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    • 专利摘要:
    The present invention is directed to a method for treating hepatitis B virus infection in humanscomprising administering a synergistically effective amount of agents having known anti-hepatitisB virus activity in combination or alternation. Specifically, the invention is directed to a methodfor treating hepatitis B virus infection comprising administering FTC in combination or alternationwith penciclovir, famciclovir or Bis-POM-PMEA. Additionally, the invention is directed to amethod for treating hepatitis B virus infection comprising administering L-FMAU in combinationor alternation with DAPD, penciclovir or Bis-POM-PMEA. The invention is further directed to amethod for treating hepatitis B virus infection comprising administering DAPD in combination oralternation with Bis-POM-PMEA.
    公开号:EP1382343A1
    申请号:EP03077543
    申请日:1999-11-02
    公开日:2004-01-21
    发明作者:Phillip A. Furman；George R. Painter；David Barry；Franck Rousseau
    申请人:Triangle Pharmaceuticals Inc；
    IPC主号:A61K31-00
    专利说明:
    [0001] This invention is in the area of methods for the treatment of hepatitis B virus (alsoreferred to as "HBV") that includes administering to a host in need thereof, an effectivecombination of nucleosides which have known anti-hepatitis B activity.
    [0002] HBV is second only to tobacco as a cause of human cancer. The mechanism bywhich HBV induces cancer is unknown, although it is postulated that it may directly triggertumor development, or indirectly trigger tumor development through chronic inflammation,cirrhosis, and cell regeneration associated with the infection.
    [0003] Hepatitis B virus has reached epidemic levels worldwide. After a two to three monthincubation period in which the host is unaware of the infection, HBV infection can lead toacute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated bloodlevels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, oftenfatal form of the disease in which massive sections of the liver are destroyed.
    [0004] Patients typically recover from acute hepatitis. In some patients, however, high levelsof viral antigen persist in the blood for an extended, or indefinite, period, causing a chronicinfection. Chronic infections can lead to chronic persistent hepatitis. Patients infected withchronic persistent HBV are most common in developing countries. By mid-1991, there wereapproximately 225 million chronic carriers of HBV in Asia alone, and worldwide, almost 300million carriers. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver, andhepatocellular carcinoma, a primary liver cancer.
    [0005] In western industrialized countries, high risk groups for HBV infection include thosein contact with HBV carriers or their blood samples. The epidemiology of HBV is verysimilar to that of acquired immune deficiency syndrome (AIDS), which accounts for whyHBV infection is common among patients with AIDS or AIDS related complex. However,HBV is more contagious than HIV.
    [0006] However, more recently, vaccines have also been produced through geneticengineering and are currently used widely. Unfortunately, vaccines cannot help those alreadyinfected with HBV. Daily treatments with α- interferon, a genetically engineered protein, hasalso shown promise, but this therapy is only successful in about one third of treated patients.Further, interferon cannot be given orally.
    [0007] A number of synthetic nucleosides have been identified which exhibit activity againstHBV. The (-)-enantiomer of BCH-189, known as 3TC, claimed in U. S. Patent 5,539,116 toLiotta, et al., has been approved by the U. S. Food and Drug Administration for the treatmentof hepatitis B. See also EPA 0 494 119 A1 filed by BioChem Pharma, Inc.
    [0008] β-2-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane ("FTC"), claimed in U.S. Patent Nos. 5,814,639 and 5,914,331 to Liotta, et al., exhibits activity against HBV. SeeFurman, et al., "The Anti-Hepatitis B Virus Activities, Cytotoxicities, and Anabolic Profilesof the (-) and (+) Enantiomers of cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine"Antimicrobial Agents and Chemotherapy, December 1992, page 2686-2692; andCheng, et al., Journal of Biological Chemistry, Volume 267(20), 13938-13942 (1992).
    [0009] U. S. Patent Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu, et al.) disclose the use of2'-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) for the treatment of hepatitis Band Epstein Barr virus.
    [0010] U. S. Patent No. 5,767,122 to Emory University and The University of GeorgiaResearch Foundation, Inc. discloses and claims enantiomerically pure β-D-dioxolanylnucleosides of the formula:
    [0011] Penciclovir (2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin-6-one;PCV) has established activity against hepatitis B. See U.S. Patent Nos. 5,075,445 and5,684,153.
    [0012] Adefovir (9-[2-(phosphonomethoxy)ethyl]adenine, also referred to as PMEA or [[2-(6-amino-9H-purin-9-yl)ethoxy]methylphosphonicacid), also has established activity againsthepatitis B. See for example U.S. Patent Nos. 5,641,763 and 5,142,051.
    [0013] Yale University and The University of Georgia Research Foundation, Inc. disclose theuse of L-FDDC (5-fluoro-3'-thia-2',3'-dideoxycytidine) for the treatment of hepatitis B virusin WO 92/18517.
    [0014] von Janta-Lipinski et al. disclose the use of the L-enantiomers of 3'-fluoro-modifiedβ-2'-deoxyribonucleoside 5'-triphosphates for the inhibition of hepatitis B polymerases (J.Med. Chem., 1998, 41,2040-2046). Specifically, the 5'-triphosphates of 3'-deoxy-3'-fluoro-β-L-thymidine(β-L-FTTP), 2',3'-dideoxy-3'-fluoro-β-L-cytidine (β-L-FdCTP), and 2',3'-dideoxy-3'-fluoro-β-L-5-methylcytidine(β-L-FMethCTP) were disclosed as effectiveinhibitors of HBV DNA polymerases.
    [0015] It has been recognized that drug-resistant variants of HBV can emerge after prolongedtreatment with an antiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in the viral lifecycle, and most typically in the case ofHBV, DNA polymerase. Recently, it has been demonstrated that the efficacy of a drugagainst HBV infection can be augmented by administering the compound in combinationwith a second, and perhaps third, antiviral compound that induces a different mutation fromthat caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, orother parameter of the drug can be altered by such combination therapy. In general,combination therapy induces multiple simultaneous stresses on the virus.
    [0016] United States Patent No. 5,808,040 discloses that L-FMAU can be administered incombination with FTC, 3TC, carbovir, acyclovir, interferon, AZT, DDI (2',3'-dideoxyinosine),DDC (2',3'-dideoxycytidine), L-DDC, L-F-DDC, and D4T.
    [0017] United States Patent No. 5,674,849 discloses the use of a nucleoside in combinationwith an oligonucleotide for the treatment of a viral disease.
    [0018] United States Patent No. 5,684,010 discloses a method for the treatment of hepatitis Bthat includes administering in combination or alternation a compound of the formula:
    [0019] WO 98/23285 discloses a method for the treatment or prophylaxis of hepatitis B virusinfections in a human or animal patient which comprises administering to the patienteffective or prophylactic amounts of penciclovir (or a bioprecursor thereof such asfamciclovir) and alpha-interferon.
    [0020] In light of the fact that hepatitis B virus has reached epidemic levels worldwide, andhas severe and often tragic effects on the infected patient, there remains a strong need toprovide new effective treatments for humans infected with the virus that have low toxicity tothe host.
    [0021] Therefore, it is an object of the present invention to provide new methods for thetreatment of human patients or other hosts infected with hepatitis B virus and relatedconditions comprising administering a synergistically effective amount of a combination ofanti-HBV agents. Summary of the Invention
    [0022] It has been discovered that certain combinations of agents with hepatitis B activity aresynergistic, and thus can provide enhanced benefits to the patient when administered in aneffective combination or alternation dosage pattern.
    [0023] In one preferred embodiment of the present invention, a method for treating HBVinfection and related conditions in humans is disclosed, comprising administering asynergistically effective amount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(FTC), preferably substantially in the form of the (-)-optical isomer, or a pharmaceutically acceptable salt, ester or prodrug thereof with Penciclovir (2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin-6-one,also referred to as "PCV").Famciclovir, or any other bioprecursor of Penciclovir, can be used in place of Penciclovir inany embodiment of this invention.
    [0024] Another preferred embodiment of the present invention is a method for treating HBVinfection and related conditions in humans, comprising administering in combination oralternation a synergistically effective amount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(FTC), preferably substantially in the form of the (-)-optical isomer, or apharmaceutically acceptable salt, ester or prodrug thereof, with 9-[2-(phosphonomethoxy)ethyl]adenine(PMEA, also referred to below as Bis-POM-PMEA orBP-PMEA ), or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in apharmaceutically acceptable carrier.
    [0025] In another preferred embodiment of the present invention, a method for treating HBVinfection and related conditions in humans is disclosed, comprising administering incombination or alternation a synergistically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester or prodrugthereof, with a compound of the formula:
    [0026] In yet another preferred embodiment of the present invention, a method for treatingHBV infection and related conditions in humans is disclosed, comprising administering asynergistically effective combination or alternation amount of2'-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester or prodrug thereof, with Penciclovir, or a pharmaceutically acceptable salt, ester or prodrug thereof,optionally in a pharmaceutically acceptable carrier.
    [0027] In still another preferred embodiment of the present invention, a method for treatingHBV infection and related conditions in humans is disclosed, comprising administering asynergistically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU),or a pharmaceutically acceptable salt, ester or prodrug thereof, with 9-[2-(phosphonomethoxy)ethyl]adenine(PMEA), or a pharmaceutically acceptable salt, ester orprodrug thereof, optionally in a pharmaceutically acceptable carrier.
    [0028] Another preferred embodiment of the present invention comprises a method fortreating HBV infection and related conditions in humans, comprising administering asynergistically effective amount of a compound of the formula:
    [0029] As used herein, the term "isolated enantiomer" refers to a nucleoside composition thatincludes approximately 95% to 100%, or more preferably, over 97% of a single enantiomerof that nucleoside.
    [0030] The terms "substantially pure form" or substantially free of its opposite enantiomerrefers to a nucleoside composition of one enantiomer that includes no more than about 5% of the other enantiomer, more preferably no more than about 2%, and most preferably less thanabout 1 % is present.
    [0031] The synergistic combination of compounds or their pharmaceutically acceptableesters or salts, are also useful in the prevention and treatment of HBV infections and otherrelated conditions such as anti-HBV antibody positive and HBV-positive conditions, chronicliver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronicpersistent hepatitis, and fatigue. These synergistic formulations can also be usedprophylactically to prevent or retard the progression of clinical illness in individuals who areanti-HBV antibody or HBV antigen positive or who have been exposed to HBV.
    [0032] The active compound can be converted into a pharmaceutically acceptable ester byreaction with an appropriate esterifying agents, for example, an acid halide or anhydride. Thecompound or its pharmaceutically acceptable derivative can be converted into apharmaceutically acceptable salt thereof in a conventional manner, for example, by treatmentwith an appropriate base. The ester or salt of the compound can be converted into the parentcompound, for example, by hydrolysis.
    [0033] The term "synergistic combination" refers to a combination of drugs which producesa synergistic effect in vivo, or alternatively, in vitro as measured according to the methodsdescribed herein. I. Active Compounds, and Physiological Acceptable Salts Thereof
    [0034] The active compounds disclosed herein are therapeutic nucleosides or cyclic oracyclic nucleoside analogs with known activity against hepatitis B. It has been discoveredthat certain combinations of nucleosides provide an advantage over monotherapy, or overother combinations. Not all combinations of the known anti-HBV drugs provide a benefit; itis often the case that drugs act antagonistically.
    [0035] The active compound can be administered as any derivative that upon administrationto the recipient, is capable of providing directly or indirectly, the parent compound, or thatexhibits activity itself. Nonlimiting examples are the pharmaceutically acceptable salts(alternatively referred to as "physiologically acceptable salts"), and the 5' and N4 cytosinyl orN6-adeninyl acylated (esterified) derivatives of the active compound (alternatively referred toas "physiologically active derivatives"). In one embodiment, the acyl group is a carboxylicacid 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, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionallysubstituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, or is a sulfonate ester such asalkyl or aralkyl sulphonyl including methanesulfonyl, phosphate, including but not limited tomono, di or triphosphate ester, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl(e.g., dimethyl-5-butylsilyl) or diphenylmethylsilyl. Aryl groups in the esters optionallycomprise a phenyl group.
    [0036] Modifications of the active compound, and especially at the N4 cytosinyl or N6adeninyl and 5'-O positions, can affect the bioavailability and rate of metabolism of theactive species, thus providing control over the delivery of the active species. Further, themodifications can affect that antiviral activity of the compound, in some cases increasing theactivity over the parent compound. This can easily be assessed by preparing the derivativeand testing its antiviral activity according to the methods described herein, or other methodsknown to those skilled in the art. Prodrugs
    [0037] Any of the anti-hepatitis B agents described herein can be administered as a prodrugto increase the activity, bioavailability, stability or otherwise alter the properties of thenucleoside. A number of hydroxyl-bound prodrug ligands are known. In general, alkylation,acylation or other lipophilic modification of the hydroxy, mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples of substituent groups thatcan replace one or more hydrogens on the hydroxyl or phosphate moiety are alkyl, aryl,steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many aredescribed in R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any ofthese can be used in combination with the disclosed nucleosides to achieve a desired effect.
    [0038] Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents thatcan be covalently incorporated into the nucleoside, preferably at the 5'-OH of the nucleosideor hydroxyl of the acyclic nucleoside analogs (such as PMEA or Penciclovir), include U.S.Patent Nos. 5,149,794 (Sep. 22, 1992, Yatvin, et al.); 5,194,654 (mar. 16, 1993, Hostetler, etal.); 5,223,263 (June 29, 1993, Hostetler, 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.).
    [0039] Foreign patent applications that disclose lipophilic substituents that can be attached tothe active compounds of the present invention, or lipophilic preparations, include WO89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273,WO/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721. II. Preparation of the Active Compounds
    [0040] The therapeutic nucleosides used in the synergistic compositions of the presentinvention and processes for preparing them are known in the art.
    [0041] β-2-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), and itsenantiomers, can be prepared by the methods disclosed in U. S. Patent Nos. 5,204,466,5,700,937, 5,728,575 and 5,827,727.
    [0042] 2'-Fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) can be prepared by themethods disclosed in U. S. Patent Nos. 5,565,438, 5,567,688 and 5,587,362 to Chu, et al.
    [0043] Methods for the preparation of the DAPD compounds, including (2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine(DAPD) are disclosed in U. S. Patent Nos.5,767,122; 5,684,010; 5,444,063, and 5,179,104.
    [0044] Pencyclovir can be prepared by the methods disclosed in U.S. Patent Nos. 5,075,445and 5,684,153.
    [0045] PMEA can be prepared by the methods disclosed in U.S. Patent Nos. 5,641,763 and5,142,051.
    [0046] Mono, di, and triphosphate derivatives of the active nucleosides can be prepared asdescribed according to published methods. The monophosphate can be prepared according tothe procedure of Imai, et al., J. Org. Chem., 34(6), 1547-1550 (June 1969). The diphosphatecan be prepared according to the procedure of Davisson, et al., J. Org. Chem., 52(9), 1794-1801(1987). The triphosphate can be prepared according to the procedure of Hoard, et al., J.Am. Chem. Soc., 87(8), 1785-1788 (1965). III. Combination Therapy
    [0047] It has been recognized that drug-resistant variants of HBV can emerge after prolongedtreatment with an antiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in the viral lifecycle, and most typically in the case ofHBV, DNA polymerase. Recently, it has been demonstrated that the efficacy of a drug against HBV infection can be prolonged, augmented, or restored by administering thecompound in combination or alternation with a second, and perhaps third, antiviral compoundthat induces a different mutation from that caused by the principle drug. Alternatively, thepharmacokinetics, biodistribution, or other parameter of the drug can be altered by suchcombination therapy. In general, combination therapy induces multiple simultaneous stresseson the virus. Example 1
    [0048] Test compounds DAPD, DXG, (-)-β-FTC, L-FMAU DMVI assay controls Untreated cells, 3TC (lamivudine), penciclovir (PCV)
    [0049] Details of the assay methodology can be found in: Korba and Gerin, Antiviral Res.19: 55-70 (1992) and Korba, Antiviral Res. 29: 49-52 (1996). The antiviral evaluations wereperformed on six separate cultures per each of four test concentrations. All wells, in allplates, were seeded at the same density and at the same time.
    [0050] Due to the inherent variations in the levels of both intracellular and extracellular HBVDNA, only depressions greater than 3.0-fold for HBV virion DNA from the average levelsfor these HBV DNA forms in untreated cells are generally considered to be statisticallysignificant [P<0.05] (Korba and Gerin, Antiviral Res. 19: 55-70, 1992). Typical values forextracellular HBV virion DNA in untreated cells range from 80 to 150 pg/ml culture medium(average of approximately 92 pg/ml).
    [0051] For reference, the manner in which the hybridization analyses were performed forthese experiments results in an equivalence of approximately 1.0 pg of extracellular HBVDNA/ml culture medium to 3 X 105 viral particles/ml.
    [0052] Toxicity analyses were performed in order to access whether any observed antiviraleffects are due to a general effect on cell viability. The method used was uptake of neutralred dye, a standard and widely used assay for cell viability in a variety of virus-host systems,including HSV and HIV. Details of the procedure are provided in the toxicity table legends. EXPERIMENTAL PARAMETERS
    [0053] Test compounds were received as solid material at room temperature in good packagecondition. Test compounds were solubilized in 100% tissue culture grade DMSO (Sigma, Corp.) at 100mM (DAPD, FTC, L-FMAU) or 50mM (DXG). Daily aliquots of testcompounds were made in individual tubes and stored at -20°C. On each day of treatment,daily aliquots of the test compounds were suspended into culture medium at roomtemperature, and immediately added to the cell cultures.
    [0054] For the antiviral test analyses, confluent cultures were maintained on 96-well flatbottomed tissue culture plates. Two separate (replicate) plates were used for each drugtreatment. A total of 3 cultures on each plate were treated with each of the dilutions ofantiviral agents (6 cultures per dilution). Cultures were treated with 9 consecutive dailydoses of the test compounds. Medium was changed daily with fresh test compounds. Onlyextracellular (virion) HBV DNA levels were followed.
    [0055] Toxicity analysis were performed in 96-well flat bottomed tissue culture plates. Cellsfor the toxicity analyses were cultured and treated with test compounds with the sameschedule and under identical culture conditions as used for the antiviral evaluations. Eachcompound was tested at 4 concentrations, each in triplicate cultures. Uptake of neutral reddye was used to determine the relative level of toxicity 24 hours following the last treatment.The absorbance of internalized dye at 510 nM (A510) was used for the quantative analysis.Values are presented as a percentage of the average A510 values (± standard deviations) in 9separate cultures of untreated cells maintained on the same 96-well plate as the testcompounds.
    [0056] Combination treatments were conducted using the primary analysis format except that6 serial 3-fold dilutions were used for each drug combination and a total of 8 separatecultures were used for each dilution of the combinations. Compounds were mixed at molarratios designed to give approximately equipotent antiviral effects based on the EC90 values.Three different molar ratios were used for each combination to allow for variability in theestimates of relative potency. These molar ratios were maintained throughout the dilutionseries. The corresponding monotherapies were conducted in parallel to the combinationtreatments using the standard primary assay format.
    [0057] For reporting purposes, the SI, EC50, EC90, and CC50 values reported for thecombination treatments are those of the first compound listed for the combination mixture.The concentrations and SI, EC50, EC90, and CC50 values of the second compound in themixture can be calculated using the molar ratio designated for that particular mixture. Further details on the design of combination analyses as conducted for this report can befound in BE Korba (1996) Antiviral Res. 29:49.
    [0058] Analysis of synergism, additivity, or antagonism were determined by analysis of thedata using the CalcuSyn™ program (Biosoft, Inc.). This program evaluates drug interactionsby use of the widely accepted method of Chou and Talalay combined with a statisticallyevaluation using the Monte Carlo statistical package. The data are displayed in severaldifferent formats including median-effect and dose-effects plots, isobolograms, andcombination index [CI] plots with standard deviations. For the latter analysis, a CI greaterthan 1.0 indicates antagonism and a CI less than 1.0 indicates synergism.
    [0059] For the toxicity analyses associated with the combination treatments, the experimentaldesign was limited by either/or the toxicity of the more toxic compound in the mixture or thestock concentrations (e.g. related to the total volume of DMSO that could be added to thecultures without inducing toxicity due to DMSO and not the test compounds). Antiviral Evaluations
    [0060] ASSAY CONTROLS: Within normal variations, levels of extracellular HBV (virion)DNA remained constant in the untreated cells over the challenge period. The positivetreatment controls, 3TC (lamivudine) [((-)β,L,2',3'-dideoxy-3' thiacytidine] and penciclovir[PCV] (both purchased from Moraveck Biochemicals, La Brea, CA), induced significantdepressions of HBV DNA replication at the concentrations used. The activities observed for3TC in these analyses were consistent with previous experiments where approximately 0.15to 0.2µM 3TC induced a 90% depression of HBV virion DNA relative to average levels inuntreated cells after 9 days of continuous treatment of 2.2.15 cells [EC90] (for example, seeKorba and Boyd, Antimicrob. Agents Chemother. (1996) 40:1282-1284). The activitiesobserved for PCV in these analyses were higher than previously reported (EC90 ofapproximately 0.7 to 0.9uM, Korba and Boyd, Antimicrob. Agents Chemother. (1996)40:1282-1284). However, the preparation of PCV used for these experiments hasconsistently produced anti-HBV activities in the range reported here in several otherindependent experiments.
    [0061] TEST COMPOUNDS: Test compound DAPD, FTC, DXG, and L-FMAU inducedsignificant and selective depressions in extracellular (virion) HBV DNA levels produced by2.2.15 cells.
    [0062] The antiviral activity of DAPD was enhanced by co-treatment with FTC. Theantiviral activity of DAPD was synergistic at a 3:1 or a 1:1 molar ratio at all but the highestconcentrations tested. As the relative concentration of FTC increased, the co-operativeeffects of the two agents decreased. At the 1:3 molar ratio, the two agents appeared to beantagonistic.
    [0063] DAPD and PCV appeared to be antagonistic at all three molar ratios and at allconcentrations.
    [0064] At the 1:10 and 1:1 molar ratios, DAPD and L-FMAU appeared to be antagonistic.At the 1:3 molar ratio (approximately equipotent potencies based on the EC90's) theinteractions of the two agents were more complex. DAPD and L-FMAU exhibitedmoderately synergistic to additive interactions at lower concentrations which progressed toincreasingly more antagonistic interactions at higher concentrations. Subsequent testing,however, indicated that DAPD is synergistic with L-FMAU.
    [0065] The antiviral activity of L-FMAU was enhanced by co-treatment with FTC. Theantiviral activity of DAPD and FTC was moderately synergistic at a 3:1 or a 10:1 molar ratioat all but the highest concentrations tested. As the relative concentration of FTC increased,the cooperative effects of the two agents decreased. At the 1:1 molar ratio, the two agentsappeared to be antagonistic.
    [0066] The antiviral activity of L-FMAU was also enhanced by co-treatment with PCV. Theantiviral activity of DAPD and PCV was weakly synergistic at a 1:1 or a 1:3 molar ratio at allconcentrations tested. As the relative concentration of PCV increased, the co-operativeeffects of the two agents decreased. At the 1:10 molar ratio, the two agents appeared to beantagonistic. Toxicity Evaluations
    [0067] No significant toxicity (greater than 50% depression of the dye uptake levels observedin untreated cells) was observed for 3TC, PCV, or any of the test compounds at theconcentrations used for the antiviral evaluations.
    [0068] None of the combination treatments appeared to enhance the toxicity profiles of eitheragent in the different mixtures. The toxicity profiles of some of the combination mixtureswas apparently higher than the corresponding monotherapies since the values are reported asa factor of the concentration of the first compound listed for each mixture. This is especially notable for the mixtures containing PCV. However, recalculation of the toxicity profiles onthe basis of the second compound (e.g. PCV) in the mixtures revealed that all of the apparenttoxicities were due to the more toxic compound and that no enhanced toxicity was present inthese combinations. Example 2
    [0069] Test compounds provided (-)-β-FTC DMVI assay controls untreated cells, 3TC (lamivudine), penciclovir (PCV)
    [0070] Details of the assay methodology were as given above. Test compounds werereceived as solid material at room temperature in good package condition. Test compoundswere solubilized in 100% tissue culture grade DMSO (Sigma, Corp.) at 100mM. Dailyaliquots of test compounds were made in individual tubes and stored at -20°C
    [0071] TEST COMPOUNDS: Test compound FTC induced significant and selectivedepressions in extracellular (virion) HBV DNA levels produced by 2.2.15 cells.
    [0072] The antiviral activity of FTC was enhanced by co-treatment with PCV. The antiviralactivity of the combination therapy was synergistic at all molar ratios tested. As the relativeconcentration of PCV increased, the cooperative effects of the two agents decreased. Toxicity Evaluations
    [0073] No significant toxicity (greater than 50% depression of the dye uptake levels observedin untreated cells) was observed for 3TC, PCV, FTC, or any of the combination treatments atthe concentrations used for the antiviral evaluations (Tables S1, T1).
    [0074] None of the combination treatments appeared to enhance the toxicity profiles of eitheragent in the different mixtures. The toxicity profiles of some of the combination mixtureswas apparently higher than the corresponding monotherapies since the values are reported asa factor of the concentration of the first compound listed for each mixture. Example 3 Combination Therapy with PMEA
    [0075] Test compounds provided PMEA, (-)-β-FTC, DAPD, L-FMAU DMVI assay controls Untreated cells, 3TC (lamivudine)
    [0076] Details of the assay methodology were as given above. Test compounds (except forbis-POM-PMEA) were received as powdered material on dry ice in good package conditionand stored at -20°C. Test compound bis-POM-PMEA was received as a 100mM solution inDMSO. Daily aliquots of test compounds were made in individual tubes and stored at -20°C.On each day of treatment, daily aliquots of the test compounds were suspended into culturemedium at room temperature, and immediately added to the cell cultures.
    [0077] TEST COMPOUNDS (PRIMARY ANALYSES): All of the test compounds inducedsignificant and selective depressions in extracellular (virion) HBV DNA levels produced by2.215 cells. However, the potencies of test compounds (-)-β-FTC, DAPD and L-FMAU werelower than that observed in earlier analyses. This was most apparent for DAPD and L-FMAU.
    [0078] Bis-POM-PMEA (BP-PMEA) + FTC. The mixture of BP-PMEA and FTCproduced an anti-HBV activity that was moderately synergistic overall. The potency of themixtures increased as the relative proportion of FTC increased. However, the most favorableoverall interactions occurred where the concentration of FTC was proportionately lower. Thesame relative degree of synergism was generally observed at all concentrations of the 30:1mixture. Relatively more synergistic interactions were observed at the lower concentrationsof the 10:1 and 3:1 mixture and moderate to strong antagonism was observed at the highestconcentrations of the 3:1 mixture.
    [0079] BP-PMEA + DAPD. The mixture of BP-PMEA and DAPD produced an anti-HBVactivity that was moderately to weakly synergistic at lower relative concentrations of DAPDand moderately to strongly antagonistic at higher relative concentrations of DAPD. Thepotency of the mixtures also decreased as the relative proportion of DAPD increased.Relatively more synergistic interactions were observe at the lower concentrations of thedifferent mixtures.
    [0080] BP-PMEA + L-FMAU. The mixture of BP-PMEA and L-FMAU produced an anti-HBVactivity that was moderately synergistic at lower relative concentrations of L-FMAU and additive to weakly antagonistic at higher relative concentrations of L-FMAU. Thepotency of the mixtures was lowest at the highest relative concentration L-FMAU (1:1 molarratio). The most favorable overall interactions were observed at the 3:1 molar ratio of thetwo compounds. Relatively more synergistic interactions were observed at the lowerconcentrations of different mixtures. Toxicity Evaluations
    [0081] No significant toxicity (greater than 50% depression of the dye uptake levels observedin untreated cells) was observed for 3TC, any of the test compounds, or any of the compoundmixtures at the concentrations used for the antiviral evaluations. None of the compoundmixtures appeared to significantly enhance toxicity. The patterns of toxicity observed for thecompound mixtures was similar to, and consistent with, that observed for the monotherapies. IV. Preparation of Pharmaceutical Compositions
    [0082] Humans suffering from any of the diseases described herein arising out of HBVinfection, can be treated by administering to the patient an effective amount of identifiedsynergistic anti-HBV agents in a combination or independent dosage form for combination oralternation therapy, optionally in a pharmaceutically acceptable carrier or diluent. The activematerials can be administered by any appropriate route, for example, orally, parenterally,intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
    [0083] The active compounds are included in the pharmaceutically acceptable carriers ordiluents in amounts sufficient to deliver to a patient a therapeutically effective amount ofcompound to inhibit viral replication in vivo, especially HBV replication, without causingserious toxic effects in the patient treated. By "inhibitory amount" is meant an amount ofactive ingredient sufficient to exert an inhibitory effect as measured by, for example, an assaysuch as the ones described herein.
    [0084] A preferred dose of the compound for all the above-mentioned conditions will be inthe range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, moregenerally 0.1 to about 100 mg per kilogram body weight of the recipient per day. Theeffective dosage range of the pharmaceutically acceptable derivatives can be calculated basedon 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 byother means known to those skilled in the art.
    [0085] The compound is conveniently administered in unit or any suitable dosage form,including but not limited to one containing 7 to 3000 mg, preferably 70 to 1400 mg of activeingredient per unit dosage form. An oral dosage of 50-1000 mg is usually convenient, moretypically 50-300 mg.
    [0086] Ideally the active ingredient should be administered to achieve peak plasmaconcentrations of the active compound of from about 0.2 to 70 µM, preferably about 1.0 to 10µM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solutionof the active ingredient, optionally in saline, or administered as a bolus of the activeingredient.
    [0087] The concentration of active compound in the drug composition will depend onabsorption, inactivation, and excretion rates of the drug as well as other factors known tothose of skill in the art. It is to be noted that dosage values will also vary with the severity ofthe 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 andthe professional judgment of the person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein are exemplary only and arenot intended to limit the scope or practice of the claimed composition. The active ingredientmay be administered at once, or may be divided into a number of smaller doses to beadministered at varying intervals of time.
    [0088] A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an edible carrier. They may beenclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipients and used in the formof tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition.
    [0089] The tablets, pills, capsules, troches and the like can contain any of the followingingredients, 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 suchas alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; aglidant 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 dosageunit form is a capsule, it can contain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings of sugar, shellac, or otherenteric agents.
    [0090] The compound 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.
    [0091] The compound or a pharmaceutically acceptable derivative or salt thereof can also bemixed with other active materials that do not impair the desired action, or with materials thatsupplement the desired action, such as antibiotics, antifungals, antiinflammatories, proteaseinhibitors, or other nucleoside or nonnucleoside antiviral agents, as discussed in more detailabove. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol orother synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; cheating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agentsfor the adjustment of tonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vials made of glass orplastic.
    [0092] If administered intravenously, preferred carriers are physiological saline or phosphatebuffered saline (PBS).
    [0093] In a preferred embodiment, the active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation ofsuch formulations will be apparent to those skilled in the art. The materials can also beobtained commercially from Alza Corporation.
    [0094] Liposomal suspensions (including liposomes targeted to infected cells withmonoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled-in the art, forexample, as described in U.S. Patent No. 4,522,811. For example, liposome formulationsmay be prepared by dissolving appropriate lipid(s) such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triiphosphate derivatives is then introduced into thecontainer. The container is then swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming the liposomal suspension.
    [0095] This invention has been described with reference to its preferred embodiments.Variations and modifications of the invention, will be obvious to those skilled in the art fromthe foregoing detailed description of the invention. It is intended that all of these variationsand modifications be included within the scope of this invention.
    权利要求:
    Claims (32)
    [1]
    Use of a synergistically effective amount of a compound of the formula
    [2]
    The method of claim 1, wherein R is OH.
    [3]
    The method of claim 1, wherein R is NH2.
    [4]
    A pharmaceutical composition comprising an effective amount of a compound of theformula
    [5]
    The pharmaceutical composition of claim 4, wherein R is NH2.
    [6]
    The pharmaceutical composition of claim 4, wherein R is OH.
    [7]
    A method for the treatment of hepatitis B virus in a human comprising administering incombination or alternation a synergistically effective amount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(β-FTC) or a pharmaceutically acceptable salt,ester, or prodrug thereof with an effective amount of a second anti-hepatitis B agentselected from the group consisting of penciclovir, famciclovir, and Bis-POM-PMEA.
    [8]
    The method of claim 7, wherein the β-FTC is in the form of the (-)-optical isomer.
    [9]
    The method of claim 8, wherein the second anti-hepatitis B agent is penciclovir.
    [10]
    The method of claim 8, wherein the second anti-hepatitis B agent is famciclovir.
    [11]
    The method of claim 8, wherein the second anti-hepatitis B agent is Bis-POMPMEA.
    [12]
    A method for the treatment of hepatitis B virus in a human comprising administering incombination or alternation a synergistically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester, orprodrug thereof with an effective amount of a second anti-hepatitis B agent selectedfrom the group consisting of a compound of the formula
    [13]
    The method of claim 12, wherein the second anti-hepatitis B agent is a compound ofthe formula
    [14]
    The method of claim 12, wherein the second anti-hepatitis B agent is a compound ofthe formula
    [15]
    The method of claim 12, wherein the second anti-hepatitis B agent is penciclovir.
    [16]
    The method of claim 12, wherein the second anti-hepatitis B agent is PMEA, or apharmaceutically acceptable salt, ester or prodrug thereof.
    [17]
    A method for the treatment of hepatitis B virus in a human comprising administering incombination or alternation a synergistically effective amount of a compound of theformula
    [18]
    The method of claim 17, wherein R is OH.
    [19]
    The method of claim 17, wherein R is NH2.
    [20]
    Use of a synergistically effective amount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(β-FTC) or a pharmaceutically acceptable salt, ester, or prodrugthereof with an effective amount of a second anti-hepatitis B agent selected from thegroup consisting of penciclovir, famciclovir, and Bis-POM-PMEA for the treatment ofhepatitis B virus.
    [21]
    Use of a synergistically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester, or prodrug thereof with an effective amount of a second anti-hepatitis B agent selectedfrom the group consisting of a compound of the formula
    [22]
    Use of a synergistically effective amount of a compound of the formula
    [23]
    A pharmaceutical composition comprising an effective amount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(β-FTC) or a pharmaceutically acceptablesalt, ester, or prodrug thereof in a synergistic combination with an effective amount of a second anti-hepatitis B agent selected from the group consisting of penciclovir,famciclovir, and Bis-POM-PMEA.
    [24]
    The composition of claim 23, wherein the second anti-hepatitis B agent is penciclovir.
    [25]
    The composition of claim 23, wherein the second anti-hepatitis B agent is famciclovir.
    [26]
    The composition of claim 23, wherein the second anti-hepatitis agent is Bis-POM-PMEA.
    [27]
    A pharmaceutical composition comprising an effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester, orprodrug thereof in a synergistic combination with an effective amount of a second anti-hepatitisB agent selected from the group consisting of a compound of the formula
    [28]
    The composition of claim 27, wherein R is NH2.
    [29]
    The composition of claim 27, wherein R is OH.
    [30]
    A pharmaceutical composition comprising an effective amount of a compound of theformula
    [31]
    The composition of claim 30, wherein R is NH2.
    [32]
    The composition of claim 30, wherein R is OH.
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    同族专利:
    公开号 | 公开日
    IL193148A|2011-09-27|
    ES2237189T3|2005-07-16|
    ES2314157T3|2009-03-16|
    EP1124562B1|2005-01-19|
    DE69939604D1|2008-10-30|
    DK1382343T3|2010-04-26|
    IL142910A|2009-06-15|
    DE69923338T2|2006-04-06|
    US20090247487A1|2009-10-01|
    DK1380303T3|2008-12-01|
    CY1108635T1|2014-04-09|
    CN1891221A|2007-01-10|
    AT287268T|2005-02-15|
    DE69923338D1|2005-02-24|
    JP2002528508A|2002-09-03|
    HK1062146A1|2004-10-21|
    AU1810600A|2000-05-22|
    ES2338642T3|2010-05-11|
    DE69942042D1|2010-04-01|
    IL193148D0|2009-02-11|
    US20030158150A1|2003-08-21|
    EP1382343B1|2010-02-17|
    KR20010082282A|2001-08-29|
    PT1382343E|2010-03-10|
    US7572800B2|2009-08-11|
    CN1173705C|2004-11-03|
    PT1380303E|2008-11-03|
    WO2000025797A1|2000-05-11|
    CY1110629T1|2015-04-29|
    EP1380303B1|2008-09-17|
    ID29471A|2001-08-30|
    EP1124562A1|2001-08-22|
    CN1666742A|2005-09-14|
    JP2011079840A|2011-04-21|
    AT408410T|2008-10-15|
    US6528515B1|2003-03-04|
    AT457734T|2010-03-15|
    CN1329497A|2002-01-02|
    EP1380303A1|2004-01-14|
    KR100632520B1|2006-10-09|
    IL142910D0|2002-04-21|
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    US10666498P| true| 1998-11-02|1998-11-02||
    US106664P||1998-11-02||
    EP99961553A|EP1124562B1|1998-11-02|1999-11-02|Combination therapy to treat hepatitis b virus|CY20101100385T| CY1110629T1|1998-11-02|2010-04-30|POLYTHERAPY FOR THERAPEUTIC VACANCY|
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