Enantioselective hydroxylation of nortriptyline in human liver microsomes, intestinal homogenate, and patients treated with nortriptyline

The enantioselectivity of hydroxylation of nortriptyline (NT) to E-10-hydroxynortriptyline (E-10-OH-NT) was studied in human liver microsomes, intestinal homogenate, and patients treated with NT. The rate of formation of (-)-E-10-OH-NT was higher than that of (+)-E-10-OH-NT both in the liver microsomes and in the intestinal homogenate. Quinidine, a prototype competitive inhibitor of the cytochrome P450IID6 ("debrisoquin hydroxylase"), inhibited the formation of (-)-E-10-OH-NT in a concentration-dependent manner in liver microsomes, while the formation of (+)-E-10-OH-NT was hardly affected. This indicates that P450IID6 catalyzes the hydroxylation of NT in a highly enantioselective manner to (-)-E-10-OH-NT in the liver. Another P450 isozyme besides IID6 seems to be responsible for the formation of the (+)-enantiomer in the liver. In intestinal homogenate, the formation of both enantiomers of E-10-OH-NT was inhibited to about the same extent by quinidine, the maximum inhibition being much less than in the liver. In the urine of six patients treated with NT, the (-)-enantiomer accounted for 91 +/- 2% of the unconjugated E-10-OH-NT, and for 78 +/- 6% of the glucuronide conjugates. The study shows that NT is hydroxylated in a highly enantioselective way, probably catalyzed by the polymorphic P450IID6, to (-)-E-10-OH-NT both in vitro in human liver as well as in vivo in patients treated with the drug.     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25?mg nortriptyline hydrochloride and collected urine for 72–80?h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54–58% of the dose and it was reduced to 25–40% when 50?mg quinidine sulphate was ingested on the first and second day.

2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced.

3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected.

4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position.

5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10-and in 2-position     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine.

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25 mg nortriptyline hydrochloride and collected urine for 72-80 h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54-58% of the dose and it was reduced to 25-40% when 50 mg quinidine sulphate was ingested on the first and second day. 2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced. 3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected. 4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position. 5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10- and in 2-position.     

The enantioselective glucuronidation of morphine in rats and humans. Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme

The formation of morphine glucuronides is enantio- and regioselective in rats and humans. In rat liver microsomes, natural (-)-morphine formed only the 3-O-glucuronide, whereas the unnatural (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions, with the 6-O-glucuronide being the principal product. In human liver microsomes, both the 3-OH-and 6-OH positions were glucuronidated with each of the enantiomers, with the 3-O-glucuronide being the major product with (-)-morphine, and the 6-OH position preferred with the (+)-enantiomer. By using a series of biochemical and biological situations such as induction by xenobiotics, ontogeny, selective inhibition and genetic deficiencies, which are considered to be diagnostic of UDP-glucuronosyltransferase heterogeneity, we determined that two UDP-glucuronosyltransferase isoenzymes were responsible for the glucuronidation of morphine in rat liver. One isoenzyme (the so-called "morphine UDP-glucuronosyltransferase") was responsible for the glucoronidation at the (-)-3-OH and (+)-6-OH positions of morphine, whereas the other formed only the (+)-morphine-3-glucuronide. Evidence from enzyme induction and the genetically deficient deficient Gunn rat suggested that bilirubin UDPGT may be responsible for the (+)-morphine-3-UDP-glucuronosyltransferase activity. In human kidney, glucuronidation of both (-)- and (+)-enantiomers at the 6-OH position was deficient, whereas the activity at the 3-OH positions was still present, which indicated the involvement of two UDP-glucuronosyltransferases in the glucuronidation of morphine in man, as well as rats.     

In vitro glucuronidation of thyroxine and triiodothyronine by liver microsomes and recombinant human UDP-glucuronosyltransferases.

Glucuronidation, which may take place on the phenolic hydroxyl and carboxyl groups, is a major pathway of metabolism for thyroxine (T4) and triiodothyronine (T3). In this study, a liquid chromatography/mass spectrometry (LC/MS) method was developed to separate phenolic and acyl glucuronides of T4 and T3. The method was used to collect the phenolic glucuronide of T4 for definitive characterization by NMR and to determine effects of incubation pH, species differences, and human UDP-glucuronosyltransferases (UGTs) involved in the formation of the glucuronides. Formation of T4 phenolic glucuronide was favored at pH 7.4, whereas formation of T4 acyl glucuronide was favored at pH 6.8. All the UGTs examined catalyzed the formation of T4 phenolic glucuronide except UGT1A4; the highest activity was detected with UGT1A3, UGT1A8, and UGT1A10, followed by UGT1A1 and UGT2B4. Formation of T3 phenolic glucuronide was observed in the order of UGT1A8 > UGT1A10 > UGT1A3 > UGT1A1; trace activity was observed with UGT1A6 and UGT1A9. UGT1A3 was the major isoform catalyzing the formation of T4 and T3 acyl glucuronides. In liver microsomes, phenolic glucuronidation was the highest in mice for T4 and in rats for T3 and lowest in monkeys for both T4 and T3. Acyl glucuronidation was highest in humans and lowest in mice for T4 and T3. Phenolic glucuronidation was higher than acyl glucuronidation for T4 in humans; in contrast, the acyl glucuronidation was slightly higher than phenolic glucuronidation for T3. UGT activities were lower toward T3 than T4 in all the species. The LC/MS method was a useful tool in studying glucuronidation of T4 and T3.     

Stereoselectivity and interaction between the glucuronidation of S-(-)- and R-(+)-propranolol in rat hepatic microsomes pretreated with different inducers

Phase II glucuronidation metabolism of side-chain propranolol was studied using microsomes from rats treated with the inducers beta-naphthoflavone (BNF) or dexamethasone (Dex). The glucuronide concentrations of propranolol enantiomers were assayed by RP-HPLC. The kinetic constants of glucuronidation, Km, Vmax and Clint were determined. There are significant differences between the R- and S-enantiomeric glucuronide in Km, Vmax and Clint P < 0.05, P < 0.01 and P < 0.05 in control microsome. There are significant differences in Km and Clint (P < 0.01 or P < 0.001) but no significant differences in Vmax (P > 0.05) between R and S-enantiomeric glucuronide in the microsomes induced with Dex and BNF. The formation of S-(-)-propranolol glucuronide was inhibited by R-(+)-propranolol from the rat microsomes pretreated with BNF and Dex. The glucuronidation metabolism of propranolol enantiomers exhibited the stereoselectivity in rat hepatic microsomes induced with BNF or Dex. Multiple UGT1A and 2B may be involved in stereoselective O-glucuronidation of propranolol enantiomers in rat liver microsomes. The glucuronides produced were in favor of the R-enantiomer. There is an interaction between the glucuronidation of R- and S-enantiomer.     

Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of human liver microsomes that we tested.     

Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides

Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs–Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10  1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.     

Involvement of UDP-Glucuronosyltransferases in the Extensive Liver and Intestinal First-Pass Metabolism of Flavonoid Baicalein

Purpose The present study aims to investigate the involvement of UDP-glucuronosyltranferase (UGT) in the extensive liver and intestinal first-pass glucuronidation of baicalein (B) in both rats and humans and also to study sulfation and P450 mediated hydroxylation of B.Materials and Methods B was incubated with liver and intestine microsome, cytosol, S9 fractions from human, rat and various human recombinant UGT isozymes, respectively. The generated metabolites were identified by HPLC/MS/MS and quantified by HPLC/UV.Results Three metabolites of B namely baicalein 7-O-glucuronide (BG), the isomer of baicalein 7-O-glucuronide (BG’), and baicalein sulfate were found. BG, the predominant metabolite of B, was extensively generated in liver and jejunum microsomes in both humans and rats. Its formation was mainly catalyzed by UGT 1A9 and also mediated by UGT 1A1, 1A3, 1A8, 1A7 and 2B15 with different kinetic profiles. UGT 1A8 mediated formation of BG’ was mainly found in human intestine and rat liver microsomes. Sulfation and P450 mediated hydroxylation of B were much less significant than glucuronidation.Conclusions Extensive liver and intestinal first-pass glucuronidation of B were found in both humans and rats. Under the current experimental conditions, UGT 1A9 and UGT 1A8 demonstrated the fastest formation rate of BG in human liver preparations and BG’ in human intestine preparations, respectively.     

Glucuronidation and sulfation of the tea flavonoid (-)-epicatechin by the human and rat enzymes.

(-)-Epicatechin (EC) is one of the flavonoids present in green tea, suggested to have chemopreventive properties in cancer. However, its bioavailability is not clearly understood. In the present study, we determined the metabolism of EC, focusing on its glucuronic acid and sulfate conjugation using human liver and intestinal microsomes and cytosol as well as recombinant UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) isoforms in comparison with that occurring in the rat. Surprisingly, EC was not glucuronidated by the human liver and small intestinal microsomes. There was also no evidence of glucuronidation by human colon microsomes or by recombinant UGT1A7, which is not present in the liver or intestine. Interestingly, in the rat liver microsomes EC was efficiently glucuronidated with the formation of two glucuronides. In contrast, the human liver cytosol efficiently sulfated EC mainly through the SULT1A1 isoform. For the intestine, both SULT1A1 and SULT1A3 contributed. Other SULT isoforms contributed little. High-performance liquid chromatography of the sulfate conjugates showed one major sulfatase-sensitive peak with all tissues. An additional minor sulfatase-resistant peak was formed by the liver and intestinal cytosol as well as with SULT1A1 but not by the Caco-2 cytosol and SULT1A3. In the rat, EC sulfation was considerably less efficient than in the human liver. These results indicate that sulfation is the major pathway in EC metabolism in the human liver and intestine with no glucuronidation occurring. There was also a large species difference both in glucuronidation and sulfation of EC between rats and humans.     

Formation of a quaternary N-glucuronide of amitriptyline in human liver microsomes

Amitriptyline N-glucuronide was isolated from urine of a patient treated with therapeutic doses of amitriptyline. The glucuronide was hydrolyzed by hot alkaline treatment and, to a lesser degree, by treatment with beta-glucuronidase. A method for the direct measurement of amitriptyline glucuronide by HPLC was developed. Human liver microsomes were shown to glucuronidate amitriptyline in the presence of UDPGA, and the activity varied 7-fold among microsomes from 13 different human livers. The glucuronidation of amitriptyline was inhibited by p-nitrophenol but not by morphine. E-10-hydroxynortriptyline, a major metabolite of amitriptyline, had only a slight inhibitory effect on the glucuronidation of amitriptyline. No significant correlation was found between the glucuronidation of amitriptyline and that of E-10-hydroxynortriptyline in the microsomes studied.     

Glucuronides of tea catechins: enzymology of biosynthesis and biological activities.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are major green tea catechins with antioxidant and anticancer activities. In this study, we characterized the glucuronidation of EGCG and EGC in human, mouse, and rat microsomes and by nine different human UGT 1A and 2B isozymes expressed in insect cells. Six EGCG and EGC glucuronides were biosynthesized, and their structures were identified for the first time. (-)-EGCG-4"-O-glucuronide was the major EGCG glucuronide formed in all incubations. The catalytic efficiency (V(max)/K(m)) for (-)-EGCG-4"-O-glucuronide formation followed the order: mouse intestine > mouse liver > human liver > rat liver > rat small intestine. The UGT-catalyzed glucuronidation of EGC was much lower than that of EGCG. The V(max)/K(m) for (-)-EGC-3'-O-glucuronide followed the following order: mouse liver > human liver > rat liver > rat and mouse small intestine. Human UGT1A1, 1A8, and 1A9 had high activities with EGCG. UGT1A8, an intestine-specific UGT, had the highest V(max)/K(m) for EGCG but low activity with EGC. Mice appeared to be more similar to humans than rats to humans in the glucuronidation of EGCG and EGC. Some of these catechin glucuronides retained the activities of their parent compounds in radical scavenging and in inhibiting the release of arachidonic acid from HT-29 human colon cancer cells. These results provide foundations for understanding the biotransformation and biological activities of tea catechins.     

Effects of a novel potential antidepressant on the behavior and cortisol levels of isolated guinea pig pups

A novel, potential antidepressant, E-6006 citrate (E-6039), dose-dependently reduced the vocalizations emitted by isolated guinea pig pups. The (+)-E-6006, but not the (-)-E-6006, enantiomer also reduced vocalizing. There were no reliable effects of E-6039 on locomotor activity, crouching, or other behavioral measures, but both E-6039 and the (+)-E-6006 enantiomer elevated plasma cortisol levels during isolation. The contrasting effects of E-6039 on vocalizations and plasma cortisol are discussed in terms of E-6039's putative ability to inhibit release of substance P. The reduction in the vocalizations of isolated guinea pig pups corroborates positive results with this drug in other antidepressant screens utilizing mice and rats, and provides further support for the potential of E-6039 as an antidepressant compound.     

Hepatic microsomal UDP‐glucuronosyltransferase (UGT) activities in the microminipig

The microminipig, a small minipig, was bred as a novel experimental animal for nonclinical pharmacology/toxicology studies by Fuji Micra Inc. (Shizuoka, Japan). Species differences in drug metabolism between humans and the microminipig need to be elucidated in more detail in order to discuss the results of nonclinical studies. Glucuronidation catalysed by UDP‐glucuronosyltransferase (UGT) is an important pathway in the metabolism of a wide variety of compounds. The aim of the present study was to identify the characteristics of hepatic UGT activity in the microminipig and compare them with those in humans and other experimental animals. This study examined in vitro UGT activities using liver microsomes from microminipigs (8 months old and 1 day old), humans, mice, rats, dogs, monkeys and minipigs. The glucuronides of estradiol, imipramine, serotonin, propofol, 3′‐azido‐3′‐deoxythymidine (AZT) and morphine, selective substrates of human UGT1A1, 1A4, 1A6, 1A9 and 2B7 (AZT and morphine), respectively, were measured using LC‐MS/MS. Estradiol‐3‐glucuronidation activity was higher in the microminipig than in humans and the other animals. High levels of estradiol‐3‐glucuronidation were observed in the microsomes of 1‐day‐old microminipigs. Imipramine‐N‐glucuronidation, a distinctive conjugation by human UGT1A4, was catalysed by microminipig liver microsomes, but not by dog liver microsomes. Although AZT‐glucuronidation activity was low in the microminipig compared with humans, morphine‐3‐glucuronidation activity in the microminipig was higher than that in humans. The UGT activities in the microminipig were similar to those in the minipig. The results of the present study have provided useful information for selecting an appropriate animal model for nonclinical studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Glucuronides of hydroxylated metabolites of amitriptyline and nortriptyline isolated from rat bile

Polar conjugates were isolated from the bile of rats given amitriptyline (AT, unlabeled or labeled with 14C), nortriptyline (NT), or 10-hydroxy (10-OH) derivatives of the drugs. The procedure involved extraction on a column of polystyrene resin, elution with methanol, and separation by preparative TLC followed by reversed phase HPLC. Individual metabolites were characterized by NMR spectroscopy and fast atom bombardment mass spectrometry and by enzymatic or acid deconjugation with subsequent identification of aglycones and glucuronic acid. Conversely, they were compared with conjugates obtained from hydroxy compounds by incubation with rat liver microsomes and UDP-glucuronic acid. Glucuronides isolated from the bile of rats given AT were derived from 2-OH-AT, (E)- and (Z)-10-OH-AT, 2-hydroxy-3-methoxy- (or 3-hydroxy-2-methoxy) AT, 10, 11-(OH)2-AT, and some of the N-demethylated analogues of these compounds. In most cases, 10-OH compounds form two diastereoisomeric glucuronides produced from the enantiomeric alcohols; 10, 11-(OH)2 metabolites occur as cis- and trans-isomers that are conjugated with glucuronic acid. Administration of synthetic (E)- and (Z)-10-OH-AT and -NT leads to the excretion of their glucuronides along with conjugates formed after demethylation and/or introduction of a second OH group. NT gives rise to 2-OH-NT glucuronide besides those conjugates derived from (E)-10-OH-NT. No glutathione conjugates could be detected.     

Treatment of depression with E-10-hydroxynortriptyline — a pilot study on biochemical effects and pharmacokinetics

The major metabolite of nortriptyline, i.e. E-10-hydroxynortriptyline (E-10-OH-NT), was given as a racemate in increasing doses from 75 to 225 mg/day to five patients with major depressive episode. Plasma concentrations of both the (–)- and (+)-enantiomers were linearly related to the doses. The mean ratio between them was 3.6±0.53, indicating stereospecific kinetics during maintenance treatment. Lumbar punctures were performed in four of the patients before and after 3 weeks of E-10-OH-NT treatment. There was a 18% mean decrease (P<0.01) in the noradrenaline metabolite HMPG in cerebrospinal fluid (CSF), supporting previous in vitro data showing that E-10-OH-NT inhibits noradrenaline uptake in vivo. During treatment, the median depression score measured by the Montgomery-Åsberg Depression Rating Scale declined from 32 to 14 (P<0.05). As the study was open, the clinical outcome is not conclusive but does not contradict the hypothesis that E-10-OH-NT has antidepressant properties. If present at all, side effects were mild and did not interfere with the treatment.     

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.     

Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.     

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。     

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.