In Vitro Inhibitory Effects of Scutellarin on Six Human/Rat Cytochrome P450 Enzymes and P-glycoprotein

Inhibition of cytochrome P450 (CYP) and P-glycoprotein (P-gp) are regarded as the most frequent and clinically important pharmacokinetic causes among the various possible factors for drug-drug interactions. Scutellarin is a flavonoid which is widely used for the treatment of cardiovascular diseases. In this study, the in vitro inhibitory effects of scutellarin on six major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six rat CYPs (CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2) activities were examined by using liquid chromatography-tandem mass spectrometry. Meanwhile, the inhibitory effects of scutellarin on P-gp activity were examined on a human metastatic malignant melanoma cell line WM-266-4 by calcein-AM fluorometry screening assay. Results demonstrated that scutellarin showed negligible inhibitory effects on the six major CYP isoenzymes in human/rat liver microsomes with almost all of the IC50 values exceeding 100 μM, whereas it showed values of 63.8 μM for CYP2C19 in human liver microsomes, and 63.1 and 85.6 μM for CYP2C7 and CYP2C79 in rat liver microsomes, respectively. Scutellarin also showed weak inhibitory effect on P-gp. In conclusion, this study demonstrates that scutellarin is unlikely to cause any clinically significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P-gp.


Results and Discussion
The substrates and inhibitors of CYPs and P-gp used in this study were in line with the FDA's guideline and previous reports [47][48][49][50]. These experimental methods have been validated in our previous study [51,52], and IC 50 values of inhibitors were in good agreement with the published values according to the acceptable degree of accuracy [47][48][49][50].
Scutellarin was evaluated for the ability to inhibit the activities of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P-gp. The IC 50 values for six CYPs in human/rat liver microsomes are presented in Table 1. P-gp inhibition is shown in Figure 2.  using calcein AM assay. A response equal to or greater than 25% of verapamil at 100 μM was considered to be positive at the tested concentration. Each data point represents the mean value (±SD) of triplicate determinations. Experimental details are given in the section of P-glycoprotein assay.

Inhibition by Scutellarin on Six CYPs in HLM
Using human liver microsomes and industry-accepted CYP substrates, scutellarin showed weak inhibitory effects on the six tested CYPs, as the IC 50 values for CYP1A2, CYP2C8, CYP2C9, CYP2D6 and CYP3A4 were in excess of 100 μM, and only 63.8 μM for CYP2C19 ( Figure 3).

Inhibition by Scutellarin on Six CYPs in RLM
Similarly, scutellarin had a weak inhibitory effect on six tested CYPs using rat liver microsome, the IC 50 values for CYP1A2, CYP2C11, CYP2D4 and CYP3A2 were in excess of 100 μM, but 63.1 and 85.6 μM for CYP2C7 and CYP2C79 (Figure 4), respectively.

Inhibition by Scutellarin on P-glycoprotein
The effect of scutellarin on P-gp was examined by verapamil (100 μM) as the positive control after results showed that scutellarin, even at highest concentration, does not show cytotoxicity on WM-266-4 cells. All of the six concentrations examined (4.1~1,000 μM) for scutellarin produced less than 25% P-gp inhibition, which suggests that scutellarin cannot influence P-gp activity.
Our previous study discovered that HEI did not exert the inhibitory effects on rat liver CYP1A2, CYP2C11 and CYP2E1, but showed weak inhibitory activities on rat liver CYP2D4 and CYP3A2 in vivo [14]. Since scutellarin is the capital effective component of HEI [12] and it is necessary to investigate the in vitro inhibitory potential of scutellarin on CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2 activities in RLM. These results showed that scutellarin (even at 100 μM) had no apparent inhibitory effects on rat CYP1A2, CYP2C11, CYP2D4 and CYP3A2 in vitro, but had weak inhibitory effects on rat CYP2C7 and CYP2C79 with IC 50 values of 63.1 and 85.6 μM. Our present in vitro results were partly in accord with our former study, but our result on rat CYP1A2 was consistent with previous report that scutellarin was a poor inhibitor on rat CYP1A2 with an IC 50 value of 108.20 ± 0.657 μM in in vitro experiments and exhibited a weak mixed-type inhibition against the activity of CYP1A2 with a K i value of 95.2 μM, Scutellarin at 30 mg/kg also weakly inhibited CYP1A2 activity in whole animal studies [53]. Because there were only the results of rat in all above studies, we studied in vitro inhibitory effects of scutellarin on human CYP1A2, CYP2C8, CYP2C9, CYP2D6 and CYP3A4. The in vitro results showed that scutellarin (even 100 μM) had no apparent inhibitory effects on human CYP1A2, CYP2C8, CYP2C9, CYP2D6 and CYP3A4 activities, but had weak inhibitory effects on human CYP2C19 activity with IC 50 values of 63.8 μM. In our studies, there are no significant difference between the IC 50 values of scutellarin on human/rat CYP2C8 and CYP2C19 which may be accepted for species differences. A compound that inhibits an isoform with IC 50 value of less than 10 μM is generally considered as a "potent" inhibitor to the isozyme, whereas the compound with IC 50 value between 10 and 50 μM is a "moderate" inhibitor [54,55]. Results from the in vitro studies can be used to predict in vivo interaction and guide the need for further in vivo study evaluation though it is not possible to directly extrapolate the in vitro results with what can occur in vivo. These viewpoints are widely accepted by researcher on CYP inhibition or drug interactions. So, we believed that scutellarin may have no significant inhibitory effects on CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4.
The WM-266-4 is an atypical cell line used in studies about drug interactions. Therefore, the method on P-gp inhibition-calceinAM assay was carefully validated in HD Biosciences Co., Ltd. and accordant with FDA's guideline. Furthermore, we found that scutellarin (even at the concentration of 1 mM) had no significant inhibitory effects on P-gp in WM-266-4 cells in this study.
Pooled human liver microsomes (HLM, LOT 32556) were purchased from BD Gentest Corporation (simple donor information see Table 2.). Pooled rat liver microsomes were prepared from three male Sprague-Dawley rats in HD Biosciences Co. (Shanghai, China). The microsomal preparations contain a protein concentration of 10 mg/mL and are stored in a −80 °C freezer before use. The human metastatic malignant melanoma cell line WM-266-4 was procured from ATCC (Manassas, VA, USA). Glucose-6-phosphate (G-6-P) and NADP + were purchased from Majorbio Co. (Shanghai, China). Glucose-6-phosphate dehydrogenase (G-6-PDH) was purchased from Calbiochem (Gibbstown, NJ, USA). The assay kit for protein determination with a biscinchoninic acid reagent was from Pierce (Rockford, IL, USA). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA) and used throughout the study. All inorganic salts were of analytical grade and were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). All organic solvents were of high performance liquid chromatography (HPLC) grade and were purchased from Sigma-Aldrich, USA.

Instrument
A Shimadzu LC-20A liquid chromatographic system equipped with a DGL-20A vacuum degasser, a dual pump, and a SIL-20A autosampler (Shimadzu, Tokyo, Japan) was used. Detection was performed using an API 4000 mass spectrometer equipped with a TurboIonSpray (ESI) Interface (Applied Biosystems, Concord, ON, Canada). The Analyst 1.5 software package (Applied Biosystems, Concord, ON, Canada) was used to control the LC-MS/MS system, and for data acquisition and processing. Fluorescence was measured by Flexstation II 96 Scanning Fluorometer (Molecular Devices, Sunnyvale, CA, USA).

LC-MS/MS Analysis
Chromatographic separations were performed using the Waters Nova-Pak ® C 18 (150 × 3.9 mm) column. The column was maintained at ambient temperature (25 °C). A post-column diverter valve was used to direct the HPLC column eluate to a waste container for the first 3.2 min of the chromatographic run and then to the ionization source. The flow rate was maintained at 0.7 mL/min and the mobile phase was used as follows: solvent A [0.1% formic acid in mixture of acetonitrile/ methanol/formic acid (50/50/0.1)] and solvent B (5 mM NH 4 Ac).
The HPLC gradient program used was as follows: (1) mobile phase B was set to 5% at 0 min; (2) a linear gradient was run to 15% B in 2.0 min; (3) a linear gradient was run to 80% B in 3.3 min; (4) a linear gradient was run to 90% B in 3.6 min; (5) a isocratic gradient was run to 90% B in 4.0 min; (6) a linear gradient was run to 15% B in 7.0 min; and (7) the solvent composition was returned to 5% B in 0.1 min for re-equilibration for 2 min. The mass transitions of the metabolites of specific substrates using API4000 LC-MS/MS were consistent with our previous papers [48,49].

P-glycoprotein Assay [49]
One vial of WM-266-4 cells was thawed and cultured in a 15 cm culture dish overnight. Cells were split the next day and transferred onto a clear bottom 96-well plate with 5.0 × 10 4 cells per well. Scutellarin was serially diluted to concentrations of 2000, 666.7, 222.2, 74.1, 24.7, and 8.2 μM with a dilution factor of 3, using culture media without serum. A volume of 50 μL of each serial dilution of scutellarin was added to the 96-well plate in triplicate. The final concentrations of scutellarin in this reaction were 1000, 333.4, 111.1, 37.1, 12.4, and 4.1 μM. Verapamil was used as a positive control at a concentration of 100 μM and 1% DMSO was used as a negative control. After 30 min of incubation time, calcein-AM diluted with 50 μL culture media without serum was added to a concentration of 1 μM and the sample was incubated for an additional 30 min. The plates were placed in the Flexstation II (Molecular Devices) and fluorescence was measured at excitation and emission wavelengths of 494 and 517 nm, respectively.

Data Analysis
The CYP inhibition potential were evaluated by measuring the formation of one or more metabolites of the test substrates. The peak area ratios of the metabolites and internal standard were acquired using the Analyst 1.5 software package (Applied Biosystems). The peak area ratios were plotted as a percentage of the relevant negative control for each reaction and the 50% inhibitory concentration (IC 50 ) values were calculated by nonlinear regression using Graphpad Prism 5.0 (San Diego, CA, USA).
The P-gp inhibitor verapamil was used as positive control at a concentration of 100 μM. The experiment was considered valid if an increase in fluorescence greater than 5-fold was observed. For P-gp inhibition, a compound with a response less than 25% of verapamil at 100 μM is normally reported as "negative" at the tested concentration. The percent response value was used to determine and report the P-gp interaction at the tested concentration. A response equal to or greater than 25% of verapamil at 100 μM was considered to be "positive" at the tested concentration.

Conclusions
In conclusion, the present study demonstrated that scutellarin showed no significant inhibitory effects on CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and P-gp, and it could not be the compound or a member of Herba Erigerontis injection which can inhibit CYP activities. Scutellarin is safe and unlikely to cause any clinically significant herb-drug interactions and thus cause the occurrence of adverse drug reactions in humans when co-adminstrated with subtrates of the six CYPs and P-gp.