Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9.

Abstract

Background: The caspase recruitment domain-containing protein 9 (CARD9) inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis β-glucan-induced proinflammatory signaling, suggesting its viability as a candidate for preliminary anti-Pneumocystis drug testing in the rodent Pneumocystis pneumonia (PCP) model.

Methods: Mice were injected intraperitoneally (IP) daily with either vehicle or BRD5529 at 0.1 or 1.0 mg/kg for 2 weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent™ for lung stiffness. Lungs, liver, and kidney were then harvested for hematoxylin and eosin (H&E) staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via enzyme-linked immunosorbent assay (ELISA) and extracellular matrix generation via quantitative polymerase chain reaction (qPCR). Blood collection postmortem was performed for blood chemistry analysis. Furthermore, administration of BRD5529 prior to the intratracheal inoculation of fungal β-glucans, which are known proinflammatory mediators via the Dectin-1-CARD9 pathway, resulted in significant reductions in lung tissue interleukin-6 and tumor necrosis factor-α, suggesting the exciting possibility of the use of this CARD9 inhibitor as an additional therapeutic tool in fungal infections.

Results: BRD5529 at both IP doses resulted in no significant changes in daily or final weight gain, and analysis of lung stiffness by flexiVent™ showed no significant differences between the groups. Furthermore, ELISA results of proinflammatory cytokines showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts were statistically similar. Examination and pathology scoring of H&E slides from lung, liver, and kidney in all groups, as well as subsequent pathology scoring, showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns.

Conclusions: In our initial general safety and toxicology assessments, BRD5529 displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.