Hybrid Katp Channel Opener and Redox Catalyst for Lung Transplantation

DESCRIPTION (provided by applicant): Radikal Therapeutics (RTX) is developing a novel cytoprotective agent (R-801) for the prevention of ischemia-reperfusion injury (IRI) following lung transplantation (LTX). In rodent models of severe redox injury, R-801profoundly reduces organ dysfunction, tissue infarction, and parenchymal inflammation. R- 801 is formed from the covalent fusion of 2 distinct moieties, each with demonstrated tissue protective properties: 1) a mito-K+-ATP channel activating moiety derived from pinacidil, and 2) a pyrrolidine nitroxide domain that acts as superoxide dismutase and catalase mimetics and a peroxynitrite decomposition catalyst. In a murine model of lethal Cl2 inhalational lung injury, R-801 given 2 h after Cl2 exposure blockedall histologic damage (plt0.01), reduced the elevation in nuclear NF-kB by 76% (plt0.0001), and restored the level of IkB to supranormal (plt10-7). Aim #1: Establish the superiority of R- 801 relative to its component functional domains in a rat model ofwarm-ischemic lung IRI. Left lungs of Sprague Dawley (SD) rats are rendered ischemic in situ for 60 min and reperfused for 4 h. Prior to ischemia, rats are treated with IV R-801 (0, 3, 10, 30 mg/kg), hydroxymethylproxyl (HMP; 30 mg/kg), pinacidil (30 mg/kg), or a combination of HMP and pinacidil (each 30 mg/kg). A sham rat undergoes thoracotomy but neither ischemia nor drug therapy. R-801 is expected to exhibit superior efficacy, relative to treatment with pinacidil, HMP, and their combination, with respect to tissue damage and inflammation. Tissue damage is assessed by examining histologic score, PMN infiltration, lipid peroxidation, protein nitrosation, PARP-1 activation, nuclear NF-kB, apoptosis, and oxygenation (PaO2). Inflammation is assessed by examining BAL for protein, PMNs, TNF- , and MIP-1 . Aim #2: Establish the efficacy of R- 801 in a syngeneic rat model of orthotopic LTX. SD donor rats are treated with R-801 or vehicle control 10 min before lung removal. After flushing with cold Perfadex spiked with R-801 or vehicle, donor lungs are stored cold for 12h before left LTX. Immediately following LTX with left donor lungs, recipients will receive R-801. Recipient rats will be evaluated for a) wet/dy weight ratio (W/D) (a measure of pulmonary edema),b) oxygenation by the graft, c) graft pulmonary vascular resistance, d) dynamic compliance, and e) lung tissue analysis for F2 -isoprostane (a measure of lipid peroxidation), histology, and immunohistochemical reactivity to 3-nitrotyrosine (3-NT) and poly(ADP-ribose). Specific analyses will be carried out at 3 time points: at 1 h post reperfusion for IkB , nuclear p50, and phosphorylation of mitogen activated protein kinases (MAPKs - ERK, p38, JNK); at 3h post reperfusion for RT-PCR to quantify lung tissuemRNA concentrations of TNF- , MIP-1 , and Bcl-2; and at 6 h post reperfusion for determination of BALF cellularity, protein concentration, TNF- , MIP-1 , IL-6, and IL1- . R-801 therapy is expected to translate into decreased primary graft dysfunction and mortality after lung transplant. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Ischemia-reperfusion injury is a major medical complication following lung transplantation and contributes to the high mortality in this population. At present there are no approved prophylactic measures. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in a clinically-relevant animal model.