Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.

No study has yet explored the effects of aschantin on the activities of human CYP and UGT enzymes. In this study, we evaluated the inhibitory effects of aschantin on the activities of eight major human CYP enzymes (CYPs: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGT enzymes (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) in pooled human liver microsomes to investigate the possibility of aschantin-drug interactions.
No study has yet explored the effects of aschantin on the activities of human CYP and UGT enzymes. In this study, we evaluated the inhibitory effects of aschantin on the activities of eight major human CYP enzymes (CYPs: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGT enzymes (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) in pooled human liver microsomes to investigate the possibility of aschantin-drug interactions.
Aschantin lowered the IC 50 values of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 by more than 2.5-fold after 30 min pre-incubation with human liver microsomes and NADPH, compared to the values obtained after no pre-incubation (Table 1 and Figure 2), indicating that aschantin is a potent mechanism-based inhibitor of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.
Aschantin was a potent mechanism-based inhibitor of CYP2C19, with a K i value of 5.8 µM, indicating that this compound should be used carefully by patients taking CYP2C19 substrates such as amitriptyline, diazepam, imipramine, lansoprazole, omeprazole, and phenytoin, to avoid drug interactions [25]. It was also a mechanism-based inhibitor of CYP2C8, with a K i value of 10.2 µM, indicating that aschantin should be used carefully by patients taking drugs metabolized by CYP2C8; such drugs include cerivastatin, paclitaxel, repaglinide, and sorafenib. Interactions with such drugs should be avoided [26].
No study has yet explored the effects of aschantin on the activities of human CYP and UGT enzymes. In this study, we evaluated the inhibitory effects of aschantin on the activities of eight major human CYP enzymes (CYPs: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGT enzymes (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) in pooled human liver microsomes to investigate the possibility of aschantin-drug interactions.

CYP Enzyme
Aschantin was a potent mechanism-based inhibitor of CYP2C19, with a Ki value of 5.8 µM, indicating that this compound should be used carefully by patients taking CYP2C19 substrates such as amitriptyline, diazepam, imipramine, lansoprazole, omeprazole, and phenytoin, to avoid drug interactions [25]. It was also a mechanism-based inhibitor of CYP2C8, with a Ki value of 10.2 µM, indicating that aschantin should be used carefully by patients taking drugs metabolized by CYP2C8; such drugs include cerivastatin, paclitaxel, repaglinide, and sorafenib. Interactions with such drugs should be avoided [26]. Aschantin exhibited potent mechanism-based inhibition of CYP2C9-catalyzed diclofenac hydroxylation (Ki, 3.7 µM), indicating that it should be used carefully by patients taking CYP2C9
The compound was also a mechanism-based inhibitor of CYP3A4, with a K i value of 12.6 µM, indicating that it should be used carefully by patients taking drugs metabolized by CYP3A4; such drugs include amlodipine, atorvastatin, cyclosporine, clarithromycin, estradiol, felodipine, lovastatin, nifedipine, ritonavir, simvastatin, and tacrolimus [25].
Herbal preparations containing aschantin may also exhibit time-dependent inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 activities. Currently, no human data on aschantin pharmacokinetics are available; such data are essential for predicting the drug-drug interaction potential of aschantin. Our in vitro results suggest that aschantin should be examined in terms of potential in vivo pharmacokinetic drug interactions caused by inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.

Materials and Reagents
Aschantin was isolated from dried flower buds of Magnolia fargesii as described previously [28] and its purity was more than 99.0%. The dried flower buds of Magnolia fargesii were obtained from Korea Plant Extract Bank, Korea Research Institute of Biology and Biotechnology. The pulverized materials (3 kg) were extracted for 3 days with 9 L of methanol three times and were evaporated. The methanolic extract (225 g) was suspended in 2 L of water and extracted successively with 1 L of hexane and1 L of chloroform. The chloroform soluble fraction (100 g) was subjected to silica gel column chromatography with gradient elution of hexane/acetone to yield fractions 1-19.

Inhibitory Effects of Aschantin on the Activities of Seven Major CYPs in Human Liver Microsomes
The degrees of inhibition (the IC 50 values) of aschantin toward CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were evaluated using pooled human liver microsomes employing a cocktail of CYP substrates and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The incubation mixtures were prepared in total volumes of 100 µL, as follows: pooled human liver microsomes (0.2 mg/mL), 1.0 mM NADPH, 10 mM MgCl 2 , 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin in DMSO (final concentrations of 0.1-100 µM, DMSO less than 1% [v/v]), and a cocktail of seven CYP probe substrates as defined in our previous report [18]. The CYP substrates were used at concentrations approximating their respective K m values: 50 µM phenacetin, 2.5 µM coumarin, 2.0 µM amodiaquine, 10 µM diclofenac, 100 µM [S]-mephenytoin, 5 µM bufuralol, and 2.5 µM midazolam. After 3 min pre-incubation at 37˝C, the reactions were initiated by addition of NADPH and incubation proceeded for 15 min at 37˝C in a shaking water bath. The reaction was stopped by placing the tubes on ice and adding 100 µL amounts of ice-cold methanol containing internal standards ( 13 C 2 , 15 N-acetaminophen for acetaminophen and N-desethylamodiaquine; d 9 -1'-hydroxybufuralol for 4'-hydroxydiclofenac; and 7-hydroxycoumarin, 4'-hydroxy-mephenytoin, 1'-hydroxybufuralol, and 1'-hydroxymidazolam). Then the incubation mixtures were centrifuged at 13,000ˆg for 4 min at 4˝C. All assays were performed in triplicate and the average values were used in calculations. To measure the mechanism-based inhibition of CYP activities, various concentrations of aschantin (0.1-100 µM) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. Each reaction was initiated by adding the seven-CYP probe substrate cocktail.
The metabolites formed from the seven substrates were simultaneously quantified using our previously described LC-MS/MS method [18]. To this end, we employed a tandem mass spectrometer (TSQ Quantum Access, Thermo Scientific, San Jose, CA, USA) coupled to a Nanospace SI-2 LC system (Shiseido, Tokyo, Japan). The column and autosampler temperatures were 50˝C and 6˝C, respectively. The mass spectrometer was equipped with an electrospray ionization (ESI) source and was operated in positive ion mode. The ESI source settings for metabolite ionization were as follows: capillary voltage, 4200 V; vaporizer temperature, 350˝C; capillary temperature 330˝C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 15

Inhibitory Effects of Aschantin on CYP2B6 in Human Liver Microsomes
The effects of aschantin on CYP2B6-catalyzed bupropion hydroxylase activity were evaluated using LC-MS/MS with pooled human liver microsomes [18]. Each incubation mixture was prepared in a total volume of 100 µL, including 1.0 mM NADPH, 10 mM MgCl 2 , 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin (0.1-100 µM), pooled human liver microsomes (0.2 mg/mL), and a CYP2B6-selective substrate (50 µM bupropion), as reported previously [18]. After 3 min of pre-incubation at 37˝C, each reaction was initiated by adding NADPH; incubation proceeded for 15 min at 37˝C in a shaking water bath. The reaction was stopped by placing the tubes on ice and adding 100 µL amounts of ice-cold d 9 -1'-hydroxybufuralol (the internal standard) in methanol. Then the incubation mixtures were centrifuged at 13,000ˆg for 4 min at 4˝C. All incubations were performed in triplicate, and the average values were used in calculations. To evaluate NADPH-dependent mechanism-based inhibition, various concentrations of aschantin (0.1-100 µM) were pre-incubated for 30 min with pooled human liver microsomes in the presence of NADPH. The reaction was initiated by adding bupropion. Hydroxybupropion levels were determined using the LC-MS/MS method described above; the SRM parameters were 256.1 > 238.0 for hydroxybupropion and 287.2 > 187.0 for d 9 -1'-hydroxybufuralol.

Inhibitory Effects of Aschantin on the Activities of Six Major UGTs in Human Liver Microsomes
The extents of aschantin on UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were evaluated by LC-MS/MS, using pooled human liver microsomes incubated with the cocktail of UGT substrates. The method was modified from that of Joo et al. [29]. Each incubation mixture was prepared in a final volume of 100 µL, as follows: pooled human liver microsomes (0.2 mg/mL), 5 mM UDPGA, 10 mM MgCl 2 , 50 mM Tris buffer (pH 7.4), various concentrations of aschantin in DMSO (final concentrations of 0.1-200 µM, DMSO less than 1% [v/v]), and the UGT enzyme-specific substrate of the cocktail set (A set: 0.5 µM SN-38, 2 µM chenodeoxycholic acid, and 0.5 µM trifluoperazine; B set: 1 µM N-acetylserotonin, 0.2 µM mycophenolic acid, and 1 µM naloxone). After 3 min of pre-incubation at 37˝C, the reactions were initiated by addition of UDPGA; incubation continued for 60 min at 37˝C in a shaking water bath. The reaction was stopped by placing the tubes on ice and adding 50 µL ice-cold acetonitrile containing internal standards (propofol glucuronide for chenodeoxycholic acid 24-acyl-β-glucuronide and mycophenolic acid glucuronide, and meloxicam for SN-38 glucuronide, trifluoperazine glucuronide, N-acetylserotonin β-D-glucuronide, and naloxone 3-β-D-glucuronide). The incubation mixtures were centrifuged at 13,000ˆg for 4 min at 4˝C. All assays were performed in triplicate and the average values were used in calculations.
The metabolites formed from the six UGT cocktail substrates were simultaneously measured using the LC-MS/MS method. A tandem mass spectrometer (TSQ Quantum Access) coupled to a Nanospace SI-2 LC system was used. The column and autosampler temperatures were 50˝C and 6˝C, respectively. The mass spectrometer was equipped with an ESI source and was operated in both positive and negative ion modes. The ESI source settings for metabolite ionization were as follows: capillary voltage, 4200 V; vaporizer temperature, 350˝C; capillary temperature 330˝C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 15 psi. Each metabolite was quantified via SRM in the negative ion (chenodeoxycholic acid 24-acyl-β-glucuronide, 567.

Mechanism-Based Inhibition of CYP Activities by Aschantin
The mechanism-based inhibitory effects of aschantin on CYP2C8, CYP2C9, CYP2C19, and CYP3A4 activities were further evaluated in human liver microsomes. The microsomes (1 mg/mL) were pre-incubated with various concentrations of aschantin in 50 mM potassium phosphate buffer (pH 7.4) in the presence of NADPH. Aliquots (10 µL) of the incubated mixtures were withdrawn at 5, 10, 15, and 20 min after incubation commenced and added to other tubes containing the CYP substrates (2 µM amodiaquine for CYP2C8, 10 µM diclofenac for CYP2C9, 100 µM [S]-mephenytoin for CYP2C19, or 2 µM midazolam for CYP3A4), 1 mM NADPH, 50 mM potassium phosphate buffer (pH 7.4), and 10 mM MgCl 2 , in 90 µL reaction mixtures. The second reaction was terminated after incubation for 10 min by adding 100 µL amounts of ice-cold methanol containing d 9 -1'-hydroxybufuralol. The incubation mixtures were centrifuged at 13,000ˆg for 4 min at 4˝C, and then 50 µL of each supernatant was diluted with 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed by LC-MS/MS, as described above.

Data analysis
IC 50 values (i.e., the concentrations of inhibitors required for 50% inhibition of the original enzyme activity) were calculated using Sigma Plot version 11.0 (Systat Software, Inc., San Jose, CA, USA). The apparent kinetic inhibitory potentials (the K i values) were estimated from the fitted curves using Enzyme Kinetics version 1.1 (Systat Software Inc.).