No public access
master's thesis
A comparison of cyp 450 inhibition methods in human liver microsomes applied in early drug discovery

Ana Delost (2016)
University of Rijeka
Department of Biotechnology
Metadata
TitleUsporedba metoda testiranja inhibicije citokroma p450 u sustavu ljudskih jetrenih mikrosoma u ranoj fazi istraživanja
AuthorAna Delost
Mentor(s)Miranda Mladinić Pejatović (thesis advisor)
Abstract
Jedan od važnih koraka u ranoj fazi istraživanja novih lijekova je procjena mogućnosti kemijskog spoja da izazove lijek-lijek interakciju (engl. drug-drug interaction, DDI). Enzimi citokromi P450 (CYP450) odgovorni su za oksidativni metabolizam velikog broja lijekova kao i metaboličku interakciju lijekova. Za najučestalije CYP450 izoforme (CYP1A2, CYP2C9, CYP2C19, CYP2D6 i CYP3A4) mogućnost inhibicije određuje se pojedinačno za svaku izoformu u in vitro sustavu ljudskih jetrenih mikrosoma korištenjem specifičnih supstrata. Sve veći broj sintetiziranih spojeva, često lipofilnih svojstava i ograničenih količina, zahtjeva otapanje u organskim otapalima koja sama mogu imati inhibitorne ili stimulatorne učinke na CYP450 enzime. U ovom je radu razvijena metoda određivanja potencijala za inhibiciju CYP450 enzima pojedinačnim pristupom (engl. single approach) za ukupno 5 izoformi u ljudskim jetrenim mikrosomima, koristeći dimetilsulfoksid (DMSO) kao organsko otapalo u polaznoj matičnoj otopini. Dobiveni rezultati su uspoređeni s prethodno razvijenom i validiranom metodom u kojoj matičnu otopinu čini metanol. U tu je svrhu korišteno 7 komercijalnih inhibitora, prema preporuci regulatornih agencija, za pojedine izoforme CYP450. IC50 vrijednosti dobivene korištenjem DMSO kao otapala dobro koreliraju sa vrijednostima dobivenim s metanolom, te potvrđuju da se on može koristiti u ovoj metodi ukoliko je njegov udio u konačnoj inkubacijskoj smjesi <0.1%. Kako bi se povećala efikasnost, u određivanju potencijala za inhibiciju CYP450 za veći broj spojeva, razvijena je tzv. metoda koktel pristupa (engl. cocktail approach) koja omogućuje istovremeno profiliranje nekoliko izoformi unutar jedne inkubacijske smjese, te omogućava brže testiranje DDI potencijala. IC50 vrijednosti dobivene iz koktel pristupa dobro koreliraju s vrijednostima iz pojedinačnog pristupa, kao i s literaturnim vrijednostima, te upućuju na dobru optimizaciju ove metode. Nadalje, rezultati potvrđuju da se metoda koktel pristupa može koristiti kao visokoprotočna metoda za predviđanje mogućih lijek-lijek interakcija velikog broja novosintetiziranih spojeva, čime se povećava efikasnost tijekom rane faze istraživanja.
KeywordsCytochromes P450 drug-drug interaction cocktail approach
Parallel title (English)A comparison of cyp 450 inhibition methods in human liver microsomes applied in early drug discovery
Committee MembersMiranda Mladinić Pejatović (committee chairperson)
Jasminka Giacometti (committee member)
Sandra Kraljević Pavelić (committee member)
GranterUniversity of Rijeka
Lower level organizational unitsDepartment of Biotechnology
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeBiotechnology in medicine
Academic title abbreviationmag. biotech. in med.
Genremaster's thesis
Language Croatian
Defense date2016-07-18
Parallel abstract (English)
One of the important steps in early drug discovery is the assessment of drug-drug interactions (DDI) potential for new chemical entities. Cytochrome P450 enzymes are responsible for the oxidative metabolism of numerous drugs, as well as their metabolic interaction. The inhibition potential for the most common CYP450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) is determined individually for each isoform in an in vitro system of human liver microsomes using specific substrates. An increasing number of new chemical entities, often lipophilic and of limited quantities, require organic solvents which can have either inhibitory or stimulatory effects on CYP450 on their own. In this work, a method was developed to assess the CYP450 inhibition potential for a total of 5 individual isoforms (single approach) in human liver microsomes, using dimetylsulfoxid (DMSO) as an organic solvent in the stock solution. Results were compared to a previously developed and validated method using a methanol stock solution. A total of seven commercially available and inhibitors, recommended by regulatory agencies, for the different CYP450 isoforms were used. IC50 values obtained using DMSO were in good agreement with those obtained using methanol, confirming that DMSO can be used in this assay providing the final concentration is <0.1%. In an effort to improve the screening efficiency for a larger number of compounds, the cocktail approach method was developed. This method enables simultaneous profiling of several isoforms within a one incubation mixture, allowing faster screening for DDI potential. IC50 values obtained in the cocktail approach correlate well with values obtained in the single approach, as well as the literature, suggesting the method was well optimized. Moreover, this confirms that the cocktail approach method can be used as a high-throughput screening tool to assess CYP450 inhibition potential for a larger number of compounds thereby increasing efficiency during the early discovery phase.
Parallel keywords (Croatian)citokromi P450 lijek-lijek interakcije koktel pristup
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:777175
CommitterLea Lazzarich