吉非替尼在荷瘤裸鼠体内的时辰药动学及其机制
发布时间：2019-02-11 08:36
【摘要】：目的:本研究通过建立Balb/c裸鼠皮下移植瘤模型,探究吉非替尼时辰给药的药动学特点,并从代谢酶、受体通路及时辰基因的角度探讨其可能的机制。方法:1.裸鼠皮下移植瘤模型的建立与随机分组:将Balb/c裸鼠置于严格的人工控制的12 h明期-12 h暗期(07:00开灯,19:00关灯)的条件下适应性饲养2周。于右上肢皮下接种HCC827细胞,建立裸鼠皮下移植瘤模型。将造模成功的裸鼠随机分为08:00、12:00、16:00、20:00、24:00及次日04:00六个时辰组。每个时辰组又随机分为给药组和模型组,并将模型组随机分为9个取血点组。另外,每个时辰点各随机加入未种瘤的正常裸鼠作为对照组。2.时辰给药及血浆、肝组织样本的获取:给药组裸鼠在相应时辰点给予1.0mg·kg-1吉非替尼混悬液,并于给药后0 h、15、30 min、1、3、5、7、9、16、24h摘眼球取血。将血液离心得到血浆并低温保存。取血后立即脱臼处死裸鼠,取肝组织冷冻保存。3.血药浓度检测:采用高效液相色谱法联合质谱(high-performance liquid chromatography coupled with tandem mass spectrometry,HPLC-MS/MS)的方法检测各个时辰点吉非替尼的血药浓度,并用Win Nonlin 6.3计算各时辰点的药动学参数。4.相关基因表达水平的检测:采用实时荧光定量PCR(quantificational real time-polymerase chain reaction,q RT-PCR)检测裸鼠肝组织中细胞色素P-450酶3a11(cytochrome P450 enzyme 3a11,cyp3a11)、cyp3a13、孕烷X受体(pregnane X receptor,PXR)、组成型雄甾烷受体(constitutive androstane receptor,CAR)及相关时辰基因的表达。结果:1.8:00给药组吉非替尼的AUC(0-24h)明显高于其他给药组,清除率明显高于其他各组。对于MRT(0-24h),8:00、24:00、4:00给药组之间没有统计学差异,但均高于12:00、16:00、20:00给药组(P0.05)。16:00、20:00给药组的AUC(0-24h)较低,其清除率较高(P0.05)。2.各时辰点模型组的cyp3a11、cyp3a13、PXR、CAR、Per1、Per2和Bmal1 m RNA的表达具有昼夜节律性。cyp3a11、PXR和CAR m RNA在16:00到24:00之间表达较高,在20:00达到峰值,cyp3a13在16:00、20:00和24:00均有较高的表达(P0.05)。Bmal1m RNA的表达在20:00时达到峰值,Per1、Per2在4:00表达较高(P0.05)。给与吉非替尼后,20:00给药组cyp3a11、PXR、CAR m RNA 24 h的总体表达水平最高。12:00给药组cyp3a13 m RNA 24 h的总体表达水平最高。Bmal1 m RNA 24 h的总体表达水平在20:00给药组最高,这与代谢酶的表达趋势相吻合。Per1 m RNA 24 h的总体表达水平在24:00、4:00给药组较高,在20:00给药组最低。Per2 m RNA 24 h的总体表达水平在8:00、12:00、24:00给药组较高,这与代谢酶的表达相反,但与血药浓度的变化相吻合。结论:吉非替尼在荷瘤裸鼠体内的药动学过程因给药时间的不同而不同,具有明显的昼夜节律,表现为明早期和暗晚期(8:00、24:00、4:00)吉非替尼总体血药浓度较高。这可能与代谢酶(cyp3a11、cyp3a13)、核受体通路基因及相关时辰基因的时辰性表达有关。
[Abstract]:Objective: to investigate the pharmacokinetic characteristics of gifitinib in nude mice with subcutaneous transplantation of Balb/c, and to explore its possible mechanism from the point of view of metabolic enzymes, receptor pathways and temporal genes. Methods: 1. The establishment and random grouping of nude mice model of subcutaneous transplanted tumor: Balb/c nude mice were kept adaptively for 2 weeks under strict manual control from 12 h bright period to 12 h dark period (07:00 on and 19:00 lights off). HCC827 cells were inoculated subcutaneously in right upper limb to establish subcutaneous transplanted tumor model in nude mice. The successful nude mice were randomly divided into six groups: 08: 00, 12: 00, 16: 00, 20: 00, 24: 00 and 04:00 the next day. Each time group was randomly divided into administration group and model group, and the model group was randomly divided into 9 blood sampling points group. In addition, normal nude mice without tumor were randomly added at each time point as control group. 2. At the same time, the nude mice in the administration group were given 1.0mg kg-1 gefitinib suspension at the corresponding time point, and the blood was taken from the eyeball at 1530 min,1,3,5,7,9,16,24h after administration. The plasma was obtained by centrifugation and stored in low temperature. Immediately after blood extraction, the nude mice were killed by dislocations, and liver tissue was cryopreserved. Determination of blood drug concentration: the plasma concentration of gefitinib was detected by high performance liquid chromatography (HPLC) and mass spectrometry (high-performance liquid chromatography coupled with tandem mass spectrometry,HPLC-MS/MS) at all time points. The pharmacokinetic parameters of each time point were calculated by Win Nonlin 6.3. 4. Detection of expression of related genes: detection of cytochrome P-450 3a11 (cytochrome P450 enzyme 3a11cyp3a11) and cyp3a13, X receptor (pregnane X receptor,PXR in liver tissues of nude mice by real-time fluorescence quantitative PCR (quantificational real time-polymerase chain reaction,q RT-PCR. Constitutive androstane receptor (constitutive androstane receptor,CAR) and its related gene expression. Results: 1.The AUC (0-24 hours) of gifitinib in the group of 8: 00 was significantly higher than that in the other groups, and the clearance rate was significantly higher than that in the other groups. For MRT (0-24 h), there was no statistical difference between the 8: 00 and 24: 00 groups, but they were higher than that of the 12: 00 (16: 00) 20: 00 group (P0.05). The AUC (0-24 h) of the 16: 00 (20: 00) group was lower than that of the control group (0-24 h). Its clearance rate was higher (P0.05). The expressions of cyp3a11,cyp3a13,PXR,CAR,Per1,Per2 and Bmal1 m RNA were circadian in each time point model group. The expression of cyp3a11,PXR and CAR m RNA was higher between 16:00 and 24:00, and reached its peak at 20:00. The expression of cyp3a13 was higher at 16: 00 and 24:00 (P0.05). The expression of Bmal1m RNA reached its peak at 20:00, and the expression of Per1,Per2 was higher at 4:00 (P0.05). After administration of Gifitinib, the overall expression level of cyp3a11,PXR,CAR m RNA was the highest in 20:00 group, the highest in 12:00 group, and the highest in Bmal1 m RNA group at 20:00. The total expression level of Per1 m RNA at 24 h was higher in 24: 00 and lowest at 20:00, while that of Per2 m RNA 24 h was higher in 8: 00 12: 00: 00: 00. This is contrary to the expression of metabolic enzymes, but consistent with changes in blood concentration. Conclusion: the pharmacokinetic process of gefitinib in tumor-bearing nude mice varies with the time of administration and has an obvious circadian rhythm, showing that the overall plasma concentration of Gifitinib is high in the early stage and dark stage (8: 00 24: 00: 4: 00). This may be related to the temporal expression of metabolic enzymes (cyp3a11,cyp3a13), nuclear receptor pathway genes and related temporal genes.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965
本文编号：2419565
[Abstract]:Objective: to investigate the pharmacokinetic characteristics of gifitinib in nude mice with subcutaneous transplantation of Balb/c, and to explore its possible mechanism from the point of view of metabolic enzymes, receptor pathways and temporal genes. Methods: 1. The establishment and random grouping of nude mice model of subcutaneous transplanted tumor: Balb/c nude mice were kept adaptively for 2 weeks under strict manual control from 12 h bright period to 12 h dark period (07:00 on and 19:00 lights off). HCC827 cells were inoculated subcutaneously in right upper limb to establish subcutaneous transplanted tumor model in nude mice. The successful nude mice were randomly divided into six groups: 08: 00, 12: 00, 16: 00, 20: 00, 24: 00 and 04:00 the next day. Each time group was randomly divided into administration group and model group, and the model group was randomly divided into 9 blood sampling points group. In addition, normal nude mice without tumor were randomly added at each time point as control group. 2. At the same time, the nude mice in the administration group were given 1.0mg kg-1 gefitinib suspension at the corresponding time point, and the blood was taken from the eyeball at 1530 min,1,3,5,7,9,16,24h after administration. The plasma was obtained by centrifugation and stored in low temperature. Immediately after blood extraction, the nude mice were killed by dislocations, and liver tissue was cryopreserved. Determination of blood drug concentration: the plasma concentration of gefitinib was detected by high performance liquid chromatography (HPLC) and mass spectrometry (high-performance liquid chromatography coupled with tandem mass spectrometry,HPLC-MS/MS) at all time points. The pharmacokinetic parameters of each time point were calculated by Win Nonlin 6.3. 4. Detection of expression of related genes: detection of cytochrome P-450 3a11 (cytochrome P450 enzyme 3a11cyp3a11) and cyp3a13, X receptor (pregnane X receptor,PXR in liver tissues of nude mice by real-time fluorescence quantitative PCR (quantificational real time-polymerase chain reaction,q RT-PCR. Constitutive androstane receptor (constitutive androstane receptor,CAR) and its related gene expression. Results: 1.The AUC (0-24 hours) of gifitinib in the group of 8: 00 was significantly higher than that in the other groups, and the clearance rate was significantly higher than that in the other groups. For MRT (0-24 h), there was no statistical difference between the 8: 00 and 24: 00 groups, but they were higher than that of the 12: 00 (16: 00) 20: 00 group (P0.05). The AUC (0-24 h) of the 16: 00 (20: 00) group was lower than that of the control group (0-24 h). Its clearance rate was higher (P0.05). The expressions of cyp3a11,cyp3a13,PXR,CAR,Per1,Per2 and Bmal1 m RNA were circadian in each time point model group. The expression of cyp3a11,PXR and CAR m RNA was higher between 16:00 and 24:00, and reached its peak at 20:00. The expression of cyp3a13 was higher at 16: 00 and 24:00 (P0.05). The expression of Bmal1m RNA reached its peak at 20:00, and the expression of Per1,Per2 was higher at 4:00 (P0.05). After administration of Gifitinib, the overall expression level of cyp3a11,PXR,CAR m RNA was the highest in 20:00 group, the highest in 12:00 group, and the highest in Bmal1 m RNA group at 20:00. The total expression level of Per1 m RNA at 24 h was higher in 24: 00 and lowest at 20:00, while that of Per2 m RNA 24 h was higher in 8: 00 12: 00: 00: 00. This is contrary to the expression of metabolic enzymes, but consistent with changes in blood concentration. Conclusion: the pharmacokinetic process of gefitinib in tumor-bearing nude mice varies with the time of administration and has an obvious circadian rhythm, showing that the overall plasma concentration of Gifitinib is high in the early stage and dark stage (8: 00 24: 00: 4: 00). This may be related to the temporal expression of metabolic enzymes (cyp3a11,cyp3a13), nuclear receptor pathway genes and related temporal genes.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965
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