Stereoselective glucuronidation of ofloxacin in rat liver microsomes.

The stereoselective glucuronidation of ofloxacin [(+/-)-OFLX], a new quinolone antibacterial agent, was studied in vitro using rat liver microsomes. OFLX glucuronidation exhibited Michaelis-Menten kinetics in rat liver microsomes. Stereoselective glucuronidation of the optical enantiomers occurred. S-(-)-OFLX glucuronide was produced 7-fold more than R-(+)-OFLX glucuronide with little or no difference in the values of KM of the enantiomers. The value of Vmax/KM for the glucuronide conjugate of S-(-)-OFLX was 8-fold greater than for the conjugate of R-(+)-OFLX. These results demonstrate that OFLX undergoes stereoselective glucuronidation in vitro. Moreover, we studied the in vivo interaction between enantiomers of OFLX in rats to clarify the effects of R-(+)-OFLX on the metabolism and disposition of S-(-)-OFLX. When the racemate [(+/-)-OFLX (20 mg/kg)] or single enantiomer [S-(-)-OFLX (10 mg/kg)] is administered iv to the rat, the serum concentrations of S-(-)-OFLX were higher after racemate administration than those after enantiomer administration, although the dose of S-(-)-OFLX was identical in both cases. These results indicate that R-(+)-OFLX may compete with S-(-)-OFLX in the in vivo glucuronidation. Furthermore, the results of the enantiomeric inhibition study showed that R-(+)-OFLX competitively inhibited S-(-)-OFLX glucuronidation in vitro with a Ki value of 2.92 mM.     

Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isoforms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The Km, Vmax and CLint values of cynomolgus monkey liver microsomes were generally higher than the S50, Vmax and CLmax values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in Vmax and CLint values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isoforms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.     

Prominent but reverse stereoselectivity in propranolol glucuronidation by human UDP-glucuronosyltransferases 1A9 and 1A10.

Propranolol is a nonselective beta-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.     

Stereoselective metabolism of the monoterpene carvone by rat and human liver microsomes

The large amounts of carvone enantiomers consumed as food additives and in dental formulations justifies the evaluation of their biotransformation pathway. The in-vitro metabolism of R-(-)- and S-(+)-carvone was studied in rat and human liver microsomes using chiral gas chromatography. Stereoselective biotransformation was observed when each enantiomer was incubated separately with liver microsomes. 4R, 6S-(-)-Carveol was NADPH-dependently formed from R-(-)-carvone, whereas 4S, 6S-(+)-carveol was produced from S-(+)-carvone. Metabolite formation followed Michaelis-Menten kinetics exhibiting a significant lower apparent Km (Michaelis-Menten Constant) for 4R, 6S-(-)-carveol compared with 4S, 6S-(+)-carveol in rat and human liver microsomes (28.4+/-10.6 microM and 69.4+/-10.3 microM vs 33.6+/-8-55 microM and 98.3+/-22.4 microM). The maximal formation rate (Vmax) determined in the same microsomal preparations yielded 30.2+/-5.0 and 32.3+/-3.9 pmol (mg protein)(-1) min(-1) in rat liver and 55.3+/-5.7 and 65.2+/-4.3 pmol (mg protein)(-1) min(-1) in human liver microsomes. Phase II conjugation of the carveol isomers by rat and human liver microsomes in the presence of UDPGA (uridine S'-diphosphogluaronic acid) only revealed glucuronidation of 4R, 6S-(-)-carveol. Vmax for glucuronide formation was more than 4-fold higher in the rat liver compared with human liver preparations (185.9+/-34.5 and 42.6+/-7.1 pmol (mg protein)(-1) min(-1), respectively). Km values, however, showed no species-related difference (13.9+/-4.1 microM and 10.2+/-2.2 microM). This study demonstrated stereoselectivity in phase-I and phase-II metabolism for R-(-)- and S-(+)-carvone and might be predictive for carvone biotransformation in man.     

氧氟沙星对映体在经不同诱导剂诱导的大鼠肝微粒体中的葡醛酸化代谢

为评价氧氟沙星（OFLX）对映体葡醛酸化代谢的立体选择性,采用手性HPLC法测定大鼠肝微粒体孵育液中OFLX对映体. 结果显示：经苯巴比妥（PB）和β-萘黄酮（BNF）诱导的不同葡醛酸转移酶（UDPGT）亚族对OFLX对映体葡醛酸化代谢有不同的影响. 在所试验的对照,PB或BNF诱导的微粒体中S-（-）-和R-（+）-OFLX之间,K_m和V_max无显著性差异;但PB组中S- （-）-和R-（+）-OFLX的K_m和V_max与对照组或BNF组相应的对映体比较有显著性差异;OFLX对映体之间的Cl_int在对照组与BNF组没有显著性差异;而在PB组则有显著性差异. 另外BNF组的Cl_int较对照组和PB组分别有显著性差异. 因此,经PB诱导的UDPGT亚族对S-和R-OFLX的Ⅱ相代谢存在立体选择性,并主要是由于其催化部位的差异引起了内在清除率的变化.     

Glucuronidation of propranolol and 4'-hydroxypropranolol. Substrate specificity and stereoselectivity of rat liver microsomal glucuronyltransferases

Liquid-chromatographic procedures were developed to assay the formation of diastereomeric glucuronides of propranolol (PG) and 4'-hydroxypropranolol (HPG) by rat liver microsomes, with identifications performed by GC/MS techniques. Propranolol was conjugated at a rate 10% of that determined for 4'-hydroxypropranolol. Glucuronyltransferase activity increased slightly (10-17%) in the presence of MgCl2. Inclusion of 0.04% Triton X-100 produced a 55% inhibition of PG formation, but increased HPG formation greater than 2-fold. Pretreatment of animals with phenobarbital resulted in a 4-fold increase in PG formation, but did not affect HPG formation unless MgCl2 was also present. Under these conditions, a 50-60% increase in HPG formation occurred. Pretreatment with 3-methylcholanthrene did not affect the formation of either glucuronide. (R)-(+)-Propranolol was glucuronidated 2-fold faster than the (S)-(-) enantiomer at substrate concentrations below 0.1 mM, and 1.3-fold faster at substrate concentrations above 2.0 mM. The estimated Vmax, 0.67 nmol/mg/min, was identical for both enantiomers. The dissociation constants were significantly different, however, being 0.57 mM for (R)-(+)-propranolol and 0.87 mM for (S)-(-)-propranolol. No stereoselectivity was observed in the formation of HPG when 4'-hydroxypropranolol was used as substrate, or when propranolol was incubated in the presence of an NADPH-generating system. Propranolol and 4'-hydroxypropranolol were used as substrate, or when propranolol was incubated in the presence of an NADPH-generating system. Propranolol and 4-hydroxypropranolol are apparently glucuronidated by different forms of rat liver glucuronyltransferase. Furthermore, propranolol glucuronidation occurs stereoselectively in vitro because of the different enzyme affinities for the enantiomers of the drug.     

Glucuronidation of propofol and its analogs by human and rat liver microsomes

Propofol (2,6-diisopropylphenol), widely used an intravenous anesthetic, is rapidly metabolized to its glucuronide in the in vivo studies. Kinetic parameters for the glucuronidation of propofol and its analogs, such as 2,5-diisopropylphenol, 2-tert-butyl-6-methylphenol, 2-tert-butyl-5-methylphenol, 2,6-dimethylphenol and 2,5-dimethylphenol, were determined in vitro using human and rat liver microsomes. 2,5-Dimethylphenol and 2-tert-butyl-6-methylphenol exhibited the highest and lowest glucuronidation rates, respectively. Substitutes at the 2,6-positions gave lower glucuronidation rates than those at the 2,5-positions in both the human and rat microsomes. 2,5-Diisopropylphenol was glucuronidated at a lower rate in human than propofol. The affinity of uridine 5'-diphosphate (UDP)-glucuronosyltransferase for disubstituted phenols, such as propofol, 2,5-diisopropylphenol, 2,5-dimethylphenol, and 2-tert-butyl-6-methylphenol, gave higher Km values in human liver microsomes than in rat ones, and lower Vmax values showed similar relationship, expect for Vmax in propofol. The alkyl group at the 6 position showed a higher Km for glucuronidation by a steric hindrance in the human and rat microsomes. Our results propose that the glucuronidation of propofol and its analogs may not be explained by only a steric hindrance.     

Enantioselective pharmacokinetics of etodolac in the rat: tissue distribution, tissue binding, and in vitro metabolism.

The nonsteroidal anti-inflammatory agent etodolac (ET) exhibits stereoselectivity in its pharmacokinetics following administration to humans and rats. To underline the factors responsible for this stereoselectivity, the tissue distribution, in vitro tissue binding, and microsomal metabolism of ET enantiomers were studied in the rat. Following iv administration of racemic ET, the S:R AUC ratios in tissues were stereoselective, and different from that in plasma. Binding of enantiomers to tissues was stereoselective, although it did not relate well with in vivo tissue distribution. Rather, the tissue distribution of enantiomers appeared to be better explained by the unbound fractions of enantiomers in plasma. With respect to in vitro glucuronidation by liver microsomes, the Vmax of S-ET was 3.4-fold greater than that of R-ET; the enantiomers possessed similar Km. There appeared to be stereoselectivity in the oxidative metabolism of ET enantiomers by liver and kidney microsomes, in favor of the R-enantiomer. The lower AUC in rat plasma of pharmacologically active S-ET as compared with its antipode is due to its relatively greater distribution to tissues, owing to a lesser degree of binding to plasma proteins, and to its higher rate of glucuronidation.     

Characterization of rat and human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac.

In the current study, the identification of the rat and human UDP-glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of diclofenac was determined. Recombinant human UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate of 166-pmol/min/mg protein, while UGT1A6 and 2B15 catalyzed the glucuronidation of diclofenac at low rates (<20-pmol/min/mg protein). Conversely, human UGT2B7 displayed a high rate of diclofenac glucuronide formation (>500 pmol/min/mg protein). Recombinant rat UGT2B1 catalyzed the glucuronidation of diclofenac at a rate of 250-pmol/min/mg protein. Rat UGT2B1 and human UGT2B7 displayed a similar, low apparent Km value of <15 microM for both UGT isoforms and high Vmax values 0.3 and 2.8 nmol/min/mg, respectively. Using diclofenac as a substrate, enzyme kinetics in rat and human liver microsomes showed that the enzyme(s) involved in diclofenac glucuronidation had a low apparent Km value of <20 microM and a high Vmax value of 0.9 and 4.3 nmol/min/mg protein, respectively. Morphine is a known substrate for rat UGT2B1 and human UGT2B7 and both total morphine glucuronidation (3-O- and 6-O-glucuronides) and diclofenac glucuronidation reactions showed a strong correlation with one another in human liver microsome samples. In addition, diclofenac inhibited the glucuronidation of morphine in human liver microsomes. These data suggested that rat UGT2B1 and human UGT2B7 were the major UGT isoforms involved in the glucuronidation of diclofenac.     

Characterization of a rat liver glucuronosyltransferase that glucuronidates the selective D1 antagonist, SCH 23390, and other benzazepines.

SCH 23390 is a novel benzazepine that selectively blocks dopamine receptors of the D1 subtype. Glucuronidation of this selective D1 antagonist was studied in vitro using rat liver microsomes. Methods to separate SCH 23390 glucuronide from SCH 23390 were developed which utilized either HPLC techniques or solvent extraction of SCH 23390 with 3-heptanone. Formation of a SCH 23390 glucuronide was confirmed upon incubation of SCH 23390 and UDPGA with naive rat liver microsomes. Liver enzyme activity for SCH 23390 glucuronidation was also enhanced after addition of the detergents, Lubrol or Triton X-100, to the naive liver microsomes. Kinetic analyses indicated an apparent Vmax and Km for UDPGA as 120.9 pmol/mg protein/min and 0.63 mM, and an apparent Vmax and Km for SCH 23390 as 282.4 pmol/mg protein/min and 0.41 microM. Further characterization of the liver enzyme responsible for the glucuronidation of SCH 23390 revealed a stereoselective substrate preference similar to that seen with the D1 dopamine receptor. Substrate inhibition studies indicated that SCH 23390, haloperidol, apomorphine, and alpha-naphthol demonstrated the highest affinity for the glucuronosyltransferase enzyme. However, (-)-sulpiride, raclopride, and endogenous substrates such as dopamine, serotonin, epinephrine, and norepinephrine demonstrated low affinity for the liver enzyme. These studies describe a rat liver glucuronosyltransferase with a unique substrate specificity toward selected dopaminergic agents. Finally, induction profiles revealed that neither phenobarbital (100 mg/kg, ip, for 3 days), beta-naphthoflavone (100 mg/kg, ip, for 4 days), nor 3-methylcholanthrene (80 mg/kg, ip, for 4 days) enhanced liver glucuronosyltransferase activity for SCH 23390 glucuronidation.     

氧氟沙星对映体在经不同诱导剂诱导的大鼠肝微粒体中的葡醛酸化代谢(英文)

为评价氧氟沙星 ( OFLX)对映体葡醛酸化代谢的立体选择性 ,采用手性 HPLC法测定大鼠肝微粒体孵育液中 OFLX对映体 .结果显示 :经苯巴比妥 ( PB)和β-萘黄酮 ( BNF)诱导的不同葡醛酸转移酶 ( UDPGT)亚族对 OFLX对映体葡醛酸化代谢有不同的影响 .在所试验的对照 ,PB或 BNF诱导的微粒体中 S- ( - ) -和 R- ( + ) - OFLX之间 ,Km 和Vmax无显著性差异 ;但 PB组中 S- ( - ) -和 R- ( + ) -OFLX的 Km 和 Vmax与对照组或 BNF组相应的对映体比较有显著性差异 ;OFLX对映体之间的 Clint在对照组与 BNF组没有显著性差异 ;而在 PB组则有显著性差异 .另外 BNF组的 Clint较对照组和 PB组分别有显著性差异 .因此 ,经 PB诱导的 UDPGT亚族对 S-和 R- OFLX的 相代谢存在立体选择性 ,并主要是由于其催化部位的差异引起了内在清除率的变化 .     

反式曲马朵在大鼠肝微粒体O-去甲基代谢中的立体选择性

目的研究反式曲马朵O-去甲基代谢的立体选择性。方法高效毛细管电泳法测定大鼠肝微粒体孵育液中反式曲马朵和O-去甲基曲马朵对映体的浓度,酶促动力学方法研究O-去甲基曲马朵对映体的生成。结果 (-)-O-去甲基曲马朵生成有较大的V_max;反式曲马朵两对映体间存在相互作用,使(+)-O-去甲基曲马朵生成的V_max明显减慢;奎宁及奎尼丁对(+)-O-去甲基曲马朵生成的抑制作用较强。结论反式曲马朵O-去甲基代谢有立体选择性,对映体间的相互作用及酶抑制剂使其立体选择性程度加强。     

Stereoselective formation of fenoterol-para-glucuronide and fenoterol-meta-glucuronide in rat hepatocytes and enterocytes

The glucuronidation of fenoterol (Berotec, Partusisten) in isolated rat hepatocytes and enterocytes was investigated. Two different glucuronides, fenoterol para-glucuronide and fenoterol meta-glucuronide, were formed in proportions, that were constant over the concentration range investigated (0-1 mM). The fraction of para-glucuronide formed was 0.40 +/- 0.01 for hepatocytes and 0.54 +/- 0.01 for enterocytes. Fenoterol consists of a racemic mixture of SS'(+)fenoterol and RR'(-)fenoterol. The maximum glucuronidation rate of the (-)enantiomer (Vmax = 3.6 +/- 0.3 nmol/min/mg in hepatic microsomes and 3.4 +/- 0.1 nmol/min/mg in intestinal microsomes) is significantly lower than the same values of the (+)isomer (Vmax = 6.7 +/- 0.8 nmol/min/mg in hepatic microsomes and 5.8 +/- 0.4 nmol/min/mg in intestinal microsomes). Kmapp-values for the (-)enantiomer were lower than for the (+)enantiomer. Similar, but less pronounced, differences in Vmax were observed in isolated cells: Vmax = 148 +/- 13 and 372 +/- 50 pmol/min/mg [(-)fenoterol in hepatocytes and enterocytes], Vmax = 173 +/- 12 and 444 +/- 57 pmol/min/mg [(+)fenoterol in hepatocytes and enterocytes]. Calculation of intrinsic metabolic clearance (Clint = Vmax/Kmapp) from the cellular data suggests that the (+)enantiomer may be more efficiently eliminated by liver metabolism in vivo than the (-)enantiomer. This can result in stereoselective first-pass metabolism of the fenoterol enantiomers.     

Involvement of human hepatic UGT1A1, UGT2B4, and UGT2B7 in the glucuronidation of carvedilol.

Carvedilol ((+/-)-1-carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol) is metabolized primarily into glucuronide conjugates. In the present study, we identified the human UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of carvedilol by thin-layer chromatography using microsomes from human liver or insect cells expressing recombinant UGT isoforms. We observed two forms of carvedilol glucuronides, namely G1 and G2, in hepatic microsomes. The glucuronidation of carvedilol was catalyzed by at least three recombinant UGT isoforms: UGT1A1, UGT2B4, and UGT2B7. UGT2B4 formed both G1 and G2, whereas UGT1A1 and UGT2B7 were responsible for the formation of glucuronide G2 and G1, respectively. The enzyme kinetics for carvedilol glucuronidation by UGT1A1, UGT2B4, and UGT2B7 in addition to human liver microsomes were examined by Lineweaver-Burk analysis. The values of Km and Vmax for human liver microsomes were 26.6 microM and 106 pmol/min/mg protein for G1, and 46.0 microM and 44.5 pmol/min/mg protein for G2, respectively. The Km values for UGT1A1, UGT2B4, and UGT2B7 for G1 and G2 (22.1-55.1 microM) were comparable to those of the liver microsomes, whereas the Vmax values were in the range of 3.33 to 7.88 pmol/min/mg protein. The Km and Vmax/Km values for UGT2B4 and UGT2B7 for G1 were similar, whereas UGT2B4 had lower Km and higher Vmax/Km values for G2 compared with those of UGT1A1. These results suggest that G1 formation is catalyzed by UGT2B4 and UGT2B7, whereas G2 is formed by UGT2B4 and UGT1A1. These three hepatic UGT isoforms may have important roles in carvedilol metabolism.     

在大鼠肝微粒体中反式曲马朵和反式氧去甲基曲马朵对映体代谢的性别差异

目的研究反式曲马朵(trans T)对映体代谢，反式氧去甲基曲马朵(Ml)对映体生成及其与葡糖醛酸结合的性别差异。方法以trans T或Ml为底物分别与大鼠肝微粒体孵育，高效毛细管电泳法测定孵育液中trans T和Ml对映体。结果与(+)-对映体相比，(-)-trans T优先代谢，(-)-Ml优先生成。在雌性大鼠肝微粒体中(-)-Ml优先与葡糖醛酸结合；Ml两对映体生成及其与葡糖醛酸结合的CL_int比值偏离1的程度较大。在雄性大鼠肝微粒体中(+)-Ml优先与葡糖醛酸结合。结论Trans T代谢，M1生成及其与葡糖醛酸结合均具立体选择性和性别差异；Ml生成及其与葡糖醛酸结合立体选择性的程度以雌性大鼠的较高。     

普萘洛尔光学异构体在经不同诱导剂诱导的大鼠肝微粒体P450系统中的代谢特征

采用正常及β萘黄酮（BNF）或苯巴比妥（PB）诱导的大鼠肝微粒体,研究了R(+)和S(-)普萘洛尔代谢酶动力学参数及立体选择性. 实验表明,3种微粒体酶的亲和力均无立体选择性,反应速度有较大差别,并表现出立体选择性. BNF组对两种对映体的催化作用较对照组增强,并对R(+)对映体有选择性;PB组的催化作用较对照组减弱,但对S(-)对映体有选择性. 实验结果提示普萘洛尔经细胞色素P450代谢时,酶活性中心的结合基团无立体选择性,而催化基团具有立体选择性.     

Regioselective glucuronidation of denopamine: marked species differences and identification of human udp-glucuronosyltransferase isoform.

Denopamine is one of the oral beta(1)-adrenoceptor-selective partial agonists. Denopamine glucuronide is the most abundant metabolite in human, rat, and dog urine when administered orally. Species differences in denopamine glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. In rat and rabbit, only the phenolic glucuronide was detected, whereas in dog and monkey, not only the phenolic glucuronide but also the alcoholic glucuronide was found. In contrast, in humans, the alcoholic glucuronide was detected exclusively. The kinetics of denopamine glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K(m) and V(max) values accounted for 2.87 +/- 0.17 mM and 7.29 +/- 0.23 nmol/min/mg protein, respectively. With the assessment of denopamine glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17), only UGT2B7 exhibited high denopamine glucuronosyltransferase activity. The K(m) value of denopamine glucuronidation in recombinant UGT2B7 microsomes was close to those in human liver and jejunum microsomes. The formation of denopamine glucuronidation by human liver, jejunum, and recombinant UGT2B7 microsomes was effectively inhibited by diclofenac, a known substrate for UGT2B7. The denopamine glucuronidation activities in seven human liver microsomes were significantly correlated with diclofenac glucuronidation activities (r(2) = 0.685, p < 0.05). These results demonstrate that the denopamine glucuronidation in human liver and intestine is mainly catalyzed by UGT2B7 and that glucuronidation of the alcoholic hydroxyl group, but not the phenolic hydroxyl group, occurs regioselectively in humans.     

Glucuronidation of propranolol by dog liver microsomes. Effects of enantiomeric inhibition and detergent treatment on stereoselectivity

The diastereomeric glucuronides of (R)- and (S)-propranolol [(R)-PG and (S)-PG] formed by incubating the racemic drug with dog liver microsomes were separated and quantitated chromatographically. Conjugation occurred stereoselectively with 3- to 4-fold more (S)-PG than (R)-PG produced. The values for the apparent Km were 1.0 and 1.7 mM, and those of the Vmax were 6.4 and 25 nmol/min/mg protein for (R)- and (S)-propranolol, respectively. Stereoselectivity was lower in parallel incubations conducted with the separate enantiomers than in incubates with the racemic substrate. The S enantiomer was found to be a noncompetitive inhibitor of (R)-PG formation, with a Ki of 1.8 mM. (S)-PG did not inhibit (R)-PG formation when added to incubates and (R)-propranolol did not inhibit the formation of (S)-PG. The presence of sodium cholate and Brij 35 stimulated glucuronidation by 60 and 120%, respectively, but did not affect the stereoselectivity even at concentrations high enough to inhibit conjugation. On the contrary, Triton X-100 and Emulgen 911 were inhibitory and produced a decrease in stereoselectivity; the magnitude of these effects increased with increasing detergent concentration.     

The activity of 1-naphthol-UDP-glucuronosyltransferase in the brain

Cerebral microsomes catalysed efficiently the glucuronidation of 1-naphthol, this formation of glucuronide being activated by treatment with Triton X-100 or digitonin. Activated microsomes from the brain of the rat conjugated 1-naphthol with an apparent Km of 95 microM and a Vmax of 5.47 nmol/hr mg protein at 30 degrees C. Microsomal uridine diphosphate (UDP)-glucuronosyltransferase activity in brain towards 1-naphthol was not significantly induced by pretreatment of animals with 3-methylcholanthrene or phenobarbital. These data suggest that UDP-glucuronosyltransferases in brain are different from the hepatic enzymes with regard to biochemical parameters and in response to inducers of drug metabolism. The hepatic UDP-glucuronosyltransferase deficiency in Gunn rats was also observed in the brain.     

Glucuronidation of 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetic acid (TA-1801A) in humans: species differences in liver and intestinal microsomes

The metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetate (TA-1801), a potent hypolipidemic agent, was studied in humans after oral administration and compared with that found in rats, rabbits, and dogs previously. Hydrolysis of the ethyl ester to produce metabolite M1 (TA-1801 active form; TA-1801A) is the first metabolic step and the subsequent biotransformation includes the glucuronidation to form the metabolite M4 and the oxidation to form the metabolites M2 and M3. The metabolism of TA-1801 in humans was qualitatively similar to that in the experimental animals studied, although species differences were seen in the amount of metabolites. M4, the glucuronide of TA-1801A was the most abundant metabolite in human urine (24.3% of the dose). In vitro studies using human liver and jejunum microsomes indicated that the TA-1801A glucuronosyltransferase activity in human jejunum microsomes was 2-fold higher than that in liver microsomes. With regard to the interspecies differences in the TA-1801A glucuronosyltransferase activities, the intrinsic clearance for the TA-1801A glucuronidation in liver microsomes was in the following order: rabbit>monkey>human=rat=dog. In jejunum microsomes, the intrinsic clearance for the TA-1801A glucuronidation was in the following order: human>monkey>rabbit>rat=dog. These results suggest that the species differences in the intestinal TA-1801A glucuronidation contribute to the species differences in the excretion rate of TA-1801A glucuronide into the urine.