Optimization of Experimental Conditions of Automated Glucuronidation Assays in Human Liver Microsomes Using a Cocktail Approach and Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry

Badée, Justine; Qiu, NaHong; Parrott, Neil; Collier, Abby C.; Schmidt, Stephan; Fowler, Stephen

doi:10.1124/dmd.118.084301

Cited by 27 publications

(37 citation statements)

References 32 publications

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“…The glucuronide formation rates of selected UGT probe substrates were determined using individual and pooled microsomal liver preparations within a single experiment using a Tecan Fluent system and under optimized experimental conditions similar to those described in our previous study . For substrates exhibiting high turnover rates, the quality and reliability of in vitro measurements were confirmed by the lack of depletion of parent substrate, which was monitored in parallel to glucuronide formation.…”

Section: Discussionmentioning

confidence: 99%

“…24 Selective or partially selective substrates of hepatic UGT isoforms were previously discussed in our recent study. 24 Oxazepam was incubated as a mixture of Sand R-stereoisomers. Stereoselective analysis demonstrated that S-oxazepam glucuronide represented the main glucuronide metabolite (50%−91% of total concentration) in the microsomal incubations (inhouse data), and therefore S-oxazepam glucuronide formation was used to assess the microsomal glucuronidation activity of UGT2B15.…”

Section: Selection and Incubation Of Ugt Probe Substratesmentioning

confidence: 93%

“…Glucuronide formation of β‐estradiol, chenodeoxycholic acid, trifluoperazine, 5‐hydroxytryptophol, propofol, gemfibrozil, zidovudine, amitriptyline, oxazepam, and testosterone was used to determine the hepatic metabolic activity of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17, respectively . Selective or partially selective substrates of hepatic UGT isoforms were previously discussed in our recent study . Oxazepam was incubated as a mixture of S‐ and R‐stereoisomers.…”

Section: Methodsmentioning

confidence: 99%

“…The glucuronidation activity assays were performed as described in our previous publication, but with individual incubation of substrates . The experimental protocol was amended using lower HLM concentrations to ensure linearity and measurability of glucuronidation activity in individual HLM preparations from adult and pediatric donors.…”

Section: Methodsmentioning

confidence: 99%

“…These include the partial selectivity of in vitro UGT probe substrates, the lack of clinically used drugs as selective probe substrates, and variations in experimental conditions across in vitro studies . To minimize experimental variability, we recently optimized the experimental conditions of an in vitro automated glucuronidation assay and discussed the selectivity of UGT probe substrates as functional markers of UGT activity in HLMs of UGT phenotyping and screening assays . This enabled a more effective performance of multidonor UGT activity studies including correlation analysis of UGT activities using multiple donors and the assessment of genetic polymorphism or ontogeny in UGT‐metabolized drug clearance.…”

mentioning

confidence: 99%

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Characterization of the Ontogeny of Hepatic UDP‐Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes

Badée

Qiu

Collier

et al. 2019

The Journal of Clinical Pharma

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An understanding of the postnatal development of hepatic UDP‐glucuronosyltransferase (UGT) enzymes is required for accurate prediction of the age‐dependent changes in pharmacokinetics of many drugs used in children. However, the maturation rate of hepatic UGT isoforms remains a major knowledge gap. This study aimed to establish the age‐associated changes in glucuronidation activity of 10 major hepatic UGT isoforms in humans, namely, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Human liver microsomes from pediatric and adult donors were incubated under optimized incubation conditions to assess the activity rates of hepatic UGT isoforms using a panel of 19 in vitro UGT probe substrates and clinically used drugs. Statistically strong correlations of glucuronidation activities allowed the ontogeny of UGT1A1, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 to be established using multiple selective UGT substrates and matched human liver microsome samples. The postnatal development of hepatic UGTs is isoform‐dependent using either individual or cross‐correlated selective isoform substrates. Maximal adult activity was reached at different times ranging from within a month (UGT1A1, UGT2B4, UGT2B7, UGT2B10, and UGT2B15), during infancy (UGT1A3, UGT1A4, and UGT1A9), to adolescence (UGT1A6 and UGT2B17). This study provides an extensive characterization of the postnatal ontogeny profiles of hepatic UGT enzymes that are instrumental for predicting drug disposition via in vitro–in vivo extrapolation algorithms and verifying pharmacokinetic predictions against in vivo observations via pediatric physiologically based pharmacokinetic modeling in pediatric patients.

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Section: Discussionmentioning

confidence: 99%

Section: Selection and Incubation Of Ugt Probe Substratesmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of the Ontogeny of Hepatic UDP‐Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes

Badée

Qiu

Collier

et al. 2019

The Journal of Clinical Pharma

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In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

Lu¹,

2020

Biopharm & Drug Disp

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Drug-drug interactions (DDIs) caused by the co-administration of multiple drugs are major safety concerns in the clinic. Several drugs have been withdrawn from the market due to perpetrator or victim DDIs. Strategies have been developed to assess DDI risks early in drug discovery to reduce DDI liabilities. High-to-medium throughput assays are available to identify undesirable scaffolds and to guide structural modifications to minimize DDIs. Definitive methods are used at later stages of drug discovery and development to provide a more accurate measurement of DDI parameters and to enable clinical translations. Physiologically based pharmacokinetic modeling and simulations are powerful tools to accurately predict DDIs and to assess risks in the clinic. Although significant advances have been made over the years, many challenges remain for clinical DDI translations. This includes DDIs involving non-cytochrome P450 enzymes, transporters, enzyme-transporter interplay, indirect effects from biologics, and pharmacodynamic based DDI. This review focuses on methods that are used to assess hepatic DDIs caused by enzyme inhibition and induction.

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Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs)

Zhou

Argikar

Miners

2021

Methods in Molecular Biology

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Optimization of Experimental Conditions of Automated Glucuronidation Assays in Human Liver Microsomes Using a Cocktail Approach and Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract: UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs.

Cited by 27 publications

References 32 publications

Characterization of the Ontogeny of Hepatic UDP‐Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes

Characterization of the Ontogeny of Hepatic UDP‐Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes

In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs)

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