创新药物M0及其代谢产物M3的临床前药代动力学研究

发布时间：2018-08-22 07:42

【摘要】：M0为第四军医大学提供的创新药物候选化合物,药效实验发现具有缺血性脑卒中的治疗及卒中后神经功能症状改善的药理作用。初步研究表明M0体内快速代谢为M3,为表征M0药代动力学特征有必要建立同时测定M0及其代谢产物M3的分析方法揭示代谢规律,为M0新药研究提供实验支持。为此本课题建立了一种高灵敏度,高选择性的LC-MS/MS方法测定全血、血浆、组织等多种生物样本中M0及其代谢产物M3的浓度,并计算相关的临床前药代动力学参数,考察给药剂量与相关药动学参数的相关性特征,评价多剂量给药后的累积效应,研究药物在各组织中分布状况,为临床合理用药提供参考依据。一、生物样品中M0及其代谢产物M3定量分析方法的建立与确证建立快速、专一、灵敏的液相色谱-串联质谱(LC-MS/MS)方法测定生物样品中创新药物M0及其代谢产物M3的浓度。由于M0在生物样品中的不稳定性,样品前处理均采用即刻处理的方法。对生物样品进行盐酸酸化后立即采用叔丁基甲醚为萃取剂对酸化后的生物样品进行液液萃取。色谱柱选用CNW Athena C18-WP(3μm,2.1×100 mm);流动相为甲醇-水(含0.2%甲酸),采用梯度洗脱程序,流动相不经分流直接进入质谱,流速为0.3 m L/min,进样量为5μL。采用ESI离子源,负离子扫描模式,MRM检测模式,用于定量的离子通道分别为m/z 323.00→263.00(M0)、m/z197.00→135.05(M3)、m/z 285.00→186.10(IS,4-羟基甲苯磺丁脲)、m/z169.00→125.05(IS,没食子酸)。生物样品中内源性物质不影响M0及M3的定量。生物样品在各自的线性范围内,线性关系良好(R20.99),日内和日间精密度(RSD)在1.20%~12.19%范围内,准确度均在-10.82%~9.60%范围内,本文建立的生物样品测定方法可应用于M0及其代谢产物M3的临床前药代动力学研究。二、M0及其代谢产物M3在SD大鼠体内药代动力学研究用建立的LC-MS/MS方法测定SD大鼠单剂量静脉注射M0 30 mg/kg、60 mg/kg、120 mg/kg及多剂量静脉注射M0 60 mg/kg后体内M0及其代谢产物M3的浓度。根据测定浓度,采用非房室模型法估算药代动力学参数。结果表明,SD大鼠单剂量静脉注射给药M0 30 mg/kg、60 mg/kg、120 mg/kg三个剂量组后M0的药代动力学药时曲线末端相消除半衰期(t1/2)分别为0.07±0.01 h、0.27±0.10 h、0.19±0.11 h。AUC0-τ分别为10692.87±2044.18 h×ng/mL、20400.11±2186.28 h×ng/m L、44604.32±8722.26 h×ng/mL。可见M0的AUC0-τ与给药剂量基本呈正相关,相关系数R2为0.8894,SD大鼠多剂量给药M0后,M0在体内没有蓄积现象产生。代谢产物M3的药代动力学药时曲线末端相消除半衰期(t1/2)分别为0.20±0.03 h、0.34±0.07 h、0.65±0.63 h。AUC0-τ分别为219.48±84.63 h×ng/m L、616.87±325.79 h×ng/mL、1250.85±208.00 h×ng/mL。可见M3的AUC0-τ与给药剂量基本呈正相关,相关系数R2为0.8111。SD大鼠多剂量给药M0后,其代谢产物M3在体内没有蓄积现象产生。三、M0及其代谢产物M3在Beagle犬体内药代动力学研究用建立的LC-MS/MS方法测定Beagle犬单剂量静脉注射M0 8.9 mg/kg、17.8mg/kg、35.6 mg/kg及多剂量静脉注射M0 17.8 mg/kg后体内M0及其代谢产物M3的浓度。根据测定的浓度,采用非房室模型法估算药代动力学参数。结果表明,Beagle犬单剂量静注给药M0 8.9 mg/kg、17.8 mg/kg、35.6 mg/kg三个剂量组后M0的药代动力学药时曲线末端相消除半衰期(t1/2)分别为1.86±0.78 h、1.50±0.13 h、2.97±0.49 h。AUC0-τ分别为1448.25±422.45 h×ng/mL、4141.48±496.34 h×ng/m L、8515.05±1536.73h×ng/m L。可见M0的AUC0-τ与给药剂量基本呈正相关,相关系数R2为0.9137,Beagle犬多剂量给药M0后,M0在体内没有蓄积现象产生。代谢产物M3的药代动力学药时曲线末端相消除半衰期(t1/2)分别为0.57±0.09 h、0.34±0.11 h、0.47±0.14 h。AUC0-τ分别为36.87±11.83 h×ng/m L、82.01±24.18 h×ng/mL、231.01±33.74 h×ng/m L。可见M3的AUC0-τ与给药剂量基本呈正相关,相关系数R2为0.9189,Beagle犬多剂量给药M0后,其代谢产物M3在体内没有蓄积现象产生。四、M0及其代谢产物M3在SD大鼠体内组织分布研究SD大鼠静脉注射给药M0 60 mg/kg后,于给药后5 min、10 min、15 min、30 min、1 h、1.5 h、2 h、3 h、4 h断颈处死,并立即取出各脏器组织,用生理盐水冲去表面浮血,滤纸吸干,用手术剪剪碎组织脏器并立即称取0.2 g,置于液氮中速冻后,再转移至-80℃冰箱保存待测。按组织前处理方式处理样品后,用已建立的LC-MS/MS法测定SD大鼠各组织中的药物浓度,并计算药物在各组织中的含量。结果表明,给药后M0快速被代谢,在第一取血点5min已检测不到M0,代谢产物M3迅速达到最大血药浓度,并随血液循环广泛地分布到血流丰富的组织脏器,其中肾脏中代谢产物M3分布最多。
[Abstract]:M0 is an innovative drug candidate provided by the Fourth Military Medical University. Pharmacodynamic experiments have shown that M0 has a pharmacological effect on the treatment of ischemic stroke and the improvement of neurological symptoms after stroke. METHODS A highly sensitive and selective LC-MS/MS method was developed for the determination of M0 and its metabolite M3 in whole blood, plasma, tissue and other biological samples. To evaluate the cumulative effect of multi-dose administration and study the distribution of drugs in various tissues, so as to provide reference for clinical rational drug use. 1. Establishment and confirmation of quantitative analysis method for M0 and its metabolite M3 in biological samples. Establishment of a rapid, specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of M0 and its metabolite M3. Concentrations of innovative drug M0 and its metabolite M3 in biological samples were determined. Due to the instability of M0 in biological samples, immediate pretreatment was used for sample preparation. After acidification of biological samples with hydrochloric acid, tert-butyl methyl ether was used as extractant for liquid-liquid extraction of acidified biological samples. CNW Athena C1 was used as chromatographic column. 8-WP (3 micron, 2.1 x 100 m m); mobile phase is methanol-water (containing 0.2% formic acid), using gradient elution procedure, the mobile phase directly enters the mass spectrometry without splitting, the flow rate is 0.3 m L/min, the sample volume is 5 muL. ESI ion source, negative ion scanning mode, MRM detection mode are used for quantitative ion channels m/z 323.00_263.00 (M0), m/z 197.00_135.05 respectively. (M3), m/z 285.00 186.10 (IS, 4-hydroxy toluene sulfonylurea), m/z 169.00 125.05 (IS, gallic acid). Endogenous substances in biological samples did not affect the quantification of M0 and M3. The linear relationship between biological samples was good (R20.99), and the intra-day and inter-day precision (RSD) ranged from 1.20% to 12.19%, with accuracy ranging from - 10.82% to 9.60%. In vivo pharmacokinetics of M0 and its metabolite M3 in SD rats The pharmacokinetic parameters of M0 and its metabolite M3 were estimated by non-compartment model according to the concentration of M0 and its metabolite M3 in vivo. AUC0-_were 10692.87 (+2044.18) h (+ng/mL), 20400.11 (+2186.28) h (+ng/mL) and 44604.32 (+8722.26) h (+ng/mL), respectively. AUC0-_of M0 was positively correlated with the dosage. The correlation coefficient R2 was 0.8894. There was no accumulation of metabolite M3 in SD rats. The terminal phase elimination half-lives (t1/2) were 0.20 (+ 0.03) h, 0.34 (+ 0.07) h, 0.65 (+ 0.63) H. AUC0-_were 219.48 (+ 84.63) h (+) / mL, 616.87 (+ 325.79) h (+) / mL, 1250.85 (+ 208.00) h (+) / mL, respectively. It was found that AUC0-_of SD M3 was positively correlated with the dosage, and the correlation coefficient R2 was 0.8111.SD M3. The pharmacokinetics of M0 and its metabolite M3 in Beagle dogs was studied by LC-MS/MS. The concentrations of M0 and its metabolite M3 in beagle dogs were determined by single-dose intravenous injection of M0 8.9 mg/kg, 17.8 mg/kg, 35.6 mg/kg and multiple-dose intravenous injection of M0 17.8 mg/kg. The pharmacokinetic parameters were estimated by non-compartment model. The results showed that the terminal phase elimination half-lives (t1/2) of the pharmacokinetic curves of beagle dogs after intravenous injection of M0 at single dose of M0 8.9 mg/kg, 17.8 mg/kg and 35.6 mg/kg were 1.86 (+0.78) h, 1.50 (+0.13) h, 2.97 (+0.49) H. AUC0-_were 1448.25 (+422.45) ng/ml, 4141 (+41) h, respectively. The AUC0-_of M0 was positively correlated with the dosage. The correlation coefficient R2 was 0.9137. There was no accumulation of M0 in beagle dogs after multiple doses of M0. The AUC0-_of M3 was positively correlated with the dosage. The correlation coefficient R2 was 0.9189. There was no accumulation of M3 and its metabolite M3 in SD rats after multiple doses of M0 in Beagle dogs. Tissue distribution of SD rats after intravenous injection of M0 60 mg/kg was studied. The rats were sacrificed at 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours and 4 hours after intravenous injection of M0 60 mg/kg. After the samples were treated by tissue pretreatment method, the drug concentration in SD rat tissues was determined by LC-MS/MS and the drug content in the tissues was calculated. The metabolite M3 was found in the kidneys of the tissues with abundant blood flow.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R969.1

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