Human Liver Microsomal Cytochrome P450 3A Enzymes Involved in Thalidomide 5-Hydroxylation and Formation of a Glutathione Conjugate

Abstract

(R)-Thalidomide was oxidized to 5-hydroxythalidomide and 5’-hydroxythalidomide by NADPH-fortified liver microsomes from humans and monkeys. (R)-Thalidomide was hydroxylated more efficiently than (S)-thalidomide. Recombinant human P450s 3A4, 3A5, and 3A7 and monkey P450s 3A8 and 3A5 (co-expressed with NADPH-P450 reductase in bacterial membranes) also catalyzed (R)-thalidomide 5-hydroxylation. Purified human P450s 2C19, 3A4, and 3A5 mediated (R)-thalidomide 5-hydroxylation at similar rates in reconstituted systems. P450 2C19 showed a rather non-saturable substrate-velocity curve; however, P450s 3A4 and 3A5 showed sigmoidal curves. P450 also oxidized 5-hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed formation of a glutathione conjugate from (R)- and (S)-5-hydroxythalidomide, catalyzed by liver microsomal P450s 3A4 and 3A5 in the presence of glutathione (assigned as a conjugate of 5-hydroxythalidomide formed on the phenyl ring). These results indicate that human P450s 3A4 and 3A5 mediate thalidomide 5-hydroxylation and further oxidation leading to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide.

Characterization of SfmD as a Heme Peroxidase That Catalyzes the Regioselective Hydroxylation of 3-Methyltyrosine to 3-Hydroxy-5-methyltyrosine in Saframycin A Biosynthesis

“…In Vitro Enzymatic Assays of SfmD-The assay conditions used to test whether SfmD is a heme-containing oxygenase are similar to the assay conditions that reported for heme-containing oxygenases in the presence of oxygen, such as conditions for cytochrome P450 (18), secondary amine oxygenase (19), heme oxygenase (20), prostaglandin H Synthase (21), and tryptophan 2, 3-dioxygenase TioF (22).…”

Thalidomide Increases Human Hepatic Cytochrome P450 3A Enzymes by Direct Activation of the Pregnane X Receptor

“…11,12 Also, the second oxidation step involves a reactive intermediate, possible an arene oxide that can be trapped by GSH to give GSH adducts. 13 Two aspects of in vivo drug interaction of thalidomide were reported: 14 an enhanced clearance of midazolam and a higher area under the curve of 4 hydroxymidazolam following pre treatment with thalidomide in humanized liver mice, presumably due to human P450 3A induction. Although induction of total P450 contents by thalidomide in rat livers have been reported, 15 apparently no interaction of thalidomide has been shown with ethinyl estradiol (P450 3A4 substrate and inhibitor) in humans.…”

Whole embryo culture: a “New” technique that enabled decades of mechanistic discoveries

“…Although the limb buds of both species appeared grossly normal, in situ hybridization revealed that the limb differentiation factor FGF-10 was severely downregulated in rabbit embryos, but not rat embryos, exposed to equivalent concentrations of thalidomide in WEC. Recent identification of a thalidomide-GSH conjugate in human liver microsomes (Chowdhury et al, 2010) provides additional support for the validity of the Hansen et al (1999) WEC findings in the mechanism of thalidomide-induced teratogenicity.…”