A humanized UGT1 mouse model expressing the UGT1A1*28 allele for assessing drug clearance by UGT1A1-dependent glucuronidation

Abstract

Humanized mice that express the human UDP-glucuronosyltransferase (UGT) 1 locus have been developed in a Ugt1-null background as a model to improve predictions of human UGT1A-dependent drug clearance. Enzyme kinetic parameters (K(m) and V(max)) and pharmacokinetic properties of three probe drugs were compared using wild-type and humanized UGT1 mice that express the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28)) Ugt1(-/-) mice]. The well characterized substrate for UGT1A1, 7-ethyl-10-hydroxy-camptothecin (SN-38), showed the greatest difference in parent drug exposure ( approximately 3-fold increase) and clearance ( approximately 3-fold decrease) in Tg(UGT1(A1*28)) Ugt1(-/-) mice after intravenous administration compared with wild-type and phenobarbital-treated animals. In contrast, the clearance of the UGT2B7 substrate (-)-17-allyl-4, 5alpha-epoxy-3, 14-dihydroxymorphinan-6-one (naloxone) was not altered in Tg(UGT1(A1*28)) Ugt1(-/-) mice. In addition, pharmacokinetic parameters with 1-(4-fluorophenyl)3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone (ezetimibe, Zetia; Merck & Co., Whitehouse Station, NJ), considered to be a major substrate for UGT1A1, showed small to no dependence on UGT1A1-directed glucuronidation. Enzyme kinetic parameters assessed for SN-38, ezetimibe, and naloxone using liver microsomes prepared from wild-type and Tg(UGT1(A1*28)) Ugt1(-/-) mice showed patterns consistent with the in vivo pharmacokinetic data. For SN-38 glucuronidation, V(max) decreased 5-fold in Tg(UGT1(A1*28)) Ugt1(-/-) mouse liver microsomes compared with microsomes prepared from wild-type mice, and decreased 10-fold compared with phenobarbital-treated Tg(UGT1(A1*28)) Ugt1(-/-) mice. These differences are consistent with SN-38 glucuronidation activities using HLMs isolated from individuals genotyped as UGT1A1*1 or UGT1A1*28. For ezetimibe and naloxone the differences in V(max) were minimal. Thus, Tg(UGT1(A1*28)) Ugt1(-/-) mice can serve as a pharmacokinetic model to further investigate the effects of UGT1A1 expression on drug metabolism.

Fig. 1.

Glucuronidation reactions investigated in this study.

Fig. 2.

Time courses of SN-38, ezetimibe, and naloxone in mice dosed intravenously (n of 2 mice/time point, and each data point shown represents the average of two determinations). Diamonds, wild-type C57BL/6 mice; squares, humanized UGT1 mice; triangles, humanized UGT1 mice with phenobarbital treatment.

Fig. 3.

Protein expression of UGT1A1 in Tg(UGT1^A1*28)Ugt1(−/−) male MLMs and HLMs. Mice were treated with phenobarbital at 100 mg/kg/day for 4 days. Left, control = Tg(UGT1^A1*28)Ugt1(−/−) mice without phenobarbital treatment; pb, Tg(UGT1^A1*28)Ugt1(−/−) mice with phenobarbital treatment. HLMs have been genotyped for expression of the UGT1A1*28 or UGT1A1*1 alleles.

Fig. 4.

Human UGT1A gene expression in Tg(UGT1^A1*28)Ugt1(−/−) mice treated with phenobarbital. Six age-matched humanized Tg(UGT1^A1*28)Ugt1(−/−) mice were treated intraperitoneally with either dimethyl sulfoxide (control) or phenobarbital (100 mg/kg) for 4 consecutive days. The day after the last dose the mice were sacrificed, and liver tissues were collected and pooled (n = 3). Liver total RNA was prepared, followed by reverse transcription-polymerase chain reaction to determine the expression levels of human UGT1A1, UGT1A3, UGTA4, UGT1A6, and UGT1A9.

Fig. 5.

Substrate concentration-dependent glucuronidation of SN-38 (A), ezetimibe (B), and naloxone (C) by HLM genotyped as UGT1A1*28/*28 (HH9 and HH81) and UGT1A1*1/*1 (HH83 and HH112) (n of 3 replicates/data points). Open circles, HH83; open triangles, HH112; open squares, HH9; open diamonds, HH81.

Fig. 6.

Substrate concentration-dependent glucuronidation of SN-38 (A), ezetimibe (B), and naloxone (C) by MLMs prepared from wild-type C57BL/6 mice (open circles), Tg(UGT1^A1*28)Ugt1(−/−) mice without phenobarbital treatment (open squares), and with phenobarbital treatment (open triangles) (n of 3 replicates/data points using pooled MLMs prepared from a minimum of three animals).