With NADPH as the electron donor, rat liver NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) catalyzes the single-electron reduction of several quinone antibiotics to a semiquinone or free radical state. The benzanthraquinones adriamycin, daunorubicin, carminomycin, 7-O-methylnogalarol, and aclacinomycin A and the N-heterocyclic quinones streptonigrin and mitomycin C are activated to free radical intermediates which can transfer their single electron to molecular oxygen to form superoxide. The overall Km range for this electron transfer is 0.4 to 42.1 X 10-4 M. We postulate that the formation of the "site-specific free radical" intermediate is central to the cytotoxic action of these antibiotics.An unusually high proportion of anticancer agents contain quinone groups. Such quinone-containing anticancer agents include adriamycin, daunorubicin, mitomycin C, streptonigrin, lapachol, and analogs of these agents. Although investigators have identified several potential biochemical sites of action for these drugs, preponderant evidence indicates that the agents act primarily by interfering with DNA and RNA replication(1-4).We have established that these quinone agents are catalytically activated to a free radical state by a microsomal system requiring NADPH as an electron donor (5, 6). Normal microsomes as well as microsomes from murine leukemia cells catalyze augmented oxygen consumption with the quinone antibiotics, indicative of free radical formation. By using effectors of this microsomal system we obtained indirect evidence that a flavoprotein catalyst of the microsomes was involved and that cytochrome P-450 was not. Iyanagi and Yamazaki (7) have shown that NADPH cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) reduces quinone substrates to semiquinones; and Goodman and Hochstein (8) reported that NADPH cytochrome P-450 reductase catalyzes production of superoxide from adriamycin. In the present paper, we describe and characterize the catalysis of drug free-radical formation bhey -tochrome P-450 reductase from rat-iver microsomes and propose a mechanism for the cytotoxic action of these agents.METHODS AND MATERIALS Adriamycin, N-dimethyladriamycin, daunorubicin, N-acetyldaunorubicin, 7-iminodaunorubicin, aclacinomycin A, mitomycin C, streptonigrin, and lapachol were kindly provided by the Drug Synthesis and Chemistry Branch and the Natural Products Branch (Division of Cancer Treatment, National Cancer Institute, National Institutes of Health). Daunorubicinol (9) and adriamycin (Fe3+)4 (10) were prepared as described. Carminomycin was provided by G. F. Gause (Institute of New Antibiotics, Moscow). Rubidazone was supplied by Rene Maral (Rhone-Poulenc, Vitry-Sur-Seine, France). Nogalamycin, 7-O-methylnogarol, and steffimycin were provided by G. Neil (Upjohn Co.); and N-trifluoroacetyladriamycin-14-valerate (AD-32) and N-trifluoroacetyladriamycin (AD-41) were supplied by M. Israel, (Farber Cancer Center, Boston, MA).NADPH cytochrome P-450 reduct...
A study designed to correlate clinical and pharmacologic observations was undertaken in 96 patients treated with adriamycin. The basic dosage schedule was 60 mg/m2 I.V. q 3 weeks. Pharmacokinetic studies showed a prolonged plasma half-life, low urinary excretion, and undetectable levels in CSF. Patients with significantly impaired liver function had marked elevation and prolongation of plasma drug levels associated with severe toxicity unless dosage was reduced by 50-7596. Of the 82 evaluable patients, 10/25 with sarcomas, 9/31 with carcinomas, and 15/26 with hematologic malignancies have achieved complete or partial remission. An additional 22/48 have improved. Six patients with solid tumors had progressive CNS disease while responding systemically. Adriamycin can he used with relative safety and high efficacy in a dosage schedule that resulted from pharmacologic studies. Dosage reduction in patients with liver disease is essential to avoid life-threatening toxicity.
This laboratory has reported previously that a cytoplasmic extract of weanling or regenerating adult rat liver (but not normal rat liver) will produce a 2.5-fold increase in the incorporation of tritiated thymidine ([3H]dThd) into liver DNA of a 34%-hepatectomized test animal. (J. Physiol. London 248: 273-284, 1975). The present study showed that hepatic stimulator substance (HSS) will stimulate DNA synthesis in normal adult rats and CF1 mice as well. The increased incorporation of [3H]dThd into DNA produced in the normal, nonhepatectomized adult rat was comparable with that induced by a 34% hepatectomy. Autoradiographic studies revealed that the [3H]dThd was incorporated into nuclear DNA and that the stimulation occurred almost exclusively in parenchymal cells. HSS was shown to be heat stable (100 degrees C for 15 min) and was precipitated but not inactivated by alcohol. Ultrafiltration and dialysis studies suggested a molecular weight slightly greater than 10,000. HSS proved to be organ specific, stimulating the liver but not the kidney, bone marrow, or spleen. HSS was found to contain no insulin, glucagon, epidermal growth factor, or peptides of the nonsuppressible insulinlike/multiplication-stimulating activity (somatomedin) group.
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