两种卡洛芬注射液在犬体内的生物等效性试验

Bioequivalence of Two Carprofen Formulations in Dogs

【目的】建立犬血浆中卡洛芬的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法,比较两种以卡洛芬为活性成分的制剂在比格犬体内的药代动力学特征,以评价其生物等效性。【方法】24只健康比格犬随机分成2组,按单剂量、双周期和双序列的交叉设计,分别皮下注射4.4 mg/kg BW受试制剂和参比制剂RIMADYL,于给药前(0 h)和给药后0.5、1、2、3、4、6、8、12、24、36、48和72 h从臂头静脉采血。对UPLC-MS/MS方法的特异性、线性、检测限、定量限、准确度、精密度、稳定性等进行考察。利用建立好的UPLC-MS/MS方法测定血浆中卡洛芬的浓度,并用WinNonlin 8.1软件对达峰时间(T_(max))、达峰浓度(C_(max))、消除半衰期(T_(1/2))、药时曲线下面积(AUC_(0-t))等药代动力学参数进行分析,判定两制剂是否等效。【结果】建立的UPLC-MS/MS分析方法在10~5000 ng/mL浓度范围内线性关系良好,相关系数(R^(2))≥0.99;检测限和定量限分别为5和10 ng/mL;高、中、低、定量限4个浓度的相对回收率在91.45%~111.61%范围内,日内和日间变异系数均<15%。参比制剂和受试制剂的T_(max)分别为(2.27±1.09)和(2.33±1.10)h;C_(max)分别为(20.02±5.24)和(19.92±5.42)μg/mL;T_(1/2)分别为(8.54±3.71)和(8.65±2.64)h;AUC_(0-t)分别为(246.78±55.94)和(249.22±53.33)μg·h/mL。受试制剂与参比制剂的药时曲线相似,且受试制剂相比参比制剂,主要药动学参数C_(max)、AUC_(0-t)和AUC_(0-∞)的90%置信区间均在80.00%~125.00%之间。【结论】本试验建立的UPLC-MS/MS分析方法准确、可靠,可用于血浆中卡洛芬浓度的测定,卡洛芬的受试制剂与参比制剂RIMADYL具有生物等效性,临床上均可用于相关疾病的治疗,本试验结果可为卡洛芬兽医临床合理用药提供参考。

【Objective】The purpose of the experiment was to develop an ultra-high-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method,investigate the pharmacokinetic characteristics of two carprofen formulations in Beagle dogs,and evaluate the bioequivalence of two formulations.【Method】Twenty-four beagle dogs were randomly allocated to two equal-sized treatment groups in a randomized dual-cycle and dual-sequence cross-over design.The test and the reference(RIMADYL)formulations were subcutaneously administered in a single dose of 4.4 mg/kg BW.Blood samples were collected from brachiocephalic vein at 0,0.5,1,2,3,4,6,8,12,24,36,48 and 72 h after administration.The specificity,linearity,detection limit,quantification limit,accuracy,precision and stability of the UPLC-MS/MS method were investigated.The concentration of carprofen in plasma was determined by UPLC-MS/MS.The pharmacokinetic parameters(T max,C max,T_(1/2)and AUC_(0-t))and bioequivalence were calculated and analyzed by WinNonlin 8.1.【Result】The methodological results showed good correlation over the concentration range of 10-5000 ng/mL with a correlation coefficient(R^(2))≥0.99.The limit of detection and the limit of quantification were 5 and 10 ng/mL,respectively.Relative recoveries of high,medium,low and quantification limit concentrations were between 91.45%-111.61%.Coefficient variations of intra-assay and inter-assay were both less 15%.The pharmacokinetic parameters of two formulations were as follows:T max were(2.27±1.09)and(2.33±1.10)h;C_(max)were(20.02±5.24)and(19.92±5.42)μg/mL;T 1/2 were(8.54±3.71)and(8.65±2.64)h;AUC_(0-t)were(246.78±55.94)and(249.22±53.33)μg·h/mL.The plasma profiles of carprofen following the administration of both formulations were similar.And the 90%CI of the average C max,AUC_(0-t)and AUC_(0-∞)of the test/reference preparation were all fell between 80.00%-125.00%.【Conclusion】The UPLC-MS/MS method established in this experiment was accurate and reliable,and could be used to determine the concentration of carprofen in plasma samples.The two formulations were bioequivalent for carprofen,and could be used clinically for the treatment of related diseases.The result of this study provided theoretical basis for clinical rational administration.