AU1133899A - Mixtures of dideoxy-nucleosides and hydroxycarbamide for inhibiting retroviral spread - Google Patents

Description

Regulatioa 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention title: MIXTURES OF DIDEOXY-NUCLEOSIDES AND HYDROXYCARBAMIDE FOR INHIBITING RETROVIRAL SPREAD The following statement is a full description of this invention, including the best method of performing it known to us: 1 Mixtures of Dideoxy-Nucleosides and Hydroxycarbamide for Inhibiting Retroviral Spread FIELD OF THE INVENTION The present invention relates to a combination of a reverse transcriptase inhibitor and hydroxycarbamide in a synergistically effective amount wherein the combination is useful in inhibiting retroviral spread.

BACKGROUND OF THE INVENTION The expression "Acquired Immune-Deficiency S dro,-" (AIDS) was first used in 1981 to describe a state of cellular immune deficiency in homosexuals, characterized by the Sappearance of opportunistic infections and Kaposi's Sarcoma evolving very aggressively (CDC (Center for Disease Control), MMWR, 30 305-308. DC, (1981)). In 1983 a retrovirus since called HIV (Human Immunodeficiency Virus type 1) was isolated among AIDS patients (Barrf -Sinoussi F. et al Science, 220: 868-870 (1983)).

Over the past several years, researchers and clinicians have gained considerable experience in studying and caring for individuals infected with HIV throughout the often prolonged course of H1V disease and AIDS. On the basis of this experience, it has become clear that ethe pathogenic mechanisms underlying HIV infection and disease are not unidimensional, but rather are extremely complex (Fauci AS., Science, 239, 617,(1988)). Any attempt to design a comprehensive therapeutic strategy for HIV disease must take this fact into account. (Fauci, 1993, Science, 262:1011-1018).

After entry of the HIV virus into cells and uncoating of the HIV particle, reverse transcription of the viral RNA genome into DNA replicas occurs. Among several forms of unintegrated viral DNA (now containing the long repeats [LTRs, at both the 5' and the 3' ends), only the two-LTR linear forms can integrate into the host genome. Such a process appears strictly dependent upon cell activation/replication of T lymphocytes, although "resting" T cells are clearly susceptible to H1V infection. (Zack J.A. et al, Cell; 61, 213- 222, (1990)). Furthermore, part of the reverse transcription process also can occur in unactivated T cells, a process that results in the accumulation of incomplete

DNA

molecules, which may persist for several hours and remain capable of being integrated into the host genome if the cell undergoes sufficient activation (Zack J.A. et al, Cell 61. 213- 222, (1990)). Therefore, infected "resting" CD4 T lymphocytes can be considered a transient viral reservoir in infected individuals (Bokrinsky M.I. et al; Science, 254, 423- 427. (1991)). These observations are of particular importance in anatomic compartments t| such as the peripheral blood and lymphoid organs, where the CD4+T cell subset represents the predominant infected cell type (Schmittman S M. et al, Science, 245, 305-308, (1989)); (Fox CH. et al J. Infect Dis; 164, 1051-1057, (1991)).

The above discussion provides a sound scientific basis for blocking the initial burst of virus replication and dissemination as well as the persistent replication throughout the course of disease by treating HIV-infected individuals with anti-retroviral agents from the earliest time that HIV infection is recognized through the entire course of infection. Unfortunately, currently available agents are only partially effective in suppressing virus replication and spread, and this effect is transient (Hirsch MS, et al New Engl. J. Med. 328 1686, (1993)).

Clear cut, but limited, benefit is seen when 3'-azido-2', 3'-dideoxythymidine or azidothymidine (AZT) is given to a patient with advanced HIV disease, and the benefits of early intervention are usually only temporary and do not result in significant long-term advantages with regard to the course of disease and death. (Fauci, 1993, Science, 262:1011-1018).

A number of 2'-3'-dideoxynucleosides have been found to be useful for the treatment or prophylaxis of retroviral infections and especially HIV and AIDS. Examples of such materials include: 3'-dideoxy-cytosine (ddC); 3'-dideoxy-adenosine (ddA); 2', 3'dideoxy-guanosine (ddG); and 3'-dideoxy-inosine (ddl) and 3' didexoxy-thymidine (ddT). See European patent application 0206497 and published PCT application number WO 87/01284.

Hydroxycarbamide (HC) was initially synthesized over 120 years ago and has been found to demonstrate activity against a broad spectrum of tumors. (Donehower, Seminars in Oncology, Vol. 19, No.3, Suppl. 9 (June) 1992: pp 11-19). Additionally, hydroxycarbamide has been used as a viricide. In published PCT application number WO 93/09718, hydroxycarbamide is taught to be useful in a hydrogel polymer coating of a blood bag in order to inhibit viral and HIV infectivity.

Gao et al (PNAS. USA, Vol. 90. pp. 8925-8928, October 1993) disclose that hydroxyurea (hydroxycarbamide) treatment of peripheral blood lymphocytes (PBLs) decreases dNTP levels and the DNA synthesis rate to vels comparable to quiescent PBLs.

The article alleges a possible use of hydroxyurea in AIDS therapy.

However, there still remains a need for more effective treatments for the suppression of retroviruses and. in particular, the prevention and/or inhibition of HIV and viral spread. By viral spread, it is intended to include the inhibition of viral replication, and also may include the ability of inhibiting the virus to infect a further host cells.

Objectives of the present invention in the search for new antiretroviral agents include: 1) the identification of compounds with less toxicity and antiviral activity greater than

AZT.

2) the development of drug combinations which provide an additive or synergistic effect and decrease the probability of drug resistant isolates.

BRIEF SUMMARY OF THE INVENTION 15 The present invention relates to a combination of a reverse transcriptase inhibitor and hydroxycarbamide in a synergistic combination wherein the synergistic combination is capable of preventing and/or inhibiting the spread of retroviruses including HIV. More specifically, the present invention relates to a method of preventing and/or inhibiting the spread of retroviruses, including HIV (HIV-1 and HIV-2), HTLV-1, HTLV-2, SIV or HSV, by exposing a cell population, including cells infected by a retrovirus such as, for example, HIV, to a synergistic combination of a reverse transcriptase inhibitor and hydroxycarbamide. Additionally, the present invention encompasses the treatment of HIVinfected and AIDS patients with a synergistic combination of a reverse transcriptase inhibitor and hydroxycarbamide in order to prevent and/or inhibit the spread of HIV in these patients.

In a preferred embodiment of the present invention, the reverse transcriptase inhibitors include dideoxynucleosides, such as, for example, ddl, ddA, ddG and ddT (DT4).

4 In particular and in the preferred combination of the present invention, it has been found that a synergistic combination of hydroxycarbamide (HC) (or HU hydroxyurea) and 3dideoxy-inosine (ddl) can be formed which is especially effective in preventing and/or inhibiting H1V spread. The preferred embodiment of the invention encompasses a composition including a pharmaceutical composition comprising a synergistic combination of ddl and HC. The pharmaceutical composition can optionally contain a pharmaceutically acceptable carrier and/or excipient and/or vehicle. The preferred method of the instant invention comprises preventing and/or inhibiting retroviral or HIV spread by treating a cell population. including cells infected with HIV, with a synergistic combination of ddl .and HC. Additionally, the preferred method comprises treating an HIV infected or AIDS patient with a synergistic combination of ddl and HC so as to prevent and/or inhibit HIV spread in the patient.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a study of the anti-viral activity of a mixture of dideoxyinosine (ddl) and hydroxycarbamide (HC) on non-activated CD4' lymphocytes infected with the HIV virus.

Figure 2 is a study of the antiviral activity of a mixture of AZT and HC.

Figure 3 illustrates the inhibition of viral spread by mixture ofHC and ddl in pre-activated peripheral blood mononuclear cells (PBMC) culture infected with the HIV virus.

DETAILED DESCRIPTION OF THE INVENTION The following examples of specific embodiments of the present invention are offered for illustrative purposes only and are not limiting with respect to the scope of the disclosure or claim coverage.

Testing of the mixture of dideoxyinosine (ddl) and hydroxycarbamide (HC) on the spread of the HIV virus was conducted under two types of conditions: a) CD4 lymphocytes purified from PBMC, and infected with HIV virus without prior activation/proliferation of these cells by phytohemagglutinin (PHA) and interleukin-2 (IL-2).

b) PBMC preactivated by PHA and IL-2, then infected with the HIV virus.

1- Example 1 The activity of the mixture of ddl and HC on non-activated CD4' lymphocytes, infected with HIV virus was studied.

Non-activated

CD

4 cells were infected, then treated for 7 days by HC, ddl or the combination of the two, then activated by PHA and IL-2 (PHA-IL-2).

Cellular viability between 90% and 100% was observed during the first seven days after infection, both for the infected control and for the infected cells treated with the two drugs separately or in combination. Comparable proliferative cellular response was observed in the presence of PHA-IL-2 for the first 3 days (days 7-9) both in the six virus infected groups and in the non-infected, non-treated donor CD-4' cells. This proliferative response is associated with cytopathic effect in the infected control group, and in the groups treated with HC alone at 0.05 and 0.15 mvM: these groups had greater than 50% loss of viability compared to the uninfected control group; this effect is due to viral replication and is accompanied by large-scale production cf p24-HIV in the culture supernatant seen at day 15 (86215 pg of p24/nml for the infected control, 75470 and 82005 for 0.05 and 0.15mM HC treatment groups, respectively).

The cytopathic effect was observed later for the cells treated with 5 uM ddl and reached substantially the same level of p 24 production as the infected control 10 days later at day (101080 pg p24/ml).

The mixture of HC at 0.05 mM with ddl at 5 pM does not substantially change the viral replication profile as compared to ddl alone (84883 pg p24/ml at day By contrast, a surprising synergistic effect is observed with the combination 0.15 mM of HC and 5 pM of ddl, where no residual viral production is detectable 1 pg p24/ml) at day 7 and day 25 despite cellular proliferation which is identical to the non-treated, noninfected control cell viability measured by MTT test).

In particular, a study has been made of the activity of a mixture of ddl and HC on nonactivated CD4 lymphocytes infected with the IIV virus. The CD4' lymphocytes were purified from PBMC with immunomagnetic beads (Dynabeads® M450). These cells were infected with the HIV-1 virus strain IUB at a multiplicity of infection of 5 000 tissue culture infectious dose (TCID) per 106 cells (241pg/ml p24 antigen equivalent of virus).

After 2 hours of virus-cell contact, the cells were washed twice and placed in the culture medium RPMI 1640 (supplemented with 10% fetal calf serum (FCS), 2mM glutamine, penicillin 100 UI/ml and Streptomycin 100tg/mi) at a density of 1.3x10 6 cells/ml. ddl was immediately added at a concentration of 5 .iM and HC at concentrations of 0.05 mM and 0.15 mM. The drugs and culture medium were partially renewed on day 4, maintaining the same concentration of each. On day 7, in order to remove the drugs, the cells were washed twice and put back in culture in the presence of PHA at a concentration of 1 pg/ml and recombinant IL-2 at a concentration of 20 U/ml. This culture was maintained until day 25, with partial renewal of the medium twice a week. The number of viable cells was quantified by a tetrazolium-based calorimetric dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method (Pauwels, R. et al J S"T Virol. Methods, 20, 309-321. (1988)), and activity is expressed as a percentage of the signal in the drug-free and virus-free control. viral replication was quantified by measuring the HIVI p24 antigen by ELISA using the Dupont de Nemours kit.

See figure 1 for the results of example 1.

Example 2 In order to determine whether or not this synergistic effect of ddl in a mixture with HC is specific to ddl, or whether a similar effect could be observed with AZT, a parallel study was conducted combining HC with azidothymidine (AZT) with surprising results.

AZT alone at 5 pm has only slight antiviral activity, less than 1 log, inhibition (10030 pg p24/ml compared to 86215 pg p24!ml for infected control), less than ddl at the same dose under the same conditions: 99.1% inhibition (766 pg p24/ml compared to 86215 pg p24/ml for infected controi). The drug concentrations used here are easily attainable in plasma (plasma concentration achievable under treatment conditions: 4 pM for AZT and pM for ddl (Yarchoan et al New Engl. J. Med. 321 726-738, (1989)).

Comparable proliferative cellular response was observed after stimulation by PHA-IL-2 in all groups. This proliferative response is associated with cytopathic effect in the infected control group, and in the groups treated with HC alone at doses of 0.05 and 0.15 mM and combined with AZT at 5pM (these groups had greater than 50% loss of viability compared 7 to the uninfected control group). The combination of HC at 0.05mM and at 0.15 mM with AZT at 5 pMl (10108 and 9166 pg p24/ml, respectively) does not modify the viral replication profile compared to AZT alone.

The results show that the synergistic etfect which eradicates HIV replication in CD4' cells non-activated by PHA-IL-2 (Example 1) is not found from the mixture of HC with AZT.

A study has been made of the activity of the combination of AZT and HC on non-activated CD4' Imnphocytes infected with the HIV virus. The CD4 lymphocytes were purified from, PBMC with immunomagnetic beads (Dynabeads® M450). These cells were infected with the WV-1 virus strain IIB at a multiplicity of infection of 5 000 tissue cuiture infectious dose (TCID) per 106 cells (24 lpg/ml p 24 antigen equivalent of virus). After 2 hours of virus cell contact, the cells were washed twice and placed in the culture medium RPMI 1640 (supplemented with 10% FCS, 2mM glutamine, penicillin 100 IU/ml and streptomycin 100 tg/ml) at a density of .3x 10 6 cells/ml. AZT was immediately added at a concentration of 5 tM and HC at a concentration of 0.05 mM and 0.15 mM. The drugs S 15 and culture medium were partially renewed on day 4, maintaining the same concentration of each. On day 7, in order to remove the drugs, the cells were washed twice and put back in culture in the presence of PHA at a concentration of 1 pg/ml and recombinant IL-2 at a concentration of 20 U/ml. This culture was maintained until day :with partial renewal of the medium twice a week. The number of viable cells was quantified by a tetrazolium-based colorimetric 3 4, 5-dimethylthiazol-2-yl)-2, diphenyltetrazolium bromide (MTT) method (Pauwels, R. et al J. Virol. Methods, 20, 309- S321, (1988)), and activity is expressed as a percentage of the signal in the drug-free and virus-free control. Viral replication was quantified by measuring the HIV-1 p2 4 antigen by ELISA using the Dupont de Nemours kit.

See figure 2 for the results of example 2.

Example 3 A further study demonstrated inhibition of viral spread in preactivated PBMC culture infected with the HIV virus, by the mixture of HC and ddl.

-e c 8 PBMC preactivated with PHA and IL-2. were infected and treated by HC at 0.15 mM: this g concentration corresponds to the IC.o (inhibitory concentration 50%) after 3 days measured by MTT test, with cellular viability >90% (this cell viability was determined by treating the cells with 2% Trypan Blue for 2 min and monitoring for dye exclusion). The combination gof 0.15 mM of HC and 10 ItM of ddl does not modify the ICso and cellular viability.

The virus replicated rapidly in the non-treated culture maintaining a stable level as from day 6 (day 6 71815; day 12 72750; day 20 62750 pg p24/ml). Treatment with ddl alone at 10 L u M and HC alone at 0.15 mM induces inhibition of 97.1% (2071 pg p24/ml) Sand 82.6%. (12500 pg p24/ml) respectively at day 6. By contrast, a major synergistic effect is observed with the combination of 10 .M I ddl and 0.15 mM HC, with an inhibition of 99.8% (100 pg p24/ml) at day 6 and no residual viral production detectable pg. p24/ml) at day 12 and day This major synergistic effect, having been demonstrated with non-activated lymphocytes.

where the combination of ddl with HC eradicates the HIV infection from the cells, is also observed here where lymphocytes are preactivated and treated with the combination of ddl and HC while the PBMC are replicating.

Viral replication by the combination of HC and ddl is eliminated in preactivated

PBMC

S. culture infected with the HIV virus. The PBMC were purified from peripheral blood by discontinuous Ficoll density gradient centrifugation. The cells were grown at a density of 1.3x106 cells/ml in RPMI 1640 medium supplemented with 10% FCS, 2mM glutamine, peniciliin 100 IU/ml and streptomycin 100 pg/ml, in the presence of PHA at a concentration of 1 ug/ml and recombinant IL-2 at 20 U/ml for 72 hours, then infected by HIV-1 strain IIB at a multiplicity of 5000 TCID for 106 cells 24 1pg/ml p24 antigen equivalent of virus).

After 2 hours of virus-cell contact, the cells were washed twice and placed in the culture medium containing IL-2 but without PHA in the presence ofddl at a concentration of pM and of HC at a concentration of 0.15 mM. These cultures were maintained for 20 days, with partial renewal of the medium and of the two drugs twice a week maintaining the initial concentration. At day 6 and day 14 fresh uninfected donor PBMC were added (5x10s/ml) to replenish aged cultures (Nature, 361:1993, 650-654). The number of viable i-- 9 cells was quantified by a tetrazolium-based calorimetric 5-dimethylthiazol-2-yl)-2, diphenyIlt -azolium bromide (MTT) method (Pauweis. R. et al J. Virol. Methods, 20:309- 321. (1988)). and activity is expressed as a percentage of the signal in the drug-free and vims-free control. Viral replication was quantified by measuring the HIV-1 p24 antigen by ELISA using the Dupont de Nemours kit.

See figure 3 for the results of example 3.

Gao et al in Proc. Nat'. Acad. Sci. USA, cited supra, teach that "human immunodeficiency virus type I (HIV-1) viral DNA synthesis in quiescent and activated peripheral blood i lymphocytes (PBLS) were studied. Incomplete viral DNA (previously demonstrated to be S 10 associated with HIV-1 virions) is carried by HIV-1 virions into quiescent and activated PBLS, contributing to the formation of an early viral DNA pool in these cells. The viral DNA is subsequently completed but only extremely slowly and inefficiently in quiescent PBLs compared to that in stimulated PBLS. We find that this correlates with significantly S:lower levels of DNTP substrates in quiescent compared to activated PBLS. At these low DNTP concentrations, HIV-1 reverse transcriptase acts in a partially distributive manner.

1 5. Increasing dNTP concentrations from the levels of quiescent PBLs to the levels of activated PBLs increases the processive action of the reverse transcriptase, which in turn stimulates rapid and efficient formation of full length DNA. Furthermore, hydroxyurea treatment of stimulated PBLs decreases the dNTP levels and the DNA synthesis rate to levels comparable to quiescent PBLs. Our data therefore indicate that low levels of dNTP may explain why HIV-1 DNA is synthesized slowly and efficiently in quiescent PBLs and suggest that pharmacologic induction of low dNTP levels represents a therapeutic approach for inhibition of HIV-1 replication." The explanation by Gao et al given for slow and inefficient HIV-1 viral DNA synthesis in quiescent PBLs may well be valid, given that viral DNA is synthesized completely in PBLs under conditions of sufficient replicatioalactivation (Fauci, 1993, Science, 262:1011- 1018-).

However, Gao et al fail to explain how HC (hydroxycarbamide or hydroxyurea] by means of its inhibitory action on ribonucleotide reductase, and the reduction of dNTP pools in activated lymphocytes, would constitute a potential use of HC in the treatment of patients

I

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IS

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sS 1' s with AIDS. At the concentration of i mM, HC partially reduces the various dNTP pools studied, see figure 4(a).

A comparison of figure and table 1 (dNTP pools in quiescent and PHA stimulated PBLs) of Gao et al. shows that under the same conditions (PBLs infected with HIV-1 in the presence and the absence of PHA) the reduction of pool levels in table I is substantially greater than the HC-induced reduction over an equivalent 48 hour period. (See table 1).

If one accepts what Gao et al hypothesize that viral DNA synthesis in "restin'g cells while slow and inefficient eventually produces complete viral DNA capable of integration, it is difficult to understand how, under the conditions described, HC could have activity in AIDS Patients. It is demonstrated in the present disclosure that quiescent cells in the presence of HC at nontoxic concentrations are incapable of preventing the production of infectious virions as measured by the H1V 1 p2 4 antigen in the supematant after stimulation by PHA and IL-2.

An explanation of the HC-induced depletion of the DNTP pools which "significantly reduces the rate of H1V-1 DNA synthesis and inhibits the completion of viral

DNA

synthesis in PHA stimulated PBLs". see figure 4(b) of Gao et al, would be that HC at the concentration of imM is cytotoxic for non-activated lymphocytes pretreated for 24 hours and activated by PHA and IL-2 for the following 48 hours in the presence of HC. Under such conditions, greater than 70% of the PBLs die due to the drug's toxicity.

Under the heading "Potential use of hydroxyurea in AIDS therapy", Gao et al state that "by depleting the cellular DNTP pool, hydroxyurea in expected to increase the therapeutic effect of nucleoside analogs 3-azido 3'-deoxythmidine, dideoxyinosine, or dideoxycytosine, which act as competitors of cellular dNTP". If this were true, one would expect that, in the treatment of infected "resting" cells where DNTP pools are found at their lowest levels, nucleoside analogs would have a major effect. However, as shown in the present disclosure, the treatment of "resting" cells infected by HIV-1 for seven days and treated by AZT alone at 5mM has only a slight effect on viral replication as measured by p 2 4 antigen. Similarly, for ddl alone, at the same concentration, and under the same conditions, viral replication is only temporarily and partially inhibited, regaining the level of the infected control at day 25. It is not, therefore, in accepting the Gao et al explanation c_ I

II

that one could have predicted an increase in the therapeutic effect in AIDS therapy by associating a nucleoside analog with HC, all the more so since the results in the present disclosuie show that a surprising synergistic etfect is observed for the association of HC and ddi, but not at all for HC and AZT.

The subject of the present invention is also a new composition for the treatment of a cell population in the presence of a retrovirus. Additionally, the invention includes a phanuaceutical composition intended, in particular, for the treatment and prevention of retroviral infections, especially those linked to HIV and AIDS wherein the composition contains a synergistic combination of hydroxycarbamide (HC) and a reverse transcriptase inhibitor, in particular a synergistic combination of a dideoxynucleoside except AZT and hydroxycarbamide, most preferably a synergistic combination of dideoxyinosine and *i hydroxycarbamide as active principle, in a pharmaceutically acceptable vehicle. The composition of the invention can also contain inert or pharmacodynamically active S. additives, carriers and/or excipients.

The pharmaceutical composition of the invention can take the form of a lyophilized powder of the active substance, to be dissolved immediately before use in a physiological solution for the purpose of injection. The medicament can then be administered parenterally, for example intravenously, intraperitoneally. in the cerebrospinal fluid, and the like. For injection, the active principle is dissolved in a physiological solution until the desired concentration for administration is obtained.

The pharmaceutical composition according to the invention can also take a form which is suitable for oral administration. For example, suitable forms are tablets, hard gelatin capsules, drag es, powders and granules. The formation of such oral forms is well-known to those skilled in the art. Any of the known formulations are useful in preparing the instant oral pharmaceutical compositions.

As regards the dosage of the medicament according to the invention, it will be clear to the artisan that the doses to be administered are variable according to the treatment period, and frequency of administration, the host and the nature and severity of the disease and that the dosages can be easily determined without any undue amount of experimentation.

~B~1~P~giZ~ B~~M 12 The compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to insure the release of a sufficient dosage unit of the present synergistic combination into the patient to provide the desired inhibition of the spread of the retrovirus. The actual dosage administered will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the patient. With these considerations in mind, the dosage of the instant synergistic combination for a particular subject can be readily determined by the physician. It might be noted that in extreme cases a dosage approaching the toxic level may be the acceptable treatment protocol.

For example, in the treatment of HIV-infected and AIDS patients, the composition can comprise from about 1 to 66 mg/Kg/day of ddl and from about greater than 5 mg/Kg/day to S. about 20 mg/Kg/day of HC.

The invention also covers the use of hydroxycarbamide (HC) and dideoxyinosine in combination with other medicinal compositions intended for the treatment of retroviral S 15 infections and tumors. Immunostimulants and immunomodulators such as for example cytokines, including IL-12 and interferon molecules can be used in combination with the present invention.

A preferred range for in vitro administration of the compositions of the present invention includes hydroxycarbamide in a concentration greater than 0.05 mM and less than or equal to 0.25mM in combination with a dideoxynucleoside except AZT such as ddl at concentrations which are generally known and used in the art. A preferred embodiment of the present invention utilizes HC at 0.15 mM and the dideoxynucleoside such as ddl in a range of between about 0.01pM to about 100IOM, preferably between about 2.5 1 M to about most preferably from about 5pM to about 10I M.

In addition the following articles provide support for the synergistic effect of reverse transciptase inhibitors and hydroxycarbamide in preventing and/or inhibiting the spread of retroviruses.

Serge D. Malley et a! (Synergistic anti-human immunodeficiency virus type 1 effect of hvdroxamate compounds with 3' -dideoxyinosine in infected resting human lymphocytes, Proc. Nail. Acad Sci. USA Vol. 90, pp 11017-11021) reports a study of the 13 anti-HIV-1 activity of the dideoxynucleoside analogs (ddNs) AZT, ddl, and ddC and two hydroxamates (R-CONHOH), d-aspartic acid P-hydroxamate (DAH) and hydroxycarbamide [hydroxyurea in infected resting peripheral blood lymphocytes (R-PBLs) in vitro.

In particular this study tests the hydroxamates alone and in various combinations with AZT, ddl and ddC in HIV-l-infected R-PBLs. at hydroxamate doses at which neither cell viability nor cellular DNA synthesis after PHA activation is impaired.

The procedure adopted in this study was as follows. R-PBLs were incubated with HIV-1 for 2 hr at 37 0 C with a multiplicity of infection of 10' TCID.o per 106 cells. Unbound virus was then eliminated by two successive washes with culture medium, and the cells were seeded at a density of 10" cells per ml. in the presence of the various drugs. On day 4, twothirds of the medium was renewed, and fresh drugs were added to maintain the initial concentration. On day 7. cells were collected and sedimented by centrifugation, and the supernatant was kept for p24 assay. After two washes to remove traces of drug, cells of each culture were put in fresh medium containing PHA (PHA 16. Wellcome; I pg/ml) and recombinant IL-2 (Eurocetus, Amsterdam: 20 units/ml). These cultures were maintained for 3 weeks (days 7-28); on days 14, 21 and 28 half of the medium was taken for p24 assay and replaced by fresh medium containing recombinant IL-2 (20 units/ml).

The viability of cells at different time points was quantified by MTT dimethylthiazol-2-yl)-2, 5-diphenylterazolium bromide] -based colorimetric assay (29).

The determination of cellular DNA synthesis at different time points was performed by sampling and further culturing 2 x 105 cells for 4hr in fresh medium containing 3 H]thymidine (5 tiCi/ml; 1 Ci 37GBq). Incorporated radioactivity was precipitated with (w'dvol) trichloroacetic acid, recovered onto glass-fibre filters, and measured in a scintillatio.a spectrometer.

The combination treatments of DAH/ddI and of HU/ddI on R-PBLs resulted in a remarkable synergistic effect with total suppression of viral production after PHA activation as early as day 14. This total viral suppression was observed with no effect on the capacity of the cells to replicate and total protection against the cytopathic effect induced by viral replication. In contrast, no synergistic effect was observed with 14 combinations of AZT or ddC with DAH or HU; viral production after activation showed a profile similar to that with AZT or ddC alone, respectively. However, a synergistic effect (equivalent to that observed with ddl and the two hydroxamates) has been observed with tpM ddC, and each of the two hydroxamates at the same concentrations unpublished data).

Francois Birion et al (Anti-HIV Activity of the Combination of Didanosine and Hvdroxvurea in HIV-l-Infected Individuals. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirolo.v. Vol. 10. pp 36-40) reports a pilot clinical trial of a drug combination known to suppress viral production in both resting and activated peripheral blood mononuclear cells (PBMC) in vitro.

This clinical trial tested the tolerance and antiviral activity of the combination of HU (hydroxyurea) and ddl (didanosine) in a small number of HIV-1 seropositive asymptomatic volunteer subjects considered to be relatively immuno-competent based on their asymptomatic status and CD4 count. The results show that 3 months of treatment was well tolerated and led to a large reduction in the viral load in the peripheral blood of all patients.

and down to nonquantifiable levels in half the population. This dramatic fall in viral load was accompanied by a substantial rise in CD4 count.

I The selection criteria used in the study were HIV-1 seropositivity. CD4 count >250 cells/mm 3 asymptomatic previously untreated with anti-HIV chemotherapy and with a hematological profile corresponding to grade 0 of the WHO classification (WHO offset publications, 48, 1979). From 85 consecutive patients seen by this team of investigating physicians, 17 patients met all inclusion criteria and were invited to participate. The first 12 subjects who accepted formed the study cohort and included nine males and three females, age range from 26 to 60 with CD4' counts between 263 and 582/mm'. Female participants agreed to use effective contraception. The treatment protocol was 90 days with 200 mg ddl twice daily on an empty stomach and 500mg HU twice daily taken at least I h after the ddI.

The results of the study showed that overall tolerance was good. No patient interrupted treatment due to clinical or biological side-effects.

C ;i.

,i ,r i Before treatment, the study failed tc detect infrctious virions in the plasma of 11 patients.

On the other hand. infectious virus was recovered from PBMC in all patients at day 0 with titers ranging from <1 to 234.2 TCIDo./10 6 cells, except for patient 8 for whom in vitro PBMC activation was required to reveal the presence of virus. After 90 days of treatment.

no infectious virus could be detected in the quantitative PBMC cultures of seven patients (TCIDdWI06 cells The PBMC of six of the seven were activated with PHA and did not result in recovery of virus. Although infectious virus obtained from quantitative PBMC cultures of the other five patients remained detectable, ranging from <1 to TCID d10 6 cells, a substantial decrease in viral titers of between 1.28 log and 1.83 log was observed, with a median decrease of 1.63 log.

Quantitation of plasma HIV RNA determined by PCR was performed before treatment with values ranging from 3,521 to 128,973 copies!ml of plasma. After 90 days of treatment, a substantial decrease of between 1.36 log and 2.92 log in RNA copy numbers was observed in six patients, with a median decline of 1.71 log. Moreover, for the other six patients, no HIV RNA could be detected, having fallen below the sensitivity threshold of the method used. By increasing the number of amplification cycles, as described above, we detected the presence of viral RNA in the plasma of patients 7 and 8, whereas results remained negative for patients 9-12.

An increase in CD4 count, in some cases very substantial, was observed for 11 of the 12 patients at day 90. Median CD4 count in the population was 343 cells/mm 3 at day 0 (median percentage of CD4 relative to total lymohocytes and 541 at day 90 (median relative percentage Individual CD4 changes at day 90 range from an increase of 577 cells to a decrease of 69 with a median increase of 120. For the subgroup of six patients whose plasma viral load became undetectable on day 90m, the median increase was 244 cells/mm 3 These results indicate that 90 days of treatment with the combination of HU and ddl is well tolerated in our population of previously untreated, asymptomatic, HIV-infected individuals, and induces a marked reduction in viral load for all patients, as quantified by infectious virus titer and plasma HIV-RNA, which, in half of the cases studied, results in the apparent complete suppression of HIV in the peripheral blood. This reduction in viral load is accompanied by a median increase in CD4 count of 120 cells/mm 3 The median r M' j ul:4 t-A bi

U.

16 CDd increase of the six subjects for whom no viral RNA was quantifiable at day 90 was 244 cells/mm'.

All of the references cited hereinabove are expressly incorporated herein, in toto, by reference thereto.

The word 'comprising' and forms of the word 'comprising' as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions which are obvious to the person skilled in the art and which do not have a material effect upon the invention.

The invention has been described with reference to specific and preferred embodiments. It will be recognised by those skilled in the art that numerous changes and substitutions may be made without departing from the spirit and scope of the invention.

Claims (24)

1. A composition useful for inhibiting the spread of a retrovirus in a cell population comprising a mixture of at least one dideoxynucleoside excluding AZT and hydroxycarbamide wherein the hydroxycarbamide is present in a synergistically effective amount with respect to the amount of the dideoxynucleoside.

2. A composition according to claim 1, wherein the dideoxynucleoside is selected from the group consisting of a ddl, a ddA. a ddG, and a ddT, but excluding AZT.

3. A composition according to claim 2, wherein the dideoxynucleoside is ddl.

4. A composition according to claim 1 or 3, wherein the retrovirus is HIV.

5. A composition according to claim or 3 wherein the retrovirus is HV- 1.

6. A composition according to claim 1 or 3, wherein the amount of dideoxynucleoside "is such as to provide a concentration of about .01 pM to 100[pM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM :and equal to or less than about 0.25mM. a

7. A composition according to claim 6, wherein the amount of dideoxynucleoside is o. such as to provide a concentration of about 2.5pM to 25pM and the amount of the 'hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25 mM.

8. A composition according to claim 6, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5uM to 10p M and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25 mM.

9. A composition according to claim 1 or 3, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5pM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM r A composition according to claim 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5tpM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM.

11. A composition according to claim I or 3. wherein the amount of dideoxynucleoside is such as to provide a concentration of about 1 OpMl and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM.

12. A composition according to claim 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 10 )tM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM.

13. A composition according to claim 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0.01lM to 100pM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM S'and equal to or less than about 0.25mM.

14. A composition according to claim 13, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 2.5pM to 25pM and the amount of the S-hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25mM.

15. A composition according to claim 13, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5pM to 10.pM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25mM.

16. A method of inhibiting spread of a retrovirus in a human cell population in the presence of the retrovirus comprising administering to the human cell population an effective spreading inhibiting amount of a composition according to anyone of claims 1 and 2.

17. A method according to claim 16, wherein the dideoxynucleoside is ddl.

18. A method according to any one of the claims 16 and 17, wherein the retrovirus is HIV. I

19. A method according to claim 18, wherein the retrovirus is HIV-1. A method according to any one of the claims 16 and 17. wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0.01M to 100 LtM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mnM and equal to or less than about 0.25mM.

21. A method according to claim 18. wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0.01pM to 100pM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25mM.

22. A method according to claim 20 or 21. wherein the amount of dideoxynucleoside is such as to provide a concentration of about 2.5p.M to 25pM and the amount of the hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25mM.

23. A method according to claim 20. wherein the amount of dideoxynucleoside is such S 15 as to provide a concentration of about 5tMl to 10M and the amount of hydroxycarbamide is such as to provide a concentration greater than 0.05mM and equal to or less than about 0.25mM.

24. A method according to claim 16 or 17, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5pM to 1 OpM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM. A method according to claim 18, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5pM and the amount of the hvdroxvcarbamide is such as to provide a concentration of about 0.15mM.

26. A method according to claim 16 or 17, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0OpM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0.15mM. i'

27. A method according to claim 18. wherein the amount of dideoxynucleoside is such as to provide a concentration of about 1 0gM and the amount of the hydroxycarbamide is such as to provide a concentration of about 0. 1 COMPAGNIE DE DEVELOPPEMENT AGUETTANT 15 January, 1999