Pharmacokinetics and metabolism of jatrorrhizine, a gastric prokinetic drug candidate

Jatrorrhizine, a protoberberine alkaloid derived from Coptis chinensis, is currently under investigation as a natural gastric prokinetic drug candidate. In vitro and in vivo studies were conducted to characterize its pharmacokinetics and metabolism. After intravenous administration, the plasma concentration kinetics and major metabolites in rats were investigated. The metabolic kinetics, key cytochrome P450 enzymes and UDP-glucuronosyltransferase isoforms (UGTs) of jatrorrhizine were studied in rat liver microsomes (RLMs). After intravenous administration, plasma jatrorrhizine concentrations showed a biphasic decline, dose-independent clearance and half-life of terminal elimination phase, and a relatively large distribution volume. The metabolic pathway for the conversion of jatrorrhizine was important for its elimination. In addition, the demethylated and glucuronidated products were found to be the major metabolites in rats. The enzyme kinetics for both demethylation and glucuronidation were fitted to the hyperbolic Michaelis-Menten equation in RLMs. CYP3A1/2 and CYP2D2 were mainly responsible for demethylation, and UGT 1A1 and 1A3 were responsible for glucuronidation in RLMs. The metabolic properties of jatrorrhizine suggest multiple metabolic pathways. These results will contribute to promote further research and development of jatrorrhizine.     

Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.     

Characterization of in vitro metabolites of cudratricusxanthone A in human liver microsomes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata, exhibits several biological activities; however, metabolic biotransformation was not investigated. Therefore, metabolites of CTXA were investigated and the major metabolic enzymes engaged in human liver microsomes (HLMs) were characterized using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). CTXA was incubated with HLMs or human recombinant CYPs and UGTs, and analysed by an LC‐MS/MS equipped electrospray ionization (ESI) to qualify and quantify its metabolites. In total, eight metabolites were identified: M1–M4 were identified as mono‐hydroxylated metabolites during Phase I, and M5–M8 were identified as O‐glucuronidated metabolites during Phase II in HLMs. Moreover, these metabolite structures and a metabolic pathway were identified by elucidation of MSⁿ fragments and formation by human recombinant enzymes. M1 was formed by CYP2D6, and M2–M4 were generated by CYP1A2 and CYP3A4. M5–M8 were mainly formed by UGT1A1, respectively. While investigating the biotransformation of CTXA, eight metabolites of CTXA were identified by CYPs and UGTs; these data will be valuable for understanding the in vivo metabolism of CTXA. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes.

Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.     

In vitro metabolic characterization, phenotyping, and kinetic studies of 9cUAB30, a retinoid X receptor-specific retinoid.

The present study was conducted to compare the in vitro phase I and phase II metabolic profiles of (2E,4E,6Z,8E)-8-(3',4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid (9cUAB30) in human, rat, and dog microsomes and to characterize and identify the associated metabolic kinetics and specific isozymes from human liver microsomes (HLM) responsible for metabolism, respectively. Data from these experiments revealed that nine (M1-M9) phase I metabolites along with a single glucuronide conjugate were observed across the species investigated. With the exception of glucuronidation, no evidence of metabolism was detected for phase II enzymes (data not shown). Significant differences between species with regard to metabolic profile, stability, and gender were noted. For the eight phase I metabolites detected in HLM, the specific isozymes responsible for the biotransformations were CYP2C8, CYP2C9, and CYP2C19, with minor contributions from CYP1A2 and CYP2B6. For the glucuronide conjugate, UGT1A9 was the major catalyzing enzyme, with a minor contribution from UGT1A3. Kinetic analysis of eight of the detected metabolites indicated that four seemed to follow classical hyperbolic kinetics, whereas the remaining four showed evidence of either autoactivation or substrate inhibition.     

Drug–drug interactions in the metabolism of imidafenacin: Role of the human cytochrome P450 enzymes and UDP-glucuronic acid transferases,and potential of imidafenacin to inhibit human cytochrome P450 enzymes

Imidafenacin (IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a newly synthesized antimuscarinic drug developed for the treatment of overactive bladder. To predict clinically relevant drug interactions in the metabolism of IM, the paper investigated: (1) the major enzymes responsible for the metabolism of IM, (2) the effects of concomitant drugs on the inhibition of metabolism of IM, and (3) the effects of IM and its metabolites on the inhibition of human cytochrome P450 (CYP). The elimination of IM and production of oxidative metabolites were mainly catalysed by recombinant CYP3A4, and the elimination of IM by human liver microsomes (HLM) was markedly inhibited by co-incubation with ketoconazole. The production of the N-glucuronide metabolite was only catalysed by recombinant UGT1A4. Clinically established CYP3A4 inhibitors including itraconazole, ketoconazole, erythromycin and clarithromycin inhibited the elimination of IM in HLM. IM and its major metabolites did not affect the activities of CYP enzymes in vitro. The results suggest that the major enzymes responsible for the metabolism of IM are CYP3A4 and UGT1A4, and oxidative metabolism of IM is reduced by concomitant administration of CYP3A4 inhibitors. In contrast, IM and its metabolites have no inhibitory effect on the CYP-mediated metabolism of concomitant drugs.     

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.     

Drug-drug interactions in the metabolism of imidafenacin: role of the human cytochrome P450 enzymes and UDP-glucuronic acid transferases, and potential of imidafenacin to inhibit human cytochrome P450 enzymes

Imidafenacin (IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a newly synthesized antimuscarinic drug developed for the treatment of overactive bladder. To predict clinically relevant drug interactions in the metabolism of IM, the paper investigated: (1) the major enzymes responsible for the metabolism of IM, (2) the effects of concomitant drugs on the inhibition of metabolism of IM, and (3) the effects of IM and its metabolites on the inhibition of human cytochrome P450 (CYP). The elimination of IM and production of oxidative metabolites were mainly catalysed by recombinant CYP3A4, and the elimination of IM by human liver microsomes (HLM) was markedly inhibited by co-incubation with ketoconazole. The production of the N-glucuronide metabolite was only catalysed by recombinant UGT1A4. Clinically established CYP3A4 inhibitors including itraconazole, ketoconazole, erythromycin and clarithromycin inhibited the elimination of IM in HLM. IM and its major metabolites did not affect the activities of CYP enzymes in vitro. The results suggest that the major enzymes responsible for the metabolism of IM are CYP3A4 and UGT1A4, and oxidative metabolism of IM is reduced by concomitant administration of CYP3A4 inhibitors. In contrast, IM and its metabolites have no inhibitory effect on the CYP-mediated metabolism of concomitant drugs.     

The UDP-glucuronosyltransferase 2B7 isozyme is responsible for gemfibrozil glucuronidation in the human liver.

Gemfibrozil, a fibrate hypolipidemic agent, is eliminated in humans by glucuronidation. A gemfibrozil glucuronide has been reported to show time-dependent inhibition of cytochrome P450 2C8. Comprehensive assessment of the drug interaction between gemfibrozil and cytochrome P450 2C8 substrates requires a clear understanding of gemfibrozil glucuronidation. However, the primary UDP-glucuronosyltransferase (UGT) isozymes responsible for gemfibrozil glucuronidation remain to be determined. Here, we identified the main UGT isozymes involved in gemfibrozil glucuronidation. Evaluation of 12 recombinant human UGT isozymes shows gemfibrozil glucuronidation activity in UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, with UGT2B7 showing the highest activity. The kinetics of gemfibrozil glucuronidation in pooled human liver microsomes (HLMs) follows Michaelis-Menten kinetics with high and low affinity components. The high affinity K(m) value was 2.5 microM, which is similar to the K(m) value of gemfibrozil glucuronidation in recombinant UGT2B7 (2.2 microM). In 16 HLMs, a significant correlation was observed between gemfibrozil glucuronidation and both morphine 3-OH glucuronidation (r = 0.966, p < 0.0001) and flurbiprofen glucuronidation (r = 0.937, p < 0.0001), two reactions mainly catalyzed by UGT2B7, whereas no significant correlation was observed between gemfibrozil glucuronidation and either estradiol 3beta-glucuronidation and propofol glucuronidation, two reactions catalyzed by UGT1A1 and UGT1A9, respectively. Flurbiprofen and mefenamic acid inhibited gemfibrozil glucuronidation in HLMs with similar IC(50) values to those reported in recombinant UGT2B7. These results suggest that UGT2B7 is the main isozyme responsible for gemfibrozil glucuronidation in humans.     

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1

1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes.

2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC–MS/MS and/or NMR analyses.

3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors.

4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible.

5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.     

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.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes

Abstract

1.?Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far.

2.?Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one.

3.?Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CL_int) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities.

4.?Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.     

Characterization of bropirimine O-glucuronidation in human liver microsomes

1. The antitumour agent bropirimine undergoes significant Phase II conjugation in vivo. Incubation of [14C]bropirimine with human liver microsomes resulted in the formation of a single product peak (M1) using high-performance liquid chromatography with radiochemical detection and was tentatively assigned as bropirimine glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 442/444 amu with liquid chromatography/mass spectrometry. Following metabolite isolation, the structure of M1 was established as bropirimine O-glucuronide by 1H-nuclear magnetic spectroscopy. 2. Studies aimed at identifying the human liver UDP-glucuronosyltransferase (UGT) enzyme(s) involved in the glucuronidation of bropirimine were carried out using recombinant human UGTs and it was determined that glucuronidation of bropirimine was catalysed by UGT1A1, UGT1A3 and UGT1A9. Bropirimine O-glucuronidation followed Michaelis-Menten kinetics and the Km and Vmax (mean +/- SD; n = 3) were 1217 +/- 205 microM and 667 +/- 188 pmol min(-1) mg(-1), respectively. 3. The activity of bropirimine O-glucuronidation by human liver microsomes was inhibited by bilirubin (40%) and with mefenamic acid (80%). Although buprenorphine extensively inhibited the activity of bropirimine O-glucuronidation by UGT1A3, the inhibition profile did not parallel that observed in HLMs. 4. The results demonstrate that UGT1A9 and to a lesser extent UGT1A1 are responsible for the majority of bropirimine O-glucuronidation in man.     

In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug

Thiocoraline is a potent new marine anti-cancer drug in vitro, which will be tested in phase I clinical studies shortly. To assess the biotransformation and the potential implications for human pharmacology and toxicology, the in vitro metabolism of thiocoraline was characterized using human plasma, human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes and human cell lines.Thiocoraline is significantly metabolized by enzymes present in human plasma; t (1/2) shifted from 25.2 h in phosphate buffered saline to 4.3 h in human plasma. Using CYP supersomes it was shown that thiocoraline is mainly metabolized by CYP3A4, with CYP1A1, CYP2C8 and CYP2C9 playing a minor role in the biotransformation (<3%). Only minor glucuronidation was observed for thiocoraline by UGT1A1 and UGT1A9 and no glucuronidation was observed in human liver S9 fraction. In addition, no glucosidation and sulfation were observed for thiocoraline in human liver cytosol and S9 fraction. However, the metabolites formed by cytochrome P450 were further conjugated by UGT, glutathione-S-transferase (GST) and sulfotransferase (ST). In contrast to the CYP metabolism observed in supersomes, no effect could be observed from the CYP3A4 inhibitors on the cytotoxicity of thiocoraline in Hep G2 cells. However, this could be due to low CYP expression levels in the Hep G2 and IGROV-1 cell line.These results provide evidence that human CYP3A4 plays a major role in the metabolism of thiocoraline in vitro and that the metabolites formed by CYP are conjugated by the phase II enzymes UGT, ST and GST.     

Drug glucuronidation in clinical psychopharmacology

Glucuronidation is a phase II metabolic process and one of the most common pathways in the formation of hydrophilic drug metabolites. At least 33 families of uridine diphosphate-glucuronosyltransferases have been identified in vitro, and specific nomenclature similar to that used to classify the cytochrome (CYP) P450 system has been established. The UGT1 and UGT2 subfamilies represent the most important of these enzymes in human drug metabolism. Factors affecting glucuronidation include the following: cigarette smoking, obesity, age, and gender. In addition, several drugs have been found in vitro to be substrates, inhibitors, or inducers of UGT enzymes. Induction or inhibition of both UGT and CYP isoforms may occur simultaneously. Some important drug interactions involving glucuronidation have been documented and others can be postulated. This review summarizes the relevant literature pertaining to drug glucuronidation and its implications for clinical psychopharmacology.     

Identification of Human UDP-Glucuronosyltransferase Responsible for the Glucuronidation of Niflumic Acid in Human Liver

Purpose To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of niflumic acid in human liver. Methods The glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). Results Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a K_m value of 16 μM, which is similar to that found with recombinant UGT1A1. The glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3β-glucuronidation activity (r=0.78, p<0.05). β-Estradiol inhibited niflumic acid glucuronidation with an IC₅₀ of 25 μM in HLMs, comparable to that for UGT1A1. Conclusions These findings indicate that UGT1A1 is the main isozyme involved in the glucuronidation of niflumic acid in the human liver.     

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.     

Non-oxidative ethanol metabolism in human hepatic cells in vitro: Involvement of uridine diphospho-glucuronosyltransferase 1A9 in ethylglucuronide production

Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation.