doktorska disertacija
Kristina Lampič (Author), Tomaž Vovk (Mentor), Matevž Pompe (Thesis defence commission member), Irena Kralj Cigić (Thesis defence commission member), Mojca Kržan (Thesis defence commission member), Helena Prosen (Co-mentor)

Abstract

Kronična vnetna črevesna bolezen, ki jo opredelimo kot ponavljajoče vnetje črevesa, se je v zadnjem stoletju razširila v globalno bolezen z naraščajočo pojavnostjo v industrijsko razvitih državah. Sodobni cilj zdravljenja je bolezen brez izrazitih simptomov, zdravljenje pa lahko glede na zahtevnost bolezni poteka v več stopnjah. Azatioprin, purinski antimetabolit, je imunosupresivno zdravilo, ki se pri zdravljenju kronične vnetne črevesne bolezni uporablja kot samostojno zdravilo, pri težjih oblikah bolezni pa tudi v kombinaciji z biološkimi zdravili iz skupine zaviralcev faktorja tumorske nekroze alfa. Gre za predzdravilo, ki z metabolno pretvorbo prehaja v dve skupini aktivnih metabolitov: 6-tiogvanin nukleotide, nosilce terapevtskega učinka in 6-metilmerkaptopurin nukleotide, ki lahko povzročijo okvaro jeter, njihov terapevtski učinek pa ni dokazan. Za optimalno zdravljenje je priporočeno prilagojeno odmerjanje zdravila glede na koncentracijo aktivnih metabolitov in aktivnost presnovnega encima tiopurin S-metiltransferaze. Analitika obeh skupin metabolitov je dobro razvita, tradicionalni biološki matrici pa sta polna kri in rdeče krvne celice (ang. red blood cells oziroma RBC). Obe matrici zahtevata invaziven odvzem krvi, priprava vzorcev je zaradi narave biološkega materiala zahtevna, stabilnost analitov pa omejena. Alternativo tradicionalnim biološkim matricam predstavljajo posušeni krvni madeži (ang. dried blood spots oziroma DBS), pri katerih kapljico krvi vzamemo iz prsta ali pete in jo prenesemo na papirček. Možna je tudi uporaba venske krvi, ki jo na papirček nanesemo s primerno pipeto. V raziskovalnem delu smo razvili in validirali metodo tekočinske kromatografije s tandemsko masno spektrometrijo (LC-MS/MS) za kvantitativno določanje metabolitov azatioprina s tehniko posušenih krvnih madežev. Metoda vključuje ekstrakcijo iz 30 μl posušenega krvnega madeža, hidrolizo in kvantitativno določitev vsebnosti 6-tiogvanina in 6-metilmerkaptopurina z metodo LC-MS/MS. Metoda je selektivna, linearna, točna in natančna v območju 50 –5300 pmol/8×108 RBC za 6-tiogvanin in 260 –5300 pmol/8×108 RBC za 6-metilmerkaptopurin. S testom integritete redčenja smo dokazali, da lahko zgornjo mejo koncentracije, pri kateri še določimo točne in zanesljive rezultate, za oba analita premaknemo do 8000 pmol/8×108 RBC, s čimer pokrijemo tako terapevstko območje za 6-tiogvanin kot spodnjo mejo toksičnega območja za 6-metilmerkaptopurin. Absolutni in relativni matrični učinek sta nizka, izkoristek ekstrakcije pa ≥ 80%. Potrdili smo, da različne vrednosti hematokrita, različni volumni nanosa in odvzem vzorca iz različnih delov krvnega madeža ne vplivajo na točnost in natančnost metode. Oba analita sta v posušenih krvnih madežih stabilna vsaj en mesec v temperaturnem območju od -80 do 40°C. Ustreznost metode smo dodatno potrdili z analizo vzorcev bolnikov. Rezultati naše in referenčne metode so primerljivi in omogočajo sprejemanje enakih odločitev pri odmerjanju zdravil. Novo razvita metoda je enostavna, zaradi številnih prednosti tehnike posušenih krvnih madežev pa predstavlja pomembno alternativo uveljavljenim metodam za terapevtsko spremljanje koncentracij metabolitov azatioprina.

Keywords

kronična vnetna črevesna bolezen;imunosupresivna zdravila;azatioprin;analizne metode;tehnika posušenih krvnih madežev;Bland Altmanova analiza;tekočinska kromatografija;masna spektrometrija;validacija metode;doktorske disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [K. Lampič]
UDC: 543.544.5:543.51:616.34-002(043.3)
COBISS: 22736131 Link will open in a new window
Views: 487
Downloads: 113
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Other data

Secondary language: English
Secondary title: Development of analytical method for determination of azathioprine metabolites in dried blood spots
Secondary abstract: Chronic inflammatory bowel disease, defined as recurrent inflammation of bowel, has spread to a global disease with increasing incidence in industrialized countries over the last century. The current goal of the treatment is the remission of the disease and it can take several stages depending on the complexity of the disease. Azathioprine, a purine antimetabolite is an immunosuppressive drug used in the treatment of chronic inflammatory bowel disease as a standalone drug or in combination with biological agents from the group of tumor necrosis factor alpha inhibitors. It is a prodrug that undergoes metabolic conversion into two groups of active metabolites: 6-thioguanine nucleotides, carriers of therapeutic effect and 6-methylmercaptopurine nucleotides, which are hepatotoxic and their therapeutic effect has not been confirmed yet. For the optimal treatment tailored therapy dosing is recommended based on the concentration of active metabolites and the genotype of the metabolic enzyme thiopurine S-methyltransferase. The analytics of both groups of metabolites is well developed and the established biological matrices are whole blood and red blood cells (RBC). Both matrices require invasive blood collection and demanding sample preparation due to the nature of the biological material. The stability of analytes is generally limited. An alternative to the established biological matrices are dried blood spots (DBS), where a drop of blood is taken from a finger or a heel and transferred to a DBS paper. It is also possible to use venous blood, which is applied to a DBS paper with a suitable pipette. In the research work we developed and validated the liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of azathioprine metabolites by the dried blood spot technique. The method involves extraction from 30 μl of dried blood spot, hydrolysis, and quantification of 6-thioguanine and 6-methylmercaptopurine by LC-MS/MS method. The method is selective, linear, accurate, and precise in the range of 50-5300 pmol/8×108 RBC for 6-thioguanine and 260-5300 pmol/8×108 RBC for 6-methylmercaptopurine. The dilution integrity test demonstrated that the upper limit of concentration can be increased up to 8000 pmol/8×108 RBC for both analytes, thus covering both the therapeutic range for 6-thioguanine and the lower limit of the toxic range for 6-methylmercaptopurine. The absolute and relative matrix effects are low and the recoveryis ≥ 80%. We confirmed that different hematocrit values, different application volume, and sampling from different parts of the blood spot does not affect the accuracy and precision of the method. Both analytes are stable in dried blood spots for at least one month in the temperature range from -80 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. The new developed method is simple and due to the many advantages of the dried blood spot technique it represents an alternative to established methods for the therapeutic drug monitoring of azathioprine metabolites.
Secondary keywords: -;
Type (COBISS): Doctoral dissertation
Study programme: 1000381
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Pages: XIII, 107 str.
ID: 11876614