Introduction

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Antifolate compounds like methotrexate (MTX) are widely used to treat leukemia (Jolivet et al., 1983), rheumatoid arthritis (Cash et al., 1994; Bannwarth et al., 1996), psoriasis (Weinstein et al., 1977), and other autoimmune diseases (Owen et al., 1979). However, long-term MTX therapy sometimes produces serious side effects such as nephrotoxicity (Jacobs et al., 1976), hepatic disorders (Dahl et al., 1971; Weinblatt et al., 1992), and lung fibrosis (Furst et al., 1990). Such toxicity is believed to be derived from long-term accumulation of the drug in the relevant organs; therefore, new derivatives of MTX with a lower potential for accumulation are desirable. Recently, an MTX derivative, N-[[4-[(2,4-diamminopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl]-L-homoglutamic acid (MX-68; Fig. 1), was synthesized. MX-68 is a novel antifolate that was specifically designed chemically not to undergo intracellular polyglutamation via substitution of the glutamic acid moiety with homoglutamic acid (Matsuoka et al., 1997). MX-68 is less hepatotoxic than MTX because of its lower polyglutamation inside the cells (Mendelsohn et al., 1996). MX-68 is more potent at suppressing collagen-induced arthritis in DBA/1J mice, whereas its inhibitory effect on cell proliferation is similar to that of MTX (Mihara et al., 1996; Mihara et al., 1997).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structures of MX-68 and MTX.

Thus, although MX-68 is a promising antifolate derivative, no information about its pharmacokinetic profile has been reported. As far as MTX disposition is concerned, a number of investigations have been performed. Although glomerular filtration principally accounts for the urinary excretion of MTX, an uptake system on the basolateral membrane has been identified in renal cortical slices that was inhibited competitively by various other anionic drugs (Nierenberg, 1983). MTX undergoes bidirectional transport within the renal tubules: an active secretory process (Chen and Chiou, 1983; Iven and Brasch, 1988) recognizes a variety of types of organic anions, whereas an active reabsorptive process specifically favors folate derivatives (Hendel and Nyfors, 1984; Selhub et al., 1987). Recently, the multispecific organic anion transporter 1 (OAT1) was isolated from the cDNA library of rat kidney (Sekine et al., 1997). OAT1 showed wide substrate selectivity to organic anions, including MTX, and seems to play a role in MTX uptake by renal tubular cells (Sekine et al., 1997). The kidney-specific transporter OAT-K1 was also identified as being localized on the brush border membranes of renal proximal tubules and it transports MTX and folate (Saito et al., 1996; Masuda et al., 1997b).

A significant amount of MTX is also eliminated by an active transport mechanism via biliary excretion and undergoes enterohepatic circulation (Willams et al., 1965; Hillman and Steinberg, 1982; Hendel and Brodthagen, 1984; Weir et al., 1985; Bannwarth et al., 1996). Biliary elimination is responsible for up to 10 to 30% of MTX excretion in humans, and it becomes even more important in patients with renal insufficiency (Shen and Azarnoff, 1978; Nuernberg et al., 1990; Songsiridej and Furst, 1990). Both uptake at the basolateral membrane into hepatocytes and subsequent excretion at the bile canalicular membrane are carrier-mediated processes (Horne, 1993, Masuda et al., 1997a). Thus, transporters located both in the liver and kidney are involved in the disposition of MTX, leading to its nonlinear pharmacokinetic profile in the body.

In the present study, we report the biliary and urinary excretion kinetics of MX-68, evaluated by changing plasma and tissue concentrations at steady state under various continuous infusion rates. Because MX-68 has two carboxyl groups in its structure, organic anion transport systems may be responsible for the membrane penetration of MX-68. In particular, to investigate the involvement of canalicular multispecific organic anion transporter (cMOAT), which has a wide spectrum of substrate specificity toward organic anions, including MTX and folates, as far as biliary excretion is concerned, we used Eisai hyperbilirubinemic rats (EHBRs) with a hereditary deficiency of cMOAT (Ougoh et al., 1986). The kinetic profile in the MX-68 excretion as well as its in vitro cytosolic binding isotherm suggested that saturation of transport systems across several plasma membranes and intracellular binding results in the nonlinear excretion profile in both liver and kidney.

	Materials and Methods

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Chemicals and Reagents. MX-68 and [¹⁴C]MX-68 were synthesized at Fuji Gotemba Research Laboratories (Chugai Pharmaceutical, Shizuoka, Japan). All other chemicals and reagents were commercial products of analytical grade.

Animals. Male Sprague-Dawley rats (SDRs) from Nisseizai (Tokyo, Japan) and EHBRs from Eisai Laboratories (Gifu, Japan) weighing 250 to 300 g were used throughout the experiments. The animals had free access to food and water. This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Intravenous Infusion Study. With the animals under light ether anesthesia, both the femoral artery and vein were cannulated with PE50 polyethylene tubing (i.d., 0.58 mm; o.d., 0.965 mm; Becton Dickinson & Co., Parsippany, NJ), for blood sampling and for infusion of MX-68, respectively, and the bile duct was cannulated with PE10 polyethylene tubing (i.d., 0.28 mm; o.d., 0.61 mm, Becton Dickinson & Co.) for bile collection. The bladder was also catheterized with polyethylene tubing (Hibiki 8, Tokyo, Japan). SDRs and EHBRs were divided into five and three groups, respectively, and MX-68 dissolved in 0.9% NaCl solution was infused for 3 h to each group at a rate of 0.067, 0.334, 1.67, 8.36, and 33.4 µmol/h/kg for SDRs and 0.067, 1.67, and 33.4 µmol/h/kg for EHBRs. The infusion rate of labeled MX-68 was fixed at 0.067 µmol/h/kg. At fixed times after initiation of the infusion, 0.4-ml arterial blood samples were collected, and plasma was obtained by centrifugation (Microfuge; Beckman Instruments, Columbia, MD). Bile was collected in preweighed 1.5-ml Eppendorf microfuge tubes at 30-min intervals for 3 h. Urine was collected in preweighed 1.5-ml Eppendorf microfuge tubes at 1-h intervals. At the urine sampling time, the remaining urine in the bladder was flushed out with 2 ml of 0.9% NaCl solution. At the end of the infusion, the liver and kidneys were removed. Aliquots of plasma (100 µl), bile (50 µl), and urine (100 µl) were mixed with a scintillation cocktail (Hionic Flour; Packard, Groningen, the Netherlands) and counted (model LS 6000SE; Beckman Instruments). Approximately 300 mg of liver and kidney tissues was solubilized in 2 ml of Soluene 350 (Packard) and mixed with a scintillation cocktail after decolorization with 0.4 ml of 30% H₂O₂ solution. The procedure for measuring radioactivity was the same as that applied to plasma, bile, and urine. In our preliminary study, the radioactivity in plasma, urine, and bile derived from parent compound and its metabolites was separately determined by radio-HPLC after the oral administration of [¹⁴C]MX-68. As far as ¹⁴C radioactivity was concerned, the parent compound accounted for 97.1 ± 1.3% in plasma at 30 min after administration and 86.9 ± 4.0 and 94.7 ± 4.1% in urine and bile during the 24-h period immediately after administration. Thus, MX-68 undergoes very little metabolism in rats, and the radioactivity is largely due to the parent compound.

Determination of Kinetic Parameters. Pharmacokinetic parameters involving the biliary and urinary excretion were calculated according to the following equations:

(1)

(2)

(3)

(4)

where C_pss, C_hss, C_kss, V_bile, and V_urine are the plasma concentrations of MX-68 (micromolar) at steady state (assessed as the plasma concentration 3 h after infusion), the concentration of MX-68 (micromolar) in the liver 3 h after infusion, the concentration of MX-68 (micromolar) in the kidney 3 h after infusion, the biliary excretion rate of MX-68 from 2.5 to 3 h (nanomoles per hour per kilogram), and the urinary excretion rate of MX-68 from 2 to 3 h (nanomoles per hour per kilogram), respectively; CL_{bile, p} and CL_{bile, h} (both in milliliters per hour per kilogram) are the biliary excretion clearance with regard to the plasma and hepatic concentrations of MX-68, respectively; and CL_{urine, p} and CL_{urine, k} (both in milliliters per hour per kilogram) are the urinary excretion clearance with regard to the plasma and renal concentrations of MX-68, respectively. To calculate C_hss and C_kss, the specific gravity of the liver was assumed to be unity. Thus, the amount of MX-68 in the liver (nanmoles per gram of liver) can be regarded as the tissue concentration (micromolar), and the units of CL_{bile, h} should be milliliters per hour per kilogram. CL_{bile, h, u} and CL_{urine, k, u} are the biliary and urinary excretion clearance with regard to the unbound concentration in the liver and kidney, respectively, and calculated as follows:

(5)

(6)

where f_h and f_k are the unbound fractions of MX-68 in the liver and kidney, respectively, and are determined as shown below.

(7)

(8)

(9)

(10)

Determination of Plasma Protein Binding. The plasma protein binding of MX-68 was determined by ultrafiltration. [¹⁴C]MX-68 dissolved in phosphate buffer (50 mM, pH 7.4) was diluted 10 times with rat plasma to give final concentrations of 0.66, 4.28, 22.3, and 148 µM. The mixture was incubated at 37°C for 30 min to ensure binding equilibrium. After incubation, 40 µl of aliquot was taken for the determination of total plasma concentration. Next, the plasma was placed in an ultrafiltration apparatus (Centrifree; Amicon, Inc., Beverly, MA) and centrifuged at 3000 rpm (RL-100; TOMY, Tokyo, Japan) for 10 min. After centrifugation, the concentration in the filtrate was determined as the unbound concentration. The plasma unbound fraction (f_p) was calculated by dividing the unbound concentration by the total plasma concentration. All of the binding was normalized with respect to the filter blank in an ultrafiltration apparatus that was assessed in the absence of plasma.

Determination of Tissue Homogenate Binding. Rat liver and kidney homogenates of 33.3% (w/v) were prepared using a Teflon homogenizer (Iuchi, Osaka, Japan) in PBS containing 100 µM NADPH (pH 7.4). This homogenate was then serially diluted with the same buffer to provide 16.6% and 8.3% homogenate. [¹⁴C]MX-68 was dissolved into 1 ml of the liver homogenates to give final MX-68 concentrations of 2.39, 6.33, 21.9, and 127 µM. These substrate concentrations were the same as those found in the liver in vivo at steady state. Similarly, [¹⁴C]MX-68 was dissolved into 1 ml of the kidney homogenates to give final MX-68 concentrations of 1.57, 3.96, 17.9, and 104 µM, which were the same as those found in the kidney in vivo at steady state. The mixture was then incubated for 10 min at 37°C. After incubation, an aliquot was taken, and the concentration was determined as the total concentration (C_t). Then, 500 µl of the mixture was placed in an ultrafiltration apparatus (Centrifree-0.5; Amicon Inc.) and centrifuged at 10,000g (RL-100; TOMY) for 10 min at 4°C. After centrifugation, the free concentration (C_f) in the filtrate was also determined. The bound concentration to the tissue (C_b) was calculated by subtracting C_f from C_t. After plotting C_b/C_f against the homogenate concentration, a straight line was obtained. The C_b/C_f ratio at 100% homogenate concentration was then extrapolated, and nonspecific adsorption was subtracted from the extrapolated value. The unbound fraction in the tissue (f_T) was then calculated according to the following equation:

(11)

where Y is C_b/C_f at 100% homogenate thus estimated.

Determination of MX-68 Binding to Liver Cytosol Fraction. The cytosol fraction was obtained after centrifugation of 33% liver homogenate at 100,000g for 60 min at 4°C. MX-68 was added to cytosol to give final MX-68 concentrations of 0.001, 0.01, 0.1, 0.3, 0.6, 4, and 85 µM. Incubation and centrifugation steps were the same as described above for homogenate samples. The MX-68 concentration bound to the cytosol (C_b) versus unbound MX-68 concentration (C_f) was fitted to the following equations:

(12)

where K_d is the equilibrium dissociation constant, and B_max is the binding capacity. The fitting was performed by the use of an iterative nonlinear least-squares method using MULTI (Yamaoka et al., 1981). The input data were weighted as the reciprocal of the observed values, and the damping Gauss-Newton method was used as the fitting algorithm.

Isolation of Brush Border Membrane Vesicles (BBMVs) from Rat Kidney Cortex and Uptake Study in BBMVs. Pairs of kidneys were removed from SDRs. BBMVs were prepared according to the methods reported by Kessler et al. (1978) with some modifications (Y. Han, Y.K., and Y.S., unpublished observations). The prepared BBMVs were suspended in transport buffer (50 mM NaCl, 1 mM MgSO₄, and 50 mM HEPES, pH 7.5) and stored at 80°C before being used for the uptake experiments. Protein concentrations were determined using a BioRad Protein assay kit with BSA as a standard (BioRad Laboratories, Richmond, CA). The uptake study was performed by a rapid filtration method as reported previously (Ishikawa et al., 1990). The transport reaction was started by rapidly mixing an aliquot (94-96 µl) of transport medium [50 mM NaCl, 1 mM MgSO₄, and 50 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.0] containing the substrate (MX-68) with the vesicle suspension (50 µg of protein in 4-6 µl). After the desired period of incubation at 25°C, the transport was stopped by the addition of 1 ml of ice-cold stop buffer containing 50 mM NaCl, 1 mM MgSO₄, and 50 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.0. The stopped reaction mixture was passed through a 0.45-µm HA filter (Millipore Corp., Bedford, MA) and then washed twice with 5 ml of stop buffer. Radioactivity retained on the filter and in the reaction mixture was determined after mixing with scintillation cocktail (Clear-sol I; Nacalai Tesque, Tokyo, Japan) in a liquid scintillation counter (LS 6000SE; Beckman Instruments). Uptake of substrates was normalized with respect to both the medium concentration of substrates and the amount of membrane protein. The kinetic parameters for MX-68 uptake by BBMV were calculated by fitting the data to the following equation:

(13)

where V₀ is the initial uptake rate of MX-68 (picomoles per minute per milligram of protein), S is the MX-68 concentration in the medium (micromolar), K_m is the Michaelis constant (micromolar), V_max is the maximum uptake rate (picomoles per minute per milligram of protein), and P_dif is the nonspecific uptake clearance (microliters per minute per milligram of protein). The fitting was performed by an iterative nonlinear least-squares method as described above.

Oral Absorption of MX-68. SDRs and EHBRs were fasted overnight. MX-68 was then administered by gastic intubation at a dose of 0.3 mg/kg dissolved in 1 ml of 0.9% NaCl solution, and the rats were placed in Bollman cages. Rats received food from 4 h after the administration of MX-68 and had free access to water throughout the experiments. Bile and urine were collected at 4, 8, 12, 24, and 48 h after administration. The radioactivity-counting procedure was the same as that used for the i.v. infusion study described above.

	Results

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Steady-State Excretion Profile of MX-68. To determine the hepatobiliary and urinary transport of MX-68 in SDRs and EHBRs, the plasma concentrations, biliary excretion, and urinary excretion of MX-68 were determined at various infusion rates ranging from 0.067 to 33.4 µmol/h/kg (Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Tissue-to-plsma concentration ratio of MX-68 at steady-state. The tissue-to-plasma concentration ratio () in the liver (A) and kidney (B) was determined by dividing the hepatic and renal concentration, respectively, by the plasma concentration (Cpss) at steady state. The tissue-to-plasma unbound concentration ratio () for each tissue was also calculated by dividing the hepatic and renal unbound concentration by the plasma unbound concentration in A and B, respectively. Each point and bar represents the mean ± S.E. of four rats. *, significantly different from the value at the lowest plasma concentration (p < .05)

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Time profile (A) and concentration dependence (B) of MX-68 uptake by renal BBMVs. A, uptake was initiated by mixing a small volume of membrane vesicle suspension equilibrated at pH 7.5 with pH 5.0 buffer containing MX-68 (0.5 µM). Data represent mean ± S.E. (n = 3). B, initial uptake rate of MX-68 at various concentrations was determined and described as an Eadie-Hofstee plot. The fitted line based on eq. 13 is also shown. Data represent mean ± S.E. (n = 3).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Oral absorption of MX-68 in SDRs (A) and EHBRs (B). After oral administration of MX-68, the cumulative amount of biliary () and urinary () excretion was followed. The sum of the excretion via both routes () was also calculated and shown as the straight curve. Each point and bar represents the mean ± S.E. of three rats.

Steady-State Distribution Profile of MX-68. The tissue-to-plasma concentration ratio and unbound concentration ratio at steady state were calculalated based on eqs. 7 to 10 and are shown in Table 1. Because the biliary excretion rate at the lowest infusion rate did not reach steady state (Table 1), kinetic parameters for the hepatic distribution (K_{p, liver} and K_{pu, liver}) at the lowest infusion rate were not calculated. The K_{p, liver} in SDRs decreased as C_pss increased (Table 1). The K_{p, liver} values in EHBRs also showed this C_pss-dependent decrease and were comparable with those in SDRs (data not shown). To exclude the involvement of intracellular binding in such hepatic distribution, the K_{pu, liver} was calculated based on eq. 8. The K_{pu, liver} also showed a steep slope around 1 to 10 µM C_pss (Fig. 2A). The K_{p, kidney} and K_{pu, kidney} in SDRs exhibited a similar C_pss-dependent decrease (Fig. 2B), and their absolute values were much higher than the K_{p, liver} and K_{pu, liver} values, respectively (Fig. 2).

Cytosol Binding of MX-68 In Vitro. To assess the binding isotherm of MX-68 in liver, the cytosolic binding was measured using liver cytosol. The concentration of MX-68 bound to the cytosol (C_b) and the concentration of unbound MX-68 (C_f) were 9.19 ± 0.04 and 0.808 ± 0.039 nM, 90.9 ± 0.3 and 9.15 ± 0.31 nM, 345 ± 5 and 155 ± 5 nM, 708 ± 28 and 292 ± 28 nM, 1.38 ± 0.04 and 0.618 ± 0.039 µM, 6.33 ± 0.28 and 3.67 ± 0.28 µM, and 115 ± 4 and 84.6 ± 3.9 µM, respectively. The binding profile showed two saturable components, and K_d1, B_max1, K_d2, and B_max2 were 0.0137, 0.151, 307, and 533 µM, respectively.

Concentration-Dependent Uptake of MX-68 by Renal BBMVs. The time course of MX-68 uptake by BBMVs at the inward H⁺ gradient is shown in Fig. 3A. Because the uptake of MX-68 was linear up to 3 min (Fig. 3A), the initial uptake velocity was assessed as the uptake for a 3-min incubation period at various concentrations (0.1-100 µM; Fig. 3B). The uptake of MX-68 by BBMVs was saturable (Fig. 3B). The kinetic parameters for MX-68 uptake were K_m = 0.236 ± 0.097 µM, V_max = 3.87 ± 0.87 pmol/min/mg protein, and P_dif = 0.705 ± 0.319 µl/min/mg protein.

Oral Absorption of MX-68. Oral absorption of MX-68 was studied in rats after bile duct ligation (Fig. 4). The amount of MX-68 excreted into bile in SDRs was 23% of the dose for 48 h after oral administration (Fig. 4A). In EHBRs, biliary excretion was markedly reduced and cumulative biliary excretion amounted to 2.2% of the dose for 48 h after oral administration, whereas 51.4% of the administered dose wasfound in urine up to 48 h (Fig. 1B). The total amount excreted into both bile and urine for 48 h was higher in EHBRs than SDRs (38.6% and 53.6% of the dose in SDRs and EHBRs, respectively; Fig. 4). In addition, urinary excretion in EHBRs was still taking place at 48 h (Fig. 4B), whereas in SDRs it seemed to be almost complete (Fig. 4A).

	Discussion

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The purpose of the present study was to clarify the excretion, distribution, and oral absorption kinetics of a novel antifolate drug, MX-68. The present experimental protocol was designed to identify any nonlinear pharmacokinetic processes by changing the MX-68 infusion rate. In SDRs over the entire C_pss range examined, CL_{bile, p} was at least twice as large as CL_{urine, p} (Table 2). Thus, biliary excretion is predominant compared with urinary excretion. A similar excretion profile was also found in a previous investigation of MTX by Bremnes et al. (1989). However, to discuss the contribution of each excretion route to the overall elimination of MX-68, we should also note the possibility of reentry into the central blood compartment via enterohepatic circulation. CL_{bile, p} decreased as C_pss increased (Table 2), indicating that a saturable transport process exists during the net biliary excretion from circulating plasma into bile. The K_{pu, liver} also decreased as the C_pss increased (Fig. 2A). Because the absolute value for CL_{bile, p} was much less than the hepatic plasma flow rate (~2 × 10³ ml/h/kg; Kato et al., 1999), this K_{pu, liver} value should represent the ratio of unbound MX-68 concentration in the liver to that in the extracellular space. Therefore, this saturation in K_{pu, liver} (Fig. 2A) suggests that the hepatic uptake process is mediated by saturable mechanism or mechanisms. This was confirmed by our separate analysis in isolated rat hepatocytes (Y. Han, Y.K., Y. Watanabe, K. Terao, Y. Asoh, and Y.S., unpublished data) that indicates the involvement of active transporters in the hepatic uptake of MX-68. Saturation of this transport system (K_m ~ 2 µM) may account for the saturation in K_{pu, liver} in vivo because saturation of K_{pu, liver} was clear around a C_pss value of 1 to 10 µM (Fig. 2A), corresponding to the unbound plasma concentration of 0.3 to 3 µM.

The absolute value of K_{pu, liver} was about unity (Fig. 2A), indicating that the concentrative uptake in vivo is actually not very effective. Therefore, the apparent concentrative distribution to the liver (K_{p, liver} > 1; Fig. 2A) should mainly result from intracellular binding. One possible reason for such a low K_{pu, liver} value is active efflux transport across the sinusoidal and/or bile canalicular membrane. The biliary excretion clearance with respect to the hepatic MX-68 concentration (CL_{bile, h}) was much lower in EHBRs than SDRs (Table 2), suggesting that biliary excretion across the bile canalicular membrane is mainly mediated by cMOAT.

Interestingly, the CL_{bile, h} increased as the C_hss increased (Table 2). To avoid the effect of intracellular binding, the CL_{bile, h, u} , which should represent the transport efficiency across the bile canalicular membrane, was also determined. Because such a C_hss-dependent increase was not found in CL_{bile, h, u} (Table 2), the nonlinearity in CL_{bile, h} (Table 2) mainly represents saturation of intrahepatocellular binding. It should also be noted that in the present study, there is no saturation of cMOAT because CL_{bile, h, u} did not decrease as the C_hss increased (Table 2). Thus, saturation in the net biliary excretion was mainly due to saturation in the uptake at the sinusoidal side.

The CL_{urine, p} also decreased as the C_pss increased (see Table 2 for this and the following values). Taking into account both the f_p (0.295), which was constant over the plasma concentration range (0.66-148 µM), and the glomerular filtration rate, determined in our preliminary study (~240 ml/h/kg), the urinary excretion clearance accounted for by glomerular filtration should be 71 ml/h/kg. This value was lower than the CL_{urine, p} (162 ml/h/kg) at the C_pss of 0.66 µM, indicating the saturable renal tubular secretion of MX-68. To examine the secretion and/or reabsorption of MX-68 at the brush border membrane, the CL_{urine, k} was also determined. Interestingly, the increase and subsequent decrease were found in CL_{urine, k} as the C_kss increased. To neglect the effect of intracellular binding, the CL_{urine, k, u} was also calculated and still increased as C_kss increased. Therefore, the increase in CL_{urine, k} cannot fully be explained by saturation of the intracellular binding. In addition, saturable uptake of MX-68 was confirmed by renal BBMVs (Fig. 3). These results suggest that MX-68 is reabsorbed at the brush border membrane. It should also be noted that a renal secretion process might also be available for MX-68 at the brush border membrane because the CL_{urine, k, u} fell as the infusion rate increased. However, to demonstrate such a renal secretion process, inhibition studies of the secretion using competing drugs must be performed. At the highest infusion rate (33.4 µmol/h/kg) in the present study, both systems may be saturated because the observed CL_{urine, p} could be almost completely accounted for by glomerular filtration. Hendel and Nyfors (1984) proposed the two conflicting renal tubular processes: reabsorption and secretion. Saturation of the reabsorption process resulted in an increase in the CL_{urine, p}, and saturation of the tubular secretion occurred at much higher plasma MTX levels resulted in the decrease in CL_{urine, p}. On the other hand, there was only a slight decrease in CL_{urine, p} of MX-68 as the C_pss increased. Thus, the overall renal excretion kinetics is simpler in the case of MX-68 compared with MTX.

During the continuous infusion of MX-68 at the lowest infusion rate (0.067 µmol/h/kg), its biliary excretion rate did not reach steady state (Table 1). Because the plasma MX-68 concentration seemed to reach a steady state even at the lowest infusion rate (Table 1), such a phenomenon may indicate the existence of a deep compartment for MX 68 in the liver from which the efflux of MX-68 should be slow. The deep compartment can be easily saturated at higher infusion rates because the biliary excretion rate at higher infusion rates (0.334-33.4 µmol/h/kg) reached steady state (Table 1). The binding isotherm in liver cytosol fraction indicated the existence of a high-affinity specific binding site. By comparing the K_d1 value for the high-affinity site (0.014 µM) with the steady-state hepatic unbound concentration (0.25-25 µM) found at various infusion rates except at the lowest one, it appears that such a high-affinity binding site should be almost completely occupied by MX-68 at these infusion rates (0.334-33.4 µmol/h/kg) because the hepatic unbound concentration was actually at least 18 times higher than the K_d1 value. Such a high-affinity site may act as a deep compartment in the liver, hindering the MX-68 concentration equilibrium between plasma and bile.

A substantial amount of MX-68 was excreted into bile and urine in SDRs after oral administration (Fig. 4). This result suggests that MX-68 can be absorbed from the gastrointestinal duct. Such oral absorption in EHBRs might be higher than that in SDRs because the total amount excreted was higher in EHBRs than in SDRs (Fig. 4), and the urinary excretion continued, increasing in EHBRs (Fig. 4B), although we should also note the possibility that the drug might be retained in the carcass. There are at least two possible explanations for this: one of them is up-regulation of the transport system for MX-68 absorption in EHBRs, whereas the other is a reduction or deficiency in the intestinal exsorption system in EHBRs. Gene expression of cMOAT in SDRs was detected in the duodenum as well as the liver, whereas its expression in the kidney is relatively small (Ito et al., 1997). In the present study, MX-68 excretion across the bile canalicular membrane in EHBRs was much lower than that in SDRs (Table 2), whereas its secretion across the renal brush border membrane was almost comparable in the two strains (Table 2). This may be reasonable if we consider the gene expression level of cMOAT in both organs. Thus, further studies should be performed to clarify whether cMOAT plays a role as a barrier system to reduce the absorption of organic anions in the intestine.