UDP-glucuronosyltransferase Enzymes Research Articles

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.

Cannabinoid-Induced Inhibition of Morphine Glucuronidation and the Potential for In Vivo Drug-Drug Interactions.

Opioids are commonly prescribed for the treatment of chronic pain. Approximately 50% of adults who are prescribed opioids for pain co-use cannabis with their opioid treatment. Morphine is primarily metabolized by UDP-glucuronosyltransferase (UGT) 2B7 to an inactive metabolite, morphine-3-glucuronide (M3G), and an active metabolite, morphine-6-glucuronide (M6G). Previous studies have shown that major cannabis constituents including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit major UGT enzymes. To examine whether cannabinoids or their major metabolites inhibit morphine glucuronidation by UGT2B7, in vitro assays and mechanistic static modeling were performed with these cannabinoids and their major metabolites including 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC), 7-hydroxy-cannabidiol (7-OH-CBD), and 7-carboxy-cannabidiol (7-COOH-CBD). In vitro assays with rUGT-overexpressing microsomes and human liver microsomes showed that THC and CBD and their metabolites inhibited UGT2B7-mediated morphine metabolism, with CBD and THC exhibiting the most potent Ki,u values (0.16 µM and 0.37 µM, respectively). Only 7-COOH-CBD exhibited no inhibitory activity against UGT2B7-mediated morphine metabolism. Static mechanistic modeling predicted an in vivo drug-drug interaction between morphine and THC after inhaled cannabis, and between THC, CBD, and 7-OH-CBD after oral consumption of cannabis. These data suggest that the co-use of these agents may lead to adverse drug events in humans.

Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes.

Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450(CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.

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.

Drug-Drug Interaction Between Cannabidiol, Cyclosporine, and Mycophenolate Mofetil: A Case Report

Kidney transplantation remains the optimal therapy for many patients with end-stage kidney disease (ESKD). Chronic pain is one of the most common and distressing symptoms among patients with ESKD, and its treatment is a complex and challenging task to accomplish. The benefits of cannabidiol (CBD) in chronic pain treatment have been reported recently. Cannabidiol is metabolized by cytochrome P450, mainly CYP3A4 and CYP2C19, and can also undergo direct conjugation via UDP-glucuronosyltransferase enzymes, with a growing body of evidence suggesting it is also a potent inhibitor or inducer of these pathways. Cannabidiol was also found to be a potent inhibitor of carboxylesterases in vitro. Because cytochrome P450 enzymes and carboxylesterases are also responsible for the clearance and activation of immunosuppressants, respectively, drug-drug interactions are likely to occur. Here, we report a pharmacokinetic drug interaction between CBD and cyclosporine and mycophenolate mofetil in a patient with ESKD with a kidney transplantation. It is thus crucial to take into account these interactions and monitor drug levels to avoid drug toxicity or a lack of efficacy. This study is in accordance with the guidelines of the Declaration of Helsinki and the Declaration of Istanbul.

Medicinal Chemistry and NMR Driven Discovery of Novel UDP-glucuronosyltransferase 1A Inhibitors That Overcome Therapeutic Resistance in Cells

The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind ∼20 fold more tightly to UGT1A-C (Kd ∼ 600 μM vs ∼30 μM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.

Comparative metabolism of fargesin in human, dog, monkey, mouse, and rat hepatocytes.

Fargesin, a bioactive lignan derived from Flos Magnoliae, possesses anti-inflammatory, anti-oxidative, anti-melanogenic, and anti-apoptotic effects. This study compared the metabolic profiles of fargesin in human, dog, monkey, mouse, and rat hepatocytes using liquid chromatography-high resolution mass spectrometry. In addition, we investigated the human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for fargesin metabolism. The hepatic extraction ratio of fargesin among the five species ranged from 0.59 to 0.78, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. During metabolism, fargesin generates three phase 1 metabolites, including fargesin catechol (M1) and O-desmethylfargesin (M2 and M3), and 11 phase 2 metabolites, including O-methyl-M1 (M4 and M5) via catechol O-methyltransferase (COMT), glucuronides of M1, M2, M4, and M5, and sulfates of M1-M5. The production of M1 from fargesin via O-demethylenation is catalyzed by CYP2C9, CYP3A4, CYP2C19, and CYP2C8 enzymes, whereas the formation of M2 and M3 (O-desmethylfargesin) is catalyzed by CYP2C9, CYP2B6, CYP2C19, CYP3A4, CYP1A2, and CYP2D6 enzymes. M4 is metabolized to M4 glucuronide by UGT1A3, UGT1A8, UGT1A10, UGT2B15, and UGT2B17 enzymes, whereas M4 sulfate is generated by multiple SULT enzymes. Fargesin is extensively metabolized in human hepatocytes by CYP, COMT, UGT, and SULT enzymes. These findings help to elucidate the pharmacokinetics and drug interactions of fargesin.

Gilbert’s syndrome as a protection against the development of other diseases

Introduction and purpose Gilbert's syndrome is a condition caused by a mutation in the gene responsible for the enzyme UDP-glucuronosyltransferase 1A1, which conjugates bilirubin in hepatocytes. Its less efficient functioning results in disorder of bilirubin metabolism which leads to increased level of this compound. It has been observed that there is interesting correlation between elevated levels of bilirubin and the risk of developing certain diseases in patients with this condition. The aim of this review is to understand the correlation between elevated bilirubin levels and the risk of developing selected diseases in individuals with Gilbert's syndrome and how to use this knowledge in innovative therapeutic options. 
 State of knowledge Bilirubin was considered a non-functional waste product of heme breakdown, used to be seen as a concerning indicator of liver disease or even a potentially harmful substance for the nervous system. However, lately it turns out that bilirubin has strong antioxidant properties, which help to scavenge free radicals and inhibit lipid oxidation. Recent studies have shown that even slightly elevated levels of bilirubin in the blood, such as those commonly found in individuals with Gilbert's syndrome may protect these patients against diseases associated with increased oxidative stress, an overactive immune response and metabolic dysfunction. 
 Conclusions As a result, individuals with Gilbert's syndrome are observed to have a lower risk of developing diseases related to oxidative stress, such as cardiovascular diseases and cancer. In these patients mortality rate compared to the population with normobilirubinemia is decreased. Based on available studies, it has also been concluded that inducing mild hyperbilirubinemia - iatrogenic Gilbert's syndrome - using appropriate substances may have health benefits.

Abstract B014: ONECUT2 activates diverse drug-resistant phenotypes in prostate cancer

Abstract BACKGROUND. Prostate cancer (PC) cells can escape from lineage confinement and be reprogrammed to a drug-resistant, high-plasticity state, however the mechanism underlying this process is not understood. Our group identified the HOX/CUT transcription factor ONECUT2 (OC2) as a mediator of metastatic castration-resistant PC (mCRPC) that can be inhibited with a novel family of small molecules. OC2 was shown to be a lineage plasticity driver that promotes neuroendocrine prostate cancer (NEPC). In this study, we sought to uncover the mechanism whereby OC2 exerts effects on lineage plasticity. METHODS. RNAseq, CUT&RUNseq, scRNAseq, forced expression, shRNA, CRISPR-Cas9, co-precipitation/western blot, PCR, mutagenesis, human PC, PC models, computational modeling. RESULTS. We found that OC2 is capable of activating multiple androgen receptor- (AR)-indifferent, drug-resistant lineages, including programs not associated with NEPC, such as upregulation of the glucocorticoid receptor (GR). Consistent with the GR findings, OC2 regulated genes on the AR cistrome previously shown to be regulated by GR in CRPC, including KLK3/PSA. OC2 activated bivalent promoters of master regulator genes via epigenetic modification, and reprogramed multiple super-enhancers, including those associated with TMPRSS2 and SRRM4. OC2-expressing cells in human CRPC included populations that express AR, NE+/stem-like+, NE-/stem-like+, and “double negative” (NE-/AR-) phenotypes. OC2 also transcriptionally upregulated UDP-glucuronosyltransferase enzymes UGT2B15 and UGT2B17, which inactivate DHT, suggesting that the emergence of phenotypic plasticity also arises from AR suppression by ligand depletion. Consistent with this, low levels of androgen correlated with OC2 activation in human CRPC tumors. Enforced OC2 promoted both enzalutamide and docetaxel resistance in PC models. CONCLUSIONS. These findings identify OC2 as an upstream mediator of many lineage-defining factors that govern drug resistance, including those associated with adenocarcinoma. Because OC2 can be inhibited pharmacologically, these results suggest that suppressing this target may inhibit the emergence of a wide range of drug resistance mechanisms arising from loss of lineage confinement. Citation Format: Chen Qian, Qian Yang, HyoYoung Kim, Yiwu Yan, Elahe Mostaghel, Leigh Ellis, Beatrice Knudsen, Salma Kaochar, Huihui Ye, Isla Garraway, Sungyong You, Michael Freeman. ONECUT2 activates diverse drug-resistant phenotypes in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B014.

(068) Low Drug-Drug interaction risk of Estetrol (E4), a Promising New Estrogen for the Treatment of Menopausal Symptoms

Abstract Introduction Polypharmacy is common among postmenopausal women and exposes them to the risks of inappropriate drug use and drug-drug interactions (DDI). Most estrogens are influenced metabolically by drugs which affect key metabolic enzymes. Objective To investigate the DDI potential of E4, several in vitro studies with metabolizing enzymes were performed. A clinical study was also conducted to evaluate the consequence of inhibiting the main enzyme responsible for E4 metabolism. Methods In vitro studies on human hepatocytes or human liver microsomes and recombinant enzymes were conducted to evaluate the role of drug metabolizing enzymes on E4 metabolism [cytochrome P450 enzymes (CYP), UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT)]. We also assessed the potential of E4 to inhibit or induce these enzymes. Finally, we performed a clinical study to determine the effect of UGT inhibition on the pharmacokinetics (PK) of E4. In this open-label two period, two-way cross-over study, 24 healthy female volunteers received two single doses of a product containing E4 15 mg once alone and once in combination with valproic acid 500 mg twice daily, a strong UGT inhibitor. The consequence of UGT inhibition was quantified by serial plasma measurements of E4 up to 168h after drug administration. Results Effects of other drugs on E4 (E4 as a substrate) CYP enzymes, a superfamily of enzymes that functions as monooxygenases did not play a significant role in the metabolism of E4. Consequently, E4 does not interact with drugs that inhibit or induce CYP enzymes. In vitro and in vivo studies showed that UGT2B7 was the main enzyme involved in E4 glucuronidation and that SULT1 was the dominant enzyme for sulfation of E4. The clinical phase 1 study confirmed the role of UGT in the metabolism of E4. UGT inhibition by valproic acid increased total E4 exposure and maximum concentration only by 1.13 and 1.36-fold, respectively. Because the effect of UGT inhibition on E4 pharmacokinetics is minimal, no clinically relevant effects are expected during coadministration of drugs that inhibit UGT activity. Effect of E4 on other drugs (interference by E4) Studies on the effect of E4 on other drugs showed that E4 neither induces nor inhibits CYP or UGT enzymes, except for a very minimal inhibition of CYP3A4, UGT1A9 and UGT2B7 isoforms. The half maximal inhibitory concentration (IC50) of these in vitro effects were several hundred times above the anticipated clinical maximum concentrations, indicating a low potential for clinically relevant DDIs. Also, there was no evidence of time-dependent inhibition of CYP enzymes. Conclusions Based on these in vitro and in vivo drug interaction studies on metabolizing enzymes, E4 is not expected to have a potential risk for DDI. Unlike other estrogens, CYP enzymes do not play a major role in the metabolism of E4 and can therefore be combined with drugs that inhibit or induce CYP or UGT enzymes without compromising its safety or efficacy. Disclosure Yes, this is sponsored by industry/sponsor: Estetra an affiliate company of Mithra Pharmaceuticals Clarification: Industry initiated, executed and funded study Any of the authors act as a consultant, employee or shareholder of an industry for: Estetra an affiliate company of Mithra Pharmaceuticals.

Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine.

Drug resistance underpins poor outcomes in many malignancies including refractory and relapsed acute myeloid leukemia (R/R AML). Glucuronidation is a common mechanism of drug inactivation impacting many AML therapies e.g. cytarabine, decitabine, azacytidine and venetoclax. In AML cells, the capacity for glucuronidation arises from increased production of the UDP-glucuronosyltransferase 1A (UGT1A) enzymes. UGT1A elevation was first observed in AML patients who relapsed after response to ribavirin, a drug used to target the eukaryotic translation initiation factor eIF4E, and subsequently in patients who relapsed on cytarabine. UGT1A elevation resulted from increased expression of the sonic-hedgehog transcription factor GLI1. Vismodegib inhibited GLI1, decreased UGT1A levels, reduced glucuronidation of ribavirin and cytarabine and re-sensitized cells to these drugs. Here, we examined if UGT1A protein levels, and thus glucuronidation activity, were targetable in humans and if this corresponded to clinical response. We conducted a Phase II trial using vismodegib with ribavirin, with or without decitabine, in largely heavily pretreated patients with high-eIF4E AML. Pre-therapy molecular assessment of patients' blasts indicated highly elevated UGT1A levels relative to healthy volunteers. Among patients with partial response, blast response or prolonged stable disease, vismodegib reduced UGT1A levels which corresponded to effective targeting of eIF4E by ribavirin. In all, our studies are the first to demonstrate that UGT1A protein, and thus glucuronidation, are targetable in humans. These studies pave the way for the development of therapies that impair glucuronidation, one of the most common drug deactivation modalities.

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

Ripretinib, a tyrosine kinase inhibitor (TKI), is the first FDA approved fourth-line therapy for adults with advanced gastrointestinal stromal tumor (GIST). Studies have shown that several TKIs for treating GIST were potent inhibitors of human UDP-glucosyltransferase (UGTs) enzymes. However, whether ripretinib affects the activity of UGTs remains unclear. The aim of this study was to investigate the effects of ripretinib on major UGT isoforms, as well as to evaluate its potential drug-drug interactions (DDIs) risk caused by the inhibition of UGTs activities. The inhibitory effects and inhibition modes of ripretinib on UGTs were systematically evaluated using high-performance liquid chromatography (HPLC) and enzyme kinetic studies, respectively. Our data showed that ripretinib exhibited potent inhibition against UGT1A1, UGT1A3, UGT1A4, UGT1A7 and UGT1A8. Enzyme kinetic studies indicated that ripretinib was not only a competitive inhibitor of UGT1A1, UGT1A4 and UGT1A7, but also a noncompetitive inhibitor of UGT1A3, as well as a mixed inhibitor of UGT1A8. The prediction results of in vitro-in vivo extrapolation (IVIVE) demonstrated that ripretinib might bring the potential risk of DDIs when combined with substrates of UGT1A1, UGT1A3, UGT1A4, UGT1A7 or UGT1A8. Therefore, special attention should be paid when ripretinib is used in conjunction with other drugs metabolized by UGTs to avoid risk of DDIs in clinic.

Systems-level transcriptional regulation of Caenorhabditis elegans metabolism.

Metabolism is controlled to ensure organismal development and homeostasis. Several mechanisms regulate metabolism, including allosteric control and transcriptional regulation of metabolic enzymes and transporters. So far, metabolism regulation has mostly been described for individual genes and pathways, and the extent of transcriptional regulation of the entire metabolic network remains largely unknown. Here, we find that three-quarters of all metabolic genes are transcriptionally regulated in the nematode Caenorhabditis elegans. We find that many annotated metabolic pathways are coexpressed, and we use gene expression data and the iCEL1314 metabolic network model to define coregulated subpathways in an unbiased manner. Using a large gene expression compendium, we determine the conditions where subpathways exhibit strong coexpression. Finally, we develop "WormClust," a web application that enables a gene-by-gene query of genes to view their association with metabolic (sub)-pathways. Overall, this study sheds light on the ubiquity of transcriptional regulation of metabolism and provides a blueprint for similar studies in other organisms, including humans.

Inhibition of Nicotine Metabolism by Cannabidiol (CBD) and 7-Hydroxycannabidiol (7-OH-CBD).

Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug-drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3'-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 μM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 μM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 μM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 μM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 μM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.

Contribution of UGT Enzymes to Human Drug Metabolism Stereoselectivity: A Case Study of Medetomidine, RO5263397, Propranolol, and Testosterone.

The enantiomeric forms of chiral compounds have identical physical properties but may vary greatly in their metabolism by individual enzymes. Enantioselectivity in UDP-glucuronosyl transferase (UGT) metabolism has been reported for a number of compounds and with different UGT isoforms involved. However, the impact of such individual enzyme results on overall clearance stereoselectivity is often not clear. The enantiomers of medetomidine, RO5263397, and propranolol and the epimers testosterone and epitestosterone exhibit more than a 10-fold difference in glucuronidation rates by individual UGT enzymes. In this study, we examined the translation of human UGT stereoselectivity to hepatic drug clearance considering the combination of multiple UGTs to overall glucuronidation, the contribution of other metabolic enzymes such as cytochrome P450s (P450s), and the potential for differences in protein binding and blood/plasma partitioning. For medetomidine and RO5263397, the high individual enzyme (UGT2B10) enantioselectivity translated into ∼3- to >10-fold differences in predicted human hepatic in vivo clearance. For propranolol, the UGT enantioselectivity was irrelevant in the context of high P450 metabolism. For testosterone, a complex picture emerged due to differential epimeric selectivity of various contributing enzymes and potential for extrahepatic metabolism. Quite different patterns of P450- and UGT-mediated metabolism were observed across species, as well as differences in stereoselectivity, indicating that extrapolation from human enzyme and tissue data are essential when predicting human clearance enantioselectivity. SIGNIFICANCE STATEMENT: Individual enzyme stereoselectivity illustrates the importance of three-dimensional drug-metabolizing enzyme-substrate interactions and is essential when considering the clearance of racemic drugs. However, translation from in vitro to in vivo can be challenging as contributions from multiple enzymes and enzyme classes must be combined with protein binding and blood/plasma partitioning data to estimate the net intrinsic clearance for each enantiomer. Preclinical species may be misleading as enzyme involvement and metabolism stereoselectivity can differ substantially.

Dimethandrolone, a Potential Male Contraceptive Pill, is Primarily Metabolized by the Highly Polymorphic UDP-Glucuronosyltransferase 2B17 Enzyme in Human Intestine and Liver.

Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%-80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17-mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

Species Difference in the Metabolism of Mulberrin in Vitro and Its Inhibitory Effect on Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Estimation of fraction of drug metabolism by a single UDP-glucuronosyl transferase enzyme using relative expression factor.

The estimation of the contributions of UDP-glucuronosyl transferase (UGT) isoforms to the overall metabolism still suffers from technical difficulties due to limited information on enzyme levels in recombinant systems and specific inhibitors, unlike the case for cytochrome P450s (CYPs). The protein expression levels of UGT in both recombinant system microsomes (RM) and human liver microsomes (HLM) were quantified using liquid chromatography-tandem mass spectrometry, and the relative expression factor (REF) value of HLM to recombinant microsomes was estimated to evaluate the fractions of drug metabolism by a single UGT enzyme (fmUGT) of UGT substrates. The REF values of UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17 were 0.228, 0.0714, 0.0665, 0.420, 0.118, and 0.0442, respectively. fmUGTs in HLM were estimated for several typical UGT substrates utilizing these values and metabolic clearances in RM. These values were comparable to the reported values estimated by various methods. This study provided useful information on REF values, which promote a robust estimation of fmUGT values for UGT substrates when evaluating the contribution of UGT isoforms to total metabolic clearance.

Identification of naturally occurring inhibitors in Xian-Ling-Gu-Bao capsule against the glucuronidation of estrogens.

Xian-Ling-Gu-Bao (XLGB) capsule, a well-known traditional Chinese medicine prescription, is widely used for the treatment of osteoporosis. It could significantly increase the levels of estrogen in ovariectomized rats and mice. However, this working mechanism has not been well elucidated. Considering that UDP-glucuronosyltransferase (UGT) enzymes are the important enzymes that inactivate and regulate estrogen activity in vivo, this study aimed to identify the bioactive compounds from XLGB against the glucuronidation of estrogens. First, thirty compounds were considered as candidate bioactive compounds based on our previous studies including pharmacological evaluation, chemical profiles, and metabolic profiles. Second, the characteristics of estrogen glucuronidation by uridine diphosphate glucuronic acid (UDPGA)-supplemented human liver microsomes (HLM), human intestine microsomes (HIM), and expressed UGT enzymes were determined, and the incubation systems of their key UGT enzymes were optimized. Then, inhibitory effects and mechanisms of XLGB and its main compounds toward the key UGT isozymes were further investigated. As a result, estrogen underwent efficient glucuronidation by HLM and HIM. UGT1A10, 1A1, and 2B7 were mainly responsible for the glucuronidation of estrone, β-estradiol, and estriol, respectively. For E1 and E2, UGT1A10 and 1A1 tended to mediate estrogen-3-O-glucuronidation, while UGT2B7 preferred catalyzing estrogen-16-O-glucuronidation. Furthermore, the incubation system for active UGT isoforms was optimized including Tris-HCl buffer, detergents, MgCl2 concentration, β-glucuronidase inhibitors, UDPGA concentration, protein concentration, and incubation time. Based on optimal incubation conditions, eleven, nine, and nine compounds were identified as the potent inhibitors for UGT1A10, 1A1, and 2B7, respectively (IC50 < 4.97 μM and Ki < 3.35 μM). Among them, six compounds (bavachin, isobavachin, isobavachalcone, neobavaisoflavone, corylifol A, and icariside II) simultaneously demonstrated potent inhibitory effects against these three active enzymes. Prenylated flavanols from Epimedium brevicornu Maxim., prenylated flavonoids from Psoralea corylifolia L., and salvianolic acids from Salvia miltiorrhiza Bge. were characterized as the most important and effective compounds. The identification of potent natural inhibitors of XLGB against the glucuronidation of estrogen laid an important foundation for the pharmacodynamic material basis.

In silico prediction of UGT-mediated metabolism in drug-like molecules via graph neural network

UDP-glucuronosyltransferases (UGTs) have gained increasing attention as they play important roles in the phase II metabolism of drugs. Due to the time-consuming process and high cost of experimental approaches to identify the metabolic fate of UGT enzymes, in silico methods have been developed to predict the UGT-mediated metabolism of drug-like molecules. We developed consensus models with the combination of machine learning (ML) and graph neural network (GNN) methods to predict if a drug-like molecule is a potential UGT substrate, and then we applied the Weisfeiler-Lehman Network (WLN) model to identify the sites of metabolism (SOMs) of UGT-catalyzed substrates. For the substrate model, the accuracy of the single substrate prediction model on the test set could reach to 0.835. Compared with the single estimators, the consensus models are more stable and have better generalization ability, and the accuracy on the test set reached to 0.851. For the SOM model, the top-1 accuracy of the SOM model on the test set reached to 0.898, outperforming existing works. Thus, in this study, we proposed a computational framework, named Meta-UGT, which would provide a useful tool for the prediction and optimization of metabolic profiles and drug design.Graphical