WO2020103175A1 - 一种康普瑞汀类衍生物冻干粉针及其制备方法 - Google Patents

一种康普瑞汀类衍生物冻干粉针及其制备方法

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WO2020103175A1
WO2020103175A1 PCT/CN2018/118108 CN2018118108W WO2020103175A1 WO 2020103175 A1 WO2020103175 A1 WO 2020103175A1 CN 2018118108 W CN2018118108 W CN 2018118108W WO 2020103175 A1 WO2020103175 A1 WO 2020103175A1
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derivative
lyophilized powder
phospholipids
derivatives
powder injection
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PCT/CN2018/118108
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French (fr)
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周建平
丁杨
袁洲
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中国药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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 invention relates to the technical field of pharmaceutical preparations and preparation thereof, in particular to a freeze-dried powder injection of compradine derivatives and a preparation method thereof.
  • Combretastatin is a cis-stilbene natural product isolated from the bark of the African bush Dwarf Willow.
  • Combretastatin A4 (hereinafter referred to as CA4) is the most cytotoxic and the most structural of all structures.
  • the anti-tumor mechanism of CA4 is mainly to inhibit tubulin polymerization, induce apoptosis and fight tumor blood vessels.
  • the target of action is similar to colchicine, but its activity is significantly better than colchicine.
  • CA4 has poor water solubility and fat solubility, and its cis-stilbene structure is more active but unstable, so there are currently three major structural modifications: 1 modification of the A ring structure; 2 modification of the B ring structure; 3 modification of carbon Carbon double bond bridge bond structure. Through structural modification, a more active CA4 derivative is obtained. The effective dose is low, and the toxic and side effects are small when used. It has huge clinical application prospects.
  • the core structure of the CA4 derivative is:
  • A is N, O or S;
  • R3 preferably has a structure such as phenyl, hydroxyl, amino, etc. that can form van der Waals forces, hydrogen bonds, etc. with phospholipids.
  • the preferred active structure is structure 1: 4- (3,5-dimethoxy) -5- (4-methoxyphenyl) oxazole, structure 2: 2-phenyl-4- (3 , 4,5-trimethoxyphenyl) -5- (4-pyridyl) thiazole, structure 3: 4- (3,5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxy Phenyl) imidazole.
  • CA4 derivatives water solubility of CA4 derivatives is still extremely poor, which limits their clinical application. Therefore, the design and preparation of prodrugs of water-soluble derivatives are currently hot research topics.
  • American OxiGene has designed and synthesized its phosphate prodrug CA4 phosphate (combretastatin A4 phosphate, CA4P), which has entered the stage of clinical experimental research.
  • CA4P improves the water solubility of CA4, it cannot be stably stored in an aqueous solution, so it needs to be prepared as a lyophilized powder needle for storage.
  • the lyophilized powder needle has the following defects: 1 CA4P lyophilized powder is easy to produce after reconstitution Degradation and poor stability; 2The phosphate form is poorly soluble in fat, and it is not easy to enter the cell to exert the drug effect, and the effective dose is high; 3The toxic and side effects in the body are large, and it is degraded and released quickly after intravenous administration, and it is eliminated quickly without slow release. Effect; 4 It is limited to intravenous administration and does not have the convenience and compliance of oral administration.
  • the method of directly preparing drugs into liposomes, solid dispersion nanoparticles, polymeric micelles and other new formulations can improve the drug itself, but it has the problems of low drug loading and unstable encapsulation rate. Its corresponding lyophilization There are similar problems with powder needles, and there are also deficiencies in the preparation process, the amount of auxiliary materials, the safety of auxiliary materials and storage.
  • An object of the present invention is to provide a phospholipid complex of a compradine derivative in order to solve the above-mentioned defects of a freeze-dried powder injection of compradine derivatives.
  • Still another object of the present invention is to provide a lyophilized powder injection of a completin derivative phospholipid complex and a preparation method thereof.
  • a compatilin derivative phospholipid complex is composed of a compastatin derivative and a phospholipid.
  • the molar ratio of the compastatin derivative to the phospholipid is 2: 1 to 1:10, preferably 1: 3 .
  • Comprentin derivatives are imidazole, oxazole and thiazole Combretastatin A4 derivatives with 3,5-dimethoxyphenyl substituted at the 4-position of ring A, preferably with phenyl, hydroxyl, amino and other phospholipids Structures that form van der Waals forces, hydrogen bonds, etc., preferably 4- (3,5-dimethoxy) -5- (4-methoxyphenyl) oxazole, 2-phenyl-4- (3,4, 5-trimethoxyphenyl) -5- (4-pyridyl) thiazole, 4- (3,5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole, Further preferred is 4- (3,5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole.
  • the phospholipids are selected from natural source phospholipids and synthetic source phospholipids;
  • the natural source phospholipids are preferably soybean lecithin, egg yolk lecithin, brain phospholipids, sphingomyelin, serine phospholipids;
  • the synthetic source phospholipids are preferably hydrogenated phospholipids, PMPC, DPPC , DMPC, DPPE, DSPE, DPPSA, and their structurally modified analogs.
  • a lyophilized powder injection of a completin derivative phospholipid complex is composed of the completin derivative phospholipid complex, a stabilizer and a lyoprotectant.
  • the stabilizer is selected from poloxamer 188, polysorbate 80, polysorbate 20 or phospholipid and any combination thereof.
  • the lyoprotectant is selected from mannitol, lactose, glucose, trehalose, sorbitol or sucrose and any combination thereof.
  • a preparation method of the commoditin derivative freeze-dried powder injection includes the following steps:
  • the reaction solvent (a) is preferably one or more selected from aromatic hydrocarbons, halogen derivatives, cyclic ethers, methylene chloride, chloroform, acetone, and ethanol, preferably ethanol.
  • the reaction concentration is preferably 3 mg / mL
  • the reaction temperature is preferably 25 ° C.
  • the recombination time is preferably 12 h.
  • the lyophilized powder injection of the compatriot derivative phospholipid complex prepared by this method can be reconstituted in 5% glucose or 0.9% saline injection.
  • the particle size after reconstitution is preferably 180.7 ⁇ 23.9nm, PDI is 0.108 ⁇ 0.021.
  • the lyophilized powder injection of the compatriot derivative phospholipid complex of the present invention is dissolved in 5% glucose or 0.9% saline injection, and can be used for intravenous injection, drip infusion or direct oral administration to treat non-small cell lung cancer, liver cancer, Colon cancer.
  • the preparation process is simple, the toxicity of the introduced organic solvent is low, and it is suitable for industrial production;
  • the same moles can be loaded with more CA4 derivatives, the drug loading is high, and the dosage of phospholipids is reduced;
  • the phospholipid structure is amphiphilic. Compared with the CA4 derivative, it can significantly increase the hydrophilicity and lipophilicity of the CA4 derivative phospholipid complex, and the lipid membrane can increase the cell's uptake of drugs;
  • Oral administration of CA4 derivative phospholipid complex can improve oral bioavailability compared with CA4 derivative phosphate
  • CA4 derivative phospholipid complex is administered intravenously. Compared with CA4 derivative phosphate, it has good stability during preparation and storage, has a certain sustained release effect and pH response, and nanoparticles have a certain passive targeting It can reduce the dosage and improve the efficacy, and overcome the defects of high dosage of CA4 derivative phosphate;
  • CA4 derivative phospholipid complex can significantly reduce the irritation of oral administration of CA4 derivative and intravenous injection of CA4 derivative phosphate.
  • Figure 1 In vitro release assay of lyophilized powder injections of compistatin derivative phospholipid complex.
  • Figure 2 Cellular uptake measurement of the commoditin derivative phospholipid complex freeze-dried powder injection.
  • Figure 3 Cytotoxicity determination of lyophilized powder injections of compistatin derivative phospholipid complex.
  • Figure 4 Determination of the oral bioavailability of the compistatin derivative phospholipid complex.
  • Compretin derivatives (4- (3,5-dimethoxy) -5- (4-methoxyphenyl) oxazole) and soybean lecithin in 50mL round
  • Compastatin derivatives (2-phenyl-4- (3,4,5-trimethoxyphenyl) -5- (4-pyridyl) thiazole) and soybean Lecithin was added to 200mL acetone in a 50mL round-bottom flask to control the drug concentration to 0.5mg / mL. After stirring until completely dissolved, the compound was left to stand at 35 ° C for 2h, and then vacuum dried to obtain the compound of compistatin derivative phospholipid. Substance, the measured recombination rate is 72%
  • Compretin derivatives (4- (3,5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and egg yolk Lecithin in a 50mL round-bottom flask, control the drug concentration to 2mg / mL, add 40mL of ethanol, stir until it is completely dissolved, and leave to compound at 50 ° C for 2h, then vacuum dry to obtain the compatriot derivative phospholipid complex
  • the measured recombination rate is 62%.
  • Compretin derivatives (4- (3,5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and egg yolk Lecithin was added to 30mL ethanol in a 50mL round-bottom flask, the drug concentration was controlled to 3mg / mL, and stirred to completely dissolve. After standing at 25 ° C for 12 hours, the compound was left to dry in vacuo to obtain the compatriot derivative phospholipid complex , The measured recombination rate is 97%.
  • Compastatin derivative phospholipid complex (Example 2), 200 mg (2%) of Tween-80, 500 mg (5%) of lactose, dissolve and disperse in 10 mL of water for injection, fill it under high pressure homogenization In 10mL vial, freeze-dried, the appearance of lyophilized powder is more fluffy, and the particle size after reconstitution is 221.6 ⁇ 45.6nm.
  • Compaq-derivin derivative phospholipid complex (Example 6), Poloxamer 188, 300 mg (3%), mannitol 500 mg (5%), dissolve and disperse in 10 mL of water for injection, and fill in It was freeze-dried in a 10mL vial, and the appearance of the lyophilized powder was shaped. The particle size after reconstitution was 180.7 ⁇ 23.9nm.
  • Cell uptake Set four groups of blank, Completin derivatives, Completin derivatives phosphate, Completin derivative phospholipid complex lyophilized powder injection (prepared in Example 9). Take A549 cells in logarithmic production phase and inoculate 5 ⁇ 10 6 cells / well in a 6-well plate. Incubate the complete culture medium in a cell incubator at 37 °C for 24 hours to make it adhere to the wall. Discard the original medium. Serum culture medium was incubated for 15 min. Each well was added with 1500 ⁇ L (containing 0.0375 ⁇ L of DMSO) diluted with serum-free medium to an effective concentration of 25 nM drug.
  • control group was added with 1500 ⁇ L (containing DMSO 0.0375 ⁇ L) of drug-free serum-free medium. Incubate at 37 ° C to investigate the relationship between uptake rate and uptake time. Incubate at different times (1h, 2h, 3h), observe the cell morphology and density, quickly discard the medium, add 4 ° C PBS to stop the cell uptake, and rinse the cells 3 times.
  • Cytotoxicity Take A549 cells in logarithmic production stage, inoculate 1 ⁇ 10 5 cells / well in 96-well plates, incubate in complete medium at 37 °C for 24 hours, remove the medium, add 100 ⁇ L of serum-free medium to each well Corresponding groups of different concentrations (drug concentrations of 2.5, 5, 10, 25, 50, 100 nM), after incubation at 37 ° C for 24 h, add 20 ⁇ L of 5 mg / mL MTT solution to each well. After incubating at 37 ° C for 4 hours, the medium was discarded, 100 ⁇ L of DMSO was added, and shaken for 10 min to dissolve formazan crystals.
  • a total of 18 rats were selected and randomly divided into the group consisting of a compatrin derivative group, a common mixture group, and a compradine derivative phospholipid complex group (prepared in Example 6).
  • gavage was given at a dose of 50 mg / kg, and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 8, 12, 24, and 36 hours after administration, 0.3 mL of whole blood was collected from the fundus venous plexus and placed in heparin In a sodium test tube, centrifuge at 10,000 rpm for 10 min, and take the upper plasma to detect the concentration of the compatrin derivative. See Figure 4.

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Abstract

本发明公开了一种康普瑞汀类衍生物冻干粉针及其制备方法。所述冻干粉针由康普瑞汀衍生物磷脂复合物、稳定剂和冻干保护剂构成，制备方法包括：康普瑞汀衍生物磷脂复合物的制备；高压均质；冷冻干燥。冻干粉针溶解于5％葡萄糖或0.9％生理盐水注射液中使用，可用于静脉注射、滴注或直接口服以治疗非小细胞肺癌、肝癌、结肠癌。本发明制备的冻干粉针药脂比高，毒副作用小，可改善康普瑞汀类衍生物的亲水性和亲脂性，具有一定缓释效果和被动靶向，能提高口服生物利用度，降低静脉给药剂量，减毒增效，复溶性良好，且该制备方法工艺过程简单，易于工业化生产。

Description

一种康普瑞汀类衍生物冻干粉针及其制备方法 技术领域
本发明涉及药物制剂及其制备技术领域，具体涉及一种康普瑞汀类衍生物冻干粉针及其制备方法。
背景技术
康普瑞汀(Combretastatin)是从非洲灌木矮柳树的树皮中分离的一种顺式二苯乙烯类天然产物，其中Combretastatin A4(以下简称为CA4)是所有结构中细胞毒性最强、结构最简单的一种化合物，其具有顺式碳碳双键连接臂连接的3，4，5－三甲氧基取代的苯环和3－羟基－4－甲氧基取代苯环的基本结构。CA4的抗肿瘤机制主要为抑制微管蛋白聚合，诱导细胞凋亡和对抗肿瘤血管作用，作用靶点与秋水仙碱类似，而活性明显优于秋水仙碱。
但是CA4水溶性和脂溶性差，且其顺式二苯乙烯结构活性更强但不稳定，因此目前主要进行3个方面的结构修饰：①修饰A环结构；②修饰B环结构；③修饰碳碳双键桥连键结构。通过结构修饰得到活性更强的CA4衍生物，起效剂量低，使用时毒副作用较小，具有巨大的临床应用前景。所述CA4衍生物，其母核结构为：
Figure PCTCN2018118108-appb-000001
其中：A为N、O或S；R3优选自具有苯基、羟基、氨基等能与磷脂形成范德华力、氢键等作用的结构。其中根据专利CN201110422678，优选活性结构为结构1:4-(3,5-二甲氧基)-5-(4-甲氧苯基)噁唑、结构2:2-苯基-4-(3,4,5-三甲氧基苯基)-5-(4-吡啶基)噻唑、结构3:4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑。
但是CA4衍生物的水溶性仍极差，限制了它们的临床应用，因此设计和制备其水溶性衍生物前药是目前的研究热点。至今国内外文献报道的CA4衍生物的研究多局限于设计和合成其磷酸盐前药。美国OxiGene公司设计并合成了其磷酸盐前药CA4磷酸盐(combretastatin A4  phosphate,CA4P)，目前已经进入临床实验研究阶段。虽然CA4P提高了CA4的水溶性，但其在水溶液中不能稳定贮存，因此需制备为冻干粉针储存，其冻干粉针存在以下几个方面的缺陷：①CA4P冻干粉复溶后易产生降解，稳定性差；②磷酸盐形式脂溶性较差，不易进入细胞而发挥药效，起效剂量高；③体内毒副作用大，静脉内给药后即降解速释，消除快，不具有缓释效果；④局限于静脉内给药，不具备口服给药的便利和顺应性。
此外，将药物直接制成脂质体、固体分散纳米粒、聚合胶束等新型制剂的方法虽可改善药物本身，但存在载药量低，包封率不稳定等问题，其相应的冻干粉针存在相似问题，且制备工艺、辅料用量、辅料安全性及储存等方面也会存在不足。
现有专利中，未见康普瑞汀类衍生物磷脂复合物相关发明。
发明内容
本发明的一个目的是为解决康普瑞汀类衍生物冻干粉针的上述缺陷,提供一种康普瑞汀类衍生物磷脂复合物。
本发明的又一个目的是提供一种康普瑞汀类衍生物磷脂复合物冻干粉针及其制备方法。
本发明的目的可通过以下技术方案实现：
一种康普瑞汀类衍生物磷脂复合物由康普瑞汀类衍生物与磷脂构成，康普瑞汀类衍生物与磷脂的摩尔比为2:1～1:10，优选为1:3。
其中康普瑞汀类衍生物为A环含4位取代的3，5-二甲氧基苯基的咪唑、噁唑及噻唑类CombretastatinA4衍生物，优选具有苯基，羟基，氨基等能与磷脂形成范德华力、氢键等作用的结构，优选4-(3,5-二甲氧基)-5-(4-甲氧苯基)噁唑、2-苯基-4-(3,4,5-三甲氧基苯基)-5-(4-吡啶基)噻唑、4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑,进一步优选4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑。
其中磷脂选自天然来源的磷脂与合成来源磷脂；所述天然来源的磷脂优选大豆卵磷脂、蛋黄卵磷脂、脑磷脂、神经鞘磷脂、丝氨酸磷脂；所述合成来源磷脂优选氢化磷脂、PMPC、DPPC、DMPC、DPPE、DSPE、DPPSA、及其结构修饰的类似物。
本发明所述的康普瑞汀类衍生物磷脂复合物在制备康普瑞汀类衍生物磷脂复合物冻干粉针中的应用。
一种康普瑞汀类衍生物磷脂复合物冻干粉针，由所述的康普瑞汀类衍生物磷脂复合物、稳定剂和冻干保护剂构成。
其中稳定剂选自泊洛沙姆188、聚山梨醇80、聚山梨醇20或磷脂及其任意组合。
其中冻干保护剂选自甘露醇、乳糖、葡萄糖、海藻糖、山梨醇或蔗糖及其任意组合。
一种所述的康普瑞汀衍生物冻干粉针的制备方法，包括以下步骤：
(1)将康普瑞汀类衍生物与磷脂溶于有机溶剂(a)中，控制反应浓度为0.5～20mg/mL，并于0～100℃温度下静置复合大于0.5h；
(2)将复合所用的有机溶剂(a)采用减压蒸发和真空干燥方法除去，得到康普瑞汀类衍生物磷脂复合物固体；
(3)将康普瑞汀类衍生物磷脂复合物分散到溶有稳定剂和冻干保护剂的注射用水中，高压均质，过膜，冷冻干燥；其中,冻干溶液各组份重量百分比为康普瑞汀类磷脂复合物0.1％～2.0％，稳定剂0～10％，冻干保护剂2～10％。
所述反应溶剂(a)优选选自芳烃、卤素衍生物、环醚、二氯甲烷、三氯甲烷、丙酮、乙醇中的一种或多种，优选为乙醇。
其中反应浓度优选为3mg/mL，反应温度优选为25℃，复合时间优选为12h。
该法制备的康普瑞汀类衍生物磷脂复合物冻干粉针可复溶于5％葡萄糖或0.9％生理盐水注射液中，复溶后粒径优选为180.7±23.9nm，PDI为0.108±0.021。
本发明康普瑞汀类衍生物磷脂复合物冻干粉针溶解于5％葡萄糖或0.9％生理盐水注射液中使用，可用于静脉注射、滴注或直接口服以治疗非小细胞肺癌、肝癌、结肠癌。
本发明提供的康普瑞汀类衍生物磷脂复合物冻干粉针，具有以下优点：
1、制备工艺简单，引入有机溶剂毒性低，适于工业生产；
2、与脂质体相比，相同的摩尔数可以装载更多CA4衍生物，载药量高，降低磷脂用量；
3、磷脂分子与特定的CA4衍生物分子的缔和作用降低药物的泄漏率；
4、磷脂结构具有两亲性，与CA4衍生物相比，能明显提高CA4衍生物磷脂复合物的亲水性和亲脂性，类脂膜能提高细胞对药物的摄取；
5、CA4衍生物磷脂复合物口服给药，与CA4衍生物磷酸盐相比，可提高口服生物利用度；
6、CA4衍生物磷脂复合物静脉注射给药，与CA4衍生物磷酸盐相比，制备和储存过程中 稳定性好，具有一定的缓释效果和pH响应，且纳米粒具有一定的被动靶向作用，可降低给药剂量，提高药效，克服CA4衍生物磷酸盐用药剂量高的缺陷；
7、CA4衍生物磷脂复合物能明显降低CA4衍生物口服及CA4衍生物磷酸盐静脉注射的刺激性。
附图说明
图1：康普瑞汀类衍生物磷脂复合物冻干粉针的体外释放测定。
图2：康普瑞汀类衍生物磷脂复合物冻干粉针细胞摄取测定。
图3：康普瑞汀类衍生物磷脂复合物冻干粉针细胞毒性测定。
图4：康普瑞汀类衍生物磷脂复合物口服生物利用度测定。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进一步详细的说明。应当理解，此处所述的具体实施例用以说明本发明，并不用于限定本发明的范围。
实施例1
精密称取摩尔质量比2:1康普瑞汀类衍生物(4-(3,5-二甲氧基)-5-(4-甲氧苯基)噁唑)和大豆卵磷脂于50mL圆底烧瓶中，加入5mL丙酮，控制药物浓度为20mg/mL，搅拌至完全溶解后，30℃下静置复合1h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为50％。
实施例2
精密称取摩尔质量比2:1康普瑞汀类衍生物(2-苯基-4-(3,4,5-三甲氧基苯基)-5-(4-吡啶基)噻唑)和大豆卵磷脂于50mL圆底烧瓶中，加入200mL丙酮，控制药物浓度为0.5mg/mL，搅拌至完全溶解后，35℃下静置复合2h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为72％
实施例3
精密称取摩尔质量比1:1康普瑞汀类衍生物(4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基) 咪唑)和蛋黄卵磷脂于50mL圆底烧瓶中，控制药物浓度为3mg/mL，加入30mL苯，搅拌至完全溶解后，40℃下静置复合2h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为62％。
实施例4
精密称取摩尔质量比1:2康普瑞汀类衍生物(4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑)和蛋黄卵磷脂于50mL圆底烧瓶中，控制药物浓度为2mg/mL，加入40mL乙醇，搅拌至完全溶解后，50℃下静置复合2h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为62％。
实施例5
精密称取摩尔质量比1:4康普瑞汀类衍生物(4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑)和脑磷脂于50mL圆底烧瓶中，加入35mL氯仿，控制药物浓度为2.5mg/mL,搅拌至完全溶解后，20℃下静置复合12h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为53％。
实施例6
精密称取摩尔质量比1:3康普瑞汀类衍生物(4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑)和蛋黄卵磷脂于50mL圆底烧瓶中，加入30mL乙醇，控制药物浓度为3mg/mL,搅拌使其完全溶解，25℃下静置复合12h后，抽真空干燥得康普瑞汀类衍生物磷脂复合物，测定复合率为97％。
实施例7
称取康普瑞汀类衍生物磷脂复合物(实施例1)100mg，Poloxamer 188 500mg(5％),葡萄糖200mg(2％)，溶解分散于10mL注射用水中，高压均质后灌装于10mL西林瓶中，冷冻干燥，冻干粉外观塌陷，测定复溶后粒径为242.1±36.9nm。
实施例8
称取康普瑞汀类衍生物磷脂复合物(实施例2)100mg，Tween-80 200mg(2％)，乳糖500mg (5％)，溶解分散于10mL注射用水中，高压均质后灌装于10mL西林瓶中，冷冻干燥，冻干粉外观较蓬松，测定复溶后粒径为221.6±45.6nm。
实施例9
称取康普瑞汀类衍生物磷脂复合物(实施例6)100mg，Poloxamer 188 300mg(3％)，甘露醇500mg(5％)，溶解分散于10mL注射用水中，高压均质后灌装于10mL西林瓶中，冷冻干燥，冻干粉外观成型，测定复溶后粒径为180.7±23.9nm。
实施例10
溶解度和表观油水分配系数的测定
采用摇瓶法(GB/T 21853-2008)测定，分别量取正辛醇预饱和的水20mL置磨口三角瓶中，各3份，分别加入过量的康普瑞汀类衍生物4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑和实施例6所述康普瑞汀类衍生物磷脂复合物，25℃振摇12h至溶解平衡，移至离心管中，2000×g离心20min。取上层液10mL，各置磨口三角瓶中，分别加入正辛醇10mL，于25℃振摇至溶解平衡，2000×g离心20min。分别移取水相、正辛醇相2mL置10mL量瓶中，用甲醇定容，测定康普瑞汀类衍生物和康普瑞汀类衍生物磷脂复合物在正辛醇一水系统中的溶解度。见表1。结果显示，康普瑞汀类衍生物磷脂复合物水中溶解度提高约8倍，LogP提高约1.2倍。
表1 康普瑞汀类衍生物及其磷脂复合物在正辛醇一水系统中的溶解度
Figure PCTCN2018118108-appb-000002
实施例11
体外释放测定。
分别取1mL康普瑞汀类衍生物磷脂复合物冻干粉针(实施例6)复溶物置于透析袋内，满足漏槽条件，两端用密封夹封口，分别放入200mL释放介质中，释放介质为含1％Tween80的pH7.4、pH6.6和pH5.5的PBS缓冲液，在37℃，100rmp搅拌下考察其释放情况。C118P 组分别于5min、10min、15min、0.5h、1h、2h、4h、6h、8h、12h，分别于0.5h、1h、2h、4h、6h、8h、12h，取2mL释放介质，同时补充等体积新鲜的37℃释放介质，样品过0.22μm滤膜，LC-MS进样，记录峰面积，计算各时间点的累积释放度，绘制释放曲线。见图1。结果显示，康普瑞汀类衍生物磷脂复合物冻干粉制剂组体外释放模型在pH 7.4和pH 6.6条件下符合药物释放一级动力学，pH 5.5条件下符合零级动力学，药物释放具有较明显的缓释效果和pH响应现象，可预见制剂在体内的药物释放具有缓释和肿瘤组织富集能力。
实施例12
细胞药效学测定
细胞摄取：设置空白、康普瑞汀类衍生物、康普瑞汀类衍生物磷酸盐、康普瑞汀类衍生物磷脂复合物冻干粉针(实施例9制备)四组。取对数生产期的A549细胞以5×10 6个/孔接种于6孔板中，完全培养液37℃细胞培养箱中培养24h，使其贴壁，弃去原有培养基，用不含血清的培养液孵育15min，每孔加入1500μL(含DMSO 0.0375μL)用无血清培养基稀释成25nM药物有效浓度，设置对照组加入1500μL(含DMSO 0.0375μL)不含药物的无血清培养基，于37℃孵育，考察摄取率与摄取时间的关系。孵育不同的时间(1h、2h、3h)，观察细胞形态和密度，快速弃去培养基，加入4℃PBS终止细胞摄取，并冲洗细胞3遍。加入200μL细胞裂解液于冰浴中震荡30min破碎细胞，12000×g离心15min，吸取20μL上清液，BCA蛋白定量，取50μL上清液，加入甲醇200μL，涡旋3min，12000×g离心5min，取上清液100uL，LC-MS测定药物含量(所有组别n＝3)。。见图2。结果显示，康普瑞汀类衍生物磷脂复合物冻干粉针组细胞摄取量高于原料药和磷酸盐组，且具有显著性，说明康普瑞汀类衍生物磷脂复合物冻干粉针由于其亲脂特性更易被细胞摄取利用。
细胞毒性：取对数生产期的A549细胞，以1×10 5个/孔接种于96孔板中，完全培养基37℃培养24h，移去培养基，每孔加入100μL无血清培养基稀释的不同浓度的对应组别(药物浓度为2.5、5、10、25、50、100nM)，于37℃孵育24h后，每孔加入20μL的5mg/mL的MTT溶液。于37℃孵育4h后，弃去培养基，加入100μL的DMSO，振摇10min以溶解甲瓒结晶，采用酶标仪与570nm处测定吸光度，计算细胞存活率。康普瑞汀类衍生物磷脂复合物细胞毒性强。见图3。结果显示，康普瑞汀类衍生物磷脂复合物冻干粉针IC 50在24h和48h各浓度下均低于原料药和磷酸盐组，且均有显著性，说明康普瑞汀类衍生物磷脂复合物冻干粉针的细胞毒性强于康普瑞汀类衍生物磷酸盐冻干粉针，可提高康普瑞汀类衍生物药物疗效，降 低康普瑞汀类衍生物的临床给药剂量。
实施例13
口服生物利用度测定
选取大鼠18只，随机分成康普瑞汀衍生物组、普通混合物组、康普瑞汀类衍生物磷脂复合物组(实施例6制备)。禁食12h后，以50mg/kg的剂量灌胃，给药后0.25、0.5、1、1.5、2、3、4、6、8、12、24、36h眼底静脉丛采全血0.3mL，置于肝素钠试管中，10000rpm离心10min，取上层血浆检测康普瑞汀类衍生物的浓度。见图4。结果显示，康普瑞汀类衍生物磷脂复合物组与原料药组相比口服生物利用度提高约7倍，半衰期延长约5倍，口服给药，克服了康普瑞汀类衍生物磷酸盐类药物只限于静脉给药的缺陷。

Claims (9)

  1. 一种康普瑞汀类衍生物磷脂复合物，其特征在于，由康普瑞汀类衍生物和磷脂构成，其中康普瑞汀类衍生物和磷脂的摩尔比为2:1～1:10。
  2. 根据权利要求1所述的康普瑞汀类衍生物磷脂复合物，其特征在于，所述康普瑞汀类衍生物为A环含4位取代的3，5-二甲氧基苯基的咪唑、噁唑及噻唑类Combretastatin A4衍生物，优选具有苯基、羟基，氨基等能与磷脂形成范德华力、氢键作用的结构；进一步优选4-(3,5-二甲氧基)-5-(4-甲氧苯基)噁唑、2-苯基-4-(3,4,5-三甲氧基苯基)-5-(4-吡啶基)噻唑、4-(3,5-二甲氧苯基)-5-(3-羟基-4-甲氧基苯基)咪唑。
  3. 根据权利要求1所述的康普瑞汀类衍生物磷脂复合物，其特征在于，所述磷脂选自天然来源的磷脂与合成来源磷脂；其中天然来源的磷脂优选大豆卵磷脂、蛋黄卵磷脂、脑磷脂、神经鞘磷脂、丝氨酸磷脂；合成来源磷脂优选氢化磷脂、PMPC、DPPC、DMPC、DPPE、DSPE、DPPSA、或其结构修饰的类似物。
  4. 权利要求1-3所述的康普瑞汀类衍生物磷脂复合物在制备康普瑞汀类衍生物磷脂复合物冻干粉针中的应用。
  5. 一种康普瑞汀类衍生物冻干粉针，其特征在于，由权利要求1-3中任一项所述的康普瑞汀类衍生物磷脂复合物、稳定剂和冻干保护剂构成。
  6. 根据权利要求5所述的康普瑞汀类衍生物冻干粉针，其特征在于，所述稳定剂选自泊洛沙姆188、聚山梨醇80、聚山梨醇20中的一种或多种。
  7. 根据权利要求1所述的康普瑞汀类衍生物冻干粉针，其特征在于，所述冻干保护剂选自甘露醇、乳糖、葡萄糖、海藻糖、山梨醇或蔗糖中的一种或多种。
  8. 一种权利要求5所述的康普瑞汀衍生物冻干粉针的制备方法，其特征在于，包括以下步骤：
    (1)将康普瑞汀类衍生物与磷脂溶于有机溶剂(a)中，控制反应浓度为0.5～20mg/mL，并于0～100℃温度下静置复合大于0.5h；
    (2)将复合所用的有机溶剂(a)采用减压蒸发和真空干燥方法除去，得到康普瑞汀类衍生物磷脂复合物固体；
    (3)将康普瑞汀类衍生物磷脂复合物分散到溶有稳定剂和冻干保护剂的注射用水中，高压均质，过膜，冷冻干燥即得所述的康普瑞汀衍生物冻干粉针；其中冻干溶液各组份重量百分比为康普瑞汀类磷脂复合物0.1～2.0％，稳定剂0～ 10％，冻干保护剂2～10％，余量为注射用水
  9. 根据权利要求6所述的制备方法，其特征在于，所述反应溶剂(a)选自芳烃、卤素衍生物、环醚、二氯甲烷、三氯甲烷、丙酮、乙醇中的一种或多种。
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