AMPLIFICATION OF ANTIBIOTIC AND/OR ANTITUMOUR ACTIVITY

Abstract: A method of amplifying the antitumour activity of phleomycin, bleomycin, tallysomycin or mixtures thereof which comprises administration of such compounds in association with an amplifying compound having two rings, each of which may be five or six membered and each of which may be heteroaromatic or aromatic. Also disclosed is a pharmaceutical preparation comprising the phleomycin, bleomycin, tallysomycin, or a mixture thereof in admixture with the amplifying compound; a phleomycin-amplifier antibiotic regime having a higher therapeutic index than that of the phleomycin alone.

Description: AMPLIFICATION OF ANTIBIOTIC AND/OR ANTITUMOUR ACTIVITY Technical Field The present invention relates to a method of amplifying the antibiotic and/or antitumour activity of a group of known antibiotic and/or antitumour agents known as the phleomycins, the bleomycins, and the tallysomycins, and mixtures thereof. Background Art The structural formulae of the phleomycins, bleomycins and the tallysomycins are as follows:- wherein R" is selected from the groups R,, R~ and R_, Phleomycin R 1 = R, Bleomycin R' = R 2 Tallysomycin R' = R 3 R" = Terminal amine The phleomycins, bleomycins and tallysomycins are copper- containing glycopeptides obtained by culturing Streptomyces vertiσillus. These materials were originally thought to be pure compounds/ however subsequent research has shown that the naturally occuring material is a mixture of amides of the corresponding acid i.e. the phleomycins are amides of phleomycinic acid etc. In addition to the naturally occurring amides additional amides may be prepared synthetically. The phleomycins, bleomycins and tallysomycins have all shown degrees of antibiotic and/or antitumour activity, however the use of the compounds has been impeded by the nephrotoxic properties of the compounds. Various attempts have been made to amplify or potentiate the activity of these antibiotic and/or antitumour compounds to the point where they may be safely used at concentrations which are sufficiently low that the adverse side effects of the compounds are avoided. The prior art amplifying or potentiating compounds are described, for instance, in Australian patents 496,392 and 524,079, and in Australian patent application 72948/81. The present invention relates to a further group of compounds which amplify or potentiate the antibiotic and/or antitumour activity of the phleomycins, bleomycins and tallysomycins. The expressions "amplify" and "potentiate" are used to mean the augmentation of the activity of the antibiotic and/or antitumour compound by a second compound such that the response produced is greater than the predictable additive effect of the two compounds. Disclosure of the Invention It is an object of the present invention to provide a method of amplifying the antibiotic and/or antitumour activity of the phleomycins. It is a further object of this invention to provide a phleomycin-amplifier composition having a higher therapeutic index than that of the phleomycin alone. The present invention consists in a method of amplifying the antibiotic and/or antitumour activity of phleomycin, bleomycin, tallysomycin, or mixtures thereof, which comprises administering the phleomycin, bleomycin, tallysomycin, or OMPI mixtures thereof in association with an amplifying compound selected from the group comprising compounds having two rings, each of which may be five membered or six membered and each of which may be heteroaromatic or aromatic, which rings are directly joined by a bond, at least one of the rings carrying at least one strongly basic side chain joined to the ring through a sulphur atom. The antibiotic and/or antitumour agent and the amplifying compound may be administered simultaneously or they may be administered separately; all that is required is that they be administered in sufficient juxtaposition in time that the antibiotic and/or antitumour activity is amplified. In a further aspect the present invention relates to a pharmaceutical preparation comprising a phleomycin, bleomycin, tallysomycin or a mixture thereof in admixture with an amplifying compound selected from the group comprising compounds having two rings, each of which may be five membered or six membered and each of which may be heteroaromatic or aromatic, which rings are directly joined by a bond, at least one of the rings carrying at least one strongly basic side chain joined to the ring through a sulphur atom. It is preferred that one of the rings of the amplifying compound is a five membered ring and one a six membered ring. It is further preferred that each of the rings is a heteroaromatic group. The hetero atoms are preferably selected from the group comprising of nitrogen and sulphur. The strongly basic side chain is preferably a nitrogen-substituted alkyl radical linked directly to its associated ring through a sulphur atom. It is particularly preferred that the nitrogen-containing substituent is a dialkyl amino group. The amplifying compound is most preferably selected from the group comprising N, N- Dimethyl-2- l'-(thien-2"-yl) pyrimidine-2'-ylthicTj ethylamine (HBr) (hereinafter called O PI "LS34"), N, N Dimethyl-2- j ' - (Pyridine-4"-yl) thiazol-2' -ylthicT] ethylamine (2HBr) (hereinafter called "BC55"), and 4-(5'-4'- dimethylamino ethylthio-1'3 '4 ' thiadiazol-2 ' -yl) pyridine (hereinafter called BC151) which compounds have the following structural formulae: "LS34" N,N-Dimethyl-2-[4'-(thien-2"-yl) pyrimidine-2 '-ylthio] ethylamine (HBr) "BC55" N,N-Dimethyl-2-[4 l - (Pyridine-4"-yl) thiazol-2'-yltio] ethylamine (2HBr) "BC151" 4- (5 ' -^f-dimethylaminoethylthio-l ' 3 ' 4 thiadiazol-2'-yl) Pyridine The antibiotic and/or antitumour compounds may be mixtures of individual phleomycins, bleomycins, or tallysomycins or may be purified members of those groups whether produced naturally or synthetically. In another embodiment mixtures of one or more of the groups of antibiotic and/or antitumour compounds may be mixed with one or more of one or both of the other groups of such compounds. Particularly good results have been obtained with phleomycin PEP i.e., phleomycin in which the amide moiety is The antibiotic and/or ' antitumour compound and the amplifying compound may be administered in any suitable form. It is preferred that intra-peritoneal injections be used. However, intra- uscular, sub-cutaneous, oral or implantation routes could also be used. Best Mode of Carrying Out the Invention The antitumour activity of the pharmaceutical preparations according to this invention is illustrated by the following examples. METHOD Swiss HICR mice were used with Ehrlich's Ascites Tumour. Tumour passages were effected by intraperitoneal - injection of 10 cells which had been prepared by ' O PI aspirating several ml of ascites fluid from a tumou -bearing mouse. An aliquot (0.1 ml.) of mouse ascites fluid was diluted 1000-fold with Eagle's Medium and the number of live cells was determined on a haemocytometer after a further 1:1 dilution with Turk's fluid. The bulk of the ascites fluid was then diluted with Eagle's medium to give a concentration of 10 live cells per ml. The mice were injected intraperitoneally with this suspension (0.1 ml. each) within one hour of collecting the ascites fluid, all preparations being handled over ice during that period. Bacto-fluid thioglycollate medium was used to test for contamination. Solutions of the phleomycins were made up in phosphate-buffered saline at pH7.2 in daily doses at the commencement of each 5 day injection period and were kept frozen until the commencement of each day's injections. The injections were given at 10.00 a.m.. Bulk solutions of amplifying compound were stored at room temperature and were injected 30 minutes after the phleomycin. In most experiments several levels of phleomycin and amplifying compound were given both alone and in combination, and several groups of test animals were injected with buffered saline alone, i.e. with the vehicle for the phleomycins and amplifying compound. s Test groups usually comprised 7 or 8 animals. Procedures were based closely on those detailed by Geran et al (1972). Cancer Chemotherapy Reports, September 1972, Vol. 3 No. 2 Part 3. All groups were retained until their median survival time had been reached and a T/C equal to or greater than 125 was considered a positive result. A T/C value of 200%, for example, indicates that the use of the compounds can increase the life-span of test animals to twice that of animals receiving phleomycin alone. T /C = median survival time (M.S.T.) of test group. median survival time (M.S.T.) of control group. The dosage of phleomycin is given as the dose per day in milligrams per kilogram of body weight (MK/d) . The dosage of amplifier is given as the total dose administered over a five day period. The results of the trials are given in Table 1. TABLE I AMPLIFIER ANTIBIOTIC LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER (days) (MK/d) (MK) BC151 PLM-PEP 4 32 4 535 >100 >312 4 33 4 535 > 50 > 151 2 22 2 535 > 100 > 454 1 26 1 535 76 292 1 21 V 1 ' 535 32 152 1 18 1 535 > 61 > 338 0.5 17 0.5 535 26 153 0.5 107 23 135 0.5 18 0.5 535 > 50 > 277 ' C PI AMPLIFIER ANTIBIOTIC LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER (days) (MK/d) (MK) BC151 PLM-CHP 4 — 50 4 535 67.5 135 4 - 27 4 535 > 100 > 370 4 - 23 4 535 > 36 >156 2 - 23 2 535 > 50 > 217 1 — 24 1 535 >100 > 416 1 - 25 1 535 58.5 234 0.5 — 22 0.5 535 49 222 LS34 PLM-PEP 2 — 31 2 30 > 69 >222 2 10 >48.5 >156 AMPLIFIER ANTIBIOTIC LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER (days) (MK/d) (MK) LS34 PLM-CHP 4 36 4 19 >50 >150 LS34 PLM-D 20 - 29.5 20 140 68.5 232 20 70 54.5 184 20 17. 5 > 62 >210 10 - 30.5 10 140 > 63 >206 0.625 - 17 0.625 35 23 135 0.625 17. 5 23.5 138 BC55 PLM-PEP 4 42 4 500 60 142 2 24 2 50 55 229 2 24 2 75 > 93 >387 ANTIBIOTIC STRUCTURE OF THE VARIABLE TERMINAL AMINE PLM-PEP NH. (CH 2 ) 3 .NH.CH.CH 3 .Ph PLM-CHP NH. (CH 2 ) 3 .NH.CgH 1]L PLM-D NH. (CH 2 ) 4 .NH(C:NH)NH 2 The results in TABLE 1 above show that there is a significant enhancement of the antitumour effect of a number of phleomycins by their use in combination with the- amplifiers disclosed herein, with a T/C value greater than 400 on at least one testing, indicating that the use of such an amplifier in combination with the phleomycin can increase the life-span to more than four times that of animals receiving phleomycin alone. Enhancement of the antitumour activity has been demonstrated in tests where the phleomycin has been administered by intraperitoneal injection and where the amplifier has been administered by subcutaneous implantation, which indicates that no initial complexes are formed between the phleomycin and the amplifier, and that the enhancement phenomenon observed is not just the result of both compounds acting on the tumour cells by a pseudo in vitro effect. <> The mechanism by which amplification of phleomycin action takes place is not known, but it is thought to be as a result of the amplifiers having an effect on the secondary structure of the double helix of DNA, thus enhancing the ability of the phleomycin to break the individual DNA strands. It will be recognised by persons skilled in the art that numerous variations and modifications may be made to the invention as described above without departing from the spirit or scope of the invention as broadly described.

Claims: CLAIMS 1. A method of amplifying the antibiotic and/or antitumour activity of phleomycin, bleomycin, tallysomycin, or mixtures thereof, which comprises administering the phleomycin, bleomycin, tallysomycin, or mixtures thereof in association with an amplifying compound selected from the group comprising compounds having two rings, each of which may be five membered or six membered and each of which may be heteroaromatic or aromatic, which rings are directly joined by a bond, at least one of the rings carrying at least one strongly basic side chain joined to the ring through a sulphur atom. 2. A method as in claim 1, wherein the phleomycin, bleomycin, tallysomycin, or mixtures thereof, is administered simultaneously with the amplifying compound. 3. A method as in claim 1, wherein the phleomycin, bleomycin, tallysomycin, or mixtures thereof, and the amplifying compound are administered consecutively in sufficient juxtaposition in time that the antibiotic and/or antitumour activity is amplified. 4. A pharmaceutical preparation comprising a phleomycin, bleomycin or a mixture thereof in admixture with an amplifying compound selected from the group comprising compounds having two rings, each of which may be five *> membered or six membered and each of which may be heteroaromatic or aromatic, which rings are directly joined by a bond, at least one of the rings carrying at least one strongly basic side chain joined to the ring through a sulphur atom. 5. A pharmaceutical preparation as in claim 4, wherein one of the rings of the amplifying compound is a five membered ring and one is a six membered ring. 6. A pharmaceutical preparation as in claim 4 or claim 5, wherein each of the rings is a heteroaromatic ring. OMPI 7. A pharmaceutical preparation as in claim 6, wherein the hetero atoms are selected from the group comprising of nitrogen and sulphur. 8. A pharmaceutical preparation as in any one of claims 4 to 7, wherein the strongly basic side chain linked to its associated ring through a sulphur atom is an alkylaminothio or dialkylaminothio group. 9. A pharmaceutical preparation as in claim 8, wherein the amplifying compound is selected from the group comprising: N,N-Dimethyl-2-[4'-(thien-2"-yl)pyrimidine-2'-ylthio] ethylamine; N,N-Dimethyl-2-[4 '- (Pyridine-4"-yl) thiazol-2'- ylthio]ethylamine and 4-(5' - d -dimethylaminoethylthio-1' ,3 4'-thiadiazol-2'-yl)pyridine. 10. A pharmaceutical preparation as in any one of claims 4 to 9 wherein the phleomycin is selected from the group comprising phleomycin PEP, phleomycin CHP and phleomycin D.

Inventors: GRIGG GEOFFREY WALTER, LAMBERTON JOHN A, GRIGG, GEOFFREY, WALTER, LAMBERTON, JOHN, A.
Applicants: COMMW SCIENT IND RES ORG, COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH OR
Application year: 1984
Main classification: A61K31
IPC codes: C07K9