IL85096A - Use of 3'-azido-3'- deoxythymidine in the treatment of prophylaxis of human retrovirus infection - Google Patents

Description

Use of 31 -azido-31 -deoxythmidine in the treatment or prophylaxis of human retrovirus infection THE WELLCOME FOUNDATION LIMITED C. 73785 Antiviral Nucleosides The present invention relates to pharmaceutical formulations containing 3 ' -azido-3 ' -deoxythymidine for use in the treatment or prophylaxis of human retroviral infections.

Retroviruses form a sub-group of RNA viruses which, in 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 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.

A species of retrovirus has now been reproducibly isolated from patients with AIDS (Acquired Immune Deficiency Syndrome) . While it has been extensively characterised, there i as yet, no agreed name for the virus, and it is currently known either as human T-cell lymphotropic virus III (HTLV III) , AIDS-associated retrovirus (ARV) , or lymphadenopathy associated virus (LAV) . It was anticipated that the name to be agreed on internationally is Acquired Immune Deficiency .Virus (AIDV) . This virus (referred to herein as AIDV) is in fact now internationally known and recognised as Human Immunodeficiency Virus (HIV) , as proposed by the International Committee on the Taxonomy of Viruses, It has been shown preferentially to infect and destroy T-cells bearing the O T 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 immune 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, and not as a direct result of AIDV infection.

Recently, AIDV (HIV) has also been recovered from _ . 4 other tissue types, including" B-celTs expressing the T 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 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 (HIV) infection. In the initial "carrier" state, the only indication of infection is the presence of anti-AIDV (HIV) 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 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, 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 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 antimoniotungstate, suramin, ribavirin and 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, 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) and Ostertag et al. (Proc. Int. Symposium in Japan, published 1974 - see CA. 82, 168523s, 1975) reported that in in vitro experiments 3 ' -azido-31 -deoxythymidine increased the production of FLV A-type particles but reduced the production of C-type particles. 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 favourably replace bromo- deoxyuridine for medical treatment of diseases caused 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) . Glinski et al . (J. Org. Chem. (1973), 38 , 4299-4305) discloses certain derivatives of 31 -azido-3 ' ^ deoxythymidine (infra) and their ability to block mammalian exoribonuclease activity.

The abovementioned publications by Ostertag et al . as well as a further publication of Ostertag namely the article published in Exp. Cell Res. 1978, 116(1), pp 31-35 (C.A. 90, 37403t) deal with substantially the same subject matter, namely the behaviour of a cell system transformed with a mouse (murine) Friend Leukaemia complex and treated inter alia with interferon and azidothymidine ( 3 ' -azido-3 ' -deoxythymidine) .

First the cell system concerned comprises chronically infected cells, i.e. all of the cells are infected and does not therefore simulate the normal in vivo process of viral infection. Also, the virus complex is not cytopathic to the host cells and again differs _from the type of human retrovirus with which the present invention is concerned (i.e. AIDV - now known as HIV) which is cytopathic to the host cell. The cell system in the references is not therefore a good indicator of the effect of an antiviral agent against acute infection, i.e. where cells are in process of becoming infected.

Secondly, as indicated above, the virus concerned in the references is a mouse virus and the effect of an agent against such a virus provides no indication of its effect against a human virus. Thus, as reported by Gross et al . , Annals of the New York Academy of Science, the antiviral agent ribavirin has an activity value against the murine Friend Leukaemia virus of 3.2 g whereas R. Yarchoan and S. Broder, Strategies for the Pharmacological Intervention against HTLV-III/LAV, pages 335-360 report that ribavirin only provided partial inhibition against (human) HTLV-III virus replication at 100 g/ml. It is therefore well established that a compound which may be active against a (non-human) animal virus will not necessarily have the same activity against a human virus.

The latter point is perhaps illustrated by the conclusion in W. Ostertag et al, Proc. Nat. Acad. Sci. , USA, No. 12, 4980-4985, on page 4984, that "In some instances, azidothymidine might favourably replace BrdUrd for medical treatment of diseases caused by DNA viruses". Despite this statement in 1974, azidothymidine was not recognized as worthy of testing as an antiviral agent until 1980 when de Clerq et al reported in Biochemical Pharmacology Volume 29, pages 1849-1851 the antiviral testing °f inter alia azidothymidine ( 3 ' -azido-3 ' -deoxythymidine) against herpes simplex and vaccinia (both representative DNA virus) and also against vesicular stomatitis virus (an RNA virus) . The compound was completely inactive against herpes simplex and vesicular stomatitis virus and also weakly active against vaccinia. This virus is not directly related to any human virus and in any event the low activity compared with the other compounds tested would clearly rule out this compound for consideration as a useful human antiviral therapeutic agent.

The present invention is based on the discovery that the above compound has potent activity against human retroviruses, which are RNA, not DNA viruses.

Thus at the priority date of the present invention, having regard to the complete state of the art, there was no realistic suggestion or teaching that the compound would be effective against human retroviruses, or even any other human viruses.

We have now discovered that 31 -azido-3 ' -deoxythmidine has a surprisingly potent activity against human retroviruses, with a particularly high activity against AIDV (HIV) as demonstrated by the experimental data referred to below. Such activity renders the compound useful in the therapy of human retroviral infections.

According to the present invention, we provide pharmaceutical formulations comprising as active ingredient 3 '-azido-3'- deoxythymidine and at least one pharmaceutical carrier therefor .

Activity of 3'-azido-3'-deoxythymidine against human retroviruses has been established in various in vitro assay systems. For example, infection o* 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-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 5000 AIDV virions per cell and cloned T4, tetanus-specific, T-helper lymphocytes, showed no decrease in cells treated with 3'-azido-3'^4eoxythymidine, while untreated cells had_decreased 5-fold. Cytopathic effects 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 the triphosphate of 3 ' -az ido- 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 CN5 in ections caused by human 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 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.

Experiments have shown that 3'-azido-3'-deaxythymidine is converted, in vivo, by the action of cellular enzymes into the 5'-monophosphate. The monophosphate is then further phosphoryiated 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 terminator in the reverse transcription of AIDV, as evidenced by its effect on avian myeloblastosis virus and Moloney murine leukaemia virus. This form also inhibits AIDV reverse transcriptase in vitro whilst having a negligible ef'ect on human DNA polymerase activity.

Examples of human retrovirus infections which may be treated or prevented by 31 -az"ido-3 ' de xythymidine using the pharmaceutical formulations according to 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 includes AIDS, AIDS-related complex and HTLV-I positive leukaemia and lymphoma. Suitable patients for treatment also include those having antibodies to AIDV, AIDV CNS infections, PGL and ARC. 3 ' -Az ido-3 1 -deoxythymidine (hereafter re ferred 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 ( inc luding subcutaneous , intramuscular , intravenous and intradermal ) . It will be appreciated that the pre ferred route will vary with the condition and age of the recipient , the nature of 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 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, 'or example, containing 5 to 1500 mg, preferably 10 to 1000 mg, and most preferably 20 to 700 mg of active ingredient per unit dosage form.

Fxperiments 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 about 75 u , preferably about 2 to 50 μΜ, most preferably about 3 to about 30 μ . This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution o* 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 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.

The pharmaceutical formulations according to the present invention may optionally include one or more further therapeutic agents. The pharmaceutically acceptable carriers thereof and optionally other carriers referred to above 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 subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be present in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the 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 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 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 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), 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, hydroxypropylmethylcelluiose 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 flavoured 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 nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophiiized) condition requiring only the- addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. 3 ' -Azido-31 -deoxythymidine may also be used in therapy in conjunction with other medicaments such as 9- [ [2-hydroxy-l- (hydroxy-methyl) ethoxy] methyl] guanine , 9- ( 2-hydroxyethoxymeth l) guanine (acyclovir), 2-amino-9 ( 2-hydroxyethoxymethyl) urine , interferon, e.g. -interferon , interleukin II, and phosphonoformate (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 increase lymphocyte numbers and/or functions as 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) and its pharmaceutically acceptable derivatives 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. Chem, A3 (15) (1978) 3044-3048; K.A. Watanabe et al. , J. Or. Chem., 45, 3274 (1980); and R.P. Glinski et al. , J. Chem. Soc . Chem. Commun. 91_ (1970) as well as the process described in the Reference Exampl described hereafter.

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 3 ' -azido-3 ' -deoxythymidine .

Fxamole 1: Tablet Formulations The 'ollowing 'ormulations A and B were prepared by wet granulation o' the ingredients with a solution o' povidone, followed by addition of magnesium stearate and compression. mo/tablet mo/tablet Formulation A Active ingredient 250 250 Lactose B.P. 210 26 Povidone B.P. 15 9 Sodium Starch Glycollate 20 12 Magnesium Stearate 5 3 500 300 Formulation B mq/tablet mq/tablet (a) Active ingredient 250 250 (b) Lactose 150 - (c) Avicel PH 101 60 26 (d) Povidone B.P. 15 9 (e) Sodium Starch Glycoilate 20 12 (f) Magnesium Stearate 5 3 500 300 Formulation C. - - mq/tablet Active ingredient 100 Lactose 200 Starch 50 ovidone 5 Magnesium stearate 4 359 The fallowing formulations, D and F, were prepared by direct compression of the admixed ingredients. The lactose used in formulation F was of the direct compression type (Dairy Crest - "Zeparox").

Formulation D ma/ta.ble-t Active Ingredient 250 Pregelatinised Starch NF15 150 400 Formulation F ma/;table,t Active Ingredient 250 Lactose 150 Avicel 100 500 Formulation 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.

HDL/LMJ/26th February 1986 mq/tablet (a) Active Ingredient 500 (b) Hydroxypropylmethylcellulose 112 (Methocel 4M Premium) (c) Lactase 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 a'ter 12 hours.

Example 2: 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 mo/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) Sodium Starch Glycollate 25 :(d) Magnesium S tearate 2_ 420 Formulation C ma/capsule (a) Active ingredient 250 (b) Macrogoi 4000 BP 350 600 Capsules were prepared by melting the Macrogoi 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

HDL/LMJ/26th February 1986 Formulation D ma/capsule 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 F (Controlled Release Capsule) _ The following controlled release capsule formulation, was prepared by extruding ingredients a, b and c using an extruder, followed by spheronisation o' the extrudate and drying. The dried pellets were then coated with release-controlling membrane (d) and 'ilied into a two-piece, hard gelatin capsule. ma/capsule (a) Active Ingredient (b) Macrocrystalline Cellulose (c) Lactose BP (d) Fthyl Cellulose Fxamole 3: Injectable Formulation Formulation A.

Active ingredient Hydrochloric acid solution, 0.1 Sodium hydroxide solution, 0.1 Sterile water The active ingredient was dissolved in most of the water (35 e -40 e 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 with sterile closures and overseals.

HDL/L J/26th February 1986 - - Formulation B, Active ingredient 0.125 g Sterile, pyrogen-free, pH 7 phosphate buff er, q.s. to 25 ml Fxamole 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 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).

Fxamole 5: Syrup- Active ingredient 0.2500 g Sorbitol Solution 1.5000 g Glycerol 2.0000 g Sodium Benzoate 0.0050 g Flavour, Peach 17.42.3169 0.0125 ml Purified Water q.s. to 5.0000 ml The active ingredient was dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution- o' 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.

Fxamole 6; Suppository ma/suppositorv Active Ingredient (63 im)* 250 Hard Fat, BP (Witepsoi H15 - Dynamit NoBel) 1770 2020 *The active ingredient was used as a powder wherein at least 90% of the particles were of 63 ym diameter or less.

One-fif th of the Witepsol H15 was melted in a steam- jacketed pan at 45 e C maximum. The active ingredient was sifted through a 200um 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 stired to ensure a homogenous mix. The entire suspension was passed through a 250ym stainless steel screen and, with continuous stirring, was allowed to cool to 40 e C. At a temperature of 38 e C to 40 e C 2.02g of the mixture was filled into suitable plastic moulds. The suppositories were allowed to cool to room temperature. - — - Example 7: Pessaries mg/pessarv Active ingredient 63 ym 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 1000 The above ingredients were mixed directly and pessaries prepared by direct compression of the resulting mixture.

Reference Example : 3 ' -Az ido-3 ' -deoxythymidine a) 2,3'-Anhydrothymidine Thymidine (85.4 g: 0.353 mol) was dissolved in 500 ml dry DMF and added to N- (2-chloro-l,l,2-trifluoroethyl)diethylamine (100.3 g; 0.529 mol) (prepared according to the method o' D.F. Ayer, J. Med. Chem. 6, 608 (1963)). This solution was heated at 70 °C for 30 minutes then poured into 950 ml ethanol (FtOH) with vigorous stirring. The product precipitated rom this solution and was filtered. The FtOH supernatant was refrigerated then filtered to yield the titTe compound, "mp. = 228 -230 e C. \ b) 3'-Azido-3'-deoxythvi-n'idine 2,3'-0-Anhydrothymidine (25 g: 0.1115 mol) and NaN-j (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 FtOAc (3 x 700 ml). The FtOAc extracts were dried over filtered and the FtOAc was removed in vacuo to yield a viscous oil. This oil was stirred with 200 ml water providing the title compound as a solid which was collected by filtration, mp = 116-118 eC Antiviral Activity (a) (i) Retrovirus - Induced Malignancy 3'-Azido-3'-deoxythymidine was administered to female BALB/c mice injected with 1.5X10^ Pfu of the RVB3 strain of Rauscher Murine Leukaemia Virus. Treatment was started 4 hours after infection at dosages of 80 mg/kg intraperitoneal.;/ 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.

Gi-LHTLV-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'-azida-3,-deoxythymidine (3y M); d) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (9μ Μ); e) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (27y M).

On day 18, total DNA was extracted 'rom each culture and 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 'ragment o* HTLV-I.

No hybridisation was observed for a), indicating a lack of virus in the unin ected 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 for T-cell receptor (J chain, with a strong signal being generated for all cultures, showing the continued presence of TM-11 for the duration of the experiment.

HDL/LMJ/26th February 1986 (b) AIDV (i) Reverse Transcriptase Activity 3'-Azida-5'-triphosphate-3'-deaxythymidine was tested in vitro against AIDV transcriptase (AIDV RT).

AIDV RT was purified from pelleted and extracted AIDV by elution through DFAF 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 Q2-I8) as ^e 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 gC^ was 10- fold greater than the activity in the presence of 0.3 mM MnC^. Maximal enzyme activity was also found in 80 to 140 mM KC1 and 60 to 100 mM NaCl. Incorporation o* [^H] dTTP was linear with respect to enzyme concentration. When tested, 3'-azido-5'-triphosphate-3'-deoxythymidine was found to be a competitive inhibitor af AIDV RT, giving a Ki of 0.04 y M when using rA-odT 18) as the template-primer. The enzyme had a Km for dTTP of 2.81μΜ, 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 leukemia virus and AIDV, showed 3'-azido- 5'-triphosphate-3'-deoxythymidine to be a terminator 0* DNA chain elongation.

In Vitro Anti-AIDV Activity 3'-Azido-3'-deoxythymidine was tested and Ound to possess activity in a number °* iH 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 *rom 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 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 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.

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^ doses of u to 5000 virions/cell] and cell survival af ter 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-fold decreased. Cell survival was also evaluated in an HTLV-I transformed, AIDV superinfected cell line derived 'rom the cells above. 3'-azido-3'-deoxythymidine at concentrations of 2.7, 0.27 and 0.13 mcg/ml totally blocked cytopathic effects at 7 days. Protective effects 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 cytophatic effect induction by a less related Haitian isolate of AIDV.

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

Claims (34)

- 22 - 85096/2 CLAIMS:

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

2. A formulation according to Claim 1, wherein the carrier is nonaqueous.

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 pharmaceutical formulation according to Claim 1 comprising 3'-azido-3'-deoxythymidine in which the formulation is a tablet or capsule.

9. A formulation according to Claim 8 providing sustained release of the active ingredient after oral administration.

10. A formulation according to Claim 7 further comprising a flavoring 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 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 1000 mg of the active ingredient.

14. A formulation according to Claim 11 wherein the unit dose is between 20 and 700 mg of the active ingredient. - 23 - 85096/2

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

16. Use of 3'-azido-3'-deoxythymidme in the manufacture of a medicament for the treatment or prophylaxis of a human retrovirus infection.

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

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

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

20. Use of 3'-azido-3'-deoxythymidine, as claimed in Claim 1 wherein the infection is an AIDV infection.

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

22. Use of 3'-azido-3'-deoxythymidine, as claimed in Claim 1 wherein the infection is an HTLV-I or HTLV-II infection.

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

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

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

26. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of human retrovirus infections.

27. 3 '-Azido-3 '-deoxythymidine for use in the treatment or prophylaxis of AIDS. - 24 - 85096/2

28. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of progressive generalized lymphadenopathy.

29. 3 '-Azido-3' -deoxythymidine for use in the treatment or prophylaxis of AIDS related complex.

30. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of an AIDV infection.

31. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a human T-cell lymphotropic virus infection.

32. 3 '-Azido-3 '-deoxythymidine for use in the treatment or prophylaxis of an HTLV-I or HTLV-II infection.

33. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of the AIDV carrier state.

34. 3'-Azido-3'-deoxythymidine for use in the treatment of a human subject having anti-AIDV antibodies. 7378S-8Claims/MC/be/30.11.1993