Novel strategies to enhance bumetanide concentrations in the brain in the treatment of neonatal seizures

Seizures are a prevalent neurodevelopmental disorder that affect up to 5/1000 newborns. Seizures result in detrimental developmental outcomes for many neonates, including mortality, disability and epilepsy. Current antiepileptic treatments, such as phenobarbital, display poor efficacy rates. Gamma-aminobutyric acid (GABA)-mediated signalling may promote excitatory neurotransmission in neonates with a birth injury due to intracellular accumulation of chloride. Bumetanide is a potential adjunct treatment for neonatal seizures, which reduces intracellular chloride concentrations by inhibiting the sodium-potassium-chloride cotransporter NKCC1. This accelerates the excitatory to inhibitory switch in GABA neurotransmission. In vitro bidirectional permeability assays were used to determine the apparent permeability of bumetanide and the potential for bumetanide efflux by organic anion transporter 3 (OAT3). Bumetanide was found to be a transported efflux substrate of human OAT3, thus OAT3 inhibition is a potential therapeutic augmentation strategy. OAT3 inhibition was investigated in vivo using an integrated intracerebral microdialysis model for the potential to augment bumetanide concentrations in the brain. Bumetanide was detected in brain extracellular fluid and this concentration increased after probenecid administration. An in vivo model of hypoxic-ischaemic neonatal seizures was established to quantify the pharmacodynamic effect of bumetanide in seizures. OAT3 inhibition enhanced the concentration of bumetanide in the brain in this model. Seizure burden in these animals was reduced significantly by phenobarbital and bumetanide and decreased further when an OAT3 inhibitor was administered. Physiologically-based pharmacokinetic modelling was employed to predict plasma and brain concentrations of bumetanide in a virtual neonatal population. The simulated brain tissue concentrations of bumetanide in neonates were below the half-maximal inhibitory concentration for the target transporter NKCC1. However, large interindividual variability and a paucity of physiological limited the accuracy of these simulations. In summary, augmentation strategies that focus on preventing bumetanide efflux from the brain via OAT3 may contribute to an improved outcome for neonates with seizures