拓扑替康反相脂质纳米粒口服制剂研究

发布时间：2018-08-27 05:47

【摘要】：拓扑替康(TPT)是水溶性半合成喜树碱(CPT)类抗癌药物,可以特异性抑制拓扑异构酶Ⅰ的功能,阻碍DNA的复制。目前拓扑替康上市剂型有拓扑替康注射用冻干粉针剂和口服胶囊剂。相比静脉注射给药而言,口服TPT临床治疗有效、方便,且具有较低的骨髓抑制作用,但由于拓扑替康活性内酯结构受pH影响,在中性及碱性条件下易水解开环为羧酸盐构型,从而失去活性,导致生物利用度降低,抗肿瘤活性下降,所以选择合适的药物载体在肠道中对其活性构型进行保护显得尤为重要,另一方面,拓扑替康口服后具有较强的胃肠道不良反应。本课题针对现有TPT口服制剂存在的问题,研究制备拓扑替康反相脂质纳米粒新剂型,目的是增加药物在消化道中的稳定性同时减少胃肠粘膜刺激,并且提高药物的口服生物利用度。通过单因素试验与正交试验,以透光率、粒径、澄清度等为指标,对反相脂质纳米粒的膜材种类、用量以及载药量进行了筛选,采用冷冻干燥工艺、以中链甘油三酸酯(MCT)为油相制备反相脂质纳米粒(RLBN-TPT)油溶液,载药量为4mg/g。通过动态光散射和透射电镜对RLBN-TPT制剂进行了表征,结果显示反相脂质纳米粒的粒径为60nm左右,呈均匀球状。RLBN-TPT制剂在模拟肠液中分散后,对其粒径和微观形态进行了观察,结果显示分散后形成均匀球形的脂质囊泡或乳滴。在体外稳定性评价研究中,建立了高效液相色谱法同时测定拓扑替康开环、闭环两种构型的含量。通过测定拓扑替康反相脂质纳米粒(RLBN-TPT)在模拟肠液中的释放,研究拓扑替康在人工肠液中的稳定性,考察反相脂质纳米粒对药物的保护作用。选用CAPCELL PAK C18 MGⅡ色谱柱(250×4.6 mm,5μm),流动相为A:0.1%三乙胺(用冰醋酸调节pH值至6.4)-B:乙腈[梯度:0~8min(88%A~60%A);8~17(60%A)],流速1.0mL/min,检测波长360nm,柱温55℃。结果表明开环、闭环构型浓度在0.25~5.00μg·mL-1范围内,浓度与峰面积呈良好线性关系,该方法回收率高,准确度好。与拓扑替康水溶液比较,反相脂质纳米粒中的拓扑替康在肠液中的稳定性明显增强,开环构型产生的速率明显减小(p0.05),表明反相脂质纳米粒对药物活性内酯形式具有一定保护作用,有效避免其在短时间内的水解开环,增强拓扑替康在模拟肠液中的稳定性。建立了HPLC法测定RLBN-TPT制剂中拓扑替康的含量,方法学研究表明,拓扑替康峰面积和浓度在0.125μmg·mL-1~125μg·mL-1范围内呈良好的线性关系,且高、中、低的回收率均在98%-102%之间,RSD为0.67%,该方法准确、精密度高、专属性强、灵敏度高,可以用于制剂中拓扑替康含量的测定;又建立了HPLC法测定RLBN-TPT制剂中拓扑替康的有关物质,方法学研究表明该方法灵敏度高、专属性强、精密度高,可以用于制剂中拓扑替康有关物质的测定。通过影响因素试验结果可知,RLBN-TPT制剂对湿、热比较敏感,所以在储存过程中应避免高温,注意防潮;加速试验表明,RLBN-TPT制剂批次间重现性较好,外观、粒径、含量和溶出度均无显著变化且无药物析出,有关物质稍有增加,本品包装及长期稳定性还需进一步考察。在药代动力学实验中,建立了LC-MS/MS法测定Wistar大鼠体内拓扑替康血药浓度,血浆中药物浓度在1.0~200 ng/mL(r20.997)范围,与峰面积比之间呈良好的线性关系,确定定量下限为1.0 ng/mL,方法回收率高,准确度好,满足Wistar大鼠体内血药浓度测定要求。以上市拓扑替康胶囊剂为参比制剂,比较了RLBN-TPT制剂在Wistar大鼠体内的药代动力学特征。结果表明,RLBNTPT制剂的Cmax和AUC0-∞分别是市售制剂的1.61倍和1.57倍(p0.05),显著提高了药物的口服生物利用度。为进一步阐明反相脂质纳米粒的吸收途径,比较了阻断淋巴吸收途径前后两种制剂的体内吸收行为,结果表明,阻断后反相脂质纳米粒AUC显著下降,下降至阻断前的30%,市售制剂下降为阻断前的63%,由此可以说明RLBN-TPT制剂通过增加药物的淋巴吸收,从而提高了药物的口服生物利用度。建立了BALB/c荷瘤裸鼠的HepG2实体瘤动物模型,结果显示,RLBN-TPT组和上市制剂的抑瘤率分别为44.08%和16.3%(p0.05),通过TUNEL染色观察肿瘤细胞凋亡、HE染色观察肿瘤组织凋亡情况,并对肿瘤组织进行病理学分析,结果表明,RLBN-TPT制剂组肿瘤细胞死亡面积更大,肿瘤组织大面积坏死。表明相比于市售制剂,RLBN-TPT制剂能够更好地抑制肿瘤生长,具有更强抗肿瘤活性。在肠道刺激性试验中,比较了RLBN-TPT制剂组与市售胶囊制剂组小鼠空肠的病变程度。由结果可知,服用市售制剂后小鼠肠黏膜层表面可见明显水肿、脱失,以及血管充血,表明制剂对胃肠道具有较高的刺激性,而服用RLBN-TPT制剂后小鼠肠壁黏表层仅偶见水肿现象,其壁黏膜层及黏膜下、浆膜层、肠壁全层结构清晰,微绒毛其柱状上皮及杯状细胞等细胞成分分布均匀,表明RLBNTPT制剂相较于市售胶囊制剂,降低了口服TPT肠道刺激性。综上所述,RLBN-TPT制剂能够显著提高拓扑替康的稳定性并减少药物对胃肠道刺激,促进了药物的淋巴吸收,提高了药物的口服生物利用度。该制剂制备工艺简单,不使用有机溶剂,辅料具有较高的生物相容性,应用前景广阔,其对淋巴转运的促进作用对淋巴密切相关的自身免疫等疾病的靶向治疗具有重要意义。
[Abstract]:Topotecan (TPT) is a water-soluble semi-synthetic camptothecin (CPT) class of anticancer drugs, can specifically inhibit the function of topoisomerase I, hinder DNA replication. Topotecan is currently available in the form of topotecan injection freeze-dried powder injection and oral capsules. Compared with intravenous administration, oral TPT is effective, convenient, and has the characteristics of DNA replication. Low bone marrow inhibition, but because topotecan active lactone structure is affected by pH, in neutral and alkaline conditions, it is easy to hydrolyze to open ring to carboxylate configuration, thus losing activity, resulting in reduced bioavailability and antitumor activity, so the selection of appropriate drug carriers in the intestinal protection of its active configuration is particularly important. On the other hand, topotecan has strong gastrointestinal adverse reactions after oral administration. In view of the existing problems of TPT oral preparations, a new dosage form of topotecan reversed-phase lipid nanoparticles was prepared. The aim was to increase the stability of the drug in the digestive tract, reduce gastrointestinal mucosal irritation and improve the oral bioavailability of the drug. Usage. Single factor test and orthogonal test were used to screen the membrane materials, dosage and drug loading of reversed phase lipid nanoparticles. The oil solution of reversed phase lipid nanoparticles (RLBN-TPT) was prepared by freeze-drying process with medium chain triglyceride (MCT) as oil phase and the drug loading was 4mg/g. RLBN-TPT preparations were characterized by dynamic light scattering and transmission electron microscopy. The results showed that the particle size of RPL-TPT was about 60 nm and was uniform spherical. After dispersing in simulated intestinal fluid, the particle size and micro-morphology of RLBN-TPT preparations were observed. The results showed that uniform spherical lipid vesicles or emulsion droplets were formed after dispersing. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of topotecan in open-loop and closed-loop configurations. The stability of topotecan in artificial intestinal fluid was studied by measuring the release of topotecan reverse-phase lipid nanoparticles (RLBN-TPT) in simulated intestinal fluid, and the protective effect of topotecan in artificial intestinal fluid was investigated. APCELL PAK C18 MG II column (250 *4.6 mm, 5 micron), mobile phase A: 0.1% triethylamine (adjusting pH to 6.4 with glacial acetic acid) - B: acetonitrile [gradient: 0-8 min (88% A-60% A); 8-17 (60% A)], flow rate 1.0 mL/min, detection wavelength 360 nm, column temperature 55 The results show that the closed-loop configuration concentration is in the range of 0.25-5.00 UG mL-1, the concentration and peak area show a good line. Compared with topotecan aqueous solution, the stability of topotecan in reverse lipid nanoparticles in intestinal fluid was significantly enhanced, and the rate of formation of ring-opening configuration was significantly reduced (p0.05), indicating that reverse lipid nanoparticles have a certain protective effect on the form of drug-active lactones, effectively avoiding its short duration. A method for the determination of topotecan in RLBN-TPT preparations by HPLC was established. The results showed that the peak area and concentration of topotecan had a good linear relationship in the range of 0.125 micron g.mL-1 to 125 micron.mL-1, and the recovery of topotecan was between 98% and 102%. RSD is 0.67%. The method is accurate, precise, specific and sensitive, and can be used for the determination of topotecan in preparations. A method for the determination of topotecan in RLBN-TPT preparations by HPLC has been established. The methodological study shows that the method has high sensitivity, specificity and precision, and can be used for the determination of topotecan in preparations. Quality determination. The results of influencing factors showed that RLBN-TPT preparation was sensitive to humidity and heat, so high temperature should be avoided during storage, and moisture should be paid attention to. In the pharmacokinetic experiment, a LC-MS/MS method was established to determine the plasma concentration of topotecan in Wistar rats. The plasma concentration of topotecan in Wistar rats ranged from 1.0 ng/mL to 200 ng/mL (r20.997), and there was a good linear relationship between the plasma concentration and the peak area ratio. The pharmacokinetic characteristics of RLBN-TPT preparations in Wistar rats were compared with those of Topotecan capsules. The results showed that CMAx and AUC0-uuuuuuuuuuuuuuuuuuuuuuuu In order to further elucidate the absorption pathway of reversed-phase lipid nanoparticles, the in vivo absorption behaviors of the two preparations before and after blocking lymphatic absorption pathway were compared. The results showed that the AUC of reversed-phase lipid nanoparticles decreased significantly after blocking, to 30% before blocking, and 63% before blocking. Thus, the RLBN-TPT preparations were available. The results showed that the inhibition rates of RLBN-TPT group and marketed preparation were 44.08% and 16.3% (p0.05), respectively. The apoptosis of tumor cells was observed by TUNEL staining, and the apoptosis of tumor tissues was observed by HE staining. Pathological analysis of tumor tissues showed that RLBN-TPT preparation group had a larger area of tumor cell death and a larger area of tumor tissue necrosis. The results showed that the surface of intestinal mucosa was evidently edema, loss, and blood vessel congestion, indicating that the preparation had a high irritation to gastrointestinal tract, while the intestinal mucosal surface of mice treated with RLBN-TPT only occasionally showed edema, and the mucosal layer and submucosal layer, serosa of intestinal wall. In conclusion, RLBN-TPT preparation can significantly improve the stability of topotecan and reduce the gastrointestinal tract irritation and promote the development of drugs. Lymphatic absorption enhances the oral bioavailability of the drug. The preparation process is simple, does not use organic solvents, excipients have high biocompatibility and broad application prospects. Its promotion of lymphatic transport is of great significance for targeted treatment of lymph-related autoimmune diseases.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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