ANTIVIRAL COMBINATIONS OF BCH-189 AND RITONAVIR

Abstract: Combinations comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof and ritonavir or a pharmaceutically acceptable derivative thereof, pharmaceutical formulations containing them and their use in the treatment of viral infection.

Description: ANTIVIRAL COMBINATIONS OF BCH-189 AND RITOVANIR The present invention relates to combinations of antiviral agents. More specifically it is concerned with combinations of 1 ,3-oxathiolane nucleoside analogues with protease inhibitors effective against HIV. Human immunodeficiency virus (HIV) causes a variety of clinical conditions including the acquired immune deficiency syndrome (AIDS) and chronic neurological disorders. The compound of formula (I) also known as BCH-189 has been described as having antiviral activity in particular against the human immunodeficiency viruses (HIVs), the causative agents of AIDS (5th Anti-Aids Conference, Montreal, Canada 5th-9th June 1989: Abstracts T.C.O.1 and M.C.P. 63; European Patent Application Publication No. 0382562). The compound of formula (I) is a racemic mixture of the two enantiomers of formulae (1-1 ) and (l-2) Although the enantiomers of the compound of formula (I) are equipotent against HIV one of the enantiomers (the(-)-enantiomer) has considerably lower cytotoxicity than the (+) enantiomer. The (-) enantiomer has the chemical name (-)-cis- -amino-1-(2-hydroxymethyl- 1 ,3-oxathiolan-5-yl)-(1 H)- pyrimidin-2-one. It has the absolute stereochemistry of the compound of formula (1-1 ) which has the name (2R,cis)- -amino-1-(2- hydroxymethyl-1 ,3-oxathiolan-5-yl)-(1 H)-pyrimidin-2-one. This compound is now known as lamivudine and 3TC™. The compound of formula (II): has the chemical name (2S, 3S, 5S)-5-(N-(N-methyl-N-((2-isopropyl-4- thiazolyl)methyl)amino)carbonyl)valinyl)amino)-2-(N- ((thiazolyl)methoxycarbonyl)amino)1 ,6-diphenyl-3-hydroxyhexane and is also known as A-84538, ABT-538 or ritonavir. The compound of formula (II) has been described as having HIV-protease inhibitor activity (International Patent Application Publication No. 94/14436). We have now found that the compound of formula (I) and, in particular its (-)- enantiomer exhibits unexpected advantages when used in combination with the compound of formula (II). In particular the compound of formula (I) shows a synergistic antiviral effect and/or a reduction in cytotoxicity when used in combination with the compound of formula (II). There is thus provided in a first aspect of the invention a combination comprising the compound of formula (I) or a pharmaceutically acceptable derivative thereof and the compound of formula (II) or a pharmaceutically acceptable derivative thereof. Preferably the compound of formula (I) is in the form of its (-)-enantiomer, lamivudine. When the Compound formula (I) is in the form of the (-)-enantiomer it will normally be provided substantially free of the corresponding (+)-enantiomer, that is to say no more than about 5% w/w of the (+)- enantiomer, preferably no more than about 2%, in particular less than about 1% w/w will be present. By "a pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, ester, or salt of such ester, of a parent compound or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) the parent compound or an antivirally active metabolite or residue thereof. It will be appreciated by those skilled in the art that the compound of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof, at functional groups in both the base moiety and at the hydroxymethyl group of the oxathiolane ring. Modification at all such functional groups are included within the scope of the invention. However of particular interest are pharmaceutically acceptable derivatives obtained by modification of the 2-hydroxymethyl group of the oxathiolane ring. Preferred esters of the compound of formula (I) include the compounds in which the hydrogen of the 2-hydroxymethyl group is replaced by an acyl function R-C- in which the non-carbonyl moiety R of the ester is selected from hydrogen, straight or branched chain alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, C< | _ alkyl or C-|_4 alkoxy); sulphonate esters such as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl); amino acid esters (e.g. L-valyl or L-isoleucyl) and mono-, di- or tri-phosphate esters. With regard to the above described esters, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 16 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group. In particular the esters may be a Cι_ιρalkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted by at least one halogen (bromine, chlorine, fluorine or iodine), Chalk !, nitro or trifluoromethyl groups. Pharmaceutically acceptable salts of the compound of formula (I) include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of 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. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and NR4+ (where R is C-j_4alkyl) salts. The advantageous effects of the compounds of formula (I) and formula (II) are realised over a wide ratio for example 1 :250 to 10:1 preferably 1 :50 to 1 :1 , particularly about 1 :20 to 1 :1. Conveniently each compound will be employed in the combination in an amount at which it exhibits antiviral activity when used alone. The combinations of the invention may contain other components in addition to the compounds of formulae (I) and (II). In particular, the combinations according to the invention may contain other antiviral agents, in particular other agents active against HIV, such as other inhibitors of HIV replication. The inhibitor may comprise any inhibitor of HIV replication no matter its method of inhibiting HIV replication. Such inhibitors include for example those which inhibit HIV reverse transcriptase, HIV protease, HIV integrase, TAT and the like. Such inhibitors include for example S'-azido-S'-deoxythymidine ( AZT, zidovudine), 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddl), N'-[1 (S)- benzyl-3-[4a(S),8a(S)-3(S)-(tert-butylcarbamoyl)decahydroisoquinoline-2-yl]- 2(R)-hydroxypropyl]-N"-(quinolin-2-ylcarbonyl)-L-asparaginamide (Ro 31 -8959) stavudine (D4T), nevirapine (BI-RG-587), loviride (α-APA), delavuridine (BHAP), (1 S, 4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2- cyclopentene-1-methanol (1592U), L-668229, MK-639, 3S-tetrahydro-3-furyl N- ((1 S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl-2-hydroxypropyl) carbamate (VX-478) and (+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2- butenyl)-imidazo(4,5,1-jk)(1 ,4)-benzodiazepin-2(1 H)thione(R-82150) or a pharmaceutically acceptable derivative thereof. Preferably the further inhibitor of HIV replication is zidovudine: it has been found that there are particular advantages associated with a triple combination of lamivudine, ritonavir and zidovudine. In a preferred embodiment the present invention accordingly provides a combination comprising lamivudine, ritonavir and zidovudine. In a particularly preferred embodiment, the present invention provides a combination of lamivudine, ritonavir and zidovudine in a ratio of about 1 :4:2. It is expected that the present combinations will be generally useful against viral infections or virus-associated tumours in humans, and the method of their use to inhibit viral infectivity or tumour growth in vitro or in vivo is also within the scope of the present invention. Thus there is provided in a further aspect a method for the treatment of a viral infection in a mammal, including man, comprising co-administration of an antiviral compound of formula (I) or a pharmaceutically acceptable derivative thereof and an inhibitor of HIV protease of formula (II) or a pharmaceutically acceptable derivative thereof and optionally zidovudine or a pharmaceutically acceptable derivative thereof. It will be appreciated that the compounds of formulae (I) and (II) may be administered either simultaneously, sequentially or in combination. If administration is sequential, the delay in administering the second of the active ingredients should not be such as to lose the benefit of the synergistic effect of the combination. Preferably administration will be simultaneous. For combinations comprising the compound of formula (I), the compound of formula (II) and zidovudine, it will be appreciated that each agent may be administered simultaneously, sequentially or in alternative paired combinations. Preferably all three agents will be administered simultaneously. In a preferred embodiment, lamivudine and zidovudine may be administered as a fixed combination, for example as a tablet. It will be appreciated by those skilled in the art that reference herein to treatment extends to prophylaxis as well as the treatment of established infections or symptoms. It will be further appreciated that the amount of a combination of the invention required for use in treatment will vary, with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. In general however a suitable dose will be in the range of from about 1 to about 100mg/kg e.g. from about 1 to about 50-mg/kg of bodyweight per day, preferably in the range of 2 to 30mg/kg/day of each of the active ingredients of the combination. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. The combination is conveniently administered in unit dosage form; for example containing 10 to 1000mg, conveniently 20 to 500mg, most conveniently 100 to 300mg of each active ingredient per unit dosage form. A preferred dosage regimen comprises administration to a human of a unit dose of lamivudine (150mg) twice per day, a unit dose of zidovudine (300mg) twice per day and two doses of ritonavir (100mg) six times per day. While it is possible that, for use in therapy, the active ingredients of the combination may be administered as the raw chemical it is preferable to present combinations as a pharmaceutical formulation. The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof and inhibitor of HIV replication together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be ' acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution, a suspension or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives. The compounds according to the invention may also be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre- filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Pharmaceutical formulations 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, and the suppositories may be conveniently formed by admixture of the active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate. For intra-nasal administration the compounds of the invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one more dispersing agents, solubilising agents or suspending agents. Liquid sprays are conveniently delivered from pressurised packs. For administration by inhalation the compounds according to the invention are conveniently delivered from an insufflator, nebuliser or a pressurised pack or other convenient means of delivering an aerosol spray. Pressurised packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurised aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or e.g. gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator. When desired the above described formulations adapted to give sustained release of the active ingredient may be employed. The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives. The compound of formula (I) may be obtained as described in European Patent Application Publication No. 0382562. Methods for the preparation of lamivudine are described in Intemational Patent Application Publication No. WO91/17159. Methods for the preparation of ritonavir are described in Intemational Patent Application Publication No. WO94/14436. Zidovudine can be prepared, for example, as described in US Patent 4,724,232. Zidovudine can also be obtained from Aldrich Chemical Co., Milwaukee, WI53233, USA. The following examples illustrate the invention but are not intended as a limitation thereof. Antiviral Activities Alone or in Combination Example 1 Compounds are first serially-diluted in 2-fold decrements in 96-well microtitre plates. Chequerboard titrations are prepared by mixing 25μl aliquots from each compound dilution both alone or in combination (to a final volume of 50μl in new 96-well microtitre plates). Aliquots of MT-4 cells (10 6 cells/μl) in RPMI 1640 growth medium are infected with HIV-1 strain RF at a moi of 2 x 10 '3 infectious doses/cell. Virus is adsorbed at room temperature for 90 minutes, after which the cells are washed in RPMI 1640 growth medium to remove unadsorbed virus and resuspended at 10 6 cells/μl in RPM1 1640 growth medium. 50μl of infected cell suspension are inoculated into wells containing compound or growth medium only. 50μl of mock-infected cell suspension are inoculated into wells not containing compound. The plates are then incubated for 7 days at 37°C in 5% CO 2 /air. After incubation, 10μl of 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) at 7.5mg/μl are added to all wells and the plates incubated for a further 90 minutes at 37° C. 150μl of 10% (v/v) Triton X-100 in isopropanol are then added and the cells resuspended. After 15 minutes at room temperature the plates are analysed in a Multiskan MC (Flow Laboratories, Irvine, UK) reader at 405nm. Conversion of yellow MTT to its formazan derivative is maximum in the uninfected untreated cells, and absent in untreated infected cells. Dose response curves are plotted for each compound alone (IC50% values) and for reciprocal titrations of each compound at a fixed concentration of the second compound and isobolograms plotted. Example 2 The human T-cell lymphotropic virus type 1 -transformed cell line MT4 is grown and infected with HIV-1 strain 3B or strain MN (Advanced Biotechnologies Inc., Columbia, Maryland) at 10 times the amount necessary to cause a 50% reduction of MT4 cell growth (10 x TCID50, 2 x 10 4 plaque forming units/cell). Mock-infected cells are also prepared. Following I hour incubation, the cells are pipetted onto 96-well dishes at 1 x 10 4 cells/well. The wells contain various concentrations of zidovudine, and peak or trough plasma levels of lamivudine and ritonavir. The infected T-lymphoblastoid cells are incubated for 5 days to allow for HIV-1 mediated growth inhibition. Plates are then treated with 28 μl of 5% Nonident P-40 (Sigma) in phosphate-buffered saline (PBS) and 60 μl samples are transferred to filter-bottomed, 96-well plates (Idexx Corp.). Plates are placed in an automated assay instrument (Idexx Screen Machine) which adds propidium iodide to each well, performs a series of washes, and determines the resulting fluoroscence (E). Fluorescence has been shown to correlate directly with cell number, allowing for the quantitation of HIV-1 mediated cytopathic effect (CPE). Uninfected cells are determined to have 0% CPE and infected untreated cells are determined to have 100% CPE. Percent inhibition of HIV-1 induced CPE and IC95S (95% inhibitory concentration) are determined. The antiviral activities of combinations comprising lamivudine and ritonavir and lamivudine, ritonavir and zidovudine are tested and compared with the active agents used alone. Both combinations are found to exhibit synergy. Cvtotoxicities of Compounds Alone or in Combination Example 3 Cvtotoxicities of lamivudine and ritonavir and lamivudine, ritonavir and zidovudine alone and in combination are compared in uninfected peripheral blood lymphocytes and an established T-lymphocyte cell line: cytotoxicity is measured using a [ 3 H]-thymidine uptake assay.

Claims: CLAIMS 1. A combination comprising a compound of formula (I): or a pharmaceutically acceptable derivative thereof and ritonavir or a pharmaceutically acceptable derivative thereof. 2. A combination as claimed in claim 1 wherein the ratio of the compound of formula (I): ritonavir is from 1 :250 to 10:1. 3. A combination as claimed in claim 1 or claim 2 wherein the compound of formula (I) is lamivudine or a pharmaceutically acceptable derivative thereof. 4. A combination as claimed in any one of claims 1 to 3 which further comprises zidovudine or a pharmaceutically acceptable derivative thereof. 5. A combination as claimed in claim 4 wherein the ratio of lamivudine:htonavir: zidovudine is 1 :4:2 by weight. 6. A pharmaceutical formulation comprising a combination as claimed in any one of claims 1 to 5 in association with one or more pharmaceutically acceptable carriers therefor. 7. A method for the treatment of a viral infection in a mammal, including man, comprising coadministration of a therapeutically effective amount of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable derivative thereof and a therapeutically effective amount of ritonavir or a pharmaceutically acceptable derivative thereof. 8. A method as claimed in claim 7 wherein the viral infection is HIV infection. 9. A method as claimed in claim 7 or claim 8 wherein the compound of formula (I) is lamivudine. 10. A method as claimed in claim 9 wherein lamivudine and ritonavir are administered simultaneously or sequentially as a synergistic combination. 11. A method as claimed in any one of claims 7 to 10 further comprising administration of a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof. 12. A method as claimed in claim 11 which comprises administration to a human of 150mg lamivudine twice per day, 300mg zidovudine twice per day and 600mg ritonavir twice per day.

Inventors: MCDADE HUGH BROWNLIE, MCDADE, HUGH, BROWNLIE
Applicants: GLAXO GROUP LTD, MCDADE HUGH BROWNLIE, GLAXO GROUP LIMITED, MCDADE, HUGH, BROWNLIE
Application year: 1996
Main classification: A61K38
IPC codes: A61K31, A61K38, A61P31, A61P37