EP1113803A1 - Combinaisons antivirales contenant de la lamivudine et de l'abacavir - Google Patents

Combinaisons antivirales contenant de la lamivudine et de l'abacavir

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Publication number
EP1113803A1
EP1113803A1 EP99947375A EP99947375A EP1113803A1 EP 1113803 A1 EP1113803 A1 EP 1113803A1 EP 99947375 A EP99947375 A EP 99947375A EP 99947375 A EP99947375 A EP 99947375A EP 1113803 A1 EP1113803 A1 EP 1113803A1
Authority
EP
European Patent Office
Prior art keywords
combination
amino
pharmaceutically acceptable
treatment
abacavir
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99947375A
Other languages
German (de)
English (en)
Inventor
Nathaniel A. Glaxo Wellcome Inc. BROWN
Lynn D. Glaxo Wellcome Inc. CONDREAY
Douglas Fraser Glaxo Wellcome plc GRAY
Marc Glaxo Wellcome Inc. RUBIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
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Glaxo Group Ltd
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Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Publication of EP1113803A1 publication Critical patent/EP1113803A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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

Definitions

  • the present invention relates to therapeutic combinations comprising (2R,cis)-4- amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl)-pyrimidi ⁇ -2-one (lamivudine) and a second therapeutic agent (-)-(1S, 4R) -4-[2-amino-6-(cyclopropylamino)-9H- purin-9-yl]-2-cyclopentene-1-methanol, (1592U89, abacavir).
  • the present invention is also concerned with pharmaceutical compositions containing said combinations and their use in the treatment of HBV infections including infections with HBV mutants bearing resistance to nucleoside and/or non- nucleoside inhibitors of the replication of the hepatitis B virus.
  • Hepatitis B is a viral disease transmitted orally or parentally by contaminated material such as blood or blood products, contaminated needles, sexually, and vertically from infected or carrier mothers to their off-spring. In those areas of the world where the disease is common, vertical transmission at an early age results in a high proportion of infected individuals becoming chronic carriers of hepatitis B. An estimated 350 million people world-wide are chronically infected with hepatitis B and as many as 150 million may die from liver disease in the absence of intervention.
  • Interferon is an immune modulator designed to boost the disease fighting ability of the immune system.
  • Lamivudine has been reported to be effective against HBV in a two year study, showing that most patients showed substantially reduced levels of viral replication with 52% maintaining undetectable levels of virus thorough to the end of the second year.
  • Abacavir is described in EP 0434450 as having potent activity against HBV. It has been shown to effectively block the replication of HBV in vitro, Antimicrobial Agents and Chemotherapy May 1997, P1082-1093.
  • the use of the combinations of the present invention may give rise to equivalent antiviral effect with reduced toxicity, or an increase in drug efficacy because synergy between compounds occurs. Lower overall drug doses will also possibly reduce the frequency of occurrence of drug resistant variants of HBV.
  • lamivudine exhibits unexpected advantages when used in combination with abacavir.
  • the combination shows a statistically significant synergistic anti-HBV effect as shown in Figure 1. It is a feature of this invention that the use of this drug combination will provide synergistic antiviral effects, more complete viral suppression, viral suppression over longer periods, limit the emergence of drug resistant HBV mutants and allow better management of drug related toxicities.
  • the use of this drug combination may also result in a decrease of the number of, for example, tablets administered a day, therefore may increase patient compliance.
  • references herein to treatment extend to prophylaxis as well as to the treatment of established infections and symptoms.
  • pharmaceutically acceptable derivative includes any pharmaceutically acceptable salt, ester or salt of such ester, of lamivudine or abacavir or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) such a compound or an antivirally active metabolite or residue thereof.
  • compositions of lamivudine, and abacavir include those derived from pharmaceutically acceptable bases, inorganic and organic acids.
  • suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene- p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonic acids.
  • Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and NR 4+ (where R is C 1-4 alkyl) salts.
  • Preferred salts of abacavir are the sulphate salts particularly the hemi-sulphate salt.
  • esters of lamivudine and abacavir are independently selected from the following group: (1) carboxylic acid esters in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted by, for example, halogen, C 1-4 _ alkyl, or C 1-4 alkoxy), or amino; (2) sulphonate esters, such as alkyl- or aralkylsulphonyl (for example, methanesulphonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl
  • any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms.
  • Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms.
  • Any aryl moiety present in such esters advantageously comprises a phenyl group. Any reference to any of the above compounds also includes a reference to a physiologically acceptable salt thereof.
  • esters are the mono-, di-, and triphosphate esters of lamivudine and abacavir (which may be optionally blocked), or any other compound which upon administration to a human subject is capable of providing (directly or indirectly) said mono-, di-, or triphosphate ester.
  • abacavir is the mono-, di-, and tri-phosphate esters of (1 R, 4S)-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]guanine (carbovir).
  • the present invention provides a combination comprising (2R,cis)-4-amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl)-pyrimidin- 2-one or a pharmaceutically acceptable derivative thereof and (-)-(1S, 4R) -4-[2- amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1 -methanol or a pharmaceutically acceptable derivative thereof.
  • the present invention further provides combinations according to the invention for use in therapy, particularly in the treatment of an HBV infection including infections resistant to nucleoside and/or non-nucleoside inhibitors of the replication of the hepatitis B virus.
  • the present invention provides a method for the treatment of a mammal, including a human, suffering from an HBV infection comprising administration of a therapeutically effective amount of a combination according to the invention.
  • the compounds of the combination may be administered simultaneously, either in the same or different pharmaceutical composition, or sequentially. If there is sequential administration, the delay in administering the second active ingredient should not be such as to lose the benefit of a synergistic therapeutic effect of the combination of the active ingredients. It will also be understood that lamivudine, and abacavir or the pharmaceutically acceptable derivatives thereof whether presented simultaneously or sequentially, may be administered individually or in any combination thereof. Lamivudine, and abacavir are preferably administered simultaneously or sequentially in separate pharmaceutical formulations, most preferably simultaneously.
  • the combination according to the invention is administered as a single combined formulation.
  • the present invention also provides the use of lamivudine in the manufacture of a medicament for administration simultaneously or sequentially with abacavir for the treatment of HBV infections.
  • the use of abacavir in the manufacture of a medicament for administration simultaneously or sequentially with lamivudine for the treatment of HBV infections is a further aspect of the invention.
  • a further aspect of the invention is a combination according to the invention wherein the lamivudine and abacavir are present in a synergistic ratio.
  • synergistic effects of the combination of lamivudine and abacavir or pharmaceutically acceptable derivatives thereof are seen over a ratio, for example, of 1 :0.5 to 1 :10 (by weight), preferably 1 :1 to 1 :8 (by weight).
  • each compound will be employed in the combination in an amount at which it exhibits anti-HBV activity when used alone.
  • the amount of a combination of lamivudine and abacavir required to be effective as an anti-HBV agent will, of course, vary and is ultimately at the discretion of the medical practitioner.
  • the factors to be considered include the route of administration and nature of the formulation, the animal's body weight, age and general condition and the nature and severity of the disease to be treated.
  • a suitable daily dose will be in the range of from about 0.1 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 0.5 to 20 mg per kilogram body weight per day, most preferably in the range of 0.5 to 2 mg per kilogram body weight per day.
  • lamivudine is administered at a level in the range 25 to 150mg per day.
  • lamivudine is conveniently administered at a level of about 100 mg per day.
  • a suitable daily dose will be in the range of from about 3 to about 120 mg per kilogram body weight of the recipient per day, preferably in the range of 1 to 90 mg per kilogram body weight per day, most preferably in the range of 5 to 60 mg per kilogram body weight per day.
  • abacavir is administered at a level in the range 200 to 800 mg per day.
  • abacavir is administered at a level of about 600 mg per day. Conveniently this can be as 300 mg twice daily.
  • the desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing from 1 to 1500 mg, preferably from 5 to 1000 mg, most preferably from 5 to 500 mg of active ingredient per unit dosage form.
  • a sub-dose of abacavir may be administered independently of a dose of a combination of the invention. Alternatively, if the condition of the recipient so requires, the dose may be administered as a continuous infusion.
  • the components of the combination which may be referred to as active ingredients may be administered for therapy to an animal e.g. a mammal including a human in a conventional manner.
  • compositions according to the present invention comprise a combination according to the invention in association with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formula and not deleterious to the recipient thereof.
  • the individual components of the combination are administered separately they are generally each presented as a pharmaceutical composition.
  • the references hereinafter to compositions refer unless otherwise stated to compositions containing either the combination or a component thereof.
  • a combination of lamivudine and abacavir or pharmaceutically acceptable derivatives thereof may conveniently be presented as a pharmaceutical composition with one or more pharmaceutically acceptable carrier thereof in a unitary dosage form.
  • a convenient unitary dosage formulation contains the active ingredients in amounts of from 1 mg to 2 g each, for example, 2 mg to 200 mg such as 25 to 150 mg of lamivudine and 200 to 800 mg of abacavir.
  • compositions may also be prescribed to the patient in "patient packs" containing the whole course of treatment in a single package, usually a blister pack.
  • Patient packs have an advantage over traditional prescriptions, where a pharmacists divides a patients supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions.
  • the inclusion of a package insert has been shown to improve patient compliance with the physicians instructions.
  • a patient pack comprising at least one active ingredient, of the combination according to the invention and an information insert containing directions on the use of the combination of the invention.
  • compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods represent a further feature of the present invention and include the step of bringing into association the active ingredients with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch giycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Moulded tablets may be made by moulding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • compositions suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Topical administration may also be by means of a transdermal iontophoretic device.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art.
  • the suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in moulds.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents; and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or daily sub-dose of the active ingredients, as herein before recited, or an appropriate fraction thereof.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents.
  • the compounds of the combination of the present invention may be obtained in a conventional manner.
  • Abacavir may be prepared by the method described in European Specification
  • Active ingredient denotes lamivudine or abacavir, multiples thereof or a physiologically functional derivative of any of the aforementioned compounds.
  • Example 1 Tablet Formulation The following formulations A, B and C are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.
  • formulations D and E are prepared by direct compression of the admixed ingredients.
  • the lactose in formulation E is of the direct compression type (Dairy Crest-"Zeparox").
  • Active Ingredient 250 Pregelatinized Starch NF15 150
  • the formulation is prepared by wet granulation of the ingredients with a solution of povidone followed by the addition of magnesium stearate and compression. mg/tablet
  • Drug release takes place over a period of about 6-8 hours and is complete after 12 hours.
  • a capsule formulation is prepared by admixing the ingredients of formulation D in Example 1 above and filling into a two-part hard gelatin capsule.
  • Formulation B (infra) is prepared in a similar manner.
  • Capsules of formulation C are prepared by melting the Macrogel 4000 B.P., dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.
  • Capsules of formulation D are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.
  • Vitamin E TPGS obtained from Eastman Chemical Co.
  • TPGS obtained from Eastman Chemical Co.
  • PEG400 polyethylene glycol 400
  • the resultant solution was heated to 65°C.
  • 1.5 kg of active ingredient was dissolved in the liquefied solution of Vitamin E TPGS and PEG 400.
  • 0.395 kg of propylene glycol at room temperature was added and mixed until a homogenous solution was formed. The solution was cooled to 28-35°C.
  • the solution was then de-gassed.
  • the mixture was preferably encapsulated at 28-35°C at a fill weight equivalent to 150 mg of volatiles-free compound, into Size 12 oblong, white opaque soft gelatin capsules using a capsule filling machine.
  • the capsule shells were dried to a constant fill moisture of 3-6% water and a shell hardness of 7-10 newtons, and placed in a suitable container.
  • the following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronization of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.
  • the active ingredient is dissolved in most of the water (35° - 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate.
  • the batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
  • the active ingredient is dissolved in the glycofurol.
  • the benzyl alcohol is then added and dissolved, and water added to 3 ml.
  • the mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml amber glass vials (type l).
  • the active ingredient is dissolved in a mixture of the glycerol and most of the purified water.
  • An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour.
  • the volume is made up with purified water and mixed well.
  • Example 6 Suppository mg/capsule suppository Active ingredient 250
  • Witepsol H15 is melted in a steam-jacketed pan at 45 C maximum.
  • the active ingredient is sifted through a 200 ⁇ m sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45° C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix.
  • the entire suspension is passed through a 250 ⁇ m stainless steel screen and, with continuous stirring, is allowed to cool to 45° C. At a temperature of 38° C to 40° C, 2.02 g of the mixture is filled into suitable, 2 ml plastic moulds. The suppositories are allowed to cool to room temperature.
  • Example 7 Pessaries mg/pessary Active Ingredient 250
  • the human hepatoblastoma cell line (Hep-G2-2.2.15) which constitutively produces infectious HBV was seeded into 96 well microtiter plates at a density of 5 x 10 3 cells per well. These cells were treated with a combination of lamivudine and abacavir on triplicate plates. Culture media containing drugs was replenished every other day for 9 days, at which time supematants were collected and analyzed for HBV content.
  • the lamivudine/abacavir combination was tested twice in triplicate in matrix fashion.
  • Experiment 1 utilised a lamivudine range of 0.008 nM to 125 nM (5-fold dilutions in columns), and abacavir, at concentrations of 40 nM to 25 ⁇ M (5 fold dilutions in rows).
  • Experiment 2 was performed with dilutions of lamivudine ranging from 0.14 nM to 100 nM (in 3-fold dilutions in columns), and a abacavir range of 250.7 nM to 25 ⁇ M (3.16 fold dilutions in rows).
  • Experiment 3 was performed using dilutions of lamivudine ranging from 0.045 nM to 100 nM (in 3- fold dilutions and a abacavir range of 0.8 nM to 25 ⁇ M (3.16 fold dilutions in rows). Both drugs were diluted in a separate 96 well microtiter plate, and subsequently transferred onto plates containing the cell monolayers. Cells are grown in 150 ⁇ l RPMI 1640 supplemented with 2 mM L-Glutamine and 10% fetal bovine serum. Prior to transfer of drug, 120 ⁇ l of media was removed from the cells, leaving 30 ⁇ l on the monolayers to prevent drying.
  • HBV detection was performed by "capturing" virus from supernatants on Anti-HBsAg coated plates, washing, denaturing to release HBV DNA, performing PCR with biotinylated primers, streptavidin capture of biotinylated PCR products with concomitant probe hybridization, addition of substrate, and reading optical densities of the colorimetric reaction. Dilutions of a standardized HBV-containing supernatant were included on every plate, and HBV DNA concentrations of test wells were calculated from this HBV standard curve. The useful range of detection is at least .045 to 45 fg of HBV DNA, where 30 copies of the genome can be reliably detected. Samples were tested in conjunction with both positive (.448 fg/ul plasmid DNA) and negative (RPMI medium supplemented with 2 mM L-Glutamine and 10% Fetal calf serum) controls.
  • the average IC50 and standard error of the IC50s for the triplicate plates were calculated using SAS nonlinear regression to fit data to the Hill equation for each concentration response curve. When only a single determination of an IC50 for a particular dose combination could be made, the average of the standard errors from adjacent concentrations was used to estimate the standard error. Fractional inhibitory concentrations (FIC50) were calculated for each combination and plotted using the isobologram representation (Berenbaum, M.C. (1985) The Expected Effect of a Combination of Agents: the General Solution. J. Theor. Biol. 114, 413-431).
  • Figure 1 shows a single isobologram, produced by combining the data from the three experiments, showing statistically significant synergism.
  • the Kj for the active form of abacavir, (-) carbovir triphosphate, (-)CBV-tp, was determined against wild type and YMDD variant HBV polymerases in an in vitro assay consisting of HBV core particles isolated from transiently transfected or transduced HepG2 cells.
  • HepG2 cells were transiently transfected with plasmid containing the HBV genome that had the wild type sequence or contained the following amino acid changes in the reverse transcriptase gene; M552I, M552V, L528M, L528MM552V, L528MM552I (Allen Ml, Deslauhers M, Andrews CW, Tipples GA, Walters KA, Tyrrell DLJ, Brown N for the Lamivudine Clinical Investigation Group, and Condreay LD. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine). HEPATOLOGY 1998; 27:1670-1677).
  • Transient transductions were performed using baculovirus containing the same constructs (Condreay JP, Witherspoon SM, Clay WC, Kost TA. (1999) Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. PNAS, 96, 127-132).
  • the core polymerase assay was a modification of a previously reported protocol (Daw ' s MG, Wilson, JE, VanDraanen NA, Miller WH, Freeman GA, Daluge SM, Boyd FL, Aulabaugh, AE, Painter, GR, Boone LR.
  • HBV core particles were isolated in the following manner. Briefly, 5 days post-transfection or transduction, the cells were harvested and pelleted. The cell pellet was resuspended in a hypotonic solution and the cells lysed with NP-40 at a final concentration of 0.5%. The nuclei were pelleted and the cell supernatant was digested with RNase and DNase. The treated lysate was pelleted through a sucrose step gradient.
  • the resulting pellet which contained HBV cores, was resuspended by sonication in a buffer containing 50 mM Tris-HCI, pH 7.4, 50 mM KCI, and 5 mM MgCI 2 . This material was stored at -80° C until used in polymerase assays.
  • Antibody-capture assay plates were prepared using the following protocol: Costar #3632(Corning, Corning, NY), were sequentially coated with 1)
  • Neutravidin (Pierce, Rockford, IL) in 50 mM sodium carbonate buffer (Sigma, St. Louis, MO), pH 9.6, 0.10 mg/ml, 0.15 ml per well for 2 hours at room temperature, then washed with 3 x 0.25 ml/well with wash buffer ,WB,( 150 mM NaCI, 100 mM Sodium Phosphate, pH 7.2, 0.02%Tween-20 (Pierce, Rockford, IL), 0.02% Sodium Azide, 0.01% Bovine Serum Albumin (Sigma, St.
  • the core polymerase assay was a modification of a previously reported protocol (Davis MG, Wilson, JE, VanDraanen NA, Miller WH, Freeman GA, Daluge SM, Boyd FL, Aulabaugh, AE, Painter, GR, Boone LR. (1996) DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs. Antiviral Res 30, 133-145). Core particle preparations were thawed on ice and resuspended by sonication prior to each assay.
  • Core particle assays contained the following: Core particles (usually 10 7 to 10 8 / 10 ul reaction) in 230 mM KCI, 33 mM Tris-HCI, pH 7.4, 8 mM MgCI 2) 0.2 % NP-40, 5 mM DTT, 7 units/ml pyruvate kinase buffer (Sigma, St. Louis, MO), 2 mM phosphoenol pyruvate buffer (Sigma, St. Louis, MO), 25 mM unlabeled dNTP's (Amersham Pharmacia Biotech, Piscataway, NJ), 33 P labelled dNTP (NENTM Life Science Products, Boston, MA, 2000 Ci/mmole) at 0 to 4 ⁇ M.
  • Assays of 10 ul were incubated at 37° C for 45 to 90 minutes in a 96 well polypropylene plate (USA Scientific, Ocala, FL), then stopped with 90 ul of the following buffer: 50 mM Tris-HCI, pH 7.4, 50 mM KCI, 5 mM MgCI 2 , 0.05% Tween-20, 2 mM sodium pyrophosphate, 0.02% sodium azide. The entire 100 ul-stopped assay was transferred to a 96-well antibody capture plate and incubated at room temperature with shaking for two hours.
  • Inhibition assays were essentially the same as a core polymerase assays except that the concentration of ⁇ 33 P-dGTP was at K m for each polymerase assayed.
  • the concentration of (-)CBV-tp ranged from 0.001 ⁇ M to 312 mM.
  • K j 's were calculated using the following formula: K .
  • the IC 50 is the inhibitory concentration of compound at which polymerase activity is 50%.
  • [S] is the concentration of competing substrate (for (-)CBV-tp it is 33 P labelled dGTP). All IC 50 's were determined at the K m for dCTP for each polymerase.

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  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne des combinaisons thérapeutiques comprenant une (2R,cis)-4-amino-1-(2-hydroxyméthyl-1,3-oxathiolan-5-yl)-pyrimidin-2-one (lamivudine) et un (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentène-1-méthanol (1592U89, abacavir). L'invention concerne également des compositions pharmaceutiques contenant ces combinaisons, ainsi que l'utilisation de ces compositions dans le traitement d'infections à virus de l'hépatite B (HBV), notamment d'infections provoquées par des mutants de ce virus, lesquels sont résistants aux inhibiteurs nucléosidiques et/ou non nucléosidiques de la réplication du virus de l'hépatite B.
EP99947375A 1998-09-18 1999-09-17 Combinaisons antivirales contenant de la lamivudine et de l'abacavir Withdrawn EP1113803A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9820417.5A GB9820417D0 (en) 1998-09-18 1998-09-18 Antiviral combinations
GB9820417 1998-09-18
PCT/EP1999/006885 WO2000016779A1 (fr) 1998-09-18 1999-09-17 Combinaisons antivirales contenant de la lamivudine et de l'abacavir

Publications (1)

Publication Number Publication Date
EP1113803A1 true EP1113803A1 (fr) 2001-07-11

Family

ID=10839131

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99947375A Withdrawn EP1113803A1 (fr) 1998-09-18 1999-09-17 Combinaisons antivirales contenant de la lamivudine et de l'abacavir

Country Status (7)

Country Link
EP (1) EP1113803A1 (fr)
JP (1) JP2002526449A (fr)
KR (1) KR20010075202A (fr)
CN (1) CN1317971A (fr)
AU (1) AU6085099A (fr)
GB (1) GB9820417D0 (fr)
WO (1) WO2000016779A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI1267880T2 (sl) 2000-02-29 2010-04-30 Bristol Myers Squibb Co Formulacija z nizko dozo entekavirja in uporaba
TW200403061A (en) * 2002-06-04 2004-03-01 Glaxo Group Ltd Pharmaceutical compositions
EP1536800B1 (fr) * 2002-06-27 2006-01-04 Medivir Ab Interaction synergique d'abacavir et d'alovudine
WO2012062835A1 (fr) * 2010-11-12 2012-05-18 Glaxo Wellcome Manufacturing Pte Ltd Nouvelles compositions pharmaceutiques

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MY104575A (en) * 1989-12-22 1994-04-30 The Wellcome Foundation Ltd Therapeutic nucleosides.
IL113432A (en) * 1994-04-23 2000-11-21 Glaxo Group Ltd Process for the diastereoselective synthesis of nucleoside analogues
MY115461A (en) * 1995-03-30 2003-06-30 Wellcome Found Synergistic combinations of zidovudine, 1592u89 and 3tc
CO4970782A1 (es) * 1997-11-13 2000-11-07 Merck & Co Inc Terapia combinada para el tratamiento del sida
GB9809213D0 (en) * 1998-04-29 1998-07-01 Glaxo Group Ltd Pharmaceutical compositions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0016779A1 *

Also Published As

Publication number Publication date
KR20010075202A (ko) 2001-08-09
GB9820417D0 (en) 1998-11-11
JP2002526449A (ja) 2002-08-20
AU6085099A (en) 2000-04-10
CN1317971A (zh) 2001-10-17
WO2000016779A1 (fr) 2000-03-30

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