The metabolism of vandetanib, a tyrosine kinase inhibitor used for treatment of symptomatic/progressive medullary thyroid cancer, was studied using human hepatic microsomes, recombinant cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). The role of CYPs and FMOs in the microsomal metabolism of vandetanib to N-desmethylvandetanib and vandetanib-N-oxide was investigated by examining the effects of CYP/FMO inhibitors and by correlating CYP-/FMO-catalytic activities in each microsomal sample with the amounts of N-desmethylvandetanib/vandetanib-N-oxide formed by these samples. CYP3A4/FMO-activities significantly correlated with the formation of N-desmethylvandetanib/ vandetanib-N-oxide. Based on these studies, most of the vandetanib metabolism was attributed to N-desmethylvandetanib/vandetanib-N-oxide to CYP3A4/FMO3. Recombinant CYP3A4 was most efficient to form N-desmethylvandetanib, while FMO1/FMO3 generated N-oxide. Cytochrome b5 stimulated the CYP3A4-catalyzed formation of N-desmethylvandetanib, which is of great importance because CYP3A4 is not only most efficient in generating N-desmethylvandetanib, but also most significant due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib. Indeed, the CYP3A4-mediated reaction exhibits kinetics of positive cooperativity and this corresponded to the in silico model, where two vandetanib molecules were found in CYP3A4-active center.

• Keywords
• cytochromes P450, flavin-containing monoxygenases, metabolism, tyrosine kinase inhibitor, vandetanib,
• MeSH
• Quinazolines chemistry   pharmacology   MeSH
• Cytochrome P-450 CYP3A chemistry   metabolism   MeSH
• Enzymes chemistry   metabolism   MeSH
• Protein Kinase Inhibitors chemistry   pharmacology   MeSH
• Microsomes, Liver metabolism   MeSH
• Rabbits MeSH
• Rats MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Mice MeSH
• Oxidation-Reduction * MeSH
• Piperidines chemistry   pharmacology   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Recombinant Proteins MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Quinazolines MeSH
• Cytochrome P-450 CYP3A MeSH
• Enzymes MeSH
• Protein Kinase Inhibitors MeSH
• Piperidines MeSH
• Antineoplastic Agents MeSH
• Recombinant Proteins MeSH
• vandetanib MeSH Browser

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.

• Keywords
• Cytochrome P450, Cytochrome b(5), DNA Adducts, Metabolism, Mouse models,
• MeSH
• DNA Adducts metabolism   MeSH
• Aryl Hydrocarbon Hydroxylases metabolism   MeSH
• Cytochrome P-450 CYP3A MeSH
• Cytochrome-B(5) Reductase deficiency   genetics   MeSH
• Cytochromes b5 deficiency   genetics   MeSH
• Ellipticines metabolism   pharmacology   MeSH
• Phenotype MeSH
• Genotype MeSH
• Hepatocytes enzymology   MeSH
• Microsomes, Liver enzymology   MeSH
• Liver enzymology   MeSH
• Activation, Metabolic MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• NADPH-Ferrihemoprotein Reductase metabolism   MeSH
• Antineoplastic Agents metabolism   pharmacology   MeSH
• Cytochrome P-450 Enzyme System metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Aryl Hydrocarbon Hydroxylases MeSH
• CYP3A protein, mouse MeSH Browser
• Cytochrome P-450 CYP3A MeSH
• Cytochrome-B(5) Reductase MeSH
• Cytochromes b5 MeSH
• Ellipticines MeSH
• ellipticine MeSH Browser
• NADPH-Ferrihemoprotein Reductase MeSH
• Antineoplastic Agents MeSH
• Cytochrome P-450 Enzyme System MeSH

ABSTRACT: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase.

• Keywords
• Coenzymes, DNA, Enzymes, High pressure liquid chromatography,
• Publication type
• Journal Article MeSH