Introduction

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Temocapril · HCl (fig. 1), is a prodrug type of ACE inhibitor that is rapidly hydrolyzed at its 2-ethyl ester group to be converted into the pharmacologically active diacid metabolite temocaprilat, which is a potent inhibitor of ACE (Oizumi et al., 1988; Sada et al., 1989a, b; 1990). Because the in vivo ACE activity, estimated by the plasma angiotensin II/angiotensin I ratio, is well correlated with the plasma concentration of temocaprilat and with systolic and diastolic blood pressure responses to angiotensin I (Delacretaz et al., 1994), it is important to clarify the pharmacokinetics of temocapril precisely from a pharmacological standpoint.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemical structures of temocapril · HCl (-{(2S,6R)-6-[(1S)-1-ethoxy-carbonyl-3-phenyl-propyl]amino-5-oxo-2-(2-thienyl)perhydro-1,4-thiazepin-4-yl}acetic acid hydrochloride) and its diacid, temocaprilat.

Many active forms of ACE inhibitors, such as captopril (Brogden et al., 1988), enalapril (Todd and Goa, 1992), cilazapril (Deget and Brogden, 1991), ramipril (Frampton and Peters, 1995) and spirapril (Noble and Sorkin, 1995), are excreted predominantly in the urine, whereas 85% to 90% of administered doses of temocapril is excreted in the feces in animal studies (Ikeda et al., 1990; Higuchi et al., 1990). In humans, 36% to 44% of the dose is excreted in the feces and 17% to 24% is excreted in the urine 48 hr after administration (Suzuki et al., 1993). These data indicate that, unlike many other ACE inhibitors, temocapril is excreted predominantly in the feces in both humans and other animals.

Especially in the treatment of patients with renal failure, the presence of an excretion route other than urinary excretion gives ACE inhibitors a pharmacokinetic and pharmacodynamic advantage. In patients with renal failure, the AUCs of captopril and enalapril are markedly increased, so it is necessary to reduce the dosage and/or to change the dosage interval, because these ACE inhibitors are eliminated primarily via renal excretion (Lowenthal et al., 1985; Sica, 1992). Although the duration of plasma ACE inhibition after administration of enalapril to patients with renal failure is markedly prolonged, the duration of ACE inhibition by temocapril in these patients is affected minimally (Oguchi et al., 1993). This observation is related to the pharmacokinetic nature of these drugs; for enalapril, the C_max value and the AUC for the active metabolite increased 6 and 13 times, respectively, in severe renal insufficiency, whereas the C_max value for temocaprilat was only slightly altered and the AUC only doubled in the same patients (Oguchi et al., 1993). Fosinopril, which is excreted in both bile and urine, has pharmacokinetic properties similar to those of temocapril in patients with renal dysfunction (Murdoch and McTavish, 1992). Thus, unlike the case for other ACE inhibitors, it may not be necessary to adjust the dosage of temocapril and fosinopril in patients according to their degree of insufficiency. No study, however, has been performed yet to examine why these two drugs are excreted predominantly into the bile.

Recently, it has been shown that some conjugated endogenous compounds, such as bilirubin glucuronides and cysteinyl leukotrienes (Frenandez-Checa et al., 1992; Ishikawa et al., 1990; Nishida et al., 1992a), and some exogenous organic anions, such as BSP and conjugated metabolites of E3040 (Kitamura et al., 1990; Takenaka et al., 1995a), are excreted into bile via an ATP-dependent primary active transporter located on the bile canalicular membrane. It has been established that the biliary excretion of some organic anions is impaired in mutant rats (TR rats or EHBR) whose cMOAT is hereditarily defective (Huber et al., 1987; Jansen et al., 1985, 1987a,b; Sathirakul et al., 1993, 1994; Shimamura et al., 1994; Takenaka et al., 1995b). Because temocaprilat has carboxy groups within its chemical structure, it is possible that this compound is the substrate for cMOAT.

In this study, we investigated the mechanism for the biliary excretion of temocaprilat across the canalicular membrane in SDR and EHBR. The results provide evidence that temocaprilat is transported by cMOAT that is defective in EHBR and that its transport is competitively inhibited by DNP-SG, a typical substrate for cMOAT.

	Materials and Methods

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Materials. [¹⁴C]Temocapril · HCl (specific activity 18 mCi/mmol) was synthesized by Daiichi Pure Chemicals Co. Ltd. (Tokyo, Japan). [¹⁴C]Temocaprilat was prepared by hydrolysis of [¹⁴C]temocapril · HCl with rat plasma followed by purification with high-performance liquid chromatography. The radiochemical purity of both compounds was more than 95%, as confirmed by thin-layer chromatography on silica gel (n-butanol:acetic acid:distilled water = 4:1:1). Unlabeled temocaprilat was synthesized in our laboratory (Yanagisawa et al., 1987). The active forms of benazapril, cilazapril, delapril, enalapril and imidapril (benazeprilat, cilazprilat, delaprilat, enalaprilat and imidaprilat) were synthesized by the Institute of Science and Technology Inc. (Tokyo, Japan). [³H]S-(2,4-dinitrophenyl)-glutathione ([³H]DNP-SG) was synthesized enzymatically using [glycine-2-³H]glutathione (DuPont New England Nuclear Corp., Boston, MA), 1-chloro-2,4-dinitrobenzene and glutathione S-transferase according to the method described previously (Kobayashi et al., 1990). Unlabeled DNP-SG was synthesized chemically by a procedure based on a method previously reported (Saxena and Henderson, 1995), and the purity was checked by high-performance liquid chromatography (more than 99%). [³H]Taurocholate was purchased from DuPont New England Nuclear. ATP, creatine phosphate and creatine phosphokinase were purchased from Sigma Chemical Co. (St Louis, MO). Male Sprague-Dawley rats and EHBR (7 weeks old) were purchased from SLC Co., Ltd. (Shizuoka, Japan). All other chemicals used were commercially available and reagent grade products.

In vivo rat study. The animals (both SDR and EHBR) were anesthetized with i.p. administered urethane (1 mg/kg) and -chlorarose (25 mg/kg), and the common bile duct was cannulated with polyethylene tubing (PE-10) to collect bile specimens. [¹⁴C]Temocapril · HCl (1 mg/kg), dissolved in N, N-dimethylacetamide and PEG400 (5:95 v/v), was injected i.v. via the femoral vein. At each time-point, blood specimens (200 µl) were collected from the jugular vein using a heparinized syringe, and the plasma was immediately separated by centrifugation. Bile specimens were collected into preweighed Eppendorf microfuge tubes at the specified intervals. The total radioactivities of plasma and bile samples were measured by scintillation spectrophotometer (LSC-3500, Aroka Co., Tokyo, Japan). The radioactivity of both temocapril and temocaprilat extracted from plasma and bile samples by ethanol was measured by silica-gel thin-layer chromatography (n-butanol: acetic acid: distilled water = 4:1:1) followed by analysis in a Bio-Image Analyzer (BAS-2000, Fuji Photo Film Co., Ltd. Tokyo, Japan). Animal experiments were carried out according to the guidelines provided by the Institutional Animal Care and Use Committee of Sankyo Co., Ltd. (Tokyo, Japan).

In vitro transport experiment using bile canalicular membrane vesicles. CMVs, prepared from both male SDR and EHBR according to the method previously reported (Kobayashi et al., 1990), were suspended in 50 mM Tris buffer (pH 7.4) containing 250 mM sucrose. Enrichment of marker enzymes (ALP, LAP and -GTPase) in CMVs compared with the liver homogenate was determined using p-nitrophenylphosphate, L-leucyl-p-diethylaminoanilide and L--glutamyl-p-N-ethyl-N-hydroxylethylaminoanilide as substrates, respectively. In addition, the orientation of the CMVs was determined by examining the nucleotide pyrophosphatase in the absence and presence of 0.2% of Triton X-100 (Böhme et al., 1994).

Data analysis. All data are represented as mean ± S.E.. The AUC up to 6 hr after administration [AUC(0-6)] for individual animals was determined by the trapezoidal method. Biliary clearance (CL_bile) was calculated by dividing the cumulative amount excreted in the bile over 6 hr [X_bile(0-6)] by AUC(0-6). Student's t test was used to determine the significance of differences. Uptake rates were fitted to the Michaelis-Menten equation using the nonlinear least-squares program PCNONLIN (version 4.2, Statistical Consultants Inc., KY) to calculate the kinetic parameters.

	Results

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In vivo rat study. The plasma concentrations (panel A) and cumulative biliary excretion (panel B) of temocaprilat after i.v. administration of temocapril · HCl in both SDR and EHBR are shown in figure 2. The bile flow rate (60 ± 6 µl/min/kg b.w.t.; n = 4) was not affected by the addition of -chloralose compared with our previous experiments in which only urethane was used (66 ± 4 µl/min/kg b.wt.; n = 4, Sathirakul et al., 1994). No other radioactivity except that of temocaprilat (more than 95% of total radioactivity) was observed in either plasma or bile on thin-layer chromatography. EHBR had higher plasma levels of temocaprilat than SDR, and the AUC(0-6) calculated from these profiles was 6 times higher in EHBR than in SDR (table 1). The cumulative biliary excretion of radioactivity in SDR was 78% of the dose by 6 hr, whereas in EHBR it was significantly less (25%). The CL_bile value in EHBR was 1/20 that in SDR. These data suggest that cMOAT that is defective in EHBR plays a role in the biliary excretion of temocaprilat.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Plasma concentration (panel A) and cumulative biliary excretion (panel B) of radioactivity after i.v. bolus administration of [14C]temocapril · HCl in SDR and EHBR. SDR () and EHBR () received the injection of [14C]temocapril · HCl, dissolved in N, N-dimethylacetamide: PEG400 (5:95 v/v), via the femoral vein at a dose of 1.0 mg/kg. Each point represents the mean ± S.E. (N = 3-4).

In vitro transport experiment using bile canalicular membrane vesicles. Because significant differences between SDR and EHBR were observed in the plasma concentrations and biliary excretion of temocaprilat in vivo, we investigated the uptake of temocaprilat into CMVs prepared from both SDR and EHBR. No significant difference was observed between SDR and EHBR in the enrichment of ALP, LAP and -GTPase in CMVs compared with liver homogenate; the enrichments of ALP, LAP and -GTPase were 29 ± 8 (n = 4), 25 ± 2 (n = 4) and 34 ± 6 (n = 4) for SDR and were 32 ± 5 (n = 3), 28 ± 4 (n = 3) and 31 ± 8 (n = 3) for EHBR, respectively. In addition, 35% and 32% of CMVs were composed of inside-out membrane vesicles for SDR and EHBR, respectively, results comparable to those reported for CMVs prepared from male Wistar rats (32%; Böhme et al., 1994). As reported previously (Takenaka et al., 1995a), the CMVs from SDR used in the present study exhibited the ATP-dependent transport for DNP-SG, whereas the CMVs from EHBR had approximately 1/100 the activity for DNP-SG. Both CMVs had comparable activity for taurocholate transport (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Time profiles for the uptake of [14C]temocaprilat into CMVs prepared from SDR (panel A) and EHBR (panel B). CMVs (20 µg protein) were incubated in the medium containing [14C]temocaprilat (30 µM) with () or without () 5 mM ATP and ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Each point represents the mean ± S.E. (N = 3). *P < .05 compared with no ATP addition.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of the uptake of [14C]temocaprilat into CMVs. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [14C]-labeled and unlabeled temocaprilat with () or without () 5 mM ATP and ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Each point represents the mean ± S.E. (N = 3).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Eadie-Hoffstee plot for the uptake of [14C]temocaprilat into CMVs in the absence and presence of DNP-SG. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [14C]temocaprilat (30 µM), ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) and 5 mM ATP with () or without () 25 µM of DNP-SG. The data for the saturable uptake of temocaprilat in the absence of DNP-SG were taken from figure 4. Each point represents the mean ± S.E. (N = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effect of DNP-SG on the uptake of [14C]temocaprilat into CMVs. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [14C]temocaprilat (30 µM), ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) and 5 mM ATP with various concentrations of DNP-SG. The bar marked ATP represents the [14C]temocaprilat uptake in the absence of ATP and DNP-SG. Each point represents the percentage of the control value (mean ± S.E.; N = 3-4).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Eadie-Hoffstee plot for the uptake of [3H]DNP-SG into CMVs in the absence and presence of temocaprilat. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [3H]DNP-SG (1 µM), ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) and 5 mM ATP with () or without () 100 µM of temocaprilat. Each point represents the mean ± S.E. (N = 3-4), except  (the raw data).

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Concentration-dependent effect of temocaprilat on the uptake of [3H]DNP-SG into CMVs. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [3H]DNP-SG (1 µM), ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) and 5 mM ATP with various concentrations of temocaprilat. The bar marked ATP represents the [3H]DNP-SG uptake in the absence of ATP and temocaprilat. Each point represents the percentage of the control value (mean ± S.E.; N = 3-4).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Effect of ACE inhibitors on the uptake of [14C]temocaprilat into CMVs. CMVs (20 µg protein) from SDR were incubated for 2 min in the medium containing [14C]temocaprilat (30 µM), ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) and 5 mM ATP with two concentrations (50 and 200 µM) of unlabeled ACE inhibitors. Each point represents the percentage of the control value (mean ± S.E.; N = 3).

	Discussion

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Although ACE inhibitors are prescribed for hypertensive patients under various circumstances, many of them are excreted predominantly in the urine. Only a few ACE inhibitors, such as temocapril (Suzuki et al., 1993) and fosinopril (Murdoch and McTavish, 1992), are excreted predominantly in the bile, and the reason why these compounds are excreted mainly in the bile is unknown. This report provides evidence that a cMOAT that is defective in EHBR plays a major role in the biliary excretion of temocaprilat. This is a unique characteristic of temocaprilat, because the active metabolites of many other ACE inhibitors have little effect on this transport system.

In the present study, we injected temocapril · HCl into SDR and EHBR and investigated the pharmacokinetics of its active form, temocaprilat, particularly focusing on its biliary excretion. Analysis by thin-layer chromatography indicated that most of the radioactivity in plasma and bile was associated with temocaprilat both in SDR and EHBR. This finding is reasonable, considering that temocapril is easily converted into temocaprilat in rat plasma and that temocapril has no other major metabolite except temocaprilat (unpublished observation). Biliary excretion of temocaprilat was impaired in EHBR, which suggests a contribution of cMOAT that is defective in EHBR.

As reported previously, the biliary excretion of DBSP, cefodizime and ICG after i.v. bolus administration is reduced in EHBR (Sathirakul et al., 1993). By analyzing the biliary excretion of these ligands with a pharmacokinetic model that incorporates the drug uptake from blood to liver, the distribution in hepatocytes and the biliary excretion, we have revealed that the transport rate via the bile canalicular membrane was severely impaired for DBSP and cefodizime in EHBR, whereas the reduced intracellular transport rate of ICG contributed more than the reduction in the canalicular membrane transport to the impaired excretion of ICG in EHBR. These results suggest that the reduced biliary excretion of temocaprilat in EHBR does not necessarily represent a contribution from the specific transport system for temocaprilat. To confirm this contribution, we prepared CMVs from both SDR and EHBR and investigated the transport mechanism of temocaprilat into CMVs.

That the uptake of temocaprilat into CMVs was stimulated with ATP only in CMVs from SDR and that it was minimally stimulated in EHBR (fig. 4) clearly suggest that temocaprilat is transported into bile via cMOAT. The transport parameter of temocaprilat (K_m = 92.5 µM) was in the same range as those of other substrates transported by this system (BSP, 31 µM; DNP-SG, 71 µM; p-nitrophenyl-glucuronide, 20 µM; bilirubin glucuronide, 71 µM) (Akerboom et al., 1991; Nishida et al., 1992a,b; Oude Elferink and Jansen, 1994). Moreover, ATP-dependent temocaprilat transport was competitively inhibited by DNP-SG (fig. 5), and its K_i value (25.8 µM) was comparable with the K_m value for DNP-SG (29.6 µM, fig. 7); DNP-SG also competitively inhibited the transport of temocaprilat (fig. 7). These results suggest that temocaprilat is predominantly transported bile by the same transporter as DNP-SG.

Among the ACE inhibitors examined in the present study, only temocaprilat was a good substrate for cMOAT. Although we do not have a good explanation for this result, the conformation of this ligand may be related to the recognition by cMOAT, since X-ray analysis revealed that the conformation of the major functional group on the thiazepinone ring in temocapril is restricted permitting higher affinity for enzyme compared with other ACE inhibitors (Yanagisawa et al., 1987).

Analysis of the transport mechanisms for the conjugated metabolites of some xenobiotics suggests the existence of a multiplicity of organic anion transporters, some of them being maintained even in EHBR (Sathirakul et al., 1994; Shimamura et al., 1994; Takenaka et al., 1995b; Yamazaki et al., 1996). The biliary excretion of conjugated metabolites (containing glucuronide moiety) of LG is reduced in EHBR (both LG-mono-glucuronide and LG-glucuronide-sulfate), and the coadministration of glycyrrhizin and DBSP significantly decreased their excretion but had no effect on the excretion of the disulfate metabolite (Shimamura et al., 1994). Another study involving the biliary excretion of E3040 metabolites also confirmed different biliary excretion mechanisms for glucuronide and sulfate metabolites (Takenaka et al., 1995b). Temocaprilat was concluded to be transported predominantly by the cMOAT that is deficient in EHBR, because the ATP-dependent uptake of temocaprilat into CMVs prepared from EHBR was minimal. Other mechanisms may exist for temocaprilat transport, however, because 1) temocaprilat is transported into bile to some extent in EHBR (fig. 2), 2) slight but significant transport stimulation with ATP is also observed in EHBR (fig. 3), 3) the transport of temocaprilat is not completely inhibited by excess concentrations of DNP-SG (fig. 8) and 4) the K_i value of temocaprilat for DNP-SG uptake is 3.7 times higher than its own K_m value (fig. 5; fig. 7).

We have to consider the uptake of temocaprilat into hepatocytes from plasma across the basolateral membrane to account for the efficient biliary excretion of temocaprilat. In our preliminary experiments with isolated hepatocytes, we found that the uptake of temocaprilat is mediated predominantly by an active transport system shared with pravastatin (unpublished observation). This specific uptake system, together with the efflux system revealed in the present study, may be responsible for the efficient excretion of temocaprilat in bile. The reduced biliary excretion of temocaprilat in EHBR (fig. 2; Table 1), however, may be accounted for largely by defective transport across the bile canalicular membrane, although we cannot refute the hypothesis that the previously described uptake system is inhibited by endogenous compound(s) in EHBR plasma. In addition, the biliary excretion of temocaprilat in humans is less extensive than that in rats; Suzuki et al. (1993) showed that 36% to 44% and 17% to 24% of the dose is excreted in feces and in urine, respectively, after p.o. administration of temocapril. This species difference might be accounted for, at least in part, by a difference in the transport process across the bile canalicular membrane, because our preliminary experiments indicated that the uptake of temocaprilat into human CMVs was significantly less than that observed in rat CMVs.

Recently, the molecular features of rat cMOAT have been clarified (Mayer et al., 1995; Paulusma et al., 1996; Büchler et al., 1996, Ito et al., 1996, in press) on the basis of the similarity in substrate specificity between cMOAT and hMRP (Leier et al., 1994, 1996; Müller et al., 1994; Jedlitschky et al., 1994, 1996; Loe et al., 1996a,b). Paulusma et al. (1996) and Büchler et al. (1996) succeeded in cloning the cDNA of cMOAT from Wistar rats, which is composed of 4623 bp, and found defective expression of this protein in TR rats and EHBR, respectively. In addition, Paulusma et al. (1996) showed that a 1-bp deletion at amino acid 393 resulted in the introduction of the stop codon at amino acid 401 in TR rats. Most recently, we cloned cMOAT from SDR liver and found that one base-pair replacement (G  A) at amino acid 588 resulted in introduction of the premature stop codon (Ito et al., in press). Because EHBR and TR are allelic mutants (Kitamura et al., 1992) and because both strains exhibit an autosomal recessive inheritance in the biliary excretion of organic anions (Jansen et al., 1985; Mikami et al., 1986; Hosokawa et al., 1992), we concluded that the impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant animals (Ito et al., in press).

In conclusion, we have confirmed the contribution of cMOAT to the biliary excretion of temocaprilat. The other ACE inhibitors examined in the present study had a low affinity for cMOAT.