胸腺五肽的酶法聚乙二醇定点修饰及其初步药学性质研究

Site-Specifically Enzymatic PEGylation of Thymopentin and Preliminary Study on its Pharmaceutical Properties

拟利用微生物转谷氨酰胺酶(m TGase)催化法对胸腺五肽(TP5)进行聚乙二醇(PEG)定点修饰,并初步测定修饰产物的半衰期与生物活性。m TGase的底物苄氧羰基-L-谷氨酰胺酰甘氨酸(ZQG)的末端羧基经碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)活化,然后与叔丁氧羰基-聚乙二醇氨(BOC-PEG-NH2)的氨基相连得到含酰胺供体的PEG-ZQG;经m TGase催化,PEG-ZQG的酰胺基与TP5共价缀合,采用Superdex30凝胶过滤柱层析纯化得到结合物,并对其进行紫外扫描、~1H NMR分析;最后测定结合物在小鼠体内的药代动力学特性和体外促脾淋巴细胞增殖活性。结果表明,1H NMR显示TP5与PEG-ZQG以1∶1成功结合,修饰位点为赖氨酸残基的侧链氨基,产率达78. 5%;修饰产物PEG-ZQG-TP5在小鼠体内的消除半衰期、曲线下面积分别是TP5的5. 9倍、4. 8倍,其对小鼠脾淋巴细胞促增殖能力与TP5相当。因此,基于m TGase催化策略能够实现对TP5的高效定点修饰,产物的体内半衰期明显延长、生物利用度显著提高,且活性保持率高。

This paper was aimed to explore site-specific modification of the thymopentin(TP5)with polyethylene glycol(PEG)by using the microbial transglutaminase(m TGase)catalytic method,and then to determine of the half-life and biological activity of the modified products.A substrate Benzyloxycarbonyl-L-glutaminylglycine(ZQG)of m TGase was activated at carboxyl group using carbodiimide/N-hydroxysuccinimide(EDC/NHS)method,then linked to the amino group of tert-butoxycarbonyl-polyethylene glycol ammonia(BOC-PEG-NH2)to yield amide-containing polymer PEG-ZQG.The amide group of PEG-ZQG was covalently conjugated to the side chain amino group of TP5 lysine residue by m TGase catalysis,followed by purification by Superdex 30 gel filtration column chromatography to obtain the purified PEG-ZQG-TP5.The PEG-ZQG and PEG-ZQG-TP5 were characterized by UV-visible(UV-vis)spectrophotometry and^1 H NMR analysis.Finally,the author tested its pharmacokinetics profiles in mice and the effect on spleen lymphocyte proliferation in vitro.PEG-ZQG was found to have the UV absorption peak at 255 nm and1 H NMR peaks atδ=7.47~7.17 ppm that was characteristic of ZQG,confirming the identity as expected.TP5 was successfully modified with PEG-ZQG by the catalysis of m TGase,with a yield of 78.5%.The1 H NMR spectrum showed that PEG-ZQG was covalently conjugated to the side chain amino group of the lysine residue of TP5 with a molar ration of 1∶1.The conjugate PEG-ZQG-TP5 increased the elimination half-life and the area under the curve(AUC)in mice to 5.9 times and 4.8 times compared with that of TP5.PEG-ZQG-TP5 showed significantly ability to promote proliferation of mouse spleen lymphocytes in vitro that was equivalent to TP5 at the same peptide concentrations.Therefore,based on the m TGase catalytic strategy,TP5 successfully conducted site-specific PEGylation with high efficiency.The resultant conjugate demonstrated significantly prolonged in vivo half-life,better bioavailability and no obvious loss in the biological activity.