雄黄水分散剂型制备及抗肿瘤功效研究

发布时间：2018-07-05 12:20

本文选题：As_4S_4 + 水分散剂型　；参考：《北京协和医学院》2017年博士论文

【摘要】：背景:白血病是一类造血干细胞恶性克隆性疾病,被归为威胁人类健康的十大恶性肿瘤,约占恶性肿瘤年新发病例的3.3%,在死亡病例中占4.1%,是39岁及以下人群恶性肿瘤致死的首要原因。根据2015年美国国家癌症协会(American Cancer Society)统计,髓系白血病占白血病年新增例的一半以上;在我国,髓系白血病约占白血病年新发病例的70%。髓系白血病根据自然病程可分为急性髓系白血病(acute myeloid leukemia,AML)和慢性髓系白血病(chronicmyeloid leukemia,CML),临床上目前主要采用化疗和造血干细胞移植的治疗方案,且以化疗最为常用。在临床治疗中化疗可分为诱导缓解治疗和缓解后治疗,常用药物主要为地西他滨(decitabine)、惠环类抗生素(anthracycline antibiotics,AA)、阿糖胞苷(cytarabine,Ara-C)等。虽然多数初诊患者经治疗后可获得完全缓解,但复发比例很高,且复发后极易对原化疗药产生耐药。因此,开发可供选择的新药对白血病的治疗具有重要意义。乳腺癌是引起全球女性死亡的首要原因。国家癌症中心2013年统计数据结果显示,我国女性乳腺癌发病率为女性十大癌症之首,死亡率为9.2/10万,严重威胁女性健康。随着乳腺癌治疗技术的不断提高,患者预后得到很大改善。大多数乳腺癌患者在早期就接受手术治疗、化疗或放疗,5年存活率可达到95%,但乳腺癌的复发转移,尤其是远处转移仍然是困扰临床医生的最大难题。化疗是对转移性乳腺癌(MBC)患者最常用和最有效的治疗手段之一。MBC的系统化疗经历了非蒽环类药的单药化疗到联合化疗、蒽环类单药化疗到联合化疗、紫杉类联合化疗以及近年来的化疗联合生物治疗,有效率从20%～40%提高到60%～80%,完全缓解(CR)率从0提高到15%。远处转移所致症状严重干扰患者日常生活,是乳腺癌致死的主要原因。因此,开发可供选择的抗乳腺癌转移新药对MBC的治疗十分重要。雄黄主要成分为四硫化四砷(As_4S_4),应用于髓系白血病的治疗已有半个多世纪,也取得了一定疗效。雄黄与临床一线化疗药物相比具有如下特点:1.对加速期和终变期CML患者有效;2.与全反式维甲酸(ATRA)、伊马替尼、阿糖胞苷、三氧化二砷(As203)和其他细胞毒性化疗药等无交叉耐药,对ATRA治疗或联合HA或DA治疗方案复发患者有效;3.具有广谱性,对CML和AML均有效,也有在急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)治疗中应用的临床报道;4.无骨髓抑制和其他严重不良反应;5.口服给药,患者顺应性好,巩固治疗期患者生存质量高。近年来,雄黄在多种实体瘤研究中表现出抗肿瘤作用。文献报道As_4S_4可诱导人实体肿瘤细胞如胃癌细胞等细胞周期阻滞和凋亡,抑制肿瘤细胞在模型小鼠体内的迁移和侵袭。研究者经球磨法制备纳米雄黄,证明其对人乳腺癌细胞MCF-7具有显著杀伤作用,且具有抗小鼠原位乳腺癌作用;也有文献将药用雄黄与(β-榄香烯联合用药发现,可有效逆转阿霉素耐药MCF-7细胞的耐药性;另有文献利用精研机研磨获得纳米雄黄,通过硝酸溶解后实验证实其可以抑制乳腺癌MCF-7细胞转移侵袭。在文献研究中,雄黄表现出了抗乳腺癌作用。作为一类含砷的矿物药物,雄黄的水分散性障碍是临床应用中面临的主要挑战,水分散性障碍限制了机体对雄黄的吸收,导致雄黄的生物利用度极低。临床上为了达到治疗所需的血药浓度和药效,不得不加大给药剂量,这既加重了患者的经济负担,也导致患者顺应性差;此外,长期大剂量服用砷类化合物也存在健康风险问题。因此,改善雄黄的水分散性,从而提高雄黄的生物利用度具有重要意义和应用价值。热熔挤出技术(Hot Melt Extrusion,HME)最早应用于塑料加工行业,自上世纪80年代开始引入制药领域,用于制备药物固体分散体,能有效提高难溶性药物的水分散性和生物利用度;具有制备过程不使用有机溶剂和一体化程度高的优点,在制药工业和科研领域受到广泛关注,但HME技术在我国的药物研究中的应用相对较少,难溶性矿物中药方面更是鲜有报道。研究方法:本论文利用HME技术,将水分散性高分子与雄黄原粉(r-As_4S_4)熔融共挤出,制备雄黄水分散剂型(e-As_4S_4)。通过动态光散射(dynamic light scattering,DLS)和扫描电镜(scanning electron microscopy,SEM)等方法对 e-As_4S_4中As_4S_4颗粒的形貌和尺寸分布进行表征分析。在细胞实验中,通过CCK-8试剂盒研究e-As_4S_4对多种实体肿瘤细胞(人黑色素瘤细胞A375、人胃癌细胞MGC803、人肺癌紫杉醇耐药株细胞A549/TAXOL、人宫颈癌细胞HeLa和小鼠乳腺癌细胞4T1)和两种白血病细胞(AML细胞系HL60细胞CML细胞系K562细胞)的杀伤作用。通过AnnexinV/PI双染流式细胞分析和Hoechst 33342/PI双染形态学观察研究e-As_4S_4对HL60细胞、K562细胞和阿霉素耐药株K562细胞K562/A02的诱导凋亡作用。在体内治疗实验中,采用NOD/SCID小鼠尾静脉注射HL60细胞建立AML模型,经灌胃给药验证e-As_4S_4的体内抗白血病功效,并与r-As_4S_4和全反式维甲酸(ATRA)进行对比;采用BALB/c小鼠皮下脂肪垫接种4T1细胞的方法建立乳腺癌小鼠模型,经灌胃给药研究e-As_4S_4对乳腺癌的治疗作用和相关机理。研究结果:理化表征结果表明,e-As_4S_4通过减小As_4S_4颗粒粒径和高分子增溶共同作用增加As_4S_4的水分散性和生物利用度。细胞实验结果表明,e-As_4S_4能有效抑制多种实体肿瘤细胞(A375、MGC803、A549/TAXOL、HeLa和4T1)和两种白血病细胞(HL60和K562)的增殖,且表现出时间和浓度依赖效应。同时,与含等量As_4S_4的r-As_4S_4相比,e-As_4S_4的细胞增殖抑制作用显著增强。e-As_4S_4可以显著诱导细胞发生凋亡,说明e-As_4S_4通过诱导细胞凋亡来发挥细胞增殖抑制作用。在低浓度下,e-As_4S_4可促进K562细胞红系分化标志性蛋白血红蛋白(hemoglobin,Hb)和血型糖蛋白A(GlycophorinA,CD235a)表达上调,诱导K562细胞发生红系分化并表现出时间和剂量依赖效应。体内实验结果证实:在AML模型小鼠治疗实验中,与含等量As_4S_4的r-As_4S_4相比,e-As_4S_4有效抑制了 HL60细胞在模型小鼠体内的增殖、减轻了 AML模型小鼠脾肿大和肝、脾髓外侵袭,显著延长了 AML模型小鼠的生存期;在乳腺癌模型小鼠治疗中,e-As_4S_4有效抑制了 4T1细胞的肝脏侵袭,减少了肺转移的发生,显著延长了乳腺癌模型小鼠的生存期。经HME加工后As_4S_4的药效显著提高。结论:经HME技术制备的雄黄水分散剂型e-As_4S_4可显著提高As_4S_4的水分散性和生物利用度,有效延长AML白血病模型小鼠和乳腺癌模型小鼠生存期,具有替代临床应用药物r-As_4S_4的潜力。e-As_4S_4在提高As_4S_4白血病治疗功效的同时,可降低长期大剂量服用As_4S_4带来的潜在健康风险,可为白血病患者提供新的药物选择。e-As_4S_4也表现出未来在其他实体肿瘤如乳腺癌等的治疗中应用的潜力。
[Abstract]:Background: leukemia is a malignant clonogenic disease of hematopoietic stem cells, which is classified as the ten major malignant tumor that threatens human health. It accounts for about 3.3% of the new annual cases of malignant tumors. It accounts for 4.1% of the deaths in the cases of death. It is the leading cause of death in the population aged 39 years and below. According to the National Cancer Association of the United States (American Cancer Society) in 2015. Myeloid leukemia accounts for more than half of the new cases of leukaemia. In China, myeloid leukemia, which accounts for new cases of leukemia, can be divided into acute myeloid leukemia (acute myeloid leukemia, AML) and chronic myeloid leukemia (chronicmyeloid leukemia, CML) based on the course of natural disease, which is mainly used in clinical practice at present. The treatment and hematopoietic stem cell transplantation is the most commonly used therapy. Chemotherapy can be divided into induced remission and post remission treatment in clinical treatment. The commonly used drugs are decitabine, anthracycline antibiotics, AA, cytarabine, Ara-C, etc. Although most of the first patients are treated by treatment After the treatment, complete remission can be achieved, but the recurrence rate is very high, and it is very easy to produce drug resistance after relapse. Therefore, the development of the new drug is of great significance for the treatment of leukemia. Breast cancer is the leading cause of the death of women in the world. The National Cancer Center 2013 statistics show that the incidence of breast cancer in our country The rate is the first ten major cancer of women, the death rate is 9.2/10 million, which is a serious threat to women's health. With the continuous improvement of the treatment technology of breast cancer, the prognosis of the patients is greatly improved. Most breast cancer patients receive surgical treatment, chemotherapy or radiotherapy in the early stage, the survival rate of 5 years can reach 95%, but the recurrence of breast cancer, especially the distant metastasis Chemotherapy is one of the most difficult problems plaguing clinicians. Chemotherapy is one of the most commonly used and most effective treatments for patients with metastatic breast cancer (MBC). Systemic chemotherapy,.MBC, has experienced non anthracycline chemotherapy to combined chemotherapy, anthracycline chemotherapy to combined chemotherapy, combined chemotherapy of Taxus, and chemotherapy combined with biological treatment in recent years. The treatment has increased from 20% to 40% to 60% ~ 80%, and the rate of complete remission (CR) from 0 to 15%. distant metastasis is a major cause of death in patients. Therefore, it is important to develop a new drug for breast cancer metastasis for the treatment of MBC. The main ingredient of realgar is four vulcanization, four arsenic (As_4S_4). The treatment of myeloid leukemia has been used for more than half a century and has achieved a certain effect. Realgar has the following characteristics compared with clinical first-line chemotherapy drugs: 1. for CML patients in the accelerated and terminal phase; 2. with all trans retinoic acid (ATRA), imatinib, arabine, three oxygenation two arsenic (As203) and other cytotoxic chemotherapeutic agents Resistance, effective for ATRA treatment or combined with HA or DA therapy; 3. has broad spectrum, effective for both CML and AML, and also in the treatment of acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL); 4. no myelosuppression and other severe adverse reactions; 5. oral administration, patient compliance, consolidation therapy In the recent years, the quality of survival is high. In recent years, realgar has demonstrated anti-tumor effect in a variety of solid tumor studies. It is reported that As_4S_4 can induce cell cycle arrest and apoptosis in human tumor cells, such as gastric cancer cells, and inhibit the migration and invasion of tumor cells in the model mice. The human breast cancer cell MCF-7 has a significant killing effect, and it has the effect of anti mouse breast cancer in situ. There are also some literature that can effectively reverse the drug resistance of doxorubicin resistant MCF-7 cells by using drug realgar and (beta elemene). Inhibition of metastasis and invasion of MCF-7 cells in breast cancer. In the literature study, realgar showed anti breast cancer effect. As a class of arsenic containing mineral drugs, the realgar water dispersive disorder is the main challenge in clinical applications. Water dispersal barriers restrict the body's absorption of Realgar and lead to the extremely low bioavailability of realgar. The amount of drug concentration and efficacy needed to achieve the treatment had to be increased, which not only aggravated the economic burden of the patients, but also resulted in poor compliance of the patients. In addition, the long-term and large dose of arsenic compounds also had health risk problems. Therefore, it is of great significance to improve the water dispersibility of Realgar and to improve the bioavailability of realgar. Hot Melt Extrusion (HME), which was first applied to the plastic processing industry, was introduced into the pharmaceutical field since 80s last century, for the preparation of drug solid dispersions, which can effectively improve the water dispersibility and bioavailability of insoluble drugs; the preparation process does not use organic solvents and has a high degree of integration. It has received extensive attention in the pharmaceutical industry and scientific research fields, but the application of HME technology in the drug research in China is relatively small, and there are few reports on the insoluble mineral medicine. Research methods: This paper uses HME technology to co extrusion the water dispersive polymer and realgar raw powder (r-As_4S_4) to prepare the dispersive male yellow water dosage form (e-As _4S_4). The morphology and size distribution of As_4S_4 particles in e-As_4S_4 were characterized by dynamic light scattering (dynamic light scattering, DLS) and scanning electron microscopy (scanning electron microscopy, SEM). In cell experiments, a variety of solid tumor cells (human melanoma cells, human beings, human beings, human beings, human melanoma cells) were studied by the CCK-8 kit. Gastric cancer cell MGC803, human lung cancer paclitaxel resistant cell A549/TAXOL, human cervical cancer cell HeLa and mouse breast cancer cell 4T1) and two leukemic cells (AML cell line HL60 cell CML cell K562 cells). Through AnnexinV/PI double dye flow cytometry and Hoechst 33342/PI double staining morphological observation of e-As_4S_4 against L60 cells, K562 cells and adriamycin resistant K562 cells K562/A02 induced apoptosis. In the in vivo treatment, AML model was established by injecting HL60 cells from the tail vein of NOD/SCID mice. The anti leukemic efficacy of e-As_4S_4 in vivo was verified by intragastric administration and compared with r-As_4S_4 and all trans retinoic acid (ATRA), and BALB/c mice were used. A mouse model of breast cancer was established by subcutaneous fat pad inoculation with 4T1 cells. The therapeutic effect and mechanism of e-As_4S_4 on breast cancer were studied by intragastric administration. The results showed that e-As_4S_4 increased the water dispersibility and bioavailability of As_4S_4 by reducing the particle size of As_4S_4 particles and the combination of polymer solubilization. The experimental results showed that e-As_4S_4 could effectively inhibit the proliferation of various solid tumor cells (A375, MGC803, A549/TAXOL, HeLa and 4T1) and two leukemia cells (HL60 and K562), and showed time and concentration dependent effects. At the same time, the inhibitory effect of e-As_4S_4 cell proliferation was significantly enhanced by the inhibitory effect of e-As_4S_4 cell proliferation. In order to induce apoptosis, e-As_4S_4 can induce cell proliferation inhibition by inducing apoptosis. Under low concentration, e-As_4S_4 can increase the expression of hemoglobin, Hb, and blood group A (GlycophorinA, CD235a) in K562 cells, and induce erythroid differentiation in K562 cells. The results of time and dose dependence showed that in the experiment of AML model mice, compared with r-As_4S_4 containing equal amount of As_4S_4, e-As_4S_4 effectively inhibited the proliferation of HL60 cells in the model mice, alleviated the splenomegaly of the AML model mice and the invasion of the liver and spleen, and significantly extended the survival time of the AML model mice. In the treatment of breast cancer model mice, e-As_4S_4 effectively inhibits the invasion of 4T1 cells and reduces the occurrence of lung metastasis, significantly prolongs the survival period of the breast cancer model mice. The effect of As_4S_4 after HME processing is significantly improved. Conclusion: the dispersion of the male yellow water dispersible dosage form of HME technology can significantly improve the water dispersion of the As_4S_4. Sex and bioavailability can effectively prolong the survival period of AML leukemia model mice and breast cancer model mice. The potential of replacing clinical drug r-As_4S_4,.E-As_4S_4, can improve the therapeutic efficacy of As_4S_4 leukemia and reduce the potential health risk of long term large dose of As_4S_4, which can provide new drugs for leukemia patients. .e-As_4S_4 also shows potential for future applications in the treatment of other solid tumors such as breast cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284;R285

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