considerably; the presence of Cytochrome C is even almost zero. These results show that Q-VD-OPh allows the mitochondrial damage caused by viral replication to be reduced considerably.

(44) B-4. Q-VD-OPh Inhibits Replication of the HIV-1 Virus.

(45) In order to determine the effect of the compound Q-VD-OPh on viral replication, the replication of the HIV-1 virus in the presence or absence of Q-VD-OPh was evaluated by quantification of the number of TCD4+ lymphocytes expressing the p24 antigen in CD4+ T lymphocytes infected by HIV and stimulated by ConA and IL-2. Analysis of the percentage of CD4+ T lymphocytes infected by HIV-1 on the 5th and 6th days post-infection (FIGS. 2C and 4A, respectively) and of the amount of the p24 viral antigen in the culture supernatants on the 6th day post-infection (FIG. 4B) show that the compound Q-VD-OPh reduces viral replication by more than 75%. The inhibition of viral replication is even stronger when the CD4+ T lymphocytes are treated both with the compound Q-VD-OPh and with another protease inhibitor, pepstatin A (pepsin inhibitor).

(46) FIGS. 2C and 4A show the count of cells expressing the p24 viral antigen by immunological labelling and flow cytometry. Five days after infection, 72.6% of the cells express the viral antigen and are therefore infected. If the infection is followed by the addition of Q-VD-OPh, only 18% of the cells are infected five days after infection.

(47) B-5. The Inhibitory Effect of Q-VD-OPh on Cell Death Caused by Viral Infection and on Viral Replication is Stronger, the Earlier it is Administered.

(48) In order to determine if the time at which the compound O-VD-OPh is administered has an impact on the inhibition of cell death caused by HIV-1 infection and on the inhibition of viral replication, Q-VD-OPh was administered to CD4+ T lymphocytes in culture either before infection with HIV-1 or after. In each case there was determined, by flow cytometry, on the 5th day post-infection, the percentage of CD4+ T lymphocytes infected by the virus by intracellular labelling of the p24 antigen (FIG. 7A) and the percentage of dead CD4+ lymphocytes (FIG. 7B).

(49) The results obtained using increasing concentrations of Q-VD-OPh (0.5; 1; 2; 5; 10 and 20 μM) administered to the CD4+ T lymphocytes after infection by HIV-1 show that the inhibition of viral replication and the inhibition of cell death caused by the viral infection are dose-dependent.

(50) The fact that a high concentration of inhibitor (20 μM) is accompanied by pronounced inhibition of cell death in response to the viral infection also emphasizes the fact that the inhibitor Q-VD-OPh is not only absolutely non-toxic for CD4+ T cells but, on the contrary, promotes survival of CD4+ T cells infected by the HIV-1 virus. Moreover, it is possible that Q-VD-OPh blocks the death of uninfected CD4+ cells (bystander effect; Hurtrel et al., 2005).

(51) Furthermore, when Q-VD-OPh is added before infection by HIV-1 (at a final concentration of 10 μM), inhibition of viral replication and inhibition of cell death are even greater than when Q-VD-OPh is added before infection by HIV-1 (in a concentration of from 0.5 to 20 μM). This shows that the effect of the inhibitor Q-VD-OPh on the viral infection and on the consequences of the viral infection is greater, the earlier it is administered. Accordingly, the inhibitor Q-VD-OPh may be used not only to treat a viral infection but also prophylactically, in order to prevent a viral infection.

(52) B-6. Q-VD-OPh Inhibits the Expression of a Group of HIV-1 Proteins in Primary CD4+ T Cells.

(53) The inventors analyzed the expression, on the 6th day post-infection, of the HIV-1 proteins in the cytosol fractions, the soluble membrane fractions and the insoluble membrane fractions of CD4+ T lymphocytes infected by the HIV-1 virus and then incubated in the presence or absence of the compound Q-VD-OPh. The expression profile obtained (see FIG. 6) shows that the inhibitor Q-VD-OPh drastically reduces the totality of HIV proteins expressed in the cytosol and in the membrane fractions of the primary CD4+ T lymphocytes. On the other hand, the presence of inhibitors does not cause different compartmentalization of the proteins nor an accumulation of the proforms. Q-VD-OPh therefore has an inhibitory effect for the expression of the totality of the viral genome but has no effect on intracellular protein traffic and therefore on the maturation of the proteins.

(54) B-7. Q-VD-OPh Inhibits the Replication of Primary HIV-1 Strains.

(55) Viral replication was analyzed by flow cytometry in CD4+ T lymphocytes infected by the HIV-1 Lai virus or with the serum of a chronic HIV+ patient containing a primary strain with X4/R5 dual tropism.

(56) In the case of the HIV-1 Lai virus, as in the case of the serum of a chronic HIV+ patient, it is noted that, when the lymphocytes infected by the virus were cultivated in the presence of the compound Q-VD-OPh, viral replication is strongly inhibited (FIG. 5A). 3.79% of infected cells treated with Q-VD-OPh, as compared with 36.46% of the cells not treated with Q-VD-OPh in the case of an infection by the strain LAI and 0.06% of infected cells treated with Q-VD-OPh in the case of an infection by a primary strain.

(57) Moreover, a quantitative analysis of cell death by measurement of mitochondrial depolarization, and of viral replication by ELISA assay of the p24 viral antigen in CD4+T lymphocytes infected with the serum of the chronic HIV+ patient show that Q-VD-OPh also causes strong inhibition of cell death (total or almost total inhibition) and of viral replication (FIG. 5B) on a primary isolate of the HIV-1 retrovirus.

(58) The inventors further analyzed the replication of five primary HIV-1 isolates taken from HIV+ patients, of R5 tropism, in the absence or presence of the inhibitor Q-VD-OPh or of ddl, a reverse transcriptase inhibitor. It is noted that, for the five primary isolates, Q-VD-OPh and ddl have a similar effect, namely strong inhibition of viral replication (see FIG. 5C). The viral strains with R5 tropism are those which are found most commonly and have the characteristic of emerging early during the infection and of persisting throughout the evolution of the disease, whereas the viral strains with X4 tropism tend to be late strains. The ability of Q-VD-OPh to inhibit viral replication more particularly of the viral strains with R5 tropism renders this molecule very interesting from a therapeutic point of view because it may be used to stop a viral infection at a very early stage.

(59) B-8. Q-VD-OPh Inhibits Replication of the SIVmac251 Virus and the Formation of Syncytia.

(60) In order to evaluate the field of application of the antiviral properties of the inhibitor Q-VD-OPh, the inventors analyzed the effect of Q-VD-OPh on replication of the SIVmac251 virus and on the formation of syncytia (FIG. 8). The cell line CEMx174 was infected with a strong dose of virus of the strain SIVmac251 (200 AID50), which corresponds to ten times the concentration used to infect monkeys. Analysis of viral production in the culture supernatant by RT-PCR on the 4th and 5th days post-infection shows that replication of the SIVmac251 virus is inhibited in the presence of the inhibitor Q-VD-OPh, in a dose-dependent manner. Moreover, when a high concentration of inhibitor (20 μM) is administered, viral replication is inhibited by more than 70%. Consequently, it appears that Q-VD-OPh inhibits replication of the HIV and SIV viruses in a similar manner.

(61) In addition, the formation of syncytia, a characteristic which would be linked to greater or lesser virulence, is likewise strongly inhibited under the effect of the inhibitor Q-VD-OPh. However, a concentration of Q-VD-OPh equal to or greater than 10 μM is required to obtain a 70% reduction in the number of syncytia 5 days after infection and treatment.

(62) These results show that Q-VD-OPh is a broad-spectrum viral replication inhibitor which allows not only HIV viruses but also SIV viruses to be blocked.

(63) B-9. Q-VD-OPh Acts in Synergy with AZT and Indinavir to Inhibit Viral Replication and Death of the CD4.sup.+ T Lymphocytes.

(64) The inventors tested the hypothesis according to which the inhibitor Q-VD-OPh might have a synergistic effect with other antiviral molecules used in combating HIV, in particular with azidothymidine (AZT), a reverse transcriptase inhibitor, and with Indinavir, a protease inhibitor. Primary CD4+ T cells were infected by an HIV-Lai virus and then stimulated with concanavalin A and IL-2. The cells were then cultivated in the presence or absence of Q-VD-OPh (0.1, 1 or 10 μM) and in the presence or absence of AZT (0.1 μM) or Indinavir (1 μM). Where the drugs were added to the culture, they were added 96 hours (d3) after infection. Cell death was then quantified by flow cytometry on the 5th day post-infection.

(65) The results obtained (FIG. 9) show that Q-VD-OPh, when used in combination with AZT or Indinavir, inhibits cell death resulting from viral infection much more strongly than Q-VD-OPh, AZT or Indinavir used alone. Consequently, Q-VD-OPh used in association with anti-HIV treatments for preventing cell death, which is a consequence of viral replication, gives rise to a synergistic effect.

C. Conclusion

(66) The totality of these works clearly shows that the compound Q-VD-OPh is a potent inhibitor of the replication of the HIV and SIV viruses. The more effective inhibitory effect of Q-VD-OPh when administered before infection (pretreatment—see Example B5) shows, moreover, that this molecule acts during the first stages of the replication cycle of the virus. The compound Q-VD-OPh is therefore of major interest for therapeutic use as an antiviral agent and in particular as an antiretroviral agent, and as an antilentiviral agent.

(67) Furthermore, the ability of Q-VD-OPh to prevent apoptosis may be an additional advantage allowing the immune response in respect of pathogenic agents to be restored in a more consistent manner.

(68) It is to be noted that the use of another broad-spectrum caspase inhibitor z-VAD-fmk does not inhibit apoptosis of those cells during viral replication (Petit et al., 2002) nor does it inhibit viral replication of HIV or the death of the T lymphocytes induced by HIV infection (Petit et al., 2002), suggesting that this new inhibitor may have a major role in the fight against this viral infection.

(69) In addition, Q-VD-OPh is capable of acting in synergy with other antiviral molecules such as AZT and Indinavir. The use of Q-VD-OPh in association with other antiviral molecules, in particular with other molecules from the range of anti-HIV agents currently available, therefore appears particularly promising.

Example 2: Comparative Analysis

A. Material and Methods

(70) Analysis by Flow Cytometry of the Fall in λφm and of the p24 Protein of the HIV Virus

(71) In order to evaluate the change in the transmembrane potential of the inner mitochondrial membrane, the cells were labelled with a fluorescent probe DIOC.sub.6 (Molecular Probes, Invitrogen), at a concentration of 40 nM, and incubated for 15 minutes at 37° C. Viral replication was evaluated by internal labelling of the p24 viral protein using an anti-p24-PE antibody (KC-57, Coulter Corp, Beckman).

(72) Immunoblot

(73) 20 μg extracts of total lysate prepared with 1% NP40 were boiled for 5 minutes in Laemmli buffer containing 2% SDS and 10% 2-β-mercaptoethanol, then deposited on 10-20% polyacrylamide gradient gels (Invitrogen). After transfer of the proteins, the immunoblots were incubated with the following primary antibodies: anticaspase-3, anticaspase-8 and anticaspase-9 (Cell Signaling), anti-PARP (Pharmingen) and anti-Tubulin (Santa-Cruz). The secondary antibodies coupled to peroxidase (horseradish) (Amersham Biosciences) allows the proteins to be revealed by chemiluminescence (ECL, Amersham) using a CCD camera (G:Box-Chemi-XT16-SynGene).

(74) Synthetic Inhibitors and Other Chemical Products

(75) The broad-spectrum caspase inhibitor Z-VAD-fmk (Calbiochem), the general caspase inhibitor: Q-VD-OPH, the caspase-8 inhibitor: Q-IETD-OPH, the caspase-3, -7 inhibitor: Q-DEVD-OPH and the caspase-9 inhibitor: Q-LEHD-OPH (MP Biomédicals, France) were used. The anti-CD95 antibody (human Fas) (clone 7C11, Immunotech) was used to induce apoptosis. The HIV antiproteases Saquinavir, Ritonavir and Indinavir are obtained from NIH. The probe CFSE (carboxyfluorescein diacetate succinimidyl ester) used for the proliferation study was obtained from Molecular Probes (Invitrogen).

(76) Proliferation Test with CFSE

(77) The PBMCs are incubated in the presence of 1 μM of CFSE for 7 minutes at 37° C. The cells are taken up at 1.Math.10.sup.6/ml and then placed in culture and activated by an anti-CD3 at 1 μg/ml (Immunotech).

B. Results

(78) B-1. Validation of New Caspase Inhibitors

(79) B-1-a. Determination of the Functionality of New Specific Caspase-3, -8 and -9 Inhibitors in Respect of Apoptosis Induced by Fas/CD95.

(80) Jurkat cells were incubated in the presence or absence of anti-CD95 at different concentrations in order to determine a dose-response curve for apoptosis. Death was evaluated by analyzing the fall in the mitochondrial transmembrane potential (% Δφm low) using the probe DIOC6, by flow cytometry (FIG. 10A). The same experiment was then conducted in the presence of 0.25 μg/ml of anti-CD95 and different caspase inhibitors. The apoptosis induced by anti-CD95 is expressed by the percentage Δφm low analyzed by flow cytometry (FIG. 10B).

(81) Jurkat cell extracts which have been treated in the presence or absence of anti-CD95 and of the different caspase inhibitors were analyzed by immunoblot for caspase-3, -8, -9 and PARP (substrate specific to caspase-3 and -7), tubulin is used as control for the deposits (FIG. 100).

(82) B-1-b. Determination of the Ability of the Specific Inhibitors to Inhibit Viral Replication and Consequently Cell Death.

(83) Primary CD4 T cells were infected by the viral strain HIV-Lai and then stimulated by ConA/IL-2 in the presence or absence of Q-VD-OPH, Q-DEVD-OPH (casp-3 inhibitor), Q-LEDH-OPH (caspase-9 inhibitor) and Q-IETD-OPH (caspase-8 inhibitor) at 10 μM for each of the inhibitors. On days 5 and 7 post-infection, the internal p24 viral protein is measured by flow cytometry after fixation and permeabilization of the CD4 T cells. The results show a weak effect on inhibition of viral replication for each of the different specific caspase inhibitors on d5 and an absence of protection at d7; Q-VD-OPH, on the other hand, inhibits replication completely (FIG. 11A). Death was evaluated by analysis of the fall in the mitochondrial transmembrane potential (% Δφm low) using the probe DIOC6 by flow cytometry (FIG. 11B). We show an absence of effect of these inhibitors, contrary to Q-VD-OPH. Extracts of primary CD4 T cells infected in the presence or absence of the different caspase inhibitors as well as QVD-OPH were analyzed by immunoblot for the HIV viral proteins (FIG. 12). The results show that the quantity of viral proteins produced in the presence of the different inhibitors is slightly lower than that of the control cultures, but that viral production is very markedly reduced in the presence of QVD-OPH. These results are in agreement with our observations regarding the detection of the p24 protein by flow cytometry.

(84) B-2. Toxic effects of the drugs

(85) B-2-a. QVD-OPH at 50 μM does not Block the Proliferation of Lymphocytes Stimulated by 1 μg/ml of Anti-CD-3.

(86) A lymphocyte proliferation study was conducted with the fluorescent probe CFSE, which allows cell division to be monitored. The PBMCs are stimulated with anti-CD3 at 1 μg/ml and incubated in the presence or absence of the HIV antiproteases Saquinavir, Ritonavir and Indinavir, at concentrations of 1 μM, 10 μM and 50 μM, as well as QVD-OPH at 10 μM, 50 μM and 100 μM. Analysis of CFSE carried out after 4 and 5 days' stimulation shows, by flow cytometry, that the HIV antiproteases block the proliferation of lymphocytes at a dose of 50 μM. QVD-OPH, on the other hand, at the same concentrations, has no effect on lymphocyte proliferation (FIG. 13).

(87) The cell toxicity was evaluated by analysis of the fall in the mitochondrial transmembrane potential (% Δφm low) using the probe DIOC6, by flow cytometry. The cells stimulated by anti-CD3 were analyzed in the same way on the day following activation. Accordingly, QVD-OPH, even at high concentrations (100 μM), has no effect on mitochondrial depolarization (compared with the control), whereas Indinavir and Saquinavir show a fall in Δφm of more than 70-80% at a dose of 50 μM (FIG. 14).

(88) B-2-b. Evaluation of the Toxicity of QVD-OPH in Respect of Lymphocytes and Monocytes.

(89) PBMCs are incubated in the presence of the different HIV antiproteases Saquinavir, Ritonavir and Indinavir, at concentrations of 1 μM, 10 μM and 50 μM, as well as QVD-OPH at 10 μM, 50 μM and 100 μM. The cell toxicity was evaluated by analysis of the fall in the mitochondrial transmembrane potential (% Δφm low) after 4 days' culture by analyzing the monocytes and lymphocytes. The effect of QVD-OPH on mitochondrial depolarization remains minimal on one or other of the populations even at a concentration of 100 μM, while the HIV antiproteases, in particular Saquinavir, show depolarization of more than 90% (FIGS. 15A and B). These results are in agreement with our previous works.

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