201204381 ….六、發明說明： 【發明所屬之技術領域】 [0001] [0002] Ο 〇 100116179 本發明與包括昇糖素的穩定即用溶液有關。此溶液適合 用於非腸道投藥，特別是適合驗自我注射。 【先前技術】 昇糖素為種與人類昇糖素相同的多狀激素，其增加血 液葡萄糖及舒緩胃腸道的平滑肌。昇糖素為-種包含29 胺基酸殘基的單鏈多肽。其不溶於水但是在小於3或大於 9. 5的pH為可溶的。因為昇糖素化性及物性穩定性之限 制，升糖素為以冷凍乾燥粉末的型式供應，其必須在與 其所供應水相稀釋劑重組後立即使用。其可於包含無菌 昇糖素的管形瓶及無_釋_注射管的_盒靜脈注 射地、肌肉内地、或是皮下地使用。管形瓶包括1毫克（1 單位）昇糖素及49毫克乳糖或〗07毫克乳糖〜水合物 (Novo nordisk)。稀釋劑注射管包括12奄克升甘由 、注射用水、及鹽酸或是僅有注射用水。昇糖素是 1 甘油 血糖性休克的情況時用做緊急藥物。所以，不、疋低 重組步驟的即用水溶液是投藥的—種較佳Μ需要任= 在緊急情況及當病患必須自我注射藥物時寺别疋 素水溶液的最大限制是其為物理及化學不穩昇糖 因為昇糖素傾向於聚集而造成小纖維與凝是 為-種由酸性ΡΗ、增加的離子強度、肽濃夜、_，此 高溫所刺激的過程。聚集特別是不能接-及/或 導在濃度大於2.5毫克/毫升持物小時所為已報 溶液之老化會造成細胞^_粉纖_ =糖素 Kathy L.De J〇ng， Bev ^ 匕考 表單編號Αοιοι 第3頁/共！ 叶0stopher 1003279836-0 201204381 M.Yip, Michael R. Defellippis, Biophys J BioFAST, 2006年6月9日）。由文獻報告可推論昇糖素 的本質對調配者要發展昇糖素溶液，特別是即用溶液存 在挑戰。 先前技藝參考文獻顯示企圖處理在製備溶液時與不溶於 水藥物相關的問題。例如，美國專利號碼US 6, 217, 886 (後文稱為‘886)揭示一種製備生物活性微胞產物的方法 ，其包括與微胞結合的一或更多生物活性兩親媒性化合 物，該方法包括步驟： a) 混合一或更多脂質，其中至少一個脂質成份共價地鍵 結至水溶性聚合物； b) 從脂質形成殺菌的穩定化微胞； c) 使用一或更多生物活性兩親性化合物在該化合物與該 微胞以更具生物活性的構形相結合的條件下與來自步驟 (b)的微胞一起培養。 美國專利號碼US ‘886及數個其他參考文獻，例如，醫 藥科學期刊，第93卷，第10期，2004年10月，第 2476-2487頁，提出DSPE-PEG 2000為一種用於不溶於 水藥物的新穎基於脂質的載體。 非離子性、兩性離子、陰離子及陽離子表面活性劑及脂 質的微胞於昇糖素及胰島素構形的影響亦已由Pasta等人 [Pasta et a 1, Biochmica et Bi ophys i ca Acta, 953 ( 1 988) 314-320 ]藉由圓二色光譜及内源蛋白螢光 進行研究。一些兩性化合物非常顯著地保護昇糖素不會 被胰蛋白酶與膜凝乳蛋白酶蛋白質水解，然而其他兩性-化合物完全不會保護或是僅些微保護該激素。文獻中已 100116179 表單編號A0101 第4頁/共19頁 1003279836-0 201204381 報導昇糖素於溫和鹼性溶液的濃度相依平衡涉及較高螺 旋度的相關型式之形成（W.B. Gratzer and G.H.201204381 .... Description of the Invention: [Technical Field of the Invention] [0001] [0002] 0002 〇 100116179 The present invention relates to a stable ready-to-use solution comprising a glycoside. This solution is suitable for parenteral administration, especially for self-injection. [Prior Art] Glycosin is a polymorphic hormone similar to human glycosides, which increases blood glucose and soothes smooth muscles of the gastrointestinal tract. Glycosin is a single-chain polypeptide comprising a 29 amino acid residue. It is insoluble in water but soluble at a pH of less than 3 or greater than 9.5. Due to the limitations of glycosidation and physical stability, glycoside is supplied in the form of a freeze-dried powder which must be used immediately after reconstitution with the aqueous phase diluent supplied thereto. It can be administered intravenously, intramuscularly, or subcutaneously in a vial containing sterile glucagon and a cartridge-free injection tube. The vial comprises 1 mg (1 unit) of glycoside and 49 mg of lactose or 07 mg of lactose to hydrate (Novo nordisk). The diluent injection tube includes 12 grams of sucrose, water for injection, and hydrochloric acid or water for injection only. Glycoglycan is used as an emergency drug in the case of glycemic shock. Therefore, the ready-to-use aqueous solution that does not detract from the recombination step is administered. It is better to use it. In the case of an emergency and when the patient must self-inject the drug, the maximum limitation of the solution is that it is physical and chemical. Stabilized sugar is a process in which small fibers and coagulation are caused by glycosides, which are caused by acidic sputum, increased ionic strength, peptide night, and _, which are stimulated by this high temperature. Accumulation, in particular, can not be connected - and / or guided at a concentration greater than 2.5 mg / ml of the hour of the reported aging of the solution will cause cells ^ _ powder fiber _ = glycan Kathy L. De J〇ng, Bev ^ 表单 test form number Αοιοι Page 3 / Total! Leaf 0stopher 1003279836-0 201204381 M.Yip, Michael R. Defellippis, Biophys J BioFAST, June 9, 2006). It can be inferred from the literature that the nature of the glycoside is a challenge for the formulator to develop a glycemic solution, especially a ready-to-use solution. Previous technical references have shown attempts to address the problems associated with water-insoluble drugs when preparing solutions. For example, U.S. Patent No. 6,217,886 (hereinafter referred to as '886) discloses a method of preparing a biologically active microcell product comprising one or more biologically active amphiphilic compounds that bind to micelles, The method comprises the steps of: a) mixing one or more lipids, wherein at least one lipid component is covalently bonded to the water soluble polymer; b) forming a bactericidal stabilized microcell from the lipid; c) using one or more biological activities The amphipathic compound is incubated with the micelle from step (b) under conditions in which the compound binds to the cell with a more biologically active configuration. U.S. Patent No. US '886 and several other references, for example, Journal of Medical Sciences, Vol. 93, No. 10, October 2004, pages 2476-2487, suggesting that DSPE-PEG 2000 is a water insoluble solution. A novel lipid-based carrier for drugs. The effects of nonionic, zwitterionic, anionic and cationic surfactants and lipid microvesicles on glycoside and insulin conformation have also been determined by Pasta et al. [Pasta et a 1, Biochmica et Bi ophys i ca Acta, 953 ( 1 988) 314-320 ] was studied by circular dichroism spectroscopy and endogenous protein fluorescence. Some amphiphilic compounds very significantly protect the glycoside from proteolysis by trypsin and chymotrypsin, whereas other amphoteric-compounds do not protect at all or only slightly protect the hormone. In the literature, 100116179 Form No. A0101 Page 4 of 19 1003279836-0 201204381 Reports that the concentration-dependent equilibrium of glycosides in mild alkaline solutions involves the formation of a correlation pattern of higher vortex (W.B. Gratzer and G.H.
Beaven, The JouTnal of Biol〇gical Chemistry, vol. 244，No. 24，PP· 6675一6679’ 1 969)。亦報 導在水溶液中的單雜畀糖素不具穩定球狀結構，構形隨 溫度或所加入變性劑濃度的變化而連續變化（Bhiny〇 Pnajipan and Walter Gratzer> European J. Biochemistry, 45， 547-553， （1974))。 意欲發展一種特別是在沒有足夠重組時間的緊急情況下 C) 適合用於自我非腸道投藥的穩疋即用昇糖素溶液，發明 者發現環糊精、糖賻、醇、類蛋白質人類血清蛋白無法 溶解昇糖素。令人驚訝地，發明者發現具有共價地連接 至聚乙二醇的二硬脂醯-磷脂醢乙醇胺的昇糖素水溶液在 5及7. 5之間的pH值對該溶液具化性及物性穩定性，特別 是在蛋白質構形方面。 【發明内容】 [0003] 〇 本發明提供一種包括醫療有效量的昇糖素及醫藥可接受 載劑的穩定，即用溶液。 本發明可摘要如下： A. —種穩定即用溶液，其包括調整至pH約5-7.5的醫 療有效量的昇糖素及聚乙二醇化二硬脂醯-磷脂醯乙醇胺 與醫藥可接受載劑。 B. 如A所敘述的穩定即用溶液，其中昇糖素濃度範圍從 0. 5毫克/毫升至5毫克/毫升。 C. 如A所敘述的穩定即用溶液微胞溶液，其中聚乙二醇化 二硬脂醯-磷脂醯乙醇胺具有約300至5000的分子量並以 100116179 表單編號A0101 第5頁/共19頁 1003279836-0 201204381 範圍從1毫克/毫升至40毫克/毫升的量存在。 D. 如A所敘述的穩定即用溶液，其中載劑包括從水性或極 性非質子溶劑（例如二甲亞颯）選出的溶劑。 E. 如D所敘述的穩定即用溶液，其中非質子溶劑以5至15% 體積的溶液體積存在。 F. 如A所敘述的穩定即用溶液，其中將溶液的pH調整至約 6.5。 G. 如A所敘述的穩定即用溶液，其中溶液更包括從脂肪酸 或兩性離子膽鹼衍生物或其混合物組成的群組選出的穩 定劑。 H. 如G所敘述的穩定即用溶液，其中膽鹼衍生物為二豆蔻 醯脂醯膽鹼並以約1毫克/毫升至2毫克/毫升的濃度存在 〇 I. 如G所敘述的穩定即用溶液，其中脂肪酸為辛酸並以約 0.1毫克/毫升至3毫克/毫升的量存在。 J. 如A所敘述的穩定即用溶液，其中醫藥可接受載劑包括 一或更多酸、缓衝劑、防腐劑、張力調整劑及其混合物 〇 K. 如J所敘述的穩定即用溶液，其中緩衝劑為從組氨酸琥 珀酸鹽、磷酸鹽、擰檬酸鹽及其混合物組成的群組選出 0 【實施方式】 [0004] 此處所使用用語‘穩定’表示溶液維持物理及化學的穩 定，特別是，溶液於在2-8°C或是較佳為在室溫的長時間 儲存（至少三個月，較佳為約6個月至12個月）維持是清澈 的。本發明溶液的物理穩定性是由記錄於650奈米的穿透 100116179 表單編號 A0101 第 6 頁/共 19 頁 1003279836-0 201204381 百分率及於420奈米的吸收值來決定。而且該溶液維持效 力，此是由在藥碘公認的生物分析方法及例如HPLC的化 學分析決定。當總不純物不超過10 %，較佳為當將溶液 於室溫儲存約三個月及/或於2-8 °C儲存約三個月或更多 時總不純物不超過10 %時，溶液可稱為穩定的。此外， 當在儲存期間結束時，例如室溫三個月或更多或是當於 2-8°C儲存約六個月或更多時，昇糖素相關物質，例如昇 糖素亞颯、D-trp (25)-昇糖素或D- phen (22)-昇糖 素不超過3 %時，溶液可稱為穩定的。 此處所使用用語‘即用’表示溶液是在一種不需額外重 組步驟即可投藥的型式，例如以冷凍乾燥型式於市場提 供的產品。昇糖素，為一種在低血糖性休克的情況時用 做緊急藥物投藥的醫療物質，因此，總是希望具有以預 先填充的注射器儲存的即用溶液。依據緊急狀況而定， 病患可自行投藥昇糖素溶液而不會延遲投藥。根據一個 較佳實施例，本發明的即用穩定微胞溶液適合用於單次 劑量非腸道自行投藥，其中組合物配藥於預先填充的注 射器。昇糖素每單次投藥的量的範圍為每毫升溶液自1毫 克至約5毫克，較佳為，每毫升1毫克。根據另一實施例 ，溶液填充於預先填充的具有自動喷射器的注射器。一 般而言，自動喷射器為彈簧驅動的並設計為以一次單一 劑量來投藥預先填充的注射器的整個内容物。可能不需 要有任何流體路徑且可能不需要與注射器内所含藥物或 生物製品有任何接觸。自動喷射器的組件可由塑膠與鋼 鐵製造，自動喷射器裝置的設計及性能特性可包括安全 機構以防止不慎作動、所使用注射針的自動包套、於前 100116179 表單編號A0101 第7頁/共19頁 1003279836-0 201204381 方裝置的開窗、鎖定片以防止一旦兩個子組件連接時自 動喷射器裝置的分開、及自行失:能以防止重複使用。 此處所使用用語‘微胞’表示溶液是在微胞型式，該溶 液目視為清澈溶液。較佳為，微胞由磷脂質穩定化。當 沒有任何類似凝膠形成/聚集的構形變化之徵兆、顯示α 螺旋結構的於CD光譜的變化及/或無論在製備後立即還是 在各種儲存條件（例如室溫或2-8° C)儲存約六個月時以微 粒物質的角度而言溶液都為清澈的，則微胞溶液可稱為 物理穩定的。此處要注意很重要的是激素構形的任何變 化為不能接受的，因為發現在投藥時此會提供較不有效 的蛋白質或是提供較少的醫療效果（參考Kathy L.De Jong, Bev Incledon， Chrostopher M.Yip， Michael R. Defel 1ippi s, Biophys J Biofast, 2006年6月Θ日）。本發明的微胞溶液適合用於非腸道投藥 。用語‘適合用於非腸道投藥’表示溶液包括賦形劑， 其量為安全的及經USFDA之類的主管機關核可用於非腸道 用途。 在本發明一個實施例，提供一種微胞溶液，其包括醫療 有效量的昇糖素及共價地鍵結至PEG (PEG-DSPE)的二硬 脂醯-磷脂醯乙醇胺及（選擇性地）一或更多穩定劑及醫藥 可接受載劑，其中溶液的pH為在約5-7. 5。本發明的即用 溶液包括濃度範圍從約0.5毫克/毫升至約5毫克/毫升的 昇糖素，而且，其包括濃度範圍從4毫克/毫升至40毫克/ 毫升的聚乙二醇化二硬脂醯-磷脂醯乙醇胺。在一個較佳 實施例，聚乙二醇化二硬脂醯-磷脂醯乙醇胺的分子量範 圍從約300至50 00道爾頓，特別是，約2000及5000道 100116179 表單編號A0101 第8頁/共19頁 1003279836-0 201204381 爾頓。 在一個實施例，杏發明的即用昇糖素溶液包括具有分子 量約30 0至5000的聚乙二醇化二硬脂醯-磷脂醯乙醇胺， 其以1毫克/毫升至8毫克/毫升的量使用。發現添加例如 二甲亞颯的極性非質子溶劑可使得穩定化昇糖素所需的 聚乙二醇化二硬脂醯-磷脂醯乙醇胺的量減少。在一些實 施例，極性非質子溶劑構成總體積的5%至15% v/v。 在一個較佳實施例，微胞溶液更包括一種傾向於存在於 微胞的疏水部分的穩定劑。較佳為，穩定劑從脂肪酸或 兩性離子膽鹼衍生物組成的群組選出。用語脂肪酸包括 得自或包含在動物或植物脂肪、油或臘中為酯化型式的 脂肪族（飽和或不飽和）一元羧酸。可用於本發明組成物 的脂肪酸或其鹽類的實例包括但不限於具有‘η’個數目 的碳原子的脂肪酸或其鹽類，其中‘η’的範圍從約4至 約28。脂肪酸可為飽和脂肪酸或不飽和脂肪酸，及其鹽 類與上述的組合。飽和脂肪酸及其鹽類可從丁酸、己酸 、辛酸、癸酸、月桂酸、肉豆蔻酸、棕櫚酸、硬脂酸、 二十烷酸、二十二烷酸、辛酸鈉、月桂酸鈉、肉豆蔻酸 鈉、棕櫚酸鈉及其類似物及/或其混合物選出。不飽和脂 肪酸及其鹽類可從肉豆蔻油酸、棕櫚油酸、油酸、亞油 酸、α亞油酸、花生四稀酸、二十碳五烯酸、芥酸、二 十二碳六烯酸、油酸鈉、花生四烯酸鈉及其類似物及/或 其混合物選出。在一個實施例，其中穩定劑為一種從二 豆蔻醯脂醯膽鹼、蛋黃磷脂醯膽鹼、二棕櫚磷脂醯膽鹼 、二硬酯酸磷酸脂膽鹼、氫化大豆磷脂醯膽鹼、二月桂 酸磷脂醯膽鹼、二油酸磷脂醯膽鹼及其溶小體產品組成 100116179 表單編號Α0101 第9頁/共19頁 1003279836-0 201204381 的群組選出的兩性離子膽鹼衍生物。在一個實施例，本 發明溶液包括濃度範圍從約1毫克/毫升至2遭:克/毫升的 二豆蔻醯脂醯膽鹼。在另一個實施例，微胞溶液包括濃 度範圍從約0. 1毫克/毫升至3毫克/毫升，特別是，1毫克 /毫升的辛酸做為穩定劑。添加這些穩定劑改善昇糖素的 穩定性，使得溶液可儲存於室溫約六個月。 本發明的即用溶液更包括醫療可接受載劑，其包括一或 更多酸、緩衝劑、防腐劑、張力調整劑及其混合物。可 用於醫療可接受載劑的緩衝劑是從組氨酸酯、琥珀酸酯 、磷酸酯、擰檬酸酯及其類似物與混合物組成的群組選 出。可加至微胞溶液的張力調整劑為從葡萄糖、蔗糖、 甘露糖醇、乳糖與甘油之類的單糖及其混合物組成的群 組選出。 然而本發明的發明者令人驚訝及意外地發現，昇糖素可 在單獨聚乙二醇化二硬脂醯-磷脂醯乙醇胺或是與二豆蔻 醯脂醯膽鹼之類的去垢劑或是脂肪酸（例如辛酸）的組合 的協助下溶解並穩定化，將pH調整至約5. 0至7. 5，較佳 為約6至7的pH。 儘管本發明大體上揭示如上文，可參考下文實例進一步 討論及說明其他方面。然而，實例僅用於說明本發明且 不應認為是本發明限制。Beaven, The Jou Tnal of Biol〇gical Chemistry, vol. 244, No. 24, PP · 6675-6679' 1 969). It has also been reported that monoterpene glycosides in aqueous solutions do not have a stable globular structure, and the configuration varies continuously with temperature or concentration of denaturant added (Bhiny〇Pnajipan and Walter Gratzer> European J. Biochemistry, 45, 547-553 , (1974)). Intended to develop a stable, ready-to-use glycosidic solution suitable for self-parenteral administration, especially in emergencies without adequate reconstitution time. The inventors found cyclodextrin, glycocalyx, alcohol, protein-like human serum. The protein cannot dissolve the glycoside. Surprisingly, the inventors have found that an aqueous solution of a glycoside having a disteocyanine-phospholipid oxime ethanolamine covalently linked to polyethylene glycol has a pH of between 5 and 7.5. Physical stability, especially in terms of protein configuration. SUMMARY OF THE INVENTION [0003] The present invention provides a stable, ready-to-use solution comprising a medically effective amount of a glycoside and a pharmaceutically acceptable carrier. The present invention can be summarized as follows: A. A stable ready-to-use solution comprising a medically effective amount of glycoside and pegylated disteganium-phospholipid ethanolamine adjusted to a pH of about 5-7.5 and a pharmaceutically acceptable carrier Agent. B. A stable ready-to-use solution as described in A, wherein the concentration of glycoside ranges from 0.5 mg/ml to 5 mg/ml. C. A stable ready-to-use solution micelle solution as described in A, wherein the PEGylated disteocyanine-phospholipid oxime ethanolamine has a molecular weight of about 300 to 5000 and is 100116179. Form number A0101 Page 5 of 19 pages 1003279836- 0 201204381 Ranges from 1 mg/ml to 40 mg/ml are present. D. A stable ready-to-use solution as described in A, wherein the carrier comprises a solvent selected from aqueous or polar aprotic solvents such as dimethyl hydrazine. E. A stable ready-to-use solution as described in D, wherein the aprotic solvent is present in a solution volume of 5 to 15% by volume. F. A stable ready-to-use solution as described in A, wherein the pH of the solution is adjusted to about 6.5. G. A stable ready-to-use solution as described in A, wherein the solution further comprises a stabilizer selected from the group consisting of fatty acids or zwitterionic choline derivatives or mixtures thereof. H. A stable ready-to-use solution as described in G, wherein the choline derivative is diterpene choline and is present in a concentration of from about 1 mg/ml to 2 mg/ml. I. A solution is used in which the fatty acid is octanoic acid and is present in an amount of from about 0.1 mg/ml to 3 mg/ml. J. A stable ready-to-use solution as described in A, wherein the pharmaceutically acceptable carrier comprises one or more acids, buffers, preservatives, tonicity adjusting agents, and mixtures thereof. K. Stable, ready-to-use solutions as described in J Wherein the buffer is selected from the group consisting of histidine succinate, phosphate, citrate, and mixtures thereof. [Embodiment] [0004] The term 'stable' as used herein means that the solution maintains physical and chemical properties. Stable, in particular, the solution is maintained clear for long periods of storage (at least three months, preferably from about 6 months to 12 months) at 2-8 ° C or preferably at room temperature. The physical stability of the solution of the present invention is determined by the percentage recorded in the 650 nm penetration 100116179 Form No. A0101, Page 6 of 19, 1003279836-0 201204381 and the absorption at 420 nm. Moreover, the solution maintains efficacy, which is determined by the biological analysis methods recognized by the drug iodine and chemical analysis such as HPLC. When the total impurities are not more than 10%, preferably when the solution is stored at room temperature for about three months and/or stored at 2-8 ° C for about three months or more, the total impurities are not more than 10%, the solution may be It is called stable. In addition, when at the end of the storage period, such as room temperature for three months or more, or when stored at 2-8 ° C for about six months or more, a glycoside-related substance, such as a glycoside, When D-trp (25)-glycoside or D-phen (22)-glycoside is not more than 3%, the solution can be said to be stable. The term "ready' is used herein to mean that the solution is in a form that can be administered without the need for an additional reconstitution step, such as a product that is commercially available in a freeze-dried form. Glycan, a medical substance used for emergency drug administration in the case of hypoglycemic shock, therefore, it is always desirable to have a ready-to-use solution stored in a pre-filled syringe. Depending on the emergency, the patient can self-administer the glycemic solution without delaying the administration. According to a preferred embodiment, the ready-to-use stable cell solution of the present invention is suitable for single-dose parenteral administration wherein the composition is dispensed into a pre-filled syringe. The amount of glycosin administered per single dose ranges from 1 mg to about 5 mg per ml of solution, preferably 1 mg per ml. According to another embodiment, the solution is filled in a pre-filled syringe with an automatic injector. In general, the autoinjector is spring driven and designed to administer the entire contents of the prefilled syringe in a single dose. It may not be necessary to have any fluid paths and may not require any contact with the drug or biological product contained in the syringe. The components of the automatic injector can be made of plastic and steel. The design and performance characteristics of the automatic injector device can include a safety mechanism to prevent inadvertent actuation, and the automatic wrapping of the needle used, in the first 100116179 Form No. A0101 Page 7 / Total Page 19, 1003279836-0 201204381 The fenestration and locking tabs of the square device prevent separation of the automatic injector device and self-disconnection when the two subassemblies are connected: to prevent repeated use. The term "microcell" as used herein means that the solution is in the microcell format and the solution is considered to be a clear solution. Preferably, the micelles are stabilized by phospholipids. When there are no signs of a change in conformation similar to gel formation/aggregation, a change in the CD spectrum showing the alpha helix structure and/or whether it is immediately after preparation or at various storage conditions (eg room temperature or 2-8 ° C) The solution is clear to be clear from the perspective of particulate matter when stored for about six months, and the microcell solution can be said to be physically stable. It is important to note here that any change in the conformation of the hormone is unacceptable because it is found to provide less effective protein or provide less medical benefit when administered (see Kathy L. De Jong, Bev Incledon). , Chrostopher M.Yip, Michael R. Defel 1ippi s, Biophys J Biofast, June 6th, 2006). The micelle solution of the invention is suitable for parenteral administration. The term "suitable for parenteral administration" means that the solution includes excipients in an amount that is safe and approved for use in parenteral applications by a competent authority such as the USFDA. In one embodiment of the invention, there is provided a microcell solution comprising a medically effective amount of a glycoside and a disteocyanine-phospholipid ethanolamine covalently bonded to PEG (PEG-DSPE) and (optionally) 5。 The pH of the solution is about 5-7. The ready-to-use solution of the present invention comprises a glycoside having a concentration ranging from about 0.5 mg/ml to about 5 mg/ml, and further comprising a pegylated distearyl glycol having a concentration ranging from 4 mg/ml to 40 mg/ml.醯-phospholipid 醯 ethanolamine. In a preferred embodiment, the molecular weight of the pegylated disteocyanine-phospholipid oxime ethanolamine ranges from about 300 to 50,000 Daltons, in particular, about 2,000 and 5,000 lanes. 100116179 Form No. A0101 Page 8 of 19 Page 1003279836-0 201204381 lton. In one embodiment, the ready-to-use glycoside solution of the apricot invention comprises a pegylated distearyl phospholipid-phosphoethanolamine having a molecular weight of from about 30 to about 5,000, which is used in an amount from 1 mg/ml to 8 mg/ml. . It has been found that the addition of a polar aprotic solvent such as dimethyl hydrazine reduces the amount of PEGylated distearyl phosphatide phosphatidylethanolamine required to stabilize the glyce. In some embodiments, the polar aprotic solvent constitutes from 5% to 15% v/v of the total volume. In a preferred embodiment, the micelle solution further comprises a stabilizer which tends to be present in the hydrophobic portion of the micelle. Preferably, the stabilizer is selected from the group consisting of fatty acids or zwitterionic choline derivatives. The term fatty acid includes an aliphatic (saturated or unsaturated) monocarboxylic acid derived or encapsulated in an animal or vegetable fat, oil or wax. Examples of the fatty acid or a salt thereof which can be used in the composition of the present invention include, but are not limited to, a fatty acid having a 'n' number of carbon atoms or a salt thereof, wherein 'η' ranges from about 4 to about 28. The fatty acid may be a saturated fatty acid or an unsaturated fatty acid, and a salt thereof in combination with the above. Saturated fatty acids and their salts can be from butyric acid, caproic acid, caprylic acid, citric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, behenic acid, sodium octanoate, sodium laurate Sodium myristate, sodium palmitate and the like and/or mixtures thereof are selected. Unsaturated fatty acids and their salts can be obtained from myristic acid, palmitoleic acid, oleic acid, linoleic acid, alpha linoleic acid, peanut tetraacene acid, eicosapentaenoic acid, erucic acid, twenty-two carbon six The olefinic acid, sodium oleate, sodium arachidonate and the like and/or mixtures thereof are selected. In one embodiment, wherein the stabilizer is one from dimethoprim choline, egg yolk phospholipid choline, dipalmitophos choline, distearate choline, hydrogenated soybean phospholipid choline, dilau Acid phospholipid choline, dioleate phosphocholine choline and its lysate products 100116179 Form No. Α0101 Page 9 of 19 Group of 1003279836-0 201204381 selected from the group of zwitterionic choline derivatives. In one embodiment, the solution of the invention comprises diterpene choline at a concentration ranging from about 1 mg/ml to about 2 g/ml. In another embodiment, the micelle solution comprises a concentration ranging from about 0.1 mg/ml to 3 mg/ml, in particular, 1 mg/ml of octanoic acid as a stabilizer. The addition of these stabilizers improves the stability of the glycemic so that the solution can be stored at room temperature for about six months. The ready-to-use solutions of the present invention further comprise a medically acceptable carrier comprising one or more acids, buffers, preservatives, tonicity adjusting agents, and mixtures thereof. Buffers which are useful as medically acceptable carriers are selected from the group consisting of histidine esters, succinates, phosphates, citrates, and the like, and mixtures thereof. The tonicity adjusting agent which can be added to the micelle solution is selected from the group consisting of monosaccharides such as glucose, sucrose, mannitol, lactose and glycerin, and mixtures thereof. However, the inventors of the present invention have surprisingly and unexpectedly discovered that glycosides can be used in the PEGylated distearyl phosphatide-phospholipid ethanolamine or the detergent such as dicarmine choline or The pH is adjusted to a pH of from about 5.0 to 7.5, preferably from about 6 to 7, with the aid of a combination of a fatty acid (e.g., octanoic acid). Although the present invention is generally disclosed as above, other aspects may be further discussed and illustrated with reference to the examples below. However, the examples are only intended to illustrate the invention and should not be construed as limiting the invention.
實例A 研究pH於昇糖素溶液的作用。使用具有分子量2000的8毫 克/毫升MPEG-DSPE2K來製備昇糖素母溶液（1毫克/毫升 )，將溶液分成3部分及調整pH為3. 0、6. 0、及9. 0。在 40°C/75% RH儲存3天後，分析這些溶液以進行昇糖素分 100116179 表單編號A0101 第10頁/共19頁 1003279836-0 201204381 Ο [0005]Example A The effect of pH on a glycemic solution was investigated. The glycerol mother solution (1 mg/ml) was prepared using a MW-2000E2K having a molecular weight of 2000. The solution was divided into 3 portions and the pH was adjusted to 3.0, 6.0, and 9.0. After storage at 40 ° C / 75% RH for 3 days, these solutions were analyzed for glycosidation 100116179 Form No. A0101 Page 10 of 19 1003279836-0 201204381 Ο [0005]
析。表1示出溶液數據，其指出物理及化學穩定性。 表1 : pH於40°C3天後昇糖素水溶液分析（HPLC)的作用 pH 3.0 60 9.0 外觀 混濁 清澈 清激 分析 73.39 107.30 7658 從此數據，顯然昇糖素與MPEG-DSPE在酸性pH (pH 3.0)為物理不穩定的；在鹼性pH (pH 9.0)為化學不穩 定的。於pH 6.0 (接近昇糖素的等電點6.2)，發現昇糖 素水溶液為更穩定的。 而且，進行pH對窄pH範圍的作用的研究。使用8毫克/毫 升MPEG-DSPE2K來製備昇糖素母溶液（1毫克/毫升），使 用檸檬酸鹽緩衝液及氫氧化鈉溶液將pH從5. 5調整至7. 5 。在30°C/65% RH儲存1個月後，分析這些溶液以進行昇 糖素及相關物r質的分析。 表2: pH於昇糖素水溶液物理化學穩定性的作用 pH 時間 物理外親 HPLC 分 析作） Μ IM 物哲 昇耱素亞 礪 B^ip(25y 異糖棄 D-i*«<22)- 昇耱素 單一最大 總共 55 相始 清激 96.31 0.28 ND 0.22 0.68 2.42 1個月/3〇eC 清激 93-77 0.59 0.02 0.04 0.98 5M~ 6.Q 初始 清激 95.86 026 ND 0.21 0.68 2.50 1铟月召〇°C 清激 91.17 0.64 0.03 0 03 1.01 一 4.16 初始 清激 97.25 0.23 ND 0.20 064 2.32 O.J 1個月/30°C 清激 94.61 0.83 ND 0.2 1.05 4.22 " 7.0 初始 清激 96.03 0.27 ND 0.21 0.67 2拓 i锢月/30eC 清激 92.62 0.91 ND 〇-〇I~~ L40 4.93 7孓 初始 清激 9491 021 ND 0.21 0.67 2.32 1锢月召o°c 请激 90.35 0.73 ND 0.01 1.52 5.22 結果（表2)顯示在使用mPEG-DSPE2000製備的溶液形式 中從pH 5. 5至pH 7· 5的類似的昇糖素物理化學穩定性 。因此可從這些初步結果歸納，當昇糖素溶液維持在5.0 100116179 表單編號A0101 第11頁/共19頁 1003279836-0 201204381 至約7. 5的pH範圍及聚乙二醇化二硬脂醯—磷脂醯乙醇胺 ’可得到一種穩定溶液。 實例1-2 如下製備一種昇糖素水溶液： 表3:昇糖素溶液的組成 實例1 實例2 ΛΧ*5ν 董以毫克表示 昇糖素 1 1 讀EG-DSPE2<K)〇 S 8 蔗耱 50 80 組氨酸 - 1.5 加入以調整至PH 60 乳緩溶液(10%) 加入α調整至PH65 碳緩氩錄毫克/毫升） - 注射用水 加入至1 毫升 ~' 將mPEG-DSPE 2000及蔗糖於室溫溶解於純水，使用乳酸 及/或碳酸氫鈉溶液（實例丨）將溶液pH調整至6. 5或是使 用組氨酸調整pH為6. 0。 使用磁攪拌器於室溫將所需量的昇糖素溶解於上述溶液 ，經由0. 8/0. 2μ PVDF過濾器於無菌區域過濾溶液並包 裝於5毫升USP-形式I玻璃管形瓶。根據Ια指導原則於 2-8T，25。(：/60猶及4(TC/75%RH保存最終產物以進 行穩定性研究。此穩定性結果示於下文。 表4 :實例1組成物的穩定性測試結果 100116179 表單煸號A0101 第12頁/共19頁 1003279836-0 201204381 穩定性條件 時間 敘述 以％表示的 分析 相關物 質(％> 於420 奈米(AU) 的歧雜 於650奈 米的％Τ 單一 總共 初始 清激溶液 101.19 0.53 1.40 0.011 99.812 2-8T 1個月 清激溶液 102.71 0.59 2.05 0.004 99.947 25°C/60%RH i锢月 请激溶液 97.57 0.58 4.16 0005 99.591 40^5%1〇1 1捆月 清激溶液 74.83 2.96 17.49 0.090 97J25 2-e°c 2個月 清激溶液 94.66 0.52 3.0 0.006 99.927 " 2個月 沉澱 SL82 1.12 836 0.025 97.328 40°C/75^®H 2锢月 πΜ 6616 3.82 31.42 0.0570 沙.506 2-8°C 3個月 清激溶液 95.49 060 3.24 0.009 99.701 25°C/60%RH 3個月 說鷇 85.22 LQ9 8.93 0248 61.279 40°C/75%RH 3個月 沉歎 39.36 6.62 31.75 0.636 26.875 2-8°C 6楢月 沉嚴 癸7-01 0.72 3.21 0.076 92.430 2-8°C 12姐月 沉澱 80.06 0,59 4.12 0.917 19.258 Ο 發現實例2的組合物於室溫是不穩定的，因為當儲存於室 溫時，混濁於33天開始出現。所以，可歸納當使用mPEG-DSPE配製昇糖素成溶液並將pH調整至5至7.5的特定範 圍時’發現溶液在2-8°C下僅穩定三個月的期間。然而， 當儲存於室溫時溶液是不穩定的。室溫穩定性由添加例 如脂肪酸或是二豆蔻醯脂醯膽鹼的穩定劑而進一步改善 〇 ^ 實例3-5 表5:本發明的組成Analysis. Table 1 shows solution data indicating physical and chemical stability. Table 1: Effect of aqueous solution of glycosidic acid (HPLC) after 3 days at 40 ° C pH 3.0 60 9.0 Appearance turbidity Clear and clear analysis 73.39 107.30 7658 From this data, it is clear that glycoside and MPEG-DSPE are at acidic pH (pH 3.0 ) is physically unstable; chemically unstable at alkaline pH (pH 9.0). At pH 6.0 (close to the isoelectric point of 2.6, the glycoside was found to be more stable). Moreover, studies of the effect of pH on a narrow pH range were performed. 。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 After storage at 30 ° C / 65% RH for 1 month, these solutions were analyzed for analysis of glycosides and related substances. Table 2: Effect of pH on the physicochemical stability of aqueous solution of glycein pH time physical external pro-HPLC analysis) Μ IM 物 哲 耱 砺 砺 ^ ^ ^ ^ 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 Alizarin single maximum total 55 phase initial stimuli 96.31 0.28 ND 0.22 0.68 2.42 1 month / 3 〇 eC Qing dynasty 93-77 0.59 0.02 0.04 0.98 5M~ 6.Q Initial stimuli 95.86 026 ND 0.21 0.68 2.50 1 Indium Call 〇°C Qingxie 91.17 0.64 0.03 0 03 1.01 A 4.16 Initial Sweeping 97.25 0.23 ND 0.20 064 2.32 OJ 1 month / 30 °C Qingxu 94.61 0.83 ND 0.2 1.05 4.22 " 7.0 Initial Sweeping 96.03 0.27 ND 0.21 0.67 2 Extension i锢月/30eC 清激92.62 0.91 ND 〇-〇I~~ L40 4.93 7孓 Initial blasting 9941 021 ND 0.21 0.67 2.32 1锢月召o°c Please stimulate 90.35 0.73 ND 0.01 1.52 5.22 Results (Table 2) Similar citrate physicochemical stability from pH 5. 5 to pH 7.5 was shown in solution form prepared using mPEG-DSPE2000. Therefore, it can be summarized from these preliminary results when the glycemic solution is maintained at 5.0 100116179 Form No. A0101 Page 11 / 19 pages 1003279836-0 201204381 to a pH range of about 7.5 and PEGylated disteganide-phospholipid醯Ethanolamine' gives a stable solution. Example 1-2 An aqueous solution of glycoside was prepared as follows: Table 3: Composition of glycosidic solution Example 1 Example 2 ΛΧ*5ν Dong expressed in milligrams of glycoside 1 1 Read EG-DSPE2<K) 〇S 8 Cane toad 50 80 histidine-1.5 was added to adjust to PH 60 milk solution (10%) Add α to pH65 carbon slow argon recorded mg/ml) - Add water to 1 ml~' Place mPEG-DSPE 2000 and sucrose in room 0。 The pH is adjusted to 6. 0. Using a histidine to adjust the pH to 6.5 or a histidine. The desired amount of glycoside was dissolved in the above solution using a magnetic stirrer at room temperature, and the solution was filtered through a 0.80 to 0.22 PVDF filter in a sterile area and packaged in a 5 ml USP-Form I glass vial. According to the Ια guiding principle at 2-8T, 25. (:/60 is still 4 (TC/75% RH preserves the final product for stability studies. The stability results are shown below. Table 4: Stability test results for the composition of Example 1 100116179 Form No. A0101 Page 12 / Total 19 pages 1003279836-0 201204381 Stability condition time describes the analysis of related substances expressed in % (% > % at 420 nm (AU) mixed with 650 nm Τ Single total initial refreshing solution 101.19 0.53 1.40 0.011 99.812 2-8T 1 month clearing solution 102.71 0.59 2.05 0.004 99.947 25°C/60%RH i锢月请激溶液97.57 0.58 4.16 0005 99.591 40^5%1〇1 1 bundle of monthly clearing solution 74.83 2.96 17.49 0.090 97J25 2-e°c 2 months clear solution 94.66 0.52 3.0 0.006 99.927 " 2 months precipitation SL82 1.12 836 0.025 97.328 40°C/75^®H 2锢月πΜ 6616 3.82 31.42 0.0570 沙.506 2- 8 ° C 3 months clear solution 95.49 060 3.24 0.009 99.701 25 ° C / 60% RH 3 months said 鷇 85.22 LQ9 8.93 0248 61.279 40 ° C / 75% RH 3 months sigh 39.36 6.62 31.75 0.636 26.875 2- 8°C 6楢月沈严癸7-01 0.72 3.21 0.076 92.430 2-8°C 12 Monthly precipitation 80.06 0,59 4.12 0.917 19.258 Ο The composition of Example 2 was found to be unstable at room temperature because turbidity began to appear at 33 days when stored at room temperature. Therefore, it can be summarized when using mPEG-DSPE to prepare liters. When the glycoside was solution and the pH was adjusted to a specific range of 5 to 7.5, the solution was found to be stable for only three months at 2-8 ° C. However, the solution was unstable when stored at room temperature. Stability is further improved by the addition of stabilizers such as fatty acids or diterpene choline. Examples 3-5 Table 5: Composition of the invention
成会 涵 1 3 4 5 — 弄棟* 1毫克 1毫克 1毫克 ~ mPEG-DSPE2000 8东克 ?毫克 inPEG'DSPESOOO - - 7奄克 蔗棒 50亳克 50奄克 50 二豆蔻醴鹿_袭 2 .毫克 - 争酸 - 1毫克 0.1奄克—' 组氨酸 1毫克 1电克 1:_奄免 隹射《水 加入直1毫丼 ;'知八至..1毫务 加入至1毫升 乳斑溶液(10%) 加入痛整盔械1$;5 加入议調整至碑 6.5 加八孿調[整至 pH6.S 碳酸細璆物毫 加入以調整至pH 6.5 加入以調整至I>H 6.5 加入辟調整至 PH6.S 表單編號A0101 第13頁/共19頁 100116179 1003279836-0 201204381 實例3:將昇糖素及二豆蔻醯脂醯膽鹼（DMPC)於室溫以 注射用水混合在一起，直到混合物成為半透明的。接著 將特定量的mPEG-DSPE 2000加至此浪合物並授拌直到變 成清澈的。將蔗糖與組氨酸加入並溶解於溶液中’將PH 調整至6-6. 5之間並以注射用水補足體積。經由〇. 2μ PVDF過濾器於無菌區域過濾溶液，將經過濾溶液填充於1 毫升預先填充的注射器（附針頭）及以塞子塞住。根據 ICH指導原則於2-8°C，25°C/60%RH保存最終溶液以進 行穩定性研究。 實例4:將mPEG-DSPE 2000溶解於WFI，將昇糖素加至 此溶液將昇糖素及藉由於室溫連續攪拌而使其溶解。將 嚴糖、組氨酸及辛酸一個一個加入及溶解，將pH調整至 6-6. 5之間及以WFI補足體積。經由0. 2μ PVDF過遽器於 無菌區域過濾、溶液，將經過渡溶液填充於1毫升預先填充 的注射器（附針頭）並以塞子塞住。根據ICH指導原則於 2-8°C及25°C/60%RH保存最終溶液以進行穩定性研究 實例5:於室溫將mPEG-DSPE 5000溶解於純化水 /Τι 磁攪拌器於室溫將所需量的昇糖素溶解於上述溶液。以 連續攪拌將蔗糖、組氨酸及辛酸一個—個加入並溶解於 上述溶液’若需要驗祕酸氫贿料/魏酸溶液調 整溶液pH至6. 5,形成清澈無色溶液。經由〇 8/〇 2 PVDF過濾器於無菌區域過濾溶液並包裴於5毫升, 式I玻璃管形瓶。 表6 :實例3及實例4溶液的穩定性結果 100116179 表單編號A0101 第14頁/共19頁 1003279836-0 201204381 實例號瑪 時閟雕（月）ΚΓ (室 m 1 — 物理终觀 實例3 ΪΜΜΜΊ 清澉溶液 溃激齡 5锢月 清雜€~~~~ 6値月m1 请液 10個月洛8°C WmmS.~~~~ 實例4 RTO錮月 清教 4W1俩月 WmSM~~~~ SmAM-^C 清_液 5個月/RT ~~~~ _勝8°€ 请澈缕载 6餾月/RT Wi$E~~~~ f俩月/2-3% #lsE~-- 發現實例3及4的昇糖素溶液為穩定的。不希望受限於任 何理論，此作用可說是因為藉由辛酸之類的脂肪酸或是 DMPC之類的兩性離子膽鹼衍生物與mPEG-DSPE合併而穩 定化微胞。將組氨酸加至此溶液以作為緩衝劑，因此， 可歸納mPEG-DSPE可改善昇糖素溶液的物·理及化學穩定 性。但是’即使當儲存於2-8eC，於僅包含^^㈠卿的 昇糖素溶液中沉澱於6個月内開始出現。然而，添加辛酸 或是DMPC之類的穩定劑可進-步改善昇糖素溶液微胞溶 液的物理及化學穩定性。 100116179 表單編號A0101 而且，將類似於實例3及實例4的溶液進行物理特徵化（於 650奈米的％穿透率及於·奈米的吸收值），在初始時間 點及在各種儲存條件（例如2-8。(：及25\/6q%rh)儲存三 個月的昇糖素的生物分析與化學分析及相關物㈣ 測定。在於室溫儲存溶液六個月之後，溶液形式的激素 的物理穩定性在構形方面㈣行昇糖h生物分析確認 > 第15頁/共19頁 1003279836-0成会涵1 3 4 5 — 栋栋* 1 mg 1 mg 1 mg~ mPEG-DSPE2000 8 East gram? mg inPEG'DSPESOOO - - 7 gram cane stick 50 gram 50 gram 50 two bean 蔻醴 deer _ 2 .mg - 争 —— - 1 mg 0.1 克 - 'Histidine 1 mg 1 gram 1: 奄 隹 隹 "Water added straight 1 丼; 'Knowledge eight to .. 1 pm added to 1 ml of milk Spot solution (10%) Add to the painful helmet 1$; 5 Add the adjustment to the monument 6.5 plus the bark adjustment [to the pH6.S carbonic acid fines added to adjust to pH 6.5 Add to adjust to I> H 6.5 Addition adjustment to PH6.S Form No. A0101 Page 13 of 19 100116179 1003279836-0 201204381 Example 3: Mixing glycosidic and dimethoprim choline (DMPC) together with water for injection at room temperature, Until the mixture becomes translucent. A specific amount of mPEG-DSPE 2000 was then added to the wave and allowed to mix until it became clear. Add sucrose to histidine and dissolve in solution. Adjust pH to between 6 and 6. 5 and make up the volume with water for injection. The solution was filtered through a μ. 2μ PVDF filter in a sterile area, and the filtered solution was filled in a 1 ml pre-filled syringe (with a needle) and stoppered. The final solution was stored at 2-8 ° C, 25 ° C / 60% RH according to ICH guidelines for stability studies. Example 4: mPEG-DSPE 2000 was dissolved in WFI, and glycosidic acid was added to this solution to dissolve the glycoside and by continuously stirring at room temperature. Adding and dissolving the sugar, histidine, and octanoic acid one by one, adjusting the pH to between 6 and 6. 5 and making up the volume with WFI. The solution was filtered through a 0.2 μμ PVDF filter in a sterile area, and the transition solution was filled in a 1 ml pre-filled syringe (with a needle) and stoppered. The final solution was stored at 2-8 ° C and 25 ° C / 60% RH according to ICH guidelines for stability studies. Example 5: Dissolve mPEG-DSPE 5000 in purified water / Τι magnetic stirrer at room temperature at room temperature The required amount of glycoside is dissolved in the above solution. The sucrose, the histidine, and the octanoic acid are added to the above solution, and the pH of the solution is adjusted to 6.5 to form a clear colorless solution. The solution was filtered through a 〇 8/〇 2 PVDF filter in a sterile area and wrapped in 5 ml of a glass vial of formula I. Table 6: Stability results of the solution of Example 3 and Example 4 100116179 Form No. A0101 Page 14 of 19 1003279836-0 201204381 Example No. Ma Shizheng (Month) ΚΓ (Room m 1 - Physical Final Example 3 ΪΜΜΜΊ Qing澉 Solution 激 激 激 € ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 -^C 清_液5月月/RT~~~~ _胜8°€ Please clear 6 months of distillation/RT Wi$E~~~~ f two months/2-3% #lsE~-- The glycemic solution of Examples 3 and 4 is stable. It is not intended to be limited by any theory, and this effect can be said to be due to a fatty acid such as caprylic acid or a zwitterionic choline derivative such as DMPC and mPEG-DSPE. Combine and stabilize the micelles. Add histidine to this solution as a buffer, therefore, it can be concluded that mPEG-DSPE can improve the physical and chemical stability of the glycoside solution. But 'even when stored at 2-8eC Precipitation starts within 6 months in the glycosidic solution containing only ^^(一)卿. However, the addition of octanoic acid or a stabilizer such as DMPC can further improve the dissolution of the glycoside solution. Physical and chemical stability of the liquid. 100116179 Form No. A0101 Moreover, the solutions similar to Examples 3 and 4 were physically characterized (% penetration at 650 nm and absorption at Na), at initial Time point and bioanalytical and chemical analysis of glycosidic acid stored in various storage conditions (eg 2-8. (: and 25\/6q% rh) for three months) and correlation (4) determination. After the month, the physical stability of the hormone in solution form is in terms of configuration (4) Glucose h bioanalysis confirmation> Page 15 of 19 1003279836-0
G 201204381 。生物分析的步驟是依據昇糖素注入的USP專論。結果分 別i列於表7及表8。 表7:根據實例3溶液的化學及生物穩定性數據 穩定性條 件 時間 昇耱素的 HMX：分 析 再耱素 的生物 分析 相關物f 於420奈 糸的吸收 值 <AU) 於650奈来 的％穿透率 異綠素亞 颯 D- M25> 昇搶素 D- pte<22)- 異耱棄 軍一最 大 總共 初始 106.84 1194 0.07 ND 0.03 0.86 1.96 25°e/60% RH i個月 98.53 1.145 0.11 ND ND 0.55 2.79 0.096 92.931 ~~~ 25°C/60% RH 2镅月 103.0 - 0.07 N0 ¢,02 0.86 5.55 0.246 91.780 2-8°C 3価月 105.80 - 0.102 ND 1.132 0.239 1.965 0.045 98.180 25°a60% RH 3個月 91J8 - 0.074 ND 0.304 2.160 8.231 0.206 89.244 2-8^ 6锢月 104.63 - 0.112 1.015 0-878 4.807 0.06 96.70 表8:根據實例4溶液的化學及生物穩定性數據 f 穩定性條伴 時間 昇糖素的 HPLC 分 析 異糖音 的生物 分析 相Μ物筲 於420 奈未的 吸故值 (AU) 於650条米 的％穿透率 昇糖素亞 壤 D- ^pQsy 葬耱素 0- |Ae(22)- 昇繚棄 單一最 大 總共 初始 106.70 1.09 0.17 ND 0.17 0J7 2.21 25°C/60%R Η 1铟月 101.05 0.89- 1.096 021 ND ND 0.96 3.04 0.027 97.218 25°C/60^ftR Η 2铟月 89.11 1.162 0.89 ND 1.21 121 7.02 - - 2-8°C 3個月 101.47 1.049- 1.068 0.36 ND ND 0.97 3.01 0.027 97.900 25°C/6〇?谈 Η 3锢月 90.98 1.02 1.03 ND 0.33 1.67 7.84 0.025 98.221 2-8°C 6個月 102.24 1.298- 1.409 0.490 ND 1.240 0.490 3_960 0.012 98.682 2-8eC 9姻月 102‘鉍 0.983- 1.013 0.75 0.01 U9 1.28 5.823 0.02 97.852 實例6 二曱亞硬之類的極性非質子溶劑之作用：將„11>別_1)外£ 3毫克/毫升溶解於注射用水中，將昇糖素加至二曱亞砜（ 相當於10%總體積）及藉由於室溫連續攪拌而溶解。將昇 糖素溶液緩慢加至mPEG-DSPE溶液並連續攪拌，將PH調 整至6·0 —7·0之間及以WFI補足體積。經由0.2μ PVDF過 濾器於無菌區域過濾溶液。 表9 :實例6溶液的穩定性結果 100116179 表單蝙號Α0101 第16頁/共19頁 1003279836-0 201204381 時間 物理外觀 總相關物質 3個月 清澈溶液 - 2個月/RT 清澈溶液 3.656% 3個月/RT 清澈溶液 4.551% 發現具較少m P E G - D S P E與少量的極性非質子溶劑的微胞 溶液於室溫儲存溶液三個月為化學穩定的。 【圖式簡單說明】 [0007] 無。 【主要元件符號說明】 [0008] 無。 100116179 表單編號A0101 第17頁/共19頁 1003279836-0G 201204381. The step of bioanalysis is based on the USP monograph on glycosidic injection. The results are listed in Tables 7 and 8 respectively. Table 7: Chemical and biological stability data for the solution according to Example 3 Stability conditions Time HMX of astaxanthin: Analysis of bioanalytical correlations of recanzein f Absorption value at 420 Naphth <AU) at 650 Nai % penetration rate isochloroanthine D-M25> Assault D-pte<22)- Different abandonment army one maximum total initial 106.84 1194 0.07 ND 0.03 0.86 1.96 25°e/60% RH i month 98.53 1.145 0.11 ND ND 0.55 2.79 0.096 92.931 ~~~ 25°C/60% RH 2镅月103.0 - 0.07 N0 ¢,02 0.86 5.55 0.246 91.780 2-8°C 3価月105.80 - 0.102 ND 1.132 0.239 1.965 0.045 98.180 25° A60% RH 3 months 91J8 - 0.074 ND 0.304 2.160 8.231 0.206 89.244 2-8^ 6锢月104.63 - 0.112 1.015 0-878 4.807 0.06 96.70 Table 8: Chemical and biological stability data for solution according to Example 4 f Stability bar HPLC analysis of time-glucagon by HPLC analysis of the sugar-staining bioassay of the sputum 筲 420 奈 奈 吸 吸 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( - |Ae(22)- 升 缭 a single maximum total initial 106.70 1.09 0.17 ND 0.17 0J7 2.21 25°C/60%R Η 1 Indium month 101.05 0.89- 1.096 021 ND ND 0.96 3.04 0.027 97.218 25°C/60^ftR Η 2 Indium month 89.11 1.162 0.89 ND 1.21 121 7.02 - - 2-8°C 3 Month 101.47 1.049- 1.068 0.36 ND ND 0.97 3.01 0.027 97.900 25°C/6〇?谈Η3锢月90.98 1.02 1.03 ND 0.33 1.67 7.84 0.025 98.221 2-8°C 6 months 102.24 1.298- 1.409 0.490 ND 1.240 0.490 3_960 0.012 98.682 2-8eC 9 marriage month 102'铋0.983- 1.013 0.75 0.01 U9 1.28 5.823 0.02 97.852 Example 6 The role of a polar aprotic solvent such as diterpenoid: „11>别_1) £3 mg/ The ml was dissolved in water for injection, and the glycoside was added to disulfoxide (equivalent to 10% of the total volume) and dissolved by continuous stirring at room temperature. The ghrelin solution was slowly added to the mPEG-DSPE solution and continuously stirred to adjust the pH to between 6.8 and 7.0 and to make up the volume with WFI. The solution was filtered through a 0.2 μ PVDF filter in a sterile area. Table 9: Stability of the solution of Example 6 100116179 Form bat number Α 0101 Page 16 / Total 19 pages 1003279836-0 201204381 Time physical appearance Total related substances 3 months clear solution - 2 months / RT Clear solution 3.656% 3 months /RT clear solution 4.551% It was found that the solution of the micelle with less m PEG - DSPE and a small amount of polar aprotic solvent was chemically stable at room temperature for three months. [Simple description of the schema] [0007] None. [Main component symbol description] [0008] None. 100116179 Form No. A0101 Page 17 of 19 1003279836-0