創(chuàng)新藥物M0及其代謝產(chǎn)物M3的臨床前藥代動力學(xué)研究
發(fā)布時間：2018-08-22 07:42
【摘要】：M0為第四軍醫(yī)大學(xué)提供的創(chuàng)新藥物候選化合物,藥效實驗發(fā)現(xiàn)具有缺血性腦卒中的治療及卒中后神經(jīng)功能癥狀改善的藥理作用。初步研究表明M0體內(nèi)快速代謝為M3,為表征M0藥代動力學(xué)特征有必要建立同時測定M0及其代謝產(chǎn)物M3的分析方法揭示代謝規(guī)律,為M0新藥研究提供實驗支持。為此本課題建立了一種高靈敏度,高選擇性的LC-MS/MS方法測定全血、血漿、組織等多種生物樣本中M0及其代謝產(chǎn)物M3的濃度,并計算相關(guān)的臨床前藥代動力學(xué)參數(shù),考察給藥劑量與相關(guān)藥動學(xué)參數(shù)的相關(guān)性特征,評價多劑量給藥后的累積效應(yīng),研究藥物在各組織中分布狀況,為臨床合理用藥提供參考依據(jù)。一、生物樣品中M0及其代謝產(chǎn)物M3定量分析方法的建立與確證建立快速、專一、靈敏的液相色譜-串聯(lián)質(zhì)譜(LC-MS/MS)方法測定生物樣品中創(chuàng)新藥物M0及其代謝產(chǎn)物M3的濃度。由于M0在生物樣品中的不穩(wěn)定性,樣品前處理均采用即刻處理的方法。對生物樣品進行鹽酸酸化后立即采用叔丁基甲醚為萃取劑對酸化后的生物樣品進行液液萃取。色譜柱選用CNW Athena C18-WP(3μm,2.1×100 mm);流動相為甲醇-水(含0.2%甲酸),采用梯度洗脫程序,流動相不經(jīng)分流直接進入質(zhì)譜,流速為0.3 m L/min,進樣量為5μL。采用ESI離子源,負離子掃描模式,MRM檢測模式,用于定量的離子通道分別為m/z 323.00→263.00(M0)、m/z197.00→135.05(M3)、m/z 285.00→186.10(IS,4-羥基甲苯磺丁脲)、m/z169.00→125.05(IS,沒食子酸)。生物樣品中內(nèi)源性物質(zhì)不影響M0及M3的定量。生物樣品在各自的線性范圍內(nèi),線性關(guān)系良好(R20.99),日內(nèi)和日間精密度(RSD)在1.20%~12.19%范圍內(nèi),準確度均在-10.82%~9.60%范圍內(nèi),本文建立的生物樣品測定方法可應(yīng)用于M0及其代謝產(chǎn)物M3的臨床前藥代動力學(xué)研究。二、M0及其代謝產(chǎn)物M3在SD大鼠體內(nèi)藥代動力學(xué)研究用建立的LC-MS/MS方法測定SD大鼠單劑量靜脈注射M0 30 mg/kg、60 mg/kg、120 mg/kg及多劑量靜脈注射M0 60 mg/kg后體內(nèi)M0及其代謝產(chǎn)物M3的濃度。根據(jù)測定濃度,采用非房室模型法估算藥代動力學(xué)參數(shù)。結(jié)果表明,SD大鼠單劑量靜脈注射給藥M0 30 mg/kg、60 mg/kg、120 mg/kg三個劑量組后M0的藥代動力學(xué)藥時曲線末端相消除半衰期(t1/2)分別為0.07±0.01 h、0.27±0.10 h、0.19±0.11 h。AUC0-τ分別為10692.87±2044.18 h×ng/mL、20400.11±2186.28 h×ng/m L、44604.32±8722.26 h×ng/mL。可見M0的AUC0-τ與給藥劑量基本呈正相關(guān),相關(guān)系數(shù)R2為0.8894,SD大鼠多劑量給藥M0后,M0在體內(nèi)沒有蓄積現(xiàn)象產(chǎn)生。代謝產(chǎn)物M3的藥代動力學(xué)藥時曲線末端相消除半衰期(t1/2)分別為0.20±0.03 h、0.34±0.07 h、0.65±0.63 h。AUC0-τ分別為219.48±84.63 h×ng/m L、616.87±325.79 h×ng/mL、1250.85±208.00 h×ng/mL。可見M3的AUC0-τ與給藥劑量基本呈正相關(guān),相關(guān)系數(shù)R2為0.8111。SD大鼠多劑量給藥M0后,其代謝產(chǎn)物M3在體內(nèi)沒有蓄積現(xiàn)象產(chǎn)生。三、M0及其代謝產(chǎn)物M3在Beagle犬體內(nèi)藥代動力學(xué)研究用建立的LC-MS/MS方法測定Beagle犬單劑量靜脈注射M0 8.9 mg/kg、17.8mg/kg、35.6 mg/kg及多劑量靜脈注射M0 17.8 mg/kg后體內(nèi)M0及其代謝產(chǎn)物M3的濃度。根據(jù)測定的濃度,采用非房室模型法估算藥代動力學(xué)參數(shù)。結(jié)果表明,Beagle犬單劑量靜注給藥M0 8.9 mg/kg、17.8 mg/kg、35.6 mg/kg三個劑量組后M0的藥代動力學(xué)藥時曲線末端相消除半衰期(t1/2)分別為1.86±0.78 h、1.50±0.13 h、2.97±0.49 h。AUC0-τ分別為1448.25±422.45 h×ng/mL、4141.48±496.34 h×ng/m L、8515.05±1536.73h×ng/m L。可見M0的AUC0-τ與給藥劑量基本呈正相關(guān),相關(guān)系數(shù)R2為0.9137,Beagle犬多劑量給藥M0后,M0在體內(nèi)沒有蓄積現(xiàn)象產(chǎn)生。代謝產(chǎn)物M3的藥代動力學(xué)藥時曲線末端相消除半衰期(t1/2)分別為0.57±0.09 h、0.34±0.11 h、0.47±0.14 h。AUC0-τ分別為36.87±11.83 h×ng/m L、82.01±24.18 h×ng/mL、231.01±33.74 h×ng/m L�？梢奙3的AUC0-τ與給藥劑量基本呈正相關(guān),相關(guān)系數(shù)R2為0.9189,Beagle犬多劑量給藥M0后,其代謝產(chǎn)物M3在體內(nèi)沒有蓄積現(xiàn)象產(chǎn)生。四、M0及其代謝產(chǎn)物M3在SD大鼠體內(nèi)組織分布研究SD大鼠靜脈注射給藥M0 60 mg/kg后,于給藥后5 min、10 min、15 min、30 min、1 h、1.5 h、2 h、3 h、4 h斷頸處死,并立即取出各臟器組織,用生理鹽水沖去表面浮血,濾紙吸干,用手術(shù)剪剪碎組織臟器并立即稱取0.2 g,置于液氮中速凍后,再轉(zhuǎn)移至-80℃冰箱保存待測。按組織前處理方式處理樣品后,用已建立的LC-MS/MS法測定SD大鼠各組織中的藥物濃度,并計算藥物在各組織中的含量。結(jié)果表明,給藥后M0快速被代謝,在第一取血點5min已檢測不到M0,代謝產(chǎn)物M3迅速達到最大血藥濃度,并隨血液循環(huán)廣泛地分布到血流豐富的組織臟器,其中腎臟中代謝產(chǎn)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R969.1
本文編號：2196446
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R969.1
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