CN111533801A - 新的特立帕肽糖基化衍生物及其应用 - Google Patents
新的特立帕肽糖基化衍生物及其应用 Download PDFInfo
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
本发明涉及医药技术领域,且公开了新的特立帕肽糖基化衍生物,具体指具有式(I)结构的多肽,具有式(I)结构的多肽及其药学上可接受的盐或酯:X1VX2EIQLMH X3LGKHL X4X5MERVEWLRKKLQ X6VHNF (I),其中,X1,X2,X5表示Ser(GlcNAc)或Ser(Glc)或Ser(Fuc)或Ser(Rib)或Ser(Gal)或Ser(GalNAc)或Ser(Lyx)或Ser(Rha)或Ser(Man)或Ser(ManNAc)或Ser(Cel)或Ser(Mal)或Ser(Suc)或Ser(Lac)或Ser(Tre)或Ser,X3,X4,X6表示Asn(GlcNAc)或Asn(Glc)或Asn(Fuc)或Asn(Rib)或Asn(Gal)或Asn(GalNAc)或Asn(Lyx)或Asn(Rha)或Asn(Man)或Asn(ManNAc)或Asn(Cel)或Asn(Mal)或Asn(Suc)或Asn(Lac)或Asn(Tre)或Asn。该新的特立帕肽糖基化衍生物及其制备方法与应用,该系列衍生物具有促进成骨细胞分化的作用,可用于治疗骨质疏松症,且不会损坏骨晶体结构,不会影响骨强度,使用更加安全可靠。
Description
技术领域
本发明涉及医药技术领域,具体为新的特立帕肽糖基化衍生物及其制备方法与应用。
背景技术
骨质疏松作为一种常见的全身性骨代谢疾病,主要表现为骨量低下、骨微结构破坏以及骨脆性增加,从而导致继发性骨折的发生,骨质疏松可发生于不同的性别与年龄阶段,但多见于绝经后妇女与老年男性,2001年美国国立卫生研究院提出骨矿密度和骨质量是影响骨强度的两个主要方面,骨质量由破骨细胞的骨吸收和成骨细胞增加基质沉积矿化的作用共同维持,若骨形成作用减弱,骨吸收作用增强,骨稳态发生改变,则引起骨质疏松,将骨质疏松症的药物治疗分为抗分解代谢药与合成代谢药,抗分解代谢药主要是包括雌激素、选择性雌激素受体调节剂、双膦酸盐类、钙、维生素D等,这些药物主要通过降低骨的更新而使骨质增加,虽能有有效预防骨折,但同时带来骨质量的降低会增加潜在的发生骨折的危险,合成代谢药如氟化物因对骨晶体结构损坏从而影响骨强度的副作用,未能广泛应用,目前,甲状旁腺激素作为调节钙磷代谢、骨代谢的重要激素之一,被认为是最有前途的合成代谢药。
特立帕肽(Teriparatide)是利用基因工程重组技术合成的人甲状旁腺激素类似物,其氨基酸序列与天然人甲状旁腺激素N末端34位氨基酸序列完全相同,该类似物对甲状旁腺激素受体有着相似的亲和力,可激活成骨细胞相同的信号通道,从而促进骨形成,该药于2002年获美国FDA批准上市,是目前唯一被批准应用的骨合成代谢制剂,甲状旁腺激素以及人甲状旁腺激素类似物特立帕肽中乙酰氨基葡萄糖糖基化的生物功能还未见相关报道,但有研究发现,糖基化能从多方面改善多肽及蛋白的性质和功能,如增加亲水性,生物利用度,限定或维持构象,增加其对水解酶抵抗力以及减小免疫原性等,发明人经过艰苦的努力,基于特立帕肽序列对其进行了不同位点的包括O-连和N-连糖基化修饰,获得了一系列新的特立帕肽糖基化衍生物,优选化合物具有较好的成骨细胞分化促进效应,为骨质疏松的治疗提供了一种新选择。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了新的特立帕肽糖基化衍生物及其制备方法与应用,具备较好的成骨细胞分化促进效应等优点,解决了现有的治疗骨质疏松的药物因对从骨晶体结构损坏而影响骨强度的副作用,未能广泛应用的问题。
(二)技术方案
为实现上述具有较好的成骨细胞分化促进效应的目的,本发明提供如下技术方案:新的特立帕肽糖基化衍生物,具体指具有式(I)结构的多肽。
具有式(I)结构的多肽及其药学上可接受的盐或酯:
X1VX2EIQLMH X3LGKH LX4X5MERVEWLRKKLQ X6VHNF (I)
其中,
X1,X2,X5表示Ser(GlcNAc)或Ser(Glc)或Ser(Fuc)或Ser(Rib)或Ser(Gal)或Ser(GalNAc)或Ser(Lyx)或Ser(Rha)或Ser(Man)或Ser(ManNAc)或Ser(Cel)或Ser(Mal)或Ser(Suc)或Ser(Lac)或Ser(Tre)或Ser。
X3,X4,X6表示Asn(GlcNAc)或Asn(Glc)或Asn(Fuc)或Asn(Rib)或Asn(Gal)或Asn(GalNAc)或Asn(Lyx)或Asn(Rha)或Asn(Man)或Asn(ManNAc)或Asn(Cel)或Asn(Mal)或Asn(Suc)或Asn(Lac)或Asn(Tre)或Asn。
式(I)中多肽片段第1位的氨基酸残基(X1)优选为Ser(GlcNAc)。
式(I)中多肽片段第3位的氨基酸残基(X2)优选为Ser(GlcNAc)。
式(I)中多肽片段第10位的氨基酸残基(X3)优选为Asn(GlcNAc)。
式(I)中多肽片段第16位的氨基酸残基(X4)优选为Asn(GlcNAc)。
式(I)中多肽片段第17位的氨基酸残基(X5)优选为Ser(GlcNAc)。
式(I)中多肽片段第30位的氨基酸残基(X6)优选为Asn(GlcNAc)。
在本文中,“本发明的特立帕肽糖基化衍生物”指的是本发明中具有式(I)所示结构多肽,在本文中,这种多肽可以称为“特立帕肽糖基化衍生物”,“多肽片段”或“本发明多肽”。
式(I)多肽的N末端的氨基和C末端的羧基以及氨基酸侧链基团可以不进行修饰,也可以在基本不影响本发明多肽活性的前提下进行修饰,如形成“药学上可接受的酯”,N末端氨基基团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-酰基修饰,C末端羧基基团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,本发明优选对式(I)多肽N末端的氨基不进行乙酰化修饰,即是-NH2,C末端的羧基进行酰胺化修饰,即是-CONH2。
本文所使用的多肽及氨基酸和化学基团的表示方法均为所属领域公认的表示方法,其中氨基酸的缩写可参照表1中的定义,特殊氨基酸以及糖氨基酸结构可参照表2中的定义,在本文中,若不特别指出,氨基酸一般指L-型的氨基酸。
表1氨基酸缩写表
表2特殊氨基酸缩写表
“药学上可接受的盐”指一些小分子酸性或碱性化合物与多肽形成的盐,一般能够增加多肽的溶解性,所形成的盐基本上不改变多肽的活性,例如,通常能与本发明多肽形成盐的酸有盐酸、磷酸、硫酸、乙酸、琥珀酸、马来酸和柠檬酸等;能与本发明多肽形成盐的碱有碱金属或碱土金属的氢氧化物、铵和碳酸盐等。
本发明多肽的成骨细胞促进作用可以通过所属领域常规的实验方法来验证,如细胞学实验等,在本发明的具体实施方式中,优选通过细胞学实验如小鼠成骨细胞样本碱性磷酸酶(ALP)染色,通过该试验,发现本发明所涉及的式(I)多肽片段都具有体外成骨细胞促进作用,能够用于骨质疏松症的治疗。
此外,本发明的另一方面提供了含有上述具有式(I)结构的多肽片段的药物组合物,其可以用于治疗骨质疏松症,该组合物可以含有本发明的多肽片段中的一种或多种,优选仅含一种特立帕肽糖基化衍生物,该组合物可以含有一种或多种药学上可接受的稀释剂、赋形剂或载体,优选该组合物为单位剂量形式,如片剂、膜剂、丸剂、胶囊(包括持续释放或延迟释放形式)、粉剂、颗粒剂、糖浆剂或乳液剂、消毒的注射用溶液、悬浮液或冻干粉末针剂、气雾剂或液体喷剂、滴剂自动注射装置或栓剂,上述活性药物组分可以与一种无毒的药物学可接受的惰性载体组合在一起,如乙醇、甘油、水或其组合,本发明式(I)的特立帕肽糖基化衍生物优选使用消毒的注射用水溶液。
本发明的药物组合物可通过所属领域技术人员所熟知的给药方式来进行给药,例如口服、直肠、舌下、肺部、透皮、离子透入、阴道及鼻内给药。
本发明的药物组合物优选胃肠道外给药,如皮下、肌内或静脉内注射
本发明合成的部分优选化合物的名称、结构式和质谱数据如表3所示。
表3部分优选化合物的名称、结构式和质谱数据
为了便于理解,以下将通过具体的实施例和附图对本发明进行描述。需要特别指出的是,这些描述仅仅是示例性的描述,并不构成对本发明范围的限制。
(三)有益效果
与现有技术相比,本发明提供了新的特立帕肽糖基化衍生物及其制备方法与应用,具备以下有益效果:
1、该新的特立帕肽糖基化衍生物及其制备方法与应用,通过
2、该新的特立帕肽糖基化衍生物及其制备方法与应用,通过
附图说明
图1为特立帕肽糖基化衍生物片段小鼠成骨细胞的ALP染色结果;
图2为表3中PTHG-2结构式示意图;
图3为表3中PTHG-4结构式示意图;
图4为表3中PTHG-9结构式示意图。
具体实施方式
下面将结合本发明的实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例一:固相合成O-连糖肽片段(PTH-2)
步骤如下:氨基酸α-氨基用9-芴基甲氧羰基(Fmoc)保护,并对氨基酸进行侧链保护:Ser,Asp,Glu,Tyr的侧链保护基为叔丁基(tBu),Lys,Trp,His的侧链保护基为叔丁氧羰基(Boc),Arg的侧链保护基为2,2,4,6,7-五甲基苯并呋喃-5-磺酰基(Pbf),Gln和Cys的侧链保护基为三苯甲基(Trt),Ser(AcNH-Beta-Glc)的侧链保护基为乙酰基(Ac),以6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)、N,N-二异丙基乙胺(DIPEA)为活化试剂,使上述保护氨基酸依次偶联,偶联每次40分钟,以20%哌啶/DMF为脱Fmoc试剂,每次10分钟,多肽连接完毕后,用10%水合肼/DMF溶液脱除乙酰基保护12小时,使用TFA/EDT/TIPs/Water(95:2:2:1,v/v/v/v)室温反应2小时,从而将其从树脂上切割下来,同时脱除侧链保护基,后用无水乙醚沉淀得到粗肽。粗肽在30分钟内以反向HPLC纯化,冻干得到纯度≥97.0%的白色冻干粉末。
实施例二:固相合成N-连糖肽片段(PTH-4,PTH-9)
步骤如下:氨基酸α-氨基用9-芴基甲氧羰基(Fmoc)保护,并对氨基酸进行侧链保护:Ser,Asp,Glu,Tyr的侧链保护基为叔丁基(tBu),Lys,Trp,His的侧链保护基为叔丁氧羰基(Boc),Arg的侧链保护基为2,2,4,6,7-五甲基苯并呋喃-5-磺酰基(Pbf),Gln和Cys的侧链保护基为三苯甲基(Trt),Asn(AcNH-Beta-Glc)的侧链保护基为乙酰基(Ac),以6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)、N,N-二异丙基乙胺(DIPEA)为活化试剂,使上述保护氨基酸依次偶联,偶联每次40分钟,以20%哌啶/DMF为脱Fmoc试剂,每次10分钟,多肽连接完毕后,用10%水合肼/DMF溶液脱除乙酰基保护12小时,使用TFA/EDT/TIPs/Water(95:2:2:1,v/v/v/v)室温反应2小时,从而将其从树脂上切割下来,同时脱除侧链保护基,后用无水乙醚沉淀得到粗肽,粗肽在30分钟内以反向HPLC纯化,冻干得到纯度≥97.0%的白色冻干粉末。
实验例:细胞生物学实验,具体为MC3T3-E1细胞(小鼠胚胎成骨细胞前体细胞)ALP染色实验
1)MC3T3-E1细胞复苏;
将配制好的生长培养基(α-MEM培养基+10%FBS培养基)放入37℃恒温水浴锅预热,从液氮罐中取出MC3T3-E1细胞冻存管,迅速放入37℃恒温水浴锅进行解冻,轻轻旋转直至细胞溶解,之后用移液枪吸取细胞悬液至15ml无菌离心管,加入5ml生长培养基,室温离心(1000rpm,6min),离心完成后,弃去培养基,重复清洗一次,清洗完成后,将细胞悬液转移至培养瓶同时加入5ml新鲜生长培养基,吹打直至细胞完全分散。将培养瓶放入二氧化碳细胞培养箱,在5%CO2,37℃条件下进行细胞培养。
2)MC3T3-E1细胞传代;
显微镜下观察培养瓶中细胞生长状况,当细胞贴壁生长、融合度达到80%且形态良好时可以进行传代培养,当细胞生长出现接触性抑制时,用移液管弃掉培养基,PBS清洗两次后加入1ml0.25%胰酶消化液,室温消化1-2min,轻轻拍打培养瓶至细胞大部分脱落,同时避免过度消化,消化过程中放到显微镜下观察,当细胞变为圆形时可停止消化,若未观察到可适当延长消化时间,消化结束后加入5ml新鲜生长培养基终止消化,吹打均匀至细胞完全脱落。转移培养瓶中液体至15ml离心管,室温离心(1000rpm,6min),离心完成后,弃去培养基,加入5ml新鲜生长培养基并将细胞悬液转移至培养瓶进行细胞培养,若需冻存,离心结束后弃去培养基,轻轻拍打培养瓶至细胞完全脱落,加入适量冻存液后转移至冻存管中,实行分阶段降温:4℃下放置15min、-20℃下放置30min、-80℃下放置24h,最后放入液氮罐中进行长期保存。
3)MC3T3-E1细胞种板;
制成的细胞悬液用血球计数板计数,根据计数结果,将细胞悬液密度进行稀释,按1×104/cm2接种于24孔板,放入二氧化碳细胞培养箱(5%CO2,37℃),样品和阴性对照培养,光学显微镜下观察细胞生长状态,当细胞贴壁生长、融合度达到90%且形态良好时,更换成骨诱导培养基,之后每48h换液一次,连续培养21天后进行相关指标检测。
4)ALP染色。
用移液枪吸净24孔板中旧培养基,每孔加入400μl清理液清洗细胞,重复操作三次,清洗完成后加入300μl固定液(4%多聚甲醛),使其完全覆盖整个细胞表面,在37℃下固定10min,用移液枪吸净24孔板中固定液,每孔加入400μl清理液清洗细胞,重复操作三次,清洗完成后加入300μl染色液,使其完全覆盖整个细胞表面,在37℃下染色30min左右,用移液枪吸净24孔板中固定液,每孔加入400μl清理液清洗细胞,重复操作三次,光学显微镜下观察细胞染色情况。
实验结果:ALP染色定性结果显示,特立帕肽糖基化衍生物均显示出良好的体外成骨细胞促进作用,结果如图1所示。
本发明的有益效果是:
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (5)
1.新的特立帕肽糖基化衍生物,其特征在于,具体指具有式(I)结构的多肽。
具有式(I)结构的多肽及其药学上可接受的盐或酯:
X1VX2EIQLMH X3LGKHL X4X5MERVEWLRKKLQ X6VHNF (I)
其中,
X1,X2,X5表示Ser(GlcNAc)或Ser(Glc)或Ser(Fuc)或Ser(Rib)或Ser(Gal)或Ser(GalNAc)或Ser(Lyx)或Ser(Rha)或Ser(Man)或Ser(ManNAc)或Ser(Cel)或Ser(Mal)或Ser(Suc)或Ser(Lac)或Ser(Tre)或Ser。
X3,X4,X6表示Asn(GlcNAc)或Asn(Glc)或Asn(Fuc)或Asn(Rib)或Asn(Gal)或Asn(GalNAc)或Asn(Lyx)或Asn(Rha)或Asn(Man)或Asn(ManNAc)或Asn(Cel)或Asn(Mal)或Asn(Suc)或Asn(Lac)或Asn(Tre)或Asn。
2.含有上述具有式(I)结构的多肽片段的药物组合物,其特征在于,该药物组合物可以含有权利要求1中任一项所述的多肽,优选仅含一种特立帕肽糖基化衍生物。
3.根据权利要求2所述的含有上述具有式(I)结构的多肽片段的药物组合物,其特征在于,其还含有药学上可接受的稀释剂、赋形剂或载体。
4.根据权利要求3所述的含有上述具有式(I)结构的多肽片段的药物组合物,其特征在于,其中所述的载体是乙醇、甘油或水中的一种或多种。
5.新的特立帕肽糖基化衍生物的应用,利用具有式(I)结构的多肽片段的药物组合物,可以用于治疗骨质疏松症。
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