舒尼替尼在荷腎癌裸鼠體內(nèi)的時辰藥理學(xué)研究
發(fā)布時間：2019-06-19 16:10
【摘要】：目的:通過皮下接種構(gòu)造ACHN人腎細(xì)胞腺癌移植瘤Balb/c裸鼠模型,探究舒尼替尼(Sunitinib,SU)時辰給藥的藥效學(xué)及藥動學(xué)特點(diǎn),并從時辰基因、代謝酶等多個方面探究其可能的藥效作用機(jī)制。方法:1.建立裸鼠腎癌模型:將4周齡的Balb/c裸鼠置于嚴(yán)格的明暗環(huán)境中適應(yīng)性飼養(yǎng)兩周,將培養(yǎng)好的ACHN人腎細(xì)胞腺癌細(xì)胞于裸鼠右上肢皮下注射,建立Balb/c裸鼠移植瘤模型。2.藥效學(xué)研究的隨機(jī)分組:將裸鼠模型隨機(jī)分為4:00、8:00、12:00、16:00、20:00、24:00六個時辰組,每個時辰組又隨機(jī)分為舒尼替尼給藥組和模型組。3.藥效學(xué)研究的時辰給藥及檢測指標(biāo):舒尼替尼給藥組分別在對應(yīng)的六個時辰點(diǎn)灌胃給予16.25mg·kg-1舒尼替尼溶液0.2m L,模型組則在同樣的時辰點(diǎn)給以等量的CB(Citrate buffer,檸檬酸鹽緩沖液,PH=4.5)。在給藥期間每三天對腫瘤的長短徑進(jìn)行一次測量以計算其體積,根據(jù)其體積變化趨勢繪制出腫瘤體積生長曲線。至第21天則分別于相應(yīng)時辰點(diǎn)處死裸鼠,剝離瘤體稱重并制作病理切片以比較各組腫瘤的病理形態(tài)。4.藥動學(xué)研究的隨機(jī)分組:將裸鼠模型隨機(jī)分為4:00、8:00、12:00、16:00、20:00、24:00六個時辰組,每個時辰組又分為舒尼替尼給藥組和模型組。每個給藥組包括15 min、30 min、1h、3h、5 h、7 h、9 h、14 h、19 h、24 h共10個取血點(diǎn)。每個時辰組設(shè)置正常裸鼠作為空白對照組。5.藥動學(xué)研究的時辰給藥及檢測指標(biāo):給藥組于六個時辰點(diǎn)分別灌胃給以舒尼替尼16.25mg·kg-1,并于給藥后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,離心取血漿置-80℃保存,采用HPLC-MS/MS(high-performance liquid chromatography coupled with tandem mass spectrometry,高效液相色譜法聯(lián)合質(zhì)譜)的方法測定不同時辰點(diǎn)舒尼替尼及其主要活性代謝物N-去乙基舒尼替尼(SU12662)的血藥濃度,并用Win Nonlin 6.3計算各時辰點(diǎn)的藥動學(xué)參數(shù)。取血后迅速處死裸鼠,并立即取肝臟于-80℃冷凍保存。6.相關(guān)基因表達(dá)水平的檢測:從肝組織中提取總RNA采用q RT-PCR(Quantificational Real Time-PCR,實(shí)時熒光定量PCR)檢測cyp3a11(cytochrome P450 enzyme 3a11,細(xì)胞色素P450酶3a11)、cyp3a13、Bmal-1(brain and muscle arnt-like protein-1,時辰基因編碼芳香烴受體核轉(zhuǎn)運(yùn)樣蛋白)、Clock(circadian locomotor output cycles kaput,生物鐘循環(huán)輸出蛋白)及時辰基因的作用位點(diǎn)Dbp(D site-binding protein,D-位點(diǎn)結(jié)合蛋白)的表達(dá)。結(jié)果:1.舒尼替尼在荷瘤裸鼠體內(nèi)的時辰藥效學(xué)具有晝夜節(jié)律性,與模型組相比,各時辰給藥組的腫瘤體積增長速度均有所減慢,腫瘤重量也有不同的減輕。16:00組的腫瘤生長曲線坡勢陡直,與其他五個給藥組相較,腫瘤體積增長迅速,抑瘤率最低;8:00、12:00給藥組與16:00及20:00給藥組相比,腫瘤體積生長緩慢,抑瘤率高,有顯著性差異(p0.05),其中8:00給藥組腫瘤受抑制情況最為明顯。病理切片結(jié)果顯示,16:00及20:00給藥組腫瘤組織細(xì)胞排列基本無間隙,邊界較為清楚,腫瘤壞死量少,炎性細(xì)胞浸潤情況輕微;8:00及12:00給藥組中大量的腫瘤細(xì)胞是壞死的,表現(xiàn)出嚴(yán)重的炎性細(xì)胞浸潤和血管破裂出血。2.舒尼替尼在荷瘤裸鼠體內(nèi)的時辰藥動學(xué)具有晝夜節(jié)律性,相比其他各時辰給藥組,16:00與20:00給藥組AUC0-24h較低,清除率高;8:00及12:00給藥組裸鼠體內(nèi)總藥物具有更高的AUC0-24h、MRT0-24h和更低的CL/F,在體內(nèi)駐留時間長,血藥濃度高,與16:00給藥組相比具有統(tǒng)計學(xué)差異(p0.05)。與藥效學(xué)研究結(jié)果相一致。3.代謝酶基因cyp3a11、cyp3a13及時辰基因Bmal-1、Clock、Dbp在荷腎癌裸鼠肝臟組織中的表達(dá)顯示出顯著的晝夜節(jié)律性。模型組代謝酶基因cyp3a11、時辰基因Bmal-1在12:00至20:00時間范圍內(nèi)表達(dá)量高,在16:00表達(dá)最高,達(dá)到峰值;cyp3a13及Clock表達(dá)量自8:00起呈現(xiàn)不斷上升的趨勢,在20:00至24:00時間段內(nèi)坡度最大,增長速率最為迅速。結(jié)合位點(diǎn)蛋白Dbp在16:00、24:00均有較高程度的表達(dá)。在不同的時間點(diǎn)給予舒尼替尼后,16:00組Bmal-1、cyp3a11總體表達(dá)量最高;24:00組Clock、Dbp及cyp3a13總體表達(dá)量最高,其中Clock及cyp3a13在20:00組也有較高的表達(dá)。時辰基因與代謝酶基因的整體表達(dá)趨勢具有一致性。結(jié)論:舒尼替尼在荷瘤裸鼠體內(nèi)的時辰藥理學(xué)具有晝夜節(jié)律性,在明早期(8:00及12:00)給藥時,腫瘤生長緩慢,抑瘤率高,抑瘤效果最好,體內(nèi)總藥物清除率低,在體內(nèi)的駐留時間長,具有更高的血藥濃度。作用機(jī)制可能為時辰基因通過作用于Dbp直接調(diào)控代謝酶的表達(dá),以影響藥物在裸鼠體內(nèi)代謝的速率,使得舒尼替尼的藥動學(xué)特點(diǎn)呈現(xiàn)晝夜節(jié)律性,亦對藥效學(xué)產(chǎn)生影響。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R965
本文編號：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R965
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王雅杰;王寧;;腫瘤分子靶向藥物分類及作用機(jī)制[J];中國實(shí)用外科雜志;2010年07期
，本文編號：2502471
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