GO-PTX和PGO-PTX的药效学及药代动力学研究
发布时间：2018-09-06 10:05
【摘要】：背景:紫杉醇是目前治疗乳腺癌和卵巢癌的一线药物,临床疗效较好。但是,紫杉醇的水溶性低是它的一大缺点,而临床使用的紫杉醇助溶剂又有一些严重的副作用,如神经毒性,肾毒性等。此课题中,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为载体,经过结构修饰及活性筛选后得到的新型紫杉醇载体药物。前期研究表明,PGO-PTX和GO-PTX具有载药量较高,水溶性较强,毒性较低的特点,在未来临床应用中具有广阔的发展前景。目的:本课题旨在对GO-PTX和PGO-PTX,从细胞和动物水平,进行抗肿瘤药效学评价及大鼠体内药代动力学研究,为该新型载体药物的更深入研究提供理论依据。方法:PGO-PTX、GO-PTX对A2780细胞生长和凋亡诱导的影响本实验采用经典的MTT(四氮唑盐)法,通过实验数据计算细胞存活率及PGO-PTX、GO-PTX药物的IC50,评价PGO-PTX、GO-PTX药物对人卵巢癌细胞株A2780的增殖抑制作用,以及PGO、GO药物载体对人卵巢癌细胞株A2780的毒性作用。同时,采用DAPI染色法检测细胞凋亡效应,通过对荧光显微镜下细胞形态学的分析,评价PGO-PTX、GO-PTX的凋亡诱导作用,得出实验结论。PGO-PTX、GO-PTX对荷瘤裸鼠移植癌的药效学研究本实验建立了人卵巢癌细胞株A2780裸鼠移植瘤模型,进行了PGO-PTX、GO-PTX药物对荷瘤裸鼠移植癌的药效学研究。20只雌性裸鼠随机剪脚趾编号,均分为四组,即模型对照组,阳性对照组,PGO-PTX药物组、GO-PTX药物组。根据不同分组,在当天及第3、5、7、9天尾静脉注射相应药物或空白溶媒,并测量瘤块大小。最后一天处死取瘤,称重。对不同时间的肿瘤体积及瘤重抑瘤率进行数据统计分析,得出实验结论。血浆中PGO-PTX、GO-PTX分析方法的建立样品处理采用甲基叔丁基醚沉淀蛋白的方法,选择硝苯地平为内标,以乙腈-水(0.05%甲酸,PH=3.5)=45:55(v/v)为流动相等度洗脱,流速为0.2 m L/min,进样量10μL。以Thermo BDS HYPERSIL C18(2.1×100 mm I.D.,2.4μm)柱进行分离;采用三重四级杆质谱仪,以电喷雾电离源作为液相和质谱连接的接口,多反应监测(MRM)模式进行正离子检测。用于定量检测的离子对为m/z 876.3→m/z 308.0(紫杉醇)和m/z 347.3→m/z 315.4(内标硝苯地平)。通过对分析方法的确证,得出实验结论。PGO-PTX、GO-PTX在大鼠体内的药代动力学研究本实验进行了PGO-PTX、GO-PTX在SD大鼠体内的药代动力学研究,为药物在动物体内的变化规律提供重要的评价参数。将12只SD大鼠随机分为三组,即紫杉醇组,PGO-PTX组、GO-PTX组,每组4只,雌雄各半。实验前一天SD大鼠禁食过夜后,每组分别以0.14 mg/kg的药物剂量,尾静脉注射14μmol/L的紫杉醇、PGO-PTX和GO-PTX。分别于给药前,及给药后0,0.038,0.25,0.5,1,2,4,6,8小时,毛细管眼眶采血。检测大鼠尾静脉注射后的血药浓度,并采用DAS 2.0统计软件计算相关药动学参数,绘制血药浓度-时间曲线,得出实验结论。结果:PGO-PTX、GO-PTX对A2780细胞生长和凋亡诱导的影响PGO-PTX、GO-PTX药物对人卵巢癌细胞株A2780的增殖有抑制作用,其IC50分别为2.59μmol/L和2.44μmol/L。与阳性药物紫杉醇注射液相比,PGO-PTX、GO-PTX药物对人卵巢癌细胞株A2780的抑制作用无显著性差异。药物载体PGO略有细胞毒性,GO没有显著的细胞毒性。同时,对给予不同浓度的PGO-PTX、GO-PTX药物后的细胞进行镜下形态学观察,发现均有不同程度的细胞边缘缺失,细胞核碎裂等变化。与阳性对照组相比,其凋亡诱导作用相当。同时,随着药物浓度的递增,凋亡诱导作用呈现增强的趋势。PGO-PTX、GO-PTX对荷瘤裸鼠移植癌的药效学研究实验结果表明,与模型对照组相比,PGO-PTX药物组、GO-PTX药物组对小鼠卵巢癌移植瘤生长均有显著的抑制作用,瘤体积有变小或不再生长的趋势,瘤重抑瘤率分别为38.5%、40%。与阳性对照组相比,PGO-PTX药物组、GO-PTX药物组与其肿瘤生长抑制作用相当。本研究结果证明PGO-PTX、GO-PTX具有一定的抗癌效果。血浆中PGO-PTX、GO-PTX分析方法的建立本实验所建立的测定大鼠血浆中PGO-PTX、GO-PTX的LC-MS/MS分析方法,样品的测定不受血浆中杂质的干扰,血浆中紫杉醇的线性范围为0.25~1000ng·m L-1,线性关系良好(R=0.9978),最低检测浓度为0.25 ng·ml-1,提取回收率高于75%,批内和批间精密度RSD均小于10%,准确度在0.53%~8.04%,待测药基质效应在87.57%~101.68%之间,内标提取回收率和基质效应分别为89.90%、97.34%。样品的室温,冻融、进样盘及长期稳定性试验结果表明,在本测定条件下,样品较稳定,未见明显降解。该方法符合生物样品分析要求,且专属性强,灵敏度高,适用于大鼠血浆药代动力学的研究。PGO-PTX、GO-PTX在大鼠体内的药代动力学研究大鼠血浆PGO-PTX、GO-PTX和紫杉醇的AUC0-t分别为4.36±0.92μg/L*h、21.48±5.81μg/L*h和24.17±4.92μg/L*h,T1/2为3.18±2.86 h、2.68±0.77 h和2.17±1.39h。实验结果表明,PGO-PTX的AUC0-t远小于紫杉醇,而GO-PTX与紫杉醇的药代动力学参数结果相似。结论:在细胞、动物药效学实验中,PGO-PTX、GO-PTX皆有良好的抗肿瘤作用;在大鼠药代动力学实验中,GO-PTX与紫杉醇的药代动力学参数结果相似。综上,GO-PTX体现出良好的药效作用及药动学特征,具有较好的应用前景。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965
本文编号：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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965
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1 薛冰洋;GO-PTX和PGO-PTX的药效学及药代动力学研究[D];郑州大学;2016年
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