CN103002906A - 甲状旁腺激素类似物及其应用 - Google Patents
甲状旁腺激素类似物及其应用 Download PDFInfo
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Abstract
披露了两种PTH类似物配体,SP-PTH-AAK和Aib-SP-PTH-AAK,如在体外和体内证明的,其对PTH受体具有长效活性。因此,这些多肽特别适用于治疗其中期望长效活性的疾病,如甲状旁腺功能减退。还披露了制备这些类似物多肽的方法。
Description
联邦政府资助研究的声明
在国家卫生研究院授予的基金DK-11794下,在美国政府的支持下,做出本发明。美国政府对本发明具有某些权利。
技术领域
通常,本发明涉及甲状旁腺激素(PTH)类似物,具体地是在PTH受体上具有长效激动剂活性的那些。这些类似物可用于治疗其中期望长效活性的疾病,如甲状旁腺功能减退。
背景技术
PTH(1-34)是骨质疏松症和PTH缺乏病症,即甲状旁腺功能减退的治疗中有效的治疗剂。甲状旁腺功能减退是由甲状旁腺的甲状旁腺激素(PTH)产生不足所表征的终身疾病(life-long disease)。因为PTH是调节钙和磷酸盐水平的关键,PTH的损失减少血液和骨中的钙水平和增加磷酸盐水平(血钙过少和血磷酸盐过多)。血钙过少导致症状如神经肌肉兴奋性,包含感觉异常、肌肉颤搐、喉部痉挛(其可导致不能说话和不能向医疗服务者(health providers)提供潜在医学病症的警报,其导致延迟的或错误的治疗)、以及可能手足抽搐和痉挛。它仅仅是内分泌紊乱,其中缺少的激素(即PTH)还没有用作治疗。
已经鉴别PTH(1-34)为甲状旁腺功能减退的骨化三醇治疗(calcitrioltherapy)的安全的和有效的替代方式,且能够在没有高钙尿症(hypercalciuria)的情况下,维持正常的血清钙水平(Winer等人,J ClinEndocrinol Metab 88:4214-4220,2003)。但是,多肽每天需要至少两次的注射,因此,已经认为在这种疾病中需要长效PTH(1-34)类似物(Winer等人,上文)。
因而,需要另外的PTH受体激动剂,特别是在PTH受体上具有长效活性的那些。
发明内容
本发明涉及PTH和PTHrP类似物的开发(发展,development)。此处描述的典型的多肽,SP-PTH-AAK和Aib-SP-PTH-AAK在体外和体内对PTH受体具有长效活性,且在中性水溶液中表现高的溶解性。因此,本发明的多肽适于其中期望长效活性的疾病的治疗,包含甲状旁腺功能减退(hypoparathyroidism)。
因此,本发明特征在于多肽(例如,分离的),或其药学可接受的盐,包含式(I)的氨基酸序列:
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-X13-X14-X15-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-X25-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile (I)
其中X01是Ser、Ala、或Aib;X03是Ser、Ala、或Aib;X08是Met、Leu、或Nle;X10是Asn、Ala、Val、Asp、Ile、Glu、或Gln;X11是Leu、Ala、Val、Met、Lys、Arg、Har、或Trp;X12是Gly、Ala、His、或Arg;X13是Lys、Ala、Leu、Gln、Arg、His、或Trp;X14是His、Leu、Arg、Phe、Trp、或Ser;X15是Ile或Leu;X16是Gln或Asn;X17是Asp或Ser;X18是Ala、Leu、Met、Glu、Ser、或Phe;X22是Ala、Phe、Glu、Ser、Leu、Asn、Trp、或Lys;X25是His、Arg、Leu、Trp、或Lys;和X26是Lys、His、Ala、Ser、Asn、或Arg;或包含式(I)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的它的片段,条件是至少一个X18不是Leu或Met,X22不是Phe,和X26不是His。
在某些实施例中,多肽包含式(II):
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-Lys-X14-X15-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-His-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile(II)
其中X01是Ser、Ala、或Aib;X03是Ser、Ala、或Aib;X08是Met、Leu、或Nle;X10是Asn、Gln、或Asp;X11是Leu、Arg、Har、或Lys;X12是Gly或Ala;X14是His、Trp、或Ser;X15是Ile或Leu;X16是Gln或Asn;X17是Asp或Ser;X18是Ala或Leu;X22是Ala或Phe;和X26是Lys或His;或包含式(II)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的它的片段。
在其他实施例中,多肽包含式(III):
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-Lys-X14-Ile-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-His-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile(III)
其中X01是Ser、Ala、或Aib;X03是Ser、Ala、或Aib;X08是Met、Leu、或Nle;X10是Asn或Gln;X11是Leu、Arg、或Har;X12是Gly或Ala;X14是His或Trp;X16是Gln或Asn;X17是Asp或Ser;X18是Ala或Leu;X22是Ala或Phe;和X26是Lys或His;或包含式(III)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的它的片段。
在任一上述多肽的具体实施例中,X01和X03是Ala;X10是Gln;X11是Arg;X12是Ala;和X14是Trp。在其他实施例中,X01是Ala;X03是Aib;X10是Gln;X11是Har;X12是Ala;和X14是Trp。在任一上述多肽中,X18可以是Ala;X22可以是Ala;和/或X26可以是Lys。
在某些实施例中,多肽基本上相同于上述描述的多肽(例如,至少90%或95%相同)(例如,其中X18和X22是Ala和其中X26是Lys)。在某些实施例中,如与SP-PTH比较,多肽在中性水溶液中(例如,pH为7.4的磷酸盐缓冲盐(PBS))表现更高的溶解性(例如,如与在酸性溶液(例如,pH为1、2、3、4,如10mM乙酸(pH为2.9))中比较,至少40%、50%、60%、70%、80%、90%、或95%溶于中性水溶液中)。在某些实施例中,多肽是生物学上活性的多肽(例如,PTH受体激动剂)。在某些实施例中,多肽以比hPTH(1-34)高的亲和力结合到人类PTH-1受体的R0状态(R0state)上。在其他实施例中,多肽的长度小于200、150、100、75、50、40、39、38、或37个氨基酸。可在它的C-末端酰胺化多肽。
在具体的实施例中,多肽包含或是以下氨基酸序列:
Ala-Val-Ala-Glu-Ile-Gln-Leu-Met-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或包含所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的它的片段。在另一个实施例中,多肽包含或是以下氨基酸序列:Ala-Val-Aib-Glu-Ile-Gln-Leu-Met-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或包含所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的它的片段。在其他实施例中,多肽包含或是选自由下列序列组成的组的氨基酸序列:
Ala-Val-Ala-Glu-Ile-Gln-Leu-N le-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;Ala-Val-Ala-Glu-Ile-Gln-Leu-Leu-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;Ala-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;和
Ala-Val-Aib-Glu-Ile-Gln-Leu-Leu-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或其包含所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
可在它们的C-末端酰胺化上述描述的任一多肽。
本发明特征也在于包含本发明的多肽的药物组合物(例如,上述或此处描述的任何多肽)和药学上可接受的载体。
在某些实施例中,通过固相合成来合成或重组地生产本发明的多肽。
本发明特征还在于用于治疗受试者的方法,其中所述受试者具有,例如,选自由甲状旁腺功能减退、血磷酸盐过多、骨质疏松症、骨折修复(fracture repair)、骨软化、关节炎、和血小板减少症组成的组的疾病。该方法包含给予受试者足够治疗疾病的量的本发明的多肽或包含本发明的多肽的药物组合物。例如,可皮下地、静脉内地、鼻内地、经肺部地、经皮地、和口服地给予多肽或药物组合物。
本发明特征还在于包含编码本发明的多肽的序列的核酸。可操作地将核酸连接到启动子上。核酸可以是载体的部分。本发明特征还在于包含载体的细胞,以及通过在其中表达编码的多肽的条件下培养细胞来生产多肽的方法。
就“受试者”来说,意味着人或非人动物(例如,哺乳动物)。
就“治疗”来说,意味着,与未经治疗的等同对照比较,在患有病症或疾病的受试者(例如,诊断具有甲状旁腺功能减退的受试者)中改善病症或疾病的至少一个症状。如通过任何标准技术测量,症状的这种减轻(例如,血液钙水平的减少或血清磷酸盐水平的增加)至少是5%、10%、20%、40%、50%、60%、80%、90%、95%、或100%。
就“纯化的多肽”或“分离的多肽”来说,意味着已经和其他成分分离开的多肽。一般,当按重量计,它至少是30%不含其他成分时,多肽是基本上纯的。在某些实施例中,按重量计,制品至少是50%、60%、75%、85%、90%、95%、96%、97%、98%、或99%不含其他成分。例如,可通过从天然来源提取;通过编码这样的多肽的重组多聚核苷酸的表达;或通过化学方法合成多肽获得纯化的多肽。可通过任何适当的方法测量纯度,例如,柱层析、聚丙烯酰胺凝胶电泳、或通过HPLC分析。
就“生物学上活性”来说,意味着,化合物或组合物(例如,此处描述的多肽)在给予细胞或动物(例如,人或非人哺乳动物)之后具有至少一种生物学上显著的效应。PTH、PTHrP、和其类似物(例如,此处描述的那些)的生物活性包含,但不限于,受体结合、cAMP或IP3生产、蛋白激酶A、蛋白激酶C、磷脂酶C、磷脂酶D、和磷脂酶A2活化、细胞内的改变(例如,增加或减少)、血浆、或尿钙或磷酸盐水平、和体内或体外骨新陈代谢或分解代谢中的改变。与适当的对照(例如,野生型多肽或它的拟表型如PTH(1-34)或PTHrP(1-36))比较,例如,本发明的生物学上活性的多肽(例如,此处描述的任何多肽),在任何生物活性中可表现增加(例如,至少5%、10%、25%、50%、100%、500%、1000%、10,000%)或减少(例如,95%、90%、75%、50%、25%、10%、5%、1%、0.1%、0.01%、或0.001%)。
就“基本上相同”来说意味着,例如,当利用下面描述的方法最佳地比对时,核酸或氨基酸序列与第二个核酸或氨基酸序列,例如,PTH或PTHrP序列或它的片段共有至少60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%序列同一性。“基本同一性(substantialidentity)”可用于涉及序列的各种类型和长度,如全长序列、抗原表位或免疫原性肽、功能结构域、编码和/或调控序列、外显子、内含子、启动子、和基因组序列。以本领域技术内的各种方式测定两个多肽或核酸序列之间的百分比同一性,例如,利用公开可用的计算机软件如Smith WatermanAlignment(Smith et al.,J Mol Biol 147:195-7,1981);如包括在GeneMatcherPlusTM,Schwarz and Dayhof(1979)Atlas of Protein Sequence and Structure,Dayhof,M.O.,Ed pp 353-358中的“Best Fit”(Smith and Waterman,Advancesin Applied Mathematics,482-489,1981);BLAST程序(Basic LocalAlignment Search Tool;(Altschul et al.,J Mol Biol 215:403-10,1990)、BLAST-2、BLAST-P、BLAST-N、BLAST-X、WU-BLAST-2、ALIGN、ALIGN-2、CLUSTAL、或Megalign(DNASTAR)软件。另外,本领域的技术人员可确定关于测量比对的适当的参数,包含在被比较的序列长度上,以达到最大比对所需要的任何算法。通常,关于蛋白质、比较序列的长度将至少是6个或8个氨基酸,优选地9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、40、50、60、70、80、90、100、125、150、200、250、300、350、400、或500个氨基酸或更多达到蛋白质的整个长度。关于核酸,通常,比较序列的长度将至少是18、21、24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、69、72、75、78、81、84、87、90、93、96、99、102、105、108、111、125、150、200、250、300、350、400、450、500、550、600、650、700、800、900、1000、1100、1200、或至少1500个核苷酸或更多达到核酸分子的整个长度。应该理解,当将DNA序列和RNA序列比较时,为了测定序列同一性的目的,胸腺嘧啶核苷相当于尿嘧啶核苷。保守的取代一般包含在下列组内的取代:甘氨酸、丙氨酸;缬氨酸、异亮氨酸、亮氨酸;天门冬氨酸、谷氨酸、天冬酰胺、谷酰胺;丝氨酸、苏氨酸;赖氨酸、精氨酸;和苯丙氨酸、酪氨酸。
就“中性pH”来说意味着约6-9的pH(例如,6.5-8.0)。具体的中性pH值包含6.5、6.6、6.8、7.0、7.2、7.4、7.6、7.8、和8.0。
从下列具体实施方式、附图、和权利要求将显然可见本发明的特征和优势。
附图说明
图1A和1B是显示PTH类似物对在R0(图1A)和RG(图1B)中的大鼠PTHR1构型的结合亲和力的图表。如示出的,SP-PTH-AAK对受体的R0和RG形式都表现最强的结合。PTH(1-34)对受体的R0形式表现最弱的结合。每一图表下方示出曲线拟合参数。
图2是显示在MC3T3-E1细胞中,对PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK的cAMP响应的图表。每一图表下方示出曲线拟合参数。
图3是在利用PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、Aib-SP-PTH-AAK、或PTH(3-34)处理之后,显示在利用cAMP-响应元件-荧光素酶基因构建体转染的MC3T3-E1细胞中的发光的图表。图表下方示出曲线拟合参数。
图4A和4B是显示cAMP刺激和R0(图4A)或RG(图4B)结合之间的相关性的图表。
图5A和5B是显示PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK对人类PTH-1受体的R0(图5A)和RG(图5B)形式的结合亲和力的图表。每一图表下方示出曲线拟合参数。
图6是显示PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK对在HKRK-B7细胞上表达的人类PTH-1受体的cAMP刺激的图表。
图7A和7B是显示在冲掉配体之后,cAMP刺激的图表。图7A显示pmol/孔的cAMP产生,以及图7B显示关于每一配体针对最大cAMP刺激而标准化的这些结果。
图8A-8E是显示接受媒介物(vehicle)、或5nmol/kg PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、或Aib-SP-PTH-AAK的0-8小时(图8A)或0-30小时(图8B)的皮下注射的小鼠中的血钙水平的图表。在图8C(0-24小时)和图8D(0-54小时)中,显示关于以10nmol/kg皮下注射肽的相似结果。也显示了在禁食条件下来自相似实验的结果(图8E)。
图9是显示用于测量血钙、血清磷酸盐水平、和在TPTX大鼠的尿中钙-对-肌氨酸酐比率的实验方案的示意图。
图10A-10E是显示在给予媒介物、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、或Aib-SP-PTH-AAK的TPTX大鼠中血钙(图10A)和血清无机磷酸盐(图10B)响应的图表。将模拟操作的大鼠(sham operated rat)显示为对照。也提供了1.25和5nmol/kg的SP-PTH与1.25、5和20nmol/kg的PTH(1-34)比较的相似的实验(图10C)。也显示了利用10nmol/kg PTH(1-34)或SP-PTH静脉注射的正常大鼠中的药物动力学概况(图10D)。也显示了在利用24.3nmol/kg hPTH(1-34)、hPTH(1-84)、或SP-PTH-AAK静脉地注射的TPTX大鼠中的药物动力学概况(图10E)。
图11A是显示在接受SP-PTH或SP-PTH-AAK静脉注射的食蟹猴(猕猴)中,用于测量血清和尿钙和磷酸盐水平的实验方案的示意图。
图11B是显示在静脉注射1nmol/kg之后,猴中的SP-PTH的血浆浓度的图表。
图12A-12C是显示在接受媒介物、SP-PTH、SP-PTH-AAK、或PTH(1-34)(*P<0.05,vs.媒介物;**P<0.01 vs.媒介物)注射的食蟹猴中的血清钙(图12A和12C)和血清磷酸盐(图12B)的图表。图12A和12B显示利用静脉地注射的0.25nmol/kg的SP-PTH或SP-PTH-AAK、或媒介物对照的结果。图12C显示在40-倍增加的PTH(1-34)剂量(10nmol/kg)皮下注射之后的血清钙浓度。
图13A和13B是显示在接受媒介物、SP-PTH、或SP-PTH-AAK(*P<0.05,vs.媒介物;**P<0.01vs.媒介物)静脉注射的食蟹猴中的尿钙(图13A)和尿磷酸盐(图13B)肌氨酸酐比率的图表。
图14是显示在接受媒介物或2.5nmol/kg或10nmol/kg的SP-PTH或SP-PTH-AAK的食蟹猴中,用于测量血清钙和血清磷酸盐水平的实验方案的示意图。
图15A-15H是显示在接受媒介物、2.5nmol/kg或10nmol/kg的SP-PTH-AAK、10nmol/kg的PTH(1-34)、或10nmol/kg的PTH(1-84)的食蟹猴中的血清和尿钙和磷酸盐水平和血清肌氨酸酐水平的图表。图15A显示血清钙水平,图15B和15C显示血清无机磷酸盐水平。图15D显示血清肌氨酸酐水平。图15E和15G显示尿钙水平,图15F和15H显示尿磷酸盐水平。
图16A和16B是显示多肽在pH为7.4的PBS溶液中的溶解性的图表。
图16C显示SP-PTH-AAK在5、25、和40℃下在磷酸盐-柠檬酸盐缓冲液中和10mM乙酸中的稳定性。与开始样品比较,通过rPHPLC测量样品中剩余的完整SP-PTH-AAK肽的量。
图17A和17B是显示在表达野生型或磷酸化-缺陷型(PD)PTH受体的小鼠中,PTH(1-34)(图17A)和M-PTH(1-28)(图17B)对血钙水平的影响的图表。
图18A和18B是显示在表达野生型或PD PTH受体的小鼠中,PTH(1-34)(图18A)和M-PTH(1-28)(图18B)对血液cAMP水平的影响的图表。
图19是显示在表达野生型或PD PTH受体的小鼠中,SP-PTH-AAK对血钙水平的影响的图表。
图20A-20D是显示在TPTX大鼠中,每天一次的SP-PTH对血清和尿Ca的影响的图表。通过利用媒介物(模拟品(sham)和TPTX)、或SP-PTH(1、2、4、或8nmol/kg)、或利用1,25(OH)2D(0.075或0.2μg/kg.orn)sc.注射,每天一次处理TPTX和模拟-对照大鼠,处理10天。在第10天最后注射之后,将大鼠放置在新陈代谢笼中,在8(图20A)和24小时(图20B)获得颈静脉血;以时间间隔0-8(图20C)和8-24小时(图20D)收集尿;平均值±s.e.m:n=5;‘~,P vs媒介物<005。
具体实施方式
本发明涉及在PTH受体上具有延长的活性的新的甲状旁腺激素(PTH)类似物。通过SP-PTH-AAK([Ala1,3,12,18,22,Gln10,Arg11,Trp14,Lys26]PTH(1-14)/PTHrP(15-36))和Aib-SP-PTH-AAK(Ala1,12,18,22,Aib3,Gln10,高Arg11,Trp14,Lys26]PTH(1-14)/PTHrP(15-36))例示这些类似物。如下面描述的,在体外,这些多肽以比PTH(1-34)和其他参考多肽高的亲和力结合到PTH-1受体(PTHR1)的非-G蛋白偶联的(、结合的,coupled)R0构型上。因此,这些多肽在培养的细胞中诱导延长的cAMP信号响应。与PTH(1-34)或其他测试类似物比较,这些多肽在实验室测试动物(小鼠、大鼠、和猴)中的血液离子化的钙水平中也表现延长的增加。由于它们在体内证实的长效性能,这些类似物对于诸如甲状旁腺功能减退的病症的治疗具有效用。
典型的多肽,SP-PTH-AAK和Aib-SP-PTH-AAK,包含基于人类PTH(1-14)序列的N-末端部分和基于人类PTHrP序列(参考下面表1)的C-末端部分,具有含有亲和力-增强的氨基酸替代(取代,substitution)的N-和C-末端部分。如下面实例中说明的,这些多肽在体外表现令人惊讶的高结合亲和力和cAMP信号效力,以及体内增强的功能效应。最后,如下面描述的,与野生型PTH(1-34)多肽比较,这些多肽表现高的溶解性。基于这些性能,这些多肽可用于其中期望延长的对PTH受体的活性的任何应用,例如,用于甲状旁腺功能减退的治疗。
PTH激动剂的R0 vs.RG结合
如PCT公开WO 2009/017809中描述的,识别了PTH受体的新的“R0”状态,其中受体不结合到它的G-蛋白上,但是能够进行激动剂结合。先前已经认为,可区分G-蛋白-偶联的受体的两种形式:结合到G-蛋白上的形式(RG)和不结合到G-蛋白上的形式(R)。GPCR信号(传导)需要通过受体直接地激活G-蛋白,例如,必须形成RG状态,且可通过激动剂配体的结合诱导这种RG形成。激动剂配体的结合诱导或稳定RG状态,反过来,RG状态稳定激动剂的高亲和力结合。在结合GTP,或非-可水解的GTP类似物,例如,GTPγS之后,将从受体分离受体-偶联的G蛋白,引起受体恢复到低亲和力状态。现在,已经公认某些GPCR,象PTHR,可形成新的状态(R0),其可在有GTPγS的情况下,因此,甚至在受体预期不结合G蛋白时,也可以高亲和力结合某些激动剂配体。基于R0状态的这种发现,PCT公开WO 2009/017809描述了,除了以高亲和力结合RG状态之外,以高亲和力结合到R0状态上的配体,比以较低的亲和力结合到R0上的配体具有更长的活性半衰期。这种延长的活性不依赖于配体在体内的生物利用度或药物代谢动力学。相应地,具有短的作用持续时间的激动剂对于受体的R0形式具有较低的亲和力。
如下面实例中描述的,与在体外的hPTH(1-34)比较,SP-PTH-AAK和Aib-SP-PTH-AAK对PTH受体的R0形式表现显著更高的结合,在体外和体内表现长效活性。因此,本发明的多肽适于用作长效PTH激动剂。
制备本发明的多肽
本发明的多肽(例如,SP-PTH-AAK和Aib-SP-PTH-AAK)适于通过液-相或固-相肽合成和通过利用组合化学(combination chemistry)的原位合成来生产。具体地,固相肽合成技术已经被成功地用于人类PTH的生产且可用于这些化合物的生产(关于指导,参考,上文的Kimura等人和Fairwell等人,Biochem.22:2691,1983)。已经在Goud等人J Bone Min Res6:781,1991中报道了以相对大规模生产人类PTH的成功。合成肽的合成方法通常涉及自动化的合成仪和作为固相的适当的树脂的应用,其中将树脂连接到期望的多肽的C-末端氨基酸上。然后通过顺序地连接下一个期望的氨基酸的适当保护形式完成N-末端方向的肽延伸,一般利用FMOC-或BOC-基化学方案,直到完成合成。然后从肽中清除保护基团,通常同时从树脂上分离肽,然后分离肽和利用传统技术纯化肽,如通过利用乙腈作为溶剂和利用三氟乙酸作为离子配对试剂进行反相HPLC。通常在许多出版物中描述这样的程序,例如可参考Stewart and Young,“Solid PhasePeptide Synthesis,”2nd Edition,Pierce Chemical Company,Rockford,IL(1984)。
也可通过本领域已知的任何方法重组地制备本发明的多肽。原核(例如,细菌)和真核(例如,酵母和哺乳动物)表达系统也可用于生产本发明的多肽,特别是其中多肽只包含基因组编码的氨基酸的情况(例如,不是Aib或Har)。
多肽修饰
此处描述的任何多肽(例如,SP-PTH-AAK和Aib-SP-PTH-AAK)可含有一个或更多修饰如N-末端或C-末端修饰。修饰包含乙酰化作用、酰化作用、ADP-核糖基化作用、酰胺化作用、黄素的共价连接、血红素部分的共价连接、核苷或核苷衍生物的共价连接、脂质或脂质衍生物的共价连接、磷脂酰肌醇的共价连接、交联、环化作用、二硫键形成、去甲基化作用、共价交联的形成、胱氨酸的形成、焦谷氨酸的形成、甲酰化(formylation)、γ-羧化作用、糖基化、GPI锚形成、羟基化作用、碘化作用、甲基化作用、肉豆蔻酰化、氧化、蛋白水解加工、磷酸化作用、异戊烯化(prenylation)、外消旋作用、硒化(selenylation)、硫酸盐化作用、的转运RNA介导的氨基酸添加到蛋白质如精氨酰化作用、和泛素化作用。参考,例如,Proteins-Structure and Molecular Properties,2nd Ed.,T.E.Creighton,W.H.Freeman and Company,New York,1993和Wold,F.,Posttranslational Covalent Modification of Proteins中第1-12页,Posttranslational Protein Modifications:Perspectives and Prospects B.C.Johnson,Ed.,Academic Press,New York,1983;Seifter et al.,MethodsEnzymol 182:626 646(1990)和Rattan et al.,Ann NY Acad Sci 663Α& 62(1992)。
本发明的任何多肽可进一步包含异源序列(融合伴侣),因此形成融合蛋白。融合蛋白可包含融合伴侣如纯化和检测标签,例如,可直接地或间接地检测的蛋白质如绿色荧光蛋白、红血球凝集素、或碱性磷酸酯酶,DNA结合结构域(例如,GAL4或LexA),基因活化结构域(例如,GAL4或VP16),纯化标签、或分泌信号肽(例如,前原胰蛋白酶(preprotyrypsin)信号序列)。在其他实施例中,融合伴侣可以是标签,如c-myc、多聚组氨酸、或FLAG。每一个融合伴侣可含有一个或更多结构域,例如,促胰岛素信号序列和FLAG标签。在其他情形中,融合伴侣是Fc蛋白质(例如,小鼠Fc或人Fc)。
治疗疾病的方法
可利用此处描述的多肽(例如,SP-PTH-AAK和Aib-SP-PTH-AAK)治疗PTH机能不良或钙或磷酸盐不平衡关联的任何疾病。多肽可用于治疗甲状旁腺功能减退、血磷酸盐过多、骨质疏松症、骨折修复、骨软化、关节炎、或血小板减少症,或可用于增加受试者的干细胞活化(mobilization)。给予的任何模式(例如,口服、静脉内的、肌肉的、眼部的、局部的(topical)、皮肤的、皮下的、和直肠的)可用于本发明的治疗方法。医师将决定用于被治疗的病人的适当的剂量,其将部分地依赖于病人的年龄和尺寸、疾病或病症的严重性、和被治疗的具体的疾病或病症。
药物组合物的配制(formulation)
可通过任何适当的方式实现此处描述的任何多肽(例如,SP-PTH-AAK和Aib-SP-PTH-AAK)的给予,其中所述方式实现治疗受试者和疾病病症的化合物浓度。可在任何适当的载体物质中含有任何适当量的多肽,通常按重量计以组合物总重量的1-95%的量存在。可在适于口服、肠胃外(例如,静脉内地或肌肉地)、直肠的、皮肤的、鼻的、阴道的、吸入的、皮肤(贴片)、眼部、或头颅内给予途径的剂型提供组合物。因而,组合物可以是,例如,片剂、安瓿、胶囊、药丸、散剂、粒剂、悬浮液、乳剂、溶液、包含水凝胶的凝胶体、软膏、油膏、膏状物、膏药、灌药(大剂量药液,drenche)、渗透递送设备(osmotic delivery device)、栓剂、灌肠剂、注射剂、移植物、喷雾、或气雾剂的形式。可依照传统的药物学实践配制药物组合物(参考,例如,Remington:The Science and Practiceof Pharmacy,20th edition,2000,ed.A.R.Gennaro,Lippincott Williams &Wilkins,Philadelphia,和Encyclopedia of Pharmaceutical Technology,eds.J.Swarbrick and J.C.Boylan,1988-1999,Marcel Dekker,New York)。
可配制药物组合物从在给予之后立即或在给予之后的任何预定时间或时间段释放活性化合物。后面类型的组合物通常被称为受控释放制剂,其包含(i)在延长的时段,在身体内形成本发明试剂基本恒定浓度的制剂;(ii)在延长的时段,在预定间隔时间(lag time)之后,在身体内形成本发明试剂的基本恒定浓度的制剂;(iii)在预先确定的时段,通过维持身体中的试剂相对不变的、有效的水平维持试剂的作用同时伴随在试剂的血浆水平中的波动关联的不期望的最小副作用的制剂(锯齿动力学模式);(iv)局部化试剂作用的制剂,例如,受控释放组合物邻近或在患病组织或器官中的空间放置;(v)达到定量给予的便利的制剂,例如,每周一次或每隔两周一次给予组合物;和(vi)通过利用载体或化学衍生物递送化合物,将试剂的作用靶向具体的靶细胞类型的制剂。以受控释放制剂的形式给予化合物对于在胃肠道中具有窄吸收窗口或相对短的生物半衰期的化合物是特别优选的。
为了获得受控释放,可进行多种策略中的任一种,其中释放的速率超过所讨论化合物的新陈代谢的速率。在一个实例中,通过各种配方参数和成分的适当的选择获得受控释放,包含,例如,各种类型的受控释放组合物和涂层。因而,利用适当的赋形剂将化合物配制到药物组合物中,其在给予之后,以受控方式释放化合物。实例包含单或多单位(unit)片剂或胶囊组合物、油溶液、悬浮液、乳剂、微胶囊、分子复合物、微球体、纳米颗粒、贴剂(patch)、和脂质体。
肠胃外组合物
可肠胃外地通过注射、灌输、或移植(皮下的、静脉内的、肌肉的、腹膜内的,等等),以剂型、制剂、或通过适当的递送设备或含有传统的、无毒的药学上可接受的载体和佐剂的植入物给予含有此处描述的多肽(例如,SP-PTH-AAK和Aib-SP-PTH-AAK)的组合物。这样的组合物的制剂和配制是药物制剂领域的技术人员所公知的。
可以单位剂型(例如,单剂量安瓿),或在含有几种剂量以及可在其中添加适当的防腐剂的小瓶中(参加下文)提供适于肠胃外应用的组合物。组合物可以是溶液、悬浮液、乳剂、输注设备、或适于移植的递送设备的形式,或它可呈现为在使用之前要利用水或另外的适当媒介物复配(复溶,reconstitute)的干粉。除了活性剂之外,组合物可包含适当的肠胃外可接受的载体和/或赋形剂。活性剂可包含在适于受控释放的微球体、微胶囊、纳米颗粒、脂质体等中。此外,组合物可包含悬浮剂、增溶剂、稳定剂、pH-调节剂、张性调节剂、和/或分散剂。
如上述指出的,依照本发明,药物组合物可以为适于无菌注射的形式。为了制备这样的组合物,将适当的活性剂溶解在或悬浮在肠胃外可接受的液体媒介物中。在可接受的媒介物和溶剂中可采用的是水,通过添加适当量的盐酸、氢氧化钠或适当的缓冲液、1,3-丁二醇、林格氏溶液、葡萄糖溶液、和等张的氯化钠溶液,将水的pH调整到适当的pH。水性制剂也可含有一种或多种防腐剂(例如,对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、或对羟基苯甲酸正丙酯)。在其中一种化合物仅难溶或微溶于水的情形中,可添加溶解增强剂或增溶剂,或溶剂可包含10-60%w/w的丙二醇等。
下列实例倾向于解释而不是限制本发明。
实例1
多肽合成
通过Massachusetts General Hospital Biopolymer Core机构合成典型的肽[Ala1,12,Aib3,Gln10,Har11,Trp14]PTH(1-14)/PTHrP(15-36)(高效Aib-PTH:Aib-SP-PTH)和[Ala1,12,18,22,Aib3,Gln10,Har11,Trp14,Lys26]PTH(1-14)/PTHrP(15-36)(高效Aib-AAK PTH:Aib-SP-PTH-AAK)。Aib和Har分别代表α-氨基异丁酸和高精氨酸。
通过Sigma Aldrich(北海道,日本)和American Peptide Company,Inc.(加利福尼亚,USA)合成典型的肽[Ala1,3,12,Gln10,Arg11,Trp14]PTH(1-14)/PTHrP(15-36)(高效PTH:SP-PTH)和[Ala1,3,12,18,22,Gln10,Arg11,Trp14,Lys26]PTH(1-14)/PTHrP(15-36)(SP-PTH-AAK)。将所有的多肽溶解在10mM乙酸中,且存储在-80℃。利用PACE方法(Pace et al.,ProteinScience 4:2411-23,1995)或通过氨基酸分析测定多肽浓度。
在下面表1中示出这些多肽中的每一个。
表1.多肽
Mc残基:Ala1,3,12、Gln10、Arg11,19、Trp14
M残基:Ala1,12、Aib3、Gln10、高Arg11、Trp14、Arg19
实例2
表征-R0/RG结合和cAMP效力
利用如PCT公开WO 2009/017809中描述的方法或相似的方法测量PTH(1-34)、SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK对PTH-1受体的R0和RG形式的结合。简要地,可通过加入不可水解的核苷类似物GTPγS协助受体的R0形式。例如,可通过利用负显性Gαs亚单位共转染细胞协助RG形式。基于放射性示踪剂配体(125I-PTH(1-34))的置换(displacement)测量结合。如图1A中示出的,与PTH(1-34)比较,对于大鼠PTH受体的R0形式,四个检验的多肽表现出大约1-2个数量级更强的结合。特别是SP-PTH-AAK在结合中表现出大于两个数量级的增加(PTH(1-34)的log EC50-9.7 vs.-7.5)。数据是四个实验的平均值,每一个实验进行两次。利用Graph-Pad Prism 4.0曲线拟合至数据。每一个图中的插图显示拟合参数。
与hPTH(1-34)比较,也在SP-PTH-AAK和Aib-SP-PTH-AAK中增加了在大鼠PTH受体上的RG结合(图1B)。
同时,利用两个不同的方法:放射性免疫测定(RIA;图2)和融合到荧光素酶基因上的cAMP响应元件(图3),评价这些多肽的cAMP刺激活性。简要地,如Okazaki等人描述的(Proc Natl Acad Sci USA105:16525-30,2008),在96-孔板中,在完整的MC3T3-E1细胞,小鼠前成骨细胞系(preosteoblastic cell line)中进行RIA cAMP分析。利用各种浓度(-12至-6 logM)的指定配体在室温中,在有IBMX的情况下将细胞处理30分钟,在其之后,清除缓冲液,通过添加50mM HCl终止反应。然后通过RIA测定HCl裂解物的cAMP含量。数据是六个实验的平均值,每一个实验进行两次。利用Graph-Pad Prism 4.0将曲线拟合数据。插图显示每一个曲线的拟合参数。
如下测量cAMP-响应元件-荧光素酶响应。在96-孔板中,利用编码融合到cAMP-响应元件(CRE)启动子上的荧光素酶基因的质粒DNA,设计以便通过cAMP/PKA途径评价信号的质粒构建体来转染MC3T3-E1细胞。在转染48小时之后,在含有媒介物(图表横坐标(plot abscissa)上的-14 logM)或各种浓度(-13至-16 logM)的指定配体的培养基中,将细胞在37℃孵育四小时。然后,利用Promega Steady-Glo试剂和PerkinElmerCo.Envision平板读数器(读板器)测量荧光素酶活性,如发光。数据是三个实验的平均值,每一个实验进行两次。在媒介物-处理的孔中获得的未处理的基值(basal value)是9434±1303数/秒。利用Graph-Pad Prism 4.0将曲线拟合数据。图中的插图显示拟合参数。
也计算了cAMP效力和R0(图4A)或RG(图4B)结合之间的相关性。观察到R0结合和cAMP效力之间的强相关性。观察到RG结合和cAMP效力之间的较低相关性。
利用人类PTH受体(图5A和5B)重复R0和RG结合实验。此处,如Dean等人,Mol Endocrinol 22:156-66,2008描述的,在从转染的COS-7细胞中制备的膜中进行R0和RG PTHR1构型的竞争性结合实验。数据是三个实验的平均值,每一个实验进行两次。利用Graph-Pad Prism 4.0将曲线拟合数据。插图显示拟合参数。与PTH(1-34)比较,如关于大鼠受体,SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK多肽表现增强的R0结合。
同时,利用人类受体测量了cAMP效力分析(图6)。此处,在含有媒介物(图表横坐标上的-11 logM),或各种浓度的(-10至-6logM)的指定配体的缓冲液中,将来源于LLC-PCK1细胞和利用人类PTHR1稳定地转染的HKRK-B7细胞孵育30分钟,且通过RIA测量细胞中的cAMP含量。SP-PTH-AAK、Aib-SP-PTH-AAK、SP-PTH、和Aib-SP-PTH表现类似于PTH(1-34)的cAMP效力。在表2中提供数据的总结。
表2.受体结合和cAMP活化数据的总结
实例3
在冲掉配体之后的cAMP响应
也检验了在配体冲掉之后,SP-PTH-AAK、Aib-SP-PTH-AAK、SP-PTH、和Aib-SP-PTH刺激cAMP活性的能力。如Okazaki等人,Proc NatlAcad Sci USA 105:16525-30,2008描述的,在96-孔板中,在MC3T3-E1细胞中进行cAMP冲掉(wash out)响应实验。在室温下,利用媒介物或指定配体(100nM)将细胞处理五分钟;每一个配体重复孔用于获得最初(值)(最大cAMP值);在这些孔中,利用配体和IBMX共孵育细胞。在最初处理五分钟之后,清除缓冲液,关于最初的孔,通过添加50mM HCl终止反应。在其他孔中,利用缓冲液将细胞漂洗三次,和如横坐标上指出的,在缓冲液中孵育各种时间;在其之后以含有IBMX的缓冲液代替缓冲液,和将细胞孵育另外五分钟。在那之后,清除缓冲液,通过添加50mMHCl终止反应。然后,通过RIA确定HCl裂解液的cAMP含量。
如图7A和7B中示出的,SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK比PTH(1-34)维持较长时期的cAMP-刺激活性,因而表明这些多肽具有长效激动剂活性。
实例4
小鼠中的研究
也分析了小鼠的血Ca++响应。利用媒介物或指定配体皮下地注射小鼠(C57 BL/6),从而给出5nmol/kg体重的最终剂量,在注射之后的时间经尾部采取血液,并利用Bayer Rapid Lab模型348血液分析仪评价Ca++浓度。如示出的,所有的多肽,包含PTH(1-34)在给予之后两个小时表现相似的钙水平(图8A)。然而,在后面的时间点上(4、6、8、和24小时),与接受PTH(1-34)或媒介物的小鼠比较(图8A和8B),接受SP-PTH、SP-PTH-AAK、Aib-SP-PTH、或Aib-SP-PTH-AAK的小鼠中增加了血钙水平。利用10nmol/kg,通过静脉注射的相似实验也得出相似的结果(图8C和8D)。如图8E中示出的,也进行了在禁食条件下开展的相似实验。
实例5
大鼠中的研究
也检验了多肽对已经经历甲状腺甲状旁腺切除术(thyroparathyroidectomy)(TPTX)的大鼠的影响。此处,从Charles River LaboratoriesJapan,Inc.(神奈川,日本)获得五-周-大雄性Crl:CD(SD)大鼠,且使其在标准的20-26℃和35-75%湿度的实验室条件下适应1周。可自由获取自来水以及标准啮齿动物食物(CE-2)(含有1.1%钙、1.0%磷酸盐、和250IU/100g维生素D3)(Clea Japan,Inc.,静冈,日本)地饲养大鼠。
在六-周-大的大鼠上进行甲状腺甲状旁腺切除术(TPTX)。在手术之后24小时,通过来自尾部静脉流血的血清离子钙(iCa)选择TPTX大鼠(<1.0mM)。在手术之后72小时,依据iCa将TPTX大鼠分成六组,每组五或六个动物。TPTX-媒介物组从尾静脉而静脉地接受1ml/kg体重的剂量的媒介物(10mM乙酸溶液)。将1.25nmol/kg的剂量的SP-PTH、SP-PTH-AAK、Aib-SP-PTH、和Aib-SP-PTH-AAK各自静脉地注射到TPTX大鼠中。
为了测量血清钙和磷酸盐,利用克他命麻痹大鼠,在各个注射之后的各种时间从颈静脉获得血液(例如,在6、8、24、48、和72小时)(图9)。通过自动分析仪(736-20 Model Hitachi,东京,日本)测定血清钙和磷。
如图10A和10B示出的,在多肽的静脉注射给予之后,在延长时期内增加了血清钙水平,并减少了血清磷酸盐水平。为了比较,也提供了比较1.25和5nmol/kg的SP-PTH与1.25、5、和20nmol/kg的PTH(1-34)的相似的实验(图10C)。这些差异不像是由药物动力学的改变介导的,如PTH(1-34)和SP-PTH在正常的大鼠(图10D)和TPTX大鼠中(图10E)种表现相似的概况(profile)。在表3中示出了这些肽在TPTX大鼠中的性能。
表3.肽药代动力学
实例6
食蟹猴中的高血钙分析(Hypercalcemic assay)
在食蟹猴中检验了SP-PTH和SP-PTH-AAK(“SP-AAK”)对血清和尿钙和磷酸盐水平的影响。简要地,测量了三-或四-岁-大的,雄性食蟹猴(HAMRI Co.,Ltd.,Ibaraki,Japan)它们的体重。将血液收集到管子中。猴接受0.3ml/kg剂量的每种肽的静脉或皮下给予。通过用25mmol/L磷酸盐-柠檬酸盐缓冲液/100mmol/L NaCl/0.05% Tween 80(pH.5.0)(PC-缓冲液)稀释调整存储液(储备液,stock solution)中的多肽浓度。在给予之前,允许立即将所有的多肽放置在冰上。各自分别地将多肽给予三个猴子的组。在给予之后1、2、4、和8小时,通过隐静脉收集血液,以便监测钙和磷水平的时间进程(图11A)。观察到SP-PTH具有大约4分钟的血浆半衰期(图11B)。
如图12A和12B示出的,在注射之后,SP-PTH或SP-PTH-AAK的注射增加了一段相当长的时间的血钙(图12A)。同样地减少了血清磷酸盐(图12B)。用于比较,图12C显示,皮下地给予的PTH(1-34)的简短(brief)钙影响的剂量是图12A和12B的实验的40-倍。与媒介物比较,在接受任一多肽的小鼠中,减少了尿钙/肌氨酸酐比率(图13A)。如图13B中示出的,也测量了尿磷酸盐/肌氨酸酐比率。
利用两个不同剂量的多肽皮下注射进行相似的实验(2.5nmol/kg或10nmol/kg;图14)。从这些实验来看,观察到利用SP-PTH和SP-PTH-AAK的血清钙以剂量依赖的方式增加(图15A)。也观察到血清无机磷酸盐的减少(图15B和15C)。图15D显示了血清肌氨酸酐水平的增加。也收集了尿钙和磷酸盐数据。这些数据来自图15A-15D中示出的相同的实验。在注射之前(T=0)的24-小时时间间隔中和在之后的时间收集尿,并评价Ca(图15E和15G)、Pi(图15F和15H)和肌氨酸酐;将[Ca]和[PI]表示成对[肌氨酸酐]的比率。图15G和15H呈现来自预注射(pre-injection)值的改变。数据是平均值±s.e.m.;n=3/组。
实例7
溶解性
在体外沉淀实验中评价SP-PTH-AAK和SP-PTH在不同的缓冲液中的相对的溶解性。关于每一个多肽,制备每一个含有50μg冻干的多肽粉末的两个小瓶;一个小瓶在pH为7.4的50μl的PBS中复配;另一个小瓶在50μl的10mM乙酸(pH2.9)中复配,以便给出最终的浓度1.0mg/ml或1.5mg/ml。在室温下一小时之后,将小瓶在15,000xg离心2分钟。清除上清液和利用Pierce BCA分析(Thermo Fischer Scientific,Rockford,Ill.)分析蛋白质含量。关于每一个多肽,将PBS样品的含量表示成相应的乙酸样品的含量的百分数。
如图16A中示出的,SP-PTH-AAK和SP-Aib-PTH-AAK多肽表现出类似于hPTH(1-34)NH2的溶解性。在图16B中显示hPTHrP、片段、和类似物的溶解度检验。也进行SP-PTH-AAK的溶解度检验。将SP-PTH-AAK在5、25、和40℃以不同肽浓度存储在50mM磷酸盐-柠檬酸盐(PC)缓冲液(pH4.0、4.5、和5.5)和10mM乙酸中4周。通过反相HPLC对完整肽的分析显示在25℃ 4周接近完全的稳定性(图16C)。在40℃,在作为主峰的肩翼(shoulder)的HPLC层析图上检测到降解的产物,可能地甲硫氨酸-氧化物衍生物。
实例8
对磷酸化-缺陷型(PD)PTH受体的影响
在表达PD PTH受体代替野生型PTH受体的“敲入”小鼠中检验SP-PTH-AAK的活性(Bounoutas等人,Endocrinology 147:4674,2006)。如Bounoutas解释的,PD受体表现不足的内化,其可导致延长的cAMP信号。
在野生型和PD小鼠中,检验野生型PTH(1-34)和更高效力的M-PTH(1-28)改变血液钙水平的能力。如图17A和17B中示出的,与野生型小鼠比较,在PD小鼠中减少或消除了血液钙水平的增加。
也在两种类型小鼠之间比较了血液cAMP水平。关于野生型PTH(1-34)和关于M-PTH(1-28),以数量级增加了血液cAMP水平,如与野生型小鼠比较,在PD小鼠中延长了持续时间(图18A和18B)。
在图19中示出了SP-PTH-AAK在野生型和PD小鼠中对血液钙水平的影响。令人惊讶地,在注射之后至少六小时,SP-PTH-AAK减少了血液钙水平,且从不升高到高于利用媒介物对照所看到的水平。
实例9
在TPTX大鼠中重复的日剂量
我们在啮齿动物和猴子中进行的单独的注射实验,在某些情形中,显示持续超过24小时的生物活性,如显著的血钙过多所指示的,我们认为这些实验中的高度延长应归于使用的高剂量。在人类治疗应用中,人们将给予仅仅足够标准化血液Ca的剂量。关键的实验是确定在重复剂量方案之后标准化sCa是否真正可行。因而,我们进行图20A-20D的实验,其中每天利用1、2、4和8nmol/kg(s.c)剂量的SP-PTH处理TPTX大鼠共10天。为了比较,使用0.075和0.2mcg/kg(口服)的剂量的1,25(OH)2-维生素-D(骨化三醇)相同的时期。在这个研究中,如它在最后注射之后8小时避免血钙过多(图20A),和它在24小时上维持在正常的sCa附近(图20B),4nmol/kg的SP-PTH剂量给出最满意的sCa控制。令人印象深刻的是,利用4nmol/kg的SP-PTH剂量,尿钙排泄在所有的时间点上都是很低的,其与利用0.2μg/kg(在8-9mg/dl范围中达到sCa所需要的剂量)的骨化三醇的升高的尿钙形成对比(图20C和20D)。
统计分析
通常将上述描述的数据表示成平均值±标准误差(SEM)。利用SAS软件确定统计显著性(Ver.5.00.010720,SAS Institute Japan,东京,日本)。将<0.05的p值的差异认为是统计上显著的。*P<0.05、**P<0.01、***P<0.001。
其他实施例
本说明书中提及的所有专利、专利申请、和公开,包括2010年5月13日提交的美国申请No.61/334,319和2010年11月18日提交的美国申请No.61/415,141合并在此以供参考,并入程度如同特别和单独地指出每一单独的专利、专利申请、或公开并入在此以供参考一样。
Claims (26)
1.一种多肽、或其药学可接受的盐,包括式(I):
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-X13-X14-X15-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-X25-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile (I)
X01是Ser、Ala、或Aib;
X03是Ser、Ala、或Aib;
X08是Met、Leu、或Nle;
X10是Asn、Ala、Val、Asp、Glu、或Gln;
X11是Leu、Ala、Val、Met、Lys、Ile、Arg、Har、或Trp;
X12是Gly、Ala、His、或Arg;
X13是Lys、Ala、Leu、Gln、Arg、His、或Trp;
X14是His、Leu、Arg、Phe、Trp、或Ser;
X15是Ile或Leu;
X16是Gln或Asn;
X17是Asp或Ser;
X18是Ala、Leu、Met、Glu、Ser、或Phe;
X22是Ala、Phe、Glu、Ser、Leu、Asn、Trp、或Lys;
X25是His、Arg、Leu、Trp、或Lys;以及
X26是Lys、His、Ala、Ser、Asn、或Arg;
或其包括式(I)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段,条件是至少一个X18不是Leu或Met,X22不是Phe,以及X26不是His。
2.根据权利要求1所述的多肽、或其药学可接受的盐,所述多肽包括式(II):
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-Lys-X14-X15-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-His-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile (II)
X01是Ser、Ala、或Aib;
X03是Ser、Ala、或Aib;
X08是Met、Leu、或Nle;
X10是Asn、Gln、或Asp;
X11是Leu、Arg、Har、或Lys;
X12是Gly或Ala;
X14是His、Trp、或Ser;
X15是Ile或Leu;
X16是Gln或Asn;
X17是Asp或Ser;
X18是Ala或Leu;
X22是Ala或Phe;以及
X26是Lys或His;
或其包括式(II)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
3.根据权利要求2所述的多肽、或其药学可接受的盐,包括式(III):
X01-Val-X03-Glu-Ile-Gln-Leu-X08-His-X10-X11-X12-Lys-X14-Ile-X16-X17-X18-Arg-Arg-Arg-X22-Phe-Leu-His-X26-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile (III)
X01是Ser、Ala、或Aib;
X03是Ser、Ala、或Aib;
X08是Met、Leu、或Nle;
X10是Asn或Gln;
X11是Leu、Arg、或Har;
X12是Gly或Ala;
X14是His或Trp;
X16是Gln或Asn;
X17是Asp或Ser;
X18是Ala或Leu;
X22是Ala或Phe;以及
X26是Lys或His;
或其包括式(III)的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
4.根据权利要求1-3的任一项所述的多肽,其中X01和X03是Ala;X10是Gln;X11是Arg;X12是Ala;以及X14是Trp。
5.根据权利要求1-3的任一项所述的多肽,其中X01是Ala;X03是Aib;X10是Gln;X11是Har;X12是Ala;以及X14是Trp。
6.根据权利要求1-5的任一项所述的多肽,其中X18是Ala;X22是Ala;或X26是Lys。
7.根据权利要求6所述的多肽,其中X18是Ala;X22是Ala;以及X26是Lys。
8.根据权利要求1-7的任一项所述的多肽,其中与SP-PTH比较,所述多肽在中性水溶液中表现更大的溶解性。
9.根据权利要求1-8的任一项所述的多肽,其中所述多肽是PTH受体激动剂。
10.根据权利要求1-9的任一项所述的多肽,其中所述多肽以比hPTH(1-34)更高的亲和力结合到人类PTH-1受体的R0状态上。
11.根据权利要求1-10的任一项所述的多肽,其中所述多肽的长度小于50个氨基酸。
12.根据权利要求1-11的任一项所述的多肽、或其药学可接受的盐,包括氨基酸序列:
Ala-Val-Ala-Glu-Ile-Gln-Leu-Met-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或其包括所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
13.根据权利要求12所述的多肽、或其药学可接受的盐,包括氨基酸序列:
Ala-Val-Ala-Glu-Ile-Gln-Leu-Met-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile。
14.根据权利要求1-11的任一项所述的多肽、或其药学可接受的盐,包括氨基酸序列:
Ala-Val-Aib-Glu-Ile-Gln-Leu-Met-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或其包括所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
15.根据权利要求14所述的多肽、或其药学可接受的盐,包括氨基酸序列:
Ala-Val-Aib-Glu-Ile-Gln-Leu-Met-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile。
16.根据权利要求1-11的任一项所述的多肽、或其药学可接受的盐,包括选自由以下组成的组的氨基酸序列:
Ala-Val-Ala-Glu-Ile-Gln-Leu-Nle-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;
Ala-Val-Ala-Glu-Ile-Gln-Leu-Leu-His-Gln-Arg-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;
Ala-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile;和
Ala-Val-Aib-Glu-Ile-Gln-Leu-Leu-His-Gln-Har-Ala-Lys-Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-Glu-Ile,
或其包含所述序列的氨基酸1-28、1-29、1-30、1-31、1-32、1-33、1-34、或1-35的片段。
17.一种药物组合物,包括权利要求1-16的任一项所述的多肽和药学上可接受的载体。
18.根据权利要求1-16的任一项所述的多肽,其中通过固相合成来合成所述多肽。
19.一种用于治疗受试者的方法,所述受试者具有选自由甲状旁腺功能减退、血磷酸盐过多、骨质疏松症、骨折修复、骨软化、关节炎、和血小板减少症所组成的组中的疾病,所述方法包括给予所述受试者足够治疗所述疾病的量的权利要求1-16的任一项所述的多肽或权利要求17所述的药物组合物。
20.根据权利要求19所述的方法,其中给予的途径选自由皮下、静脉内、鼻内、经肺部、经皮肤、以及口服所组成的组。
21.一种包括编码权利要求1-16的任一项所述的多肽的序列的核酸。
22.根据权利要求21所述的核酸,可操作地连接到启动子上。
23.一种载体,包括权利要求22所述的核酸。
24.一种细胞,包括权利要求23所述的载体。
25.一种制造多肽的方法,包括在表达所述多肽的条件下培养权利要求24所述的细胞。
26.根据权利要求25所述的方法,进一步包括纯化所述多肽。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US33431910P | 2010-05-13 | 2010-05-13 | |
US61/334,319 | 2010-05-13 | ||
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WO2011143406A2 (en) | 2010-05-13 | 2011-11-17 | The General Hospital Corporation | Parathyroid hormone analogs and uses thereof |
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KR102611820B1 (ko) | 2016-09-29 | 2023-12-07 | 아센디스 파마 본 디지즈 에이/에스 | 낮은 피크 대 트로프 비를 가진 pth 화합물 |
GB201706781D0 (en) | 2017-04-28 | 2017-06-14 | Univ Sheffield | Parathyroid hormone fusion polypeptide |
EP3655104A4 (en) | 2017-07-15 | 2021-03-17 | The Regents of the University of California | OSTEOADSORPTIVE FLUOROGENIC SUBSTRATE FROM CATHEPSIN K FOR IMAGING OSTEOCLAST ACTIVITY AND MIGRATION |
EP3765061A4 (en) * | 2018-03-16 | 2022-01-26 | The General Hospital Corporation | PARATHORMON-POLYPEPTIDE CONJUGATES AND METHODS OF USE |
WO2021030196A1 (en) * | 2019-08-09 | 2021-02-18 | Phasebio Pharmaceuticals, Inc. | Elp fusion proteins comprising parathyroid hormone for controlled and sustained release |
MX2022007371A (es) * | 2020-01-13 | 2022-07-12 | Ascendis Pharma Bone Diseases As | Tratamiento del hipoparatiroidismo. |
IL301411A (en) * | 2020-09-28 | 2023-05-01 | Ascendis Pharma Bone Diseases As | Improving the physical and mental condition of patients with hypoparathyroidism |
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