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INTRODUCTION: Bumetanide, a loop diuretic, was identified as a candidate drug for repurposing for Alzheimers disease (AD) based on its effects on transcriptomic apolipoprotein E signatures. Cross-sectional analyses of electronic health records suggest that bumetanide is associated with decreased prevalence of AD; however, temporality between bumetanide exposure and AD development has not been established. METHODS: We evaluated Medicare claims data using Cox proportional hazards regression to evaluate the association between time-dependent use of bumetanide and time to first AD diagnosis while controlling for patient characteristics. Multiple sensitivity analyses were conducted to test the robustness of the findings. RESULTS: We sampled 833,561 Medicare beneficiaries, 60.8% female, with mean (standard deviation) age of 70.4 (12). Bumetanide use was not significantly associated with AD risk (hazard ratio 1.05; 95% confidence interval, 0.99-1.10). DISCUSSION: Using a nationwide dataset and a retrospective cohort study design, we were not able to identify a time-dependent effect of bumetanide lowering AD risk. HIGHLIGHTS: Bumetanide was identified as a candidate for repurposing for Alzheimers disease (AD). We evaluated the association between bumetanide use and risk of AD. We used Medicare data and accounted for duration of bumetanide use. Bumetanide use was not significantly associated with risk of AD.

BackgroundExaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice.MethodsWe used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos.ResultsWe demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons.ConclusionsThis further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Purpose: This scoping review aims to map the effects of dosage levels, dosage intervals, duration of exposure, and serum concentration levels of gentamicin, amikacin, vancomycin, furosemide, and bumetanide on newborn hearing. Method: Using PubMed, Scopus, and Ovid databases (January 2010-2022), a scoping review was conducted to identify studies on ototoxic drug exposure in neonates. The review included articles that described details on ototoxic drug exposure and hearing status, dosage levels, duration of exposure, and serum concentration levels. The search results were summarized using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews. Results: Out of 4,395 entries, 28 were selected for inclusion in the scoping review. The studies were separated according to the exposed drugs: gentamicin, amikacin, vancomycin, furosemide, bumetanide, and a combination of drugs. Four out of five studies on amikacin exposure revealed an increased association with ototoxicity and abnormal trough levels. Six of seven studies on gentamicin exposure reported elevated trough concentration levels in a small number of infants, but no studies reported hearing loss. Two out of four studies on vancomycin exposure reported a dose-dependent risk for infants to develop hearing loss. Conclusions: Gentamicin exposure in neonates has been extensively studied and considered relatively safe, except in cases of elevated peak or trough concentration levels. Amikacin exposure was reported to be more ototoxic, as the elevation of trough concentration levels was associated with refer results in hearing. Loop-diuretic exposure demonstrated a significant ototoxic effect. When used with other ototoxic medications, vancomycin is said to have a greater effect on ototoxicity. [ABSTRACT FROM AUTHOR]

Background: Cardiac surgery associated acute kidney injury (CS-AKI) is common. Urine response to loop diuretic and urine neutrophil gelatinase associated lipocalin (uNGAL) are separately associated with CS-AKI. We aimed to determine whether urine response to loop diuretic and uNGAL together were associated with postoperative day 2–4 CS-AKI. Methods: Two-center prospective observational study (ages 0–18 years). uNGAL (8–12 h after admission) (ng/mL) and urine response to loop diuretic (6 h for bolus furosemide and 12 h for infusion bumetanide) (mL/kg/hr) were measured. All diuretic doses were converted to furosemide equivalents. The primary outcome was day 2–4 CS-AKI. Patients were sub-phenotyped using a priori cutoffs (uNGAL + ≥ 100 ng/mL and UOP + < 1.5 mL/kg/hr) and optimal cutoffs (uNGAL + ≥ 127 ng/mL and UOP + ≤ 0.79 mL/kg/hr): 1) uNGAL–/UOP–, 2) uNGAL–/UOP + , 3) uNGAL + /UOP–, and 4) uNGAL + /UOP +. Multivariable regression was used to assess the association of uNGAL, UOP and each sub-phenotype with outcomes. Results: 476 patients were included. CS-AKI occurred in 52 (10.9%). uNGAL was associated with 2.59-fold greater odds (95%CI: 1.52–4.41) of CS-AKI. UOP was not associated with CS-AKI. Compared with uNGAL + alone, uNGAL + /UOP + improved prediction of CS-AKI using a priori and optimal cutoffs respectively (AUC 0.70 vs. 0.75). Both uNGAL + /UOP + (IQR OR:4.63, 95%CI: 1.74–12.32) and uNGAL + /UOP– (IQR OR:5.94, 95%CI: 2.09–16.84) were associated with CS-AKI when compared with uNGAL–/UOP–. Conclusions: uNGAL is associated with CS-AKI. The sub-phenotype association was largely driven by uNGAL. Future studies standardizing diuretic dose and timing may be needed to refine the combined performance for clinical decision making. [ABSTRACT FROM AUTHOR]

The early onset epilepsies encompass a heterogeneous group of disorders, some of which result in drug‐resistant seizures, developmental delay, psychiatric comorbidities, and sudden death. Advancement in the widespread use of targeted gene panels as well as genome and exome sequencing has facilitated the identification of different causative genes in a subset of these patients. The ability to recognize the genetic basis of early onset epilepsies continues to improve, with de novo coding variants accounting for most of the genetic etiologies identified. Although current disease‐specific and disease‐modifying therapies remain limited, novel precision medicine approaches, such as small molecules, cell therapy, and other forms of genetic therapies for early onset epilepsies, have created excitement among researchers, clinicians, and caregivers. Here, we summarize the main findings of presentations and discussions on novel therapeutic strategies for targeted treatment of early onset epilepsies that occurred during the Workshop on Neurobiology of Epilepsy (WONOEP XVI, Talloires, France, July 2022). The presentations discussed the use of chloride transporter inhibitors for neonatal seizures, targeting orexinergic signaling for childhood absence epilepsy, targeting energy metabolism in Dravet syndrome, and the role of cannabinoid receptor type 2, reversible acetylcholinesterase inhibitors, cell therapies, and RNA‐based therapies in early life epilepsies. [ABSTRACT FROM AUTHOR]

Objective: Following hypoxic–ischemic (HI) brain injury, neuronal cytoplasmic chloride concentration ([Cl−]i) increases, potentially contributing to depolarizing γ‐aminobutyric acid (GABA) responses, onset of seizures, and the failure of antiepileptic drugs that target inhibitory chloride‐permeable GABAA receptors. Post‐HI seizures characteristically begin hours after injury, by which time substantial accumulation of [Cl−]i may have already occurred. In immature neurons, a major pathway for Cl− influx is the reversible Na+‐K+‐2Cl− cotransporter NKCC1. Methods: Spontaneous neuronal network, neuronal [Cl−]i, and GABA activity were determined in hippocampal preparations from neonatal Clomeleon and SuperClomeleon/DLX‐cre mice to test whether blocking NKCC1 earlier after oxygen–glucose deprivation (OGD) injury would more effectively ameliorate the increase in [Cl−]i, ictallike epileptiform discharges (ILDs), and the failure of the GABAergic anticonvulsant phenobarbital. Results: In vitro, murine intact hippocampi were free of ILDs for 12 h after preparation. Transient OGD resulted in a gradual increase in [Cl−]i, depolarizing action of GABA, and facilitation of neuronal network activity. Spontaneous ILDs began 3–5 h after injury. Blocking NKCC1 with 2–10 μmol·L−1 bumetanide reduced [Cl−]i equally well when applied up to 10 h after injury. Whereas phenobarbital or bumetanide applied separately were less effective when applied later after injury, ILDs were successfully suppressed by the combination of phenobarbital and bumetanide regardless of the number of prior ILDs or delay in application. Significance: The present age‐specific group studies demonstrate that after OGD, NKCC1 transport activity significantly contributes to progressive [Cl−]i accumulation, depolarizing action of GABA, and delayed onset of ILDs. In this neonatal model of neuronal injury and ILDs, earlier treatment with bumetanide alone more efficiently recovered control baseline [Cl−]i and depressed epileptiform discharges. However, there was no time dependency to the anti‐ictal efficacy of the combination of phenobarbital and bumetanide. These in vitro results suggest that after perinatal injury, early pre‐emptive treatment with phenobarbital plus bumetanide would be as efficacious as late treatment after seizures are manifest. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) results in acute damage and triggers secondary injury responses with sustained neuronal loss and dysfunction. However, the underlying mechanisms for these delayed neuronal pathologies are not entirely understood. SCI results in the swelling of spinal neurons, but the contribution of cell swelling to neuronal loss and functional deficits after SCI has not been systematically characterized. In this study, we devised a three-dimensional image analysis pipeline to evaluate spinal neurons, examining their types, quantities, volumes, and spatial distribution in a double-lateral hemisection SCI mouse model. We found that both excitatory and inhibitory neurons swell and are lost, albeit with distinct temporal patterns. Inhibitory neurons demonstrated marked swelling and decline in number on day 2 after SCI, which resolved by day 14. In contrast, excitatory neurons maintained persistent swelling and continued cell loss for at least 35 days after SCI in mice. Excitatory neurons exhibited sustained expression of the Na+-K+-Cl− cotransporter 1 (NKCC1), whereas inhibitory neurons down-regulated the protein by day 14 after SCI. Treatment with a Food and Drug Administration–approved NKCC1 inhibitor, bumetanide, mitigated swelling of excitatory neurons and reduced their loss in the secondary injury phase after SCI. The administration of bumetanide after SCI in mouse improved locomotor recovery, with functional benefits persisting for at least 4 weeks after treatment cessation. This study advances our understanding of SCI-related pathology and introduces bumetanide as a potential treatment to mitigate sustained neuronal swelling and enhance recovery after SCI. Editor's summary: Spinal neuron swelling occurs after spinal cord injury (SCI), but the mechanisms, dynamics, and relationship of neural swelling to long-term locomotor deficits remain unclear. Here, Li et al. visualized neuronal swelling after SCI in mice using whole-mount tissue and a three-dimensional image analysis pipeline. They found that both excitatory and inhibitory neurons undergo swelling and loss after SCI, but with differing kinetics. Targeting overall tissue edema did not reduce neuronal swelling; however, inhibition of the Na+-K+-Cl− cotransporter 1 (NKCC1) with the FDA-approved drug bumetanide reduced excitatory neuron swelling. Bumetanide given in the first 4 weeks after injury improved recovery of motor function in the presence of excitatory neurons. These findings in mice suggest that excitatory neuronal swelling may be a therapeutic target for reducing functional deficits after SCI. —Molly Ogle [ABSTRACT FROM AUTHOR]

Aim: We aimed to explore whether the combination of CLP290 and bumetanide maximally improves neuropathic pain following spinal cord injury (SCI) and its possible molecular mechanism. Methods: Rats were randomly divided into five groups: Sham, SCI + vehicle, SCI + CLP290, SCI + bumetanide, and SCI + combination (CLP290 + bumetanide). Drug administration commenced on the 7th day post‐injury (7 dpi) and continued for 14 days. All rats underwent behavioral assessments for 56 days to comprehensively evaluate the effects of interventions on mechanical pain, thermal pain, cold pain, motor function, and other relevant parameters. Electrophysiological assessments, immunoblotting, and immunofluorescence detection were performed at different timepoints post‐injury, with a specific focus on the expression and changes of KCC2 and NKCC1 proteins in the lumbar enlargement of the spinal cord. Results: CLP290 and bumetanide alleviated SCI‐associated hypersensitivity and locomotor function, with the combination providing enhanced recovery. The combined treatment group exhibited the most significant improvement in restoring Rate‐Dependent Depression (RDD) levels. In the combined treatment group and the two individual drug administration groups, the upregulation of potassium chloride cotransporter 2 (K+‐Cl−cotransporter 2, KCC2) expression and downregulation of sodium potassium chloride cotransporter 1 (Na+‐K+‐Cl−cotransporter 1, NKCC1) expression in the lumbar enlargement area resulted in a significant increase in the KCC2/NKCC1 ratio compared to the SCI + vehicle group, with the most pronounced improvement seen in the combined treatment group. Compared to the SCI + vehicle group, the SCI + bumetanide group showed no significant paw withdrawal thermal latency (PWTL) improvement at 21 and 35 dpi, but a notable enhancement at 56 dpi. In contrast, the SCI + CLP290 group significantly improved PWTL at 21 days, with non‐significant changes at 35 and 56 days. At 21 dpi, KCC2 expression was marginally higher in monotherapy groups versus SCI + vehicle, but not significantly. At 56 dpi, only the SCI + bumetanide group showed a significant difference in KCC2 expression compared to the control group. Conclusion: Combined application of CLP290 and bumetanide effectively increases the ratio of KCC2/NKCC1, restores RDD levels, enhances GABAA receptor‐mediated inhibitory function in the spinal cord, and relieves neuropathic pain in SCI; Bumetanide significantly improves neuropathic pain in the long term, whereas CLP290 demonstrates a notable short‐term effect. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) presents significant challenges in pediatric care, often remaining underrecognized. This paper provides an overview of pediatric AKI, highlighting its epidemiology, pathophysiology, diagnosis, predisposing conditions, and treatment. AKI in children stems from diverse causes, including renal tubular damage, vasoconstriction, and inflammation. Diagnosis relies on traditional markers such as serum creatinine and urine output, alongside emerging biomarkers such as Cystatin C, NGAL, KIM-1, IL-18, TIMP-2 and IGFBP7, urinary calprotectin, URBP4, L-FABP, and clusterin. Various pediatric conditions predispose to AKI, including type 1 diabetes, pneumonia, bronchiolitis, appendicitis, gastroenteritis, COVID-19, multisystem inflammatory syndrome, sickle cell disease, and malignancies. Treatment entails supportive care with fluid management and, in severe cases, renal replacement therapy. Timely recognition and management are essential to mitigating adverse outcomes. Enhanced awareness and integration of novel biomarkers could improve pediatric AKI care, warranting further research for better diagnosis and management. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the safety and tolerability of STP1, a combination of ibudilast and bumetanide, tailored for the treatment of a clinically and biologically defined subgroup of patients with Autism Spectrum Disorder (ASD), namely ASD Phenotype 1 (ASD-Phen1). We conducted a randomized, double-blind, placebo-controlled, parallel-group phase 1b study with two 14-day treatment phases (registered at clinicaltrials.gov as NCT04644003). Nine ASD-Phen1 patients were administered STP1, while three received a placebo. We assessed safety and tolerability, along with electrophysiological markers, such as EEG, Auditory Habituation, and Auditory Chirp Synchronization, to better understand STP1's mechanism of action. Additionally, we used several clinical scales to measure treatment outcomes. The results showed that STP1 was well-tolerated, with electrophysiological markers indicating a significant and dose-related reduction of gamma power in the whole brain and in brain areas associated with executive function and memory. Treatment with STP1 also increased alpha 2 power in frontal and occipital regions and improved habituation and neural synchronization to auditory chirps. Although numerical improvements were observed in several clinical scales, they did not reach statistical significance. Overall, this study suggests that STP1 is well-tolerated in ASD-Phen1 patients and shows indirect target engagement in ASD brain regions of interest. [ABSTRACT FROM AUTHOR]

Therapeutics discovery and development for Alzheimers disease (AD) has been an area of intense research to alleviate memory loss and the underlying pathogenic processes. Recent drug discovery approaches have utilized in silico computational strategies for drug candidate selection which has opened the door to repurposing drugs for AD. Computational analysis of gene expression signatures of patients stratified by the APOE4 risk allele of AD led to the discovery of the FDA-approved drug bumetanide as a top candidate agent that reverses APOE4 transcriptomic brain signatures and improves memory deficits in APOE4 animal models of AD. Bumetanide is a loop diuretic which inhibits the kidney Na+-K+-2Cl- cotransporter isoform, NKCC2, for the treatment of hypertension and edema in cardiovascular, liver, and renal disease. Electronic health record data revealed that patients exposed to bumetanide have lower incidences of AD by 35%-70%. In the brain, bumetanide has been proposed to antagonize the NKCC1 isoform which mediates cellular uptake of chloride ions. Blocking neuronal NKCC1 leads to a decrease in intracellular chloride and thus promotes GABAergic receptor mediated hyperpolarization, which may ameliorate disease conditions associated with GABAergic-mediated depolarization. NKCC1 is expressed in neurons and in all brain cells including glia (oligodendrocytes, microglia, and astrocytes) and the vasculature. In consideration of bumetanide as a repurposed drug for AD, this review evaluates its pharmaceutical properties with respect to its estimated brain levels across doses that can improve neurologic disease deficits of animal models to distinguish between NKCC1 and non-NKCC1 mechanisms. The available data indicate that bumetanide efficacy may occur at brain drug levels that are below those required for inhibition of the NKCC1 transporter which implicates non-NKCC1 brain mechansims for improvement of brain dysfunctions and memory deficits. Alternatively, peripheral bumetanide mechanisms may involve cells outside the central nervous system (e.g., in epithelia and the immune system). Clinical bumetanide doses for improved neurological deficits are reviewed. Regardless of mechanism, the efficacy of bumetanide to improve memory deficits in the APOE4 model of AD and its potential to reduce the incidence of AD provide support for clinical investigation of bumetanide as a repurposed AD therapeutic agent.

Abstract Background The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+–K+–2Cl−–1 (NKCC1) and K+–2Cl−–2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development. Methods We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA. Results We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway. Conclusions Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

Background: The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+–K+–2Cl−–1 (NKCC1) and K+–2Cl−–2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development. Methods: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA. Results: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway. Conclusions: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes. [ABSTRACT FROM AUTHOR]

Bumetanide is used widely as a tool and off‐label treatment to inhibit the Na‐K‐2Cl cotransporter NKCC1 in the brain and thereby to normalize intra‐neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood–brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5‐fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC50 (2.4 μM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain. [ABSTRACT FROM AUTHOR]

Excessive water consumption is an extremely rare and potential asthma risk factor with very few cases reported in the literature. Common triggers of asthma include genetic factors, smoking, allergens, and viral respiratory infections. The adult patient with asthma reportedly drank too much water and was unable to get relief from his asthma while hospitalized. The patient's asthma was better controlled with the use of diuretics and control of the patient's fluid intake and output. This case explores asthma induced by excessive drinking of water. [ABSTRACT FROM AUTHOR]

Increases in the current threshold occur in optic nerve axons with the application of infra-red laser light, whose mechanism is only partly understood. In isolated rat optic nerve, laser light was applied near the site of electrical stimulation, via a flexible fibre optic. Paired applications of light produced increases in threshold that were reduced on the second application, the response recovering with increasing delays, with a time constant of 24 s. 3-min duration single applications of laser light gave rise to a rapid increase in threshold followed by a fade, whose time-constant was between 40 and 50 s. After-effects were sometimes apparent following the light application, where the resting threshold was reduced. The increase in threshold was partially blocked by 38.6 mM Li+ in combination with 5 μ M bumetanide, a manoeuvre increasing refractoriness and consistent with axonal depolarization. Assessing the effect of laser light on the nerve input resistance ruled out a previously suggested fall in myelin resistance as contributing to threshold changes. These data appear consistent with an axonal membrane potential that partly relies on temperature-dependent electroneutral Na+ influx, and where fade in the response to the laser may be caused by a gradually diminishing Na+ pump–induced hyperpolarization, in response to falling intracellular [Na+]. [ABSTRACT FROM AUTHOR]

Determining a drug's bioavailability and bioequivalence is important for developing and approving a drug product. The procedure supports applications for generic drug products and novel therapeutic substances, makes important decisions regarding safety and efficacy, and measures a drug's concentration in biological matrices. This study aimed to develop and validate a specific, simple, sensitive, and accurate method using liquid chromatography–tandem mass spectrometry (LC–MS) for measuring bumetanide (BUM) in human plasma. Chromatographic separation was achieved using a Hypurity C18 column (4.6 × 50 mm, 5 μm) under isocratic conditions, and LC–MS detected positive ionization acquisition modes. Protonated precursor to product ion transitions were observed at m/z 365.08 → 240.10 and 370.04 → 244.52 for BUM and internal standard, respectively. The linear range of BUM in plasma samples was 3.490–401.192 ng/mL. The inter‐precision value ranged from 1.76% to 4.75%. The inter‐accuracy value ranged from 96.46% to 99.95%. The method was adequately validated per the U.S. Food and Drug Administration guidelines, and the results were within permissible bounds. The Cmax and Tmax values were ~53.097 ± 13.537 ng/mL and 1.25 (0.67–5.00) h, respectively. The new approach showed satisfactory results for studying BUM in human plasma with potential use in pharmacokinetic and bioequivalence investigations. [ABSTRACT FROM AUTHOR]

Convergent evidence has suggested a significant effect of bumetanide exposure on brain connectivity in patients with autism spectrum disorder (ASD), yet the characteristics of favorable treatment outcome remains largely unknown. In this work, we aimed to elucidate the primary group distinctions in the temporal pattern of dynamic functional connectivity (dFC) estimated from task based EEG among individuals with ASD and healthy controls (HC) and explore the effect of bumetanide on brain network states. Through a systematic analysis, we investigated the ASD-related intrinsic alterations of dFC at baseline, followed by a longitudinal study to examine the influence of bumetanide treatment on these abnormalities by comparing patients at baseline and follow-up. EEG data was recorded from 9 individuals with ASD and 9 HC at baseline. After 3 months of bumetanide treatment, EEG data was re-recorded from the individuals with ASD. Utilizing phase locking value, sliding-window analysis, and K-means clustering, we obtained dFC for the alpha, beta, and gamma bands, and observed three repeating brain states. Two temporal metrics of connectivity state expression including fraction of time (FT) and dwell time (DT) were calculated. The results showed that in the alpha and gamma bands, FT and DT in ASD groups before bumetanide treatment were higher in the strong connection state and lower in the weak connection state compared to the HC group. Also, the use of bumetanide led to a significant decrease in the difference between the HC and ASD groups, although the adjustment made in the gamma band was more significant.

IntroductionAutism spectrum disorder (ASD) is characterised by difficulty with social communication and restricted, repetitive patterns of behaviour. This study aimed to improve understanding of the ASD patient experience with the treatment (bumetanide) regarding the changes in core symptoms and to assess changes considered as meaningful. To achieve this, qualitative interviews were conducted with caregivers of patients in two phase 3 clinical trials (NCT03715153; NCT03715166) of a novel ASD treatment.MethodsCaregivers were invited to participate in one interview after completion of the pivotal phase 3 study; for those of them who continued treatment after study completion, a second interview was held 3 months after trial completion. The interviews were conducted by qualitative researchers and followed a semi-structured interview guide. The interviews focused on patients’ ASD symptoms and their impact on their daily life before enrolment, and on any symptom changes patients experienced during the trial.ResultsOut of the 13 eligible patients’ caregivers, 11 were interviewed up to two times at clinical sites in the UK, Spain, and Italy. The caregivers reported impairments in a wide range of skills: deficits in communication and social interaction; restricted, repetitive patterns of behaviour, interests, or activities; and cognitive, emotional, and motor impairments. Compared to before the trial initiation, caregivers also reported improvements in the following domains: communication, interaction with others, cognition, aggression, emotions, repetitive movements, eating, and sleeping.ConclusionThe exit interviews provided a rich source of qualitative data, allowing a deeper understanding of caregivers’ and patients’ experience of the disease and allowing us to understand what constitutes a meaningful change. These data also helped identify important experiences that may inform the patient-reported outcome measurement strategy for future trials in ASD.

Background: Acting on the main target of dopaminergic cells, the striatal γ‐aminobutyric acid (GABA)‐ergic cells, might be a new way to treat persons with Parkinson's disease (PD). Objective: The objective of this study was to assess the efficacy of bumetanide, an Na–K–Cl cotransporter (NKCC1) inhibitor, to improve motor symptoms in PD. Methods: This was a 4‐month double‐blind, randomized, parallel‐group, placebo‐controlled trial of 1.75 to 3 mg/day bumetanide as an adjunct to levodopa in 44 participants with PD and motor fluctuations. Results: Compared to the baseline, the mean change in OFF Movement Disorder Society Unified Parkinson's Disease Rating Scale Part III score after 4 months of treatment (primary endpoint) did not improve significantly compared with placebo. No changes between participants treated with bumetanide and those treated with placebo were observed for most other outcome measures. Despite no relevant safety signals, bumetanide was poorly tolerated. Conclusions: There was no evidence in this study that bumetanide has efficacy in improving motor symptoms of PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A salt of vandetanib, namely, 4‐({4‐[(4‐bromo‐2‐fluorophenyl)amino]‐6‐methoxyquinazolin‐7‐yl}methoxy)‐1‐methylpiperazin‐1‐ium 2‐(butylamino)‐4‐phenoxy‐6‐sulfamoylbenzoate acetonitrile monosolvate, C22H25BrFN4O2+·C17H19N2O5S−·C2H3N, composed of kinase inhibitor vandetanib and sulfamyl diuretic bumetanide in a 1:1 molar ratio, is reported. There is proton transfer between the piperidine ring of vandetanib and the carboxyl group of bumetanide to form the salt. In the vandetanib cation, the arene and pyrimidine rings are not coplanar, their planes subtending a dihedral angle of 60.47 (14)°. The roles of the intermolecular interactions in the crystal packing were clarified using Hirshfeld surface analysis, and two‐dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H...H (40.5%), O...H/H...C (20.7%), C...H/ H...C (18.8%) and N...C/C...N (9.0%) contacts. [ABSTRACT FROM AUTHOR]

Equipped with an early social predisposition immediately post-birth, humans typically form associations with mothers and other family members through exposure learning, canalized by a prenatally formed predisposition of visual preference to biological motion, face configuration, and other cues of animacy. If impaired, reduced preferences can lead to social interaction impairments such as autism spectrum disorder (ASD) via misguided canalization. Despite being taxonomically distant, domestic chicks could also follow a homologous developmental trajectory toward adaptive socialization through imprinting, which is guided via predisposed preferences similar to those of humans, thereby suggesting that chicks are a valid animal model of ASD. In addition to the phenotypic similarities in predisposition with human newborns, accumulating evidence on the responsible molecular mechanisms suggests the construct validity of the chick model. Considering the recent progress in the evo-devo studies in vertebrates, we reviewed the advantages and limitations of the chick model of developmental mental diseases in humans. [ABSTRACT FROM AUTHOR]

• Patients with HypoPP experience disabling attacks of weakness. • Animal models showed promising data in the use of bumetanide in HypoPP. • Bumetanide was not efficacious in reversing an attack of hand weakness in HypoPP. • This study supports further investigation of bumetanide as a therapeutic agent. The aim of this study was to establish whether bumetanide can abort an acute attack of weakness in patients with HypoPP. This was a randomised, double-blind, cross-over, placebo-controlled phase II clinical trial. Focal attack of weakness was induced by isometric exercise of ADM followed by rest (McManis protocol). Participants had two study visits and received either placebo or 2 mg bumetanide at attack onset (defined as 40 % decrement in the abductor digiti minimi CMAP amplitude from peak). CMAP measurements assessed attack severity and duration. Nine participants completed both visits. CMAP percentage of peak amplitudes in the bumetanide (40.6 %) versus placebo (34.9 %) group at 1hr following treatment did not differ significantly (estimated effect difference 5.9 % (95 % CI: (-5.7 %; 17.5 %), p = 0.27, primary outcome). CMAP amplitudes assessed by the area under the curve for early (0–2hr post-treatment) and late (2–4 h post-treatment) efficacy were not statistically different between bumetanide and placebo (early effect estimate 0.043, p = 0.3; late effect estimate 0.085, p = 0.1). Two participants recovered from the attack following bumetanide intake; none recovered following placebo. Bumetanide was well tolerated but not efficacious to rescue a focal attack in an immobilised hand in the majority of patients, although data supports further studies of this agent. [ABSTRACT FROM AUTHOR]

Poorly water-soluble drugs represent a challenge for the pharmaceutical industry because it is necessary to find properly tuned and efficient systems for their release. In this framework, organic–inorganic hybrid systems could represent a promising strategy. A largely diffused inorganic host is hydroxyapatite (HAP, Ca10(PO4)6(OH)2), which is easily synthesized with different external forms and can adsorb different kinds of molecules, thereby allowing rapid drug release. Hybrid nanocomposites of HAP nanorods, obtained through hydrothermal synthesis, were prepared with two model pharmaceutical molecules characterized by low and pH-dependent solubility: meloxicam, a non-steroidal anti-inflammatory drug, and bumetanide, a diuretic drug. Both hybrids were physically and chemically characterized through the combined use of X-ray powder diffraction, scanning electron microscopy with energy-dispersive spectroscopy, differential scanning calorimetry, and infrared spectroscopy measurements. Then, their dissolution profiles and hydrophilicity (contact angles) in different media as well as their solubility were determined and compared to the pure drugs. This hybrid system seems particularly suitable as a drug carrier for bumetanide, as it shows higher drug loading and good dissolution profiles, while is less suitable for meloxicam, an acid molecule. [ABSTRACT FROM AUTHOR]

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Nitrosamine compounds are classified as potential human carcinogens, the origin of these impurities can be broadly classified in two categories, nitrosamine impurity found in drug products that are not associated with the Active Pharmaceutical Ingredient (API), such as N-nitrosodimethylamine (NDMA) or nitrosamine impurities associated with the API, such as nitrosamine drug substance-related impurities (NDSRIs). The mechanistic pathway for the formation of these two classes of impurities can be different and the approach to mitigate the risk should be tailored to address the specific concern. In the last couple of years number of NDSRIs have been reported for different drug products. Though, not the only contributing factor for the formation of NDSIRs, it is widely accepted that the presence of residual a nitrites/nitrates in the components used in the manufacturing of the drug products can be the primary contributor to the formation of NDSRIs. Approaches to mitigate the formation of NDSRIs in drug products include the use of antioxidants or pH modifiers in the formulation. The primary objective of this work was to evaluate the role of different inhibitors (antioxidants) and pH modifiers in tablet formulations prepared in-house using bumetanide (BMT) as a model drug to mitigate the formation of N -nitrosobumetanide (NBMT). A multi-factor study design was created, and several bumetanide formulations were prepared by wet granulation with and without sodium nitrite spike (100 ppm) and different antioxidants (ascorbic acid, ferulic acid or caffeic acid) at three concentrations (0.1%, 0.5% or 1% of the total tablet weight). Formulations with acidic and basic pH were also prepared using 0.1 N hydrochloric acid and 0.1 N sodium bicarbonate, respectively. The formulations were subjected to different storage (temperature and humidity) conditions over 6 months and stability data was collected. The rank order of N -nitrosobumetanide inhibition was highest with alkaline pH formulations, followed by formulations with ascorbic acid, caffeic acid or ferulic acid present. In summary, we hypothesize that maintaining a basic pH or the addition of an antioxidant in the drug product can mitigate the conversion of nitrite to nitrosating agent and thus reduce the formation of bumetanide nitrosamines. [ABSTRACT FROM AUTHOR]

Neonatal hypoxia–ischemia (HI) is one of the main causes of tissue damage, cell death, and imbalance between neuronal excitation and inhibition and synaptic loss in newborns. GABA, the major inhibitory neurotransmitter of the central nervous system (CNS) in adults, is excitatory at the onset of neurodevelopment and its action depends on the chloride (Cl−) cotransporters NKCC1 (imports Cl−) and KCC2 (exports Cl−) expression. Under basal conditions, the NKCC1/KCC2 ratio decreases over neurodevelopment. Thus, changes in this ratio caused by HI may be related to neurological disorders. The present study evaluated the effects of bumetanide (NKCC cotransporters inhibitor) on HI impairments in two neurodevelopmental periods. Male Wistar rat pups, 3 (PND3) and 11 (PND11) days old, were submitted to the Rice-Vannucci model. Animals were divided into 3 groups: SHAM, HI-SAL, and HI-BUM, considering each age. Bumetanide was administered intraperitoneally at 1, 24, 48, and 72 h after HI. NKCC1, KCC2, PSD-95, and synaptophysin proteins were analyzed after the last injection by western blot. Negative geotaxis, righting reflex, open field, object recognition test, and Morris water maze task were performed to assess neurological reflexes, locomotion, and memory function. Tissue atrophy and cell death were evaluated by histology. Bumetanide prevented neurodevelopmental delay, hyperactivity, and declarative and spatial memory deficits. Furthermore, bumetanide reversed HI-induced brain tissue damage, reduced neuronal death and controlled GABAergic tone, maintained the NKCC1/KCC2 ratio, and synaptogenesis close to normality. Thereby, bumetanide appears to play an important therapeutic role in the CNS, protecting the animals against HI damage and improving functional performance. [ABSTRACT FROM AUTHOR]

The efficacy of bumetanide (oral liquid formulation 0.5 mg bid) as a treatment for the core symptoms of autism spectrum disorders in children and adolescents aged 7–17 years is being investigated in an international, randomised, double-blind, placebo-controlled phase III study. The primary endpoint is the change in Childhood Autism Rating Scale 2 (CARS2) total raw score after 6 months of treatment. At baseline, the 211 participants analysed are broadly representative of autistic subjects in this age range: mean (SD) age, 10.4 (3.0) years; 82.5% male; 47.7% with intelligence quotient ≥ 70. Mean CARS2 score was 40.1 (4.9) and mean Social Responsiveness Scale score was 116.7 (23.4). Final study results will provide data on efficacy and safety of bumetanide in autistic children and adolescents. [ABSTRACT FROM AUTHOR]

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance. [ABSTRACT FROM AUTHOR]