30.Wk4Assgn

PHARMACOGENETICS

Cytochrome P450 2D6 genotype affects the pharmacokinetics of controlled-release paroxetine in healthy Chinese subjects: comparison of traditional phenotype and activity score systems

Rui Chen1 & Haotian Wang2 & Jun Shi3 & Kai Shen1 & Pei Hu1

Received: 19 December 2014 /Accepted: 27 April 2015 /Published online: 13 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Purpose This study evaluated the effects of cytochrome P450 (CYP) 2D6 polymorphisms on the pharmacokinetics of controlled-release paroxetine in healthy Chinese subjects and used paroxetine as a tool drug to compare the performance of traditional phenotype and activity score systems. Methods Pharmacokinetic data were evaluated in 24 subjects who received a single oral dose of 25 mg controlled-release paroxetine. Plasma paroxetine concentrations were measured by LC-MS/MS. CYP2D6 genotypes were tested by PCR and direct DNA sequencing. Subjects were classified by two sys- tems of phenotype prediction. In the traditional phenotype system, subjects were classified as extensive metabolizers or intermediate metabolizers; in the activity score system, sub- jects were divided into four activity groups. Analysis of vari- ance testing was applied to estimate the effects of CYP2D6 polymorphisms on the pharmacokinetics of paroxetine. Results With the traditional phenotype system, significant dif- ferences were observed in the following pharmacokinetic param- eters of paroxetine: t1/2, Cmax, AUC0–t, AUC0–inf, Vz/F, and CL/F (all P<0.05). The AUC or exposure of paroxetine was about 3.5- fold higher in the intermediate metabolizer group than in the extensive metabolizer group. With the activity score system, significant differences were observed in the t1/2, Cmax, AUC0–t, AUC0–inf, Vz/F, and CL/F among the four different activity score

groups (all P<0.05). We found that the AUC of paroxetine de- creased by around one half as the activity score increased by 0.5. Conclusion The pharmacokinetics of controlled-release par- oxetine after a single administration was affected by CYP2D6 polymorphisms. Both the traditional phenotype and the activ- ity score systems performed well and distinguished subjects with different drug exposures. The activity score system pro- vided a more detailed classification for the subjects.

Keywords CYP2D6 polymorphisms . Paroxetine .

Phenotype . Activity score

Introduction

Paroxetine is a selective serotonin reuptake inhibitor (SSRI) that is currently used worldwide for depression, panic disor- der, obsessive-compulsive disorder, social anxiety disorder, generalized anxiety disorder, and post-traumatic stress disor- der [1]. Two studies observed that the plasma concentration of paroxetine might be associated with therapeutic response, and that such concentrations could potentially be used to predict therapeutic response [2, 3]. Gilles et al. defined an upper threshold of paroxetine serum concentration above which re- sponse to treatment was unlikely [2]. Yasui-Furukori also sug- gested that plasma paroxetine concentrations were negatively associated with improvement, and that responses occurred below an upper threshold of paroxetine [3].

Paroxetine is extensively metabolized in humans; its me- tabolism involves oxidation, methylation, and conjugation. Renal clearance of the compound is negligible [4]. CYP2D6 is most likely to be the major contributor to paroxetine metab- olism in humans [5] and is also likely the major contributor to the previously reported wide inter-individual variation in the pharmacokinetics of paroxetine [4].

* Pei Hu [email protected]

1 Clinical Pharmacology Research Center, Peking Union Medical College Hospital, 41 Damucang Alley, Xicheng District, Beijing 100032, China

2 Beijing Tsinghua Changguang Hospital, Beijing, China 3 Roche Innovation Center Shanghai, Shanghai, China

Eur J Clin Pharmacol (2015) 71:835–841 DOI 10.1007/s00228-015-1855-6

CYP2D6 is also one of the most well-known and widely investigated polymorphic enzymes in human, owing to the wide inter-individual and inter-ethnic variations in the activity of this enzyme [6]. To date, more than 100 allelic variants of CYP2D6 have been identified [7]. Among these variants are fully functional alleles, reduced function alleles, nonfunction- al alleles, and gene duplications of functional CYP2D6 alleles. This allelic diversity results in a wide range of CYP2D6 ac- tivities, from ultra-rapid metabolism to no metabolism. Asians exhibit a marked shift toward slower CYP2D6 activity on a population basis. This can be explained by the high frequency (around 42 %) of the CYP2D6*10 reduced function allele in Asians; the frequency of CYP2D6*10 ranges between 3 and 7 % in other populations [8, 9].

The four traditional phenotypes of CYP2D6, based on the use of a probe substrate to challenge a population, are stratified and include poor (PM), intermediate (IM), extensive (EM), and ultra-rapid (UM) metabolizer phenotypes [10]. To date, there is no universally defined and accepted method of genotype grouping or classification. Gaedigk et al. [10] introduced the activity score system in 2008. It has since gained acceptance among the community and has also been adopted by the Clin- ical Pharmacogenetics Implementation Consortium (CPIC) [8]. This system, in essence, assigns each allele a value that approx- imates its function, and the sum of the values assigned to each allele is the activity score. Functional alleles with activity levels comparable to the CYP2D6*1 reference allele are given a value of 1. Reduced function and nonfunctional alleles receive values of 0.5 and 0, respectively. Gene duplications score double the value given for their single counterparts [9, 10].

Although some studies have reported the effects of CYP2D6 polymorphisms on the pharmacokinetics of paroxe- tine [11–14], an association between CYP2D6 activity scores and the pharmacokinetics of paroxetine has never been report- ed. Very few studies have compared the traditional phenotype and activity score systems. In this study, we investigated the effects of CYP2D6 polymorphisms on the pharmacokinetics of controlled-release paroxetine in healthy Chinese subjects, and used paroxetine as a tool drug to compare the two systems of phenotype prediction.

Materials and methods

Subjects and study design

The study protocol was approved by the ethics committee of Peking Union Medical College Hospital, and all subjects pro- vided written informed consent. A total of 24 healthy Chinese men and women, aged 19–45 years, with a body mass index (BMI) between 19 and 25 kg/m2 were enrolled in the study after a medical screening. The subject would be defined Chinese only if his/her parents and grandparents were all Chinese. All

subjects were in good health based on physical examination, electrocardiogram, blood pressure monitoring, and routine clin- ical laboratory tests (complete blood count, blood chemistry, and urinalysis), and were free of any clinically significant dis- eases that could interfere with the study evaluation. Subjects were not allergic to paroxetine or heparin. The subjects stayed in the ward and were provided standardized food until 96 h after paroxetine administration. The consumption of alcohol, grape- fruit juice, caffeine-containing drinks, and indole- and flavones- containing foods was not permitted in the period from 24 h prior to paroxetine administration until after the final blood sample of the study was collected. The subjects were instructed to abstain from taking any medication for at least 1 week prior to paroxetine administration as well as during the study period.

Study procedures

All subjects were admitted to the clinical trial center on the evening before the day on which the drug was administered. On day 1, all subjects were given a single oral dose of a 25 mg controlled-release paroxetine tablet (25 mg/tablet, GlaxoSmithKline Co., Ltd.) with 200 mL of water. An angiocatheter with a diluted heparin solution lock was inserted into a vein in the antecubital area. Blood samples were col- lected prior to drug administration (baseline) and then at 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 32, 48, 72, and 96 h after drug administration. Blood samples (7 mL) were centrifuged within 1 h; plasma samples were stored at −20 °C until the analysis.

Genotyping of CYP2D6

Peripheral blood samples were collected and DNA was ex- tracted by total genomic DNA isolation using a Wizard TM Genomic Purification Kit (Promega, USA). The DNA was dissolved in 100 μL of DNA hydration solution and stored at −70 °C. All subjects recruited in this study were genotyped by DNA sequencing analysis for CYP2D6*1, *2, *5, *10, *14, *41, which are frequently found in Asians and have clinical consequences [15]. CYP2D6*3, *4, *6, *9 and *2xN were not tested in this study. For detection of CYP2D6 genotypes, a 20 μL PCR reaction was performed. Primers were designed with the primer3 software [16]. The PCR am- plifications were performed with SolGent™ f-Taq DNA po- lymerase. The reaction mixture contained 14.5 μL of water, 2.0 μL of 10×f-taq buffer, 0.1 μL of enzyme mix (5 U/μL), 0.4 μL of dNTP mixture (10 mmol/L), 0.8 μL of forward primer (10 mmol/L), 0.8 μL of reverse primer (10 mmol/L), and 1.0 μL of genomic DNA (50 ng/mL). Cycling conditions were as follows: 3 min at 94 °C, followed by 35 cycles of 94 °C for 20 s, 62 °C for 30 s, 72 °C for 20 min, and a final extension of 5 min at 72 °C. The PCR products were then analyzed by direct sequencing using the ABI PRISIM BigDye Terminator Cycle Sequencing Kit and an ABI Prism 3100

836 Eur J Clin Pharmacol (2015) 71:835–841

Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) to detect CYP2D6*10 (100C>T) [17]. The CYP2D6*5 allele was identified using the long-PCR method with minor modifications as described previously [18].

Bioanalytic methodology

The plasma paroxetine concentrations were measured using a previously reported method [19, 20]. In brief, a rapid, simple, and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to determine paroxetine levels in the human plasma sam- ples collected in the present study. An aliquot of 0.2 mL of plasma or standard was mixed with 50 μL of the internal standard, vortexed, alkalinized with 0.4 mL of 2 % ammonia water, shaked for 1 min, loaded onto a Waters Oasis HLB 96- Well solid-phase extraction cartridge, eluted with 0.2 mL of 90 % methanol, evaporated to dryness at 40 °C under nitro- gen, reconstituted with 150 μL of mobile phase, and assayed. Calibration curves for paroxetine were constructed over the range from 0.02 to 50 ng/mL. Correlation coefficients were greater than 0.990. The mean recovery of the drug from plas- ma ranged from 79.6 to 102.2 %. Intraday and interday coef- ficients of variation were less than 10.6 and 11.3 %, respec- tively. The lower limit of quantification was 0.02 ng/mL.

Pharmacokinetic analysis

Pharmacokinetic parameters were obtained through non- compartmental analysis using Phoenix WinNonlin version 6.3 (Pharsight Corp., Mountain View, CA, USA). Pharmaco- kinetic parameters determined after single dose included area under the plasma concentration-time curve from time 0 to 96 h (AUC0-t), AUC from time 0 to infinity (AUC0-inf), maximum plasma concentration (Cmax), time to maximum plasma con- centration (Tmax), apparent clearance (CL/F), apparent volume of distribution (Vz/F), and terminal elimination half-life (t1/2). The Cmax and Tmax were estimated directly from the observed plasma concentration-time data. The AUC0-t was calculated using the log-trapezoidal rule. AUC0-inf was calculated as AUC0-inf=AUC0-t+Ct/Ke, where Ct is the last plasma concen- tration measured and Ke is the elimination rate constant; Ke was determined using linear regression analysis of the logarithm-linear part of the plasma concentration-time curve. The t1/2 was calculated as ln2/Ke.

Statistical analysis

Both a traditional phenotype and an activity score system were used to evaluate the effects of CYP2D6 polymorphisms on the pharmacokinetics of paroxetine. In the traditional phenotype system, individuals carrying two functional alleles (e.g., 2D6*1), or one functional and one reduced function allele

(e.g., 2D6*10), were classified as EMs. Individuals carrying two reduced function alleles, one functional and one nonfunc- tional allele (e.g., 2D6*5), or one reduced function and one nonfunctional allele were classified as IMs. Individuals carry- ing two nonfunctional alleles were classified as PMs. Contro- versies exist regarding the classification of subjects carrying two reduced function alleles or those carrying one functional allele and one nonfunctional allele. Here, we preferred to clas- sify these subjects as intermediate metabolizers according to a recent review [21]. The activity score system used in this study was based on a previously reported scheme [10] where scores were summed for each 2D6 allele. A score of 0 was given to a nonfunctional allele (2D6*5); 0.5 for reduced func- tion alleles (2D6*10, *14, and *41); and 1.0 for functional alleles (2D6*1 and *2). The activity score of a given subject was calculated as the sum of the values assigned to the two 2D6 alleles.

The pharmacokinetic parameters were analyzed with anal- ysis of variance (ANOVA) to estimate the effects of CYP2D6 polymorphisms on the pharmacokinetics of controlled-release paroxetine. A P value less than 0.05 was considered to be statistically significant. ANOVA was performed with SPSS (version 19.0, SPSS™).

Results

Demographic characteristics

Eleven genotypes were identified in the 24 subjects: CYP2D6*1/*1 (n = 2), CYP2D6*1/*2 (n = 2), CYP2D6*1/*5 (n = 1), CYP2D6*1/*10 (n = 2), CYP2D6*1/*41 (n = 1), CYP2D6*2/*10 (n = 1), CYP2D6*2/*14B (n = 1), CYP2D6*5/*10 (n = 4), CYP2D6*10/*10 (n=8), CYP2D6*10/*14B (n=1), and CYP2D6*10/*41 (n=1). The allele frequencies of CYP2D6*1, *2, *5, *10, *14B, and *41 were 20.83, 8.33, 10.42, 52.08, 4.17, and 4.17 %, respectively.

All of the subjects completed the study without clinically important adverse effects. Their ages, heights, and weights are presented in Table 1. The genotypes of subjects were classi- fied with the two aforementioned systems (Table 1). There were no significant differences in the demographic data among the classification groups.

Effects of CYP2D6 polymorphisms on the pharmacokinetics of paroxetine

With the traditional phenotype system, the pharmacokinetic parameters and plasma concentration-time profiles of paroxe- tine after a single administration were compared between the different phenotype groups. The Cmax of paroxetine in the EM group was 3.2-fold lower than that in the IM group (P=

Eur J Clin Pharmacol (2015) 71:835–841 837

0.002). Both the AUC0-t and the AUC0-inf values of paroxetine in the EM group were 3.5-fold lower than those in the IM group (P=0.012 for AUC0-t and 0.013 AUC0-inf). The Vz/F and CL/F ratios for paroxetine in the EM group were 3.8- and 5.55-fold higher, respectively, than those in the IM group (P= 0.003 and 0.001) (Table 2, Fig. 1).

With the activity score system, the pharmacokinetic pa- rameters and plasma concentration-time profiles of paroxe- tine were compared among the different activity score groups. Two trends were apparent: as the activity scores increased from 0.5 to 2, the t1/2, Cmax, AUC0-t, and AUC0-inf values decreased; as the activity scores increased, the Vz/F and CL/F values increased (all P<0.05). The t1/2, Cmax, and CL/F values of the score 0.5 group were signif- icantly different from those of the score 1.5 and score 2 groups. The Cmax and CL/F values of the score 1 group were significantly different from those of the score 1.5 and the score 2 groups. The Vz/F values of subjects of the score 1.5 group were significantly different from those of the score 0.5 and score 1 groups. The AUC values of the score 0.5 group were significantly different from those of the other three groups (Table 2, Fig. 2).

Discussion

To date, few studies have evaluated the use of the activity score system in Asian populations, and there are very few such studies that describe pharmacokinetic profiles. Typically, large sample sizes are needed to evaluate phenotype prediction sys- tems; these are often based on metabolic ratios from urine. However, it is hard to conduct large sample size studies when pharmacokinetic profiles are described. In the present study, paroxetine was used as a tool drug to compare traditional phenotype and activity score systems based on pharmacoki- netic profiles and pharmacokinetic parameters in Chinese sub- jects. The two systems of phenotype prediction both provided good classifications for the subjects, and could distinguish subjects with different drug exposures. With the traditional phenotype system, the AUC of paroxetine was about 3.5-fold higher in the IM group than in the EM group. With the activity score system, the subjects were classified further into four groups; AUC values decreased by about one half as the activ- ity score increased by 0.5. The activity score system provided a more detailed classification for the subjects as compared with the traditional phenotype system.

Table 1 Baseline demographic characteristics of the population in this study based on two CYP2D6 phenotype systems

Characteristic CYP2D6 traditional genotype classification CYP2D6 activity score system

All (n=24) EM (n=9) IM (n=15) Score 0.5 (n=4) Score 1 (n=11) Score 1.5 (n=5) Score 2 (n=4)

Age (years) 25.6±5.5 24.8±6.2 26.1±5.3 26.1±6.9 26.1±5.0 22.1±1.4 28.2±8.5

Weight (kg) 60.9±6.0 58.9±6.6 62.0±5.5 64.0±2.7 61.3±6.2 57.6±5.5 60.6±8.4

Height (m) 1.7±0.1 1.7±0.1 1.7±0.1 1.7±0.1 1.7±0.1 1.6±0.1 1.7±0.1

BMI (kg/m2) 21.7±2.0 21.6±2.3 21.8±1.8 22.1±2.5 21.7±1.7 21.5±2.1 21.7±2.8

Gender Female=10 Female=5 Female=6 Female=1 Female=5 Female=3 Female=1

Male=14 Male=5 Male=9 Male=3 Male=6 Male=2 Male=3

Table 2 Pharmacokinetic parameters of paroxetine after a single oral dose of 25 mg controlled-release paroxetine in healthy Chinese subjects based on two CYP2D6 phenotype systems

Parameters Traditional phenotype Activity score

EM (n=9) IM (n=15) P* 0.5 (n=4) 1 (n=11) 1.5 (n=5) 2 (n=4) P*

t1/2 (h) 10.5±1.4 13.2±2.5 0.007 14.9±2.0 12.6±2.5 10.5±1.7 10.4±1.2 0.017

Tmax (h) 9.3±2.0 9.3±1.6 1.000 8.5±1.9 9.6±1.5 9.6±1.7 9.0±2.6 0.705

Cmax (ng/mL) 2.8±2.5 8.8±4.8 0.002 12.2±6.3 7.6±3.7 2.7±2.9 2.8±2.2 0.005

AUC0-t (h•ng/mL) 66.2±87.0 231.2±166.6 0.012 382.1±255.1 176.4±83.3 83.6±115.4 44.5±36.2 0.005

AUC0-inf a (h•ng/mL) 67.2±87.9 235.2±173.1 0.013 392.6±267.3 177.9±83.5 84.8±116.7 45.2±36.1 0.005

Vz/F (L) 14170.9±10112.5 3730.9±5179.9 0.003 2124.9±1781.1 4314.9±5932.3 15340.8±11653.2 12708.4±9301.0 0.029

CL/F (L/h) 994.0±758.5 179.1±184.5 0.001 100.6±81.9 207.7±205.7 1099.2±912.5 862.6±618.4 0.008

EM extensive metabolizer, IM intermediate metabolizer

*P<0.05 was considered statistically significant. P values were overall P values a The ranges of AUC0-inf are (11.0, 284.5) and (31.1, 724.6) h•ng/mL for EM and IM groups, respectively; and are (115.5, 724.6), (31.1, 321.6), (11.0, 284.5), and (14.6, 97.1) h•ng/mL for four groups from activity score 0.5 to 2, respectively

838 Eur J Clin Pharmacol (2015) 71:835–841

Numerous CYP2D6 alleles have been identified, and their frequencies vary across different populations [22]. CYP2D6*10 is a major variant with a frequency of around 42 % in Asian populations [8], with a frequency of 38.1 % in Japanese and [23] 40–51 % in Chinese [15, 24, 25]. In the present study, the frequency of the CYP2D6*10 allele was 52.08 %, similar to previously reported frequencies in Chinese [15, 24, 25]. The high frequency of CYP2D6*10 results in high frequency of CYP2D6 IM individuals among Chinese as compared with the very low frequency of CYP2D6 IM indi- viduals among Caucasians [9]. Beyond the fact that genotype and phenotype distributions have ethnic diversity, the relation- ship between CYP2D6 genotype and phenotype (i.e., meta- bolic ratios or pharmacokinetic parameters) has been shown to be dependent on ethnicity [9, 10]. Therefore, the relationship between CYP2D6 genotype and paroxetine concentrations may be different in Asians than in other ethnic groups.

The goal of genotype testing in clinical practice is to link genotypes to individual treatment response and to adjust treat- ment doses accordingly. However, the predictive value of CYP2D6 genotyping at the individual level is limited, given that there are considerable overlaps in pharmacokinetics

between phenotype groups. Take paroxetine as an example: Table 2 shows the range of AUC0-inf in each group; the over- laps between the groups are obvious. The systems of pheno- type prediction, however, still provide information about the probability that a given individual will present with a particu- lar phenotype [10]. The activity score system has gained ac- ceptance among community, since it was introduced by Gaedigk to streamline genotype interpretation and improve phenotype prediction in 2008 [8]. The translation of genotype data using the activity score system has the potential to sim- plify CYP2D6 phenotype prediction, but more studies are needed to evaluate and improve this system. First, different reduced function alleles have differing CYP2D6 activity levels, e.g., CYP*10 and *14 may have different enzyme ac- tivities but, as reduced function alleles, they are both given a value of 0.5. Revising such score values up or down may benefit phenotype prediction. Second, it has been demonstrat- ed that the prediction of phenotype from genotype is ethnicity dependent, so it is conceivable that the value for certain alleles could be different in Asians than in other ethnicities. More data from Asian subjects will be needed to establish corrected activity scores for Asian populations.

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Figure 1 Mean (SD) plasma concentration-time profiles of paroxetine after a single oral dose of 25 mg controlled-release paroxetine in different CYP2D6 phenotype groups

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Figure 2 Mean (SD) plasma concentration-time profiles of paroxetine after a single oral dose of 25 mg controlled-release paroxetine in different CYP2D6 activity score groups

Eur J Clin Pharmacol (2015) 71:835–841 839

Phenotype prediction or dose adjustment may be less im- portant when a medicine has a broad therapeutic index. For some medicines, e.g., paroxetine, repeating dosing will dimin- ish the effect of genetic polymorphisms on paroxetine concen- trations due to the autoinhibition of CYP2D6 [26]. Differ- ences in the steady state of paroxetine levels between groups are more relevant with regard to clinical response [13]. Parox- etine was used as a tool drug in the present study to evaluate the performance of the activity score system in Chinese; par- oxetine levels in the steady state after repeated dosing was not evaluated here. Alleles not tested for here or unidentified ge- netic variations of CYP2D6 could lead to phenotype misclas- sification, especially when a subject carries allele(s) with low frequencies. The frequency of PMs is very low among Asians (0–2 %) [9]; we did not have a PM subject in the present study. Gene duplications were not tested in the present study. Given that the frequency of CYP2D6 gene duplications in Asians is 1–2 % [8], it is unlikely that any of the 24 subjects had this polymorphism.

Conclusion

The pharmacokinetics of controlled-release paroxetine after a single administration was affected by CYP2D6 polymor- phisms. Both the traditional phenotype and the activity score systems provided good classification to distinguish subjects with different drug exposures. The activity score system pro- vided a more detailed classification for the subjects.

Acknowledgments The work was supported by a grant from the Na- tional Program on Key Research Project of New Drug Innovation (No. 2012ZX09303006-002). All authors declare no conflict of interest.

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