WO2021110004A1 - 紫杉醇弱酸性衍生物主动载药脂质体及其制备与应用 - Google Patents

紫杉醇弱酸性衍生物主动载药脂质体及其制备与应用 Download PDF

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WO2021110004A1
WO2021110004A1 PCT/CN2020/133184 CN2020133184W WO2021110004A1 WO 2021110004 A1 WO2021110004 A1 WO 2021110004A1 CN 2020133184 W CN2020133184 W CN 2020133184W WO 2021110004 A1 WO2021110004 A1 WO 2021110004A1
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paclitaxel
drug
liposome
derivative
weakly acidic
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French (fr)
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王永军
于江
何仲贵
周双
刘丹
李金花
刘洪卓
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沈阳药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention belongs to the field of liposome drug delivery, and relates to active drug-carrying liposomes of weak acid derivatives of paclitaxel, and preparation and application thereof.
  • Paclitaxel is a first-line chemotherapeutic agent, and it is currently one of the most effective drugs for the clinical treatment of breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer and other tumors.
  • paclitaxel is a fat-soluble drug and is insoluble in water. Therefore, the commercial solution of paclitaxel, Taxol, is made with polyoxyethylene castor oil and absolute ethanol as a mixed solvent. Due to the use of surfactants and organic solvents, severe allergic reactions and toxic side effects occurred during the application process, which greatly restricted its clinical application. In recent years, nanotechnology-based drug delivery systems have been favored by researchers.
  • Paclitaxel is a fat-soluble drug.
  • Paclitaxel liposomes (Lipusu) use its fat-soluble properties to load paclitaxel into the phospholipid bilayer through passive drug loading.
  • the encapsulation rate of the preparation obtained in this way is low, and the drug is encapsulated in the phospholipid bilayer, which is unstable, and is prone to sudden drug release in the body, which causes safety problems, and the efficacy is not improved significantly, and it is stored for a long time. It is easy to leak, which limits its clinical application. Therefore, although nanotechnology has greatly improved the drugability of paclitaxel, there are still many problems in actual clinical applications.
  • Active drug-loaded liposomes as a drug delivery vehicle, have received extensive attention from researchers. They have good stability, long circulation time in the body, can regulate the distribution of drugs in the body, and accumulate more in the tumor site, reducing damage to normal tissues. The damage, so as to achieve the purpose of increasing efficiency and reducing toxicity.
  • a variety of liposome drugs have been marketed in the world, such as Onivyde TM , Wait.
  • the active drug loading method mainly relies on pH gradient or metal ion gradient for drug loading.
  • the active drug loading technology is generally suitable for water-soluble drugs that are weakly acidic or alkaline or can coordinate with metal ions. It is difficult to use this for poorly soluble drugs.
  • This method is used to obtain liposomes with high encapsulation efficiency.
  • the development of solvent-assisted active drug loading technology provides the possibility to achieve active drug loading for insoluble drugs.
  • the method is to dissolve the poorly soluble drugs in an organic solvent miscible with water.
  • the organic solvent can increase the solubility of the drugs and increase the permeability of the membrane, thereby realizing the active encapsulation of the poorly soluble drugs.
  • paclitaxel which has neither weak acid or alkalinity nor complex with metal ions, it is difficult to achieve active encapsulation in the prior art. Therefore, we need to find more suitable methods to improve the drug delivery efficiency of paclitaxel.
  • the present invention provides an active drug-loaded liposome of paclitaxel weakly acidic derivative and a preparation method thereof.
  • the liposome actively encapsulates the weakly acidic derivative of paclitaxel in the liposome. In phase, it can improve the stability of the preparation and the drug loading, increase the drug-resistant dose, enhance the anti-tumor effect, and reduce the toxic and side effects.
  • the present invention provides a weakly acidic derivative of paclitaxel.
  • the weakly acidic derivative uses paclitaxel as a raw material and is connected to anhydrides or dibasic acids of saturated or unsaturated carbon chains of different lengths through esterification reactions, so that the drugs exhibit different Strength of weak acidity;
  • R 1 represents a spacer group, which can be a C1-C8 saturated alkane carbon chain, a C2-C8 alkene carbon chain or contains heteroatoms such as O, S, N, Se, C1-C8 alkane carbon chain of Si;
  • R 1 is preferably C1-C6 alkane or C2-C6 alkene
  • R 1 is preferably C1-C4 alkane or C2-C4 alkene
  • R 1 is preferably C2-C3 alkane or C4 alkene
  • the pKa of the weakly acidic derivative of the drug should satisfy 3 or more and 12 or less;
  • the weakly acidic paclitaxel derivative of the present invention is suitable for active loading into the aqueous phase of liposomes.
  • the weakly acidic derivative of paclitaxel can be stabilized in the internal water environment by electrostatic interaction with cations or formation of insoluble precipitates of metal cations in the internal water environment.
  • the present invention provides an active drug-loaded liposome of paclitaxel weakly acidic derivative, and a preparation method and application thereof:
  • Active drug-loaded liposomes of weakly acidic derivatives of paclitaxel include the following components: drugs, phospholipids, cholesterol, PEGylated phospholipids, calcium acetate and buffer salts.
  • the molar ratio of cholesterol content to all phospholipids is 10-45%, preferably 30-45%, and the mass ratio of the drug to the lipid material (phospholipid+cholesterol) is 1:20-1:5.
  • the phospholipids are natural phospholipids such as egg yolk lecithin (EPC), soybean phospholipids, sphingomyelin, hydrogenated soybean phospholipids (HSPC), or synthetic phospholipids such as distearoyl phosphatidyl choline (DSPC), dipalmitoyl phosphatidyl choline One or two of alkali (DPPC), dimyristoylphosphatidylcholine (DMPC), and distearoylphosphatidylglycerol (DSPG), preferably hydrogenated soybean phospholipid (HSPC) or distearoylphosphatidyl One or two of Choline (DSPC);
  • EPC egg yolk lecithin
  • soybean phospholipids soybean phospholipids
  • sphingomyelin hydrogenated soybean phospholipids
  • synthetic phospholipids such as distearoyl phosphatidyl choline (DSPC), dipalmitoyl phosphatidyl choline One or two of
  • the PEGylated phospholipid is one or more of DSPE-MPEG 1000, DSPE-MPEG 2000 , and DSPE-MPEG 5000 , preferably DSPE-MPEG 2000 ;
  • DSPC distearoylphosphatidylcholine
  • cholesterol DSPE-MPEG 2000
  • DSPC distearoylphosphatidylcholine
  • the present invention provides a method for preparing active drug-loaded liposomes of weakly acidic derivatives of paclitaxel, and the specific steps are as follows:
  • step (2) The gradient material is added to the film obtained in step (1), hydrated, and the particle size is reduced by an extrusion device to obtain a blank liposome with a uniform particle size.
  • the gradient substance described in step (2) is an acetate or phosphate solution, preferably an acetate solution, more preferably a calcium acetate solution, with a concentration of 50-200 mM.
  • the drug solution described in step (4) is an organic solution of drugs, wherein the organic solvent can be ethanol, dimethyl sulfoxide (DMSO), methanol, acetonitrile, acetone, N,N-dimethylformamide (DMF) , Preferably dimethyl sulfoxide (DMSO) and ethanol, the amount of organic solvent can be 2%-50% (V/V), preferably 5%-20%.
  • the organic solvent can be ethanol, dimethyl sulfoxide (DMSO), methanol, acetonitrile, acetone, N,N-dimethylformamide (DMF) ,
  • the amount of organic solvent can be 2%-50% (V/V), preferably 5%-20%.
  • the liposome of the present invention encapsulates the weakly acidic derivative of paclitaxel in an active drug loading mode, and encapsulates the drug in the aqueous phase of the liposome. It has a high encapsulation rate (>95%) and a high drug loading capacity.
  • the liposomes of the present invention have uniform particle size, good stability, not easy to leak, simple preparation process, and are suitable for industrial scale-up production.
  • Figure 1 shows the NMR and mass spectra of PTX-SA, PTX-GA, and PTX-DA in Examples 1 and 2 of the present invention.
  • Figure 2 is a mass spectrum of paclitaxel-thioglycolic acid derivative in Example 3 of the present invention.
  • Figure 3 is a graph of cytotoxicity in Example 7 of the present invention.
  • Figure 4 is a graph showing the drug-time curve of liposomes in the in vivo pharmacokinetic test of Example 8 of the present invention.
  • Figure 5 is a tissue distribution diagram of liposomes in Example 9 of the present invention.
  • Fig. 6 is a graph showing changes in tumor volume and body weight in the in vivo pharmacodynamic test of liposomes in Example 10 of the present invention.
  • Example 1 Synthesis of paclitaxel-succinic acid (PTX-SA) and paclitaxel-glutaric acid (PTX-GA) linked by ester bonds
  • Sepharose Sephadex G-50 is pre-equilibrated with 120 mM sodium sulfate aqueous solution, and the calcium acetate in the aqueous phase of the blank liposome is removed by column chromatography to form a transmembrane ion gradient;
  • PTX-SA and PTX-GA are dissolved in DMSO to prepare Into the mother liquor of 5mg/ml, the DMSO solution of the drug was added dropwise to the blank liposome according to the ratio of drug-to-lipid to mass ratio of 1:10, and incubated at 65°C for 30min with stirring. After the end, the drug loading was terminated in an ice bath.
  • Example 6 The influence of the concentration of the internal water phase on the encapsulation efficiency of weakly acidic derivatives
  • Sepharose Sephadex G-50 is pre-equilibrated with 120 mM sodium sulfate aqueous solution, and the calcium acetate in the aqueous phase of the blank liposome is removed by column chromatography to form a transmembrane ion gradient;
  • PTX-SA is dissolved in DMSO to prepare 5 mg/ml According to the ratio of the drug-to-lipid ratio of 1:10, the DMSO solution of the drug was added dropwise to the blank liposome, and the solution was stirred at 65°C for 30 min. After the end, the drug loading was terminated in an ice bath.
  • Example 7 In vitro cytotoxicity test
  • the MTT method (thiazole blue) was used to investigate the cytotoxicity of drugs and preparations on breast cancer cells (4T1) and prostate cancer cells (RM-1).
  • the specific operation steps are as follows: digest cells in good condition with trypsin, dilute the cell suspension with fresh medium to a concentration of 1 ⁇ 10 4 cells/mL, and then add 100 ⁇ L of cell suspension (1000 cells/mL) to a 96-well plate. Hole), stay in the incubator overnight, discard the old medium, add 200 ⁇ L of different concentrations of paclitaxel (PTX), paclitaxel-succinic acid (PTX-SA), paclitaxel-succinic acid lipid to each well In the culture medium of PTX-SA LPs, the control group was added with fresh medium without medicine.
  • PTX paclitaxel
  • PTX-SA paclitaxel-succinic acid
  • Example 8 In vivo pharmacokinetic test
  • Dosing regimen and blood sample collection A total of 6 rats were randomly divided into two groups, three in each group, fasted 12 hours before the start of the experiment, and the weight of each rat was weighed. Tail vein injection was given to each group of rats with paclitaxel solution (Taxol) and paclitaxel-succinic acid liposomes. The equivalent dose of paclitaxel injected to each rat was 4 mg/kg.
  • Example 9 In vivo tissue distribution test
  • mice 4T1 cells in the logarithmic growth phase were digested with trypsin and inoculated subcutaneously on the right side of BALB/C mice to establish a mouse breast cancer tumor model.
  • the tumor-bearing mice were randomly divided into two groups, nine mice in each group: (1) paclitaxel solution (8mg/kg); (2) paclitaxel-succinic acid liposome ( (Equivalent dose of paclitaxel), 6 hours after tail vein injection, 12 hours and 48 hours after administration, three mice were sacrificed, the heart, liver, spleen, lung, kidney and tumor tissues were taken out, washed with normal saline, and filter paper The water was sucked dry and stored in a refrigerator at -80°C.
  • the drug content in the organs and tumor tissues of the tumor-bearing mice was determined by UPLC-MS/MS. The result is shown in Figure 4. It can be seen from the results that compared with the solution, the liposome group has more drugs accumulated in the tumor site, and with the prolonged administration time, the drug has relatively more accumulation in the tumor site.
  • the 4T1 cells in the logarithmic growth phase were trypsinized, centrifuged, and the cells were resuspended in sterile PBS. After mixing, 200 ⁇ L of cell suspension was inoculated into the right subcutaneously of BALB/c mice to establish a mouse breast cancer tumor model . Closely observe the state of the mice and the growth of the tumor.
  • the tumor volume is about 100mm 3
  • the tumor-bearing mice are divided into 4 groups according to the tumor volume, 5 in each group, and PBS (blank control) and paclitaxel are injected into the tail vein.
  • paclitaxel-succinic acid liposome equivalent to paclitaxel 8mg/kg
  • paclitaxel-succinic acid liposome equivalent to paclitaxel 30mg/kg.
  • the dose was administered once two days apart, and a total of four doses were administered.
  • mice The tumor volume and body weight change curves of mice are shown in Figure 5.
  • the experimental results show that compared with paclitaxel solution, paclitaxel-succinic acid liposome has better anti-tumor effect, can significantly increase the drug resistance dose without causing toxic side effects, and the high-dose group has better anti-tumor effect obvious.

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Abstract

一种紫杉醇弱酸性衍生物主动载药脂质体及其制备方法与应用。涉及紫杉醇的弱酸性衍生物，所述的弱酸性衍生物是以紫杉醇为原料，通过酯化反应与不同长度的饱和或不饱和碳链的酸酐或者二元酸相连，使药物呈现不同强度的弱酸性；所述紫杉醇弱酸性衍生物结构可以用以下通式（I）表示：其中R 1如权利要求书和说明书所述。上述紫杉醇衍生物，通过pH梯度法将其主动包封于脂质体的内水相中，减少了聚氧乙烯蓖麻油，吐温80等表面活性剂的使用，避免了过敏反应的产生。所制备的脂质体包封率>95％，载药量高，稳定性好，在体内长循环，并且能够显著提高耐药剂量，从而达到增效、减毒的目的。

Description

紫杉醇弱酸性衍生物主动载药脂质体及其制备与应用 技术领域
本发明属于脂质体药物递送领域，涉及紫杉醇弱酸性衍生物主动载药脂质体及其制备与应用。
背景技术
紫杉醇(Paclitaxel，PTX)为一线化疗药，是目前临床治疗乳腺癌、非小细胞肺癌、卵巢癌，前列腺癌等多种肿瘤的最有效药物之一。但紫杉醇为脂溶性药物，不溶于水，因此紫杉醇的市售溶液剂泰素(Taxol)以聚氧乙烯蓖麻油和无水乙醇作为混合溶媒制成的。由于表面活性剂和有机溶剂的使用，使得在应用过程中产生严重的过敏反应及毒副作用，极大地限制了其临床应用。近年来，基于纳米技术的药物递送系统受到广大研究者的青睐，其中有很多被应用于紫杉醇的药物递送，并且有很多已经应用于临床实践中，包括紫杉醇脂质体(力朴素)，紫杉醇白蛋白纳米粒，紫杉醇纳米胶束等。紫杉醇为脂溶性药物，紫杉醇脂质体(力朴素)就是利用其脂溶性通过被动载药方式将紫杉醇载入磷脂双分子层。但这种方式所得的制剂包封率较低，且药物包载于磷脂双分子层，不稳定，易于在体内发生药物突释现象，进而产生安全性问题，且疗效改善不明显，并且长期储存易于泄漏，进而限制了其在临床上的应用。因此，虽然纳米技术极大地提高了紫杉醇的成药性，但在实际临床应用中仍然存在很多问题。
主动载药脂质体作为一种药物递送载体受到研究者的广泛关注，其具有稳定性好，体内循环时间长，能够调控药物在体内的分布，更多的蓄积在肿瘤部位，减少对正常组织的损伤，从而达到增效减毒的目的。目前，世界上已有多种脂质体药物上市，如
Figure PCTCN2020133184-appb-000001
Onivyde TM，
Figure PCTCN2020133184-appb-000002
等。主动载药法主要依靠pH梯度或者金属离子梯度进行载药，然而主动载药技术一般适用于弱酸弱碱性或能够与金属离子进行配位的水溶性药物，对于难溶性药物来说难以用这种方法获得高包封率的脂质体。溶剂辅助主动载药技术的发展为难溶性药物实现主动载药提供了可能。该方法是将难溶性药物溶解于与水混溶的有机溶剂中，有机溶剂能够增加药物溶解度并且提高膜的渗透性，进而实现了难溶性药物的主动包封。然而，对于紫杉醇这种既没有弱酸弱碱性又不具有与金属离子络合的药物来说，现有技术很难实现其主动包封。因此，我们需要寻找更合适的方法来提高紫杉醇的药物递送效率。
发明内容
针对现有技术存在的不足，本发明提供了一种紫杉醇弱酸性衍生物主动载药脂质体及其制备方法，所述脂质体是将紫杉醇弱酸性衍生物主动包封于脂质体内水相中，提高制剂稳定性及载药量，增大耐药剂量，增强抗肿瘤效果，减少毒副作用。
本发明采用的技术方案如下：
本发明提供了紫杉醇的弱酸性衍生物，所述的弱酸性衍生物是以紫杉醇为原料，通过酯化反应与不同长度的饱和或不饱和碳链的酸酐或者二元酸相连，使药物呈现不同强度的弱酸性；
上述紫杉醇弱酸性衍生物结构可以用以下通式表示：
PTX-O-CO-R 1-COOH
其中，-O-CO-为连接酯键，R 1表示间隔基团，可以为C1-C8的饱和烷烃碳链，C2-C8的烯烃碳链或含有杂原子如O、S、N、Se、Si的C1-C8烷烃碳链；
进一步的，R 1优选C1-C6的烷烃或C2-C6的烯烃；
进一步的，R 1优选C1-C4的烷烃或C2-C4烯烃；
再进一步的，R 1优选C2-C3的烷烃或C4的烯烃；
药物弱酸性衍生物具有的pKa应满足大于等于3，小于等于12；
根据上述的紫杉醇弱酸性衍生物可以是但不限于以下结构：
Figure PCTCN2020133184-appb-000003
本发明的紫杉醇弱酸性衍生物适合主动装载到脂质体内水相中。
进一步的，紫杉醇弱酸性衍生物在内水相环境中可以和阳离子通过静电相互作用或者金属阳离子形成难溶性沉淀而稳定在内水相环境中。
本发明提供一种紫杉醇弱酸性衍生物主动载药脂质体及其制备方法和应用：
紫杉醇弱酸性衍生物主动载药脂质体，包括以下组分：药物，磷脂，胆固醇，PEG化磷脂，醋酸钙和缓冲盐。其中胆固醇含量占所有磷脂组成的摩尔比为10-45％，优选为30-45％，药物与脂质材料(磷脂+胆固醇)的质量比为1:20～1：5。
所述的磷脂为天然磷脂如蛋黄卵磷脂(EPC)、大豆磷脂、鞘磷脂、氢化大豆磷脂(HSPC)，或合成磷脂如二硬酯酰基磷脂酰胆碱(DSPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、二硬脂酰磷脂酰甘油(DSPG)中的一种或两种，优选为氢化大豆磷脂(HSPC)或二硬酯酰基磷脂酰胆碱(DSPC)中的一种或两种；
PEG化磷脂为DSPE-MPEG 1000、DSPE-MPEG 2000、DSPE-MPEG 5000中的一种或几种，优选为DSPE-MPEG 2000；
进一步地，优选二硬酯酰基磷脂酰胆碱(DSPC)，胆固醇，DSPE-MPEG 2000组合。
本发明提供了紫杉醇弱酸性衍生物主动载药脂质体的制备方法，具体步骤如下：
(1)将磷脂、胆固醇、PEG化磷脂等溶解于适量有机溶剂中，减压旋转蒸发除去有机溶剂，得到均匀的薄膜。
(2)将梯度物质加入到步骤(1)中得到的薄膜中，水化，通过挤出设备降低粒度，得到粒径均一的空白脂质体。
(3)通过柱层析、超滤等方法，用缓冲盐除去脂质体外水相中的梯度物质，建立跨膜动力梯度，得到梯度空白脂质体。
(4)将药物溶液与梯度空白脂质体于相变温度之上进行搅拌孵育，即得到载药脂质体，除去其中剩余的有机溶剂，即得最终载药脂质体产品。
步骤(2)中所述的梯度物质为醋酸盐或磷酸盐溶液，优选为醋酸盐溶液，进一步优选为醋酸钙溶液，浓度为50-200mM。
步骤(4)中所述的药物溶液为药物的有机溶液，其中有机溶剂可以为乙醇，二甲基亚砜(DMSO)，甲醇，乙腈，丙酮，N,N-二甲基甲酰胺(DMF)，优选为二甲基亚砜(DMSO)和乙醇，有机溶剂的用量可以为2％-50％(V/V)，优选为5％-20％。
本发明的主要优点：
(1)本发明的脂质体，以主动载药方式包载紫杉醇弱酸性衍生物，将药物包封于脂质体内水相，具有包封率高(>95％)，载药量高的特点。
(2)本发明的脂质体粒径均一，稳定性好，不易泄露，制备工艺简单，适宜工业放大生产。
(3)本发明的脂质体，体内药动实验证明能够明显延长药物在血浆中的半衰期和生物利用度。
(4)本发明的脂质体，体内药效学试验证明，其具有更好的抗肿瘤效果，且最大耐药剂量更高，毒副作用更小。
附图说明：
图1为本发明实施例1，2中PTX-SA，PTX-GA，PTX-DA核磁图和质谱图。
图2为本发明实施例3中紫杉醇-硫代羟基乙酸衍生物质谱图。
图3为本发明实施例7中细胞毒性图。
图4为本发明实施例8中脂质体的体内药动试验药时曲线图。
图5为本发明实施例9中脂质体的体内组织分布图。
图6为本发明实施例10中脂质体的体内药效学试验肿瘤体积变化及体重变化图。
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但并不因此将发明限制于所述的实施例范围之内。
实施例1：酯键相连的紫杉醇-丁二酸(PTX-SA)和紫杉醇-戊二酸(PTX-GA)的合成
将紫杉醇，DMAP，丁二酸酐或戊二酸酐置于茄形瓶中，加入无水二氯甲烷溶解，室温反应5小时，TLC监测反应(二氯甲烷:甲醇＝10:1，0.1％冰醋酸)，反应完毕后，得到白色油状粘稠液体，将其溶于二氯甲烷中，用0.5M HCl洗两次，水洗一次，蒸干，通过柱层析法以二氯甲烷:甲醇100：1的洗脱溶剂(含千分之一冰醋酸)洗脱进行分离纯化，即得白色固体。
采用质谱和核磁共振氢谱( 1H-NMR)来确定衍生物的结构，选用溶剂为CDCl 3，结果如图1A和图1B，波谱解析结果如下：
PTX-SA:1H NMR(400MHz,Chloroform-d)δ8.07(m,2H,Ar-H),7.68(d,J＝7.8Hz,2H,Ar-H),7.54(t,J＝7.4Hz,1H,Ar-H),7.44(td,J＝7.4,3.3Hz,3H,Ar-H),7.32(m,7H,Ar-H),7.01(dd,J＝18.7,8.9Hz,1H,-NH-),6.22(s,1H,10-H),6.17(m,1H,13-H),5.92(dd,J＝9.2,3.1Hz,1H,3'-H),5.61(d,J＝7.0Hz,1H,2-H),5.46(d,J＝3.2Hz,1H,2'-H),4.89(m,1H,5-H),4.36(dd,J＝11.1,6.7Hz,1H,7-H),4.23(d,J＝8.5Hz,1H,20α-H),4.14(d,J＝8.4Hz,1H,20β-H),3.72(d,J＝7.0Hz,1H,3-H),2.61–2.52(m,4H,-OCCH2CH2-CO-),2.52(m,1H,6α-H),2.37(s,3H,4-COCH3),2.31(m,2H,14-H),2.14(s,3H,10-COCH3),1.84(m,3H,18-CH3),1.84(m,1H,6β-H),1.61(s,3H,19-CH3),1.15(s,3H,17-CH3),1.06(s,3H,16-CH3).
PTX-GA:1H NMR(400MHz,Chloroform-d)δ8.14(m,2H,Ar-H),7.73(d,J＝7.8Hz,2H,Ar-H),7.61(t,J＝7.4Hz,1H,Ar-H),7.52(td,J＝7.4,3.3Hz,3H,Ar-H),7.38(m,7H,Ar-H),7.20(dd,J＝18.7,8.9Hz,1H,-NH-),6.30(s,1H,10-H),6.26(m,1H,13-H),6.01(dd,J＝9.2,3.1Hz,1H,3'-H),5.69(d,J＝7.0Hz,1H,2-H),5.49(d,J＝3.2Hz,1H,2'-H),4.97(m,1H,5-H),4.44(dd,J＝11.1,6.7Hz,1H,7-H),4.29(d,J＝8.5Hz,1H,20α-H),4.20(d,J＝8.4Hz,1H,20β-H),3.82(d,J＝7.0Hz,1H,3-H),2.56(m,1H,6α-H),2.47(s,3H,4-COCH3),2.42(dt,J＝7.4,3.6Hz,2H,- OCCH2CH2CH2-CO-),2.38(m,2H,14-H),2.31(t,J＝7.1Hz,2H,-OCCH2CH2CH2-CO-),2.22(s,3H,10-COCH3),1.94(m,3H,18-CH3),1.86(m,1H,6β-H),1.86(dd,2H,-OCCH2CH2CH2-CO-),1.66(s,3H,19-CH3),1.21(s,3H,17-CH3),1.13(s,3H,16-CH3).
实施例2：酯键相连的紫杉醇-反式-二丁烯-1，4-二甲酸(PTX-DA)的合成
将紫杉醇，DMAP，反式-二丁烯-1，4二甲酸置于茄形瓶中，加入无水二氯甲烷溶解，冰水浴条件下搅拌40min，加入EDCI，继续搅拌1h，然后转移至室温反应2小时，薄层色谱监测反应(二氯甲烷:甲醇＝10:1，0.1％冰醋酸)，反应完毕后，用0.5M HCl洗两次，水洗一次，蒸干得白色固体。通过柱层析法以依次以二氯甲烷:甲醇100：1、50：1的洗脱溶剂(含千分之一冰醋酸)洗脱进行分离纯化，得白色固体。
采用质谱和核磁共振氢谱( 1H-NMR)来确定衍生物的结构，选用溶剂为CDCl 3，结果如图1C，波谱解析结果如下：
PTX-DA:1H NMR(400MHz,Chloroform-d)δ8.06(m,2H,Ar-H),7.66(d,J＝7.8Hz,2H,Ar-H),7.53(t,J＝7.4Hz,1H,Ar-H),7.44(td,J＝7.4,3.3Hz,3H,Ar-H),7.33(m,7H,Ar-H),6.95(dd,J＝18.7,8.9Hz,1H,-NH-),6.22(s,1H,10-H),6.17(m,1H,13-H),5.91(dd,J＝9.2,3.1Hz,1H,3'-H),5.61(d,J＝7.0Hz,1H,2-H),5.56-5.51(s,2H,-CH＝CH-),5.44(d,J＝3.2Hz,1H,2'-H),4.89(m,1H,5-H),4.36(dd,J＝11.1,6.7Hz,1H,7-H),4.23(d,J＝8.5Hz,1H,20α-H),4.13(d,J＝8.4Hz,1H,20β-H),3.74(d,J＝7.0Hz,1H,3-H),3.10-2.94(4H,-OCCH2CH＝CHCH2-CO-),2.48(m,1H,6α-H),2.37(s,3H,4-COCH3),2.26(m,2H,14-H),2.15(s,3H,10-COCH3),1.84(m,3H,18-CH3),1.81(m,1H,6β-H),1.61(s,3H,19-CH3),1.15(s,3H,17-CH3),1.06(s,3H,16-CH3).
实施例3：酯键相连的紫杉醇-硫代羟基乙酸衍生物的合成
将紫杉醇，DMAP，硫代羟基乙酸酐置于茄形瓶中，加入无水二氯甲烷溶解，室温反应5小时，TLC监测反应(二氯甲烷:甲醇＝10:1，0.1％冰醋酸)，反应完毕后，得到白色油状粘稠液体，将其溶于二氯甲烷中，用0.5M HCl洗两次，水洗一次，蒸干，通过柱层析法以二氯甲烷:甲醇100：1的洗脱溶剂(含千分之一冰醋酸)洗脱进行分离纯化，即得白色固体。
采用质谱来确定衍生物的结构，结果如图2。
实施例4：化合物稳定性比较
将合成的纯度>99％的PTX-SA，PTX-GA，PTX-DA置于4℃冰箱保存，分别在放置10天，20天，30天后通过高效液相色谱分别进行纯度测定，比较稳定性。
结果如表1所示，PTX-SA和PTX-GA具有良好的化学稳定性，而PTX-DA不稳定，30天内药物降解约20％。
表1 PTX-SA，PTX-GA，PTX-DA的放置10天，20天，30天纯度百分比
化合物 0天 10天 20天 30天
PTX-SA 99.4％ 99.2％ 99.3％ 99.1％
PTX-GA 99.2％ 99.0％ 98.9％ 98.7％
PTX-DA 99.3％ 94.5％ 87.6％ 79.8％
实施例5：紫杉醇弱酸性衍生物主动载药脂质体的制备
将DSPC、胆固醇、DSPE-PEG2000按照质量比68.1:22.2:1.2置于500mL茄形瓶中，加入氯仿使固体溶解，40℃下减压旋转蒸发成膜，加入120mM醋酸钙水溶液，于65℃旋转水化30min，在氮气流保护下分别通过孔径为400nm、200nm和100nm的聚碳酸酯膜各十次，获得粒径均一的空白脂质体。琼脂糖凝胶SephadexG-50用120mM硫酸钠水溶液预平衡，通过柱层析除去空白脂质体外水相的醋酸钙，以形成跨膜离子梯度；将PTX-SA、PTX-GA用DMSO溶解，配制成5mg/ml的母液，按照药脂比质量比1:10的比例，向空白脂质体中滴加药物的DMSO溶液，于65℃下搅拌孵育30min，结束后冰浴终止载药。
结果表明，PTX-SA载药后脂质体粒径约为122.6nm，测得包封率约为97.2％；而PTX-GA载药脂质体测得包封率仅为67.6％；因此不同碳链的酸修饰紫杉醇对脂质体包封率有影响。
实施例6：内水相浓度对弱酸性衍生物包封率的影响
将DSPC、胆固醇、DSPE-PEG2000按照质量比68.1:22.2:1.2置于500mL茄形瓶中，加入氯仿使固体溶解，40℃下减压旋转蒸发成膜，加入50mM、120mM和200mM醋酸钙水溶液，于65℃旋转水化30min，在氮气流保护下分别通过孔径为400nm、200nm和100nm的聚碳酸酯膜各十次，获得粒径均一的空白脂质体。琼脂糖凝胶SephadexG-50用120mM硫酸钠水溶液预平衡，通过柱层析除去空白脂质体外水相的醋酸钙，以形成跨膜离子梯度；将PTX-SA用DMSO溶解，配制成5mg/ml的母液，按照药脂比1:10的比例，向空白脂质体中滴加药物的DMSO溶液，于65℃下搅拌孵育30min，结束后冰浴终止载药。
结果表明：随着内水相浓度增加，PTX-SA脂质体包封率逐渐增加，当内水相醋酸钙浓度达到120mM时，包封率不再增加，因此选取120mM作为紫杉醇弱酸性衍生物的最优内水相浓度。
实施例7：体外细胞毒试验
采用MTT法(噻唑蓝)考察药物及制剂对乳腺癌细胞(4T1)，前列腺癌细胞(RM- 1)的细胞毒性。
具体操作步骤如下：用胰酶将状态良好的细胞消化下来，用新鲜培养基将细胞悬液稀释至浓度为1×10 4cells/mL，然后向96孔板加入100μL的细胞悬液(1000cells/孔)，于培养箱中过夜贴壁，弃去旧培养基，向各孔中加入200μL不同浓度的含有紫杉醇(PTX)，紫杉醇-丁二酸(PTX-SA)，紫杉醇-丁二酸脂质体(PTX-SA LPs)的培养液，对照组加入不含药的新鲜培养液，培养24小时或48小时后，每孔加入20μL浓度为5mg/mL的MTT溶液继续培养4小时。取出后弃去孔内溶液并用滤纸吸干，每孔加入200μLDMSO溶解生成的蓝紫色结晶，于振荡器上振摇10min使之完全溶解，用酶标仪测570nm波长下的吸光度值，并利用公式计算细胞抑制率，通过GraphPadPrism5.0计算各组IC50值。结果如图2，表1，表2所示。
表2紫杉醇，紫杉醇弱酸性衍生物及其脂质体对4T1细胞的半数致死浓度
Figure PCTCN2020133184-appb-000004
表3紫杉醇，紫杉醇弱酸性衍生物及其脂质体对RM-1细胞的半数致死浓度
Figure PCTCN2020133184-appb-000005
实施例8：体内药动试验
给药方案与血样采集共6只大鼠随机分为两组，每组三只，于实验开始前12h禁食禁水并称量每只大鼠的体重。尾静脉注射分别给予每组大鼠紫杉醇溶液剂(泰素)、紫杉醇-丁二酸脂质体，每只大鼠注射的紫杉醇等效剂量为4mg/kg。分别于给药后0.083h，0.25h，0.5h，1h，2h，4h，8h，12h，24h和48h时采集血样至涂有肝素的EP管中，13000rpm离心10min，取上清储存于-20℃中，使用UPLC-MS/MS测定大鼠血浆中紫杉醇和紫杉醇-丁二酸的浓度，使用DAS2.0软件计算药动参数。实验结果如图3，表3所示。根据实验结果可知，所制备的主动载药脂质体能够显著延长药物的半衰期，提高生物利用度。
表4紫杉醇及紫杉醇-丁二酸药动学参数
Figure PCTCN2020133184-appb-000006
实施例9：体内组织分布试验
将对数生长期的4T1细胞用胰酶进行消化接种于BALB/C小鼠右侧皮下，建立小鼠乳腺癌肿瘤模型。当肿瘤体积达到200-300mm 3左右时，将荷瘤小鼠随机分成两组，每组九只：(1)紫杉醇溶液剂(8mg/kg)；(2)紫杉醇-丁二酸脂质体(等效紫杉醇剂量)，尾静脉注射给药后6小时，12小时和48小时后分别处死三只小鼠，取出心、肝、脾、肺、肾和肿瘤组织，用生理盐水冲洗干净，用滤纸将水分吸干，储存于-80℃冰箱，使用UPLC-MS/MS测定荷瘤小鼠各器官和肿瘤组织中药物的含量。结果如图4所示。根据结果可以看出，与溶液剂相比，脂质体组药物在肿瘤部位蓄积更多，并且随着给药时间的延长，药物在肿瘤部位的蓄积相对更多。
实施例10：体内药效试验
将对数生长期的4T1细胞用胰酶消化，离心，用无菌PBS将细胞重悬，混匀后吸取200μL细胞悬液接种于BALB/c小鼠右侧皮下，建立小鼠乳腺癌肿瘤模型。密切观察小鼠状态及肿瘤生长情况，当肿瘤体积为100mm 3左右时将荷瘤小鼠按照肿瘤体积大小平均分为4组，每组5只，分别尾静脉注射给予PBS(空白对照)，紫杉醇溶液剂(8mg/kg)，紫杉醇-丁二酸脂质体(等效紫杉醇8mg/kg)，紫杉醇-丁二酸脂质体(等效紫杉醇30mg/kg)。间隔两天给药一次，共计给药四次，每天测量小鼠肿瘤体积及体重变化，小鼠肿瘤体积＝(肿瘤长径×肿瘤短径 2)/2。
小鼠肿瘤体积，体重变化曲线如图5所示。实验结果表明，与紫杉醇溶液剂相比，紫杉醇-丁二酸脂质体具有更好的抗肿瘤效果，在不产生毒副作用的情况下能够显著提高耐药剂量，并且高剂量组抗肿瘤效果更明显.

Claims (11)

  1. 紫杉醇弱酸性衍生物，其特征在于，所述的弱酸性衍生物结构通式如下所示：
    PTX-O-CO-R 1-COOH
    其中，-O-CO-为连接酯键，R 1表示间隔基团，可以为C1-C8的饱和烷烃碳链，C2-C8的烯烃碳链或含有杂原子如O、S、N、Se、Si的C1-C8烷烃碳链。
  2. 根据权利要求1所述的紫杉醇弱酸性衍生物，其特征在于，R 1为C1-C6的烷烃或C2-C6的烯烃。
  3. 根据权利要求1所述的紫杉醇弱酸性衍生物，其特征在于，修饰位点C-2’位羟基可以替换为C-7位羟基。
  4. 根据权利要求1-3任何一项所述的紫杉醇弱酸性衍生物，其特征在于，所述的紫杉醇弱酸性衍生物为如下结构：
    Figure PCTCN2020133184-appb-100001
  5. 包含权利要求1-4任何一项所述的紫杉醇弱酸性衍生物脂质体，其特征在于，包括以下组分：药物，磷脂，胆固醇，PEG化磷脂，醋酸盐溶液和缓冲盐，其中胆固醇含量占所有磷脂组成的摩尔比为10-45％，优选为30-45％，药物与脂质材料的质量比为1:20～1：5，所述的脂质材料为磷脂和胆固醇的总和。
  6. 根据权利要求5所述的主动载药脂质体，其特征在于，所述的磷脂为天然磷脂如卵磷脂、大豆磷脂、鞘磷脂、氢化大豆磷脂，或合成磷脂如二硬酯酰基磷脂酰胆碱、二棕榈酰基磷脂酰胆碱、二肉豆蔻酰磷脂酰胆碱、二硬脂酰磷脂酰甘油中的一种或两种，优选为氢化大豆磷脂或二硬酯酰基磷脂酰胆碱中的一种或两种。
  7. 根据权利要求5所述的主动载药脂质体，其特征在于，PEG化磷脂为DSPE-MPEG 1000、DSPE-MPEG 2000、DSPE-MPEG 5000中的一种或几种，优选为DSPE-MPEG 2000。
  8. 根据权利要求5所述的主动载药脂质体的制备方法，其特征在于，
    (1)将磷脂、胆固醇、PEG化磷脂等溶解于适量有机溶剂中，减压旋转蒸发除去有机溶剂，得到均匀的薄膜。
    (2)将梯度物质加入到步骤(1)中得到的薄膜中，水化，通过挤出设备降低粒度，得到粒径均一的空白脂质体。
    (3)通过柱层析、超滤等方法，用缓冲盐除去脂质体外水相中的梯度物质，建立跨膜动力梯度，得到梯度空白脂质体。
    (4)将药物溶液与梯度空白脂质体于相变温度之上进行搅拌孵育，即得到载药脂质体，除去其中剩余的有机溶剂，即得最终脂质体产品。
  9. 根据权利要求8所述的制备方法，其特征在于，步骤(2)中所述的梯度物质为醋酸盐或磷酸盐溶液，优选为醋酸盐溶液，进一步优选为醋酸钙溶液，浓度为50-200mM。
  10. 根据权利要求8所述的制备方法，步骤(4)中所述的药物溶液为药物的有机溶液，其中有机溶剂可以为乙醇，二甲基亚砜(DMSO)，甲醇，乙腈，丙酮，N,N-二甲基甲酰胺(DMF)，优选为二甲基亚砜(DMSO)和乙醇，有机溶剂的用量可以为2％-50％(V/V)，优选为5％-20％。
  11. 权利要求1-4中任何一项所述的紫杉醇弱酸性衍生物或权利要求6-7中任何一项所述的紫杉醇弱酸性衍生物主动载药脂质体在药物传递系统和制备抗肿瘤药物中的应用。
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