Effect of Genetic Polymorphisms of CYP2C19 on the Steady-state Serum Concentrations of Valproic Acid in Chinese Han Patients With Schizophrenia

Valproic acid is an anticonvulsant, which is also widely used for treating psychiatric disorders. Some clinical trials have demonstrated benets of valproic acid augmentation therapy in schizophrenia. Interindividual variability in valproic acid dose and serum concentration may reect functional consequences of genetic polymorphisms in genes encoding drug-metabolizing enzymes. The aim of this study was to determine the relationship between serum concentrations of valproic acid and single nucleotide polymorphisms of the cytochrome P450 (CYP) 2C19 gene in patients with schizophrenia. All patients had been receiving xed dose of valproic acid for at least 2 weeks. The daily doses were 0.5– 1.5 g. No other drugs except olanzapine were coadministered. Serum concentrations of valproic acid were measured using the ultra-high performance liquid chromatography method with mass-spectrometric detection. The CYP2C19 (CYP2C19*2 G681A and CYP2C19*3 G636A) genotypes were identied by real-time PCR analyses. The mean concentration/dose ratios of valproic acid was signicantly higher in patients with 1 (P = 0.029) or 2 (P = 0.007) mutated alleles for CYP2C19 than in those without mutated alleles. The mean concentration/dose ratios of valproic acid was signicantly higher in patients with CYP2C19 *1/*2 genotype (P = 0.029) or CYP2C19 *2/*3 genotype (P = 0.014) than in those with CYP2C12 *1/*1 genotype. The ndings of this study suggest that CYP2C19 genotypes play an important role in controlling steady-state serum concentrations of valproic acid in Chinese Han population.


Introduction
Over the last 40 years, a variety of adjunctive treatments have been used to treat schizophrenia (G. W. Christison et al., 1991). These are often used in addition to antipsychotics, in an attempt to alleviate the symptoms of schizophrenia such as hallucinations and delusional beliefs, although they have been used instead of antipsychotics. Valproate /valproic acid has been used for people whose psychosis did not respond to traditional therapy. Valproic acid is traditionally used as an anticonvulsant drug and is also used for affective disorders, especially for the treatment of acute mania (A. Cipriani et al., 2013). Furthermore it is thought to have anti-aggressive effects and it may reduce impulsive behaviour, which might be useful for some people with schizophrenia (L. Citrome et al., 2000). There is large interindividual variability in its pharmacokinetics and pharmacodynamics (Y. Ghodke-Puranik et al., 2013, J. Methaneethorn, 2018. Therefore, its serum concentration needs to be monitored as a guide of dose adjustment during the course of therapy. The enzyme cytochrome P450 (CYP) 2C19 is involved in the metabolism of valproic acid. Several mutated alleles of the CYP2C19 locus that cause decreased enzyme activity, that is, CYP2C19*2 (G681A, splicing defect) and CYP2C19*3 (G636A, W212X, premature stop codon) have been reported (K. Kurose et al., 2012, U. M. Zanger andM. Schwab, 2013). The steady-state serum concentration of valproic acid is signi cantly dependent on the mutated alleles. However, there are large overlaps in these steady-state serum concentrations among the different genotype groups and considerable interindividual variations in the values within each genotype. In previous published studies, body weight, age, gender and comedications had signi cant in uence on VPA distribution volume (J. Ding et al., 2015, H. Nakashima et al., 2015, N. Ogusu et al., 2014. Although various genetic polymorphisms were associated with the increase or decrease of VPA serum concentration (T. Budi et al., 2015, X. M. Chu et al., 2012, W. Feng et al., 2016, Y. Guo et al., 2012, C. C. Hung et al., 2011, D. Jiang et al., 2009, T. K. Kiang et al., 2006, S. Mei et al., 2017, L. Tan et al., 2010, only two study identi ed the quantitative relationship between genetic polymorphisms CYP2C19 and VPA distribution volume in epileptic patients (D. Jiang et al, 2009, S. Mei et al., 2018. Therefore, we aimed to investigate the effects of various factors including CYP2C19 polymorphisms, age, gender, body mass index (BMI) and duration of schizophrenia on the steady-state serum concentrations of valproic acid in Chinese Han patients with schizophrenia, which might be useful for VPA dose adjustment in clinical practice.

Patients
The subjects were 139 Chinese Han patients with schizophrenia (52 males and 87 females) who all ful lled the criteria for schizophrenia, according to the ICD-10. They were physically healthy without any history of substance abuse, neurological disorder, delirium, or dementia and without any clinically signi cant ndings, including a clinical laboratory examination, electrocardiography, and electroencephalography. The mean ± SD of age and BMI value were 34.03 ± 12.85 years and 23.80 ±3.77, respectively. 10 were smokers (≥ 10 cigarettes/day), whereas the remainders were nonsmokers. None was a heavy drinker. This study was approved by the Ethics Committee of the Hangzhou 7 th people's hospital, and all the patients had given written informed consent to participate in this study.

Drug treatment
All the subjects had received valproic acid for at least 2 weeks. It has been shown that plasma concentrations of valproic acid reach steady state by 2 weeks after repeated oral administration. The daily dose was xed and was given twice a day. The patients were receiving no drugs except olanzapine (2.5-20 mg/d). Female patients did not receive oral contraceptives. Patients' adherence was con rmed by the nursing staff or their families. Blood samples were taken at 8 AM.

Analytical methods
Serum concentrations of valproic acid were measured using the ultra-high performance liquid chromatography method with mass-spectrometric detection by Zhejiang BIOZON Medical Co.,Ltd. The lowest limit of detection for valproic acid was 2.5 μg/ml using 0.4 ml of serum, and the interassay coe cient of variation was 7.98% at a concentration of 50 μg/ml. The steady-state serum concentrations was adjusted by the doses of valproic acid, and the concentration/dose (C/D) ratios were used in statistical analyses. DNA was isolated from peripheral leucocytes using a nucleic acid extraction and puri cation kit (Kuangyuan, Suzhou, China). The *2 and *3 alleles of CYP2C19 were identi ed by PCR analyses using Human CYP2C19 genotyping kit (Kuangyuan, Suzhou, China).

Statistical analyses
The serum C/D ratios of valproic acid of patients were compared among different genotypic groups or different metabolic type groups using one way ANOVA. Multiple regression analyses were used to detect the correlation between serum C/D ratios of valproic acid and several factors, including the polymorphisms of CYP2C19, age, gender, BMI values, smoke and duration of schizophrenia. A P value of less than 0.05 was regarded as statistically signi cant. SSPS 19.0 for Windows was used for these statistical analyses.

Discussion
Genetic polymorphism may be an important source of interindividual variability in the pharmacokinetics and pharmacodynamics of valproic acid. In this study, the mean serum C/D ratios of valproic acid increased in accordance with the number of the mutated alleles for CYP2C19. It is the data that strongly implied that the mean serum C/D ratios of valproic acid increases in a gene dose-dependent manner. The mean concentration/dose ratios of valproic acid were signi cantly higher in patients with 1 or 2 mutated alleles for CYP2C19 than in those without mutated alleles. The result was assistant with previous report in epileptic (L. Citrome et al, 2000).
Otherwise, multiple regressin analyses showed that the serum C/D ratios of valproic acid were not only correlated with the number of mutated alleles for CYP2C19 but also associated with the BMI values. One study showed that loss-of-function CYP2C19 polymorphisms were associated with an increased BMI values (M. Noai et al., 2016). This may explained the association between BMI values and the serum C/D ratios of valproic acid in our study. And some studies reported the effect of age on the metabolism of valproic acid (U. A. Argikar and R. P. Remmel, 2009, S. J. Miyagi and A. C. Collier, 2011), showed that there was no statistically signi cant difference in the rate of VPA metabolism. Another study showed that serum valproic acid levels was signi cantly increased with younger age (L. Ben Mahmoud et al., 2017). These results are inconsistent. A larger population sample size is needed to further verify the results.
This study has several limitations. The dose of valproic acid was not uni ed. Data of clinical responses related with the observed differences in the steady-state serum concentrations of valproic acid were lacking. The possibility of committing type II errors cannot be ruled out because of the low power.

Conclusions
The ndings of this study suggest that CYP2C19 genotypes play an important role in controlling the steady-state serum concentrations of valproic acid in Chinese Han subjects.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of the Hangzhou Seventh People's Hospital. The purpose and importance of the study were explained to each participant before they proceeded into actual activities. Informed consent was obtained from all participants.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this published article.

Competing interests
There are no con icts of interest to report for any of the authors.

Funding
Funding for this study was provided by Grant 2015C03054 from the Key Research Project of the Zhejiang Province, Science and Technology Project of Medicine and Health of Zhejiang Province of China (2020KY222) and Zhejiang Provincial Natural Science Foundation of China (LY17H090013).

Authors' contributions
Wang S, Song M, Shi J were responsible for study design, Ju X, Liu J, Wang C, Shi J were responsible for recruiting the subjects, Wang S, Li J, Yan P, Ju X were responsible for collecting clinical data and performing the clinical rating. Wang S, Li J, Yan P were responsible for statistical analysis, and manuscript. All authors contributed to and have approved the nal manuscript.