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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Developmental Research Laboratories (S.S., H.S., T.K., Y.M., K.H., T.B.) and Discovery Research Laboratories (K.M., K.N., S.H.), Shionogi & Co., Ltd., Osaka, Japan; Department of Clinical Evaluation of Pharmacotherapy, Kobe University Graduate School of Medicine, Hyogo, Japan (N.O.); and Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K., Y.S.)
Received October 30, 2006; accepted April 26, 2007.
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; Shepherd et al., 1995). The hypocholesterolemic agents most commonly used are the HMG-CoA reductase inhibitors (so-called statins). Statins have a potent hypocholesterolemic action and few side effects, resulting in good patient compliance.
As alternative pharmacological targets for the hypocholesterolemic effect, inhibition of intestinal absorption of cholesterol and bile acids has attracted attention. Ezetimibe was found to be a potent inhibitor of Niemann-Pick C1-like 1, playing a significant role in the cholesterol absorption in the small intestine (Garcia-Calvo et al., 2005), and combination therapy with statins increased the effectiveness of LDL cholesterol reduction (Gazi and Mikhailidis, 2006). Inhibitors of the ileal apical sodium-dependent bile acid transporter (ASBT/SLC10A2), a membrane transporter responsible for the absorption of bile acids (Craddock et al., 1998), include 2164U90 (Lewis et al., 1995), S-8921 (Hara et al., 1997), SC-435 (West et al., 2002), S 0960 (Schlattjan et al., 2003), and R-146224 (Kitayama et al., 2006). Bile acids are synthesized from cholesterol in the liver, and they are secreted into the bile. More than 90% of the bile acids are reabsorbed from the intestinal lumen in the ileum, and they are removed from the blood by the first-pass effect in the liver, leading to enterohepatic circulation (Hofmann, 1993). The bile acid pool is closely regulated; thus, interruption of the enterohepatic circulation of bile acids would lead to an increase in the biosynthesis of bile acids from cholesterol (Packard and Shepherd, 1982). Indeed, treatment with ASBT inhibitors increases the fecal excretion of bile acids (Lewis et al., 1995; Hara et al., 1997; Huff et al., 2002; Kitayama et al., 2006), resulting in an induction of hepatic cholesterol 7
-hydroxylase (CYP7A), a rate-limiting enzyme of bile acid synthesis (Higaki et al., 1998; Huff et al., 2002). ASBT inhibitors exhibit a hypocholesterolemic effect (2164U90, S-8921, R-146224, and SC-435). This is partly ascribed to the increase in the bile acid synthesis in the liver and partly to the increase in the uptake of plasma LDL cholesterol through the up-regulation of LDL receptors due to partial depletion of hepatic cholesterol, because SC-435 or S-8921 treatment causes induction of hepatic LDL receptors (Higaki et al., 1998; Huff et al., 2002). This latter finding is due to the partial depletion of hepatic cholesterol (Packard and Shepherd, 1982).
S-8921 is a water-insoluble compound with a clog P of 5.5. Among the ASBT inhibitors, S-8921 is a weak inhibitor of ASBT with an IC50 value of 66 µM (Hara et al., 1997), whereas others range from 1.2 nM to 10 µM (Lewis et al., 1995; West et al., 2002; Schlattjan et al., 2003; Kitayama et al., 2006). Unlike 2164U90 and SC-435, which are competitive inhibitors of ASBT (Root et al., 1995; Hallén et al., 2002), the inhibition of ASBT by S-8921 is a mixture of competitive and noncompetitive processes (Hara et al., 1997). Furthermore, it has been found that the glucuronide conjugate of S-8921 (S-8921G) is a more potent inhibitor of taurocholate (TCA) absorption than the parent compound (J. Ono, S. Sakamoto, T. Ichihashi, M. Izawa, Y. Yano, T. Mizui, and K. Hirano, unpublished data). S-8921G is the major metabolite of S-8921 in the bile following oral administration, and rat bile after oral administration of S-8921 inhibits the ileal absorption of TCA (J. Ono, S. Sakamoto, T. Ichihashi, M. Izawa, Y. Yano, T. Mizui, and K. Hirano, unpublished data). The absolute bioavailability of S-8921 was only a few percent in rats and dogs (Yamaguchi et al., 1998). After oral dosing of [14C]S-8921 in rats, the urinary and biliary excretion of the total radioactivity was approximately 1 to 2% and 20 to 30% dose, respectively, and, even in intravenous dosing, most of the total radioactivity was excreted into the bile, although the unchanged form of S-8921 was undetectable in the bile (Yamaguchi et al., 1998). Enterohepatic circulation of the total radioactivity of S-8921 was observed to a small extent (6% dose) (Yamaguchi et al., 1998). In our preliminary study, S-8921G was barely deconjugated, and it was absorbed when S-8921G was instilled into the ileal loop (S. Sakamoto, unpublished observation). However, most of the radioactivity in the feces after oral administration of [14C]S-8921 was accounted for by S-8921 (K. Miyata, K. Nomura, and S. Hara, unpublished observation). Thus, it is now considered that S-8921G is converted to S-8921 by bacterial flora in the cecum and/or the large intestine, and part of the S-8921 is reabsorbed, which may result in the long-acting pharmacological effect.
The purpose of the present study was to show the importance of S-8921G in the pharmacological action of S-8921. In vivo pharmacological and pharmacokinetic properties of S-8921 were compared between normal and UDP-glucuronosyltransferase-1 (UGT1)-deficient rats (Gunn rats) (Burchell et al., 1995). In addition, we compared the inhibition potencies of S-8921 and S-8921G against human ASBT (hASBT) using cDNA-transfected cells.
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Pharmacokinetic Study of S-8921 and S-8921G in Rats. S-8921 was suspended in 5% gum arabic solution for administration. Rats were given a single oral dose of 10 mg/kg S-8921. The biliary excretion was determined in a separate experiment. For the plasma concentration study (n = 5/group; mean body weight, 310 g for Wistar rats and 292 g for Gunn rats), 5 ml of blood was collected from a jugular vein cannula at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing. After blood sampling, the blood collected was replaced by 25 U/ml heparin-saline. Plasma was separated immediately, and it was stored at –80°C until quantitative determination. For the biliary excretion study (n = 5/group; mean body weight, 300 g for Wistar rats and 300 g for Gunn rats), bile was collected at 0 to 6 h and 6 to 24 h after dosing in a bottle containing 1 M acetic acid, pH 2.3 to 2.4, on ice. S-8921 and S-8921G were stable in the bile. Bile samples were stored at –80°C until quantitative determination. The concentrations of S-8921 and S-8921G in the plasma and bile were determined by LC/MS/MS after solid phase extraction using an SPEC·C2 disk cartridge column (ANSYS Technologies, Inc., Lake Forest, CA). The recovery of S-8921 and S-8921G by solid phase extraction was more than 90%. The eluent was centrifuged, and 30 µl of the supernatant or the diluted supernatant was injected into the LC/MS/MS system.
The LC/MS/MS system consisted of a model Alliance 2690 LC apparatus (Waters, Milford, MA) equipped with a model TSQ API2 performance pack (ThermoQuest, Waltham, MA). The analytical column was a YMC-Pak Pro C18 column (5 µm, 35 mm, 2.0 mm i.d.; YMC, Kyoto, Japan) kept in a column oven at 30°C. The mobile phase consisted of solvent A (0.1% acetate in water) and solvent B (0.1% acetate in acetonitrile). The flow rates from 0.0 to 1.1 min, from 1.1 to 3.2 min, from 3.2 to 8.0 min, and from 8.0 to 9.5 min were set to 1.0, 0.5, 0.3, and 1.0 ml/min, respectively. After a 1.1-min isocratic period at 30% B, a linear gradient was started toward 95% B at 3.0 min, kept at that composition until 7.5 min, and followed by an 8-min linear gradient period toward 30% B. The mass spectrometer was operated using the electrospray ionization source in the negative selected reaction-monitoring mode. The divert valve between 0 and 3.2 min, between 3.2 and 7.4 min, and between 7.4 and 9.5 min was set to on (wasted), off (introduced into the electrospray ionization interface), and on (wasted), respectively. Monitoring ions were 539.1 (precursor) 
477.1 (product) and 715.2 (precursor) 539.3 (product) for S-8921 and S-8921G, respectively. The lower limit of quantitation for S-8921 and S-8921G was 0.5 and 0.5 ng/ml in plasma specimens and 10 and 20 ng/ml in bile specimens, respectively. d9-S-8921 and d9-S-8921G were used for the internal standard of LC/MS/MS analysis. The bias values from back-calculated concentrations were within –6.4 to +10.4% (S-8921) and –3.2 to +5.7% (S-8921G), and the calibration curves in plasma and bile had good linearity in the range of 0.5 to 150 and 10 to 500 ng/ml, respectively. The intra- and interassay bias and relative standard deviation (RSD) for S-8921 ranged from –7.2 to +19.2% and less than 10.5%, respectively. Conversely, the intra- and interassay bias and RSD for S-8921G ranged from –8.7 to +5.7% and less than 8.3%, respectively. All validation items complied with the acceptance criteria.
Pharmacological Effect of S-8921 and S-8921G in Rats. Animals were divided into the following eight dosing groups, so that each group (n = 8) had a similar baseline serum cholesterol concentration: 1) control, 5% gum arabic solution; 2) S-8921, 0.1 mg/kg; 3) S-8921, 1 mg/kg; 4) S-8921, 10 mg/kg; 5) S-8921G, 0.1 mg/kg; 6) S-8921G, 1 mg/kg; 7) S-8921G, 10 mg/kg; and 8) 5% gum arabic solution. S-8921 and S-8921G were suspended in 5% gum arabic solution. During the experiment, the animals were fed either an ordinary pellet diet (CA-1; CLEA Japan, Inc.) or a high-cholesterol diet [CA-1 containing 1% (w/w) cholesterol and 0.5% (w/w) sodium cholate; CLEA Japan, Inc.]. Normal rats (Wistar rats) in dosing groups 1 to 7 were fed a high-cholesterol diet, and those in group 8 were fed an ordinary diet. Gunn rats were divided into groups 1, 4, 7, and 8. There was no statistically significant difference in basal body weights among the groups before the treatment with test compounds, and the mean body weight of normal rats and Gunn rats in each group was 302 to 312 g and 303 to 316 g, respectively.
S-8921 or S-8921G was orally administered once a day in the morning for 7 days. Blood samples (5 ml) were collected from the abdominal aorta under pentobarbital anesthesia the next morning following the final administration, and serum was separated by centrifugation at 4°C. Serum total cholesterol was determined from the mean value of the enzymatic method using a commercial kit (Pureauto S CHO-N; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) on the day of blood sampling. A high-density lipoprotein (HDL) fraction was prepared from a 200-µl serum sample stored at 4°C, according to the method reported by Goldstein et al. (1983) with some modification. In brief, the serum (initial density = 1.006 g/ml) was adjusted to a final density of 1.063 g/ml by adding solid potassium bromide. Then, the serum was centrifuged at 42,000 rpm for 4 h at 10°C. After centrifugation, the upper fraction (80 µl) was aspirated to obtain the lower fraction that was designated as the HDL fraction (its density was assumed to be more than 1.063 g/ml). The serum HDL cholesterol level was calculated by multiplying the cholesterol level in the HDL fraction by the volume ratio (=0.6) of the HDL fraction to the serum sample. The cholesterol level in the HDL fraction was quantified using an automated measurement apparatus (model 7070; Hitachi, Tokyo, Japan). The serum non-HDL cholesterol level was calculated by subtracting the HDL cholesterol level from the total cholesterol level.
Construction of Stably Transfected HEK293 Cells Expressing hASBT. The hASBT gene was isolated by PCR using human ileum cDNA (Multiple Tissue cDNA panels; Clontech, Mountain View, CA). The gene was amplified using a forward primer containing a c-Myc site (5'-CACCATGCAGAAGCTGATCTCAGAGGAGGACCTGATGAATGATCCGA-3') and a reverse primer containing an ApaI site (5'-TTGTGGGCCCACTTGATGTCTAC T-3'). PCR product was cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA). Then, the pENTR/D-TOPO vector was digested with NotI and ApaI, and the ASBT cDNA was ligated into the NotI and ApaI sites of the pcDNA5/FRT expression vector (Invitrogen). hASBT-expressing HEK293 cells (hASBT-HEK) were constructed by the cotransfection of hASBT-pcDNA5/FRT and pOG44 vector (Invitrogen) into HEK293 cells (Flp-In-293 cell line; Invitrogen) using Fu-GENE6 (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's instructions, and by selection with 100 µg/ml hygromycin B (Invitrogen) for 2 weeks. We checked that transfection of ASBT was successful by reverse transcription-PCR.
Inhibitory Effect on TCA Uptake into hASBT-HEK Cells. hASBT-HEK and mock-control cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml hygromycin B (Invitrogen) at 37°C with 5% CO2 and 95% humidity. The cells were then seeded in 12-well plates at a density of 1.5 x 105 cells/well. After a 3-day culture period, the cells were washed twice and preincubated for 15 min at 37°C with transport buffer (137 mM NaCl, 4.17 mM KHCO3, 0.44 mM KH2PO4, 0.34 mM K2HPO4, 10 mM HEPES, 5.55 mM D-glucose, 1.26 mM CaCl2, 0.49 mM MgCl2, and 0.41 mM MgSO4, pH 7.4). In the Na+-free buffer, choline chloride was used instead of NaCl.
Uptake was initiated by replacing transport buffer with 600 µlof substrate solution ([3H]TCA: 1, 5, and 10 µM) or substrate solution containing S-8921 (10, 100, or 200 µM) or S-8921G (10, 30, or 100 nM). After incubation for 30 s, the substrate solution was removed, and the cells were washed three times with 1 ml of ice-cold transport buffer and dissolved in 500 µl of 2 N NaOH. Aliquots (800 µl) were transferred to scintillation vials after adding 500 µl of 2 N HCl. The radioactivity associated with the cells and incubation buffer was measured in a liquid scintillation counter (LS6000SE; Beckman Coulter, Fullerton, CA) after adding 5 ml of scintillation fluid (Clearsol I; Nacalai Tesque) to the scintillation vials. The remaining 20 µl of cell lysate was used to determine the protein concentration by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Glucuronidation with Human UGT Supersomes. The reaction mixture (0.5 ml) consisted of 0.1 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1 mg of protein/ml human UGT Supersomes, 0.0125 mg of Brij 58, and 50 µM[14C]S-8921. The reaction was started by addition of 5 mM UDP-glucuronic acid and incubated for 1 h at 37°C. Then, the reaction was stopped by addition of 0.5 ml of ice-cold acetonitrile to the reaction mixture, and the mixture was kept in ice water for 15 min. Next, the reaction mixture was centrifuged at 3000 rpm for 10 min. Then, 50 µl of supernatant was injected into the LC/mass spectrometer fitted with a radiometric detector. The incubation was performed in duplicate.
An HPLC method for the analysis of S-8921 and its metabolites was developed using a Symmetry C8 column (5 µm, 150 mm, 4.6 mm i.d.; Waters). The mobile phase consisted of 0.1% acetic acid in distilled water and methanol. The programmed elution was isocratic with 63% methanol for the first 25 min, with a linear change to 73% methanol over the next 5 min, and then the program was maintained at 73% for 25 min. The eluent was monitored at 274 nm throughout the 55-min run time. The LC/mass spectrometer with a radiometric detector consisted of the following components: pump and autosampler (Agilent 1100 series; Agilent Technologies, Palo Alto, CA), Symmetry C8 analytical column (5 µm, 150 mm, 4.6 mm i.d.; Waters), LCQ DECA mass spectrometer (ThermoQuest), and radiometric detector (Packard Flow Scintillation analyzer; PerkinElmer Life and Analytical Sciences).
Absorption of S-8921 from the Loop in the Rat Jejunum and Ileum. The Sprague-Dawley rats were anesthetized with ethyl urethane. For the plasma collection group (n = 6; mean body weight, 406 g), a cannula was inserted into the portal vein for the collection of samples. For the bile collection group (n = 6; mean body weight, 380 g), both portal vein and bile duct cannulation was performed. Then, the animals were kept on a warm plate at 37°C. A closed loop (10 cm) of the jejunum or the terminal ileum was made by ligation at both ends. S-8921 (100 µM; 0.5 ml) dissolved in PEG400 and saline containing 0.1% DMSO [25/75 (v/v)] was instilled into the loop. At predetermined times after dosing, an aliquot of blood was collected from the portal vein. The blood was immediately centrifuged at 14,000g for 3 min at room temperature to obtain plasma. At the end of the experiment, the loop was washed with saline and the washing solution was collected. Plasma, bile, and washing solution were frozen for storage until assay. S-8921G was barely absorbed and deconjugated to S-8921 when S-8921G (10 µM; 0.5 ml) dissolved in PEG400 and saline containing 0.1% DMSO [25/75 (v/v)] were instilled into the ileal loop.
For the measurement of S-8921 or S-8921G, an aliquot (100 µl) of plasma, bile, and washing solution was added to 220 µl of acetonitrile. Then, the mixture was centrifuged at 14,000g for 3 min at room temperature, and the supernatant (20 µl) was subjected to HPLC analysis under the following chromatographic conditions: pump model LC-10AD, UV detector model SPD-10AVvp (
= 260 nm), and controller model SCL-10Avp (Shimadzu, Kyoto, Japan); Cosmosil 5C18 AR-II column (150 mm, 3.0 mm i.d.; Nacalai Tesque); mobile phase, A: 0.1% acetate in water and B: 0.1% acetate in acetonitrile [A/B = 70/30–15/85 (v/v)]; and flow rate, 0.4 ml/min. The concentrations were determined by a nonvalidated HPLC method without an internal standard. The calibration curves for S-8921 and S-8921G were in the range 0.04 to 10 and 0.02 to 5 µM, respectively. The linearity of the analytical analysis was more than 0.99. The intra-assay bias and RSD at 0.4 and 0.2 µM for S-8921 and S-8921G ranged from –3.6 to 5.0% and from 14.3 to 9.2%, respectively.
Uptake of [14C]S-8921G into Freshly Isolated Rat Hepatocytes. Rat hepatocytes were prepared as described by Nezasa et al. (2003). Rat hepatocytes were preincubated in Krebs-Henseleit buffer for 10 min at either 37 or 4°C. To measure the uptake in the absence of Na+, sodium chloride and sodium bicarbonate in Krebs-Henseleit buffer were replaced with choline chloride and choline bicarbonate, respectively. The uptake study was initiated by adding an equal volume of substrate solution to the cell suspension. After incubation for 15 s to 5 min, the reaction was terminated by separating the cells from the reaction medium by centrifugal filtration (Yamazaki et al., 1993). After an overnight incubation in 2 N NaOH to dissolve the hepatocytes, the centrifuge tube was cut, and each compartment was transferred to a vial. The compartment containing the dissolved hepatocytes was neutralized with 50 µl of 2 N HCl, mixed with scintillation cocktail (Pico-Flour 40; PerkinElmer Life and Analytical Sciences), and the radioactivity was measured in a liquid scintillation counter (Tri-Carb 3100; PerkinElmer Life and Analytical Sciences). The concentration dependence of [14C]S-8921G uptake was investigated at 3.4 to 1000 µM after incubation in Krebs-Henseleit buffer for 30 s at 37°C. In addition, the inhibitory effect of various compounds [unlabeled S-8921G, 1 mM; bromosulfophthalein (BSP), 100 µM; estrone 3-sulfate, 100 µM; TCA, 300 µM; and S-8921, 100 µM] on 10 µM[14C]S-8921G uptake by hepatocytes was determined at 15 and 45 s.
Data Analysis. Pharmacokinetic analysis was performed using WinNonlin (Pharsight, Mountain View, CA) based on a noncompartment model with uniform weighting.
The apparent inhibition constant (Ki) for S-8921 and S-8921G was determined by WinNonlin using the equation for noncompetitive inhibition:
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The kinetic parameters for hepatic uptake of [14C]S-8921G were calculated according to the following equation:
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Statistical Analysis. Statistical analysis for significant differences was performed using the Mann-Whitney test with the SAS system (SAS Institute, Cary, NC). Statistical significance was defined as P < 0.01 or P < 0.05.
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of S-8921G was 2.6-fold higher than that of S-8921 (Table 1). S-8921 was not detected in the bile of normal rats until 24 h after oral administration, but 12.7 ± 2.5% dose was excreted into the bile as S-8921G (Table 1). The plasma level of S-8921 in Gunn rats was very high compared with that of normal rats, and the AUC0- of Gunn rats was approximately 9-fold higher than that of normal rats (Fig. 2b; Table 1). In Gunn rats, S-8921G was not detected in the plasma except for one point in one rat, and neither S-8921 nor S-8921G was detected in the bile of Gunn rats.
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Glucuronidation of S-8921 with Human UGT Supersomes. [14C]S-8921 was incubated with nine human UGT Supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) in the presence of UDP-glucuronic acid. S-8921 was converted to S-8921G, by incubating with UGT1A1, -1A3-, -1A8-, -1A9-, and -1A10-expressed microsomes (Table 2).
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Effect of S-8921 and S-8921G on Serum Cholesterol in Gunn Rats. Feeding a high-cholesterol diet resulted in higher serum non-HDL cholesterol levels in both normal and Gunn rats (Fig. 3; control 5% GA). Serum non-HDL cholesterol in hypercholesterolemic normal rats treated with S-8921 (1 and 10 mg/kg p.o.) or S-8921G (1 and 10 mg/kg p.o.) was significantly reduced in comparison with the control group (Fig. 3a). In contrast, HDL cholesterol was slightly increased (Fig. 3a). In hypercholesterolemic Gunn rats, there was no significant reduction in serum non-HDL cholesterol by S-8921 (10 mg/kg p.o.) (Fig. 3b). However, S-8921G (10 mg/kg p.o.) significantly reduced serum non-HDL cholesterol (Fig. 3b).
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S-8921G Concentration in the Portal Vein of Rats. The concentrations of S-8921 and S-8921G in the portal vein were measured after S-8921 was applied to the loop of the jejunum and ileum. S-8921G was detected in the portal vein at 5 min after administration of 100 µM S-8921 into the intestinal loop, and the plasma concentration of S-8921G in the portal vein was higher than that of S-8921 at all time points (Fig. 5). In addition, S-8921G was detected in the lumen of both the jejunum and ileum. The concentrations of S-8921G in the lumen of the jejunum and ileum at 45 min after dosing were 7.60 ± 1.36 and 1.23 ± 0.99 µM (mean ± S.D.; n = 3), respectively. When S-8921 was applied to the loop of the jejunum in rats following bile duct cannulation, the concentration of S-8921G in the bile and intestinal lumen at 45 min after dosing was 1.68 ± 0.59 and 2.27 ± 0.95 µM (mean ± S.D.; n = 3), respectively. The volume of the bile collected during 45 min and in the intestinal lumen was 0.4 to 0.7 ml and 0.5 ml, respectively. Taking this into consideration, the cumulative amounts of S-8921G recovered in the bile and intestinal lumen were 1.1 and 1.1 nmol 45 min after dosing and 16 and 3.1 nmol after 6 h, respectively.
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Plasma concentrations of S-8921G after single oral administration of S-8921 were higher than those of S-8921 in normal rats (Fig. 2), and S-8921G was excreted into the bile in normal rats (Table 1). In Gunn rats, all the UGT1 isoforms of which are hereditarily deficient due to the mutation in the common region (Iyanagi, 1991), the plasma concentration of S-8921 was much greater than that in normal rats, and, particularly, S-8921G was barely detectable in both plasma and bile (Fig. 2; Table 1). It is likely that S-8921G is mainly produced by UGT1 isoforms, and glucuronidation is the major mechanism limiting the oral absorption and elimination of S-8921 from the systemic circulation. It should be noted that the detailed pharmacokinetic analysis of S-8921 has not been performed in Gunn rats; thus, the possibility of adaptive changes in the metabolic enzymes and transporters cannot be excluded; these may also be part of the mechanism underlying an increase in the plasma concentration of S-8921. In vivo results were supported by an in vitro metabolism study using UGT Supersomes, which elucidated that UGT1 isoforms, except UGT1A4 and UGT1A6, converted S-8921 to its glucuronide (Table 2). Among these isoforms, UGT1A1, UGT1A8, and UGT1A10 are expressed in the small intestine, whereas UGT1A1, UGT1A3, and UGT1A9 are expressed in the liver (Strassburg et al., 2000; Tukey and Strassburg, 2000). These UGT1 isoforms could be involved in the intestinal and hepatic glucuronidation of S-8921, although the contribution of these isoforms to the glucuronidation of S-8921 remains to be elucidated in future studies.
The hypocholesterolemic effect of S-8921 was examined in hypercholesterolemic rats. Feeding the high-cholesterol diet increased non-HDL cholesterol in rats (Fig. 3). The serum non-HDL cholesterol level following repeated oral administration of either S-8921 or S-8921G in hypercholesterolemic normal rats was significantly reduced compared with the control group, whereas HDL cholesterol was slightly increased by the treatment (Fig. 3a). In contrast, repeated oral administration of S-8921 had no significant effect in hypercholesterolemic Gunn rats, whereas S-8921G significantly reduced the serum non-HDL cholesterol level (Fig. 3b). Accordingly, glucuronidation is the critical step for the pharmacological action of S-8921 in rats, although S-8921 itself is also an inhibitor of ASBT. This was supported by the in vitro finding that S-8921G is a 6000-fold more potent inhibitor of hASBT than S-8921 (Fig. 4). In general, the glucuronides of xenobiotics are less active than their parent compounds, and glucuronidation is recognized as one of the major detoxification pathways. N-O-Glucuronides of hydroxamic acids, acyl glucuronides of carboxylic acids, morphine 6-O-glucuronide, retinoid glucuronides, and D-ring glucuronides of estrogens are known to have greater pharmacological or toxicological activities than their parent compounds (Ritter, 2000). S-8921G is the first case in which glucuronidation of a phenolic compound results in more potent pharmacological activity than the parent compound.
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The mechanism underlying the asymmetrical efflux of S-8921G in the jejunum remains unknown. Because the recovery was greater in the bile than in the lumen during the intestinal absorption study, the efflux transport across the basolateral membrane will be more effective than the luminal efflux. In the small intestine, Mrp3 is expressed on the basolateral membrane of the intestinal epithelial cells (Rost et al., 2002), and we found that the basolateral membrane vesicles from rat intestine exhibited ATP-dependent uptake of glucuronide conjugates (Shoji et al., 2004). In the small intestine, Mrp3 protein is expressed in the whole segment, but the expression level is higher in the mid-to-distal region than the proximal region (Zelcer et al., 2006), whereas the mRNA expression level exhibits the opposite behavior (Maher et al., 2005). For the efflux transport of glucuronide conjugates in the luminal side, MRP2 and BCRP have been shown to mediate the intestinal efflux of the glucuronide conjugates of xenobiotics (Adachi et al., 2005). Mrp2 expression decreases from the proximal to the distal region of small intestine (Maher et al., 2005), whereas Bcrp exhibits a complementary expression pattern (Tanaka et al., 2005). Further studies are necessary to investigate the involvement/contribution of these ATP-binding cassette transporter(s) in the efflux transport of S-8921G along with the glucuronidation activity.
The uptake of S-8921G was characterized using freshly isolated rat hepatocytes. The uptake of S-8921G was saturable and exhibited sodium dependence (Fig. 6). The uptake was inhibited by organic anions, such as BSP, estrone 3-sulfate, and TCA (Table 3). These results suggest that S-8921G absorbed into the portal vein is removed by hepatic organic anion transporters. Na+-taurocholate cotransporting polypeptide (SLC10A1) is responsible for the sodium-dependent uptake of bile acids (Hagenbuch and Meier, 1994), whereas organic anion transporting polypeptides (OATP/SLCO), such as Oatp1a1, Oatp1a4, and Oatp1b2 in rodents, and OATP1B1 and OATP1B3, account for the sodium-independent uptake of a variety of organic anions (Hagenbuch and Meier, 2003). Thus, these transporters may be responsible for the hepatic uptake of S-8921G. Because MRP2 is well known as the transporter involved in the biliary excretion of many glucuronides (Kusuhara and Sugiyama, 2001), it can be speculated that MRP2 is responsible for the biliary excretion of S-8921G. Further studies are necessary to identify the transporters that play key roles in the intestinal and hepatic disposition of S-8921G.
In conclusion, S-8921G produced by UGT1 isoforms (UGT1A1, UGT1A3, UGT1A8, UGT1A9, and UGT1A10) is a very potent inhibitor of hASBT, and it plays an essential role in the hypocholesterolemic effect of S-8921. The schematic diagram of the pharmacokinetics of S-8921 is shown in Fig. 7. After oral administration of S-8921, S-8921 is metabolized to its glucuronide by UGT1 isoforms in the intestine. A part of S-8921G is directly excreted into the intestinal lumen, and it inhibits ASBT in the ileum. In addition, S-8921 and its glucuronide are absorbed into the portal vein. S-8921G is taken up into the liver by transporter(s), and S-8921 is metabolized to its glucuronide by UGT1 isoforms in the liver. S-8921G, excreted into the bile, is also involved in the inhibition of ASBT in the ileum. Inhibition of ASBT leads to a reduction in serum total cholesterol, particularly non-HDL cholesterol (i.e., LDL cholesterol).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: LDL, low-density lipoprotein; ASBT, apical sodium-dependent bile acid transporter; S-8921, methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate; S-8921G, S-8921 glucuronide; TCA, taurocholate; GA, gum arabic; LC, liquid chromatography/chromatograph; MS/MS, tandem mass spectrometry; RSD, relative standard deviation; HDL, high-density lipoprotein; HEK, human embryonic kidney; PCR, polymerase chain reaction; h, human; UGT, UDP glucuronosyltransferase; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; AUC, area under the curve; BSP, bromosulfophthalein; Mrp/MRP, multidrug-resistance protein; BCRP/Bcrp, breast cancer resistance protein; OATP/Oatp, organic anion transporting protein; 2164U90, (–)-(3R,5R)-trans-3-butyl-3-ethyl-2,3,4,5-tetrahydro-5-phenyl-1,4-benzothiazepine 1,1-dioxide; SC-435, 1-[4-[4[(4R,5R)-3,3-dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2]octane methanesulfonate; S 0960, 7, 12-dihydroxy-3
-(3', 7', 12'-trihydroxy-5'-cholan-24-amido)-5-cholan-24-oic acid; R-146224, 1-{7 - [(1 - (3,5-diethoxyphenyl)-3-{[(3,5-difluoro-phenyl)(ethyl)amino]carbonyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide.
Address correspondence to: Dr. Yuichi Sugiyama, Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp
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