ME论文MEHPβCD安全性评价药代动力学药效学维拉帕米

1、【关键词】2-ME HP-CD 安全性评价 药代动力学 药效学 维拉帕米【英文关键词】2-ME HP-CD safety evaluation pharmacokinetics pharmacodynamics verapamil2-ME论文：2-甲氧基雌二醇/羟丙基-环糊精注射剂的研究【中文摘要】2-甲氧基雌二醇(2-ME)是一种雌激素的代谢产物,药理活性研究表明2-ME可以对抗血管生成并抑制肿瘤细胞增殖,具有抗肿瘤的作用。2-ME是一种水难溶性药物,在水中的溶解度非常低(约0.2gml-1),其口服吸收效率低下,且无规律。增加药物的溶解度,选择一种合适的剂型,通过一定的给药途径作用于机体,

2、以便于提高临床用药的安全性和疗效。因此本研究筛选出安全性高,对2-ME增溶效果好的羟丙基-环糊精(HP-CD)为辅料,制备成2-ME/HP-CD注射剂。首先进行了2-ME包合物的处方及制备工艺研究,对比了HP-CD和磺丁基-p-环糊精(SBE-CD)对2-ME的增溶作用,在综合考虑安全性及成本后,选择HP-CD为包合材料,得到优化制备工艺为：采用50%的HP-CD的水溶液超声法包合2-ME原料药,超声功率为450W,包合时间为5min。最后2-ME的溶解度达到33.6mmoll-1,即10.147mgml-1,在水中溶解度提高4.6万倍,得到有临床应用前景的2-ME/HP-CD注射剂。对2-M

3、E/HP-CD注射剂进行初步安全性评价,进行了急性毒性、刺激性和溶血性研究,结果显示2-ME/HP-CD注射剂无肾毒性、溶血性和刺激性,注射给药的安全性优于2-ME对照液组。考察了2-ME/HP-CD注射剂在小鼠体内的药物代谢动力学,结果表明2-ME/HP-CD注射剂在小鼠体内成二室模型,分布半衰期(t1/2)为(3.0840.12)min,消除半衰期(t1/2)为(10.722.31) min,AUC0240min为(175.425)gml-1min,2-ME在体内分布及消除较快。高剂量维拉帕米和2-ME联合给药后,维拉帕米增加了2-ME的AUC和MRT,而清除率(CL)下降。说明高剂量维拉

4、帕米能对2-ME/HP-CD注射剂体内药物动力学特征产生影响。2-ME/HP-CD注射剂在家犬体内采用静脉滴注的方式给药,研究其药物代谢动力学特征,结果显示2-ME/HP-CD注射剂在家犬体内呈二室模型。分布半衰期(t1/2)为(8.7742.02)min,消除半衰期(t1/2p)为(18.362.04)min, AUC0120min为(131.8623)gml-1min,和在小鼠体内的药动学参数比较,t1/2和t1/2都有所延长,2-ME/HP-CD注射剂在家犬和小鼠体内的药动学特征有差异,人体的药动学特征可能和家犬的药动学特征更为类似,家犬的药动学特征可以为人体临床试验提供参考。如何长期维

5、持2-ME在体内的有效血药浓度是2-ME/HP-CD注射剂今后研究的关键。建立了BALB/c近交系小鼠乳腺癌移植模型,以肿瘤体积变化、抑瘤率的计算和肺脏转移情况为检测指标,初步研究了2-ME/HP-CD注射剂的体内抗肿瘤效果。结果显示2-ME/HP-CD注射剂对小鼠乳腺癌有抑制作用,抑制率为20.9%-36.7%,表明2-ME体内肿瘤抑制效率差于文献报道的体外细胞实验结果,其原因有待深入研究。以鼠乳腺癌4T1、人肺腺癌细胞株SPC-A1、人前列腺癌PC-3为研究对象,通过体外细胞实验,采用SRB法考察2-ME和2-ME/HP-p-CD注射剂对三种肿瘤细胞的生长抑制作用,同时考察维拉帕米(ver

6、apamil)对三种肿瘤细胞的生长抑制作用,利用IC50的变化和协同指数探讨对2-ME抗癌作用的影响。证实2-ME/HP-CD注射剂和2-ME具有同样的体外抗肿瘤作用,钙拮抗剂维拉帕米能增强2-ME和2-ME/HP-CD注射剂对肿瘤细胞的抗癌活性,其增效机制可能与影响P-糖蛋白(P-gp)的外排功能有关,也可能和其他分子生物机制有关,有待于从分子生物水平继续深入研究。【英文摘要】2-methoxyestradiol (2-ME) is a metabolite of estrogen, and pharmacological studies have shown efficacy agains

7、t angiogenesis, inhibition toward tumor cell proliferation and anti-tumor effects.2-ME is a drug with very low solubility in water (about0.2gml-1), low oral absorption efficiency and no regular pattern, so it is crucial to select a suitable dosage form and route of administration to increase the sol

8、ubility of the drug and improve the safety of clinical medicine in order to achieve better clinical efficacy. Therefore, cyclodextrin HP-CD was screened out in this study to be safe and with good solubilization effect toward2-ME, then was used to prepare the2-ME/HP-CD injection.First, the design and

9、 preparation of2-ME injection were studied, in which HP-CD and SBE-CD were tested and compared for their effects to enhance the solubility of2-ME. And considering the safety and cost, HP-CD was selected as the clathrate agent. The optimized preparation procedures were as follows:50%HP-P-CD aqueous s

10、olution was used for packaging2-ME, the ultrasonic power was450W and the inclusion time was5min. Under these conditions, the solubility of2-ME was increased to33.6mmoll-1, which is10.147mgml-1,4.6times more than the solubility in water, thus the clinically promising2-ME/HP-CD injection was prepared.

11、To evaluate the safety of the2-ME/HP-CD injection, acute toxicity, stimulation and hemolytic experiments were conducted, and it had been observed that the injection of2-ME/HP-CD was with no renal toxicity, hemolytic and stimulation, and the safety of this injection was superior to2-ME control soluti

12、on group.Pharmacokinetics of2-ME/HP-CD injection were investigated in mice, indicating that2-ME/HP-CD injection was established into a two-compartment model in mice, with the distribution half-life t1/2 and elimination half-life ti/2p (3.080.12) min and (10.722.31) min, respectively, the AUC0240min

13、(175.4 23)gml-1min, and was found that the in vivo distribution and elimination of2-ME declined rapidly. When2-ME was combined with Verapamil, its AUC and MRT showed an increasing trend, and CL showed a downward trend, indicating that Verapamil had impacts on the in vivo pharmacokinetics of2-ME/HP-C

14、D injection.2-ME/HP-CD injection was used for intravenous administration in dogs, and pharmacokinetic characteristics was studied. The injection was proved to be two compartment model, with the half-life (t1/2)(8.772.02) min, elimination half-life (t1/2)(18.362.04) min and AUC0120min (131.8623)gml-1

15、min. Comparing to the kinetic parameters in mice, t1/2 and t1/2 were both lengthened to some extent, but there were significant differences in the kinetic parameters between dogs and mice. And as the kinetic parameters of dogs were more similar to human, thus they could be used as references for hum

16、an clinical researches. And how to maintain the effective blood concentration of2-ME in vivo became the key factor for future studies of2-ME/HP-CD injection.In vivo pharmacodynamics of2-ME/HP-CD injection were applied for a preliminary study, and BALB/c inbred mice was transplanted breast cancer cel

17、ls to investigate the inhibition rate, tumor volume change and lung metastases, which proved2-ME/HP-P-CD injection had a certain therapeutic effect toward murine breast cancer with a inhibition rate of20.9%-36.7%, indicating that the in vitro inhibition effect was superior to the in vivo result, and

18、 the reasons needed to be further studied.As for vitro experiments, mouse breast cancer4T1, human lung adenocarcinoma cell line SPC-A1and human prostate cancer PC-3cells were used as study objectives, SRB staining assay was applied to examine the tumor cell growth inhibition effect of2-ME and2-ME/HP

19、-CD injection, and when combined with verapamil, changes of IC50and the synergy index were used to explore the anticancer effects of2-ME. The injection of2-ME/HP-CD and2-ME were confirmed to have the same anti-tumor effect, and the calcium antagonist verapamil was also found to improve the anticance

20、r activity of2-ME and2-ME/HP-CD injection, of which the mechanism may be related to the efflux function of P-glycoprotein (P-gp) or other metabolisms needed to be further studied from the aspects of molecular biology.【目录】2-甲氧基雌二醇/羟丙基-环糊精注射剂的研究 摘要 4-6 Abstract 6-8 目录 9-14 前言 14-16 第一章 HP-CD及其包合物的应用进展

21、 16-22 1 -CD的特性 16-17 2 HP-CD的特性 17-18 2.1 增加药物的稳定性和溶解度 18 2.2 减少药物的毒副作用 18 2.3 促进药物的释放,提高生物利用度 18 3 影响包合物形成的因素 18-19 4 包合效果的评价指标 19-21 5 结论 21-22 第二章 2-ME/HP-CD注射剂的处方工艺研究 22-34 1 实验材料 22-23 1.1 仪器设备 22 1.2 材料和试剂 22-23 2 实验方法 23-25 2.1 2-ME测定方法的建立 23-24 2.2 处方工艺的研究 24-25 3 实验结果 25-32 3.1 2-ME测定方法的建立

22、 25-28 3.2 制备工艺的研究 28-32 4 讨论 32-33 5 结论 33-34 第三章 2-ME/HP-CD注射剂的初步安全性评价 34-43 1 实验材料 34-35 1.1 仪器设备 34 1.2 材料和试剂 34-35 1.3 实验动物 35 2 实验方法 35-38 2.1 制剂的配制 35 2.2 急性毒性实验 35-36 2.3 血管刺激性实验 36 2.4 溶血实验 36-38 3. 实验结果 38-42 3.1 急性毒性实验 38-40 3.2 血管刺激性实验结果 40 3.3 溶血性实验结果 40-42 4 讨论 42 5 结论 42-43 第四章 2-ME/H

23、P-CD注射剂在小鼠体内的药代动力学研究 43-55 1 实验材料 43-44 1.1 仪器和设备 43 1.2 材料和试剂 43-44 1.3 实验动物 44 2 实验方法 44-46 2.1 色谱条件 44 2.2 血浆样品处理 44 2.3 方法学考察 44-45 2.4 实验方案 45-46 2.5 数据处理 46 2.6 统计学分析 46 3 实验结果 46-53 3.1 方法专属性 46-47 3.2 标准曲线的制备 47-48 3.3 精密度试验 48 3.4 方法回收率 48-49 3.5 稳定性试验 49 3.6 血浆中药物浓度 49-50 3.7 药时曲线 50-52 3.

24、8 药动学参数 52-53 4 讨论 53-54 5 结论 54-55 第五章 2-ME/HP-CD注射剂在家犬体内的药代动力学研究 55-66 1 实验材料 55-56 1.1 仪器设备 55-56 1.2 材料和试剂 56 1.3 实验动物 56 2 实验方法 56-58 2.1 色谱条件 56 2.2 血浆样品处理 56 2.3 方法学考察 56-57 2.4 实验方案 57-58 3. 实验结果 58-65 3.1 方法专属性 58-59 3.2 标准曲线的制备 59-60 3.3 精密度试验 60 3.4 方法回收率 60-61 3.5 稳定性试验 61 3.6 给药剂量及样品制备 61-62 3.7 血浆中药物浓度的测定 62 3.8 药时曲线 62-63 3.9 药动学参数 63-65 4 讨论 65 5 结论 65-66 第六章 2-ME/HP-CD注射剂的体内药效学的初步研究 66-74 1 实验材料 66-67 1.1 仪器设备 66 1.2 材料