The Pharmacogenomics Journal (2011) 11, 359–367 & 2011 Macmillan Publishers Limited. All rights reserved 1470-269X/11 www.nature.com/tpj ORIGINAL ARTICLE

Association between CYP2C19*17 and of , and in Dutch hospitalized patients

A de Vos1, J van der Weide1,2 Polymorphisms in genes coding for metabolizing enzymes, such as 1,2 the enzymes CYP2C19 and CYP2D6, can lead to and HM Loovers failure and side effects. In earlier studies, the novel variant CYP2C19*17 1Department of Clinical Chemistry, St Jansdal increased metabolism of several CYP2C19 substrates. The objective of this Hospital, Harderwijk, The Netherlands and study was to evaluate the impact of CYP2C19*17 on the metabolism of 2Psychiatric Hospital Meerkanten, Ermelo, amitriptyline (AT), citalopram (CIT), and clomipramine (CLOM). Six-hundred The Netherlands and seventy-eight patients were included in this study, based on availability of DNA and serum levels of parent drug and main metabolite. We Correspondence: Dr J van der Weide, Department of Clinical investigated the relationship between CYP2C19 genotypes and metabolic Chemistry, St Jansdal Hospital, P.O. Box 138, parameters, including serum levels corrected for dose and metabolic ratio Harderwijk 3840 AC, The Netherlands. (MR). The CYP2C19*17 allele was significantly associated with decreased MR E-mail: [email protected] for CIT (CYP2C19*1/*17 mean MR ¼ 2.3, compared with CYP2C19*1/*1 mean MR ¼ 2.8) and AT (CYP2C19*17/*17 mean MR ¼ 0.8, compared with CYP2C19*1/*1 mean MR ¼ 3.7 in the CYP2D6*1/*1 subgroup). Furthermore, significant association of CYP2D6 genotype with AT, CIT, and CLOM metabolism was observed. No clear correlation was found between CYP2C19 genotype and CLOM metabolism. This study confirms the increased activity of the CYP2C19*17 allele and shows increased metabolism of that are metabolized by CYP2C19, including AT and CIT. However, the clinical relevance of CYP2C19*17 is probably limited for AT, CIT, and CLOM. The Pharmacogenomics Journal (2011) 11, 359–367; doi:10.1038/tpj.2010.39; published online 8 June 2010

Keywords: pharmacogenetics; ; ; cytochrome P450; poly- morphism

Introduction

Although a wide range of different drug classes is available, about 30–50% of patients will not respond satisfactorily to treatment with standard psychiatric . Part of the variation in response and adverse effects is attributed to interindividual differences in metabolic activity. Cytochrome P450 (CYP450) enzymes are involved in the metabolism of many drugs, the family members CYP2C19 and CYP2D6 account for the metabolism of 30–40% of all . Many studies have shown clear correlations between metabolic ratios (MRs) of psychotropics and CYP2C19 and CYP2D6 genotypes.1–8 More than 20 poly- morphisms have been described for CYP2C19.9 In Caucasians, the homozygous Received 14 December 2009; revised 30 March 2010; accepted 27 April 2010; presence of the non-functional CYP2C19*2 allele accounts for 75–93% of poor published online 8 June 2010 metabolizers. The higher percentage of Asian poor metabolizers is attributed to CYP2C19*17 and metabolism of A de Vos et al 360

influences the metabolism of the three antidepressants. Furthermore, the number of patients outside therapeutic range were investigated to provide more direct information on the clinical consequences of CYP2C19 polymorphisms, in particular CYP2C19*17.

Materials and methods

Patients All adult patients of the Psychiatric Hospital Meerkanten (Ermelo, the Netherlands) treated with AT, CIT, and/or CLOM in the period 1998–2008 for which both data on serum levels of parent drug, serum levels of main metabolite (NT, DCIT, and DCLOM, respectively), and DNA samples were available were included in this study (n ¼ 678). Information on age, gender, CYP2D6 genotype, presence of CYP2C19*2, and serum levels of antidepressant were col- Figure 1 Main metabolic pathway for amitriptyline (AT), citalopram lected. To allow correction of serum levels for dose, data on (CIT), and clomipramine (CLOM). The metabolites , prescribed daily dose of AT, CIT, or CLOM were collected for desmethyl citalopram, and desmethyl clomipramine are abbreviated the days preceding blood collection. Information on daily to NT, DCIT, and DCLOM, respectively. dose was available for 460 patients. Information on co- medication was not collected. The use and publication of data on medication, CYP2C19, and CYP2D6 genotype and the non-functional CYP2C19*3 variant.10 As a substantial serum levels for research purposes has been approved by the part of the interindividual differences in CYP2C19 activity psychiatric hospital’s research board. For this study, patients can be explained by polymorphisms of CYP2C19, genotyp- were not subjected to any procedures besides routine clinical ing of CYP2C19 could potentially increase treatment practice. Patients are informed on the use of data for research efficacy of CYP2C19-dependent drugs. However, subjects purposes, and data are excluded from research on objection. carrying neither the CYP2C19*2 nor CYP2C19*3 allele still show large interindividual differences in CYP2C19 activity. CYP2C19 and CYP2D6 genotyping Part of these differences might be caused by the polymor- Patients were routinely screened for CYP2C19*2, CYP2D6*3, phism CYP2C19*17, which increased CYP2C19 expression *4, *5, *6, and multiplication. Blood was collected in the levels in vitro.11 Moreover, the CYP2C19*17 polymorphism EDTA. DNA was isolated using the QIAamp has been associated with increased metabolism of ome- DNA Blood MiniKIT (Qiagen, Hilden, Germany). Anoni- prazole, , and .12–14 The main mized DNA samples and whole blood were routinely stored metabolic pathway of citalopram (CIT) is at À70 1C for research purposes. CYP2C19*2, CYP2D6*3, *4, into desmethyl-CIT (DCIT) by CYP2C19, CYP3A4, and and *6 determinations were performed as single-tube tetra- CYP2D6 (Figure 1).1,15–19 The metabolite DCIT, which is primer assays using two control primers and two allele- about 10 times less active than CIT itself, is metabolized into specific primers (see Supplementary Appendix A and B). PCR didesmethyl-CIT by CYP2D6.15,19 The main metabolic reactions were performed using 50–250 ng DNA, 0.2 mM pathway of the active drug amitriptyline (AT) is demethyla- dNTPs, 1.0–2.5 U Thermoprime Plus DNA polymerase tion to the nortriptyline (NT) by (Abgene House, Epsom, UK) and 1.0–1.7 Â ReddyMix PCR CYP2C19 and subsequent to 10-hydroxy-NT buffer in a final volume of 25 ml. PCR products were by CYP2D6 (Figure 1).3,20–24 Clomipramine (CLOM) is visualized by agarose gel electrophoresis. To detect demethylated into N-desmethyl-CLOM (DCLOM) by CYP2C19*17, a real-time allelic discrimination assay was several CYP450 enzymes, including CYP2C19, CYP3A4, developed. On the basis of a publication by Sim et al.,11 a and CYP1A2.25,26 Hydroxylation of the active metabolite 470 base pair region of the CYP2C19 promotor was DCLOM is catalyzed by CYP2D6 (Figure 1).25–27 AT, CIT, amplified using the primer pair CYP2C19-forward and and CLOM are frequently prescribed antidepressants in the CYP2C19-reverse (see Supplementary Appendix A for pri- Psychiatric Hospital Meerkanten (Ermelo, the Netherlands). mers, probes and PCR cycling conditions). Since November Together, they represent one-third of all antidepressants 2007, detection of CYP2C19*2 was performed by melt-curve prescribed in this institute. We set out to assess the analysis (see Supplementary Appendix A for primers, probes, additional value of CYP2C19*17 genotyping in a psychiatric and PCR cycling conditions). This melt-curve analysis assay setting. Data on serum levels of AT, CIT, and CLOM and was developed in the laboratory of Twente Hospital (ZGT) by their main metabolites were collected. Several metabolic A de Vos and Dr RGHJ Maatman (unpublished data). In parameters were tested for association with CYP2C19*17, addition, since November 2007, detection of CYP2D6*3,*4, including MR and serum level corrected for dose (C/D), and *6 was performed using real-time allelic discrimination providing information on the extend in which CYP2C19*17 assays (see Supplementary Appendix B for primers, probes

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and PCR cycling conditions). These real-time assays were was performed comparing intra-assay values (samples of also developed in the laboratory of Twente Hospital (ZGT) patients with low, medium, and high serum levels measured by A de Vos and Dr RGHJ Maatman (unpublished data). All 10  within one assay) and inter-assay results (measuring real-time allelic discrimination assays and melt-curve assays samples of patients with low, medium, and high serum were performed using 20–50 ng DNA, 0.2 mM dNTPs, 1.25 U levels 6–7  in independent assays). Variance coefficient Taq Full Hot Start DNA Polymerase (Clontech Laboratories, values were 0.1 (both intra- and inter-assay) for AT and Mountain View, CA, USA) and 1  PCR buffer in a final CLOM and 0.04 (intra-assay) and 0.06 (inter-assay) for CIT. volume of 25 ml. Amplification, detection, and melt-curve Reference samples were obtained from the Dutch Associa- analysis were performed using the Rotorgene 6000 cycler tion for Quality Assessment in Therapeutic Drug Monitoring (Corbett, San Francisco, CA, USA). Detection of CYP2D6 and Clinical Toxicology (KKGT). The assays were subjected deletion and multiplication (CYP2D6*5 and CYP2D6xN, to regular external quality controls. respectively) was performed using 50–150 ng DNA, 0.36 mM dNTPs, 1.35 mM Mg(OAc)2, 1.3 U rTth DNA Polymerase XL Chemicals (Applied Biosystems, Carlsbad, CL, USA) and 0.9  3.3xXL The HPLC purified primers and probes used in real-time PCR Buffer II (see Supplementary Appendix B for primers and were supplied by Biolegio, the Netherlands. All other PCR cycling conditions). PCR products were visualized by primers were supplied by Sigma-Aldrich, St Louis, MO, agarose gel electrophoresis. All routinely performed assays USA. Primer and probe stocks (100 mM in water) were stored were validated using references samples and were subject to at À20 1C. Acetonitrile (cat. nr. 14291), di-isopropylether regular external quality controls. Reference samples (cat. nr. 867), (cat. nr. 11727), ethylacetate (cat. nr. CYP2C19*1/*1,*1/*17, and *17/*17 to validate the 868), heptane (cat. nr. 4390), isoamylalcohol (cat. nr. 979), CYP2C19*17 assay were kindly provided by Dr RHN van potassium dihydrogen phosphate (cat. nr. 4873), sodium Schaik (Erasmus MC Rotterdam, the Netherlands). hydroxide (cat. nr. 6498), and tert-butylamine (cat. nr. 1546) were supplied by Merck. The internal standards protriptyli- HPLC ne.HCl (cat. nr. P8813) and (cat. nr. Therapeutic drug monitoring was routinely performed D5676) were supplied by Sigma-Aldrich, and amitriptyline. for all patients. Blood samples for TDM were drawn HCl (cat. nr. Lu 00-020-C) was supplied by 12–16 h post-medication to ensure measurement of trough (, ). medication levels at steady state. Serum was stored at À20 1C within 6 h of collection. Serum levels of AT, CIT, Statistics and CLOM and their metabolites were determined by HPLC Statistical analyses were performed using SPSS for Windows within 14 days of collection. Liquid–liquid extraction was (SPSS version 15.0, Chicago, IL, USA). Cases were grouped performed with heptane/ethylacetate/isoamylalcohol/2 M according to genotype. A subset of data showed a significant NaOH (157:40:3:16 vol/vol/vol/vol) solution (AT and difference in variances, a lack in normality of residuals, and CLOM) or with di-isopropylether/isoamylalcohol/2 M NaOH large differences in sample size. Transformation to log values (99/1/8 vol/vol/vol) solution (CIT). Concentrations of AT, did not eliminate these difference in variances and residuals. CLOM, and their metabolites were determined using a Therefore, data were analyzed with the non-parametric Chromsper guardcolumn (SS 10  2 mm, silica) and two in Mann–Whitney to test the significance of differences between tandem Chromsper 5Si cartridge columns (100  3 mm, 5u) mean values of a specific subgroup and the preceding (Varian, Palo Alto, CA, USA) with ethanol/water/tert- subgroup, as well as the reference subgroup (CYP2C19*1/*1 butylamine (200:50:0.06 vol/vol/vol) as the mobile phase, and/or CYP2D6*1/*1). MRs were calculated as the ratio flow rate 0.5 ml min–1, UV detection at 254 nm. Since 2007, between steady-state concentrations of parent drug (AT, CIT, concentrations of AT, CLOM, and their metabolites were or CLOM) and the main metabolite (NT, DCIT, and DCLOM, analyzed on a Inertsil ODS 3 column (150 cm  4.6 mm, 4u) respectively). For each patient, the average of all MRs and C/

(Aurora Borealis Control) with 67 mM KH2PO4 pH 3/ Ds available were used. Group averages were calculated using acetonitrile (130/70 vol/vol) as the mobile phase, flow rate the averages for individual patients. The study population was 0.8 ml min–1 (AT) or 1.3 ml min–1 (CLOM), UV detection at divided into six subgroups based on CYP2C19 genotype 254 nm. This method was an adaptation of a method (*1/*1, *1/*17, *17/*17, *1/*2, *2/*2,and*2/*17). Association described by Aymard et al.28 The two methods produced studies with respect to CYP2D6 genotype were performed comparable concentration values. Concentrations of CIT using the semi-quantitative gene dose (SGD) system.3 Avalue and metabolite were determined by analysis of samples on a of 0 was appointed to non-functional alleles (CYP2D6*3,*4, Hypersil BDS C18 column (100 cm  4.6 mm, 3u) (Aurora *5,*6,and*4xN), a value of 1 to functional alleles (CYP2D6*1)

Borealis Control) with 50 mM KH2PO4 pH 3.7/acetonitrile and a value of 2 to gene multiplication of functional alleles (70/30 vol/vol), flow rate 0.8 ml min–1, UV detection at (CYP2D6*1xN). The SGD value is the sum of values of both 240 nm. This method was an adaptation of a method alleles, resulting in four subgroups (SGD ¼ 0, 1, 2, and 3). described by Rop et al.29 Internal standards were protripty- Patients with gene duplication in combination with alleles line (AT), amitriptyline (CLOM), and desmethylclozapine that differ in functionality (for example (CYP2D6*1/*4)xN), (CIT). Detection limits were 5 mgl–1 for AT, CLOM, and were excluded, as these patients could not be assigned to metabolites, and 1 mgl–1 for CIT and metabolite. Validation a specific SGD group.

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Results Increased age was associated with increased MR, increased C/D, and decreased daily dose for AT, CIT, and CLOM (data Group characteristics and CYP2C19*17 allele frequency not shown). The relationship between parent drug concen- A total of 678 adult patients using AT, CIT, or CLOM were tration and dose was non-linear for AT, CIT, and CLOM; a included in this study. Patients were screened for trend was observed for higher levels with higher dose, CYP2C19*17 using a real-time PCR method and data on whereas the C/D value decreased with increased doses (data CYP2C19*2 and CYP2D6 genotype were collected. Genotype not shown). frequencies and group characteristics are shown in Table 1. The overall allele frequency of CYP2C19*17 was 0.22. The Amitriptyline frequency of CYP2C19*17 observed in this Dutch popula- A total of 150 patients were included in the association tion of psychiatric patients is similar to the frequencies study for AT. Information on daily dose was available for 86 observed in Polish peptic ulcer patients (0.27) and in patients. The presence of CYP2C19*17 was associated with

Swedish (0.18), Norwegian (0.22), and Ethiopian (0.18) lower AT levels and MRAT/NT values (Table 2; Figure 2). This psychiatric patients, but higher than the frequency association did not reach significance. The presence of observed in Chinese psychiatric patients (0.04).11,12,30 CYP2C19*2 was significantly associated with higher AT

levels and MRAT/NT values. No significant associations were observed between CYP2C19 genotype and NT levels, AT þ NT levels, C/D values or daily dose. CYP2D6 genotype Table 1 Demographics affected AT metabolism in earlier studies, as well as in this study (see Supplementary Appendix C, 3,23). Therefore, the Total study AT CIT CLOM CYP2C19 a study population was grouped based on both population receivers receivers receivers and CYP2D6 genotype (Table 3). The association of CYP2C19*17 with MR reached significance within the Gender AT/NT CYP2D6*1/*1 subgroup (SGD ¼ 2). The sum of the active Male 246 (36%) 56 (37%) 120 (36%) 87 (36%) Female 432 (64%) 94 (63%) 218 (64%) 157 (64%) compounds AT and NT is used to follow-up AT therapy. CYP2C19 genotype did not significantly affect the propor- Age tion of subjects with AT þ NT serum levels below therapeutic 18–40 227 (33%) 38 (25%) 108 (32%) 103 (42%) range (100–300 mgl–1) (Table 3). The concentration of the 41–64 274 (40%) 81 (54%) 105 (31%) 114 (47%) active metabolite NT, rather then AT itself, has been asso- 65+ 177 (26%) 31 (21%) 125 (37%) 28 (11%) ciated with side effects of AT therapy.22 Both CYP2C19*17 and CYP2D6 non-functional alleles were significantly Genotypes associated with higher proportion of patients with NT serum *1/*1 289 (43%) 60 (40%) 143 (42%) 109 (45%) levels above therapeutic range (50–150 mgl–1). *1/*17 191 (28%) 45 (30%) 90 (27%) 67 (27%) *17/*17 31 (5%) 8 (5%) 18 (5%) 10 (4%) Citalopram *2/*2 14 (2%) 5 (3%) 6 (2%) 5 (2%) *1/*2 107 (16%) 21 (14%) 57 (17%) 37 (15%) Group characteristics of the 338 patients included in the *2/*17 46 (7%) 11 (7%) 24 (7%) 16 (7%) association study for CIT are shown in Table 4. Information on daily dose was available for 223 patients. The presence Abbreviations: AT, amitriptyline; CIT, citalopram; CLOM, clomipramine. of CYP2C19*17 was significantly associated with lower aTotal number of patients less than sum of AT, CIT, and CLOM receivers because of patients that received more than one of the investigated antidepressants in the MRCIT/DCIT values, whereas the presence of CYP2C19*2 was period 1998–2008. significantly associated with higher MRCIT/DCIT values and

Table 2 Characteristics of the CYP2C19 subgroups for AT

*1/*1 *1/*17 *17/*17 *2/*2 *1/*2 *2/*17 Total

AT (mgl–1) 77 (15–240) 69 (15–175) 67 (25–120) 142 (75–208)a 94 (15–255) 71 (28–132) 78 (15–255) NT (mgl–1) 71 (10–238) 62 (10–185) 83 (25–190) 45 (15–104) 63 (15–144) 47 (23–78) 65 (10–238) AT+NT (mgl–1) 148 (30–295) 131 (60–295) 151 (55–201) 186 (93–313) 157 (35–358) 119 (30–364) 144 (30–364) Dose (mg per day) 101 (25–300) 114 (25–225) 143 (75–300) 108 (88–138) 103 (25–175) 101 (40–150) 108 (25–300) n ¼ 30 n ¼ 29 n ¼ 5 n ¼ 3 n ¼ 14 n ¼ 5 n ¼ 86 C/Db 1.1 (0.3–4.4) 0.8 (0.2–4.2) 0.6 (0.3–1.2) 1.5 (0.9–2.1) 1.3 (0.3–5.7) 1.1 (0.5–1.9) 1.0 (0.2–5.7) a c MRAT/NT 1.4 (0.1–4.0) 1.4 (0.3–4.0) 0.9 (0.5–1.6) 4.4 (2.0–7.7) 1.7 (0.7–4.3) 1.7 (0.9–2.8) 1.5 (0.1–7.7)

Abbreviations: AT, amitriptyline; C/D, corrected for dose; MR, metabolic ratio; NT, nortriptyline. Shown in brackets is the range of values (min–max). aSignificantly different from CYP2C19 subgroup CYP2C19*1/*1 (Po0.05). bNumber of subjects is restricted to subjects for which information on daily dose was available. cSignificantly different from preceding CYP2C19 subgroup (Po0.05).

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higher C/D values (Table 4; Figure 2). Strikingly, the subgroup CYP2C19*2/*2 showed significantly higher mean dose compared with CYP2C19*1/*1. Though this mean value is based on four subjects, it might indicate unsatisfac- tory response at lower doses, leading to increased doses in an attempt to obtain a more satisfactory response. No signi- ficant differences were observed between CYP2C19 geno- type and CIT levels, but higher CIT levels did correlate with decreasing predicted activity (as predicted from genotype) (Table 4). The relationship between CYP2C19 genotype and CIT levels was also reflected in the proportion of patients with serum levels outside therapeutic range (30–130 mgl–1). The CYP2C19*17 allele was significantly associated with lower proportions of patients with CIT levels above therapeutic range (Table 4). No significant associations were observed between CYP2C19 genotype and DCIT levels. CYP2D6 has been shown in earlier studies to Figure 2 Overview of the metabolic ratios for amitriptyline (MRAT/NT), metabolize both CIT and DCIT (Figure 1).31 In this study, citalopram (MRCIT/DCIT), and clomipramine (MRCLOM/DCLOM) for the increasing SGD values were significantly associated with different CYP2C19 genotypes. Significant differences (P 0.05) between o decreasing levels of both CIT and DCIT (see Supplementary genotype groups and either CYP2C19*1/*1 or the preceding subgroup are indicated by an asterisk. Appendix D).

Table 3 Characteristics of the CYP2D6/CYP2C19 subgroups for AT

*1/*1 *1/*17 *17/*17 *2/*2 *1/*2 *2/*17

Number of subjects SGD ¼ 06 1 100 1 0 SGD ¼ 123142 3 106 SGD ¼ 226235 2 105 SGD ¼ 31 2 0 0 0 0

Dosea SGD ¼ 0 119 (3) 100 (1) 65 (5) — 150 (1) — SGD ¼ 1 108 (10) 150 (1) 106 (17) 138 (1) 82 (7) 88 (4) SGD ¼ 2 123 (15) 154 (3) 105 (7) 94 (2) 119 (6) 150 (1) SGD ¼ 3 25 (1) — 175 (1) — — —

b MRAT/NT SGD ¼ 0 1.4±0.8 1.4 1.1±1.0c — 1.3 — SGD ¼ 1 1.1±0.6c 1.1±0.6 0.8±0.1 4.8±2.9d 1.2±1.1c 1.6±0.7 SGD ¼ 2 1.6±0.6 1.5±0.9 0.8±0.4d 3.7±1.4d 2.2±0.8 1.7±0.4 SGD ¼ 3 3.2 2.1±0.1 — — — —

AT+NTo100e SGD ¼ 0 17 (1/6) 0 (0/1) 20 (2/10) — 0 (0/1) — SGD ¼ 1 22 (5/23) 29 (4/14) 50 (1/2) 0 (0/3) 30 (3/10) 50 (3/6) SGD ¼ 2 42 (11/26) 35 (8/23) 20 (1/5) 50 (1/2) 40 (4/10) 40 (2/5) SGD ¼ 3 0 (0/1) 50 (1/2) — — — —

NT4150e SGD ¼ 0 0 (0/6) 0 (0/1) 30 (3/10)c — 0 (0/1) — SGD ¼ 1 0 (0/23) 7 (1/14) 0 (0/2) 0 (0/3) 0 (0/10) 0 (0/6) SGD ¼ 2 0 (0/26) 4 (1/23) 20 (1/5)d 0 (0/2) 0 (0/10) 0 (0/5) SGD ¼ 3 0 (0/1) 0 (0/2) — — — —

Abbreviations: AT, amitriptyline; NT, nortriptyline; SGD, semi-quantitative gene dose. aShown in brackets is the number of subjects. bThe MR values are shown with standard deviation. cSignificantly different from CYP2D6*1/*1 subgroup (SGD ¼ 2; Po0.05). dSignificantly different from CYP2C19 subgroup CYP2C19*1/*1 (Po0.05). eShown in brackets is the number of subjects outside therapeutic range/total number of subjects.

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Table 4 Characteristics of the CYP2C19 subgroups for CIT

*1/*1 *1/*17 *17/*17 *2/*2 *1/*2 *2/*17 Total

CIT (mgl–1) 80 (7–291) 68 (14–240) 63 (14–144) 97 (42–150) 89 (14–267) 93 (24–235) 79 (7–291) DCIT (mgl–1) 32 (4–147) 34 (6–117) 31 (6–99) 31 (14–69) 30 (6–136) 34 (11–110) 32 (4–147) Dose (mg per day) 30 (10–60) 29(10–60) 33 (15–60) 41 (33–50)a 27 (10–60)b 32 (15–60) 30 (10–60) n ¼ 96 n ¼ 61 n ¼ 13 n ¼ 4 n ¼ 35 n ¼ 14 n ¼ 223 C/Dc 2.6 (0.4–8.0) 2.5 (0.8–7.4) 1.9 (1.3–3.1) 2.4 (1.1–3.2) 3.6 (0.9–7.1)a 3.5 (1.2–12.6) 2.8 (0.4–12.6) a a MRCIT/DCIT 2.8 (0.6–8.7) 2.3 (1.0–7.2) 2.4 (1.3–6.9) 4.3 (1.6–7.5) 3.2 (0.7–8.4) 3.0 (1.3–6.1) 2.7 (0.6–8.7) CITo30 (% subjects) 10 (15/143) 12 (11/90) 17 (3/18) 0 (0/6) 9 (5/57) 4 (1/24) 10 (35/338) CIT4130 (% subjects) 17 (25/143) 8 (7/90)a 6 (1/18) 33 (2/6) 19 (11/57) 13 (3/24) 14 (49/338)

Abbreviations: C/D, corrected for dose; CIT, citalopram; DCIT, desmethyl-CIT; MR, metabolic ratio. Shown in brackets is the range of values (min–max). aSignificantly different from CYP2C19 subgroup CYP2C19*1/*1 (Po0.05). bSignificantly different from preceding CYP2C19 subgroup (Po0.05). cNumber of subjects is restricted to subjects for which information on daily dose was available.

Table 5 Characteristics of the CYP2C19 subgroups for CLOM

*1/*1 *1/*17 *17/*17 *2/*2 *1/*2 *2/*17 Total

CLOM (mgl–1) 145 (20–508) 127 (15–315) 153 (20–340) 202 (93–400) 169 (40–565) 154 (55–305) 146 (15–565) DCLOM (mgl–1) 157 (20–548) 154 (10–420) 109 (10–255) 96 (63–133) 162 (15–435) 174 (40–350) 155 (10–548) Dose (mg per day) 120 (38–300) 108 (25–225) 84 (50–150) 167 (150–200) 115 (25–200) 110 (50–225) 115 (25–300) n ¼ 70 n ¼ 47 n ¼ 4 n ¼ 3 n ¼ 20 n ¼ 7 n ¼ 151 C/Da 1.3 (0.3–4.5) 1.5 (0.2–4.9) 1.4 (0.5–3.3) 2.1 (0.9–4.0) 1.6 (0.5–4.4) 1.8 (0.6–2.6) 1.46 (0.2–4.9) b MRCLOM/DCLOM 1.4 (0.2–8.6) 1.1 (0.2–3.2) 1.7 (0.6–4.5) 2.5 (1.7–5.2) 1.5 (0.4–9.6) 1.2 (0.4–2.7) 1.4 (0.2–9.6) CLOMo50 5 (5/109) 7 (5/67) 20 (2/10)b 0 (0/5) 3 (1/37) 0 (0/16) 5 (13/244) CLOM+DCLOM4400 19 (21/109) 24 (16/67) 20 (2/10) 20 (1/5) 27 (10/37) 31 (5/16) 23 (55/244)

Abbreviations: C/D, corrected for dose; CLOM, clomipramine; DCLOM, N-desmethyl-CLOM; MR, metabolic ratio. Shown in brackets is the range of values (min–max). aNumber of subjects is restricted to subjects for which information on daily dose was available. bSignificantly different from CYP2C19 subgroup CYP2C19*1/*1 (Po0.05).

Clomipramine increasing SGD values correlated with decreasing

Group characteristics of the 244 patients included in the MRCLOM/DCLOM values (see Supplementary Appendix E). association study for CLOM are shown in Table 5. The mean However, even within the separate CYP2D6 genotype age of subgroup CYP2C19*17/*17 was significantly lower subgroups, no additional significant differences in CLOM compared with the other genotype groups (data not shown). levels, DCLOM levels, MRCLOM/DCLOM values, or C/D values Information on daily dose was available for 151 patients. were observed between the different CYP2C19 genotypes The presence of CYP2C19*17 was not significantly asso- (data not shown). ciated with the majority of the variables investigated; the relationship between the CYP2C19 genotypes and ASCOC values for CYP2C19*2 and CYP2C19*17 either CLOM or DCLOM levels does not show a clear trend In the SGD system described by Steimer et al.,3 the SGD (Table 5). The MRCLOM/DCLOM for subgroup CYP2C19*2/*2 value of CYP2D6 alleles is based on the allele-specific change was significantly different from the MRCLOM/DCLOM for of concentration on identical background (ASCOC). We CYP2C19*1/*1 (Table 5; Figure 2). However, daily dose was calculated the ASCOC values of CYP2C19 alleles for the data relatively high for this subgroup. The homozygous presence collected on AMI, CIT, and CLOM (Table 6). Positive values, of CYP2C19*17 was significantly associated with higher indicating decreased metabolic activity, were observed for proportions of patients with CLOM levels below therapeutic CYP2C19*2 for all genotype comparisons, with similar range (Table 5). However, daily dose was relatively low for average values for AT, CIT, and CLOM ( þ 21, þ 18 and this subgroup. Within the CYP2D6*1/*1 subgroup, the þ 18%, respectively). Negative values, indicating increased differences in daily dose reached significance for subgroup metabolic activity, were observed for CYP2C19*17 for most CYP2C19*17/*17 compared with CYP2C19*1/*1 (data not genotype comparisons, with similar average values for AT, shown). CYP2D6 genotype has been correlated in earlier CIT, and CLOM (À11, À10, and À8%). These individual studies with CLOM metabolism.25–27 In this study, CYP2D6 differences in concentration were not significant, except genotype was significantly associated with MRCLOM/DCLOM; one. Combining all data for each antidepressant, the ASCOC

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Table 6 Relative concentration allele increases CYP2C19 activity toward , imi- pramine and es-CIT.12–14,32 Sim et al.11 have shown with an Genotype vs reference genotype ASCOC value (%) electrophoretic mobility shift assay that CYP2C19*17 intro- duces an extra transcription factor-binding site in the AT promotor region of CYP2C19, resulting in an increased *2 *1/*2 (n ¼ 21) vs *1/*1 +22 CYP2C19 enzyme activity in vitro. We observed significant a *2/*2 (n ¼ 5) vs *1/*2 +51 associations between CYP2C19*17 and lower MR for AT and *2/*17 (n ¼ 11) vs *1/*17 +4 CIT, but not CLOM. The majority of other parameters Total *2 vs *1 +21 *17 *1/*17 (n ¼ 45) vs *1/*1 À10 concerning AT and CIT metabolism show no significant *17/*17 (n ¼ 8) vs *1/*17 À1 associations with CYP2C19*17. Nevertheless, they do show a *2/*17 (n ¼ 11) vs *1/*2 À23 trend toward lower levels of parent drug. These data support Total *17 vs *1 À11 the function of CYP2C19*17 in CIT metabolism, and indicate an effect of CYP2C19*17 on AT metabolism. The CIT combined data on CYP2C19*17 and CLOM metabolism (in *2 *1/*2 (n ¼ 57) vs *1/*1 +11 particular, the total ASCOC value and proportion of subjects *2/*2 (n ¼ 6) vs *1/*2 +8 below therapeutic range) might indicate a minor effect of *2/*17 (n ¼ 24) vs *1/*17 +37 CYP2C19*17 on CLOM metabolism. However, this effect is Total *2 vs *1 +18a more likely caused by the significantly lower daily dose and *17 *1/*17 (n ¼ 90) vs *1/*1 À15 CYP2C19 17/ 17 *17/*17 (n ¼ 18) vs *1/*17 À7 lower mean age of the * * subgroup. We do *2/*17 (n ¼ 24) vs *1/*2 +4 not know the reason for the lower mean age in the Total *17 vs *1 À10 CYP2C19*17/*17 subgroup. The more evident association between AT and CIT metabolic parameters and CYP2C19*2, CLOM compared with CYP2C19*17, could be caused by a smaller *2 *1/*2 (n ¼ 37) vs *1/*1 +17 relative effect of CYP2C19*17 on CYP2C19 activity. This is *2/*2 (n ¼ 5) vs *1/*2 +18 reflected in the smaller ASCOC values obtained for *2/*17 (n ¼ 16) vs *1/*17 +21 CYP2C19*17. In earlier studies, an SGD value of 0 and 1 a Total *2 vs *1 +18 has been assigned to CYP2C19*2 and CYP2C19*1, respec- *17 *1/*17 (n ¼ 67) vs *1/*1 À12 tively. On the basis of the smaller effect of CYP2C19*17,we *17/*17 (n ¼ 10) vs *1/*17 +20 propose an SGD value of 1.5 for CYP2C19*17. The genotype *2/*17 (n ¼ 16) vs *1/*2 À9 Total *17 vs *1 À8 subgroup CYP2C19*2/*17 would then be appointed an SGD Total *2 vs *1 +19a value of 1.5, predicting lower CYP2C19 activity compared *17 vs *1 À10a with CYP2C19*1/*1. The MR data for AT and CIT did indeed indicate lower CYP2C19 activity for CYP2C19*2/*17 com- Abbreviations: AT, amitriptyline; CIT, citalopram; CLOM, clomipramine. pared with CYP2C19*1/*1, as did the C/D data for CIT. The aSignificant difference (Po0.05). restricted number of significant associations between CYP2C19*2 or CYP2C19*17 and metabolic parameters is likely due to the presence of alternative metabolic pathways. value for CYP2C19*2 was significantly higher than 0% for CYP2D6 is one of the enzymes involved in the metabolic CIT and CLOM. Combining all data for all three anti- pathways for AT, CIT, and CLOM. Its influence is shown in , the ASCOC was significantly higher than 0% this study by the significant associations between CYP2D6 for CYP2C19*2, and significantly lower than 0% for genotype and parent drug levels or metabolite levels. The CYP2C19*17. combination of decreasing NT levels with increasing SGD values, but no correlation with AT levels, supports the involvement of CYP2D6 in AT metabolism as depicted in Discussion Figure 1. Similarly, the association of CYP2D6 genotype with CIT and DCIT levels supports the involvement of CYP2D6 CYP2C19 is involved in the metabolism of several anti- genotype in both CIT and DCIT degradation. According to depressants, including AT, CIT, and CLOM (Figure 1). Serum published data, CYP2D6 hydroxylates both CLOM and levels indicate large interindividual differences in CYP2C19 DCLOM (Figure 1).25–27 The involvement of CYP2D6 activity. These differences are to some extend explained by genotype in CLOM degradation is not supported by the the occurrence of the non-functional alleles CYP2C19*2 and present data, but the data do support the indicated CYP2C19*3. The impact of CYP2C19*2 on the metabolism metabolism of DCLOM. of CYP2C19-dependent antidepressants is supported in this study by the association of CYP2C19*2 with increased AT Clinical relevance of CYP2C19*17 and CIT levels and increased MRAT/NT and MRCIT/DCIT values. On the basis of relative serum levels, dose recommendations These data also support the assumption that CYP2C19 has can be provided for specific genotype groups.4,33,34 How- clinically relevant activity toward the parent compounds AT ever, the clinical benefit of dose adjustments depends and CIT. Earlier studies have shown that the CYP2C19*17 on both the extend of deviation in serum levels and the

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dose–response relationship. As suggested earlier,14 the 3 Steimer W, Zopf K, von AS, Pfeiffer H, Bachofer J, Popp J et al. Allele- CYP2C19*17 allele might be associated with sub-therapeutic specific change of concentration and functional gene dose for the levels for those drugs that are extensively metabolized by prediction of steady-state serum concentrations of amitriptyline and nortriptyline in CYP2C19 and CYP2D6 extensive and intermediate CYP2C19. AT, CIT, and CLOM were selected as commonly metabolizers. Clin Chem 2004; 50: 1623–1633. used antidepressants being metabolized by CYP2C19. For 4 Kirchheiner J, Nickchen K, Bauer M, Wong ML, Licinio J, Roots I et al. AT, CYP2C19 genotype did affect the ratio of AT and NT Pharmacogenetics of antidepressants and : the contri- levels, but not the sum of AT and NT levels. This is reflected bution of allelic variations to the phenotype of drug response. Mol Psychiatry 2004; 9: 442–473. in the lack of association of CYP2C19 genotype with daily 5 Brockmoller J, Meineke I, Kirchheiner J. of mirtaza- dose and with AT þ NT levels outside therapeutic range. pine: enantioselective effects of the CYP2D6 ultra rapid metabolizer Consequently, CYP2C19*17 genotyping is not likely to be genotype and correlation with adverse effects. Clin Pharmacol Ther clinically relevant in AT therapy. Caution might be neces- 2007; 81: 699–707. 6 Scordo MG, Spina E, Dahl ML, Gatti G, Perucca E. Influence of CYP2C9, sary with respect to NT levels, as CYP2C19*17 is associated 2C19 and 2D6 genetic polymorphisms on the steady-state plasma with higher proportion of patients with NT levels above concentrations of the of and norfluoxetine. therapeutic range. With respect to CLOM, no clear associa- Basic Clin Pharmacol Toxicol 2005; 97: 296–301. tions were observed between CYP2C19 genotype and CLOM 7 Charlier C, Broly F, Lhermitte M, Pinto E, Ansseau M, Plomteux G. Polymorphisms in the CYP 2D6 gene: association with plasma levels, likely because of the small contribution of CYP2C19 concentrations of fluoxetine and . Ther Drug Monit 2003; to overall CLOM metabolism. Therefore, CYP2C19*17 25: 738–742. genotyping is not likely to improve response to CLOM 8 Veefkind AH, Haffmans PM, Hoencamp E. serum levels and therapy either. The CYP2C19*17 allele was significantly CYP2D6 genotype. Ther Drug Monit 2000; 22: 202–208. 9 http://www.cypalleles.ki.se/ 2009. Ref Type: Generic. associated with lower proportions of patients with CIT levels 10 Goldstein JA, Ishizaki T, Chiba K, de Morais SM, Bell D, Krahn PM above therapeutic range, and a non-significant increase in et al. Frequencies of the defective CYP2C19 alleles responsible for proportion of patients at sub-therapeutic range. However, the poor metabolizer phenotype in various Oriental, the correlation between CIT levels and CYP2C19 was not Caucasian, Saudi Arabian and American black populations. 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