Abstract
Many cytokines and hormones are potent immunoregulatory substances and messenger molecules that control important body functions. Judged from their effects in various biological assays, these cytokines and hormones appear to be excellent candidates for treating a large number of diseases. However, despite active development by the pharmaceutical and biotechnology industries, only a few cytokines and hormones have been approved for therapeutic applications. These disappointing results are not due to the lack of inherent biological potency of the cytokine and hormone molecules but to the insufficient bioavailability of the injected cytokines and hormones in patients. In the present study, we employ site-specific mutagenesis methods and chemical modification techniques to increase the lipophilicity of the IFNalpha2a and ACTH and thereby improve their pharmacokinetic properties. Because large numbers of red cells and large quantity of albumin exist in the blood circulation, they can potentially serve as reservoir for lipophilic therapeutics and hence lengthen their pharmacokinetic properties. Ser163 of IFNalpha2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFNalpha2a (S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFNalpha2a were purified by gel filtration chromatography. The biological activities of IFNalpha2a (S163C) and lipophilized IFNalpha2a (S163C) were similar to that of IFNalpha2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in MDBK cells. However, lipophilized IFNalpha2a (S163C) bound to human serum albumin and cell membranes more readily than IFNalpha2a. Phe39 of ACTH was also substituted by Cys and modified by conjugating with iodoacetamide derivatives of lipophilic groups. The biological activities of lipophilized ACTHs (F39C) were higher than unmodified ACTH. Lipophilized ACTHs (F39C) bound more tightly to human serum albumin and cell membranes in vitro and had longer serum half-lives in vivo than native ACTH. These results indicate that the pharmacokinetic properties of proteins and peptides can be improved by site-specific substitution with cysteine residues and subsequent conjugation with lipophilic moieties.