title: Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (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.
authors.affiliation: Department of Pharmacology and Anesthesiology, University of Padua, Largo Meneghetti 2, I-35131 Padua, Italy. luigi.quintieri@unipd.it
authors.forename: Luigi
authors.initials: L
authors.lastname: Quintieri
authors.forename: Cristina
authors.initials: C
authors.lastname: Geroni
authors.forename: Marianna
authors.initials: M
authors.lastname: Fantin
authors.forename: Rosangela
authors.initials: R
authors.lastname: Battaglia
authors.forename: Antonio
authors.initials: A
authors.lastname: Rosato
authors.forename: William
authors.initials: W
authors.lastname: Speed
authors.forename: Paola
authors.initials: P
authors.lastname: Zanovello
authors.forename: Maura
authors.initials: M
authors.lastname: Floreani
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D002250:Carbon Radioisotopes; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D019073:HT29 Cells; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008819:Mice, Nude; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: C502356:3'-deamino-3'',4'-anhydro-(2''-methoxy-3''-oxy-4''-morpholinyl)doxorubicin; D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; C066998:methoxy-morpholinyl-doxorubicin; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human