CN1118143A - 运用赋形剂对有机溶剂处理的多肽的稳定化方法 - Google Patents
运用赋形剂对有机溶剂处理的多肽的稳定化方法 Download PDFInfo
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- CN1118143A CN1118143A CN94191235A CN94191235A CN1118143A CN 1118143 A CN1118143 A CN 1118143A CN 94191235 A CN94191235 A CN 94191235A CN 94191235 A CN94191235 A CN 94191235A CN 1118143 A CN1118143 A CN 1118143A
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Abstract
在此公开的是运用赋形剂对经有机溶剂处理的干的或水性多肽稳定化的方法和组成制剂,其中,将多肽与分子量小于70,000KD的多元醇混和。
Description
此发明是有关运用赋形剂稳定经有机溶剂处理过的,干的或是水性的多肽类制剂。
多肽类药物制剂在配制及贮存过程中易变性与降解。多元醇已被用干稳定水性制剂的蛋白质或其它大分子及其水溶液的空气干燥或冷冻干燥。
美国专利4,297,344公开了通过添加选择性的氨基酸,例如:甘氨酸,丙氨酸,羟基脯氨酸,谷氨酸和氨基丁酸;或添加糖,例如:单糖,低聚糖,或糖醇对凝血因子II和VIII,抗凝血酶III,和血纤维蛋白溶酶原的抗热变性稳定化技术。
欧洲专利应用公报0,303,746号公开了以多元醇对生长激素进行稳定化的技术,这些多元醇包括:非还原糖类,糖醇,糖酸,季戊四醇,乳糖,水溶性葡聚糖,和聚蔗糖,氨基酸,在生理pH环境下具有带电侧基的氨基羧聚合物,以及胆盐。
欧洲专利应用公报0,193,917号公开了一种以蛋白质与糖的络合物水溶液为特征的生物活性缓释剂。
澳大利亚专利应有公报AU—A—30771/89号公开了应有甘氨酸和甘露糖醇稳定生长激素。
美国专利5,096,885公开了一种含有甘氨酸,甘露糖醇,非离子型表面活性剂的缓冲液的冻干人生长激素(hGH)制剂。
Pharm.Res.8:285~291,1991综述了用聚乙二醇对蛋白质进行稳定化的方法。
应有海藻糖和其它多元醇稳定水性系统干燥过程中的蛋白质的实例包括以下几则:
美国专利4,891,319公开了一种水性系统中的敏感蛋白及其他大分子的保存方法;即添加海藻糖,在室温和大气压下干燥。美国专利5,149,653揭示了在海藻糖存在下,于冰冻状态或室温干燥保存水性系统中活的病毒的方法。
多元醇还被用于稳定干的药物制剂,例如:1989年5月归档的WO 8903671公开的:于药物极细粉末混悬于油性介质的混合物中,加入稳定剂,诸如:明胶,清蛋白,葡聚糖或海藻糖。
用诸如二氯甲烷的有机溶剂处理多肽提出了一个多肽变性的问题。因此,本发明的目的是提供一种用有机溶剂处理的多肽共水性制剂的稳定化方法。
本发明的另一目的是对有机溶剂处理过的干燥多肽的稳化。
本发明的又一目的是提供一种用胶束包裹多肽的稳化方法。
本发明还有一目的是提供一种用于控释制剂的经赋形剂稳化的多肽,其中多肽是经有机溶剂处理过的。
本发明的一个方面是一种多肽经有机溶剂处理时的抗变性稳定化方法,此方法包括将一种多元醇掺入多肽中,其中多元醇的分子量不超过70,000KD。
本发明的另一个方面是一种多肽的配制方法,包含:
a)在多肽的水溶液中加入一种分子量小于70,000KD的多元醇;
b)用有机溶剂处理水溶液中的多肽。
本发明的另一方面是用于控释的干多肽的配制,包含:
a)将多肽与赋形剂混合,该赋形剂是分子量低于70,000KD的多元醇;
b)将上述处理过的多肽以有机溶剂处理。
本发明的另一方面是一种多肽控释剂的组成,包含:多肽与赋形剂混合,赋形剂为分子量小于70,000KD的多元醇,此混合物以有机溶剂处理,包埋于聚合物骨架。A.定义
此外所用的“多元醇”一词指至少有2个羟基结合于碳原子的碳氢化合物。此多元醇也可能含有其它官能团。用于实施本发明的多元醇实例包括诺如甘露糖醇和海藻糖的糖醇和聚醚。
此处的“聚醚”一词指至少含3个醚键的碳氢化合物。此聚醚可能含有其它官能团。用于实施本发明的聚醚包括聚乙二醇(PEG)。
此处的“干多肽”一词指经诸如冷冻干燥等干燥过程处理过的多肽,其中至少50%的水份已除去。
此处“包埋”一词指一种将治疗剂,如多肽,配制到适用于控释的制剂组成中的方法,用于本发明的包埋材料包括乳酸和羟基乙酸的聚合物或是共聚物,或此类聚合物和/或共聚物的混合物,通常将此类物质称为“聚交酯”。
此处“掺和”一词指于所涉多肽中加入赋形剂的方法,例如:将干的试剂混和,或是将干的试剂与另一种试剂的溶液或混悬液混和,或是将试剂的水性制剂混和。
此处的“赋形剂”一词指一种加入药物制剂中的非治疗剂以提供所需稠度与稳定性的作用。
此处“有机溶剂”一词可指含有含碳化合物的任何溶剂。例如:二氯甲烷,乙酸乙酯,二甲基亚砜,四氢呋喃,二甲基甲酰胺和乙醇。
以有机溶剂“处理”多肽在此处指将有机溶剂与干多肽混合,或将多肽水性制剂与有机溶剂制成乳浊液,在多肽水性制剂和有机溶剂之间形成界面,或用有机溶剂从水性制剂中萃取多肽。
“多肽”在此一般指含有10个以上氨基羧的肽类和蛋白质。B.一般方法
通常,在用有机溶剂处理之前,干多肽和多肽水性制剂都可以与赋形剂混和以获得稳定化效果。多肽水性制剂可以是多肽的混悬液或溶液。虽然可以加入干的赋形剂,但典型的是将赋形剂的水性制剂加入到多肽的水性制剂中,或反过来加。多肽和赋形剂的水性制剂也可以用冷冻干燥或其它方法干燥。如此得到的干燥制剂可在有机溶剂处理前重新溶解成水性制剂。
用于稳定多肽的典型赋形剂为分子量低于70,000KD的多元醇。例如:海藻糖,甘露糖醇,和聚乙二醇。通常,海藻糖对多肽的典型重量比为100∶1到1∶100,1∶1到1∶10较好,1∶3至1∶4更好。甘露糖醇与多肽的典型重量比为100∶1到1∶100,1∶1到1∶10较好,1∶1到1∶2更好。PEG对多肽的典型重量比为100∶1到1∶100,优先选用1∶1到1∶10。最佳比例的选择以能够最大限度地溶解多肽同时引起最小变性的赋形剂浓度为准。
本发明中的制剂可包含一种防腐剂,一种或多种缓冲液,多种赋形剂,例如,除了海藻糖或甘露糖醇再加聚乙二醇(PEG),或加入诸如吐温的非离子型表面活性剂。非离子型表面活性剂包括:聚山梨酯类,如,聚山梨酯20或80等,和泊洛沙姆,如泊洛沙姆184或180,普朗克尼多元醇类,以及其它乙烯/聚丙烯的嵌段共聚物等。用量以能够形成一个稳定的水性制剂为宜,通常在约0.1%(w/v)到30%(w/v)范围内。
缓冲液包括:磷酸盐,Tris,柠檬酸盐,琥珀酸盐,乙酸盐,或组氨酸缓冲液。缓冲液浓度在2mM至100mM范围内。优先选用的缓冲剂包括琥珀酸钠和磷酸钾缓冲液。
防腐剂包括:苯酚,苯甲醇,甲酚,对羟苯甲酸甲酯,对羟苯甲酸丙酯,苯扎氯氨和氯化苄乙铵。虽然防腐剂的种类与浓度影响不大,仍优先选择0.2~0.4%(w/v)的苯酚和0.7~1%(w/v)的苯甲醇。
通常,本发明中的制剂中可含有其它组份,用量以不破坏制剂的稳定性和适合于有效、安全用药为宜。例如:为本行专家所熟知的其他药用赋形剂可是本目的制剂组成的一部份。其中包括如填充剂,辅助缓冲剂,螯合剂,抗氧剂,潜溶剂等。具体的实例有:三羟基甲胺盐(“Tris缓冲剂)和乙二胺四乙酸二钠。
所涉多肽包括:糖基化和非糖基化的多肽,例如:生长激素,干扰素,病毒蛋白,如HIV蛋白酶和gp120。
本发明稳定化的多钛可制成缓释制剂,特别是那些以有机溶剂处理为通用制备步骤的缓释制剂。缓释制剂的适宜例子有:含多肽的流水性聚合物的半透性固体骨架,这种骨架为一定形状的制品如膜剂,或微囊。丝释骨架的实例包括聚酯,水凝胶[例如:聚(2—羟乙基—甲基丙烯酸酯),正如Langer等在J.Biomed.Mouster.Res,1981年15期167~277页和Chem.Tech.,1982年12期98~105页所述,或聚(乙烯醇)],聚交酯(U.S.Pat.No.3,73,919,EP.58,481),L—谷氨酸和γ—乙基—L—谷氨酸(Sidman,et al.,Biopolymers,22:547—556[1983])的共聚物,非降解型乙烯乙酸乙烯酯(Lnger,et al.,Supra),可降解型乳酸—乙醇酸共聚物,例如Lupron DepotTM(可注射微球体,含有乳酸与乙醇酸的共聚物和亮氨基脯氨酸乙酯),和聚—D—(—)—3—羟基丁酸(EP133,98)。如乙烯乙酸乙烯酯和乳酸—乙醇酸这些聚合物能持释放分子100多天,而某些水凝胶释放多肽时间较短。当包埋多肽在体内停留一段相当长的时间时,可因暴露于37℃的湿气发生变性或凝聚,结果导致生物活性的丧失和可能造成致免疫性的改变。合理的策略是根据所涉及的机制采用不同的多肽稳定化方法。例如:如果发现凝聚的机制是硫—二硫化合物相互转化过程中分子内二硫键的形成,可通过对巯基残基的修饰,从酸性溶液中冷冻干燥,控制水份含量,使用合适的添加剂,研制特殊的聚合物骨架制剂达到稳定化的作用。
缓释配位体类似物或抗体制剂也包含了脂质包裹的多肽化合物。含有多肽的脂质体的制备有以下方法:DE 3,218,121;Epstein,et al.,Proc.Natl.Aced.Sci.USA.77:4030—4034(1980);EP52,322;EP36,676;EP88,046;EP143,949;EP142,641;日本专利公报83—118008;U.S.Pat.4,485,045号和U.S.Pat.4,544,545号;和EP102,324。通常,这些脂质体为小单层脂质体(约200~800埃),其中脂为含量大于30mol%胆甾醇,选用的配比调至最佳配位体类似物治疗具有增加的循环时间的脂质体被公开在U.S.Pat.No.5,013,556。
实施例1
水性制剂的稳定化
将重组的人类生长激素(hGH)和重组的人类γ干扰素(hIFN—γ)与多种赋形剂混和以分析赋形剂对在有机溶剂,二氯甲烷中的稳定化作用。最佳配方一般是能获得最大的溶解多肽浓度及在二氯甲烷处理后能有最大的溶解多肽浓度及在二氯甲烷处理后能有最大的天然多肽的回收量的配方。hGH在每一种溶液中的最大溶解度是向溶液中连续加入用碳酸氢铵缓冲液冻干的hGH测得,溶解度限度规定为再加入多肽即出现沉淀的浓度。hIFN—γ的最大溶解度是向赋形剂浓溶液加入hIFH—γ的浓储备液(264mg/ml hIFN—γ,10mM琥珀酸钠,pH5)测得。在这种条件下任何制剂均不能观察到明显的hIFN—γ溶解度极限,但是储备液的长时间存放会因为pH值的提高而产生沉淀(最终的pH值为6)。两种多肽配方在二氯甲烷中稳定性的测定方法为:在1ml的二氯甲烷中加入100μl的多肽制剂。然后将混和物超声30分钟。然后,以50ml的不含赋形剂的缓冲液(hGH:50mMpH8的磷酸缓冲液;hIFN—γ:10mM的琥珀酸缓冲液,pH5)进行萃取。溶解多肽的回收量通过测定紫外吸收测得,单体多肽量通过大小排阻层析法估算。
分别测定了两种多肽与海藻糖,甘露糖醇,羧甲基纤维素(CMC),吐温—20表面活性剂,葡聚糖,明胶,以及聚乙二醇混和后的稳定性。过去对hGH的研究表明,含有等重量比的多肽与甘露糖醇的制剂能使多肽稳定不变性。并可获得多肽的最大溶解度,为200mg/ml(100mM的磷酸缓冲液,200mg/ml的甘露糖醇,pH8)。当溶液中海藻糖与多肽的重量比为1∶4和1∶3时,可得溶解多肽的最高浓度,分别为400mg/ml和300mg/ml。此外,当这些制剂中冻干的多肽经二氯甲烷处理后,可获得溶解单体hGH的完全回收。含有甘露糖醇或甘露糖醇与PEG的hGH制剂可获得相似的单体hGH的回收率,但防止变性所需的重量比(赋形剂/多肽)比海藻糖制剂的大(甘露糖醇:1∶1;甘露糖醇/PEG:1∶1或1∶2;海藻糖:1∶3或1∶4)(表1)。所以,海藻糖可在低浓度条件下提供较高的hGH溶解度及在二氯甲烷中的抗变性作用。不加赋形剂时,hGH的溶解度要低得多(约106mg/ml),而且多肽较易变性。
表I hGH水性制剂的二氯甲烷试验
a.所有溶液含10mM的NaPO4缓冲液,pH8。b.以278nm处紫外吸收测得的从二氯甲烷中萃取的多肽的量(占总量的分数)乘以由SEC—HPLC回收所得的单体的分数。多肽以最大溶解度时处理。c.最大溶解度定义为能溶于每一种缓冲液的纯hGH最大量。
制剂a | 回收的溶解单体b(mg/ml) | 最大溶解度c(mg/ml) |
100mg/ml PEG(3350MW) | 12.2 | 96.4 |
50mg/ml PEG | 34.7 | 89.6 |
10mg/ml PEG | 37.2 | 128.3 |
100mg/ml甘露糖醇 | 66.0 | 98.0 |
50mg/ml甘露糖醇 | 46.2 | 106 |
10mg/ml甘露糖醇 | 56.0 | 106 |
100mg/ml葡聚糖 | 34.6 | 112.6 |
50mg/ml葡聚糖 | 64.6 | 146.1 |
10mg/ml葡聚糖 | 38.4 | 167.1 |
2%CMC | 44.7 | 91.9 |
100mg/ml海藻糖 | 113.9 | 267.3 |
50mg/ml海藻糖 | 82.8 | 275 |
10mg/ml海藻糖 | 92.0 | 267.3 |
4mg/ml PEG(3350MW),90mq/ml甘露糖醇 | 102.6 | 243.7 |
10mg/ml PE(3350MW),90mg/ml甘露糖醇 | 104.0 | 184 |
20mg/ml PEG(3350MW),80mg/ml甘露糖醇 | 139.9 | 240 |
2%Gelatin | 21.9 | 70.5 |
100mg/ml PEG(1000MW) | 69.2 | 131.5 |
50mg/ml PEG | 84.3 | 246.5 |
10mg/ml PEG | 126.5 | 226.3 |
4mg/ml PEG(1000MW)96mg/ml海藻糖 | 122.3 | 230.3 |
10mg/ml PEG(1000MW)90mg/ml海藻糖 | 58.4 | 218.7 |
20mg/ml PEG(1000MW)80mg/ml海藻糖 | 75.3 | 207.5 |
无赋形剂 | 65.0 | 106.3 |
对hIFN—γ的研究显示,甘露糖醇与海藻糖都是最好的试验赋形剂。当甘露糖醇按重量比(赋形剂/多肽)1∶3使用时,经二氯甲烷处理后,溶液中溶解的二聚物量(由大小排阻层析法测定)与原料中的相同。但是,甘露糖醇制剂所得的总的溶解多肽的回收率低于60%。与之相反,具有1∶2.5重量比的海藻糖制剂其总的溶解多肽的回收率为80%,同时溶解的二聚物的分数与原料相同(由大小排阻层析法测定,即天然SEC—HPLC)。不加赋形剂的多肽制剂经二氯甲烷处理后,保留原溶解的二聚物的10%(由天然SEC—HPLC测得),总的溶解多肽回收率低于60%。如果在0.2m的NaPO4/0.1%SDS,pH6.8的环境中进行大小排阻层析阻测定(表示为SDSSEC—HPLC),二氯甲烷处理前后,所有制剂中的单体含量大于99%。
所有含自Tween20表面活性剂的制剂,经二氯甲烷处理后,观察到两种多肽的单体多肽回收率都显著降低。虽然,未对其它表面活性剂进行研究,但很可能,诸如Tween20表面性剂的疏水分子能稳定变性多肽,而诸如甘露糖醇和海藻糖的糖类则能稳定天然多肽。
表II hIFN—γ水性制剂的二氯甲烷试验
a.所有含有赋形剂的溶液含134mg/ml的hIFN—γ,10mM的琥珀酸钠,pH5“无赋形剂”制剂含256.3mg/ml的蛋白质,10mM的琥珀酸钠,pH5。b.由二氯甲烷中萃取的多肽量(占总量的分数)由280nm处的紫外吸收测得。c.完整二聚体的量由SEC—HPLC自然方法测定。用SEC—HPLC SDS法分析时,所有制剂得到大于99%的单体。d.溶解二聚体的浓度根据回收的溶解多肽量与二聚体所占分数(自然SEC—HPLC法)测得。*.上述制剂中多肽浓度为62.8mg/ml。
制 剂a | %溶解的多肽回收百分率b | %完整二聚物c | 溶解的二聚体d |
0.01%Tween20* | 36.1 | 49.0 | 11.1 |
0.01%Tween20*62mg/ml甘露糖醇 | 59.0 | 69.0 | 25.6 |
5mg/ml甘露糖醇 | 58.3 | 72.7 | 56.8 |
50mg/ml甘露糖醇 | 62.9 | 83.4 | 70.3 |
5mg/ml海藻糖 | 117 | 34.2 | 53.6 |
50mg/ml海藻糖 | 75.6 | 61.3 | 62.1 |
1%CMC | 78.2 | 62.5 | 65.5 |
赋形剂 | 51.6 | 6.0 | 7.9 |
实施例II
包埋用多肽开品与水性制剂的稳定化
在研制重组型人生长激素的长效制剂的过程中,研究了用于hGH缓释的可生物降解型聚合物骨架的应用。本应用实例中所使用的聚合物为乳酸和乙醇酸的共聚物,通常称作聚(乳酸/乙醇酸)或是PLGA。欲将hGH混合于这种聚合物,PLGA必需溶解于与水不互溶的溶剂中。最常用的溶剂为二氯甲烷,它可溶解PLGA且不与水互溶。
通常,将多肽加入含有PLGA的二氯甲烷溶液以制备hGH—PLGA微球。在最初的研究中,多肽以研磨的冻干粉加入。多肽加入之后,将二氯甲烷溶液短暂均化后再加乳化浴中。这一过程的结果是二氯甲烷的提出,同时形成含有hGH的PLGA微囊。然后通过研究多肽从微球中的释放过程以测定多肽结合入微球的完整性。用大小排阻层析法(SEC—HPLC)及其它技术检测hGH的释放。排阻层析的结果显示,多肽从PLGA微球中的释放形式为:原单体,聚集体,以及介于单体与二聚体之间洗出的一种未知结构。对这种未知结构的广泛研究显示这是一种hGH的构象变体。此外,用二氯甲烷处理hGH后可得到相同的聚集体与构象变体。因此,在该过程中使用二氯甲烷可导致hGH的变性与聚集。
一个成功的长效制剂要求hGH以原型单体由PLGA微球体中释放。在以前的研究中,人们研究了几种有机溶剂以替代二氯甲烷。研究表明,hGH易被好几种有机溶剂破坏。由于二氯甲烷具有制备PLGA微球所要求的溶剂性质(即水不互溶性,PLGA的溶解等)而其它有机溶剂并不显著改善hGH的稳定性,二氯甲烷仍被选作形成PLGA微球体的溶剂。用于溶剂研究和合成过程中的多肽均在pH7的碳酸氢铵缓冲液中配制的冷冻干燥。所以,本研究的目的是开发能在制备PLGA微球体过程中稳定hGH的制剂处方。A.方法
1.hGH制剂的配制
为开发二氯甲烷稳定的处方,将在碳酸氢铵缓冲液中冻干的多肽用适宜缓冲液重新溶解。不溶的多肽用13,000rpm离心1分钟除去。
在下述的每一冷冻干燥过程中,hGH的浓度为10mg/ml。没有测定这些处方的残留湿度,采用的冷冻周期均相同。
使用加压冲击研磨机将冻干蛋白质研磨成细颗粒的hGH。
2.hGH制剂的二氯甲烷试验。
向二氯甲烷溶液中加入hGH以测定其对hGH稳定性的影响。对固体hGH而言,多肽量(mg)对有机溶剂体积(ml)的加量比为40mg/ml。对水性hGH而言,将10ml的hGH缓冲液加到1ml的二氯甲烷中以分析每个缓冲系统对二氯甲烷中hGH多肽稳定化的影响。加入多肽后,样品于47KHz超声溶器中,(Cole.Parmer.,Mod-el 08849—00)振荡30秒以模拟微球制备过程中的均化步骤。如果处方能在此试验中稳定hGH不变性,则进一步在二氯甲烷中均化进行分析。超声或均化以后,用5mM,pH8的NaHPO4将多肽稀释50倍,从二氯甲烷中萃取出多肽。通过多肽的浓度测定(278nm处紫外吸收)和大小排阻层析法(SEC—HPLC)测定这一步萃取出的多肽的量与质。具有最大的单体多肽回收率且不形成构象变体及大于二聚体的聚集体的配方为优选配方。B.结果
1.hGH稳定化的赋形剂研究
对碳酸氢铵冻干hGH的早期研究研究了多肽的不同缓冲液不同pH条件的溶解性。由研究可知,hGH在5~10mM,pH8的磷酸缓冲液中,溶解度最大,稳定性最好。所以,后继实验均以hGH在这一缓冲液中进行。
首先试图通过在处方的缓冲液中加Tween 80表面活性剂以阻止hGH的聚集。如表III所示,水性制剂的二氯甲烷试验表明,低浓度的Tween表面活性剂(0.1%的Tween 80表面活性剂)与10mg/ml的甘露糖醇可获得良好的溶解单体多肽回收率。但,在这一实验中获得hGH最佳结果的是含有10mg/ml的甘露糖醇而不含Tween80表面活性剂的配方(5mM NaPO4,pH8)。缓冲液中较高浓度Tween表面活性剂导致聚集作用的增加与溶解多肽回收率的下降。表III中各配方对hGH的稳定化作用均优于碳酸氢铵冻干后研磨的多肽粉末。
表III hGH水性剂的二氯甲烷试验
溶解多肽占总的重量分数 | ||||||
制剂配方a | %多肽回收率b | %面积回收率c | %三聚体 | %二聚体 | %中间体 | %单体 |
1%Tween80 | 85.7 | 90.0 | 0.5 | 3.4 | 1.1 | 94.9 |
0.1%Tween80 | 70.9 | 98.3 | 2.0 | 3.6 | 1.8 | 92.6 |
1%Tween8010mg/ml甘露糖醇 | 65.0 | 97.8 | 3.3 | 3.4 | 3.4 | 90.0 |
0.1%Tween8010mg/ml甘露糖醇 | 70.9 | 98.3 | 0.0 | 2.2 | 0.0 | 97.8 |
10mg/ml PEG(3350MW) | 97.6 | 101.1 | 0.0 | 2.6 | 0.0 | 97.4 |
10mag/ml PEG10mg/ml甘露糖醇 | 76.4 | 97.7 | 1.7 | 2.8 | 1.6 | 93.9 |
5mM NaPO4pH8 | 55.3 | 99.4 | 0.0 | 3.2 | 0.0 | 96.8 |
5mM NaPO4,pH810mg/ml甘露糖醇 | 91.7 | 99.8 | 0.0 | 1.8 | 0.0 | 98.2 |
a.所有溶液均含5mM NaPO4缓冲液,pH8
b. 278nm处紫外吸收法测二氯甲烷中多肽的萃出量(占总量的分数)。
c.二氯甲烷处理后提取入缓冲液的多肽的SEC—HPLC测定结果。
固体hGH制剂的二氯甲烷试验情况如表IV所示。结果表明具有最佳稳定化效果的配方是5mM KPO4缓冲液,2.5mg/ml的海藻糖。
表IV固态rhGH制剂的二氯甲烷试验
a.所有样品干rhGH 10mg/ml及所示缓冲液与赋形剂冻干。b. 278nm处紫外吸收法测蛋白质从二氯甲烷中的萃出量。c.二氯甲烷处理后提取入缓冲液的蛋白质的SEC—HPIC测定结果。d.固体冻干制剂在二氯甲烷中25,000rpm,均化1分钟。进一步的研究以考察表面活性剂是否能稳定二氯甲烷与多肽的界面。因此,Tween表面活性剂加入二氯甲烷相,并与固体hGH混和(KPO4缓冲液,pH8)。如表V所示,于二氯甲烷相加入Tween表面活性没有改善固体hGH(KPO4缓冲液,pH8)的稳定性。此外,二氯甲烷相添加表面活性剂,Span80表面活性剂,也不能提高固相hGH(KPO4缓冲液pH8)的稳定性。继续以Tween加入二氯甲烷相,都不能获得较稳定的固态hGH制剂(甘露糖醇,KPO4缓冲液,pH8)。这些实验结果联同水性制剂的研究结果表明,由于Tween的表面活性剂促进聚集体的形成,降低二氯甲烷处理的hGH的溶解度,因而不宜在制剂中使用。
溶解蛋白总重量的分数 | ||||||
制剂配方a | %多肽回收率b | %面积回收率c | %三聚体 | %二聚体 | %中间体 | %单体 |
研磨固体 | ||||||
NH4CO3 | 44.5 | 85.4 | 7.5 | 5.9 | 7.3 | 79.2 |
5mM NaPO4,pH8 | 85.7 | 100. | 0.0 | 2.1 | 0.0 | 97.8 |
5mM NaPO4,pH810mg/ml甘露糖醇 | 87.6 | 100. | 0.0 | 3.0 | 0.0 | 97.0 |
均化固体d | ||||||
5mM KPO4,pH8,2.5mg/ml海藻糖 | 97.3 | 100. | 0.0 | 2.2 | 0.0 | 97.8 |
5mM NaPO4,pH8,10mg/ml甘露糖醇 | 96.8 | 100. | 0.0 | 2.0 | 0.0 | 98.0 |
0.3M琥珀酸钠盐,10mg/ml甘露糖醇pH7, | 94.3 | 100. | 0.0 | 4.2 | 0.0 | 95.8 |
表V二氯甲烷相中Tween表面活性剂对固态hGH稳定性的影响
a. 278nm处紫外吸收测定二氯甲烷中多肽的萃出量(占总量的分数)。b.二氯甲烷处理后缓冲液萃取多肽的SEC—HPLC结果。
溶解多肽占总的重量分数 | ||||||
MeCl2中Tween含量 | %多肽回收率a | %面积回收率b | %三聚体 | %二聚体 | %中间体 | %单体 |
0.01%Tween80 | 40.8 | 98.7 | 5.2 | 13.0 | 0.0 | 81.8 |
0.1%Tween80 | 40.8 | 102.9 | 8.0 | 14.0 | 0.0 | 77.9 |
1%Tween80 | 53.8 | 97.3 | 7.0 | 11.6 | 0.0 | 81.4 |
为了增加微球体中多肽的负载量,必须将赋形剂的含量减至最小。所以,缓冲液中(10mM NaHPO4,pH8)使用含10mg/ml hGH的低浓度甘露糖醇(2和5mg/ml)同时对此水溶液测定二氯甲烷处理后的多肽稳定性。上述甘露糖醇浓度所得溶解单体比10mg/ml甘露糖醇的分配方低20%。甘露糖醇浓度大幅下降的代价是释放多肽质量的降低。也尝试采用低浓度的其它赋形剂。于水性制剂中(10mg/ml hGH,10mM NaHPO4缓冲液,pH8)使用0.5,2,5mg/ml的羧甲纤维素(CMC)。0.5mg/ml的CMC所得的溶解单体分数与10mg/ml的甘露糖醇相同,但水相中多肽的回收率低于15%。还尝试了用CMC与甘露糖醇的等量混和物(各取1mg/ml和2.5mg/ml)以提供低浓度条件下的稳定性。每种赋形剂用2.5mg/ml的结果与10mg/ml的甘露糖醇制剂相当。所以,将含有0.5mg/ml CMC和2.5mg/ml等量的CMC与甘露糖的配方冻干以研究其在微囊化中的用途。
为评价水性形式使用的制剂,每种冻干物料均需重新溶解至最大溶解度,即再也无法溶解加入的多肽时的浓度。本实验中hGH最大浓度是于10mg/ml甘露糖醇冻干的制剂。该制剂成功地用5mMNaHPO4,pH8的缓冲液重新溶解至hGH浓度达200mg/ml(200mg/ml甘露糖醇,100mM的KPO4缓冲液)而没有多肽沉淀产生。无赋形剂的配方(KPO4,pH8)的hGH的溶解度次之,为165mg/ml。但,试图重新溶解含CMC与CMC/甘露糖醇的制剂至高多肽浓度没有成功。上述两种情况下,浓度大于100mg/ml时,制剂都形成糊状。CMC和CMC/甘露糖醇制剂形成的糊剂的二氯甲烷试验表明,与甘露糖醇的配方相比,其多肽回收率显著下降(少于75%),但溶解单体的分数大于95%。由于凝胶样制剂利于稳定其中的内水相,也尝试采用另一种增稠剂明胶。为了维持较低的赋形剂浓度的同时又能使最终制剂为凝胶(200mg/ml hGH),以0.5mg/ml的明胶,10mg/ml的hGH,10mM KPO4缓冲液,pH8试验明胶配方。该制剂的二氯甲烷试验所得溶解单体的回收率与10mg/ml的甘露糖醇制剂相当。因此,该制试也被冷冻干燥后进行进一步分析。该含200mg/ml hGH(10mg/ml明胶,100mM KPO4,pH8)的冻干多肽的重新溶解形成性质类似于具有相同hGH浓度的CMC/甘露糖醇和CMC制剂的糊剂。
实施例III
rhGH制剂在乙酸乙酯中的稳定性
蛋白质在可生物降解型聚合物中的微囊化常需用有机溶剂以溶解聚合物。聚合物,典型的为PLGA,聚交酯(PLA)或聚乙二醇(PGA),首先溶解于不完全与水混溶的有机溶剂。此过程中的常用有机溶剂是二氯甲烷和乙酸乙酯。这两种溶剂具有非常不同的物理与化学性质,因而有必要分别检测rhGH制剂在两种溶剂中的稳定性。
rhGH在乙酸乙酯中稳定性的试验方法与上述实例中检验对二氯甲烷中的研究中所用的方法类似。将冻干的固体rhGH(碳酸氢铵制剂)加入各配方中制得10mg/ml的rhGH溶液。如表VI所示,制剂用pH8的5mM的KPO4缓冲液配制,其中含不同的赋形剂,PEG(3350MW),甘露糖醇,海藻糖和Tween20,或混合赋形剂。将每一rhGH制剂(100μl)加到1ml的乙酸乙酯中,超声30秒以形成乳剂。将此乳浊液与10ml pH8,5mM的KPO4缓冲液混和得到稀释100倍的rhGH稀释液。rhGH被缓冲萃取后由大小排阻HPLC进行分析。几种制剂的溶解蛋白的回收率超过100%表明,加到乳浊液中的蛋白质的量要比由精确测定体积所得的估计量(0.1ml×10mg/ml=1mg)大。此外,溶解蛋白回收率与单体的回收量普遍比用二氯甲烷处理的相同处方的rhGH要高。总之,溶解蛋白回收率大于含海藻糖(1或2mg/ml),海藻糖与PEG(各含10mg/ml),甘露糖醇与PEG(各含10mg/ml)和甘露糖醇与Tween20(各含10mg/ml)的制剂。但,只含海藻糖(1或2mg/ml)和甘露糖醇与Tween20(各含10mg/ml)的制剂也有高的单体含量(大于97%)。含甘露糖醇/Tween20的制剂经两次乳化微囊化过程不能得到合适的溶解度,而且它要求赋形剂与蛋白质的重量比达4∶1。所以,本实验中的最优制剂配方为含1mg/ml海藻糖的配方(赋形剂与蛋白质的重量比为1∶10,且具有较高的rhGH溶解度)。
表VI rhGH水性制剂的乙酸乙酯试验
a.所有开始测试溶液均含10mg/ml的rhGH和pH8的5mM的KPO4缓冲液。三个rhGH浓度低于10mg/ml的配方除外(不加赋形剂:9.39mg/ml;10mg/ml甘露糖醇/10mg/ml PEG:7.84mg/ml;10mg/ml的甘露糖醇:9.71mg/ml)。b.乙酸乙酯处理后经缓冲液萃取的蛋白质的SEC—HPLC测定结果。溶解蛋白质回收百分率的定义为:相应浓度的样品和对照品的峰面积所得的浓度之比乘100%。对照品rhGH浓度由275nm处紫外吸收法测定,样品rhGH浓度根据在全过程中所用稀释液按保存品的100倍稀释液计算(1ml EtAC中含0.1ml保存品,再加入到10ml的缓冲液中)。
制剂配方a | %溶解蛋白回收率b | 溶解蛋白质占总的重量分数 | ||
%大聚集体 | %二聚物 | %单体 | ||
无赋形剂 | 98.9 | 2.3 | 3.2 | 94.5 |
10mg/mL PEG(3350MW) | 99.8 | 2.7 | 2.3 | 94.9 |
5mg/mL PEG | 108.5 | 1.7 | 3.0 | 95.2 |
2mg/mL PEG | 107.2 | 1.8 | 3.8 | 94.3 |
10mg/mL甘露糖醇 | 96.6 | 1.7 | 3.6 | 94.7 |
2mg/mL甘露糖醇 | 86.3 | 4.1 | 3.8 | 92.2 |
10mg/mL海藻糖 | 100.1 | 1.8 | 4.5 | 93.7 |
2mg/mL海藻糖 | 119.8 | 0.4 | 2.0 | 97.7 |
1mg/mL海藻糖 | 111.1 | 0.6 | 2.3 | 97.1 |
10mg/mL PEG(3350MW)10mg/mL海藻糖 | 115.6 | 3.8 | 2.9 | 93.3 |
2mg/mL PEG(3350MW)2mg/mL海藻糖 | 93.0 | 0.8 | 3.1 | 96.1 |
1mg/mL PEG(3350MW)1mg/mL海藻糖 | 95.8 | 4.5 | 3.3 | 92.2 |
10mg/mL PEG(3350MW)10mg/mL甘露糖醇 | 116.3 | 1.2 | 2.5 | 96.3 |
2mg/mL PEG(3350MW)2mg/mL甘露糖醇 | 106.5 | 1.7 | 2.7 | 95.6 |
0.1%Tween2010mg/mL甘露糖醇 | 122.8 | 0.8 | 1.6 | 97.6 |
实施例IV
喷雾干燥rhGH制剂在有机溶剂中的稳定性
使用复乳化的微囊化技术(水包油包水)的终产物只能装载中等量的药物。药物装载量受药物在水中的溶解度和能加入有机溶剂中的聚合物的水性药物体积的限制。每克聚合物大于0.5ml药物的装入体积常典型地导至释放过程开始时药物从囊中喷出速释。为克服这一困难,可以用固体药物制剂代替其水溶液。这样,利用一个水包油包固体的过程可用于制备具有高的药品装载量(大于10%)和低至中等起始喷出速释效应的微球体。
用于微囊化的固体制剂必须在有机溶剂中稳定而且具有相对微球体(30—100μm)较小的体积(1—5μm),这样才能获得较高的装载量和较低的喷出速释效应。对于蛋白类制剂,获取干而细小的固体的一个方法是喷雾干燥。最近,Mummenthaler等在《Parm Re.》11(1):12—20,1994上报导了喷雾干燥rhGH制剂的制备过程。由于rhGH容易因相界面作用,例如气—液界面,而变性,在rhGH的喷雾干燥必须用rhGH制剂中的表面活性剂进行。但是,如前所述,有些表面活性剂的存在会降低rhGH在二氯甲烷中的稳定性。对于在前述中已被证实对水性rhGH制剂具有最佳稳定化效果的含有不同表面活性剂与海藻糖的喷雾干燥rhGH制剂分别进行在二氯甲烷和乙酸乙酯中的稳定性试验。
喷雾干燥rhGH制剂按表VII所列配方制备。喷射时,溶液的进口温度90℃,流速5ml/min,喷嘴处气速600L/hr,干燥气速36,000L/hr。从喷雾干燥器的过滤器和旋风收集器中收集干燥的rhGH。产品颗粒直径约为5μm。
经喷雾干燥的rhGH粉末分别以二氯甲烷或乙酸乙酯处理后试验其稳定性。将相当于10mg rhGH的干粉加入装在3cc玻璃试管中的2ml有机溶剂。混悬液经微细均化刀片,10,000rpm,均化30秒。混合后将20μl的均化混悬液加入980ml的pH8的5mM的KPO4缓冲液中以抽提蛋白质。提出的蛋白浓度由278nm处紫外吸收法测定,样品同时接受大小排阻层析分析。如表VII所示,当用二氯甲烷处理时,不含表面活性剂制剂的聚集程度最大。这可能是前述喷雾干燥rhGH所观察到的干燥过程中rhGH表面变性的结果。无论加入Tween20或是PEG(3350MW)于制剂中,二氯甲烷处理样品中的集聚都减少,但总回收率仍很低而且单体含量远低于90%。与此相反,如果含表面活性剂的相同的喷雾干燥制剂用乙酸乙酯处理,其聚集量可忽略不计,而且单体rhGH回收完全。因此,含有海藻糖和Tween20或PEG(3350)的rhGH冻干制剂在乙酸乙酯中稳定,但不能防止蛋白质在二氯甲烷中变性。
表VII
喷雾干燥的rhGH固态制剂在二氯甲烷与乙酸乙酯中的稳定性
a.蛋白总回收率定义为有机溶剂处理后缓冲液提取的蛋白量除以计算加入缓冲液的蛋白量(0.02ml×5mg/ml)。b.有机溶剂处理后由缓冲液萃取的蛋白质的SEC—HPLC分析结果。溶解蛋白的回收百分率定义为以样品和参照标准品的总峰面积所得浓度之比乘100%在288nm处紫外吸收法测样品与对照的rhGH浓度。
溶解蛋白 | |||||
制剂配方 | 总回收率% | 溶解蛋白回收率% | 大聚集体% | 三聚体% | 单体% |
二氯甲烷试验 | |||||
15mg/mL rhGH3.75mg/mL海藻糖 | -- | -- | 12.3 | 8.8 | 75.4 |
10mg/mL rhGH2.5mg/mL海藻糖0.2%Tween20 | 56.5 | 65.2 | 1.9 | 13.3 | 78.1 |
10mg/mL rhGH2.5mg/mL海藻糖0.2%PEG(3350MW) | 50.7 | 56.7 | 3.9 | 12.3 | 77.7 |
乙酸乙酯试验 | |||||
10mg/mL rhGH2.5mg/mL海藻糖0.2%Tween20 | 111.7 | 126.8 | 0.9 | 0.0 | 99.2 |
10mg/mL rhGH2.5mg/mL海藻糖0.2%PEG(3350MW) | 114.3 | 125.5 | 1.1 | 0.0 | 98.9 |
5.0mg/mL rhGH1.25mg/mL海藻糖0.2%Tween20 | 106.8 | 110.4 | 0.3 | 3.0 | 96.7 |
实施例V喷雾冷冻干燥rhGH制剂在二氯甲烷与乙酸乙酯中的稳定性
高温喷雾干燥会对蛋白质造成损伤并可能形成中空的蛋白质颗粒(Mummenthaler et al.,Pharm,Res.11(1):12—20(1994))。而且,要收集微囊化所需的细小颗粒(1—5μm)十分困难,而且其总收率通常很低(低于50%)。喷雾冷冻干燥可以替代高温喷雾干燥。经冷冻干燥的rhGH制剂可形成细小颗粒(2—3μm),这些颗粒又容易破碎成更小的颗粒(小于1μm)。这类固态制剂很适宜于聚合物骨架微囊化,因为它可以提高均化分散固体蛋白(因为是微粒混悬液可降低喷出速释效应)的装载量(在30~100μm的微球体中可包入更多的固体)。
按表VII所列配方进行喷雾冷冻干燥。同样,为保证喷雾过程中rhGH的稳定性需加入表面活性剂,但那些不易因界面作用而变性的蛋白质可不加表面活性剂。如同高温喷雾干燥(Mummenthaler eta1.,Pham.Res.11(1):12—20 1994)一样,喷雾冷冻干燥rhGH时,溶液流速5mL/min,操作气速600L/hr。溶液喷射到一装有液氮的敞口金属平皿中。喷雾后,将平皿放入-30℃预冷的冷冻干燥器中。液氮挥放,蛋白质冷冻干燥(初级干燥:-30℃,100mTorr,52hrs;二级干燥:5C,100MTorr,18hr)。将最终得到的粉末装入密闭玻璃瓶备用。
然后用二氯甲烷和乙酸乙酯处理对喷雾冷冻干燥rhGH粉末进行稳定性试验。将相当于10mg rhGH量的喷雾冷冻干燥粉末加入装在3cc玻璃试管中的2ml有机溶剂中。用微细均化刀片以10,000rpm转速,均化混悬液30秒。然后将20μl的均化混悬液加入980μl pH8的5mM的KPO4缓冲液中以抽提蛋白质。在278nm处紫外吸收测萃取液中的蛋白浓度,同时还用大小排阻层析法分析样品。如表VII所示,与前述水性制剂所观察到的结果相同,在二氯甲烷中,含PEG的喷雾冷冻干燥制剂化合Tween20的更稳定。但,两种制剂的单体rhGH回收率都不高。若用乙酸乙酯处理这两种制剂均可获得单体蛋白的完全回收。制剂中的海藻糖可防止有机溶剂(乙酸乙酯)的变性作用,而表面活性剂则可防止喷雾冷冻干燥过程中蛋白质的表面变性。因此,同时含有海藻糖和表面活性剂的喷雾冷冻干燥制剂可获取从乙酸乙酯中的完全回收率。
表VIII
喷雾冷冻干燥固体rhGH制剂在
二氯甲烷和乙酸乙酯中的稳定性
a.蛋白质总回收率定义为有机溶剂处理后由缓冲液萃取的蛋白量除以计算所得的加入缓冲液中的蛋白量(0.02ml×5mg/ml)。b.有机溶剂处理后由缓冲液萃取的蛋白质的SEC—HPLC分析结果。溶解蛋白的回收百分率定义为以样品和参照标准品的峰面积所得浓度之比乘100%在278nm处紫外吸收法测样品与参照的浓度。
溶解蛋白 | |||||
制剂配方 | 总回收率% | 溶解蛋白回收率% | 大聚集体% | 二聚体% | 单体% |
二氯甲烷试验 | |||||
5mg/mL rhGH1.25mg/mL海藻糖0.2%Tween20 | 37.2 | 34.0 | 6.2 | 8.3 | 85.5 |
5mg/mL rhGH1.25mg/mL海藻糖0.2%PEG(3350MW) | 68.8 | 66.8 | 2.3 | 15.8 | 78.8 |
乙酸乙酯试验 | |||||
5mg/mL rhGH1.25mg/mL海藻糖0.2%Tween20 | 94.6 | 117.7 | 0.5 | 0.9 | 98.7 |
5mg/mL rhGH1.25mg/mL海藻糖0.2%PEG(3350MW) | 97.7 | 104.7 | 0.6 | 0.0 | 99.4 |
Claims (32)
1.一种抗有机溶剂变性作用的多肽稳定化方法,此方法包括多肽与多元醇混合,其中多元醇的分子量小于70,000KD,以形成混合物;此混合物再用有机溶剂处理。
2.按照权利要求1所述的方法,其特征在于此多肽为生长激素。
3.按照权利要求2所述的方法,其特征在于此生长激素为人生长激素。
4.按照权利要求1所述的方法,其特征在于此多肽为γ干扰素。
5.按照权利要求1所述的方法,其特征在于有机溶剂为二氯甲烷。
6.按照权利要求1所述的方法,其特征在于有机溶剂为乙酸乙酯。
7.按照权利要求1所述的方法,其特征在于多元醇为海藻糖。
8.按照权利要求1所述的方法,其特征在于多元醇为甘露糖醇。
9.按照权利要求1所述的方法,其特征在于多元醇为聚乙二醇。
10.按照权利要求1所述的方法,其特征在于多肽是干燥制品。
11.按照权利要求10所述的方法,其特征在于多肽为冷冻干燥制品。
12.按照权利要求7所述的方法,其特征在于海藻糖与多肽的重量比范围为100∶1到1∶100。
13.按照权利要求7所述的方法,其特征在于海藻糖与多肽的重量比范围为1∶1到1∶10。
14.按照权利要求7所述的方法,其特征在于海藻糖与多肽的重量比范围为1∶3到1∶4。
15.按照权利要求8所述的方法,其特征在于甘露糖醇与多肽的重量比为100∶1到1∶100。
16.按照权利要求8所述的方法,其特征在于甘露糖醇与多肽的重量比为1∶1到1∶10。
17.按照权利要求8所述的方法,其特征在于甘露糖醇与多肽的重量比为1∶1到1∶2。
18.一种配制多肽的方法包括以下步骤:
a)将多肽水溶液与分子量小于70,000KD的多元醇混合;并且
b)用有机溶剂处理该多肽水溶液
19.按照权利要求18所述的方法,其特征在于步骤a)所得产物是干的,用水性制剂重新溶解。
20.如权利要求18所述的方法,其特征在于还包括配制控释多肽制剂。
21.一种配制干燥的控释多肽制剂的方法包含以下步骤:
a)将多肽与一种赋形剂混合,其中所述赋形剂为分子量小于70,000KD的多元醇;
b)用有机溶剂处理步骤a)所得的产物。
22.按权利要求21所述的方法,其特征在于还包括将多肽包埋于聚合物骨架中。
23.一种按权利要求21所述方法制备的一种包埋组成制剂。
24.一种控释多肽组成制剂包括:一种掺有赋形剂的多肽,其中赋形剂为分子量小于70,000KD的多元醇,其中掺有赋形剂的多肽经有机溶剂处理后包埋于聚合物骨架中。
25.按权利要求24所述的组成制剂,其中掺有赋形剂的多肽在一种水性制剂中。
26.按权利要求24所述的组成制剂,其中掺有赋形剂的多肽为干品。
27.按权利要求24所述的组成制剂,其中掺有赋形剂的多肽为冷冻干燥制品。
28.按权利要求24所述的组成制剂,其中聚合物为聚交酯。
29.按权利要求24所述的组成制剂,还包括一种缓冲液。
30.按权利要求29所述的组成制剂,其中缓冲液为磷酸缓冲液。
31.按权利要求29所述的组成制剂,其中缓冲液为琥珀酸缓冲液。
32.按权利要求29所述的组成制剂,还包含一种防腐剂。
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- 1994-02-17 AT AT94909647T patent/ATE197550T1/de active
- 1994-02-17 US US08/256,187 patent/US5589167A/en not_active Expired - Lifetime
- 1994-02-17 CZ CZ0212795A patent/CZ296649B6/cs not_active IP Right Cessation
- 1994-02-17 NZ NZ262634A patent/NZ262634A/en not_active IP Right Cessation
- 1994-02-17 CN CN94191235A patent/CN1108823C/zh not_active Expired - Lifetime
- 1994-02-17 RU RU95118293A patent/RU2143889C1/ru active
- 1994-02-17 ES ES94909647T patent/ES2153418T3/es not_active Expired - Lifetime
- 1994-02-17 EP EP94909647A patent/EP0686045B1/en not_active Expired - Lifetime
- 1994-02-20 IL IL10871394A patent/IL108713A/en not_active IP Right Cessation
- 1994-02-23 ZA ZA941239A patent/ZA941239B/xx unknown
-
1995
- 1995-05-15 US US08/442,241 patent/US5753219A/en not_active Expired - Lifetime
-
1996
- 1996-09-24 US US08/719,196 patent/US5804557A/en not_active Expired - Lifetime
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2001
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100400031C (zh) * | 2000-11-29 | 2008-07-09 | 伊藤火腿株式会社 | 粉末制剂及其制备方法 |
CN100336557C (zh) * | 2005-05-11 | 2007-09-12 | 北京双鹭药业股份有限公司 | 一种生长抑素的水溶液制剂、其制备方法及应用 |
CN100337684C (zh) * | 2005-05-11 | 2007-09-19 | 北京双鹭药业股份有限公司 | 一种胸腺五肽的水溶液制剂、其制备方法及应用 |
CN100342909C (zh) * | 2005-05-11 | 2007-10-17 | 北京双鹭药业股份有限公司 | 一种胸腺素α1的水溶液制剂、其制备方法及应用 |
CN108169499A (zh) * | 2017-11-30 | 2018-06-15 | 成都斯马特科技有限公司 | 一种基于生化试剂盘进行凝血分析的方法 |
CN112739366A (zh) * | 2018-09-14 | 2021-04-30 | 卡拉治疗学股份有限公司 | κ阿片受体激动剂的口服配制品 |
Also Published As
Publication number | Publication date |
---|---|
CN1108823C (zh) | 2003-05-21 |
WO1994019020A1 (en) | 1994-09-01 |
IL108713A0 (en) | 1994-05-30 |
US5589167A (en) | 1996-12-31 |
NZ262634A (en) | 1997-02-24 |
RU2143889C1 (ru) | 2000-01-10 |
CZ212795A3 (en) | 1996-02-14 |
AU6241294A (en) | 1994-09-14 |
IL108713A (en) | 1999-12-22 |
CZ296649B6 (cs) | 2006-05-17 |
US5804557A (en) | 1998-09-08 |
DE69426292D1 (de) | 2000-12-21 |
US5753219A (en) | 1998-05-19 |
DK0686045T3 (da) | 2001-03-05 |
EP0686045A1 (en) | 1995-12-13 |
ATE197550T1 (de) | 2000-12-15 |
AU685784B2 (en) | 1998-01-29 |
ZA941239B (en) | 1995-08-23 |
EP0686045B1 (en) | 2000-11-15 |
GR3035383T3 (en) | 2001-05-31 |
DE69426292T2 (de) | 2001-05-17 |
PT686045E (pt) | 2001-04-30 |
JP3698721B2 (ja) | 2005-09-21 |
JPH08507064A (ja) | 1996-07-30 |
ES2153418T3 (es) | 2001-03-01 |
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