Severe illness caused by Coronavirus disease 2019 (COVID-19) is characterized by an overexuberant inflammatory response resulting in acute respiratory distress syndrome (ARDS) and progressive respiratory failure (1). Rhesus theta (θ) defensin-1 (RTD-1) is a macrocyclic host defense peptide exhibiting antimicrobial and immunomodulatory activities. RTD-1 treatment significantly improved survival in murine models of a severe acute respiratory syndrome (SARS-CoV-1) and endotoxin-induced acute lung injury (ALI) (2, 3). This investigation aimed to characterize the preclinical pharmacokinetics and safety of intravenous (i.v.) RTD-1. Based on the lack of adverse findings, the no-observed-adverse-effect-level (NOAEL) was established at 10 mg/kg/day in rats and 15 mg/kg/day in monkeys. Analysis of single ascending dose studies in both species revealed greater than dose-proportional increases in area under the curve (AUC 0-∞ ), (e.g. 8-fold increase from 5 mg/kg to 20 mg/kg in rats) suggestive of nonlinear PK. The volume of distribution (Vss) ranged between 550 and 1,461 mL/kg, indicating extensive tissue distribution, which was validated in a biodistribution study of [ 14 C]-RTD-1 in rats. Based on interspecies allometric scaling, the predicted human clearance and Vss are 6.48 L/h and 28.0 L for an adult (70 kg). To achieve plasma exposures associated with therapeutic efficacy established in a murine model of ALI, the estimated human equivalent dose (HED) is between 0.36 and 0.83 mg/kg/day. The excellent safety profile demonstrated in these studies, and the efficacy observed in the murine models support the clinical investigation of RTD-1 for the treatment of COVID-19 or other pulmonary inflammatory diseases.

Vicious cycles of chronic airway obstruction, lung infections with Pseudomonas aeruginosa, and neutrophil-dominated inflammation contribute to morbidity and mortality in cystic fibrosis (CF) patients. Rhesus theta defensin-1 (RTD-1) is an antimicrobial macrocyclic peptide with immunomodulatory properties. Our objective was to investigate the anti-inflammatory effect of RTD-1 in a murine model of chronic P. aeruginosa lung infection. Mice received nebulized RTD-1 daily for 6 days. Bacterial burden, leukocyte counts, and cytokine concentrations were evaluated. Microarray analysis was performed on bronchoalveolar lavage fluid (BALF) cells and lung tissue homogenates. In vitro effects of RTD-1 in THP-1 cells were assessed using quantitative reverse transcription PCR, enzyme-linked immunosorbent assays, immunoblots, confocal microscopy, enzymatic activity assays, and NF-κB-reporter assays. RTD-1 significantly reduced lung white blood cell counts on days 3 (−54.95%; p = 0.0003) and 7 (−31.71%; p = 0.0097). Microarray analysis of lung tissue homogenates and BALF cells revealed that RTD-1 significantly reduced proinflammatory gene expression, particularly inflammasome-related genes (nod-like receptor protein 3, Mediterranean fever gene, interleukin (IL)-1α, and IL-1β) relative to the control. In vitro studies demonstrated NF–κB activation was reduced two-fold (p ≤ 0.0001) by RTD-1 treatment. Immunoblots revealed that RTD-1 treatment inhibited proIL-1β biosynthesis. Additionally, RTD-1 treatment was associated with a reduction in caspase-1 activation (FC = −1.79; p = 0.0052). RTD-1 exhibited potent anti-inflammatory activity in chronically infected mice. Importantly, RTD-1 inhibits inflammasome activity, which is possibly a downstream effect of NF-κB modulation. These findings support that this immunomodulatory peptide may be a promising therapeutic for CF-associated lung disease.