**1. 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 [1–3]. 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] activities. In our previous study, after orally administering 12.9 mg/kg SPN to rats, the maximum

plasma concentration (Cmax) was approximately 13.1 ng/mL at 60 min [8]. Thus, although conclusive results are lacking, SPN is likely to be a promising drug candidate.

**Figure 1.** Chemical structure of (-)-sophoranone (SPN).

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 [11–13]. 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.

#### **2. Materials and Methods**

#### *2.1. 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, MgCl2, 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 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.

#### *2.2. 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 Km 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 N2 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<sup>+</sup>, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, 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 [12,19–21].

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.

#### *2.3. Determination of the Ki of (-)-Sophoranone on CYP2C9 Activity in Human Liver Microsomes*

Among the nine tested CYP enzymes, SPN showed the lowest IC50 value for CYP2C9 (Table 2). Based on the IC50 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.


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.

**Table 1.** Optimized mass parameters for the detection of metabolites of the nine P450-probe substrates and internal standard used in the cocktail assays.


**Table 2.** IC50 values of well-known CYP inhibitors and SPN in reversible inhibition studies using a cocktail assay (*n* = 3).

Data represent the mean ± standard deviation of triplicate. a The remaining activities at the highest concentration tested, 50 μM, were greater than 80%.

#### *2.4. 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.

#### *2.5. 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<sup>5</sup> 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 (Papp) was calculated using the following equation.

$$\mathbf{P\_{app}} = (\mathbf{V\_{r}/C\_{0}}) \times (1/\mathbf{A}) \times (\text{[Drug]}/\mathbf{t})$$

where, Vr is the volume of medium in the receiver chamber, C0 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.

#### *2.6. E*ff*ects 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 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 CO2.

#### *2.7. 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 cuto ff 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 bu ffer, phosphate-bu ffered 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.
