CN1176947C - 用于治疗骨质疏松症的甲状旁腺激素类似物 - Google Patents
用于治疗骨质疏松症的甲状旁腺激素类似物Info
- Publication number
- CN1176947C CN1176947C CNB971981612A CN97198161A CN1176947C CN 1176947 C CN1176947 C CN 1176947C CN B971981612 A CNB971981612 A CN B971981612A CN 97198161 A CN97198161 A CN 97198161A CN 1176947 C CN1176947 C CN 1176947C
- Authority
- CN
- China
- Prior art keywords
- lys
- leu
- analogue
- seq
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Abstract
本发明描述了在骨修复时具有增强的活性和增高的生物有效性的人甲状旁腺激素(hPTH)类似物。所述的肽是hPTH-(1-31)的衍生物;它们通过例如Glu22和Lys26之间或Lys26和Asp30之间形成内酰胺环化。并且,天然的Lys27可被Leu或其它疏水性残基,如Ile、正亮氨酸、Met、Val、Ala、Trp或Phe替换。典型地,这些类似物具有增强的刺激大鼠骨肉瘤细胞腺苷酰环化酶活性,在卵巢切除的大鼠模型中表现增强的骨修复活性。这些类似物也表现增强的活性和生物有效性,这一点可以由它们在大鼠中的降血压效应证明。也描述了使降血压活性和骨生成活性相互关联的分析方法。
Description
发明领域
本发明涉及人甲状旁腺激素的类似物,并发现它们在治疗骨质疏松症时有效。
发明背景
骨质疏松症是老年人,尤其是老年妇女残疾的首要原因。最近认识到人甲状旁腺激素(hPTH)及其某些类似物是骨生长的刺激素,它们在治疗骨质疏松症时有用。骨质疏松症是导致总骨质减少和骨脆性增强的渐进性疾病。这常常导致负重骨骼的自发性骨折和影响活动损伤典型的身体和精神退化。绝经后骨质疏松症由雌激素消失引起,雌激素消失引发骨代谢的长期加速,增加旧骨骼吸收和新骨骼形成之间的不平衡。这可导致负重骨骼变薄,多孔性增加和小梁减少。骨质疏松症与甲状腺功能亢进、甲状旁腺功能亢进、库兴氏综合症和某些类固醇药物的使用有关。历史上的治疗法包括增加饮食钙、雌激素疗法以及增加维生素D剂量,但主要是用诸如抑制成骨细胞骨吸收的抗吸收剂之类的试剂。
甲状旁腺激素(PTH)由甲状旁腺产生,是血钙水平的主要调节剂。PTH是多肽并且可以按Erickson和Merrifield,“蛋白质”,Neurath等编辑,学院出版社,纽约,1976,257页中公布的方法制备合成的多肽,并按Hodges等(1988),肽研究,1,19或Atherton,E.和Sheppard,R.C.,“固相肽合成”,IRL出版社,牛津,1989中的方法进行修饰。
当血钙低于正常水平时甲状旁腺释放PTH,并通过骨钙再吸收、通过增加从肠中吸收钙、通过增加肾小管中从原尿重新吸收钙来提高钙水平。尽管连续输注低水平的PTH可以从骨中清除钙,但是间歇注射同样低剂量的PTH确实可以促进骨生长。
Tregear,美国专利4,086,196描述了人PTH类似物并声称在体外细胞试验中,开始的27至34位氨基酸在促进腺苷酰环化酶方面最有效。Rosenblatt,美国专利号4,771,124公开了其中Trp23被苯丙氨酸、亮氨酸、正亮氨酸(Nle)、缬氨酸、酪氨酸、β-萘基丙氨酸或α-萘基丙氨酸替换的hPTH类似物作为PTH拮抗剂的特性。这些经修饰的hPTH类似物也去掉了氨基端的2位和6位氨基酸,导致在治疗骨质疏松症时丧失大部分兴奋剂活性。设计这些类似物作为PTH和PTH相关多肽的抑制剂。这些类似物据称可能在治疗与一些肿瘤相关的高血钙时有用。
Pang等,WO 93/06845(公开于1993年4月15日)描述了hPTH的类似物,其中涉及用多种氨基酸包括丙氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、蛋氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换Arg25、Lys26、Lys27。认为这些类似物在治疗骨质疏松症时有效并对血压和平滑肌影响极小,但没有来自动物或人体实验的支持证据。
PTH通过激活两个第二信号系统,即Gs-蛋白激活的腺苷酰环化酶(AC)和Gq-蛋白激活的磷脂酶Cβ起作用。后者导致膜结合蛋白激酶Cs(PKC)活性的刺激作用。已表明PKC活性需要PTH的29至32位残基〔Jouishomme等(1994),骨矿物质研究杂志,9,(1179-1189)〕。已确定骨生长的增加,即在治疗骨质疏松时有用的效应,与肽序列提高AC活性的能力相偶联。已表明天然PTH序列具备所有这些能力。hPTH-(1-34)序列通常表示为(A):
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
Gln Asp Val His Asn Phe-OH
下面的线性类似物(B),hPTH-(1-31)-NH2(下面表1中包含了它的数据)只具有刺激AC的活性,并已证明它在卵巢切除的大鼠模型的骨丢失重建中有全部的活性〔Rixon,R.H.等(1994),骨矿物质研究杂志,9,1179-1189;Whitfield等(1996),Calcified Tissue Int.,58,81-87;Willick等,美国专利号5,556,940,授于1996年9月17日〕:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
Gln Asp Val-NH2
上述分子(B),可具有自由的羧基末端而不是所示的酰胺末端。
本发明的目的是生产新型PTH类似物,它们具有更好的代谢稳定性、增加的骨重建活性、增加的AC活性和最小的临床副作用。
发明概述
根据本发明的一个方面,提供了具有以下氨基酸序列的人甲状旁腺激素hPTH及其药学上可接受的盐。
R-NH-R1-Val-Ser-Glu-Ile-Gln-Leu-R2-His-Asn-Leu-Gly-Lys-R3-R4-R5-R6-R7-
Glu-Arg-Val-R8--Trp-Leu-R9--R10--R11-Leu-R12-Asp--Y
其中,
R=氢或任何直链或支链烷基、酰基或芳香基,
R1=Ser、Ala或α-氨基丁酸,
R2=Met、Nle、Leu、Ile、Val、Phe或Trp,
R3=His、Lys或Glu,
R4=Leu、Lys、Arg或Glu,
R5=Asn、Lys、Org、Hci、Asp、Arg、DLys、Ser、Gly或Glu,
R6=Ser、Lys、Asp、Arg或Glu,
R7=Met、Nle、Leu、Ile、Val、Phe或Trp,
R8=Glu、Lys、Cys、Orn或Asp,
R9=Arg、Lys、His、Cys、Glu或鸟氨酸(Orn),
R10=Arg、Lys、Orn、Gln、Cys、Glu或Asp,
R11=Lys、Leu、Ala、Tyr、Orn、瓜氨酸、α-氨基丁酸、Nle、Ile、Met、Gln、Asp、或侧链具有2-10个碳原子的直链或支链α-氨基脂肪酸或其在脂肪链末端带有氨基、羧基、乙酰氨基、胍基或脲基的类似物,
R12=Gln、Arg、Cys、Glu、Asp、Lys或Orn,
R13=Asp、Cys或Lys,
X=OH、NH2,并且
Y=X,Val-X,Val-His-X,Val-His-Asn-X,Val-His-Asn-Phe-X,Val-His-Asn-Phe-Val-X,Val-His-Asn-Phe-Val-Ala-X和Val-His-Asn-Phe-Val-Ala-Leu-X,在从氨基酸对22和26、26和30、27和30、和25和29中选择的一个或两个氨基酸对之间环化,条件是当R9是Lys或Orn和R12是Glu或Asp时在25和29位之间环化;其中环化作用之一在氨基酸对22和26之间或氨基酸25和29之间。
盐类的例子包括无机酸盐,有机酸盐如甲酸、乙酸、酒石酸和柠檬酸,无机碱的盐如钠和铵和有机碱的盐如三乙胺、乙胺和甲胺。
根据本发明的另一个特征是环化受内酰胺形成的影响,涉及所选氨基酸对天然残基22和26或26和30之间侧链的偶联。也包括其它形式的环化如二硫键的形成,例如在含有Cys的类似物Cys22-Cys26和Cys26-Cys30之间二硫键的形成。
已发现多种氨基酸替换也有效。Lys27可以被Leu或其它天然存在的疏水性或极性残基替换。另一个因素是残基与受体适合的程度。Ala与Leu不同是非疏水性的。通常认为Lys和Tyr是极性的,但它们与受体有疏水作用。例如,Lys的折叠可使其疏水部分与受体其它疏水残基作用并使NH2与溶剂接触。这样的替换包括鸟氨酸、瓜氨酸、α-氨基丁酸、丙氨酸、正亮氨酸、异亮氨酸和酪氨酸,或任何直链或在侧链上有2-10个碳原子的支链α-氨基脂肪酸,任何在脂肪链末端有极性或带电基团的此类类似物。极性或带电基团的例子包括氨基,羧基,乙酰氨基,胍基和脲基。尽管看来Leu27是最好的替换,但是看来许多27位的其它替换也可保留几乎全部活性并且也具有所需特征如增加的蛋白水解酶稳定性或水溶性,预测Ile、正亮氨酸、Met和鸟氨酸是活性最高的。
替换使激素受体结合区α-螺旋稳定化。这一点已通过内酰胺类似物的圆二色谱与线型分子[Leu27]-hPTH-(1-31)-NH2的圆二色谱的比较得到证实。圆二色谱对α-螺旋二级结构的存在高度敏感,并且此技术已用于证明hPTH片段中α-螺旋的存在[Neugebauer等(1991),生物化学31,2056-2063]。此外,已表明hPTH-(20-34)-NH2中上述内酰胺的形成使α-螺旋稳定[Neugebauer等(1994),国际蛋白质多肽研究杂志,43,555-562]。在hPTH-(1-31)-NH2的21位和31位残基之间有一个潜在的两亲α-螺旋,有数据表明此螺旋的疏水面与PTH受体相互作用[Neugebauer,W.等(1995),生物化学,34,8835-8842;Gardella,T.J.等(1993),内分泌学,132,2024-2030]。
已发现最为有效的环化涉及内酰胺的形成,例如在Glu22和Lys26或Lys26和Asp30残基之间内酰胺的形成。也可能是其它的环化如Lys27和Asp30之间的环化,但是已发现这种内酰胺对α-螺旋有一定去稳定作用。
更具体地,PTH片段的受体结合研究已表明14-34残基内有主要结合区域。1我们认为残基17-29α-螺旋本身与PTH受体结合,并且此α-螺旋的两亲部分的疏水面与受体结合。2此模型与受体结合区域类似物的研究结果一致。3
NMR研究表明即使由圆二色性(CD)确定的高度螺旋化模式肽也有许多非螺旋构象。因此,受体结合肽激素如PTH的结构不可能从溶液中的自由结构可靠地推断出来。只使用有限的肽激素类似物以限制肽可能的构象状态数量8。hPTH序列的测定表明在17-29位残基内有3个可能的盐桥,它们或使α-螺旋稳定或使其不稳定。预计位于Glu22和Lys26及Lys26和Asp30之间的两个盐桥使α-螺旋稳定,位于Lys27和Asp30之间的盐桥使α-螺旋不稳定。4在这些残基对之间形成内酰胺将限制hPTH在该螺旋区内的可能构象。而且,预计其中的两个内酰胺,Glu22-Lys26和Lys26-Asp30,使位于α-螺旋两亲部分极性面的α-螺旋结构稳定。预计第三个内酰胺,Lys27-Asp30,至少部分地使α-螺旋不稳定,并涉及位于两亲螺旋疏水面的Lys27残基。如果Lys或Orn替代25位的Arg并且Glu或Asp替代Gln25,那么也可发生25和29之间的环化。
Leu替换Lys27可导致两亲螺旋的疏水面有疏水性更强的残基。这引起ROS细胞系中刺激腺苷酸环化酶的活性升高。本领域的熟练技术人员应当理解,上面讨论的其它这样的替代很可能会产生具有相同或更高活性的类似物。
预期氨基酸残基替换和任一内酰胺形成的综合效应是稳定α-螺旋、提高生物活性并保护分子的这个区域不受蛋白水解。在希望进一步保护此肽免受外切蛋白水解降解的意义上,优选C末端存在酰胺基,尽管某些肽酶仍可水解它们。[Lesile,F.M.和Goldstein,A.(1982),神经肽2,185-196]。
已发现形成其它氨基酸替换也是有效的。具体地说,我们已按照日本专利公开61-24598,用天然存在的疏水残基Nle替换对氧化敏感的8和18位的Met残基。根据授与Nakagawa等的美国专利5,393,869,预计其它诸如Leu,Ile,Val,phe和Trp的疏水残基也是有用的。
也考虑到反转内酰胺。例如,我们已证实Lys22-Glu26转换的有效性。因此预计可以在26-30和27-30之间有效地形成相似的转换。
除了前述Cys-Cys,已在优选的22-26内酰胺位点进行另一替换,即Asp22-Orn26以举例说明形成大小不同的环化/环是有效的。
在美国专利5,393,869(Nakagawa等)和在美国专利5,434,246(Fukuda等)中报道了某些替换的hPTH类似物具有重要的腺苷酸环化酶活性并且可能具有增强的对于蛋白水解攻击的稳定性,具体地,
1.Ser-1替换为Aib(α-氨基丁酸)
2.Lys-27替换为Gln(据报道有2.5倍的腺苷酸环化酶活性)
3. 14,15,16,17位残基全部或部分替换为Lys(据报导,大大地提高活性多达8倍,这可能归因于水溶性的提高。预计它们在体内对胰蛋白酶样酶更不稳定)。他们要求此四肽(14-17位残基,包含首尾两个残基)至少含有一个水溶性氨基酸,例如His-14或Lys-14;Leu-15,Lys-15或Arg-15;Asn-16,Orn-16,Hci(高瓜氨酸)-16,Asp-16,Arg-16,Lys-16,Dlys-16,Ser-16或Gly-16;和17-Ser,17-Lys,17-Asp或17-Arg。例如也可包含Glu。
4.Arg25替换为His以使蛋白酶攻击降至最低。既然我们的内酰胺,尤其与27位Leu或另一疏水氨基酸形成的内酰胺可变得有些不溶解并且也可能已变得难于溶解,那么可以期望相同的替换可用于我们的内酰胺。
本领域的技术人员也将可以理解尽管1-31h-PTH环是优选的,但是从此处得到的数据可以预计1-30至1-37的环状片段也将是有效的。特别是,文献中没有证据表明直至37的附加氨基酸会影响激素的生物学特性,特别是已给出此处包含的确定的1-34数据。
根据本发明所述的内酰胺可以按照下述已知的方法制备,可以在各种情况下如治疗骨质疏松症和一般骨折时用于刺激骨生长、骨重建和促进骨愈合。
附图简述
图1表示天然人PTH残基1-31(SEQ ID.NO:1)的结构;
图2表示[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ ID NO:3)的结构;
图3表示[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2(SEQ ID NO:4)的结构;
图4表示根据本发明所述的类似物对ROS17/2细胞腺苷酰环化酶的刺激活性;
图5表示在卵巢切除(OVX)后第8周末制备的代表性骨组织学切片,说明hPTH-(1-31)NH2及其内酰胺衍生物在卵巢切除的(OVX)Sprague-Dawley大鼠中防止骨损失和刺激骨生长的不同能力;
图6表示对照动物和经hPTH类似物处理的动物的骨小梁体积,动物是起初严重骨衰竭的大鼠。卵巢切除的第9周开始处理动物。[Leu27]环[Glu22-Lys26]hPTH-(1-31)-NH2是最有效的片段,使骨骼恢复到正常对照大鼠的数值;并且
图7表示正常的、卵巢切除的(OVX)、模拟的以及用hPTH-(1-31)-NH2、[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2和[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2处理的大鼠股骨小梁的厚度;
图8表示施用0.8nmol/100g剂量的hPTH-(1-31)-NH2、[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2或[Leu27]环(Lys26-Asp30)hPTH-(1-31)-NH2之后血压降低的最大值以及血压达到最大降低值的时间。皮下(空心柱)或静脉内(影线柱)施用多肽。
图9表示[Leu27]-hPTH-(1-31)-NH2(SEQ ID NO:5)的结构。
图10表示环(Lys27-Asp30)-hPTH-(1-31)-NH2(SEQ ID NO:6)的结构。
图11表示[Leu27]环(Glu22-Lys26)-hPTH-(1-30)-NH2(SEQ ID NO:7)的结构。
图12表示根据本发明所述的各种类似物刺激腺苷酰环化酶的活性。
图13表示hPTH(1-31)NH2和hPTH(1-30)NH2及其环状类似物的降血压作用。
图14是说明不同剂量线形hPTH(1-31)-NH2对骨生长影响的图。
图15表示[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-OH(SEQ ID NO:8)的结构。
图16表示[Leu27]环(Glu22-Lys26)-hPTH-(1-34)-NH2(SEQ ID NO:9)的结构。
图17表示[Leu27]环(Glu22-Lys26)-hPTH-(1-34)-OH(SEQ ID NO:10)的结构。
图18表示[Ala27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ IDNO:11)的结构。
图19表示[Nle27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ IDNO:12)的结构。
图20表示[Nle8,18;Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQID NO:13)的结构。
图21表示环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ ID NO:14)的结构。
图22表示[Ile27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ ID NO:15)的结构。
图23表示[Tyr27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ IDNO:16)的结构。
图24表示α-乙酰基[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(SEQ ID NO:17)的结构。
图25表示[Leu27]环(Lys22-Glu26)-hPTH-(1-31)-NH2(SEQ IDNO:18)的结构。
图26表示[Leu27]环(Asp22-Orn26)-hPTH-(1-31)-NH2(SEQ IDNO:19)的结构。
图27表示[Cys22;Cys26;Leu27]环(Cys22-Cys26)-hPTH-(1-31)-NH2(SEQ ID NO:20)的结构。
图28表示[Cys26;Cys30;Leu27]环(Cys26-Cys30)-hPTH-(1-31)-NH2(SEQ ID NO:21)的结构。
激素类似物的制备
由R.B.Merrified发展的固相肽合成技术(“固相肽合成”,酶学进展,32,221-296,1969;此处引用作为参考)被广泛成功地用于诸如甲状旁腺激素的多肽合成。此策略是基于将肽的氨基酸的羧基端连到固相载体上,然后将顺序的氨基酸以高产率连接上去。以保护基团可以除去而肽不会从固相载体去除的方式保护N末端α氨基基团。此处所用的化学方法涉及原始Merrifield方法的改良,称为Fmoc方法。Fmoc(9-芴甲氧羰基)基团可通过温和碱性条件除去,同时留下碱稳定的侧链保护基团,并且不影响与载体的连接。这种技术在E.Atherton和R.C.Sheppard,“固相肽合成:实用方法”IRL出版社,纽约,N.Y.中描述,此处引用作为参考。
实施例1 线性hPTH-(1-31)酰胺类似物的合成和纯化
在偶联过程中,氨基酸的α-氨基受到9-芴甲氧羰基(Fmoc)的保护。用1-羟基苯并三唑(HOBt),2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基脲四氟硼酸盐(TBTU)和二异丙基乙胺(DIPEA)的混合物进行偶联。使用4倍过量的活化氨基酸用双偶联法加上Asn,Gln,His,Val和Ile残基。加上Arg和Gly的偶联时间从30分钟增加到60分钟。用人工操作将第一残基(Val31)偶联到载体(Tentagel*R,Rapp Polymere,Tubingen,Germany)上。所有其它步骤在PerSeptive Biosystems*Model9050 Plus自动肽合成仪上进行。侧链保护如下:Arg(2,2,5,7,8-五甲基苯并二氢吡喃-6-磺酰基);Glu,Asp和Ser(t-丁基);His,Gln和Asn(三苯甲基);Trp(t-丁氧羰基)。
从N-末端Ser去除Fmoc后,用DCM洗涤肽-树脂,然后用7.5ml试剂K[6.19ml三氟乙酸(TFA),0.38ml H2O,90%苯酚/水,苯甲硫醚和0.19ml1,2-乙二硫醇]通过于20℃振荡4小时从树脂上切下肽。通过过滤并加入t-丁基甲醚沉淀来回收切下的肽混合物。通过离心收集沉淀,并用t-丁基甲醚洗两次,然后通过真空离心使之干燥。
将粗制品溶于14ml 15%乙腈/水,0.1%TFA中并在Vydac*C18柱(10μ,1×25cm)上层析。用含于0.1%TFA中的乙腈水溶液(14-40%)的1%/分钟梯度洗脱产品。在Vydac*C18柱(10μ,0.4×25cm)上通过分析型高压液相色谱(HPLC)测定终产品的纯度,并用电喷射质谱仪(VG Quattro)分析分子量。这样得到的hPTH-(1-31)-NH2的数据列于下面表1中。
实施例2 环状类似物的合成和纯化
[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2肽按实施例1描述的方法合成。在26位和22位分别用Lys-Alloc和Glu-OAII替换。加入Fmoc-Ser17后,将肽-树脂从柱上移至反应管(Minivial*,AppliedScience)中,并在氩气中悬浮于1.7ml含四重(三苯膦)钯(0.24mmol),5%乙酸和2.5%N-甲基吗啉(NMM)的二氯甲烷(DCM)溶液中,然后于20℃振荡6小时以除去烯丙基和alloc保护基团[Sole,N.A.等(1993),于肽:化学、结构和生物学中,Smith,J.和Hodges,R.(编辑),ESCOM,第93-94页,此处引用作为参考]。然后用含0.5%二乙基二硫代氨基甲酸酯(DEDT),0.5%NMM的DMF(50ml)洗肽-树脂,再用DMF(50ml)和DCM(50ml)洗。肽(0.06mmol)通过与2ml含0.06mmol 1-羟基-7-氮杂苯并三唑(HOAt)/0.12mmol NMM的DMF一起于20℃振荡14小时环化[Carpino,L.A.(1993)美国化学学会杂志,115,4397-4398]。将肽-树脂过滤,然后用DMF洗一次,并重新装回柱中,用DMF洗涤直至悬液中的气泡消失。其余的合成按实施例1中的方法进行,只是不去掉N-末端的Fmoc基。用前述的试剂K将Fmoc-肽从树脂上切割下来。按实施例1中的方法进行HPLC,在最终HPLC之前去掉Fmoc基。
以相似的方法制备类似物[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2。
实施例3 腺苷酰环化酶测定
在具分化能力的成骨细胞样ROS17/2大鼠骨肉瘤(RUS)细胞系上测定hPTH类似与受体结合及其活化腺苷酰环化酶偶联的信号机制的能力。已知此活性与类似物在卵巢切除的大鼠中重建骨质的能力紧密偶联。刺激腺苷酰环化酶的活性通过用[3H]-腺嘌呤预标记细胞ATP库,然后测量与特定类似物接触后开始的10分钟期间由[3H]-ATP产生的[3H]-环AMP的量来评估。这基于在Whitfied等,细胞生理学杂志,150,299-303,1992中描述的方法,此处引用作为参考。
腺苷酰环化酶结果在下面表2中表示为使AC活性达到最大增量的一半时所需的浓度。此数据也在图4中表示。图4中,实心圈代表hPTH-(1-31)-NH2的腺苷酸环化酶刺激活性,而[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2和[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2的活性分别由空心三角和实心三角表示。
实施例4用卵巢切除的大鼠模型测定hPTH类似物的合成代谢活性剂量:
剂量基于对hPTH(1-31)-NH2的剂量反应数据。使用几种剂量(0.8,0.6,0.4和0.2nmole/100g体重)并以再生或治疗评价而不是预防模式来测试hPTH-(1-31)NH2的骨重建能力。11将3月龄的大鼠切除卵巢后放9周以使其大部分非板层股骨的骨小梁严重衰竭,然后开始6周的每日注射。9周结束时,它们已丧失了75%的股骨骨小梁量。图14表示了由4种不同剂量片段注射36次产生的板层沉积引起的平均小梁厚度的剂量依赖性增加。此片段也可在大得多的大鼠中刺激骨小梁生长。因此,用0.6nmole/100g体重中度剂量的hPTH-(1-31)NH2注射36次能使卵巢切除后预衰竭9周的1岁龄大鼠的股骨骨小梁平均小梁厚度明显高于正常起始值,它们与hPTH-(1-84)一样有效。相应地,在后面的试验中使用0.6nmole/100g体重剂量。
此方法的详细描述在Rixon等,骨骼和矿物质研究杂志,9,1179-1189,1994和Whitfield等,Calcified Tissue Int.,58,81-87,1996中给出,此处引用作为参考。正常的,模拟OVX(卵巢切除的),和OVX Sprague-Dawley大鼠(3月龄;255-260g)购自Charles River实验室(St.Constant,QC)。将这些大鼠以8只动物/组随机分组并随意食用Purina大鼠饲料和水。没有意外死亡。从OVX后第2周末开始到OVX后第8周末结束,每天一次每周六次对动物进行皮下注射(即36次注射)。8只模拟OVX和8只OVX对照大鼠接受36次载体注射(含0.001N HCl的0.15N NaCl),而8只OVX大鼠接受含于载体中的0.6nmole/100g体重的片段。OVX后第8周末,取出并分离洗净股骨并在骨干中部切成两半,丢弃近端部分。除骨骺后,将每个半股骨纵向切成两部分并冲出骨髓。
片段的骨重建能力从经各种处理的动物的远端半股骨小梁平均厚度(面积/周长)的变化来判断。为了测量平均小梁厚度,将每只大鼠的两个脱矿质半股骨脱水并包埋于石蜡中。从每块骨的中间平面切割纵向的10μm切片并用Sanderson快速骨染料染色(Surgipath MedicalIndustries,Inc,Winnipeg,MB,加拿大)。用M4成像系统和来自成像研究公司(St.Catherines,ON,加拿大)的骨形成度量软件测量平均小梁厚度。
在OVX后第8周末制备的代表性组织学切片表示于图5中。此结果进一步以柱形图形式表示于图7中。柱形表示正常的、卵巢切除的(OVX)、模拟的、hPTH-(1-31)-NH2、[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2和[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2的小梁厚度值。与线性类似物hPTH-(1-31)-NH2相比,[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2表现特别高的活性。此线性类似物在骨修复中表现充分的活性,但只使用一条信号(AC活化的)途径。因此,预计环形类似物与其线性类似物一样比更长类似物如hPTH-(1-34)或hPTH-(1-84)的不利临床副作用更少,并且环形类似物活化两条细胞信号机制。
实施例5 hPTH类似物对骨小梁严重衰竭大鼠的骨重建作用
在这个骨恢复的第二个例子中,比较了内酰胺片段重建严重衰竭骨小梁的能力。在此实验中,6周每天一次0.6nmole/100g年轻性成熟大鼠体重的肽注射推迟到OVX后第9周末。此时,已丧失75%骨小梁。在图6中可以看到,[Leu27]环[Glu22-Lys26]-hPTH-(1-31)NH2是最有效的片段。它可将骨小梁体积恢复到正常大鼠的数值。
实施例6 hPTH类似物的降血压效应
腹膜内注射戊巴比妥钠(65mg/kg体重)麻醉雌性Sprague-Dawley大鼠(体重超过290g)。用YS1402半导体温度计(Yellow SpringsInstrument Co.,Inc.Yellow Springs,OH)监测直肠温度并在整个实验过程中将温度保持在36.0到38.5℃之间。也用YSI banjo半导体温度计监测耳廓温度。使尾部动脉暴露并插入Jelco 25-gIV导管(Johnson和Johnson Medical Inc.,Arlington,TX)并连接到Statham压力转导器上,用Biopac Systems MP100监测器(Harvard Instruments,Saint Laurent,QC,加拿大)以数字方式记录来自压力转导器的信号。也使股骨静脉暴露以便静脉注射PTH或其某一片段。手术后,使大鼠稳定8分钟,之后将PTH或其某一片段(溶解于含0.001N HCl的酸性盐水中)注射入股静脉或腹部皮下。静脉注射12分钟或皮下注射22分钟后收集数据。图8表示经皮下(空心柱)或静脉(影线柱)途经注射0.8hmol/100g剂量的[Leu27]环(Glu22-Lys26)hPTH-(1-31)-NH2或[Leu27]环(Lys26-Asp30)hPTH-(1-31)-NH2后,血压下降的最大值和达到最大下降值的时间。与[Leu27]环(Lys26-Asp30)hPTH-(1-31)-NH2相比,[Leu27]环(Glu22-Lys26)hPTH-(1-31)-NH2类似物表现增高的生物有效性。这可由皮下注射后使血压降到最低所需时间更短得多来证明。与hPTH-(1-31)-NH2相比,两种环形类似物经皮下注射时都表现增强的降血压效应。因此,预计每种环形内酰胺类似物经皮下注射时具有比其线性类似物更好的特性。这些特性包括由对蛋白酶抗性增强和/或从脂类环境转运能力增加而导致的更大生物有效性。从脂类环境转运能力的增加归功于靠近激素C末端的两亲螺旋的稳定作用。
进一步结果:
为了说明这些结果,按下述识别这些肽:
hPTH-(1-31)-NH2(1);[Leu27]-hPTH-(1-31)-NH2(2)(图9);[Leu27]环(Glu22-Lys26)-hPTH-(1-31)-NH2(3)(图2);[Leu27]环(Lys26-Asp30)-hPTH-(1-31)-NH2(4);环(Lys27-Asp30)-hPTH-(1-31)-NH2(5)(图10);hPTH-(1-30)-NH2(6);[Leu27]hPTH-(1-30)-NH2(7);[Leu27]环(Glu22-Lys26)-hPTH-(1-30)-NH2(8)。
腺苷酰环化酶活性。
我们曾报道[Leu27]-hPTH-(1-34)-NH2在ROS细胞系中刺激AC的活性比hPTH-(1-34)-NH2更高。5我们也发现肽2(EC50,11.5±5.2nM)比天然序列1(EC50,19.9±3.9nM)更有活性(图12)。图示为肽1(■);2(□);3(△);4(▲);5(O)。Glu22和Lys26之间内酰胺的形成(3)引起更高的AC刺激活性,EC50值为3.3±0.3nM(图12)。因此,环化和以Leu替换Lys27的净效应是活性提高6倍。相反,Lys26和Asp30(4)或Lys27和Asp30(5)之间内酰胺的形成使腺苷酰环化酶刺激活性比其线性序列更弱。因此,26-30内酰胺(4)具有比其线形形式稍低的活性,EC50为17.0±3.3nM,而2的EC50为11.5±5.2nM。27-30内酰胺(5)更明显地降低原来线形肽的活性,EC50为40.3±4.4nM,而1的EC50为19±3.9nM。
我们曾报道hPTH-(1-30)-NH2(6)具有与类似物1相近的AC刺激活性(EC50,20nm)。我们现在发现[Leu27]-hPTH-(1-30)-NH2(7)和环(Glu22-Lys26)-hPTH-(1-30)-NH2(8)(图11)具有与肽6相似的AC刺激活性。
降血压效应:
图13说明了线形(6和7)和环形内酰胺类似物(3和8)的降血压效应。显示的是用0.8nmol/100g体重的类似物注射大鼠后得到的血压最大下降值(上部)以及血压高达到最大下降值所费的时间(下部)。静脉(阴影柱)或皮下注射(空心柱)类似物(从左至右,肽8,7,6,3)。Val31的去除导致所有类似物6,7和8皮下注射的肽转运到血管系统的速度明显(P<0.05)下降(图13)。肽6和8不论是经皮下注射还是静脉注射,其总血压实际下降值与除肽5以外的其它类似物没有明显的差异(P>0.05)。
降血压效应与骨生成活性的相关性实验
如实施例6所述和图8所示,皮下注射hPTH类似物后观察到的快速降血压效应与骨生成活性相关。相应地,此实验可以在不杀死实验动物的条件下用于筛选可能的具骨生成效应的hPTH类似物并排除不降血压的构建体。
作为背景,我们使用一系列独特的骨生成和非骨生成的AC-,AC/PLC-或PLC-活化的PTH片段来明确证实只有AC刺激降低血压并比较这些片段经皮下或静脉注射雌性大鼠时的降血压作用。我们已证明降血压效应只由AC-或AC/PLC-刺激片段引发,而且是对皮下注射相对小的降血压反应而不是对静脉注射大得多的反应与OVX大鼠中hPTH-(1-31)NH2、hPTH-(1-34)和hPTH-(1-84)的骨生成活性相关,但与hPTH-(1-30)NH2无关,hPTH-(1-30)NH2刺激AC并降低血压但不刺激骨形成。
具体地,hPTH-(1-84)及其hPTH-(1-34)、hPTH-(1-31)NH2和hPTH-(1-30)NH2片段在注射入股骨静脉约1分钟内使麻醉的雌性Sprague-Dawley大鼠的尾部动脉血压下降42.4-67.1%,而在皮下注射后2-19分钟仅使血压下降8.5-36.2%。hPTH-(1-84)和hPTH-(1-34)可在它们的靶细胞中刺激腺苷酰环化酶和磷脂酶C,但是降血压效应必须特异地由腺苷酰环化酶活化刺激,因为仅能刺激腺苷酰环化酶的hPTH-(1-30)NH2和hPTH-(1-31)NH2经静脉注射时是高效降血压的,而仅能刺激磷脂酶C的hPTH-(7-84)经静脉注射时不明显降血压。既然PTH的骨生成作用由腺苷酰环化酶刺激作用介导,那么预计可以用降血压反应筛选可能的具骨生成作用的新型PTH构建体。的确,皮下注射的hPTH-(1-31)NH2、hPTH-(1-34)和hPTH-(1-84)的骨生成活性与其降血压活性紧密相关,其中hPTH-(1-34)比另外两个分子的降血压作用更强、更明显。hPTH-(1-31)NH2和hPTH-(1-84)同样具骨生成作用和降血压作用。然而这种相关性对于hPTH-(1-30)NH2来说却不存在,hPTH-(1-30)NH2与hPTH-(1-31)-NH2和hPTH-(1-34)几乎同样强地刺激AC并与hPTH-(1-31)-NH2同样多地降低血压但不刺激骨形成。不过,明显降低动脉压的能力是骨生成PTH和PTH片段的共同特征,因而是PTH片段体内生物活性可快速测定的基本指示标记。
更具体地,hPTH-(1-34)和hPTH-(1-31)-NH2在0.8nmole/100g体重时具最大降血压作用。
此降血压反应伴随着大鼠耳朵和爪子的短暂(20-30分钟)变红,这可以作为有用的目测定性标记。
尽管刺激AC或AC/PLC的片段经静脉注射时具有有效的降血压作用,但是与刺激AC/PLC的hPTH-(1-84)引起的快速(0.9±0.08分钟)43.2±5.8mmHg下降和刺激AC的hPTH-(1-34)NH2引起的同样快速的51.6±3.3mmHg下降相比,静脉注射0.8nmol/100g体重仅能刺激PLC的hPTH-(7-84)时,非常缓慢(9.8±2.0分钟)并且仅仅轻微地使尾部动脉血压降低3.28±1.0mmHg。
皮下注射刺激AC或AC/PLC的片段比静脉注射相同剂量引起的尾部动脉血压下降更慢得多并且更小得多。例如,皮下注射0.8nmol/100g体重的hPTH-(1-34)时使血压下降27.9±3.6mmHg,而静脉注射使血压下降50.9±5.6mmHg。皮下注射相同剂量的hPTH-(1-31)NH2使血压下降11.1±1.6mmHg,而静脉注射使血压下降51.6±3.3mmHg。
由静脉注射hPTH-(1-84)、hPTH-(1-34)和hPTH-(1-31)NH2引起的尾部动脉血压快速而大幅的下降没有明显不同,但是皮下注射时,hPTH-(1-34)的效率至少是其它两种分子的两倍(P<0.01)。
影响皮下注射的PTH或PTH片段的降血压作用和骨生成作用的因素之一是它们在失活前首先从注射点迁移到维管结构靶位点然后迁移到骨的能力。这种能力由注射后尾部动脉血压下降到最小值所需的时间反映出来。从C末端切掉31-84位残基使PTH分子变短并消除了PTH刺激PLC的能力而未减小其刺激AC的能力,并使尾部动脉血压达到最小值所需时间降低九倍。再去掉一个残基将hPTH-(1-30)NH2使血压下降到最小值所需的时间从hPTH-(1-31)NH2的2.0±0.31分钟大幅度地提高到13.8±2.3分钟。然而,重要的是注意到hPTH-(1-31)NH2降低血压所需时间不比hPTH-(1-84)长。
目前hPTH-(1-31)NH2在研究中的使用已提供了最直接的证据表明降血压作用完全源于AC活化,hPTH-(1-31)NH2是第一个能与hPTH-(1-34)一样强地刺激AC而不活化PLC和刺激膜相关的PKC活性的构建体。因此,尽管静脉注射0.8nmol/100g体重仅能刺激PLC/PKS的hPTH-(7-84)不能明显地影响尾部动脉血压,但是静脉注射相同剂量的hPTH-(1-31)NH2与hPTH-(1-34)同样多地降低血压。
因为AC也在OVX大鼠中介导PTH引发的刺激皮层和骨小梁形成,所以在完整雌性大鼠中激发降血压反应似乎是PTH构建体可能的骨生成能力的可简单快速测量的指示标记。另一方面,一个片段如果不能降低动脉血压,就将其从进一步评估中除去,这避免了在OVX大鼠中进行不必要的、昂贵、长期的骨生成能力测定。对静脉注射PTH的降血压反应与骨生成能力无关。然而,对皮下注射hPTH-(1-31)NH2,hPTH-(1-34)和hPTH-(1-84)的小得多的降血压反应确实与这些分子的骨生成作用平行,其中hPTH-(1-34)具有最强的降血压和骨生成作用,其它两个分子效力彼此相同,但在降低血压和刺激骨形成方面不如hPTH-(1-34)有效。但是对于hPTH-(1-30)NH2不存在这种相关性,它几乎与hPTH-(1-31)NH2和hPTH-(1-34)一样强地刺激AC并与hPTH-(1-31)NH2同样多降低血压但不刺激骨形成11。这种骨生成能力缺乏的原因不明。这不能仅仅归因于皮下注射hPTH-(1-30)NH2时,需要更多时间降低血压,因为皮下注射hPTH-(1-84)需要同样多的时间降低血压但仍具很强的骨生成能力。明显地,骨生成反应依赖于多种不同因素,它们联合使足够的活性激素或激素片段能从注射点到达表达PTH受体的成熟成骨细胞。刺激AC和血压降低的hPTH-(1-30)NH2不能促进骨形成是不稳定性更高[AC刺激的EC50稍高,即是20nm而不是hPTH-(1-31)NH2和hPTH-(1-34)的16nm]和从注射点进入循环的时间长的综合结果。
尽管hPTH-(1-30)NH2不具有骨生成能力,但是明显降低动脉压力的能力仍然是骨生成性PTH的共同特性,因而是PTH片段可能的体内生物活性的可快速测定的指示标记。另外降血压反应也可作为骨生成性PTH构建体通过口腔或其它非注射途径施用后到达靶位置能力的有效指示标记。
对每一次注射的降血压反应带来的不适和其它可能的后发症可能限制骨质疏松症病人接受PTH或PTH片段的长期治疗。幸运地是,间歇性皮下注射PTH目前已用于刺激骨形成并且这些分子经皮下注射比经静脉注射引起的降血压作用更小。这一点我们以前也已讨论。hPTH-(1-31)NH2具有另外的优势即刺激AC而不刺激PLC及任何PLC引发的潜在的Ca2+和PKC介导的副作用。
实施例7
环(Lys27-Asp30)-hPTH-(1-31)-NH2(5)按照与[Leu27]环(Glu22-Lys26)-hPTH(1-31)-NH2合成相似的方式合成。产品具有>95%的估计纯度,分子量为3700.64(±0.38)(预测的M+1=3700.14)。对此肽进行测序以证实内酰胺的位置。
[Leu27]-环(Glu22-Lys26)-hPTH-(1-30)-NH2(8)按照肽3的合成方式合成,但不需用人工操作将Val加到支持体上。产品具有>97%的估计纯度,分子量为3586.14(±0.19)(预测的M+1=3685.99)。
本发明的类似物可以施用给需要它的恒温动物,特别是通过胃肠外、体表或直肠给药,或通过呼吸或通过口腔运送。此类似物可以传统地配制成胃肠外剂量形式,其中将约1至约300mg每单位剂量与传统载体、赋形剂、粘合剂、防腐剂、稳定剂、颜料等所谓已由药学实践接受的试剂混合。
对于胃肠外施用,用超细的30直径注射器针头皮下无痛注射,需要给药1-2ml,每天不超过一次,根据疾病的严重性而定,持续1-2年。所注射的物质会在无菌等渗水溶液或悬浮液[优选地含诸如苯酚的防腐剂或诸如乙二胺四乙酸(EDTA)的助溶剂]中包含一种本发明的物质。在可接受的载体和溶剂之中,可以使用的是水、微酸化水、林格氏溶液和等渗氯化钠溶液。另外,传统上使用无菌固定油作为溶剂或悬浮介质。合成的甘油单酯、甘油双酯、脂肪酸(如油酸)可在注射制剂中用作固定油。
对于直肠施用,本发明的类似物可以通过与合适的非刺激性赋形剂,如可可脂或聚乙二醇混合制成栓剂形式。
对于体表使用,本发明的类似物可制成软膏、凝胶、溶液、悬浮液或皮肤粘性胶布。
对于吸入剂,可以按照例如PCT公开的申请号WO 94/07514中所述的方法制备。
每日的剂量不应超过0.05mg/kg体重或约3.5mg/70kg的人,并依赖于特定化合物的活性及受治疗对象的年龄、体重、性别和病情。如众所周知的,可与其携带材料混合得到单一剂量的活性成分的量将依赖于受治疗的对象和施用的特定模式。
下面列表显示的类似物目录都是根据上述方法制备的。
表1PTH类似物的分子量
SEQID | 类似物 | 分子量(测定值) | 分子量(预测值)(M+1) |
1 | hPTH-(1-31)-NH2 | 3717.77(±0.13) | 3717.14 |
3 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3685.46(±0.46) | 3685.12 |
7 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 | 3586.14(±0.19) | 3585.99 |
4 | [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 | 3685.61(±0.36) | 3685.12 |
6 | cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 | 3700.64(±0.38) | 3700.14 |
8 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-OH | 3685.96(±0.07) | 3686.12 |
9 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)-NH2 | 4083.62(±0.33) | 4083.52 |
10 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)-OH | 4084.14(±0.87) | 4084.52 |
11 | [Ala27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3642.70(±0.76) | 3643.04 |
12 | [Nle27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3684.57(±0.99) | 3685.12 |
13 | [Nle8,18;Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3649.50(0.59) | 3649.04 |
14 | cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3699.66(±0.49) | 3700.14 |
15 | [I1e27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3684.76(±0.74) | 3685.12 |
表1PTH类似物的分子量
SEQID | 类似物 | 分子量(测定值) | 分子量(预测值)(M+1) |
16 | [Tyr27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 | 3734.47(±0.80) | 3735.14 |
17 | α-acetyl-[Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3726.25(±0.42) | 3727.12 |
18 | [Leu27]cyclo(Lys22-Glu26)-hPTH-(1-31)-NH2 | 3684.67(±0.52) | 3685.12 |
19 | [Leu27]cyclo(Asp22-Om26)-hPTH-(1-31)-NH2 | 3656.90(±0.67) | 3657.12 |
20 | [Cys22;Cys26;Leu27]cyclo(Cys22-Cys26)hPTH(1-31)NH2 | 3651.26±0.31 | 3650.1 |
21 | [Cys26;Cys30;Leu27]cyclo(Cys26-Cys30)hPTH(1-31)NH2 | 3663.61±0.16 | 3664.13 |
表2肽类似物的腺苷酰环化酶(AC)活性
SEQ ID | 类似物 | 腺苷酰环化酶(EC50) |
1 | hPTH-(1-31)-NH2 | 20 |
3 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 3 |
7 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 | 20 |
4 | [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 | 3*,17** |
6 | cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 | 40 |
8 | Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-OH | 7 |
9 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)-NH2 | 8 |
10 | [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)-OH | 6 |
11 | [Ala27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 9 |
12 | [Nle27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 9 |
13 | [Nle8,18;Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 6 |
14 | cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 17 |
15 | [Ile27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 9 |
表2肽类似物的腺苷酰环化酶(AC)活性
SEQ ID | 类似物 | 腺苷酰环化酶(EC50) |
16 | [Tyr27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 9 |
17 | α-acetyl-[Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 | 24 |
18 | [Leu27]cyclo(Lys22-Glu26)-hPTH-(1-31)-NH2 | 4 |
19 | [Leu27]cyclo(Asp22-Om26)-hPTH-(1-31)-NH2 | >200 |
20 | [Cys22;Cys26;Leu27]cyclo(Cys22-Cys26)hPTH(1-31)NH2 | 16 |
21 | [Cys26;Cys30;Leu27]cyclo(Cys26-Cys30)hPTH(1-31)NH2 | 24 |
*图4
**图12
参考文献
下面公开的参考文献在此处引用作为参考。
<1>Caulfield,M.P.;McKee,R.L.;Goldman,M.E.;Duong,L.T.;Fisher,J.E.;Gay,C.T.;DeHaven,P.A.;Levy,J.J.;Roubini,E.;Nutt,R.F.;Chorev,M.;Rosenblatt,M.;牛肾甲状旁腺激素(PTH)受体对两种不同氨基酸序列具有相同亲和力-PTH和PTH相关蛋白的受体结合区域定位于13-34区;内分泌学;1990;127;83-87。
<2>Neugebauer,W.;Barbier,J.-R.;Sung,W.L.;Whitfield,J.F.;Willick,G.E.;C端截短的人甲状旁腺激素类似物的溶液结构和腺苷酰环化酶刺激活性;生物化学;1995;34;8835-8842。
<3>Gardella,T.J.;Wilson,A.K.;Keutmann,H.T.;Oberstein,R.;Potts,J.T.;Kronenberg,H.M.和Nussbaum,S.R.;通过定点突变和类似物设计来分析甲状旁腺激素主要的受体结构区;内分泌学;1993;132;2024-2040。
<4>Marqusee,S.;Baldwin,R.L.;Glu---Lys+盐桥对从头设计的短肽中螺旋的稳定作用;美国国家科学院院报;1987;84;8898-8902。
<5>Surewicz,W.K.;Neugebauer,W.;Gagnon,L.;MacLean,S.;Whitfield,J.F.;Willick,G.E.;人甲状旁腺激素结构-功能关系:两亲螺旋的基本作用;于肽:化学、结构和生物学中;1993;Smith,J.;Hodges,R.编辑;ESCOM Leiden,The Netherlands,1993,第556-558页。
<6>Whitfield,J.F.;Morley,P.;Willick,G.E.;Ross,V.;Barbier,J.R.;Isaacs,RJ.;Ohannessianbarry,L.;新型甲状旁腺激素片段hPTH-(1-31)NH2(Ostabolin)对卵巢切除大鼠股骨骨小梁生长的刺激;Calcified Tissue Int.;1996;58;81-87。
<7>Whitfield,J.F.;Morley,P.;甲状旁腺激素的骨重建小片段:骨质疏松症的新型治疗剂;药物科学趋势;1995;16;382-386。
序列表
(1)基本信息
(i)申请人:
(A)名称:National Reearch Council of Canada
(B)街道:Intellectual Property Services
Montreal Road,Bldg.M-58,Room EG-12
(C)城市:Ottawa
(D)州名:Ontario
(E)国家:Canada
(F)邮政编号(Zip):K1A OR6
(ii)发明名称:用于治疗骨质疏松症的甲状旁腺激素类似物
(iii)序列数:33
(iv)计算机可读形式
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,版本#1.30
(v)目前申请资料:
(A)申请号:PCT/CA97/00547
(B)递交日:1997年8月1日
(vi)在先申请资料:
(A)申请号:08/691,647
(B)递交日:1996年8月2日
(2)关于SEQ ID NO:1的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:1:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
34
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:2的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:3的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:3:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:4的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:4:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:5的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:5:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:6的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:6:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:7的信息
(i)序列特征
(A)长度:30个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:7:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp
20 25 30
(2)关于SEQ ID NO:8的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:8:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:9的信息
(i)序列特征
(A)长度:34个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:9:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val His
20 25 30
Asn phe
(2)关于SEQ ID NO:10的信息
(i)序列特征
(A)长度:34个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:10:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val His
20 25 30
Asn Phe
(2)关于SEQ ID NO:11的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:11:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Ala Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:12的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:12:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Xaa Leu Gln Asp Val
20 25 30
39
(2)关于SEQ ID NO:13的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:13:
Ser Val Ser Glu Ile Gln Leu Xaa His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Xaa Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:14的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:14:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:15的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:15:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Ile Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:16的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:16:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Tyr Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:17的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:17:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Mat Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:18的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:18:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Lys Trp Leu Arg Glu Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:19的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:19:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Asp Trp Leu Arg Xaa Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:20的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:20:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Cys Trp Leu Arg Cys Leu Leu Gln Asp Val
20 25 30
(2)关于SEQ ID NO:21的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:环形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:21:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Cys Leu Leu Gln Cys Val
20 25 30
(2)关于SEQ ID NO:22的信息
(i)序列特征
(A)长度:34个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:22:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn phe
(2)关于SEQ ID NO:23的信息
(i)序列特征
(A)长度:31个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:23:
Xaa Val Ser Glu Ile Gln Leu Xaa His Asn Leu Gly Lys Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Glu Arg Val Xaa Trp Leu Xaa Xaa Xaa Leu Xaa Asp Xaa
20 25 30
(2)关于SEQ ID NO:24的信息
(i)序列特征
(A)长度:4个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:24:
Val His Asn Xaa
1
(2)关于SEQ ID NO:25的信息
(i)序列特征
(A)长度:5个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:25:
Val His Asn phe Xaa
1 5
(2)关于SEQ ID NO:26的信息
(i)序列特征
(A)长度:6个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:26:
Val His Asn phe Val Xaa
1 5
(2)关于SEQ ID NO:27的信息
(i)序列特征
(A)长度:7个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:27:
Val His Asn Phe Val Ala Xaa
1 5
(2)关于SEQ ID NO:28的信息
(i)序列特征
(A)长度:8个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:28:
Val His Asn Phe Val Ala Leu Xaa
1 5
(2)关于SEQ ID NO:29的信息
(i)序列特征
(A)长度:4个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:29:
His Lys Lys Lys
1
(2)关于SEQ ID NO:30的信息
(i)序列特征
(A)长度:4个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:30:
His Leu Lys Lys
1
(2)关于SEQ ID NO:31的信息
(i)序列特征
(A)长度:4个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:31:
Lys Lys Lys Lys
1
(2)关于SEQ ID NO:32的信息
(i)序列特征
(A)长度:4个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:32:
His Leu Lys Ser
1
(2)关于SEQ ID NO:33的信息
(i)序列特征
(A)长度:5个氨基酸
(B)类型:氨基酸
(C)链型:
(D)拓扑结构:线形
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:33:
Val Leu Asn Phe Xaa
1 5
Claims (43)
1.人甲状旁腺激素hPTH类似物及其药物学上可接受的盐,具有以下氨基酸序列
R-NH-R1-Val-Ser-Glu-Ile-Gln-Leu-R2-His-Asn-Leu-Gly-Lys-R3-R4-R5-R6-R7-Glu-Arg-Val-R8-Trp-Leu-R9-R10-R11-Leu-R12-R13-Y
其中
R=氢原子,
R1=Ser、Ala或α-氨基丁酸,
R2=Met、Nle、Leu、Ile、Val、Phe或Trp,
R3=His、Lys或Glu,
R4=Leu、Lys、Arg或Glu,
R5=Asn、Lys、Orn、Hci、Asp、Arg、DLys、Ser、Gly或Glu,
R6=Ser、Lys、Asp、Arg或Glu,
R7=Met、Nle、Leu、Ile、Val、Phe或Trp,
R8=Glu、Lys、Cys,Orn或Asp,
R9=Arg、Lys、Orn、His、Cys或Glu,
R10=Arg、Lys、Orn、Gln、Cys、Glu或Asp,
R11=Lys、Leu、Ala、Tyr、Orn、瓜氨酸、α-氨基丁酸、Nle、Ile、Met、Gln、Asp、或侧链具有2-10个碳原子的直链或支链α-氨基脂肪酸或其在脂肪链末端带有氨基、羧基、乙酰氨基、胍基或脲基的类似物,
R12=Gln、Arg、Cys、Glu、Asp、Lys或Orn,
R13=Asp、Cys或Lys,
X=OH、或NH2,并且
Y=X,Val-X,Val-His-X,Val-His-Asn-X,Val-His-Asn-Phe-X,Val-His-Asn-Phe-Val-X,Val-His-Asn-Phe-Val-Ala-X和Val-His-Asn-Phe-Val-Ala-Leu-X,
在从氨基酸对22和26、26和30、27和30、和25和29中选择的一个或两个氨基酸对之间环化,条件是当R9是Lys或Orn和R12是Glu或Asp时在25和29之间环化;其中环化作用之一在氨基酸22和26之间或氨基酸25和29之间。
2.根据权利要求1所述的类似物,其中环化作用是以内酰胺形式或二硫键形式。
3.根据权利要求2所述的类似物,其在氨基酸对22和26、26和30、当R9是Lys或Orn且R12是Glu或Asp时的25和29、或当R11和R13是Lys或Asp时的27和30之间以内酰胺的形式环化,或在Cys对22和26、26和30或25和29之间以二硫键形式环化。
4.根据权利要求2所述的类似物,其中内酰胺是22-26内酰胺。
5.根据权利要求4所述的类似物,其中内酰胺是Glu22-Lys26内酰胺。
6.根据权利要求2所述的类似物,其中内酰胺是25-29内酰胺。
7.根据权利要求1-6中的任意一项所述的类似物,其中R是H,而X是NH2。
8.根据权利要求1-6中的任意一项所述的类似物,其中R11是Lys或Leu。
9.根据权利要求8所述的类似物,其中R2和R7是Met或正亮氨酸。
10.根据权利要求9所述的类似物,其中R1是Ser,R2是Met,R7是Met,R8是Glu,R9是Arg,R10是Lys,R12是Gln,并且R13是Asp。
11.根据权利要求10所述的类似物,其中R3是His或Lys,R4是Leu、Lys或Arg,R5是Asn、Orn、Hci、Asp、Arg、Lys、D-Lys、Ser或Gly,并且R6是Ser、Lys、Asp或Arg。
12.根据权利要求11所述的类似物,其中R3-R6是His-Lys-Lys-Lys,His-Leu-Lys-Lys,Lys-Lys-Lys-Lys或His-Leu-Lys-Ser。
13.根据权利要求10所述的类似物,其中Y=X。
14.根据权利要求10所述的类似物,其中Y=Val-X。
15.根据权利要求10所述的类似物,其中Y=Val-Leu-Asn-Phe-X。
16.根据权利要求1所述的类似物,具有SEQ ID NO:6。
17.根据权利要求1所述的类似物,具有SEQ ID NO:7。
18.根据权利要求1所述的类似物,具有SEQ ID NO:8。
19.根据权利要求1所述的类似物,具有SEQ ID NO:9。
20.根据权利要求1所述的类似物,具有SEQ ID NO:10。
21.根据权利要求1所述的类似物,具有SEQ ID NO:11。
22.根据权利要求1所述的类似物,具有SEQ ID NO:12。
23.根据权利要求1所述的类似物,具有SEQ ID NO:13。
24.根据权利要求1所述的类似物,具有SEQ ID NO:14。
25.根据权利要求1所述的类似物,具有SEQ ID NO:15。
26.根据权利要求1所述的类似物,具有SEQ ID NO:16。
27.根据权利要求1所述的类似物,具有SEQ ID NO:17。
28.根据权利要求1所述的类似物,具有SEQ ID NO:18。
29.根据权利要求1所述的类似物,具有SEQ ID NO:19。
30.根据权利要求1所述的类似物,具有R11是Lys,Leu,Asp,Gln,Ala,Nle,Ile或Tyr。
31.根据权利要求1所述的类似物,其是具有SEQ ID NO:1的人甲状旁腺激素hPTH-(1-31)的类似物,其中27位是Lys或由Leu、Ala、Tyr、Orn、瓜氨酸、α-氨基丁酸、Nle、Ile、Met、Gln、Asp、或侧链具有2-10个碳原子的直链或支链α-氨基脂肪酸或其在脂肪链末端带有氨基、羧基、乙酰氨基、胍基或脲基的类似物替换,并且在Glu22和Lys26之间环化形成内酰胺。
32.根据权利要求31所述的类似物,其中27位残基是Leu。
33.根据权利要求31所述的类似物,其中27位残基选自由Ile、正亮氨酸、Met和α-氨基丁酸组成的群体。
34.根据权利要求31-33的任意一项所述的类似物,具有C-端酰胺末端或C-端羧基末端。
35.根据权利要求31所述的类似物,具有SEQ ID NO:3。
36.一种施用于需要它的恒温动物的组合物,它包含与药学上可接受的载体或赋形剂结合的根据权利要求31所述的类似物。
37.根据权利要求36所述的组合物,其中类似物的27位残基是Leu。
38.根据权利要求36所述的组合物,其中类似物的27位残基选自由Ile、正亮氨酸、Met和α-氨基丁酸组成的群体。
39.根据权利要求36所述的组合物,其中类似物具有SEQ ID NO:3。
40.根据权利要求31所述的类似物在制备用于恒温动物增加腺苷环化酶活性、降血压或骨生成的药物中的用途。
41.根据权利要求40所述的用途,其中类似物的27位残基是Leu。
42.根据权利要求40所述的用途,其中类似物的27位残基选自由Ile、正亮氨酸、Met和α-氨基丁酸组成的群体。
43.根据权利要求40所述的用途,其中类似物具有SEQ ID NO:3。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/691,647 US5955425A (en) | 1996-08-02 | 1996-08-02 | Parathyroid hormone analogues for the treatment of osteoporosis |
US08/691,647 | 1996-08-02 | ||
US4056097P | 1997-03-14 | 1997-03-14 | |
US60/040,560 | 1997-03-14 |
Publications (2)
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CN1231676A CN1231676A (zh) | 1999-10-13 |
CN1176947C true CN1176947C (zh) | 2004-11-24 |
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CNB971981612A Expired - Fee Related CN1176947C (zh) | 1996-08-02 | 1997-08-01 | 用于治疗骨质疏松症的甲状旁腺激素类似物 |
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EP (1) | EP0915911B1 (zh) |
JP (3) | JP4122061B2 (zh) |
KR (1) | KR100500859B1 (zh) |
CN (1) | CN1176947C (zh) |
AT (1) | ATE253078T1 (zh) |
AU (1) | AU723728B2 (zh) |
BG (1) | BG103128A (zh) |
BR (1) | BR9711002B1 (zh) |
CA (1) | CA2261564C (zh) |
CZ (1) | CZ34799A3 (zh) |
DE (1) | DE69725850T2 (zh) |
DK (1) | DK0915911T3 (zh) |
EE (1) | EE9900039A (zh) |
ES (1) | ES2207740T3 (zh) |
FI (1) | FI990126A (zh) |
GE (1) | GEP20033095B (zh) |
IS (1) | IS4962A (zh) |
LV (1) | LV12293B (zh) |
NZ (1) | NZ333809A (zh) |
PT (1) | PT915911E (zh) |
RU (1) | RU2203286C2 (zh) |
SI (1) | SI9720057A (zh) |
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Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
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SK151599A3 (en) * | 1997-05-14 | 2000-09-12 | Rhone Poulenc Rorer Pharma | Peptide parathyroid hormone analogs |
WO2000023594A1 (en) * | 1998-10-22 | 2000-04-27 | The General Hospital Corporation | BIOACTIVE PEPTIDES AND PEPTIDE DERIVATIVES OF PARATHYROID HORMONE (PTH) AND PARATHYROID HORMONE-RELATED PEPTIDE (PTHrP) |
ATE458054T1 (de) | 1998-11-25 | 2010-03-15 | Gen Hospital Corp | Menschliches parathyroidhormon, modifikationen, herstellung und verwendung |
EP1141237B1 (en) | 1998-12-31 | 2005-11-02 | The General Hospital Corporation | Pth receptor and screening assay utilizing the same |
EP1221963A4 (en) * | 1999-07-28 | 2006-03-22 | Univ Pennsylvania | METHODS OF INHIBITING OSTEOCLASTOGENESIS |
US6316410B1 (en) * | 1999-09-22 | 2001-11-13 | National Research Council Of Canada | Parathyroid hormone analogues for the treatment of osteoporosis |
AU2002339843B2 (en) | 2001-07-23 | 2007-12-06 | The General Hospital Corporation | Conformationally constrained parathyroid hormone (PTH) analogs |
CA2472472A1 (en) | 2002-01-10 | 2003-07-24 | Osteotrophin Llc | Treatment of bone disorders with skeletal anabolic drugs |
CA2511966A1 (en) * | 2002-11-01 | 2004-07-22 | Amgen Inc. | Modulators of receptors for parathyroid hormone and parathyroid hormone-related protein |
US8088734B2 (en) * | 2003-01-21 | 2012-01-03 | Unigene Laboratories Inc. | Oral delivery of peptides |
AU2006343306A1 (en) * | 2005-09-06 | 2007-11-15 | Zelos Therapeutics, Inc. | Parathyroid hormone analogues and methods of use |
JP2010501476A (ja) * | 2006-07-31 | 2010-01-21 | ゼロス セラピューティクス, インコーポレイテッド | 副甲状腺ホルモン類似体およびその使用方法 |
KR101108354B1 (ko) | 2006-10-13 | 2012-01-25 | 일라이 릴리 앤드 캄파니 | Pth 수용체 조절제로서의 페길화된 pth 및 그의 용도 |
CA2694667C (en) | 2007-08-01 | 2018-10-30 | The General Hospital Corporation | Screening methods using g-protein coupled receptors and related compositions |
JP5941040B2 (ja) | 2010-05-13 | 2016-06-29 | ザ ジェネラル ホスピタル コーポレイション | 副甲状腺ホルモン類似体およびその使用 |
CN105440126B (zh) * | 2015-12-31 | 2019-10-25 | 南京工业大学 | 治疗骨质疏松的α-螺旋多肽及其制备方法与应用 |
WO2019018238A1 (en) | 2017-07-15 | 2019-01-24 | The Regents Of The University Of California | CATHEPSIN K OSTEOADSORBENT FLUOROGEN SUBSTRATE FOR THE IMAGING OF OSTEOCLAST ACTIVITY AND MIGRATION |
EP3856837A1 (en) | 2018-09-26 | 2021-08-04 | Borealis AG | Propylene copolymer composition with excellent optical and mechanical properties |
JP7342310B2 (ja) | 2019-05-28 | 2023-09-12 | 国立大学法人東北大学 | 電源装置、超伝導装置、超伝導デバイス、及び超伝導デバイスの製造方法 |
CN113549144A (zh) * | 2021-06-30 | 2021-10-26 | 华南理工大学 | 一种特立帕肽hPTH(1-34)的生产和纯化方法 |
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US5317010A (en) * | 1991-10-10 | 1994-05-31 | Peter K. T. Pang | Parathyroid hormone analogues substituted at AA 25, 26, 27, and use in osteoporosis treatment |
US5814603A (en) * | 1992-06-12 | 1998-09-29 | Affymax Technologies N.V. | Compounds with PTH activity |
AU672790B2 (en) * | 1992-07-15 | 1996-10-17 | Novartis Ag | Variants of parathyroid hormone and its fragments |
CA2126299C (en) * | 1994-06-20 | 2000-12-12 | Gordon E. Willick | Parathyroid hormone analogues for the treatment of osteoporosis |
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