CN102271713A - 神经降压肽衍生的分枝肽及其用途 - Google Patents
神经降压肽衍生的分枝肽及其用途 Download PDFInfo
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- CN102271713A CN102271713A CN2009801537891A CN200980153789A CN102271713A CN 102271713 A CN102271713 A CN 102271713A CN 2009801537891 A CN2009801537891 A CN 2009801537891A CN 200980153789 A CN200980153789 A CN 200980153789A CN 102271713 A CN102271713 A CN 102271713A
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Abstract
Description
发明领域
本发明涉及衍生自神经降压肽(NT)序列的体内稳定的分枝肽。所述的肽可以与功能单位结合,特异性地靶向于癌细胞。因此它们可用于肿瘤的诊断和/或治疗。
背景技术
在经典的化学治疗中的一个主要问题是大多数抗癌剂甚至对于正常细胞的非特异性的毒性。因此,特异性地靶向于肿瘤是癌症治疗和诊断研究中的重要挑战。
目前,创新的肿瘤特异性治疗遵循了针对肿瘤细胞上特异性表达或过表达的肿瘤相关蛋白的策略。
对于在大量原发性人类癌症中表达的不同内源性调节肽的受体的观察,揭示了使用合成肽来实现肿瘤选择性靶向的新前景1-3。
神经降压肽(NT)是13个氨基酸的肽,其具有在中枢神经系统中的神经递质或神经调质和在外周的局部激素的双重功能。NT受体在严重的恶性肿瘤中过表达,所述的肿瘤例如小细胞肺癌、结肠癌、胰腺癌和前列腺癌。NT稳定的类似物已经在几年前被建议用于肿瘤治疗4-10,考虑到在这些肿瘤中NT受体的高发生率和密度,NT仍然被认为是外分泌胰腺癌的基于肽的治疗的最有可能的候选物11。超过75%的所有导管的胰腺癌过表达NT受体,而正常的胰腺组织、胰腺炎和内分泌胰腺则没有此现象12。
靶向于肿瘤受体是解决癌症化学治疗的非特异性毒性问题的基础,并且是通过放射性同位素进行肿瘤定位的重要工具。但是,肽的体内应用仍然受到其短半衰期的很大限制。
本发明的发明人以前证明了以分枝的树状聚体形式合成肽,可得到能够保持肽的生物学活性并且对于体液的蛋白水解活性具有高抗性的分子,因此相对于单体肽具有显著增加的半衰期13-14。
本发明涉及衍生自NT的体内稳定的树状聚体肽。所述的肽还与功能单位结合,用来靶向于癌细胞。特别的是,NT4(8-13)与光敏剂Che6或化学治疗分子MTX结合,显示了对肿瘤细胞的特异性,并且对健康细胞无毒性,从而克服了经典化学治疗在对小鼠全身给药时的继发效应15。
在本发明中,除了分子的特异性以外,还观察到结合分子与游离的未偶联的药物相比活性增加。本发明的分子是从发明人合成的大量类似物库中筛选出的。该‘载体’肽(神经降压肽)是其中最佳的载体。例如,促黄体激素释放激素(LHRH),也称为促性腺素释放激素(GnRH)(QHWSYGLRPG,SEQ ID No.3),表现出低于NT的体外结合特性。此外,对每种新分子选择了在本发明中使用的连接体,因为它们对分子活性影响很大。例如,NT(8-13)4-β-Ala-生物素与NT(8-13)4相比显示了与NT受体较松动的结合活性;而NT4-peg-生物素则保持了与NT4相当的IC50值。NT(1-13)4-荧光素和NT(8-13)4-荧光素能够比NT(8-13)4-β-Ala-K(PEG-荧光素)更有效地染色癌细胞和组织,并且荧光团也更为稳定。最后,基于官能团选择化学治疗部分,例如有选择地和单一地用于偶联。键的强度根据药物本身的作用位点和模式进行调节,选择最佳的结合。例如6-巯基嘌呤通过‘不可裂解’的连接体与神经降压肽结合,其完全是无毒性的,而通过‘可裂解’的连接体与神经降压肽结合的该药物是有活性的,但是所得的活性不足以认为值得进一步开发。类似地,单星素衍生物也不是本发明化合物的第一选择。总之,这些特别的特征使本发明的目的独特并且优于任何可能的类似物。
发明概述
本发明的目的是具有通式A或B的多聚体分子:
其中
n=1-5,
其中m=9-12并且p=1-5,
其中q=1-5并且m=9-12;
并且Z=NH2或OH。
优选的是,该肽包含以下氨基酸序列:QLYENKPRRPYIL(神经降压肽,SEQ ID No.1)、pyroELYENKPRRPYIL(SEQ ID No.2)或RRPYIL(神经降压肽8-13,即SEQ ID No.1的氨基酸8至氨基酸13)。
优选的是,本发明的多聚体分子用于医药用途。
还优选作为抗肿瘤剂。
更优选的是,肿瘤是结肠癌或胰腺癌或前列腺癌。本发明的进一步的目的是本发明的多聚体分子在制备药物中的用途。
优选药物具有抗肿瘤活性。
本发明的进一步的目的是本发明的多聚体分子在肿瘤诊断中的用途。
优选的是,肿瘤是结肠癌或胰腺癌或前列腺癌。本发明的进一步的目的是药物组合物,该药物组合物包含本发明的多聚体分子或其药学上可接受的和有效的盐以及稀释剂和/或溶剂和/或载体和/或赋形剂和/或溶媒。
本发明的进一步的目的是治疗方法,该方法包括施用或暴露于本发明的多聚体分子。
优选治疗是抗肿瘤。
本发明的进一步的目的是诊断方法,该方法包括施用或暴露于本发明的多聚体分子。
优选诊断是肿瘤诊断。还优选肿瘤是结肠癌或胰腺癌或前列腺癌。
现在通过涉及以下附图的非限制性实施例来描述本发明:
图1.合成四分枝的NT(8-13)4-PEG-K(PEG-荧光素)[NT4(8-13)-FLUO]1、NT(8-13)4-βAla-K(PEG-苯丁酸氮芥)[NT4(8-13)-CLB]2、四分枝的NT(8-13)-βAla-K(PEG-5-氟脱氧尿苷)[NT4(8-13)-5-FdU]3、四分枝的NT(8-13)-βAla-K(PEG-6-巯基嘌呤)[NT4(8-13)-6-MP]4、四分枝的NT(8-13)-βAla-K(PEG-考布他汀)[NT4(8-13)-CBTST]5、四分枝的NT(8-13)-βAla-K(PEG-单星素)[NT4(8-13)-Mon]6、四分枝的NT(8-13)-βAla-K(PEG-替拉扎明)[NT4(8-13)-TPZ]7、四分枝的NT(8-13)-βAla-K(PEG-Asp-DOTA)[NT4(8-13)-DOTA]8以及四分枝的NT(8-13)-βAla-K(PEG-考布他汀醚)[NT4(8-13)-O-CBTST]9和四分枝的NT(8-13)-βAla-K(PEG-单星素醚)[NT4(8-13)-O-Mon]10。NT(1-13)4类似物携带氨基酸序列pyroELYENKPRRPYIL(SEQ ID No.2)。
图2.肿瘤细胞系中的结合与内化。HT-29、PC-3和PANC-1暴露于NT(1-13)4-荧光素30分钟(时间0)。在时间0和在培养基中37℃温育1和2小时后拍照。用凝集素-Cy3(红)染色细胞膜,并且用DAPI(蓝)染色细胞核。
图3.药物结合缓慢释放分枝NT在HT-29、PC-3和PANC-1细胞上的细胞毒性。左起组A):NT(8-13)结合甲氨蝶呤(MTX),游离MTX;不相关的肽(U4)结合MTX,A1:NT(1-13)结合MTX。左起组B):NT(8-13)结合苯丁酸氮芥-化合物2-(CLB),游离CLB;不相关的肽(U4)结合CLB。左起组C):NT(8-13)结合考布他汀-醚-化合物9-(O-CBTST);游离CBTST;不相关的肽(U4)结合CBTST。左起组D):NT(8-13)结合单星素-醚-化合物10-(MON),游离MON;不相关的肽(U4)结合MON。左起组E):NT(8-13)结合替拉扎明-化合物7-(TPZ),游离TPZ;不相关的肽(U4)结合TPZ。
图4.药物结合快速释放分枝NT在HT-29、PC-3和PANC-1细胞上的细胞毒性。左起组F):NT(8-13)结合5-氟脱氧尿苷-化合物3-(5FdU),游离5FdU,不相关的肽(U4)结合5FdU,F1:NT(1-13)结合5FdU。左起组G):NT(8-13)结合考布他汀酯-化合物5-(CBTST),游离CBTST,不相关的肽(U4)结合CBTST。
图5.用NT(1-13)4-荧光素染色的结肠腺癌和胰腺腺癌(K)以及相应的健康组织(H)。用ImageJ软件对共聚焦显微镜图片(A)进行像素分布的分析,并且报告为RGB系统的绿色标度的像素数。(B)对于结肠(n=17)与胰腺(n=12)人类样品,比较正常样品(浅灰)和癌症样品(灰)的平均荧光值。
图6.箱线图。每个类别的箱代表四分位距(第25-75百分位数),并且箱内的黑线是中值。底部和顶部的棒分别表示第10和90百分位数。离群值用空心圈表示;指出了两组间的p值。
图7.小鼠中的肿瘤生长降低。A,注射了NT(1-13)-5FdU的小鼠(组1)、注射了游离5FdU的小鼠(组2)和注射了盐水的小鼠(组3);B,肿瘤生长的抑制计算为未处理的小鼠(组3,视为100%)和用NT(1-13)-5FdU和游离5FdU(组1和2)处理的小鼠肿瘤体积的差异,C,肿瘤重量。
发明详述
肽的合成
合成了四分枝的NT(8-13)-PEG-K(PEG_荧光素)[NT4(8-13)-Fluo]1、NT(8-13)-PEG-苯丁酸氮芥[NT4(8-13)-CLB]2、四分枝的NT(8-13)-PEG-5-氟脱氧尿苷[NT4(8-13)-5-FdU]3、四分枝的NT(8-13)-PEG-6-巯基嘌呤[NT4(8-13)-6-MP]4、四分枝的NT(8-13)-PEG-考布他汀[NT4(8-13)-CBTST]5、四分枝的NT(8-13)-PEG-单星素[NT4(8-13)-Mon]6、四分枝的NT(8-13)-PEG-替拉扎明[NT4(8-13)-TPZ]7、四分枝的NT(8-13)-PEG-DOTA [NT4(8-13)-DOTA]8和四分枝的NT(8-13)-PEG-考布他汀醚[NT4(8-13)-O-CBTST]9,和四分枝的NT(8-13)-PEG-单星素醚[NT4(8-13)-O-Mon]10(图1),应用在Novasyn TGR树脂上的Fmoc-Lys(Dde)-OH作为第一氨基酸和β-Ala作为第二氨基酸,其中[NT4(8-13)-Fluo]1的第二氨基酸为Fmoc-PEG-OH,而不是β-Ala。接着如上构建四聚体,但是用Boc-Arg(Pbf)-OH作为神经降压肽序列的最后一个氨基酸,以使最后两个偶联步骤在侧链臂上选择性地发生。一旦完成氨基酸序列,就用肼除去Dde保护基团,并且将中间体与PEG偶联。在从PEG除去Fmoc后,将化合物与在连接体上携带游离羧基的功能单位(W)偶联。
NT4(8-13)-FLUO 1、NT4(8-13)-CLB 2、NT4(8-13)-TPZ 7、NT4(8-13)-DOTA 8和NT4(8-13)-O-CBTST 9以及NT4(8-13)-O-Mon 10与分枝的载体通过酰胺键连接,该酰胺键来自荧光团或药物上存在的游离羧基和肽上的氨基(图1)。另一方面,NT4(8-13)-5-FdU 3、NT4(8-13)-CBTST5和NT4(8-13)-Mon 6与载体肽通过双功能连接体结合,所述双功能连接体在肽侧给出酰胺键,并且在药物侧给出酯键。NT(8-13)4-6-MP与载体肽通过双功能连接体结合,所述双功能连接体在肽侧给出酰胺键并且在6-MP侧给出硫代-烯醇键。这三种结合排列产生不同的药物释放特性,即NT4(8-13)-FLUO 1、NT4(8-13)-CLB 2、NT4(8-13)-TPZ 7、NT4(8-13)-DOTA 8、NT4(8-13)-O-CBTST 9和NT4(8-13)-O-Mon 10几乎不释放FLUO、CLB、TPZ、DOTA、CBTST和Mon。而NT4(8-13)-5-FdU3、NT4(8-13)-CBTST 5、NT4(8-13)-Mon 6和NT(8-13)4-6-MP 4容易从加合物中释放FdU、CBTST、Mon和6-MP。
如上所述,合成了带有序列pyroELYENKPRRPYIL(SEQ ID No.2)的四分枝的肽。
如上所述,合成了带有序列AcDDHSVA(SEQ ID No.4)的不相关的分枝的肽并且用作对照。
肽的内化和药物释放
分枝的结合的肽1-10通过用于偶联分枝肽和功能单位的连接体表现出差异。连接体在此处针对其从载体肽释放功能单位的能力而被认为是快速释放或缓慢释放。
5FdU、单星素和考布他汀的释放
将106个PANC-1细胞与NT结合的分枝肽NT(8-13)4-5FdU 3、NT4(8-13)-CBTST 5、NT4(8-13)-O-CBTST 9、NT4(8-13)-Mon 6和NT4(8-13)-O-Mon 10(100μM)以及与不相关的分枝肽(100μM)在37℃温育不同的时间间隔。将细胞离心,洗涤,接着在冻融后于水中裂解。在加入NT(8-13)4作为内标后,将上清液和裂解的细胞通过质谱进行分析,使用Ettan MALDI-Tof质谱仪,反射模式,加速电压20kV。
NT4(8-13)-O-CBTST 9和NT4(8-13)-O-Mon 10在细胞培养基中逐渐降低,而在细胞裂解物中增加,其在1小时后出现,在温育48小时后达到最大浓度。不相关的四分枝的肽在48小时后在细胞培养基中发现保持完整,并且在裂解的细胞中未检测到。
NT(8-13)4-5FdU在细胞裂解物和细胞培养基中逐渐降低。不相关的四分枝的肽结合5FdU[(AcDDHSVA)4-5FdU]在细胞裂解物中未发现,并且在培养基中保持完整4小时。实际上,NT(8-13)4-5FdU和(AcDDHSVA)4-5FdU受到5-FdU水解的挑战,其从酯连接体释放,因此它们显示了较短的半衰期。
NT4(8-13)-CBTST 5、NT4(8-13)-Mon 6在2小时后通过水解酯键释放CBSTS和MON,而醚结合的药物9和10在上清液或细胞裂解物中发现保持完整。
从NT(8-13)4-6-MP释放6-MP
将NT(8-13)4-6-MP在37℃在磷酸盐缓冲液(pH=7.4)中在1、5和15当量的GSH存在下温育。接着将粗的混合物以不同时间间隔注射到HPLC中,以测定6-MP的释放。NT(8-13)4-6-MP在1当量GSH存在下在135分钟后释放了86%的6-MP,并且在5当量GSH存在下在30分钟后释放100%。
接着将功能化的分枝肽划分为快速释放或缓慢/不释放的:NT4(8-13)-Fluo 1、NT4(8-13)-CLB 2、NT4(8-13)-TPZ 7、NT4(8-13)-DOTA8、NT4(8-13)-O-CBTST 9和NT4(8-13)-O-Mon为缓慢释放的加合物,而NT4(8-13)-5-FdU 3、NT4(8-13)-CBTST 5、NT4(8-13)-Mon 6和NT4(8-13)-6-MP 4为快速释放的。
分枝的NT肽结合功能单位的体外活性
在人类癌细胞系中的肽结合和内化
四分枝的NT(8-13)结合生物素(NT(8-13)4-PEG-生物素)的肽结合和内化通过共聚焦显微镜在人结肠腺癌(HT29)、人胰腺癌(Panc-1)和人前列腺癌(PC3)细胞系中进行分析15。
本发明发现NT(1-13)4-荧光素和NT(8-13)4-荧光素特异性结合三种细胞系,所述细胞系表达NT受体(图2)。将细胞铺板,生长24小时,在37℃用3%BSA的TBS溶液封闭30分钟,接着与所述的肽(2μM,在TBS-0.3%BSA中)温育,与4倍摩尔过量的单体类似物进行比较。在单体肽的试验中在缓冲液中加入蛋白酶抑制剂混合物。细胞在培养基中于37℃生长1、2或4小时,接着用4%福尔马林固定,并且质膜用凝集素-Cy3(0.5μg/mL,在TBS-0.3%BSA中)染色,并且细胞核用4,6-二脒基-2-苯基吲哚(DAPI)(1μg/ml,在TBS-1%BSA中)染色。通过共聚焦激光显微镜(LeicaTCS SP5)拍照。
偶联的肽的内化在1小时内完成。肽在细胞内在18小时之内降解15。在NT(1-13)和NT(8-13)四分枝肽之间未检测到细胞结合或内化速率的差异。单体的NT(1-13)-荧光素(M)没有给出信号。
四分枝的肽结合功能单位通过NT受体结合癌细胞系的能力、快速内化至细胞的能力和在4小时后仍能被检测到的能力,显示了它们在治疗应用中的重要性。
药物结合的NT4在不同肿瘤细胞系中的细胞毒性
众所周知,临床实践中采用的经典的化学治疗对不同肿瘤具有不同活性。这是由于癌细胞对药物具有天然的抗性,该抗性由不同机制引起,包括细胞降低药物的摄入量或增加排出量、在细胞内增加药物的失活或增强对DNA烷化剂产生的DNA损伤的修复。
以前报道过的细胞毒性试验由本发明的作者在HT-29上进行15,证明了将甲氨蝶呤(MTX)或光敏剂氯e6与四分枝的NT(8-13)结合,产生了类似前药的分子。所述分子不再通过相应游离药物的机制转运穿过质膜,并且可以仅仅通过肽受体的结合而‘活化’,因此显著减低了非特异性的药物毒性。
引入新的肽受体介导的药物的细胞内化机制,可能绕过细胞抗性的天然机制。作者们接着选择了几种在经典的肿瘤化学治疗中经常采用的不同分子:甲氨蝶呤(MTX)、苯丁酸氮芥(CLB)、6-巯基嘌呤和5-氟-2′-脱氧尿苷(5-FdU),或新出现的药物考布他汀(CBTST)16、单星素(MON)17、替拉扎明(TPZ)18。这些分子作为游离药物或与四分枝肽结合,在HT-29、PANC-1和PC-3肿瘤细胞系中进行试验(图3和4)。详细来说,将HT-29、PANC-1或PC-3细胞以2.5×104个/孔铺板至96-孔微量板中。铺板24小时后,加入不同浓度的游离或NT结合药物,从0.15-30μmol/L。使细胞生长6天,不更新培养基。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物来评价生长抑制。使用GraphPaD prism 3.02软件通过非线性回归分析来计算EC50值。所得到的最佳EC50值(表示为摩尔浓度)是:HT29上NT(8-13)4-CLB为1.9e-006,HT29上NT(8-13)4-5FdU为3.3e-007,PC3上NT(8-13)-TPZ为1.4e-7,PANC-1上NT(8-13)4-CBTST为4.7e-007,HT29上NT(1-13)4-5FdU为1.1e-007。
所有药物结合NT4的细胞毒性在三种细胞系上进行了试验,并且与相应的游离药物和不相关的四分枝肽(U4)同样结合相同药物的细胞毒性进行比较。
5-FdU、单星素和CBTST是从加合物中快速释放的。随后在HT-29、PANC-1和PC-3细胞中试验了快速释放药物结合四分枝肽的细胞毒性,试验中将细胞暴露在游离或NT结合药物中1小时,洗涤并且温育6天,或者可选择的是与肽温育6天,而不进行另外的洗涤。进行另外的洗涤是为了避免游离药物在接下来的6天中扩散到细胞内。
与四分枝的NT(8-13)结合显著修饰了药物活性,其可能源自细胞和药物特征的组合,包括:i)细胞对药物的敏感性;ii)药物作用机制;iii)细胞对药物的抗性机制;iv)膜转运结合药物的效率。
正如所希望的,游离药物的活性彼此差异很大,不同细胞系间差异也很大(图3和4,左起第二组)。大体而言,与四分枝的NT结合可以产生以下结果:
-药物特异性增强,
-药物活性增强,
-游离药物的特异性和活性都增强,或者没有改善。
在某些情况中,例如MTX和CLB在PC-3中,与分枝的NT结合可以绕过天然细胞对药物的抗性(图3),使细胞从完全不敏感变换到充分的响应。分枝肽载体的一个毋庸置疑的优点是其靶点特异性,这是由任何药物当与不相关的分枝肽偶联时对这三种细胞系缺乏活性所证实的(参见图3和4的所有左起第三组).
四分枝的NT(8-13)-PEG-6-巯基嘌呤[NT4(8-13)-6-MP]4和四分枝的NT(8-13)-PEG-单星素[NT4(8-13)-Mon]6在与母体游离药物比较时没有得到改善。快速释放的四分枝NT的结果非常令人感兴趣,因为在5-FdU和CBTST的情况中,药物的活性由于结合分枝的NT4而明显得到增强(图4,F和G组)。比较缓慢释放和快速释放分子的CBTST获得的结果,表明快速释放分子可以比缓慢释放化合物更有活性。
令人感兴趣的是,不受药物影响的细胞,例如PC-3对于MTX、CLB和5-FdU,当药物与分枝NT结合时可以变得对其敏感(图4,A、B和F组)。
通过转变为肽受体介导的机制来改变膜转运机制,可以深刻地修饰药物从细胞外向细胞内的转运。而且,与分枝肽的结合可能损害了药物从细胞内向细胞外排出的机制,使结合药物陷入靶细胞内。这对于过表达NT受体并且对经典化学治疗没有响应的肿瘤治疗来说是极为重要的。
使用荧光团结合NT4来分析外科切除的人类肿瘤样品
为了验证本发明的NT分枝肽作为可能的靶向剂用于治疗结肠腺癌或胰腺腺癌,对四分枝NT肽与人肿瘤手术样品的结合相对于与健康组织的结合进行了分析和定量。收集手术切除的16个结肠肿瘤和12个胰腺肿瘤。将肿瘤样品与距离肿瘤边缘5cm远的同一患者的健康组织进行比较。对同一活检的系列切片通过苏木素/曙红(H&E)光学显微镜和荧光共聚焦显微镜进行分析。具体而言,将样品包埋在组织Tek中并且储存在液氮中。将使用2800Frigocut N(Reichert-Jung,Depew,NY)获得的10μm厚的切片在37℃干燥30分钟,在室温下用4%福尔马林固定15分钟并且在甘氨酸0.1M中于4℃温育12小时。在37℃用FBS封闭30分钟,随后在室温下与NT(1-13)4-荧光素(1μg/mL,在TBS-0.3%BSA中)温育30分钟。每步都紧接着用TBS洗涤。最后,将切片与4,6-二脒基-2-苯基吲哚(DAPI)(1μg/mL,在TBS-1%BSA中)温育5分钟。每步都紧接着用TBS洗涤。使用不相关的荧光素结合四分枝肽作为对照。类似的单体肽用作比较分析。通过共聚焦激光显微镜(Leica TCS SP5)分析肽结合,对于荧光素,吸收波长488nm并且发射波长500-540,对于DAPI,吸收波长405并且发射波长420-460。所有图片都使用ImageJ软件(NIH)处理。所得电子数据报告为RGB系统的绿色范围中的像素分布。
对于每个样品,这能够将肿瘤和健康组织的免疫荧光信号翻译为表示RGB系统范围内的绿色染色的平均值的数字(图5,组B)。
当用NT(1-13)4-荧光素处理时,来自结肠腺癌和胰腺腺癌的肿瘤组织显示了相对于同一患者的正常组织显著更高的荧光发射(图5,组A)。NT(1-13)4与任何组织样品的结合和NT(8-13)4与同一样品的结合相等。
16个结肠癌中有14个在组织学上被鉴定为腺癌并且2个被鉴定为腺瘤。后者的K/H值对应于K/H分级的最低范围。对于胰腺样品,12个样品中有11个是腺癌并且一个是淋巴瘤,即它具有非常不同的细胞来源。该样品在所有检验的手术样品中具有最低的K/H。对于结肠或胰腺,未发现肿瘤分期与受体表达(K/H值)的相关性。这意味着即使在疾病的早期阶段,K和H组织间的差异也是统计学上相关的。这对于不仅由肿瘤细胞表达而且由它们过表达的肿瘤标记物来说是非常重要的一点。因此,NT受体可以作为用分枝肽早期治疗的靶点(表1,图6)。
表1:结肠和胰腺肿瘤样品的临床特征
结肠癌
胰腺癌
根据K和H的RGB值的平均值计算K/H。
图例:NOS:未有特定说明。分级=基于细胞学观察进行肿瘤分级。TNM(肿瘤的国际分期):T=肿瘤尺寸;N=涉及的淋巴结数目,M=转移数目。
进行统计学分析来评价来自所有收集的手术样品的健康和肿瘤组织之间的肽结合的差异显著性,淋巴瘤除外,因为它相对于结肠癌和胰腺癌而言具有完全不同的细胞来源。如箱形图(图6)所示,对于胰腺癌和结肠癌,与其健康部分相比,观察到信号的显著差异。对于健康和肿瘤样品之间的比较,经双侧检验,胰腺的显著性水平为p<0.01并且结肠的显著性水平为p<0.02。该结果表明这些肽可以区别健康和癌症样品,因此它们可用作肿瘤标记物。
该结果对于结肠癌和胰腺外分泌癌通过NT-分枝肽进行可能的治疗是非常有希望的。肿瘤活检成像能够对基于NT的靶向治疗的效力进行治疗前的评估,其可以根据测定的对每个患者肿瘤与健康组织中分枝肽结合的差异来评价。而且,在健康和肿瘤组织之间清楚的区别信号表明分枝的NT可能在结肠癌和胰腺癌的特异性诊断中起到显著的作用。
分枝NT肽结合功能单位的体内活性
小鼠的肿瘤生长减少
给在右胁腹带有HT29肿瘤的裸鼠注射6个剂量的NT(1-13)-5FdU。其中选择化合物NT(1-13)-5FdU用于体内试验,是考虑到其在HT29上与类似的肽偶联MTX相比的体外细胞毒性。
具体而言,对CD-1雌性裸鼠(Charles River Laboratories,Inc.),5-6周龄(平均重量20g),在右胁腹进行皮下注射1×106个HT29细胞。当肿瘤达到直径3-4mm(肿瘤接种5天后)时,将小鼠随机分配在组中(每组4只小鼠),并且在尾静脉重复注射(在首次注射后0、30、70、140、190、240小时)500μL的在0.9%NaCl中的下述溶液:(a)1mg/mL NT(1-13)4-5FdU(3.05μmol/kg);(b)30μg/mL 5FdU(3.05μmol/kg);和(c)0.9%NaCl。
用测径器每日测量肿瘤体积,采用以下公式:体积=长度*宽度2*π/6。在肿瘤接种后21天,即在最后一次治疗后6天,处死小鼠,取出肿瘤并且称重。试验按照当地伦理委员会对癌症研究中使用动物的批准来进行。涉及动物使用的规程符合所有保护用于研究目的的动物的法规,包括UKCCCR(1998),联合王国的癌症研究协调委员会对于试验性瘤形成的动物福利的指南,第2版,Br J Cancer 77:1-10。使用Student’s t检验进行统计学分析(p<0.05)。
在最后一次治疗(240小时)后,用NT(1-13)-5FdU治疗的小鼠的平均肿瘤体积是用游离5FdU或盐水治疗的动物的约50%(图7A)。在最后一次治疗后5天(360小时)测定肿瘤重量,用药物结合肽治疗的小鼠的肿瘤重量比对照组少40%(图7C)。游离的5-FdU在10天的时间范围内以3μg/kg的剂量给药6次,仅仅得到了对肿瘤生长的10%抑制。
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Claims (15)
2.根据权利要求1的多聚体分子,其中肽包含以下氨基酸序列:QLYENKPRRPYIL(SEQ ID No.1)、pyroELYENKPRRPYIL(SEQ ID No.2)或RRPYIL(SEQ ID No.1的8-13位氨基酸)。
3.根据权利要求1或2的多聚体分子,其用于医药用途。
4.根据权利要求1或2的多聚体分子,其作为抗肿瘤剂。
5.根据权利要求5的多聚体分子,其中肿瘤是结肠癌或胰腺癌或前列腺癌。
6.根据权利要求1的多聚体分子在制备药物中的用途。
7.根据权利要求6的用途,其中药物具有抗肿瘤活性。
8.根据权利要求1的多聚体分子在肿瘤诊断中的用途。
9.根据权利要求7或8的用途,其中肿瘤是结肠癌或胰腺癌或前列腺癌。
10.药物组合物,该药物组合物包含根据权利要求1-5中任一项的多聚体分子或其药学上可接受的和有效的盐以及稀释剂和/或溶剂和/或载体和/或赋形剂。
11.治疗方法,该方法包括施用或暴露于根据权利要求1的多聚体分子。
12.根据权利要求11的方法,其中治疗是抗肿瘤。
13.诊断方法,该方法包括施用或暴露于根据权利要求1的多聚体分子。
14.根据权利要求13的方法,其中诊断是肿瘤诊断。
15.根据权利要求12或14的方法,其中肿瘤是结肠癌或胰腺癌或前列腺癌。
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