CN1296381C - 血管内皮细胞生长因子(vegf)肽及其抑制血管生成的用途 - Google Patents
血管内皮细胞生长因子(vegf)肽及其抑制血管生成的用途 Download PDFInfo
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Abstract
具有部分或全部氨基酸序列QKRKRKKSRYKSWSVP(其为VEGF的部分)的肽具有抑制血管生成的能力。
Description
发明领域
本发明涉及具有潜在治疗益处的VEGF(血管内皮细胞生长因子)肽片段。
发明背景
VEGF是分泌型多肽,为胚胎发生时形成血管所必须,且在多种疾病状态的血管生成中起重要作用。在多种细胞类型中组织缺氧和一些细胞因子上调VEGF的表达,并由此引起多种体内和体外生物活性,包括:内皮细胞的分化、增殖、迁移和存活,血管通透性增加,单核细胞迁移及内皮生产血管舒张因子NO和前列环素的增加。VEGF引起的NO和前列环素生产又出现于血管发生和VEGF的一些血管保护性作用中,包括增加通透性,和抑制血管内膜(intimal)血管平滑肌细胞增生和血栓。
人VEGF存在由不同的mRNA剪接产生的121,145,165,189和206个氨基酸的多种同工型,其中VEGF121,VEGF145和VEGF165已知为分泌型且具有生物活性。已经鉴定了VEGF的两种不同的蛋白质酪氨酸激酶受体,Flt-1(VEGFR1)和KDR/Flk-1(VEGFR2)。认为KDR/Flk-1是主要介导VEGF在内皮细胞中的促有丝分裂作用和在体内的血管生成作用;Flt-1在内皮细胞中的作用未知。非-酪氨酸激酶跨膜蛋白,已经鉴定了neuropilin-1(NP-1)作为VEGF的另一受体,其特异结合VEGF165,并增强VEGF165同VEGFR2的结合。NP-1在调节VEGF的生物功能中的作用在很大程度上未知。
发明简述
意外地发现VEGF的片段具有抗血管生成作用。其可能具有VEGF拮抗物活性。
根据本发明,具有氨基酸序列QKRKRKKSRYKSWSVP(SEQ IDNo.1)的肽,其中任何Y残基至少可以由其等配物或片段或衍生物或同源物代替;这样的肽抑制血管生成。
该序列对应VEGF中的氨基酸125-140。本发明也包括该序列的变体,其中在无意外结构变化时保持新活性,即抑制血管生成。因此,所给序列可以包括使该肽相对稳定的同配或同源代替或衍化。
发明详述
本发明的肽可以通过已知方法合成。以下给出实施例。其可以已知方式配制并应用。该肽的抗-血管生成活性意味着其可用于治疗癌症。
该氨基酸序列的关键特点可能是碱性残基如K和R的存在和排列。本发明的肽优选包括1,2或更多KK,KR或RK序列,例如由非碱性残基间隔。
本发明的典型肽具有至少4个氨基酸。可以多达6或8个氨基酸,但是优选尽可能短。
本发明的肽可以是环化的,例如尾对尾,或通过取代/引入2个Cys残基。环化步骤已知;见,例如,Tam等人,JACS 113:6657-62(1991)。也可以用其他环化,例如,Mitsunobu或烯烃复分解环闭合。环肽可能表现增强的特性。
如上所示,本发明的肽包括对给定序列的修饰。这些修饰为本领域技术人员熟知。同配代替包括Abu代替Cys(当肽中需要保持偶数个Cys残基进行环化时,这可能有益),Phe代替Tyr及不同的烷基/芳基取代。
在一个氨基酸残基中,从C原子到N原子的取代基移位来产生对蛋白水解有更强耐受的肽,和其他修饰,已知并属于本发明的范围。
本发明肽的抗-血管生成特性可由下述步骤确定。活性水平优选至少等于作为初始测试对象的两种线性12肽,即,QKRKRKKSRYKS(V125-136;SEQ ID No.2)和RKKSRYKSWSVP(V129-140;SEQ ID No.3)。本发明的进一步的实例是SEQ ID Nos.4-21;也见图2。
本发明肽的活性意味其可用于治疗其中血管生成是重要病理因素的疾病。这些包括眼的血管生成疾病如糖尿病的视网膜病,或者其中可能有血管生成的疾病如ARDS(衰老相关的黄斑变性)。血管生成起重要作用的其他疾病有肿瘤生长,或子宫内膜易位,和例如牛皮癣的皮肤病。该肽可用于治疗具体的癌症,包括卡波济氏肉瘤(其发生于AIDS患者),皮肤和眼的恶性黑色素瘤,和卵巢、乳腺、肺、胰腺、前列腺、直肠的实体恶性肿瘤及表皮样癌(epidermoid cancer)。其也可用于治疗具有血管成分作为炎症状况的风湿性关节炎。
用于治疗用途,可由本领域已知步骤,使用本领域已知成分配制并施用本发明的肽。本领域技术人员可根据具体要求,如对象的状态,化合物的作用强度,给药途径等因素选择适合的肽剂量。
以下实施例解说本发明。
缩写说明
MBHA,甲基二甲苯基胺;
Fmoc,9-芴基甲氧基-羰基;
Ala,苯丙氨酸;Arg,精氨酸;Asn,天冬酰氨;Asp,天冬氨酸;Abu,氨基丁酸;Cys,半胱氨酸;Gln,谷氨酰氨;Glu,谷氨酸;Gly,甘氨酸;His,组氨酸;Ile,异亮氨酸;Leu,亮氨酸;Lys,赖氨酸;Met,甲硫氨酸;Phe,苯丙氨酸;Pro,脯氨酸;Ser,丝氨酸;Thr,苏氨酸;Trp,色氨酸;Tyr,酪氨酸;Val,缬氨酸;Pbf,2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基;Pmc,2,2,5,7,8-五甲基苯并二氢吡喃-6-磺酰基;Trt,三苯甲基;tBu,叔丁基;Boc,丁氧基羰基;Pybop,苯并三唑-1-基氧三吡咯烷膦六氟磷酸(benzotriazol-1-yloxytris pryrolidino phosphonium hexafluoro phosphate);NMM,N-甲基吗啉;DCM,二氯甲烷;DME,二甲基甲酰胺;TFA,三氟乙酸;HPLC,高效液相色谱;LC-MS,液相色谱-质谱;AAA,氨基酸分析;DMSO,二甲基亚砜;VEGF,血管内皮生长因子;TBME,叔丁基甲基酯。
肽合成
用固相方法在自动AMS 422多肽合成仪上合成所有的肽。应用Rink Amide MBHA树脂(0.59和0.68mmol/g负载)和N-Fmoc策略正交保护要环化的衍生物的Cys侧链。例如,以25微摩尔规模合成QKRKRKKSRYKS(V125-136)和RKKSRYKSWSVP(V129-140)并进行一次30分钟的碱偶联。树脂和氨基酸衍生物,Fmoc-Ala-OH.H2O,Fmoc-Arg(Pbf/Pmc)-OH,Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH,Fmoc-Cys(Trt)-OH,Fmoc-Gln(Trt)-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Gly-OH,Fmoc-His(Trt)-OH,Fmoc-Ile-OH,Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH,Fmoc-Met-OH,Fmoc-Phe-OH,Fmoc-Pro.H2O,Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH,Fmoc-Trp(tBu)-OH,Fmoc-Tyr(tBu)-OH和Fmoc-Val-OH购自Calbiochem-NovabiochemUK Lid.(伯明汉,英国)或Alexis(伯明汉,英国)。
用Pybop(Calbiochem-Novabiochem)和NMM(RathburnChemicals,Walkerburn,苏格兰)作偶联剂通过活性酯方法将各氨基酸顺序偶联成从C到N生长的肽链。用DMF中20%的哌啶去N-Fmoc保护基,然后依次用DMF和DCM清洗。合成各肽以后,以Rink AmideMBHA树脂取代为基础,用4倍过量的乙酸(0.7摩尔,RathburnChemicals)进行自动乙酰化。将偶联剂,Pybop,NMM和所有的氨基酸溶解在DMF中(0.7M,以Rink Amide MBHA树脂取代为基础,4倍过量),除氨基酸Fmoc-His(Trt)-OH和Fmoc-Phe-OH以外。将这些被保护的氨基酸衍生物溶解在N-甲基吡咯烷中。所用所有溶剂都是HPLC级质量。
用90%TFA在室温下将肽从树脂上切下并同时去保护,存在有5%苯硫基甲烷,2.5%水和2.5%乙二硫醇吸收产生的反应性阳离子。过滤切割混合物并在冰冷的TBME中沉淀。将剩下的树脂用切割试剂洗涤一次,过滤并和先前的部分合并。离心后收集沉淀,用冰冷的TBME洗涤3次并在室温干燥过夜。将粗肽溶解在15%的乙酸水溶液中并以2天冻干(-40℃,6mbar)。
纯化及性质鉴定
由分析型LC-MS用惠普HPLC装置,模式1100,在Micromass的Quatro LC质谱仪上分析粗肽,使用分析型反相柱(柱Alltech HypersilPEP反相柱,10nm,C8,5μ(250×4.6mm)0-50%乙腈,20分钟。在酰胺键吸收的215nm波长以流速1ml/min监控分离。用制备型反相HPLC(Gilson)纯化粗肽,在215nm监控并以流速20ml/min洗脱。使用与LC-MS分析相同的流动相。使用Alltech Hypersil PEP反相柱,10nm,C8,8μ(250×22mm)纯化粗肽。用0-50%乙腈在20分钟内洗脱。使用高效液相色谱(LC-MS)的类似物纯度大于95%并具有所期望的氨基酸分析。
用多种不同的梯度洗脱肽,并以215nm监控。有机相,乙腈和水相都含有0.1%TFA和3%的1-丙醇。梯度和流速如下所列。百分数显示有机相的比例。0-50%,20分钟,流速1ml/min。
用肽抑制VEGF刺激的PG12生产
在12孔板预先用肽V125-136和V129-140以所示浓度孵育汇合的HUVEC30分钟,然后或者不添加任何东西继续孵育30分钟,或者暴露于VEGF(25ng/ml)1小时。一些细胞在无肽情况下孵育,无(C,对照)或有25ng/ml的VEGF。收集上清并用酶免疫测试测量6-酮-前列腺素Fla。结果示于图一,其中条棒代表平均百分数:由重复测定得到的未刺激对照水平的6-酮-前列腺素Fla(100%)的±SEM。从两个独立实验得到相似结果。
125I-VEGF165结合实验
用PBS洗涤2遍并在4℃用稀释于结合介质(DMEM,25mM HEPES,pH7.3,含有0.1%BSA)的且存在0.03nM或0.1nM 125I-VEGF165(1200-1800Ci/mmol,Amersham)的多种浓度的肽孵育24孔板中汇合的HUVEC。在4℃孵育2小时以后,用冷PBS洗涤4次,并用0.25M的NaOH,0.5%SDS裂解,并且测量裂解液结合的放射活性。在存在100倍过量的未标记VEGF时确定非特异性的结合。从总结合减去非特异结合得到特异性结合。所有结合实验进行3次。
测试12聚体V125-136的一系列截短的衍生物。结果显示10聚体RKRKKSRYKS和9聚体QKRKRKKSR同12聚体有相似的活性(图2)这说明N-或C-末端截短可能不影响活性,且生物活性所需的最小序列是7聚体RKRKKSR。
体外血管生成试验
体外血管生成试验基于同人成纤维细胞共培养时人内皮细胞衍生的小管形成(TCS Biologicals),用于研究所选多肽对血管生成的作用。在试验开始时和分别在第4,7,和9天加入培养基时加入肽,连同VEGF和其他因子。在第11天,在室温用70%的乙醇同培养物混合30分钟然后对内皮细胞附着分子CD31进行免疫染色。用初级抗体(鼠抗人CD31)在37℃孵育1小时,用封闭缓冲液(PBS含1%BSA)洗涤3次,然后用2级抗体(缀合有碱性磷酸酶的羊抗鼠IgG)在37℃孵育1小时。在蒸馏水中洗涤3次,最后用底物溶液在37℃孵育细胞10分钟。用配有x4物镜的倒置Zeiss显微镜(Axiovert CFL25)观察并照像。孵育每个实验对照孔,其中仅有VEGF,VEGF加20μM苏拉明,一种已知的VEGF诱导的血管生成的抑制剂,以及无其他添加。结果出示于图3,其中A,由未处理的对照在11天释放的VEGF,通过特异性免疫试验测量;B,未处理的对照;C,VEGF;D,肽121-132;E,存在肽121-132时的VEGF;F,肽125-136;和G,存在肽125-136时的VEGF。
本实验进一步的结果示于图4。这些结果是关于7聚体RKRKKSR(SEQ ID No.21),并显示其抑制血管生成的潜力同12聚体相似。
序列表
<110>Ark Therapeutics Ltd.
<120>血管内皮细胞生长因子(VEGF)肽及其抑制血管生成的用途
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Claims (5)
1.一种抑制血管生成的肽,其特征在于该肽由氨基酸序列QKRKRKKSRYKS组成,或者由该氨基酸序列的N端缺失了1-2个氨基酸和/或C端缺失了1-3个氨基酸的序列组成。
2.根据权利要求1的肽,其为环状形式。
3.根据任一前述权利要求的肽的用途,用于生产抑制血管生成的药物。
4.权利要求1-2任一项的肽在生产用于治疗眼的血管生成疾病的药物中的用途。
5.根据权利要求4的用途,其中所述眼的血管生成疾病为糖尿病性视网膜病。
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GB0026134D0 (en) * | 2000-10-25 | 2000-12-13 | Eurogene Ltd | Peptides and their use |
ATE476512T1 (de) * | 2002-01-09 | 2010-08-15 | Suntory Holdings Ltd | Zuckertransferase gnt-v mit angiogener wirkung |
EP1812030A4 (en) * | 2004-10-14 | 2009-01-14 | Sopherion Therapeutics Inc | ANTI-ANGIOGENIC PEPTIDES AND METHODS OF USE THEREOF |
CN1314705C (zh) * | 2005-06-03 | 2007-05-09 | 中国药科大学 | 高效抑制血管生成多肽及其制备方法和应用 |
CN101443031A (zh) * | 2006-05-15 | 2009-05-27 | 瓦利奥有限公司 | 治疗用肽的新应用 |
CN102066404B (zh) * | 2008-04-23 | 2013-11-13 | 诺娃细胞科技公司 | 血管生成肽 |
CN107540740B (zh) * | 2017-08-25 | 2021-06-15 | 海南医学院第一附属医院 | 一种化合物、制备方法及其mri成像应用 |
CN114213546B (zh) * | 2021-12-03 | 2023-05-05 | 华中科技大学同济医学院附属协和医院 | 靶向vegfr的裂解肽偶联体及其应用 |
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WO2001027136A2 (fr) * | 1999-10-12 | 2001-04-19 | Centre National De La Recherche Scientifique (Cnrs) | Peptides ayant une activite de stimulation de la reponse immunitaire et de regeneration tissulaire |
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GB0026134D0 (en) * | 2000-10-25 | 2000-12-13 | Eurogene Ltd | Peptides and their use |
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MXPA03003516A (es) | 2003-08-07 |
HUP0301523A3 (en) | 2006-11-28 |
EP1328539A1 (en) | 2003-07-23 |
CA2426736A1 (en) | 2002-05-02 |
AU1071302A (en) | 2002-05-06 |
PL362084A1 (en) | 2004-10-18 |
CN1612893A (zh) | 2005-05-04 |
US7223835B2 (en) | 2007-05-29 |
KR100866431B1 (ko) | 2008-10-31 |
HUP0301523A2 (hu) | 2003-10-28 |
WO2002034767A1 (en) | 2002-05-02 |
NO20031845D0 (no) | 2003-04-24 |
US20040054143A1 (en) | 2004-03-18 |
KR20030081320A (ko) | 2003-10-17 |
JP2004512342A (ja) | 2004-04-22 |
GB0026134D0 (en) | 2000-12-13 |
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AU2002210713B2 (en) | 2005-03-03 |
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