Cyclic conversion of the novel Src kinase inhibitor [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) and Its N-oxide metabolite by flavin-containing monoxygenases and cytochrome P450 reductase.
[7-(2,6-Dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) is a novel multi-targeted Src family kinase inhibitor with demonstrated anticancer activity in preclinical species. Potent kinase inhibition is associated with TG100435 and its major N-oxide metabolite [7-(2,6-dichlorophenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine (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 (P450s) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole, suggesting only FMO- and CYP3A4-mediated metabolism. TG100855 formation was markedly inhibited (~90%) by methimazole or heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retroreduction of TG100855 back to TG100435 was observed. The N-oxidation reaction was approximately 15 times faster than the retroreduction reaction. Interestingly, the retroreduction of TG100855 to TG100435 in recombinant P450 or liver microsomes lacked inhibition by the P450 inhibitors. TG100435 formation in the human liver microsomes or recombinant P450 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, whereas cytochrome P450 reductase seems to be responsible for the retroreduction reaction.
Kousba
Ahmed
A
Department of Pharmaceutical Property Assessment, TargeGen Inc, San Diego, CA 92121, USA. akousba@targegen.com
Soll
Richard
R
Yee
Shiyin
S
Martin
Michael
M
D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008713:Methimazole / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011759:Pyrrolidines / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014227:Triazines / Q000378:metabolism* / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
C522500:(7-(2,6-dichlorophenyl)-5-methylbenzo(1,2,4)triazin-3-yl)-(4-(2-(1-oxypyrrolidin-1-yl)ethoxy)phenyl)amine; C522499:(7-(2,6-dichlorophenyl)-5-methylbenzo(1,2,4)triazin-3-yl)-(4-(2-pyrrolidin-1-ylethoxy)phenyl)amine; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011759:Pyrrolidines; D011994:Recombinant Proteins; D014227:Triazines; D008713:Methimazole; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D019061:src-Family Kinases; D007654:Ketoconazole