IE903879A1 - Use of a benzodiazepine and a phenylpyrrylketone derivative - Google Patents

Abstract

The compounds 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(1H)-one, 2-glycinamido-5-chlorophenyl 2-pyrryl ketone and pharmaceutically utilisable salts thereof have anti-retroviral activity and are, consequently, suitable for the treatment and prophylaxis of infectious diseases caused by retroviruses.

Description

The invention is based on the finding that the compounds of the group consisting of 7-chlor0-5-(2-pyrryl)-3H-l.4-benzodiazepin-2(1H)-one (hereinafter Compound A), described in USP 3 405 122, 2-glycinamido-5-chlorophenyl(2-pyrryl) ketone (herein10 after Compound B), described in USP 3 407 211, and pharmaceutically acceptable salts thereof, are useful for the treatment or prophylaxis of infectious diseases caused by retrovirus, specially for alleviating the cytopathic destructive effects of retroviral diseases, particularly those caused by the retrovirus HIV. specially HIV 1.

Pharmaceutically acceptable salts may be salts of organic acids such as lactic, acetic, malic or p-toluenesul20 fonic acid, or of mineral acids such as hydrochloric or sulfuric acid.

The invention relates to the use of the above compounds and salts in the treatment of infectious diseases, such as AIDS and ARC (AIDS Related Complex). These diseases are caused by retrovirus, particularly HIV, specially HIV-1 and HIV-2. The above compounds and salts are particularly useful for alleviating the cytopathic destructive effects of the retroviral diseases, but they can also be administered to asymptomatic HIV-infected patients.

The invention further relates to antiviral compositions in dosage unit form, suitable particularly for oral or parenteral administration, for the treatment of the above diseases, and containing a therapeutically effective amount Me/27.8.90 - 2 of one of the above compounds or salts and, optionally, a therapeutically effective amount of a second anti-HIV-compound, specially of a reverse transcriptase inhibitor (anti-RT agent), such as ddC, AZT, ddl or ddA.

The invention also relates to commercial packages containing as active pharmaceutical ingredient one of the above compounds A and B or a salt thereof and, optionally, a second anti-HIV compound, together with instructions for the 1Q use thereof.

The invention further relates to a method of treatment of the above infectious diseases caused by retrovirus, by administering a compound A or B or a pharmaceutically acceptable salt thereof, in daily dosage individually determined by the attending physician, particularly in an anti-virally effective amount of 0.1 to 10 mg/kg body weight per day in one or more doses, preferably in an amount of 1 to 3 mg/kg body weight given one or two times per day, specially wherein the therapeutic blood level achieved is 0.05 to 10, preferably 0.1 to 5 μΜ. This dosage may be administered in one or more doses at various intervals, such as 2, 4, 6, 8, 12 or 24 hours.

The above compounds exhibit anti-retroviral activity, particularly as inhibitor of the transactivator protein TAT (anti-TAT agents). When an anti-RT agent, such as ddC is also administered, this can be done in a range of 0.05 μΜ to 1.0 μΜ, particularly of 0.005 to 0.25 mg/kg body weight. The preliminary dose ranges for oral administration can be of 0.001 to 0.25 mg/kg given in one or more doses at intervals of 2, 4, 6, 8, 12 hours and so on. Currently, 0.01 mg/kg body weight of ddC are given every 8 hours. When given in combined therapy, the anti-RT agent may be given at the same time as the anti-TAT agent or the dosing may be staggered. The two drugs may also be combined in a composiIE 903879 - 3 tion; the doses of each may then be less than when used as a single agent.

The antiviral compositions of the invention contain at least one of the above anti-TAT agents and, optionally, other therapeutic agents, e.g. an anti-RT agent, together with one or more pharmaceutically acceptable carriers. These carriers include those well known in the art as suitable for oral, rectal, nasal, topical, buccal, sublingual, vaginal. o or parenteral (including subcutaneous, intramucular, intravenous, and intradermal) administration. Examples of compositions of the invention are solutions of the active ingredient(s) in water, saline, or orange juice; capsules, e.g. soft gelatine capsules; sachets or tablets, each Ί5 containing a pre-determined amount of theactive ingredient, e.g. as granules; solutions or suspensions in an aqueous liquid or in an oil-in-water emulsion or a water-in-oil liquid emulsion. Tablets may include one or more of lactose, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose sodium, magnesium stearate, stearic acid and other excipients, colorants and pharmacologically compatible carriers. Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gells, pastes, foams, or spray formulas containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions which may contain anti-oxidants, buffers. - 4 bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The compounds of the present invention were tested for anti-TAT activity in an assay comprising the following steps (a) putting both the expression of the Secreted Alkaline Phosphatase (SeAP) gene and the viral transactivator TAT gene under the control of the HIV promoter LTR responsive to the action of the HIV transactivator TAT; (b) transfecting cultured mammalian cells with plasmids which contain the gene constructs of (a) above and cause cellular production of the transactivating factor TAT and SeAP; (c) adding the agent to be tested, here compounds A and B as defined above; and determining the amount of SeAP produced by measuring SeAP enzymatic activity, whereby inhibition of SeAP production correlates with the anti-TAT inhibition activity. In this assay, the inhibition of SeAP positively correlates with anti-TAT activity. The greater the ability of an agent to inhibit SeAP, the greater is its anti-TAT activity. - 5 Specifically with respect to the results Figure 1 and Figure 2 for compounds A and B. run as follows: reported in the assay was At 24 hours post transfection. 1, 10 and 100 μΜ of test compound were added to the culture media of COS cells transfected with two plasmids, one containing the reporter gene which codes for SeAP under control of HIV-LTR, and the other containing the TAT gene also under control of HIV-LTR.

The alkaline phosphatase activity of the media was assayed 48 hours after addition of the test compounds. The results are set forth in Figures 1 and 2 which represent three independent assays of the test compounds. The anti-TAT activity is measured by the percent inhibition of SeAP gene expression under the control of HIV-LTR. The cytotoxicity of the compound was tested in a parallel assay in which SeAP gene expression is put under control of Rous Sarcoma Virus (RSV)-LTR which does not respond to TAT. The results set forth in Figures 1 and 2 show that Compounds A and B are specific inhibitors of TAT and do not exhibit nonspecific cytotoxicity.

The Compounds A and B were also tested for the inhibition of HIV-cytopathic effect and the reduction of cell-associated viral antigens in an experiment run as follows : High titer virus stocks (HIV-1 NIT strain) were grown in CD4+ CR10 cells in RMPI-1640 media (Gibco Laboratories) supplemented with 10% fetal calf serum and 0.1 mg/ml Gentamicin. The collected media were filtered and the virus isolates were concentrated 100 fold and stored at -80°C.

CD4+ CEM cells, propagated in the same medium, were incubated for 60 minutes at 37°C with diluted stock virus at MOI=1. Cells were washed with phosphate buffered saline and 5 resuspended in the medium at 2 x 10 cells/ml. Various quantities of Compounds A and B were added. Three days after - 6 infection, the number of live cells were counted by trypan blue exclusion (Proc. Natl. Acad. Sci. 83. 1986, 1911). At the same time, aliquots of cells were fixed with acetone and stained with antibodies from AIDS patients, followed by a second staining with fluorescein-conjugated goat anti-human IgG (Cappel). Cells stained with the fluorescent antibody were counted using a fluorescence microscope and the results were expressed as percentage of the total number of cells counted (J. Med. Virol. 19, 1986, 325).

For cytotoxicity testing, uninfected CEM cells were treated with Compounds A and B at similar concentrations and toxicity of the compounds was measured by the live-cell count. Two compounds. AZT and ddC, with known potency for the treatment of AIDS, were tested in parallel with Compound A. To test the effect of a longer-term treatment with Compound A, the compound-treated cells above were replated at day 3 in media containing the compound and the assay was repeated at day 6. Results of the assays are shown in Figures 3 and 4 for compound A and in Figure 5 for compound B.

Compound A was also tested for anti-viral activity in the p24 viral antigen capture assay: The Enzyme Linked Immuno-absorbant Assay (ELISA) was used to test Compound A for anti-HIV activity by measuring virus replication in CD4+ T-lymphocytes, by detecting viral p24 antigen released into the tissue culture supernatant of infected cells.

High titer virus stocks [HIV/HTLV III. RF strain] were grown in H9 cells in RPMI-1640 (Flow Laboratories) supplemented with 10% fetal calf serum, penicillin (100 IU/ml) and streptomycin (100 ug/ml). Cell debris were removed by centrifugation and the supernatant stored at -70°C. The host cell line used in the anti-viral assays was the C8166 CD4+ T-cell line which is particularly susceptible to infection with HIV. Cells were incubated with 10 TCID_„, HIV/HTLV 0 III (RF) at 37°C for 90 minutes and then washed with PBS.

Cell aliquots (2 x 10 ) were resuspended in 1.5 ml growth medium and appropriate amounts of Compound A added and incubated at 37°C. At 72 hours post-infection, the supernatants from the cell cultures were assayed for p24 antigen The antigen capture assays were carried out on supernatants taken from infected cell cultures. In each assay a positive control was included, together with a supernatant control taken from uninfected cells. The plates were read using a spectrophotometric detection system. In two separate assays Compound A was found to exhibit anti-viral activity, approximately 0.1 μΜ and 6 μΜ of Compound A yielding 50 percent inhibition of viral replication.

The following galenical compositions containing the active ingredients as defined above, can be prepared in a manner known per se: Tablet formulation: Ingredients Compound A or B Lactose Pregelatinized starch Oral liquid formulation Ingredients Compound A or B Methylparaben Sucrose Flavoring agent Citrate buffer Purified water q.s. mg/tablet mg 180 mg mg mq/formulation .0 mg 20.0 mg q.s. q.s. q.s. .0 ml •E 903879 Tablet formulations: Ingredients mg/tablet mg/tablet Compound A or B 20 mg 20 mg 5 ddC 5 mg Starch 40 mg 40 mg Avicel 80 mg 80 mg Lactose 269 mg 274 mg Magnesium Stearate 2 mg 2 mg 10 416 mg 416 mg Tablet formulation: Ingredients mg/tablet 15 Compound A or B 20 mg ddC 5 mg Lactose 175 mg Pregelatinized starch 15 mg Microcrystalline cellulose 72 mg 20 Modified starch 10 mg Magnesium stearate 3 mg 300 mg Soft gelatine capsule formulations: Ingredients mq/capsule Compound A or B 20 mg ddC 0 or 5 mg Ethoxylated Fatty acids 500 mg PEG 4000 100 mg Vegetable Oils q.s. to 1.0 ml - 9 Oral liquid formulation Ingredients Compound A or B ddC Methylparaben Propylparaben Sucrose Flavoring agent Citrate buffer Purified water q.s.

Parenteral formulation: Ingredients Compound A or B ddC Propylene glycol Emulphor Water for Injection q.s mg/ml 4.0 mg 1.0 mg 2.0 mg 0.2 mg 100.0 q.s q.s. .0 mg 1.0 ml mg/ml 20.0 mg .0 mg 20.0 mg 2.0 mg 1.0 ml

Claims (7)

Claims

1. The use of a compound of the group consisting of 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(1H)-one, 5 2-glycinamido-5-chlorophenyl(2-pyrryl)ketone and pharmaceutically acceptable salts thereof, for the manufacture of pharmaceutical preparations for the treatment or prophylaxis of infectious diseases caused by retrovirus, specially for alleviating the cytopathic 10 destructive effects of retroviral diseases, particularly those caused by the retrovirus HIV, specially HIV 1.

2. Antiviral compositions in dosage unit form suitable for oral or parenteral administration for the treatment or 15 prophylaxis according to claim 1. containing a therapeutically effective amount of a compound according to claim l and, optionally, a therapeutically effective amount of a second anti-HIV compound, specially of a reverse transcriptase inhibitor, particularly ddC.

3. Commercial packages containing as active pharmaceutical ingredient a compound according to claim 1 and, optionally, a second anti-HIV compound according to claim 2, together with instructions for the use thereof as claimed in 25 claim 1.

4. A method of treatment or prophylaxis of infectious diseases caused by retrovirus, specially for alleviating the cytopathic destructive effects of retroviral diseases. 30 particularly those caused by the retrovirus HIV, specially HIV 1, by administering a compound according to claim 1 in daily dosage individually determined by the attending physician, particularly in an anti-virally effective amount of 0.1 to 10 mg/kg body weight per day in one or more doses, 35 preferably in an amount of 1 to 3 mg/kg body weight given - 11 one or two times per day, specially wherein the achieved therapeutic blood level is 0.05 to 10, preferably 0.1 to 5 μΜ.

5. Use according to claim 1, substantially as hereinbefore described.

6. An antiviral composition according to claim 2, substantially as hereinbefore described.

7. A commercial package according to claim 3, substantially as hereinbefore described. Dated +his the 26th day of October, 1990 F. R. KELLY & CO. BY: EXECUTIVE 27 Clyde Road TRUE COPY 5 Sheets Sheet 1 F. HOFFMANN-LA ROCHE AG