TWI250877B - Process for producing liposome suspensions and products containing liposome suspensions produced thereby - Google Patents
Process for producing liposome suspensions and products containing liposome suspensions produced thereby Download PDFInfo
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- TWI250877B TWI250877B TW92119528A TW92119528A TWI250877B TW I250877 B TWI250877 B TW I250877B TW 92119528 A TW92119528 A TW 92119528A TW 92119528 A TW92119528 A TW 92119528A TW I250877 B TWI250877 B TW I250877B
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- Prior art keywords
- liposome
- mixture
- peg
- drug
- group
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- 238000000034 method Methods 0.000 title claims abstract description 84
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Abstract
Description
1250877 玖、發明說明：1250877 玖, invention description:
t潑^明戶斤屬之^技：冷員I 發明領域 本發明是有關於一用以製造一脂質體懸浮液的方法’ 5以及含有由該方法所製得的脂質體懸浮液的產物（特別是 脂質體-包封的藥物）。BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a method for producing a liposome suspension '5 and a product containing a liposome suspension prepared by the method ( Especially liposome-encapsulated drugs).
L ^tT 發明背景 自1960年代，英國的Alec D· Bangham博士揭露脂質體 10 的概念與技術以來，脂質體已成為藥物傳遞系統（drug delivery system)技術領域中的一個重要研究目標。脂質體 主要是由碌脂質（phospholipids)所構成’主要是利用填脂質 具有由一非極性（non-polar)或疏水性（hydrophobic)的長形 結構以及一位在該長形結構的一端的極性（Polar)或親水性 15 (hydrophilic)的端部結構所造成的兩性特性（ampholytic character)，藉此，具有該親水性結構被包覆在内的核心區 域的微胞（micellae)可被形成。呈微胞狀的脂質體可具有一 小至20 nm的粒徑，而於藥物製造上適用作為將具有生物活 i生的物質帶入至人類或動物體内的載體。 20 脂質體依粒徑以及包覆層數可被區分為：粒徑落在大 約0.1至1 μιη之範圍内的多層囊泡（multilamellar vesicle， MLV);粒徑落在大約0.1-1 μπι之範圍内的募層囊泡 (oligolamellar vehicle，0LV);粒徑落在20至 100 nm之範圍 内的單層小囊泡（small unilamellar vesicle，SUV);粒徑約為 1250877 1000 nm之單層大胞囊（large unilamellar vesicle，LUV);以 及粒徑落在大約1至2 μπι之範圍内並且内部含有數個較小 囊泡的大囊泡。有關脂質體的一般性回顧，可以參見，例 如 R.R.C. New，ed. Liposomes: a practical approach (1990)， 5 Oxford °L ^tT BACKGROUND OF THE INVENTION Since the 1960s, Dr. Alec D. Bangham of the United Kingdom has revealed the concept and technology of liposome 10, liposomes have become an important research target in the field of drug delivery systems. Liposomes are mainly composed of phospholipids' mainly using a lipid-filled lipid having a non-polar or hydrophobic elongated structure and a polarity at one end of the elongated structure. The ampholytic character caused by the hydrophilic or hydrophilic end structure, whereby micelles having a core region in which the hydrophilic structure is coated can be formed. The microcytic liposomes may have a particle size as small as 20 nm, and are useful as a carrier for bringing a substance having a biological activity into a human or an animal. 20 The size of the liposome and the number of coating layers can be distinguished as: multilamellar vesicle (MLV) with a particle size falling within the range of about 0.1 to 1 μηη; the particle size falls within the range of about 0.1-1 μm Oligolamellar vehicle (0LV); small unilamellar vesicle (SUV) with a particle size falling within the range of 20 to 100 nm; single-layered large cell with a particle size of approximately 1250877 1000 nm Large unilamellar vesicle (LUV); and large vesicles with a particle size falling within the range of about 1 to 2 μπι and containing several smaller vesicles inside. For a general review of liposomes, see, for example, R.R.C. New, ed. Liposomes: a practical approach (1990), 5 Oxford °
關於脂質體的製備，目前已知的方法有，例如，水合 法（hydration)、超音波處理法（ultrasonification)、透析與稀 釋法（dialysis and dilution processes)、逆相蒸發法（reverse-phase evaporation)(參見 ，例如， US 4,235,871) 、 界面活性 10 劑處理法（surfactant treatment)、脫水-復水法（dehydration-rehydration) 或乾燥 -復原法 （dry-reconstitution) 、;東結 及融解 法（freeze and thaw)(參見，例如 US 6,355,267)、孔擠壓法 (pore extrusion)以及高壓均質法（high pressure homogeni-zation)等等（參見，例如，d.Z)· M.M 15 Standish，J.C. Watkins, J. Mol. Biol.，13，238-252，1965; R.R.C. New，ed. Liposomes: a practical approach (1990)， Oxford; G. Gregoriadis，ed· Liposome Technology，Vol· 1， 1993, CRC·， H. Talsma and D.J.A. Crommelin, Pharmaceutical Technology，96-106，Oct. 1992)。在這查已 20 知方法當中，孔擠壓法與高壓均質法被認為較有產業上的 潛力來供大規模製造脂質體。而若進一步考慮經濟效益， 孔擠壓法被認為是更佳的選擇。 US 4,737,323揭露利用擠壓法來製造一脂質體懸浮液 的方法，該脂質體具有均勻的尺寸以及一不大於〇·4 Pm的 1250877 平均粒徑，該方法包括下列步驟··提供一含有尺寸不一的 脂質體之懸浮液，該等脂質體有一主要部分是具有大於ι 〇 pm的尺寸；以及在加塵下令該懸浮液通經_個不對稱的陶 究過濾器，該過濾器的内表面孔徑大於所欲的平均脂質體 5尺寸且不大於大約丨·0 μιη。依據此件美國專利的揭示，一 有機溶劑，例如該案實施例丨所示的氯仿（chl〇r〇f〇rm”被 用來溶解脂質組份。接著，該有機溶劑在真空或一惰性氣 體下被移除，而形成-由脂質所構成的乾薄膜。繼而有一 水性基質（aqueous medium)被加至該薄膜上以使脂質膨脹 10開來，因而於該水性基質内形成尺寸不一的脂質體。 授予 Steven Lehigh 的 US 5,004,611 與 US 5,〇53,217 揭示 用以製備含有一具生物活性的化合物的脂質體之一水性分 散體的方法，其基本上係由下列步驟所構成：將呈一重量 比例為40 · 1至1 : 20的組份（a)至少一部分的雙層形成膜脂 15 質（bilayer forming membrane lipid)以及組份（b)至少一基本 上是由一為該脂質的溶劑之水可互溶的有機液體所構成的 非水性液體混合以足量的水，藉此，脂質體被自發性地形 成，該具生物活性的化合物係在水的加入之前、當時戍之 後被加入，並且其最終存在的數量足以使得該具生物活性 20的化合物之一生物有效劑量被締合以脂質體。依據這兩件 美國專利案的實施例1的揭示，首先在50〜60°C下製備出— 預-脂質體組成物（pro-liposome composition)，其中該脂質 組份（a)(例如卵碌脂）先被溶於該組份（b)(例如乙醇）内；接 著將水分成兩個部分來予以加入，其中第一個部分的水内 1250877 可含有該具生物活性的化合物（例如葡萄糖），而第二部分的 水被用來製造出最後的配方。該預-脂質體組成物經過在氮 氣下予以平衡後被冷卻至25°C，接著分兩個階段來加入碟 酸鹽緩衝液並予以振盪，如此即形成散浮有内含該具生物 5 活性的化合物的脂質體之水性分散體。因此，依據此二件 美國專利案的揭示，所用的乙醇會以一特定數量被包含在 所形成的脂質體水性分散體内。 授予 Francis C· Szoka，Jr.的 US 5,077,057、US 5,277,914以及US 5,549,910揭示用以製造一脂質體懸浮液 10的方法’該脂質體具有所界定的粒徑且包封有一有用的化 合物，該化合物於水、醇或鹵化烴溶劑内展現出一不良的 溶解度。依據此三件美國專利案的揭示，一溶解度不佳的 化合物連同一包封數量的脂質被溶於一非質子性溶劑（諸 如DMS0)内’而若有需要，可額外地使用一低級烷醇（例如 15乙醇）來幫助脂質的溶解。所形成的混合物接而被擠壓或注 射至一合適的水性溶液内並予以攪拌，即形成脂質體懸浮 液。所用的擠壓裝置可為一注射筒、一有孔的盤或管或其 他可提供大約0.05〜5 mm的孔洞之合適裝置。特別地，此 三件美國專利案的實施例2例示多索如必辛（doxorubicin)的 20包封，其中多索如必辛被溶於DMSO内，並被加入至一含有 卵磷脂酸甘油（EPG):卵磷脂醯膽鹼（EPC) ··膽固醇（7:3:6) 的乙醇溶液内。接著，脂質-多索如必辛混合物在3(rc下被 注射至一由 140 mMNaCl-10 mM Tris-HCl，pH 4_0所構成的 水相内’繼而將所形成的脂質懸浮液透析，並利用管柱層 1250877 析法將被脂質體包封的多索如必辛分離出。此實施例所製 得的脂質體粒徑為227 nm，且有41.2%的多索如必辛被包封 在脂質體内。 另外，對於含有親脂性、溶解性差的活性化合物（特別 5 是具有如EP-A-560 138中所揭示的化學式的二氫吡啶化合 物）的冷涞-乾燥脂質體製品（freeze-dried liposomal preparations)，US 5,653,998 (對應於TW 359616)揭示使用 具特定化學式的短鏈脂肪酸（含有不超過10個碳原子）或其 鹽類來作為安定劑，以供形成將該親脂性、溶解性差的活 10 性化合物包封在磷脂膜内的可供非經腸道投藥安定的脂質 體藥學製品。 於授予Martin C. Woodle等人的US 5,013,556中所揭示 的脂質體包含有1〜20 wt%之經一聚烧基醚（polyalkylether) 予以衍生化的親水疏水兩重性脂質（amphipathic lipid)，此 15 可以經聚乙二醇予以衍生化的磷脂醯乙醇胺（phosphatidyl ethanolamine derivatized with polyethyleneglycol)為例。該 經衍生化的脂質可以使脂質體的循環期間增進數倍。 於授予Francis J· Martin等人的US 5,213,804的實施例 10中進一步揭示利用US 5,013,556中所揭示的脂質體來製 20 備多索如必辛脂質體，其中選用的脂質組份以選定的比例 被溶於氯仿内，並予以添加配於氯仿/乙醇溶液内的α -生育 酚（alpha-tocopherol)，然後氣仿溶劑被移除而形成一脂質薄 膜。該脂質薄膜接而以一含有1 mM desferal的125 mM硫酸 銨水溶液予以水合，而該水合處理係藉由使用液態氮與一 1250877 ’里水洽之冷凍-融解法來進行。之後，進行擠壓處理來調整 貝體的尺寸’繼而於5%蔗糖溶液内進行透析。一多索如 必辛〉谷液接而與經透析的脂質體製備物混合，並於60°C的 水’合下予以振搖培育，而使得多索如必辛被包封於脂質體 5 内。 杈予 Yechezkel Barenolz等人的 US 5，192,549 中揭示將 親水疏水兩重性藥物裝填至脂質體内的方法，其中包括 在一錢溶液内形成脂質體，隨後將存在於脂質體的外部水 相内的銨移除或稀釋，因而於脂質體的内部與外部水相之 1〇間生成—pH梯度，而使得該親水疏水兩重性藥物被攝入脂 貝體内，而且該親水疏水兩重性藥物存在於脂質體内部之 最終數量要比存在於脂質體外部之數量為高。 以上所提到的所有文獻、專利前案以及當中所引述的 文獻的全部揭露内容在此被併入本案以作為參考資料。 15 就申請人所知，在上述文獻或專利前案中所揭示的方 法中：㈣在操作上涉及到氯仿或其他有毒的有機溶劑的 移除；有的在溶液注射法（s〇lvent injecti〇n)上有諸多限制而 不利於無菌製劑的操作，特別是所採用的溶液轉速、注射 速率、溶劑比例、注射針孔徑等操作條件都會非常容易地 影響到微脂體的均-性以及品質；有的在擠壓㈣usi〇n) 過程中會使操作壓力高達150〜250 psi，而更非為所欲的是 擠壓速率非常低（約為0.1〜0.2 L/min·)，若要付諸實際量 產’這會使得製作過程冗長而完全不符合無菌針劑的生產 要求。有鑒於上述文獻或專利前案中所揭示的方法無一者 10 1250877 能應用於脂質體懸浮液的大規模生產上，本技藝仍存在有 一需要來發展一快速且經濟的方法以供脂質體的大規模生 產。 就申請人的所知，上述文獻或專利前案中所揭示的方 5 法有的在操作上涉及到氯仿或其他有毒的有機溶劑的移 除，且無一者能應用於脂質體懸浮液的大規模生產上。因 此，本技藝仍存在有一需要來發展一快速且經濟的方法以 供脂質體的大規模生產。 【發明内容】 10 發明概要 因此，為了能夠大規模生產脂質體懸浮液，且不涉及 有毒的有機溶劑的移除，在第一個方面，本發明提供一種 用以製造一脂質體懸浮液的方法，其包含下列步驟： (a)將下列組份加入至一適量的一醇溶劑内以形成一 15 混合物： (i) 一選自於下列群組中的磷脂化合物：卵磷脂 (lecithin)、構脂醯膽驗（phosphatidylcholines， PC)、礙脂醯乙醇胺（phosphatidylethanolamines， PE)、鱗脂 Si甘油（phosphatidylglycerols，PG)、 20 鱗脂醯肌醇（phosphatidylinositols)、神經顆構脂 (sphingomyelins，SM)、鱗脂酸（phosphatidic acids)，前述化合物的二（C12-C18)醯基衍生物， 以及此等之一組合， (ii) 膽固醇（cholesterol)，以及 11 1250877 (in) —選自於下列群組中的聚乙二醇_衍生的化合物 (PEG-dedved Compound) ··聚乙二醇—脂醯乙醇 胺（PEG_PE)、甲氧基聚乙二醇_磷脂醯乙醇胺 (mPEG-PE)，前述化合物的二（Ci2-Ci8)酸基衍生 5 物，以及此等之一組合， 其中組份⑴、（ii)與（iii)的數量是呈5〜1〇: 2〜1〇: i 的莫耳比例，而且組份（i)、（ii)與（iii)的總量相對於 該醇溶劑的數量是呈1 : 5以上（w/v)的比例； (b)令得自步驟（a)的混合物直接與一硫酸銨水溶液相 1〇 混合，其中該得自步驟⑷的混合物與該硫酸銨 ((NH4)2S〇4)水溶液的數量係呈1 : 2〜10 (v/v)之比 例； (C)令步驟（b)所形成的混合物進行一孔擠壓處理 (pore-extrusion treatment)； 15 以一 〜15%蔗糖水溶液來透析從步驟（c)所得到 的經孔擠壓處理的混合物，藉此，—含有脂質體粒 子散浮在内的脂質體懸浮液被得到。 由本案方法所製得的脂質體懸浮液可被用來包封一親 水疏水兩重性藥物，因此，在第二個方面，本發明提供一 20種用以製造一脂質體-包封的藥物的方法，其包含下列步 驟1 k疋的藥物與一由上述方法所製得的脂質體懸浮 液相混合，藉此，該親水疏水兩重性藥物被包封在該脂質 體懸浮液中的脂質體粒子内。 本發明之其他目的、特徵及優點，在參照以下詳細說 12 1250877 明與較佳實施例後，將變得明顯。 t實方式u 較佳實施例之詳細說明（發明的詳細說明） 申請人發現，目前被本技藝的業者用來生產脂質體懸 5 浮液或脂質體-包封的藥物之方法，在製造過程上常常涉及 到要將用來溶解脂質組份的有害有機溶劑（諸如氯仿或 DMSO等等）移除之步驟，且這些方法幾乎都無法做到脂質 體的大規模生產。為克服這些問題，申請人研究出一改良 方法，其在操作上不需要用到諸如氯仿或DMSO的有害有機 10 溶劑，且其可大規模地生產出所欲的脂質體懸浮液，進而 供應用於一選定藥物的包封。 於是，本發明提供一種用以製備一脂質體懸浮液的方 法，其包含下列步驟： (a)將下列組份加入至一適量的一醇溶劑内以形成一混 15 合物： (i) 一選自於下列群組中的磷脂化合物：卵磷脂 (lecithin)、填脂酸膽驗（phosphatidylcholines， PC)、填脂酸乙醇胺（phosphatidylethanolamines， PE)、填脂醯甘油（phosphatidylglycerols，PG)、 20 填脂酸肌醇（phosphatidylinositols)、神經勒鱗脂 (sphingomyelins, SM)、麟脂酸（phosphatidic acids)，前述化合物的二（C12-C18)醯基衍生物， 以及此等之一組合， (ii) 膽固醇（cholesterol)，以及 13 1250877 (iii) 一選自於下列群組中的聚乙二醇-衍生的化合物 (PEG-derived compound):聚乙二醇_碟脂醯乙醇 胺（PEG-PE)、甲氧基聚乙二醇_磷脂醯乙醇胺 (mPEG-PE)，前述化合物的二（cacy醯基衍生 物，以及此等之一組合， 其中組份⑴、（ϋ)與（iii)的數量是呈5〜1〇 : 2〜1〇 : 1 的莫耳比例，而且組份⑴、（ii)與（iii)的總量相對於 該醇溶劑的數量是呈1 : 5以上（w/v)的比例； (b)令得自步驟（a)的混合物直接與一硫酸銨水溶液相混 合，其中該得自步驟（a)的混合物與該硫酸銨 ((NHASCU)水溶液的數量係呈i ·· 2〜10 (v/v)之比 例； ⑷令步驟（b)所形成的混合物進行一孔擠壓處理 (pore-extrusion treatment)； 15 ⑷ 以一 5%〜15%蔗糖水溶液來透析從步驟（c)所得到的 經孔擠壓處理的混合物，藉此，一含有脂質體粒子 散浮在内的脂質體懸浮液被得到。 20 在本案方法的步驟（a)中所用的醇溶劑是一可與水互溶 的無毒性醇溶劑，且較佳地是選自於下賴構成的群組： 脂族醇（諸如甘油、丙二醇）、乙醇、異丙醇、甲醇、乙 、及此等 < ⑽合物。在本發明的_個較佳具體例中，在 本案方法的步驟（a)中所用的醇溶劑是乙醇。Regarding the preparation of liposomes, currently known methods are, for example, hydration, ultrasonification, dialysis and dilution processes, reverse-phase evaporation. (See, for example, US 4,235,871), surfactant-active surfactant treatment, dehydration-rehydration or dry-reconstitution, east knot and melt method (freeze and Thaw) (see, for example, US 6,355,267), pore extrusion, and high pressure homogeni-zation, etc. (see, for example, dZ)·MM 15 Standish, JC Watkins, J. Mol. Biol., 13, 238-252, 1965; RRC New, ed. Liposomes: a practical approach (1990), Oxford; G. Gregoriadis, ed· Liposome Technology, Vol· 1, 1993, CRC·, H. Talsma and DJA Crommelin, Pharmaceutical Technology, 96-106, Oct. 1992). Among the methods known for this, the pore extrusion method and the high pressure homogenization method are considered to have industrial potential for large-scale production of liposomes. If further economic benefits are considered, the hole extrusion method is considered to be a better choice. No. 4,737,323 discloses a method for producing a liposome suspension by extrusion, which has a uniform size and a 1250877 average particle size of not more than 〇4 Pm, the method comprising the following steps: providing a containment size a suspension of liposomes having a major portion having a size greater than 1 μm; and allowing the suspension to pass through an asymmetrical ceramic filter under dusting, the inner surface of the filter The pore size is greater than the desired average liposome 5 size and no greater than about 丨·0 μιη. According to the disclosure of this U.S. patent, an organic solvent such as chloroform (chl〇r〇f〇rm) as shown in Example 该 of the present invention is used to dissolve the lipid component. Then, the organic solvent is in a vacuum or an inert gas. The lower layer is removed to form a dry film composed of lipids. An aqueous medium is then applied to the film to expand the lipids 10, thereby forming lipids of different sizes in the aqueous matrix. U.S. Patent No. 5,004,611 and U.S. Patent No. 5,217, the disclosure of which is incorporated herein by reference to the entire entire entire entire entire entire entire entire entire disclosure a component having a ratio of 40 · 1 to 1: 20 (a) at least a portion of a bilayer forming membrane lipid and a component (b) at least one substantially consisting of a solvent for the lipid A non-aqueous liquid composed of a water-miscible organic liquid is mixed with a sufficient amount of water whereby the liposome is spontaneously formed, before the addition of water It is then added and is ultimately present in an amount sufficient to cause the biologically effective dose of one of the biologically active compounds 20 to be associated with the liposomes. According to the disclosure of Example 1 of these two U.S. patents, first at 50 to 60 Preparing a pre-liposome composition at ° C, wherein the lipid component (a) (eg, egg fat) is first dissolved in the component (b) (eg, ethanol); The water is divided into two parts, wherein the first part of the water 1250877 may contain the biologically active compound (eg glucose) and the second part of the water is used to make the final formulation. The liposome composition is cooled to 25 ° C after being equilibrated under nitrogen, and then added to the discate buffer in two stages and shaken, thus forming a compound having the biological 5 activity dispersed therein. An aqueous dispersion of liposomes. Thus, according to the disclosure of these two U.S. patents, the ethanol used will be included in the aqueous dispersion of the liposome formed in a specific amount. Granted to Francis C. Szoka A method for making a liposome suspension 10 is disclosed in US Pat. No. 5,077,057, the disclosure of which is incorporated herein by reference. A poor solubility is exhibited in the hydrocarbon solvent. According to the disclosure of the three U.S. patents, a poorly soluble compound is dissolved in an aprotic solvent (such as DMS0) with the same amount of encapsulated lipids. It is desirable to additionally use a lower alkanol (e.g., 15 ethanol) to aid in the dissolution of the lipid. The resulting mixture is then extruded or injected into a suitable aqueous solution and agitated to form a liposomal suspension. The squeezing means used may be a syringe, a perforated disc or tube or other suitable means for providing a hole of about 0.05 to 5 mm. In particular, Example 2 of the three U.S. patents illustrates a 20-package of doxorubicin in which doxorubicin is dissolved in DMSO and added to a glycerol containing lecithin ( EPG): Lecithin choline (EPC) · · Cholesterol (7:3:6) in ethanol solution. Next, the lipid-dose succinimide mixture was injected into the aqueous phase consisting of 140 mM NaCl-10 mM Tris-HCl, pH 4_0 at 3 rc, and then the formed lipid suspension was dialyzed and utilized. The column layer 1250877 was isolated by liposome-encapsulated doxorubicin. The liposome prepared in this example had a particle size of 227 nm, and 41.2% of the polysoxin was encapsulated in In addition, for liposome-dried liposome preparations containing a lipophilic, poorly soluble active compound (particularly 5 as a dihydropyridine compound having the chemical formula disclosed in EP-A-560 138) (freeze- Dried liposomal preparations), US 5,653,998 (corresponding to TW 359616) discloses the use of short-chain fatty acids of a specific chemical formula (containing no more than 10 carbon atoms) or salts thereof as stabilizers for the formation of such lipophilic, poorly soluble The liposome disclosed in US 5,013,556 to Martin C. Woodle et al. contains 1 to 20 wt% of the liposome disclosed in US Patent No. 5,013,556 to Martin C. Woodle et al. Polycondensation (polyalkylether) Derivatized hydrophilic hydrophobic double lipid (amphipathic lipid), which can be exemplified by phosphatidyl ethanolamine derivatized with polyethyleneglycol. The derivatized lipid can make lipid In the case of Example 10 of US Pat. No. 5,213,804, the disclosure of which is incorporated herein by reference. The lipid component is dissolved in chloroform at a selected ratio, and alpha-tocopherol is added to the chloroform/ethanol solution, and then the solvent is removed to form a lipid film. This was followed by hydration with a 125 mM aqueous solution of ammonium sulphate containing 1 mM desferal, which was carried out by a freeze-thaw method using liquid nitrogen with a 1,250,877 lis. The size of the shell' is then dialyzed in a 5% sucrose solution. The liposome preparations were mixed and incubated with water at 60 ° C under shaking to allow doxorubicin to be encapsulated in liposomes 5. U.S. Patent No. 5,192,549 to Yechezkel Barenolz et al. A method of filling a hydrophilic hydrophobic dual drug into a liposome, comprising forming a liposome in a solution of money, and subsequently removing or diluting the ammonium present in the outer aqueous phase of the liposome, thereby being internal to the liposome A pH gradient is generated between the outer aqueous phase, so that the hydrophilic hydrophobic dual drug is taken up into the liposome, and the final amount of the hydrophilic hydrophobic dual drug present in the liposome is greater than the amount existing outside the liposome. High. All of the documents, patents, and references cited above are incorporated herein by reference. 15 To the best of the applicant's knowledge, in the methods disclosed in the above mentioned documents or patents: (iv) the removal of chloroform or other toxic organic solvents involved in operation; some in solution injection (s〇lvent injecti〇) n) There are many restrictions that are not conducive to the operation of sterile preparations, especially the operating conditions such as the speed of the solution, the injection rate, the solvent ratio, and the pore size of the injection needle can easily affect the homogeneity and quality of the liposome. Some may cause operating pressures of up to 150 to 250 psi during extrusion (four) usi〇n), and the more desirable is that the extrusion rate is very low (about 0.1 to 0.2 L/min·), to pay The actual mass production 'this will make the production process lengthy and completely inconsistent with the production requirements of aseptic injection. In view of the fact that none of the methods disclosed in the above documents or patents can be applied to large-scale production of liposome suspensions, there is still a need in the art to develop a rapid and economical method for liposomes. Mass production. As far as the Applicant is aware, some of the methods disclosed in the above documents or patents involve the removal of chloroform or other toxic organic solvents in operation, and none of them can be applied to liposome suspensions. Mass production. Therefore, there is still a need in the art to develop a rapid and economical method for the mass production of liposomes. SUMMARY OF THE INVENTION [Invention] Accordingly, in order to enable large-scale production of liposome suspensions without involving removal of toxic organic solvents, in a first aspect, the present invention provides a method for producing a liposome suspension It comprises the following steps: (a) adding the following components to an appropriate amount of monool solvent to form a 15 mixture: (i) a phospholipid compound selected from the group consisting of lecithin, Phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), 20 phosphatidylinositols, sphingomyelins (SM), Phosphadidic acids, di(C12-C18) mercapto derivatives of the foregoing compounds, and combinations of these, (ii) cholesterol (cholesterol), and 11 1250877 (in) - selected from the group consisting of PEG-dedved Compound · Polyethylene glycol-lipid ethanolamine (PEG_PE), methoxy polyethylene glycol _ phospholipid 醯 ethanolamine (mPEG-PE) a di(Ci2-Ci8) acid group derived from the above compound, and a combination thereof, wherein the amounts of the components (1), (ii) and (iii) are 5~1〇: 2~1〇: i Mohr ratio, and the total amount of components (i), (ii) and (iii) is a ratio of 1:5 or more (w/v) with respect to the amount of the alcohol solvent; (b) obtained from the step ( The mixture of a) is directly mixed with an aqueous solution of ammonium monosulfate, wherein the amount of the mixture obtained from the step (4) and the aqueous solution of the ammonium sulfate ((NH4)2S〇4) is 1: 2 to 10 (v/v). (C) subjecting the mixture formed in the step (b) to a pore-extrusion treatment; 15 dialysis against the pores obtained in the step (c) by using a -15% aqueous solution of sucrose The treated mixture, whereby a liposome suspension containing the dispersion of liposome particles is obtained. The liposome suspension prepared by the method of the present invention can be used to encapsulate a hydrophilic hydrophobic dual drug, and therefore, in a second aspect, the present invention provides a method for producing a liposome-encapsulated drug. The drug comprising the following step 1 k疋 is mixed with a liposome suspension prepared by the above method, whereby the hydrophilic hydrophobic dual drug is encapsulated in the liposome particles in the liposome suspension. Other objects, features and advantages of the present invention will become apparent from the description and appended claims appended claims DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (Detailed Description of the Invention) Applicants have discovered that methods currently used by the art to produce liposomal suspension 5 suspensions or liposome-encapsulated drugs are in the manufacturing process. The step of removing the harmful organic solvent (such as chloroform or DMSO, etc.) used to dissolve the lipid component is often involved, and almost no such method can achieve mass production of the liposome. In order to overcome these problems, the Applicant has developed an improved method which does not require the use of a harmful organic 10 solvent such as chloroform or DMSO, and which can produce a desired liposome suspension on a large scale, and then supply it for use. Encapsulation of a selected drug. Accordingly, the present invention provides a method for preparing a liposome suspension comprising the steps of: (a) adding the following components to an appropriate amount of a monol solvent to form a mixed 15-compound: (i) Phospholipid compounds selected from the group consisting of lecithin, phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), 20 Phosphatidylinositols, sphingomyelins (SM), phosphatidic acids, di(C12-C18)indolyl derivatives of the foregoing compounds, and combinations thereof, (ii) Cholesterol (cholesterol), and 13 1250877 (iii) a PEG-derived compound selected from the group consisting of polyethylene glycol _ 醯 醯 ethanolamine (PEG-PE), Methoxy polyethylene glycol _ phospholipid oxime ethanolamine (mPEG-PE), bis (cacy fluorenyl derivative of the aforementioned compound, and a combination thereof, wherein the amounts of components (1), (ϋ) and (iii) are 5~1〇: 2~1〇: 1 Molar ratio, and the total amount of components (1), (ii) and (iii) is a ratio of 1:5 or more (w/v) with respect to the amount of the alcohol solvent; (b) obtained from step (a) The mixture is directly mixed with an aqueous solution of ammonium monosulfate, wherein the amount of the mixture obtained from the step (a) and the aqueous solution of ammonium sulfate ((NHASCU) is in a ratio of i ·· 2 to 10 (v/v); (4) The mixture formed in the step (b) is subjected to a pore-extrusion treatment; 15 (4) dialysis of the pore-extruded mixture obtained in the step (c) with a 5% to 15% aqueous sucrose solution, Thereby, a liposome suspension containing the liposome particles is obtained. 20 The alcohol solvent used in the step (a) of the method of the present invention is a water-miscible non-toxic alcohol solvent, and preferably It is selected from the group consisting of aliphatic alcohols (such as glycerin, propylene glycol), ethanol, isopropanol, methanol, ethylene, and the like (10). In the preferred embodiment of the present invention The alcohol solvent used in step (a) of the process of the present invention is ethanol.
較佳地，被使用於本案方法步驟⑷中的組份⑴是選自 於下列所構成的組群：磷脂醯膽驗（Pc)、二月桂酿PC 14 1250877 (dilauroyl PC)、二肉莖蔻醯PC (dimyristoyl PC)、二棕櫚醯 PC (dipalmitoyl PC)、二硬脂酸 PC (Distearoyl phosphatidylcholine，DSPC)、二油醯PC (dioleoyl PC)、二 亞麻油醯PC (dilinoleoyl PC)、1-棕櫚醯-2_油醯PC 5 (l_palmitoyl-2-oleoyl PC)，以及此等之一組合。在本發明 的一個較佳具體例中，被使用於本案方法步驟（a)中的組份 ⑴是DSPC。 較佳地，被使用於本案方法步驟（a)中的組份（iii)是選 自於下列所構成的組群·· PEG-2000-PE、PEG-3000-PE、 10 PEG-4000-PE、PEG-5000-PE、mPEG-2000 PE、mPEG-3000 PE、mPEG_4000 PE、mPEG-5000 PE，前述化合物的二 (Ci2_Ci8)驢基衍生物，以及此等之一組合。 該等二（Ci2-C is)隨基衍生物之實例包含’但不限於， PEG-2000-DSPE、PEG-3000-DSPE、PEG-4000-DSPE、PEG-15 5000-DSPE、1,2-二醯基-SN-甘油基-3-磷脂醯乙醇胺-N-[甲 氧基（聚 6:，）-2000](l，2-diacyl-SN-glycen>3-phosphatidylethanolamine-N-[methoxy (poly ethylene glycol)-2000])、1，2-二醯基-SN-甘油基-3-磷脂醯乙醇胺-N-[甲氧基 (聚乙二醇）-3000]，其中該醯基是肉菫蔻醯、棕櫚醯基、硬 20 脂醯或油醯。在本發明的一個較佳具體例中，被使用於本 案方法步驟（a)中的組份（iii)是PEG-2000-DSPE。 較佳地，被使用於本案方法步驟（a)中的組份（i)、（ii) 與（iii)的總量相對於該醇溶劑的數量是呈1 : 5以上（w/v)的 比例，且更佳為1 : 7〜10 (w/v)。 15 1250877 在本發明的-個更佳㈣例中，於本案方法的步驟⑷ 中，使用DSPC、膽@醇以及PEG_2隊DspE來作為脂質體 粒子的構成組份並將此等溶於乙醇溶劑内。 本案方法的步驟（a)係在一為“〜乃它的溫度範圍下被 5進行，較佳為55〜65t：，更佳為6(TC。 在本發明的一個較佳具體例中，Dspc、膽固醇以及 peg-2〇o〇-DSPE被溶於乙醇内，並於一個6〇t的水浴内被 均勻混合。 在本案方法步驟（b)中，不像習知方法要使用一注射器 1〇來將脂質/有機溶劑混合物以一小數量分次地注射至一水 性溶液内，俾以形成一月旨質體散浮液，可在攪拌下將硫酸 錢水溶液直接加入至得自步驟⑷的混合物内，《是將得自 步驟（a)的混合物直接加入至硫酸銨水溶液内。 車乂佳地，本案方法步驟（b)是在一為45〜7〇。〇的溫度範 15圍下被進行，較佳為55〜，更佳為6(TC。在此操作溫 度下，於混合物内初形成的脂質體粒子的雙層脂質膜結構 疋鬆散的，且該等脂質體粒子會將少量的硫酸銨水溶液包 封在内。 較佳地，被使用於本案方法步驟（b)中的硫酸銨水溶液 2〇具有一當量濃度為〇.2〜〇·8Ν，更佳為〇·4〜〇·6Ν。 較佳地，在本案方法步驟（…中，該得自步驟的混合 物與硫酸銨水溶液的數量係呈1:2〜1〇(v/v)之比例，更佳 為 1 : 4 〜8 (v/v) 〇 較佳地，在本案方法步驟（c)中，該孔擠壓處理係藉由 16 1250877 令步驟⑻所形成的混合物通經—個具_孔㈣圍為 0.45 μηι的擠壓器而被進行。 適用於本發明的㈣n可為任—種可提供大約〇〇5〜 0.45 μηι的擠遂孔#的注射筒、褒設有過遽膜的㈣裳置、 5有孔的盤或管或是其他合適的褒置。該過滤膜可為陶究過 濾膜或聚碳酸酯過濾膜。 較佳地’在本案方法步驟（c)中，於步驟（b)所形成的混 合物先被通經-擠壓孔徑較大的過滤器，繼而通經一擠壓 孔徑較小的過濾器而被進行兩階段的擠壓。在本發明的一 10個較佳具體例中’在所用的操作温度（例如，6〇。〇下，於步 驟(b)所形成的混合物先被通經_擠壓孔徑為G1叫的過滤 器’繼而通經-擠壓孔徑為0.05 μιη的過遽器而被進行兩階 段的擠壓。 較佳地，本案方法步驟⑷係在環温下被進行，這時脂 15貝體粒子的雙層月日質膜結構會變成緊密的，而使得被包封 在月曰貝體粒子内的硫酸銨水溶液不會因為透析處理而從脂 質體粒子的内部全部地流失至嚴糖溶液内。 由本案方法步驟⑷所得到的脂質體懸浮液可被馬上應 用亦可被直接低溫保存（例如，保存在5°C下），或是經冷 2〇珠乾燥處理後再予儲存於低溫（例如，下以供曰後使用。 與習知方法相較，依據本發明的脂質體懸浮液製備方 法可^相對低壓（約4〇〜14〇psi)下進行，而使操作速率得以 提昇（、”勺為2〜1 〇 L/minute)。因此，本發明能以一快速 且經濟的方法來供脂質體的大規模生產。 17 1250877 依據本案方法而被製得的脂質體懸浮液可供應用於藥 物、化妝品等產業上，特別是被用來包封一選定的藥物， 包括，但不限於，anthracycline類抗生素、喜樹鹼 (Camptothecin)類抗癌藥。 5 因此，本發明亦提供一種用以製造一脂質體-包封的藥 物的方法，其包括下列步驟：令一選定的藥物與一由上述 方法所製得的脂質體懸浮液相混合，藉此，該藥物被包封 在該脂質體懸浮液中的脂質體粒子内。 較佳地，依據本案方法而被製得的脂質體懸浮液被用 10 來包封夕索如必辛（doxorubicin)、多諸如必辛 (daunorubicm)、依瑞諾特肯（irin〇tecan)以及維諾瑞賓 (vinorelbine)。 較佳地’該藥物以一為超過9〇叫藥物/μιη〇ι脂質的數 1被包封在脂質體内，更佳為12〇〜18〇盹藥物/μιη〇1脂質。 15 較佳地，該藥物與脂質體懸浮液的混合是在一為45〜 7〇°C的溫度範圍下被進行，較佳為55〜65它，又更佳為6〇 C °於該溫度下，脂質體粒子的雙層脂質膜結構會變成較 鬆散的’而容許該藥物進入至脂質體粒子的内部，進而有 可能與被包封在内硫酸銨形成離子鍵結。之後，混合物被 20降溫至環溫’這時脂質體粒子的雙層脂質膜結構即再度成 為緊密的，藉此，被包封在内的藥物即不會滲漏出去。 較佳地’在進行藥物包封時，可同時使用一製藥上可 接又的添加劑，例如：低溫保護劑（cry〇pr〇tect〇rs)，包括多 凡醇（諸如甘油）、單醣（諸如葡萄糖）、雙醣（諸如蔗糖、乳 18 1250877 糖、海藻糖）以及蛋白質或胺基酸（諸如組胺酸）；安定劑， 諸如抗氧化劑形式的安定劑，例如丁基化羥基苯甲醚 (butylated hydroxyanisole，BHA)、丁 基化經基甲苯 (butylated hydroxytoluene，ΒΗΤ)、α -生育紛（alpha-5 tocopherol)暨其鹽類、抗壞血酸暨其鹽類與酯類，較佳為抗 壞血酸暨其鹽類；pH-調節劑，諸如緩衝物、酸或鹼，特別 是抗壞血酸與氫氧化納；渗透活性劑，諸如甘油、甘露糖 醇與葡萄糖。 在本發明的一個較佳具體例中，依據本案方法而被製 10 得的脂質體懸浮液被用來包封多索如必辛（doxorubicin)，同 時使用組胺酸作為安定劑。 本發明將就下面實施例來作進一步說明，但應瞭解的 是，該等實施例僅為例示說明之用，而不應被解釋為本發 明的實施之限制。 15 實施例 實驗材_料的製備： A. 硫酸銨水溶液： 將158g的硫酸銨（Showa Co.，Japan)加入至注射用水 内並調整體積至6L，接而以Posydine (Whatman Inc·， 20 Germany)薄膜（142mm，0.22 μηι)予以過濾、並保存在6°C下 備用。在使用前，將該硫酸銨水溶液的溫度調整至60°C。 B. 9%(w/w)蔗糖溶液： 將4500 g的蔗糖加入至適量注射用水内溶解並稀釋至 50 L，以Posydine薄膜（142 mm，0.22 μπι)予以過濾後備用。 19 1250877 C.組胺酸-蔗糖溶液： 將12.6 g的組胺酸單水合物（histidine monohydrate) (Ajinomoto Co.，Japan)溶於 90 mL 的 9%蔗糖溶液内，以 IN NaOH將pH值調至6.2〜6.6之間後，加入9%蔗糖溶液至 5 3 00 mL並混合均勻，繼而以Posydine薄膜（47 mm，0.22 μηι) 予以過濾後備用。 實施例1·脂質體懸浮液的製備·· 於一玻璃容器内，將 PEG-2000-DSPE (16 g)(Genzyme Co·，America)、膽固醇（15·2 g)(N〇F Co.，Japan)以及 DSPC 10 (46.4 g)(NOF Co·，Japan)加入至600 mL的乙醇内，並在加 熱至一大約為60°C之溫度下混合，以使此等形成一個均勻 混合液。 上述混合液在大約60°C下被持續攪拌，並予以直接加 入4 L的先前已備妥且溫度維持為60°C的硫酸銨水溶液 15 内。所形成的混合物接著於60°C下使用一個1.5 L過濾組 (Advantec Toyo Kaisha，Ltd·，Japan)來進行下列的孔擠壓處 理： (1)於該過濾組内裝配一個0·1 μηι 142mm濾膜，並將 混合物過濾10次；以及 20 (2)將一個0.05 μιη 142 mm濾、膜改裝配於該過濾組 内，再將混合物過濾10次， 其中擠壓壓力被控制為3〜10 kg/cm2 (流速約為2〜10 L/min.) 〇 從上述孔擠壓處理所收集到4500 mL的過濾液於環溫 20 1250877 下利用一個 30 KD 的空心管（Hollow Fiber，A/G Technology， UFP-3 0-C-6A，30,000 NM，4800 cm2)以及大約 30 L 的先前 已備妥的9% (w/w)蔗糖溶液來進行透析，而收集到容積約 為3000 mL之不含乙醇的脂質體懸浮液。 5 產品品質分析 對於實施例1所製得的脂質體懸浮液，取出一樣品並 以一 GC分析儀（Varian，Inc·，America)來進行分析，結果顯 示該樣品内的脂質體懸浮液不含乙醇；另取一樣品並以 Particle size analyzer(Beckman coulter, Inc·)來進行粒徑 10 (particle size)分析，檢測結果顯示該樣品内所含有的脂質 體之平均粒徑為72.9 nm。 實施例2.製備脂質體包封的多索如必辛（liposome-encapsulated doxorubicin) = 將實施例1所得到的3000 mL脂質體懸浮液加入至一 15 個内裝有8000 mg的多索如必辛HC1 (doxorubicin HC1，紅 色粉末）的玻璃容器内，繼而加入200 mL先前製備的組胺 酸_蔗糖溶液。所形成的混合物被置於一為60°C的水浴中並 予以振搖歷時30分鐘。之後，將混合物冷卻至大約35°C， 並以先前製備的9%蔗糖溶液予以稀釋至4 L並混合均勻。 20 由此即得到所欲的脂質體-包封的多索如必辛（liposome-encapsulated doxorubicin) 。 所 形成的 產物被 進一步 分裝於 無菌玻璃小管（sterile glass vials)内，而製作成含有2.0 mg 多索如必辛HCl/mL的注射製品（injection preparation)。 產品品質分析 21 1250877 對於實施例2所製得的脂質體_包封的多索如必辛注射 衣，觀察被封裝在玻璃小管内的内含物的色澤，結果發 現該等玻璃小管都是包含外觀看來呈橘紅色至紅色的注射 液。取適量樣品以一 HPLC分析儀（Waters c〇，Amedca)來 進步與標準品作比較，結果顯示測試樣品内所含的多索 如必辛HC1的滞留時間（retenti〇n time)與洗提圖譜⑷此⑽ Pwfile)跟標準品所具者相一致。 另外分析實施例2所製得的脂質體_包封的多索如必辛 10Preferably, the component (1) used in the step (4) of the method of the present invention is selected from the group consisting of phospholipids (Pc), PC 14 1250877 (dilauroyl PC), and two meat stems.醯PC (dimyristoyl PC), dipalmitoyl PC, Distearoyl phosphatidylcholine (DSPC), dioleoyl PC, dilinoleoyl PC, 1-palm醯-2_油醯PC 5 (l_palmitoyl-2-oleoyl PC), and a combination of these. In a preferred embodiment of the invention, the component (1) used in step (a) of the method of the present invention is a DSPC. Preferably, the component (iii) used in the step (a) of the method of the present invention is selected from the group consisting of PEG-2000-PE, PEG-3000-PE, 10 PEG-4000-PE. PEG-5000-PE, mPEG-2000 PE, mPEG-3000 PE, mPEG_4000 PE, mPEG-5000 PE, a di(Ci2_Ci8) mercapto derivative of the aforementioned compound, and a combination thereof. Examples of such two (Ci2-Cis) derivatives include 'but are not limited to, PEG-2000-DSPE, PEG-3000-DSPE, PEG-4000-DSPE, PEG-15 5000-DSPE, 1,2- Dimercapto-SN-glyceryl-3-phospholipid oxime ethanolamine-N-[methoxy(poly6:,)-2000](l,2-diacyl-SN-glycen>3-phosphatidylethanolamine-N-[methoxy ( Polyethylene glycol)-2000]), 1,2-dimercapto-SN-glyceryl-3-phospholipid oxime ethanolamine-N-[methoxy(polyethylene glycol)-3000], wherein the thiol group is meat菫蔻醯, palm 醯, hard 20 醯 or oil 醯. In a preferred embodiment of the invention, component (iii) used in step (a) of the method of the invention is PEG-2000-DSPE. Preferably, the total amount of components (i), (ii) and (iii) used in step (a) of the method of the present invention is 1:5 or more (w/v) with respect to the amount of the alcohol solvent. The ratio, and more preferably 1: 7 to 10 (w/v). 15 1250877 In a more preferred (four) example of the present invention, in step (4) of the method of the present invention, DSPC, cholesteryl alcohol, and PEG_2 team DspE are used as constituent components of the liposome particles and dissolved in an ethanol solvent. . Step (a) of the method of the present invention is carried out by 5 in a temperature range of "~, but it is preferably 55 to 65t:, more preferably 6 (TC.) In a preferred embodiment of the present invention, Dspc Cholesterol and peg-2〇o〇-DSPE are dissolved in ethanol and uniformly mixed in a 6 〇t water bath. In step (b) of the method, a syringe is not used as in the conventional method. The lipid/organic solvent mixture is injected into the aqueous solution in small portions in a small amount to form a January plastid dispersion, and the aqueous solution of sulfuric acid can be directly added to the mixture obtained from the step (4) under stirring. Inside, "the mixture obtained from step (a) is directly added to the aqueous solution of ammonium sulfate. In the ruthless manner, step (b) of the method is carried out at a temperature of 15 to 〇. Preferably, it is 55~, more preferably 6 (TC.) at this operating temperature, the bilayer lipid membrane structure of the liposome particles initially formed in the mixture is loose, and the liposome particles will have a small amount of sulfuric acid. The aqueous ammonium solution is encapsulated. Preferably, it is used in the method step (b) The aqueous ammonium sulfate solution has an equivalent concentration of 〇.2~〇·8Ν, more preferably 〇·4~〇·6Ν. Preferably, in the method step (..., the mixture obtained from the step) The amount of the aqueous solution with ammonium sulfate is in a ratio of 1:2 to 1 Torr (v/v), more preferably 1:4 to 8 (v/v). Preferably, in the method (c) of the method, The hole extrusion treatment is carried out by a mixture formed by the step (8) of 16 1250877, which is carried out by an extruder having a hole of 0.45 μm. The (4) n which is suitable for the present invention can be provided for any kind.注射5~ 0.45 μηι squeezed hole # of the syringe, 褒 褒 褒 ( 四 四 四 四 四 四 四 四 、 、 、 、 、 、 、 、 、 、 、 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是 或是Or a polycarbonate filter membrane. Preferably, in step (c) of the method, the mixture formed in step (b) is first passed through a filter having a larger pore size, and then passed through an extrusion orifice. The smaller filter is subjected to a two-stage extrusion. In one of the ten preferred embodiments of the invention 'at the operating temperature used (e.g., 6 Torr. Next, the mixture formed in the step (b) is first subjected to two-stage extrusion by passing through a filter having a pore size of G1 and then passing through a pressurizer having a pore diameter of 0.05 μm. Preferably, the method step (4) of the present method is carried out at a ring temperature, at which time the double-layered plasma membrane structure of the lipid 15 shell particles becomes compact, and the aqueous ammonium sulfate solution encapsulated in the moon shell particles is made. It will not be completely lost from the inside of the liposome particles to the strict sugar solution due to dialysis treatment. The liposome suspension obtained by the method step (4) of the present method can be applied immediately or can be directly cryopreserved (for example, stored at 5°). C)), or after drying by cold 2 beads, store it at low temperature (for example, use it for later use). Compared with the conventional method, the preparation method of the liposome suspension according to the present invention can be carried out at a relatively low pressure (about 4 Torr to 14 psi), and the operation rate can be improved ("the spoon is 2~1 〇L/ Minutes. Therefore, the present invention can provide large-scale production of liposomes in a rapid and economical manner. 17 1250877 The liposome suspension prepared according to the method of the present invention can be applied to industries such as pharmaceuticals and cosmetics, in particular Is used to encapsulate a selected drug, including, but not limited to, anthracycline antibiotics, camptothecin anticancer drugs. 5 Therefore, the present invention also provides a liposome-encapsulated A pharmaceutical method comprising the steps of: mixing a selected drug with a liposome suspension prepared by the above method, whereby the drug is encapsulated in liposome particles in the liposome suspension Preferably, the liposome suspension prepared according to the method of the present invention is encapsulated with 10 for doxorubicin, more such as daunorubicm, and irin〇tecan. Vinore (vinorelbine) Preferably, the drug is encapsulated in the liposome with a number of more than 9 药物 called drug/μιη〇ι lipid, more preferably 12 〇 18 〇盹 drug/μιη 〇 1 lipid. Preferably, the mixing of the drug with the liposome suspension is carried out at a temperature ranging from 45 to 7 ° C, preferably from 55 to 65, and more preferably at a temperature of 6 ° C. The double-layered lipid membrane structure of the liposome particles becomes looser and allows the drug to enter the interior of the liposome particles, which in turn may form an ionic bond with the encapsulated ammonium sulfate. Thereafter, the mixture is 20 The temperature of the double-layered lipid membrane of the liposome particles is once again compacted, whereby the encapsulated drug does not leak out. Preferably, when the drug is encapsulated, At the same time, a pharmaceutically acceptable additive such as cryoprotectant (cry〇pr〇tect〇rs), including polyalcohol (such as glycerol), monosaccharide (such as glucose), disaccharide (such as sucrose, milk 18) 1250877 sugar, trehalose) and protein or amino acid A histidine, a stabilizer such as an antioxidant, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (α), alpha-fertility (alpha) -5 tocopherol) and its salts, ascorbic acid and its salts and esters, preferably ascorbic acid and its salts; pH-adjusting agents such as buffers, acids or bases, especially ascorbic acid and sodium hydroxide; osmotic activity Agents such as glycerol, mannitol and glucose. In a preferred embodiment of the invention, the liposome suspension prepared according to the method of the present invention is used to encapsulate doxorubicin while using histidine as a stabilizer. The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting. 15 EXAMPLES Experimental material preparation: A. Ammonium sulfate aqueous solution: 158 g of ammonium sulfate (Showa Co., Japan) was added to water for injection and the volume was adjusted to 6 L, followed by Posydine (Whatman Inc., 20 Germany The film (142 mm, 0.22 μηι) was filtered and stored at 6 ° C until use. The temperature of the aqueous ammonium sulfate solution was adjusted to 60 ° C before use. B. 9% (w/w) sucrose solution: 4500 g of sucrose was added to an appropriate amount of water for injection and diluted to 50 L, and filtered with a Posydine film (142 mm, 0.22 μm) for use. 19 1250877 C. Histidine-sucrose solution: 12.6 g of histidine monohydrate (Ajinomoto Co., Japan) was dissolved in 90 mL of 9% sucrose solution, and the pH was adjusted with IN NaOH. After Between 6.2 and 6.6, a 9% sucrose solution was added to 5 3 00 mL and mixed well, followed by filtration with a Posydine film (47 mm, 0.22 μηι). Example 1·Preparation of liposome suspension·· In a glass container, PEG-2000-DSPE (16 g) (Genzyme Co., America), cholesterol (15·2 g) (N〇F Co., Japan) and DSPC 10 (46.4 g) (NOF Co., Japan) were added to 600 mL of ethanol and mixed at a temperature of about 60 ° C to form a homogeneous mixture. The above mixture was continuously stirred at about 60 ° C and directly added to 4 L of an aqueous ammonium sulfate solution 15 which was previously prepared and maintained at a temperature of 60 ° C. The resulting mixture was then subjected to the following pore extrusion treatment at 60 ° C using a 1.5 L filter set (Advantec Toyo Kaisha, Ltd., Japan): (1) A 0·1 μηι 142 mm was assembled in the filter set. Filter the membrane and filter the mixture 10 times; and 20 (2) a 0.05 μιη 142 mm filter, membrane modified into the filter set, and then filter the mixture 10 times, wherein the extrusion pressure is controlled to 3~10 kg /cm2 (flow rate is about 2~10 L/min.) 4 4500 mL of filtrate collected from the above hole extrusion treatment is used at a ring temperature of 20 1250877 using a 30 KD hollow tube (Hollow Fiber, A/G Technology , UFP-3 0-C-6A, 30,000 NM, 4800 cm2) and approximately 30 L of previously prepared 9% (w/w) sucrose solution for dialysis, with a volume of approximately 3000 mL collected A liposomal suspension of ethanol. 5 Product quality analysis For the liposome suspension prepared in Example 1, a sample was taken and analyzed by a GC analyzer (Varian, Inc., America), and the results showed that the liposome suspension in the sample did not contain Ethanol; another sample was taken and subjected to particle size analysis using a Particle size analyzer (Beckman Coulter, Inc.). The results showed that the average particle size of the liposome contained in the sample was 72.9 nm. Example 2. Preparation of liposome-encapsulated doxorubicin = 3000 mL of liposome suspension obtained in Example 1 was added to a 15 DOS containing 8000 mg of Doxor In a glass container of octane HC1 (doxorubicin HC1, red powder), 200 mL of the previously prepared histidine-sucrose solution was added. The resulting mixture was placed in a water bath at 60 ° C and shaken for 30 minutes. Thereafter, the mixture was cooled to about 35 ° C and diluted to 4 L with a previously prepared 9% sucrose solution and mixed well. 20 Thus, the desired liposome-encapsulated doso-encapsulated doxorubicin is obtained. The resulting product was further dispensed into sterile glass vials to prepare an injection preparation containing 2.0 mg of doxorubicin HCl/mL. Product quality analysis 21 1250877 For the liposome-encapsulated doxorubicin injection prepared in Example 2, the color of the contents encapsulated in the glass tubules was observed, and it was found that the glass vials were all included. The appearance appears to be an orange-red to red injection. Take an appropriate amount of sample and compare it with the standard by an HPLC analyzer (Waters c〇, Amedca). The results show the retention time and elution profile of doxorubicin HC1 contained in the test sample. (4) This (10) Pwfile) is consistent with the standard. Further analysis of the liposome prepared in Example 2 - encapsulated doxorubicin 10
主射製品的藥物包覆率，而發現測試樣品的藥物包覆率係 為队㈣。而以 Partiele size analyzer (BeekmanThe drug coverage rate of the main shot product, and the drug coverage rate of the test sample was found to be the team (4). Partiele size analyzer (Beekman
Inc.)所進行的粒徑（particle size)檢測顯示，樣品内所含有 的脂質體平均粒徑為91.0 nm。The particle size measurement performed by Inc. showed that the average particle size of the liposome contained in the sample was 91.0 nm.
將實施例2所製得的脂質體_包封的多索如必辛注射製 品儲存於2〜η：下歷經一段達30個月㈣間後，該等製品 15經檢測試驗被確認是安定的（表υ，而且該等製品在脂質體 的粒徑上亦無產生明顯的變化。The liposome-encapsulated doxorubicin injection preparation prepared in Example 2 was stored under 2~η: after a period of 30 months (four), and the products 15 were confirmed to be stable after the test. (Table υ, and the products did not show significant changes in the particle size of the liposomes.
於本說明書中被引述之所有專利和文獻以其整體被併 入本案作為參考資料。若有所衝突時，本案詳細說明（包含 界定在内）將佔上風。 22 20 1250877 雖然本發明已參考上述特定的具體例被描述，明顯地 在不背離本發明之範圍和精神之下可作出很多的修改和變 化。因此意欲的是，本發明僅受如隨文檢附之申請專利範 圍所示者之限制。 5 【圖式簡單說明】 (無圖） 【圖式之主要元件代表符號表】All patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including the definition) will prevail. Although the present invention has been described with reference to the specific embodiments described above, many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only by the scope of the appended claims. 5 [Simple description of the diagram] (No diagram) [The main components of the diagram represent the symbol table]
23twenty three
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| TWI391149B (en) * | 2010-05-18 | 2013-04-01 | Univ Southern Taiwan | Nanopegylated liposome and method for making the same |
| US9480712B2 (en) | 2013-10-24 | 2016-11-01 | Industrial Technology Research Institute | Biomedical composition |
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| US9480712B2 (en) | 2013-10-24 | 2016-11-01 | Industrial Technology Research Institute | Biomedical composition |
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