ANTI-HBV PYRIMIDINE NUCLEOSIDE

The present invention relates to a 5-prop-l-ynyl-l-(5-0-trimethylace- tyl-3-D-arabinofuranosyl)uracil nucleoside and physiologically and pharmaceutically acceptable salts thereof, for use in the treatment or prophylaxis of hepatitis B viral infections.

World wide, hepatitis B Virus (HBV) is a viral pathogen of major consequence. It is most common in Asian countries, and prevalent in sub-Saharan Africa. The virus is aetiologically associated with primary hepatocellular carcinoma and is thought to cause 80% of the world\'s liver cancer. In the United States more than ten thousand people are hospitalised for HBV illness each year, an average of 250 die with fulminant disease. The United States currently contains an estimated pool of 500,000 to 1-million infectious carriers. Chronic active hepatitis will develop in over 25% of carriers, and often progresses to cirrhosis. It is estimated that 5000 people die from HBV related cirrhosis each year in the USA, and that perhaps 1000 die from HBV-related liver cancer. Even when a universal HBV vaccine is in place, the need for effective anti-HBV compounds will continue. The large reservoir of persistently infected carriers, estimated at 220 million worldwide, will receive no benefit from vaccination and will continue at high risk of HBV induced liver disease. This carrier population serves as the source of infection of susceptible individuals perpetuating the instance of disease particularly in endemic areas or high risk groups such as IV drug abusers and homosexuals. Thus, there is a great need for effective antiviral agents, both to control the chronic infection and reduce progression to hepatocellular carcinoma.

Clinical effects of infection with HBV 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 may be more prolonged leading to persistent chronic liver disease as

outlined above. In "Viral Infections of Humans" (second edition, Ed., Evans, A.S. (1982) Plenum Publishing Corporation, New York), Chapter 12 describes in some detail, the aetiology of viral hepatitis in ec ions.

European patent publication no. 22384A1 discloses a general class of nucleosides for the therapeutic or prophylatic treatment of infections caused by a retrovirus, including HIV, or by hepatitis B virus.

It has now been found that 5-prop-l-ynyl-l-(5-0-trimethylacetyl-/S-D- arabinofuraπosyl)uracil previously disclosed in our co-pending European Patent Publication No. 0375164 for its use against certain herpes viruses, has advantageous properties for the treatment or prophylaxis of retroviral or retroviral-like infections, particularly hepatitis B infections. Our co-pending European Patent Application No. 91305147.0 discloses the parent compound l-(^-D-arabinofuranosyl)- 5-prop-l-ynyl- uracil and esters thereof for use in the treatment of hepatitis particularly hepatitis B viral infections.

One feature of the invention provides use of 5-prop-l-ynyl-l-(5-0-tri- methylacetyl-0-D-arabinofurano«yl)ur*cil, i.e. the compound of formula

(in its enol or keto form) or a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a hepatitis B viral infections.

The invention also provides a method for the treatment or prophylaxis of a mammal (particularly a human) having a hepatitis B viral

infection comprising administration to said mammal of an effective amount of the compound of 5-prop-l-ynyl-l-(5-0-trimethylacetyl-^-D- arabinofuranosyl)uracil or a physiologically acceptable salt thereof.

Yet another feature of the invention is an anti-hepatitis B pharmaceu¬ tical formulation comprising 5-prop-l-ynyl-l-(5-0-trimethylacetyl-^-D- arabinofuranosyl)uracil or a physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Yet a further feature of the invention is the use of 5-prop-l-ynyl-l- (5-0-trimethylacetyl-3-D-arabinofuranosyl)uracil, or a physiologically acceptable salt thereof in the treatment or prophylaxis of a hepatitis B viral infection.

Hepatitis B viral conditions which may be treated in accordance with the invention have been discussed in the introduction hereinbefore.

By the use of the compounds of the invention an advantageous therapeutic effect for the treatment or prophylaxis of hepatitis B infections can be achieved. Thus, for example, such compounds are readily absorbed from the gastro-intestinal tract and can therefore be administered by the oral route to achieve high plasma levels of the parent nucleoside.

The physiologically acceptable salts of the invention include base salts, for example derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) and ammonium and NX, (wherein X is C. , alkyl) salts. By physiologically acceptable salts we mean to include pharmaceutically acceptable salts.

The compound of the invention 5-prop-l-ynyl-l-(5-0-trimethylacetyl-^- D-arabinofuranosyl)uracil or a physiologically salt thereof) may be administered to a mammal including a human ("the recipient") by any route appropriate to the clinical condition to be treated; suitable routes include oral, rectal, nasal, topical (including buccal and

sublingual) , vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) . It will be appreciated that the preferred route may vary, for example, according to the age, weight and sex of the recipient and the nature and severity of the condition to be treated.

The amount of a compound of the invention required for the treatment of a hepatitis viral infection will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician. In general, however, for each of the conditions, a suitable, effective dose will be in the range 1 to lOOmg per kilogram body weight per day and most preferably in the range 5 to 30 mg per kilogram body weight per day; an optimum dose is about 15 mg per kilogram body weight per day. The effective dose may be presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 2000 mg, preferably 20 to 500 mg and most preferably 100 to 400 mg of a compound of the invention per unit dosage form. Alternatively, if the condition of the recipient so requires, the dose may be administered as a continuous infusion.

The compounds of the invention may be administered for the treatment or prophylaxis of viral infections alone or in combination with other therapeutic agents, for example, with other antiviral agents such as 9-(2-hydroxy-ethoxymethyl)guanιne (acyclovir) used to treat herpes viral infections in particular HSV and most notably those involving the HSV (1) variety, with 3\'-deoxy-3\'-azidothymidine (zidovudine) or a 2\' ,3\'-dideoxynucleoside for example 2\' ,3\'-dideoxy cytidine, 2\' ,3\'-dideoxyinosine, 2\' ,3\'-dideoxyadenosine or 2\' ,3\'-dideoxy guanosine, used to treat retroviral infections in particular Human Immunodefeciency Virus (HIV) infections, interferons particularly α-interferon and soluble proteins such as CD4, or any other agents

such as analagesics or antipyretics which when in combination with a compound of the invention provide a beneficial therapeutic effect.

A medicament for use in the invention preferably in the form of a pharmaceutical formulation comprising at least one compound of the invention ("the active ingredient"), together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipients thereof.

Formulations of the invention include those suitable for administra¬ tion by any of the aforementioned routes which may conveniently be presented in unit dosage form prepared by any of the methods well known in the art of pharmacy. Such methods include the. step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, If necessary, shaping the product.

Formulations of the invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; as an edible foam or whip; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, or paste or may be contained within liposomes.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active Ingredient in a free-flowing form such as a powder or granules, optionally mixed with

a binder (for example povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, dlsintegrant (for example sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) , surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets are optionally coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein, using for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile, or to be soluble or effervescent when added to liquid.

A capsule may be made by filling a loose or compressed powder on an appropriate filling machine, optionally with one or more additives. Examples of suitable additives include binders such as povidone; gelatin, lubricants, inert diluents and disintegrants as for tablets. Capsules may also be formulated to contain pellets or discrete sub-units to provide slow or controlled release of the active ingredient. This can be achieved by extruding and spheronising a wet mixture of the drug plus an extrusion aid (for example microcrys- talline cellulose) plus a diluent such as lactose. The spheroids thus produced can be coated with a semi-permeable membrane (for example ethyl cellulose, Eudragit WE30D) to produce sustained release properties.

An edible foam or whip formulation ideally comprises; 50-70% of an edible oil, particularly a vegetable oil, including corn oil, peanut oil, sunflower oil, olive oil and soybean oil; 2-10% of one or more surfactants particularly lecithin, polyols, polyol polymer esters including glyceryl fatty acid esters, polyglyceryl fatty acid esters (e.g. decaglycerol tetraoleate) , or sorbitan fatty acid esters (e.g. sorbitan monostearate) ; 1-4% of a propellant which is suitable for ingestion, notably a compressed gas propellant especially nitrogen, nitrous oxide or carbon dioxide, or a gaseous hydrocarbon especially propane, butane or isobutane; 0.5-30% of one or more viscosity

modifiers of particle size in the range 10-50 microns in diameter, particularly powdered sugars or colloidal silicon dioxide; and optionally 0.5-1% of one or more suitable, non-toxic colourings, flavourings or sweetners. The active ingredient is preferably present in such formulations in a concentration of 10-46%, advantageously 30%. An edible foam or whip formulation as described above may be prepared in a conventional manner, for example by mixing the edible oil, surfactant(s) and any other soluble ingredients, adding the viscosity modifier(s) and milling the mixture to form a uniform dispersion and suspension. The active ingredient is blended into the milled mixture until evenly dispersed. Finally, a metered quantity of propellant is incorporated to the mixture after said mixture has been measured into a suitable dispensing container.

For infections of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base or as a water-in-oil base.

If desired, the aqueous phase of the cream base may include, for example, at least 40-45% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.

The oily phase of an emulsion formulation according to the invention may comprise merely an emulsifier (otherwise known as an emulgent) , but desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stablilizer. It is also preferred to include both an oil and a fat. Together, the emulsifer(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stear te and sodium lauryl sulphate.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. The cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also Include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. The

ingredient is preferably rresent in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured material, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert material such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or higher fatty alcohol (e.g. hard wax, European Pharmacopoeia) or triglycerides and saturated fatty acids (e.g. Witepsol) .

Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

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

Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include

suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for 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 dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The compounds of the invention may be prepared by any method known in the art for the preparation of similar compounds (examples include methods described in UK Patent Specification No. 1 601 020, EP Publications Nos. 61283 and 27065, or Robins M.J. and Barr, P.J. J. Org. Chem. (1983) 48, 1854-1862), as well as the processes described In the Examples given hereinafter.

Compounds of the present invention may be prepared as follows:

A. reacting a compound of formula (II)

[i.e. l-(3-D-arabinofuranosyl)-5-prop-l-ynyluracil]

with a compound serving to provide the trimethylacetyl group at the 5-position of the sugar moiety; or

reacting a compound of formula (III)

wherein B is a purine or pyrimidine base (other than 5-prop-l-ynyl-uracil) , with 5-prop-l-ynyluracil; and optionally thereafter or simultaneously therewith, where the resulting compound is a compound of formula (I), converting it into a salt thereof; or where the resulting compound is a salt, converting it into a different salt or a compound of formula (I) . Thereafter the active compound can be brought into association with a carrier and formulated as an anti-HBV pharmaceutical formulation.

With regard to process A, a compound of formula (I) may be prepared from a compound of formula (II) , the preparation of which is described in EP Publication No. 272065, for example by

acylation using an appropriate acylating agent such as trimethylacetylhalide (e.g. chloride) or trimethylacetic anhydride, advantageously in the presence of a base such as pyridine or triethylamine which may also serve as a solvent medium for the reaction at a temperature in the range of 0 -70 C advantageously 0°-30°C, preferably 0-15 C. The ratio of acylating agent to compound of formula (II) is preferably about 1.2:lw/w.

Again, a compound of formula (I) may be prepared from a compound of formula (II) by transesterification using an appropriate ester of trimethylacetic acid (e.g. the methyl ester) In the presence of a base such as pyridine or triethylamine which may also serve as a solvent medium for the reaction.

In addition, the esterification reaction may be carried out for - example in a solvent such as pyridine or dimethylformamide in the presence of a coupling agent such as N,N-dicyclohexylcarbodiI- mide, optionally in the presence of a catalytic base such as 4-dime hylamInopyridine using the acylating agents referred to above. Subsequently, the ester obtained as reaction product may be isolated in conventional manner.

With regard to process B, group B is preferably a purine or pyrimidine base capable of donating the esterified sugar to a 5-prop-l-ynyluracil base using for example an enzyme such as a phosphorylase enzyme in the presence of a phosphate salt at a pH of 5.0 - 9.0 and a temperature of 15 -90 C, advantageously 40°-60°C.

Salts according to the invention may also be prepared in conventional manner for example by reaction of a compound of formula (II) with an appropriate base to form the corresponding base salt followed by acylation. Again, salts may be prepared by reaction of the esterified compound with a base such as sodium

hydride to form the corresponding sodium salt. Other derivatives according to the invention can also be prepared in conventional manner.

The following examples are for illustration of the present invention and should not be considered as limiting in any way:-

Example 1

5-Prop-l-vnyl-l-(5-0-trimethylacetyl--3-D-arabinofuranosy l)uracil

To a stirred solution of l-(/9-D-arabinofuranosyl)-5-prop-l-ynyluracil (0.28g, lmmol, synthesised by the method described in EP Publication No. 272065) in dry pyridine (5ml) at 0 C under dry nitrogen, was added dropwise a solution of trimethylacetylchloride (0.15ml, 0.14g), 1.2mmol) in dry dichloromethane (5ml) over a period of 10 minutes. The mixture was stirred at 0 C for 90 minutes then at room temperature for 2 hours. The solvent was evaporated under reduced pressure, and residual pyridine co-evaporated with portions of ethanol (3 x 25ml) to give an oil. Chromatographic separation on a silica gel column eluting with 8% methanol/dichlorome hane gave pure product which was triturated with ether to give a white solid identified as the title compound. Mpt: 204-210°C.

Analysis Calc: C 55.74, H 6.011, N 7.65% Found: C 55.95, H 6.006, N 7.525%

5(d.DMS0) 11.55(lH,6s,NH), 7.58(lH,s,H-6) , 6.0(lH,d,H-l\' ) , 5.72(lH,d, 0H-2\'), 5.62(lH,m,OH-3\'), 4.4-4.13-(2H,m,H-5\' ) , 4.08-3.89(3H,m,H-2\' , H-3\',H-4\'), 1.97(3H.s,C-(CH ₃ ), 1.19ppm(9H,s,tBu) .

Example 2

Sodium salt of 5-Prop-l-vnyl-l-(5-trimethylacetyl--3-D-arabinofurano- svDuracil

A suspension of sodium hydride (0.05g of 80% w/v suspension in oil, 1.66mmol, (washed several times with dry tetrahydrofuran) in dry tetrahydrofuran (4ml) was added to a stirred solution of 5-prop-l-ynyl-l-(5-trimethylacetyl- -D-arabinofuranosyl)uracil (0.06g, 1.64mmol) in dry tetrahydrofuran, ensuring complete exclusion of moisture. The solvent was evaporated after 1 hour to give O.lg of the required sodium salt.

The following Examples illustrate pharmaceutical formulations according to the invention in which the "Active Ingredient" is the compound of formula (I) .

Example A t a m c Solution

Active Ingredient

Sodium chloride, analytical grade

Thiomersal

Purified water to pH adjusted to

Example B: Tablet Formulations

The following formulations A, B and C are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

Formulation A

(a) Active ingredient

(b) Lactose B.P.

(c) Povidone B.P.

(d) Sodium Starch Glycollate

(e) Magnesium Stearate

Formulation B

(a) Active ingredient

(b) Lactose

(c) Avicel PH 101

(d) Povidone B.P.

(e) Sodium Starch Glycollate

(f) Magnesium Stearate

Formulation C m%/tablet

Active ingredient 100

Lactose 200

Starch 50

Povidone 5

Magnesium stearate 4

359

The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direct compression type.

Formulation D mg/tablet

Active Ingredient 250

Pregelatinised Starch NF15 150

400

Formulation E mg/tablet

Active Ingredient 250

Lactose 150

Avicel 100

500

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients (belov) with a solution of povidone followed by the addition of magnesium stearate and compression.

mg/tablet

(a) Active Ingredient 500

(b) Hydroxypropylmethylcellulose 112 (Methocel K4M Premium)

(c) Lactose B.P. 53

(d) Povidone B.P.C. 28

(e) Magnesium Stearate 7

700

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

Example C: Capsule Formulations Formulation A

A capsule formulation is prepared by admixing the uncompressed ingredients of Formulation D in Example B above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium Starch Glycollate 25

(d) Magnesium Stearate 2

420

Formulation C mg/capsule

(a) Active ingredient 250

(b) Macrogol 4000 BP 350

600

Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

Formulation D mg/capsule

Active ingredient 250

Lecithin 100

Arachis Oil 100

450

Capsules are prepared by dispersing the active Ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared by extruding ingredients (a), (b) , and (c) below, using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release- controlling membrane (d) and filled into a two-piece, hard gelatin capsule.

mg/caosule

(a) Active Ingredient 250

(b) Microcrystalline Cellulose 125

(c) Lactose BP 125

(d) Ethyl Cellulose 13

513

Iniectable Formulation

Example D:

Active ingredient 0.200 g

Sterile, pyrogen free phosphate buffer (pH 7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35- 40 C) , then made up to volume and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Example E : Intramuscular Injection

Active Ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glycofurol 75 1.45 g

Water for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1) .

Example F : Syrup Suspension

Active ingredient 0.2500 g

Sorbitol Solution 1.5000 g

Glycerol 2.0000 g

Dispersible Cellulose 0.0750 g

Sodium Benzoate 0.0050 g

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose) . The two dispersions are mixed and made up to the required volume with the purified water.

Example H : Suppository mp/suppositorv

Active Ingredient (63μm) 250

Hard Fat, BP (Witepsol H15 - Dynamit Nobel) 1770

2020

* The active ingredient is used as a powder wherein at least 90% of the particles are of 63μm diameter or less.

One-fifth of the Witepsol H15 is melted in a steam- acketed pan at 45°C maximum. The active ingredient is sifted through a 200μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45 C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250μm stainless steel screen and, with continuous stirring, is allowed to cool to 40 C. At a temperature of 38°C to 40°C 2.02g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

e e e

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

o c ormu at on

The active compound is dissolved in a mixture of purified water and glycerol and heated to 70 C. The remaining ingredients are heated ttooggeetthheerr aatt 7700 CC.. TThhee ttwwoo ppaarrttss .are added together and emulsified. Cooled and filled into containers.

Since 5-prop-l-ynyl-l-(5-0-trimethylacetyl- -D-arabinofuranosyl)uracil is converted iu vivo to the parent compound

l-(^-D-arabinofuranosyl)-5-prop-l-ynyluracil, the antiviral tests were carried out on the parent compound, although bioavailabilty tests were carried out on the compound (and its salts) of the invention.

Antiviral Activity

The human HBV producer cell 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 characteristics of the HBV chronically infected hepatocyte. It is infectious as demonstrated by the ability to cause disease in chimpanzees. This cell line was utilized in vitro to identify compounds with anti-HBV activity.

Monolayer cultures were treated with l-100μM of the active compound l-(/9-D-arabinofuranosyl)-5-prop-l-ynyluracil, for ten days. Supernatant media containing extracellular virion DNA (Dane particles) were harvested on day ten, treated with proteinase K (1 mg/mL) and sodium dodecyl sulfate (1%), and incubated at 50°C for one hour. DNA was extracted with equal volumes of phenol followed by chloroform and then precipitated by ammonium acetate and propanol. The DNA precipitate was dissolved and collected on nitrocellulose using the procedure of Schleicher and Schuell (S & S, 10 Optical Ave., Keene, NH 03431, Publication #700, 1987), and treated as described 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 ammonium 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 effect of the drug was determined by measuring a reduction of the amount of Dane particles extruded into the culture medium (extracellular DNA) and a similar decrease in the intracellular replicative intermediates (intracellular replicative form) .

TABLE 1

Percent (Reduction) Inhibition of Hepatitis B Virus DNA at lOOuM 1-(θ-D-arabinofuranosyl)-5-prop-l-vnyluracil

Expt.No. Released into Extracellular Intracellular

Media Replicative Form DNA

87.9% 92.2% 79.1% 75.5%

TABLE 2

Percent (Reduction) Inhibition of Hepatitus B Virus DNA in Extracellular Media

Concentration of l-( -D-arabino Expt. 5 Expt. 6 furanosyl)-5-prop-l-ynyluracil

lOOμM 94.4 98.5

(88) (95.3)

33μM 87.4 89.1

llμM N.I. 71.5 _.

37μM 55.3 N.I.

N.I. - no inhibition detected

In parentheses is % reduction in intracellular hepatitis B virus DNA replicative form.

TABLE 3

Percent Inhibition of Hepatitis B Specific Intracellular Replicative Form (RF) DNA. Expt.7.

Concentration of lμM lOμM lOOμM

1-(^-D-arabinofuranosyl)

-5-prop-l-ynynluracil 35% 33% 42%

Determination of Oral Bioavailabilitv

Long Evans Rats were administered the compound of Example 1 and the parent compound by gavage at a dose of 50mg/kg. The urine was collected for 24 and 48 hours post-dose, ultrafiltered, and analysed by reverse-phase high-pressure liquid chromatography. The oral bioavallablllty of the Example 1 compound and the parent compound was expressed as the percent of the dose exreted in the urine over the 48 hour period of collection, as l-( -D-arabinofuranosyl)-5-prop-l-ynyl- uracil, i.e. the parent compound.

Compound Urinary Recovery

(% of dose)

Example 1 64.89

Parent Compound 9.70

Toxic ity

Cell toxicity is assessed in a cell growth inhibition assay. Subconfluent cultures of Vero cells grown on 96-well microtiter dishes are exposed to different dilutions of drug, and cell viability determined daily on replicate cultures using uptake of a tetrazolium day (MTT) . The concentration required for a 50% inhibition of cell viability at 96 hours is termed CCID ₅ _.

Example CCID-.(μM) at

96 hr

477