Oxidation of the N-terminal gly-residue of peptides: Stress study of pexigauan acetate in a drug formulation

Purpose. The purpose of this study was to identify four major degradation p roducts, which were formed during a stress study of pexiganan (a 22-mer pep tide) in a 1% formulation. Methods. The degradation products were isolated and characterized by LC/MS, tryptic and aminopeptidase digests. Results. One of the degradation products was shown to be des-gly(1)-pexigan an. The other three are structural isomers of N-glyoxylyl-des-gly(1)-pexiga nan. These isomers undergo reversible inter-conversions, as well as decompo se irreversibly to des-gly(1)-pexiganan. Thus, all the impurities were form ed from a single oxidation product of pexiganan, N-glyoxylyl-des-gly(1)-pex iganan. The aldehyde group of the glyoxylyl residue and the NH-amide of the adjacent isoleucine residue form a piperazinedione derivative of des-gly(1 )-pexiganan. This heterocyclic compound rearranges to other tautomers or ba ck to the N-glyoxylyl compound (see Fig. 3). Tryptic digests of the three d egradation products showed that their N-terminal segment produced N-glyoxyl yl-I-G-K whereas the N-terminal segment of pexiganan produced G-I-G-K. All the other tryptic-digest segments were identical to those formed in pexigan an. The LC/MS of the N-terminal segment and of synthetic N-glyoxylyl-I-G-K were identical. The enzymatic resistance of the three impurities to undergo aminopeptidase-M cleavage further supported the conclusion that their N-te rminal amino residues are substituted. Conclusions. After a year under stress conditions 1% pexiganan cream lost a bout 15% of the active component to oxidative-deamination,(4) where the N-t erminal glycine residue was oxidized to N-glyoxylyl-des-gly(1)-pexiganan. T he other nine E-amino lysine-residues of the peptide stayed intact. This ox idation product inter-converted and formed two additional impurities, tauto mers of piperazinedionyl-des-gly(1)-pexiganan, and decomposed to des-gly(1) -pexiganan, the forth impurity.