Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Purpose: Nemorubicin (3′-deamino-3′-[2″( S )-methoxy-4″-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3′-deamino-3″,4′-anhydro-[2″( S )-methoxy-3″( R )-oxy-4″-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. Experimental Design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P 450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro -cultured human tumor cell lines and tumor-bearing mice, respectively. Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro , and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. Conclusions : CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo .