The polymorphic variants DRD2 rs1800497 and ABCB1 3435C>T are associated with antipsychotic safety parameters in adolescents with an acute psychotic episode: the results of a pilot study

Children and adolescents are more likely than adults to experience adverse side effects when taking antipsychotics. Pharmacogenetic testing allows one to more accurately choose the initial dose of a drug. The genes of pharmacokinetic factors have been shown to be of high prognostic value for the safety of antipsychotics in adults. Patients Udvalg Undersњgelser Akathisia genotyped for 1236C>T, 2677G>T/A, 3435C>T, and safety of antipsychotics in adolescents with an acute psychotic episode. are associated with antipsychotic safety parameters in adolescents with an acute psychotic episode: the results of a pilot study.

Introduction. Schizophrenia is a disabling mental disorder, often manifested as an acute psychotic episode, the treatment of which is the first stage of treatment of the underlying disease [1]. The first line treatment for the acute psychotic episode are antipsychotics.
Children and adolescents are more likely to have adverse adverse side effects (ADEs) of antipsychotics than adults. It was shown that types of ADEs in response to antipsychotics in children were generally similar in adults with the first psychotic episode, but children are more prone to metabolic disorders [2,3].
All of that determines the importance of personalized choice of antipsychotics in the treatment of acute psychotic episode in children and adolescents. Nowadays, there are no clinical guidelines on the use of pharmacogenetic testing for pre-scribing of antipsychotics in patients with an acute psychotic episode. But a certain evidence base, mainly for adult patients, confirms the importance of genes of pharmacokinetic factors -in particular, CYP2D6. CYP2D6 polymorphisms change the activity of the corresponding isoenzyme, which may affect the safety of antipsychotics [4]. The role of other genes encoding isoenzymes of cytochrome P450 family are less important in selection of antipsychotics [4]. But their effect on serum levels of antipsychotics was confirmed by the studies done on Asians [5,6]. Another important pharmacogenetic marker studied as an antipsychotic safety predictor is the ABCB1 gene encoding the transport protein P-glycoprotein (P-gp). ABCB1 polymorphisms lead to a decreasing of P-gp activity, which in turn affects the higher toxicity of drugs taken [7]. There were studies confirmed the role of ABCB1 polymorphisms in the safety of antipsychotics in adults [8,9]. Studies of pharmacodynamic genetic factors, in particular dopamine and serotonin receptor genes, have shown inconsistent results [10,11].
To date, quite a number of pharmacogenetic studies on the efficacy and safety of antipsychotics in children and adolescents have been conducted. But among them, there were not enough studies that include patients with an acute psychotic episode [12]. Other studies which confirmed the role of CYP2D6 in the safety of antipsychotics predominantly included adult patients with schizophrenia [13,14].
Given the lack of work involving adolescents experiencing acute psychotic episodes who were prescribed antipsychotics, new data are required. In our study, we aimed to study the predictive role of polymorphic variants of pharmacokinetic factor genes for early signs of ineffectiveness and intolerance of antipsychotics in adolescents with an acute psychotic episodes. Purpose: To establish possible associations of pharmacokinetic and pharmacodynamic factors' genes with effectiveness and safety of antipsychotics in adolescents experiencing acute psychotic episodes during 28 days of treatment.

Study sample
We included 36 adolescents in the study and monitored the participants' condition for 28 days. Patients were included in the study within 1 to 3 days after admission to a hospital. Each patient and his parent(s) signed the informed consent to be involved in the study. Informed consent included permission to publish results of the study. Personal information or identifiers of study participants were not included in database. All patients were Russian by self-identification.
All patients received antipsychotics as their main therapy. Some patients had antipsychotic's change during 28 days of observation. In the first 14 days, antipsychotics were replaced in 6 patients due to lack of efficacy. Between the 14th and 28th days, the main antipsychotics were replaced in 8 patients: in 4 cases due to poor tolerance, in 4 cases due to inefficacy. We had no influence on the changes in psychopharmacotherapy.
As the main antipsychotic, first-generation antipsychotics (FGA) were used more often. In addition, additional antipsychotics were added in several cases. Twenty-five patients took trihexyphenydil during 14 days and 24 patients took it between 14 and 28 days.
Doses of antipsychotics were converted to the chlorpromazine equivalent to unify further analysis. __ [15].
To assess the effectiveness of antipsychotics in dynamics, special scales were used: Children's Global Assessment Scale (CGAS) [16], Positive and Negative Symptoms Scale (PANSS) [17]. The mental state was assessed 3 times: when the patient was included in the study, on the 14th and 28th days of observation.
The safety of psychopharmacotherapy was assessed with UKU Side Effects Rating Scale (UKU SERS) [ Laboratory part A buccal epithelium scrape was collected from each patient on the day of inclusion in the study to genotype. The biomaterial was transported to the laboratory and stored at -77 degrees Celsius. DNA from buccal epithelium was performed by sorbent method. The isolated DNA was stored at -77 degrees Celsius.

Statistical analysis
Statistical analysis was performed with IBM SPSS Statistics 21.0. Given the small sample size, we used non-parametric criteria (Mann-Whitney, Kruskal-Wallis) to compare continuous variables between groups. We compared the frequencies of the categorical variables with each other by Pearson's Chi-square, and the exact Fisher's criterion (for 2x2 comparisons). Bonferroni's correction of multiple comparisons was introduced. The limit of significance was set to 0.05.
In our analysis, we did not exclude patients with concurrent drugs used besides the main antipsychotic. But the concurrent use of psychopharmacotherapy was taken into account during analysis to prevent a bias.

Results
Clinical and demographic characteristics of the patients included in the study are presented in Table 1.
There were no associations of clinical and demographic characteristics of patients with effectiveness of psychopharmacotherapy.
The further analysis concerned the pharmacogenetic part of the research: establishment of possible associations of genetic polymorphisms with the psychopharmacotherapy effectiveness and safety.
The distribution of genotypes of all polymorphisms corresponded to the distribution according to Hardy-Weinberg equilibrium. (p>0.05) ( Table 2).
Clinical and demographic parameters were compared between carriers of different genotypes of genetic polymorphisms to determine their comparability.
Significant differences in the PANSS scale and some of its subscales scores between the carriers of CYP2D6*4, HTR2A rs6313 and ABCB1 3435 C>T were established (Table 3). There were no differences in CGAS score depending on genetic polymorphisms.
We did not revealed differences in first or second generation antipsychotics prescription between carriers of different genotypes of genetic polymorphisms.
Doses of antipsychotics between carriers of different genetic polymorphisms were analyzed. Statistically significant differences were obtained only for CYP2D6*10 and DRD2 rs1800497. The revealed associations are presented in Table 4.
The analysis of the concurrent pharmacotherapy revealed the following: 10 carriers of the C-allele of HTR2A rs6313 took mood stabilizer between 14 and 28 days, which significantly differed from carriers of TT homozygotes, none of which received mood stabilizer during this period (p=0.016).
It has been established that the decrease in the total PANSS scale score for 28 days was significantly lower for the carriers

Analysis of the effectiveness of antipsychotics depending on pharmacodynamic gene's polymorphisms
No significant differences were found in the CGAS and PANSS scales' scores mean change during the observation period for HTR2A rs6313 and DRD4 rs1800955 polymorphisms' carriers.
Significant differences in the PANSS "Positive Symptoms" on 14th and 28th days as well as the total PANSS score on day 28 for DRD2 rs1800497 carriers were found (Figure).

Analysis of the effectiveness and safety of antipsychotics depending on pharmacokinetic and pharmacodynamic gene's polymorphisms
The carriage of ABCB1 3435C>T and DRD2 rs1800497 was associated with a significant increased UKU SERS and it's several subscales scores (Table 5). Other polymorphisms included in the study did not demonstrate any significant associations with the average scores of UKU SERS, BARS and SAS scales.
The analysis of associations of genetic polymorphisms with frequency of individual adverse drug reactions showed negative results.

Discussion
Differences of initial PANSS scores between carriers of genetic polymorphisms did not influence on mental state changes during treatment. The objectives of the study did not include an interpretation of possible reasons why PANSS scores were initially higher in carriers of certain genotypes.
It was found that only CYP2D6*4 and DRD2 rs1800497 showed significant associations with the effectiveness of the treatment of adolescents experiencing acute psychotic episode. CYP2D6*4 which is associated with slower metabolism of the CYP2D6 isoenzyme, associated with poorer treatment outcomes. That is a controversial result, because reducing the rate of antipsychotics' metabolism should increase their serum levels. At the same time, patients with CYP2D6*4 have a higher risk to Nevrologiya (-6,5) [-10,25; -3,75] vs (-3) [-6,5;-2]; p=0,028) and the 28th day (-11) [-13; -9,5] vs (-5) [-9; -3,5]; p=0,001) compared to the initial state. Also those patients with DRD2 rs1800497 had more prominent decreasing of PANSS total score on the 28th day of therapy (-26)  develop schizophrenia than the general population [21]. Consequently, a less pronounced decreasing of the PANSS scores may be due to the increased resistance of symptoms to treatment by CYP2D6*4. Carriers of DRD2 rs1800497 had better response to psychopharmacotherapy. It was known that DRD2 rs1800497 leads to decreased density of dopamine receptors in the striatum area, as well as to a deterioration of ligand binding [22]. Positive psychotic symptoms are associated with dopaminergic activity. Increased activity of striatum dopaminergic neurons is one of the pathophysiological mechanisms of psychosis [22]. Thus, the DRD2 rs1800497 carriage can facilitate the reduction of positive psychotic symptoms by antipsychotics. It should be separately noted that the degree of initial expression of psychosis was not influenced by the carrying of DRD2 rs1800497. Our findings were consistent with other studies confirming the effect of DRD2 rs1800497 on improving the response to antipsychotics [23,24]. But not all studies have found positive results. Escamilla et al. (2018) did not revealed of significant associations DRD2 rs1800497 with a response to antipsychotics in adult schizophrenics [25], The negative result was also shown by Kang et al. (2015) for amisulphride [26], and by Vehof et al. (2012) for second-generation antipsychotics [27]. But carriers of DRD2 rs1800497 in our study initially received much higher doses of antipsychotics. However, differences in dosages disappeared after the first week of therapy. This fact may question the isolated effect of the DRD2 rs1800497 carrier on the reduction of positive symptoms at 14th day: it may have been due to a more aggressive start of psychopharmacotherapy. It is doubtful that the high initial dose had an effect on the reduction of psychotic symptoms on the 28th day of observation. There were no objective reasons why DRD2 rs1800497 carriers were initially prescribed a higher dose of antipsychotics.
We found significant associations of ABCB1 3435C>T and DRD2 rs1800497 with the safety of antipsychotics on 14th day. By 28th day, the average scores of UKU SERS, SAS and BARS scales were not significantly associated with the carriage of genetic polymorphisms.
Presence of ABCB1 3435C>T can lead to reduced P-gp activity, which leads to increased concentration of protein-transporter's substrates in blood. Thus, the deteriorating tolerance of antipsychotics in ABCB1 3435C>T carriers looks reasonable.
The results obtained in our study are consistent with earlier published data on the impact of CYP2D6 and ABCB1 on the safety of antipsychotics in children [8] and adults [28]. The polymorphism ABCB1 3435C>T was also previously shown to be substantial for riperidone safety [8]. In our study, that polymorphism was also associated with decreased tolerance of psychopharmacotherapy. Indirect confirmation of the significance of the selected polymorphisms was also the fact that CYP2D6*10 and ABCB1 led to an increase in serum levels of some antipsychotics, in particular, riperidone, aripiprasol, clozapine [29][30][31].
The association DRD2 rs1800497 with increased score of UKU subscale «Neurological disturbances» obtained in our study looks logical, as extrapyramid symptoms on antipsychotics depend on the activity of dopamine receptors. Reduced expression of dopamine receptors related to DRD2 rs1800497 polymorphism may lead to excessive blockage of the nigrostriar pathway by antipsychotics, increasing the risk of extrapyramidal symptoms. The predictive role of pharmacodynamic factor's genes for antipsychotic safety has previously been demonstrated in adult patients [32,33].
Most of research studied the pharmacogenetics of antipsychotics in children was done not on patients with acute psychotic episodes [12]. Consequently, those studies will differ in terms of both doses and delivery times compared to ours. Given the small number of published studies with similar designs, comparison of current research with other pediatric samples was not possible. But few studies done on patients experiencing first psychotic episodes (youths) confirmed the importance of CYP2D6 and ABCB1 genes to predict the safety of antipsychotics [34,35].
The design of our study did not allow us to assess the longterm side effects of antipsychotics: in particular, weight gain. We did not measure the level of prolactin, which can also be attributed to study limitations. But at the same time, our research focuses on a very important problem -the prediction of the effectiveness and safety of antipsychotics during the acute treatment stage of psychosis. The results presented are preliminary, as the study is ongoing and the sample of patients is getting larger. The associations identified are significant for replication on a larger sample of patients.
Conclusion. As a result of the study it was found that CYP2D6*4 was associated with less reduction of positive psychotic symptoms during treatment with antipsychotics in adolescents with acute psychotic episodes. In contrast, DRD2 rs1800497 led to a more significant decrease in the severity of total psychotic symptoms on 28th day and positive symptoms -on 14th and 28th days as compared to the initial state.
Polymorphisms ABCB1 3435C>T and DRD2 rs1800497 were significant predictors of antipsychotics' unsafety during the first 14 days of treatment.
It is planned to continue the study and to increase the sample of patients as well as the panel of genetic polymorphisms.