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Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

¹ Temple University;
² Howard Florey Institute;
³ Semmelweis University

(RECEIVED January 3, 2008; ACCEPTED April 8, 2008)

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro exhibiting a half-life of approximately 100 minutes as documented by reversed-phase chromatography. Indeed, after a 30-minute incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 minutes. Nevertheless, the major early metabolite, a full single chain fragment was detectable until 90 minutes, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered 3 times at 20 mg/kg to mice infected with a sublethal dose (over LD50) of an extended spectrum βlactamase producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a three-week period did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.

Keywords: Structure/function studies; Isolation, Characterization, Chromatography; Mass Spectrometry; Active site/binding site/epitope mapping; Peptide/fragment isolation; Kinetics; Synthesis of Peptides and Proteins

⁴ E-mail: lotvos{at}comcast.net

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