GC-072: A Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens.

Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care, but in the case of septicemia left untreated it can range from 50 - 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for two weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24 or 339 LD50 challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line, oral treatment of melioidosis.

Translocation of ricin across polarized human bronchial epithelial cells.

Due to widespread availability, toxicity, and potential for use as a bioterrorism agent, ricin is classified as a category B select agent. While ricin can be internalized by a number of routes, inhalation is particularly problematic. The resulting damage leads to irreversible pulmonary edema and death. Our study describes a model system developed to investigate the effects of ricin on respiratory epithelium. Human bronchial epithelial (HBE) cells were cultured on collagen IV-coated inserts until polarized epithelial cell monolayers developed. Ricin was added to the apical or basal medium and damage to the cell monolayer was then assessed. Within a few hours after exposure, the cell monolayer was permeable to paracellular passage of the toxin. A mouse anti-ricin antibody neutralized ricin and prevented cellular damage as long as the antibody was present before the addition of toxin. These studies suggested that effective therapeutic agents or antibodies neutralizing ricin biological activity must be present at the apical surface of epithelial cells. The in vitro system developed here provides a method by which to screen potential therapeutics for protecting lung epithelial cells against ricin intoxication.

Flow cytometric analysis of micronuclei in peripheral blood reticulocytes III. An efficient method of monitoring chromosomal damage in the beagle dog.

Erythrocyte-based micronucleus tests have traditionally analyzed bone marrow because splenic filtration in most species removes micronucleated cells from peripheral blood. We have evaluated a flow cytometric method for monitoring micronucleated reticulocyte frequencies (%MN-RET) in the peripheral blood of beagle dogs treated with cyclophosphamide (CP) and have found that analysis of micronucleated reticulocytes (MN-RETs) in peripheral blood is a suitable surrogate for bone marrow analysis. The three-color flow cytometric method uses anti-CD71 labeling to identify reticulocytes and Plasmodium berghei-containing erythrocytes as a calibration standard. The spontaneous %MN-RET determined by flow cytometry was 0.31 +/- 0.09% (n = 22) for peripheral blood, compared with 0.38 +/- 0.13% (SD, n = 12) for bone marrow, and 0.27 +/- 0.08% (n = 12) for peripheral blood by microscopic scoring with acridine orange staining. The kinetics of appearance and disappearance of MN-RETs in blood were determined by collecting daily samples after iv treatment with CP. The maximum frequency occurred approximately 48 h after dosing. Frequencies of MN-RETs in peripheral blood at steady state following daily CP treatment were 55-68% of corresponding bone marrow values assessed by microscopy and 55-112% as assessed by flow cytometry. This difference is presumably due to splenic removal, which appears slightly less stringent than that previously reported for CP-treated Sprague-Dawley rats. Responses in bone marrow and peripheral blood were highly correlated and similar to or greater than those reported in mice and rats at equitoxic doses.