次血红素短肽化合物体外稳定性研究

发布时间：2018-04-26 09:05

本文选题：多肽类药物 + 体外稳定性　；参考：《吉林大学》2014年硕士论文

【摘要】：多肽类药物在当今医药市场占有非常重要的地位，在治疗如糖尿病，肿瘤，癌症，疫苗，精神性疾病等方面发挥重大作用。多肽类药物具有活性高，良好的特异性及靶向性，代谢产物主要为氨基酸、毒副作用小、不易造成积累性中毒，，与传统药物相比，成本更低，渗透性更强。但其自身固有的缺点也限制其发展，稳定性差，容易被各种酶解，降解速度快，体内半衰期短，口服利用度低，需频繁注射给药，加重了患者身心、经济的负担。在过去的几十年里，研究人员已经运用大量策略来增加多肽对肽酶的抵抗性，如化学修饰、定点突变、延长肽链、引入非天然氨基酸、自组装修饰、通过二硫键环化等。通过对多肽进行修饰或改造，来提高多肽类药物的稳定性，是当前研究的热门课题。血红素是由原卟啉和铁离子所形成的配合物，参与多种生理生化反应，具有重要的生理功能。本实验室根据设计合成了一个以血红素为辅基的过氧化物酶：次血红素六肽（Deuterohemin-AlaHisThrValGluLys，DhHP-6），具有很高的过氧化物酶活性，比活力可达到天然微过氧化物酶MP-11的93％。实验结果表明，DhHP-6具有延长线虫寿命，有效清除体内自由基，抗氧化抗衰老，抗心肌氧化损伤等作用，但关于稳定性方面的研究较少。本实验根据文献报道和实验室前期工作，通过固相肽合成手段将次血红素基团与四条稳定性较差，容易降解的多肽偶联，经过HPLC分析，MS检测，分离纯化，可以得到纯度高达98%以上，物理化学性质稳定的次血红素短肽化合物。结合HPLC，MS分析检测，对形成的次血红素短肽化合物在体外血浆，模拟胃液，模拟肠液中的稳定性进行研究。实验结果表明，在体外血浆中，线性肽在1~5min内迅速降解殆尽，次血红素短肽化合物在与血浆共孵育24h后只降解40%左右，稳定性大大提高，半衰期明显延长；在多肽类药物非常容易降解的胃肠道模拟液中，次血红素短肽化合物也有着非常良好的表现，在体外模拟胃液实验中，次血红素短肽的半衰期可以达到5min以上；在模拟肠液实验中，次血红素短肽的半衰期更是可以达到2h以上，说明次血红素基团可以有效的提高多肽的稳定性，延长半衰期，对肽序列具有一定的保护作用。本实验有助于研究含有卟啉环的金属卟啉多肽与蛋白的设计与应用，为提高多肽类药物的稳定性提供了一条新思路，具有潜在的临床意义。
[Abstract]:Polypeptide drugs play an important role in the treatment of diabetes, cancer, cancer, vaccines, psychiatric diseases and so on. Polypeptide drugs have high activity, good specificity and targeting, the main metabolites are amino acids, less toxic side effects, not easy to cause accumulative poisoning, compared with traditional drugs, the cost is lower and the permeability is stronger. However, its inherent shortcomings also limit its development, its stability is poor, it is easy to be hydrolyzed by various enzymes, the degradation rate is fast, the body half life is short, the oral utilization is low, and it needs to be injected frequently, which increases the burden of body, mind and economy of the patients. In the past few decades, researchers have used a large number of strategies to increase the resistance of peptides to peptidases, such as chemical modification, site-directed mutation, prolongation of peptide chains, introduction of unnatural amino acids, self-assembly modification, cyclization through disulfide bonds, and so on. It is a hot topic to improve the stability of polypeptide drugs by modifying or modifying peptides. Heme is a complex formed by protoporphyrin and iron ions, which participates in many physiological and biochemical reactions and has important physiological functions. According to the design, a peroxidase, Deuterohemin-Ala-ThrValGluLys-DhHP-6, was synthesized in our laboratory. It has a high peroxidase activity, and its specific activity can reach 93% of that of natural microperoxidase (MP-11). The results showed that DhHP-6 had the effects of prolonging the life of the worm, effectively scavenging free radicals in vivo, anti-aging and anti-oxidative injury of myocardium, but little research on the stability of DhHP-6. In this experiment, according to the literature reports and laboratory work, the heme group was coupled with four peptides with poor stability and easy degradation by solid phase peptide synthesis, and then detected by HPLC and then purified by MS. The subheme short peptide compounds with purity above 98% and stable physical and chemical properties can be obtained. The stability of subheme short peptide compounds in plasma, gastric juice and intestinal fluid was studied by HPLC- MS. The results showed that the linear peptide was degraded rapidly in 1~5min in vitro, the degradation of heme short peptide was only about 40% after incubation with plasma for 24 h, the stability was greatly improved, and the half-life was prolonged. The heme short peptide compounds also have a very good performance in the gastrointestinal mimics which are easily degraded by polypeptide drugs. The half-life of sub-heme short peptides can reach 5min in vitro. In the experiment of simulated intestinal fluid, the half-life of the sub-heme short peptide can reach more than 2 hours, which indicates that the heme group can effectively improve the stability of the peptide, prolong the half-life, and protect the peptide sequence to a certain extent. This study is helpful to study the design and application of metalloporphyrin polypeptides and proteins containing porphyrin ring. It provides a new way to improve the stability of polypeptide drugs and has potential clinical significance.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91

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