Limited influence of UGT1A1*28 and no effect of UGT2B7*2 polymorphisms on UGT1A1 or UGT2B7 activities and protein expression in human liver microsomes

Abstract

What is already known about this subject • The UGT1A1*28 polymorphism is known to reduce UGT1A1 enzyme activities, via an extra TA repeat in the promoter. • However, a gap exists with regard to a comprehensive assessment of the influence of this genotype on variability in enzyme activity. • There is equivocal evidence on the functional relevance of the UGT2B7*2 polymorphism on UGT2B7 enzyme activities. What this study adds • Using comprehensive approaches to measure enzyme activities and protein expression levels, the UGT1A1*28 polymorphism is shown to contribute to only 40% of the variability in enzyme activities for UGT1A1. • A novel, nonproprietary method for genotyping UGT1A1*28 is provided. • Definitive evidence is provided to conclude there is no effect of the UGT2B7*2 polymorphism on zidovudine glucuronidation activity. Aims UGT1A1 and UGT2B7 are enzymes that commonly contribute to drug glucuronidation. Since genetic factors have been suggested to contribute to variability in activities and expression levels of these enzymes, a quantitative assessment of the influence of the major genotypes (UGT1A1*28 or UGT2B7*2) on enzyme activities was conducted. Methods Using a bank of microsomal samples from 59 human livers, the effect of UGT1A1*28 or UGT2B7*2 polymorphisms were investigated on rates of estradiol 3‐glucuronidation (a marker of UGT1A1 enzyme activity) or zidovudine glucuronidation (a marker of UGT2B7 enzyme activity) and levels of immunoreactive protein for each enzyme. Glucuronidation rates for both enzymes were measured at Km/S50 and 10 times Km/S50 concentrations. Results UGT1A1 and UGT2B7 enzyme activities varied up to 16‐fold and sixfold, respectively. Rates at Km/S50 concentration closely correlated with rates at 10 times Km/S50 concentration for both enzymes (but not at 1/10th Km for UGT2B7). Enzyme activities correlated with relative levels of immunoreactive protein for UGT1A1 and UGT2B7. Furthermore, rates of zidovudine glucuronidation correlated well with rates of glucuronidation of the UGT2B7 substrate gemcabene, but did not correlate with UGT1A1 enzyme activities. For the UGT1A1*28 polymorphism, consistent with levels of UGT1A1 immunoreactive protein, mean UGT1A1 activity was 2.5‐ and 3.2‐fold lower for TA6/TA7 (P < 0.05) and TA7/TA7 (P < 0.001) genotypes in comparison with the TA6/TA6 genotype. Conclusions Relative to the observed 16‐fold variability in UGT1A1 activity, these data indicate only a partial (approximately 40%) contribution of the UGT1A1*28 polymorphism to variability of interindividual differences in UGT1A1 enzyme activity. For the UGT2B7*2 polymorphism, genotype had no influence on immunoreactive UGT2B7 protein or the rate of 3'‐azido‐3'‐deoxythymidine glucuronidation.

Raloxifene Glucuronidation in Human Intestine, Kidney, and Liver Microsomes and in Human Liver Microsomes Genotyped for the UGT1A1*28 Polymorphism

“…To confirm this finding also by in vitro experiments, human liver microsomes genotyped for the UGT1A1*28 polymorphism were used. This in vitro system should provide a useful tool to allow a more comprehensive prediction of UGT1A1 metabolism and to gain an insight into the effects of the UGT1A1*28 polymorphism (Peterkin et al, 2007;Yoder Graber et al, 2007;Zhang et al, 2007). The influence of UGT1A1*28 polymorphism on glucuronidation of raloxifene to M1 and M2 was confirmed in our study and was especially prominent in the case of M1 formation, where UGT1A1 *1/*1, *1/*28, and *28/*28 differed significantly.…”

Genetic Polymorphisms in the TATA Box and Upstream Phenobarbital-Responsive Enhancer Module of the UGT1A1 Promoter Have Combined Effects on UDP-Glucuronosyltransferase 1A1 Transcription Mediated by Constitutive Androstane Receptor, Pregnane X Receptor, or Glucocorticoid Receptor in Human Liver

“…SNPs of Ϫ2353CϾT and Ϫ997GϾA have no effects on UGT1A1 mRNA levels in this liver sample population. These findings are consistent with previous reports on the effects of these genetic polymorphisms on UGT1A1 transcription (Ramírez et al, 2008), UGT1A1 activity and protein expression in liver microsomes (Peterkin et al, 2007), bilirubin metabolism (Sugatani et al, 2002), and drug metabolism of irinotecan (Innocenti et al, 2002;Kitagawa et al, 2005).…”

Effect of Rifampin and Rifabutin on the Pharmacokinetics of Lersivirine and Effect of Lersivirine on the Pharmacokinetics of Rifabutin and 25-O-Desacetyl-Rifabutin in Healthy Subjects

“…A formal analysis comparing lersivirine PKs between men and women has not been conducted; however, preliminary data suggest that the difference between gender and age is not significant, after adjusting for weight and race. These data and the fact that genetic variants of both CYP3A and UGT2B7, enzymes predominantly responsible for lersivirine metabolism, have been shown not to be of clinical relevance (6,13,14,25) suggest that the results of this study are generalizable to other populations.…”