WO2020173475A1 - 一种双药物共递送系统及其制备方法与应用 - Google Patents
一种双药物共递送系统及其制备方法与应用 Download PDFInfo
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- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
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- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to the field of medical technology, in particular to a dual-drug co-delivery system, its preparation method and its application in the preparation of anti-tumor drugs. Background technique
- Chemotherapy is a treatment that uses chemical drugs to prevent the proliferation, infiltration, and metastasis of cancer cells, and eventually kills cancer cells. It is the main treatment for most solid tumors and hematological tumors.
- the chemicals used in chemotherapy are called chemotherapeutics. Due to the emergence of chemotherapeutic drug resistance and dose-dependent side effects, a single drug can no longer meet clinical needs, so a combination of multiple chemotherapeutic drugs is used to solve this problem. Although the combined use of multiple chemotherapeutic drugs can reduce the toxic side effects and the possibility of drug resistance caused by a single drug, there are problems such as the number of administrations and the difficulty of determining the dose distribution plan.
- a chemotherapeutic drug co-delivery system is an effective method to solve the above-mentioned problems.
- the co-delivery system encapsulates two or more chemotherapeutic drugs in a carrier, and uses the synergy between the drugs to enhance their killing effect on tumor cells.
- the advantages of drug co-delivery system are: 1) Two or more chemotherapeutic drugs exert a synergistic effect to produce stronger anti-tumor effect in a smaller dose, reduce toxic side effects, and avoid resistance The production of drug properties; 2) The time and space of drug release can be controlled to coordinate the pharmacokinetics and pharmacodynamics of different drugs; 3) The number of administrations can be reduced, and patient compliance can be improved; 4) A single dose can avoid Uncertainty of dose distribution when multiple drugs are administered.
- Drug co-delivery systems mainly use liposomes, polymer micelles, solid lipid nanoparticles, and inorganic nanomaterials such as mesoporous cassia nanoparticles, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, etc., as drug co-delivery carriers.
- drugs delivered such as the delivery of two small-molecule chemotherapeutics that act on different targets to maximize tumor cell killing effect and reduce the possibility of drug resistance; another example is the co-delivery of chemotherapeutics and siRNA, Using RNA interference technology to silence drug-resistant genes and interfere with cell signaling pathways to overcome drug resistance; another example is the co-delivery of chemotherapeutic drugs and imaging agents to achieve targeted drug delivery and non-invasive tumor imaging.
- CPX-351 The delivery system (CPX-351) has entered Phase III clinical trials in November 2014. Tests have shown that CPX-351 is effective for elderly patients with newly-treated or relapsed acute myeloid leukemia (AML). It can be used as a remission induction therapy for patients who have failed standard induction therapy, or as a consolidation therapy for patients who have received stem cells. Patients who have been transplanted or have not undergone stem cell transplantation.
- AML relapsed acute myeloid leukemia
- lipid materials it not only needs to use a variety of lipid materials, but also needs to be prepared in a copper gluconate/triethylamine buffer system to make the drugs cytarabine and daunorubicin It forms a complex with the copper ions in the buffer system (containing organic solvents) in a certain molar ratio to ensure the retention time of the drug in the liposome.
- the liposome co-delivery system involves the use of organic solvents, and therefore has the problem of high risk of toxic and side effects.
- the purpose of the present invention is to address the technical defects existing in the prior art.
- it provides a dual-drug co-delivery system that has strong anti-tumor effects, low toxic and side effects, and can be used for relapsed and refractory leukemia.
- the dual-drug co-delivery system is made of raw materials including a block copolymer, homoharringtonine and adriamycin hydrochloride; the block copolymer is selected from polyethylene caprolactam-polyvinyl acetate-polyethylene glycol (Soluplus), methoxy polyethylene glycol-polycaprolactone (mPEG-PCL), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE_PEG 2 ”), polyoxyethylene polyoxypropylene ether One or more of block copolymers and polylactic acid-glycolic acid copolymers (PLGA), preferably Soluplus.
- a block copolymer is selected from polyethylene caprolactam-polyvinyl acetate-polyethylene glycol (Soluplus), methoxy polyethylene glycol-polycaprolactone (mPEG-PCL), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (
- the ratio of homoharringtonine, adriamycin hydrochloride, and block copolymer in parts by weight is (1-10): (1-40): (4-200); preferably (1-5): (3-15): (45-200).
- the average hydration diameter of the system is 20-200 nm, preferably 60-180 nm.
- the molecular weight ratio of polyethylene caprolactam, polyvinyl acetate and polyethylene glycol in the block copolymer Soluplus is 57:30:13.
- the present invention provides a method for preparing the aforementioned dual-drug co-delivery system, which includes preparing micelles loaded with homoharringtonine and loading doxorubicin.
- the micelles loaded with homoharringtonine are obtained by mixing and stirring homoharringtonine, a block copolymer and a phosphate buffer.
- the specific preparation process includes the following steps:
- the block copolymer is dissolved in a phosphate buffer to obtain a clear solution D;
- (2) Mix the clear solution D obtained in step (1) with the micelles (micelle C) loaded with homoharringtonine, and add an aqueous solution of adriamycin hydrochloride (preferably, adriamycin) under heating and stirring.
- adriamycin hydrochloride preferably, adriamycin
- the concentration of doxorubicin hydrochloride in the hydrochloride aqueous solution is 3-8 mg/mL) to obtain liquid E;
- the heating is heated to 25-70 ° C.
- the stirring speed is 100-1000 r/min.
- the present invention provides the application of the aforementioned dual-drug co-delivery system in the preparation of drugs for inhibiting tumor growth-related diseases or drugs for treating leukemia.
- the tumor growth-related diseases are tumors for which the combination of homoharringtonine and anthracycline antibiotics are effective, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, cervical cancer and the like.
- the acute myeloid leukemia is a relapsed, refractory AML1-ET0 acute myeloid leukemia.
- the block copolymer as a carrier in the dual drug co-delivery system provided by the present invention is an amphiphilic high molecular polymer, which can form a hydrophilic shell-hydrophobic core structure in solution: amphiphilic block copolymer
- the micelles are formed in the phosphate buffer, and the two drugs homoharringtonine (hydrophobic) and adriamycin hydrochloride (hydrophilic) are assembled into the hydrophobic core of the micelle through a two-step assembly method.
- the formation of a dual drug co-delivery system can achieve combined administration.
- the dual-drug co-delivery system of the present invention can also exert a different therapeutic effect on tumors by adjusting the ratio of the two drugs in the micelles.
- the dual drug co-delivery system of the present invention has a stronger killing effect on a variety of hematological tumors and solid tumor cells.
- the tumor cells include acute myeloid leukemia cells, chronic myeloid leukemia cells, cervical cancer cells, and breast cancer cells.
- the system can also be used as a drug for the treatment of relapsed and refractory AML1-ET0 acute myeloid leukemia, with excellent effects; especially: in leukemia cell experiments, compared with the combined use of adriamycin and homoharringtonine
- the dual-drug co-delivery system of the present invention can more effectively inhibit the proliferation of myeloid leukemia cells; in animal experiments, it has a better therapeutic effect on acute myeloid leukemia AML1-ET0 mice, and is effective in prolonging the survival period of mice. It can also reduce side effects.
- the dual drug co-delivery system of the present invention can be used in the treatment of tumor growth inhibition and leukemia.
- the dual-drug co-delivery system of the present invention does not use any organic solvents in the preparation process, has high safety, and meets the requirements of clinical medication; the preparation process is simple and suitable for industrial production. Description of the drawings
- Figure 1 shows the particle size distribution curve of the dual-drug co-delivery system of the present invention in phosphate buffer
- Figure 2 shows a photograph of the phosphate buffer of the dual-drug co-delivery system of the present invention
- Figure 3 is a bar graph showing the effect of the dual-drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 8:1) on the viability of HL60/A cells;
- Figure 4 is a graph showing the effect of the dual-drug co-delivery system (the weight ratio of DOX and HHT is 8:1) on U937R cell viability of the present invention
- Figure 5 is a graph showing the effect of the dual-drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 15:1) on the viability of K562 cells;
- Figure 6 is a graph showing the effect of the dual-drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 6:1) on HeLa cell viability;
- Figure 7 is a graph showing the effect of the dual-drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 4: 1) on the vitality of MCF7 cells;
- Fig. 8 is a graph showing the prolonged survival period of leukemia mice by the dual drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 9:1);
- Figure 9 is a bar graph showing that the dual drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 9:1) reduces the number of leukemia cells in mice;
- Figure 10- Figure 11 shows a bar graph of the dual-drug co-delivery system of the present invention (the weight ratio of DOX and HHT is 9:1) reducing toxic and side effects. Concrete lungs
- Homoharringtonine is a biologically active ingredient extracted from plants of the genus Cephalotaxus. It can inhibit the proliferation of leukemia cells and induce apoptosis.
- Doxorubicin hydrochloride is a broad-spectrum anti-tumor drug that exhibits anti-tumor effects by destroying DNA structure.
- Homoharringtonine and doxorubicin (DOX) hydrochloride are hydrophobic and hydrophilic drugs, respectively, and can be used clinically for the combined treatment of acute myeloid leukemia.
- DOX doxorubicin
- the liposome co-delivery system has a complicated preparation process and requires the use of a variety of lipid materials and the addition of organic solvent methanol to make homoharringtonine first encapsulated in liposomes, and then further encapsulate adriamycin hydrochloride by electrostatic action salt.
- organic solvent methanol in the liposome co-delivery system will bring toxic and side effects.
- the liposome co-delivery system is designed to inhibit the growth of solid tumor HeLa cells.
- the liposomes are relatively stable and usually used The high permeability and retention effect of solid tumors accumulate and release drugs in tumor tissues, but cannot effectively kill leukemia cells in the circulating blood.
- the liposome co-delivery system accumulates in the bone marrow, it is difficult to alleviate the inhibitory effect of the drug itself on the growth of bone marrow cells, and the treatment of acute myeloid leukemia requires the participation of bone marrow cells, so the liposome co-delivery system is uncomfortable. It is used in the treatment of acute myeloid leukemia.
- Polymer micelles are a type of co-delivery drug carriers that have received widespread attention. Because their general size is tens of nanometers, they are also called nanomicelles. They are composed of amphiphilic polymers at a suitable concentration and temperature. It is formed spontaneously in the solvent system.
- the drug is encapsulated in the hydrophobic core of the micelle through the hydrophilic-hydrophobic interaction. Therefore, it is usually used as a co-delivery drug carrier for two hydrophobic drugs; its advantage is that it does not affect the activity of the drug itself and is significantly hydrophobic
- the drug is solubilized, the preparation method is simple, and no organic solvent is required.
- conventionally designed polymer micelles are difficult to apply to hydrophilic drugs, and it is especially difficult to load both hydrophobic and hydrophilic drugs into the hydrophobic core of the micelle at the same time.
- doxorubicin hydrochloride is water-soluble and homoharringtonine is hydrophobic, it is generally understood that it is difficult to use polymer micelles as the co-delivery drug carrier for the above-mentioned dual drugs.
- Different drugs are encapsulated in the hydrophobic core of the micelle.
- two drugs with different hydrophilic and hydrophobic properties are encapsulated into polymer micelles without using organic solvents, and the polymer micelles are used to form a co-delivery system.
- the dual-drug co-delivery system of the present invention uses amphiphilic block copolymers as the carrier, which can form a hydrophilic shell-hydrophobic core structure micelle in an aqueous solution; the homoharringtonine is first encapsulated by hydrophobic interaction
- doxorubicin hydrochloride is assembled into the hydrophobic core of the micelle to form the dual-drug co-delivery system of the present invention, that is, doxorubicin hydrochloride is combined with the amphiphilic block copolymer. Co-delivery of salt and homoharringtonine.
- the block copolymer selected in the dual drug co-delivery system of the present invention is selected from polyethylene caprolactam-polyvinyl acetate-polyethylene glycol (for example, a product under the trade name of Soluplus), methoxy polyethylene glycol-polyhexyl Lactone (mPEG-PCL), Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE_PEG 2 ”), polyoxyethylene polyoxypropylene ether block copolymer, polylactic acid-glycolic acid copolymer (PLGA ), preferably Soluplus.
- Available block copolymers are commercially available, with an average molecular weight of 1,000 to 150,000.
- the invention also provides the application of the dual-drug co-delivery system formed by adriamycin hydrochloride and homoharringtonine in the preparation of tumor treatment drugs.
- the dual-drug co-delivery system of the present invention is particularly useful as a drug for the treatment of relapsed and refractory acute myeloid leukemia, including AML1-ET0 acute myeloid leukemia, which is characterized by t(8; 21) (q22; q22) Chromosome translocation, which is common in M2 in FAB typing, is present in approximately 12% to 15% of AML patients; the AML 1 gene on chromosome 21 q22 and the ET0 gene on chromosome 8 q22 are rearranged to form an AML1-ET0 fusion gene.
- AML1-ET0 acute myeloid leukemia which is characterized by t(8; 21) (q22; q22)
- Chromosome translocation which is common in M2 in FAB typing, is present in approximately 12% to 15% of AML patients; the AML 1 gene on chromosome 21 q22 and the ET0 gene on chromosome 8 q22 are rearranged to form
- the cell lines used in the following examples were purchased from the National Experimental Cell Resource Sharing Platform. Unless otherwise specified, the phosphate buffers used in the following examples are all 1X PBS solutions (pH 7.4); the solvents of the aqueous solutions used are all ultrapure water.
- Example 1 Preparation of micelles loaded with harringtonine
- PLGA 10 KD-40 KD, the content ratio of polylactic acid and glycolic acid is 50:50
- Soluplus commercially available from BASF, Germany, molecular weight 120 KD, polyethylene caprolactam , The molecular weight ratio of polyvinyl acetate and polyethylene glycol is 57: 30: 13;
- the average hydration diameter of micelle F of Example 1 is 80 nm (as shown in Figure 1), the drug loading of doxorubicin is 5%, and the encapsulation efficiency is 75%; the performance parameters of other examples are shown in Table 2 and Tables 3.
- Table 3 Dual-drug co-delivery system with different ratios of DOX and HHT
- Experiment 1 The inhibitory effect of a single free drug, a combination of two free drugs and a dual drug co-delivery system on the proliferation of a variety of hematological tumors and solid tumor cells in vitro
- HL60/A cells HL60 cells resistant to adriamycin
- the specific implementation method is as follows: In Corning 96-well U-shaped plate, each well uses 100
- the experimental group includes: 1) Single HHT group (H): HHT is dissolved with dimethyl sulfoxide (DMSO) to obtain 10 mg/mL mother liquor; 2) Single D0X group (D): Adriamycin hydrochloride is dissolved in water to obtain 5 mg/mL mother liquor; 3) Free drug combination group (DH): mother liquor 1) and 2) were diluted with culture medium and mixed, the weight ratio of D0X to HHT was 8:1; 4)
- the dual-drug co-delivery system of the present invention has similar effects on U937R (cytarabine-resistant strain, a drug-resistant cell line of acute myeloid leukemia screened by the inventor in the laboratory), K562, HeLa and MCF7 cells.
- U937R cytarabine-resistant strain, a drug-resistant cell line of acute myeloid leukemia screened by the inventor in the laboratory
- K562, HeLa and MCF7 cells The culture of each cell The conditions are shown in Table 4, and the results are shown in Figure 4- Figure 7.
- the HHT concentration in the dual-drug co-delivery system is 20 ng/mL and the DOX concentration is 120 ng/mL
- a single DOX, single HHT, and free drug combination is under the action of the dual-drug co-delivery system in Example 5 of the present invention.
- the cell survival rates were 90%, 41%, 35%, and 30%.
- the HHT concentration in the dual-drug co-delivery system is 25 ng/mL and the DOX concentration is 100 ng/mL
- single DOX, single HHT, free drug combination and the dual-drug co-delivery system in Example 4 of the present invention
- the cell survival rates were 92%, 50%, 43%, and 27%, respectively.
- mice used in this experiment are 6-8 weeks old female C57 mice (from the Institute of Basic Medicine, Chinese Academy of Medical Sciences) Animal Experiment Center), raised in an SPF environment.
- the results of Figure 8 show that the terminal survival period of mice in the free drug combination group is 52 days, and the survival rate of mice in the dual drug co-delivery system group of the present invention is as high as 9/10 at 52 days.
- the dual drug co-delivery system group of the present invention is free The survival time of mice in the drug combination group was longer, and the difference was significant.
- the experiment process is the same as experiment two.
- mice After a course of treatment, the mice were sacrificed, and the ratio of GFP positive cells in peripheral blood (PB) and bone marrow (Bone) was measured by flow cytometry, and the white blood cells (WBC) in peripheral blood (WBC) were measured by a blood cell analyzer (Mindray BC-5120) The number of red blood cells (RBC) and platelets (PLT), the results are shown in Figure 10 and Figure 11.
- Figure 10 shows that after treatment, the percentage of leukemia cells in the peripheral blood of the DHM group decreased from 40% in the DH group to 10%; the percentage of leukemia cells in the bone marrow decreased from 50% in the DH group to 5%, indicating that DHM can significantly reduce peripheral blood and The proportion of leukemia cells in the bone marrow.
- DHM group DH group WBC decreased from 10 X 10 9 / L to 5 X 10 9 / L, RBC increased from 5.
- the dual-drug co-delivery system provided by the present invention has a killing effect on a variety of hematological tumors and solid tumor cells, at the same time, it can reduce toxic and side effects, can inhibit tumor growth and be used for leukemia treatment; no organic solvent is used in the preparation process, and the preparation process Simple and suitable for industrial applications.
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Abstract
一种双药物共递送系统及其制备方法与应用,由包括嵌段共聚物、高三尖杉酯碱和阿霉素盐酸盐的原料制得,该共递送系统对多种血液肿瘤和实体肿瘤细胞都具有更强的杀伤作用,可为抑制肿瘤生长和白血病治疗提供可行的药物递送系统,前景广阔。
Description
一种双药物共递送系统及其制备方法与应用
技术领域
本发明涉及医药技术领域, 特别涉及一种双药物共递送系统、 其制备方法及在制 备抗肿瘤药物方面的应用。 背景技术
化疗是利用化学药物阻止癌细胞的增殖、 浸润、 转移, 直至最终杀灭癌细胞的治 疗方式, 是大部分实体肿瘤和血液系统肿瘤的主要治疗方式。 化疗过程中使用的化学 药物称为化疗药物。 由于化疗药物耐药性的出现和剂量依赖性的毒副作用, 单一用药 已无法满足临床需求, 于是使用多种化疗药物联用来解决此问题。 多种化疗药物的联 合使用虽然能够降低单一用药导致的毒副作用和产生耐药的可能性, 但存在给药次数 多、 剂量分配方案不容易确定等问题。
化疗药物共递送系统是解决上述问题的有效方法, 该共递送系统是将两种或以上 化疗药物包裹在一个载体中, 利用药物间协同作用增强其对肿瘤细胞的杀伤效果。 与 单一用药和联合用药相比, 药物共递送系统的优势在于: 1)两种或多种化疗药物发挥 协同作用, 在较小的剂量下产生更强的抗肿瘤效应, 降低毒副作用, 避免耐药性的产 生; 2) 可控制药物释放的时间和空间, 以协调不同药物的药物代谢动力学和药效学; 3) 减少给药次数, 改善病人依从性; 4) 单一给药剂量, 避免多种药物给药时剂量分 配的不确定性。
药物共递送系统主要使用脂质体、 聚合物胶束、 固体脂质纳米粒、 以及无机纳米 材料如介孔桂纳米粒、 金纳米粒、 磁性纳米粒、 碳纳米管等作为药物共递送载体。 递 送的药物可有多种类型, 如递送两种作用于不同靶标的小分子化疗药物, 以达到最大 的肿瘤细胞杀伤效应, 降低产生药物耐受的可能性; 又如共递送化疗药物和 siRNA, 利用 RNA干扰技术沉默耐药基因, 干扰细胞信号通路, 从而克服耐药; 再如共递送化 疗药物和成像剂, 实现靶向药物递送和非侵袭性的肿瘤成像。
现有的药物共递送系统的研宄多集中于实体肿瘤的化疗治疗,包括乳腺癌、肝癌、 宫颈癌、 前列腺癌、 肺癌、 脑瘤、 头颈部鳞状细胞癌、 胰腺癌、 胃癌、 黑色素瘤、 卵 巢癌等。 由于血液肿瘤种类很多, 且每一个种类中的亚型十分复杂, 对治疗其的药物 种类也有很多限制, 使得治疗血液肿瘤的药物不如治疗实体肿瘤的药物多, 因此迄今 适用于血液肿瘤, 如白血病、淋巴瘤、骨髓异常增生综合症等的药物共递送系统很少, 在化疗中面临更大的挑战。 除少量基础研宄外, 目前可用于制备治疗血液肿瘤的药物 共递送系统的药物仅有阿糖胞苷和柔红霉素, 由其形成药物共递送系统为脂质体共递
送系统 (CPX-351), 已于 2014年 11月进入三期临床试验。 试验显示 CPX-351对初治 或复发的急性髓系白血病 (acute myeloid leukemia, AML) 老年患者有效, 可以作为 缓解诱导疗法用于标准诱导疗法无效的患者, 或者作为巩固疗法用于已接受了干细胞 移植或未进行过干细胞移植的患者。 然而, 上述脂质体共递送系统的制备工艺复杂, 不仅需要使用多种脂质材料,而且需要在葡萄糖酸铜 /三乙胺缓冲体系中制备, 以使药 物阿糖胞苷和柔红霉素与缓冲体系 (含有有机溶剂) 中的铜离子按照一定的摩尔比形 成复合物, 从而保证药物在脂质体中的滞留时间。 该脂质体共递送系统涉及有机溶剂 的使用, 因而存在毒副作用风险高的问题。
因此, 本领域急需开发出适用于血液肿瘤, 尤其是复发性、 难治性白血病的新型 药物共递送系统。 发明内容
本发明的目的是针对现有技术中存在的技术缺陷, 第一方面, 提供一种具有强抗 肿瘤作用、 低毒副作用、 且可用于复发性、 难治性白血病的双药物共递送系统, 该双 药物共递送系统由包括嵌段共聚物、 高三尖杉酯碱和阿霉素盐酸盐的原料制得; 所述 嵌段共聚物选自聚乙烯己内酰胺 -聚醋酸乙烯酯-聚乙二醇(Soluplus)、 甲氧基聚乙二 醇-聚己内酯 (mPEG-PCL)、 二硬脂酰基磷脂酰乙醇胺-聚乙二醇 2000 ( DSPE_PEG2。。。)、 聚氧乙烯聚氧丙烯醚嵌段共聚物、 聚乳酸-羟基乙酸共聚物 (PLGA) 中的一种或几种, 优选 Soluplus。
其中,高三尖杉酯碱、阿霉素盐酸盐、嵌段共聚物按重量份数比为(1-10):(1-40): (4-200); 优选 (1-5) : (3-15) : (45-200)。
所述系统的平均水合直径为 20-200 nm, 优选 60-180 nm。
所述嵌段共聚物 Soluplus中聚乙烯己内酰胺、聚醋酸乙烯酯和聚乙二醇的分子量 比为 57 : 30 : 13。
第二方面, 本发明提供一种制备上述双药物共递送系统的方法, 包括制备载有高 三尖杉酯碱的胶束和加载阿霉素。
优选的, 所述载有高三尖杉酯碱的胶束是由高三尖杉酯碱、 嵌段共聚物和磷酸盐 缓冲液混合搅拌得到的。 具体制备过程包括以下步骤:
1) 将嵌段共聚物和高三尖杉酯碱加入磷酸盐缓冲液中, 加热搅拌得到液体 B;
2) 过滤步骤 1) 得到的液体 B, 滤液即为载有高三尖杉酯碱的胶束, 称胶束 C。 所述加载阿霉素是将所述载有高三尖杉酯碱的胶束与含有嵌段共聚物的磷酸盐缓 冲液混合, 再加入阿霉素盐酸盐水溶液搅拌。 具体包括以下步骤:
(1) 将嵌段共聚物溶于磷酸盐缓冲液中, 得到澄清溶液 D;
(2) 将步骤 (1) 得到的澄清溶液 D与载有高三尖杉酯碱的胶束 (胶束 C) 混合, 加热搅拌下滴加阿霉素盐酸盐水溶液 (优选的, 阿霉素盐酸盐水溶液中阿霉素盐酸盐 的浓度为 3-8 mg/mL), 得到液体 E;
(3) 将步骤 (2) 得到的液体 E过滤, 滤液即为所述双药物共递送系统。
所述加热均为加热至 25-70°C。
所述搅拌的速度均为 100-1000 r/min。
第三方面, 本发明提供上述双药物共递送系统在制备抑制肿瘤生长相关疾病药物 或治疗白血病药物中的应用。
所述肿瘤生长相关疾病为高三尖杉酯碱和蒽环类抗生素联合应用有效的肿瘤, 包 括但不限于急性髓系白血病、 慢性髓系白血病、 宫颈癌等。
所述急性髓系白血病为复发性、 难治性的 AML1-ET0急性髓系白血病。
本发明提供的双药物共递送系统中作为载体的嵌段共聚物为两亲性的高分子聚合 物,在溶液中可形成亲水性壳-疏水性核结构:两亲性的嵌段共聚物在磷酸盐缓冲液中 形成胶束, 通过两步组装方法, 先后将两种药物高三尖杉酯碱 (疏水性) 和阿霉素盐 酸盐 (亲水性) 组装进胶束疏水核中, 形成双药物共递送系统可实现联合给药。 本发 明的双药物共递送系统还可通过调整胶束中两种药物的比例从而对肿瘤发挥有差别的 治疗作用。 本发明的双药物共递送系统对多种血液肿瘤和实体肿瘤细胞都具有更强的 杀伤作用, 肿瘤细胞包括急性髓系白血病细胞、 慢性髓系白血病细胞、 宫颈癌细胞、 乳腺癌细胞。该系统还可用作治疗复发性、难治性的 AML1-ET0急性髓系白血病的药物, 效果优异; 特别是: 在白血病细胞实验中, 与联合使用阿霉素和高三尖杉酯碱相比, 本发明的双药物共递送系统能更有效地抑制髓系白血病细胞的增殖; 在动物实验中, 对急性髓系白血病 AML1-ET0小鼠具有更好的治疗效果,在延长小鼠生存期的同时还能 降低毒副作用。 本发明的双药物共递送系统可用于抑制肿瘤生长和白血病的治疗中。
本发明双药物共递送系统在制备过程中不使用任何有机溶剂, 安全性高, 满足临 床用药要求; 制备工艺简单, 适于工业化生产。 附图说明
图 1所示为本发明的双药物共递送系统在磷酸盐缓冲液中的粒径分布曲线图; 图 2所示为本发明的双药物共递送系统的磷酸盐缓冲液的照片;
图 3所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 8 : 1) 对 HL60/A 细胞活力影响的柱状图;
图 4所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 8 : 1) 对 U937R 细胞活力影响的曲线图;
图 5所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 15 : 1) 对 K562 细胞活力影响的曲线图;
图 6所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 6 : 1) 对 HeLa细 胞活力影响的曲线图;
图 7所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 4 : 1) 对 MCF7细 胞活力影响的曲线图;
图 8所示为本发明的双药物共递送系统(D0X和 HHT的重量比为 9 : 1)延长白血病 小鼠生存期的曲线图;
图 9所示为本发明的双药物共递送系统(D0X和 HHT的重量比为 9 : 1)降低小鼠体 内白血病细胞数量的柱状图;
图 10-图 11所示为本发明的双药物共递送系统 (D0X和 HHT的重量比为 9 : 1) 降 低毒副作用的柱状图。 具体实肺式
高三尖杉酯碱 (HHT) 是从三尖杉属植物中提取出的一种生物活性成分, 能抑制 白血病细胞的增殖,诱导细胞凋亡。阿霉素盐酸盐是一种广谱抗肿瘤药,通过破坏 DNA 结构表现出抗肿瘤效应。 高三尖杉酯碱和阿霉素 (DOX) 盐酸盐分别为疏水和亲水性 药物, 在临床上可用于急性髓系白血病的联合治疗。 但是, 两种药物在急性髓系白血 病治疗中分别给药, 会导致患者依从性差; 此外, 由于药代动力学不同, 两种药物在 体内的浓度无法保持设定的比例。
在这两种药物的共递送系统方面, 目前仅有一种脂质体共递送系统的文献报道 (Shim G , Lee S , Choi J , et al. Liposomal Co-Delivery of Omacetaxine Mepesuccinate and Doxorubicin for Synergistic Potentiation of Antitumor Activity. Pharmaceutical Research, 2014, 31(8):2178-2185.), 该递送系统使用脂质体作为载体, 粒径为 126 nm, 在小鼠皮下移植宫颈癌细胞(HeLa细胞) 的模型中具有抑制肿瘤生长的作用。该脂质 体共递送系统制备过程复杂, 需要使用多种脂质材料, 并加入有机溶剂甲醇使高三尖 杉酯碱首先被包裹到脂质体中, 然后通过静电作用进一步包裹阿霉素盐酸盐。 该脂质 体共递送系统中存在有机溶剂甲醇会带来毒副作用, 除此之外, 该脂质体共递送系统 是针对抑制实体肿瘤 HeLa细胞生长进行设计的, 脂质体比较稳定, 通常利用实体瘤 的高通透性和滞留效应在肿瘤组织中蓄积和释放药物, 而不能有效杀伤循环血液中的 白血病细胞。 而且, 该脂质体共递送系统如果在骨髓中累积, 难以减轻药物本身对骨 髓细胞生长的抑制作用, 而急性髓系白血病的治疗需要骨髓细胞的参与, 因而该脂质 体共递送系统病不适于在急性髓系白血病治疗中应用。
聚合物胶束是一类受到广泛关注的共递送药物载体, 因其一般尺寸在几十纳米, 所以也将其称为纳米胶束, 是由两亲性聚合物在合适的浓度和温度下于溶剂体系中自 发形成, 通过亲 -疏水作用, 将药物包裹在胶束的疏水核中, 因此通常作为两种疏水药 物的共递送药物载体; 其优势是不影响药物本身的活性, 显著地对疏水药物进行增溶, 制备方法简单, 不需要使用有机溶剂。 但是, 常规设计的聚合物胶束对于亲水性药物 则难以应用, 特别是难以将疏水和亲水性药物同时负载到胶束的疏水核中。
由于阿霉素盐酸盐是水溶性的, 而高三尖杉酯碱是疏水性的, 因此, 按通常理解 难以采用聚合物胶束作为上述双药的共递送药物载体, 把两种亲疏水性质不同的药物 包裹到胶束的疏水核中。 本发明通过使用筛选出的独特高分子作为载体, 在不使用有 机溶剂的前提下将亲疏水性质不同的两种药物包裹到聚合物胶束中, 利用聚合物胶束 形成共递送系统。
本发明的双药物共递送系统选用两亲性的嵌段共聚物作为载体, 其在水溶液中可 形成亲水性壳-疏水性核结构的胶束;通过疏水作用先将高三尖杉酯碱包裹在胶束的疏 水核中,再将阿霉素盐酸盐组装进胶束的疏水核中,形成本发明的双药物共递送系统, 即用两亲性嵌段共聚物将阿霉素盐酸盐和高三尖杉酯碱共递送。
本发明的双药物共递送系统中选用的嵌段共聚物选自聚乙烯己内酰胺-聚醋酸乙 烯酯-聚乙二醇 (例如商品名为 Soluplus 的商品)、 甲氧基聚乙二醇-聚己内酯 (mPEG-PCL)、 二硬脂酰基磷脂酰乙醇胺-聚乙二醇 2000 (DSPE_PEG2。。。)、 聚氧乙烯聚 氧丙烯醚嵌段共聚物、 聚乳酸-羟基乙酸共聚物 (PLGA) 中的一种或几种, 优选 Soluplus。 可用的嵌段共聚物均可商购, 平均分子量在 1000-150000。
本发明还提供了阿霉素盐酸盐和高三尖杉酯碱形成的双药物共递送系统在制备治 疗肿瘤药物方面的应用。
本发明的双药物共递送系统尤其可用作治疗复发性、 难治性的急性髓系白血病的 药物, 包括 AML1-ET0急性髓系白血病, 其表征为 t(8; 21)(q22; q22)染色体易位, 常见于 FAB分型中的 M2,存在于大约 12 %〜 15 % AML患者中;染色体 21 q22上的 AML 1 基因和染色体 8 q22上的 ET0基因重排形成 AML1-ET0融合基因。 以下结合具体实施例, 更具体地说明本发明的内容, 并对本发明作进一步阐述, 但这些实施例绝非对本发明进行限制。
除非特别指明, 以下实施例中所用的细胞系均购自国家实验细胞资源共享平台。 除非特别指明, 以下实施例中所用的磷酸盐缓冲液均为 1 X PBS溶液(pH7. 4); 所 用水溶液的溶剂均为超纯水。
实施例一: 制备载有髙三尖杉酯碱的胶束
(1) 将 15. 0-100. 0 mg 嵌段共聚物和 5. 0 mg高三尖杉酯碱溶于 4 mL磷酸盐缓 冲液中, 搅拌得到高分子混悬液 A; 嵌段共聚物选自聚乙烯己内酰胺-聚醋酸乙烯酯 - 聚乙二醇 (Soluplus)、 甲氧基聚乙二醇 (mPEG) -聚己内酯 (PCL) (mPEG-PCL, 分子 量: 4000 D)、二硬脂酰基磷脂酰乙醇胺(DSPE) -聚乙二醇 2000(PEG2。。。) (DSPE_PEG2。。。, 分子量: 2807 D)、 聚氧乙烯聚氧丙烯醚嵌段共聚物 (购自德国巴斯夫公司, 分子量 9840 D-14600 D, 聚氧乙烯和聚氧丙烯的含量比为 80 : 20)、 聚乳酸-羟基乙酸共聚物
(PLGA, 10 KD-40 KD,聚乳酸和羟基乙酸的含量比为 50 : 50) 中的一种或几种, 优选 Soluplus (可商购, 购自德国巴斯夫公司, 分子量 120 KD, 聚乙烯己内酰胺、 聚醋酸 乙烯酯和聚乙二醇的分子量比为 57 : 30 : 13);
(2) 将高分子混悬液 A在 25-7CTC加热、 100-1000 r/min搅拌 20-120 min, 获 得溶液 B;
(3) 将溶液 B室温静置约 1小时, 至其呈澄清透明状, 然后用 0. 22 p m聚醚砜 水系滤膜过滤以除去未包裹进胶束的游离药物, 即得载有高三尖杉酯碱的胶束 C; 所 得到的载有高三尖杉酯碱的胶束 C平均水合直径在 70 nm, 载药量为 14%, 包封率为 64% o
按照以上方法, 制备得到一系列载有高三尖杉酯碱的胶束 C, 仅是调整了使用的 原料组成和制备参数, 具体见表 1。
表 1 载有高三尖杉酯碱的胶束 C的制备参数
( 1 ) 将 50. 0-200. 0 mg嵌段聚合物溶于 3 mL磷酸盐缓冲液中, 搅拌至完全溶解, 得到澄清溶液 D;
( 2 )将阿霉素盐酸盐用水溶解,配制成浓度为 3-8 mg/mL的阿霉素盐酸盐水溶液;
( 3 )将澄清溶液 D与实施例 1得到的载有高三尖杉酯碱的胶束 C混合, 25-70 °C 加热、 100-1000 r/min搅拌下滴加 1 mL步骤 ( 2 ) 得到的阿霉素盐酸盐水溶液, 搅拌 20-120 min, 得到溶液 E; 载有高三尖杉酯碱的胶束 C的聚合物与步骤 ( 1 ) 中所用的 聚合物相同;
( 4) 将溶液 E室温静置, 用 0. 22 |J m聚醚砜水系滤膜过滤以除去未包裹进胶束 的游离药物, 得到同时包载高三尖杉酯碱和阿霉素的胶束 F, 即为本发明的双药物共 递送系统, 见图 2 , 为红色澄清溶液。
按照以上方法, 制备得到一系列双药物共递送系统, 仅是调整了加载阿霉素的参 数, 具体见表 2。 通过改变向澄清溶液 D中加入载有高三尖杉酯碱的胶束 C的体积, 可得到不同 D0X和 HHT比例的双药物共递送系统, 其平均水合直径如表 3所示。
表 2 加载阿霉素的参数
实施例 1的胶束 F平均水合直径在 80 nm (如图 1所示), 阿霉素的载药量为 5%, 包封率为 75%; 其它实施例的性能参数见表 2和表 3。
表 3 DOX和 HHT比例不同的双药物共递送系统
以 HL60/A细胞 (对阿霉素耐药的 HL60细胞) 为例, 具体实施方法如下: 在 Corning 96孔 U型板中, 每孔使用 100 |J L RPMI-1640培养基 (含 10%胎牛血 清和 1%青链霉素) 培养 5 X 104个细胞。 实验组包括: 1) 单一 HHT组 (H): HHT用二 甲基亚砜 (DMS0) 溶解得到 10 mg/mL母液; 2) 单一 D0X组 (D): 阿霉素盐酸盐用水 溶解得到 5 mg/mL母液; 3) 游离药物联合组 (DH): 母液 1) 和 2) 分别用培养基稀释 后混合得到, D0X与 HHT的重量比为 8 : 1 ; 4) 本发明的双药物共递送系统组 (DHM): 将实施例 1-6的双药物共递送系统分别用培养基稀释后得到。对照组每孔加入 100 H L 培养基; 实验组每孔加入 100 kl L稀释后的药物溶液, 使 HHT终浓度为 5 ng/mL、 10 ng/mL、 25 ng/mL、 50 ng/mL、 100 ng/mL; 对应的 DOX终浓度为 40 ng/mL、 80 ng/mL、 200 ng/mL、 400 ng/mL、 800 ng/mL。 将 Corning 96孔 U型板中的细胞孵育 24 h后用 PBS清洗细胞, 向每孔加入 110 M L CCK8稀释液, 孵育 2 h, 用酶标仪测定 450和 630 nm波长下的吸光度值(0D值), 按照公式 1计算细胞存活率, 以实施例 1的结果为例, 见图 3。
公式 1 : 细胞存活率 =0D4M-63。 (实验组) / OD45O-63O (对照组) X 100%
图 3结果表明,当双药物共递送系统中 HHT浓度为 25 ng/mL, D0X浓度为 200 ng/mL 时, 单一 D0X对 HL60阿霉素耐药株无细胞毒性, 单一 HHT作用下细胞存活率为 56%,
游离药物联合作用下细胞存活率为 54%, 双药物共递送系统作用下细胞存活率为 40%, 因此相同药物浓度下, 本发明双药物共递送系统比单一药物、 游离药物联合能够更加 有效地抑制肿瘤细胞的增殖。
本发明的双药物共递送系统对 U937R (阿糖胞苷耐药株, 发明人所在实验室筛选 的急性髓系白血病耐药细胞株)、 K562、 HeLa和 MCF7细胞也有类似效果, 各细胞的培 养条件见表 4, 结果见图 4 -图 7。
对于 U937R细胞, 当双药物共递送系统中 HHT浓度为 15 ng/mL、 D0X浓度为 120 ng/mL时, 单一 D0X、 单一 HHT、 游离药物联合、 本发明实施例 1双药物共递送系统作 用下细胞存活率分别为 55%、 59%、 34%、 21%。
对于 K562细胞,当双药物共递送系统中 HHT浓度为 25 ng/mL、D0X浓度为 375 ng/mL 时, 单一 D0X、 单一 HHT、 游离药物联合、 本发明实施例 7双药物共递送系统作用下细 胞存活率分别为 80%、 59%、 35%、 27%。
对于 HeLa细胞,当双药物共递送系统中 HHT浓度为 20 ng/mL、D0X浓度为 120 ng/mL 时, 单一 D0X、 单一 HHT、 游离药物联合、 本发明实施例 5双药物共递送系统作用下细 胞存活率分别为 90%、 41%、 35%、 30%。
对于 MCF7细胞,当双药物共递送系统中 HHT浓度为 25 ng/mL、D0X浓度为 100 ng/mL 时, 单一 D0X、 单一 HHT、 游离药物联合、 本发明实施例 4双药物共递送系统作用下细 胞存活率分别为 92%、 50%、 43%、 27%。
表 4 肿瘤细胞的培养条件
实验二: 游离药物联合、 双药物共递送系统对 AML1-ET0小鼠生存期的影响 本实验所采用的小鼠为 6-8周大的雌性 C57鼠 (来自中国医学科学院基础医学研 宄所的动物实验中心), 在 SPF级环境中饲养。 用 200 M L乙二胺四乙酸钠盐 (EDTA) / PBS悬浮 1 X 106个 AML1-ET0小鼠原代脾脏细胞 (GFP标记), 通过尾静脉注射到经 亚致死辐照 (450 cGy) 的 C57小鼠体内。 移植第七天从小鼠尾静脉取血, 用流式细胞 仪检测外周血白血病细胞阳性率, 确认小鼠发病。 从移植后的第 8天开始, 腹腔注射
以下各组药物: (1) 游离药物高三尖杉醋碱 0. 4 mg/kg、 阿霉素盐酸盐 3. 6 mg/kg, 作 为 DH组; (2)本发明实施例 8的双药物共递送系统(D0X与 HHT的重量比为 9 : 1), 作 为 DHM组; (3)无菌 PBS作为溶剂对照组(con组); (4)无菌 PBS溶解的 Soluplus 72 mg/kg作为 Solupus组。 连续给药四天, 停药七天, 为一个疗程; 结果见图 8。
图 8结果表明,游离药物联合组小鼠终末生存期为 52天,本发明双药物共递送系 统组小鼠在 52天时存活率高达 9/10, 本发明的双药物共递送系统组比游离药物联合 组小鼠的生存期更长, 且差异显著。
67天时检测 DHM组存活小鼠外周血 (PB)、 脾 (spleen)、 骨髓 (bone) 的 GFP阳 性细胞比例, 各小鼠不同脏器的白血病细胞浸润结果见图 9。 图 9结果表明, 本发明 双药物共递送系统可延长小鼠生存期, 同时能减少各器官白血病细胞的浸润。 实验三: 游离药物联合、 双药物共递送系统对 AML1-ET0小鼠的药效
实验过程同实验二。
治疗一个疗程后处死小鼠, 用流式细胞仪检测外周血 (PB) 和骨髓 (Bone) GFP 阳性细胞比例, 同时使用血细胞分析仪 (迈瑞公司 BC-5120型号) 检测外周血中白细 胞 (WBC)、 红细胞 (RBC) 和血小板 (PLT) 数量, 结果见图 10和图 11。
图 10结果表明,治疗后 DHM组外周血中白血病细胞比例从 DH组的 40%降低到 10%; 骨髓中白血病细胞比例从 DH组的 50%降低到 5%,表明 DHM能显著降低外周血和骨髓中 白血病细胞比例。 图 11结果表明, 治疗后 DHM组 WBC从 DH组的 10 X 109/L降低至 5 X 109/L, RBC从 DH组的 5. 9 X 1012/L升高至 6. 4 X 1012/L, PLT从 DH组的 9. 3 X 10n/L 升高至 1. 2 X 107L; 说明 DHM治疗在减少白血病细胞的同时, 没有降低红细胞和血小 板的数量, 表明其对骨髓细胞没有抑制作用, 在发挥治疗效果的同时还能降低毒副作 用。
IDIk应用性
本发明提供的双药物共递送系统对多种血液肿瘤和实体肿瘤细胞都具有杀伤作 用, 同时能降低毒副作用, 可抑制肿瘤生长和用于白血病治疗; 制备过程中不使用任 何有机溶剂, 制备工艺简单, 适于工业应用。
Claims
1、一种双药物共递送系统, 由包括嵌段共聚物、 高三尖杉酯碱和阿霉素盐酸盐的 原料制得;所述嵌段共聚物选自聚乙烯己内酰胺 -聚醋酸乙烯醋-聚乙二醇(Soluplus)、 甲氧基聚乙二醇-聚己内酯 (mPEG-PCL)、 二硬脂酰基磷脂酰乙醇胺-聚乙二醇 2000
(DSPE-PEG2。。。)、 聚氧乙烯聚氧丙烯醚嵌段共聚物、 聚乳酸-羟基乙酸共聚物 (PLGA) 中的一种或几种, 优选 Soluplus。
2、根据权利要求 1所述双药物共递送系统,其中, 高三尖杉酯碱、阿霉素盐酸盐、 嵌段共聚物按重量份数比为(1-10):(1-40):(4-200);优选(1-5) :(3-15) :(45-200)。
3、 根据权利要求 1 或 2 所述双药物共递送系统, 所述系统的平均水合直径为 20-200 nm, 优选 60-180 nm。
4、 根据权利要求 1-3任一所述双药物共递送系统, 所述嵌段共聚物 Soluplus中 聚乙烯己内酰胺、 聚醋酸乙烯酯和聚乙二醇的分子量比为 57 : 30 : 13。
5、 根据权利要求 4所述双药物共递送系统, 高三尖杉酯碱、 阿霉素盐酸盐、 嵌段 共聚物 Soluplus按重量份数比为(1-10): (1-40): (4-200); 优选(1-5) : (3-15) :
(45-200)。
6、一种制备权利要求 1-5任一所述双药物共递送系统的方法,包括制备载有高三 尖杉酯碱的胶束和加载阿霉素;
优选的, 所述载有高三尖杉酯碱的胶束是由高三尖杉酯碱、 嵌段共聚物和磷酸盐 缓冲液混合搅拌得到。
7、根据权利要求 6所述方法,所述加载阿霉素是将所述载有高三尖杉酯碱的胶束 与含有嵌段共聚物的磷酸盐缓冲液混合, 再加入阿霉素盐酸盐水溶液搅拌得到。
8、根据权利要求 6或 7所述方法,所述载有高三尖杉酯碱的胶束的具体制备过程 包括以下步骤:
1) 将嵌段共聚物和高三尖杉酯碱加入磷酸盐缓冲液中, 加热搅拌得到液体 B;
2) 过滤步骤 1) 得到的液体 B, 滤液即为载有高三尖杉酯碱的胶束, 称胶束 C。
9、 根据权利要求 6-8任一所述方法, 所述加载阿霉素具体包括以下步骤:
(1) 将嵌段共聚物溶于磷酸盐缓冲液中, 得到澄清溶液 D;
(2) 将步骤 (1) 得到的澄清溶液 D与载有高三尖杉酯碱的胶束 (胶束 C) 混合, 加热搅拌下滴加阿霉素盐酸盐水溶液 (优选的, 阿霉素盐酸盐水溶液中阿霉素盐酸盐 的浓度为 3-8 mg/mL), 得到液体 E;
(3) 将步骤 (2) 得到的液体 E过滤, 滤液即为所述双药物共递送系统。
10、 根据权利要求 8或 9所述方法, 所述加热均为加热至 25-70°C。
11、 根据权利要求 8或 9或 10所述方法, 所述搅拌的速度均为 100-100() r/min。
12、 权利要求 1-5任一所述双药物共递送系统在制备抑制肿瘤生长相关疾病药物 或治疗白血病药物中的应用。
13、根据权利要求 12所述应用,所述肿瘤生长相关疾病为高三尖杉酯碱和蒽环类 抗生素联合应用有效的肿瘤, 包括但不限于急性髓系白血病、 慢性髓系白血病、 宫颈 癌等。
14、根据权利要求 13所述应用, 所述急性髓系白血病为复发性、难治性的急性髓 系白血病。
15、 根据权利要求 14所述应用, 所述急性髓系白血病为 AML1-ET0急性髓系白血 病,其表征为 t (8; 21) (q22; q22)染色体易位,常见于 FAB分型中的 M2 ,染色体 21 q22 上的 AML 1基因和染色体 8 q22上的 ET0基因重排形成 AML1-ET0融合基因。
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