CA1340519C - Antiviral nucleosides - Google Patents

Abstract

3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a retrovirus.

Description

- l 1340S13 The present invention relates to 3 -azido-3 -deoxythymidine and its use in the treatment or prophylaxis of human retroviral infections.

This Application is a divisional of Canadian Patent Application Serial No.
556,981 filed January 20, 1988.
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 lor 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 vircually 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. HTi_V-I infections are especially widespread and are responsible for many deaths world wide each year.

A species of retrovirus has now been reproducibly isolated from patients with AIDS. This virus is now identified as Human Immunodeficiency Virus (referred to herein as HIV) and has been shown preferentially to infect and destroy T-cells bearing the 01~4 surFace marker. The virus 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 HIV infectian.

Recently, HIV 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 13~0~19 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 HIV infection. In the initial'carrier' state, the only indication of infection is the presence of anti- 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 of~en never progress to the second stage characterised by persistent generalised Iymphadenopathy (PGI_). It is currently estimated that about 20% of PGL patients progress to a more sdvanced condition known as 'AIDS related complex' (ARC). Physical symptoms associated with ARC may include general malaise, increased temperature and chronic in~ections. 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 uryent need to develop ways to combat these viruses.

Various drugs have now been proposed as 'cures' for AIDS. These includeantimoniotungstate, suramin, ribavirin and isoprinosine, which are either somewhat toxic or have shown no mart<ed anti-retroviral activity. As the HIV
genome is incorporated into the host cell DNA after infection and is virtually invulnerable to attack in this ~tate, it will persist 8S long as the host cell survives, causing new infection in the meantime. Thus, any treatment of AIDS
would have to be for an extended period, possibly life, requiring substances with an acceptable toxicity.

Reports have described the testing of compounds against various retroviruses for example Friend Leukaemia Virus (FLV) a murine retrovirus. For instance Krieg et al. (Exp. Cell Res.
116 (1978) 21-29) found that 3'-azido-3'-deoxythymidine affected the release of C-type particles and increased the formation of A-type particles of a FLV complex in in vitro experiments and Ostertag et al. (Proc. Nat. Acad. Sci. (1974) 71 4980-85) stated that on the basis of antiviral activity related to the above FLV complex and a lack of cellular toxicity 3'-azido-3'-deoxythymidine "might favourably replace bromodeoxyuridine for medical treatment of ~liceases caused by DNA viruses". However De Clerq et al. (Biochem. Pharm. (1980) 29 1849-1851) ~- ~'' ~hed six years later that 3'-azido-3'-deoxythymidine had no ,11 /7~t.h F~hr~ l~rv 1 9R~

appreciable activity against any viruses used in their tests, including vacclnia, 1 9 HSVI and varicella zoster virus (VZV). Glinski et al. (J. Org. Chem. (1973), 3~,4299-4305) discloses certain derivatives of 3'-azido-3'-deoxythymidine ~ and their ability to block mammalian exoribonuclease activity.

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

Thus, in a first aspect of the present invention, there is provided a compound of formula (I): ~

'~ ~
. ~ N (I) Ho ~7 (i.e. 3'-azido-3'-deoxythymidine) for use in the treatment or prophylaxis of human retrovirus infections.
The compound of formula (I) is hereafter referred to as the compound according to the invention.

Activity of 3'-azido-3'-deoxythymidine against human retroviruses has been established in various in vitro assay systems. For example, infection of the H9 human iymphoblastoid cell-line by HIV is effectively prevented by concentrations of 3'-azido-3'-deoxythymidine as low as 0.013 mcg/ml up to 20 hours after infection. HIV 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 HIV virions per cell and cloned T4, tetanus-specific, T-helper lymphocytes, showed no decrease in cells treated with 3'-azido-3'-deoxythymidine, while untreated cells had decreased ~-fold. Cytopathic effects were also completely blocked in the same cell-line transformed by HTLV-I and super-infected with HIV .

Other studies using purified HIV reverse transcriptase have shown that the activity of this enzyme is blocked by the triphosphate of 3'-azido-3'-deoxythymidine by a competitive inhibition mechanism.
HDL/LMJ/26th February 1986 , . . .... .

1 3 4 0 ~ 1 g Phase I clinical trials have also shown that 3'-azido-3'-deoxythymidine is capable of crossing the blood/brain barrier in clinically effective quantities. This property is both unusual and valuable for the treatment and prophylaxis of CNS
infections caused by human retroviruses.

The ability of 3'-azido-3'-deoxythymidine tn modify the cnurse of retrovirus-lnduced 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 HTI V-I at concentrations as low as û.8 mcg/ml.

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

The present invention further provides a method for the treatment or prophylaxisof AIDS in a human subject which comprises administering to the said human subject an effective amount of a compound according to the invention.

The present invention also includes a method for the treatment or prophylaxis ofinfections caused by retroviruses in a human subject, which comprises administering to the said human subject an effective antiviral amount of a compound according to the invention.

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

Fxamples of human retrovirus infections which may be treated or prevented in accordance with the present invention include T-cell lymphGtropic retroviruses (HTLV), especially HTLV~I, HTLV-II and AIDV (HTLV-III). Clinical conditions that may be treated or prevented in accordance with the invention include AIDS, AIDS-related complex and HTLV-I positive leukaemia and lymphoma. Sui~able patients for treatment also include those having antibodies to HIV, AIDV C~IS
infections, PGL and ARC.

13~0~1~
~xperiments have shown that 3'-azido-3'-deoxythymidine is converted, in vo, by the action of cellular enzymes into the 5'-monophosphate. The monophosphate is then further phosphorylated by other enzymes to form the triphosphate via the diphosphate, and other studies have demonstrated that it is the triphosphate form of 3'-azido-3'-deoxythymidine which is believed to be the effective chain terminator in the reverse transcription of HIV, as evidenced by its effect on avian myeloblastosis virus and MoLoney murine leukaemia viru3. This form also inhibits HIV reverse transcriptase in vitro whilst having a negligible ef,~ect on human DNA polymerase activity.

13~0513 3'-Azido-3'-deoxythymidine (hereafter referred to as the active ingredient), may be administered to humans for prophylaxis or treatment of retroviral infections by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal).It will be appreciated that the preferred route wiLl vary with the condition andage of the recipient, the natùre 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 or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit_dosaye forms, for example, containing 5 to 1500 mg, preferably 10 to 1000 mg, and most preferably 20 to 700 m~ 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 IIM, preferably about 2 to 50 IIM, most preferably about 3 to about 30 lIM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to about 100 mg/kg o' 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.

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 .~

1340~19 the present invention comprise at least one active ingredient, as above defined,togethsr with one or more acceptable carriers thereof and optionally other therapeutic agents. Fach carrier must be "acceptable" in the sense of belng compatible with the other in~redients 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 ~ormulations may conveniently be présented 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 ingredient with the carrier w hich 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; a~ a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-ln-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Oral formulations may further include other agents conventional in the art, such as sweeteners, flavouring agent~ 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 te.g- sodium starch glycol!ate, cross-linked povidone, cross-linkedsodium 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, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.

HDL/I_MJ/26th February 1986 -a-1340~19 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 apprnpriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile sus;oensions 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 (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Fxtemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dasage 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.

The administered ingredients may also be used in therapy in conjunction with other medicaments such as 9-L[2-hydroxy-1-(hydroxy-methyl)ethoxy]methyl]guanine, 9-(2-hydroxyethoxymethyl)guanine (acyclovir), 2-amino-9-(2-hydroxyethoxymethyl)purine, interferon, e.g., a-interferon, interleukin II, and phosphonoformate (Foscarnet) or in conjunction with other immune modulating therapy including bone marrow or Iymphocyte transplants or medications such as levamisol or thymosin which serve to increase Iymphocyte r~umbers and/or function as is appropriate.

It should be understood that in addition to the ingredients particularly mentioned .
HDL/I_MJ/26th February 1986 13~0~19 above the formulations of this invention may include other agents conventional in the art of formulation.

The compound of formula (I) may be prepared in 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 ai., J. Org. Chem, 43 (15) (1978) 3044-3048; K.A. Watanabe e~ al., J. Org. Chem., 45, 3274 (19B0); and R.P. Glinski et al., J. Chem. Soc. Ghem. Commun., 915 (197û~, as well as the processes described in Reference Example 8 hereinafter.

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

Reference Example 1: Tablet Formulations The followiny 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.
mq/tablet~ mq/tablet Formulation A

(a) Active ingredient 250 250 (b) Lactose B.P. 210 26 (c) Povidone B.P. 15 9 (d) Sodium Starch Glycollate 20 12 (e) Magnesium Stearate 5 3 HDL/I_MJ/26th February 19~6 Formulation B 13 ~ O ~;19 mq/tabletmQ/tablet (a) Active ingredient 250 250 (b) I_actose 150 -' (c) Avicel*PH 101 60 26 (d) Povidone B.P. 15 9 (e) Sodium Starch Glycollate 20 12 (f) Magnesium Stearate 5 3 Formulation C.
mq/tablet Active ingredient 1nO
Lactose 200 5tarch 50 Povidone 5 Magnesium stearate 4 The following formulations, D and E, 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
m~/tablet Active Ingredient 250 Pregelatinised Starch NF15 150 Formulation F
mq/tablet Active Ingredient 250 Lactose 150 Avicel * 100 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.
I IDLhMJ/26th February 1986 *trade mark .

mq/tablet (a) Active Ingredient 50013 4 0 5 13 (b) Hydroxypropylmethylcellulose 11Z
(Methocel~4M Premium) (c) Lactose B.P. 53 (d) Povidone B.P.C. 28 (e) Magnesium Stearate Drug release took place over a period of about 6-8 hours and was complete after 12 hours.

Reference ExamPle 2: C~rsu'Q Formulations Formulation A

A capsule formulation was prepared ~y 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
mq/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) Sodium Starch Glycollate 25 (d) Magnesium Stearate 2 ~ormulation C
mq/capsule (a) Active ingredient 250 (b) Macrogol*4000 BP 350 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.

*trade mark HDL/I_MJ/26th February 1986 - ~ -Formulatlon D
m~/capsule Active ingredient 250 Lecithin 100 Arachis Oil 100 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 r the extrudate and drying. The dried pellets were then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.

mq/capsule (a) Active lngredient 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 125 (d) Ethyl Cellulose 13 Reference ExamPle 3: Iniectable Forrnulation Formulation A.

Active ingredient 0.2009 Hydrochloric acid solution, 0.1M q.s. to pH 4.0 to 7.0 Sodium hydroxide solution, 0.1M q.s. to pH 4.0 to 7.0 Sterile water q.s. to 10ml The active ingredient was dissolved in most of the water (35 ~-40-C) and the pH
adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch was then made up to volume with the water and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals.

HDL/I_MJ/26th February l9a6 Formulation B. 1 ~ 4 0 ~19 Active ingredient 0.125 9 Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml Reference ExamPle 4: Intramuscular iniection Active Ingredient 0.20 9 Benzyl Alcohol 0.10 9 Glycofurol 75 1.45 9 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).

Reference ExamPle 5: In~redients Active ingredient 0.2500 9 Sorbitol Solution 1.5000 9 Glycerol 2.0000 9 Sodium Benzoate; 0.0050 9 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 thepurified water. An aqueous solution of the sodium benzoate was then added to thesolution, followed by addition of the sorbitol solution and finally the flavour. The volume was made up with purified water and mixed well.

Reference ExamPle 6: SuPPositorY

mq/suppository Active lngredient (63~m)* 250 Hard Fat, BP (Witepsol**H15 - Dynamit Nobel) 1770 **trade mark .

*The active ingredient was used as a powder wherein at least 90% of the particles were of 631~m diameter or less.

One-fifth of the Witepsol Hl5 was melted in a steam-jacketed pan at 45-C maximum.
The active ingredient was sifted through a 200)Jm 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 Hl5 was added to the suspension and stired to ensure a homogenous mix. The entire suspension was passed through a 250~m stainless steel screen and, with continuous stirring, wasallowed to cool to 40~C. At a temperature of 38~C to 40-C 2.029 of the mixture was filled into suitable plastic moulds. The suppositories were allowed to cool to room temperature.

Rerer,:~ce ExamPle 7: Pessanes mq/pessary Active ingredient 63~m 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 The above ingredients were mixed directly and pessaries prepared by direct compression of the resulting mixture.

Z'HDL/I_MJ/26th February l9a6 Reference ExamPle 8: 3'-Azido-3'-deox~thymidine 1~40513 a) 2l3'-Anhydrothymidine .
Thymidine (85.4 9: 0.353 mol) was dissolved in 500 ml dry DMF and added to N-(2-chloro-1,1,2-trifluoroethyl)diethylamine (100.3 9; 0.529 mol) (prepared according to the method of D.~. 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 from this solution and was filtered. The FtOH supernatant was refrigerated then filtered to yield the title compound. mp. = 228 -230~C.

b) 3'-Azido-3'-deoxythymidine 2,3'-0-Anhydrothymidine (25 9: 0.1115 mol) and NaN3 (29 9, 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 nf water. The aqueous solution was extracted with FtOAc (3 x 700 ml). The FtOAc extracts were dried over Na2SO4, 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 ~ C

HDL/LMJ/26th February 1986 .

-Example : Antiviral Activity (a) (i) Retrovirus - Induced Maliqnancy 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 af ter 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 HTl_V-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 (31-M);

d) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (91JM);

e) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (271JM).

On day 1~, total DNA was extracted from each culture and digested with Bam H1 togenerate 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 H1 fragment ofHTLV-I.

No hybridisation was observed for a), indicating a lack of virus in the uninfected control. A strong signHl 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.

Fach culture was also probed with a probe for T-cell receptor ~ 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 19û6 ~.. _.

(b) HIV 13 l~S~3 (i) Reverse Transcriptase Activity 3'-Azido-5'-triphosphate-3'-deoxythymidine was tested in vitro agalnst HIV
transcriptase (HIV RT) HIV RT was purified from pelleted and extracted HIV 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, HIV 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 MgC12 was 10-fold greater than the activity in the presence of 0.3 mM MnCl2. Maximal enzyme activity was also found in 80 to 140 mM KCl and 60 to 100 mM NaCl.
Incorporation of [3H] dTTP was linear with respect to enzyme concentration.
When tested, 3'-azido-5'-triphosphate-3'-deoxythymidine was found to be a competitive inhibitor of HIV RT, giving a Ki of 0.04 I~M when using rA-odT (12 18) as the template-primer. The enzyme had a Km for dTTP of 2.811~M, 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 HIV, showed 3'-azido-5'-triphosphate-3'-deoxythymidine to be a terminator of DNA chain elongation.

(ii~ In Vitro Anti-HlV 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 HIV of the H9 and U937 human Iymphoblastoid 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 Iymphocytes 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 sntiviral activity even when added as late as 20 hours after initial HIV infection. Inhibition of viral replication was also evident when the drug was present in the media only during the 20 hour period of virus HDL/LMJ/26th February 1986 ., , -18- 1340~13 absorption. Effects were seen at 0.13 and 0.013 mcg/ml. 3'-Azido-3'-deoxythymidine exhibited no direct anti-RT activity against purified HIV
virions. Similarly, the drug had little or no effect on the production and release of virions from the chronically infected H9 HIV cell line.

(iii) Preventinq Infection bY HIV

The ability of 3'-azido-3'-deoxythymidine to block infection of cells by HIV wasdetermined as follows.

Cloned T4 positive tetanus specific T helper Iymphocytes were infected with a pool of HIV isolates [at challenge 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/ml3'-azido-3'-deoxythymidine, while untreated, infected cells were 5-fold decreased. Cell survival was also evaluated in an HTI V-I transformed, HIV
superinfected cell line derived from the cells above. 3'-azido-3'-deoxythymidineat 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 concentraion also effectively prevented cytopathic effect induction by a less related Haitian isolate of HIV

Example 18: Toxicity Assay 3'-Azido-3'-deoxythymidine was administered to both mice and rats.- The LD50 value was in excess of 750 mg/kg in both species.

HDL/LMJ/26th February 1986

Claims (46)

1. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a retrovirus infection in a human.

2. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of AIDS in a human.

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

4. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of AIDS related complex in a human.

5. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of an HIV infection in a human.

6. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of a T-cell lymphotropic virus infection in a human.

7. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of an HTLV-I or HTLV-II
infection in a human.

8. 3'-Azido-3'-deoxythymidine for use in the treatment or prophylaxis of the HIV carrier state in a human.

9. 3'-Azido-3'-deoxythymidine for use in the treatment of a human subject having anti-HIV
antibodies.

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

11. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment of AIDS
in a human.

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

13. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment or prophylaxis of an HIV infection in a human.

14. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment or prophylaxis of a T-cell lymphotropic virus infection in a human.

15. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment or prophylaxis of an HTLV-I or HTLV-II infection in a human.

16. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment or prophylaxis of the HIV carrier state in a human.

17. Use of 3'-azido-3'-deoxythymidine for the manufacture of a medicament for the treatment or prophylaxis of a human subject having anti-HIV
antibodies.

18. An anti-retroviral pharmaceutical composition comprising an acceptable, anti-retrovirally effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

19. A composition according to claim 18, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

20. A composition according to claim 18, containing 20 to 700 mg of said deoxythymidine, per unit dosage form.

21. An anti-AIDS pharmaceutical composition comprising an acceptable, anti-AIDS effective amount of 3'-azido-3'-deoxythymidine in association with a pharmaceutically acceptable carrier.

22. A composition according to claim 21, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

23. A composition according to claim 21, containing 20 to 700 mg of said deoxythymidine, per unit dosage form.

24. An anti-PGL pharmaceutical composition comprising an acceptable, anti-PGL effective amount of 3'-azido-3'-deoxythymidine in association with a pharmaceutically acceptable carrier.

25. A composition according to claim 19, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

26. An anti-AIDS related complex pharmaceutical composition comprising an acceptable anti-AIDS related complex effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

27. A composition according to claim 26, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

28. An anti-AIDS infection pharmaceutical composition comprising an acceptable anti-AIDS
infection effective amount of 3'-azido-3'-deoxythymidine in association with a pharmaceutically acceptable carrier.

29. A composition according to claim 28, containing 20 to 700 mg of said deoxythymidine, per unit dosage form.

30. An anti-T-cell lymphotropic virus infection pharmaceutical composition comprising an acceptable anti-T-cell lymphotropic virus infection effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

31. A composition according to claim 30, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

32. An anti-HTLV-I or HTLV-II infection pharmaceutical composition comprising an acceptable anti-HTLV-I or HTLV-II infection effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

33. A composition according to claim 32, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

34. An anti-AIDV carrier state pharmaceutical composition comprising an acceptable, anti-AIDV
carrier state effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

35. A composition according to claim 34, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

36. An anti-AIDV antibodies pharmaceutical composition comprising an acceptable anti-AIDV
antibodies effective amount of 3'-azido-3'-deoxythymidine, in association with a pharmaceutically acceptable carrier.

37. A composition according to claim 36, containing 5 to 1500 mg of said deoxythymidine, per unit dosage form.

38. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of a retrovirus infection in a human.

39. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of AIDS in a human.

40. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of PGL.

41. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of an AIDV infection in a human.

42. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of a T-cell lymphotropic virus infection in a human.

43. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of an HTLV-I or HTLV-II
infection in a human.

44. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of the AIDV carrier state in a human.

45. Use of 3'-azido-3'-deoxythymidine for the treatment or prophylaxis of a human subject having anti-AIDV antibodies.

46. In a system for the treatment of AIDS, the improvement wherein 3'-azido-3'-deoxythymidine is employed.