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.

Department of Analytical Environmental and Forensic Sciences MRC PHE Centre for Environment and Health King's College London 150 Stamford Street London SE1 9NH UK

Department of Biochemistry Faculty of Science Charles University Albertov 2030 CZ 12843 Prague 2 Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 CZ 61300 Brno Czech Republic

Department of Oncology 2nd Medical Faculty Charles University and University Hospital Motol 5 Uvalu 84 1 CZ 150 06 Prague 5 Czech Republic

Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 1 CZ 150 06 Prague 5 Czech Republic

NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in partnership with Public Health England and Imperial College London 150 Stamford Street London SE1 9NH UK

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Apoferritin/Vandetanib Association Is Long-Term Stable But Does Not Improve Pharmacological Properties of Vandetanib

Identification of Enzymes Oxidizing the Tyrosine Kinase Inhibitor Cabozantinib: Cabozantinib Is Predominantly Oxidized by CYP3A4 and Its Oxidation Is Stimulated by cyt b5 Activity