A new PEG-beta-alanine active derivative for releasable protein conjugation.

A new PEGylating agent, PEG-betaAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines. Furthermore, the conjugates obtained with PEG-betaAla-NHCO-OSu showed an interesting slow release of polymer chains upon incubation under physiological conditions. Further investigations determined that the PEG chains released are those coupled to histidine residues, and this finally yields less PEGylated species as well as free protein. This release allows a partial recovery of protein activity that is often remarkably and permanently reduced after stable PEGylation, and it occurs in water or blood without the involvement of enzymes. On the other hand, the rate of PEG release, tuned by the chemical structure of this new PEGylating agent, is not too high, and therefore, the achievement of a desired prolongation of protein half-life in vivo is still feasible. The pharmacokinetics of hGH-PEG6k-betaAla conjugate was compared to that of native hGH in rats and monkeys, and the blood residence times were increased by 10- and 7-fold, respectively. The conjugate potency was evaluated in hypophysectomized rats demonstrating a superior pharmacodynamic profile with respect to native hGH.