Title:  Drug combination for the treatment of viral diseases

United States Patent:  6,576,622

Issued:  June 10, 2003

Inventors:  Strair; Roger (Skillman, NJ); Medina; Daniel (Monmouth Junction, NJ); Tung; Peter (New Haven, CT)

Assignee:  University of Medicine & Dentistry of New Jersey (Newark, NJ)

Appl. No.:  929249

Filed:  September 10, 1997

This invention pertains to a method for treating a human with human immunodeficiency virus infection which comprises administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor, or pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

Sanctuary Growth of HIV in the Presence of AZT

Human immunodeficiency virus resistance to the non-nucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, AZT-resistant HIV generally emerges in patients only after more prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, replication-defective HIV encoding reporter genes were utilized. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of cell lines derived from such infected cells has demonstrated decreased accumulation of AZT, increased phosphorylation of thymidine to TTP, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line.

Early HIV Breakthrough Infection in the Presence of Stavudine

By utilizing replication-defective HIV encoding reporter genes, applicants have demonstrated that early HIV breakthrough infection in the presence of Stavudine results from infection of cells which are refractory to the antiviral effects of the drug. In addition, applicants demonstrate that the combination of Stavudine and Floxuridine has potent antiviral activity in cells refractory to the antiviral activity of Stavudine alone. Data is presented indicating that the predominant mechanism of HIV breakthrough early after the initiation of stavudine (d4T) is related to the inefficacy of d4T as an antiviral agent in a subset of the host population. This inefficacy is demonstrated to be independent of the presence of HIV with genetic drug resistance and has also been demonstrated in several cell types and with retroviruses other than HIV. These results may help explain several features of the in vivo and in vitro selection of d4T resistant virus and, contribute to an understanding of the mechanisms responsible for HIV kinetics after the initiation of antiviral drugs.

The present invention relates to a method for treating a human with human immunodeficiency virus infection. The method comprises administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor. Thymidine analogs, such as 3'-azido-3'-deoxythymidine (AZT), are prodrugs in the treatment of acquired immunodeficiency syndrome. 3'-Azido-3'-deoxythymidine is converted by cellular enzymes to 3'-azido-3'-deoxythymidine monophosphate (AZTMP). The monophosphate is then converted by cellular enzymes to 3'-azido-3'-deoxythymidine diphosphate (AZTDP) and 3'-azido-3'-deoxythymidine triphosphate (AZTTP). In human cells infected with HIV, 3'-azido-3'-deoxythymidine triphosphate is an inhibitor of the viral reverse transcriptase necessary for viral replication. Some cells, however do not efficiently metabolize AZT to the triphosphate and may overproduce the natural thymidine triphosphate, which competes with the antiviral activity of AZTTP. Studies have demonstrated that these cells contribute to the early failure of the antiviral activity of AZT. By coadministering a thymidylate synthase inhibitor with the thymidine analog, applicants have found that that the thymidine analog is a more effective inhibitor of HIV replication. The thymidylate synthase inhibitor may function by resulting in lower levels of thymidine triphosphate to compete with the phosphorylated thymidine analog reverse transcriptase inhibition.

In another embodiment, the method further comprises administering to the human a therapeutically effective amount of a folate antagonist together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidine analog. In yet another embodiment, the method further comprises administering to the human a therapeutically effective amount of hydroxyurea together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidylate synthase inhibitor. In still yet another embodiment, both the folate antagonist and hydroxyurea may be administered with the thymidine analog and the thymidylate synthase inhibitor.

Use of Floxuridine to Modulate the Antiviral Activity of AZT

Recent clinical studies have demonstrated that early HIV replication after initiation of AZT is generally a consequence of the replication of AZT-sensitive virus (29). A prior in vitro analysis of this early breakthrough replication in the presence of AZT has demonstrated the infection of cells in which AZT was an ineffective antiviral agent (31). A metabolic characterization of these cells has led to the development of a novel combination therapy designed to potentiate the antiviral efficacy of AZT. The present invention describes the antiviral effects of the combination of floxuridine and AZT. This combination suppresses early viral breakthrough, lowers the IC₅₀ of AZT, and has particular antiviral efficacy in the subset of cells that are infected with AZT-sensitive virus in the presence of AZT. The antiviral efficacy of this combination in peripheral blood mononuclear cells suggests potential clinical utility.

In an attempt to explain the ability of genetically-sensitive HIV to replicate in the presence of AZT, applicants have initially utilized recombinant replication-defective HIV to quantitate infection in the presence of AZT (31). In those studies, replication-defective HIV encoding a selectable marker was used to infect target cells in the presence of 10 .mu.M AZT. The cells infected with the defective HIV were isolated by expression of the selectable marker. A subset of these infected cells was demonstrated to be readily infected with another HIV in the presence of 10 .mu.M AZT. These cells were persistently refractory to the antiviral effects of AZT and were demonstrated to have excessive phosphorylation of thymidine to TTP, increased thymidine kinase activity and decreased accumulation of AZTTP.

These data suggested that a component of early infection with AZT-sensitive HIV in the presence of AZT was a consequence of the infection of cells which were refractory to the antiviral effects of AZT. Some of these cells had metabolic factors resulting in reduced AZTTP/TTP ratios in the cells. These data also suggest that it may be possible to overcome this reduced antiviral efficacy of AZT by biochemical modulation of TTP pool sizes. One way to potentially modulate these cells is with fluoropyrimidines such as 5-fluorodeoxyuridine (FUdR). These compounds are known to reduce cellular thymidine pools by the inhibition of thymidylate synthase.

In the present invention, applicants demonstrate the suppression of early HIV infection in the presence of AZT with FUdR. FUdR will be shown to potentiate the antiviral effects of AZT in whole cell populations (including peripheral blood mononuclear cells [PBMC]) as well as in subsets of. cells isolated by infection with recombinant HIV in the presence of AZT. Infection of these latter cells will be shown to be extremely sensitive to combined AZT-FUdR therapy.

The term "prodrug", as used herein refers to compounds which undergo biotransformation prior to exhibiting their pharmacological effects. The chemical modification of drugs to overcome pharmaceutical problems has also been termed "drug latentiation." Drug latentiation is the chemical modification of a biologically, active compound to form a new compound which upon in vivo enzymatic attack will liberate the parent compound. The chemical alterations of the parent compound are such that the change in physicochemical properties will affect the absorption, distribution and enzymatic metabolism. The definition of drug latentiation has also been extended to include nonenzymatic regeneration of the parent compound. Regeneration takes place as a consequence of hydrolytic, dissociative, and other reactions not necessarily enzyme mediated. The terms prodrugs, latentiated drugs, and bioreversible derivatives are used interchangeably. By inference, latentiation implies a time lag element or time component involved in regenerating the bioactive parent molecule in vivo. The term prodrug is general in that it includes latentiated drug derivatives as well as those substances which are converted after administration to the actual substance which combines with receptors. The term prodrug is a generic term for agents which undergo biotransformation prior to exhibiting their pharmacological actions.

As set out above, the present invention relates to a method for treating a human with human immunodeficiency virus infection which comprises administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor.

The thymidine analogs, and prodrugs thereof, which may be employed in the present invention are compounds which act as inhibitors of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus. In general, the thymidine analogs are prodrugs which are converted by cellular enzymes to their respective active monophosphates, diphosphates, and/or triphosphates which are inhibitors of viral reverse transcriptase. Nonlimiting examples of thymidine analogs may be selected from the group consisting of 3'-azido-3'-deoxythymidine, and D4T. In a preferred embodiment, the thymidine analog is 3'-azido-3'-deoxythymidine.

3'-Azido-3'-deoxythymidine (AZT, azidothymidine, zidovudine, Retrovir.TM.), is an antiretroviral drug active against human immunodeficiency virus. 3'-Azido-3'-deoxythymidine is an inhibitor of the replication of retroviruses including HIV also known as HTLV 111, LAV, or ARV. 3'-Azido-3'-deoxythymidine is a thymidine analog in which the 3'-hydroxy (--OH), group of thymidine is replaced by an azido (--N₃) group. Cellular thymidine kinase converts 3'-azido-3'-deoxythymidine into AZT monophosphate. The monophosphate is further converted into AZT diphosphate by cellular thymidylate kinase and to the AZT triphosphate derivative by other cellular enzymes. 3'-Azido-3'-deoxythymidine triphosphate interferes with the HIV viral RNA dependent DNA polymerase (reverse transcriptase) and thus, inhibits viral replication. 3'-Azido-3'-deoxythymidine is useful in treating humans identified as having HIV infection. 3'-Azido-3'-deoxythymidine is disclosed in J. R. Horwitz et al., J. Org. Chem. 29, July 1964, pp. 2076-2078; M. Imazawa et al., J. Org. Chem., 43(15) 1978, pp. 3044-3048; also see Biochemical Pharmacology, Vol. 29, pp. 1849-1851; C. J. Kreig et al., Experimental Cell Research 116 (1978) pp. 21-29; W. Ostertag et al, Proc. Nat. Acad. Sci. U.S.A. 71 (1974).

The thymidine analogs which act as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, may be administered as the free base or in the form of a pharmaceutically acceptable salt, e.g., an alkali metal salt such as sodium or potassium, an alkaline earth salt or an ammonium salt (all of which are hereinafter referred to as a pharmaceutically acceptable base salt). The salts of the thymidine analog are converted to the free base after being administered to the human and are thus prodrugs.

The amount of thymidine analog which acts as an inhibitor of viral reverse transcriptase present in the therapeutic compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of thymidine analog is that amount necessary to inhibit viral reverse transcriptase. All prodrugs or precursors are administered to a human in a therapeutically effective amount sufficient to generate an effective amount of the compound which inhibits viral reverse transcriptase necessary for viral replication of human immunodeficiency virus. In general, a suitable effective dose of the thymidine analog or its pharmaceutically acceptable basic salts will be in the range of about 5 mg to 250 mg per kilogram body weight of recipient per day, preferably in the range of 7.5 mg to 100 mg per kilogram body weight per day, and most preferably in the range 10 mg to 40 mg per kilogram body weight per day.

The thymidylate synthase inhibitors, and prodrugs thereof, which may be employed in the present invention are compounds which are antimetabolites which interfere with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibit the formation of ribonucleic acid (RNA). In general, the thymidylate synthase inhibitors inhibit the synthesis of, thymidine triphosphate so that the phosphorylated thymidine analog which acts as an inhibitor of the viral reverse transcriptase can compete more effectively with thymidine triphosphate and will more effectively inhibit viral reverse transcriptase necessary for viral replication of human immunodeficiency virus. Nonlimiting examples of thymidylate synthase inhibitors may be selected from the group consisting of 5-fluorouracil, 5-fluoro-2-pyrimidone (a prodrug of 5-fluorouracil), and floxuridine. Preferably, the thymidylate synthase inhibitor is floxuridine. These drugs may inhibit HIV infection by other mechanisms as well.

5-Fluorouracil (5-FU) is a fluorinated pyrimidine antineoplastic antimetabolite. The metabolism of 5-fluorouracil in the anabolic pathway blocks the methylation reaction of deoxyuridylic acid to thymidylic acid and interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and growth, the effect of fluorouracil may be to create a thymine deficiency which provokes unbalanced growth and death of the cell. The effects of DNA and RNA deprivation are most marked on those cells which grow more rapidly and which take up fluorouracil at a more rapid pace.

Floxuridine (FUdr) is a fluorinated pyrimidine antineoplastic antimetabolite. Chemically, floxuridine is 2'-deoxy-5-fluorouridine. FUdr produces the same toxic and antimetabolic effects as does 5-fluorouracil. The primary effect is to interfere with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibit the formation of ribonucleic acid (RNA). Derivatives of 5-fluorouracil and floxuridine may also be incorporated into DNA or RNA.

The amount of thymidylate synthase inhibitor present in the therapeutic compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of thymidylate synthase inhibitor is that amount necessary to improve the antiviral efficacy of the thymidine analog so that the phosphorylated thymidine analog which acts as an inhibitor of the viral reverse transcriptase can compete more effectively in the inhibition of viral reverse transcriptase necessary for the replication of HIV. In general, a suitable effective dose of the thymidylate synthase inhibitor or its pharmaceutically acceptable salts will be in the range of about 0.01 mg to 25 mg per kilogram body weight of recipient per day, preferably in the range of 0.01 mg to 10 mg per kilogram body weight per day, and most preferably in the range 0.01 mg to 5 mg per kilogram body weight per day.

As set out above, the method of the present invention may further comprise administering to a human a therapeutically effective amount of a folate antagonist together with the thymidine analog which acts as an inhibitor of viral reverse transcriptase and the thymidylate synthase inhibitor to modulate the effects of the thymidine analog. The folate antagonists, and prodrugs thereof, which may be employed in the present invention are compounds which are antimetabolites which interfere with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibit the formation of ribonucleic acid (RNA). Nonlimiting examples of folate antagonists may be selected from the group consisting of methotrexate and trimetraexate. Preferably, the folate antagonist is methotrexate.

Methotrexate (Amethopterin) is an antimetabolite used in the treatment of certain neoplastic diseases, severe psoriasis, and adult rheumatoid arthritis. Chemically methotrexate is N-[4-[[(2,4-diamino-6-pteridinyl)-methyl]methylamino]benzoyl]-L-glutamic acid. Methotrexate inhibits dihydrofolic acid reductase. Dihydrofolates must be reduced to tetrahydrofolates by this enzyme before they can be utilized as carriers of one carbon groups in the synthesis of purine nucleotides and thymidylate. Therefore, methotrexate interferes with DNA synthesis, repair, and cellular replication.

The amount of folate antagonist present in the therapeutic compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of folate antagonist is that amount necessary to modulate the effects of the thymidine analog. In general, a suitable effective dose of folate antagonist or its pharmaceutically acceptable salts will be in the range of about 0.05 mg to 25 mg per kilogram body weight of recipient per day, preferably in the range of 0.05 mg to 10 mg per kilogram body weight per day, and most preferably in the range of 0.05 mg to 4 mg per kilogram body weight per day.

As set out above, the method of the present invention may further comprise administering to a human a therapeutically effective amount of hydroxyurea, and prodrugs thereof, together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidylate synthase inhibitor. Hydroxyurea has the structural formula H₂ N--CO--NHOH. The precise mechanism by which hydroxyurea produces cytotoxic effects is not known but it is believed that hydroxyurea causes an immediate inhibition of DNA synthesis without interfering with the synthesis of ribonucleic acid or of protein.

The amount of hydroxyurea present in the therapeutic compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of hydroxyurea is that amount necessary to modulate the effects of the thymidylate synthase inhibitor. In general, a suitable effective dose of hydroxyurea or its pharmaceutically acceptable salts will be in the range of about 5 mg to 250 mg per kilogram body weight of recipient per day, preferably in the range of 7.5 mg to 100 mg per kilogram body weight per day, and most preferably in the range 10 mg to 40 mg per kilogram body weight per day.

Administration may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous and intradermal), with oral or parenteral being preferred. The preferred route may vary with the condition and age of the recipient.

While it is possible for the administered ingredients to be administered alone, it is preferable to present them as part of a pharmaceutical formulation. The formulations of the present invention comprise the administered ingredients, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of mixing the ingredients to be administered with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administration 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.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations suitable for topical administration include lozenges comprising the ingredients in a flavored base, 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 ingredient to be administered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered and a pharmaceutically acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

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 nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous, or oily solutions of the active ingredient.

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

Formulations suitable for parenteral administration include aqueous and non-aqueous 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 multidose containers, for example, sealed 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.

Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, or an appropriate fraction thereof, of the administered ingredient.

Claim 1 of 11 Claims

We claim:

1. A method for treating a human with human immunodeficiency virus infection which comprises administering to the human a synergistic therapeutic combination of a therapeutically effective amount of a 3'-azido-3'-deoxythymidine, and floxuridine, or pharmaceutically acceptable salts thereof.
 

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