| Objective:1. To establish a sensitive and selective High Performance Liquid Chromatography tandem mass-spectrometric method (HPLC-MS/MS) method for the simultaneously determination of LER, BEN and benazeprilat in plasma.2. To investigate the interaction of LER and BEN on the pharmacokinetics after long-term administration of two drugs combination in rats.3. To study the effect of LER on the excretion of BEN and benazeprilat in rat urine and faeces after long-term administration of two drugs combination in rats.4. To investigate the effect of BEN on metabolism of LER in liver microsomes.Methods:1. Plasma samples were extracted by solid phase extraction (SPE) with methanol, and5μL was injected onto the LC-MS/MS. LER, BEN, benazeprilat and diazepam (used as internal standard, I.S.) were separated on a Diamonsil ODS column C18(2)(5μm,150mm×4.6mm) at column temperature of30℃by acetonitrile-0.1%acetic acid (50:50, v/v) with a gradient flow rate of0.6mL/min (0-5min);5-5.1min,1mL/min;1mL/min (5.1-7.5min);7.5-7.6min,0.6mL/min;0.6mL/min (7.6-8.5min). Mass spectrometer was operated using electrospay ionization (ESI) with positive ionization mode at following parameters:nebulizer gas of40psi, spray gas of9L/min. The turbo ion spray source temperature was set at350℃, and the capillary voltage was4000V. The following ion transitions (m/z):612.3→280.1for LER,425.2-351.2for BEN,397.2→351.2for benazeprilat and285.1→193.1for I.S. were selected for quantification in multiple reactions monitoring (MRM) mode, with a scan time of0.2s per transition. The fragment voltages for LER, BEN, benazeprilat and I.S. were120V,90V,90V and130V, respectively. Nitrogen was used as collision gas with energy of20eV,25eV,20eV and34eV for LER, BEN, benazeprilat and I.S.2. Twenty four wistar rats were randomly divided into four groups:the LER group:LER administered dose of5mg/kg, qd; the BEN low dose group:LER (5mg/kg)+BEN (5 mg/kg), qd; BEN medium group:LER (5mg/kg)+BEN (10mg/kg), qd; BEN high dose group:LER (5mg/kg)+BEN (20mg/kg), qd. Each group was administered for7consecutive days. Blood samples were collected on the day7at0,0.25,0.50,1.0,1.5,2.0,3.0,4.0,6.0,8.0,12.0and24.0h. After disposition of plasma samples, LER was determined by the established LC-MS/MS method, and main pharmacokinetic parameters were calculated by DAS2.0and statistically analyzed.Twenty four wistar rats were randomly divided into four groups:the BEN group:BEN administered dose of10mg/kg, qd; the LER low dose group:BEN (10mg/kg)+LER (2.5mg/kg), qd; LER medium group:BEN (10mg/kg)+LER (5mg/kg), qd; LER high dose group:BEN (10mg/kg)+LER (10mg/kg), qd. Each group was administered for7consecutive days. Blood samples were collected on the day7at0,0.083,0.167,0.25,0.50,1.0,1.5,2.0,3.0,4.0,6.0,8.0,12.0and24.0h. After disposition of plasma samples, BEN and benazeprilat were determined by the established LC-MS/MS method, and main pharmacokinetic parameters were calculated by DAS2.0and statistically analyzed.3. A sensitive and selective High Performance Liquid Chromatography tandem mass-spectrometric (HPLC-MS/MS) method for the simultaneously determination of BEN and benazeprilat in rat uirne and faeces was established and validated. Twenty four wistar rats were randomly divided into four groups. The rats were administered as the part two described. On the day6, the rats were placed in metabolic cages. The urine and faeces were collected at0-0.5,0.5-1,1-2,2-4,4-6,6-8,8-12,12-24h. After disposition of rat urine and faeces samples, BEN and benazeprilat were determined in the established LC-MS/MS method, and the cumulative excretion of BEN and benazeprilat were calculated according to the volume of the collected urine of each period.4. Liver microsomes (0.5mg/mL) and LER (1600ng/mL) were incubated with or without ketoconazole (1μmol/L) or BEN (1,5,10,20,50μmol/L) for30min, and the reaction was terminated. The concentration of LER in the incubation medium was analyzed by LC-MS/MS to study the effect of BEN on the activity of CYP3A and LER motabolism in rat microsomes.Results:1. The linear range of the calibration curve for determination of LER in plasma by LC-MS/MS method was1～2000ng/mL, and the LLOQ was1ng/mL. The mean matrix effects were in the range of94.34%～97.19%, and absolute recoveries were in the range of71.94%～73.95%; The linear range of the calibration curve for determination of BEN in plasma by LC-MS/MS method was1～2000ng/mL, and the LLOQ was1ng/mL. The mean matrix effects were in the range of95.78%～99.03%, and absolute recoveries were in the range of92.42%～92.81%; The linear range of the calibration curve for determination of benazeprilat in plasma by LC-MS/MS method was1～2000ng/mL, and the LLOQ was1ng/mL. The mean matrix effects were in the range of95.84%～100.98%, and absolute recoveries were in the range of91.90%～94.95%. Both intra-day and inter-day precisions (RSD) were less than15%. LER, BEN and benazeprilat were stable after storage at-20℃for30d, two freeze thaw cycles and storage at room temperature for8h after extraction.2. Compared with the LER monotherapy group, the LER plus low, medium and high (5,10,20mg/kg) BEN, AUC0-24h of LER were increased3.43%,8.35%and9.34%, respectively. AUC0-∞of LER were increased3.4%,8.7%and9.91%, respectively. But there were not statistically significant. The mean residence time (MRT) of LER were extend. The LER peak concentration (Cmax) were decreased with the increasing dosage of BEN. In LER plus10mg/kg and20mg/kg BEN group, the Cmax of LER were reduced by17.33%(p<0.05) and12.34%(p<0.05).Compared with the BEN monotherapy group, the BEN plus low and medium (2.5,5mg/kg) LER, AUC0-24h of BEN were increased3.42%and6.04%respectively. AUC0-∞of BEN were increased3.35%and6.69%, respectively. In BEN plus high LER (10mg/kg), AUC0-24h of BEN were increased9.63%(p<0.05), AUC0-∞of BEN were increased9.86%(p <0.05). The exposure of BEN was significant increased by high doses of LER. The other parameters did not change significantly. AUCo-24h of benazeprilat were reduced by0.82%,4.11%and7.32%, respectively. AUCo-∞,of benazeprilat were reduced by0.26%,3.64%and6.22%, respectively. There was no statistical difference among groups.3. The wistar rat were long-time (7days) administered BEN (10mg/kg) and BEN combined with low, medium and high (2.5,5,10mg/kg) doses of LER. The24h cumulative excretions of BEN in rat urine of each group were281.510±52.407,310.057±65.610,305.272±63.328,287.742±48.986ng, respectively. The BEN excretion of each group was not significant difference after statistical paired t-test. The24h cumulative excretions of benazeprilat in rat urine of each group were6289.125±1721.376,7144.304±2601.19,6010.824±1104.396,6066.845±992.641ng. The benazeprilat excretion of each group was not significant difference after statistical paired t-test. LER does not affect the excretion of BEN and benazeprilat in the urine. The combination of LER and BEN does not cause the accumulation of BEN and benazeprilat in vivo.4. The established LC-MS/MS method was suitable for determination of LER in rat microsomes incubation system with good linearity in1～2000ng/mL (R2>0.99). The LER (1600ng/mL) metabolic inhibition rate of ketoconazole (1μmol/L) was44.9%. The BEN (1,5,10,20,50μmol/L) did not inhibit the metabolism of LER in rat liver microsomes (IC50>50μmol/L).Conclusions:Healthy wistar rat long-time (7d) administered BEN and LER, the exposure of LER in vivo did not change significantly. The exposure of BEN and benazeprilat did not change significantly with the low and medium dose LER. The exposure of BEN was significantly increased by high dose LER. Urine test showed that LER did not affect the excretion of BEN and benazeprilat in the rat urine. In vitro incubated liver microsomes test showed that BEN had no inhibitory effect on the metabolism of LER. |
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