HU201678B - Process for producing pharmaceutical compositions against human retrovirus, comprising 3'-azido-3'-deoxythymide derivatives - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising 3'-azido-3'-deoxytimidine as a pharmaceutically acceptable base or a 5'-ester derivative thereof, such as triphosphate or acetate, by the use of the known methods for the preparation of a pharmaceutically acceptable liquid or solid carrier. and / or mixing with other excipients and converting into a pharmaceutical composition suitable for treating, optionally, slow-release, human-induced disease caused by human retort. EN 201 678 B Scope of description: 9 pages, 1 drawing -1-

Description

FIELD OF THE INVENTION The present invention relates to a method of treating or preventing human retroviral infections containing pharmaceutically acceptable derivatives of 3'-azido-3'-doxoxytimidine.

Retroviruses are a subset of RNA viruses that, in order to replicate themselves, must first "reverse engineer" the RNA genomes 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-II are each retroviruses 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 isolated from patients with AIDS (Acquired Immune Deficiency Syndromc). While the virus has been extensively characterized, it has no accepted name to date and is generally known as either human T-cell lymphotropic virus III (HTLV-I1I), AIDS-associated retrovirus (ARV), or lymphadnopathy-associated virus (LAV). It is suggested that as long as the virus has no internationally accepted name, it will be labeled as Immunodeficiency Virus (AIDV). This virus (hereafter referred to as AIDV) has been found to be particularly capable of infecting and killing T-cells carrying the OKT ⁴ surface marker and is now generally accepted as the causative 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. Thus, the true cause of death of AIDS victims is mild infection, such as pneumonia or virus-induced cancer, and not the direct consequence of AIDV infection.

Recently, AIDV has been recovered from other tissue types, including T * marker B cells, macrophages and non-blood derived tissues in the central nervous system (CNS). The latter infection has been recognized in patients with the classic AIDS symptom, 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 a needle. 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 advanced stage known as "AIDS-related Elemental Group" (ARC). Physical Symptoms Associated with ARC: Pseudonym 2

John's weakness, temperature rise 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 "cure" of AIDS. These include anti-tungstenramate, suramine, ribavirin and isoprinosine, which are either somewhat toxic or have no remarkable antiretroviral activity. As the AIDV genome fuses with the host cell DNA after infection, it is virtually impossible to overcome in this state, so it can exist as long as the host cell is alive and may cause another infection. Thus, any treatment for AIDS may last for a long time, perhaps over a lifetime, which would require substances of acceptable toxicity.

Testing of various anti-rat retroviruses, such as Frien Leukemia Virus (FLV), has been described in the literature. For example, Kreig et al., Exp. Cell. Gap. 116,21-29 (1978)], it was found that 3'-azido-3'-deoxythymidine (AZT) influences the release of C-type particles of the FLV complex and increases the formation of A-type particles in in vitro assays and Ostertag et al., Proc. Nat. Acai. Sci., 71, 4980-85 (1974)], found that 3'-azido-3'-deoxythymidine was found to be beneficial in the therapeutic treatment of diseases caused by DNA viruses with bromodeoxyuridine, due to its lack of antiviral activity and cellular toxicity substitute ". Although De Clerq et al., Biochem. Pharm. 29, 1849-1851 (1980)] six years later, using their assay, found that 3'-azido-3'-deoxythymidine had no detectable activity against any virus, including pox, HVSI and Varicella. Zoster Virus (VZV). Glinski et al., J. Med. Org. Chem., 38, 4299-4305 (1973)] describe certain derivatives of? 3'-azido-3'-deoxythymidine and their ability to block mammalian exoribonuclease activity without direct therapeutic significance.

However, none of these references mentions any therapeutic effect from which AZT and its derivatives may be inferred for human therapeutic use. In support of this, the publications cited are discussed in more detail below:

Biochem. In Pharma (29, 1980, 1849-1851) against 3'-azido-3'-deoxythymidine (AZT) against HSU-1 and vaccinia virus (both DNA viruses), against vesicular stomatitis virus (RNA virus), and the anti-tumor effect of mouse L1210 on leukemia cells was investigated. Based on the experimental data presented, activity against HSU and vesicular stomatitis viruses could not be determined (1D _SO : 200 µg / ml), while vaccinia virus values were 40-100 pg / ml ID _S0 . However, comparing the latter with the values obtained for the other test compounds, AZT exhibits at least 100-fold lower activity, such as the known anti-herpes virus Ara-A and less active than five other compounds. It is noteworthy that the authors do not mention the OCTs in the evaluation of the test results, that is, the active compounds, which obviously jc-23

EN 201 678 Β that no antiviral activity has been found for the compound. Particularly noteworthy is the lack of activity against vesicular stomatitis virus, as it is also an RNA virus like human retroviruses. Lack of activity also indicates ID ₅₀ = 280 µg / ml for L1210 cells. On the basis of the foregoing, it can be concluded that no guidance has been provided in this publication on the possible antiviral or antirumor effect of AZT.

Exp. Gap. (776, pp. 21-12-1978) and Proc. Nat. Acad. Sci. USA (77,1974,4980-85) reports the activity of certain compounds, including AZT, against mouse Frien Leukemia Virus (ELV) 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 this experience in human medicine.

Regarding (a), the publications describe that when AZT is administered, the release of C-type ELV particles from the cells is reduced and the production of FLV A-type particles within the cell is increased, which A-type particles are intracellular to C-viruses. endogenous or exogenous forms. Studies have shown that AZT does not inhibit the replication of type A particles. For C-type particles, test results show that AZT inhibits the release of such particles but cannot prevent their recurrence. In this way, even when the release of the C-type particles is dilated, the viral particles within the cell can still infect fresh cells, for example by cell contact. There is no scientifically acceptable evidence in the above publications to prove the inhibition of virus replication, that is to say, these publications should not provide any basis for the therapeutic use of said compound in human. The experiments involved the use of a very specific cell culture of animal origin, which is unrelated to human medicine, especially 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 between them or the experience gained from one to be used against another.

The fact that structural differences do indeed influence the susceptibility of viruses to antiviral (antiviral) treatments is evidenced by ribavirin test results. According to Shannon's study [Annals of the New York Academy of Sciences, 472-507 (p. 480)], this compound had an ED ₅₀ of 3.2 µg / ml against Gross murine leukemia virus (MLV), while Yarchoan, Brodcr et al. Strategies for the Pharmacological Intervention Against HTLV-III / LAV ', page 353, National Cancer Institute, Bethesda, 1987) were able to demonstrate only a partial inhibitory effect (100 pg / ml) in the in vitro assay of HIV. HIV (Human Immunodeficiency Virus) is the current accepted name for the AIDS virus.

We have discovered that pharmaceutically acceptable, known esters and salts of 3'-azido-3'-deoxythymidine have surprisingly significant activity against human retroviruses, particularly high activity against AIDV, as evidenced by the studies reported below. Such activity makes it possible to use the compound in the treatment of human retroviral infections.

Accordingly, the present invention relates to pharmaceutical compositions containing a compound of formula (I), pharmaceutically acceptable basic salts of 3'-azido-3'-deoxythymidine and 5'-triphosphate or acetate, prepared according to known methods for the treatment of human retroviral infections. process. In the context of the present invention, the term pharmaceutically acceptable basic salt refers to derivatives that, directly or indirectly, are converted to 3'-azido-3'-doxoxytimidine when administered to a patient. These include triphosphate and acetate esters, as well as basic salts, for example the sodium salt.

The term "produced by known methods" encompasses methods published prior to the priority date of the present invention.

The activity of 3'-azido-3'-desxitimidine 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 g / ml of 3'-azido-3'-deoxythymidine, even 20 hours after infection. U937 also inhibits AIDV infection at similarly low levels in human limboflastoid cells, PHA-stimulated white blood cells, and cultured peripheral blood lymphocytes. In addition, in the 10-day experiment, up to 5,000 AIDV viruses per cell and T4-cloned tetanus-specific T-helper lymphocytes did not show cell reduction when treated with 3'-azido-3'-deoxythymidine, whereas untreated cells showed a 5-fold decrease. The cytopathic effect of the same cell line transformed with HTLV-I and superinfected with AIDV is also completely blocked

Clinical studies have shown 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 the administration of 3'-azido-3'-deoxythymidine to intravenously administered Rauscher appears to be a leukemia virus, HTLV-I. inhibits epelinomegaly induced by the equivalent of murine. In further experiments, 3'-azido-3'-deoxythymidine showed inhibition of HTLV-I repetition at concentrations below 0.8 µg / 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 pharmaceutical compositions of the invention include T cell lymphotropic retroviral (HTLV), particularly HTLV-I,

HU 201 678 Β

HTLV-H and AIDV (HTLV-III) infections. Clinical conditions that can be treated or prevented by the pharmaceutical compositions of 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 5'-monosulfate by cellular enzymes. Monophosphate can then be further phosphorylated to other phosphorylated enzymes via diphosphate, and is a triphosphate form of 3'-azido-3'-deoxythymidine, which is a chain termination effective in reverse transcription of AIDV, such as avian myeloblastosis and murine leukemia virus It reflected. This form also inhibits AIDV reverse transcriptase, whereas it has negligible activity on human DNA polymerase activity.

Pharmaceutically acceptable derivatives of 3'-azido-3'-deoxythymidine (hereinafter referred to as the active ingredient) may be administered to a human by any suitable route, including oral, rectal, nasal, topical (including oral and sublingual), to prevent or treat retroviral infections, 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 will range from 3 to 20 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 form, for example, in the form of 10-1500 mg, preferably 20-1000 mg, and most preferably 50-700 mg of active ingredient per dosage unit.

Based on experiments with 3'-azido-3'-deoxythymidine, it is recommended that dosages be administered to give a maximum concentration of the active ingredient in the nucleus of about 1-75 pm, preferably about 2-50 pmol, and most preferably about 3-30 pmol. pm. This condition can be achieved, for example, by intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally in the form of a pill containing about 1 to 100 mg / kg of the active ingredient. The desired blood level can also be maintained by continuous infusion of about 0.015.0 mg / kg / hr or between about 0.5-15 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 contain at least one active ingredient as defined above with one or more acceptable carriers. The pharmaceutical compositions of the invention may also be mixed with other known active ingredients. 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 compositions include those for oral, rectal, nasal, topical (including oral and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may be conveniently 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.

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 pores 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 a pill, lithium or paste. Oral formulations may also conventionally contain other agents such as sweeteners, flavoring agents, and emollients.

The tablets may be made by compression or compression, optionally with one or more accessory ingredients. Compressed tablets may be formulated with suitable binders (povidone, gelatin, hydroxypropylmethylcellulose), inert diluents, preservatives, disintegrating agents (e.g., sodium starch glycolate, crosslinked carboxylate, methylcellulose), treated with surfactants or dispersants, by compression in the form of loose powders or granules. Compressed tablets may be made by molding in a suitable machine a mixture of a polish blend 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, for example, using varying proportions of hydroxypropyl methylcellulose to provide the desired release profile.

Formulations suitable for topical administration in the mouth, containing the active ingredient in a flavoring agent, 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 suppositories and may be formulated with a suitable material, such as cocoa butter or salicylate.

Formulations suitable for vaginal administration may be used such as pcs., Tampon, cream, gel, paste, foam or spray, 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 selected recipients; and aqueous and non-aqueous sterile suspensions containing suspending agents and disintegrating agents. Preparations in unit dose or multi-dose containers, sealed containers, half-4Ί

They may also be packaged in ampoules or vials and stored under freeze-drying (lyophilization) conditions, to which only sterile liquid carrier such as water must be added immediately prior to use. Immediate injection solutions and suspensions may be prepared from sterile supples, granules and tablets as described above.

Preferred unit dosage formulations are those containing a daily dose or unit of active ingredient, or a suitable portion thereof, as above.

The added 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-hydroxyethoxymethyl). -guanine (acyclovir), 2-amino-9- (2-hydroxyethoxymethyl) purine, interferon such as α-interferon, interleukin II and phosphonoformate (Foscamet) or other immunomodulatory therapies such as bone marrow or lymphocyte transplantation, or therapies such as levamisole or thymosin to increase lymphocyte count 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.

Pharmaceutically acceptable derivatives of the compounds of formula (I) are 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 (1987);

K. A. Natanabe et al; J. Org. Chem. 45, 3274 (1980) and R. P. Glinski et al., J. Chem. Soc. Chem. Commun. 914. (1970); Zaitseva et al., Bioorg. Khim. 10 (5) 670-680 (1984), or analogously thereto, and may be prepared by the methods described in the Examples of the invention.

3'-Azido-3'-oxo-thymidine is converted into a pharmaceutically acceptable phosphate and / or and other esters, for example, by reaction with a furfurylating agent, such as phosphorus oxychloride, or a suitable esterifying agent, such as an acid halide or anhydride. The esters of the compound of Formula I may be converted into their pharmaceutically acceptable salts by conventional means, e.g. For example, the ester or salt of 3'-azido-3'-deoxythymidine can be converted to its parent compound by hydrolysis.

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" as used in the examples means a pharmaceutically acceptable salt or ester of a compound of formula (I).

Example 1

To a solution of 5 '-Acetyl-3'-azido-3'-deoxythymidine g in 3'-azido-3'-deoxythymidine and 50 mL of pyridine was added acetyl chloride (2.1 molar equivalent) at room temperature. The reaction mixture was stirred for 2 hours and kept at 0-15 ° C for 20 hours. The reaction mixture was poured into ice water with stirring, and the aqueous layer was added to dean. The oily product was dissolved in water and extracted five times with water, twice with 0.5N hydrochloric acid and twice with water, and dried over anhydrous magnesium sulfate. The solution was filtered and concentrated in vacuo. The residual oil was dissolved in chloroform, poured onto a silica gel column and chromatographed with 2% methanol in chloroform. The fraction containing product was evaporated and the oil was chromatographed again with ethyl acetate: hexane (6: 4). The product fraction was evaporated in vacuo to give a white solid. Melting point: 96-98 ° C.

Analysis results:

Calculated: C, 46.60; H, 4.89%; N, 22.65%;

Found: C, 46.67%; H, 4.94%; N, 22.59%.

EXAMPLE 2 j-O-Anhydro-3 '-azido-3' -deoxythymithithiol

3.0 g of 3'-azido-3'-deoxythymidine (11.2 mmol) are mesylated in 2 mL of dried pyridine with 2.7 mL of methanesulfonyl chloride. After stirring for 1 hour at 5 ° C, the reaction mixture was poured into ice water and the precipitate was filtered. The product (3'-azido-5'-methyl-thymidine) in 75 ml of dimethylformamide was treated with 0.78 potassium carbonate (5.6 mmol) in an oil bath at 80 ° C for 6 hours and then poured into ice water. The product is extracted from water with ethyl acetate. The solvent was then removed in vacuo and the resulting oil was chromatographed on silica gel, eluting with chloroform: methanol (9: 1). The title compound is obtained as a solid after evaporation of the appropriate fraction.

Melting point: 184-186 ° C.

Example 3

3'-azido-3 '-deoxythymidine

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) [D. E. Ayer: J. Mer. Chem. 6, 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.

Melting point: 228-230 ° C.

b) 3'-Azido-3'-deoxythymidine g is 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 anhydrous 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.

Example 4

Monosodium salt of 3'-azido-3'-deoxythymidine

Almost 1 g of 3'-azido-3'-deoxythymidine was dissolved in 50 ml of distilled water and the solution was adjusted to pH 12 with 1N sodium hydroxide. About half of the solution is freeze-dried. The title compound is obtained as a lyophilized powder.

Analysis calculated for C ₁₀ H ₁₂ N ₅ NaO ₄ 6/10 N ₂ O *: C, 40.03%; H, 4.43%; N, 23.34%; Na, 7.66%; Found: C, 39.88%; H, 4.34%; N, 23.23%; Well 7.90%.

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Example 5

Preparation of 5'-monophosphate of 3'-azido-3'-deoxythymidine

Dissolve 0.5 g of 3'-azido-3'-deoxythymidine (1.87 mmol) in 5 ml of triethyl phosphate and cool to -5 ° C. While stirring vigorously, 0.685 mL of phosphorus oxychloride (7 mmol) was added all at once and the solution was kept at -10 ° C for 22 hours. A sample of the reaction mixture is taken up to which concentrated ammonium hydroxide is added. TLC (cellulose, n-propanol: water 7: 3) showed no starting material remaining, single fluorescent spot, Rf lower than nucleoside. The reaction mixture was poured into ice-water (20 mL), placed in an ice bath and adjusted to pH 7.5 with 2N sodium hydroxide. The basic mixture was extracted once with chloroform and once with water. The aqueous phase was again adjusted to pH 7.5 and the residual organic solvent was removed in vacuo. The material was kept at -10 ° C until purification as follows.

To make deactivated carbon, 100 g of coconut carbon (50-200 mesh) are washed with 500 mL of IN hydrochloric acid, 3 L of water, 15 mL of 95% ethanol containing 3% toluene, 600 mL of 85% methanol and finally copiously with water. Deactivated charcoal (12 mL of precipitated wet carbon) was added to the monophosphate side (0.72 g, 1.1 mmol, 30 mL) with stirring. The supernatant was decanted and the charcoal washed with 150 mL of water. The carbon was eluted from the carbon by washing with 120 mL of 1.5 M ammonium hydroxide in 50% ethanol. The solution was filtered through a 0.22 micron filter, concentrated to 10 ml in vacuo, filtered on an Amicon Centriflo CF-25 membrane and lyophilized. Diammonium 3'-azido-3'-deoxythymidine-5'-monophosphate is obtained as a solid. The compound is described as a nuclozoside 5'-monophosphate (described as the ability of a 5'-nucleotidase to decompose into a nucleoside).

Example 6

3'-azido-5'-triphosphate-3'-deoxythymidine and 3'-azido-5 '-diphosphate-3-deoxythymidine

a) bis (tri-n-butylammonium) triphosphate

The DOW 50 (Dowcx ion exchange resin, DOW Chemical Laboratories) pyridinium resin column was prepared by pouring 40 ml of resin onto a 25 cm diameter column and washing with water until the wash water was colorless. Pyrophosphate decahydrate (1.12 g, 2.51 mmol) was dissolved in water (30 mL) and poured onto the column. The column was eluted with water and the fraction containing 125 ml of UV absorbent was combined. The solution was concentrated in vacuo to 10 ml and 1.2 ml of tri-n-butylamine was added. The mixture was concentrated in vacuo and the residue was evaporated four times with pyridine. The product was stored at -5 ° C in a refrigerator.

b) Hydrogen form of 3 'azido-5' monophosphate 3 'deoxythymidine

The hydrogen form of the monophosphate was prepared by dissolving 0.1 g of ammonium salt (0.283 mmol) obtained in Example 5 in 6 mL of water and passing the solution through 1.5 mL (10 equiv) of DOW 50 H ⁺ column.

c) a derivative of 3'-azido-3'-deoxythymidine phosphorus morpholidate

The hydrogen form of the monophosphate from step (b) (0.283 mmol) was dissolved in water (9 mL), morpholine (99 µL, 1.13 mmol, 4 eq) was added and the solution was heated to reflux. Dicyclohexylcarbodiimide (0.234 g, 1.13 mmol, 4 eq.) Was dissolved in 5 ml of tert-butanol which was added over 3 hours. The reaction mixture was heated at reflux overnight, then cooled to room temperature, filtered and the solvent removed in vacuo. Ethanol was added to the residue and concentrated in vacuo four times. The residue was dissolved in methanol and the phosphorus morpholidate was precipitated by addition of ether. The precipitate was worked up four times with ether and dried in a rotary evaporator. The title compound is obtained.

d) 3'-Azido-3'-deoxythymidine-5'-triphosphate

The phosphorus morpholidate derivative obtained in step c) is dried in vacuo four times by removing pyridine. The bis (tri-n-butylammonium) pyrophosphate obtained in step a) is also dried under vacuum to remove pyridine. Phosphorus morpholidate is dissolved in 5 ml of pyridine and poured into a flask containing the pyrophosphate reagent. The reaction was allowed to proceed at room temperature overnight. The pyridine is then removed in vacuo, water is added to the residue, and it is removed three times in vacuo. The residue was frozen.

The residue was thawed and dissolved in 50 ml of water. The solution was poured onto a DEAE Scphadex A-25 column (1 x 10 cm) treated with 50 ml of ammonium bicarbonate solution. The phosphate is eluted with 300 ml of 50-800 mM linearly increasing concentrations of ammonium bicarbonate. The fractions containing the diphosphate nucleotide and the triphosphate nucleotide were collected separately, and the diphosphate and triphosphate fractions were each dried in vacuo, redissolved in water and dried, and then redissolved in water and lyophilized.

Example 7

Enzymatic synthesis of 3'-azido-5'-triphosphate-3'-deoxythymidine

5'-triphosphate is synthesized from 5'-diphosphate using pyruvate kinase and nucleosidadephosphate kinase. The reaction mixture was 6 mM 3'-azido-TDP, 12 nM adenosine triphosphate, 40 mM magnesium chloride, 40 mM potassium pipcrazine-N, N-bis (2-ethanesulfonic acid), PIPES fupper pH 6, 8), contains 30 mM phosphocnolpyruvate, 40 IU / ml nuclucoside diphosphate kinase and 100 IU / ml pyrufate kinase in a final volume of 5 ml. The reaction mixture was incubated at 37 ° C for 5 days and then poured onto a DEAE Sephadex A-25 column (2.5 x 10 cm) treated with ammonium bicarbonate. Nucleotides were eluted with increasing concentrations of 100-1000 mM ammonium bicarbonate. The triphosphate containing fractions were collected and evaporated to dryness in vacuo. The compound was further purified using a preparative HPLC column (Whatman Inc., Magnum 9 SAX) and eluted with increasing concentrations of 10-100 mM potassium phosphate (pH 3.5). The resulting compound was further purified using the former DEAE Scphadex A-25 column. The fractions containing tetramammonium 3'-azido-3'-oxo-thymidine-5'-triphosphate were collected, dried in vacuo, redissolved in water and lyophilized to give the title compound.

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Example 8

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) mgltabletta mgltabletta (a) an active ingredient 250 250 (b) Lactose B.P. 210 26 (c) In Povidone B.P. 15 9 d) sodium starch glycolate 20 12 (e) magnesium stearate 5 500 3 300 Formulation B) mg / tabletla mgltabletta (a) an active ingredient 250 250 b) lactose 150 - c) Avicel PH 101 60 26 (d) Povidone B.P. 15 9 e) sodium starch glycolate 20 12 (f) magnesium stearate 5 5

500 300

Formulation C mg / tablet

(a) Active ingredient

(b) lactose 200

(c) starch

(d) Povidone 5

(e) magnesium starch 4

359

The following adjustments (d) and (E) are made by direct pressing of the components.

D) Formulation mgtkapszula (a) an active ingredient 250 (b) pregelatinised starch NF 15 150

400

Formulation E mg / capsule

(a) active ingredient

(b) lactose (Dairy Crest - "Zcparox") 150

(c) Avicel 100

500

Formulation F (Controlled Release Formulations) The formulation is wet pressed with a solution of the following ingredients, povidone, and then added with magnesium stearate.

mgllablelta

(a) an active ingredient 500 b) hydroxypropyl methylcellulose 112 (Methoccl K4M Premium) c) Lactose B.P. 53 (d) In Povidone B.P.C. 28 (e) magnesium stearate 7

700

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

Example 9: 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 mgt capsule

(a) active ingredient

b) LactoseB.P. 143

c) sodium starch glycolate 25

(d) magnesium stearate;

420

Formulation C mg / capsule

(a) active ingredient

b) Macrogol 4000 BP 350

600

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

Formulation D mg / capsule

(a) active ingredient

b) lccitin 100

(c) arachidic 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 ingredients a), b), c) into pellets and drying the extrudate. The dry pellet cake is coated with release membrane d) and filled into a hard gelatin capsule.

mg / capsule

(a) active ingredient

(b) microcrystalline cellulose 125

c) Lactose BP 125

d) ethylcellulose 13

513

Example 10 (injectable formulations)

Formulation A Active Ingredient 0.200 g Hydrochloric Acid Solution 0.1 M Appropriate Volume 4.0 to 7.0 Sodium Hydroxide Solution 0.1 M Appropriate Volume 4.0 to 7.0 Sterile Water To 10 ml

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, 25 ml required

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HU 201 678 Β

Example 11 (intramuscular injection) active ingredient 0.20 g benzyl alcohol 0.10 g glycofurol 75 1.45 g water for injection, required volume 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 passed through a sterile microporous filter into a 3 ml brown sterile vial (type 1) and sealed.

Example 72 (Mixture) Active Ingredient 0.2500g Sorbitol Solution 1.5000g Glycerol 2.0000g Sodium Benzoate 0.0050g Flavoring Peach 17.42.3169 0.01225ml Sterile Water Required 5,0000ml- from

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 13 (Suppository) mg / Suppository.

active ingredient (63 pm) ⁺ 250

Hari Fát, 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 filtered 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 H15 is added to the slurry and mixed to a homogeneous mixture. The whole slurry was passed through a 250 µm stainless steel sieve and allowed to cool to 40 ° C with constant stirring, then 2.02 g of the mixture was poured into a suitable plastic mold at 38-40 ° C.

Example 14 (Pessarium) mglpessarium active ingredient 63 pm 250 anhydrous dextrose 380 potato starch 363 magnesium stearate 7

1000

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

Example 75

Antiviral activity

(a) (i) Harm caused by retrovirus

Female BALB / c mice infected with 1.5x10 * Rauschcr Murinc Leukacmia Virus (strain RVB 3) are given 3'-azido-3'-deoxythymidine. Treatment is initiated at an intraperitoneal dose of 80 mg / kg 4 hours post-infection and administered either orally in drinking water at 0.5 mg / ml or 1.0 mg / ml every 8 hours. For such treatment, it has been found to inhibit spleen cell infection and subsequent development of splenomegaly, as well as suppress viremia.

ü) HTLV

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

a) TM-11 cells alone;

b) TM-11 cells and MJ tumor cells;

c) TM-11 cells, MJ tumor cells and 3'-azido-3'-deoxythymidine (3 µM / pmol);

d) TM-11 cells, MJ tumor cells and 3'-azido-3'-deoxythymidine (9 µM / pmol);

e) TM-11 cells, MJ tumor cells and 3'-azido-3'-deoxythymidine (27 µM / pmol).

At day 18, DNA from each culture was extracted and digested with Bam HI to produce an HTLV-I genomic fragment, free of any host adjoining sequences, and having a standard molecular weight of 3.3 kD. Digestion is then assayed with isotope lambda-ΜΤ-2, a standard assay for HTLV for the detection of BamHI framgens.

No hybridization was observed at (a), indicating 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 at d) -ncl or e) there is no hybridization, which indicates complete destruction of the virus.

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

b) A77) V

i) Reverse transcriptase activity

3'-Azido-5'-triphosphate-3'-oxyoxytimidine was tested in vitro with 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 stable for at least two months when stored per ml in 60% glycerol containing 1 mg bovine serum albumin. Using rA-odT ₍₁₂ _ ₁₈₎ was Template primer, AIDV RT the pH optimum of 7.0 to 7.3, the optimum of 0.3 mM MnCl ₂ and MgCl ₂ optimum of 5 mM. The activity in the presence of 5 mMMgCl ₂ is ten times greater than in the presence of 0.3 mM MnCl ₂ . Maximum enzyme activity was also found at 80140 mM KCl and 60-100 mM NaCl. The intake of fH) 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 µM when using rA-odT ₍ 12-18 ₎ as template primer. The enzyme from the TTP, has a value _m of 2.81 pM s, supposing that 3'-azido-5'-triphosphate-3'-deoxythymidine binds more strongly to the enzyme than the dTPP. Further experiments on avian micloblastosis virus, Moloney murine leukemia virus, and AIDV RT show that 3'-azido-5'-triphosphate deoxythymidine is a terminator of the DNA chain length.

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HU 201 678 Β 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. Therapeutic drug was measured by reverse transcriptase (RT) activity assay in a solution of infected, uninfected and drug treated cells. 3'-Azido-3'-deoxythymidine effectively blocks A1DV infection of the H9 and U937 human lymphoblastoid cell lines at concentrations of 2.7-0.0013 pcg / ml. Similarly, it inhibits normal PHA infection stimulated by white blood cells and cultured peripheral blood lymphocytes at drug concentrations below 0.013 pcg / 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 virus infection of the sensitive cell, but most of the antiviral activity is still present when AIDV infection 20 hours after the start. Inhibition of viral recurrence was also evident when the drug was present in the cell only around 20 hours after viral absorption. The effect was seen at 0.13 and 0.013 pcg / ml. 3'-Azido-3'-Doxoxytimidine has no direct anti-RT activity against the 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 is determined as follows:

Cloned T4-positive tetanus-specific T-helper lymphocytes were infected with isolated AIDV virus [max. 5000 viruses / cell dose-triggered] and after infection, cell survival was monitored. After 10 days in culture, 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 above cells and highly infected with AIDV. 3'-Azido-3'-deoxylimidine, at concentrations of 2.7,0.27 and 0.13 pg / ml, completely blocks the cytopathic effect by day 7. There was a protective effect in the infection caused by the cell-free virus and the cell-associated virus. 3'-Azido-3'-deoxythymidine, at a concentration of 0.27 pg / ml, is also effective in inhibiting the cytopathic effect induced by the less related Haiti-isolated AIDV.

Example 16

Toxicity study

3'-Azido-3'-deoxythymidine was administered to both mice and rats. The LD * in both species was above 750 mg / kg.

Example 17

Activity against HIV (Human Immunodeficiency Virus)

Examining anti-HIV activity of 3-azido-3'-deoxythymidine-5'-acetate found that the IC _S0 value (the concentration that kills 50% of the virus) 3 pmol.

Claims (14)

PATENT CLAIMS CLAIMS 1. A process for the preparation of a pharmaceutical composition comprising 3'-azido-3'-deoxythymidine as its active ingredient, or a 5'-ester derivative thereof, such as a triphosphate or acetate, characterized in that the active ingredient is known in a pharmaceutically acceptable liquid or solid carrier. and / or mixed with other excipients and formulated for administration, optionally to slow-release human retrovirus-mediated diseases. 2. A process according to 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. The method of claim 1, 2 or 7, wherein the composition is formulated as a tablet or capsule. The process according to claim 7, characterized in that the composition is prepared in a slow release form, preferably with a hydroxymethylcellulose coating, 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 6, characterized in that the active ingredient is used in a unit dosage form or in a 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 method of claim 10, wherein the active ingredient is present in an amount of from 10 mg to 1000 mg. 14. A10. The process of claim 1, wherein the active ingredient is present in an amount of from 20 mg to 700 mg.