150 : Physico-chemical properties, pharmacokinetics and pharmacodynamics of pegylated cytokines
Introduction: Covalent attachment of poly (ethylene glycol) (PEG) to therapeutic proteins can be used to enhance of pharmacological effectiveness of drug. In comparison with unmodified analogs, PEGylated proteins have better physical and thermal stability, greater protection against proteolytic cleavage, reduced immunogenicity and toxicity and improved pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Methods: Physicochemical properties of PEGylated proteins were analyzed in comparison with unmodified proteins by SDS–PAGE, SEC-HPLC, RP-HPLC, mass spectrometry, peptide mapping, circular dichroism, fluorescence spectroscopy and in vitro specific activity. Preclinical studies were conducted with animals to determine toxicities, safety and PK/PD characteristics of new PEG- conjugates. Results: The series of new conjugates of human recombinant cytokines (IFN, G-CSF) were prepared with PEGs of different structure and lengths specifically attached to the N-terminal amino group of the proteins. The methods of single step chromatographic purification to produce mono PEGylated proteins were developed. The functional activity of the PEGylated cytokines depended on molecular weight of the attached PEGs and was 30%–80% of the activity of unmodified proteins. Fluorescent spectra and CD spectra for PEG-proteins and their native analogs were nearly superimposed across the near- to far-UV spectrum, suggesting that pegylation had no significant effect on the secondary or tertiary conformation of the cytokines. It was shown that the trypsin resistance and thermal stability were significantly improved in PEGylated proteins. The native proteins aggregated rapidly in 100–200 min, whereas the PEG-proteins showed no aggregation more for 24 h. The results of preclinical studies have demonstrated that PEGylated proteins have significantly better pharmacological properties than their unmodified analogs. The systemic clearance of PEGylated cytokines decreased 20–30-fold and the serum half-life increased 12–16-fold compared to unconjugated proteins. Conclusions : The two PEG-cytokine conjugates were selected for further studies in clinical trials.