Interaction Between Clopidogrel and Panax Notoginseng Saponins

Background: Panax notoginseng saponins (PNS) is commonly used in combination with clopidogrel in clinic. This study was to investigate the effect of PNS on the pharmacokinetics of clopidogrel active metabolite CAMD and the effect on the activities of clopidogrel metabolic enzymes in rats. Methods: In pharmacokinetics studies, the rats were divided into clopidogrel and combination groups, and continuously administered for seven days. The concentration of CAMD was determined by LC-MS/MS. In enzymes studies, the rats were divided into control and PNS groups. After administration for seven days, CYP2C19 and CYP3A4 activities were measured by the metabolic rate of the specic substrate in rat liver microsomes. The activity of CES1 enzyme was determined by double antibody sandwich method. Results: PNS signicantly increased AUC 0- ∞ of CAMD from 43.1±11.6 to 72.0±25.1 h·ng/mL (p<0.05). Combination group had lower CL/F and Vd/F than clopidogrel group (p<0.05). PNS signicantly decreased the activity of CYP3A4 and CES1 (p<0.01), but no signicant effect on CYP2C19. Conclusions: The combination use of PNS and clopidogrel produced a signicant increase in the AUC of clopidogrel active metabolite CAMD. PNS could inhibit the activity of CYP3A4 and CES1 enzyme in rat.

Meng reported that in patients undergoing percutaneous coronary intervention (PCI), the ADP-induced platelet aggregation inhibition rate in the PNS-clopidogrel group was signi cantly higher than that in the clopidogrel group [3]. Zhao reported PNS Injection could reduce platelet aggregation in acute cerebral infarction patients with clopidogrel resistance [4]. These studies indicated that PNS and clopidogrel had signi cant interactions, however the exact mechanism was unclear. This paper was designed to nd some evidence on synergistic effect mechanism of PNS and clopidogrel, whether PNS can increase the exposure of active metabolite of clopidogrel, whether PNS may affect metabolic enzymes activity of clopidogrel? Taking these factors into consideration, we performed the study on the effect of PNS on the pharmacokinetics of clopidogrel active metabolite and the effect of PNS on metabolic enzymes.
According to the metabolic characteristics of clopidogrel, the metabolic enzymes mainly aimed at CYP2C19, CYP3A4 and CES1.

LC-MS/MS Analysis
The concentrations of CAMD, 4-hydroxymephenytoin and 6b-hydroxytestosterone were determined by modi ed LC-MS/MS methods. Separations were performed on a UPLC BEH C18 column (1.7 µm, 2.1 mm i.d.×50 mm). The ow rate of the mobile phase was 0.3 ml/min, with a gradient ranging from 10 to 95% methanol containing 0.1% formic acid in a 3-min run. The mass spectrometric analysis was carried out on an electrospray ionization (ESI) source in positive ion mode, and the quanti cation was performed Effect of PNS on the pharmacokinetics of clopidogrel active metabolite Ten rats were randomly divided into two groups: the clopidogrel group and the combination group. For oral dosing, clopidogrel bisulfate and PNS dissolved in a 0.1% CMC-Na solution were continuously administered to the rats for seven days by gavage at doses of 30 mg/kg and 40 mg/kg, respectively. The combination group received PNS 15 min prior to clopidogrel administration. On the seventh day, blood samples (150mL) were collected into 1.5-mL pretreated EDTA centrifuge tubes from the fossa orbitalis vein before (0 hour) and after clopidogrel bisulfate administration at 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 hours. Immediately after collection, 2μL of 500 mM MPB in acetonitrile was added to each blood sample to derivatize the active metabolite of clopidogrel [5]. The blood samples were gently mixed. Then, the samples were centrifuged at 4000 rpm for 10 min at 4 °C, and the separated plasma samples were stored at −80℃until assay. All frozen standards and samples were thawed on wet ice before homogenization. A 50 mL aliquot of each plasma sample and 150 mL of loratadine methanol solution (IS) were transferred into a 1.5 mL centrifuge tube. The mixture was vortex-mixed for 2 min and centrifuged at 20000 rpm for 15min. Then the supernatant was transferred to the LC-MS/MS for analysis. The concentration of clopidogrel active metabolite CAMD was determined. The research ethics committee of our hospital approved the protocol followed in this study. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [6].

Effect of PNS on rat liver enzymes
The rats were divided into control and PNS (40 mg/kg/d) groups. The rats were administrated orally for seven days. Liver microsomes were prepared by calcium precipitation method [7]. The microsomes preparations were stored at -80 ºC until used. Protein concentrations were determined by the Lowry method [8], with bovine serum albumin as the standard.
CYP2C19 and CYP3A4 Enzyme activity assay (S)-mephenytoin and testosterone were chosen as the typical substrates for CYP2C19 and CYP3A4. The incubation was performed in 0.1 mL of incubation mixture containing 50mmol (s)-mephenytoin or 40 mmol testosterone, the microsomes protein was 0.5 mg/mL. The metabolic reaction was stopped by adding 0.3 mL of methanol (contain IS loratadine) to the incubation mixture at 30 min (for mephenytoin) and 10min (for testosterone), respectively. The contents were vortex-mixed and centrifuged. An aliquot of 5µL of supernatant was injected into the LC-MS/MS system. The concentrations of 4hydroxymephenytion and 6β-hydroxytestosterone were determined.

CES1 Enzyme activity assay
The concentration of CES1 in rat hepatic microsomes was determined by double antibody sandwich method. Puri ed rat CES1 antibody was used to coat microtiter plate wells and make solid-phase antibody. Rat CES1 was successively added to the wells, then combined with horseradish peroxidase (HRP) labeled antibody to form the antibody-antigen-enzyme-antibody complex. After thorough washing, tetramethylbenzidine (TMB) substrate was added for color development, then TMB substrate was converted to blue by HRP enzyme, and to the nal yellow by sulphuric acid solution. The color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of CES1 in the samples was then calculated by the standard curve.

Effect of PNS on the pharmacokinetics of CAMD
The effect of PNS on the pharmacokinetics of CAMD following oral administration was investigated in rats. As shown in Table 1 and Figure 2, when coadministered with PNS for seven days, the C max and AUC 0-∞ of CAMD increased from 15.8±5.5 to 34.7±28.9 ng/mL (p>0.05) and 43.1±11.6 to 72.0±25.1 h·ng/mL (p<0.05), respectively. Combination group had lower CL/F and Vd/F than clopidogrel group (p<0.05) . As shown in Figure 3, compared to the control group (4.0±0.1 nmol/mg protein/min), there was a signi cant decrease in the CYP3A4 activity (the formation of 6β-hydroxytestosterone) in rat hepatic microsomes pretreated PNS (3.5±0.1 nmol/mg protein/min) (p<0.01).
Effect of PNS on CES1 Enzyme activity As shown in Figure 4, compared to the control group (34.3±0.6 pg/mg protein), there was a signi cant decrease in the CES1 activity in rat hepatic microsomes pretreated PNS (30.4±1.3 pg/mg protein) (p<0.01).

Discussion
The use of traditional Chinese medicine has gained increasing acceptance all over the world due to its multi-target and multi-level function characteristic. PNS is a common used tradition Chinese medicine in clinic. The interaction between PNS and western medicine needs attention. Among the relevant causes of drug-drug interactions, the effect of drugs on metabolic enzymes have been identi ed as one of the main reasons. According to the literature reviewed, some studies have reported the effect of PNS on metabolic enzymes. Chen reported PNS could signi cantly induce CYP1A2 and CYP2E1 enzyme activity, mRNA expression and CYP2E1 protein expression leve1 [9]. A vitro studies revealed that PNS could inhibit CYP3A activity [10]. Several studies had actually found that PNS could induce CYP1A2 activity [11,12]. In our study of the effect of PNS on the pharmacokinetics of clopidogrel active matabolite CAMD, coadministration with PNS for seven days signi cantly increased the exposure of CAMD approximately 1.7-fold, and Cl/F was decreased to 58.4% of clopidogrel alone. In the study of the effect of PNS on metabolic enzymes, pretreated PNS could inhibit CYP3A4 enzyme activity in rat livers. This couldn't explain why PNS can increase CAMD exposure.
Qi reported PNS had a weak inhibition effect on CES, but concentraion -dependent inhibition on CES2 in vitro [13]. A study by Sun showed PNS were demonstrated to inhibit the CES activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of CES1 and CES2, whereas exhibited minor effect on the mRNA expression [14]. A review showed that more than 50 natural inhibitors of CES1 or CES2, including phenolic compounds, triterpenoids and tanshinones, while inducers of CES1 and CES2 were less reported [15]. In our study, we found that pretreated 40mg/kg/d PNS could inhibit CES1 enzyme activity in rat livers, thereby reduce the inactivation of clopidogrel and indirectly increased the concentration of active metabolite CAMD.
We know that inhibition of CYP enzyme can reduce the activation of clopidogrel, while inhibition of CES1enzyme can reduce the production of inactive metabolites of clopidogrel. Therefore, the synergistic mechanism between PNS and clopidogrel is complex and multifaceted, the effect on CES1 enzymes probably be more signi cant. In subsequent studies, it is necessary to carry out experiments on the effects of notoginsenoside R1, ginsenoside Rb1, Rg1, Rd and Re, the main components of PNS on CES1 enzyme.

Conclusions
The combination use of PNS and clopidogrel produced a signi cant increase in the AUC of clopidogrel active metabolite CAMD. PNS could inhibit CYP3A4 and CES1 enzyme activity in rat liver. The synergistic mechanism of clopidogrel and PNS is probably related to the inhibition of CES1 enzyme.   Effect of PNS on CYP2C19 enzyme activity in rat microsomes As shown in Figure 3, compared to the control group (4.0±0.1 nmol/mg protein/min), there was a signi cant decrease in the CYP3A4 activity (the formation of 6β-hydroxytestosterone) in rat hepatic microsomes pretreated PNS (3.5±0.1 nmol/mg protein/min) (p<0.01).

Figure 2
Effect of PNS on CYP2C19 enzyme activity in rat microsomes As shown in Figure 3, compared to the control group (4.0±0.1 nmol/mg protein/min), there was a signi cant decrease in the CYP3A4 activity (the formation of 6β-hydroxytestosterone) in rat hepatic microsomes pretreated PNS (3.5±0.1 nmol/mg protein/min) (p<0.01).

Figure 3
Effect of PNS on CYP3A4 enzyme activity in rat microsomes Effect of PNS on CES1 enzyme activity in rat microsomes Effect of PNS on CES1 enzyme activity in rat microsomes