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Effects of Cytochrome P450 (CYP) 2C19 Genotypes on Steady-State Plasma Concentrations of Escitalopram and its Desmethyl Metabolite in Japanese Patients With Depression

Background: Plasma concentrations of the S-enantiomer of citalopram were different between extensive and poor CYP2C19 metabolizers in healthy subjects and depressed patients. However, most studies applied dose-corrected concentrations. Thus, we studied the effects of polymorphisms of the CYP2C19 gene on raw plasma drug concentrations in Japanese patients with depression. Methods: Subjects in this study consisted of 412 depressed patients receiving 5, 10, 15, or 20 mg of escitalopram once a day. Plasma concentrations of escitalopram and desmethylescitalopram were quantified using HPLC. CYP2C19 genotypes were identified using polymerase chain reaction methods. Results: There were no differences in the steady-state plasma concentrations of escitalopram or desmethylescitalopram in each dose group (5, 10, 15, or 20 mg of escitalopram) among CYP2C19 genotype groups. However, 1-way analysis of variance showed significant effects of CYP2C19 genotypes on the dose-adjusted plasma concentration of escitalopram but not in the dose-adjusted plasma concentration of desmethylescitalopram. Analysis of covariance including age, sex, and body weight showed significant effects of CYP2C19 genotypes on the dose-adjusted plasma concentration of escitalopram and the ratio of desmethylescitalopram to escitalopram. Conclusions: These findings suggest that the CYP2C19 variants are associated with steady-state plasma concentrations of escitalopram to some extent but are not associated with desmethylescitalopram.


INTRODUCTION
Escitalopram is the therapeutically active S-enantiomer of racemic citalopram, a commonly prescribed selective serotonin reuptake inhibitor. Escitalopram and citalopram have been used to treat patients with anxiety and major depressive disorders. [1][2][3] Escitalopram exerts a highly selective, potent, and dose-dependent inhibitory effect on human serotonin transport. 1 By inhibiting the reuptake of serotonin into presynaptic nerve endings, it enhances the activity of serotonin in the central nervous system. 4 Escitalopram is approved at clinical doses of 10 and 20 mg (5 mg in certain subpopulations or as an initial dose). When taken orally, escitalopram reaches t max within 5 hours and is 56% bound to protein. Escitalopram reaches steady-state concentrations in the blood within 1-2 weeks. 5,6 Escitalopram is primarily metabolized by cytochrome P450 (CYP) 2C19, which is a highly polymorphic enzyme known to cause interindividual differences in pharmacokinetics. 5,6 In a previous study, we demonstrated that compared with the extensive metabolizers of mephenytoin after racemic citalopram was given, 7 the AUC of the S-enantiomer of citalopram was significantly higher in the poor metabolizers of mephenytoin.
Approximately 15%-25% of the East Asian population consists of poor metabolizers (PM) of S-mephenytoin, whereas the PM frequency in whites is less than 5% (Man et al, 2010). 8 Most poor metabolism by CYP2C19 is attributable to the CYP2C19*2 and *3 alleles. 9,10 The allele frequencies of the CYP2C19*2 and CYP2C19*3 alleles were 0.30-0.35 and 0.11-0.13 in Asians, respectively, whereas they were 0.15 and 0.001 in whites, respectively. [8][9][10] Although several studies indicate the relationship between the CYP2C19 genotypes and the steady-state plasma concentration of escitalopram in the Asian and white population, [11][12][13][14] there is still little information about these associations in the Japanese population. In addition, these studies' design was only dose corrected. Therefore, we examined the impact of the CYP2C19 genotypes on the raw (noncorrected) steady-state plasma concentration of escitalopram in depressed Japanese patients at each dose. Furthermore, various factors other than CYP2C19 genotypes such as age, body weight, and sex difference were examined.

Subjects
The subjects of this study consisted of 412 depressed Japanese outpatients (121 men and 291 women) who fulfilled the criteria for major depressive disorders according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. The mean 6 SD (range) age and body weight were 43. 1

Protocol
The subjects received 5, 10, 15, or 20 mg of escitalopram once a day (8-10 PM) for 2-52 weeks. The elimination half-lives of escitalopram and desmethylescitalopram were previously reported to be 26 hours and 20-29 hours, respectively. 5 Therefore, plasma concentrations of these compounds already reached steady state in all the subjects before initiating the study. We confirmed adherence from patients. In the case in which the plasma drug concentration of escitalopram could not be detected, the data were excluded from the study (n = 10). We excluded the patients who had potential hepatic/renal dysfunction. The drugs coadministered were zolpidem 5-10 mg/d in 38 cases, brotizolam 0.25 mg/d in 26 cases, flunitrazepam 1-2 mg/d in 12 cases, lorazepam 1-3 mg/d in 19 cases, alprazolam 0.8-2.4 mg/d in 18 cases, and sennoside 24 mg/d in 56 cases.

Assays for Escitalopram and Desmethylescitalopram
Blood samples (5 mL) were obtained 14-16 hours after the last dose of escitalopram. The plasma samples were frozen and maintained at 2208C until analysis. The plasma concentrations of escitalopram and desmethylescitalopram were analyzed in duplicates using highperformance liquid chromatographic (HPLC) methods that were developed in our laboratory. 15 In brief, the extraction procedure was as follows: 500 mL of 0.5 M NaOH, 100 mL of internal standard solution (trifluperidol 200 mcg/mL), and 100 mL of methanol were added to 2000 mL of plasma. Tubes were vortexed for 10 seconds, and 5 mL of n-heptane-chloroform (70:30, vol/vol) was added as an extraction solvent. After 10 minutes of shaking, the mixture was centrifuged at 2500g for 10 minutes at 48C, and the organic phase was evaporated in vacuum conditions at 408C until dry (TAITEC VC-960; Shimadzu, Kyoto, Japan). The residue was dissolved in 500 mL in the mobile phase, and 400 mL was injected onto the HPLC system. The HPLC system consisted of Shimadzu LC-10AT high-pressure pumps, a Shimadzu CTO-10AVP column oven, a Shimadzu Workstation CLASS-VP chromatography integrator (Kyoto, Japan), a Shimadzu SPD-10AVP (Kyoto, Japan), a Shimadzu SIL-10ADVP (500-mL injection volume) (Tokyo, Japan), and a column (STR-ODS II C18 150 · 4.6, 3 mm) (Shimadzu, Tokyo, Japan). The mobile phase was composed of phosphate buffer (0.02 M, pH = 4.6), acetonitrile, and perchloric acid (60%) (57.25: 42.5: 0.25, vol/vol/vol). The lower limits of detection were 1.0 ng/mL, and the values of the interassay coefficient of variation were less than 8.7% at all concentrations (2.5-150 ng/mL) of the calibration curve for escitalopram and desmethylescitalopram.

Data Analyses
The comparison of plasma drug concentrations among CYP2C19 genotypes was performed using 1-way analysis of variance. Analysis of covariance was used for analyses of the effects of age, sex, body weight, and CYP2C19 genotype on plasma drug concentrations. Dummy variables for sex were as follows; male = 1, female = 2. A P value of 0.05 or less was regarded statistically significant. SPSS 22.0 for Windows SPSS, Japan Inc, Tokyo was used for these statistical analyses.
Analysis of covariance showed that CYP2C19 genotypes and age were correlated with the steady-state plasma concentration of escitalopram, whereas CYP2C19 genotypes and sex were correlated with the metabolite ratio (Tables 2 and 3).

DISCUSSION
The results of this study suggested that there was a significant correlation between the number of mutated alleles for CYP2C19 and the steady-state plasma concentration of escitalopram corrected by dose. However, no significant difference was found in the steady-state plasma concentration of escitalopram in the patients receiving 5, 10, 15, or 20 mg of escitalopram, probably because of the large variability in steady-state plasma concentrations of escitalopram. This finding is in accordance with previous studies. [11][12][13][14] Therefore, CYP2C19 genotypes have an impact, to some extent, on the steady-state plasma concentration of escitalopram in depressed patients. A recent meta-analysis showed that compared with subjects with the homo EM, the exposure to (es) citalopram increased by 95% (95% CI 40-149, P , 0.0001) in the PM and 30% (95% CI 4-55, P , 0.05) in the hetero EM. 16 Our study indicated a 73% increase in the PM and a 20% increase in the hetero EM, which was shown as a weaker effect of CYP2C19. However, another study reported 276%-230% in PM and 97%-78% in hetero EM in 541 patients, showing a much stronger effect of CYP2C19. 14 In the meta-analysis, 800 patients were included, whereas the number of subjects in this study and that in the study by Waade et al 14 were over 400 and 500, respectively. Therefore, repeated analyses together with additional studies should be conducted to calculate the true effect.
Contrary to our prediction, compared between CYP2C19 genotypes, no differences were found at each dose. These findings may be explained by the large interindividual variability in the plasma concentrations of escitalopram. An in vitro study showed that not only CYP2C19 but also CYP3A4 is involved in the N-demethylation of escitalopram. 17 It has been shown that there is a great interindividual variation in the activity of CYP3A4. 18 Therefore, the variability in CYP3A4 possibly influenced the variability in plasma concentrations of escitalopram. In addition,   deamination and dehydrogenation to a propionic acid derivative, N-oxidation, and glucuronide conjugations other than N-demethylation were confirmed. 19,20 These minor metabolic pathways could confound the results and might have been activated after repeated doses. Finally, a steady-state plasma concentration in repeated doses was influenced by various factors, such as compliance, drugdrug and/or drugfood interaction, and renal and hepatic impairment.
A significant correlation was found between the steadystate plasma concentration of escitalopram and age. This is a novel finding, but it is not surprising because renal function tends to decrease with advanced age. A previous study showed that in patients with mild to moderate renal impairment [creatinine clearance (CL CR ) 10-53 mL/min], the t 1/2 of a single dose of citalopram increased by approximately 35%, with oral and renal clearance being approximately 15% and 40% lower than that in healthy volunteers, respectively. 6 Therefore, careful dose setting and possibly therapeutic drug monitoring are needed not only in CYP2C19 PM but also in elderly patients. However, Waade et al 14 showed that for CYP2C19 PMs, compared with those ,40 years, a 1.4-fold higher mean concentration/dose ratio of escitalopram was observed in patients .65 years, but the difference was not statistically significant (P = 0.1). In the other CYP2C19 phenotype subgroups, similar degrees of differences in mean concentration/dose ratios of escitalopram were found to be significant among patients aged .65 versus ,40 years (P , 0.02). 14 Furthermore, in this study, the metabolite ratio was significantly associated with CYP2C19 but also with sex. Waade et al 14 showed that metabolic ratios in women were 15% lower than those in men. Although we do not have a clear explanation for the sex difference, the different CYP3A4 activity between the sexes may be attributable to our findings because CYP3A4 is a major enzyme catalyzing N-demethylation of escitalopram. 21 There are several limitations to our study. First, our sample had a small number of patients with PM because genotypes were retrospectively identified, which has some methodological weaknesses. These issues include uncertain or unconfirmed information regarding partial adherence and the concurrent use of potentially interacting drugs or herbal agents. Second, we did not detect CYP2C19*17, which may be contributing to steady-state plasma concentrations of escitalopram 22,23 because it has been shown to be very rare (0.3%-1.3%) in Japanese populations. [24][25][26]

CONCLUSIONS
The present study suggests that CYP2C19 genotypes and age are determinants affecting dose-adjusted escitalopram concentrations. Particularly careful administration is necessary in elderly CYP2C19 PM patients.