Experimental Antitumor Activity Against Murine Tumor Model

[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 FixÃ¢mes, FranÃ§oisLavelle, 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.]; RhÃ´ne-Poulenc SantÃ©,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 mechanism of action of antitumor triazenes (4) and nitrosoureas (5). Although mitozolomide is a derivative of a novel ring system 8-Carbamoyl-3-(2-chlorÅ“thyl)imidazo[5,1-tf|-1,2,3,5-tetrazin- (6), a crystallographic analysis (7) confirms a familial relationship 4(3H)-one (mitozolomide) demonstrates curative action against to a series of imidazotriazenes, DTIC, MTIC, BCTIC, and MCTIC a range of murine tumor model systems. At single doses of (Chart 1); like these agents, the new compound can degrade by between 20 and 40 mg/kg, the latter of which approximates the a variety of different pathways (6) each of which can release 10% lethal dose value in mice, the compound elicited cures reactive and cytotoxic moieties. Studies on the mode of action against the L1210 and P388 leukemias irrespective of the route of mitozolomide (8-10) have confirmed this relationship and of tumor and/or drug administration; in these tests, animals strongly suggest that the new agent is a prodrug of the mono- receiving 105 cells i.p. survived >60 days after treatment. Potent chloroethyltriazene MCTIC. In corroboration, chemical investi effects were also observed against the TLX5 lymphoma (s.c.) gations have confirmed (6) that mitozolomide ring opens to and B16 melanoma (i.p.). In other experiments, 7 of 10 animals implanted with 2 x 10s Lewis lung carcinoma cells survived >60 MCTIC under mildly alkaline conditions (Chart 2). BCTIC (Chart 1), also a potential prodrug of MCTIC, has been days while 10 of 10 animals survived >60 days after implantation found previously to be a potent antitumor agent in experimental of the Colon 26 tumor. Potent inhibition of the solid tumor models murine systems (11) but proved to be clinically disappointing. was also observed with complete cures of the Colon 38, M5076 This lack of activity may have been because of its propensity to sarcoma, and ADJ/PC6A plasmacytoma. In cross-resistance cyclize rapidly to an inactive triazolinium salt (11,12). Certainly, studies, the compound was ineffective against an L1210 leuke its inherent instability and that of the monochloroethyltriazene mia made resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea and (MCTIC), which is also active against murine tumors (13), has against a TLX5 lymphoma resistant to dimethyltriazenes but thwarted clinical development of these antineoplastic drugs. In cured animals bearing the L1210 leukemia with derived resist comparison, mitozolomide has a half-life of 1.2 h in humans (14), ance to cyclophosphamide. which is in good agreement with values obtained from previous animal experiments (15), is acid stable (6), and thus presents itself as an alternative candidate to the aforementioned imida INTRODUCTION zotriazenes with potential for use in humans. The synthesis and screening of the novel agent mitozolomide6 We report here the activity of mitozolomide in vivo against a (Chart 1) in our laboratories stemmed from our chemical interest number of murine tumors including variants resistant to nitrosou in molecules bearing NNN linkages in either cyclic (1) or acyclic reas, triazenes, and cyclophosphamide. These studies demon strate that this new agent possesses potent broad-spectrum (2) arrangements and bicyclic heterocycles with bridgehead ni trogen atoms (3) and from our pharmacological studies on the activity.

1This is Part 6 of the series "Antitumor Imidazotetrazines" (Part 5 is rÃ©f.7). 2 Recipient of grants from the Cancer Research Campaign, United Kingdom, in MATERIALS AND METHODS partial support of this research. 3To whom correspondence and requests for reprints should be addressed. Drugs. Mitozolomide was synthesized in our laboratories (6). BCNU, 4 Recipient of a grant from the Science and Engineering Research Council, in trans-i -<2-chloroethyl)-3-(4-methylcyclohexyl)-1 -nitrosourea, and cyclo partial support of this work. Present address. Laboratory of Molecular Pharmacol phosphamide were gifts from the National Cancer Institute, Bethesda, ogy, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20205. MD. Several injection vehicles for mitozolomide were used: Vehicle 1, 'Recipient of National Cancer Institute Contract N01-CM-07350, under which 10% DMSO:90% arachis oil; Vehicle 2, suspension in carboxymethyl- antitumor results from the Institut Jules Bordet were obtained. "The generic name "mitozolomide" refers to 8-carbamoyl-3-(2-chloro- cellulose; Vehicle 3, suspension in 0.9% NaCI solution containing Tween 80. BCNU, frans-1 -(2-chloroethyl)-3-{4-metnylcyclohexyl)-1 -nitrosourea, ethyl)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 cyclophosphamide, and DTIC were dissolved in 10% DMSO:90% arachis used are: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; DTIC, 5-(3,3-dimethyltriazen- oil. 1-yl)imidazole-4

CANCER RESEARCH VOL. 45 JULY 1985 3008

CONH BD2F,), and CBA/Ca mice were used at Screening Center A and obtained from Bantin and Kingman, Ltd. (Hull, United Kingdom). BALB/c x DBA/2 F, (hereafter called CD2F,), BD2F,, DBA/2, BALB/ ,N c, and C57BL/6 mice used at Screening Centers B and C were obtained If XCH2CH2CI from Charles River Breeding Laboratories and IFFA-CREDO Breeding 0 Mitozolomide Laboratories (France). Toxicity Studies. Studies of the acute toxicity of mitozolomide by CONH, single injection of the drug in DMSO:arachis oil (1:9) were performed in BD2F!, CBA/Ca, BALB/c, and CD2Fi mice. Groups of 5 to 10 mice, of defined sex and weighing from 20 to 22 g, were given i.p. or i.v. injections, N V-NH and deaths were noted over a period of 30 days. Lethal dose values and their 95% confidence limits were calculated according to the method of Litchfield and Wilcoxon (21). R1=R2rMe (DTIC)

R1 = Me,R2= H (MTIC) RESULTS R1 = R2=CH2CH2Cl (BCTIC) The activity of mitozolomide against the L1210 leukemia, and R^CHjCHjCl.R^H (MCTIC) other murine tumor models, is shown in Table 1. The drug has Chart 1. Structure of mitozokxnide, DTIC, MTIC, BCTIC, and MCTIC. curative activity against the i.p.-implanted L1210 tumor on single or repeat dosage schedules when administered i.p. or p.o. However, it was less effective against i.v.- or i.c.-implanted cells following p.o. administration. Bioavailability of the p.o.-adminis CONH, CONH2 tered drug was evidently high, as reported previously (15), since H20 the results of dosing with the drug i.p. or p.o. were essentially K identical. Mitozolomide was markedly active against other survival time models (Table 1), with cures elicited in mice inoculated with the CONH2 Mitozolomide i.p. P388 leukemia, the s.c. TLX5 lymphoma, and the Colon 26 tumor. The Lewis lung carcinoma was sensitive to the drug, with C 2 a high percentage of cures being obtained with either a chronic VNH daily schedule or single injection; some cures were even obtained MCTIC when a single delayed dosage was used. The drug produced significant increases in survival time in mice inoculated with the 816 melanoma, but only occasional cures were obtained. HO ClCH2CH2NCN-CH2CH2Cl A feature of the tumor-inhibitory activity of mitozolomide is its pronounced activity against the murine solid tumor models Lewis lung carcinoma, Colon 38, M5076 reticulum cell sarcoma, and the ADJ/PC6A plasmacytoma (Table 2); however, only marginal BCNU CICH2CH2-N=C=0 activity was observed against the CD8F, mammary tumor. The drug not only markedly inibited the i.m. primary Lewis lung Chart 2. Decomposition pathways for mitozolomideand BCNU. carcinoma but also eradicated pulmonary mÃ©tastases(Table 2). The effects of early or delayed treatment (Chart 3) of the M5076 reticulum cell sarcoma illustrate the striking activity of the drug C, Institut Jules Bordet, Brussels, Belgium; D, other National Cancer against this tumor. Both treatments produced a final equivalent Institute Laboratories. response. Tumor Systems. The murine L1210 leukemia, P388 leukemia, B16 Cross-resistance studies with the drug using L1210 lines melanoma, BALB/c x DBA/8 F, (hereafter called CD8F,) mammary tumor, Lewis lung carcinoma, Colon 26 tumor, and Colon 38 tumor were sensitive and resistant to BCNU, BCTIC, and cyclophosphamide originally obtained from Dr. A. Bogden, Mason Research Institute, (Table 1) revealed that mitozolomide was completely cross- Worcester, MA, and used for antitumor testing in accordance with the resistant with BCNU and BCTIC but not cross-resistant with protocols described by the National Cancer Institute (16). The JLX5 cyclophosphamide. The TLX5 lymphoma resistant to dimethyl- lymphoma and M5076 reticulum cell sarcoma were passaged and used triazenes such as DTIC was cross-resistant to the action of the as described previously (17, 18). The assay used to determine lung drug. The activity of mitozolomide against the National Cancer mÃ©tastaseswas that described by Wexler (19). Institute murine tumor panel is compared with that of other The ADJ/PC6A plasmacytoma was maintained s.c. in BALB/c mice. widely used agents in Table 3. For antitumor assays with this tumor, a protocol identical to that for the In acute toxicity tests of mitozolomide, there were no signifi M5076 tumor was used (17,18). The L1210- and P388-resistant tumor variants were obtained from Dr. cant strain differences and no sex differences observed in mor A. Bogden, Mason Research Institute. The TLX5 line resistant to the tality (Table 4). The 10% lethal dose value in BD2F! mice was triazenes was initially made resistant to ethyl-5-(3,3-dimethyltriazen-1- 44 mg/kg, whereas the dose required to obtain cures in a number yl)-2-phenylimidazole-4-carboxylate as described previously (20). of different tumor systems using this strain was 20 mg/kg (see Animals. BALB/c, DBA/2, C57BL x DBA/2 F, (hereafter called Table 1).

CANCER RESEARCH VOL. 45 JULY 1985 3009

labte 1 ActivitymodelsInoculum oÃmitozolomideagainst murine tumor survival time TreatmentExperi- Screening of Dose (mg/ dian death survivors/to mentRoute1* Tumor Center* No.kg/day)10* of cells Route injection Vehicle* 100%c'd130>740204130112100>700>700>700188100>571>571164138100138>732>732184148100386214186143100>39012010084>594>594163134100>545>545>545>545147100153>612276182100>667>66715611610067>496161174114100204179146100Long-term day14.4/10.5>60/>60>24.8/16.510.6/10.59.0/9.18.1/8.1>52.3/>56>56/>56>56/>56>20.4/15.08.1/8.0>60/>60>60/>6017.0/17.214.4/14.510.4/10.511.2/11.3>60/>60>60/>6015.0/15.112.0/12.18.3/8.221.8/27.0>23.1/15.012.7/13.09.7/10.07.0/7.032.1/>3912.1/12.010.4/10.07.6/8.5>60/>60>60/>6016.4/16.513.2/13.510.0/10.1>60/>60>60/>60>60/>6046.2/>6016.0/16.210.8/11.015.0/15.0>47.7/>60>36.3/27.014.8/17.89.5/9.8>60/>60>48.5/>6013.9/14.010.7/10.49.4/9.08.0/8.155.0/>6019.8/19.519.6/21.013.6/13.812.0/12.1>36.2/24.521.2/21.517.4/17.512.0/12.0Range9-22>6015->6010-1298-96->56>56>5613->566-10>60>6016-1814-1510-1110-12>60>6014-1611-138-106-3113-369-159-11714-3911-1310-116-9>60>6013-2012-159-11>60>606024->6015-1710-1113-1723->6022->6012-216-11>6016->6010-169-129-11835->6018-2215-2313-1411-1318->6019-2316-2011-13T/C"xtalno.0/5 i.p.2 L1210 leukemia A 804020105010*i.p. 1 1 (60)'5/51/50/50/50/55/7

i.p.3 L1210 B 80402010010*P.O. 1 2 (56)7/77/71/70/105/5

Â¡.p.4 L1210 A 20107.55.0010*i.p. 1-5 1 (60)5/50/50/50/50/6

502512.56.253.12010si.p. 1-9 3 i.p.5 L1210 C (60)6/66/60/60/60/250/8

i.v.6 L1210 B 502512.56.250103p.o. 1 2 (36)3/80/80/80/106/10

i.e.7 L1210 B 2010010e p.o. 1 2 (39)0/100/100/5

i.p.8 P388 leukemia A 8040201050106i.p. 1 1 (60)5/55/50/50/50/55/5

i.p.9 P388 A 20107.55.02.5010*i.p. 1-5 1 (60)5/55/53/50/50/50/6

i.p.10 P388 C 502512.56.25010"i.p. 1-5 3 (60)4/62/60/60/435/5

i.p.11 P388 A 402010502 p.o. 1 3 (60)7/100/100/100/50/5

s.c.12 TLX5 lymphoma A 80402010502x 105 i.p. 3 1 (60)4/50/50/50/50/202/5

TLX5 A s.c.Days x 105 i.p. 3-7 1 16840Mean/me (60)0/50/50/5

CANCER RESEARCH VOL. 45 JULY 1985 3010

ContinuedExperi Tabte 1â€” TreatmentScreening Days of (mg/ dian death survivors/to Vehicle*B16Tumor Center* Route No. of cells Route injection 100%Â°'"9620821615814510061>299159121103100228117117100>326>326100>219190100156144122111100>900156100101125106102100112102100100Long-term ment131415161718192021Inoculum kg/day)502512.56.253.120502512.56.253.1202010505025040200402010501050502512.56.25016840Mean/meday13.7/14.8>29.8/32.032.6/33.325.2/24.422.1/22.315.6/15.412.0/12.3>55.4/>6036.3/32.026.6/24.319.2/20.819.3/20.136.6/41.021.1/21.019.9/21.118.5/18.052.9/>6246.1/>6218.9/19.0>68/>68>54.5/59.033.5/31talno.0/10 3breiLewismelanoma C i.p. 0.5 ml of 1:10 i.p. 1-9 (60)1/100/100/100/100/100/10

3nomaLewislung 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/BCNU26 B i.p. 1:10 dilution p.o. 7 (68)4/100/100/10

.015.1/14.012.9/13.010.6/11.010.2/10.09.3/9.0>81.0/>81.014.9/14.09.2/9.08.5/8.910.8/11.09.2/9.38.5/9.08.7/8.811.4/11.510.4/10.510.2/10.310.2/10.3Range10-1616->6025-3522-3215-2912-226-1838->6024->6022->6018-2411-2714-4418-2316-2117-2116->6210->6216-23>6834->6824-4014-1711-1410-119-119-10>8112-198-107-910-118-106-98-1011-1210-1110-1110-11T/C"x

1L1210/cyclo- B i.p. 105 i.p. 1 (60)0/100/100/100/1010/10

2phospnamideL1210/BCTICB 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 1Dose (60)0/50/50/5

* See "Materials and Methods." " T/C, treated versus control. c Based on medianvalues. 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 Â¡.p.,Day1; 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 Studies on the mechanism of action of mitozolomide and of The antitumor activity reported here for mitozolomide sug its chemistry support the hypothesis that the new agent is a gests the drug to be among the most potent of those screened prodrug of the monochloroethyltriazene MCTIC (Chart 1) (6). against the National Cancer Institute murine tumor panel (Tables MCTIC is a chloroethylating agent (6), chemically comparable to 1 to 3). The drug is effective against the majority of murine 2-chloroethyldiazohydroxide, which has been suggested to be models irrespective of the site of tumor implantation. The activity of mitozolomide p.o. (Table 1) is an encouraging result, congruent 7E. S. Newlandsef al. Phase 1 clinical trial of mitozolomide(CCRG81010; M & with the findings of high bioavailability reported in preclinical (14) B 39565; NSC (353451), Cancer Treatment Reports.

CANCER RESEARCH VOL. 45 JULY 1985 3011

Table 2 Activity of mitozolomide against murine solid tumors TreatmentScreen-Inoculum vol vol ume index of inhibi Experi- ingCen- Days of Dose(mg/ ume0(cu (T/c'x tion of mÃ© mentkg/day)1e Tumor ter" Route No. of cells Route injection Vehicle mm)1000 100%)14 tastases97 Lewis lung carcinoma B i.m. 5x10" i.p. 1-4 1 20 10502 -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+1.1+1.3+1.5-2.0-1.4-1.0+0.2Tumor170052007000208819641100158319700003564621374582913861831Tumor2474100002147100005100000057100414576100%96710

502512.56.2503Coton 38 carcinoma C s.c. Fragment i.p. 2,9 3

M50768coma ovarian sar- A i.m. 10* i.p. 1-17 1 4204

ADJ/PC6A20toma plasmacy- A i.m. 10* i.p. 14 1 1052.51.2505

CD8F,50nocarcinoma mammaryade- D s.c. Homogenate i.p. 1 3 2512.50AW6(g)-2.6

a See "Materials and Methods." 6 Changein mean body weight between first and second weight evaluationdays. 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 tumoror 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 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 mÃ©tastases. Experiment 3: CCNU,40 mg/kg i.p., Day 1; T/C x 100% = 0. Experiment 4: CCNU,40 mg/kg Â¡.p.,Day1; T/C x 100% = 0.

resistant with BCNU but not with cyclophosphamide. Cyclophos- 6.0 phamide, although reacting with DNA to produce cross-links, does so in a manner different from those of the nitrosoureas (23) DAYS 22,29,36 and mitozolomide (8, 9), and the cross-resistance pattern re - 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 2.0 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 10 20 30 W 50 60 70 here, which is similar to that of the nitrosoureas, and its mecha

DAYS AFTER IMPLANTATION nism of action (8-10), speculation regarding its clinical potential Chart 3. Activity of mitozolomide against the early and advanced M5076 retic- in comparison with the nitrosoureas should additionally take ulum cell sarcoma. M5076 cells (10") were injected i.m. into the left hind legs of account of particular differences which exist between mitozolom groups of 5 female BD2F, mice on Day 0. Drug (10 mg/kg) was administered on ide and the nitrosoureas. For instance, mitozolomide, unlike 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; â€¢,Day22; T, Days 22, 29, and 36. 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 formed on decomposition of antitumor 2-chloroethylnitrosoureas, deleterious side effects which have been attributed, tentatively, such as BCNU (Chart 2) (22). Not surprisingly then, mechanistic to carbamoylation (26). Additionally, unlike certain nitrosoureas, studies on mitozolomide have shown it to interact with DNA in a the drug shows a large differential cytotoxicity between human manner similar to that of the nitrosoureas (8, 9). This similarity cell lines which are either normal or transformed (9). Finally, with the nitrosoureas extends to its broad spectrum of antitumor mitozolomide has a chemical and biological half-life longer than action and to the patterns of cross-resistance observed between that of BCNU (6,14, 22, 27) and thus is advantaged with regard mitozolomide and other drugs (Table 1). Mitozolomide is cross- to its pharmacodynamics.7 Comparison of the activity of this new

CANCER RESEARCH VOL. 45 JULY 1985 3012

Table3 Activity of mitozolomideana other drugs in the National Cancer Institute murine tumor panel (28) 26 adeno- DrugMitozolomide leukemia>150leukemia-H->175melanomaInactive>150carcinomaInactive>150tumorInactivecarcinomaInactivemammary-H-

Nitrogen mustard BCNU DTIC Cisplatin Cyclophosphamide Methotrexate Inactive>150 AdriamycinT/C Inactive<42CD8F,

activity criteria:(++)L1210 >125P388 >120B16 >125LL >140Colon >130C38 <42

Table 4 carbamoyl-3-{2-chloroethyl)imidazo[5,1

CANCER RESEARCH VOL. 45 JULY 1985 3013

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. Experimental Antitumor Activity against Murine Tumor Model Systems of 8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1- d ]-1,2,3,5-tetrazin-4(3H)-one (Mitozolomide), a Novel Broad-Spectrum Agent

John A. Hickman, Malcolm F. G. Stevens, Neil W. Gibson, et al.

Cancer Res 1985;45:3008-3013.

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