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

Abstract

Cardiopulmonary bypass (CPB) procedures are frequently associated with massive inflammatory responses, resulting in a high rate of morbidity and mortality in routine cardiac operations. One recognized 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 following protamine reversal of heparin anticoagulation in the CPB procedures, we proposed that poly(ethylene glycol) (PEG)-modified protamine could retain the heparin-neutralization ability and yet diminish the induced complement activation by the formed heparin-protamine complexes (HPC), thereby providing highly improved pharmacological properties. PEGylation of protamine was carried out by utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. Size exclusion chromatography (SEC), reverse-phase high performance liquid chromatography (RP-HPLC), and matrix-assisted laser desorption mass spectrometry (MALDI-MS) were used to assess the conjugation stiochiometry, the purity of the conjugates, and the site of PEG modification, respectively. The heparin-neutralizing activity was determined by using heparin affinity chromatography and various biological assays including 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 of 1:1. Only one conjugation site for PEG that was located at the N-terminal end of protamine was obtained. In the biological evaluations, the PEG-modified protamine displayed a full retention of the heparin-neutralizing ability of protamine and a significantly reduced activity in complement activation following its complexation with heparin. Results from studies of the particle size and zeta potential indicated that the PEG-modified protamine formed substantially smaller aggregates with heparin, rendering them less effective in triggering the size-dependent complement responses. As with protamine, PEG-modified protamine exhibited an enhanced aqueous solubility, therefore attaining significantly improved pharmaceutical properties. These preliminary results suggested that the PEG-modified protamine conjugate might serve as a potential protamine substitute with improved therapeutic and pharmaceutical properties in heparin reversal.

Serum albumin–protamine conjugate for biocompatible platform for targeted delivery of therapeutic macromolecules

“…We argue that the immunogenicity associated with protamine use would also be attenuated upon conjugation with albumin. This argument is supported by earlier observation that conjugation of PEG molecules to protamine attenuates complement activation and subsequent immunogenic response 19 because of the shielding effects created by PEG. As albumin, being several-folds larger than protamine, is conjugated to N-terminus of protamine, a similar nonimmunogenic profile is expected.…”

“…As protamine does not contain any lysyl residues, the only site to introduce the SPDP linker at neutral to slightly alkaline condition is the N-terminal proline, a secondary amine. 19 However, addition of SPDP in either ethanol or DMF caused precipitation of protamine when the reaction media was phosphate buffered saline, which was later solved performing the reaction in 2M NaCl stabilized with PEG-600. FPLC chromatographic data demonstrate that the purification method effectively removed unreacted SPDP [ Fig.…”

Anticoagulant Properties of a New Anticoagulant Diethyl Citrate in Rabbit Model

“…However, all these methods exhibit limitations [1][2][3][4]. For example, low molecular heparin has high cost and bleeding risk; no heparin dialysis needs high blood flow, which can cause blood coagulation in dialyser and blood vessel, resulting in dialysis failure; low doses heparin dialysis requires a blood flow rate of over 180 mL/min, and the incidence of secondary hemorrhage is about 13%; when using protamine to neutralize heparin, it is very hard to accurately adjust the protamine/heparin ratio, and the intravenous application of protamine may cause low blood pressure, allergy and even ventricular fibrillation [5]; prostacyclin interferes hemodynamics and can lead to severe hypotension; the resource of recombinant hirudin reagents is limited. Therefore, when Et 2 Cit or Na 3 Cit was used as an anticoagulation, the pH increase of dialysate, that is, dialysis in alkaline conditions, could not only achieve the purpose of anticoagulant, but also avoid the occurrence of dialysis acidosis, which would improve the survival rate and quality of life.…”

Ternary Complex of Plasmid DNA with Protamine and γ-Polyglutamic Acid for Biocompatible Gene Delivery System

“…However, protamine was reported to cross-link with the complement protein and induce toxicity clinically such as thrombocytopenia and granulocytopenia responses. 9,10) The toxicity was related to its total cationic charge. 11) In the previous reports, we successfully constructed the safe ternary complex of pDNA with polyethyleneimine and γ-polyglutamic acid (γ-PGA) with high gene expression.…”