Oxidation of the N-terminal gly-residue of peptides: stress study of pexiganan acetate in a drug formulation.
OBJECTIVE
The purpose of this study was to identify four major degradation products, which were formed during a stress study of pexiganan (a 22-mer peptide) 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-glyl-pexiganan. The other three are structural isomers of N-glyoxylyl-desgly1-pexiganan. These isomers undergo reversible inter-conversions, as well as decompose irreversibly to des-gly1-pexiganan. Thus, all the impurities were formed from a single oxidation product of pexiganan, N-glyoxylyl-des-gly1-pexiganan. The aldehyde group of the glyoxylyl residue and the NH-amide of the adjacent isoleucine residue form a piperazinedione derivative of des-gly1-pexiganan. This heterocyclic compound rearranges to other tautomers or back to the N-glyoxylyl compound (see Fig. 3). Tryptic digests of the three degradation products showed that their N-terminal segment produced N-glyoxylyl-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 pexiganan. 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-terminal amino residues are substituted.


CONCLUSIONS
After a year under stress conditions 1% pexiganan cream lost about 15% of the active component to oxidative-deamination, where the N-terminal glycine residue was oxidized to N-glyoxylyl-desgly1-pexiganan. The other nine epsilon-amino lysine-residues of the peptide stayed intact. This oxidation product inter-converted and formed two additional impurities, tautomers of piperazinedionyl-des-gly-pexiganan, and decomposed to des-gly1-pexiganan, the forth impurity.
Feibush
B
B
Magainin Pharmaceuticals, Inc., Plymouth Meeting, Pennsylvania 19462, USA. bfeibush@hotmail.com
Snyder
B C
BC
D000287:Administration, Topical; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000494:pharmacology; D000890:Anti-Infective Agents / Q000737:chemistry* / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides; D002626:Chemistry, Pharmaceutical; D002845:Chromatography; D004800:Enzymes, Immobilized; D006023:Glycoproteins / Q000378:metabolism*; D006038:Glyoxylates / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009824:Ointments; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D004800:Enzymes, Immobilized; D006023:Glycoproteins; D006038:Glyoxylates; D009824:Ointments; D010446:Peptide Fragments; D010455:Peptides; D000626:Aminopeptidases; D014357:Trypsin; C093828:pexiganan