WO1996041634A2

WO1996041634A2 - Use of a non-nucleoside reverse transcriptase inhibitor in association with nucleoside inhibitors for the treatment of hiv infection

Info

Publication number: WO1996041634A2
Application number: PCT/EP1996/002540
Authority: WO
Inventors: Paolo La Colla; Maria Elena Marongiu; Giovanna Piras
Original assignee: Sardinian Antiviral Research Consortium - Sarc S.C.R.L.
Priority date: 1995-06-13
Filing date: 1996-06-12
Publication date: 1996-12-27
Also published as: WO1996041634A3; IT1281502B1; ITCA950009A0; ITCA950009A1; AU6355296A

Abstract

The use of a non-nucleoside reverse transcriptase inhibitor is described, in particular of derivatives of 6-benzyl-4-oxopyrimidine having general formula (I), in which X is selected from the group consisting of O, S; R is selected from the group consisting of alkyl from C1 to C4, cycloalkyl C5 and C6; R', R' and Z, equal or different among them may be H, alkyl from C1 to C4 considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives, in association with nucleoside inhibitors selected from the group consisting of AZT, d4T and their phosphodiester or phosphotriester derivatives, for the treatment of HIV infection. Pharmaceutical compositions useful for the treatment of HIV infection consisting of the association of different components are also described.

Description

USE OF A NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR IN ASSOCIATION

WITH NUCLEOSIDE INHIBITORS FOR THE TREATMENT OF HIV INFECTION

FIELD OF THE INVENTION

The present invention refers to the use of a non-nucleoside reverse transcriptase inhibitor, and in particular of derivatives of 6-benzyl-

4-oxopyrimidine (dihydro alkoxybenzyl oxopyrimidine-DABO) having general formula

in which X is selected from the group consisting of 0 , S ; R is selected from the group consisting of alkyl from C-^ to C^, cycloalkyl from Cc to Cg ; R' , R" and Z, equal or different among them may be H, alkyl from C-^ to C^ considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives , in association with nucleoside inhibitors selected from the group consisting of 3 ' ~azido-2 ' , 3 ' -dideoxythymidine (AZT) , 2 * , 3 ' dideoxy-2 ' , 3 ' didehydro thymidine ( d*IT) and their phosphodiester or phosphotriester derivatives , if necessary associated also to dideoxy inosine ( ddl ) or dideoxy cytosine ( ddC ) , for the preparation of pharmaceutical compositions active in the treatment of HIV infection. Pharmaceutical compositions useful for the treatment of HIV infection consisting of the association of the two or three components are also described. PRIOR ART The acquired immunodeficiency syndrome (AIDS) is a pathology of viral origin, caused by the human immunodeficiency virus (Human Immunodeficiency Virus - HIV) , and it is characterized by the progressive compromising of immunologic defences. As it is a lethal infection with pandemic development, the search for new therapeutic strategies, more efficacious than those at present available in slowing the disease progression in the affected subjects and the infection diffusion in the population has been given a substantial boost. Among the various steps of HIV replication cycle, the process of retrotranscription of the genome from RNA to double-chain DNA by means of the "reverse transcriptase" enzyme is, probably, the step studied in greatest detail.

The reverse transcriptase has three functions: the DNA polymerase RNA- dependent one, the DNA polymerase DNA-dependent one and the RNAse-H one.

Several antiviral agents blocking the HIV replication having as selective target the reverse transcriptase are known. They may be divided in two different classes: the nucleoside reverse transcriptase inhibitors (NRTI) and the so called non-nucleoside reverse transcriptase inhibitors (NNRTI) .

To the first class in particular belong the AZT, d4T, ddl and ddC compounds, all already known and used in the clinic in the therapy of AIDS. Once phosphorylated, they inhibit the synthesis of the viral DNA competing with the physiologic nucleosides for the specific site in the reverse transcriptases of HIV-1 and HIV-2 and they act as terminators of the chain of the DNA on the way to be synthesized as they lack the hydroxyl in the C3' of the sugar. To the NNRTI class belong several compounds having very different chemical structures such as for example HEPT, TIBO, Nevirapine, BHAP, TSAO, etc., all specific inhibitors of the reverse transcriptase of HIV-1 but not of HIV-2, which are defined in detail in the publications shown below.

They do not need the matabolization from cellular enzymes and they interact with the reverse transcriptase in a site differing from the triphosphate nucleosides site. Compounds belonging to the NNRTI class are for example described in the following publications: Miyasaka T. et al. (1988). A novel lead for specific anti-HIV-1 agents: l-[ (2-hydroxyetohoxy) -methyl]-6- (phenylthio)thy ine. Journal of Medicinal Chemistry 3 .2507- 509; Pauwels R. et al. (1990). Potent and selective inhibition of HIV-1 replication in vitro by a novel series of TIBO derivatives. Nature 343.470-474; Merluzzi V. J. et al. (1990). Inhibition of HIV-1 replication by a non-nucleoside reverse transcriptase inhibitor. Science 250.l4ll-l4l3; Romero D. L. et al. (1991)- Non-nucleoside reverse transcriptase inhibitors that potentially and specifically block human immunodeficiency virus type 1 replication. Proceeding of the National Academy of Science USA 88.8806-8810; Camarasa M-J. et al. 3'-Spiro nucleosides, a new class of specific human immunodeficiency virus type 1 inhibitors synthesis and antiviral activity of [2',5'~ bis-0-(tert-butyldimethylsilyl)-beta-D-xylo-and-ribofuranosyl]-3'- spiro-5"-[4"-amino-l", 2"-oxathiole-2", 2"-dioxide](TSA0)pyrimidine nucleosides. J Med Chem 1992; 35:2721-7.

The clinical experience has demonstrated that the prolonged use of reverse transcriptase, either nucleoside or non-nucleoside, inhibitors produces, on the one hand the onset of serious toxic effects, on the other the development of resistant viruses.

The use in association of a nucleoside analog with a NNRTI has been reported by Chow et al., Nature 36l, 1993₁ where however it has been described the inefficacy against HIV in long term cultures.

The present invention allows to overcome the above described drawbacks as it allows to obtain the extinction of the HIV infection not only as a result of prolonged treatments, but also as a result of limited duration treatments, and therefore not much toxic. The present invention refers to the use of a non-nucleoside reverse transcriptase inhibitor, and in particular of a derivative of 6- benzyl-4-oxopyrimidine (DABO) having general formula (I)

in which X is selected from the group consisting of 0, S; R is selected from the group consisting of alkyl from C-^ to C^ , cycloalkyl from Cc to Cg; R' , R" and Z, equal or different among them may be H, alkyl from C-^ to C^ considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives, in association with a nucleoside inhibitor selected from the group consisting of AZT, d4T and their phosphodiester or phosphotriester derivatives, if necessary associated also to ddl or ddC, for the preparation of pharmaceutical compositions active in the treatment of HIV infection. The present invention refers also to the pharmaceutical compositions useful for the treatment of HIV infection constituted of the association of a non-nucleoside inhibitor with one or two nucleoside inhibitors, as defined above. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents the long term cytotoxicity of a high DABO and AZT concentration;

Figure 2A represents the p24 antigen levels in the treatments with DABO (5 uM) or ATZ (0.16 μM) alone in comparison with DABO + ATZ association;

Figure 2B represents the cytopathic effect in the treatments with DABO or AZT alone in comparison with DABO + AZT assciation; Figure 3A represents the p24 antigen levels in the treatments with DABO (5 uM) or AZT (0.08 μM) alone in comparison with DABO + AZT association;

Figure 3B represents the cytopathic effect in the treatments with DABO or AZT alone in comparison with DABO + AZT association; Figure 4A represents the p24 antigen levels in the treatments with DABO (50 μM) or AZT (2.5 μM) alone in comparison with DABO + AZT association;

Figure 4B represents the cytopathic effect in the treatments with DABO or AZT alone in comparision with DABO + AZT association; Figure 5 represents the viral DNA sequences in the treatment with AZT (2.5 μM) in comparison with DABO (10 μM) + AZT (2.5 μM) association. DETAILED DESCRIPTION OF THE INVENTION

In order to show the utility of the compositions according to the present invention against the HIV infections, we report for illustrative purpose some of the results obtained by us, relating to cytotoxicity and anti-HIV activity of a compound representative of the class of the substituted 6-benzyl-4-oxopyrimidines , i.e. 2- cicloexylthio-6-(3'-methyl)benzyl-3,4-dihydro-4-oxo-5-methyl- pyrimidine, and a compound representative of the class of nucleoside inhibitors, i.e. 3'-azido-2', 3'-dideoxythymidine (AZT), individually used or, according to the invention, in association in different ratios, in models of in vitro infection able to estimate either the cytotoxicity or the efficacy of the long term anti-HIV action. The evaluation of cytotoxicity and antiviral activity has been carried out in MT-4 cells, line of T4 lymphocytes permissive for the replication of the HIV viruses. The cells have been cultivated in RPMI 1640 added with 10% fetal calf serum (FCS) , penicillin 100 U/ml and streptomycin 100 μg/ml. The cultures have been incubated at 37 °C in a % CO2 atmosphere and periodically controlled in order to verify the absence of mycoplasmas contamination.

The virus used in antiviral activity tests (HIV-1, strain Hlg) has been obtained from supernatant of H9/III_B cronically infected cells. The stock virus solutions have been titrated in C8166 and maintained at -80 °C until the moment of use.

EVALUATION OF LONG TERM CYTOTOXICITY

For the estimation of cytotoxicity, 50 μl of RPMI containing 1x10 MT- 4 cells have been added, in 96 well multiplates, to 50 μl of RPMI containing or not scalar dilutions of the compounds under examination, alone and in association. Then the cultures have been incubated at 37 'C . Each 4 days the whole culture has been suspended again in a new medium, containing the same concentrations of inhibitors, in order to take again the cultures to a density (lxlO-'/ml) such that their exponential growth is allowed.

The cells viability has been determined by a colorimetric method based on the use of a tetrazolium salt, the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium-bromide (MTT) , which is transformed by the succinic dehydrogenase mitocondrial enzyme in a product having blue color (formazan) whose amount in our experimental conditions, turns out to be proportional to the number of vital cells. EVALUATION OF LONG TERM ANTI-HIV EFFICACY For the estimation of the antiviral activity, we have reproduced in vitro two different conditions.

The first one with the aim to verify the possibility to block an acute infection process in progress, if necessary extinguishing the infection; the second one predictive of a treatment aimed to prevent the spreading of the infection from an infected cell to healthy bystander cells. Therefore, MT-4 cells, seeded at a density equal to lxl0⁶/ml, have been infected for an hour at 20 °C with lxlO⁶ CCID_5Q (1 CCIDJ-Q = 2 - 50 infecting viruses). After the removal of the inoculum, the cells have been washed three times and then suspended again at a density equal to 2xl0-^>/ml as such or after dilution 1:100 in normal cells. Briefly, 0 ul of RPMI containing 1x10 MT-4 cells have been added, in 96 well multiplates, to 0 ml of RPMI containing or not scalar dilutions of the compounds under examination, alone or in association. Then the cells have been incubated at 37 °C. In case of survival, each 4 days the whole culture has been suspended again in a new medium containing the same concentrations of inhibitors, in order to take again the cells to a density (lxlO-⁵ /ml) such that their exponential growth is allowed. If necessary, after a determined treatment period, specified case by case, duplicate cultures have been suspended again lacking the inhibitors and propagated till the thirty-second day in an inhibitor free culture medium (reverted cultures) .

Each four days from the end of the infection, the estimation of the anti-HIV activity of the different inhibitors, used alone or in the different associations, has been carried out on the base of the following parameters: - determination of the levels of p24 viral antigen;

- estimation of protection from the development of the virus-induced cytopathic effect;

- determination of the titre of infecting virus;

- search for viral DNA sequences. The p24 antigen levels have been estimated by an immunoenzymatic test. The protection from the development of the cytopathic effect has been determined by the above described MTT colorimetric method. The presence of viral nucleic acids has been estimated by "polymerase chain reaction" (PCR) using primers specific for the gag and pol genes of HIV-1 (strain IHg) . The amplification products have been displayed on 2% agar gel.

ADVANTAGES OF THE USED PROCEDURE

Either in the cytotoxicity tests or in the antiviral activity ones, after a first transfer of the whole culture from the well of the 6 well multiplate (culture volume 100 μl) to the well of the 24 well multiplate (culture volume 1 ml) , and a second transfer from the latter to a 50 ml Falcon flask (culture volume 10 ml), from the third passage on only 1x10 total cells (suspended again at lxlO^/ml) have been transplanted from flask to flask.

This procedure allows to assure density conditions optimal for the exponential growth of the cells (10 initial cells multiply till producing, on the twelfth day, 4.1x10' cells) and to maintain in the culture all the cells originally infected for a period of time of twelve days.

BIOLOGICAL ACTIVITY RESULTS

Cytotoxicity The long term cytotoxicity of a high DABO and AZT concentration is shown in Fig. 1.

As it is possible to notice, the maximum not cytotoxical DABO and AZT concentrations as a result of the prolonged treatment with the single compounds for 32 days are, respectively, >100 μM and 2.5 μM. Moreover the two compounds are without cytotoxicity when are used in association for 32 days at the doses of 50 μM (DABO) and 2.5 μM (AZT)

(results not shown).

Antiviral efficacy in cultures having low density of infected cells

Without inhibitors the HIV infection spreads producing, on the fourth day after the infection, p24 antigen levels greater than 950 ng/ml

( Fig . 2A) and cytopathologic effect with death of every cell in culture (Fig. 2B) .

In these experimental conditions, the treatment with 5 μM DABO and

0.16 μM AZT, individually used, inhibits the viral replication only for the first 4 days. Whereupon the viral replication starts again and one has an increase in the p24 antigen levels and development of the cytopathic effect within the next 4-8 days. On the contrary, in the cultures treated with 5 uM DABO in association with 0.16 μM AZT, the p24 antigen amount quickly decreases and it is no more detectable for the duration of the experiment (Fig. 2A) . In this case one has a complete protection from the development of the cytopathic effect and the cells multiply exponentially as in the non infected controls (Fig. 2B).

It is interesting to notice that the suspension of the treatment with the association on the twelfth day after the infection, does not determine a restarting of the viral replication. This is testified by the absence of the p24 antigen (Fig. 2A) , by the fact that the cells keep vital and able to exponentially multiply (Fig. 2B) and by the lack of infecting viruses (not shown data) .

Identical results have been obtained with the following associations: DABO (0.6-1.2-2.5-5 and 10 μM) + AZT 0.32 μM; DABO (2.5 and 10 μM) + AZT 0.16 μM; DABO 10 μM + AZT 0.08 μM.

In Fig. 3 it is possible to observe that associations of 5 μM DABO with 0.08 μM AZT, are efficacious in zeroing the p24 levels (Fig. 3A) , in protecting the infected cells from the development of the cytopathic effect (Fig. 3B) and in preventing the appearance of infecting viruses (not shown data) provided that they are continuously present in the culture medium.

On the contrary, if the treatment is suspended, one has the renewal of the viral replication with production of p24 antigen, development of cytopathic effect and formation of infecting viruses. Results quite similar to this one have been obtained also with the associations DABO 1.2 μM + AZT 0.16 μM and DABO 2.5 μM + AZT 0.08 μM. Moreover analogous results have been obtained using DABO+AZT+ddl or ddC associations. Therefore, the results shown in the figures 1, 2 and 3 demonstrate that associations of DABO with AZT according to the present invention, which allow to reach an hematic concentration of DABO ranging from 0.6 to 20 μM, preferably ranging from 0.6 to 10 uM, and of AZT ranging from 0.08 to 5 μM, preferably varying from 0.08 to 0.32 μM, are not cytotoxical and are efficacious in inhibiting the HIV multiplication.

Within the above indicated concentration ratios there are also associations characterized by the capability of totally extinguishing the HIV infection.

Efficacy in cultures having a high density of infected cells In these experimental conditions, the HIV-1 virus quickly replicate in not treated controls. Within 2 days from the infection p24 antigen levels equal to 950 ng/ml (Fig. 4A) are reached and death by cytopathic effect of the cells in culture is observed (Fig. 4B) . In such a case, not even the treatment with high doses of DABO (50 uM) prevents the viral replication with production of p24 antigen and development of the cytopathic effect. On the contrary, in the cultures treated with 2.5 uM AZT the p24 antigen amount decreases and keeps for the duration of the experiment at levels three thousands times lower than those of the not treated controls (Fig. 4A) . In presence of 2.5 μM AZT one has also a total protection from the development of the cytopathic effect and the cells multiply exponentially as in the not infected controls (Fig. 4B) . However, the suspension of the treatment even after considerable time (twentieth day) from the infection determines a restarting of the viral replication, as testified by the raising of the p24 levels (Fig. 4A) , by the decrease of the cells viability after the development of cytopathic effect (Fig. 4B) and by the production of infecting virus with a titre equal to that of the not treated control (6. x10^/ml) . In presence of associations of 10 μM DABO with 2.5 uM AZT, the p24 antigen amount quickly decreases and it is no more detectable for the duration of the experiment (Fig. 4A) . Moreover one has a total protection from the development of the virus-induced cytopathic effect and the cells multiply exponentially as in the not infected controls (Fig. 4B). However, contrary to what happens with 2.5 μM AZT, the suspension of the treatment even after only 8 days does not determine a restarting of the viral replication. This is testified by the absence of the p24 antigen, by the fact that the cells are vital and able to exponentially multiply and by the lack of infecting viruses. Therefore, the results shown in Fig. 4 demonstrate that not cytotoxical DABO and AZT associations are efficacious in extinguishing the HIV infection even as a result of a short term treatment. Cells of some samples of the experiments in Fig. 4 have been submitted to PCR in order to evidentiate the possible presence of viral DNA sequences.

As it is possible to notice in Fig. 5, such sequences are present for the duration of the experiment in the cells treated with 2.5 μM AZT. In the cells treated with the 10 uM DABO + 2.5 μM AZT association for 20 days, or in those treated for only 8 days and then suspended again in inhibitors free culture medium, said sequences are lacking, testifying that the culture has been sterilized. On the base of what has been found one therefore may conclude that DABO + AZT associations are able to extinguish the HIV infection in cultures having either low or high density of infected cells and therefore show their utility either in the therapy of patients with proclaimed HIV, or in the precocious treatment of seropositive subjects and in the prophylaxis of the primary infection.

Moreover, DABO + AZT associations (the latter at doses equal to the maximum hematic levels reachable in therapy) are able to extinguish the HIV infection in cultures having a high density of infected cells as a result of a treatment limited in time. In therapy this may reflect in discontinuous treatments able to reduce the toxicity of the drugs and their selective pressure towards pharmaco-resistant mutants.

Claims

CLAIMS 1. Use of a non-nucleoside reverse transcriptase inhibitor in association with a nucleoside reverse transcriptase inhibitor selected from the group consisting of AZT, d4T and their phosphodiester or phosphotriester derivatives, for the preparation of pharmaceutical compositions active in the treatment of HIV infection.

2. Use as claimed in claim 1, characterized in that the association also comprises dideoxy inosine (ddl) or dideoxy cytosine (ddC) .

3- Use as claimed in claims 1 and 2, characterized in that the non- nucleoside inhibitor is a derivative of 6-benzyl-4-oxopyrimidine (DABO) having general formula (I)

in which X is selected from the group consisting of 0, S; R is selected from the group consisting of alkyl from C^ to C^, cycloalkyl from C- to Cg; R' , R" and Z, equal or different among them may be H, alkyl from C-^ to C_/j considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives. 4. Use as claimed in claim 3, characterized in that the derivative of 6-benzyl-4-oxopyrimidine is 2-cicloexylthio-6-(3'-methyl)benzyl-3,

4- dihydro-4-oxo-5-methyl-pyrimidine.

5 - Use as claimed in claims from 1 to 4 , characterized in that the dose of the non-nucleoside inhibitor allows to reach a hematic concentration ranging from 0.6 to 20 μM. 6. Use as claimed in claim 5 , characterized in that the dose of the non-nucleoside inhibitor allows to reach a hematic concentration ranging from 0.

6 to 10 μM.

7. Use as claimed in claims from 1 to 4, characterized in that the dose of the nucleoside inhibitor allows to reach a hematic concentration ranging from 0.08 to 5 μ .

8. Use as claimed in claim 7, characterized in that the dose of the nucleoside inhibitor allows to reach a hematic concentration ranging from 0.08 to 0.32 μM.

9- Use as claimed in claim 4, characterized in that the dose of 2- cicloexylthio-6-(3'-methyl)benzyl-3,4-dihydro-4-oxo-5-methyl- pyrimidine allows to reach a hematic concentration equal to 5 μM and the nucleoside inhibitor is AZT and it allows to reach a hematic concentration equal to 0.16 uM.

10. Use as claimed in claim 4, characterized in that the dose of 2- cicloexylthio-6-(3'-methyl)benzyl-3,4-dihydro-4-oxo-5-methyl- pyrimidine allows to reach a hematic concentration equal to 10 μM and the nucleoside inhibitor is AZT and it allows to reach a hematic concentration equal to 2.5 μM.

11. Pharmaceutical compositions active in the treatment of the HIV infection comprising a non-nucleoside reverse transcriptase inhibitor in association with a nucleoside reverse transcriptase inhibitor selected from the group consisting of AZT, d4T and their phosphodiester or phosphotriester derivatives.

12. Compositions as claimed in claim 11, characterized in that the association also comprises dideoxy inosine (ddl) or dideoxy cytosine (ddC).

13. Compositions as claimed in claims 11 and 12, characterized in that the non-nucleoside inhibitor is a derivative of 6-benzyl-4- oxopyrimidine (DABO) having general formula (I)

in which X is selected from the group consisting of 0, S; R is selected from the group consisting of alkyl from C-^ to C^, cycloalkyl from Cc to Cg; R' , R" and Z, equal or different among them may be H, alkyl from C-^ to C/ considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives.

14. Compositions as claimed in claim 13, characterized in that the derivative of 6-benzyl-4-oxopyrimidine is 2-cicloexylthio-6-(3'- methyl)benzyl-3, -dihydro-4-oxo-5-methyl-pyrimidine.

15- Compositions as claimed in claim 11, characterized in that the dose of the non-nucleoside inhibitor ranges from 0.6 to 20 μM.

16. Compositions as claimed in claim 15, characterized in that the dose of the non-nucleoside inhibitor ranges from 0.6 to 10 μM.

17. Compositions as claimed in claim 11, characterized in that the dose of the nucleoside inhibitor ranges from 0.08 to 5 uM.

18. Compositions as claimed in claim 17, characterized in that the dose of the nucleoside inhibitor ranges from 0.08 to 0.32 μM.

19. Compositions as claimed in claim 14, characterized in that the dose of 2-cicloexylthio-6-(3'-methyl)benzyl-3, -dihydro-4-oxo-5- methyl-pyrimidine is equal to 5 μM and the nucleoside inhibitor is AZT in a dose equal to 0.16 μM.

20. Compositions as claimed in claim 14, characterized in that the dose of 2-cicloexylthio-6-(3'-methyl)benzyl-3,4-dihydro-4-oxo-5~ methyl-pyrimidine is equal to 10 μM and the nucleoside inhibitor is AZT in a dose equal to 2.5 μM.