Probe substrate and enzyme source-dependent inhibition of UDP-glucuronosyltransferase ( UGT ) 1 A 9 by wogonin

Background: Drug-metabolizing enzymes (DMEs) inhibition based drug-drug interaction and herb-drug interaction severely challenge the R&D process of drugs or herbal ingredients. Objective: To evaluate the inhibition potential of wogonin (an important flavonoid isolated from the root of Scutellaria baicalensis) towards one of the most important phase II DMEs, UDP-glucuronosyltransferase (UGT) 1A9. Methods: Both recombinant UGT1A9-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and human liver microsomes (HLMs)-catalyzed propofol glucuronidation reaction were used as two different probe reactions. Results: Wogonin noncompetitively inhibited recombinant UGT1A9-catalyzed 4-MU glucuronidation, and exerted competitive inhibition towards HLMs-catalyzed propofol glucuronidation. The inhibition kinetic parameters (K i ) were calculated to be 3.2 μM and 52.0μM, respectively. Conclusion: Necessary monitoring was needed when wogonin was co-administered with the clinical drugs mainly undergoing UGT1A9-mediated glucuronidation elimination. Additionally, probe reactions-dependent inhibition of wogonin towards the activity of UGT1A9 should be paid attention when translating these in vitro data into in vivo situation.


Introduction
Since the beginning of the herbs' utilization from ancient times, the utilization of herbal medicines has a history of about 4000 years 1 .With the increasing utilization of herbal remedies, more and more cases on herbal adverse effects are reported.Herb-drug interaction has been frequently reported, and the coadministered herbs might affect the pharmacokinetic behaviouts of many important clinical drugs, including warfarin, aspirin, digoxin and cisplatin 2 .The drugs can be efficiently eliminated by phase I and phase II drug metabolizing enzymes (DMEs)mediated elimination system.Herbal components can change the activity of all these DMEs through the good interaction with them.When these herbs are co-administered with the drugs mainly undergoing the DMEs inhibited by herbal components, the exposed concentration of drugs might exceed the minimal toxicity dose in the therapeutic window, and then induce severe toxicity.
The glucuronidation through the conjugation with glucuronic acid represents the main elimination pathway of clinical drugs, and the inhibition of this important pathway might induce the severe drugdrug interaction.For example, the exposed concentration of zidovudine (the probe substrate of UGT2B7) can increase by 31% and 74% due to the inhibition of glucuronidation pathway by atovaquone and fluconazole, respectively 3,4 .Some previous literatures have indicated that some herbal components can exhibit the strong inhibition towards UGT isoforms.For example, the most abundant flavonoid component quercetin inhibited the activity of UGT1A1 and UGT1A9 5 .In vitro experiment showed that the activity of UGT1A1, 1A6, 1A9, 2B7 and 2B15 can be inhibited by silybin 6 .The herbal components deoxyschizandrin and schisantherin A have been demonstrated to have the potential to induce herb-drug interaction through the strong inhibition towards UGT1A3 7 .Fang et al. investigatd the stucture-activity relationship for the ginsenosides' inhibition towards UGT isoforms, elucidating the potential UGTs-inhibition based ginseng-clinical drugs interactions 8 .
Wogonin, an important flavonoid isolated from the root of Scutellaria baicalensis Georgi, has been reported to exhibit multiple biochemical and pharmacological activities, including antioxidant 9 , anti-viral 10 , antithrombotic 11 , and anti-inflammatory activities 12 .The present study aims to study the inhibition of wogonin towards one of the most important UGT isoforms, UGT1A9.In vitro incubation system was performed.Given that the probe reactions-dependent behaviour for the inhibition of DMEs by compounds exists, two kinds of probe reactions were carried out, including recombinant UGT1A9-catalyzed 4methylumbelliferone (4-MU) glucuronidation and human liver microsomes (HLMs)-catalyzed glucuronidation.

Evaluation of wogonin's inhibition towards UGT1A9-catalyzed 4-MU glucuronidation
The inhibition of wogonin towards recombinant UGT1A9-catalyzed 4-MU glucuronidation was evaluated as previously described 13 .The mixture (200µl total volume) contained recombinant 0.05 mg/ml of UGT1A9, 5 mM UDPGA, 5 mM MgCl 2 , 50 mM Tris-HCl buffer (pH 7.4), and 4-MU (30µM) in the absence or presence of different concentrations of wogonin.Wogonin was dissolved in DMSO and the final concentration of DMSO was 0.5% (v/v).After 5 min pre-incubation at 37 o C, the UDPGA was added in the mixture to initiate the reaction.Incubation time was 30 min for UGT1A9.The reactions were quenched by adding 100 ìl acetonitrile with 7-hydroxycoumarin (100µM) as internal standard.The mixture was centrifuged at 20,000×g for 10 min and an aliquot of supernatant was transferred to an auto-injector vial for HPLC analysis as previously described 8 .

Determination of inhibition kinetic parameters
Inhibition kinetic parameters (K i ) were determined utilizing various concentrations of 4-MU (or propofol) in the presence of different concentrations of wogonin.Dixon and Lineweaver-Burk (L-B) plots were adapted to determine the inhibition type, and second plot of slopes from Lineweaver-Burk plot vs. wogonin concentrations was utilized to calculate K i value.

Results
The inhibition of wogonin towards recombinant UGT1A9-catalyzed 4-MU glucuronidation reaction was evaluated, and the results were shown in figure 1. Wogonin exhibited dose-dependent inhibition towards 4-MU glucuronidation (figure 1A), and both Dixon plot (figure 1B) and Lineweaver-Burk plot (figure 1C) showed that the inhibition type was best fit to the noncompetitive inhibition.The second plot (figure 1D) was used to calculate the inhibition kinetic parameter (K i ) to be 3.2 ìM.Furthermore, human liver microsomes (HLMs)-catalyzed propofol glucuronidation reaction was employed to evaluate the wogonin's inhibition towards UGT1A9 activity.

Discussion
Several phenolic hydroxyl groups exist in the structure of wogonin, and previous studies have indicated that the phenolic hydroxyl groups-containing compounds are the good substrates of UGT isoforms, including arbidol 15 and magnolol 16 .The UGT isoforms involved in the metabolic elimination of wogonin have been identified.UGT1A9 was regarded as the main UGT isoform involved in the glucuronidation of wogonin in the liver, and UGT1A10 was the major UGT isoform responsible for wogonin's glucuronidation in the intestine 17 .All these information indicated the possibly good interaction between wogonin and UGT1A9.
In this study, wogonin showed inhibitory potential towards the activity of UGT1A9, regardless of the enzyme sources and probe substrates.However, the inhibition type and potential showed the difference when selecting difference enzyme sources and probe substrates.Wogonin noncompetitively inhibited recombinant UGT1A9catalyzed 4-MU glucuronidation, and competitively inhibited human liver microsomes (HLMs)-catalyzed propofol glucuronidation reaction.When choosing UGT1A9-catalyzed 4-MU glucuronidation reaction as the probe reaction, the inhibition potential was stronger.This kind of enzyme sources and probe substrates-dependent inhibition behaviour has been observed in the previous literatures 18 .UGT1A9 is one of the most important UGT isoforms in liver, kidney and intestine, and take part in the metabolism of many xenobiotics.For example, UGT1A9 is the major UGT isoform involved in the ethanol glucuronidation 19 .UGT1A9 showed extensive activity towards the glucuronidation of acetaminophen 20 .Additionally, UGT1A9 has been demonstrated to be responsible for the glucuronidation of many clinical drugs or drug candidates, including noscapine and sorafenib 21,22 .Therefore, the potential drug-drug interaction exists for the co-administration of wogonin and the clinical drugs mainly undergoing UGT1A9-catalyzed metabolic elimination.

Conclusion
Necessary monitoring was needed when wogonin was co-administered with the clinical drugs mainly undergoing UGT1A9-mediated glucuronidation elimination.Additionally, probe reactions-dependent inhibition of wogonin towards the activity of UGT1A9 should be paid attention when translating these in vitro data into in vivo situation.