本文選題：As_4S_4 + 水分散劑型　； 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：背景:白血病是一類造血干細胞惡性克隆性疾病,被歸為威脅人類健康的十大惡性腫瘤,約占惡性腫瘤年新發(fā)病例的3.3%,在死亡病例中占4.1%,是39歲及以下人群惡性腫瘤致死的首要原因。根據(jù)2015年美國國家癌癥協(xié)會(American Cancer Society)統(tǒng)計,髓系白血病占白血病年新增例的一半以上;在我國,髓系白血病約占白血病年新發(fā)病例的70%。髓系白血病根據(jù)自然病程可分為急性髓系白血病(acute myeloid leukemia,AML)和慢性髓系白血病(chronicmyeloid leukemia,CML),臨床上目前主要采用化療和造血干細胞移植的治療方案,且以化療最為常用。在臨床治療中化療可分為誘導緩解治療和緩解后治療,常用藥物主要為地西他濱(decitabine)、惠環(huán)類抗生素(anthracycline antibiotics,AA)、阿糖胞苷(cytarabine,Ara-C)等。雖然多數(shù)初診患者經治療后可獲得完全緩解,但復發(fā)比例很高,且復發(fā)后極易對原化療藥產生耐藥。因此,開發(fā)可供選擇的新藥對白血病的治療具有重要意義。乳腺癌是引起全球女性死亡的首要原因。國家癌癥中心2013年統(tǒng)計數(shù)據(jù)結果顯示,我國女性乳腺癌發(fā)病率為女性十大癌癥之首,死亡率為9.2/10萬,嚴重威脅女性健康。隨著乳腺癌治療技術的不斷提高,患者預后得到很大改善。大多數(shù)乳腺癌患者在早期就接受手術治療、化療或放療,5年存活率可達到95%,但乳腺癌的復發(fā)轉移,尤其是遠處轉移仍然是困擾臨床醫(yī)生的最大難題�；熓菍D移性乳腺癌(MBC)患者最常用和最有效的治療手段之一。MBC的系統(tǒng)化療經歷了非蒽環(huán)類藥的單藥化療到聯(lián)合化療、蒽環(huán)類單藥化療到聯(lián)合化療、紫杉類聯(lián)合化療以及近年來的化療聯(lián)合生物治療,有效率從20%～40%提高到60%～80%,完全緩解(CR)率從0提高到15%。遠處轉移所致癥狀嚴重干擾患者日常生活,是乳腺癌致死的主要原因。因此,開發(fā)可供選擇的抗乳腺癌轉移新藥對MBC的治療十分重要。雄黃主要成分為四硫化四砷(As_4S_4),應用于髓系白血病的治療已有半個多世紀,也取得了一定療效。雄黃與臨床一線化療藥物相比具有如下特點:1.對加速期和終變期CML患者有效;2.與全反式維甲酸(ATRA)、伊馬替尼、阿糖胞苷、三氧化二砷(As203)和其他細胞毒性化療藥等無交叉耐藥,對ATRA治療或聯(lián)合HA或DA治療方案復發(fā)患者有效;3.具有廣譜性,對CML和AML均有效,也有在急性淋巴細胞白血病(acute lymphoblastic leukemia,ALL)治療中應用的臨床報道;4.無骨髓抑制和其他嚴重不良反應;5.口服給藥,患者順應性好,鞏固治療期患者生存質量高。近年來,雄黃在多種實體瘤研究中表現(xiàn)出抗腫瘤作用。文獻報道As_4S_4可誘導人實體腫瘤細胞如胃癌細胞等細胞周期阻滯和凋亡,抑制腫瘤細胞在模型小鼠體內的遷移和侵襲。研究者經球磨法制備納米雄黃,證明其對人乳腺癌細胞MCF-7具有顯著殺傷作用,且具有抗小鼠原位乳腺癌作用;也有文獻將藥用雄黃與(β-欖香烯聯(lián)合用藥發(fā)現(xiàn),可有效逆轉阿霉素耐藥MCF-7細胞的耐藥性;另有文獻利用精研機研磨獲得納米雄黃,通過硝酸溶解后實驗證實其可以抑制乳腺癌MCF-7細胞轉移侵襲。在文獻研究中,雄黃表現(xiàn)出了抗乳腺癌作用。作為一類含砷的礦物藥物,雄黃的水分散性障礙是臨床應用中面臨的主要挑戰(zhàn),水分散性障礙限制了機體對雄黃的吸收,導致雄黃的生物利用度極低。臨床上為了達到治療所需的血藥濃度和藥效,不得不加大給藥劑量,這既加重了患者的經濟負擔,也導致患者順應性差;此外,長期大劑量服用砷類化合物也存在健康風險問題。因此,改善雄黃的水分散性,從而提高雄黃的生物利用度具有重要意義和應用價值。熱熔擠出技術(Hot Melt Extrusion,HME)最早應用于塑料加工行業(yè),自上世紀80年代開始引入制藥領域,用于制備藥物固體分散體,能有效提高難溶性藥物的水分散性和生物利用度;具有制備過程不使用有機溶劑和一體化程度高的優(yōu)點,在制藥工業(yè)和科研領域受到廣泛關注,但HME技術在我國的藥物研究中的應用相對較少,難溶性礦物中藥方面更是鮮有報道。研究方法:本論文利用HME技術,將水分散性高分子與雄黃原粉(r-As_4S_4)熔融共擠出,制備雄黃水分散劑型(e-As_4S_4)。通過動態(tài)光散射(dynamic light scattering,DLS)和掃描電鏡(scanning electron microscopy,SEM)等方法對 e-As_4S_4中As_4S_4顆粒的形貌和尺寸分布進行表征分析。在細胞實驗中,通過CCK-8試劑盒研究e-As_4S_4對多種實體腫瘤細胞(人黑色素瘤細胞A375、人胃癌細胞MGC803、人肺癌紫杉醇耐藥株細胞A549/TAXOL、人宮頸癌細胞HeLa和小鼠乳腺癌細胞4T1)和兩種白血病細胞(AML細胞系HL60細胞CML細胞系K562細胞)的殺傷作用。通過AnnexinV/PI雙染流式細胞分析和Hoechst 33342/PI雙染形態(tài)學觀察研究e-As_4S_4對HL60細胞、K562細胞和阿霉素耐藥株K562細胞K562/A02的誘導凋亡作用。在體內治療實驗中,采用NOD/SCID小鼠尾靜脈注射HL60細胞建立AML模型,經灌胃給藥驗證e-As_4S_4的體內抗白血病功效,并與r-As_4S_4和全反式維甲酸(ATRA)進行對比;采用BALB/c小鼠皮下脂肪墊接種4T1細胞的方法建立乳腺癌小鼠模型,經灌胃給藥研究e-As_4S_4對乳腺癌的治療作用和相關機理。研究結果:理化表征結果表明,e-As_4S_4通過減小As_4S_4顆粒粒徑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。細胞實驗結果表明,e-As_4S_4能有效抑制多種實體腫瘤細胞(A375、MGC803、A549/TAXOL、HeLa和4T1)和兩種白血病細胞(HL60和K562)的增殖,且表現(xiàn)出時間和濃度依賴效應。同時,與含等量As_4S_4的r-As_4S_4相比,e-As_4S_4的細胞增殖抑制作用顯著增強。e-As_4S_4可以顯著誘導細胞發(fā)生凋亡,說明e-As_4S_4通過誘導細胞凋亡來發(fā)揮細胞增殖抑制作用。在低濃度下,e-As_4S_4可促進K562細胞紅系分化標志性蛋白血紅蛋白(hemoglobin,Hb)和血型糖蛋白A(GlycophorinA,CD235a)表達上調,誘導K562細胞發(fā)生紅系分化并表現(xiàn)出時間和劑量依賴效應。體內實驗結果證實:在AML模型小鼠治療實驗中,與含等量As_4S_4的r-As_4S_4相比,e-As_4S_4有效抑制了 HL60細胞在模型小鼠體內的增殖、減輕了 AML模型小鼠脾腫大和肝、脾髓外侵襲,顯著延長了 AML模型小鼠的生存期;在乳腺癌模型小鼠治療中,e-As_4S_4有效抑制了 4T1細胞的肝臟侵襲,減少了肺轉移的發(fā)生,顯著延長了乳腺癌模型小鼠的生存期。經HME加工后As_4S_4的藥效顯著提高。結論:經HME技術制備的雄黃水分散劑型e-As_4S_4可顯著提高As_4S_4的水分散性和生物利用度,有效延長AML白血病模型小鼠和乳腺癌模型小鼠生存期,具有替代臨床應用藥物r-As_4S_4的潛力。e-As_4S_4在提高As_4S_4白血病治療功效的同時,可降低長期大劑量服用As_4S_4帶來的潛在健康風險,可為白血病患者提供新的藥物選擇。e-As_4S_4也表現(xiàn)出未來在其他實體腫瘤如乳腺癌等的治療中應用的潛力。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R284;R285

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5 葛廣哲;樹，

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