Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50) values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (K i) value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a K i value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index) value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions.


Introduction
Functional dyspepsia is a very common chronic gastrointestinal disorder, with a prevalence of 40% using the more liberal criteria [1]. Although the etiology and pathogenesis of functional dyspepsia are poorly understood, pathophysiologic abnormalities such as delayed gastric emptying and impaired gastric accommodation have been reported in 30-40% of functional dyspepsia patients [2][3][4][5]. Medications used for the treatment of functional dyspepsia have been developed based on the pathophysiological mechanisms associated with functional dyspepsia; those medications include prokinetic agents, which are now restricted due to serious adverse effects and failure to confirm their efficacy. Therefore, it is necessary to develop safer and more effective drugs for the treatment of functional dyspepsia.
DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber from the roots of Corydalis yanhusuo W. T. Wang and Pharbitidis semen from the seed of Pharbitis nil Choisy, and it is used as an oral therapy (Motilitone ) for the treatment of functional dyspepsia (FD) in Republic of Korea since May 2011 [6][7][8]. Pharbitidis semen has been used as a folk medicine for its analgesic effect on abdominal disorders. Corydalis tubers from the roots of Corydalis yanhusuo W. T. Wang have long been 2 Evidence-Based Complementary and Alternative Medicine used as a herbal drug for their analgesic and antiulcer effects [9][10][11]. Corydaline and tetrahydroberberine, isoquinoline alkaloids of Corydalis tubers, promote gastric emptying and facilitate gastric accommodation [12,13]. DA-9701 has been demonstrated to show strong gastroprokinetic effects and a safety profile superior to cisapride and mosapride [7,8]. The gastroprokinetic effects of DA-9701 might be mediated by the induction of pacemaker currents in the interstitial cells of Cajal (ICC) [6]. DA-9701 also has been shown to have antagonistic effects on the D2 receptor and agonistic effects on 5-HT4, 5-HT1a, and 5-HT1b receptors [7,8].
To the best of our knowledge, no previous study has reported the effects of Corydalis tubers and Pharbitidis semen, the component herbs of DA-9701, on human CYP and UGT enzymes. In this study, the effects of DA-9701 and its component herbs, Corydalis tubers and Pharbitidis semen, on the activities of seven major human CYP enzymes and four major human UGTs, 1A1, 1A4, 1A9, and 2B7, were examined using pooled human liver microsomes in order to evaluate the possibility of drug interactions of DA-9701.

Preparation of
Corydalis tuber extract and Pharbitidis semen extract were prepared as previously described [13]. Briefly, dried Corydalis tuber and Pharbitidis semen were separately extracted with 50% aqueous ethanol three times at room temperature for 48 h. The solvents were removed under vacuum. The marker compounds including corydaline from Corydalis tuber extract and chlorogenic acid from Pharbitidis semen extract were also determined by HPLC [8]. The two raw materials were purchased in China.

Inhibitory Effects of DA-9701 and Its Component Herbs on Seven Major CYP Activities in Human Liver
Microsomes. Inhibitory potencies (IC 50 values) of DA-9701, Corydalis tuber extract, and Pharbitidis semen extract were determined using CYP assays in the presence and absence of DA-9701, Corydalis tuber extract, and Pharbitidis semen extract using pooled human liver microsomes (H161, Gentest). Phenacetin O-deethylase, coumarin 7-hydroxylase, amodiaquine N-deethylase, diclofenac 4-hydroxylase, (S)mephenytoin 4-hydroxylase, bufuralol 1 -hydroxylase, and midazolam 1 -hydroxylase activities were determined as probe activities for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A, respectively, using cocktail incubation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) [22]. The incubation mixtures were prepared in a total volume of 200 μL as follows: pooled human liver microsomes (0.25 mg/mL), 3.3 mM MgCl 2 , 50 mM potassium phosphate buffer (pH 7.4), and a cocktail of probe substrates and various concentrations of DA-9701, Corydalis tuber extract, or Pharbitidis semen extract (final concentrations of 1-200 μg/mL with an acetonitrile concentration less than 0.5% v/v). The substrates were used at concentrations approximately equal to their respective K m values: 50 μM phenacetin, 2.5 μM coumarin, 2.5 μM amodiaquine, 10 μM diclofenac, 100 μM (S)mephenytoin, 5 μM bufuralol, and 2.5 μM midazolam. After a 3-min preincubation period at 37 • C, the reactions were initiated by addition of an NADPH (final concentration of 1.3 mM) generating system and incubated for 20 min at 37 • C in a shaking water bath. After incubation, the reaction was stopped by placement of the tubes on ice and Evidence-Based Complementary and Alternative Medicine 3 addition of 100 μL of ice-cold methanol containing internal standards ( 13 C 2 , 15 N-acetaminophen for acetaminophen and N-desethylamodiaquine, d 5 -7-hydroxycoumarin for 7-hydroxycoumarin, 13 C 6 -4-hydroxydiclofenac for 4hydroxydiclofenac, d 3 -4-hydroxy-mephenytoin for 4hydroxymephenytoin and 1 -hydroxymidazolam, d 9 -1hydroxybufuralol for 1 -hydroxybufuralol). The incubation mixtures were then centrifuged at 13, 000 × g for 5 min. All incubations were performed in triplicate, and the mean values were used. For evaluation of NADPH-dependent mechanism-based inhibition of CYP activities, various concentrations of DA-9701, Corydalis tuber extract, or Pharbitidis semen extract (1-200 μg/mL) were preincubated for 30 min with human liver microsomes in the presence of NADPH. The reaction was started by addition of a cocktail of CYP probe substrates.
All seven metabolites produced from the cocktail incubation of CYP isoform-specific substrates were simultaneously determined by LC/MS/MS [22]. The system consisted of a tandem mass spectrometer (TSQ Quantum Access, ThermoFisher Scientific, San Jose, CA, USA) coupled with a Nanospace SI-2 LC system (Tokyo, Japan). Separation was performed on an Atlantis dC18 column (5 μm, 2.1 mm i.d. ×100 mm, Waters, MA, USA) using the gradient elution of a mixture of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.25 mL/min: 10% mobile phase B for 1 min, 10% to 95% mobile phase B for 1 min, and 95% mobile phase B for 5 min. The column and autosampler temperatures were 50 and 6 • C, respectively. After 1.5 min, the LC eluent was diverted from waste to the mass spectrometer fitted with the electrospray ionization (ESI) source and operated in positive ion mode. ESI source settings for ionization of the metabolites were as follows: electrospray voltage, 5.0 kV; vaporizer temperature, 420 • C; capillary temperature 360 • C; sheath gas pressure, 35 psi; auxiliary gas pressure, 10 psi. Quantification was performed by selected reaction monitoring (SRM) of the [M + H] + ion and the related product ion for each metabolite. SRM transitions for the metabolites and internal standards are summarized in our previous paper [22]. Analytical data were processed using Xcalibur software (Thermo Fisher Scientific).

Inhibitory Effects of DA-9701 and Its Component
Herbs on Four UGT Activities in Human Liver Microsomes. The inhibitory potencies (IC 50 values) of DA-9701, Corydalis tuber extract, or Pharbitidis semen extract were also determined with UGT assays in the presence and absence of DA-9701, Corydalis tuber extract, or Pharbitidis semen extract using pooled human liver microsomes. Etoposide glucuronidation, trifluoperazine glucuronidation, propofol glucuronidation, and azidothymidine glucuronidation activities were determined as probe activities for UGT1A1, UGT1A4, UGT1A9, and UGT2B7, respectively, using LC/MS/MS [23]. Incubation mixtures were prepared in a total volume of 100 μL as follows: pooled human liver microsomes (0.2 mg/mL for etoposide, trifluoperazine, and azidothymidine; 0.1 mg/mL for propofol), 25 μg/mL alamethicin, 10 mM MgCl 2 , 50 mM tris buffer (pH 7.4), each UGT-isoform specific probe substrate (200 μM etoposide for UGT1A1, 5 μM trifluoperazine for UGT1A4, 10 μM propofol for UGT1A9, or 100 μM azidothymidine for UGT2B7), and various concentrations of DA-9701, Corydalis tuber extract, or Pharbitidis semen extract (final concentrations of 1-200 μg/mL with acetonitrile concentration less than 0.5% v/v). Reactions were initiated by addition of UDPGA (final concentration of 5 mM), and incubations were carried out at 37 • C in a shaking water bath for 30 min. Reactions were terminated by addition of 100 μL of ice-cold methanol containing an internal standard (100 ng/mL ezetimibe for etoposide glucuronide and propofol glucuronide; 30 ng/mL meloxicam for trifluoperazine glucuronide and azidothymidine glucuronide). The incubation mixtures were centrifuged at 13, 000 × g for 5 min, followed by dilution of 40 μL of the supernatant with 60 μL of water. The aliquot (5 μL) was injected onto LC/MS/MS. All incubations were performed in duplicate and the mean values were used.
Glucuronides produced from UGT isoform-specific substrates were, respectively, determined by LC/MS/MS [23]. Separation was performed on an Atlantis dC18 column (5 μm, 2.1 mm i.d. ×100 mm, Waters, MA, USA) using the gradient elution of a mixture of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.25 mL/min: 10% mobile phase B for 2 min, 10% to 95% mobile phase B for 4 min. The column and autosampler temperatures were 50 and 6 • C, respectively. After 3.0 min, the LC eluent was diverted from waste to the mass spectrometer fitted with an ESI source. ESI source settings for ionization of trifluoperazine glucuronide and azidothymidine glucuronide in positive ion mode were as follows: electrospray voltage, 5.0 kV; vaporizer temperature, 420 • C; capillary temperature 360 • C; sheath gas pressure, 35 psi; auxiliary gas pressure, 10 psi. ESI source settings for ionization of etoposide glucuronide and propofol glucuronide in negative ion mode were as follows: electrospray voltage, −4.0 kV; vaporizer temperature, 420 • C; capillary temperature 360 • C; sheath gas pressure, 35 psi; auxiliary gas pressure, 10 psi. Quantification was performed by SRM of the [M + H] + ion for trifluoperazine glucuronide and azidothymidine glucuronide or [M − H] − for etoposide glucuronide and propofol glucuronide and the related product ion for each metabolite. SRM transitions for the metabolites and internal standard are summarized in our previous paper [23]. Analytical data were processed using Xcalibur software (Thermo Fisher Scientific).

Results and Discussion
The inhibitory effects of DA-9701, Corydalis tuber extract, and Pharbitidis semen extract on seven major human CYP isoforms were evaluated using cocktail CYP probe substrates in human liver microsomes. CYP2D6-catalyzed bufuralol 1 -hydroxylation activity was inhibited by DA-9701 and Corydalis tuber extract with IC 50 values of 25.9 and 15.8 μg/mL, respectively, but not by Pharbitidis semen extract (Table 1) Although corydaline and palmatine inhibited CYP2D6 isozyme, they are not the major constituents causing the inhibition of CYP2D6 activity, because their contents in DA-9701 (corydaline, 0.85 μg/mg and palmatine, 0.13 μg/mg DA-9701) and Corydalis tuber extract (corydaline, 1.12 μg/mg and palmatine, 0.18 μg/mg Corydalis tuber extract) are very low. Therefore, other constituents may contribute to the inhibition of CYP2D6 activity by DA-9701 and Corydalis tuber extract. Further studies are needed to explore how the constituents of those compounds contribute to the inhibition of human CYP2D6 isozyme. The inhibitory potencies of DA-9701, Corydalis tuber extract, and Pharbitidis semen extract were not significantly affected after a 30-min period of preincubation with human liver microsomes in the presence of NADPH ( Table 1), suggesting that a mechanismbased inhibitory component was not present in DA-9701, Corydalis tuber extract, and Pharbitidis semen.
Volume per dose index (VDI) is defined as the volume in which one dose would be diluted to obtain the corresponding IC 50 concentration, as described by Strandell et al. [24], to determine the potential for in vivo inhibition of herbal preparations. If VDI value for a herbal preparation approaches 4 L, corresponding to human blood volume, the potential enzyme interaction of herbal preparation with pharmaceuticals should be further investigated [24]. Since a recommended human dose for DA-9701 is 30 mg, VDI value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, suggesting that DA-9701 may not be a potent CYP2D6 inhibitor.  In an inhibition study, the apparent K i value is a better parameter for defining the interaction of the inhibitor with a particular enzyme. K i values and inhibition types (competitive, noncompetitive, uncompetitive, or mixed) for DA-9701 and Corydalis tuber extract were initially estimated by graphical methods such as Lineweaver-Burk plot and Dixon plot, but they were ultimately determined by nonlinear leastsquare regression analysis for the best enzyme inhibition model using Enzyme kinetics software. DA-9701 showed noncompetitive inhibition for CYP2D6-catalyzed bufuralol 1 -hydroxylation, with a K i value of 6.3 μg/mL (Table 2, Figure 1). Corydalis tuber extract competitively inhibited CYP2D6-catalyzed bufuralol 1 -hydroxylation, with a K i value of 3.7 μg/mL (Table 2, Figure 1). The inhibitory potential of corydaline on CYP2D6 activity (K i value of 10.1 μg/mL (27.3 μM)) [23] was not found to be strong, as compared with the inhibition produced by quinidine [K i value of 12 ng/mL (0.038 μM)], a selective inhibitor of CYP2D6 [25,26].
CYP2D6 catalyzes oxidation of a wide range of substrates, including desipramine, dextromethorphan, haloperidol, S-metoprolol, nortryptyline, paroxetine, and tamoxifen (http://medicine.iupui.edu/clinpharm/ddis/). Compared to quinidine, a known inhibitor of CYP2D6-catalyzed bufuralol 1 -hydroxylation [25,26,28], DA-9701 is a weak inhibitor (K i , 12 ng/mL versus 6.3 μg/mL). On the basis of an IC 50 value and VDI value of 1.16 L for a 30 mg dose, DA-9701 will be expected to have weak, if any, inhibition of CYP2D6catalyzed metabolism in vivo. However, no pharmacokinetic data on DA-9701 are available. False prediction of in vivo drug-drug interactions from in vitro data may be occurred as a result of mechanism-based inhibition, plasma protein binding of inhibitors, production of inhibitory metabolites, and/or hepatic uptake. Therefore, the results need to be confirmed in clinical studies.

Conclusions
In conclusion, the effects of DA-9701 and its component herbs, Corydalis tuber extract and Pharbitidis semen extract, on seven CYPs and four UGTs were evaluated across a wide range of substrates using human liver microsomes in vitro. Pharbitidis semen extract showed no inhibition of seven CYPs and four UGTs in human liver microsomes. CYP2D6 activity was moderately inhibited by DA-9701 and Corydalis tuber extract during incubation with NADPH in human liver microsomes. On the basis of a K i value and VDI value, DA-9701 will be expected to have weak, if any, inhibition of CYP2D6-catalyzed metabolism in vivo. Those results suggest that high uptake of botanical drug DA-9701 or Corydalis tuber extract may cause an interaction with drugs metabolized by CYP2D6 in some individuals. It is important to note, however, that the inhibition of CYP activities in vitro does not necessarily translate into drug interactions in clinical situations. Clinical trials to evaluate the inhibitory effects of DA-9701 on CYP2D6 remain to be conducted.