The Evaluation of CP-001 (a Standardized Herbal Mixture of Houttuynia cordata, Rehmannia glutinosa, Betula platyphylla, and Rubus coreanus) for Cytochrome P450-Related Herb-Drug Interactions

In the present study, the effect of CP-001, a standardized herbal mixture of Houttuynia cordata, Rehmannia glutinosa, Betula platyphylla, and Rubus coreanus, on cytochrome P450 (CYP) enzyme-mediated drug metabolism was investigated in vitro to evaluate the potential for herb-drug interactions. CP-001 was tested at concentrations of 1, 3, 10, 30, and 100 μg/mL. A CYP-specific substrate mixture was incubated with CP-001 in human liver microsomes, and the metabolites generated by each CYP-specific metabolic reaction were measured by liquid chromatography-tandem mass spectrometry. CP-001 seemed to slightly inhibit some CYP isozymes, but the IC50 values for all CYP isozymes were greater than 100 μg/mL. Furthermore, CP-001 did not exhibit time-dependent CYP inhibitory activities, indicating that it does not act as a mechanism-based inactivator of CYP enzymes. In conclusion, the effects of CP-001 on CYP isozyme activities were negligible at the concentrations tested. Therefore, the likelihood of herbal mixture-drug interaction is considered minimal.


Introduction
Herbal medicines are increasingly being used as alternative medicines worldwide. Consequently, it is very likely that some patients will take herbal medicines in combination with prescription or conventional medications, which may lead to unexpected adverse effects caused by herb-drug interactions. There may be many mechanisms underlying various herbdrug interactions, but the representative mechanism is an alteration of the absorption, metabolism, or clearance of coadministered drugs by the herb. Many herbs can induce or inhibit drug metabolizing enzymes such as cytochrome P450 (CYP450), altering the pharmacokinetics of the coadministered drugs and subsequently reducing their pharmacological effects or causing toxicity [1,2]. For these reasons, evaluations of herb-drug interactions associated with drug metabolizing enzymes are necessary to ensure the safety of the concomitant use of herbal medicines.
CP-001 is a standardized herbal mixture of Rehmannia glutinosa, Houttuynia cordata, Rubus coreanus, and Betula platyphylla. R. glutinosa has traditionally been used to treat hemostasis, activate blood circulation, and improve kidney function in East Asian countries [2]. It has been reported that R. glutinosa has antiallergy effects [3] and anti-inflammatory functions [4][5][6]. H. cordata has been used to treat inflammatory diseases in traditional oriental medicine, and it has been reported to possess several pharmacological activities such as anti-inflammatory, antiviral, and anticancer effects [7][8][9]. R. coreanus is a type of raspberry, the fruits of which have traditionally been used for the treatment of asthma and allergy in Asian countries and have anti-inflammatory and antioxidative activities.  Recently, in our pharmacological assay, CP-001 exhibited a synergistic therapeutic effect on atopic dermatitis (AD). This herbal mixture is currently under development as a therapeutic agent for the treatment of AD. Therefore, a preclinical evaluation of potential herb-drug interactions was required. In this study, the effects of CP-001 on CYP450mediated drug metabolism was evaluated to predict the CYP450-related herb-drug interactions.

CYP Inhibition
Assay. The CYP inhibition assay was performed with 0.5 mg/mL human liver microsomes in a final incubation volume of 0.2 mL. The incubation mixtures consisted of various concentrations of CP-001 (1, 3, 10, 30, and 100 g/mL in methanol), an NADPH-generating system (NGS) containing 0.1 M glucose-6-phosphate, 10 mg/mL -NADP + , and 1.0 U/mL glucose-6-phosphate dehydrogenase and a substrate mixture (Table 1)  To test the possibility of mechanism-based inactivation, 0.5 mg/mL human liver microsomes was preincubated with various concentrations of CP-001 (1, 3, 10, 30, and 100 g/mL) in 0.1 M potassium phosphate buffer (pH 7.4) for 30 min in the presence of NGS. After the preincubation, the substrate mixture was added, and the solution was continuously incubated for 30 min. The rest of the procedure was performed as described previously.

Sample Preparation.
The incubation mixtures were passed through activated Sep-Pak C 18 cartridges (96-well OASIS HLB extraction cartridge, Waters, Milford, MA, USA). The cartridges were activated with methanol (1 mL) and 0.1% acetic acid in distilled water (2 mL). After sample loading, the cartridges were washed twice with 1 mL 0.1% acetic acid in distilled water and eluted with 1 mL methanol. After the evaporation of the eluates under nitrogen gas, the residue was redissolved in 100 L of reconstitution buffer (0.1% formic acid in distilled water : acetonitrile = 85 : 15), and 5 L aliquots were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS).

LC-MS/MS Analysis.
The LC-MS/MS system consisted of an Agilent 1260 series binary pump HPLC system (Agilent Technologies, Palo Alto, CA, USA) and an Agilent 6460 triple quad mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ionization (ESI) source. A Fortis C 8 column (2.1 mm × 100 mm, 5 m; Fortis Technologies Ltd., Cheshire, England, UK) was used for the separation. The column temperature was maintained at a constant 40 ∘ C using a thermostatically controlled column oven. The HPLC mobile phases consisted of 0.1% formic acid in distilled water (A) and 90% acetonitrile in 0.1% formic acid (B). A gradient program was used for the HPLC separation with a flow rate of 0.2 mL/min. The solvent composition was initially set at 15% B, gradually increased to 85% Bover 3 min, and maintained for 1.5 min, and then the column was reequilibrated for 3.5 min. The entire column eluent was introduced directly into the mass spectrometer. Nitrogen was used both as the nebulizing gas at 20 psi and as the drying gas at a flow rate of 10 L/min at 300 ∘ C. The mass spectrometer was operated in positive ion mode. Multiple reaction monitoring (MRM) detection was employed. The precursor-product ion pairs (Q1/Q3) used in MRM mode were presented in Table 1.

Results and Discussion
The inhibitory effects of CP-001 on CYP-specific metabolic activities were evaluated in human liver microsomes. The assay system was tested with well-known selective inhibitors of CYP isozyme (positive controls). The remaining activity of the CYP isozymes after the treatment of each selective inhibitor was as follows: 5.0% for CYP1A2 (furafylline); 8.2% for CYP2A6 (methoxsalen); 5.3% for CYP2C9 (sulfaphenazole); 12.7% for CYP2C19 (ticlopidine); 2.5% for CYP2D6 (quinidine); and 4.5% for CYP3A4 (ketoconazole). All the inhibitors selectively inhibited the corresponding CYP marker activity. When CP-001 was evaluated at concentrations of 1, 3, 10, 30, and 100 g/mL, CP-001 showed minimal inhibitory effects on all CYP isozymes tested (Table 2), with estimated IC 50 values above 100 g/mL, although the enzyme activity of CYP2C19 was slightly inhibited in a concentrationdependent manner. When CP-001 was tested after preincubation with human liver microsomes, the extent of inhibition on several CYP isozyme activities was somewhat different, but the IC 50 values were all above 100 g/mL (Table 3), comparable to those without pre-incubation. Therefore, CP-001 is considered to have negligible effects on CYP-mediated drug metabolism as a mechanism-base inhibitor as well as a competitive inhibitor.
There have been several reports on the effects of the herbs contained in CP-001 on CYP catalytic activities [10][11][12]. Regarding H. cordata, decreases in the activities of CYP1A1, CYP2C11, and CYP2E1 were observed in 24 h oxidized frying oil-fed rats after feeding with a diet containing H. cordata [10]. However, this study was not conducted to evaluate the herb-drug interaction but rather to evaluate chemopreventive potential as those CYP enzymes could be involved in the activation of precarcinogens or other chronic diseases. R. glutinosa was reported to inhibit CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 in vitro in a concentration-dependent manner, but significant inhibition was only observed at a relatively high concentration (1 mg/mL), which is difficult to reach in a clinical setting [11]. There was a report on the effects of R. coreanus on CYP3A activity, but the effect was not so considerable [12]. The effects of B. platyphylla on CYPmediated drug metabolism have not yet been reported.
In conclusion, we have evaluated the inhibitory potential of CP-001 on human CYP enzyme activities in vitro as a part of preclinical ADME studies. The present results indicate that CP-001 may not interact with coadministered drugs by modulating CYP-mediated metabolism. However, the results of in vitro tests are not necessarily consistent with in vivo findings. Therefore, continuous monitoring of herb-drug interactions along with pharmacokinetics studies may be required through subsequent clinical stages.