Short Communication Cholesterol-Lowering Effect of Ezetimibe in Uridine Diphosphate Glucuronosyltransferase 1A-Deficient (Gunn) Rats

Ezetimibe (EZE) selectively blocks intestinal cholesterol absorption by interacting with Niemann-Pick C1 Like 1 (NPC1L1). After administration, EZE is extensively metabolized in liver and intestine to its phenolic glucuronide form (EZE-G) by uridine diphosphate glucuronosyltransferases (UGTs), among which UGT1A1 and 1A3 exhibit highest activity. EZE-G is excreted into bile and undergoes extensive enterohepatic recirculation. Considering the pharmacokinetic properties of EZE and an in vitro binding study showing the high affinity binding of EZE-G to NPC1L1, glucuronidation by UGTs has been believed to be essential for the pharmacological efficacy of EZE. To study the role of glucuronidation by UGTs for the cholesterol-lowering effect of EZE, in vitro and in vivo studies were performed using Gunn rats, which hereditarily lack the expression of UGT1A enzymes. The biliary excreted amount of EZE-G was reduced by 73% up to 3 h after administration of EZE (0.3 mg/kg) in Gunn rats, which is consistent with the reduction of in vitro EZE glucuronidation activity found in liver and intestinal microsome from Gunn rats. These results indicate that the formation of EZE-G in Gunn rats is much lower than that in Wistar rats. However, in vivo study showed that 0.3 mg/kg EZE, which is the clinically relevant dose, reduced cholesterol absorption in both Wistar and Gunn rats to nearly the same degree and the dose dependence was not significantly different between Wistar and Gunn rats at the range 0.001 0.3 mg/kg. These results indicate that a deficiency of UGT1A activity does not necessarily alter the cholesterol-lowering effect of EZE in rats at therapeutic doses. Ezetimibe (EZE) (Fig. 1) (van Heek et al., 2000) is the first member of a new class of compounds that selectively block intestinal cholesterol absorption (Kosoglou et al., 2005) by interacting with NiemannPick C1 Like 1 (NPC1L1), the recently identified sterol transporter (Altmann et al., 2004; Yamanashi et al., 2007). EZE is extensively metabolized in liver and intestine to its phenolic glucuronide form (EZE-G) by uridine diphosphate glucuronyltransferases (UGTs), of which UGT1A1 and 1A3 exhibit the highest activity, whereas UGT2B isozymes have a lower activity (van Heek et al., 2000; Patrick et al., 2002; Ghosal et al., 2004). After glucuronidation, EZE-G is excreted into bile and undergoes extensive enterohepatic recirculation. This process has been suggested to contribute to the pharmacological action of EZE (van Heek et al., 2000). In accordance with this hypothesis, it was demonstrated that the cholesterol-lowering activity was reduced in TR– rats, which genetically lack the expression of ATP-binding cassette transmembrane transporter C2 (ABCC2) and showed the elevated plasma EZE-G levels and reduced fecal excretion of EZE and EZE-G (Oswald et al., 2006c). Furthermore, in vitro binding assay revealed that EZE-G can bind to NPC1L1 with high affinity (Garcia-Calvo et al., 2005). Considering these data, it is likely that glucuronidation by UGTs is essential for the pharmacological action of EZE. To examine this hypothesis, we decided to use Gunn rats, which hereditarily lack the expression of UGT1A isozymes and, therefore, exhibit a hyperbilirubinemic phenotype (Iyanagi, 1991). Since EZE glucuronidation is predominantly mediated by UGT1A1 and 1A3 isozyme (Ghosal et al., 2004), the EZE glucuronidation capacity in Gunn rats may be much lower than that of control Wistar rats, and therefore, Gunn rats may be suitable for studying the role of glucuronidation. In the present study, we examined whether the hereditary defect in the expression of UGT1A family enzymes results in the reduced cholesterol-lowering effect of EZE by performing in vitro and in vivo experiments. Materials and Methods Materials. EZE was purchased from Sequoia Research Products Ltd. (Pangbourne, UK). [1 ,2 (n)-H]Cholesterol (specific activity, 46.0 Ci/mmol) was obtained from GE Healthcare (Piscataway, NJ). Cholesterol was purchased from Wako Pure Chemicals (Osaka, Japan). L-Phosphatidylcholine and uridine diphosphate glucuronic acid (UDPGA) were obtained from Nacalai Tesque (Osaka, Japan). Sodium taurocholate was obtained from SigmaAldrich (St. Louis, MO). C-labeled UDPGA (180 mCi/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). All other chemicals used in this study were either of analytical or molecular biological grade. C-labeled and unlabeled EZE-G was synthesized according to the previously reported method (Zaks and Dodds, 1998). Formation of unlabeled EZE-G was confirmed by mass spectrometry and H nuclear magnetic resonance spectroscopy. Male Gunn (genotype j/j) and Wistar rats were obtained from SLC Inc. (Shizuoka, Japan). All animals used in this study were housed in temperatureand humidity-controlled animal cages with a 12-h dark/light cycle and with free access to water and normal animal chow (MF; Oriental Yeast, Tokyo, Japan). Preparation of Liver and Intestinal Microsomes and Enzyme Assay. For the preparation of microsomes, male Gunn (260 280 g, n 3) and Wistar rats Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.015628. ABBREVIATIONS: EZE, ezetimibe; EZE-G, ezetimibe phenolic glucuronide; NPC1L1, Niemann Pick C1 Like 1; UGT, uridine-diphosphate glucuronosyltransferase; UDPGA, uridine diphosphate glucuronic acid; HPLC, high performance liquid chromatography; ABC, ATP-binding cassette transmembrane transporter; DPM, degradations per minute. 0090-9556/07/3509-1455–1458$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 9 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 15628/3244734 DMD 35:1455–1458, 2007 Printed in U.S.A. 1455 at A PE T Jornals on July 7, 2017 dm d.aspurnals.org D ow nladed from (245 260 g, n 3) were used. Liver and intestinal microsomes were prepared as described previously (Omura and Sato, 1964; Fasco et al., 1993), and the protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as standard. Enzyme assay was performed as follows (Ghosal et al., 2004). Microsomal incubations were carried out with 100 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, microsomes (0.1 mg protein/ml for liver and 0.2 mg protein/ml for intestine), 5 mM saccharolactone, 25 g/ml alamethicin, 2 mM UDPGA, and 0.5 50 M EZE. Before addition of UDPGA, microsomes were incubated for 15 min on ice to maximize the UGT activity. After 5 min of preincubation at 37°C, UDPGA was added and the incubation was continued for 2.5 10 min. The enzyme reaction was terminated by addition of 10 l of 70% perchloric acid and the analytical procedure described below was carried out. Analysis of EZE and EZE-G in Microsome Incubations. The concentrations of EZE and EZE-G were determined by HPLC as described previously (van Heek et al., 2000; Oswald et al., 2006b) with some modification. The analysis was performed on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with a 5m YMC-Pack C18 column (250 4.6 mm; YMC, Kyoto, Japan). The mobile phase was 0.1% phosphoric acid/acetonitrile 60:40 and the flow rate was set at 1.0 ml/min. The analytical column was maintained at 40°C and eluents were continuously monitored at 232 nm. Specimens were prepared as follows. After termination of the reaction by adding 10 l of 70% perchloric acid, 20 l of 3.5 M potassium chloride and 10 l of internal standard solution (0.05 mg/ml 4-hydroxychalcone dissolved in methanol) were added. Then, specimens were vortexed, centrifuged at 15,000g for 15 min, and 40 l of supernatant was directly injected onto the HPLC. Biliary Excretion Study Using Wistar and Gunn Rats. Male Wistar (220 253 g, n 3) and male Gunn rats (235 248 g, n 3) were used for biliary excretion study. EZE was solubilized with ethanol (20 mg/ml) and was added directly to the blank rat plasma to give final concentrations of 0.3 mg/ml. After overnight (18-h) fasting, rats were anesthetized with pentobarbital (50 mg/kg i.p.) and bile duct was cannulated. After cannulation, rats received an intravenous administration of 1 ml/kg EZE-containing plasma from the jugular vein. After EZE dosing, bile specimens were collected for 3 h. Bile specimens were weighted and 25l aliquots were subjected to the EZE assay. Analysis of EZE and Total EZE-G in Bile Specimens. The amount of EZE and EZE-G in bile specimens was determined as follows. Bile specimens (25 l) were collected in a 10-ml glass tube and diluted with 1 ml of 0.3 M acetate buffer (pH 5.4). Then, -glucuronidase solution (50 l, 22,000 units/ ml; Wako Pure Chemicals) was added in each tube and the mixture was incubated for 2 h at 60°C. For determination of unconjugated EZE, 50 l of water was added instead of -glucuronidase. After incubation, 5 M HCl (1 ml), internal standard solution (25 l) and ethyl acetate (8 ml) were added and tubes were shaken for 15 min. Then, tubes were centrifuged (3000 rpm, 10 min) and organic layer was transferred to the other tube. Then the organic layer was washed with 5% (w/v) sodium hydrogen carbonate (1 ml) and evaporated under dry nitrogen stream. The residue was reconstituted in 100 l of acetonitrile and 50 l was injected onto the HPLC system. The HPLC condition is the same as that in the in vitro enzyme assay. Preparation of Cholesterol Emulsion. Cholesterol emulsion was prepared as described previously (Tso et al., 1980). In brief, stock lipid solutions were mixed to give a final concentration of 13.3 mM triolein, 2.6 mM cholesterol, 3 mM L-phosphatidylcholine, and 3 Ci/ml [H]cholesterol. Solvents were evaporated and 19 mM sodium taurocholate (dissolved in phosphate-buffered saline) was added to give the required lipid concentration. Then the mixture was sonicated three times for 5 min each, using an ultrasonic homogenizer (UP 200H; Hielscher Ultrasonics, Teltow, Germany) to produce a stable emulsion. Acute Cholesterol Absorption Study Using Wistar and Gunn Rats. Male Wistar (180 233 g) and male Gunn rats (203 265 g) were used for in vivo study. An acute cholesterol absorption study was conducted as described previously (van Heek et al., 2000) with minor modification. EZE ethanol solution (20 mg/ml) was added directly to the blank rat plasma to give final concentrations of 0.003, 0.01, 0.03, 0.1, and 0.3 mg/ml. Fasted rats (18 h) were anesthetized with pentobarbital (50 mg/kg i.p.; Dainippon Pharmaceutical, Osaka, Japan) and an intraduodenal cannula was inserted as described previously (Tso et al., 1980). After cannulation, rats received intravenously 1 ml/kg blank plasma or EZE-containing plasma from jugular vein. Immediately after drug dosing, 5 ml/kg cholesterol emulsion was delivered directly into the intestine via the duodenal cannula. Three hours after cholesterol loading, rats were sacrificed; then, plasma was isolated and directly analyzed for [H]cholesterol in duplicate. Livers were also taken, weighed, and minced. Aliquots of 50 200 mg of tissue were solubilized using Solvable reagent (PerkinElmer Life and Analytical Sciences) and subjected to [H]cholesterol assay. Intestinal mucosa was scraped from upper intestine (approximately 40 cm from the pylorus), homogenized with 5 volumes of ice-cold phosphate-buffered saline, and directly analyzed for [H]cholesterol in duplicate. Protein concentrations of homogenate were determined using the BCA protein assay kit (Pierce). For plasma and liver, data are expressed as percentage of administered radioactivity per total plasma volume or total liver. Total plasma volume was assumed to be 4% of the body weight as described elsewhere (Hawk and Leary, 1995). For the intestinal mucosa, data are shown as H DPM per mg of protein. Determination of Plasma Protein Binding of EZE-G. For the determination of plasma protein binding of EZE-G in Wistar and Gunn rats, [C]EZEG was synthesized according to the method described by Zaks and Dodds (1998) by incubating EZE and [C]UDPGA with rat liver microsome. Specific activity of [C]EZE-G was 180 mCi/mmol. The purity of [C]EZE-G was confirmed by thin-layer chromatography with radioactivity detection. Plasma protein binding was determined by ultrafiltration. C-labeled and unlabeled EZE-G was added to plasma specimens from Wistar and Gunn rats to produce the final concentrations of 5, 20, 50, and 200 ng/ml. After incubation for 60 min at 37°C, specimens underwent ultrafiltration using a Centrifree device (Millipore, Billerica, MA) according to the manufacturer’s protocol and the filtrates were directly analyzed for C radioactivity. Results and Discussion Plasma EZE concentration is known to be much lower than that of EZE-G in both humans and rats (Patrick et al., 2002; Oswald et al., 2006c), and one of the reasons to account for this difference may be the rapid glucuronidation of EZE in liver and intestine by UGTs. In addition, detailed studies have shown that most ( 80%) of the intraduodenally or intravenously administered EZE was recovered in bile within 2 3 h and the main form ( 90%) was EZE-G (van Heek et al., 2000). In addition, it is believed that the pharmacological action of EZE-G is much more potent than that of EZE (van Heek et al., 2000). In the present study, we conducted an in vivo experiment in Gunn rats, which hereditarily lack the expression of UGT1A family members. Because human UGT1A1 and 1A3 are the major enzymes mediating the EZE phenolic glucuronidation (Ghosal et al., 2004), we hypothesized that Gunn rats may exhibit reduced EZE glucuronidation activity, resulting in a reduced cholesterol-lowering effect of EZE. First, we conducted in vitro metabolism experiments using liver and intestinal microsomes from both Wistar and Gunn rats to confirm the EZE glucuronidation capacity of Gunn rats. The results of metabolism experiments are shown in Fig. 2. Due to the limit of detection, we could not determine the glucuronidation rate of EZE at concentrations lower than 0.5 M. It was found that the glucuronidation rate of EZE by liver and intestinal microsomes from Gunn rats was reduced by 60 80% compared with that from Wistar rats at EZE concentrations FIG. 1. Chemical structure of EZE (R H) and EZE-G (R glucuronide). 1456 YAMAMOTO ET AL. at A PE T Jornals on July 7, 2017 dm d.aspurnals.org D ow nladed from