KR20010075202A - Antiviral Combinations Comprising Lamivudine and Abacavir - Google Patents

Abstract

The present invention relates to (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one (lamibudine) and (-)-(1S , 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-l-methanol (1592U89, Abakavir) will be. The present invention also provides a pharmaceutical composition comprising said combination, and an HBV infection comprising infection with a HBV mutant that is resistant to nucleosides and / or non-nucleoside inhibitors against hepatitis B virus replication. It relates to the use in the treatment.

Description

Antiviral Pharmaceutical Formulations Comprising Lamivudine and Abacavir {Antiviral Combinations Comprising Lamivudine and Abacavir}

Hepatitis B is a viral disease transmitted orally or parenterally by contaminated substances, such as blood or blood products, by contaminated saliva, by sexual intercourse, and by a vertical relationship from the infected or carrier mother to the child. In areas where the disease is common, transmission by vertical relationships in childhood results in a high percentage of infected people becoming chronic carriers of hepatitis B. An estimated 350 million people worldwide are chronically infected with hepatitis B, and as many as 150 million people can die of liver disease without treatment.

Currently, the only solution established for the treatment of hepatitis B is the repeated injection of interferon, which may be accompanied by an unpleasant side effect and shows a long-term sustained response only in less than one third of the subjects treated. Interferons are immunomodulators intended to promote disease resistance to the immune system.

Lamivudine has been reported to be effective against HBV in a two-year study, with a substantial reduction in viral replication levels in most patients, and in 52% of the cases, the virus could not be detected sufficiently by the end of the second year. Was found to be maintained.

EP 0434450 describes abakavir as having potent activity against HBV. It has been shown to effectively inhibit HBV replication in vitro (Antimicrobial Agents and Chemotherapy May 1997, P1082-1093).

The present invention provides (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one (lamibudine) and a second therapeutic agent (- )-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopentene-1-methanol (1592U89, Abakavir) To a formulation for use. The invention also provides for the treatment of HBV infections, including infections with pharmaceutical compositions containing such combinations, and HBV mutants that are resistant to nucleosides and / or non-nucleoside inhibitors against hepatitis B virus replication. To its use in

The use of the pharmaceutical formulations of the present invention may result in synergism between the compounds, resulting in reduced toxicity, ie increased drug efficacy, while exhibiting equivalent antiviral effects. In addition, reducing the overall drug dosage will likely reduce the incidence of drug resistant HBV strains.

We have found that lamivudine presents unexpected benefits when used in combination with abacavir. In particular, as shown in FIG. 1, such pharmaceutical agents exhibit a statistically significant synergistic anti-HBV effect. It is a feature of the present invention that the use of such pharmaceutical agents provides synergistic antiviral effects, ie more complete viral inhibition than for longer periods of time, limits the occurrence of drug resistant HBV mutants and better handles drug related toxicity. To make it possible. In addition, the use of such pharmaceutical formulations may, for example, reduce the number of daily dose tablets and thus increase patient compliance.

As will be appreciated by those skilled in the art, the term treatment encompasses the prevention as well as the treatment of identified infections and symptoms.

As used herein, the term “pharmaceutically acceptable derivative” refers to such compounds or antiviral active metabolites or residues thereof when administered to a salt or recipient of any pharmaceutically acceptable salt, ester or ester of lamivudine or abakavir. Any other compound capable of providing (directly or indirectly).

Pharmaceutically acceptable salts of lamivudine and abakavir include those derived from pharmaceutically acceptable bases, inorganic acids and organic acids. Examples of suitable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid , Malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Other acids, such as oxalic acid, are not inherently pharmaceutically acceptable, but may be useful in the preparation of salts useful as intermediates to obtain the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from suitable bases include alkali metals (eg sodium), alkaline earth metals (eg magnesium), ammonium and NR ₄ where R is C _1-4 -alkyl.

Preferred salts of abakavir are sulfates, in particular hemisulphate.

Preferred esters of lamivudine and abacavir are independently selected from the following groups: (1) The bicarbonyl moiety of the carboxylic acid moiety of the ester group is linear or branched alkyl (e.g. methyl, n-propyl, t-butyl or n-butyl), cycloalkyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. halogen, C _1-4 Carboxylic ester selected from -alkyl or phenyl optionally substituted with C _1-4 -alkoxy), or amino; (2) sulfonate esters such as alkyl- or aralkylsulfonyl (eg methanesulfonyl); (3) amino acid esters (eg, L-valyl or L-isoroyl); And (4) phosphonate esters. In such esters, unless otherwise specified, it is advantageous for any alkyl moiety present to contain 1 to 18 carbon atoms, in particular 1 to 6 carbon atoms, more particularly 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters advantageously contains 3 to 6 carbon atoms. Any aryl moiety present in such esters is advantageously made up of phenyl groups. In addition, all references to any of the above compounds also include references to physiologically acceptable salts thereof.

Particularly preferred esters are mono-, di-, and triphosphate esters of lamivudine and abakavir (which can optionally be blocked), or when administered to humans (directly or indirectly) ) Any other compound that can be provided.

More preferred derivatives of abakavir are mono-, di- and triphosphate esters of (1R, 4S) -9- [4- (hydroxymethyl) -2-cyclopenten-1-yl] guanine (carbovir) .

Thus, according to one feature, the invention provides (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one or a pharmaceutical thereof Acceptable derivatives and (-)-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopentene-1-methanol or a pharmaceutical thereof It provides a pharmaceutical formulation comprising an acceptable derivative.

The pharmaceutical formulation may be referred to as the pharmaceutical formulation of the present invention below.

The invention also provides a pharmaceutical formulation of the invention for use in therapy, especially for the treatment of HBV infections, including infections resistant to nucleosides and / or non-nucleoside inhibitors of hepatitis B virus replication. To provide.

In another aspect, the present invention provides a method of treating a HBV infected mammal, including a human, comprising administering a therapeutically effective amount of a pharmaceutical formulation of the invention.

The compounds of the pharmaceutical formulations of the invention may be administered simultaneously or sequentially in the same or different pharmaceutical compositions. In the case of continuous administration, the delay time upon administration of the second active ingredient should be such that it does not lose the benefit of the synergistic therapeutic effect of the pharmaceutical formulation of the active ingredients. In addition, lamivudine and abacavir or pharmaceutically acceptable derivatives thereof may be administered alone or in any mixture thereof, whether provided simultaneously or sequentially. Lamivudine and abakavir are preferably administered simultaneously or sequentially in separate pharmaceutical formulations, with simultaneous administration being particularly preferred.

Preferably, the pharmaceutical preparations according to the invention are preferably administered in a single mixed preparation.

The present invention also provides the use of lamivudine in the manufacture of a pharmaceutical formulation for simultaneous or continuous administration of abacavir for the treatment of HBV infection. Yet another feature of the present invention is the use of abakavir in the manufacture of a pharmaceutical formulation for simultaneous or successive administration of lamivudine for the treatment of HBV infection.

A further feature of the present invention is the pharmaceutical formulation of the present invention, wherein lamivudine and abakavir are present in synergistic proportions.

The synergistic effect of the pharmaceutical preparations of lamivudine and abakavir or pharmaceutically acceptable derivatives thereof is shown, for example, at a ratio of 1: 0.5 to 1:10 (weight ratio), preferably 1: 1 to 1: 8 (weight ratio). .

Conveniently, each compound will be used in the pharmaceutical formulation of the present invention in an amount that exhibits anti-HBV activity when used alone.

The amount of pharmaceutical preparation of lamivudine and abakavir required to be effective as an anti-HBV agent will of course vary, but ultimately is at the discretion of the practitioner. Factors to be considered include the route of administration and the nature of the formulation, the body weight, age and general symptoms of the animal, and the nature and depth of the therapeutic disease.

In general, a suitable daily dose of lamivudine is about 0.1 to about 50 mg / kg (body weight) / day, preferably 0.5 to 20 mg / kg (body weight) / day, most preferably 0.5 to 2 mg / kg (weight) / day.

Preferably, lamivudine is administered at a level of 25 to 150 mg / day.

Preferably, lamivudine is conveniently administered at a level of about 100 mg / day.

A suitable daily dose of abacavir is about 3 to about 120 mg / kg (body weight) / day, preferably 1 to 90 mg / kg (body weight) / day, most preferably 5 to 60 mg / kg ( Body weight) / day.

Preferably, abacavir is administered at a level of 200 to 800 mg / day.

Preferably, abacavir is administered at a level of about 600 mg / day. It may be convenient to administer 300 mg twice a day.

Unless stated otherwise, all weights of active ingredients are calculated in terms of the drug itself. In the case of pharmaceutically acceptable derivatives of lamivudine and abakavir, or solvates thereof, the values will increase in proportion. The desired dosage is preferably divided into two, three, four, five, six or more doses at appropriate intervals throughout the day. Such divided doses may be administered in unit dosage forms, eg, in unit dosage forms containing 1 to 1500 mg, preferably 5 to 1000 mg, most preferably 5 to 500 mg of active ingredient per unit dosage form. have. The divided doses of abacavir may be administered independently of the dosage of the pharmaceutical formulation of the present invention. Alternatively, if the patient's symptoms are needed, the dose may be administered as a continuous infusion.

Components of the pharmaceutical formulation, which may be called active ingredients, may be administered therapeutically to an animal, such as a mammal, including a human, in a conventional manner.

The active ingredients of the pharmaceutical preparations can be administered by the raw compound itself, but it is preferable to provide them in a pharmaceutical composition. Pharmaceutical compositions according to the invention comprise a combination of the invention in combination with one or more pharmaceutically acceptable carriers or excipients and any other therapeutic agent. The carrier (s) is to be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. When the individual components of the pharmaceutical formulations of the invention are administered separately, they are generally provided as respective pharmaceutical compositions. Reference to the composition below means compositions containing the combinations or components thereof of the invention unless otherwise stated.

Pharmaceutical formulations of lamivudine and abakavir or pharmaceutically acceptable derivatives thereof may be conveniently provided in pharmaceutical compositions with one or more pharmaceutically acceptable carriers thereof in unit dosage form. Convenient unit dosage forms contain 1 mg to 2 g of active ingredient each, for example 2 mg to 200 mg (eg 25 to 150 mg) lamivudine and 200 to 800 mg abacavir.

In addition, the pharmaceutical composition may be prescribed to the patient in a “patient active controlled pack” containing the entire therapeutic drug in a single package, typically a blister pack. Patient Active Adjustable Packs allow patients to always have access to package instructions contained in Patient Active Adjustable Packs, which are not typically available in conventional prescriptions, compared to conventional prescriptions in which the dispenser dispenses a large amount of pharmaceutical composition to a patient. There is an advantage in that it exists. Inclusion of package instructions has been shown to improve patient compliance with physician instructions.

It is an additional feature of the present invention that the administration of the pharmaceutical formulation of the invention using a single patient active controlled pack or patient active controlled pack of each composition comprising a package instruction to instruct the patient to use the invention correctly I will understand the point.

According to another feature of the present invention, there is provided a patient active controlled pack comprising an information manual containing instructions on the use of at least one active ingredient of the pharmaceutical formulation of the invention and a mixture of the invention.

As another feature, the present invention provides a dual pack comprising separately administered lamivudine and abakavir or a pharmaceutically acceptable derivative thereof.

The 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 of the methods well known in the art of pharmacy. These methods are a further feature of the invention and comprise the step of mixing the active ingredient with a carrier comprising one or more accessory ingredients. Generally, formulations are prepared by uniformly and intimately mixing the active ingredients with liquid carriers or finely divided solid carriers or both, and then molding the product, if necessary.

Compositions of the invention suitable for oral administration are provided as discrete units such as capsules, caplets, cachets or tablets containing a predetermined amount of active ingredient; Provided in 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. In addition, the active ingredient may be provided as a macrocyclic, delayed or paste.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may optionally be used in a suitable machine to freely bind the active ingredients in free flowing form, such as powders or granules, to binders (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (e.g. sodium glycol starch, Crosslinked povidone, crosslinked sodium carboxymethyl cellulose), surfactants or dispersants, and mixed together to produce compression. Molded tablets can be made by molding a blend of the powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and formulated to provide slow or controlled release of the active ingredients, for example using hydroxypropylmethylcellulose in various ratios to provide the desired release profile. Tablets may optionally be provided with enteric skin so that they are released from the intestine rather than from the stomach.

Preferably, the pharmaceutical formulation of the present invention is administered orally.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavor base, typically sucrose and gum arabic or tragaganth; Pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic; And water-containing agents comprising the active ingredient in a suitable liquid carrier. Compositions for rectal administration may be presented as suppositories using a suitable substrate, including, for example, cocoa butter or salicylate.

In addition, topical administration may use a transdermal iontophoretic device.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations which contain, in addition to the active ingredient, carriers known in the art as suitable.

Most preferably, a pharmaceutical composition suitable for rectal administration wherein the carrier is a solid is provided as a unit dosage suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently prepared by mixing the active formulation with a softening or molten carrier (s), then cooling and molding in a mold.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injections which may contain antioxidants, buffers, fungicides, and solutes that render the formulation isotonic with the blood of the intended recipient; And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickeners; And liposomes or other particulate systems whose compounds are intended to target blood components or one or more organs. The formulations may be provided in single dose or multidose closed containers, such as ampoules and vials, and are freeze dried (freeze dried), requiring only the addition of a sterile liquid carrier, such as injection water, immediately before use. Can be stored as. Immediately injectable solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind mentioned above.

Preferred unit dosage forms are those containing the active ingredient or a suitable fraction thereof in a daily or divided dose as described above.

In addition to the components specifically mentioned above, the formulations of the present invention may comprise other substances of ordinary skill in the art relating to the form of the formulation, for example those suitable for oral administration may be used as sweeteners, thickeners and flavoring agents. It may include additional materials.

The compounds of the pharmaceutical preparations of the present invention can be obtained by conventional methods.

Methods for preparing lamivudine are described in WO91 / 17159 and WO95 / 29174, which are incorporated herein by reference.

Abakavir may be prepared by the methods described in EP 0434450, PCT Application PCT / GB / 4500225, PCT Application PCT / GB95 / 02014, or US Patent 5,034,394, which are incorporated herein by reference. Quote for reference.

The following examples are intended to illustrate, and are not intended to limit the scope of the invention in any way. The term "active ingredient" refers to lamivudine or abakavir, both compounds, or physiologically functional derivatives of these compounds.

Example 1: Tablet

Formulations A, B and C below were prepared by wet granulating the components with a povidone solution, followed by the addition of magnesium stearate and compression.

Formulation A

mg / tablet

Active ingredient 250

Lactose B.P. 210

Povidone B.P. 15

Glycol Sodium Starch 20

Magnesium stearate 5

500

Formulation B

mg / tablet

Active ingredient 250

Lactose B.P. 150

Avicel PH 101 60

Povidone B.P. 15

Glycol Sodium Starch 20

Magnesium stearate 5

500

Formulation C

mg / tablet

Active ingredient 250

Lactose B.P. 200

Starch 50

Povidone 5

Magnesium stearate 4

359

Formulations D and E below were prepared by direct compression of the mixed ingredients. Lactose in Formulation E is of direct compression (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 (sustained release formulation)

This formulation was prepared by wet granulating the components with a povidone solution, then adding magnesium stearate and compressing.

mg / tablet

Active ingredient 500

Hydroxypropylmethylcellulose 112

(Methocell K4M Premium)

Lactose B.P. 53

Povidone B.P. 28

Magnesium Stearate 7

700

Drug release occurred over about 6-8 hours and was complete after 12 hours.

Example 2: Capsule Formulation

Formulation A

Capsule formulations were prepared by mixing the components of Formulation D of Example 1 above into a two-part hard gelatin capsule. Formulation B below was prepared in a similar manner.

Formulation B

mg / capsules

Active ingredient 250

Lactose B.P. 143

Glycol Sodium Starch 25

Magnesium stearate 2

420

Formulation C

mg / capsules

Active ingredient 250

Macrogel 4000 BP 350

600

Capsule Formulation C was prepared by melting Macrogel 4000 B.P., dispersing the active ingredient in the melt, and filling the melt in two parts hard gelatin capsules.

Formulation D

mg / capsules

Active ingredient 250

Lecithin 100

Peanut oil 100

450

Capsule Formulation D was prepared by dispersing the active ingredient in lecithin and peanut oil and filling the dispersion into soft elastic gelatin capsules.

Formulation E

mg / capsules

Active ingredient 150.0

Vitamin E TPGS 400.0

Polyethylene Glycol 400 NF 200.5

Propylene Glycol USP 39.5

4 kg of vitamin E TPGS (obtained from Eastman Chemical Co.) was heated until liquefied at 50 ° C. To the liquefied vitamin E TPGS is added 2.005 kg of polyethylene glycol 400 (PEG 400) heated to 50 ° C. (low aldehyde, <10 ppm; obtained from Union Carbide or Dow Chemical Co.) and mixed until a homogeneous solution is formed. It was. The resulting solution was heated to 65 ° C. 1.5 kg of active ingredient was dissolved in a liquefied solution of vitamin E TPGS and PEG 400. 0.395 kg of propylene glycol at room temperature was added and mixed until a homogeneous solution was formed. The solution was cooled to 28-35 ° C. The gas of the solution was then removed. Preferably, the mixture was placed in an elliptical white opaque soft gelatin capsule of size 12 using a capsule filling machine at 28-35 ° C. with the same fill weight as 150 mg of volatile compounds. The capsule shells were dried until a constant fill moisture of 3-6% water and shell hardness of 7-10 N were obtained and placed in a suitable container.

Formulation F (sustained release capsule)

The sustained release capsule formulations below were prepared by extruding components a, b and c using an extruder, followed by spheroonization of the extrudate and drying. The dried pellets were then coated with a sustained release membrane (d) and filled into two-part hard gelatin capsules.

mg / capsules

(a) Active Ingredient 250

(b) microcrystalline cellulose 125

(c) lactose B.P. 125

(d) ethyl cellulose 13

513

Example 3: Injection Formulations

Formulation A

mg

Active ingredient 200

Amount required to adjust to hydrochloric acid solution (0.1 M) or pH 4.0 to 7.0

Sodium Hydroxide Solution (0.1 M)

Amount to aseptic volume 10 ml

The active ingredient was dissolved in most water (35-40 ° C.) and the pH was adjusted to 4.0-7.0 by addition of hydrochloric acid or sodium hydroxide as needed. The batch was then added to the required volume by adding water to the batch, filtered through a sterile microporous filter and placed in 10 ml of sterile amber glass vials (Type 1) and sealed with sterile stoppers and outer lids.

Formulation B

125 mg of active ingredient

Aseptic pyrogenic phosphate buffer (pH 7) volume to 25 ml

Example 4 Intramuscular Injection

200 mg active ingredient

0.10 g of benzyl alcohol

Glycofurol 75 1.45 g

Amount to bring the volume of injection to 3.00 ml

The active ingredient was dissolved in glycofurol. Benzyl alcohol was then added to dissolve and water was added to bring the volume to 3 ml. The mixture was then filtered through a sterile microporous filter and sealed in 3 ml of sterile amber glass vials (Type 1).

Example 5: Syrup

250 mg active ingredient

1.50 g of sorbitol solution

Glycerol 2.00 g

0.005 g sodium benzoate

Flavoring (Peach 17.42.3169) 0.0125 ml

Volume to 5.00 ml of purified water

The active ingredient was dissolved in a mixture of glycerol and most purified water. Subsequently, an aqueous sodium benzoate solution was added to this solution, followed by the addition of the sorbitol solution, and finally the flavoring agent. Make the required volume with purified water and mix well.

Example 6: Suppositories

mg / capsule suppository

Active ingredient 250

Hard fat, BP (Witthesol H15-Dynamit Nobel) 1770

2020

One fifth of Whitepsol H15 was melted at up to 45 ° C. in a steam jacketed pan. The active ingredient was sieved through a 200 μm sieve and added to the molten substrate and mixed until a smooth dispersion was obtained using Silverson with a cutting head. The mixture was kept at 45 ° C. and the remaining Uittsol H15 was added to the suspension and stirred to make a homogeneous mix. The entire suspension was passed through a 250 μm stainless steel screen and cooled to 45 ° C. with continued stirring. At a temperature of 38 to 40 ° C., 2.02 g of the mixture was charged to a suitable 2 ml plastic mold. Suppositories were cooled to room temperature.

Example 7 Pessary

mg / pessari

Active ingredient 250

Anhydrous Dextrose 380

Potato Starch 363

Magnesium Stearate 7

1000

The ingredients were mixed directly and the mixture was compressed directly to make pessaries.

Biological data

Example 8

Human interstitial tumor cell lines (Hep-G2-2.2.15) that essentially produce infectious HBV were seeded in 96-well microtiter plates at a density of 5 × 10 ³ cells / well. These cells were treated with a mixture of lamivudine and abakavir on three plates. The drug containing medium was supplemented every other day for 9 days, and each time the supernatant was collected and analyzed for HBV content.

The lamivudine / avacavir mixture was tested twice in triplicates. Test 1 used lamivudine ranging from 0.008 nM to 125 nM (5 fold dilution, column) and abacavir at a concentration of 40 nM to 25 μM (5 fold dilution, row). Test 2 was performed with lamivudine dilution from 0.14 nM to 100 nM (3 fold dilution, column) and abacavir from 250.7 nM to 25 μM (3.16 fold dilution, row). Test 3 was performed using lamivudine dilution from 0.045 nM to 100 nM (3 fold dilution) and abacavir from 0.8 nM to 25 μM (3.16 fold dilution, row). Both drugs were diluted in separate 96-well microtiter plates and then transferred onto plates containing cell monolayers. The cells were grown in 150 μl RPMI 1640 supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Before transferring the drug, 120 μl of medium was removed from the cells and 30 μl was left in the monolayer to prevent drying. 90 μl of drug free fresh medium was added, followed by 30 μl of 5 × drug dilution. Lamivudine and Abakavir were each tested at the same concentration on their plates. Data was normalized to the values obtained with non-drug treated cells and expressed as percentage of control for analysis.

Methods used to detect HBV are described in Jansen RW, Johnson LC, Averett, DR.High-Capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay.Antimicrob Agents Chem 1993; 37 ( 3): 441-447). In brief, HBV detection “captures” the virus from the supernatant on an anti-HBsAg coated plate, releases HBV DNA by denaturation, performs PCR with biotinylated primers, and uses accompanying probe hybridization. Biotinylated PCR product was captured by streptavidin, the substrate was added, and the optical density of the colorimetric reaction was read. Dilutions of the normalized HBV containing supernatant were included in all plates and the HBV DNA concentration of the test wells was calculated from this HBV standard curve. A useful range of detection is at least 0.045 to 45 fg of HBV DNA, where 30 copies of the genome could be reliably detected. Samples were tested with positive (0.448 fg / μl plasmid DNA) and negative (RPMI medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum) controls.

The standard error of the mean IC50 and IC50 for the three plates was calculated by fitting data to the Hill equation for each concentration response curve using SAS nonlinear regression. When only one IC50 could be measured for a particular dose mixture, the standard error was calculated using the average of the standard errors from adjacent concentrations. Fractional inhibitory concentrations (FIC50) were calculated for each mixture and isobologram representation (Berenbaum, MC (1985) The Expected Effect of a Combination of Agents: the General Solution. J. Theor. Biol. 114, 413 -431). To assess the statistical significance for synergy or antagonism, each sum of paired FIC50 values was compared to theory 1 using an unpaired t-test. P values less than 0.05 were considered statistically significant. Because each test uses a different test concentration range (or range that can be used by the isobologram method), the comparison of P values between each test should be interpreted with great care. In some cases, not all concentrations tested can support the calculation of IC 50 because the response is inhibited to over 50% of the control for all doses.

1 is a single isobologram obtained by combining the data from the three tests and shows that there is a statistically significant synergy.

Example 9

Wild type and YMDD in an in vitro assay comprising HBV core particles isolated from transiently transfected or transduced HepG2 cells with K _i of (-) carbovir triphosphate ((-) CBV-tp), the active form of abakavir The variant HBV polymerase was determined. HepG2 cells have wild-type sequences or amino acid changes in reverse transcription genes (M552I, M552V, L528M, L528MM552V, L528MM552I (Allen MI, Deslauriers 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) were transiently transfected with plasmids containing the HBV genome. Was performed using a baculovirus containing the same structure (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) Core polymerase assays were performed by modifying previously reported protocols (Davis MG, Wilson, JE, VanDraanen NA, Miller WH, Freeman GA, Daluge SM, Boyd F). L, 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). In the same way, briefly, 5 days after transfection or transduction, cells were harvested and pelleted. Cell pellets were resuspended in hypotonic solution and cells were lysed with NP-40 to a final concentration of 0.5%. Nuclei were pelleted and cell supernatants were digested with RNase and DNase. Treated lysates were pelleted via a sucrose step gradient. The HBV core containing pellets thus obtained were resuspended by sonication in a buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM KCl and 5 mM MgCl ₂ . This material was stored at −80 ° C. until used for polymerase analysis. Antibody capture assay plates were prepared using the following protocol: Costa # 3632 (Costar # 3632; Corning, Corning, NY) was prepared with 50 mM sodium carbonate buffer (Sigma, St. Louis, MO, pH 9.6). 0.10 mg / ml of Neutravidin (Neutravidin, Pierce, Rockfield, Ill.) At 0.15 ml / well for 2 hours at room temperature, followed by washing buffer (WB) (150 mM NaCl, 100 mM sodium phosphate) (pH 7.2), 0.02% Tween-20 (manufactured by Pierce, Rockfield, Ill.), 0.02% sodium azide, 0.01% bovine serum albumin (manufactured by Sigma, St. Louis, MO)) washed with ml / well; Successively, 2) coated with 0.15 ml / well for 1 hour at room temperature with biotinylated goat anti-rabbit IgG (Pearce, Rockford, Ill.) Diluted to 0.0015 mg / ml in WB, followed by 3 with WB. Washed with 0.25 ml / well; Successively, 3) coated with 0.15 ml / well for 1 hour at room temperature with rabbit anti-HBV core antigen antibody (Zymed, San Francisco, CA) diluted 1: 300 in WB. Plates were washed with 2 x 0.25 ml / well with WB and WB was added at 0.25 ml / well at the end of the wash. The plate was stored in a humidification chamber at 4 ° C. Corepolymerase assays were performed by modifying previously reported protocols (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). The core particle preparation was thawed in the refrigerator and resuspended by decomposition by sonication before each analysis. The core particle analyte contained the following components: 230 mM KCl, 33 mM Tris-HCl, pH 7.4, 8 mM MgCl ₂ , 0.2% NP-40, 5 mM DTT, 7 units / ml Pyruvate Kinase Buffer (US) Sigma, St. Louis, MO), 2 mM phosphoenol pyruvate buffer (Sigma, St. Louis, MO), 25 mM unlabeled dNTP (Amersham Pharmacia Biotech, Piscataway, NJ), ³³ P Labeling dNTP (NEN , US Boston, Massachusetts Life Science Products Products, 2000 Ci / mmole) in the 0 to 4 μM of the core particles (usually 10 ⁷ to 10 ^8/10 ul reaction). 10 ul of analyte was incubated at 37 ° C. for 45-90 minutes in 96-well polypropylene plate (USA Scientific, Ocala, Fla.) And stopped with 90 ul of the following buffer: 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl ₂ , 0.05% Tween-20, 2 mM Sodium Pyrophosphate, 0.02% Sodium Azide. A total of 100 ul of stopped analyte was transferred to a 96-well antibody capture plate and incubated for 2 hours at room temperature with shaking. After antibody capture, wells were washed 5 times with the following buffer: 150 mM NaCl, 100 mM sodium phosphate pH 7.2, 0.05% Tween-20, 2 mM sodium pyrophosphate, 0.02% sodium azide. Excess buffer was removed by tapping the plate on the blotter paper. 170 ul of Wallac Optiphase “Supermix” (Wallac Inc., Gaithersburg, Midland, USA) was added to each well. Plates were counted with a Wallac 1450 Microbeta Plus Liquid Scintillation Counter (Wallac Inc., Gaithersburg, Midland, USA). Inhibition analysis ^It was essentially the same as the core polymerase assay except that the concentration of ³³ P-dGTP was the K _m of each polymerase analyzed. The concentration of (−) CBV-tp was 0.001 μM to 312 mM. K _i was calculated using the following equation: K _i = IC ₅₀ / (1+ [S] / K _m ). IC ₅₀ is the inhibitory concentration of the compound at which the polymerase activity is 50%. [S] is the concentration of the competing substrate (for (-) CBV-tp, ³³ P labeled dGTP). All IC _50s were determined at the K _m of dCTP for each polymerase. The IC ₅₀ of (−) CBV-tp was evaluated by using a nonlinear regression method by fitting the obtained concentration response curve to the Hill equation y = (Vmax * x ^ n) / (K ^ n + x ^ n). The calculation was performed using the RoboSage Add-in from Microsoft Excel.

(-) CBV-tp: Fold change of IC ₅₀ , K _i , and K _i HBV Polymerase (-) CBV-tp IC ₅₀ (μM) (-) CBV-tp K _i (μM) (-) CBV-tp K _i 2 times change wt 0.21 0.10 One LMMV 2.69 1.34 13 MI 8.70 4.35 42 LMMI 2.54 1.27 12 LM 0.67 0.33 3 MV 4.64 2.32 22 * HBV genome with mean LMMV-L528MM552V amino acid mutation in two independent analyzes HBV genome with MI-M552I amino acid mutation HBV genome with LMMI-L528MM552I amino acid mutation HBV genome with LM-L528M amino acid mutation HBV with MV-M552V amino acid mutation Genome

Claims (23)

(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 pharmaceutical formulation of claim 1, wherein the compounds are present in a proportion that exhibits synergy. The pharmaceutical formulation according to claim 2, wherein the ratio is in the range of 1: 0.5 to 1:10 by weight of the active ingredient. The pharmaceutical formulation of claim 3 wherein the ratio is from 1: 1 to 1: 8. The pharmaceutical preparation according to any one of claims 1 to 4 for use as a medicament. A pharmaceutical formulation comprising the pharmaceutical formulation of claim 1 and at least one pharmaceutically acceptable carrier. The formulation of claim 6 which is in unit dosage form. The formulation according to claim 6 or 7, which is for oral administration. The formulation according to claim 6, comprising 25 to 150 mg lamivudine and 200 to 800 mg abacavir. The formulation of claim 9 comprising 100 mg lamivudine and 300 mg abacavir. (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 A method of treating an HBV infected mammal, including a human, comprising administering an effective amount. 12. The method of claim 11, wherein said pharmaceutical formulation is claimed in any one of claims 1-4. 13. The method of claim 11 or 12, wherein said pharmaceutical formulation is administered concurrently. 13. The method of claim 11 or 12, wherein said pharmaceutical formulation is administered continuously. 13. The method of claim 11 or 12, wherein said pharmaceutical formulation is administered as a single pharmaceutical formulation. The method of claim 11, wherein the HBV infection is resistant to nucleoside and / or non-nucleoside inhibitors of hepatitis B virus replication. With (-)-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-1-methanol for treating HBV infection Use of (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one in the manufacture of a medicament for simultaneous or continuous administration. For simultaneous or continuous administration with (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one to treat HBV infection Use of (-)-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-1-methanol in the manufacture of a medicament. (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one or a pharmaceutically acceptable thereof for treating an HBV infection Derivative and second therapeutic agent (-)-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopentene-1-methanol or its Use of a pharmaceutical formulation comprising a pharmaceutically acceptable derivative. Use of a pharmaceutical formulation as claimed in any one of claims 1 to 4 for treating an HBV infection. (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolane for treating HBV infection resistant to nucleosides and / or non-nucleoside inhibitors -5-yl) -pyrimidin-2-one or a pharmaceutically acceptable derivative thereof and the second therapeutic agent (-)-(1S, 4R) -4- [2-amino-6- (cyclopropylamino) -9H Use of a pharmaceutical preparation comprising -purin-9-yl] -2-cyclopenten-l-methanol or a pharmaceutically acceptable derivative thereof. Use of a pharmaceutical formulation as claimed in any one of claims 1 to 4 for the treatment of HBV infection resistant to nucleosides and / or non-nucleoside inhibitors of hepatitis B virus replication. (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -pyrimidin-2-one or (-)-(1S, 4R) -4- Instructions for combining two or more active ingredients together with at least one active ingredient selected from [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-1-methanol Patient active adjustable pack with information manual included.