Introduction

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Z-335¹ is a new orally active thromboxane A₂ (TXA₂) receptor antagonist, which is in phase II of clinical trials. Z-335 inhibits the specific binding of [³H]SQ-29548 to human platelets and also inhibits human platelet aggregation induced by the thromboxane A₂ receptor agonist U-46619. Z-335 ameliorates experimental thrombosis without prolonging bleeding time (Tanaka et al., 1998a,b). These observations suggest that the target organ of Z-335 is the intravascular compartment (platelet) and that the pharmacological action of Z-335 will be regulated by the plasma concentration of the drug. In rats, Z-335 exhibits concentrative uptake into liver and is eliminated by biliary excretion (Kawabata et al., 1999). In addition, on the pharmacokinetic study in healthy male volunteers, recovery of the unchanged drug in the urine within 24 h is minimal. These results indicate that Z-335 would be mainly taken up into liver, followed by metabolism or biliary excretion.

Hepatic elimination is one of the major pathways involved in detoxification of xenobiotics, and hepatic uptake is the initial process for the elimination of xenobiotics mediated by metabolism or biliary excretion. Recently, the molecular mechanism underlying the hepatocellular uptake of organic anions has been clarified. In these studies, sodium/taurocholate cotransporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) have been cloned as transporters responsible for the Na⁺-dependent uptake of bile acids and Na⁺-independent uptake of organic anions, respectively (Meier, 1995). Furthermore, oatp2 and oatp4 have been identified as homologs of oatp1 (Noé et al., 1997; Cattori et al., 2000). On the other hand, in human liver, OATP-A (Kullak-Ublick et al., 1995), OATP-B, (Tamai et al., 2000), liver-specific organic anion transporter (LST-1/OATP-2, OATP-C; Abe et al., 1999; Hsiang et al., 1999; Tamai et al., 2000) and OATP-8 (König et al., 2000) have also been identified as homologs of oatp1.

It has been suggested that these transporters are responsible for a large proportion of the hepatic uptake of endogenous and/or exogenous organic anions. In addition, we must consider the drug-drug interactions on the uptake process when these transporters regulate the hepatic elimination of xenobiotic compounds. Therefore, clarifying the contribution of these transporters to the elimination pathways of xenobiotic compounds is important.

To elucidate the transport system that mediates Z-335 uptake into the liver, we investigated the uptake characteristics of Z-335 in isolated rat hepatocytes. Furthermore, we estimated the extent of hepatic uptake of Z-335 in intact rats by using the kinetic parameters obtained from isolated hepatocytes.

	Materials and Methods

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Chemicals. Z-335 (Fig. 1) and ID910096 (internal standard) were synthesized, and pravastatin was extracted from Mevalotin (Sankyo Co., Ltd., Tokyo, Japan), in the central research laboratories of ZERIA Pharmaceutical Co., Ltd. [¹⁴C]Z-335 (827 MBq/mmol) was synthesized by Daiichi Pure Chemicals Co., Ltd. Estradiol 17-glucuronide, estrone 3-sulfate, taurocholate, cholate, glycocholate, ibuprofen, digoxin, quinidine, quinine, probenecid, pancuronium, cimetidine, and rotenone were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and bromosulfophthalein (BSP) were purchased from Aldrich (Milwaukee, WI). All other chemicals were of reagent grade.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Structure of Z-335.

Animals. Male Sprague-Dawley rats (230-300 g) from Charles River Japan, Inc. (Kanagawa, Japan) were used.

In vivo Experiments. Rats were anesthetized with pentobarbital (50 mg/kg), and the femoral artery and vein were cannulated with heparinized polyethylene tubing (PE50; BD Biosciences, Franklin Lakes, NJ). The hepatic vein was also cannulated according to the method of Yokota et al. (1976). Z-335 dissolved in physiological saline was infused through the femoral vein cannula at a flow rate of 20 µl/min. The infusion rate was set at 50 nmol/min/kg. At specific times, arterial and hepatic venous blood was collected in polyethylene tubes, and samples were centrifuged to separate plasma.

Measurement of Z-335. The plasma samples (50 µl) were added to methanol (1 ml). After centrifugation of the mixture, the supernatant was evaporated to dryness under nitrogen gas in a water bath at 40°C and dissolved in 100 µl of mobile phase. The plasma concentration of Z-335 was determined by using high-performance liquid chromatography with a reversed-phase column (Capcell Pak MF C₈; Shiseido, Tokyo, Japan). The following instruments were used for the high-performance liquid chromatography assay: solvent delivery system, LC-9 A (Shimazu, Kyoto, Japan); auto injector, SIL-6 B (Shimazu); UV detector, SPD-10 A (Shimazu). UV absorbance was detected at 230 nm. The mobile phase consisted of 0.1 M KH₂PO₄, pH 7.0/CH₃CN/isopropanol (85:11:4), pumped at a flow rate of 0.9 ml/min.

Cell Preparation. Rat hepatocytes were isolated by the procedure of Baur et al. (1975). After isolation, hepatocytes were suspended (2 mg of protein/ml) in albumin-free Krebs-Henseleit buffer supplemented with 12.5 mM HEPES, pH 7.4. Cell viability was routinely checked by the trypan blue exclusion test, and only hepatocytes exhibiting more than 90% viability were used.

Uptake Study. Uptake of [¹⁴C]Z-335 (5 µM) was initiated by adding the ligand to the preincubated (37°C for 3 min) cell suspension. At designated times, the reaction was terminated by separating the cells from the medium with a centrifugal filtration technique (Schwenk, 1980). Briefly, 200-µl aliquots were placed into 0.4-ml centrifuge tubes containing 50 µl of 2 M NaOH covered by silicone mineral oil (density, 1.015). The samples were then centrifuged for 10 s in a tabletop Microfuge (Beckman Coulter, Inc., Fullerton, CA). The hepatocytes passed through the oil layer and into the alkaline solution of 2 M NaOH. After the cells had dissolved in the alkaline solution, the tube was sliced with a razor blade, and both compartments (the medium side and the cell side) were transferred into vials containing scintillator (Ultima Gold XR; Packard Instrument Co., Ltd., Downer Grove, IL). The radioactivity in medium and cells was measured by a liquid scintillation counter (2000CA, 2900; Packard Instrument Co., Ltd.). The time course of Z-335 uptake was plotted in terms of the cell to medium concentration ratio (C/M ratio). Initial uptake velocity was calculated using linear regression on points taken at 30 and 60 s.

Determination of Kinetic Parameters. The kinetic parameters for Z-335 uptake were estimated according to the following equation:

(1)

where v₀ is the initial uptake velocity of Z-335 (nanomoles per minute per milligram of protein), S is the Z-335 concentration in the medium (micromolar), K_m is the Michaelis constant (micromolar), V_max is the maximum uptake velocity (nanomoles per minute per milligram of protein), and P_dif is the nonspecific uptake clearance (microliters per minute per milligram of protein). The above equation was fit to the uptake data sets by a nonlinear least-squares method using a MULTI program (Yamaoka et al., 1981) to obtain estimates of kinetics parameters.

(2)

Estimation of Hepatic Uptake Clearance Extrapolated from the in Vitro Data and in Vivo Hepatic Clearance for Z-335. Based on the kinetic parameters obtained by the fitting procedure described, the permeability-surface product (PS_influx) (milliliters per minute per 250-g rat) was calculated with the following equation:

(3)

where 1 g of liver = 1.25 × 10⁸ cells () (Lin et al., 1980), 1 mg of protein = 1 × 10⁶ cells (), and the 250-g rat = 11.1 g of liver () (Sugita et al., 1982).

(4)

(5)

(6)

(7)

(8)

(9)

	Results

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Time Course and Concentration Dependence of Uptake of Z-335 into Isolated Rat Hepatocytes. The time course of [¹⁴C]Z-335 (5 µM) uptake by isolated rat hepatocytes is shown in Fig. 2. The C/M ratio of [¹⁴C]Z-335 reached 21.2 at 0.5 min and 71.7 at 5 min. The concentration dependence of the initial uptake velocity (Fig. 3) indicated a two-component process. The estimated kinetic parameters (mean ± S.E.) were as follows: K_m = 45.6 ± 5.2 µM, V_max = 4.1 ± 1.2 nmol/min/mg of protein, and P_dif = 8.3 ± 4.8 µl/min/mg of protein (Table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Time course of [14C]Z-335(5 µM) uptake by isolated rat hepatocytes. Open circles represent apparent C/M ratio (the cellular uptake amount divided by the extracellular Z-335 concentration). Each point and vertical bar give mean ± S.E. from nine determinations in three separate preparations.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of Z-335 uptake by isolated rat hepatocytes. The relationship between initial uptake velocity (v0) and concentration of Z-335 in the incubation medium. Each open circle and vertical bar represent mean ± S.E. from three different preparations. The solid line is the least-square fit of date to eq. 1. The dotted line represents nonspecific diffusion calculated with value of nonspecific uptake clearance (Pdif). Closed circles, which were calculated by subtracting nonspecific diffusion from total uptake (open circles), represent saturable uptake. Dashed line is theoretical curve of saturable uptake.

Effect of Temperature and Metabolic Inhibitors on the Initial Uptake Velocity of Z-335. The initial uptake velocity of Z-335 exhibited marked temperature dependence; the uptake decreased to 19% at 0°C and to 45% at 27°C. Rotenone, an inhibitor of mitochondrial respiration, and FCCP, an oxidative phosphorylation uncoupler, significantly decreased the uptake of Z-335.

Effect of Metabolic Inhibitors on the Initial Uptake Velocity of Z-335. The initial uptake velocity of [¹⁴C]Z-335 declined along with an expected reduction in cellular ATP level. The uptake velocities at 30 min were 37% of control values with FCCP and 49% with rotenone (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of metabolic inhibitors on initial uptake of [14C]Z-335. Each point and vertical bar represent mean ± S.E. of four determinations. Hepatocytes were preincubated at 37°C for 3 min. At time 0, the metabolic inhibitor (2 µM FCCP, open circles; 30 µM rotenone, closed circles) was added. At the time indicated, the initial uptake was determined.

Na⁺ Dependence of Z-335 Uptake into Isolated Rat Hepatocytes. The Na⁺ dependence of Z-335 uptake was studied by replacing the Na⁺ in the medium by choline. Eadie-Hofstee plots for Z-335 uptake in Na⁺- or choline-containing medium are shown in Fig. 5. When sodium was replaced by choline, the estimated kinetic parameters (mean ± S.E.) were as follows: K_m = 35.8 ± 5.4 µM, V_max = 2.3 ± 0.4 nmol/min/mg protein, and P_dif = 7.3 ± 3.5 µl/min/mg of protein. Consequently, replacement of sodium by choline did not significantly decrease uptake (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Eadie-Hofstee Plot of sodium-dependent and -independent uptake of [14C]Z-335 into isolated rat hepatocytes. Each point and vertical bar represent mean ± S.E. from three different preparations. Isolated rat hepatocytes were incubated in sodium-containing medium (open circles) or choline-containing medium (sodium replaced by choline, closed circles) during the uptake experiment.

Inhibition of the Initial Uptake Velocity of Z-335 by Organic Anions, Bile Acids, Organic Cations, and Neutral Compounds. To investigate the specificity of Z-335 uptake, the effects of other substrates on the uptake of Z-335 were studied. Z-335 uptake was decreased by the addition of estradiol 17-glucuronide, estrone 3-sulfate, taurocholate, cholate, and glycocholate, with K_i values of 9.3, 31.0, 47.3, 43.0, and 60.2 µM, respectively (Fig. 6, A and B; Table 2). The inhibitory effects of pravastatin and BSP on Z-335 uptake were also observed, with K_i values of 52.9 and 7.0 µM, respectively (Fig. 6C; Table 2). In addition, inhibitory effects of quinidine, quinine, and pancuronium on Z-335 uptake were observed, with K_i values of 65.7, 57.2, and 280.0 µM, respectively (Fig. 6D; Table 2). Furthermore, Z-335 uptake was inhibited by the addition of probenecid, with a K_i value of 67.6 µM. The inhibition of Z-335 uptake by digoxin (a neutral compound), ibuprofen, and cimetidine (classified as a type I organic cation; Nakamura et al., 1994) was also estimated. Digoxin, ibuprofen, and cimetidine had slight effects on Z-335 uptake at relatively higher concentrations, with K_i values of 161.3, 159.9, and 752.0 µM, respectively (Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Inhibition of [14C]Z-335 (5 µM) uptake by conjugated steroids (A), bile acids (B), organic anions(C), and organic cations (D). Each point and vertical bar represent mean ± S.E. from five different preparations. A, v0 of [14C]Z-335 as a function of Estradiol 17-glucuronide (0.5-500 µM, open circles) concentration in medium. B, v0 of [14C]Z-335 as a function of taurocholate (1-1000 µM, open circles), cholate (1-1000 µM, closed circles), and glycocholate (1-1000 µM, open triangles) concentration in medium. C, v0 of [14C]Z-335 as a function of BSP (1-1000 µM, open circles) and pravastatin (1-1000 µM, closed circles) concentration in medium. D, v0 of [14C]Z-335 as a function of quinidine (1-1000 µM, open circles) and quinine (1-1000 µM, closed circles) concentration in medium.

Extrapolation of in Vivo Clearance from in Vitro Uptake Parameters. The in vivo clearance parameters were calculated by using eqs. 6, 7, 8, and 9. CL_tot, CL_h, and CL_{int, app} were estimated to be 3.0, 2.8, and 315.7 ml/min/250-g rat, respectively (Table 3). Furthermore, PS_influx in a 250-g rat was extrapolated from the in vitro uptake parameters (eq. 3). CL_{h, well} and CL_{h, PT}, based on PS_influx, were also calculated by using a well stirred model (eq. 4) and a parallel-tube model (eq. 5). The extrapolated PS_influx was 132.3 ml/250-g rat, and thus CL_{h, well} and CL_{h, PT} were calculated to be 1.2 and 1.3 ml/min/250-g rat, respectively (Table 3).

	Discussion

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The uptake of Z-335 is highly concentrative (C/M ratios were 21.2 at 30 s and 71.7 at 5 min), temperature-dependent, and sensitive to metabolic inhibitors, suggesting an energy-dependent uphill transport. In a previous article, the intracellular ATP concentration was decreased rapidly to <8% at 30 min by addition of FCCP (2 µM) or rotenone (30 µM) (Yamazaki et al., 1993). In the presence of metabolic inhibitors, despite the decreased cellular ATP, uptake remained at 37% (FCCP) and 49% (rotenone) of the control value, respectively. Organic anion transporting polypeptides (e.g., oatp1 and oatp2) have been reported to transport endogenous and exogenous substrates by an ATP-independent transport system, and it was suggested that oatp1 and oatp2 are glutathione exchangers (Li et al., 1998, 2000). These findings may be relevant to our observation that ATP-independent uptake contributes a part of the total uptake of Z-335. Furthermore, the concentration dependence of initial uptake velocity (Fig. 3) indicated a two-component process, one saturable component, with a K_m value of 45.6 µM and a V_max value of 4.1 nmol/min/mg of protein, and a nonspecific diffusion clearance, with a P_dif value of 8.3 µl/min/mg of protein. The contribution of the carrier-mediated uptake to the total uptake [(V_max/K_m)/(V_max/K_m + P_dif.)] was estimated to be 91%, and therefore, the uptake of Z-335 into liver would occur mainly by the carrier-mediated uptake system.

Sodium/taurocholate cotransporting polypeptide (ntcp) has been identified in rat liver and is expressed in rat hepatocyte sinusoidal membranes (Stieger et al., 1994). In our study, when the Na⁺ in the medium was replaced by choline, the V_max value was reduced slightly, but uptake clearance (V_max/K_m + P_dif.) remained at approximately 70% of that in Na⁺-containing medium. This finding suggests that the contribution of Na⁺-dependent uptake was slight and that Z-335 is taken up into liver mainly by an Na⁺-independent uptake system.

The system responsible for the uptake of Z-335 was investigated by the addition of conjugate steroids (e.g., estradiol 17-glucuronide and estrone 3-sulfate), bile acids (e.g., taurocholate, cholate, and glycocholate), and exogenous organic anions (e.g., pravastatin and BSP) to the incubation medium. Previously, it has been suggested that hepatic uptake of these substrates is mediated by oatp1, oatp2, and oatp4 (Meier, 1995; Noé et al., 1997; Tokui et al., 1999; Cattori et al., 2000). The uptake of Z-335 was inhibited by these substrates, with K_i values similar to their K_m values, suggesting that Z-335 shares a common carrier protein with these substrates and might also be taken up via organic anion transport systems, such as oatp1, oatp2, and oatp4. Furthermore, the K_i value of digoxin, which is specifically transported by oatp2 (Noé et al., 1997), for Z-335 uptake was 161.3 µM, considerably higher than the K_m value of digoxin (expression system for oatp2, 0.24 µM; Noé et al., 1997; hepatocytes, 0.695 µM; Hedman and Meijer, 1998), indicating that Z-335 and digoxin are taken up by different carrier proteins. Therefore, the contribution of oatp2 to the uptake of Z-335 would be negligible.

In transport measurements with isolated hepatocytes, more hydrophobic organic cations, such as quinidine, quinine, and cyanine 863, have previously been classified as type II cations (Groothuis and Meijer, 1996). In addition, it has been postulated that these cations are transported by a transport system different from that involved in transport of type I organic cations, such as tetraethylammonium and choline (Elferink et al., 1995). Furthermore, a cation transporter (rOCT1) has been sequenced from a cDNA library of rat kidney (Gründemann et al., 1994) and is expressed in the basolateral membranes of renal proximal tubules and in the sinusoidal membranes of hepatocytes (Koepsell, 1998). In hepatocytes, rOCT1 transports type I cations across the basolateral membrane, whereas type II cations are taken up by a different transport system (Nagel et al., 1997). van Montfoort et al. (1999) demonstrated that type II cations were transported by rat oatp1, oatp2, and human OATP, whereas type I cations were not transported by these systems. The uptake of Z-335 was inhibited by quinidine, quinine, and pancuronium, which are classed as type II cations, with estimated K_i values of 65.7, 57.2, and 280.0 µM, respectively. Furthermore, the inhibitory effect of cimetidine, a type I cation (Nakamura et al., 1994), on the uptake of Z-335 was slight, with a K_i value (752.0 µM) 20 times the K_m value of cimetidine uptake (32 µM; Nakamura et al., 1994). Furthermore, Hedman and Meijer (1998) reported that quinidine and quinine exhibited a stereoselective inhibitory effect on the hepatic uptake of digoxin and that quinine was more potent than quinidine as an inhibitor of digoxin uptake. However, stereoselective inhibition of Z-335 uptake by quinidine and quinine was not observed in our study. In addition, the weak inhibition by digoxin supports a slight involvement of oatp2.

Recently, Sugiyama et al. (2001) demonstrated that probenecid was nonselective inhibitor for oatp1, 2, oat1, and oat3 transfectants in LLC-PK1 cells. In addition, taurocholate was a selective inhibitor for the oatp family. The uptake of Z-335 was inhibited significantly by probenecid, with a K_i value of 67.6 µM, and taurocholate. Taken together, the data suggest that Z-335 uptake might involve the oatp family, possibly oatp1, and not the oat family.

After Z-335 is taken up into rat liver, it has been mainly metabolized to taurine conjugate. Also, Z-335 and its metabolite are mainly eliminated by biliary excretion. Accurate evaluation of hepatic clearance is very important for predicting the pharmacological effect and/or side effects of this drug. Therefore, in this study, the hepatic distribution of Z-335 at steady state was directly evaluated by measuring the plasma concentration in both arterial and hepatic venous blood. In addition, the rate-limiting process involved in hepatic elimination of Z-335 was also examined by comparison of the in vivo apparent hepatic intrinsic clearance (CL_{int, app}), with the kinetic parameters obtained from isolated hepatocytes. The total body clearance (CL_tot) at steady state was estimated to be 3.0 ml/min/250-g rat, and CL_tot was similar to hepatic clearance (CL_h) (2.8 ml/min/250-g rat). Furthermore, the calculated CL_{int, app} for Z-335 was 315.7 ml/min/250-g rat (Table 3). On the other hand, based on in vitro parameters, the calculated PS_influx was 132.3 ml/min/250-g rat, which was comparable to CL_{int, app}. These results indicate that only the influx process influences CL_{int, app}; therefore, hepatic intrinsic clearance of Z-335 at steady state is rate-limited by the uptake process.

In conclusion, hepatic intrinsic clearance of Z-335 at steady state is rate-limited by the uptake process since Z-335 is efficiency taken up by an active transport mechanism, probably involving oatp1 and/or oatp4, followed by metabolism or biliary excretion.