Glucuronidation Activity 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.

Glucuronidation of tizoxanide, an active metabolite of nitazoxanide, in liver and small intestine: Species differences in humans, monkeys, dogs, rats, and mice and responsible UDP-glucuronosyltransferase isoforms in humans

Tizoxanide (TZX) is an active metabolite of nitazoxanide (NTZ) originally developed as an antiparasitic agent, and is predominantly metabolized into TZX glucuronide. In the present study, TZX glucuronidation by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice, and recombinant human UDP-glucuronosyltransferase (UGT) were examined. The kinetics of TZX glucuronidation by the liver and intestinal microsomes followed the Michaelis–Menten or biphasic model, with species-specific variations in the intrinsic clearance (CLint). Rats and mice exhibited the highest CLint values for liver microsomes, while mice and rats were the highest for intestinal microsomes. Among human UGTs, UGT1A1 and UGT1A8 demonstrated significant glucuronidation activity. Estradiol and emodin inhibited TZX glucuronidation activities in the human liver and intestinal microsomes in a dose-dependent manner, with emodin showing stronger inhibition in the intestinal microsomes. These results suggest that the roles of UGT enzymes in TZX glucuronidation in the liver and small intestine differ extensively across species and that UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. This study presents the relevant and novel-appreciative report on TZX metabolism catalyzed by UGT enzymes, which may aid in the assessment of the antiparasitic, antibacterial, and antiviral activities of NTZ for the treatment of various infections.

After 50 Years of Hepatic Clearance Models, Where Should We Go from Here? Improvements and Implications for Physiologically Based Pharmacokinetic Modeling.

There is overwhelming preference for application of the unphysiologic, well-stirred model (WSM) over the parallel tube model (PTM) and dispersion model (DM) to predict hepatic drug clearance, CLH , despite that liver blood flow is dispersive and closer to the DM in nature. The reasoning is the ease in computation relating the hepatic intrinsic clearance ( CLint ), hepatic blood flow ( QH ), unbound fraction in blood ( fub ) and the transmembrane clearances ( CLin and CLef ) to CLH for the WSM. However, the WSM, being the least efficient liver model, predicts a lower EH that is associated with the in vitro CLint ( Vmax / Km ), therefore requiring scale-up to predict CLH in vivo. By contrast, the miniPTM, a three-subcompartment tank-in-series model of uniform enzymes, closely mimics the DM and yielded similar patterns for CLint versus EH , substrate concentration [S] , and KL / B , the tissue to outflow blood concentration ratio. We placed these liver models nested within physiologically based pharmacokinetic models to describe the kinetics of the flow-limited, phenolic substrate, harmol, using the WSM (single compartment) and the miniPTM and zonal liver models (ZLMs) of evenly and unevenly distributed glucuronidation and sulfation activities, respectively, to predict CLH For the same, given CLint ( Vmax and Km ), the WSM again furnished the lowest extraction ratio ( EH,WSM = 0.5) compared with the miniPTM and ZLM (>0.68). Values of EH,WSM were elevated to those for EH, PTM and EH, ZLM when the Vmax s for sulfation and glucuronidation were raised 5.7- to 1.15-fold. The miniPTM is easily manageable mathematically and should be the new normal for liver/physiologic modeling. SIGNIFICANCE STATEMENT: Selection of the proper liver clearance model impacts strongly on CLH predictions. The authors recommend use of the tank-in-series miniPTM (3 compartments mini-parallel tube model), which displays similar properties as the dispersion model (DM) in relating CLint and [ S ] to CLH as a stand-in for the DM, which best describes the liver microcirculation. The miniPTM is readily modified to accommodate enzyme and transporter zonation.

Physiologically Based Pharmacokinetic Modeling to Predict the Impact of Liver Cirrhosis on Glucuronidation via UGT1A4 and UGT2B7/2B4-A Case Study with Midazolam.

Hepatic impairment, due to liver cirrhosis, decreases the activity of cytochrome P450 enzymes (CYPs). The use of physiologically based pharmacokinetic (PBPK) modeling to predict this effect for CYP substrates has been well-established, but the effect of cirrhosis on uridine-glucuronosyltransferase (UGT) activities is less studied and few PBPK models have been reported. UGT enzymes are involved in primary N-glucuronidation of midazolam and glucuronidation of 1'-OH-midazolam following CYP3A hydroxylation. In this study, Simcyp was used to establish PBPK models for midazolam, its primary metabolites midazolam-N-glucuronide (UGT1A4) and 1'-OH midazolam (CYP3A4/3A5), and the secondary metabolite 1'-OH-midazolam-O-glucuronide (UGT2B7/2B4), allowing to simulate the impact of liver cirrhosis on the primary and secondary glucuronidation of midazolam. The model was verified in noncirrhotic subjects before extrapolation to cirrhotic patients of Child-Pugh (CP) classes A, B, and C. Our model successfully predicted the exposures of midazolam and its metabolites in noncirrhotic and cirrhotic patients, with 86% of observed plasma concentrations within 5th-95th percentiles of predictions and observed geometrical mean of area under the plasma concentration curve between 0 hours to infinity and maximal plasma concentration within 0.7- to 1.43-fold of predictions. The simulated metabolic ratio defined as the ratio of the glucuronide metabolite AUC over the parent compound AUC (AUCglucuronide/AUCparent, metabolic ratio [MR]), was calculated for midazolam-N-glucuronide to midazolam (indicative of UGT1A4 activity) and decreased by 40% (CP A), 48% (CP B), and 75% (CP C). For 1'-OH-midazolam-O-glucuronide to 1'-OH-midazolam, the MR (indicative of UGT2B7/2B4 activity) dropped by 35% (CP A), 51% (CP B), and 64% (CP C). These predicted MRs were corroborated by the observed data. This work thus increases confidence in Simcyp predictions of the effect of liver cirrhosis on the pharmacokinetics of UGT1A4 and UGT2B7/UGT2B4 substrates. SIGNIFICANCE STATEMENT: This article presents a physiologically based pharmacokinetic model for midazolam and its metabolites and verifies the accurate simulation of pharmacokinetic profiles when using the Simcyp hepatic impairment population models. Exposure changes of midazolam-N-glucuronide and 1'-OH-midazolam-O-glucuronide reflect the impact of decreases in UGT1A4 and UGT2B7/2B4 glucuronidation activity in cirrhotic patients. The approach used in this study may be extended to verify the modeling of other uridine glucuronosyltransferase enzymes affected by liver cirrhosis.

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

In vitro glucuronidation of 17β-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. The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. 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. 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.

Glucuronidation dynamics of curcumin and tetrahydrocurcumin for differential structures and chemical reactivities in human liver microsome and uridine diphosphate glucuronosyltransferase 2B7

THC is the main metabolite of curcumin with better bioactivity. This study aimed to explore the factors that cause differences in the bioactivity of curcumin and THC. We analyzed the metabolic activities of curcumin and THC and the factors responsible for the differences in their activities by glucuronidation activity assay, LC-MS, HPLC, homologous sequence comparisons, and molecular docking. Curcumin has higher metabolic activity than THC in HLM and UGT2B7, while the keto-enol isomers of curcumin and THC were distinctly different under different pH, and their structural transformations were hypothesized. Furthermore, UGT1A and UGT2B are differential sequences of curcumin and THC in UGTs. The binding sites and patterns of curcumin and THC in UGT2B7 are markedly different. In summary, the difference in keto-enolic interconversion isomerism between curcumin and THC is the main factor causing the difference in their activities, which provides a scientific basis for the development of curcumin.

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.

A Comprehensive Bioinformatic Analysis of RNA-seq Datasets Reveals a Differential and Variable Expression of Wildtype and Variant UGT1A Transcripts in Human Tissues and Their Deregulation in Cancers.

The UGT1A locus generates over 60 different alternatively spliced transcripts and 30 circular RNAs. To date, v2 and v3 transcripts are the only variant UGT1A transcripts that have been functionally characterized. Both v2 and v3 transcripts encode the same inactive variant UGT1A proteins (i2s) that can negatively regulate glucuronidation activity and influence cancer cell metabolism. However, the abundance and interindividual variability in the expression of v2 and v3 transcripts in human tissues and their potential deregulation in cancers have not been comprehensively assessed. To address this knowledge gap, we quantified the expression levels of v1, v2, and v3 transcripts using RNA-seq datasets with large cohorts of normal tissues and paired normal and tumor tissues from patients with six different cancer types (liver, kidney, colon, stomach, esophagus, and bladder cancer). We found that v2 and v3 abundance varied significantly between different tissue types, and that interindividual variation was also high within the same tissue type. Moreover, the ratio of v2 to v3 variants varied between tissues, implying their differential regulation. Our results showed higher v2 abundance in gastrointestinal tissues than liver and kidney tissues, suggesting a more significant negative regulation of glucuronidation by i2 proteins in gastrointestinal tissues than in liver and kidney tissues. We further showed differential deregulation of wildtype (v1) and variant transcripts (v2, v3) in cancers that generally increased the v2/v1 and/or v3/v1 expression ratios in tumors compared to normal tissues, indicating a more significant role of the variants in tumors. Finally, we report ten novel UGT1A transcripts with novel 3' terminal exons, most of which encode variant proteins with a similar structure to UGT1A_i2 proteins. These findings further emphasize the diversity of the UGT1A transcriptome and proteome.

Glucuronidation Pathways of 5- and 7-Hydroxypropranolol: Determination of Glucuronide Structures and Enzyme Selectivity

Propranolol, a non-selective beta-blocker medication, has been utilized in the treatment of cardiovascular diseases for several decades. Its hydroxynaphthyl metabolites have been recognized to possess varying degrees of beta-blocker activity due to the unaltered side-chain. This study achieved the successful separation and identification of diastereomeric glucuronic metabolites derived from 4-, 5-, and 7-hydroxypropranolol (4-OHP, 5-OHP, and 7-OHP) in human urine. Subsequently, reaction phenotyping of 5- and 7-hydroxypropranolol by different uridine 5’-diphospho-glucuronosyltransferases (UGTs) was carried out, with a comparison to the glucuronidation of 4-hydroxypropranolol (4-OHP). Among the 19 UGT enzymes examined, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1, and UGT2A2 were found to be involved in the glucuronidation of 5-OHP. Furthermore, UGT1A6 exhibited glucuronidation activity towards 7-OHP, along with the aforementioned eight UGTs. Results obtained by glucuronidation of corresponding methoxypropranolols and MS/MS analysis of 1,2-dimethylimidazole-4-sulfonyl (DMIS) derivatives of hydroxypropranolol glucuronides suggest that both the aromatic and aliphatic hydroxy groups of the hydroxypropranolols may be glucuronidated in vitro. However, the analysis of human urine samples collected after the administration of propranolol leads us to conclude that aromatic-linked glucuronidation is the preferred pathway under physiological conditions.

B-316 Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations

Abstract Background Ethanol is utilized in many pharmaceutical formulations and in the treatment of methanol poisoning. It is also consumed recreationally and is the most abused drug globally. About 95% of consumed ethanol is metabolized by alcohol dehydrogenase into acetaldehyde, followed by conversion of the acetaldehyde into acetic acid by acetaldehyde dehydrogenase. Less than one percent is metabolized via nonoxidative pathways, which may include: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates the glucuronidation pathway has been reported to be underdeveloped and matures with age. In this work, the patterns and concentrations of phase II nonoxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), were assessed in random urine collections of pediatric, adult, and geriatric patients. Methods Test results (n = 63498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) at our facility were utilized for this study. For both analytes, the lower limit of positivity was 100 ng/mL. The age range under consideration was 0–100 years. Data was partitioned into age groups: 0–17 years, 18–80 years and 81–100 years. The EtG and EtS concentrations across the partitions were compared. Results Across all ages, 60%–65% of patients had both EtG and EtS present in urine. Between 5%–10% had EtG present without EtS, and 25%–35% of patients had neither of the ethanol metabolites present. In all age groups there was a low positivity rate of EtS without the presence of EtG (0%–1%). All age partitions showed a similar pattern with the highest percentages of patients showing the presence of both metabolites, and the lowest percentage of patients showing the presence of EtS only. Markedly, there were no pediatric patient samples that had the sulfated metabolite present in the absence of the glucuronidated metabolite; this was statistically significantly different than this proportion in adults (Fisher’s Exact test, P = 0.025). This finding suggests greater glucuronidation activity in the pediatric population. Conclusion From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure. This work also shows that glucuronidation may be the predominant pathway for ethanol metabolism in pediatric populations. Although the level and frequency of ethanol exposure, as well as the time between exposure and sampling were unknown for the patients studied, the data offers insights into the relative distribution of EtG and EtS in random urine samples for pediatric and adult populations.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

Heterodimerization of Human UDP-Glucuronosyltransferase 1A9 and UDP-Glucuronosyltransferase 2B7 Alters Their Glucuronidation Activities

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role as prominent phase II metabolic enzymes, mediating the glucuronidation of both endobiotics and xenobiotics. Dimerization greatly modulates the enzymatic activities of UGTs. In this study, we examined the influence of three mutations (H35A, H268Y, and N68A/N315A) and four truncations (signal peptide, single transmembrane helix, cytosolic tail, and di-lysine motif) in UGT2B7 on its heterodimerization with wild-type UGT1A9, using a Bac-to-Bac expression system. We employed quantitative fluorescence resonance energy transfer (FRET) techniques and co-immunoprecipitation assays to evaluate the formation of heterodimers between UGT1A9 and UGT2B7 allozymes. Furthermore, we evaluated the glucuronidation activities of the heterodimers using zidovudine and propofol as substrates for UGT2B7 and UGT1A9, respectively. Our findings revealed that the histidine residue at codon 35 was involved in the dimeric interaction, as evidenced by the FRET efficiencies and catalytic activities. Interestingly, the signal peptide and single transmembrane helix domain of UGT2B7 had no impact on the protein-protein interaction. These results provide valuable insights for a comprehensive understanding of UGT1A9/UGT2B7 heterodimer formation and its association with glucuronidation activity. SIGNIFICANCE STATEMENT: Our findings revealed that the H35A mutation in UGT2B7 affected the affinity of protein-protein interaction, leading to discernable variations in fluorescence resonance energy transfer efficiencies and catalytic activity. Furthermore, the signal peptide and single transmembrane helix domain of UGT2B7 did not influence heterodimer formation. These results provide valuable insights into the combined effects of polymorphisms and protein-protein interactions on the catalytic activity of UGT1A9 and UGT2B7, enhancing our understanding of UGT dimerization and its impact on metabolite formation.

Investigating the Contribution of Major Drug-Metabolising Enzymes to Possum-Specific Fertility Control.

The potential to improve the effectiveness and efficiency of potential oestrogen-based oral contraceptives (fertility control) for possums was investigated by comparing the inhibitory potential of hepatic CYP3A and UGT2B catalytic activity using a selected compound library (CYP450 inhibitor-based compounds) in possums to that of three other species (mouse, avian, and human). The results showed higher CYP3A protein levels in possum liver microsomes compared to other test species (up to a 4-fold difference). Moreover, possum liver microsomes had significantly higher basal p-nitrophenol glucuronidation activity than other test species (up to an 8-fold difference). However, no CYP450 inhibitor-based compounds significantly decreased the catalytic activity of possum CYP3A and UGT2B below the estimated IC50 and 2-fold IC50 values and were therefore not considered to be potent inhibitors of these enzymes. However, compounds such as isosilybin (65%), ketoconazole (72%), and fluconazole (74%) showed reduced UGT2B glucuronidation activity in possums, mainly at 2-fold IC50 values compared to the control (p < 0.05). Given the structural features of these compounds, these results could provide opportunities for future compound screening. More importantly, however, this study provided preliminary evidence that the basal activity and protein content of two major drug-metabolising enzymes differ in possums compared to other test species, suggesting that this could be further exploited to reach the ultimate goal: a potential target-specific fertility control for possums in New Zealand.

Development of a bioprinter-based method for incorporating metabolic competence into high-throughput in vitro assays.

The acceptance and use of in vitro data for hazard identification, prioritization, and risk evaluation is partly limited by uncertainties associated with xenobiotic metabolism. The lack of biotransformation capabilities of many in vitro systems may under- or overestimate the hazard of compounds that are metabolized to more or less active metabolites in vivo. One approach to retrofitting existing bioassays with metabolic competence is the lid-based Alginate Immobilization of Metabolic Enzymes (AIME) method, which adds hepatic metabolism to conventional high-throughput screening platforms. Here, limitations of the lid-based AIME method were addressed by incorporating bioprinting, which involved depositing S9-encapsulated microspheres into standard 384-well plates with requisite cofactors for phase I and II hepatic metabolism. Objectives of this study included: 1) compare the lid-based and AIME bioprinting methods by assessing the enzymatic activity of a common cytochrome P450 (CYP) enzyme, 2) use biochemical assays with the bioprinting method to characterize additional measures of phase I and II metabolic activity, and 3) evaluate the bioprinting method by screening 25 chemicals of known metabolism-dependent bioactivity in the VM7Luc estrogen receptor transactivation (ERTA) assay. A comparison of the two methods revealed comparable precision and dynamic range. Activity of additional CYP enzymes and glucuronidation was observed using the AIME bioprinting method. The ERTA experiment identified 19/21ER-active test chemicals, 14 of which were concordant with expected biotransformation effects (73.7%). Additional refinement of the AIME bioprinting method has the potential to expand high-throughput screening capabilities in a robust, accessible manner to incorporate in vitro metabolic competence.

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.

Profiling of Urinary Glucuronidated Bile Acids across Age Groups.

We investigated the age-dependent changes in urinary excretion of glucuronidated bile acids at the C-3 position. Bile acid 3-glucuronides accounted for 0.5% of urinary bile acids in neonates, and the proportion of bile acid 3-glucuronides plateaued at 1-3 years of age. The 3-glucuronides of secondary bile acids were first secreted at 3 months of age, the same time as the establishment of the gut bacterial flora in infants. A considerable portion of bile acid 3-glucuronides were present as non-amidated forms. Our results indicate dynamic hepatic enzyme activity in which the levels of uridine 5'-diphospho-glucuronosyltransferases (UGTs) differ by age group, with higher glucuronidation activity of UGTs towards nonamidated bile acids than amidated bile acids.

Activation of Alternative Bilirubin Clearance Pathways Partially Reduces Hyperbilirubinemia in a Mouse Model Lacking Functional Ugt1a1 Activity.

Bilirubin is a heme catabolite and Ugt1a1 is the only enzyme involved in the biological elimination of bilirubin. Partially functional or non-functional Ugt1a1 may result in neuronal damage and death due to the accumulation of unconjugated bilirubin in the brain. The understanding of the role of alternative bilirubin detoxification mechanisms that can reduce bilirubin toxicity risk is crucial for developing novel therapeutic strategies. To provide a proof-of-principle showing whether activation of alternative detoxification pathways could lead to life-compatible bilirubin levels in the absence of Ugt1a1 activity, we used Ugt1−/− hyperbilirubinemic mice devoid of bilirubin glucuronidation activity. We treated adult Ugt1−/− mice with TCPOBOP, a strong agonist of the constitutive androstane receptor (CAR). TCPOBOP treatment decreased plasma and liver tissue bilirubin levels by about 38%, and resulted in the transcriptional activation of a vast array of genes involved in bilirubin transport and metabolism. However, brain bilirubin level was unaltered. We observed ~40% degradation of bilirubin in the liver microsomes from TCPOBOP treated Ugt1−/− mice. Our findings suggest that, in the absence of Ugt1a1, the activation of alternative bilirubin clearance pathways can partially improve hyperbilirubinemic conditions. This therapeutic approach may only be considered in a combinatorial manner along with other treatments.

Effects of common genetic variants of human uridine diphosphate glucuronosyltransferase subfamilies on irinotecan glucuronidation

The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6–UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

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.