HU197209B - Process for producing pharmaceutical compositions containing 3'-azido-3'-deoxytimidine of antiviral activity againt human retrovirus - Google Patents

Abstract

Pharmaceutical formulation comprises (1) 3'-azido-3'-deoxythymidine (I), or its pharmaceutically acceptable derivs. as active ingredients and (2) an acceptable carrier. The formulations are pref. administered by injection (partic. using sterile water as the carrier) or orally as tablets or capsules. Derivs. of (I), (A), which are converted in vivo to (I), or to its active metabolites or decomposition prods., are new, excluding the following: monophosphate, disodium-monophosphate, 2-cyanoethyl-monophosphate, triphosphate, p-toluenesulphonate, acetate, methanesulphonate and Ph3C-derivs., and cpds. where 5'-C atom is attached to another nucleotide or nucleoside deriv. Specified (A) are the 5'-(diphosphate,3-methylbutyrate, octanoate, palmitate, 3-chlorobenzoate, benzoate, hydrogensuccinate and pivalate) esters of (I).

Description

The present invention relates to pharmaceutical compositions containing 3'-azido-3'-deoxythymidine, which are useful in the treatment or prevention of human retroviral infections.

Retroviruses are a subset of RNA viruses that, in order to replicate themselves, must first translate 'the genomes of RNAs into DNA (transcription' usually describes the synthesis of RNA from DNA). As the viral genome fuses with the host cell genome in the form of DNA, it creates a favorable position for the host cell's transcription / translation mechanism to replicate itself. When united, viral DNA is virtually indistinguishable from host DNA, and in this state the virus can exist as long as the cell is alive. Because virtually invulnerable in this form, all treatments must be directed to a different stage of the life cycle and must necessarily continue until all virus-carrying cells die.

HTLV-I and HTLV-Π are each retroviral and are known to cause human leukemia. HTLV-I infection is extremely widespread and has many fatalities worldwide every year.

A species of retrovirus has recently been reproducibly isolated from patients with AIDS (Acquired Immune Deficiency Syndrome). While the virus has been extensively characterized, it has no accepted name to date and is generally known either as human T-cell lymphotropic virus III (HTLV-III), AIDS-associated retrovirus (ARV), or lymphadenopathy-associated virus (LAV). We forward that until the virus has an internationally accepted name, it is labeled as immunodeficiency virus (AIDV) or called human retrovirus · This virus (hereafter referred to as AIDV) has been found to be particularly capable of infecting and killing T-cells carrying OKT ⁴ it is now generally accepted as an agent of AIDS. The patient gradually loses this T cell pool, upsets the overall immune system, reduces the ability to fight other infections, and predisposes them to infections, which often become fatal, so the true cause of AIDS deaths is mild infection such as pneumonia. , or cancer caused by the virus and not a direct consequence of AIDV infection.

Recently, AIDV virus has also been recovered from other tissue types, including T * marker B cells, macrophages and non-blood derived tissues in the central nervous system (CNS). This latter infection has been recognized in patients with the classic AIDS symptom, which is associated with progressive demyelination, leading to atrophy and symptoms such as encephalopathy, progressive dysarthria, asthma and disorientation.

AIDV infection has at least four clinical manifestations. In the initial carrier condition, the only indication for infection is the presence of anti-AIDV antibody in the bloodstream. The view is that this carrier is capable of transmitting infection, for example by blood transfusion, sexual contact, or the use of needles. The carrier condition often never reaches the second stage, which is characterized by persistent generalized lymph node disease (PGL). It is currently estimated that about 20% of PGL patients are in the more advanced stage known as .AIDS-related syndrome (ARC). Physical symptoms associated with ARC include general weakness, increase in thermo-temperature and chronic infection. These conditions usually reach the final fatal stage of AIDS, when the patient has completely lost the ability to fight the infection.

The existence of these human retroviruses and others has only recently been recognized, and the diseases they are associated with are imminent, and it is therefore urgently necessary to develop an appropriate method of killing these viruses.

Various drugs are suggested for the treatment of AIDS. These include antimony tungsten, suramine, ribavirin and isopininosin, which are either slightly toxic or have no remarkable antiretroviral activity. As the AIDV genome fuses with the host cell DNA after ferration, it is virtually indestructible in this state, so it may exist as long as the host cell is alive and may cause another infection, so any treatment of AIDS for a long time, maybe a lifetime which would require substances of acceptable toxicity.

Testing of various retroviruses, such as Friend Leukemia Virus (FLV), for rat (mouse) retrovirus, has been described in the literature. For example, Krieg et al. (Exp. Cell. Sl. 116, 21-29 (1978)) found that 3'-azido-3'-deoxythymidine influences the release of type C particles of the FLV complex and increases particle formation in in vitro assays and Ostertag et al. (Proc. Nat. Acad. Sci. 71, 4980-85 (1974)) found that 3'-azido-3 'was lacking in antiviral activity and cellular toxicity against FLV. -deoxythymidine may be a useful substitute for bromodeoxyuridine in the therapeutic treatment of diseases caused by .DNA viruses. Although De Clerq et al. (Biochem. Pharm. 29, 1849-1851 (1980)), six years later, using their test, it was found that 3'-azido-3'-deoxythymidine had no detectable activity, including chicken pox, HSVI and Varicella Zoster Vi3 rus (VZV). Glinski et al., J. Org. Chem. 38, 4299-4305 (1973), disclose certain derivatives of 3'-azido-3'-deoxythymidine (AZT) and their ability to block mammalian exoribonuclease activity, with direct therapeutic relevance. no.

None of these references, however, mentions any therapeutic effect from which AZT could be inferred for human therapeutic use. In support of this, the publications cited are discussed in more detail below:

Biochem. Pharma (29, 1980, 1849-1851) reported the use of 3'-azido-3'-deoxythymidine (AZT) against HS ^TT- 1 and vaccinia virus (both DNA viruses), vesicular stomatitis virus (RNA virus), and antitumor activity of mouse L 1210 against leukemia cells. Based on the experimental data presented, HS ^TT and vesicular stomatitis virus activity could not be determined (ID 50:> 200 µm / ml), while vaccinia virus was measured at 40-100 mg / ml ID 50. However, comparing the latter with the values obtained for the other test compounds, AZT shows at least 100-fold lower activity, such as the known anti-herpesvirus Ara-A and less active than five other compounds. It is noteworthy that the authors make no mention of AZT in the evaluation of the test results, that is, the active compounds, which obviously means that no antiviral activity has been found for this compound. Particularly noteworthy is the lack of activity against vesicular stomatitis virus, which, like human retroviruses, is also an RNA virus. The lack of activity is also indicated by the ID 50 = 280 Mg / ml for L 1210 cells. Based on all of these, it can be stated that no guidance was provided in this publication regarding the potential antiviral or antitumor activity of AZT.

Exp. Cell. Gap. (116, 21-29, 1978) and Proc. Nat. Acad. Sci. USA (71, 1974, 4980-85) reports the activity of certain compounds, including AZT, against mouse Friend Leukemia Virus (FLV) cells. There are two fundamental points to be made with regard to the articles: One is (a) the experience gained with the special rat system and the other (b) the applicability of these experiences in human medicine.

As for (a), the publications describe that when AZT is administered, the release of FLV C-type particles from the cells is reduced and the production of FLV-type A particles within the cell is increased, which is an A-type of C-type viruses. intracellular endogenous or exogenous forms. Studies have shown that AZT does not inhibit the replication of type A particles. In the case of C-type particles 4, the test results only show that AZT inhibits the release of such particles but cannot prevent their recurrence. Thus, even if the release of the C-type particles is inhibited, the viral particles within the cell are still able to infect fresh cells, for example by cell contact. There is no scientific acceptable evidence in the above publications to prove the inhibition of viral replication, that is to say, these publications could not provide any guidance for the therapeutic use of said compound in human. The experiments used a very specific cell culture of animal origin, which is unrelated to human medicine, and in particular to the evaluation of antiviral agents.

In relation to (b) above, the following are mentioned:

Based on the current state of scientific knowledge and knowledge, there is no basis for making findings regarding the mouse FLV virus in human viruses, e.g. could be applied or utilized in connection with the AIDV virus. The differences between FLV and AIDV viruses, both biochemically and virologically, are such that they do not allow any comparison to be made or the experience gained from one to be used with another.

The fact that structural differences actually influence the susceptibility of viruses to antiviral (antiviral) treatments is evidenced by ribavarin test results. Shannon's study (Annals of the New York Academy of Sciences, pp. 472-507 (page 480)) showed an ED 50 value of this compound of 3.2 mg / ml against Gross murine leukemia virus (MLV), while Yarchoan, Broder et al. For the Pharmacological Intervention Against HTLV-III / LAV, page 353, National Cancer Institute, Bethesda, 1987, only a partial inhibitory effect (100 mg / ml) of HIV was detected in an in vitro assay of HIV (Human Immunodeficiency Virus). Currently accepted name of virus.

We have discovered that 3'-azido-3'-deoxythymidine has surprisingly significant activity against human retroviruses, particularly high activity AIDV, as demonstrated in the following studies. Such activity makes it possible to use the compound in the treatment of human retroviral infections.

Accordingly, the present invention relates to a process for the manufacture of a medicament for the treatment of human retroviral infections comprising the compound of formula (I), 3 * -azido-3'-deoxythymidine. The compound of formula (I) is hereinafter referred to as the compound of the invention.

The activity of 3'-azido-3'-deoxythymidine against human retroviruses was tested in various in vitro systems. For example, the H9 human lymphoblastoid cell line is effectively inhibited by AIDV at concentrations below 0.013 mg / ml of 3'-azido-3'-deoxythymidine, even 20 hours after infection. U937 also inhibits AIDV infection at similarly low levels in human lymphoblastoid cells, PHA-steroid white blood cells, and cultured peripheral blood lymphocytes. In addition, in a 10-day experiment, up to 5,000 AIDV viruses are pei cells and no T4-cloned tetanus-specific T-helper lymphocyte is treated with 3'-azido-3'-deoxythymidine, whereas untreated cells show a 5-fold decrease. The cytopathic effect of the same cell line transformed with HTLV-I and superinfected with AIDV was also completely blocked.

Clinical studies show that the activity of purified AIDV reverse transcriptase is blocked by 3'-azido-3'-deoxythymidine triphosphate by a competitive inhibitory mechanism.

The Phase I clinical trial also shows that 3'-azido-3'-deoxythymidine is able to break through the blood brain barrier in a clinically effective amount. This property is both unusual and valuable for the treatment and prevention of human retroviral CNS infection.

The ability of 3'-azido-3'-deoxythymidine to modulate the influence of retrovirus-induced malignancy was demonstrated in a mouse model in which 3'-azido-3'-deoxythymidine was administered by intravenous administration of Rauscher Murine Leukemia Virus, HTLV-I. murine equivalent, inhibits splenomegaly induced by. In further experiments, 3'-azido-3'-deoxythymidine showed inhibition of HTLV-I repetition at concentrations below 0.8 Mg / ml in vitro. Thus, a further advantageous feature of the present invention is the use of the compounds of the invention in the manufacture of a medicament for the treatment or prevention of human retroviral infections.

Human retroviral infections that can be treated or prevented by the present invention include, for example, T cell lymphotropic retroviral (HTLV) infections, particularly HTLV-I, HTLV-Π and AIDV (HTLV-III). Clinical conditions that can be treated or prevented by the present invention include cases of AIDS, AIDS-related syndrome, and HTLV-I positive leukemia and lymphoma. Eligible patients include those with antibodies to AIDV, AIDV CNS infections, PGL, and ARC.

Experiments show that 3'-azido-3'-deoxythymidine can be converted in vivo to δ'-monophosphate by cellular enzymes. Monophosphate can then be further phosphorylated with other enzymes via diphosphate to triphosphate and, as discussed in other studies, is a form of 3'-azido-3'-deoxythymidine triphosphate, a potent chain terminator for reverse transcription of AIDV. myeloblastosis virus and Moloney murine leukemia virus. This form also inhibits AIDV reverse transcriptase, whereas it has negligible activity on human DNA polymerase activity.

3'-Azido-3'-deoxythymidine or pharmaceutical compositions containing 3'-azido-3'-deoxythymidine or for the prevention of retroviral infections or. for administration to a human by any suitable route, such as oral, rectal, nasal, topical (including oral and sublingual), vaginal and parenteral (such as subcutaneous, intramuscular, intravenous and intradermal). It will be understood that the preferred route of administration will vary according to the condition and age of the patient, the nature of the infection and the active ingredient chosen.

Generally, the dosage ranges from 3.0 to 120 mg / kg body weight per day, preferably from 6 to 90 mg / kg body weight, and most preferably from 15 to 60 mg / kg body weight per day. Preferably, the desired dose may be administered in divided doses of two, three, four, five, six or more daily, at appropriate intervals. These doses may be administered in unit dosage forms, for example, in the form of 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dose.

Based on experiments conducted with 3'-azido-3'-deoxythymidine, it is recommended that dosages be administered to produce a maximum concentration of active ingredient in the nucleus of about 1-75 μΜ, preferably about 2-50 μΜ, most preferably about 3-30 μ -. may. This condition can be achieved, for example, by intravenous injection of 0.1% solution of the active ingredient in saline or by oral administration in the form of a pill containing about 1100 mg / kg of active ingredient. The desired blood level can also be maintained by continuous infusion of about 0.01 to 5.0 mg / kg / hr or between about 0.4 to 15 mg / kg of active ingredient between infusions.

Although the active ingredient may be administered alone, it is preferable to use pharmaceutical compositions. The compositions of the present invention comprise at least one active ingredient as defined above, in combination with one or more acceptable carriers, 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 deleterious to the recipient thereof. The formulations include those suitable for oral, rectal, nasal, topical (including oral and sublingual), vaginal or subcutaneous (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of combining the active ingredient with a carrier comprising one or more accessory ingredients. In general, the formulations may be prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and, if necessary, shaping the product. 15

Formulations of the present invention for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the ingredient; such as powders or granules; or as oil-in-water liquid emulsions or water-in-oil liquid emulsions. The active ingredient may also be administered in the form of pills, lithium or paste. Oral formulations may also typically contain other agents such as sweeteners, flavoring agents, and thickeners.

The tablets may be made by compression or compression, optionally with one or more accessory ingredients. Compressed tablets may be formulated with suitable excipients (povidone, gelatin, hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrating agents (e.g., sodium sulfurizing glycolide, cross-linked sodium carboxymethylcellulose), treated with surfactants or dispersants, may be prepared by compression in the form of loose powders or granules.

Compressed tablets may be made by compressing a powder mixture moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored, and may be formulated so as to permit slow or controlled release of the active ingredient, for example, using varying proportions of hydroxypropyl methylcellulose to provide the desired release profile.

In many formulations for topical administration, lozenges containing the active ingredient in flavoring agents, usually sugar, acacia, tragacanth; pastilles comprising the active ingredient in an inert material such as gelatin and glycerol or in sugar and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository, and may be formulated with a suitable material, such as cocoa butter or salicylate.

oo

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

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions containing antioxidants, buffers, bacteriostatic agents and solutes which render the composition isotonic with the blood of the selected recipient; and aqueous and non-aqueous sterile suspensions containing suspending agents and thickening agents. The compositions may also be presented in unit dosage form or in multiple doses in sealed containers, such as ampoules or vials, and may be stored under freeze-drying (lyophilization) conditions, to which only a sterile liquid carrier such as water is required. Immediate 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 of active ingredient, or a suitable portion thereof, as above.

The administered ingredients may also be used in combination with other medicaments used in medicine, such as

9 - [(2-Hydroxy-1-hydroxymethyl-ethoxy) methyl] guanine, 9- (2-hydroxyethoxyethyl) guanine (acyclovir), 2-amino-9- (2- hydroxyethoxymethyl) purine, interferon such as interferon alpha, interleukin II and phosphonoformate (Foscarnet), or other immunomodulatory therapies such as bone marrow or lymphocyte transplantation or therapies such as levamisole or thymosin which are lymphocytes number and / or function.

It is to be understood that, in particular, in addition to the above ingredients, the formulations of the invention may also contain other agents commonly used in such formulations.

The compound of formula (I) is known in the art, for example, J.R. Horwitz et al., J. Org. Chem. 29, 2076-78 (1964); M. Imazawa et al., J. Org. Chem. 43 (15, 3044-3048 (1978); K. Watanabe et al., J. Org. Chem. 45, 3274 (1980) and RP Glinski et al., J. Chem. Soc. Chem. Commun. 914, (1970)). , in the literature, or analogously thereto, and in the examples of the invention.

The compound of formula (I) of the present invention may also be prepared by:

A) a compound of formula II wherein:

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M is a precursor group of a 3'-azido group, preferably, for example, a halogen atom, or a derivative thereof (e.g., a C 1 -C 4 ester or a salt) is reacted with an agent or conditions necessary to convert said precursor group to the desired azido group;

obsession

B) a compound of Formula III wherein:

R is a hydroxyl group or a precursor group of a group of pharmaceutically acceptable derivatives, such as an ester or an ether group, preferably an acetoxy or triphenylmethoxy group, and is reacted with an agent or conditions necessary to convert said precursor group to the desired desired group, e.g.

C) reacting a compound of formula IV, or a functionally equivalent derivative thereof, with a compound, such as a compound of formula IVa, for the introduction of the desired ribofuranosyl ring in the 1-position of the compound of formula IV; or

D) a compound of Formula V: wherein:

R ¹ is a hydroxy group, or R group as defined above, is preferably reacted with an agent or conditions, such as an alkali metal azide, to convert a compound of the present invention;

and then, or at the same time, perform the following conversion:

if a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine is formed, then that derivative is converted to the compound of formula (I), preferably by hydrolysis.

From the process of the invention described above, it will be appreciated that the precursor compounds of formulas II and III, as well as the agents and conditions mentioned above, are selected from those known in the synthetic chemistry of nucleosides. The following examples of the conversion procedures are provided as guidance and it is understood that they may be modified in conventional manner depending on the desired compounds of formula (I). Namely, where a transformation is described which otherwise results in an undesirable reaction of the labile group, such a group may be protected in a conventional manner and the protecting group may be removed after the conversion. Thus, in Process A, the compound of formula II in which M is selected from, for example, halogen (e.g., chlorine), hydroxy, or oranosulfonyloxy, such as trifluoromethylsulfonyloxy, methanesulfonyloxy or -toluenesulfonyloxy.

The compound of formula (I) can be prepared by reacting a compound of formula (II) in which the M group is in a threo configuration (such as chlorine) (in which the 5'-hydroxy group is preferably protected, for example, by trityl), for example lithium or sodium azide. For example, the 3 * -treo halogen (e.g., chlorine) starting material can be prepared by reaction of the corresponding 3'-erythrohydroxy compound with triphenylphosphine and carbon tetrachloride, or alternatively, with organosulfinyl halide (e.g., trifluoromethyl). sulfonyl chloride) to form the corresponding 3'-erythroorganosulfonyloxy compound which can then be halogenated.

Alternatively, the 3'-threohydroxy compound of formula (II) can be treated, for example, with triphenylphosphine, carbon tetrabromide and lithium azide to form the corresponding 3'-erythro-azido compound. After removal of all protecting groups, for example, it can be reacted as described above.

With respect to process AB), R is a protected hydroxy group, for example an ester group of the above-mentioned type in relation to formula (I), in particular an acetoxy group, or an ether group such as a trialkylsilyloxy group such as tert-butyldimethylsilyloxy or aralkoxy such as triphenylmethoxy. Such groups may be converted, for example, by hydrolysis to the desired hydroxy group or by esterification to an alternative ester group of 3'-azido-3'-deoxythymidine.

For process C), this can be accomplished, for example, by reaction of the corresponding pyrimidine of formula IV, or a salt or derivative thereof, with a compound of formula IVa, wherein

A is a cleavable group such as acetoxy or benzoyloxy or a halogen such as chlorine, and

B is an optionally protected hydroxyl group such as toluenesulfonyloxy followed by deprotection.

Regarding procedure D)

R ¹ may be a precursor group for R in the compound of formula III.

The 3'-azido-3'-deoxythymidine can then be prepared, for example, by reaction with an alkali metal azide such as lithium azide, preferably in a suitable solvent such as aqueous dimethylformamide, followed by acidic or basic hydrolysis, preferably under mild conditions.

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The following examples are offered by way of illustration only and are not intended to limit the scope of the invention in any way. The term "active ingredient" used in the examples refers to the compound of formula (I).

Example '-Azi do-3'-deoxymethiimidine

a) 2,3'-Anhydrothymidine

85.4 g of thymidine (0.353 mol) are dissolved in 500 ml of dried dimethylformamide and 100.3 g of N- (2-chloro-1,1,2-trifluoroethyl) diethylamine (0.529 mol) are added (DE Ayer). (J. Mer. Chem. 6, 608 (1963)). The solution was heated at 70 ° C for 30 minutes and then poured into 950 ml of ethanol with vigorous stirring. The product precipitated from this solution is filtered. The upper ethanol layer was frozen and filtered to give the title compound. Mp 228-230 ° C.

b) 3'-Azido-3'-deoxyturiticin was suspended in a mixture of 2,3'-O-anhydro-thymidine (0.1115 mole) and 29 g of sodium azide (0.446 mole) in 250 ml of dimethylformamide and 38 ml of water, The reaction mixture was heated at reflux for 5 hours and then poured into 1 liter of water. The aqueous solution was extracted with ethyl acetate (3 x 700 mL). The organic extract was dried over sodium sulfate, filtered and the ethyl acetate removed in vacuo. The resulting viscous material was mixed with 200 mL of water and the title compound was filtered as a solid. Melting point: 116-118 ° C.

Manufacture of pharmaceutical preparations

Example 1

Tablet formulation

The following formulations A) and B) are prepared by granulating the ingredients moistened with a solution of povidone and then pressing after the addition of magnesium stearate.

Formulation A) mg / tablet mg / tablet

(a) an active ingredient 250 250 (b) Lactose B.P. 210 26 (c) In Povidone B.P. 15 9 (d) sodium starch; stem-glycolic acid 20 12 (e) magnesium stearate 5 3 500 300

Formulation B mg / tablet mg / tablet

(a) an active ingredient 250 250 (b) lactose 150 - (c, Avicel PH 101 60 26 (d) In Povidone B.P. 15 9 (e) sodium starch; stem-glycolic acid 20 12 (f) magnesium stearate 5 3 500 300 Formulation C mg / tablet (a) an active ingredient 100 (b) lactose 200 (c) starch 50 (d) Povidone 5 (e) magnesium stearate 4

359

The following formulations D) and E) are prepared by direct compression of the ingredients- s. Formulation D) mg / capsule (a) an active ingredient 250 (b) pre-gelatinized starch NF 15 150 400 Formulation E) mg / capsule (a) an active ingredient 250 (b) lactose (Dairy Crest - .Zeparox ') 150 (c) Avicel 100 500

Formulation F (controlled release formulation)

The product is wet granulated with a solution of povidone,

followed by extrusion after the addition of magnesium stearate. mg / tablet (a) an active ingredient 500 (b) hydroxypropylmethyl; cellulose (Methocel K4M Premium) 112 (c) Lactose B.P. 53 (d) In Povidone B.P.C. 28 (e) magnesium stearate 7 700

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The drug is released within 6-8 hours and is complete after 12 hours.

Example 2: Formulation of capsules

Formulation A)

The capsules are prepared by mixing the ingredients of formulation D) of Example 1 and filling them into a hard gelatin capsule. The following formulation B) is also carried out in a similar manner.

Formulation B) mg / capsule (a) an active ingredient 250 (b) Lactose B.P. 143 (c) sodium starch glycols 25 (d) magnesium stearate 2

420

Formulation C mg / capsule (a) an active ingredient 250 (b) Macrogol 4000 BP 350 600

The capsules are prepared by melting Macrogol 4000 BP, dispersing the active ingredient in a melt, and filling the melt into a hard gelatin capsule.

Formulation D mg / capsule (a) active ingredient 250 (b) lecithin 100 (c) arachid oil 100

450

Capsules are prepared by dispersing the active ingredient in lecithin and arachid oil and filling the dispersion into a soft plastic gelatin capsule.

Formulation E (controlled-release capsule)

The following controlled release capsules are formulated by extruding components (a), (b), (c) into an extruder, pelletizing and drying the extrudate. The dry pellets are coated with the release-controlling membrane (d) and filled into a hard gelatin capsule.

mg / capsule (a) active ingredient 250 (b) microcrystalline

cellulose 125 Lactose BP 125 ethylcellulose 13

513

Example 3 (injectable formulations)

Formulation A)

active ingredient 0.200 yrs hydrochloric acid solution 0.1 M right quantity 4.0 Toi To pH 7.0 sodium hydroxide solution 0.1 M right quantity 4.0 Toi To pH 7.0 sterile water suitable for 10 ml quantity

The active ingredient is dissolved in most of the water (35-40 ° C) and the pH is adjusted to 4.0 to 7.0 with hydrochloric acid or sodium hydroxide as required. The reconstituted solution is made up to volume with water, filtered through a sterile microporous filter into a 10 mL brown vial (Type 1), sealed with a sterile stopper and sealed with a protective layer.

Formulation B Active Ingredient 0.125 g sterile, pyrogen-free, pH 7 phosphate buffer, required volume per 25 ml

Example 4 (intramuscular injection) Active ingredient 0.20 g benzyl alcohol 0.10 g glycofurol 75 1.45 g water for injection, required amount 3.00 ml.

The active ingredient is dissolved in glycofurol, then benzyl alcohol is added, dissolved and made up to 3 ml with water. The solution is filtered through a sterile microporous filter and filled into a 3 ml brown sterile vial (type 1) and sealed.

Example 5 (Mixture)

active ingredient 0.2500g sorbitol solution 1.5000g glycerol 2.0000g Sodium benzoate 0.0050g flavoring, Peach 17.42.3169 0.0125 ml sterile water quantity required To 5,000 ml

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The active ingredient is dissolved in glycerol and most of the sterile water. An aqueous solution of sodium benzoate, then the sorbitol solution and finally the flavoring are added. The volume is adjusted to the appropriate volume with sterile water and mixed well.

Example 6 (Suppository) mg / Suppository Active Ingredient (63 pn) * 250

Hard Wood, BP (Witepsol

H15 - Dynamit Nobel) 1770

2020 + The active ingredient is used as a powder, at least 90% of which is 63 pm in diameter or less.

One-fifth of the Witepsol H15 in a double-walled steam cooker, max. Melt at 45 ° C. The active ingredient is sieved through a 200 µm sieve, added to the molten material, mixed and comminuted until a uniformly uniform dispersion is obtained. The mixture is kept at 45 ° C, the remaining Witepsol H 15 is added to the slurry and mixed to a homogeneous mixture. The whole slurry was passed through a 250 µm stainless steel screen and allowed to cool to 40 ° C with constant stirring, then poured into a suitable plastic mold at a temperature of 38-40 ° C. The suppository was allowed to cool to room temperature.

Example 7 (Pessary) mg / pessary active ingredient 63 pm 250 anhydrous dextrose 380 potato 363 magnesium stearate 7

1000

These ingredients are mixed directly and the pessary is obtained by direct compression of the mixture.

Biological examples

Example 1

Antiviral activity (a) (i) Retroviral damage

BALB / c female mice, which

Infected with 1.5 ⁴ 10 ⁴ Rauscher Murine Leu10 Chemistry Virus (strain RVB3)

3'-Azido-3'-deoxythymidine was added. Treatment is initiated 4 hours after infection with an intraperitoneal dose of 80 mg / kg and all

For 8 hours, it is administered orally in drinking water at a dose of 0.5 mg / ml or 1.0 mg / ml. For such treatment, it has been found to inhibit spleen cell infection and subsequent development of splenomegaly, as well as suppress viremia.

(ii) lITLV

TM-11 cells (T cell clone sensitive to HTLV-I infection) were co-cultured with irradiated HTLV-I producing MJ tumor cells.

hands on: (the) Ib, TM-11 cells alone; TM-11 cells and MJ tumor cells; 20 (C) TM-11 cells, MJ tumor cells and 3'-azido-3'-deoxythymidine (3 µM); (D) TM-11 cells, MJ tumor cells and 3'-azido-3'-deoxythymidine (9 µM); 25 (E) TM-11 cells, MJ tumor cells, and 3'-azido-3'-deoxythymidine (27 pM).

On day 18, NDS from each culture was extracted and digested with Bam HI to produce an HTLV-I genomic fragment, free of any host adjoining sequence, and having a standard molecular weight of 3.3 kD. Digestion is then assayed with isotopically labeled lambda-MT-2, a standard assay for the recognition of the BamHI fragment of HTLV-I.

No hybridization was observed at (a), which indicates the absence of virus in the uninfected control. (B) shows a strong signal in the untreated infected control. A weak signal is observed at (c), which indicates incomplete destruction of the virus, and (d) or (e), no hybridization, which indicates complete destruction of the virus.

Each culture was also assayed for a T cell receptor beta chain assay. Each of the 45 cultures has a strong signal that indicates the continued presence of TM-11 during the experiment.

(b) AIDV (i) Reverse Tunctriptase Activity

In vitro, 3'-azido-5'-triphosphate-3'-deoxytynidine was administered by AIDV transcriptase (AIDV

RT).

AIDV RT was purified from pelleted and extracted AIDV by eluting on DEAE and phosphocellulose columns. Enzyme activity is linear for 60 minutes and θ® stable for at least two months when stored in 60% glycerol containing 1 mg of wine serum albumin per ml. Using rA-odT (22 -18) as a template primer, the AIDV RT had an optimum pH of 7.0-7.3, a MnCl2 optimum of 0.3 mM and a MgCl2 optimum of ^6: 15-5 mM. The activity was in the presence of 5 mM MgCl 2

-918 ten times greater than 0.3 mM MnCh. Maximum enzyme activity was also found at 80-140 mM KCl and 60-100 mM NaCl. The intake of [ ³ H] dTTP was linear with respect to the enzyme concentration. In the test, 3'-azido-5'-triphosphate-3'-deoxythymidine was found to be a competitive inhibitor of AIDV RT with a Ki of 0.04 μΜ when rA-odT (i2-is) as template primer We are used. The enzyme for dTTP is one

Has a value of 2.81 μΜ k ·, believing that 3'-azido-5'-triphosphate-3'-deoxythymidine binds more strongly to the enzyme than dTPP. Further experiments on avian myeloblastosis virus, Moloney murine leukemia virus, and AIDV RT have shown that 3'-azido-5'-triphosphate-3'-deoxythymidine is a terminator of the DNA chain length.

(ii) In vitro anti-AIDV activity

3'-Azido-3'-deoxythymidine has been tested and found to be active in a number of in vitro assays. The therapeutic effect was measured by assaying for reverse transcriptase (RT) activity in a solution of infected, uninfected and drug-treated cells. 3'-Azido-3'-deoxythymidine effectively blocks AIDV infection of the H9 and U937 human lymphoblastoid cell lines at concentrations between 2.7 and 0.0013 μηι / ml. Similarly, it inhibits normal PHA infection stimulated by white blood cells and cultured peripheral blood lymphocytes at drug concentrations below 0.013 pg / ml. In an experiment with and without drug, H9 cell shows that 3'-azido-3'-deoxythymidine is most effective when. it is present at the time of infection of the susceptible cell, but most of the antiviral activity is still maintained if administered 20 hours after the onset of AIDV infection. Inhibition of viral recurrence was also evident when the drug was only absorbed by the virus.

He was present in the cell around 20 o'clock. The effect was seen at 0.13 and 0.013 pg / ml. 3'-Azido-3'-deoxythymidine has no direct anti-RT activity against purified AIDV virus. Similarly, the drug has little or no effect on virus production and release in the chronically infected H9 AIDV cell line.

(iii) Inhibition of AIDV infection

The ability of 3'-azido-3'-deoxythymidine to block AIDV infection of cells was determined as follows:

Cloned T <positive tetanus-specific T-helper lirafocytes were infected with isolated AIDV virus (induced by up to 5,000 virus / cell doses) and checked for cell survival after infection. After 10 days, viral cytopathic effect was not seen on infected T cells treated with 8'8 and 1.3 pg / ml 3'-azido-3'-deoxythymidine, whereas untreated infected cells showed a 5-fold decrease. Cell survival was also evaluated in the HTLV-I transformed cell line derived from the former cells and highly infected with AIDV. At concentrations of 2.7, 0.27 and 0.13 pg / ml, 3'-azido-3x-deoxythymidine completely blocks the cytopathic effect by day 7. Both infection with cellular virus and cell associated virus showed protective effects. 3'-Azido-3'-deoxythymidine, at a concentration of 0.27 pg / ml, is also effective in inhibiting the cytopathic effect caused by the less related Haiti-isolated AIDV.

Example 2

Toxicity study

3'-Azido-3'-deoxythymidine was administered to both mice and rats. LD50 values were above 750 mg / kg in both species.

Claims (14)

PATENT CLAIMS CLAIMS 1. A process for the preparation of a pharmaceutical composition comprising 3'-azido-3'-deoxythymidine as an active ingredient, comprising mixing the active ingredient 3'-azido-3'-deoxythymidine prepared according to known methods with a known pharmaceutically acceptable liquid or solid carrier and / or other excipient. and converting it into a dosage form suitable for administration, optionally to slow-release human retrovirus-mediated diseases. 2. The process of claim 1 wherein the liquid carrier is a non-water solvent. A process according to claim 1 or 2, characterized in that sterile ingredients are used. The method of claim 3, wherein the composition is prepared by injection. 5. The method of claim 4, wherein the composition is in the form of a closed vial. The process according to claim 3 or 4, wherein the liquid carrier is sterile water. 7. The method of claim 1 or 2, wherein the composition is formulated for oral administration. 8. The method of claim 1, 2 or 7, wherein the composition is formulated as a tablet or capsule. -1 021 9. The process of claim 7, wherein the composition is formulated in a slow release form, preferably coated with hydroxymethylcellulose. 10. A process according to claim 7, wherein the flavoring agent is also used. 11. A process according to any one of claims 1 to 5, characterized in that the active ingredient is administered in unit dosage form or in multiple thereof. 12. The process of claim 10, wherein the active ingredient is present in an amount of from 5 mg to 1500 mg. 13. The process according to claim 10, wherein the active ingredient is present in an amount of from 10 mg to 1000 mg. 14. The method of claim 10, wherein the active ingredient is present in an amount of from 20 mg to 700 mg.