Recombinant UDP-glucuronosyltransferases Research Articles

In Vitro Ciclopirox Glucuronidation in Liver Microsomes from Humans and Various Experimental Animals.

Ciclopirox is a widely used antifungal drug, redisposition of which has drawn increasing attentions due to multiple promising activities. The drug undergoes extensive glucuronidation, which acts as a major obstacle in the ongoing novel application and still remains poorly understood. The current study aims to phenotype ciclopirox glucuronidation pathway and as well to decipher the related species differences. Ciclopirox glucuronidation was investigated in liver microsomes from humans (HLM) and various experimental animals. Assays with recombinant uridine diphosphate glucuronosyltransferases (UGTs), enzyme kinetic analyses and selective inhibitors were used to determine the role of individual UGTs in ciclopirox glucuronidation. HLM is highly active in ciclopirox glucuronidation withMichaelis-Menten constant (Km),maximum velocity (Vmax), and intrinsic clearance (CLint)values of 139 μM, 7.89 nmol/min/mg, and 56 μL/min/mg, respectively. UGT1A9 displays by far the highest activity, whereas several other isoforms (UGT1A6, UGT1A7, and UGT1A8) catalyze formation of traced glucuronides. Further kinetic analysis demonstrates that UGT1A9 has a closed Km value (167 μM) to HLM. UGT1A9 selective inhibitor (magnolol) can potently inhibit ciclopirox glucuronidation in HLM with the IC50 value of 0.12 μM. The reaction displays remarkable differences across liver microsomes from mice, rats, cynomolgus monkey, minipig, and beagle dog, with the CLint values in the range of 26-369 μL/min/mg. In addition, ciclopirox glucuronidation activities of experimental animals' liver microsomes were less sensitive to magnolol than that of HLM. Ciclopirox glucuronidation displays remarkable species differences with UGT1A9 as a dominant contributor in humans. It is suggested that the pharmacological or toxicological effects ofciclopirox may be UGT1A9 and species dependent.

Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Ciprofol is primarily glucuronidated by UGT1A9 and predicted not to cause drug-drug interactions with typical substrates of CYP1A2, CYP2B6, and CYP2C19

Ciprofol is a novel intravenous anesthetic agent. Its major glucuronide metabolite, M4, is found in plasma and urine. However, the specific isoforms of UDP-glucuronosyltransferases (UGTs) that metabolize ciprofol to M4 remain unknown. This study systematically characterized UGTs that contribute to the formation of M4 using human liver microsomes (HLM), human intestinal microsomes (HIM), and human recombinant UGTs. The inhibitory potential of ciprofol and M4 against major human UGTs and cytochrome P450 enzymes (P450s) was also explored. In vitro-in vivo extrapolation (IVIVE) and physiologically-based pharmacokinetic (PBPK) simulations were performed to predict potential in vivo drug-drug interactions (DDIs) caused by ciprofol. Glucuronidation of ciprofol followed Michaelis-Menten kinetics in both HLM and HIM with apparent Km values of 345 and 412 μM, Vmax values of 2214 and 444 nmol min−1·mg protein−1, respectively. The in vitro intrinsic clearances (CLint = Vmax/Km) for ciprofol glucuronidation by HLM and HIM were 6.4 and 1.1 μL min−1·mg protein−1, respectively. Human recombinant UGT studies revealed that UGT1A9 is the predominant isoform mediating M4 formation, followed by UGT1A7, with UGT1A8 playing a minor role. Ciprofol competitively inhibited CYP1A2 (Ki = 12 μM) and CYP2B6 (Ki = 4.7 μM), and noncompetitively inhibited CYP2C19 (Ki = 29 μM). No time-dependent inhibition by ciprofol was noted for CYP1A2, CYP2B6, or CYP2C19. In contrast, M4 showed limited or no inhibitory effects against selected P450s. Neither ciprofol nor M4 inhibited UGTs significantly. Initial IVIVE suggested potential ciprofol-mediated inhibition of CYP1A2, CYP2B6, and CYP2C19 inhibition in vivo. However, PBPK simulations showed no significant effect on phenacetin, bupropion, and S-mephenytoin exposure or peak plasma concentration. Our findings are pertinent for future DDI studies of ciprofol as either a perpetrator or victim drug.

Potential risk of drug-drug interactions of ponatinib via inhibition against human UDP-glucuronosyltransferases

Ponatinib is an efficient oral tyrosine kinase inhibitor (TKI) for T315I-positive Ph + ALL and T315I-positive chronic myeloid leukemia (CML) or BCR-ABL when no other TKIs can be prescribed. In this research, we evaluated the inhibitory effects of ponatinib on human recombinant UDP-glucuronosyltransferases (UGTs) and predicted the magnitude of potential drug-drug interaction (DDI) risk of co-treatment with ponatinib and UGTs substrates by using in vitro-in vivo extrapolation (IVIVE) method. Our study presented that ponatinib showed a broad-spectrum inhibition against UGTs. Particularly, ponatinib exhibited potent inhibitory effects towards UGT1A7, UGT1A1, and UGT1A9 with IC50 values of 0.37, 0.41, and 0.89 μM, respectively, which might lead to clinically significant DDI.

Drug-drug interaction potentials of tucatinib inhibition of human UDP-glucuronosyltransferases

Tucatinib is known as a tyrosine kinase inhibitor (TKI), which has been commonly approved for the treatment of adult patients with advanced unresectable or metastatic HER2-positive breast cancer. However, there haven't been systematic study about the inhibition of tucatinib on UDP-Glucuronosyltransferases (UGTs) and the potential risk of drug-drug interactions (DDIs). In present study, we aimed to systematically investigate the inhibition of tucatinib on recombinant human UGTs and pooled human liver microsomes (HLMs), and to quantitatively evaluate its potential risk of DDIs by in vitro-in vivo extrapolation (IVIVE). Our data indicated that tucatinib exhibited extensive inhibition on recombinant UGTs. Tucatinib was a weak inhibitor of UGT1A4, 2B4 and 2B7; tucatinib possessed a strong inhibitory effect on UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B15 and UGT2B17, with IC50 values of 0.53 μM–15.50 μM. Especially, it also potently inhibited estradiol and SN-38 glucuronidation in HLMs with IC50 values of 46.83 μM and 1.33 μM. The quantitative prediction of DDIs risk indicated that the co-administration of tucatinib with drugs mainly metabolized by hepatic or intestinal UGTs (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B15 and UGT2B17) might result in potential DDIs risk through inhibition of glucuronidation. More attention should be paid to the influence of tucatinib on UGTs in liver and intestine to avoid unnecessary clinical DDIs risk.

Assessing the contribution of UGT isoforms on raltegravir drug disposition through PBPK modeling

This work aimed to develop a physiologically based pharmacokinetic (PBPK) model for raltegravir accounting for UDP-glucuronosyltransferase (UGT) metabolism to assess the effect of UGT gene polymorphisms. Raltegravir elimination was evaluated using Km and Vmax values from human recombinant systems and UGT tissue scalar considering liver, kidney, and intestine. The predicted/observed ratios for raltegravir PK parameters were within a 2-fold error range in UGT1A1 poor and normal metabolizers, except in Asian UGT1A1 poor metabolizers. This PBPK modeling approach suggests that UGT1A3 is the main contributor to raltegravir's metabolism. UGT1A3 and UGT1A1 gene polymorphisms might have an additive effect on raltegravir's drug disposition and response. The final model accounting for hepatic, renal, and intestinal UGT metabolism, biliary clearance, and renal excretion improved model predictions compared with the previously published models. This PBPK model with the quantitative characterization of raltegravir elimination pathways can support dose adjustments in different clinical scenarios.

Cabozantinib Carries the Risk of Drug-Drug Interactions via Inhibition of UDPglucuronosyltransferase (UGT) 1A9.

Cabozantinib is a multiple receptor tyrosine kinases inhibitor (TKI) approved to treat progressive, metastatic medullary thyroid cancer, advanced renal cell carcinoma, and hepatocellular carcinoma. Drugdrug interactions (DDIs) for cabozantinib have been identified involving the role of cytochromes P450. Although the previous study reported that cabozantinib showed a slight inhibition of UDP-glucuronosyltransferase (UGT) 1A1 at the highest concentration tested, there are no reports on the potential for UGTs-mediated-DDIs. Hence, the current study aims to address this knowledge gap. This study aimed to investigate the inhibitory effect of cabozantinib on human UGTs and to quantitatively evaluate the DDI potential via UGT inhibition. The inhibitory effects of cabozantinib on UGTs were determined by measuring the formation rates for 4- methylumbelliferone (4-MU) glucuronide and trifluoperazine N-glucuronide using recombinant human UGT isoforms in the absence or presence of cabozantinib. Inhibition kinetic studies were conducted to determine the type of inhibition of cabozantinib on UGTs and the corresponding inhibition constant (Ki) value. In vitro-in vivo extrapolation (IVIVE) was further employed to predict the potential risk of DDI in vivo. Cabozantinib displayed potent inhibition of UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15. Cabozantinib exhibited noncompetitive inhibition towards UGT1A1 and 1A3 and inhibition towards UGT1A7 and 1A9. The Ki,u values (mean ± standard deviation) were calculated to be 2.15±0.11 μM, 0.83±0.05 μM, 0.75±0.04 μM and 0.18 ± 0.10 μM for UGT1A1, 1A3, 1A7 and 1A9, respectively. Co-administration of cabozantinib at the clinically approved dose of 60 mg/day or 140 mg/day may result in approximately a 26% to 60% increase in the systemic exposure of drugs predominantly cleared by UGT1A9, implying a high risk of DDIs. Cabozantinib has the potential to cause DDIs via the inhibition of UGT1A9; therefore, additional attention should be paid to the safety of the combined use of cabozantinib and drugs metabolized by UGT1A9.

Comparative analysis of bilirubin glucuronidation activity in canine and human primary hepatocytes using a 3D culture system.

Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs.

Contributions of UDP-Glucuronosyltransferases to Human Hepatic and Intestinal Metabolism of Ticagrelor and Inhibition of UGTs and Cytochrome P450 Enzymes by Ticagrelor and its Glucuronidated Metabolite.

Ticagrelor is the first reversibly binding, direct-acting, oral P2Y12 receptor inhibitor. The contribution of UDP-glucuronosyltransferases (UGTs) enzymes to the metabolism of ticagrelor to its glucuronide conjugation, ticagrelor-O-glucuronide, in human liver microsomes (HLM) and human intestinal microsomes (HIM), was well characterized in the current study. The inhibition potential of human major UGTs by ticagrelor and ticagrelor-O-glucuronide was explored. The inhibitory effects of ticagrelor-O-glucuronide on cytochrome P450s (CYPs) enzymes were investigated as well. Ticagrelor glucuronidation exhibits substrate inhibition kinetics in both HLM and HIM with apparent Km values of 5.65 and 2.52 μM, Vmax values of 8.03 and 0.90 pmol min−1·mg protein−1, Ksi values of 1,343.0 and 292.9 respectively. The in vitro intrinsic clearances (V max/K m) for ticagrelor glucuronidation by HLM and HIM were 1.42 and 0.36 μl min−1·mg protein−1, respectively. Study with recombinant human UGTs suggested that multiple UGT isoforms including UGT1A9, UGT1A7, UGT1A3, UGT1A4, UGT1A1, UGT2B7 and UGT1A8 are involved in the conversion of ticagrelor to ticagrelor-O-glucuronide with UGT1A9 showing highest catalytic activity. The results were further supported by the inhibition studies on ticagrelor glucuronidation with typical UGT inhibitors in pooled HLM and HIM. Little or no inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 by ticagrelor and ticagrelor-O-glucuronide was noted. Ticagrelor-O-glucuronide also exhibited limited inhibitory effects toward CYP2C8, CYP2D6 and CYP3A4. In contrast, ticagrelor-O-glucuronide weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activity with apparent IC50 values of 45.0, 20.0 and 18.8 μM, respectively. The potential of ticagrelor-O-glucuronide to cause drug-drug interactions warrant further study.

Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 μM and 0.51 ± 0.063 μM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT: Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

Identification of Human UDP-Glucuronosyltransferase and Sulfotransferase as Responsible for the Metabolism of Dotinurad, a Novel Selective Urate Reabsorption Inhibitor.

Dotinurad, a novel selective urate reabsorption inhibitor, is used to treat hyperuricemia. In humans, orally administered dotinurad is excreted mainly as glucuronide and sulfate conjugates in urine. To identify the isoforms of UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) involved in dotinurad glucuronidation and sulfation, microsome and cytosol fractions of liver, intestine, kidney, and lung tissues (cytosol only) were analyzed along with recombinant human UGT and SULT isoforms. Dotinurad was mainly metabolized to its glucuronide conjugate by human liver microsomes (HLMs), and the glucuronidation followed the two-enzyme Michaelis-Menten equation. Among the recombinant human UGT isoforms expressed in the liver, UGT1A1, UGT1A3, UGT1A9, and UGT2B7 catalyzed dotinurad glucuronidation. Based on inhibition analysis using HLMs, bilirubin, imipramine, and diflunisal decreased glucuronosyltransferase activities by 45.5%, 22.3%, and 22.2%, respectively. Diflunisal and 3'-azido-3'-deoxythymidine, in the presence of 1% bovine serum albumin, decreased glucuronosyltransferase activities by 21.1% and 13.4%, respectively. Dotinurad was metabolized to its sulfate conjugate by human liver cytosol (HLC) and human intestinal cytosol (HIC) samples, with the sulfation reaction in HLC samples following the two-enzyme Michaelis-Menten equation and that in HIC samples following the Michaelis-Menten equation. All eight recombinant human SULT isoforms used herein catalyzed dotinurad sulfation. Gavestinel decreased sulfotransferase activity by 15.3% in HLC samples, and salbutamol decreased sulfotransferase activity by 68.4% in HIC samples. These results suggest that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7, whereas its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. SIGNIFICANCE STATEMENT: The identification of enzymes involved in drug metabolism is important to predicting drug-drug interactions (DDIs) and interindividual variability for safe drug use. The present study revealed that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7 and that its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. Therefore, dotinurad, a selective urate reabsorption inhibitor, is considered safe for use with a small risk of DDIs and low interindividual variability.

Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

Metabolism of 2,3-Dehydrosilybin A and 2,3-Dehydrosilybin B: A Study with Human Hepatocytes and Recombinant UDP-Glucuronosyltransferases and Sulfotransferases.

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

Identification of human uridine diphosphate-glucuronosyltransferase isoforms responsible for the glucuronidation of 10,11-dihydro-10-hydroxy-carbazepine.

To determine the kinetics of the formation of 10,11-dihydro-10-hydroxy-carbazepine (MHD)-O-glucuronide in human liver microsomes (HLMs), human intestine microsomes (HIMs), human kidney microsomes (HKMs) and recombinant human UDP-glucuronosyltransferase (UGTs), and identify the primary UGT isoforms catalyzing the glucuronidation of MHD. The kinetics of the glucuronidation of MHD was determined in HLMs, HIMs as well as HKMs. Screening assays with 13 recombinant human UGTs, inhibition studies and correlation analysis were performed to identify the main UGTs involved in the glucuronidation of MHD. MHD-O-glucuronide was formed in HLMs, HIMs as well as HKMs, HLMs showed the highest intrinsic clearance of MHD. Among 13 recombinant human UGTs, UGT2B7 and UGT1A9 were identified to be the principal UGT isoforms mediating the glucuronidation of MHD, while UGT1A4 played a partial role. In addition, inhibition studies and correlation analysis further confirmed that UGT2B7 and UGT1A9 participated in the formation of MHD-O-glucuronide. MHD could be metabolized by UGTs in the liver, intestine and kidney, and the hepatic glucuronidation was the critical metabolic pathway. UGT2B7 and UGT1A9 were the primary UGT isoforms mediating the formation of MHD-O-glucuronide in the liver.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17β-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

Metabolism of flutamide in diet control fischer 344 and brown Norway x F 344 rats, and its hydroxylation and conjugation by human CYP450s and UDP-glucuronosyltransferases

In our current report, flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)-phenyl] propanamide) treated diet control and non limited-fed Fischer 344 (F344) and Brown Norway (BN) x F344 rats showed that diet control reduces spontaneous and flutamide-induced hyperplasia((1)). In this continued study, we found that serum concentrations of active metabolite of flutamide, 2-hydroxyflutamide (OH-flu), were 181 ± 26.6 ng/mL and 68 ± 8.0 ng/mL (p<0.05), in non-limited fed and diet control F344 rats. In BN x F344 rats, the serum concentrations of 2-OH-flutamide were 232 ± 57 ng/mL and 52 ± 6 ng/mL (p<0.05) in non-limited fed and diet control animals. In diet control groups, liver microsomes from flutamide-treated F344 rats showed high 7-ethoxyresorufin O-deethylase (EROD) activity, while 7-Benzoxyresorufin O-dealkylase (BROD) activity was not affected significantly. Both rat and human liver microsomes showed flutamide oxidation activity. Human liver microsomes showed 10 times higher activity than rat liver microsomes (0.673 ± 0.04 vs 0.063 ± 0.008 nmol OH-flu/min/mg protein). Microsomes from human tissues such as colon, colon cancer, kidney, bladder, pancreas, prostate, prostate cancer, or ovarian cancer, showed no or non-detectable activity for flutamide hydroxylation. CYP450 1A1, 1A2, 1B1 and 2C19 from human lymphoblastoid cell lines, oxidized flutamide to OH-flu in vitro with activities from 0.118 ± 0.005 to 0.275 ± 0.010 nmol/min/mg protein. Microsomes isolated from human kidney, colon, and liver showed UDP-glucuronosyltransferase (UGT) activity for glucuronidation of OH-flu. Human kidney showed the highest activity. Several human recombinant UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) also showed glucuronidation activity for OH-flu. It was found that UGT1A6 was more active than other human UGTs.

Characterizations of Human UDP-Glucuronosyltransferase Enzymes in the Conjugation of p-Cresol.

p-Cresol is a uremic toxin that is formed by intestinal microbiota and extensively conjugated by first-pass metabolism. p-Cresol glucuronide exerts various forms of cellular toxicity in vitro and is accumulated in the plasma of subjects with kidney disease, where associations with adverse cardiovascular and renal outcomes are evident. The objective of this study was to determine the contributions of human UDP-glucuronosyltransferase (UGT) enzymes in the formation of p-cresol glucuronide. Utilizing commonly expressed hepatic or renal human recombinant UGTs (ie, hrUGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17), hrUGT1A6 and hrUGT1A9 exhibited the highest catalytic activities in the generation of p-cresol glucuronide. The kinetics of p-cresol glucuronide formation in hrUGT1A6 and pooled human liver microsomes were best described by the Hill equation and in hrUGT1A9 and pooled human kidney microsomes by substrate inhibition. Using inhibitory and selective UGT inhibitors (ie, acetaminophen or amentoflavone for UGT1A6 and niflumic acid for UGT1A9), UGT1A6 was identified the predominant enzyme responsible for p-cresol glucuronide production in pooled human liver (78.4%-81.3% contribution) and kidney (54.3%-62.9%) microsomes, whereas UGT1A9 provided minor contributions (2.8% and 35.5%, respectively). The relative contributions of UGT1A6 (72.6 ± 11.3%, mean ± SD) and UGT1A9 (5.7 ± 4.1%) in individual human liver microsomes from 12 adult donors were highly variable, where an inverse association (R = -.784, p = .003) between UGT1A6 contribution and UGT1A9 probe substrate activity (ie, mycophenolic acid) was evident. Our novel findings provide valuable tools for conducting further mechanistic studies and for designing clinical interventions to mitigate the toxicities associated with p-cresol glucuronide.

Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes

20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite which is known to increase platelet aggregation and cardiovascular risk. In this study, nine non-steroidal anti-inflammatory drugs (NSAIDs) selected by chemical structures were screened to determine their effects on the glucuronidation of 20-HETE using human liver microsomes (HLMs). Then, the combined effects of the selected NSAID and genetic polymorphisms in UDP-glucuronosyltransferase (UGT) were investigated. Among the tested NSAIDs, diclofenac was the strongest inhibitor of 20-HETE glucuronidation with an IC50 value of 3.5 μM. Celecoxib, naproxen, mefenamic acid, ibuprofen, and indomethacin showed modest inhibition with IC50 values of 77, 91, 190, 208, and 220 μM, respectively, while acetylsalicylic acid, rofecoxib, and meloxicam did not inhibit 20-HETE glucuronidation. Glucuronidation of 20-HETE by UGT2B7 and UGT1A9 recombinant enzymes was significantly inhibited by indomethacin, mefanemic acid, diclofenac, ibuprofen, naproxen, and celecoxib (P < 0.001). In addition, diclofenac exhibited a competitive inhibition mechanism with the Km value of 20-HETE glucuronidation increasing from 23.5 μM to 62 μM in the presence of 3.5 μM diclofenac. Diclofenac further decreased 20-HETE glucuronidation in HLMs carrying UGT2B7*2 alleles compared with the wild-type HLMs. The results from this study would be useful in understanding the alteration of 20-HETE levels in relation to NSAID and UGT genetic polymorphisms.

DUPLICATE: Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes

DUPLICATE: Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes

Glucuronidation of Abiraterone and Its Pharmacologically Active Metabolites by UGT1A4, Influence of Polymorphic Variants and Their Potential as Inhibitors of Steroid Glucuronidation.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ4-Abi (D4A) and 5α-Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5α-Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5α-Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients.