AU639273B2 - Use of a benzodiazepine and a phenylpyrrylketone derivative - Google Patents

Description

FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION S F Ref: 142657

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: F.Hoffmann-La Roche AG 124 Grenzacherstrasse CH-4002, Basel

SWITZERLAND

Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Use of a Benzodiazepine and a Phenylpyrrylketone Derivative The following statement is a full description of this invention, including the best method of performing it known to me/us

S

5845/3 1- RAN 4430/38K Abstract The compounds 7-chloro-5-(2-pyrryl)--3H-1,4-benzodiazepin-2(1H)--one, 2-glycinamido-5-chlorophenyl(2-pyrry.)ketone and pharmaceutically acceptable salts thereof exhibit 10 anti-retroviral act3.vity and are accordingly useful for the treatment or prophylaxis of infectious diseases caused by retrovirus.

a 1A RAN 4430/38K The invention is based on the finding that the compounds of the group consisting of 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(1H)-one (hereinafter Compound described in USP 3 405 122, 2-glycinamido-5-chlorophenyl(2-pyrryl)ketone (herein- 10 after Compound described in USP 3 407 211, and pharmaceutically acceptable salts tnereof, are useful for the treatment or prophylaxis of infectiout diseases caused by retrovirus, specially for alleviating the S. 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-toluenesul- 20 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 25 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 10 use thereof.

The invention further relates to a method of treatment S 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, specir'ly wherein the therapeutic blood level achieved is 0.05 to 10, preferably 0.1 to 5 ILM. This dosage may be administered in one or more doses at various intervals, such as 2, 4, 6, 8, 12 or 24 hours.

25 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 1LM to 1.0 iM, 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 composi- 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, 10 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 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. Forrmlations 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 10 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: 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: transfecting cultured mammalian cells with plasmids which contain the gene constructs of above and cause cellular production of the transactivating factor TAT and 25 SeAP; 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 reported in Figure 1 and Figure 2 for compounds A and B, the assay was run as follows: At 24 hours post transfection, 1, 10 and 100 -M 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.

10 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 S* activity is measured by the percent inhibition of SeAP gene 15 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 20 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 25 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 0

C.

CD4 CEM cells, propagated in the same medium, were incubated for 60 minutes at 37 0 C with diluted stock virus at MOI=1. Cells were washed with phosphate buffered saline and resuspended in the medium at 2 x 105 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 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 S 20 Figures 3 and 4 for compound A and in Figure 5 for compound

B.

lo, 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 0 C. The host cell line used in the anti-viral assays was the C8166 CD4 7 T-cell line which is particularly susceptible to infection with HIV. Cells were incubated with 10 TCID50, HIV/HTLV III (RF) at 37 0 C for 90 minutes and then washed with PBS.

Cell aliquots (2 x 105) were resuspended in 1.5 ml growth medium and appropriate amounts of Compound A added and incubated at 37 0 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 iM and 6 uM of Compound A yielding 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 20 mg 180 mg 15 mg mq/formulation 20.0 mg 20.0 mg q.s.

q.s.

q.s.

5.0 ml 8 Tablet formulations: Ingredients Compound A or B ddC Starch Avicel Lactose Magnesium Stearate mq/tablet 20 mg 5 mg 40 mg 80 mg 269 mg 2 mg 416 mg mq/tablet 20 mg 40 mg 80 mg 274 mg 2 mq 416 mg Tablet formulation: Ingredients Compound A or B ddC Lactose Pregelatinized starch Microcrystalline cellulose Modified starch Magnesium stearate mq/tablet 20 mg 5 mg 175 mg 15 mg 72 mg 10 mg 33 mg 300 mg Soft gelatine capsule formulations:

I

Ingredients Compound A or B ddC Ethoxylated Fatty acids PEG 4000 Vegetable Oils q.s. to mq/capsule 20 mg 0 or 5 mg 500 mg 100 mg 1.0 ml 9 oral liquid formulation: ingredients compound A -or B ddC Methylparaben Propylparaben Sucrose Flavoring agent 10 citrate buffer Purified water q.s.

mg/ml 4. 0 mg 1.0 mg 2.0 mg 0.2 mg 100.0 q. S.

q. s.

5.0 mg 1.0 ml 6 4 6 6

I

6

I.

Parenteral formulation: Ingredients Compound A or B ddC Propylene glycol Emul1p ho r Water for Injection q.s.

Mcr/ml 20.0 mg 5. 0 mg 20.0 Mg 2.0 mg 1. 0 ml

Claims (2)

1. The use of a compound of the group consisting of

7-chloro-5-*(2-pyrryl)-3H-l,4-benzodiazepin-2(1H)-one, 2-glycinamido-5-chlorophenyl(2-pyrryl)ketone and pharma- ceutically 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 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 prophylaxis according to claim 1, containing a therapeuti- cally effective amount of a compound according to claim 1 and, optionally, a therapeutically effective amount of a second anti-HIV compound, specially of a reverse transcrip- tase inhibitor, particularly ddC. 3. Commercial packages containing as active pharmaceuti- cal ingredient a compound according to claim 1 and, optionally, a second anti-HIV compound accordi.g to claim 2, together with instructions for the use thereof as claimed in claim 1. I 4. A method of treatment or prophylaxis of infectious diseases caused by retrovirus, specially for alleviating the cytopathic destruotive effects of retroviral diseases, 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, 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 iM. Antiviral compositions In dosage unit form suitable for oral or parenteral administration substantially as hereinbefore described with reference to any one of the Examples. DATED this TWENTY-SECOND day of OCTOBER 1990 F.Hoffmann-La Roche AG bO fm Patent Attorneys for the Applicant SPRUSON FERGUSON GSA/2640M