Materials and chemicals
PNS was purchased from Chengdu DeSiTe Biological Technology Co., Ltd. Clopidogrel bisulfate, testosterone, (S)-mephenytoin, buspirone, loratadine, 6b-hydroxytestosterone, 4-hydroxymephenytoin, trinatric isocitric acid, isocitric dehydrogenase, b-nicotinamide adenine dinucleotide phosphate (NADP) and b-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) were purchased from Sigma (St Louis, Mo, USA). The 2-bromo-3’-methoxyacetophenone (MPB) , CAMD were purchased from the Toronto Research Chemicals.
The concentrations of CAMD, 4-hydroxymephenytoin and 6b-hydroxytestosterone were determined by modified LC-MS/MS methods. Separations were performed on a UPLC BEH C18 column (1.7 µm, 2.1 mm i.d.×50 mm). The flow 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 quantification was performed using multiple reaction monitoring (MRM) Mode (the ion pair of CAMD at 504.0→354.1, 4-hydroxymephenytoin at 235.1→150.1, 6b-hydroxytestosterone at 305.4→269.3, loratadine (IS) at 383.2→267.1.
Male Sprague-Dawley (SD) rats were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science (Shanghai, China).
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 . 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 .
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 . The microsomes preparations were stored at -80 ºC until used. Protein concentrations were determined by the Lowry method , 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 4-hydroxymephenytion and 6β-hydroxytestosterone were determined.
CES1 Enzyme activity assay
The concentration of CES1 in rat hepatic microsomes was determined by double antibody sandwich method. Purified 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 final 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.