HU203971B - Process for producing synergetic combination pharmaceutical compositions comprising 3'-azido-3'-deoxythymidine and one more active ingredient - Google Patents

Abstract

According to the invention, the 3'-azido-3'-deoxytimidine and acyclovir, α-interferon, dipyridamole, probenicide, dilazep or 6 - [(4-nitrobenzyl) thio] -9-3-D-ribofuranosyl purine are synergistic. are formulated into pharmaceutical compositions for the prevention or treatment of human retroviral infections. Scope of description: 10 pages EN 203 971 -1-

Description

This invention relates to a synergistic combination pharmaceutical composition comprising 3'-azido-3'-deoxythymidine and an additional active ingredient for the treatment or prevention of viral infections.

More particularly, the present invention relates to pharmaceutical compositions comprising 3'-azido-3'-deoxythymidine and acyclovir, α-interferon, dipyridamole, probenicide, dilazep or 6 - [(4-nitrobenzyl) thio] -9-pD-ribofuranosylpurine. the preparation.

In the field of chemotherapy against viruses, there are few drugs capable of effectively combating the virus itself. This shows that it is difficult to attack the virus while leaving uninfected cells intact. It has long been assumed that due to the extreme parasitic nature of viruses, all host conditions for viral replication are provided by the host cell. It has recently been established that certain stages of the virus life cycle, which vary from species to species, are determined by the virus itself and may be susceptible to attack in stages where its functions are sufficiently different from those of the host cell. However, due to the high degree of similarity between the functions of the virus and the host cell, it has proved very difficult to determine effective treatment.

Therefore, compounds found to be suitable for the treatment of viral infections are generally somewhat toxic to the host cell. Ideal medication is non-toxic at concentrations effective against the virus, but if this is not possible, the compound used as the drug should have a good therapeutic ratio, i.e. the concentration at which the treatment is toxic should be substantially higher than that at which the antiviral effect is detectable. .

One of the groups of viruses that has gained particular importance today is retroviruses. Retroviruses are a subset of RNA viruses; in order to first replicate their genome's RNA must be "reverse" transcribed into DNA (transcription usually refers to the synthesis of RNA from DNA). Once in DNA form, the viral genome can be incorporated into the host cell genome, enabling the virus to take advantage of all the benefits of the host cell's transcription / translation system for its own replication. Once incorporated, viral DNA is virtually indistinguishable from host cell DNA and, in this state, the virus may persist for as long as the host cell lives. Because the virus is virtually invulnerable in this form, any treatment should be directed to a different stage of the viral life cycle and must necessarily last until all cells infected with the virus are killed.

Both HTLV-I and HTLV-II are retroviruses known to cause human leukemia. HTLV-I infections are particularly widespread and cause many deaths each year worldwide.

A retrovims species has also been reproducibly isolated from AIDS patients. While the old man has already been thoroughly labeled2, there is still some controversy about his internationally acceptable designation. It is currently known as human T-cell lymphotropic vim III (HTLVIII), AIDS-related retrovirus (ARV), or lymphadenopathy-related vermi (LAV), but is expected to be known internationally as human immunodeficiency virus (HVV). (The former internationally used abbreviations are derived from the proper English names.) This virus (hereinafter referred to as HIV) has been prone to infection and killing, in particular, of T cells carrying OKT4 surface macrophages, and is now generally considered a disease of AIDS. The patient gradually loses his / her T cells, causing the overall immune system to be disturbed, the ability to fight other infections, and the patient to become susceptible to opportunistic infections, which are often fatal. Thus, the cause of death of AIDS victims is usually an opportunistic infection, such as pneumonia or cancer that can be caused by vomiting, and is not necessarily a direct consequence of HIV infection. Other conditions associated with HIV infection include thrombocytopenia purpura and Kaposi's sarcoma.

More recently, HIV has been recovered from other types of tissue, including B cells expressing the T4 marker, macrophages, and non-blood-derived central nervous system tissues. This central nervous system infection is not necessarily linked to classical AIDS, and has also been shown in asymptomatic HIV-infected patients. CNS HV infection is accompanied by a progressive decline in neural formation leading to atrophy and symptoms such as encephalopathy, progressive articulation disorder, movement disorder, and disorientation. Other conditions associated with HIV infection include asymptomatic carrier status, advanced general lymph node disease (PGL), and AIDS-related symptomatic complex (ARC).

It has been found that certain chronic neurological infections are caused by retroviruses. Examples of such diseases are human multiple sclerosis, viral infection of goat arthritis encephalitis and visna-maedi infection in sheep.

It has been reported that some compounds have been tested for their effectiveness against various retroviruses, such as Friend's leukemia virus (FLV) and rat retrovims.

Krieg et al., Exp. Cell. Sl., 116, 21-29 (1978)], for example, was found to be active against FLV in the in vitro assay of 3'-azido-3'-deoxythymidine. Ostertag et al., Proc. Nat. Acad. Sci., 71, 4980-85 (1974)] suggested that 3'-azido-3'-dideoxyethimidine, due to its antiviral activity on FLV and in the absence of cellular toxicity, "could positively replace bromodeoxyuridine in the treatment of diseases caused by DNA viruses. ". De Clerq et al., Biochem. Pharm. 29, 1849-51 (1980)], however, found 6 years later that 3'-azido3'-deoxythymidine did not show significant activity against one of the viruses used in its assays.

Neither is it against DNA viruses such as vaccinia, HSVI and varicella zoster virus (VZV).

Combinations of 3'-azido-3'-deoxythymidine with other therapeutic agents have been found to be useful in the treatment of viral infections, particularly retroviral infections, including HIV.

Accordingly, the present invention relates to a process for the manufacture of a medicament for the prevention or treatment of human retroviral infections. The compositions are prepared by synergistically forming 3'-azido-3'-deoxythymidine with acyclovir,? -Interferon, dipyridamole, probenicid, dilazep or 6 - [(4-nitrobenzyl) thio] -9-3-D-ribofuranosyl-. with purine active ingredient in a molar ratio of 20: 1 to 1: 20 and formulated with carrier and / or other excipients.

The combination compositions of the present invention are particularly effective against the following viruses: human T cell lymphotropic viruses (HTLV), particularly HTLV-I, HTLV-II and HTLV-III (HIV); feline leukemia virus, equine infectious anemia, caprine arthritis and other lentivines; and other human attack viruses, such as hepatitis B virus, Epstein-Barr virus (EBV), and multiple sclerosis (MS).

The combination compositions of the present invention have been found to be effective in treating Kaposi's sarcoma (KS) and thrombocytopenia purpura (TP).

The action of 3'-azido-3'-deoxythymidine against infections caused by a wide variety of varicose veins is clearly of great medical importance, and the novel mode of action of this compound allows it to be used in combination therapy to reduce the development of resistance. A particular advantage of such combination therapies is that other therapeutics described below have an unexpected synergistic effect on 3'-azido-3'-deoxythymidine.

It has been found that the combination of 3'-azido-3'-deoxythymidine and the above therapeutic agents results in a synergistic therapeutic effect that is disproportionate to that of the agents used. Significantly less of each agent is required for treatment, thus increasing the therapeutic ratio and reducing the risk of toxic effects of the compounds used.

By synergistic ratio is meant the ratio of 3'-azido-3'-deoxythymidine to another therapeutically active agent which results in a greater therapeutic effect than the sum of the therapeutic effects of each component.

Note that although there is usually an optimum ratio that results in a maximum synergistic effect, a synergistic effect can be obtained even when one of the two substances is used in very small amounts so that any ratio of the synergists has a synergistic effect. A synergistic effect is achieved when the two agents are present in a ratio of 20: 1 to 1:20, preferably 10: 1 to 1:10.

These compositions are hereinafter referred to as the compositions of the invention.

The compositions of the invention may conveniently be presented in the form of a single pharmaceutical composition or as separate active ingredients, for example, in the form of tablets or injection combinations administered at the same time or at separate times to achieve the desired therapeutic effect.

The probenicide is a particularly potent combination with 3'-azido-3'-deoxythymidine because it has both renal secretion activity and glucuronidation inhibitory activity.

It will be appreciated that the compounds and compositions of the present invention may be used in combination with other immunomodulatory therapies, such as bone marrow and lymphocyte transplantation.

Some of the retroviral infections described above, such as AIDS, are often associated with opportunistic infections. In this regard, it is noted that, for example, the combination of 3'-azido-3'-deoxythymidine (AZT) and acyclovir has been shown to be particularly effective in treating AIDS patients with opportunistic herpes infection.

3'-Azido-3'-deoxythymidine is one of the active ingredients of the above combinations and can be used in any conventional manner for therapeutic purposes, such as oral, rectal, nasal, topical (including oral and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal applications). It is noted that the preferred route of administration will vary with the condition and age of the patient and the nature of the infection.

Generally, a suitable dosage will be from 3.0 to 120 mg / kg body weight / day, preferably from 6 to 90 mg / kg body weight / day, more preferably from 15 to 60 mg / kg body weight / day. Preferably, the desired dose is administered in portions of 2, 3, 4, 5,6 or more, at appropriate intervals throughout the day. A single dose may be formulated in a unit dosage form, for example, in a unit dose containing from 10 to 1500 mg, preferably from 20 to 1000 mg, more preferably from 50 to 700 mg, of the active ingredient.

Studies with 3'-azido-3'-deoxyimidine show that a dose should be administered which achieves peak plasma concentrations of 1-75 µmol / l, preferably 2-50 µmol / l, more preferably 3-30 µmol / l. a. This value can be achieved, for example, by intravenous injection of 0.1-5%, optionally saline, or by oral administration of a pill containing 1-100 mg / kg of active ingredient. The desired blood drug level can be achieved by continuous infusion, from 0.01 to 5.0 mg / kg / hour, or by repeated infusions from 0.4 to 15 mg / kg.

The combination compositions of the present invention may be administered in a similar manner as described above to provide the desired therapeutic dose of the active ingredient and the additional therapeutic agent. The dosage of the combination compositions will depend on the condition being treated, the particular agent, the additional therapeutic agents and other clinical factors, including the patient's body weight and condition, and the route of administration of the combination. As mentioned above, a

HU 203 971 A

The 3'-azido-3'-deoxythymidine and the additional therapeutic agent may be administered simultaneously, for example in the form of a single pharmaceutical composition, or separately, e.g. as separate pharmaceutical compositions, and generally the compositions may be administered topically, orally, rectally or parenterally (e.g. , subcutaneous or intramuscular).

Combinations in which the second agent is a nucleoside transport inhibitor may be administered in the usual manner. However, for oral administration, the required dose is from 1 to 200 mg / kg body weight / day, preferably from 5 to 50 mg / kg body weight / day. For parenteral administration, 1 to 100 mg / kg body weight / day, preferably 2 to 30 mg / kg body weight / day are required. The amount of nucleoside transport inhibitor present in the combination composition will depend on the amount of 3'-azido-3'-deoxythymidine and should be sufficient to effectively inhibit nucleoside transport, preferably from 0.1 to 100 mg / kg / day, more preferably from 1 to 20 mg / kg / day. mg / kg body weight / day.

Combination formulations of the present invention wherein the second agent is a renal secretion inhibitor or a glucuronidation inhibitor, or both, can be administered to the patient in a conventional manner. However, for oral administration, the required dose is from 1 to 200 mg / kg body weight / day, preferably from 5 to 50 mg / kg body weight / day. For parenteral administration, 1 to 100 mg / kg body weight / day, preferably 2 to 30 mg / kg body weight / day are generally sufficient. The amount of inhibitor of renal secretion and / or glucuronidation present in the composition will depend on the amount of active ingredient, the appropriate dosage being 4-100 mg / kg body weight / day, preferably 5-60 mg / kg body weight / day, more preferably 10-40 mg / kg body weight / day. Sun.

Combinations thereof prepared according to the invention, which contain the active ingredient of 5-250 mg / kg body weight / day of a second active substance, interferon célszemen and preferred effective dose of the interferon 3xl0 ⁶ 10xl0 ⁶ IU / m ² body surface area / day, preferably ⁶ 4xlO ^oh -6xlO IU / m ² body surface area / day. Suitable dosage regimens of active ingredient and interferon are 5 mg / kg body weight / day and 3x10® IU of interferon / m ² body surface area / day to 250 mg of active ingredient / kg body weight / day and 10x10 ⁶ ne interferon / m ² body surface area / day, preferably 5 mg active ingredient / kg body weight / day and 4x10 ⁶ IU interferon / m ² body surface area / day 100 mg active ingredient / kg body weight / day and 6x10 ⁶ IU interferon / m ² body surface area / day.

Interferon alpha is conveniently administered by injection, for example, by subcutaneous, intramuscular or intravenous injection, while the active ingredient is preferably administered orally or by injection, but may be administered by any of the routes described above. The interferon and the active ingredient may be administered together or separately, the daily dose preferably being divided into several daily doses for each active ingredient.

For combinations according to the invention having a second active ingredient, a 2 ', 3'-dideoxynucleoside, a preferred effective oral dose is 2.550 mg active ingredient / kg body weight / day, preferably 510 mg active ingredient / kg body weight / day; a preferred effective oral dose of the second therapeutic nucleoside is 5-100 mg nucleoside / kg body weight / day, preferably 15-75 mg nucleoside / kg body weight / day. For oral administration, the ratio of the active ingredient to the second therapeutic nkleoside is preferably in the range 1: 1 to 1:10, more preferably 1: 21: 8.

In the case of intravenous administration, a suitable dosage range of the active ingredient is generally from 1.5 to 15 mg / kg body weight / day, and a suitable effective dosage range of a therapeutic nucleoside is from 5 to 30 mg / kg body weight / day. For intravenous administration, the preferred ratio of active ingredient to second therapeutic nucleoside is in the range 2: 1 to 1:20, preferably 1: 2 to 1:10.

Both components of the combination are preferably administered orally or by injection, may be administered together or separately. The daily dose of both agents can be administered in several divided doses.

Although the active ingredient can be administered alone, it is preferably formulated as a pharmaceutical composition. The pharmaceutical compositions of the present invention comprise at least one of the active ingredients described above, together with one or more acceptable carriers, and optionally other therapeutic agents. All excipients used should be "acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The compositions may be oral, rectal, nasal, topical (oral or sublingual), vaginal or parenteral (subcutaneous, intramuscular, intravenous or intradermal). The compositions may be formulated in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. A typical step in such methods is, for example, mixing the active ingredient with a carrier which comprises one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers, or both, and if necessary, shaping the product.

According to the present invention, compositions for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; in the form of powder or granules; as a solution or as a suspension in an aqueous or non-aqueous liquid; as an oil-in-water or water-in-oil liquid emulsion. The active ingredient may be formulated as a pill, syrup or paste.

The tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be formulated in a suitable compression machine using the active ingredient in free-flowing form such as powder or granules, optionally with a binder (e.g. povidone, gelatin, hydroxypropylmethylcellulose), glidant, inert diluent,

The disintegrating agent, disintegrating agent ((e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactant or dispersant is formulated in a suitable preformed tableted, anhydrous powder. Tablets may be coated or labeled, if desired, and may be formulated so as to provide slow or controlled release of the active ingredient, e.g. profile.

Formulations for topical administration in the mouth may be prepared in the form of lozenges containing the active ingredient in a flavored form, for example, usually with sucrose, acacia or tragacanth; pastilles comprising the active ingredient together with an inert carrier such as gelatin and glycerol or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Formulations for rectal administration are prepared in the form of suppositories using a suitable carrier such as cocoa butter or salicylate.

Formulations for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or as sprays containing in addition to the active ingredient such carriers as are conventionally employed in the preparation of like compositions.

Formulations for parenteral administration may be aqueous or nonaqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the patient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be in unit dosage form or may be presented in multi-dose containers, for example ampoules and vials, in freeze-dried (lyophilized) form which may be prepared by the addition of a sterile liquid carrier such as water for injection before use. In use, solutions and suspensions for injection may be prepared from sterile powders, granules and tablets, the preparation of which is described above.

Preferred unit dosage formulations are those containing the daily dose in a unit, or a portion thereof, as described above, or an appropriate proportion thereof.

Formulations suitable for veterinary use may also be prepared in the same manner as is customary in the manufacture of a veterinary composition. Such veterinary preparations may be, for example

(a) for oral administration (for example, liquid preparations such as aqueous or non-aqueous solutions or suspensions); tablets or large tablets, powders, granules or granules which may be mixed with feed, tongues to be applied to the tongue;

b) formulations for parenteral administration, for example, by sibutaneous, intramuscular or intravenous injection, such as sterile solutions or suspensions; or (if necessary) intramammary injections where the suspension or solution passes through the udder into the udder;

(c) topical preparations such as creams, ointments or sprays applied to the skin; or

(d) vaginal preparations such as pessaries, creams or foams.

It will be appreciated that the above compositions are suitable for use in combination with the present invention in a single or separate formulation, and are prepared in a manner similar to that described above.

In addition to the components described above in detail, the compositions of the present invention may contain other substances conventionally used in the preparation of the compositions depending on the type of preparation to be prepared, for example, oral compositions may contain additional sweetening, thickening and flavoring agents.

1-3 below. Formulation examples include the preparation of pharmaceutical formulations containing 3'-azido-3'-des, oxytimidine and another active ingredient indicated in the example,

. Formulation example 1 injection preparation acyclovir Weight (mg) 400 3'-Azido-3'-deoxythi midin 200 1 M sodium hydroxide solution need Sterile water supplement by To 10 ml

The above formulation is prepared by adding the therapeutic nucleoside to a 1M sodium hydroxide solution and mixing until complete dissolution. 3'-azido-3'-deoxythymidine is then added to the solution and dissolved. Make up to 10 ml with sterile water. The solution was filtered through a sterile filter and filled into sterile vials and freeze-dried.

Formulation Example 2 Preparation of tablets

acyclovir Weight (mg) 500 3'-Azido-3'-deoxythiic midin 125 povidone 14 Sodium starch glycolate 25 Magnesium stearate 6 670

HU 203 971 A

To prepare the above composition, the therapeutic nucleoside and 3'-azido-3'-deoxythymidine are mixed with sodium starch glycolate and granulated with a povidone solution. The granules are dried and then mixed with magnesium stearate and pressed.

Formulation example 3 Preparation of capsules

acyclovir Mass (mg) 250.0 3'-azido-3'-deoxythymidine 62.5 Lactose 170.5 Sodium starch glycolate 15.0 Magnesium stearate 2.0 500.0

The components are mixed together and filled into hard gelatine capsules.

Formulation Example 4 illustrates the preparation of a composition comprising 3'-azido-3'doxythymidine and interferon as active ingredients.

Formulation example 4

For the preparation of an injection

Interferon 3 mega units 'azido-3' deoxythymidine 200 mg

Sterile pH 7 buffer supplemented to 50 ml

Interferon and 3'-azido-3'-deoxythymidine are dissolved in sterile water. The solution is stained on a sterile filter and filled into sterile vials.

The following 5-8. Formulation Examples 2 to 3 describe the preparation of formulations containing the active ingredient 3'-azido-3-oxo-oxymethimidine and dipyridamole as the nucleoside transport inhibitor.

Formulation Example 5 Preparation of tablets

Nucleoside transport inhibitor Weight (mg) 300 3 '-azido-3' -deoxythymidine 200 Lactose 105 Starch 50 Poly (vinyl pinolidinon) 20 Magnesium stearate 10 Gross weight 685

The active ingredients are mixed with lactose and starch and granulated with a solution of polyvinylpyrrolidinone moistened. The granules are dried, sieved, mixed with magnesium stearate and then compressed into tablets.

Formulation Example 6 Preparation of capsules

Weight (mg) Nucleoside transport inhibitor 300 3 '-azido-3' -deoxythymidine 100 Lactose 100 Sodium starch glycolate 10 Poly (vinyl pyrrolidinone); 10 Magnesium stearate 3 Gross weight 523

The active compounds are mixed with lactose and sodium starch glycolate and granulated with polyvinylpyrrolidinone solution. The granules are dried, sieved and mixed with magnesium stearate and filled into hard gelatine capsules.

Formulation example 7

Preparation of cream

Nucleoside transport inhibitor Weight (mg) 7.50 3'-azido-3'-deoxythymidine 5.00 Glycerol 2.00 Cetostearyl alcohol 6.75 Sodium laureth sulfate 0.75 White soft paraffin 12.50 Liquid paraffin 5.00 Chloro-cresol 0.10 Purified water supplement To 100.00

The active compounds were dissolved in a mixture of purified water and glycerol and heated to 70 ° C. The other components were heated together to 70 ° C. The two heated portions are mixed and ergulated. The mixture is then cooled and filled into suitable vessels.

Formulation Example 8

Preparation of intravenous injections

Weight (mg) (1) 3'-Azido-3'-deoxythymidine 200

Nucleoside transport inhibitor 300

Glycerin 200

Sodium hydroxide solution in an amount sufficient to adjust the pH to 7.0-7.5

Water for injection is made up to 10 ml

Glycerol is added to a portion of the water for injection. The two active ingredients are mixed and the pH of the mixture is adjusted to 7.0-7.5 with sodium hydroxide solution. Make up to 10 ml with water for injections. The solution is aseptically stained with sterile solution, filled into sterile vials and sealed.

HU 203 971 A

Weight (mg) (2) 3'-Azido-3'-deoxythymidine 100

Nucleoside transport inhibitor 150

Mannit 125

Sodium hydroxide solution in an amount sufficient to adjust the pH to 8.0-9.0

Water for injection is made up to 2.5 ml

The active compounds and mannitol are dissolved in a portion of the water for injection. The pH of the solution was adjusted to 8.0-9.0 with sodium hydroxide solution and made up to 2.5 ml with water for injection. The solution is then aseptically filtered to sterile, filled into sterile vials and freeze-dried. The vials were sealed under atmospheric nitrogen gas, sealed with a sterile closure and sealed with a metal collar.

The next 9-11. Examples 3 to 8 describe the preparation of compositions containing 3'-azido-3'-deoxythymidine and a probenicide as a glucuronidation inhibitor and a renal secretion inhibitor.

Formulation Example 9 Preparation of tablets

3'-azido-3 'deoxythymidine Weight (mg) 100 Glucuronidation / renal secretion inhibitor 200 Lactose 105 Starch 50 Poly (vinyl pyrrolidinone); 20 Magnesium stearate 10 Gross weight 485

The active compounds are mixed with lactose and starch and granulated with a solution of polyvinylpyrrolidinone moistened. The granules are dried, sieved, mixed with magnesium stearate and then compressed into tablets.

Formulation example 10 Preparation of capsules

3'-azido-3 'deoxythymidine Weight (mg) 100 Glucuronidation / renal secretion inhibitor 100 Lactose 100 Sodium starch glycolate 10 Poly (vinyl pyrrolidinone); 10 Magnesium stearate 3 Gross weight 323

The active compounds are mixed with lactose and sodium starch glycolate and granulated with a solution of polyvinylpyrrolidinone moistened. The granules are dried, sieved, mixed with magnesium stearate and filled into hard gelatine capsules.

Formulation example 11 Preparation of intravenous injections Weight (mg) (1) 3'-Azido-3'-deoxythymidine 200 Glucuronidation / renal secretion inhibitor 300 Glycerol 200 Sodium hydroxide Sufficient to adjust pH to 7.2-7.5 amount Water for injections additionally 10 Ml Glycerol is added to a portion of the water for injection. The two active ingredients are added and the solution is adjusted to pH 7.0-7.5 with sodium hydroxide solution. The volume of the solution is then made up to 10 ml with water for injection, the solution is aseptically filtered, filled into sterile ampoules and sealed. Weight (mg) (2) 3'-Azido-3'-deoxythymidine 100 Glucuronidation / renal kivá- inhibition of libido 150 mannitol ¹²⁵ Sodium hydroxide Enough to adjust pH 8.0-9.0. amount Water for injections in addition to 2.5. " per ml f

The active ingredient and mannitol are dissolved in a portion of the water for injection. The solution is adjusted to pH 8.0-9.0 with sodium hydroxide solution and made up to volume with water for injection. Under aseptic conditions, the solution is filtered sterile, filled into sterile vials, and the water removed by freeze-drying. The vials were sealed under atmospheric pressure of nitrogen gas, sterile sealed and sealed with a metal collar.

Test Example 1

Clinical examination

In a patient with AIDS, 3'-azido-3'-deoxythymidine (AZT) is administered orally on day 1 and day 2 for 8 hours at a dose of 2 mg / kg body weight. On days two and three, patients are also given 500 mg of probenicide (PB) every 6 hours, with a single dose of AZT on day three. The peak (C _maj ) and minimum (C _min ) levels of AZT on the third day after probenicide administration were significantly higher than the corresponding first-day levels, resulting in a decrease in total body clearance (C17F) of one third and The mean half-life of AZT was prolonged (from 0.88 to 1.73 hours) during PB treatment. Mean urinary AZT glucuronide / AZT ratio decreased significantly during PB treatment, from 7.3 to 2.4. The essential pharmacokinetic parameters of AZT before and after PB administration are shown in Table I. (The effect of probenicide on its own is negligible compared to that of AZT.)

HU 203 971 A

/. table

Serial number of patients AUC (Micromoles) c '' Ttiax (Micromoles) c · '' Ttuit (hour Umol) Ϊ max (hours) Cfeljes / P 1mI / min / 70kg) Half-life (hours) 1. OCT / PB 10.41 6.13 0.15 0.25 829.50 1.84 2. OCT / PB 10.00 6.46 0.27 0.50 921.00 1.61 Average 10.21 6.30 0.21 0.38 875.25 1.73 ± SD 0.29 0.23 0.08 0.18 64.70 0.16 1. THERE 3.70 3.74 0.00 0.50 2333.80 0.87 2. THERE 3.04 2.51 0.00 0.31 3027.00 0.89 Average 3.37 3.13 0.00 0.41 2680.40 0.88 ± SD 0.47 0.87 0.00 0.13 480.17 0.01

Test Example 2

Antiviral activity

II. Table 3 shows the enhancement of the effect of 3'-azido-3'-deoxythymidine (AZT) against Friend leukemia virus (FLV) by nucleoside transport inhibitors such as dipyridamole, dilazep and 6 - [(4-nitrobenzyl) thio] 9-P -ribofuranosyl) -purin. FG-10 cells were plated on a plate and infected with FLV 24 hours later. One hour after infection, the test compounds or combinations are added at known concentrations. The plates are incubated for 3 days, the medium is replaced with fresh McCoy 5A medium, and the incubation is continued for 3 days. AII. Table I shows the concentration of test compounds or combinations that inhibit plaque formation by 50%. Neither dipyridole (at a concentration of 10 pmol / l) nor dilazep (at a concentration of 5 μτηόΐ / l) exhibit any detectable antiviral activity.

//. spreadsheet

A compound / composition Azidothymidine ED ₅₀ (nmol / l) AZT 5 AZT + 1 pmol / l dipyrimidine 1 AZT + 5 µmol / l dipyrimidine 0.5 AZT + 10 pmol / l dipyrimidine 0.2 AZT + 5 µmol / l dilazep 0.5 AZT + 5 p / 1 6 - [(4-nitrobenzyl) thio] -9-β-D-ribofuranosyltitrine 3

Test Example 3

In vitro use of AZTIacyclovir combination

Against HfV

The in vitro activity of AZT and acyclovir (ACV) combinations against HIV was investigated.

ACV alone showed low activity, at the highest concentration tested, 16 µg / ml, provided less than 30% protection, while AZT provided 100% protection at 8 pmol / l.

AIII. Table 2 shows the combinations of the two drugs that provide 100% protection.

The results show that ACV synergizes approximately three-fold with the antiviral activity of AZT.

///. spreadsheet

ACV (µg / ml) AZT (pmol / 1) - 0 8 0.5 2 1 1 4 0 8

4. Test Example

In vitro use of AZTI interferon combinations

Against HIV

Peripheral blood mononuclear cells (PBMCs) obtained from 40 heparinized blood of healthy, HIV seronegative, voluntary donors were obtained by Ficoll-Hypaque sedimentation. Cells were treated with 10 pg / ml phytohemagglutinin (HPA) and R PM1 supplemented with 20% fetal calf eyes (FCS), antibiotics, 1-glutamine and 10% interleukin-2 (IL-2) (Electronucleonics, Bethesda, MD). Cultured in 1640 medium. 4-6 days after PHA treatment, the cells are resuspended in 25 ml flasks containing 5 ml of medium at a concentration of ⁴ x 105 cells / ml and exposed to the following drugs and virus. The day of infection with the vincts is denoted as day 0. On day 4, fresh medium was added. Then every 3-4. On day 1, a portion of the cell suspension is removed for analysis and replaced with fresh cell-free medium. Experiments 2 and 4 are completed after 14 days, experiments 1 and 3 are continued for 16 days.

Aliquots of virus cultures are aliquots of cell-free cell supernatant of HIV-infected H9 cells frozen at -70 ° C. An infectious dose (TCID ₅₀ ) of 50% tissue culture (TCID ₅₀ ) was 10 ⁵ / ml.

HU 203 971 A

Four separate studies were performed using PBMCs from different donors. Our results are shown in Table IV. is shown in Table II. In the first experiment, both drugs were added after the cells were exposed to the virus. An aliquot of 40x10 ⁶ cells was resuspended in 20 ml of medium containing 10 ⁵ TCID 50 virus, washed after 3 hours with virus-free medium, and resuspended in virus-free medium. 2-4. In the experiments, cells were incubated for 24 hours with or without recombinant interferon alpha (rIFNaA), then AZT was added and exposed to the virus. The virus was added directly to the culture in a small volume of medium and not washed. The virus vaccines of the experiments 2, 3 and 4, 4x10 ^3, 10 ³ and 2xl0 ³ TCID 50 value. The concentration of the drugs used is always adjusted at the original concentration when changing the medium.

Double dilutions of the specified concentrations of rlFNaA and AZT in each experiment were tested in duplicate. All doses of rIFNaA and AZT used in the combinations were tested individually to provide reference results.

In each experiment, at 2 concentrations, 2 cultures were used for the infected and uninfected controls. In Experiment 1, 3.2 µM AZT and 128 U / ml rIFNaA were used, and double dilution of this combination was tested in 5 replicates.

In Experiments 2 and 3, 0.16 µM AZT and 128 U / ml rIFNaA were used, and double dilution of this combination was tested in 3-4 replicate assays. In Experiment 4, 0.08 µM AZT and 128 U / ml rIFNaA were used, and a double dilution of this combination was used in 2 replicates.

After approximately one week, the cultures are assayed for viral load every 3-4 days. In cells, HIV antigens were assayed by indirect immunofluorescence; supernatant reverse transcriptase (RT) activity and viral yield were assayed and determined by radioimmunoassay (RIA) to determine the content of HIV p24 antigen.

Chou and Talalay (Advances in Enzyme Reguladon, 1984, 22, 27-55) use a multiple drug effect analysis procedure to calculate a combined drug effect.

The data were evaluated by the isobologram geometric method for drug drug interactions. The concentration providing the desired effect (e.g., 50% inhibition) is plotted on the horizontal axis, and the concentration giving the same effect on the vertical axis is plotted. The line connecting these points is drawn and the concentration of the agents used in the combination having the same effect is indicated. If this point falls below the former line, the combination is considered to have a synergistic effect.

In Experiment 1, all AZT concentrations used were inhibitory, making it impossible to judge the effect of the combination. synergistic interactions between the two agents were consistently observed in the experiments. (See Tables IV-IX). Synergism was present at every degree of watery replication used, and was maintained even when negligible rIFNaA alone was used. Synergy calculations were performed by analyzing multiple drug effects of RT data for Experiments 2-4, Viral Yield data for Experiments 1 and 3, and RIA data for Experiment 4. The result of the isobologram process also shows synergism.

ARC. spreadsheet

The examination number Call a vaccine method Added CALL (TCIDjo) Time of drug administration rlFNaA AZT First THE 10 ⁵ 0 0 Second B 4x10 ³ -24 hours 0 Third B 10 ³ -24 hours 0 4th B 2X10 ³ -24 hours 0,

at 37 ° C a) 20 ml of medium containing the "A" method 40xl0 ⁶ cells are suspended IQ * TCIDjq HIV t, 4 »h and then washed and re / suspended.

Method B The indicated amount of virus is added to 2x10 ⁶ cells in culture medium, and the cells are not subsequently washed.

Table V.

Experiment 2. Day 10 Effect of rlFNaA and AZT on Mean RTs (cpm / 10 ⁶ cells x 10 ³ ) rlFNaA (E / ml)

AZT (JlmóV 1 0 8 16 32 64 128 0 205 173 150 193 169 151 0.01 159 85 0.02 110 42 0.04 71 10 0.08 31 3 0.16 7 1

VI. spreadsheet

Experiment 3, Day 13 Effect of rlFNaA and AZT on Mean RT (cpm / 10 ⁶ cells x 10 ³ ) rlFNaA (E / ml)

AZT (Mol / l) 0 16 32 64 128 0 157 143 117 117 130

HU 203 971 A

AZT (Union l / l) 0 16 32 64 128 0.02 51 10 0.04 10 0 0.08 2 0 0.16 5 0

VII. spreadsheet

Experiment 4, ll. Effect of day rlFNaA and AZT on mean RT values (cpni / 10 ⁶ cells x 10 ³ ) rlFNaA (E / ml)

AZT (Gmol / l) 0 32 64 128 0 32 5 4 2 0.02 8 1 0.04 4 0 0.08 1 0

Vili. spreadsheet

Experiment 3. Day 13 Effect of rlFNaA and AZT on viral yield (TCID ₅₀ / ml) rlFNaA (E / ml)

AZT (Mol / 1) 0 16 32 64 128 0 10 ⁵ · ⁷ 10 ⁵ · ⁶ 10 ⁵ · ^{3 51} 10 ⁵ ° 0.02 10 ⁴ · ⁸ 10 ¹ · ⁹ 0.04 10 ³ CIO ¹ ' ⁹ 0.08 10 ¹ · ⁹ CIO ¹ · ⁹

PATENT CLAIMS

Claims (3)

PATENT CLAIMS CLAIMS 1. A method of preventing or treating human retroviral infections 5 for the preparation of compositions comprising 3'-azido-3'-deoxythymidine and aeyclovir, a-interferon, dipyridamole, probenicide, dilazep or 6 - [(4-nitrobenzyl) - in a molar ratio of 20: 1 to 1: 20. thio] -9- [5-D-ribofuranosyl purine individually 10 or mixed with carrier and / or other excipients to form a composition. (Priority: 15/9/1986) A process for the preparation of a synergistic pharmaceutical composition for the prophylaxis or treatment of human retroviral infections according to claim 1, characterized in that 3'-azido-3'-deoxythymidine and dipyridamole, dilazep or 6 - [(4-nitrobenzyl) thio] -9β-D-ribofuranosylpurine, alone or in combination 20 with carrier and / or other excipients. (Priority: 4/4/1986) 3. The method of claim 1, wherein the 3'-azido-3'-deoxythymidine is 3'-azido-3'-deoxythymidine and a probenicide in a molar ratio of 20: 1 to 1:20, for the prevention or treatment of human retroviral infections. mixed with carrier and / or other excipients to form a composition.