CA2055433A1 - Method of treatment of hepatitis - Google Patents

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

WO90t1407~PCT/US90/02685 ~'METHOD OF TREATMENT OF HEPATITIS 2 0 ~ ~ ~ 3 FIELD OF THE INVENTION
The present invention relates to a method for treating hepatitis B.

Chronic infection which the hepatitis B virus (HBV) affects approximately 5~ of the world's population.
Chronic carriers o~ hepatitis B are at a increased risk of morbidity and mortality due to chronic liver disease, 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 ~-interferon has shown yreat promise in a subset of patients treated for prolonged perlod of time, the response rates overall have, unfortunately, been disap-po~ntlngly low.
rrhe human hepatiki~ B virus ia a member o~ a ~amily o~ viru~ known a~ hepadnavlru~e~. O~h~r virus~s ln this ~amily ar~ thc woodchuck hepat~ti~ vlru~, the ground squirrel h~patitl~ v.lru~, ~nd the duc}; h~pat1ti~
virus. 'rhese animal viruses hav@ been invaluable models ~or characterization of hepadnaviruses and delineation o~
their unu~ual replicative cycle. These viruses replic~te asymmetrically through an RNA template which re~uires 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 30 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 o~ t~e present invention -to overcome the above-mentioned deficiencies ln the prlor ~rt.

WO90~14n79 PCT/US90/02685 ~5~3~ 2 - ~
It is another object of the present invention to provide methods for treating hepatitis B.
It i5 further object of the present invention to provide compositions for tre ting 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, inhib~ts 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 oP viral polymerases, in particular, reverse transcriptases. DDC is a nucleoside analogue, and i~ appears to prev~nt the ~ormation oE normal phosphodiester linkages once ~t become~ .incorpora-ted .LntQ
A growing DN~ chain 'rhl~ proco.3 leads to "ch~ tenmin~
tion." DDC ha~ a high aP~lnity ~or rQverse transcriptase, and, there~ore, may inhibit replication of hepatitis B
virus by preventing reverse transcription from the pregeno-mic RNA template. This inter~erence 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 l shows the ln 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 in~ected with duck hepatitis B virus who received ~DC or no treatment.
Figure 3 shows changes in mean serum duck hepatitis B virus DNA levels among Peking ducks chronically - , : . , : , : : .~ - :: . : , , . : , . .
,.: :................ . . : . : ' ' .

Wogo/14079 PCT/US90/0268;
~ 2~a~33 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.

2', 3' dideoxycytidine can be used for treating hepatitis B in patients 90 infected. The DDC i5 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.
~n ~ DDC triphosphate had little e~ect on DNA polymerase activit~ o~ clther duck hapatlti~ B vlru~ or human hepatlti~ ~ viru~. ~rev.lous ras~arch~r~ hav~ u~d ~he ~n ~i~ a~say ~to ~a~s antlvlral ~atlvlt~ in hepatitis B, c~. Nord@n~elt, et al., ~ ~h- ~l~iQ}~
Scand. Sect. _ 87:75-76, 1979; and He~s, et al., Antimic.
Aaents Chemo. lg~ 50, 19~ lowever, it has now been di~covered that khis assessment may be unreliable as ~
means of screening antiviral agents. The ~NA 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 c~ntrast, 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 de~rees of inhibition of both DNA polymerase activity and duck hepatitis B DNA were similar (67~ and 69~, respectively and were comparab~e to the degrees o~ inhib.ition of ~he~e ~ar~ers reparted in s~udies of other an~iviral agents use~ in trea~ment of chronic hepatitis B. The antiviral effect was only part-ial, however, in that no duck became completely negative ~090/l4079 PCT/VS90/02685 ?~5~13-~ 4 _ ~
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 si~ilar 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 ~ollowing withdrawal of theseagents, serum levels o~ duck hepatitis B virus often rebound to above pretreatment levels (Hirota, et al., Hepatology 7:24-28, }987).
The effect of 2', 3'-dideoxycytidine was assessed in 16 Pekiny ducks chronically in~ected with the duck hepatitis ~ virus ~DHBV). Nine ducks were given DDC at the rate o~ 11 mg/m2 intravqnou~ly evary ~ix hours, and ~v~n ducks received no tre~tmcnt. Scrum D~IBV ~NA ~nd DN~
polymaras~ act.Lvity d~crea~d ln Qv~ry duek tr~at~d wl~h DDC. The m~an lnhibition oE DNA polym~r~8Q ~nd D~IBV DNA on the third day o~ treatment me~sured 6~ (p~0.01) and 73~
(p~0.01), respec~ively. The inhibition of DNA polymerase persisted a~ter 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 ~1 of serum pooled ~rom DHBV carrier ducks. The inoculum contain~d approx.i-mately 1.3 x 10~ vlrions per ml. serum as estimated by slot blot hybridization.

WO90/14079 2 ~ 5 a ~ 3 3 .

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 5iX days. Two ducks were given adenine arabinoside monopho~phate ~Ara-AMP; vidara-bine monophosphate: Parke, Davis, Detroit, Michigan) at arate o~ 400 and 1000 my/mZ intramuscularly twice daily for six days. Seven ducks received no treatment. Blood was drawn from a wing vein be~ore treatment, twice during treatment (days 3 and 6), and twice thereafter (days 10 and 18). Liver biopsies were per~ormed under general anesthe-sia before and on the last day o~ treatment. Tis~ue was processed for light microscopy. Sections ~or D~lBV DNA
determination w~re ~rozen .immcdiat~ly and ~torcd at -70C
until re~u:ired.
~ c ~ v~
Serum DNA polymerase activity was determined by measuring 3~- thymidine incorporation into puri~ied Dane particles by the method of Feinberg, et al., ~nalYt~
Biochem. 132:6-13, 1983. The ln ~ e~ects o~ 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 37C, and the DNA polymerase reaction was then performed~
DHBV DNA was analyzed by molecular hybridization ~sing 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., ~kl~-, to a speci~ic activity of 3 x la~
to 1 x 10 cpm/~g.
DHBV DNA was detected in serum ~nd liver tissue by slot blot analysis. For analysis of DHBV DNA in serum, W~0/14079 PCT/US90/02fi8;
2 ~ 5 3 ~ - 6 - ~
10~1 of serum was denatured with 1 ~l of 1 M NaOH for five minutes. The mixture was then neutralized by adding 90 ~1 of 1 M ammonium acetate. For analysis o~ 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 ~g/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 ab~olute ethanol and diluted to a con-centration of ~pproximately 2 mg o~ 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 membr~ne was air dried and baked in a v~cuum oven at 80'C ~or two hours and hybr~dlzed at ~0C with the D~IBV ~NA prob~. ~h~
hybridizcd m~mbran~ w~re @xpo~ed to X-ray ~llm ~or ~
and 72 hour~, and the r~ultln~ a~ltoracllo~relm~ w~r~ ~ann~d using Zcnith 9c~nnincJ ~en~l~om~t~r. Th@ amount Or ~ V
DNA was quanti~ied by camparing the autoradiographic signals for each sample with those o~ 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 hybridiization. ~en 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, J. 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.
PHARMACOKINE~TIC STUDY OF DDC LEVELS
Serial serum levels of DDC were monitored in one duck after the initial dose o~ the drug was administered.
Blood was drawn before an IV bolus o~ ~C an~ ~0 minute~, 1, 2, 3, and 6 hours therea~ter. DDC was measured in sera by high performance liquid chromatography.
.

Woso/14079 PCT/US90/0268S

20~3~
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 T~IP}IOSPHA~E ON DNA POLYMER~SE
~ t~o, DDC triphosphate had little e~fect 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 ~M and less than 20% inhibition at 100 ~M DDC. At this concentration, cellular DN~ polymerase activity is also inhiblted by DDC.

Pl~sma lev~ls o~ DDC a~tor an XV bolu~ o-~ 2.5 my (11 mg/m2) were ~6 ~M ~t tcn mi~utos ~nd loss that 1 ~M at six hours (data not shown). The estimated peak level of DDC was 60 mM and the estimate half~ e was approximately thirty minutes.
IN VIVO EFFECTS OF DDC ON DUCKS CH~ONICALLY ~NFECTED
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 o~ nine ducks treated with DDC continued to show greater than 50~ inhibi~ion ~welv~ day~ a~ter stopping ~reatment (mean inhibition on day 18 - 55~).

WO~0/l4079 PCT/US90/0268~
20~5~33 8 - ~ ~
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 d~y 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.

Tre~tes~ with 2 ' 3'-D~deoKy~tidine tDDC)_or Adenine Arabinoside MonoPhosphate (Ara-AMPl Duck Hepatitis B Virus DNA (pa/10 ul~
Time DDC Ara-AMP Control (9) (2) ~7) Pre 13.1 t 1.3~.3 and g.7 3.0 ~ .L.5 Day 3 .6 ~ 1.6*0.6 ~nd 8.6 3.9 ~ ~.6 Ds~y 6 ~ l.fi and ~.~ 3.0 Day 10 3.1 L 1.33.9 ~d 1.7 ~.~ t 1.~
Day 1~ 8.3 ~ 1.71.0 and 5.~ 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 wlth 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 chan~es in DNA polymeras~s levels correslated with succesaive ahange~ in DN~ polymeras~ l~v~15 on days 3, 6, and 10 o~ treatmeSnt.
Results o~ measuring DHBV DNA by slot blot analysis o~ total cellular DN~ extracted ~rom liver bio-~ 9 ~ 205~3 psies before and on the last day of treatment with DDC areshown in Figure 4. Three ducks showed a marked inhibition of DHBV DNA after treatment with DDC taverage 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 ~igure 5, on the lePt are slot blot analyses of total D~BV 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 o~ DHBV DNA found in liver before (lanes 1 and 2) and a~ter treatment ~lanes 3 anc~ ~). DNA
in lanes 2 and ~ were digestod with EcoRl.
~ variety o~ hi~toloyLc lo~ions w~re oblervod by light micro~copy in :Ln~ccted duck~. 'rhose ine~udQd m~cro~
vesicul~r stca~o~is and chron.Lc portal ln~llkrat~. Amony DDC treated ducks ~h@re wer@ no Gorrela~lons noted betwQen response to treatment and chanyes in histologic lesions.
DDC, or 2', 3,'- dideoxycytidine, comprises a pyrimidine nuclcoside with the ribo~e moiety o~ the mole~
cule in the 2', 3'-dideoxy configuration, as illustrated below:

~ N

OC~ , H~ H2 1 .~
~ ' ` '.

, W~90/14~79 PCT/US90/02685 20~ 3~33 - lO - ~
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., phenoxymethyl), aryl (e.g., phenyl optionally substituted by halogen, C1 4 alkyl or Cl,4 alkoxy);
sulfonate esters such as alkyl- or aralkylsul~onyl (e.g., methanesulfo~yl); and mono-, di~, and triphosphate esters.
The compounds as described ~bove also include pharmaceutically acceptable salts thereo~. Unless other-wise specified, any alkyl moiety present advantageously contains ~rom 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters pre~erably comprises a phenyl group, including a substi-tuted phenyl yroup.
Examples o~ pharmaceutically acceptablc ~a:lts andpharmaceutically ac~ptable derivatives o~ th~ aompound~
which can b@ u~e :In tr~atincJ hepatLtis B nocord.lnFJ to the pre~ent invention .Lnclude b~e s~lt~ sueh a~ tho~@ d@rlved ~rom a base such as alkali metal ~soclium, l~thium, potas-sium), alkaline earth metal (magnesium) salts, ammonium and NXb where X is C14 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 C14 alkyl and X is halo-gen).
Speci~ic examples of pharmaceutically acceptable derivatives o~ the compound of ~ormula ~ that ~Ay be u~ed in accordance wlth the present invention include the monosodium salt and the following 5' esters: monophos-phate, disodium monophosphate, diphosphate, triphosphate, WO~o/14079 PCT/US90/02685 - 2~
acetate, 3-methylbutyratej octanoate, palmitate, 3-chloro benzoate, 4-methylbenzoate, hydrogen succinate, pivalate, and methylate.
Also included within the scope of this invention 5 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 o~ providing, either directly or indirectly, a nucleoside analogue as described above, or an 10 antivirally actlve metabolite or residue thereof. All o~
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 15 invention to b~ administered alone in solution. ~lowever, the ackive ingredient may be used or administer~d in a ?
pharmaceutlcal ~ormulation. These ~ormulation~ com-pri~e the nucleos:Lde or derlvi3tlv~ thereo~ togethcr w:lkh on~ or more pharmaceukically acceptable c:arri~r~ and/or oth~r 20 ther~peutic ag~nts. A~. lnclud~d wlthln th@ ~cop~ o~ the present invention, "acceptable" is defined as being com-patible with other ingredients of the formulation and not injurlous to the patient or host cell.
The administration o~ DDC to treat hepatitis B
25 can be accomplished by a variety of means of administra-tion. Whatever administrative method is chosen should result in circulating levels of the DDC within a range of i about 0.01 ~M to about 2.0 ~M. A range of approximately 0.05 to aboùt 0.5 mg/kg administered ever four hours is 30 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 35 to ameliorate or inhibit toxic side effects.
The pharmaceutical ~ormulations according to the present invention may conveniently be administered in unit dosage form and may be prepared by any methods known in the woso/l4079 PCT/US90/02685 ~ 12 -pharmaceutical art. Determination of the effective amounts to be included in the dosage forms within the skill of the art.
ThP 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 u~ed pharmaceutically. Preferably the prepa-rations, par~icularly tho~e 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 Pro-tn ~bout O.l to 99 percent, and pre~erably ~rom about 25-85 porcent, by weight, o~ DDC, togethor with the ~2xcipienk.
The pharmaceutlcal preparations oP the p~ent lnvontion arc manu~actured ;Ln a manner whleh 1.~ lt~el~
known, Por example, by -me~.~ns o~ aanvcn~lon~l mixing, granulatiny, dragee-m~kin~J, dis~olving, or lyophilizing processes. Thus, pharmaceutical preparations ~or oral use can be obtained by combining the active compounds with solid excipients, optically grinding a resulting ~ixture, 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, g~m tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcel-lulose, and/or polyvinyl pyrrolidone. I~ de~i~ed, 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 thereo~ such a5 :` : .. ; . . . . .
! ~

WO90/14079 PCr/US90/02685 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, lacyuer solutions, and suitable ory~nic solvents ur solvent mixtures. In order to produce coakinys resistant to gastrlc juices, solutions of suitable cellulos~ 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, ~or identl~ication or in order to charact~rize di~Perent combinations o~ act~ve compound doses.
Other pharm~c~u~.ical preparations wh:lch c~n b~
used orally includ~ pueh~ c~psu.L~ macle o~ cJ~lakln/ a~
well as 50~t, Bealed capsul~s m~c10 o~ ~J~latin and a pla~-ticizer such as glycerol or sorbitol. The pu~h-~i~ cap-sules can contain the active compounds in the form of granules which may be mixed with ~illers 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 triglyderides, paraffin hydrocarbons, polyethylene glycols or higher alkanols. In aqdition, it is also possible to u~e gelatin rectal capsul~ which consist o~ a combination o~ the active compounds with a base. Possible base materials include, for example, liquid ~090/14079 PCT/US90tO2685 ~ 3 - 14 -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 10 inarraa~e the vlscos.ity o~ the su~pension 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 i5 well known that "unshielded" tri-phosphates c~nnot be used as druys b~cau~e triphosphate compound~ do not pen~trat~ cell mcmbranes. ~h~r~;~or~, th@
triphosphate der.tvat.ives Or th.ls inv~ntlon ma~ ba d~llvar~d by m~an~ o~ lLpor,orn~s, ~mall partla.les ~abol1~t ~ M ko ~0 abou~ ~ ~M in diam~t~r) which can ~v~ as an ;lntrac@ll-1lar transport system to d@l$ver normAlly non-absorb~ble drugs across the cell membrane. Such use oP liposomes for drug delivery is well known in the art, and is based upon the ability o~ a phospholipid to ~orm 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 h~patitis 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 ~orm o~ pessaries, kampons, creams, ~ls, pastes, foams, or sp~ay ~ormulations containing, in addi~

~ .

V~90/l407~ PCT/US90/0268~
~ 2 ~5~ 3 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 khe kind prèviously desGribed.
~ n treatlng hepatit~ 5 B according to the presen-t 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 accordlny to the pre ent ~nvention.
The PorQgoing desarlption o~ the spQcl~ mbodi-ments will so ~ully reveal the g~ner~l naturQ o~ the invention that oth@rs c~n, by applyin~J aurr@nt knowlcdgQ~
readily modi~y And/or aélapt eOr v~rlous appllcal:larls ~uc:h specific embodiments without departiny ~rom the generic concept, and therefore such adaptations and modifications are intended to be comprehended wikhin 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 (24)

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/kc administered from four to twelve times daily.

7. The method according to claim 1 wherein the 2', 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.