Recombinant UDP-glucuronosyltransferases Research Articles

Influence of substrates on the in vitro kinetics of steviol glucuronidation and interaction between steviol glycosides metabolites and UGT2B7

Steviol glycosides, a natural sweetener, may perform bioactivities via steviol, their main metabolite in human digestion. The metabolising kinetics, i.e. glucuronidation kinetics and interaction between steviol glycosides or their metabolites and metabolising enzyme, are important for understanding the bioactivity and cytotoxicity. The present study investigated kinetics of steviol glucuronidation in human liver microsome and a recombinant human UDP-glucuronosyltransferases isomer, UGT2B7, along with molecular docking to analyse interaction between UGT2B7 and steviol or glucose. The active pocket of UGT2B7 is consisted of Arg352, Leu347, Lys343, Phe339, Tyr354, Lys355 and Leu353. The influence of stevioside, rebaudioside A, glucose and some chemotherapy reagents on the glucuronidation was also studied. The predicted hepatic clearence suggested that steviol could be classified as high-clearence drug. The steviol glycosides did not affect the glucuronidation of steviol notably.

Involvement of UDP-glucuronosyltransferases in higenamine glucuronidation and the gender and species differences in liver

ObjectivesHigenamine (HG), an active ingredient of Aconite root in Chinese herbal medicine, is mainly metabolized by UDP-glucuronosyltransferases (UGT). However, the systematic glucuronidation of HG in humans remains unclear. The purpose of this study was to investigate the glucuronidation of HG. Methods12 recombinant human UGT (rUGT) isozymes were used to characterize the HG glucuronidation. Liver microsomes from male and female mice, rats, guinea pigs, dogs, and humans were used to determine the species and gender differences using liquid chromatography-mass spectrometry. Key findingsOne monoglucuronide was detected in reactions catalyzed by rUGT1A6, rUGT1A8, rUGT1A9, also human and dog liver microsomes. UGT1A9 is the most important glucuronosyltransferase that metabolizes HG. Because carvacrol, a specific inhibitor of UGT1A9, can significantly decrease the glucuronidation of HG in Human liver microsomes and UGT1A9. HG metabolism by UGT1A9 described in Michaelis-Menten kinetics (Km=15.4 mM,Vmax=2.2 nmol/mg/min) and glucuronidation in liver microsomes were species dependent. Gender did not affect the kinetic parameters among species except in rats. ConclusionsUGT1A9 is a major isoenzyme responsible for the glucuronidation of HG in Human liver microsomes (HLMs). Dog may be an appropriate animal model to evaluate HG metabolism.

Glucuronidation and its impact on the bioactivity of [6]-shogaol.

-shogaol (6S) from ginger has been reported to have diverse bioactivities and can be widely metabolized in animals and humans; however, the impact of glucuronidation on its bioactivity is still largely unknown. This study investigates the glucuronidation of 6S and its effect on cell cytotoxicity and Nrf2-inducing activities of 6S. The glucuronidated metabolite of 6S, 4-O-monoglucuronide 6S (6S-G), was synthesized and characterized for the first time. Glucuronidation of 6S in humans was studied using microsomes of the liver and intestine and recombinant UDP-glucuronosyltransferase (UGTs). The kinetics of 6S glucuronidation by human liver and intestinal microsomes followed the substrate inhibition kinetics model. The intrinsic glucuronidation clearance (CLint ) of 6S in human liver microsomes was higher than that in human intestine microsomes. Among the recombinant UGTs examined, UGT1A1, 1A3, 1A6, 1A8, 1A10, 2B7, 2B15, and 2B17 exhibited glucuronidation activity toward 6S, with UGT2B7 being the most potent one. Compared with 6S, the glucuronidation of 6S largely eliminated its cell cytotoxicity against human colon cancer cell lines HT-116 and HT-29, and its Nrf2-inducing activity. The findings from current study provide foundations for understanding the role of glucuronidation in biotransformation and biological activities of 6S.

Hepatic glucuronidation of 4-tert-octylphenol in humans: inter-individual variability and responsible UDP-glucuronosyltransferase isoforms.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. The purpose of this study was to assess inter-individual variability in and the possible roles of UGT isoforms in hepatic 4-tOP glucuronidation in the humans. 4-tOP glucuronidation activities in the liver microsomes and recombinant UGTs of humans were assessed at broad substrate concentrations, and kinetics were analyzed. Correlation analyses between 4-tOP and diclofenac or 4-hydroxybiphenyl activities in pooled and individual human liver microsomes were also performed. Typical CLint values were 17.8mL/min/mg protein for the low type, 25.2mL/min/mg protein for the medium type, and 47.7mL/min/mg protein for the high type. Among the recombinant UGTs (13 isoforms) examined, UGT2B7 and UGT2B15 were the most active of catalyzing 4-tOP glucuronidation. Although the K m values of UGT2B7 and UGT2B15 were similar (0.36 and 0.42µM, respectively), the CLint value of UGT2B7 (6.83mL/min/mg protein) >UGT2B15 (2.35mL/min/mg protein). Strong correlations were observed between the glucuronidation activities of 4-tOP and diclofenac (a probe for UGT2B7) or 4-hydroxybiphenyl (a probe for UGT2B15) with 0.79-0.88 of Spearman correlation coefficient (r s) values. These findings demonstrate that 4-tOP glucuronidation in humans is mainly catalyzed by hepatic UGT2B7 and UGT2B15, and suggest that these UGT isoforms play important and characteristic roles in the detoxification of 4-tOP.

Metabolism studies on hydroxygenkwanin and genkwanin in human liver microsomes by UHPLC-Q-TOF-MS

Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.

Human UDP-glucuronosyltransferase UGT1A4 forms tertiary N-glucuronides predominately with the energetically less favored tautomer of substituted 1H-indazole (benzpyrazole)

BackgroundHuman UDP-glucuronosyltransferase UGT1A4 is reported to convert a range of tertiary amine substrates to the respective quaternary glucuronides. In this study, we report on the in vitro glucuronidation of a substituted 1H-indazole (benzpyrazole), a transient receptor potential ankyrin 1 antagonist.MethodsDepending on the mammalian liver used for production, two peaks with different UV spectra and slightly different MS/MS spectra were found in the LC-MS/MS analysis. An optimized HPLC method gave a baseline separation of the two glucuronides. Preparation and isolation allowed to assign their structure by 1H-, 13C-, and 15N-NMR.ResultsBoth nitrogens of this structure were accessible to glucuronidation resulting in the respective tertiary glucuronides. As expected, the N-glucuronidation proceeded with marked species difference. Surprisingly, the glucuronide produced by human liver homogenate and by the majority of liver preparations from 11 animal species was attached at the energetically less favorable 2-position, indicating the regioselectivity of the respective enzyme. Among 13 recombinant human UDP-glucuronosyltransferases tested only the isoenzyme 1A4 showed significant formation of both glucuronides with the energetically less favorable N-glucuronide in 33-fold excess. An MS/MS fragmentation mechanism was proposed for a fragment loss of 134 Da that differentiated the spectra of the two glucuronides.ConclusionsThis investigation shows that assumptions on the structure of metabolites based on stability of tautomers may be wrong. A novel meachanism to form tertiary N-glucuronides was proposed.

Hepatic and renal metabolism of genistein: An individual-based model to predict glucuronidation behavior of genistein in different organs

The present study aims to develop an individual-based model to predict the glucuronidation characteristics of genistein in 45 human livers. The model was validated in 18 human kidneys. Ten UDP-glucuronosyltransferases (UGTs) were expressed in liver abundantly. Among them, UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 contributed 95.6% to genistein glucuronidation in human liver, with significant correlation between gene expression of the five UGTs and the glucuronidation of genistein. The genistein glucuronidation differences between individuals were varied enormously; meanwhile the expression variations can explain 24.7% total metabolic variation in livers. In kidney, UGT1A6, UGT1A9 and UGT2B7 were amply expressed. Their gene expressions and glucuronidation rates of genistein had high correlations. Expression variations of UGT1A9/2B7 can explain up to 40.9% of the total variation of genistein glucuronidation in kidney. The model was therefore established based on UGT expression between individuals taking into account an important assessment which is the ratio of metabolic rate of each recombinant UGT isoform. Excellent linear correlations between predicted and observed glucuronidation rate revealed that the model could predict metabolic activity of genistein using quite a small size of tissue. In conclusion, the individual-based model can be employed for predicting individual glucuronidation behavior of genistein in different organs.

Inhibition of human UDP-glucuronosyltransferase enzymes by lapatinib, pazopanib, regorafenib and sorafenib: Implications for hyperbilirubinemia

Kinase inhibitors (KIs) are a rapidly expanding class of drugs used primarily for the treatment of cancer. Data relating to the inhibition of UDP-glucuronosyltransferase (UGT) enzymes by KIs is sparse. However, lapatinib (LAP), pazopanib (PAZ), regorafenib (REG) and sorafenib (SOR) have been implicated in the development of hyperbilirubinemia in patients. This study aimed to characterise the role of UGT1A1 inhibition in hyperbilirubinemia and assess the broader potential of these drugs to perpetrate drug–drug interactions arising from UGT enzyme inhibition. Twelve recombinant human UGTs from subfamilies 1A and 2B were screened for inhibition by LAP, PAZ, REG and SOR. IC50 values for the inhibition of all UGT1A enzymes, except UGT1A3 and UGT1A4, by the four KIs were <10μM. LAP, PAZ, REG and SOR inhibited UGT1A1-catalysed bilirubin glucuronidation with mean IC50 values ranging from 34nM (REG) to 3734nM (PAZ). Subsequent kinetic experiments confirmed that REG and SOR were very potent inhibitors of human liver microsomal β-estradiol glucuronidation, an established surrogate for bilirubin glucuronidation, with mean Ki values of 20 and 33nM, respectively. Ki values for LAP and PAZ were approximately 1- and 2-orders of magnitude higher than those for REG and SOR. REG and SOR were equipotent inhibitors of human liver microsomal UGT1A9 (mean Ki 678nM). REG and SOR are the most potent inhibitors of a human UGT enzyme identified to date. In vitro–in vivo extrapolation indicates that inhibition of UGT1A1 contributes significantly to the hyperbilirubinemia observed in patients treated with REG and SOR, but not with LAP and PAZ. Inhibition of other UGT1A1 substrates in vivo is likely.

Introduction of an N-Glycosylation Site into UDP-Glucuronosyltransferase 2B3 Alters Its Sensitivity to Cytochrome P450 3A1-Dependent Modulation.

Our previous studies have demonstrated functional protein–protein interactions between cytochrome P450 (CYP) 3A and UDP-glucuronosyltransferase (UGT). However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis. A baculovirus-Sf-9 cell system for expressing CYP3A1 and UGT2B3/UGT2B3(S316N) was established using a Bac-to-Bac system. Glycosylation of UGT2B3(S316N) was demonstrated in this expression system. The microsomal activity of recombinant UGT was determined using 4-methylumbelliferone as a substrate. The effect of CYP3A1 co-expression on UGT function was examined by comparing the kinetic profiles between single (UGT alone) and double expression (UGT plus CYP) systems. The kinetics of the two expression systems fitted a Michaelis-Menten equation. When the 4-MU concentration was varied, co-expression of CYP3A1 lowered the Vmax of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N), the Vmax in the dual expression system was higher than that in the single expression system. The data obtained demonstrate that the introduction of N-glycosylation to UGT2B3 alters its sensitivity to CYP3A1-dependent modulation while CYP3A1 enhanced UGT2B3(S316N) activity, and wild-type UGT2B3 was suppressed by CYP3A1. These data suggest that N-glycosylation of UGT is one of the determinants regulating the interaction between CYP3A and UGT.

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.

The inhibitory effects of nor-oleanane triterpenoid saponins from Stauntonia brachyanthera towards UDP-glucuronosyltransferases

The inhibition of UDP-glucuronosyltransferases (UGTs) by herbal components might be an important reason for clinical herb-drug interaction (HDI). The inhibitory effects on UGTs via nor-oleanane triterpenoid saponins, which were the bioactive ingredients from Stauntonia brachyanthera, a traditional Chinese folk medicines with highly biological values, were evaluated comprehensively with recombinant UGT isoforms as enzyme source and a nonspecific substrate 4-methylumbelliferone (4-MU) as substrate. The results showed that there are seven compounds, 2, 3, 4, 8, 9, 13 and 14, respectively, exhibited potential inhibitions towards UGT1A1, UGT1A3 and UGT1A10 among all 23 compounds isolated from the plants. The IC50 values were 17.1μM, 13.5μM, 9.5μM, 15.7μM, 16.3μM, 1.1μM, and 0.3μM, respectively. Data fitting using Dixon and Lineweaver-Burk plots demonstrated that the inhibition of UGT1A10, UGT1A1 and UGT1A3 was best fit to noncompetitive type and competitive, respectively. The inhibition kinetic parameter (Ki) was calculated to be 39μM, 17μM, 3.3μM, 10μM, 9.3μM, 0.19μM, and 0.016μM, respectively. All these experimental data suggested that HDI might occur when compounds containing herbs were co-administered with drugs which mainly undergo UGTs-mediated metabolism.

Glucuronidation of mono(2-ethylhexyl) phthalate in humans: roles of hepatic and intestinal UDP-glucuronosyltransferases.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP), which is an endocrine-disrupting chemical. In the present study, MEHP glucuronidation in humans was studied using recombinant UDP-glucuronosyltransferases (UGTs) and microsomes of the liver and intestine. Among the recombinant UGTs examined, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, and UGT2B7 glucuronidated MEHP. The kinetics of MEHP glucuronidation by UGT1A3, UGT1A7, UGT1A8, UGT1A10, UGT2B4, and UGT2B7 followed the Michaelis-Menten model, whereas that by UGT1A9 fit the negative allosteric model. CLint values were in the order of UGT1A9>UGT2B7>UGT1A7>UGT1A8≥UGT1A10>UGT1A3>UGT2B4. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model. Diclofenac (20µM) and raloxifene (20µM) decreased CLint values to 43 and 36% that of native microsomes, respectively. The kinetics of MEHP glucuronidation by intestine microsomes fit the biphasic model. Diclofenac (150 and 450µM) decreased CLint values to 32 and 13% that of native microsomes for the high-affinity phase, and to 28 and 21% for the low-affinity phase, respectively. Raloxifene (2.5 and 7.0µM) decreased CLint values to 35 and 4.1% that of native microsomes for the high-affinity phase, and to 48 and 53% for the low-affinity phase, respectively. These results suggest that MEHP glucuronidation in humans is catalyzed by UGT1A3, UGT1A9, UGT2B4, and/or UGT2B7 in the liver, and by UGT1A7, UGT1A8, UGT1A9, UGT1A10, and/or UGT2B7 in the intestine, and also that these UGT isoforms play important and characteristic roles in the detoxification of DEHP.

Metabolism studies on prim-O-glucosylcimifugin and cimifugin in human liver microsomes by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Prim-O-glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti-inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O-glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP-glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of ursolic acid

This study aims to characterize the glucuronidation kinetics of ursolic acid (UA) in human liver microsomes (HLMs) and intestinal microsomes (HIMs) and identify the main UDP-glucuronosyltransferases (UGTs) involved. In our present study, only one type of UA glucuronide was observed after incubation with HLMs and HIMs respectively and was identified as a UA hydroxyl O-glucuronide. The glucuronidation of UA can be shown in HLMs and HIMs with Km values of 3.29 ± 0.16 and 3.74 ± 0.22 μM and Vmax values of 0.33 ± 0.03 and 0.42 ± 0.03 nmol/min/(mg protein). Among the 12 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of UA [Km values of 2.58 ± 0.12 and 4.66 ± 0.60 μM, Vmax values of 0.72 ± 0.01 and 1.00 ± 0.06 nmol/min/(mg protein)]. The chemical inhibition study showed that the IC50 for hecogenin inhibition of UA glucuronidation was 51.79 ± 4.32 μM in HLMs. And chenodeoxycholic acid inhibited UA glucuronidation in HLMs with an IC50 of 28.26 ± 2.91 μM. In addition, UA glucuronidation in a panel of eight HLM was significantly correlated with telmisartan glucuronidation (r2 = 0.7660, p < 0.01) and trifluoperazine glucuronidation (r2 = 0.5866, p < 0.01) respectively. These findings collectively indicate that UGT1A3 and UGT1A4 were the main enzymes responsible for the glucuronidation of UA in human.

Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition.

Although there is evidence for an important role of UGT2B10 in the N-glucuronidation of drugs and other xenobiotics, the inhibitor selectivity of this enzyme is poorly understood. This study sought primarily to characterize the inhibition selectivity of UGT2B10 by UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors used for reaction phenotyping, and 34 antidepressant and antipsychotic drugs that contain an amine functional group. Initial studies demonstrated that cotinine is a highly selective substrate of human liver microsomal UGT2B10. The kinetics of cotinine N-glucuronidation by recombinant UGT and human liver microsomes (± bovine serum albumin) were consistent with the involvement of a single enzyme. Of the UGT enzyme-selective inhibitors employed for reaction phenotyping, only the UGT2B4/7 inhibitor fluconazole reduced recombinant UGT2B10 activity to an appreciable extent. The majority of antidepressant and antipsychotic drugs screened for effects on UGT2B10 inhibited enzyme activity with IC50 values <100 µM. The most potent inhibition was observed with the tricyclic antidepressants amitriptyline and doxepin and the tetracyclic antidepressant mianserin, and the structurally related compounds desloratadine and loratadine. Molecular modeling using a ligand-based approach indicated that hydrophobic and charge interactions are involved in inhibitor binding, whereas spatial features influence the potency of UGT2B10 inhibition. Respective mean Ki,u (± S.D.) values for amitriptyline, doxepin, and mianserin inhibition of human liver microsomal UGT2B10 were 0.61 ± 0.05, 0.95 ± 0.18, and 0.43 ± 0.01 µM. In vitro-in vivo extrapolation indicates that these drugs may perpetrate inhibitory drug-drug interactions when coadministered with compounds that are cleared predominantly by UGT2B10.

In Vitro Evaluation of the Effect of 7-Methyl Substitution on Glucuronidation of Daphnetin: Metabolic Stability, Isoform Selectivity, and Bioactivity Analysis

The C-8 phenol group is essential to exert the bioactivities of daphnetin, but it is readily conjugated with glucuronic acid prior to excretion. In this study, daphnetin-7-methylether (7M-DNP) was used to investigate the effect of 7-methyl substitution on daphnetin glucuronidation in human/rat liver (HLM/RLM) and intestine (HIM/RIM) microsomes, and recombinant UDP-glucuronosyltransferases (UGTs). Compared with daphnetin, the Vmax /Km values of 7M-DNP via 8-O-glucuronidation were 2.1-fold lower in HLM, 1.7-fold lower in HIM, and 2.4-fold lower in RLM, suggesting an improvement in metabolic stability. Different from daphnetin 8-O-glucuronidation exclusively catalyzed by UGT1A6 and UGT1A9, UGT1A1, -1A3, -1A7, -1A8, and -1A9 showed glucuronidation activity toward 7M-DNP. Kinetics studies, chemical inhibition, and the relative activity factor approach were used to demonstrate that UGT1A9 was mainly responsible for the reaction in HLM, whereas UGT1A1 was a primary contributor in HIM. The Vmax /Km values of 7M-DNP glucuronidation in HLM and HIM were 0.61-0.74-fold lower than those of rat, suggesting the differences between the two species. The bioactivity analysis demonstrated that 7M-DNP had an anti-inflammatory activity comparable to that of daphnetin. These findings indicated that the outcomes of 7-methyl substitution on daphnetin might be positive, but this should be confirmed in future in vivo studies.

In Vitro Characterization of the Human Liver Microsomal Kinetics and Reaction Phenotyping of Olanzapine Metabolism.

Olanzapine (OLZ) is an atypical antipsychotic used in the treatment of schizophrenia and related psychoses. The metabolism of OLZ is complex and incompletely characterized. This study aimed to elucidate the enzymes and pathways involved in the metabolism of OLZ and to determine the kinetics of OLZ oxidation and glucuronidation by human liver microsomes, recombinant cytochrome P450 (rP450) enzymes, and recombinant UDP-glucuronosyltransferase (rUGT) enzymes. An ultra-performance liquid chromatography-mass spectrometry method was developed and validated to quantify OLZ, its four oxidative metabolites (N-desmethyl-OLZ, 2-hydroxymethyl-OLZ, 7-hydroxy-OLZ, and OLZ-N-oxide), and two N-glucuronides (OLZ-10-N-glucuronide and OLZ-4'-N-glucuronide). Consistent with previous reports, UGT1A4, CYP1A2, and flavin-containing monooxygenase 3 play major roles in catalyzing the formation of OLZ-10-N-glucuronide, 7-hydroxy-OLZ, and OLZ-N-oxide, respectively. In addition, a previously uncharacterized major contribution of CYP2C8 to OLZ-N-demethylation was demonstrated. The kinetics of OLZ metabolite formation (Km and Vmax) by human liver microsomes, rP450 enzymes, and rUGT enzymes were characterized in the presence of bovine serum albumin [2% (w/v)]. Consistent with the known effect of bovine serum albumin on CYP1A2, CYP2C8, and UGT1A4 activities, Km values reported here are lower than previously reported values for OLZ metabolic pathways. In addition to CYP1A2-mediated OLZ-N-demethylation, these results suggest that other P450 enzymes, particularly CYP2C8, contribute significantly to oxidative OLZ metabolism through catalysis of OLZ-N-demethylation.

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9

1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9).2. Although the Km and CLint values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the Km or S50, Vmax and CLint or CLmax values for the 4′-glucuronidation of liver and intestinal microsomes between humans and monkeys.3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4′-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys.4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

Inhibition of Human UDP-Glucuronosyltransferase Enzymes by Canagliflozin and Dapagliflozin: Implications for Drug-Drug Interactions.

Canagliflozin (CNF) and dapagliflozin (DPF) are the first sodium-glucose cotransporter 2 inhibitors to be approved for clinical use. Although available evidence excludes clinically significant inhibition of cytochromes P450, the effects of CNF and DPF on human UDP-glucuronosyltransferase (UGT) enzymes are unknown. Here, we report the inhibition of human recombinant UGTs by CNF and DPF, along with the Ki values for selected recombinant and human liver microsomal UGTs. CNF inhibited all UGT1A subfamily enzymes, but the greatest inhibition was observed with UGT1A1, UGT1A9, and UGT1A10 (IC50 values ≤ 10 µM). DPF similarly inhibited UGT1A1, UGT1A9, and UGT1A10, with IC50 values ranging from 39 to 66 µM. In subsequent kinetic studies, CNF inhibited recombinant and human liver microsomal UGT1A9; Ki values ranged from 1.4 to 3.0 µM, depending on the substrate (propofol/4-methylumbelliferone) enzyme combination. Ki values for CNF inhibition of UGT1A1 were approximately 3-fold higher. Consistent with the activity screening data, DPF was a less potent inhibitor of UGT1A1 and UGT1A9. The Ki for DPF inhibition of UGT1A1 was 81 µM, whereas the Ki values for inhibition of UGT1A9 ranged from 12 to 15 µM. Based on the in vitro Ki values and plasma concentrations reported in the literature, DPF may be excluded as a perpetrator of DDIs arising from inhibition of UGT enzymes, but CNF inhibition of UGT1A1 and UGT1A9 in vivo cannot be discounted. Since the sodium-glucose cotransporter 2 inhibitors share common structural features, notably a glycoside moiety, investigation of drugs in this class for effects on UGT to identify (or exclude) potential drug-drug interactions is warranted.

In vitro metabolism of canagliflozin in human liver, kidney, intestine microsomes, and recombinant uridine diphosphate glucuronosyltransferases (UGT) and the effect of genetic variability of UGT enzymes on the pharmacokinetics of canagliflozin in humans.

O-glucuronidation is the major metabolic elimination pathway for canagliflozin. The objective was to identify enzymes and tissues involved in the formation of 2 major glucuronidated metabolites (M7 and M5) of canagliflozin and subsequently to assess the impact of genetic variations in these uridine diphosphate glucuronosyltransferases (UGTs) on in vivo pharmacokinetics in humans. In vitro incubations with recombinant UGTs revealed involvement of UGT1A9 and UGT2B4 in the formation of M7 and M5, respectively. Although M7 and M5 were formed in liver microsomes, only M7 was formed in kidney microsomes. Participants from 7 phase 1 studies were pooled for pharmacogenomic analyses. A total of 134 participants (mean age, 41 years; men, 63%; white, 84%) were included in the analysis. In UGT1A9*3 carriers, exposure of plasma canagliflozin (Cmax,ss , 11%; AUCτ,ss , 45%) increased relative to the wild type. An increase in exposure of plasma canagliflozin (Cmax,ss , 21%; AUCt,ss , 18%) was observed in participants with UGT2B4*2 genotype compared with UGT2B4*2 noncarriers. Metabolites further delineate the role of both enzymes. The pharmacokinetic findings in participants carrying the UGT1A9*3 and UGT2B4*2 allele implicate that UGT1A9 and UGT2B4 are involved in the metabolism of canagliflozin to M7 and M5, respectively.