胸腺素α1及其系列结构修饰物的体外肝匀浆稳定性评价
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摘要
目的考察并评价胸腺素α1(Tα1)不同位点修饰PEG聚合物的体外肝匀浆酶稳定性,为修饰物的结构优化提供数据支持。方法应用反相高效液相色谱法(RP-HPLC)研究Tα1及其结构修饰物TC系列和PT系列在大鼠肝匀浆中的体外代谢性质,检测波长均为200 nm。结果多肽药物的浓度在4.0～800 mg/ml范围内与其峰面积呈良好线性关系(r>0.9990),绝对回收率>85%,日内相对标准偏差(RSD)<1.50%,日间RSD<2.10%。各修饰物的半衰期依次为PT系列>Tα1>TC系列。结论 Cys替换的TC系列结构修饰物的半衰期均TC系列,表明Cys位点PEG化后修饰物更加稳定。
Objective To investigate the enzymatic degradation stability of peptide drug Tα1 and its PEG modified analogsatdifferentsites,andprovidedatasupportforstructuraloptimizationtowardsmodifiedpeptidedrugs.Methods Reversed phase-high performance liquid chromatography(RP-HPLC)was used to investigate in vitro metabolisms of Tα1 and its structural modified analogs TC series and PT series in liver homogenates of rats. The separation of all the peptide drugs and their digested fragments was achieved at the detection wavelength of 200 nm.Results A good linear range was achieved between 4.0 and 800 μg/ml(r >0.9990)for Tα1 and its structural modified analogs,i.e.,TC and PT series. The recoveriesofallpeptidedrugsweregreaterthan85%inliverhomogenatesystems.Therelativestandardderivations(RSDs)of intra-dayandinter-daywerelessthan1.50%and2.10%,respectively.Therevealedorderofdigestedhalf-livesofthepeptide drugs was PT series>Tα1>TC series. Conclusion All the half-lives of TC series are shorter than that of Tα1,indicated that the TC series could be more easily degraded than Tα1 in this liver homogenate system. Meanwhile,all the half-lives of PT series are longer than those of TC series,which indicates the peptides become more stable when modified by PEG.
Objective To investigate the enzymatic degradation stability of peptide drug Tα1 and its PEG modified analogsatdifferentsites,andprovidedatasupportforstructuraloptimizationtowardsmodifiedpeptidedrugs.Methods Reversed phase-high performance liquid chromatography(RP-HPLC)was used to investigate in vitro metabolisms of Tα1 and its structural modified analogs TC series and PT series in liver homogenates of rats. The separation of all the peptide drugs and their digested fragments was achieved at the detection wavelength of 200 nm.Results A good linear range was achieved between 4.0 and 800 μg/ml(r >0.9990)for Tα1 and its structural modified analogs,i.e.,TC and PT series. The recoveriesofallpeptidedrugsweregreaterthan85%inliverhomogenatesystems.Therelativestandardderivations(RSDs)of intra-dayandinter-daywerelessthan1.50%and2.10%,respectively.Therevealedorderofdigestedhalf-livesofthepeptide drugs was PT series>Tα1>TC series. Conclusion All the half-lives of TC series are shorter than that of Tα1,indicated that the TC series could be more easily degraded than Tα1 in this liver homogenate system. Meanwhile,all the half-lives of PT series are longer than those of TC series,which indicates the peptides become more stable when modified by PEG.
引文
[1]Fosgerau K,Hoffmann T.Peptide therapeutics:current status and future directions[J].Drug Discov Today,2015,20(1):122-128.
[2]Liu W,Tan JJ,Mehryar MM,et al.Peptide HIV fusion inhibitors:modifications and conjugations[J].Med Chem Comm,2014,5(10):1472-1482.
[3]Liu HM,Liu XF,Yao JL,et al.Utilization of combined chemical modifications to enhance the blood-brain barrier permeability and pharmacological activity of endomorphin-1[J].J Pharmacol Exp Ther,2006,319(1):308-316.
[4]刘兴东,孟庆斌,孟庆国.抗菌肽的分子设计研究进展[J].军事医学,2014,38(5):396-399.
[5]Rodriguez MC,Cudic M.Optimization of physicochemical and pharmacological properties of peptide drugs by glycosylation[J].Methods Mol Biol,2013,1801:107-136.
[6]Qie JJ,Ma JB,Wang LY,et al.Site-directed PEGylations of thymosin alpha 1 analogs and evaluation of their immunoactivity[J].Chin J Chem,2009,27(4),805-809.
[7]Feng WY,Chan KK,Covey JM.Electrospray LC-MS/MS quantitation,stability,and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201(NSC 710295)in the mouse[J].J Pharmaceut Biomed,2002,28(3-4):601-612.
[8]Tuthill CW,Rudolph A,Li Y,et al.Quantitative analysis of thymosinα1 in human serum by LC-MS/MS[J].AAPS Pharm Sci Tech,2000,1(2):E11.
[9]李志红,赵永芳.胸腺素含量的ELISA测定法[J].氨基酸和生物资源,1999,21(1):21-23.
[10]周克夫,章军,陈天圣,等.胸腺素α1抗体的制备和鉴定[J].中国免疫学杂志,2001,17(1):43-47.
[11]黄小花,周克夫,章军,等.抗胸腺素α1(Tα1)多克隆抗体的纯化及转基因藻中Tα1的检测[J].厦门大学学报(自然科学版),2004,43(S1):122-125.
[2]Liu W,Tan JJ,Mehryar MM,et al.Peptide HIV fusion inhibitors:modifications and conjugations[J].Med Chem Comm,2014,5(10):1472-1482.
[3]Liu HM,Liu XF,Yao JL,et al.Utilization of combined chemical modifications to enhance the blood-brain barrier permeability and pharmacological activity of endomorphin-1[J].J Pharmacol Exp Ther,2006,319(1):308-316.
[4]刘兴东,孟庆斌,孟庆国.抗菌肽的分子设计研究进展[J].军事医学,2014,38(5):396-399.
[5]Rodriguez MC,Cudic M.Optimization of physicochemical and pharmacological properties of peptide drugs by glycosylation[J].Methods Mol Biol,2013,1801:107-136.
[6]Qie JJ,Ma JB,Wang LY,et al.Site-directed PEGylations of thymosin alpha 1 analogs and evaluation of their immunoactivity[J].Chin J Chem,2009,27(4),805-809.
[7]Feng WY,Chan KK,Covey JM.Electrospray LC-MS/MS quantitation,stability,and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201(NSC 710295)in the mouse[J].J Pharmaceut Biomed,2002,28(3-4):601-612.
[8]Tuthill CW,Rudolph A,Li Y,et al.Quantitative analysis of thymosinα1 in human serum by LC-MS/MS[J].AAPS Pharm Sci Tech,2000,1(2):E11.
[9]李志红,赵永芳.胸腺素含量的ELISA测定法[J].氨基酸和生物资源,1999,21(1):21-23.
[10]周克夫,章军,陈天圣,等.胸腺素α1抗体的制备和鉴定[J].中国免疫学杂志,2001,17(1):43-47.
[11]黄小花,周克夫,章军,等.抗胸腺素α1(Tα1)多克隆抗体的纯化及转基因藻中Tα1的检测[J].厦门大学学报(自然科学版),2004,43(S1):122-125.