CN113150065B - 一种合成肽及其应用 - Google Patents
一种合成肽及其应用 Download PDFInfo
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- CN113150065B CN113150065B CN202110266149.4A CN202110266149A CN113150065B CN 113150065 B CN113150065 B CN 113150065B CN 202110266149 A CN202110266149 A CN 202110266149A CN 113150065 B CN113150065 B CN 113150065B
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Abstract
本发明公开了一种合成肽及其应用,属于药物技术领域。其化学式为:(Gly)x‑(beta‑Ala‑L‑His)y‑(Gly)z、(Gly)x‑(beta‑Ala‑L‑1‑Methyl‑His)y‑(Gly)z或者(Gly)x‑(beta‑Ala‑L‑3‑Methyl‑His)y‑(Gly)z;y等于或者大于1,x和z为0‑3且不同时为零。本发明的合成肽创造性地把甘氨酸、肌肽、鹅肌肽或蛇肌肽等组合起来,开发的镶嵌式合成肽出乎意料地通过静脉注射透过血脑屏障进入大脑,有效地在治疗神经系统疾病中发挥了作用。本发明只需要3mg/kg的合成多肽静脉注射剂量,即可达到显著的治疗效果。
Description
技术领域
本发明属于药物技术领域,特别涉及一种合成肽及其应用。
背景技术
甘氨酸是化学结构最简单的氨基酸。尽管结构简单,甘氨酸在中枢神经系统中却是一种重要的抑制性神经递质,在控制神经元兴奋性方面发挥重要作用。动物实验发现甘氨酸具有明显的神经保护作用,在一些临床试验中也同样发现其可以改善认知功能障碍和痴呆。
肌肽(L-Carnosine),是一种由β-丙氨酸和L-组氨酸两种氨基酸组成的二肽。肌肽具有很强的抗氧化能力,对人体有益。肌肽已被证实可清除在氧化应激过程中使细胞膜的脂肪酸过度氧化而形成的活性氧自由基(ROS)以及α-β不饱和醛。肌肽具有抗炎、抗糖化、抗氧化和螯合作用,可作为一种非处方食品补充剂,在预防和辅助治疗心血管疾病和神经退行性等慢性疾病方面具有良好的前景。 在动物实验里,肌肽的神经保护机制可以防止永久性脑部缺血。肌肽还是一种重要的细胞内抗氧化剂。肌肽不仅无毒,而且具备强的抗氧化性,因此以它作为一种新型的食品添加剂与药用试剂已经引起广泛关注。肌肽参与细胞内的过氧化反应,除了具有抑制细胞膜的过氧化过程,还能抑制细胞内的相关的过氧化反应。1900年,俄国学者 Gulewitsch最早发现了肌肽, 之后各国学者又在不同的肌肉组织中还提取分离了其它组氨酸二肽衍生物,如鹅肌肽(Anserine),其是一种由β-丙氨酸和1-甲基-L-组氨酸两种氨基酸组成的二肽, 以及鲸肌肽或蛇肌肽(Balenine, 又称Ophidine,),蛇肌肽则是由β-丙氨酸和3-甲基-L-组氨酸组成的二肽。不同动物的这些肌肽和肌肽衍生物的含量也不同,不同物种之间这几种组氨酸二肽的含量及比例也各不相同,具有一定的特异性。除了在肌肉组织中存在着组氨酸二肽以外,这些二肽还存在于别的组织中, 如脑组织。这些肌肽衍生物具有水溶性和较强的而且具有显著的抗氧化、抗衰老、降尿酸等功能,在食品工业中已用作天然的抗氧化剂和降尿酸食疗,也同样具有一定的神经保护功能。
尽管甘氨酸和肌肽以及肌肽衍生物都有一定的脑保护作用,但是它们往往需要较大的剂量,比如甘氨酸外周给要量高达(800mg/kg),这可导致副反应严重,也不方便使用,导致它们的实际临床应用有限。单独使用肌肽、鹅肌肽和蛇肌肽等效果也不好,与最新的NA-1药物的治疗效果差距非常大。
发明内容
本发明提供了一种合成肽,它是由甘氨酸、beta-丙氨酸、组氨酸、1-甲基组氨酸和3-甲基组氨酸中的几种组成,尤其是由甘氨酸,肌肽,鹅肌肽(Anserine),甚至蛇肌肽等形成了一系列具有兼具肌肽,鹅肌肽或蛇肌肽与甘氨酸这一结构特征的多肽化合物。这些肽类出乎意外地容易透过血脑屏障,通过静脉途径给药展示了很好的生物活性,从而在治疗神经系统疾病,尤其是脑损伤,脑卒中方面显示了广阔的前景。所述技术方案如下:
一方面,本发明实施例提供了一种合成肽,其化学式为:
(Gly)x-(beta-Ala-His)y-(Gly)z或者
(Gly)x-(beta-Ala-1-Methyl-His)y-(Gly)z或者
(Gly)x-(beta-Ala-3-Methyl-His)y-(Gly)z;
式中,x 为0-3的整数(如为0、1、2和3),y 为1-3的整数(如为1、2和3),z为0-3的整数(如为0、1、2和3),但x和z不同时为零。
其中,本发明中氨基酸缩写简称如下:
甘氨酸:Gly;beta-丙氨酸:β-Ala或beta-Ala;L-组氨酸:His;1-甲基组氨酸: 1-Methyl-His或(1-Methyl-His);3-甲基组氨酸:3-Methyl-His或(3-Methyl-His)。
其中,对于式(Gly)x-(beta-Ala-His)y-(Gly)z,其化学式可以选自以下任意一种:
(a)beta-Ala-His-Gly;
(b)Gly-beta-Ala-His;
(c)beta-Ala-His-Gly-Gly;
(d)Gly-Gly-beta-Ala-His;
(e)Gly-beta-Ala-His-Gly;
(f)Gly-Gly-beta-Ala-His-Gly-Gly;
(g)beta-Ala-His-beta-Ala-His-Gly;
(h)Gly-beta-Ala-His-beta-Ala-His;
(i)beta-Ala-His-beta-Ala-His-Gly-Gly;
(j)Gly-Gly-beta-Ala-His-beta-Ala-His;
(k)Gly-beta-Ala-His-beta-Ala-His-Gly;
(l)Gly-Gly -beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly;
(m) beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly-Gly;
(n) Gly-Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His;
(o) Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly。
其中,对于式(Gly)x-(beta-Ala-1-Methyl-His)y-(Gly)z,其化学式可以选自以下任意一种:
(a)beta-Ala-(1-Methyl-His)-Gly;
(b)Gly-beta-Ala-(1-Methyl-His);
(c)beta-Ala-(1-Methyl-His)-Gly-Gly;
(d)Gly-Gly-beta-Ala-(1-Methyl-His);
(e)Gly-beta-Ala-(1-Methyl-His)-Gly;
(f)Gly-Gly-beta-Ala-(1-Methyl-His)-Gly-Gly;
(g)beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly;
(h)Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His);
(i)beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly-Gly;
(j)Gly-Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His);
(k)Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly;
(l)Gly-Gly -beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly;
(m) beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly-Gly-Gly;
(n) Gly-Gly-Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His);
(o) Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly-Gly。
其中,对于(Gly)x-(beta-Ala-3-Methyl-His)y-(Gly)z,其化学式可以选自以下任意一种:
(a)beta-Ala-(3-Methyl-His)-Gly;
(b)Gly-beta-Ala-(3-Methyl-His);
(c)beta-Ala-(3-Methyl-His)-Gly-Gly;
(d)Gly-Gly-beta-Ala-(3-Methyl-His);
(e)Gly-beta-Ala-(3-Methyl-His)-Gly;
(f)Gly-Gly-beta-Ala-(3-Methyl-His)-Gly-Gly;
(g)beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly;
(h)Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His);
(i)beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly-Gly;
(j)Gly-Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His);
(k)Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly;
(l)Gly-Gly -beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly;
(m) beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly-Gly-Gly;
(n) Gly-Gly-Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His);
(o) Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly-Gly。
优选地,本合成肽的化学式可以选自以下任意一种:
(a)beta-Ala-His-Gly;
(b)Gly-beta-Ala-His;
(c)beta-Ala-His-Gly-Gly;
(d)Gly-Gly-beta-Ala-His;
(e)Gly-beta-Ala-His-Gly;
(f)Gly-Gly-beta-Ala-His-Gly-Gly。
更优选地,本合成肽的化学式可以选自以下任意一种:
(a)beta-Ala-L-His-Gly;
(b)Gly-beta-Ala-L-His。
另一方面,本发明还提供了前述合成肽或其盐在制备药物(尤其是治疗神经系统疾病药物)中的应用。
具体地,前述合成肽或其盐在制备治疗缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病或其它神经退行性疾病药物等中的应用。优选地,前述合成肽在制备治疗缺血性脑卒中药物中的应用。
其中,前述药物为注射剂。具体地,注射剂为粉针剂或者注射液。进一步地,前述药物采用静脉的方式给药。前述药物也可以作为活性成分制备其它剂型,以便于相应的医疗应用。作为神经保护用药,开发的制剂可以包括口服给药和舌下给药制剂,作为脑卒中现场急救用药,开发的制剂可以包括喷雾给药,肛门给药等制剂,便于没有行动能力的患者得到救治。又一方面,本发明还提供了通过固相合成制备本发明各类合成肽的方法,但其中的一些肽用液相合成更加方便。多肽药物成盐是改善药物分子理化性质、提高其成药性的常见手段之一,前述药物可以是任何形式的盐。
为了确定本专利提供的合成肽在神经系统疾病方面的应用,本发明用 SD 大鼠作为实验对象,采用脑中动脉阻断法(MCAO)制备脑缺血大鼠模型,缺血 1-2小时后静脉注射药物,22-24 小时对每只动物做行为学观察和评分。行为学观察之后,将实验大鼠进行安乐死并取出其大脑,脑组织切片,TTC染色后进行量化分析,计算脑梗死体积%。
本发明的合成肽创造性地把甘氨酸、肌肽、鹅肌肽或蛇肌肽等组合起来,开发的镶嵌式合成肽出乎意料地通过静脉注射透过血脑屏障,进入大脑,有效地在治疗神经系统疾病中发挥了作用。该类合成肽优选地应用于治疗缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病及其他神经退行性疾病。本发明只需要 3mg/kg 的合成多肽静脉注射剂量,出乎意料地可达到显著的治疗效果,与已有临床活性的标准多肽NA1达到了同样的效果。
该合成肽的为甘氨酸以及肌肽,鹅肌肽或蛇肌肽组成,其全部为天然的氨基酸或肽,安全可靠。
同时本发明的合成肽便于合成,合成过程中还可以使用肌肽,鹅肌肽或蛇肌肽的衍生物作为合成片段,代替对应的两个氨基酸来进一步简化目标肽的合成。另外其中涉及的一些短肽,如beta-Ala-His-Gly,Gly-beta-Ala-His,beta-Ala-His-Gly-Gly,Gly-Gly-beta-Ala-His,Gly-beta-Ala-His-Gly,beta-Ala-(1-Methyl-His)-Gly,Gly-beta-Ala-(1-Methyl-His),beta-Ala-(1-Methyl-His)-Gly-Gly,Gly-Gly-beta-Ala-(1-Methyl-His),Gly-beta-Ala-(1-Methyl-His)-Gly,beta-Ala-(3-Methyl-His)-Gly,Gly-beta-Ala-(3-Methyl-His),beta-Ala-(3-Methyl-His)-Gly-Gly,Gly-Gly-beta-Ala-(3-Methyl-His)和Gly-beta-Ala-(3-Methyl-His)-Gly更是可以通过液相合成而得,成本更低,便于以后规模化生产与产品质量监管。
附图说明
图1是模型组的脑组织的切片图;
图2是S1组的脑组织的切片图;
图3是S8组的脑组织的切片图;
图4是S9组的脑组织的切片图;
图5是假手术组的脑组织的切片图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实例1:beta-Ala-His-Gly的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-Gly-OH偶联得到Fmoc-Gly-CTC树脂。
2.用20%哌啶脱除Fmoc-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-His(Trt)-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将beta-Ala偶联至Fmoc-His-Gly-CTC树脂上。
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例2:Gly-beta-Ala-His的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由固相载体2-CTC树脂和Fmoc-His(Trt)-OH偶联得到Fmoc-His(Trt) -CTC树脂。
2.用20%哌啶脱除Fmoc-His(Trt) -CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-beta-Ala -OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将Gly偶联至Fmoc-beta-Ala -His-CTC树脂上
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例3:Gly-beta-Ala-His-beta-Ala-His-Gly的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-Gly-OH偶联得到Fmoc-Gly-CTC树脂。
2.用20%哌啶脱除Fmoc-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-His(Trt)-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将beta-Ala, His,beta-Ala和Gly依次偶联至Fmoc-His-Gly-CTC树脂上。
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例4:beta-Ala-His-Gly-Gly的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-Gly-OH偶联得到Fmoc-Gly-CTC树脂。
2.用20%哌啶脱除Fmoc-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3. 称取3倍过量的Fmoc-Gly-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4. 用20%哌啶脱除Fmoc-Gly-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
5.称取3倍过量的Fmoc-His(Trt)-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
6. 用20%哌啶脱除Fmoc-His(Trt)-Gly-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
7.称取3倍过量的Fmoc-beta-Ala-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
8. 用20%哌啶脱除Fmoc保护基,脱除干净后用DMF洗涤干净。
9.裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
10.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
11.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例5:Gly-Gly-beta-Ala-His的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-His(Trt)-OH偶联得到Fmoc-His(Trt)-CTC树脂。
2.用20%哌啶脱除Fmoc-His(Trt)-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-beta-Ala-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将Gly和Gly偶联至Fmoc-His-Gly-CTC树脂上。
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例6:Gly-beta-Ala-His-Gly的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-Gly-OH偶联得到Fmoc-Gly-CTC树脂。
2.用20%哌啶脱除Fmoc-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-His(Trt)-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将beta-Ala和Gly依次偶联至Fmoc-His-Gly-CTC树脂上。
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例7:Gly-Gly-beta-Ala-His-Gly-Gly的合成:
1.在活化剂系统(HoBT,DIC)的存在下,由树脂固相载体2-CTC树脂和Fmoc-Gly-OH偶联得到Fmoc-Gly-CTC树脂。
2.用20%哌啶脱除Fmoc-Gly-CTC上的Fmoc保护基,脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-Gly-OH,与三倍过量的活化剂,加入少量DMF充分溶解,溶解完之后加入至洗涤干净的树脂中,反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤,将His, beta-Ala,Gly和Gly依次偶联至CTC树脂上。
5.合成完毕之后进行裂解,裂解试剂配比为TFA:EDT:Tis:TA:苯甲醚:H2O=80:5:1:5:5:4(体积比),按1g肽树脂需10ml裂解试剂配比,室温下裂解约2h(120 r/min),裂解之后用冰甲基叔丁基醚沉淀,下层沉淀即为粗品。
6.取上步的粗肽溶解,在制备型HPLC上用0.1%TFA/乙腈纯化。
7.纯化完之后进行冻干,冻干结束后取出粉末进行分装质检。
实例8:同样的办法也可以合成如下多肽:
beta-Ala-His-Gly-Gly;
Gly-Gly-beta-Ala-His;
Gly-beta-Ala-His-Gly;
beta-Ala-His-beta-Ala-His-Gly;
Gly-beta-Ala-His-beta-Ala-His;
beta-Ala-His-beta-Ala-His-Gly-Gly;
Gly-Gly-beta-Ala-His-beta-Ala-His;
beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly;
Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His;
beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly;
Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His;
beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly;
Gly-Gly-beta-Ala-His-beta-Ala-His beta-Ala-His;
beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly-Gly;
Gly-Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His;
Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly;
beta-Ala-His-beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly-Gly-Gly;
Gly-Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His-beta-Ala-His;
Gly-Gly-beta-Ala-His-beta-Ala-His-beta-Ala-His-beta-Ala-His-Gly;
beta-Ala-(1-Methyl-His)-Gly;
Gly-beta-Ala-(1-Methyl-His);
beta-Ala-(1-Methyl-His)-Gly-Gly;
Gly-Gly-beta-Ala-(1-Methyl-His);
Gly-beta-Ala-(1-Methyl-His)-Gly;
beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly;
Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His);
beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly-Gly;
Gly-Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His);
Gly-beta-Ala-(1-Methyl-His)-beta-Ala-(1-Methyl-His)-Gly;
beta-Ala-(3-Methyl-His)-Gly;
Gly-beta-Ala-(3-Methyl-His);
beta-Ala-(3-Methyl-His)-Gly-Gly;
Gly-Gly-beta-Ala-(3-Methyl-His);
Gly-beta-Ala-(3-Methyl-His)-Gly;
beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly;
Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His);
beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly-Gly;
Gly-Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His);
Gly-beta-Ala-(3-Methyl-His)-beta-Ala-(3-Methyl-His)-Gly。
实例9:动物实验方法
小动物监护仪:深圳市荣显达科技有限公司,VT200。
SD大鼠:中国食品药品鉴定研究院,180-200 g。
线栓:迈越生物(M8507)。
TTC染色液:源叶生物(R24053)。
动物模型操作步骤:
对实验用SD大鼠进行称重,使用水合氯醛麻醉。麻醉后对大鼠四肢进行固定,仰卧,连接小动物监护仪,对大鼠体温、血压和心率等重要生理指标进行监护。对大鼠颈部进行脱毛,用75%酒精棉球消毒后对实验大鼠颈部进行长度2cm左右的正中切口,通过钝性分开大鼠颌下腺,分离过程中尽量避免对腺体的破坏,然后钝性分离出实验大鼠左侧的颈总动脉(CCA),沿着CCA向上小心分离出颈内动脉(ICA)和颈外动脉(ECA)。分离过程中避免损伤迷走神经。使用两个动脉夹分别将CCA和ICA夹闭,剪断ECA远端并将硅胶线栓从ECA “端口”插入,插到ICA动脉夹处短暂松一下动脉夹并迅速将线栓头端插过动脉夹后再次夹闭ICA,然后轻轻松开ICA上的动脉夹并不断使线栓插入,直到线栓黑标插过ECA和ICA的分叉,线栓头端堵住大脑中动脉后用缝合线将ECA“端口”和线栓紧紧系紧,以防止大鼠醒后线栓“退出”或出血。松开并拿掉CCA和ICA上的动脉夹,使组织恢复原位,并滴加适量的青霉素以防伤口感染。使用医用带线缝合针进行缝合,封好之后再次用碘伏消毒。观察小动物监护仪判断实验大鼠指标是否正常,然后将大鼠放在小动物电热毯上使其保持体温直至苏醒放置动物笼中。
给药流程:
将待测药物配成3mg/mL的溶液。在结扎后1-2 h尾静脉给药。
给药时将大鼠用固定器固定,利用1 mL注射器按照3 mg/kg剂量吸取待测药物,然后对大鼠进行尾静脉推注,注射时缓缓推注以降低实验大鼠的心肺负荷。
动物分为不同的试验小组,每组六只。
模型组:结扎后尾静脉给等量的生理盐水;
药物组S1(NA1), S8(beta-Ala- His-Gly)和S9(Gly-beta-Ala- His) :结扎后尾静脉给药;
假手术组:分离颈内动脉(ICA)和颈外动脉(ECA)后不做结扎处理,尾静脉给等量的生理盐水。
行为学观察:
结扎后22-24 h对每只动物做行为学观察和评分。
按照如下评分表,让未参与该实验的技术员盲评。
0分:正常直线行走;
1分:前肢无力;
2分:后肢无力;
3分:轻度转圈;
4分: 严重转圈;
5分:偏瘫。
TTC染色和量化分析:
行为学观察之后,将实验大鼠进行安乐死并取出其大脑。将脑组织横切成6片2 mm厚的切片,再移至TTC染色液中,37℃孵箱中避光孵育10 min,拍照。然后将TTC染色后的脑组织和剩余的少量未进行TTC染色的脑组织-20℃保存。
使用Image J软件对TTC染色后的照片做量化分析。
脑梗死体积%=(总梗死面积*切片厚度)/(总脑切片面积*切片厚度)*100 %
实验结果:
1.1、TTC量化结果如表1所示:
表1
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 平均值 |
模型组 | 0.407 | 0.402 | 0.392 | 0.420 | 0.377 | 0.340 | 0.390 |
S1(NA1) | 0.187 | 0.218 | 0.153 | 0.145 | 0.107 | 0.134 | 0.157 |
S8 | 0.236 | 0.172 | 0.086 | 0.197 | 0.150 | 0.191 | 0.172 |
S9 | 0.239 | 0.206 | 0.208 | 0.263 | 0.171 | 0.095 | 0.197 |
假手术组 | 0.048 | 0.038 | 0.042 | 0.051 | 0.039 | 0.044 | 0.044 |
从表1的结果可见,注射生理盐水的脑缺血大鼠,梗死体积 0.390±0.028;注射 3mg/kg beta-Ala-L-His-Gly(S8)的脑缺血大鼠,梗死体积 0.172±0.051;注射 3 mg/kgGly-beta-Ala- His(S9)的脑缺血大鼠,梗死体积 0.197±0.059;作为比较,静脉给于3mg/kg NA1的脑缺血大鼠,梗死体积 0.157±0.040。作为对照的假手术组的结果则为梗死体积 0.044±0.005。与生理盐水比较,其它三组有明显的生物活性且S8和S9与S1没有统计学区别, 其中,NA1(Nerinetide)是一种神经保护剂 (Michael Tymianski and JonathanD. Garman. Model systems and treatment regimes for treatment of neurologicaldisease. 2015, US Patent, US8940699B2),可干扰突触后密度蛋白95(PSD-95),通过终止细胞内NO自由基的产生来实现,在临床前缺血性脑卒中模型中可减少动物实验性(猕猴)脑缺血再灌注的梗死面积,并改善其功能预后, 因此是一个很好的阳性对照产品。Hill博士研究了静脉注射新型神经肽NA-1(2.6mg/kg)用于血管内血栓切除术的急性缺血性脑卒中(AIS)患者的疗效和安全性, 结果表明使用NA1治疗可改善患者预后。(Michael D Hillet al. Efficacy and safety of nerinetide for the treatment of acute ischaemicstroke (ESCAPE-NA1): a multicentre, double-blind, randomised controlledtrial.Lancet. Published online February 20,2020)。
1.2行为学测评结果如表2所示:
表2
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 平均值 | 标准误差 |
模型组 | 4 | 4 | 3 | 5 | 4 | 3 | 3.8 | 0.8 |
S1 | 2 | 2 | 2 | 2 | 1 | 3 | 2.0 | 0.6 |
S8 | 3 | 2 | 1 | 2 | 2 | 3 | 2.2 | 0.8 |
S9 | 3 | 3 | 2 | 2 | 1 | 1 | 2.0 | 0.9 |
假手术组 | 0 | 0 | 0 | 0 | 0 | 0 | 0.0 | 0.0 |
从表2可以看出本发明提供的合成肽具有生理活性。注射生理盐水的脑缺血大鼠,行为学评分为3.8±0.8;注射 3 mg/kg beta-Ala-His-Gly的脑缺血大鼠,评分为2.2±0.8;注射 3 mg/kg Gly-beta-Ala- His的脑缺血大鼠,评分为2.0±0.9;作为比较,静脉给于3 mg/kg NA1的脑缺血大鼠,评分为2.0±0.6。作为对照的假手术组的结果则为1.0±0.0。与生理盐水比较,其它三组有明显的生物活性,且S8和S9与S1没有统计学区别。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种合成肽,其特征在于,其化学式选自以下任意一种:
(a)beta-Ala-His-Gly;
(b)Gly-beta-Ala-His。
2.权利要求1所述的合成肽或其盐在制备药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述合成肽或其盐在制备治疗缺血性脑卒中、出血性脑卒中或脑创伤药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述药物为注射剂、口服给药、舌下给药、喷雾给药或肛门给药。
5.根据权利要求4所述的应用,其特征在于,所述药物为粉针剂或者注射液。
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