Preclinical Pharmacokinetics and Safety of Intravenous RTD-1.

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 (A. Gupta, M. V. Madhavan, K. Sehgal, N. Nair, et al., Nat Med 26:1017-1032, 2020, https://doi.org/10.1038/s41591-020-0968-3). 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) (C. L. Wohlford-Lenane, D. K. Meyerholz, S. Perlman, H. Zhou, et al., J Virol 83:11385-11390, 2009, https://doi.org/10.1128/JVI.01363-09; J. G. Jayne, T. J. Bensman, J. B. Schaal, A. Y. J. Park, et al., Am J Respir Cell Mol Biol 58:310-319, 2018, https://doi.org/10.1165/rcmb.2016-0428OC). This investigation aimed to characterize the preclinical pharmacokinetics (PK) 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 the area under the curve extrapolated to infinity (AUC0-∞) (e.g., 8-fold increase from 5 mg/kg to 20 mg/kg in rats) suggestive of nonlinear PK. The volume of distribution at steady state (Vss) ranged between 550 and 1,461 mL/kg, indicating extensive tissue distribution, which was validated in a biodistribution study of [14C]RTD-1 in rats. Based on interspecies allometric scaling, the predicted human clearance and Vss are 6.48 L/h and 28.0 L, respectively, 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 treatment of COVID-19 or other pulmonary inflammatory diseases.

Rhesus θ-Defensin-1 Attenuates Endotoxin-induced Acute Lung Injury by Inhibiting Proinflammatory Cytokines and Neutrophil Recruitment.

Acute lung injury (ALI) is a clinical syndrome characterized by acute respiratory failure and is associated with substantial morbidity and mortality. Rhesus θ-defensin (RTD)-1 is an antimicrobial peptide with immunomodulatory activity. As airway inflammation and neutrophil recruitment and activation are hallmarks of ALI, we evaluated the therapeutic efficacy of RTD-1 in preclinical models of the disease. We investigated the effect of RTD-1 on neutrophil chemotaxis and macrophage-driven pulmonary inflammation with human peripheral neutrophils and LPS-stimulated murine alveolar macrophage (denoted MH-S) cells. Treatment and prophylactic single escalating doses were administered subcutaneously in a well-established murine model of direct endotoxin-induced ALI. We assessed lung injury by histopathology, pulmonary edema, inflammatory cell recruitment, and inflammatory cytokines/chemokines in the BAL fluid. In vitro studies demonstrated that RTD-1 suppressed CXCL8-induced neutrophil chemotaxis, TNF-mediated neutrophil-endothelial cell adhesion, and proinflammatory cytokine release in activated murine alveolar immortalized macrophages (MH-S) cells. Treatment with RTD-1 significantly inhibited in vivo LPS-induced ALI by reducing pulmonary edema and histopathological changes. Treatment was associated with dose- and time-dependent inhibition of proinflammatory cytokines (TNF, IL-1ß, and IL-6), peroxidase activity, and neutrophil recruitment into the airways. Antiinflammatory effects were demonstrated in animals receiving RTD-1 up to 12 hours after LPS challenge. Notably, subcutaneously administered RTD-1 demonstrates good peptide stability as demonstrated by the long in vivo half-life. Taken together, these studies demonstrate that RTD-1 is efficacious in an experimental model of ALI through inhibition of neutrophil chemotaxis and adhesion, and the attenuation of proinflammatory cytokines and gene expression from alveolar macrophages.

Pharmacokinetics of Tedizolid in Plasma and Sputum of Adults with Cystic Fibrosis.

Over the past decade, the prevalence of infections involving methicillin-resistant Staphylococcus aureus (MRSA) in patients with cystic fibrosis (CF) has increased significantly. Tedizolid (TZD) demonstrates excellent activity against MRSA and a favorable safety profile. The pharmacokinetics of several antibiotics have been shown to be altered in CF patients. The purpose of this study was to characterize the pharmacokinetics of tedizolid in this population. Eleven patients with CF were randomized to receive tedizolid phosphate at 200 mg orally or intravenously once daily for 3 doses with a minimum 2-day washout, followed by crossover to the remaining dosage form. Plasma and expectorated sputum were collected following the third dose of each dosage form for analysis. Population pharmacokinetic analysis was performed using the maximum likelihood expectation maximization method, and the disposition of TZD was described by a two-compartment model. The sputum concentrations exceeded the unbound plasma concentrations with an estimated mean sputum-to-unbound plasma penetration ratio of 2.88 (coefficient of variation, 50.3%). The estimated population mean ± standard deviation of total clearance, central volume of distribution, and bioavailability were 9.72 ± 1.62 liters/h, 61.6 ± 6.94 liters, and 1.04 ± 0.232, respectively. The total clearance was higher in CF patients than in healthy volunteers; however, it was similar to published data for patients with complicated skin and skin structure infections (cSSSIs). This study demonstrates that the oral bioavailability of tedizolid is excellent in patients with CF and that the plasma pharmacokinetics are similar to those reported for patients with cSSSIs.

Anti-Inflammatory Effects of RTD-1 in a Murine Model of Chronic Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB, Inflammasome Gene Expression, and Pro-IL-1ß Biosynthesis.

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-1ß) 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-1ß 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.