Abstract
The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma Cmax, 0.06–0.88 mM). Gemcabene showed similar affinity (S50) for recombinant UGTs (0.92–1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC50) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC50 for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 μM) compared with recombinant UGT2B7 (27.4 μM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3′,4′,5′-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r2 = 0.722, P < 0.0001; n = 24) with rates of glucuronidation of the UGT2B7 probe substrate 3′-azido-3′-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.
Gemcabene (Fig. 1) increases high-density lipoproteins while lowering serum triglycerides in humans (Bays and Stein, 2003). Gemcabene is cleared almost exclusively by glucuronidation, with 78% of a radioactive dose recovered in human urine, consisting of 61% glucuronide and 17% unchanged parent drug (Pfizer Global Research and Development, unpublished observations). The primary aim of the present study was to identify the major human UDP-glucuronosyltransferase (UGT) isoform(s) catalyzing the glucuronidation of gemcabene.
To date, at least 17 human UGTs have been identified, and 12 are commercially available. Seventeen have been cloned and sequenced, and several of their cDNAs have been heterologously expressed using cell systems to demonstrate substrate specificity (Mackenzie et al., 1997). Similar to cytochrome P450s, individual UGTs have distinct, yet overlapping, substrate specificities (Radominska-Pandya et al., 1999; Tukey and Strassburg, 2000). Historically, phenotyping of drug glucuronidation has received much less attention than cytochrome P450-mediated metabolism, due to a relative lower risk of UGT-mediated drug-drug interactions, compared with cytochrome P450s (Williams et al., 2004). The consequent lack of attention to glucuronidation has led to the slower discovery of selective probe substrates and inhibitors useful in the phenotyping of UGT-catalyzed reactions (Miners et al., 2004). However, recent advances now make it possible to conduct UGT reaction phenotyping experiments with significantly greater confidence in the resulting data (Court, 2004; Williams et al., 2005). These efforts would help to predict the influence of interindividual variation in glucuronidation due to polymorphisms in the encoded proteins or modulation of protein activity on drug bioavailability, pharmacokinetics, and efficacy.
Definitive cytochrome P450 reaction phenotyping studies typically use three approaches to assess the relative contributions of each P450 to the metabolism of the test compound (Bjornsson et al., 2003). These include use of 1) recombinant enzymes, 2) selective chemical inhibition of human liver microsomal metabolism, and 3) correlation analyses using a phenotyped human liver bank (Bjornsson et al., 2003). In the case of P450s, relative hepatic expression levels are known. However, the absence of knowledge on the relative expression levels of UGTs in human liver microsomes or recombinantly expressed enzyme systems makes it difficult to quantitatively extrapolate or estimate the relative contributions of individual UGTs to glucuronidation of the drug in question (Clarke, 1998).
Chemical structures of gemcabene, gemcabene glucuronide, internal standard, S-flurbiprofen, and HMF.
The strategy described in this article to determine the UGTs responsible for gemcabene metabolism is based on the typically used procedures for definitive cytochrome P450 reaction phenotyping (Court, 2004). We were able to identify the major UGT form(s) contributing to gemcabene glucuronidation by 1) characterizing recombinant UGT-catalyzed gemcabene glucuronidation, 2) identifying selective recombinant UGT inhibitors, and 3) phenotyping a human liver bank for activities of relevant UGTs.
Materials and Methods
Chemicals and Reagents. Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Flavones were purchased from INDOFINE Chemical Company, Inc. (Hillsborough, NJ). HPLC-grade methanol and acetonitrile were purchased from Mallinckrodt (St. Louis, MO). Gemcabene [6-(5-carboxy-5-methyl-hexyloxy)-2,2-dimethyl-hexanoic acid (PD0072953)], gemcabene glucuronide [6-[6-(5-carboxy-5-methyl-hexyloxy)-2,2-dimethyl-hexanoyloxy]-3,4,5-trihydroxy-tetrahydro-pyran-2-carboxylic acid (PD0329728)], and internal standard [6-(5-carboxy-5-methyl-heptyloxy)-2-ethyl-2-methyl-hexanoic acid (PD0200455)] were all synthesized by the Department of Chemistry, Pfizer Global Research and Development (Ann Arbor, MI; Fig. 1).
Biological Reagents. Pooled human liver microsomes (mixed gender, n = 60), individual liver microsomes from a panel of 24 donors, and recombinant forms of the following UGTs prepared from baculovirus expression systems: UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 (SUPERSOMES), were purchased from BD Gentest (Woburn, MA).
Gemcabene Glucuronidation Assay. Gemcabene was incubated at 37°C with 50 mM Tris-HCl (pH 7.1), 5 mM UDPGA, 5 mM MgCl2, 1 mM saccharolactone (d-glucaric acid-1,4-lactone monohydrate; HLM only), 50 μg alamethicin/mg of protein, and pooled HLM (1.25 mg/ml) or recombinant UGT (0.1–0.5 mg/ml) following a 15-min preincubation on ice. The incubation times varied between 15 and 240 min. The final incubation volume was 0.2 ml. Reactions were initiated by the addition of UDPGA and were terminated by the addition of 0.1 ml of acetonitrile, containing internal standard, and placed on ice. For incubations where inhibitors were included, preincubation was for 5 min at 37°C prior to reaction initiation. Quenched samples were centrifuged, and the supernatants were analyzed by HPLC-mass spectrometry. Incubation times and protein concentrations were also established in preliminary studies with both HLMs and expressed UGTs to be within initial linear rate conditions. Glucuronide concentrations were determined using authentic gemcabene glucuronide with a standard curve prepared in complete incubation matrix, including UDPGA. Enzyme activities were calculated and expressed as nanomoles per minute per milligram of protein. All experiments were conducted in triplicate, and results were reported as a mean ± standard deviation.
Gemcabene Glucuronide Analysis. HPLC-mass spectrometry analyses were performed with an Agilent 1100 HPLC system (Agilent, Palo Alto, CA) coupled to an LCQ Advantage mass spectrometer (Finnigan-MAT, San Jose, CA), with a Finnigan atmospheric pressure ionization source operated in the electrospray ionization mode tuned to unit mass resolution. Chromatographic separation was achieved with a C18 column (2.0 × 150 mm, 5 μm, Luna; Phenomenex, Torrance, CA). The mobile phases were 10 mM ammonium acetate (pH 4.5) in water (solution A) and acetonitrile (solution B). The solvent program consisted of the following: an initial isocratic mobile phase mix (15% solution B) for 3 min, followed by a linear gradient from 15 to 80% solution B over 3 min, held for 4 min, and returned to initial conditions over 1 min with a flow rate of 0.4 ml/min. The mass spectrometer was operated in the positive ion mode with single-ion monitoring for gemcabene glucuronide, m/z 477, and m/z 329 for the internal standard. The capillary was operated at 200°C, the spray voltage was set to 4.5 kV, and nitrogen was used as a drying gas at a sheath pressure of 20 psi.
AZT Glucuronidation Assay and Analysis. Incubation conditions were similar to the gemcabene glucuronidation assay except AZT (1.5 mM final concentration), HLM (0.25 mg/ml) protein, 30-min incubation time; and the UV response was quantitated by comparison to a standard curve of AZT-glucuronide using 3-acetamidophenol as the internal standard. Analyses of AZT glucuronide activity were performed on an Agilent Series 1100 platform HPLC system (Agilent) using a Phenomenex Aqua 5 μm C18 150 × 4.6 mm-high-performance liquid chromatography column (Phenomenex). The mobile phase solution A was 20 mM phosphate buffer (pH 2.2), and solution B was acetonitrile. Initial conditions were 84% A/16% B pumped at 1.0 ml/min. A linear gradient from 16 to 65% B between 0 and 7.1 min was used, followed by 4.9-min re-equilibration at 16% B.
Estradiol Glucuronidation Assay and Analysis. Incubation conditions were similar to the gemcabene assay except β-estradiol (25 μM final concentration), HLM (0.50 mg/ml) protein 30-min incubation time; and the response was quantitated by comparison to a standard curve of estradiol-3-glucuronidation using α-naphthylglucuronide as the internal standard. After 30 min, the reaction was stopped by the addition of 50 μl of formic acid (25% v/v in buffer). Subsequently, 10 μl of α-naphthylglucuronide (internal standard) was added to each sample. The resulting supernatant was analyzed by HPLC-UV. Analysis was performed on a PerkinElmer Series 200 autosampler and liquid chromatography pump equipped with a PerkinElmer 785A UV-visible detector and NCI 900 network chromatography interface (PerkinElmer Life and Analytical Sciences, Boston, MA), using an Agilent Zorbax SB-C18, 5 μm, 150 × 4.6-mm high-performance liquid chromatography column (Agilent). The mobile phase solution A was 0.1% trifluoroacetic acid, and solution B was acetonitrile/0.1% trifluoroacetic acid (90:10, % v/v). Initial conditions were 71% A/29% B pumped at 1.0 ml/min. A linear gradient from 29 to 60% B between 0 and 8 min was used, followed by 1 min at 100% B and then a 4-min re-equilibration at 29% B.
Enzyme Kinetic Analysis. Substrate concentration (S) and velocity (v) data were fitted to the appropriate enzyme kinetic model by nonlinear least-squares regression analysis (Sigmaplot; SPSS, Chicago, IL) (Bjornsson et al., 2003) to derive the enzyme kinetic parameters Vmax (maximal velocity) and Km (substrate concentration at half-maximal velocity). The Michaelis-Menten model (eq. 1) or the substrate activation models (eq. 2) (Segel, 1975), which incorporates the Hill coefficient (n), were used: 
The best fit (to eq. 1 or eq. 2) was based on the following criteria, including visual inspection of the data plots (Michaelis-Menten and Eadie-Hofstee), distribution of the residuals, size of the sum of the squared residuals, and the standard deviation of the estimates. The IC50 estimate for the inhibition of gemcabene glucuronidation was determined by nonlinear curve fitting with GraphPad Prism (GraphPad Software, San Diego, CA).
Velocity versus substrate concentration (top panel) and Eadie-Hofstee plot (bottom panel) of the kinetics of gemcabene glucuronidation in pooled human liver microsomes. The top panel shows a sigmoidal curve, whereas the bottom panel shows the curved plot diagnostic of homotropic activation kinetics. This observation appears to have no in vivo relevance, as gemcabene was shown to demonstrate linear pharmacokinetics in humans.
Results
Kinetics of Gemcabene Glucuronidation in Human Liver Microsomes. Gemcabene glucuronidation best fit a model described by Hill kinetics and the Eadie-Hoftsee plot (Fig. 2), indicating homotropic activation. The S50 was 1.37 ± 0.12 mM, and the Vmax was 6.04 ± 0.38 nmol/min/mg of protein in pooled human liver microsomes (Table 1).
Kinetic parameters for gemcabene glucuronidation in human liver microsomes and recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17
Values are mean ± standard deviation of three experiments. HLM and recombinant UGTs were pretreated with alamethacin (50 μg/mg protein) and incubated with varying concentrations of gemcabene to determine kinetic parameters as described under Materials and Methods. The data were fitted to the Hill equation (S50).
Gemcabene Glucuronidation by Recombinant UGTs. From the 12 recombinant UGTs (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17) investigated, three (UGT1A3, UGT2B7, and UGT2B17) demonstrated the ability to catalyze gemcabene glucuronidation (Fig. 3). Further experiments were conducted to characterize enzyme kinetics (Table 1 and Fig. 4). Similar to the kinetics observed with human liver microsomes, Eadie-Hofstee plots indicated homotropic activation kinetics for gemcabene glucuronidation by UGT1A3, UGT2B7, and UGT2B17, and the observed S50 values were similar to those observed for human liver microsomes (Table 1).
Inhibitors of Gemcabene Glucuronidation. Initial screening studies (data not shown) investigated the potential of the following compounds (at 1, 10, and 100 μM concentration) to inhibit HLM-, recombinant UGT1A3-, recombinant UGT2B7-, and recombinant UGT2B17-catalyzed gemcabene glucuronidation: naproxen, S-flurbiprofen, 7′,2′,4′-trimethoxyflavone, 5,6,7,3′,4′,5′-hexamethoxyflavone (HMF), 3,5,7,3′,4′-quercetinpentamethylether, 5,6,7-trihydroxyflavone, 6,4′-dimethoxyflavone, oxazepam, β-estradiol, diclofenac, silibinin, chloramphenicol, and hecogenin. These studies suggested that HMF and S-flurbiprofen were selective inhibitors of UGT1A3- and UGT2B7-catalyzed gemcabene glucuronidation, respectively. Subsequent studies were conducted in HLM and in expressed UGT1A3, 2B7, and 2B17 to quantitatively assess the inhibitory potential of S-flurbiprofen and HMF to inhibit gemcabene glucuronidation (Fig. 5) and to estimate the relative contributions of individual UGTs to the glucuronidation of gemcabene in human liver microsomes. Substrate (gemcabene) concentrations approximated the S50 value for gemcabene glucuronidation catalyzed by HLM (1.4 mM) and by recombinant UGT1A3 (0.92 mM), recombinant UGT2B7 (1.45 mM), and recombinant UGT2B17 (1.37 mM). S-Flurbiprofen was more selective at inhibiting recombinant UGT2B7-catalyzed gemcabene glucuronidation (IC50 = 27.4 ± 2.9 μM) compared with recombinant UGT1A3 (IC50 > 1000 μM) and recombinant UGT2B17 (IC50 = 653 ± 158 μM) enzymes (Table 2). The IC50 for inhibition of gemcabene glucuronidation by recombinant UGT2B7 was similar to that observed for inhibition of HLM glucuronidation (IC50 = 60.6 ± 4.9 μM).
Inhibition of gemcabene glucuronidation in human liver microsomes and recombinant UGTs with S-flurbiprofen and HMF
Values are mean ± standard deviation of three experiments. IC50 values for the inhibition of gemcabene glucuronidation by S-flurbiprofen and HMF were determined using substrate concentrations approximating the S50 values determined in HLM (1.37 mM) and each recombinant UGT isozyme (1A3, 0.92 mM; 2B7, 1.45 mM; and 2B17, 1.37 mM).
Correlation of Gemcabene Glucuronidation with AZT and Estradiol-3-Glucuronidation in HLM. Rates of gemcabene glucuronidation in microsomes from 24 individual human livers correlated well (r2 = 0.722, P < 0.0001) with rates of glucuronidation of the UGT2B7 probe substrate AZT (Fig. 6, top) but not with rates of glucuronidation of β-estradiol (Fig. 6, bottom) (r2 < 0.0001, P = 0.984) at the 3-position, a marker of UGT1A1 activity (Senafi et al., 1994).
Discussion
The strategy for this study was to use a combination of experimental methods, including HLM and recombinant UGTs, to identify and characterize the UGT isoforms involved in gemcabene glucuronidation. To achieve this objective, initial studies were performed with HLM to determine enzyme kinetic parameters and, accordingly, the appropriate gemcabene concentration (S50 = 1.37 mM) for subsequent identification of recombinant UGT activities in a panel of commercially available recombinant UGTs. Since only three recombinant UGTs demonstrated the potential to glucuronidate gemcabene, further experiments focused on attempts to differentiate their relative contributions (comparison of enzyme kinetic parameters between recombinant UGTs and HLM, identification of selective inhibitors, and correlation of probe substrate reaction rates in a human liver bank). In combination, the data presented indicate that UGT2B7 is the primary enzyme contributing to glucuronidation of gemcabene. Since glucuronidation is the major clearance mechanism for the compound, hepatic UGT2B7 appears to be the major determinant of gemcabene elimination in humans.
Gemcabene is glucuronidated by recombinant UGT1A3, UGT2B7, and UGT2B17. Glucuronidation was measured at 1.37 mM (triplicate data from two experiments). CONT, control vector.
Enzyme kinetics for UGT1A3- (○, top panel), UGT2B7- (○, middle panel), and UBT2B17- (○, bottom panel) catalyzed gemcabene glucuronidation. Top panels show velocity versus substrate concentration. Bottom panels show Eadie-Hofstee plots diagnostic of homotropic activation.
Characterization of the enzyme kinetic parameters for gemcabene glucuronidation did not differentiate the contribution of recombinant UGT1A3, recombinant UGT2B7, or recombinant UGT2B17 in gemcabene glucuronidation. The observation of homotropic activation kinetics for each recombinant UGT was in agreement with the kinetics observed for gemcabene glucuronidation in HLM. Additionally, S50 values were similar for the recombinant UGTs (0.92–1.45 mM) compared with HLM (1.37 mM). The relative hepatic expression levels of UGTs in the liver are unknown, although there is evidence that UGT1A3, UGT2B7, and UGT2B17 (Strassburg et al., 1997) are expressed in the liver. The observation of homotropic activation in HLM has no apparent influence on gemcabene clearance in humans, which was linear between the total plasma Cmax of 0.06 and 0.88 mM following multiple-dose administration (Pfizer Global Research and Development, unpublished observations). Since exposure increased in a dose-proportional manner up to 0.88 mM (Pfizer Global Research and Development, unpublished observations), the in vivo Km value appears to be higher than the highest plasma concentration, which is consistent with the in vitro observations.
Experiments conducted to characterize selective inhibitors of recombinant UGTs identified S-flurbiprofen as an effective inhibitor of both recombinant UGT2B7- and HLM-catalyzed gemcabene glucuronidation with similar IC50. The lack of inhibitory potency of S-flurbiprofen on recombinant UGT1A3- and recombinant UGT2B17-catalyzed gemcabene glucuronidation (IC50 > 600 μM) indicates significant potential for UGT2B7 to contribute to gemcabene glucuronidation in HLM. The selective inhibitory effects of HMF on recombinant UGT1A3- but not recombinant UGT2B7-, recombinant UGT2B17-, or HLM-catalyzed gemcabene glucuronidation provide further evidence that UGT2B7 plays a major role in gemcabene glucuronidation.
A human liver bank phenotyped for UGT1A1 and UGT2B7 provided a third approach to assess the relative contributions of these enzymes to the glucuronidation of gemcabene. The observation that gemcabene glucuronidation correlated with UGT2B7 (AZT glucuronidation) reaction rates and not UGT1A1 (estradiol-3-glucuronidation) reaction rates provides further support that UGT2B7 is the major contributor to gemcabene glucuronidation in HLM. The observation that a correlation was found with the UGT2B7 phenotype, but not with a distinctly different UGT activity, rules out differences in liver quality as the basis for the correlation.
Inhibition of HLM- (○), recombinant UGT1A3- (□), recombinant UGT2B7- (•), or recombinant UGT2B17- (▪) catalyzed gemcabene glucuronidation by S-flurbiprofen (left panel) or HMF (right panel). The data presented in the semilogarithmic plots indicate that S-flurbiprofen has similar potency (IC50<70 μM) toward inhibition of recombinant UGT2B7 or HLM, and is less potent (IC50> 600 μM) toward UGT1A3 or UGT2B17. Additionally, HMF has much greater inhibitory potency (IC50 < 30 μM) toward UGT1A3 compared with recombinant UGT2B7, recombinant UGT2B17, or HLM (IC50 > 100 μM) in each case.
In combination, the selective inhibitor and correlation analysis data suggest that UGT2B7 contributes to approximately 70% of the glucuronidation of gemcabene in HLM. As previously indicated, the magnitude of drug-drug interactions for glucuronidated drugs is typically low (Williams et al., 2004). Based on a high in vitro Km, the potential for similar drug-drug interactions for gemcabene would also be expected to be low. Screening efforts to identify a selective inhibitor also indicated low inhibitory potential for UGT2B7-catalyzed glucuronidation, in comparison to that observed for the most potent inhibitors of cytochrome P450 enzymes, S-flurbiprofen demonstrated the lowest in vitro IC50, at 24 μM, whereas some cytochrome P450 enzymes have potent (submicromolar) IC50 values (Ekins et al., 2003).
In summary, the combination approach described above, which is typically taken for definitive cytochrome P450 reaction phenotyping, can also be used effectively for UGT reaction phenotyping. Further efforts to characterize selectivity of UGT inhibitors would help optimize this process. The data generated using this approach indicates that UGT2B7 is the major UGT enzyme contributing to glucuronidation of gemcabene in HLM.
Correlation between gemcabene glucuronidation (S50 concentration, 1.37 mM) and UGT2B7-catalyzed AZT glucuronidation (top panel) or UGT1A1-catalyzed estradiol-3-glucuronidation (bottom panel) samples from a human liver bank (n = 24).
Acknowledgments
We thank Simon E. Ball for helpful discussions.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.005108.
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ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; HPLC, high-performance liquid chromatography; UDPGA, uridine 5′-diphosphoglucuronic acid; HLM, human liver microsome(s); AZT, zidovudine, 3′-azido-3′-deoxythymidine; HMF, 5,6,7,3′,4′,5′-hexamethoxyflavone.
- Received April 8, 2005.
- Accepted June 17, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
