Bumetanide

Major depressive disorder (MDD), the most prevalent psychiatric disorder worldwide, is often linked to changes in GABAergic neurotransmission. K⁺/Cl^- cotransporter 2 (KCC2) plays a key role in maintaining proper chloride levels within neurons, influencing the hyperpolarization caused by GABA_A receptor (GABA_AR) activation in mature neurons. However, the impact of KCC2-modulated GABAergic neurotransmission on depressive behaviors remains unclear. In this study, we investigated the role of KCC2 in a mouse model of depression. Our findings revealed a significant decrease in KCC2 expression in the dorsal hippocampus (dHip), accompanied by a depolarizing shift of the GABA reverse potential and a reduction in the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Knockdown of KCC2 expression increased the susceptibility to stress in mice. Conversely, overexpression of KCC2 or treatment with bumetanide reversed the chronic social defeat stress (CSDS)-induced changes in E_GABA and deficits of GABAergic neurotransmission, contributing to the improvement of depression-like behaviors. Additionally, extracellular vesicles (EVs) derived from bumetanide-treated HEK293T cells and engineered with acetylcholine receptor (AChR)-specific rabies virus glycoprotein (RVG) peptides, when injected via the tail vein, were found to alleviate depression symptoms. These results suggest that KCC2-mediated chloride homeostasis plays a key role in depression pathology and that bumetanide can be used to treat depression.

Down syndrome (DS) is a major cause of genetically defined intellectual disability, characterised by low IQ and cognitive deficits. Among the neurobiological causes of these deficits, disruptions in gamma-aminobutyric acid (GABA)ergic signalling in the hippocampus and an excitation/inhibition imbalance are thought to impair learning and memory. Evidence suggests that GABA A receptor-mediated signalling in DS is significantly depolarising and potentially excitatory rather than predominantly hyperpolarising and inhibitory, and is accompanied by increased hippocampal expression of the cation-chloride cotransporter NKCC1. The treatment with the NKCC1 inhibitor bumetanide restored GABAergic signals, synaptic plasticity, hippocampus-dependent memory and sleep quality in adult DS mice. We hypothesise that the use of bumetanide, by re-establishing GABAergic signals, may improve memory, learning and psychological characteristics in children and adolescents with DS.

The present study is a randomised, double-blind, placebo-controlled trial to evaluate the efficacy of a 3-month treatment with the drug bumetanide (0.02 mg/kg two times per day or placebo) on memory and psychological functioning in children and adolescents with DS. We also aim to identify possible predictors and biological and genetic markers related to treatment.The target recruitment is 64 children and adolescents with DS (aged 10–17 years). Seven visits are scheduled for each participant, during which outcome and safety measures are assessed through various clinical and instrumental investigations.Outcome measures include neuropsychological and psychological assessments, as well as biomarkers. Neuropsychological measures consist of tests evaluating cognitive level, memory and executive functions. Psychological measures include parent-reported questionnaires on psychopathological and behavioural symptoms, quality of life, sleep and adaptive functioning. Biomarkers encompass electroencephalogram (EEG) and genomic, transcriptomic, proteomic and metabolomic evaluations from blood and urine samples.Safety measures include physical, nephrological, cardiological and audiometric evaluations, as well as blood and urine analysis, EEG, ECG and a pregnancy test (if applicable). Neuropsychological, psychological, biomarker and safety measures are collected at baseline (visit 1 (V1)), at the end of treatment (V6) and during follow-up (V7), which is scheduled for 2 months after the end of treatment. Interim psychological and safety assessments are conducted at 1 week (V2), 2 weeks (V3), 1 month (V4) and 2 months (V5) after the start of treatment. We expect bumetanide treatment to improve visual long-term memory skills (primary outcome). Additionally, we expect improvements in total scores and subdomain scores across other visual, verbal and spatial long-term memory tasks, as well as executive functions, psychopathological measures, adaptive functioning, sleep quality and quality of life scores (secondary outcomes). Furthermore, we expect potential differences in genomic, transcriptomic, proteomic and metabolomic profiles between patients who respond to therapy and those who do not.

Ethical approval for this study was granted by the Bambino Gesù Children’s Hospital Ethics Committee (process number 1042_OPBG_2016) and the Italian Medicines Agency. Substantial Amendment No 5 (ES5) was approved on 20 September 2023 by the Italian National Paediatric Ethics Committee and involved a revision and integration of the study protocol, now updated to version 8.1, in line with the recommendations of the Data Safety Monitoring Board.This study is being conducted in accordance with the Declaration of Helsinki. The present study protocol adheres to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines and was prepared using the SPIRIT 2025 Checklist. In accordance with Good Clinical Practice, written informed consent is obtained from all participants’ parents or caregivers, and assent is obtained from participants when possible. The main features of the study will be presented at both international and national conferences, during scientific meetings, in presentations to families and on social media using documentation approved by competent authorities.

The study was prospectively registered in the EudraCT portal on 16 August 2016, prior to the enrolment of the first participant on 11 January 2023 ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-005780-16/IT ). In accordance with Regulation (EU) 536/2014 for pharmacological interventional trials, the study was subsequently transitioned to the Clinical Trials Information System (EU CT No 2024-519342-71-00), with the initial record entered on 23 December 2024, and the transition authorised on 3 February 2025 ( https://euclinicaltrials.eu/search-for-clinical-trials/?lang=en&EUCT=2024-519342-71-00 ).

EudraCT 2015-005780-16.