2014
DOI: 10.1124/dmd.114.058818
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In Vitro Assay of Six UDP-Glucuronosyltransferase Isoforms in Human Liver Microsomes, Using Cocktails of Probe Substrates and Liquid Chromatography–Tandem Mass Spectrometry

Abstract: UDP-glucuronosyltransferase (UGT)-mediated drug-drug interactions are commonly evaluated during drug development. We present a validated method for the simultaneous evaluation of drugmediated inhibition of six major UGT isoforms, developed in human liver microsomes through the use of pooled specific UGT probe substrates (cocktail assay) and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The six probe substrates used in this assay were estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3… Show more

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Cited by 43 publications
(34 citation statements)
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“…Several LC-MS/MS-based P450 (Dierks et al, 2001;Kim et al, 2005;Smith et al, 2007;Zambon et al, 2010;Na et al, 2011;Pillai et al, 2013) or UGT (Gagez et al, 2012;Joo et al, 2014;Seo et al, 2014) The optimum incubation conditions for the assessment of enzyme activities are different between the P450 and UGT enzymes. The microsomal incubation of P450 enzymes was conducted in phosphate buffer, whereas UGT enzymes were incubated in Tris-HCl buffer.…”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…Several LC-MS/MS-based P450 (Dierks et al, 2001;Kim et al, 2005;Smith et al, 2007;Zambon et al, 2010;Na et al, 2011;Pillai et al, 2013) or UGT (Gagez et al, 2012;Joo et al, 2014;Seo et al, 2014) The optimum incubation conditions for the assessment of enzyme activities are different between the P450 and UGT enzymes. The microsomal incubation of P450 enzymes was conducted in phosphate buffer, whereas UGT enzymes were incubated in Tris-HCl buffer.…”
Section: Discussionmentioning
confidence: 99%
“…The resultant P450-mediated metabolites are determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Kim et al, 2005;Na et al, 2011;Spaggiari et al, 2014). Recently, three screening methods for UGT enzyme activities using cocktail incubation and tandem mass spectrometry also have been reported (Gagez et al, 2012;Joo et al, 2014;Seo et al, 2014).…”
Section: Introductionmentioning
confidence: 99%
“…The metabolic activities of individual HLMs (n ¼ 14) toward niclosamide, phenacetin (a probe substrate for CYP1A2) (Zhou et al, 2009), b-estradiol (a probe substrate for UGT1A1) (Seo et al, 2014) and chenodeoxycholic acid (a probe substrate for UGT1A3) (Seo et al, 2014) were determined, respectively, according to the metabolism assay protocols as described above. Phenacetin (10 mM) was incubated with NADPH-supplemented individual HLM (0.25 mg/ml) for 20 min.…”
Section: Activity Correlation Analysismentioning
confidence: 99%
“…The incubation for glucuronidation was adapted from a previously published method to allow for a 0.5 mL incubation volume [17]. The final incubation mixture contained 0.1 M phosphate buffer (pH 7.4), 0.25 mg/mL microsomal protein concentration, 25 μg/mL alamethicin, 50 mM Tris-HCl, 10 mM MgCl 2 , and 5,000 ng/mL of VX-970.…”
Section: Methodsmentioning
confidence: 99%