Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs).

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs' mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

Mechanisms and emerging strategies for irinotecan-induced diarrhea.

Irinotecan (also known as CPT-11) is a topoisomerase I inhibitor first approved for clinical use as an anticancer agent in 1996. Over the past more than two decades, it has been widely used for combination regimens to treat various malignancies, especially in gastrointestinal and lung cancers. However, severe dose-limiting toxicities, especially gastrointestinal toxicity such as late-onset diarrhea, were frequently observed in irinotecan-based therapy, thus largely limiting the clinical application of this agent. Current knowledge regarding the pathogenesis of irinotecan-induced diarrhea is characterized by the complicated metabolism of irinotecan to its active metabolite SN-38 and inactive metabolite SN-38G. A series of enzymes and transporters were involved in these metabolic processes, including UGT1A1 and CYP3A4. Genetic polymorphisms of these metabolizing enzymes were significantly associated with the occurrence of irinotecan-induced diarrhea. Recent discoveries and progress made on the detailed mechanisms enable the identification of potential biomarkers for predicting diarrhea and as such guiding the proper patient selection with a better range of tolerant dosages. In this review, we introduce the metabolic process of irinotecan and describe the pathogenic mechanisms underlying irinotecan-induced diarrhea. Based on the mechanisms, we further outline the potential biomarkers for predicting the severity of diarrhea. Finally, based on the current experimental evidence in preclinical and clinical studies, we discuss and prospect the current and emerging strategies for the prevention of irinotecan-induced diarrhea.

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

As a new type of oral tyrosine kinase inhibitor, entrectinib can act on multiple targets and exert efficacy and has been approved for the treatment of non-small cell lung cancer (NSCLC) and solid tumors. However, whether entrectinib affects the activities of recombinant human UDP-glucuronosyltransferases (UGTs) remains unclear. Herein, we aimed to investigate the inhibitory effects of entrectinib on human UGTs and to assess the potential risk of causing drug-drug interactions (DDIs) based on the inhibition against UGTs. High-performance liquid chromatography (HPLC) was used to evaluate the inhibitory effects of entrectinib on UGTs according to the product formation rate of UGT substrate with or without entrectinib, and the inhibition kinetics experiment was conducted to assess the inhibitory type of entrectinib on UGTs. Our results showed that entrectinib exhibited extensive inhibitory effects on most human UGTs, and especially inhibited the activities of UGT1A7, UGT1A8, and UGT2B15 with Ki (Inhibition constant) of lower than 5 µM (0.95-4.38 µM). Furthermore, the results from quantitative prediction research suggested that the combination of entrectinib at 600 mg/day with substrates primarily metabolized by hepatic UGT2B15 or intestinal UGT1A7 and UGT1A8 might cause clinical DDIs. Thus, special attention should be paid to avoid adverse reactions induced by DDIs when co-administration of entrectinib and drugs metabolized by UGTs.

Uridine diphosphate glucuronosyltransferase 1A1 prevents the progression of liver injury.

BACKGROUND: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear. AIM: To determine the role and mechanism of UGT1A1 in liver damage progression. METHODS: We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study. RESULTS: Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism. CONCLUSION: UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Anthraquinone biocolourant dermocybin is metabolized whereas dermorubin is not in in vitro liver fractions and recombinant metabolic enzymes.

Fungal anthraquinones dermocybin and dermorubin are attractive alternatives for synthetic dyes but their metabolism is largely unknown. We conducted a qualitative in vitro study to identify their metabolism using human liver microsomes and cytosol, as well as recombinant human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes. Additionally, liver microsomal and cytosolic fractions from rat, mouse and pig were used. Following incubations of the biocolourants with the enzymes in the presence of nicotinamide adenine dinucleotide phosphate, UDP-glucuronic acid, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or S-adenosyl methionine (SAM) to enable CYP oxidation, glucuronidation, sulfonation or methylation, we observed several oxidation and conjugation metabolites for dermocybin but none for dermorubin. Human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A7 catalysed dermocybin oxidation. The formation of dermocybin glucuronides was catalysed by human UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10 and 2B15. Human SULT1B1, 1C2 and 2A1 sulfonated dermocybin. Dermocybin oxidation was faster than conjugation in human liver microsomes. Species differences were seen in dermocybin glucuronidation between human, rat, mouse and pig. In conclusion, many CYP and conjugation enzymes metabolized dermocybin, whereas dermorubin was not metabolized in human liver fractions in vitro. The results indicate that dermocybin would be metabolized in humans in vivo.

Activation of the YY1-UGT2B7 Axis Promotes Mammary Estrogen Homeostasis Dysregulation and Exacerbates Breast Tumor Metastasis.

Metastasis is the most common pathway of cancer death. The lack of effective predictors of breast cancer metastasis is a pressing issue in clinical practice. Therefore, exploring the mechanism of breast cancer metastasis to uncover reliable predictors is very important for the clinical treatment of breast cancer patients. In this study, tandem mass tag quantitative proteomics technology was used to detect protein content in primary breast tumor tissue samples from patients with metastatic and nonmetastatic breast cancer at diagnosis. We found that the high expression of yin-yang 1(YY1) is strongly associated with poor prognosis in high-grade breast cancer. YY1 expression was detected in both clinical tumor tissue samples and tumor tissue samples from mammary-specific polyomavirus middle T antigen overexpression mouse model mice. We demonstrated that upregulation of YY1 expression was closely associated with breast cancer metastasis and that high YY1 expression could promote the migratory invasive ability of breast cancer cells. Mechanistically, YY1 directly binds to the UGT2B7 mRNA initiation sequence ATTCAT, thereby transcriptionally regulating the inhibition of UGT2B7 expression. UGT2B7 can regulate the development of breast cancer by regulating estrogen homeostasis in the breast, and the abnormal accumulation of estrogen, especially 4-OHE2, promotes the migration and invasion of breast cancer cells, ultimately causing the development of breast cancer metastasis. In conclusion, YY1 can regulate the UGT2B7-estrogen metabolic axis and induce disturbances in estrogen metabolism in breast tumors, ultimately leading to breast cancer metastasis. Disturbances in estrogen metabolism in the breast tissue may be an important risk factor for breast tumor progression and metastasis SIGNIFICANCE STATEMENT: In this study, we propose for the first time a regulatory relationship between YY1 and the UGT2B7/estrogen metabolism axis and explore the molecular mechanism. Our study shows that the YY1/UGT2B7/estrogen axis plays an important role in the development and metastasis of breast cancer. This study further elucidates the potential mechanisms of YY1-mediated breast cancer metastasis and the possibility and promise of YY1 as a predictor of cancer metastasis.

Activation of Cryptic Donor Splice Sites within the UDP-Glucuronosyltransferase (UGT)1A First-Exon Region Generates Variant Transcripts That Encode UGT1A Proteins with Truncated Aglycone-Binding Domains.

The human UDP-glucuronosyltransferases (UGTs) have crucial roles in metabolizing and clearing numerous small lipophilic compounds. The UGT1A locus generates nine UGT1A mRNAs, 65 spliced transcripts, and 34 circular RNAs. In this study, our analysis of published UGT-RNA capture sequencing (CaptureSeq) datasets identified novel splice junctions that predict 24 variant UGT1A transcripts derived from ligation of exon 2 to unique sequences within the UGT1A first-exon region using cryptic donor splice sites. Of these variants, seven (1A1_n1, 1A3_n3, 1A4_n4, 1A5_n1, 1A8_n2, 1A9_n2, 1A10_n7) are predicted to encode UGT1A proteins with truncated aglycone-binding domains. We assessed their expression profiles and deregulation in cancer using four RNA sequencing (RNA-Seq) datasets of paired normal and cancerous drug-metabolizing tissues from large patient cohorts. Variants were generally coexpressed with their canonical counterparts with a higher relative abundance in tumor than in normal tissues. Variants showed tissue-specific expression with high interindividual variability but overall low abundance. However, 1A8_n2 showed high abundance in normal and cancerous colorectal tissues, with levels that approached or surpassed canonical 1A8 mRNA levels in many samples. We cloned 1A8_n2 and showed expression of the predicted protein (1A8_i3) in human embryonic kidney (HEK)293T cells. Glucuronidation assays with 4-methylumbelliferone (4MU) showed that 1A8_i3 had no activity and was unable to inhibit the activity of 1A8_i1 protein. In summary, the activation of cryptic donor splice sites within the UGT1A first-exon region expands the UGT1A transcriptome and proteome. The 1A8_n2 cryptic donor splice site is highly active in colorectal tissues, representing an important cis-regulatory element that negatively regulates the function of the UGT1A8 gene through pre-mRNA splicing. SIGNIFICANT STATEMENT: The UGT1A locus generates nine canonical mRNAs, 65 alternately spliced transcripts, and 34 different circular RNAs. The present study reports a series of novel UDP-glucuronosyltransferase (UGT)1A variants resulting from use of cryptic donor splice sites in both normal and cancerous tissues, several of which are predicted to encode variant UGT1A proteins with truncated aglycone-binding domains. Of these, 1A8_n2 shows exceptionally high abundance in colorectal tissues, highlighting its potential role in the first-pass metabolism in gut through the glucuronidation pathway.

Uridine 5'-Diphospho-glucuronosyltransferase 1A3 (UGT1A3) Prediction of Hepatic Clearance of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) Substrate Telmisartan by Glucuronidation Using In Vitro-In Vivo Extrapolation (IVIVE).

BACKGROUND AND OBJECTIVE: The prediction of pharmacokinetic parameters for drugs metabolised by cytochrome P450 enzymes has been the subject of active research for many years, while the application of in vitro-in vivo extrapolation (IVIVE) techniques for non-cytochrome P450 enzymes has not been thoroughly evaluated. There is still no established quantitative method for predicting hepatic clearance of drugs metabolised by uridine 5'-diphospho-glucuronosyltransferases (UGTs), not to mention those which undergo hepatic uptake. The objective of the study was to predict the human hepatic clearance for telmisartan based on in vitro metabolic stability and hepatic uptake results. METHODS: Telmisartan was examined in liver systems, allowing to estimate intrinsic clearance (CLint, in vitro) based on the substrate disappearance rate with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Obtained CLint, in vitro values were corrected for corresponding unbound fractions. Prediction of human hepatic clearance was made from scaled unbound CLint, in vitro data with the use of the well-stirred model, and finally referenced to the literature value of observed clearance in humans, allowing determination of the essential scaling factors. RESULTS: The in vitro scaled CLint, in vitro by UGT1A3 was assessed using three systems, human hepatocytes, liver microsomes, and recombinant enzymes. Obtained values were scaled and hepatic metabolism clearance was predicted, resulting in significant clearance underprediction. Utilization of the extended clearance concept (ECC) and hepatic uptake improved prediction of hepatic metabolism clearance. The scaling factors for hepatocytes, assessing the in vitro-in vivo difference, changed from sixfold difference to only twofold difference with the application of the ECC. CONCLUSIONS: The study showed that taking into consideration hepatic uptake of a drug allows us to obtain satisfactory scaling factors, hence enabling the prediction of in vivo hepatic glucuronidation from in vitro data.

Detection of uridine diphosphate glucuronosyltransferase 1A1 for pancreatic cancer imaging and treatment via a "turn-on" fluorescent probe.

Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is expressed ubiquitously in cancer cells and can metabolize exogenous substances. Studies show higher UGT1A1 levels in pancreatic cancer cells than normal cells. Therefore, we need a method to monitor the activity level of UGT1A1 in pancreatic cancer cells and in vivo. Here, we report a fluorescent probe, BCy-panc, for UGT1A1 imaging in cells and in vivo. Compared with other molecular probes, this probe is readily prepared, with high selectivity and sensitivity for the detection of UGT1A1. Our results show that BCy-panc rapidly detects UGT1A1 in pancreatic cancer. In addition, there is an urgent need for evidence to clarify the relationship between UGT1A1 and pancreatic cancer development. The present investigation found that the increase of UGT1A1 by chrysin was effective in inducing apoptosis in pancreatic cancer cells. These results indicate that the synergistic effect of chrysin and cisplatin at the cellular level is superior to that of cisplatin alone. The UGT1A1 level may be a biomarker for early diagnosis of cancer. Meanwhile, UGT1A1 plays a crucial role in pancreatic cancer, and the combination of chrysin and cisplatin may provide effective ideas for pancreatic cancer treatment.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. AIM OF THE STUDY: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. MATERIALS AND METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-ß-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 µM, 9.05 µM, 3.87 µM, and 7.00 µM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. CONCLUSION: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Kinetic Characterization of Estradiol Glucuronidation by Liver Microsomes and Expressed UGT Enzymes: The Effects of Organic Solvents.

BACKGROUND AND OBJECTIVE: In vitro glucuronidation of 17ß-estradiol (estradiol) is often performed to assess the role of uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) in xenobiotic/drug metabolism. The objective of this study was to determine the effects of four commonly used organic solvents [i.e., dimethyl sulfoxide (DMSO), methanol, ethanol, and acetonitrile] on the glucuronidation kinetics of estradiol, which can be glucuronidated at C3 and C17 positions. METHODS: The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. RESULTS: In human liver microsomes (HLM), methanol, ethanol, and acetonitrile significantly altered estradiol glucuronidation kinetics with increased Vmax (up to 2.6-fold) and CLmax (up to 2.8-fold) values. Altered estradiol glucuronidation in HLM was deduced to be attributed to the enhanced metabolic activities of UGT1A1 and UGT2B7, whose activities differ at the two glucuronidation positions. The effects of organic solvents on estradiol glucuronidation were glucuronidation position-, isozyme-, and solvent-specific. Furthermore, both ethanol and acetonitrile have a greater tendency to modify the glucuronidation activity of estradiol in animal liver microsomes. CONCLUSION: Organic solvents such as methanol, ethanol, and acetonitrile showed great potential in adjusting the glucuronidation of estradiol. DMSO is the most suitable solvent due to its minimal influence on estradiol glucuronidation. Researchers should be cautious in selecting appropriate solvents to get accurate results when assessing the metabolism of a new chemical entity.

Role of microsomal metabolism in bromfenac-induced cytotoxicity.

This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity.

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.

Efficient Production of Flavonoid Glucuronides in Escherichia coli Using Flavonoid O-Glucuronosyltransferases Characterized from Marchantia polymorpha.

As a model liverwort, Marchantia polymorpha contains various flavone glucuronides with cardiovascular-promoting effects and anti-inflammatory properties. However, the related glucuronosyltransferases have not yet been reported. In this study, two bifunctional UDP-glucuronic acid/UDP-glucose:flavonoid glucuronosyltransferases/glucosyltransferases, MpUGT742A1 and MpUGT736B1, were identified from M. polymorpha. Extensive enzymatic assays found that MpUGT742A1 and MpUGT736B1 exhibited efficient glucuronidation activity for flavones, flavonols, and flavanones and showed promiscuous regioselectivity at positions 3, 6, 7, 3', and 4'. These enzymes catalyzed the production of a variety of flavonoid glucuronides with medicinal value, including apigenin-7-O-glucuronide and scutellarein-7-O-glucuronide. With the use of MpUGT736B1, apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide were prepared by scaled-up enzymatic catalysis and structurally identified by NMR spectroscopy. MpUGT742A1 also displayed glucosyltransferase activity on the 7-OH position of the flavanones using UDP-glucose as the sugar donor. Furthermore, we constructed four recombinant strains by combining the pathway for increasing the UDP-glucuronic acid supply with the two novel UGTs MpUGT742A1 and MpUGT736B1. When apigenin was used as a substrate, the extracellular apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide production obtained from the Escherichia coli strain BB2 reached 598 and 81 mg/L, respectively. Our study provides new candidate genes and strategies for the biosynthesis of flavonoid glucuronides.

Bile duct ligation elevates 5-HT levels in cerebral cortex of rats partly due to impairment of brain UGT1A6 expression and activity via ammonia accumulation.

Hepatic encephalopathy (HE) is often associated with endogenous serotonin (5-HT) disorders. However, the reason for elevated brain 5-HT levels due to liver failure remains unclear. This study aimed to investigate the mechanism by which liver failure increases brain 5-HT levels and the role in behavioral abnormalities in HE. Using bile duct ligation (BDL) rats as a HE model, we verified the elevated 5-HT levels in the cortex but not in the hippocampus and striatum, and found that this cortical 5-HT overload may be caused by BDL-mediated inhibition of UDP-glucuronosyltransferase 1A6 (UGT1A6) expression and activity in the cortex. The intraventricular injection of the UGT1A6 inhibitor diclofenac into rats demonstrated that the inhibition of brain UGT1A6 activity significantly increased cerebral 5-HT levels and induced HE-like behaviors. Co-immunofluorescence experiments demonstrated that UGT1A6 is primarily expressed in astrocytes. In vitro studies confirmed that NH4Cl activates the ROS-ERK pathway to downregulate UGT1A6 activity and expression in U251 cells, which can be reversed by the oxidative stress antagonist N-acetyl-l-cysteine and the ERK inhibitor U0126. Silencing Hepatocyte Nuclear Factor 4α (HNF4α) suppressed UGT1A6 expression whilst overexpressing HNF4α increased Ugt1a6 promotor activity. Meanwhile, both NH4Cl and the ERK activator TBHQ downregulated HNF4α and UGT1A6 expression. In the cortex of hyperammonemic rats, we also found activation of the ROS-ERK pathway, decreases in HNF4α and UGT1A6 expression, and increases in brain 5-HT content. These results prove that the ammonia-mediated ROS-ERK pathway activation inhibits HNF4α expression to downregulate UGT1A6 expression and activity, thereby increasing cerebral 5-HT content and inducing manic-like HE symptoms. This is the first study to reveal the mechanism of elevated cortical 5-HT concentration in a state of liver failure and elucidate its association with manic-like behaviors in HE.

Promiscuity and Quantitative Contribution of UGT2B17 in Drug and Steroid Metabolism Determined by Experimental and Computational Approaches.

Uridine 5'-diphospho-glulcuronosyltransferase 2B17 (UGT2B17) is important in the metabolism of steroids and orally administered drugs due to its high interindividual variability. However, the structural basis governing the substrate selectivity or inhibition of UGT2B17 remains poorly understood. This study investigated 76 FDA-approved drugs and 20 steroids known to undergo glucuronidation for their metabolism by UGT2B17. Specifically, we assessed the substrate selectivity for UGT2B17 over other UGT enzymes using recombinant human UGT2B17 (rUGT2B17), human intestinal microsomes, and human liver microsomes. The quantitative contribution of intestinal UGT2B17 in the glucuronidation of these compounds was characterized using intestinal microsomes isolated from UGT2B17 expressors and nonexpressors. In addition, a structure-based pharmacophore model for UGT2B17 substrates was built and validated using the studied pool of substrates and nonsubstrates. The results show that UGT2B17 could metabolize 23 out of 96 compounds from various chemical classes, including alcohols and carboxylic acids, particularly in the intestine. Interestingly, amines were less susceptible to UGT2B17 metabolism, though they could inhibit the enzyme. Three main pharmacophoric features of UGT2B17 substrates include (1) the presence of an accessible -OH or -COOH group near His35 residue, (2) a hydrophobic functional group at ∼4.5-5 Šfrom feature 1, and (3) an aromatic ring ∼5-7 Šfrom feature 2. Most of the studied compounds inhibited UGT2B17 activity irrespective of their substrate potential, indicating the possibility of multiple mechanisms. These data suggest that UGT2B17 is promiscuous in substrate selectivity and inhibition and has a high potential to produce significant variability in the absorption and disposition of orally administered drugs.

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.

Cytochrome P450 2B6 and UDP-Glucuronosyltransferase Enzyme-Mediated Clearance of Ciprofol (HSK3486) in Humans: The Role of Hepatic and Extrahepatic Metabolism.

Ciprofol (HSK3486) is a novel intravenous agent for general anesthesia. In humans, HSK3486 mainly undergoes glucuronidation to form M4 [fraction of clearance (fCL): 62.6%], followed by the formation of monohydroxylated metabolites that further undergo glucuronidation and sulfation to produce M5-1, M5-2, M5-3, and M3 (summed fCL: 35.2%). However, the complete metabolic pathways of HSK3486 in humans remain unclear. In this study, by comparison with chemically synthesized reference standards, three monohydroxylated metabolites [M7-1, 4-hydroxylation with an unbound intrinsic clearance (CLint,u) of 2211 µl/min/mg; M7-2, ω-hydroxylation with a CLint,u of 600 µl/min/mg; and M7-3, (ω-1)-hydroxylation with a CLint,u of 78.4 µl/min/mg] were identified in human liver microsomes, and CYP2B6 primarily catalyzed their formation. In humans, M7-1 was shown to undergo glucuronidation at the 4-position and 1-position by multiple UDP-glucuronosyltransferases (UGTs) to produce M5-1 and M5-3, respectively, or was metabolized to M3 by cytosolic sulfotransferases. M7-2 was glucuronidated at the ω position by UGT1A9, 2B4, and 2B7 to form M5-2. UGT1A9 predominantly catalyzed the glucuronidation of HSK3486 (M4). The CLint,u values for M4 formation in human liver and kidney microsomes were 1028 and 3407 µl/min/mg, respectively. In vitro to in vivo extrapolation analysis suggested that renal glucuronidation contributed approximately 31.4% of the combined clearance. In addition to HSK3486 glucuronidation (M4), 4-hydroxylation (M7-1) was identified as another crucial oxidative metabolic pathway (fCL: 34.5%). Further attention should be paid to the impact of CYP2B6- and UGT1A9-mediated drug interactions and gene polymorphisms on the exposure and efficacy of HSK3486. SIGNIFICANCE STATEMENT: This research elucidates the major oxidative metabolic pathways of HSK3486 (the formation of three monohydroxylated metabolites: M7-1, M7-2, M7-3) as well as definitive structures and formation pathways of these monohydroxylated metabolites and their glucuronides or sulfate in humans. This research also identifies major metabolizing enzymes responsible for the glucuronidation (UGT1A9) and oxidation (CYP2B6) of HSK3486 and characterizes the mechanism of extrahepatic metabolism. The above information is helpful in guiding the safe use of HSK3486 in the clinic.

Cannabidiol and cannabidiolic acid: Preliminary in vitro evaluation of metabolism and drug-drug interactions involving canine cytochrome P-450, UDP-glucuronosyltransferase, and P-glycoprotein.

Phytocannabinoid-rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)-mediated drug metabolism and canine P-glycoprotein-mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor-dependent depletion of CBD (but not CBDA) and UDP-glucuronosytransferase cofactor-dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP-glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (ß-naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid-drug inhibition experiments showed inhibition (IC50 = 4.6-8.1 µM) of tramadol metabolism via CYP2B11-mediated N-demethylation (CBD and CBDA) and CYP2D15-mediated O-demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12-mediated midazolam 1'-hydroxylation (IC50 > 10 µM). CBD and CBDA were not substrates or competitive inhibitors of canine P-glycoprotein. Results for cannabinoid-enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid-drug interactions involving certain CYPs (but not P-glycoprotein). Confirmatory in vivo studies are warranted.