Year: 2023
Type: dissertation
Abstract: Introduction: Mental disorders such as schizophrenia and bipolar disorder are a substantial burden not only for the patient, but also the society.Drug treatment is not optimal and development of new psychotropic drugs are not progressing as fast as needed.Many antidepressants and antipsychotics (a combination widespread in the psychiatric field) are metabolized by the highly polymorphic CYP2D6 enzyme.Hence, it causes interindividual drug responses, leading to adverse drug reactions and decreased efficiency in some patients.Risperidone, metabolized by CYP2D6, is commonly used for treatment of schizophrenia, in which studies have shown that an increased metabolism (ultrarapid metabolizers) causes insufficient treatment and decreased (poor and intermediate metabolizer) an overexposure to medicine leading to side effects and toxic outcomes.Objective: This study aims to investigate newly identified mutations in the gene coding for the drug metabolizing enzyme CYP2D6 and whether these mutations may contribute to inter-individuality in drug response of CYP2D6 dependent substrates.The aim is to find out how the various mutations affect the catalytic activity of the enzymes.Questions to be answered are what the in-silico predictions for the mutations are and if in vitro experiments can confirm these predictions.Method: Newly identified mutations within the pharmacogenomic project Genetic Precision Medicine were first analysed using bioinformatic tools.This to identify rsnumber, prevalence and amino acid exchange.The mutations were further analysed using ANNOVAR to obtain ADME-scores (administration, distribution, metabolism, excretion) with an optimized prediction framework tool, this to determine if mutations were deleterious or functional neutral.Mutations of interest were then investigated in vitro.Site directed mutagenesis was performed to obtain mutated CYP2D6 expression plasmids which were transfected into HEK293 cells.Harvested transfected cells were incubated with CYP2D6 substrate, bufuralol for 2.5 and 5 h.The production of hydroxylated bufuralol was measured using HPLC and a mobile phase of 30 % acetonitrile and 70 % perchloric acid.Catalytic activity for mutated CYPD6 were compared with normal function CYP2D6.1.Levels of apoprotein of the different mutated CYP2D6 variants and CYP2D6.1 were also measured with western blot.Results: All four analysed mutations showed a decreased or absent bufuralol metabolite production.D301N gave no metabolite, R133C 2 %, A305T 7 % and L314M 34 % activity of normal function CYP2D6 enzyme.Metabolite production was significantly lower, p<0.01 for A305T and L314M, and p<0.001 for R133C and D301N in comparison to CYP2D6.The western blot with HEK293 transfected cells showed an expression of mutant CYP2D6 protein for all mutants, and no expression for empty pCMV2 plasmid (mock).The in vitro experiments were in accordance to the in-silico prediction tool, which predicted the mutations as deleterious.Discussion and conclusion: The result from this study indicates that the newly identified mutations cause a reduced or absent CYP2D6 enzyme activity.Depending on CYP2D6 diplotype, an individual could be classified as poor/intermediate metabolizers for many psychotropic drugs, such as risperidone.This would require either a change of dose or a change of treatment to avoid adverse drug reactions and toxic outcomes.The in-silico prediction tool predicted the catalytic activity of the mutations fairly well, which was confirmed with the in vitro experiments.Being able to identify a patient's genomic data with the help of predictive tools and experimental validation can lead to optimal treatment and decrease the cost and suffering for patients and society in form of for example reduced adverse drug reactions and hospitalisation.
Author: Akcan, Martina
Language: English
Subfield: Pharmacology
Domain: Life Sciences
Open Access status: green
Funders: Karolinska Institutet, Umeå Universitet
OpenAlex ID: W7019419083