[Animal Modeling]-Pharmacokinetic Evaluation of Miniature Pig Coronary Model
In the miniature pig coronary artery model study, pharmacokinetic evaluation is an important indicator for evaluating new drug-eluting stents. These include in vitro pharmacokinetic studies and tissue drug concentration monitoring. As an example of introducing this system to everyone, consider a miniature pig coronary artery model experiment using sirolimus-eluting stents.
1. Reagent (1) Standard sirolimus, batch number: 070101, molecular weight: 914.2 Da.
(2) DMORPM (internal standard) standard, molecular weight: 884.2 Da.
(3) Alcohol: Burdick & Jackson, HPLC grade, batch number: G90G1H.
(4) Acetic acid: Beijing Chemical Reagent Company, analytical grade, batch number: 20060512,
(5) Ethyl acetate: Beijing Chemical Reagent Company, analytical grade, batch number: 060119.
(6) Ammonium acetate: Beijing Yili Fine Chemical Co., Ltd., analytical grade, batch number: 20010323,
(7) Sodium acetate in water: Beijing Yili Fine Chemical Co., Ltd., analytical grade, batch number: 951022,
(8) Water: Deionized water prepared with MilliQ purification system.
(9) Acetonitrile (chromatographic grade): Honeywell, USA.
(10) Tetrahydrofuran (chromatographic grade): Honeywell, USA.
2, main equipment
(1) The HPLC-MS/MS system is composed of 2690 high performance liquid chromatograph (Waters) and API-3000 triple quadrupole mass spectrometer (Applied Biotechnology, USA), equipped with an electrospray ion source.
(2) MilliQ water purifier (Barley Instruments (Shanghai) Co., Ltd.).
(3) Low-temperature high-speed centrifuge (Hereus, Germany).
(4) Coming320 pH meter (Corning, USA).
(5) Mettler AX105 analytical balance (METTLER Toldo, Switzerland).
3. Analytical conditions HPLC conditions: Select Alltech Company Alltima C18 2.1 mm x 50 mm (3μm) chromatographic column; column temperature is 51°C; mobile phase is 80% methanol and 20% 10 mmol/L ammonium acetate (pH = 5.1) . ); The flow rate is 0.2 ml/each sample analysis takes 5 minutes. Tandem mass spectrometry analysis conditions: electrospray ionization (ESI) and multiple reaction monitoring (MRM) mass spectrometry scanning methods in positive ionization mode to determine that the mother-daughter pair of sirolimus and the internal standard mass-to-charge ratio were 931.7 (M + H4 +) → 864.7 and 901.8 (M + H4 +) → 834.8.
Four. In vitro pharmacokinetic analysis process (1) The composition of the reference substance solution: accurately weigh an appropriate amount of sirolimus reference substance, dilute it with acetonitrile water (65:35), and contain 20μg solution per ml. I will. It was found (2) Preparation of test solution: 6 sirolimus eluting stents without polymer carriers and 6 sirolimus eluting stents with polymer carriers were immersed in 5ml and 7% bovine serum albumin, respectively in. Shake at 37°C and 80r/min constant temperature. After 2 hours, 1 day, 7 days and 14 days, the scaffold was taken out and placed in a centrifuge tube. Accurately add 5 ml of methanol, leave it for 4 hours, sonicate for 5 minutes, and then use 10 μl or 20 μl as the test solution. (3) Chromatographic conditions and system applicability: octadecylsilane bonded silica gel is used as the filler, methanol-acetonitrile-water (63:14:13) is the mobile phase, the detection wavelength is 280m, and the flow rate is 1 ml/min. The column temperature is 40°C. Use the reference solution as the system suitability test solution, accurately measure 20μl and inject it into the liquid chromatograph, the elution order is sirolimus B isomer and sirolimus C isomer. The retention time of sirolimus B isomer must be 15-22 minutes, the theoretical plate number based on the sirolimus B isomer peak must be 2000 or more, and sirolimus B and C must be separated isomer. The degree must be 1.2 or higher.
(4) Measurement: Sampling the test solution and reference solution according to HPLC (2000 Chinese Pharmacopoeia Appendix VD) and recording the peak area. According to the external standard method and the sum of the peak area of sirolimus 8, C isomer, the drug content of the test product was calculated.
Fives. In vivo measurement of sirolimus intake in coronary artery tissue
(1) Preparation of sirolimus stock solution
1) Preparation of sirolimus standard curve stock solution (1 mg/ml): the company's standard product, which can accurately measure 10.13 mg of sirolimus, transfer it to a 10 ml volumetric flask, and dissolve it by adding an appropriate amount of methanol. Dilute with methanol to obtain 1 mg/ml mother liquor (ie mother liquor), and store it in a refrigerator at -30°C for later use. Do it. Accurately absorb 0.1 ml of the above stock solution, transfer it to a 10 ml volumetric flask, dilute to the mark with pure water to form a standard curve of 10 μg/ml standard curve, and store it in a refrigerator at -30°C for future use.
2) Preparation of sirolimus quality control stock solution (1 mg/ml): accurately weigh 10.12 mg sirolimus standard solution, transfer to a 10 ml volumetric flask, add appropriate amount of methanol to dissolve, and make up the volume with methanol. Dilute to a 1 mg/ml stock solution and store in a refrigerator at -30°C for later use. Accurately absorb 0.1 ml of the above stock solution, transfer it to a 10 ml volumetric flask, dilute with methanol to obtain a 10μg/ml standard solution for quality control, and store it in a refrigerator at -30°C for future use. Accurately draw 1 ml of the above 10μg/ml quality control working solution, transfer it to a 100 ml volumetric flask, dilute with blank arterial homogenate to mark (arterial weight: water volume = 1:4 g/ml), 100g/ml quality control working solution Store in a refrigerator at -30°C for later use. (2) Preparation of internal standard stock solution: accurately weigh out 5.05 mg internal standard stock solution, transfer it into a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve, and dilute to the mark to obtain 500 μg/ml stock solution. And store it at -30. Use the refrigerator. Accurately absorb 0.2 ml of the above mother liquor, transfer to a 10 ml volumetric flask, dilute with methanol to obtain an internal standard of 10μg/ml, and store it in a refrigerator at -30°C for future use. Accurately absorb 1 ml of the above 10μg/ml internal standard substance, transfer it to a 100 ml volumetric flask, dilute it with methanol to 100g/ml internal standard substance, and store it in a refrigerator at 4°C for later use. I will.
(3) Preparation of standard curve of arterial homogenate and quality control sample: select 10 healthy piglets, establish venous access, sacrifice bleeding, immediately open the chest cavity, remove the heart, remove the coronary artery tissue and carefully separate it. Weigh the tissue accurately, put it in a micro-tissue homogenizer (10000r/min), homogenize it, and then store it in a refrigerator at -30°C.
According to the following table, use pig blank arterial homogenate as the solvent, low, medium and high 3 standard solutions with concentrations of 0.2, 0.5, 2, 5, 10, 20, 50 and 100g/ml quality control sample preparation curve sample ( Low concentration 0.5g/ml, medium concentration 20g/ml, high concentration 80g/ml). Separate the above samples and store them in a refrigerator at -30°C for future use.
Accurately measure 8ml of blank porcine arterial homogenate (arterial weight: water volume = 1:4g/ml), transfer to a 100ml volumetric flask, dilute to the mark with purified water, mix well, and store it in a refrigerator at -30℃ for later use in. ..Accurately measure 8 ml of blank porcine artery homogenate, transfer to a 100 ml volumetric flask, dilute to the mark with pure water, mix well, and store in a refrigerator at -30°C for future use. Thaw the artery homogenate sample at room temperature, mix for 10 seconds, take 500μl artery homogenate sample, add 100μl internal standard substance (100g/ml), and mix for 30 seconds. Add 2 ml of 0.1 mol/L sodium acetate and mix for 30 seconds. Add 4 ml of ethyl acetate, mix for 40 seconds and shake for 10 minutes. After centrifugation at 3850r/min for 10 minutes at 4°C, the upper organic phase was transferred to a glass test tube and dried at 40°C under N2 flow. Add 200μl of compound solution (methanol: 10 mM ammonium acetate = 2:1, v:v), mix for 1 minute, and then filter with a microfilter (0.22μm). Measure under the HPLC-MS/MS conditions described in 3.6, record the mass chromatogram and peak area of the compound, and use the internal standard method for quantification.
(4) Contents and methods of evaluation
1) Specificity: The samples used for specific evaluation include blank porcine arterial homogenate samples (double blank), blank arterial homogenate (blank) containing internal standard working fluid, including quality control. Porcine artery samples and homogeneous samples of unknown concentration. In the above method, the obtained mass spectrum is determined by the retention time of the interference peak. Evaluation criteria: The peak area of the interference peak should be less than 20% of the minimum quantification limit peak area.
2) Standard curve: Process and measure the standard curve sample according to the above method. The linear fit was performed with the ratio of the peak area of sirolimus to the internal standard as Y and the concentration as X (weighting factor 1/x2). The curve regression equation of the standard curve is Y = aX + b. Evaluation criteria: correlation coefficient r≥0.99; the accuracy of the minimum quantitative concentration sample (RSD%) is within 20%; the accuracy (RE%) is within ±20%; the accuracy of other standard curve samples (RSD%) is 15% The accuracy (RE%) must be within ±15%.
3) Precision and accuracy: 3 types of quality control samples, from low to high concentration: LQC (low concentration quality control sample), MQC (medium concentration quality control sample), HQC (high concentration quality control sample) 3.6 and 3.9 in use The described method processes five samples and continuously determines them in three batches to calculate intra-day and intra-day precision (RSD%) and precision (RE%). Evaluation criteria: accuracy (RSD%) must be within 15%, accuracy (RE%) must be within ±15%.
动物造模,冠脉模型
2021-10-07
448