201125578 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種經包覆之第九凝血因子緩釋郵丨型 【先前技術】 亦可能 種减脅生命 第九凝血因子(Factor IX)係體内凝血系統的其中 絲胺酸蛋白酶。此類蛋白的缺乏將產生血液;疑固Z & f 礙，導致亦被稱為聖誕節病的B型血友病。患有只，早 病的患者較正常人易出血及受傷或手術後發生癒入不 一般易出血的部位在肌肉或肘、膝和踩關節腔内，不良 在腦和脊髓、咽喉或腸道内。B型血友病係 的終身性疾病。 通常，治療B型血友病患者的方法為靜脈注射如市售 商品BeneFIX®(Wyeth)的第九凝血因子濃縮劑，以更換喪失 或突變的蛋白。然而，胜肽藥物會因迅速被蛋白分解酵素 分解或被抗體中和而縮短其半衰期和循環時間，此將降低 其療效。 將蛋白i ^^乙一醇化(PEGylation)係一種在蛋白質化 學中常用的技術’用意是在增強保護蛋白藥物不受蛋白分 解酵素的分解。例如，PCT國際專利申請案w〇 2009/083187A1提供一種化學改良血液凝固因子ιχ(凝血因 子IX)，其包含第九凝血因子活性肽區(Αρ區），其中該Αρ 區包含一共彳貝鍵偶合水溶親水性聚合物。在pcT申請案的 一較佳具體實施例中，該水溶親水性聚合物藉由第九凝血 201125578 因子的Asn- 157及/或Asn-167與第九凝血因子相連接。在 另一較佳具體實施例中，該水溶親水性聚合物係藉由第九 凝血因子的 Ser-158、Thr- 159、Thr-163、Thr-169、Ser-171、201125578 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a coated ninth coagulation factor sustained release type (previous technique) and may also be a life-threatening ninth factor (Factor IX) A serine protease of the in vivo coagulation system. The lack of such proteins will produce blood; suspected Z & f, leading to hemophilia B, also known as Christmas disease. Patients with only early disease are more likely to bleed and become injured or become more likely to be bleeding after surgery. They are in the muscles or elbows, knees and stepped joints, and are in the brain and spinal cord, throat or intestines. A lifelong disease of the hemophilia type B. Typically, a method of treating patients with hemophilia B is intravenous injection of a ninth clotting factor concentrate such as the commercially available BeneFIX® (Wyeth) to replace lost or mutated proteins. However, peptide drugs can shorten their half-life and cycle time by being rapidly broken down by proteolytic enzymes or neutralized by antibodies, which will reduce their efficacy. The protein PEGylation is a technique commonly used in protein chemistry, which is intended to enhance the protection of proteinaceous proteins from protein proteolytic enzymes. For example, PCT International Patent Application No. 2009/083187 A1 provides a chemically modified blood coagulation factor ιχ (coagulation factor IX) comprising a ninth coagulation factor active peptide region (Αρ region), wherein the Αρ region comprises a total of mussel bond coupling Water soluble hydrophilic polymer. In a preferred embodiment of the pcT application, the water-soluble hydrophilic polymer is linked to the ninth coagulation factor by Asn-157 and/or Asn-167 of the ninth coagulation 201125578 factor. In another preferred embodiment, the water-soluble hydrophilic polymer is Ser-158, Thr-159, Thr-163, Thr-169, Ser-171, of the ninth coagulation factor.
Thr-172、Ser_174 或 Thr_ 179,特別指藉由 Ser_158、ThM63、 Ser-171或Ser-174而與第九凝血因子相連接，該水溶親水 性聚合物一般係指PEG。然而，這種化學性的PEG添加方 式將不可逆地改變該多肽的結構，因而可能導致無法預期 的副作用。 【發明内容】 本發明的特徵在於一種粉末狀新穎醫藥劑型，其包含被 親脂性生物可分解聚合物或共聚物包覆而形成微小球之人 類第九凝血因子，該包覆可提供緩釋人類第九凝血因子之功 效，且在冷凍乾燥後無殘留任何有機溶液。 在一態樣中，本發明提供一種粉末狀醫藥劑型，包含治 療有效量之人類第九凝血因子(hFIX)，其被親脂性生物可分 解聚合物或共聚物包覆而形成微小球，因而使該醫藥劑型提 供緩釋hFIX及延長生物活性的功效，其中該親脂性生物可 分解聚合物或共聚物係選自由磷脂質、卵磷脂、聚乙醇酸 (PGA)、Μ乳酸乙醇酸)(plga)、聚（r -麩胺酸）、聚乙稀 酉义(PVA)、聚糙胺酸、聚己内醋（p〇iyCapr〇iact〇ne)、聚 酐（polyanhydrides)、聚胺基酸、聚二氧六環酮 (polydioxanone)、聚沒基丁酯(p〇iyhydroxybutyrate)、聚石粦 腈(polyphophazenes)、聚 g旨型聚胺醋（p〇iyesterurethane)、 聚叛基苯氧丙烷-共聚癸二酸 4 201125578 聚原酸酯 (polycarbosyphenoxypropane-cosebacic acid) ^ (polyorthoester)，及其組合所構成的群組。 在其他態樣中，本發明提供用於製造本發明之 型的方法，包含： 4樂劑 (a) 混合水溶液内之治療有效量人類第九凝血因子 與有機溶液内之親脂性生物可分解聚合物或共 IX) 獲得第一乳劑； 〜物以 (b) 混合該第一乳劑與界面活性劑溶液以獲得第二 以及 n (c) 揮發該有機溶劑，接著過濾、清洗及冷凍乾燥該第_ 乳劑而獲得粉末狀醫藥劑型； 人~~ 因此在該粉末狀醫藥劑型内，hFIX被親脂性生物可分解聚 合物或共聚物包覆而形成微小球，其具有緩釋hFIX及延長 生物活性的功效。 在下列的描述中詳細說明本發明的一或多項具體實施 例。從下列圖示和數種具體實施例的詳細說明，以及從申 請專利範圍附件將可更彰顯本發明的其他特色及優點。 【實施方式】 除非另有說明’否則此處使用之全部技術和科學名詞 與熟習本領域之技術者通常所瞭解的意義相同。 此處所使用的冠詞「一」係指該冠詞之语法咬*句的 或多於一個（即，至少一個）。 0 本發明提供一種粉末狀醫藥劑塑’其包含治療有冰量 201125578 之人類第九凝血因子(hFIX)，其被親脂性生物可分解聚人斗 或共聚物包覆而形成微小球，因而使該醫藥劑型可4 hFIX及延長生物活性。該hFIx可為天然或重級之人^釋 九凝血因子。根據本發明，該醫藥劑型被用於治療B 第 友病。 ^'成 在本文中「治療」一詞意指治癒、緩和或改善該 體的出血性疾病。 ~ 在本文中「微小球」一詞意指直徑在微米範園的小球 狀顆粒。在本發明的一具體實施例中，該微小球具有從〇 ^ 至1〇〇微米的直徑。本發明的微小球可進一步被冷凍乾燥 成粉末狀以易於傳送、操作和投藥。 在本文中「生物可分解聚合物或共聚物」一詞意指該 聚合物或共聚物可在體内被酵素、化學及/或物理處理而被 分解或崩解形成較小的化學物質。 在本文中「親脂性」一詞意指生物可分解聚合物或共 聚物可溶解於脂、油、脂肪及有機溶劑。本發明中’親脂 性生物可分解聚合物或共聚物係選自由磷脂質、卵構脂、 聚乙醇酸(PGA)、聚（乳酸乙醇酸)(PLGA)、聚（<r -麩胺酸）、 聚乙烯酸（PVA)、r -聚麵胺酸、聚己内酉旨 (polycaprolactone)、聚酐(polyanhydrides)、聚胺基酸、水 二氧六環酮（polydioxanone)、聚經基丁西曰 (polyhydroxybutyrate)、聚構腈(polyphophazenes)、聚醋裂 聚胺3旨（polyesterurethane)、聚鼓基苯氧丙烧-共聚癸一 & (polycarbosyphenoxypropane-cosebacic acid)、聚原酉文酉曰 6 201125578 (polyorthoester) ’及其組合所構成的群組。在本發明的一實 例中，該親脂性生物可分解聚合物或共聚物係為pLGA，濃 度為例如約1毫克/毫升至1000毫克/毫升，較佳為9〇毫克 /毫升。 視乳酸交醋與乙醇酸交酯用於聚合反應的比例可獲得 不同型的PLGA:其通常以使用的單體比例來表示（例如 PLGA 75 . 25表示該共聚物的組成物為75%的乳酸及25〇/〇 的乙醇酸）。根據本發明，該單體的比例為pLGA 1〇/9〇至 PLGA 90/10。在一具體實施例中，pLGA 75/25 或 pLGA 85/15係用於製造本發明的醫藥劑型。 通# ’微小球内hFIX的裝載率或包埋率為至少8〇%總 質ϊ比。裝载劑量密庠視用於不同目的而不同，以及視通 常根據病人個別藥物動力學的給藥法而定。熟習本領域之 技術者可根據標準技術調整該包埋率。 在本文中「有效量」一詞意指可使被投藥生物體產生 治療效果的所需藥量。如熟習本領域之技術者所瞭解，有 效罝可視投藥途徑、所使用賦形劑及共同使用其他藥物的 可忐性而不同。該醫藥劑型較佳為經由靜脈、肌肉内或皮 下投藥。 医，醫藥劑型可進一步包含一或多種可被溶解於水溶液 的醤藥上可接受載劑。此類載劑包括，但不侷限於：食鹽 水、緩衝溶液、右旋糖、水、甘油、乙醇，及其組合。 本發明亦提供一種用於製造本發明之醫藥劑型的方 法，包含： 201125578 (a) 此s水,奋液内之治療有效量人類第九凝血因子(πιχ) 溶液内之親脂性生物可分解聚合物或共聚物以 獲侍第一乳劑； (b) 此合该第—乳化劑與界面活性劑溶液以獲得 劑；以及 一 ()揮發4有機溶劑，接著過濾、清洗及冷康乾燥該第二 乳劑而獲得粉末狀醫藥劑型； 因此在該粉末狀醫藥劑型内，Μχ被親雜生物 合物或共聚物包覆而形成微小球，其具有緩釋hFlX以及延 長生物活性的功效。 7康本七明，5玄祕小球被製成水包油包水(w/o/w 的形^。言亥hFIX以0_001%至90%第一水溶液質量比的濃 度被/合解於第—水溶液内。在本發明的—具體實施例中， 該hFIX的重量為20毫克。接著將該含有hFIX的第一水溶 液以1 . 1至1 : 100(體積：體積）的比例混合含有至少一親 脂性生物可分解聚合物或絲物的有機溶液，崎得第一 乳劑。在本發明的—具體實施例中，該水溶 液的比例為1 : 10。 另钱/合 根據本發明，形成s亥有機溶液的有機溶劑係選自由一 氣曱烷、氣仿、醋酸乙酯、L4-二氧陸圜、二曱基甲醯= _F)、二甲亞石風_s〇)、甲苯和四氫吱喃（thf)所構成 的群組。在本發明的-具體實施例中，該有機 曱烷。 一乳 用於製造該第一乳劑的溶液可視需要進一步含有至少 8 201125578 一種適於製造該主乳劑的界面活性劑。在一具體實施例 中，該一或多種界面活性劑係選自由脂肪酸甘油酯、脂肪 酸蔗糖酯、脂肪酸山梨糖醇酯、脂肪酸丙二醇酯'單及雙 月曰肪I甘油一乙酿酒石酸醋、驗式碌酸紹納、p〇iys〇rbate 20、polysorbate 60、polysorbate 65、polysorbate 80、經丙 基纖維素、羥丙基曱基纖維素、擰檬酸單及雙甘油酯、酒 石酸單及雙甘油酯、乳酸單及雙甘油酯、乙氧基單及雙甘 油酯、磷酸單鈉鹽衍生物單及雙甘油酯、琥珀酸單酸甘油 酯、脂肪酸聚甘油酯、交酯化蓖麻酸聚甘油酯、乳酸硬脂 酸納、乳酸硬脂酸納、脂肪酸鹽類、聚氧乙稀(2〇)山梨醇酐 單棕櫚酸酯、polysorbate40、聚氧乙烯(20)單硬脂酸酯、聚 氧乙烯(20)山梨醇酐三硬脂酸醋、Triton X-1 〇〇、Tween 40、 I乙一醇200〜800、月桂基硫酸納、酵乙氧基化合物、烧基 酚乙氧基醇、烷基多聚糖苷，及其組合所構成的群組。 根據本發明’該第一乳劑與界面活性劑溶液混合而獲 得第二乳劑。在本發明的一具體實施例中，該界面活性劑 可為’但不侷限於脂肪酸甘油目旨、脂肪酸蔬糖g旨' 脂肪酸 山梨糖醇酯、脂肪酸丙二醇酯、單及雙脂肪酸甘油二乙醯 酒石酸酯、驗式填酸銘鈉、polysorbate 20、polysorbate 60、 polysorbate 65、polysorbate 80、羥丙基纖維素、羥丙基曱 基纖維素、檸檬酸單及雙甘油酯、酒石酸單及雙甘油酯、 乳酸單及雙甘油酯、乙氧基單及雙甘油酯、磷酸單鈉鹽衍 生物單及雙甘油酯、琥珀酸單酸甘油酯、脂肪酸聚甘油酯、 交酯化蓖麻酸聚甘油酯、乳酸硬脂酸鈉、乳酸硬脂酸鈉、 201125578 脂肪酸鹽類、聚氧乙烯（20)山梨醇酐單棕櫚酸酯、 P〇lyS〇rbate40、聚氧乙烯(20)單硬脂酸酯、聚氧乙烯(2〇曰)山 梨醇酐三硬脂酸酯、Triton x-100、Tween 4〇、聚乙二醇 200〜800、月桂基硫酸鈉、醇乙氧基化合物、烷基酚乙氧基 醇、烧基多聚糖苦，或其組合。在本發明的一具體實施例 中，該界面活性劑係為PVA，於第二水溶液内的濃度約， 例如0.1%至1.0% ’較佳為0.5〇/〇。 根據本發明，該界面活性劑溶液與該第一乳劑係以^ . 1至1000 : 1的比例以獲得第二乳劑。在本發明的—實例 —“ 〇為· 1。该第二乳劑在揮發有機溶劑之後，> =由本技㈣域巾的習知方法麟、清洗及冷;東 :传粉末狀醫藥劑型。該揮發時間可被設定為0.1至24、丨 9一具體貫施例中，該揮發時間較佳為3$^ 小日寻。 4 两分散 虱夕種不可混合液體，例如將油乳化入水， :由任何已知或常用方法或技術 本發明，可利用r 根據Thr-172, Ser_174 or Thr_179, in particular, is linked to a ninth coagulation factor by Ser_158, ThM63, Ser-171 or Ser-174, which is generally referred to as PEG. However, this chemical PEG addition will irreversibly alter the structure of the polypeptide and may result in unpredictable side effects. SUMMARY OF THE INVENTION The present invention features a powdery novel pharmaceutical dosage form comprising a human ninth coagulation factor coated with a lipophilic biodegradable polymer or copolymer to form microspheres, the coating providing sustained release to humans The efficacy of the ninth coagulation factor, and no residual organic solution after lyophilization. In one aspect, the invention provides a powdered pharmaceutical dosage form comprising a therapeutically effective amount of a human ninth coagulation factor (hFIX) coated with a lipophilic biodegradable polymer or copolymer to form microspheres, thereby The pharmaceutical dosage form provides sustained release of hFIX and prolongs biological activity, wherein the lipophilic biodegradable polymer or copolymer is selected from the group consisting of phospholipids, lecithin, polyglycolic acid (PGA), lactic acid glycolic acid (plga) , poly(r-glutamic acid), polyethylene sulphur (PVA), poly-aramidic acid, polycaprolactone (p〇iyCapr〇iact〇ne), polyanhydrides, polyamino acids, poly Polydioxanone, p〇iyhydroxybutyrate, polyphophazenes, polypyrene urethane, polyphenol phenoxypropane-copolymer Diacid 4 201125578 Polycarbosyphenoxypropane-cosebacic acid ^ (polyorthoester), and combinations thereof. In other aspects, the invention provides a method for making a form of the invention comprising: 4 agents (a) a therapeutically effective amount of a human ninth coagulation factor in a mixed aqueous solution and a lipophilic biodegradable polymerization in an organic solution Or IX) obtaining a first emulsion; (b) mixing the first emulsion with a surfactant solution to obtain a second and n (c) volatilizing the organic solvent, followed by filtering, washing, and lyophilizing the first In the powdered pharmaceutical dosage form, hFIX is coated with a lipophilic biodegradable polymer or copolymer to form microspheres, which have sustained release of hFIX and prolong biological activity. . One or more specific embodiments of the present invention are described in detail in the following description. Other features and advantages of the present invention will become apparent from the Detailed Description of the Drawings. [Embodiment] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The article "a" as used herein refers to the grammatical bite of the article or more than one (ie, at least one). The present invention provides a powdered pharmaceutical preparation comprising a human ninth blood coagulation factor (hFIX) treated with a milky amount of 201125578, which is coated with a lipophilic biodegradable polycap or copolymer to form microspheres, thereby The pharmaceutical dosage form can be used for 4 hFIX and prolongs biological activity. The hFIx can be a natural or heavy person to release the nine coagulation factors. According to the present invention, the pharmaceutical dosage form is used to treat B autism. ^'成 In the context of the word "treatment" is meant to cure, alleviate or ameliorate the bleeding disorders of the body. ~ In this paper, the term "microball" means small spherical particles of diameter in the micrometer range. In a specific embodiment of the invention, the microspheres have a diameter from 〇^ to 1〇〇 microns. The microspheres of the present invention can be further freeze-dried into a powder for easy delivery, handling and administration. The term "biodegradable polymer or copolymer" as used herein means that the polymer or copolymer can be decomposed or disintegrated into smaller chemicals by enzymatic, chemical and/or physical treatment in the body. The term "lipophilic" as used herein means that the biodegradable polymer or copolymer is soluble in fats, oils, fats and organic solvents. In the present invention, the 'lipophilic biodegradable polymer or copolymer is selected from the group consisting of phospholipids, egg fats, polyglycolic acid (PGA), poly(lactic acid glycolic acid) (PLGA), poly(<r-glutamic acid) ), polyvinyl acid (PVA), r-polyaminic acid, polycaprolactone, polyanhydrides, polyamino acids, polydioxanone, polypyridyl Polyhydroxybutyrate, polyphophazenes, polyester urethane, polycarbosyphenoxypropane-cosebacic acid, polyglycoside 6 201125578 (polyorthoester) 'Group of its combination. In one embodiment of the invention, the lipophilic biodegradable polymer or copolymer is pLGA at a concentration of, for example, from about 1 mg/ml to about 1000 mg/ml, preferably 9 mg/ml. Different types of PLGA can be obtained depending on the ratio of lactic acid to glycolide and glycolide for polymerization: it is usually expressed in terms of the proportion of monomers used (for example, PLGA 75. 25 means that the composition of the copolymer is 75% lactic acid). And 25 〇 / 〇 of glycolic acid). According to the invention, the ratio of the monomers is from pLGA 1 〇 / 9 〇 to PLGA 90/10. In a specific embodiment, pLGA 75/25 or pLGA 85/15 is used to make the pharmaceutical dosage form of the invention. The loading rate or embedding rate of hFIX in the microspheres is at least 8〇% of the total mass ratio. The loading dose is used for different purposes and is usually determined by the individual drug kinetics of the patient. Those skilled in the art can adjust the embedding rate according to standard techniques. As used herein, the term "effective amount" means the amount of the drug required to produce a therapeutic effect in the administered organism. As will be appreciated by those skilled in the art, the effectiveness will vary depending on the route of administration, the excipients used, and the susceptibility to the co-use of other drugs. The pharmaceutical dosage form is preferably administered intravenously, intramuscularly or subcutaneously. The medical dosage form may further comprise one or more peony acceptable carriers which are soluble in the aqueous solution. Such carriers include, but are not limited to, salt water, buffer solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The invention also provides a method for the manufacture of a pharmaceutical dosage form of the invention, comprising: 201125578 (a) a therapeutically effective amount of a lipophilic biodegradable polymerization in a human ninth coagulation factor (πιχ) solution Or a copolymer to obtain the first emulsion; (b) the first emulsifier and the surfactant solution to obtain the agent; and one () to volatilize the 4 organic solvent, followed by filtration, washing and cooling to dry the second The emulsion is obtained as a powdered pharmaceutical dosage form; therefore, in the powdered pharmaceutical dosage form, the ruthenium is coated with a heteropolybiotide or a copolymer to form microspheres, which have a sustained release of hFlX and an effect of prolonging biological activity. 7 Kang Ben Qi Ming, 5 mysterious small balls were made into water-in-oil water (w / o / w shape ^. Yan Hai hFIX with 0_001% to 90% of the first aqueous solution mass ratio of the concentration / solution In the first aqueous solution, in the embodiment of the present invention, the weight of the hFIX is 20 mg. Then, the first aqueous solution containing hFIX is mixed at a ratio of 1.1 to 1:100 (volume:volume) to contain at least A lipophilic biodegradable organic solution of a polymer or silk, the first emulsion is obtained. In a specific embodiment of the invention, the ratio of the aqueous solution is 1:10. Further money/combination according to the invention, forming s The organic solvent of the organic solution is selected from the group consisting of monooxane, gas, ethyl acetate, L4-dioxane, dimercaptomethyl hydrazine = _F), dimethyl sulphide _s〇, toluene and tetrahydrogen. A group of th ( (thf). In a particular embodiment of the invention, the organodecane. The solution used to make the first emulsion may further comprise at least 8 201125578 a surfactant suitable for the manufacture of the primary emulsion. In one embodiment, the one or more surfactants are selected from the group consisting of fatty acid glycerides, fatty acid sucrose esters, fatty acid sorbitol esters, fatty acid propylene glycol esters, single and bimonthly fat I glycerol monoethyl phthalate, Sulphuric acid, p〇iys〇rbate 20, polysorbate 60, polysorbate 65, polysorbate 80, propyl cellulose, hydroxypropyl decyl cellulose, citric acid mono and diglycerides, tartaric acid mono and diglycerol Ester, lactic acid mono- and diglycerides, ethoxy mono- and di-glycerides, monosodium phosphate derivatives mono- and diglycerides, succinic acid monoglycerides, fatty acid polyglycerides, lactide ricinoleic polyglycerol Ester, sodium lactic acid stearate, sodium lactic acid stearate, fatty acid salts, polyoxyethylene (2〇) sorbitan monopalmitate, polysorbate 40, polyoxyethylene (20) monostearate, polyoxygen Ethylene (20) sorbitan tristearate, Triton X-1 oxime, Tween 40, I ethyl alcohol 200~800, sodium lauryl sulfate, ethoxylate, phenol ethoxylated alcohol, alkane A group of glycosides, and combinations thereof. According to the invention, the first emulsion is mixed with a surfactant solution to obtain a second emulsion. In a specific embodiment of the present invention, the surfactant may be 'but not limited to fatty acid glycerol purpose, fatty acid vegetable sugar g' fatty acid sorbitol ester, fatty acid propylene glycol ester, mono- and di-fatty acid glycerol diethyl hydrazine Tartaric acid ester, sodium sulphate, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, hydroxypropyl cellulose, hydroxypropyl decyl cellulose, citric acid mono- and diglycerides, tartrate mono- and diglycerides , lactic acid mono and diglycerides, ethoxy mono and diglycerides, monosodium phosphate derivatives mono and diglycerides, succinic acid monoglycerides, fatty acid polyglycerides, lactide ricinoleic acid polyglycerides , sodium lactic acid stearate, sodium lactic acid stearate, 201125578 fatty acid salts, polyoxyethylene (20) sorbitan monopalmitate, P〇lyS〇rbate40, polyoxyethylene (20) monostearate, Polyoxyethylene (2〇曰) sorbitan tristearate, Triton x-100, Tween 4〇, polyethylene glycol 200~800, sodium lauryl sulfate, alcohol ethoxylate, alkylphenol ethoxylate Base alcohol, burnt-polysaccharide bitter, or a combination thereofIn a specific embodiment of the invention, the surfactant is PVA and the concentration in the second aqueous solution is about 0.1% to 1.0% by weight, preferably 0.5 Å/Å. According to the present invention, the surfactant solution and the first emulsion are in a ratio of from 0.1 to 1000:1 to obtain a second emulsion. In the present invention - the example - "〇 is 1. The second emulsion is after volatilizing the organic solvent, > = the conventional method of the art (4) domain towel Lin, washing and cold; East: powdered pharmaceutical dosage form. The volatilization time can be set to 0.1 to 24, 丨9. In a specific embodiment, the volatilization time is preferably 3$^ small day search. 4 two dispersions of non-mixable liquids, such as emulsifying oil into water, by: Any known or commonly used method or technique of the invention may be utilized based on r
八， 』用10至500瓦功率的超音波處理0.0J 分鐘。在本發日+ I 3〇 ^ 月的一較佳具體實施例中，可利用75瓦方 的超音波虛1。 力率 Α 2〜3分鐘。任何習知本領域的技術者可柄祕 不同裝載劑詈―、 」根據 衣執㈠里或不同材料進行重複實驗以選擇適當 件。本發明藉+ 田的條 a稭由下列實例作進一步描述’其被提供作Λ 明而非限制之目的。 勺說 實例 '製備經包4之人類重组第九凝血因子的緩释镟小球 重組人類第九凝血因子(rhFIX)係來自於提供自台灣動 10 201125578 物科技研究所(ATIT)的基因轉殖豬乳。收集該豬乳後，於4 C之下以3,000 X g離心20分鐘以移除脂肪。脫脂乳以填酸 鹽缓衝液沈澱及於4°C之下以22860 X g離心1〇分鐘以獲得 含rhFIX的區份。利用篩孔大小30 kD之聚;ε風高分子薄膜 (TAMI)的超濾法進一步濃縮乳清部分。接著藉由q瓊脂糖 凝膠快速流動層析(Amersham製藥科技)和肝素瑷脂糖凝膠 柱(Amersham製藥科技)捕捉及純化rhFIX。使用奈米過遽 作為病毒移除步驟。 以1 : 10 (體積/體積）比例混合溶解於蒸餾水的rhFIX 及90毫克/毫升溶解於二氯曱烧的聚乳酸聚乙醇酸 (PLGA)’然後以75瓦超音波將該混合物振盪2〜3分鐘而獲 得第一乳劑。進一步將該第一乳劑與0.5°/〇 PVA以1 : 30(體 積/體積）比例混合，然後以75瓦超音波振盪2〜3分鐘而獲 得第二乳劑。將該第二乳劑中之有機溶劑揮發3〜4小時之 後，進行過濾、清洗及冷凍乾燥而獲得包覆有rhFIX之 PLGA微小球。於處理過程中收集過濾溶液和清潔液，並將 兩者混合成「組合溶液」以用於測定包埋率和藥物劑量。 實例2 :藉由艟外模擬生理試驗測定rhFIX的釋藥動能和 活性 藉由將製造自實施例1的緩釋微小球加入維持在37°C 的生理緩衝液(0.5% Tween 20，PBS，pH 7.4)内以進行該模 擬生理試驗。每天收集少許等量緩衝液以測定從微小球釋 出之rhFIX活性。藉由活化部分凝血活酶時間（ActivatedEight, 』Using ultrasonic power of 10 to 500 watts for 0.0J minutes. In a preferred embodiment of the present day + I 3 〇 ^ month, an ultrasonic imaginary of 75 watts can be utilized. Force rate Α 2~3 minutes. Anyone skilled in the art can handle different types of loading agents 、-, "repeated experiments according to the clothing (1) or different materials to select the appropriate parts. The invention is further described by the following examples, which are provided for purposes of illustration and not limitation. Spoon says the example 'Preparation of the recombinant human ninth coagulation factor of the recombinant human ninth coagulation factor. The recombinant human ninth coagulation factor (rhFIX) is derived from the gene transfer provided by Taiwan Institute of Technology (ATIT). Pig milk. After collecting the pig's milk, it was centrifuged at 3,000 x g for 20 minutes under 4 C to remove fat. The skim milk was precipitated in an acid salt buffer and centrifuged at 22860 X g for 1 minute at 4 ° C to obtain a fraction containing rhFIX. The whey fraction was further concentrated by ultrafiltration of ε wind polymer film (TAMI) using a mesh size of 30 kD. The rhFIX was then captured and purified by q-Sepharose Rapid Flow Chromatography (Amersham Pharmaceutical Technology) and Heparin Delipogel Column (Amersham Pharmaceutical Technology). Use nanometer 遽 as a virus removal step. Mix rhFIX dissolved in distilled water and 90 mg/ml of polylactic acid polyglycolic acid (PLGA) dissolved in dichlorohydrazine in a ratio of 1:10 (vol/vol) and then oscillate the mixture with a 75 watt ultrasonic wave 2~3 The first emulsion was obtained in minutes. Further, the first emulsion was mixed with 0.5 ° / 〇 PVA in a ratio of 1: 30 (volume / volume), and then shaken at 75 watts for 2 to 3 minutes to obtain a second emulsion. After the organic solvent in the second emulsion was volatilized for 3 to 4 hours, it was filtered, washed, and freeze-dried to obtain a PLGA microsphere coated with rhFIX. The filtration solution and the cleaning solution were collected during the treatment, and the two were mixed into a "combination solution" for determination of the embedding rate and the drug dosage. Example 2: Determination of release kinetic energy and activity of rhFIX by an external simulated physiological test by adding a sustained-release microsphere manufactured from Example 1 to a physiological buffer maintained at 37 ° C (0.5% Tween 20, PBS, pH) The simulated physiological test was performed within 7.4). A small amount of buffer was collected daily to determine the rhFIX activity released from the microspheres. By activating partial thromboplastin time (Activated
Partial Thromboplastin Time，aPTT)試驗測定該 rhFIX 活 201125578 性’同時利用FIX缺乏血漿作為對照。 該aPTT試驗係用於測定樣本内FIX活性的一種凝血時 間試驗。簡言之，將固定量(25微升）的血漿混合50微升 aPTT試劑及在37t反應1分鐘。然後將50微升的0.025 莫耳濃度(M)CaCl2加入該樣本内，藉由自動血塊凝結分析 儀(TECO Coatron M4)於37°C在波長660奈米之下進行4分 鐘的分析，其原理係測量該樣本在凝結時光散射的變化。 體外測定的結果示於第1圖。 如第1圖所示，發現在體外測定中釋出自本發明劑型 的rhFIX仍保留其活性’且逐漸從該PLGA85/15(第4批） 和PLGA75/25(第3批）的劑型被釋出達四天。 實例3 :從微小球釋出rhFIX之生物活性的體内試驗 使用B型血友病小鼠模式以測定根據本發明劑型及未 包覆之rhFIX之緩釋比較。將50IU/公斤rhFIX投予至每組 /、隻的B型血友病小鼠(R333Q-hFIX小鼠係獲贈於Darrel W. Stafford博士，生物系，美國北卡羅來納大學，Chapd mu) 以進行測定。分別經由尾靜脈之靜脈注射及背部之皮下 才又予未包覆之rhFIX及本發明的劑型。投藥之後於不同時 間點從各小鼠採集血液以及離⑽獲得血漿樣本。藉由上 述的aPTT檢測法測定其活性。;疑血時間超㉟12〇秒、時被視 為弱或無活性的rhFix。詳細資料整理於表丨，以平均土標 準偏差表示，並以獨立t檢定法(SPSS 12 0版，claritas& 司）進行統相估。p<g.()5之值被視為具有統計學上的顯著 意義。 201125578 如表1所示，被投予未包覆之rhFlX處理組，其經由 所投入rhFIX活性使凝血時間縮短的效果約可維持48小時 (2天），但在72小時(3天）之後變得不顯著。反之，經投予 本發明劑型後的處理組，其rhFIX的生物活性可延長至至 少120小時(5天），並於168小時(7天)後才變得不顯著。The Partial Thromboplastin Time, aPTT) assay determined that the rhFIX activity was 201125578 sex' while using FIX deficient plasma as a control. The aPTT test is a coagulation time test for determining FIX activity in a sample. Briefly, a fixed amount (25 microliters) of plasma was mixed with 50 microliters of aPTT reagent and reacted for 1 minute at 37t. Then 50 μl of 0.025 molar concentration (M) CaCl 2 was added to the sample and analyzed by an automatic clot coagulation analyzer (TECO Coatron M4) at 37 ° C for 4 minutes at a wavelength of 660 nm. The change in light scattering of the sample during condensation is measured. The results of the in vitro assay are shown in Figure 1. As shown in Fig. 1, it was found that rhFIX released from the dosage form of the present invention retained its activity in an in vitro assay and was gradually released from the formulations of PLGA85/15 (fourth batch) and PLGA75/25 (third batch). Up to four days. Example 3: In vivo assay for release of biological activity of rhFIX from microspheres The hemophilia B model was used to determine a sustained release comparison of the dosage form according to the invention and uncoated rhFIX. 50 IU/kg rhFIX was administered to each group/type of hemophilia B mice (R333Q-hFIX mice were received from Dr. Darrel W. Stafford, Department of Biology, University of North Carolina, Chapd mu) Determination. The uncoated rhFIX and the dosage form of the present invention were separately administered via intravenous injection of the tail vein and subcutaneous in the back. Blood was collected from each mouse at various time points after administration and plasma samples were obtained from (10). The activity was measured by the aPTT assay described above. When the suspected blood time exceeds 3512 〇 seconds, it is regarded as weak or inactive rhFix. Details are compiled in Tables, expressed as mean soil standard deviations, and evaluated by the independent t-test (SPSS version 12, claritas & division). The value of p<g.()5 is considered to be statistically significant. 201125578 As shown in Table 1, the uncoated rhFlX treatment group was administered, and the effect of shortening the clotting time via the rhFIX activity was maintained for about 48 hours (2 days), but changed after 72 hours (3 days). Not significant. On the contrary, the biological activity of rhFIX can be prolonged to at least 120 hours (5 days) after administration to the treatment group of the present invention, and becomes insignificant after 168 hours (7 days).
如上所述，其結論為本發明的醫藥劑型可緩釋比打X 及於體内維持刪X至少5天以上的生物活性。因此，其 1射頻率可被降低至每5或7天—次，將可明顯地減少b =血友病患者的負擔。此外，該粉末狀劑型通常可提供較 佳的化學穩定性以及可被儲存—段較長的時間。 本發明雖然已根據目前認為最可行及較佳具體實施例 說明’但是必需瞭解本發明非僅侷限於該揭示的具體 广例。反之’其擬包括中請專利範圍之精神和範圍内的 種改良和類似配置，而根據廣義解釋其涵蓋全部此類的 改良和類似構造。 13 201125578 【圖式簡單說明】 熟習本領域之技術者在審視下列詳細說明和附圖之後 可更輕易地瞭解本發明的上述態樣和優點，其為： 第1圖顯示根據本發明之醫藥劑型的hFIX活性，其係 藉由aPTT檢測法於不同時間測定第3批(PLGA75/25， 4.6IU/毫克)和第4批(PLGA85/15，4.6IU/毫克）劑型的結果。 【主要元件符號說明】 益 14As described above, it is concluded that the pharmaceutical dosage form of the present invention has a sustained release ratio of X and maintains X activity in the body for at least 5 days or more. Therefore, the frequency of one shot can be reduced to every 5 or 7 days, which will significantly reduce the burden of b = hemophilia patients. In addition, the powdered dosage form generally provides better chemical stability and can be stored for a longer period of time. The present invention has been described in terms of what is presently considered to be the most feasible and preferred embodiments. Instead, it is intended to include modifications and similar configurations within the spirit and scope of the scope of the patent application, and it is intended to cover all such modifications and similar constructions. 13 201125578 BRIEF DESCRIPTION OF THE DRAWINGS The above aspects and advantages of the present invention will be more readily understood by those skilled in the art after reviewing the following detailed description and the accompanying drawings in which: FIG. 1 shows a pharmaceutical dosage form according to the present invention. hFIX activity, which was determined by the aPTT assay at different times for the third batch (PLGA 75/25, 4.6 IU/mg) and the fourth batch (PLGA 85/15, 4.6 IU/mg) dosage form. [Main component symbol description] Benefit 14