Lack of Correlation between In Vitro and In Vivo Studies on the Inhibitory Effects of (‒)-Sophoranone on CYP2C9 is Attributable to Low Oral Absorption and Extensive Plasma Protein Binding of (‒)-Sophoranone.

(‒)-Sophoranone (SPN) is a bioactive component of Sophora tonkinensis with various pharmacological activities. This study aims to evaluate its in vitro and in vivo inhibitory potential against the nine major CYP enzymes. Of the nine tested CYPs, it exerted the strongest inhibitory effect on CYP2C9-mediated tolbutamide 4-hydroxylation with the lowest IC50 (Ki) value of 0.966 ± 0.149 μM (0.503 ± 0.0383 μM), in a competitive manner. Additionally, it strongly inhibited other CYP2C9-catalyzed diclofenac 4′-hydroxylation and losartan oxidation activities. Upon 30 min pre-incubation of human liver microsomes with SPN in the presence of NADPH, no obvious shift in IC50 was observed, suggesting that SPN is not a time-dependent inactivator of the nine CYPs. However, oral co-administration of SPN had no significant effect on the pharmacokinetics of diclofenac and 4′-hydroxydiclofenac in rats. Overall, SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. The very low permeability of SPN in Caco-2 cells (Papp value of 0.115 × 10−6 cm/s), which suggests poor absorption in vivo, and its high degree of plasma protein binding (>99.9%) may lead to the lack of in vitro–in vivo correlation. These findings will be helpful for the safe and effective clinical use of SPN.


Introduction
(-)-Sophoranone (SPN; Figure 1), a major bioactive flavonoid isolated from the roots of Sophora tonkinensis, is used in traditional Chinese medicine for the treatment of acute pharyngolaryngeal infections and sore throat [1][2][3]. It exhibits anti-inflammatory effects by inhibiting nitric oxide production in macrophages [4] and 5-lipoxygenase activity [3]. Several studies have also demonstrated its other biological activities, such as anti-cancer [5], anti-diabetic diabetic [6], and immunomodulatory [7]  Drug-drug interactions can increase the likelihood of treatment failure or the frequency and severity of adverse events [9]. Thus, drug-drug interaction assessment is a critical component of new drug discovery and development as well as clinical practice [9,10]. The majority of known drug interactions occur because of inhibition of drug-metabolizing enzymes [11][12][13]. Among all drug-metabolizing enzymes, the cytochrome P450 (CYP) superfamily plays an important role in the oxidation of almost 90% of currently used drugs [14]. Among at least 57 human cytochrome P450 enzymes identified to date, 9 hepatic P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) have shown to play predominant roles in the metabolism of drugs and other xenobiotics [12]. Therefore, the inhibitory potential of SPN on the nine major CYP enzymes should also be investigated.
There are a few reports on the in vitro and in vivo inhibitory effects of SPN on CYP enzymes. In rats, oral administration of 5 g/kg S. tonkinensis extract over 14 days was found to increase the plasma concentrations of metoprolol, omeprazole, and bupropion. This might be attributed to the inhibition of the activities of rat CYP enzymes, CYP2D6, CYP2C19, and CYP2B6 [15]. However, these results could not directly reflect the in vivo inhibitory potential of SPN on CYP enzymes due to multiple components of the extract. Several flavonoids, including SPN, have been found to inhibit CYP3A4-mediated reactions in vitro [16].
However, currently, there is limited information about SPN's in vitro inhibitory potentials, especially on the other eight CYP enzymes, thereby warranting further in vitro and in vivo investigations to improve our understanding of drug interactions with SPN. Using human liver microsomes in this study, we evaluated SPN's potential to inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in a reversible and time-dependent manner. We report herein that SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. To explain this lack of correlation between in vitro and in vivo results, we performed plasma protein binding of SPN and permeability test using Caco-2 cells.

Chemicals and Reagents
Pooled human liver microsomes from 150 donors (75 males; 75 females) were purchased from Corning Life Sciences (Woburn, MA, USA), and (-)-sophoranone (99.7% purity; SPN) was supplied by SK Chemicals Ltd. (Sungnam, Gyeonggi-do, Korea). β-Nicotinamide adenine dinucleotide phosphate disodium salt (NADP), glucose 6-phosphate disodium salt hydrate, glucose 6-phosphate dehydrogenase, MgCl 2 , and all chemicals including the specific substrates, its metabolites, and well-known inhibitors of nine P450s were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), Santa Cruz Biotechnology (Dallas, TX, USA), or Cayman Chemicals (Ann Arbor, MI, USA) unless stated otherwise. The purity of all purchased compounds was higher than 97.0%. HPLC-grade Pharmaceutics 2020, 12, 328 3 of 17 acetonitrile and methanol were obtained from Burdick & Jackson Company (Morristown, NJ, USA). Caco-2 cells were supplied by the Korean Cell Line Bank (Seoul, Korea) and cultured according to the supplier's recommendations. Transwell (24-well, 6.5 mm polycarbonate inserts, 0.4-µm pore) and cell culture reagents were purchased from Corning Life Sciences. Heparinized human plasma was obtained from donors at the Severance Hospital of Yonsei University Health System (Seoul, Korea) and stored at −80 • C prior to use.

Reversible Inhibition of (-)-Sophoranone towards the Nine CYP Isoforms in Human Liver Microsomes
The inhibitory effects of SPN on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were evaluated in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay), as previously described [17,18] with a slight modification. Concentrations of each CYP probe in Table 1 were used close to their reported K m values [17,18].
Briefly, a 90-µL incubation mixture, including pooled human liver microsomes (final concentration 0.1 mg/mL), 50 mM phosphate buffer (pH 7.4), each CYP-probe substrate cocktail set, and SPN (0-50 µM), was pre-incubated for 5 min at 37 • C. SPN was dissolved in methanol and spiked into the incubation mixture to a final concentration of 0.5% methanol. All P450-selective substrates (except coumarin due to solubility) were dissolved in methanol and serially diluted with methanol to the required concentrations, and the organic solvent was subsequently evaporated under a gentle stream of N 2 gas to minimize the effects of organic solvents on CYP activities. On the other hand, coumarin dissolved in 50 mM phosphate buffer (pH 7.4) was directly added into the mixed tube. The reaction was initiated by adding 10-µL aliquot of NADPH-generating system (1.3 mM NADP + , 3.3 mM glucose 6-phosphate, 3.3 mM MgCl 2 , and 0.4 unit/mL glucose-6-phosphate dehydrogenase) before 15 min incubation at 37 • C in a shaking water bath. After incubation, the reactions were stopped by adding 200 µL of ice-cold acetonitrile containing 2 µM chlorpropamide as an internal standard. The incubation mixtures were centrifuged (16,000× g, 15 min) and 5 µL of the supernatant was injected into the LC-MS/MS system. All incubations were performed in triplicate, and the data are shown as the mean ± standard deviation. Incubation samples containing well-known CYP inhibitors for each isozyme ( Table 2) in parallel were included to compare inhibitory effects, all of which appear on the US FDA list of recommended or accepted in vitro inhibitors [12,[19][20][21].
Additionally, to determine whether the inhibition of CYP2C9 by SPN was substrate specific, we also examined SPN's inhibitory effects on other CYP2C9-specific biotransformation pathways (i.e., diclofenac 4 -hydroxylation and losartan oxidation) in human liver microsomes [22,23]. Diclofenac and losartan were used at 5 µM, respectively, and other procedures were similar to those of cocktail assays.

Determination of the K i of (-)-Sophoranone on CYP2C9 Activity in Human Liver Microsomes
Among the nine tested CYP enzymes, SPN showed the lowest IC 50 value for CYP2C9 (Table 2). Based on the IC 50 values, the K i values of SPN on CYP2C9 activity were determined. Briefly, K i values were obtained by incubating various concentrations of two CYP2C9 probe substrates (50, 100, and 150 µM tolbutamide; or 2, 5, and 10 µM diclofenac) in the presence of 0−5 µM SPN or 0−2 µM sulfaphenazole, a well-known typical CYP2C9 inhibitor. Other procedures were similar to those of the reversible inhibition studies. All incubations were performed in triplicate, and the data are shown as the mean ± standard deviation. The optimized ion spray voltage was 4 kV and a nebulizing gas flow of 3 L/min, heating gas flow of 10 L/min, an interface temperature of 300 • C, desolvation line temperature of 250 • C, heating block temperature of 400 • C, and a drying gas flow rate of 10 L/min. a ESI, electrospray ionization mode; b CE, collision energy.

Time-Dependent Inactivation of (-)-Sophoranone toward the Nine CYP Isoforms in Human Liver Microsomes
Pooled human liver microsomes (1 mg/mL) were incubated with SPN (0−50 µM) for 30 min at 37 • C in the absence or presence of an NADPH-generating system (i.e., the "inactivation incubation"). After inactivation incubation, aliquots (10 µL) were transferred into fresh incubation tubes (final volume 100 µL) containing an NADPH-generating system and each P450-selective substrate cocktail set. The reaction mixtures were incubated for 15 min at 37 • C in a shaking water bath. After incubation, the reactions were stopped by adding 200 µL of ice-cold acetonitrile containing 2 µM chlorpropamide, as an internal standard. The incubation mixtures were centrifuged (16,000× g, 15 min) and 5 µL of the supernatant was injected into the LC-MS/MS system. All incubations were performed in triplicate, and the data are shown as the mean ± standard deviation.

Caco-2 Cell Permeability of (-)-Sophoranone
Caco-2 cell permeability was assessed to predict the oral absorption of SPN. Cell culture and transport studies were performed as previously described [24,25]. Briefly, for the bi-directional transport studies, the cells were seeded at a density of 1 × 10 5 cells/well, and the cell medium was replaced until they formed confluent monolayers. On the 25th day, the cell monolayers were washed with pre-warmed HBSS buffer. The bi-directional permeability assay was instigated by adding 10 µM for propranolol, or 10 µM and 50 µM for SPN in HBSS to an apical well (200 µL) for apical (A) to basolateral (B) transport or to a basolateral insert (800 µL) for the B to A transport. Before the experiment, the integrity of the cell monolayers was evaluated by measuring the transepithelial electrical resistance using a Millicell ohmmeter. After 2 h incubation at 37 • C, samples were withdrawn from both sides, respectively. All samples were stored at −80 • C until LC-MS/MS analysis, and all experiments were performed in triplicate.
The apparent permeability coefficient (P app ) was calculated using the following equation.
where, V r is the volume of medium in the receiver chamber, C 0 is the donor compartment concentration at time zero, A is the area of the cell monolayer, t is the treatment time of the drug, and [Drug] is the drug concentration in the receiver chamber.

Effects of (-)-Sophoranone on the Pharmacokinetics of Diclofenac in Rats
In this study, we investigated whether SPN, an in vitro potent inhibitor of CYP2C9, affects the pharmacokinetics of diclofenac in rats. Male Sprague-Dawley rats (8 weeks, 270-290 g) were purchased Pharmaceutics 2020, 12, 328 6 of 17 from Orient Bio (Sungnam, Gyeonggi-do, Korea), and the protocol for pharmacokinetic interaction studies in rats was approved by the Institutional Animal Care and Use Committee (IACUC-CUK) at The Catholic University of Korea (Approval No. 2019-021, approved 31 May 2019). The procedures used for housing and handling were previously reported [18]. Before administration, rats were fasted for 12 h with free access to water. The carotid arteries of each rat were cannulated with a polyethylene tube (Clay Adams, Franklin Lakes, NJ, USA) for blood sampling. Each rat was individually housed in a rat metabolic cage and allowed to recover from anesthesia for 4-5 h prior to the start of the experiment. The rats were divided into two groups: (1) diclofenac alone (n = 6) and (2) SPN and diclofenac co-administration (n = 6). SPN was suspended in dimethylsulfoxide:PEG400:distilled water (5:60:35, v/v/v) and administered by oral gavage at a dose of 75 mg/kg in a volume of 5 mL/kg. Fifteen minutes after oral administration of SPN, 2 mg/kg diclofenac was dissolved in normal saline and administered by oral gavage. Approximately 0.25 mL of blood from each rat was collected into an Eppendorf tube before diclofenac dosing (0 min), and at 3, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min post-dosing. The blood samples were immediately centrifuged at 13,000× g for 5 min at 4 • C. The plasma samples were divided into two Eppendorf tubes by 50 µL and stored at −80 • C until LC-MS/MS analysis. After the experiments, the rats were euthanized with CO 2 .

Determination of the Unbound Fraction of (-)-Sophoranone in Plasma and Human Liver Microsomes
The plasma or liver microsomal protein bindings were performed using a rapid equilibrium dialysis device and cellulose membranes with a molecular weight cutoff of 8000 (Thermo Scientific, Rockford, IL, USA) [17]. The rat and human plasma samples (200 µL) containing SPN at 10 and 50 µM, respectively, were dialyzed against a dialysis buffer, phosphate-buffered saline (PBS, 400 µL). The loaded dialysis plate was covered with sealing tape, placed on an orbital shaker at approximately 200 rpm, and incubated at 37 • C for 4 h. Thereafter, samples (100 µL) from both PBS and plasma chambers were collected and mixed with an equal volume of blank plasma and PBS, respectively. All samples were stored at −80 • C until LC-MS/MS analysis. The unbound fraction of SPN in human (or rat) plasma was calculated by dividing the SPN concentration in PBS by that in plasma.
The human liver microsomal incubation mixtures (final concentration 0.1 mg/mL) without NADPH generating system were used to determine the unbound fraction of SPN. Other procedures were similar to those of plasma protein binding assay.

In Vitro Samples
Metabolites of nine P450-selective substrates were analyzed using a Shimadzu Nexera X2 UPLC system coupled to an LCMS-8050 triple quadruple mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with an electrospray ionization interface as previously described with a slight modification [17,18]. Separation was performed on a reversed-phase column (Luna C 18 , 50 mm × 2.0 mm i.d.; 3 µm particle size; Phenomenex, Torrance, CA, USA) maintained at 40 • C. The mobile phase consisted of distilled water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), with a flow rate of 0.5 mL/min. The gradient elution program used was as follows: (1) Mobile phase A was set to 95% at 0 min, (2) a linear gradient was run to 5% in 2.6 min, and (3) a linear gradient was run to 95% in 3.0 min and re-equilibrated for 2 min. The total run time was 5 min. The optimized compound-dependent parameters of the metabolites of the nine P450-selective substrates and the internal standard are listed in Table 1. Three-day validations were performed to confirm the effectiveness of the LC-MS/MS system for simultaneous determination of the nine P450-selective substrate metabolites at the respective ranges of 0.01-10 µM in blank microsomal incubation mixtures. We found that the precision (≤12.1%) and accuracy (95.4-110.2%) values were within acceptable ranges. Supplemental Figure S1 shows the representative LC-MS/MS chromatograms of a human liver microsomal incubation sample containing nine P450-selective metabolites and an internal standard. The auto-optimized mass transitions were m/z 312 > 231 and m/z 437 > 207.1 for quantification of 4 -hydroxydiclofenac and losartan carboxylic acid, respectively. HPLC conditions were the same as those in the cocktail assay.

In Vivo Samples
The plasma concentrations of diclofenac and 4 -hydroxydiclofenac were determined by a previously reported LC-MS/MS method [26] with some modifications. Briefly, 50 µL aliquots of plasma were extracted with 300 µL aliquots of acetonitrile containing chlorpropamide (internal standard), followed by LC-MS/MS (Shimadzu Corporation). Chromatographic separation was performed on a Phenomenex Luna C 18 column (100 × 2.00 mm; 3.0 µm). The isocratic mobile phase consisted of 0.1% formic acid in distilled water (A) and 0.1% formic acid in acetonitrile (B) (45:55, v/v), with a flow rate of 0.3 mL/min. The transitions were m/z 296.0 > 214.0 for diclofenac, m/z 312 > 231 for 4 -hydroxydiclofenac, and m/z 277 > 111 for the internal standard. The data acquisition was computed using LabSolutions LCMS Ver.5.6 (Shimadzu Corporation). The calibration curves for diclofenac and 4 -hydroxydiclofenac were linear (r ≥ 0.996) over the concentration range of 20-5000 ng/mL.
The LC-MS/MS condition for the determination of SPN in plasma was the same with a previously reported method [27]. The calibration curve for SPN was linear (r ≥ 0.995) over the concentration range of 1-250 ng/mL.

Analysis of Inhibition Kinetics and Pharmacokinetic Parameters
The IC 50 values were calculated via nonlinear least-squares regression analysis from logarithmic plots of inhibitor concentration versus percentage of activity remaining after inhibition, using SigmaPlot (ver. 14.0; Systat Software Inc, Chicago, IL, USA). The K i values were determined from the equations for a single substrate single inhibitor model and the software available in the SigmaPlot Enzyme Kinetics module. Competitive, non-competitive, uncompetitive, or mixed inhibition models were evaluated and ranked according to the best fit based on Akaike Information Criterion (AIC) values. For visual inspection, the data were presented as Dixon plots.
Pharmacokinetic parameters were calculated by a non-compartmental analysis using WinNonlin Professional software (version 5.2, Pharsight Corp., Mountain View, CA, USA) that used the total area under the plasma concentration-time curve from time zero to infinity (AUC ∞ ) or the last measured time (AUC t ). The logarithmic trapezoidal rule was used during the declining plasma level phase and the linear trapezoidal rule was used for the rising plasma-level phase. The peak plasma concentration (C max ) and time to reach C max (T max ) were read directly from the experimental data. Statistically significant differences were recognized at p < 0.05.
To determine whether the inhibitory effects of SPN on CYP2C9 was substrate specific, we examined the inhibitory effects on other CYP2C9-specific biotransformation pathways (i.e., diclofenac 4′-hydroxylation and losartan oxidation) and found that SPN also markedly inhibited their activities, with IC50 values of 0.879 ± 0.0888 μM and 0.455 ± 0.0486 μM, respectively, ( Figure 3).  To determine whether the inhibitory effects of SPN on CYP2C9 was substrate specific, we examined the inhibitory effects on other CYP2C9-specific biotransformation pathways (i.e., diclofenac 4 -hydroxylation and losartan oxidation) and found that SPN also markedly inhibited their activities, with IC 50 values of 0.879 ± 0.0888 µM and 0.455 ± 0.0486 µM, respectively, (Figure 3). Pharmaceutics 2020, 12

Determination of the Ki of (-)-Sophoranone for CYP2C9 Activity
Based on the lowest IC50 value for CYP2C9, to characterize the type of reversible inhibition of CYP2C9 by SPN, enzyme kinetic experiments were performed in the presence of various concentrations of SPN and tolbutamide, or diclofenac. Otherwise, identical samples containing a known potent CYP2C9 inhibitor (sulfaphenazole), were included in the analysis. Representative Dixon plots of CYP2C9 inhibition by SPN and sulfaphenazole in human liver microsomes are shown in Figure 4, and the Ki values are summarized in Supplemental Table S1. Using a nonlinear regression analysis, SPN demonstrated competitive inhibition against CYP2C9-catalyzed tolbutamide hydroxylation or diclofenac hydroxylation, with calculated Ki values of 0.503 ± 0.0383 μM and 0.587 ± 0.0470 μM ( Figure 4A,B). Sulphafenazole competitively inhibited CYP2C9 with a Ki value of 0.267 ± 0.0170 μM ( Figure 4C), which was similar to a previously reported value [28].

Determination of the K i of (-)-Sophoranone for CYP2C9 Activity
Based on the lowest IC 50 value for CYP2C9, to characterize the type of reversible inhibition of CYP2C9 by SPN, enzyme kinetic experiments were performed in the presence of various concentrations of SPN and tolbutamide, or diclofenac. Otherwise, identical samples containing a known potent CYP2C9 inhibitor (sulfaphenazole), were included in the analysis. Representative Dixon plots of CYP2C9 inhibition by SPN and sulfaphenazole in human liver microsomes are shown in Figure 4, and the Ki values are summarized in Supplemental Table S1. Using a nonlinear regression analysis, SPN demonstrated competitive inhibition against CYP2C9-catalyzed tolbutamide hydroxylation or diclofenac hydroxylation, with calculated K i values of 0.503 ± 0.0383 µM and 0.587 ± 0.0470 µM ( Figure 4A,B). Sulphafenazole competitively inhibited CYP2C9 with a K i value of 0.267 ± 0.0170 µM ( Figure 4C), which was similar to a previously reported value [28].

Determination of the Ki of (-)-Sophoranone for CYP2C9 Activity
Based on the lowest IC50 value for CYP2C9, to characterize the type of reversible inhibition of CYP2C9 by SPN, enzyme kinetic experiments were performed in the presence of various concentrations of SPN and tolbutamide, or diclofenac. Otherwise, identical samples containing a known potent CYP2C9 inhibitor (sulfaphenazole), were included in the analysis. Representative Dixon plots of CYP2C9 inhibition by SPN and sulfaphenazole in human liver microsomes are shown in Figure 4, and the Ki values are summarized in Supplemental Table S1. Using a nonlinear regression analysis, SPN demonstrated competitive inhibition against CYP2C9-catalyzed tolbutamide hydroxylation or diclofenac hydroxylation, with calculated Ki values of 0.503 ± 0.0383 μM and 0.587 ± 0.0470 μM ( Figure 4A,B). Sulphafenazole competitively inhibited CYP2C9 with a Ki value of 0.267 ± 0.0170 μM ( Figure 4C), which was similar to a previously reported value [28].

Time-Dependent Inactivation of (-)-Sophoranone towards the Nine CYP Isoforms in Human Liver Microsomes
The IC 50 shift method incorporating a dilution is one of the most efficient and convenient methods for evaluating time-dependent inhibitory effects. A shift in IC 50 to a lower value ("shift") with pre-incubation indicates time-dependent inactivation [29][30][31]. After 30 min pre-incubation of SPN with human liver microsomes in the presence of NADPH, no obvious shift in IC 50 was observed for inhibition of the nine CYPs ( Figure 5), suggesting that SPN is not a time-dependent inactivator for the nine CYPs.

Time-Dependent Inactivation of (-)-Sophoranone towards the Nine CYP Isoforms in Human Liver Microsomes
The IC50 shift method incorporating a dilution is one of the most efficient and convenient methods for evaluating time-dependent inhibitory effects. A shift in IC50 to a lower value ("shift") with pre-incubation indicates time-dependent inactivation [29][30][31]. After 30 min pre-incubation of SPN with human liver microsomes in the presence of NADPH, no obvious shift in IC50 was observed for inhibition of the nine CYPs ( Figure 5), suggesting that SPN is not a time-dependent inactivator for the nine CYPs.   Figure 5. Time-dependent inhibition curves of SPN on the nine major P450 activities in human liver microsomes using substrate cocktails after 30 min pre-incubation with the presence (•) or absence (○) of an NADPH-generating system. Data are the mean ± standard deviation of triplicate incubations.

Effects of (-)-Sophoranone on the Pharmacokinetics of Diclofenac in Rats
We conducted pharmacokinetic studies to investigate the effects of SPN on the pharmacokinetics of diclofenac in rats. Findings in the literature on the dried S. tonkinensis herbs indicate that a recommended daily dose for an adult human with the body weight of 60 kg were to be 6-10 g [32], which correlated to the equivalent dose ranges in rats, 0.620-1.03 g/kg [33]. He et al. [2] reported that the average contents of SPN in various S. tonkinensis samples were found to be approximately 2.53 mg/g (0.0253%). Reflecting this content, the dosage in rats, 0.620-1.03 g/kg of the dried herb, might be consistent with 15.7-26.1 mg/kg in terms of SPN. Thus, in this study, the SPN dose of 75 mg/kg was used in rats, which is approximately 2.87-to 4.87-fold greater than the recommended human dose.
The mean plasma concentration-time profiles of diclofenac and 4-hydroxydiclofenac after oral administration of diclofenac (2 mg/kg) in the absence or presence of oral co-administration of SPN (75 mg/kg) in rats are illustrated in Figure 6, and the relevant pharmacokinetic parameters are shown in Table 3. The plasma levels of diclofenac and 4-hydroxydiclofenac were similar in both groups ( Figure 6A,B). Likewise, no significant differences were observed in any other pharmacokinetic parameter of diclofenac and 4 -hydroxydiclofenac ( Table 3). The in vivo marker for CYP2C9 activity, expressed as the molar AUC ratio of 4 -hydroxydiclofenac to diclofenac, was not significant (0.799 ± 0.167 versus 0.904 ± 0.0534; p value of 0.215) in the presence or absence of SPN (Table 3). In the treatment group with co-administration of SPN, the C max of SPN was found to be 33.7 ± 14.8 ng/mL (0.0732 ± 0.0321 µM) at approximately 60-75 min post-dose ( Figure 6C). Given the K i values of SPN on CYP2C9 activity (0.503 ± 0.0383 µM for tolbutamide hydroxylation and 0.587 ± 0.0470 µM for diclofenac hydroxylation), the plasma concentrations of SPN are too low to inhibit CYP2C9-mediated metabolism of diclofenac in vivo. Overall, the co-administration of SPN did not alter the pharmacokinetics of diclofenac and 4 -hydroxydiclofenac.

Effects of (-)-Sophoranone on the Pharmacokinetics of Diclofenac in Rats
We conducted pharmacokinetic studies to investigate the effects of SPN on the pharmacokinetics of diclofenac in rats. Findings in the literature on the dried S. tonkinensis herbs indicate that a recommended daily dose for an adult human with the body weight of 60 kg were to be 6-10 g [32], which correlated to the equivalent dose ranges in rats, 0.620-1.03 g/kg [33]. He et al. [2] reported that the average contents of SPN in various S. tonkinensis samples were found to be approximately 2.53 mg/g (0.0253%). Reflecting this content, the dosage in rats, 0.620-1.03 g/kg of the dried herb, might be consistent with 15.7-26.1 mg/kg in terms of SPN. Thus, in this study, the SPN dose of 75 mg/kg was used in rats, which is approximately 2.87-to 4.87-fold greater than the recommended human dose.
The mean plasma concentration-time profiles of diclofenac and 4-hydroxydiclofenac after oral administration of diclofenac (2 mg/kg) in the absence or presence of oral co-administration of SPN (75 mg/kg) in rats are illustrated in Figure 6, and the relevant pharmacokinetic parameters are shown in Table 3. The plasma levels of diclofenac and 4-hydroxydiclofenac were similar in both groups ( Figure 6A,B). Likewise, no significant differences were observed in any other pharmacokinetic parameter of diclofenac and 4′-hydroxydiclofenac ( Table 3). The in vivo marker for CYP2C9 activity, expressed as the molar AUC ratio of 4′-hydroxydiclofenac to diclofenac, was not significant (0.799 ± 0.167 versus 0.904 ± 0.0534; p value of 0.215) in the presence or absence of SPN (Table 3). In the treatment group with co-administration of SPN, the Cmax of SPN was found to be 33.7 ± 14.8 ng/mL (0.0732 ± 0.0321 μM) at approximately 60-75 min post-dose ( Figure 6C). Given the Ki values of SPN on CYP2C9 activity (0.503 ± 0.0383 μM for tolbutamide hydroxylation and 0.587 ± 0.0470 μM for diclofenac hydroxylation), the plasma concentrations of SPN are too low to inhibit CYP2C9-mediated metabolism of diclofenac in vivo. Overall, the co-administration of SPN did not alter the pharmacokinetics of diclofenac and 4′-hydroxydiclofenac.

Determination of the Unbound Fraction of (-)-Sophoranone in Plasma and Human Liver Microsomes
SPN was extensively bound to plasma proteins, regardless of species. The free fractions (%) of SPN at 10 and 50 µM in human plasma were 0.0457 ± 0.00612% and 0.0927 ± 0.0400%, respectively, (n = 3, each). Similarly, when 10 and 50 µM SPN were added to the rat plasma, the free fractions were 0.0380 ± 0.0102% and 0.0531 ± 0.0149%, respectively, (n = 3, each). After adding 10 and 50 µM SPN to rat and human plasma, free fractions remained relatively unchanged, suggesting that SPN has no binding saturation in plasma.
SPN also exhibited marked non-specific bindings to human liver microsomes, although to a lesser extent than those in human plasma. The unbound fractions of SPN at 10 and 50 µM were calculated to be 0.621 ± 0.0405% and 0.724 ± 0.170%, respectively (n = 3, each), at a microsomal protein concentration of 0.1 mg/mL.

Discussion
This study focused on the in vitro and in vivo inhibitory effects of SPN on human CYPs, especially CYP2C9. We screened the inhibitory effects of SPN on the major human CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in human liver microsomes. Of the nine tested CYP isoforms, SPN exerted the strongest inhibitory effect on CYP2C9 activity, with the lowest IC 50 value of 0.966 ± 0.149 µM (Table 2; Figure 2). In addition to CYP2C9, SPN mildly inhibited several CYP enzymes, with potency ranked in the order CYP2C8 > CYP2C19; the IC 50 values were 13.6 ± 3.15 µM and 16.8 ± 3.21 µM, respectively (Table 2; Figure 2). Although the IC 50 values could not been calculated, SPN also appears to weakly inhibit CYP2D6 and CYP3A4; the residual enzyme activities at the highest tested concentration (50 µM) were 53.9 ± 3.53% and 53.3 ± 4.00%, respectively ( Figure 2). No apparent inhibition of the other CYPs (CYP1A2, CYP2A6, CYP2B6, and CYP2E1) was observed (Figure 2). SPN also strongly inhibited other CYP2C9-catalyzed diclofenac 4 -hydroxylation and losartan oxidation activities (Figure 3). The inhibition mechanisms of SPN on CYP2C9-catalyzed tolbutamide 4-hydroxylation and diclofenac 4 -hydroxylation activities were both competitive, with K i values of 0.503 ± 0.0383 µM and 0.587 ± 0.0470 µM, respectively. Pre-incubation of SPN for 30 min with human liver microsomes and an NADPH-generating system did not alter the inhibition potencies against the nine CYPs, suggesting that SPN is not a time-dependent inactivator.
The reversible inhibition of SPN-mediated CYP3A4 activity was less consistent with the published literature. Li et al. [16] reported that among 44 tested flavonoids, SPN inhibited CYP3A4-catalyzed bufalin 5 -hydroxylation activity with a K i value of 2.17 ± 0.29 µM. They only focused on the in vitro inhibitory potentials of several flavonoids against CYP3A4 activity. To the best of our knowledge, to date, bufalin has not been used as the in vitro probe substrate for the CYP3A4 activity, and the reference material of 5 -hydroxybufalin is not commercially available. Because of the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4-mediated drug-drug interaction studies, including midazolam, nifedipine, and testosterone [34]. In that study, when other CYP3A4 substrates were tested, the ranges of IC 50 values by SPN were reported to be 5.62-38.4 µM [16]. Additionally, we examined the inhibitory effect on another CYP3A4-catalyzed testosterone 6β-hydroxylation and found that SPN also inhibited the activity with an IC 50 value of 31.5 ± 4.79 µM, which showed a higher percentage inhibition compared to midazolam (data not shown). Altogether, the in vitro CYP3A4 inhibition by SPN seemed to be substrate-specific.
Generally, alterations in the activities of hepatic CYPs through in vitro inhibition or induction represent the major mechanisms underlying pharmacokinetic drug-drug interactions [11][12][13]. It has been estimated that CYP2C9 is responsible for the metabolic clearance of up to 15-20% of all drugs undergoing phase I metabolism, including clinically important drugs such as S-warfarin, phenytoin, tolbutamide, losartan, and several anti-inflammatory drugs [23,35]. Considering that SPN is a potent CYP2C9 inhibitor in vitro, there may be potential for herb-drug interactions between SPN and CYP2C9 substrates after concomitant oral administration.
Using the in vitro reversible inhibition results, a clinical drug-drug interaction risk was initially predicted by the basic static model approach, as recommended by the EMA [36] and US FDA [37] with calculating the R 1 value (R 1 = 1 + [I max,u /K i,u ]), which representing the predicted AUC ratio in the presence or absence of inhibitor. Where, I max,u (C max,u ) is maximal free plasma concentration of the inhibitor and K i,u is the unbound in vitro inhibition constant. However, little information is yet to be reported on the C max values of SPN after oral administration of SPN. As stated in the Introduction, from our previous study, the C max of SPN was reported to be 13.1 ng/mL in rats after oral dosing of 12.9 mg/kg SPN in rats [8]. Thus, we investigated whether SPN affects the pharmacokinetics of diclofenac and 4 -hydroxydiclofenac, produced by hepatic CYP2C9 enzyme, in rats. In the group that received co-administration of SPN (75 mg/kg), the C max of SPN was found to be 33.7 ± 14.8 ng/mL (0.0732 ± 0.0321 µM) at 60-75 min ( Figure 6C). These results suggest that SPN has low oral bioavailability. The calculated values of I max, u and K i,u for SPN used in this study were 0.0420 ± 0.0184 nM and 3.39 ± 0.258 nM (3.95 ± 0.316 nM for diclofenac 4 -hydroxylation), respectively. Considering these values, the R 1 value of SPN for the inhibition of CYP2C9 in vitro was calculated as 1.0124 (K i , u for tolbutamide 4-hydroxylation) or 1.0106 (K i, u for diclofenac 4 -hydroxylation) which are both below the EMA and US FDA cut-off criteria of R 1 , 1.02 [36,37], indicating that the potential for clinically relevant drug interaction-mediated CYP2C9 inhibition by SPN may be low and no clinical interaction studies are warranted. In our results, also no significant differences were observed in any of the other pharmacokinetic parameters of diclofenac and 4 -hydroxydiclofenac in rats in the absence or presence of oral co-administration of SPN at a dose of 75 mg/kg (Table 3). Furthermore, the molar metabolic conversion ratio, expressed as AUC 4 -hydroxydiclofenac /AUC diclofenac , which indicated a causal factor for the evaluation of the capacities of CYP2C9 activity in vivo, did not show significant differences (0.799 ± 0.167 versus 0.904 ± 0.0534) in both groups (Table 3).
To explain the lack of in vitro-in vivo correlation, we assessed two factors that could limit the accuracy of in vitro models in predicting metabolic drug interactions in vivo, which were SPN's degree of plasma protein binding and its permeability in Caco-2 cells. We found that SPN was extensively bound in both human and rat plasma proteins (>99.9%) with a mean unbound fraction value of 0.0574% in the range of 10 and 50 µM. Thus, taking the plasma protein binding of SPN into account, the unbound maximum concentrations of SPN in plasma might be 0.0420 ± 0.0184 nM, which is much lower than the unbound K i values of SPN in vitro. Some drugs that indicate in vitro-in vivo discrepancy because of high plasma protein bindings have been reported [38][39][40]. Tolfenamic acid strongly inhibited CYP1A2 in vitro but not in vivo because of high plasma protein binding (99.7%) [38]. Montelukast is a very potent inhibitor of CYP2C8 in vitro with K i values ranging from 0.0092-0.15 µM [41]. However, in humans, montelukast has had no effect on the pharmacokinetics of the CYP2C8 substrates, pioglitazone [39] and rosiglitazone [40]. The high degree of protein binding of montelukast in plasma (>99.7%) is similar to that of tolfenamic acid and explicitly explains the lack of its in vivo interaction, irrespective of its strong inhibitor potency in vitro. The Caco-2 cell model is widely used to predict the absorption across the intestinal barrier, and a good correlation between its oral absorption in humans and its apparent permeability (P app ) across the Caco-2 cell barrier has been shown [24,25]. A recent study has provided some updated guidelines on how permeability values might correlate with human oral absorption: Low permeability (0-20% absorbed) is correlated to P app values < 1-2 × 10 −6 cm/s; moderate permeability (20-80% absorbed) to P app values < 2-10 × 10 −6 cm/s; and high permeability (80-100% absorbed) to P app values > 10 × 10 −6 cm/s [42]. Propranolol had >90% human absorption and exhibited high permeability with a P app value of (26.8 ± 3.31) × 10 −6 cm/s in our assay. SPN exhibited a very low permeability with mean P app values of 0.115 × 10 −6 cm/s (0.429% of propranolol P app ) and 0.172 × 10 −6 cm/s (0.642% of propranolol P app ) at 10 and 50 µM, respectively, indicating that it is poorly absorbed in vivo. SPN was not a substrate for efflux transporters, that is, P-gp and BCRP, as the efflux ratio (B-to-A/A-to-B) is less than 2.
Overall, SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. This apparent discrepancy is due to the extensive plasma protein binding and very low permeability of SPN, which resulted in poor oral absorption. These approaches could help in making more reliable in vitro-in vivo extrapolations about the risk of in vivo inhibition potential. In conclusion, these findings have provided useful information on the safe and effective use of SPN in clinical practice.

Supplementary Materials:
The following is available online at http://www.mdpi.com/1999-4923/12/4/328/s1, Figure S1: Typical LC-MS/MS chromatograms for the formed metabolites of the nine CYP-specific probe in human liver microsomes and their internal standard in the positive electrospray ionization mode. Table S1: K i values and inhibition types for CYP2C9 by SPN and sulfaphenazole in human liver microsomes (n = 3).