On the mechanism of degradation of oxytocin and its analogues in aqueous solution

Abstract

Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys(1) and Cys(6) residues. Although OT is one of the most commonly used peptidic drugs, the mechanism of its degradation in aqueous solution and the identity of its degradants have not been fully elucidated. To investigate the pathways and products of OT degradation in slightly acidic to neutral solutions, we prepared the peptides: OT, [D-Cys(1)]OT, a series of N-alkylated OT analogues, [[(13)C3,(15) N]Cys(1)]OT, and OT where each sulfur atom was systematically replaced by either methylene, (34)S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and (13)C-NMR. Our findings suggest that the degradation begins with β-elimination of the disulfide linkage to form a putative intermediate linear peptide containing an S-thiocysteine (a persulfide) in position 6 and a dehydroalanine in position 1. This intermediate persulfide appears to donate a sulfur atom to an intact OT molecule to form OT trisulfide and higher monomeric polysulfides, while the dehydroalanine residue is hydrolyzed with loss of the N-terminal amino group to yield a linear N-pyruvoylated octapeptide containing a reduced Cys(6). Based on the MS and (13)C-NMR data of the products from degradation of [[(13)C3,(15)N]Cys(1)]OT, we postulate that the ultimate degradation products of OT are dimers composed of two pyruvoylated octapeptides held together by one disulfide bridge between the two Cys(6) residues and by one more, non-reducible, linkage resulting from an aldol-type condensation between the two N-terminal pyruvoyl groups.

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

“…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. However, it is restricted in use where there is no guaranteed refrigeration supply chain as problems arise with the aqueous stability of the peptide, particularly at elevated temperatures . As with the dipeptide [Gly‐Tyr], the only amine available for polymer conjugation on the peptide structure is the N ‐terminal amine, and therefore polymer attachment occurs site specifically.…”

“…However, it is restricted in use where there is no guaranteed refrigeration supply chain as problems arise with the aqueous stability of the peptide, particularly at elevated temperatures. [41,42] As with the dipeptide [Gly-Tyr], the only amine available for polymer conjugation on the Macromol. Rapid Commun.…”

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

“…In these, often remote areas, deficiencies in simple medical resources and infrastructure such as cold chain transportation and storage can exacerbate complications that arise during pregnancy, delivery and recovery. This is because oxytocin is chemically unstable in aqueous solution especially in hot and humid conditions . Degradation severely diminishes therapeutic efficacy, to a point where it is quite common for levels of oxytocin, in samples labelled oxytocin, to contain zero active peptide in many countries where cold chain storage is not available.…”

“…The disulfide bond (Cys 1 ‐Cys 6 ) present is complicit in the instability of oxytocin due to its propensity to undergo dimerization, β‐elimination, oxidation and polysulfide formation . It is also an attractive site for modification with polymers containing disulfide reactive end‐groups such DSM or arsenic.…”

Physicochemical and Formulation Developability Assessment for Therapeutic Peptide Delivery—A Primer

“…Under stressed conditions, chemical and physical degradation of oxytocin were observed (56,57), with deamidation products observed below pH 2 and above pH 9. Based on kinetic analysis, pH 4.5 was identified as the most stable solution condition (58).…”