Novispirin G10-Induced Lung Toxicity in aKlebsiella pneumoniaeInfectionModel

Abstract

Mammalian cathelicidins are a class of innate antimicrobial peptides isolated from leukocytes and epithelial cells that aid host defense against bacterial infections. Synthetic analogs of cathelicidins offer the promise of potent broad-spectrum antimicrobial efficacy. We developed a combined lung infection and ex vivo whole-blood assay model to characterize the toxicity and efficacy of synthetic cathelicidin-derived peptides. Male C57BL/6 mice were administered saline or Klebsiella pneumoniae by intratracheal instillation. Five hours later, the Klebsiella-infected mice were instilled with saline, tobramycin (1 mg/kg of body weight or 10 mg/kg), novispirin G10 (0.4 mg/kg), or a combination of tobramycin (1 mg/kg) and G10 (0.4 mg/kg). At 24 h, bronchoalveolar lavage fluid (BAL) was collected for analysis of culturable bacteria and for markers of inflammation and lung toxicity. Blood samples were analyzed for circulating cytokines. Recovery of Klebsiella from the lung, recruitment of neutrophils, and production of interleukin-6 (IL-6) in BAL samples were highly correlated (r ‫؍‬ 0.68 and 0.84, respectively; P < 0.01). Animals treated with G10 or G10 plus tobramycin had increased hemoglobin (P < 0.001) and protein (P < 0.001) levels compared to those for Klebsiella-infected or tobramycin-alonetreated animals. The levels of circulating IL-6 in mice infected with Klebsiella were 1000-to 10,000-fold higher than in the noninfected controls. The highest levels of IL-6 were measured in mice given G10 alone or in combination with tobramycin. These studies demonstrated that G10 was relatively nontoxic in saline-treated mice but was highly toxic in mice infected with Klebsiella. This finding establishes the importance of investigating candidate antimicrobial agents in an in vivo infection model.The search for new and effective antimicrobial agents to treat bacterial or fungal infections has focused in recent years on components of the innate defense systems of plants and animals. Peptides isolated and purified from phagocytic cells have been especially promising as agents against multidrugresistant bacteria and special-niche bacteria associated with chronic conditions such as cystic fibrosis (6,7,10,14,17,27). A class of antimicrobial peptides that has been particularly well studied is the mammalian-derived cathelicidins. These peptides share a highly conserved N-terminal domain identical to a cathelin protein (hence the class name) but are structurally diverse at the C terminus, which also determines the antimicrobial activity of the peptide (14,27,28,29). Lipid A binding and endotoxin neutralization are also determined by the C terminus (2,5,11,12,15,18,29).The members of one class of promising antimicrobial peptides resemble the 18 amino acids of the N terminus of SMAP-29, derived from sheep neutrophils (16,19). While showing a broad spectrum of antimicrobial activity and low toxicity in ovine models, the parent compound, SMAP-29, exhibits unacceptably high hemolytic and cytotoxic activity in human cells (1,16). A series ...

Effects of Intratracheal Administration of Novispirin G10 on a Rat Model of Mucoid Pseudomonas aeruginosa Lung Infection

“…Novispirin G10, the product of one of these searches, has been demonstrated to rapidly kill both grampositive and gram-negative bacteria, including mucoid strains (19,28). In the present study, novispirin G10 effectively killed our mucoid and nonmucoid strains of the gram-negative bacterium P. aeruginosa in vitro, in accordance with the literature (4,19,28,34). Our animal studies showed that two intratracheal administrations of novispirin G10 given within 3 h post intratracheal bacterial challenge dramatically decreased the bacterial load in the lungs during the early time points (days 3 and 5) relative to the placebo control group.…”

“…Ovispirin 1 and novispirin G10 are octadecapeptide derivatives of sheep myeloid antimicrobial peptide 29 that possess substantial activity against gram-positive and gram-negative bacteria (19,28). Although novispirin G10 differs from ovispirin 1 only by containing an isoleucine-to-glycine substitution at residue position 10, this modification leads to significantly lower in vitro cytotoxicity (4,28). It has been demonstrated that novispirin G10 can effectively kill P. aeruginosa in burn wounds (28).…”

Pegylation of Antimicrobial Peptides Maintains the Active Peptide Conformation, Model Membrane Interactions, and Antimicrobial Activity while Improving Lung Tissue Biocompatibility following Airway Delivery

“…Dosing solutions also contained a nominal dose of ϳ0.2 Ci [ 14 C] mannitol and 200 g 4-kDa fluorescein isothiocyanate (FITC) dextran (FD4000) serving as transport probes to monitor pulmonary membrane integrity. Dosing solutions were administered into the airways of the IPRL as a coarse spray through a single actuation of the pressurized metered dose inhaler, delivering 5.5 cm 3 propellant vapor. Serial aliquots were sampled from the recirculating perfusate at predetermined times and centrifuged (13,000 ϫ g, 10 min, 4°C) to remove trace blood cells, and the supernatant was stored at Ϫ20°C until assay.…”

“…In the few in vivo experimental studies that have examined lung antimicrobial efficacy of airway-administered AMPs (3,6,11,23,47), the effectiveness in clearing lung infection has been variable. While such mixed success likely reflects multifactorial differences in the modes of action of the various AMPs, the microbial burden, and the character of the lung infection model itself, another critical determinant about which little is known is the pulmonary disposition of AMPs following local delivery.…”

Design of Host Defence Peptides for Antimicrobial and Immunity Enhancing Activities

“…However subtle toxicities have never been examined although there is a feeling in the field that cationic peptides are toxic when administered systemically. For example it was demonstrated with the cathelicidin variant novispirin G10 that it demonstrated lung toxicity (as revealed by IL-6 induction), in the context of a Klebsiella infection but not in uninfected mice [111]. One approach to overcoming toxicity that has been demonstrated with indolicidin is to formulate it in liposomes which permits it to be used in systemic protection [112].…”