Evaluation of 5-hydroxytryptophol and other endogenous serotonin (5-hydroxytryptamine) analogs as substrates for UDP-glucuronosyltransferase 1A6.

Serotonin is a specific in vitro substrate for human UDP-glucuronosyltransferase (UGT) 1A6. In this study, the contribution of UGT1A6 to the glucuronidation of endogenous structural analogs of serotonin, including 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin, was evaluated using available recombinant human UGT isoforms, human liver microsomes, and liver microsomes from animals that do not express functional UGT1A6 (Gunn rats and cats). Only UGT1A6 and UGT1A9 were found to glucuronidate 5-hydroxytryptophol at a concentration of 2 mM, although the glucuronidation rate with UGT1A6 was over 10 times that of UGT1A9. K(m) values for human liver microsomes (156, 141, and 134 microM) were most similar to that of expressed UGT1A6 (135 microM) but vastly different from that of UGT1A9 (3674 microM). 5-Hydroxytryptophol glucuronidation by human liver microsomes (n = 54) correlated well with serotonin glucuronidation (R(s) = 0.83) and UGT1A6 protein content (R(s) = 0.85). 5-Hydroxytryptophol also competitively inhibited serotonin glucuronidation by human liver microsomes (K(i) = 291 microM) and UGT1A6 (K(i) = 200 microM). N-acetylserotonin was glucuronidated most extensively by UGT1A6, although UGT1A9 and UGT1A10 showed moderate catalysis. 6-Hydroxymelatonin was glucuronidated largely by UGT1A9 and UGT1A10 but not at all by UGT1A6. Gunn rat liver glucuronidation rates for serotonin, 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin were 11, 5, 32, and 3%, respectively, of that of normal rat liver. Cat liver microsomes did not glucuronidate serotonin, whereas relatively low activities were observed for the other indole substrates. In conclusion, these results indicate that human UGT1A6 plays a predominant role in the glucuronidation of 5-hydroxytryptophol and N-acetylserotonin, whereas 6-hydroxymelatonin is not a substrate for this enzyme.     

Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9.

(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. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.     

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.     

The glucuronidation of Delta4-3-Keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs): 6alpha- and 21-hydroxyprogesterone are selective substrates for UGT2B7.

The stereo- and regioselective glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-glucuronidation and the high-affinity component of 16alpha-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.     

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.     

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin.

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.     

Stereoselective glucuronidation and hydroxylation of etodolac by UGT1A9 and CYP2C9 in man

1. In vitro metabolic studies with etodolac were performed. S- and R-etodolac were converted to the acylglucuronide and hydroxylated metabolites by UDP-glucuronosyltransferase (UGT) and cytochrome P450 in microsomes. However, the stereoselectivities of UGT and P450 for the isomers were opposite. S-etodolac was glucuronidated preferentially than R-etodolac by UGT. In contrast, R-etodolac was hydroxylated preferentially than S-etodolac by P450. 2. Of several human P450 enzymes, CYP2C9 had the greatest activity for hydroxylation of R-etodolac. Sulfaphenazole, an inhibitor of CYP2C9, and anti-CYP2C9 antibody inhibited the hydroxylation of R-etodolac in human liver microsomes. CYP2C9 therefore contributes to the stereoselective hydroxylation of R-etodolac. 3. Of several human UGT enzymes, UGT1A9 had the greatest activity for glucuronidation of S-etodolac. Propofol and thyroxine, inhibitors of UGT1A9, inhibited the glucuronidation of S-etodolac in human liver microsomes. Therefore, UGT1A9 is mainly responsible for the stereoselective glucuronidation of S-etodolac. 4. Because S-etodolac was metabolized more rapidly than R-etodolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolac substantially influenced the overall metabolism of S- and R-etodolac in man.     

Glucuronidation of etoposide in human liver microsomes is specifically catalyzed by UDP-glucuronosyltransferase 1A1.

A metabolite formed by incubation of human liver microsomes, etoposide, and UDP-glucuronic acid was identified as etoposide glucuronide by liquid chromatography-tandem mass spectrometry analysis. According to the derivatization with trimethylsilylimidazole (Tri-Sil-Z), it was confirmed that the glucuronic acid is linked to an alcoholic hydroxyl group of etoposide and not to a phenolic group. Among nine recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9. UGT1A10, UGT2B7, and UGT2B15), only UGT1A1 exhibited the catalytic activity of etoposide glucuronidation. The enzyme kinetics in pooled human liver microsomes and recombinant UGT1A1 microsomes showed a typical Michaelis-Menten plot. The kinetic parameters of etoposide glucuronidation were K(m) = 439.6 +/- 70.7 microM and V(max) = 255.6 +/- 19.2 pmol/min/mg of protein in human liver microsomes and K(m) = 503.2 +/- 110.2 microM and V(max) = was 266.5 +/- 28.6 pmol/min/mg of protein in recombinant UGT1A1. The etoposide glucuronidation in pooled human liver microsomes was inhibited by bilirubin (IC(50) = 31.7 microM) and estradiol (IC(50) = 34 microM) as typical substrates for UGT1A1. The inhibitory effects of 4-nitrophenol (IC(50) = 121.0 microM) as a typical substrate for UGT1A6 and UGT1A9, imipramine (IC(50) = 393.8 microM) as a typical substrate for UGT1A3 and UGT1A4, and morphine (IC(50) = 109.3 microM) as a typical substrate for UGT2B7 were relatively weak. The interindividual difference in etoposide glucuronidation in 13 human liver microsomes was 78.5-fold (1.4-109.9 pmol/min/mg of protein). The etoposide glucuronidation in 10 to 13 human liver microsomes was significantly correlated with beta-estradiol-3-glucuronidation (r = 0.841, p < 0.01), bilirubin glucuronidation (r = 0.935, p < 0.01), and the immunoquantified UGT1A1 protein content (r = 0.800, p < 0.01). These results demonstrate that etoposide glucuronidation in human liver microsomes is specifically catalyzed by UGT1A1.     

Substrate-dependent modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1) by propofol in recombinant human UGT1A1 and human liver microsomes

Our previous study has shown that propofol, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A9, activated the glucuronidation of 4-methylumbelliferone (4-MU) by recombinant UGT1A1 in a concentration-dependent manner. In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a K(m) value of 101 microM in the absence of propofol. In the presence of 200 microM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the V(max) value increased to 1.5-fold, while the K(m) value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3beta-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3beta-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC(50) values of 59 and 228 microM, respectively. In addition, a known UGT1A1 modulator, 17alpha-ethynyloestradiol, stimulated oestradiol 3beta-glucuronidation slightly at a concentration of 5 microM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5-100 microM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate.     

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen

AIMS: To characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. METHODS: Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. RESULTS: Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K(m) values (+/-SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 +/- 13 microm (16, 43) and 473 +/- 108 microm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K(m) (72 microm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. CONCLUSION: UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.     

13-hydroxy- and 13-oxooctadecadienoic acids: novel substrates for human UDP-glucuronosyltransferases.

Although there are numerous studies of glucuronidation of endogenous compounds, information on the glucuronidation of fatty acids is lacking. In the present studies, both linoleic acid (LA) and its biologically active oxidized derivatives, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-oxooctadecadienoic acid (13-OXO), have been shown to be effective substrates for human liver UDP-glucuronosyltransferases (UGT) and recombinant UGT2B7. LA (carboxyl glucuronide) and 13-OXO (carboxyl glucuronide, unproven) were actively glucuronidated by human liver microsomes (HLM) and human recombinant UGT2B7 with similar activities, in the range of 2 nmol/mg. min. The hydroxyl derivative of LA, 13-HODE, was glucuronidated at both the hydroxyl and carboxyl functions with carboxyl glucuronidation predominating (ratio of COOH/OH, 2:1). For all substrates, the K(m) for formation of the carboxyl-linked glucuronide was in the range of 100 to 200 microM while that for the hydroxyl-linked glucuronide was somewhat lower (>100 microM). This is the first demonstration of glucuronidation of LA and its oxidized derivatives, 13-HODE and 13-OXO, by HLM and recombinant UGT2B7.     

Enantiomer Selective Glucuronidation of the Non‐Steroidal Pure Anti‐Androgen Bicalutamide by Human Liver and Kidney: Role of the Human UDP‐Glucuronosyltransferase (UGT)1A9 Enzyme

Bicalutamide (Casodex^®) is a non‐steroidal pure anti‐androgen used in the treatment of localized prostate cancer. It is a racemate drug, and its activity resides in the (R)‐enantiomer, with little in the (S)‐enantiomer. A major metabolic pathway for bicalutamide is glucuronidation catalysed by UDP‐glucuronosyltransferase (UGT) enzymes. While (S)bicalutamide is directly glucuronidated, (R)bicalutamide requires hydroxylation prior to glucuronidation. The contribution of human tissues and UGT isoforms in the metabolism of these enantiomers has not been extensively investigated. In this study, both (R) and/or (S)bicalutamide were converted into glucuronide (‐G) derivatives after incubation of pure and racemic solutions with microsomal extracts from human liver and kidney. Intestinal microsomes exhibited only low reactivity with these substrates. Km values of liver and kidney samples for (S)bicalutamide glucuronidation were similar, and lower than values obtained with the (R)‐enantiomer. Among the 16 human UGTs tested, UGT1A8 and UGT1A9 were able to form both (S) and (R)bicalutamide‐G from pure or racemic substrates. UGT2B7 was also able to form (R)bicalutamide‐G. Kinetic parameters of the recombinant UGT2B7, UGT1A8 and UGT1A9 enzymes support a predominant role of the UGT1A9 isoform in bicalutamide metabolism. Accordingly, (S)bicalutamide inhibited the ability of human liver and kidney microsomes to glucuronidate the UGT1A9 probe substrate, propofol. In conclusion, the present study provides the first comprehensive analysis of in vitro bicalutamide glucuronidation by human tissues and UGTs and identifies UGT1A9 as a major contributor for (R) and (S) glucuronidation in the human liver and kidney.     

Sequential metabolism of 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) by cytochrome P450 and UDP-glucuronosyltransferase in human liver microsomes.

Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) was examined using a recombinant enzyme system and human liver microsomes. We analyzed the glucuronidation of 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) by rat CYP1A1 expressed in yeast microsomes and human UGT expressed in baculovirus-infected insect cells. Multiple UGT isozymes showed glucuronidation activity toward 8-hydroxy-2,3,7-triCDD (8-OH-2,3,7-triCDD), which was produced by CYP1A1. Of these UGTs, UGT1A1, 1A9, and 2B7, which are constitutively expressed in human livers, showed remarkable activity toward 8-OH-2,3,7-triCDD. The apparent kinetic parameters of glucuronidation, K(m) and k(cat), were estimated to be 0.8 microM and 1.8 min(-1), respectively, for UGT1A1, 0.8 microM and 1.8 min(-1), respectively, for UGT1A9, and 3.9 microM and 7.0 min(-1), respectively, for UGT2B7. In human liver microsomes with NADPH and UDP-glucuronic acid, 2,3,7-triCDD was first converted to 8-OH-2,3,7-triCDD, then further converted to its glucuronide. We compared the ability of 10 human liver microsomes to metabolize 2,3,7-triCDD and observed a significant difference in the glucuronidation of 2,3,7-triCDD that originated from the difference of the P450-dependent hydroxylation of 2,3,7-triCDD.     

In vitro characterisation of human renal and hepatic frusemide glucuronidation and identification of the UDP-glucuronosyltransferase enzymes involved in this pathway

In order to gain insights into the renal and hepatic glucuronidation of frusemide (FSM), this study: (i) characterised the kinetics of FSM glucuronidation by human liver microsomes (HLM) and human kidney cortical- (HKCM) and medullary- (HKMM) microsomes, and (ii) identified the human UDP-glucuronosyltransferase enzyme(s) involved in this pathway. HLM, HKCM and HLMM efficiently glucuronidated FSM. FSM glucuronide (FSMG) formation followed Michaelis-Menten kinetics in all tissues. While the mean K(m) for FSMG formation by HKMM (386 +/- 68 microM) was lower than the K(m) values for HLM (988 +/- 271 microM) and HKCM (704 +/- 278 microM), mean V(max)/K(m) values were comparable for the three tissues. A panel of recombinant UGT enzymes was screened for the capacity to glucuronidate FSM. UGT 1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 metabolised FSM. Of the renally and hepatically expressed enzymes, comparison of kinetic parameters suggests a predominant role of UGT1A9 in FSM glucuronidation, although UGT1A1 may also contribute to FSMG formation by HLM. Consistent with these observations, the UGT1A selective inhibitors phenylbutazone and sulfinpyrazone decreased FSMG formation by HLM, HKCM and HKMM by 60-80%, whereas the UGT2B7 selective inhibitor fluconazole reduced FSM glucuronidation by < or =20%. The ability of HKCM and HKMM to form FSMG supports the proposition that the kidney is the main organ involved in FSM glucuronidation in vivo, although a role for hepatic metabolism remains a possibility in renal dysfunction. The data further demonstrate the potential importance of both the medulla and cortex in renal drug metabolism and detoxification.     

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.     

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.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K(m)?=?45.1 μM, V(max)?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K(m)?=?81.3 μM, V(max)?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC(50)) values of 173.6 and 76.2 μM, respectively. Enzyme kinetics was also performed in HIMs. The K(m) value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K(m)?=?58.6 μM, V(max)?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.     

Glucuronidation of the dietary fatty acids, phytanic acid and docosahexaenoic acid, by human UDP-glucuronosyltransferases.

Linoleic acid has recently been shown to be glucuronidated in vitro by human liver and intestinal microsomes and recombinant UGT2B7. In the present study, the dietary fatty acids (FA), phytanic acid (PA), and docosahexaenoic acid (DHA) have been used as substrates for human UDP-glucuronosyltransferases (UGTs). Both compounds were effectively glucuronidated by human liver microsomes (HLM; 1.25 +/- 0.36 and 1.12 +/- 0.32 nmol/mg x min for PA and DHA, respectively) and UGT2B7 (0.71 and 0.53 nmol/mg x min). Kinetic analysis produced relatively low K(m) values for PA with both HLM and UGT2B7 (149 and 108 microM, respectively). The K(m) for DHA glucuronidation by HLM (460 microM) was considerably higher than that for UGT2B7 (168 microM), suggesting the involvement in microsomes of other UGT isoforms in addition to UGT2B7. Glucuronidation of PA and DHA by gastrointestinal microsomes from 16 human subjects was determined. In general, both PA and DHA were glucuronidated by gastric and intestinal microsomes, and activity toward both substrates was lowest in the stomach, increased in the small intestine, and lower in the colon. However, there were large interindividual variations in UGT activity toward both substrates in all segments of the intestine, as has been seen with other substrates. Thus, PA and DHA are effective in vitro substrates for human liver, gastric and intestinal microsomes, and glucuronidation may play a role in modulating the availability of these FA as ligands for nuclear receptors.     

Altered AZT (3'-azido-3'-deoxythymidine) glucuronidation kinetics in liver microsomes as an explanation for underprediction of in vivo clearance: comparison to hepatocytes and effect of incubation environment.

Human liver microsomes are a reagent commonly used to predict human hepatic clearance of new chemical entities via phase 1 metabolism. Another common metabolic pathway, glucuronidation, can also be observed in human liver microsomes, although the scalability of this process has not been validated. In fact, several groups have demonstrated that clearance estimated from liver microsomes with UDP-glucuronic acid typically underpredicts the actual in vivo clearance more than 10-fold for compounds that are predominantly glucuronidated. In contrast, clearance predicted using human hepatocytes, for these same compounds, provides a more accurate assessment of in vivo clearance. We sought to characterize the kinetics of glucuronidation of the selective UGT2B7 substrate AZT (3'-azido-3'-deoxythymidine), a selective UGT2B7 substrate, in human liver microsomes (HLMs), recombinant UGT2B7, and human hepatocytes. Apparent Km values in these three preparations were 760, 490, and 87 microM with apparent Vmax values highest in hepatocytes. The IC50 for ibuprofen against AZT glucuronidation, when run at its Km concentration in HLMs and hepatocytes, was 975 and 170 microM respectively. Since incubation conditions have been shown to modulate glucuronidation rates, AZT glucuronidation was performed in various physiological and nonphysiological buffer systems, namely Tris, phosphate, sulfate, carbonate, acetate, human plasma, deproteinized human liver cytosol, and Williams E medium. The data showed that carbonate and Williams E medium, more physiologically relevant buffers, yielded the highest rates of AZT glucuronidation. Km observed in HLM/carbonate was 240 microM closer to that found in hepatocytes, suggesting that matrix differences might cause the kinetic differences observed between liver preparations. Caution should be exercised when extrapolating metabolic lability via glucuronidation or inhibition of UGT enzymes from human liver microsomes, since this system appears to underpredict the degree of lability or inhibition, respectively, due in part to an apparent decrease in substrate affinity.     

Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases

Mitiglinide (MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus. In the present study, a potential metabolic pathway of MGN, via carboxyl-linked glucuronic acid conjugation, was found. MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. Kinetic analysis indicated that MGN from four species had the highest affinity for the rabbit liver microsomal enzyme (K(m)=0.202 mM) and the lowest affinity for the dog liver microsomal enzyme (K(m)=1.164 mM). The metabolic activity (V(max)/K(m)) of MGN to the carboxyl-glucuronidation was in the following order: rabbit>dog>rat>human. With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7), only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity. The K(m) values of MGN glucuronidation in recombinant UGT1A3 and UGT2B7 microsomes were close to those in human liver microsomes. The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin (substrate for UGT1A3) and diclofenac (substrate for UGT2B7), respectively. The MGN glucuronidation activities in 15 human liver microsomes were significantly correlated with quercetin (r(2)=0.806) and diclofenac glucuronidation activities (r(2)=0.704), respectively. These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.