METABOLIC ADVERSE EFFECTS INDUCED BY SECOND GENERATION ANTIPSYCHOTIC DRUGS IN PEDIATRIC PATIENTS: PHARMACOGENETIC PROFILING AND MECHANISTIC ANALYSIS OF GENETIC VARIANTS EFFECT IN VITRO MODELS

Second-generation antipsychotics (SGAs) are increasingly used in pediatric patients both in and offlabel. The rise in SGA prescription rates may be due to the perception of improved safety compared with first-generation antipsychotics. However, SGA use is often associated with rapid weight gain and metabolic syndrome as adverse drug reactions (ADRs). Interestingly, these side effects are not observed in all SGA-treated children, suggesting that some underlying genetic factors may predispose an individual to develop these adverse conditions. Recent studies have identified single nucleotide polymorphisms (SNPs) in genes encoding for enzymes involved in drug metabolism (CYP450) and SGA receptors in the central nervous system (CNS), which partly explain the inter-patient variability in ADR development. With this project, we intend to define the role of SNPs in genes related to the pharmacodynamics of SGA, expressed in peripheral tissues, to evaluate their involvement in the mechanisms responsible for weight change and metabolic ADRs. To this end in a cohort of 209 paediatric patients affected by disruptive behavioural disturbance who are prescribed SGAs (risperidone and aripiprazole), we have assessed the association of pharmacodynamics genetic variants with the occurrence of changes in body mass index (BMI) Zscore. This allows us to evaluate if the presence of specific variant alleles increases or decreases the risk of weight changes and metabolic adverse events. Tested SNPs will be selected from a comprehensive meta-analysis by Zhang e al (2016) of associated gene variants with antipsychotic-related weight gain. Between the 15 SNPs studied, the variants statistically significant associated with BMI changes are rs6318, rs3813929, rs5181147 in 5- Hydroxytryptamine Receptor 2C (HTR2C), rs1799732, rs6275, rs7131056 in D2 dopamine receptor (DRD2) and rs489693 in melanocortin-4-receptor (MCR4). We consider that the identification of these SNPs might provide useful input for personalized and individualized early intervention. Another goal of this project is to examine the direct effect of SGAs and validate the role of selected SNP at the peripheral level by performing in vitro experiments. Dysregulation in adipose tissue homeostasis has been suggested as a plausible mechanism by which antipsychotics induced metabolic alteration. To investigate the impact of SGAs on dysfunctional adipose tissue we decided to use the SW872 liposarcoma cell model. 6 First, we performed a detailed characterization of the model establishing the cell culture condition for the adipogenic differentiation process. Then, to clarify the mechanisms by which SGAs induce weight gain we studied the risperidone effect on the adipogenesis of SW872. We observed that risperidone directly promoted adipocyte differentiation and lipid accumulation. To better understand the phenotype of risperidone-induced adipocytes, next we assess its impact on some mitochondria parameters. We observed that risperidone's effect on mitochondrial phenotype can more faithfully recapitulate the white adipocyte profile observed in human adipose tissue. In line with this, our data also showed that Hoxc4, a key marker of white adipocytes, is highly expressed in risperidone adipocytes. From these results, we hypothesize that the induction of white adipocytes may be a possible mechanism by which risperidone induces weight gain as a side effect. Finally, to validate in the adipocyte cell line the impact of SNPs on differentiation, we have set up an in vitro model system for the expression of the genetic variants, by choosing the site-directed mutagenesis approach to modified the DNA sequence of the genes of interest . We selected two SNPs: rs6318 for the 5-HT2C gene and rs6275 for the DRD2 gene, identified in our population study as related to BMI changes after SGAs administration and already evaluated in correlation with weight gain, metabolic ADRs, and antipsychotic treatment with a focus on SNC expression . Using our adipocyte differentiation protocol, we showed that the modified cells could be differentiated into adipocytes therefore providing a relevant model for studying the SNP role in SGAs-induced adipogenesis.