Consequences of inhibition of bumetanide metabolism in rodents on brain penetration and effects of bumetanide in chronic models of epilepsy

Abstract

The diuretic bumetanide, which acts by blocking the Na-K-Cl cotransporter (NKCC), is widely used to inhibit neuronal NKCC1, particularly when NKCC1 expression is abnormally increased in brain diseases such as epilepsy. However, bumetanide poorly penetrates into the brain and, in rodents, is rapidly eliminated because of extensive oxidation of its N-butyl sidechain, reducing the translational value of rodent experiments. Inhibition of oxidation by piperonyl butoxide (PBO) has previously been reported to increase the half-life and diuretic activity of bumetanide in rats. Here we studied whether inhibition of bumetanide metabolism by PBO also increases brain levels of bumetanide in rats, and whether this alters pharmacodynamic effects in the kindling model of epilepsy. Furthermore, we studied the effects of PBO in mice. Mice eliminated bumetanide less rapidly than rats (elimination half-life 47 min vs. 13 min). Pretreatment with PBO increased the half-life in mice to average values (70 min) previously determined in humans, and markedly elevated brain levels of bumetanide. In rats, the increase in plasma and brain levels of bumetanide by PBO was less marked than in mice. PBO significantly increased the diuretic activity of bumetanide in rats and, less effectively, in mice. In epileptic mice, bumetanide (with PBO) did not suppress spontaneous seizures. In the rat kindling model, bumetanide (with or without PBO) did not exert anticonvulsant effects on fully kindled seizures, but dose-dependently altered kindling development. These data indicate that PBO offers a simple means to enhance the translational properties of rodent experiments with bumetanide, particularly when using mice.

In vivo effects of bumetanide at brain concentrations incompatible with NKCC1 inhibition on newborn DGC structure and spontaneous EEG seizures following hypoxia-induced neonatal seizures

“…As a consequence of the rapid metabolism and poor brain penetration in rodents, it was difficult to reach brain concentrations in the NKCC1-inhibiting range following systemic administration of bumetanide (Brandt et al, 2010;Cleary et al, 2013;Loscher et al, 2013;Puskarjov et al, 2014;Topfer et al, 2014). The peak brain concentrations of rats subjected with hypoxiainduced neonatal seizures in the present study only amounted to $2.17 ng/g, which was equal $6.4 nM after 3-week bumetanide treatment.…”

“…100-300 nM) (Russell, 2000;Hannaert et al, 2002). To reach such high brain concentrations, systemic bumetanide doses in the range of $10-15 mg/kg would be required (Brandt et al, 2010;Topfer et al, 2014). Thus, the presently observed antiepileptic effects of bumetanide at a low dose may be a consequence secondary to the effects mediated by targets outside the blood-brain barrier (BBB)-protected CNS or alternatively were primary consequences of CNS-located target(s) other than NKCC1.…”

The effect of organic anion transporter 3 inhibitor probenecid on bumetanide levels in the brain: an integrated in vivo microdialysis study in the rat

“…Given that physicochemical properties are likely to limit BBB permeation of bumetanide, it is pertinent to explore potential drug delivery strategies to augment bumetanide transport into the brain, with a view to fully exploring the potential clinical use of NKCC1‐blockade in the CNS. Previous studies that focussed on increasing BBB penetration have employed lipophilic prodrugs of bumetanide and inhibitors of bumetanide metabolism [34,35]. In this study, we investigate the effect of oat3 inhibition, which represents a novel potential augmentation strategy, on brain levels of bumetanide.…”

Acute and Chronic Efficacy of Bumetanide in an in vitro Model of Posttraumatic Epileptogenesis

“…The ability of bumetanide to cross the intact blood brain barrier and the relatively short half-life of diuretic activity in rodents may contribute to the lower efficacy of bumetanide in some in vivo models of chronic experimental epilepsy [52]. Clinical trials of bumetanide to date have been restricted to acute neonatal brain injuries, where the blood brain barrier is not likely to be intact [53, 54].…”