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In cystic fibrosis (CF), reduced HCOsub3/subsup-/supsecretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHsubASL/sub) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCOsub3/subsup-/supand Clsup-/suptransport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHsubASL/sub. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHsubASL/sub. This response was HCOsub3/subsup-/supdependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed twoiWNK/iisoforms in secretory cells and ionocytes, and knockdown of eitheriWNK1/ioriWNK2/iincreased CF pHsubASL/sub. WNK inhibition decreased Clsup-/supsecretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Clsup-/supsubstitution also alkalinized CF pHsubASL/sub. These data suggest that WNK kinases influence the balance between transepithelial Clsup-/supversus HCOsub3/subsup-/supsecretion. Moreover, reducing basolateral Clsup-/supentry may increase HCOsub3/subsup-/supsecretion and raise pHsubASL/sub, thereby improving CF host defenses.

A recent Phase II randomized, controlled trial of bumetanide as an adjunctive treatment for neonatal seizures showed a robust efficacy signal and no evidence of toxicity. Concerns regarding bumetanide as an adjunctive anticonvulsant are addressed here. An adequately powered multi-institutional trial is needed to accurately determine efficacy.

Neuronal excitation-inhibition (E/I) imbalances are considered an important pathophysiological mechanism in neurodevelopmental disorders. Preclinical studies on tuberous sclerosis complex (TSC), suggest that altered chloride homeostasis may impair GABAergic inhibition and thereby E/I-balance regulation. Correction of chloride homeostasis may thus constitute a treatment target to alleviate behavioral symptoms. Recently, we showed that bumetanide—a chloride-regulating agent—improved behavioral symptoms in the open-label study Bumetanide to Ameliorate Tuberous Sclerosis Complex Hyperexcitable Behaviors trial (BATSCH trial; Eudra-CT: 2016-002408-13). Here, we present resting-state EEG as secondary analysis of BATSCH to investigate associations between EEG measures sensitive to network-level changes in E/I balance and clinical response to bumetanide. EEGs of 10 participants with TSC (aged 8–21 years) were available. Spectral power, long-range temporal correlations (LRTC), and functional E/I ratio (fE/I) in the alpha-frequency band were compared before and after 91 days of treatment. Pre-treatment measures were compared against 29 typically developing children (TDC). EEG measures were correlated with the Aberrant Behavioral Checklist-Irritability subscale (ABC-I), the Social Responsiveness Scale-2 (SRS-2), and the Repetitive Behavior Scale-Revised (RBS-R). At baseline, TSC showed lower alpha-band absolute power and fE/I than TDC. Absolute power increased through bumetanide treatment, which showed a moderate, albeit non-significant, correlation with improvement in RBS-R. Interestingly, correlations between baseline EEG measures and clinical outcomes suggest that most responsiveness might be expected in children with network characteristics around the E/I balance point. In sum, E/I imbalances pointing toward an inhibition-dominated network are present in TSC. We established neurophysiological effects of bumetanide although with an inconclusive relationship with clinical improvement. Nonetheless, our results further indicate that baseline network characteristics might influence treatment response. These findings highlight the possible utility of E/I-sensitive EEG measures to accompany new treatment interventions for TSC.Clinical Trial RegistrationEU Clinical Trial Register, EudraCT 2016-002408-13 (www.clinicaltrialsregister.eu/ctr-search/trial/2016-002408-13/NL). Registered 25 July 2016.

Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (

We present the secondary-analysis of neurocognitive tests in the ‘Bumetanide in Autism Medication and Biomarker’ (BAMBI;EUDRA-CT-2014-001560-35) study, a randomized double-blind placebo-controlled (1:1) trial testing 3-months bumetanide treatment (≤ 1 mg twice-daily) in unmedicated children 7–15 years with ASD. Children with IQ ≥ 70 were analyzed for baseline deficits and treatment-effects on the intention-to-treat-population with generalized-linear-models, principal component analysis and network analysis. Ninety-two children were allocated to treatment and 83 eligible for analyses. Heterogeneous neurocognitive impairments were found that were unaffected by bumetanide treatment. Network analysis showed higher modularity after treatment (mean difference:-0.165, 95%CI:-0.317 to − 0.013,p = .034) and changes in the relative importance of response inhibition in the neurocognitive network (mean difference:-0.037, 95%CI:-0.073 to − 0.001,p = .042). This study offers perspectives to include neurocognitive tests in ASD trials.

The evident genetic, pathological and clinical heterogeneity of Alzheimer’s disease (AD) poses challenges for traditional drug development. We conducted a computational drug-repurposing screen for drugs to treat apolipoprotein E4 (APOE4)-related AD. We first established APOE genotype-dependent transcriptomic signatures of AD by analyzing publicly available human brain databases. We then queried these signatures against the Connectivity Map database, which contains transcriptomic perturbations of more than 1,300 drugs, to identify those that best reverse APOE genotype-specific AD signatures. Bumetanide was identified as a top drug for APOE4-related AD. Treatment of APOE4-knock-in mice without or with amyloid β (Aβ) accumulation using bumetanide rescued electrophysiological, pathological or cognitive deficits. Single-nucleus RNA sequencing revealed transcriptomic reversal of AD signatures in specific cell types in these mice, a finding confirmed in APOE4 induced pluripotent stem cell (iPSC)-derived neurons. In humans, bumetanide exposure was associated with a significantly lower AD prevalence in individuals over the age of 65 years in two electronic health record databases, suggesting the effectiveness of bumetanide in preventing AD. Through computational drug repurposing, Taubes et al. identified bumetanide as a potential drug for APOE4-related Alzheimer’s disease (AD). The effectiveness of bumetanide was validated in AD mouse models and via real-world health record databases.

Sevoflurane acts as a gamma-aminobutyric acid subtype A receptor agonist and can induce widespread apoptosis of immature dentate granule cells in postnatal day 21 mice. The dentate granule cells of postnatal day 21 mice undergo a developmental stage when gamma-aminobutyric acid (GABA) shifts from inducing the depolarization of neurons to causing hyperpolarization. However, it is unclear whether sevoflurane induces apoptosis of immature granule cells by facilitating the depolarization or hyperpolarization of neurons.We utilized bumetanide, an Nasup+/sup-Ksup+/sup-2Clsup-/supcotransporter isoform 1 (NKCC1) antagonist, to determine whether the NKCC1-mediated GABA depolarization of neurons plays a role in sevoflurane-induced neuroapoptosis. We also investigated whether sevoflurane exposure is related to long-term cognitive dysfunction in postnatal day 21 mice and explored the possible protective effects of bumetanide.Bumetanide attenuated the sevoflurane-induced apoptosis of dentate granule cells in postnatal day 21 mice. Exposure to sevoflurane at postnatal day 21 mice did not affect their motor ability or anxiety level, and it had no effect on spatial learning or memory functions. However, sevoflurane exposure at postnatal day 21 impaired the pattern separation ability in the contextual fear discrimination test; bumetanide mitigated this effect of sevoflurane as well.Bumetanide attenuates sevoflurane-induced apoptosis and is a promising prospect for protecting against anesthesia-induced neurotoxicity in the developing brain.

A pharmacological study was conducted to analyze the impact of inhibitors of Na+-H+ antiporters (amiloride 100 µM) and cation-chloride-cotransporters (bumetanide 200 µM) on two cultivars of the African rice species (Oryza glaberrima Steud) differing in salt resistance (TOG5307: salt-resistant and TOG5949: salt-sensitive) exposed to 75 mM NaCl during 3 days. Amiloride increased Na+ accumulation in roots and leaves to a higher extent in salt-resistant TOG5307 than in salt-sensitive TOG5949. Bumetanide reduced Cl− accumulation in both cultivars as well as K+ accumulation in TOG5307 and Na+ accumulation in TOG5949, suggesting that the cation-chloride-cotransporter in O. glaberrima does not necessarily strictly behave as a Na+:K+:2Cl− transporter. Inhibitors mainly acted on the absorption step but had low impact on root-to-shoot translocation process. The salt-resistant cultivar TOG5307 was able to efficiently regulate Na+ uptake and to cope with high concentration of accumulated toxic ions, as demonstrated by a higher cell viability index and a higher concentration of protein and photosynthetic pigments in NaCl-exposed plants comparatively to salt-sensitive TOG5949.

In heart failure (HF) patients, current European Society of Cardiology (ESC) guidelines recommend the use of three loop diuretics (furosemide, torasemide, bumetanide) in order to not only reduce HF hospitalizations but also improve symptoms and exercise capacity in patients with signs and/or symptoms of congestion. In addition, for the first time in hypertensive patients, European Society of Hypertension (ESH) guidelines recommend the use of torasemide. This review aimed to summarize the mode of action of loop diuretics, to present their pharmacokinetic characteristics, and to discuss their place in the management of arterial hypertension and heart failure, with special emphasis however on torasemide.

The main purpose of this study was to develop a simple precise, rapid and accurate UV-visible spectrophotometric method for determination of Bumetanide in spiked human urine by extracting the Bumetanide from spiked human urine using ethyl acetate after extraction it was scanned between 200-400nm by using UV detector and its absorbance maxima was found to be 222nm.The calibration curve was linear in the range of 1-17 µg/ml. .the recovery and assay studies of bumetanide were within 93-94.85% indicating that the proposed method can be estimation of bumetanide.

Objective: Recent trials have indicated positive effects of bumetanide in autism spectrum disorder (ASD). We tested efficacy of bumetanide on core symptom domains using a single center, parallel-group, participant-randomized, double-blind, placebo-controlled phase-2 superiority trial in a tertiary hospital in the Netherlands. Method: Unmedicated children aged 7 to 15 years with ASD and IQ ≥55 were block-randomized 1:1 to oral-solution bumetanide versus placebo, titrated to a maximum of 1.0 mg twice daily for 91 days (D91), followed by a 28-day wash-out period. The primary outcome was difference in Social Responsiveness Scale−2 (SRS-2) total score at D91, analyzed by modified intention-to-treat with linear mixed models. Results: A total of 92 participants (mean age 10.5 [SD 2.4] years) enrolled between June 2016 and December 2018. In all, 47 children were allocated to bumetanide and 45 to placebo. Two participants dropped out per treatment arm. After 91 days, bumetanide was not superior to placebo on the primary outcome, the SRS-2 (mean difference −3.16, 95% CI = −9.68 to 3.37, p = .338). A superior effect was found on one of the secondary outcomes, the Repetitive Behavior Scale−Revised (mean difference −4.16, 95% CI = −8.06 to −0.25, p = .0375), but not on the Sensory Profile (mean difference 5.64, 95% CI = −11.30 to 22.57, p = .508) or the Aberrant Behavior Checklist Irritability Subscale (mean difference −0.65, 95% CI = −2.83 to 1.52, p = .552). No significant wash-out effect was observed. Significant adverse effects were predominantly diuretic effects (orthostatic hypotension (17 [36%] versus 5 [11%], p = .007); hypokalemia (24 [51%] versus 0 [0%], p < .0001), the occurrence of which did not statistically influence treatment outcome. Conclusion: The trial outcome was negative in terms of no superior effect on the primary outcome. The secondary outcomes suggest efficacy on repetitive behavior symptoms for a subset of patients. Clinical trial registration information: Bumetanide in Autism Medication and Biomarker Study (BAMBI); https://www.clinicaltrialsregister.eu/; 2014-001560-35.

Mechanisms of saliva formation by wombat parotid glands were investigated in anaesthetized wombats at two levels of cholinergically-stimulated flow viz. mid-range (30–40% maximum flow) and maximum flow using ion-transport and carbonic-anhydrase inhibitors. Bumetanide (0.005–0.1 mmol l−1 carotid plasma) progressively reduced mid-range flow by 52 ± 3.4% (mean ± SEM). Concurrently, saliva [Cl] decreased, [Na] and [HCO3] increased but HCO3 excretion was unaltered. Salivary flow during high-rate cholinergic stimulation was 31 ± 1.1% of the pre-bumetanide maximum. During mid-range stimulation, SITS (0.075 mmol l−1) was without effect whereas 0.75 mmol l−1 stimulated transient increases in fluid output. The higher SITS concentration caused no alterations to flow or electrolyte concentrations during maximal stimulation. Carotid plasma [amiloride] (0.05 mmol l−1) caused immediate falls in flow rate of 20–30% followed by progressive recovery over 25 min to levels above pre-amiloride flow rates despite plasma [amiloride] increasing tenfold. Concurrently, salivary [Na] and [Cl] rose to equal plasma concentrations and [K] fell by 50% indicating blockade of acinar Na/H exchangers and luminal Na channels in the ducts. Increased salivary osmolarity caused the flow recovery. Saliva flow during maximum cholinergic stimulation was reduced by 38–46%. The depression of flow was interpreted as resulting from competition between amiloride and acetylcholine for access to the muscarinic receptors. Plasma [acetazolamide] (0.35–2.5 mmol l−1) did not alter saliva outflow during mid-range or maximum flow regimes whereas salivary [Cl] increased and [HCO3] decreased consistent with reduced anion exchange resulting from inhibition of carbonic anhydrase. Combined with bumetanide, acetazolamide (1.5 mmol l−1) reduced flow by an additional 18–22% relative to bumetanide alone thereby demonstrating that acinar HCO3 synthesis supported a limited proportion of saliva formation and that some HCO3 secretion was independent of carbonic anhydrase activity.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl−secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-inducedIsc(IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl−- or nominally HCO3−-free buffer. In the case of Cl−, the dependency was restricted to the presence of Cl−in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl−-cotransporter, only numerically reducedIPropsuggesting that a large part of this current must be carried by an ion other than Cl−. In the aboral caecum,IPropwas significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IPropsignificantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3−-cotransporters in caecum, which might underlie the observed Na+dependency. These results suggest that propionate sensing in caecum is coupled to HCO3–secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

The secondary active Na-K-Cl cotransporter 1 (NKCC1) promotes electroneutral uptake of two chloride ions, one sodium ion and one potassium ion. NKCC1 regulates Cl− homeostasis, thus being implicated in transepithelial water transport and in neuronal excitability. Aberrant NKCC1 transport is linked to a variety of human diseases. The loop diuretic drugs bumetanide, furosemide, azosemide and ethacrynic acid target NKCC1, but are characterized by poor selectivity leading to severe side effects. Despite its therapeutic importance, the molecular details of the NKCC1 inhibition mechanism remain unclear. Using all-atom simulations, we predict a putative binding mode of these drugs to the zebrafish (z) and human (h) NKCC1 orthologs. Although differing in their specific interactions with NKCC1 and/or monovalent ions, all drugs can fit within the same cavity and engage in hydrophobic interactions with M304/M382 in z/hNKCC1, a proposed ion gating residue demonstrated to be key for bumetanide binding. Consistent with experimental evidence, all drugs take advantage of the K+/Na+ ions, which plastically respond to their binding. This study not only provides atomic-level insights useful for drug discovery campaigns of more selective/potent NKCC1 inhibitors aimed to tackle diseases related to deregulated Cl− homeostasis, but it also supplies a paradigmatic example of the key importance of dynamical effects when drug binding is mediated by monovalent ions.

Background The co-administration of loop diuretics with thiazide diuretics is a therapeutic strategy in patients with hypertension and volume overload. The aim of this study was to assess the efficacy and safety of treatment with bumetanide plus chlorthalidone in patients with chronic kidney disease (CKD) stage 4–5 KDIGO. Methods A double-blind randomized study was conducted. Patients were randomized into two groups: bumetanide plus chlorthalidone group (intervention) and the bumetanide plus placebo group (control) to evaluate differences in TBW, ECW and ECW/TBW between baseline and 30 Days of follow-up. Volume overload was defined as ‘bioelectrical impedance analysis as fluid volume above the 90th percentile of a presumed healthy reference population. The study’s registration number was NCT03923933. Results Thirty-two patients with a mean age of 57.2 ± 9.34 years and a median estimated glomerular filtration rate (eGFR) of 16.7 ml/min/1.73 m2 (2.2–29) were included. There was decreased volume overload in the liters of total body water (TBW) on Day 7 (intervention: -2.5 vs. control: -0.59, p = 0.003) and Day 30 (intervention: -5.3 vs. control: -0.07, p = 0.016); and in liters of extracellular water (ECW) on Day 7 (intervention: -1.58 vs. control: -0.43, p p p = 0.073) and Day 30 (intervention: -26.1 vs. control: -10, p = 0.028) and in diastolic blood pressure on Day 7 (intervention: -8.5 vs. control: -2.25, p = 0.059) and Day 30 (intervention: -13.5 vs. control: -3.4, p = 0.018). Conclusion In CKD stage 4–5 KDIGO without renal replacement therapy, bumetanide in combination with chlorthalidone is more effective in treating volume overload and hypertension than bumetanide with placebo.

Background A range of neurological pathologies may lead to secondary hydrocephalus. Treatment has largely been limited to surgical cerebrospinal fluid (CSF) diversion, as specific and efficient pharmacological options are lacking, partly due to the elusive molecular nature of the CSF secretion apparatus and its regulatory properties in physiology and pathophysiology. Methods CSF obtained from patients with subarachnoid hemorrhage (SAH) and rats with experimentally inflicted intraventricular hemorrhage (IVH) was analyzed for lysophosphatidic acid (LPA) by alpha-LISA. We employed the in vivo rat model to determine the effect of LPA on ventricular size and brain water content, and to reveal the effect of activation and inhibition of the transient receptor potential vanilloid 4 (TRPV4) ion channel on intracranial pressure and CSF secretion rate. LPA-mediated modulation of TRPV4 was determined with electrophysiology and an ex vivo radio-isotope assay was employed to determine the effect of these modulators on choroid plexus transport. Results Elevated levels of LPA were observed in CSF obtained from patients with subarachnoid hemorrhage (SAH) and from rats with experimentally-inflicted intraventricular hemorrhage (IVH). Intraventricular administration of LPA caused elevated brain water content and ventriculomegaly in experimental rats, via its action as an agonist of the choroidal transient receptor potential vanilloid 4 (TRPV4) channel. TRPV4 was revealed as a novel regulator of ICP in experimental rats via its ability to modulate the CSF secretion rate through its direct activation of the Na+/K+/2Cl− cotransporter (NKCC1) implicated in CSF secretion. Conclusions Together, our data reveal that a serum lipid present in brain pathologies with hemorrhagic events promotes CSF hypersecretion and ensuing brain water accumulation via its direct action on TRPV4 and its downstream regulation of NKCC1. TRPV4 may therefore be a promising future pharmacological target for pathologies involving brain water accumulation.

Background Bumetanide is a selective NKCC1 chloride importer antagonist which is being repurposed as a mechanism-based treatment for neurodevelopmental disorders (NDDs). Due to their specific actions, these kinds of interventions will only be effective in particular subsets of patients. To anticipate stratified application, we recently completed three bumetanide trials each focusing on different stratification strategies with the additional objective of deriving the most optimal endpoints. Here we publish the protocol of the post-trial access combined cohort study to confirm previous effects and stratification strategies in the trial cohorts and in new participants. Method/design Participants of the three previous cohorts and a new cohort will be subjected to 6 months bumetanide treatment using multiple baseline Single Case Experimental Designs. The primary outcome is the change, relative to baseline, in a set of patient reported outcome measures focused on direct and indirect effects of sensory processing difficulties. Secondary outcome measures include the conventional questionnaires ‘social responsiveness scale’, ‘repetitive behavior scale’, ‘sensory profile’ and ‘aberrant behavior scale’. Resting-state EEG measurements will be performed at several time-points including at Tmax after the first administration. Assessment of cognitive endpoints will be conducted using the novel Emma Tool box, an in-house designed battery of computerized tests to measure neurocognitive functions in children. Discussion This study aims to replicate previously shown effects of bumetanide in NDD subpopulations, validate a recently proposed treatment prediction effect methodology and refine endpoint measurements. Trial registration EudraCT: 2020–002196-35, registered 16 November 2020, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-002196-35/NL

Bumetanide falls in the class of loop diuretics like furosemide. The effects of both have been extensively researched and are well documented. In medical practice furosemide is more widely used in patients both orally and through intravenous administration.

Benzodiazepines (BZDs) such as Zolpidem can produce a temporary revival of patients who have been akinetic and apathic for years. The mechanisms underlying this “awakening” reaction are suggested globally to be related to an activation of gamma-aminobutyric acid (GABA) inhibitory systems. However, brain trauma or cerebro-vascular infarcts, like many other pathological insults, are associated with a shift of the polarity of GABA from inhibition to excitation consequently to an increase of intracellular chloride concentration ([Cl−]i) levels. Experimental and clinical observations suggest that BZDs generate paradoxical reactions in these conditions, hence the transient “awakening”. The NKCC1 (Na-K-2Cl co-transporter isoform 1) chloride importer antagonist Bumetanide restores low [Cl−]i levels and an efficient inhibitory drive. It is therefore suggested that the administration of Bumetanide might provide a persistent “awakening” by shifting GABAergic actions from excitation to inhibition and attenuating the mechanism underlying the apathic/akinetic state.

A wide variety of genetic and non-genetic pathologies share serious psychiatric symptoms, which determine a poor quality of life for patients and their families. To evaluate whether bumetanide, a drug initially developed as a diuretic and currently analyzed for a new indication in patients with severe neuropsychiatric pathologies, could improve the disruptive and self-injurious behaviors secondary to Tuberous Sclerosis Complex (TSC) and characteristic of the autistic spectrum the case of this 6-year-old patient is considered. Following preclinical and clinical evidence of the efficacy of bumetanide in Tuberous Sclerosis and other neurodevelopmental disorders, the drug may alleviate the psychiatric manifestations (TAND) of Tuberous Sclerosis pathology. This would allow avoiding the excessive prescription of antipsychotic drugs indicated to control disruptive behaviors. Methodology: The Administracion Nacional de Medicamentos, Alimentos y Tecnologia Medica (ANMAT) approval was requested for compassionate use since it is not an approved drug in Argentina for this indication. The patient was evaluated with laboratory tests at T0, T1, T2, T3 and T4, corresponding to the basal moments, of 7 days, 30 days, 60 days and 90 days after starting the medication. Likewise, behavior was evaluated with the Aberrant Behavior Checklist (ABC) at the same times described. CARS was used for autistic characteristics and TAND Checklist for psychiatric disorders, both at the beginning. Bumetanide was administered at 1 mg/day and increased to 1.5 mg/day one month after the first dose. Results: We observed, in this case, the primary endpoint, irritability, showed moderate improvement. On the contrary, hyperactivity, attention, sociability and better connection through gaze experienced and evident improvement. Regarding isolation scales and stereotyped behaviors, an important improvement was found after 3 months of treatment with bumetanide, an antagonist NKCC1, evaluated through the Aberrant Behavior Checklist (ABC). On the other hand, no remarkable adverse effects were observed.

Objective . The role of Cl – -transport in ATP-dependent regulation of contractile activity of rat pulmonary artery (PA) smooth muscle cells was studied. Design and methods . The study was performed on endotheliumdenuded ring segments of the PA of male Wistar rats. Mechanical tension was measured using organ bath technique. Contractions of the PA segments were induced by high-potassium solution (30 mM KCl), hyposmotic solution (40 mM NaCl), as well as restoration of the medium osmolarity (120 mM NaCl) after incubation in hyposmotic solution. Inhibitor of Na + , K + , 2Cl – cotransport (NKCC) bumetanide (10 μM, 30 minutes preincubation), nonselective Cl – -channel blocker SITS (100 μM) and Ca2 + -activated Cl – channels blocker niflumic acid (NA, 10 μM) were used to modulate the Cl – -transport. Results . ATP (10–500 μM) did not affect vascular tone of PA segments incubated in Krebs solution (120 mM NaCl), while 500 and 1000 μM ATP led to the development of transient contractions, the amplitude of which decreased in the presence of bumetanide, SITS and NA. Incubation of PA segments in a hyposmotic medium (40 mM NaCl) caused the development of transient contraction. Subsequent recovery of the medium osmolarity (120 mM NaCl) induced another transient contraction — isosmotic striction. ATP (500 μM) eliminated the relaxation phase of hyposmotic striction, and completely suppressed the development of isosmotic striction. Bumetanide did not affect the action of ATP during isosmotic striction, but restored the relaxation phase of hyposmotic striction. SITS and NA eliminated the effect of ATP on hypo- and isosmotic striction. Conclusions . The constrictive effect of ATP on the smooth muscle cells of the PA is associated with the activation of mechanisms of transmembrane Cl – -redistribution, in which NKCC and Ca2 + -activated Cl – channels are involved.

There are currently no approved pharmacological treatments to improve social reciprocity and limit repetitive and rigid behaviors in autism spectrum disorder (ASD). We describe the design of two Phase III studies evaluating the efficacy/safety of bumetanide oral liquid formulation in ASD. These are international, multicenter, randomized, double-blind, placebo-controlled studies in children and adolescents with ASD aged 7 to 17 years (n = 200; study 1), or younger children with ASD aged 2 to 6 years (n = 200; study 2). The primary endpoint of each is change in Childhood Autism Rating Scale 2 total raw score after 6 months. These studies could contribute to the first pharmacological treatment to improve social reciprocity and limit repetitive and rigid behaviors in children and adolescents with ASD. Supplementary Information The online version contains supplementary material available at 10.1007/s10803-020-04709-8.

The current study evaluated the analgesic effects of bumetanide as an adjunctive treatment in managing neuropathic pain following spinal cord injury. The peripheral expression level of Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) assessed as a possible biomarker indicating central underlying mechanisms.This open-label, single-arm, pilot trial of bumetanide (2 mg/day) is an add-on treatment conducted in 14 SCI patients for 19 weeks. The whole duration consisted of three phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, nine patients completed the study. The primary outcome variables were the endpoint pain score measured by the numeric rating scale (NRS), and the short-form McGill Pain Questionnaire. Secondary endpoints included the Short-Form Health Survey that measures the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels.Bumetanide treatment significantly reduced average pain intensity according to the NRS and the short form of the McGill Pain Questionnaire scores. The baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P 0.05). Through the current study, pain improvement accompanied by the more significant mean change from the baseline for the overall quality of life.These data might be a piece of preliminary evidence for the analgesic effect of bumetanide on neuropathic pain and could support the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

N-acetyl-L-cysteine (NAC) mucolytic and antioxidant role is well known, but the effect on epithelial ion transport has not been yet described. The aim of the study was to evaluate the short-term and prolonged influence of NAC on ion transport in the epithelium. The experiment was performed on 108 fragments of rabbit tracheae. Fragments were divided into four groups: inhibited sodium (I) and chloride (II) transport, NAC with inhibited sodium (III) and NAC with inhibited chloride (IV) transport. The changes in electrophysiological parameters were measured in stationary conditions and during mechanical-chemical stimulation after immediate (15 s) and prolonged (60 min) N-acetylcysteine administration on the tissue. Each 15-second stimulation caused repeatable changes in the electric potential of the tissue. In trachea fragments with blocked chloride ion transport, significantly lower (P

Bumetanide, a sulfamyl loop diuretic, is used for the treatment of edema in association with congestive heart failure. Being a polar, anionic compound at physiologic pH, bumetanide uptake and efflux into different tissues is largely transporter-mediated. Of note, organic anion transporters (SLC22A) have been extensively studied in terms of their importance in transporting bumetanide to its primary site of action in the kidney. The contribution of one of the less-studied bumetanide transporters, monocarboxylate transporter 6 (MCT6; SLC16A5), to bumetanide pharmacokinetics (PK) and pharmacodynamics (PD) has yet to be characterized. The affinity of bumetanide for murine Mct6 was evaluated using Mct6-transfected Xenopus laevis oocytes. Furthermore, bumetanide was intravenously and orally administered to wild-type mice (Mct6(+/+)) and homozygous Mct6 knockout mice (Mct6(−/−)) to elucidate the contribution of Mct6 to bumetanide PK/PD in vivo. We demonstrated that murine Mct6 transports bumetanide at a similar affinity compared with human MCT6 (78 and 84 μM, respectively, at pH 7.4). After bumetanide administration, there were no significant differences in plasma PK. Additionally, diuresis was significantly decreased by ∼55% after intravenous bumetanide administration in Mct6(−/−) mice. Kidney cortex concentrations of bumetanide were decreased, suggesting decreased Mct6-mediated bumetanide transport to its site of action in the kidney. Overall, these results suggest that Mct6 does not play a major role in the plasma PK of bumetanide in mice; however, it significantly contributes to bumetanide’s pharmacodynamics due to changes in kidney concentrations. SIGNIFICANCE STATEMENT: Previous evidence suggested that MCT6 transports bumetanide in vitro; however, no studies to date have evaluated the in vivo contribution of this transporter. In vitro studies indicated that mouse and human MCT6 transport bumetanide with similar affinities. Using Mct6 knockout mice, we demonstrated that murine Mct6 does not play a major role in the plasma pharmacokinetics of bumetanide; however, the pharmacodynamic effect of diuresis was attenuated in the knockout mice, likely because of the decreased bumetanide concentrations in the kidney.

The article presents the consensus materials of the European Society of Cardiology on the use of diuretics in chronic heart failure (CHF). Diuretics represent an important class of drugs for the treatment of heart failure from the perspective of evidence based medicine. Much attention in the consensus s paid to the algorithm of using diuretics, in particular loop diuretics, in acute decompensation of CHF. Clinical and pharmacological advantages and disadvantages of the main drugs of loop diuretics (furosemide, torasemide, bumetanide) in CHF are discussed. There was analyzed the effectiveness of low and high doses of loop diuretics, the method of administration (bolus or continuous infusion) in terms of effective diuretic therapy and prevention of tolerance to diuretics, as well as the impact on the prognosis of survival during decompensation of heart failure. It also discusses modern approaches to the diagnostic criteria for assessing the effectiveness of diuretic therapy in acute decompensation of CHF, and shows the possibility of determination the level of excretion of Na + ions in the urine as a surrogate marker of the effectiveness of diuretic therapy to optimize the dosage regimen of diuretics. An algorithm of diuretic therapy is proposed (the choice of dosages and regimens for using loop diuretics) for patients with acute decompensation of heart failure, including intravenous use of loop diuretics in an optimal increasing dose for the first 24 hours before stopping stagnation with a further transition to a maintenance dose of loop diuretic for euvolemia status and combined diuretic therapy with thiazides to obtain synergism. The recommendations for correction of electrolyte disturbances against the background of diuretic therapy is considered. Loop diuretics are recommended for CHF to prevent signs and symptoms of congestion: this is the only group of drugs with a level of recommendation as class I in patients with heart failure with a reduced or preserved ejection fraction.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1−/− and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1−/− lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1−/− cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (−/− cells. HP recovery was shown to be absent in TRPV1−/− lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1−/− mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

The specific NKCC1 cotransporter antagonist, bumetanide, attenuates the severity of Autism Spectrum Disorders (ASD), and many neurodevelopmental or neurodegenerative disorders in animal models and clinical trials. However, the pervasive expression of NKCC1 in many cell types throughout the body is thought to challenge the therapeutic efficacy of bumetanide. However, many peripheral functions, including intestinal, metabolic, or vascular, etc., are perturbed in brain disorders contributing to the neurological sequels. Alterations of these functions also increase the incidence of the disorder suggesting complex bidirectional links with the clinical manifestations. We suggest that a more holistic view of ASD and other disorders is warranted to account for the multiple sites impacted by the original intra-uterine insult. From this perspective, large-spectrum active repositioned drugs that act centrally and peripherally might constitute a useful approach to treating these disorders.

Bumetanide, a drug being studied in autism spectrum disorder (ASD) may act to restore gamma-aminobutyric acid (GABA) function, which may be modulated by the immune system. However, the interaction between bumetanide and the immune system remains unclear. Seventy-nine children with ASD were analysed from a longitudinal sample for a 3-month treatment of bumetanide. The covariation between symptom improvements and cytokine changes was calculated and validated by sparse canonical correlation analysis. Response patterns to bumetanide were revealed by clustering analysis. Five classifiers were used to test whether including the baseline information of cytokines could improve the prediction of the response patterns using an independent test sample. An immuno-behavioural covariation was identified between symptom improvements in the Childhood Autism Rating Scale (CARS) and the cytokine changes among interferon (IFN)-γ, monokine induced by gamma interferon and IFN-α2. Using this covariation, three groups with distinct response patterns to bumetanide were detected, including the best (21.5%, n = 17; Hedge’s g of improvement in CARS = 2.16), the least (22.8%, n = 18; g = 1.02) and the medium (55.7%, n = 44; g = 1.42) responding groups. Including the cytokine levels significantly improved the prediction of the best responding group before treatment (the best area under the curve, AUC = 0.832) compared with the model without the cytokine levels (95% confidence interval of the improvement in AUC was [0.287, 0.319]). Cytokine measurements can help in identifying possible responders to bumetanide in ASD children, suggesting that immune responses may interact with the mechanism of action of bumetanide to enhance the GABA function in ASD.

Death rattle, which could often be associated with a pulmonary fluid overload, occurs in 25% to 90% of dying patients. The co-administration of scopolamine (anticholinergic drug) and bumetanide (loop diuretic) could be considered in order to avoid unnecessary fluid overload at end-stage of life. The objective of this study was to investigate the physical and chemical stabilities of the admixture bumetanide and scopolamine in order to prepare them in advance by a centralized intravenous additive service in-hospital pharmacy. The stability of the lowest (LOW) concentration was evaluated on five polypropylene syringes containing the admixture bumetanide (Burinex, 2 mg/4 mL) and scopolamine (0.25 mg/mL) at 41.67 µg/mL and 5.21 µg/mL. The highest (HIGH) concentration with 125 µg/mL of bumetanide and 31.25 µg/mL of scopolamine was evaluated on five polypropylene syringes. All syringes were stored for 18 days at 5°C ± 3°C. Periodic samples were visually and microscopically examined to observe any particle appearance or color change. The pH and absorbance at 3 wavelengths (350 nm, 410 nm, and 550 nm) were monitored. The concentrations were measured by ultra-high performance liquid chromatography-photodiode array detection, using a newly developed method. During the 18 days of test, there was no change in color or appearance of opacity, turbidity, or precipitation, and the pH remained stable. Mean concentrations of bumetanide and scopolamine at LOW and HIGH concentrations after 18 days remained statistically unchanged. The lower limits of the 95% confidence intervals of both molecules at LOW and HIGH concentrations remained higher than a 90% threshold of concentration, indicating the mixture was chemically stable. Degradation rates of bumetanide and scopolamine content at LOW and HIGH concentrations should not exceed a maximum of 0.70% every 10 days. This study was the first to show that the admixture of bumetanide and scopolamine is physically and chemically stable at two concentrations used in a palliative-care unit. This combination available in ready-to-use polypropylene syringes presents numerous advantages for patient's comfort and safety.

Volume transmission plays an essential role in CNS function, with neurotransmitters released from synapses diffusing through the extracellular space (ECS) to distant sites. Changes in the ECS volume fraction (α) will influence the diffusion and the concentration of transmitters within the ECS. We have recently shown that neuronal activity evoked by physiological photic stimuli results in rapid decreases in ECS α as large as 10% in the retina. We now characterize the cellular mechanisms responsible for this ECS shrinkage. We find that block of inwardly rectifying K

Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl− across cell membranes. NKCC1 mediates trans-epithelial Cl− secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.

In his editorial, Kevin Staley criticizes our recent work demonstrating the lack of effect of bumetanide in a novel model of neonatal seizures. The main points in our response are that (1) our work is on an asphyxia model, not one on "hypercarbia only"; (2) clinically relevant parenteral doses of bumetanide applied in vivo lead to concentrations in the brain parenchyma that are at least an order of magnitude lower than what would be sufficient to exert any direct effect-even a transient one-on neuronal functions, including neonatal seizures; and (3) moreover, bumetanide's molecular target in the brain is the Na-K-2Cl cotransporter NKCC1, which has vital functions in neurons, astrocytes, and oligodendrocytes as well as microglia. This would make it impossible even for highly brain-permeant NKCC1 blockers to specifically target depolarizing and excitatory actions of gamma-aminobutyric acid in principal neurons of the brain, which is postulated as the rationale of clinical trials on neonatal seizures.

Alzheimer’s disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation–chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl−]i by accumulating and extruding Cl−, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

Alzheimer's disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation-chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABA

BackgroundTreatment development for neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) is impeded by heterogeneity in clinical manifestation and underlying etiologies. Symptom traits such as aberrant sensory reactivity are present across NDDs and might reflect common mechanistic pathways. Here, we test the effectiveness of repurposing a drug candidate, bumetanide, on irritable behavior in a cross-disorder neurodevelopmental cohort defined by the presence of sensory reactivity problems.MethodsParticipants, aged 5–15 years and IQ ≥ 55, with ASD, ADHD, and/or epilepsy and proven aberrant sensory reactivity according to deviant Sensory Profile scores were included. Participants were randomly allocated (1:1) to bumetanide (max 1 mg twice daily) or placebo tablets for 91 days followed by a 28-day wash-out period using permuted block design and minimization. Participants, parents, healthcare providers, and outcome assessors were blinded for treatment allocation. Primary outcome was the differences in ABC-irritability at day 91. Secondary outcomes were differences in SRS-2, RBS-R, SP-NL, BRIEF parent, BRIEF teacher at D91. Differences were analyzed in a modified intention-to-treat sample with linear mixed models and side effects in the intention-to-treat population.ResultsA total of 38 participants (10.1 [SD 3.1] years) were enrolled between June 2017 and June 2019 in the Netherlands. Nineteen children were allocated to bumetanide and nineteen to placebo. Five patients discontinued (n = 3 bumetanide). Bumetanide was superior to placebo on the ABC-irritability [mean difference (MD) −4.78, 95%CI: −8.43 to −1.13, p = 0.0125]. No effects were found on secondary endpoints. No wash-out effects were found. Side effects were as expected: hypokalemia (p = 0.046) and increased diuresis (p = 0.020).ConclusionDespite the results being underpowered, this study raises important recommendations for future cross-diagnostic trial designs.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder with few medication options. Bumetanide, an FDA-approved diuretic, has been proposed as a viable candidate to treat core symptoms of ASD, however, neither the brain region related to its effect nor the cell-specific mechanism(s) is clear. The availability of nanoparticles provides a viable way to identify pharmacological mechanisms for use in ASD. Here, we found that treatment with bumetanide, in a systemic and medial prefrontal cortex (mPFC) region-specific way, attenuated social deficits in BTBR mice. Furthermore, using poly (ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles [NP(bumetanide)], we showed that the administration of NP(bumetanide) in a mPFC region-specific way also alleviated the social deficits of BTBR mice. Mechanistically, the behavioral effect of NP(bumetanide) was dependent on selective microglia-specific targeting in the mPFC. Pharmacological depletion of microglia significantly reduced the effect of nanoencapsulation and depletion of microglia alone did not improve the social deficits in BTBR mice. These findings suggest the potential therapeutic capabilities of nanotechnology for ASD, as well as the relevant link between bumetanide and immune cells.

Neonatal stroke is the second cause of acute symptomatic neonatal seizures after hypoxic-ischemic encephalopathy. The aim of this systematic review is to determine which drug among those available represents the best therapeutic choice for treatment of secondary seizures due to neonatal stroke.We performed a systematic review searching on PubMed the keywords "Neonatal", "Stroke", "Seizures" and "Treatment". Search was limited only to English language with no time limit. Last literature search was done on May 30, 2022.We selected 5 articles involving a total of 52 full-term neonates. In 96.1% the first line treatment was phenobarbital and in 3.9% was used phenobarbital associated with midazolam from the seizure onset but in all of these cases it was necessary to introduce further medications for controlling the seizures. As second line treatment was used lidocaine (response rate of 53.3%), midazolam (response rate of 15.38%) bumetanide (response rate of 100%), and fosphenytoin (no response). As third line treatment was used lidocaine (response rate of 87.5%), Midazolam (response rate of 60%), levetiracetam and clonazepam (response rate of 100%).Our review shows that the use of ASMs that act throughout a gabaergic mechanism are inadequate in controlling seizures secondary to neonatal stroke in full-term newborns. Very effective seems to be lidocaine and levetiracetam with an apparent safer profile in short and long term. Bumetanide shows promising results, but they need to be confirmed by phase 3 studies.

Children with neurodevelopmental disorders (NDDs), such as autism spectrum disorder and attention-deficit/hyperactivity disorder, vary greatly in mechanism and clinical presentation. This heterogeneity, however, is largely denied or ignored in trials and has led to a deadlock in treatment development. The aim of this dissertation was to improve this situation by developing stratified trial designs and improving clinical endpoints. We tested these innovations in the context of a novel, mechanism-based treatment option for NDDs. This treatment is based upon the repurposing of the diuretic bumetanide, a chloride importer antagonist. A large body of (pre)clinical evidence has indicated that elevated chloride levels may cause hyperexcitability in the brain, that may be treated by bumetanide in a yet unknown subset of NDDs. The studies tested behavioral, neurocognitive and genetic stratification techniques to find these responsive subsets. We further conducted a parent-driven approach to develop patient reported outcome measures (PROMs) to improve clinical endpoint measurement. Overall, we found that bumetanide can have a positive effect on behavioral manifestations, such as irritable and repetitive behaviors. Genetic stratification seems especially promising to enhance effectiveness in trial designs. The PROM study showed that the use of generic item banks may be a solution to assess consequences of sensory issues across NDDs as a powerful translational endpoint. In conclusion, these findings highlight the need for a shift from the traditional diagnosis-driven approach to a stratified and etiology-driven approach in NDD clinical trials.

The behavioral phenotypes emerge from cognitive architecture comprising attention, executive functions, and primary communication skills that all have shown remarkable deficits in Down's Syndrome (DS). These states arise from the proper functional interactions of the contributing neurotransmission and neuromodulation systems and other coding platforms. Gamma-aminobutyric acid (GABA) is an integral part of the neural interaction and regulation networks that its reverse action leads to broad detrimental consequences. This inhibitory substance needs an appropriate balance of co-transporters that largely shape the ionic milieu. Bumetanide, a specific NKCC1 inhibitor used for an eighteen-month interval, showed promising effects in restoring some behavior deficits in a fourteen-year-old boy diagnosed with genetically confirmed mosaic Down's Syndrome.

Two orthologues of the gene encoding the Na+-Cl− cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl− cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

Background: The therapeutic effect of bumetanide on autism spectrum disorder (ASD) seems to be controversial. To obtain better evidence on the efficacy of bumetanide, a systematic review and meta-analysis were performed.Methods: Randomized, placebo-controlled trials (RCTs) of bumetanide treatment in children with ASD were identified through systematic review from database inception to January 17, 2021. Subsequently, a meta-analysis was carried out to examine the effect of bumetanide on the severity of symptoms of ASD as assessed by the Childhood Autism Rating Scale (CARS) and Social Responsive Scale (SRS); core symptoms according to criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 of the American Psychiatric Association [social affect (SA), restricted, repetitive patterns of behavior, interests, or activities (RRB) and sensory symptoms]; and the therapeutic effect as assessed by Clinical Global Impressions-Efficacy (CGI-E).Results: In total, six RCTs involving 496 participants with ASD were identified in our study. The results showed that bumetanide could significantly improve the severity of the ASD symptoms measured by CARS and SRS. There was also evidence that bumetanide had positive effect on the core symptoms of ASD such as the SA and RRB, but there was no statistically significant effect on sensory symptoms. A significant positive effect on CGI-E scores in ASD patients was also observed.Conclusion: Our meta-analysis provided some support that bumetanide could improve the symptoms of children with ASD. However, additional large-scale longitudinal studies that provide clearer information and better control for confounding factors are needed to confirm our findings.

Background There is little evidence on the use of subcutaneous furosemide infusions in heart failure patients, especially in the community setting (Beattie & Johnson, 2012). Integrated working is key to smooth the transition from management in the hospital to community settings, particularly for patients who are increasingly fatigued with short prognoses but who may still benefit from parenteral therapy. Aim To retrospectively evaluate clinical effectiveness of subcutaneous furosemide used in the community setting at end-of-life. Method We reviewed case notes of patients treated with continuous subcutaneous furosemide by the community palliative care team (2019–2021). A standardised proforma was used to assess patient demographics, indications, clinical outcomes and barriers/facilitators to the process. Results 16 patients received a total 22 interventions. Median age 77(50-94), 11(69%) male, primary HF (Heart failure) diagnosis 6 HFrEF, 5 HFpEF,2 RHF, 2 Valvular, 1 unknown aetiology. Phase of Illness, 13 unstable and 9 deteriorating with AKPS 30-60%. Oral diuretics included loop diuretics (furosemide 15/22 and bumetanide 6/22), aldosterone receptor antagonists (spironolactone 4/22), mineralocorticoid receptor antagonists (eplerenone 11/22) and thiazides (indapamide 1/22). Majority of interventions had stage 3 renal function (16/22 episodes). Patients were discussed with cardiology or palliative care consultant. Median dose given was 160mg (range 80-240mg) for 11(1-33) days. Outcomes 15/22 showed symptomatic improvement, 6/22 had side-effects including worsening renal function, hypotension, hypokalaemia and site reaction needing antibiotics. 21/22 interventions avoided a hospital admission; 12/16 patients achieved preferred place of death (1 was admitted to hospital; 3 alive). Barriers to community administration included: challenges obtaining medications, burden of a continuous sub cutaneous infusion [CSCI]. Conclusion There is a clear benefit for individual patients, allowing those in last year of life to spend as much time as possible at home. We plan to develop a decision support tool to aid wider community decision making, facilitating timely use of subcutaneous furosemide, therefore maintaining effective symptom control in community settings.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl– concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl– fluorescent probe whose fluorescence intensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl– concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

New crystalline forms of bumetanide, namely, four cocrystals, two salts, and one salt-cocrystal were crystallized. Urea and lactams such as valerolactam, caprolactam, and N-methyl caprolactam forme...

Introduction Autism Spectrum Disorder (ASD) is characterized by several impairments in communications and social interactions, as well as restricted interests or stereotyped behaviors. Interventions applied for this disorder are based on multi-modal approaches, including pharmacotherapy. No definitive cure or medication has been introduced so far; therefore, researchers still investigate potential drugs for treating ASD. One of the new medications introduced for this purpose is bumetanide. The present article aimed to review the efficacy of this drug on the core symptoms of ASD and its potential side effects. Methods We searched all papers reported on pharmacokinetics, pharmacodynamics, efficacy, and adverse effects of bumetanide on animal models and humans with ASD. The papers were extracted from the main databases of PubMed, Web of Science, and Scopus. Results The findings revealed that cortical neurons have high Chloride ion (Cl-)i and excitatory actions of gamma-aminobutyric acid in the valproic acid animal model with ASD and mice with fragile X syndrome. Bumetanide, which has been introduced as a diuretic, is also a high-affinity-specific Na+-K+-Cl- cotransporter (NKCC1) antagonist that can reduce Cl- level. The results also indicate that bumetanide can attenuate behavioral features of autism in both animal and human models. Moreover, the studies showed that such medication could activate fusiform face area in individuals with ASD while viewing emotional faces. Also, recent findings suggest that a dose of 1 mg/d of this drug, taken twice daily, might be the best compromise between safety and efficacy. Conclusion Recent studies provided some evidence that bumetanide can be a novel pharmacological agent in treating core symptoms of ASD. Future studies are required to confirm the efficacy of this medication in individuals with ASD.