Investigation On The Mechanism Of Fufang Danshen Dripping Pills-Clopidogrel Synergistic Effects

OBJECTIVEThe coronary heart disease,as well as associated cardiovascular disease(CVD)is the leading killer of human being with high mortality and fatality.Anti-platelet therapy with clopidogrel(CPG)is first choice to prevent a thero thrombotic event in CVD patients.Despite its obvious advantages,many clinical observation suggest that approximately 5-40%of patients treated with conventional doses of the drug display inadequate responses,which may lead to serious cardiovascular event.The Chinese traditional medicine for treating CAD,especially for activating blood circulation to dissipate blood stasis,is commonly used as mainstream.Fufang Danshen Dripping Pill(FDDP)is a famous traditional chinese medicine(TCM)recipe,containing Slvia Miltiorrhiza(SM),Radix Notoginseng(NG),Borneol(BN),and has been used for the treatment of CADs.And many prescriptions of TCM hospital for treatment of some CADs usually contain FDDP and CPG,but the synergistic effect remains unclear.As for the synergistic effect of Chinese and West medicine combination,most reaseach are maily centrated on the genic polymorphism of drug metabolizing enzymes,as well as the pharmacokinetics and pharmacodynamics(PK-PD)of single component.To our knowledge,there was no publication describing a synergistic effect of Chinese and West medicine combination by modulating plasma protein binding rate,metabolism target and PK-PD.METHODThe synergistic effects of CPG-FDDP combination by modulating the plasma protein binding rate,metabolism target,PK,PD and in silico phamarcology is investigated as follows:1.The ginsenoside Rgl and Rbl from FDDP,which account for the CVD activity were selected as analyst.the HPLC and equilibrium dialysis were firstly employed to determine the concentration of the analyst both in dialysate(PBS)and blank serum(bovine serum for Rgl,human serum for Rbl respectivly.).The differences in protein-binding rate between each anlyst alone and combination with CPG group were then compared.Finally,consisting with in-sinico molecular docking and homology modeling procedure,molecular simulation technique was used to display the competition mechanism with each plasma protein.2.The CES1?Cytochrome P450 3A4?CYP450 2C19?CYP450 1A2?CYP4502B6 which involved in CPG metabolism were selected,the chemical composition database of FDDP were then constructed and finally the computational pharmacological effects between the chemical composition database and CPG metabolism target was investigated to demonstrated the synergistic effects modulating the above metabolism enzyme.3.The ginsenoside Rg1,as well as CPGS(the CPG inactive metabolite)were selected as analyst.SD rats were treated with oral CPG alone at a dosage of 30 mg/kg(or FDDP at dosages of 324 mg/kg)and the two drug combination for 21 days before sample collection.The concentrations of Rgl(or CPGS)in the blood plasma samples of the first cycle(single dose)and the second cycle(for 21d)were determined using a validated high-performance liquid chromatography method.The differences in PK between alone and combination group were finally compared to explore the PK synergy.4.The coagulation parameters(consisted by prothrombin time,activated partial thromboplastin time,fibrinogen concentration and thrombin time),thrombosis in arteriovenous shunt and P2Y12 target were for corresponding drugs for 21 continuous days.And the hemorheology?haemostatic and blood corpuscle index for 100 continuous days.The above index was choosed to explore the synergistic pharmacodynamics of the FDDP-CPG combination.5.Based on each target's biological roles in CVD disease,27 CVDT protein were firstly selected;chemical composition database of FDDP were then constructed and the effects between the chemical composition database and CVDT target were explored by molecular simulation method.The interaction between bioactive component and its associated target was finally visualization analysis by network pharmacology.RESULTS1.The serum protein binding rate of ginsenoside Rg1 at low,middle and high concentrations(0.3125?1.2500?5.0000 mg·L-1)were 11.16±4.82?13.42±4.21?14.61 ±3.42%respectively.when together with CPG,the binding rates were 6.53±2.32?9.23±4.54?12.12±3.72%respetivly.The protein binding rate of Rbl in serum PBS and human plasma at low(0.6250 mg·L-1)?middle(1.2500 mg·L-1)?high(5.0000 mg·L-1)concentrations were 58.17±3.82?57.43 ±3.21?55.63±3.42%respectively.when combination with CPG,the value were decline to 46.54±3.35?49.25 ± 3.56?48.15 ± 3.76%respectively.The molecular simulation results suggested that the Rgl and Rbl compound have competitive synergistic effects with bovine?human plasma protein respectly.2.1MX1(CES1)?3NXU(CYP450 3A4)?4GQS(CYP450 2C19)?2HI4(CYP4501A2)?3IBD(CYP450 2B6)and THA?RIT?OXU?Chlorzoxazone?CPZ were used as receptors and cutoff for each target respectively,the No.of hits with potentially positive activities with each metabolism enzymes target from FDDP were 29?8?31?51?44 respectively.The computational pharmacological studies were accordance with the referenced cocktail experiment results.3.The PK results of Rgl:compared with the FDDP alone group,the value of Ke decreased,while the T1/2 ??AUC?Tmax?Cmax?MRT increased in the combination group.the above PK results is consistent with the first chapter.The PK results of CPGS:in the first cycle,some parameters such as Tmax?Cmax is similar to the reference.Compared with CPG alone group,the T1/2 ??Tmax and MRT in combination group increased,while Cmax?AUC decreased.For the two cycles in each CPG alone group,the Cmax?AUC(0-tn)?AUC(0-?)of CPGS significantly increased after 21 d dose.compared with the alone group in second cycle,T1/2 ??Cmax?AUC(0-tn)?AUC(0-?)?MRT(0-tn)of CPGS in combination group decreased.4.The 21 d Short-term results showed:compared with the alone group,the bleeding time of coagulation parameters was prolonged,the thrombosis in arteriovenous shunt were inhibited in the combination groups.The molecular docking showed that 23 bioactive compounds contained in FDDP was able to inhibit target P2Y12.the 100d long-term results suggested:The leukocyte?red blood cells?platelet parameters of blood cell index and blood viscosity was reduced,and the bleeding time of haemostatic parameters(TT?APTT?PT)was prolonged in the medication group.The above effect was much better in the combination groups.There were statistical differences between the combination and FDDP alone group(p<0.05),and no statistical differences between the combination and CPG alone group.5.The bioactive components in FDDP had good drug(lead)like characteristics.Target PAFR and 5LO were constructed and validated by homology modeling and ramachandran plot pespectly.The virtual screening results showed that compounds from SM,NG mainly interacted with coagulation and inflammation targets.The component-target network analysis results indicated that many predicted targets were hit by only one candidate compounds,but most can be modulated by multiple components rather than single ingredient,suggesting FDDP is of "target-rich and lead-poor" imbalance.CONCLUSION1.The protein binding rate of ginsenoside Rgl and Rbl were decreased when administration with CPG through equilibrium dialysis and molecular simulation investigation.2.The FDDP has interaction effect with CPG metabolism enzymes target(CES1?Cytochrome P450 3A4?CYP450 2C19?CYP450 1A2?CYP450 2B6)on the overall level.3.Combination with CPG has partial effect on the pharmacokinetic character of Rgl from FDDP,in Clinical CVD patients should take long-term medication as to maintain steady blood concentration for anti-platelet effect.The activity of CES1 could be partly inhibited by FDDP,which would result in the decline of CPGS production.The concentration-time course of CPGS was altered slightly by FDDP,and FDDP decreased the peak plasma concentration and area under the curve of CPGS.4.The PD results demonstrated the synergistic effects of CPG and FDDP by modulating coagulation parameters?thrombosis?blood rheology?blood parameters index.5.The results gave a molecular description of the pharmacodynamics material basis for the systematical in-silico pharmacology on CVDT.Above all,the whole research demonstrated the synergistic effects of CPG and FDDP by modulating plasma protein binding,metabolic enzymes,PK and PD synergy.