Thiolytically Cleavable Dithiobenzyl Urethane-Linked Polymer–Protein Conjugates as Macromolecular Prodrugs: Reversible PEGylation of Proteins
| Autor: | Samuel Zalipsky, Charles Engbers, Nasreen Mullah, Maria U. Hutchins, Radwan Kiwan |
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| Rok vydání: | 2007 |
| Předmět: |
Male
Polymers Molecular Conformation Biomedical Engineering Pharmaceutical Science Bioengineering Urethane Polyethylene Glycols Rats Sprague-Dawley chemistry.chemical_compound PEG ratio Animals Organic chemistry Moiety Prodrugs Sulfhydryl Compounds Chromatography High Pressure Liquid Pharmacology Bioconjugation Chemistry Organic Chemistry Benzene Combinatorial chemistry Rats Cross-Linking Reagents Covalent bond Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization PEGylation Muramidase Lysozyme Ethylene glycol Biotechnology Conjugate |
| Zdroj: | Bioconjugate Chemistry. 18:1869-1878 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc7001902 |
| Popis: | New thiolytically cleavable dithiobenzyl (DTB) urethane-linked conjugates of methoxypoly(ethylene glycol) (mPEG) and a model protein, lysozyme, were prepared and thoroughly characterized. In contrast to our earlier communication [Zalipsky, et al. (1999) Bioconjugate Chem. 10, 703], in the current study we used a more sterically hindered form of para-DTB urethane linkage containing a methyl group on the alpha-carbon to the disulfide moiety. The new reagent for covalent attachment of mPEG-DTB to amino groups of proteins was synthesized via a seven-step process. As a result of PEG conjugation, the lysozyme was shown to completely lose its bacterial cell wall-lysing activity. However, activity was almost fully restored upon cysteine-mediated cleavage of the PEG component. The conjugate decomposition process was monitored by RP-HPLC and by ion spray LC-MS, which showed the formation of the p-mercaptobenzyl urethane-lysozyme intermediate, and ultimately its conversion to the unmodified lysozyme as the sole protein component. Pharmacokinetic evaluation of (125)I-labeled cleavable and noncleavable PEG-lysozyme given intravenously in rats revealed similar clearance patterns; both cleared in a significantly slower manner compared to that of the native protein. However, subcutaneous administration of the same conjugates showed a significantly larger AUC of the cleavable conjugate, indicating that some cleavage of the DTB urethane may have occurred. Although the DTB-linked PEG-lysozyme exhibited almost the same plasma clearance as the noncleavable counterpart, hinting that methyl-DTB linkage might be stable in the bloodstream, SDS-PAGE examination of the conjugate incubated in plasma showed decomposition at least partially mediated by albumin. These results suggest the potential of PEG-DTB-proteins as macromolecular prodrugs capable of generating fully active native proteins under in vivo conditions. |
| Databáze: | OpenAIRE |
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