7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.

ABSTRACT:(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R-and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R-and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Oxazepam is a 1,4-benzodiazepine derivative that is used in clinical practice for its anxiolytic, sedative, and anticonvulsant effects (Greenblatt et al., 1980(Greenblatt et al., , 1981. In humans, this drug is cleared from the body almost exclusively by hepatic glucuronidation, followed by urinary excretion (Abernethy et al., 1983). Oxazepam is formulated as a racemic preparation of S-and R-stereoisomers although the S-enantiomer is thought to be much more active as a benzodiazepine receptor agonist compared with the R-enantiomer (Mohler et al., 1978). Conjugation occurs via the hydroxyl group attached to the asymmetric 3-carbon position yielding diastereomeric glucuronides that are readily separated by routine high pressure liquid chromatography (HPLC 1 ) (Mascher et al., 1984;Patel et al., 1995a).Interindividual variability in the pharmacokinetics and metabolism of (R,S)-oxazepam have been investigated in human volunteers (Patel et al., 1995a). S-Oxazepam glucuronide was found to be formed preferentially over R-oxazepam glucuronide with S/R glucuronide diastereomeric ratios in the plasma and urine of volunteers averaging 3.5 Ϯ 0.6 and 3.9 Ϯ 0.8, respectively. Interestingly, in 2 of 11 subjects (18%), the S/R ratio in the urine was relatively low (Ͻ1.9). Since the plasma clearance of oxazepam in these individuals was also very low (Ͻ0.6 ml/min/kg) compared with other individuals (0.9 -1.4 ml/min/ kg), it was concluded that these differences probably were the result of slower S-oxazepam clearance by glucuronidation in a significant minority of the study population (i.e., a "slow metabolizer" phenotype). Although pharmacodynamic measurements were not made, the relatively slow elimination of oxazepam would be expected to result in prolonged sedation in these individuals.In vitro studies using human liver microsomes (HLMs) showed a similar picture in that S-oxazepam glucuronide was the predominant metabolite (S/R ratios averaging 4.0), and 4 of 37 livers displayed relatively slow oxazepam glucuronidation activities coinciding with low S/R metabolite ratios (Ͻ2.0) (Patel et al., 1995a). Enzyme kinetic analysis showed that the low glucuronidation activity was associated with higher apparent K m values and lower V max values for S-oxazepam glucuronidation in the four atypical livers compared with ...

UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3 end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99 -amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that isoform 2 is differentially expressed in liver, kidney, colon, and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions. Conclusion: Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway. (HEPATOLOGY 2007;45:128-138.)

The UGT1A isoforms 2 act as negative modulators of their isoform 1 homologs in microsome preparations, revealing a new regulatory mechanism of the glucuronidation pathway. Findings further provide the first direct evidence of a novel alternative splicing mechanism at the 3' end of the UGT1 locus that further increases the number of proteins derived from this single gene.