A novel combined strategy for the physical PEGylation of polypeptides

Poly(ethylene glycol) (PEG) may be covalently conjugated to peptide drugs to overcome their rapid clearance but in doing so their potency can be lost. Here, a non-covalent approach was used to conjugate PEG bearing a terminal cholanic moiety (mPEG(5kDa)-cholane) to a 28 amino acid peptide, vasoactive intestinal peptide (VIP). Palmitoylation of the peptide was essential to facilitate physical interaction via a single binding site involving two mPEG(5kDa)-cholane molecules with an affinity constant of similar to 3.10(4) M-1; these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12 nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH 7.4 (4 mu g/mL) and markedly increased peptide solution stability at 25 degrees C over 30 days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an similar to 10% decrease in solubility. The predominantly a-helical conformation of the peptide alone at pH 5-6.5 was lost at pH 7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an similar to 10% loss of a-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1 mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG(5kDa)-cholane dramatically increased peptide concentration in the systemic circulation. This is the first demonstration of non-covalent PEGylation of acylated peptides, an important biologic class, which improves in vitro and in vivo properties, and thereby may prove an alternative to covalent PEGylation strategies.