The purpose of this study was to generate, by real-time PCR, a quantitative expression level profile of the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A and 2B, in 26 adult and 3 fetal tissues, for better understanding of their roles in xenobiotic and endobiotic metabolism. Adult liver contained the highest level of combined UGTs mRNA, and UGT2B4 was the most abundant isoform in this tissue (40% of total). Other well expressed hepatic UGTs, in decreasing order of mRNA level, were 1A9, 2B7, 1A4, 2B10, 1A1, 1A6, 2B11, 2B15, 1A3, 2A3, 2B17 and 2B28. UGT2B4 was by far the most abundant isoform in the fetal liver (90% of total). The combined UGT mRNA expression in both adult and fetal olfactory epithelium was high, about 20% the adult hepatic level. Interestingly, a large developmental change was found in this tissue from high UGT2A1 and UGT2A2 expression in the fetus to UGT1A6 in the adult. The most abundantly expressed UGTs in the small intestine were 2A3, 1A10, 1A1, 1A6 and 2B7, while 1A10 and 2A3 predominated in the colon. The results provide the most comprehensive data to date on the tissue distribution of the human UGTs.

Bupropion is primarily metabolized in human liver by cytochrome P450 (CYP) 2B6, an isoform that shows high interindividual variability in expression and catalysis. The aim of this study was to identify mechanisms underlying this variability through comprehensive phenotype-genotype analysis of a well-characterized human liver bank (n = 54). There was substantial variability in microsomal bupropion hydroxylation activities (over 45-fold) and CYP2B6 protein content (over 288-fold), with excellent correlation between protein and activity values (rs = 0.88). CYP2B6 mRNA levels showed less variability (13-fold) and poorer correlation (rs = 0.44) to CYP2B6 protein resulting from 20-30% of livers that contained substantial CYP2B6 mRNA, but low CYP2B6 protein. Livers were genotyped for the common coding polymorphisms (Q172H, K262R and R487C) and 14 additional variations identified by sequencing of the gene promoter to -3000 bp. Of 14 haplotypes that were inferred, *1A (reference), *1H (-2320t>c; -750t>c) and *6B (-1456t>c; -750t>c; Q172H; K262R) were most common with frequencies of 0.28, 0.20 and 0.26, respectively. Alcohol use history (P = 0.011) and *6B haplotype (P = 0.011) were identified as significant predictors of bupropion hydroxylation. A consideration of the effects of these variables on CYP2B6 mRNA and protein levels suggests that alcohol use is associated with enhanced CYP2B6 gene transcription, but the presence of at least one *6B allele reduces this effect on bupropion hydroxylation at the post-transcriptional level. In conclusion, the results of this study indicate that interindividual variability in bupropion hydroxylation is a consequence of interactions between environmental and genetic influences on CYP2B6 gene function.

Cytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.

Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K m value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K m value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.Nicotine is not carcinogenic by itself and might even have some beneficial therapeutic effects in some neurological diseases. Nonetheless, it is the major perpetrator of tobaccorelated diseases because nicotine addiction drives smokers to pursue the habit despite the known health hazards. Nicotine concentration in the blood increases sharply during cigarette smoking and then decreases rapidly because of metabolism and clearance, driving the addicted individual to reach for another cigarette. Hence, better understanding of nicotine metabolism can assist the development of treatments to reduce the health risks associated with nicotine addiction.Cytochrome P450 monooxygenase 2A6 (CYP2A6) catalyzes nicotine oxygenation and plays an important role in nicotine metabolism (Hukkanen et al., 2005;Nakajima and Yokoi, 2005). However, there is more to nicotine metabolism than CYP2A6 because both nicotine and its primary oxidation metabolite, cotinine, undergo direct N-glucuronidation at the aromatic nitrogen (Fig.

AimsThe influence of ageing on the pharmacokinetics of zolpidem, an extensively prescribed hypnotic medication, was evaluated in healthy human volunteers. Methods A series of 16 elderly (age: 61-85 years) and 24 young (age: 22-42 years) volunteers received single 5 mg oral doses of zolpidem tartrate. Serum zolpidem concentrations were determined by HPLC with fluorescence detection in samples drawn during 8 h after dosage. The effect of testosterone on zolpidem biotransformation was evaluated in vitro using human liver microsomes. Possible induction of CYP3A protein expression and function was studied in cultured human hepatocytes. Results Among men, apparent oral clearance of zolpidem was decreased in elderly compared to young subjects (3.8 vs 11.0 ml min -1 kg -1 , P < 0.01), C max was increased (93 vs 40 ng ml -1 , P < 0.01), and half-life increased (2.7 vs 1.5 h, P < 0.03). Among women, zolpidem oral clearance was decreased in the elderly (3.0 vs 5.8 ml min -1 kg -1 , P < 0.02), C max increased (108 vs 60 ng ml -1 , P < 0.001), with no difference in t 1 / 2 (2.3 vs 2.4 h). Among male subjects, free serum testosterone concentrations were lower in the elderly (10.5 vs 19.0 pg ml -1 , P < 0.01), and were significantly correlated with zolpidem clearance ( r 2 = 0.46, P < 0.001). Multiple regression analysis indicated a greater relative contribution of serum testosterone than age to the oral clearance of zolpidem among men. In human liver microsomes, co-incubation of zolpidem (10 m M ) with varying concentrations of testosterone produced activation of biotransformation of zolpidem to its principal hydroxylated metabolite. Maximum activation was achieved at equimolar concentrations of testosterone (10 m M ). However, testosterone did not induce immunoactive CYP3A4 expression or catalytic function in cultured human hepatocytes. Conclusions The increased C max and lower oral clearance of zolpidem in the elderly are consistent with recommendations of lower clinical doses of zolpidem in the elderly. Our clinical and in vitro data both suggest that reduced free serum testosterone may have a modulatory role in age-dependent changes in zolpidem pharmacokinetics in men.

The objective of this study was to use recombinant enzymes and human liver microsomes (HLMs) to comprehensively evaluate the functional impact of the three most common nonsynonymous polymorphisms (S7A, T181A, and R184S) identified in the human UDP glucuronosyltransferase (UGT) 1A6 gene. In addition to the known allozymes, other possible amino acid variants were expressed in human embryonic kidney (HEK)293 cells to enable structure-function analysis. Initial studies using different substrates (serotonin, 5-hydroxytryptophol, 4-nitrophenol, acetaminophen, and valproic acid) showed similar results with 2-fold higher glucuronidation by UGT1A6*2 (S7A/ T181A/R184S) compared with UGT1A6*1 (reference), and intermediate activities for other variants. Enzyme kinetic analyses with the UGT1A6-specific substrate (serotonin) showed 50% lower K m values for all R184S variants and 2-fold higher V max values for both S7A/T181A variants compared with UGT1A6*1. Furthermore, intrinsic clearance (V max /K m ) values were highest for the UGT1A6*2 allozyme (2.3-fold over UGT1A6*1), resulting from additive effects of higher enzyme affinity and activity. As expected, K m values of *1/*1 genotyped HLMs (5.4 Ϯ 0.2 mM) were similar to recombinant UGT1A6*1 (5.8 Ϯ 0.6 mM). Conversely, *2/*2 HLMs showed higher K m values (7.0 Ϯ 0.3 mM) rather than the lower K m values displayed by recombinant UGT1A6*2 (3.6 Ϯ 0.3 mM), suggesting that this allozyme may display different enzyme kinetic behavior in HLMs compared with HEK293 cells. At best, these polymorphisms were predicted to account for 15 to 20% of the observed 13-fold variability in glucuronidation of UGT1A6 substrates by HLMs, indicating that there are likely other genetic or environmental factors responsible for the majority of this variation.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• UDP-glucuronosyltransferase (UGT) 2B15 is a major drug glucuronidation enzyme expressed in human liver.• Oxazepam is an isoform-selective probe drug that is being used for in vitro studies of UGT2B15.• The most common UGT2B15 missense polymorphisms (D85Y and K523T) are correlated with variable oxazepam glucuronidation in human liver bank samples.• UGT2B17 is also expressed in liver and has high sequence homology and substrate specificity overlap with UGT2B15. WHAT THIS STUDY ADDS• UGT2B15 D85Y polymorphism is identified as a major determinant of oxazepam disposition, accounting for as much as 34% of interindividual variability in oxazepam apparent oral clearance.• An effect of the UGT2B15 K523T or the UGT2B17 deletion polymorphisms on oxazepam disposition could not be detected.• Provides evidence supporting the use of oxazepam as an isoform-selective in vivo probe for studies of variability in UGT2B15 activity. AIMSAlthough in vitro studies indicate that oxazepam is an isoform-selective substrate probe for UDP-glucuronosyltransferase 2B15, the utility of this drug as an in vivo probe is uncertain. The main aim of this study was to determine whether common missense polymorphisms in the UGT2B15 gene (D85Y and K523T) are associated with altered oxazepam pharmacokinetics and pharmacodynamics. We also determined the possible influence of a common deletion polymorphism in the gene encoding UGT2B17, which shows substantial substrate specificity overlap with UGT2B15. METHODSThirty healthy male subjects were administered 15 mg of oxazepam by mouth followed by plasma oxazepam concentration monitoring for 36 h, and pharmacodynamic testing for 8 h. Genotypes were determined by genomic polymerase chain reaction and commercial 5′-nuclease assays. RESULTSAllele frequencies for D85Y, K523T, UGT2B17del were 47%, 23% and 19%, respectively. Median oxazepam apparent oral clearance was significantly lower in 85YY subjects (1.62 ml min -1 kg -1) compared with 85DD subjects (3.35 ml min -1 kg -1; P = 0.003, Student-Newman-Keuls test), whereas 85DY subjects were intermediate (2.34 ml min -1 kg -1; P = 0.018 vs. 85DD, P = 0.034 vs. 85YY). Regression analysis indicated that UGT2B15 D85Y genotype accounted for 34% of interindividual variability. However, neither UGT2B15 K523T nor UGT2B17del was associated with altered oxazepam disposition. Furthermore, no differences in pharmacodynamic measures, including quantitative electroencephalography, digit-symbol substitution test, self-or observer-rated visual analogue scales, could be demonstrated for any of the polymorphisms evaluated. CONCLUSIONSThese results identify UGT2B15 D85Y as a major determinant of oxazepam clearance, and indicate that oxazepam may be useful as an in vivo probe for glucuronidation by UGT2B15.

1 Mast cells derive from the bone marrow and are responsible for the development of allergic and possibly in¯ammatory reactions. Mast cells are stimulated by immunoglobulin E (IgE) and speci®c antigen, but also by a number of neuropeptides such as neurotensin (NT), somatostatin or substance P (SP), to secrete numerous pro-in¯ammatory molecules that include histamine, cytokines and proteolytic enzymes. 2 Chondroitin sulphate, a major constituent of connective tissues and of mast cell secretory granules, had a dose-dependent inhibitory eect on rat peritoneal mast cell release of histamine induced by the mast cell secretagogue compound 48/80 (48/80). This inhibition was stronger than that of the clinically available mast cell`stabilizer' disodium cromoglycate (cromolyn). Inhibition by chondroitin sulphate increased with the length of preincubation and persisted after the drug was washed o, while the eect of cromolyn was limited by rapid tachyphylaxis. 3 Immunologic stimulation of histamine secretion from rat connective tissue mast cells (CTMC) was also inhibited, but this eect was weaker in umbilical cord-derived human mast cells and was absent in rat basophilic leukemia (RBL) cells which are considered homologous to mucosal mast cells (MMC). Oligo-and monosaccharides were not as eective as the polysaccharides. 4 Inhibition, documented by light and electron microscopy, involved a decrease of intracellular calcium ion levels shown by confocal microscopy and image analysis. Autoradiography at the ultrastructural level showed that chondroitin sulphate was mostly associated with plasma and perigranular membranes.5 Chondroitin sulphate appears to be a potent mast cell inhibitor of allergic and nonimmune stimulation with potential clinical implications.