GO-PTX和PGO-PTX的藥效學及藥代動力學研究
發(fā)布時間：2018-09-06 10:05
【摘要】：背景:紫杉醇是目前治療乳腺癌和卵巢癌的一線藥物,臨床療效較好。但是,紫杉醇的水溶性低是它的一大缺點,而臨床使用的紫杉醇助溶劑又有一些嚴重的副作用,如神經(jīng)毒性,腎毒性等。此課題中,氧化石墨烯紫杉醇(graphene oxide-paclitaxel,GO-PTX)和功能化的氧化石墨烯紫杉醇(functionalized graphene oxide-paclitaxel,PGO-PTX)是以氧化石墨烯和功能化的氧化石墨烯為載體,經(jīng)過結(jié)構(gòu)修飾及活性篩選后得到的新型紫杉醇載體藥物。前期研究表明,PGO-PTX和GO-PTX具有載藥量較高,水溶性較強,毒性較低的特點,在未來臨床應用中具有廣闊的發(fā)展前景。目的:本課題旨在對GO-PTX和PGO-PTX,從細胞和動物水平,進行抗腫瘤藥效學評價及大鼠體內(nèi)藥代動力學研究,為該新型載體藥物的更深入研究提供理論依據(jù)。方法:PGO-PTX、GO-PTX對A2780細胞生長和凋亡誘導的影響本實驗采用經(jīng)典的MTT(四氮唑鹽)法,通過實驗數(shù)據(jù)計算細胞存活率及PGO-PTX、GO-PTX藥物的IC50,評價PGO-PTX、GO-PTX藥物對人卵巢癌細胞株A2780的增殖抑制作用,以及PGO、GO藥物載體對人卵巢癌細胞株A2780的毒性作用。同時,采用DAPI染色法檢測細胞凋亡效應,通過對熒光顯微鏡下細胞形態(tài)學的分析,評價PGO-PTX、GO-PTX的凋亡誘導作用,得出實驗結(jié)論。PGO-PTX、GO-PTX對荷瘤裸鼠移植癌的藥效學研究本實驗建立了人卵巢癌細胞株A2780裸鼠移植瘤模型,進行了PGO-PTX、GO-PTX藥物對荷瘤裸鼠移植癌的藥效學研究。20只雌性裸鼠隨機剪腳趾編號,均分為四組,即模型對照組,陽性對照組,PGO-PTX藥物組、GO-PTX藥物組。根據(jù)不同分組,在當天及第3、5、7、9天尾靜脈注射相應藥物或空白溶媒,并測量瘤塊大小。最后一天處死取瘤,稱重。對不同時間的腫瘤體積及瘤重抑瘤率進行數(shù)據(jù)統(tǒng)計分析,得出實驗結(jié)論。血漿中PGO-PTX、GO-PTX分析方法的建立樣品處理采用甲基叔丁基醚沉淀蛋白的方法,選擇硝苯地平為內(nèi)標,以乙腈-水(0.05%甲酸,PH=3.5)=45:55(v/v)為流動相等度洗脫,流速為0.2 m L/min,進樣量10μL。以Thermo BDS HYPERSIL C18(2.1×100 mm I.D.,2.4μm)柱進行分離;采用三重四級桿質(zhì)譜儀,以電噴霧電離源作為液相和質(zhì)譜連接的接口,多反應監(jiān)測(MRM)模式進行正離子檢測。用于定量檢測的離子對為m/z 876.3→m/z 308.0(紫杉醇)和m/z 347.3→m/z 315.4(內(nèi)標硝苯地平)。通過對分析方法的確證,得出實驗結(jié)論。PGO-PTX、GO-PTX在大鼠體內(nèi)的藥代動力學研究本實驗進行了PGO-PTX、GO-PTX在SD大鼠體內(nèi)的藥代動力學研究,為藥物在動物體內(nèi)的變化規(guī)律提供重要的評價參數(shù)。將12只SD大鼠隨機分為三組,即紫杉醇組,PGO-PTX組、GO-PTX組,每組4只,雌雄各半。實驗前一天SD大鼠禁食過夜后,每組分別以0.14 mg/kg的藥物劑量,尾靜脈注射14μmol/L的紫杉醇、PGO-PTX和GO-PTX。分別于給藥前,及給藥后0,0.038,0.25,0.5,1,2,4,6,8小時,毛細管眼眶采血。檢測大鼠尾靜脈注射后的血藥濃度,并采用DAS 2.0統(tǒng)計軟件計算相關(guān)藥動學參數(shù),繪制血藥濃度-時間曲線,得出實驗結(jié)論。結(jié)果:PGO-PTX、GO-PTX對A2780細胞生長和凋亡誘導的影響PGO-PTX、GO-PTX藥物對人卵巢癌細胞株A2780的增殖有抑制作用,其IC50分別為2.59μmol/L和2.44μmol/L。與陽性藥物紫杉醇注射液相比,PGO-PTX、GO-PTX藥物對人卵巢癌細胞株A2780的抑制作用無顯著性差異。藥物載體PGO略有細胞毒性,GO沒有顯著的細胞毒性。同時,對給予不同濃度的PGO-PTX、GO-PTX藥物后的細胞進行鏡下形態(tài)學觀察,發(fā)現(xiàn)均有不同程度的細胞邊緣缺失,細胞核碎裂等變化。與陽性對照組相比,其凋亡誘導作用相當。同時,隨著藥物濃度的遞增,凋亡誘導作用呈現(xiàn)增強的趨勢。PGO-PTX、GO-PTX對荷瘤裸鼠移植癌的藥效學研究實驗結(jié)果表明,與模型對照組相比,PGO-PTX藥物組、GO-PTX藥物組對小鼠卵巢癌移植瘤生長均有顯著的抑制作用,瘤體積有變小或不再生長的趨勢,瘤重抑瘤率分別為38.5%、40%。與陽性對照組相比,PGO-PTX藥物組、GO-PTX藥物組與其腫瘤生長抑制作用相當。本研究結(jié)果證明PGO-PTX、GO-PTX具有一定的抗癌效果。血漿中PGO-PTX、GO-PTX分析方法的建立本實驗所建立的測定大鼠血漿中PGO-PTX、GO-PTX的LC-MS/MS分析方法,樣品的測定不受血漿中雜質(zhì)的干擾,血漿中紫杉醇的線性范圍為0.25~1000ng·m L-1,線性關(guān)系良好(R=0.9978),最低檢測濃度為0.25 ng·ml-1,提取回收率高于75%,批內(nèi)和批間精密度RSD均小于10%,準確度在0.53%~8.04%,待測藥基質(zhì)效應在87.57%~101.68%之間,內(nèi)標提取回收率和基質(zhì)效應分別為89.90%、97.34%。樣品的室溫,凍融、進樣盤及長期穩(wěn)定性試驗結(jié)果表明,在本測定條件下,樣品較穩(wěn)定,未見明顯降解。該方法符合生物樣品分析要求,且專屬性強,靈敏度高,適用于大鼠血漿藥代動力學的研究。PGO-PTX、GO-PTX在大鼠體內(nèi)的藥代動力學研究大鼠血漿PGO-PTX、GO-PTX和紫杉醇的AUC0-t分別為4.36±0.92μg/L*h、21.48±5.81μg/L*h和24.17±4.92μg/L*h,T1/2為3.18±2.86 h、2.68±0.77 h和2.17±1.39h。實驗結(jié)果表明,PGO-PTX的AUC0-t遠小于紫杉醇,而GO-PTX與紫杉醇的藥代動力學參數(shù)結(jié)果相似。結(jié)論:在細胞、動物藥效學實驗中,PGO-PTX、GO-PTX皆有良好的抗腫瘤作用;在大鼠藥代動力學實驗中,GO-PTX與紫杉醇的藥代動力學參數(shù)結(jié)果相似。綜上,GO-PTX體現(xiàn)出良好的藥效作用及藥動學特征,具有較好的應用前景。
[Abstract]:BACKGROUND: Paclitaxel is the first-line drug for the treatment of breast and ovarian cancer with good clinical efficacy. However, the low water solubility of paclitaxel is one of its major drawbacks, and the paclitaxel cosolvent used in clinic has some serious side effects, such as neurotoxicity and nephrotoxicity. GO-PTX and functionalized graphene oxide-paclitaxel (PGO-PTX) are novel taxol carrier drugs, which are prepared by structural modification and activity screening with graphene oxide and functionalized graphene oxide as carriers. OBJECTIVE: To evaluate the antitumor pharmacodynamics of GO-PTX and PGO-PTX at the cellular and animal levels and to study the pharmacokinetics in rats, so as to provide theoretical basis for further study of the new carrier drugs. The effects of PGO-PTX and GO-PTX on the proliferation and apoptosis of human ovarian cancer cell line A2780 were evaluated by MTT assay. The cell viability and IC50 of PGO-PTX and GO-PTX were calculated. The toxicity of PGO and GO drug carriers on human ovarian cancer cell line A2780 was evaluated. At the same time, DAPI staining was used to detect the apoptosis effect. The apoptosis inducing effect of PGO-PTX and GO-PTX was evaluated by the analysis of cell morphology under fluorescence microscope. The pharmacodynamics of PGO-PTX and GO-PTX on transplanted cancer in nude mice was studied. The pharmacodynamics of PGO-PTX and GO-PTX drugs on transplanted cancer in nude mice were studied.Twenty female nude mice were randomly divided into four groups: model control group, positive control group, PGO-PTX drug group, GO-PTX drug group.According to different groups, the corresponding drugs or blank solvents were injected into tail vein on the same day and the third, fifth, seventh and ninth day, and the tumor size was measured. On the last day, the tumor was sacrificed and weighed. The tumor volume and tumor inhibition rate at different time were statistically analyzed and the experimental conclusion was drawn. In order to elute the mobile phase equivalently, the flow rate was 0.2 m L/min and the injection volume was 10 u L. The ion pairs used for quantitative detection were M. Pharmacokinetics of PGO-PTX and GO-PTX in SD rats were studied. Pharmacokinetics of PGO-PTX and GO-PTX in SD rats were studied. The results provided the pharmacokinetics of PGO-PTX and GO-PTX in SD rats. 12 SD rats were randomly divided into three groups: paclitaxel group, PGO-PTX group, GO-PTX group, 4 rats in each group, half male and half female. The serum concentration of PGO-PTX and GO-PTX were measured after tail vein injection in rats, and the pharmacokinetic parameters were calculated by DAS 2.0 statistical software. The concentration-time curves of PGO-PTX and GO-PTX were drawn. Results: PGO-PTX and GO-PTX affected the growth and apoptosis of A2780 cells. The IC50 of strain A2780 was 2.59 and 2.44 micromol/L, respectively. Compared with paclitaxel injection, PGO-PTX and GO-PTX had no significant inhibitory effect on human ovarian cancer cell line A2780. The drug carrier PGO had slight cytotoxicity and GO had no significant cytotoxicity. Compared with the positive control group, the induction of apoptosis was similar. At the same time, with the increase of drug concentration, the induction of apoptosis was enhanced. PGO-PTX, GO-PTX on tumor-bearing nude mice transplanted cancer drugs. The results showed that compared with the model control group, PGO-PTX drug group, GO-PTX drug group had a significant inhibitory effect on the growth of ovarian cancer xenografts in mice. The tumor volume tended to decrease or regenerate. The tumor weight inhibition rate was 38.5% and 40% respectively. Compared with the positive control group, the PGO-PTX drug group, GO-PTX drug group and its tumor growth were significantly inhibited. The results of this study showed that PGO-PTX and GO-PTX had certain anticancer effects. The method of determination of PGO-PTX and GO-PTX in rat plasma was established. The method of LC-MS/MS for the determination of PGO-PTX and GO-PTX in rat plasma was established. The determination of PGO-PTX and GO-PTX in rat plasma was not interfered by impurities in plasma. The linear range of paclitaxel in plasma was 0.25-100. The linear relationship was good (R = 0.9978), the minimum detection concentration was 0.25 ng ml 1, and the extraction recovery was higher than 75%. The precision of RSD was less than 10%, the accuracy was 0.53%~8.04%, the matrix effect was 87.57%~101.68%, the recovery of internal standard and matrix effect were 89.90% and 97.34%, respectively. The results of disc and long-term stability tests showed that the samples were stable and no obvious degradation was observed. The method was suitable for the study of pharmacokinetics of rat plasma. The pharmacokinetics of PGO-PTX, GO-PTX and purple in rat plasma were studied. AUC0-t of taxol was 4.36.92 ug/L * h, 21.48.81 ug/L * h and 24.17.92 ug/L * h, T1/2 was 3.18.86 h, 2.68.77 h and 2.17.39 h, respectively. The results showed that AUC0-t of PGO-PTX was much smaller than that of paclitaxel, while the pharmacokinetic parameters of GO-PTX were similar to that of paclitaxel. The pharmacokinetic parameters of GO-PTX and paclitaxel were similar in the pharmacokinetic experiment of rats. In conclusion, GO-PTX showed good pharmacodynamic effect and pharmacokinetic characteristics and had a good application prospect.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R965
本文編號：2226006
[Abstract]:BACKGROUND: Paclitaxel is the first-line drug for the treatment of breast and ovarian cancer with good clinical efficacy. However, the low water solubility of paclitaxel is one of its major drawbacks, and the paclitaxel cosolvent used in clinic has some serious side effects, such as neurotoxicity and nephrotoxicity. GO-PTX and functionalized graphene oxide-paclitaxel (PGO-PTX) are novel taxol carrier drugs, which are prepared by structural modification and activity screening with graphene oxide and functionalized graphene oxide as carriers. OBJECTIVE: To evaluate the antitumor pharmacodynamics of GO-PTX and PGO-PTX at the cellular and animal levels and to study the pharmacokinetics in rats, so as to provide theoretical basis for further study of the new carrier drugs. The effects of PGO-PTX and GO-PTX on the proliferation and apoptosis of human ovarian cancer cell line A2780 were evaluated by MTT assay. The cell viability and IC50 of PGO-PTX and GO-PTX were calculated. The toxicity of PGO and GO drug carriers on human ovarian cancer cell line A2780 was evaluated. At the same time, DAPI staining was used to detect the apoptosis effect. The apoptosis inducing effect of PGO-PTX and GO-PTX was evaluated by the analysis of cell morphology under fluorescence microscope. The pharmacodynamics of PGO-PTX and GO-PTX on transplanted cancer in nude mice was studied. The pharmacodynamics of PGO-PTX and GO-PTX drugs on transplanted cancer in nude mice were studied.Twenty female nude mice were randomly divided into four groups: model control group, positive control group, PGO-PTX drug group, GO-PTX drug group.According to different groups, the corresponding drugs or blank solvents were injected into tail vein on the same day and the third, fifth, seventh and ninth day, and the tumor size was measured. On the last day, the tumor was sacrificed and weighed. The tumor volume and tumor inhibition rate at different time were statistically analyzed and the experimental conclusion was drawn. In order to elute the mobile phase equivalently, the flow rate was 0.2 m L/min and the injection volume was 10 u L. The ion pairs used for quantitative detection were M. Pharmacokinetics of PGO-PTX and GO-PTX in SD rats were studied. Pharmacokinetics of PGO-PTX and GO-PTX in SD rats were studied. The results provided the pharmacokinetics of PGO-PTX and GO-PTX in SD rats. 12 SD rats were randomly divided into three groups: paclitaxel group, PGO-PTX group, GO-PTX group, 4 rats in each group, half male and half female. The serum concentration of PGO-PTX and GO-PTX were measured after tail vein injection in rats, and the pharmacokinetic parameters were calculated by DAS 2.0 statistical software. The concentration-time curves of PGO-PTX and GO-PTX were drawn. Results: PGO-PTX and GO-PTX affected the growth and apoptosis of A2780 cells. The IC50 of strain A2780 was 2.59 and 2.44 micromol/L, respectively. Compared with paclitaxel injection, PGO-PTX and GO-PTX had no significant inhibitory effect on human ovarian cancer cell line A2780. The drug carrier PGO had slight cytotoxicity and GO had no significant cytotoxicity. Compared with the positive control group, the induction of apoptosis was similar. At the same time, with the increase of drug concentration, the induction of apoptosis was enhanced. PGO-PTX, GO-PTX on tumor-bearing nude mice transplanted cancer drugs. The results showed that compared with the model control group, PGO-PTX drug group, GO-PTX drug group had a significant inhibitory effect on the growth of ovarian cancer xenografts in mice. The tumor volume tended to decrease or regenerate. The tumor weight inhibition rate was 38.5% and 40% respectively. Compared with the positive control group, the PGO-PTX drug group, GO-PTX drug group and its tumor growth were significantly inhibited. The results of this study showed that PGO-PTX and GO-PTX had certain anticancer effects. The method of determination of PGO-PTX and GO-PTX in rat plasma was established. The method of LC-MS/MS for the determination of PGO-PTX and GO-PTX in rat plasma was established. The determination of PGO-PTX and GO-PTX in rat plasma was not interfered by impurities in plasma. The linear range of paclitaxel in plasma was 0.25-100. The linear relationship was good (R = 0.9978), the minimum detection concentration was 0.25 ng ml 1, and the extraction recovery was higher than 75%. The precision of RSD was less than 10%, the accuracy was 0.53%~8.04%, the matrix effect was 87.57%~101.68%, the recovery of internal standard and matrix effect were 89.90% and 97.34%, respectively. The results of disc and long-term stability tests showed that the samples were stable and no obvious degradation was observed. The method was suitable for the study of pharmacokinetics of rat plasma. The pharmacokinetics of PGO-PTX, GO-PTX and purple in rat plasma were studied. AUC0-t of taxol was 4.36.92 ug/L * h, 21.48.81 ug/L * h and 24.17.92 ug/L * h, T1/2 was 3.18.86 h, 2.68.77 h and 2.17.39 h, respectively. The results showed that AUC0-t of PGO-PTX was much smaller than that of paclitaxel, while the pharmacokinetic parameters of GO-PTX were similar to that of paclitaxel. The pharmacokinetic parameters of GO-PTX and paclitaxel were similar in the pharmacokinetic experiment of rats. In conclusion, GO-PTX showed good pharmacodynamic effect and pharmacokinetic characteristics and had a good application prospect.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R965
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1 薛冰洋;GO-PTX和PGO-PTX的藥效學及藥代動力學研究[D];鄭州大學;2016年
，本文編號：2226006
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