QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.013607v1 35/9/1518    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoon, Y.-J. Articles by Shin, J.-G. Search for Related Content PubMed PubMed Citation Articles by Yoon, Y.-J. Articles by Shin, J.-G.

Characterization of Benidipine and Its Enantiomers' Metabolism by Human Liver Cytochrome P450 Enzymes

Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (Y.-J.Y., K.-B.K., H.K., K.-A.S., H.-S.K., I.-J.C., K.-H.L., J.-G.S.); Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan, Korea (E.-Y.K., J.-G.S.); and Frontier Inje Research for Science and Technology, Inje University, Busan, Korea (K.-H.L.)

(Received October 29, 2006; accepted May 24, 2007)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)- and (+)- isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL_int) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)- isomer was similar to those from the (+)- isomer (1.9 ± 0.1 versus 2.3 ± 2.3 µl/min/pmol P450 and 0.5 ± 0.2 versus 0.6 ± 0.6 µl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL_int values of CYP3A4-mediated metabolite formation from (-)- isomer were similar to those from (+)- isomer (17.7 versus 14.4 µl/min/pmol P450, respectively). The total CL_int values of CYP3A5-mediated metabolite formation from (-)- isomer were also similar to those from (+)- isomer (8.3 versus 11.0 µl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

Pharmacokinetic studies in rats (Kobayashi and Kobayashi, 1998) and humans (Kobayashi et al., 1997) have shown that benidipine is well absorbed but exhibits low absolute bioavailability as a result of marked first-pass metabolism. Accordingly, various metabolites were identified in these species (Kobayashi et al., 1997; Kobayashi and Kobayashi, 1998). In our previous study involving human plasma, we found that the plasma concentrations of (+)--benidipine were consistently higher than those of the (-)- isomer after dosing of 8 mg of racemic benidipine (Kang et al., 2005). The C_max and area under the curve to infinity values of (+)--benidipine (1.47 ± 0.75 ng/ml and 2.48 ± 1.18 ng · h/ml, respectively) were higher than those of (-)--benidipine (0.75 ± 0.46 ng/ml and 1.34 ± 0.48 ng · h/ml, respectively) by 1.96- and 1.85-fold, respectively (p < 0.001). However, the mechanism of stereoselective disposition of benidipine in vivo has not yet been elucidated.

In the present study, we examined the metabolic pathways of benidipine using human liver microsomal fractions and in particular identified the cytochrome P450 (P450) isoforms responsible for each metabolic pathway. Furthermore, to understand the mechanism of enantioselective pharmacokinetics of benidipine in vivo, we also investigated the stereoselectivity on benidipine metabolism in vitro.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Metabolic pathway of benidipine by HLMs.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

Identification of Benidipine Metabolites in HLMs. The incubation mixtures, containing 0.25 mg of pooled HLMs (H161, Gentest) and benidipine (100 µM), were reconstituted in 100 mM phosphate buffer (pH 7.4) and prewarmed for 5 min at 37°C. The reaction was initiated by adding the NADPH-regenerating system (1.3 mM ß-nicotinamide adenine dinucleotide phosphate, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, and 1.0 U/ml glucose-6-phosphate dehydrogenase) and further incubated (final volume of 250 µl) for 30 min at 37°C in a shaking water bath. The control incubations were conducted with heat-denatured microsomal preparations (80°C for 10 min). The reaction was terminated by the addition of 100 µl of acetonitrile on ice. The incubation mixtures were then centrifuged at 10,000g for 5 min at 4°C. Aliquots of the supernatant were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the identification of the metabolites.

Metabolism of Benidipine and Its Enantiomers in HLMs or cDNA-Expressed P450 Isoforms. The optimal conditions for microsomal incubation were determined in the linear range for the formation of metabolites of benidipine. In all the experiments, racemic benidipine and its enantiomers were dissolved and serially diluted with methanol to the required concentrations, and the final concentration of organic solvent did not exceed 1% in the final incubation mixtures. The incubation mixtures containing either 5 µl of microsomes (5 mg of protein/ml of stock, prepared from four different human liver microsomal preparations) or 25 µl of cDNA-expressed P450 (diluted to 20 pmol/ml with 100 mM phosphate buffer, pH 7.4) and various concentrations of benidipine or its enantiomers (0-200 µM) were reconstituted in 100 mM phosphate buffer (pH 7.4) and prewarmed for 5 min at 37°C. The reaction was initiated by adding the NADPH-regenerating system and further incubated (final volume of 250 µl) for 5 min at 37°C in a shaking water bath. The reaction was terminated by placing the incubation tubes on ice and by immediately adding 100 µl of acetonitrile. After adding the internal standard (amlodipine, 10 µM), the mixture was centrifuged at 10,000g for 5 min at 4°C, and aliquots of the supernatant were injected into an LC/MS/MS system.

LC/MS/MS Analysis of Benidipine and Its Metabolites. For the identification of benidipine and its metabolites, a tandem quadrupole mass spectrometer (QTrap 4000 LC/MS/MS, Applied Biosystems, Foster City, CA), coupled with an Agilent 1100 series high-performance liquid chromatography system (Agilent, Wilmington, DE), was used. The separation was performed on an XTerraMSC₁₈ column (2 mm i.d. x 30 mm, 2.5 µm, Waters, Milford, MA) using the mobile phase that consisted of acetonitrile and water (40:60, v/v) at a flow rate of 0.2 ml/min. For identification of the metabolites, mass spectra were recorded by electrospray ionization with a positive mode. The turbo ion spray interface was operated at 5500 V and 500°C. The operating conditions were optimized by flow injection of an analyte and were determined as follows: nebulizing gas flow, 40 psi; curtain gas flow, 10 psi; and collision energy, 50 eV. Quadruples Q1 and Q3 were set on unit resolution.

Multiple-reaction monitoring mode using specific precursor/product ion transition was used for the quantification. Detection of the ions was performed by monitoring the transitions of m/z 416315 for N-desbenzylbenidipine (collision energy 24 eV), 50491.2 for dehydrobenidipine (collision energy 95 eV), and 409238 for amlodipine (IS, collision energy 15 eV). Peak areas for all the components were automatically integrated using Analyst software (version 1.4, Applied Biosystems). The lower limits of quantification for the two metabolites were 0.2 nM. The interassay precision for the analyte was less than 15%.

Chemical Inhibition Studies with HLMs. The inhibitory effects of known P450 isoform-selective inhibitors on the formation of N-desbenzylbenidipine and dehydrobenidipine were evaluated to determine the P450 isoform(s) involved in the metabolic pathway. The formation ratio of N-desbenzylbenidipine and dehydrobenidipine from benidipine racemate or enantiomers was determined from the reaction mixtures incubated in the presence or absence of known P450 isoform-selective inhibitors (Kim et al., 2006; Lee et al., 2006). The P450 isoform-selective inhibitors used were furafylline (10 µM) for CYP1A2, coumarin (100 µM) for CYP2A6, triethylenethiophosphoramide (5 µM) for CYP2B6, montelukast (0.1 µM) for CYP2C8, sulfaphenazole (10 µM) for CYP2C9, S-benzylnirvanol (1 µM) for CYP2C19, quinidine (10 µM) for CYP2D6, diethyldithiocarbamate (10 µM) for CYP2E1, ketoconazole (1 µM) for CYP3A, and azamulin (5 µM) for CYP3A. SKF 525-A (100 µM) was also used as nonselective P450 inhibitor. FMO was heat-deactivated by incubating tubes (0.1 mg of microsomal protein and phosphate buffer) without NADPH-generating system in a water bath set at 45°C for 5 min to elucidate whether FMO is responsible for benidipine metabolism. Except for the addition of P450 isoform-selective inhibitors, all the other incubation conditions were similar to those described previously (Shin et al., 1999, 2002).

Correlation Experiments. Benidipine (5 µM) was incubated with microsomes from 15 different livers to test the correlation of benidipine metabolism with the activity of individual P450s. The activities of each P450 isoform were determined using mixture incubation and tandem mass spectrometry, as described previously (Kim et al., 2005). Isoform-specific reaction markers were used to determine the activity of each P450: phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), paclitaxel 6-hydroxylation (CYP2C8), tolbutamide 4-methylhy-droxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), dextro-methorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation (CYP3A). The correlation coefficients between the formation rates of benidipine metabolites and the activity of each P450 isoform in the different HLMs were calculated by parametric regression analysis (SAS version 8.01, SAS Institute Inc., Cary, NC). A p value less than 0.05 was considered statistically significant.

Data Analysis. Results are expressed as mean ± S.D. of estimates obtained from four different liver microsomes in duplicate experiments. In the microsomal incubation studies, the apparent kinetic parameters of benidipine biotransformation (K_m and V_max) were determined by fitting a one-enzyme Michaelis-Menten equation [V = V_max · S/(K_m + S)] or a Hill equation [V = V_max · Sⁿ/(K_m + Sⁿ)]. The calculated parameters were the maximum rate of formation (V_max), the Michaelis constant (apparent K_m), the intrinsic clearance (CL_int = V_max/apparent K_m), and Hill coefficient (n). Calculations were performed using WinNonlin software (Pharsight, Mountain View, CA). The percentages of inhibition were calculated by the ratio of the amounts of metabolites formed with and without the specific inhibitor. In the incubation study using cDNA-expressed P450 isoforms, a Hill equation model was fitted to the unweighted data on the formation rate of both metabolites to estimate the enzyme kinetic parameters. The models that best fit were selected based on the dispersion of residuals and standard errors of the parameter estimates.

View larger version (22K): [in this window] [in a new window]   FIG. 2. MS/MS spectra of benidipine (A) and its two metabolites (B and C) obtained by LC/MS/MS analysis of the human liver microsomal incubates of benidipine in the presence of NADPH-generating system.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Identification of Benidipine Metabolites in HLMs. After the incubations of benidipine with HLMs in the presence of an NADPH-generating system, the benidipine and its two metabolites (N-desbenzylbenidipine and dehydrobenidipine) were profiled, characterized, and tentatively identified using LC/MS analysis (Fig. 1). Other metabolites that were observed in plasma, urine, and feces after p.o. administration of benidipine to humans were not found in the human liver microsomal incubation sample. LC/MS/MS analysis of the unchanged benidipine and its two metabolites produced the informative and prominent product ions for structural elucidation. MS/MS spectrum of benidipine, having a protonated molecular ion [M + H]⁺ at m/z 506, showed major fragment ions at m/z 174 (the loss of benzylpiperidine group), m/z 315 (carboxyl group cleavage), and m/z 91 (the loss of benzyl group) (Fig. 2). Major metabolites N-desbenzylbenidipine and dehydrobenidipine were identified by cochromatography and the MS/MS spectral data of the authentic compounds (Fig. 2). The mass spectrum of N-desbenzylbenidipine has a protonated molecular ion peak [M + H]⁺ at m/z 416, suggesting loss of benzyl molecule from benidipine ([M + H]⁺, m/z 506). The MS/MS spectrum of N-desbenzylbenidipine also showed a fragmentation pattern similar to the parent compound (Fig. 2). Dehydrobenidipine gave an [M + H]⁺ molecular ion at m/z 504, suggesting loss of hydrogen atoms from the parent compound. The MS/MS spectrum of dehydrobenidipine by fragmenting m/z 504 through collision gave the characteristic daughter ions at m/z 91, 174, 285, and 313, suggesting the possible oxidation of the dihydropyridine ring (Fig. 2).

Identification of the P450 Isoforms Involved in the Metabolism of Benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of dehydrobenidipine and N-desbenzylbenidipine. Heat inactivation of the FMO activity did not inhibit benidipine metabolism, whereas SKF 525-A did inhibit benidipine metabolism (Table 1), suggesting that P450 is the enzyme responsible for benidipine metabolism. The rates of formation of metabolite were proportional to incubation times up to 10 min and protein concentration up to 0.3 mg/ml at 5 min. A P450 isoform-selective inhibition study was performed to evaluate which P450 isoforms are involved in the metabolism of benidipine in HLMs (Fig. 3). Among the 10 inhibitors tested, ketoconazole, a well known CYP3A-selective inhibitor (Bourrie et al., 1996), inhibited N-desbenzylbenidipine and dehydrobenidipine formation from benidipine. After treatment of 1 µM ketoconazole, the level of N-desbenzylbenidipine and dehydrobenidipine formation from benidipine enantiomers markedly decreased to 7 to 17% and 26 to 31% of that in the controls, respectively. It is difficult to explain why ketoconazole showed partial inhibition on dehydrobenidipine formation of benidipine with the present data. However, CYP3A has multiple binding sites and therefore sometimes shows partial inhibition (partial inhibition of erythromycin N-demethylation by testosterone in microsomes) (Wang et al., 1997). Therefore, further studies would be needed to clarify partial inhibition by ketoconazole. Benidipine metabolism was also inhibited by azamulin, another CYP3A inhibitor (Streel et al., 2002). Correlations between the rate of benidipine metabolism and the activities of P450s in HLMs are summarized in Table 2. The formation rates of dehydrobenidipine and N-desbenzylbenidipine from benidipine were significantly correlated with the activity of CYP3A (Fig. 4).

View larger version (40K): [in this window] [in a new window]   FIG. 3. Stereoselective inhibition of known P450 isoform-selective inhibitors on N-debenzylation and dehydrogenation from racemic, (+)--, and (-)--benidipine in human liver microsomal incubations. Pooled HLMs (0.1 mg/ml, Gentest H161) were incubated with 5 µM benidipine enantiomers or racemate in the absence or presence of various chemical inhibitors at 37°C for 5 min. The chemical inhibitors used were as follows: furafylline (FURA, 10 µM) for CYP1A2, coumarin (COUM, 100 µM) for CYP2A6, triethylenethiophosphoramide (TEPA, 5 µM) for CYP2B6, montelukast (MONT, 0.1 µM) for CYP2C8, sulfaphenazole (SULF, 10 µM) for CYP2C9, S-benzylnirvanol (S-BEN, 1 µM) for CYP2C19, quinidine (QUIN, 10 µM) for CYP2D6, diethyldithiocarbamate (DETC, 10 µM) for CYP2E1, ketoconazole (KETO, 1 µM) for CYP3A, and azamulin (AZAM, 5 µM) for CYP3A. Data shown are averages of remaining activity relative to the control metabolite formation rate estimated from duplicate experiments.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Correlation analysis between the known P450 enzyme activities and the rate of formation of dehydrobenidipine (A) and N-desbenzylbenidipine (B) from racemic benidipine in 15 HLMs.

Dehydrobenidipine and N-desbenzylbenidipine formation from benidipine and its enantiomers (1 and 10 µM) was also studied using the human cDNA-expressed P450 isoforms CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 (Fig. 5). CYP3A4 and CYP3A5 metabolized benidipine more efficiently than any other P450, with little contribution of CYP2A6 and CYP2D6. To determine the relative contributions of four P450 isoforms to benidipine metabolism in HLMs, we estimated the percentage of the net reaction rate from the abundance-adjusted simulation of each P450 isoform in HLMs. This simulation also shows that CYP3A is the major P450 enzyme responsible for benidipine metabolism. The percentage of net reaction by CYP2A6 and CYP2D6 is lower than 3.6 and 2.3%, respectively, whereas that by CYP3A is higher than 90.0%. The levels of involvement of other recombinant P450 isoforms in the formation of benidipine metabolites were negligible.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Representative plot of the formation of N-desbenzylbenidipine and dehydrobenidipine from racemic benidipine and two enantiomers by cDNA-expressed P450 isoforms. Human cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were incubated with 1 µM (A) and 10 µM (B) benidipine at 37°C for 5 min. Data shown are averages of duplicate experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Kinetics for the formation rate of N-desbenzylbenidipine (A) and dehydrobenidipine (B) from racemic benidipine and two enantiomers in HLMs. An increasing concentration of benidipine (0-200 µM) was incubated with HLMs (0.1 mg/ml) and an NADPH-generating system for 5 min at 37°C. Data points are the averages obtained from four different HLMs.

Next, we examined the enzyme kinetic parameters for the formation of N-desbenzylbenidipine and dehydrobenidipine from benidipine enantiomers (0-200 µM) on incubation with cDNA-expressed human CYP3A4 and CYP3A5 (Fig. 7). Under the experimental conditions used, the metabolism of benidipine by these P450 isoforms was also best described by a Hill equation (Table 4). CYP3A4 and CYP3A5 metabolized both benidipine enantiomers to N-desbenzylbenidipine and dehydrobenidipine (Table 4). The CYP3A4-catalyzed N-debenzylation of benidipine enantiomers showed lower K_m and higher V_max than those of the dehydrogenation, resulting in a higher CL_int of N-desbenzylbenidipine formation than dehydrobenidipine formation (12.3-16.1 versus 1.6-2.1 µl/min/pmol CYP3A4, respectively). CYP3A5 also showed similar tendency as in the case of CYP3A4.

View larger version (29K): [in this window] [in a new window]   FIG. 7. The formation rate of N-desbenzylbenidipine and dehydrobenidipine from benidipine enantiomers in recombinant human CYP3A4 (A) and CYP3A5 (B). An increasing concentration of benidipine (0-200 µM) was incubated with recombinant CYP3A and an NADPH-generating system for 5 min at 37°C. Each data point represents the average of triplicate incubations.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The metabolism studies of benidipine have been reported for various organisms such as rats and humans (Kobayashi et al., 1988a,b, 1997). As the result of previous research, a lot of metabolites were identified, and major metabolic pathways were N-debenzylation, dehydrogenation, ester hydrolysis, decarboxylation, and so on. In the present study, we determined that the major metabolites of human liver microsomal incubation were N-desbenzylbenidipine and an oxidized metabolite of the dihydropyridine ring (dehydrobenidipine) (Fig. 1). Benidipine was metabolized by HLMs in the presence of an NADPH-generating system but was not metabolized in the absence of NADPH. This result suggests that benidipine metabolism in humans is P450-dependent.

Owing to a common structural feature, N-debenzylation and oxidation of dihydropyridine ring pathways have been consistently reported as routes for other dihydropyridine calcium antagonists such as barnidipine (Teramura et al., 1997b) and efonidipine (Nakabeppu et al., 1995). Estimated intrinsic clearance, based on the V_max/K_m for the two primary metabolic pathways, suggests N-debenzylation is the more dominant metabolic pathway of benidipine than oxidation of the dihydropyridine ring. The CL_int of both benidipine enantiomers indicated that N-desbenzyl metabolite formation contributes to 79% of benidipine metabolism and dehydro metabolite formation contributes to 21% (Table 3).

Therefore, it is considered that benidipine is catalyzed by the same mechanism as that proposed for other structural analogs (Guengerich and Bocker, 1988). Our present findings were further supported by the reports that P450s catalyze oxidation of the dihydropyridine ring (Baarnhielm and Hansson, 1986) and that nifedipine and felodipine, typical 1,4-dihydropyridine-type calcium antagonists, are oxidized by the CYP3A4 isoform (Bocker and Guengerich, 1986; Guengerich et al., 1991). The N-debenzylation pathway was specifically dependent on a particular substituent present in benidipine, and barnidipine, a similar structural analog, is also metabolized by the CYP3A enzyme (Teramura et al., 1997a).

We provide evidence that benidipine metabolism is catalyzed by the CYP3A subfamily. First, formation rates of N-desbenzylbenidipine and dehydrobenidipine were inhibited (68-78%) by ketoconazole and azamulin, potent CYP3A-selective inhibitors (Fig. 3). Second, expressed human CYP3A4 and CYP3A5 metabolized benidipine to its two metabolites, whereas other P450 isoforms did not (Fig. 5). Third, midazolam 1'-hydroxylation activity (a marker of CYP3A activity) in 15 individual HLMs exhibited high correlation with the formation rate of the metabolites (r = 0.66-0.80) (Table 2). The significant correlation we observed between the activity of CYP1A2 and benidipine metabolism in the panel of HLMs tested may not be caused by the actual involvement of CYP1A2 in benidipine metabolism. Because our inhibition and recombinant experiments do not support a significant role of CYP1A2 in benidipine metabolism, the observed significant correlation between benidipine metabolism and CYP1A2 is probably derived from the significant correlation between the activity of CYP1A2 and CYP3A (Pearson r = 0.57; p = 0.02) in the bank of human livers tested. It is interesting that the two major metabolic pathways of benidipine are catalyzed by the same CYP3A subfamily despite their different metabolic mechanisms, but this may be explained by the fact that CYP3A has a broad substrate specificity (Spatzenegger and Jaeger, 1995; Patki et al., 2003). Taken together, these results suggest that CYP3A is the major enzyme involved in the metabolism of benidipine at concentrations in the usual experimental ranges.

In general, the stereoselective pharmacokinetics of dihydropyridine calcium antagonists, such as felodipine, nimodipine, nitrendipine, and nilvadipine, in animals and humans has been extensively reported (Niwa et al., 1988; Soons et al., 1993) and reviewed (Tokuma and Noguchi, 1995; Inotsume and Nakano, 2002). The stereoselective pharmacokinetics can be explained by the rate-limiting step of a single P450 enzyme and similar stereoselectivity for these structurally related drugs (Niwa et al., 1989; Eriksson et al., 1991). At present, only limited pharmacokinetic data of the benidipine enantiomers are available. The preliminary data from a study in humans (Tokuma and Noguchi, 1995; Kang et al., 2005) suggest that the plasma concentrations of the (+)- enantiomer were higher than those of the (-)- enantiomer. Therefore, we evaluated the possibility of enantioselective metabolism of benidipine using HLMs and recombinant P450 isoforms as examples of felodipine (Eriksson et al., 1991) and nilvadipine (Niwa et al., 1989). In this in vitro study, however, no significant differences were found for the estimated Michaelis-Menten parameters V_max, K_m, or CL_int (Table 3). Enzyme kinetic study using cDNA-expressed CYP3A4 and CYP3A5 also did not show any stereoselective differences on benidipine metabolism (Table 4). Considering no enantioselective metabolism of benidipine in this in vitro study, the higher plasma levels of (+)--benidipine than (-)--benidipine could be caused by enantioselectivity in plasma protein binding and/or by the enantioselectivity in the absorption via drug transporters such as P-glycoprotein. For example, the free fraction value of semotiadil (R isomer), a calcium antagonist, is 0.7 times less than that of levosemotiadil (S isomer) in human plasma (Rodriguez Rosas et al., 1997). However, the protein binding of benidipine in human serum was almost constant at about 99% over a concentration range of 1 to 10,000 ng/ml (Kobayashi and Kobayashi, 1999). Therefore, the effect of enantioselective protein binding on enantioselective disposition of benidipine would not be important.

In conclusion, this study shows that the primary metabolic pathway of benidipine in HLMs is N-debenzylation and oxidative dehydrogenation. The P450 isoform-specific chemical inhibition study, correlation study, and incubation study of cDNA-expressed P450 enzymes showed that CYP3A4 and CYP3A5 were responsible for the formation of two metabolites from benidipine. In addition, benidipine did not show stereoselectivity on metabolism. Therefore, detailed studies for elucidation of the mechanism of enantioselective disposition of benidipine in vivo are required.

	Acknowledgments

 
We thank Kyowa Hakko Kogyo Co. (Shizuoka, Japan) for providing the benidipine and its metabolites.

	Footnotes

 
doi:10.1124/dmd.106.013607.

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Ministry of Science and Technology (M10300000370-06J0000-37010) and by a grant of the Korea Health 21R&D Project, Ministry of Health & Welfare, R.O.K. (A030001).

ABBREVIATIONS: P450, cytochrome P450; SKF 525-A, 2'-diethylaminoethyl 2,2-diphenylpentanoate hydrochloride; FMO, flavin monooxygenase; HLM, human liver microsome); LC/MS/MS, liquid chromatography/tandem mass spectrometry; CL_int, intrinsic clearance.

Address correspondence to: Jae-Gook Shin, Department of Pharmacology and PharmacoGenomics Research Center, 633-165, Gaegum-Dong, Busanjin-Gu, Busan 614-735, South Korea. E-mail: phshinjg{at}inje.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 


Baarnhielm C and Hansson G (1986) Oxidation of 1,4-dihydropyridines by prostaglandin synthase and the peroxidic function of cytochrome P-450. Demonstration of a free radical intermediate. Biochem Pharmacol 35: 1419-1425.[CrossRef][Medline]

Bocker RH and Guengerich FP (1986) Oxidation of 4-aryl- and 4-alkyl-substituted 2,6-dimethyl-3,5-bis(alkoxycarbonyl)-1,4-dihydropyridines by human liver microsomes and immunochemical evidence for the involvement of a form of cytochrome P-450. J Med Chem 29: 1596-1603.[CrossRef][Medline]

Bourrie M, Meunier V, Berger Y, and Fabre G (1996) Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther 277: 321-332.[Abstract/Free Full Text]

Eriksson UG, Lundahl J, Baarnhielm C, and Regardh CG (1991) Stereoselective metabolism of felodipine in liver microsomes from rat, dog, and human. Drug Metab Dispos 19: 889-894.[Abstract]

Guengerich FP and Bocker RH (1988) Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines. J Biol Chem 263: 8168-8175.[Abstract/Free Full Text]

Guengerich FP, Brian WR, Iwasaki M, Sari MA, Baarnhielm C, and Berntsson P (1991) Oxidation of dihydropyridine calcium channel blockers and analogues by human liver cytochrome P-450 IIIA4. J Med Chem 34: 1838-1844.[CrossRef][Medline]

Inotsume N and Nakano M (2002) Stereoselective determination and pharmacokinetics of dihydropyridines: an updated review. J Biochem Biophys Methods 54: 255-274.[CrossRef][Medline]

Kang W, Lee DJ, Liu KH, Sunwoo YE, Kwon KI, Cha IJ, and Shin JG (2005) Analysis of benidipine enantiomers in human plasma by liquid chromatography-mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. J Chromatogr B Analyt Technol Biomed Life Sci 814: 75-81.[CrossRef][Medline]

Karasawa A, Kubo K, Shuto K, Oka T, and Nakamizo N (1988) Antianginal effects of the new calcium antagonist benidipine hydrochloride in anesthetized rats and spontaneously hypertensive rats. Electrocardiographic study. Arzneimittelforschung 38: 1702-1707.[Medline]

Kim H, Yoon YJ, Kim H, Kang S, Cheon HG, Yoo SE, Shin JG, and Liu KH (2006) Characterization of the cytochrome P450 enzymes involved in the metabolism of a new cardioprotective agent KR-33028. Toxicol Lett 166: 105-114.[CrossRef][Medline]

Kim MJ, Kim H, Cha IJ, Park JS, Shon JH, Liu KH, and Shin JG (2005) High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 19: 2651-2658.[CrossRef][Medline]

Kobayashi H and Kobayashi S (1998) Absorption, distribution, metabolism and excretion of 14C-labelled enantiomers of the calcium channel blocker benidipine after oral administration to rat. Xenobiotica 28: 179-197.[CrossRef][Medline]

Kobayashi H and Kobayashi S (1999) Enantiomer-enantiomer interaction of a calcium channel antagonist, benidipine hydrochloride, during liver metabolism in the rat. Eur J Drug Metab Pharmacokinet 24: 121-126.[Medline]

Kobayashi H, Kobayashi S, Dalrymple PD, Wood SG, and Chasseaud LF (1997) Absorption, metabolism and excretion after oral administration of a new Ca antagonist, 14C-benidipine hydrochloride to man. Xenobiotica 27: 597-608.[CrossRef][Medline]

Kobayashi H, Ohishi T, Nishiie H, Kobayashi S, Inoue A, Oka T, and Nakamizo N (1988a) Absorption, distribution and excretion after oral administration of 14C-benidipine hydrochloride in rats and dogs. Arzneimittelforschung 38: 1742-1746.[Medline]

Kobayashi H, Ohishi T, Nishiie H, Kobayashi S, Inoue A, Oka T, and Nakamizo N (1988b) Absorption, distribution and excretion of 14C-benidipine hydrochloride after repeated administration to rats. Arzneimittelforschung 38: 1747-1749.[Medline]

Lee HK, Moon JK, Chang CH, Choi H, Park HW, Park BS, Lee HS, Hwang EC, Lee YD, Liu KH, et al. (2006) Stereoselective metabolism of endosulfan by human liver microsomes and human cytochrome P450 isoforms. Drug Metab Dispos 34: 1090-1095.[Abstract/Free Full Text]

Muto K, Kuroda T, Kawato H, Karasawa A, Kubo K, and Nakamizo N (1988) Synthesis and pharmacological activity of stereoisomers of 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid methyl 1-(phenylmethyl)-3-piperidinyl ester. Arzneimittelforschung 38: 1662-1665.[Medline]

Nakabeppu H, Nakajima A, Kamikawaji Y, and Shinozaki Y (1995) Identification of efonidipine hydrochloride metabolites in rats. Arzneimittelforschung 45: 766-770.[Medline]

Niwa T, Tokuma Y, Nakagawa K, and Noguchi H (1989) Stereoselective oxidation of nilvadipine, a new dihydropyridine calcium antagonist, in rat and dog liver. Drug Metab Dispos 17: 64-68.[Abstract]

Niwa T, Tokuma Y, Nakagawa K, Noguchi H, Yamazoe Y, and Kato R (1988) Stereoselective oxidation and plasma protein binding of nilvadipine, a new dihydropyridine calcium antagonist, in man. Res Commun Chem Pathol Pharmacol 60: 161-172.[Medline]

Patki KC, Von Moltke LL, and Greenblatt DJ (2003) In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes P450: role of CYP3A4 and CYP3A5. Drug Metab Dispos 31: 938-944.[Abstract/Free Full Text]

Rodrigues AD (1999) Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 57: 465-480.[CrossRef][Medline]

Rodriguez Rosas ME, Shibukawa A, Ueda K, and Nakagawa T (1997) Enantioselective protein binding of semotiadil and levosemotiadil determined by high-performance frontal analysis. J Pharm Biomed Anal 15: 1595-1601.[CrossRef][Medline]

Shimada T, Yamazaki H, Mimura M, Inui Y, and Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270: 414-423.[Abstract/Free Full Text]

Shin JG, Park JY, Kim MJ, Shon JH, Yoon YR, Cha IJ, Lee SS, Oh SW, Kim SW, and Flockhart DA (2002) Inhibitory effects of tricyclic antidepressants (TCAs) on human cytochrome P450 enzymes in vitro: mechanism of drug interaction between TCAs and phenytoin. Drug Metab Dispos 30: 1102-1107.[Abstract/Free Full Text]

Shin JG, Soukhova N, and Flockhart DA (1999) Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6. Drug Metab Dispos 27: 1078-1084.[Abstract/Free Full Text]

Soons PA, Mulders TM, Uchida E, Schoemaker HC, Cohen AF, and Breimer DD (1993) Stereoselective pharmacokinetics of oral felodipine and nitrendipine in healthy subjects: correlation with nifedipine pharmacokinetics. Eur J Clin Pharmacol 44: 163-169.[CrossRef][Medline]

Spatzenegger M and Jaeger W (1995) Clinical importance of hepatic cytochrome P450 in drug metabolism. Drug Metab Rev 27: 397-417.[Medline]

Streel B, Laine C, Zimmer C, Sibenaler R, and Ceccato A (2002) Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry. J Biochem Biophys Methods 54: 357-368.[CrossRef][Medline]

Teramura T, Fukunaga Y, Van Hoogdalem EJ, Watanabe T, and Higuchi S (1997a) Examination of metabolic pathways and identification of human liver cytochrome P450 isozymes responsible for the metabolism of barnidipine, a calcium channel blocker. Xenobiotica 27: 885-900.[CrossRef][Medline]

Teramura T, Watanabe T, Higuchi S, and Hashimoto K (1997b) Metabolism and pharmacokinetics of barnidipine hydrochloride, a calcium channel blocker, in man following oral administration of its sustained release formulation. Xenobiotica 27: 203-216.[CrossRef][Medline]

Tokuma Y and Noguchi H (1995) Stereoselective pharmacokinetics of dihydropyridine calcium antagonists. J Chromatogr A 694: 181-193.[CrossRef][Medline]

Wang RW, Newton DJ, Scheri TD, and Lu AY (1997) Human cytochrome P450 3A4-catalyzed testosterone 6 beta-hydroxylation and erythromycin N-demethylation. Competition during catalysis. Drug Metab Dispos 25: 502-507.[Abstract/Free Full Text]

Wrighton SA, Brian WR, Sari MA, Iwasaki M, Guengerich FP, Raucy JL, Molowa DT, and Vandenbranden M (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 38: 207-213.[Abstract]

Yamada S, Kimura R, Harada Y, and Nakayama K (1990) Calcium channel receptor sites for (+)-[3H]PN 200-110 in coronary artery. J Pharmacol Exp Ther 252: 327-332.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. K. Bae, S. Cao, K.-A. Seo, H. Kim, M.-J. Kim, J.-H. Shon, K.-H. Liu, H.-H. Zhou, and J.-G. Shin Cytochrome P450 2B6 Catalyzes the Formation of Pharmacologically Active Sibutramine (N-{1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}-N,N-dimethylamine) Metabolites in Human Liver Microsomes Drug Metab. Dispos., August 1, 2008; 36(8): 1679 - 1688. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.013607v1 35/9/1518    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoon, Y.-J. Articles by Shin, J.-G. Search for Related Content PubMed PubMed Citation Articles by Yoon, Y.-J. Articles by Shin, J.-G.