Farmakogenetika tiopurin-S-metiltransferaze

doktorska disertacija

Miha Milek (Avtor), Irena Mlinarič-Raščan (Mentor)

Povzetek

Farmakogenetika raziskuje genetske osnove, ki vplivajo na različne odzive posameznikov na zdravljenje. Cilj farmakogenetike je izboljšati učinkovitost in zmanjšati toksičnost terapije pri določenem posamezniku in na ta način omogočiti individualizirano zdravljenje. Tiopurinske učinkovine se uporabljajo pri zdravljenju hematoloških malignosti in avtoimunskih bolezni ter imajo nizek terapevtski indeks, zato se pogosto pojavijo življenjsko nevarni stranski učinki. Za svoje delovanje tiopurini potrebujejo metabolno aktivacijo, količina citotoksičnih in neaktivnih metabolitov pa je odvisna od aktivnosti polimorfnega encima tiopurin-S-metiltransferaze (TPMT). Na podlagi posameznikovega genotipa TPMT lahko prilagajamo odmerek tiopurinskih učinkovin in na ta način preprečimo pojav stranskih učinkov ter izboljšamo potek zdravljenja. V okviru doktorske disertacije smo raziskali povezanost genetskih polimorfizmov in encimske aktivnosti TPMT v slovenski populaciji. Ugotovili smo, da se genotipske frekvence najpogostejših polimorfizmov TPMT*3A (4,1%), TPMT*3C (0,5%) in TPMT*3B (0,3%) ujemajo z vrednostmi pri drugih populacijah Kavkazijcev. Heterozigotni posamezniki z enim mutiranih alelom so imeli nižjo aktivnost TPMT kot posamezniki z divjim tipom alelov. S statistično analizo smo določili mejo med skupinama posameznikov s srednjo in visoko aktivnostjo (9.82 pmol/107 RBC h-1) ter ugotovili, da na podlagi genotipa pravilno napovemo encimsko aktivnost TPMT pri 91,6 % posameznikov. Na ta način smo zasnovali farmakogenetske smernice in vzpostavili analizne metode, primerne za identifikacijo bolnikov s spremenjenim odzivom na tiopurine, pri katerih je potrebno znižanje doze. Zaradi nepopolnega ujemanja genotipa in fenotipa TPMT smo želeli ugotoviti dodatne faktorje, ki vplivajo na aktivnost TPMT in prekomerno toksičnost tiopurinov. V ta namen smo s pomočjo modelne celične linije za akutno limfoblastno levkemijo (ALL) preiskali molekularni mehanizem regulacije aktivnosti TPMT preko metilnega donorja S-adenozilmetionina (SAM). Ugotovili smo, da SAM preko stabilizacije proteinske strukture TPMT prepreči citostatični in citotoksični učinek 6-merkaptopurina (6-MP), zakasni apoptozo, sproženo s 6-MP, ter zniža koncentracijo znotrajceličnih citotoksičnih metabolitov. Potencialni vpliv SAM na toksičnost tiopurinov in pojav stranskih učinkov pri bolnikih z ALL smo ugotovili tudi v retrospektivni študiji, s katero smo dokazali visoko stopnjo povezave med pojavom hematotoksičnosti in prisotnostjo polimorfizmov, ki so povezani z nizko aktivnostjo TPMT in 5,10-metilentetrahidrofolat reduktazo (MTHFR), encimom, ki sodeluje pri biosintezi SAM. Z identifikacijo SAM kot pomembnega modulatorja aktivnosti TPMT in citotoksičnosti 6-MP, lahko pričakujemo upoštevanje novih genetskih in drugih kazalcev pri optimizaciji zdravljenja s tiopurini. V nadaljevanju smo vzpostavili modelne humane celične linije HEK293, HepG2 in Jurkat s stabilnim povečanim izražanjem TPMT za proučevanje endogene funkcije encima. Kot dostavni sistem za vnos plazmida z vključkom zaporedja cDNA za TPMT smo uporabili umetne virusne delce (AVP), ki so omogočili stabilno izražanje fuzijskega rekombinantnega proteina EGFP-TPMT in povečano aktivnost TPMT. Dalje smo ugotovili, da v celicah HEK293 in HepG2 s povečanim izražanjem TPMT poteka hitrejša presnova znotrajceličnega SAM, količina znotrajceličnega S-adenozilhomocisteina (SAH) pa je znižana, medtem ko metilacijski potencial ni spremenjen. Vpliva povečane ekspresije TPMT na celokupno metilacijo DNA v odsotnosti inhibicije DNA-metiltransferaz nismo ugotovili, medtem ko smo v celicah HepG2 odkrili značilno znižane vrednosti reducirane oblike glutationa. Dosedanje raziskave kažejo potencialno endogeno vlogo TPMT pri procesih, ki vplivajo na presnovo SAM, transmetilacijo in oksidativni status celice. Aktivnost TPMT je eden glavnih faktorjev pri presnovi tiopurinskih učinkovin, saj vpliva na učinkovitost zdravljenja in pojav neželenih učinkov. Poleg genetskih polimorfizmov na aktivnost TPMT vpliva tudi količina endogenega metilnega donorja SAM, katerega količina ima potencialen vpliv na pojav prekomerne toksičnosti in odziv posameznikov na tiopurinske učinkovine. Nadaljnje študije bodo razjasnile potencialno endogeno vlogo TPMT pri znotrajcelični presnovi, kar je pomembno za identifikacijo različnih fenotipov, ki nastanejo kot posledica različnih vrednosti aktivnosti encima.

Ključne besede

farmakogenetika;farmakogenomika;tiopurin-S-metiltransferaza;genetski polimorfizmi TPMT;vloga, SAM;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2008
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	UL BF - Biotehniška fakulteta
Založnik:	[M. Milek]
UDK:	577
COBISS:	2503281
Št. ogledov:	265
Št. prenosov:	58
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	ǂThe ǂpharmacogenetics of thiopurine-S-methyltransferase
Sekundarni povzetek:	Pharmacogenetics studies the genetic basis of inter-individual differences in drug response. The goal of pharmacogenetic testing is to individualize drug treatment by identifying relevant factors which influence drug efficacy and lower the therapy-related toxicity in individual patients. Thiopurines are prodrugs used in the treatment of haematological malignancies and autoimmune diseases. Due to their narrow therapeutic index, they frequently exhibit life-threatening side-effects. They require metabolic activation, in order to achieve their cytotoxic effect. The amount of cytotoxic and inactive metabolites largely depends on the activity of thiopurine S-methyltransferase (TPMT), a highly polymorphic drug metabolizing enzyme. TPMT genotyping is the basis for dosage adjustment protocols to prevent thiopurine-related side-effects and improve treatment outcome. In presented dissertation, the correlation of TPMT genetic polymorphisms and enzyme activity in the Slovenian population was evaluated. The genotypic frequencies of the most common polymorphisms TPMT3A (4.1 %), TPMT3C (0.5 %) and TPMT*3B (0.3 %) were in accord with the values observed in other Caucasian populations. Heterozygous individuals with a single mutant allele exhibited lower enzyme activity than wild-type individuals. Statistic analysis was employed to determine the cut-off value (9.82 pmol/107 RBC h-1) between intermediate and high TPMT activity groups. The correlation between TPMT genotype and enzyme activity was 91.6 %. With the aid of relevant analytical methods, the pharmacogenetic basis for the identification of patients with altered response to thiopurines requiring dosage adjustment protocols was established. Due to the incomplete TPMT genotype-to-phenotype correlation observed in several studies, we examined the potential factors involved in additional regulation of TPMT activity and unfavourable thiopurine toxicity. Using a model cell line for acute lymphoblastic leukemia (ALL), we delineated the molecular mechanism of the effect of methyl donor S-adenosylmethionine (SAM) on TPMT activity. We found that the addition of exogenous SAM prevents 6-MP induced cytotoxicity, delays the onset of 6-MP triggered apoptosis and lowers the concentration of cytotoxic metabolites. The potential effect of SAM on thiopurine toxicity was confirmed in the retrospective study, where we established the correlation between hematotoxic events in ALL patients and the presence of TPMT genetic polymorphisms, as well as low-activity mutations in the MTHFR gene, coding for an important enzyme involved in the biosynthesis of SAM. By identifying SAM as an important modulator of TPMT activity and 6-MP cytotoxicity, novel rationalization of therapy may apply. Further, to delineate the endogenous function of TPMT, we generated human HEK293, HepG2 and Jurkat cell lines exhibiting stable TPMT overexpression. The gene delivery system termed artificial viral particles (AVP) was used to transfect the cells with a plasmid containing TPMT cDNA insert, enabling stable overexpression of the recombinant fusion EGFP-TPMT protein and elevated TPMT activity. In addition, we found that TPMT overexpression in HEK293 and HepG2 cells results in faster intracellular SAM turnover, while the concentration of intracellular S-adenosylhomocysteine (SAH) is decreased, maintaining the cellular methylation potential. The effect of TPMT overexpression on global DNA methylation was not observed in the absence of DNA methyltransferase inhibition, while significantly lower amounts of reduced glutathione were found in HepG2 cells with stable TPMT overexpression. These results show that TPMT might be involved in endogenous processes affecting the cellular SAM turnover, transmethylation reactions and oxidative status. TPMT activity is one of the major factors influencing thiopurine drug metabolism, drug efficacy and unwanted toxicity. In addition to TPMT genetic polymorphisms, other factors involved in the regulation of endogeous methyl donor SAM may influence TPMT activity, and consequently, thiopurine toxicity and drug response. Further studies will clarify the endogenous role of TPMT in intracellular metabolism, which is crucial for the identification of different phenotypes resulting from significant inter-individual differences in TPMT activity.
Vrsta dela (COBISS):	Doktorska disertacija
Komentar na gradivo:	Univ. Ljubljana, Fak. za farmacijo
Strani:	140 f.
ID:	12895809