技术领域technical field
本发明属于药物制剂领域，涉及一种新藤黄酸脂质体注射剂及其制备方法，更具体地说，本发明涉及一种有助于避免体内的网状内皮系统吞噬并可以稳定贮存的一种新藤黄酸脂质体注射剂及其制备方法。The invention belongs to the field of pharmaceutical preparations, and relates to a new gambogic acid liposome injection and a preparation method thereof. More specifically, the invention relates to a kind of drug that helps avoid reticuloendothelial system phagocytosis in the body and can be stored stably. New gambogic acid liposome injection and its preparation method.
背景技术Background technique
新藤黄酸是从中药藤黄中分离出的一种具有较高抗肿瘤活性的化合物，其抗菌谱较广，且毒性较低，能选择性地作用肿瘤细胞，而对正常动物造血系统和免疫系统没有影响，机体耐受良好，对白血病L1210，S180腹水癌，Lewis肺癌，La795肺腺癌，胰腺癌，胃癌，肝癌等实体肿瘤均有较好的抑制作用，其中新藤黄酸的抗肿瘤活性约为藤黄酸的2倍，新藤黄酸能有效阻滞癌细胞在G0~G1期，进而抑制肿瘤细胞的生长，促进肿瘤细胞的凋亡，提示新藤黄酸是一种非常有潜力的癌症治疗药物。Neogambogic acid is a compound with high anti-tumor activity isolated from the traditional Chinese medicine Gamboge. It has a wide antibacterial spectrum and low toxicity. It has no effect on the system and is well tolerated by the body. It has a good inhibitory effect on leukemia L1210 , S180 ascites cancer, Lewis lung cancer, La795 lung adenocarcinoma, pancreatic cancer, gastric cancer, liver cancer and other solid tumors. The tumor activity is about twice that of gambogic acid. Neogambogic acid can effectively block cancer cells in the G0 ~G1 phase, thereby inhibiting the growth of tumor cells and promoting the apoptosis of tumor cells, suggesting that neogambogic acid is a very Potential cancer therapy drug.
新藤黄酸分子式：C38H46O9，分子量631.0，结构式：Neogambogic acid molecular formula: C38 H46 O9 , molecular weight 631.0, structural formula:
脂质体(liposome)系指将药物包封于类脂质双分子层内而形成的微型泡囊体，是一种人工膜。在水中磷脂分子亲水头部插入水中，脂质体疏水尾部伸向空气，搅动后形成双层脂分子的球形脂质体，直径25~1000nm不等。当两性分子如磷脂和鞘脂分散于水相时，分子的疏水尾部倾向于聚集在一起，避开水相，而亲水头部暴露在水相，形成具有双分子层结构的的封闭囊泡，称为脂质体。脂质体可用于转基因，或装载药物，利用脂质体可以和细胞膜融合的特点，将药物送入细胞内部。脂质体的制备方法有：注入法、薄膜分散法、超声波分散法、逆向蒸发法。Liposome refers to a microvesicle formed by encapsulating drugs in a lipid bilayer, and is an artificial membrane. In the water, the hydrophilic head of phospholipid molecules is inserted into the water, and the hydrophobic tail of liposomes extends to the air. After agitation, spherical liposomes with bilayer lipid molecules are formed, with diameters ranging from 25 to 1000 nm. When amphiphilic molecules such as phospholipids and sphingolipids are dispersed in the aqueous phase, the hydrophobic tails of the molecules tend to gather together and avoid the aqueous phase, while the hydrophilic heads are exposed to the aqueous phase, forming closed vesicles with a bilayer structure , called liposomes. Liposomes can be used to transgene, or load drugs, and use the characteristics of liposomes that can fuse with cell membranes to deliver drugs into cells. The preparation methods of liposomes include injection method, film dispersion method, ultrasonic dispersion method and reverse evaporation method.
自从1965年Bangham发现脂质体后，1971年Gregoriadis和Rymen首次报道将脂质体作为药物载体，上世纪70年代末脂质体开始作为蒽环类抗肿瘤药物的有效载体，脂质体是类似生物膜结构的双分子小囊，是具有单个或多个双层磷脂膜的囊泡，其主要成分是磷脂，磷脂分子中含磷酸基团的部分具有强烈极性(亲水性)，碳氢链具有非极性(疏水性)。具有下述优点：1）体内可被生物降解，免疫原性小；2）水溶和脂溶性药物都可包埋运载，药物缓释，药效持续时间长；3）通过细胞内吞融合作用，脂质体可直接将药物送人细胞内，避免使用高浓度游离药物从而降低不良反应；4）正常组织毛细血管壁完整，大部分的脂质体不能渗透，而肿瘤生长部位毛细血管的通透性增加，使脂质体药物聚集量增加，并由于药物的缓释，直接用于肿瘤部位，增加了治疗效果。脂质体药物进人体循环后主要被网状内皮系统(reticuloendothelialsystem,RES)中的白细胞、单核细胞及巨噬细胞吞噬，这对于RES肿瘤的治疗有特殊的意义。早期脂质体药物的应用因稳定性差、药物易渗漏、储存期限短、组织靶向性差和易被RES迅速清除等受到限制；现在在其表面包裹了高分子物质聚乙二醇的脂质体因其组成中含有亲水性聚合物，可提高其表面的亲水性。聚乙二醇的脂质体可以阻止血浆蛋白吸附，调理于脂质体表面，减少RES的吞噬吸收，逃避免疫系统的拦截，延长了药物在体内的循环时间，所以通常又称之为长循环脂质体。国内最早上市的脂质体药物是南京绿叶思科药业有限公司生产的注射用紫杉醇脂质体，随后，陆续有常州金远药业制造有限公司、上海复旦张江生物医药股份有限公司、石药集团中奇制药技术（石家庄）有限公司生产的盐酸多柔比星脂质体注射液，以及上海新先锋药业有限公司、上海新亚药业有限公司生产的注射用两性霉素B脂质体。Since Bangham discovered liposomes in 1965, Gregoriadis and Rymen first reported liposomes as drug carriers in 1971. In the late 1970s, liposomes began to be used as effective carriers for anthracycline antitumor drugs. Liposomes are similar to Bimolecular vesicles of biomembrane structure are vesicles with single or multiple double-layered phospholipid membranes, and their main components are phospholipids. The chains are non-polar (hydrophobic). It has the following advantages: 1) It can be biodegraded in the body and has low immunogenicity; 2) Both water-soluble and fat-soluble drugs can be embedded and carried, and the drug is released slowly, and the drug effect lasts for a long time; 3) Through endocytosis and fusion, Liposomes can directly deliver drugs into cells, avoiding the use of high concentrations of free drugs to reduce adverse reactions; 4) normal tissue capillary walls are intact, most liposomes are impermeable, and the permeability of capillaries at tumor growth sites Increased liposome drug aggregation, and due to the sustained release of drugs, directly used in the tumor site, increasing the therapeutic effect. After liposome drugs enter the human circulation, they are mainly phagocytized by leukocytes, monocytes and macrophages in the reticuloendothelial system (RES), which has special significance for the treatment of RES tumors. The application of early liposome drugs was limited by poor stability, easy drug leakage, short storage period, poor tissue targeting and easy to be quickly cleared by RES; now lipids with macromolecular substances polyethylene glycol are wrapped on their surface The body can improve the hydrophilicity of its surface because of its composition containing hydrophilic polymers. Polyethylene glycol liposomes can prevent the adsorption of plasma proteins, regulate the surface of liposomes, reduce the phagocytosis and absorption of RES, avoid the interception of the immune system, and prolong the circulation time of drugs in the body, so it is usually called long circulation. Liposomes. The earliest liposome drug listed in China was Paclitaxel liposome for injection produced by Nanjing Luye Cisco Pharmaceutical Co., Ltd., followed by Changzhou Jinyuan Pharmaceutical Manufacturing Co., Ltd., Shanghai Fudan Zhangjiang Biomedical Co., Ltd., CSPC Doxorubicin hydrochloride liposome injection produced by Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd., and amphotericin B liposome for injection produced by Shanghai New Pioneer Pharmaceutical Co., Ltd. and Shanghai Xinya Pharmaceutical Co., Ltd.
新藤黄酸为亮黄色无定形粉末，mp65~68℃，在乙醇、甲醇等有机溶剂中溶解，水中不溶，溶于有机溶剂和稀碱液，具有光学活性，对三氯化铁呈蓝色，由于新藤黄酸在水溶液中溶解度低，注射后对机体刺激性大，体内半衰期短，限制了它在临床上的应用，因此需要用到现代制剂技术有效地提高新藤黄酸的溶解度，降低刺激性，延长药物在体内的作用时间以及局部聚集量，更好的发挥抗癌作用，发明人前期公开的专利（授权公告号为CN101947204A）所述新藤黄酸固体脂质纳米粒及其制备方法，虽然解决了新藤黄酸的水溶性、刺激性等问题，但是其在稳定性和载药量稍有欠缺，容易受到湿度、温度、光照的影响而氧化分解；发明人前期公开的专利（授权公告号为CN101879137B）所述一种隐形脂质体的制备方法，该发明涉及一种隐形脂质体的制备方法，该方法采用二次冻干法，制备成新藤黄酸隐形脂质体，但是该制备方法需要二次冻干，生产工艺复杂不适合工业化生产，生产成本也高，生产工艺中存在有机溶剂去除不净等缺点；安徽医药，13(6):596～598,2009发明人公开了新藤黄酸脂质体冻干粉的制备及其包封率测定，这篇文章研究了新藤黄酸脂质体冻干粉，但是该脂质体没有加入长循环的聚乙二醇化脂，用该方法制备的新藤黄酸脂质体进入机体内容易被体内肝脾网状内皮系统所吞噬。所述这些均给新藤黄酸在实际生产和临床应用中带来限制，所以本发明是在前期专利或研究的基础上制备的一种新藤黄酸脂质体注射剂，本发明提高了新藤黄酸的溶解度与在机体内的生物利用度，减少对机体的刺激性，延长药物在机体内作用时间等效果。本发明提供的新藤黄酸脂质体注射剂，具有以下优点：Neogambogic acid is bright yellow amorphous powder, mp65~68℃, soluble in ethanol, methanol and other organic solvents, insoluble in water, soluble in organic solvents and dilute lye, optically active, blue to ferric chloride, Due to the low solubility of gambogic acid in aqueous solution, it is highly irritating to the body after injection, and its half-life in the body is short, which limits its clinical application. Therefore, it is necessary to use modern preparation technology to effectively improve the solubility of gambogic acid and reduce irritation. , prolong the action time of the drug in the body and the local aggregation amount, and better exert the anticancer effect. The new gambogic acid solid lipid nanoparticles and its preparation method described in the inventor's previously published patent (authorized announcement number is CN101947204A), although It solves the problems of water solubility and irritation of new gambogic acid, but it is slightly lacking in stability and drug loading, and is easily oxidatively decomposed by humidity, temperature, and light; the inventor’s previously disclosed patent (authorized announcement number It is a preparation method of stealth liposome described in CN101879137B), the invention relates to a preparation method of stealth liposome, the method adopts secondary freeze-drying method to prepare new gambogic acid stealth liposome, but the preparation The method requires secondary freeze-drying, the production process is complex and not suitable for industrialized production, and the production cost is also high. There are shortcomings such as organic solvent removal in the production process; Anhui Medicine, 13(6):596-598, 2009. Preparation of lyophilized powder of gambogic acid liposome and determination of its encapsulation efficiency. This article studies the lyophilized powder of new gambogic acid liposome, but the liposome does not add long-circulating PEGylated lipid. The new gambogic acid liposome prepared by the method is easily swallowed by the reticuloendothelial system of the liver and spleen in the body. These all bring restriction to new gambogic acid in actual production and clinical application, so the present invention is a kind of new gambogic acid liposome injection prepared on the basis of previous patent or research, the present invention has improved new gambogic acid The solubility and bioavailability in the body can reduce the irritation to the body and prolong the action time of the drug in the body. The new gambogic acid liposome injection provided by the invention has the following advantages:
1.本发明的新藤黄酸脂质体提高新藤黄酸的溶解度，降低刺激性，延长药物在体内的作用时间以及局部聚集量，更好的发挥抗癌作用，降低了新藤黄酸的毒副作用，提高了机体的耐受性；1. The new gambogic acid liposome of the present invention improves the solubility of new gambogic acid, reduces irritation, prolongs the action time and local aggregation of drugs in the body, better exerts anticancer effect, and reduces the toxic and side effects of new gambogic acid , improve the tolerance of the body;
2.本发明的制剂可克服固体脂质纳米粒放置过程中药物外排，包封率低的现象，新藤黄酸脂质体载药量更高、性能更稳定，制剂可稳定贮存；2. The preparation of the present invention can overcome the phenomenon of drug efflux and low encapsulation efficiency during the placement of solid lipid nanoparticles, and the new gambogic acid liposome has higher drug loading and more stable performance, and the preparation can be stored stably;
3.与新藤黄酸的乳剂、固体脂质纳米粒等相比，本发明的制剂通过长循环辅料纳米粒表面进行修饰，有助于避免体内肝脾网状内皮系统的吞噬，且具有缓释功能，可以延长药物在体内的循环时间。3. Compared with the emulsion of neogambogic acid, solid lipid nanoparticles, etc., the preparation of the present invention is modified by the surface of long-circulation excipient nanoparticles, which helps to avoid the phagocytosis of the liver and spleen reticuloendothelial system in vivo, and has sustained release Function, can prolong the circulation time of drugs in the body.
发明内容Contents of the invention
针对上述现有技术及我们自己分离纯化的新藤黄酸药物，本发明提供一种新藤黄酸的新型载药体——新藤黄酸脂质体，本发明所述的新藤黄酸脂质体载药量更高、性能更稳定，制剂可稳定贮存。本发明的另一目的在于提供一种制备本发明的新藤黄酸脂质体的制备方法。For the above-mentioned prior art and the new gambogic acid medicines separated and purified by ourselves, the present invention provides a novel drug carrier of new gambogic acid——new gambogic acid liposome, and the new gambogic acid liposome of the present invention carries The dosage is higher, the performance is more stable, and the preparation can be stored stably. Another object of the present invention is to provide a method for preparing the new gambogic acid liposome of the present invention.
本发明将新藤黄酸包裹于脂质体的类似生物膜结构的双分子小囊中，增加了新藤黄酸的溶解度，极大地减小了刺激性，制剂在一定程度上达到更明确的靶向、控释等效果，并且物理化学性质稳定。In the present invention, neogambogic acid is encapsulated in bimolecular vesicles of a liposome similar to a biomembrane structure, which increases the solubility of neogambogic acid, greatly reduces irritation, and the preparation achieves a clearer targeting to a certain extent. , controlled release and other effects, and the physical and chemical properties are stable.
为实现上述目的，本发明提供了一种新藤黄酸脂质体，其基本上由治疗有效量的由新藤黄酸、磷脂材料、胆固醇、脂类辅料、有机酸或硫酸铵、糖、缓冲剂、注射用水等组成。To achieve the above object, the invention provides a new gambogic acid liposome, which basically consists of a therapeutically effective amount of new gambogic acid, phospholipid material, cholesterol, lipid adjuvant, organic acid or ammonium sulfate, sugar, buffer , water for injection, etc.
具体而言，在上述技术方案中，本发明提供的新藤黄酸脂质体含有下列成分：Specifically, in the above-mentioned technical scheme, new gambogic acid liposome provided by the invention contains following composition:
新藤黄酸0.01～10%Neogambogic acid 0.01～10%
磷脂材料0.005～5%Phospholipid material 0.005～5%
胆固醇0.001～2%Cholesterol 0.001～2%
聚乙二醇化脂0.005～1.5%PEGylated fat 0.005～1.5%
有机酸或硫酸铵0.0025～2.5%Organic acid or ammonium sulfate 0.0025～2.5%
糖0.1～25%Sugar 0.1～25%
缓冲剂0.1～15%Buffer 0.1～15%
其余为注射用水组成加至适量The rest is composed of water for injection and added to an appropriate amount
优选的配方组成如下：The preferred formula consists of the following:
新藤黄酸10gNeogambogic acid 10g
氢化大豆卵磷脂66gHydrogenated Soy Lecithin 66g
胆固醇10gCholesterol 10g
聚乙二醇化脂8gPEGylated fat 8g
有机酸或硫酸铵5gOrganic acid or ammonium sulfate 5g
糖800gSugar 800g
缓冲剂50gBuffer 50g
注射用水加至1000mLAdd water for injection to 1000mL
所述磷脂材料选自蛋黄卵磷脂、氢化蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、二月桂酰卵磷脂、二肉豆蔻酰卵磷脂、二棕榈酰卵磷脂、二硬脂酰卵磷脂、1-肉豆蔻酰-2-棕榈酰卵磷脂、1-棕榈酰-2-肉豆蔻酰卵磷脂、1-棕榈酰-2-硬脂酰卵磷脂、1-硬脂酰-2-棕榈酰卵磷脂、二油酰基卵磷脂、二肉豆蔻酰磷脂酸、二棕榈酰磷脂酸、二肉豆蔻酰磷脂酞乙醇胺、二棕榈酰磷脂酞乙醇胺、脑磷脂酰丝氨酸、脑神经鞘磷脂、二棕搁酞神经鞘磷脂、二硬脂酰神经鞘磷脂、二硬脂酰磷脂酰乙醇胺的一种或几种，但不限于此。The phospholipid material is selected from egg yolk lecithin, hydrogenated egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dilauroyl lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, 1-myristoyl-2-palmitoyl lecithin, 1-palmitoyl-2-myristoyl lecithin, 1-palmitoyl-2-stearoyl lecithin, 1-stearoyl-2-palmitoyl lecithin Phospholipids, dioleoyl lecithin, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, brain phosphatidylserine, brain sphingomyelin, dipalmitoyl phthalein One or more of sphingomyelin, distearoyl sphingomyelin, and distearoyl phosphatidylethanolamine, but not limited thereto.
所述聚乙二醇化脂选自PEG分子量为1000~10000Da的硬脂酸酯、PEG分子量为1000~10000Da维生素E琥珀酸酯，甲氧基PEG2000-二硬脂酰磷脂酰乙醇铵、胆固醇-PEG、甘油二脂-PEG、DSPE多臂PEG或DSPE多臂PEG，PEG硬脂酸酯与分子量为200~10000Da的PEG的混合物，Myrj类，Brij类，PEG—二硬脂酰基磷脂酰乙醇胺、PEG—二棕榈酰磷脂酰胆碱、PEG—二棕榈酰磷脂酰乙醇胺，神经节苷脂，聚丙烯酰胺，壳聚糖，聚乙二醇聚十六烷基腈基丙酸酯，分子量为200~10000Da的PEG、PVP或PVA的一种或几种任意组合，但不限于此。The PEGylated fat is selected from stearate with a PEG molecular weight of 1000~10000Da, vitamin E succinate with a PEG molecular weight of 1000~10000Da, methoxy PEG2000-distearoylphosphatidylethanolamine, cholesterol-PEG , diglyceride-PEG, DSPE multi-arm PEG or DSPE multi-arm PEG, a mixture of PEG stearate and PEG with a molecular weight of 200~10000Da, Myrj class, Brij class, PEG-distearoylphosphatidylethanolamine, PEG —Dipalmitoylphosphatidylcholine, PEG—dipalmitoylphosphatidylethanolamine, ganglioside, polyacrylamide, chitosan, polyethylene glycol polyhexadecyl cyanopropionate, molecular weight 200~ One or any combination of 10000 Da PEG, PVP or PVA, but not limited thereto.
糖选自海藻糖、蔗糖、甘露醇、葡萄糖、氯化钠、乳糖、山梨醇、木糖醇、葡聚糖、聚乙二醇、聚乙烯吡咯烷酮、右旋糖酐、甘油或甘氨酸的一种或几种任意组合，但不限于此。One or more sugars selected from trehalose, sucrose, mannitol, glucose, sodium chloride, lactose, sorbitol, xylitol, dextran, polyethylene glycol, polyvinylpyrrolidone, dextran, glycerol or glycine Any combination, but not limited to.
缓冲剂选自组氨酸或甘氨酸，但不限于此。The buffer is selected from histidine or glycine, but not limited thereto.
本发明还提供了一种新藤黄酸脂质体的制备方法，该方法包括以下步骤：The present invention also provides a kind of preparation method of new gambogic acid liposome, this method comprises the following steps:
（1）将新藤黄酸与权利要求1的磷脂材料、胆固醇、脂类辅料加热熔融或有机溶剂溶解，制成脂质溶液；(1) heating and melting new gambogic acid and the phospholipid material, cholesterol, and lipid excipients of claim 1 or dissolving them in an organic solvent to make a lipid solution;
（2）将脂质溶液置旋转蒸发器旋转薄膜蒸发，制成脂质薄膜；(2) Evaporate the lipid solution in a rotary evaporator to make a lipid film;
（3）将脂质薄膜以含有权利要求1的有机酸或硫酸铵、糖、缓冲剂水溶液水合，震荡，或将（1）中的脂质溶液直接与水溶液震荡混合，制成脂质分散水溶液；(3) Hydrate the lipid film with an aqueous solution containing an organic acid or ammonium sulfate, sugar, and buffer according to claim 1, and oscillate, or directly mix the lipid solution in (1) with an aqueous solution to prepare a lipid-dispersed aqueous solution ;
（4）对脂质分散水溶液进行超声、匀质乳化、微射流、挤压过滤处理，从而制得新藤黄酸脂质体。(4) Ultrasound, homogeneous emulsification, micro-fluidization, extrusion and filtration are performed on the lipid-dispersed aqueous solution to prepare new gambogic acid liposomes.
可进一步对制得的新藤黄酸脂质体进行冻干处理或置于4℃下密封保存，其中对所得到的新藤黄酸脂质体进行冻干处理的工艺为：将1~40%（重量比）的冻干保护剂溶于本发明的新藤黄酸脂质体的水分散体中，该冻干保护剂选自海藻糖、蔗糖、甘露醇、葡萄糖、氯化钠、乳糖、山梨醇、木糖醇、葡聚糖、聚乙二醇、聚乙烯吡咯烷酮、右旋糖酐、甘油或甘氨酸的一种或几种任意组合，但不限于此，以保证形成冻干粉晶型较好，然后分装在西林瓶中，并置于冷冻干燥机中-45℃预冻2.5h，然后在5℃/h升温至-50℃，维持10h，再以5℃/h升温至0℃，维持8h，最后升温至10℃保温10h后出箱，加塞密封即可。The obtained new gambogic acid liposomes can be further freeze-dried or placed in sealed storage at 4°C, wherein the process for freeze-drying the obtained new gambogic acid liposomes is: adding 1 to 40% ( weight ratio) of the lyoprotectant dissolved in the aqueous dispersion of the new gambogic acid liposomes of the present invention, the lyoprotectant is selected from trehalose, sucrose, mannitol, glucose, sodium chloride, lactose, sorbitol , xylitol, dextran, polyethylene glycol, polyvinylpyrrolidone, dextran, glycerin or glycine in any combination of one or more, but not limited to this, to ensure that the formation of freeze-dried powder crystals is better, and then divide Packed in vials, and placed in a freeze dryer at -45°C for 2.5 hours, then heated to -50°C at 5°C/h, maintained for 10 hours, then raised to 0°C at 5°C/h, maintained for 8 hours, Finally, the temperature was raised to 10°C and kept for 10 hours, and then it was taken out of the box and sealed with a stopper.
所述有机溶剂选自二氯甲烷、氯仿、丙酮、异丙酮、甲醇、乙醇、乙醚、四氢呋喃、碳酸二甲酯、叔丁醇、三氯叔丁醇、乙酸乙酯、丙二醇、二甲基砜、石油醚或异丙醇的一种或几种任意组合，但不限于此。The organic solvent is selected from dichloromethane, chloroform, acetone, isopropanone, methanol, ethanol, ether, tetrahydrofuran, dimethyl carbonate, tert-butanol, chlorobutanol, ethyl acetate, propylene glycol, dimethyl sulfone , petroleum ether or isopropanol or any combination of several, but not limited thereto.
具体实施方式：detailed description:
下面再以实施方式对本发明进一步说明和给出实施细节。In the following, the present invention will be further described and implementation details will be given in the form of implementation.
实施例1：Example 1:
.将10g新藤黄酸与氢化大豆卵磷脂66g、胆固醇10g、聚乙二醇化脂8g置旋转蒸发器加热熔融，旋转，制成脂质薄膜；将脂质薄膜以含硫酸铵5g、蔗糖800g、甘氨酸5g水溶液水合，震荡，制成脂质分散水溶液；对脂质分散水溶液进行超声、匀质乳化、微射流、挤压分别用0.80μm、0.45μm、0.25μm的微孔滤膜过滤处理，从而制得新藤黄酸脂质体混悬液；向含有7mg乳糖的西林瓶中加入1mL新藤黄酸脂质体混悬液，放入真空冷冻干燥机中冷冻干燥（见冻干曲线1），即制成的新藤黄酸脂质体注射剂冻干粉针剂。.10g new gambogic acid and hydrogenated soybean lecithin 66g, cholesterol 10g, and polyethylene glycol fat 8g are put in a rotary evaporator to heat and melt, and rotate to make a lipid film; the lipid film is prepared with ammonium sulfate 5g, sucrose 800g, Glycine 5g aqueous solution is hydrated and oscillated to make a lipid dispersion aqueous solution; the lipid dispersion aqueous solution is subjected to ultrasonication, homogeneous emulsification, micro-jet, and extrusion and is treated with microporous membrane filtration of 0.80 μm, 0.45 μm, and 0.25 μm respectively, thereby Prepare new gambogic acid liposome suspension; add 1mL new gambogic acid liposome suspension to the cillin bottle containing 7mg lactose, put into vacuum freeze dryer and freeze-dry (see freeze-drying curve 1), namely The new gambogic acid liposome injection freeze-dried powder injection made.
冻干曲线1：Freeze-drying curve 1:
工序升温速度时间（h）温度(℃)Process heating speed time (h) temperature (°C)
(1)预冻3室温至-30℃～-50℃(1) Pre-freeze 3 room temperature to -30℃～-50℃
(2)保温1.5-50℃(2) heat preservation 1.5-50 ℃
(3)抽真空0.5-30℃～-50℃(3) Vacuuming 0.5-30℃～-50℃
(4)升温5℃/小时6升温至-5℃(4) Heating 5°C/hour 6 to -5°C
(5)升温5℃/小时4-5℃升温至0℃(5) Heat up 5°C/hour 4-5°C to 0°C
(6)升温5℃/小时30℃至10℃(6) Heating 5°C/hour 30°C to 10°C
(7)升温5℃/小时310℃至25℃(7) Heating 5°C/hour 310°C to 25°C
(8)保温225℃(8) Heat preservation at 225°C
实施例2：Example 2:
将10g新藤黄酸与氢化大豆卵磷脂66g、胆固醇10g、聚乙二醇化脂8g置旋转蒸发器加热熔融，旋转，制成脂质薄膜；将脂质薄膜以含硫酸铵5g、蔗糖800g、甘氨酸5g水溶液水合，震荡，制成脂质分散水溶液；对脂质分散水溶液进行超声、匀质乳化、微射流、挤压分别用0.80μm、0.45μm、0.25μm的微孔滤膜过滤处理，向含有10mg乳糖的西林瓶中加入1mL混悬液，放入真空冷冻干燥机中冷冻干燥（见冻干曲线2），即制成的新藤黄酸脂质体注射剂冻干粉针剂。Put 10g of neogambogic acid, 66g of hydrogenated soybean lecithin, 10g of cholesterol, and 8g of pegylated fat into a rotary evaporator to heat and melt, and rotate to make a lipid film; 5g of aqueous solution was hydrated and shaken to make lipid dispersion aqueous solution; the lipid dispersion aqueous solution was subjected to ultrasonication, homogeneous emulsification, micro-jet, and extrusion, respectively, and filtered through microporous membranes of 0.80 μm, 0.45 μm, and 0.25 μm, to the Add 1mL of the suspension to a vial containing 10mg of lactose, put it into a vacuum freeze dryer and freeze-dry (see freeze-drying curve 2), and the new gambogic acid liposome injection freeze-dried powder injection is prepared.
冻干曲线2：Freeze-drying curve 2:
工序升温速度时间（h）温度(℃)Process heating speed time (h) temperature (°C)
(1)预冻3室温至-30℃～-50℃(1) Pre-freeze 3 room temperature to -30℃～-50℃
(2)保温1.5-30℃～-50℃(2) Heat preservation 1.5-30℃～-50℃
(3)抽真空0.5-30℃～-50℃(3) Vacuuming 0.5-30℃～-50℃
(4)升温5-10℃/小时6升温至-5℃(4) Heat up 5-10°C/hour 6 to -5°C
(5)升温5-10℃/小时4-5℃升温至0℃(5) Heat up 5-10°C/hour 4-5°C to 0°C
(6)升温5-10℃/小时30℃至10℃(6) Heating 5-10°C/hour 30°C to 10°C
(7)升温5-10℃/小时310℃至25℃(7) Heating 5-10°C/hour 310°C to 25°C
(8)保温225℃(8) Heat preservation at 225°C
实施例3：Example 3:
将10g新藤黄酸与氢化大豆卵磷脂66g、胆固醇10g、聚乙二醇化脂8g置旋转蒸发器加热熔融，旋转，制成脂质薄膜；将脂质薄膜以含硫酸铵5g、蔗糖800g、甘氨酸5g水溶液水合，震荡，制成脂质分散水溶液；对脂质分散水溶液进行超声、匀质乳化、微射流、挤压分别用0.80μm、0.45μm、0.25μm的微孔滤膜过滤处理，向含有10mg乳糖的西林瓶中加入1mL混悬液，放入真空冷冻干燥机中冷冻干燥（见冻干曲线3），即制成的新藤黄酸脂质体注射剂冻干粉针剂。Put 10g of neogambogic acid, 66g of hydrogenated soybean lecithin, 10g of cholesterol, and 8g of pegylated fat into a rotary evaporator to heat and melt, and rotate to make a lipid film; 5g of aqueous solution was hydrated and shaken to make lipid dispersion aqueous solution; the lipid dispersion aqueous solution was subjected to ultrasonication, homogeneous emulsification, micro-jet, and extrusion, respectively, and filtered through microporous membranes of 0.80 μm, 0.45 μm, and 0.25 μm, to the Add 1mL of the suspension to a vial containing 10mg of lactose, put it into a vacuum freeze dryer and freeze-dry (see freeze-drying curve 3), and the new gambogic acid liposome injection freeze-dried powder injection is prepared.
冻干曲线3：Freeze-drying curve 3:
工序升温速度时间（h）温度(℃)Process heating speed time (h) temperature (°C)
(1)预冻3室温至-30℃～-50℃(1) Pre-freeze 3 room temperature to -30℃～-50℃
(2)保温1.5-30℃～-50℃(2) Heat preservation 1.5-30℃～-50℃
(3)抽真空0.5-30℃～-50℃(3) Vacuuming 0.5-30℃～-50℃
(4)升温5-10℃/小时6升温至-5℃(4) Heat up 5-10°C/hour 6 to -5°C
(5)升温5-10℃/小时4-5℃升温至0℃(5) Heat up 5-10°C/hour 4-5°C to 0°C
(6)升温5-10℃/小时30℃至10℃(6) Heating 5-10°C/hour 30°C to 10°C
(7)升温5-10℃/小时310℃至25℃(7) Heating 5-10°C/hour 310°C to 25°C
(8)保温225℃(8) Heat preservation at 225°C
实施例4：Example 4:
将15mg新藤黄酸和120mg单硬脂酸溶于丙酮中，300mg卵磷脂以少量乙醇溶解，两部分再混合，构成有机相。将400mg泊洛沙姆-188和200mg吐温-80溶于注射用水中，构成水相。将70-80℃有机相缓慢滴入同温度下（70-80℃）的水相中，恒温搅拌1个小时，取出在0-2℃水相中搅拌1小时，即得新藤黄酸脂质体注射剂混悬液，用0.45μm的微孔滤膜过滤，向含有10mg甘露醇的西林瓶中加入1mL混悬液，放入真空冷冻干燥机中冷冻干燥（见冻干曲线4），即制成的新藤黄酸脂质体注射剂冻干粉针剂。Dissolve 15mg neogambogic acid and 120mg monostearic acid in acetone, dissolve 300mg lecithin with a small amount of ethanol, and mix the two parts again to form the organic phase. Dissolve 400mg of poloxamer-188 and 200mg of Tween-80 in water for injection to form the aqueous phase. Slowly drop the organic phase at 70-80°C into the water phase at the same temperature (70-80°C), stir at constant temperature for 1 hour, take it out and stir it in the water phase at 0-2°C for 1 hour to obtain new gambogic acid lipids Injection suspension, filtered through a 0.45 μm microporous membrane, added 1 mL of the suspension to a vial containing 10 mg of mannitol, and placed in a vacuum freeze dryer for freeze-drying (see freeze-drying curve 4). The new gambogic acid liposome injection lyophilized powder injection.
冻干曲线4：Freeze-drying curve 4:
工序升温速度时间（h）温度(℃)Process heating speed time (h) temperature (°C)
(1)预冻3室温至-30℃～-50℃(1) Pre-freeze 3 room temperature to -30℃～-50℃
(2)保温1.5-30℃～-50℃(2) Heat preservation 1.5-30℃～-50℃
(3)抽真空0.5-30℃～-50℃(3) Vacuuming 0.5-30℃～-50℃
(4)升温5-10℃/小时6升温至-5℃(4) Heat up 5-10°C/hour 6 to -5°C
(5)升温5-10℃/小时4-5℃升温至0℃(5) Heat up 5-10°C/hour 4-5°C to 0°C
(6)升温5-10℃/小时30℃至10℃(6) Heating 5-10°C/hour 30°C to 10°C
(7)升温5-10℃/小时310℃至25℃(7) Heating 5-10°C/hour 310°C to 25°C
(8)保温225℃(8) Heat preservation at 225°C
实施例5：Example 5:
将5mg新藤黄酸和75mg单硬脂酸溶于丙酮中，150mg卵磷脂以少量乙醇溶解，两部分再混合，构成有机相。将600mg泊洛沙姆-188和300mg吐温-80溶于注射用水中，构成水相。将70-80℃有机相缓慢滴入同温度下（70-80℃）的水相中，恒温搅拌1个小时，取出在0-2℃水相中搅拌1小时，，即得新藤黄酸脂质体注射剂混悬液，用0.45μm的微孔滤膜过滤，向含有10mg甘露醇的西林瓶中加入1mL混悬液，放入真空冷冻干燥机中冷冻干燥（见冻干曲线5），即制成的新藤黄酸脂质体注射剂冻干粉针剂。Dissolve 5mg neogambogic acid and 75mg monostearic acid in acetone, dissolve 150mg lecithin with a small amount of ethanol, and mix the two parts again to form the organic phase. Dissolve 600mg of poloxamer-188 and 300mg of Tween-80 in water for injection to form the aqueous phase. Slowly drop the organic phase at 70-80°C into the water phase at the same temperature (70-80°C), stir at constant temperature for 1 hour, take it out and stir in the water phase at 0-2°C for 1 hour, to obtain new gambogic acid ester Plastid injection suspension is filtered with a 0.45 μm microporous membrane, 1 mL of the suspension is added to a vial containing 10 mg of mannitol, and the suspension is placed in a vacuum freeze dryer to freeze-dry (see freeze-drying curve 5), namely The new gambogic acid liposome injection freeze-dried powder injection made.
冻干曲线5：Freeze-drying curve 5:
工序升温速度时间（h）温度(℃)Process heating speed time (h) temperature (°C)
(1)预冻3室温至-30℃～-50℃(1) Pre-freeze 3 room temperature to -30℃～-50℃
(2)保温1.5-30℃～-50℃(2) Heat preservation 1.5-30℃～-50℃
(3)抽真空0.5-30℃～-50℃(3) Vacuuming 0.5-30℃～-50℃
(4)升温5-10℃/小时6升温至-5℃(4) Heat up 5-10°C/hour 6 to -5°C
(5)升温5-10℃/小时4-5℃升温至0℃(5) Heat up 5-10°C/hour 4-5°C to 0°C
(6)升温5-10℃/小时30℃至10℃(6) Heating 5-10°C/hour 30°C to 10°C
(7)升温5-10℃/小时310℃至25℃(7) Heating 5-10°C/hour 310°C to 25°C
(8)保温225℃(8) Heat preservation at 225°C
实施例6：Embodiment 6:
新藤黄酸脂质体注射剂冻干粉的外观和色泽的考察：Investigation on the appearance and color of the new gambogic acid liposome injection lyophilized powder:
取例5中得到的新藤黄酸脂质体注射剂冻干粉制剂，肉眼观察均为均匀的淡黄色，呈疏松块状物，表面细腻光洁，具有足够的强度。The new gambogic acid liposome injection freeze-dried powder preparation obtained in Example 5 was uniformly light yellow in the form of loose lumps with a fine and smooth surface and sufficient strength.
实施例7：Embodiment 7:
新藤黄酸脂质体注射剂冻干粉的再分散性的考察：Investigation on the redispersibility of new gambogic acid liposome injection freeze-dried powder:
取例5中得到的新藤黄酸脂质体注射剂冻干粉制剂，加蒸馏水1mL，轻轻振摇，30s内分散为均匀的淡黄色新藤黄酸脂质体胶体溶液，表明冻干剂再分散性良好。Get the new gambogic acid liposome injection lyophilized powder preparation obtained in Example 5, add 1mL of distilled water, shake gently, and disperse into a uniform light yellow new gambogic acid liposome colloid solution within 30s, indicating that the freeze-dried agent is redispersed sex is good.
实施例8：Embodiment 8:
新藤黄酸脂质体注射剂冻干粉的pH值的考察：Investigation of the pH value of the new gambogic acid liposome injection lyophilized powder:
取例5中得到的新藤黄酸脂质体注射剂冻干粉制剂，加入蒸馏水分散后，测定pH值，三批样品测定结果分别为7.18，7.22，7.20，平均pH值为7.20，于冻干前相比pH值变化不大，冻干剂偏弱酸性。Get the new gambogic acid liposome injection freeze-dried powder preparation obtained in example 5, after adding distilled water to disperse, measure the pH value, three batches of sample measurement results are respectively 7.18, 7.22, 7.20, and the average pH value is 7.20, before freeze-drying Compared with the change of pH value, the freeze-dried agent is weakly acidic.
| Application Number | Priority Date | Filing Date | Title |
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| CN201410622327.2ACN105616354A (en) | 2014-11-07 | 2014-11-07 | Neogambogic acid liposome injection and preparation method thereof |
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| CN201410622327.2ACN105616354A (en) | 2014-11-07 | 2014-11-07 | Neogambogic acid liposome injection and preparation method thereof |
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| CN105616354Atrue CN105616354A (en) | 2016-06-01 |
| Application Number | Title | Priority Date | Filing Date |
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| CN201410622327.2APendingCN105616354A (en) | 2014-11-07 | 2014-11-07 | Neogambogic acid liposome injection and preparation method thereof |
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| CN (1) | CN105616354A (en) |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20160601 | |
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