吉非替尼在荷瘤裸鼠體內(nèi)的時辰藥動學(xué)及其機制
發(fā)布時間：2019-02-11 08:36
【摘要】：目的:本研究通過建立Balb/c裸鼠皮下移植瘤模型,探究吉非替尼時辰給藥的藥動學(xué)特點,并從代謝酶、受體通路及時辰基因的角度探討其可能的機制。方法:1.裸鼠皮下移植瘤模型的建立與隨機分組:將Balb/c裸鼠置于嚴格的人工控制的12 h明期-12 h暗期(07:00開燈,19:00關(guān)燈)的條件下適應(yīng)性飼養(yǎng)2周。于右上肢皮下接種HCC827細胞,建立裸鼠皮下移植瘤模型。將造模成功的裸鼠隨機分為08:00、12:00、16:00、20:00、24:00及次日04:00六個時辰組。每個時辰組又隨機分為給藥組和模型組,并將模型組隨機分為9個取血點組。另外,每個時辰點各隨機加入未種瘤的正常裸鼠作為對照組。2.時辰給藥及血漿、肝組織樣本的獲取:給藥組裸鼠在相應(yīng)時辰點給予1.0mg·kg-1吉非替尼混懸液,并于給藥后0 h、15、30 min、1、3、5、7、9、16、24h摘眼球取血。將血液離心得到血漿并低溫保存。取血后立即脫臼處死裸鼠,取肝組織冷凍保存。3.血藥濃度檢測:采用高效液相色譜法聯(lián)合質(zhì)譜(high-performance liquid chromatography coupled with tandem mass spectrometry,HPLC-MS/MS)的方法檢測各個時辰點吉非替尼的血藥濃度,并用Win Nonlin 6.3計算各時辰點的藥動學(xué)參數(shù)。4.相關(guān)基因表達水平的檢測:采用實時熒光定量PCR(quantificational real time-polymerase chain reaction,q RT-PCR)檢測裸鼠肝組織中細胞色素P-450酶3a11(cytochrome P450 enzyme 3a11,cyp3a11)、cyp3a13、孕烷X受體(pregnane X receptor,PXR)、組成型雄甾烷受體(constitutive androstane receptor,CAR)及相關(guān)時辰基因的表達。結(jié)果:1.8:00給藥組吉非替尼的AUC(0-24h)明顯高于其他給藥組,清除率明顯高于其他各組。對于MRT(0-24h),8:00、24:00、4:00給藥組之間沒有統(tǒng)計學(xué)差異,但均高于12:00、16:00、20:00給藥組(P0.05)。16:00、20:00給藥組的AUC(0-24h)較低,其清除率較高(P0.05)。2.各時辰點模型組的cyp3a11、cyp3a13、PXR、CAR、Per1、Per2和Bmal1 m RNA的表達具有晝夜節(jié)律性。cyp3a11、PXR和CAR m RNA在16:00到24:00之間表達較高,在20:00達到峰值,cyp3a13在16:00、20:00和24:00均有較高的表達(P0.05)。Bmal1m RNA的表達在20:00時達到峰值,Per1、Per2在4:00表達較高(P0.05)。給與吉非替尼后,20:00給藥組cyp3a11、PXR、CAR m RNA 24 h的總體表達水平最高。12:00給藥組cyp3a13 m RNA 24 h的總體表達水平最高。Bmal1 m RNA 24 h的總體表達水平在20:00給藥組最高,這與代謝酶的表達趨勢相吻合。Per1 m RNA 24 h的總體表達水平在24:00、4:00給藥組較高,在20:00給藥組最低。Per2 m RNA 24 h的總體表達水平在8:00、12:00、24:00給藥組較高,這與代謝酶的表達相反,但與血藥濃度的變化相吻合。結(jié)論:吉非替尼在荷瘤裸鼠體內(nèi)的藥動學(xué)過程因給藥時間的不同而不同,具有明顯的晝夜節(jié)律,表現(xiàn)為明早期和暗晚期(8:00、24:00、4:00)吉非替尼總體血藥濃度較高。這可能與代謝酶(cyp3a11、cyp3a13)、核受體通路基因及相關(guān)時辰基因的時辰性表達有關(guān)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R965
本文編號：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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R965
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