WO1998031375A9

WO1998031375A9 - Enhanced suppression of hiv-1 by the combination of cytidine nucleoside analogues and ctp synthase inhibitors

Info

Publication number: WO1998031375A9
Application number: PCT/US1998/000784
Authority: WO
Filing date: 1998-01-20
Publication date: 1998-12-03

Abstract

A method to increase potency of cytidine-based anti-HIV drugs using CTP synthase inhibitors, and to overcome resistance of human immunodeficiency virus (HIV) to cytidine-based anti-HIV drugs using CTP synthase inhibitors is disclosed.

Description

ENHANCED SUPPRESSION OF HIV-1 BY THE COMBINAΗON OF CYΗDINE NUCLEOSIDE ANALOGUES AND CTP SYNTHASE INHIBITORS

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a method to increase activity of cytidine-based anti-HIV drugs and to overcome resistance of human immunodeficiency virus (HIV) to cytidine-based anti-HIV drugs by administration to a patient in need thereof a cytidine-based anti-HIV drug in combination with CTP synthase inhibitors . More specifically, the invention relates to a method where 2' ,3'-dideoxycytidine (ddC) or β-L-2 ' , 3 ' -dideoxy-3 ' - thiacytidine (3-TC) are administered in combination with the CTP synthase inhibitors 3-deazauridine and/or cyclopentenyl cytosine.

Related Art Acquired immune deficiency syndrome, or AIDS, is a fatal disease which has reached epidemic proportions among certain high risk groups. Several features of AIDS make therapy extremely difficult. The main target of the AIDS virus, now known as HIV, or human immunodeficiency virus, is the T4 lymphocyte, a white blood cell that marshals the immune defenses. This depletion of T4 cells in AIDS causes a severe depression of the immune response, so that a compound which is to be effective against AIDS must modify virus effect without much help from host immunity. Furthermore, the virus also affects cells in the central nervous system, where it is protected by the blood-brain barrier from compounds that might otherwise be effective against the virus. In infecting its host, the HIV binds to specific cell-surface receptor molecules. The virus penetrates the cell cytoplasm and sheds its protein coat, thereby baring its genetic material, a single strand of RNA. A viral enzyme, reverse transcriptase, accompanies the RNA. The virus, which is a retrovirus, reverse transcribes the RNA into DNA. Ultimately, DNA copies of the HIV genome become integrated into the chromosomes of the host cell.

This integrated viral genome, known as a provirus, may remain latent until the host cell is stimulated, such as by another infection. The proviral DNA is then transcribed into mRNA, which directs the synthesis of viral proteins. The provirus also gives rise to other RNA copies that will serve as the genetic material of viral progeny. The proteins and the genomic RNA congregate at the cell membrane and assemble to form new HIV particles, which then break off from the cell. Two HIV genes, tat and trt/art, appear to control this burst of replication, which destroys the cell. These genes code for small proteins that boost the transcription of proviral DNA and the synthesis of viral proteins . Several compounds have been shown to reduce the activity of reverse transcriptase in vitro. The reverse transcription is the step that is essential to viral replication and irrelevant to host cells. It has been found that HIV replication is considerably slower in the presence of compounds such as suramin, antimoniotungstate, phosphonoformate, and a class of nucleoside analogues known as dideoxynucleosides .

Nucleoside analogues are a class of synthetic compounds that resemble the naturally occurring nucleosides, which are chemical precursors of DNA and RNA. A nucleoside comprises a single-or double-ring base linked to a five-carbon sugar molecule. An analogue differs from the naturally-occurring nucleoside in large or small features of the base or the sugar. An enzyme that normally acts on a nucleoside in the course of viral replication can also bind to the nucleoside analogue. Because the nucleoside and the analogue differ, however, binding to the analogue can incapacitate the enzyme, thereby disrupting a molecular process crucial to viral replication.

Of the synthetic nucleoside analogues, zidovudine (AZT) , didanosine (ddl) and zalcitabine (ddC) , have been extensively studied and used on a large clinical scale (Clumeck, 1993, J Antimicrob Chemother 32 Suppl A pp 133- 138). Additionally, the U.S. Food and Drug Administration

(FDA) has approved the marketing of five new drugs for the treatment of HIV infection. These include stavudine (D4T) and lamivudine (3-TC) , which are nucleoside analogues similar to AZT, ddl and ddC . The other three are protease inhibitors, a new class of anti-HIV drugs (Med Lett Drugs Ther, 1996, 38(972): 35-37).

3-TC, also called L-2 ' , 3 ' -dideoxy-3 ' -thiacytidine, and ddC, also called 2 ' , 3 ' -dideoxycytidine, are the major cytidine analogue anti-HIV drugs currently in clinical use in patients with AIDS. 3-TC in particular, a beta-L(-) nucleoside analog, was shown to synergistically inhibit replication of HIV in vitro when combined with 3'-azido-3'- deoxythymidine (AZT) without added toxicity (Bridges et al . , 1996, Bichem Pharmacol 51(6): 731-736). In fact, in a recent phase I/II study of 3-TC therapy in asymptomatic and mildly symptomatic HIV-infected patients, 3-TC exhibited an excellent safety profile and had antiretroviral activity at all dosages studied (van Leeuwen et al, 1995, J Infect Dis 171(5) : 1166-1171) .

Due to the high mutation rate of the HIV virus, strains that emerge resistant to nucleoside analogue therapy are a constant problem in therapeutic protocols. Thus, co-administration of two or more analogue drugs has been extensively examined in an attempt to keep HIV replication under control. However, mutant strains having resistance to multiple nucleoside analogues continue to be described (Gu et al, 1995, Proc Natl Acad Sci USA 92(7): 2760-2764). In fact, one study demonstrated that HIV from AZT-naive patients' lymphocytes was more sensitive to the inhibitory effect of either 3-TC or ddC than was highly AZT-resistant HIV obtained from AZT-treated patients' cells, indicating partial cross-resistance between thymidine and cytidine analogs (Mathez et al, 1993, Antimicrob Agents Chemother 37(10): 2206-2211).

Virus resistance encountered in multiple drug therapies is indicative that a stronger combination of drugs is required for the long-term treatment of patients infected with HIV. We have found that the activities of 3- TC and ddC are markedly (6 to 9-fold) increased by low levels of CTP synthase inhibitors such as 3-deazauridine (3-DU) and cyclopentenyl cytosine (CPE-C) . In addition, the potency of 3-TC against the replication of a recombinant HIV mutant multiply cross-resistant to AZT, ddl , ddC, D4T, ddG and protease inhibitors such as KNI, was increased approximately 50-fold by combination of 3-TC with a low level of 3-DU. The potency of ddC against the replication of the 3-TC-resistant mutant HIV-1₁₈₄ was likewise increased markedly by a combination of ddC with a low level of 3-DU.

The benefits of anti-HIV nucleoside analogue drugs in combination with CTP synthase inhibitors has not previously been described. Although articles have appeared on the enhancement of arabinosylcytosine (an anti-tumor drug) by 3-deazauridine (Barlogie et al . , 1981, Cancer Res. 41: 1227-1235) and cyclopentenyl cytosine (Grem et al., 1990, Cancer Res. 50: 7279-7284), the combined drug treatment disclosed in the present invention is a novel and highly beneficial development in the battle against HIV.

Consequences of using such combinations would be increased therapeutic effectiveness of 3-TC or ddC, including significant dose-reduction and inhibition of growth of HIV mutants and hence of clinical drug resistance.

SUMMARY OF THE INVENTION

The present invention relates to a composition comprising a cytidine-based nucleoside analogue and a CTP synthase inhibitor wherein the combination provides increased prevention or inhibition of the replication and spread of retroviruses including HIV relative to the effects of the nucleoside analogue alone.

Another object of the present invention relates to a method of preventing and/or inhibiting the replication and spread of a retrovirus, by exposing a cell population, including cells infected by the retrovirus, to a composition comprising a combination of a nucleoside analogue and a CTP synthase inhibitor. The term "retrovirus" is inclusive of any virus that utilizes reverse transcriptase in its life cycle and would therefore be susceptible to the antiviral activity of nucleoside analogues, including, for example, HIV (HIV-1 and HIV-2), HTLV-1, HTLV-2 or SIV. Also encompassed are viruses such as HBV that, although not technically classified as a "retrovirus", utilize a reverse transcriptase and are therefore susceptible to the antiviral activity of nucleoside analogues .

The present invention also encompasses methods of treating HIV-infected and AIDS patients with a composition comprising a nucleoside analogue and a CTP synthase inhibitor in order to prevent and/or inhibit the replication and spread of HIV in these patients. Since the administration of a CTP synthase inhibitor could benefit patients already receiving therapy with a nucleoside analogue drug alone, improvements to such a therapeutic regiment are also claimed.

In a preferred embodiment of the present invention, the nucleoside analogue is a cytidine analogue such as ddC or 3-TC. The CTP synthase inhibitor is preferably 3- deazauridine (3-DU) or cyclopentenyl cytosine (CPE-C) . In particular, we have found that the combination of either ddC or 3-TC and 3-DU are especially effective in preventing and/or inhibiting HIV replication and spread.

The preferred embodiments of the invention include pharmaceutical compositions comprising the combination of either ddC or 3-TC and either 3-DU or CPE-C. The pharmaceutical compositions can optionally contain a pharmaceutically acceptable carrier and/or vehicle. The preferred method of the invention comprises preventing and/or inhibiting retroviral or HIV replication and spread by treating a cell population, including cells infected with HIV, with such a composition. Additionally, the preferred method comprises treating an HIV infected or AIDS patients with such a composition so as to prevent and/or inhibit HIV replication and spread in the patient. The term "treatment" encompasses administration of compounds propylactically to prevent or suppress an undesired condition, and therapeutic administration to eliminate or reduce the extent or symptoms of the condition. Treatment according to the invention may be for a human or an animal infected with a retrovirus, or it may include application in vitro to a cell culture or extracellular media. Treatment may be by systemic administration to a patient or locally to an affected site. The compositions of the present invention, i.e., compositions comprising a cytidine-based nucleoside analog and a CTP synthase inhibitor, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents. Specific examples of pharmaceutically acceptable derivatives of the compounds included in the composition that may be used in accordance with the present invention include the monosodium salt and the following 5'^' esters: monophosphate; disodium; monophosphate; diphosphate; triphosphate; acetate; 3-methyl-butyrate; octanoate; palmitate; 3-chloro benzoate; benzoate; 4-methyl benzoate; hydrogen succinate; pivalate; and mesylate.

Also included within the scope of this invention are the pharmaceutically acceptable salts, esters, salts of such esters, nitrile oxides, or any other covalently linked or non-linked compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a nucleoside analog as described above, or an anti-virally active metabolite or residue thereof.

It is possible for the nucleoside and the synthase inhibitor of the present invention to each be administered alone in solution. However, in the preferred embodiment, the active ingredient (s) may be used or administered in a pharmaceutical formulation. These formulations comprise at least one active ingredient (the nucleoside or the synthase inhibitor or both) , together with one or more pharmaceutically acceptable carriers and/or other therapeutic agents. As included within the scope of this invention, "acceptable" is defined as being compatible with other ingredients of the formulation and not injurious to the patient or host cell. These carriers include those well known to practitioners in the art as suitable for oral, rectal, nasal, topical, buccal, sublingual, vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration.

In the present case, it will be appreciated that the compounds according to the invention may also be used in the manufacture of pharmaceuticals for the treatment or prophylaxis of viral infections. The administration of the composition to humans suffering from AIDS, under conditions which effectively interrupt or suppress activity of the HIV virus, can be accomplished by one or more of several means of administration. Formulations of the present invention suitable for oral administration (including sustained release formulations) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid; in an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electurary or paste. Tablets may, if desired, be enteric coated. Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing in addition to the active ingredients such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. The administered ingredients may also be used in therapy in conjunction with other anti-viral drugs and biologicals, or in conjunction with other immune modulating therapy including bone marrow or lymphocyte transplants or medications . In the preferred embodiment, whatever administrative method is chosen should result in effective circulating levels of each compound. An "effective amount" of the composition is such as to produce the desired effect in a patient which can be monitored using several end-points known to those skilled in the art. For example, such effects could be monitored in terms of a therapeutic effect, e.g., alleviation of some symptom associated with the disease being treated, or further evidence involving decrease in detectable virus or increase in CD4+ T cell count. These methods are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan.

Since a constant supply of the nucleoside analogue is required to inhibit virus replication, oral administration in the form of a tablet or capsule is preferred. In order to achieve this, the preliminary dosage range for oral administration is broad, since it is^" expected that dose modifications might need to be made in individual patients to ameliorate or forestall toxic side effects.

The preliminary dosage ranges are lower than what is currently known as the clinical standard for each of the compounds of the invention. Since the CTP synthase inhibitor will potentiate the activity of the nucleoside analogue, comparatively less analogue should be required than what is currently excepted. For example, the standard dosage for ddC alone currently averages a 0.75 mg tablet three times a day. The dose range for ddC when coadministered with either CPE-C or 3-DU is between 0.05 to 1.0 mg three times a day depending on the individual patient. Likewise, the standard dosage for 3-TC currently averages 150 mg twice a day. The dose range for 3-TC when coadministered with either CPE-C or 3-DU is between 10 to 200 mg twice a day depending on the patient.

The dosage ranges for the CTP synthase inhibitors will also be less than what is currently known in the art with regard to these drugs. As previously discussed, although

CTP synthase inhibitors have not been used for the treatment of HIV, both CPE-C and 3-DU have been applied as anti-tumor drugs. However, as demonstrated in the examples to follow, the quantity of these drugs that is needed to potentiate the activity of the nucleoside analogue is significantly less than the quantity required for antitumor activity. The dosage range for CPE-C according to the present invention is 1 to 25 mg twice a day, and the dosage range for 3-DU according to the present invention is 5 to 250 mg twice a day. Again, the exact dosage within each range will depend on the individual. In addition, dosages will be otpimized to achieve circulating plasma concentrations within the following ranges for each preferred drug: 3-TC, 0.002 to 1.000 μM; 3-DU, 0.5 to 10.0 μM; ddC, 0.01 to 1.00 μM; and CPE-C, 0.001 to 0.020 μM.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is better understood by reading the following detailed description with reference to the accompanying figures, in which like reference numerals refer to like elements throughout, and in which: Fig. 1 is a graph illustrating the potentiating effect of 3-DU at three concentrations (0.5, 1 and 2 μM) on 3-TC activity (also at three concentrations) against an HIV-1 clinical strain. Drug susceptibility was determined using PHA-stimulated PBM cells from an HIV-seronegative blood donor. A 10² 50% tissue culture infectious dose (TCID₅₀) of virus stock was used to infect one million cells. Concentration of the HIV p24 protein was determined on day 7 by radioimmunoassay .

Fig. 2 is a graph illustrating the potentiating effect of 3-DU at three concentrations on ddC activity (also at three concentrations) against an HIV-1 clinical strain using the same cells and detection method at day 7 as for Fig. 1.

Fig. 3 is a graph illustrating the effect of 3-DU alone on PHA-treated PBM cells at day 6. Cell viability was determined by trypan blue-exclusion.

Fig. 4 is a graph illustrating the effect of 3-DU on 3-TC activity against an HIV-1 mutant (HIV-1₁₈₄) resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitor KNI272, using the same cells and detection method at day 7 as for

Fig. 1.

Fig. 5 is a graph illustrating the effect of 3-DU on ddC activity against the HIV mutant, using the same cells and detection method at day 7 as for Fig. 1. Fig. 6 is a graph demonstrating the effect of CPE-C at two concentrations (40 nM and 100 nM) on 3-TC activity against an HIV-1 clinical strain using the same cells and detection method at day 7 as for Fig. 1.

Fig. 7 is a graph demonstrating the effect of CPE-C alone on PHA-treated PMB cells at concentrations ranging from 0 to 100 nM) . PHA-stimulated PBM cells (3 X 10⁷) were incubated with CPE-C at various concentrations in 15 ml culture medium. Cell viability was determined at day 6 with trypan blue-exclusion. Fig. 8 is a graph showing the effect of 2 μM 3-DU on cellular dNTP pools in PHA-treated PBM cells after up to 50 hours in culture. The concentration^" of dNTPs was determined using a known enzymatic assay (Sherman et al, 1989, Anal Biochem 180: 222-226).

Fig. 9 is a graph demonstrating the effect of various concentrations of 3-DU (0 to 2.0 μM) after 20 hours incubation on the activity of deoxycytidine kinase (dCK) activity in PHA-treated PMB cells. dCK activity was determined using a known assay (Gao et al, 1995, Proc. Natl. Acad. Sci. USA 92: 8333-8337). Fig. 10 is a graph showing the time-dependent activation of dCK by 2 μM 3-DU in PHA-treated PMB cells. dCK activity was determined after 10, 20, 30, 40 and 50 hours of culture.

Fig. 11 is a diagram summarizing the potentiating effect of the CTP synthase inhibitor. As shown in the figure, inhibition of CTP synthase causes a fall in dCTP levels . Such a decrease causes a compensatory increase in deoxycytidine kinase, thereby leading to increased levels of the phosphorylated cytidine-based nucleotides .

DETAILED DESCRIPTION OF THE INVENTION In describing preferred embodiments of the present invention illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose .

Examples

Source of virus :

The HIV-1 clinical strain used for in vitro studies was isolated from a patient with advanced HIV-1 infection prior to antiviral therapy. The multi-resistant HIV-1 mutant was produced by recombination of HIV-l_{62(75/77rll6ιl51}

(which is resistant to the reverse transcriptase inhibitors ddC, AZT, ddl, ddG and D4T) (Shirasaka et al, 1995, Proc Natl Acad Sci USA 92: 2398-2402) with HIV-1₄₃₁, which is resistant to the protease inhibitor KNI . The reverse transcriptase mutations at codons A62V, V75I, F77L, F116Y, and Q151M originated during AZT/ddC combination therapy. Mutant HIV-1₁₈₄, resistant to 3-TC, contains a mutation widely known in the art (Gao et al, 1993, Antimicrob Agents Chemother 37: 1390-1392). Both the 431 and 184 mutants were constructed by site-directed mutagenesis in our lab using a plasmid vector encoding the HIV-1 genome, which was then used to transfect COS cells for the production of virus .

Source of cells: Peripheral blood mononuclear (PBM) cells were isolated from the blood of HIV-seronegative donors and stimulated with PHA using standard conditions known in the art.

Source of nucleoside analogs:

3-TC has been licensed to Glaxo-Wellcome and is sold under the trade name Epivir. ddC has been licensed to

Hoffman-LaRoche and is sold under the trade name Hivid.

Source of CTP synthase inhibitors:

Methods of synthesizing CPE-C have been disclosed by Marquez et al . in U.S. Patent No. 4,975,434. 3-DU is commercially available from Sigma Chemicals (Cat. No. D-

6011) . In addition, both 3-DU and CPE-C may be obtained from the pharmaceutical resources branch at the National Cancer Inst. in Bethesda, MD.

Example 1 : Effect of 3-DU on 3-TC Activity Against Clinical HIV

To determine the potentiating effect of 3-DU on 3-TC activity, one million PHA-stimulated PBM cells were infected with 10² (TCID₅₀) of a purified HIV-1 clinical strain. Low concentrations of 3-DU ranging from 0 to 2 μM were incubated with the cells. On day 7, the concentration of the HIV-1 p24 protein was determined using a radioimmunoassay that is commercially available from DuPont (Boston, Mass., Cat No. 189174, Lot No. 189174).

As is shown in Fig. 1, the concentration of p24 in the cell cultures was decreased from approximately 140 ng/ml in untreated cultures to about 100 ng/ml in cultures treated with 0.1 μM 3-TC alone. The addition of 3-DU at a concentration of 0.5 μM resulted in a 25% decrease of detectable HIV-1 p24 in the presence of 0.1 μM 3-TC, while at concentrations of 1 and 2 μM 3-DU, respectively, the amount of HIV-1 p24 was reduced to less than 10 ng/ml in the presence of 0.1 μM 3-TC.

This data suggests that potentiation of 3-TC activity against clinical HIV-1 in culture can be increased at least 3-fold at this concentration when 1 μM 3-DU is also administered. Also, while 0.02 μM 3-TC gave only a slight reduction in detectable p24 protein after 7 days (less than

10%), the combined addition of 0.02 μM 3-TC and 2 μM 3-DU resulted in more than a 50% reduction in detectable virus. Note that 3-DU exhibited negligible antiviral activity at the applied dosage levels when administered alone, and little cytotoxicity as demonstrated in Fig. 3. Example 2 : Effect of 3-DU on ddC activity against clinical HIV

The potentiating effect of 3-DU on the cytidine analogue ddC was also tested using the clinical HIV-1 isolate. The experimental conditions were the same as for Example 1 except 3-DU was tested at concentrations 0.1, 0.5 and 1 μM.

As shown in Fig. 2, ddC alone reduced detectable viral p24 protein from approximately 105 ng/ml in untreated control cultures to about 75 ng/ml at day 7 when supplied at a concentration of 0.04 μM. A concentration of 0.01 μM of ddC alone had a negligible antiviral effect. However, detectable viral protein in cultures with 0.01 μM ddC was reduced by about 20% to 40% in the presence of varying concentrations of 3-DU (0.1 to 1.0 μM) . Strikingly, a concentration of 1 μM 3-DU in the presence of 0.04 μM ddC reduced detectable viral protein to less than 10% of what was seen with ddC alone at this concentration. Again, only very slight differences in the amount of detectable protein are seen when 3-DU is given without ddC, suggesting that the increased antiviral effect is due to the potentiation of ddC activity by 3-DU and not to additive antiviral effects . Example 3: Effect of 3-DU on 3-TC activity against HIV-1 multi-resistant mutant

The potentiating effect of 3-DU on 3-TC activity was also tested using an HIV mutant strain resistant to AZT, ddl, ddC, D4T, ddG and KNI272. Experimental conditions were the same as described in Example 1 (Radioimmunoassay Lot No. 189179). 3-DU was tested at concentrations ranging from 0 to 2 μM, each in the presence of 0 to 100 nM of 3- TC. As shown in Fig. 4, 3-TC alone was mildly effective at reducing detectable p24 protein from the multi-resistant virus. Based on the data, we have estimated that a critical dose of 586 nM 3-TC would reduce virus production by 50%. However, in cultures also treated with 3-DU, a drastic improvement is seen in the reduction of detectable protein. Although 3-DU did exhibit some antiviral activity alone, the expected critical dose of 3-TC required for a 50% reduction dropped from 568 nM in cultures treated only with 3-TC to 11 nM in the presence of 1 μM 3-DU and to less than 2 nM in the presence of 2 μM 3-DU.

These findings are particularly significant since, as previously discussed, a mutated virus resistant to one or more nucleoside analogues may demonstrate partial cross- resistance to other nucleoside analogues (Mathez et al, 1993) . This partial cross-resistance may lower the effect of other nucleoside analogues. A lower activity for a nucleoside analogue drug may mean that the concentration required for in vivo treatment may be above the limits of safety. However, these findings suggest that, as long as some virus susceptibility remains to a cytidine analogue, that effect may be potentiated by the administration of a CTP synthase inhibitor.

Example 4 : Effect of 3-DU on ddC activity against a 3-TC- resistant HIV-1 mutant (HIV-1₁₈₄)

The potentiating effect of 3-DU on ddC activity against the mutant HIV-1₁₈₄ was also tested using the same experimental conditions. 3-DU was again tested at concentrations of 0, 1 and 2 μM, each in the presence of 0, 1, 10 and 40 nM ddC .

As shown in Fig. 5, low concentrations of 3-DU inhibited HIV-1₁₈₄ replication and potentiated ddC activity in a dose- dependent manner. In fact, ddC alone did not inhibit virus replication at any of the concentrations tested. However, in the presence of either 1 or 2 μM 3-DU, increasing inhibition was observed with increasing concentrations of ddC, again supportive of the crucial finding that 3-DU can potentiate even partial activity of a nucleoside analogue against a mutant virus that may be highly resistant to other related and perhaps cross- reactive analogues. Example 5: Effect of CPE-C on 3-TC activity against clinical HIV

The potentiating effect of CPE-C was also tested using the assay described in Example 1. Again, one million PHA- stimulated PBM cells were infected with 10² TCID₅₀ infectious doses in the presence of either 0, 40 nM, or 100 nM CPE-C and various concentrations of 3-TC at day 0. On day 7, the concentration of viral p24 protein was measured using the radioimmunoassay as described above (Assay Lot No. 189179) .

As shown in Fig. 6, at the concentrations of 40 and 100 nM, CPE-C showed significant antiviral activity on its own without any 3-TC present. In addition, CPE-C at the tested concentrations demonstrated significant cytotoxicity (Fig. 7). New data, however, suggests that it is possible to achieve potentiation of analogue activity in vitro using this assay at lower CPE-C concentrations and thus lower cytotoxicity.

Example 6: Effect of 3-DU on dNTP pools To examine the direct effect of 3-DU on cellular dNTP pools, PHA-stimulated PBM cells were incubated with 3-DU at 2 μM in 20 ml of culture medium for 20 hours. Cellular dNTPs were then extracted and the concentration was determined using an enzymatic assay originally developed at Burroughs Wellcome (Sherman et al, 1989, Anal Bioche 180: 222-226) . Although not required in this particular experiment, a modification of this assay has also been published that corrects for the presence of dideoxy nucleotides (Gao et al, 1994, Anal Biochem 222: 116-122). As shown in Fig. 8, only dCTP pools decline over a period of 50 hours, as would be expected as a consequence of the inhibition of CTP synthase.

Example 7 : Effect of 3-DU on deoxycytidine kinase (dCK) activity As dCTP levels rise, a concomitant increase in dCK activity would be expected due to feedback stimulation of the salvage pathway. To examine the effect of 3-DU on dCK activity, PHA-stimulated PBM cells were incubated with 3-DU at the indicated concentrations (see FIG. 9) for 20 hours. Cellular proteins were extracted and dCK activity was measured using a published assay (Gao et al, 1995, "Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2 ' , 3 ' -dideoxynucleotide activities against the replication of human immunodeficiency virus", Proc Natl Acad Sci USA 92: 8333-8337; see the top of page 8334).

As shown in Fig. 9, increasing concentrations of 3-DU resulted in a dose-dependent increase in dCK activity in PBM cells after 20 hours in culture. Fig. 10 demonstrates the time-dependent activation of dCK and indicates that, although levels of dCK rise without the presence of 3-DU, the concentration of dCK peaks at approximately 30 hours in untreated cells, while the concentration of dCK in cells treated with 2 μM 3-DU continues to rise even at 50 hours. Additionally, the concentration of dCK in treated cells after 30 hours of culture is indeed higher than in untreated cells, indicating that, as expected, 3-DU treatment leads to higher levels of dCK by decreasing cellular pools of dCTP.

Fig. 11 summarizes how CTP synthase inhibition acts to potentiate the activity of the anti-viral nucleoside analogue. Since deoxycytidine kinase is responsible for the initial phosphorylation step of ddC and 3-TC, the effect of increased levels of dCK is a concomitant increase in mono-phosphate and thus in tri-phosphate derivatives of the antiviral drugs, thereby increasing the potency and speed with which the nucleoside analogues are recognized by the viral reverse transcriptase.

The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. Modifications and variations of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings.^" It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

The following citations are herein incorporated in their entirety by reference:

US PATENT DOCUMENTS

4,879,277 11/7/89 Mitsuya et al . 514/49

5,565,437 10/15/96 Marquez et al . 514/45

5,026,687 06/25/91 Yarchoan et al . 514/45

4,975,434 12/04/90 Marquez et al . 514/274

OTHER REFERENCES Clumeck, 1993, J Antimicrob Chemother 32 Suppl A pp 133-13?

Med Lett Drugs Ther, 1996, 38(972): 35-37 Bridges et al., 1996, Biche Pharmacol 51(6): 731-736 van Leeuwen et al, 1995, J Infect Dis 171(5): 1166-1171 Gu et al, 1995, Proc Natl Acad Sci USA 92(7): 2760-2764 Mathez et al, 1993, Antimicrob Agents Chemother 37(10):

2206-2211

Barlogie et al . , 1981, Cancer Res 41: 1227-1235 Gre et al . , 1990, Cancer Res 50: 7279-7284 Sherman et al, 1989, Anal Bioche 180: 222-226 Gao et al, 1995, Proc Natl Acad Sci USA 92: 8333-8337

Shirsaka et al, 1995, Proc Natl Acad Sci USA 92: 2398-2402 Gao et al, 1993, Antimicrob Agents Chemother 37: 1390-1392 Balzarini et al . , 1987, Mol Pharmacol 32(6): 798-806

Claims

WHAT IS CLAIMED TS:

1) A composition comprising (i) a cytidine nucleoside analogue effective to prevent or inhibit the multiplication of a retrovirus or virus that uses a reverse transcriptase, and (ii) a CTP synthase inhibitor.

2) The composition of claim 1 wherein the retrovirus or virus is selected from the group consisting of HIV-1, HIV-2, HTLV-1, HTLV-2, SIV or HBV.

3) The composition of claim 2 wherein the retrovirus is HIV-1.

4) The composition of claim 1 wherein the cytidine analog is 2 ' , 3 ' -dideoxycytidine .

5) The composition of claim 1 wherein the cytidine analogue is L-2 ' , 3 ' -dideoxy-thiacytidine .

6) The composition of any of claims 1-5 wherein the CTP synthase inhibitor is 3-deazauridine .

7) The composition of any of claims 1-5 wherein the CTP synthase inhibitor is cyclopentenyl cytosine.

8) A method of preventing or inhibiting the replication and spread of a retrovirus or virus that uses a reverse transcriptase, comprising exposing a cell population to a composition comprising a cytidine nucleoside analogue and a CTP synthase inhibitor such that replication and spread of said retrovirus or virus is prevented or inhibited. 9) The method of claim 8 wherein the^" cell population is infected by said retrovirus or virus .

10) The method of claim 8 wherein said retrovirus or virus is selected from the group consisting of HIV-1, HIV-2, HTLV-1, HTLV-2, SIV or HBV.

11) The method of claim 10 wherein the retrovirus is HIV-1.

12) The method of claim 8 wherein the cytidine analog is 2 ' , 3 ' -dideoxycytidine .

13) The method of claim 8 wherein the cytidine analogue is L-2 ' , 3 ' -dideoxy-thiacytidine .

14) The method of any of claims 8-13 wherein the CTP synthase inhibitor is 3-deazauridine .

15) The method of any of claims 8-13 wherein the CTP synthase inhibitor is cyclopentenyl cytosine.

16) A method of treating a patient infected with a retrovirus or virus that utilizes a reverse transcriptase with a composition comprising a cytidine nucleoside analogue and a CTP synthase inhibitor such that the replication and spread of said retrovirus or virus is prevented or inhibited.

17) The method of claim 16 wherein said retrovirus or virus is selected from the group consisting of HIV-1, HIV-2, HTLV-1, HTLV-2, SIV or HBV.

18) The method of claim 17 wherein said retrovirus is HIV- 1. 19) The method of claim 16 wherein the cytidine analogue is 2 ' , 3 ' -dideoxycytidine .

20) The method of claim 16 wherein the cytidine analogue is L-2 ' , 3 ' -dideoxy-thiacytidine .

21) The method of any of claims 16-20 wherein the CTP synthase inhibitor is 3-deazauridine .

22) The method of any of claims 16-20 wherein the CTP synthase inhibitor is cyclopentenyl cytosine.

23) A method of improving the anti-viral activity of a cytidine nucleoside analogue drug in a patient, comprising administering to a patient previously exposed to a retrovirus or a virus that utilizes a reverse transcriptase and who is currently undergoing nucleoside analogue therapy an appropriate dose of a CTP synthase inhibitor.

24) The method of claim 23 wherein the retrovirus or virus is selected from the group consisting of HIV-1, HIV-2, HTLV-1, HTLV-2, SIV or HBV.

25) The method of claim 24 wherein the retrovirus is HIV-1.

26) The method of claim 23 wherein the cytidine analog is 2 ', 3 ' -dideoxycytidine .

27) The method of claim 23 wherein the cytidine analogue is L-2 ' , 3 ' -dideoxy-thiacytidine .

28) The method of any of claims 23-27 wherein the CTP synthase inhibitor is 3-deazauridine. 29) The method of any of claims 23-27 wherein the CTP synthase inhibitor is cyclopentenyl cytosine.