Abstract
AbstractProlonging serum half-lives of protein/peptide drugs in vivo is important in drug delivery systems. Site-specific chemical modification is one of the approaches. By choosing a suitable site of proteins and controlling the reaction condition, we can obtain the homogeneous product without affecting the activity of proteins. Because of the high reactivity of thiol group, cysteine is a good choice to react with some sulfhydryl-specific modifiers. Here, we mutated a phenylalanine39 of ACTH to cysteine (ACTH-F39C). The free sulfhydryl group of cysteine then reacted with two kinds of chemical modifiers—iodoacetamide derivatives with long carbon chains and methoxy-polyethylene glycol (m-PEG) maleimide derivatives. After lipophilic modification, the in vitro biological activities of these adducts were not disturbed compared to ACTH wild type. In ELISA and flow cytometry experiments, F39C-12CH3 is associated more strongly with plasma membranes and serum albumins than the unmodified one. We injected unmodified ACTH and the lipophilic adducts to mice by intramuscular injection. The maintaining percentage in mice serum of F39C-12CH3 is 3-folds higher than F39C after 6 hours. In PEG modification, the in vitro activities of F39C-PEG20K and F39C-PEG40K were slightly decreased. After 24 hours，the remaining percentage of F39C-PEG20K and F39C-PEG40K in mice serum is 6-folds higher than F39C. We concluded that chemical modifications to change the lipophilicity or molecular size of proteins/peptides could prolong the serum half-life in vivo.