WO2004009595A1 - Combination therapy with 1,3-dioxolanes and inosine monophosphate dehydrogenase inhibitors - Google Patents

Combination therapy with 1,3-dioxolanes and inosine monophosphate dehydrogenase inhibitors Download PDF

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Publication number
WO2004009595A1
WO2004009595A1 PCT/US2003/021109 US0321109W WO2004009595A1 WO 2004009595 A1 WO2004009595 A1 WO 2004009595A1 US 0321109 W US0321109 W US 0321109W WO 2004009595 A1 WO2004009595 A1 WO 2004009595A1
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dxg
hiv
dapd
purine nucleoside
pharmaceutically acceptable
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PCT/US2003/021109
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French (fr)
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Phillip A. Furman
Katyna Borroto-Esoda
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Triangle Pharmaceuticals, Inc.
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Priority to AU2003261114A priority Critical patent/AU2003261114A1/en
Publication of WO2004009595A1 publication Critical patent/WO2004009595A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • the present invention is pharmaceutical compositions and methods for the treatment or prophylaxis of human immunodeficiency virus (HIV) infection and/or hepatitis B virus (HBV) infection in a host comprising administering such compositions.
  • HIV human immunodeficiency virus
  • HBV hepatitis B virus
  • HIV human immunodeficiency virus
  • Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and most typically in the case of HIV, reverse transcriptase, protease or DNA polymerase.
  • efficacy of a drug against HIV infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug.
  • the pharmacokinetics, metabolism, biodistribution or other parameter of the drug can be altered by such combination or alternation therapy.
  • combination therapy is typically over alternation therapy because it induces multiple simultaneous pressures on the virus.
  • alternation therapy is typically over alternation therapy because it induces multiple simultaneous pressures on the virus.
  • mutations will be induced in the HIV-I genome by a given drug, whether the mutation is permanent or transient, or how an infected cell with a mutated HIV-] sequence will respond to therapy with other agents in combination or alternation. This is exacerbated by the fact that there is a paucity of data on the kinetics of drag resistance in long-term cell cultures treated with modem antiretr virai agents.
  • HIV replication is dependent upon the host cell to provide the necessary substrates for viral replication, including deoxynucleoside triphosphates for the reverse transcription of viral RNA into double stranded DNA by the HIV-encoded reverse transcriptase (HIV-RT).
  • Nucleoside reverse transcriptase inhibitors (NRTls) of the HIV- RT such as AZT, 3TC, ddl, D4T, and more recently abacavir, have formed the comer stone of anti-HIV therapy. These NRTls must first be converted to their S'-triphospbates and must be able to successfully compete with the natural nucleotides of the host cell to effectively inhibit virus replication.
  • Hydroxyurea and more recently mycophenolic acid, have been used to target enzymes involved in the de n ⁇ vo synthesis of deoxynucleotides. Hydroxyurea has been used to target cellular ribonucleotide reductase that, through a complex pattem of regulation, provides the appropriate supply of the four deoxynucleotides needed for the synthesis of DNA. Hydroxyurea may exert its effect by decreasing the endogenous deoxynucleotide pools thereby resulting in a relative increase in the intracellular concentration of the 5'- triphosphate of the NRTI.
  • HIV-I variants resistant to 3'-a2ido-3'-deo ⁇ ythymidine (AZT), 2 ⁇ 3'- dideoxyinosine (DD1) or 2',3'-dideoxycytidine (DDC) have been isolated from patients receiving long term monotherapy with these drugs (Larder BA, et at. Science 1989;243:1731-4; St Clair MH, et al. Science 1991;253:1557-9; St Clair MH, et al. Science 1991;253:1557-9; and Fitzgibbon JE, el al. Antimicrob Agents Chemoiher 1 92;36:153-7).
  • NRTIs nonnucleoside reverse transcrtptase inhibitors
  • HBV Hepatitis B virus
  • HBV induces cancer The mechanism by which HBV induces cancer is unknown, although it is postulated that h may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis and cell regeneration associated with the infection.
  • Hepatitis B virus has reached epidemic levels worldwide. After a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed. Patients typically recover from acute viral hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic infection. Chronic infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries.
  • HBV infection chronic persistent hepatitis can cause fatigue, cirrhosis of the liver and hepatocellular carcinoma, a primary liver cancer.
  • high risk groups for HBV infection include those in contact with HBV carriers or their blood samples.
  • the epidemiology of HBV is in fact very similar to that of acquired immunodeficiency syndrome, which accounts for why HBV infection is common among patients with AIDS or HIV-associated infections.
  • HBV is more contagious than HIV.
  • a human serum-derived vaccine has also been developed to immunize patients against HBV.
  • Vaccines have been produced through genetic engineering, While the vaccine has been found effective, production of the vaccine is troublesome because the supply of human serum from chronic carriers is limited, and the purification procedure is long and expensive. Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. In addition, the vaccine does not help the patients already infected with the virus.
  • the biologically active species of many nucleosides is the triphosphate form, which inhibits
  • DNA polymerase or reverse transcriptase or causes chain termination.
  • 3TC has been approved for the treatment of hepatitis B (sold under the name Lamivu ine by GlaxoWellcome).
  • hepatitis B sold under the name Lamivu ine by GlaxoWellcome.
  • TTC ⁇ -2-Hydroxymethyl-5-(5-fluor cytosin-l-yI)-l,3 ⁇ oxathiolane (TTC), claimed in U. S. Patent Nos. 5,814,639 and 5,914,331 to Liotta et al., exhibits activity against HBV.
  • Furman et al. 'The Anti-Hepatitis B Virus Activities, Cytotoxicities, and
  • Penciclovir (PCV; 2-amino-l,9-dihydro-9- ⁇ 4-hydroxy-3-(hydroxymethyl)butyI ⁇ - 6H-purin-6-one) has established activity against hepatitis B. See U.S. Patent Nos. 5,075,445 and 5,684,153.
  • Adefovir (9- ⁇ 2-(pho$ ⁇ honomethoxy)ethyl ⁇ adenine, also referred to as PMEA or
  • L-FDDC (5-fluoro-3 '-thia-2',3 '-dideoxycytidine) for the treatment of hepatitis B virus in WO 92/18517.
  • WO 96740164 filed by Emory University, UAB Research Foundation, and the Centre National de la mecanic Scientifique (CNRS) discloses a number of ⁇ -L-2',3'- dideoxy ⁇ ucleosides for the treatment of hepatitis B.
  • WO 95 07287 also filed by Emory University, UAB Research Foundation, and the Centre National de la mecanic Scientifique (CNRS) discloses 2' or 3' deoxy and 2 ⁇ 3'-dideoxy- ⁇ -L-pentofuranosyl nucleosides for the eatment of HIV infection.
  • W095/32984 discloses lipid esters of nucleoside monophosphates as immuno- suppresive drugs.
  • DE 4224737 discloses cytosine nucleosides and their pharmaceutical uses.
  • U.S. Patent No. 5,703,058 discloses (5-carboximido or 5-fluoroM2',3'- unsaturated or 3 '-modified) pyrimidine nucleosides for the treatment of HIV or HBV.
  • Lin et al. discloses the synthesis and antiviral activity of various 3'-azido analogues of ⁇ -D nucleosides in J. Med. Chem. 31(2), 336-340 (1988).
  • WO 00/03998 filed by Idenix Pharmaceuticals, Ltd. discloses methods of preparing substituted 6-benzy -oxopyrimIdines, and the use of such pyrimidines for the treatment of HIV.
  • a method for the treatment of hepatitis B infection in humans and other host animals includes administering an effective amount of a biologically active 2'-deoxy- ⁇ -L- erythro-pentofura ⁇ onucleoside (alternatively referred to as ⁇ -L-dN or a ⁇ -L-2'-dN) or a pharmaceutically acceptable salt or prodrug thereof, including ⁇ -L-deoxyribothymidine (B-L-dT), ⁇ -L-deoxyribocytidi ⁇ e ( ⁇ -L-dC), ⁇ -L-deoxyribouridine ( ⁇ -L-dU), ⁇ -L- deoxyribo-guanosine ( ⁇ -L-dG), ⁇ -L-deoxyriboadenosine (
  • (+/- dioxolane-T) exhibits a modest activity against HIV (ECso of 20 ⁇ M in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 ⁇ M.
  • R is OH, CI, NH: or H, or a pharmaceutically acceptable salt or derivative of the compounds optionally in a pharmaceutically acceptable carrier or diluent.
  • the compound wherein R is ehloro is referred to as (-M2R,4R)-2-arnino-6-chloro-9-[(2- hydroxymcthyl)-l,3-dioxolan-4-yl]purine.
  • the compound wherein R is hydroxy is (•)- (2R,4R)-9-[(2-hydroxy-methyl l,3-dioxolan-4-yl]guani ⁇ e.
  • the compound wherein R is amino is (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l,3-dioxolan-4-yl]adenine.
  • the compound wherein R is hydrogen is (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l,3- dioxolan-4yl]purine.
  • Amdoxovir [DAPD, (-)- ⁇ -D-2,6-diaminopurine dioxolane)]is an aqueous, soluble and bioavailable pro-drug that is rapidly absorbed and converted in vivo to DXG, (-)- ⁇ -
  • D-dioxolane guanosi ⁇ e (Furman et. al., Gu et. al.). (-)-(2R.4R).2-amino-9-[(2- hydroxymcthyl)-l,3-dioxolan-4-yl]adenine (DAPD) is a selective inhibitor of HIV-1 replication in vitro as a reverse transcriptase inhibitor (RTI). DAPD is thought to be deaminated in vivo by adenosine deaminase, a ubiquitous enzyme, to yield (-)- ⁇ -D- dioxolane guanine (DXG), which is subsequently converted to the corresponding 5'- triphosphate (DXG-TP).
  • DXG deadenosine deaminase
  • DXG ' TP is a potent inhibitor of the HIV reverse transcriptase (HIV-RT) with a Ki of 0.019 ⁇ M.
  • HIV-RT HIV reverse transcriptase
  • Ribavirin (l- ⁇ -D-ribofuranosyl-l,2,4-triazole-3-carboxamide) is a synthetic, non- interferon-inducing. broad spectrum antiviral nucleoside analog sold under the trade name Virazole (The Merck Index, 1 lth edition, Editor: Budavart, S., Merck & Co., Inc., Rahway, NJ, pl304, 1 89).
  • U.S. Patent No. 3,798,209 and RE29,835 disclose and claim ribavirin.
  • Ribavirin is a purine analog with a broad spectrum of antiviral activity (Sidwell et. al.).
  • RBV monophosphate is also an inhibitor of IMPDH and has been shown to enhance the anti-HIV activity of didanosine in vitro (Palmer and Cox).
  • MPA and hydroxyurea the synergistic effects of RBV in combination with specific NRTls is attributed to changes in the intracellular dNTP pools.
  • Ribavirin In the United States, ribavirin was first approved as an aerosol form for the treatment of a certain type of respiratory virus infection in children. Ribavirin is structurally similar to guanosi ⁇ e, and has in vitro activity against several DNA and RNA viruses including Flavmridae (Gary L. Davis Gastroenteroloev ] )8:S104-Sl 14, 2000).
  • Ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA (Gary L. Davis Gastroenterolop ⁇ 1 18:S104- SI 14, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. It is being studied in combination with DDI as an anti-HIV treatment. More recently, it has been shown to exhibit activity against hepatitis A, B and C. Since the beginning of the
  • ribavirin As an anti-HIV treatment, however, when used as a monotherapy, several controlled studies have shown that ribavirin is not effective against HIV. It has no effect on T4 cells, T8 cells or p24 antigen.
  • Mycophenolic acid (6-(4-hydroxy-6-mcthoxy-7-mcthyl-3-oxo-5- ⁇ hthalanyl)-4- methyl-4-hexanoic acid) is known to reduce the rate of de novo synthesis of guanosine monophosphate by inhibition of inosine monophosphate dehydrogenase ("IMPDH"). It also reduces lymphocyte proliferation.
  • IMPDH inosine monophosphate dehydrogenase
  • Lymphocytes and monocytes rely on the de-novo pathway of guanosine synthesis.
  • MPA selectively inhibits lymphocyte and monocyte proliferation.
  • MPA has been shown to inhibit HIV replication in these cells presumably through reduction of dGTP pools (Ichimura and Levy, Chapuis et. al).
  • MPA has been shown to increase the in im anti-HIV activity of abacavir when used in combination (Margolis et. al.).
  • MMF prodrug mycophenolate mofetil
  • mycophenolic arid has a synergistjc effect when combined with Abacavir (Ziagen) in vitro.
  • Mycophenolic acid depletes guanosine, one of the essential DNA building blocks.
  • Abacavir is an analog of guanosine and as such, must compete with the body's natural production of guanosine in order to have a therapeutic effect.
  • mycophenolic acid improves Abacavir's uptake by the cell.
  • scientistss have determined that the combination of mycophenolic acid and Abacavir is highly active against Abacavir-resistant virus.
  • 4,686,234 describes various derivatives of mycophenolic acid, its synthesis and uses in the treatment of autoimmune disorders, psoriasis, and inflammatory diseases, including, in particular, rheumatoid arthritis, tumors, viruses, and for the ueatment of allograft rejection.
  • Jt is another object of the present invention to provide methods and compositions to eat drug resistant strains of HBV.
  • a drug resistant strain of HIV or exhibits the behavior of drug-naive virus when given the combination of a ⁇ -D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor.
  • the HIV strain is resistant to a ⁇ -D-l,3-dioxolanyl nucleoside. It has further been discovered that certain combinations of ⁇ -D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor are useful to treat hepatitis B virus (HBV) infections in a host.
  • HBV hepatitis B virus
  • the present invention is directed to compositions and methods for the treatment or prophylaxis of HIV, and in particular to a drug-resistant strain of HIV, including but not limited to a DAPD and/or DXG resistant strain of HIV, in an infected host, and in particular a human, comprising administering an effective amount of a ⁇ -D-
  • R is H, OH, CI, NH. or NR'R*; R' and R* are independently hydrogen, alkyl or cycloalkyl, and R J is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodi ⁇ g, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
  • IMPDH inosine monophosphate dehydrogenase
  • compositions and methods are provided for the treatment or prophylaxis of HBV, and in particular to a drug-resistant strain of HBV, including but not limited to a ⁇ DAPD and/or DXG resistant strain of HBVri ⁇ an nfected host, and particular a human, comprising administering an effective amount of a ⁇ -D-1,3- dioxolanyl purine nucleoside of the formula:
  • R is H, OH, CI, NH or NR'R 2 ;
  • R 1 and R 2 are independently hydrogen, alkyl or cycloalkyl, and
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
  • IMPDH inosine monophosphate dehydrogenase
  • the ⁇ -D-I,3-dioxolanyl purine nucleoside is of the formula:
  • R ⁇ is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or
  • ⁇ -D-l,3-dioxolanyl purine nucleoside is of the formula:
  • R J is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug,
  • ⁇ -D-l,3-dioxolanyl purine nucleoside is of the formula:
  • R 3 is H, alkyL aryl, acyl, phosphate, including monophosphate, diphosphate or uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid. or its pharmaceutically acceptable salt or prodrug.
  • the ⁇ -D-l ,3-dioxolanyl purine nucleoside is of the formula:
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
  • ⁇ -D-l,3-dioxolanyl purine nucleoside is of the formula:
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
  • an enantiomerically enriched ⁇ -D-l,3-dioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with an IMPDH inhibitor, for example ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, selenazofiirin, 5-ethynyl-I- ⁇ -D-rib ⁇ furanosylimidazole-4-carboxamide (EICAR), or (S)-N-3-(3-(3-methoxy-4-oxazol-5-yI-phenyl)-ureido]-benzyl-carbamic acid tetrahydrofuran-3-yi-ester (VX-497), which effectively decreases the EC$o for DXG when tested against wild type or mutant strains of HIV- 1.
  • an IMPDH inhibitor for example ribavirin, mycophenolic acid, benzamide riboside, tiazo
  • the IMPDH inhibitor is mycophenolic acid.
  • the IMPDH inhibitor is ribavirin.
  • the nucleoside is administered in combination with the IMPDH inhibitor.
  • the nucleoside may be DAPD.
  • the enantiomerically enriched ⁇ -D-l,3-dioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with a compound that reduces the rate of de novo synthesis of guanosine or deoxyguanosine nucleotides.
  • DAPD is administered in combination or alternation with ribavirin or mycophenolic acid which reduces the rate of de nov synthesis of guanosine nucleotides.
  • an enantiomerically enriched ⁇ -D-l,3-dioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with a compound that effectively increases the intracellular concentration of DXG-TP.
  • DAPD is administered in combination or alternation with ribavirin or mycophenolic acid that effectively increases the intracellular concentration of DXG-TP. It has also been discovered that, for example, this drug combination can be used to treat DAPD-resistant and DXG-resistant strains of HIV. DAPD and DXG resistant strains of HIV, after ueatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
  • DAPD DAPD
  • IMPDH inhibitor that effectively reverses drug resistance observed in HIV-1 mutant suains.
  • the enantiomerically enriched ⁇ D-l,3-dioxoIanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with an IMPDH inhibitor that effectively reverses DAPD or DXG drug resistance observed in HIV-I mutant strains.
  • an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents are administered together.
  • the dosages will depend on such factors as abso ⁇ tion, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • TfieseTJosage ranges can b ⁇ modif ⁇ edls ⁇ esireaTb acrtieve a desiredTesult.
  • the disclosed combination and alternation regiments are useful in the prevention and treatment of HIV infections and other related conditions such as AlDS-related complex (ARC), persistent generalized lymphadenopathy (PGL), AlDS-related neurological conditions, anti-HIV antibody positive and HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia pu ⁇ urea and opportunistic infections.
  • these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HIV antibody or HIV-antigen positive or who have been exposed to HIV.
  • Figure J is a bar graph illustrating the EC» Hi ⁇ M of various concentrations of MPA on the activity of DAPD, DXG, and abacavir in peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Figure 2 is a bar graph illustrating the EC M in ⁇ M of various concentrations of RBV on the activity of DAPD, DXG, and abacavir in PBMC.
  • Figure 3 is a graph showing the EC50 in ⁇ M of MPA and RBV on the antiviral activity of DAPD against drug resistant mutant strains.
  • Figure 4 is a graph showing the EC ⁇ in ⁇ M of MPA and RBV on the antiviral activity of DXG against drug resistant HIV variants.
  • Figure 5 is a graph showing the concentration of DXG-TP (pmole/J 0* cells) with increasing amounts of MPA in PBMC.
  • Figure 6 is a graph showing various concenuations of DXG (pmole/10* cells) with or without 0-25 ⁇ M of MPA and the intracellular concentration of DXG-TP and dGTP measured by liquid chromatography mass specwomeiry in PBMC.
  • Figure 7 is a graph of the DXG-TP ( ⁇ mole/10* cells) concentrations in DAPD incubated PBM cells.
  • Figure 8 is a graph of DXG-TP (pmole/10 6 cells) concentrations in DXG incubated PBM cells.
  • a drug resistant strain of HIV exhibits the behavior of drug-naive virus when given the combination of a ⁇ -D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor.
  • the HIV strain is resistant to a ⁇ -D-l,3-dioxolanyl nucleoside,
  • the present invention is directed to compositions including a combination of a ⁇ - D-I,3-dioxolanyl purine nucleoside and an IMPDH inhibitor, and methods of use of the compositions for the treatment or prophylaxis of HIV or HBV, and in particular to drug- resistant strains of HIV or HBV, such as DAPD and/or DXG resistant strains, in a host, for example a mammal, and in particular a human.
  • the ⁇ -D-1,3- dioxolanyl purine nucleoside and/or the IMPDH inhibitor is enantiomerically enriched.
  • IMPDH catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate
  • IMP xanthosine-5'-monophosphate
  • XMP xanthosine-5'-monophosphate
  • IMPDH inosine monophosphate dehydrogenase
  • the present invention is directed to compositions and methods for the treatment or prophylaxis of HIV, and in particular to drug-resistant strains of HIV, such as DAPD and/or DXG resistant strains of HIV, in a host, for example a mammal, and in particular a human, comprising administering an effective amount of an enantiomerically enriched ⁇ -D-!,3-di ⁇ xolany] purine nucleoside of die formula:
  • R is H, OH, CI, NH2 orNR'R 2 ;
  • R 1 and R 2 are independently hydrogen, alkyl or cycloalkyl, and
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid or its pha ⁇ naceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogcnase (IMPDH) inhibitor.
  • IMPDH inosine monophosphate dehydrogcnase
  • compositions and methods are provided for the ueatment or prophylaxis of HBV, and in particular to a drug-resistant strain of HBV, including but not limited to a DAPD and or DXG resistant strain of HBV, in an infected host, and in particular a human, comprising administering an effective amount of a ⁇ -D-1,3- dioxolanyl purine nucleoside of the formula:
  • R is H, OH, CI, NHj or NR'R 2 ;
  • R 1 and R 2 are independently hydrogen, alkyl or cycloalkyl, and
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
  • IMPDH inosine monophosphate dehydrogenase
  • ⁇ -D-l,3-dioxo!anyl purine nucleoside is of the formula:
  • R ⁇ is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
  • ⁇ -D-I,3-dioxoIanyl purine nucleoside is of the formula:
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or Uiphosphate or a stabilized phosphate derivative, a phospholipid, or an ether-Iipid, or its pha ⁇ naceutically acceptable salt or prodrug.
  • ⁇ -D-l,3-dioxolanyl purine nucleoside is of the formula:
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
  • ⁇ -D-l,3-dioxolanyl purine nucleoside is of the formula:
  • R 3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or
  • ⁇ -D-l,3-dioxo!anyl purine nucleoside is of the formula
  • R 3 is H, alkyl, atyl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an cther-lipld, or its pharmaceutically acceptable salt or prodrug.
  • the enantiomerically enriched ⁇ -D-l-3-dioxolanyl purine nucleoside e.g., DAPD
  • an IMPDH inhibitor for example, ribavirin, mycophenolic acid, be ⁇ zamide riboside, tiazofurin, selenazofurin, 5-ethynyl-l- ⁇ -D-rib ⁇ furan ⁇ Sylimidazole-4-carboxamide (EICAR), or (S)- N-3-[3-(3-meihoxy-4-oxazol-5-yl-pr»enyl)-ureido]-berizyl arbamic acid teUahydrofuran- 3-yI-ester (VX-497), which effectively decreases the EC» for DXG when tested against wild type or mutant suains of HI V-J or HBV.
  • EICAR 5-ethynyl-l- ⁇ -D-rib ⁇ furan ⁇ Sylimid
  • Mycophenolate mofetii is a prodrug which is converted to mycophenolic acid (MPA) in vivo. Also within the scope of the invention are the compositions including MPA and methods of use disclosed herein, wherein MPA is replaced with the mycophenolate mofetii prodrug.
  • the IMPDH inhibitor is mycophenolic acid.
  • the IMPDH inhibitor is ribavirin-
  • the nucleoside is administered in combination with the IMPDH inhibitor.
  • the nucleoside is DAPD.
  • the enantiomerically enriched ⁇ -D-l-3-dioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with a compound that reduces the rate of de now synthesis of guanosine and deoxyguanosine nucleotides.
  • DAPD is administered in combination or alternation with ribavirin or mycophenolic acid which reduces the rate ofde novo synthesis of guanosine nucleotides.
  • the enantiomerically enriched ⁇ -D-l,3- Jioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with a compound that effectively increases the inttacellular concemration of DXG-TP.
  • DAPD is administered in combination or alternation with ribavirin or mycophenolic acid that effectively increases the intracellular concen ation of DXG-TP.
  • this drug combination can be used to ueat DAPD-resistant and DXG-resistant strains of HIV or HBV.
  • DAPD and DXG resistant strains of HIV after treatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
  • the enantiomerically enriched ⁇ -D-l,3-dioxolanyl purine nucleoside, and in particular DAPD is administered in combination or alternation with an IMPDH inhibitor that effectively reverses drug resistance observed in HIV-I or HBV mutant strains.
  • the enantiomerically enriched ⁇ -D-I,3-dioxolanyl purine nucleoside, and in panicular DAPD is administered in combination or alternation with an IMPDH inhibitor that effectively reverses DAPD or DXG drug resistance observed in HIV- 1 or HBV mutant strains.
  • optically active and racemic forms may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism.
  • the present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein.
  • the optically active forms can be prepared by, for example, resolution of the racemic form by recrystailization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution.
  • the compounds are provided in substantially pure form (i.e. approximately 95% pure or greater).
  • Optically active forms of the compounds can be prepared using any method known in the art, including by resolution of the racemic form by recrystailization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
  • Examples of methods to obtain optically active materials include at least the following. i) physical separation of crystals - a technique whereby macroscopic crystals of the individual enantiomers are manually separated.
  • This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization - a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enz y matic resolutions - a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmeuic synthesis - a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmeuic synthesis - a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.
  • the stationary phase can be made of
  • 25 chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;
  • the barrier typically separates two iscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.
  • Chiral chromatography including simulated moving bed chromatography, is used in one embodiment.
  • a wide variety of chiral stationary phases are commercially available.
  • protected refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes
  • oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
  • halo includes chloro, bromo, iodo and fluoro.
  • alkyl refers to a saturated suaighf, branched, or cyclic, primary, secondary or tertiary hydrocarbon of typically C t to Cio, and specifically includes methyl, trifluoromethyl, CCIj, CFCI 2 , CFjCI, ethyl, CHiCFi, CFjCFj, propyl, Isopropyl, cyclopropyl, butyl, isobutyl, r-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
  • the term includes both substituted and unsubstituted alkyl groups.
  • Moieties with which the alkyl group can be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, niuo, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis. John Wiley and Sons, Second Edition, 1991, hereby inco ⁇ orated by reference.
  • lower alkyl refers to a Ci to C 4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, lower alkyl is . Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is .
  • aryl refers to phenyl, biphenyl, or naphthyl, and phenyl.
  • the term includes both substituted and unsubstituted moieties.
  • the aryl group can be substituted with one or more moieties selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylami ⁇ o, arylamino, alkoxy, aryloxy, nitro, cya ⁇ o, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et at., Protective Croups in Organic Synthesis. John Wiley and Sons, Second Edition, 1 1.
  • acyl refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from suaight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted wiih halogen (e.g., F, CI, Br or I), C
  • halogen e.g., F, CI, Br or I
  • alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxyuityl, substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl.
  • Aryl groups in the esters optimally comprise a phenyl group.
  • the lerm "lower acyl" refers to an acyl group in which the non-carbonyl moiety is lower alkyl.
  • enantiomerically enriched is used throughout the specification to describe a compound which includes approximately 95%, 96%, 97% or 98%, and even at least about 99% or more of a single enantiomer of that compound.
  • D or L a nucleoside of a particular configuration
  • the term "host,” as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and a human. Alternatively, the host can be carrying a part of the viral genome, whose replication or function can be altered by the compounds of the present invention.
  • the term host specifically refers to infected cells, cells transfected with all or part of the viral genome and animals, in particular, primates (including chimpanzees) and humans. In most animal applications of the present invention, the host is a human patient.
  • Veterinary applications in certain indications, however, are clearly anticipated by the present invention (such as simian immunodeficiency virus in chimpanzees).
  • prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention.
  • Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, aikylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylatcd to produce the active compound.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.
  • the compounds of this invention either possess antiviral activity, or are metabolized to a compound that exhibits such activity.
  • any of the compounds as disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts
  • administration of the compound as a pharmaceutically acceptable salt may be appropriate.
  • pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, ethanesulfonate, acetate, citrate, malonate, tartarate, succinatc, benzoate, ascorbate, ⁇ -ketoglutarate and ⁇ - glycerophosphate.
  • Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate and carbonate salts, hydrobromate and phosphoric acid.
  • Pha ⁇ naceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • alkaline earth metal salts of carboxylic acids can also be made.
  • nucleosides described herein can be administered as a nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside.
  • a number of nucleotide prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the hydroxyl group of the compound or of the mono, di or iphosphate of the nucleoside will increase the stability of the nucleotide.
  • substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.
  • acyl refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxy ethyl, aryl including phenyl optionally substituted with halogen, G to C ⁇ alkyl or C, to C ⁇ alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, uityl or monomethoxyuityl, substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl).
  • the active nucleoside or other hydroxyl containing compound can also be provided as an ether lipid (and particularly a 5 '-ether lipid or a 5'-phosphoethcr lipid for a nucleoside), as disclosed in the following references, which are incorporated by reference herein: Kucera, L.S., N. Iyer, E. Leakc, A. Raben, Modest E.K., D.L.W., and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-I production and induce defective virus formation.” AIDS Res. Hum. Retro Viruses. 6:491-501; Piantadosi, C, J- Marasco C.J., S.L.
  • Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside or other hydroxyl or amine containing compound, e.g., at the 5'-OH position of the nucleoside or lipophilic preparations include U.S. Patent Nos. 5,149,794 (Sep. 22, 1992, Yatvin et al.);
  • nucleotide prodrugs are described in the following references: Ho, D.H.W. (1973) "Disuibution of Kinase and deaminase of l ⁇ -D- arabinofuranosylcytosine in tissues of man and muse.” Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotide analogues," In: De Clercq
  • S-acyl-2-thioethyI group also referred to as "SATE".
  • compositions Humans or other hosts infected with HIV or HBV, and in particular, an infection caused by a drug resistant strain of HIV or HBV, can be treated by administering to the patient an effective amount of the defined ⁇ -D-I,3-dioxolanyl nucleoside, and in particular, DAPD or DXG, in combination or alternation with an IMPDH inhibitor, including ribavirin or mycophenolic acid, or a pharmaceutically acceptable salt or ester thereof in the presence of a pharmaceutically acceptable carrier or diluent.
  • the active materials can be administered by any appropriate route, for example, orally, parenterally, enterally, intravenously, i ⁇ tradermally, subcutaneously, topically, nasally, rectally, in liquid, or solid form.
  • the active compounds are included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount of compound to inhibit viral replication in vivo, especially HIV or HBV replication, without causing serious toxic effects in the treated patient.
  • inhibitory amount is meant an amount of active ingredient sufficient to exert an inhibitory effect as measured by, for example, an assay such as the ones described herein.
  • a dose of the compound for all the above-mentioned conditions will be in the range from about 1 to 50 mg/kg, or 1 to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per kilogram body weight of the recipient per day.
  • the effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent nucleoside to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the -derivative,-or-by-otber-means known to those skilled n the art.
  • the compounds are conveniently administered in unit any suitable dosage form, including but not limited to one containing 7 to 3000 mg, or 70 to 1400 mg of active ingredient per unit dosage form.
  • An oral dosage of 50 to 1 00 mg is usually convenient
  • At least one of the active ingredients, or the combination of active ingredients is administered to achieve peak plasma concentrations of the active compound of from about 0.2 to 70 mM, or about 1.0 to 10 M. This may be achieved, for example, by the intravenous injection of a 0.1 to 10 % solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient.
  • the dosage form may be for example about 0.1 to 1000 mg of a ⁇ -D-l,3-dioxolanyl purine nucleoside and about 0.1 to 1000 mg of an IMPDH inhibitor.
  • a further example of dosage form is about 0.1 to 500 mg of a ⁇ -D-l,3-dioxolanyl purine nucleoside and about 0.1 to 500 mg of an IMPDH inhibitor.
  • IMPDH inhibitor Another example of dosage form is from about 0.1 to 50 mg of a ⁇ -D- 1,3-dioxoIanyl purine nucleoside and 0.1 to 50 mg of an IMPDH inhibitor.
  • a further example of dosage form is from about 0.1 to 10 g of ⁇ -D-l,3-dioxolanyl purine nucleoside and 0.1 to 10 g of IMPDH inhibitor.
  • the dosage form may be for example from about 0.1 to 1000 mg of DAPD or DXG and 0.1 to 1000 mg of MPA, RBV, or their prodrugs.
  • a further example of dosage form is about 0.1 to 500 mg of DAPD or DXG and 0.1 to 500 mg of MPA, RBV, or their prodrugs.
  • Another example of dosage form is from about 0.1 to 50 mg of DAPD or DXG and 0.1 to 50 mg of MPA, RBV, or their prodrugs or salts.
  • a further example of dosage form is from about 0.1 to 10 mg of DAPD or DXG and 0.1 to 10 mg of MPA, RBV, or their prodrugs or salts.
  • An example of a dose range is about 0.1 to 100 mg/kg of body weight per day of a ⁇ -D-l,3-dioxolanyl purine nucleoside and about 0.1 to 100 mg/kg of body weight per day of an IMPDH inhibitor.
  • Another example of a dose range is about 1 to 50 mg/kg of body weight per day of a ⁇ -D-I,3-dioxolanyl purine nucleoside and about I to 50 mg/kg of body weight per day of an IMPDH inhibitor.
  • Another example of a dose range is about 1 to 20 mg/kg of body weight per day of a ⁇ -D-!,3-diox ⁇ lanyl purine nucleoside and about 1 to 20 mg kg of body weight per day of an IMPDH inhibitor.
  • the concentration of active compound in the drug composition will depend on abso ⁇ tion, distribution, metabolism and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the adminisuation of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • a mode of administration of the active compound is oral.
  • Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, tioches, or capsules.
  • Pharmaceutically compatible bind agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • dosage unit forms can comain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
  • the compounds can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the compounds or their pharmaceutically acceptable derivative or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, anti-fungals, anti- inflammatories, protease inhibitors, or other nucleoside or non-nucleoside antiviral agents, as discussed in more detail above.
  • Solutions or suspensions used for parental, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of lonicity such as sodium chloride or dextrose.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, abso tion promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • compositions may be prepared by mixing the drug with a suitable non-initiating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-initiating excipient such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and micro-encapsulated delivery systems.
  • a controlled release formulation including implants and micro-encapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polyiactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also as pharmaceutically acceptable carriers, these may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container.
  • An aqueous solution of the active compound or its monophosphate, diphosphate, and/or uiphosphate derivatives is then introduced into the container.
  • the container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
  • an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents are administered together.
  • the dosages will depend on such factors as absorption, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Examples of suitable dosage ranges can be found in the scientific literature and in the Physicians Desk Reference. Many examples of suitable dosage ranges for other compounds described herein are also found in public literature or can be identified using known procedures. These dosage ranges can be modified as desired to achieve a desired result.
  • the disclosed combination and alternation regiments are useful in the prevention and treatment of HIV infections and other related conditions such as AlDS-related complex (ARC), persistent generalized Jymphadenopathy (PGL), AlDS-related neurological conditions, anti-HIV antibody positive and HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia pu ⁇ urea and opportunistic infections.
  • these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical Illness in individuals who are anti-HIV antibody or HIV-antigen positive or who have been exposed to HIV.
  • this drug combination can be used to treat HIV, and in particular DAPD-resista ⁇ l and DXG-resistant strains of HIV.
  • DAPD and DXG resistant strains of HIV after ueatment with the disclosed drug combination, exhibit characteristics of drug-narve virus.
  • the anti-HIV activity of the composition comprising a ⁇ -D-1 ,3-dioxolane nucleoside and an inosine monophosphate dehydrogenase inhibitor, or the pharmaceutically acceptable salts or prodrugs of the composition, can be enhanced by administering one or more other effective anti-HIV agent.
  • the composition can be administered in combination and or alternation with a reverse transcriptase inhibitor (a "RTI”), which can be either a synthetic nucleoside (a "NRTI”) or a non- nucleoside compound (a "NNRTI”); a protease inhibitor; a fusion binding inhibitor (such as a pyrophosphate analog).
  • a reverse transcriptase inhibitor a "RTI”
  • NRTI synthetic nucleoside
  • NRTI non- nucleoside compound
  • protease inhibitor such as a pyrophosphate analog
  • antiviral agents that can be used in combination or alternation with the compounds (or compositions including a 1 ,3-dioxolane and an IMPDH inhibitor), disclosed herein for HIV therapy include cis-2-hydroxymethyJ-5-(5-fluorocytosin-l-y
  • 1,3-oxathiolane FTC
  • the (-)-enantiomer of 2-hydroxymethyl-5-(cytosin-l-yl)-l,3- oxathiolane 3TC
  • DXG ⁇ -D- dioxolanyl-
  • Potease inhibitors include Crixivan (Merck), nelf avir (Agouron), ritonavir (Abbott), saquinavir (Roche), DMP-266 (Sustiva) and DMP-450 (DuPont Merck).
  • compositions of the present invention include (I S,4R)-4-[2-amino-6 ⁇ yclopropyl-amino)-9H-purin-9-ylJ-2-cyclopentene-l -methanol succinate ("J592", a carbovir analog; GlaxoWellcome); 3TC: (-)- ⁇ -L-2',3'-dideoxy-3'- thiacytidine (GlaxoWellcome); a-APA R 18893' a-n ⁇ tro-anilino-phenylacetamide; A- 77003; C2 symmetry-based protease inhibitor (Abbott); A-75925: C2 symmetry-based protease inhibitor (Abbott); AAP-BHAP: bishetero-aryipiperazine analog (Upjohn); ABT-538: C2 symmeUy-based protease inhibitor (Abbott); AzddU:
  • BILA 2185 N-(l,l-dimethyiethyl)-l-I2S-[[2-2,6-dimethyl- phenoxy)-l-oxoethyl]amino]-2R-hydroxy-4-phenylbutyl]4R-pyridinylthio)-2-piperidine- carboxamide (BioMega/Bochringer-Ingelheim); BM+51.0836: thiazolo-iso-indolinone derivative; BMS 186,318: aminodiol derivative HIV-1 protease inhibitor (Bristol-Myers- Squibb); d4API: 9-[2,5-dihydro-5-(phosphonomethoxy)-2-ruranel]adenine (Gilead); d4C: 2',3'-didehydro-2',3'-dideoxycytidine; d4T: 2',3'-dide
  • UC-781 N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furan-carbothio- amide
  • UC-82 N-[4-chloro-3-(3-methyl-2-butenyloxy)phenylJ-2-methyl-3-thiophene- carbothioa ide
  • VB 11,328 hydroxyethyl-s ⁇ lphonamide protease inhibitor (Vertex);
  • VX-478 hydroxyethylsulphonamide protease inhibitor (Vertex);
  • XM 323 cyclic urea protease inhibitor (Dupont Merck).
  • Compounds, or concentrations of IMPDH inhibitor compounds may be antagonistic in combination with 1,3-dioxolanes, such as MAP and RBV, and thus may not be preferred under certain circumstances.
  • an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents arc administered together.
  • the dosages will depend on such factors as absorption, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the adminisuation of the compositions.
  • suitable dosage ranges can be found in the scientific literature and in the Physicians Desk Reference. Many examples of suitable dosage ranges for ther compounds described herein are also found in public literature or can be identified using known procedures. These dosage ranges can be modified as desired to achieve a desired result.
  • this drug combination can be used to treat HBV, and in particular DAPD-resistant and DXQ-resistant strains of HBV.
  • DAPD and DXG resistant strains of HBV after treatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
  • the anti-hepatitis B viral activity of the composition comprising a ⁇ -D-1,3- dioxolanc nucleoside and an inosine monophosphate dehydrogenase inhibitor, or the pharmaceutically acceptable salts or prodrugs of the composition, can be enhanced by administering one or more other effective anti-HBV agent.
  • composition can be administered in combination and or alternation with 3TC, ⁇ -2- hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3-oxathiolane ("FTC"), L-FMAU, interferon, famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil; Hepsera); lobucavir or ganciclovir.
  • FTC ⁇ -2- hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3-oxathiolane
  • L-FMAU L-FMAU
  • interferon interferon
  • famciclovir penciclovir
  • BMS-200475 bis pom PMEA (adefovir, dipivoxil; Hepsera); lobucavir or ganciclovir.
  • compositions according to the present invention can be administered in combination or alternation with one or more other antiviral, anti-HIV, anti-HBV, anti- HCV or anti-herpetic agent or interferon, anti-cancer, antiproliferative or antibacterial agents, including other compounds of the present invention.
  • Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the metabolism, catabolism or inactivation of other compounds and as such, are co-administered for this intended effect
  • Ribavirin was analyzed in vitro for activity against HIV-1 and for its effects on the in vitro anti-HIV activity of two dGTP analogues, DAPD and DXG. RBV was also evaluated for cytotoxicity in the laboratory adapted cell line MT2 and in peripheral blood mo ⁇ onuclear cells (PBMC). RBV is an inhibitor of the enzyme IMP dehydrogenase. This enzyme is part of the pathway utilized by cells for the de novo synthesis of GTP.
  • RBV was tested for cytotoxicity on the laboratory adapted T-cell line MT2 and in PBMCs using a XTT based assay.
  • the XTT (2,3-bis(2-methoxy-4-nitro-5- suIfoxyphenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) assay is an in vitro colorimetric cyto-protection assay. Reduction of XTT by mitochondria dchydrogen ⁇ ses results in the cleavage of the tetrazolium ring of XTT, yielding orange formazan crystals, which are soluble in aqueous solution. The resultant orange solution was read in a spectrophotometer at a wavelength of 450nM.
  • RBV was prepared in 100% DMSO at a final concentration of lOOmM.
  • a 2mM solution of RBV was prepared in cell cuhure media (RPMI supplemented with 10% fetal calf serum. L-Glutamine Img/ml and 20ug/ml gentamicin) followed by 2 fold serial dilutions on a 96 well plate.
  • Cells were added to the plat at 3xl0 4 /well (MTX) and 2xl s /well .PBMC) and the plates were incubated for 5 days w jT'C in a 5% CO. incubator (addition of the cells to the plate diluted the compound to a final high concentration of ImM).
  • XTT was added to each well and incubated at 37°C for 3 hours followed by the addition of acidified isopropanol.
  • the plate was read at 450 ⁇ m in a 96 well plate reader.
  • a dose response curve was generated using the abso ⁇ tion values of celts grown in the absence of compound as 100% protection.
  • RBV was not toxic in these assays at concentration of up to I M, as shown in Table 1.
  • XXTAss y RBV was tested for activity against the xxLAl strain of HIV-1 in the laboratory adapted cell line MT2. Dilutions of RBV were made in cell culture media in a 96 well plate; the highest concentration tested was 100 ⁇ M. Triplicate samples of compound were tested. MT2 cells were infected with xxLAl at a multiplicity of infection (MOI) of
  • the infected cells were plated at 3.0 x 10 well into a 96 well plated containing drug dilutions and incubated for 5 days at 37*C in CO..
  • the antiviral activity of RBV was determined using the XTT assay described above. This method has been modified into a susceptibility assay and has been used in a variety of In vitro antiviral tests and is readily adaptable lo any system with a tytic virus (Weislow, O.S., et. al.1989).
  • a dose response curve is generated by plotting % protection on the Y axis and drug concentration on the X axis. From this curve ECso values were determined. RBV was not active against HIV-1 in these assays at any of the concentrations tested.
  • P24Ass ⁇ y RBV was also tested for activity against the xxLAl strain of HIV-1 in PBMCs using a p24 based ELISA assay.
  • cell supernatants were incubated on microelisa wells coated with antibodies to HIV-1 p24 core antigen.
  • anti- HIV- 1 conjugate labeled with horseradish peroxidase was added.
  • the labeled antibody bound to the solid phase antibody/antigen complexes previously formed.
  • Addition of the tetramethylbenzidine substrate results in blue color formation. The color turned yellow when the reaction is stopped.
  • the plates were then analyzed on a plate reader set at 490 nm.
  • the absorbance is a direct measurement of the amount of HIV-1 produced in each well and a decrease in color indicates decreased viral production. Dilutions of RBV were made in cell culture media in a 96 well plate, the highest conee ⁇ uation of RBV tested was 100 ⁇ M. PBMC were obtained from HIV-1 negative donors by banding on
  • Ficoll gradients stimulated with phytohemaglutinin (PHAP) for 48 hours prior to infection with HIV-I, and infected with virus for 4 hours at 37°C at a MOI of 0.001.
  • Infected cells were seeded into 96 well plates containing 3-fold serial dilutions of RBV. Plates were incubated for 3 days at 37°C. The concentration of virus in each well was determined using the NEN ⁇ 24 assay. Using the abso ⁇ tion values of the cell controls as
  • a dose response curve is generated by plotting percent protection on the Y axis and drug concentration on the X axis. From this curve, ECjo values were determined.
  • RBV inhibited HIV-1 replication in PBMCs with a median ECso of 20.5 ⁇ M ⁇ 11.8.
  • Combination assays were performed using varying concentrations of DAPD, DXG, Abacavir and AZT alone or with a fixed concentration of RBV. Five fold serial dilutions of test compound were performed on 96 well plated with the following drug concentrations: DAPD 100 ⁇ M, DXG 50 ⁇ M. Abacavir 20 ⁇ M and AZT 10 ⁇ M ' . The concentrations of RBV used were 1, 5, 10, 20, 40 and 60 ⁇ M. Assays were performed in the MT2 cell line as described above in the XXT sensitivity assay section. Addition of 40 and 60 ⁇ M RBV, in combination with the compounds listed above, as found to be toxic in these assays, therefore, ECjo values for the compounds were determined in the presence and absence of 1, 5, 10 and 20 ⁇ M RBV (Table 2).
  • Addition of 20 ⁇ M RBV had the greatest effect on the antiviral activity of DAPD and DXG with a 14-2 and 12 fold decrease in the apparent EC50 values respectively. Addition of RBV had no effect (less than 2 fold difference in the apparent EC 5 0) on the activity of Abecavir. Addition of 20 ⁇ M RBV resulted in a greater than 6-fold increase in the apparent EC W of AZT indicating that the combination is antagonistic with respect to inhibition of HIV. Similar results were obtained with the addition of 1, 5 and 10, ⁇ M RBV, although to a lesser extent than that observed with the higher concentration of RBV.
  • the effect of RBV on the activity of DAPD and DXG against mutant stiains of HIV was also analyzed (Table 4).
  • the restraint strains analyzed included viruses created by site directed mutagenesis, K65R and L74V, as well as a recombinant virus containing mutations at positions 98S, 116Y, I 1M and 215Y.
  • the wild type backbone in which these mutants were created, xxLAI was also analyzed for comparison.
  • the concentrations of DAPD and DXG tested were as described in the above MT2/XTT combination assay section. RBV was tested in combination with DAPD and DXG at a fixed concenuation of 20 ⁇ M.
  • the mutant viruses tested all demonstrated increased EC. ⁇ values (greater than four fold) for both DAPD and DXG indicating resistance to these compounds.
  • Addition of 20 ⁇ M RBV decreased the ECjo values of DAPD and DXG against these viruses.
  • the EC M values determined for DAPD and DXG in the presence of 20 ⁇ M RBV were at least 2.5-fold lower than those obtained for the wild type virus.
  • Combination assays were also performed in PBMCs using varying concentrations of DAPD, DXG, Abacavir and AZT alone or with a fixed concentration of RBV. Compound dilutions and assay conditions were as described above. The concentrations of RBV used were 1, 5, 10, 20, 40 and 60 ⁇ M. Addition of 40 and 60 ⁇ M RBV, in combination with the compounds listed above, was found to be toxic in these assays. The ECJO values determined for the compounds in the presence and absence of 1, 5, 10 and 20 ⁇ M RBV are shown in Table 5.
  • RBV inhibited the replication of HIV-1 in PBMCs with an ECso of 20.5 ⁇ M. Ribavirin was not toxic to these cells at concentrations up to 1 mM resulting in a therapeutic index of >48. Addition of 20 ⁇ M RBV to DAPD, DXG and Abacavir completely inhibited HIV replication in PBMCs at all the concentrations tested but had little effect on the activity of AZT. Addition of lower concentrations of RBV also had a significant effect on the activity of DAPD, DXG and Abacavir. In the MT2 cell line, RBV was not active against HIV replication.
  • MPA Mycophenolic acid
  • PBMCs using a XTT based assay using a XTT based assay.
  • the XTT (2,3-bis(2-fl»ethoxy-4-nitro-5-sulfophenyl)- 5[(phenyIamino)carbonyl]-2H-tetrazolium hydroxide) assay is an in vitro colorimeuic cyto-protection assay. Reduction of XTT by mitochondria dehydrogenases results in the cleavage of the tetrazolium ring of XTT, yielding orange fom-azan crystals, which are soluble in aqueous solution. The resultant orange solution is read in a spec ophoTometcr at a wavelength of 450nM.
  • MPA was prepared in 100% DMSO at a final concenUation of lOO M.
  • a 200 ⁇ M solution of MPA was prepared in cell culture media (RPMI supplemented with 10% fetal calf serum, L-Glutamine lmg/ml and 20ug/ml gentamicin) followed by 2 fold serial dilutions on a 96 well plate.
  • Cells were added to the plat at 3xl0 4 well (MTX) and 2xlO J /well (PBMC) and the plates were incubated for 5 days at 37 C in a 5% CO- incubator (addition of the cells to the plate diluted the compound to a final high concentration of lOO ⁇ M).
  • XTT was added to each well and incubated at 37*C for 3 hours followed by the addition of acidified isopropanol.
  • the plate was read at 450nm in a 96 well plate reader.
  • a dose response curve was generated using the absorption values of cells grown in the absence of compound as 100% protection.
  • MPA was toxic in both cell lines with a 50% cytotoxic does (CCJO) of 5.7 ⁇ M in the MT2 cell line and 4.5 ⁇ M in PBMC. See Table 7.
  • MPA was tested for activity against the xxLAl strain of HIV-1 in the laboratory adapted cell line MT2. Dilutions of MPA were made in cell culture media in a 96 well plate; the highest concentration tested was 1 ⁇ M. Triplicate samples of compound were tested.
  • MT2 cells were infected with xxLAI at a multiplicity of infection (MOI) of 0.03 for 3 hours at 37°C In 5% CO ⁇ . The infected cells were plated at 3.0 x IOVwell into a 96 well plated containing drug dilutions and incubated for 5 days at 37°C in COj.
  • the antiviral activity of MPA was determined using the XTT assay described above. This method has been modified into a susceptibility assay and has been used in a variety of in vitro antiviral tests and is readily adaptable to any system with a lytic virus (Weislow,
  • MPA was also tested for activity against the xxLAI strain of HIV-I in PBMCs using a p24 based Elisa assay In this assay, cell supernatants are incubated on microelisa wells coated with antibodies to HI V-l p24 core antigen. Subsequently, anti-
  • HIV- 1 conjugate labeled with horse radish peroxidase is added.
  • the labeled antibody binds to the solid phase antibody/antigen complexes previously formed.
  • Addition of the tetramethylbenzidine substrate results in blue color formation. The color turns yellow when the reaction is stopped.
  • the plates are then analyzed on a plate reader set at 490 nm. The absorbance is a direct measurement of the amount of HIV-1 produced in each well and a decrease in color indicates decreased viral production. Dilutions of MPA were made in cell culture media in a 96 well plate, the highest concentration of MPA tested was 1 ⁇ M.
  • PBMC peripheral blood mononuclear cells
  • Infected cells were seeded into 96 well plates containing 4-fold serial dilutions of MPA Plates were incubated for 3 days at 37 ⁇ C The concentration of virus in each well was determined using the NEN p24 assay. Using the absorption values of the cell controls as lOwTprotection and drug free, vimsTnfectwTcells as 0% " profectiori, a dose response curve is generated by plotting % protection on the Y axis and drug concentration on the X axis. From this curve EC 5 0 values were determined.
  • MPA inhibited HIV-1 replication in PBMCs with a median EC$o of 95 nM ⁇ 29.
  • Combination assays were performed using varying concentrations of DAPD, DXG, Abacavir, AZT and FTC alone or with a fixed concentration of MPA.
  • Five fold serial dilutions of test compound were performed on 96 well plated with the following drug concentrations: DAPD - 100 ⁇ M, DXG - 50 ⁇ M, Abacavir - 20 ⁇ M and AZT - 10 ⁇ M, and FTC - 10 ⁇ M.
  • the concentrations of MPA used were 1, 0.5, 0.25, 0.1, and 0.01 ⁇ M.
  • Assays were performed in the MT2 cell line as described in section 3.1.
  • Table 9 illustrates the fold differences in ECso values obtained for each of the compounds in combination with 0.1 and 0.25 ⁇ M MPA.
  • the effect of MPA on the activity of DAPD and DXG against mutant strains of HIV was also analyzed (Table 10).
  • the restraint strains analyzed included viruses created by site directed mutagenesis, K65R and L74V, as well as a recombinant virus containing mutations at positions 98S, 1 16Y. 151M and 215 Y.
  • the wild type backbone In which these mutants were created, xxLAI was also analyzed for comparison.
  • the concentrations of DAPD and DXG tested were as described In section 4.1.
  • MPA was tested in combination with DAPD and DXG at a fixed concenUation of 0.25 ⁇ M.
  • DAPD and DXG were active against all of the wild type strains of HIV tested.
  • the mutant viruses tested all demonstrated increased EC50 values for both DAPD and DXG indicating resistance to these compounds.
  • Addition of 0-25 ⁇ MPA decreased the ECso values of DAPD and DXG against these viruses.
  • These values determined for DAPD and DXG in the presence of 025 ⁇ M MPA were similar to those obtained for the wild type virus.
  • Combination assays were also performed in PBMCs using varying concentrations of DAPD- DXG, Abacavir, AZT and FTC alone or with a frxed concentration of MPA.
  • Mycophenolic acid inhibited the replication of HIV-1 in PBMCs with an EC» of 0.095 ⁇ M. CCso value obtained for MPA in these cells were 4.5 ⁇ M resulting in a therapeutic index of 47.
  • Addition of 0.25 ⁇ M MPA to DAPD, DXG and Abacavir completely inhibited HIV replication in PBMCs at all the concentrations tested but had little effect on the activity of AZT and FTC (less than 2 - fold change in ECso- Addition of lower concentrations of MPA also had a significant effect on the activity of DAPD, DXG but had little effect on the activity of Abacavir, AZT and FTC. In the MT2 cell line, MPA was not active against HIV replication.
  • PBMC peripheral blood mononuclear cells
  • the bioanalytical method for the analysis of DXG-TP from peripheral blood mononuclear cells utilizes ion-pair solid phase extraction (SPE) and ion-pair HPLC coupled to electrospray ionization (ESI) mass spectrometry.
  • SPE solid phase extraction
  • ESI electrospray ionization
  • Pelleted PBMC samples containing approximately 0.5 x 10 7 cells are diluted with a solution containing the internal standard (2 ⁇ S'-dideoxycytidine-S'- uiphosphate (ddCTP)) and the DXG-TP and ddCTP are selectively extracted using ion-pair SPE on a C-l 8 cartridge.
  • ddCTP S'-dideoxycytidine-S'- uiphosphate
  • the DXG-TP and ddCTP are separated with mkrobore ion-pair HPLC on a Waters Xterra MS C18 analytical column with retention times of about 10 minutes.
  • the compounds of interest are detected in the positive ion mode by ES1-MS/MS on a Micromass Quattro LC Uiple quadrupole mass spectrometer.
  • DAPD DAPD
  • DXG and FTC emtricitabine
  • Glaxo Cells Cytotoxicity and activity assays were performed in the T-cell line MT2 and in peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Viruses were created by subcloning the HIV protease and RT coding sequences obtained from patient plasma HIV RNA into a modified version of the plasmid xxLAI. Amplification and cloning strategies have been previously described in the art. Recombinant viruses contained mutations in the HIV-RT at positions A98S, F116Y, Q151M, T2J5Y and D67N, T69D, K70R, K103N, M184V, T215Y, K219Q.
  • Viruses containing mutations at positions K65R, L74V, Q151M and K65R Q1S1M of the HIV-RT were generated by site directed mutagenesis of the xxLAI plasmid using the QuickCha ⁇ ge Site-Directed Mutagenesis kit from Stratagene. Genotypic analysis of the recombinant viruses and of viruses obtained by site directed mutagenesis was performed by didcoxy sequencing using ABI Prism 377* technology.
  • Anti-HIV assays were performed using two different assay methodologies.
  • a cytotoxicity-based assay (XTT) was performed to evaluate activity in MT2 cells and an HIV-1 p24 based enzyme linked immunoabsorbant assay (ELISA) (Organon Teknika Corporation) was performed to evaluate activity in PBMCs,
  • ELISA enzyme linked immunoabsorbant assay
  • MT2 cells were infected with cither the mutant virus or wild type LAI at a multiplicity of infection (MOI) of 0.03 in RPMI 1640 medium containing 10 % fetal bovine serum, 20 ⁇ g ml gentamicin (Life Technologies), and 2 ⁇ g/ml polybrene (Sigma) for 2 hours at 37°C.
  • MOI multiplicity of infection
  • test compounds were tested in triplicate at five-fold serial dilutions.
  • the infected cells were cultured for 5 days in the presence of test compounds.
  • XTT was added and the plates were analyzed.
  • a dose response curve for each individual compound was generated using the abso ⁇ tion values of the uninfected cell controls as 100% protection and no drug; virus infected cells as 0% protection. From the dose response curve, an ECso (50% effective concentration) inhibited viral induced CPE by 50%.
  • PBMC peripheral blood mononuclear cells
  • ELISA assay for the detection of HIV-I p24 core antigen.
  • PBMCs were infected with HIV- 1 virus at en MOI of 0.001 for 4 hours at 37°C, and plated in the presence of drugs as described above. Infected cells were cultured for 4 days. On day four, the amount of HIV-1 p24 was determined in each well. Uninfected cells were used as background and readings from the virus control were considered 100% infection.
  • Cytotoxicity Assays MT2 cells and PHAP-stimulated human PBMCs were seeded at densities of 3 X 10 4 and I X 10 s cells/well, respectively, in 96-well plates containing two-fold serial dilutions of DAPD or DXG.
  • a fixed concentration of RBV (I, 5, 10, 20, 40, and 60 ⁇ M) or MPA (0.01, 0.1 0-25 0.5 and 1 ⁇ M) was added to the DAPD/DXG dilutions. The cultures were incubated for 5 days at
  • DNA from each sample was denatured by adding an equal volume of denaturing buffer (0 8 M NaOH, 20 mM EDTA) and heating to 100" C for 10 min.
  • the DNA samples were blotted onto positive-charged nylon membranes washed once with 0.4 M NaOH followed by a single wash with 2X SSPE.
  • the DNA was cross-linked to the membrane by exposing to UV light.
  • a specific ⁇ 2 P-probe encompassing nucleotide position 4212-4242 of human mtDNA was used to quantitate the level of mtDNA in each sample.
  • Cellular DNA was quantitated using a 32P-probe specific for human glyceraldehyde 3-phosphate dehydrogenase.
  • the quantity of mtDNA was normalized to the amount of cellular DNA resulting in relative mtDNA levels.
  • the relative levels of mtDNA from the treated cells were compared to the relative mtDNA levels of the untreated cell controls.
  • the data from 3 experiments were combined and the data reported as the mean + the standard deviation.
  • Mycophenolic acid and RBV were tested for activity against the LAI strain of
  • Activity of DAPD and DXG in Combination with MPA and RBV The effect of MPA and RBV on the activity of DAPD and DXG in the MT2 XTT and PBMC/p24 assays as described above was evaluated. The combination of MPA and RBV with abacavir or AZT was also evaluated.
  • Abacavir was used as a positive conUol; the active form of abacavir (carbovir-triphosphate) is also a guanosine analogue and has been shown to have synergistic anti-HIV activity when combined with MPA.
  • ACT was used as a negative control.
  • the combination of RBV and AZT is antagonistic with regards to HIV replication, possibly as a consequence of increased levels of oTTP which acts as a feedback inhibitor of thymidine kinase activity.
  • addition of low concentrations of MPA (0.01 ⁇ M) had no effect on the anti-HJV activity of any of the compounds tested.
  • a direct relationship between the concenUation of MPA or RBV and the level of synergy seen in combination with DAPD and DXG was observed.
  • the combination of 0.25 ⁇ M MPA with DAPD or DXG produced the greatest results (16.7- and 10.5-fold increase in the activity of DAPD and DXG respectively) without any cytotoxicity.
  • addition of 0.25 ⁇ M MPA resulted in a 2.3 fold decrease in the activity of AZT. Little effect was noted on the activity of abacavir (less than 2-fold) at any of the MPA concentrations tested.
  • DAPD and DXG were previously reported to have little effect on mitochondrial DNA (mtDNA) synthesis.
  • MtDNA synthesis was evaluated in HepG2 cells incubated with 0.25 ⁇ M MPA or 20 ⁇ M RBV alone and in combination with DAPD or DXG to determine if the presence of an IMPDH inhibitor would effect mtDNA synthesis.
  • RBV concenuations up to 80 ⁇ M did not affect mtDNA synthesis or cell growth, whereas concentrations of MPA greater than 0.25 ⁇ M caused a dose dependent increase in cell death and decrease in mtDNA synthesis. Consistent with previously reported results,
  • DAPD did not cause a significant reduction in mitochondrial DNA synthesis at clinically relevant concentrations.
  • the combination of 20 ⁇ M RBV and DAPD did cause an inhibition of mtDNA synthesis at the higher concentrations of DAPD.
  • MtDNA synthesis was not appreciably affected when cells were exposed to 0.25 ⁇ M MPA and DAPD concentration up to 25 ⁇ M.
  • DXG did not affect mtDNA synthesis at the highest concentration used in this study (50 ⁇ M).
  • the addition of MPA did not alter the lack of inhibition of mtDNA synthesis seen with DXG.
  • the combination of DXG and RBV did appear to cause some inhibition of mtDNA synthesis at the highest
  • Table 3a Effect of increasing concentrations of MPA and RBV on the activity of DAPD and DXG against virus containing the K65R/QI 51M mutations.
  • Table 4a shows the effects on mitochondrial function of DAPD, DXG, MPA and RBV (%mt DNA ⁇ SD relative to controls )
  • Dioxolane guansine is a nucleoside analog of deoxyguanosine that has potent activity in vitro and in vivo against wild-type HIV-1 and viruses that carry mutations which confer resistance to AZT, 3TC, and to non-n ⁇ cleoside reverse transcriptase inhibitors. Resistance to DAPD/DXG was conferred by mutations at positions 65, 74, and 151 in the RT. The ability of two inhibitors of IMPDH, RBV and MPA, to enhance the anti-viral activity of DAPDDXG against both wild-type and resistant virus was confirmed. Both RBV and mycophenolic acid when combined with DAPD or DXG gave a strong synergistic anti-HIV response against wild-type virus.
  • the L74V, K65R and QI5IM mutations have all been shown " to conftr variouT ⁇ evels of resistance to DXG7
  • the combination of 25 ⁇ MPA oT20 " ⁇ M RBV resulted in a decrease in EC» values of DXG for these mutants to within two- fold of the values obtained for wild-type virus
  • addition of increasing concentrations of MPA (up to 0.2 ⁇ M) or RBV (up to 10 ⁇ M) resulted in a greater than 50-fold increase in the activity of both DAPD and DXG against the highly DXG resistant K65R/Q1 SIM mutant.
  • DAPD this combination can be of particular use in salvage therapy particularly when combined with multiple anti-retroviral agents.
  • the combination of DAPD and MPA against HIV may be used in the salvage setting.
  • PBMC Peripheral Blood Mononuclear Cells
  • FBS gentamicin & Fetal Bovine Serum
  • PBMC peripheral blood mononuclear cells
  • Samples are processed through a wash procedure and resuspended in a methanol solution for analysis of uiphosphates. Analysis ofiriphosph ⁇ tes Sample Prep: Samples are sonicated then processed with ion pairing solid phase extraction (SPE) to isolate nucleotides.
  • SPE solid phase extraction
  • SPE extract is analyzed for nucleotide triphosph tes wiu positive ion electrospray ionization tandem mass spectrometry coupled to ionpairing high-performance liquid chromatography.
  • DXG-TP Quantitative range: 0.0064 - 1.3 pmoles/10* cells
  • DAPD-TP Lower limit of detection: 0.0088 pmoles/10 6 cells

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Abstract

Compositions, including a combination of a β-D-1,3-dioxolanyl nucleoside and an IMPDH inhibitor are provided that can be used to treat HIV and HBV in a host. In some embodiment, a drug resistant strain of HIV and HBV exhibit the behavior of drug-naïve virus when given the In one nonlimiting embodiment, the HIV strain is resistant to a ß-D-1,3-dioxolanyl nucleoside. In another nonlimiting embodiment, the HBV strain is resistant to a β- D-1,3-dioxolanyl nucleoside.

Description

COMBINATION THERAPY WITH 1,3-DIOXOLANES AND INOSINE MONOPHOSPHATE DEHYDROGENASE INHIBITORS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Application No. 60/393,935, filed July 3, 2002, the disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention is pharmaceutical compositions and methods for the treatment or prophylaxis of human immunodeficiency virus (HIV) infection and/or hepatitis B virus (HBV) infection in a host comprising administering such compositions.
BACKGROUND OF THE INVENTION
It has been recognized that drug-resistant variants of HIV (human immunodeficiency virus) can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and most typically in the case of HIV, reverse transcriptase, protease or DNA polymerase. Recently, it has been demonstrated that the efficacy of a drug against HIV infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug.
Alternatively, the pharmacokinetics, metabolism, biodistribution or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically over alternation therapy because it induces multiple simultaneous pressures on the virus. One cannot predict, however, what mutations will be induced in the HIV-I genome by a given drug, whether the mutation is permanent or transient, or how an infected cell with a mutated HIV-] sequence will respond to therapy with other agents in combination or alternation. This is exacerbated by the fact that there is a paucity of data on the kinetics of drag resistance in long-term cell cultures treated with modem antiretr virai agents.
HIV replication is dependent upon the host cell to provide the necessary substrates for viral replication, including deoxynucleoside triphosphates for the reverse transcription of viral RNA into double stranded DNA by the HIV-encoded reverse transcriptase (HIV-RT). Nucleoside reverse transcriptase inhibitors (NRTls) of the HIV- RT such as AZT, 3TC, ddl, D4T, and more recently abacavir, have formed the comer stone of anti-HIV therapy. These NRTls must first be converted to their S'-triphospbates and must be able to successfully compete with the natural nucleotides of the host cell to effectively inhibit virus replication. Reducing the level of endogenous nucleotides can shift the competition in favor of the nucleotide analogue. Hydroxyurea, and more recently mycophenolic acid, have been used to target enzymes involved in the de nσvo synthesis of deoxynucleotides. Hydroxyurea has been used to target cellular ribonucleotide reductase that, through a complex pattem of regulation, provides the appropriate supply of the four deoxynucleotides needed for the synthesis of DNA. Hydroxyurea may exert its effect by decreasing the endogenous deoxynucleotide pools thereby resulting in a relative increase in the intracellular concentration of the 5'- triphosphate of the NRTI. In vitro, synergistic anti-HIV activity was observed between ddl and hydroxyurea. In addition, HIV variants resistant to ddl are significantly more sensitive to the drug in the presence of hydroxyurea. The combination of hydroxyurea and ddl has been extensively used in a number of clinical studies and the results from these studies confirm the efficacy of the combination.
HIV-I variants resistant to 3'-a2ido-3'-deoχythymidine (AZT), 2\3'- dideoxyinosine (DD1) or 2',3'-dideoxycytidine (DDC) have been isolated from patients receiving long term monotherapy with these drugs (Larder BA, et at. Science 1989;243:1731-4; St Clair MH, et al. Science 1991;253:1557-9; St Clair MH, et al. Science 1991;253:1557-9; and Fitzgibbon JE, el al. Antimicrob Agents Chemoiher 1 92;36:153-7). Mounting clinical evidence indicates that AZT resistance is a predictor of poor clinical outcome in both children and adults (Mayers DL. Lecture at the Thirty- second lnterscience Conference on Antimicrobial Agents and Chemotherapy. (Anaheim, CA. 1992); Tudor- illiams G, St Clair MH, McKlnney RE, et al Lancet 1992;339:15-9; Ogino MT, Dankner WM, Spector SA. J Pediatr 1993;123:1-8; Crumpacker CS, D'Aquila RT, Johnson VA, et al Third Workshop on Viral Resistance. (Gaithersburg, MD. 1993); and Mayers D, and the RV43 Study Group. Third Workshop on VTraT Resistance. (Gaithersburg, MD. 1993)). The rapid development of HIV-I resistance to nonnucleoside reverse transcrtptase inhibitors (NNRTIs) has also been reported both in cell culture and in human clinical trials (Nunberg JH, Schleif WA, Boots EJ, et <Λ. J Virol 1991;65(9):4887-92; Richman D, Shih CK, Lowy 1, et al. Proc Natl Acad Sci (USA) I991;88 :11241-5, MeHors JW, Dutsch an GE, l GJ, Tramontano E, Winkler SR, Cheng YC. Mol Pharm 1 92; 1:446-51; Richman DD and the ACTG 164/168 Study
Team. Second International HIV-1 Drug Resistance Workshop. (Noordwijk, the Netherlands. 1993); and Saag MS, Emini EA, Laskin OL, et al. N Engl J Med 1993;329:1065-1072). In the case of the NNRTI L'697,661, drug-resistant HIV-1 emerged within 2-6 weeks of initiating therapy in association with the return of viremia to pretreatment levels (Saag MS, Emini EA, Laskin OL, et al. N Engl J Med
1993;329:1065-1072). Breakthrough viremia associated with the appearance of drug- resistant strains has also been noted with other classes of HIV-I inhibitors, including protease inhibitors (Jacobsen H, Crβig CJ, Duncan IB, Haenggi M, Yasargil K, Mous J. Third Workshop on Viral Resistance. (Gaithersburg, MD. 1993)). This experience has led to the realization that the potential for HIV-1 drug resistance must be assessed early on in the preclinical evaluation of all new therapies for HIV-1.
Hepatitis B virus ("HBV") is second only to tobacco as a cause of human cancer.
The mechanism by which HBV induces cancer is unknown, although it is postulated that h may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis and cell regeneration associated with the infection.
Hepatitis B virus has reached epidemic levels worldwide. After a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed. Patients typically recover from acute viral hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic infection. Chronic infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver and hepatocellular carcinoma, a primary liver cancer. In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is in fact very similar to that of acquired immunodeficiency syndrome, which accounts for why HBV infection is common among patients with AIDS or HIV-associated infections. However, HBV is more contagious than HIV.
Daily treatments with α-interferon, a genetically engineered protein, have shown promise. A human serum-derived vaccine has also been developed to immunize patients against HBV. Vaccines have been produced through genetic engineering, While the vaccine has been found effective, production of the vaccine is troublesome because the supply of human serum from chronic carriers is limited, and the purification procedure is long and expensive. Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. In addition, the vaccine does not help the patients already infected with the virus.
An essential step in the mode of action of purine and pyrimidine nucleosides against viral diseases, and in particular, HBV and HIV, is their metabolic activation by cellular and viral kinases, to yield the mono-, di- and triphosphate derivatives. The biologically active species of many nucleosides is the triphosphate form, which inhibits
DNA polymerase or reverse transcriptase, or causes chain termination.
A number of synthetic nucleosides have been identified which exhibit activity against HBV. The (-)-enantiomer of BCH-189 (2',3'-dideoxy-3'-thiacytidine), known as
3TC has been approved for the treatment of hepatitis B (sold under the name Lamivu ine by GlaxoWellcome). See EPA 0494 1 1 Al filed by BloChem Pharma, Inc. β-2-Hydroxymethyl-5-(5-fluor cytosin-l-yI)-l,3<oxathiolane (TTC), claimed in U. S. Patent Nos. 5,814,639 and 5,914,331 to Liotta et al., exhibits activity against HBV. See Furman et al., 'The Anti-Hepatitis B Virus Activities, Cytotoxicities, and
Anabolic Profiles of the (-) and (+) Enantiomers of cis-5-Fluoro-l-{2-(Hydroxymethyl)- l,3-oxathiolane-5-yl)-Cytosine" Antimicrobial Agents and Chemotherapy. December 1992, page 2686-2692; and Cheng, et al., Journal of Biological Chemistry. Volume 267(20)7Ϊ3938.13942~(I992)."
U.S. Patent Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu, et al.) disclose the use of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) for the treatment of hepatitis B and Epstein Barr virus.
Penciclovir (PCV; 2-amino-l,9-dihydro-9-{4-hydroxy-3-(hydroxymethyl)butyI}- 6H-purin-6-one) has established activity against hepatitis B. See U.S. Patent Nos. 5,075,445 and 5,684,153. Adefovir (9-{2-(pho$ρhonomethoxy)ethyl}adenine, also referred to as PMEA or
{{2-(6-amino-9H-purin-9-yl)ethoxy}methy]phosρhonic acid), has been approved for ueatment of hepatitis B infected patients (sold by Gilead Sciences, Inc. under the name Hepsera; see for example U.S. Patent Nos.5,641 ,763 and 5, 142,0 1.)
Yale University and The University of Georgia Research Foundation, Inc. disclose the use of L-FDDC (5-fluoro-3 '-thia-2',3 '-dideoxycytidine) for the treatment of hepatitis B virus in WO 92/18517.
Other drugs explored for the treatment of HBV include adenosine arabinoside, thymosin, acyclovir, phosphonofoimate, zidovudmc, (+ cyanidanol, quinacrinε, and 2'- fluoroarabinosyl-5-iodouracil. U.S. Patent Nos. 5,444,063 and 5,684,010 to Emory University disclose the use of enantiomerically pure β-D-l,3-dioxolane purine nucleosides to treat hepatitis B
WO 96740164 filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses a number of β-L-2',3'- dideoxyπucleosides for the treatment of hepatitis B. WO 95 07287 also filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses 2' or 3' deoxy and 2\3'-dideoxy-β-L-pentofuranosyl nucleosides for the eatment of HIV infection.
W096/I 512 filed by Geπencor International, Inc., and Lipitek, Inc., discloses the preparation of L-ribofuranosy] nucleosides as antitumor agents and virucides. W095/32984 discloses lipid esters of nucleoside monophosphates as immuno- suppresive drugs.
DE 4224737 discloses cytosine nucleosides and their pharmaceutical uses.
Tsai et al., in Biochem. Pharmacol. 1994, 48(7), 1477-81, disclose the effect of the anti-HIV agent 2,-β-D-F-2'r3,-dideoxynucleoside analogs on the cellular content of mitochondrial DNA and lactate production.
Galvez, J. Chem. Inf. Comput. Sci. 1994, 35(5), 1198-203, describes molecular computation of β-D-3'-azido-2,,3,-dideoxy-5-fluorocytidlne.
Mahmoudian, Pharm. Research 1991, 8(\), 43-6, discloses quantitative structure- activity relationship analyses of HIV agents such as β-D-3'-azido-2',3'-dideoxy-5- fluorocytidine.
U.S. Patent No. 5,703,058 discloses (5-carboximido or 5-fluoroM2',3'- unsaturated or 3 '-modified) pyrimidine nucleosides for the treatment of HIV or HBV.
Lin et al., discloses the synthesis and antiviral activity of various 3'-azido analogues of β-D nucleosides in J. Med. Chem. 31(2), 336-340 (1988).
WO 00/03998 filed by Idenix Pharmaceuticals, Ltd. discloses methods of preparing substituted 6-benzy -oxopyrimIdines, and the use of such pyrimidines for the treatment of HIV.
Idenix Pharmaceuticals disclosed the use of 2'-deoxy-β-L-erythropεnto- furanonucleosides, and their use in the treatment of HBV in WO 00/09531. A method for the treatment of hepatitis B infection in humans and other host animals is disclosed that includes administering an effective amount of a biologically active 2'-deoxy-β-L- erythro-pentofuraπonucleoside (alternatively referred to as β-L-dN or a β-L-2'-dN) or a pharmaceutically acceptable salt or prodrug thereof, including β-L-deoxyribothymidine (B-L-dT), β-L-deoxyribocytidiπe (β-L-dC), β-L-deoxyribouridine (β-L-dU), β-L- deoxyribo-guanosine (β-L-dG), β-L-deoxyriboadenosine (β-L-dA) and β-L- deoxyriboinosine (β-L-dl), administered either alone or in combination, optionally in a pharmaceutically acceptable carrier. 5' and N4 (cytidine) or N* (adcnosine) acylated or alkylated derivatives of the active compound, or the S'-phospholipid or S'-ether lipids were also disclosed.
1-3-DioxoIanvl Nucleosides
The success of various synthetic nucleosides in inhibiting the replication of HIV/HBV in vivo or in vitro has led a number of researchers to design and test nucleosides that substitute a heteroatom for the carbon atom at the 3'-position of the nucleoside. Norbeck, et al., disclosed that (+/-)- l-{(2-β,4-β)-2-(hydroxymethyl)-4- dioxoIanyl]-thymine (referred to as (+/- dioxolane-T) exhibits a modest activity against HIV (ECso of 20 μM in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 μM. Tetrahedron Letters 30 (46), 6246, (1989).
On April 11, 1988, Bernard Belleau, Dilip Dixit, and Nghe Nguyen-Ba at BioChem Pharma filed patent application U.S.S.N. 07/179,615 which disclosed a generic group of racemic 2-substituted-4-substUuted-l,3-dioxolane nucleosides for the treatment of HIV. The '615 patent application matured into European Patent Publication No. 0 337 713; U.S. Patent No. 5,041,449; and U.S. Patent No. 5,270-315 assigned to BioChem
Pharma, Inc.
On December 5, 1990, Chung K. Chu and Raymond F. Schinazi filed U.S.S.N. 07/622,762, which disclosed an asymmetric process for the preparation of enantiomerically enriched β-D-l,3-dioxolane nucleosides via stereospecijlc synthesis, and certain nucleosides prepared thereby, including (-M2R,4R)-9-[(2-hydroxymethyl)- l,3-dioIoan-4-yl]guanine (DXG), and its use to treat HIV. This patent application issued as U.S. Patent No.5,179,104.
Figure imgf000008_0001
DXG On May 21, 1991, Tarek Mansour, et al., at BioChem Pharma filed U.S.S.N. 07/703,379 directed to a method to obtain the enantiomers of 1,3-dioxolane nucleosides using a stereoselectivc synthesis that includes condensing a l,3^i6xolane intermediate" covalently bound to a chiral auxiliary with a silyl Lewis acid. The corresponding application was filed in Europe as EP 0 515 156.
On August 25, 1992, Chung K. Chu and Raymond F. Schinazi filed U.S.S.N. 07 935,515, disclosing certain enantiomerically enriched β-D-dioxoIanyl purine compounds for the treatment of humans infected with HIV of the formula:
Figure imgf000009_0001
wherein R is OH, CI, NH: or H, or a pharmaceutically acceptable salt or derivative of the compounds optionally in a pharmaceutically acceptable carrier or diluent. The compound wherein R is ehloro is referred to as (-M2R,4R)-2-arnino-6-chloro-9-[(2- hydroxymcthyl)-l,3-dioxolan-4-yl]purine. The compound wherein R is hydroxy is (•)- (2R,4R)-9-[(2-hydroxy-methyl l,3-dioxolan-4-yl]guaniπe. The compound wherein R is amino is (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l,3-dioxolan-4-yl]adenine. The compound wherein R is hydrogen is (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l,3- dioxolan-4yl]purine. This application issued as U.S. Patent Nos. 5,925,643 and 5,767,122.
In 1992, Kim et al., published an article teaching how to obtain (-)-L-β- dioxolane-C and (-t-)-L-β-dioxolane-T from 1,6-anhydro-L-β-glυcopyranose. Kim et al.,
Potent anti-HIV and anti-HBV Activities of (-H-β ioxolane-C and (+)-L-β-Dioxolane- Tand Their Asymmetric Syntheses, Tetrahedron Letters Vol 32(46), pp 5899-6902.
On October 28, 1992, Raymond Schinazi filed U.S.S.N. 07/967,460 directed to the use of the compounds disclosed in U.S.S.N. 07/935,515 for the treatment of hepatitis B. This application has issued as U.S. Patent Nos. 5,444,063; 5,684,010; 5,834,474; and
5,830,898. In 1993, Siddiqui, et al., at BioChem and Glaxo published that cis-2,6- diaminopurine dioxolanc can be deaminated selectively using adenosine deaminase.
Siddiqui, et al., Antiviral Optically Pure dioxolane Purine Nucleoside Analogues, Bioorganic ά Medicinal Chemistry Letters, Vol. 3 (8), pp 1543-1546 (1993). PCT/CA99/01229 and U.S. Patent No. 6,358,963, also by Shire, disclose certain
1,3-dioxolane purine nucleosides and their use to treat viral infections. PCT/CAOO/00212 and U.S. Patent No. 6,511,983, by Shire, disclose pharmaceutical combinations of antiviral agents.
Amdoxovir [DAPD, (-)-β-D-2,6-diaminopurine dioxolane)]is an aqueous, soluble and bioavailable pro-drug that is rapidly absorbed and converted in vivo to DXG, (-)-β-
D-dioxolane guanosiπe (Furman et. al., Gu et. al.). (-)-(2R.4R).2-amino-9-[(2- hydroxymcthyl)-l,3-dioxolan-4-yl]adenine (DAPD) is a selective inhibitor of HIV-1 replication in vitro as a reverse transcriptase inhibitor (RTI). DAPD is thought to be deaminated in vivo by adenosine deaminase, a ubiquitous enzyme, to yield (-)-β-D- dioxolane guanine (DXG), which is subsequently converted to the corresponding 5'- triphosphate (DXG-TP). Biochemical analysis has demonstrated that DXG'TP is a potent inhibitor of the HIV reverse transcriptase (HIV-RT) with a Ki of 0.019 μM. In vitro, the anti-HIV activity seen upon treatment with DAPD is almost entirely due to the generation of DXG by the action of adenosine deaminase.
Figure imgf000010_0001
DAPD
Ribavirin
Ribavirin (l-β-D-ribofuranosyl-l,2,4-triazole-3-carboxamide) is a synthetic, non- interferon-inducing. broad spectrum antiviral nucleoside analog sold under the trade name Virazole (The Merck Index, 1 lth edition, Editor: Budavart, S., Merck & Co., Inc., Rahway, NJ, pl304, 1 89). U.S. Patent No. 3,798,209 and RE29,835 disclose and claim ribavirin.
Ribavirin (RBV) is a purine analog with a broad spectrum of antiviral activity (Sidwell et. al.). RBV monophosphate is also an inhibitor of IMPDH and has been shown to enhance the anti-HIV activity of didanosine in vitro (Palmer and Cox). As with MPA and hydroxyurea, the synergistic effects of RBV in combination with specific NRTls is attributed to changes in the intracellular dNTP pools.
In the United States, ribavirin was first approved as an aerosol form for the treatment of a certain type of respiratory virus infection in children. Ribavirin is structurally similar to guanosiπe, and has in vitro activity against several DNA and RNA viruses including Flavmridae (Gary L. Davis Gastroenteroloev ] )8:S104-Sl 14, 2000).
Ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA (Gary L. Davis Gastroenterolopγ 1 18:S104- SI 14, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. It is being studied in combination with DDI as an anti-HIV treatment. More recently, it has been shown to exhibit activity against hepatitis A, B and C. Since the beginning of the
AIDS crisis, people have used ribavirin as an anti-HIV treatment, however, when used as a monotherapy, several controlled studies have shown that ribavirin is not effective against HIV. It has no effect on T4 cells, T8 cells or p24 antigen.
The combination of IFN and ribavirin for the treatment of HCV infection has been reported to be effective in the treatment of IFN naϊve patients (Battaglia, A.M. et al., Ann. Pharmacother. 34:487-494, 2000). Results are promising for this combination treatment both before hepatitis develops or when histological disease is present (Berenguer, M. et al. Aniivir. Ther. 3(Suppl. 3):125-136, 1998). Side effects of combination therapy include hemolysis, flulike symptoms, anemia, and fatigue (Gary L. Davis. Gastroentcrology 1 18:S104-Sl 14, 2000).
Figure imgf000012_0001
RIBAVIRIN
MycopheBolie Acid
Mycophenolic acid (6-(4-hydroxy-6-mcthoxy-7-mcthyl-3-oxo-5-ρhthalanyl)-4- methyl-4-hexanoic acid) is known to reduce the rate of de novo synthesis of guanosine monophosphate by inhibition of inosine monophosphate dehydrogenase ("IMPDH"). It also reduces lymphocyte proliferation.
Lymphocytes and monocytes rely on the de-novo pathway of guanosine synthesis. MPA selectively inhibits lymphocyte and monocyte proliferation. In addition, MPA has been shown to inhibit HIV replication in these cells presumably through reduction of dGTP pools (Ichimura and Levy, Chapuis et. al). MPA has been shown to increase the in im anti-HIV activity of abacavir when used in combination (Margolis et. al.). Several pilot clinical studies have been conducted to evaluate the effect of MPA (given as the prodrug mycophenolate mofetil, MMF) (Coull, Chapuis and Margolis) in combination with HAART. Overall, therapy with MMF resulted in a transient decrease in HIV viral load in the majority of patients. In addition, there were no significant changes in the levels of CD4+ cells following 24 weeks of treatment.
Figure imgf000012_0002
YCOPHENOLIC ACID
Scientists have shown that mycophenolic arid has a synergistjc effect when combined with Abacavir (Ziagen) in vitro. Mycophenolic acid depletes guanosine, one of the essential DNA building blocks. Abacavir is an analog of guanosine and as such, must compete with the body's natural production of guanosine in order to have a therapeutic effect. By depleting naturally occurring guanosine, mycophenolic acid improves Abacavir's uptake by the cell. Scientists have determined that the combination of mycophenolic acid and Abacavir is highly active against Abacavir-resistant virus. However, notably the combination of mycophenolic acid and zidovudine or stavudine was antagonistic, likely due to the inhibition of thymidine phosphorylation by mycophenolic acid. 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, California, September 26-29, 1999. Heredia, A., et al. (1999), J. Acquir Immune Defic Syndr.; 22:406-7. Margolis, et al.,(1999) J Acquir Immune Defic Syndr., 21:362-370. U.S. Patent No. 4,686,234 describes various derivatives of mycophenolic acid, its synthesis and uses in the treatment of autoimmune disorders, psoriasis, and inflammatory diseases, including, in particular, rheumatoid arthritis, tumors, viruses, and for the ueatment of allograft rejection.
On May 5, 1995, Morris et al., in U.S. Patent No. 5,665,728, disclosed a method of preventing or treating hypeφroliferative vascular disease in a mammal by administering an antiproliferative effective amount of rapamycin alone or in combination with mycophenolic acid.
IMPDH
In the_de novo_purine synthesis
Figure imgf000014_0001
responsible for the conversion of inosine monophosphate (IMP) to guanosine monophosphate (GMP), which is normally converted to GDP, GTP, and dGTP. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to
NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Studies with various cell types, including lymphocytes, have shown that inhibiting IMPDH causes a reduction in the intercellular levels of GTP and dGTP. Mycophenolate mofctil is a prodrug which is rapidly converted to mycophenolic acid (MPA), a potent and reversible uncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH). RBV monophosphate, the active metabolite of the antiviral agent RBV, is a substrate mimic of IMP and as such functions as a competitive inhibitor of IMPDH. It has been postulated that the mechanism by which IMPDH inhibitors enhance the antiviral activity of certain purine nucleoside analogues is through the reduction of the levels of dGTP thereby shifting the advantage of the competition between dGTP and the 5'-nucleoside analogue triphosphate for the HIV-RT in favor of the nucleotide analog.
In light of the global threat of the HIV epidemic, it is an object of the present invention to provide new methods and compositions for the ueatment of HIV. It is another object of the present invention to provide methods and compositions to treat drug resistant strains of HIV.
In light of the fact that hepatitis B virus has reached epidemic levels worldwide, and has severe and often uagic effects on the infected patient, there remains a strong need to provide new effective pharmaceutical agents to treat humans infected with the virus that have low toxicity to the host.
Therefore, it is another object of the present invention to provide compositions and methods for the treatment of human patients or other hosts infected with HBV.
Jt is another object of the present invention to provide methods and compositions to eat drug resistant strains of HBV. SUMMARY OF THE INVENTION
It has been unexpectedly found that a drug resistant strain of HIV or exhibits the behavior of drug-naive virus when given the combination of a β-D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor. In one nonlimiting embodiment, the HIV strain is resistant to a β-D-l,3-dioxolanyl nucleoside. It has further been discovered that certain combinations of β-D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor are useful to treat hepatitis B virus (HBV) infections in a host.
The present invention, therefore, is directed to compositions and methods for the treatment or prophylaxis of HIV, and in particular to a drug-resistant strain of HIV, including but not limited to a DAPD and/or DXG resistant strain of HIV, in an infected host, and in particular a human, comprising administering an effective amount of a β-D-
1,3-dioxolanyl purine nucleoside of the formula:
Figure imgf000015_0001
wherein R is H, OH, CI, NH. or NR'R*; R' and R* are independently hydrogen, alkyl or cycloalkyl, and RJ is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodiυg, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
Alternatively, compositions and methods are provided for the treatment or prophylaxis of HBV, and in particular to a drug-resistant strain of HBV, including but not limited to a~DAPD and/or DXG resistant strain of HBVriή an nfected host, and particular a human, comprising administering an effective amount of a β-D-1,3- dioxolanyl purine nucleoside of the formula:
Figure imgf000016_0001
wherein R is H, OH, CI, NH or NR'R2; R1 and R2 are independently hydrogen, alkyl or cycloalkyl, and R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
In one embodiment, the β-D-I,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000016_0002
wherein R} is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or
Uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
In another embodiment, the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000017_0001
wherein RJ is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug,
In yet another embodiment, the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000017_0002
wherein R3 is H, alkyL aryl, acyl, phosphate, including monophosphate, diphosphate or uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid. or its pharmaceutically acceptable salt or prodrug.
In yet another embodiment, the β-D-l ,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000017_0003
wherein R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
In yet another embodiment, the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000018_0001
wherein R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug. ln one embodiment, an enantiomerically enriched β-D-l,3-dioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with an IMPDH inhibitor, for example ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, selenazofiirin, 5-ethynyl-I-β-D-ribθfuranosylimidazole-4-carboxamide (EICAR), or (S)-N-3-(3-(3-methoxy-4-oxazol-5-yI-phenyl)-ureido]-benzyl-carbamic acid tetrahydrofuran-3-yi-ester (VX-497), which effectively decreases the EC$o for DXG when tested against wild type or mutant strains of HIV- 1.
In one embodiment, the IMPDH inhibitor is mycophenolic acid. In another embodiment of the invention, the IMPDH inhibitor is ribavirin. In yet another embodiment, the nucleoside is administered in combination with the IMPDH inhibitor. In particular, the nucleoside may be DAPD.
In another embodiment, the enantiomerically enriched β-D-l,3-dioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with a compound that reduces the rate of de novo synthesis of guanosine or deoxyguanosine nucleotides. In another embodiment, DAPD is administered in combination or alternation with ribavirin or mycophenolic acid which reduces the rate of de nov synthesis of guanosine nucleotides.
In yet another embodiment, an enantiomerically enriched β-D-l,3-dioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with a compound that effectively increases the intracellular concentration of DXG-TP.
In yet another embodiment, DAPD is administered in combination or alternation with ribavirin or mycophenolic acid that effectively increases the intracellular concentration of DXG-TP. It has also been discovered that, for example, this drug combination can be used to treat DAPD-resistant and DXG-resistant strains of HIV. DAPD and DXG resistant strains of HIV, after ueatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
Therefore, in yet another embodiment of the present invention, the enantiomerically enriched β-D-l,3-dioxoIanyl purine nucleoside, and in panicular
DAPD, is administered in combination or alternation with an IMPDH inhibitor that effectively reverses drug resistance observed in HIV-1 mutant suains.
In yet another embodiment of the present invention, the enantiomerically enriched β~D-l,3-dioxoIanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with an IMPDH inhibitor that effectively reverses DAPD or DXG drug resistance observed in HIV-I mutant strains.
In general, during alternation therapy, an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents are administered together. The dosages will depend on such factors as absoφtion, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Examples of suitable dosage ranges can be found in the scientific literature and in the Physicians Desk Reference- Many examples of suitable dosage ranges for other compounds described herein are also found in public literature or can be identified using known procedures. "TfieseTJosage ranges can b¥modifιedls^esireaTb acrtieve a desiredTesult.
The disclosed combination and alternation regiments are useful in the prevention and treatment of HIV infections and other related conditions such as AlDS-related complex (ARC), persistent generalized lymphadenopathy (PGL), AlDS-related neurological conditions, anti-HIV antibody positive and HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia puφurea and opportunistic infections. In addition, these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HIV antibody or HIV-antigen positive or who have been exposed to HIV.
BRIEF DESCRIPTION OF THE FIGURES
Figure J is a bar graph illustrating the EC» Hi μM of various concentrations of MPA on the activity of DAPD, DXG, and abacavir in peripheral blood mononuclear cells (PBMC).
Figure 2 is a bar graph illustrating the ECM in μM of various concentrations of RBV on the activity of DAPD, DXG, and abacavir in PBMC.
Figure 3 is a graph showing the EC50 in μM of MPA and RBV on the antiviral activity of DAPD against drug resistant mutant strains.
Figure 4 is a graph showing the EC∞ in μM of MPA and RBV on the antiviral activity of DXG against drug resistant HIV variants. Figure 5 is a graph showing the concentration of DXG-TP (pmole/J 0* cells) with increasing amounts of MPA in PBMC.
Figure 6 is a graph showing various concenuations of DXG (pmole/10* cells) with or without 0-25 μM of MPA and the intracellular concentration of DXG-TP and dGTP measured by liquid chromatography mass specwomeiry in PBMC. Figure 7 is a graph of the DXG-TP (ρmole/10* cells) concentrations in DAPD incubated PBM cells. Figure 8 is a graph of DXG-TP (pmole/106 cells) concentrations in DXG incubated PBM cells.
DETAILED DESCRIPTION OF THE INVENTION It has been unexpectedly found that a drug resistant strain of HIV exhibits the behavior of drug-naive virus when given the combination of a β-D-l,3-dioxolanyl nucleoside and an IMPDH inhibitor. In one nonlimiting embodiment, the HIV strain is resistant to a β-D-l,3-dioxolanyl nucleoside,
The present invention is directed to compositions including a combination of a β- D-I,3-dioxolanyl purine nucleoside and an IMPDH inhibitor, and methods of use of the compositions for the treatment or prophylaxis of HIV or HBV, and in particular to drug- resistant strains of HIV or HBV, such as DAPD and/or DXG resistant strains, in a host, for example a mammal, and in particular a human. In one embodiment, the β-D-1,3- dioxolanyl purine nucleoside and/or the IMPDH inhibitor is enantiomerically enriched. IMPDH catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate
(IMP) to xanthosine-5'-monophosphate (XMP), which is a necessary step in guanosine nucleotide synthesis. It has been discovered that reduction of intracellular deoxy- guanosine 5'-triphosphate (dGTP) levels through inhibition of inosine monophosphate dehydrogenase (IMPDH) effectively increases the inUacellular concentration of DXG-TP thereby augmenting inhibition HIV replication. This alone, however, cannot explain the unexpected sensitivity of a drug resistant form of HIV to a β-D-l,3-dioxolanyl purine nucleoside administered in the presence of an IMPDH inhibitor.
Therefore, the present invention is directed to compositions and methods for the treatment or prophylaxis of HIV, and in particular to drug-resistant strains of HIV, such as DAPD and/or DXG resistant strains of HIV, in a host, for example a mammal, and in particular a human, comprising administering an effective amount of an enantiomerically enriched β-D-!,3-diθxolany] purine nucleoside of die formula:
Figure imgf000022_0001
wherein R is H, OH, CI, NH2 orNR'R2; R1 and R2 are independently hydrogen, alkyl or cycloalkyl, and R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid or its phaπnaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogcnase (IMPDH) inhibitor.
Alternatively, compositions and methods are provided for the ueatment or prophylaxis of HBV, and in particular to a drug-resistant strain of HBV, including but not limited to a DAPD and or DXG resistant strain of HBV, in an infected host, and in particular a human, comprising administering an effective amount of a β-D-1,3- dioxolanyl purine nucleoside of the formula:
Figure imgf000022_0002
wherein R is H, OH, CI, NHj or NR'R2; R1 and R2 are independently hydrogen, alkyl or cycloalkyl, and R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable carrier or diluent, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor.
In one embodiment, the β-D-l,3-dioxo!anyl purine nucleoside is of the formula:
Figure imgf000023_0001
wherein R} is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
In another embodiment, the β-D-I,3-dioxoIanyl purine nucleoside is of the formula:
Figure imgf000023_0002
wherein R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or Uiphosphate or a stabilized phosphate derivative, a phospholipid, or an ether-Iipid, or its phaπnaceutically acceptable salt or prodrug.
In yet another embodiment, the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000024_0001
wherein R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
In yet another embodiment, the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000024_0002
wherein R3 is H, alkyl, aryl, acyl, phosphate, including monophosphate, diphosphate or
Uiphosphate or a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, or its pharmaceutically acceptable salt or prodrug.
In yet another embodiment, the β-D-l,3-dioxo!anyl purine nucleoside is of the formula
Figure imgf000024_0003
wherein R3 is H, alkyl, atyl, acyl, phosphate, including monophosphate, diphosphate or triphosphate or a stabilized phosphate moiety, a phospholipid, or an cther-lipld, or its pharmaceutically acceptable salt or prodrug. ln one embodiment, the enantiomerically enriched β-D-l-3-dioxolanyl purine nucleoside, e.g., DAPD, is administered in combination or alternation with an IMPDH inhibitor, for example, ribavirin, mycophenolic acid, beπzamide riboside, tiazofurin, selenazofurin, 5-ethynyl-l-β-D-ribθfuranθSylimidazole-4-carboxamide (EICAR), or (S)- N-3-[3-(3-meihoxy-4-oxazol-5-yl-pr»enyl)-ureido]-berizyl arbamic acid teUahydrofuran- 3-yI-ester (VX-497), which effectively decreases the EC» for DXG when tested against wild type or mutant suains of HI V-J or HBV.
Mycophenolate mofetii is a prodrug which is converted to mycophenolic acid (MPA) in vivo. Also within the scope of the invention are the compositions including MPA and methods of use disclosed herein, wherein MPA is replaced with the mycophenolate mofetii prodrug. In one embodiment, the IMPDH inhibitor is mycophenolic acid. In another embodiment of the invention, the IMPDH inhibitor is ribavirin- In one embodiment, the nucleoside is administered in combination with the IMPDH inhibitor. In another embodiment, the nucleoside is DAPD.
In another embodiment, the enantiomerically enriched β-D-l-3-dioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with a compound that reduces the rate of de now synthesis of guanosine and deoxyguanosine nucleotides.
In a embodiment, DAPD is administered in combination or alternation with ribavirin or mycophenolic acid which reduces the rate ofde novo synthesis of guanosine nucleotides. in yet another embodiment, the enantiomerically enriched β-D-l,3- Jioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with a compound that effectively increases the inttacellular concemration of DXG-TP. In yet another embodiment, DAPD is administered in combination or alternation with ribavirin or mycophenolic acid that effectively increases the intracellular concen ation of DXG-TP.
It has also been discovered that, for example, this drug combination can be used to ueat DAPD-resistant and DXG-resistant strains of HIV or HBV. DAPD and DXG resistant strains of HIV, after treatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
Therefore, in yet another embodiment of the present invention, the enantiomerically enriched β-D-l,3-dioxolanyl purine nucleoside, and in particular DAPD, is administered in combination or alternation with an IMPDH inhibitor that effectively reverses drug resistance observed in HIV-I or HBV mutant strains.
In yet another embodiment of the present invention, the enantiomerically enriched β-D-I,3-dioxolanyl purine nucleoside, and in panicular DAPD, is administered in combination or alternation with an IMPDH inhibitor that effectively reverses DAPD or DXG drug resistance observed in HIV- 1 or HBV mutant strains.
Stereochemistry
Compounds of the present invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. The present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. The optically active forms can be prepared by, for example, resolution of the racemic form by recrystailization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution.
In one embodiment of the invention, the compounds are provided in substantially pure form (i.e. approximately 95% pure or greater).
Optically active forms of the compounds can be prepared using any method known in the art, including by resolution of the racemic form by recrystailization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
Examples of methods to obtain optically active materials include at least the following. i) physical separation of crystals - a technique whereby macroscopic crystals of the individual enantiomers are manually separated.
This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization - a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions - a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmeuic synthesis - a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmeuic synthesis - a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which may be achieved using chiral catalysts or chiral auxiliaries; vi) diastereomer separations - a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer, vii) first- and second-order asymmetric transformations - a technique whereby diastereomers from the racemate equilibrate to yield a
5 preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired 10 enantiomer is then released from the diastereomer; viii) kinetic resolutions - this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic
15 reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors - a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course
20 of the synthesis; x) chiral liquid chromatography - a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase
(including via chiral HPLC). The stationary phase can be made of
25 chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; x>) chiral gas chromatography - a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column 30 containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents - a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiϋ) transport across chiral membranes - a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two iscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.
Chiral chromatography, including simulated moving bed chromatography, is used in one embodiment. A wide variety of chiral stationary phases are commercially available.
Definitions The term "protected" as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
The term halo, as used herein, includes chloro, bromo, iodo and fluoro. The term alkyl, as used herein, unless otherwise specified, refers to a saturated suaighf, branched, or cyclic, primary, secondary or tertiary hydrocarbon of typically Ct to Cio, and specifically includes methyl, trifluoromethyl, CCIj, CFCI2, CFjCI, ethyl, CHiCFi, CFjCFj, propyl, Isopropyl, cyclopropyl, butyl, isobutyl, r-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups. Moieties with which the alkyl group can be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, niuo, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis. John Wiley and Sons, Second Edition, 1991, hereby incoφorated by reference.
The term lower alkyl, as used herein, and unless otherwise specified, refers to a Ci to C4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, lower alkyl is . Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is . The term aryl, as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl, and phenyl. The term includes both substituted and unsubstituted moieties. The aryl group can be substituted with one or more moieties selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamiπo, arylamino, alkoxy, aryloxy, nitro, cyaπo, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et at., Protective Croups in Organic Synthesis. John Wiley and Sons, Second Edition, 1 1.
The term acyl refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from suaight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted wiih halogen (e.g., F, CI, Br or I), C| to C4 alkyl or Ci to C. alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxyuityl, substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the esters optimally comprise a phenyl group. The lerm "lower acyl" refers to an acyl group in which the non-carbonyl moiety is lower alkyl.
The term "enantiomerically enriched" is used throughout the specification to describe a compound which includes approximately 95%, 96%, 97% or 98%, and even at least about 99% or more of a single enantiomer of that compound. When a nucleoside of a particular configuration (D or L) is referred to in this specification, it is presumed that the nucleoside is an enantiomerically enriched nucleoside, unless otherwise stated.
The term "host," as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and a human. Alternatively, the host can be carrying a part of the viral genome, whose replication or function can be altered by the compounds of the present invention. The term host specifically refers to infected cells, cells transfected with all or part of the viral genome and animals, in particular, primates (including chimpanzees) and humans. In most animal applications of the present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly anticipated by the present invention (such as simian immunodeficiency virus in chimpanzees).
Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, aikylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylatcd to produce the active compound. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. The compounds of this invention either possess antiviral activity, or are metabolized to a compound that exhibits such activity.
Pharmaceutically Acceptable Salts and Prodrugs
In cases where any of the compounds as disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, ethanesulfonate, acetate, citrate, malonate, tartarate, succinatc, benzoate, ascorbate, α-ketoglutarate and α- glycerophosphate. Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate and carbonate salts, hydrobromate and phosphoric acid.
Phaπnaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal
(for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
Any of the nucleosides described herein can be administered as a nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside. A number of nucleotide prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the hydroxyl group of the compound or of the mono, di or iphosphate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.
Any of the compounds which are described herein for use in combination or alternation therapy can be administered as an acylated prodrug, wherein the term acyl refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxy ethyl, aryl including phenyl optionally substituted with halogen, G to C< alkyl or C, to C< alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, uityl or monomethoxyuityl, substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl).
The active nucleoside or other hydroxyl containing compound can also be provided as an ether lipid (and particularly a 5 '-ether lipid or a 5'-phosphoethcr lipid for a nucleoside), as disclosed in the following references, which are incorporated by reference herein: Kucera, L.S., N. Iyer, E. Leakc, A. Raben, Modest E.K., D.L.W., and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-I production and induce defective virus formation." AIDS Res. Hum. Retro Viruses. 6:491-501; Piantadosi, C, J- Marasco C.J., S.L. Morris-Natschke, K.L. Meyer, F. Gumus, J.R. Surles, K.S. lshaq, L S. Kucera, N. Iyer, C.A. Wallen, S. Piantadosi, and E.J. Modest. 1991. "Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV activity." J. Med Chem. 34:1408.1414; Hosteller, K.Y., D.D. Richman, DΛ. Carson, L.M. Stuhmiϋer, G M. T. van Wijk, and H. van den Bosch. 1992. "Greatly enhanced inhibition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'«deoxythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3-- leoxythymidiπe." Antimicrob. Agents
Chemoiher. 36:2025.2029; Hosteller, .Y., L.M. Stuhmiller, H.B. Lenting, H. van den Bosch, and D.D. Richman, 1990. "Synthesis and antiretroviral activity of phospholipid analogs of azJdothy idine and other antiviral nucleosides." J. Biol, Chem. 265:61127.
Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside or other hydroxyl or amine containing compound, e.g., at the 5'-OH position of the nucleoside or lipophilic preparations, include U.S. Patent Nos. 5,149,794 (Sep. 22, 1992, Yatvin et al.);
5,194,654 (Mar. 16, 1993, Hosteller et al., 5,223-263 (June 29, 1993. Hosteller et al.);
5,256,641 (Oct. 26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler et al.); 5,463,092 (Oct. 31, 1995, Hostetler et al ); 5,543,389 (Aug. 6, 1996, Yatvin et al.);
5,543,390 (Aug. 6, 1996, Yatvin et al.); 5,543,391 (Aug. 6, 1996, Yatvin et al.); and
5,554,728 (Sep. 10, 1996; Basava et al.), all of which are incorporated herein by reference. Foreign patent applications that disclose lipophilic substituents that can be attached to the nucleosides of the present invention, or lipophilic preparations, include WO 89/02733, W0 9000555, W0 91/16920, WO 91/18914, W0 93/00910, W0
94/26273, W0 96/15132, EP 0 350287, EP 93917054.4, and W0 91/19721.
Nonlimiting examples of nucleotide prodrugs are described in the following references: Ho, D.H.W. (1973) "Disuibution of Kinase and deaminase of lβ-D- arabinofuranosylcytosine in tissues of man and muse." Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotide analogues," In: De Clercq
(Ed.), Advances in Antiviral Drug Design. Vol. I, JAI Press, pp. 179-231; Hong, C.I., Nechaev, A., and West, CR. (1979a) "Synthesis and antitumor activity of l-β-D- arabino-furanosylcytosiπe conjugates of cortisol and co^tisoπe.,, Bicohem. Bioph s. Rs. Commun. 88, 1223-1229; Hong, C.I., Nechaev, A., Kirisits, AJ. Buc heit, D.J. and West, CR. (1980) "Nucleoside conjugates as potential aπtitυmor agents. 3. Synthesis and antitumor activity of l-(β-D-arabinofuranosyl) cytosine conjugates of corticosteriods and selected lipophilic alcohols." J. Med. Chem. 28, 171-177; Hosteller, K.Y.,
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Antimicrobial Agents Chemother. 38, 2792-2797; Hunston, R.N., Jones, A. A. McGuigan, C, Walker, R.T., Balzarini, J., and DeClercq, E. (1984) "Synthesis and biological properties of some cyclic phosphouiesters derived from 2'-deoxy-5- fluorouridine." J. Med. Chem.27, 440-444; Ji, Y.H., Moog, C, Schmitt, G., BischorT, P. and Luu, B. (1 90); "Monophosphoric acid esters of 7-β-hydroxycholesterol and of pyrimidine nucleoside as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity." J. Med. Chem.33 2264-2270; Jones, A.S., McGuigan, C, Walker, R.T., Balzarini, J. and DeClercq, E. (1984) "Synthesis, properties, and biological activity of some nucleoside cyclic phosphoramidates." J. Chem. Soc. Perkin Trans. I, 1471-1474; Juodka, B.A. and Smrt, J. (1974) "Synthesis of diribonucleoside phosph (P→N) amino acid derivatives." Coll. Czech. Chem. Comm. 39, 363-968; Kataoka, S-, Imai, J., Ya aji, N., Kato, M., Saito. M„ Kawada, T. and I ai, S. (1989) "Alkylated cAMP derivatives; selective synthesis and biological activities." Nucleic Acids Res. Sym. Ser. 21, 1-2; Kataoka, S., Uchida, "(cAMP) benzyl and methyl triesters." Heterocycles 32, 1351-1356; Kinchington, D„ Harvey, JJ., O'Connor, T.J., Jones,
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Figure imgf000037_0001
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Pharmaceutical Compositions Humans or other hosts infected with HIV or HBV, and in particular, an infection caused by a drug resistant strain of HIV or HBV, can be treated by administering to the patient an effective amount of the defined β-D-I,3-dioxolanyl nucleoside, and in particular, DAPD or DXG, in combination or alternation with an IMPDH inhibitor, including ribavirin or mycophenolic acid, or a pharmaceutically acceptable salt or ester thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, enterally, intravenously, iπtradermally, subcutaneously, topically, nasally, rectally, in liquid, or solid form.
The active compounds are included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount of compound to inhibit viral replication in vivo, especially HIV or HBV replication, without causing serious toxic effects in the treated patient. By "inhibitory amount" is meant an amount of active ingredient sufficient to exert an inhibitory effect as measured by, for example, an assay such as the ones described herein. A dose of the compound for all the above-mentioned conditions will be in the range from about 1 to 50 mg/kg, or 1 to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per kilogram body weight of the recipient per day. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent nucleoside to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the -derivative,-or-by-otber-means known to those skilled n the art.
The compounds are conveniently administered in unit any suitable dosage form, including but not limited to one containing 7 to 3000 mg, or 70 to 1400 mg of active ingredient per unit dosage form. An oral dosage of 50 to 1 00 mg is usually convenient
In one embodiment, at least one of the active ingredients, or the combination of active ingredients, is administered to achieve peak plasma concentrations of the active compound of from about 0.2 to 70 mM, or about 1.0 to 10 M. This may be achieved, for example, by the intravenous injection of a 0.1 to 10 % solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient.
In compositions comprising a β-D-l-3-dioxolanyl purine nucleoside, or salt or prodrug, end an IMPDH inhibitor, or salt or prodrug, the dosage form may be for example about 0.1 to 1000 mg of a β-D-l,3-dioxolanyl purine nucleoside and about 0.1 to 1000 mg of an IMPDH inhibitor. A further example of dosage form is about 0.1 to 500 mg of a β-D-l,3-dioxolanyl purine nucleoside and about 0.1 to 500 mg of an
IMPDH inhibitor. Another example of dosage form is from about 0.1 to 50 mg of a β-D- 1,3-dioxoIanyl purine nucleoside and 0.1 to 50 mg of an IMPDH inhibitor. A further example of dosage form is from about 0.1 to 10 g of β-D-l,3-dioxolanyl purine nucleoside and 0.1 to 10 g of IMPDH inhibitor. In compositions comprising DAPD, or DXG and MPA, RBV, or their salts or prodrugs, the dosage form may be for example from about 0.1 to 1000 mg of DAPD or DXG and 0.1 to 1000 mg of MPA, RBV, or their prodrugs. A further example of dosage form is about 0.1 to 500 mg of DAPD or DXG and 0.1 to 500 mg of MPA, RBV, or their prodrugs. Another example of dosage form is from about 0.1 to 50 mg of DAPD or DXG and 0.1 to 50 mg of MPA, RBV, or their prodrugs or salts. A further example of dosage form is from about 0.1 to 10 mg of DAPD or DXG and 0.1 to 10 mg of MPA, RBV, or their prodrugs or salts.
An example of a dose range is about 0.1 to 100 mg/kg of body weight per day of a β-D-l,3-dioxolanyl purine nucleoside and about 0.1 to 100 mg/kg of body weight per day of an IMPDH inhibitor. Another example of a dose range is about 1 to 50 mg/kg of body weight per day of a β-D-I,3-dioxolanyl purine nucleoside and about I to 50 mg/kg of body weight per day of an IMPDH inhibitor. Another example of a dose range is about 1 to 20 mg/kg of body weight per day of a β-D-!,3-dioxσlanyl purine nucleoside and about 1 to 20 mg kg of body weight per day of an IMPDH inhibitor.
The concentration of active compound in the drug composition will depend on absoφtion, distribution, metabolism and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the adminisuation of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. A mode of administration of the active compound is oral. Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, tioches, or capsules. Pharmaceutically compatible bind agents, and/or adjuvant materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, H can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can comain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The compounds can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The compounds or their pharmaceutically acceptable derivative or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, anti-fungals, anti- inflammatories, protease inhibitors, or other nucleoside or non-nucleoside antiviral agents, as discussed in more detail above. Solutions or suspensions used for parental, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of lonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
If administered inuavenously, carriers are physiological saline or phosphate buffered saline (PBS). If administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, abso tion promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. If rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-initiating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
In a embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and micro-encapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polyiactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also as pharmaceutically acceptable carriers, these may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or uiphosphate derivatives is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
Anti-HIV Agents That Can Be Used in Combination and/or Alternation With the Composition of the Present Invention
In general, during alternation therapy, an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents are administered together. The dosages will depend on such factors as absorption, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Examples of suitable dosage ranges can be found in the scientific literature and in the Physicians Desk Reference. Many examples of suitable dosage ranges for other compounds described herein are also found in public literature or can be identified using known procedures. These dosage ranges can be modified as desired to achieve a desired result.
The disclosed combination and alternation regiments are useful in the prevention and treatment of HIV infections and other related conditions such as AlDS-related complex (ARC), persistent generalized Jymphadenopathy (PGL), AlDS-related neurological conditions, anti-HIV antibody positive and HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia puφurea and opportunistic infections. In addition, these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical Illness in individuals who are anti-HIV antibody or HIV-antigen positive or who have been exposed to HIV.
It has been discovered that, for example, this drug combination can be used to treat HIV, and in particular DAPD-resistaπl and DXG-resistant strains of HIV. DAPD and DXG resistant strains of HIV, after ueatment with the disclosed drug combination, exhibit characteristics of drug-narve virus. The anti-HIV activity of the composition comprising a β-D-1 ,3-dioxolane nucleoside and an inosine monophosphate dehydrogenase inhibitor, or the pharmaceutically acceptable salts or prodrugs of the composition, can be enhanced by administering one or more other effective anti-HIV agent. For example, the composition can be administered in combination and or alternation with a reverse transcriptase inhibitor (a "RTI"), which can be either a synthetic nucleoside (a "NRTI") or a non- nucleoside compound (a "NNRTI"); a protease inhibitor; a fusion binding inhibitor (such as a pyrophosphate analog).
Examples of antiviral agents that can be used in combination or alternation with the compounds (or compositions including a 1 ,3-dioxolane and an IMPDH inhibitor), disclosed herein for HIV therapy include cis-2-hydroxymethyJ-5-(5-fluorocytosin-l-y|)-
1,3-oxathiolane (FTC); the (-)-enantiomer of 2-hydroxymethyl-5-(cytosin-l-yl)-l,3- oxathiolane (3TC); carbovir, acyclovir, foscamet, interferon, AZT, DDl, DDC, D4T, CS- 87 (3'-azido-2\3'-dideoxy-uridine), and β-D-dioxolane nucleosides such as β-D- dioxolanyl-guanine (DXG), β-D-dioxolanyl-2,6-diaminopurine (DAPD), and β-D- dioxoIanyl-6-chIoropurine (ACP) and MKC-442 (6-benzyl-l-(ethoxymethyl)-5- isopropy] uracil. Potease inhibitors include Crixivan (Merck), nelf avir (Agouron), ritonavir (Abbott), saquinavir (Roche), DMP-266 (Sustiva) and DMP-450 (DuPont Merck).
A more comprehensive list of compounds that can be administered in combination and/or alternation with the compositions of the present invention include (I S,4R)-4-[2-amino-6<yclopropyl-amino)-9H-purin-9-ylJ-2-cyclopentene-l -methanol succinate ("J592", a carbovir analog; GlaxoWellcome); 3TC: (-)-β-L-2',3'-dideoxy-3'- thiacytidine (GlaxoWellcome); a-APA R 18893' a-nϊtro-anilino-phenylacetamide; A- 77003; C2 symmetry-based protease inhibitor (Abbott); A-75925: C2 symmetry-based protease inhibitor (Abbott); AAP-BHAP: bishetero-aryipiperazine analog (Upjohn); ABT-538: C2 symmeUy-based protease inhibitor (Abbott); AzddU:3'-azido-2',3'- dideoxyuridinc; AZT: 3,-azido-3'-deoxythymidine (GlaxoWellcome); AZT-p-ddl: 3'- azido-3'-deoxythymidilyl-(5\5 2\3'-dideoxyinosinic acid (Ivax); BHAP: bisheteroaryl-piperazine; BILA 1906: N-{lS-[[[3-[2S-{(l,l-dimethylethyl)amino]- carbonyl)-4R]-3-pyrldinylmethyl)thioJ-l-piperidinyl]-2R-hydroxy-lS-(phenylmethyl)- propyl]amino]-carbonyl]-2-methylpropyl}-2-quiπolinecarboxamide (Bio Mega/
Boehringer-Ingelheim); BILA 2185: N-(l,l-dimethyiethyl)-l-I2S-[[2-2,6-dimethyl- phenoxy)-l-oxoethyl]amino]-2R-hydroxy-4-phenylbutyl]4R-pyridinylthio)-2-piperidine- carboxamide (BioMega/Bochringer-Ingelheim); BM+51.0836: thiazolo-iso-indolinone derivative; BMS 186,318: aminodiol derivative HIV-1 protease inhibitor (Bristol-Myers- Squibb); d4API: 9-[2,5-dihydro-5-(phosphonomethoxy)-2-ruranel]adenine (Gilead); d4C: 2',3'-didehydro-2',3'-dideoxycytidine; d4T: 2',3'-didehydro-3'-deoxythymidine (Bristol-Myers-Squibb); ddC; 2',3'-<Jideoxycvtidine (Roche); ddl: 2\3'-dideoxyinosine (Bristol-Myers-Squibb); DMP-266: a l,4-dihydro-2H-3, l-benzoxazin-2-onc; DMP-450: {[4R-(4-a,5-a,6-b,7-b)]-hexahydro-5,6-bis(hydroxy)-l ,3-bis(3-amino)phenyl]-methyl)- 4,7-bis-(phenylmethyl)-2H-l,3-diazepin-2-one)-bismesylate (Avid); EBU-dM:5-ethyl-l- ethoxymethyI-6-(3,5-dimethylbenzyl)-uracil; E-EBU: 5-ethyl-l -ethoxymethyl-6- benzyluracil; DS: dextran sulfate; E-EPSeU:l-(ethoxymethyl)-(6-phenylselenyl)-5- ethyluracil; E-EPU: l-(ethoxymethy|)-(6-prιenyl-thiθ)-5-ethyluracil; FTC:β-2\3'. dideoxy-5-fluoro-3 '-thiacytidine (Triangle); HBY097:S-4-isopropoxy-carbonyl-6- methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(lH)-thione; HEPT: l-[(2- hydroxyethoxy)methyl]-6-(phenytthio)thymine; HlV-l :human immunodeficiency virus type 1; JM2763: l,P-(l-3-propaned.yl)-bis-l,4,8,l 1-tetraaza-cycloteUadecaπe (Johnson Matthey); JMSIOO-.L -fl^-phenylenebi^tmeΛhylene^-biS-l^.β.ll-tetTϊuizacyclotctra- decane(Johnson Matthey); KNI-272: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid- containing uϊpepiide; L-697,593;5-eu^yl-6-methyI-3-(2-phtώlimido-eTltyϊ) yridiS 2(lH)-one; L-735,524:hydr xy-amino-pentane amide HIV-1 protease inhibitor (Merck); L-697,661; 3-{[(-4,7-dichloro-l,3-benzoxazol-2-yl)methyl]amino -5-etbyl-6-methyl- pyridin-2(l H)-one; L-FDDC: (-)-β-L-5-fluoro-2',3'-dideoxycytidine; L-FDOC;(-)-β-L- 5-fluoro-dioxolane cytosine; MKC442:6-benzyl-l-ethoxymethyl-5-isopropyluracil (I- EBU; Triangle/Mitsubishi); Nevirapine-.l l-cyclc-propyl-5,1 l-dihydro-4-methyl-6H- dipyridol-P-Σ-b^'.S'-ej-diazepin-β-oπe (Boehringer-Ingelheim); NSC648400: 1 - beπzyIθxymethyl-5-ethyl-6-(8lpha-pyridylthio)uracil (E-BPTU); P9941: 12- pyridylacetyl-IlePheAla-y(CHOH)l2 (Dupont Merck); PFA: phosphonoformate (foscaroet; AsUa); PMEA: 9-(2-phosphonylmethoxyethyl)adenine (Gilead); PMPA: (R)- 9-(2-phosphonylmethoxyproρyl)adenine (Gilead); Ro 31-8959: hydroxyethylamine derivative HIV-1 protease inhibitor (Roche); RP1-3I2: peptidyl protease inhibitor, 1- [(3s)-3-(n-alpha-berizyloxycarbonyl)-l-asparginyl)-amino-2-hydroxy-4-phenylbutyryl]-n- tert-butyl-l-proline amide; 2720: 6-chloro-3,3-dimethyi-4-(isopropenyloxycarbonyl)-3.4- dihydro-quinoxaIin-2-(lH)-thione; SC-52151: hydroxy-ethylurea isostere protease inhibitor (Searle); SC-55389A: hydroxycthyl-urea Isostere protease inhibitor (Searle); TIBO R82150: (+)-(5S>4,5,6,7-teUahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo- [4,5,l-jk]-[l,4]-benzodiazepin-2(lH)-thione (Janssen); TIBO 82913: (+H5S)-4,5,6,7,- tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-buteπyl)imidazo(4,5,ljkHL4]' enz< - diazepin-2( lH)-thione (Janssen); TSAO-mSTiP'.S'-bis-O- ert-butyldimethylsilylj-S'- spiro-5,-(4,-amino-r,2,-oxathiole-2',2'-dioxide)]-β-D-pento-furanosyI-N3-methyl- thy ine; U90152:l-t3-[(l-methylethyl)-amino]-2-pyridiπyl]-4-([5-[(methylsulphonyl)- amino]-lH-indol-2yl]carbony!]piperaziπe; UC: thiocarboxanllide derivatives (Uniroyal);
UC-781 : N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furan-carbothio- amide; UC-82: N-[4-chloro-3-(3-methyl-2-butenyloxy)phenylJ-2-methyl-3-thiophene- carbothioa ide; VB 11,328: hydroxyethyl-sυlphonamide protease inhibitor (Vertex); VX-478:hydroxyethylsulphonamide protease inhibitor (Vertex); XM 323: cyclic urea protease inhibitor (Dupont Merck).
Compounds, or concentrations of IMPDH inhibitor compounds, such as AZT: 3'- azido-3'-deoxythymidiπe (GlaxoWellcome); may be antagonistic in combination with 1,3-dioxolanes, such as MAP and RBV, and thus may not be preferred under certain circumstances.
AnnVHBV Ageβts That Can Be Used in Combination and/or Alternation With the Composition of the Present Invention In general, during alternation therapy, an effective dosage of each agent is administered serially, whereas in combination therapy, effective dosages of two or more agents arc administered together. The dosages will depend on such factors as absorption, bio-distribution, metabolism and excretion rates for each drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the adminisuation of the compositions. Examples of suitable dosage ranges can be found in the scientific literature and in the Physicians Desk Reference. Many examples of suitable dosage ranges for ther compounds described herein are also found in public literature or can be identified using known procedures. These dosage ranges can be modified as desired to achieve a desired result.
It has been discovered that, for example, this drug combination can be used to treat HBV, and in particular DAPD-resistant and DXQ-resistant strains of HBV. DAPD and DXG resistant strains of HBV, after treatment with the disclosed drug combination, exhibit characteristics of drug-naive virus.
The anti-hepatitis B viral activity of the composition comprising a β-D-1,3- dioxolanc nucleoside and an inosine monophosphate dehydrogenase inhibitor, or the pharmaceutically acceptable salts or prodrugs of the composition, can be enhanced by administering one or more other effective anti-HBV agent. For example, the composition can be administered in combination and or alternation with 3TC, β-2- hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3-oxathiolane ("FTC"), L-FMAU, interferon, famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil; Hepsera); lobucavir or ganciclovir. In addition, compositions according to the present invention can be administered in combination or alternation with one or more other antiviral, anti-HIV, anti-HBV, anti- HCV or anti-herpetic agent or interferon, anti-cancer, antiproliferative or antibacterial agents, including other compounds of the present invention. Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the metabolism, catabolism or inactivation of other compounds and as such, are co-administered for this intended effect
EXAMPLES
Example 1
I. Ribavirin in Combination with DAPD
Ribavirin (RBV) was analyzed in vitro for activity against HIV-1 and for its effects on the in vitro anti-HIV activity of two dGTP analogues, DAPD and DXG. RBV was also evaluated for cytotoxicity in the laboratory adapted cell line MT2 and in peripheral blood moπonuclear cells (PBMC). RBV is an inhibitor of the enzyme IMP dehydrogenase. This enzyme is part of the pathway utilized by cells for the de novo synthesis of GTP.
i) Cytotoxicity Assays:
RBV was tested for cytotoxicity on the laboratory adapted T-cell line MT2 and in PBMCs using a XTT based assay. The XTT (2,3-bis(2-methoxy-4-nitro-5- suIfoxyphenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) assay is an in vitro colorimetric cyto-protection assay. Reduction of XTT by mitochondria dchydrogenαses results in the cleavage of the tetrazolium ring of XTT, yielding orange formazan crystals, which are soluble in aqueous solution. The resultant orange solution was read in a spectrophotometer at a wavelength of 450nM. RBV was prepared in 100% DMSO at a final concentration of lOOmM. For the cytotoxicity assays, a 2mM solution of RBV was prepared in cell cuhure media (RPMI supplemented with 10% fetal calf serum. L-Glutamine Img/ml and 20ug/ml gentamicin) followed by 2 fold serial dilutions on a 96 well plate. Cells were added to the plat at 3xl04/well (MTX) and 2xl s/well .PBMC) and the plates were incubated for 5 days w jT'C in a 5% CO. incubator (addition of the cells to the plate diluted the compound to a final high concentration of ImM). At the end of the 5-day incubation, XTT was added to each well and incubated at 37°C for 3 hours followed by the addition of acidified isopropanol. The plate was read at 450πm in a 96 well plate reader. A dose response curve was generated using the absoφtion values of celts grown in the absence of compound as 100% protection.
RBV was not toxic in these assays at concentration of up to I M, as shown in Table 1.
Table 1. Cytotoxicity of RBV
Figure imgf000048_0001
ii) Sensitivity Assays
XXTAss y RBV was tested for activity against the xxLAl strain of HIV-1 in the laboratory adapted cell line MT2. Dilutions of RBV were made in cell culture media in a 96 well plate; the highest concentration tested was 100 μM. Triplicate samples of compound were tested. MT2 cells were infected with xxLAl at a multiplicity of infection (MOI) of
0.03 for 3 hours at 37t in 5% C02. The infected cells were plated at 3.0 x 10 well into a 96 well plated containing drug dilutions and incubated for 5 days at 37*C in CO.. The antiviral activity of RBV was determined using the XTT assay described above. This method has been modified into a susceptibility assay and has been used in a variety of In vitro antiviral tests and is readily adaptable lo any system with a tytic virus (Weislow, O.S., et. al.1989). Using ihe absoφtion values of the cell controls as 100% protection and no drug, virus infected cells as 0% protection, a dose response curve is generated by plotting % protection on the Y axis and drug concentration on the X axis. From this curve ECso values were determined. RBV was not active against HIV-1 in these assays at any of the concentrations tested.
P24Assμy RBV was also tested for activity against the xxLAl strain of HIV-1 in PBMCs using a p24 based ELISA assay. In this assay, cell supernatants were incubated on microelisa wells coated with antibodies to HIV-1 p24 core antigen. Subsequently, anti- HIV- 1 conjugate labeled with horseradish peroxidase was added. The labeled antibody bound to the solid phase antibody/antigen complexes previously formed. Addition of the tetramethylbenzidine substrate results in blue color formation. The color turned yellow when the reaction is stopped. The plates were then analyzed on a plate reader set at 490 nm. The absorbance is a direct measurement of the amount of HIV-1 produced in each well and a decrease in color indicates decreased viral production. Dilutions of RBV were made in cell culture media in a 96 well plate, the highest coneeπuation of RBV tested was 100 μM. PBMC were obtained from HIV-1 negative donors by banding on
Ficoll gradients, stimulated with phytohemaglutinin (PHAP) for 48 hours prior to infection with HIV-I, and infected with virus for 4 hours at 37°C at a MOI of 0.001. Infected cells were seeded into 96 well plates containing 3-fold serial dilutions of RBV. Plates were incubated for 3 days at 37°C. The concentration of virus in each well was determined using the NEN ρ24 assay. Using the absoφtion values of the cell controls as
100% protection and drug free, virus infected cells as 0% protection, a dose response curve is generated by plotting percent protection on the Y axis and drug concentration on the X axis. From this curve, ECjo values were determined.
RBV inhibited HIV-1 replication in PBMCs with a median ECso of 20.5 μM ± 11.8.
in) Combination Assays
The effects of RBV on the in vitro anti-HIV-1 activity of DAPD and DXG were evaluated using the MT2/XTT and PBMC/p24 assays described above. The effects of RBV on the activity of Abacavir and AZT were also analyzed. MT2 XTTassμys_
Combination assays were performed using varying concentrations of DAPD, DXG, Abacavir and AZT alone or with a fixed concentration of RBV. Five fold serial dilutions of test compound were performed on 96 well plated with the following drug concentrations: DAPD 100 μM, DXG 50 μM. Abacavir 20 μM and AZT 10 μM'. The concentrations of RBV used were 1, 5, 10, 20, 40 and 60 μM. Assays were performed in the MT2 cell line as described above in the XXT sensitivity assay section. Addition of 40 and 60 μM RBV, in combination with the compounds listed above, as found to be toxic in these assays, therefore, ECjo values for the compounds were determined in the presence and absence of 1, 5, 10 and 20 μM RBV (Table 2).
Table 2. Effects of RBV on the antiviral activity of DAPD, DXG, Abacavir and AZT in MT2 cells
Mean ECso values (μM)
Figure imgf000050_0001
■■ number of replicates
Addition of 1, 5, 10 and 20 μM RBV decreased the EC50 values obtained for DAPD and DXG. Table 3 illustrates the fold differences in ECW values obtained for each of the compounds in combination RBV.
Table 3. Fold differences in EC» values in combination with RBV in MT2 cells
Figure imgf000050_0002
Figure imgf000051_0001
Addition of 20 μM RBV had the greatest effect on the antiviral activity of DAPD and DXG with a 14-2 and 12 fold decrease in the apparent EC50 values respectively. Addition of RBV had no effect (less than 2 fold difference in the apparent EC50) on the activity of Abecavir. Addition of 20 μM RBV resulted in a greater than 6-fold increase in the apparent ECW of AZT indicating that the combination is antagonistic with respect to inhibition of HIV. Similar results were obtained with the addition of 1, 5 and 10, μM RBV, although to a lesser extent than that observed with the higher concentration of RBV.
iv) DAPD Resistant HIV-I mutants
The effect of RBV on the activity of DAPD and DXG against mutant stiains of HIV was also analyzed (Table 4). The restraint strains analyzed included viruses created by site directed mutagenesis, K65R and L74V, as well as a recombinant virus containing mutations at positions 98S, 116Y, I 1M and 215Y. The wild type backbone in which these mutants were created, xxLAI, was also analyzed for comparison. The concentrations of DAPD and DXG tested were as described in the above MT2/XTT combination assay section. RBV was tested in combination with DAPD and DXG at a fixed concenuation of 20 μM. The mutant viruses tested all demonstrated increased EC.σ values (greater than four fold) for both DAPD and DXG indicating resistance to these compounds. Addition of 20 μM RBV decreased the ECjo values of DAPD and DXG against these viruses. The ECM values determined for DAPD and DXG in the presence of 20 μM RBV were at least 2.5-fold lower than those obtained for the wild type virus. These results are summarized in Table 4.
Table 4. Effects of RBV on the antiviral activity of DAPD and DXG: Resistant Virus
EC$o values (nM)
Figure imgf000051_0002
Figure imgf000052_0001
* lRBV] = 20 μM b indicates fold difference from WT
PBMC/p24 assays
Combination assays were also performed in PBMCs using varying concentrations of DAPD, DXG, Abacavir and AZT alone or with a fixed concentration of RBV. Compound dilutions and assay conditions were as described above. The concentrations of RBV used were 1, 5, 10, 20, 40 and 60 μM. Addition of 40 and 60 μM RBV, in combination with the compounds listed above, was found to be toxic in these assays. The ECJO values determined for the compounds in the presence and absence of 1, 5, 10 and 20 μM RBV are shown in Table 5.
Table 5. Effects of RBV on the antiviral activity of DAPD, DXG, Abacavir and AZT m PMBCs
Mean ECso values (μM)
Figure imgf000052_0002
*= number of replicates
Addition of 1 μM RBV resulted in a slight decrease (less than 3-fold) in the ECJO of DAPD and DXG and Abacavir, but had no effect on the EC50 value obtained for AZT. These effects became more pronounced with increasing concentrations of RBV. Table 6 illustrates the fold differences in ECso values obtained for each of the compounds in combination with 1, 5, 10 and 20 μM RBV.
Table 6. Fold differences in EC., values with RBV
Figure imgf000052_0003
Figure imgf000053_0001
RBV inhibited the replication of HIV-1 in PBMCs with an ECso of 20.5 μM. Ribavirin was not toxic to these cells at concentrations up to 1 mM resulting in a therapeutic index of >48. Addition of 20 μM RBV to DAPD, DXG and Abacavir completely inhibited HIV replication in PBMCs at all the concentrations tested but had little effect on the activity of AZT. Addition of lower concentrations of RBV also had a significant effect on the activity of DAPD, DXG and Abacavir. In the MT2 cell line, RBV was not active against HIV replication. Addition of 20 μM RBV decreased the apparent ECso of DAPD and DXG, 14.2 and 12-foM respectively. Addition of 20 μM RBV had no effect on the activity of Abacavir and resulted in a 6-fold increase in the apparent EC50 of AZT indicating that the combination is antagonistic with respect to inhibition of HIV. Similar results were obtained in MT2s with the addition of 5 and 10 μM RBV, although to a lesser extent than that observed with the higher concentration of RBV. When tested against mutant strains of HIV-I, the combination of 20 μM RBV with DAPD or DXG decreased the ECso values of these compounds to less than those observed with wild type virus, i.e. the previously resistant virus strains are now sensitive to inhibition by DAPD and DXG. Weislow, O.S., R. Kiser, D.L. Fine, J. Bβder, R.H. Shoemaker, and M.R. Boyd. 1989. New soluble formazan assay for HIV-1 cytopathic effects: Application to high-flux screening of synthetic and natural products for AIDS- antiviral activity. J. of NCI. 81 :577-586.
π. Mycophenolic Acid in Combination with DAPD
Mycophenolic acid (MPA) was analyzed in vitro for activity against HIV-1 and for its effects on the in vitro anti-HIV activity of two dGTP analogues, DAPD and DXG. MPA was also evaluated for cytotoxicity in the laboratory adapted cell line MT2 and in peripheral blood mononuclear cells (PBMC). MPA is an inhibitor of the enzyme IMP dehydrogenase. This enzyme is part of the pathway utilized by cells for the de-novo synthesis of GTP. Combination assays were also performed with Abacavir, AZT and FTC.
i) Cytotoxicity Assays; MPA was tested for cytotoxicity on the laboratory adapted T-cell line MT2 and in
PBMCs using a XTT based assay. The XTT (2,3-bis(2-fl»ethoxy-4-nitro-5-sulfophenyl)- 5[(phenyIamino)carbonyl]-2H-tetrazolium hydroxide) assay is an in vitro colorimeuic cyto-protection assay. Reduction of XTT by mitochondria dehydrogenases results in the cleavage of the tetrazolium ring of XTT, yielding orange fom-azan crystals, which are soluble in aqueous solution. The resultant orange solution is read in a spec ophoTometcr at a wavelength of 450nM. MPA was prepared in 100% DMSO at a final concenUation of lOO M. For the cytotoxicity assays, a 200μM solution of MPA was prepared in cell culture media (RPMI supplemented with 10% fetal calf serum, L-Glutamine lmg/ml and 20ug/ml gentamicin) followed by 2 fold serial dilutions on a 96 well plate. Cells were added to the plat at 3xl04 well (MTX) and 2xlOJ/well (PBMC) and the plates were incubated for 5 days at 37 C in a 5% CO- incubator (addition of the cells to the plate diluted the compound to a final high concentration of lOOμM). At the end of the 5-day incubation, XTT was added to each well and incubated at 37*C for 3 hours followed by the addition of acidified isopropanol. The plate was read at 450nm in a 96 well plate reader. A dose response curve was generated using the absorption values of cells grown in the absence of compound as 100% protection.
MPA was toxic in both cell lines with a 50% cytotoxic does (CCJO) of 5.7 μM in the MT2 cell line and 4.5 μM in PBMC. See Table 7.
Table 7. Cytotoxicity of MPA
Figure imgf000054_0001
ii) Sensitivity Assays XXTAssay
MPA was tested for activity against the xxLAl strain of HIV-1 in the laboratory adapted cell line MT2. Dilutions of MPA were made in cell culture media in a 96 well plate; the highest concentration tested was 1 μM. Triplicate samples of compound were tested. MT2 cells were infected with xxLAI at a multiplicity of infection (MOI) of 0.03 for 3 hours at 37°C In 5% CO∑. The infected cells were plated at 3.0 x IOVwell into a 96 well plated containing drug dilutions and incubated for 5 days at 37°C in COj. The antiviral activity of MPA was determined using the XTT assay described above. This method has been modified into a susceptibility assay and has been used in a variety of in vitro antiviral tests and is readily adaptable to any system with a lytic virus (Weislow,
O.S., et. al. 1989). Using the absorption values of the cell controls as 100% protection and no drug, virus infected cells as 0% protection, a dose response curve is generated by plotting % protection on the Y axis and drug concentration on the X axis. From this curve ECso values were determined. MPA was not active against HIV-1 in these assays at any of the concentrations tested.
P24 Assay
MPA was also tested for activity against the xxLAI strain of HIV-I in PBMCs using a p24 based Elisa assay In this assay, cell supernatants are incubated on microelisa wells coated with antibodies to HI V-l p24 core antigen. Subsequently, anti-
HIV- 1 conjugate labeled with horse radish peroxidase is added. The labeled antibody binds to the solid phase antibody/antigen complexes previously formed. Addition of the tetramethylbenzidine substrate results in blue color formation. The color turns yellow when the reaction is stopped. The plates are then analyzed on a plate reader set at 490 nm. The absorbance is a direct measurement of the amount of HIV-1 produced in each well and a decrease in color indicates decreased viral production. Dilutions of MPA were made in cell culture media in a 96 well plate, the highest concentration of MPA tested was 1 μM. PBMC were obtained from HJV-J negative donors by banding on Ficoll gradients, stimulated with phytohemagluiinin (PHAP) for 48 hours prior to infection with HIV-1, and infected with virus for 4 hours at 37βC at a MOI of 0.OOJ.
Infected cells were seeded into 96 well plates containing 4-fold serial dilutions of MPA Plates were incubated for 3 days at 37βC The concentration of virus in each well was determined using the NEN p24 assay. Using the absorption values of the cell controls as lOwTprotection and drug free, vimsTnfectwTcells as 0%"profectiori, a dose response curve is generated by plotting % protection on the Y axis and drug concentration on the X axis. From this curve EC50 values were determined.
MPA inhibited HIV-1 replication in PBMCs with a median EC$o of 95 nM ± 29.
Hi) Combination assays:
The effects of MPA on the in vitro anti-HlV-1 activity of DAPD and DXG were evaluated using the MT2/XTT and PBMC/p24 assays described above. The effects of MPA on the activity of Abacavir, AZT and FTC were also analyzed.
MT2 XTT assays
Combination assays were performed using varying concentrations of DAPD, DXG, Abacavir, AZT and FTC alone or with a fixed concentration of MPA. Five fold serial dilutions of test compound were performed on 96 well plated with the following drug concentrations: DAPD - 100 μM, DXG - 50 μM, Abacavir - 20 μM and AZT - 10 μM, and FTC - 10 μM. The concentrations of MPA used were 1, 0.5, 0.25, 0.1, and 0.01 μM. Assays were performed in the MT2 cell line as described in section 3.1. Addition of I and 0.5 μM MPA, in combination with the compounds listed above, was found to be toxic in these assays, therefore, ECso values for the compounds were determined in the presence and absence of 0.25, 0.1, and 0.01 μM MPA (Table 8).
Table 8. Effects of MPA on the antiviral activity of DAPD, DXG, Abacavir, AZT, and FTC in MT2 cells Mean EC» values (μM)
Figure imgf000056_0001
Figure imgf000057_0001
•= number of replicates
Addition of 0.01 μM MPA had no effect on the ECjo values obtained for any of the compounds. Table 9 illustrates the fold differences in ECso values obtained for each of the compounds in combination with 0.1 and 0.25 μM MPA.
Table 9. Fold Differences in ECso Values in Combination with MPA in MT2 cells
Figure imgf000057_0002
Addition of 0-25 μM MPA had the greatest effect on the antiviral activity of DAPD and DXG with a 16.7 and 10.5 fold decrease in the apparent EC$o values respectively. Addition of 0.25 μM MPA had little effect on the activity of Abacavir and FTC, less than a 2 fold decrease in the apparent ECso, and resulted in a 2.3 fold increase in the apparent ECso of AZT indicating that the combination is antagonistic with respect to inhibition of HIV. Similar results were obtained with the addition of 0.1 μM MPA, although to a lesser extent than that observed with the higher concentration of MPA.
iv) DAPD Resistant HIV-I mutants
The effect of MPA on the activity of DAPD and DXG against mutant strains of HIV was also analyzed (Table 10). The restraint strains analyzed included viruses created by site directed mutagenesis, K65R and L74V, as well as a recombinant virus containing mutations at positions 98S, 1 16Y. 151M and 215 Y. The wild type backbone In which these mutants were created, xxLAI, was also analyzed for comparison. The concentrations of DAPD and DXG tested were as described In section 4.1. MPA was tested in combination with DAPD and DXG at a fixed concenUation of 0.25 μM. DAPD and DXG were active against all of the wild type strains of HIV tested. The mutant viruses tested all demonstrated increased EC50 values for both DAPD and DXG indicating resistance to these compounds. Addition of 0-25 μ MPA decreased the ECso values of DAPD and DXG against these viruses. These values determined for DAPD and DXG in the presence of 025 μM MPA were similar to those obtained for the wild type virus.
Table 10. Effects of MPA on the Antiviral Activity of DAPD and DXG: Resistant Virus
EC50 values (μM)
Figure imgf000058_0001
" [MPA] = 0.25 μM b indicates fold difference from WT
PBMC/p24 assays
Combination assays were also performed in PBMCs using varying concentrations of DAPD- DXG, Abacavir, AZT and FTC alone or with a frxed concentration of MPA.
Compound dilutions and assay conditions were as described above. The concentrations of MPA used were 1, 0.5, 0.25, 0.1, and 0.01 μM. Addition of 1 and 0-5 μM MPA, in combination with the compounds listed above, was found to be toxic in these assays. The ECso values determined for the compounds in the presence and absence of 0.25, 0.1, and 0.01 μM MPA are shown in Table 11.
Table 11. Effects of MPA on the antiviral activity of DAPD, DXG, Abacavir, AZT, and FTC in PMBCs
Mean ECso values (μM)
Figure imgf000058_0002
Figure imgf000059_0001
*= number of replicates
Addition of 0.01 urn MPA decreased the EC50 for DAPD and DXG but had no effect on the EC50 values obtained for Abacavir, AZT and FTC (less than 2 fold Change in ECso). Addition of 0.1 and 0.25 μM MPA decreased the ECso for DAPD, DXG and Abacavir, but had no effect on the ECso values obtained for AZT and FTC. Table 12 illustrates the fold differences in ECso values obtained for each of the compounds in combination with 0.01, 0.1 and 0-25 μM MPA.
Table 12. Fold Differences in ECso Values with MPA
Figure imgf000059_0002
Mycophenolic acid inhibited the replication of HIV-1 in PBMCs with an EC» of 0.095 μM. CCso value obtained for MPA in these cells were 4.5 μM resulting in a therapeutic index of 47. Addition of 0.25 μM MPA to DAPD, DXG and Abacavir completely inhibited HIV replication in PBMCs at all the concentrations tested but had little effect on the activity of AZT and FTC (less than 2 - fold change in ECso- Addition of lower concentrations of MPA also had a significant effect on the activity of DAPD, DXG but had little effect on the activity of Abacavir, AZT and FTC. In the MT2 cell line, MPA was not active against HIV replication. Addition of 0.25 μM MPA decreased the apparent EC50 of DAPD and DXG, 16.7 and 10.5 - fold respectively. Addition of 0.25 μM MPA had linle effect on the activity of Abacavir and FTC and resulted in a 2.3 - fold increase in the apparent EC» of AZT indicating that the combination is antagonistic with respect to inhibition of HIV. Similar results were obtained in MT2s with the addition of 0.1 μM MPA, although to a lesser extent than that observed with the higher concentration of MPA. When tested against mutant strains of HIV-1, the combination of ~0.2TμM MPA with DAPTJfor DXG decreased
Figure imgf000060_0001
less than those observed with wild type virus, i,e. the previously resistant virus strains are now sensitive to inhibition by DAPD and DXG.
iv) Concentration of DXG-TP in PBMCs
The effect of mycophenolic acid on the intracellular concentration of DXG- triphosphate (DXG-TP) was evaluated in peripheral blood mononuclear cells (PBMC). PBMC were obtained from HIV negative donors, stimulated with phytohemagluttinin, and incubated at 37 °C in complete media supplemented with various concentrations of DXG (5 μM or 50 μM) in the presence or absence of 0.25 μM mycophenolic acid. PBMC were harvested following 48 or 72 hours of incubation and the inuacellular DXG- TP levels determined by LC-MS-MS as described below. Addition of 0.25 μM mycophenolic acid increased the median concentration of intracellular DXG-TP by 1.7- fold as compared to the levels in cells incubated with DXG alone.
The bioanalytical method for the analysis of DXG-TP from peripheral blood mononuclear cells utilizes ion-pair solid phase extraction (SPE) and ion-pair HPLC coupled to electrospray ionization (ESI) mass spectrometry. Pelleted PBMC samples containing approximately 0.5 x 107 cells are diluted with a solution containing the internal standard (2\ S'-dideoxycytidine-S'- uiphosphate (ddCTP)) and the DXG-TP and ddCTP are selectively extracted using ion-pair SPE on a C-l 8 cartridge. The DXG-TP and ddCTP are separated with mkrobore ion-pair HPLC on a Waters Xterra MS C18 analytical column with retention times of about 10 minutes. The compounds of interest are detected in the positive ion mode by ES1-MS/MS on a Micromass Quattro LC Uiple quadrupole mass spectrometer.
While analyzing DXG-TP PBMC samples, six point, 1/x2 weighted, quadratic calibration curves, ranging from 0.008 to 1.65pmoies/106 cells, are used to quantitate samples. Typically, quality conuol (QC) samples, at two concenUations (0.008 and 1.65pmo.es/10* cells), are analyzed in duplicate in each analytical run to monitor the accuracy of the method. The bioanalytical method has a reproducible extraction efficiency of approximately 80%. The limit of quantitation (LOQ) is 0.008pmoles/} ' cells. Tlie range of the assay is~0.008 to1τ6Spτnoles/106 cells.
Example 2
The effect of MPA and RBV on the anti-HIV activity of DAPD and DXG against wild type and drug resistant variants of HIV-1 was examined. When tested against wild- type virus, using either human PBMC or MT2 cells, both MPA and RBV decreased the ECso value for DXG by at least 10-fold. In contrast, both MPA and RBV increase the ECso value for AZT and had little or no effect on the activity of abacavir or FTC when tested in the MT2 cell line. MPA and RBV completely reversed the resistance to DXG observed with HIV isolates containing the L74V, K65R, or Q151M mutations, which confer partial resistance to DAPD and DXG. Similarly, when tested against a mutant virus fully resistant to inhibition by DXG (K65R/QI51M, ECso = 80 μM) MPA and RBV reduced the ECso for DXG to within 5-fold of wild type. The combination of 0.25 μM MPA or 20 μM RBV with DAPD or DXG did not increase the cytotoxicity of these drugs. In addition, when tested at physiologically relevant concentrations, neither MPA nor RBV demonstrated mitochondrial toxicity alone or in combination with DAPD or DXG. These studies support a role for the use of IMPDH inhibitors in combination therapy with amdoxovir in the treatment of HIV.
The effects of MPA and RBV on the in vitro activity of DAPD and DXG demonstrated that when DAPD or DXG is combined with MPA or RBV a suong synergistic anti-HIV response occurred in vitro. Moreover, since when cells infected with mutant virus resistant to DAPD or DXG were exposed to these combinations the ECso for DAPD and DXG reverted to wild-type values, these resuhs suggest that the combination of MMF and DAPD is useful for treating experienced patients who are not responding to current NRTI-containing regimens.
i) MATERIALS AND METHODS
Reagents: DAPD, DXG and FTC (emtricitabine) were synthesized at Triangle Pharmaceuticals Inc. AZT (azidothymidine) and abacavir were provided by Glaxo
Figure imgf000062_0001
Cells: Cytotoxicity and activity assays were performed in the T-cell line MT2 and in peripheral blood mononuclear cells (PBMC). Cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum and 20 μg/mL gentamicin (Life Technologies). PBMC were obtained from HIV-seronegative donors by banding on Ficoll (Amersham Pharmacia Biotech) and were activated by phytohemaglutinin, PHAP, (Sigma-Aldrich) for two days prior to infection. Cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 20 μg/mL gentamicin and 10% 1L2.
Viruses: Recombinant viruses were created by subcloning the HIV protease and RT coding sequences obtained from patient plasma HIV RNA into a modified version of the plasmid xxLAI. Amplification and cloning strategies have been previously described in the art. Recombinant viruses contained mutations in the HIV-RT at positions A98S, F116Y, Q151M, T2J5Y and D67N, T69D, K70R, K103N, M184V, T215Y, K219Q.
Viruses containing mutations at positions K65R, L74V, Q151M and K65R Q1S1M of the HIV-RT were generated by site directed mutagenesis of the xxLAI plasmid using the QuickChaπge Site-Directed Mutagenesis kit from Stratagene. Genotypic analysis of the recombinant viruses and of viruses obtained by site directed mutagenesis was performed by didcoxy sequencing using ABI Prism 377* technology.
Antiviral Assays: Anti-HIV assays were performed using two different assay methodologies. A cytotoxicity-based assay (XTT) was performed to evaluate activity in MT2 cells and an HIV-1 p24 based enzyme linked immunoabsorbant assay (ELISA) (Organon Teknika Corporation) was performed to evaluate activity in PBMCs, For the XTT assay, MT2 cells were infected with cither the mutant virus or wild type LAI at a multiplicity of infection (MOI) of 0.03 in RPMI 1640 medium containing 10 % fetal bovine serum, 20 μg ml gentamicin (Life Technologies), and 2 μg/ml polybrene (Sigma) for 2 hours at 37°C. Following infection, cells were seeded into 96-well plates at 3 x \04 cells/well, containing test compounds. Within each 96-well plate, test compounds were tested in triplicate at five-fold serial dilutions. The infected cells were cultured for 5 days in the presence of test compounds. On day five, XTT was added and the plates were analyzed. A dose response curve for each individual compound was generated using the absoφtion values of the uninfected cell controls as 100% protection and no drug; virus infected cells as 0% protection. From the dose response curve, an ECso (50% effective concentration)
Figure imgf000063_0001
inhibited viral induced CPE by 50%. Anti-HIV activity was also assessed using human peripheral blood mononuclear cells (PBMC) and an ELISA assay for the detection of HIV-I p24 core antigen. PBMCs were infected with HIV- 1 virus at en MOI of 0.001 for 4 hours at 37°C, and plated in the presence of drugs as described above. Infected cells were cultured for 4 days. On day four, the amount of HIV-1 p24 was determined in each well. Uninfected cells were used as background and readings from the virus control were considered 100% infection.
Dose response curves and EC50 values were determined as described above.
The effect of MPA and RBV on the in vitro anti-HIV activity of DAPD, DXG, abacavir, FTC, and AZT was evaluated using the MT2 XTT and PBMC/p24 assays described above. Combination assays were performed by varying concentrations of the test compounds alone or with a fixed concentration of MPA or RBV.
Cytotoxicity Assays: MT2 cells and PHAP-stimulated human PBMCs were seeded at densities of 3 X 104 and I X 10s cells/well, respectively, in 96-well plates containing two-fold serial dilutions of DAPD or DXG. For combination cytotoxicity assays, a fixed concentration of RBV (I, 5, 10, 20, 40, and 60 μM) or MPA (0.01, 0.1 0-25 0.5 and 1 μM) was added to the DAPD/DXG dilutions. The cultures were incubated for 5 days at
37°C in a humidified 5% CO- atmosphere and were then incubated with XTT (2,3-bis(2- methoxy*4-niuo-5-sulfophenyl)-5[(phenylammino)carbonyI]-2H-tetrazolium hydroxide) for 3 hours. Cytotoxicity was determined by comparing ueated cultures with the untreated conuol. Mitocbσndria DNA Assay: The effect of amdoxovir and DXG alone or in combination with MPA or RBV on mitochondria! DNA synthesis (mtDNA) was assessed using HepG2 cells. Cells were seeded (5 x 104 cells/well) into 12-well tissue culture plates and incubated for 4 days at 37° m a humidified 5% CO? atmosphere with RPMI 1640 (BRL) supplemented with 10% fetal calf serum and 2 mM gluta ine. At day 4, the medium was replaced with medium containing various concenuations of test compound. For combination experiments the concentrations of MPA and RBV were held constant (0.25 μM MPA and 20 uM RBV) and the concentrations of DAPD and DXG were varied (0.01, 0.1, 1, 10, 25, and 50 μM DAPD or DXG). The cells were incubated for 2 weeks and during the incubation period medium was changed every other day. At the end of the two weeks cells were collected and total cellular DNA was ex acted using the Dneasy Kit (Qiagen). DNA from each sample was denatured by adding an equal volume of denaturing buffer (0 8 M NaOH, 20 mM EDTA) and heating to 100" C for 10 min. The DNA samples were blotted onto positive-charged nylon membranes washed once with 0.4 M NaOH followed by a single wash with 2X SSPE. The DNA was cross-linked to the membrane by exposing to UV light. A specific ϊ2P-probe encompassing nucleotide position 4212-4242 of human mtDNA was used to quantitate the level of mtDNA in each sample. Cellular DNA was quantitated using a 32P-probe specific for human glyceraldehyde 3-phosphate dehydrogenase. The quantity of mtDNA was normalized to the amount of cellular DNA resulting in relative mtDNA levels. The relative levels of mtDNA from the treated cells were compared to the relative mtDNA levels of the untreated cell controls. The data from 3 experiments were combined and the data reported as the mean + the standard deviation.
II) Results
Anti-HIV Activity and cytotoxicity of Mycophenolic Acid and RBV: Mycophenolic acid and RBV were tested for activity against the LAI strain of
HIV-1 virus using the MT2/XTT and PBMC/p24 assays described above. Neither MPA nor RBV demonsuated anti-HIV activity when tested in the MT2/XTT assays. The highest concenUation of MPA and RBV tested was 1 μM and 100 μM, respectively. However, both MPA and RBV demonstrated activity against HIV replication when tested in the PBMC/p24 assay. Median ECso values of 0.095 μM and 20 5 μM were determined for MPA and RBV, respectively. Inhibition of HIV replication is presumed to occur due to inhibition of cell proliferation caused by decreased levels of dGTP (Ichimura and Levy) and not by a direct effect of MPA on HIV. Using steady state kinetic analysis whh wild type HIV-RT MPA alone did not directly inhibit HIV-RT. Mycophenolic acid and RBV were also tested for cytotoxicity in the MT2 cell line and PBMC as described above. MPA was found to be toxic to both cell lines with CCso values (concentration required for 50% cell death) of 5.7 μM (MT2) and 4.5 μM (PBMC). RBV was not found to be toxic to either cell line at concentrations up to 1 mM. Results are shown in Table la.
Table la. Cytotoxicity and antiviral activity of MPA and RBV
MT2 PBMC
Figure imgf000065_0001
* No activity observed at sub-cytotoxic levels
Activity of DAPD and DXG in Combination with MPA and RBV: The effect of MPA and RBV on the activity of DAPD and DXG in the MT2 XTT and PBMC/p24 assays as described above was evaluated. The combination of MPA and RBV with abacavir or AZT was also evaluated. Abacavir was used as a positive conUol; the active form of abacavir (carbovir-triphosphate) is also a guanosine analogue and has been shown to have synergistic anti-HIV activity when combined with MPA. ACT was used as a negative control. The combination of RBV and AZT is antagonistic with regards to HIV replication, possibly as a consequence of increased levels of oTTP which acts as a feedback inhibitor of thymidine kinase activity. In the MT2 cell line, addition of low concentrations of MPA (0.01 μM) had no effect on the anti-HJV activity of any of the compounds tested. A direct relationship between the concenUation of MPA or RBV and the level of synergy seen in combination with DAPD and DXG was observed. The combination of 0.25 μM MPA with DAPD or DXG produced the greatest results (16.7- and 10.5-fold increase in the activity of DAPD and DXG respectively) without any cytotoxicity. By contrast, addition of 0.25 μM MPA resulted in a 2.3 fold decrease in the activity of AZT. Little effect was noted on the activity of abacavir (less than 2-fold) at any of the MPA concentrations tested.
Similar experiments were performed with RBV in combination with DAPD, DXG, abacavir and AZT. In these assays, the addition of RBV (up to 20 μM) had no effect on anti-HIV activity of abacavir. By contrast, the addition of 20 μM RBV caused a decrease in the apparent EC50 values of DAPD and DXG by 14.3 and 11.7-fold respectively. Combining 20 μM RBV with AZT resulted in a >6-fold increase in the ECjo value of AZT. The results obtained from the combination studies using HIV- infected MT2 cells are summarized ln Table 2a.
Table 2a. Effect of MPA and RBV on the activity of NRTls in MT2 cells
Figure imgf000066_0001
* EC50 β me EC50 in combination
The effects of MPA and RBV on the anti-HIV activity of DAPD, DXG, abacavir, and AZT were also evaluated in PBMC. At the lowest concentration of MPA tested, 0.01 μM, there was no change in the ECso values observed for abacavir or AZT. By contrast, addition of 0.0) μM MPA resulted in a 4.6- and 9.3-fold increase in the activity of DAPD and DXG, respectively. Increasing the MPA concenUation to 0.25 μM caused little change in the ECSo values of AZT but resulted in a dramatic increase in the activity of DAPD, DXG and abacavir (Figure 1). Likewise, RBV had no effect on the anti-HIV activity of AZT but caused a significant increase in the activity of DAPD, DXG, and abacavir (Figure 2).
Activity of DAPD and DXG tn Combination with MPA and RBV Against Drug Resistant Variants.
The effect of MPA and RBV on the activity of DAPD and DXG against strains of HIV-1 containing mutations that confer resistance to these analogues was also analyzed. Resistant virus included laboratory isolates containing the K65R and L74V mutations created by site directed mutagenesis in the LAI backbone. These mutations have been shown to develop rn vitro when virus was passed in the presence of increasing concentrations of DXG. The QI51M mutation also was shown to cause moderate resistance to DXG. A laboratory strain containing the K65R/Q151 M double mutation as a well as a recombinant virus containing mutations at positions A98S, 116Y, 151M and 15Y were tested, Combination assays were performed with a fixed concentration of MPA (0-25 μM) or RBV (20 μM). Both viruses demonstrated increased ECso values for DAPD and DXG, with the K65R Q151M mutant being the most resistant (>10 fold increase in ECJO). However, in the presence of 025 μM MPA or 20 μM RBV the ECso values determined for DAPD were within two-fold of the values obtained for wild-type virus (Figure 3). Likewise, the combination of MPA or RBV with DXG resulted in a > 10-fold increase in activity against the mutant strains (Figure 4).
The effects of MPA and RBV on the activity of DAPD and DXG against the highly resistant K6SR/QI 1M mutant were also evaluated in PBMC Addition of increasing concentrations of MPA (up to 0.2 μM) or RBV (up to 10 μM) resulted in a greater than 50-fold increase in the activity of both DAPD and DXG (Table 2a).
Mitochondria! toxicity of DAPD and DXG in combination with MPA or RBV:
DAPD and DXG were previously reported to have little effect on mitochondrial DNA (mtDNA) synthesis. MtDNA synthesis was evaluated in HepG2 cells incubated with 0.25 μM MPA or 20 μM RBV alone and in combination with DAPD or DXG to determine if the presence of an IMPDH inhibitor would effect mtDNA synthesis. RBV concenuations up to 80 μM did not affect mtDNA synthesis or cell growth, whereas concentrations of MPA greater than 0.25 μM caused a dose dependent increase in cell death and decrease in mtDNA synthesis. Consistent with previously reported results,
DAPD did not cause a significant reduction in mitochondrial DNA synthesis at clinically relevant concentrations. However, the combination of 20 μM RBV and DAPD did cause an inhibition of mtDNA synthesis at the higher concentrations of DAPD. MtDNA synthesis was not appreciably affected when cells were exposed to 0.25 μM MPA and DAPD concentration up to 25 μM. DXG did not affect mtDNA synthesis at the highest concentration used in this study (50 μM). Furthermore, the addition of MPA did not alter the lack of inhibition of mtDNA synthesis seen with DXG. The combination of DXG and RBV did appear to cause some inhibition of mtDNA synthesis at the highest
DXG concenUation tested (50 μM). Incubating cells alone or in combination with either MPA or RBV did not result in any reduction of mtDNA synthesis. Dideoxycytidine was used as a positive control in these studies. Results are shown in Table 3a.
Table 3a. Effect of increasing concentrations of MPA and RBV on the activity of DAPD and DXG against virus containing the K65R/QI 51M mutations.
Figure imgf000068_0001
Table 4a shows the effects on mitochondrial function of DAPD, DXG, MPA and RBV (%mt DNA ± SD relative to controls )
Table 4a,
Figure imgf000068_0002
DAPD 9418 9213 7215 6713 4611
DAPD + MPA* 74118 96 8 9714 7814 531
DAPD + RBV* 7311 79±6 63 + 13 29111 1916
DXG 9519 79±6 82±8 7918 72119
DXG + MPA 126116 12218 96 ±9 96111 98
DXG + RBV 8315 6915 69111 61116 55113 ddC 9915 3011 1511 till 12+1
SμM 10 μM 20 μM 40 μM 80 μM
RBV 9319 102111 8819 84 12 801
0.1 μM 025 μM 0.5 μM IμM 5μM
MPA 75115 72112 51116 44123 39120
•MPA = 0.25 μM, RBV = 20 μM
Conclusion
Dioxolane guansine (DXG) is a nucleoside analog of deoxyguanosine that has potent activity in vitro and in vivo against wild-type HIV-1 and viruses that carry mutations which confer resistance to AZT, 3TC, and to non-nυcleoside reverse transcriptase inhibitors. Resistance to DAPD/DXG was conferred by mutations at positions 65, 74, and 151 in the RT. The ability of two inhibitors of IMPDH, RBV and MPA, to enhance the anti-viral activity of DAPDDXG against both wild-type and resistant virus was confirmed. Both RBV and mycophenolic acid when combined with DAPD or DXG gave a strong synergistic anti-HIV response against wild-type virus. When tested against LAI, addition of MPA or RBV resulted in a greater than 10-fold increase in the anti-HIV activity of DXG. The concentrations of RBV required to produce these effects were higher than those required for MPA. MPA at a concentration of 0.25 μM completely inhibited HIV replication in PBMC when combined with DXG. This concenUation is below that required to inhibit T-ccll proliferation. Neither MPA nor RBV was cytotoxic at any of the concentrations tested and there was no enhanced mitochondrial toxicity. The combination of either IMPDH inhibitor restored the ability of DXG to inhibit otherwise resistant HIV. The L74V, K65R and QI5IM mutations have all been shown " to conftr variouTϊevels of resistance to DXG7 The combination of 25 Ϊ MPA oT20" μM RBV resulted in a decrease in EC» values of DXG for these mutants to within two- fold of the values obtained for wild-type virus When tested in PBMC, addition of increasing concentrations of MPA (up to 0.2 μM) or RBV (up to 10 μM) resulted in a greater than 50-fold increase in the activity of both DAPD and DXG against the highly DXG resistant K65R/Q1 SIM mutant.
These results support the use of MPA or RBV in combination therapy with DAPD for the treatment of HIV infection. Due to the favorable resistance profile of
DAPD, this combination can be of particular use in salvage therapy particularly when combined with multiple anti-retroviral agents. The combination of DAPD and MPA against HIV may be used in the salvage setting.
Example 3
The effects of MPA on intracellular DXG-TP and dGTP levels in PBMC was evaluated.
Cell Preparation
Peripheral Blood Mononuclear Cells (PBMC), from HIV negative donors were processed using standard techniques by banding on a Ficoll gradient. PBMCs were thawed and resuspended in complete media (containing glutamine, gentamicin & Fetal Bovine Serum, (FBS)) with 10% IL224 hours prior to use.
Compound Incubation
PBMC were incubated in complete media + 10% IL2 (37°C, 5% CO.) at a concentration of 2x10* cells/ml. The concenUation of MPA in media ranged from I μM
- 0.0625 μM. The concentrations of DXG and DAPD were 50 μM and 100 μM respectively, unless otherwise noted. Incubations ranged from Time 0 to 24 hour. Samples are processed through a wash procedure and resuspended in a methanol solution for analysis of uiphosphates. Analysis ofiriphosphøtes Sample Prep: Samples are sonicated then processed with ion pairing solid phase extraction (SPE) to isolate nucleotides.
Sample Analysis: The SPE extract is analyzed for nucleotide triphosph tes wiu positive ion electrospray ionization tandem mass spectrometry coupled to ionpairing high-performance liquid chromatography.
Quantitative and Detection Limits: dGTP: Quantitative range: 0.016 - 0.79 pmoles/106 cells
DXG-TP: Quantitative range: 0.0064 - 1.3 pmoles/10* cells
DAPD-TP: Lower limit of detection: 0.0088 pmoles/106 cells
The results are shown in Figure 5. Reduction of intracellular deoxyguanosine triphosphate (dGTP) levels through inhibition of IMPDH may effectively increase the intracellular concentration of DXG-TP, thereby augmenting inhibition HIV replication PBMCs were incubated with various concentrations of DXG with or without 0.25 μM of MPA and the intracellular concenUation of DXG-TP and dGTP measured by
Liquid Chromatography Mass Specuometry (LC-MS-MS). The results are shown in Figure 6. Intracellular DXG-TP levels increased in a linear fashion with increasing DXG concentrations (5 to 500 M). Addition f MPA resulted in an approximate 4-fold increase in DXG-TP levels at all concentrations of DXG tested. Maximum DXG-TP levels were observed following 2 hours of incubation with DXG, followed by a steady decline. In the presence of MPA, peak DXG-TP levels were also observed at 2 hours, however, elevated levels were maintained for up to 8 hours. Inuacellular dGTP levels decrease approximately 2-fold in cells incubated with MPA. The increased activity observed in vitro with the combination of DXG and MPA may be due to increased concentrations of the active compound DXG-TP. The data suggest a role for the use of
MPA in combination therapy with arndoxovir for the ueatment of HIV.
The results are further shown In Figure 7 which is a graph of the DXG-TP (pmole/1 6 cells) concentrations in DAPD incubated PBM cells; and Figure 8 which is a graph of DXG-TP (pmole/106 cells) concentrations in DXG incubated PBM cells. The results of the effects of MPA on DXG-TP and dGTP levels in PBMC are further shown in Table lb below. Table lb. Effect of MPA on DXG-TP and dGTP levels in PBMC*
Figure imgf000072_0001
•All incubaiioΛ. were performed for 6 hours at 37'C In 5% CO, ND- Not Determined Treating PBMC with increasing concentrations of MPA results in a linear increase in the intracellular DXG-TP levels. Increasing the concentration of DXG in the extracellular media results in a linear increase in the intracellular DXG-TP levels. These results were augmented by addition of 0.25 μM MPA. Addition of 0.25 μM MPA for 6 hours resulted in an average 4-fold increase in the intracellular DXG-TP levels and a 25% decrease in the endogenous levels of dGTP. Following 6 hours of incubation with
DAPD or 2 hours of incubation with DXG, DXGTP levels peaked, followed by a steady decline. The addition of 0.25 μM MPA extended the time of peak DXG-TP.
These results indicate that the increased activity observed in vitro with the combination of DXG and MPA is due to increased concentrations of the active compound DXG-TP.
This invention has been described with reference to certain embodiments. Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the Invention. It is intended that alt of these variations and modifications be included within the scope of this invention.
7)

Claims

What is claimed is:
I. A method for the ueatment of hepatitis B virus (HBV) in an infected host comprising administering an effective amount of a β-D-l,3-dioxo.anyl purine nucleoside of the formula:
Figure imgf000073_0001
wherein R is H, OH, CI, NH2 or NR'R2; R1 and R2 are independently hydrogen, alkyl or cycloalkyl; and R1 is H, alkyl, aryl, acyl, phosphate, a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, In combination or alternation with an inosine monophosphate dehydrogeπase (IMPDH) inhibitor, or a pharmaceutically acceptable salt or prodrug of either compound.
2. The method of claim 1, wherein the β-D-l,3-dioxoIanyl purine nucleoside is of the formula:
Figure imgf000073_0002
wherein RJ is H, alkyl, aryl, acyl, or phosphate or a pharmaceutically acceptable salt or prodrug.
3. The method of claim I, wherein the β-D-l,3-d-θxolanyl purine nucleoside is of the formula:
Figure imgf000074_0001
whcrein R* is H, alkyl, aryl, acyl, phosphate or a phaπnaceutically acceptable salt or prodrug.
4. The method of claim 1, wherein the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000074_0002
wherein R3 is H, alkyl, aryl, acyl, or phosphate, or a pharmaceutically acceptable salt or prodrug.
5. The method of claim I, wherein the β-D-l,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000074_0003
wherein R3 is H, alkyl, aryl, acyl, phosphate or a pharmaceutically acceptable salt or prodrug.
6". The method of claim 1 , wherein the β-D-1 ,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000075_0001
wherein R3 is H, alkyl, aryl, acyl, or phosphate or a pharmaceutically acceptable salt or prodrug.
7. The method of claim 1 , wherein the β-D-1, 3-dioxolaπyl purine nucleoside is (-)-(2R,4R)-2-amino-9-I(2-hydroxymethyl)-l ,3-dioxolan-4-yl)-adenine (DAPD).
8. The method of claim 1 , wherein the β-D- 1 ,3-dioxolanyl purine nucleoside is (-H2R,4R 9-[(2-hydroxymethyl)-l,3-dioxolan-4-y|]-guanine (DXG)-
9. The method of claim 1 , wherein the IMPDH inhibitor is selected from the group consisting of ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, selenazofurin, 5-ethynyl-l-β-D-riboruranosylimidazole-4-caιboxamide (EICAR) and (S)-N-3-[3^3-metIraxy-4-oxazol-5-yl-phenyl)-ureido]-beπzyl-carbamic acid terrahydrofυran-3-yl-ester (VX-497).
10. The method of claim 9, wherein the IMPDH inhibitor is mycophenolic acid.
11. The method of claim 9, wherein the IMPDH inhibitor is ribavirin.
12. The method of claim I , wherein the host is a human.
13. The method of claim I , wherein the HBV is a drug-resistant s ain.
14. The method of claim 13, wherein the HBV suain is resistant to one or more of the compounds (-)-(2R,4R)-2-aminθ'9-l(2-hydroxymethyl)-l,3-dioxoIan^-ylJ- adenine (DAPDJ rϊd (-R2R74R)-9-[(2-hydrdxyrneWyl)-
(DXG).
15. The method of claim I, wherein at least one of the β-D-I,3-dioxolanyl nucleoside and the IMPDH inhibitor is enantiomerically enriched.
16. The method of claim 1 wherein at least one of the β-D-1, 3-dioxolanyl nucleoside and the IMPDH inhibitor is administered in a pharmaceutically acceptable carrier or diluent.
17. A method for the treatment of a human immunodeficiency virus in an infected host comprising administering an effective amount of a β-D-1, 3-dioxolanyl purine nucleoside of the following formulae:
Figure imgf000076_0001
wherein R3 is H, alkyl, aryl, acyl, phosphate, a stabilized phosphate moiety, a phospholipid, or an ether-Iipid, in combination or alternation with an inosine monophosphate dehydrogeπase (IMPDH) inhibitor, or a pharmaceutically acceptable salt or prodrug of either compound.
18. The method of claim 1 , wherein the IMPDH inhibitor is selected from the group consisting of ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, le!eTw∞tΛϊπr 5^thynyl-I-βϊD^
(S)-N-3-[3-(3-methoxy-4-oxazol-5-yl-phenyl)-ureido]-benzyl-carbamic acid tetrahydrofuraπ-3-yl-ester (VX-497).
19. The method of claim 18, wherein the IMPDH inhibitor is mycophenolic acid.
20. The method of claim 18, wherein the IMPDH inhibitor is ribavirin.
21. The method of claim 17, wherein the host is a human.
22. The method of claim 17, wherein the HIV is a drug-resistant suain.
23. The method of claim 22, wherein the HIV strain is resistant to one or more of the compounds (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l,3-dioxolaπ-4-yl]- adenine (DAPD) and (>H2R,4R)-9-[(2-hydroxymethyl)-l,3-dioxoIan-4-yl]-guanrne (DXG).
24. The method of claim 17, wherein at least one of the β-D-1, -dioxolanyl purine nucleoside and the IMPDH inhibitor is enantiomerically enriched.
25. The method of claim 17, wherein at least one of the β-D-1, 3-dioxolanyl purine nucleoside and the IMPDH inhibitor is administered in a pharmaceutically acceptable carrier or diluent.
26. Use of a β-D-1, 3-dioxolan l purine nucleoside of the formula;
Figure imgf000078_0001
wherein R is H, OH, Cl, H2 or NR'R2; R1 and R2 are independently hydrogen, alkyl or cycloalkyl, and R} is H, alkyl, aryl, acyl or phosphate in combination with an inosine monophosphate dehydrogenase (IMPDH) inhibitor or a pharmaceutically acceptable salt or prodrug of either compound in the manufacture of a medicament for the treatment of hepatitis B virus (HBV) in an infected host.
27. The use of claim 26, wherein the β-D-1, 3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000078_0002
wherein R >3 : is H, alkyl, aryl, acyl, or phosphate or a pharmaceutically acceptable salt or prodrug.
28. The use of claim 26, wherein the β-D-1, 3-dloxolanyl purine nucleoside is of the formula:
Figure imgf000078_0003
wherein R3 is H, alkyl. a l. *c l> phosphate or a pharmaceutically acceptable salt or prodrug.
29. The use of claim 26, wherein the β-D-1, 3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000079_0001
wherein R3 is H, alkyl, aryl, acyl, or phosphate, or a pharmaceutically acceptable salt or prodrug.
30. The use of claim 26, wherein the β-D-1 ,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000079_0002
wherein R3 is H, alkyl, aryl, acyl, phosphate or a pharmaceutically acceptable salt or prodrug.
31. The use of claim 26, wherein the β-D-1 ,3-dioxolanyl purine nucleoside is of the formula:
Figure imgf000079_0003
wherein R3 is H, alkyl, aryl, acyl, or phosphate or a pharmaceutically acceptable salt or prodrug.
32. The use of claim 26, wherein the β-D-1, 3-dioxolanyl purine nucleoside is (-)-(2R,4R 2-amino-9-l(2-hydroxymethyl)-l,3-dioxo)an-4-yl]-adenine (DAPD).
33. The use of claim 26, wherein the β-D-1 ,3-dioxolanyl purine nucleoside is (-)-(2R,4R)-9-[(2-hydroxymethyI)-1 ,3-dioxolan-4-yl]-guanine (DXG).
34. The use of claim 26, wherein the IMPDH inhibitor is selected from the group consisting of ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, selenazofurin, 5-ethyπyl-l-β-T>-ribofuranosylimida2θle-4-carboxamide (ElCAR) and (S)-N-3-{3-(3-methoxy-4-oxa2θl-5-yI-ρhenyl)-ureido]-benzyl-carbamic cid teuahydrofuran-3-yl-ester (VX-497).
35. The use of claim 34, wherein the IMPDH inhibitor is mycophenolic acid.
36. The use of claim 34, wherein the IMPDH inhibitor is ribavirin.
37. The use of claim 26, wherein the HBV is a drug-resistant strain.
38. The use of claim 37, wherein the HBV strain is resistant to one or more of the compounds (-)-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-l ,3-dioxolan-4-yl]-adenine (DAPD) and (-H2R,4R)-9-[(2-hydroxymethyl)-l,3^ioxolen-4-yl]-guanine (DXG).
39. The use of claim 26, wherein at least one of the β-D-1 ,3-dioxolanyl nucleoside and the IMPDH inhibitor is enantiomerically enriched.
40. The use of claim 26, wherein at least one of the β-D-1, 3-dioxolanyl nucleoside and the IMPDH inhibitor is administered in a pharmaceutically acceptable carrier or diluent.
41. Use of a β-D-1 ,3-dioxolanyl purine nucleoside of the following formulae:
Figure imgf000081_0001
wherein R3 is H, alkyl, aryl, acyl, or phosphate, in combination or alternation with an inosine monophosphate dehydrogenase (IMPDH) inhibitor or a pharmaceutically acceptable salt or prodrug of either compound in the manufacture of a medicament for the treatment of a human immunodeficiency virus in an infected host.
42. The use of claim 41, wherein the IMPDH inhibitor is selected from the group consisting of ribavirin, mycophenolic acid, benzamide riboside, tiazofurin, selenazofurin, 5-ethynyl-l-β-D-ribofuranosyIimidazole-4-carboxamide (EICAR) and (S)-N-3-[3-(3-methoxy-4-oxazol-5-yl-phenyl)-ureido]-benzyl-carbamic acid tetrahydrofuran-3-yl-ester (VX-497).
43. The use of claim 42, wherein the IMPDH inhibitor is mycophenolic acid.
44. The use of claim 42, wherein the IMPDH inhibitor is ribavirin.
45. The use of claim 41 , wherein the HI V is a drug-resistant stiain.
46. The use of claim 45, wherein the HTV strain is resistant to one or more of the compounds (-)-(2R,4R)-2-amino-9-[(2-hydroxymcthyl)- 1 ,3-dioxolan-4-y^adenine DAPD and (-)-(2R74RV9-[(2^hydrrotymethyl)-
47. The use of claim 41 , wherein at least one of the β-D-1 ,3-dioxolanyl purine nucleoside and the IMPDH inhibitor is enantiomerically enriched.
48. The use of claim 41, wherein at least one of the β-D-l,3-dioxolanyl purine nucleoside and the IMPDH inhibitor is administered in a pharmaceutically acceptable carrier or diluent.
PCT/US2003/021109 2002-07-03 2003-07-03 Combination therapy with 1,3-dioxolanes and inosine monophosphate dehydrogenase inhibitors WO2004009595A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009793A1 (en) * 1992-10-28 1994-05-11 Emory University ENANTIOMERICALLY PURE β-D-DIOXOLANE NUCLEOSIDES WITH SELECTIVE ANTI-HEPATITIS B VIRUS ACTIVITY
WO2000051615A1 (en) * 1999-03-03 2000-09-08 University Of Maryland Biotechnology Institute Synergistic combinations of guanosine analog reverse transcriptase inhibitors and inosine monophosphate dehydrogenese inhibitors and uses therefor
US6358963B1 (en) * 1998-12-23 2002-03-19 Biochem Pharma Inc. Antiviral nucleoside analogues

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009793A1 (en) * 1992-10-28 1994-05-11 Emory University ENANTIOMERICALLY PURE β-D-DIOXOLANE NUCLEOSIDES WITH SELECTIVE ANTI-HEPATITIS B VIRUS ACTIVITY
US6358963B1 (en) * 1998-12-23 2002-03-19 Biochem Pharma Inc. Antiviral nucleoside analogues
WO2000051615A1 (en) * 1999-03-03 2000-09-08 University Of Maryland Biotechnology Institute Synergistic combinations of guanosine analog reverse transcriptase inhibitors and inosine monophosphate dehydrogenese inhibitors and uses therefor

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