Effects of Acute Ebselen Add-on Treatment on Resting State Function Connectivity in Depressed Patients with Inadequate Response to Antidepressants

Effects of Acute Ebselen Add-on Treatment on Resting State Function Connectivity in Depressed Patients with Inadequate Response to Antidepressants | Research Square Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of Acute Ebselen Add-on Treatment on Resting State Function Connectivity in Depressed Patients with Inadequate Response to Antidepressants Fitri Fareez Ramli, Luca M Villa, Marieke A.G. Martens, Nisha Singh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8880141/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jun, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Ebselen has been shown to induce a positive emotional processing bias and brain neurochesmistry in healthy volunteers. However, little is known about its effects on resting-state functional connectivity (rs-FC) in depressed patients who show inadequate responses to standard antidepressants. The study aimed to investigate the effect of ebselen as an add-on treatment on rs-FC in this population. We conducted a randomised, double-blind, placebo-controlled trial involving depressed patients who were not responsive to their current treatment. Participants received either ebselen 600 mg twice daily or placebo for seven days. Resting-state functional magnetic resonance imaging was conducted at baseline and on day seven. Resting-state data were analysed using seed-based analysis and independent component analysis. Greater changes in rs-FC between the pregenual anterior cingulate cortex and the cerebellum were observed in ebselen than in placebo, but became negligible after corrections for multiple comparisons. We identified treatment effects within visual networks and between visual and sensorimotor networks, however, these did not survive correction for multiple comparisons. Short-term ebselen treatment in depressed patients with inadequate antidepressant responses did not produce significant changes in rs-FC. These findings may reflect limitations related to the short treatment duration and small and heterogenous sample. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience antidepressant depression ebselen lithium-mimetic Figures Figure 1 Figure 2 Figure 3 1. Introduction In the search for a lithium-mimetic agent that possesses the therapeutic benefits of lithium while minimising its adverse effects, ebselen has emerged as a promising candidate due to its similar mechanisms of action to lithium. A growing body of preclinical and clinical research in mood disorders has established a strong mechanistic foundation supporting the potential use of ebselen in bipolar disorder and treatment-resistant depression [ 1 – 7 ]. Ebselen has been shown to reduce myo-inositol levels potentially through inhibition of inositol monophosphatase (IMPase) in both preclinical models and human experimental studies [ 1 , 2 , 4 – 7 ]. IMPase is a key enzyme within the phosphoinositide signalling pathway, a second messenger system utilised by several serotonergic receptors (5-HT), including 5-HT 2A and 5-HT 2C [ 4 ]. Both ebselen and lithium have shown to induce behavioural and molecular changes associated with 5HT 2A agonists [ 4 , 7 ]. Interestingly, ebselen also has been shown to attenuate molecular changes induced by the 5HT 2C agonist [ 8 ], suggesting various modulatory role within serotonergic signalling. In human studies involving healthy volunteers, ebselen administration reduced myo-inositol in the anterior cingulate cortex (ACC), a key brain region involved in emotion processing. These neurochemical changes coincided with a positive shift in emotion processing, suggesting a potential use of ebselen as a mood-regulating agent in mood disorders [ 1 , 5 , 6 ]. Functional magnetic resonance (fMRI) imaging provides valuable mechanistic insights into the neural underpinnings of psychiatric disorders and their treatment. One variation of fMRI is resting-state fMRI, which enables an assessment of functional connectivity - a temporal correlation of the activity of brain regions - when an individual is awake yet not engaged in any external tasks. Patients with depression who are resistant to multiple treatments have been shown to exhibit altered resting-state functional connectivity within the affective and cognitive control networks compared with treatment-responsive counterparts [ 9 ]. Notably, ketamine treatment in patients with treatment-resistant depression has been associated with normalisation of hyperconnectivity within the default mode network, a core network implicated in maladaptive self-referential processing and rumination. Collectively, these findings suggest that alterations or normalisation of resting-state functional connectivity may provide mechanistic insight into how pharmacological interventions exert therapeutic effects in depression [ 10 – 12 ]. To our knowledge, no previous studies have examined the effects of ebselen on resting-state functional connectivity in a clinical population. Therefore, the present study aimed to investigate the effects of acute ebselen administration as an add-on treatment on resting-state functional connectivity in depressed patients with inadequate responses towards standard antidepressants. A secondary objective was to explore the association between changes in neurometabolite concentrations of myo-inositol, glx, and glutamate in the ACC and alterations in resting-state functional connectivity between the ebselen and placebo groups. 2. Methods We conducted a double-blind, randomised, placebo-controlled, experimental trial involving patients with major depressive episodes who were still depressed despite adequate duration and dose of antidepressants. This study was a part of the larger study reported before (for more details, refer to [ 13 ]). The study was approved by the National Research Ethics Service Committee [NRES], South-Central Oxford (D:20/SC/0151) and was performed in accordance with the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. All participants gave their written informed consent before the study. We screened participants using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (DSM-5) (SCID-5). We included patients with major depressive disorder and a Hamilton Depression Rating Scale score of ≥ 14 who had inadequate responses to their current antidepressant treatment. The participants were required to have been on their current antidepressant at a therapeutic dose for at least four weeks. The participants were excluded if they had: history of or current psychosis, bipolar disorder and emotionally unstable personality disorder; history of significant substance dependence (including alcohol) over the past six months; clinically significant risk of suicide; current or history of electroconvulsive therapy for the current depressive episode; failure to respond to standard pharmacological augmentation treatments (lithium and atypical antipsychotics); severe claustrophobia; body mass index outside the range of 18–36 kg/m 2 ; contraindications to magnetic resonance imaging scanning; current pregnancy and lactation; participation in a study involving emotional processing tasks or interventional medication within the last three months. We did not exclude the participants with medical conditions on prescribed medications if these did not compromise the safety or affect data quality. Premenopausal women and male participants engaging in sexual activity with a risk of pregnancy were required to use highly effective contraception during the study and for 30 days after receiving the study medication. Male participants were required not to donate sperm during this period. Participants were randomised to receive either ebselen 600 mg or placebo twice daily for seven days, stratified based on sex and number of antidepressant treatments (1–2 vs > 2). The MRI scans (structural, proton magnetic resonance spectroscopy (MRS) and functional MRI) were conducted at baseline and on day seven of treatment. 2.1 Data acquisition The structural, MRS and resting-state functional MRI (fMRI) scans in that order were conducted at the Oxford Centre for Human Brain Activity, University of Oxford, using 3T SIEMENS MAGNETOM with a 32-channel receive array head coil (Siemens, Erlangen, Germany). Structural images were acquired using a T1- Magnetization Prepared - RApid Gradient Echo (MPRAGE) image with a voxel resolution of 1.0 x 1.0 x 1.0 mm with a repetition time (T R ) = 1900 ms, echo time (T E ) = 3.96 ms, flip angle = 8 o , slice thickness = 1 mm, 192 slices, field-of-view (FoV) read = 256 mm). A detailed description of proton MRS pre-processing can be found in another publication (refer to [ 13 ]). Briefly, an 8-ml voxel was manually placed in the ACC of the MPRAGE image acquired [ 14 ]. The adjustments of first- and second-order shims were conducted using gradient echo shimming [ 15 ]. We acquired the spectra using the semi-Localization by Adiabatic Selective Refocussing sequence (T E = 28 ms, T R = 3 s, 128 averages) with variable power radiofrequency pulses with optimized relaxation delay (VAPOR)[ 16 ]. Before the resting-state functional MRI scan, participants were instructed to look at the cross mark in the centre of the white background and reminded to stay awake during the fMRI scan (voxel size of 2.4 x 2.4 x 2.4 mm, T R = 735 ms, T E = 39 ms, flip angle = 52 o , slice thickness = 2.4 mm, 192 slices, FoV read = 210 mm, 800 volumes, and 64 slices). Gradient echo phase and magnitude field maps were also acquired for correcting any distortions (2.4 x 2.4 x 2.4 mm, T R = 600 ms, T E1 /T E2 : 4.50/6.96 ms, flip angle = 46 o ). 2.2 Resting-state fMRI pre-processing and statistical analysis The data were pre-processed and analysed using FSL (version 6.6.1, FMRIB Analysis Group, University of Oxford, UK) [ 17 ]. Individual subject data were pre-processed (brain extractions of T1-MPRAGE and fieldmap magnitude images, creations of fieldmap images). Further, the Multivariate Exploratory Linear Optimized Decomposition into Independent Components (MELODIC, version 3.15) function in FSL was used to perform the individual subject independent component analysis (ICA), including motion correction, unwarping of effective echo-planar imaging data (EPI echo spacing = 0.64 ms, EPI T E = 39, unwarp direction = -y, percentage signal loss threshold = 10), co-registration of EPI data to the structural scan using boundary-based registration, and normalization into Montreal Neurological Institute (MNI) standard space using both linear and non-linear registrations (warp resolution = 10 mm, resampling resolution = 2 mm) [ 18 ]. We also applied temporal filtering with a high-pass filter cut-off of 100 s (0.005 Hz), spatial smoothing with a Gaussian kernel of full-width at half-height (FWHM) of 5 mm, variance time course, automatic dimensionality estimation and IC map threshold of 0.5. We performed a data quality check on the first-level analysis. The following criteria were used to indicate acceptable motion: movement ≤ 2.4 mm in translation along x, y, or z planes and ≤ 3 o (equivalent to 0.052 radians] rotation in any direction [pitch, roll or yaw) [ 19 ]. The study-specific training dataset was created by manually labelling ten randomly selected individual datasets into signal and noise [ 20 ]. The Functional MRI of the Brain (FMRIB)’s ICA-based Xnoiseifier (FIX) was used to train the study-specific dataset. We selected the best threshold of 20, with a true positive ratio of 97.3 and a true negative ratio of 89.9, to denoise all data. Subsequently, seed-based functional connectivity analysis and independent component analysis were conducted separately. For seed-based analyses, the pregenual ACC was chosen as a seed due to the significant effects of ebselen in reducing myo-inositol and glx (a composite measure of glutamate+glutamine) levels in the ACC which coincided with a positive shift in emotional processing [ 21 , 22 ]. In line with these previous studies, our MRS data, used for association testing in this study, were acquired from a voxel located in the ACC [ 13 ]. Also, increased connectivity between the ventromedial prefrontal cortex (of which ACC is a part) and the amygdala was associated with clinical improvement in bipolar mood disorder patients receiving lithium monotherapy, indicating that ACC might be sensitive to the effect of a lithium-mimetic agent ebselen [ 23 ]. The pregenual ACC mask was created from Automated Anatomical Labelling atlas 3 [ 24 ]. Dual regression was conducted to produce subject-specific timeseries and spatial maps [ 25 – 27 ]. FSL voxel-based morphometry (FSLVBM) was used for quantifying grey matter volumes, which were included as a covariate for resting-state functional connectivity analyses [ 28 – 30 ]. The general linear model was used to compare the changes (post-treatment minus baseline parameters) in functional connectivity between ebselen and placebo with grey matter volumes included as a covariate. We utilised the FSL randomise to analyse the data using permutation-based analysis (number of permutations = 5000) using threshold-free-cluster-enhancement and a family-wise error corrected p-value threshold of < 0.05 [ 30 – 32 ]. The whole brain resting-state fMRI analysis (group ICA) was also conducted to determine changes in resting-state functional connectivity across the whole brain. We utilised the MELODIC to temporally concatenate the de-noised functional data and decompose them into independent components of 25. Spatial mixture modelling estimates that the average number of individual IC maps is typically about 25 [ 26 ]. The potential networks were identified by visual inspections. The correlation between the identified network and published resting state networks [ 26 , 33 ] was analysed using the FSL cross-correlation tool (threshold of 0.3). Dual regression was conducted to produce subject-specific timeseries and spatial maps [ 25 – 27 ]. Similarly, the permutation-based analysis (number of permutations = 5000) as described above was conducted with grey matter volumes as a covariate [ 30 – 32 ]. Parameter estimates of changes in resting-state functional connectivity measures were extracted for all significant clusters. 2.3 Proton Magnetic Resonance Spectroscopy Pre-processing and Analysis The LCModel in MATLAB 2016 was used to measure concentrations of myo-inositol, glx, and glutamate, with total creatine (creatine and phosphocreatine) as reference [ 34 ]. The cerebrospinal fluid, white matter, and grey matter percentages were measured to correct metabolite concentrations [ 35 ]. The correlation between changes in neurometabolite concentrations (myo-inositol, glx and glutamate) in the ACC and changes in resting-state functional connectivity between the pregenual ACC and other brain regions were conducted using permutation-based analysis, with metabolite levels as a covariate. 2.4 Statistical analysis for patient baseline characteristics Descriptive data were presented as means ± standard error of means (SEMs) for continuous data and count and percentages for categorical data. Independent t-tests and chi-square tests were used to analyse continuous and categorical data, respectively. 3. Results Forty-five participants aged 18–66 (37.1 + 2.0 years) were included in the final analysis. There were no significant differences between groups in age ( t (43) = 0.007, p = 0.994), sex (p = 0.371 ( χ 2 )), number of antidepressant treatments (p = 0.944 ( χ 2 )) and baseline HAM-D ( t (43) = 0.213, p = 0.833). 3.1 Seed-based analysis of changes in resting-state functional connectivity between pregenual ACC and other brain regions and its correlations with neurometabolite changes in ACC Three participants were excluded due to significant movements of > 2.4 mm in translation along x, y, or z planes or 3 o (equivalent to 0.052 radians) rotation in any direction (pitch, roll, or yaw) [19]. A greater increase in resting-state functional connectivity between the pregenual ACC and the right cerebellum (number of significant voxels = 4, MNI 27, 41, 21) was observed in the ebselen group but became non-significant after correction for multiple comparisons (Figure 1). We did not observe any significant correlations between changes in neurometabolite concentrations of myo-inositol, glx and glutamate in the ACC and changes in resting-state functional connectivity (between the pregenual ACC and other brain regions) between treatment groups. Group Independent Component Analysis (ICA) Nineteen components were identified as resting-state networks (RSNs) of interest corresponding to previously identified canonical RSNs (26,33,36), and six components were identified as noise (physiological and head movement). All the identified RSNs are presented in Figure 2. Group comparisons revealed significant treatment effects within visual networks and between visual networks and sensorimotor networks (Figure 3, Table 1). However, the effects did not survive correction with multiple RSNs (corrected p-value = 0.001, 19 RSNs x 2 t -tests comparisons). Table 1.  Changes in resting-state functional connectivity in the ebselen and placebo groups after 7 days of treatment within visual networks and between visual and sensorimotor networks [n=45]. Reference network Network with significant voxel Cluster size (number of voxels) Peak p-value Peak voxel (MNI coordinates) Contrast Visual Somatosensory area (cluster 1) 30 0.008 63, 51, 61 Ebselen > Placebo Visual Somatosensory area (cluster 2) 25 0.025 65, 52, 67 Ebselen > Placebo Visual Visual area 1 0.049 37, 20, 41 Ebselen < Placebo 4. Discussion A short-term ebselen treatment increased changes in resting-state functional connectivity between the pregenual ACC and cerebellum, as well as between visual and sensorimotor networks but reduced changes in functional connectivity within the visual cortex. However, the effects did not survive correction for multiple comparisons. In addition, no significant correlations were observed between changes in ACC neurochemical concentrations over treatment course and a change in resting-state functional connectivity in the pregenual ACC. Negligible findings might be attributable to several factors, including the short treatment duration, heterogeneity in concurrent antidepressant regimens, small sample size, and the distinct clinical characteristics of this depression cohort. Although short-term ebselen administration has been shown to induce significant neurochemical changes in the same cohort [ 13 ], a longer duration of ebselen treatment might be required to elicit substantial changes in large-scale brain functional connectivity. There are lines of evidence supporting changes in functional connectivity by antidepressants after 3–5 hours. However, the studies reporting these findings typically involved healthy volunteers or unmedicated major depressive disorder patients [ 37 , 38 ]. In contrast, our participants were receiving ongoing antidepressant treatment; albeit ineffective, this treatment may have reduced the sensitivity to detect additional connectivity changes following short-term ebselen administration. The divergent effects of ebselen on emotion processing observed between healthy volunteers [ 1 , 6 ] and our current population [ 13 ] may partly explain the limited impact on resting-state functional connectivity. Prior studies reported a positive emotional bias shift in the FERT following ebselen administration in healthy volunteers [ 1 , 6 ]. However, a similar effect was not replicated in our current cohort, as reported separately [Ramli et al. [ 13 ]. The absence of significant differences in emotion processing in the FERT between healthy volunteers and depressed patients with inadequate responses towards standard antidepressants may reflect the extent to which ebselen can modulate associated functional connectivity patterns [ 39 ]. Another possible explanation is that short-term ebselen administration does not substantially influence other emotion processing domains, specifically emotion encoding and memory. Previous research in healthy volunteers has shown that short-term ebselen administration does not significantly affect these components of emotion processing [ 1 ]. While ebselen shares overlapping mechanisms of action with lithium, emerging evidence suggests that lithium may exert additional effects on emotion encoding, as demonstrated in a recent pilot study [ 40 ]. The presence of negative biases in emotion encoding and memory observed in our patient cohort compared to healthy volunteers suggests that ebselen may not exert its effects through modulation of these particular emotion processing domains [ 39 ]. The negligible effects of ebselen on resting-state functional connectivity within the limbic system and cortical regions may partly account for the present findings. However, the absence of significant effects should be interpreted cautiously. The current study employed resting-state functional MRI which reflects spontaneous brain activity at rest and the emotion encoding and memory aspects were assessed separately. Therefore, no inferences can be drawn regarding functional connectivity during task-based functional MRI involving emotional processing tasks. Given that ebselen is considered a lithium-mimetic agent, it is informative to consider the literature on the lithium’s effects on resting-state functional connectivity. It is also possible that our cohort included patients who were relatively non-responsive to ebselen, thereby reducing the power to detect treatment-related differences. Supporting this possibility, a previous study has shown that lithium responders and non-responders exhibit distinct brain activations patterns, with responders exhibiting higher activation within frontal and cingulate regions during a task-based fMRI paradigm involving emotional processing [ 41 ]. Such interindividual variability may similarly apply to ebselen responsiveness. However, this measure was not assessed in the present study. Although ebselen and lithium share overlapping mechanisms of actions, they likely exert their effects through partially distinct neurological pathways. Both agents can influence glutamatergic neurotransmission. However, the underlying mechanisms are different. Ebselen appears to modulate glutamatergic signalling by reducing synaptic glutamate levels through inhibition of glutaminase [ 4 , 42 ], while lithium is thought to attenuate glutamatergic transmission by inhibiting N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx [ 43 ]. Moreover, ebselen exhibits greater potency than lithium in inhibiting IMPase and shows higher selectivity for IMPase relative to glycogen synthase kinase-3ß [ 2 ], suggesting important pharmacodynamic differences between the two agents. Several limitations should be acknowledged. These include small sample size, heterogeneity of concurrent antidepressant treatments, lack of baseline fMRI data prior to initiation of standard antidepressant treatment, and short treatment duration. Additionally, the lack of a healthy volunteer comparison group limits direct comparisons between disease and non-disease states. Nevertheless, to our knowledge, this is the first study to examine the effects of ebselen on resting-state functional connectivity in a clinical population. As such, despite the largely negative findings, this study contributes novel and valuable insights to the growing body of ebselen research and informs the designs of future studies investigating lithium-mimetic agents in mood disorders. Future studies should prioritise larger, adequately powered samples with longer treatment durations, stratification by treatment response, and the inclusion of unmedicated depressed patients or healthy control groups. These designs would allow for better elucidation of the neurofunctional effects of ebselen and its potential role as a mood-stabilising or antidepressant agent. Acute ebselen treatment as an add-on therapy to standard antidepressants did not produce significant changes in resting-state functional connectivity in depressed patients who had inadequate response to standard antidepressant treatment. Although ebselen showed effects on resting-state functional connectivity between the pregenual ACC and the cerebellum, as well as within visual network and between visual and sensorimotor networks, these findings did not survive correction for multiple comparisons. These findings may reflect limitations related to the short treatment duration, small and heterogenous sample, and the complexity of treatment-resistant depression. Declarations Authors contributions PJC, CJH and NS conceived the study. BRG, PJC, FFR, LMV, and MAGM designed the study. PJC acquired the funding. FFR, BRG, LMV, and PJC contributed to data collection. FFR, LMV, BRG, and MAGM analysed the data. FFR wrote the original manuscript. BRG, PJC, CJH, FFR, LMV, MAGM and NS revised the article and contributed to the final version of the manuscript. All authors have reviewed and approved the final manuscript. Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Funding This research was funded by the Medical Research Council (grant number MR/K022202), Sound Pharmaceuticals, and by the NIHR Oxford Health Biomedical Research Centre. Dr. Ramli is supported by Yayasan Khazanah and National University of Malaysia. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Competing interests PJC holds a patent on behalf of Oxford University for the use of ebselen in patients with treatment resistant depression. CJH was supported by the NIHR Oxford Health Biomedical Research Centre; she has received consultancy fees from P1vital, Lundbeck, Servier, UCB, Zogenix, J&J, IESO outside of the current work. BRG received consultancy fees from Tiefenbacher Pharmaceuticals. FFR declares no conflicts of interest. LMV is an employee of Qynapse. Ethical approval The study was approved by the National Research Ethics Service Committee [NRES], South-Central Oxford [ID:20/SC/0151]. Informed consent Informed consent was obtained from all participants before the study. Acknowledgements: This study has been delivered through the National Institute for Health and Care Research (NIHR) Oxford Health Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the [name of the research funder], the NIHR or the Department of Health and Social Care. We thank all the participants, as well as Jon Campbell, Michael Sanders, David Parker, Juliet Semple and Nicola Aikin for expert radiographic assistance, and care of the participants during scanning. References Singh, N., Sharpley, A.L., Emir, U.E., Masaki, C., Herzallah, M.M., Gluck, M.A. et al. Effect of the putative lithium mimetic ebselen on brain myo-inositol, sleep, and emotional processing in humans. 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