Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4.
Modulation of UDP-glucuronosyltransferase 2B7 (UGT2B7)-catalyzed morphine glucuronidation by cytochrome P450 (P450) was studied. The effects of P450 isozymes on the kinetic parameters of UGT2B7-catalyzed glucuronidation of the morphine 3-hydroxyl group were examined by simultaneous expression of UGT2B7 and either CYP3A4, -1A2, or -2C9 in COS-1 cells. Although coexpression of CYP3A4 with UGT2B7 had little effect on Vmax, the Km was increased by about 9.8-fold compared with the UGT2B7 single expression system. The other P450 isozymes (CYP1A2 and CYP2C9) had some effects on Km and Vmax values. Immunoprecipitation of UGT from solubilized human liver microsomes resulted in coprecipitation of CYP3A4 with UGT2B7. The protein-protein interaction between CYP3A4 and UGT2B7 was further confirmed by overlay assay using glutathione S-transferase-CYP3A4 fusion protein. Addition of CYP3A4 to untreated COS microsomes expressing UGT2B7 had no or minor effects on morphine glucuronidation. In contrast, the formation of morphine-3-glucuronide by detergent-treated microsomes from COS-1 cells expressing UGT2B7 was reduced by CYP3A4, whereas the formation of the 6-glucuronide was enhanced. These results strongly suggest that 1) the glucuronidation activity of UGT2B7 toward morphine is specifically modulated by interaction with CYP3A4 in microsomal membranes and that 2) CYP3A4 alters UGT2B7 regioselectivity so that the ratio of morphine activation/detoxication is increased. This study provides the first evidence that P450 is not only involved in oxidation of drugs but also modulates the function of UGTs.
Takeda
Shuso
S
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Ishii
Yuji
Y
Iwanaga
Megumi
M
Mackenzie
Peter I
PI
Nagata
Kiyoshi
K
Yamazoe
Yasushi
Y
Oguri
Kazuta
K
Yamada
Hideyuki
H
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D009020:Morphine / Q000493:pharmacokinetics*; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D020719:Glucuronides; D011993:Recombinant Fusion Proteins; D009020:Morphine; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase