PEG-Modified Protamine with Improved Pharmacological/Pharmaceutical Properties as a Potential Protamine Substitute: Synthesis and in Vitro Evaluation

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• 作者：Li-Chien Chang ; Hsiao-Feng Lee ; Meng-Ju Chung ; and Victor C. Yang
• 刊名：Bioconjugate Chemistry
• 出版年：2005
• 出版时间：January 2005
• 年：2005
• 卷：16
• 期：1
• 页码：147 - 155
• 全文大小：146K
• 年卷期：v.16,no.1(January 2005)
• ISSN：1520-4812

文摘

Cardiopulmonary bypass (CPB) procedures are frequently associated with massive inflammatoryresponses, resulting in a high rate of morbidity and mortality in routine cardiac operations. Onerecognized attribute of these deleterious responses is the synergic effect of heparin and protamine,which elicit the activation of the complement system in vivo. To circumvent such toxic effects followingprotamine reversal of heparin anticoagulation in the CPB procedures, we proposed that poly(ethyleneglycol) (PEG)-modified protamine could retain the heparin-neutralization ability and yet diminishthe induced complement activation by the formed heparin-protamine complexes (HPC), therebyproviding highly improved pharmacological properties. PEGylation of protamine was carried out byutilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. Size exclusion chromatography (SEC),reverse-phase high performance liquid chromatography (RP-HPLC), and matrix-assisted laserdesorption mass spectrometry (MALDI-MS) were used to assess the conjugation stiochiometry, thepurity of the conjugates, and the site of PEG modification, respectively. The heparin-neutralizingactivity was determined by using heparin affinity chromatography and various biological assaysincluding the plasma-activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa methods.The potency in inducing complement activation was examined in vitro using the CH₅₀ hemolytic assay.The PEG-modified protamine was successfully synthesized with a PEG/protamine stiochiometry of1:1. Only one conjugation site for PEG that was located at the N-terminal end of protamine wasobtained. In the biological evaluations, the PEG-modified protamine displayed a full retention of theheparin-neutralizing ability of protamine and a significantly reduced activity in complement activationfollowing its complexation with heparin. Results from studies of the particle size and zeta potentialindicated that the PEG-modified protamine formed substantially smaller aggregates with heparin,rendering them less effective in triggering the size-dependent complement responses. As withprotamine, PEG-modified protamine exhibited an enhanced aqueous solubility, therefore attainingsignificantly improved pharmaceutical properties. These preliminary results suggested that the PEG-modified protamine conjugate might serve as a potential protamine substitute with improvedtherapeutic and pharmaceutical properties in heparin reversal.