METABOLIZAM AGLIKONA ODABRANIH FLAVONOIDA POSREDOVAN HUMANIM JETRENIM CITOKROMIMA P450

Flavonoidi su velika i heterogena skupina polifenolnih spojeva niske molekulske mase koje ljudi hranom svakodnevno unose, a organizmu predstavljaju ksenobiotike kojih se nastoji riješiti. Među najvažnijim mehanizmima koje ljudski organizam ima na raspolaganju za eliminaciju ksenobiotika jest oksidativni metabolizam koji je najvećim dijelom posredovan citokromima P450. Iako su rezultati istraživanja bioloških učinaka in vitro obećavajući, flavonoidi često ne ostvaruju svoj farmakološki potencijal in vivo zbog slabe bioraspoloživosti. Fiziološki mehanizmi djelovanja i metabolička sudbina flavonoida još su uvijek vrlo slabo istraženi. Stoga je cilj ovoga rada bio karakterizirati oksidativni metabolizam većeg broja odabranih flavonoida posredovan humanim citokromima P450. U tu svrhu ispitano je 30 odabranih aglikona flavonoida u inkubacijama s humanim jetrenim mikrosomima i rekombinantnim citokromima P450. Nastanak metabolita praćen je tekućinskom kromatografijom spregnutom sa spektrometrijom masa visoke razlučivosti. U 15 od 30 analiziranih flavonoida primijećen je metabolizam posredovan citokromima P450 1A2, 2C19, 2D6, 2E1 i/ili 3A4. Detektirani metaboliti nastali su reakcijama aromatske hidroksilacije ili O-demetilacije ili kombinacijom ovih reakcija. Vrijednosti konstante specifičnosti (kcat/Km) svih primijećenih metaboličkih reakcija kretale su se u širokom rasponu (0,0015-2,9) × 106 M–1 min–1 što upućuje na visoku selektivnost humanih jetrenih citokroma P450 prema flavonoidima. Enzim koji je daleko najučinkovitije metabolizirao ispitane flavonoide bio je CYP1A2 što sugerira da je zbog mogućih interakcija potreban dodatni oprez kod unosa flavonoida i istovremene primjene lijekova koji se primarno metaboliziraju putem CYP1A2. U određenoj su mjeri opisana i jedinstvena strukturna obilježja odgovorna za metabolizam posredovan citokromima P450 koja omogućuju predviđanje mogućih metaboličkih putova i za flavonoide koji nisu bili predmetom ovog istraživanja. Dobiveni rezultati omogućuju bolje razumijevanje metabolizma flavonoida što može pomoći u prevladavanju niske bioraspoloživosti flavonoida in vivo i usmjeriti napore istraživača prema pronalaženju flavonoida s povoljnim terapeutskim i metaboličkim profilom. Flavonoids are large and heterogeneous group of polyphenolic compounds with low molecular mass that people ingest in everyday diet, but represent xenobiotics which human organism tends to eliminate. Among most important mechanisms that human body has at disposal for elimination of xenobiotics is oxidative metabolism mainly mediated by cytochromes P450. Although results of in vitro studies are promising, due to low bioavailability flavonoids often do not demonstrate their pharmacological potential in vivo. Physiological mechanisms of act as well as metabolic fate of flavonoids are still poorly understood. The objective of this work was to characterize oxidative metabolism mediated by human cytochromes P450 of larger number of flavonoid compounds. For that purpose, 30 selected flavonoid aglycones were examined in incubations with human liver microsomes and recombinant cytochromes P450. The formation of metabolites was monitored by liquid chromatography coupled with high resolution mass spectrometry. Out of 30 analyzed flavonoids, 15 was susceptible to oxidative metabolism mediated by cytochromes P450 1A2, 2C19, 2D6, 2E1 and/or 3A4. The detected metabolites were formed by aromatic hydroxylation or O-demethylation, or combination of these reactions. The values of catalytic effectiveness (kcat/Km) of all observed metabolic reactions were obtained in very wide range from 0.0015 to 2.9 × 106 M–1 min–1 which indicates high selectivity of human liver cytochromes P450 to flavonoids. The dominant enzyme responsible for the observed metabolic reactions was CYP1A2 suggesting that potential interactions of flavonoids with drugs primarily metabolized by CYP1A2 should be considered. Specific structural characteristics responsible for metabolism mediated by cytochromes P450 were determined to some extent thus enabling prediction of possible metabolic pathways for flavonoids other than those analyzed in this study. These results contribute to better understanding of flavonoid metabolism thus helping to overcome their low bioavailability in vivo and steer the efforts of researchers towards detecting flavonoids with desirable therapeutic and metabolic profiles.