Cancer Research Campaign Experimental Chemotherapy Group, Department of ... 'Recipient of National Cancer Institute Contract N01-CM-07350, under which.
[CANCER RESEARCH
45, 3008-3013,
July 1985]
Experimental Antitumor Activity against Murine Tumor Model Systems of 8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-ci]-1,2,3,5-tetrazin-4(3H)-one (Mitozolomide), a Novel Broad-Spectrum Agent1 John A. Hickman,2 3 Malcolm F. G. Stevens,2 Neil W. Gibson,4 Simon P. Langdon,2 Christian Fixâmes, FrançoisLavelle, Ghanem Atassi,5 Edward Lunt, and Robert M. Tilson Cancer Research Campaign Experimental Chemotherapy Group, Department of Pharmaceutical Sciences, Aston University, Birmingham B4 7ET, England [J. A. H., M. F. G. S., S. P. L, N. W. G.]; Rhône-Poulenc Santé,Centre de Recherches de Vitry, 94407, Vitry Sur Seine Cedex, France [C. F., F. L]; Institut Jules Bordet, 1000 Bruxelles, Belgium [G. A.]; and May and Baker, Ltd., Dagenham, Essex RMW 7XS, England [E. L, R. M, T.]
ABSTRACT 8-Carbamoyl-3-(2-chlorœthyl)imidazo[5,1-tf|-1,2,3,5-tetrazin4(3H)-one (mitozolomide) demonstrates curative action against a range of murine tumor model systems. At single doses of between 20 and 40 mg/kg, the latter of which approximates the 10% lethal dose value in mice, the compound elicited cures against the L1210 and P388 leukemias irrespective of the route of tumor and/or drug administration; in these tests, animals receiving 105 cells i.p. survived >60 days after treatment. Potent effects were also observed against the TLX5 lymphoma (s.c.) and B16 melanoma (i.p.). In other experiments, 7 of 10 animals implanted with 2 x 10s Lewis lung carcinoma cells survived >60 days while 10 of 10 animals survived >60 days after implantation of the Colon 26 tumor. Potent inhibition of the solid tumor models was also observed with complete cures of the Colon 38, M5076 sarcoma, and ADJ/PC6A plasmacytoma. In cross-resistance studies, the compound was ineffective against an L1210 leuke mia made resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea and against a TLX5 lymphoma resistant to dimethyltriazenes but cured animals bearing the L1210 leukemia with derived resist ance to cyclophosphamide.
INTRODUCTION The synthesis and screening of the novel agent mitozolomide6 (Chart 1) in our laboratories stemmed from our chemical interest in molecules bearing NNN linkages in either cyclic (1) or acyclic (2) arrangements and bicyclic heterocycles with bridgehead ni trogen atoms (3) and from our pharmacological studies on the
1This is Part 6 of the series "Antitumor Imidazotetrazines" (Part 5 is réf.7). 2 Recipient of grants from the Cancer Research Campaign, United Kingdom, in partial support of this research. 3To whom correspondence and requests for reprints should be addressed. 4 Recipient of a grant from the Science and Engineering Research Council, in partial support of this work. Present address. Laboratory of Molecular Pharmacol ogy, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20205. 'Recipient of National Cancer Institute Contract N01-CM-07350, under which antitumor results from the Institut Jules Bordet were obtained. "The generic name "mitozolomide" refers to 8-carbamoyl-3-(2-chloroethyl)imidazo]5,1-d]-1,2,3,5-tetrazin-4(3H)-one, also designated CCRG 81010, M & B 39565, and NSC 353451 and formerly known as azolastone. The abbreviations used are: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; DTIC, 5-(3,3-dimethyltriazen1-yl)imidazole-429915912 kg/day)502512.56.253.120502512.56.253.120201050502504020040201050105050 day13.7/14.8>29.8/32.032.6/33.325.2/24.422.1/22.315.6/15.412.0/12.3> no.0/10 tal
3breiLewis melanoma
C
i.p.
0.5 ml of 1:10
i.p.
1-9
(60)1/100/100/100/100/100/10
3nomaLewis lung card-
C
i.v.
2x10*
i.p.
1-9
(60)7/102/101/100/100/505/10
2Lewislung
B
i.v.
10*
p.o.
2
(44)0/100/100/108/10
2Colonlung
B
i.V.
10*
p.o.
5
(62)6/100/1010/10
2L1210/BCNU 26
B
i.p.
p.o.
7
(68)4/100/100/10
1L1210/cyclo-
B
i.p.
105
i.p.
1
2phospnamideL1210/BCTIC B
i.p.
Mf
i.p.
1-5
(81)0/100/100/10
1TLX5/triazene
D
i.p.
10"
i.p.
1-9
(60)0/100/100/100/100/5
A
s.c.
2X105
i.p.
3-7
1:10 dilution
.015.1/14.012.9/13.010.6/11.010.2/10.09.3/9.0>81.0/>81.014.9/14.09.2/ (60)0/100/100/100/1010/10
1Dose
(60)0/50/50/5
* See "Materials and Methods." " T/C, treated versus control. c Based on median values. d Long-term survivors included. 9 Positive controls were used in some experiments. Experiment 1: BCNU,20 mg/kg i.p., Day 1; T/C x 100% >740; 5 of 5 long-term survivors on Day 60. Experiment 4: 5 -fluorouracil, 20 mg/kg/day i.p., Days 1 to 9; T/C x 100% = 195. Experiment 5: MeCCNU,50 mg/kg p.o., Day 1; T/C x 100% >514; 8 of 8 long-term survivors on Day 36. Experiment 6: CCNU, 20 mg/kg p.o., Day 1; T/C x 100% >390; 6 of 10 long-term survivors on Day 39. Experiment 7: BCNU, 40 mg/kg ¡.p.,Day 1; T/C x 100% >594; 5 of 5 long-term survivors on Day 60. Experiment 9: 5-fluorouracil, 20 mg/kg/day i.p., Days 1 to 5; T/C x 100% = 171. Experiment 11: BCNU, 40 mg/kg i.p. Day 3; T/C x 100% = 166; 0 of 5 long-term survivors on Day 60. Experiment 14: cyclophosphamide,90 mg/kg i.p., Day 1; T/C x 100% >253; 9 of 10 long-term survivors on Day 60. Experiment 15: MeCCNU,40 mg/kg p.o., Day 2; T/C x 100% >244; 8 of 10 long-term survivors on Day 44. Experiment 16: MeCCNU,50 mg/kg P.O.,Day 5; T/C x 100% >216; 4 of 10 long-term survivors on Day 62. Experiment 17: MeCCNU,40 mg/kg p.o., Day 7; T/C x 100% >219; 7 of 10 long-term survivors on Day 68. Experiment 18: BCNU, 40 mg/kg i.p., Day 1; T/C x 100% = 144; 0 of 10 long-term survivors. ' Numbers in parentheses, day of evaluation.
and clinical7 pharmacokinetic studies.
DISCUSSION The antitumor activity reported here for mitozolomide sug gests the drug to be among the most potent of those screened against the National Cancer Institute murine tumor panel (Tables 1 to 3). The drug is effective against the majority of murine models irrespective of the site of tumor implantation. The activity of mitozolomide p.o. (Table 1) is an encouraging result, congruent with the findings of high bioavailability reported in preclinical (14) CANCER
Studies on the mechanism of action of mitozolomide and of its chemistry support the hypothesis that the new agent is a prodrug of the monochloroethyltriazene MCTIC (Chart 1) (6). MCTIC is a chloroethylating agent (6), chemically comparable to 2-chloroethyldiazohydroxide, which has been suggested to be 7E. S. Newlands ef al. Phase 1 clinical trial of mitozolomide (CCRG 81010; M & B 39565; NSC (353451), Cancer Treatment Reports.
RESEARCH VOL. 45 JULY 1985 3011
ANTITUMOR
IMIDAZOTETRAZINES
Table 2 Activity of mitozolomide against murine solid tumors TreatmentScreen-Inoculum Experiment kg/day)1e
Tumor
ingCenter"
Route
Lewis lung carcinoma
B
i.m.
No. of cells
Route
5x10"
Days of injection Vehicle
i.p.
1-4
1
vol ume0(cu
Dose(mg/
mm)1000
vol ume index (T/c'x
of inhibi tion of mé 100%)14 tastases97
20
-0.80+2.0-2.4+1.9+2.5+2.3+2.3-1.0+1.9+0.9+1.9+1.1-0.30+1. 1700520070002088196411001583197000035646 24741000021471000051000000571 96710
10502
502512.56.2503 Coton 38 carcinoma
C
s.c.
M5076 8coma ovarian sar-
A
i.m.
ADJ/PC6A 20toma plasmacy1052.51.2505
A
CD8F, 50nocarcinoma mammary ade-
D
Fragment
i.p.
2,9
3
10*
i.p.
1-17
1
i.m.
10*
i.p.
14
1
s.c.
Homogenate
i.p.
1
3
4204
2512.50AW6(g)-2.6
a See "Materials and Methods." 6 Changein mean body weight between first and second weight evaluation days. These days were as follows: Experiment 1, Days 0 and 8; Experiment 2, Days 2 and 20; Experiment 3, Days 1 and 17; Experiment 4, Days 14 and 19; and Experiment 5, Days 21 and 28. Tumor volumes are given as mean values (Experiments 1, 3, and 4), median values (Experiment 2), or the median change (Experiment 5). Days for evaluation of or volume were: Day 21, Experiment 1; Day 20, Experiment 2; Day 24, Experiments 3 and 4; or Day 28, Experiment 5. Ten animals per group were used in tumor Experiments 1, 2,3, and 5; 5 animals per group were used in Experiment 4. T/C, treated versus control. * Positive controls were used in some experiments. Experiment 1: DTIC, 200 mg/kg/day i.p., Days 1 to 4; T/C x 100% 50%; 62% inhibition of métastases. Experiment 3: CCNU, 40 mg/kg i.p., Day 1; T/C x 100% = 0. Experiment 4: CCNU, 40 mg/kg ¡.p., Day 1; T/C x 100% = 0.
resistant with BCNU but not with cyclophosphamide. Cyclophosphamide, although reacting with DNA to produce cross-links, does so in a manner different from those of the nitrosoureas (23) and mitozolomide (8, 9), and the cross-resistance pattern re
6.0 DAYS
-
22,29,36
6.0
ported here may reflect this difference in mechanism. Differences in tumor sensitivity to the nitrosoureas and cyclophosphamide have been reported previously (25). The haloethylnitrosoureas offered great therapeutic promise when they were first screened against a range of murine tumors and were found to have a potent broad-spectrum activity (24). This promise was largely unfulfilled in the clinic for reasons that are unclear. Given the activity spectrum of mitozolomide reported here, which is similar to that of the nitrosoureas, and its mecha nism of action (8-10), speculation regarding its clinical potential
2.0
10
20
DAYS
30
AFTER
W
50
60
70
IMPLANTATION
Chart 3. Activity of mitozolomide against the early and advanced M5076 reticulum cell sarcoma. M5076 cells (10") were injected i.m. into the left hind legs of groups of 5 female BD2F, mice on Day 0. Drug (10 mg/kg) was administered on the stated day(s) shown by i.p. injection. Points, mean tumor volumes; bars, SE. D, control; O, Day 1; V, Days 1, 8, and 15; •, Day 22; T, Days 22, 29, and 36.
formed on decomposition of antitumor 2-chloroethylnitrosoureas, such as BCNU (Chart 2) (22). Not surprisingly then, mechanistic studies on mitozolomide have shown it to interact with DNA in a manner similar to that of the nitrosoureas (8, 9). This similarity with the nitrosoureas extends to its broad spectrum of antitumor action and to the patterns of cross-resistance observed between mitozolomide and other drugs (Table 1). Mitozolomide is crossCANCER
in comparison with the nitrosoureas should additionally take account of particular differences which exist between mitozolom ide and the nitrosoureas. For instance, mitozolomide, unlike those haloalkyl nitrosoureas which demonstrate potent antitumor activity against the murine screens, does not decompose to a carbamoylating agent (6,10) and may therefore lack some of the deleterious side effects which have been attributed, tentatively, to carbamoylation (26). Additionally, unlike certain nitrosoureas, the drug shows a large differential cytotoxicity between human cell lines which are either normal or transformed (9). Finally, mitozolomide has a chemical and biological half-life longer than that of BCNU (6,14, 22, 27) and thus is advantaged with regard to its pharmacodynamics.7 Comparison of the activity of this new
RESEARCH VOL. 45 JULY 1985 3012
ANTITUMOR
IMIDAZOTETRAZINES
Table3
Activity of mitozolomide ana other drugs in the National Cancer Institute murine tumor panel (28) adeno26 DrugMitozolomide leukemia>150leukemia-H->175 melanomaInactive>150 carcinomaInactive>150 tumorInactive carcinomaInactive mammary-HNitrogen mustard BCNU DTIC Cisplatin Cyclophosphamide Methotrexate AdriamycinT/C
Inactive>150 Inactive125P388
>120B16
>125LL
Table 4 Acute toxicity of mitozolomide in mice
Mouse strainBD2F, BALB/c CBA/CaRoute
of in jection (sin gle injection)i.p.
10.
(mg/kg)44
(mg/kg)50 (37-53)" (45-55) 42 (28-63) 57 (43-76) Female i.p. FemaleLD,oa 45(41-50)LO.» 55 (49-62) i.p.SexFemale
CBA/Ca
i.v. Female 73(57-93) i.v. Male 61 (48-78) CD-1 i.v. Female 57(48-68) " LD,o, 10% lethal dose; LD«,, 50% lethal dose. " Numbers in parentheses, 95% confidence limits.
11. • 12.
92(83-102) 74 (65-84) 73(66-80)
13.
14.
drug with that of other widely used agents against the National Cancer Institute murine tumor panel (Table 3) shows that it is equivalent to BCNU, c/s-platinum, Adriamycin, and cyclophos-
15. 16.
phamide and more active than DTIC. The drug, shortly to begin Phase 1 trials in the United States and Phase 2 trials in the United Kingdom, therefore also offers a challenging prospective test for the predictive value of these widely used screening systems. A report of the outcome of the United Kingdom Phase 1 trials has been submitted by us for publication.7
17. 18. 19.
REFERENCES 1. Stevens, M. F. G. The medicinal chemistry of 1,2,3-triazines. Prog. Med. Chem., 13: 205-269,1976. 2. Vaughan, K., and Stevens, M. F. G. Monoalkyltriazenes.Chem. Soc. Rev., 7: 377-397,1978. 3. Baig, G. U., Stevens, M. F. G., Stone, R., and Lunt, E. Triazines and related products. Part 24. Synthesis of pyrazol-4-ylidenehydrazinoimidazolesby hydrazinolysis of imidazo[5,1-c][1,2,4]triazines and 2-arylazoimidazolesby diazonium coupling reactions. J. Chem. Soc. Perkin Trans. 1,1811-1819,1982. 4. Farina, P., Gescher, A., Hickman, J. A., Morton, J. K., D'Incaici, M., Ross, D.,
5. 6. 7.
8. 9.
Stevens, M. F. G., and Torti, L. Studies of the mode of action of antitumour triazenes and triazines—IV. The metabolism of l-^acetylphenyiy^.S-dimethyltriazene. Biochem. Pharmacol.,37:1887-1892,1982. Gibson, N. W., and Hickman, J. A. The rote of isocyanates in the toxicity of antitumour haloalkylnitrosoureas.Biochem. Pharmacol.,37:2795-2800,1982. Stevens, M. F. G., Hickman, J. A., Stone, R., Gibson, N. W., Lunt, E., Newton, C. G., and Baig, G. U. Antitumor imidazotetrazines.1. Synthesis and chemistry of 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-140Colon >130C38
carbamoyl-3-{2-chloroethyl)imidazo[5,1
