Stability Enhancing N-Terminal PEGylation of Oxytocin Exploiting Different Polymer Architectures and Conjugation Approaches

Abstract

Oxytocin, a cyclic nine amino acid neurohypophyseal hormone therapeutic, is effectively used in the control of postpartum hemorrhaging (PPH) and is on the WHO List of Essential Medicines. However, oxytocin has limited shelf life stability in aqueous solutions, particularly at temperatures in excess of 25 °C and injectable aqueous oxytocin formulations require refrigeration (<8 °C). This is particularly problematic in the hot climates often found in many developing countries where daytime temperatures can exceed 40 °C and where reliable cold-chain storage is not always achievable. The purpose of this study was to develop N-terminal amine targeted PEGylation strategies utilizing both linear PEG and polyPEG "comb" polymers as an effective method for stabilizing solution formulations of this peptide for prolonged storage in the absence of efficient cold-chain storage. The conjugation chemistries investigated herein include irreversible amine targeted conjugation methods utilizing NHS ester and aldehyde reductive amination chemistry. Additionally, one reversible conjugation method using a Schiff base approach was explored to allow for the release of the native peptide, thus, ensuring that biological activity remains unaffected. The reversibility of this approach was investigated for the different polymer architectures, alongside a nonpolymer oxytocin analogue to monitor how pH can tune native peptide release. Elevated temperature degradation studies of the polymer conjugates were evaluated to assess the stability of the PEGylated analogues in comparison to the native peptide in aqueous formulations to mimic storage conditions in developing nations and regions where storage under appropriate conditions is challenging.

Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein

“…15, 16 However, conjugating these polymers does not necessarily improve protein stability during storage. There are few recent examples of protein-stabilizing polymers such as zwitterionic carboxybetaine polymer for enzymes, 8, 17 cationic dendrimer for proline-specific endopeptidase (PEP), 18 and branched or “comb” PEG for oxytocin, 19, 20 but these have been mainly limited to enzyme or small peptide stabilization. There is still a tremendous need and interest in developing new PEG alternatives that can confer both increased half-life and storage stability for a variety of proteins and stressors.…”

Comb Poly(Oligo(2‐Ethyl‐2‐Oxazoline)Methacrylate)‐Peptide Conjugates Prepared by Aqueous Cu(0)‐Mediated Polymerization and Reductive Amination

“…Previously, our groups have utilized reductive amination chemistry for the conjugation of linear and comb PEG polymers containing aldehyde functionality onto the therapeutic peptide oxytocin (cyclic [Cys‐Tyr‐Ile‐Gln‐Asn‐Cys]‐Pro‐Leu‐Gly) . Oxytocin is an important therapeutic recommended globally as an uterotonic for the prevention of postpartum hemorrhaging and is classified by the World Health Organization as an essential medicine.…”

“…PEGylated conjugates (including comb‐shaped P(OEGA) architectures) have previously been shown to possess better thermal stability than native equivalents under high thermal stressing . In order to evaluate the potential benefits that conjugation of comb‐shaped P(OEtOxMA) might have on the stability of oxytocin, accelerated high temperature thermal studies were conducted.…”

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

“…Copyright 2015, American Chemical Society. Bottom: Adapted with permission . Copyright 2016, American Chemical Society.…”

“…Alternatively, the N ‐terminus was targeted for modification with linear PEG and comb polymers of OEG(M)A and oligo(2‐ethyl‐2‐oxazoline) methacrylate (OEtOxMA) through NHS‐ester and aldehyde (CHO) functional end‐groups from which both irreversible and reversible conjugates could be formed . Comb polymers were synthesized from functional initiators using either Cu 0 ‐wire mediated polymerization (NHS‐POEGA, CHO‐POEGA) or aqueous SET‐LRP (CHO‐POEGMA, CHO‐P(OEtOxMA).…”