Pharmacogenomics is a scientific discipline that studies the links between genetic diversity and a patient's response to drug treatment. Pharmacogenomics aims to improve clinical outcomes by optimising drug therapy in the light of genetic polymorphisms that, among other things, may influence the activity of metabolic enzymes and consequently the concentration of the active substance in the plasma and/or at the target site. One of the groups of drugs for which the use of pharmacogenomics is very important due to the narrow therapeutic window and complex metabolism are the thiopurines. This group of cytotoxic and immunosuppressive DNA antimetabolites includes 6-mercaptopurine (6-MP), which is indicated for the treatment of acute lymphoblastic leukaemia (ALL). Activation of this prodrug requires a cascade of enzymatic reactions, of which inosine 5'-monophosphate dehydrogenase (IMPDH) has the strongest effect on the rate of formation of the active metabolite. Therefore, inter-individual differences in IMPDH activity could influence differences in 6-MP dosing and the efficacy of ALL treatment. The aim of this MSc thesis was to set up, optimise and validate a high-performance liquid chromatography (HPLC) method for the determination of IMPDH activity in haemolysates via the measurement of xanthosine monophosphate and adenosine monophosphate. We found that the composition of the mobile phase and the pH of the mobile phase buffer, the method of precipitation of the proteins in the haemolysate, and the time of incubation of the haemolysate with the substrate have the greatest influence on the quality of the analysis. After optimisation and validation of the method, IMPDH activity was determined in haemolysates from paediatric ALL patients who were on 6-MP maintenance therapy at the time of collection. We examined the associations between IMPDH activity and previously identified genetic polymorphisms in the IMPDH1 and IMPDH2 genes, the relative cumulative dose of 6-MP and the concentration of 6-MP metabolites. We found that the rs2278293 polymorphism was statistically significantly correlated with IMPDH activity. Patients with at least one variant T allele had higher activity than homozygotes with the CC genotype, confirming and complementing the clinical data known so far. We were unable to demonstrate associations between IMPDH activity and other parameters. As possible improvements, we would like to confirm the optimisation and validation in another laboratory, to perform the study in a larger sample of patients and to confirm the proven association in a larger population of healthy individuals. This could allow the measurement of IMPDH activity and the determination of the polymorphism to be introduced into clinical practice to adjust the dosage of thiopurines and other IMPDH-metabolised drugs.
|
