Received for publication June 22, 2007.
Revised September 14, 2007.
Accepted for publication September 19, 2007.

Cyclic conversion of the Novel SRC Kinase Inhibitor TG100435 and its N-oxide metabolite by Flavin-containing Monoxygenases and Cytochromes P450 Reductase

Abstract

TG100435 is a novel multi-targeted Src family kinase inhibitor with demonstrated anti-cancer activity in preclinical species. Potent kinase inhibition is associated TG100435 and its major N-oxide metabolite (TG100855). The objectives of the current study were to identify the hepatic enzyme(s) responsible for (1) the total metabolic flux of TG100435, (2) the formation of TG100855 and (3) the subsequent metabolism of TG100855. Flavin-containing monooxygenases (FMO) and cytochrome P450 monooxygenases (CYP) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole suggesting FMO and only CYP3A4 mediated metabolism. TG100855 formation was markedly inhibited (~90%) by methimazole and heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retro-reduction of TG100855 back to TG100435 was observed. The N-oxidation reaction was approximately 15-times faster than the retro-reduction reaction. Interestingly, the retro-reduction of TG100855 to TG100435 in recombinant CYP or liver microsomes lacked inhibition by the CYP inhibitors. TG100435 formation in the human liver microsomes or recombinant CYP increased as a function of cytochrome P450 reductase activity suggesting potential involvement of cytochrome P450 reductase. The results of this in vitro study demonstrate the potential of TG100435 and TG100855 to be interconverted metabolically. FMO seem to be the major N-oxidizing enzymes while cytochrome P450 reductase appears to be responsible for the retro-reduction reaction.

Key words: cytochrome P450, flavin-containing monooxygenase, human CYP enzymes, microsomes