Mechanism of cytochrome P450 3A4 inhibition mediated by flavonoids

Flavonoidi su spojevi koji se u većim količinama nalaze u biljkama. Čovjek prehranom svakodnevno konzumira flavonoide. Enzimi citokrom P450 su najvažniji enzimi koji sudjeluju u metabolizmu lijekova i lipofilnih ksenobiotika. Najveći broj lijekova na tržištu metabolizira se putem CYP3A4 enzima. Poznato je da flavonoidi mogu stupati u reakcije s CYP3A4 enzimom, pri čemu ga mogu inhibirati. Važno je razjasniti dosad nepoznat mehanizam inhibicijskog učinka flavonoida na CYP3A4 enzim. Ispitana je vrsta inhibicije kojom akacetin, apigenin, krizin i pinocembrin inhibiraju aktivnost CYP3A4 enzima. Inaktivacijska kinetika flavonoida određena je uz testosteron i nifedipin kao marker supstrate. Određeni su osnovni parametri enzimske inaktivacije (konstanta inhibicije, konstanta brzine inaktivacije, učinkovitost inaktivacije i polovica maksimalne inhibitorne koncentracije). Ispitana je pseudoireverzibilna inhibicija enzima uz pomoć hemokrom-piridin testa. Primjenom glutationa, hvatača slobodnih radikala, nastojala se utvrditi struktura reaktivnih intermedijera odgovornih za inaktivaciju enzima. Apigenin je uzrokovao o metabolizmu ovisnu inhibiciju CYP3A4 enzima (ostatna aktivnost enzima 10,6 ± 1,3%). Za krizin je utvrđena IC50 vrijednost od 0,6 ± 0,5 μM, konstanta inhibicije Ki = 0,6 ± 0,3 μM te učinkovitost inhibicije 0,108 min-1 μM-1. Za apigenin je utvrđena konstanta brzine inaktivacije, kinact = 0,11 ± 0,01 μM-1. Svi flavonoidi su smanjili koncentraciju hema, a krizin najviše (ostatna koncentracija hema 5,5%, odnosno 2,9%). Nije uočena statistički značajna razlika između ostatne aktivnosti enzima nakon dijalize s i bez dodatka kalijevog heksacijanoferata. Reaktivni međuprodukti flavonoida nisu uočeni na spektrometru masa. Svi flavonoidi pokazuju o metabolizmu ovisnu inhibiciju CYP3A4 enzima, pri čemu se krizin pokazao kao najsnažniji inhibitor. Flavonoidi se kovalentno vežu za hem te na taj način dovode do inaktivacije enzima. Niti jedan flavonoid nije pokazao inhibiciju pseudoireverzibilnog karaktera na CYP3A4 enzim. Reaktivni međuprodukti flavonoida nisu uočeni zbog niskih koncentracija ili nestabilnosti produkata.Flavonoids are compounds found in larger amounts in plants. People consume flavonoids in their diet on a daily basis. Cytochrome P450 enzymes are the most important enzymes involved in the metabolism of drugs and lipophilic xenobiotics. Most drugs on the market are metabolized by the CYP3A4 enzyme. It is known that flavonoids can react with the CYP3A4 enzyme, whereby they can inhibit it. It is important to elucidate the hitherto unknown mechanism of the inhibitory effect of flavonoids on the CYP3A4 enzyme.The type of inhibition by which acacetin, apigenin, chrysin and pinocembrin inhibit CYP3A4 enzyme activity was investigated. The inactivation kinetics of flavonoids were determined with testosterone and nifedipine as substrates. The basic parameters of enzyme inactivation (Ki, kinact, inactivation efficiency and IC50 concentration) were determined. Pseudoireversible inhibition of the enzyme was examined using the hemochromopyridine test. The use of glutathione sought to determine the structure of reactive intermediates responsible for enzyme inactivation.Apigenin caused metabolism based inhibition of the CYP3A4 enzyme (residual enzyme activity 10.6 ± 1.3%). For chrysin, an IC50 value of 0.6 ± 0.5 μM, Ki = 0.6 ± 0.3 μM, and an inhibition efficiency of 0.108 min-1 μM-1 were recorded. An inactivation rate constant, kinact = 0.11 ± 0.01 μM-1, was recorded for apigenin. All flavonoids reduced the concentration of heme, chrysin being the most potent (residual concentration of heme was 5.5% and 2.9%, respectively). No statistically significant difference was observed between the residual enzyme activity after dialysis with and without the addition of potassium hexacyanoferrate. Reactive intermediates of flavonoids were not observed.All flavonoids show metabolism-dependent inhibition of the CYP3A4 enzyme, with chrysine proving to be the most potent inhibitor. Flavonoids bind covalently to heme, thus leading to enzyme inactivation. None of the flavonoids showed pseudo-irreversible inhibition of the CYP3A4 enzyme. Reactive intermediates of flavonoids were not observed due to low concentrations or product instability