(CANCER RESEARCH 45, 1778-1786, April 1985] Activity of Mitozolomide (NSC 353451), a New Imidazotetrazine, against Xenografts from Human Melanomas, Sarcomas, and Lung and Colon Carcinomas Oy stein Fodstad, ' Steina r Aamdal,2 Alexander Pihl, and Michael R. Boyd Norsk Hydros Institute for Cancer Research, Montebello, Oslo 3, Norway [0. F., S. Aa., A. P.] and Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205 [0. F., M. R. B.¡ ABSTRACT In this investigation, we first tested the anticancer activity of mitozolomide against cells from different xenografted cancers in The chemosensitivity of human tumor xenografts to mitozo- a HTCF assay in vitro. When a pronounced inhibition of colony lomide, 8-carbamoyl-3-{2-chloroethyl)imidazo[5-1 -d]-1,2,3,5-te- formation was observed, we next examined in the HTCF assay trazin-4(3H)-one, was studied in 3 different assay systems. In the efficiency of the drug on a panel of tumors for each histolog concentrations of 1 to 500 ¿/g/ml,mitozolomide completely inhib ical type. Since in vitro test systems have inherent limitations ited the colony-forming ability in soft agar of cell suspensions (23), we also measured the in vivo effect of mitozolomide on the from sarcomas, melanomas, lung and colon cancers, and a same tumors, using the 6-day subrenal capsule assay in immu- mammary carcinoma. When a panel of tumors of the different nocompetent mice (1-3), as well as s.c. growing tumors in histological types was tested for its sensitivity to mitozolomide athymic, nude mice (5-7, 10, 16, 18). In all 3 assay systems, in vitro, in the 6-day subrenal capsule assay in conventional mice, marked anticancer activity against human melanomas, sarco and, in some cases, as s.c. growing tumors in nude mice, good mas, lung cancers, and colon cancers was observed. agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. MATERIALS AND METHODS The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor Animals size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, and an Male C57BL/6 x DBA/2 F, (hereafter called B6D2F,) mice, 6 to 12 osteosarcoma were cured of their tumors. The approach here weeks old, were used for the subrenal capsule assay. BALB/c athymic, used for evaluating the effect of a new drug on human cancers nude mice were purchased from the Laboratory Breeding and Research may be useful for selecting the tumor types which primarily Center, Gl, Bomholtgaard, Ry, Denmark. NMRI nude mice were bred in our own nude mouse facility. The nude mice were housed in laminar-air should be studied in clinical trials. The results indicate that clinical flow rooms at constant temperature (24-26 °C)and humidity (30 to responses to mitozolomide may be anticipated in sarcoma, mel 50%). The cages and bedding for the nude mice were sterilized by anoma, small cell lung cancer, and possibly in colon cancer. autoclaving, and the food by 7-irradiation. Tumors INTRODUCTION Altogether, 18 human tumor lines were used, all of which were In a systematic study of imidazotetrazines, a novel agent, maintained as s.c. growing xenografts in athymic, nude mice (NMRI or mitozolomide3 (previously Azolastone; NSC 353451 ), with potent BALB/c strain). Two lung cancer lines (H-249 and N-417) were kindly activity against murine tumors, has been discovered by the provided by Desmond N. Carney, National Cancer Institute-Navy Medical Cancer Research Campaign Experimental Chemotherapy Group, Oncology Branch, Bethesda, MD. One lung adenocarcinoma, T 402, was University of Aston, Birmingham, United Kingdom (8, 9, 22). The obtained from Nathan 0. Kaplan, University of California, San Diego, CA. mechanism of action of mitozolomide has not yet been eluci Of the 2 colon cancers, Co-115 was obtained from B. Sordat, Epalinges/ dated, but evidence has been presented that the formation of Lausanne, Switzerland, and one, WiDr, as well as the mammary cancer interstrand DNA cross-links may be involved (8, 9,12). line MDA-MB 231, were obtained from American Type Culture Collection, Rockville, MD. The other tumors (5 melanoma, 2 osteosarcoma, 3 soft In view of the promising effects of mitozolomide in murine tissue sarcoma, and 2 lung cancer lines) were established in this labo systems (11, 22), we initiated experiments to see whether mito ratory from tumor biopsies of patients at the Norwegian Radium Hospital. zolomide might be active also against human cancers and, if so, to establish which of the tumor types examined were most Drugs sensitive, to aid in the planning of subsequent clinical trials. Mitozolomide (Chart 1) was a gift from M. G. Stevens, Cancer Re ' To whom requests for reprints should be addressed, at Norsk Hydros Institute search Campaign, Experimental Chemotherapy Group, Department of for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Pharmacy, University of Aston in Birmingham, United Kingdom. Abrin Norway 2 Fellow of the Norwegian Cancer Society. was prepared in our laboratory (15). The other drugs were commercial 'The abbreviations used are: mitozolomide, 8-carbamoyl-3-(2-chloro- preparations obtained from the Department of Pharmacy, The Norwegian ethyl)imidazo|5.1-ci]-1.2.3,5-tetrazin-4(3H)-one (also designated NSC 353451, and Radium Hospital. MSB 39565); CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CDDP, c/s-di- Mitozolomide was dissolved in a minimal amount of dimethyl sulfoxide chlorodiammineplatinum (II); ADM, doxorubicin; HTCF, human tumor colony-form and kept refrigerated in glass tubes. Immediately before use, the drug ing; SRC, subrenal capsule; FVR, fractional volume reduction; SCLC, small cell lung carcinoma. was diluted in 0.9% NaCI solution (saline) to appropriate concentrations Received 10/5/84; revised 12/26/84; accepted 12/28/84. and injected into the animals in volumes of 0.2 ml. CCNU was dissolved CANCER RESEARCH VOL. 45 APRIL 1985 1778 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. ACTIVITY OF MITOZOLOMIDE AGAINST HUMAN TUMORS CONH, Xenografts in Athymic Mice. The nude mouse experiments were carried out as described previously (5, 6). Mice with progressively grow ing s.c. xenografts were divided into groups of 8 to 10 animals each. N When the tumors had reached a mean diameter of 6 to 8 mm, the animals were treated with the drugs in the doses indicated in the legends to charts. The maximum tolerable dose of mitozolomide, given i.v. 3 to 4 times on a once-weekly schedule, was found to be 40 mg/kg/dose. The tumor size was assessed by measuring twice weekly 2 perpen Chart 1. Molecular formula of mitozolomide. dicular diameters. By subtracting, for each diameter, the value at the start of treatment, the mean tumor diameter differences (±SE), were in Cremophor EL (Sigma) and injected i.v. into B6D2F, mice and i.p. into calculated (6). The results are presented as growth curves where the nude mice, whereas the other drugs were used as aqueous solutions mean tumor diameter difference for each treated and untreated group is and given i.v. in volumes of 0.2 to 0.4 ml. plotted versus time. At the start of treatment this value is by definition zero. Assay of Anticancer Activity The effect of treatment was expressed numerically as the FVR (21), defined by the expression: HTCF Assay. Human tumors serially transplanted in athymic, nude mice were mechanically disaggregated by the use of a Stomacher 80 mean Vc FVR = logio Lab-Blender (Seward Surgical, London, United Kingdom). For the sar mean V, comas, an enzymatic procedure (digestion with collagenase, 1 mg/ml, and 1000 IU hyaluronidase, 37 °Cfor 1 h) was included before the Vc and V, are the relative tumor volumes in the control group and the mechanical disaggregation. The cell suspensions were filtered through a treated group, respectively, at a given time (usually the time when the 4- to 5-^m nylon mesh, centrifuged, and resuspended in serum-contain tumor diameter difference between control and treated tumors was ing Ham's F-10 medium. The cultivation in soft agar and the chemosen- maximal). FVR gives the number of decades by which the treated tumor sitivity testing were performed by a modification of the procedure of volume is reduced below the control value. Thus, e.g., a FVR value of Courtenay and Mills (4) as described previously (23). The cells were 1.0 implies that the mean volume of the treated tumors is reduced to incubated for 1 h with 4 different drug concentrations. Triplicate cultures one-tenth of that of the control tumors (21). The tumor volume was were prepared in culture tubes containing rat RBC, and the tubes were calculated according to the formula: incubated in an atmosphere containing 5% O2, 5% CO2, and 90% N2. After incubation for 5 days, 2 ml of serum-containing medium were added 7T/6(MDf to each tube, and the incubation was continued for another 1 to 3 weeks. Colonies of more than 30 cells were scored by means of a Zeiss stereo where MD is the mean tumor diameter (6). microscope. The plating efficiency was defined as the number of colonies formed as a percentage of the number of viable cells plated. Four different concentrations of each compound, over a 3-log range, RESULTS were used. Since the 50% lethal dose value of mitozolomide after a single i.v. dose was found to be 80 mg/kg, i.e., 5 times that of ADM, we Initial Screening.