RLS-0071 inhibits acute lung injury in a novel 2-hit model

Rationale: Acute lung injury (ALI) is often a complication of viral infection (SARS-CoV-2, Influenza, RSV) that leads to significant morbidity and mortality. A novel rat two-hit ALI model was developed that uses a complement stimulus for the second hit, leading to a robust immune-mediated ALI, characterized by leukopenia, neutrophil infiltration of the lung tissue as well as neutrophil extracellular trap (NETs) formation and cytokine storm. ReAlta's lead compound, RLS-0071, is a PEGylated peptide with dual acting anti-inflammatory activity that inhibits classical and lectin complement activation, as well as neutrophil-mediated NETosis in vitro. The efficacy of RLS-0071 on ALI, complement activation, NET formation and inflammatory cytokine release was assessed in this preclinical animal model. Methods: Wistar rats were infused intravenously with lipopolysaccharide (LPS) (1st hit), followed by incompatible red blood cells (RBC) 30 min later (2-hit). At 4 hours, blood was collected to assess C5a levels, free DNA (NET biomarker) and cytokine levels. Lung tissue was recovered for histological analysis. RLS-0071 administration prior to RBC transfusion (prophylaxis dosing) and at fixed times (30 seconds to 180 minutes) after 2-hit (rescue dosing) was examined to assess the impact of the compound on lung histology, C5a, free DNA as well as inflammatory cytokine levels. Results: Scoring of hematoxylin and eosin (H&E) lung sections showed a significant reduction in lung injury with single prophylaxis dose of RLS-0071 given 30 minutes before the second hit preserving lung histology in the animals. Analysis of the sections in which the images were converted to black and white pixels quantified by ImageJ (NIH) were used to derive a lung injury score (0-5) as shown below. RLS-0071 delivered as prophylaxis or rescue significantly reduced neutrophil infiltration, C5a levels, free DNA levels in the blood and pro inflammatory cytokines IL-1b, IL-6, TNFα and IL-17. Rescue dosing demonstrated that RLS-0071 could significantly prevent lung damage when delivered up to 180 minutes after the 2-hit. Conclusion: Animals receiving RLS-0071 showed lung tissue architecture comparable to controls. RLS-0071 demonstrated the ability to prevent downstream C5a proinflammatory signaling via complement modulation consistent with the observed reduction of pro-inflammatory cytokines production such as IL-1, IL-6, and TNFα. RLS-0071 further modulates the innate inflammatory response by inhibiting NETosis displaying synergistic mechanism of actions in this rat model of ALI.