title: A novel combined strategy for the physical PEGylation of polypeptides.
abstract: 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 (mPEG5kDa-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 mPEG5kDa-cholane molecules with an affinity constant of ~3·10(4)M(-1); these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH7.4 (4μg/mL) and markedly increased peptide solution stability at 25°C over 30days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an ~10% decrease in solubility. The predominantly α-helical conformation of the peptide alone at pH5-6.5 was lost at pH7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an ~10% loss of α-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG5kDa-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.
authors: Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy.
authors: Elena
authors: E
authors: Ambrosio
authors: Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy.
authors: Michela
authors: M
authors: Barattin
authors: Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy.
authors: Sara
authors: S
authors: Bersani
authors: MedImmune Ltd., Formulation Sciences, Granta Park, Cambridge CB21 6GH, UK.
authors: Saif
authors: S
authors: Shubber
authors: MedImmune Ltd., Formulation Sciences, Granta Park, Cambridge CB21 6GH, UK.
authors: Shahid
authors: S
authors: Uddin
authors: MedImmune Ltd., Formulation Sciences, Granta Park, Cambridge CB21 6GH, UK.
authors: Christopher F
authors: CF
authors: van der Walle
authors: Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy.
authors: Paolo
authors: P
authors: Caliceti
authors: Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy. Electronic address: stefano.salmaso@unipd.it.
authors: Stefano
authors: S
authors: Salmaso
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002757:Cholanes / Q000737:chemistry*; D005612:Freeze Drying; D008297:Male; D010455:Peptides / Q000097:blood* / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D017207:Rats, Sprague-Dawley; D012995:Solubility; D014660:Vasoactive Intestinal Peptide / Q000097:blood* / Q000737:chemistry*
chemical_list: D002757:Cholanes; D010455:Peptides; D014660:Vasoactive Intestinal Peptide; D011092:Polyethylene Glycols
keywords: Acylation; Bioavailability; Formulation; Self-assembly; Stability