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Biomarkers that can guide cancer therapy based on patients’ individual cancer molecular signature can enable a more effective treatment with fewer adverse events. Data on actionable somatic mutations and germline genetic variants, studied by personalized medicine and pharmacogenomics, can be obtained from tumor tissue or blood samples. As tissue biopsy cannot reflect the heterogeneity of the tumor or its temporal changes, liquid biopsy is a promising alternative approach. In recent years, extracellular vesicles (EVs) have emerged as a potential source of biomarkers in liquid biopsy. EVs are a heterogeneous population of membrane bound particles, which are released from all cells and accumulate into body fluids. They contain various proteins, lipids, nucleic acids (miRNA, mRNA, and DNA) and metabolites. In cancer, EV biomolecular composition and concentration are changed. Tumor EVs can promote the remodeling of the tumor microenvironment and pre-metastatic niche formation, and contribute to transfer of oncogenic potential or drug resistance during chemotherapy. This makes them a promising source of minimally invasive biomarkers. A limited number of clinical studies investigated EVs to monitor cancer progression, tumor evolution or drug resistance and several putative EV-bound protein and RNA biomarkers were identified. This review is focused on EVs as novel biomarker source for personalized medicine and pharmacogenomics in oncology. As several pharmacogenes and genes associated with targeted therapy, chemotherapy or hormonal therapy were already detected in EVs, they might be used for fine-tuning personalized cancer treatment.

Kljub velikemu napredku zdravljenja, ki ga je prinesel pojav bioloških zdravil, ostajajo enostavne sintezne zdravilne učinkovine steber zdravljenja tako rakavih kot avtoimunskih bolezni. V to skupino sodijo tiopurini, ki se kot predzdravila pogosto uporabljajo pri zdravljenju akutne limfoblastne levkemije ter vnetne črevesne bolezni. Zaradi ozkega terapevtskega okna je pomembno, da je odmerjanje tiopurinov prilagojeno posamezniku, kar predstavlja kompleksen izziv sodobne terapije. Velik vpliv na izid zdravljenja s tiopurini ima encim tiopurin-S-metiltransferaza (TPMT). Interindividualna raznolikost odziva na tiopurine je namreč lahko posledica prisotnosti genskih polimorfizmov, metabolnih dejavnikov, ki stabilizirajo TPMT, prav tako pa je lahko rezultat interakcij z zdravilnimi učinkovinami v sočasni terapiji. Poglaviten namen raziskovalnega dela v sklopu doktorske disertacije je bil doprinesti k učinkoviti in varni terapiji s tiopurini, zato smo si zastavili sledeče cilje: poglobiti razumevanje delovanja encima TPMT in njegove endogene vloge, najti nove genetske dejavnike odziva na tiopurine ter opredeliti molekularne mehanizme interakcij med zdravilnimi učinkovinami, ki se pojavljajo ob sočasnem jemanju s tiopurini. V sklopu raziskovanja endogene vloge smo se osredotočili na selenove spojine, saj je znano, da TPMT poleg tiopuinov katalizira tudi reakcijo metilacije njihovih selenovih analogov. Med najpomembnejše selenove spojine, ki jih najdemo v organizmu, sodi selenocistein, ki je kot gradnik selenoproteinov udeležen pri ključnih procesih za vzdrževanje homeostaze v celicah. Z uporabo spektroskopije STD NMR in merjenjem fluorescence triptofanov v TPMT smo pokazali, da se selenocistein veže na humani rekombinantni TPMT. Analiza molekulskega sidranja je predpostavila lego selenocisteina v aktivnem mestu TPMT, ki bi bila relevantna za reakcijo metilacije. Ob prisotnosti encima in donorja metilne skupine, S-adenozilmetionina (SAM), smo potrdili encimsko katalizirano nastajanje metiliranega produkta Se-metilselenocisteina. To dokazuje doslej neznano vlogo encima kot selenocistein-Se-metiltransferaze. Pri iskanju novih bioloških označevalcev odziva na tiopurine smo se najprej osredotočili na raziskovanje lastnosti gena za TPMT. Ugotovili smo, da ima natančna določitev sekvence DNA v promotorski regiji, ki vsebuje variabilna števila tandemskih ponovitev (VNTR), velik pomen. Motivi z zaporedjem ABnC so namreč v t.i. vezavnem nesorazmerju s klinično pomembnimi polimorfizmi TPMT*3C in TPMT*3A. Motiv AB2C se pojavlja skupaj z alelom TPMT*3C, medtem ko se motivi z večjim številom ponovitev B (AB3C, AB4C, AB5C ter celo AB7C) pojavljajo skupaj z alelom TPMT*3A. Motiv AB7C je prvič dokazan na naši populaciji in opisan v pričujoči publikaciji. Omenjena polimorfizma sta klinično relevantna, zato bi bilo lahko v prihodnosti določanje motivov VNTR enako dober biološki označevalec pri uravnavanju odmerka tiopurinov, kot so polimorfizmi posameznih nukleotidov (SNP). Pri nadaljnjem iskanju bioloških označevalcev za napovedovanje odziva smo uporabili model limfoblastoidnih celičnih linij (LCL), na katerih smo določali občutljivost na 6-merkaptopurin in 6-tiogvanin. Ugotovili smo, da je bila aktivnost TPMT pričakovano odvisna od najpogostejših SNP v genu, a ni korelirala z dovzetnostjo celic LCL na 6-merkaptopurin, kar bi sicer pričakovali na osnovi in vivo delovanja. To spoznanje kaže na obstoj drugih nepoznanih pomembnih dejavnikov, ki celicam v prisotnosti citotoksičnih spojin omogočajo preživetje. Kot tovrstni kazalec rezistence oz. preobčutljivosti celic LCL na tiopurine se je izkazal nivo izražanja gena za prenašalni P-glikoprotein, ABCB1. V klinični praksi se tiopurini pogosto uporabljajo hkrati z drugimi zdravilnimi učinkovinami, način medsebojnega vplivanja pa pogosto ni znan, zato smo s pomočjo sodobnih in silico metod raziskovali mehanizme interakcij s sicer znanimi substrati in inhibitorji TPMT. Povzeli smo biokemijske značilnosti vezave na TPMT in kinetične karakteristike pretvorbe substratov ter klinične posledice terapije tiopurinov sočasno z inhibitorji TPMT. Z molekulskim sidranjem inhibitorjev v vezavna mesta TPMT smo dopolnili vpogled v molekularne osnove vezave. Še posebno smo se osredotočili na interakcijo TPMT s sulfasalazinom, derivatom aminosalicilne kisline, ki se uporablja pri zdravljenju vnetne črevesne bolezni in se je v in vitro študijah izkazal kot močan inhibitor TPMT. Z merjenjem spremembe fluorescence triptofanov v humanem rekombinantnem TPMT smo dokazali vezavo sulfasalazina na encim. Naša in silico analiza je pokazala dobro sidranje sulfasalazina v vezavno mesto za ko-faktor SAM, a tudi potencialno vezavo na vezavno mesto za substrat. To smo eksperimentalno potrdili tudi z encimsko kinetiko, ki je pokazala inhibicijo mešanega tipa. Rezultati tega doktorskega dela podajajo pomembne nove izsledke o TPMT in tiopurinih z biokemijskega in farmakološkega vidika. Dokaz vpletenosti TPMT v metabolizem selenovih spojin predstavlja dobro izhodišče za natančnejše določanje endogene vloge TPMT ter vpetosti TPMT v patološke procese preko selenovega metabolizma. Z odkritjem povezave med genskimi polimorfizmi TPMT*3 in motivi ABnC v promotoju TPMT ter z identifikacijo vpliva izražanja ABCB1 na rezistenco 6-merkaptopurina smo razširili nabor potencialnih označevalcev dovzetnosti na tiopurine, kar bo v klinični praksi doprineslo k izboljšanju napovedi izida zdravljenja s tiopurini. Despite great therapeutic advances in the field of biological agents, small synthetic molecules still represent an important therapeutic pillar in management of cancer as well as autoimmune disorders. Among such drugs, thiopurines have been commonly used in the treatment of acute lymphoblastic leukaemia and inflammatory bowel disease. Due to their narrow therapeutic index, individualized approach in application of these drugs is crucial, which represents a complex challenge of the current medicine. One of the most important factors influencing thiopurine therapy is thiopurine S-methyltransferase (TPMT), as interindividual differences in response to thiopurines often result from genetic polymorphisms in its gene, metabolic factors stabilizing TPMT, or drug interactions accompanying concomitant treatment. The overall goal of the research presented in this thesis was to contribute to efficacy and safety of thiopurine therapy. Therefore, we aim to elucidate the function and endogenous role of TPMT, further, to identify novel genetic factors of the response to thiopurines, and to define molecular mechanisms of the drug interactions in thiopurine therapy. To unravel the endogenous substrate of TPMT, we focused on selenium compounds, since esides thiopurines, selenopurines are also known TPMT substrates. One of the most important selenium compounds natively present in biological systems is selenocysteine - a key building block of selenoproteins, which are involved in numerous important biological processes in maintaining homeostasis of cells. By STD NMR spectroscopy and fluorescence measurement of tryptophan residues, we demonstrated binding of selenocysteine (Sec) to human recombinant TPMT. The molecular docking analysis positioned Sec into the active site of TPMT with orientation suited for the methylation reaction. With 77Se NMR we finally detected Se-methylselenocysteine (MeSec), produced in the enzymatically catalysed reactionin the presence of the TPMT and the methyl donor S-adenozylmethionine, suggesting the previously unknown role of the enzyme as selenocisteine Se-methyltransferaze. In the pursuit of novel biological markers of response to thiopurines, we first characterized the gene encoding TPMT. We recognised a crucial importance of the exact DNA sequence in the TPMT promoter containing variable number of tandem repeats (VNTR). Motifs ABnC are in complete linkage disequilibrium with clinically established genetic polymorphisms TPMT*3C and TPMT*3A. The motif AB2C is in statistically significant complete linkage disequilibrium with the TPMT*3C allele, and the motifs with three or more B repeats, including a previously unreported VNTR allele AB7C, appear together with TPMT*3A alleles. Since TPMT*3 alleles are clinically relevant, the motifs ABnC represent a viable indirect pharmacogenomic marker in future thiopurine treatment. In our further search for biomarkers predicting the response to thiopurines, we tested the susceptibility to 6-mercaptopurine and 6-thioguanine on the established in vitro model, the lymphoblastoid cell lines (LCL). While the activity of TPMT expectedly correlated with the common genetic polymorphisms, lower TPMT activity did not correlate with enhanced cytotoxicity to 6-mercaptopurine (6MP), which is in contrast to the established correlation in vivo. This finding indicates the importance of other unknown factors that mask the influence of TPMT. In fact, higher levels of ABCB1 expression in LCL predicted their lower sensitivity to 6MP, suggesting this transporter as a potential biological marker for the resistance to 6-mercaptopurine. We further examined mechanisms of the interaction between thiopurines and other drugs often applied concomitantly in the therapy. Their mutual influence remains unexplained, therefore, we first reviewed the biochemical properties of the binding to TPMT, kinetic characteristics of substrate methylation and the clinical outcome of concomitant therapy, and complemented by in silico analysis. The molecular docking and chemical structures of known TPMT inhibitors elucidated the molecular basis for the binding to TPMT. In this context, we specifically focused on the interaction of TPMT by sulfasalazine, a derivative of 5-aminosalicylic acid, which is used as the first line treatment of intestinal bowel disease and has been recognised as a potent TPMT inhibitor. By measuring the change in fluorescence of tryptophan residues in human recombinant TPMT, we confirmed the binding of sulfasalazine and TPMT. Our in silico analysis proposed the primary binding of sulfasalazine to the co-factor binding site, but also indicated possible binding to the substrate binding site. We experimentally proved this by determining the mixed inhibition of the enzyme. The results of this doctoral thesis provide important findings on TPMT and thiopurines from the biochemical and pharmacological point of view. The evidence of involvement of TPMT in the metabolism of selenium compounds represents a good starting point for further delineation of the endogenous role of TPMT and the inclusion of TPMT in pathological processes through selenium metabolism. By discovering the relationship between the genetic polymorphisms TPMT*3 and the ABnC motifs in the TPMT promoter region, and by identifying the impact of ABCB1 expression on the 6-mercaptopurine resistance, we expanded the range of potential markers of susceptibility to thiopurins, which will contribute to future improvements of the outcome of thiopurine treatment.

Direktni oralni antikoagulansi (DOAK) posljednjih su godina pokazali rastući trend propisivanja zbog povoljne farmakokinetike i farmakodinamike bez potrebe za rutinskim nadzorom koagulacije. Svoj antikoagulantni učinak ostvaruju izravnom inhibicijom trombina (dabigatran) ili izravnom inhibicijom aktiviranog čimbenika X (rivaroksaban, apiksaban i edoksaban). Iako DOAK-i stupaju u manje interakcija s drugim lijekovima u usporedbi s varfarinom, kombinacija DOAK-a i mnogih lijekova zahtijeva oprez te u nekim slučajevima i klinički nadzor. Nedavne studije dokumentirale su interindividualnu varijabilnost u razinama DOAK-a u plazmi i odgovoru pacijenta na lijek. Iako klinički i biokemijski čimbenici mogu dovesti do interindividualnih varijabilnosti prisustvo genskih varijanti ili interakcije lijekova mogu doprinijeti tim razlikama. Potrebno je razumjeti ulogu farmakogenomike prvenstveno polimorfizam gena metaboličkih enzima i prijenosnika lijekova u interindividualnoj varijabilnosti četiri najčešće propisivana DOAK-a. Cilj istraživanja: Pregledno prikazati kako farmakogenetička predispozicija, prvenstveno polimorfizmi gena CYP3A4/5 i CES1, koji kodiraju metaboličke enzime, te ABCB1 i ABCG2, koji kodiraju transportne proteine, uz interakcije lijekova u politerapiji mogu doprinijeti interindividualnoj varijabilnost razina DOAK-a u plazmi, te učinkovitosti i sigurnosti njihove primjene. Materijali i metode: Prilikom izrade ovog rada provedena je opsežna pretraga nedavno objavljenih izvornih i preglednih znanstvenih radova, kliničkih ispitivanja, in vitro studija, terapijskih smjernica i publikacija stručnih društava. Naglasak je na polimorfizmima farmakogena bitnih za farmakokinetiku DOAK-a: gena CYP3A4/5 i CES1, za enzime uključene u biotransformaciju, te gena ABCB1 i ABCG2, za transportere lijekova i njihovom doprinosu u interindividualnoj varijabilnosti i bioraspoloživosti DOAK-a, te rizicima razvoja neželjenih učinaka prvenstveno krvarenja. Pretražena je baza podataka PubMed, Web of Science, Cochrane, Google Schoolar koristeći ključne riječi: apiksaban, CES1, CYP3A4, CYP3A5, dabigatran, direktni oralni antikoagulansi, edoksaban, farmakogenetika, farmakogenomika, genske varijante, interakcije gen-lijek, interakcije lijek-lijek, 4 krvarenje, novi oralni antikoagulansi, polimorfizam gena transportera ABCB1, ABCG2, rivaroksaban, SNP, tromboembolijski događaji u razdoblju od zadnjih 10-ak godina. Prikazani su interesantni slučajevi pacijenata iz kliničke prakse koji su zbog farmakogenetičke predispozicije i interakcije lijekova razvili nuspojave DOAK-a u vidu krvarenja. Raspravljene su prisutne dileme, te predočeno trenutno stanje u Hrvatskoj uspoređeno sa situacijom u Europi i drugdje u svijetu. Rezultati: Nekoliko varijanti gena CES1 i ABCB1 se smatra potencijalno odgovornim za nastanak interindividualnih varijabilnosti DOAK-a. Utvrđeno je da su varijante gena CES1 rs2244613 i rs8192935 povezane s nižim trough koncentracijama dabigatrana i nižim rizikom od krvarenja. S druge strane varijanta gena ABCB1 rs1045642 povezuje s povećanim vršnim koncentracijama dabigatrana i rivaroksabana, te povećanom učestalošću krvarenja. Farmakogenetička analiza varijanti gena ABCB1 rs2032582 (C.2677G> T) i rs104562 (C3435C> T) mogla bi biti opravdana i od koristi kod pacijenata na terapiji rivaroksabanom. Da bi se spriječili neželjeni učinci tijekom terapije apiksabanom kod nositelja nefunkcionalnih alela gena CYP3A5 potrebano je pažljivo odabrati dozu i pratiti nuspojave. Postoji veliki rizik od razvoja neželjenih učinaka lijeka kad se edoksaban kombinira s lijekovima koji inhibiraju CYP izoenzime u homozigotnih nositelja nefunkcionalnih alela za gen CYP3A5. Zaključak: Polimorfizam gena zajedno s istovremenom primjenom inhibitora/induktora transportnih proteina ili enzima odgovornih za metabolizam lijeka može povećati rizik od nastanka štetnih događaja vezanih uz promjenu DOAK-a. Due to good pharmacokinetics and pharmacodynamics without the requirement for routine coagulation monitoring, direct oral anticoagulants (DOAK) have seen an increase in prescribing in recent years. DOAC achieves its anticoagulant effect by direct inhibition of thrombin (dabigatran) or direct inhibition of activated factor X (rivaroxaban, apixaban and edoxaban). Although DOACs interact less with other drugs compared to warfarin, the combination of DOAC and many drugs requires caution and clinical supervision in some cases. Recent studies have documented interindividual variability in DOAC plasma levels and patient response to the drug. Although clinical and biochemical factors may lead to interindividual variability, the presence of genetic variants or drug interactions may contribute to these differences. The role of pharmacogenomics, namely polymorphism of metabolic enzyme genes and drug transporters, in the interindividual variability of the four most often prescribed DOACs, must be understood. Objectives: Demonstrate how pharmacogenetic predisposition, primarily polymorphisms of CYP3A4 / 5 metabolic enzyme genes, and CES1 and ABCB1 and ABCG2 transport proteins with drug interactions in polytherapy, may contribute to interindividual variability in DOAC plasma levels, and their efficiency and safety. Materials and methods: During the preparation of this paper, an extensive search of recently published original and reviewed scientific papers, clinical trials, in vitro studies, therapeutic guidelines and publications of professional societies was conducted. The emphasis is on pharmacogen polymorphisms that are important for DOAC pharmacokinetics: CYP3A4 / 5 and CES1 genes, for enzymes involved in biotransformation, and ABCB1 and ABCG2 genes, for drug transporters and their contribution to interindividual variability and bioavailability of DOACs, effects primarily of bleeding. The database PubMed, Web of Science, Cochrane, Google Schoolar were searched using keywords: apixaban, CES1, CYP3A4, CYP3A5, dabigatran, direct oral anticoagulants, edoxaban, pharmacogenetics, pharmacogenomics, gene variants, drug-drug interactions, gene-drug interactions, bleeding, new oral anticoagulants, polymorphism of transporter genes ABCB1, ABCG2, rivaroxaban, SNP, thromboembolic events in the period of the last 10 years. Interesting cases of patients from clinical practice who have developed DOAC side effects in the form of bleeding due to pharmacogenetic predisposition and drug interactions are presented. The present dilemmas were discussed, and the current situation in Croatia was compared to the situation in Europe and elsewhere in the world. Results: Several variants of the CES1 and ABCB1 genes are thought to be potentially responsible for the occurrence of interindividual variability in DOAC. Variants of the CES1 rs2244613 and rs8192935 genes were found to be associated with lower trough out dabigatran concentrations and a lower risk of bleeding. On the other hand, the ABCB1 gene variant rs1045642 is associated with increased peak concentrations of dabigatran and rivaroxaban, and increased bleeding frequency. Pharmacogenetic analysis of the ABCB1 gene variants rs2032582 (C.2677G>T) and rs104562 (C3435C>T) could be justified and useful in patients on rivaroxaban therapy. To prevent side effects during apixaban therapy in carriers of non-functional alleles of the CYP3A5 gene, the dose should be carefully selected and adverse reactions monitored. There is a high risk of developing adverse drug reactions when edoxaban is combined with drugs that inhibit CYP isoenzymes in homozygous carriers of non-functional alleles for the CYP3A5 gene. Conclusion: Gene polymorphism in combination with the concomitant use of inhibitors / inducers of transport proteins or enzymes responsible for drug metabolism may increase the risk of adverse events associated with alterations in DOACs.

Precision medicine approaches based on pharmacogenomics are now being successfully implemented to enable physicians to predict more efficient treatments and prevention strategies for a given disease based on the genetic background of the patient. This approach has already been proposed for vaccines, but research is lagging behind the needs of society, and precision medicine is far from being implemented here. While vaccinomics concerns the effectiveness of vaccines, adversomics concerns their side effects. This area has great potential to address public concerns about vaccine safety and to promote increased public confidence, higher vaccination rates, and fewer serious adverse events in genetically predisposed individuals. The aim here is to explore the contemporary scientific literature related to the vaccinomic and adversomic aspects of the three most-controversial vaccines: those against hepatitis B, against measles, mumps, and rubella, and against human Papilloma virus. We provide detailed information on the genes that encode human leukocyte antigen, cytokines and their receptors, and transcription factors and regulators associated with the efficacy and safety of the Hepatitis B and Measles, Mumps and Rubella virus vaccines. We also investigate the future prospects of vaccinomics and adversomics of a COVID-19 vaccine, which might represent the fastest development of a vaccine ever.

Uvod rada: Kardiovaskularne bolesti (KVB), vodeći su uzrok smrtnosti u cijelome svijetu, pa tako i u Hrvatskoj. Farmakoterapija KVB uključuje čestu istodobnu primjenu antikoagulantnih/antiagregacijskih lijekova, hipolipemika, antihipertenziva, antiaritmika, ali i nesteroidnih protuupalnih lijekova kao i lijekova za liječenje anksioznosti te depresije. Porast kroničnih bolesti u starijoj populaciji te propisivanje velikog broja različitih lijekova uzrokuje višestruki trošak zdravstvene zaštite. Polifarmacija je povezana s većom vjerojatnošću razvoja nuspojava koje su prepoznate kao važan uzrok morbiditeta, smrtnosti i troškova zdravstvene zaštite. I do 30 % hospitalizacija pacijenata starijih od 70 godina povezuje se s nuspojavama lijekova. Farmakogenomika (PGx) otkriva moguću povezanost varijacija gena, prvenstveno onih koje mogu utjecati na apsorpciju, distribuciju, metabolizam i izlučivanje lijeka, s neučinkovitosti kao i s razvojem ozbiljnih nuspojava na primjenjenu terapiju. Dosadašnja PGx istraživanja kardiovaskularnih (KV) lijekova omogućila su prijenos saznanja o varijantama pojedinih farmakogena u kliničku praksu tj. izradu smjernica za doziranje varfarina, pojedinih inhibitora HMG- CoA reduktaze (statini) i klopidogrela. Za mnoge novije KV lijekove broj PGx istraživanja je ograničen i nema jednoznačnih zaključaka. Prvenstveno se to odnosi na direktne oralne antikoagulanse (DOAK-e), druge antiagregacijske lijekove - inhibitore agregacije trombocita iz skupine antagonista P2Y12 receptora, statine, većinu antihipertenziva i antiaritmika. Postoji potreba za istraživanjem kompleksnog uzroka nastanka interakcija i nuspojava koje u svom dizajnu proučava ulogu većeg broja farmakogena te njihovu interakciju s kliničkim čimbenicima, s posebnim naglaskom na procjenu važnosti interakcija lijek-lijek-gen. Nova PGx saznanja o KV lijekovima mogu značajno doprinijeti odabiru optimalnog lijeka i doze te minimalizaciji rizika razvoja nuspojava, rezultirajući značajnom zdravstvenom i ekonomskom koristi za pacijente i društvo u cjelini. Svrha rada: Ovaj projekt osigurava multidiciplinaran pristup znanstvenom imperativu u području PGx. Rezultati obrade svih podataka ispitanika sa i bez nuspojava na primjenu DOAK-a, antiagregacijskih lijekova i statina će identificirati varijante gena koje moduliraju farmakokinetiku (metabolizam, transport) te mogu biti odgovorne za varijabilnu bioraspoloživost i nuspojave navedenih lijekova poglavito u politerapiji. Također će rezultati analiza interakcija lijek-lijek te lijek-lijek-gen omogućiti identifikaciju kombinacija KV i drugih lijekova u terapiji i pridruženih varijanti farmakogena koje mogu značajno utjecati na ukupnu izloženost KV lijekovima te predstavljaju rizične čimbenike za razvoj nuspojava. Bitno postignuće ovog projekta će biti i edukacija svih dionika zdravstvene skrbi o PGx. Metode: Ovaj projekt je planiran kao prospektivno istraživanje „ugnježđenih slučajeva i kontrola“ (engl. nested case-control). Istraživanje traje 60 mjeseci i uključit će ukupno 1200 konsekutivno uključenih ispitanika koji zadovoljavaju uključne kriterije: dob 18 godina ili više ; novopostavljenu indikaciju za primjenu bilo kojeg od lijekova od interesa za koju je predviđena najmanje 3-mjesečna primjena i potpisan informirani pristanak. Ispitanici se prate tijekom redovitih kontrolnih pregleda. Lijekovi od interesa su: DOAK-i, antiagregacijski lijekovi i statini ; kao monoterapija ili bez ograničenja u odnosu na bilo koju drugu istodobnu farmakoterapiju. Slučajevi obuhvaćaju ispitanike kojima se tijekom praćenja uoče nuspojave: krvarenja koja zadovoljavaju kriterije „velikog“ ili „ne-velikog, klinički relevantnog krvarenja (za antikoagulanse i inhibitore agregacije trombocita) ; lezije poprečno-prugastih mišića ili jetre (za statine) ; bilo koja druga ozbiljna nuspojava. Za DOAK-e i antitrombocitne lijekove, nuspojava se smatra i pojava vaskularnih incidenata tj. neučinkovitost lijeka. Kontrolnu skupinu čine oni ispitanici u kojih se tijekom trajanja istraživanja ne uoči razvoj nuspojava. Uzorkuju se biološki uzorci za biokemijske, hematološke, koagulacijske standardne parametre i farmakogenetičke analize relevantnih gena ovisno o primijenjenoj terapiji. Na kraju istraživanja provest će se statistička obrada podataka koja će se temeljiti na univarijatnoj i multivarijatnoj analizi svih promatranih parametara. Rezultati - tijek istraživanja: Etička povjerenstva Medicinskog fakulteta Sveučilišta u Zagrebu i Kliničkog bolničkog centra Zagreb su odobrila istraživanje koje financira Hrvatska zaklada za znanost pod šifrom UIP-2020- 02-8189. Istraživanje je počelo 15. prosinca 2020. te je do travnja 2022. godine uključeno više od 500 ispitanika. Očekivani znanstveni i klinički doprinos: Očekujemo da će rezultati projekta donijeti nova znanstvena saznanja o povezanosti farmakogena s učinkovitosti i sigurnosti primjene KV lijekova specifično u populaciji ispitanika s komorbiditetima i politerapijom. Zbog toga će doprinijeti definiranju populacije pacijenata za koje je od kliničke značajnosti provesti analizu farmakogena. Rezultati projekta mogu dati poticaj za primjenu farmakogenetičkih saznanja u kliničkoj medicini s ciljem individualizacije farmakoterapije i razvijanja modela racionalnog upravljanja farmakoterapijom.

Cilj istraživanja: Cilj ovoga rada jest procijeniti postoji li i koliki je potencijal farmakogenetike u području optimizacije imunosupresivne terapije u transplantaciji bubrega. Nadalje, cilj je i procijeniti ulogu farmakogenetike u ovom kliničkom području. Hipoteza je da genetičko nasljeđe može značajno izmijeniti međusobne interakcije imunosupresivnih lijekova i organizma. Materijali i metode: Učinjen je pregled dostupne relevantne literature, a posebna je pozornost pri tome bila posvećena istraživanju udžbeničke literature i stručnih smjernica. Potom je slijedilo pretraživanje bibliografskih baza podataka (PubMed) i baza podataka sintetiziranog znanja (PharmGKB), koje je uključivalo sljedeće ključne pojmove u različitim kombinacijama: farmakogenomika, imunosupresivni lijekovi, transplantacija bubrega, polimorfizam, mikofenolat, CNI, ciklosporin, takrolimus, mTOR inhibitor, sirolimus, everolimus, CYP3A4, CYP3A5, farmakodinamika, prijenosnici. U obzir je uzimana literatura objavljena u razdoblju od 2010. do 2020., uz manje iznimke vezane za udžbenike i pojedina pionirska istraživanja. Rezultati: Pregledana su značajnija istraživanja u kojima je metodologija temeljena na kombiniranju informacije o dozi lijeka s podacima dobivenima genotipizacijom primatelja bubrežnog presatka za polimorfizme pojedinačnih nukleotida (engl. Single-Nucleotide Polymorphism, SNP) gena koji kodiraju metaboličke enzime i prijenosnike imunosupresivnih lijekova, kao i pregledni radovi koji se na takva i slična istraživanja referiraju. Pronađeno je kako smjernice konzorcija za implementaciju kliničke farmakogenetike (engl. Clinical Pharmacogenetics Implementation Consortium, CPIC) otvaraju mogućnost da genotipom gena natporodice citokroma P450, porodica 3, potporodica A, člana 5 (engl. Cytochrome P450 Family 3 Subfamily A Member 5, CYP3A5) usmjeravano doziranje takrolimusa poboljša kliničke ishode jer se njime može brže nakon transplantacije dostići ciljne koncentracije takrolimusa u krvi, čak i kada se primjenjuje titracija terapijskim praćenjem lijeka (engl. Therapeutic Drug Monitoring, TDM). Brže postizanje ciljnih koncentracija može potencijalno umanjiti rizik odbacivanja presatka uslijed premale ili toksičnost uslijed prevelike izloženosti lijeku. U smjernicama se oprezno pristupa ideji zamjenjivanja TDM-a genotipizacijom CYP3A5, zbog toga što drugi faktori (poput demografskih, interakcija lijek-lijek, genetičkih varijacija koje bi utjecale na farmakodinamiku) također mogu utjecati na doziranje takrolimusa. Takrolimus je jedini takav imunosupresiv, ukoliko zanemarimo smjernice za doziranje azatioprina (prema genotipu tiopurin metiltransferaze (engl. thiopurine S-methyltransferase, TPMT)) čija je primjena u transplantaciji već dugi niz godina zanemariva. Daleko je manje farmakogenetički istraživana farmakodinamika imunosupresiva primjenjivanih u transplantaciji bubrega, a po pitanju jedinog farmakodinamičkog polimorfizma koji je značajnije istraživan, onoga gena koji kodira enzim inozin-5'-monofosfat-dehidrogenazu (engl. inosine-5'-monophosphate dehydrogenase, IMPDH), nije postignut širi konsenzus po pitanju odabira farmakogenetičkog biljega i njegove kliničke primjene. Zaključak: Trenutno postoje ograničenja za rutinsku kliničku primjenu farmakogenomike u odabiru imunosupresivnog liječenja za bolesnike kandidate za transplantaciju bubrega. Izazov u farmakogenomici imunosupresivnih lijekova nepostojanje je dostatnih kliničkih dokaza o prednosti provedbe doziranja na temelju testova genotipizacije. Da bi se znanstvene spoznaje o važnosti polimorfizama primijenile u individualizaciji terapije nužno je provesti dodatne kliničke studije o utjecaju farmakogenetike na preživljenje presatka ili na opće stanje bolesnika. Objašnjenje za poziciju koju farmakogenomika trenutno zauzima u području imunosupresivne terapije u transplantaciji bubrega, djelomično može biti u tomu da se većina najčešće upotrebljavanih imunosupresiva metabolizira putem enzima natporodice citokroma P450, porodica 3, potporodica A, član 4 (engl. Cytochrome P450 Family 3 Subfamily A Member 4, CYP3A4) čija je ekspresija neovisno o genotipu vrlo varijabilna inter- i intraindividualno te je složenost predviđanja farmakokinetike neminovna. Na to, između ostalog, utječu lijek-lijek interakcije, varijacije u genima koji utječu na aktivnost CYP3A te epigenski faktori. Objectives: The aim of this thesis is to assess whether there is, and to what extent, a potential for pharmacogenetics in the field of immunosuppressive therapy optimization in kidney transplant recipients. The aim is also to determine what role pharmacogenetics currently plays in this area. The hypothesis is that genetic inheritance can significantly alter the interactions between immunosuppressive drugs and the organism. Materials and methods: A review of the available relevant literature was made, and special attention was paid to the research of textbook literature and professional guidelines. This was followed by a search of bibliographic databases (PubMed) and synthesized knowledge databases (PharmGKB), which included the following key terms in different combinations: pharmacogenomics, immunosuppressive drugs, kidney transplantation, polymorphism, mycophenolate, CNI, cyclosporine, tacrolimus, mTOR inhibitor, sirolimus, everolimus, CYP3A4, CYP3A5, pharmacodynamics, transporters. The literature published in the period from 2010 to 2020 was taken into account, with minor exceptions related to textbooks and individual pioneering research. Results: Significant studies in which the methodology is based on combining drug dose information with data obtained by renal graft recipients genotyping for Single-Nucleotide Polymorphisms (SNPs) in genes encoding metabolic enzymes and transporters of immunosuppressive drugs have been reviewed, as well as review papers referring to such and similar studies. Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines have been found to open the possibility that Cytochrome P450 Family 3 Subfamily A Member 5, CYP3A5 genotype-directed dosing of tacrolimus improves clinical outcomes because it could help reach the target tacrolimus blood concentrations faster after transplantation, even when therapeutic drug monitoring (TDM) titration is applied. Achieving target concentrations faster can potentially reduce the risk of graft rejection due to too low or toxicity due to too high drug exposure. The guidelines cautiously approach the idea of replacing TDM with CYP3A5 genotyping, because other factors (such as demographics, drug-drug interactions, genetic variations that would affect the pharmacodynamics) may also affect tacrolimus dosing. Tacrolimus is the only immunosuppressive drug for which guidelines on dosing according to genotype exist, disregarding the guidelines for dosing of azathioprine (according to thiopurine S-methyltransferase (TPMT) genotype), use of which in transplantation has been negligible for many years. The pharmacodynamics of immunosuppressants used in kidney transplantation is far less researched, and for the only pharmacodynamic polymorphism that has been significantly researched, the inosine-5'-monophosphate dehydrogenase gene (IMPDH), a consensus on the choice of pharmacogenetic marker and its clinical application has yet to be reached. Conclusion: Currently, significant limitations exist for the routine application of pharmacogenomics in the selection of immunosuppressive treatment for kidney transplant recipients. A challenge in the pharmacogenomics of immunosuppressive drugs is the lack of sufficient clinical evidence on the benefit of dosing based on genotyping tests. In order to apply scientific knowledge about the importance of polymorphisms in therapy individualization of kidney transplant candidates, it is necessary to conduct additional clinical studies on the impact of pharmacogenetics on graft survival or general patient condition. The explanation for the current position of pharmacogenomics in the field of immunosuppressive therapy in renal transplantation may be partly consequence of the fact that most commonly used immunosuppressants are metabolized by the enzyme Cytochrome P450 Family 3 Subfamily A Member 4, CYP3A4, since expression of this enzyme is, regardless of genotype, highly variable inter- and intraindividually. This is influenced, among other things, by drug-drug interactions, variations in genes that affect CYP3A activity, and epigenetic factors

Methotrexate is used as first-line treatment of moderate to severe psoriasis. Despite the marked variability in treatment outcomes, no pharmacogenetic markers are currently used for personalised management of therapy. In this retrospective study, we investigated the effects of genetic predisposition on efficacy and toxicity of low-dose methotrexate in a cohort of 137 patients with moderate to severe plaque psoriasis. We genotyped 16 polymorphisms in genes for enzymes involved in the folate-methionine pathway and in methotrexate transport, and analysed their association with treatment efficacy and toxicity using classification and regression tree analysis and logistic regression. The most pronounced effect observed in this study was for GNMT rs10948059, which was identified as a risk factor for inadequate efficacy leading to treatment discontinuation. Patients carrying at least one variant allele had ~7-fold increased risk of treatment failure compared to patients with the wild-type genotype, as shown by the classification and regression tree analysis and logistic regression (odds ratio [OR], 6.94; p = 0.0004). Another risk factor associated with insufficient treatment responses was DNMT3b rs2424913, where patients carrying at least one variant allele had a 4-fold increased risk of treatment failure compared to patients with the wild-type genotype (OR, 4.10; p = 0.005). Using classification and regression tree analysis, we show that DNMT3b rs2424913 has a more pronounced role in patients with the variant GNMT genotype, and hence we suggest an interaction between these two genes. Further, we show that patients with the BHMT rs3733890 variant allele had increased risk of hepatotoxicity (OR, 3.17; p = 0.022), which is the most prominent reason for methotrexate discontinuation. We also show that variants in the genes for methotrexate transporters OATP1B1 (rs2306283/rs4149056 SLCO1B1 haplotypes) and ABCC2 (rs717620) are associated with increased risk of treatment failure. The associations identified have not been reported previously. These data suggest that polymorphisms in genes for enzymes of the methionine cycle (which affect cell methylation potential) might have significant roles in treatment responses to methotrexate of patients with psoriasis. Further studies are warranted to validate the potential of the pharmacogenetic markers identified. Soavtorji: Irena Mlinarič-Raščan, Pij B. Marko, Maruška Marovt, Tanja Gmeiner, Alenka Šmid. Št. članka: 111456. Bibliografija: str. 9-11. Abstract. ARRS European Regional Develop- ment Fund, European Union - Slovenia

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. 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 TPMT*3A (4.1 %), TPMT*3C (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.

CYP2C9 jedan je od najvažnijih i najobilnije eksprimiranih enzima sustava citokrom P450 (CYP) u ljudskim jetrima. Odgovoran je za biotransformaciju oko 15 % klinički važnih lijekova, uključujući kumarinske antikoagulante (varfarin), antikonvulzive (fenitoin, valproična kiselina), oralne antidijabetike (gliklazid, tolbutamid), blokatore angiotenzinskih-II receptora (kandesartan, losartan), nesteroidne protuupalne lijekove (NSAID) te fluvastatin i siponimod. Gen CYP2C9 visoko je polimorfan te je, do danas, identificirano preko 60 različitih alela, među kojima najmanje njih 20 dokazano mijenja aktivnost genskog produkta, što doprinosi velikoj interindividualnoj varijabilnosti terapijskog odgovora. S obzirom na učinak polimorfizama na smanjenu aktivnost enzima te njihovu relativno veliku učestalost u bjelačkoj populaciji, najvažniji su haplotipovi CYP2C9*2 i CYP2C9*3. Navedeni aleli rezultiraju smanjenim metabolizmom većine supstrata CYP2C9 i većom izloženosti lijeku te posljedično povećanim rizikom razvoja neželjenih reakcija, osobito kod primjene lijekova s uskim terapijskim rasponom. U svrhu otkrivanja pojedinaca s povećanim rizikom razvoja neželjenih reakcija i prije početka liječenja, provodi se genotipizacija CYP2C9 na temelju koje se zaključuje o metaboličkom fenotipu. Tri su moguća fenotipa CYP2C9: spori (PM), intermedijarni (IM) i normalni (NM) metabolizatori. U bjelačkoj populaciji spori metabolizatori čine 3-5 %. Ovakav personalizirani pristup omogućuje pravovremenu primjenu odgovarajuće terapije i bolji ishod za bolesnika. U ovome su radu prikazana dosadašnja saznanja o utjecaju genskih varijacija CYP2C9 na terapijski učinak i sigurnost lijekova-supstrata CYP2C9 te je opisan praktični pristup validaciji metode za genotipizaciju CYP2C9 u kliničkoj praksi. CYP2C9 is one of the most important and abundantly expressed enzymes of the cytochrome P450 system in the human liver. It is responsible for the metabolism of approximately 15 % of clinically important drugs, including coumarin anticoagulants (warfarin), anticonvulsants (phenytoin, valproic acid), oral antidiabetic agents (gliclazide, tolbutamide), angiotensin II receptor antagonists (candesartan, losartan), nonsteroidal anti-inflammatory drugs (NSAIDs), fluvastatin and siponimod. CYP2C9 gene is highly polymorphic and, to this day, over 60 different alleles have been identified, among which at least 20 have been proven to alter its gene product activity, which contributes to significant inter-individual variability of therapeutic response. Due to the decreasing effect on enzyme activity and the relatively high prevalence in Caucasians, CYP2C9*2 and CYP2C9*3 are the most important haplotypes. These alleles result in reduced metabolism for most CYP2C9 substrates, increased drug exposure, and consequently increased risk of adverse drug reactions, especially when administered drugs have a narrow therapeutic index. To identify individuals with increased risk of adverse drug reactions (ADRs) even before the treatment initiation, preemptive CYP2C9 genotyping is performed, and metabolic phenotype is inferred based on these results. There are three possible CYP2C9 phenotypes: poor metabolizer (PM), intermediate metabolizer (IM), and normal metabolizer (NM). In the Caucasian population, poor metabolizers make 3-5 %. This personalized approach enables in time selection of appropriate therapy and a better outcome for a patient. In this paper, current knowledge on the influence of CYP2C9 gene variations on the efficacy and safety of drugs metabolized by the CYP2C9 enzyme and a practical approach to validation of methods for CYP2C9 genotyping in clinical practice is presented.

Prethodna istraživanja pokazala su pozitivan utjecaj korištenja farmakogenomike u svakodnevnoj kliničkoj praksi. Primjerice, farmakogenomika omogućava farmaceutskoj industriji razvoj lijekova koji ciljano djeluju na specifične bolesti i poremećaje u organizmu, čime se povećava terapijski učinak, a smanjuje mogućnost oštećenja zdravog tkiva. Istraživanja su pokazala kako farmakogenomika može pridonijeti pravilnom odabiru lijeka na osnovu genet-skog profila pojedinog pacijenta, čime se ubrzava proces liječenja te smanjuje rizik od nastanka nuspojava. Među¬tim, za optimalnu integraciju farmakogenomike u kliničku praksu, nužna je i odgovarajuća edukacija zdravstvenih djelatnika o farmakogenomici. Zbog navedenog, cilj ovog istraživanja bio je ispitati stavove studenata farmacije, medicine i dentalne medicine na Sveučilištu u Splitu o farmakogenomici. Istraživanje je bilo ustrojeno kao anketno presječno istraživanje i provedeno je u listopadu 2020. godine. U istraživanju je sudjelovalo ukupno 503 studenta studija farmacije, medicine i dentalne medicine na Sveučilištu u Splitu. Rezultati su pokazali da je samo 3 % stu¬denata navelo da pati od neke kronične nasljedne bolesti. Nadalje, rezultati istraživanja su pokazali da bi 58, 2 % studenata razmotrilo genetsko testiranje ukoliko bi na taj način mogli saznati koje bolesti će potencijalno razviti u budućnosti. Analizira rezultata istraživanja pokazala je da su studenti farmacije iskazali sveukupno pozitivnije stavove o farmakogenomici u usporedbi sa studentima medicine i dentalne medicine. Čak 80, 7 % studenata farmacije smatra da bi farmakogenomika trebala biti važan dio kurikuluma u usporedbi sa 44, 3 % studenata dentalne medicine i 49, 6 % studenata medicine (p

The community pharmacist challenges with the dispensing of medicines for the pain treatment, whether they are prescribed or recommended as over-the-counter medicines in self-treatment. It is very important to know pharmacodynamic and pharmacokinetic characteristics of individual drugs, but also to understand other factors that may influence their efficacy and safety. The fact that everyone differs due to the influence of genes, but also in impact of environmental factors on (de)activation genes, results in different responses to drugs. Implementations of personalized therapy concept in pharmaceutical care is a huge challenge for pharmacists as well as for the other health-care providers. Pharmacists, with their specific knowledge and skills related to drugs are in position to tailor personalized therapy approach. This paper presents the pharmacological characteristics of nonsteroidal anti-inflammatory drugs with emphasis on the impact of specific genetic variations on their efficacy and safety.

Akutna limfoblastna leukemija (ALL) predstavlja najčešću malignu bolest dečjeg doba. Akutna limfoblastna leukemija je klonalna maligna bolest hematopoetskog tkiva, koja se odlikuje klonalnom proliferacijom limfoblasta. a na osnovu njihovih imunofenotipskih karakteristika može se podeliti na B-ćelijske i T-ćelijske. Najčešći oblik akutne limfoblastne leukemije kod dece je B ćelijska prekursorska leukemija. Najčešće se javlja u uzrastu od druge do pete godine života i nešto je češća kod dečaka. Optimalna primena antileukemijskih lekova u sklopu jasno definisanih terapijskih protokola i precizna stratifikacija bolesnika u grupe rizika, uz maksimalnu suportivnu terapiju doveli su do značajnog porasta ukupnog preživljavanja koje sada iznosi i do 90%. Lečenje leukemije kod dece se sastoji iz četiri faze, od kojih je poslednja i najduža, faza održavanja u čijem sklopu se primenjuje lek iz grupe tiopurina – 6-merkaptopurin (6-MP). Toksični efekti ovog leka, pre svega mijelosupresija, kod izvesnog broja dece mogu dovesti do životno ugrožavajućih infekcija kao i do značajnog odlaganja terapije i povećanja rizika za nastanak recidiva bolesti. Enzim tiopurin S-metiltransferaza (TPMT) je najznačajni enzim za inaktivaciju 6-MP i poznato je da deca koja imaju sniženu aktivnost enzima, imaju izraženije toksične efekte i zahtevaju primenu manjih dozu 6-MP. Individualizovana terapija na osnovu TPMT genotipa je postala standard u lečenju akutne limfoblastne leukemije u dece. Osim enzima TPMT, postoji još nekoliko metaboličkih puteva u inaktivaciji i transportu 6-MP koji mogu uticati na povećanu toksičnost tokom primene leka. Inozin trifosfat pirofosfataza je drugi enzim, kodiran od strane ITPA gena, koji inaktiviše 6-MP, i nedovoljna aktivnost ovog enzima takođe doprinosi pojavi izražene mijelotoksičnosti tokom primene 6-MP. P-glikoprotein, kodiran od strane ABCC4 gena, i Multidrug Resistance Protein 4, kodiran od strane ABCB1 gena su transmembranski proteini koji funkcionišu kao efluks pumpe i izbacuju ksenobiotike iz ćelija. Povećana odnosno smanjena aktivnost ovih proteina može dovesti do smanjenog terapijskog efekta lekova odnosno do povećane toksičnosti tokom primene 6-MP. Cilj ovog istraživanja je bio da utvrdi učestalost varijacija u genima TPMT, ITPA, ABCC4 i ABCB1 kod dece obolele od akutne limfoblastne leukemije i njihov uticaj na intenzitet toksičnih efekata 6-MP kao i na ukupno preživljavanje... Acute lymphoblastic leukemia is the most common childhood malignancy. Acute lymphoblastic leukemia is malignant clonal disease of hematopoetic tissue and it is characterized by clonal proliferation of lymphoblasts, which can be of B-cell or T-cell origin. The most prevalent type of acute lymphoblastic leukemia in children is B-cell precursor leukemia. It is most commonly seen in children between age of two and five and there is slight male predominance. Optimal use of antileukemic drugs in the settings of clearly defined therapeutic protocols and precise risk stratification with maximal supportive therapy have led to significant improvement in overall survival, which is now near 90%. Leukemia treatment consists of four phases and last and longest phase is maintenance phase. In this phase, the most important drug is 6-mercaptopurine (6-MP) which belongs to the group of thiopurine drugs. 6-MP induced toxicity, myelosupression as the most prominent one, can in one number of children lead to life-threatening infections as well as significant therapy delay which contributes to increased risk for relapse. Enzyme thiopurine S-methyltransferase (TPMT) is most important inactivation enzyme for 6-MP and it is well established that children with TPMT deficiency have more pronounced toxicity and require lower dose of 6-MP. Personalized therapy based on TPMT genotype has become standard in treatment of acute lymphoblastic leukemia in children. Besides inactivation pathway that utilizes TPMT there are several other metabolic pathways for inactivation and transport mechanisms that can influence toxicity profile of 6-MP. Inosine triphosphate pirophosphatase (ITPA), which is coded by ITPA gene, is one of those 6-MP inactivation enzymes which, when deficient, can contribute to profound myelotoxicity. P-glycoprotein, coded by ABCC4 gene, and Multidrug Resistance Protein 4, coded by ABCB1 gene, are transmembrane transporters that works as efflux-pumps and removes xenobiotics from cells. Increased or reduced activity of these efflux-pumps can lead to reduced therapeutic effect or increased toxicity of 6-MP. Goal of this study was to determine frequency of variations in genes TPMT, ITPA, ABCC4 and ABCB1 in children with acute lymphoblastic leukemia and their influence on 6-MP induced toxicity as well as on overall survival...

Budućnost medicine je u ranoj dijagnostici te preventivnom liječenu. Personalizirana je medicina bitan dio te budućnosti jer u svojem konceptu sadrži individualan pristup svakoj osobi te prilagodbu terapije sukladno genomu pojedinca. Da genetika utječe na učinkovitost terapije lijekovima, prepoznato je u prošlome stoljeću, otkada se broj studija iz dana u dan povećava. Optimizacija terapije bolesnika prema njegovom genomu te proučavanje učinaka gena na nastajanje nuspojava, glavna je zadaća nove discipline koja je važan dio personalizirane medicine – farmakogenomike. Njezin razvoj napreduje zahvaljujući sve novijim metodama molekularne dijagnostike. Sve većim brojem farmagenomskih testiranja nastaje i puno genetskih podataka koje je potrebno na pravilan način organizirati te jasno povezati s njihovim odnosima s pojedinim lijekovima. Zato u tome sudjeluje velik broj disciplina i struka, a jedna od važnijih je bioinformatika koja omogućuje preglednost i dostupnost svih podataka i smjernica za uporabu lijekova široj populaciji. Smjernice i preporuke za korištenje lijekova neminovne su u pokušajima veće implementacije u kliničku praksu, isto kao i edukacija liječnika, zdravstvenog osoblja, ali i bolesnika. Brojne radne skupine rade na skupljanju takvih vrsta podataka, prikupljanju studija i relevantnih dokaza, kako bi ti podatci bili u javnim bazama podataka. Jedna od važnijih takvih grupa je Clinical Pharmacogenetics Implementation Consortium (CPIC). Primjena farmakogenomskih testiranja još uvijek nije dovoljna u medicinskoj praksi, najveći utjecaj ostvaruje u liječenju karcinoma, a jedan od boljih primjera ulaska farmakogenomike u svakodnevno liječenje jest doziranje lijeka varfarina u kardioloških bolesnika. Veliku potrebu za takvim liječenjem pokazuje i pedijatrija koja je suočena s velikim brojem štetnih utjecaja lijekova na djecu, ali nažalost, primjena farmakogenomike je daleko za spoznajama koje imamo. Postoje još brojna ograničenja implementacije u kliničku praksu kao što su cijene i dostupnost testova, vrijeme izdavanja nalaza ili zaštite podataka. Cilj ovog rada je prikazati dostupne baze podataka te dio smjernica i preporuka za korištenje takvih vrsta podataka. Prikazat će se današnja primjena farmakogenomike u liječenju te ograničenja, ali i potencijali za njezinu širu primjenu u praksi u budućnosti.

Cilj rada je kreirati alat koji će potencijalno pomoći liječniku odabir najbolje terapije, te uštedjeti velike svote novaca svima uključenima u proces izrade lijeka. Kao polazne točke u kreiranju ovoga rada koristile su se dvije baze podataka. Spojili smo genetske karakteristike staničnih linija raka i kemijske strukture lijekova. Nad tim podacima kreirali smo model algoritma slučajne šume, te algoritme hrbatne regresije i regresije najmanjeg kuta. Pokazalo se da se algoritam slučajne šume ponaša najbolje, i naš model ima bolje rezultate ($ R^2 $ metriku) od konkurentnog rada. Pokazalo se, također, da se model ne ponaša dovoljno dobro u slučaju predviđanja rezultata za novonastali, dosad neviđeni, lijek. Zbog toga, daljnji fokus istraživanja će biti na kreiranju modela koji će se moći snalaziti u danim okolnostima. The main goal was to create tool that would, potentially, be able to help doctors in therapy decision making, as well as saving a lot of money during the development of treatment methods. As starting points in this paper two databases were used. We merged genetic characteristics of cell lines and chemical structures of medicine. We applied random forest algorithm on these data. We also trained the ridge regression and the least angle regression. It was shown that the random forest algorithm showed best results, and that my results were better than the previous work. I showed that, also, my model does not manage to work well on predicting results on previously unknown medication. The future research will be focused on creating a model that will work well in such use case.

Bez sumnje je da budućnost medicine stremi ka vrhunskim metodama rane dijagnostike i individualno dizajniranom liječenju, stoga je Komisija EU 2016. među svoje ključne prioritete u zdravstvu uvrstila koncept personalizirane medicine i osnavala Internacionalni konzorcij za personaliziranu medicinu (International Consortium of Personalized Medicine, ICPerMed), sa zadaćom da sinkronizira rad svih članica EU na standardiziranju dijagnostičkih i terapijskih postupaka vezanih uz personaliziranu medicinu u čijem radu sudjeluje i Republika Hrvatska. Ključna područja personalizirane medicine su identifikacija gena i molekularnih biljega povezanih s bolestima; farmakogenomika (procjena omjera koristi i rizika pri odabiru i doziranju pojedinih lijekova, predviđanje nuspojava) te procjena rizika za nastanak najčešćih bolesti moderne civilizacije i posljedično liječenje. Primjena rezultata farmakogenomike u konceptu personalizirane medicine ovisi o sposobnosti kliničkih laboratorija da kliničaru u pravom trenutku podastre precizne, točne, korisne informacije, koje bi omogućile propisivanje pravog lijeka u pravoj dozi za svakog pojedinog bolesnika. Ovaj pregled pobliže će prikazati domete farmakogenomike koja predstavlja značajnu kariku u personaliziranoj medicini i omogućuje transformaciju medicine od reaktivne ka proaktivnoj medicini te predstaviti preporuke CIPC-a (Clinical Pharmacogenetics Implementation Consortium) za testiranje odnosa gen/lijek 19 najčešćih lijekova koji se koriste u Hrvatskoj a u svrhu osiguranja odgovarajučeg liječenja.

Polifarmacija predstavlja značajan rizik za interakcije lijekova, lijekova i bolesti te lijekova i gena. Primjena farmakogenetičkih testiranja je još uvijek nedostatna. Posebna pažnja u farmakogenomici posvećena je enzimima citokroma P450 (CYP), koji sudjeluju u metabolizmu 70% -90% svih propisivanih lijekova. Najčešći enzimi CYP koji sudjeluju u metabolizmu lijekova su CYP2D6, CYP2C9, CYP2C19, CYP3A4 / 5. Prema genotipovima možemo predvijeti fenotipove od slabog do vrlobrzog metabolizatora lijekova-supstrata. Osim enzima uključenih u metabolizam sve više istraživanja uključuje polimorfizam gena prijenosnika lijekova preko različitih membrane, koji mogu značajno modulirati farmakokinetiku i interakcije. Dvije glavne obitelji prijenosnika relevantne za interakcije lijek-lijek-gen su ABC i SLC. Postoje dobro dokumentirani podaci o interakcijama lijekova na razini prijenosnika ABCB1, ABCC2, ABCG2, SLCO1B1 .

Napredak molekularnih dijagnostičkih metoda rezultirao je spoznajama da su u moduliranju varijabilnosti odgovora na primjenjenu farmakoterapiju uključeni brojni geni, te je uveden naziv farmakogenomika. Sa sve brojnijim farmakogenetičkim saznanjima o povezanosti kako pojedinih varijacija gena, tako i kombinacija genotipova s odgovorom na primjenu lijekova bilo u smislu izostanka terapijskog učinka ili nastanka ozbiljnih nuspojava pojavljuje se koncept personalizirane medicine. Udruživanjem spoznaja o farmakokinetici (FK) i farmakodinamici (FD) sa saznanjima iz farmakogenomike trajno unaprjeđujemo farmakoterapiju mijenjajući paradigmu po kojoj isti lijek i doza vrijede za sve, naglašavajući individualan pristup svakom pacijentu. Sve je više primjera koji dokazuju prednosti ovakovog individualiziranog pristupa u odnosu na pristup koji se temelji na rezultatima farmakokinetičkih studija na populacijskoj razini. Nova saznanja o utjecaju farmakogenomike na FK/FD kod sve većeg broja lijekova potaknula su i istraživanja razvoja lijekova prikladnih za specifične podskupine bolesnika, te se na tu temu povećao istovremeni interes farmaceutske industrije, akademske zajednice, zdravstvenih profesionalaca te regulatornih tijela.

CILJ ISTRAŽIVANJA Cilj je ovog specijalističkog rada pregledno prikazati podatke o farmakogenomici (PGx) implementirane u informacije o lijeku nacionalno odobrenih lijekova u Republici Hrvatskoj ako za njih postoji smjernica Konzorcija za implementaciju kliničke farmakogenetike (CPIC) i/ili smjernica Nizozemske farmakogenetičke radne skupine (DPWG). Dodatno je cilj prikazati podatke o farmakogenomici koje je utvrdila platforma The Pharmacogenomics Knowledge Base (PharmGKB) u informacijama o lijeku za lijekove odobrene u Europskoj uniji centraliziranim postupkom od strane Europske komisije (CP lijekovi) te za lijekove koje je u Sjedinjenim Američkim Državama odobrila Uprava za hranu i lijekove (FDA lijekovi), a koji su objavljeni na stranici PharmGKB‐a. MATERIJALI I METODE Pretražene su informacije o lijeku lijekova odobrenih nacionalnim postupkom u Republici Hrvatskoj dostupne u javnoj bazi Agencije za lijekove i medicinske proizvode (HALMED) za koje postoje CPIC i DPWG smjernice objavljene na stranici PharmGKB‐a. Podaci su analizirani s obzirom na vrstu regulatornog postupka kojim je lijek odobren, terapijsku skupinu lijekova, vrstu i broj različitih genomskih biomarkera navedenih u sažetku opisa svojstava lijeka kao i dio sažetka opisa svojstava lijeka u kojem se podatak nalazi te vrstu informacije prema PGx definiciji. Također, analizirana je i prisutnost genomskog biomarkera u uputi o lijeku. CP i FDA lijekovi analizirani su prema razini PGx podatka određenoj od strane PharmGKB‐a, vrsti genomskog biomarkera u informacijama o lijeku te ATK oznaci. REZULTATI Ako se u obzir uzima zasebno rješenje, analizirano je ukupno 265 nacionalnih lijekova. Utvrđeno je da je 14 djelatnih tvari sadržavalo podatak o farmakogenomici (PGx) informativnog karaktera, 11 PGx podatak koji je nalagao intervenciju, a 3 PGx podatak koji je preporučivao genetičko testiranje. Za dvije djelatne tvari nije postojao PGx podatak. Analizom vrste biomarkera koji je bio naveden u informacijama o lijeku utvrđeno je da se u nešto više od 50% slučajeva radilo o biomarkeru CYP2D6. Drugi po učestalosti bio je CYP2C19, koji se nalazio u informacijama o lijeku za 8 djelatnih tvari. Zatim su slijedili HLA‐B, koji se nalazio u informacijama o lijeku za 2 djelatne tvari te HLA‐A, CYP2C9, DPYD, TPMT i VKORC1 za po jednu djelatnu tvar. Najveći broj podataka o farmakogenomici nalazio se u dijelovima 4.5, 5.2 i 4.4 sažetka opisa svojstava lijeka. PGx podatak u uputi o lijeku bio je prisutan kod 7 djelatnih tvari. Najveći broj djelatnih tvari bio je iz skupine lijekova koji djeluju na živčani sustav. Analizirano je 88 djelatnih tvari CP lijekova te 243 djelatne tvari FDA lijekova. U informacijama o lijeku CP lijekova utvrđeno je 49 različitih biomarkera, a u informacijama o lijeku FDA lijekova njih 86. Najveći dio CP lijekova imao je podatak o farmakogenomici informativnog karaktera (40%) ili onaj koji je zahtijevao testiranje (39%), dok je najveći dio FDA lijekova imao podatak o farmakogenomici koji je nalagao intervenciju (44%) ili je bio informativnog karaktera (34%). Najveći broj CP i FDA lijekova pripadao je skupini za liječenje zloćudnih bolesti i imunomodulatora. ZAKLJUČAK Prema analiziranim podacima, velik broj podataka o farmakogenomici u odobrenim informacijama o lijeku isključivo je informativnog karaktera. Potrebno je uključivanje podataka koji omogućuju implementaciju farmakogenomike u kliničku praksu. Rezultati ovog rada u skladu su s rezultatima ispitivanja provedenih u drugim zemljama, koji također nalažu bolju implementaciju podataka o farmakogenomici u informacije o lijeku. OBJECTIVES The objective of this paper is to give an overview of the implementation of pharmacogenomic (PGx) information into the product information (PI) of nationally approved medicinal products in the Republic of Croatia for which there is a Clinical Pharmacogenetics Implementation Consortium (CPIC) and/or Royal Dutch Association for the Advancement of Pharmacy ‐ Pharmacogenetics Working Group (DPWG) guideline. An additional objective is to present an overview of pharmacogenomic information implemented in the product information of the medicinal products approved by the European Commission in the European Union (CP products) and medicinal products approved in the United States by the Food and Drug Administration (FDA products), as determined by the Pharmacogenomics Knowledge Base (PharmGKB) and published at PharmGKB website. MATERIAL AND METHODS The analysis included the product information of nationally approved medicinal products in Croatia listed in the publicly available database on the Agency for medicinal products and medical devices (HALMED) website, for which CPIC and DPWG guidelines were published on the PharmGKB website. The data were analyzed according to the marketing authorization procedure, therapeutic class, type and number of different genomic biomarkers present in the Summary of Product Characteristics (SmPC), section of the SmPC containing the information, as well as the type of information according to PGx definition. The presence of genomic biomarkers in the Patient Information Leaflet was also analyzed. CP products and FDA products were analyzed for the level of PGx information determined by PharmGKB, the type of genomic biomarker included in the product information and the ATC code. RESULTS Taking into account individual marketing authorizations, the total of 265 nationally approved medicinal products were analyzed. It was established that the PIs for 14 active substances contained informative pharmacogenomic (PGx) information, 11 had actionable PGx and 3 recommended genetic testing. Two active substances did not include a PGx. The analysis of the biomarker type stated in the product information showed that CYP2D6 was mentioned in 50% of the cases, followed by CYP2C19 in the product information for 8 active substances, HLA‐B in the product information for 2 active substances, and HLA‐A, CYP2C9, DPYD, TPMT and VKORC1, each mentioned in the product information of 1 active substance. Pharmacogenomic information was mostly included in the SmPC sections 4.5, 5.2 and 4.4. PGx information was included in the patient leaflets for 7 active substances. The majority of active substances analyzed belonged to the Nervous System group. Overall, 88 active substances of CP products and 243 active substances of FDA products were analyzed. For CP products, there were 49 different biomarkers included in the product information, in comparison with 86 for FDA products. Most of the CP products contained informative pharmacogenomic information (40%) or PGx requiring testing (39%), whereas most of the FDA products had actionable (44%) or informative (34%) pharmacogenomic information. Most of the CP and FDA products belonged to antineoplastic and immunomodulating agents. CONCLUSION According to results of this paper, a large amount of pharmacogenomic information in the approved product information is of informative nature only. There is a need to include information which would enable the implementation of pharmacogenomics in the clinical practice. The results of this study correspond with the results of studies conducted in other countries, which also propose the need for a better implementation of pharmacogenomic data in product information.

CYP2D6 je najopsežnije istražen polimorfni enzim koji sudjeluje u metabolizmu lijekova. Gen CYP2D6 je visoko polimorfan te je do danas otkriveno barem 112 alelnih varijanti koje kodiraju za inaktivni enzim, enzim sa smanjenom, normalnom ili povećanom enzimskom aktivnosti. Ovisno o kombinaciji alela i sposobnosti metabolizma lijekova, razlikujemo četiri metabolička fenotipa. Fenotip brzog metabolizma (EM) se smatra normalnom karakteristikom najvećeg dijela populacije, a javlja se u osoba s dva potpuno aktivna alela. Pojedinci s deficitom enzima (5 – 10 % pripadnika bijele rase) klasificiraju se kao spori metabolizatori (PM). Karakterizira ih nedostatak aktivnog alela te posljedično smanjena aktivnost enzima kodiranog tim alelom što rezultira nakupljanjem lijeka u organizmu i povećanim rizikom od pojave neželjenih učinaka lijeka. Među ostalima je enzimska aktivnost veoma varijabilna te se kreće od ekstremno visoke u vrlo brzih metabolizatora do smanjene u srednjih (intermedijarnih) metabolizatora. Osim značajne interindividualne varijbilnost, zabilježena je i značajna međuetnička varijabilnost u raspodjeli alelnih varijanti CYP2D6 koja podrazumijeva i postojanje "populacijski specifičnih" alelnih varijanti. Usprkos niskoj zastupljenosti enzima CYP2D6 (2 – 4 %) u odnosu na druge jetrene enzime CYP, ovaj enzim sudjeluje u biotransformaciji otprilike 20 – 25 % lijekova dostupnih na tržištu. Enzim CYP2D6 sudjeluje u metabolizmu velikog broja klinički značajnih lijekova, uključujući opioide (kodein, tramadol), antidepresive (amitriptilin, imipramin, fluoksetin, mianserin, venlafaksin), antipsihotike (aripiprazol, haloperidol, olanzapin, risperidon), antitustike (dekstrometorfan), β-blokatore (metoprolol, bufuralol, propranolol), protutumorske lijekove (tamoksifen) i antiemetike (ondansetron i tropisetron). Genotipizacija CYP2D6 je jedan od alata koji se koriste prilikom provođenja personaliziranog liječenja, što rezultira povećanjem djelotvornosti terapije i smanjenjem troškova liječenja. Procjenjuje se da bi takav personalizirani pristup liječenju na temelju genetskih značajki mogao biti relevantan za terapiju s 10 – 20 % lijekova. U ovom radu se razmatra utjecaj genetske varijabilnosti CYP2D6 na klinički ishod i terapijski učinak lijekova-supstrata CYP2D6. CYP2D6 is the most extensively studied polymorphic drug-metabolizing enzyme. CYP2D6 gene is highly polymorphic with at least 112 allelic variants discovered so far which encode an inactive enzyme, enzyme with reduced, normal or increased enzyme activity. Subjects are classified into four metbolic phenotype groups, depending on combination of alleles and the ability to metabolise xenobiotics. Extensive metabolism (EM) is considered to be a characteristic of the normal population and is found in subjects with two fully functional alleles. Individuals who are poor metabolisers have no active alleles and, therefore, lack functional enzyme activity which, consequently, results in accumulation of specific drug substrates and adverse drug reaction (ADR) occurrence. Among other subjects, enzyme activity is highly variable and ranges from extremely high in ultrarapid metabolizers to reduced in intermediate metabolizers. Apart from significant interindividual variability, a significant interethnic variability in distribution of CYP2D6 allelic variants has also been described, including existence of ‘population-specific’ allelic variants. Although CYP2D6 constitutes only 2 – 4 % of the total hepatic CYPs, it is one of the most important drug metabolizing enzymes due to its role in biotransformation of approximately 25 % of clinically used drugs. CYP2D6 enzyme is involved in the metabolism of numerous clinically significant drugs including opioids (codeine, tramadol), antidepressants (amitriptyline, imipramine, fluoxetine, mianserin, venlafaxine), antipsychotics (aripiprazole, haloperidol, olanzapine, risperidone), antitussives (dextromethorphan), beta-blockers (metoprolol, bufuralol, propranolol), anticancer drugs (tamoxifen) and antiemetic drugs (ondansetron i tropisetron). Genotyping of CYP2D6 is one of the tools used for implementation of personalized drug treatment which results in increased drug efficacy and decreased cost of ADR treatment. It is estimated that such personalized gene-based treatment could be relevant for 10 – 20 % of all drug therapy. In this thesis, the impact of the CYP2D6 genetic variability on the clinical outcome and therapeutic and/or adverse effects of drug-substrates of CYP2D6 is reviewed.

Anastrozole is the first choice treatment in postmenopausal women with hormone-dependent breast cancer (HR-BC). Cytokine P450 (CYP) and UDP-Glucuronosyltransferase (UGT) have a key role in deactivating and eliminating anastrozole. The negative effect of anastrozole on bone metabolism has been demonstrated, while the effect on Wnt inhibitors is still being investigated. The aim of the study was to determine the circulating levels of Wnt inhibitors in HR-BC patients treated with anastrozole and compare them with patients with breast cancer prior to the introduction of the adjuvant hormonal therapy. We also investigated the association of Wnt inhibitors with bone mineral density (BMD) and dietary and life habits of participants. Single nucleotide polymorphisms (SNPs) in genes encoding CYP and UGT may play a key role in individual responses to anastrozole. The aim was to determine the frequency of mutated CYP3A4*1B, CYP3A5*3 and UGT1A4*2 alleles and to investigate their correlation with the outcome of adjuvant hormonal treatment and the occurrence of side effects. Participants and methods: Cross- sectional study included 141 postmenopausal HR- BC patients, of whom 85 were treated with 1 mg of anastrozole, and 56 were included prior to the introduction adjuvant hormonal therapy. Serum concentrations of Wnt inhibitors were measured using commercially available ELISA assays. BMD ; g/cm2 was measured by dual-energy x-ray absorptiometry (DXA) imaging (Lunar Prodigy, GE Healthcare, SAD) and a self- reported questionnaire was used to investigate the lifestyle and eating habits of the examinees. Genomic DNA was isolated from peripheral blood samples using commercially available kit (QIAamp DNA Blood Midi Kit, Qiagen, Hilden, Germany). A real-time PCR was used for the detection of polymorphisms, with the application of the specific TaqMan® SNP Genotyping Assay (Applied Biosystems). Results: Serum levels of sclerostin were significantly higher in the group of participants treated with anastrozole (31.8 pmol/l) compared to the w/o anastrozole therapy group (24.1 pmol/l, p

Clopidogrel is an antithrombotic medicament from the group of thienopyrids, used for the prevention of blood cloths by the patients with acute coronary syndrome, patients after an ischemic stroke, myocardial infarction or by the patients with proven lower limb ischemia. Clopidogrel is a "prodrug", which means that it needs to be metabolised into an active metabolite before it stars working. The cytochrome P450 2C19 plays the main role in the bioactivation of clopidogrel. Individual enzymes of cytochrome complex are coded by genes, which are polymorphic. The function of P450 2C19 can differ by individuals, from the reason of gene polymorphism, therefore the bioactivation of clopidogrel can differ as well. There are few alleles of cytochrome P450 CYP2C19, which are connected with different therapeutic reactions on the submission of clopidogrel. Individuals with the allele CYP2C19*17 have an increased concentration of active metabolite and are at a higher risk of bleeding complications. The carriers of CYP2C19*2 a CYP2C19*3 alleles have a decreased metabolisation of clopidogrel into an active metabolite, which leads to insufficient antiaggregate effect and to increased appearance of cardiovascular complications. The allele CYP2C19*1 is connected with a fully functioning metabolism. In the group of 512 patients, we detected 191 carriers of the genotype *1/*1, 79 carriers of the genotype *2/*1, 12 carriers of the genotype *2/*2, 1 carrier of the genotype *3/*1, 43 carriers of the genotype *17/*17, 146 carriers of the genotype *17/*1 and 40 carriers of the genotype *17/*2. The stratification of the patients according to genotype CYP2C19 has a significant meaning for adjusting the right treatment strategy and it is a prevention of stroke and the risk of bleeding.

Individual variation in response to drugs is an important clinical problem, which ranges from failure to respond to the drug, over adverse reactions to drugs, to interactions among drugs being administered concurrently. Numerous findings indicate that the differences in the patients response on the same drug are caused by genetic variations. This is the subject of pharmacogenetics. Although pharmacogenetics generally equated with the concept of pharmacogenomics, pharmacogenetics is primarily related to variations in a single gene that influence the on drug response, while pharmacogenomics is a broader term, which studies how all of the genes (the genome) can influence responses to drugs. In focus of this paper will be individual variation in response to drugs arising from single nucleotide polymorphisms in genes encoding the drug target proteins, enzymes that metabolize drugs, drug transporters, and polymorphisms of genes responsible for toxicity and hypersensitivity to drugs. Determination of pharmacogenetic profile of patients could point out patients who are at increased risk of adverse drug effects (for which drug should be applied at lower doses or other drugs can be used) and those in which are likely to achieve the desired therapeutic effect, and so to enable individualization of therapy. Individualne varijacije u odgovoru na lekove važan su klinički problem i mogu dovesti do potpunog odsustva reakcije na lek i pojave neželjenih reakcija na lekove. Brojni nalazi ukazuju da su razlike u odgovoru bolesnika na isti lek uslovljene genetskim varijacijama, i predmet su istraživanja farmakogenetike. Iako se farmakogenetika uglavnom izjednačuje sa pojmom farmakogenomika, farmakogenetika se uglavnom odnosi na varijacije u jednom genu koje utiču na odgovor na lek, dok je farmakogenomika šira oblast, koja ispituje kako svi geni u genomu povezani sa metabolizmom određenog leka mogu uticati na odgovor na dati lek. U ovom radu prevashodno će biti opisane individualne varijacije u odgovoru na lekove koje nastaju usled jednonukletidnih polimorfizama u genima koji kodiraju ciljne proteine lekova, enzime koji metabolišu lekove, transportere lekova, kao i polimorfizmi gena koji su odgovorni za toksičnost i preosetljivost na lekove. Određivanje farmakogenetskog profila bolesnika moglo bi da ukaže na bolesnike koji su u povećanom riziku od pojave neželjenih efekata lekova (kod kojih bi trebalo da se primene niže doze ili drugi lekovi) i na one kod kojih će se najverovatnije postići željeni terapijski efekat, odnosno da omogući individualizaciju terapije.

Farmakogenetika je disciplina koja proučava interindividualne razlike u slijedu DNA, a koje dovode do varijabilnog odgovora na terapiju lijekovima. Krajnji cilj farmakogenetike jest uspostava liječenja na principima tzv. personalizirane medicine, u kojoj se svakoj osobi, odnosno bolesniku, pristupa kao pojedincu. Način na koji se to postiže je da mu se metodama molekularne dijagnostike odredi farmakogenetski status i tako pruži mogućnost prilagodbe liječenja po njegovoj mjeri. Cilj ovog rada je sustavno prikazati ažurne informacije i smjernice farmakogenetskih principa liječenja, približiti ih i učiniti dostupnima kliničarima i drugim korisnicima, te tako potaknuti širu upotrebu farmakogenetike u kliničkoj praksi. Poseban naglasak je stavljen na kliničku interpretaciju smjernica na kojima se temelji odabir i doziranje lijekova s varijabilnim učinkom i nuspojavama, a koje ovise o rezultatu farmakogenetskog testiranja. Osim sustavnog prikaza smjernica iz relevantnih izvora koje su danas dostupne i primjenjive te prikaza potencijala farmakogenetike i farmakogenomike u fazama razvoja lijeka, načinjen je i kritički osvrt farmakogenetskih načela s obzirom na ograničenja koja otežavaju njihovo uvođenje u kliničku praksu. Među tim ograničenjima su cijena molekularnih testova, njihova relativna nedostupnost te složenost interpretacija rezultata. Ovaj je rad osnova za u skoroj budućnosti planiranu izradu web stranice – priručnika na hrvatskom jeziku, koja bi kliničarima i drugim zainteresiranim korisnicima omogućila jednostavniji pristup informacijama o farmakogenetskim načelima liječenja te dostupnosti pojedinih pretraga. U zaključku, farmakogenetika i farmakogenomika pružaju velike mogućnosti za poboljšanje kvalitete zdravstvene skrbi za svakog bolesnika. Međutim, one imaju smisla dok su ekonomski isplative, tj. određivanje terapeutskog profila ne prelazi financijska sredstva koja bi bila utrošena korištenjem konvencionalnih terapeutskih postupaka. Pharmacogenetics is a discipline that studies interindividual differences in DNA which cause a variable drug therapy response. The ultimate goal of pharmacogenetics is the establishment of treatment on the principles of so-called Personalized medicine, in which every person or patient is managed as an individual. The way in which it is achieved - is to determine its pharmacogenetic status by molecular diagnostic methods and thus provide the possibility of adjusting the treatment to each patient’s needs. The aim of this paper is to systematically present pharmacogenetic principles up-to-date information and treatment recommendations, to make them available to clinicians and other users, thus promoting the use of pharmacogenetics in clinical practice. Particular emphasis has been put on the clinical interpretation of the guidelines which are the basis of drug choice and dosage, with variable outcomes and side effects that depend on the pharmacogenetic testing result. In addition to a systematic overview of the relevant sources available and applicable today, as well as the pharmacogenetics and pharmacogenomic potential in drug development phases, a critical review of the pharmacogenetic principles has been made, with regard to the constraints that make them difficult to be introduced into clinical practice. Among these limitations are the price of molecular tests, their relative inaccessibility, and the complexity of the interpretation of results. This work is the basis for the construction of a Croatian language website - manual in the near future, which would provide easier access to information on pharmacogenetic treatment principles and specific tests availability to clinicians and other interested users. In conclusion, pharmacogenetics and pharmacogenomics provide great opportunities to improve the quality of health care for each patient. However, they make sense while being economically viable, i.e. determining a therapeutic profile does not exceed the financial resources that would be spent using conventional therapeutic procedures.

Shizofrenija označuje skupino psihotičnih motenj, ki sodijo med hude duševne motnje, nevrodegenerativne in progresivne narave. Prevalenca shizofrenije v splošni populaciji je od 0.5 do 1.0 %, značilno pa se pojavi pri mladih odraslih (moški: med 17. in 25. letom starosti, ženske: med 25. in 35. letom). Pri shizofreniji se pojavijo motnje mišljenja, zaznavanja, čustvovanja in vedenja, kakor tudi kognitivne motnje. Značilen je delno ali popolnoma okrnjen stik z realnostjo. Zdravimo jo na različne načine, najpogostejša zdravila pri shizofreniji so antipsihotiki. V raziskavi smo preučevali vpliv polimorfizmov adenozinskih (ADORA) in dopaminskih (DRD) receptorjev na pojav produktivne psihopatološke simptomatike pri shizofreniji, kakor tudi vpliv na nastanek ekstrapiramidne simptomatike ob prejemanju antipsihotične terapije. Naša raziskava je potekala v dveh delih. V eno skupino so bili vključeni pacienti v akutni fazi bolezni, prva epizoda ali akutni relaps shizofrenije, v drugo skupino pa pacienti na vzdrževalnem zdravljenju v stabilni fazi shizofrenije. Pacienti so se z vključitvijo v raziskavo strinjali s podpisom. V obeh vzorcih smo ocenjevali psihopatološko in ekstrapiramidno simptomatiko ob prejemanju antipsihotikov. Pri vseh bolnikih smo opravili genotipizacijo na skupno 22 izbranih polimorfizmih v ADORA1, ADORA2A, ADORA3, DRD1 in DRD2. Polimorfizmi so bili izbrani s pomočjo podatkovne zbirke SNP ameriškega National Center for Biotechnology Information (NCBI), HapMap, ter »tagger pairwise« metodo programskega kompleta HaploView. Vse dobljene podatke smo statistično obdelali s programskim paketom STATISTICA 7 za Windows. Za preliminarno preverjanje statistične pomembnosti porazdelitve kvantitativnih kliničnih spremenljivk med različnimi genotipi smo uporabili analizo variance (ANOVA), analizo kovariance pa smo opravili z metodo MANCOVA. Preverili smo Hardy-Weinbergovo ravnotežje, kakor tudi vezavno neravnovesje (LD) med SNP. Analizo haplotipov smo opravili s programom R-project. V raziskavo je bilo vključenih 74 pacientov v akutni fazi shizofrenije in 127 pacientov v neakutni fazi, ki so prejemali vzdrževalno antipsihotično terapijo. S psihopatološko simptomatiko pri shizofreniji so bili povezani polimorfizmi ADORA1 rs3766566, ADORA2A rs2236624 in ADORA2A rs2298383, ter haplotipi ADORA1 CTCAACG, ADORA3 TGTTGT, DRD1 TG in DRD1 CA. Z ekstrapiramidnimi neželenimi učinki antipsihotikov so bili povezani polimorfizmi ADORA2A rs35320474 (tardivne diskinezije), ADORA2A rs5751876 (akatizija), DRD1 rs4532 (akatizija) in DRD1 rs5326 (akatizija), ter haplotipi ADORA1 CTTGATG (akatizija), ADORA1 CTCAACG (akatizija), ADORA2A TCCTG (parkinsonizem), ADORA3 TGTTGT (parkinsonizem), ADORA3 CACTAC (akatizija), ADORA3 CACTAT (akatizija in tardivne diskinezije), DRD1 TG (parkinsonizem, akatizija, tardivne diskinezije), DRD1 TA (akatizija) in DRD1 CG (akatizija). Naša raziskava predstavlja prvo tovrstno študijo polimorfizmov adenozinskih receptorjev v možganih pri akutnih in neakutnih pacientih s shizofrenijo, s katero smo ugotovili, da bi lahko opazovani polimorfizmi v ADORA1, ADORA2A in ADORA3 z nevromodulacijo vplivali tako na psihopatološko simptomatiko, kot na ekstrapiramidno simptomatiko, povzročeno z antipsihotiki. Schizophrenia is a severe mental disorder of neurodegenerative and progressive nature. The prevalence of schizophrenia in general population is 0.5-1.0 %, and it typically strikes individuals in late adolescence or early adulthood (age span 17 – 25 and 25 – 35 for males and females, respectively). The symptoms are classified as positive (delusions, hallucinations), negative (loss of motivation, drive, diminished social interactions and appropriate behavior, flattening of mood, difficulties in abstract thinking), and other (formal thought disorder, depression, anxiety, poor concentration, poor sleep, cognitive impairment). It is usually treated with antipsychotics. We studied the effect of polymorphisms of adenosine (ADORA) and dopamine (DRD) receptors on schizophrenia psychopathology, as well as the occurence of extrapyramidal adverse effects of antipsychotics. There is a substantial body of evidence that biochemically adenosine modulates neurotransmission of dopamine that is an established key-neurotransmitter for the development of psychosis. Additionally, it has been shown that adenosine and dopamine receptors are co-localised as heterodimers on neuron cell membranes. The study was conducted in two parts. One sample consisted of acute patients (first episode or acute relapse of psychosis), and the second sample consisted of non-acute schizophrenia patients that were receiving long-term antipsychotic treatment and were in a stable phase of the illness. All patients agreed to participate with a written informed consent. All included patients were assessed for psychopathology, extrapyramidal symptoms, and were subsequently genotyped for 22 selected SNPs (using NCBI, HapMap and tagger-pairwise HaploView) in ADORA1, ADORA2A, ADORA3, DRD1 and DRD2. Data was statistically analysed using ANOVA, MANCOVA, Hardy-Weinberg equilibrium, and linkage disequlibrium. The haplotype analysis was conducted using R-project programme. We included 74 acute and 127 non-acute patients. Schizophrenia psychopathology was associated with SNPs ADORA1 rs3766566, ADORA2A rs2236624, and ADORA2A rs2298383, and haplotypes ADORA1 CTCAACG, ADORA3 TGTTGT, DRD1 TG, and DRD1 CA. Extrapyramidal adverse effects were associated with ADORA2A rs35320474 (involuntary movements), ADORA2A rs5751876 (akathisia), DRD1 rs4532 (akathisia), and DRD1 rs5326 (akathisia), and with haplotypes ADORA1 CTTGATG (akathisia), ADORA1 CTCAACG (akathisia), ADORA2A TCCTG (parkinsonism), ADORA3 TGTTGT (parkinsonism), ADORA3 CACTAC (akathisia), ADORA3 CACTAT (akathisia and involuntary movements), DRD1 TG (parkinsonism, akathisia, involuntary movements), DRD1 TA (akathisia) in DRD1 CG (akathisia). This is the first comprehensive study of adenosine receptors' polymorphisms in (human) brain in acute and non-acute schizophrenia patients that has shown associations between ADORA1, ADORA2A and ADORA3 with psychopathology and adverse effects of antipsychotics.

/SUMMARY Background: The clinical efficacy of tamoxifen therapy may be modified by the drug-metabolizing enzymes and transporting molecules involved into the pharmacokinetics of tamoxifen. The aim of this study was to evaluate the association of CYP2D6, ABCB1 polymorhisms and comedication with efficacy and safety of tamoxifen treatment. Methods: Totally 258 women with hormonal positive breast carcinoma were retrospectively evaluated in relation to CYP2D6, ABCB1 polymorphisms and comedication. Results: CYP2D6 polymorphisms or co-medication affecting CYP2D6 activity demonstrated no statistically significant effect on the efficacy of tamoxifen therapy or adverse event incidence; there was only a trend towards shortening the time to event (TTE) in CYP2D6 poor metabolizers. ABCB1 polymorphism rs2032582 was not associated with clinical outcomes, while a trend towards an increase of TTE in variant allele carriers was noted. The ABCB1 polymorphism rs1045642 demonstrated statistical significance in premenopausal patients (p = 0.0012, HR 0.69 (95% CI 0.21 to 2.31), and its significant association was noted with gynaecological /vasomotor adverse events (p = 0.0221, HR = 1.0588), with no evidence of the influence on the incidence and onset of venous complications. Conclusions: Although this work did not show...

Razvoj nove discipline farmakogenetike/farmakogenomike omogućio je bolje razumijevanje genetičke predispozicije za razvoj neočekivanih reakcija na primijenjeni lijek i posljedično unaprijedio individualizaciju farmakoterapije. Postoje genetički određene interindividualne razlike u metaboličkom i transportnom kapacitetu, a time i kinetici lijekova i drugih ksenobiotika. Osim nuspojava lijekova koje su ovisne o dozi važne su nuspojave posredovane imunosnim reakcijama, a povezane su s varijabilnosti u sustavu HLA. Individualna osjetljivost prema toksičnim učincima farmakoterapije, ovisna o biotransformacijskim procesima, povezuje se s genskim polimorfizmima enzima faze I. i II. te transporterima ABC i SLC. Genski polimorfizmi također mogu imati ključnu ulogu u interakcijama lijekova. Povezivanje farmakogenomike s farmakovigilancijom ima važnu ulogu i u praćenju pojavnosti nuspojava poslije stavljanja lijeka na tržište. Regulatorna tijela uvođenjem farmakogenomičkih informacija (informacije o genomskim biljezima) u Sažetku o proizvodu (SPC) nastoje istaknuti ulogu i važnost genomskih varijabilnosti u farmakoterapiji. Također se navodi kojoj kategoriji te farmakogenetičke informacije pripadaju, tj. smatra li se testiranje obvezatnim, ima li ulogu preporuke ili je informativnog karaktera. Radi prikupljanja i implementacije znanja ustanovljeni su i različiti konzorciji među kojima prednjači Clinical Pharmacogenetics Implementation Consortium – CPIC, kao zajednički projekt The Pharmacogenomics Knowledge Base (PharmGKB) i The Pharmacogenomics Research Network koji je okupio eminentne stručnjake iz područja farmakogenetike/farmakogenomike. Glavni je cilj CPIC-a izdavanje recenziranih, ažuriranih, temeljenih na dokazima, slobodno dostupnih uputa i smjernica za primjenu lijeka prema rezultatu farmakogenetičkih analiza. Te će smjernice svakako ubrzati prijenos znanja u kliničku primjenu., The development of a new discipline, pharmacogenetics/pharmacogenomics, has enabled a better understanding of genetic predispositions for unexpected drug reaction developments, and consequently it improves the individualization of pharmacotherapy. There are certain genetic individual variations in metabolic and transport capacity, and thus the kinetics of drugs and other xenobiotics. In addition to adverse drug reactions (ADR) that are dose-dependent, significant ADR are mediated by immune reactions and are associated with variations in the HLA system. The individual sensitivity to the pharmacotherapy toxic effects, dependent on biotransformation processes, is associated with the genetic polymorphism of the phase I and phase II enzymes and ABC and SLC transporters. Genetic polymorphisms can also play a key role in drug interactions. Connecting pharmacogenomics to pharmacovigilance has a significant role in monitoring ADR during the post-marketing period. Regulatory authorities highlight the role and importance of genomic variability in pharmacotherapy by introducing pharmacogenomics (information on genomic markers) in the SPC. It also states into which category pharmacogenetics information belongs, whether pharmacogenetic testing is required, recommended or informative. In order to collect and implement pharmacogenomic knowledge different consortia have been established, including prestige Clinical Pharmacogenetics Implementation Consortium (CPIC), as a joint project of The Pharmacogenomics Knowledge Base (PharmGKB) and the Pharmacogenomics Research Network, which brought together eminent experts in the field of pharmacogenetics/pharmacogenomics. The main objectives of CPIC’s is to provide peer-reviewed, updated, evidence-based, freely available recommendations and guidelines for drug use according to results of pharmacogenetic analysis. These guidelines will certainly accelerate the knowledge transfer to the clinical use.

Interindividual variability in drug metabolism is an important cause of adverse drug reactions and variability in drug efficiency. Polymorphisms of cytochrome P450 (CYPs) genes have a significant effect on drug metabolism and toxicity. This review brings an update about how genetic polymorphisms of CYP2C8 and CYP2C9 enzymes affect the disposition and clinical outcomes of ibuprofen and diclofenac, two of the most common pain relievers. The most common side effects associated with the influence of CYP2C8*3 and CYP2C9*2*3 variants on ibuprofen and diclofenac pharmacokinetics are hepatotoxicity and gastrointestinal bleeding. CYP genotyping may therefore identify patients at increased risk of these adverse reactions, and these patients could have their doses adjusted or start receiving another NSAID that does not share the same metabolic pathways with ibuprofen or diclofenac. However, before genotyping is introduced into regular clinical practice, more research is needed to evaluate the effectiveness of this strategy in improving treatment with ibuprofen and diclofenac., Interindividualne razlike u metabolizmu mogu biti važan čimbenik nastanka nuspojava te varijabilnosti u učinkovitosti lijeka. Polimorfizmi gena koji kodiraju metaboličke enzime citokrome P450 (CYP) mogu imati značajan učinak na metabolizam lijeka i toksičnost. Ovaj pregled donosi spoznaje o tome kako polimorfizam enzima CYP2C8 i CYP2C9 utječe na bioraspoloživost i kliničke ishode liječenja ibuprofenom i diklofenakom, koji se svrstavaju među najčešće propisivane nesteroidne protuupalne lijekove. Hepatotoksičnost i gastrointestinalno krvarenje najčešće su nuspojave povezane s utjecajem varijanti CYP2C8*3 i CYP2C9*2*3 na farmakokinetiku ibuprofena i diklofenaka. Na osnovi rezultata genotipizacije CYP-a mogu biti prepoznati pacijenti koji imaju povećani rizik od razvoja nuspojava te im je nužno prilagoditi dozu lijeka ili odabrati drugi lijek koji ne dijeli isti metabolički put. Osim enzima CYP, značajan utjecaj imaju i polimorfizmi gena koji kodiraju fazu II metabolizma, osobito enzimi UGT, te transporteri, poput ABCC2, koji mogu modulirati ne samo transport na barijeri jetre i žuči nego i izlučivanje bubrezima. Stoga je u budućim istraživanjima nužan poligenski pristup. Prije uvođenja genotipizacije u redovitu kliničku praksu potrebno je provesti daljnja istraživanja koja će uključivati veće fenotipski dobro definirane skupine ispitanika za procjenu učinkovitosti ove strategije u poboljšanju liječenja ibuprofenom i diklofenakom. Zbog značajne međuetničke razlike u učestalosti polimorfizama gena CYP istraživanja treba provesti među različitim rasama i populacijama.

Cilj istraživanja Cilj ovoga specijalističkoga rada je prikupiti, kroz pretraživanje znanstvene i stručne literature, i kritički procijeniti relevantne podatke koji se odnose na utjecaj genskih varijanti onih gena čiji proteinski produkti utječu na učinkovitost terapije statinima. Materijali i metode Tijekom izrade i pisanja ovoga specijalističkoga rada sustavno je pretražena znanstvena i stručna literature iz područja farmakogenetike, farmakogenomike, farmakoterapije i kardiologije, kako domaća tako i strana. Kao literaturni izvori korišteni su i odgovarajući priručnici, knjige i udžbenici koji se kao nastavna literatura koriste na studijima medicine i farmacije. Za detaljniji opis pojedinih lijekova iz skupine inhibitora hidroksi-metil-glutaril-koenzim-A-reduktaze korišteni su podaci iz Sažetka opisa svojstava lijeka te baze interakcija Lexicomp i Drugs.com. Od dostupnih baza podataka pretraživane su baze podataka Medline i PubMed za ključne riječi: inhibitori hidroksi-metil-glutaril-koenzim-A-reduktaze, statini, farmakogenetika, farmakogenomika, genski polimorfizam. Zaključak Do danas je identificirano više gena čiji proteinski produkti imaju utjecaja na farmakokinetiku i farmakodinamiku statina. Za kliničku praksu najznačajnije su varijante (polimorfizmi) onih gena koji kodiraju proteine uključene u farmakokinetiku statina tj. apsorpciju, distribuciju, metabolizam i izlučivanje, a to su enzimi iz porodice CYP-a, P-glikoprotein i organski anionski transportni polipeptid (OATP). Lovastatin, atorvastatin i simvastatin se najviše metaboliziraju putem CYP3A4 i CYP3A5, a fluvastatin putem CYP2C9 i CYP2C8. Metabolizam ovih lijekova može biti djelomično posredovan i putem CYP2D6 ili nekoliko glukuronozil-transferaza (UGT1A1, UGT1A3, UGT2B7). Pravastatin i rosuvastatin imaju minimalnu interakciju s metaboličkim enzimima i uglavnom se izlučuju nepromijenjeni. Zbog toga se smatra da su pravastatin i rosuvastatin najmanje pod utjecajem genskih varijacija metaboličkih enzima. Kod pacijenata koji su na politerapiji značajno se povećava vjerojatnost neželjenih interakcija lijekova, posebice u slučajevima onih pacijenata koji su zbog genskih polimorfizama spori ili vrlo brzi metabolizatori. Ako se kod takvih pacijenata istovremeno sa statinima primjenjuju i lijekovi koji su inhibitori ili induktori enzima iz obitelji CYP-a, mogućnost nuspojava i toksičnosti značajno raste. Polimorfizam 3435T na MDR1 ili ABCB1 genu povezan je sa smanjenom ekspresijom P-glikoproteina. Inhibicija i indukcija P-glikoproteina je novije otkriven mehanizam koji utječe na interakcije lijekova. Osobe koje su nositelji T/C ili C/C genotipa kod polimorfizma gena SLCO1B1 (rs4149056 (521T>C)) imaju lošiji odgovor na terapiju statinima te imaju veći rizik za razvoj nuspojava za razliku od osoba koji su nositelji T/T genotipa. Genski polimorfizmi koji imaju utjecaj na farmakodinamiku statina od manjeg su kliničkog značaja. Poznavanje genskih polimorfizama ne može apsolutno prognozirati kako će pacijent reagirati na propisanu terapiju, ali može biti dobar putokaz za opreznije doziranje pojedinih lijekova i učestalije praćenje rizičnih pacijenta. Objectives The aim of this work is to search the literature and collect a large number of data for assesment of the impact of gene polymorphisms of the genes whose protein products influence the effectiveness of statin therapy. Materials and Methods During the writing and preparation of this thesis, literature by Croatian and foreign authors in the fields of pharmacogenetics, pharmacogenomics, pharmacotherapy and cardiology was systematically reviewed. Manuals, books and textbooks, which are used in studies of pharmacy and medicine were also used. For a more detailed description of certain drugs from the group of inhibitors of hydroxy-methyl-glutaryl-coenzyme-A-reductase, data from the SPC, and base interaction Lexicomp and Drugs.com were used. From available databases, the databases Medline and PubMed were searched for the following keywords: hydroxy-methyl-glutaryl-coenzyme-A-reductase inhibitors, statins, pharmacogenetics, pharmacogenomics, gene polymorphism. Conclusion Several genes whose protein products may affect pharmacokinetics and pharmacodynamics of statins have beeen identified so far. In clinical practice, it is the variants (polymorphisms) of genes that encode proteins involved in the pharmacokinetics of statins , i.e. , absorption, distribution, metabolism and excretion, that are the most important. They are enzymes of the CYP family, P-glycoprotein and organic anion transporting polypeptide (OATP). Lovastatin, atorvastatin and simvastatin are mostly metabolised by cytochrome CYP3A4 and CYP3A5, and fluvastatin through CYP2C9 and CYP2C8. The metabolism of these drugs may be partially mediated through the CYP2D6 or several glucuronosyltransferase (UGT1A1, UGT1A3, UGT2B7). Pravastatin and rosuvastatin have minimal interaction with metabolic enzymes and are mainly excreted unchanged. Therefore, it is considered that pravastatin and rosuvastatin are least affected by genetic variations of metabolic enzymes. In patients with polytherapy the possibility of drug interactions increases significantly, especially in the case of patients who are, due to genetic polymorphism, slow or very fast metabolizers. If inhibitors or inducers of CYP enzymes are coadministrated with statins, the possibility of side effects and toxicity increases significantly. 3435T polymorphism of the MDR1 (ABCB1) gene is associated with reduced expression of P-glycoprotein. Inhibition and induction of P-glycoprotein is a recently discovered mechanism that may affect drug interactions. People who are carriers of the T/C or C/C genotype of SLCO1B1 gene polymophysm (rs4149056 (521T>C)) have a poorer response to therapy with statins, and are at greater risk of developing side effects than carriers of the T/T genotype. Genetic polymorphisms that have an impact on pharmacodinamics of statins are clinically less significant. Knowledge of the genetic polymorphisms can not absolutely predict how a patient will respond to prescribed therapy, but can be a good indicator of the need for cautious dosage of certain drugs and frequent monitoring of patients at risk.

Mercaptopurine (6-mercaptopurine) together with azathioprine and 6-thioguanine belong to a group of widely used chemotherapeutics and immunosuppressants. However, insufficient therapy outcome or severe adverse effects such as myelosuppression are still being reported. Technological progress in DNA and RNA sequencing facilitates effective identification of causative genes responsible for the therapy failure, i.e., description of genetic variants for enzymes involved in metabolism of physiological purines as well as thiopurine drugs. Variants of these enzymes may substantially alter concentrations of cytotoxic forms of thiopurines, which affect therapy success rates. Currently, a number of mutations in genes that play role in thiopurine metabolism have been annotated. Nevertheless, molecular mechanisms underlying the effect of these mutations are not fully elucidated. Knowledge of 3D structure for these enzymes may shed light on the effect of the genetic variants to protein function and mechanisms modulating therapeutic efficacy of thiopurines. This thesis focuses mainly on biochemical and structural characterization of thiopurine-S-methyltransferase, fosfatase NUDT15 and cytosolic 5′-nucleotidase II. It summarizes current state of knowledge and emphasizes the importance of structural biology methods for...

Primjena statina u posljednja dva desetljeća značajno je smanjila pobol i smrtnost od kardiovaskularnih bolesti (KVB) koje u svojoj podlozi imaju aterosklerozu. Taj se učinak prvenstveno temelji na smanjenju serumskog kolesterola male molekularne mase (LDL-kolesterol) čija se razina efikasnije snižava većim dozama statina. U brojnim studijama dokazana je korist od statina u primarnoj i sekundarnoj prevencijii KVB, a smanjenje rizika kardiovaskularnih incidenata linearno ovisi u smanjenju serumskog LDL-kolesterola. Međutim, postoji velika interindividualna varijabilnost u odgovoru na terapiju statinima. Uz brojne poznate čimbenike kao što su dob, spol, funkcija jetre i bubrega, istodobna primjena drugih lijekova, novija istraživanja pružaju dokaze da su genetske varijacije dobrim dijelom odgovorne za razlike u terapijskoj učinkovitosti statina. Sve donedavno farmakogenetičke studije su se uglavnom bavile pojedinačnim genima u istraživanju ovih učinaka. Kako su studije s ciljnim genima mogle objasniti samo mali dio opaženih varijacija s inkonzistentnim rezultatima pristupilo se studijama cijelog genoma te je nastala nova znanost – farmakogenomika. Akumulacija spoznaja o povezanosti genoma s bolestima i interakciji genoma i lijekova vodi prema personaliziranoj medicini koja je usmjerena k pojedinoj osobi primjenom preventivnih i prediktivnih DNA biljega. Cilj ovog rada je dati pregled spoznaja o farmakogenetici statina, rezultate studija cijeloga genoma i perspektive razvoja farmakogenomike i individualizirane medicine., Over the last two decades the incidence and mortality of cardiovascular diseases (CVD) have been significantly lowered by use of statins. Low density lipoprotein (LDL) cholesterol is the main goal of statin therapy which efficacy is dose dependent. The results of many studies have shown benefits of statins in primary and secondary prevention of CVD and linear relationship between risk reduction of cardiovascular events and serum cholesterol rate lowering have been observed. However, there is large interindividual variability in response to statin therapy. There are many factors involved in variations in lipid responses to statins such as age, gender, renal and hepatic function and interaction with other drugs. The results of recent studies have shown genetic variations as a key role player in variability of statin responses. Until recently, pharmacogenetic studies have used mainly candidate gene approaches to investigate these effects. Since candidate gene studies explain only a small part of the observed variation and results have often been inconsistent, genome-wide association (GWA) studies have been introduced giving birth to pharmacogenomics. Accumulation of knowledge on genom-disease and genomedrug interactions leads to personalized medicine focused to individual person by use of preventive and predictive DNA markers. The aims of this review are to present facts on phamacogenetics of statins, results of GWA studies and future perspectives on pharmacogenomics and its translation into individualized medicine.

Farmakogenomika proučava odnos između genetičkog sklopa individue i njegovog odgovora na lekove i jedan je od stubova personalizovane medicine. Dosadašnji princip lečenja da se standardna doza leka daje svim pacijentima sa istom dijagnozom po unapred utvrđenom protokolu se napušta. Za veliki broj pacijenta ta doza leka često nije efikasna i/ili sigurna za upotrebu. Cilj farmakogenomičkih studija je da identifikuju farmakogenomičke markere, varijacije u genomu koje mogu pouzdano da predvide odgovor na terapiju, što je osnov za individualizaciju terapije. Model sistemi bolesti za analizu farmakogenomičkih markera korišćeni u ovom radu su dečja akutna limfoblastna leukemija (ALL) i reumatoidni artritis (RA). Lečenje ovih bolesti uključuje imunosupresivne i citotoksične lekove 6-merkaptopurin (6-MP), metotreksat (MTX), antibiotik baktrim, antimikotik nistatin, kao i anti-TNF lekove. Genetičke varijacije koje modulišu metaboličke puteve povezane sa ovim lekovima su kandidati za farmakogenomičke markere. Cilj ove studije je da ispita učestalosti genetičkih varijanti u genima TPMT, ITPA, ABCB1, ABCC4, TYMS, MTHFR, SLC19A1, DHFR, TNF i IL-6, kao i da oceni farmakogenomički potencijal ovih varijanti u srpskoj populaciji. Biće ispitana i uloga ovih farmakogenomičkih markera kao faktora rizika za razvoj dečje ALL. Ispitaće se i uticaj terapije održavanja, gde okosnicu terapije čine lekovi 6-MP i MTX, kao i pola i uzrasta dece sa ALL na ekspresiju gena TPMT. Biće funkcionalno okarakterisane varijante u genu TPMT, potencijalni modulatori ekspresije gena TPMT, sa posebnom pažnjom na ulogu VNTR regiona u promotoru gena TPMT. U studiju je bilo uključeno 174 pedijatrijskih ALL pacijenata, 73 RA pacijenata i 104 kontrolnih zdravih ispitanika. Genetičke varijacije u svim gorepomenutim genima su određene metodama baziranim na PCR-u... Pharmacogenomics is focused on exploring the relation between the genomic signature of an individual and their drug response. It is the basis for implementation of personalized medicine. The old-fashioned therapeutic paradigm of »one protocol dose fits all patients with the same diagnosis« is getting abandoned. The standard drug dose is often not efficient and/or safe for many of patients. Pharmacogenomic studies identify pharmacogenomic markers, genomic variations that could reliably predict the drug response, which is the basis for therapy individualization. In order to analyze pharmacogenomic markers, childhood acute lymphoblastic leukemia (ALL) and rheumatoid arthritis (RA) are used as disease model systems. ALL and RA therapy protocols include cytotoxic and immunosuppressive drugs 6-mercaptopurine (6-MP) and methotrexate (MTX), antibiotic bactrim and antimycotic nystatin, as well as anti-TNF drugs. Genetic variations that modulate metabolic pathways related to these drugs are candidate pharmacogenomic markers. The aim of this study is to analyze frequencies of genetic variants in TPMT, ITPA, ABCB1, ABCC4, TYMS, MTHFR, SLC19A1, DHFR, TNF and IL-6 genes in Serbian population and to evaluate the pharmacogenomic potential of these variants. Also, the role of these pharmacogenomic markers as risk factors for development of childhood ALL will be assessed. Influence of the maintenance therapy, which includes 6-MP and MTX as most important drugs, as well as the age and gender of patients will be analyzed in regard to TPMT gene expression. Functional assays will be carried out in order to identify potential modifiers of TPMT expression with a special focus on VNTR region in promoter of TPMT gene. In this study, 174 pediatric ALL patients, 73 RA patients and 104 healthy subjects were enrolled. Genetic variants in above-mentioned genes were detected using PCR-based methodology...

Farmakogenomika istražuje genetičku osnovu raznolikosti u učinkovitosti i nuspojavama lijeka (NL) s glavnim ciljem pronalaženja biomarkera koji će predvidjeti varijabilnost na farmakokinetskoj razini u vidu varijabilnog transporta i/ili metabolizma lijeka, te na farmakodinamskoj razini gdje su varijabilnosti receptora ali i drugih ciljnih molekula djelovanja lijeka odgovorne za razvoj neželjenih učinaka. NL mogu rezultirati značajnim pobolom pa i smrtnošću a njihovo zbrinjavanje predstavlja signifikantan trošak za zdravstveni sustav. 90 % NL nastaje pri primjeni preporučenih doza. Uz to prema objavljenim podacima NL su odgovorne za 7% hospitalizacija a broj se penje i >30% u starijoj populaciji. U mnogim farmakoterapijskim područjima primjenjuju se standardni načini doziranja. Liječenje je uglavnom usmjereno prema bolesti a manje prema pojedinom bolesniku i njegovim specifičnostima. Istraživanja u farmakogenomici su rezultirala primjerima o važnom utjecaju naslijeđa na učinkovitost i toksične učinke lijeka. Ipak široka primjena saznanja iz farmakogenomike s ciljem individualizacije i optimizacije farmakoterapije još nije sasvim zaživjela. Posljednjih godina osnovano je više društava s ciljem promicanja znanja o farmakogenetičkim biomarkerima, te je sve više smjernica kako pretvoriti nalaze genetičkih analiza u algoritme za odabir najprikladnijeg lijeka i doze za određene fenotipske skupine bolesnika. Među najprestižnijim internacionalnim društvima je Clinical Pharmacogenetics Implementation Consortium (CPIC) za implementaciju farmakogenetike u svakodnevnu kliničku praksu, te američko društvo National Academy of Clinical Biochemistry (NACB) koje prednjači u izdavanju uputa Guidelines and Recommendations for Laboratory Analysis and Application of Pharmacogenetics to Clinical Practice. Osim genotipizacije metaboličkih enzima faze I. prvenstveno CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, DPYD i faze II. TPMT, UGT1A1, NAT2, SULT u novije vrijeme posebna se pažnja pridaje ispitivanju uloge transportnih proteina ABC i SLC u moduliranju bioraspoloživosti lijeka. Među značajnijim primjerima su smjernice za doziranje varfarina prema nalazima genotipizacije CYP2C9 i VKORC1, klopidogrela i CYP2C19, kodeina, tamoksifena i tricikličkih antidepresiva prema CYP2D6 i CYP2C19, 5-fluorouracila i DPYD, fenitoina i CYP2C9, azatioprina i TPMT, irinotekana i UGT1A1, statina i SLCO1B1. U sustavu HLA dokazana je predispozicija nositelja alela HLA- B*5701 za razvoj preosjetljivosti na abakavir, HLA- B*5801 se povezuju s preosjetljivosti na alopurinol. HLA-A*3101 je odgovoran za preosjetljivost na karbamazepin koja je zabilježena u različitim populacijama, dok je alel HLA-B*1502 specifičan za azijske populacije.