IE880209L - Pharmaceutical formulation - Google Patents

Description

4 4 The invention relates to pharmaceutical!/ acceptable derivatives of 3'-azido-3'-deoxythymidine and in k particular to a pharmaceutical formulation comprising the derivatives, a process for the preparation of the 5 pharmaceutical formulations, novel pharmaceutically acceptable derivatives, processes for the preparation of the derivatives and their use in the. treatment or prophylaxis of human retroviral infections.

Retroviruses form a sub-group of RNA viruses which, in 10 order to replicate, must first "reverse transcribe" the RNA of their genome into DNA ("transcription" conventionally describes the synthesis of RNA from DNA).

Once in the form of DNA, the viral genome is incorporated into the host cell genome, allowing it to take full IS 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 ' 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-I and HTLV-II are both retroviruses and are known to be causative agents of leukaemia in man. HTLV-I infections are especially widespread and are responsible 5 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 virus, and it is currently known either as human T-cell 10 lymphotropic virus III (HTLV III), AIDS-associated retrovirus (ARV), or lymphadenopathy associated virus (LAV). It is anticipated that the name to be agreed on internationally is acquired immune deficiency virus (AIDV)■ This virus (referred to herein as AIDV) has been 15 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 of the iranune system, reducing his ability to combat other 20 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, 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 discovered in patients expressing classical AIDS symptoms 5 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 10 indication of infection is the presence of anti-AIDV antibodies in the blood-stream. It is believed that such "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 15 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, 20 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 25 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. These include antinoniotungstate, suramin, ribavirin and 5 isoprinosine, 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 state,- it will persist as long as the host cell survives, 10 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 15 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 experiments, and Ostertag et al. (Proc. Mat. Acad. Sci. 20 (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 by DNA viruses". However, De Clerq et al. (Biochem. 25 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). Glinski et al. {J. Org. Chem. (1973), 38. 4299-4305) discloses certain derivatives of 3'-azido-3 ' -5 deoxythymidine (infra) and their ability to block mammalian exoribonuclease activity. He have now discovered that 3'-azido-3'-deoxythymidine has a surprisingly potent activity against human retroviruses, with a particularly high activity against AIDV as 10 demonstrated by the experimental data referred to below. Such activity renders the compound useful in the therapy of human retroviral infections. 3'-azido-3'-deoxythymidine is a compound of the formula I O a pharmaceutical formulation comprising, as active ingredient, a pharmaceutically acceptable derivative of 3 '-azido-3'-deoxythymidine and a pharmaceutically acceptable carrier therefor.

In another aspect the invention provides a process for the preparation of the formulation according to the invention comprising bringing a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine into association 5 with a pharmaceutically acceptable carrier.

IP In a further as p e ct the invention provides ■« pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine which, upon administration to a human subject, are capable of providing (directly or indirectly) 10 3'-azido-3'-deoxythymidine, or an anti-retrovirally active metabolite or residue therefor, other than the following 5'-derivatives, namely the monophosphate, disodium monophosphate, 2-cyanoethyl monophosphate, triphosphate, p-toluene sulphonate, acetate, methanesulphonate and 15 triphenylmethyl derivatives and where the 5'-C of 3'-azido-3'-deoxythymidine is linked to a further nucleotide or nucleoside derivative.

Activity of 3'-azido-3'-deoxythymidine against human retroviruses has been established in various in vitro 20 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 * 0.013 mcg/ml up to 20 hours after infection. AIDV infection of U937 human lymphoblastoid cells, PHA-25 stimulated white blood cells and cultured peripheral blood lymphocytes is also prevented at similarly low concentrations. In addition, 10-day challenge experiments using up to 500 AIOV virions per cell and cloned T4, tetanus-specific, T-helper lymphocytes, showed no decrease in cells treated with 3'-azido-3'-deoxythymidine, while 5 untreated cells had decreased 5-fold. Cytopathic effects v were also completely blocked in the same cell-line 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 10 the triphosphate of 3'-azido-31-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 15 is both unusual and valuable for the treatment and prophylaxis of CNS infections caused by human retroviruses.

The ability of 3 '-azido-3 1-deoxythymidine to modify the course of retrovirus-induced malignancy has been 20 demonstrated in a mouse model, whereby administration of 3'-azido-3'-deoxythymidine prevented splenomegaly caused ♦ by intravenously administered Rauscher Murine Leukaemia Virus, the murine equivalent of HTLV-I. In further experiments, 3'-azido-3'-deoxythymidine has been shown to 25 inhibit the in vitro replication of HTLV-I at concentrations as low as 0.8 mcg/ml.

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

By "a pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound which, upon administration to a human subject, is capable of providing (directly or 15 indirectly) 31-azido-31-deoxythymidine or an anti-retrovirally active metabolite or residue thereof. An example of a non-ester compound is the derivative wherein the 5' -C- and 2-C-atoms are linked by an oxygen atom to form an anhydro group.

Preferred esters of the compound of formula (I) include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain alkyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl 25 (e.g. phenyl optionally substituted by halogen, Cj^ alkyl or Cx-4 alkoxy); sulphonate esters such as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl); and mono-, di-or tri-phosphate esters. With regard to the above-described esters, unless otherwise specified, any alkyl 5 moieties present in such esters advantageously contain 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group. Any reference to any of the above compounds also includes a reference to a 10 pharmaceutically acceptable salt thereof.

Experiments have shown that 31-azido-3'-deoxythymidine is converted, in vivo, by the action of cellular enzymes into the 5'-monophosphate. The monophosphate is then further phosphorylated by other enzymes to form the triphosphate 15 via the diphosphate, and other studies have demonstrated that it is the triphosphate form of 3*-azido-3*-deoxythymidine which iB believed to be the effective chain terminator in the reverse transcription of AIDV, as evidenced by its effect on avian myeloblastosis virus and 20 Moloney murine leukaemia virus. This form also inhibits AIDV reverse transcriptase in vitro whilst having a negligible effect on human DNA polymerase activity.

Examples of pharmaceutically acceptable salts of the compound of formula (I) and its pharmaceutically 25 acceptable derivatives 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 NX] (wherein X is C1-4 alkyl).

Specific examples of pharmaceutically acceptable derivatives of the compound of formula (I) that may be 5 used in accordance with the present invention include the monosodium salt and the following 5' esters: monophosphate; disodium monophosphate; diphosphate; triphosphate; acetate; 3-methyl-butyrate; octanoate; palmitate; 3-chloro benzoate; benzoate; 4-methyl benzoate; 10 hydrogen succinate; pivalate; and mesylate.

A pharmaceutically acceptable derivative of 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 15 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 recipient, the nature of 20 the infection and the chosen active ingredient.

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 '25 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or ■ore 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 5 mg, preferably 10 to 1000 mg, and most preferably 20 to 700 mg of active ingredient per unit dosage form.

Experiments with 3'-azido-3'-deoxythymidine suggest that a dose should be administered to achieve peak plasma concentrations of the active compound of from about 1 to 10 about 75 vM, preferably about 2 to 50 While it is possible for the active ingredient to be 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 25 carriers thereof and optionally other therapeutic agents. 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 5 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 step of bringing into association the active 10 ingredient with th 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 15 necessary shaping the product.

Formulations of the present 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 20 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 emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Oral formulations may further include other agents 25 conventional in the art, such as sweeteners, flavouring agents and thickeners.

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 5 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-linked povidone, cross-linked sodium carboxymethyl cellulose), 10 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 aB to provide slow or controlled release of 15 the active ingredient therein using, for example, hydroxypropylmethylcedllulose in varying proportions to provide the desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in 20 a favoured basis, usually sucrose and acacia or tragacanth; pastilleB 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 5 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, 10 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 agentB. The formulations may be presented in unit-dose or multi-dose 15 sealed containers, for example, 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 20 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 unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active 25 ingredient.

The administered ingredients may also be used in therapy in conjunction with other medicaments such as 9-[[2-hydroxy-l-(hydroxy-methyl)ethoxy3methyl]guanine, 9-(2-hydroxyethoxymethyl)guanine (acyclovir), 2-nmino-9-(2-5 hydroxyethoxymethyl)purine, interferon, e.g.,a-interferon, interleukin II, and phosphonof ormate (Foscarnet) or in conjunction with other immune modulating therapy including bone marrow or lymphocyte transplants or medications such as levamisol or thymosin which serve to 10 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 15 the art of formulation.

The invention also provides a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine for use in the treatment of prophylaxis of human retrovirus infections particularly AIDS.

Pharmaceutically acceptable derivatives of a compound of formula I may be prepared in 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; M. Imazawa et al., J. Org. 25 Chem. 43 (15) (1978) 3044-3048; K.A. Watanabe et al., J.

Org. Chem., 45, 3274 (1980); and R.P. Glinski et al., J. Chem. Soc. Chem. Commun., 915 (1970), as well as the processes described in the Examples.

In a further aspect the invention provides a process for 5 the preparation of the pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine according to the invention comprising either A) reacting a compound of the formula: HO (wherein H represents a precursor group for the 3'-azido 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 0 (B) reacting a compound of formula: CH, (II) (III) (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 desired group: or (C) reacting a compound of formula A CH, (IV) or a functional equivalent thereof, with a compound serving to introduce the desired ribofuranosyl ring at the 1-position of the compound of formula (IV); or (D) reacting a compound of formula CH, (wherein R1 is hydroxy or R as defined above), with an agent under conditions serving to convert the said compound into 3 '-azido-3'-deoxythymidine or a pharmaceutically acceptable derivative thereof; (V) and (i). when 3 '-azido-31-deoxythymidine is formed, converting the said compound into a pharmaceutically acceptable derivative thereof, 5 or (ii) when a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine is formed, optionally converting the said derivative simultaneously or thereafter into 3 '-azido-3'-deoxythymidine or a 10 different derivative thereof.

In the above-described process according to the invention, 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 15 are known in the art of 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 20 a 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 M in the 25 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 (1), a 5 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-halogen (eg chlorine) starting. 10 material may be obtained for example by reaction of the corresponding 3'-erythro-hydroxy compound with for example tri.phenylphosphine and carbon tetrachloride, or alternatively by treatment with organosulphonyl halide (eg trif1uoromethanesulphonyl chloride) to form a 15 corresponding 31-erythro-organosulphonyloxy compound which is then halogenated. Alternatively a 3'-threo-hydroxy compound of formula (II) may be treated, for exanqsle with triphenylphosphine, carbon tetranibromide and lithium aside to form the corresponding 3'-erythro azido compound. 20 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, 25 or an ether group such as a trialkylsilyloxy group, e.g. t,-butyldimethylsilyloxy or an aralkoxy group e.g. 21 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'-deoxythymmidine.

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 acetoxy or 10 benzoyloxy or halo, eg chloro group and B represents an optionally protected hydroxy group eg a p-toluenesulphonyloxy group), and subsequently removing any protecting groups.

Regarding process (D), R1 may represent a precursor group 15 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, advantageously in an appropriate solvent such as moist DMF followed by acid or base hydrolysis advantageously under 20 mild conditions. 3 '-Azido-3 '-deoxythymidine may be converted into a pharmaceutically acceptable phosphate, or other eater by reaction with respectively a phosphorylating agent, e.g. POCI3 or an appropriate esterifying agent, e.g. an acid halide -or anhydride. The compound of formula (I)., including esters thereof, may be converted into pharmaceutically acceptable salts thereof in conventional manner, e.g. by treatment with an appropriate base. An ester or salt of 3'-azido-3'-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 pharmaceutically acceptable derivative of a compound of formula (I).

Example 1; Tablet Fornulations 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.

FOr—ilation A (a) Active ingredient (b) Lactose B.P. (c) Povidone B.P. w/Ublet 250 210 15 (d) Sodium Starch Glycollate 20 (e) Magnesium Stearate 5 n/ tablet 250 26 9 12 3 500 300 For—ilation B I/table* (a) Active ingredient 250 (b) Lactose 150 (c) Avicel PH 101 60 (d) Povidone B.P. 15 15 (e) Sodium Starch Glycollate 20 (f) Magnesium Stearate 5 mm/ tablet 250 26 9 12 3 500 300 Formulation C lag/tablet Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesium stearate 4 359 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/capsule 250 Pregelatinised Starch NF15 150 400 Formulation E mg/capsule Avicel Active Ingredient Lactose 250 150 100 500 Fwulation F (Controlled Release Formulation) The formulation was prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. (a) Active Ingredient teg/tablet 500 (b) Hydroxypropylmethyl- cellulose (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.

Kxanple 2t 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.

Formulation B ■g/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) Sodium Starch Glycollate 25 (d) Magnesium Stearate 2 420 Fonsulatlon c an/capsule (a) Active ingredient 250 (b) Macrogol 4000 BP 350 600 Capsules were 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.

Formlation p ■q/capaule Active ingredient 250 Lecithin 100 Arachis Oil 100 450 Capsules were 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 was 5 prepared by extruding ingredients a, b and c using an extruder followed by spheronisation of the extrudate and drying. The dried pellets were then coated with release-controlling meinbrane (d) and filled into a two-piece, hard gelatin capsule. mq/capsule (a) Active Ingredient 250 (b) Macrocrystalline Cellulose 125 (c) Lactose BP 125 (d) Ethyl Cellulose 13 513 Example 3s 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. ■. to 10ml The active ingredient was dissolved in most of the water (35°-40°c) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as 5 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 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 4: 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 was dissolved in the glycofurol. The benzyl alcohol was then added and dissolved, and water 20 added to 3 ml. The mixture was then filtered through a sterile micropore filter and sealed in sterile 3 ml amber glass vials (type 1).

Exwple 5» Inqredlent-B Active ingredient Sorbitol Solution Glycerol Sodium Benzoate Flavour, Peach 17.42.3169 Purified Hater 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 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 ■g/suppository Active ingredient (63um)* 250 Hard Fat, BP (Witepsol H15 - Dynamit NoBel) 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 Witepsol H15 was melted in a steam-jacketed pan at 45°C maximum. The active ingredient was sifted through a 200|im 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 HIS was added to the suspension and stirred , to ensure a homogenous mix. The entire suspension was passed through a 250i*n 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 7t ' Pessaries ■g/pessary Active ingredient 63pm 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 1000 The above ingredients were mixed directed and pessaries prepared by direct compression of the resulting mixture.

Example 8 3' -Azldo-3' —deoxy-5' -0-otrtanovltJiY"',|',,i "** To a solution of 3'-azido-3'-deoxy thymidine in pyridine (0°c), octanoyl chloride (1.2 equivalents) was added. The reaction was allowed to warm to room temperature. When tic (CHCI3 :MeOH :20:1, on silica gel) indicated complete reaction, the solution was poured onto ice water. The aqueous phase was decanted. The resulting oil was chromatographed on silica gel eluted with CHCHl3:MeOH. The title compound was obtained as an oil by evaporation 5 of the solvent from the appropriate fractions.

CHN:cal. C-54.95 H-6.92 N-17.80 fnd. C-54.82 H-6.96 N-17.66 07.46(d,lH.J5|e°lHz,6H). a 6•13(t,1H,1'H) , a 4. 5-4.2(m,3H,3'H and 5'CH2) o4.0-3.8(m.lH,4lH), 02 . 3-2.1 (m, 4H, 2 ' H and (CH2)1 of octanoyl)), ol.8l(d,3H,J5r6=1.0Hz, 5CH3), a1-5-0.6(m,13,H, 5' octanoyl (CH2)5CH3) Bxanle 9 5 * —Acetyl-3 * azldo-3 * -deoxythymidine To a solution of 31-azido-31-deoxythymidine (20g) in pyridine (50ml) at ambient temperature, acetyl chloride (2.1 equivalents) was added. The reaction was stirred for two hours and kept at 0 to 5°C for 20 hours. It was poured onto ice water with stirring. The aqueous phase 20 was decanted. The oily product was dissolved in water and extracted with water (5 times), 0.5 N hydrochloric acid, water (2x), and dried over magnesium sulphate. The solution was filtered and evaporated in vacuo. The residual oil was dissolved in chloroform, applied to a 25 silica gel column, and flash chromatographed using 2% methanol in chloroform. Fractions with product were evaporated and the oil was chromatographed again using ethyl acetate: hexane (6:4 v/v). Fractions with product were evaporated in vacuo to a white solid. m.p. 96-98°C cal. C-46.60 H-4.89 N-22.65 fhd. C-46.67 H-4.94 S-22.59 Example 10 The following compounds were prepared according to the 10 procedure of Example 8 or 9 as appropriate from the appropriate acid halid or anhydride. 3•-Azido-S *-0-benzoyl-3'-deoxythymidine cal. C-S3.68 fnd. C-53.81 m.p. 54-59°C 3'-Azido-3' -deoxy-5'fi-pivaloylthymidine cal. C-51.27 H-6.03 N-19.93 fnd. C-51.07 H-6.05 N-19.83 m.p. 99-100°C 3'-Acido-3'-deoxy-5-fl-(3-methylbutyryl)thymidine cal. C-50.24 H-6.13 N-19.53 fnd. C-50.27 H-6.11 H-19.49 H-4.77 11-18.41 H-4.72 N-18.46 -32A- 67.46(d.lH.J,.t=1.2H«.6H), 66.13(t,lH,l'H), 64.55-4.15(m,3H,3'H and S'CHZ), 83.8-4.15(m,lH,4'H), 62.4-1.78(m,3H,2'H and 5' methine), 61.80(d,3H,Js.6~1.2Hz.5CH,), 60.9(d,6H,J-6.4Hz, methyls on 5' butyryl) 5 3' -Azido-3'-deoxy-5-Q-palmitoylthymidine cal. C-61.67 H-8.57 S-13.85 fnd. C-61.85 H-8.59 N-13.75 87.45(d, 1H, J3.6=»1.0Hz,6H) , 66.12(t,lH,l'H), S4.S-4.05(m,3H,3'H and 5'CH2), 64.0-3.8(m,lH,4'H), 62.35-2.11(m,4H,2'H and (CHZ)1 10 of palmitoyl), 61.8 d, 3H, Js.(=*l. 0Hz, 5CH,), 61.35-0.6(m,29H,5' -palmitoyl (CH2) 13CH3) 3'-Azido-3'-deoxy-5-O-toluyl thymidine Cal. C-56.10; H-4.97; N-18.17 Fnd. C-5S.88; H-5.00; N-18.09 15 m.p. 73°C NMR taken in DMSO-d( NMR; 67.95-7.29 (m,5H; bH,cH,6H), 66.16 (t,lH,l'H), 64.6-4.4 (m,3H,3'H,5'H), 64.2-4.0 (m,lH,4'H), 62.39 (s,3H,dCH3), 61.63 (s,3H,5CH,) 20 3'-Azido-3'-deoxythymidine 5'-0-(hydrogen succinate) Cal. C-44.98; H-4.83; N-17.96 Fnd. C-44.90; H-4.77; N-17.85 NMR taken in DMSO-dj NMR; 67.46 (s,lH,6H), 66.13 (m,lH,l'H), 64.48-4.40 (m,lH,3'H), 25 64.34-4.20 (m,2H,5'H), 63.99-3.94 (m,lH,4'H), 61.78 (s,3H,5CHs) 3' -Azido-3'-deoxy-5'-mesylthymidine Cal. C-38.25; H-4.37; N-20.28; S-9.28 Fnd. C-38.15; H-4.38; N-20.19jS-9.30 m.p. 253°C (dec.) NMR taken in DMSO-d6 NMR: a7.49(d,lH,J6f5«l:0 Hz, 6H). a6 . 1 5 (t.lH.Ji.Hz, 1*H), 0 4.54-4.41 (m, 3H; 3 ' H, 5 ' H) , cr4.14-4.02 (m,lH,4'H). 03.24 (s,3H,5'-mesyl CH3), a 1.79 (d. 3H.J5#6»1.0 Hz, 5CH3) 3' -Azido-S' -0- (3-chlorobenzoyl) -3' -deoxythymidine Cal. C-50.31; H'3.97; N-17.26; Cl-8.74 Fnd. C-50.16; H-4.03; N-17.13; Cl-8.66 NMR taken in DMSO-dg NMR: oil.37 (b,1H,3-NH), a 7.98-7.43 (m,5H;5'- phenyl,6H), o6.17 (dd,lH); J11,2a'•1 H*» Jl',2b'"7-2 Hz, l'H), o4.68-4.48 (m,3H;3'H,5'H), 04.14-4.11 (m,1H,4'H), 02.48-2.41 (m,2H,2'H), 01.64 (d,3H,J5f6-1.2 Hz, 5CH3) Sxawple XI 2.5' —O—Anhydro-3 * -agido-3' -deoxythvldlne 3'-Azido-3'-deoxythymidine (3.0g, 11.2 mMol) was mesylated by the addition of methanesulphonyl chloride (2.7 nL) to a solution of the starting material' in dry pyridine (2mL). The reaction was allowed to proceed at 5°C for one hour, then poured onto ice water. The precipitate was collected 15 by filtration. The product (3'-azido-5'-mesylthymidine) was reacted with potassium carbonate (0.78 g, 5.6 mMol) in DMF (75 mL). The reactants were heated in an 80°C oil bath for six hours, then poured into ice water.. The product was extracted from the water with ethyl acetate. 20 The solvent was removed in vacuo and the resultant oil was flash chromatographed on silica gel by elution with CHCl3:Me0H (9:1 v/v). The title compound was obtained as a solid after evaporation of the solvent from the appropriate fractions. m.p. - 184 - 186°C gxawple 12 3 * -Azido-3 * -daonrthMriJliw a) 2.3' -Anhydrothvnld ine Thymidine (85.4 g: 0.353 mol) was dissolved in 500 ml dry DHF and added to N-(2-ch1oro- 1 , 1 , 2 -trifluoroethyl)diethylamine (100.3 gs 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. (b) 3'-Agido-3 * -deoxythymidine 2,3' -0-Anhydrothymidine (25 g: 0.1115 mol) and NaN3 (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, nip « 116-118°C.

E»agle 13 Honoaodlam Salt of 3 '-A« ido-3 ' -deoxythymidine Approximately lg of 3 1-azido-3'-deoxythymidine was dissolved in 50mL of distilled water. The pH was adjusted 5 to 12 with IN NaOH. Approximately half of the solution was freeze-dried. The title compound was obtained as a lyophilised powder.

Analysis calculated for CioH12N5Na04 6/10 N2O cal: C-40.03; H-4.43; N-23.34; Na-7.66 10 fnd: C-39.88; H-4.34; N-23.23; Na-7.90 SxMple 14 Preparation of 5' -H(aopho«|)hat« of 3' -Asldo-3 * —deoxythyldlne 3'-Azido-3'-deoxythymidine (0.5 g, 1.87 mmol) was dissolved in 5 mL of triethyl phosphate and the mixture 15 was cooled to -5°C. Phosphorus oxychloride (0.685 mL, 7mmol) was added in one portion to the rapidly stirred solution which was then maintained at -10°C for 22 hours. An aliquot was removed and added to concentrated ammonium hydroxide. Analysis of this sample on TLC (cellulose, n-20 PrOHtHjO, 7:3 v/v) showed no remaining starting material and a single fluorescent spot with lower mobility than the nucleoside. The reaction mixture was poured onto 20mL of ice and water. This was placed in an ice bath and the pH of the solution was adjusted to a value of 7.5 by the addition of 2N NaOH. The basic mixture was extracted once with chloroform and once with ether. The aqueous layer was again adjusted to give a pH of 7.5 and concentrated in vacuo to remove residual organic solvent. The material 5 was stared at -10°C until purified as follows: Deactivated charcoal was prepared by washing coconut charcoal (50-200 mesh, 100 g) with 500 mL of 1 N HC1, 3 L of water, 35 mL of 3% toluene in 95% ethanol, 600 mL of 95% ethanol and finally extensively with water. 10 Deactivated charcoal (12 mL of settled wet charcoal) was added with stirring to the monophosphate solution (0.72 g, 1.8 mmol, 30 mL). The supernatant was decanted and the charcoal was washed with 150 mL of water. The nucleotide was eluted from the charcoal by washing with 120 mL of 1.5 15 M ammonium hydroxide in 50% ethanol. This solution was filtered through a 0.22 micron filter, concentrated in vacuo to 10 mL, filtered through a Amicon Centriflo CF- 25 membrane, and lyophilized to yield the diammonium 3'-azido-3'-deoxythymidine-5'-monophosphate as a solid. This 20 compound was characterised as a nucleoside 5'-monophosphate by the ability of 5'-nucleotidase to degrade it to the nucleoside.

Bxainple 15 3 * -Agido-5 *-trlpho»phat»-3 * -fleoivthvMld ine a n d - 3 ' -Aitido-5 ' - <8 i phoi pha t e - 3 ' -deoxvthvaildlne (a) Bis (trl-n-butylaaoionluia) pyrophosphate A column of DOW 50 ("Dowex" ion exchange resin - DON Chemical Laboratories) pyridinium resin was prepared by pouring 40 mL of resin into a 25 cm diameter column and washed with water until no more colour eluted.

' Pyrophosphate decahydrate (1.12 g, 2.51 mM) was 10 dissolved in 30 mL of water and applied to the column.

The column was eluated with water. A 125 mL fraction of the eluant which contained UV absorbing material was collected. The volume was reduced to 10 mL in vacuo and tri-n-butylamine (1.2 mL) was added. The 15 volume was reduced in vacuo and the residue was dried by coevaporation with pyridine four times. The product was stored in a freezer (-5°C). (b) Hydrogen Form of 3 ' -Agldo-5 ' -monophoshate-3 1 -deoxythymidine The hydrogen form of the monophosphate was prepared by passing the anmonium salt obtained in Example 14_ (0.1 g. 0.283 mMol) dissolved in 6 mL of water, through a 1.5 mL (10 eq.) column of DOW 50 H+. -as- tc) IPteosphoromorpholidate Derivative of 3'-Agjdo-3' -fleomhyaidlng In 9 mL of water was dissolved 0.283 mMol of the hydrogen form of the monophosphate obtained in stage b) . Morpholine (99 PL, 1.13 mMol, 4 eq.) was added and the Bolution heated to reflux. Oicyclohexyl carbodiimide (0.234 g, 1.13 mMol, 4 eq.) dissolved in t-butanol (5 mL) was added over a three hour period. The reaction was refluxed overnight. The reaction was cooled to room temperature, filtered, and the solvents removed in vacuo. Ethanol was added and evaporated in vacuo four times. The residue was dissolved in methanol and the phosphoromorpholidate precipitated by the addition of ether. The precipitate was triturated with ether four times and dried on a rotary evaporator. The title compound was obtained. (d) 3*-htldo-3'-deoxythyi.dine-5'-triphosphate The phosphoromorpholidate derivative obtained in stage c), was dried by a removal of pyridine in vacuo four times. The bis (n-Bu)3N pyrophosphate obtained in stage a) was also dried by removal of pyridine in vacuo. The phosphoromorpholidate was dissolved in pyridine, 5 mL, and added to the vessel containing the pyrophosphate reagent. The reaction was allowed to continue overnight at room temperature. The pyridine was removed in vacuo. Water was added to the residue and removed in vacuo three times. The residue was frozen.

The residue was thawed and dissolved in 50 mL of water. 5 The solution was applied to a column (1 x 10 cm) of DFAE Saphadex A-25 which had ben equilibrated with 50 mM ammonium bicarbonate. The phosphates were eluted with a 300 nL linear gradient of 50-800 mM ammonium bicarbonate. The fractions containing the diphosphate nucleotide were 10 pooled as were those containing the triphosphate nucleotide. The pooled diphosphate and triphosphate fractions were each dried in vacuo, redissolved in water, dried again, redissolved in water and lyophilized.

KTO)1» 16 Enzymatic Synthesis of 3*-A»ldo-5'-15 triphosphate-3'-deoxvthymidine The 5'-triphosphate was synthesized from the 5 ' -diphosphate using pyruvate kinase and nucleoside diphosphate kinase. The reaction mixture contained: 6mM 3'-azido TOP, 12 mM adenosine triphosphate, 40 mM MgCl2> 20 40 mM potassium piperazine-N,N'-bis(2-ethanesulphonic acid) PIPES buffer (pH 6.8), 30 mM phosphoenolpyruvate, 40 IU/ml nucleoside diphosphate kinase and 100 IU/ml pyruvate kinase in a final volume of 5 mL. The reaction mixture was incubated at 37°C for 5 days. The reaction mixture 25 was applied to a column (2.5 x 10 cm) of DEAE Sephadex A-25 riiich had been equilibrated with ammonium bicarbonate.

The nucleotides were eluted with a gradient of 100 - 1000 mM ammonium bicarbonate. Fractions containing the triphosphate were pooled, and evaporated to t«dryness in vacuo. The compound was further purified using a 5 preoperative HPLC column (Whatman, Inc., Magnun 9 SAX) eluted with a gradient of 10 - 100 mM potassium phosphate, pH 3.5. The resulting compound was further purified using a DFAE Sepahdex A-25 column as above. The fractions containing the tetraamonium 3' -azido-3 '-deoxythymidine-10 51-triphosphate were pooled, dried in vacuo, redissolved in water and lyophilized to yield the title compound.

K»m»l« 17 Antiviral Activity (a) (i) retrovirus — Induced Malignancy 3'-Azido-3'-deoxythymidine was administered to female BALB/c mice infected with 1.5X104 Pfu of the RVB3 strain of Rauscher Murine Leukaemia Virus. Treatment was started 4 hours after infection at dosages of 80 mg/kg intraperitoneally 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 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 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 cells and 3"-a*ido-3'-10 deoxythymidine (9pM); e) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (27PM).

On day 18, total DNA was extracted from each culture and digested with Bam HI to generate a fragment of the HTLV-I 15 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.

Ny hybridisation was observed for a), indicating a lack of 20 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) indicating complete extermination of the virus.

Each culture was also probed with a probe lor T-cell receptor B 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) Reverse 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 MnClj. Maximal enzyme activity was also found in 80 to 140 mM KCl and 60 to 100 mM NaCl. . Incorporation of C3h3 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-I8) as the template-primer. The enzyme had a Km for dTTP of 2.81 UM, suggesting that 3 *-azido-5*-triphosphate-3* -deoxythymidine binds tighter to the enzyme than does dTTP. Further experiments with the RT's of avian myeloblastosis virus, Moloney murine leukaemia virus and AIDV, showed 3 '-azido-5'-triphosphate-3'-deoxythymidine to be a terminator of DNA chain elongation. (ii)In Vitro AntJL-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 PHA 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 5 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'-10 Azido-3 '-deoxythymidine exhibited no direct anti-RT activity 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. (iii)Preventing Infection by AIDV The ability of 3'-azido-3'-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 20 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 3'-azido-3'~ deoxythymidine, while untreated, infected cells were 5-25 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 5 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. 8mw»1« 17 Toxicity Assay 3 1 -Azido-3' -deoxythymidine was administered to both mice and rats. . The LD50 value was in excess of 750 mg/kg in both species.

Claims (43)

1.-46- CIAIHS X. A pharmaceutical formulation comprising, as active ingredient, a pharmaceutically acceptable derivative of 3 1-azido-3'-deoxythymidine and a pharmaceutically acceptable carrier therefor.

2. A formulation according to claim 1 wherein the carrier is other than water.

3. A formulation according to claim 1 or 2 which is sterile.

4. A formulation according to claim 3 adapted for administration by injection.

5. A formulation according to claim 4 contained in a sealed vial.

6. A formulation according to either of claims 3 and 4 wherein the carrier is sterile water.

7. A formulation according to claim 1 or 2 adapted for oral administration.

8. A formulation according to claim 1, 2 or 7 in the form of a tablet or capsule. -47-

9. A formulation according to claim 1, 2 or 3 providing sustained release of the active ingredient after oral administration.

10. A formulation according to claim 7 further comprising 5 a flavouring agent.

11. A formulation according to any of claims 1 to 10 comprising a unit dose of the active ingredient, or a multiple thereof.

12. A formulation according to claim 11 wherein the unit 10 dose is between 5 and 1500 mg of the active ingredient.

13. A formulation according to claim 11 wherein the unit dose is between 10 and lOOOmg of the active ingredient.

14.IS 14. A formulation according to claim 11 wherein the unit dose is between 20 and 700mg of the active ingredient.

15. A process for the preparation of a formulation according to any of claims 1 to 14 which comprises bringing a pharmaceutically acceptable derivative of 20 3 ' -azido-3 ' -deoxythymidine into association with the said pharmaceutically acceptable carrier. -48-

16. Pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine which, upon administration to a human subject, are capable of providing (directly or indirectly) 3'-azido-3'-deoxythymidine, or an anti-5 retrovirally active metabolite or residue thereof, other than the following 5'-derivatives, namely the monophosphate, disodium monophosphate, 2-cyanoethyl monophosphate, triphosphate, p-toluene sulphonate, acetate, methanesulphonate and triphenylmethyl 10 derivatives and where the 5'-C of 3'-azido-3'- deoxythymidine is linked to a further nucleotide or nucleoside derivative.

17. Pharmaceutically acceptable derivatives as claimed in claim 16 in the form of pharmaceutically acceptable 15 salts, esters or salts of such esters of 3'-azido-3'- deoxythymidine.

18. A derivative as claimed in.claim 16 selected from the group consisting of the 5'-diphosphate, 5'-(3-methy1-butyrate), 51-octanoate, 5 ' -palmitate, 5 ' - ( 3 - 20 chlorobenzoate), 5'-benzoate, 5'-hydrogen succinate, and 5'-pivalate esters of 3'-azido-3'-deoxythymidine.

19. A process for the preparation of any of the compounds according to any of claims 16 to 18 comprising either -49- (A) reacting a compound of formula! ® HO (II) (Wherein H represents a precursor group for the 31-atido group) or a derivative (e.g. an ester or salt) thereof, with an agent or under conditions serving jto convert the said precursor grouip into the desired azido group; or 0 (B) reacting a compound of formula R (III) (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 10 to convert the said precursor group into the corresponding desired group: or (C) reacting a compound of formula (IV) -50- or a functional equivalent thereof, with a compound serving to introduce the desired ribofuranosyl ring at the 1-position of the compound of formula (IV); or 0 (D) reacting a compound of formula CH, (V 5 (wherein R1 is hydroxy or R as defined above), with an agent under conditions serving to convert the said compound into 3'-azido-3'-deoxythymidine or a pharmaceutically acceptable derivative thereof; and 10 (i) when 3 '-azido-31-deoxythymidine is formed, converting the said compound into a pharmaceutically accceptable derivative thereof. (ii) when a pharmaceutically acceptable derivative of 15 3 ' -azido-3'-deoxythymidine is formed, optionally converting the said derivative simultaneously or thereafter into 31 -azido-3 ' -deoxythymidine or a differnt derivative thereof. -51-

20. A pharmaceutically acceptable derivative of 3' -azido-31 -deoxythymidine for use in the treatment or - prophylaxis of human retrovirus infections.

21. A pharmaceutically acceptable derivative of 3'-Azido-5 3 ' -deoxythymidine for use in the treatment or prophylaxis of AIDS.

22. A pharmaceutically acceptable derivative of 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of progressive generalised 10 lymphadenopathy.

23. A pharmaceutically acceptable derivative of 3'-Azido-3' -deoxythymidine for use in the treatment or prophylaxis of AIDS related complex.

24. A pharmaceutically acceptable derivative of 3'-Azido-15 3 ' -deoxythymidine for use in the treatment or prophylaxis of an AIDV infection.

25. A pharmaceutically acceptable derivative of 3' -Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a human T-cell lymphotroic virus 20 infection.

26. A pharmaceutically acceptable derivative of 3*-Asides' -deoxythymidine for use in.the treatment or;-52-;prophylaxis of an HTLV-I or HTLV-II infection.;

27. A pharmaceutically acceptable derivative of 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of the AIDV carrier state.;

28. A pharmaceutically acceptable derivative of 3*-Asides' -deoxythymidine for use in the treatment of an human subject having anti-AIDV antibodies.

29. A pharmaceutical formulation substantially as hereinbefore described with reference to the Examples.

30. A process for preparing a pharmaceutical formulation substantially as hereinbefore described with reference to the Examples.

31. Pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine substantially as hereinbefore described with reference to the Examples.

32. A process for preparation of pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine substantially as hereinbefore described with reference to the Examples. - 53 -

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

34. Use of a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of AIDS.

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

36. Use of a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine in the manufacture of . a medicament for the treatment or prophylaxis of AIDS related complex. 15

37. Use of a pharmaceutically acceptable derivative of 3' -azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of an AIDV infection.

38. Use of a pharmaceutically acceptable derivative of 3'- 20 azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of a human T-cell lymphotropic virus infection. - 54 -

39. Use of a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of an HTLV-I infection. 5

40. Use of a pharmaceutically acceptable derivative of 3' -a z ido-3' -deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of an HTLV-I positive leukaemia or lymphoma.

41. Use of a pharmaceutically acceptable derivative of 3'- 10 azido-3'-deoxythymidine in the manufacture of a medicament for the treatment or prophylaxis of the AZOV carrier state.

42. Use of a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine in the manufacture of a 15 medicament for the treatment of a human subject having anti-AIDV antibodies.

43. Use of a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine as claimed in any of Claims 33 to 42 the form of a pharmaceutically acceptable salt, ester 20 or salt of such ester of 3'-azido-3'-deoxythymidine. - 55 - ttae of a pharmaceutically acceptable derivative of 3' -azido-3'-deoxythymidine substantially as hereinbefore described with reference to the Examples. Dated this 14th day of March, 1986 CRUICKSHANX & CO. Agents for the Applicant 1 Holies Street Dublin 2.