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

Hye Hyun Yoo

In Sook Kim

Seong-Gyu Ko

Sun-A Kim

Evidence-Based Complementary and Alternative Medicine

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Evidence-based Complementary and Alternative Medicine

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 ofHouttuynia cordata,Rehmannia glutinosa,Betula platyphylla, andRubus coreanus, on cytochrome P450 (CYP) enzyme-mediated drug metabolism was investigatedin vitroto 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 IC50values 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.</jats:p

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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013, Article ID 824270, 4 pageshttp://dx.doi.org/10.1155/2013/824270Research ArticleThe Evaluation of CP-001 (a Standardized Herbal Mixture ofHouttuynia cordata, Rehmannia glutinosa, Betula platyphylla,and Rubus coreanus) for Cytochrome P450-RelatedHerb-Drug InteractionsHye Hyun Yoo,1 Sun-A Kim,1 In Sook Kim,1 and Seong-Gyu Ko21 Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan,Gyeonggi-do 426-791, Republic of Korea2 Laboratory of Clinical Biology and Pharmacogenomics, Department of Preventive Medicine, College of Oriental Medicine,Kyung Hee University, Seoul 130-701, Republic of KoreaCorrespondence should be addressed to Seong-Gyu Ko; epiko@khu.ac.krReceived 5 April 2013; Revised 11 June 2013; Accepted 13 June 2013Academic Editor: Bo-Hyoung JangCopyright © 2013 Hye Hyun Yoo et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.In the present study, the effect of CP-001, a standardized herbal mixture of Houttuynia cordata, Rehmannia glutinosa, Betulaplatyphylla, and Rubus coreanus, on cytochrome P450 (CYP) enzyme-mediated drug metabolism was investigated in vitro toevaluate the potential for herb-drug interactions. CP-001 was tested at concentrations of 1, 3, 10, 30, and 100𝜇g/mL. A CYP-specific substrate mixture was incubated with CP-001 in human liver microsomes, and the metabolites generated by each CYP-specificmetabolic reaction weremeasured by liquid chromatography-tandemmass spectrometry. CP-001 seemed to slightly inhibitsome CYP isozymes, but the IC50values for all CYP isozymes were greater than 100 𝜇g/mL. Furthermore, CP-001 did not exhibittime-dependent CYP inhibitory activities, indicating that it does not act as a mechanism-based inactivator of CYP enzymes. Inconclusion, the effects of CP-001 on CYP isozyme activities were negligible at the concentrations tested. Therefore, the likelihoodof herbal mixture-drug interaction is considered minimal.1. IntroductionHerbal medicines are increasingly being used as alternativemedicines worldwide. Consequently, it is very likely thatsome patients will take herbalmedicines in combinationwithprescription or conventional medications, which may lead tounexpected adverse effects caused by herb-drug interactions.There may be many mechanisms underlying various herb-drug interactions, but the representative mechanism is analteration of the absorption, metabolism, or clearance ofcoadministered drugs by the herb. Many herbs can induce orinhibit drug metabolizing enzymes such as cytochrome P450(CYP450), altering the pharmacokinetics of the coadminis-tered drugs and subsequently reducing their pharmacologicaleffects or causing toxicity [1, 2]. For these reasons, evaluationsof herb-drug interactions associated with drug metabolizingenzymes are necessary to ensure the safety of the concomitantuse of herbal medicines.CP-001 is a standardized herbal mixture of Rehmanniaglutinosa, Houttuynia cordata, Rubus coreanus, and Betulaplatyphylla. R. glutinosa has traditionally been used to treathemostasis, activate blood circulation, and improve kidneyfunction in East Asian countries [2]. It has been reported thatR. glutinosa has antiallergy effects [3] and anti-inflammatoryfunctions [4–6]. H. cordata has been used to treat inflam-matory diseases in traditional oriental medicine, and it hasbeen reported to possess several pharmacological activitiessuch as anti-inflammatory, antiviral, and anticancer effects[7–9]. R. coreanus is a type of raspberry, the fruits of whichhave traditionally been used for the treatment of asthma andallergy in Asian countries and have anti-inflammatory andantioxidative activities.2 Evidence-Based Complementary and Alternative MedicineTable 1: Information on the CYP-specific substrates used and the metabolites monitored in this study.P450 isozyme Marker substrate Concentration Metabolites monitored Q1/Q3CYP 1A2 Phenacetin 40𝜇M Acetaminophen 152.1/110.1CYP 2A6 Coumarin 2.5 𝜇M 7-OH-Coumarin 162.9/106.9CYP 2D6 Dextromethorphan 5𝜇M Dextrorphan 258.3/157.1CYP 2C9 Diclofenac 10𝜇M 4-OH-Diclofenac 312.2/230.9CYP 2C19 (±)-Mephenytoin 160𝜇M 4-OH-Mephenytoin 235/150.1CYP 3A4 Midazolam 2.5 𝜇M 1-OH-Madazolam 343.1/325.1Internal standard Terfenadine 472.4/436.4Recently, in our pharmacological assay, CP-001 exhibiteda synergistic therapeutic effect on atopic dermatitis (AD).This herbal mixture is currently under development as atherapeutic agent for the treatment of AD. Therefore, apreclinical evaluation of potential herb-drug interactions wasrequired. In this study, the effects of CP-001 on CYP450-mediated drug metabolism was evaluated to predict theCYP450-related herb-drug interactions.2. Materials and Methods2.1. Chemicals and Reagents. CP-001 was provided byHanpoong Pharm & Foods Co., Ltd. (Jeonju, Rebiblicof Korea). CP-001 was standardized to contain 1.25%catalpol, 0.26% quercitrin, and 0.42% ellagic acid. Pooledhuman liver microsomes were purchased from BD Gen-test (Woburn, MA, USA). Glucose-6-phospate, 𝛽-NADP+,glucose-6-phospate dehydrogenase, phenacetin, coumarin,diclofenac, mephenytoin, dextromethorphan, midazolam,furafylline, methoxsalen, sulfaphenazole, ticlopidine, quini-dine, ketoconazole, and terfenadine were obtained fromSigma Chemical Co. (St. Louis, MO, USA). Formic acidwas purchased fromMerck (Darmstadt, Germany). All othersolvents used were of HPLC grade and were purchasedfrom J. T. Baker (Phillipsburg, NJ, USA). Distilled waterwas prepared using a MilliQ purification system (Millipore,Billerica, MA, USA).2.2. CYP Inhibition Assay. The CYP inhibition assay wasperformed with 0.5mg/mL human liver microsomes in afinal incubation volume of 0.2mL. The incubation mixturesconsisted of various concentrations of CP-001 (1, 3, 10, 30,and 100𝜇g/mL in methanol), an NADPH-generating system(NGS) containing 0.1M glucose-6-phosphate, 10mg/mL 𝛽-NADP+, and 1.0U/mL glucose-6-phosphate dehydrogenaseand a substrate mixture (Table 1) in 0.1M potassium phos-phate buffer (pH 7.4). The incubation mixture was incubatedat 37∘C without NGS for 5min and then continuously incu-bated for 30minwithNGS in awater bath.Well-known selec-tive CYP inhibitors were tested as positive controls (10 𝜇Mfurafylline for CYP1A2; 20𝜇M methoxsalen for CYP2A6;50𝜇M sulfaphenazole for CYP2C9; 20𝜇M ticlopidine forCYP2C19; 50 𝜇M quinidine for CYP2D6; 5 𝜇M ketoconazolefor CYP3A4). After the incubation, the reaction was stoppedby adding 400 𝜇L of 0.1% acetic acid containing 4𝜇L of aninternal standard solution (16 𝜇M terfenadine in DMSO).To test the possibility of mechanism-based inactivation,0.5mg/mL human liver microsomes was preincubated withvarious concentrations of CP-001 (1, 3, 10, 30, and 100 𝜇g/mL)in 0.1M potassium phosphate buffer (pH 7.4) for 30min inthe presence of NGS. After the preincubation, the substratemixture was added, and the solution was continuously incu-bated for 30min.The rest of the procedure was performed asdescribed previously.2.3. Sample Preparation. The incubation mixtures werepassed through activated Sep-Pak C18cartridges (96-wellOASISHLB extraction cartridge,Waters,Milford,MA,USA).The cartridges were activated with methanol (1mL) and 0.1%acetic acid in distilled water (2mL). After sample loading,the cartridges were washed twice with 1mL 0.1% acetic acidin distilled water and eluted with 1mL methanol. After theevaporation of the eluates under nitrogen gas, the residuewas redissolved in 100𝜇L of reconstitution buffer (0.1%formic acid in distilled water : acetonitrile = 85 : 15), and 5 𝜇Laliquots were subjected to liquid chromatography-tandemmass spectrometry (LC-MS/MS).2.4. LC-MS/MS Analysis. The LC-MS/MS system consistedof an Agilent 1260 series binary pump HPLC system (AgilentTechnologies, Palo Alto, CA,USA) and anAgilent 6460 triplequad mass spectrometer (Agilent Technologies, Palo Alto,CA, USA) equipped with an electrospray ionization (ESI)source. A Fortis C8column (2.1mm × 100mm, 5𝜇m; FortisTechnologies Ltd., Cheshire, England, UK) was used for theseparation. The column temperature was maintained at aconstant 40∘C using a thermostatically controlled columnoven.The HPLCmobile phases consisted of 0.1% formic acidin distilled water (A) and 90% acetonitrile in 0.1% formic acid(B). A gradient program was used for the HPLC separationwith a flow rate of 0.2mL/min. The solvent compositionwas initially set at 15% B, gradually increased to 85% Bover3min, and maintained for 1.5min, and then the columnwas reequilibrated for 3.5min. The entire column eluent wasintroduced directly into themass spectrometer. Nitrogen wasused both as the nebulizing gas at 20 psi and as the drying gasat a flow rate of 10 L/min at 300∘C.Themass spectrometer wasoperated in positive ion mode. Multiple reaction monitoring(MRM) detection was employed. The precursor-product ionpairs (Q1/Q3) used in MRMmode were presented in Table 1.Evidence-Based Complementary and Alternative Medicine 3Table 2: The effects of CP-001 on the CYP enzyme activities without preincubation.P450 isozymeRemaining activities (% of control)IC50CP-001 (𝜇g/mL)1 3 10 30 100CYP1A2 101.6 116.1 87.5 95.6 78.3 >100CYP2A6 83.9 81.4 85.4 88.8 94.2 >100CYP2D6 84.5 90.1 88.1 80.4 75.2 >100CYP2C9 122.0 130.1 93.6 111.5 108.0 >100CYP2C19 79.4 87.1 81.8 76.8 62.0 >100CYP3A4 87.8 96.1 86.3 88.8 85.2 >100Table 3: The effects of CP-001 on the CYP enzyme activities with preincubation.P450 isozymeRemaining activities (% of control)IC50CP-001 (𝜇g/mL)1 3 10 30 100CYP1A2 111.4 98.8 79.3 83.5 70.6 >100CYP2A6 116.1 106.8 104.9 101.5 96.9 >100CYP2D6 107.3 109.6 129.2 109.3 98.7 >100CYP2C9 83.7 96.8 75.5 76.4 69.8 >100CYP2C19 117.5 109.7 106.3 94.2 75.1 >100CYP3A4 111.6 109.9 102.3 89.9 75.7 >1003. Results and DiscussionThe inhibitory effects of CP-001 on CYP-specific metabolicactivities were evaluated in human liver microsomes. Theassay system was tested with well-known selective inhibitorsof CYP isozyme (positive controls). The remaining activityof the CYP isozymes after the treatment of each selectiveinhibitor was as follows: 5.0% for CYP1A2 (furafylline);8.2% for CYP2A6 (methoxsalen); 5.3% for CYP2C9 (sul-faphenazole); 12.7% for CYP2C19 (ticlopidine); 2.5% forCYP2D6 (quinidine); and 4.5% for CYP3A4 (ketoconazole).All the inhibitors selectively inhibited the correspondingCYPmarker activity. When CP-001 was evaluated at concentra-tions of 1, 3, 10, 30, and 100 𝜇g/mL, CP-001 showed minimalinhibitory effects on all CYP isozymes tested (Table 2), withestimated IC50values above 100𝜇g/mL, although the enzymeactivity of CYP2C19 was slightly inhibited in a concentration-dependentmanner.WhenCP-001 was tested after preincuba-tion with human liver microsomes, the extent of inhibitionon several CYP isozyme activities was somewhat different,but the IC50values were all above 100 𝜇g/mL (Table 3),comparable to those without pre-incubation. Therefore, CP-001 is considered to have negligible effects on CYP-mediateddrug metabolism as a mechanism-base inhibitor as well as acompetitive inhibitor.There have been several reports on the effects of theherbs contained in CP-001 on CYP catalytic activities [10–12].Regarding H. cordata, decreases in the activities of CYP1A1,CYP2C11, and CYP2E1 were observed in 24 h oxidized fryingoil-fed rats after feeding with a diet containing H. cordata[10]. However, this study was not conducted to evaluate theherb-drug interaction but rather to evaluate chemopreventivepotential as those CYP enzymes could be involved in theactivation of precarcinogens or other chronic diseases. R.glutinosawas reported to inhibit CYP1A2,CYP2C9,CYP2D6,CYP2E1, and CYP3A4 in vitro in a concentration-dependentmanner, but significant inhibition was only observed at arelatively high concentration (1mg/mL), which is difficult toreach in a clinical setting [11]. There was a report on theeffects of R. coreanus on CYP3A activity, but the effect wasnot so considerable [12].The effects of B. platyphylla on CYP-mediated drug metabolism have not yet been reported.In conclusion, we have evaluated the inhibitory potentialof CP-001 on human CYP enzyme activities in vitro as apart of preclinical ADME studies.The present results indicatethat CP-001 may not interact with coadministered drugs bymodulating CYP-mediated metabolism. However, the resultsof in vitro tests are not necessarily consistent with in vivofindings. Therefore, continuous monitoring of herb-druginteractions along with pharmacokinetics studies may berequired through subsequent clinical stages.AcknowledgmentThis study was supported by a Grant of the TraditionalKoreanMedicine R&DProject of theMinistry for Health andWelfare, Republic of Korea (B110017 and B110043).References[1] B. J. Gurley, S. F. Gardner, M. A. Hubbard et al., “CytochromeP450 phenotypic ratios for predicting herb-drug interactions inhumans,”Clinical Pharmacology andTherapeutics, vol. 72, no. 3,pp. 276–287, 2002.4 Evidence-Based Complementary and Alternative Medicine[2] R.-X. Zhang, M.-X. Li, and Z.-P. Jia, “Rehmannia glutinosa:review of botany, chemistry and pharmacology,” Journal ofEthnopharmacology, vol. 117, no. 2, pp. 199–214, 2008.[3] Y.-Y. Sung, T. Yoon, J. Y. Jang, S.-J. Park, and H. K. Kim,“Topical application of Rehmannia glutinosa extract inhibitsmite allergen-induced atopic dermatitis in NC/Nga mice,”Journal of Ethnopharmacology, vol. 134, no. 1, pp. 37–44, 2011.[4] P.-S. Wu, S.-J. Wu, Y.-H. Tsai, Y.-H. Lin, and J. C.-J. Chao, “Hotwater extracted Lycium barbarum and Rehmannia glutinosainhibit liver inflammation and fibrosis in rats,” The AmericanJournal of Chinese Medicine, vol. 39, no. 6, pp. 1173–1191, 2011.[5] H. Kim, E. Lee, S. Lee, T. Shin, Y. Kim, and J. Kim, “Effectof Rehmannia glutinosa on immediate type allergic reaction,”International Journal of Immunopharmacology, vol. 20, no. 4-5,pp. 231–240, 1998.[6] H.-M. Kim, C.-S. An, K.-Y. Jung, Y.-K. Choo, J.-K. Park,and S.-Y. Nam, “Rehmannia glutinosa inhibits tumour necrosisfactor-𝛼 and interleukin-1 secretion from mouse astrocytes,”Pharmacological Research, vol. 40, no. 2, pp. 171–176, 1999.[7] H.M. Lu, Y. Z. Liang, L. Z. Yi, and X. J.Wu, “Anti-inflammatoryeffect ofHouttuynia cordata injection,” Journal of Ethnopharma-cology, vol. 104, no. 1-2, pp. 245–249, 2006.[8] W. Li, P. Zhou, Y. Zhang, and L.He, “Houttuynia cordata, a noveland selective COX-2 inhibitor with anti-inflammatory activity,”Journal of Ethnopharmacology, vol. 133, no. 2, pp. 922–927, 2011.[9] T.-Y. Lin, Y.-C. Liu, J.-R. Jheng et al., “Anti-enterovirus 71activity screening of Chinese herbs with anti-infection andinflammation activities,” The American Journal of ChineseMedicine, vol. 37, no. 1, pp. 143–158, 2009.[10] Y.-Y. Chen, C.-M. Chen, P.-Y. Chao, T.-J. Chang, and J.-F.Liu, “Effects of frying oil and Houttuynia cordata thunb onxenobiotic-metabolizing enzyme system of rodents,” WorldJournal of Gastroenterology, vol. 11, no. 3, pp. 389–392, 2005.[11] P. M. Y. Or, F. F. Y. Lam, Y. W. Kwan et al., “Effects ofRadix Astragali and Radix Rehmanniae, the components ofan anti-diabetic foot ulcer herbal formula, on metabolismof model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4probe substrates in pooled human livermicrosomes and specificCYP isoforms,” Phytomedicine, vol. 19, no. 6, pp. 535–544, 2012.[12] H. Kim, Y.-J. Yoon, J.-H. Shon, I.-J. Cha, J.-G. Shin, and K.-H.Liu, “Inhibitory effects of fruit juices on CYP3A activity,” DrugMetabolism and Disposition, vol. 34, no. 4, pp. 521–523, 2006.Submit your manuscripts athttp://www.hindawi.comStem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014MEDIATORSINFLAMMATIONofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Behavioural NeurologyEndocrinologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Disease MarkersHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalOncologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Oxidative Medicine and Cellular LongevityHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014PPAR ResearchThe Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofObesityJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Computational and  Mathematical Methods in MedicineOphthalmologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Diabetes ResearchJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Research and TreatmentAIDSHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Gastroenterology Research and PracticeHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Parkinson’s DiseaseEvidence-Based Complementary and Alternative MedicineVolume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

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

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The Evaluation of CP-001 (a Standardized Herbal Mixture of Houttuynia cordata

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