Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.

Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 µg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.

Effects of the NKCC1 inhibitors bumetanide, azosemide, and torasemide alone or in combination with phenobarbital on seizure threshold in epileptic and nonepileptic mice.

The sodium-potassium-chloride (Na-K-Cl) cotransporter NKCC1 is found in the plasma membrane of a wide variety of cell types, including neurons, glia and endothelial cells in the brain. Increased expression of neuronal NKCC1 has been implicated in several brain disorders, including neonatal seizures and epilepsy. The loop diuretic and NKCC inhibitor bumetanide has been evaluated as an antiseizure agent alone or together with approved antiseizure drugs such as phenobarbital (PB) in pre-clinical and clinical studies with varying results. The equivocal efficacy of bumetanide may be a result of its poor brain penetration. We recently reported that the loop diuretic azosemide is more potent to inhibit NKCC1 than bumetanide. In contrast to bumetanide, azosemide is not acidic, which should favor its brain penetration. Thus, azosemide may be a promising alternative to bumetanide for treatment of brain disorders such as epilepsy. In the present study, we determined the effect of azosemide and bumetanide on seizure threshold in adult epileptic mice. A structurally related non-acidic loop diuretic, torasemide, which also blocks NKCC1, was included in the experiments. The drug effects were assessed by determing the maximal electroshock seizure threshold (MEST) in epileptic vs. nonepileptic mice. Epilepsy was induced by pilocarpine, which was shown to produce long-lasting increases in NKCC1 in the hippocampus, whereas MEST did not alter NKCC1 mRNA in this region. None of the three loop diuretics increased MEST or the effect of PB on MEST in nonepileptic mice. In epileptic mice, all three diuretics significantly increased PB's seizure threshold increasing efficacy, but the effect was variable upon repeated MEST determinations and not correlated with the drugs' diuretic potency. These data may indicate that inhibition of NKCC1 by loop diuretics is not an effective means of increasing seizure threshold in adult epilepsy.

Phenobarbital and midazolam suppress neonatal seizures in a noninvasive rat model of birth asphyxia, whereas bumetanide is ineffective.

OBJECTIVE: Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in ~50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. METHODS: Intermittent asphyxia was induced for 30 minutes by exposing the rat pups to three 7 + 3-minute cycles of 9% and 5% O2 at constant 20% CO2 . Drug treatments were administered intraperitoneally either before or immediately after asphyxia. RESULTS: All untreated rat pups had seizures within 10 minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30 mg/kg but not 15 mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1 mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3 mg/kg) alone or in combination with phenobarbital (15 or 30 mg/kg) exerted no significant effect on seizure occurrence. SIGNIFICANCE: The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.

The search for brain-permeant NKCC1 inhibitors for the treatment of seizures: Pharmacokinetic-pharmacodynamic modelling of NKCC1 inhibition by azosemide, torasemide, and bumetanide in mouse brain.

Because of its potent inhibitory effect on the Na+-K+-2Cl- symporter isotype 1 (NKCC1) in brain neurons, bumetanide has been tested with varying results for treatment of seizures that potentially evolve as a consequence of abnormal NKCC1 activity. However, because of its physicochemical properties, bumetanide only poorly penetrates into the brain. We previously demonstrated that NKCC1 can be also inhibited by azosemide and torasemide, which lack the carboxyl group of bumetanide and thus should be better brain-permeable. Here we studied the brain distribution kinetics of azosemide and torasemide in comparison with bumetanide in mice and used pharmacokinetic-pharmacodynamic modelling to determine whether the drugs reach NKCC1-inhibitory brain concentrations. All three drugs hardly distributed into the brain, which seemed to be the result of probenecid-sensitive efflux transport at the blood-brain barrier. When fractions unbound in plasma and brain were determined by equilibrium dialysis, only about 6-17% of the brain drug concentration were freely available. With the systemic doses (10 mg/kg i.v.) used, free brain concentrations of bumetanide and torasemide were in the NKCC1-inhibitory concentration range, while levels of azosemide were slightly below this range. However, all three drugs exhibited free plasma levels that would be sufficient to block NKCC1 at the apical membrane of brain capillary endothelial cells. These data suggest that azosemide and torasemide are interesting alternatives to bumetanide for treatment of seizures involving abnormal NKCC1 functionality, particularly because of their longer duration of action and their lower diuretic potency, which is an advantage in patients with seizures.

Lack of antidepressant effects of burst-suppressing isoflurane anesthesia in adult male Wistar outbred rats subjected to chronic mild stress.

Post-ictal emergence of slow wave EEG (electroencephalogram) activity and burst-suppression has been associated with the therapeutic effects of the electroconvulsive therapy (ECT), indicating that mere "cerebral silence" may elicit antidepressant actions. Indeed, brief exposures to burst-suppressing anesthesia has been reported to elicit antidepressant effects in a subset of patients, and produce behavioral and molecular alterations, such as increased expression of brain-derived neurotrophic factor (BDNF), connected with antidepressant responses in rodents. Here, we have further tested the cerebral silence hypothesis by determining whether repeated exposures to isoflurane anesthesia reduce depressive-like symptoms or influence BDNF expression in male Wistar outbred rats (Crl:WI(Han)) subjected to chronic mild stress (CMS), a model which is responsive to repeated electroconvulsive shocks (ECS, a model of ECT). Stress-susceptible, stress-resilient, and unstressed rats were exposed to 5 doses of isoflurane over a 15-day time period, with administrations occurring every third day. Isoflurane dosing is known to reliably produce rapid EEG burst-suppression (4% induction, 2% maintenance; 15 min). Antidepressant and anxiolytic effects of isoflurane were assessed after the first, third, and fifth drug exposure by measuring sucrose consumption, as well as performance on the open field and the elevated plus maze tasks. Tissue samples from the medial prefrontal cortex and hippocampus were collected, and levels of BDNF (brain-derived neurotrophic factor) protein were assessed. We find that isoflurane anesthesia had no impact on the behavior of stress-resilient or anhedonic rats in selected tests; findings which were consistent-perhaps inherently related-with unchanged levels of BDNF.

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti-seizure efficacy.

Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B.

The Na+-K+-2Cl- cotransporter NKCC1 plays a role in neuronal Cl- homeostasis secretion and represents a target for brain pathologies with altered NKCC1 function. Two main variants of NKCC1 have been identified: a full-length NKCC1 transcript (NKCC1A) and a shorter splice variant (NKCC1B) that is particularly enriched in the brain. The loop diuretic bumetanide is often used to inhibit NKCC1 in brain disorders, but only poorly crosses the blood-brain barrier. We determined the sensitivity of the two human NKCC1 splice variants to bumetanide and various other chemically diverse loop diuretics, using the Xenopus oocyte heterologous expression system. Azosemide was the most potent NKCC1 inhibitor (IC50s 0.246 µM for hNKCC1A and 0.197 µM for NKCC1B), being about 4-times more potent than bumetanide. Structurally, a carboxylic group as in bumetanide was not a prerequisite for potent NKCC1 inhibition, whereas loop diuretics without a sulfonamide group were less potent. None of the drugs tested were selective for hNKCC1B vs. hNKCC1A, indicating that loop diuretics are not a useful starting point to design NKCC1B-specific compounds. Azosemide was found to exert an unexpectedly potent inhibitory effect and as a non-acidic compound, it is more likely to cross the blood-brain barrier than bumetanide.

Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.