The Pharmacogenomics Journal (2014), 1–7 © 2014 Macmillan Publishers Limited All rights reserved 1470-269X/14 www.nature.com/tpj
ORIGINAL ARTICLE
Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolic ratios and stereoselective metabolism in forensic autopsy cases L Karlsson1, A-L Zackrisson2, M Josefsson2,3, B Carlsson4, H Green1,2 and FC Kugelberg1,2 We investigated whether polymorphisms in the CYP2D6 and CYP2C19 genes influence the metabolic ratios and enantiomeric S/R ratios of venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,Odidesmethylvenlafaxine (DDV) in blood from forensic autopsy cases. In all, 94 postmortem cases found positive for VEN during toxicological screening were included. The CYP2D6 genotype was shown to significantly influence the ODV/VEN (P = 0.003), DDV/NDV (P = 0.010) and DDV/ODV (P = 0.034) ratios. The DDV/ODV (P = 0.013) and DDV/VEN (P = 0.021) ratios were significantly influenced by the CYP2C19 genotype. The S/R ratios of VEN were significantly influenced by both CYP2D6 and CYP2C19 genotypes. CYP2D6 poor metabolizers (PMs) had lower S/R VEN ratios and CYP2C19 PMs had high S/R ratios of VEN in comparison. Our results show that the CYP2D6 genotype influences the O-demethylation whereas CYP2C19 influences the N-demethylation of VEN and its metabolites. In addition, we show a stereoselective metabolism where CYP2D6 favours the R-enantiomer whereas CYP2C19 favours the S-enantiomer. The Pharmacogenomics Journal advance online publication, 23 September 2014; doi:10.1038/tpj.2014.50
INTRODUCTION It is well known that pharmacogenetic polymorphisms affect the biotransformation and clinical outcome for many drugs and contribute to interindividual differences in the therapeutic response. The most common source of genetic variations is singlenucleotide polymorphisms. The cytochrome P450 (CYP) isozymes exhibit polymorphic genetic variability and have profound effect on the metabolism for many antidepressants.1 The clinically most relevant polymorphisms in psychiatric disorders are found in CYP2D6 and CYP2C192,3 since the drugs most frequently used are predominantly metabolized by these enzymes in one or several phases of their elimination process.4 Several postmortem studies have focused on the effect of the polymorphisms in these genes.5–10 Levo et al.8 found a correlation between genotype and parental/metabolite ratios in postmortem blood for tramadol. In healthy volunteers, the plasma concentration of tramadol was higher in CYP2D6 poor metabolizer (PM) individuals than in extensive metabolizers (EMs).11 Positive correlations between metabolites and the number of active alleles of CYP2D6 and CYP2C19 in a postmortem material have also been reported for amitriptyline.7 In addition, the elimination of psychiatric drugs has been shown to be stereoselective. For example, the enantiomeric disposition of mianserine is dependent on CYP2D6 activity with higher S/R ratio in PMs than in EMs indicating a stereoselective metabolism where the S-enantiomer is favoured.12 CYP2D6 has also been shown to be involved in the metabolism of methadone with a stereoselectivity towards the R-enantiomer.13
Venlafaxine (VEN), a racemic mixture (50:50) of the R- and S-enantiomers, is an antidepressant that selectively inhibits reuptake of noradrenaline and serotonin, and slightly inhibits reuptake of dopamine.14 However, the S-enantiomer inhibits serotonin reuptake, whereas the R-enantiomer is a potent inhibitor of both serotonin and noradrenaline reuptake.15,16 On the basis of in vitro studies, CYP2D6, CYP2C9, CYP2C19 and CYP3A4 have been shown to be involved in the metabolism of VEN.17 VEN is metabolized into its major active metabolite O-desmethylvenlafaxine (ODV) by CYP2D6 and CYP3A4 catalyzes the metabolism to its minor metabolite N-desmethylvenlafaxine (NDV).18 These two metabolites are then further metabolized to N,O-didesmethylvenlafaxine (DDV).19 Clinical studies show that both CYP2D6 and CYP2C19 influence the concentrations of VEN and ODV and that CYP2D6 PM individuals have an increased risk of cardiovascular toxicity20 and other adverse effects.21 The CYP2D6 enzyme also influences the enantiomeric ratios of VEN6 and CYP2D6 has been reported to display stereoselectivity towards the R-enantiomer.22 Several studies have focused on the demethylation of VEN into ODV.21,23–26 It has been suggested that CYP2C19 have a role in the demethylation of VEN21 but so far, the possible influence of CYP2C19 has not been fully elucidated. We have investigated whether the disposition of VEN and its three main metabolites (ODV, NDV and DDV) in a forensic autopsy material is affected by the CYP2D6 and CYP2C19 genotypes. In addition, we studied the disposition of the enantiomers of VEN and metabolites as well as the predictive values of the genotype on the metabolic ratios (M/P ratios).
1 Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; 2Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden; 3Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden and 4 Division of Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health, Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden. Correspondence: Dr L Karlsson or Dr FC Kugelberg, Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping SE-581 85, Sweden. E-mail: [email protected] or [email protected] Received 10 February 2014; revised 27 June 2014; accepted 8 July 2014
Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolism L Karlsson et al
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MATERIALS AND METHODS Case selection Ninety-four forensic autopsy cases in which VEN was found in femoral blood were selected for the study. The samples, collected from the National Board of Forensic Medicine in Sweden, were routinely screened for a wide range of prescription drugs and ethanol. The samples were analysed by an enantioselective method and genotyped for CYP2D6 and CYP2C19. The cases were classified using the ICD (International Classification of Disease) codes by the responsible pathologist. Additional information related to each of the cases (age, gender and circumstances surrounding death) was provided from the forensic pathology and toxicology databases at the Swedish National Board of Forensic Medicine. The study was approved by the Regional Ethical Review Board in Linköping, Sweden (No. M87-08).
Enantioselective analysis of VEN The concentration of the S- and R-enantiomers of VEN and its three metabolites (ODV, NDV and DDV) in femoral blood was determined by using a LC/MS/MS method as described previously.6,27 Briefly, the samples were extracted using Isolute C8 columns 100 mg (International Sorbent Technology, Hengoed, UK) After elution and evaporation with nitrogen at 50 °C, the analytes were reconstituted in 100 μl mobile phase (tetrahydrofuran:ammonium acetate, 10 mM, pH 6; 10:90 v/v) and 2 μl was injected. The LC/MS/MS system consisted of an Acquity liquid chromatography system (Waters, Milford, MA, USA) and an API 4000 tandem quadrupole instrument equipped with an electrospray interface (Turbo VTM source, ® TurbolonSpray probe) operating in positive ion mode (Applied Biosystem/ MSD Sciex, Toronto, ON, Canada). Chromatographic separation was performed on a Chirobiotic-V column (5 μm particle size, 250 × 2.1 mm: Astec/Supelco, Sigma-Aldrich, St. Louis, MO, USA) protected with a 5- μm in-line filter (VICI AB Int., Schenkon, Switzerland) at a mobile phase flow rate of 0.2 ml min − 1. Data were processed using the Analyst 1.4.2 Software (Applied Biosystem/MSD Sciex).
Genotyping for CYP2C19 and CYP2D6 Genotyping for CYP2D6 and CYP2C19 was performed with PCR followed by Pyrosequencing as described previously.10,28 For CYP2D6, four of the most important non-functional alleles *3, *4, *5 and *6, were included (http:// www.cypalleles.ki.se). The copy number variation was determined to identify multiple gene copies (that is, active alleles such as CYP2D6*1xN or inactive alleles such as CYP2D6*4xN) and to identify the whole gene deletion (CYP2D6*5). For CYP2C19, the three main non-functional alleles *2, *3 and *4 and the gain-of-function allele *17 were included (http://www. cypalleles.ki.se). On the basis of the results from the genotype analyses, the cases were classified into four predicted phenotype groups for CYP2D6 and CYP2C19, respectively. A carrier of only non-functional alleles was termed as poor metabolizer (PM). A heterozygous carrier of one active allele (*1) in combination with one non-functional allele was termed as intermediate metabolizer (IM). A carrier of two active alleles (*1/*1) or for CYP2C19, one non-functional and one gain-of-function allele (that is, *2/*17) was termed as extensive metabolizer (EM). For CYP2D6, a carrier of more than two active alleles (*1xN/*1) and for CYP2C19, a carrier of one active and one gain-of-function allele (*1/*17) or two gain-of-function alleles (*17/*17) was termed as ultra-rapid metabolizer (UM).
Statistical analysis Data are presented as mean ± s.d. or median and the 10th and 90th percentiles. The influence of the genotypes on the metabolic ratios and the S/R ratios was determined using multivariate ANOVA. A P-value of o0.05 was considered as statistically significant. All statistical analysis was performed using StatView® (version 5.0; SAS® Institute, Cary, NC, USA).
RESULTS General data The material comprises 94 VEN-positive postmortem cases, 35 women and 59 men with ages ranging between 18 and 86 years (median 49 years). Most of the cases were polydrug cases and the median number of drugs was 4 (range 1–11). In 13 cases, VEN was the only drug identified, and none of these cases were classified as intoxications. The most frequently found drugs in addition to VEN The Pharmacogenomics Journal (2014), 1 – 7
were ethanol, zopiclone, mirtazapin and diazepam. The manner of death was classified as suicide in 50 cases, natural in 15 cases, accidental in 22 cases and undetermined/other in 7 cases (for details, see Supplementary Information Supplementary Table S1). The distribution of genetic variation for CYP2D6 was 7 PM, 39 IM, 43 EM and 2 UM (3 cases were undetermined). For CYP2C19, 2 PM, 14 IM, 48 EM and 27 UM were found (3 cases were undetermined). Analytical results, including total (S+R) concentrations, enantiomeric S/R ratios as well as total (S+R) metabolite/parent drug (M/P) ratios, are presented in Table 1. The median concentrations of VEN, ODV NDV and DDV in all cases were 0.56, 0.48, 0.08 and 0.1 μg g − 1, respectively. Association between CYP genotypes and VEN metabolic ratios The CYP2D6 genotype significantly influenced the M/P ratios of ODV/VEN (P = 0.003), DDV/NDV (P = 0.010) and DDV/ODV (P = 0.034) (Figure 1). Median ODV/VEN ratios for CYP2D6 were 0.22 for PM, 0.75 for IM, 1.78 for EM and 2.03 for UM. The DDV/ODV (P = 0.013) and DDV/VEN (P = 0.021) ratios were significantly influenced by the CYP2C19 genotype. CYP2C19 also influenced the NDV/VEN ratio but did not reach statistical significance (P = 0.061). Median NDV/VEN ratios for CYP2C19 were 0.01 for PM, 0.14 for IM, 0.17 for EM and 0.25 for UM, clearly showing a trend. Association between CYP genotypes and enantiomeric ratios of VEN and its metabolites The S/R ratios of VEN were significantly influenced by both CYP2D6 and CYP2C19 genotypes (P = 0.009 and P = 0.015, respectively) (Figure 2). CYP2D6 significantly influenced both the ODV S/ R ratios and the NDV S/R ratios (P = o 0.0001 and P = 0.003, respectively). The S/R ratios of DDV were significantly influenced by CYP2D6 (P = 0.0002). Receiver operating characteristic curves To evaluate the predictive value of the genotype on the metabolic ratio, we created receiver operating characteristic (ROC) curves and calculated the area under the curve. The area under the curve
Table 1. Total (S+R) concentration, enantiomeric (S/R) drug concentration ratios and metabolite/parent drug concentration ratios (M/P) n
10th percentile
Median
90th percentile
Total (S+R) concentration (μg g −1) VEN 94 0.127 ODV 89 0.126 NDV 93 0.016 DDV 94 0.023
0.562 0.484 0.082 0.102
2.364 1.224 0.739 0.296
S/R concentration ratios VEN 94 ODV 89 NDV 90 DDV 94
0.518 0.729 0.473 0.688
0.968 0.894 0.807 1.030
1.692 2.795 1.441 1.490
M/P concentration ratios ODV/VEN 89 NDV/VEN 93 DDV/VEN 94 DDV/ODV 89 DDV/NDV 93
0.193 0.042 0.039 0.071 0.142
0.977 0.184 0.188 0.193 1.218
3.478 0.722 0.708 0.395 4.186
Abbreviations: DDV, N,O-didesmethylvenlafaxine; NDV, N-desmethylvenlafaxine; ODV, O-desmethylvenlafaxine; VEN, venlafaxine.
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Figure 1. The metabolic ratios (metabolite/parent drug concentration ratios) for venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in femoral blood after dividing the cases into CYP2D6 genotypes and CYP2C19 genotypes. Values are mean ± s.d. (CYP2D6: n = 7 PM, n = 39 IM, n = 43 EM and n = 2 UM and CYP2C19: n = 2 PM, n = 14 IM, n = 48 EM and n = 27 UM). EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; UM, ultra-rapid metabolizer.
for ODV/VEN ratios was 0.8936 with a P-value of 0.001 (Figure 3). Using the ROC curve to get a cutoff for skewed ODV/VEN ratios (ratios of below 0.35) resulted in that 26% of cases having a skewed ratio were PM for CYP2D6, compared with 1.5% PM in the © 2014 Macmillan Publishers Limited
remaining cases. The ROC curve for the effect of CYP2C19 genotype on NDV/VEN ratio had an area under the curve of 0.9972 with a P-value of 0.017. The cases with the lowest NDV/VEN ratio were all PM for CYP2C19. The Pharmacogenomics Journal (2014), 1 – 7
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Figure 2. Enantiomeric (S/R) concentration ratios for venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in femoral blood after dividing the cases into CYP2D6 genotypes and CYP2C19 genotypes. Values are mean ± s.d. (CYP2D6: n = 7 PM, n = 39 IM, n = 43 EM and n = 2 UM and CYP2C19: n = 2 PM, n = 14 IM, n = 48 EM and n = 27 UM). EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; UM, ultra-rapid metabolizer.
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Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolism L Karlsson et al
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Figure 3. Receiver operating characteristic (ROC) curves for metabolic ratios (metabolite/parent drug concentration ratios) of venlafaxine (VEN) and the metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) divided into genotype/phenotype groups for CYP2D6 and CYP2C19, respectively.
DISCUSSION In this study, we present a detailed investigation on the demethylation of VEN and its major metabolites ODV and NDV. We found that the CYP2D6 genotype influences the O-demethylation whereas CYP2C19 influences the N-demethylation of VEN and its metabolites. In addition, we observed a stereoselective metabolism where CYP2D6 favours the R-enantiomer whereas CYP2C19 favours the S-enantiomer. The major metabolic pathway of VEN is O-demethylation, accounting for ~ 55% of an oral dose excreted as ODV.29 The metabolism of VEN has been the focus of several studies and the main effort has been on CYP2D6. An in vitro study with microsomes from human liver and CYP2D6 transformed yeast found that CYP2D6 catalyzes the O-demethylation of VEN into ODV and that CYP3A4 was involved in the N-demethylation of VEN into NDV. No indication that CYP2D6 was involved in the N-demethylation of VEN was shown.18 Another in vitro study showed that the CYP2C9, CYP2C19 and CYP2D6 enzymes have a role in the metabolism of VEN into ODV (using human liver microsomes and CYP microsomes) with CYP2D6 being the main enzyme (relative contribution of 1, 10 and 89%, respectively).17 In the same study, CYP2C9, CYP2C19 and CYP3A4 were found to be involved in the metabolism of VEN into NDV (relative contribution of 31, 33 and 36%, respectively). The formation of DDV from ODV has been suggested to be dependent on CYP3A4.30 We found that the demethylation of VEN into ODV is influenced by the CYP2D6 genotype, with lowest ODV/VEN ratios for PM individuals and highest ODV/VEN ratios for UM individuals (Figure 1). A correlation between the metabolic ratio (ODV/VEN) and CYP2D6 genotype has been shown in clinical studies with lowest ratios for PMs.21,23–26 In addition, McAlpine et al.21 found an association between ODV/VEN ratio and CYP2C19 genotype. CYP2D6 PM individuals have also been found to have an increased risk of developing side effects.24 Otton et al.18 speculated that NDV is O-demethylated by CYP2D6 into DDV which is in line with our results. We show that the CYP2D6 genotype influences the demethylation of NDV into DDV, with PM individuals having the lowest DDV/NDV ratio and UM individuals showing the highest ratio (Figure 1). Taken together, CYP2D6 seems to be involved in the O-demethylation of VEN into ODV and NDV into DDV, which is not entirely surprising since it is the same chemical reaction in both metabolism steps. All M/P ratios are dependent on both the formation and degradation of the metabolite. For example, the © 2014 Macmillan Publishers Limited
ODV/VEN ratio is influenced by CYP2D6 in terms of the formation of ODV. However, our data also suggest that CYP2C19 has a possible effect on the ODV/VEN ratio by an impact on the degradation of ODV. We show that the CYP2C19 genotype influenced the demethylation of VEN into NDV and ODV into DDV. Fogelman et al.17 found that CYP2C9, CYP2C19 and CYP3A4 are involved in the formation of NDV with CYP2C19 having the highest intrinsic clearance. Even though CYP2C19 was found to have the highest intrinsic clearance, the high abundance of CYP3A4 will enlarge the importance of this enzyme. Hermann et al.23 reported an influence of CYP2D6 on the NDV concentration. They found a higher formation of NDV in individuals with one non-functional CYP2D6 allele compared with EM, most likely due to higher fraction of VEN being metabolized to NDV and less NDV being metabolized into DDV when CYP2D6 activity is impaired. This is in accord with our data on both CYP2C19 and CYP2D6. Two other studies found the highest concentration of NDV in CYP2D6 PMs and the lowest concentration in UMs.24,26 None of the three studies has investigated the influence of CYP2C19, which we here find influencing the demethylation of VEN into NDV and ODV into DDV. However, the contribution of CYP3A4 in the demethylation of VEN and its metabolites is still unclear mainly due to the lack of relevant genotype analysis and has not been investigated in the present study. The lack of genotype for CYP3A4 in the present study is a limitation. Our ROC curve analysis shows that the CYP2D6 and CYP2C19 genotypes are useful predictors for skewed VEN metabolism. A large proportion of the individuals with low ratios can be explained by these genotypes showing that these pharmacogenetic analyses are important when studying metabolic ratios in both in clinical and forensic setting. There is a limited knowledge about the stereoselective metabolism of VEN and its metabolites. To date, it is known that CYP2D6 has been found to be stereoselective towards the R-enantiomer of VEN.22 This finding is in line with our present and previous data showing that CYP2D6 PM individuals have a low VEN S/R ratio in combination with a high ODV S/R ratio.6 Otton et al.18 reported that the S-enantiomer of VEN was preferentially N-demethylated. Our data suggest that this stereoselectivity is CYP2C19 mediated with PM individuals having high VEN S/R ratio in combination with low ODV S/R ratio. Stereoselective metabolism of other drugs has been reported. For methadone, CYP2B6 has been reported to favour the The Pharmacogenomics Journal (2014), 1 – 7
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ACKNOWLEDGMENTS This work was supported by grants from the Swedish Research Council, the Lions Research Foundation and the National Board of Forensic Medicine in Sweden.
REFERENCES
Figure 4. Metabolism of venlafaxine (VEN) into its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV).
S-enantiomer whereas CYP2C19 favours the R-enantiomer.31 The (+)-enantiomer of omeprazole is metabolized by CYP2C19 and the (− )-enantiomer is partly metabolized by this enzyme but mainly by CYP3A4.32 Stereoselective metabolism could have an important role, influencing pharmacokinetics, pharmacodynamics and toxicity.33 Chiral considerations are especially relevant for drugs where the enantiomers display different properties like in the case with VEN where the S-enantiomer inhibits serotonin reuptake while the R-enantiomer inhibits both serotonin and noradrenaline reuptake.15,16 A change in the S/R ratios of the enantiomers caused by differences in CYP2D6 and/or CYP2C19 activity might therefore affect the clinical outcome. Other factors to consider when analyzing forensic data, besides genetic variation, are information about age, ingested dose and time of sampling in relation to the time when the drug was taken, which are rarely known, and metabolic drug interactions. In the present study, in most cases other drugs were found with an average of four drugs. Drug interactions may affect the pharmacokinetics of VEN since many of the drugs found are metabolized by the same enzymes as VEN. Drug interactions involving VEN may have occurred in cases included in the present study. Further, the possibility that VEN undergoes postmortem drug redistribution has to be considered.34 Even if redistribution has occurred, it is likely that the parent drug and the metabolites redistribute at the same extent. On the basis of the findings in the present study, together with previous studies,17,18,30 Figure 4 displays a conclusion of the metabolism of VEN. VEN is O-demethylated into ODV via CYP2D6, CYP2C9 and CYP2C19. VEN is also N-demethylated into NDV by CYP3A4, CYP2C19 and CYP2C9. ODV and NDV are then further N-demethylated and O-demethylated into DDV via CYP2C19 and CYP2D6, respectively. CYP2D6 is stereoselective and prefers the R-enantiomer whereas CYP2C19 prefers the S-enantiomer. In clinical practice, the sum of VEN and ODV concentrations is usually used as the therapeutic concentration.35,36 The present data demonstrate the importance of analysing not only ODV, but also NDV in order to make a correct interpretation of the genotype influence. When looking at the total demethylation of VEN into DDV, DDV/VEN ratio, our data show that both CYP2D6 and CYP2C19 genotypes are influencing the transformation. In clinic, CYP2C19 may have a more therapeutic relevance than CYP2D6 since it metabolizes both active compounds, that is, VEN and ODV, but the CYP2D6 genotype has a significant role for the enantiomeric ratios. CONFLICT OF INTEREST The authors declare no conflict of interest.
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7 24 Shams ME, Arneth B, Hiemke C, Dragicevic A, Muller MJ, Kaiser R et al. CYP2D6 polymorphism and clinical effect of the antidepressant venlafaxine. J Clin Pharm Ther 2006; 31: 493–502. 25 van der Weide J, van Baalen-Benedek EH, Kootstra-Ros JE. Metabolic ratios of psychotropics as indication of cytochrome P450 2D6/2C19 genotype. Ther Drug Monit 2005; 27: 478–483. 26 Veefkind AH, Haffmans PM, Hoencamp E. Venlafaxine serum levels and CYP2D6 genotype. Ther Drug Monit 2000; 22: 202–208. 27 Kingback M, Josefsson M, Karlsson L, Ahlner J, Bengtsson F, Kugelberg FC et al. Stereoselective determination of venlafaxine and its three demethylated metabolites in human plasma and whole blood by liquid chromatography with electrospray tandem mass spectrometric detection and solid phase extraction. J Pharm Biomed Anal 2010; 53: 583–590. 28 Zackrisson AL, Lindblom B. Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing. Eur J Clin Pharmacol 2003; 59: 521–526. 29 Howell SR, Husbands GE, Scatina JA, Sisenwine SF. Metabolic disposition of 14C-venlafaxine in mouse, rat, dog, rhesus monkey and man. Xenobiotica 1993; 23: 349–359.
30 Sproule BA, Hazra M, Pollock BG. Desvenlafaxine succinate for major depressive disorder. Drugs Today (Barc) 2008; 44: 475–487. 31 Gerber JG, Rhodes RJ, Gal J. Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19. Chirality 2004; 16: 36–44. 32 Tybring G, Bottiger Y, Widen J, Bertilsson L. Enantioselective hydroxylation of omeprazole catalyzed by CYP2C19 in Swedish white subjects. Clin Pharmacol Ther 1997; 62: 129–137. 33 Smith SW. Chiral toxicology: it's the same thing...only different. Toxicol Sci 2009; 110: 4–30. 34 Jaffe PD, Batziris HP, van der Hoeven P, DeSilva D, McIntyre IM. A study involving venlafaxine overdoses: compararsion of fatal and therapeutic concentrations in postmortem specimens. J Forensic Sci 1999; 44: 193–196. 35 Baumann P, Hiemke C, Ulrich S, Eckermann G, Gaertner I, Gerlach M et al. The AGNP-TDM expert group consensus guidelines: therapeutic drug monitoring in psychiatry. Pharmacopsychiatry 2004; 37: 243–265. 36 Hiemke C, Baumann P, Bergemann N, Conca A, Dietmaier O, Egberts K et al. AGNP consensus guidelines for therapeutic drug monitoring in psychiatry: update 2011. Pharmacopsychiatry 2011; 44: 195–235.
Supplementary Information accompanies the paper on the The Pharmacogenomics Journal website (http://www.nature.com/tpj)
© 2014 Macmillan Publishers Limited
The Pharmacogenomics Journal (2014), 1 – 7
ORIGINAL ARTICLE
Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolic ratios and stereoselective metabolism in forensic autopsy cases L Karlsson1, A-L Zackrisson2, M Josefsson2,3, B Carlsson4, H Green1,2 and FC Kugelberg1,2 We investigated whether polymorphisms in the CYP2D6 and CYP2C19 genes influence the metabolic ratios and enantiomeric S/R ratios of venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,Odidesmethylvenlafaxine (DDV) in blood from forensic autopsy cases. In all, 94 postmortem cases found positive for VEN during toxicological screening were included. The CYP2D6 genotype was shown to significantly influence the ODV/VEN (P = 0.003), DDV/NDV (P = 0.010) and DDV/ODV (P = 0.034) ratios. The DDV/ODV (P = 0.013) and DDV/VEN (P = 0.021) ratios were significantly influenced by the CYP2C19 genotype. The S/R ratios of VEN were significantly influenced by both CYP2D6 and CYP2C19 genotypes. CYP2D6 poor metabolizers (PMs) had lower S/R VEN ratios and CYP2C19 PMs had high S/R ratios of VEN in comparison. Our results show that the CYP2D6 genotype influences the O-demethylation whereas CYP2C19 influences the N-demethylation of VEN and its metabolites. In addition, we show a stereoselective metabolism where CYP2D6 favours the R-enantiomer whereas CYP2C19 favours the S-enantiomer. The Pharmacogenomics Journal advance online publication, 23 September 2014; doi:10.1038/tpj.2014.50
INTRODUCTION It is well known that pharmacogenetic polymorphisms affect the biotransformation and clinical outcome for many drugs and contribute to interindividual differences in the therapeutic response. The most common source of genetic variations is singlenucleotide polymorphisms. The cytochrome P450 (CYP) isozymes exhibit polymorphic genetic variability and have profound effect on the metabolism for many antidepressants.1 The clinically most relevant polymorphisms in psychiatric disorders are found in CYP2D6 and CYP2C192,3 since the drugs most frequently used are predominantly metabolized by these enzymes in one or several phases of their elimination process.4 Several postmortem studies have focused on the effect of the polymorphisms in these genes.5–10 Levo et al.8 found a correlation between genotype and parental/metabolite ratios in postmortem blood for tramadol. In healthy volunteers, the plasma concentration of tramadol was higher in CYP2D6 poor metabolizer (PM) individuals than in extensive metabolizers (EMs).11 Positive correlations between metabolites and the number of active alleles of CYP2D6 and CYP2C19 in a postmortem material have also been reported for amitriptyline.7 In addition, the elimination of psychiatric drugs has been shown to be stereoselective. For example, the enantiomeric disposition of mianserine is dependent on CYP2D6 activity with higher S/R ratio in PMs than in EMs indicating a stereoselective metabolism where the S-enantiomer is favoured.12 CYP2D6 has also been shown to be involved in the metabolism of methadone with a stereoselectivity towards the R-enantiomer.13
Venlafaxine (VEN), a racemic mixture (50:50) of the R- and S-enantiomers, is an antidepressant that selectively inhibits reuptake of noradrenaline and serotonin, and slightly inhibits reuptake of dopamine.14 However, the S-enantiomer inhibits serotonin reuptake, whereas the R-enantiomer is a potent inhibitor of both serotonin and noradrenaline reuptake.15,16 On the basis of in vitro studies, CYP2D6, CYP2C9, CYP2C19 and CYP3A4 have been shown to be involved in the metabolism of VEN.17 VEN is metabolized into its major active metabolite O-desmethylvenlafaxine (ODV) by CYP2D6 and CYP3A4 catalyzes the metabolism to its minor metabolite N-desmethylvenlafaxine (NDV).18 These two metabolites are then further metabolized to N,O-didesmethylvenlafaxine (DDV).19 Clinical studies show that both CYP2D6 and CYP2C19 influence the concentrations of VEN and ODV and that CYP2D6 PM individuals have an increased risk of cardiovascular toxicity20 and other adverse effects.21 The CYP2D6 enzyme also influences the enantiomeric ratios of VEN6 and CYP2D6 has been reported to display stereoselectivity towards the R-enantiomer.22 Several studies have focused on the demethylation of VEN into ODV.21,23–26 It has been suggested that CYP2C19 have a role in the demethylation of VEN21 but so far, the possible influence of CYP2C19 has not been fully elucidated. We have investigated whether the disposition of VEN and its three main metabolites (ODV, NDV and DDV) in a forensic autopsy material is affected by the CYP2D6 and CYP2C19 genotypes. In addition, we studied the disposition of the enantiomers of VEN and metabolites as well as the predictive values of the genotype on the metabolic ratios (M/P ratios).
1 Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; 2Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden; 3Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden and 4 Division of Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health, Sciences, Linköping University, County Council of Östergötland, Linköping, Sweden. Correspondence: Dr L Karlsson or Dr FC Kugelberg, Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping SE-581 85, Sweden. E-mail: [email protected] or [email protected] Received 10 February 2014; revised 27 June 2014; accepted 8 July 2014
Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolism L Karlsson et al
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MATERIALS AND METHODS Case selection Ninety-four forensic autopsy cases in which VEN was found in femoral blood were selected for the study. The samples, collected from the National Board of Forensic Medicine in Sweden, were routinely screened for a wide range of prescription drugs and ethanol. The samples were analysed by an enantioselective method and genotyped for CYP2D6 and CYP2C19. The cases were classified using the ICD (International Classification of Disease) codes by the responsible pathologist. Additional information related to each of the cases (age, gender and circumstances surrounding death) was provided from the forensic pathology and toxicology databases at the Swedish National Board of Forensic Medicine. The study was approved by the Regional Ethical Review Board in Linköping, Sweden (No. M87-08).
Enantioselective analysis of VEN The concentration of the S- and R-enantiomers of VEN and its three metabolites (ODV, NDV and DDV) in femoral blood was determined by using a LC/MS/MS method as described previously.6,27 Briefly, the samples were extracted using Isolute C8 columns 100 mg (International Sorbent Technology, Hengoed, UK) After elution and evaporation with nitrogen at 50 °C, the analytes were reconstituted in 100 μl mobile phase (tetrahydrofuran:ammonium acetate, 10 mM, pH 6; 10:90 v/v) and 2 μl was injected. The LC/MS/MS system consisted of an Acquity liquid chromatography system (Waters, Milford, MA, USA) and an API 4000 tandem quadrupole instrument equipped with an electrospray interface (Turbo VTM source, ® TurbolonSpray probe) operating in positive ion mode (Applied Biosystem/ MSD Sciex, Toronto, ON, Canada). Chromatographic separation was performed on a Chirobiotic-V column (5 μm particle size, 250 × 2.1 mm: Astec/Supelco, Sigma-Aldrich, St. Louis, MO, USA) protected with a 5- μm in-line filter (VICI AB Int., Schenkon, Switzerland) at a mobile phase flow rate of 0.2 ml min − 1. Data were processed using the Analyst 1.4.2 Software (Applied Biosystem/MSD Sciex).
Genotyping for CYP2C19 and CYP2D6 Genotyping for CYP2D6 and CYP2C19 was performed with PCR followed by Pyrosequencing as described previously.10,28 For CYP2D6, four of the most important non-functional alleles *3, *4, *5 and *6, were included (http:// www.cypalleles.ki.se). The copy number variation was determined to identify multiple gene copies (that is, active alleles such as CYP2D6*1xN or inactive alleles such as CYP2D6*4xN) and to identify the whole gene deletion (CYP2D6*5). For CYP2C19, the three main non-functional alleles *2, *3 and *4 and the gain-of-function allele *17 were included (http://www. cypalleles.ki.se). On the basis of the results from the genotype analyses, the cases were classified into four predicted phenotype groups for CYP2D6 and CYP2C19, respectively. A carrier of only non-functional alleles was termed as poor metabolizer (PM). A heterozygous carrier of one active allele (*1) in combination with one non-functional allele was termed as intermediate metabolizer (IM). A carrier of two active alleles (*1/*1) or for CYP2C19, one non-functional and one gain-of-function allele (that is, *2/*17) was termed as extensive metabolizer (EM). For CYP2D6, a carrier of more than two active alleles (*1xN/*1) and for CYP2C19, a carrier of one active and one gain-of-function allele (*1/*17) or two gain-of-function alleles (*17/*17) was termed as ultra-rapid metabolizer (UM).
Statistical analysis Data are presented as mean ± s.d. or median and the 10th and 90th percentiles. The influence of the genotypes on the metabolic ratios and the S/R ratios was determined using multivariate ANOVA. A P-value of o0.05 was considered as statistically significant. All statistical analysis was performed using StatView® (version 5.0; SAS® Institute, Cary, NC, USA).
RESULTS General data The material comprises 94 VEN-positive postmortem cases, 35 women and 59 men with ages ranging between 18 and 86 years (median 49 years). Most of the cases were polydrug cases and the median number of drugs was 4 (range 1–11). In 13 cases, VEN was the only drug identified, and none of these cases were classified as intoxications. The most frequently found drugs in addition to VEN The Pharmacogenomics Journal (2014), 1 – 7
were ethanol, zopiclone, mirtazapin and diazepam. The manner of death was classified as suicide in 50 cases, natural in 15 cases, accidental in 22 cases and undetermined/other in 7 cases (for details, see Supplementary Information Supplementary Table S1). The distribution of genetic variation for CYP2D6 was 7 PM, 39 IM, 43 EM and 2 UM (3 cases were undetermined). For CYP2C19, 2 PM, 14 IM, 48 EM and 27 UM were found (3 cases were undetermined). Analytical results, including total (S+R) concentrations, enantiomeric S/R ratios as well as total (S+R) metabolite/parent drug (M/P) ratios, are presented in Table 1. The median concentrations of VEN, ODV NDV and DDV in all cases were 0.56, 0.48, 0.08 and 0.1 μg g − 1, respectively. Association between CYP genotypes and VEN metabolic ratios The CYP2D6 genotype significantly influenced the M/P ratios of ODV/VEN (P = 0.003), DDV/NDV (P = 0.010) and DDV/ODV (P = 0.034) (Figure 1). Median ODV/VEN ratios for CYP2D6 were 0.22 for PM, 0.75 for IM, 1.78 for EM and 2.03 for UM. The DDV/ODV (P = 0.013) and DDV/VEN (P = 0.021) ratios were significantly influenced by the CYP2C19 genotype. CYP2C19 also influenced the NDV/VEN ratio but did not reach statistical significance (P = 0.061). Median NDV/VEN ratios for CYP2C19 were 0.01 for PM, 0.14 for IM, 0.17 for EM and 0.25 for UM, clearly showing a trend. Association between CYP genotypes and enantiomeric ratios of VEN and its metabolites The S/R ratios of VEN were significantly influenced by both CYP2D6 and CYP2C19 genotypes (P = 0.009 and P = 0.015, respectively) (Figure 2). CYP2D6 significantly influenced both the ODV S/ R ratios and the NDV S/R ratios (P = o 0.0001 and P = 0.003, respectively). The S/R ratios of DDV were significantly influenced by CYP2D6 (P = 0.0002). Receiver operating characteristic curves To evaluate the predictive value of the genotype on the metabolic ratio, we created receiver operating characteristic (ROC) curves and calculated the area under the curve. The area under the curve
Table 1. Total (S+R) concentration, enantiomeric (S/R) drug concentration ratios and metabolite/parent drug concentration ratios (M/P) n
10th percentile
Median
90th percentile
Total (S+R) concentration (μg g −1) VEN 94 0.127 ODV 89 0.126 NDV 93 0.016 DDV 94 0.023
0.562 0.484 0.082 0.102
2.364 1.224 0.739 0.296
S/R concentration ratios VEN 94 ODV 89 NDV 90 DDV 94
0.518 0.729 0.473 0.688
0.968 0.894 0.807 1.030
1.692 2.795 1.441 1.490
M/P concentration ratios ODV/VEN 89 NDV/VEN 93 DDV/VEN 94 DDV/ODV 89 DDV/NDV 93
0.193 0.042 0.039 0.071 0.142
0.977 0.184 0.188 0.193 1.218
3.478 0.722 0.708 0.395 4.186
Abbreviations: DDV, N,O-didesmethylvenlafaxine; NDV, N-desmethylvenlafaxine; ODV, O-desmethylvenlafaxine; VEN, venlafaxine.
© 2014 Macmillan Publishers Limited
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Figure 1. The metabolic ratios (metabolite/parent drug concentration ratios) for venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in femoral blood after dividing the cases into CYP2D6 genotypes and CYP2C19 genotypes. Values are mean ± s.d. (CYP2D6: n = 7 PM, n = 39 IM, n = 43 EM and n = 2 UM and CYP2C19: n = 2 PM, n = 14 IM, n = 48 EM and n = 27 UM). EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; UM, ultra-rapid metabolizer.
for ODV/VEN ratios was 0.8936 with a P-value of 0.001 (Figure 3). Using the ROC curve to get a cutoff for skewed ODV/VEN ratios (ratios of below 0.35) resulted in that 26% of cases having a skewed ratio were PM for CYP2D6, compared with 1.5% PM in the © 2014 Macmillan Publishers Limited
remaining cases. The ROC curve for the effect of CYP2C19 genotype on NDV/VEN ratio had an area under the curve of 0.9972 with a P-value of 0.017. The cases with the lowest NDV/VEN ratio were all PM for CYP2C19. The Pharmacogenomics Journal (2014), 1 – 7
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Figure 2. Enantiomeric (S/R) concentration ratios for venlafaxine (VEN) and its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in femoral blood after dividing the cases into CYP2D6 genotypes and CYP2C19 genotypes. Values are mean ± s.d. (CYP2D6: n = 7 PM, n = 39 IM, n = 43 EM and n = 2 UM and CYP2C19: n = 2 PM, n = 14 IM, n = 48 EM and n = 27 UM). EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; UM, ultra-rapid metabolizer.
The Pharmacogenomics Journal (2014), 1 – 7
© 2014 Macmillan Publishers Limited
Influence of CYP2D6 and CYP2C19 genotypes on venlafaxine metabolism L Karlsson et al
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Figure 3. Receiver operating characteristic (ROC) curves for metabolic ratios (metabolite/parent drug concentration ratios) of venlafaxine (VEN) and the metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) divided into genotype/phenotype groups for CYP2D6 and CYP2C19, respectively.
DISCUSSION In this study, we present a detailed investigation on the demethylation of VEN and its major metabolites ODV and NDV. We found that the CYP2D6 genotype influences the O-demethylation whereas CYP2C19 influences the N-demethylation of VEN and its metabolites. In addition, we observed a stereoselective metabolism where CYP2D6 favours the R-enantiomer whereas CYP2C19 favours the S-enantiomer. The major metabolic pathway of VEN is O-demethylation, accounting for ~ 55% of an oral dose excreted as ODV.29 The metabolism of VEN has been the focus of several studies and the main effort has been on CYP2D6. An in vitro study with microsomes from human liver and CYP2D6 transformed yeast found that CYP2D6 catalyzes the O-demethylation of VEN into ODV and that CYP3A4 was involved in the N-demethylation of VEN into NDV. No indication that CYP2D6 was involved in the N-demethylation of VEN was shown.18 Another in vitro study showed that the CYP2C9, CYP2C19 and CYP2D6 enzymes have a role in the metabolism of VEN into ODV (using human liver microsomes and CYP microsomes) with CYP2D6 being the main enzyme (relative contribution of 1, 10 and 89%, respectively).17 In the same study, CYP2C9, CYP2C19 and CYP3A4 were found to be involved in the metabolism of VEN into NDV (relative contribution of 31, 33 and 36%, respectively). The formation of DDV from ODV has been suggested to be dependent on CYP3A4.30 We found that the demethylation of VEN into ODV is influenced by the CYP2D6 genotype, with lowest ODV/VEN ratios for PM individuals and highest ODV/VEN ratios for UM individuals (Figure 1). A correlation between the metabolic ratio (ODV/VEN) and CYP2D6 genotype has been shown in clinical studies with lowest ratios for PMs.21,23–26 In addition, McAlpine et al.21 found an association between ODV/VEN ratio and CYP2C19 genotype. CYP2D6 PM individuals have also been found to have an increased risk of developing side effects.24 Otton et al.18 speculated that NDV is O-demethylated by CYP2D6 into DDV which is in line with our results. We show that the CYP2D6 genotype influences the demethylation of NDV into DDV, with PM individuals having the lowest DDV/NDV ratio and UM individuals showing the highest ratio (Figure 1). Taken together, CYP2D6 seems to be involved in the O-demethylation of VEN into ODV and NDV into DDV, which is not entirely surprising since it is the same chemical reaction in both metabolism steps. All M/P ratios are dependent on both the formation and degradation of the metabolite. For example, the © 2014 Macmillan Publishers Limited
ODV/VEN ratio is influenced by CYP2D6 in terms of the formation of ODV. However, our data also suggest that CYP2C19 has a possible effect on the ODV/VEN ratio by an impact on the degradation of ODV. We show that the CYP2C19 genotype influenced the demethylation of VEN into NDV and ODV into DDV. Fogelman et al.17 found that CYP2C9, CYP2C19 and CYP3A4 are involved in the formation of NDV with CYP2C19 having the highest intrinsic clearance. Even though CYP2C19 was found to have the highest intrinsic clearance, the high abundance of CYP3A4 will enlarge the importance of this enzyme. Hermann et al.23 reported an influence of CYP2D6 on the NDV concentration. They found a higher formation of NDV in individuals with one non-functional CYP2D6 allele compared with EM, most likely due to higher fraction of VEN being metabolized to NDV and less NDV being metabolized into DDV when CYP2D6 activity is impaired. This is in accord with our data on both CYP2C19 and CYP2D6. Two other studies found the highest concentration of NDV in CYP2D6 PMs and the lowest concentration in UMs.24,26 None of the three studies has investigated the influence of CYP2C19, which we here find influencing the demethylation of VEN into NDV and ODV into DDV. However, the contribution of CYP3A4 in the demethylation of VEN and its metabolites is still unclear mainly due to the lack of relevant genotype analysis and has not been investigated in the present study. The lack of genotype for CYP3A4 in the present study is a limitation. Our ROC curve analysis shows that the CYP2D6 and CYP2C19 genotypes are useful predictors for skewed VEN metabolism. A large proportion of the individuals with low ratios can be explained by these genotypes showing that these pharmacogenetic analyses are important when studying metabolic ratios in both in clinical and forensic setting. There is a limited knowledge about the stereoselective metabolism of VEN and its metabolites. To date, it is known that CYP2D6 has been found to be stereoselective towards the R-enantiomer of VEN.22 This finding is in line with our present and previous data showing that CYP2D6 PM individuals have a low VEN S/R ratio in combination with a high ODV S/R ratio.6 Otton et al.18 reported that the S-enantiomer of VEN was preferentially N-demethylated. Our data suggest that this stereoselectivity is CYP2C19 mediated with PM individuals having high VEN S/R ratio in combination with low ODV S/R ratio. Stereoselective metabolism of other drugs has been reported. For methadone, CYP2B6 has been reported to favour the The Pharmacogenomics Journal (2014), 1 – 7
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ACKNOWLEDGMENTS This work was supported by grants from the Swedish Research Council, the Lions Research Foundation and the National Board of Forensic Medicine in Sweden.
REFERENCES
Figure 4. Metabolism of venlafaxine (VEN) into its metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV).
S-enantiomer whereas CYP2C19 favours the R-enantiomer.31 The (+)-enantiomer of omeprazole is metabolized by CYP2C19 and the (− )-enantiomer is partly metabolized by this enzyme but mainly by CYP3A4.32 Stereoselective metabolism could have an important role, influencing pharmacokinetics, pharmacodynamics and toxicity.33 Chiral considerations are especially relevant for drugs where the enantiomers display different properties like in the case with VEN where the S-enantiomer inhibits serotonin reuptake while the R-enantiomer inhibits both serotonin and noradrenaline reuptake.15,16 A change in the S/R ratios of the enantiomers caused by differences in CYP2D6 and/or CYP2C19 activity might therefore affect the clinical outcome. Other factors to consider when analyzing forensic data, besides genetic variation, are information about age, ingested dose and time of sampling in relation to the time when the drug was taken, which are rarely known, and metabolic drug interactions. In the present study, in most cases other drugs were found with an average of four drugs. Drug interactions may affect the pharmacokinetics of VEN since many of the drugs found are metabolized by the same enzymes as VEN. Drug interactions involving VEN may have occurred in cases included in the present study. Further, the possibility that VEN undergoes postmortem drug redistribution has to be considered.34 Even if redistribution has occurred, it is likely that the parent drug and the metabolites redistribute at the same extent. On the basis of the findings in the present study, together with previous studies,17,18,30 Figure 4 displays a conclusion of the metabolism of VEN. VEN is O-demethylated into ODV via CYP2D6, CYP2C9 and CYP2C19. VEN is also N-demethylated into NDV by CYP3A4, CYP2C19 and CYP2C9. ODV and NDV are then further N-demethylated and O-demethylated into DDV via CYP2C19 and CYP2D6, respectively. CYP2D6 is stereoselective and prefers the R-enantiomer whereas CYP2C19 prefers the S-enantiomer. In clinical practice, the sum of VEN and ODV concentrations is usually used as the therapeutic concentration.35,36 The present data demonstrate the importance of analysing not only ODV, but also NDV in order to make a correct interpretation of the genotype influence. When looking at the total demethylation of VEN into DDV, DDV/VEN ratio, our data show that both CYP2D6 and CYP2C19 genotypes are influencing the transformation. In clinic, CYP2C19 may have a more therapeutic relevance than CYP2D6 since it metabolizes both active compounds, that is, VEN and ODV, but the CYP2D6 genotype has a significant role for the enantiomeric ratios. CONFLICT OF INTEREST The authors declare no conflict of interest.
The Pharmacogenomics Journal (2014), 1 – 7
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The Pharmacogenomics Journal (2014), 1 – 7
