Strong and Selective Inhibitory Effects of the Biflavonoid Selamariscina A against CYP2C8 and CYP2C9 Enzyme Activities in Human Liver Microsomes.

Like flavonoids, biflavonoids, dimeric flavonoids, and polyphenolic plant secondary metabolites have antioxidant, antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. However, there is limited data on their effects on cytochrome P450 (P450) and uridine 5'-diphosphoglucuronosyl transferase (UGT) enzyme activities. In this study we evaluate the inhibitory potential of five biflavonoids against nine P450 activities (P450s1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A) in human liver microsomes (HLMs) using cocktail incubation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most strongly inhibited P450 activity was CYP2C8-mediated amodiaquine N-dealkylation with IC50 ranges of 0.019~0.123 μM. In addition, the biflavonoids-selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone-noncompetitively inhibited CYP2C8 activity with respective Ki values of 0.018, 0.083, 0.084, 0.103, and 0.142 μM. As selamariscina A showed the strongest effects, we then evaluated it against six UGT isoforms, where it showed weaker inhibition (UGTs1A1, 1A3, 1A4, 1A6, 1A9, and 2B7, IC50 1.7 μM). Returning to the P450 activities, selamariscina A inhibited CYP2C9-mediated diclofenac hydroxylation and tolbutamide hydroxylation with respective Ki values of 0.032 and 0.065 μM in a competitive and noncompetitive manner. However, it only weakly inhibited CYP1A2, CYP2B6, and CYP3A with respective Ki values of 3.1, 7.9, and 4.5 μM. We conclude that selamariscina A has selective and strong inhibitory effects on the CYP2C8 and CYP2C9 isoforms. This information might be useful in predicting herb-drug interaction potential between biflavonoids and co-administered drugs mainly metabolized by CYP2C8 and CYP2C9. In addition, selamariscina A might be used as a strong CYP2C8 and CYP2C9 inhibitor in P450 reaction-phenotyping studies to identify drug-metabolizing enzymes responsible for the metabolism of new chemicals.


Introduction
Flavonoids are polyphenolic secondary metabolites that are common in the plant kingdom and are ingested by humans in their food [1]. Flavonoids are grouped into various classes based on structure. These classes are: anthocyanidins, chalcones, flavanones, flavones, flavonols, isoflavonoids, and biflavonoids [2]. Many pharmacological benefits have been ascribed to flavonoids, including antioxidant, anti-inflammatory, anti-cancer, antiviral, and hepatoprotective effects [3,4].
Having flavonoids in your diet may reduce the risk of atherosclerosis, cardiovascular disease, diabetes mellitus, osteoporosis, and certain cancers [4,5]. Because of flavonoids' benefits and wide distribution, their intake has risen steadily in recent years in the West and Asia. Daily intake of flavonoids has been estimated at 100 mg/day in the Asian population because of the high consumption of soy products [6,7]. On the other hand, daily intake of flavonoids has been estimated to be in the range of 20-50 mg/day in Western populations [8]. Further intake of flavonoids through dietary supplements and plant extracts with prescribed drugs is common. The vast body of literature describes the significant interactions between flavonoid herbs and therapeutic drugs.
Several flavonoids are substrates for cytochrome P450 (P450) and uridine 5'diphosphoglucuronosyl transferase (UGT) enzymes [2], suggesting that flavonoids could inhibit the activities of these enzymes. A number of studies have demonstrated that flavonoids are potent inhibitors of CYP1A2, CYP3A, and UGT1A1 in vitro [5,8]. For example, the flavone tangeretin competitively inhibits the activity of CYP1A2 with a Ki value as low as 68 nM in human liver microsomes (HLMs) [9]. It also inhibits UGT1A1-mediated estradiol glucuronidation with an IC50 value of 1 M [10]. The flavonols quercetin and kaempferol inhibit the metabolism of nifedipine and felodipine by CYP3A4 in HLMs at concentrations larger than 10 M. [11]. Animal studies show that oral quercetin increases the bioavailability of oral doxorubicin [12]. These results can be attributed to the reduced first-pass metabolism of doxorubicin due to quercetin-induced inhibition of CYP3A and/or enhanced doxorubicin absorption in the gastrointestinal tract via quercetin-induced inhibition of P-glycoprotein (P-gp). Surya Sandeep et al. (2014) reported that naringenin significantly increases the bioavailability of orally administered felodipine, a P-glycoprotein and CYP3A4 substrate drug, in rats, through the inhibition of intestinal P-gp and CYP3A4 [13]. Alnaqeeb et al. (2019) reported that quercetin and guava leaf extracts in combination with warfarin exert a greater increase on warfarin's Cmax and International Normalized Ratio values than when used alone, indicating the inhibition of CYP2C8, 2C9 and 3A4, major warfarin-metabolizing enzymes [14]. Biflavonoids, formed by the covalent bond between two monoflavonoids, are a subclass of flavonoid [15]. They are secondary metabolites, but are limited to several species in plants such as Ginkgo biloba, Selaginella species, Hypericum perforatum, and Garcinia kola [16]. Befitting their status as flavonoids, they have anti-cancer, anti-microbial, antiviral, and anti-inflammatory properties [16]. In contrast to the extensive studies on drug interaction with flavonoids, data on the inhibitory effects of biflavonoids on P450 and UGT enzymes are rare, though biflavonoids are taken in the form of dietary supplements (e.g., Ginkgo biloba extract [17]). The inhibitory potential of amentoflavone, the major biflavonoid in Cupressus funebris, against P450 and UGT enzymes was only recently reported [18,19].
In this study, we evaluate the inhibitory effects of five biflavonoids-selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone ( Figure 1)-on nine P450 enzymes using HLMs. We further investigate the ability of selamariscina A, which most strongly inhibited CYP2C8 and CYP2C9 activities, to inhibit six UGT isoforms. Furthermore, the inhibition mechanism and kinetic parameters (Ki) were determined for selamariscina A and compared with those of montelukast, a well-known selective CYP2C8 inhibitor [20].
We isolated selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone from Selaginella tamariscina (Beauv.), which were collected at Yen Tu Mountain, Uong Bi town, Quang Nihn province, Vietnam. The five compounds were purified and examined by HPLC to get 95% purity. Their chemical structures were identified by analyzing their NMR data, which were in good agreement with those published in a previous report [21,22].

Kinetic Characterization of Five Biflavonoids on CYP2C8 in Human Liver Microsomes
To determine the inhibition mechanism and constants (Ki values) of the five biflavonoids against CYP2C8 activity, different concentrations of biflavonoids (0, 0.002, 0.005, 0.02, 0.05, and 0.2 μM for selamariscina A; 0, 0.05, 0.02, 0.05, 0.2 and 0.5 μM for the other four biflavonoids) were added to reaction mixtures containing different concentrations of amodiaquine (0.1, 0.4 and 1 μM). The other conditions were the same as in the cytochrome P450 inhibition study.

Time-dependent Inhibition Assay
The time-dependent inhibition of selamariscina A against CYP2C8 and CYP2C9 enzymes was evaluated using an IC50 shift method. Selamariscina A was pre-incubated at six concentrations (0, 0.002, 0.005, 0.02, 0.05, and 0.2 μM) with HLMs in the presence of an NDAPH generation system for 30 min at 37 C. The reaction was initiated by adding 1 μM amodiaquine or 10 μM diclofenac and further incubated for 10 min. Incubation was terminated by adding 50 μL of ice-cold acetonitrile containing 7 nM trimipramine. After centrifugation, aliquots of supernatants were analyzed by LC-MS/MS.

Inhibitory Effect of Selamariscina A against Human UGT Activity
The ability of selamariscina A to inhibit the metabolism of six UGT enzyme probe substrates was evaluated using previously developed methods with minor modifications [28]. The microsomal incubation was performed by dividing the non-interactive substrate cocktail sets (set A included SN-38 for UGT1A1, CDCA for UGT1A3 and TFP for UGT1A4 while set B included N-SER for UGT1A6, MPA for UGT1A9 and NX for UGT2B7) ( Table 2). In brief, HLMs (0.25 mg/mL) were activated by incubation in the presence of alamethicin (25 μg/mL) for 15 min on ice. After the addition of UGT probe substrates and inhibitor (0, 0.5, 2, 5, 20 and 50 μM), the incubation mixtures were pre-incubated at 37 C for 5 min. After pre-incubation, 5 mM UDPGA was added to initiate a reaction, and further incubated for 60 min at 37 C. The reaction was stopped by adding 50 μL of ice-cold acetonitrile containing 250 nM estrone glucuronide (IS). After centrifugation at 18,000 g (5 min, 4 C), aliquots of supernatants were analyzed by LC-MS/MS. All microsomal incubations were conducted in triplicate.

Data Analysis
We analyzed the data with Shimadzu LabSolution LC-MS software. The IC50 values were calculated by WinNonlin software (Pharsight, Mountain View, CA, USA). The type of inhibition and the apparent kinetic parameters for inhibitory activity (Ki) were determined by following several criteria: visual inspection of Dixon plots, Lineweaver-Burk double reciprocal plots, and secondary plots of Lineweaver-Burk plots versus biflavonoid concentrations, the size of the sum of squares of the residuals, Akaike Information Criteria values, the S.E. and 95% confidence interval of the parameter estimates from the nonlinear regression analysis [29] using the WinNonlin software. The models tested included competitive, competitive partial, noncompetitive, noncompetitive partial, uncompetitive, uncompetitive partial, and mixed-type inhibition.

Comparison of the Selectivity of Selamariscina A and Montelukast for CYP2C8 Inhibition
Montelukast has been used to inhibit CYP2C8 in reaction-phenotyping studies [20]. We reevaluated its inhibitory potential against the nine P450 isoforms in this study using HLMs (XTreme 200, XenoTech). Montelukast strongly inhibited CYP2C8 activity with an IC50 value of 0.52 M, but it showed weak inhibition on the other eight P450 enzymes (IC50  9.73 M) ( Table 3). The IC50 value for the CYP2C8 isoform (IC50 = 0.52 M at 0.25 mg/mL microsomal protein concentration) was similar to previously reported values (IC50 = 0.18 M at 0.3 mg/mL microsomal protein concentration) [20]. However, montelukast showed more than 25 times weaker inhibition than selamariscina A (IC50 = 0.019 M at 0.25 mg/mL microsomal protein concentration). At 0.5 M selamariscina A concentration, approximately 25 times greater than the Ki value, selamariscina A was found to inhibit CYP2C8 and CYP2C9 by 92.8% and 88.6% respectively, and only slightly affected the enzyme activities of the other P450 isoforms (Figure 3). Selamariscina A at 0.5 M concentration inhibited none of the other P450 isoform-specific activities above 21.8% in HLMs, indicating that selamariscina A could be used as a selective CYP2C8 and CYP2C9 inhibitor in P450 phenotyping studies. Montelukast at 0.5 M concentration, a well-known selective CYP2C8 inhibitor [20], showed moderate inhibition on CYP2C8 by 52.7% in pooled HLMs. At 5 M concentration, montelukast inhibited CYP2C8 by 86.1% in HLMs; however, it also inhibited CYP2C9 and CYP2B6 activities by 31.0% and 20.4%, respectively in pooled HLMs. Montelukast (5 M) showed negligible inhibition on the other six P450 isoforms. Selamariscina A could be useful as a strong CYP2C8 and CYP2C9 inhibitor in P450 reactionphenotyping studies.

Evaluation of Drug Interaction Potential of Selamariscina A
It was estimated that an in vivo interaction potential via the inhibition of P450 would likely occur if the ratio of inhibitor Cmax/Ki exceeded one and would be possible if it was between 0.1 and 1.0 [40,41]. Based on amentoflavone's maximum concentrations (0.041 and 0.063 M) in rat blood after a single oral dose of Selaginella doderleinii Hieron extracts (200 mg/kg; contents: 103.82 mg/g amentoflavone, 37.52 mg/g robustaflavone, 44.4 mg/g 2,"3'-dihydro-3',3"-biapigenin, 53.4 mg/g 3',3"-binaringenin, and 35.1 mg/g delicaflavone) [42] and Selaginella doderleinii Hieron extracts (600 mg/kg) [43], the respective values of Cmax/Ki were 0.49 and 0.76 from the data of pooled HLMs (Ki = 0.083 M), indicating that amentoflavone has possible drug interaction potential with CYP2C8 substrate drugs [44]. Recently, nanotechnology-based delivery systems such as liposomes have been developed for improving oral bioavailability [42]. The values of Cmax (0.22 M) of amentoflavone after administration of liposome-based Selaginella doderleinii Hieron extracts (200 mg/kg) were 5.4 times higher than those of the control [42], resulting in a Cmax/Ki value of 2.65, indicating that amentoflavone has drug interaction potential. In the case of selamariscina A, the present study provides the first published data on its pharmacokinetics in animals and humans. Therefore, it is difficult to estimate the drug interaction potential of selamariscina A for humans. However, selamariscina A might have drug interactions with CYP2C8 substrate drugs such as cerivastatin [45], paclitaxel [46], and rosiglitazone [47] because its CYP2C8 inhibitory potential was more than 4.5 times stronger than that of amentoflavone. Therefore, in vivo studies are necessary to determine whether drug interactions between selamariscina A and CYP2C8 or CYP2C9 substrates have clinical relevance.

Conclusion
In conclusion, we report that selamariscina A is a strong CYP2C8 and CYP2C9 inhibitor. When evaluated for amodiaquine O-deethylation and diclofenac hydroxylation inhibitory activity against CYP2C8 and CYP2C9, as well as seven other P450s, it exhibited above 50-fold selectivity. Like montelukast and sulfaphenazole, selamariscina A could be useful as a strong CYP2C8 and CYP2C9 inhibitor in P450 phenotyping studies when HLMs are the enzyme source. Additionally, selamariscina A might cause clinically relevant pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP2C8 or CYP2C9. These in vitro findings provide primary data for future in vivo animal and clinical studies on risk prediction related to the interaction of drugs with herbs.