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Research ArticleArticle

Characterization of Niflumic Acid as a Selective Inhibitor of Human Liver Microsomal UDP-Glucuronosyltransferase 1A9: Application to the Reaction Phenotyping of Acetaminophen Glucuronidation

John O. Miners, Kushari Bowalgaha, David J. Elliot, Pawel Baranczewski and Kathleen M. Knights
Drug Metabolism and Disposition April 2011, 39 (4) 644-652; DOI: https://doi.org/10.1124/dmd.110.037036

Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50–100 μM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with Ki values ranging from 0.10 to 0.40 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (Ki range 14–18 μM), whereas competitive inhibition (Ki 62 μM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 μM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean Vmax for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 μM NFA. Mean Km values decreased in parallel with Vmax, although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

Introduction

The importance of conjugation with glucuronic acid (“glucuronidation”) as an elimination and detoxification mechanism for xenobiotics and endogenous compounds has become increasingly recognized over the last two decades. In particular, glucuronidation contributes to the clearance of structurally diverse drugs from all therapeutic classes (Miners and Mackenzie, 1991; Kiang et al., 2005). The glucuronidation reaction is catalyzed by UDP-glucuronosyltransferase (UGT) and, consistent with its broad substrate profile, UGT is known to exist as an enzyme “superfamily” in humans. Eighteen human UGT proteins that use UDP-glucuronic acid as cofactor have been characterized to date, and these have been classified in three subfamilies, UGT1A, UGT2A, and UGT2B, based on amino acid sequence identity (Mackenzie et al., 2005). Of the 11 UGT proteins expressed to a significant extent in liver current evidence suggests that UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15 are of the greatest importance in the hepatic metabolism of drugs, although UGT1A3, UGT2B4, and UGT2B10 also contribute to drug glucuronidation (Kiang et al., 2005; Miners et al., 2010a).

Whereas most human UGT enzymes glucuronidate low molecular mass aliphatic alcohols and phenols, enzyme substrate selectivity occurs with increasing structural complexity of the aglycone (Miners et al., 2006, 2010a). Likewise, the individual enzymes tend to differ in terms of inhibitor selectivity and hence patterns of drug-drug interactions (Kiang et al., 2005; Miners et al., 2010b). Moreover, activities appear to be variably affected by age, alcohol consumption, cigarette smoking, diet, disease states, ethnicity, genetic polymorphism, and hormonal factors (Miners and Mackenzie, 1991; Miners et al., 2002, 2004; Guillemette 2003; Court, 2010).

Given these features of human UGT enzymes, attention has focused on the identification of enzyme-selective substrate and inhibitor probes for the reaction phenotyping of drug glucuronidation reactions. UGT enzyme-selective substrates have been identified for the main human hepatic drug-metabolizing UGTs, and these may be used in activity correlation studies in a panel of human liver microsomes (HLM) or hepatocytes (Court, 2005; Miners et al., 2006, 2010a). However, enzyme-selective inhibitors represent a more valuable experimental tool for reaction phenotyping because the extent of inhibition of the glucuronidation of a test compound by HLM or hepatocytes reflects the contribution of the individual UGT enzymes to metabolic clearance. In contrast to the ready availability of substrate probes, only two UGT enzyme selective inhibitors have been identified to date. Hecogenin (10 μM) is a highly selective inhibitor of UGT1A4 (Uchaipichat et al., 2006a), and fluconazole (2500 μM) is a moderately selective inhibitor of UGT2B7 (Uchaipichat et al., 2006b), although more recent studies suggest similar inhibition of UGT2B4 (Raungrut et al., 2010). [Isolongifolol and a phenyl-substituted secondary alcohol derived from this compound have been reported to be potent, selective inhibitors of recombinant UGT2B7 (Bichlmaier et al., 2007), although experimental conditions for selective inhibition of human liver microsomal UGT2B7 appear not to have been characterized.]

Although UGT1A1 appears to be the principal enzyme involved in the glucuronidation of the nonsteroidal anti-inflammatory drug niflumic acid (NFA) (Mano et al., 2006a), it has been demonstrated that this compound potently inhibits 4-methylumbelliferone glucuronidation by recombinant UGT1A9 (Mano et al., 2006b; Gaganis et al., 2007). In addition, it has been reported that NFA inhibits the glucuronidation of the predominantly UGT1A9 substrate mycophenolic acid with HLM as the enzyme source (Vietri et al., 2002). However, high concentrations of NFA may also inhibit other UGT enzymes (Gaganis et al., 2007). To investigate the potential utility of NFA as an inhibitor probe for the reaction phenotyping of drug glucuronidation reactions, this study characterized the concentration dependence of UGT enzyme inhibition by NFA and the mechanism of inhibition. The results demonstrated that, under appropriate experimental conditions, NFA selectively inhibits UGT1A9 and, to a lesser extent, UGT1A1 and UGT2B15.

After demonstration of UGT enzyme selectivity, NFA inhibition was applied to the reaction phenotyping of acetaminophen (APAP; paracetamol) glucuronidation by HLM. Glucuronidation accounts for approximately 65% of the metabolic clearance of a therapeutic dose of APAP in humans (Miners et al., 1984). Although APAP is one of the most widely used drugs worldwide, the relative contribution of individual UGT enzymes to APAP glucuronidation is yet to be completely resolved. Whereas APAP glucuronidation is generally attributed to UGT1A1, UGT1A6, and UGT1A9 (Bock et al., 1993; Court et al., 2001), more recently Mutlib et al. (2006) proposed a significant role for UGT2B15. Thus, NFA inhibition was used here to further explore the relative contribution each of these enzymes to APAP glucuronidation by HLM.

Materials and Methods

Materials.

Acetaminophen β-d-glucuronide (APAPG), alamethicin (from Trichoderma viride), APAP, codeine (COD), 4-methylumbelliferone (4MU), 4-methylumbelliferone β-d-glucuronide, β-estradiol (β-E2), β-estradiol-3-β-d-glucuronide, propofol (PRO), testosterone (TST), trifluoperazine (TFP), UDP-glucuronic acid (UDPGA) (sodium salt), and zidovudine (AZT) were purchased from Sigma-Aldrich (Sydney, Australia). Codeine 6-O-β-d-glucuronide and deferiprone 3-O-β-d-glucuronide (DEFG) were purchased from Toronto Research Chemicals (North York, ON, Canada). Lamotrigine (LTG) and lamotrigine N2-β-d-glucuronide were a gift from the Wellcome Research Laboratories (Beckenham, UK), and deferiprone (DEF) was kindly supplied by Dr. John Connelly (ApoPharma Inc., Toronto, ON, Canada). NFA was a gift from the Squibb Institute for Medical Research (Princeton, NJ). Supersomes expressing UGT2B4, UGT2B7, and UGT2B15 were purchased from BD Biosciences (San Jose, CA). Solvents and other reagents used were of analytical reagent grade.

Human Liver Microsomes and Recombinant UGTs.

HLM were prepared from the hepatic tissue of five donors (HL7, HL10, HL12, HL13, and HL40) by differential ultracentrifugation according to Bowalgaha et al. (2005) and, where indicated, pooled by mixing equal protein amounts. Livers were obtained from the tissue “bank” of the Department of Clinical Pharmacology, Flinders Medical Centre. Approval for the use of human liver tissue for in vitro drug metabolism studies was obtained from the Flinders Clinical Research Ethics Committee. HLM were activated by preincubation with the pore-forming agent alamethicin (50 μg/mg microsomal protein) before use in incubations, as described by Boase and Miners (2002).

Human UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, and 2B17 cDNAs were stably expressed in a human embryonic kidney cell line (HEK293T) (Uchaipichat et al., 2004). After growth to at least 80% confluence, cells were harvested and washed in phosphate-buffered saline. Cells were subsequently lysed by sonication using a Vibra Cell VCX 130 Ultrasonics Processor (Sonics and Materials, Newtown, CT). Lysates were centrifuged at 12,000g for 1 min at 4°C, and the supernatant fraction was separated and stored in phosphate buffer (0.1 M, pH 7.4) at −80°C until use. Given the relatively higher expression of UGT2B4, UGT2B7, and UGT2B15 in Sf cells, Supersomes expressing these proteins were used for activity and inhibition studies. Expression of all UGT proteins was demonstrated by immunoblotting with a commercial UGT1A antibody (BD Gentest, Woburn, MA) or a nonselective UGT antibody (raised against purified mouse Ugt) (Uchaipichat et al., 2004) and by activity measurement (see below).

NFA Inhibition of Recombinant UGT Enzyme Activities.

NFA was screened for inhibition of UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 activities using the nonselective UGT substrate 4MU as the probe. Incubations were performed at a 4MU concentration corresponding to the apparent Km or S50 of each enzyme (Uchaipichat et al., 2004), in the absence and presence of NFA (1, 10, and 100 μM). NFA stock solutions were prepared in dimethyl sulfoxide such that the final concentration of solvent in incubations was 1% (v/v), which has a negligible or minor effect on most UGT activities (Uchaipichat et al., 2004). An equivalent volume of dimethyl sulfoxide was included in control incubations. Protein concentrations and incubation time varied for each enzyme and were as described by Uchaipichat et al. (2004). Incubations, in a total volume of 200 μl, contained MgCl2 (4 mM), UDPGA (5 mM), HEK293T cell lysate or Supersomes, and 4MU in phosphate buffer (0.1 M, pH 7.4). Reaction mixtures were preincubated at 37°C for 5 min before initiation of the reaction by addition of cofactor (UDPGA). Incubations were performed in air at 37°C (shaking water bath) for the specified time for each enzyme (Uchaipichat et al., 2004) and then were terminated by the addition of 70% of perchloric acid (2 μl). Mixtures were vortex-mixed and centrifuged at 4000g for 10 min. Formation of 4MU glucuronide was quantified by HPLC following the procedure described by Lewis et al. (2007).

Inhibition of UGT1A4 by NFA was assessed with LTG as the probe substrate according to Rowland et al. (2006). Incubations, in a total volume of 200 μl, contained MgCl2 (4 mM), UDPGA (5 mM), HEK293 cell lysate expressing UGT1A4 (1 mg/ml), LTG (1500 μM), and NFA (0, 10, and 100 μM). LTG 2-glucuronide was measured by HPLC (Rowland et al., 2006). Effects of NFA on UGT2B4 activity were determined with COD as the substrate (Raungrut et al., 2010). In brief, incubations in a total volume of 100 μl contained MgCl2 (4 mM), UDPGA (5 mM), Supersomes expressing UGT2B4 (1 mg/ml), COD (2000 μM), and NFA (0, 1, 10, and 100 μM). Incubations were performed at 37°C for 120 min. Codeine 6-O-β-d-glucuronide formation was quantified by HPLC (Raungrut et al., 2010).

NFA Inhibition of Human Liver Microsomal UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15/17 Activities.

NFA was screened for inhibition of human liver microsomal UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15/17 activities using β-E2, TFP, DEF, PRO, AZT, and TST as the respective probe substrates (Miners et al., 2010a). Inhibition, relative to control activities, was characterized for three NFA concentrations (1, 10, and 100 μM; see above) at probe substrate concentrations corresponding to the approximate Km or S50 for each compound with HLM as the enzyme source: 40 μM β-E2 (Soars et al., 2003), 60 μM TFP (Uchaipichat et al., 2006a), 4000 μM DFP (K. Bowalgaha and J. O. Miners, unpublished data), 125 μM PRO (Rowland et al., 2008), 1000 μM AZT (Boase and Miners, 2002), and 5 μM TST (Bowalgaha et al., 2007). Studies were performed with pooled HLM from five livers (HL7, HL10, HL12, HL13, and HL40). Microsomal protein concentrations, incubation times, and HPLC conditions for the TFP, PRO, AZT, and TST glucuronidation assays were as described by Uchaipichat et al. (2006a), Rowland et al. (2008), Boase and Miners (2002), and Bowalgaha et al. (2007), respectively.

For experiments investigating inhibition of β-E2 3-glucuronidation with pooled HLM as the enzyme source, incubations contained pooled HLM protein (0.2 mg/ml), phosphate buffer (0.1 M, pH 7.4), MgCl2 (4 mM), UDPGA (5 mM), and β-E2, with and without NFA, in a total volume of 200 μl. Incubations were performed for 30 min at 37°C, and β-estradiol-3-β-d-glucuronide formation was quantified by HPLC according to Udomuksorn et al. (2007).

Incubations characterizing human liver microsomal DEF 3-glucuronidation activity contained DEF (4000 μM), HLM protein (0.5 mg/ml), MgCl2 (4 mM), and UDPGA (5 mM) in phosphate buffer (200 μl; 0.1 M, pH 7.4). Reactions were performed for 60 min and 37°C, after which time samples were treated with 70% HClO4 (2 μl). After centrifugation (4000g for 10 min), a 15-μl aliquot of the supernatant fraction was injected on to the HPLC column. HPLC was performed using an 1100 series instrument (Agilent Technologies, Sydney, Australia) fitted with a Nova-Pak C18 analytical column (3.9 × 150 mm, 5 μm particle size; Waters, Milford, MA). Analytes were detected by UV absorption at 280 nm. The mobile phase was composed of 5 mM heptane sulfonic acid in water (pH 2.2) (A) and acetonitrile (B), delivered at a flow rate of 1 ml/min. Initial conditions were 98% A-2% B increasing linearly to 83% A-17% B over 10 min, which was held constant for 1.5 min before returning to the initial conditions. DEFG and DEF eluted at 4.9 and 8.8 min, respectively. DEFG was quantified by reference to a standard curve constructed using the authentic compound. Overall within-day assay reproducibility was assessed by measuring DEFG formation at low (0.5 mM) and high (10 mM) substrate concentrations on nine occasions with pooled HLM as the enzyme source. Within-day coefficients of variation were 3.5 and 1.6% at 0.5 and 10 mM, respectively.

Kinetic Characterization of NFA Inhibition of UGT1A1, UGT1A9, and UGT2B15.

Mechanisms of inhibition and inhibitor constants for NFA inhibition of UGT1A1 and UGT1A9 were determined with the individual recombinant enzymes and with pooled HLM, whereas inhibition of UGT2B15 was characterized only with the recombinant enzyme (due to the lack of convenient selective probes suitable for measurement of microsomal enzyme activity). Incubation conditions were as described in previous sections, unless specified otherwise. Control activities were determined in the absence of NFA in all experiments.

UGT1A1.

Studies with recombinant UGT1A1 were conducted with 4MU and β-E2 as the probe substrates, whereas experiments with HLM as the enzyme source used only β-E2. Experiments to determine the inhibitor constants with UGT1A1 as the enzyme source included four NFA concentrations (5, 10, 15, and 20 μM) at each of three β-E2 (7.5, 15, and 40 μM) and 4MU (30, 60, and 120 μM) concentrations. Likewise, studies with pooled HLM used four concentrations of NFA (5, 10, 20, and 50 μM) at each of three concentrations of β-EST (15, 30, and 60 μM).

UGT1A9.

Experiments to characterize NFA inhibition of UGT1A9 were as described for UGT1A1, except that PRO was used as the UGT1A9-selective substrate with HLM as the enzyme source. Four NFA concentrations (0.2, 0.4, 0.6, and 0.8 μM) at each of three 4MU (5, 10, and 15 μM) and PRO (20, 40, and 100 μM) concentrations were used to determine the inhibitor constants with UGT1A9 as the enzyme source. Inhibition experiments with pooled HLM similarly used four NFA concentrations (0.25, 0.5, 1, and 1.5 μM) at each of three PRO concentrations (60, 100, and 200 μM).

UGT2B15.

NFA inhibition of UGT2B15 glucuronidation was investigated using Supersomes expressing UGT2B15 as the enzyme source and 4MU as the substrate. Incubation and assay conditions were as described under UGT1A1. The Supersomes protein concentration and incubation time were 0.25 mg/ml and 90 min, respectively. Experiments to determine inhibitor constants included four NFA concentrations (20, 40, 60, and 80 μM) at each of three 4MU concentrations (50, 150, and 300 μM).

APAP Glucuronidation Assay and NFA Inhibition of APAP Glucuronide Formation.

APAP glucuronidation kinetic studies were performed with microsomes from three separate human livers (H12, H13, and H40) and the individual recombinant UGTs shown to glucuronidate this compound in activity screening experiments. Linearity of product formation with respect to incubation time and protein concentration for HLM and expressed UGT enzymes was confirmed before kinetic studies. Incubations, in a total volume of 200 μl contained phosphate buffer (0.1 M, pH 7.4), MgCl2 (4 mM), UDPGA (5 mM), HLM (1 mg/ml), and APAP (12 concentrations in the range of 0.05 to 40 mM). Reactions were performed for 80 min at 37°C (shaking water bath), terminated by the addition of 70% HClO4 (2 μl), and then vortex-mixed and centrifuged (4000g for 10 min). An identical procedure was used for kinetic studies with recombinant UGTs, except that the incubation time was increased to 120 min. A 100-μl aliquot of the supernatant fraction was mixed with 15 μl of 1 M sodium acetate to increase the pH of samples before analysis by HPLC. HPLC was performed using an 1100 series instrument (Agilent Technologies) fitted with a Synergi Hydro reverse-phase C18 analytical column (3 × 150 mm, 4 μm particle size; Phenomenex, Sydney, Australia). The mobile phase comprised glacial acetic acid-acetonitrile-water (1:5:94). Analytes were eluted at a flow rate of 1 ml/min and detected by UV absorption at 243 nm. Under these conditions, retention times for APAPG and APAP were 1.7 and 3.5 min, respectively. APAPG concentrations in incubation mixtures were quantified by comparison of peak areas with those of an APAPG standard curve. Within-day overall assay reproducibility for the APAP glucuronidation assay was assessed by measuring the rate of APAPG formation in nine separate incubations of pooled HLM; coefficients of variation were 5.2 and 3.0% for APAP concentrations of 0.25 and 10 mM, respectively.

The kinetics of human liver microsomal APAP glucuronidation across the substrate concentration range used in kinetic studies (12 concentrations in the range of 0.05 to 40 mM) were further characterized in the absence and presence of three NFA concentrations (2.5, 50, and 100 μM). Inhibition experiments were conducted with microsomes from the same three livers used in the kinetic studies (namely, HL12, H13, and H40). NFA inhibition of APAP glucuronidation by recombinant UGT1A1, 1A6, 1A9, and 2B15 was determined with four NFA concentrations (2.5, 5, 50, and 100 μM) at APAP concentrations corresponding to the Km or S50 for each enzyme (see Results).

Data Analysis.

All kinetic and inhibition experiments were performed in duplicate, and data points represent the mean of duplicate estimates (<10% variance). APAP glucuronidation kinetic data were modeled using the Michaelis-Menten equations for single and two-enzyme reactions, the Hill equation, and the substrate inhibition equation (for expressions, see Uchaipichat et al., 2004) using EnzFitter (version 2.0; Biosoft, Cambridge, UK). Likewise, the mechanism of inhibition and Ki values for NFA inhibition of UGT activities were determined by fitting the expressions for competitive, noncompetitive, uncompetitive, and mixed inhibition to experimental data using EnzFitter. (Given the importance of mechanism of inhibition to the results and their interpretation, equations corresponding to each mechanism are given below.) Goodness of fit of all expressions was assessed from comparison of the parameter S.E. of fit, coefficient of determination (r2), 95% confidence intervals, and F statistic.

For competitive inhibition: Embedded Image where Vmax is maximal velocity, [S] is substrate concentration, Km is the Michaelis constant, and Ki is the inhibitor constant (for the EI complex).

For noncompetitive inhibition: Embedded Image where Ki is the inhibitor constant for the EI and ESI complexes.

For mixed (competitive-noncompetitive) inhibition: Embedded Image where Ki and Ki′ are the inhibitor constants for the EI and ESI complexes, respectively. Ki′ is related to Ki by a factor, α (>1); that is, EI has a lower affinity for the substrate than does the enzyme (Segel, 1993).

Results

NFA Inhibition of Recombinant and Human Liver Microsomal UGT Enzyme Activities.

The selectivity of NFA as an inhibitor of human UGTs was characterized initially with recombinant proteins. 4MU was used as the probe substrate for the inhibition studies with UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17. Because UGT1A4 and UGT2B4 exhibit low or negligible activity toward 4MU (Uchaipichat et al., 2004; Kubota et al., 2007), LTG and COD were used as the respective substrates (Rowland et al., 2006; Raungrut et al., 2010). NFA was a potent inhibitor of UGT1A7 and UGT1A9. The activity of both enzymes was abolished by 10 μM NFA (Fig. 1). UGT1A1, 1A6, 1A8, 1A10, 2B15, and 2B17 activities were inhibited by approximately 30 to 90% at an added NFA concentration of 100 μM, whereas UGT1A3, 1A4, 2B4, and 2B7 were inhibited by ≤25%. Although inhibition of UGT1A7, UGT1A8, and UGT1A10 is notable, especially the potent effect of NFA on UGT1A7, inhibition of these enzymes was not studied further because expression in liver and kidney is minor or negligible (Ohno and Nakajin, 2009).

Fig. 1.

Effects of NFA (0, 1, 10, and 100 μM) on the activities of recombinant human UGT enzymes. 4MU was used as the probe substrate, except for UGT1A4 (LTG) and UGT2B4 (COD). The 4MU, LTG, and COD concentrations correspond to the known Km or S50 values for each substrate and enzyme. Each bar represents the mean of duplicate measurements.

The results of inhibition screening studies of human liver microsomal UGT activities were broadly consistent with the recombinant enzyme data. Glucuronidation of PRO, a selective marker of hepatic UGT1A9 activity, was inhibited by 72 and 95% at NFA concentrations of 1 and 10 μM, respectively (Fig. 2). NFA at 100 μM caused 36 to 81% inhibition of β-E2 (UGT1A1), DEF (UGT1A6), and TST (UGT2B15/17) glucuronidation but minimal inhibition of TFP (UGT1A4) and AZT (UGT2B7) glucuronidation (Fig. 2). The inhibition of human liver microsomal PRO glucuronidation across the NFA concentration range investigated was somewhat lower than the inhibition observed with 4MU glucuronidation by recombinant UGT1A9. This result is not unexpected because long-chain unsaturated fatty acids released from the microsomal membrane during the course of an incubation contribute significantly to the inhibition observed with HLM as the enzyme source (Rowland et al., 2008).

Fig. 2.

Effects of NFA (0, 1, 10, and 100 μM) on the activities of human liver microsomal UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15/17 activities determined with β-E2, TFP, DEF, PRO, AZT, and TST as the respective probe substrates. Concentrations of each substrate correspond to their published Km values with HLM as the enzyme source. Experiments were performed with pooled HLM from five livers (HL7, HL10, HL12, HL13, and HL40), and each bar represents the mean of duplicate measurements.

Kinetic Characterization of NFA Inhibition of Recombinant and Human Liver Microsomal UGT Activities.

Kinetic studies to determine values of the inhibitor constant were performed with those recombinant UGTs and human liver microsomal UGT activities exhibiting IC50 values <200 μM in the inhibition screening studies (i.e., UGT1A1, UGT1A9, and UGT2B15) (Figs. 1 and 2). Inhibition experiments with recombinant UGT1A1, UGT1A9, and UGT2B15 all used 4MU as the substrate. In addition, β-E2 and PRO were used as selective substrate probes for the kinetic characterization of NFA inhibition of recombinant and human liver microsomal UGT1A1 and UGT1A9, respectively. Of note, NFA was a potent inhibitor of the glucuronidation of 4MU and PRO by UGT1A9 and human liver microsomal PRO glucuronidation. In all cases, data were best described by the expression for mixed (competitive-noncompetitive) inhibition (Fig. 3). Derived Ki values were in the range of 0.10 to 0.40 μM (Table 1). As indicated above, higher Ki values are expected with HLM as the enzyme source because of the added inhibitory effects of membrane long-chain unsaturated fatty acids. Derived Ki′ (dissociation constant for the ESI complex) values were 3- to 7-fold higher than the corresponding Ki values.

Fig. 3.

Dixon plots for NFA inhibition of 4MU glucuronidation by recombinant UGT1A9 (A) and PRO glucuronidation by recombinant UGT1A9 (B) and pooled HLM (C). Each point represents the mean of duplicate measurements, and lines are from model-fitting.

View this table:

Derived kinetic parameters for niflumic acid inhibition of recombinant and human liver microsomal UGT1A1, UGT1A9, and UGT2B15 activities

Data are presented as parameter ± S.E. of parameter fit.

As for the inhibition pattern observed for UGT1A9, NFA was a mixed (competitive-noncompetitive) inhibitor of human liver microsomal and UGT1A1-catalyzed β-E2 glucuronidation (Fig. 4), although the similar Ki and Ki′ values obtained for NFA inhibition of the UGT1A1-catalyzed reaction (Table 1) are consistent with only a marginally better fit with the equation for mixed (compared with noncompetitive) inhibition. The effect of NFA on 4MU glucuronidation by UGT1A1 was best modeled using the equation for noncompetitive inhibition (Fig. 4A). The derived Ki values for NFA inhibition of UGT1A1-catalyzed 4MU and β-E2 glucuronidation (namely, 14–18 μM) were approximately 2 orders of magnitude higher than those observed for UGT1A9 (Table 1). Given the lack of a convenient selective substrate probe for UGT2B15 suitable for studies with HLM, NFA inhibition of this enzyme was assessed with the recombinant protein and 4MU as substrate. Of interest, NFA inhibition of UGT2B15 was competitive (Fig. 5; Table 1), compared with the mixed or noncompetitive inhibition observed with the other enzymes investigated here.

Fig. 4.

Dixon plots for NFA inhibition of 4MU glucuronidation by recombinant UGT1A1 and β-E2 glucuronidation by recombinant UGT1A1 and pooled HLM. Each point represents the mean of duplicate measurements, and lines are from model-fitting.

Fig. 5.

Dixon plot for NFA inhibition of 4MU glucuronidation by recombinant UGT2B15. Each point represents the mean of duplicate measurements, and lines are from model-fitting.

APAP Glucuronidation and Inhibition by NFA.

The 14 recombinant UGT enzymes investigated here were screened for the ability to glucuronidate APAP (0.5, 5, and 50 mM). Activities were determined using a HEK293 lysate/Supersomes protein concentration of 1 mg/ml and an incubation time of 120 min. UGT1A1, 1A6, 1A9, and 2B15 were the only hepatically expressed enzymes that glucuronidated APAP (data not shown). APAP glucuronidation by UGT1A1 and UGT1A9 exhibited sigmoidal kinetics, which was modeled using the Hill equation (Fig. 6). In contrast, the Michaelis-Menten and substrate inhibition equations provided the best fit for APAP glucuronidation by UGT1A1 and UGT2B15, respectively, although deviation from substrate inhibition kinetics was observed for UGT2B15-catalyzed APAPG formation at low substrate concentrations. Derived kinetic parameters are given in Table 2. NFA inhibition of APAP glucuronidation by recombinant UGT1A1, 1A6, 1A9, and 2B15 was determined at four NFA concentrations (2.5, 5, 50, and 100 μM) at the APAP concentration corresponding to the Km or S50 for each of these enzymes. Consistent with the inhibition data shown in Figs. 1, 2, and 3, 2.5 μM NFA essentially abolished APAP glucuronidation by UGT1A9 (Fig. 7).

Fig. 6.

Eadie-Hofstee plots for APAP glucuronidation by recombinant UGT1A1 (A), UGT1A6 (B), UGT1A9 (C), and UGT2B15 (D). Each point represents the mean of duplicate measurements, and curves are from model-fitting.

View this table:

Derived kinetic parameters for acetaminophen glucuronidation by recombinant human UGT enzymes

Data are presented as parameter ± S.E. of parameter fit.

Fig. 7.

Effect of NFA (0, 1, 10, and 100 μM) on APAP glucuronidation by recombinant UGT1A1, 1A6, 1A9, and 2B15. APAP concentrations corresponded to the Km or S50 for each enzyme. Each bar represents the mean of duplicate measurements.

APAP glucuronidation kinetics was characterized in the presence of increasing concentrations of NFA (0, 2.5, 50, and 100 μM) using microsomes from three livers (HL12, HL13, and HL40) (Fig. 8; Table 3). Although there was a suggestion of deviation from hyperbolic kinetics at low substrate concentrations (Fig. 8), as has been observed by other investigators (Court et al., 2001; Mutlib et al., 2006), the single-enzyme Michaelis-Menten equation provided the best fit to experimental data (on the basis of goodness-of-fit parameters; see Data Analysis). Mean Vmax values for human liver microsomal APAP glucuronidation decreased (relative to control) by 20, 35, and 40% with increasing NFA concentrations between 2.5 and 100 μM. Km values also tended to decrease with increasing NFA concentration, with a 26% decrement in mean Km between 0 and 100 μM NFA (17 versus 12.6 μM).

Fig. 8.

Representative velocity versus substrate concentration (A) and Eadie-Hofstee (B) plots for APAP glucuronidation by HLM (HL40) in the presence of increasing concentrations of NFA (0, 2.5, 50, and 100 μM). Each point represents the mean of duplicate measurements, and curves/lines are from model-fitting.

View this table:

Effect of niflumic acid (2.5, 50, and 100 μM) on the kinetics of APAP glucuronidation by HLM

Kinetic parameters were derived from fitting with the Michaelis-Menten equation. Data are presented as parameter ± S.E. of parameter fit.

Discussion

Enzyme-selective inhibitors arguably represent the most valuable experimental tool for the reaction phenotyping of drug and xenobiotic metabolism reactions in vitro. The extent of inhibition of the glucuronidation or indeed any other pathway (e.g., oxidative metabolism by cytochromes P450) of a test compound by HLM or hepatocytes by an enzyme-selective inhibitor under appropriate experimental conditions reflects the contribution of that UGT enzyme to in vitro intrinsic clearance (Miners et al., 2010a). The present study has demonstrated that a low concentration (2.5 μM) of NFA selectively inhibits UGT1A9, whereas inhibition by 50 μM NFA primarily provides a measure of the combined contributions of UGT1A9 and UGT1A1 to a glucuronidation reaction (see the following discussion).

Although NFA appears to be metabolized by UGT1A1 (Mano et al., 2006a), NFA was shown here to be a potent inhibitor of UGT1A9. Ki values ranged from 0.10 to 0.40 μM, depending on the substrate and enzyme source (recombinant enzyme or HLM). It should be noted that the inhibitor constants derived for NFA inhibition of PRO glucuronidation by HLM may be overestimated by as much as 1 order of magnitude because of the combined inhibitory effects of long-chain unsaturated fatty acids released from the microsomal membrane during the course of an incubation (Rowland et al., 2007, 2008). Ki values generated using recombinant UGT1A9 are also likely to be overestimated but to a lesser extent (Rowland et al., 2008). Although the “true” Ki values may be estimated from experiments performed in the presence of bovine serum albumin, which sequesters the inhibitory fatty acids, NFA binds extensively to albumin, thereby precluding the addition of bovine serum albumin to incubations. The Ki for NFA inhibition of 4MU glucuronidation by recombinant UGT1A9 found here is in reasonable agreement with the value (0.03 μM) reported by Mano et al. (2006b), although the previous study assumed competitive inhibition.

Substantially less inhibition was observed with UGT enzymes other than UGT1A9. Ki values for NFA inhibition of human liver microsomal UGT1A1 and UGT2B15 were 15 and 62 μM, respectively. On the basis of data given in Figs. 1 and 2, IC50 values for NFA inhibition of UGT1A3, 1A4, 1A6, 2B4, 2B7, and 2B17 exceed 200 μM. Substituting the Ki values obtained here for UGT1A1, UGT1A9, and UGT2B15 in the expression for the reduction in enzyme activity at a given inhibitor concentration [I] (i.e., vi/v = 1/{1 + [I]/Ki}) predicts that 2.5 μM NFA inhibits UGT1A9 and UGT1A1 by 86 and 14%, respectively, with negligible effects on UGT2B15. At a concentration of 50 μM, NFA is predicted to inhibit UGT1A9, UGT1A1, and UGT2B15 by 95, 77, and 45%, respectively, whereas at 100 μM NFA-predicted inhibition of UGT1A9, UGT1A1, and UGT2B15 is 99, 87, and 62%, respectively.

Apart from UGT2B15, which was competitively inhibited by NFA, the mechanism of inhibition of the UGTs characterized here was noncompetitive or mixed (competitive-noncompetitive). The usual model of noncompetitive inhibition assumes that the substrate and inhibitor bind randomly, reversibly, and independently at two distinct sites, the ESI complex is nonproductive, and the dissociation constant of each compound is unaffected by the binding of the other (Segel 1993). Mixed competitive-noncompetitive inhibition requires the dissociation constant for the ESI complex to be higher than that for EI. Of importance, Vmax is reduced by both mechanisms, but binding affinity is decreased only for mixed inhibition. The notion of multiple substrate and modifier binding sites is consistent with the results of recent kinetic studies with recombinant and human liver microsomal UGTs (Williams et al., 2002; Stone et al., 2003; Uchaipichat et al., 2004, 2008; Zhou et al., 2010). The different mechanisms of UGT1A1 inhibition with β-E2 and 4MU as the substrates may reflect differing binding orientations of these compounds within the UGT1A1 active site or potential contribution of UGT1A3 to the human liver microsomal reaction (Lépine et al., 2004; Miners et al., 2010a). However, Ki values for NFA inhibition were comparable with both substrates. Likewise, Ki values for NFA inhibition of UGT1A9 were close in value with both 4MU and PRO as the substrates. As noted previously, the higher Ki observed for human liver microsomal UGT1A9 probably reflects the greater inhibitory effect of long-chain unsaturated fatty acids with this enzyme source (Rowland et al., 2007, 2008).

Despite APAP being one of the most widely used drugs worldwide, the relative contribution of individual UGT enzymes to APAP glucuronidation is yet to be completely resolved. Initial studies with recombinant UGT enzymes demonstrated that UGT1A1, UGT1A6, and UGT1A9 exhibit the highest activity toward APAP (Bock et al., 1993; Court et al., 2001). However, UGT2B15 has also been shown to catalyze APAP glucuronidation (Mutlib et al., 2006). Consistent with these reports, UGT1A1, 1A6, 1A9, and 2B15 were all shown here to glucuronidate APAP (Fig. 6), and APAP glucuronidation kinetic models differed among enzymes. Like these authors we found that APAP glucuronidation by UGT1A1 and UGT2B15 followed sigmoidal and substrate inhibition kinetics, respectively. Likewise, our data for UGT1A9 are in agreement with the sigmoidal kinetics reported by Court et al. (2001). Whereas Mutlib et al. (2006) assumed hyperbolic kinetics for UGT1A9, the Eadie-Hofstee plot shown in their report strongly suggests atypical (sigmoidal) kinetics. In contrast with both Court et al. (2001) and Mutlib et al. (2006), who reported substrate inhibition kinetics for APAP glucuronidation by UGT1A6, we observed hyperbolic kinetics (Fig. 6). The reasons for discrepancies between studies are not clear but may arise from differences in expression systems (HEK293 cells here for UGT1A enzymes versus Supersomes in the other studies) and the relative expression of recombinant UGTs, kinetic modeling approaches, substrate concentration ranges used in kinetic studies, and the interpretation of glucuronidation kinetic data (Miners et al., 2010a). Although it is noteworthy that Km/S50 values determined here for APAP glucuronidation by UGT1A1, UGT1A6, and UGT1A9 are in very close agreement with those reported by Court et al. (2001), the rank order of Vmax values differed.

On the basis of correlations between the rates of APAP, bilirubin (UGT1A1), and propofol (UGT1A9) glucuronidation and immunoreactive UGT1A6 activity in a panel of HLM along with the kinetic parameters for APAP glucuronidation by recombinant UGT1A1, UGT1A6, and UGT1A9, Court et al. (2001) predicted that UGT1A9 accounted for approximately 60% of APAP glucuronidation in humans. This model further predicted that the relative contributions of UGT1A1 and UGT1A6 to APAP glucuronidation increased and decreased, respectively, with increasing substrate concentration. However, it should be noted that comparisons of UGT activities between enzyme sources may be problematic. In particular, differences can occur in the kinetic mechanism and derived constants for the same reaction catalyzed by different enzyme sources (e.g., HLM and a recombinant UGT) (Bowalgaha et al., 2005) and for the same recombinant UGT enzyme expressed in different laboratories (Itäaho et al., 2008). The former point is further exemplified here: APAP glucuronidation by HLM exhibits hyperbolic kinetics, whereas atypical kinetics were observed with UGT1A1, UGT1A9, and UGT2B15.

The effects of increasing concentrations of NFA on UGT1A9, UGT1A1, and UGT2B15 activities provides an alternative approach to assessing the relative contributions of each of these enzymes and UGT1A6 to APAP glucuronidation by HLM. NFA at 2.5 μM, which essentially abolishes UGT1A9 activity (including UGT1A9-catalyzed APAP glucuronidation) (Fig. 7), reduced the Vmax for human liver microsomal APAP glucuronidation by 20%. The Vmax was reduced by 35 and 40% in the presence of 50 and 100 μM NFA, respectively, concentrations that result in major inhibition of UGT1A1 and UGT2B15. Km values similarly tended to decrease in parallel with Vmax as the NFA concentration was increased. Given that UGT1A1, 1A6, 1A9, and 2B15 are the only enzymes that contribute to hepatic APAP glucuronidation, the NFA inhibition data suggest that UGT1A6 is the major contributor to APAP glucuronidation across a wide substrate concentration range (Fig. 8B), with smaller contributions from UGT1A1, UGT1A9, and UGT2B15.

In summary, it has been demonstrated that NFA at a low concentration (2.5 μM) selectively inhibits human liver microsomal UGT1A9. Higher concentrations (50–100 μM) further differentiate UGT1A1 and UGT2B15. Along with hecogenin and fluconazole, which are respective selective inhibitors of UGT1A4 and UGT2B7 (Miners et al., 2010a), the availability of NFA as an inhibitor “probe” for UGT1A9 provides a valuable experimental resource for the reaction phenotyping of human liver microsomal drug glucuronidation. For example, the comparative effects of increasing NFA concentrations on APAPG formation by HLM and recombinant UGT enzymes suggest that UGT1A6 is the main enzyme involved in the hepatic glucuronidation of APAP.

Authorship Contributions

Participated in research design: Miners, Baranczewski, and Knights.

Conducted experiments: Bowalgaha and Elliot.

Performed data analysis: Miners and Bowalgaha.

Wrote or contributed to the writing of the manuscript: Miners, Bowalgaha, Baranczewski, and Knights.

Other: Miners and Knights acquired funding for the research.

Footnotes

  • This work was supported by the National Health and Medical Research Council of Australia.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.037036.

  • ABBREVIATIONS:

    UGT
    UDP-glucuronosyltransferase
    HLM
    human liver microsomes
    NFA
    niflumic acid
    APAP
    acetaminophen (N-acetyl-p-aminophenol)
    APAPG
    acetaminophen β-d-glucuronide
    COD
    codeine
    4MU
    4-methylumbelliferone
    β-E2
    β-estradiol
    PRO
    propofol
    TST
    testosterone
    TFP
    trifluoperazine
    UDPGA
    UDP-glucuronic acid
    AZT
    zidovudine
    DEFG
    deferiprone 3-O-β-d-glucuronide
    LTG
    lamotrigine
    DEF
    deferiprone
    HPLC
    high-performance liquid chromatography
    EI
    enzyme-inhibitor
    ESI
    enzyme-inhibitor substrate
    HEK
    human embryonic.

  • Received November 3, 2010.
  • Accepted January 18, 2011.

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Characterization of Niflumic Acid as a Selective Inhibitor of Human Liver Microsomal UDP-Glucuronosyltransferase 1A9: Application to the Reaction Phenotyping of Acetaminophen Glucuronidation

John O. Miners, Kushari Bowalgaha, David J. Elliot, Pawel Baranczewski and Kathleen M. Knights
Drug Metabolism and Disposition April 1, 2011, 39 (4) 644-652; DOI: https://doi.org/10.1124/dmd.110.037036
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