Characterization and Optimization of D-β-Hydroxybutyrate and Melatonin, a Treatment for Hemorrhagic Shock

Abstract

Hemorrhagic Shock is the leading cause of preventable death after trauma. A combination of 4 M D-β-hydroxybutyrate and melatonin (BHB/M) improves survival in preclinical hemorrhagic shock models. This dissertation is concerned with the characterization and optimization of this promising treatment. We conducted multiple experiments to evaluate the safety, optimize the dosing, administration and formulation, and to unravel the mechanism of action behind BHB/M. Our results indicate that intravenous infusion is the preferred route of administration, while intraosseous infusion was associated with decreased drug serum concentrations and increased mortality in a porcine hemorrhagic shock and trauma model. We also showed that lowering the melatonin concentration in combination with 4 M BHB decreased the efficacy of the treatment. Another objective was to define the maximum tolerated dose of 4 M BHB/43 mM M in porcine hemorrhagic shock with polytrauma. Our experiments indicate that administering increased volumes of 4 M BHB/43 mM M increased mortality in pigs exposed to hemorrhagic shock and injury. Death was likely a result of the interplay of hemorrhagic shock, injury, and BHB/M-induced hypernatremia and fluid translocation. Adverse effects were reversible in the surviving 2x 4 M BHB/43 mM M-treated pigs, and we concluded that this is the maximum tolerated dose of the treatment. Dimethyl sulfoxide (DMSO), used to solubilize melatonin in the original treatment solution, may exert adverse effects. We therefore developed and evaluated two novel BHB/M formulations void of DMSO. These formulations were lyophilized to increase drug stability and simplify drug preparation in the field. Treatment efficacy was retained in a rat hemorrhagic shock model, and novel formulations induced significantly lower hemolysis than the original formulation. Lastly, we conducted experiments to unravel the mechanism of action behind BHB/M in an in vitro ischemia/reperfusion model. We hypothesized that BHB/M increases survival of hemorrhagic shock and trauma by improving mitochondrial function during blood loss and resuscitation. However, treatment with BHB/M did not induce significant differences in ATP levels, mitochondrial ROS production or respiration in H9c2 cells exposed to oxygen glucose deprivation and reoxygenation, suggesting that treatment effects may not be mediated through changes in mitochondrial function or oxidative stress.