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.

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.

The authors regret that the description of the synthesis of bumepamine in the above-mentioned article lacked an important aspect and is therefore incorrect.The correct synthesis of bumepamine is given below. The authors would like to apologise for any inconvenience caused. Synthesis of bumepamineBumetanide (1) (500 mg, 1.37 mmol) was dissolved in 9 mL dry N, N-dimethylformamide and N,N-diisopropylethylamine (765 μL, 4.39 mmol), followed by addition of aniline (250 μL, 2.74 mmol). The mixture was cooled on an ice-bath. COMU (707 mg, 1.65 mmol) was added in one portion and the mixture was gradually warmed to room temperature and stirred for 16 h. The reaction was quenched with saturated aqueous NaHCO 3 solution and extracted twice with ethyl acetate (EtOAc). The combined organic layers were washed with water, brine and dried over Na 2 SO 4 . The crude product was purified by column chromatography (toluene:EtOAc:Et 3 N, 3:2:0.01). The desired product (2; the phenylamide of bumetanide) (540 mg, 1.22 mmol) was obtained in 89% yield as a slightly yellow solid. 1 H NMR (400 MHz, d 4 -MeOH) δ 7.79 (d, J = 2.1 Hz, 1H), 7.68-7.71 (m, 2H), 7.50 (d, J = 2.1 Hz, 1H), 7.36-7.40 (m, 2H), 7.28-7.32 (m, 2H), 7.14-7.19 (m, 1H), 7.05-7.09 (m, 1H), 6.93-6.96 (m, 2H), 3.17 (t, J = 6.7 Hz, 2H), 1.40-1.48 (m, 2H), 1.13-1.21 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). 13 C NMR (100 MHz) d 4 -calculated for C 23 H 26 N 3 O 4 S [M+H] + 440.1644, found 440.1646.Next, the phenylamide of bumetanide (2) (250 mg, 0.57 mmol) was dissolved in 12 mL tetrahydrofuran and borane dimethylsulfide complex (90 μL, 0.95 mmol) was added at room temperature. The reaction mixture was stirred for 16 h at 70°C and cooled to room temperature. Since some starting material was still present, an additional amount of the borane dimethylsulfide complex (90 μL, 0.95 mmol) was added and the reaction was stirred for 5 h at 70°C. The reaction mixture was cooled to room temperature and then quenched with half-saturated aqueous NaHCO 3 solution. The mixture was extracted three times with EtOAc and the combined organic layers were dried over Na 2 SO 4 . The crude product was purified by column chromatography (CH 2 Cl 2 :MeOH, 50:1). The obtained oily substance was dried under vacuum for 4 hours and then dissolved in 10 mL dry diethyl ether (Et 2 O). 2M HCl in Et 2 O (135 μL, 0.27 mmol) was added and the flask left to stand for 10 minutes. The salt that was formed was filtered, washed three times with Et 2 O and the desired product (3; bumepamine) (90 mg, 0.21 mmol) was obtained as a slightly beige solid in 37% yield. 1 H NMR (400 MHz, d 4 -MeOH) δ 7.52-7.60 (m, 3H), 7.44-7.46 (m, 2H), 7.33 (d, J = 2.0 Hz, 1H), 7.68-7.30 (m, 2H), 7.04-7.08 (m, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.86-6.89 (m, 2H), 4.64 (s, 2H), 3.00 (t, J = 6.8 Hz, 2H), 1.29-1.37 (m, 2H), 1.06-1.15 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H). 13 C NMR (100 MHz) d 4 -

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.

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