IE880210L - Antiviral nucleosides - Google Patents

Description

./' \ :-?™\ ; V- '• s' i *. • * / The invention relates to 3'-arido-3'-deoxythymidine and in particular to the use of this compound particularly the use in the treatment of a human retrovirus infection.

Retroviruses form a sub-group of RHA viruses which, in 5 order to replicate, must first "reverse transcribe" the RNA of their genome into DNA ("transcription" conventionally describes the synthesis of RHA from DHA). Once in the form of DHA, the viral genome is incorporated into the host cell genome, allowing it to take full 10 advantage of the host cell's transcription/translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host's DNA and, in this state, the virus may persist for as long as the cell lives. As it is virtually 15 invulnerable to attack in this form, any treatment must be directed at another state of the life cycle and will, of necessity, have to be continued until all virus-carrying cells have died.

HTLV-l and HTLV-1I are both retroviruses and are known to 20 be causative agents of leukaemia in man. HTLV-X infections are especially widespread and are responsible for many deaths world-wide each year.

A species of retrovirus has now been reproducibly isolated from patients with AIDS. While it has been extensively characterised, there is, as yet, no agreed name for the 5 virus, and it is currently known either as human T-cell <■ lymphotropic virus III (HTLV III), AIDS-associated i retrovirus (ARV), or lymphadenopathy associated virus (LAV). It is anticipated that the name to be agreed on internationally is acquired immune deficiency virus 10 (AIDV). This virus (referred to herein as AIDV) has been shown preferentially to infect and destroy T-cells bearing the OKT4 surface marker and is now generally accepted as the aetiologic agent of AIDS. The patient progressively loses this set of T-cells, upsetting the overall balance 15 of the inmune system, reducing his ability to combat other infections, and predisposing him to opportunistic infections which frequently prove fatal. Thus, the usual cause of death in AIDS victims is by opportunistic infection, such as pneumonia or virally induced cancers, 20 and not as a direct result of AIDV infection.

Recently, AIDV has also been recovered from other tissue types, including B-cells expressing the T4 marker, macrophages and non-blood associated tissue in the central nervous system (CNS). This latter infection has been r 25 discovered in patients expressing classical AIDS symptoms and is associated with progressive demyelination, leading to wasting and such symptoms as encephalopathy, progressive dysarthria, ataxia and disorientation.

There are at least four clinical manifestations of AIDV infection. In the initial "carrier" state, the only 5 indication' of infection is the presence of anti-AIOV antibodies in the blood-stream. It is believed that Buch "carriers" are capable of passing on the infection, e.g. by blood transfusion, sexual intercourse or used syringe needles. The carrier state may often never progress to 10 the second stage characterised by persistent generalised lymphadenopathy (PGL). It is currently estimated that about 20% of PGL patients progress to a more advanced condition known as "AIDS related complex" (ARC)• Physical symptoms associated with ARC may include general malaise, 15 increased temperature and chronic infections. This condition usually progresses to the final, fatal AIDS condition, when the patient completely loses the ability to fight infection.

The existence of these human retroviruses and others has 20 only recently been recognised and, as the diseases with which they are linked are of a life-threatening nature, there exists an urgent need to develop ways to combat these viruses.

Various drugs have now been proposed as "cures" for AIDS. 25 These include antimoniotungstate, suramin, ribavirin and J iaoprinosine, which are either somewhat toxic or have shown no marked anti-retroviral activity. As the AIDV genome is incorporated into the host cell DNA after infection and is virtually invulnerable to attack in this 5 state, it will persist as long as the host cell survives, causing new infection in the meantime. Thus, any treatment of AIDS would have to be for an extended period, possibly life, requiring substances with an acceptable toxicity.

Reports have described the testing of compounds against various retroviruses, for example Friend Leukaemia Virus (FLV), a murine retrovirus. For instance Krieg et al. (Exp. Cell Res., 116 (1978) 21-29) found 3'-azido-3'-deoxythymidine to be active against FLV in in vitro 15 experiments, and Ostertag et al. (Proc. Nat. Acad. Sci. (1974) 71^, 4980-85) stated that, on the basis of antiviral activity related to FLV and a lack of cellular toxicity, 3'-azido-3'-deoxythymidine "might favorably replace bromodeoxyuridine for medical treatment of diseases caused 20 by DNA viruses". However, De Clerq et al. (Biochem. Pharm. (1980) 29, 1849-1851)established, six years later, that 3 '-azido-3 '-deoxythymidine had no appreciable activity against any viruses used in their tests, including vaccinia, HSVI and varicella zoster virus (VZV). 25 Glinski et al. (J. Org. Chem. (1973), 38, 4299-4305) discloses certain derivatives of 31-aiido-31 -deoxythymidine (infra) and their ability to block mammalian exoribonuclease activity. . We have now discovered that 3 '-azido-3'-deoxythymidine has a surprisingly potent activity against human retroviruses, with a particularly high activity against AIDV as demonstrated by the experimental data referred to below. Such activity renders the compound useful in the therapy of human retroviral infections.

Thus, in a first aspect of the present invention, there is 0 or prophylaxis of human retrovirus infections. The compound of formula (I) is hereafter referred to as the compound according to the invention.

According to another aspect the invention provides the use of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of a human retrovirus infection.

Activity of 3'-azido-3'-deoxythymidine against human retroviruses has been established in various in vitro assay systems. For example, infection of the H9 human lymphoblastoid cell-line by AIDV is effectively prevented by concentrations of 3'-azido-3'-deoxythymidine as low as 5 0.013 mcg/ml up to 20 hours after infection. AIDV infection of U937 human lymphoblastoid cells, PHA-stimulated white blood cells and cultured peripheral blood lymphocytes is also prevented at similarly low concentrations. In addition, 10-day challenge experiments 10 using up to 500 AIDV virions per cell and cloned T4, tetanus-specific, T-helper lymphocytes, showed no decrease in cells treated with 3'-azido-3'-deoxythymidine, while untreated cells had decreased 5-fold. Cytopathic effects were also completely blocked in the same cell-line 15 transformed by HTLV-I and super-infected with AIDV.

Other studies using purified AIDV reverse transcriptase have shown that the activity of this enzyme is blocked by the triphosphate of 3'-azido-3 '-deoxythymidine by a competitive inhibition mechanism.

Phase I clinical trials have also shown that 3'-azido-3'-deoxythymidine is capable of crossing the blood/brain barrier in clinically effective quantities. This property is both unusual and valuable for the treatment and prophylaxis of CNS infections caused by human 25 retroviruses.

The ability of 3' -azido-3'-deoxythymidine to modify the course of retrovirus-induced malignancy has been demonstrated in a mouse model, whereby administration of 3'-azido-3'-deoxythymidine prevented splenomegaly caused 5 by intravenously administered Rauscher Murine Leukaemia Virus, the murine equivalent of HTLV-I. In further experiments, 3'-azido-3 '-deoxythymidine has been shown to inhibit the in vitro replication .of HTLV-I at concentrations as low as 0.8 mcg/ml.

Thus, in a further, preferred aspect of the present invention, there is provided the use of a compound according to the invention in the manufacture of a medicament for the treiatment or prophylaxis of human retrovirus infections.

The present invention further provides a compound according to the invention for use in the treatment or prophylaxis of AIDS or a virus infection as identified above.

Examples of human retrovirus infections which may be 5 treated or prevented in accordance with the present invention include T-cell lymphotropic retroviruses (HTLV.), especially HTLV-I, HTLV-II and AIDV (HTLV-III). Clinical conditions that may be treated or prevented in accordance with the invention include AIDS, AIDS-related complex and 10 HTLV-I positive leukaemia and lymphoma. Suitable patients for treatment also include those having antibodies to AIDV, AIDV CNS infections, PGL and ARC.

Experiments have shown that 3'-axido-3'-deoxythymidine is converted, in vivo, by the action of cellular enzymes into 15 the 5•-monophosphate. The monophosphate is then further phosphorylated by other enzymes to form the triphosphate via the diphosphate, and other studies have demonstrated that it is the triphosphate form of 3'-azido-3'-deoxythymidine which is believed to be the effective chain 20 terminator in the reverse transcription of AIDV, as evidenced by its effect on avian nyeloblastosis virus and Moloney murine leukaemia virus. This form also inhibits AIDV reverse transcriptase in vitro whilst having a negligible effect on human DHA polymerase activity.

Examples of pharmaceuticals acceptable salts of the compound of formula (I) include base salts, eg derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NXj (wherein X is C^.4 alkyl). 3'-Azido-3'-deoxythymidine, (hereafter referred to as the active ingredient), may be administered to humans for prophylaxis or treatment of retroviral infections by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the and recipient k the nature of the infection.

In general a suitable dose will be in the range of 3.0 to 120 mg per kilogram body weight of the patient per day, preferably in the range of 6 to 90 mg per kilogram body weight per day and most preferably in the range 15 to 60 mg per kilogram body weight per day. The desired dose is 20 preferably presented as two, three, four, five, six or ■tore sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 5 to 1500 mg, preferably 10 to 1000 mg, and most preferably 20 to 25 700 mg of active ingredient per unit dosage form.

Experiments with 31-asido-3'-deoxythymidine suggest that a dose should be administered to achieve peak plasma concentrations of the active compound of from about 1 to about 75 pM, preferably about 2 to 50 VH, most preferably 5 about 3 to about 30 VH. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to about 100 mg/kg of the active ingredient. Desirable blood 10 levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg/kg of the active ingredient.

While it is possible for the active ingredient to be 15 administered alone it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic agents. 20 Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including 25 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 iri 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 5 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 suitable for oral administration may be 10 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 or non-aqueous liquid;or as an oil-in-water liquid emulsion or a water-in-oil liquid 15 emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Oral formulations may further Include other agents conventional in the art, such as sweeteners, flavouring agents and thickeners.

A tablet- may be made by compression or moulding, 20 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 25 cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. 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 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, hydroxypropylmethylceollulose in varying proportions to provide the desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a favoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient 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 comprising for example cocoa butter or a salicylate.

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 isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation 5 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 sealed containers, for example, ampoules and vials, and 10 may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, inmediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets 15 of the kind previously described.

Preferred unit dosage 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. 3'-Azido-3'-deoxythysldine may also be used in therapy in conjunction with other medicaments such as 9-E[2-hydroxy-l-(hydroxy-methyl)ethoxy]methyl]guanine, 9- (2-hydroxyethoxymethyl)guanine (acyclovir), 2-amino-9-(2-hydroxyethoxymethy1)purine, interferon, e.g.,a-25 interferon, interleukin II, and phosphonoformate (Foscaraet) or in conjunction with other immune modulating therapy including bone marrow or lymphocyte transplants or medications such as levamisol or thymosin which serve to increase lymphocyte numbers and/or function as is appropriate.

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 of formulation.

The compound of formula (I) may be prepared in 10 conventional manner, for example as described in the following references, or by methods analogous thereto: J.R. Horwitz et al.. J. Org. Chem. 29, (July 1964) 2076-78; H. Imazawa et al., J. Org. Chem, 43 (15) (1978) 3044-3048; K.A. Watanabe et al. . J. Org. Chem., 4!>, 3274 15 (1980); and R.P. Glinski et al., J. Chem. Soc. Chem.. Commun., 915 (1970), as well as the processes described in the Examples.

A process for the preparation of a compound of formula (I) comprises (A) reacting a compound of formula: o CH 3 (II) N HO (wherein H represents a precursor group for the 3'-arido group) or a derivative (e.g. an ester or salt) thereof, with an agent or under conditions serving to convert the said precursor group into the desired azido group; or (wherein R represents a precursor group for the hydroxy group, or for a pharmaceutically acceptable derivative group thereof) with an agent or under conditions serving to convert the said precursor group into the corresponding 10 desired group; or (B) reacting a compound of formula: 0 N (III) N., (C) reacting a compound of formula or a functional equivalent thereof, with a compound serving to introduce the desired ribofuranosyl ring at the l-position of the compound of formula (IV); or O (D) reacting a compound of formula CH, (wherein R* is hydroxy or R as defined above), with an agent or under conditions serving to convert the said compound into a compound according to the invention; (V! and thereafter, or simultaneously therewith, when a pliarmaceutically acceptable derivative of 3'-&zido-3'-10 deoxythymidine is formed, converting the said derivative into the compound of formula (I).

In the above-described process it will be appreciated that the precursor compounds of formulae (II) and (III), as well as the above-mentioned agents and conditions, will be selected from those that are known in the art of 5 nucleoside synthetic chemistry. Examples of such conversion procedures are described hereinafter for guidance and it will be understood that they can be modified in conventional manner depending on the desired compound of formula (I). In particular, where a 10 conversion is described which would.otherwise result in the undesired reaction of labile groups then such groups may be protected in conventional manner, with subsequent removal of the protecting groups after completion of the conversion. Thus, in process (A), the group H in the 15 compound of formula (II) may represent, for example, a halogen (e.g. chlorine), hydroxy or organosulphonyloxy e.g. trifluoromethylsulphonyloxy, methanesulphonyloxy or p-toluene sulphonyloxy radical.

For the preparation of the compound of formula (I), a 20 compound of formula (II) in which the group M is a halogen (eg chloro) group in the threo configuration (in which the 5'-hydroxy is advantageously protected, eg with a trityl group) may be treated for example with lithium or sodium aside. The 3'-threo-haloqen (eg chlorine) starting 25 material may be obtained for example by reaction of the corresponding 31-erythro-hvdroxy compound with for example triphenylphosphine and carbon tetrachloride, or alternatively by treatment with organosulphonyl halide (eg trifluoromethanesulphonyl chloride) to form a corresponding 3'-erythro-organosulpihonyloxy compound which is then halogenated. Alternatively a 3'-threo-hvdroxv compound of formula (II) may be treated, for example with triphenylphosphine, carbon tetrambromide and lithium azide to form the corresponding 3'-erythro azido compound. Removal of any protecting group may then subsequently be effected, e.g. as above.

With regard to process (B), R may represent a protected hydroxy group e.g. an ester grouping of the type referred to above in relation to formula (I) particularly acetoxy, or an ether group such as a trialkylsilyloxy group, e.g. t-butyldimethylsilyloxy or an aralkoxy group e.g. triphenylmethoxy. Such groups may be converted for example by hydrolysis to the desired hydroxy group or, by transesterification, be converted to an alternative ester group of 3'-azido-3'-deoxythymidine.

With regard to process (C), this may be effected for example by treating the appropriate pyrimidine of formula (IV) or a salt or protected derivative thereof, with a compound of formula (wherein A represents a leaving group, e.g. an acatoxy or benzoyloxy or halo, eg chloro group and B represents an optionally protected hydroxy group eg a p-toluenesulphonyloxy group), and subsequently removing any 5 protecting groups.

Regarding process (0), R1 may represent a precursor group as described above for R in formula (III). 3*-Azido-3'-deoxythymidine may then, for example, be obtained by reaction with an alkali metal azide, e.g. lithium azide, 10 . advantageously in an appropriate solvent such as moist DtyF followed by acid or base hydrolysis advantageously under mild conditions.

An ester or salt of 3' -azido-3 1 -deoxythymidine may be converted into the parent compound, e.g. by hydrolysis.

The following Examples are intended for illustration only and are not intended to limit the scope of the invention in any way. The term "active ingredient" as used in the Examples means a compound of formula (I).

Example It Tablet Formulations The following formulations A to C were prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression. aw/tablet Mg/tablet Formulation > (a) Active ingredient 250 250 (b) Lactose B.P. 210 26 5 (c) Povidone B.P. 15 9 (d) Sodium Starch Glycollate 20 12 (e) Magnesium Stearate 5 3 500 300 Porraulation. B aw/tablet w/ublet (a) Active ingredient 250 250 (b) Lactose 150 - (c) Avicel PH 101 60 26 (d) Povidone B.P. 15 9 15 (e) Sodium Starch Glycollate 20 12 (f) Magnesium Stearate 5 3 500 300 Formulation c nq/tablet Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesium stearate 4 J55 The following formulations, D and E, were prepared by direct compression of the admixed ingredients. The lactose used in formulation E was of the direct compression type (Dairy Crest - "Zeparox").

Formulation D Active Ingredient mg/capaule 250 Pregelatinised Starch NF15 150 400 Fonwulation a Active Ingredient Lactose nq/capsule 250 150 Avicel 100 500 Formulation F (Controlled Release Formulation) The formulation was prepared by wet granulation of the 20 ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. nq/tablet (a) Active Ingredient (b) Hydroxypropylmethyl-cellulose 500 (Methocel K4M Premium) 112 . (c) Lactose B.P 53 (d) Povidone B.P.C. 28 (e) Magnesium Stearate 7 700 Drug release took place over a period of about 6-8 hours and was complete after 12 hours.

Exatle 2i Capsule Formulations Formulation A A capsule formulation was prepared by admixing the ingredients of Formulation D in Example 1 above and filling into a two-part hard gelatin capsule. Formulation B (infra) was prepared in a similar manner.

Fotwlatlon B mq/eapaul« (a) Active ingredient 250 (b) Lactose B.P. 143 5 (c) Sodium Starch Glycollate 25 (d) Magnesium Stearate 2 420 Formulation C 10 aw/capsule (a) Active ingredient 250 (b) Macrogol 4000 BP 350 600 Capsules were prepared by melting the Macrbgol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

Formulation D Active ingredient Lecithin Arachis Oil 450 mg/capsule 250 100 100 Capsules were prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Fonwilation 8 (Controlled Release Capsule) The following controlled release capsule formulation was prepared by extruding ingredients a, b and c using a extruder followed by spheronisation of the extrudate and drying. The dried pellets were then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. (a) Active Ingredient ■q/capsule 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 15 (d) Ethyl Cellulose 125 13 513 Example 3i Injectable Formulation Formulation A.

Active ingredient 0.200g Hydrochloric acid solution 0.1M q.s. to pH 4.0 to 7.0 Sodium hydroxide solution 0.1M q.s. to pH 4.0 to 7.0 Sterile water q.s. to 10ml The active ingredient was dissolved in most of the water 10 (3S°-40°C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch was then made up to volume with the water and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed 15 with sterile closures and overseals.

Formulation B Active ingredient 0.125 g Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml Example 4i Intramuscular injection Active ingredient 0.20 g Benzyl Alcohol 0.10 g Glycofurol 75 1.45 g Hater for Injection q.s. to 3.00 ml The active ingredient was dissolved in the glycofurol. The benzyl alcohol was then added and dissolved, and water added to 3 ml. The mixture was then filtered through a sterile micropore filter and sealed in sterile 3 ml anA>er glass vials (type 1).

Example 5t Ingredients Active ingredient Sorbitol Solution Glycerol 10 Sodium Benzoate Flavour, Peach 17.42.3169 Purified Water q.s. to 0.2500 g 1.5000 g 2.0000 g 0.0050 g 0.0125 ml 5.0000 ml The active ingredient was dissolved in a mixture of the glycerol and most of the purified water. An aqueous 15 solution of the sodium benzoate was then added to the solution, followed by addition of the sorbitol solution and finally the flavour. The volume was made up with purified water and mixed well.

Example 6i Suppository Active ingredient (63pm)* Hard Fat, BP (Witepsol H15 Dynamit NoBel) ■a/suppository 250 1770 2020 * The active ingredient was used as a powder wherein at. least 90% of the particles were of 63pm diameter or less.

One-fifth of the Witep'sol H15 was melted in a steam-jacketed pan at 45°c maximum. The active ingredient was sifted through a 200ym sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion was achieved. Maintaining the mixture at 45°C, the remaining Witepsol H15 was added to the suspension and stirred to ensure a homogenous mix.

The entire suspension was passed through a 250Um stainless steel screen and, with continuous stirring, was allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02g of the mixture was filled into suitable plastic moulds. The suppositories were allowed to cool to room temperature.

Example 7 8 Pessaries mq/pessarv Active ingredient 63pm Anhydrate Dextrose Potato Starch 250 380 363 Magnesium Stearate 7 1000 The above ingredients were mixed directed and pessaries prepared by direct compression of the resulting mixture.

Bxanple 8 3' -A»ido-3' -deoxythymidine 2.3' -Anhvdrothymldlne Thymidine (85.4 g: 0.353 mol) was dissolved in 500 ml dry DMF and added to H-(2-chloro-l,1,2-trifluoroethyl)diethylamine (100.3 g; 0.529 mol) (prepared according to the method of D.E. Ayer, J. Med. Chem. 6, 608 (1963)). This solution was heated at 70®c for 30 minutes then poured into 950 ml ethanol (EtOH) with vigorous stirring. The product precipitated from this solution and was filtered. The EtOH supernatant was refrigerated then filtered to yield the title compound, mp. =» 228-230°C. 3 *-A»ldo-3'-daoxvthvnldlne 2,3' -0-Anhydrothymidine (25 g: 0.1115 mol) and NaNj (29 g, 0.446 mol) was suspended in a mixture of 250 ml DMF and.38 ml water. The reaction mixture was refluxed for 5 hours at which time it was poured into 1 liter of water. The aqueous solution was extracted with EtoAc (3 x 700 ml). The EtOAc extracts were dried over NajSO^ filtered and the EtOAc was removed in vacuo to yield a viscous oil. This oil was stirred with 200 ml water providing the title compound as solid which was collected by filtration, mp = 116-118°C.

Eim»1« 9 Antiviral Activity (a) (i) Retrovirus - Induced Malignancy 3'-Azido-3'-deoxythymidine was administered to female BALB/c mice infected with 1.5X10* Pfu of the RVB3 5 strain of Rauscher Murine Leukaemia Virus. Treatment was started 4 hours after infection at dosages of 80 mg/kg intraperitonealXy every 8 hours or 0.5 or 1.0 mg/ml orally in drinking water. Such treatment was found to prevent infection of spleen cells and 10 subsequent development of splenomegaly and also suppressed viraemia. (ii) HTLV-I TM-11 cells (T-cell clone susceptible to HTLV-I infection) were co-cultivated with irradiated, HTLV-I 15 producer MJ-tumour cells as follows: a) TM-11 cells only; b) TM-11 cells and MJ-tumour cells c) TM-11 cells, MJ-tumour cells and 3'-azido-3 '-deoxythymidine (3pM): d) TM-11 cells, MJ-tumour cellB and 3'-azido-3'- deoxythymidine (9yH): e) TM-11 cells, MJ-tumour cells and 3"-azido-3'- deoxythymidine (27yM).

On day 18, total DNA was extracted from each culture and 5 digested with Bam HI to generate a fragment of the HTLV-I genome, independent of any host flanking sequence and having a standard molecular weight of 3.3 kD. The digest was then probed with radio-labelled lambdha MT-2, a standard probe recognising the Bam HI fragment of HTLV-I.

No hybridisation was observed for a), indicating a lack of virus in the uninfected control. A strong signal was seen for b), the untreated, infected control. A weak signal was observed with c), indicating incomplete eradication of the virus, and no hybridisation was noted in d) or e) 15 indicating complete extermination of the virus.

Each culture was also probed with a probe for T-cell receptor 3 chain, with a strong signal being generated for all cultures, showing the continued presence of TM-11 for the duration of the experiment. (b) AIDV (i) H»ver»e Transcriptase Activity 3"-Azido-5'-triphosphate-3'-deoxythymidine was tested in vitro against AIDV transcriptase (AIDV RT).

AIDV RT was purified from pelleted and extracted AIDV by elution through DFAE and phosphocellulose columns. The enzyme activity was linear through 60 minutes and stable for at least 2 months when stored in 60% glycerol and 1 mg bovine serum albumin per ml. Using rA-odT (12-18) as the template-primer, AIDV RT had a pH optimum of 7.0 to 7.3, a MnCl2 optimum of 0.3 mM and a MgCl2 optimum of 5 mM. The activity in the presence of 5 mM MgCl2 was 10-fold greater than the activity in the presence of .0.3 mM MnCl2. Maximal enzyme activity was also found in 80 to 140 mM KC1 and 60 to 100 mM NaCl. Incorporation of C^h3 dTTP was linear with respect to enzyme concentration. When tested, 3'-azido-5'-triphosphate-3'-deoxythymidine was found to be a competitive inhibitor of AIDV RT, giving a Ki of 0.04 M when using rA-odT (12-18) as the template-primer. The enzyme had a Km for dTTP of 2.81 l£(, suggesting that 3 '-azido-5 '-triphosphate-3 '-deoxythymidine binds tighter to the enzyme than does dTTP. Further experiments with the RT1 s of avian myeloblastosis virus, Moloney murine leukaemia virus and AIDV, showed 3 1 -azido-5 '-tr iphosphate-3 ' -deoxythymidine to be a terminator of DMA chain elongation. (ii)In Vitro Anti-AIPV Activity 3'-Azido-3'-deoxythymidine was tested and found to possess activity in a number of in vitro assay systems. Drug effects were measured by assaying reverse transcriptase (RT) activity in the supernates from infected, uninfected, and drug treated cells. 3'-Azido-3'-deoxythymidine effectively blocked the infection by AIDV of the H9 and U937 human lymphoblastoid cell lines at concentrations from 2.7 to 0.0013 mcg/ml. Similarly, infection of normal FHA stimulated white blood cells and cultured peripheral blood lymphocytes was inhibited at drug concentrations as low as 0.013 mcg/ml. Drug addition and subtraction experiments in H9 cells revealed that 3'-azido-3'-deoxythymidine was most effective when present at the time of virus infection of susceptible cells, but still retained most of its antiviral activity even when added as late as 20 hours after initial AIDV infection. Inhibition of viral replication was also evident when the drug was present in the media only during the 20 hour period of virus absorption.

Effects were seen at 0.13 and 0.013 mcg/ml. 3'-Azido-3'-deoxythymidine exhibited no direct anti-RT tctivity against purified AIDV virions. Similarly, the drug had little or no effect on the production and release of virions from the chronically infected H9 AIDV cell line. (ili)Preventing Infection by AIDV The ability of 3 ' -azido-31 -deoxythymidine to block infection of cells by AIDV was determined as follows.

Cloned T4 positive tetanus specific T helper lymphocytes were infected with a pool, of AIDV isolates [at challenge 10 doses of up to 5000 virions/cell] and cell survival after infection was monitored. After 10 days in culture no viral cytopathic effects were seen in infected T cells treated with 8.8 and 1.3 mcg/ml 31-azido-3'-deoxythymidine, while untreated, infected cells were 5-15 fold decreased. Cell survival was also evaluated in an HTLV-I transformed, AIDV superinfected cell line derived from the cells above. 3'-azido-31-deoxythymidine at concentrations of 2.7, 0.27 and 0.13 mcg/ml totally blocked cytopathic effects at 7 days. Protective effects 20 were seen in infections induced by both cell free virions and cell associated virus. 3'-Azido-3'-deoxythymidine at 0.27 mcg/ml concentration also effectively prevented cytopathic effect induction by a less related Haitian isolate of AIDV.

Bxaaple 10 Toxicity Assay 3 '-Azido-3 '-deoxythymidine was administered to both mice and rats. The LD50 value was in excess of 750 mg/kg in both species.

Claims (21)

1. Use of 3 ' -azido-3' -deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of a human retrovirus infection. 5

2. Use of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of AIDS.

3. Use of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of 10 PGL.

4. Use of 3 '-azido-3' -deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of AIDS-related complex.

5. Use of 3'-azido-3'-deoxythymidine, as claimed in claim 15 1 wherein the infection is an AIDV infection.

6. Use of 3'-azido-3'-deoxythymidine, as claimed in claim 1 wherein the infection is a human T-cell lymphotropic virus infection.

7. 20 Use of 3'-azido-3'-deoxythymidine, as claimed in claim 1 wherein the infection is an HTLV-I or HTLV-II infection. -37-

8. Use of 3'-azido-3'-deoxythymidine, as claimed in claim 1 wherein the infection' is an HTLV-I positive leukaemia or lymphoma.

9. Use of 3 '-azido-3 ' -deoxythymidine in the manufacture 5 of a medicament for the treatment or prophylaxis of the AIDV carrier state.

10. Use of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment of an human subject ' having anti-AIDV antibodies. 10

11. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of human retrovirus infections.

12. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of AIDS.

13. 3'-Azido-3'-deoxythymidine for use in the treatment or 15 prophylaxis of progressive generalised lymphadenopathy.

14. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of AIDS-related complex. 20

15. 3'-Azido-3'-deoxythymidine for use in prophylaxis of an AIDV infection. the treatment or -38-

16. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a human T-cell lymphotropic virus infection.

17. 31-Azido-3'-deoxythymidine for use in the treatment or 5 prophylaxis of an HTLV-I or HTLV-II infection.

18. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of the AIDV.carrier state.

19. 3'-Azido-3'-deoxythymidine for use in the treatment of an human subject having anti-AIDV antibodies.

20. The use of 3'-azido-3'-deoxythymidine substantially as 10 hereinbefore described with reference to the accompanying Examples.

21. 3 *-azido-3 ' -deoxythymidine for the use substantially as hereinbefore described with reference to the Examples.;Dated this 14th day of March 1986 cmcyslmK * co., BY:JLpjJ nJ\sy_9— EXECUTIVE Agents for the Applicant 1, Holies Street. Dublin 2.