WO2000016754A2

WO2000016754A2 - Antiviral combinations comprising lamivudine and lobucavir

Info

Publication number: WO2000016754A2
Application number: PCT/EP1999/006884
Authority: WO
Inventors: Nathaniel A. Brown; Lynn D. Condreay; Douglas Fraser Gray; Marc Rubin
Original assignee: Glaxo Group Limited
Priority date: 1998-09-18
Filing date: 1999-09-17
Publication date: 2000-03-30
Also published as: GB9820416D0; WO2000016754A3; AU6191999A

Abstract

The present invention relates to therapeutic combinations comprising (2R,cis)-4- amino-1- (2-hydroxymethyl -1,3-oxathiolan -5-yl) -pyrimidin-2 -one (lamivudine) and a second therapeutic agent [1R- (1α,2β,3α)] -2-amino -9-[2,3-bis (hydroxymethyl) cyclobutyl] -1,9 -dihydro -6H- purin- 6-one, (lobucavir). 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.

Description

Antiviral Combinations

The present invention relates to therapeutic combinations comprising (2R,cis)-4- amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl)-pyrimidin-2-one (lamivudine) and a second therapeutic agent [1R-(1α,2β,3α)]-2-amino-9-[2,3- bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H-purin-6-one, (lobucavir). 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.

Currently, the only established approach to treatment of hepatitis B is repeated injections of interferon, which may be associated with unpleasant side effects, and produces a long lasting response in only one third or less of those treated. 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.

Lobucavir has been shown to be active against HBV in vitro and is in phase II clinical trials for the treatment of patients with chronic hepatitis B. The use of combinations of the invention may give rise to 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.

We have now found that lamivudine exhibits unexpected advantages when used in combination with lobucavir. In particular the combination shows a statistically significant synergistic anti-HBV effect. It is a feature of this invention that the use of this drug combinations will provide synergistic antiviral effects, more complete viral suppression, viral suppression over longer periods, limit the emergence of drug resistant HBV mutants. The use of combinations of the invention may also result in a decrease of the number of, for example, tablets administered a day, therefore may increase patient compliance.

As will be appreciated by those skilled in the art references herein to treatment extend to prophylaxis as well as to the treatment of established infections and symptoms.

Pharmaceutically acceptable salts of lamivudine, and lobucavir include those derived from pharmaceutically acceptable inorganic and organic acids. 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 acid, while not in themselves pharmaceutically acceptable may be useful in the preparation of salts useful as intermediates in obtaining 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^ alkyl) salts. Preferred esters of lamivudine and lobucavir 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 alkyl, or C₁ 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); and (4) phosphonate esters. In such esters, unless otherwise specified, 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.

Particularly preferred esters are the mono-, di-, and triphosphate esters of lamivudine (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.

Thus according to one aspect, 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 [1 R-(1α,2β,3α)]- 2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H-purin-6-one or a pharmaceutically acceptable derivative thereof.

Combinations as described above may herein after be referred to as combinations according to the invention.

As used herein "pharmaceutically acceptable derivative" includes any pharmaceutically acceptable salt, ester or salt of such ester, of lamivudine, lobucavir 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.

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.

According to another aspect, 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.

It will be appreciated that 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 lobucavir or the pharmaceutically acceptable derivatives thereof whether presented simultaneously or sequentially, may be administered individually or in any combination thereof. Lamivudine, and lobucavir are preferably administered simultaneously or sequentially in separate pharmaceutical formulations, most preferably simultaneously.

Preferably 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 lobucavir for the treatment of HBV infections. The use of lobucavir 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 lobucavir are present in a synergistic ratio.

The synergistic effects of the combination of lamivudine and lobucavir or pharmaceutically acceptable derivatives thereof are seen over a ratio, for example, of 1 :1 to 1 :20 (by weight), preferably 1 :2 to 1 :10 (by weight).

Conveniently 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 lobucavir 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.

In general for lamivudine 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.

The compound is conveniently administered at a level of about 100 mg per day.

For lobucavir a suitable daily dose will be in the range of from about 0.01 to about 15 mg per kilogram body weight of the recipient per day, preferably in the range of 2 to 12 mg per kilogram body weight per day.

Conveniently lobucavir is administered at a level of about 400 mg or 800 mg per day. Conveniently this can be as 200 mg twice daily or 200 mg four times daily.

Unless otherwise indicated all weights of active ingredients are calculated in terms of the drug ger se. In the case of a pharmaceutically acceptable derivatives of lamivudine and lobucavir, or a solvate of any thereof the figures would be increased proportionately. 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 lobucavir 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.

While it is possible for the active ingredients of the combination to be administered as the raw chemical it is preferable to present them as a pharmaceutical composition. Pharmaceutical 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. When 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 lobucavir 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 100 to 1000 mg of lobucavir.

Pharmaceutical 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.

It will be understood that the administration of the combination of the invention by means of a single patient pack, or patients packs of each composition, within a package insert diverting the patient to the correct use of the invention is a desirable additional feature of this invention.

According to a further aspect of the invention there is provided a patient pack comprising of 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.

According to another aspect the invention provides a double pack comprising in association for separate administration lamivudine and lobucavir or pharmaceutically acceptable derivatives thereof.

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. In general, 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 glycollate, 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.

Preferably the combinations according to the invention are administered orally.

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.

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. 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, bacte ostats 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.

It should be understood that in addition to the ingredients particularly mentioned above, the 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.

Methods for the preparation of lamivudine are described in International Patent Application Numbers WO91/17159, and WO 95/29174 incorporated herein by reference.

Lobucavir may be prepared by methods disclosed in EP 0358 154, and EP 0736 533 incorporated herein by reference.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way. "Active ingredient" denotes lamivudine or lobucavir, 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.

Formulation A mg/tablet

Active Ingredient 250

Lactose B. P. 210

Povidone B. P. 15

Sodium Starch Glycollate 20

Magnesium Stearate 5

500 Formulation B mg/tablet

Active Ingredient 250 Lactose B.P. 150 Avicel PH 101 60 Povidone B.P. 15 Sodium Starch Glycollate 20 Magnesium Stearate 5

500

Formulation C mg/tablet

Active Ingredient 250 Lactose B.P. 200 Starch 50 Povidone 5 Magnesium Stearate 4

359

The following 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").

Formulation D mg/tablet Active Ingredient 250

Pregelatinized Starch NF15 150

400

Formulation E mg/tablet

Active Ingredient 250 Lactose B.P. 150

Avicel 100

500

Formulation F (Controlled Release Formulation)

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

Active Ingredient 500

Hydroxypropylmethylcellulose 112

(Methocel K4M Premium)

Lactose B.P. 53

Povidone B.P. 28

Magnesium Stearate 7

700 Drug release takes place over a period of about 6-8 hours and is complete after 12 hours.

Example 2: Capsule Formulations

Formulation A

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.

Formulation B mg/capsule

Active Ingredient 250

Lactose B.P. 143

Sodium Starch Glycollate 25

Magnesium Stearate 2

420

Formulation C mg/capsule

Active Ingredient 250

Macrogel 4000 B.P. 350

600 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.

Formulation D mg/capsule

Active Ingredient 250

Lecithin 100 Arachis Oil 100

450

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.

Formulation E mg/capsule

Active Ingredient 150.0

Vitamin E TPGS 400.0

Polyethylene Glycol 400 NF 200.5

Propylene Glycol USP 39.J

Four (4) kilograms (kg) of Vitamin E TPGS (obtained from Eastman Chemical Co.) was heated at 50°C until liquefied. To the liquified Vitamin E TPGS, 2.005 kg of polyethylene glycol 400 (PEG400) (low aldehyde, <10 ppm, obtained from Union Carbide or Dow Chemical Co.) heated to 50°C was added and mixed until a homogeneous solution was formed. 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.

Formulation F (Controlled Release Capsule)

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. mg/capsule

(a) Active Ingredient 250

(b) Microcrystalline Cellulose 125

(c) Lactose B.P. 125

(d) Ethyl Cellulose 13

513 Example 3: Injectable Formulation

Formulation A mg Active Ingredient 200 Hydrochloric Acid Solution 0.1M or

Sodium Hydroxide Solution 0.1M q.s. to pH 4.0 to 7.0

Sterile water q.s. to 10 ml

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.

Formulation B

Active Ingredient 125 mg

Sterile, Pyrogen-free, pH 7 Phosphate Buffer, q.s. to 25 ml

Example 4: Intramuscular Injection

Active Ingredient 200 mg Benzyl Alcohol 0.10 g

Glycofurol 75 1.45 g

Water for injection q.s. to 3.00 ml

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 1).

Example 5: Syrup Active Ingredient 250 mg

Sorbitol Solution 1.50 g

Glycerol 2.00 g Sodium Benzoate 0.005 g

Flavor, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.00 ml

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 sorbital solution and finally the flavor. The volume is made up with purified water and mixed well.

Example 6: Suppository mg/capsule suppository

Active Ingredient 250

Hard Fat, B.P. (Witepsol H15-Dynamit Nobel) 1770

2020

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C maximum. The active ingredient is sifted through a 200μm 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 250μm 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

Anhydrate Dextrose 380

Potato Starch 363

Magnesium Stearate 7

1000

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

Biological Data

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 10³ cells per well. These cells were treated with a combination of lamivudine and lobucavir on triplicate plates. Culture media containing drugs was replenished every other day for 9 days, at which time supernatants were collected and analyzed for HBV content.

The lamivudine/lobucavir combination was tested twice in triplicate in matrix fashion. In both experiments lamivudine was tested at concentrations of range of 0.14 nM to 100 nM (3-fold dilutions in columns), and lobucavir, at concentrations of 10 nM to 1.0 μM (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 μl RPMI 1640 supplemented with 2 mM L-Glutamine and 10% fetal bovine serum. Prior to transfer of drug, 120 μl of media was removed from the cells, leaving 30 μl on the monolayers to prevent drying. 90 μl of fresh media without drug was added, followed by the addition of 30 μl of 5X drug dilutions. Lamivudine and lobucavir were each tested on their respective plates individually at the same concentrations. Data were normalised to values obtained with non-drug treated cells, and expressed as a percent of control for analysis. The method used for detection of HBV has been previously described (Jansen RW, Johnson LC, Averett, DR. High-Capacity in vitro assessment of anti- hepatitis B virus compound selectivity by a vi on-specific polymerase chain reaction assay. Antimicrob Agents Chem 1993; 37 (3): 441-447.). Briefly, 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). To assess statistical significance of synergy or antagonism, an unpaired t-test was used to compare each sum of paired FIC50 values with the theoretical value of 1. P values less than 0.05 were considered statistically significant. Comparison of P values between experiments must be interpreted with great care, as the experiments utilized different test concentration ranges (or ranges useable by the isobologram method). In some cases not all concentrations tested could support calculation of an IC50, since response was inhibited to a greater extent than 50 percent of control for all doses.

Figure 1 shows a single isobologram, produced by combining the data from both the experiments, showing but statistically significant synergism.

Claims

1. 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 [1 R-(1 α,2β,3α)]-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-1 ,9- dihydro-6H-purin-6-one or a pharmaceutically acceptable derivative thereof .

2. A combination according to claim 1 wherein the therapeutic agents are present in a synergistic ratio.

3. A combination according to claim 2 wherein the ratio is in the range 1 :1 to 1 :20 by weight of active ingredients.

4. A combination according to claim 3 wherein the ratio of 1 :2 to 1 :10

5. A combination according to any one of claims 1 to 4 for use in medicine.

6. A pharmaceutical formulation comprising a combination according to any one of claims 1 to 4 in association with one or more pharmaceutically acceptable carriers therefor.

7. A formulation according to claim 6 in unit dosage form.

8. A formulation according to any one of claims 6 to 7 suitable for oral administration.

9. A formulation according to any one of claims 6 to 8 comprising between 25 to 150 mg of lamivudine and 100 to 1000 mg lobucavir.

10. A formulation according to claim 9 comprising 100 mg of lamivudine and 200 mg lobucavir.

11. A formulation according to claim 9 comprising 50 mg of lamivudine and 200 mg lobucavir.

12. A method for the treatment of a mammal, including a human, with an

HBV infection comprising administration of a therapeutically effective amount of 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 [1 R-(1α,2β,3α)]-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H- purin-6-one or a pharmaceutically acceptable derivative thereof.

13. A method as claimed in claim 12 wherein the combination is as claimed in any of claims 1 to 4.

14. A method according to claim 12 or claim 13 wherein the combination is administered simultaneously.

15. A method according to claim 12 or claim 13 wherein the combination is administered sequentially.

16. A method according to claim 12 or claim 13 wherein the combination is administered as a single combined formulation.

17. A method as claimed in any one of claims 12 to 16 for the treatment of an HBV infection resistant to nucleoside and/or non-nucleoside inhibitors of the replication of the hepatitis B virus

18. Use of (2R,cis)-4-amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl)- pyrimidin-2-one in the manufacture of a medicament for administration either simultaneously or sequentially with [1 R-(1α,2β,3α)]-2-amino-9-[2,3- bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H-purin-6-one, for the treatment of an HBV infection.

19. Use of [1R-(1 ,2β,3α)]-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]- 1 ,9-dihydro-6H-puhn-6-one in the manufacture of a medicament for administration either simultaneously or sequentially with (2R,cis)-4-amino-1-(2- hydroxymethyl-1 ,3-oxathiolan-5-yl)-pyrimidin-2-one for the treatment of an HBV infection.

20. Use of 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 a second therapeutic agent [1 R-(1α,2β,3α)]-2-amino-9-[2,3- bis(hydroxymethyl)cyclobutyl]-1,9-dihydro-6H-purin-6-one or a pharmaceutically acceptable derivative thereof for the treatment of an HBV infection.

21. Use of a combination as claimed in any one of claims 1 to 4 for the treatment of an HBV infection.

22. Use of 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 a second therapeutic agent [1 R-(1α,2β,3α)]-2-amino-9-[2,3- bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H-purin-6-one or a pharmaceutically acceptable derivative thereof, for the treatment of an HBV infection resistant to nucleoside and/or nonnucleoside inhibitor.

23. Use of a combination as claimed in any one of claims 1 to 4 for the treatment of an HBV infection resistant to nucleoside and/or nonnucleoside inhibitor of the replication of the hepatitis B virus.

24. A patient pack comprising of at least one active ingredient selected from (2R,cis)-4-amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl)-pyrimidin-2-one, or [1 R-(1α,2β,3α)]-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-1 ,9-dihydro-6H- purin-6-one and an information insert containing directions on the use of both active ingredients together in combination.