Introduction

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Cibenzoline succinate (Fig. 1), (±)-2-(2,2-diphenylcyclopropyl)-2-imidazoline succinate, is a class I (sodium channel blockers) antiarrhythmic drug that also exhibits certain class III (potassium channel blockers) and IV (calcium channel blockers) arrhythmic activity (Harron et al., 1992). This drug is used as a racemic mixture of R(+)- and S()-cibenzoline, and S()-enantiomer is approximately twice more potent than the R(+)-enantiomer (Loh et al., 1986). After oral dosing of [¹⁴C]-cibenzoline to healthy volunteers, the administered radioactivity recovered in the urine was 85%. Approximately 56% of this radioactivity was found to be unchanged cibenzoline, and three metabolites representing 9 to 14% of the total radioactivity were identified in the urine: p-hydroxycibenzoline (M1 in Fig. 1) and 4,5-dehydrocibenzoline (M2) in both free and conjugated forms, and p-hydroxybenzophenone (Massarella et al., 1986). Additionally, the optical activity of the cibenzoline isolated from human urine samples indicated a limited extent of stereoselective metabolism of the S()-enantiomer in man, suggesting that the R(+)-enantiomer was preferentially metabolized. Furthermore, Massarella et al. (1991) reported that coadministration of cimetidine significantly increased the cibenzoline plasma concentration and prolonged the half-life of cibenzoline, suggesting that cimetidine may lower the clearance of cibenzoline by inhibition of hepatic oxidative metabolism. The metabolites M1 and M2 have been detected in urine after dosing to rats and dogs (Loh et al., 1986), and the stereoselective disposition of cibenzoline in rats is still unknown.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Metabolic pathway of cibenzoline in man and animals. Asterisk indicates the position of 14C label.

Cytochrome P450s (CYP)¹comprise a subfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals, including drugs, carcinogens, and steroids (Gonzalez, 1990; Guengerich, 1992; Niwa et al., 1998). Additionally, the oxidation of the N- and S-oxidation of xenobiotics including drugs is generally known to be mediated by CYPs and flavin-containing monooxygenases (FMO) (Ziegler, 1988). Assessment of the human CYP and/or FMO enzyme(s) involved in the biotransformation of a drug can be useful in defining the characteristics of its pharmacokinetic behavior, particularly if polymorphic or highly variable expressed enzymes are involved, and in the prediction of metabolic drug interactions. This study describes an in vitro investigation into the stereoselective metabolism of cibenzoline in human and rat liver microsomes, and the characterization of the human enzymes responsible for their biotransformation.

Experimental Procedures

Materials. [¹⁴C]Cibenzoline, cibenzoline succinate and its metabolites (Fig. 1), and S()- and R(+)-cibenzoline were supplied by UPSA (Rueil-Malmaison, France). NADP⁺, glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), and sulfaphenazole were purchased from Sigma Chemical Co. (St. Louis, MO). S-Mephenytoin, ketoconazole, and furafylline were obtained from Ultrafine Chemicals Ltd. (Manchester, UK). Quinidine sulfate and 17-methyltestosterone were purchased from Nacalai Tesque Inc. (Kyoto, Japan) and Wako Pure Chemicals Industries (Osaka, Japan), respectively. [¹⁴C]Cibenzoline enantiomers were obtained by separation by a stereoselective HPLC method using Sumichiral OA-4400 column (8- × 250-mm, Sumica Chemical Analysis Service, Osaka, Japan) with hexane/ethanol/acetonitrile/acetic acid (680:80:40:2.5) as a mobile phase. Radiochemical and chemical purities of the enantiomers were >97%, and the specific activities were 618 to 633 MBq/mmol.

Human Liver Microsomes and Microsomal Fraction Specifically Expressing Human CYP. Pooled microsomes from 12 human livers were obtained from Human Biologic Inc. (Phoenix, AZ). Individual microsomes from 12 different human livers (coded HG3, HG6, HG23, HG30, HG42, HG43, HG56, HG66, HG70, HG89, HG93, and HG112) were obtained from Gentest Corp. (Woburn, MA), and marker enzyme activities and immunoquantified levels of individual CYPs were provided with the kit (Gentest Corp.). Microsomes from baculovirus-infected insect cells (BTI-TN-5B1-4) expressing human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11, and control microsomes from wild-type insect cells were purchased from Gentest Corp.

Preparation of Rat Liver Microsomes. Male and female Sprague-Dawley rats (7-8 weeks old) weighing 210 to 300 g were purchased from Clea Japan Inc. (Tokyo, Japan). To induce enzyme activities, male rats were treated (i.p.) daily for 3 days with phenobarbital sodium (PB, in saline) 80 mg/kg, -naphthoflavone (BNF, in corn oil) 40 mg/kg, dexamethasone (DEX, in corn oil) 100 mg/kg, or clofibrate (CLO, in corn oil) 400 mg/kg. The animals received the solution at a dosing volume of 2 ml/kg. The pretreated animals were sacrificed 24 h after the last injection. Rat liver microsomes were prepared as described by Sugiura et al. (1974).

Analytical Procedures. All experiments were performed under incubation conditions leading to linear reaction rates versus protein and concentration and time. For the standard assay, the final reaction mixture (0.5 ml) contained 100 mM phosphate buffer (pH 7.4), 2 mM NADP⁺, 10 mM G6P, 5 mM MgCl₂, 1 U/ml G6PDH, 5 µM substrate [¹⁴C-labeled S()- or R(+)-cibenzoline], and liver microsomes (1 mg/ml for human and 0.4 mg/ml for rat). This substrate concentration can be compared with a peak plasma level of 582 ng/ml (2.2 µM) after an oral dosing of cibenzoline (130 mg) to humans (Massarella et al., 1988). Kinetic experiments for estimation of K_m and V_max of metabolic production were conducted using concentrations of S()- or R(+)-cibenzoline between 1.2 and 1010 µM. The reaction was started by adding the NADPH-generating system (2 mM NADP⁺, 10 mM G6P, 5 mM MgCl₂, and 1 U/ml G6PDH), and the incubation was carried out at 37°C for 30 min (for human) or 10 min (for rat). The reaction was stopped by the addition of 0.25 ml of acetonitrile. The mixtures were shaken and centrifuged at 10,700 g for 5 min. The metabolites in the supernatant were determined by HPLC. The HPLC system consisted of a System Gold model 125 programmable solvent module, a System Gold model 166 detector (Beckman Instruments, Inc., Fullerton, CA), and an analytical column Inertsil ODS-3 (5 µm, 4.6-mm i.d., 150 mm; GL Science, Inc., Tokyo, Japan) equipped with a TSKguardgel ODS-80TM cartridge (3.2-mm i.d., 15 mm; Tosoh Co., Tokyo, Japan). The column temperature was set at 35°C. The mobile phase was 50 mM phosphate buffer (pH 6.0)/acetonitrile (90:10) as eluent A and acetonitrile as eluent B; flow rate was 0.8 ml/min. Gradient conditions were 0 to 5 min, 0% B; 5 to 10 min, 0 to 10% B (linear gradient); 10 to 30 min, 10% B; 30 to 35 min, 10 to 25% B (linear gradient); 35 to 50 min, 25% B; and 50 to 55 min, 25 to 0% B (linear gradient). The eluate was collected every 30 s, and the radioactivity in the eluate was counted with a Packard liquid scintillation counter (Packard Instrument Co., Meriden, CT) after mixing with 4.6 ml of Hionicfluor (Packard Instrument Co.).

Chemical Inhibition of Cibenzoline Metabolism. The following CYP selective inhibitors were examined for their effect on the metabolism of cibenzoline by human or rat liver microsomes: furafylline (CYP1A2) (Sesardic et al., 1990; Newton et al., 1995), sulfaphenazole (CYP2C9) (Back et al., 1988; Baldwin et al., 1995; Newton et al., 1995), quinidine (CYP2D6) (Otton et al., 1988; Newton et al., 1995), and ketoconazole (CYP3A4) (Baldwin et al., 1995; Newton et al., 1995). Inhibition by furafylline was examined by adding cibenzoline enantiomers after preincubation at 37°C for 10 min. Metabolites were determined as described above.

Metabolism of Cibenzoline by Recombinant Human CYPs. Microsomes from insect cells containing recombinant CYP (50 pmol/ml) were incubated with cibenzoline enantiomers (5 µM) and the NADPH-generating system at 37°C for 30 min. Metabolites were determined as described above.

Kinetic Study. The rate of the metabolism of cibenzoline enantiomers was analyzed by a nonlinear least-squares program MULTI (Yamaoka et al., 1981) in which unweighted raw data were fitted to the model equation. The following Michaelis-Menten equation was used to analyze the relation between rate and substrate concentration:
where V, S, K_m, and V_max are the velocity of the metabolite formation, the substrate concentration, the apparent Michaelis-Menten constant, and the maximum velocity of metabolism, respectively.

	Results

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Chromatographic Analysis. Typical HPLC chromatograms obtained after 30 min of incubation of human liver microsomes with 5 µM cibenzoline enantiomers are illustrated in Fig. 2. Four additional chromatographic peaks, labeled M1, M2, M3, and M4, were observed, and the retention times of M1 and M2 were similar to those of p-hydroxycibenzoline and dehydrocibenzoline, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   HPLC chromatogram of cibenzoline and its metabolites by human liver microsomes. [14C]S()-Cibenzoline () or [14C]R(+)-cibenzoline () at 5 µM substrate concentration was incubated with human liver microsomes at 37°C for 30 min in the presence of NADPH-generating system.

Cibenzoline Metabolism by Human Liver Microsomes. The metabolism of cibenzoline enantiomers was dependent on microsomal protein content and incubation time, and the kinetics of the oxidation process was studied under optimal conditions of microsomal protein concentration (1 mg/ml) and incubation time (30 min). The data were fitted to Michaelis-Menten kinetics, and apparent kinetic characteristics, K_m and V_max values for the formation of M1, M2, M3, and M4 from each enantiomer were summarized in Table 1. The apparent K_m values for the formation of M1 from S()- and R(+)-cibenzoline were 123 and 1.58 µM, and the V_max values were 4.42 and 1.29 pmol/mg of protein/min, respectively. Therefore, the V_max/K_m value for R(+)-cibenzoline was 23-fold greater than that of S()-cibenzoline. The V_max values for the formation of M3 and M4 from S()-cibenzoline were 1.9 to 2.2 times greater than those for R(+)-cibenzoline, whereas there were little differences in K_m values: the R(+)/S()-enantiomer ratios of V_max/K_m were 0.39 to 0.53. On the other hand, the V_max, K_m, and V_max/K_m values for the M2 formation from R(+)-cibenzoline were similar to those for S()-cibenzoline; the R(+)/S() enantiomer ratio of V_max/K_m was 0.83. The sums of intrinsic clearance (CL_int, V_max/K_m) for the formation of four main metabolites from S()- and R(+)-cibenzoline were 1.47 and 1.64 µl/min/mg, respectively.

Correlation Studies. Metabolic activities of cibenzoline enantiomers were determined among 12 human liver microsomal samples (Table 2). The formation of M1 from S()-cibenzoline was not determined due to the poor turnover of this metabolite in human liver microsomes. The activities of cibenzoline metabolism were determined at 5 µM as a substrate concentration, except that the formation of M2 was determined at 100 µM cibenzoline enantiomers as a substrate concentration, because the M2 formation was not detectable in some samples of human liver microsomes when determined at 5 µM substrate concentration. The M1 formation from R(+)-cibenzoline was highly correlated with bufuralol 1'-hydroxylation and CYP2D6 content. On the other hand, the formation of M2, M3, and M4 from S()- and R(+)-cibenzoline was highly correlated with testosterone 6-hydroxylation and CYP3A4 content. The formation of M2, M3, and M4 from S()- and R(+)-cibenzoline except for M4 formation from S()-cibenzoline was also significantly correlated with S-mephenytoin N-demethylation as well as CYP2B6 content.

Chemical Inhibition of Cibenzoline Metabolism. The effect of various chemical inhibitors on the metabolism of S()- and R(+)-cibenzoline was investigated in human liver microsomes (Table 3). When determined at 5 µM, cibenzoline enantiomers as a substrate concentration, sulfaphenazole and furafylline, which were inhibitors of CYP2C9 and CYP1A2, respectively, had little or no effect on any pathway of cibenzoline metabolism. Ketoconazole, which is a potent inhibitor of CYP3A4/5 (Baldwin et al., 1995; Newton et al., 1995), was found to have a strong inhibitory effect on the formation of M2, M3, and M4 from both enantiomers. Quinidine, a potent inhibitor of CYP2D6 (Otton et al., 1988; Newton et al., 1995), inhibited the M1 formation from R(+)-cibenzoline, and partially inhibited the M2 formation from both enantiomers and M4 formation from R(+)-cibenzoline. When determined at 100 µM substrate concentration, M2 formation was inhibited by 68 to 84% by ketoconazole, although the inhibition by quinidine was not observed.

Metabolism of Cibenzoline Enantiomers in Microsomes from Cells Transfected with Human CYP cDNAs. The metabolism of cibenzoline enantiomers by microsomes from cells expressing human CYPs was investigated (Fig. 3). The p-hydroxylation reaction (M1 formation) of R(+)-enantiomer was catalyzed by CYP2D6, although M1 was not formed from S()-cibenzoline by CYP2D6. The M1 formation from both enantiomers by other CYPs was not detected. The formation of M2 from both enantiomers was catalyzed most efficiently by CYP2D6, followed by CYP2A6, 3A4, and 3A5 in S()-cibenzoline, and by CYP3A4 in R(+)-cibenzoline. The formation of M3 and M4 was catalyzed most efficiently by CYP2D6 and CYP3A4. CYP2B6 did not show the cibenzoline metabolic activities except for the slight activity of M3 formation from S()-cibenzoline. The metabolism of cibenzoline enantiomers by CYP1A1, 1A2, 1B1, 2C8, 2C9, 2C18, 2E1, and 4A11 was not observed (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Metabolic activities of cibenzoline enantiomers in microsomes from cells transfected with human CYP cDNAs. [14C]S()-cibenzoline (A) or [14C]R(+)-cibenzoline (B) at 5 µM substrate concentration was incubated with microsomes from cells transfected with human CYP cDNAs (50 pmol/ml) at 37°C for 30 min in the presence of NADPH-generating system. Results are means of two experiments.

Effect of Gender and Inducers on the Metabolism of Cibenzoline Enantiomers in Rat Liver Microsomes. The metabolism of cibenzoline enantiomers by liver microsomes from male and female rat and from PB, BNF, CLO, or DEX-pretreated male rat is summarized in Table 4. In male rat liver microsomes, M1 was predominantly formed from R(+)-cibenzoline, followed by M3. The main products from S()-cibenzoline were M1 and M3. The activity of M1 formation from R(+)-cibenzoline was 6 times higher than that from S()-cibenzoline. The formation of M2 and M4 was detectable, but much lower than M1 and M3. No marked gender-related differences were observed in the metabolic activities from either enantiomer except that M1 formation from S()-cibenzoline in female rat was 1.7 times higher than that in male rat, and that M3 formation from S()- and R(+)-cibenzoline in male rat was 1.6 to 2.5 times higher than that in female rat.

Effect of Quinidine and Ketoconazole on the Metabolism of Cibenzoline Enantiomers by Male Rat Liver Microsomes. The effect of ketoconazole and quinidine on the formation of the main metabolites, M1 and M3, from S()- and R(+)-cibenzoline in rat liver microsomes was investigated (Table 5). Ketoconazole, a potent inhibitor of CYP3A (Salphati and Benet, 1999; Yamano et al., 1999), inhibited the M1 and M3 formation from both enantiomers more than 74%, and quinidine, a potent inhibitor of CYP2D (Chow et al., 1999), inhibited their formation by 44 to 59%.

	Discussion

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It has been reported that, after oral administration to humans and animals, cibenzoline was excreted as unchanged cibenzoline, and the metabolites including p-hydroxycibenzoline (M1) and 4,5-dehydrocibenzoline (M2), in both free and conjugated forms, and p-hydroxybenzophenone (Massarella et al., 1986). Additionally, limited stereoselective metabolism of S()-enantiomer in man was noted (Massarella et al., 1986). In this study, we have examined the stereoselective metabolism of cibenzoline by hepatic microsomes from humans and rats; p-hydroxycibenzoline (M1), 4,5-dehydrocibenzoline (M2), and two unknown metabolites (M3 and M4) were observed in human liver microsomes. The CL_int (V_max/K_m) of the M1 formation from R(+)-cibenzoline was 23-fold greater than that of S()-cibenzoline in human liver microsomes, whereas the R(+)/S()-enantiomer ratios of CL_int for the M2, M3, and M4 formation were 0.39 to 0.83. The total CL_int for the formation of the four main metabolites from S()- and R(+)-cibenzoline was 1.47 and 1.64 µl/min/mg, respectively (Table 1), suggesting that the total CL_int in R(+)-enantiomer was slightly greater than that in S()-enantiomer in human liver microsomes. After oral dosing of [¹⁴C]cibenzoline to healthy volunteers, the administered radioactivity recovered in the urine was 85%, and approximately 56% of the dosed radioactivity was found to be unchanged cibenzoline (Massarella et al., 1991). Additionally, it has been reported that 30 to 65% of a cibenzoline dose was excreted in urine unchanged; total clearance (CL) ranged from 30 to 50 l/h, and renal clearance from 17 to 30 l/h (Harron et al., 1992), indicating that renal clearance is more than 50% of CL. Therefore, it is speculated that the stereoselective disposition of cibenzoline in vivo is mainly due to the stereoselective excretion in the urine. In rat liver microsomes, M1 and M3 were the predominant metabolites, and M1 formation from R(+)-enantiomer was six times greater than that from S()-enantiomer (Table 4). Further detailed studies on the relationship between the in vitro CL_int and in vivo CL of cibenzoline enantiomers in rat will be required.

Human liver microsomes and the technique of correlation with marker activities, selective chemical inhibition, and cDNA expression have been used to study the metabolism of cibenzoline enantiomers. The M1 formation from R(+)-cibenzoline was highly correlated with bufuralol 1'-hydroxylation and CYP2D6 content (Table 2), and was inhibited by quinidine, a potent inhibitor of CYP2D6 (Table 3). Additionally, the investigation with microsomes from cells expressing human CYPs shows that CYP2D6 catalyzed the M1 formation of R(+)-enantiomer, whereas the M1 formation from both enantiomers by other CYPs was not detected (Fig. 3). Therefore, these results strongly suggested that the M1 formation from R(+)-cibenzoline was catalyzed by CYP2D6.

The formation of M3 and M4 from S()- and R(+)-cibenzoline was highly correlated with testosterone 6-hydroxylation and CYP3A4 content (Table 2). Ketoconazole, which is a potent inhibitor of CYP3A4/5, had a strong inhibitory effect on the formation of these metabolites from both enantiomers, whereas inhibition by other inhibitors examined was not observed except that M4 formation from R(+)-cibenzoline was inhibited by quinidine by 45% (Table 3). On the other hand, the formation of M3 and M4 was catalyzed most efficiently by recombinant CYP2D6 and CYP3A4 (Fig. 3). Shimada et al. (1994) reported that the relative CYP3A4/5, 2C8/9/18/19, 1A2, 2E1, and 2A6 contents in 60 human samples are 29, 18, 13, 7, and 4%, respectively, and CYP2D6 level is only 2%, suggesting that the CYP3A4/5 content is more than 10-fold of CYP2D6 content. Based on the respective proportion of these CYP isoforms and their specific turnover for the M2 formation determined on recombinant CYPs, as well as the results in the correlation with marker activities and selective chemical inhibition, CYP3A4 appears to be mainly involved in the formation of M3 and M4, except that both CYP3A4 and CYP2D6 catalyze the M4 formation from R(+)-cibenzoline.

The formations of M2 from S()- and R(+)-cibenzoline at 100 µM substrate concentration were highly correlated with testosterone 6-hydroxylation and CYP3A4 content (Table 2). Ketoconazole had a strong inhibitory effect on the M2 formation from both enantiomers at both 5 and 100 µM as a substrate concentration, and the M2 formation was inhibited by quinidine by 46 to 52% at 5 µM cibenzoline enantiomers, although the inhibition by quinidine was not observed at 100 µM substrate concentration. In the investigation with recombinant CYPs, the M2 formation from both enantiomers was catalyzed most efficiently by CYP2D6, followed by CYP2A6, 3A4, and 3A5 in S()-cibenzoline, and by CYP3A4 in R(+)-cibenzoline. These results suggest that both CYP2D6 and CYP3A4 catalyze the M2 formation at a lower concentration (5 µM) and that CYP3A4 plays the major role at a higher concentration (100 µM).

Although recombinant CYP2B6 was not capable of metabolizing cibenzoline enantiomers except for the slight activity of M3 formation from S()-cibenzoline (Fig. 3), there was a significant correlation between the formation of M2, M3, and M4 and S-mephenytoin N-demethylation as well as CYP2B6 content (Table 2). This may be derived from the fact that there was a significant correlation between CYP2B6 and CYP3A4 contents (r² = 0.546, P < .01, data not shown) and between testosterone 6-hydroxylase activity and S-mephenytoin N-demethylase activity (r² = 0.694, P < .001, data not shown) in the panel of liver microsomes. Therefore, CYP2B6 does not appear to be involved principally in the metabolism of cibenzoline.

In male rat liver microsomes, M1 was predominantly formed from R(+)-cibenzoline, followed by M3, and the main products from S()-cibenzoline were M1 and M3. The formation of M3 and M4 from S()-cibenzoline was increased by pretreatment with DEX, and M3 formation from S()-cibenzoline was also increased by PB- and BNF-pretreatment. Additionally, M2 formation from R(+)-cibenzoline was increased by CLO-pretreatment, whereas other activities were not affected or were decreased by pretreatment of these inducers. Additionally, ketoconazole, a potent inhibitor of CYP3A (Salphati and Benet, 1999; Yamano et al., 1999), inhibited the M1 and M3 formation from both enantiomers more than 74%, and quinidine, a potent inhibitor of CYP2D (Chow et al., 1999), inhibited their formation by 44 to 59%, suggesting that CYP3A and CYP2D play a major role in cibenzoline metabolism in untreated male rats.

In conclusion, we have demonstrated that the individual enantiomers of cibenzoline are metabolized to four metabolites, including p-hydroxycibenzoline (M1) and dehydrocibenzoline (M2) in human and rat liver microsomes, and that these metabolic steps are catalyzed principally by CYP3A and/or CYP2D.