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OtherDRUG METABOLISM AND DISPOSITION

Temocaprilat, a Novel Angiotensin-Converting Enzyme Inhibitor, is Excreted in Bile via an ATP-dependent Active Transporter (cMOAT) That is Deficient in Eisai Hyperbilirubinemic Mutant Rats (EHBR)

Hitoshi Ishizuka, Kumiko Konno, Hideo Naganuma, Kunihiro Sasahara, Yukinori Kawahara, Kayoko Niinuma, Hiroshi Suzuki and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics March 1997, 280 (3) 1304-1311;

Abstract

Temocapril · HCl (α-{(2S,6R)-6-[(1S)-1-ethoxy-carbonyl-3-phenyl-propyl]amino-5-oxo-2-(2-thienyl)perhydro-1,4-thiazepin-4yl}acetic acid hydrochloride) is a novel prodrug of an angiotensin-converting enzyme (ACE) inhibitor. Unlike many other ACE inhibitors, its pharmacologically active metabolite,temocaprilat, is excreted predominantly in bile. To investigate the mechanism for the biliary excretion of temocaprilat, we performed in vivo and in vitro experiments using mutant Eisai hyperbilirubinemic rats EHBR) whose canalicular multispecific organic anion transporter (cMOAT) is hereditarily defective. Biliary clearance of temocaprilat after i.v. administration of [14C]temocapril · HCl (1.0 mg/kg) in EHBR was significantly lower than that in Sprague-Dawley rats (5.00 ml/min/kg for Sprague-Dawley rats vs. 0.25 ml/min/kg for EHBR). The uptake of temocaprilat into canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley rats was stimulated in the presence of ATP, whereas little stimulation was observed in CMVs from EHBR. The initial uptake rate of ATP-dependent transport of temocaprilat showed saturation kinetics; we obtained an apparentV max value of 1.14 nmol/min/mg protein and aKm value 92.5 μM. ATP-dependent transport of temocaprilat was competitively inhibited by 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT with an inhibition constant (Ki ) of 25.8 μM. The Km value for the uptake of 2,4-dinitrophenyl-S-glutathione into CMVs (Km = 29.6 μM) was consistent with thisKi value. In addition, the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione was inhibited by temocaprilat in a concentration-dependent manner. Active forms of some ACE inhibitors (benazepril, cilazapril, delapril, enalapril and imidapril) did not affect the transport of temocaprilat into CMVs even at concentrations as high as 200 μM. These data suggest that temocaprilat is effectively excreted in bile via cMOAT that is deficient in EHBR and that many of other ACE inhibitors have low affinity for cMOAT.

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.

Figure 1
Figure 1

Chemical structures of temocapril · HCl (α-{(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 (Brogdenet 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 primarilyvia 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 (Kitamuraet 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

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 glutathioneS-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 α-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 inN, 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 γ-GTPase) in CMVs compared with the liver homogenate was determined using p-nitrophenylphosphate,l-leucyl-p-diethylaminoanilide andl-γ-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).

The transport study was performed using the rapid filtration technique described in a previous report (Ishikawa et al., 1990). Transport medium (10 mM Tris, 250 mM sucrose, 10 mM MgCl2 · 6H2O, pH 7.4) containing radiolabeled compound (35 μl) with or without unlabeled substrate was preincubated at 37°C for 3 min and then was rapidly mixed with 5 μl CMVs suspension (20 μg protein) with or without 5 mM ATP and ATP-regenerating system (10 mM creatine phosphate and 100 μg/ml creatine phosphokinase). In some instances, ATP was replaced by AMP. The transport reaction was stopped by the addition of 1 ml of ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl, 10 mM Tris-HCl (pH 7.4). The stopped-reaction mixture was filtered through a 0.45-μm GVWP filter (Millipore Corp., Bedford, MA) and then was washed twice with 5 ml of the stop-solution. Radioactivity retained on the filter was determined using a liquid scintillation counter (LSC-3500, Aroka Co., Tokyo, Japan). In some studies, the compounds were dissolved in ethanol whose final concentration was less than 2%. In this case, a control experiment was also performed in the presence of ethanol. Uptake was normalized with respect to the amount of membrane protein.

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 (CLbile) was calculated by dividing the cumulative amount excreted in the bile over 6 hr [Xbile(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

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 α-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 CLbile 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.

Figure 2
Figure 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).

View this table:
Table 1

Pharmacokinetic parameters after i.v. administration of [14C]temocapril · HCl in SDR and EHBR

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 γ-GTPase in CMVs compared with liver homogenate; the enrichments of ALP, LAP and γ-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%; Böhmeet 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).

The time courses of the uptake of [14C]temocaprilat (30 μM) into CMVs are shown in figure 3. The uptake into CMVs prepared from SDR in the presence of ATP and ATP-generating system increased linearly up to 5 min, whereas without ATP, the uptake was minimal. Minimal stimulation by ATP was observed in the CMVs from EHBR, though this stimulation by ATP was statistically significant at 5 and 10 min (P < .05). Using CMVs from SDR, we measured the concentration dependence of the initial uptake rate of temocaprilat (fig. 4). The initial uptake rate was calculated 2 min after the reaction started. Although the initial uptake rate in the absence of ATP increased linearly with concentration, the rate in the presence of ATP showed saturation. The Eadie-Hoffstee plot for the ATP-dependent uptake of temocaprilat (fig. 5) provided aV max value of 1.14 ± 0.18 nmol/min/mg protein and a Km value of 92.5 ± 0.2 μM (mean ± computer-calculated S.D.).

Figure 3
Figure 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.

Figure 4
Figure 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).

Figure 5
Figure 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).

We then investigated the effect of DNP-SG on temocaprilat uptake. Although the ATP-dependent [14C]temocaprilat uptake (30 μM) was reduced in a concentration-dependent manner by unlabeled DNP-SG (10–100 μM), the uptake remained at approximately 60% of the control value even if the concentration of DNP-SG increased up to 1000 μM (fig. 6); the excess concentration of DNP-SG did not reduce the uptake of temocaprilat to that observed in the absence of ATP (approximately 35% of the control value). The Eadie-Hoffstee plot of the uptake of temocaprilat in the presence of DNP-SG (25 μM) revealed competitive inhibition (fig. 5), and theKi value for DNP-SG was calculated to be 25.8 μM.

Figure 6
Figure 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).

Because DNP-SG competitively inhibited the uptake of temocaprilat, we investigated the effect of temocaprilat on the uptake of [3H]DNP-SG. Although temocaprilat (100 μM) competitively inhibited the uptake of [3H]DNP-SG (fig.7), 200 μM temocaprilat, a concentration approximately 2 times as high as the Km value (92.5 μM) for its own transport (fig. 5), inhibited the [3H]DNP-SG transport by only 35% (fig. 8). The parameters for the uptake of DNP-SG were calculated to give a V maxvalue of 1.90 nmol/min/mg protein and a Km value of 29.6 μM (fig. 7); this Km value was comparable with the inhibition constant for temocaprilat (Ki = 25.8 μM, fig. 5). On the other hand, theKm value (92.5 μM) for temocaprilat shown in figure 5 was 3.7 times less than the inhibition constant for DNP-SG calculated from figure 7 (Ki = 343 μM). These data suggest that temocaprilat and DNP-SG share, in part, a common transporter.

Figure 7
Figure 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).

Figure 8
Figure 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).

In these kinetic studies, we examined the uptake of ligands in medium free of ATP in order to determine the ATP-independent uptake. In addition, we performed the transport experiments in medium in which AMP were present and found that the effect of AMP was minimal; the uptakes of [3H]DNP-SG and [14C]temocaprilat at 2 min in the presence of AMP were 3.2 ± 0.2 and 6.4 ± 0.3 μl/mg protein (n = 3), respectively, values not significantly different from those obtained in the absence of AMP, 3.1 ± 0.3 and 6.3 ± 0.1 μl/mg protein (n= 3), respectively). Therefore, the kinetic parameters described previously would be affected minimally by taking these blank values into account.

The active forms of other ACE inhibitors, such as benazeprilat, cilazaprilat, delaprilat, enalaprilat and imidaprilat, had no effect on the transport of temocaprilat into CMVs (fig. 9) at 50 and 200 μM, which are close to the Km value of temocaprilat. These data suggest that temocaprilat has a higher affinity for organic anion transporter than do other active metabolites of ACE inhibitors and that it has a unique biliary excretion mechanism distinct from the excretion mechanisms of other ACE inhibitors.

Figure 9
Figure 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

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 (Suzukiet 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 (Sathirakulet 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 bilevia cMOAT. The transport parameter of temocaprilat (Km = 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; Nishidaet al., 1992a,b; Oude Elferink and Jansen, 1994). Moreover, ATP-dependent temocaprilat transport was competitively inhibited by DNP-SG (fig. 5), and its Ki value (25.8 μM) was comparable with the Km 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 Ki value of temocaprilat for DNP-SG uptake is 3.7 times higher than its own Km 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; Suzukiet 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; Mülleret al., 1994; Jedlitschky et al., 1994, 1996; Loeet al., 1996a,b). Paulusma et al. (1996) andBüchler 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.

Footnotes

  • Send reprint requests to: Hitoshi Ishizuka, Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan.

  • Abbreviations:
    cMOAT
    canalicular multispecific organic anion transporter
    CMVs
    canalicular membrane vesicles
    SDR
    Sprague-Dawley rats
    EHBR
    Eisai hyperbilirubinemic rats
    DNP-SG
    2,4-dinitrophenyl-S-glutathione
    ACE
    angiotensin-converting enzyme
    ALP
    alkaline phosphatase
    LAP
    leucine aminopeptidase
    γ-GTPase
    γ-glutamyl transpeptidase
    Cmax
    maximum concentration
    AUC
    area under the curve
    Tmax
    time to maximum concentration
    CLbile
    biliary clearance
    DBSP
    dibromosulfophthalein
    ICG
    indocyanine green
    LG
    liquiritigenin
    E3040
    6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole
    hMRP
    human multidrug resistance-associated protein
    ABC
    ATP-binding cassette
    PCR
    polymerase chain reaction
    • Received July 9, 1996.
    • Accepted November 8, 1996.

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Temocaprilat, a Novel Angiotensin-Converting Enzyme Inhibitor, is Excreted in Bile via an ATP-dependent Active Transporter (cMOAT) That is Deficient in Eisai Hyperbilirubinemic Mutant Rats (EHBR)

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