舒尼替尼在荷肾癌裸鼠体内的时辰药理学研究
发布时间：2019-06-19 16:10
【摘要】：目的:通过皮下接种构造ACHN人肾细胞腺癌移植瘤Balb/c裸鼠模型,探究舒尼替尼(Sunitinib,SU)时辰给药的药效学及药动学特点,并从时辰基因、代谢酶等多个方面探究其可能的药效作用机制。方法:1.建立裸鼠肾癌模型:将4周龄的Balb/c裸鼠置于严格的明暗环境中适应性饲养两周,将培养好的ACHN人肾细胞腺癌细胞于裸鼠右上肢皮下注射,建立Balb/c裸鼠移植瘤模型。2.药效学研究的随机分组:将裸鼠模型随机分为4:00、8:00、12:00、16:00、20:00、24:00六个时辰组,每个时辰组又随机分为舒尼替尼给药组和模型组。3.药效学研究的时辰给药及检测指标:舒尼替尼给药组分别在对应的六个时辰点灌胃给予16.25mg·kg-1舒尼替尼溶液0.2m L,模型组则在同样的时辰点给以等量的CB(Citrate buffer,柠檬酸盐缓冲液,PH=4.5)。在给药期间每三天对肿瘤的长短径进行一次测量以计算其体积,根据其体积变化趋势绘制出肿瘤体积生长曲线。至第21天则分别于相应时辰点处死裸鼠,剥离瘤体称重并制作病理切片以比较各组肿瘤的病理形态。4.药动学研究的随机分组:将裸鼠模型随机分为4:00、8:00、12:00、16:00、20:00、24:00六个时辰组,每个时辰组又分为舒尼替尼给药组和模型组。每个给药组包括15 min、30 min、1h、3h、5 h、7 h、9 h、14 h、19 h、24 h共10个取血点。每个时辰组设置正常裸鼠作为空白对照组。5.药动学研究的时辰给药及检测指标:给药组于六个时辰点分别灌胃给以舒尼替尼16.25mg·kg-1,并于给药后15 min、30 min、1h、3h、5 h、7 h、9 h、14 h、19 h、24 h摘眼球取血0.5m L,离心取血浆置-80℃保存,采用HPLC-MS/MS(high-performance liquid chromatography coupled with tandem mass spectrometry,高效液相色谱法联合质谱)的方法测定不同时辰点舒尼替尼及其主要活性代谢物N-去乙基舒尼替尼(SU12662)的血药浓度,并用Win Nonlin 6.3计算各时辰点的药动学参数。取血后迅速处死裸鼠,并立即取肝脏于-80℃冷冻保存。6.相关基因表达水平的检测:从肝组织中提取总RNA采用q RT-PCR(Quantificational Real Time-PCR,实时荧光定量PCR)检测cyp3a11(cytochrome P450 enzyme 3a11,细胞色素P450酶3a11)、cyp3a13、Bmal-1(brain and muscle arnt-like protein-1,时辰基因编码芳香烃受体核转运样蛋白)、Clock(circadian locomotor output cycles kaput,生物钟循环输出蛋白)及时辰基因的作用位点Dbp(D site-binding protein,D-位点结合蛋白)的表达。结果:1.舒尼替尼在荷瘤裸鼠体内的时辰药效学具有昼夜节律性,与模型组相比,各时辰给药组的肿瘤体积增长速度均有所减慢,肿瘤重量也有不同的减轻。16:00组的肿瘤生长曲线坡势陡直,与其他五个给药组相较,肿瘤体积增长迅速,抑瘤率最低;8:00、12:00给药组与16:00及20:00给药组相比,肿瘤体积生长缓慢,抑瘤率高,有显著性差异(p0.05),其中8:00给药组肿瘤受抑制情况最为明显。病理切片结果显示,16:00及20:00给药组肿瘤组织细胞排列基本无间隙,边界较为清楚,肿瘤坏死量少,炎性细胞浸润情况轻微;8:00及12:00给药组中大量的肿瘤细胞是坏死的,表现出严重的炎性细胞浸润和血管破裂出血。2.舒尼替尼在荷瘤裸鼠体内的时辰药动学具有昼夜节律性,相比其他各时辰给药组,16:00与20:00给药组AUC0-24h较低,清除率高;8:00及12:00给药组裸鼠体内总药物具有更高的AUC0-24h、MRT0-24h和更低的CL/F,在体内驻留时间长,血药浓度高,与16:00给药组相比具有统计学差异(p0.05)。与药效学研究结果相一致。3.代谢酶基因cyp3a11、cyp3a13及时辰基因Bmal-1、Clock、Dbp在荷肾癌裸鼠肝脏组织中的表达显示出显著的昼夜节律性。模型组代谢酶基因cyp3a11、时辰基因Bmal-1在12:00至20:00时间范围内表达量高,在16:00表达最高,达到峰值;cyp3a13及Clock表达量自8:00起呈现不断上升的趋势,在20:00至24:00时间段内坡度最大,增长速率最为迅速。结合位点蛋白Dbp在16:00、24:00均有较高程度的表达。在不同的时间点给予舒尼替尼后,16:00组Bmal-1、cyp3a11总体表达量最高;24:00组Clock、Dbp及cyp3a13总体表达量最高,其中Clock及cyp3a13在20:00组也有较高的表达。时辰基因与代谢酶基因的整体表达趋势具有一致性。结论:舒尼替尼在荷瘤裸鼠体内的时辰药理学具有昼夜节律性,在明早期(8:00及12:00)给药时,肿瘤生长缓慢,抑瘤率高,抑瘤效果最好,体内总药物清除率低,在体内的驻留时间长,具有更高的血药浓度。作用机制可能为时辰基因通过作用于Dbp直接调控代谢酶的表达,以影响药物在裸鼠体内代谢的速率,使得舒尼替尼的药动学特点呈现昼夜节律性,亦对药效学产生影响。
[Abstract]:Objective: To study the pharmacodynamics and pharmacokinetics of the administration of sunitinib (SU) in the model of Balb/ c nude mice with ACHN human renal cell adenocarcinoma by subcutaneous inoculation, and to explore the possible mechanism of drug effect from several aspects such as time gene and metabolic enzyme. Method:1. The model of renal cell carcinoma in nude mice was established: the 4-week-old Balb/ c nude mice were placed in a strict dark and dark environment for two weeks, and the cultured ACN human renal cell adenocarcinoma cells were injected subcutaneously into the right upper extremity of the nude mice to establish a Balb/ c nude mice model. Pharmacodynamic studies were randomly divided into four groups:4:00,8:00,12:00,16:00,20:00,24:00, and each time group was randomly divided into sunitinib group and model group. The time-to-dose and the detection index of the pharmacodynamic study: the sunitinib group was given 16.25 mg 路 kg-1 sunitinib in the corresponding six time points, and the model group was given the same amount of CB (Citrate buffer, citrate buffer, PH = 4.5) at the same time. The length and length of the tumor were measured once every three days during the administration to calculate the volume of the tumor and the tumor volume growth curve was plotted according to its volume change trend. To the 21st day, the nude mice were sacrificed at the corresponding time points, and the tumor bodies were weighed and the pathological sections were made to compare the pathological morphology of each group of the tumors. The model of the nude mice was randomly divided into 4:00,8:00,12:00,16:00,20:00,24:00, and each time group was divided into the group of sunitinib and the model group. Each treatment group consisted of 15 min,30 min,1 h,3 h,5 h,7 h,9 h,14 h,19 h and 24 h. The normal nude mice were set in each hour group as the blank control group. The time-to-date and the detection index of the pharmacokinetic study were as follows: the administration group was given the administration of sunitinib 16.25 mg 路 kg-1 at six time points, and the blood was taken at 15 min,30 min,1 h,3 h,5 h,7 h,9 h,14 h,19 h and 24 h after administration, and the blood was taken for 0.5 m L. The plasma concentration of sunitinib and its main active metabolite N-deethylsunitinib (SU12662) was determined by HPLC-MS/ MS (high-performance liquid chromatography and mass spectrometry). The pharmacokinetic parameters of each time point were calculated with Win Nonlin 6.3. The nude mice were sacrificed quickly after the blood was taken, and the liver was immediately frozen at-80.degree. C. for 6 years. Detection of related gene expression levels: The total RNA extracted from the liver tissue was detected by q-RT-PCR (quantitative real-time-PCR, real-time fluorescence quantitative PCR), cytochrome P450 enzyme 3a11, cytochrome P450 enzyme 3a11, cyp3a13, Bmal-1 (brain and muscle arnt-like protein-1, time-gene-encoded aromatic receptor nuclear transport-like protein), Clock (circle-like locomotor output cydes kaput, The expression of Dbp (D-site-binding protein) and D-site-binding protein (D-site-binding protein) of the biological clock cycle output protein) and the hour gene. Results:1. Compared with the other five treatment groups, the growth rate of the tumor in the tumor-bearing nude mice has a circadian rhythm, and the growth rate of the tumor in the tumor-bearing nude mice is slower than that of the model group, and the weight of the tumor is also reduced. The tumor volume grew rapidly, the tumor inhibition rate was the lowest, and the tumor volume was slow, the tumor inhibition rate was high, and there was a significant difference (p0.05), of which the tumor was most obvious in the 8:00 treatment group compared with the treatment group of 16:00 and 20:00. The results of the pathological sections show that the tumor cells in the 16:00 and 20:00 treated groups have no gaps, the boundary is clear, the tumor necrosis is small, the infiltration of the inflammatory cells is mild, and the large number of tumor cells in the 8:00 and 12:00 administration groups are necrotic, Serious inflammatory cell infiltration and vascular rupture and bleeding were shown. The pharmacokinetics of sunitinib in the nude mice with tumor-bearing nude mice had a circadian rhythm, and the AUC0-24h, the clearance rate, the 8:00 and the 12:00 administration group had higher AUC0-24h, MRT0-24h and lower CL/ F compared with the other time-group treatment group,16:00 and 20:00 treatment group, and the residence time in vivo was long. The plasma concentration was high, which was statistically different from the 16:00 treatment group (p0.05). In accordance with that results of the pharmacodynamics study.3. The expression of cyp3a11, cyp3a13 and time-specific Bmal-1, Clock, and Dbp in the liver of the nude mice bearing renal cell carcinoma showed significant circadian rhythm. The expression of cyp3a11 in the model group was high in the time range of 12:00 to 20:00, the highest expression was reached at 16:00, the peak value was reached. The expression of cyp3a13 and Clock was increased from 8:00 to 24:00, and the growth rate was the most rapid. The binding site protein Dbp has a higher degree of expression at 16:00 and 24:00. After sunitinib was given at different time points, the total expression of Cyp3a11 was the highest in the 16:00 group, and the overall expression of Clock, Dbp and cyp3a13 was the highest in the 24:00 group, where Clock and Cyp3a13 had higher expression in the 20:00 group. And the whole expression trend of the time gene and the metabolic enzyme gene is consistent. Conclusion: The time and pharmacology of sunitinib in the tumor-bearing nude mice have a circadian rhythm. In the early stage (8:00 and 12:00), the tumor growth is slow, the tumor-inhibiting rate is high, the tumor-inhibiting effect is the best, the total drug clearance in the body is low, the residence time in the body is long, and the blood concentration is higher. The mechanism of action may act as an hour gene to directly regulate the expression of the metabolizing enzyme by acting on the Dbp so as to influence the rate of metabolism of the drug in the nude mice, so that the pharmacokinetics of the sunitinib presents a circadian rhythm and has an effect on the pharmacodynamics.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
本文编号：2502471
[Abstract]:Objective: To study the pharmacodynamics and pharmacokinetics of the administration of sunitinib (SU) in the model of Balb/ c nude mice with ACHN human renal cell adenocarcinoma by subcutaneous inoculation, and to explore the possible mechanism of drug effect from several aspects such as time gene and metabolic enzyme. Method:1. The model of renal cell carcinoma in nude mice was established: the 4-week-old Balb/ c nude mice were placed in a strict dark and dark environment for two weeks, and the cultured ACN human renal cell adenocarcinoma cells were injected subcutaneously into the right upper extremity of the nude mice to establish a Balb/ c nude mice model. Pharmacodynamic studies were randomly divided into four groups:4:00,8:00,12:00,16:00,20:00,24:00, and each time group was randomly divided into sunitinib group and model group. The time-to-dose and the detection index of the pharmacodynamic study: the sunitinib group was given 16.25 mg 路 kg-1 sunitinib in the corresponding six time points, and the model group was given the same amount of CB (Citrate buffer, citrate buffer, PH = 4.5) at the same time. The length and length of the tumor were measured once every three days during the administration to calculate the volume of the tumor and the tumor volume growth curve was plotted according to its volume change trend. To the 21st day, the nude mice were sacrificed at the corresponding time points, and the tumor bodies were weighed and the pathological sections were made to compare the pathological morphology of each group of the tumors. The model of the nude mice was randomly divided into 4:00,8:00,12:00,16:00,20:00,24:00, and each time group was divided into the group of sunitinib and the model group. Each treatment group consisted of 15 min,30 min,1 h,3 h,5 h,7 h,9 h,14 h,19 h and 24 h. The normal nude mice were set in each hour group as the blank control group. The time-to-date and the detection index of the pharmacokinetic study were as follows: the administration group was given the administration of sunitinib 16.25 mg 路 kg-1 at six time points, and the blood was taken at 15 min,30 min,1 h,3 h,5 h,7 h,9 h,14 h,19 h and 24 h after administration, and the blood was taken for 0.5 m L. The plasma concentration of sunitinib and its main active metabolite N-deethylsunitinib (SU12662) was determined by HPLC-MS/ MS (high-performance liquid chromatography and mass spectrometry). The pharmacokinetic parameters of each time point were calculated with Win Nonlin 6.3. The nude mice were sacrificed quickly after the blood was taken, and the liver was immediately frozen at-80.degree. C. for 6 years. Detection of related gene expression levels: The total RNA extracted from the liver tissue was detected by q-RT-PCR (quantitative real-time-PCR, real-time fluorescence quantitative PCR), cytochrome P450 enzyme 3a11, cytochrome P450 enzyme 3a11, cyp3a13, Bmal-1 (brain and muscle arnt-like protein-1, time-gene-encoded aromatic receptor nuclear transport-like protein), Clock (circle-like locomotor output cydes kaput, The expression of Dbp (D-site-binding protein) and D-site-binding protein (D-site-binding protein) of the biological clock cycle output protein) and the hour gene. Results:1. Compared with the other five treatment groups, the growth rate of the tumor in the tumor-bearing nude mice has a circadian rhythm, and the growth rate of the tumor in the tumor-bearing nude mice is slower than that of the model group, and the weight of the tumor is also reduced. The tumor volume grew rapidly, the tumor inhibition rate was the lowest, and the tumor volume was slow, the tumor inhibition rate was high, and there was a significant difference (p0.05), of which the tumor was most obvious in the 8:00 treatment group compared with the treatment group of 16:00 and 20:00. The results of the pathological sections show that the tumor cells in the 16:00 and 20:00 treated groups have no gaps, the boundary is clear, the tumor necrosis is small, the infiltration of the inflammatory cells is mild, and the large number of tumor cells in the 8:00 and 12:00 administration groups are necrotic, Serious inflammatory cell infiltration and vascular rupture and bleeding were shown. The pharmacokinetics of sunitinib in the nude mice with tumor-bearing nude mice had a circadian rhythm, and the AUC0-24h, the clearance rate, the 8:00 and the 12:00 administration group had higher AUC0-24h, MRT0-24h and lower CL/ F compared with the other time-group treatment group,16:00 and 20:00 treatment group, and the residence time in vivo was long. The plasma concentration was high, which was statistically different from the 16:00 treatment group (p0.05). In accordance with that results of the pharmacodynamics study.3. The expression of cyp3a11, cyp3a13 and time-specific Bmal-1, Clock, and Dbp in the liver of the nude mice bearing renal cell carcinoma showed significant circadian rhythm. The expression of cyp3a11 in the model group was high in the time range of 12:00 to 20:00, the highest expression was reached at 16:00, the peak value was reached. The expression of cyp3a13 and Clock was increased from 8:00 to 24:00, and the growth rate was the most rapid. The binding site protein Dbp has a higher degree of expression at 16:00 and 24:00. After sunitinib was given at different time points, the total expression of Cyp3a11 was the highest in the 16:00 group, and the overall expression of Clock, Dbp and cyp3a13 was the highest in the 24:00 group, where Clock and Cyp3a13 had higher expression in the 20:00 group. And the whole expression trend of the time gene and the metabolic enzyme gene is consistent. Conclusion: The time and pharmacology of sunitinib in the tumor-bearing nude mice have a circadian rhythm. In the early stage (8:00 and 12:00), the tumor growth is slow, the tumor-inhibiting rate is high, the tumor-inhibiting effect is the best, the total drug clearance in the body is low, the residence time in the body is long, and the blood concentration is higher. The mechanism of action may act as an hour gene to directly regulate the expression of the metabolizing enzyme by acting on the Dbp so as to influence the rate of metabolism of the drug in the nude mice, so that the pharmacokinetics of the sunitinib presents a circadian rhythm and has an effect on the pharmacodynamics.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
【参考文献】
相关期刊论文 前1条
1 王雅杰;王宁;;肿瘤分子靶向药物分类及作用机制[J];中国实用外科杂志;2010年07期
，本文编号：2502471
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