Preclinical evaluation of injectable reduced hydroxocobalamin as an antidote to acute carbon monoxide poisoning

Abstract

Background: Current management of acute inhalational carbon monoxide (CO) toxicity includes hyperbaric or normobaric O2 therapy. However, efficacy has not been established. The purpose of this study was to establish therapeutic proof of concept for a novel injectable antidote consisting of the combination of hydroxocobalamin and ascorbic acid into a reduced form (B12r) as demonstrated by clinically significant increase (>500 ppm) in CO2 production, reduced carboxyhemoglobin (COHgb) half-life (COHgb t1/2), and increased cerebral O2 delivery and attenuation of CO-induced microglial damage in a preclinical rodent model of CO toxicity.

Methods: B12r-mediated conversion of CO to CO2 and COHgb t1/2 in human blood were measured by gas analysis and Raman resonance spectroscopy. Rats were exposed to either air or CO and then injected with saline or B12r. Cognitive assessment was tested in a Morris water maze. Brain oxygenation was measured with Licox. Brain histology was assessed by fluorescent antibody markers and cell counts.

Results: B12r resulted in significant CO2 production (1,170 ppm), compared with controls. COHgb t1/2 was reduced from 33 minutes (normal saline) to 17.5 (p < 0.001). In rat models, severe CO-induced brain hypoxia (PbtO2, 18 mm Hg) was followed by significant reduction in τ25 to 12 minutes for B12r rats versus 40 minutes for normal saline-treated rats (p < 0.0001). There was major attenuation of CO-induced microglial damage, although cognitive performance differences were minimal.

Conclusion: Our preclinical data suggest that the novel synergism of hydroxocobalamin with ascorbic acid has the potential to extract CO through conversion to CO2, independently of high-flow or high-pressure O2. This resulted in a clinically significant off-gassing of CO2 at levels five to eight times greater than those of controls, a clinically significant reduction in COHgb half-life, and evidence of increased brain oxygenation and amelioration of myoglial damage in rat models. Reduced hydroxocobalamin has major potential as an injectable antidote for CO toxicity.

Fig. 1

Comparative CO₂ production (ppm/min) over 30 min induced by B_12r antidote (hydroxocobalamin B₁₂ +ascorbic acid) added to whole human blood containing 50% COHgb. The shaded area is the difference between CO₂ produced from ascorbic acid alone, and that produced by B_12r. Little or no CO₂ was produced by infusion of normal saline (NS) or oxidized hydroxocobalamin.

Fig. 2

Brain oxygen tension (Pb_tO₂, mmHg) measured in 30 Sprague-Dawley rats exposed to medical air (AIR) or CO, and injected with either saline (NS) or antidote (B12r). Solid lines are fitted equations; dotted lines show estimated threshold τ₂₅ for each CO-exposure treatment.

Fig. 3

Median path efficiencies (straight line distance/observed swim path length) for rats exposed to medical air (SHAM) or CO and injected with either NS (CO-NS) or antidote (COB12) and tested in a Morris Water maze over 8 days.

Fig. 4

Immunochemistry of brain tissue of rats exposed to medical air (CONTROL) or CO with either antidote (B12r) or NS control solution. A. Mean (SD) microglial and astrocyte cell counts of GFAP and Iba-1 tagged cells; B. Relative myelination: Myelinated axons are indicated by white arrows. Note the paucity of myelin in the CO poisoned rats (center panel); C. Microglial activation. Yellow arrows highlight activated microglia based on increased Iba-1 staining density, thickened processes, and loss of arborization.

Fig. 4

Immunochemistry of brain tissue of rats exposed to medical air (CONTROL) or CO with either antidote (B12r) or NS control solution. A. Mean (SD) microglial and astrocyte cell counts of GFAP and Iba-1 tagged cells; B. Relative myelination: Myelinated axons are indicated by white arrows. Note the paucity of myelin in the CO poisoned rats (center panel); C. Microglial activation. Yellow arrows highlight activated microglia based on increased Iba-1 staining density, thickened processes, and loss of arborization.