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.

Intracellular chloride ion concentration in differentiating neuronal cell and its role in growing neurite.

Chloride ion (Cl-) is one of the most abundant anions in our body. Increasing evidence suggests that Cl- plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl- concentrations ([Cl-]i) and the length of growing neurites. We measured [Cl-]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl-]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl-]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl-]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl-]i in neurite tip region is crucial for efficient neurite outgrowth.

Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability, and altered GABAergic transmission through Cl(-)-permeable GABAA receptors (GABAARs) contributes considerably to learning and memory deficits in DS mouse models. However, the efficacy of GABAergic transmission has never been directly assessed in DS. Here GABAAR signaling was found to be excitatory rather than inhibitory, and the reversal potential for GABAAR-driven Cl(-) currents (ECl) was shifted toward more positive potentials in the hippocampi of adult DS mice. Accordingly, hippocampal expression of the cation Cl(-) cotransporter NKCC1 was increased in both trisomic mice and individuals with DS. Notably, NKCC1 inhibition by the FDA-approved drug bumetanide restored ECl, synaptic plasticity and hippocampus-dependent memory in adult DS mice. Our findings demonstrate that GABA is excitatory in adult DS mice and identify a new therapeutic approach for the potential rescue of cognitive disabilities in individuals with DS.

Chloride cotransporter NKCC1 inhibitor bumetanide protects against white matter injury in a rodent model of periventricular leukomalacia.

BACKGROUND: Periventricular leukomalacia (PVL) is a major form of preterm brain injury. Na(+)-K(+)-Cl(-) 1 cotransporter (NKCC1) expression on neurons and astrocytes is developmentally regulated and mediates Cl(-) reversal potential. We hypothesized that NKCC1 is highly expressed on oligodendrocytes (OLs) and increases vulnerability to hypoxia-ischemia (HI) mediated white matter injury, and that the NKCC1 inhibitor bumetanide would be protective in a rodent PVL model. METHODS: Immunohistochemistry in Long-Evans rats and PLP-EGFP transgenic mice was used to establish cell-specific expression of NKCC1 in the immature rodent brain. HI was induced on postnatal day 6 (P6) in rats and the protective efficacy of bumetanide (0.3 mg/kg/i.p. q12h × 60 h) established. RESULTS: NKCC1 was expressed on OLs and subplate neurons through the first 2 postnatal weeks, peaking in white matter and the subplate between P3-7. Following HI, NKCC1 is expressed on OLs and neurons. Bumetanide treatment significantly attenuates myelin basic protein loss and neuronal degeneration 7 d post-HI. CONCLUSION: Presence and relative overexpression of NKCC1 in rodent cerebral cortex coincides with a period of developmental vulnerability to HI white matter injury in the immature prenatal brain. The protective efficacy of bumetanide in this model of preterm brain injury suggests that Cl(-) transport is a factor in PVL and that its inhibition may have clinical application in premature human infants.

Comparing vibrational spectra of free bumetanide and its solutions.

Bumetanide, which is known as a potent diuretic, is currently under investigation for its potential anti-epileptic effects in neonatal seizures. The purpose of this study was to examine the molecular structure of bumetanide both in the free form and its solutions via vibrational spectra (FT-IR, FT-Raman spectroscopies) and quantum chemical calculations. FT-IR and FT-Raman spectra of the title compound were recorded for the solid phase and the solutions of DMSO and ethanol. Optimized molecular geometry and vibrational wavenumbers of bumetanide were calculated by DFT/B3LYP functional with 6-31G(d,p), 6-31G++(d,p) and 6-311++G(d,p) basis sets. The assignment of the vibrational modes were performed based on total energy distribution (TED). The same calculations were performed for the molecule in DMSO and ethanol solutions using the polarizable conductor continuum model (CPCM) method. Lastly, probable donor-acceptor interactions of the molecule were examined with NBO analysis in different media. In all forms of bumetanide (the free molecule and the other solvents), some significant changes were observed in the dihedral angles and the vibrational frequencies.

Mechanochemical synthesis of bumetanide-4-aminobenzoic acid molecular cocrystals: a facile and green approach to drug optimization.

Molecular cocrystals are of growing interest in pharmaceutics for their improved physicochemical properties. Their mechanochemical synthesis is very promising, being easy, cheap, and "green". Here, for the first time, we report on cocrystallization of bumetanide, a diuretic and natriuretic active principle, and 4-aminobenzoic acid. The synthesis is performed both by wet and dry grinding. The cocrystal formation was investigated with a wide range of techniques, including solid-state NMR, IR, XRD, microscopy, and thermal analysis. Wet and dry grinding procedures led to different cocrystal polymorphs. In particular, the dry method gave a cocrystal by powder amorphization and subsequent crystallization. DFT calculations at the B3LYP/6-31+G(d,p) level of theory shed light on the H-bond scheme at the basis of cocrystal formation. The cocrystals showed improved solubility and dissolution rate with respect to the drug alone. This could guarantee a faster absorption and a better bioavailability of the active principle.

A novel prodrug-based strategy to increase effects of bumetanide in epilepsy.

OBJECTIVE: There is considerable interest in using bumetanide, a chloride importer Na-K-Cl cotransporter antagonist, for treatment of neurological diseases, such as epilepsy or ischemic and traumatic brain injury, that may involve deranged cellular chloride homeostasis. However, bumetanide is heavily bound to plasma proteins (~98%) and highly ionized at physiological pH, so that it only poorly penetrates into the brain, and chronic treatment with bumetanide is compromised by its potent diuretic effect. METHODS: To overcome these problems, we designed lipophilic and uncharged prodrugs of bumetanide that should penetrate the blood-brain barrier more easily than the parent drug and are converted into bumetanide in the brain. The feasibility of this strategy was evaluated in mice and rats. RESULTS: Analysis of bumetanide levels in plasma and brain showed that administration of 2 ester prodrugs of bumetanide, the pivaloyloxymethyl (BUM1) and N,N-dimethylaminoethylester (BUM5), resulted in significantly higher brain levels of bumetanide than administration of the parent drug. BUM5, but not BUM1, was less diuretic than bumetanide, so that BUM5 was further evaluated in chronic models of epilepsy in mice and rats. In the pilocarpine model in mice, BUM5, but not bumetanide, counteracted the alteration in seizure threshold during the latent period. In the kindling model in rats, BUM5 was more efficacious than bumetanide in potentiating the anticonvulsant effect of phenobarbital. INTERPRETATION: Our data demonstrate that the goal of designing bumetanide prodrugs that specifically target the brain is feasible and that such drugs may resolve the problems associated with using bumetanide for treatment of neurological disorders.

Diuretics prime plant immunity in Arabidopsis thaliana.

Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

Sensitive phosphorimetric determination of bumetanide in human urine with its inhibition effect on the formation of [Fe-morin]3+ complex.

A novel solid substrate-room temperature phosphorimetry (SS-RTP) was developed for determination of bumetanide (BMTN). It was validated by determining selectivity, linearity, accuracy, precision, and signal to noise ratio (S/N) for analysis. And all the experiments presented in this work were based on that BMTN inhibited the formation of [Fe-morin](3+) ([FeR](3+)) complex by the reaction between Fe(3+) and R, which led to severe quenching of room temperature phosphorescence (RTP) signal. The rate constant of the reaction (k) was 2.44 x 10(-4) s(-1), the activation energy (E) was 21.39 kJ mol(-1). Detection limit of this method (LD, 5.0 ag spot(-1), corresponding concentration was 1.2 x 10(-14) g mL(-1)) was evaluated and compared with other methods, indicating better sensitivity for BMTN determination using this technique. And due to the high sensitivity of the method, it has been successfully applied to determine BMTN in human urine samples. The linear range was from 0.040 pg mL(-1) to 4.0 pg mL(-1), allowing wide determined range of BMTN. Meanwhile, the mechanism of this method was also discussed.

An environmentally friendly reflectometric method for bumetanide determination in pharmaceuticals.

This paper describes a green analytical procedure for the determination of bumetanide using diffuse reflectance spectroscopy. The proposed method is based on reflectance measurements of a violet compound produced from a spot test reaction between bumetanide and p-dimethylaminocinnamaldehyde (p-DAC) in an acid medium, using filter paper as a solid support. The best conditions for the reaction have been found by experimental design methodologies. All reflectance measurements were carried out at 525 nm, and the linear range was from 1.37 x 10(-4) to 1.37 x 10(-3) mol L(-1), with a correlation coefficient of 0.998. The detection limit was estimated to be 3.98 x 10(-5) mol L(-1). Five commercial medicines containing bumetanide were analyzed by the proposed method. No interferences were observed from the common excipients present in pharmaceutical formulations. The results were favorably compared with those obtained by the United States Pharmacopoeia procedure at 95% confidence level.

Investigation of the photochemical properties and in vitro phototoxic potential of bumetanide.

The photophysical and photochemical properties of bumetanide, a sulfonamide diuretic drug, have been investigated and the photodegradation of the drug on exposure to UV-B and UV-A radiation monitored by fluorimetric and chromatographic (HPLC) methods. The main photoproducts were isolated by solid-phase extraction and liquid chromatographic procedures, and their structure elucidated by 1H-NMR and mass spectrometry. Bumetanide generates only extremely small quantities of singlet oxygen (1O2) upon irradiation in the presence of oxygen. The in vitro 3T3 N RU phototoxicity test yielded no evidence of phototoxic action.

Effect of curing conditions and plasticizer level on the release of highly lipophilic drug from coated multiparticulate drug delivery system.

The study aimed to investigate the effect of triethyl citrate (TEC) plasticizer level (10, 15, and 20%), curing temperature (40, 50, and 60 degrees C) and time (0 to 168 h) on the release of a highly lipophilic drug bumetanide from pellets coated with methacrylate ester copolymer (Eudragit RS). Bumetanide was layered onto sugar pellets followed by coating with 6% Eudragit RS with and without hydroxypropyl methyl cellulose (HPMC) seal coat using Wurster Fluid Bed equipment. Coated pellets were stored for 3 months at room temperature and the release was tested in USP purified water. At 10% TEC level, increasing curing time and temperature lead to slower drug release. At 15 and 20% TEC levels, curing initially decreased drug release followed by increase in the release at longer curing time and higher temperature. Drug release from coated pellets plasticized with 15% TEC and completely cured followed zero order kinetic models. At plasticizer level of 20%, bumetanide release from the completely cured pellets was better modeled using the Higuchi's equation reflecting possible drug migration during curing. Storage led to an increase in drug release. The use of HPMC seal coat stabilized drug release after storage. It was concluded that bumetanide migration into Eudragit RS film coat was the main cause of the accelerated release after curing and storage. The drug migration during storage at room temperature was prevented by seal coating the pellets with HPMC.

Effect of the location of hydrogen abstraction on the fragmentation of diuretics in negative electrospray ionization mass spectrometry.

The diuretic agents bumetanide, xipamide, indapamide, and related compounds were investigated in order to determine the effect of different ionization sites on their collisionally activated dissociation and the corresponding fragmentation pathways. Therefore, analytes were selectively alkylated, and structural analogues as well as deuterium labeled compounds synthesized, which contain a reduced number of ionizable hydrogen atoms. Thus, specific hydrogen abstractions and their correlated dissociation routes of the negatively charged molecules were eliminated, providing evidence for the influence of the location of ionization on product ion spectra. Fragment ions such as m/z 78 indicate ionization at the commonly present sulfamoyl residue of diuretics but does not exclude additional ionization sites. Product ion spectra of the investigated diuretic agents proved to be composed by fragmentations initiated from different hydrogen abstractions. Moreover, the generation of radical anions by collision-activated dissociation of even-electron precursor ions was observed, the generation of which is discussed by proposed fragmentation pathways.

Rat NKCC2/NKCC1 cotransporter selectivity for loop diuretic drugs.

It is generally assumed that bumetanide possesses some selectivity for the renal Na-K-Cl cotransporter NKCC2, although the results are scarce in the literature and comparisons were done with extra-renal NKCC1 at its basal, almost silent state. Here we investigated NKCC2/NKCC1 selectivity of loop diuretic drugs (bumetanide, piretanide and furosemide) as a function of the NKCC1 activated state (NKCC1 was activated by hypertonic media). NKCC2 activity was measured in isolated rat medullary thick ascending limb (mTAL) and NKCC1 in rat thymocytes and erythrocytes. When NKCC2 was compared with NKCC1at its activated state, all three diuretic drugs inhibited NKCC2 and NKCC1 with the same potency (bumetanide pIC50=6.48, 6.48 and 6.47; piretanide pIC50=5.97, 5.99 and 6.29; and furosemide pIC50=5.15, 5.04 and 5.21 for mTAL NKCC2, erythrocyte NKCC1 and thymocyte NKCC1, respectively). Basal NKCC1 exhibited a lower diuretic sensitivity, although with marked differences depending on the diuretic drug and the cell type in consideration and with the notable exception of furosemide in erythrocytes. Molecular modelling showed that bumetanide and piretanide possess four potentially active groups, of which three are shared with furosemide at similar intergroup distances. Of these three common groups, one should not bind to basal NKCC1 in thymocytes. The fourth (phenoxy) group (absent in furosemide) confers higher lipophilicity and should not bind to basal NKCC1 in erythrocytes. In conclusion, loop diuretics had no NKCC2/NKCC1 selectivity, when NKCC1 is measured at its activated state. Basal NKCC1 has a reduced diuretic sensitivity, of very different magnitude depending on the diuretic drug and cell type in consideration.

Selective inhibition of Cl(-) conductance in toad skin by blockers of Cl(-) channels and transporters.

We compared the effects exerted by two classes of Cl(-) transport inhibitors on a Cl(-)-selective, passive anion transport route across the skin of Bufo viridis, the conductance (G(Cl)) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G(Cl) with IC(50) of 6 +/- 1, 54 +/- 12, and 607 +/- 125 microM, respectively; they had no effect on the cAMP-induced G(Cl). The voltage for half-maximal activation of G(Cl) (V(50)) increased compared with controls, but effects on the maximal G(Cl) at more positive clamp potentials were small. Cl(-) channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G(Cl) with IC(50) of 8.3 +/- 1.2, 10.5 +/- 0.6, 16.5 +/- 3.4, and 36.5 +/- 11.4 microM, respectively, and also inhibited the cAMP-induced G(Cl), albeit with slightly larger IC(50). V(50) was not significantly changed compared with controls; the maximal G(Cl) was strongly reduced. We conclude that the pathway for Cl(-) is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.

Application of multiple response optimization technique to extended release formulations design.

The purpose of the present study was to apply response surface methodology and multiple response optimization utilizing superimposed contour diagrams to design extended release formulations with a preplanned release profile. Bumetanide solution was layered on sugar pellets followed by coating with various coating formulations according to a central composite statistical design using a Glatt GPCG 1 Wurster Fluid Bed. The coated pellets were tested for their release profiles and the percent of drug released after 1, 4 and 8 h were used to describe and optimize the release process. Statistical models describing the percent bumetanide released after 1, 4 and 8 h were developed and the corresponding contour diagrams were superimposed to predict the coating formulation parameters expected to produce target release profile. The pellets coated with the designed coating formulation were tested for their release profiles in comparison to the target profile. According to the model-independent approach using similarity factor (F2), the dissolution profile of the designed formulation and the expected target profile were found to be similar. It was concluded that response surface methodology and multiple response optimization could be successfully used to design and optimize extended release formulations with desired preplanned release profile.

Solubilization and stability of bumetanide in vehicles for intranasal administration, a pilot study.

The solubility of bumetanide in vehicles of various polarities, suitable for intranasal administration in acute situations, has been investigated. The solubility at 4 degrees C in glycofurol and polyethylene glycol 200 was high (167 and 143 mg/mL, respectively), decreasing exponentially with addition of phosphate buffer or coconut oil. Vehicles containing coconut oil and glycofurol did not seem to improve the solubility relative to mixtures between glycofurol and buffer. Adequate solubility (approximately 50 mg/mL) was achieved in vehicles containing about 80% cosolvent. The stability of bumetanide was studied at 5 degrees C and 57 degrees C. No degradation was observed at low temperature. At high temperature, bumetanide decomposes in nonaqueous vehicles with half-lifes ranging from 69 to 400 days, but sufficient stability may be obtained by adjustment of pH to 7.4. It may be concluded that it is possible to prepare a clinically relevant formulation for intranasal delivery of bumetanide.

Evaluation of bumetanide as a matrix for prompt fragmentation matrix-assisted laser desorption/ionization and demonstration of prompt fragmentation/post-source decay matrix-assisted laser desorption/ionization mass spectrometry.

Bumetanide was evaluated as a matrix for prompt fragmentation matrix-assisted laser desorption/ionization (MALDI) experiments on peptides. Both the MALDI mass spectrum and some physical properties of bumetanide were compared with those of some commonly used matrices. Bumetanide was then evaluated for prompt fragmentation MALDI by comparing the prompt fragmentation produced using bumetanide with that for alpha-cyano-4-hydroxycinnamic acid and 2, 5-dihydroxybenzoic acid. Bumetanide was also evaluated for use in a mixed matrix with alpha-cyano-4-hydroxycinnamic acid. A 4 : 1 matrix mixture was found to be useful for the post-source decay analysis of a prompt fragment ion. The prompt fragmentation/post-source decay analysis experiment is demonstrated on a prompt fragment ion from the oxidized b-chain of insulin.

Determination of dissociation constants of loop diuretics in acetonitrile-water mixtures.

The dissociation pK values of the representative loop diuretics furosemide, bumetanide and ethacrynic acid in 10, 30, 40, 50 and 70% (w/w) acetonitrile-water mixtures at 298.15 K were determined, according to the rules and procedures endorsed by IUPAC. The variation in pK values over the whole composition range studied can be explained by tacking into account the preferential solvation of ionizable substances in acetonitrile-water mixtures. With a view to determining the pK values of the loop diuretics studied in any of the binary solvent acetonitrile-water mixtures, correlations of pK values and different bulk properties of the solvent were examined, and the linear solvation energy relationships method, LSER, has been applied. The pK values were then correlated with the pi*, alpha and beta solvatochromic parameters of acetonitrile-water mixtures. The resulting equations allowed us to calculate pK values for the loop diuretics in any acetonitrile-water mixture up to 70% (w/w) acetonitrile.