Clopidogrel, a second generation thienopyridine P2Y12 inhibitor, has been the standard-of-care for percutaneous coronary intervention (PCI) and/or acute coronary syndrome (ACS) [
Clopidogrel is an inactive prodrug that requires enzymatic conversion by a number of carboxylesterases (CESs) enzymes and cytochrome P450 (CYP) enzymes [
Formation of the active CAMD metabolite.
Our previous study found that multiple dose (30 days) of clopidogrel altered hepatic CES1A in rats, which resulted in elevating the serum inactive carboxylic metabolite [
Xuesaitong (XST, Chinese drug Z20050467), extracted from
The 2-bromo-3’-methoxyacetophenone- (MPB-) derivatized clopidogrel thiol metabolite (CAMD, lot No. 5-MNZ-195-23), MPB (lot No. 151910100), and internal standard (IS, guanosine, lot No. 111977-201501) were purchased from the Toronto Research Chemicals, Sigma, and the Chinese National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), respectively. XST tablets (0.5 g/tablet, lot No. DHB1606, expiring before May 2018), containing 13.6 mg/g ginsenoside Rg1, 8.6 mg/g ginsenoside Rd, and 7.8 mg/g ginsenoside R1, were manufactured by the Yunnan Dali Medicine Factory (Yunnan, China). Clopidogrel tablets (25 mg/tablet, lot No. AA20150207, expiring before Jan 2018) were manufactured by Shenzhen Salubris Pharmaceuticals Co. (Shenzhen, China). Acetonitrile and methanol were HPLC grade (Merck, USA). The ultrapure water used for UHPLC-MS/MS was from a Milli-Q water purification system (Millipore, USA).
The RNAiso Plus reagent, PrimeScript™ II 1st Strand cDNA Synthesis Kit, SYBR Premix Ex Taq™ II kit and primer were provided by the TaKaRa Biotechnology Company (Takara, Japan).
Thirty male SD rats, body weights 220-300 g (License No. SCXK, Jiangsu Province, China, 2014-0007) were purchased from Suzhou Industrial Park Aier Matt Technology Co. Ltd. (Suzhou, China). All rats were pathogen-free and acclimated for at least one week. The rats were housed in an environmentally controlled room with a temperature of 20±2°C, light from 06:30 h to 18:30 h, and humidity of 60 ±5%. All rats were fed standard rodent chow and water ad libitum. This procedure was approved by the Animal Ethics Committee of the Nanjing University of Chinese Medicine.
Three groups were randomly assigned according to a parallel study. The control group consisted of 6 rats (oral administration of equal volumes of saline) for mRNA analysis. The clopidogrel group received 30 mg/kg of clopidogrel orally that comprised 12 rats, with 6 designated for mRNA analysis and 6 for the pharmacokinetic study. The combination group had 12 rats, with 6 allocated for mRNA analysis and 6 for the pharmacokinetics, and all orally received both clopidogrel at 30 mg/kg and XST at 50 mg/kg. The rats in each group were treated for 30 days. The rats fasted for 12 h before the experiment but had unlimited access to water. The drugs were suspended in saline before oral administration to rat.
All rats were continuously intragastrically fed each drug for 1 month, as described in “animal treatment”. For the pharmacokinetic analysis, 6 rats each were selected from the clopidogrel and combination group. Blood plasma samples of approximately 150
The remaining 18 rats from the three groups were used for mRNA analysis. After overnight fasting, the rats were sacrificed under anesthesia by i.p. administration of a 0.4 mL/100 g dose of a 10% chloral hydrate solution. The samples were finally collected as follows. The rat livers were removed and weighed. Portions of the liver samples were stored at −80°C for further mRNA biochemical assays [
Master stock solutions were prepared by individually dissolving CAMD and IS in methanol at free-base equivalent concentrations of 1 000
All frozen standards and samples were thawed on wet ice before homogenization. A 50
Chromatographic separations were performed with an Agilent HPLC 1290 system (Agilent, USA) consisting of a quaternary pump, an online degasser, and an autosampler. The chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase analytical column (110 Å, 3
Detection of the analytes and IS was performed on a G6430 tandem quadrupole mass spectrometer (Agilent, USA) with an electrospray ionization (ESI) interface in positive ion mode. Multiple reaction monitoring (MRM) was used to monitor precursor to product ion transitions of
The method was validated in terms of linearity, accuracy and precision, selectivity, matrix effect (ME), recovery, and stability according to the guidelines for bioanalytical method development recommended by the US Food and Drug Administration and related literature for CAMD detection [
The blood samples in the pharmacokinetic analysis were prepared, and the CAMD concentrations were assessed by the validated LC-MS/MS method. Pharmacokinetic parameters were calculated using the Drug and Statistic (DAS) 3.0 pharmacokinetic software (Chinese Pharmacological Association, Anhui, China).
Total RNA was extracted from 100 mg of the livers using the RNAiso Plus reagent (Takara, Japan) [
Each value obtained from experiments was expressed as the mean ± SE, n = 6. The mean comparisons for each group from the pharmacokinetic and mRNA Expression were performed using Student’s
Schrodinger Maestro 8.5 was used to investigate the molecular simulation. The XST bioactive components (ginsenoside Rg1, Rd and notoginsenoside R1) and the CES1A protein (PDB ID 1MX1) were prepared with LigPrep and protein preparation wizard, respectively. Then, the above materials were subjected to Glide based three-tiered in silico target screening strategy by two stages of the docking protocol, High Throughput Virtual Screening (HTVS), and Standard Precision (SP).
The protein precipitation sample preparation in combination with UHPLC–MS/MS detection provided good selectivity for the CAMD analytes and the IS. Figure
Chromatograms of CAMD and IS. (a) A blank rat plasma sample; (b) a blank plasma spiked with CAMD and IS at the LLOQ; (c) a rat plasma sample after an oral administration of XST (50 mg/kg) and clopidogrel (30 mg/kg) at intervals of 0.5 h.
The assay was validated over the nominal concentration range of 0.64-66.00 ng/mL. The calibration curve correlation coefficients (
Mean matrix effect and recovery of CAMD in rat plasma (
Concentration (ng/mL) | Matrix effect (%) | RSD (%) | Recovery (%) | RSD (%) |
---|---|---|---|---|
1.80 | 92.40 | 4.90 | 90.20 | 5.10 |
30.00 | 101.20 | 6.70 | 93.10 | 6.30 |
49.50 | 98.30 | 5.30 | 92.30 | 2.30 |
The extraction recoveries of the analytes from plasma at the three QC concentration levels were 90.20%-93.10%. The matrix effects at three QC levels were in the range of 92.40%-101.20% with RSD values below 6.70%. In this assay, the intra- and interday precisions were measured to be below 7.10% and 6.30%, respectively, with relative errors from −2.30% to 5.30% (Table
Precision and accuracy of CAMD assay in rat plasma (
Concentration | Intra-day | Inter-day | ||
---|---|---|---|---|
Precision | Accuracy | Precision | Accuracy | |
0.64 | 7.10 | 4.30 | 4.80 | 3.30 |
1.80 | 6.20 | -2.30 | 6.30 | 4.40 |
30.00 | 5.60 | 5.30 | 2.90 | 3.70 |
49.50 | 6.90 | 3.80 | 4.90 | 4.90 |
The analytes were stable in the plasma samples for at least 6 h at room temperature or on ice. No significant degradation was observed when extracted plasma samples were kept at 4°C in the autosampler for up to 24 h.
The validated UHPLC–MS/MS assay was successfully applied to the quantitation of CAMD in rat plasma samples. The mean plasma concentration-time profiles are illustrated in Figure
Pharmacokinetic parameters of CAMD after intragastric administration of clopidogrel alone or coadministration of clopidogrel and XST to rats.
Parameter | Clopidogrel group( | Combination group( |
---|---|---|
| | |
| | |
| | |
| | |
| | |
| | |
Vd/F (L) | | |
CL/F (L/h) | | |
T1/2 | | |
Mean plasma concentration–time profiles of CAMD in rats (n = 6) after continuous oral administration of clopidogrel (30 mg/kg) with or without XST (50 mg/kg).
The
In the present investigation, it was found that the pharmacokinetic parameters of CAMD in combinational group were different from those in clopidogrel rats. Clopidogrel and XST coadministration appreciably increased the Cmax, AUC, and MRT of CAMD (the active thiol metabolite). The above results indicated that combination with XST could increase the plasma concentrations of CAMD in rats.
The expression of the CES1A enzyme mRNA was measured by Real-Time RT-PCR in rat liver to evaluate the impact of the XST on clopidogrel hydrolysis. As shown in Figure
The relative expression of CES1A mRNA in rat livers after 30 days of administration in each group, as determined by RT-PCR.
The docking score of ginsenoside Rg1, Rd, and notoginsenoside R1 against CES1A protein from the molecular simulation were, respectively, -6.54, -7.12, and -8.13. Once the ligand-receptor complex formed, it adapted to the most stable conformation. The active site of the CES1A protein revealed that several molecular interactions could be considered responsible for the observed affinity. Hydrogen bonds could be found between the protein residue GLU815, LYS866, LUE838, and the XST ligand. These results suggest that the activity of the CES1A metabolic enzyme activity may be partially inhibited by XST. The molecular simulation results were consistent with the previous CES1A mRNA expression results.
Above all, great changes have took place in both pharmacokinetic parameters and CES1A metabolic enzyme aspect (by mRNA expression and molecular protein inhibition). Possible reasons for XST-clopidogrel interaction are complex and diverse, including gastrointestinal lesions that cause changes in drug absorption, changes in transporters responsible for uptake, efflux, and elimination, and changes in the metabolic enzymes which alter the clopidogrel metabolic rate.
Clopidogrel is an inactive prodrug that requires enzymatic conversion by carboxylesterases (CESs) and cytochrome P450 (CYP) enzymes. The liver is the organ responsible for drug metabolism enzymes. Patients have reduced hepatic metabolism of clopidogrel, via the CESs and P450 enzyme group. Currently, most of herbal medicines are administered via the oral route. While previous data showed that multiple dose of clopidogrel induced accumulation of the inactive clopidogrel carboxylic acid metabolite, the accumulation phenomenon was reduced by combination with the Chinese medicine FDDP [
Drug transporters also have a critical role in controlling drug exposure. Transporters are proteins facilitating the passage of drugs across biological barriers encountered during drug metabolism, among which P-glycoprotein (P-gp) can expel various drugs, resulting in multidrug resistance, and is likely to play a critical role in the uptake and absorption of substrate drugs. The intestinal absorption of ginsenoside Rg1, Rd, and notoginsenoside R1 (the main component of XST), is enhanced by the inhibition of P-gp activity. All of the above may contribute to the changes in pharmacokinetic behavior of CAMD in rats compared with when combination with XST. However, the proposed hypotheses still need further investigation and validation.
Sensitive UHPLC-MS/MS and RT-PCR technique were successfully used to characterize the clopidogrel and XST herb-drug interaction in rats. Clopidogrel and XST coadministration appreciably increased the Cmax, AUC, and MRT of CAMD (the active thiol metabolite) and decreased the CES1A mRNA expression. Animal studies indicated that clopidogrel and XST coadministration produced significant herb-drug interactions in pharmacokinetic and metabolic enzyme aspect. In a word, 30-day dose of coadministration altered hepatic CES1A, and this resulted in elevated serum CAMD levels. Decreased CES1A mRNA expression and elevated serum CAMD levels were due to the XST combination.
Sprague Dawley
Xuesaitong dispersible tablets
Carboxylesterase 1 A
Clopidogrel thiol metabolite derivative
Ultra-high-performance liquid chromatography-tandem mass spectrometry
The 2-bromo-3’-methoxyacetophenone.
The data used to support the findings of this study are available from the corresponding author upon request.
We declare that there are no conflicts of interest associated with this manuscript and there has been no financial support that could have influenced the outcome.
Shitang Ma and Guoliang Dai equally contributed to this work.
The authors are grateful for the financial support provided by the National Natural Science Foundation of China (Grants nos. 81403114 and 81403268), the talent fund of the Education Department of Anhui Province (Grant no. gxfx2017076), the Anhui Science and Technology University (Grant no. SPWD201602), and the Public Welfare Technology Application Research Linkage Project of Anhui Province (Grant no. 1704f0704062).