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), trifluoperazine (UGT1A4), 4-hydroxyindole (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). In a cocktail incubation, UGT1A1, UGT1A9, and UGT2B7 activities were substantially inhibited by other substrates. This interference could be eliminated by dividing substrates into two incubations: one containing estradiol, trifluoperazine, and 4-hydroxyindole, and the other containing chenodeoxycholic acid, propofol, and naloxone. Incubation mixtures were pooled for the simultaneous analysis of glucuronyl conjugates in a single LC-MS/MS run. The optimized cocktail method was further validated against single-probe substrate assays using compounds known to inhibit UGTs. The degree of inhibition of UGT isoform activities by such known inhibitors in this cocktail assay was not substantially different from that in single-probe assays. This six-isoform cocktail assay may be very useful in assessing the UGT-based drug-interaction potential of candidates in a drug-discovery setting.

Simultaneous Screening of Activities of Five Cytochrome P450 and Four Uridine 5′-Diphospho-glucuronosyltransferase Enzymes in Human Liver Microsomes Using Cocktail Incubation and Liquid Chromatography–Tandem Mass Spectrometry

“…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.…”

“…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).…”

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1

“…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.…”

LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma

“…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.…”