MX2, a Morpholino Anthracycline, as a New Antitumor Agent against Drug- sensitive and Multidrug-resistant Human and Murine Tumor Cells1

MX2, a new morpholino anthracycline, showed similar or superior chemotherapeutic effects to Adriamycin (ADM) against several experi mental murine tumors, i.v. administration of MX2 against L1210-bearing mice induced a prolongation of life-span by twice or more compared to ADM. MX2 was equally or slightly more effective against Lewis lung carcinoma and colon adenocarcinomas 26 and 38 than ADM when either drug was given i.v. The antitumor activity of MX2 against human tumor xenografts was similar to that of ADM, and the compound was effective against three out of four gastric adenocarcinomas, one out of two nonsmall-cell lung carcinomas, and two out of two mammary adenocarcino mas. In particular, this compound exhibited a marked effect against MX1, a human mammary adenocarcinoma. MX2, in contrast to ADM, was effective against sublines of P388 leukemia resistant to ADM or aclacinomycin A in vivo as well as in vitro. A maximum percentage increase in life-span of about 90% was obtained in mice bearing these resistant tumors. MX2 is a unique anthracycline antibiotic effective on drugsensitive as well as multidrug-resistant murine and human cells. INTRODUCTION MX2, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin, is a new morpholino anthracycline, and its parent compound is obtained from the culture broth of strain RNM134-13, a blocked mutant of Actinomadura roseoviolacea 1029-AV1. It is modified at the amino sugar of its 3' position by morpholino derivation (1). This compound shows a similar antitumor activity to ADM3 against i.p.-inoculated P388 leu kemia by i.v. administration, and exhibits nearly equal activity by p.o. administration to that obtained by i.v. administration (1, 2). MX2 induced the same level of myelosuppression as ADM at the same doses during 28-day repeated injection using a rat model4. The subacute cardiocytotoxicity, however, was found to be much weaker than that of ADM using a rabbit model.5 The major dose limiting factor of MX2 seems to be a myelosuppression, but not a cardiocytotoxicity. MX2 may be used for longer time than ADM. In this study, we examined the antitumor activities of MX2 against various experimental murine tumors and human tumor xenografts. MX2 showed a superior activity to ADM against LI210 leukemia, Lewis lung carcinoma, and colon adenocar cinomas 26 and 38. This drug also showed a comparable activity to ADM against human tumor xenografts. We have also ex amined the antitumor activity of MX2 against drug-resistant Received 5/2/88: revised 8/19/88; accepted 8/29/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education. Science and Culture, Japan. 2To whom requests for reprints should be addressed. 'The abbreviations used are: ADM, Adriamycin; ACR. aclacinomycin A; MMC. mitomycin C: P388/ADM, P388 leukemia resistant to Adriamycin: P388/ ACR. P388 leukemia resistant to aclacinomycin A; P388/MMC, P388 leukemia resistant to mitomycin C; PBS, Dulbecco's phosphate-buffered saline; HBSS, Hanks' balanced salt solution; ILS, percentage increase in life-span; /<",„. concen tration of drug necessary to reduce growth by 50%. ' Unpublished data. 5Abstract of the 45th Annual Meeting of the Japanese Association for Cancer Research. Sapporo, October, 1986. P388 leukemias in vitro and in vivo. MX2 was effective against P388/ADM, P388/ACR, and P388/MMC in vitro and in vivo, whereas ADM was not effective against P388/ADM and P388/ ACR, and was inferior to MX2 against P388/MMC in vivo. MX2 seems to warrant consideration for further development as a new chemotherapeutic agent. MATERIALS AND METHODS Drugs. MX2 was prepared as described previously (1) and its hydrochloride was used in this study. The structure of MX2-HCI is shown in Fig. 1. ADM and MMC formulated for clinical use were obtained from Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. ACR was from Sanraku Co., Ltd., Tokyo, Japan. Drugs were dissolved in 0.9% NaCl solution. Animals. Male DBA/2Cr and C57BL/6J, and female BALB/c, C57BL/6J x DBA/2Cr F, (hereafter called BD2F,) and BALB/c x DBA/2Cr Fi (hereafter called CD2F|) mice were obtained from Charles River Japan, Inc., Tokyo, Japan. Female BALB/c-nu/n«/athymic nude mice were obtained from Nihon Clea Inc., Tokyo, Japan. Murine Tumors. P388 leukemia, L1210 leukemia, Lewis lung carci noma, and colon adenocarcinomas 26 and 38 were kindly provided by the National Cancer Institute, NIH, Bethesda, MD. These tumors were maintained according to the protocol of the National Cancer Institute. LI210 leukemia was maintained in male DBA/2Cr mice; colon ade nocarcinoma 26 was maintained in female BALB/c mice; and Lewis lung carcinoma and colon adenocarcinoma 38 were maintained in male C57BL/6J mice. The subline of P388 leukemia resistant to ADM (P388/ADM) was obtained from the National Cancer Institute, NIH, Bethesda, MD. The sublines of P388 leukemia resistant to ACR (P388/ACR) and MMC (P388/MMC) were kindly provided by Dr. M. Inaba in this center. These three tumors were maintained in female CD2F| mice. Evaluation of Antirumor Activity against Murine Tumors. A sample, 0.1 ml, of cell suspension in PBS containing 10* LI 210 leukemia cells was inoculated i.p. or i.v. into female CD2F| mice on Day 0. MX2 and ADM were given i.v. on Days 1, 5, and 9. Antitumor activity was determined by comparing the mean survival time of the test group (T) with that of the control group (C) and was expressed as an increase in life-span [(T/C-1) x 100%]. Tumor-free survivors, which had no tumor macroscopically or histopathologically in abdominal cavity, in blood, or at the inoculation site on Day 60, were excluded from calculation. Tumor fragments (2x2x2 mm) of Lewis lung carcinoma and colon adenocarcinoma 26 were implanted s.c. in the flank of female BD2Fi mice and CD2F| mice on Day 0, respectively. A portion, 0.2 ml, of tumor brei of colon adenocarcinoma 38, prepared by passing the tumor pieces through an 18-gauge needle and diluted in HBSS (33% w/v) was inoculated s.c. into the flank of female BD2I , mice on Day 0. MX2 and ADM were given i.v. on Days 1, 5, and 9 for Lewis lung carcinoma or on Days 1, 8, 15, and 22 for colon adenocarcinoma 26 and 38. Tumor diameters were measured with calipers on Day 14 for Lewis lung carcinoma, on Day 13 for colon adenocarcinoma 26, or on Day 21 for colon adenocarcinoma 38. The tumor volume (r) was calculated as follows: v = '/iaft2, where a and b are long and short diameter of the tumor mass in mm. Antitumor activity was determined by: (a) the percentage mean tumor volume compared to controls taking controls as 100%; and (b) ILS calculated by the median survival time in com parison to controls. Cell suspension, 0.2 ml, in HBSS containing IO6cells of the sublines of resistant P388 leukemia cells was inoculated into female CD2Fi 6653 on April 12, 2017. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from ANTITUMOR ACTIVITY OF MX2