Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP) enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs) 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

Honokiol was found to be a potent noncompetitive inhibitor of CYP1A2 with a K i value of 1.2 μM, indicating that honokiol should be used carefully with drugs metabolized by CYP1A2 such as antipsychotics (clozapine and haloperidol), antiathmatics (theophylline and zileuton), and antidepressants (amitriptyline and clomipramine) in order to avoid drug interactions [46]. Further, several natural compounds including luotonin [52], mollugin [53], and astragaloside IV [54] have been shown to strongly inhibit CYP1A2.
Herbal preparations containing honokiol may also affect CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9 activities. At present, there is no available data on the pharmacokinetics of honokiol in humans, which are indispensable for the prediction of the drug-drug interaction potential of honokiol. These in vitro results suggest however that honokiol should be examined for potential pharmacokinetic drug interactions in vivo due to its inhibition of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9 activities based on K i values of 0.3-4.9 μM.

Inhibitory Effect of Honokiol on Eight Major CYP Activities in Human Liver Microsomes
The inhibitory potential (IC 50 values) of honokiol on CYP activities was evaluated in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The incubation mixtures were prepared in a total volume 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 honokiol (0.05-100 μM) and a cocktail mixture of seven CYP probe substrates or bupropion, a CYP2B6-selective substrate, as reported previously [50]. Honokiol was dissolved in acetonitrile. The substrates were used at concentrations approximately equal to or less than that of their respective K m values: 50 μM phenacetin for CYP1A2, 2.5 μM coumarin for CYP2A6, 50 μM bupropion for CYP2B6, 2.5 μM amodiaquine for CYP2C8, 10 μM diclofenac for CYP2C9, 100 μM [S]-mephenytoin for CYP2C19, 5 μM bufuralol for CYP2D6, and 2.5 μM midazolam for CYP3A4. After a 3 min preincubation at 37 °C, the reactions were initiated by addition of an NADP generating system and incubated for 15 min at 37 °C in a shaking water bath. The reaction was then stopped by placement of the tubes on ice and adding 100 μL of ice-cold methanol containing internal standards ( 13 C 2 , 15 N-acetaminophen for acetaminophen and N-deethylamodiaquine, and d 9 -1-hydroxybufuralol for 4-hydroxydiclofenac, 4-hydroxybupropion, 7-hydroxycoumarin, 4-hydroxymephenytoin, 1'-hydroxybufuralol, and 1'-hydroxymidazolam). The incubation mixtures were then centrifuged at 13,000 × g for 4 min. All incubations were performed in triplicate, and average values were used.
For evaluation of time-dependent inhibition of CYP activities, various concentrations of honokiol (0.05-100 μM) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. The reaction was initiated by the addition of the cocktail containing seven CYP probe substrates and bupropion.
The metabolites formed from the seven CYP cocktail substrates were simultaneously determined according to our previously described LC-MS/MS method with minor modification [50]; the concentration of 4-hydroxybupropion for CYP2B6 activity was quantified separately by LC-MS/MS. A tandem mass spectrometer (TSQ Quantum Access, Thermo Scientific, San Jose, CA, USA) coupled with a Nanospace SI-2 LC system (Tokyo, Japan) was used. The column and autosampler temperatures were 50 °C and 6 °C, respectively. The mass spectrometer was equipped with an electrospray ionization (ESI) source and operated in positive ion mode. The ESI source settings for ionization of the metabolites were as follows: capillary voltage, 4200 V; vaporizer temperature, 350 °C; capillary temperature 330 °C; sheath gas pressure, 35 psi; auxiliary gas pressure, 15 psi. Quantification was performed by selected reaction monitoring (SRM) of [M+H] + ions and the related product ions for each metabolite. SRM transitions for the metabolites and internal standards have been described previously by our group [50]. Analytical data were processed using Xcalibur ® software (Thermo Scientific).

Data Analysis
IC 50 values (concentration of the inhibitor causing a 50% inhibition of the original enzyme activity) were calculated using Sigma Plot 8.0 (Systat Software, Inc., San Jose, CA, USA). The apparent kinetic parameters for inhibitory potential (K i values) were estimated from the fitted curves using Enzyme Kinetics Ver. 1.1 software (Systat Software Inc.).

Conclusions
The effect of honokiol on eight CYPs and four UGTs was determined across a wide range of substrates and honokiol concentrations using human liver microsomes. CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9 activities were potently inhibited by honokiol upon incubation in microsomes. Honokiol weakly inhibited CYP2B6-catalyzed bupropion hydroxylation, CYP2D6-catalyzed bufuralol 1'-hydroxylation, CYP3A-catalyzed midazolam hydroxylation, UGT1A1-catalyzed 17β-estradiol 3-glucuronidation, and UGT2B7-catalyzed azidothymidine glucuronidation in a dose-dependent manner. These results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9. However, it is important to note that the inhibition of CYP activities in vitro does not necessarily translate into drug interactions in clinical situations. Thus, clinical trials to evaluate the inhibitory effects of honokiol on CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9 should be conducted.