CA2102120A1 - Use of a 1,3-oxathlolan derivative for the treatment of hepatitis b infection - Google Patents

Abstract

The use of a 1,3-oxathiolane nucleoside analogue and pharmaceutically acceptable derivatives thereof for the treatment of hepatitis B virus infections is disclosed. Pharmaceutical formulations are also provided.

Description

W O 9~/lg246 2 l ~ 212 ~ PCT/GB92/00803 U5E OF A 1,3--O~A~O~ND~RI~ATIUE FOR T9E. TREATMENT
OF HEPATTTIS B INFECTIoN

The present inventio~ relates to the use of a 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative and physiologically functional derivatives thereof for the trea~ment of hepatitis B viral infections.

Hepatitis B virus (HBV) is a viral pathogen of major worldwide imp~rtance. HBV is most common i~ Asian Countries, and prevalent in sub-Saharan Africa. The ~irus is etiologically associated with primary hepatocellular carcinoma and is thou~ht to cause 804 of the world's livcr cancer. In the United S~ates more than ten thousand people are hospitalized for HBV illness each year, an average of 250 die with fulminant disease. The United States currently contains an estimated pool of 500,000 - 1 million infectious carrisrs. Chronic active hepatitis will develop in over 25~ of carriers and of~en progresses to cirrhosis. It is est-;mated that 5000 people die from HBV-related cirrhosis each year in the U.S.A. and ~hat perhaps 1000 die from HBV-related liver cancer. Even when a universal HBV vaccine is in place, the need ~.Eor effective anti-HBV compounds will continue. The lar~e reservoir of perslstenely infected carriers, estimated at 220 million worldwide, will receive no benefit from vaccination and will continue at high risk for HBV-induced liver disease. This carrier population serves as the source of infection ~f susceptible individuals perpetuating the instance of disease partieularly in endemic areas or high risk groups such as i.v. drug abusers and homosexuals. Thus, there is a great need for effective antiviral a~ents, both to control the chronic infection a~d reduce progression to hepatoc~llular carcinoma.
'~
Clinical effects of infection with ffBV range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication .

of the virus is usually controlled by the immune response, with a course of recovery lasting weeks or months in humans, but infection ~ay be more severe leading to persistent chronic liver disease as W O ~2/19246 PCT/~B92/008~

2:102120 outlined above. In "Viral Infections of Humans" ~second edition, Ed.
Evans, A.S. (1982) Plenum Publishing Corporation, New York), Chapter 12 describes the etiology of viral hepatitis in~ections.

European Patent Specification 0 382 526 discloses certain 1,3-oxathio-lane nucleoside analogues which are effective in inhibiting the replication of human immunodeficiency virus (HIV~.

Wa have now surprlsingly found that a 1-(2-(hydroxymeehyl)-1,3-oxathi-olan-5-yl)-cytosine derivative of formula I
.. 2 ~ R .

N ~.
(I~

s ` .

(wherein R is a hydrogen a~om or a Cl 3 alkyl group~

and physiologically functional deri~ati~es thereof have potent acti~ity against HBV.
:
A preferred compound of formula (I) is that in which R is a hydrogen atom, namely: 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine.

It should be noted that the compound of formula (I) contains two chiral centers and therefore exists in the for~ of two palrs of optical isomers (i.e. enantiomers) and mixtures thereof includîng racemic mixtures. Thus the compound of formula (I) may be either a cis- or a ~rans- isomer or mixtures thereof. Each cis- and trans-isomer can exist as one of two enantiomers or mixtures thereof including racemic mixtures. All such isomers and mixtures thereof including racemic mixtures are within the scope of the in~ention. The SUBSTiTUTE SHEET

W O 92/19246 2 1~ ~ ~ 2 ~ PCT/GB92/00803 cls- isomers of the compound of formula (I) are preferred. The ::
invention includes the optical and geometric isomers and all -:
tautomeric forms of the compound of f~rmula (I); of the cis isomers, the (-) isomer, ie. (-)-cis-1-(2-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine, is especially preferred.

According to one feature of the present i~v4ntîon we provide a compound of formula (I) or a physiologically unctional derivative -~
thereof for use in the treatment or prophylaxis of a hepatitis B virus :~
infection. Accordin~ to a further feature of the present in~ention we provide the use of compound of formula (I) or a physiologically funct~onal derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of a hepa~itis B virus infection. ~:
In a further aspect of the presen~ in~ention there is included a method for the treatment or prophylaxis of a hepatitis B virus infection in a host, for example, a mammal such as a human which comprises treating the host with a therapeutically effective amount of a compound of formula ~I~ or a physiologically functional derivative thereof.
~,.

By "physiologically functional derivative" i5 meant a pharmaceutically acceptable salt, ester or salt of an ester of a compound of formula ~I) or any other compo~nd which upon administration t~ the recipient, is capable of providing (directly or indirectly) the said compound o~
formula (I) or an active metabolite Qr residue thereo.

Preferred esters in accordance with the invention include carboxyIic :~
acid esters in which the non-car~onyl moiety of the carboxylic acid portion of the~ester grouping is selected from straight or bxanehed chain alkyl e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), arylalkyl (e.g.. benzyl), aryloxyalkyl ~e.g.
phenoxymethyl), and aryl (e.g. phenyl); sulfonate esters such as alkyl- or arylalkylsulfonyl (e.g. methanesulfonyl); amino acid es~ers (e.g. L-valyl or L-isoleucyl); dicarboxylic acid esters (e.g-hemisuccinate); and 5'- mono- di- or tri-phosphate esters. The W O 92/19246 PCT/GB92tO08~
~11)2120 phosphate esters may be further esterified by, for example, a C1 20 alcohol or reacti~e der;vative thereof, or by a 2,3-di(C6 24)acyl glycerol.

Any alkyl moiety present in such esters advanta~eously contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group optionally s~bstituted e.g. by halo~en, Cl 4 alkyl, C1 4 alkoxy or nitro.

In the abo~e-described esters, the cytosine amino ~roup may be present in the form of an amide, e.g. -NHCOR whexe R is Cl 6 alkyl or aryl (e.~. phenyl optionally substituted by halogen, Cl 4 alkyl, Cl 4 alkoxy, nitro or hydroxyl~.

Examples of pharmaceutically acceptable salts according to the invention includ~ base salts, e.g. derived from an approp7iate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesiu~) salts, ammonium and NX4 (wherein X is Cl 4 alkyl).
Pharmac2utically acceptabIe acld addition salts inc}ude salts of organic carboxylic acids such as acetic, lactic, ~artaric, malic, isethionic, lactobionic and succinic aeids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic aoids and inorganio acids such as hydrochlGric, sulfuric, phosphoric and sulfamic acids.
'~

The amount of the compound of formula (I) (hereinafter also referred to as the "actlve ingredient") or physiologically functional deri~ative thereof which is requîred in a medication to aehieve the desired effect will depend on a number of factors, in particular the specific application, the nature of the particular compound used, the mode of administration and the condition of the patient. In general a suitable dose will be in the range of 3.0 to 120 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg per kilogram body weight per day and most preferably in the range 15 ~1~2~20 to 60 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses adm}nistered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, c~ntaining lO to 1500 mg, preferably 20 to lO00 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form.

Ideally, the active ingredient shGuld be ad~inistered to achieve peak plasma concentrations of the active ingredient of from about l to about 75~M, preferably about 2 to SO~M, most preferably about 3 to about 30~M. This may be achieved, for example 7 by the intra~enous injection of a O.l to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about l to about lO0 m~/kg of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about O.Ol to about 5.0 mg/kg/h~ur or by intermittent infusions containing about 0.4 to about 15 mg/kg of the actîve ingredient.

In the manufactl~re of a medicament accoxding to the i~vention, hereinafter referred to as a "formulation", the compound of formula (I) or a physiologically functional derivative thereof herein as "active ingredient', is typically admixed with, la~ , one or more pharmaceutically acceptable carriers or excipient~ and optionally other therapeutic agents.

The formulations include those suitable for oral, rectal, nasal~
topical (includin~ transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the s~ep of bringing into association the active ingredient with the carrier which c~nstitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely W 0 92~19246 ` - 6 - P ~ /GB92/OOX~:~

Zlf~2120 divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administratîon may be presented as discrete units such as capsules; cachets or tablets each containing a predetermined amount of the aceive ingredient; as a powder or granules; as a solution or suspension in an aqueous or no~-aqueous liquid; or as an oil-in-water liquid emulsion or a water-ln-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free~
flowing form such as a powder or granules, optionally mixed with a binder ~e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-lin~ed povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent. Molded~ ~
tablets may be made by molding in a suitahle machine a mixture of ~he ~-powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example hydroxypropyl~ethyl cellulose in varying proportions to pr~ovide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gu~ other than the stomach.

Formulations suitable for oral use as described above may also include buffering agents designed to neutralize stomach acidity. Such buffers may be chosen from a ~ariety of organic or inorganic agents such as weak acids or bases admixed with their conjugated salts.

Formulations suitable for topical ad~inistration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually W O 92/tg246 21 a ~ 1 ~ 0 P ~ /GB92/00803 sucrose and a~acia or tragacanth; pastilles comprisi~g the active ingredien~ in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
'~
Formulations for rectal administration may be presented as a suppository with a suitable base comprisin~ for-example cocoa butter or a salicyl~e.

Formulations suitable for vaginal administration may be presented as pessari~s, tampons, creams, gels pastes, foams or spray formulations . containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations sui~able for parenteral administration include aqueous and non-aqueous isotonic sterile injections solu~ions which may contain nti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bl.ood of the intended recipient; and aqu ous and non-~queous sterile suspensions which may include suspending agents ; and thickening agents, as liposomes or other micropar~iculate systems which are clesigned to targe~ the comp~unds to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, -ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the steri1e liquid carrier~
for example water 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.
,.
Preferred unit dosa~e formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

W O 92/19246 PCT/GB92tOO~

21(32~2~
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in ~uestion, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.

The compound of formula (I) may be prepared for ex~mple in accordance with the processes described in European Patent Specification O 382 526 or by processes analogous thereto.

Thus, ~he compound of formula I may be prepared for example by:

a~ reacting an optionally protacted 5-R-cytosine compound (wherein R
is as defined above) with a l,3-oxathiolane of $ormula IIA

~o L
\ J ~IIA~
s wherein Rl is hydrogen or a hydroxy protecting group and L is a leaving group; or b) reacting a compound of formula IIB

~ R

N
1 (IIB) SUBSTITUTE SHEET

W O 92/19246 2 ~ 0 2 ~ 2 ~ PCT/GB92/00803 (wherein R and Rl are as d~fined above) with an agent serving to con~ert the oxo group in the 4-position o~ the uracil ring to an amino group; any remaining protecting groups being removed for example by acid or base hydrolysis to produce the desired product.

'~ith regard to process a), the hydxoxy protecting group includes protecting groups such as acyl (e.g. acetyl), arylacyl (e.g. benzoyl or substituted benzoyl), trityl or monomethoxytrityl, benzy1 or substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. The 5-R-cytosine compound may be optionally protected with silyl, e.g. trimethyl silyl groups. Such groups may be removed in con~entional manner. The leaving group L is a leavin~
group typical of those known~ iD the art o~ nucleoside:chemistry e.g.
halogen such as chlorine or bromine, alkoxy such ~as: me:thoxy or ethoxy or acyl such as acetyl or benzoyl.

The reac~ion~in~proc~ess~a) may be:effected in an crganic solvent (e.g.
l,2-dichloroethane or~acetoni~trile~ in the presence~ f a Lewis:: acid such as stannic chloride:or trimethylsilyl triflate.

Compouhds of:~formu1a~IIA~ may~be:~obtained ~rom :a:~suitab1y protected 2-hydroxyaceta1dehyde of formu1a 1~
:
~ RlOCh~c~o :, wherein Rl is defined above, as described In Can. J~ ~esearch~, 3,;~l2g ~ -(1933)~ and European Patent Specification 0 382 526. Reaction of compounds of formula III with a mercaptoacetal HSCH2CH(OR52,~ wherein R ~.
is Cl 4 alkoxy such a5 ~SCH2CH(0C2H5)~, known in the art (Chem. Ber.
85:924-932, 1952), yields compounds: of formula::IIA wherein L is: OR
(a1koxy) e.g. methoxy or e~hoxy. Al:cernati~e1y, compounds of formula IIA, whereir. L is aIk~xy, may be converted :to compounds of formula IIA wherein L is halogen or acyl by methods known in the~ arC
of carbohydrate chemistry. : i-;~

W O 92~l9246 PCT/GB92/00~r~

21~2~2'3 Compounds of formula III may be prepared from 1,2-0-isopropylidene glycerol by introduction of Rl (e.g. trisubstieu~ed silyl, benzyl or trityl) and removal of the isopropylidene group with mild acid (e.g.
aqueous formic or acetic acid) or zinc bromide in acetonitrile, followed by oxidation of the alcohol group with aqueous periodate.

With regard to process b), the compound of ormula IIB is advantageously treated with 1,2,4-triazole, advantageously together with 4-chlorophenyl dichlorophosphate, t~ form the corresponding 4-(1,2,4-triazoylyl) compound which is then converted to the desired 4-amino (cytldine) compound by reaction with for example methanol.

The starting materials of for~ula IIB be prepared for example by reaction of an appropriate (optionally protec~ed) base with a compound of formula IIA in an analogous~manner to that described in process a).

SeparatiQn of the (+)-cis and (~)-trans isomers f~r example ln a protected form, ~y ~e accomplished by chromatograp~y on silica gel with mixtures of organic solYents such as ethyl acetate/m~thanol, ethylacetate/hexane or dichloromethane/methanol. Any protecting group may then be rem~ved usin~ the appropriate reagent ~or each group.

Resolution of the ~+)-enantiomers may be accomplished enzymatically with an esterase such as pig liver esterase (Sigma Chemical o., St. Louis, MO 63178~ wherein one enantiomer of a 5'-acyl derivative of the compound of formula I is de-esterified. After separation, the remaining esterified compound of formula I may be reacted under basic conditions (e.g. NH3 in methanol or NaOMe in methannl) to give the individual (~) and (-)-enantiomers.

Alternatively, the (+)-cis or (+)-tran~i iso~ers of the compound of formula I may be reacted with a phosphorylating agent (e.g.
phosphorous oxychloride) in an inert solvent such as triethylphos-phate, acetonitrile or 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimi-dinone to yield the 5'-monophosphate derivative or a salt thereof.

~vo 92~g246 ~ 1 2 0 PCTJC~92/00~03 Treatment of the 5'-monophosphate derivative with an enzyme such as snake venom 5'-nucleotidase preferentially hydrolyzes one of the enantiomers. Separation and s~bsequent dephosphorylation of the other 5'-monophosphat~ enantiomer yields the individual (+)- and (-)-enantiomers of the co~pound of formula I.

The compound of formula (I) may be converted into a pharmaceutically acceptable ester by reaction with an appropriate esterlfying agent, for example, an acid halide or anhydride. The compound of formula (I) may be converted into a pharmaceutically acceptable salt thereof in a conventional manner, for example, by treatment with an appropriate base. An ester or salt of a compound of formula (I) may be converted into the parent compound, for example, by hydrolysis.

For a better understandin~ of the invention, the following Exa~ples are given by way of illustration.

Example 1: 1-(2~ ~ydroxYmeth~ l 3-~xa~hi~l~n~ 5-ylh=chD~L~A
: .
Method A: (+)-Cis and (+~-trans-2-ben~oyloxymethyl-5-(N4-acetyl-cyto-sin-l-yl)-1,3-oxathiolane are prepared and separated~ to the (+)-cis -~nd (+)trans isomers as described in European Patent (EP) ~-Specification 0 382 526. The N4-acetyl and 2-benzoyl groups are removed wieh dimethylamine in ethanol, and the~product, (~)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-S-yl)cytoslne, Ls isolated.
..
Method B: (+)-Cis and (~)-trans-2-benzoyloxymethyl-5-(uracii-1-yl~-1,3-oxathiolane are prepared as desoribed in EP 0 382 ~26. After deprotection of the 2-hydroxyl group with saturated methanolic ~:
ammonia, the isomers are separated on silica gel using EtOAc/MeOH as eluant (EP 0 382 526j. The (+)-cis isomer is reacted with acetic anhydride in pyridine at room temperature to give the 2-acetate. ~-Solvent is removed in Yacuo at ~30C. The 2-acetate is then dissol~rd in CHC13 and washed with aqueous sodium bicarbonate. The separated ~;
organic layer is dried, and CHC13 is evaporated in vacuo, Coslversion ~

W O 92/l9246 PCT/GB92/008~ ~

of the uracil base to the cytosine base is carried out by preparation of the 4-(1,2,4-triazol-1-yl) derivati~e according t~ the methods of C.B. Reese, J~Chem.Soc., Perkins 1, 1171, 1984 and W.L. Sung, Nucleic Acids Res. 9:6139, 1981, using 1,~,4-triazole and 2 equivalents o~
4-chloro- phenyldichlorophosphate in dry pyridine at ambient temperature. This con~ersion is followed by reaction with methanol previously saturated with ammonia at 0C, a~d the 2-acetate is hydrolyzed to giYe (+)-cis-1-(2-(hydroxyme~hyl)-1,3-oxathiolan-5-yl)cytosine.

Example 2- 1-(2~ d~roxvmethyl-1,3-oxathi~l~n-~-Yl~-5-meth~ylc~tosine .. ~
(+)-Cis and (~)-trans 2-benzoyloxymethyl-5-(thymin-N-yl~-1,3-oxathio-lane are prepared and separated to the (+)-cis and (+)-trans isomers as described in European Pa~ent Specification 0 382 526. The (+j-cis isomer is reacted with acetic anhydride in pyridine at room temperature to give the 2-acetate. Solvent is removed in vacuo at <300C. The 2-acetate is then dissolved in CHCl3 washed with aqueous sodium bicarbonate. The separated organic layer is dried, and CHCl3 is evaporated tn vacuo. Conver~ion of the thymine base to the 5-methylcytosine base is carried out by preparation of the 4-(1,2,4-triazol-1-yl) deri~ative according to the methods of C.B.
Reese, J.Chem.Soc., Perkins 1, 1171, 1984 and W.L. Sung, N~cleic Acids Res. 9:6139, 1981, using 1,2,4-triazole and 2 equivalents of 4-chlorophenyldichlorophosphate in dry pyridine at ambient temperature. This conversion is followed by reaction with methanol previously saturated with ammonia at 0 C, and the 2-acetate is hydrolyzed to give (+)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-methylcytosine.
,;, Pharmaceutical Formulations In the following formulation Examples, the "Active Ingredient" is 1-(2-(hydroxy~ethyl~-1,3-oxathiolan-5-yl)cytosine.

~VO 92/l9246 21~ O P ~ /GB92/00803 Exa~le 3 Tablet Formulations The following formulation~ A, B and C are prepared by wet granulativn o the ingredients with a solution o povidone, followed by addition of magnPsium stearate and compression.

Formulation A
mg~table~ ~g~tablet ~a) Active ingredient 250 250 (b) Lactose B.P. 210 26 :~.
(c) Povidone B.P. 15 9 (d) Sodium Starch Glycollate 20 12 (e) Magnesium Stearate 5 3 Formulation B
mg~tablet mgitablet (a) Active ingredient 250 250 (b) Lactose 150 (c) Avicel PH 101 60 :26 : (d) Povidone B.P. 15 9 (e) Sodium Starch Glycollate 20~ 12 (f) Ma~nesium Stearate __~ 3 ~.
.

~ : , W ~ 9~/1g246 PCT/GB92/008~.

Formulation C
., . .. _ m~tablet Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesium Stearate 4 The following formulations, D and E, are prepared ~y dlreet compression of the admixed ingredients. The lactose in formulation E
is of the direct compression type (Dairy Crest - "Zeparox"j.

Formul~ation D
mg~tablet :
Active ingredient 250 ;~:
Pregela inized Starch NF15 150~
~: ~ ' , ~; ~
: Formulation_E : :~
~ee/tablet~ :

: Acti~e ingredient 250 . .
Lactose 150 : :
Avicel ~ 100 Formulation F [Cont~r.olled ~elease Formula~ion) :

The formulation is prepared by ~et granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. :;

r6~ ",,~ r:",.,,"~",.~,~",,.~,,6","h".,~,,"j~~;~ " ~ ,;

W O 92~19246 2 1 ~ 2 ~ 2 ~ PCT~GB92~00803 m~tablet (a) Active ingredient 500 ~b? Hydr~xypropylmethylcellulose 112 (Methocel K4M Premiu~) (c~ Lactose B.P. 53 (d) Povidone B.P. 28 (e) Magnesium Stearate Drug release takes place over a period of~ about 6-8 hours and is complete after 12 hours.

Example 4 CaRsule F~rmulations ~;

Formulation_-A
:'' A cap~ule formulation is prepared by admixing the ~ingredients of ~Formula~ion D in Example 3:above and filling into~a two-part hard gelatin capsule. Formulatlon B (infra~ ~is~prepared :in ::a~ ~slDil~ar manner.

: ~
Formulation B
mg/ca ule (a) Active ingredient250 (b) Lactose B.P. 143 (c) Sodiu~ Starch Glycoll~ate 25 ~d) Magnesium Stearate ~ 2 : 420 W O 92/19246 PCT/~B9V008~
2102 ~ 16 -Formulation C
mg~capsule (a) Active ingredient 250 (b) Macrogol 4000 B.P 350 600 ~

Formulation D -mg/capsule Active ingredient 250 Lecithin 100 Arachis Oil 100 450 ;~

Capsules of formulation D are prepared by dispersing the active ingredient in the Iecithin and arachis oil and filling the dispersion into soft, elastic gelat1n capsules.

Formulati~n E fC~L~L ~ 5~:l 5 L~
,...
The following controlled release capsule formulation ~is prepared by extruding in~redients a, b and c using an extruder, followed by spheronization of the extrudate and drying. The dried pellets are then coated with controlled-release membrane (d) and filled into a two-piece, hard gelatin capsule.

mg/capsule :~;

(a) Actîve ingxedient ~ 250 ~ -(b) MicrocrystalIine Ceilulose 125 (c) Lactose B.P. 125 : :, ~d) Ethyl Cellulose 13 513 :~
: ' '~

VVO 9~/19246 l7PCT/GB92/00803 Example 5 ,~
Injectable Formulation Fo~rmulation A

Active ingredient 0.200 g Hydrochloric acid solution, 0.1 M, or Sodium hydroxide solution, 0.1 M q.s. to pH 4.0 to 7.0 Sterile water q.s. to 10 mL

The active ingredient is dissolved in most of the water (35~C-40C) ., and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up ~o volume with the water and filtered through a sterile micropore filter into a sterile 10 mL~amber glass ~ial (type 1) and sealed ~ h sterile closures and overseals.

,, Formulation B ~
'. .
Active ingredient 0.125 -~
Sterile, pyrogen-free, pH 7 phosphate Buffer, : q.s. to 25 mL

Exa~mpl~
Intramus"c~lar iniection ;, Acti~e ingredient 0.20 g ::~ -Benzyl Alcohol . 0.10 g Glycofurol 75 1.45 g , Benzy~ Alcohol q.s, t~ 3.00 mL '~
~'~
The active ingredient is dissolved in the glycofurol. The benzyl alcohol ic then added and d~ssolved, and water added to 3 mL. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 mL amber glass vials (type 1~.

W O ~/19246 PCT/GB9~/008 21~212U

Example 7 Syrup Active in~redient 0.25 g Sorbi~ol Solution 1.50 g Glycerol 2.00 g Sodi~m Benzoate O.005 ~
Flavor, Peach 17.420316 9 0.0125 mL
Purified Water ~.s. to 5.00 mL

The acti~e ingredient is dissolved in a mixture of the ~lycerol and most of the purified water. An aqueous solution of the sodium benzoate is then added to the solution, ~ollowed by addition of the sorbitol solution and finally the flavor. The volume is made up with purified water and mixed well.

Example 8 Suppos tgrX ~, m~/suppvsitory ~.

Active ingredient 250 Hard Fat, B.P. (Witepsol H15 - Dynamit Nobel) 1770 One-fifth of the ~itepsol H1 is melted in a steam-jacketed pan at 45C maximum. The active ingredient is sifted through a 200 ~M sieve and added to the molten base with mixing, using a Silverson fiY~ted with a cutting head, until smooth dispersion is achieved. Maintaining the mixture at 45C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homo~enous mix. The en~ire suspension is passed through a 250 M stainless steel screen and, with continuo~s stirring, is allowed to cool to 40C. At a temperature of 38C ~o 40C, 2.02 g of the mixture is filled into suitable, 2 mL
plasY~ic molds. The suppositories are allowed tc cool to room tempera~ure.

. W O 92/19246 2 1 a ,~ ~ 2 0 PCT/GB92/00803 Example 9 e~

Active ingredient 250 ;
Anhydrate Dextrose 380 Potato Starch 363 Ma~nesium Stearate 1000 ~

The above ingredients are mixed directly ànd pessaries prepared by direct compression of thr~ resulting mixture. ~

Antiviral Activitv ~ ainst ~Hepatitis B Virus ~H.BV~ ~:

The compounds, 1-~2 hydroxymethyl3-1,3-oxathiolan-5-yl)cytosine and 1-(2-(2-hydroxy~ethyl~-1,3-oxat~iolan-5-yl)-5-methylcytosine, were tested as described below.

The human HBV producer oell line of HepG2, 2.2.15, described and characterized by Sells et al., PNAS 84:1005, 1987 and J. Virol.
62:2836, 1988, has been shown to share many characteris~ics of~the HBV ~:
chronically infected hepatocyte. It is infectious as demonstrated by the ability to cause disease in chimpanzees. This c211 line was utilized in vi~ro to identify compounds with anti^HBV ac~ivity.

T~ test compounds for antiviral activity, monolayer cultures were trea~ed with compound, 50-200~M for ten days. Supexnatant media containing extracellular virion DNA (Dane particles) were harvested on :~
dsys ~hree, six and ten, treated with proteinase K (1 mg~mL) and sodium dodecyl sulfate (1%), and incubated at 50C for one hour. DNA
was extracted with equal volumes of phenol followed by chloroform and then precipita~ed by ammonium acetate and propanol. The DNA
precipitate was dissolved and collected on nitrocellulose using the W O 92~19246 PCT/GB92~00 procedure of Schleicher and Schuell (S ~ S, 10 Optical Ave., Keene, NH 03431, Publication No. 700, l9B7), and treated as desoribed by Southern, J. Mol. Biol. 98:503, 1975. Cells were harvested, and the intracellular DNA was obtained after cell lysis with guanidine isothiocyanate. The intracellular DNA was handled in the same manner as the extracellular DNA. After precipitation by ~mmonium acetate and propanol, the intracellular DNA precipitate was dissolved, cut by restriction endonuclease, Hind III, applied to agarose gel and then treated as described by Southern to determine the quantity of replicative intermediate forms. The antiviral effec~ of the compound was determined by measuring at least a 100-fold reduction of the amoun~ of Dane particles extruded into ~he culture medium and a similar decrease in the intracellular replicative intermediates.

The results are given below:

W O 92/19246 PCT/GB9~/OOY~
210'~120 - 21 -Effect of l (~ IEy__oxYmethyl~ 3-oxathio (Compound Al and l-(2-(Hvdroxymethyl)-l.3-oxathiolan-5-y~
5-meth~lcytosine ~Compound B)_on HBV P=roductiGn in 2.2,15 Cell Cultures _ . .
Intracellular HBY PNA*
(p~/~g cell DNA) :
Replica- HBV DNA in Culture Medium Treatment tive (pg/mL)+
Compound Integra- Mono- inter- Day Day Day Day (~M) ted mer- mediate 0 3 6 lO
- ~, A. untreated l.l 2.2 88 88 29 46 llO
cells l.l 2.0 67 67 ~32 52 51 .' 25(A) l.l l.O l 50 lO l O
l.O 0.4 0.3 65 28 3 O
25~B) l.l l.O 3 76 12 O O
1.3 0.6 ~ 6~ ~, O O
';~
: 2. untreated l.O 2.l 92 :71 33 120 150 cells l.O 1.8 63 64 130 :43 120 .;
~ -25(A) l.2 0.9 l lSO 36 lO ~ O `~;
- l.l ~ ~.7 l :120 64 6 0 25(B) l.O 2.l l 46 41 13 0 l.O l.l 2 58 42 24 l ~ . - .. .

* Analysis of intracellular HBV DNA (Dane particles) was 24 hours following the 10th day of treatment. ;
. .:
+ A "zero" indicates an undectectable level of HBV DNA, sensitivity -cutoff was 0,1 pg/mL.

Claims (13)

1. Use of a compound of formula (I) (wherein R is a hydrogen atom or a C1-3 alkyl group) or a physiologically functional derivative thereof in the manufacture of a medicament for the treatment or prophylaxis of a hepatitis B
virus infection.

2. Use as claimed in claim 1 wherein the compound of formula (I) is 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine.

3. Use as claimed in claim 1 wherein the compound of formula (I) is 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-methyllcytosine.

4. Use as claimed in claim 1 wherein the physiologically functional derivative is a pharmaceutically acceptable salt or ester of the compound of formula (I).

5. Use as claimed in any of the preceding claims wherein the said medicament is in the form of a dosage unit.

6. Use as claimed in claim 5 wherein the said dosage unit contains 10 to 1500mg of the compound of formula (I) or a physiologically functional derivative thereof.

7. Use as claimed in claim 5 or claim 6 wherein the said dosage unit is a tablet or capsule.

8. A compound of formula (I) (as defined in claim 1) or a physiologically functional derivative thereof for use in the treatment or prophylaxis of a hepatitis B virus infection.

9. 1-(2-(Hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine for use in the treatment or prophylaxis of a hepatitis B virus infection.

10. 1-(2-(Hydroxymethyl)-1,3-oxathiolan-5-yl)-5-methyllcytosine for use in the treatment or prophylaxis of a hepatitis B virus infection.

11. A method of treating a human having a hepatitis B virus infection comprising the administration to said human of an effective anti-hepatitis B treatment amount of a compound of formula (I) (as defined in claim 1) or a physiologically functional derivative thereof to said human.

12. A method as claimed in claim 11 in which the said compound of formula (I) is 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-meth-ylcytosine.

13. A method as claimed in claim 11 in which the said compound of formula (I) is 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine.