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Abstract：
OBJECTIVE: In the present study, the effect of sildenafil on the pharmacokinetics of metformin was studied in experimental rats, and we also postulated the molecular mechanism by performing molecular docking studies.
METHODS: Analysis of metformin and sildenafil (SIL) from rat plasma was done by high performance liquid chromatography. Optimum chromatographic separation and quantification of MET, SIL and Cetirizine was achieved on Phenomenex EVO C18 column with triethyl amine (0.3%): Methanol: Acetonitrile (70:05:25 v/v) as mobile phase maintaining flow rate of 1 ml/min, the detector was tuned at 224 nm. The extraction of MET and sildenafil from rat plasma was achieved by solid-phase extraction using Strata-X cartridges. The method was validated as per the ICH guidelines. For docking studies, the crystal structure of organic cation transporter 1 (OCT1) protein and multidrug and toxin extrusion (MATE) protein (5XJJ) were downloaded from the PubChem database. The docking study was performed by PyRx virtual screening software, and the results were analyzed by BIOVIA Discovery Studio.
RESULTS: The validation of HPLC method was done, intraday and interday precision study of HPLC method demonstrated %RSD values less than 5%, the extraction recovery for MET and SIL were near to 80 % for low, medium and high QC samples. The plasma stability of MET and SIL showed % RSD values <10% for low, medium, and high QC samples. A sensitivity study for MET and SIL in rat plasma suggested a lower limit of quantification values of 8 and 10 ng/mL, respectively. The pharmacokinetic parameters were recorded, Cmax of experimental and control rats was 611.2 and 913.2 ng/mL; t1/2 1.66 and 1.98, AUC (0-t) 1637.5 and 2727.24, AUC (0-∞) 1832.38 and 2995.24 for MET. The results suggested that the Cmax of MET in experimental rats (MET + SIL) was 33.07% lower than the control (MET only) and also the t1/2 was 0.32 h shorter. Docking analysis suggested a higher binding affinity of sildenafil with MATE protein (5XJJ) compared to OCT1, suggesting possible involvement of MATE family proteins for pharmacokinetic alterations of MET.
CONCLUSIONS: The HPLC and solid-phase extraction method were developed and applied successfully for the pharmacokinetics of MET and SIL. Intake of SIL altered the pharmacokinetics of MET in rats. Molecular docking studies suggested the involvement of MATE family proteins for alterations of MET pharmacokinetics.
METHODS: Analysis of metformin and sildenafil (SIL) from rat plasma was done by high performance liquid chromatography. Optimum chromatographic separation and quantification of MET, SIL and Cetirizine was achieved on Phenomenex EVO C18 column with triethyl amine (0.3%): Methanol: Acetonitrile (70:05:25 v/v) as mobile phase maintaining flow rate of 1 ml/min, the detector was tuned at 224 nm. The extraction of MET and sildenafil from rat plasma was achieved by solid-phase extraction using Strata-X cartridges. The method was validated as per the ICH guidelines. For docking studies, the crystal structure of organic cation transporter 1 (OCT1) protein and multidrug and toxin extrusion (MATE) protein (5XJJ) were downloaded from the PubChem database. The docking study was performed by PyRx virtual screening software, and the results were analyzed by BIOVIA Discovery Studio.
RESULTS: The validation of HPLC method was done, intraday and interday precision study of HPLC method demonstrated %RSD values less than 5%, the extraction recovery for MET and SIL were near to 80 % for low, medium and high QC samples. The plasma stability of MET and SIL showed % RSD values <10% for low, medium, and high QC samples. A sensitivity study for MET and SIL in rat plasma suggested a lower limit of quantification values of 8 and 10 ng/mL, respectively. The pharmacokinetic parameters were recorded, Cmax of experimental and control rats was 611.2 and 913.2 ng/mL; t1/2 1.66 and 1.98, AUC (0-t) 1637.5 and 2727.24, AUC (0-∞) 1832.38 and 2995.24 for MET. The results suggested that the Cmax of MET in experimental rats (MET + SIL) was 33.07% lower than the control (MET only) and also the t1/2 was 0.32 h shorter. Docking analysis suggested a higher binding affinity of sildenafil with MATE protein (5XJJ) compared to OCT1, suggesting possible involvement of MATE family proteins for pharmacokinetic alterations of MET.
CONCLUSIONS: The HPLC and solid-phase extraction method were developed and applied successfully for the pharmacokinetics of MET and SIL. Intake of SIL altered the pharmacokinetics of MET in rats. Molecular docking studies suggested the involvement of MATE family proteins for alterations of MET pharmacokinetics.
摘要：
目的：在本研究中，在实验大鼠中研究了西地那非对二甲双胍药代动力学的影响，我们还通过进行分子对接研究推测了分子机制。
方法：采用高效液相色谱法分析大鼠血浆中的二甲双胍和西地那非（SIL）。MET的最佳色谱分离和定量，SIL和西替利嗪在PhenomenexEVOC18色谱柱上获得，三乙胺（0.3％）：甲醇：乙腈（70：05：25v/v）作为流动相，流速为1ml/min，检测器在224nm处调谐。使用Strata-X盒通过固相萃取从大鼠血浆中提取MET和西地那非。该方法按照ICH指南进行验证。对于对接研究，从PubChem数据库下载了有机阳离子转运蛋白1（OCT1）蛋白和多药和毒素挤出（MATE）蛋白（5XJJ）的晶体结构。对接研究通过PyRx虚拟筛选软件进行，结果由BIOVIADiscoveryStudio进行分析。
结果：完成了HPLC方法的验证，高效液相色谱法的日内和日间精密度研究表明%RSD值小于5%，在低的情况下，MET和SIL的提取回收率接近80%，中等和高QC样品。MET和SIL的血浆稳定性显示％RSD值<10％，中等,和高QC样品。对大鼠血浆中MET和SIL的敏感性研究表明，定量值的下限为8和10ng/mL，分别。记录药代动力学参数，实验和对照大鼠的Cmax分别为611.2和913.2ng/mL；MET的t1/21.66和1.98，AUC（0-t）1637.5和2727.24，AUC（0-∞）1832.38和2995.24。结果表明，实验大鼠（METSIL）的METCmax比对照组（仅MET）低33.07％，t1/2也短0.32h。对接分析表明，与OCT1相比，西地那非与MATE蛋白（5XJJ）的结合亲和力更高，表明MATE家族蛋白可能参与MET的药代动力学改变。
结论：建立了高效液相色谱和固相萃取方法，并成功应用于MET和SIL的药代动力学研究。SIL的摄入改变了MET在大鼠体内的药代动力学。分子对接研究表明，MATE家族蛋白参与了MET药代动力学的改变。
方法：采用高效液相色谱法分析大鼠血浆中的二甲双胍和西地那非（SIL）。MET的最佳色谱分离和定量，SIL和西替利嗪在PhenomenexEVOC18色谱柱上获得，三乙胺（0.3％）：甲醇：乙腈（70：05：25v/v）作为流动相，流速为1ml/min，检测器在224nm处调谐。使用Strata-X盒通过固相萃取从大鼠血浆中提取MET和西地那非。该方法按照ICH指南进行验证。对于对接研究，从PubChem数据库下载了有机阳离子转运蛋白1（OCT1）蛋白和多药和毒素挤出（MATE）蛋白（5XJJ）的晶体结构。对接研究通过PyRx虚拟筛选软件进行，结果由BIOVIADiscoveryStudio进行分析。
结果：完成了HPLC方法的验证，高效液相色谱法的日内和日间精密度研究表明%RSD值小于5%，在低的情况下，MET和SIL的提取回收率接近80%，中等和高QC样品。MET和SIL的血浆稳定性显示％RSD值<10％，中等,和高QC样品。对大鼠血浆中MET和SIL的敏感性研究表明，定量值的下限为8和10ng/mL，分别。记录药代动力学参数，实验和对照大鼠的Cmax分别为611.2和913.2ng/mL；MET的t1/21.66和1.98，AUC（0-t）1637.5和2727.24，AUC（0-∞）1832.38和2995.24。结果表明，实验大鼠（METSIL）的METCmax比对照组（仅MET）低33.07％，t1/2也短0.32h。对接分析表明，与OCT1相比，西地那非与MATE蛋白（5XJJ）的结合亲和力更高，表明MATE家族蛋白可能参与MET的药代动力学改变。
结论：建立了高效液相色谱和固相萃取方法，并成功应用于MET和SIL的药代动力学研究。SIL的摄入改变了MET在大鼠体内的药代动力学。分子对接研究表明，MATE家族蛋白参与了MET药代动力学的改变。
