Alterations in the biosynthesis of the free radicals nitric oxide (NO) and superoxide anion are generally accepted to contribute to widespread tissue injury in sepsis via their induction of microvacular ischemia and direct organ toxicity. To correct this free radical imbalance, Radikal Therapeutics (RTX) is developing a first- in-class small molecule drug (R-100), a bifunctional redox-based technology formed from the covalent linkage of 2 chemical moieties: 1) an organic nitrovasodilator that releases NO, and 2) a pyrrolidine nitroxide that acts as a trifunctional catalyst of reactive oxygen species degradation: a superoxide dismutase mimetic, a catalase mimic that detoxifies hydrogen peroxide, and a peroxynitrite decomposition catalyst. In an LD100 murine model of endotoxinemia, resuscitation by R-100 starting 1 h after lipopolysaccharide (LPS) challenge produces 100% survival, accompanied by near complete protection against end-organ injury. We hypothesize that R-100 is superior to the sum of its two component functionalities and that the covalent fusion of these two properties into a single molecular entity creates a strong commercial prospect for therapy of sepsis. RTX will test this hypothesis by carrying out endotoxemic studies to establish the dose-response, time-window, mechanism of action, and safety in mice of intraperitoneal (IP) administered R-100. Aim #1: R-100 at 3 dose levels will be compared to vehicle control 1 h post lipopolysaccharide (LPS) challenge, in order to establish the lowest dose providing optimal outcome ("LDPOO"). Tissue levels of R-100 and metabolites from heart, lung, kidney, and liver will be measured, in order to relate plasma and organ drug uptake. Aim #2: the LDPOO dose of R-100 will be compared to equimolar doses of hydroxymethylproxyl ("HMP", the nitroxide component of R-100), isososorbide mononitrate ("ISMN", a classic monofunctional NO donor), and the combination of HMP and ISMN, in order to verify that a bifunctional compound (R-100) is superior to a mixture of its component functionalities. Treatment will be initiated 1 h after LPS challenge. Aim #3: We will determine the duration of the therapeutic time window by introducing R-100 1, 2, 4, and 8 h after LPS challenge. Serum and tissue will be examined 48 h post LPS dosing in each of the above Aims for determination of morphologic and biochemical endpoints, including lipid peroxidation (F21-isoprostane, GSH:GSSG ratio), neutrophil infiltration, 3- nitrotyrosine (3-NT), nitrite/nitrate, and poly(ADP-ribose) formation, BUN, creatinine, NGAL, AST, ALT, histology injury score, and serum concentrations of TNF-1, HMGB-1, IL-6, TRP14, Trx1, LC8, I:B1, IL-12, MIP-11, CXCL9, and CXCL10. Aim #4: Over a 4 h period, we will monitor peripheral arterial blood pressure and heart rate in anesthetized endotoxinemic mice treated with vehicle control or R-100 administered 1 h after LPS challenge, in order to confirm the hemodynamic neutrality of the treatment regimen. We expect that R- 100 will be superior to HMP and ISMN, alone and in combination, and will be effective when initiated 6 h after LPS challenge, thereby verifying its utility as a potential therapeutic for clinical sepsis.
Public Health Relevance: Septic shock resulting from acute bowel perforation and infection is a major cause of mortality. At present, there is no approved therapy for this condition and prognosis is uniformly poor. We are developing a novel drug that targets the basic mechanisms of septic shock, and has proven effective in experimental models of septic inflammation. We will now test this agent in a series of investigations in order to determine the optimal dose, to confirm the mechanism of action, and to establish the window of opportunity after disease onset within which therapy may be initiated.
