Flavokawain A Inhibits Cytochrome P450 In In Vitro Metabolic And Inhibitory Investigations

Background and Purpose: Flavokawain A(FKA), the major chalcone in kava extracts, exhibits strong anti-proliferative and apoptotic effects against human urinary bladder cancer and prostate cancer. The previous pharmacokinetic studies of FKA in vivo showed that it was mainly distributed in liver, kidney and prostate tissues. As is known to all, the majority drugs were metabolized in the liver by cytochrome P450 enzymes. In this paper, we investigated the effect of FKA on CYP450 enzymes activities and further clarified the inhibitory types of FKA on CYP450 enzymes. Besides, the study about identifing the CYP450 enzymes responsible for the metabolism of FKA was also performed. Through the above research, the results could provide experimental data for the metabolism of FKA and evidence about drug-drug interaction when the drugs were co-administrated with FKA. In addition, it also provides ideas for the further development and utilization of chalcones.Methods:1. The study about the effects of FKA on CYP450 isoforms Probe-based assays were used to characterize the inhibitory effects of FKA on CYP450 isoforms(CYP1A2, CYP2D6, CYP3A2, CYP2C9, CYP2C19) and IC50 values were determined. The mechanism of CYP450 enzyme inhibition caused by FKA and molecular docking study were performed to further explore the binding site of FKA on CYP450 isoforms.2. The study on the enzyme kinetics of FKA Incubation time, protein concentration and substrate concentration were used as investigating objects to screen the optimal conditions for the incubation system of FKA. Substrate deletion approach was used to measure kinetic parameters(Vmax, Km, CLint) for microsomal enzyme.3. The study on the metabolic pathway of FKA To identify the CYP450 isoforms that contributed to the metabolic pathway of FKA, the inhibitory effects of known CYP450 isoform-selective inhibitors(a-napthoflavone, quinidine, sulfamethoxazole, omeprazole, ketoconazole) on the metabolite rate of FKA were evaluated. FKA was added and incubated with inhibitors in rat liver microsomes, the metabolic rate of FKA in samples with or without inhibitors was calculated to study the effect of different inhibitors on the metabolism of FKA.4. The study on the metabolites of FKA The metabolites of FKA in rat urine samples were indentied by QE-UPLC @BEH C18,Waters(100mm×2.1mm 1.7μm)chromatographic column. The initial mobile phase condition was as follows:A,water; B,acetonitrile. The flow rate was fixed at 0.3mL/min. The mass spectrometer was operating in full-sacn MS[200-700] with a positive mode. The relevant parameters are as listed: spray voltage, 4.2 kV;capillary temperature 300 °C; sheath gas, 40; auxiliary gas, 10; sweep gas, 3.Results:1. FKA showed significant inhibition on CYP2D6, CYP2C9, CYP3A2 and CYP1A2 activities with IC50 values of 20.39, 60.22, 69.55, 102.65μmol/L, respectively.2. The results of inhibition mechanism of FKA on CYP450 enzymes showed that the inhibitory types were mixed-inhibition, competitive inhibition, uncompetitive inhibition and noncompetitive inhibition separately for CYP2D6, CYP1A2, CYP2C9 and CYP3A2 enzymes.3. The results of molecular docking analysis revealed that three residues(Phe120, Arg221 and Asp301) were critical for the binding of FKA. The hydrogen binding interaction and π-π stacking interaction between functional group of FKA and the major residues significantly increase the stability of FKA-CYP2D6 complex.4. The protein concentration of 1.0 mg/mL, FKA(25μmol/L) and incubation time of 20 min were found to be optimal conditions. Based on the Michaelis-Menten equation, Km, Vmax and Clint were 22.79μmol/L, 2.12μmol/min/mg protein, and 0.093L/min/mg protein.5. In the chemical inhibition assay, the results showed that sulfamethoxazole, ketoconazole and a-naphthoflavone had potent inhibitory effects on the metabolism of FKA in the rat microsome incubation system. The inhibition rate was 80.11%, 50.45% and 30.11%, respectively.6. Through collecting the rat urine samples, the metabolites were identified based on developed LC-MS/MS methods. Major phase I metabolites were generated by demethylation and hydroxylation of FKA.Conclusion:1. FKA had no influence on CYP2C19 enzyme, but had a potent inhibitory effect for CYP2D6 enzyme and weak inhibitory effects on CYP3A2, CYP2C9 and CYP1A2 enzymes.2. CYP3A2 was the principal CYP isoform contributing to the metabolism of FKA. Meanwhile CYP2C9 and CYP1A2 enzymes were also relevant CYP isoforms for the metabolism of FKA.3. When FKA was co-administrated with the drugs which were metabolized by CYP2D6, CYP3A2 and CYP2C9 enzymes, the dosage and time of these drugs should be paid full attention to avoid the occurrence of drug-drug interaction. However, the potential drug-drug interaction should be further investigated in in vivo studies, such as human liver microsomes or recombinant experiments to identify the potential drug interactions of FKA with cytochrome P450 enzymes.