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R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity
Drug Metabolism and Pharmacokinetics 30 (2015) 257e262
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Regular article
Serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity Kosuke Doki a, Yukio Sekiguchi b, Keisuke Kuga b, Kazutaka Aonuma b, Masato Homma a, * a b
Department of Pharmaceutical Sciences, Faculty of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2014 Received in revised form 4 March 2015 Accepted 2 April 2015 Available online 11 April 2015
The aims of this study were to clarify whether the ratio of S- to R-flecainide (S/R ratio) in the serum flecainide concentration was associated with the stereoselectivity of flecainide metabolism, and to investigate the effects of the cytochrome P450 (CYP) 2D6 (CYP2D6) genotype and CYP2D6 inhibitor on the serum flecainide S/R ratio. In vitro studies using human liver microsomes and cDNA-expressed CYP isoforms suggested that variability in the serum flecainide S/R ratio was associated with the stereoselectivity of CYP2D6-mediated flecainide metabolism. We examined the serum flecainide S/R ratio in 143 patients with supraventricular tachyarrhythmia. The S/R ratio was significantly lower in intermediate metabolizers and poor metabolizers (IMs/PMs) than in extensive metabolizers (EMs) identified by the CYP2D6 genotype. The cut-off value for the S/R ratio to allow the discrimination between CYP2D6 EMs and IMs/PMs was 0.99. The S/R ratio in patients with co-administration of bepridil, a potent CYP2D6 inhibitor, was lower than 0.99, regardless of the CYP2D6 genotype status. Other factors, including age, sex, body weight, and renal function, did not affect the serum flecainide S/R ratio. This study suggests that the serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity on coadministration of a CYP2D6 inhibitor.
Keywords: Flecainide S/R ratio CYP2D6 Genotype CYP2D6 inhibitor
Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction Flecainide is a substrate of cytochrome P450 (CYP) 2D6, a highly polymorphic enzyme [1] and a chiral compound administered as a racemic mixture for the treatment of various supraventricular tachyarrhythmias [25]. Flecainide is principally metabolized to mO-dealkylated flecainide (MODF) by hepatic CYP2D6 and CYP1A2 enzymes and is subsequently oxidized to m-O-dealkylated lactam (Fig. 1), although 30% of the dosed amount is excreted in urine as the unchanged form [68]. Impaired CYP2D6 activity reduces flecainide clearance by 21% in intermediate metabolizers (IMs) and by 42% in poor metabolizers (PMs) [9,10]. The CYP2D6-mediated oxidation phenotype appears to be a determinant of stereoselective flecainide metabolism [1114]. Impairment of CYP2D6 activity preferentially increases the serum concentration of R-flecainide compared with that of S-flecainide
* Corresponding author. Tel.: þ81 298 53 3859; fax: þ81 298 96 7025. E-mail addresses: [email protected] (K. Doki), [email protected] (Y. Sekiguchi), [email protected] (K. Kuga), [email protected] (K. Aonuma), [email protected] (M. Homma).
(Fig. 1) [1114]. Nevertheless, whether CYP2D6 or CYP1A2 is responsible for the metabolism of flecainide enantiomers remains unclear. The ratio of S- to R-flecainide (S/R ratio) in the serum flecainide concentration may reflect CYP2D6 activity in arrhythmic patients receiving flecainide therapy, because the serum flecainide S/R ratio is lower in healthy subjects with impaired CYP2D6 activity than in those with normal CYP2D6 activity. We previously reported that the CYP2D6 genotype affected the serum flecainide S/R ratio in healthy subjects and patients receiving flecainide therapy [14]. However, the cut-off value for the serum flecainide S/R ratio when discriminating between CYP2D6 extensive metabolizers (EMs) and IMs/PMs remains unclear. In the present study, we examined the effects of CYP2D6 and CYP1A2 on the metabolism of flecainide enantiomers in an in vitro study using human liver microsomes and cDNA-expressed CYP isoforms to clarify the changes in the serum flecainide S/R ratio due to altered metabolic enzyme activity. We also investigated the effects of the CYP2D6 genotype and CYP2D6 inhibitor on the serum flecainide S/R ratio in routine therapeutic drug monitoring samples of patients with supraventricular tachyarrhythmia.
http://dx.doi.org/10.1016/j.dmpk.2015.04.001 1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The chemical structures of flecainide and m-O-dealkylated flecainide (MODF). Chiral center in the molecule is indicated by an asterisk. Solid and dashed arrows represent major and minor elimination pathways of flecainide enantiomers, respectively. R-flecainide is preferentially metabolized by CYP2D6 compared to S-flecainide.
2. Methods 2.1. Chemicals and microsomes Flecainide acetate, MODF, and an internal standard [N-(2piperidinylmethyl)-2,3-bis(2,2,2-trifluoroethoxy)benzamide acetate] were kindly supplied by Eisai Co. (Tokyo, Japan). S- and Rflecainide, bufuralol hydrochloride, and 10 -hydroxy bufuralol were purchased from Santa Cruz Biotechnology Inc. (CA, USA). Propranolol hydrochloride, bepridil hydrochloride, quinidine, potassium hexafluorophosphate, and perchloric acid were purchased from Wako Pure Chemicals (Osaka, Japan). Pooled microsomes from 50 human livers, microsomes from a baculoviruseinsect cell line expressing CYP2D6.1 (wild-type), CYP1A2, CYP2C9, CYP2C19, CYP3A4, control microsomes, and the NADPH-regeneration system solution were obtained from BD Gentest (Woburn, MA, USA). 2.2. In vitro study of flecainide metabolism All experiments were conducted such that product formation increased linearly with the incubation time and protein concentration. After pre-incubation for 5 min, incubations were performed in duplicate at 37 C in a shaking water bath for 30 min. Incubation mixtures contained microsome protein (1 mg/mL), flecainide enantiomer (1e800 mM), and an NADPH-regeneration system [NADP (1.4 mM), glucose 6-phosphate (3.3 mM), MgCl2 (3.3 mM), glucose-6-phosphate dehydrogenase (2 U/mL), final concentrations] in 200 mL of 100-mM potassium phosphate buffer (pH 7.4). The incubations for microsomes (20 pmol CYP) prepared from a baculoviruseinsect cell line expressing CYP2D6.1, CYP1A2, CYP2C9, CYP2C19 and CYP3A4 were performed as described above, except that the incubation time was 10 min in CYP2D6.1 and the concentration ranges of flecainide enantiomer were from 1 to 50 mM for CYP2D6.1, 25e400 mM for CYP1A2, and 25 mM for other CYPs. The reaction was terminated by the addition of 500 mL of ice-cold acetonitrile. After addition of 50 mL of an internal standard (1 mg), the mixtures were vortexed and centrifuged at 8960 g for 5 min. The supernatant was evaporated to dryness under a stream of nitrogen at 45 C. The residue was reconstituted with 100 mL of a mobile phase solution, and 40 mL was injected into a highperformance liquid chromatography (HPLC) system. MODF in the microsomal incubation mixture was determined by HPLC on a conventional octadecylsilyl silica column and a fluorescence detector, as previously described [15]. The coefficients of variations (CV) in intra-day and inter-day assays were 3.0%e4.2% and 3.3%e 6.7%, respectively. 2.3. In vitro inhibition study of bufuralol 10 -hydroxylation Bufuralol 10 -hydroxylation activity was assayed in microsomes (2-pmol CYP) prepared from a baculoviruseinsect cell line
expressing CYP2D6.1. Chemical inhibition of bufuralol 10 -hydroxylation was examined by incubating bufuralol (10 mM) in the presence of bepridil or quinidine (reference CYP2D6 inhibitor). The incubations were performed as described above, except that the incubation time was 5 min without pre-incubation. The reaction was terminated by addition of 20 mL of ice-cold perchloric acid. After the addition of 100 mL of an internal standard (1 mM propranolol), the mixtures were vortexed and centrifuged at 8960 g for 5 min. The 20 mL supernatant was injected into an HPLC system. In the microsomal incubation mixture, 10 -hydroxy bufuralol was quantified using HPLC on a conventional octadecylsilyl silica column and a fluorescence detector, following a previously described method but with minor modifications [16]. The mobile phase solution, comprising 2 mM perchloric acid and acetonitrile (70:30, v/ v), was pumped at a flow rate of 1.2 mL/min. The analytical column was maintained at 40 C. Excitation and emission wavelengths were set at 252 and 302 nm, respectively. The CVs in intra-day assays were less than 1%. 2.4. Patients Patients receiving oral flecainide for supraventricular tachyarrhythmias were enrolled during an outpatient visit to our hospital (Table 1). Exclusion criteria were a history of unstable angina or myocardial infarction; recent cardiac surgery; a high-degree atrioventricular nodal block; pacemaker-dependent rhythms; permanent atrial fibrillation; Brugada's syndrome; concomitant therapy, including other class I anti-arrhythmic drugs or amiodarone; and left ventricular dysfunction (i.e., ejection fraction of <50% and/ or a history of heart failure). The patients had received oral flecainide (0.7e5.3 mg/kg/day as flecainide acetate). Patients received the following drugs as needed: digoxin, b-blockers (carvedilol, atenolol, carteolol, nadolol, bisoprolol, metoprolol, and propranolol), Ca2þ antagonists (bepridil, verapamil, diltiazem, nifedipine, amlodipine, nisoldipine, and nicardipine), angiotensin-converting enzyme inhibitors (enalapril, lisinopril, and temocapril), angiotensin II receptor blockers (valsartan, telmisartan, olmesartan, losartan, and candesartan), anti-thrombotics (warfarin, aspirin, and ticlopidine), H2-blockers (famotidine and ranitidine), proton pump inhibitors (lansoprazole, rabeprazole), or other drugs (lipiddecreasing drugs and HMG-CoA reductase inhibitors). Estimated glomerular filtration rate (eGFR) was calculated for each patient from their serum creatinine value (SCr) and their age using the following equation [17]: eGFR (mL/min/1.73 m2) ¼ 194 age0.287 SCr1.094 (if female then, 0.739) This study was approved by the Ethics Committee of the University of Tsukuba. Informed consent was obtained from all patients who participated in the study.
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Table 1 Kinetic parameters for MODF formation by human liver microsomes and cDNA-expressed CYPs. R-flecainide
S-flecainide
Km
Vmax
CLint
Km
Vmax
CLint
(mM)
(pmol/min)*
(mL/min)*
(mM)
(pmol/min)*
(mL/min)*
Pooled microsome High-affinity 1.3 ± 1.1 component (CYP2D6) Low-affinity 406.8 ± 47.2 component (CYP1A2) Microsomes containing cDNA-expressed CYP CYP2D6*1 2.8 ± 0.3 CYP1A2 106.4 ± 15.0
0.5 ± 0.1
0.369
2.3 ± 5.8
0.2 ± 0.2
0.092
9.0 ± 0.4
0.022
403.1 ± 58.8
10.6 ± 0.6
0.026
29.8 ± 0.9 16.3 ± 0.9
10.764 0.153
7.2 ± 0.5 37.8 ± 7.6
30.6 ± 0.7 8.0 ± 0.4
4.236 0.212
* Vmax and CLint were expressed as per mg protein for human liver microsomes or per nmol CYP for microsomes containing cDNA-expressed CYP. Kinetic parameters were calculated from a fitted curve by non-linear regression (mean ± SE).
2.5. Determination of flecainide enantiomers in serum Patients had received flecainide for at least 2 months before blood withdrawal was performed for determining their serum flecainide concentration. To determine flecainide trough levels, blood collections were performed between 8:30 AM and 11:00 AM during outpatient visits. On sample collection days, the patients postponed taking their morning flecainide until after the blood collection. Serum samples were separated from whole blood and stored at 20 C until analysis. Serum concentrations of flecainide enantiomers were determined using an HPLC equipped with a polysaccharide-based chiral column (Chiralpak AS-RH) and a fluorescence detector, as previously described [14]. The CVs in intra-day and inter-day assays were 1.8%e5.8% and 3.4%e7.5%, respectively. No chiral inversion between S- and R-flecainide was observed experimentally in incubation mixtures using plasma or human liver microsome at 37 C for 12 h. 2.6. Genotyping of CYP2D6 Patients were genotyped for CYP2D6*1, *2, *4, *5, *10, *14, *21, *36, and CYP2D6xN using allele-specific polymerase chain reaction (PCR) with mismatch primers and step-down PCR [18]. We designated the CYP2D6*1 and *2 alleles as EM alleles; *10 as an IM allele; and *4, *5, *14, *21, and *36 as PM alleles. We used these categories to assign the following CYP2D6 genotype groups: EMs (EM/EM, EM/ IM, EM/PM), IMs (IM/IM, IM/PM), and PMs (PM/PM). 2.7. Data and kinetic calculations Enzyme kinetic parameters were calculated using SigmaPlot (ver. 10.0; Systat Software, Richmond, CA, USA). Kinetic analyses were obtained from an EadieeHofstee plot. The kinetics were described by one of the two following models: 1-enzyme MichaeliseMenten model: V ¼ Vmax$S/(Km þ S) 2-enzyme MichaeliseMenten model: V ¼ Vmax1$S/(Km1 þ S) þ Vmax2$S/(Km2 þ S), where V is the velocity of the reaction at substrate concentration S, Vmax is the maximum velocity, and Km is the substrate concentration at which the reaction velocity is 50% of Vmax. Intrinsic clearance of the in vitro incubation was calculated as CLint ¼ Vmax/Km. The half-maximal inhibitory concentration (IC50) was determined by analyzing the logarithmic plot of the inhibition of the inhibitor
concentration versus the percentage of activity remaining after inhibition. A receiver operating characteristic (ROC) curve was generated to distinguish between CYP2D6 EMs and IMs/PMs. The sensitivity (the proportion of true positive IMs/PMs) and specificity (the proportion of true negative) were calculated from the data across all possible cut-off values within the range of the S/R ratio. We determined the optimal cut-off value by maximizing the Youden index, which is defined as (sensitivity þ specificity 1). 2.8. Statistical analyses Data are expressed as numbers or mean ± SD. The influence of continuous parameters on the serum flecainide S/R ratio was evaluated as the dichotomous parameter divided by the mean value, except that the cut-off value for eGFR was based on the diagnostic criterion for chronic kidney disease. The serum flecainide S/R ratio and concentration-to-dose ratio were compared between two patient groups using Student's t-test or the ManneWhitney U-test. A P-value of <0.05 was considered statistically significant. 3. Results 3.1. Stereoselectivity of MODF formation in human liver microsomes and cDNA-expressed CYPs MichaeliseMenten and EadieeHofstee plots supported a twoenzyme model and biphasic kinetics of MODF formation from Sand R-flecainide in pooled human liver microsomes (Fig. 2). The estimated Km, Vmax, and CLint values of MODF formation are shown in Table 1. CLint for the high-affinity component was 4-fold higher in R-flecainide than in S-flecainide, whereas for the low-affinity component, it was only slightly higher in S-flecainide than in Rflecainide. MODF formation was also examined in microsomes containing cDNA-expressed CYP2D6.1 and CYP1A2. The estimated Km, Vmax, and CLint values of MODF formation are shown in Table 1. CLint for CYP2D6.1 was 2.5-fold higher in R-flecainide than in S-flecainide, whereas for CYP1A2, it was only slightly higher in S-flecainide than in R-flecainide. Other CYP isoforms showed no or weak activity of MODF formation. 3.2. Inhibitory effect of bepridil on bufuralol 10 -hydroxylation in vitro The inhibitory effect of bepridil on CYP2D6 activity was examined using the probe reaction bufuralol 10 -hydroxylation. Bufuralol
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Fig. 2. Kinetic profiles of m-O-dealkylated flecainide (MODF) formation from (a) R-flecainide and (b) S-flecainide in pooled human liver microsomes with an inset EadieeHofstee plot demonstrating the biphasic kinetics. Data are the means of duplicate determinations.
Table 2 Patients' characteristics.
Table 3 Influences of patients' characteristics on the serum flecainide S/R ratio.
Characteristics Number of patients Sex (male/female) Age (years) Body weight (kg) CYP2D6 genotype EMs IMs/PMs Current smoking Co-administration of bepridil Liver and kidney function AST (IU/L) ALT (IU/L) Serum creatinine (mg/dL) eGFR (mL/min) Flecainide dose (mg/kg/day) Serum concentration (ng/mL) S-flecainide R-flecainide S/R ratio
Characteristics 143 112/31 61 ± 13 66 ± 11 118 25 13 21 24 ± 8 23 ± 10 0.84 ± 0.20 71 ± 17 2.3 ± 0.8 162 ± 78 169 ± 81 0.96 ± 0.10
Data presented as number or mean ± SD. EMs, extensive metabolizers; IMs/PMs, intermediate metabolizers and poor metabolizers; eGFR, estimated glomerular filtration rate.
10 -hydroxylation activity in microsomes containing cDNAexpressed CYP2D6.1 was inhibited by bepridil with the IC50 of 0.016 mM. The IC50 of quinidine, a typical CYP2D6 inhibitor, was 0.008 mM. 3.3. Patients One hundred and forty-three patients treated with oral flecainide for supraventricular tachyarrhythmias were included in our analysis of serum flecainide S/R ratios. The demographic, physiological, and CYP2D6 genetic characteristics are summarized in Table 2. The CYP2D6 genotype was categorized as CYP2D6 EMs (n ¼ 118) or IMs/PMs (n ¼ 25). Twenty-one patients were coadministered bepridil, a potent CYP2D6 inhibitor. 3.4. Evaluation of the serum flecainide S/R ratio Trough serum concentrations of S- and R-flecainide were 162 ± 78 ng/mL and 169 ± 81 ng/mL, respectively (Table 2). The serum flecainide S/R ratio was 0.96 ± 0.10, and it ranged from 0.71 to 1.26. We examined whether the serum flecainide S/R ratio was influenced by age, sex, body weight, eGFR, daily dose, the CYP2D6 genotype, or co-administration of bepridil (Table 3). No differences
Age <60 yr 60 yr Sex Male Female Body weight <66 kg 66 kg eGFR <60 mL/min 60 mL/min Dose <2.3 mg/kg 2.3 mg/kg CYP2D6 genotype EMs IMs/PMs Co-administration of bepridil Yes No
S/R ratio
P-value
0.97 ± 0.10 0.96 ± 0.10
0.74
0.96 ± 0.10 0.99 ± 0.10
0.14
0.97 ± 0.10 0.96 ± 0.10
0.55
0.95 ± 0.11 0.97 ± 0.10
0.32
0.98 ± 0.11 0.95 ± 0.09
0.04
0.98 ± 0.10 0.90 ± 0.07
0.0001
0.87 ± 0.07 0.98 ± 0.09
0.000001
Data presented as mean ± SD. eGFR, estimated glomerular filtration rate; EMs, extensive metabolizers; IMs/PMs, intermediate metabolizers and poor metabolizers.
were found in serum flecainide S/R ratios between patients aged <60 and 60 years, males and females, body weight of <66 and of 66 kg, and eGFR of <60 and of 60 mL/min. The serum flecainide S/R ratio significantly differed between patients with a daily dose of <2.3 and 2.3 mg/kg (0.98 ± 0.11 vs. 0.95 ± 0.09, P ¼ 0.04), CYP2D6 genotype groups (EMs; 0.98 ± 0.10 vs. IMs/PMs; 0.90 ± 0.07, P ¼ 0.0001), and with and without co-administration of bepridil (0.87 ± 0.07 vs. 0.98 ± 0.09, P ¼ 0.000001). We examined the cut-off value for the serum flecainide S/R ratio in the CYP2D6 phenotype by comparing CYP2D6 EMs and IMs/PMs in patients without co-administration of bepridil (EMs; 1.00 ± 0.09 vs. IMs/PMs; 0.91 ± 0.07, P ¼ 0.00004; Fig. 3a). An ROC curve was generated to select the optimal cut-off for the serum flecainide S/R ratio to discriminate between CYP2D6 EMs and IMs/PMs. The optimal cut-off value was 0.99, which led to the highest Youden index based on the sensitivity of 0.95 and specificity of 0.54. Among the CYP2D6 EMs, the flecainide dose was significantly higher in patients with a serum flecainide S/R ratio of <0.99 than in those with a serum flecainide S/R ratio of 0.99 (2.7 ± 0.8 vs. 2.1 ± 0.7 mg/ kg, P ¼ 0.0001; Fig. 3b). The serum flecainide S/R ratio in patients with coadministration of bepridil was less than 0.99 (0.87 ± 0.07) and
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Fig. 3. The effect of the CYP2D6 genotype on the serum flecainide S/R ratio in patients without and with co-administration of bepridil (a). Solid and dotted lines are the mean value and cut-off value (0.99), respectively. The relationship between the daily dose of flecainide and serum flecainide S/R ratio in CYP2D6 EMs patients without co-administration of bepridil (b). The dotted line is the cut-off value (0.99).
did not differ between CYP2D6 EMs and IMs/PMs (Fig. 3a). The concentration-to-dose ratio, which was calculated as the total serum flecainide trough concentrations divided by the dose per kilogram of body weight, was 1.3-fold higher in patients with coadministration of bepridil than those without co-administration (192 ± 84 vs. 143 ± 55, P ¼ 0.002). 4. Discussion This study revealed that the serum flecainide S/R ratio was associated with the CYP2D6 genotype and changes in CYP2D6 activity. A lower serum flecainide S/R ratio corresponded to impaired CYP2D6 activity derived from the CYP2D6 IMs/PMs genotype or coadministration of the potent CYP2D6 inhibitor (Table 3; Fig. 3a). Other factors, including age, female sex, body weight, and renal function, were important for estimating flecainide clearance in the population pharmacokinetic analysis [10,19], but they did not affect the serum flecainide S/R ratio (Table 3). These results suggested that the serum flecainide S/R ratio provided specific information regarding CYP2D6 activity during therapeutic drug monitoring of flecainide. MODF formation in pooled liver microsomes is mediated by high- and low-affinity enzymes, which are associated with CYP2D6 and CYP1A2, respectively [8]. The present in vitro study revealed that the enzyme with a high-affinity for MODF formation was responsible for the stereoselectivity of flecainide metabolism. A high-affinity component in pooled human liver microsomes and recombinant CYP2D6.1 produced a faster MODF formation during the incubation of R-flecainide than during the incubation of S-flecainide (Fig. 2; Table 1). In contrast, MODF formation with the lowaffinity component in pooled human liver microsomes and recombinant CYP1A2 was slightly faster during the incubation of Sflecainide than during the incubation of R-flecainide (Fig. 2; Table 1). These findings suggest that impaired CYP2D6 activity was associated with a reduction in R-flecainide metabolism, resulting in a lower serum flecainide S/R ratio (Fig. 1). We determined an optimal cut-off value of the serum flecainide S/R ratio for the discrimination between CYP2D6 EMs and IMs/PMs (Fig. 3a). The ROC curve analysis showed that the optimal cut-off value was 0.99. Most of the CYP2D6 IMs/PMs genotype was identified as the IMs/PMs phenotype using this cut-off value. We could not determine the cut-off value of the S/R ratio for the discrimination between CYP2D6 IMs and PMs because the study population included only one CYP2D6 PMs with the lowest serum flecainide S/R ratio of 0.75 (Fig. 3a). The serum flecainide S/R ratio for the CYP2D6
EMs genotype overlapped between the EMs and IMs/PMs phenotype. This may be due to the variability in the activity of CYP2D6mediated metabolism of flecainide in CYP2D6 EMs as well as other CYP2D6 substrates, metoprolol and dextromethorphan [20,21]. The non-linearity [22] resulting from the saturation of CYP2D6mediated metabolism may partially explain the overlapping S/R ratio because patients with a lower S/R ratio (<0.99) had received a higher flecainide dose (Fig. 3b). A higher S/R ratio may be found in CYP2D6 ultrarapid metabolizers (UMs) who show excessively high CYP2D6 enzyme activity with multiple copies of the CYP2D6 gene, although CYP2D6 UMs were not included in the present study. We further examined the effect of concomitant bepridil administration on the serum flecainide S/R ratio because the present in vitro study indicated that the inhibitory effect of bepridil on CYP2D6 activity was comparable to that of quinidine, a reference CYP2D6 inhibitor. Patients who received co-administration of bepridil showed a serum flecainide S/R ratio lower than the cut-off value, regardless of the CYP2D6 genotype status (Fig. 3a). The lower S/R ratio was due to the inhibitory effect of bepridil on the CYP2D6-mediated metabolism of flecainide. We considered that a serum flecainide S/R ratio of <0.99 in EMs with co-administration of bepridil was attributed to the phenocopying phenomenon [23e25], in which co-administration of potent CYP2D6 inhibitors induces the conversion of the CYP2D6 EMs phenotype to the PMs phenotype. Therefore, the serum flecainide S/ R ratio may be a useful tool to assess the drug interaction between flecainide and CYP2D6 inhibitors in addition to the CYP2D6 genotype. This suggests that the serum flecainide S/R ratio reflects realistic CYP2D6 activity rather than CYP2D6 genotyping. The serum flecainide S/R ratio may not be associated with flecainide efficacy and safety because a similar anti-arrhythmic activity was reported between the enantiomers. A merit for determining the ratio is to assess CYP2D6 activity corresponding to the genetic polymorphisms and drug interaction on CYP2D6. We previously reported that the CYP2D6 genotype is a useful determinant for age-related decline in flecainide clearance [19]. Because CYP2D6 genotyping requires the analysis of many mutant alleles to assess enzyme activity [1], the serum flecainide S/R ratio is a convenient determinant to assess enzyme activity in routine therapeutic drug monitoring. In conclusion, the serum flecainide S/R ratio is a useful tool to assess CYP2D6 activity in patients with arrhythmia receiving flecainide therapy. The present results suggest that the serum flecainide S/R ratio reflects the effects of the CYP2D6 genotype and coadministration of a CYP2D6 inhibitor on CYP2D6 activity.
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Acknowledgments We are extremely grateful to Eisai Co. Ltd. for providing flecainide, MODF, and the internal standard used in the assay. We acknowledge the assistance provided by Ms. Katori K, Ms. Edo T, Mr. Kawata T, Ms. Shimada S, Ms. Ohhata H, Mr. Hashimoto N, Mr. Shirayama Y, and Ms. Kobayashi. References [1] Zhou SF. Polymorphism of human cytochrome P450 2D6 and its clinical significance: part I. Clin Pharmacokinet 2009;48:689e723. [2] Crozier IG, Ikram H, Kenealy M, Levy L. Flecainide acetate for conversion of acute supraventricular tachycardia to sinus rhythm. Am J Cardiol 1987;59:607e9. [3] Kreeger RW, Hammill SC. New antiarrhythmic drugs: tocainide, mexiletine, flecainide, encainide, and amiodarone. Mayo Clin Proc 1987;62:1033e50. [4] Homma M, Kuga K, Doki K, Katori K, Yamaguchi I, Sugibayashi K, et al. Assessment of serum flecainide trough levels in patients with tachyarrhythmia. J Pharm Pharmacol 2005;57:47e51. [5] Doki K, Homma M, Kuga K, Aonuma K, Kohda Y. SCN5A promoter haplotype affects the therapeutic range for serum flecainide concentration in Asian patients. Pharmacogenet Genomics 2013;23:349e54. [6] Conard GJ, Ober RE. Metabolism of flecainide. Am J Cardiol 1984;53:41Be51B. [7] McQuinn RL, Quarfoth GJ, Johnson JD, Banitt EH, Pathre SV, Chang SF, et al. Biotransformation and elimination of 14C-flecainide acetate in humans. Drug Metab Dispos 1984;12:414e20. [8] Doki K, Homma M, Kuga K, Aonuma K, Kohda Y. Effects of CYP2D6 genotypes on age-related change of flecainide metabolism: involvement of CYP1A2mediated metabolism. Br J Clin Pharmacol 2009;68:89e96. [9] Mikus G, Gross AS, Beckmann J, Hertrampf R, Gundert-Remy U, Eichelbaum M. The influence of the sparteine/debrisoquin phenotype on the disposition of flecainide. Clin Pharmacol Ther 1989;45:562e7. [10] Doki K, Homma M, Kuga K, Kusano K, Watanabe S, Yamaguchi I, et al. Effect of CYP2D6 genotype on flecainide pharmacokinetics in Japanese patients with supraventricular tachyarrhythmia. Eur J Clin Pharmacol 2006;62:919e26. [11] Gross AS, Mikus G, Fischer C, Hertrampf R, Gundert-Remy U, Eichelbaum M, et al. Stereoselective disposition of flecainide in relation to the sparteine/ debrisoquine metaboliser phenotype. Br J Clin Pharmacol 1989;28:555e66. [12] Gross AS, Mikus G, Fischer C, Eichelbaum M. Polymorphic flecainide disposition under conditions of uncontrolled urine flow and pH. Eur J Clin Pharmacol 1991;40:155e62.
[13] Birgersdotter UM, Wong W, Turgeon J, Roden DM. Stereoselective geneticallydetermined interaction between chronic flecainide and quinidine in patients with arrhythmias. Br J Clin Pharmacol 1992;33:275e80. [14] Doki K, Sekiguchi Y, Kuga K, Aonuma K, Kohda Y, Homma M. Stereoselective analysis of flecainide enantiomers using reversed-phase liquid chromatography for assessing CYP2D6 activity. Biomed Chromatogr 2014;28:1193e8. [15] Doki K, Homma M, Kuga K, Watanabe S, Yamaguchi I, Kohda Y. Simultaneous determination of serum flecainide and its metabolites by using high performance liquid chromatography. J Pharm Biomed Anal 2004;35: 1307e12. [16] Crespi CL, Chang TK, Waxman DJ. CYP2D6-dependent bufuralol 10 -hydroxylation assayed by reverse-phase ion-pair high-performance liquid chromatography with fluorescence detection. Methods Mol Biol 2006;320:121e5. [17] Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta K, et al. Revised equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis 2009;53: 982e92. [18] Ishiguro A, Kubota T, Soya Y, Sasaki H, Yagyu O, Takarada Y, et al. Highthroughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction. Anal Biochem 2005;337:256e61. [19] Doki K, Homma M, Kuga K, Aonuma K, Kohda Y. CYP2D6 genotype affects agerelated decline in flecainide clearance: a population pharmacokinetic analysis. Pharmacogenet Genomics 2012;22:777e83. [20] Zineh I, Beitelshees AL, Gaedigk A, Walker JR, Pauly DF, Eberst K, et al. Pharmacokinetics and CYP2D6 genotypes do not predict metoprolol adverse events or efficacy in hypertension. Clin Pharmacol Ther 2004;76:536e44. [21] Jurica J, Bartecek R, Zourkova A, Pindurova E, Sulcova A, Kasparek T, et al. Serum dextromethorphan/dextrorphan metabolic ratio for CYP2D6 phenotyping in clinical practice. J Clin Pharm Ther 2012;37:486e90. R, Frabetti L, Ambrosioni E, et al. [22] Boriani G, Strocchi E, Capucci A, Calliva Flecainide: evidence of non-linear kinetics. Eur J Clin Pharmacol 1991;41: 57e9. €rike K, et al. In[23] Lohmann PL, Rao ML, Ludwig M, Griese EU, Zanger UM, Mo fluence of CYP2D6 genotype and medication on the sparteine metabolic ratio of psychiatric patients. Eur J Clin Pharmacol 2001;57:289e95. [24] Ieiri I, Yamada S, Seto K, Morita T, Kaneda T, Mamiya K, et al. A CYP2D6 phenotype-genotype mismatch in Japanese psychiatric patients. Pharmacopsychiatry 2003;36:192e6. [25] Gardiner SJ, Begg EJ. Pharmacogenetics, drug-metabolizing enzymes, and clinical practice. Pharmacol Rev 2006;58:521e90.
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Regular article
Serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity Kosuke Doki a, Yukio Sekiguchi b, Keisuke Kuga b, Kazutaka Aonuma b, Masato Homma a, * a b
Department of Pharmaceutical Sciences, Faculty of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2014 Received in revised form 4 March 2015 Accepted 2 April 2015 Available online 11 April 2015
The aims of this study were to clarify whether the ratio of S- to R-flecainide (S/R ratio) in the serum flecainide concentration was associated with the stereoselectivity of flecainide metabolism, and to investigate the effects of the cytochrome P450 (CYP) 2D6 (CYP2D6) genotype and CYP2D6 inhibitor on the serum flecainide S/R ratio. In vitro studies using human liver microsomes and cDNA-expressed CYP isoforms suggested that variability in the serum flecainide S/R ratio was associated with the stereoselectivity of CYP2D6-mediated flecainide metabolism. We examined the serum flecainide S/R ratio in 143 patients with supraventricular tachyarrhythmia. The S/R ratio was significantly lower in intermediate metabolizers and poor metabolizers (IMs/PMs) than in extensive metabolizers (EMs) identified by the CYP2D6 genotype. The cut-off value for the S/R ratio to allow the discrimination between CYP2D6 EMs and IMs/PMs was 0.99. The S/R ratio in patients with co-administration of bepridil, a potent CYP2D6 inhibitor, was lower than 0.99, regardless of the CYP2D6 genotype status. Other factors, including age, sex, body weight, and renal function, did not affect the serum flecainide S/R ratio. This study suggests that the serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity on coadministration of a CYP2D6 inhibitor.
Keywords: Flecainide S/R ratio CYP2D6 Genotype CYP2D6 inhibitor
Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction Flecainide is a substrate of cytochrome P450 (CYP) 2D6, a highly polymorphic enzyme [1] and a chiral compound administered as a racemic mixture for the treatment of various supraventricular tachyarrhythmias [25]. Flecainide is principally metabolized to mO-dealkylated flecainide (MODF) by hepatic CYP2D6 and CYP1A2 enzymes and is subsequently oxidized to m-O-dealkylated lactam (Fig. 1), although 30% of the dosed amount is excreted in urine as the unchanged form [68]. Impaired CYP2D6 activity reduces flecainide clearance by 21% in intermediate metabolizers (IMs) and by 42% in poor metabolizers (PMs) [9,10]. The CYP2D6-mediated oxidation phenotype appears to be a determinant of stereoselective flecainide metabolism [1114]. Impairment of CYP2D6 activity preferentially increases the serum concentration of R-flecainide compared with that of S-flecainide
* Corresponding author. Tel.: þ81 298 53 3859; fax: þ81 298 96 7025. E-mail addresses: [email protected] (K. Doki), [email protected] (Y. Sekiguchi), [email protected] (K. Kuga), [email protected] (K. Aonuma), [email protected] (M. Homma).
(Fig. 1) [1114]. Nevertheless, whether CYP2D6 or CYP1A2 is responsible for the metabolism of flecainide enantiomers remains unclear. The ratio of S- to R-flecainide (S/R ratio) in the serum flecainide concentration may reflect CYP2D6 activity in arrhythmic patients receiving flecainide therapy, because the serum flecainide S/R ratio is lower in healthy subjects with impaired CYP2D6 activity than in those with normal CYP2D6 activity. We previously reported that the CYP2D6 genotype affected the serum flecainide S/R ratio in healthy subjects and patients receiving flecainide therapy [14]. However, the cut-off value for the serum flecainide S/R ratio when discriminating between CYP2D6 extensive metabolizers (EMs) and IMs/PMs remains unclear. In the present study, we examined the effects of CYP2D6 and CYP1A2 on the metabolism of flecainide enantiomers in an in vitro study using human liver microsomes and cDNA-expressed CYP isoforms to clarify the changes in the serum flecainide S/R ratio due to altered metabolic enzyme activity. We also investigated the effects of the CYP2D6 genotype and CYP2D6 inhibitor on the serum flecainide S/R ratio in routine therapeutic drug monitoring samples of patients with supraventricular tachyarrhythmia.
http://dx.doi.org/10.1016/j.dmpk.2015.04.001 1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The chemical structures of flecainide and m-O-dealkylated flecainide (MODF). Chiral center in the molecule is indicated by an asterisk. Solid and dashed arrows represent major and minor elimination pathways of flecainide enantiomers, respectively. R-flecainide is preferentially metabolized by CYP2D6 compared to S-flecainide.
2. Methods 2.1. Chemicals and microsomes Flecainide acetate, MODF, and an internal standard [N-(2piperidinylmethyl)-2,3-bis(2,2,2-trifluoroethoxy)benzamide acetate] were kindly supplied by Eisai Co. (Tokyo, Japan). S- and Rflecainide, bufuralol hydrochloride, and 10 -hydroxy bufuralol were purchased from Santa Cruz Biotechnology Inc. (CA, USA). Propranolol hydrochloride, bepridil hydrochloride, quinidine, potassium hexafluorophosphate, and perchloric acid were purchased from Wako Pure Chemicals (Osaka, Japan). Pooled microsomes from 50 human livers, microsomes from a baculoviruseinsect cell line expressing CYP2D6.1 (wild-type), CYP1A2, CYP2C9, CYP2C19, CYP3A4, control microsomes, and the NADPH-regeneration system solution were obtained from BD Gentest (Woburn, MA, USA). 2.2. In vitro study of flecainide metabolism All experiments were conducted such that product formation increased linearly with the incubation time and protein concentration. After pre-incubation for 5 min, incubations were performed in duplicate at 37 C in a shaking water bath for 30 min. Incubation mixtures contained microsome protein (1 mg/mL), flecainide enantiomer (1e800 mM), and an NADPH-regeneration system [NADP (1.4 mM), glucose 6-phosphate (3.3 mM), MgCl2 (3.3 mM), glucose-6-phosphate dehydrogenase (2 U/mL), final concentrations] in 200 mL of 100-mM potassium phosphate buffer (pH 7.4). The incubations for microsomes (20 pmol CYP) prepared from a baculoviruseinsect cell line expressing CYP2D6.1, CYP1A2, CYP2C9, CYP2C19 and CYP3A4 were performed as described above, except that the incubation time was 10 min in CYP2D6.1 and the concentration ranges of flecainide enantiomer were from 1 to 50 mM for CYP2D6.1, 25e400 mM for CYP1A2, and 25 mM for other CYPs. The reaction was terminated by the addition of 500 mL of ice-cold acetonitrile. After addition of 50 mL of an internal standard (1 mg), the mixtures were vortexed and centrifuged at 8960 g for 5 min. The supernatant was evaporated to dryness under a stream of nitrogen at 45 C. The residue was reconstituted with 100 mL of a mobile phase solution, and 40 mL was injected into a highperformance liquid chromatography (HPLC) system. MODF in the microsomal incubation mixture was determined by HPLC on a conventional octadecylsilyl silica column and a fluorescence detector, as previously described [15]. The coefficients of variations (CV) in intra-day and inter-day assays were 3.0%e4.2% and 3.3%e 6.7%, respectively. 2.3. In vitro inhibition study of bufuralol 10 -hydroxylation Bufuralol 10 -hydroxylation activity was assayed in microsomes (2-pmol CYP) prepared from a baculoviruseinsect cell line
expressing CYP2D6.1. Chemical inhibition of bufuralol 10 -hydroxylation was examined by incubating bufuralol (10 mM) in the presence of bepridil or quinidine (reference CYP2D6 inhibitor). The incubations were performed as described above, except that the incubation time was 5 min without pre-incubation. The reaction was terminated by addition of 20 mL of ice-cold perchloric acid. After the addition of 100 mL of an internal standard (1 mM propranolol), the mixtures were vortexed and centrifuged at 8960 g for 5 min. The 20 mL supernatant was injected into an HPLC system. In the microsomal incubation mixture, 10 -hydroxy bufuralol was quantified using HPLC on a conventional octadecylsilyl silica column and a fluorescence detector, following a previously described method but with minor modifications [16]. The mobile phase solution, comprising 2 mM perchloric acid and acetonitrile (70:30, v/ v), was pumped at a flow rate of 1.2 mL/min. The analytical column was maintained at 40 C. Excitation and emission wavelengths were set at 252 and 302 nm, respectively. The CVs in intra-day assays were less than 1%. 2.4. Patients Patients receiving oral flecainide for supraventricular tachyarrhythmias were enrolled during an outpatient visit to our hospital (Table 1). Exclusion criteria were a history of unstable angina or myocardial infarction; recent cardiac surgery; a high-degree atrioventricular nodal block; pacemaker-dependent rhythms; permanent atrial fibrillation; Brugada's syndrome; concomitant therapy, including other class I anti-arrhythmic drugs or amiodarone; and left ventricular dysfunction (i.e., ejection fraction of <50% and/ or a history of heart failure). The patients had received oral flecainide (0.7e5.3 mg/kg/day as flecainide acetate). Patients received the following drugs as needed: digoxin, b-blockers (carvedilol, atenolol, carteolol, nadolol, bisoprolol, metoprolol, and propranolol), Ca2þ antagonists (bepridil, verapamil, diltiazem, nifedipine, amlodipine, nisoldipine, and nicardipine), angiotensin-converting enzyme inhibitors (enalapril, lisinopril, and temocapril), angiotensin II receptor blockers (valsartan, telmisartan, olmesartan, losartan, and candesartan), anti-thrombotics (warfarin, aspirin, and ticlopidine), H2-blockers (famotidine and ranitidine), proton pump inhibitors (lansoprazole, rabeprazole), or other drugs (lipiddecreasing drugs and HMG-CoA reductase inhibitors). Estimated glomerular filtration rate (eGFR) was calculated for each patient from their serum creatinine value (SCr) and their age using the following equation [17]: eGFR (mL/min/1.73 m2) ¼ 194 age0.287 SCr1.094 (if female then, 0.739) This study was approved by the Ethics Committee of the University of Tsukuba. Informed consent was obtained from all patients who participated in the study.
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Table 1 Kinetic parameters for MODF formation by human liver microsomes and cDNA-expressed CYPs. R-flecainide
S-flecainide
Km
Vmax
CLint
Km
Vmax
CLint
(mM)
(pmol/min)*
(mL/min)*
(mM)
(pmol/min)*
(mL/min)*
Pooled microsome High-affinity 1.3 ± 1.1 component (CYP2D6) Low-affinity 406.8 ± 47.2 component (CYP1A2) Microsomes containing cDNA-expressed CYP CYP2D6*1 2.8 ± 0.3 CYP1A2 106.4 ± 15.0
0.5 ± 0.1
0.369
2.3 ± 5.8
0.2 ± 0.2
0.092
9.0 ± 0.4
0.022
403.1 ± 58.8
10.6 ± 0.6
0.026
29.8 ± 0.9 16.3 ± 0.9
10.764 0.153
7.2 ± 0.5 37.8 ± 7.6
30.6 ± 0.7 8.0 ± 0.4
4.236 0.212
* Vmax and CLint were expressed as per mg protein for human liver microsomes or per nmol CYP for microsomes containing cDNA-expressed CYP. Kinetic parameters were calculated from a fitted curve by non-linear regression (mean ± SE).
2.5. Determination of flecainide enantiomers in serum Patients had received flecainide for at least 2 months before blood withdrawal was performed for determining their serum flecainide concentration. To determine flecainide trough levels, blood collections were performed between 8:30 AM and 11:00 AM during outpatient visits. On sample collection days, the patients postponed taking their morning flecainide until after the blood collection. Serum samples were separated from whole blood and stored at 20 C until analysis. Serum concentrations of flecainide enantiomers were determined using an HPLC equipped with a polysaccharide-based chiral column (Chiralpak AS-RH) and a fluorescence detector, as previously described [14]. The CVs in intra-day and inter-day assays were 1.8%e5.8% and 3.4%e7.5%, respectively. No chiral inversion between S- and R-flecainide was observed experimentally in incubation mixtures using plasma or human liver microsome at 37 C for 12 h. 2.6. Genotyping of CYP2D6 Patients were genotyped for CYP2D6*1, *2, *4, *5, *10, *14, *21, *36, and CYP2D6xN using allele-specific polymerase chain reaction (PCR) with mismatch primers and step-down PCR [18]. We designated the CYP2D6*1 and *2 alleles as EM alleles; *10 as an IM allele; and *4, *5, *14, *21, and *36 as PM alleles. We used these categories to assign the following CYP2D6 genotype groups: EMs (EM/EM, EM/ IM, EM/PM), IMs (IM/IM, IM/PM), and PMs (PM/PM). 2.7. Data and kinetic calculations Enzyme kinetic parameters were calculated using SigmaPlot (ver. 10.0; Systat Software, Richmond, CA, USA). Kinetic analyses were obtained from an EadieeHofstee plot. The kinetics were described by one of the two following models: 1-enzyme MichaeliseMenten model: V ¼ Vmax$S/(Km þ S) 2-enzyme MichaeliseMenten model: V ¼ Vmax1$S/(Km1 þ S) þ Vmax2$S/(Km2 þ S), where V is the velocity of the reaction at substrate concentration S, Vmax is the maximum velocity, and Km is the substrate concentration at which the reaction velocity is 50% of Vmax. Intrinsic clearance of the in vitro incubation was calculated as CLint ¼ Vmax/Km. The half-maximal inhibitory concentration (IC50) was determined by analyzing the logarithmic plot of the inhibition of the inhibitor
concentration versus the percentage of activity remaining after inhibition. A receiver operating characteristic (ROC) curve was generated to distinguish between CYP2D6 EMs and IMs/PMs. The sensitivity (the proportion of true positive IMs/PMs) and specificity (the proportion of true negative) were calculated from the data across all possible cut-off values within the range of the S/R ratio. We determined the optimal cut-off value by maximizing the Youden index, which is defined as (sensitivity þ specificity 1). 2.8. Statistical analyses Data are expressed as numbers or mean ± SD. The influence of continuous parameters on the serum flecainide S/R ratio was evaluated as the dichotomous parameter divided by the mean value, except that the cut-off value for eGFR was based on the diagnostic criterion for chronic kidney disease. The serum flecainide S/R ratio and concentration-to-dose ratio were compared between two patient groups using Student's t-test or the ManneWhitney U-test. A P-value of <0.05 was considered statistically significant. 3. Results 3.1. Stereoselectivity of MODF formation in human liver microsomes and cDNA-expressed CYPs MichaeliseMenten and EadieeHofstee plots supported a twoenzyme model and biphasic kinetics of MODF formation from Sand R-flecainide in pooled human liver microsomes (Fig. 2). The estimated Km, Vmax, and CLint values of MODF formation are shown in Table 1. CLint for the high-affinity component was 4-fold higher in R-flecainide than in S-flecainide, whereas for the low-affinity component, it was only slightly higher in S-flecainide than in Rflecainide. MODF formation was also examined in microsomes containing cDNA-expressed CYP2D6.1 and CYP1A2. The estimated Km, Vmax, and CLint values of MODF formation are shown in Table 1. CLint for CYP2D6.1 was 2.5-fold higher in R-flecainide than in S-flecainide, whereas for CYP1A2, it was only slightly higher in S-flecainide than in R-flecainide. Other CYP isoforms showed no or weak activity of MODF formation. 3.2. Inhibitory effect of bepridil on bufuralol 10 -hydroxylation in vitro The inhibitory effect of bepridil on CYP2D6 activity was examined using the probe reaction bufuralol 10 -hydroxylation. Bufuralol
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Fig. 2. Kinetic profiles of m-O-dealkylated flecainide (MODF) formation from (a) R-flecainide and (b) S-flecainide in pooled human liver microsomes with an inset EadieeHofstee plot demonstrating the biphasic kinetics. Data are the means of duplicate determinations.
Table 2 Patients' characteristics.
Table 3 Influences of patients' characteristics on the serum flecainide S/R ratio.
Characteristics Number of patients Sex (male/female) Age (years) Body weight (kg) CYP2D6 genotype EMs IMs/PMs Current smoking Co-administration of bepridil Liver and kidney function AST (IU/L) ALT (IU/L) Serum creatinine (mg/dL) eGFR (mL/min) Flecainide dose (mg/kg/day) Serum concentration (ng/mL) S-flecainide R-flecainide S/R ratio
Characteristics 143 112/31 61 ± 13 66 ± 11 118 25 13 21 24 ± 8 23 ± 10 0.84 ± 0.20 71 ± 17 2.3 ± 0.8 162 ± 78 169 ± 81 0.96 ± 0.10
Data presented as number or mean ± SD. EMs, extensive metabolizers; IMs/PMs, intermediate metabolizers and poor metabolizers; eGFR, estimated glomerular filtration rate.
10 -hydroxylation activity in microsomes containing cDNAexpressed CYP2D6.1 was inhibited by bepridil with the IC50 of 0.016 mM. The IC50 of quinidine, a typical CYP2D6 inhibitor, was 0.008 mM. 3.3. Patients One hundred and forty-three patients treated with oral flecainide for supraventricular tachyarrhythmias were included in our analysis of serum flecainide S/R ratios. The demographic, physiological, and CYP2D6 genetic characteristics are summarized in Table 2. The CYP2D6 genotype was categorized as CYP2D6 EMs (n ¼ 118) or IMs/PMs (n ¼ 25). Twenty-one patients were coadministered bepridil, a potent CYP2D6 inhibitor. 3.4. Evaluation of the serum flecainide S/R ratio Trough serum concentrations of S- and R-flecainide were 162 ± 78 ng/mL and 169 ± 81 ng/mL, respectively (Table 2). The serum flecainide S/R ratio was 0.96 ± 0.10, and it ranged from 0.71 to 1.26. We examined whether the serum flecainide S/R ratio was influenced by age, sex, body weight, eGFR, daily dose, the CYP2D6 genotype, or co-administration of bepridil (Table 3). No differences
Age <60 yr 60 yr Sex Male Female Body weight <66 kg 66 kg eGFR <60 mL/min 60 mL/min Dose <2.3 mg/kg 2.3 mg/kg CYP2D6 genotype EMs IMs/PMs Co-administration of bepridil Yes No
S/R ratio
P-value
0.97 ± 0.10 0.96 ± 0.10
0.74
0.96 ± 0.10 0.99 ± 0.10
0.14
0.97 ± 0.10 0.96 ± 0.10
0.55
0.95 ± 0.11 0.97 ± 0.10
0.32
0.98 ± 0.11 0.95 ± 0.09
0.04
0.98 ± 0.10 0.90 ± 0.07
0.0001
0.87 ± 0.07 0.98 ± 0.09
0.000001
Data presented as mean ± SD. eGFR, estimated glomerular filtration rate; EMs, extensive metabolizers; IMs/PMs, intermediate metabolizers and poor metabolizers.
were found in serum flecainide S/R ratios between patients aged <60 and 60 years, males and females, body weight of <66 and of 66 kg, and eGFR of <60 and of 60 mL/min. The serum flecainide S/R ratio significantly differed between patients with a daily dose of <2.3 and 2.3 mg/kg (0.98 ± 0.11 vs. 0.95 ± 0.09, P ¼ 0.04), CYP2D6 genotype groups (EMs; 0.98 ± 0.10 vs. IMs/PMs; 0.90 ± 0.07, P ¼ 0.0001), and with and without co-administration of bepridil (0.87 ± 0.07 vs. 0.98 ± 0.09, P ¼ 0.000001). We examined the cut-off value for the serum flecainide S/R ratio in the CYP2D6 phenotype by comparing CYP2D6 EMs and IMs/PMs in patients without co-administration of bepridil (EMs; 1.00 ± 0.09 vs. IMs/PMs; 0.91 ± 0.07, P ¼ 0.00004; Fig. 3a). An ROC curve was generated to select the optimal cut-off for the serum flecainide S/R ratio to discriminate between CYP2D6 EMs and IMs/PMs. The optimal cut-off value was 0.99, which led to the highest Youden index based on the sensitivity of 0.95 and specificity of 0.54. Among the CYP2D6 EMs, the flecainide dose was significantly higher in patients with a serum flecainide S/R ratio of <0.99 than in those with a serum flecainide S/R ratio of 0.99 (2.7 ± 0.8 vs. 2.1 ± 0.7 mg/ kg, P ¼ 0.0001; Fig. 3b). The serum flecainide S/R ratio in patients with coadministration of bepridil was less than 0.99 (0.87 ± 0.07) and
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Fig. 3. The effect of the CYP2D6 genotype on the serum flecainide S/R ratio in patients without and with co-administration of bepridil (a). Solid and dotted lines are the mean value and cut-off value (0.99), respectively. The relationship between the daily dose of flecainide and serum flecainide S/R ratio in CYP2D6 EMs patients without co-administration of bepridil (b). The dotted line is the cut-off value (0.99).
did not differ between CYP2D6 EMs and IMs/PMs (Fig. 3a). The concentration-to-dose ratio, which was calculated as the total serum flecainide trough concentrations divided by the dose per kilogram of body weight, was 1.3-fold higher in patients with coadministration of bepridil than those without co-administration (192 ± 84 vs. 143 ± 55, P ¼ 0.002). 4. Discussion This study revealed that the serum flecainide S/R ratio was associated with the CYP2D6 genotype and changes in CYP2D6 activity. A lower serum flecainide S/R ratio corresponded to impaired CYP2D6 activity derived from the CYP2D6 IMs/PMs genotype or coadministration of the potent CYP2D6 inhibitor (Table 3; Fig. 3a). Other factors, including age, female sex, body weight, and renal function, were important for estimating flecainide clearance in the population pharmacokinetic analysis [10,19], but they did not affect the serum flecainide S/R ratio (Table 3). These results suggested that the serum flecainide S/R ratio provided specific information regarding CYP2D6 activity during therapeutic drug monitoring of flecainide. MODF formation in pooled liver microsomes is mediated by high- and low-affinity enzymes, which are associated with CYP2D6 and CYP1A2, respectively [8]. The present in vitro study revealed that the enzyme with a high-affinity for MODF formation was responsible for the stereoselectivity of flecainide metabolism. A high-affinity component in pooled human liver microsomes and recombinant CYP2D6.1 produced a faster MODF formation during the incubation of R-flecainide than during the incubation of S-flecainide (Fig. 2; Table 1). In contrast, MODF formation with the lowaffinity component in pooled human liver microsomes and recombinant CYP1A2 was slightly faster during the incubation of Sflecainide than during the incubation of R-flecainide (Fig. 2; Table 1). These findings suggest that impaired CYP2D6 activity was associated with a reduction in R-flecainide metabolism, resulting in a lower serum flecainide S/R ratio (Fig. 1). We determined an optimal cut-off value of the serum flecainide S/R ratio for the discrimination between CYP2D6 EMs and IMs/PMs (Fig. 3a). The ROC curve analysis showed that the optimal cut-off value was 0.99. Most of the CYP2D6 IMs/PMs genotype was identified as the IMs/PMs phenotype using this cut-off value. We could not determine the cut-off value of the S/R ratio for the discrimination between CYP2D6 IMs and PMs because the study population included only one CYP2D6 PMs with the lowest serum flecainide S/R ratio of 0.75 (Fig. 3a). The serum flecainide S/R ratio for the CYP2D6
EMs genotype overlapped between the EMs and IMs/PMs phenotype. This may be due to the variability in the activity of CYP2D6mediated metabolism of flecainide in CYP2D6 EMs as well as other CYP2D6 substrates, metoprolol and dextromethorphan [20,21]. The non-linearity [22] resulting from the saturation of CYP2D6mediated metabolism may partially explain the overlapping S/R ratio because patients with a lower S/R ratio (<0.99) had received a higher flecainide dose (Fig. 3b). A higher S/R ratio may be found in CYP2D6 ultrarapid metabolizers (UMs) who show excessively high CYP2D6 enzyme activity with multiple copies of the CYP2D6 gene, although CYP2D6 UMs were not included in the present study. We further examined the effect of concomitant bepridil administration on the serum flecainide S/R ratio because the present in vitro study indicated that the inhibitory effect of bepridil on CYP2D6 activity was comparable to that of quinidine, a reference CYP2D6 inhibitor. Patients who received co-administration of bepridil showed a serum flecainide S/R ratio lower than the cut-off value, regardless of the CYP2D6 genotype status (Fig. 3a). The lower S/R ratio was due to the inhibitory effect of bepridil on the CYP2D6-mediated metabolism of flecainide. We considered that a serum flecainide S/R ratio of <0.99 in EMs with co-administration of bepridil was attributed to the phenocopying phenomenon [23e25], in which co-administration of potent CYP2D6 inhibitors induces the conversion of the CYP2D6 EMs phenotype to the PMs phenotype. Therefore, the serum flecainide S/ R ratio may be a useful tool to assess the drug interaction between flecainide and CYP2D6 inhibitors in addition to the CYP2D6 genotype. This suggests that the serum flecainide S/R ratio reflects realistic CYP2D6 activity rather than CYP2D6 genotyping. The serum flecainide S/R ratio may not be associated with flecainide efficacy and safety because a similar anti-arrhythmic activity was reported between the enantiomers. A merit for determining the ratio is to assess CYP2D6 activity corresponding to the genetic polymorphisms and drug interaction on CYP2D6. We previously reported that the CYP2D6 genotype is a useful determinant for age-related decline in flecainide clearance [19]. Because CYP2D6 genotyping requires the analysis of many mutant alleles to assess enzyme activity [1], the serum flecainide S/R ratio is a convenient determinant to assess enzyme activity in routine therapeutic drug monitoring. In conclusion, the serum flecainide S/R ratio is a useful tool to assess CYP2D6 activity in patients with arrhythmia receiving flecainide therapy. The present results suggest that the serum flecainide S/R ratio reflects the effects of the CYP2D6 genotype and coadministration of a CYP2D6 inhibitor on CYP2D6 activity.
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