Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP) enzyme activities in human liver microsomes were evaluated using liquid chromatography-tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1), CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1), and CYP3A4-catalyzed midazolam 1′-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1) in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1′-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

A 30-min pre-incubation of dimethyllirioresinol, epimagnolin A, eudesmin, or magnolin with human liver microsomes and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) did not cause the IC 50 value shift of eight CYP enzymes (Figures 2-5), indicating that dimethyllirioresinol, magnolin, epimagnolin A, or eudesmin may not be mechanism-based inhibitors. However, 30 min pre-incubation of human liver microsomes with fargesin and NADPH lowered the IC 50 values of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C19-catalyzed [S]-mephenytoin 4 -hydroxylation, and CYP3A4-catalyzed midazolam 1 -hydroxylation activities by more than 2.5-fold in comparison with the IC 50 values obtained without pre-incubation (34.9 vs. 4.0 µM for CYP2C8, 30.2 vs. 1.6 µM for CYP2C19, and >100 vs. 17.9 µM for CYP3A4) ( Figure 6, Table 1), indicating that fargesin causes potent mechanism-based inhibition of CYP2C8, CYP2C19, and CYP3A4 enzymes in human liver microsomes.  (Figure 6, Table 1), indicating that fargesin causes potent mechanism-based inhibition of CYP2C8, CYP2C19, and CYP3A4 enzymes in human liver microsomes.  In the study of enzyme inhibition, the inhibitor concentration causing half maximal inactivation (Ki value) and the inhibition mode define the interaction of an inhibitor with a  In the study of enzyme inhibition, the inhibitor concentration causing half maximal inactivation (K i value) and the inhibition mode define the interaction of an inhibitor with a particular enzyme.

Discussion
In this study, the in vitro inhibitory effects of bioactive tetrahydrofurofuranoid lignans such as dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on 8 major CYP enzymes were, for the first time, evaluated in pooled human liver microsomes. Dimethyllirioresinol exhibited weak inhibition of CYP2C8 and CYP2C19 activities without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 in human liver microsomes ( Figure 2). Magnolin, epimagnolin A, and eudesmin showed no the reversible and time-dependent inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities at 100 μM in human liver microsomes (Figures 3-5). These results indicate that dimethyllirioresinol, epimagnolin A, eudesmin, and magnolin without a methylenedioxy ring in the chemical structure may not be CYP inhibitors in human liver microsomes. However, fargesin containing a methylenedioxyphenyl moiety in the chemical structure showed moderate reversible inhibition of CYP2C8, CYP2C9, and CYP2C19 activities (IC50 values of 34.9, 30.8, and 30.2 μM, respectively) and the potent time-dependent inhibition of CYP2C19, CYP2C8, and CYP3A4 activities (IC50 values of 1.6, 4.0, and 17.9 μM, respectively) in human liver microsomes. Aschantin, a chemical derivative of fargesin, with a methylenedioxyphenyl moiety also exhibited the reversible and time-dependent inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 activities in human liver microsomes [32]. These results indicate that CYP inhibitory capacity of tetrahydrofurofuranoid lignans depends on the presence of a methylenedioxyphenyl moiety. Other methylenedioxyphenyl compounds such as myristicin and podophyllotoxin exhibited mechanism-based inactivation of CYP1A2 and CYP3A4, respectively, in human liver microsomes [36,37].
Seven metabolites were simultaneously determined using a tandem mass spectrometer (TSQ Quantum Access; Thermo Scientific, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source coupled to a NANOSPACE SI-2 LC system (Shiseido, Tokyo, Japan). The column and autosampler temperatures were 50 and 6 • C, respectively. The ESI source settings for the ionization of metabolites were as follows: polarity, positive ion mode; capillary voltage, 4200 V; capillary temperature, 330 • C; vaporizer temperature, 350 • C; auxiliary gas pressure, 15 psi; and sheath gas pressure, 35 psi. Selected reaction monitoring (SRM) mode with the molecular ion and the intensive product ion was used for the quantification of each metabolite and internal standard, as follows: For the evaluation of the inhibitory effects of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on CYP2B6-catalyzed bupropion hydroxylation, each incubation mixture in a total volume of 100 µL contained 50 mM potassium phosphate buffer (pH 7.4), 10 mM MgCl 2 , pooled human liver microsomes (0.2 mg/mL), 50 µM bupropion, and various concentrations of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, or magnolin in acetonitrile (0.1-100 µM), according to our previous report [33]. After 3 min pre-incubation at 37 • C, the reaction mixtures were incubated with the addition of NADPH in a shaking water bath for 15 min at 37 • C. The reaction was stopped by adding 100 µL of ice-cold d 9 -1 -hydroxybufuralol (internal standard) in methanol. The mixtures were centrifuged at 13,000× g for 4 min at 4 • C. All incubations were performed in triplicate, and the average values were used for the subsequent calculations. For the measurement of time-dependent inhibition, pooled human liver microsomes were pre-incubated with various concentrations of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, or magnolin in acetonitrile (0.1-100 µM) and NADPH for 30 min at 37 • C. Then, the reaction mixtures were incubated with the addition of NADPH and bupropion for 15 min at 37 • C. The control reaction was performed by the addition of acetonitrile instead of the test compounds. Hydroxybupropion concentrations were quantified using the LC-MS/MS method described above; the SRM transitions for hydroxybupropion and d 9 -1 -hydroxybufuralol were 256.1 > 238.0 and 287.2 > 187.0, respectively.

Kinetic Analysis of CYP2C9 Inhibition by Fargesin
To determine the K i values and inhibition mode of fargesin for CYP2C9, various concentrations of fargesin (0-16 µM) and diclofenac (2-20 µM) were incubated with human liver microsomes (0.1 mg/mL), 10 mM MgCl 2 , 1 mM NADPH, 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 100 µL for 10 min at 37 • C. The reaction was stopped by adding 100 µL of ice-cold d 9 -1'-hydroxybufuralol in methanol (10 ng/mL), and the mixtures were centrifuged at 13,000× g for 4 min. 50 µL of the supernatant was diluted with 50 µL of water, and aliquots (5 µL) were analyzed by LC-MS/MS.

Mechanism-Based Inhibition of CYP2C8, CYP2C19, and CYP3A4 Activities by Fargesin
The mechanism-based inhibition potency of fargesin against human liver microsomal CYP2C8, CYP2C19, and CYP3A4 activities was evaluated. Human liver microsomes (1 mg/mL) were pre-incubated with various concentrations of fargesin and NADPH in 50 mM potassium phosphate buffer (pH 7.4). Aliquots (10 µL) of the pre-incubation mixtures were collected at 5, 10, 15, and 20 min after the pre-incubation and transferred to new tubes containing CYP substrates (2 µM amodiaquine for CYP2C8, 100 µM [S]-mephenytoin for CYP2C19, or 2 µM midazolam for CYP3A4), 10 mM MgCl 2 , 1 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4) in 90 µL reaction mixtures. The incubation was proceeded for 10 min and stopped by adding 100 µL of ice-cold d 9 -1 -hydroxybufuralol in methanol. The mixtures were centrifuged at 13,000× g for 4 min at 4 • C, and 50 µL of each supernatant was diluted with 50 µL of water. Aliquots (5 µL) were analyzed by LC-MS/MS, as described above.

Data Analysis
The IC 50 values were calculated using SigmaPlot ver. 11.0 (Systat Software, Inc., San Jose, CA, USA). K i , k inact , and the inhibition mode were determined using Enzyme Kinetics ver. 1.1 (Systat Software, Inc.).