WO1990014079A1

WO1990014079A1 - Method of treatment of hepatitis

Info

Publication number: WO1990014079A1
Application number: PCT/US1990/002685
Authority: WO
Inventors: Jay H. Hoofnagle; Samuel Broder; Hiroaki Mitsuya; Robert Yarchoan
Original assignee: United States Department of Commerce
Current assignee: United States Department of Commerce
Priority date: 1989-05-15
Filing date: 1990-05-15
Publication date: 1990-11-29
Anticipated expiration: 1991-11-15
Also published as: CA2055433A1; EP0472595A1; AU5659990A; EP0472595A4; JPH04501854A

Abstract

According to the present invention, hepatitis B can be treated by administering 2',3'-dideoxycytidine to a patient infected with hepatitis B. The 2',3'-dideoxycytidine, following anabolic phosphorylation, inhibits the reverse transcriptase of the hepatitis B virus.

Description

METHOD OF TREATMENT OF HEPATITIS

FIELD OF THE INVENTION The present invention relates to a method for treating hepatitis B. BACKGROUND OF THE INVENTION

Chronic infection which the hepatitis B virus

(HBV) affects approximately 5% of the world's population.

Chronic carriers of hepatitis B are at a increased risk of morbidity and mortality due to chronic liver disease _r and a proportion of these will ultimately develop cirrhosis and/or hepatocellular carcinoma. At present, there is no therapy of proven benefit for chronic hepatitis B. Although o^-interferon has shown great promise in a subset of patients treated for prolonged period of time, the response rates overall have, unfortunately, been disap¬ pointingly low.

The human hepatitis B virus is a member of a family of viruses known as hepadnaviruses. Other viruses in this family are the woodchuck hepatitis virus, the ground squirrel hepatitis virus, and the duck hepatitis B virus. These animal viruses have been invaluable models for characterization of hepadnaviruses and delineation of their unusual replicative cycle. These viruses replicate asymmetrically through an RNA template which requires reverse transcriptase activity, cf. Summers, Cell 29:403- 415, 1982.

The 2', 3'-dideoxynucleosides are nucleosides which recently have been shown to have potent antiviral activity against the reverse transcriptase activity of the human immunodeficiency virus, HIV, as described by Mitsuya, et al. in Proc. Natl. Acad. Sci. USA 1986; 83:1911-1915. The most potent of these analogues is 2' , 3'-dideoxycy- tidine, or DDC, which inhibits HIV in cell culture in concentrations as low as 10 nM. SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the above-mentioned deficiencies in the prior art. It is another object of the present invention to provide methods for treating hepatitis B.

It is further object of the present invention to provide compositions for treating hepatitis B. According to the present invention, hepatitis B can be treated by administering 2' , 3 '-dideoxycytidine to a patient infected with hepatitis B. The 2* , 3'- dideoxycytidine, following anabolic phosphorylation, inhibits the reverse transcriptase of the hepatitis B virus.

While the exact mechanisms of the antiviral activity of the compositions according to the present invention are unknown, it is believed that the mechanism of action of DDC is inhibition of viral polymerases, in particular, reverse transcriptases. DDC is a nucleoside analogue, and it appears to prevent the formation of normal phosphodiester linkages once it becomes incorporated into a growing DNA chain. This process leads to "chain termina¬ tion." DDC has a high affinity for reverse transcriptase, and, therefore, may inhibit replication of hepatitis B virus by preventing reverse transcription from the pregeno- mic RNA template. This interference in replication would lead to a decrease in serum levels of virus and a gradual fall in the amounts of hepatitis B virus DNA in the liver. DDC is particularly attractive as antiviral agent because it is absorbed orally and has comparatively minimal side effects under the conditions used.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the in vitro effects of 2' , 3'- dideoxycytidine triphosphate on the DNA polymerase reaction of the human and duck hepatitis B viruses.

Figure 2 shows changes in mean serum DNA polymerase activity among Peking ducks chronically infected with duck hepatitis B virus who received DDC or no treatment.

Figure 3 shows changes in mean serum duck hepatitis B virus DNA levels among Peking ducks chronically infected with duck hepatitis B virus who received either DDC or no treatment.

Figure 4 shows liver duck hepatitis B virus DNA levels in ducks before and after treatment with DDC. Figure 5 shows autoradiograms of duck hepatitis

B virus DNA analyses done on liver tissue taken before and on the sixth day of treatment with DDC.

DETAILED DESCRIPTION OF THE INVENTION

2' , 3 '-dideoxycytidine can be used for treating hepatitis B in patients so infected. The DDC is well absorbed orally, and is generally well tolerated. In humans, the dose-limiting toxicity has been a peripheral neuropathy which can be significantly reduced by lowering the dose. In vitro DDC triphosphate had little effect on

DNA polymerase activity of either duck hepatitis B virus or human hepatitis B virus. Previous researchers have used the in vitro assay to assess antiviral activity in hepatitis B, cf. Nordenfelt, et al., Acta Path. Microbiol. Scand. Sect. B 82:75-76, 1979; and Hess, et al., Antiπtic. Agents Chemo. JL :44-50, 1981. However, it has now been discovered that this assessment may be unreliable as a means of screening antiviral agents. The DNA polymerase activity measured in serum from humans and ducks infected with hepadnaviruses may represent only one of the viral enzymes necessary for replication, and this activity may be relatively resistant to inhibition.

In contrast, in ducks chronically infected with duck hepatitis B virus, DDC exhibited potent antiviral activity when given for six days in doses similar to those used in human antiviral trials, cf. Yarchoan, et al., Lancet 1:76-81, 1988. The degrees of inhibition of both DNA polymerase activity and duck hepatitis B DNA were similar (67% and 69%, respectively and were comparable to the degrees of inhibition of these markers reported in studies of other antiviral agents used in treatment of chronic hepatitis B. The antiviral effect was only part¬ ial, however, in that no duck became completely negative for duck hepatitis B DNA or DNA polymerase activity, and levels of these viral markers began to rise soon after the DDC therapy was stopped. These findings are similar to those reported with other antiviral agents used in chronic hepatitis B. A promising finding following DDC administra¬ tion, however, was that some inhibition of DNA polymerase activity and duck hepatitis B DNA was still observed for as long as twelve days after therapy was stopped. This observation is contrary to findings with adenine arabino- side and acyclovir, wherein following withdrawal of these agents, serum levels of duck hepatitis B virus often rebound to above pretreatment levels (Hirota,_. et al., Hepatology 7:24-28, 1987) .

The effect of 2 ' , 3 '-dideoxycytidine was assessed in 16 Peking ducks chronically infected with the duck hepatitis B virus (DHBV) . Nine ducks were given DDC at the rate of 11 mg/m² intravenously every six hours, and seven ducks received no treatment. Serum DHBV DNA and DNA polymerase activity decreased in every duck treated with DDC. The mean inhibition of DNA polymerase and DHBV DNA on the third day of treatment measured 64% (p<0.01) and 73%

(p<0.01), respectively. The inhibition of DNA polymerase persisted after treatment was stopped, and four ducks continued to show greater than 50% inhibition twelve days after stopping treatment. DHBV DNA, which was measured in total cellular DNA extracted from liver biopsies obtained before and on the last day of treatment with DDC, showed an average inhibition of 96% in three ducks treated with DDC, but showed no decrease in the remaining five ducks. DUCKS AND TREATMENT SCHEDULE

An initial group of twenty ducks chronically infected with DHBV were supplemented later with twenty ducklings who were obtained within a day of hatching and inoculated intraperitoneally with 100 μl of serum pooled from DHBV carrier ducks. The inoculum contained approxi¬ mately 1.3 x 10⁸ virions per ml. serum as estimated by slot blot hybridization. The ducks were maintained and screened at monthly intervals for persistence of DHBV DNA and DNA polymerase activity in serum. At approximately four months of age, eighteen ducks with high levels of DNA polymerase activity were selected for study.

Nine ducks with the highest levels of DNA polyme¬ rase activity were administered DDC in a dose of 11 mg/m² intravenously every six hours for six days. Two ducks were given adenine arabinoside monophosphate (Ara-AMP; vidara- bine monophosphate: Parke, Davis, Detroit, Michigan) at a rate of 400 and 1000 mg/m² intramuscularly twice daily for six days. Seven ducks received no treatment. Blood was drawn from a wing vein before treatment, twice during treatment (days 3 and 6) , and twice thereafter (days 10 and 18) . Liver biopsies were performed under general anesthe¬ sia before and on the last day of treatment. Tissue was processed for light microscopy. Sections for DHBV DNA determination were frozen immediately and stored at -70°C until required. Seroloqic Assays

Serum DNA polymerase activity was determined by measuring ³H- thymidine incorporation into purified Dane particles by the method of Feinberg, et al., Analyt. Biochem. 132:6-13, 1983. The in vitro effects of DDC as a nucleotide analogue on DHBV and HBV were assessed using the DNA polymerase reaction. A range of concentrations of DDC triphosphate were incubated with purified Dane particles for one hour at 37°C, and the DNA polymerase reaction was then performed. DHBV DNA was analyzed by molecular hybridization using a 3.0 kb, full-length DHBV DNA clone in CACYC184. The DHBV DNA insert was freed from plasmid A49 by digestion with EcoRl and agarose gel electrophoresis. The DHBV DNA was radiolabelled with ³²P using the random primer method of Feinberg, et al., ibid. , to a specific activity of 3 x 10⁷ to 1 x 10 cpm/μg.

DHBV DNA was detected in serum and liver tissue by slot blot analysis. For analysis of DHBV DNA in serum, lOμl of serum was denatured with 1 μl of 1 M NaOH for five minutes. The mixture was then neutralized by adding 90 μl of 1 M ammonium acetate. For analysis of DHBV DNA in liver biopsy specimens, approximately 100 mg of minced liver was homogenized in 10 ml of ice cold 50 mm Tris, pH 8.5, 10 mM EDTA and 1% SDS. The crude liver homogenate was digested with proteinase K (200 μg/mk) for two hours at 50°C. Total cellular DNA was prepared by two extractions with a mixture of phenol and chloroform (1:1) in Tris pH 7.5. DNA was precipitated with absolute ethanol and diluted to a con¬ centration of approximately 2 mg of DNA/ml in TE buffer.

One hundred microliters of the DNA sample prepared from serum or liver was spotted onto a nitrocellu¬ lose filter premoistened with 1 M ammonium acetate using a slot blot apparatus and vacuum manifold. The membrane was air dried and baked in a vacuum oven at 80°C for two hours and hybridized at 40°C with the DHBV DNA probe. The hybridized membranes were exposed to X-ray film for 5, 24, and 72 hours, and the resulting autoradiograms were scanned using Zenith Scanning Densitometer. The. amount of DHBV DNA was quantified by comparing the autoradiographic signals for each sample with those of known amounts of cloned DHBV DNA dotted on the same filter diluted in normal serum or normal duck liver homogenate. Liver tissue DHBV DNA was also analyzed by

Southern hybridization. Ten micrograms of total cellular DNA was subjected to horizontal slab gel electrophoresis in 1% agarose and transferred to nitrocellulose paper by the method of Southern, . Mol. Biol. 98.:503-517, 1975; as modified by Wahl, et al., Proc. Natl. Acad. Sci. USA 76: 3683-3687, 1979. Hybridization and autoradiography were carried out as described above.

PHARMACOKINETIC STUDY OF DDC LEVELS Serial serum levels of DDC were monitored in one duck after the initial dose of the drug was administered. Blood was drawn before an IV bolus of DDC and 10 minutes, 1, 2, 3, and 6 hours thereafter. DDC was measured in sera by high performance liquid chromatography. STATISTICAL ANALYSES Data were compared using Student's test, the Shapiro-Wilk test for normal distribution, and Spearman's rank correlation coefficient. Mean and standard deviations of serum DNA polymerase levels were calculated after logarithmic transformation of the data. Changes in serum and liver levels of these viral makers were expressed as percent inhibition of the pretreatment levels.

IN VITRO EFFECTS OF DDC TRIPHOSPHATE ON DNA POLYMERASE I_n vitro, DDC triphosphate had little effect on the DNA dependent DNA polymerase activity of either HBV or DHBV, as shown in Figure 1. There was no inhibition of either viral DNA polymerase activity at concentrates below 10 μM and less than 20% inhibition at .100 μM DDC. At this concentration, cellular DNA polymerase activity is also inhibited by DDC.

PHARMACOKINETIC STUDIES OF PLASMA DDC

LEVELS AFTER AN IV BOLUS Plasma levels of DDC after an IV bolus of 2.5 mg (11 mg/m²) were 46 μM at ten minutes and less that 1 μM at six hours (data not shown) . The estimated peak level of DDC was 60 mM and the estimate half-life was approximately thirty minutes.

IN VIVO EFFECTS OF DDC ON DUCKS CHRONICALLY INFECTED Antiviral therapy was tolerated well, and all ducks survived therapy and liver biopsy. No duck showed obvious evidence of drug toxicity.

Serum levels of DHBV DNA polymerase decreased in all nine ducks given DDC, but in none of the controls, as shown in Figure 2. The mean inhibition of DNA polymerase activity measured on the third day of treatment was 64%. The difference between the pretreatment and day 3 value was statistically significant (p< 0.01). The inhibition of DNA polymerase persisted after treatment was stopped, and four of nine ducks treated with DDC continued to show greater than 50% inhibition twelve days after stopping treatment (mean inhibition on day 18 = 55%) . Serum levels of DHBV DNA also decreased in all nine ducks during therapy, but in none of the controls, as shown in the Table. The mean percentage inhibition of DHBV DNA levels was 73% of day 3 of treatment (p<0.01) , as shown in Figure 3. The inhibition of serum levels of this viral marker persisted for at least twelve days after stopping DDC therapy.

TABLE Changes in Serum DHBV DNA Levels in Peking Ducks Treated with 2' 3 '-Dideoxycytidine fPDC) or Adenine Arabinoside Monophosphate (Ara-AMP)

Duck Hepatitis B Virus DNA fpg/10 ul)

Time DDC Ara-AMP Control

⁼ (9) (2) (7)

Pre 13.1 ± 1.3 4.3 and 9. Υ 3.0 ± 1.5

Day 3 .6 ± 1.6* 0.6 and 8.6 3.9 ± 1.6

Day 6 4.6 ± 1.4 1.6 and 2.9 3.0 ± 1.6

Day 10 8.1 ± 1.3 3.9 and 1.7 4.4 ± 1.4

Day 18 8.3 ± 1.7 1.0 and 5.8 2.9 ± 1.5

Data expressed as geometric mean (± relative standard error) .

* p<.01 compared to pre values by Student's paired t test.

Treatment of two ducks with Ara-AMP yielded results similar to those reported by others, cf. Hirota, et al., op_. cit. DNA polymerase and DHBV DNA levels decreased by 71% and 100% during therapy, as shown in the Table, but levels of these viral markers rapidly rose to greater than pretreatment values within four days of stopping the intramuscular injections.

Pretreatment DNA polymerase levels correlated with DHBV DNA levels in ducks treated with DDC (p = 0.01) . In addition, successive changes in DNA polymerase levels correlated with successive changes in DNA polymerase levels on days 3, 6, and 10 of treatment.

Results of measuring DHBV DNA b slot blot analysis of total cellular DNA extracted from liver bio- psies before and on the last day of treatment with DDC are shown in Figure 4. Three ducks showed a marked inhibition of DHBV DNA after treatment with DDC (average inhibition, 96%), three showed mild inhibition (mean, 7.7%), and two ducks demonstrated a 60% increase. Southern blot analysis of liver DHBV from before and on the last day of treatment showed that the decrease in total DHBV DNA was attributable to a global decrease in DHBV DNA replicative intermediates, as shown in Figure 5. In Figure 5, on the left are slot blot analyses of total DHBV DNA. On the right are Southern blot analyses showing the molecular weight of cloned DHBV (approximately 3.0 kilobases [kb]) and the molecular weights of the replicative intermediates of DHBV DNA found in liver before (lanes 1 and 2) and after treatment (lanes 3 and 4) . DNA in lanes 2 and 4 were digested with EcoRl.

A variety of histologic lesions were observed by light microscopy in infected ducks. These included macro- vesicular steatosis and chronic portal infiltrates. Among DDC treated ducks there were no correlations noted between response to treatment and changes in histologic lesions.

DDC, or 2', 3,'- dideoxycytidine, comprises a pyrimidine nucleoside with the ribose moiety of the mole¬ cule in the 2', 3'-dideoxy configuration, as illustrated below:

The DDC may be in the form of carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain alkyl, alkoxyalkyl (e.g. ,methoxymethyl) , aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethy1) , aryl (e.g., phenyl optionally substituted by halogen, C,_₄ alkyl or C,_ alkoxy) ; sulfonate esters such as alkyl- or aralkylsulfonyl (e.g., methanesulfonyl) ; and mono-, di-, and triphosphate esters. The compounds as described above also include pharmaceutically acceptable salts thereof. Unless other¬ wise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters preferably comprises a phenyl group, including a substi- tuted phenyl group.

Examples of pharmaceutically acceptable salts and pharmaceutically acceptable derivatives of the compounds which can be use in treating hepatitis B according to the present invention include base salts such as those derived from a base such as alkali metal (sodium, lithium, potas¬ sium) , alkaline earth metal (magnesium) salts, ammonium and NX₄ where X is C,_₄ alkyl. Physiologically acceptable salts containing a hydrogen atom or any amino group include salts of organic carboxylic acids such as acetic, lactic, tar- taric, maleic, isothionic, lactobionic, and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesul- fonic, benzenesulfonic, and p-toluenesulfonic acid, and inorganic acids such as hydrochloric, sulfuric, phosphoric, and sulfamic acids. Physiologically acceptable salts of a compound containing any hydroxy group include the anion of said compound in combination with a suitable cation such as Na⁺, NHY₄ ⁺, and HX₄ ⁺ (wherein X is C,_₄ alkyl and X is halo¬ gen) .

Specific examples of pharmaceutically acceptable derivatives of the compound of formula 1 that may be used in accordance with the present invention include the monosodium salt and the following 5' esters: monophos¬ phate, disodium monophosphate, diphosphate, triphosphate, acetate, 3-methylbutyrate, octanoate, palmitate, 3-chloro benzoate, 4-methylbenzoate, hydrogen succinate, pivalate, and methylate.

Also included within the scope of this invention are the pharmaceutically acceptable salts, esters, salts of such esters, nitrile oxides, or any other covalently linked or non-linked compound which, upon administration to the recipient, is capable of providing, either directly or indirectly, a nucleoside analogue as described above, or an antivirally active metabolite or residue thereof. All of these compounds are active and relatively nontoxic at con¬ centrations of sufficient potency for effective inhibition of viral infectivity and replication.

It is possible for the nucleoside of the present invention to be administered alone in solution. However, the active ingredient may be used or administered in a pharmaceutical formulation. These formulations comprise the nucleoside or derivative thereof together with one or more pharmaceutically acceptable carriers and/or other therapeutic agents. As included within the scope of the present invention, "acceptable" is defined as being com¬ patible with other ingredients of the formulation and not injurious to the patient or host cell.

The administration of DDC to treat hepatitis B can be accomplished by a variety of means of administra¬ tion. Whatever administrative method is chosen should result in circulating levels o the DDC within a range of about 0.01 μM to about 2.0 μM. A range of approximately 0.05 to about 0.5 g/kg administered ever four hours is considered to be a virustatic range in humans. In order to achieve this, the preliminary dosage range for oral admin¬ istration may be broader, being, for example, 0.001-0.50 mg/kg administered every four hours. It is recognized that dosage modifications may be required in individual patients to ameliorate or inhibit toxic side effects.

The pharmaceutical formulations according to the present invention may conveniently be administered in unit dosage form and may be prepared by any methods known in the pharmaceutical art. Determination of the effective amounts to be included in the dosage forms within the skill of the art.

The pharmaceutical compositions according to the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the DDC into preparations which can be used pharmaceutically. Preferably the prepa¬ rations, particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for admin¬ istration by injection or orally, contain from about 0.1 to 99 percent, and preferably from about 25-85 percent, by weight, of DDC, together with the excipient.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optically grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste using, for example, maize starch, wheat starch, rice starch, potato starch, and the like; gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcel- lulose, and/or polyvinyl pyrrolidone. If desired, disin¬ tegrating agents may be added such as the above-mentioned starches and carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Auxiliaries are, for example, flow- regulating agents and lubricants, such as silica, talc, stearic acid or salts thereof such as magnesium or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentra¬ ted sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethy¬ lene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetyl-cellulose phthalate or hydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.

Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plas- ticizer such as glycerol or sorbitol. The push-fit cap¬ sules can contain the active compounds in the form of granules which may be mixed with fillers as such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of combinations of the active ingredient with a suppository base. Suitable suppository bases include natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocar¬ bons.

Suitable formulations for parenteral administra¬ tion include aqueous solutions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lypophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

In the present invention, the hepatitis B may be treated by directly delivering the triphosphate derivative to the patient. It is well known that "unshielded" tri- phosphates cannot be used as drugs because triphosphate compounds do not penetrate cell membranes. Therefore, the triphosphate derivatives of this invention may be delivered by means of liposomes, small particles (about 25 μM to about 1 μM in diameter) which can serve as an intracellular transport system to deliver normally non-absorbable drugs across the cell membrane. Such use of liposomes for drug delivery is well known in the art, and is based upon the ability of a phospholipid to form bilayers spontaneously in aqueous environments.

One methods of forming the liposomes is by agitating phospholipids in aqueous suspensions at high frequencies. This results in the formation of closed vesicles characteristic of liposomes. Once inside the cells, the triphosphate derivatives act to eliminate the replication of the hepatitis B virus. Since the tri¬ phosphate has been shown to be active inside the cells, and to be the active. form therein, the liposome is clearly a method of choice for delivery of these drugs. Formulations suitable for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing, in addi- tion to the active ingredient, such carriers as are known in the art to be appropriate.

The formulations according to the present inven¬ tion may be in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a lyophi- lized condition requiring only the addition of the sterile liquid carrier for injections immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

In treating hepatitis B according to the present invention, the medication is generally administered two to six times a day. In order to improve oral bioavailability, it is often preferable to add a common buffer such as sodium acetate to a solution containing 2' , 3 '-dideoxycy¬ tidine according to the present invention.

The foregoing description of the specific embodi¬ ments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.

Claims

WHAT IS CLAIMED IS:

1. A method for treating hepatitis B comprising administering to a patient infected with hepatitis B an effective amount of 2' 3'-dideoxycytidine. 2. The method according to claim 1 wherein the

2' , 3'-dideoxycytidine is in the form of a triphosphate salt.

3. The method according to claim 1 wherein the 2* , 3'-dideoxycytidine is in a pharmaceutically acceptable carrier.

4. The method according to claim 3 wherein the carrier is normal saline.

5. The method according to claim 3 wherein carrier is a liposome.

6. The method according to claim 1 wherein the

2' , 3 '-dideoxycytidine is administered in a dosage range of from about 0.03 to about 0.5 mg/kg administered from four to twelve times daily.

7. The method according to claim 1 wherein the 2¹, 3'-dideoxycytidine is administered orally.

8. The method according to claim 1 wherein the 2', 3•-dideoxycytidine is administered intravenously.

9. The method according to claim 1 wherein the 2', 3 '-dideoxycytidine is administered rectally.

10. The method according to claim 1 wherein the

2' , 3'-dideoxycytidine is in the form of a lyophilized powder and is administered intranasally.

11. The method of claim 1 wherein the 2' , 3'- dideoxycytidine is administered intramuscularly.

12. A composition comprising 2', 3'-dideoxycy¬ tidine in a pharmaceutically acceptable carrier.

13. The composition of claim 12, wherein the 2' , 3'-dideoxycytidine is in the form of a triphosphate salt.

14. The use of 2' , 3'-dideoxycytidine for the treatment of hepatitis B infection.

15. The use according to claim 14 wherein the 2' , 3'-dideoxycytidine is in the form of a triphosphate salt.

16. The use according to claim 14 wherein the 2', 3'-dideoxycytidine is in a pharmaceutically acceptable carrier.

17. The use according to claim 14 wherein the carrier is normal saline.

18. The use according to claim 14 wherein carrier is a liposome.

19. The use according to claim 14 wherein the 2' , 3'-dideoxycytidine is administered in a dosage range of from about 0.03 to about 0.5 mg/kg administered from four to twelve times daily.

20. The use according to claim 14 wherein the 2', 3'-dideoxycytidine is administered orally.

21. The use according to claim 14 wherein the 2', 3 '-dideoxycytidine is administered intravenously.

22. The use according to claim 14 wherein the 2', 3'-dideoxycytidine is administered rectally.

23. The use according to claim 14 wherein the 2', 3 '-dideoxycytidine is in the form of a lyophilized powder and is administered intranasally.

24. The use of claim 14 wherein the 2', 3'- dideoxycytidine is administered intramuscularly.