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Določanje aktivnosti inozin-5’-monofosfat dehidrogenaze s tekočinsko kromatografijo visoke ločljivosti pri bolnikih z akutno limfoblastno levkemijo
ID Kac, Anže (Author), ID Mlinarič-Raščan, Irena (Mentor) More about this mentor... This link opens in a new window, ID Urbančič, Dunja (Comentor)

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Abstract
Farmakogenomika je znanstvena veda, ki se ukvarja z ugotavljanjem povezav med genetsko raznolikostjo in odzivom bolnika na zdravljenje z zdravili. Cilj farmakogenomike je izboljšati klinične izide z optimizacijo zdravljenja z zdravili glede na genetske polimorfizme, ki med drugim lahko vplivajo na aktivnost presnovnih encimov in posledično na koncentracijo aktivne učinkovine v plazmi in/ali na tarčnem mestu. Ena izmed skupin zdravil, pri katerih je zaradi ozkega terapevtskega okna in zapletenega metabolizma uporaba farmakogenomike zelo pomembna, so tiopurini. V to skupino citotoksičnih in imunosupresivnih antimetabolitov DNA sodi 6-merkaptopurin (6-MP), ki je med drugim indiciran za zdravljenje akutne limfoblastne levkemije (ALL). Za aktivacijo tega predzdravila je potrebna kaskada encimskih reakcij, od katerih na hitrost nastanka aktivnega presnovka najmočneje vpliva inozin-5'-monofosfat dehidrogenaza (IMPDH). Razlike med posamezniki v aktivnosti IMPDH bi torej lahko vplivale na razlike v odmerjanju 6-MP in učinkovitosti zdravljenja ALL. Namen magistrske naloge je bil postaviti, optimizirati ter validirati metodo tekočinske kromatografije visoke ločljivosti (HPLC) za določanje aktivnosti IMPDH v hemolizatih na podlagi merjenja ksantozin monofosfata in adenozin monofosfata. Ugotovili smo, da imajo največji vpliv na kvaliteto analize sestava mobilne faze in pH pufra v mobilni fazi, metoda obarjanja proteinov v hemolizatu in čas inkubacije hemolizata s substratom. Po optimizaciji in validaciji metode smo aktivnost IMPDH določili v hemolizatih pediatričnih pacientov z ALL, ki so bili v času odvzemov na vzdrževalnem zdravljenju s 6-MP. Preverili smo povezave med aktivnostjo IMPDH ter predhodno določenimi genetskimi polimorfizmi v genih IMPDH1 in IMPDH2, relativnim kumulativnim odmerkom 6-MP in koncentracijo metabolitov 6-MP. Ugotovili smo, da polimorfizem rs2278293 statistično značilno korelira z aktivnostjo IMPDH. Bolniki z vsaj enim variantnim alelom T so imeli višjo aktivnost kot homozigoti z genotipom CC, kar potrjuje in dopolnjuje do sedaj poznane klinične podatke. Povezav med aktivnostjo IMPDH in ostalimi parametri nismo uspeli dokazati. Kot možne izboljšave navajam potrditev optimizacije in validacije v drugem laboratoriju, izvedbo študije na večjem vzorcu pacientov in potrditev dokazane povezave na večji populaciji zdravih posameznikov. S tem bi lahko merjenje aktivnosti IMPDH in določanje omenjenega polimorfizma uvedli v klinično prakso za prilagoditev odmerjanja tiopurinov in tudi drugih zdravilnih učinkovin, ki se presnavljajo z ali delujejo preko IMPDH.

Language:Slovenian
Keywords:Farmakogenomika, metabolizem zdravil, inozin-5'-monofosfat dehidrogenaza, tiopurini, akutna limfoblastna levkemija, tekočinska kromatografija visoke ločljivosti.
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2024
PID:20.500.12556/RUL-158584 This link opens in a new window
Publication date in RUL:16.06.2024
Views:355
Downloads:46
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Secondary language

Language:English
Title:Determination of inosine-5’-monophosphate dehydrogenase activity by high-performance liquid chromatography in patients with acute lymphoblastic leukemia
Abstract:
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.

Keywords:Pharmacogenomics, drug metabolism, inosin-5'-monophosphate dehydrogenase, thiopurines, acute lymphoblastic leukemia, high-performance liquid chromatography.

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