Abstract Body: Introduction: Carbon monoxide (CO) poisoning is the most common human poisoning and a major cause of death and disability with over 50,000 cases annually in the U.S. Currently, no antidote is approved, and treatment is limited to high-flow oxygen and sparsely available hyperbaric oxygen therapy. These approaches have limited efficacy in preventing severe outcomes like brain injury, neurocognitive deficits, and death. Our group has synthesized a cyclodextrin-based hemoprotein-mimetic small molecule designed for IV admin in CO poisonings to sequester CO rapidly from red blood cells (RBCs) and tissues.

Hypothesis: This small-molecule scavenger will meet key biophysical parameters for rapid and stable CO binding including high thermal stability, slow autooxidation, favorable CO kinetic parameters, high solubility, and rapid CO sequestration from carboxyhemoglobin (HbCO).

Methods: Using UV/Vis and stopped-flow spectroscopy combined with spectral deconvolution, we characterized the stability, diatomic binding/release kinetics, and autooxidation of our lead synthetic scavenger. We examined competitive anaerobic CO scavenging from cell-free HbCO. We performed ex vivo CO saturated RBC Hb transfer assays under aerobic atmosphere to quantify the rate and efficiency of CO transfer from native hemoglobin.

Results: Our scavenger exhibits high structural stability (T_m>75°C or 12M urea). In air the oxy-form of the compound exhibits slow autoxidation (t_1/2 = 73.8 min, 22°C). The compound readily scavenges CO from cell-free HbCO (t_1/2 = 65.4 s, 22°C). It efficiently sequesters CO from RBC HbCO under aerated conditions (t_1/2 = 32 s, 22°C) with ~76% CO scavenger saturation. Interestingly, the binding constants of NO and CO do not change with their respective concentration (k_obs = 1.12 s^-1 for both, 37°C), suggesting an intriguing structural effect where cyclodextrin is ‘gate-keeping’ the binding site for diatomics like CO, NO, and oxygen. This effect likely is responsible for the favorable physiological parameters of the compound.

Conclusion: While the capping structure of our small molecule scavenger imposes a kinetic barrier to extremely rapid CO binding, it performs well at sequestering CO from HbCO. Even at a fraction of the size, this synthetic scavenger maintains many favorable properties of a full hemoprotein equivalent including stability, solubility, and slow autoxidation, allowing for higher concentrations and represents promising antidote for in vivo testing.