AMPLIFIED ANTIBIOTIC AND ANTITUMOR AGENTS

Abstract: The antibacterial and antitumor activity of certain specific members of the groups of compounds known as phleomycin, bleomycin and tallysomycin are amplified by administration in association with caffeine, dimethylthiocyanine or pyronine Y. The invention makes the active compounds effective at very low concentrations thereby avoiding the deleterious side effects normally associated with these compounds. The active compounds according to the present invention which show particularly advantageous characteristics when amplified as described above are those in which the phleomycin, bleomycin or tallysomycin is in an amide form having a substituent amine group selected from the following:

Description: __MP IΓ_-SD TIBIOTIΠ P&TD A ΓTUPΌMOR ASSETS Technical Field The present invention relates to a method for amplifyin the activity of certain compounds having anti biotic and antitumor properties. Bakckground Art It has been known for some time that certain compounds obtained by the culture of streptomyces verticillus and related species show antibiotic and antitumor properties. These include phleomycin, bleomycin and tallysomycin. Subsequent research showed that each of these materials were in fact mixtures of amides of the relevant acid i.e. phleomycinic acid, bleomycinic acid and tallysomycinic acid. The components of the originally isolated mixtures have been isolated and new amides have since been synthesised. While many of the known amides of phleomycinic, bleomycinic and tallysomycinic acids are known to have antibacterial and antitumor properties it is also known that many of them have unfortunate side effects such as nephrotoxicity. The present applicants Australian patent number 496,392 and Australian patent application 35175/78 disclose that certain compounds are capable of amplifying the activity of certain of the above amides. This has allowed use of these compounds in reduced, and therefore less harmful doses. The present invention concerns an extension of this concept. Disclosure of the Invention The present invention consists in a method of amplifying the antibiotic or antitumor activity of an amide of an acid selected from the group comprising phleomycinic acid, bleomycinic acid and tallysomycinic acid in which the substituted amine moiety is selected from the group comprising compounds having the structural formulae:- NH NH 2 . (CH 2 ) 4 .NH . (CH 2 ) 4 , NH . C.NH. (CH 2 ) 2 NH NH 2 . CCH 2 ) 5 .NH . (CH 2 ) 3 , NH . C.NH. (CH- -. NH 2 . (CH 2 ) 4 .NH . (CH 2 ) 3 , NH . which comprises administering any one of the said amides in association with an amplifying compound selected from the group comprising caffeine, dimethylthiocyanine and Pyronine The acid residues of the amides to which the present invention relates are known and their structure has been published and for this reason their structural formula is no reproduced here. In each of these compounds there is a terminal carboxylic group which in the compounds to which th present invention relates has been reacted with an amine moiety to give an amide compund. The amides may be obtained serai synthetically by culturing Streptomyces verticillus in the presence of an appropriate amine compound which will be incorporated into the amide produced by the organism or alternatively, some other amide may be synthetically manipulated to produce the desired compound. It should be noted that the phleomycin and bleomycin referred to in the prior art have been a mixture of amides but these known mixtures principally contained amides other than those to which the present invention relates. It is no possible to predict a priori the effect of any given amplifier upon any given amide. The selection according to the present invention is based upon the degree of ampli ication achieved by the amplifier on the designated amide and the nephrotoxicity for the combination of amplifier and amide. The amplifying compounds are caffeine, dimethylthiocyanine and Pyronine Y. Caffeine is 1, 3, 7 trimethyl xanthene and has the elemental formula ^ 8 H 1 0 °2 N 4* H 2° ancl p y ron i ne Y is a brand name of 6-( imethylamino) - 3H - xanthene - 3 - ylidene dimethylammonium chloride. The weight ratio of amide to activator to achieve optimal activation must be determined by routine testing on a case by case basis. It has been found experimentally that ratios of from 1:0.002 up to 1:30 and above have given significant enhancement under the particular circumstances of the experiment. The amindes of use in this invention have been given abbreviated names for ease of reference and in this specification the following abbreviations will be used:- Amine Moietv Abbreviation NH 2 .(CH 2 ) 3 NH CH (CH. «0> PEP NH. (CH-) 3 NH(CH 2 ) 3 NH CHPP NH ^_^ NH 2 (CH 2 ) 4 NH(CH 2 ) 4 NH C NH (CH 2 ) 2 Q AAGPe Thus bleomycin having the CHP amine moiety is called bleomycin CHP and the other acid amides are similarly described. The amide compound may be administered simultaneously with the activator compound or alternati ely it may be • -PE ad inistered some time in advance of the activator compound. Best Mode of Carrying out the Invention The antibacterial and antitumor activity of the amide/activator combinations of this invention will now be illustrated in detail by reference to specific examples. Method Random bred albino Sprague Dawley rats were used with Walker 256 carcinona Sarcoma. Tumour passages were effected by intraperitoneal injection of 10 cells which had been prepared by aspirating several ml of ascites fluid from a rat which had been carrying an ascites tumour for 7 days. This was given two 2-mϊnute treatments with Boyle's fluid (9 vols. 0.83% NH 4 C1, 1 vol. Tris buffer, pH7.2). The cell suspension was counted on a haemocytometer using Turk's fluid 7 and then diluted with Eagle's medium to 10 live cells per ml, and then 0.1 ml was injected intraperitoneally. Bacto-fluid Thioglycollate Medium was used to test for contamination. Solutions of the phleomycins, bleomycins or tallysomycins were made up in phosphate-buf ered 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 9 am and 4 p . Bulk solutions of amplifier were stored at room temperature and were injected 30 minutes after the active compound. In most experiments several levels of active compound and amplifier 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 active compound and amplifier. Test groups usually comprised 7 or 8 animals. Procedures were based closely on t-hose detailed by Giran 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 marked and a T/C (2) equal to or greater than 125 was considered a positive result. T/C » median survival time (MJS.T.)of test group median survival time (M.S.T.) of control group T/C (1) is the T/C where the control group is the sali vehicle only group and T/C (2) is the T/C where the control group is whicheve of the relative unamplified amide group or the amplifier on group has the higher M.S.T. value. The results are given in Table 1. The table also includes results for trials in which the potentiator was administered (i) via the drinking water, and (ii) from Alzet minipumps implanted under the skin. Tables 2 and 3 give results for similar tests carried out using mice infected with Ehrlich's Ascites Tumor, or wit Mouse Sarcoma 180. TABLE 1 TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER (DAYS) CD CZ ) CuKD) CuKD) EXPERIMENT 1A Phleomycin PEP 3840 24.0 266 Phleomycin PEP + Caffeine 3840 106.1 100 1111 C 416 Phleomycin PEP + Caffeine 3840 10.61 100 1111 C 416 Phleomycin PEP 1280 21.0 233 Phleomycin PEP + Caffeine 1280 106.1 100 1111 C 476 Phleomycin PEP + Caffeine 1280 10.61 60.0 667 C 285 Caffeine 106.1 8.0 89 Caf eine 10.61 8.0 89 Saline Vehicle Only 9.0 EXPERIMENT IB Phleomycin CHP 1000 21.0 280 Phleomycin CHP + Caffeine 1000 1061 21.0 280 ( 100 Phleomycin CHP + Caffeine 1000 10.61 16.0 213 C 76 Phleomycin CHP 250 9.0 120 Phleomycin CHP + Caffeine 250 1061 9.0 . 120 ( 100 Phleomycin CHP + Caffeine 250 10.61 100.0 1330 (1111 Phleomycin CHP 125 9.0 120 Phleomycin CHP + Caffeine 125 1061 7.0 93 ( 78 Phleomycin CHP + Caffeine 125 10.61 100.0 1330 (1111 Caffeine 1061 8.0 107 Caffeine 10.61 7.0 93 TABLE 1 Ccont.) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD C2) CuKD) CuKD) Saline Vehicle Only 7.5 EXPERIMENT 1C Phleomycin CHPP 1000 20.5 293 Phleomycin CHPP t Pyronin Y 1000 7575 27.5 393 C134) Phleomycin CHPP 250 12.0 171 Phleomycin CHPP + Pyronin Y 250 7575 17.0 243 C142) Pyronin Y - 7575 10.0 143 - Saline Vehicle Alone - - 7.0 - - EXPERIMENT ID Phleomycin CHP 1000 19.0 237 Phleomycin CHP 500 10.0 125 Phleomycin CHP + Dimethylthiocyanine 500 675 18.0 225 (180)* Phleomycin CHP 250 9.0 112 Phleomycin CHP + Dimethylthiocyanine 250 675 17.0 212 (188)* Phleomycin CHP 125 9.0 112 Phleomycin CHP + Dimethylthiocyanine 125 675 14.0 175 ' (155)* TABLE 1 Ccont.) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) Cl) (2) CuKD) CuKD) Dimethylthiocyanine 675 9 .0 112 Saline Vehicle Only - - 8.0 - - - EXPERIMENT IE Phleomycin CHP 250 - 14.0 133 - Phleomycin CHP + Caffeine in Minipumps 250 106, .1 100.0 952 c 714 Phleomycin CHP + Caffeine in Minipumps 250 10. .61 20.5 195 c 146 Phleomycin CHP 125 ' 13.5 129 - Phleomycin CHP + Caffeine in Minipumps 125 106. .1 12.0 114 1 ( ' 88 Phleomycin CHP + Caffeine in Minipumps 125 10. .61 26.0 248 ( 193 Caffeine in Minipumps - 106. .1 10.0 95 - Caffeine in Minipumps — 10. ,61 10.0 95 Saline Vehicle Only - - 10.5 - - EXPERIMENT IF Phleomycin AAGPE 300 - 10.5 175 - Phleomycin AAGPE + Dimethylthicicyanine 300 675 14.0 233 ( 133 # Phleomycin AAGPE 150 10 . 5 175 TABLE 1 Ccont.) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD C2) CuKD) CuKD) Phleomycin AAGPE + Di ethy11hioc anine 150 675 17.5 292 C 16 Phleomycin AAGPE 75 - 8.5 141 - Phleomycin AAGPE + Dimethylthiocyanine 75 675 11.5 191 C 13 Phleomycin AAGPE 38 - 7.5 125 - Phleomycin AAGPE + Dimethyl hiocyanine 38 675 9.5 158 C 127 Dimethylthiocyanine - 675 6.0 100 - Saline Vehicle Only - - 6.0 - - EXPERIMENT 1G Bleomycin CHP alone 1000 30.0 300 Bleomycin CHP + Dimethylthiocyanine 1000 675 100 1000 C 333 Saline Vehicle only 10.0 5 - -10- TABLE 2 MOUSE - EHRLICH'S ASCITES TUMOR Antibiotic and Amplifier by Intraperitoneal Injection CUnless Indicated Otherwise) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD (2) CuKD) CuKD) • EXPERIMENT 2A Phleomycin AAGPE 300 - 29 .5 131 - Phleomycin AAGPE + Caffeine 300 106 .1 44 .5 197 Ciso)* Phleomycin AAGPE + Caffeine 300 10. 61 30, .5 135 C103) Phleomycin AAGPE ISO - 26, .0 IIS - Phleomycin AAGPE + Caffeine ISO 106 .1 27, .0 120 (104) Phleomycin AAGPE + Caffeine 150 10. 61 52. .0 231 (200)* Caffeine - 106 .1 21. .0 93 - Caffeine - 10. 61 21. .0 93 - Saline Vehicle Only - - 22. . • 5 - - Phleomycin AAGPE 600 - 21. ,0 113 - EXPERIMENT 2B Phleomycin AAGPE 300 20.5 110 Phleomycin AAGPE + Caffeine in drinking water 300 1061 20.0 108 (97) Phleomycin AAGPE + Caffeine in drinking water 300 106.1 20.5 110 TABLE 2 Ccont.) MOUSE - EHRLICH'S ASCITES TUMOR Antibiotic and Amplifier by Intraperitoneal Injection CUnless Indicated Otherwise) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD (2) CuKD) CuKD) Phleomycin AAGPE + Caffeine in drinking water 300 10.61 20.0 108 C97) Phleomycin AAGPE ISO 19.0 102 Phleomycin AAGPE + Caffeine in drinking water 150 1061 24.5 132 C129)* Phleomycin AAGPE + Caffeine in drinking water 150 106.1 20.5 110 (108) Phleomycin AAGPE + Caffeine in drinking water 150 10.61 19.0 102 Caffeine in drinking water 1061 20.0 108 Caffeine in drinking water 106.1 21.0 113 Caffeine in drinking water 10.61 22.0 119 Saline Vehicle Only 18.5 EXPIREMENT 2C Phleomycin AC-AC-GPe 2700 32.0 171 Phleomycin ACrAC-rGPe + Dimethylthiocyanine 2700 2025 24.0 135 (75) TABLE 2 C cont . ) MOUSE - EHRLICH'S ASCITES TUMOR Antibiotic and Amplifier by Intraperitoneal Injection CUnless Indicated Otherwise) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD (2) CuKD) CuKD) Phleomycin AC 5 AC 3 GPe Dimethylthiocyanine 2700 675 40.0 222 C125)* Phleomycin AC 5 AC 3 GPe 900 27.0 150 Phleomycin AC 5 AC 3 GPe + Dimethylthiocyanine 900 2025 33.0 183 (122) Phleomycin AC 5 AC 3 GPe + Dimethylthiocyanine 900 675 58.0 322 (214)* Phleomycin ACgAC j GPe 300 24.0 133 Phleomycin AC j AC j GPe + Dimethylthiocyanine 300 2025 30.0 166 (125)* Phleomycin ACgAC j GPe + Dimethylthiocyanine 300 675 22.0 122 (91) Phleomycin ACgAC j GPe 100 20.0 111 Phleomycin AC j AC j GPe Dimethylthiocyanine 100 2025 20.0 111 (100) Phleomycin AC j AC j GPe Dimethylthiocyanine 100 675 18.0 100 (90) Phleomycin AC-AC 3 GPe 33 20.0 111 Phleomycin ACrAC- j GPe + Dimethylthiocyanine 33 2025 20.0 111 (100) Phleomycin ACς-AC-GPe + Dimethylthiocyanine 33 675. 20.0 111 (100) Dimethylthiocyanine 2025 20.0 111 675 20.0 111 TABLE 3 MOUSE SARCOMA 180 Antibiotic and Amplifier by Intraperitoneal Injection CUnless Indicated Otherwise) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C BLEOMYCIN AMPLIFIER CDAYS) CD (2) CuKD) CuKD) ' EXPERIMENT 3A Bleomycin CHP 1000 25.0 156 Bleomycin CHP + Caffeine 1000 1942 61.0 381 C244)* Bleomycin CHP 250 18.0 112 Bleomycin CHP + Caffeine 250 1942 29.0 181 (161)* Caffeine - 1942 17.0 106 - Saline Vehicle Only - - 16.0 - - EXPERIMENT 3B TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER (DAYS) CD (2) (uKD) (uKD) Phleomycin AAGPE 300 • -18.0 106 Phleomycin AAGPE + Dimethylthiocyanine 300 675 24.0 141 (135)* Dimethylthiocyanine - 675 17.0 100 - Saline Vehicle Only - - 17.0 - - EXPERIMENT 3C Phleomycin CHP 300 17.0 100 Phleomycin CHP * + Dimethylthiocyanine 300 675 22.0 129, C129)* _ TABLE 3 Ccon .) MOUSE SARCOMA 180 Antibiotic and Amplifier by Intraperitoneal Injection CUnless Indicated Otherwise) TREATMENT LEVEL OF LEVEL OF M.S.T. T/C PHLEOMYCIN AMPLIFIER CDAYS) CD (2) CuKD) CuKD) Phleomycin CHP 100 15.0 88 Phleomycin CHP + Dimethylthiocyanine 100 675 20.0 118 C133)* Dimethylthiocyanine 675 -17.0 100 Saline Vehicle Only 17.0

Claims: CLAIMS 1. A method of amplifying the antibiotic or antitumor activity of an amide of an acid selected from the group comprising phleomycinic acid, bleomycinic acid and tallysomycinic acid in which the substituted amine moiety is selected from the group comprising compounds having the structural formulae:- NH 2 . (CH 2 ) 3 •NH \ ' NH 2 . CCH 2 ) 3 . NH . CHCCH-.) <_- NH 2 . CCH 2 ) 3 .NH . CCH 2 ) 3 . m NH NH 2 . (CH 2 ) 4 .NH . (CH 2 ) 4 , NH . C.NH. (CH 2 ) NH NH 2 . (CH 2 ) 5 .NH . (CH 2 ) 3 , NH . C.NH. (CH ∑ ) NH 2 . (CH 2 ) 4 .NH . (CH 2 ) 3 , NH . which comprises administering any one of the said amides in association with an amplifying compound selected from the group comprising caffeine, dimethylthiocyanine and Pyronine Y 2. A method as claimed in claim 1 in which the amide is an amide of phleomycinic acid. 3. A method as claimed in claim 1 in which the amide is an amide of bleomycinic acid. 4. A method as claimed in any one of claims 1 to 3 in which the amide and the amplifying compound are administered in a weight ratio of from 1:0.002 to 1:30. 5. A method as claimed in any one of claims 1 to.4 in which the amine moiety is selected from the group comprising compounds having the structure formulae:- and 6. claims 1 to 5 in which the amplifying compound is caffeine. 7. A method of amplifying the antibiotic or antitumor activity of an amide of an acid selected from the group comprising phleomycinic acid, bleomycinic acid and tallysomycinic acid in which the substituted amine moiety is selected from the group comprising compounds having the structural formulae:- NH. CCH 2 ) 3 . NH . CHCCH 3 ) -(^ NH. CCH 2 ) 3 .NH . CCH 2 ) 3 . NH NH NH 2 . CCH 2 ) 4 .NH . CCH 2 ) 4 , NH . C.NH. ' CCH 2 ) £ -/Q NH . NH 2 . CCH 2 ) 5 .NH . CCH 2 ) 3 , NH . C.NH. CCH 2 ) 2 Q) NH 2 . CCH 2 ) 4 .NH . CCH 2 ) 3 , NH . substantially as hereinbefore described with reference to the accompanying examples. ' C PI

Inventors: GRIGG G, LAMBERTON J
Applicants: COMMW SCIENT IND RES ORG, GRIGG G, LAMBERTON J, COMMW SCIENT & IND RES ORG
Application year: 1981
Main classification: A61K31
IPC codes: A61K31, C07K9