Human UDP-glucuronosyltransferase isoforms involved in haloperidol glucuronidation and quantitative estimation of their contribution

A major metabolic pathway of haloperidol is glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT). In this study, we found that two glucuronides were formed by the incubation of haloperidol with human liver microsomes (HLM) and presumed that the major and minor metabolites (>10-fold difference) were O- and N-glucuronide, respectively. The haloperidol N-glucuronidation was catalyzed solely by UGT1A4, whereas the haloperidol O-glucuronidation was catalyzed by UGT1A4, UGT1A9, and UGT2B7. The kinetics of the haloperidol O-glucuronidation in HLM was monophasic with K(m) and V(max) values of 85 μM and 3.2 nmol · min?1 · mg?1, respectively. From the kinetic parameters of the recombinant UGT1A4 (K(m) = 64 μM, V(max) = 0.6 nmol · min?1 · mg?1), UGT1A9 (K(m) = 174 μM, V(max) = 2.3 nmol · min?1 · mg?1), and UGT2B7 (K(m) = 45 μM, V(max) = 1.0 nmol · min?1 · mg?1), we could not estimate which isoform largely contributes to the reaction. Because the haloperidol O-glucuronidation in a panel of 17 HLM was significantly correlated (r = 0.732, p < 0.01) with zidovudine O-glucuronidation, a probe activity of UGT2B7, and the activity in the pooled HLM was prominently inhibited (58% of control) by gemfibrozil, an inhibitor of UGT2B7, we surmised that the reaction would mainly be catalyzed by UGT2B7. We could successfully estimate, using the concept of the relative activity factor, that the contributions of UGT1A4, UGT1A9, and UGT2B7 in HLM were approximately 10, 20, and 70%, respectively. The present study provides new insight into haloperidol glucuronidation, concerning the causes of interindividual differences in the efficacy and adverse reactions or drug-drug interactions.     

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.     

Role of Human UDP-Glucuronosyltransferases in the Biotransformation of the Triazoloacridinone and Imidazoacridinone Antitumor Agents C-1305 and C-1311: Highly Selective Substrates for UGT1A10

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 μl · min(-1) · mg protein(-1), for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 μl · min(-1) · mg protein(-1). In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.     

Identification of UDP-glucuronosyltransferases responsible for the glucuronidation of darexaban, an oral factor Xa inhibitor, in human liver and intestine

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K(m) value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a K(m) value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K(m) value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K(m) for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.     

Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.     

Quantitative Prediction of Human Intestinal Glucuronidation Effects on Intestinal Availability of UDP-Glucuronosyltransferase Substrates Using In Vitro Data

We investigated whether the effects of intestinal glucuronidation on the first-pass effect can be predicted from in vitro data for UDP-glucuronosyltransferase (UGT) substrates. Human in vitro intrinsic glucuronidation clearance (CL(int, UGT)) for 11 UGT substrates was evaluated using pooled intestinal microsomes (4.00-4620 μl · min(-1) · mg(-1)) and corrected by the free fraction in the microsomal mixture (CLu(int), (UGT) = 5.2-5133 μl · min(-1) · mg(-1)). Eleven UGT substrates were stable against intestinal cytochrome P450, indicating intestinal glucuronidation has a main effect on human intestinal availability. Oral absorbability intestinal availability (F(a)F(g)) values were calculated from in vivo pharmacokinetic parameters in the literature (F(a)F(g) = 0.01-1.0). It was found that CLu(int, UGT) and human F(a)F(g) have an inverse relationship that can be fitted to a simplified intestinal availability model. Experiments using Supersomes from insect cells expressing UGT isoforms showed that the substrates used were conjugated by various UGT isoforms. These results suggest that combining the simplified intestinal availability model and in vitro conjugation assay make it possible to predict human F(a)F(g) regardless of UGT isoform.     

Characterization of hepatic and intestinal glucuronidation of magnolol: application of the relative activity factor approach to decipher the contributions of multiple UDP-glucuronosyltransferase isoforms

Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.     

Udp-glucuronosyltransferase 2b7 is the major enzyme responsible for gemcabene glucuronidation in human liver microsomes.

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.     

Identification of the UDP-glucuronosyltransferase isoforms involved in mycophenolic acid phase II metabolism.

Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil is primarily metabolized by glucuronidation. The nature of UDP-glucuronosyltransferases (UGTs) involved in this pathway is still debated. The present study aimed at identifying unambiguously the UGT isoforms involved in the production of MPA-phenyl-glucuronide (MPAG) and MPA-acylglucuronide (AcMPAG). A liquid chromatography-tandem mass spectrometry method allowing the identification and determination of the metabolites of mycophenolic acid was developed. The metabolites were characterized in urine and plasma samples from renal transplant patients under mycophenolate mofetil therapy and in vitro after incubation of mycophenolic acid with human liver (HLM), kidney (HKM), or intestinal microsomes (HIM). The UGT isoforms involved in MPAG or AcMPAG production were investigated using induced rat liver microsomes, heterologously expressed UGT (Supersomes), and chemical-selective inhibition of HLM, HKM, and HIM. The three microsomal preparations produced MPAG, AcMPAG, and two mycophenolate glucosides. Among the 10 UGT isoforms tested, UGT 1A9 was the most efficient for MPAG synthesis with a K(m) of 0.16 mM, close to that observed for HLM (0.18 mM). According to the chemical inhibition experiments, UGT 1A9 is apparently responsible for 55%, 75%, and 50% of MPAG production by the liver, kidney, and intestinal mucosa, respectively. Although UGT 2B7 was the only isoform producing AcMPAG in a significant amount, the selective inhibitor azidothymidine only moderately reduced this production (approximately -25%). In conclusion, UGT 1A9 and 2B7 were clearly identified as the main UGT isoforms involved in mycophenolic acid glucuronidation, presumably due to their high hepatic and renal expression.     

Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP-glucuronosyltransferases. HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP-glucuronic acid or UDP-glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. HKM produced a second metabolite when incubated with MPA and UDP-glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. All recombinant UGTs investigated were able to glucuronidate MPA with K:(M:) values ranging from 115.3 to 275.7 microM l(-1) and V(max) values between 29 and 106 pM min(-1) mg protein(-1). Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.     

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.     

Inhibition of genistein glucuronidation by bisphenol A in human and rat liver microsomes

Genistein is a natural phytoestrogen of the soybean, and bisphenol A (BPA) is a synthetic chemical used in the production of polycarbonate plastics. Both genistein and BPA disrupt the endocrine system in vivo and in vitro. Growing concerns of altered xenobiotic metabolism due to concomitant exposures from soy milk in BPA-laden baby bottles has warranted the investigation of the glucuronidation rate of genistein in the absence and presence (25 μM) of BPA by human liver microsomes (HLM) and rat liver microsomes (RLM). HLM yield V(max) values of 0.93 ± 0.10 nmol · min(-1) · mg(-1) and 0.62 ± 0.05 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively. K(m) values for genistein glucuronidation by HLM in the absence and presence of BPA are 15.1 ± 7.9 μM and 21.5 ± 7.7 μM, respectively, resulting in a K(i) value of 58.7 μM for BPA. Significantly reduced V(max) and unchanged K(m) in the presence of BPA in HLM are suggestive of noncompetitive inhibition. In RLM, the presence of BPA resulted in a K(i) of 35.7 μM, an insignificant change in V(max) (2.91 ± 0.26 nmol · min(-1) · mg(-1) and 3.05 ± 0.41 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively), and an increase in apparent K(m) (49.4 ± 14 μM with no BPA and 84.0 ± 28 μM with BPA), indicative of competitive inhibition. These findings are significant because they suggest that BPA is capable of inhibiting the glucuronidation of genistein in vitro, and that the type of inhibition is different between HLM and RLM.     

Effect of aging on glucuronidation of valproic acid in human liver microsomes and the role of UDP-glucuronosyltransferase UGT1A4, UGT1A8, and UGT1A10

Valproic acid (VPA) is a widely used anticonvulsant that is also approved for mood disorders, bipolar depression, and migraine. In vivo, valproate is metabolized oxidatively by cytochromes P450 and beta-oxidation, as well as conjugatively via glucuronidation. The acyl glucuronide conjugate (valproate-glucuronide or VPAG) is the major urinary metabolite (30-50% of the dose). It has been hypothesized that glucuronidation of antiepileptic drugs is spared over age, despite a known decrease in liver mass. The formation rates of VPAG in a bank of elderly (65 years onward) human liver microsomes (HLMs) were measured by liquid chromatography/tandem mass spectrometry and compared with those in a younger (2-56 years) HLM bank. In vitro kinetic studies with recombinant UDP-glucuronosyltransferases (UGTs) were completed. A 5- to 8-fold variation for the formation of VPAG was observed within the microsomal bank obtained from elderly and younger donors. VPAG formation ranged from 6.0 to 53.4 nmol/min/mg protein at 1 mM substrate concentration (n=36). The average velocities at 0.25, 0.5, and 1 mM VPA were 7.0, 13.4, and 25.4 nmol/min/mg protein, respectively, in the elderly HLM bank. Rates of VPAG formation were not significantly different in the HLM bank obtained from younger subjects. Intrinsic clearances (V(max)/K(m)) for several cloned, expressed UGTs were determined. UGT1A4, UGT1A8, and UGT1A10 also were found to catalyze the formation of VPAG in vitro. This is the first reported activity of these UGTs toward VPA glucuronidation. UGT2B7 had the highest intrinsic clearance, whereas UGT1A1 demonstrated no activity. In conclusion, our investigation revealed no differences in VPAG formation in younger versus elderly HMLs and revealed three other UGTs that form VPAG in vitro.     

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms

1.?Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast.

2.?Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes?>80% of activity at bisphenol-A concentrations under 5?μM, while UGT1A9 contributes up to 50% of activity at higher concentrations.

3.?Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p?=?0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes.

4.?Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p?=?0.006).

5.?UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.     

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

1. 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.

2. 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.

3. 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 (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. 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.

     

In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.     

Method for predicting human intestinal first-pass metabolism of UGT substrate compounds

As intestinal glucuronidation has been suggested to generate the low oral bioavailability (F) of drugs, estimating its effects would be valuable for selecting drug candidates. Here, we investigated the absorption and intestinal availability (F(a)F(g)) in animals, and intrinsic clearance via UDP-glucuronosyltransferase (UGT) in intestinal microsomes (CL(int,UGT)) for three drug candidates possessing a carboxylic acid group, in an attempt to estimate the impact of intestinal glucuronidation on F and select potential drug candidates with high F in humans. The F(a)F(g) values of the three test compounds were low in rats and monkeys (0.16-0.51), and high in dogs (≥0.81). Correspondingly, the CL(int,UGT) values were high in rats and monkeys (101-731 μL/min/mg), and low in dogs (≤?59.6 μL/min/mg). A good inverse correlation was observed between F(a)F(g) and CL(int,UGT), suggesting that intestinal glucuronidation was a major factor influencing F(a)F(g) of these compounds. By applying this correlation to F(a)F(g) in humans using human CL(int,UGT) values (26.9-114 μL/min/mg), compounds 1-3 were predicted to have relatively high F(a)F(g). Our approach is expected to be useful for estimating the impact of intestinal glucuronidation on F in animals and semiquantitatively predicting human F for drug candidates.     

Human UDP‐Glucuronosyltransferases 1A1, 1A3, 1A9, 2B4 and 2B7 are Inhibited by Diethylstilbestrol

Inhibition of UDP‐glucuronosyltransferases (UGTs) can result in many undesired side effects. Diethylstilbestrol (DES), a synthetic oestrogen famous for its multiple toxicities, was once widely administered to women in high dosages and now still gains application in clinics. This study investigated in vitro inhibitory effects of DES on catalytic activities of human UGTs, aiming at disclosing new potential toxic mechanisms on the basis of interactions between DES and metabolizing enzymes. DES (10 μM) could decrease activities of UGT1A1, 1A3, 1A9, 2B4 and 2B7 in catalysing 4‐methylumbelliferone (4‐Mu) glucuronidation. Further kinetic analyses showed that inhibition of these UGTs followed competitive (UGT1A1 and 1A9), mixed (UGT1A3 and 2B4) and non‐competitive (UGT2B7) mechanisms, with K_i values ranging from 0.91 to 4.1 μM. The inhibition potentials of UGT1A9 and 2B7 in human liver microsomes (HLM) were further tested by employing propofol and zidovudine as probe substrates, respectively. The inhibition of human liver microsomal UGT1A9 followed mixed mechanism, with the K_i value of 3.5 μM and α of 4.1. On the other hand, DES displayed non‐competitive inhibition against UGT2B7 in HLM, with the K_i value of 9.8 μM. The risks of in vivo inhibition of human UGTs were also predicted by calculation of plasma C/K_i values. Results suggest that DES can trigger in vivo inhibition of UGT1A1, 1A3, 1A9, 2B4 and 2B7 after the intravenous administration in high doses.     

Almokalant glucuronidation in human liver and kidney microsomes: evidence for the involvement of UGT1A9 and 2B7

1. Almokalant, a class III antiarrythmic drug, is metabolized to form isomeric glucuronides identified in human urine. Synthesis of the total glucuronide was studied in human liver and kidney microsomes. Recombinant UDP-glucuronosyltransferases (UGTs) were screened for activity and kinetic analysis was performed to identify the isoform(s) responsible for the formation of almokalant glucuronide in man. 2. From a panel of recombinant isoforms used, both UGT1A9 and 2B7 catalysed the glucuronidation of almokalant. The Km values in both instances were similar with 1.06 mM for the 1A9 and 0.97 mM for the 2B7. Vmax for 1A9 was fourfold higher than that measured for UGT2B7, 92 compared with 21 pmol min(-1) mg(-1), respectively, but UGT1A9 was expressed at approximately twofold higher level than the UGT2B7 in the recombinant cell lines. Therefore, the contribution of UGT2B7 to almokalant glucuronidation could be as significant as that of UGT1A9 in man. 3. Liver and kidney microsomes displayed similar Km values to the cloned expressed UGTs, with the liver and kidney microsomes at 1.68 and 1.06 mM almost identical to the 1A9. 4. The results suggest a significant role for UGT1A9 and 2B7 in the catalysis of almokalant glucuronidation.     

Structure- and isoform-specific glucuronidation of six curcumin analogs

1.?In the present study, we aimed to characterize the glucuronidation of six curcumin analogs (i.e. RAO-3, RAO-8, RAO-9, RAO-18, RAO-19, and RAO-23) derived from galangal using human liver microsomes (HLM) and twelve expressed UGT enzymes.

2.?Formation of glucuronide was confirmed using high-resolution mass spectrometry. Single glucuronide metabolite was generated from each of six curcumin analogs. The fragmentation patterns were analyzed and were found to differ significantly between alcoholic and phenolic glucuronides.

3.?All six curcumin analogs except one (RAO-23) underwent significant glucuronidation in HLM and expressed UGT enzymes. In general, the methoxy group (close to the phenolic hydroxyl group) enhanced the glucuronidation liability of the curcumin analogs.

4.?UGT1A9 and UGT2B7 were primarily responsible for the glucuronidation of two alcoholic analogs (RAO-3 and RAO-18). By contrast, UGT1A9 and four UGT2Bs (UGT2B4, 2B7, 2B15 and 2B17) played important roles in conjugating three phenolic analogs (RAO-8, RAO-9, and RAO-19). Interestingly, the conjugated double bonds system (in the aliphatic chain) was crucial to the substrate selectivity of gastrointestinal UGTs (i.e. UGT1A7, 1A8 and 1A10).

5.?In conclusion, glucuronidation of six curcumin analogs from galangal were structure- and isoform-specific. The knowledge should be useful in identifying a curcumin analog with improved metabolic property.