CN1281369A - 胰高血糖素样肽-1晶体 - Google Patents
胰高血糖素样肽-1晶体 Download PDFInfo
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- CN1281369A CN1281369A CN98812093A CN98812093A CN1281369A CN 1281369 A CN1281369 A CN 1281369A CN 98812093 A CN98812093 A CN 98812093A CN 98812093 A CN98812093 A CN 98812093A CN 1281369 A CN1281369 A CN 1281369A
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
本发明提供单一的四方平棒状或片状晶体型胰高血糖素样肽-1相关的分子、其制备方法、组合物及应用方法。所述晶体制剂显示体内作用时间延长,可用于治疗糖尿病、肥胖症及相关病症。
Description
发明领域
因为本发明用于药物研究和开发,所以涉及肽化学。本发明提供单一的四方平棒状或片状晶体型胰高血糖素样肽-1相关的分子、其制备方法、组合物及这些改进的晶形的用途。
发明背景
GLP-1于1987首次鉴定为肠降血糖素激素,它是由160个氨基酸的前体蛋白前胰高血糖素原的蛋白酶解而天然形成的37个氨基酸的肽。GLP-1由应答食物摄食的肠L细胞分泌,已经发现GLP-1刺激胰岛素分泌(促胰岛作用),引起细胞摄入葡萄糖,使血清葡萄糖水平降低(参见例如Mojsov,S.,Int.J.Peptide Protein Research,40:333-343(1992))。GLP-1活性低。随后在第6与7位置之间内源切割产生生物活性更强的GLP-1(7-37)OH肽。约80%如此产生的GLP-1(7-37)OH在C末端酰胺化并在L细胞中去除末端甘氨酸残基,并常规称为GLP-1(7-36)NH2。与其相当同源或衍生自或基于这些天然型GLP-1的分子在本说明书中一般称为GLP。
游离酸型、酰胺型GLP和许多为数众多的已知GLP的生物效应和代谢更新相似,有希望作为治疗糖尿病、肥胖症和相关病症的药物。然而,许多GLP的缺点是其生物半衰期非常短,部分短至3-5分钟,这使其作为药物的应用未引起注意。目前,认为二肽酶Ⅳ(DPP-Ⅳ)的活性容易使许多GLP失活,并是造成所观察到的非常短的血清半衰期的部分原因。胃肠外给予后快速吸收和清除也是其原因。因此需要找到延长这些有希望药物的作用的方法。
一种方法是修饰这类分子以保护其不被体内的DPP-Ⅳ酶切。例如,参见美国专利第5,512,549号。在胰岛素领域,早己经知道可以通过将晶体蛋白制剂给到皮下组织(subcutis)可以达到延长作用时间,皮下组织像仓库一样长时间释放出可溶性蛋白。
已经由盐溶液产生GLP-1(7-37)OH的不均一微晶簇,在用锌和/或间甲苯酚晶体浸泡处理后进行检测(Kim和Haren,Pharma.Res.第12卷第11期(1995)。此外,已经由含锌或鱼精蛋白的磷酸盐溶液制备含针状晶体和非晶型沉淀的GLP(7-36)NH2粗晶体悬液(Pridal等,International Journal of Pharmaceutics第136卷,第53-59页(1996))。此外,EP 0 619 322 A2描述了通过混合pH7-8.5缓冲液中的蛋白液和一定的盐和低分子量聚乙二醇(PEG)的组合物制备微晶形GLP-1(7-37)OH。但是,这类晶体簇和粗悬浮液用于制备长效GLP药用制剂欠理想,因为它们是松散结合的趋向捕获杂质的不均一晶体簇或非晶态晶态悬浮液并难以重复性生产和给予。
最意外的是,发现各种GLP的四方平棒状或片状单晶可以由母液在宽范围pH条件下重复形成,所述母液含有溶解于缓冲液的GLP和一种C1-3醇或可任选的一种单糖或双糖。所产生的平棒状或片状单晶优于或明显优于本领域已知的GLP-1(7-37)OH晶体簇或粗悬浮液。
本发明的四方平棒状或片状单品较所述已知不均一晶体簇不易捕获杂质,因此可以以更高收率生产和更多次重复性给药。本发明的晶体组合物在药学上是有吸引力的,因为它们较晶体簇或非晶态晶态悬浮液较长时间相对不均一并保持悬浮态,而所述晶体簇或非晶态晶态悬浮液趋于快速沉降、聚集或一起结成块、堵塞注射器针头和一般增加不可预期的给药。最重要的是,本发明的晶体组合物显示延长的、一致的和可重复的药代力学,所述药代动力学通过加入锌、采用常规晶体浸泡处理技术或者将锌含于结晶液中可以加以调节。
发明概述
本发明包括制备胰高血糖素样肽-1相关分子(GLP)的棒状或片状单晶的方法,包括制备含纯化GLP、含一种醇或单糖或双糖的缓冲剂和任选的硫酸铵或锌的结晶液。在另一实施方案中,要求保护具有选自GLP-1类似物、GLP-1衍生物、DPP-Ⅳ保护的GLP、GLP-1肽类似物、生物合成的GLP-1类似物的四方平棒状或片状形态的GLP晶体。本发明还包括大致均一的GLP晶体组合物、药物制剂和制备这类制剂的方法、以及治疗糖尿病、肥胖症和相关病症的方法。
发明详述
按照本领域习惯,GLP-1(7-37)OH的氨基末端已经指定为7号残基及羧基末端为37号残基。该命名法延用到其它GLP。当未具体说明时,通常认为C末端是传统羧基型。GLP-1(7-37)OH的氨基酸序列和制备是本领域众所周知的。参见美国专利第5,120,712号,该专利内容通过引用结合到本文中。为了方便读者以下提供所述序列。His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-COOH (SEQ ID NO:1)
“GLP-1类似物”定义为相对于GLP-1(7-37)具有一个或多个氨基酸置换、缺失、倒位或插入的分子,并可包括d-氨基酸型。许多GLP-1类似物是本领域已知的,包括但不限于GLP-1(7-34)、GLP-1(7-35)、GLP-1(7-36)NH2、Gln9-GLP-1(7-37)、d-Gln9-GLP-1(7-37)、Thr16-Lys18-GLP-1(7-37)、和Lys18-GLP-l(7-37)、Gly8-GLP-1(7-36)NH2、Gly8-GLP-1(7-37)OH、Val8-GLP-1(7-37)OH、Met8-GLP-1(7-37)OH、乙酰-Lys9-GLP-1(7-37)、Thr9-GLP-1(7-37)、D-Thr9-GLP-1(7-37)、Asn9-GLP-1(7-37)、D-Asn9-GLP-1(7-37)、Ser22-Arg23-Arg24-Gln26-GLP-1(7-37)、Arg23-GLP-1(7-37)、Arg24-GLP-1(7-37)、α-甲基-Ala8-GLP-1(7-36)NH2和Gln8-Gln21-GLP-1(7-37)OH等。
符合本发明的其它GLP-1类似物用下式描述。R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2 (SEQ ID NO:2)其中:R1选自L-组氨酸、D-组氨酸、去氨基组氨酸、2-氨基组氨酸、β-羟基组氨酸、高组氨酸、α-氟甲基组氨酸和α-甲基组氨酸;X选自Ala、Gly、Val、Thr、Met、Ile和α-甲基-Ala;Y选自Glu、Gln、Ala、Thr、Ser和Gly;Z选自Glu、Gln、Ala、Thr、Ser和Gly;而R2选自NH2和Gly-OH。
GLP-1类似物在WO 91/11457中也有描述,包括具有至少一种选自以下修饰的GLP-1(7-34)、GLP-1(7-35)、GLP-1(7-36)或GLP-1(7-37)、或其酰胺型、以及其药学上可接受的盐:
(a)甘氨酸、丝氨酸、半胱氨酸、苏氨酸、天冬酰胺、谷氨酰胺、酪氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、蛋氨酸、苯丙氨酸、精氨酸或D-赖氨酸取代26位和/或34位的赖氨酸;或甘氨酸、丝氨酸、半胱氨酸、苏氨酸、天冬酰胺、谷氨酰胺、酪氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、蛋氨酸、笨丙氨酸、赖氨酸或D-精氨酸取代36位的精氨酸;
(b)抗氧化氨基酸取代3l位的色氨酸;
(c)至少一种以下取代:酪氨酸取代16位的缬氨酸;赖氨酸取代18位的丝氨酸;天冬氨酸取代21位的谷氨酸;丝氨酸取代22位的甘氨酸;精氨酸取代23位谷氨酰胺;精氨酸取代24位的丙氨酸;和谷氨酰胺取代26位的赖氨酸;和
(d)至少一种以下取代:甘氨酸、丝氨酸或半胱氨酸取代8位的丙氨酸;天冬氨酸、甘氨酸、丝氨酸、半胱氨酸、苏氨酸、天冬酰胺、谷氨酰胺、酪氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、蛋氨酸或苯丙氨酸取代9位的谷氨酸;丝氨酸、半胱氨酸、苏氨酸、天冬酰胺、谷氨酰胺、酪氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、蛋氨酸或苯丙氨酸取代10位的甘氨酸;谷氨酸取代15位的天冬氨酸;和
(e)甘氨酸、丝氨酸、半胱氨酸、苏氨酸、天冬酰胺、谷氨酰胺、酪氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、蛋氨酸、或苯丙氨酸、或D-或N-乙酰化或烷基化形成的组氨酸取代7位的组氨酸;其中在取代是(a)、(b)、(d)和(e)的情况下,被取代的氨基酸可任选地是D-型氨基酸,而7位被取代的氨基酸可任选地是N-乙酰化或N-烷基化氨基酸。
“GLP-1衍生物”定义为具有GLP-1(7-37)或GLP-1类似物的氨基酸序列的分子,但是另外还具有其一个或多个以下部位的化学修饰:氨基酸侧基、α-碳原子、末端氨基或末端羧酸基团。化学修饰包括但不限于加入化学部分、产生新键和去除化学部分。氨基酸侧基的修饰包括但不限于赖氨酸ε-氨基的酰化;精氨酸、组氨酸或赖氨酸的N-烷基化;谷氨酸或天冬氨酸羧酸基团的烷基化;和谷氨酸或天冬氨酸的脱酰胺作用。所述末端氨基的修饰包括但不限于脱氨基、N-低级烷基、N-二-低级烷基和N-酰化修饰。所述末端羧基的修饰包括但不限于酰胺、低级烷基酰胺、二烷基酰胺和低级烷基酯修饰。低级烷基是C1-C4烷基。此外,可以用一般熟练的蛋白化学家已知的保护基团保护一种或多种侧基或末端基团。氨基酸的α-碳原子可以单甲基化或二甲基化。
在美国专利第5,188,666号中要求保护其它GLP-1衍生物,该专利特别通过引用结合到本文。这类分子选自具有以下氨基酸序列的肽:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X (SEQ ID NO:3)和其药学上可接受的盐,其中X选自Lys-COOH和Lys-Gly-COOH;和所述肽的衍生物,其中所述肽选自:所述肽的药学上可接受低级烷基酯;和选自酰胺、低级烷基酰胺和低级二烷基酰胺的所述肽的药学上可接受的酰胺。
另一类符合用于本发明的GLP-1衍生物包括在美国专利第5,512,549号中要求保护的化合物,所述专利特别通过引用结合到本文中,所述衍生物用下式描述:R1-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala-Trp-Leu-(SEQ ID NO:4)其中R1选自4-咪唑丙酰基、4-咪唑乙酰基或4-咪唑-α,α-二甲基-乙酰基;R2选自C6-C10非分支的酰基或不存在;R3选自Gly-OH或NH2;而Xaa是Lys或Arg,它们可以用于本发明。
“DPP-Ⅳ保护的GLP”是指抗DPP-Ⅳ作用的GLP-1类似物。这些类似物包括在8位具有一个修饰的或d氨基酸残基的类似物。这些类似物还包括在8位具有Gly或l氨基酸残基Val、Thr、Met、Ser、Cys或Asp的生物合成GLP-1类似物。其它DPP-Ⅳ保护的GLP包括脱氨基His7衍生物。
“GLP-1肽类似物”定义为不包括酰化型的GLP-1类似物或衍生物。
“生物合成的GLP-1类似物”定义为仅含有天然氨基酸的任何GLP-1类似物或衍生物,因此能够由包括重组细胞和重组生物的活细胞表达。
“治疗”定义为克制疾病、病症或紊乱对病人进行的处理和护理,包括给予本发明化合物以防止所述症状或并发症的发生、缓解所述症状或并发症,或消除所述疾病、病症或紊乱。因此,治疗糖尿病包括在需要其治疗的病人中维持生理需要的血糖水平。
用所述要求保护的方法制备的本发明的平棒状或片状GLP晶体在某种程度上其大小和形状可以变化。一般而言,其大小范围约2-25μm×10-150μm,并且是厚约0.5-5μm的平面晶体。这些单晶由单个成核点形成,并不以本领域已知的多尖峰的星状晶体簇出现。
根据本发明公开的序列信息和固相蛋白合成技术水平,可以通过化学合成获得GLP。然而,用本领域技术人员熟悉的技术,通过酶切使胰高血糖素原片段化可以获得某些GLP。此外,众所周知的重组DNA技术可以用来表达符合本发明的GLP。
多肽的固相化学合成的原理是本领域众所周知的,可以在本领域的一般教科书中找到,例如Dugas,H和Penney,C.,BioorganicChemistry(1981)Springer-Verlag,New York,第54-92页,Merrield,J.M,Chem.Soc.,85:2149(1962),和Stewart和Young,Solid PhasePeptide Synthesis,第24-66页,Freeman(San Francisco,1969)。
同样,分子生物学技术水平为一般技术人员提供另一种用其可以获得GLP的方法。尽管可以用固相肽合成法、重组法或使胰高血糖素原片段化生产GLP,但是当生产生物合成的GLP-1类似物时重组法最好,因为得率可能更高。
用于本发明目的,优选GLP-1肽类似物和生物合成GLP-1肽类似物。更优选所述DPP-Ⅳ保护的GLP,更加优选生物合成GLP-1肽类似物。另一组优选的GLP-1肽类似物是在8位含有单个氨基酸取代的肽类似物,所述取代氨基酸可以包括d氨基酸残基和修饰的氨基酸残基。更优选的生物合成GLP-1肽类似物是在8位含有单个氨基酸取代的肽类似物,更优选在8位含有Gly或l氨基酸残基Val、Thr或Met的GLP-1肽类似物。
本发明提供由母液生产单一的四方棒状GLP晶体的方法。在pH约6-7、优选约6.4±约0.2的低至中性pH范围条件下,所述结晶液或母液含GLP终浓度约1-10mg/ml,优选2-7mg/ml。
许多含有一种醇或单糖或双糖的常规缓冲液适于实施本发明。优选10-50mM Tris、乙酸铵、乙酸钠或Bis-Tris。醇浓度范围约2-15%(v/v),优选3-13%。优选的醇选自甲醇、乙醇、丙醇或甘油,最优选乙醇。
可任选地在所述母液中加入约1%(w/v)硫酸铵,一般这可使晶体的收率提高。技术人员将会认识到在所述母液中加入诸如叠氮化钠的防腐剂和其它该类防腐剂以防止细菌生长的益处。
在另一实施方案中,单糖或双糖可以以相同的重量体积比取代乙醇。适用于本发明要求保护的方法的单糖或双糖尽管优选海藻糖,但是包括海藻糖、甘露醇、葡萄糖、赤藓糖、核糖、半乳糖、果糖、麦芽糖、蔗糖和乳糖。
在本发明再一实施方案中,可以在pH范围约7-10、优选pH约7.2-9.7的中性或高pH的含锌环境下实施所述方法。在这些条件下,所述GLP浓度范围约1-20mg/ml,优选约2-10mg/ml。以与GLP的摩尔比计的总锌范围为约0.5-1.7,优选0.6-1.5。
在这类具有锌的中性或高pH条件下,合适的缓冲剂和盐浓度范围约10-100mM甘氨酸和0-200mM NaCl,优选40-60mM甘氨酸和0-150mM NaCl。优选缓冲剂是甘氨酸、天冬氨酸和Tris。所述醇或糖条件如前所述。
在所述母液制备后,让其在约15-37℃、优选约18-25℃静置12-48小时直到发生结晶。然后可以将所述晶体转移或进行处理而对晶体形态学无任何明显有害影响,表明这类晶体可以储藏较长时间而不会导致结构损害。
在另一实施方案中,在形成所述晶体后,可以通过向所述母液直接加入药学上可接受的赋形剂、载体、防腐剂和稀释剂制备药物制剂。在该实施方案中,结晶和随后的加入均在无菌条件下操作。锌可以直接加入所述母液以实现锌掺入所述晶体。防腐剂可以加入所述母液以提供适于由同一容器多次注射的晶体制剂。其它诸如抗氧化剂、缓冲剂、调节pH的酸和碱、等渗剂等的赋形剂也可以在形成所述晶体后直接加入所述母液。
在又一实施方案中,本发明提供单一的四方平棒状或片状GLP晶体的均一组合物。在本说明书公开和要求保护的所述方法之前,不可能获得这类组合物。本发明的所述组合物可用于生产方法并用以制备作用时间延长的药用制剂,所述制剂用于治疗糖尿病、肥胖症和相关病症。
采用常规晶体浸泡技术,所述要求保护的GLP晶体和组合物可任选地用锌处理。通过将所述晶体在约0.5mg/ml锌溶液中浸泡,形成用来延长所给予的GLP的作用时间的晶体复合物。此外,通过改变所述锌浓度,可以改变所述复合物组合物使得作用时间延长或缩短。
如前所述,本发明提供药用制剂,所述药用制剂包含四方平棒状或片状GLP单晶和一种或多种药学上可接受的稀释剂、载体或赋形剂。可以配制用于胃肠外给予的所述晶体,以用于治疗性或预防性治疗糖尿病、肥胖症或相关病症。例如,本发明所述晶体可以与常规药用载体和赋形剂混合。包含所述要求保护的晶体的制剂含有所述活性GLP约0.5-50mg/ml,更具体而言约1.0-10mg/ml。此外,本发明的所述晶体可以单独给予或与其它抗糖尿病药物联合给予。对于皮下或肌内给药制剂,本发明所述晶体的无菌制剂可以以原初或修饰的结晶母液的悬浮剂给予或以诸如无热原蒸馏水、生理盐水或5%葡萄糖液的药用稀释剂的悬浮剂给予。可以制备本发明所述晶体的合适制剂,并可以以水性基质或药学上可接受的油性基质(例如诸如油酸乙酯的长链脂肪酸的酯)中的悬浮剂给予。
药学上可接受的防腐剂(例如对羟基苯甲酸烷基酯,特别是对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯或对羟基苯甲酸丁酯或氯代丁醇、苯酚或间甲苯酚)优选加入所述制剂以使得可以多剂量应用。
所述制剂也可以含有等渗剂,该等渗剂是生理可耐受并赋予所述制剂合适张力以防止水净流过所述细胞膜。诸如甘油的化合物以已知浓度常规用于此目的。其它可能的等渗剂包括盐(例如NaCl)、萄萄糖、甘露醇和乳糖。甘油是优选的等渗剂。对于胃肠外制剂,所述等渗剂的浓度范围是本领域已知的,而对于甘油则优选为约16mg/mL-约25mg/mL。
所述制剂也可以含有药学上可接受的缓冲剂以控制所述pH在需要的水平。理想的是在给予后pH达到所述病人可接受的程度,而且在该pH条件下所述制剂在物理和化学上均是非常稳定的。所述pH最好控制在微酸至微碱性pH,例如在约pH5至pH9之间。更优选所述pH在约pH6至pH8之间。缓冲剂包括但不限于柠檬酸盐、醋酸盐、磷酸盐或Tris、或碱性氨基酸(例如赖氨酸或精氨酸),已知所述缓冲剂在这些pH范围是药学上可接受的。于所述范围pH下用于缓冲的其它药学上可接受的缓冲剂是本领域已知的。缓冲剂的选择和浓度在本领域技术人员中是众所周知的。
实施例1
pH6.4和1.0%硫酸铵
称取Val取代8位Ala的化学合成的GLP-1(7-37)OH类似物12.5mg置入3.0ml玻璃管制瓶中,并用2.0ml Tris-HCl、O.02%NaN3、pH6.4处理以产生pH3.6下的澄清溶液。用2N NaOH将该溶液的pH调至8.7,然后用1N HCl调低至pH6.4。在该pH调节期间该溶液保持澄清。将该溶液通过0.22微米Millex GV13针筒式滤器(Millipore,Bedford Ma)过滤到新的3.0ml玻璃管制瓶。根据在280nm的吸光度和在1cm厚测定管中1.0mg/ml V8-GLP-1溶液的消光系数为2.015测定,所述V8-GLP-1母液的浓度为4.76mg/ml。所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含2.0%硫酸铵的10mM Tris-HCl、0.02% NaN3、pH6.4的缓冲液。密封该玻璃管制瓶,轻轻回荡,然后置于18℃。36小时后于200X放大下鉴定出结晶簇。为了定量,取出部分所述母液并于16,000×g离心。如上所述,由在280nm的吸光度测定保留在所述澄清上清液中的V8-GLP-1含量。通过从初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为63.9%。
实施例2
pH6.4和1%乙醇以及1.0%硫酸铵
按实施例1,将所述V8-GLP-1母液的O.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含2.0%硫酸铵和2.0%乙醇的10mM Tris-HCl、0.02%NaN3、pH6.4的缓冲液。然后按实施例1处理和评估所述溶液。该样品产生结晶簇和少量四方单晶。得率为73.1%。
实施例3
pH6.4和5%乙醇以及1.0%硫酸铵
按实施例1,将所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含2.0%硫酸铵和10.0%乙醇的10mM Tris-HCl、0.02% NaN3、pH6.4缓冲液。然后按实施例1处理和评估所述溶液。该样品产生结晶簇、四方单晶和部分棒状晶体。得率为80.3%。
实施例4
pH6.4和10%乙醇以及1.0%硫酸铵
按实施例1,将所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含2.0%硫酸铵和20.0%乙醇的10mM Tris-HCl、0.02%NaN3、pH6.4的缓冲液。然后按实施例1处理和评估所述溶液。该样品产生四方单晶和棒状晶体。得率为81.9%。
实施例5
pH6.4和1%乙醇
按实施例1,将所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含2.0%乙醇的10mMTris-HCl、0.02%NaN3、pH6.4的缓冲液。然后按实施例1处理和评估所述溶液。该样品产生痕量晶体簇。得率为8.8%。
实施例6
pH6.4和5%乙醇
按实施例1,将所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含10.0%乙醇的10mMTris-HCl、0.02% NaN3、pH6.4的缓冲液。然后按实施例1处理和评估所述溶液。该样品产生晶体簇、四方单晶和棒状晶体。得率为39.1%。
实施例7
pH6.4和10%乙醇
按实施例1,将所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入0.25ml含20.0%乙醇的10mMTris-HCl、0.02% NaN3、pH6.4的缓冲液。然后按实施例1处理和评估所述溶液。该样品产生四方单晶和棒状晶体。得率为55.5%。
实施例8
药代动力学
称取生物合成V8-GLP-1 28mg置入玻璃管制瓶并分散于4.5ml10mM醋酸铵中以产生pH为5.6的混浊液。用5N NaOH调节pH至9.5使该物质完全溶解,并在用2N HCl将pH降至64后仍然保持完全溶解。将该溶液通过0.22微米Millex GVl3针筒式滤器过滤到新的玻璃管制瓶中,以使总体积为4.3ml。根据所述母液的20×稀释液在280nm的吸光度和用在1cm厚测定管中1mg/ml V8-GLP-1溶液的消光系数为2.015测定,所述V8-GLP-1液的浓度为5.51mg/ml。所述V8-GLP-1母液的0.25ml等分样品转移至2.0ml玻璃管制瓶。于该溶液加入10mM醋酸铵、2.0%硫酸铵、20%乙醇、pH6.4的沉淀缓冲液4.3ml。密封该玻璃管制瓶,轻轻回荡溶液,然后置于18℃。72小时后于200X放大下鉴定四方单晶。通过低速离心从所述母液取出晶体,然后再悬浮于10mM醋酸铵、16mg/ml甘油、pH5.5的缓冲液(缓冲液A)中,使其浓度约4.0mg/ml。部分所述母液于16,000×g离心。由在280nm的吸光度测定保留在所述澄清上清液中的V8-GLP-1含量。通过从初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量所述结晶得率。该结晶得率为83%。
将以上述相似方式制备、经计算的等份4.0mg/ml V8-GLP-1晶体悬浮液转移至五个玻璃管制瓶中,用缓冲液A稀释至浓度略高于2.5mg/ml V8-GLP-1的最终目的浓度。于该结晶悬浮液加入等份的ZnCl2母液(33.4mg/ml Zn的缓冲液A溶液),以使锌终浓度为0.5、1.0、1.5或24mg/ml。轻轻回荡该悬浮液并置于5℃ 18小时。至此每个管制瓶中的V8-GLP-1终浓度为2.5mg/ml的目的浓度。18小时后将所述晶态V8-GLP-1锌悬浮液转移至室温,使其通过30号针,并用1N HCl调节至pH6.0。
以与上述相似的方式,首先用0.15mg/ml锌处理,从而制备0.1mg/ml锌结晶V8-GLP-1悬浮液。于5℃ 18小时后用低速离心分离所述锌处理的晶体,并转移至缓冲液B(含0.1mg/ml锌的缓冲液A)。用缓冲液B将该悬浮液的V8-GLP-1终浓度调节为2.5mg/ml目的浓度。使该悬浮液通过30号针,所述pH用1N NaOH升高至6.0。
在过夜禁食的小猎犬中测试上述5种晶态V8-GLP-1锌悬浮液,每种悬浮液均为2.5mg/ml V8-GLP-1并含锌0.5、1.0、1.5或2.4mg/ml。每一只动物在零时接受晶态V8-GLP-1锌悬浮液24纳摩尔/Kg的单次皮下注射。在预定时间从动物取动脉血标本(1.5ml),转移至用EDTA预处理并含有40ul抑胰肽酶的试管中,然后离心。分离每个标本的血浆部分并贮藏于-80℃直至分析。在各个标本中的免疫反应性V8-GLP-1的血浆浓度用RIA法测定。表1显示各个悬浮液在24小时内产生的免疫反应性V8-GLP-1血浆水平。
表1:
在狗血浆中的免疫反应性V8-GLP-1水平(皮摩尔浓度)
0.1 mg/ml锌(n=5) | 0.5mg/ml锌(n=5) | 1.0mg/ml锌(n=3) | 1.5mg/ml锌(n=5) | 2.4mg/ml锌(n=5) | ||||||
时间(hrs) | JB-GLP-1(PM ) | SEM | V8-GLP-1(pM) | SEM | V8-GLP-1(pM) | SEM | V8-GLP-1(pM) | SEM | V8-GLp-1(pM) | SEM |
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
1.5 | nd | nd | 123 | 41 | 21 | 4 | 7 | 5 | 5 | 5 |
3.0 | nd | nd | 132 | 38 | 38 | 7 | 40 | 13 | 27 | 21 |
4.5 | nd | nd | 196 | 51 | 108 | 41 | 76 | 34 | 83 | 37 |
6.0 | 301 | 57 | 264 | 79 | 140 | 55 | 142 | 47 | 143 | 60 |
7.5 | nd | nd | 265 | 71 | 184 | 57 | 198 | 61 | 179 | 63 |
9.0 | nd | nd | 344 | 94 | 220 | 61 | 252 | 64 | 214 | 66 |
10.5 | nd | nd | 302 | 80 | 231 | 78 | 250 | 66 | 225 | 50 |
12.0 | nd | nd | 282 | 78 | 236 | 76 | 267 | 60 | 238 | 42 |
13.5 | nd | nd | 238 | 54 | 241 | 97 | 286 | 74 | 236 | 38 |
15.0 | nd | nd | 263 | 67 | 273 | 118 | 325 | 114 | 246 | 28 |
16.5 | nd | nd | 235 | 51 | 234 | 106 | 275 | 77 | 218 | 25 |
18.0 | nd | nd | 210 | 47 | 184 | 62 | 254 | 59 | 211 | 23 |
19.5 | nd | nd | 221 | 54 | 209 | 120 | 278 | 57 | 173 | 9 |
21.0 | nd | nd | 215 | 54 | 219 | 115 | 301 | 48 | 178 | 13 |
22.5 | nd | nd | 224 | 54 | 193 | 72 | 232 | 23 | 167 | 9 |
24.0 | 190 | 30 | 210 | 51 | 187 | 72 | 227 | 34 | 166 | 25 |
30.0 | nd | nd | nd | nd | nd | nd | nd | nd | 171 | 24 |
SEM=平均值的标准误差
nd=未检测
实施例9
pH9.4和5%海藻糖以及锌
称取冻干的生物合成V8-GLP-16.8mg,置入3.0ml玻璃管制瓶中。然后加入25mM甘氨酸盐酸盐、150mM NaCl、5%海藻糖、pH9.0的缓冲液1.0ml溶解所述肽。然后用5N NaOH调节所述溶液至pH10.3。轻轻搅拌该溶液的同时,加入10mg/ml氯化锌水溶液9.0ul,用2N HCl调节pH至9.4。根据所述溶液的10X稀释液在280nm的吸光度测定,所述V8-GLP-1终浓度为5.4mg/ml。然后用0.22μmMillex GV13针筒式滤器过滤溶液。将该瓶盖上盖,轻轻回荡,然后置于室温。24小时后于430X放大下鉴定出V8-GLP-1晶体簇和长方形单晶,并估计为约40μm长、15μm宽和3μm厚。取出部分母液并于16,000xg离心。根据在280nm的吸光度,测定保留在所述澄清上清液中的V8-GLP-1含量。由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为89.8%。在无海藻糖的结晶实验中未观察到小长方形晶体形态学。
实施例10
pH9.4和10%甘露醇以及锌
称取冻干的生物合成V8-GLP-16.8mg,置入3.0ml玻璃管制瓶中。然后用25mM甘氨酸盐酸盐、150mM NaCl、10%甘露醇、pH9.0的缓冲液1.0ml处理,并分散产生澄清溶液。用5N NaOH调节所述溶液至pH10.3。轻轻搅拌该溶液的同时,加入10mg/ml氯化锌水溶液9.0μl,用2N HCl调节pH至9.4。根据所述结晶溶液的10X稀释液在280nm的吸光度测定,V8-GLP-1终浓度为5.31mg/ml。然后用0.22μm Millex GV13针筒式滤器过滤溶液。将该瓶盖盖,轻轻回荡,然后置于室温。24小时后,于430X放大下鉴定出V8-GLP-1小长方形片状晶体,并估计为约10-30μm长和10μm宽。按实施例9测定得率。该样品显示结晶得率为35%。
实施例11
pH9.4和锌
制备1-ml等份在50mM甘氨酸-150mM NaCl缓冲液pH9.0中的3mg/ml V8-GLP-1溶液。于该溶液加入20.85mg/ml氯化锌水溶液7.5μl,然后将pH调节回升至pH9.0。轻轻回荡后,将3-ml玻璃管制瓶中的该澄清样品贮藏于室温1天。此后在显微镜下放大90X检查到该结晶沉淀物,显示小片状晶体簇。为了定量结晶得率,将全部悬浮液通过0.2μm滤器(Gelman Science,Ann Arbor,Michigan)。通过用1.0mg/ml V8-GLP-1溶液在1cm厚测定管中的消光系数为2.015,在280nm波长下进行光谱评估,定量保留在所述澄清滤液中的V8-GLP-1含量。通过由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为5.6%。
实施例12
pH9.0和10%乙醇以及锌
按实施例11制备1-ml等份V8-GLP-1溶液,不同的是在加入氯化锌液前于该溶液加入110μl无水乙醇。该样品以80.6%得率产生大四方晶体和部分晶体簇。
实施例13
pH9.5和10%乙醇以及锌
将在50mM甘氨酸-150mM NaCl缓冲液pH10.5中的10mg/mlV8-GLP-1溶液通过无菌0.2μm Acrodisc滤器(Gelman Science,AnnArbor,Michigan)。于500μl该溶液加入500μl 50mM甘氨酸-150mMNaCl缓冲液pH9.0。然后于该溶液加入110μl无水乙醇,再加入7.5μl20.85mg/ml氯化锌水溶液。用少量1N HCl调节所述溶液至pH9.5。轻轻回荡后,将该终溶液封装入3-ml玻璃管制瓶内,贮藏于室温2天,单一的长达150μm长、约25μm宽和不足5μm厚的V8-GLP-1结晶片以72%得率产生。
实施例14
pH7.9和8.5%甘露醇以及锌
制备在50mM甘氨酸pH9.5缓冲液中的4mg/ml V8-GLP-1,然后将其通过0.2μm滤器(Gelman Science,Ann Arbor,Michigan)。于1-ml该溶液加入100μl无水乙醇,然后加入60μl 2.08mg/ml氯化锌水溶液。用少量0.1N HCl调节所述溶液至pH8.0。轻轻回荡后,将该终溶液封装入3-ml玻璃管制瓶内,贮藏于室温2小时。然后用少量0.1N HCl调节该澄清液的pH至7.86,继续在室温贮藏二天。显微镜检察显示中等大小、单一的四方片状晶体和部分晶体簇。通过用1mg/ml V8-GLP-1溶液在1cm厚测定管中的消光系数为2.015,在280nm波长光谱评价,定量保留在所述澄清上清液中的V8-GLP-1含量。通过由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为92.2%。
实施例15
pH8.3和10%甘露醇以及锌
制备在100mM甘氨酸pH10.5缓冲液中的7mg/ml V8-GLP-1,然后将其通过0.2μm滤器(Gelman Science,Ann Arbor,Michigan)。于0.5ml该溶液加入0.4ml水。然后加入100μl无水乙醇,再加入约6μl20.86mg/ml氯化锌水溶液。用少量0.1N HCl调节所述溶液至pH8.33。轻轻回荡后,将该终溶液封装入3-ml玻璃管制瓶内,贮藏于室温1天。显微镜检察显示小型、单一的四方片状晶体和部分晶体簇。通过用1mg/ml V8-GLP-1溶液在1cm厚测定管中的消光系数为2.015,在280nm波长进行光谱评价,定量保留在所述澄清上清液中的V8-GLP-1含量。通过由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为92.4%。
实施例16
pH7.4和8.6%乙醇以及锌
制备在50mM甘氨酸pH9.0缓冲液中的4mg/ml V8-GLP-1,然后将其通过0.2μm滤器(Gelman Science,Ann Arbor,Michigan)。于5ml该溶液加入500μl无水乙醇,再加入300μl 2.08mg/ml氯化锌水溶液。加入少量N HCl用来调节该溶液至pH7.40。轻轻回荡后,将该终溶液封装入10-ml玻璃管制瓶内,贮藏于室温2天。显微镜检察显示中等大小的单一的四方晶体。通过用1mg/ml V8-GLP-1溶液在1cm厚测定管中的消光系数为2.015,在280nm波长进行光谱评价,定量保留在所述澄清上清液中的V8-GLP-1含量。通过由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该样品显示结晶得率为85.0%。
实施例17
pH6.4和5%乙醇以及1.0%硫酸铵
称取V8-GLP-1(12.5mg)置入20ml玻璃管制瓶中。加入2.0ml含150mM NaCl的10mM醋酸铵缓冲液pH6.4。通过用5N NaOH调节至pH9.5使该混浊液变澄清,然后用2N HCl降低至pH6.4。该澄清溶液通过0.22μm Millex GV13针筒式滤器(Millipore,Bedford,MA)过滤到一个新的20ml玻璃管制瓶中。通过用1.0mg/ml V8-GLP-1溶液在1cm厚测定管中的消光系数为2.015,用280nm的吸光度测定所述V8-GLP-1母液的浓度。将该蛋白浓度调节为5.0mg/ml。制备含10mM醋酸铵、150mM NaCl、2%硫酸铵和10%乙醇的pH6.4沉淀溶液,并通过0.22μm Millex GV13针筒式滤器过滤。将2ml所述沉淀溶液缓慢加入玻璃管制瓶中的2ml所述V8-GLP-1母液中。轻轻涡该瓶,并于室温温育2天。观察到四方片状晶体得率为92%。
用1N HCl将所述晶体悬浮液的pH调节为5.5,加入氯化锌至终深度为0.15mg/ml。于室温锌浸泡过夜后,用1N NaOH调节该悬浮液pH至7.5,加入防腐剂间甲苯酚至浓度为3.16mg/ml。本实施例表明,必要时,直接制备药用的GLP-1晶体的防腐制剂,而不需要中间步骤中的离心或过滤分离所述晶体。
实施例18
pH7.6和8.5%乙醇以及锌
制备在50mM甘氨酸pH9.0缓冲液中的4mg/ml V8-GLP-1,然后将其通过0.2μm滤器(Gelman Science的Acrodisc,Ann Arbor,Michigan)。于10ml该溶液加1ml无水乙醇,之后加入600μl 6.7mg/ml醋酸锌(2-水化物)水溶液。加入2%醋酸100μl,使得pH约7.6。轻轻回荡后,将该终溶液封装入20-ml玻璃管制瓶内,贮藏于室温24小时。显微镜检察显示中等大小的单一的四方晶体。然后于该全部溶液加入含3.5ml的14 mg/ml间甲苯酚水溶液和55μl 2%醋酸的溶液3.555ml,产生终pH约7.2的悬浮液。轻轻回荡后,将该悬浮液封装入20-ml玻璃管制瓶内,贮藏于室温24小时。显微镜检察再次显示中等大小的单一的四方晶体。
于室温下离心等份样品5分钟后,通过与V8-GLP-1标准液的HPLC分析比较,由稀释样品的HPLC分析确定保留在所述澄清上清液中V8-GLP-1含量。通过由初始液V8-GLP-1水平减去所述上清液V8-GLP-1水平定量结晶得率。该防腐的V8-GLP-1制剂显示结晶得率为97.7%。
实施例19
晶体稳定性
按实施例8所述,于18℃在10nM醋酸铵、1%硫酸铵、10℃乙醇缓冲液pH6.4中制备V8-GLP-1四方单晶。通过低速离心由母液取出所述晶体,再悬浮于10mM醋酸铵、16mg/ml甘氨酸、pH5.5缓冲液至V8-GLP-1浓度约4.9mg/ml。
低速离心2ml该悬浮液,并用移液管去除所述上清液。将该沉淀再悬浮于4ml含0.1mg/ml锌的10mM醋酸铵、16mg/ml甘氨酸pH5.5缓冲液中。让该晶体悬浮液于4℃浸泡于所述锌溶液中过夜。
将锌浸泡的晶体悬浮液分四个1-ml等份。低速离心这些悬浮液,用移液管去除其上清液。在10mM醋酸铵、16mg/ml甘油、0.1mg/ml锌缓冲液pH6.0中制备四份晶体悬浮液。再用0.1N NaOH将pH调节至pH7.4和/或如表2所示,加入间甲苯酚至终浓度3.16mg/ml以选择样品。再将每种悬浮液一分为二以贮藏于室温(约22℃)和4℃,如表2所示提供总计8个测试样品。
10天后,在所述显微镜下检察所述晶体悬浮液。然后用HPLC评估所述悬浮滤液以定量所述结晶悬浮液中的可溶性V8-GLP-1。该HPLC结果报告于表2中。
表2:
贮藏10天后在晶体悬浮液中的可溶性V8-GLP-1
样品 | pH | 贮藏温度 | mg/ml间甲苯酚 | 用HPLC分析的可溶性V8-GLP-1 |
A | 6.0 | 4℃ | 0 | 0.19% |
B | 7.4 | 4℃ | 0 | 0.10% |
C | 6.0 | 4℃ | 3.16 | 0.05% |
D | 7.4 | 4℃ | 3.16 | 0.06% |
E | 6.0 | 22℃ | 0 | 0.16% |
F | 7.4 | 22℃ | 0 | 0.08% |
G | 6.0 | 22℃ | 3.16 | 0.03% |
H | 7.4 | 22℃ | 3.16 | 0.04% |
该实验表明,防腐或非防腐晶体制剂在10天后低于0.2%的所述V8-GLP-1肽被溶解。
显微镜下所述晶体悬浮液显示,所述单一的四方晶体的聚集或团聚在pH7.4时少于pH6.0,4℃时少于22℃。所述间甲苯酚似乎对晶体聚集无明显影响。另外的实验显示,在得自或所述初始结晶母液或随后加入物的晶体悬浮液中存在3%以上的乙醇,明显降低所述晶体在防腐和非防腐制剂中的团聚倾向。其它试验表明,尽管所述晶体在存在间甲苯酚的情况下相对稳定,但是它们在5%苯酚存在下更不稳定,逐渐使非晶形物质形成。
其它晶体稳定性试验表明,在pH制备的V8-GLP-1晶体在化学上非常稳定,在5℃或室温下贮藏长达2个月后用HPLC分析未观察到降解峰。
按实施例16所述在甘氨酸缓冲液中制备的晶体的稳定性试验表明,当加入间甲苯酚至3.16mg/ml的水平时保藏于原初母液中的V8-GLP-1晶体不稳定。该试验使得所述晶体仅在1天后溶解。可以通过在加入防腐剂前加入锌(由氯化锌溶液)可有效阻止该组合物中的晶体不稳定性。
序列表>Eli Lilly and Company>胰高血糖素样肽-1晶体>X-10242 PCT>PCT/US 98/26480򗶮-12-14>US 60/069728򗶭-12-16ɰ>PatentIn Ver.2.0ɭᡗ>PRT>Homo SapiensɭHis Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly20 25 30ɮᡕɚl2>PRT>人工序列>>人工序列的描述:合成的突变型人序列>>在13位的Xaa是Glu、Gln、Ala、Thr、Ser或Gly;在19位的Xaa是Glu、Gln、Ala、Thr、Ser或Gly;而在29位的Xaa是Gly或不存在。ɮGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly Gln Ala1 5 10Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa20 25ɯɚl1ᡕ>PRT>人工序列>>人工序列的描述:合成的突变型人序列>ᡔ位的Xaa是Lys或不存在;29位的Xaa是Gly或不存在;如果28位的Xaa不存在,则29位的Xaa必须不存在。ɯHis Ala G1u Gly Thr phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile A1a Trp Leu Val Xaa Xaa20 25ɚl0ɰᡖɚl2>PRT>人工序列>>人工序列的描述:合成的突变型人序列>ᡋ位的Xaa是Lys或Arg;30位的Xaa是Gly或不存在;27位的Lys可以被酰化ɰAla Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln1 5 10 15Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa20 25 30
Claims (23)
1.用于制备胰高血糖素样肽-1相关分子(GLP)的棒状或片状单晶的方法,所述方法包括制备含GLP、缓冲剂、醇或单糖或双糖和可任选的硫酸铵或锌的结晶液。
2.权利要求1的方法,其中所述GLP终浓度约1-10mg/ml,所述GLP选自GLP-1类似物、GLP-1衍生物、双肽酶Ⅳ(DPP-Ⅳ)保护的GLP、GLP-1肽类似物或生物合成的GLP-1类似物;其中所述缓冲剂约10-50mM和pH约6-7,并选自Tris、醋酸铵、醋酸钠或Bis-Tris;其中所述醇或单糖或双糖选自甲醇、乙醇、丙醇、甘油、海藻糖、甘露醇、葡萄糖、赤藓糖、核糖、半乳糖、果糖、麦芽糖、蔗糖和乳糖;和可任选地其中存在1%硫酸铵。
3.权利要求1的方法,其中锌总量与所述GLP的摩尔比为O.5-1.7,所述GLP终浓度约1-20mg/ml,所述GLP选自GLP-1类似物、GLP-1衍生物、DPP-Ⅳ保护的GLP、GLP-1肽类似物或生物合成的GLP-1类似物;其中所述缓冲剂约10-100mM和pH约7-10,并选自甘氨酸、天冬氨酸或Tris;其中所述醇或单糖或双糖选自甲醇、乙醇、丙醇、甘油、海藻糖、甘露醇、葡萄糖、赤藓糖、核糖、半乳糖、果糖、麦芽糖、蔗糖和乳糖。
4.权利要求1-3的任一项的方法,其中所述GLP选自DPP-Ⅳ保护的GLP或生物合成的GLP。
5.权利要求14的任一项的方法,其中所述GLP是选自Val-8-GLP-1(7-37)OH、Thr-8-GLP-1(7-37)OH、Gly-8-GLP-1(7-37)OH或Met-8-GLP-1(7-37)OH的DPP-Ⅳ保护的GLP。
6.权利要求1-5的任一项的方法,具有将所述GLP晶体浸泡于含锌溶液的另一步骤。
7.具有四方平棒状或片状形态学的GLP晶体,选自GLP-1类似物、GLP-1衍生物、DPP-Ⅳ保护的GLP、GLP-1肽类似物或生物合成的GLP-1类似物。
8.权利要求7的晶体,其中所述GLP选自DDP-Ⅳ保护的GLP或生物合成的GLP。
9.权利要求7的晶体,其中所述GLP选自Val-8-GLP-1(7-37)OH、Thr-8-GLP-1(7-37)OH、Gly-8-GLP-1(7-37)OH或Met-8-GLP-1(7-37)OH。
10.无论何时用权利要求1-6中任一项的方法制备的GLP晶体。
11.大致均一的GLP晶体组合物。
12.权利要求11的组合物,其中所述GLP晶体选自GLP-1类似物、GLP-1衍生物、DPP-Ⅳ保护的GLP、GLP-1肽类似物或生物合成的GLP-1类似物。
13.权利要求11-12中任一项的组合物,其中所述GLP选自DPP-Ⅳ保护的GLP或生物合成的GLP-1。
14.权利要求11-13中任一项的组合物,其中所述GLP选自Val-8-GLP-1(7-37)OH、Th-8-GLP-1(7-37)OH、Gly-8-GLP-1(7-37)OH或Met-8-GLP-1(7-37)OH。
15.一种药用制剂,它包含权利要求7-11的任一项中要求保护的GLP晶体和一种或多种药学上可接受的稀释剂、载体或赋形剂。
16.权利要求15的药用制剂,其中所述制剂通过加入至所述结晶后母液和/或改变所述结晶后母液而制备,而不需要从所述母液分离所述GLP晶体。
17.一种在需要其治疗的哺乳动物中治疗糖尿病、肥胖症或相关病症的方法,它包括给予所述哺乳动物权利要求7-10中任一项的GLP晶体。
18.一种在需要其治疗的哺乳动物中治疗糖尿病、肥胖症或相关病症的方法,它包括给予所述哺乳动物权利要求11-14中任一项的组合物。
19.一种在需要其治疗的哺乳动物中治疗糖尿病、肥胖症或相关病症的方法,它包括给予所述哺乳动物权利要求15-16中任一项的药用制剂。
20.权利要求7-10的任一项中要求保护的GLP晶体,用于治疗糖尿病、肥胖症或相关病症。
21.权利要求11-14的任一项中要求保护的大致均一的GLP组合物,用于治疗糖尿病、肥胖症或相关病症。
22.大致如前所述、参照任一所述实施例的GLP晶体。
23.大致如前所述、参照任一所述实施例的大致均一的GLP晶体组合物。
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EP2280020B1 (en) | 1999-06-29 | 2016-02-17 | MannKind Corporation | Pharmaceutical formulations comprising a peptide complexed with a diketopiperazine |
PT1257577E (pt) | 2000-01-27 | 2004-08-31 | Lilly Co Eli | Processo para solubilizar compostos peptido 1 do tipo glucagon |
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AU2001228327A1 (en) * | 2000-01-31 | 2001-08-14 | Novo-Nordisk A/S | Crystallisation of a glp-1 analogue |
WO2001087322A2 (en) * | 2000-05-17 | 2001-11-22 | Bionebraska, Inc. | Peptide pharmaceutical formulations |
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- 1998-12-14 HU HU0100046A patent/HUP0100046A2/hu unknown
- 1998-12-14 KR KR1020007006521A patent/KR20010033146A/ko not_active Application Discontinuation
- 1998-12-14 AU AU18218/99A patent/AU1821899A/en not_active Abandoned
- 1998-12-14 IL IL13637798A patent/IL136377A0/xx unknown
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- 1998-12-14 JP JP2000538710A patent/JP2002508332A/ja not_active Withdrawn
- 1998-12-14 CZ CZ20002104A patent/CZ20002104A3/cs unknown
- 1998-12-14 EP EP98310245A patent/EP0926159A3/en not_active Withdrawn
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- 1998-12-14 CN CN98812093A patent/CN1281369A/zh active Pending
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- 1998-12-14 TR TR2000/01726T patent/TR200001726T2/xx unknown
- 1998-12-14 WO PCT/US1998/026480 patent/WO1999030731A1/en not_active Application Discontinuation
- 1998-12-14 BR BR9813658-5A patent/BR9813658A/pt not_active IP Right Cessation
- 1998-12-15 PE PE1998001225A patent/PE20000059A1/es not_active Application Discontinuation
- 1998-12-15 AR ARP980106356A patent/AR019806A1/es unknown
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2000
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CN108042794A (zh) * | 2018-01-05 | 2018-05-18 | 北京博康健基因科技有限公司 | 重组促胰岛素分泌素的稳定制剂及其制备方法 |
CN108042794B (zh) * | 2018-01-05 | 2022-01-25 | 北京博康健基因科技有限公司 | 重组促胰岛素分泌素的稳定制剂及其制备方法 |
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SV1998000147A (es) | 1999-08-18 |
CA2315243A1 (en) | 1999-06-24 |
ID25534A (id) | 2000-10-12 |
EP0926159A3 (en) | 2003-11-26 |
KR20010033146A (ko) | 2001-04-25 |
NZ505182A (en) | 2002-05-31 |
TR200001726T2 (tr) | 2000-10-23 |
US6555521B2 (en) | 2003-04-29 |
NO20003081L (no) | 2000-08-08 |
BR9813658A (pt) | 2000-10-10 |
CZ20002104A3 (cs) | 2001-08-15 |
HRP20000409A2 (en) | 2000-10-31 |
PE20000059A1 (es) | 2000-02-09 |
WO1999030731A1 (en) | 1999-06-24 |
CO5040043A1 (es) | 2001-05-29 |
ZA9811466B (en) | 2000-06-14 |
US20030045464A1 (en) | 2003-03-06 |
IL136377A0 (en) | 2001-06-14 |
NO20003081D0 (no) | 2000-06-15 |
EA200000652A1 (ru) | 2000-12-25 |
JP2002508332A (ja) | 2002-03-19 |
HUP0100046A2 (hu) | 2001-05-28 |
PL341210A1 (en) | 2001-03-26 |
US6380357B2 (en) | 2002-04-30 |
USRE41133E1 (en) | 2010-02-16 |
EP0926159A2 (en) | 1999-06-30 |
AU1821899A (en) | 1999-07-05 |
AR019806A1 (es) | 2002-03-20 |
US20010014666A1 (en) | 2001-08-16 |
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