Chemicals and reagents
Tolbutamide was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 4-hydroxytolbutamide and 6-hydroxychlorzoxazone were obtained from Toronto Research Chemicals Inc. (North York, Canada). Dextromethorphan, dextrorphan and chlorzoxazone were supplied by Sigma-Aldrich Co. (St Louis, MO, USA). Testosterone was obtained from International Laboratory Limited (San Bruno, CA, USA). 6β-hydroxytestosterone was purchased from BD Biosciences Co. (Woburn, MA, USA). Phenacetin, cortisone acetate, EB and EE were from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). NADPH was obtained from Roche Diagnostics GmbH (Mannheim, Germany). All other reagents were of HPLC or analytical grade.
Preparation of rat liver microsomes
Wistar rats (180 ± 20 g, male) were supplied by the Animal Experimental Center of Harbin Medical University (Harbin, China), which was fully accredited by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of the Harbin Medical University (Permit Number: HMUIRB20120011), and the rats were handled in a manner that met all the recommendations formulated by the National Society for Medical Research and Guidelines for the Care and Use of Laboratory Animals. Rat liver microsomes were prepared from liver tissue by differential ultra-centrifugation [19], packed and stored at -80°C for further analysis. These operations were carried out in the ice. Protein concentrations of the microsomes were determined by the method of Bradford [20].
Cytochrome P450 probe substrate assays
Tolbutamide and 4-methyhydroxylation assay for CYP2C9
The incubation system of CYP2C9 in vitro contained phosphate buffer (100 mM, pH7.4), liver microsomal protein (0.5 mg · mL-1), MgCl2 (10 mM), tolbutamide (90 μM) and eleutherosides in a final volume of 200 μL. Pre-incubated 5 min, the reaction was initiated by adding NADPH (1 mM concentration in incubation) and the incubation systems were incubated at 37°C for 60 min. After incubation, 50 μL ice-cold acetonitrile was added to terminate the reaction, and phenacetin of a final concentration 20 μM was added as internal standard. With 5 min suspension, the mixture was centrifuged for 30 min at 12000 r · min-1. The supernatant of 20 μL was analyzed by the Waters HPLC system 2010 (Waters, USA, with 600 pump, 996PAD UV detector and Millipore Systems). Tolbutamide, 4-hydroxytolbutamide and phenacetin were separated on a Diamonsil C18 reverse phase column (5 μm, 4.6 mm × 200 mm). The column temperature was set to 35°C. The mobile phase, at a flow rate of 1 mL · min-1, consisted of methanol and 0.1% acetic acid (55:45, v/v). UV detection was at wavelength of 229 nm. The organic solvent which is at low concentration (≤0.5%) in all incubation systems wouldn’t affect the activity of enzymes. The yield of corresponding metabolites was calculated by referring to a standard curve constructed based on known concentrations of the pure metabolites.
Dextromethorphan and O-demethylation assay CYP2D6
Incubation conditions were the same as Section Tolbutamide and 4-methyhydroxylation assay for CYP2C9. The liver microsomal protein was 1.0 mg · mL-1 and tolbutamide was replaced by 25 μM dextromethorphan. Reactions were terminated by 80 μL ice-cold acetonitrile and internal standard phenacetin (final concentration of 50 μM) was added, the denatured protein was removed by centrifuged at 12000 r · min-1 for 30 min. The supernatant of 20 μL was injected into the HPLC system, with the mobile phase of methanol, water, phosphate and triethylamine (42:58:0.15:0.3, v/v/v/v) at a flow rate of 1 mL · min-1, detection was at wavelength of 280 nm.
Chlorzoxazone and 6-hydroxylation assay for CYP2E1
Each incubation mixture (200 μL) included liver microsomal protein (0.75 mg. mL-1), MgCl2 (10 mM) in 100 mM phosphate buffer (pH7.4) and 25 μM chlorzoxazone. With 5 min pre-incubation, all reactions were initiated by addition of NADPH (1 mM) and were carried out in 37°C water bath for 30 min, and then were stopped by addition of 150 μL ice-cold acetonitrile and internal standard (80 μM phenacetin). After centrifugation at 12000 r · min-1 for 30 min, 20 μL of the supernatant was injected into the HPLC system, and eluted with methanol–water (47:53) at a flow rate of 1.0 mL · min-1, UV absorbance was monitored at 287 nm.
Testosterone and 6β-hydroxylation assay for CYP3A4
Testosterone solution (in methanol, final concentration of 100 μM) was evaporated to dryness under nitrogen in 40°C water bath, then additional reagents were added to give a final incubation volume of 200 μL: liver microsomal protein (0.5 mg · mL-1) in 50 mM sodium phosphate buffer (pH7.4) and MgCl2 (10 mM). Following a 5 min pre-incubation, reactions were started with addition of NADPH (1 mM). Following 30 min incubations at 37°C, reactions were stopped with organic solution (280 μL ice-cold acetonitrile), and cortisone acetate was added as internal standard with final concentration of 12.5 μM. The mixture was centrifugated at 12000 r · min-1 for 30 min, and the supernatant of 20 μL was injected into the HPLC, with UV detection at 245 nm. Mobile phase consisted of methanol and water (65:35, v/v), and the flow rate was 1.0 mL · min-1.
Determination of Km and Vmax
The apparent Km (Michaelis constant) and Vmax (maximum reaction velocity) values were determined in a range of concentrations of probe drugs. The concentrations were as follows: tolbutamide 3.5~600.0 μM, dextromethorphan 3.5~400.0 μM, chlorzoxazone 5.0~300.0 μM, and testosterone 12.5~500.0 μM. The other incubation conditions were the same as Section Cytochrome P450 probe substrate assays.
Determination of effects of EB and EE on CYP450 activity
To evaluate whether EB and EE affect the activity of CYP450, the probe substrate reaction assays were performed with EB or EE at concentrations of 0, 2, 10, 25, 50, 150, 300 μM under the conditions described earlier, with triplicate incubations for each concentration. The concentrations of respective probe substrates were selected according to Km established in the enzyme kinetic assays described above. The IC50 values (concentration of inhibitor causing 50% inhibition of enzyme activity) were determined based on the concentration-inhibition curves.
Assay for enzymatic kinetic parameters
The exact inhibition constants (Ki values) were measured and the modes of inhibition were determined for the components exhibiting IC50 values of less than 200 μM. The Ki values were determined in a range of concentrations of probe substrates (approximately Km/2, Km, 2Km and 4Km) and different concentrations of EB (0, 100, 200, 300 μM) and EE(0, 100, 200, 300 μM). Dixon and Lineweaver-Burk plots and the second plots showed the data graphically for interpretation of inhibition mode. All experiments were separately performed in three times.
