Linear and Branched Bicin Linkers for Releasable PEGylation of Macromolecules:  Controlled Release in Vivo and in Vitro from Mono- and Multi-PEGylated Proteins

Abstract

The utility of PEGylation for improving therapeutic protein pharmacology would be substantially expanded if the authentic protein drugs could be regenerated in vivo. Diminution of kinetic constants of both enzymes and protein ligands are commonly encountered following permanent bioconjugation with poly(ethylene glycol) polymers. In further development of releasable linker technology, we investigated an amino PEG anchimeric prodrug system, based on either the linear or branched bicin3 (BCN3) linkage, one promising representative of several aliphatic ester structures synthesized from N-modifed bis-2-hydroxyethylglycinamide (bicin). Protein models included an enzyme, lysozyme, and a receptor ligand, interferon-beta-1b, for preparation of linear or branched mono- and multi-PEGylated conjugates as inactive PEG-BCN3 prodrugs. The kinetics of protein release, both in plasma (in vitro) and in mice (in vivo), correlated with the number of PEG attachments, and the plasma half-lives of PEG release spanned a duration of hours to days within the therapeutically relevant window. Capillary electrophoresis, SDS-PAGE, mass determination, and enzymatic and antiviral activity determinations demonstrated regeneration of equivalent native proteins from the inactive PEG-BCN3 conjugates. Pharmacokinetic analysis of the PEGylated interferon-beta-1b administered subcutaneously in mice demonstrated an over 20-fold expansion of the area under the curve exposure of bioactive protein when compared to native protein.

Releasable PEGylation of Mesothelin Targeted Immunotoxin SS1P Achieves Single Dosage Complete Regression of a Human Carcinoma in Mice

“…As shown in Chart 1, four unconventional releasable linker designs were evaluated. The bis-N-2-hydroxyethylglycine (Bicin3), diglycolic acid (DGA2), and reversible nitrogen linker (RNL-8a) linker chemistries have been recently described with regard to their mechanism of controlled release from PEGylated proteins (39)(40)(41)44). The DGA2 (fast release) and RNL-8a (slow release) linkers are both aromatic designs wherein an initial ester cleavage triggers a classical 1,6-benzyl elimination reaction which releases the original amine and results in linker degradation.…”

“…The mechanisms of controlled release of authentic native protein from the linker and PEG have been described for both the aromatic-based DGA2 and RNL-8a linkers (40,41) and the aliphatic Bicin3 linker (39,44).…”

Polymers for Protein Conjugation

“…Other future directions in the development of PEGylation include: (i) releasable PEGs, which are polymers linked to the protein through cleavable linkers that allows the slow release of the conjugated protein in its full active form [17][18][19][20][21][22][23]; (ii) non-covalent PEGylation, which can be mediated through either hydrophobic interactions or the formation of coordination complexes [24][25][26]; and (iii) new PEG forms; in fact, the classic linear or branched structures of PEG can be further modified to obtain new copolymers with improved features, such as the comb shape PEGs [27][28][29].…”

Funcionalidade e caracterização das propriedades físico-químicas, biológicas e estruturais da uricase modificada por PEGlação.

“…Desde então, a tecnologia de PEGlação tem se desenvolvido e expandido e atualmente existem inúmeros métodos químicos e enzimáticos para conjugação de PEG a biomoléculas (ZALIPSKY, 1995;ROBERTS;BENTLEY;HARRIS, 2002;ZHAO et al, 2006;BALAN et al, 2007;FONTANA et al, 2008). …”

“…Esses métodos são geralmente baseados em uma molécula de disparo que, por reação enzimática ou hidrólise, libera um grupo que catalisa a separação do polímero, liberando a proteína nativa (LEE et al, 2001;PELEG-SHULMAN, 2004;ZHAO et al, 2006). Escolhendo cuidadosamente os grupos químicos do PEG ativado, a proteína modificada permanece na corrente sanguínea por longo tempo e uma liberação lenta e controlada da proteína nativa é obtida (ZALIPSKY et al, 2007).…”