WO2017114186A1 - 特利加压素脂质体及其制备方法 - Google Patents

特利加压素脂质体及其制备方法 Download PDF

Info

Publication number
WO2017114186A1
WO2017114186A1 PCT/CN2016/110336 CN2016110336W WO2017114186A1 WO 2017114186 A1 WO2017114186 A1 WO 2017114186A1 CN 2016110336 W CN2016110336 W CN 2016110336W WO 2017114186 A1 WO2017114186 A1 WO 2017114186A1
Authority
WO
WIPO (PCT)
Prior art keywords
terlipressin
liposome
aqueous phase
phospholipid
liposome according
Prior art date
Application number
PCT/CN2016/110336
Other languages
English (en)
French (fr)
Inventor
张伟明
颜携国
陶安进
袁建成
Original Assignee
深圳翰宇药业股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳翰宇药业股份有限公司 filed Critical 深圳翰宇药业股份有限公司
Publication of WO2017114186A1 publication Critical patent/WO2017114186A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/095—Oxytocins; Vasopressins; Related peptides
    • 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

Definitions

  • the invention relates to the field of polypeptides, in particular to terlipressin liposomes and a preparation method thereof.
  • Terlipressin is triglycine lysine vasopressin acetate, a novel synthetic vasopressin analog. In the early 1980s, it was produced by the Swedish company Huiling. It is a prostaglandin prodrug. The glycyl group in the molecule after injection into the blood is catalytically hydrolyzed by the enzyme to produce a continuous low level of vasopressin. Its chemical name is N- ⁇ -triglycyl-8-lysine-vasopressin, and its structure is as shown in formula I:
  • the initial injection dosage is 2mg.
  • Each 1 mg of injection powder was dissolved in 5 ml of sodium chloride injection, and intravenous injection was performed slowly (more than 1 minute), and blood pressure and heart rate were observed.
  • the maintenance dose is 1-2 mg intravenously every 4 hours for 24-36 hours until bleeding is controlled.
  • Protein peptide drugs have large molecular mass, poor stability, low bioavailability, and half of biological organisms. Short-lived, easily decomposed by enzymes in the body, and their application in the field of therapeutics is limited. Due to the short half-life of terlipressin, the prior art overcomes common injections by preparing a drug to grow circulating liposomes, but ordinary liposomes have a shorter duration of action in vivo. Therefore, it is of great practical significance to provide a liposome liposome and a preparation method thereof for prolonging its half-life and improving the sustained-release effect.
  • the present invention provides a terlipressin liposome and a preparation method thereof.
  • the liposome provided by the present invention can obtain a slower release prescription by adjusting the prescription, and prolongs the action time in vitro and in vivo. It was found that the isoelectric point of terlipressin is alkaline, and the internal PH is weakly alkaline by adjusting the internal pH of the liposome. The drug is deposited under weak alkaline conditions, and the drug can be slowly released, thereby achieving better. Slow release effect.
  • the present invention provides the following technical solutions:
  • the present invention provides a terlipressin liposome having a pH of from 7.9 to 8.9 in the aqueous phase.
  • the essential component phospholipid and terlipressin form a phospholipid bilayer in the aqueous phase to constitute a liposome.
  • the solution constituting the aqueous phase is not particularly limited, and for example, etc.
  • Other physiologically active substances such as amino acids, vitamins and the like may also be added to the aqueous solution.
  • the invention is not limited thereto, and is within the scope of the invention.
  • the inner and/or outer aqueous phase of the terlipressin liposome comprises a monosaccharide, an oligosaccharide, a water soluble polysaccharide, a glycol, a small molecule polyol A mixture of one or more of a polymer polyol, an organic acid, an amino acid, a PBS phosphate buffer solution, sodium chloride, sodium hydroxide, sodium hydrogencarbonate, sodium carbonate, ammonium sulfate.
  • Phospholipids are essential substances constituting the lipid bilayer of the liposome and are essential components for accomplishing the object of the present invention.
  • the phospholipid substance for forming a liposome any natural or synthetic phospholipid substance may be used as long as it can form a lipid bilayer, and a phospholipid substance or a mixture of a plurality of phospholipid substances may be used.
  • the lipid of the terlipressin liposome comprises a phospholipid, one or both of a PEGylated phospholipid (DSPE-PEG, PEG-PE) The above mixture.
  • the monosaccharide of the terlipressin liposome is selected from the group consisting of erythrose, xylose, glucose, mannose, galactose or fructose;
  • the oligosaccharide is selected from the group consisting of sucrose, maltose, lactose, lactulose, trehalose, malt, and cyclodextrin;
  • the water-soluble polysaccharide is selected from the group consisting of water-soluble dextrin, cellulose or a derivative of starch;
  • the diol, the small molecule polyol is selected from a mixture of one or more of ethylene glycol, propylene glycol, glycerin, xylitol, sorbitol or mannitol;
  • the high molecular polyol is polyethylene glycol or a pharmaceutically acceptable derivative thereof, polyvinyl alcohol.
  • the PEGylated phospholipid (DSPE-PEG) in the terlipressin liposome is phosphatidylethanolamine, polyethylene glycol of distearoylphosphatidylethanolamine One or a mixture of two or more of the derivatives.
  • the PEGylated phospholipid (DSPE-PEG) in the terlipressin liposome is selected from the group consisting of DSPE-PEG-2000, DSPE-PEG-5000, DSPE-PEG1000-NHS , PEG-PE.
  • the invention is not limited thereto, and is within the scope of the invention.
  • the mass ratio of the terlipressin to the lipid is 1: (10 to 100). Preferably 1: (10 to 30).
  • the lipid further comprises cholesterol; the mass ratio of the cholesterol to the phospholipid is 1: (1 to 10). Preferably 1: (1 to 3).
  • the concentration of the phospholipid in the organic phase of the terlipressin liposome is from 10 to 100 mg/mL in an organic solvent; the organic solvent is selected from the group consisting of chloroform, acetone, Chloroform or ether. Preference is given to chloroform or diethyl ether.
  • the PBS phosphate buffer contains components such as disodium hydrogen phosphate and sodium dihydrogen phosphate, and dipotassium hydrogen phosphate dipotassium hydrogen phosphate.
  • the present invention also provides the terlipressin liposome comprising the following steps:
  • the aqueous phase is mixed with the organic phase to prepare a W/O colostrum, dried, and purified.
  • methods of preparing W/O colostrum include methods of sonication, high speed dispersion, emulsion homogenizer, nozzle atomization, and the like.
  • Laboratory preparation is often emulsified using a high-speed disperser or a vortex mixer; the W/O colostrum is dried on a rotary evaporator, the organic solvent is removed, and a salt solution suitable for storage and physiologically acceptable is added to remove free liposomes.
  • Drugs such as gel filtration, dialysis, ultracentrifugation, ultrafiltration, microcolumn centrifugation, etc. Finally, the free drug in the liposome is removed by ultrafiltration or dialysis membrane.
  • Liposomes are spheroids composed of phospholipids and other amphiphilic substances dispersed in water and encapsulated by one or more layers of concentric lipid bilayer membranes. Liposomes were first used as drug carriers by Rahman et al.
  • the commonly used preparation methods of liposome mainly include film dispersion method, reverse phase evaporation method, injection method, ultrasonic dispersion, etc., but for protein polypeptides, a higher encapsulation efficiency is obtained, and a reverse encapsulation method can obtain a higher encapsulation ratio. Film dispersion method, etc.
  • the reverse phase evaporation method was originally proposed by Szoka.
  • the preparation method mainly dissolves the membrane such as phospholipid in an organic solvent, and ultrasonically oscillates for a short time until a stable W/O emulsion is formed, and then the organic solvent is removed by evaporation under reduced pressure to reach a colloidal state.
  • the buffer salt was added dropwise, and the gel on the wall of the vessel was detached by rotary evaporation, and then evaporation was continued under reduced pressure to prepare an aqueous suspension, and the unencapsulated drug was removed to obtain a large unilamellar liposome.
  • This method can wrap a large volume of water, and is generally suitable for encapsulating water-soluble drugs, macromolecular biological active substances, and the like.
  • phospholipid can be selected, PEGylated phospholipid (DSPE-PEG) such as phosphatidylethanolamine, polyethylene glycol derivative of distearoylphosphatidylethanolamine, or the like, internal water phase/outer
  • DSPE-PEG PEGylated phospholipid
  • the aqueous phase can be selected from sucrose, mannitol, lactose, sorbitol, glucose, citric acid, sodium chloride, arginine, lysine, PBS buffered salt, etc.
  • liposomal phospholipids can also be added with or without adjustment.
  • Molecular layer membrane fluidity of cholesterol can be selected, PEGylated phospholipid (DSPE-PEG) such as phosphatidylethanolamine, polyethylene glycol derivative of distearoylphosphatidylethanolamine, or the like, internal water phase/outer
  • the aqueous phase can be selected from sucrose, mannitol, lactose,
  • the unmodified liposome After the ordinary liposome enters the circulatory system, the unmodified liposome mostly migrates to the mononuclear phagocytic system (MPS) rich parts such as the liver and spleen, and a small amount is taken up by the lung, bone marrow and kidney; and the hepatocyte membrane receptor Phospholipid negative bases that are directly exposed to the surface are recognized and thus are first engulfed by hepatocytes, so most of the liposomes have been phagocytosed before reaching the target site, failing to achieve an effective therapeutic concentration at the target site.
  • MPS mononuclear phagocytic system
  • the addition of polyethylene glycol can effectively improve this problem.
  • the long-circulating liposome can enhance the hydrophilicity of the liposome membrane due to the presence of the surface PEG layer, reduce the interaction between the plasma protein and the liposome membrane, and prevent the condensation of the liposome, which can be avoided to some extent.
  • the uptake of liposomes by the reticuloendothelial system allows the liposomes to have a longer half-life in vivo.
  • the basis for the development of doxorubicin long-circulating liposomes is the application of PEGylated long-circulating liposomes. Since the composition of the liposome contains PEG-DSPE, the effect is to prevent plasma proteins from adsorbing on the surface of the liposome, thereby reducing the uptake of MPS.
  • the present invention provides a terlipressin liposome having a pH of from 7.9 to 8.9 in the aqueous phase.
  • the use of the liposome preparation solves the shortcomings of short drug action time.
  • the isoelectric point of terlipressin is alkaline. By adjusting the pH value inside the liposome, the internal pH is weakly alkaline, and the drug is deposited under weak alkaline conditions, and the drug can be slowly released, thereby achieving better sustained release. effect.
  • the liposome provided by the invention can maintain a long action time, the blood drug concentration is stable, and the incidence of adverse reactions is low.
  • the invention discloses a terlipressin liposome and a preparation method thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the present invention aims to provide a liposome capable of effectively prolonging the release time of terlipressin in vivo, which composition contains the active ingredient terlipressin, and obtains a composition suitable for the patient's needs by selecting an appropriate prescription and process. .
  • phospholipid may be selected or the like or a combination thereof; PEGylated phospholipid (DSPE-PEG) such as phosphatidylethanolamine, polyethylene glycol derivative of distearoylphosphatidylethanolamine, or the like, such as DSPE - PEG-2000, DSPE-PEG-5000, DSPE-PEG1000-NHS, etc.; the inner aqueous phase/external aqueous phase can be selected from sucrose, mannitol, lactose, sorbitol, glucose, citric acid, sodium chloride, arginine, Lysine, PBS buffer salt, sodium hydroxide, sodium hydrogencarbonate, sodium carbonate, amine sulfate, etc.; cholesterol with liposome phospholipid bilayer membrane fluidity can also be selectively added or not.
  • PEGylated phospholipid such as phosphatidylethanolamine, polyethylene glycol derivative of distearoylphosphatidylethanolamine, or the like,
  • the present invention provides a liposome formulation having a drug to lipid ratio of from 1:10 to 1:100, particularly preferably from 1:10 to 1:30; a mass ratio of cholesterol to phospholipid of 1:10 to 1:1, particularly preferably 1:3 to 1:1; the phospholipid is used in an organic solvent in an amount of 10 mg/ml to 100 mg/ml; and the organic solvent may be chloroform, acetone, chloroform, diethyl ether or the like, preferably chloroform. Ether or a combination thereof.
  • the preparation process generally comprises: mixing the aqueous phase containing the drug with the organic phase of the lipid to prepare the W/O colostrum, and the organic solvent in the organic phase is generally a volatile solvent, usually chloroform or a mixture of chloroform and diethyl ether.
  • the lipid is a phospholipid, cholesterol, or the like.
  • the method for preparing the colostrum can be selected from the methods for preparing the emulsion, including ultrasonic, high-speed dispersion, emulsion homogenizer, nozzle atomization, and the like.
  • Laboratory preparation is often emulsified using a high-speed disperser or a vortex mixer; the W/O colostrum is dried on a rotary evaporator, the organic solvent is removed, and a salt solution suitable for storage and physiologically acceptable is added to remove free liposomes.
  • Drugs note that there are gel filtration, dialysis, ultracentrifugation, ultrafiltration, microcolumn centrifugation and so on.
  • the free drug in the liposome is removed by ultrafiltration or dialysis membrane.
  • the present invention provides a terlipressin liposome having a pH of from 7.9 to 8.9 in the aqueous phase.
  • the use of the liposome preparation solves the shortcomings of short drug action time.
  • the isoelectric point of terlipressin is alkaline.
  • the pH value inside the liposome is weakly alkaline, and the drug is deposited under weak alkaline conditions, and the drug can be slowly released, thereby achieving better sustained release. effect.
  • the liposome provided by the invention can maintain a long action time, the blood drug concentration is stable, and the incidence of adverse reactions is low.
  • the terlipressin liposome provided by the present invention and the raw materials and reagents used in the preparation method thereof are commercially available.
  • the aqueous phase was added, and the pH was set to 8.0 from sodium hydrogencarbonate and sodium hydroxide.
  • the crude liposome was obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by dialysis membrane method. That is, a liposome solution of the drug is obtained.
  • the aqueous phase is the liposome solution from which the drug is obtained.
  • the aqueous phase is added, and the pH of the sodium carbonate and citric acid is between 7.9 and 8.0.
  • the crude liposome is obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by dialysis membrane method. That is, a liposome solution of the drug is obtained.
  • the aqueous phase is added, and the pH of the ammonium sulfate and sodium hydroxide is between 8.8 and 8.9.
  • the crude liposome is obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by dialysis membrane method. That is, a liposome solution of the drug is obtained.
  • the aqueous phase is added, and the pH is set between 8.0 and 8.5 by ammonium sulfate and sodium hydroxide.
  • the crude liposome is obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by dialysis membrane method. That is, a liposome solution of the drug is obtained.
  • aqueous phase containing the active ingredient, DSPE-PEG2000 2g, cholesterol 1g dissolved in chloroform 20mL (oil phase), placed in a round bottom flask, rotary evaporation to remove the organic phase
  • the aqueous phase is added, and the pH is adjusted from 7.0 to 7.1 by ammonium sulfate and sodium hydroxide.
  • the crude liposome is obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove free water by dialysis membrane method. Phase, that is, a liposome solution of the drug is obtained.
  • aqueous phase containing the active ingredient, DSPE-PEG2000 2g, cholesterol 1g dissolved in chloroform 20mL (oil phase), placed in a round bottom flask, rotary evaporation to remove the organic phase , adding aqueous phase, prepared from ammonium sulfate and sodium hydroxide
  • the pH is between 10.2 and 10.3, and a rough liposome is obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by a dialysis membrane method to obtain a liposome solution of the drug.
  • the aqueous phase was added, and the pH was adjusted to 7.5 by ammonium sulfate and sodium hydroxide.
  • the crude liposome was obtained by vortexing.
  • the particle size was first reduced by a homogenizer, and the free aqueous phase was removed by ultrafiltration, and then passed through 0.22.
  • a filter of ⁇ m that is, a liposome solution of the drug is obtained.
  • the aqueous phase was added, and the pH was adjusted to 9.2 by ammonium sulfate and sodium hydroxide.
  • the crude liposome was obtained by vortexing, and then homogenized and passed through a 0.22 ⁇ m filter membrane to remove the free aqueous phase by dialysis membrane method. A liposome solution of the drug is obtained.
  • the obtained liposome was placed in a stoppered test tube, and 4 ml of human plasma containing 0.01% NaN3 was added, fixed on the stirring slurry of the dissolution apparatus, and immersed in the aqueous solution of the dissolution cup, the temperature of the water solution was maintained at 37 ° C, and the slurry was stirred at 100 rpm. Turn. 0.5 ml was sampled at the set time. The obtained sample was added with 4.5 ml of physiological saline, centrifuged at 10000*g for 10 min, the supernatant was discarded, and the precipitate was dissolved in an appropriate amount of acidified methanol. After filtration, HPLC was determined to obtain lipid at each time point. The amount of drug released in the body, the following formula calculates the percentage of drug release at each time point:
  • Drug release (%) (amount of total drug encapsulated in liposome - amount of drug not released) / amount of drug encapsulated in liposome x 100%.
  • the test results are shown in Table 2:
  • Example 11 Determination of blood concentration in vivo
  • In vivo blood concentration 108 rats were randomly divided into 9 groups, 12 in each group, and samples were given intravenously according to Examples 1 to 5 and Comparative Examples 1 to 4, respectively, and then 0.5 d after administration. 1d, 2d, 4d, 8d blood collection, in vitro heparin anticoagulation, centrifugation of plasma at 8000r.min -1 , cryopreservation.
  • the plasma was taken into a centrifuge tube, the protein was precipitated by adding methanol, vortexed and mixed, and then centrifuged. The supernatant was treated with an acetonitrile solvent, and the blood concentration was measured by HPLC. The results are shown in Table 3:

Abstract

本发明提供了一种特利加压素脂质体，其内水相的pH值为7.9～8.9。还提供了特利加压素脂质体的制备方法。

Description

特利加压素脂质体及其制备方法
本申请要求于2015年12月31日提交中国专利局、申请号为201511031763.3、发明名称为“特利加压素脂质体及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及多肽领域，特别涉及特利加压素脂质体及其制备方法。
背景技术
特利加压素为三甘氨酰基赖氨酸加压素醋酸盐，是一种新型人工合成的血管加压素类似物。二十世界八十年代初由瑞典辉凌公司生产上市，为加压素的前体药物，在注射入血液后分子中的甘氨酰基被酶催化水解而产生持续低水平的加压素。其化学名称为N-α-三甘氨酰-8-赖氨酸-加压素，其结构如式Ⅰ所示：
主要用于肝硬化静脉曲张出血的止血，现临床广泛应用于肝肾综合征、肝硬化腹水、感染性休克、烧伤、急性肝功能衰竭、心脏骤停等的治疗。目前市场上的翰唯，起始注射用量为2mg。每1mg注射粉针剂用5ml氯化钠注射液溶解，缓慢进行静脉注射(超过1分钟)，同时对血压及心率观测。维持剂量为每4小时静脉给药1-2mg，延续24-36小时，直至出血得到控制。
蛋白多肽类药物分子质量大，稳定性差，生物利用度低，体内生物半 衰期短，极易被生物体内的酶分解等缺点，使他们在治疗学领域的应用受到局限。由于特利加压素半衰期短，现有技术通过把药物制备成长循环脂质体，克服普通注射剂，但是普通脂质体在体内作用时间较短。因此提供一种特利加压素脂质体及其制备方法，以延长其半衰期，提高缓释效果具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种特利加压素脂质体及其制备方法。本发明提供的脂质体通过调节处方可以获得释放更为缓慢的处方，延长在体内外作用时间。实验发现由于特利加压素等电点偏碱性，通过调节脂质体内部PH，使内部PH呈弱碱性，药物在弱碱性条件下沉积，药物能够缓慢释放，从而达到更好的缓释效果。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种特利加压素脂质体，其内水相的pH值为7.9～8.9。
在脂质体中，必要成分磷脂和特利加压素是在水相中形成磷脂双层从而构成脂质体的，本发明中，对构成水相的溶液不加特别限制，例如可以采用等渗或者非等渗的药物成分的水溶液、缓冲液、生理盐水、葡萄糖水溶液等。水溶液中也可以加入其它生理活性物质如氨基酸、维生素等。均在本发明的保护范围之内，本发明在此不做限定。
在本发明的一些具体实施方案中，所述特利加压素脂质体的内水相和/或外水相包括单糖、低聚糖、水溶性多糖、二元醇、小分子多元醇、高分子多元醇、有机酸、氨基酸、PBS磷酸缓冲盐溶液、氯化钠、氢氧化钠、碳酸氢钠、碳酸钠、硫酸铵中的一种或两者以上的混合物。
磷脂是构成脂质体脂质双层的必须物质，是完成本发明目的的必须成分。作为用来形成脂质体的磷脂类物质，只要可以形成脂质双层，可以使用任何天然或者合成的磷脂类物质，也可以使用一种磷脂类物质或者多种磷脂类物质的混合物。
在本发明的一些具体实施方案中，所述特利加压素脂质体的脂质包括磷脂，所述磷脂为PEG化磷脂(DSPE-PEG，PEG-PE)中的一种或两者 以上的混合物。
在本发明的一些具体实施方案中，所述特利加压素脂质体的所述单糖选自赤藓糖、木糖、葡萄糖、甘露糖、半乳糖或果糖；
所述低聚糖选自蔗糖、麦芽糖、乳糖、乳果糖、海藻糖、麦芽三塘和环糊精；
所述水溶性多糖选自水溶性糊精、纤维素或淀粉的衍生物；
所述二元醇、所述小分子多元醇选自乙二醇、丙二醇、甘油、木糖醇、山梨醇或甘露醇中的一种或两者以上的混合物；
所述高分子多元醇为聚乙二醇或其可药用的衍生物、聚乙烯醇。
在本发明的一些具体实施方案中，所述特利加压素脂质体中所述所述PEG化磷脂(DSPE-PEG)为磷脂酰乙醇胺，二硬脂酰磷脂酰乙醇胺的聚乙二醇衍生物中的一种或两者以上的混合物。
在本发明的一些具体实施方案中，所述特利加压素脂质体中所述PEG化磷脂(DSPE-PEG)选自DSPE-PEG-2000，DSPE-PEG-5000，DSPE-PEG1000-NHS，PEG-PE。均在本发明的保护范围之内，本发明在此不做限定。
在本发明的一些具体实施方案中，所述特利加压素与所述脂质的质量比为1:(10～100)。优选1:(10～30)。
在本发明的一些具体实施方案中中脂质还包括胆固醇；所述胆固醇与所述磷脂的质量比为1:(1～10)。优选1:(1～3)。
在本发明的一些具体实施方案中，所述特利加压素脂质体的有机相中所述磷脂在有机溶剂中的浓度为10～100mg/mL；所述有机溶剂选自氯仿、丙酮、氯仿或乙醚。优选氯仿或乙醚。
在本发明中，PBS磷酸盐缓冲液，其成分包括磷酸氢二钠和磷酸二氢钠，磷酸氢二钾合磷酸二氢钾。
本发明还提供了所述特利加压素脂质体，包括如下步骤：
制备获得含有特利加压素的水相，调整pH值为7.9～8.9；
制备获得含有脂质的有机相；
取所述水相与所述有机相混合，制备获得W/O初乳，干燥，纯化。
在本发明的一些具体实施方案中，制备获得W/O初乳的方法包括超声、高速分散、乳匀机、喷嘴雾化等方法。实验室制备常采用高速分散器或涡旋混合器乳化；W/O初乳在旋转蒸发器上干燥，除去有机溶剂，加入适于储存和生理上可以接受的盐溶液，除去脂质体中游离药物，有凝胶过滤法，透析法，超速离心法，超滤法，微柱离心法等。最后通过超滤或透析膜除去脂质体中的游离药物。
脂质体是由磷脂和其它两亲性物质分散于水中，由一层或多层同心的脂质双分子膜包封而成的球状体。最早由Rahman等人将脂质体作为药物载体应用。脂质体常用制备方法主要有薄膜分散法，反相蒸发法，注入法，超声波分散等，但是对于蛋白多肽，要获得较高的包封率，逆向蒸发法可以获得较高的包封率，薄膜分散法等。
反相蒸发法最初由Szoka提出，制备方法主要将磷脂等膜材溶于有机溶剂中，短时超声振荡，直至形成稳定的W/O乳液，然后减压蒸发除掉有机溶剂，达到胶态后，滴加缓冲盐，旋转蒸发使器壁上的凝胶脱落，然后在减压下继续蒸发，制得水性混悬液，除去未包入的药物，即得大单层脂质体。此法可包裹较大的水容积，一般适用于包封水溶性药物，大分子生物活性物质等。
本发明提供的脂质体，磷脂可以选择，，PEG化磷脂(DSPE-PEG)如磷脂酰乙醇胺，二硬脂酰磷脂酰乙醇胺的聚乙二醇衍生物等或其组合，内水相/外水相可以选择蔗糖，甘露醇，乳糖，山梨醇，葡萄糖，柠檬酸，氯化钠，精氨酸，赖氨酸，PBS缓冲盐等；还可以选择性加入或不加调节脂质体磷脂双分子层膜流动性的胆固醇。
普通脂质体进入循环系统后，未经修饰的脂质体大部分运转至肝脏和脾脏等单核吞噬细胞系统(MPS)丰富的部位，少量被肺，骨髓及肾摄取；而肝细胞膜受体对直接暴露于表面的磷脂负电基进行识别，因而首先被肝细胞吞噬，因此大部分脂质体在到达靶部位之前已被吞噬，无法在靶部位达到有效的治疗浓度。聚乙二醇的加入可有效改善这一问题。长循环脂质体由于表面PEG层的存在，可增强脂质体膜的亲水性，减少血浆蛋白与脂质体膜的相互作用，阻止脂质体的凝聚说融合，可在一定程度上避 免网状内皮系统对脂质体的摄取，使得脂质体在体内具有较长的半衰期。阿霉素长循环脂质体得以发展的基础正是PEG化长循环脂质体的应用。由于脂质体的组成中含有PEG-DSPE，作用是阻止血浆蛋白吸附于脂质体的表面，从而减少MPS的摄取。
本发明提供了一种特利加压素脂质体，其内水相的pH值为7.9～8.9。使用该脂质体制剂解决了药物作用时间短的缺点。特利加压素等电点偏碱性，通过调节脂质体内部pH值，使内部pH呈弱碱性，药物在弱碱性条件下沉积，药物能够缓慢释放，从而达到更好的缓释效果。
本发明提供的脂质体后可以维持较长作用时间，血药浓度平稳，不良反应发生率低。
具体实施方式
本发明公开了一种特利加压素脂质体及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明旨在提供一种能有效延长特利加压素在体内释放时间的脂质体，该组合物含有活性成分特利加压素，通过选择合适的处方和工艺获得适合病人需求的组合物。
本发明提供的脂质体，磷脂可以选择等或其组合；PEG化磷脂(DSPE-PEG)如磷脂酰乙醇胺，二硬脂酰磷脂酰乙醇胺的聚乙二醇衍生物等或其组合，如DSPE-PEG-2000，DSPE-PEG-5000，DSPE-PEG1000-NHS等；内水相/外水相可以选择蔗糖，甘露醇，乳糖，山梨醇，葡萄糖，柠檬酸，氯化钠，精氨酸，赖氨酸，PBS缓冲盐，氢氧化钠，碳酸氢钠，碳酸钠，硫酸胺等；还可以选择性加入或不加调节脂质体磷脂双分子层膜流动性的胆固醇。
在一些实施例中，本发明提供的脂质体处方，药脂比从1：10到1：100，特别优选的是1：10到1：30；胆固醇与磷脂的质量比为1：10到1：1，特别优选的是1：3到1：1；磷脂在有机溶剂中的用量为10mg/ml到100mg/ml；有机溶剂可以使用氯仿，丙酮，氯仿，乙醚等，优选的是氯仿，乙醚或其组合。
主要通过以下技术放案实现：
制备工艺过程一般包括：含有药物的水相与脂质的有机相混合制备W/O初乳，有机相中的有机溶剂一般采用易挥发的溶剂，通常为氯仿或氯仿与乙醚的混合液。脂质为磷脂、胆固醇等。制备初乳的方法可以从制备乳剂的方法中选择，包括超声、高速分散、乳匀机、喷嘴雾化等方法。实验室制备常采用高速分散器或涡旋混合器乳化；W/O初乳在旋转蒸发器上干燥，除去有机溶剂，加入适于储存和生理上可以接受的盐溶液，除去脂质体中游离药物，注意有凝胶过滤法，透析法，超速离心法，超滤法，微柱离心法等。
最后通过超滤或透析膜除去脂质体中的游离药物。
本发明提供了一种特利加压素脂质体，其内水相的pH值为7.9～8.9。使用该脂质体制剂解决了药物作用时间短的缺点。特利加压素等电点偏碱性，通过调节脂质体内部pH值，使内部pH呈弱碱性，药物在弱碱性条件下沉积，药物能够缓慢释放，从而达到更好的缓释效果。本发明提供的脂质体后可以维持较长作用时间，血药浓度平稳，不良反应发生率低。
缩写及英文对应的含义请见表1：
表1缩写及英文含义
缩写及英文 含义
DSPE-PEG 二硬脂酰基磷脂酰乙醇胺-聚乙二醇
PEG-PE 磷脂酰乙醇胺-聚乙二醇
HSPC 氢化磷脂
SPC 大豆磷脂
本发明提供的特利加压素脂质体及其制备方法中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：
将药物特利加压素500mg溶于20mL水相中得到含有活性成分的水相；
DSPE-PEG2000 2g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由碳酸氢钠与氢氧化钠配制得pH在8.0之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
实施例2：
将药物特利加压素5mg溶于20mL水相中得到含有活性成分的水相；
PEG-PE 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由硫酸铵与氢氧化钠，蔗糖配制的pH在8.4～8.5之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
实施例3：
将药物特利加压素5mg溶于20mL水相中得到水相；
DSPE-PEG2000 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由碳酸钠与柠檬酸配制的pH在7.9～8.0之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
实施例4：
将药物特利加压素5mg溶于20mL水相中得到含有活性成分的水相；
DSPE-PEG2000 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由硫酸铵与氢氧化钠配制的pH在8.8～8.9之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
实施例5：
将药物特利加压素5mg溶于20mL水相中得到含有活性成分的水相；DSPE-PEG2000 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由硫酸铵与氢氧化钠配制得pH在8.0～8.5之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
对比例1：
将药物特利加压素5mg溶于20mL水相中得到含有活性成分的水相，DSPE-PEG2000 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相，加入水相，由硫酸铵与氢氧化钠配制得pH在7.0～7.1之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
对比例2：
将药物特利加压素5mg溶于20mL水相中得到含有活性成分的水相，DSPE-PEG2000 2g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相，加入水相，由硫酸铵与氢氧化钠配制的 pH在10.2～10.3之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
对比例3：
将药物特利加压素50mg溶于30mL水相中得到含有活性成分的水相；
大豆磷脂3g、胆固醇3g溶于氯仿-乙醚(1：1)30mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由硫酸铵与氢氧化钠配制得pH在7.5之间，涡旋得到粗略的脂质体，先通过均质机缩小粒径，使用超滤法除去游离的水相，再通过0.22μm的滤膜，即得到药物的脂质体溶液。
对比例4：
将药物特利加压素300mg溶于20mL水相中得到含有活性成分的水相；
氢化磷脂3g、胆固醇1g溶于氯仿20mL(油相)中，置圆底烧瓶中，旋转蒸发除去有机相；
加入水相，由硫酸铵与氢氧化钠配制得pH在9.2之间，涡旋得到粗略的脂质体，先均质再通过0.22μm的滤膜，使用透析膜法除去游离的水相，即得到药物的脂质体溶液。
实施例10：药物释放度的检测
取得到的脂质体置于具塞试管中，加入4ml含0.01％NaN3的人血浆，固定于溶出仪的搅拌浆上并浸入溶出杯的水液中，水液温度保持37℃，搅拌浆100rpm转动。于设定的时间取样0.5ml，所得样品加入4.5ml生理盐水，10000*g离心10min，弃去上清液，沉淀溶于适量酸化甲醇中，过滤后，HPLC测定，即得各时间点脂质体中未释放的药物量，以下式计算各个时间点的药物释放百分数：
药物释放(％)＝(包封于脂质体中的总药物量-未释放的药物量)/包封于脂质体中的药物量×100％。检测结果见表2：
表2：不同实施例制备的样品的释放度
Figure PCTCN2016110336-appb-000002
结论：从表1可以看出使用DSPE-PEG，氢化磷脂，大豆磷脂制备的脂质体释放时间依次缩短，氢化磷脂刚性交普通磷脂强，药物不容易从囊中泄露有关，而DSPE-PEG相对较长，可能与DSPE-PEG是两亲型聚合物，在脂质体表面交错覆盖成致密的构象云，形成较厚的立体位阻层从而比较稳定有关，而在脂质体内水相呈弱碱性条件下，脂质体释放时间更加延长，从实施例1-5说明只有内水相的pH值在一定范围内才有较好的缓释效果，对比实施例1与2可以发现内水相的pH值为7.9～8.9之间具有较好的缓释效果。
证明了发明者的思路，即由于特利加压素本身呈碱性，内水相呈碱性，药物呈沉淀沉降，从而缓慢释放。
实施例11：体内血药浓度的测定
体内血药浓度：取大鼠108只，随机分成9组，每组各12只，分别静脉注射给予按实施例1～5、对比例1～4样品，然后在给药后分别于0.5d， 1d，2d，4d，8d采血，体外肝素抗凝，以8000r.min-1离心血浆，低温保存。
取血浆至离心管中，加入甲醇沉淀蛋白，涡旋混合后离心，将上清液用乙腈溶剂后，进样，用HPLC测定血药浓度。结果见表3:
表3：不同样品的大鼠体内动物数据
Figure PCTCN2016110336-appb-000003
从表3结果可以看出，实施例1样品在大鼠体内的达峰时间(2d)相对实施例4(4d)较短，实施例2的血药浓度较为平缓，即实施例2释放时间较长，提示在实施例2中加入通过调节PH具有一定的阻碍活性成分迁移的作用，从而达峰浓度较低，释放时间更长，即有缓释释放效果。对比例3和对比例4也证明了氢化磷脂较普通磷脂有相对较长的释放时间，但均较DSPE-PEG较快，实施例1-5说明只有内水相的pH值在为7.9～8.9之间具有较好的缓释效果，对比实施例1与2也佐证了这个发明。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种特利加压素脂质体，其特征在于，其内水相的pH值为7.9～8.9。
  2. 根据权利要求1所述的特利加压素脂质体，其特征在于，其内水相和/或外水相包括单糖、低聚糖、水溶性多糖、二元醇、小分子多元醇、高分子多元醇、有机酸、氨基酸、PBS磷酸缓冲盐溶液、氯化钠、氢氧化钠、碳酸氢钠、碳酸钠、硫酸铵中的一种或两者以上的混合物。
  3. 根据权利要求1或2所述的特利加压素脂质体，其特征在于，其脂质包括磷脂，所述磷脂为或PEG化磷脂中的一种或两者以上的混合物。
  4. 根据权利要求2或3所述的特利加压素脂质体，其特征在于，所述单糖选自赤藓糖、木糖、葡萄糖、甘露糖、半乳糖或果糖；
    所述低聚糖选自蔗糖、麦芽糖、乳糖、乳果糖、海藻糖、麦芽三塘和环糊精；
    所述水溶性多糖选自水溶性糊精、纤维素或淀粉的衍生物；
    所述二元醇、所述小分子多元醇选自乙二醇、丙二醇、甘油、木糖醇、山梨醇或甘露醇中的一种或两者以上的混合物；
    所述高分子多元醇为聚乙二醇或其可药用的衍生物、聚乙烯醇。
  5. 根据权利要求3或4所述的特利加压素脂质体，其特征在于，所述磷脂选自；
    PEG化磷脂为磷脂酰乙醇胺，二硬脂酰磷脂酰乙醇胺的聚乙二醇衍生物中的一种或两者以上的混合物。
  6. 根据权利要求3至5任一项所述的特利加压素脂质体，其特征在于，所述PEG化磷脂选自DSPE-PEG-2000，DSPE-PEG-5000，DSPE-PEG1000-NHS。
  7. 根据权利要求3至6任一项所述的特利加压素脂质体，其特征在于，所述特利加压素与所述脂质的质量比为1:(10～100)。
  8. 根据权利要求3至7任一项所述特利加压素脂质体，其特征在于，所述脂质还包括胆固醇；所述胆固醇与所述磷脂的质量比为1:(1～10)。
  9. 根据权利要求3至8任一项所述的特利加压素脂质体，其特征在 于，其有机相中所述磷脂在有机溶剂中的浓度为10～100mg/mL；所述有机溶剂选自氯仿、丙酮、氯仿或乙醚。
  10. 根据权利要求1至9任一项所述的特利加压素脂质体，其特征在于，包括如下步骤：
    制备获得含有特利加压素的水相，调整pH值为7.9～8.9；
    制备获得含有脂质的有机相；
    取所述水相与所述有机相混合，制备获得W/O初乳，干燥，纯化。
PCT/CN2016/110336 2015-12-31 2016-12-16 特利加压素脂质体及其制备方法 WO2017114186A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201511031763.3 2015-12-31
CN201511031763.3A CN106924715A (zh) 2015-12-31 2015-12-31 特利加压素脂质体及其制备方法

Publications (1)

Publication Number Publication Date
WO2017114186A1 true WO2017114186A1 (zh) 2017-07-06

Family

ID=59224473

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/110336 WO2017114186A1 (zh) 2015-12-31 2016-12-16 特利加压素脂质体及其制备方法

Country Status (2)

Country Link
CN (1) CN106924715A (zh)
WO (1) WO2017114186A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114469733A (zh) * 2021-10-13 2022-05-13 成都科建生物医药有限公司 一种阿霉素脂质体的制备装置与制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110585170A (zh) * 2019-09-17 2019-12-20 南京赛弗斯医药科技有限公司 一种采用3d打印制备的醋酸特利加压素注射用缓释微球及其制备方法
CN114177147B (zh) * 2021-12-15 2023-05-23 福建省闽东力捷迅药业股份有限公司 一种注射用特利加压素的制备方法以及制得的注射用特利加压素

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101301465A (zh) * 2008-04-16 2008-11-12 山东农业大学 禽转移因子纳米脂质体及其制备方法
CN104761618A (zh) * 2015-01-06 2015-07-08 苏州天马医药集团天吉生物制药有限公司 特利加压素的纯化制备方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006009825A1 (en) * 2004-06-17 2006-01-26 Virun, Inc. Compositions comprising a mucoadhesive protein and an active principle for mucosal delivery of said agents
CN101404980A (zh) * 2006-01-24 2009-04-08 耐百生物制药有限公司 大分子微球的制备技术
CN101327190B (zh) * 2008-07-29 2011-04-27 北京大学 一种供注射用的抗肿瘤长循环靶向脂质体
CN101653416B (zh) * 2009-09-22 2012-05-30 北京大学 一种整合素介导的肿瘤双重靶向脂质体及其制备方法
BR112013003823A2 (pt) * 2010-08-20 2016-06-28 Cerulean Pharma Inc conjugados de peptídeo terapêutico-políemro, partículas, composições, e métodos relacionados
CN103505418A (zh) * 2012-06-29 2014-01-15 国家纳米科学中心 一种药物载体和药物组合物及其制备方法
CN102935068B (zh) * 2012-10-19 2014-12-17 浙江海正药业股份有限公司 一种包载水溶性药物的脂质体制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101301465A (zh) * 2008-04-16 2008-11-12 山东农业大学 禽转移因子纳米脂质体及其制备方法
CN104761618A (zh) * 2015-01-06 2015-07-08 苏州天马医药集团天吉生物制药有限公司 特利加压素的纯化制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114469733A (zh) * 2021-10-13 2022-05-13 成都科建生物医药有限公司 一种阿霉素脂质体的制备装置与制备方法
CN114469733B (zh) * 2021-10-13 2023-06-09 成都科建生物医药有限公司 一种阿霉素脂质体的制备装置与制备方法

Also Published As

Publication number Publication date
CN106924715A (zh) 2017-07-07

Similar Documents

Publication Publication Date Title
JP4885715B2 (ja) イリノテカン製剤
CN102188377B (zh) 包载药物脂质体的制备方法
US20040147578A1 (en) Use of lipoaminoacids as absorption promoters in a pharmaceutical composition
JP2012031200A (ja) 被覆薬物送達製剤
WO2017114186A1 (zh) 特利加压素脂质体及其制备方法
WO2023030524A1 (zh) 靶向动脉粥样硬化脂质体纳米载体递送系统及其制备方法
CN101670112A (zh) 一种稳定的白蛋白脂质载药系统及其制备方法
CN1951400A (zh) 蟾酥纳米脂质体及其制备方法
US10071084B2 (en) Nanoparticle, method of preparating the same, and use of the nanoparticle
JPH07505408A (ja) 治療及び診断用組成物及びその製造方法，ならびにその用途
CN114652683A (zh) 一种Mdivi-1纳米长循环脂质体及其制备方法和应用
CN109381429B (zh) 一种白细胞膜修饰的紫杉醇靶向缓释脂质体及其制备方法
JP2006273812A (ja) リポソーム製剤の製造方法
JPWO2005123078A1 (ja) 水難溶性カンプトテシン含有リポソーム製剤
CN105534905B (zh) 一种含有恩替卡韦的多囊脂质体及其制备方法
Qi et al. Comparative pharmacokinetics and antitumor efficacy of doxorubicin encapsulated in soybean-derived sterols and poly (ethylene glycol) liposomes in mice
JPWO2005021012A1 (ja) ゲムシタビン封入薬剤担体
Yuan et al. Ligand-free high loading capacity ursolic acid self-carried nanovesicles enable hepatocyte targeting via absorbing apolipoproteins
CN101244040B (zh) 薯蓣皂苷脂质体及其制剂的制法与用途
CN106389332B (zh) 一种长循环的氧化石蒜碱有机酸复合物新制剂
WO2018214944A1 (zh) 用c(RGD-ACP-K)修饰的长循环脂质体
WO2007014150A2 (en) Method of administering liposomes containing oligonucleotides
CN110464835A (zh) 一种胰岛素柔性微粒及其制剂
TWI786706B (zh) 包含陽離子脂質體的組成物於抑制皂素溶血的用途
KR100996975B1 (ko) 혈류 내 순환시간 증가를 위한 단백질로 수식된 리포솜 및이의 제조방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16880976

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16880976

Country of ref document: EP

Kind code of ref document: A1