Study On Effects Of Danshen Capsules On The Pharmacokinetics And Pharmacodynamics Of Clopidogrel In Healthy Volunteers

With the increasing uses of herbal medicines from traditional natural products,the combination with therapeutic drugs is becoming more and more common in china.However,the potential herb-drug interactions are not fully understood.Thus,it is very necessary to investigate the potential herb-drug interactions during combined administration to ensure the rational and safe use of drugs.Danshen（Salvia miltiorrhiza Bunge）is widely and traditionally used in the prevention and treatment of cardiovascular,cerebrovascular,and hepatic diseases in Asian countries especially in China.Danshen is often co-administrated with other drugs in order to improve therapeutic efficacies.Thus,the possible herb-drug interactions must be taken precaution to avoid severe damage in clinic.In fact,a number of herb-drug interactions leading to adverse outcome were reported when Danshen was co-administered with therapeutic agents.For instance,Danshen gave rise to gross anticoagulation and bleeding complications when it was combined with warfarin for anticoaglant treatment.Nowadays,the most commercially available preparations from Danshen extract are formulated with the ethanol extract,in which the diterpenoid tanshinones including cryptotanshinone,tanshinone IIA,and tanshinone I are the major components,accounting for approximately95%of the total amount.It was reported that tanshinones played significant roles in the inhibition and induction of several CYP450 isozymes.Therefore,the possible CYP-related herb-drug interactions should be paid extra attention during the utilization of tanshinones-rich Danshen preparations in clinic.Clopidogrel bisulfate,an inactive thienopyridine prodrug,is 85%hydrolyzed in vivo by esterases to an inactive carboxylic acid metabolite.The remaining drug undergoes oxidative biotransformation to its active thiol metabolite by a 2-step,CYP-dependent process in which CYP3A4 and CYP2C19 were involved.It was reported that several drugs such as atorvastatin and omeprazole,due to their common requirements for CYP3A4or CYP2C19 metabolism,had the competitive inhibition to the metabolism of clopidogrel,decreasing the conversion of the clopidogrel prodrug to the active metabolite and increasing the risk for cardiovascular events because of inadequate platelet P2Y12 receptor inhibition.Inversely,St.John’s wort（SJW）,a CYP2C19 and CYP3A4 inducer,enhances the CYP metabolic activity and antiplatelet effect of clopidogrel in hyporesponders.So far,very little is known about whether the Danshen capsules has a modulatory effect on the pharmacokinetic and pharmacodynamics of clopidogrel in vivo.In the present study,the effects of multi-dose administration of Danshen capsules on the pharmacokinetic behaviors as well as the anti-platelet aggregation activity were investigated in healthy volunteers,aiming to provide valuable information for the use of Danshen preparations in clinical practice.Part one Establishment of Qualitative and Quantitative Analysis Methods of Danshen Capsules（One）Determination of cryptotanshinone and tanshinoneⅡA in danshen capsules by LC-MS/MSObjective:To develop a LC-MS/MS method for the determination of cryptotanshinone and tanshinone IIA in Danshen capsules.Methods:The samples were separated on a Symmetry C18（150 mm×4.6mm i.d.,5μm）column using acetonitrile and water（containing of 0.1%formic acid）as mobile phase in gradient elution mode.The mass spectrometer was operated under the positive ion mode with the ESI source.Scanning analysis was carried out using positive ion mode and multiple reaction monitoring（MRM）.m/z 297.1→251.2 for cryptotanshinone and m/z295.1→277.1 tanshinone IIA.Results:The calibration curves for cryptotanshinone and tanshinone IIA were linear over the range of 0.6256²0.02 ng/mL,0.6325²0.24 ng/m L,respectively.And the precision for cryptotanshinone and tanshinone IIA was2.1%and 1.8%,respectively.The average recovery was in the range of96.23¹01.44%.Conclusion:The analysis method is sensitive,simple and suitable enough to be applied for the determination of cryptotanshinone and tanshinone IIA in Danshen capsules.（Two）Determination of cryptotanshinone and tanshinoneⅡA in human plasma by LC-MS/MSObjective:To develop a LC-MS/MS method for the determination of cryptotanshinone and tanshinone IIA in human plasma.Methods:The plasma samples were protein precipitated with methano1.After sample preparation,the samples were separated on a Symmetry C18（150 mm×4.6 mm i.d.,5μm）column using acetonitrile and water（with 0.1%formic acid）as mobile phase in gradient elution mode.The mass spectrometer was operated under the positive ion mode with the ESI source.And the detection was performed by multiple reaction monitoring（MRM）of the transition of m/z 297.1→251.2 for cryptotanshinone,m/z295.1→277.1 tanshinone IIA and m/z 360.9→233.0 fenofibrate.Results:The linear relationship for cryptotanshinone and tanshinone II A was good in the concentration range of 0.5005¹00.1ng/mL and 0.5060¹01.2ng/mL,and the intra-day and inter-day precision（RSD）were less than 8.3%.The average recovery rate for cryptotanshinone and tanshinone IIA at low,medium and high levels were greater than 88.3%.Part Two The Choice of Healthy Subjects and the Determination ofClopidogrel and Its Active Metabolite in Human Plasma by HPLC-MS/MS（One）The choice of healthy subjects and its genetic type detectionObjective:Recruited healthy subjects openly and carried on the gene test to determine the genotypes and metabolic type.Methods:The plan was approved by Hebei Medical University Second Hospital Medical Ethics Committee.The recruitment advertisement was posted on the website to recruit healthy subjects openly.The sequencing reaction general kit was used for genetic testing subjects.Results:20 healthy subjects was selected and recruited.The age of the healthy subjects are within the scope of the 25³0 years old,BMI within the scope of the 19²5 kg/m².All of the 20 healthy subjects are extensive metabolizer and intermediate metabolizer.Conclusion:This study selected 20 volunteers with extensive and intermediate metabolism,so as to research the effects Danshen capsules on the pharmacokinetics and pharmacodynamics of clopidogrel.（Two）Determination of clopidogrel and its active metabolite in human plasma by HPLC-MS/MSObjective:To establish a HPLC-MS/MS method for the determination of clopidogrel and its active metabolites in human plasma.Methods:2-Bromo-1-（3-methoxyphenyl）ethanone（MPB）was used as a derivatization agent.The plasma treatment procedure involved single-step protein precipitation to determine plasma concentrations of bulk drug and active metabolites.A Diamonsil C18 column was used,with the mobile phase of acentonitrile:1 mmol/L ammonium acetate solution containing 0.1%formic acid（80:20）,with ticlopidine as the internal standard.The mass spectrometer was operated under the positive ion mode with the ESI source.And the detection was performed by MRM mode with the transitions of m/z322.2→m/z 212.0（clopidogrel）,m/z 504.4→m/z 354.1（active metabolites derivative）and m/z 264.2→m/z 154.1（ticlopidine）,respectively.Results:The calibration curves were linear over a concentration range of0.093²3.3 ng/m L for clopidogrel,0.32⁸0.0 ng/mL for CAMD.The lower limits of quantitation were 0.093 ng/m L and 0.32 ng/mL,respectively.The recovery rate ranged from 83.4%⁸5.2%and 81.9%⁸4.0%for the analytes,and the relative standard deviations for intra-and inter-batch precision were all less than 7%.In stability tests,clopidogrel and CAMD were found to be stable in plasma under various storage conditions.Conclusion:The method is simple and quick,with high repeatability,sensitivity and specificity,which can be used to analyze clinical samples and determine the pharmacokinetics of clopidogrel as well as its active metabolites.Part Three Effects of Danshen Capsules on the Pharmacodynamics of Clopidogrel in Healthy VolunteersObjective:To investigate the interaction between Danshen capsules and clopidogrel from the pharmacokinetic point of view.Methods:In 20 healthy volunteers,clopidogrel 300 mg was orally taken once daily for 7 days;then was orally administration of Danshen capsules on day 8 for 6 consecutive days.On the 14th day,four tablets of Danshen capsules were orally administered,and then clopidogrel 300 mg was orally administered.Blood samples were collected before taking clopidogrel and at different times after taking the drug.The plasma concentrations of clopidogrel and its active metabolites were determined by HPLC-MS/MS.Results:Co-administration of multiple doses of Danshen capsules caused an increase in apparent oral clearance of clopidogrel and its metabolite by96.5%and 73.7%,a corresponding decline in C_max by 41.7%and 32.9%,and a decline in AUC_（0,∞）by 49.3%and 41.5%in human volunteers,respectively.Conclusion:It was shown that the oral co-administration of Danshen extract significantly altered the pharmacokinetic profiles,including the CL/F,C_max and AUC_（0,∞）of clopidogrel and CAMD.That is,Danshen capsule can induce the metabolism of clopidogrel.Part Four Effects of Danshen Capsules on the Anti-platelet Aggregation of Clopidogrel in Healthy VolunteersObjective:To investigate the interaction between Danshen capsules and clopidogrel from anti-platelet aggregation of clopidogrel.Methods:In 20 healthy volunteers,clopidogrel 300 mg was orally taken once daily for 7 days;then was orally administration of Danshen capsules on day 8 for 6 consecutive days.On the 14th day,four tablets of Danshen capsules were orally administered,and then clopidogrel 300 mg was orally administered.Blood samples were collected before taking clopidogrel and at different times after taking the drug.Platelet aggregation rate was tested with turbidometric platelet aggregometry.Results:The platelet aggregation rate between the two periods of Danshen capsules application were compared,and a significant difference was found.Co-administration of multiple doses of Danshen capsules caused a decrease in platelet aggregation rate of clopidogrel.Conclusion:It was shown that the oral co-administration of Danshen extract significantly altered the platelet aggregation rate of clopidogrel.There was an interaction between Danshen capsules and clopidogrel.Part Five Effect of Cryptotanshinone and TanshinoneⅡA in Danshen Capsules on the Activity of CYP3A4 in Human Liver CellsObjective:To investigate the effects of tanshinone IIA and cryptotanshinone in Danshen capsules on cytochrome P450 enzyme and CYP3A4 expressions in human liver cells.Then analyze the drug-drug interaction so as to lay a foundation for clinical reasonable combination of Danshen capsules and provide theoretical basis for clopidogrel.Methods:The effects of tanshinone IIA and cryptotanshinone on CYP4A4 activity in human liver were investigated by incubation method of liver microsome.The effects of tanshinone IIA and cryptotanshinone on the expression of CYP3A4 protein in human liver cells were investigated by Western blotting at 48 h after administration.Results:Compared with the blank control group,two components of cryptotanshinone and tanshinone IIA enhanced CYP3A4 activity after liver microsome incubation,and the results were statistically significant at 20 min（P<0.05）.Cryptotanshinone and tanshinone IIA induced the expression of CYP3A4 protein in liver cells after the intervention of cryptotanshinone and tanshinone IIA for 48 h.Conclusion:The two active ingredients in Danshen capsules can induce CYP3A4 activity,and increase the CYP3A4 expression in liver cells.Therefore,Danshen capsules can induce the metabolism of clopidogrel,which suggests that the drug-drug interaction should be concerned in clinic.