Neuronal drug screening method and new applications for dilazep and penbutolol

Abstract

The present invention relates to the field of drug development and neuropharmacology. One aspect of this invention involves a method for screening substances that increase the stability of excitatory synaptic connections under destabilizing conditions. This method entails exposing a tested compound to an in vitro differentiated neuron culture along with a destabilization compound, followed by incubation with the test compound. Subsequently, a postsynaptic parameter is measured in the neurons, and this measurement is compared to that of neurons not exposed to the tested substance. This high-content screening system represents a significant advancement in neuropharmacology and drug discovery, offering automated and high-throughput capabilities for assessing the effects of chemical compounds on neuronal connections in vitro. Another aspect of the invention pertains to the use of dilazep, penbutolol, and their salts for alleviating and treating symptoms associated with depressive disorders.

Description

[0001] Description

[0002] Title of Invention: Neuronal Drug Screening Method and New Applications for Dilazep and Penbutolol

[0003] BACKGROUND

[0004] Depressive disorder is a common mental disorder. According to the World Health Organization (WHO), it is the second leading cause of disability in the world and is estimated to take first place by 2030 (Executive Board, 2012). Early reports from 2020 and subsequent reports show that the COVID-19 pandemic has significantly contributed to the deterioration of mental health worldwide (Ettman et al., 2022; Hou et al., 2021 ; Lorenzoni et al., 2022).

[0005] According to the current medical classification, to be diagnosed with depression, a person should have experienced 5 out of 9 symptoms in the last 2 weeks. The symptoms mentioned include: (1) depressed mood; (2) loss of interest or pleasure; (3) significant change in weight or appetite; (4) insomnia or excessive sleepiness; (5) psychomotor agitation or retardation; (6) fatigue or loss of energy; (7) feeling of worthlessness; (8) decreased ability to think, concentrate, or indecision; and (9) suicidal ideation (American Psychiatric Association, 2013). It is worth mentioning that depression often co-occurs with other diseases, sharing common biological causes and environmental factors. The most common comorbidities include cardiac, cancer, neurological, metabolic and inflammatory diseases (Gold et al., 2020). Even though we have various forms of treatment for depression, such as psychotherapy and pharmacotherapy (Karrouri et al., 2021), many patients are classified as treatment-resistant (Souery et al., 2006). Some studies show that patients with treatment-resistant depression have an increased risk of suicide and a decreased standard of living (Bergfeld et al., 2018; Mrazek et al., 2014). Moreover, the increase in the incidence of depression is disproportionately faster than the development of new antidepressant drugs and therapies (Goodwin et al., 2022). Hence, there is a constant need to search for new drugs and treatment methods that give hope for curing more patients.

[0006] Most antidepressants are based on the monoamine hypothesis of depression. The mechanism of action of most antidepressants is correlated with increasing the levels of serotonin, dopamine and norepinephrine. The first group of drugs is tricyclic antidepressants (TCAs). These drugs increase levels of serotonin, norepinephrine, and, to a lesser extent, dopamine (Gillman, 2007). Due to the serious side effects of TCA drugs, they are currently used in patients who do not respond to less toxic therapies (Gillman, 2007; Khalid and Waseem, 2023). Another type of antidepressant drugs are monoamine oxidase inhibitors (MAOIs). MAOI drugs increase the levels of serotonin, norepinephrine and dopamine in synapses by inhibiting their breakdown by monoamine oxidase. Iproniazid was the first MAOI used to treat depression (Pare and Sandler, 1959). However, nonselective and irreversible binding resulted in serious side effects such as hypotension and liver damage (Davison, 1957). Both TCAand MAOI drugs are the first generation of antidepressants, which were preceded by more selective drugs of subsequent generations. Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed drugs (Marken and Munro, 2000). They increase serotonin activity by inhibiting serotonin reuptake from the synaptic cleft. Overtime, it turned out that SSRIs support plastic processes and neurogenesis (Malberg et al., 2000; Wang et al., 2008). SSRIs have proven to be much less toxic and have reduced the risk of overdose (Lane et al., 1995). There are also groups of drugs whose simultaneous therapeutic target is two neurotransmitters. These include norepinephrine and dopamine reuptake inhibitors (NDRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs). NDRI drugs increase the levels of dopamine and norepinephrine (Stahl et al., 2004), SNRIs serotonin and norepinephrine (Lambert and Bourin, 2002). The latest generation drug is ketamine, for which the first clinical trials began in 2000 (Berman et al., 2000). It has been proven effective in some treatment-resistant patients (Murrough et al., 2013; Serafini et al., 2014). In 2020, it was proven that ketamine (an enantiomer of ketamine) in the form of an intranasal spray, in combination with other antidepressants, significantly reduces depressive symptoms (Fu et al., 2020).

[0007] It is estimated that approximately 30% of depressed patients are resistant to conventional antidepressants (Kennedy and Giacobbe, 2007; McLachlan, 2018). Despite the heterogeneity of the group of cases, treatment-resistant depression (TRD) is most often defined as a situation in which the patient does not achieve remission while using two different antidepressants (Voineskos et al., 2020).

[0008] Neuronal connections, which consist of dendritic and axonal trees and the synapses they form, determine the proper functioning of the brain. During development, there is a period of dynamic changes, both the formation and removal of dendrites and connections between neurons. In the mature brain, plastic changes in dendrites and neuronal connections are significantly limited, but not impossible. There are several examples of the processes that can change structuraly the the connections between neurons in a physiological (e.g. learning, lactation, aging). However, changes of neuronal network can both induce and accompany disease states. The most visible effects of destabilization of neuronal connections can be observed in the case of neurodevelopmental, neurpsychiatric and neurodegenerative diseases. Particular attention was paid to the morphology of dendritic trees and dendritic spines, that are morphological coralates of excitatory synapses in many neurons, in patients diagnosed with intellectual disability and / or autism. Postmortem analysis of brain sections has shown a reduction in dendrites in the prefrontal cortex (Mukaetova-Ladinska et al., 2004) and hippocampus (Raymond et al., 1996) of such patients. At the same time, an increased density of dendritic spines is observed in certain brain areas (Hutsler and Zhang, 2010). Pathological changes in dendrites, dendritic spines and synapses may also occur in the mature, relatively morphologically stable brain. Dendritic loss combined with regenerative changes can be initiated following stroke or brain injury (Brown, 2010; Kuriakose and Xiao, 2020; Merino- Serrais et al., 2023; Slujitoru et al., 2012). In the case of neurodegenerative diseases, a loss of neuronal connections is observed (Anderton et al., 1998; Flood, 1991) and a loss of the ability to make plastic changes within them (Golovyashkina et al., 2015; Hoover et al., 2010; Ittner et al., 2010; Jaworski et al., 2011). The last discussed group of diseases related to the destabilization of neuronal connections in the mature brain are neuropsychiatric disorders. Those best characterized in terms of changes in neuronal connections include schizophrenia and depression. Postmortem brain sections from patients diagnosed with schizophrenia show a reduction in dendritic branching in some cortical areas (Moyer et al., 2015).

[0009] A variety of research methods are used in the context of discovering new antidepressants. These methods can be divided into three main categories: knowledge-based methods, computational methods, and experimental methods (Immadisetty et al., 2013). Each of these categories plays an important role in the process of identifying potential antidepressant drugs.

[0010] Knowledge-based methods use existing chemical and pharmacological knowledge to identify chemicals or molecular compounds that may have the potential to counteract depression. These approaches are based on research on existing drugs and the mechanisms of action of chemicals. Examples of knowledge-based methods are structure-activity relationship (SAR) analysis and molecular hybridization. SAR uses existing knowledge about the structure and activity of chemical substances to optimize antidepressants (Wong et al., 1974). Molecular hybridization allows combining the features of different chemical compounds into one, potentially better drug (Nolan et al., 2011 ; Viegas-Junior et al., 2007). Computational methods use advanced computer technologies and mathematical models to predict potential compounds or substances that may be effective in the treatment of depression (Leelananda and Lindert, 2016; Sadybekov and Katritch, 2023). The technique used that combines knowledge-based methods with computational methods is virtual screening (VS). VS uses computational capabilities to evaluate millions of existing chemical structures for their potential activity on specific molecular targets, which helps filter potential compounds (Sheridan, 2008; Willett, 2003; Zhang and Muegge, 2006). Experimental methods are practical studies of chemical substances in the laboratory and on animals or humans. They allow for the assessment of the impact of substances on mechanisms related to depression and the identification of the best drug candidates.

[0011] An interesting experimental approach is high-throughput screening (HTS), which allows the testing of many compounds thanks to the use of process automation (Mayr and Fuerst, 2008). HTS studies can be based on in silico models (Bajorath, 2002), on in vitro models such as cell cultures (Inglese et al., 2007; Mayr and Fuerst, 2008; Szymanski et al., 2011), on in vivo models, usually such as Danio rerio (Delvecchio et al., 2011). A meta-analysis (conducted until 2016) showed that 15 existing drugs found 20 new applications, correlated with antidepressant effects (Caban et al., 2017). Drug repositioning allows for the reduction of drug development costs and time to market. An important aspect is also the availability of pharmacokinetic and toxicological characteristics of the drug (Padhy and Gupta, 2011). It is estimated that the standard process for introducing a new drug to the market is 10-17 years, and drug repositioning takes 3-8 years (Rao et al., 2022). In recent years, there have been an increasing number of companies offering libraries of chemical compounds / drugs that are also used in the clinic. A promising approach in drug repositioning (in the context of depression) seems to be testing compounds / drugs from commercially available libraries of chemical compounds in HTS systems based on biological processes. It is, therefore, a promising drug repositioning strategy that accelerates the availability of effective therapies to patients.

[0012] SUMMARY OF THE INVENTION

[0013] In one aspect, the current invention discloses a method that aims to identify compounds that can counteract the effects of synaptic destabilization, potentially leading to the discovery of novel therapies or treatments for conditions characterized by synaptic instability. The method involves screening for compounds that enhance synapse stability in cultured neurons by adding a synaptic destabilizer and at least one compound to neuron cultures, while another set of neuron cultures is treated only with the destabilizer as a control. Selected properties of postsynaptic compartments in both groups are monitored to assess synaptic stability. The objective is to identify compounds that induce synapse stabilization comparable to or exceeding that observed in neurons not exposed to the destabilizer. This screening approach aims to discover potential therapeutic compounds that can mitigate synaptic destabilization, offering insights into developing treatments for conditions associated with synaptic dysfunction in neuronal networks.

[0014] In other aspects, the invention discloses dilazep and penbutolol as well as salts thereof for use in prevent or treat depressive disorders that comprise major depressive disorder, single episode depressive disorder, dysthymic disorder, mixed depressive and anxiety disorder, premenstrual dysphoric disorder, persistent depressive disorder, premenstrual dysphoric disorder, substance / medication-induced depressive disorder, depressive disorder due to another medical condition and treatment-resistant depressive disorder in patients needed thereof.

[0015] The current invention also discloses the use of these compounds in fixed-dose combination formulations or as separate formulations for concurrent administration, specifically aimed at treating depressive disorders by utilizing fixed-dose combinations or separate but concurrent formulations.

[0016] Additionally, the invention comprises the incorporation of one or more additional active ingredients selected from a group that encompasses antidepressants, anxiolytics, mood stabilizers, antipsychotics, and combinations thereof.

[0017] Objects of the present invention are presented in more detail in the following Description and Examples.

[0018] DESCRIPTION OF THE FIGURES: Figure 1 : In vitro treatment of neurons with chemical compounds during screening. (A) Treatment scheme for neuronal cultures. Cells were treated at 16 and 18 day in vitro (DIV) and then fixed at 21 DIV. (B) Final concentrations of compounds in neuronal dishes. (C) Procedure for preparing the appropriate concentration of compounds and treating neurons cultured in vitro. 2.5 mM compounds were taken from the derivative dish (left panel) and a pooled working solution (middle panel) containing GBZ with the test compound suspended in the neuronal culture medium was created. Then 15 pl of the mixture was added to neuronal cultures, obtaining the following final concentrations: 2,5 pm tested compound, 10 urn gabazine (GBZ, destabilizer) in - 0.1% DMSO. As control, cells were treated either with 0.1% DMSO or 10 pm GBZ in 0.1% DMSO in culture medium.

[0019] Figure 2: Sequential analysis block diagram used to determine excitatory parameters of synaptic connections and total GluA1 intensity. Neurons cultured in vitro were fixed in 21 DIV and then immunofluorescently stained with anti-Bassoon and anti-GluA1 antibodies. Image acquisition was performed at 40x magnification using an Opera Phenix® microscope. (A) Analysis area with immunofluorescently labelled Bassoon and GluA1 proteins. (B) Determination of synapses by Bassoon immunofluorescence. Selection filters were applied to determine the signal contrast. Points representing synapses were thus obtained. (C) Measurement of GluA1 immunofluorescence intensity at previously determined synapses. (D) Measurement of GluA1 immunofluorescence intensity throughout the entire image (in and out of synapses).

[0020] Figure 3: Diagram for determining the level of destabilization of excitatory synapses and normalization of data. (A) Density plot of GluA1 immunofluorescence intensity at the synapse under destabilizing conditions. From this graph, the destabilization level was determined, which is the value of the GluA1 immunofluorescence signal for 30% of the synapses with the highest intensity. (B) Plots of GluA1 immunofluorescence density at the synapse for the test conditions. The percentage of synapses above the destabilization level, determined for data collected under destabilizing conditions, was checked for neurons in a specific culture dish. Left graph shows the effect of increasing synapse saturation at GluA1 is highlighted. The right graph represents a decrease in synapse saturation in GluA1. (C) Diagram of normalization of immunofluorescence data of GluA1 at the synapse and total GluA1. The purpose of the normalization was to allow different replicates to be compared, and the results for the two different parameters to be combined. To this end, each value (for GBZ, CTR, TEST conditions) was divided by the corresponding value from the destabilizing (GBZ) conditions. In this way, the score from the destabilizing conditions was equalized to 1 , and the other conditions were multiple (fold change).

[0021] Figure 4: High-throughput screening analysis of compounds testing the potential stabilization of excitatory synaptic connections under destabilizing conditions in vitro. The figure shows data obtained from screening for stabilizing effects of tested compounds on the excitatory synaptic connections under destabilizing conditions. Images were obtained using an Opera Phenix® microscope at 40x magnification. The data were obtained from 3 or 4 biological repeats for each compound tested. Each biological replicate consisted of cells cultured in two separate culture wells. Each experiment contained a control variant (CTR, 0.1% DMSO), a destabilization variant (GBZ, 10 pM GBZ in 0.1% DMSO) and a test condition (2.5 pM test compound and 10 pM GBZ in 0.1% DMSO). (A) Scheme of the experiment in which effect of compounds from the Prestwick library (ZW) were tested for ability to stabilizie excitatory synaptic connections under destabilizing conditions (GBZ). (B) Diagram showing numbers of tested, preselected and finaly selected compounds promoting excitatory synaptic connections. (C) Representative image of in vitro cultured hippocampal neuron with immunofluorescently stained GluA1 for measurement of GluA1 immunofluorescence intensity in individual synapses. (D) Results of quantitative analysis of for normalized of GluA1 fluorescence intensity at the synapse in control (CTR) and destabilizing (GBZ) conditions carried out during screening. Data are presented as median (± MinMax) The analysis was based on data obtained from 460 wells (for CTR) and 485 wells (for GBZ) of in vitro cultured neurons. **** - p < 0.0001 , by Mann-Whitney test. (E) Graph showing the stabilizing effect of the selected compounds on excitatory synaptic connections (based on the parameter of GluA1 intensity at the synapse), median ± MinMax (normalized data). The destabilization level was determined based on data from all experiments for the destabilization condition (dotted line). The level of selection was determined based on the median from the control conditions (solid line), above which 120 compounds were selected for further analysis. The chart also delineates antipsychotic and antidepressant compounds.

[0022] Figure 5: Characterization of the specificity of the compound's synaptic effect on increasing GluA1 intensity in screening. The data show a comparison of the fluorescence intensity of synaptic GluA1 and total GluA1 . Microscopic images were acquired at 40x magnification using an Opera Phenix® microscope. The data obtained were from 3 or 4 biological repeats for each compound tested. Each biological repetition consisted of two separate culture wells. Each experiment contained a control variant (CTR, 0.1% DMSO), a destabilization variant (GBZ, 10 pM GBZ + 0.1% DMSO) and a test condition (2.5 pM test compound [ZW] + 10 pM GBZ + 0.1% DMSO). (A) Representative image of the neuron cultured in vitro immunofluorescently stained for GluA1 . (B) Quantitative analysis of normalized total GluA1 fluorescence intensity in neurons in control (CTR) and in destabilizing (GBZ) conditions. Data are presented as median (±MinMax); Data were obtained from 460 wells for CTR and 485 wells for GBZ of neurons cultured in vitro. **** - p < 0.0001 by Mann-Whitney test. (C) Graph presents a comparison of median GluA1 fluorescence intensities at the synapse (full circles) and total (empty circles) ± MinMax (normalized data) for top 120 hits from Prestwick library. The graph also delineates the level of destabilization for both variants , the level of control for GluA1 intensity at the synapse and total GluA1 .

[0023] Figure 6: In vivo effects of selected compounds on depression-like behavior in tail suspension test (TST). (A) TST scheme (left panel) and doses of compounds used.30 min before the TST, the animals were intraperitoneally administered the test compounds in 0.1% DMSO. The compounds were prepared in solvent, in such a way as not to exceed the administered volume of 10 pl / 1 g of mouse body weight. The control group (CTR) received solvent - 0.1% DMSO, while the reference group received imipramine (30 mg / kg in 0.1% DMSO). B) The graph shows the time spent in immobility by phase of the estrous cycle of each mouse. (C) The graph shows a comparison of the averages between the control group (CTR) and the positive control group (CTR+) in the TST verifying the immobility time for each tested animal (D) The graph shows the comparison of immobility times between between animals of the control group (CTR) and animals that received tested compounds. Each point on the graph represents a single mouse, the column graph represents average values ± SEM., *** - p < 0,001 , ** - p < 0,01 , ns non significant by t-test (for parametric data) or Mann- Whitney test (for non-parametric data). Group sizes were: proestrus - 28 mice; estrus - 15 mice; diestrus - 14 mice; CTR - 10 mice; CTR+ - 8 mice; Dilazep - 7 mice; Penbutolol - 8 mice.

[0024] DETAILED DESCRIPTION:

[0025] The presented invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0027] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0028] The disclosed method provides a reliable and efficient means to screen compounds for their ability to enhance synapse stability in neuron cultures. The high-throughput screening method disclosed herein represents a valuable tool for drug discovery and development in the field of neuropharmacology. The method facilitates the identification of novel compounds capable of modulating excitatory synaptic connections, paving the way for new therapeutic interventions in neurological disorders and synaptic dysfunctions comprising depressive disorder.

[0029] The method comprises the following steps:

[0030] I. Preparation of Neuron Cultures: Neuron cultures are prepared using standard techniques and maintained in appropriate growth media.

[0031] II. Addition of Synaptic Destabilizer and Test Compounds: Neuron cultures are divided into two groups: one group is exposed to a synaptic destabilizer and at least one test compound, while the other group is exposed to the synaptic destabilizer alone.

[0032] III. Monitoring Postsynaptic Compartment: The postsynaptic compartments of individual neurons in in vitro culture in both groups is monitored. Changes in synaptic stability are indicated by alterations in postsynaptic compartment measured e.g. by a fluorescent signal.

[0033] IV. Data Analysis: The obtained data is analyzed to quantify the degree of synapse stabilization induced by the test compounds compared to control synapses lacking the test compound. A compound is identified as a potential enhancer of synapse stability when induces a stabilization of the synapses at least at the level of neurons treated with no destabiliser.

[0034] In one embodiment, the method comprises culturing neuronal cells derived from any sources in suitable dishes under conditions conducive to synaptic development and maintenance.

[0035] In the preferred embodiment, the method comprises the use of rat primary in vitro cultures enriched in dissociated hippocampal neurons. In other embodiment, the method comprises the use of rodent or primate primary neurons, neuronal and neuron-like cell lines, stem cell-derived neurons, genetically modified neurons, dissociated neuronal cultures, organotypic brain slice cultures and brain organoids. The neurons used in the present invention may be of various origins, including mammalian origin (such as rodents, human beings, primates, etc.), chicken, zebrafish, etc.

[0036] Furthermore, in another particular embodiment, the present invention comprises adding of treating several different isolated neuronal cell populations with the candidate compound(s) in parallel. The invention thus also encompasses the use, in parallel, of several populations of isolated neurons.

[0037] The present method relates to a screening method for evaluating synaptic stability potential, suitable for identifying compounds capable of modulating synaptic stability and enhancing excitatory neurotransmission. The screening method according to the present invention is useful for searching for compounds that can serve as a prophylactic or therapeutic agent for neuropsychiatric disorders, such as depressive disorder. The disclosed invention is particularly adapted for the screening of large numbers of compounds, such as combinatorial libraries of compounds. Indeed, the disclosed invention provides materials and methods allowing efficient and simple screening of several compounds in short periods of time. In particular, the disclosed methods can be partially or fully automated, thereby allowing efficient and simultaneous screening of large sets of compounds.

[0038] The method comprises the induction of synaptic destabilization. In a further embodiment, the method involves inducing conditions that destabilize synaptic connections by adding an appropriate destabilizing compound or inducing them by other methods.

[0039] As used herein, “synaptic destabilization” refers to the disruption or impairment of the structural and functional integrity of synapses. In a preferred embodiment, the method comprises induction of synaptic destabilization using a chronic treatment with an antagonist of GABA-A receptors. In a more preferred embodiment, the method comprises induction of synaptic destabilization using gabazine salt, solvate, clathrate, polymorph, or co-crystal thereof.

[0040] In other embodiment, the method comprises the induction of synaptic destabilization using chemical treatment, genetic, chemogenetic or optogenetic manipulations, and / or electrophysiological manipulations.

[0041] In other embodiment, the method comprises the induction of synaptic destabilization using synaptic blockers, neurotoxins, and glutamate receptor antagonists.

[0042] In alternative embodiments, synaptic destabilizers comprise A 40 (Beta-Amyloid 40), Ap42 (Beta-Amyloid 42), Ketamine, Phencyclidine (PCP), MK-801 , 6-Hydroxydopamine (6-OHDA), 1-Methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP), Rotenone, Paraquat, Alpha-synuclein, Huntingtin protein, Islet amyloid polypeptide (IAPP), Anti-NMDA receptor antibodies, Anti- synaptic vesicle protein antibodies, Sarcosine, Org 25935, Tumor necrosis factor-alpha (TNF- a), lnterleukin-1 beta (IL-1 P), lnterleukin-6 (IL-6), Reactive oxygen species (ROS), Botulinum toxin A, Botulinum toxin B, Synaptophysin analogs, Synapsin analogs

[0043] As indicated above, the current invention is particularly suited for simultaneous (optionally parallel) testing of several different candidate compounds.

[0044] As used herein, the term “compound” refers to compounds and / or compositions that are to be screened for their ability to modulate synaptic stability. Test compounds may include a wide variety of different compounds, including chemical compounds, mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. Compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Compounds can be obtained from libraries, such as natural product libraries, combinatorial libraries or FDA approved libraries. As used herein, “synapses” are asymmetric communication junctions formed between two neurons, or, at the neuromuscular junction (NMJ) between a neuron and a muscle cell. Chemical synapses enable cell-to-cell communication via secretion of neurotransmitters, whereas in electrical synapses signals are transmitted through gap junctions, specialized intercellular channels that permit ionic current flow at the mature NMJ, pre- and postsynaptic membranes are separated by a synaptic cleft containing extracellular proteins that form the basal lamina. At the chemical synapses (neuron to neuron or neuron to muscle) synaptic vesicles are clustered at the presynaptic release site, while their receptors are clustered at the postsynaptic membrane. The pre- and postsynaptic sites are separated by the so-called synaptic cleft and anchored by specialized scaffold proteins.

[0045] Immunofluorescence staining techniques are employed to label synaptic structures, receptors, or integral components of the receptor complex. In one embodiment, antibodies recognizing synaptic markers such as PSD-95, synaptophysin, or Bassoon, along with antibodies against GluA1 receptors, facilitate visualization and quantification of excitatory synaptic connections and GluA1 receptor intensity.

[0046] In one embodiment, the method comprises monitoring postsynaptic activity. In a more preferred embodiment, the method comprises monitoring postsynaptic activity using confocal fluorescence microscopy. Examplary embodiment of the method involves the use of a specialized microscope, such as the Opera Phenix® High-Content Screening System, with a 40x water objective and dedicated software for image acquisition and analysis. By implementing this embodiment, researchers can efficiently screen chemical compounds and assess their effects on synaptic stability in neuronal cultures, with potential applications in drug discovery and neurobiological research.

[0047] In the disclosed embodiment, the software identifies synaptic puncta, measures their spatial distribution and intensity, and calculates postsynaptic activity using parameters comprising fluorescence intensity of postsynaptic marker.

[0048] The method disclosed comprises the use of postsynaptic biomarkers for measuring postsynaptic activity. The list of biomarkers that can be used comprise: Synaptophysin, Synaptotagmin, Synapsin, PSD-95, SNAP-25, VAMP, Syntaxin, Bassoon, Piccolo, Neuroligin, Neurexin, Glutamate receptors, GABA receptors, Dopamine receptors, Serotonin receptors, Acetylcholine receptors, Transporters, Brain-derived neurotrophic factor (BDNF), Nerve growth factor (NGF), Glial cell line-derived neurotrophic factor (GDNF), Neurotrophin-3 (NT- 3), Neurotrophin-4 / 5 (NT-4 / 5), FM dyes, Arc / Arg3.1 , c-fos, GCaMP genetic calcium ion sensors, phosphorylated synapsin, phosphorylated NMDA receptors, miR-132, miR-134, miR- 138, miR-124, miR-125, Reelin, Thrombospondins, Tenascins, Glypicans, Heparan sulfate proteoglycans, Shank proteins (Shankl , Shank2, Shank3), Homer proteins (Homerl , Homer2, Homer3), AMPA receptor subunits (GluA1 , GluA2, GluA3, GluA4), NMDA receptor subunits (GluN1 , GluN2A, GluN2B, GluN2C, GluN2D), mGluR receptors (mGluRI , mGluR2, mGluR3, mGluR5), Neurogranin, ProSAP / SPAR, Growth-associated protein 43 (GAP-43), Synaptopodin, Neuroplastin, Glutamate decarboxylase (GAD), Vesicular glutamate transporter (VGLUT), Tyrosine hydroxylase (TH), Choline acetyltransferase (ChAT), Gephyrin, CaMKII,.. Probably some more channels like potassium channels should be added here. Also staining with compounds selectively higligting F-actin etc. I can think of some other proteins used for staining of both compartments.

[0049] A major component of the new drug discovery paradigm is a continually growing family of fluorescent and luminescent reagents that are used to measure the temporal and spatial distribution, content, and activity of intracellular ions, metabolites, macromolecules, and organelles. Classes of these reagents include labeling reagents that measure the distribution and amount of molecules in living and fixed cells, environmental indicators to report signal transduction events in time and space, and fluorescent protein biosensors to measure target molecular activities within living cells. Those skilled in this art will recognize a wide variety of fluorescent reporter molecules that can be used in the present invention, including, but not limited to, fluorescently labeled biomolecules such as proteins, phospholipids and DNA hybridizing probes. Similarly, fluorescent reagents specifically synthesized with particular chemical properties of binding or association have been used as fluorescent reporter molecules Fluorescently labeled antibodies are particularly useful reporter molecules due to their high degree of specificity for attaching to a single molecular target in a mixture of molecules as complex as a cell or tissue.

[0050] Additionally, cells can be genetically engineered to express reporter molecules, such as green fluorescent protein (GFP), coupled to a protein of interest as previously described.

[0051] Once in the cell, the fluorescent probes accumulate at their target domain as a result of specific and high affinity interactions with the target domain or other modes of molecular targeting such as signal-sequence-mediated transport. Fluorescently labeled reporter molecules are useful for determining the location, amount and chemical environment of the reporter.

[0052] In alternative embodiments, the method comprises measurement of synaptic stability can be done using methods comprising electrophysiology, immunohistochemistry assays, synaptic density and morphology analysis or live imaging of synapses.

[0053] As used herein, synaptic stability refers to the maintenance of the structural and functional integrity of synapses, which are the connections between neurons where communication occurs. In one embodiment, synaptic stability is measured using marker proteins. In alternative embodiments, synaptic stability comprises measuring synapse structural integrity, molecular composition, activity-dependent plasticity, and neurotransmitter regulation. For example, candidate agents may be identified by known pharmacology, by structure analysis, by rational drug design using computer-based modelling, and the like.

[0054] In proffered embodiment of the present invention, a cell screening system is provided comprising a high magnification fluorescence optical system having a microscope objective, an XY stage adapted for holding a plate with an array of locations for holding cells and having a means for moving the plate to align the locations with the microscope objective and a means for moving the plate in the direction to effect focusing; a digital camera; a light source having optical means for directing excitation light to cells in the array of locations and a means for directing fluorescent light emitted from the cells to the light detector; and a computer means for receiving and processing digital data from the digital camera wherein the computer means includes: a digital frame grabber for receiving the images from the camera, a display for user interaction and display of assay results, digital storage media for data storage and archiving, and means for control, acquisition, processing and display of results.

[0055] Screening for large numbers of compounds for activity with respect to a particular biological function requires preparing arrays of cells for parallel handling of cells and reagents. In one embodiment, method comprises but is not limited to the use of 96 well microtiter plates for compatibility with current automated loading and robotic handling systems. The smaller format of a microplate increases the overall efficiency of the system by minimizing the quantities of the reagents, storage and handling during preparation and the overall movement required for the scanning operation.

[0056] As used herein, “control synapses” are synapses in which the synaptic destabilization was induced without adding the test compound. Validation of destabilization control can be performed by measuring synaptic stability in synapses where synapse destabilization was not conducted.

[0057] According to the invention synaptic stability can be determined on a single synapse basis or as a mean value of synapses under tested conditions in comparison with a mean value of control synapses. If the synaptic stability is determined on a single synaptic basis, determining synaptic stability comprises that a synaptic stability markers are used that can be analyzed by image acquiring methods comprising of any type of fluorescence microscopy, comprising confocal microscopy, spinning disc microscopy, two photon microscopy and light sheet microscopy or transmission light microscopy comprising bright-field microscopy, dark-field microscopy, phase contrast microscopy, polarization microscopy.

[0058] Differential interference contrast optics microscopy. This way not only information about total synaptic stability is obtained but also which synapses in specific are stabilised after treatment with the test compounds. In another embodiment the synaptic stability is determined not on a single synapsis basis, but as the mean value of appropriate number of synapses treated with the respective test compound. This value can be expressed as a fold increase in comparison with control synapses.

[0059] Anti-depressant effects of screened compounds may be assessed further using well- established rodent behavioral assays such as the tail suspension test, the forced swim test or response to mild immobilization tests or mild unpredictable stress or any other type of stress paradigm or test used by a person skilled in art.

[0060] The terms "screen" and "screening" and the like as used herein mean to test an agent to determine if it has a particular action or efficacy.

[0061] The terms "identification", "identify", "identifying" and the like as used herein means to recognize an agent as being effective for a particular use.

[0062] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e. , the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0063] There are no fixed values when a test compound is considered to have synaptic stability enhancement potential or not. This depends on a plurality of factors such as the origin of the neuronal cells, the type of synaptic destabilization inducer, the test compound, the concentration, the general condition of the neuronal cells, the exact culture and assay conditions and other factors. Evaluation of synaptic stability enhancement potential must always be viewed with regard to control synapses. A person skilled in the art will be able to determine the result above which the tested compound will be considered to have synaptic stability enhancement potential or not.

[0064] One skilled in the art will recognize that, both in vivo and in vitro trials using suitable, known and generally accepted cell and / or animal models are predictive of the ability of a test compound to treat or prevent a given disorder.

[0065] In another aspect, the present disclosure relates to a method for treating depressive disorder in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of dilazep, also known as 35898-87-4, F8KLC2BD5Z, CHEMBL126075, having the SMILES code:

[0066] COC1=CC(=CC(=C1OC)OC)C(=O)OCCCN2CCCN(CC2)CCCOC(=O)C3=CC(=C(C(=C3)O C)OC)OC and described by a chemical formula: and salts thereof.

[0067] In another aspect, the present disclosure relates to a method for treating depressive disorder in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of penbutolol, also known as 38363-40-5, (-)-Penbutolol, Betapressin, Levatol, having the SMILES code:

[0068] CC(C)(C)NCC(COC1=CC=CC=C1C2CCCC2)O and described by a chemical formula: and salts thereof.

[0069] For use in subjects, the salts of the compounds of this invention refer to non-toxic pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate.

[0070] On one aspect, the present invention is directed to methods for the treatment of “depressive disorders”, which may also be called “depression” interchangeably. As used herein, the term “depressive disorders” comprises but is not limited to all depressive disorders included in the International Classification of Diseases, (ICD-11 , https: / / icd.who.int / browse / 2024- 01 / mms / en#1563440232) and Diagnostic And Statistical Manual Of Mental Disorders (DSM- 5-TR, American Psychiatric Association. (2013), https: / / doi.org / 10.1176 / appi.books.9780890425596) such as major depressive disorder, single episode depressive disorder, dysthymic disorder, mixed depressive and anxiety disorder, premenstrual dysphoric disorder, persistent depressive disorder, premenstrual dysphoric disorder, substance / medication-induced depressive disorder, depressive disorder due to another medical condition other specified depressive disorder and unspecified depressive disorder.

[0071] Depressive disorders can be characterized by sadness, loss of interest in activities, and decreased energy. Other symptoms include loss of confidence and self-esteem, inappropriate guilt, thoughts of death and suicide, diminished concentration, and disturbance of sleep and appetite. A variety of somatic symptoms can also be present. Though depressive feelings are common, especially after experiencing setbacks in life, depression is diagnosed only when the symptoms reach a threshold and last at least two weeks. Depressive disorders can vary in severity from mild to very severe, and includes unipolar and bipolar depressive disorder, as well as seasonal affective disorder (SAD). Depressive disorder is typically also characterized into eight basic dimensions i.e., Pessimism, Weak Concentration, Sleep Problems, Anhedonia, Fatigue, Loneliness, Low Self-esteem, and Somatic Complaints to define the profile of children's and adolescents depression. Depressive disorder can occur as an idiopathic disease (with no somatic disease associated with it), or it can be a psychiatric symptom of a somatic disorder, especially a number of neurodegenerative disorders.

[0072] Scales known in the art to be administered in assessing levels of depressive disorder include:

[0073] (1) Hamilton Depressive disorder Rating Scale 28-ltem: primary outcome measure (Hamilton M. J., Neurol Neurosurg Psychiatry 1960; 23:56-62; Hamilton M., Br J Social Clin Psychology 1967; 6:278-296).

[0074] (2) Columbia-Suicide Severity Rating Scale (Posner K. et al., Am J Psychiatry.2011 ; 168(12): 1266-77).

[0075] (3) Clinical Global Improvement Scale' Severity and Improvement (Guy W. Clinical Global Impression (CGI) ECDEU Assessment manual for Psychopharmacology. Rockville, Md.: U.S. Dept Health Education and Welfare 1976).

[0076] (4) Quick Inventory of Depressive Symptoms, Self-Report version (Trivedi M. H. et al., Psychol Med. 2004; 34(1):73-82).

[0077] (5) Concise Health Risk Tracking (Trivedi M. H. et al., J Clin Psychiatry. 2011 ;72(6):757-64).

[0078] (6) MGH Cognitive and Physical Functioning Questionnaire (Fava M. et al., Psychother Psychosom. 2009; 78(2):91-7).

[0079] (7) Quality of Life Enjoyment and Satisfaction Questionnaire (Endicott J. et al., Psychopharmacol Bull. 1993; 29(2):321 -6).

[0080] (8) Brief Psychiatric Rating Scale (Andersen J. et al. Psychopathology. 1989; 22(2-3): 168-76; Hafkenscheid A., Acta Psychiatr Scand. 1991 ; 84(3):294-300). (9) Only at baseline and end of Phase 3-Neurocognitive Test Battery: immediate and delayed verbal recall, speed of comprehension, digit span forward and backward, N-backtest, and trailmaking task (Trandafir A. et al., Schizophr Res. 2006; 81 (2-3):217-26).

[0081] One skilled in the art will further recognize that in the methods and dosing regimens of the present invention, the maintenance of the antidepressant response in a subject may be determine by for example, a clinician, physician, psychiatrist, psychologist, or other suitable medical professional. Additionally, maintenance of the antidepressant response may be established by for example, an absence of relapse of the depressive disorder (or one or more symptoms of the depressive disorder), an absence of the need for additional or alternate treatment(s) for the depressive disorder, an absence of the worsening of the depressive disorder, an absence of the need for hospitalization for a suicidal attempt or to prevent suicide.

[0082] As used herein, the term "subject" refers to a warm-blooded animal, such as a human that would benefit biologically, medically or in quality of life from the treatment. The subject can be mammals and non-mammals. Examples of the mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of the non-mammals include, but are not limited to, birds, fish and the like. In one embodiment, the subject is human. It may be a human who has been diagnosed as in need of treatment for a disease or disorder disclosed herein. As used herein, the term "treat," "treating," or "treatment" of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the subject. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder, either physically (e.g., through stabilization of a discernible symptom), physiologically, (e.g., through stabilization of a physical parameter), or both. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0083] As used herein, the term “administering” includes all means of introducing the compounds and compositions described herein to the subject. Examples of suitable methods of administration include, but are not limited to, oral, intravenous (iv), intranasal (in) intramuscular (im), subcutaneous (sc), transdermal, and rectal. Compounds may also be administered directly to the nervous system including, but not limited to, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and / or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and / or catheters with or without pump devices. The compositions of Ocompounds may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0084] The terms "prevent", "prevention", and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.

[0085] The term "in need thereof’ would be a subject known or suspected of having or being at risk of depressive disorder, or a disease or condition that causes neurodegeneration and / or a synaptic dysfunction.

[0086] A subject in need of treatment would be one that has already developed the disease. A subject in need of prevention would be one with risk factors of the disease.

[0087] The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject. The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

[0088] The present invention further comprises pharmaceutical compositions containing dilazep, penbutolol, or a combination thereof with a pharmaceutically acceptable carrier. Pharmaceutical compositions containing one or more of the compounds of the invention described herein as the active ingredient can be prepared by intimately mixing the compound or compounds with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending upon the desired route of administration (e.g., oral, parenteral). Thus for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives comprise water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral preparations, such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Solid oral preparations may also be coated with substances such as sugars or be enteric-coated so as to modulate major site of absorption. For parenteral administration, the carrier will usually consist of sterile water and other ingredients may be added to increase solubility or preservation.

[0089] Injectable suspensions or solutions may also be prepared utilizing aqueous carriers along with appropriate additives.

[0090] To prepare the pharmaceutical compositions of this invention, one or more compounds of the present invention as the active ingredient is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending of the form of preparation desired for administration, e.g., oral or parenteral such as intramuscular. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed.

[0091] Thus, for liquid oral preparations, such as for example, suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders; disintegrating agents and the like.

[0092] Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, through other ingredients, for example, for purposes such as aiding solubility or for preservation, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient necessary to deliver an effective dose as described above. The pharmaceutical compositions herein will contain, per unit dosage unit, e.g., tablet, capsule, powder, injection, suppository, teaspoonful and the like, of from about 0.001-1000 mg and may be given at a dosage of from about 0.01-200.0 mg / kg / day, preferably from about 0.1 to 100 mg / kg / day, more preferably from about 0.1-50 mg / kg / day, or any range therein. The dosages, however, may be varied depending upon the requirement of the subjects, the severity of the condition being treated and the compound being employed. The use of either daily administration or post-periodic dosing may be employed.

[0093] Therapeutically effective dosage levels and dosage regimens for disclosed compounds may be readily determined by one of ordinary skill in the art. For example, therapeutic dosage amounts and regimens for pharmaceutical agents approved for sale are publicly available, for example as listed on packaging labels, in standard dosage guidelines, in standard dosage references such as the Physician's Desk Reference (Medical Economics Company or online at http: / / www.pdrel.com) or other sources.

[0094] One skilled in the art will recognize that wherein the present invention is directed to methods of prevention, a subject in need of thereof (i.e. a subject in need of prevention) shall include any subject (preferably a mammal, more preferably a human) who has experienced or exhibited at least one symptom of the disorder, disease or condition to be prevented. Further, a subject in need thereof may additionally be a subject (preferably a mammal, more preferably a human) who has not exhibited any symptoms of the disorder, disease or condition to be prevented, but who has been deemed by a physician, clinician or other medical profession to be at risk of developing said disorder, disease or condition. For example, the subject may be deemed at risk of developing a disorder, disease or condition (and therefore in need of prevention or preventive treatment) as a consequence of the subject's medical history, including, but not limited to, family history, pre-disposition, co-existing (comorbid) disorders or conditions, genetic testing, and the like.

[0095] As used herein, the term “comprising one or more additional therapeutics”, “co-therapy”, “combination therapy”, “adjunctive treatment”, “adjunctive therapy” and “combined treatment” shall mean treatment of a subject in need thereof by administering dilazep, penbutolol or a combination thereof in combination with one or more additional therapeutic comprising antidepressants, anxiolytics, mood stabilizers, antipsychotics, and combinations thereof by any suitable means, simultaneously, sequentially, separately or in a single pharmaceutical formulation. Where the compounds and the other therapeutics are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds and additional therapeutics may be administered via the same or different routes of administration. Examples of suitable methods of administration include, but are not limited to, oral, intravenous (iv), intranasal (in) intramuscular (im), subcutaneous (sc), transdermal, and rectal. Compounds and additional therapeutics may also be administered directly to the nervous system including, but not limited to, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and / or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and / or catheters with or without pump devices. The compounds and additional therapeutics may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0096] As used herein, unless otherwise noted, the term “additional therapeutic” shall mean any pharmaceutical agent which can be used to treat depressive disorder. Suitable examples include, but are not limited to mono-amine oxidase inhibitors such as phenelzine, tranylcypromine, moclobemide, and the like; tricyclics such as imipramine, amitriptyline, desipramine, nortriptyline, doxepin, protriptyline, trimipramine, chlomipramine, amoxapine, and the like; tetracyclics such as maprotiline, and the like; non-cyclics such as nomifensine, and the like; triazolopyridines such as trazodone, and the like; serotonin reuptake inhibitors such as fluoxetine, sertraline, paroxetine, citalopram, citolapram, escitolapram, fluvoxamine, and the like; serotonin receptor antagonists such as nefazadone, and the like; serotonin noradrenergic reuptake inhibitors such as venlafaxine, milnacipran, desvenlafaxine, duloxetine and the like; noradrenergic and specific serotonergic agents such as mirtazapine, and the like; noradrenaline reuptake inhibitors such as reboxetine, edivoxetine and the like; atypical antidepressants such as bupropion, and the like; natural products such as Kava-Kava, St. John's Wort, and the like; dietary supplements such as s-adenosylmethionine, and the like; and neuropeptides such as thyrotropin-releasing hormone and the like; compounds targeting neuropeptide receptors such as neurokinin receptor antagonists and the like; and hormones such as triiodothyronine, and the like. Preferably, the antidepressant is selected from the group consisting of fluoxetine, imipramine, bupropion, venlafaxine and sertaline. Wherein the present invention is directed to therapy with a combination of agents, “therapeutically effective amount” shall mean that amount of the combination of agents taken together so that the combined effect elicits the desired biological or medicinal response. For example, the therapeutically effective amount of combination therapy comprising dilazep and a serotonin reuptake inhibitor would be the amount of dilazep and the amount of the serotonin reuptake inhibitor that when taken together or sequentially have a combined effect that is therapeutically effective, more preferably where the combined effect is synergistic. Further, it will be recognized by one skilled in the art that in the case of combination therapy with a therapeutically effect amount, the amount of each component of the combination individually may or may not be therapeutically effective.

[0097] Compounds of this invention may be administered in any of the foregoing compositions and according to dosage regimens established in the art whenever treatment of depressive disorder is required.

[0098] Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound or combinations used, the mode of administration, the strength of the preparation and the advancement of the disease condition. In addition, factors associated with the particular subject being treated, including subject's sex, age, weight, diet, time of administration and concomitant diseases, will result in the need to adjust dosages.

[0099] The following Examples are set forth to aid in the understanding of the invention, and are not intended and should not be construed to limit in any way the invention set forth in the claims which follow thereafter.

[0100] EXAMPLES:

[0101] Example 1 :

[0102] Treatment of primary neuronal cultures with chemical compounds during in vitro screening

[0103] To analyze the excitatory synaptic connections, rat hippocampal neurons obtained from embryonic day 16 embryos were cultured on 96-well dishes according to the standard laboratory protocols (e.g., Jaworski et al., 2005; Urbanska et al. ,2012). Chemical compounds (2.5 pM final concentration) along with Gabazine (GBZ, 10 pM) or GBZ alone were administered to the neurons at 16 and 18 days in vitro (DIV). Cells were then cultured untill 21 DIV and fixed. Fixed cells were next stained immunofluorescently to visualize synaptic connections as described below which afterward were analyzed using an Opera Phenix high- throughput microscope (see below for more technical details). A scheme of the experiment is shown in Figure 1 . On each 96-well dish there were 38 experimental variants. The first, a negative control variant, consisted of neurons treated with solvent DMSO (with a final concentration of 0.1%). The second variant was neurons treated with 10 pM gabazine (GBZ), which mimicked the conditions of destabilization of neuronal connections. The other variants were neurons treated with the tested chemicals at a final concentration of 2.5 pM administered with 10 pM GBZ and solvent. Stable final concentrations of the compounds were obtained as shown in Figure 1C. Briefly, a pre-prepared medium containing GBZ dissolved in DMSO was spread in a 96-well dish and the test compounds (from the derivative dish with compounds at 2.5 mM concentration) were added to it. In this way, a pooled working solution was created. In order not to change the final concentration of the used compounds, 15 pl of the neuronal culture media from each well was replaced with 15 pl of the mixture of GBZ and the test compound was added to the neuronal cultures (2 wells per test compound in each neuronal dish). An analogical approach was used for adding to neurons 15 pl of the medium-solvent mixture (negative control conditions, S wells in each dish) and 15 pl of the GBZ-solvent mixture in the medium (destabilizing conditions, 3 wells in each dish).

[0104] Immunofluorescence staining and slide preparation Cells were fixed in 21 DIVfor 10 min. in room temperature with 80 pl of fixation buffer (4% PFA in phosphate buffer supplemented with 4% sucrose) per well . Next fixed cells were washed 3 times with PBS and incubated overnight at 4°C with the mouse anti-Bassoon primary and rabbit anti-AMPA 1 (GluA1) diluted in xxxx at 1 :400 and 1 :300, respectively and incubated overnight. The next day, cells were washed 3 times, 10 min each wash, with PBS and incubated with donkey anti-mouse IgG Alexa Fluor® 488 and donkey anti-rabbit Alexa Fluor® 568 secondary antibodies diluted 1 :500 in xyz. The wells were then washed once with PBS and incubated for 10 min with Hoechst 33258 dye diluted in PBS (XYZ final concentration) to visualize cell nuclei. Finally, the specimens were washed twice with PBS and the cells were left in 150 pl of PBS per well. Cells prepared this way were stored at 4°C in the dark until analysis (no longer than a week).

[0105] Image acquisition

[0106] Acquisition of microscopic images of neurons Hoechst 33258 stained nuclei and immunofluorescently labelled Bassoon and GluA1 was carried out with an Opera Phenix microscope, using a 40x water objective. Dedicated Harmony 4.9 software was used for both image registration and analysis. From 96-well dish, where neurons were cultured, an image of each well , which consisted of 25 fields of view located in its center, was acquired. The laser intensity setting was adjusted for each staining individually, but the value remained constant throughout the given biological repeat. During image acquisition, a darkened chamber was used to hold the analyzed dishes and a robotic arm (plate handler II, PerkinElmer) was used to transfer the dishes from the chamber to the microscope. After setting the appropriate acquisition parameters, the dishes were positioned in the chamber in a specific order. The arm was then programmed to sequentially move each plate into the interior of the microscope, put it back in place after image registration and move the next plate. In that way, images of 5 to 7 96-well dishes from single experiment repetitionwere recorded overnight.

[0107] Analysis of stability of excitatory synaptic connections and total GluA1 intensity

[0108] Harmony 4.9 software, based on sequential analysis blocks, was used to analyze the images for assessing stability of excitatory synapses. Each culture dish was accompanied by a plan naming each well in the dish according to the experimental variant. Fig. 2 shows an analysis scheme to characterize excitatory synaptic connections. The analysis began with step 1 (Fig. 2A), in which an analysis area with immunofluorescently labeled Bassoon and GluA1 proteins was selected. Step 2 (Fig. 2B) consisted of determining points / areas (representing the synapse) based on Bassoon protein staining. These points had a specific radius (1.12 pm), thus determining not only the presynaptic side indicated by Bassoon imunofluresnet puncta, but also its surroundings containing both postsynaptic part of the synapse. Then, in step 3 (Fig. 2C), the IF signal intensity of the GluA1 protein within the previously delineated synapses was measured. In the resulting data, each synapse point was measured separately (values were not summed). In Step 4 (Figure 2D) GluA1 intensity across the entire area (in and out of synapses) was measured. In this way, all cells were analyzed, and the result was the sum of the intensities of all fields of one well.

[0109] Processing of data obtained during the screening test

[0110] The raw data obtained required further processing to obtain the final results. The following section describes the processing of raw data obtained from the analysis of excitatory synaptic connections (GluA1 intensity at the synapse), total GluA1 intensity.. Analysis of synaptic connections and total GluA1 intensity after exporting data from Harmony 4.9 was analyzed using RStudio software (R version 4.2.1). A proprietary script was created in RStudio to extract and visualize relevant data. The first analyzed parameter was the intensity of GluA1 immunofluorescence at the synapse, where the effect of destabilization (GBZ) was compared to the effect of the tested compound under destabilization conditions. In this way, it was determined whether the compound decreased or increased the number of synapses saturated with the AMPA receptor containing the GluA1 subunit. Next, the intensity of GluA1 immunofluorescence was determined at each synapse separately fora density distribution analysis. This analysis began by creating a density plot for the destabilizing conditions, where the number of synapses assigned to a given GluA1 intensity value was determined (Fig. 3A). To ensure correctness and robustness of analysis (confidence of correct GluA1 staining), the following steps focused only on synapses with the highest GluA1 intensity / saturation. Therefore, it was checked what values of GluA1 immunofluorescence intensity charcterized 30% of synapses with the highest intensity under destabilizing conditions. Based on this, the level of destabilization was determined. Given the variability between cultures, the destabilization level was a unique value for each individual biological replicate. Based on the destabilization value (i.e., the specific value of GluA1 intensity for GBZ treatment), it was checked whether there would be an increase in the percentage of synapses at the highest GluA1 intensity under the test conditions (Fig. 3B). If there was more than 30% synapses above the destabilization level, this meant an increased number of synapses saturated with GluA1. Such a result was interpreted as a stabilizing effect of the tested compound on excitatory synaptic connections under destabilizing conditions. If the value was below 30%, it meant a lower number of excitatory synapses and no stabilizing effect of the tested compound. The second parameter analyzed was the total GluA1 immunofluorescence intensity (i.e., in and out of the synapse). In this case, since the data obtained was the average value of GluA1 intensity in the whole well, it was not analyzed by density plots. Analysis of this parameter aimed to answer the question of the nature of the compound's action, namely whether it has a synapse-specific effect or increases GluA1 expression in the whole cell.

[0111] Results:

[0112] Identification of compounds that stabilize excitatory synaptic connections in neurons cultured in vitro under induced destabilization conditions using high-throughput screening.

[0113] For the screening experiment, the Prestwick library was used, which contains 1520 compounds of great chemical diversity and with a wide range of therapeutic applications, most of which are compounds approved for use in the clinic. During the screening, each plate analyzed contained 3 control wells (final concentrations of 0.1% DMSO), 3 wells from destabilizing conditions (final concentrations of 10 pM GBZ in 0.1% DMSO), and 2 test wells (after administration of GBZ along with the tested compound at a concentration of 2.5 pM). Each compound was tested at least in 3 biological replicates. A schemeof the experimental steps performed (consistent with the previously established model) is shown in Figure 4A. In order to extract the maximum amount of information on the effect of the compound, the analysis focused on several parameters, as described in the following subsections. The first parameter analyzed was the fluorescence intensity of GluA1 in the synapse. This was the primary parameter for selecting compounds for further analysis. The second parameter was the total GluA1 fluorescence intensity. The comparison of the GluA1 fluorescence parameter at the synapse and total GluA1 fluorescence was intended to determine whether the effect of the chemical compound is specific to synapses.

[0114] Screening the effects of chemical compounds on the stability of excitatory synaptic connections

[0115] To verify whether the tested compound stabilized excitatory synaptic connections, the parameter of GluA1 fluorescence intensity at the synapse was analyzed. After measuring GluA1 fluorescence intensity at synapses in all tested variants, the data were normalized within each dish separately for subsequent direct comparison. Replicates in which the induction of destabilizing conditions failed (no difference between negative control and GBZ administration) were discarded at this stage. The data were then ranked from the highest GluA1 fluorescence intensity values based on the median value for each variant. Given that there were as many as 950 compounds above the level of destabilization (above the median from GBZ), the group was narrowed down, focusing only on those compounds that increased synapse enrichment in GluA1 to the highest degree. A cutoff level was set at the median of the negative control group. 136 compounds were identified as passing this treshold. Taking into account the fact of automatic analysis, devoid of experimenter verification, additional analysis of microscopic images was performed for cell status for each of the 136 preselected compounds (Fig. 4.8B). As a result, of the preselected 136 compounds, 16 were rejected due to clearly visible toxicity. Further analyses were performed for 120 pre-selected compounds, as summarized in Figure 4.8D. Among the identified compounds, 20% (25 of 120) have antidepressant or antipsychotic activity. These compounds are highlighted in the graph. Such a high proportion of compounds with antidepressant or antipsychotic characteristics confirms that the developed model of induced destabilization of neuronal connection networks in vitro is useful for identifying compounds with antidepressant characteristics in the screening system. The selected 120 compounds that promoted excitatory neuronal connections were further characterized as described below to select the most optimal compounds for further in vivo experiments.

[0116] Screening the effect of chemical compounds on total GluA1 intensity

[0117] Studies have shown that the number of excitatory AMPA receptors (including the GluA1 subunit), among other things, is regulated by the activity of the neuronal network (Malinov and Malenka, 2002). The studied model of destabilization of excitatory synaptic connections is associated with reduced saturation of the postsynaptic part with the AMPA receptor (more precisely, the GluA1 subunit), which is most likely the reason for the reduced activity of the excitatory network. Given the phenomenon of receptor movement in and out of the synapse (), to understand better candidate compound impact on synapse stabilization, it seemed important to answer the question of whether the tested compound acted explicitly to increase the saturation of the synapse in GluA1 , or whether it is a more general phenomenon of increasing the total fluorescence intensity of GluA1 in the cell. Based on available microscopic images, an analysis of the total fluorescence intensity of GluA1 was performed to obtain information determining the nature of the effect caused by a particular chemical compound. The analysis proved that inducing destabilizing conditions significantly reduces both synaptic and total GluA1 fluorescence intensity (Fig. 5A-D). It should be noted that the total value consists of both synaptic and extrasynaptic fluorescence, so it cannot be ruled out that the effect of reducing the total signal largely depends on the decrease in signal within synapses. Figure 5E compares the measurement of GluA1 fluorescence intensity at the synapse and total fluorescence for 120 selected compounds. Most of the compounds, as many as 84, increased both synaptic and total GluA1 fluorescence signal (above the CTR at both parameters). The group of 33 compounds was characterized by specific enrichment of GluA1 only at synapses (above CTR in synaptic GluA1 , but below CTR in total intensity). The remaining 3 compounds, although they increased the GluA1 signal at the synapse above the CTR, did not reverse the GBZ destabilizing effect on total intensity (below the level of destabilization (GBZ) of total intensity)

[0118] Example 2:

[0119] Study of the effects of selected chemical compounds (in vitro) promoting excitatory synaptic connections on an in vivo animal model.

[0120] Animal living conditions

[0121] Female mice of the C57BL / 6 strain provided by the Medical University of Bialystok, Center for Experimental Medicine (CMD) were used for the behavioral tests. In accordance with the decision issued by the Second Local Ethical Commission in Warsaw (LKE WAW2 / 140 / 2022), 60 female C57BL / 6 mice were used in the behavioral test conducted. Animals from CMD were delivered at the age of 11 weeks and then placed in breeding facilities at the International Institute of Molecular and Cellular Biology (IIMCB) in Warsaw. Upon arrival at IIMCB, the animals underwent a 7- day quarantine, during which they were subjected to veterinary observation and adaptation to housing conditions. After quarantine, the animals were moved to the experimental area (holding room) and separated by 4 individuals per cage (15 cages in total). The breeding premises met the requirements of the Decree of the Minister of Agriculture and Rural Development of December 14, 2016, regarding the minimum requirements to be met by the center and the minimum requirements for the care of animals maintained at the facility. The breeding rooms were maintained with a 12 / 12 light cycle, a relative humidity of 55% ± 10% and a temperature of 22°C ± 2°C. GM500 (Sealsafe® PLUS) cages were used - floor area 501 cm2 in the IVC (Individually Ventilated Cages) system. The cages provided 75- fold air exchange per hour and environmental enrichment, which consisted of nesting material and plastic houses. The animals received rodent feed and water ad libitum.

[0122] Preparation of chemical compounds tested on animals

[0123] Based on the in vitro screening, 2 compounds (dilazep and penbutolol) were selected for testing their potential antidepressant effects. The dose of the administered drug was decided based on literature data and information provided by Prestwick. The compounds were dissolved in a solvent (0.1% DMSO) immediately before the intraperitoneal injection. Appropriate concentrations of the compounds (see Figure 6) were prepared to be administered in a volume of 10 pl / 1 g of mouse body weight. In the behavioral experiment conducted, a control group (intraperitoneal administration of 0.1% DMSO) was tested in addition to the groups of mice treated with the compounds. A reference group was also used, in which mice were administered imipramine (30 mg / kg body weight), which shows a positive effect in the behavioral test conducted (Ripoll et al., 2003).

[0124] Tail suspension test

[0125] The Tail suspension test (TST) is a standard test that examines stress-related helplessness and the development of depression. Thirty minutes after intraperitoneal administration of the chemical compound, the cage with the mice was moved to the experimental room. The mice (each individually; at least 7 mice per experimental condition) were then suspended by their tails using 0.8 cm wide adhesive tape at a distance of 1 cm from the end of the tail to a metal bar located about 70 cm from the ground. The design allowed the suspension and measurement of the activity of 4 animals at the same time, as walls separated each mouse from the others. In order to standardize the conditions of the behavioral test, the experimental room was darkened, and the only light source was an LED ring light (Mozos, MLM-RING) located at the height of the camera (ELPUSB4KHDR01- MFV). The animal's behavior was recorded for 6 minutes after suspension. After the test was performed, the mice were returned to their living conditions in the groups they were in before the test was performed. After 24 hours, mice were given a vaginal swab (according to McLean et al., 2012), which was then given to a veterinarian to assess the estrous cycle of individual mice.

[0126] Analysis of results

[0127] The analysis of the results of the behavioral test consisted of measuring how long the mouse remained immobile. Analysis of the raw data (recorded videos) was carried out using a proprietary script based on automatic analysis of animal movement, the time (in seconds) in which the mouse remained motionless over 6 minutes was counted. Additional step involved verification of the obtained data by the experienced researcher to avoid inclusion of false movements, i.e. events interpreted by the script as a movement, when however, the mice remain inactive). Next, curated data data obtained from experimental groups (treated with candidate compounds) and reference group were compared with the control group. The reduction in time spent motionless was interpreted as an antidepressant / antianxiety effect.

[0128] Results:

[0129] Identification of compounds with antidepressant potential in vivo

[0130] Dilazep and Penbutolol were selected from the in vitro screening for subsequent in vivo analysis. It was decided that the compounds would be administered as a single dose, and then their fast-acting effect would be verified. A scheme of the behavioral test is shown in Figure 6A. After quarantine and following the habituation procedure, the animals were intraperitoneally injected with properly prepared compounds. The mice were then placed in a holding cage for 30 minutes, after which TST was performed, recording their activity for 6 minutes. The control group received solvent alone (CTR; 0.1% DMSO), the reference group (CTR +) received imipramine (30 mg / kg body weight in 0.1% DMSO). The doses for the other compounds are presented in Figure 6A. In addition, 24 hours after the test, a vaginal swab was taken to determine the estrous cycle of individual mice. The parameter analyzed in TST was the immobility time. During the data analysis, 3 recordings were disqualified due to an incorrectly performed test. Since females were used in the experiment, the analysis began by testing whether the phase of the estrous cycle affects the time spent motionless. Figure 6B shows the time spent in immobility of individual mice by phase of the estrous cycle (proestrus, estrus, diestrus), where no significant differences were observed between groups. The analysis proved that the administration of imipramine significantly reduced the time in the immobility of the animals compared to the control group (Figure 6C). Figure 6D shows a comparison of the effects of the tested compounds to the control group. Both dilazep and penbutolol statistically significantly reduced mice immobility times, showing their potential antidepressive effect.

[0131] REFERENCES

[0132] 1 . Executive Board, 130. (2012). Global burden of mental disorders and the need for a comprehensive, coordinated response from health and social workers at the country level: Report by the Secretariat (s. Executive Board 130th session, provisional agenda item 6.2). World Health Organization.

[0133] 2. Ettman, C. K., Cohen, G. H., Abdalla, S. M., Sampson, L., Trinquart, L., Castrucci, B. C., Bork, R. H., Clark, M. A., Wilson, I., Vivier, P. M., i Galea, S. (2022). Persistent depressive symptoms during COVID-19: A national, population-representative, longitudinal study of U.S. adults. Lancet Regional Health. Americas, 5, 100091 . https: / / doi.Org / 10.1016 / j.lana.2O21 .100091

[0134] 3. Hou, W. K., Li, T. W., Liang, L., Liu, H., Ettman, C. K., Hobfoll, S. E., Lee, T. M.-C., i Galea, S. (2021 ). Trends of depression and anxiety during massive civil unrest and COVID-19 in Hong Kong, 2019-2020. Journal of Psychiatric Research, 145, 77-84. https: / / d0i.0rg / l 0.1016 / j.jpsychires.2021 .11 .037

[0135] 4. Lorenzoni, G., Azzolina, D., Maresio, E., Gallipoli, S., Ghidina, M., Baidas, S., Berchialla, P., Giron, M. C., Silano, M., Gregori, D., i #PRESTOinsieme Study Group. (2022). Impact of the COVID-19 lockdown on psychological health and nutritional habits in Italy: Results from the #PRESTOinsieme study. BMJ Open, 12(4), e048916. https: / / doi.Org / 10.1136 / bmjopen-2021- 048916

[0136] 5. American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders: DSM-5. (5th edition). American Psychiatric Association.

[0137] Gold, S. M., Kohler- Forsberg, O., Moss-Morris, R., Mehnert, A., Miranda, J. J., Bullinger, M., Steptoe, A., Whooley, M. A., i Otte, C. (2020). Comorbid depression in medical diseases.

[0138] Nature Reviews Disease Primers, 6(1 ), Article 1. https: / / doi.org / 10.1038 / s41572-020-0200-2

[0139] 6. Karrouri, R., Hammani, Z., Benjelloun, R., i Otheman, Y. (2021 ). Major depressive disorder: Validated treatments and future challenges. World Journal of Clinical Cases, 9(31), 9350- 9367. https: / / doi.Org / 10.12998 / wjcc.v9.i31 .9350

[0140] 7. Souery, D., Papakostas, G. I., i Trivedi, M. H. (2006). Treatment-resistant depression. The Journal of Clinical Psychiatry, 67 Suppl 6, 16-22.

[0141] Bergfeld, I. O., Mantione, M., Figee, M., Schuurman, P. R., Lok, A., i Denys, D. (2018). Treatment-resistant depression and suicidality. Journal of Affective Disorders, 235, 362- 367. https: / / d0i.0rg / l 0.1016 / j.jad.2O18.04.016 8. Mrazek, D. A., Hornberger, J. C., Altar, C. A., i Degtiar, I. (2014). A review of the clinical, economic, and societal burden of treatment-resistant depression: 1996-2013. Psychiatric Services (Washington, D.C.), 65(8), 977-987. https: / / doi.org / 10.1176 / appi.ps.201300059

[0142] 9. Goodwin, R. D., Dierker, L. C., Wu, M., Galea, S., Hoven, C. W., i Weinberger, A. H. (2022). Trends in U.S. Depression Prevalence From 2015 to 2020: The Widening Treatment Gap. American Journal of Preventive Medicine, 63(5), 726-733. https: / / doi.Org / 10.1016 / j.amepre.2022.05.014

[0143] 10. Gillman, P. K. (2007). Tricyclic antidepressant pharmacology and therapeutic drug interactions updated. British Journal of Pharmacology, 151 (6), 737-748. https: / / doi.Org / 10.1038 / sj.bjp.0707253

[0144] 11 . Khalid, M. M., i Waseem, M. (2023). Tricyclic Antidepressant Toxicity. W StatPearls. StatPearls Publishing. http: / / www.ncbi.nlm.nih.gov / books / NBK430931 /

[0145] Pare, C. M. B., i Sandler, M. (1959). A CLINICAL AND BIOCHEMICAL STUDY OF ATRIAL OF IPRONIAZID IN THE TREATMENT OF DEPRESSION. Journal of Neurology, Neurosurgery, and Psychiatry, 22(3), 247-251 .

[0146] Davison, A. N. (1957). The mechanism of the irreversible inhibition of rat-liver monoamine oxidase by iproniazid (marsilid). The Biochemical Journal, 67(2), 316-322. https: / / doi.org / 10.1042 / bj0670316

[0147] 12. Marken, P. A., i Munro, J. S. (2000). Selecting a Selective Serotonin Reuptake Inhibitor: Clinically Important Distinguishing Features. Primary Care Companion to The Journal of Clinical Psychiatry, 2(6), 205-210.

[0148] 13. Malberg, J. E., Eisch, A. J., Nestler, E. J., i Duman, R. S. (2000). Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 20(24), 9104-9110. https: / / doi.org / 10.1523 / JNEURCSCI.20-24-09104.2000

[0149] 14. Wang, J.-W., David, D. J., Monckton, J. E., Battaglia, F., i Hen, R. (2008). Chronic Fluoxetine Stimulates Maturation and Synaptic Plasticity of Adult-Born Hippocampal Granule Cells. Journal of Neuroscience, 28(6), 1374-1384. https: / / d0i.0rg / l 0.1523 / J NEUROSC I.3632- 07.2008

[0150] 15. Lane, R., Baldwin, D., i Preskorn, S. (1995). The SSRIs: Advantages, disadvantages and differences. Journal of Psychopharmacology (Oxford, England), 9(2 Suppl), 163-178. https: / / doi.Org / 10.1177 / 0269881195009002011

[0151] 16. Stahl, S. M., Pradko, J. F., Haight, B. R., Modell, J. G., Rockett, C. B., i Learned-Coughlin, S. (2004). A Review of the Neuropharmacology of Bupropion, a Dual Norepinephrine and Dopamine Reuptake Inhibitor. Primary Care Companion to The Journal of Clinical Psychiatry, 6(4), 159-166.

[0152] 17. Lambert, O., i Bourin, M. (2002). SNRIs: Mechanism of action and clinical features. Expert Review of Neurotherapeutics, 2(6), 849-858. https: / / doi.Org / 10.1586 / 14737175.2.6.849

[0153] 18. Berman, R. M., Cappiello, A., Anand, A., Oren, D. A., Heninger, G. R., Charney, D. S., i Krystal, J. H. (2000). Antidepressant effects of ketamine in depressed patients. Biological Psychiatry, 47(4), 351-354. https: / / d0i.0rg / l 0.1016 / 50006-3223(99)00230-9

[0154] 19. Murrough, J. W., losifescu, D. V., Chang, L. C., Al Jurdi, R. K., Green, C. E., Perez, A. M., Iqbal, S., Pillemer, S., Foulkes, A., Shah, A., Charney, D. S., i Mathew, S. J. (2013). Antidepressant Efficacy of Ketamine in Treatment-Resistant Major Depression: A Two-Site Randomized Controlled Trial. American Journal of Psychiatry, 170(10), 1134-1142. https: / / d0i.0rg / l 0.1176 / appi.ajp.2O13.13030392 20. Serafini, G., Howland, R. H., Rovedi, F., Girardi, P., i Amore, M. (2014). The role of ketamine in treatment-resistant depression: A systematic review. Current Neuropharmacology, 12(5), 444-461 . https: / / doi.Org / 10.2174 / 1570159X12666140619204251

[0155] 21 . Fu, D.-J., lonescu, D. F., Li, X., Lane, R., Lim, P., Sanacora, G., Hough, D., Manji, H., Drevets, W. C., i Canuso, C. M. (2020). Esketamine Nasal Spray for Rapid Reduction of Major Depressive Disorder Symptoms in Patients Who Have Active Suicidal Ideation With Intent: Double-Blind, Randomized Study (ASPIRE I). The Journal of Clinical Psychiatry, 81 (3), 6605. https: / / doi.org / 10.4088 / JCP.19m13191

[0156] 22. Kennedy, S. H., i Giacobbe, P. (2007). Treatment resistant depression — Advances in somatic therapies. Annals of Clinical Psychiatry: Official Journal of the American Academy of Clinical Psychiatrists, 19(4), 279-287. https: / / doi.org / 10.1080 / 10401230701675222

[0157] 23. McLachlan, G. (2018). Treatment resistant depression: What are the options? BMJ (Clinical Research Ed.), 363, k5354. https: / / doi.org / 10.1136 / bmj.k5354

[0158] 24. Voineskos, D., Daskalakis, Z. J., i Blumberger, D. M. (2020). Management of Treatment- Resistant Depression: Challenges and Strategies. Neuropsychiatric Disease and Treatment, 16, 221-234. https: / / doi.org / 10.2147 / NDT.S198774

[0159] 25. Mukaetova-Ladinska, E. B., Arnold, H., Jaros, E., Perry, R., i Perry, E. (2004). Depletion of MAP2 expression and laminar cytoarchitectonic changes in dorsolateral prefrontal cortex in adult autistic individuals. Neuropathology and Applied Neurobiology, 30(6), 615-623. https: / / doi.Org / 10.1111 / j.1365-2990.2004.00574.x

[0160] 26. Raymond, G. V., Bauman, M. L., i Kemper, T. L. (1996). Hippocampus in autism: A Golgi analysis. Acta Neuropathologica, 91 (1 ), 117-119. https: / / doi.org / 10.1007 / s004010050401

[0161] 27. Hutsler, J. J., i Zhang, H. (2010). Increased dendritic spine densities on cortical projection neurons in autism spectrum disorders. Brain Research, 1309, 83-94. https: / / d0i.0rg / l 0.1016 / j.brainres.2009.09.120

[0162] 28. Brown, D. A. (2010). Muscarinic acetylcholine receptors (mAChRs) in the nervous system: Some functions and mechanisms. Journal of Molecular Neuroscience: MN, 41 (3), 340-346. https: / / doi.org / 10.1007 / s12031-010-9377-2

[0163] 29. Kuriakose, D., i Xiao, Z. (2020). Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. International Journal of Molecular Sciences, 21 (20), 7609. https: / / d0i.0rg / l 0.3390 / ijms21207609

[0164] 30. Merino-Serrais, P., Plaza-Alonso, S., Hellal, F., Valero-Freitag, S., Kastanauskaite, A., Munoz, A., Plesnila, N., i DeFelipe, J. (2023). Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke. Cerebral Cortex, 33(4), 1074-1089. https: / / doi.org / 10.1093 / cercor / bhac121

[0165] 31 . Slujitoru, A. S., Enache, A. L., Pintea, I. L., Rolea, E., Stocheci, C. M., Pop, O. T., i Predescu, A. (2012). Clinical and morphological correlations in acute ischemic stroke. Romanian Journal of Morphology and Embryology = Revue Roumaine De Morphologie Et Embryologie, 53(4), 917-926.

[0166] 32. Anderton, B. H., Callahan, L., Coleman, P., Davies, P., Flood, D., Jicha, G. A., Ohm, T., i Weaver, C. (1998). Dendritic changes in Alzheimer’s disease and factors that may underlie these changes. Progress in Neurobiology, 55(6), 595-609. https: / / doi.org / 10.1016 / s0301- 0082(98)00022-7

[0167] 33. Flood, D. G. (1991 ). Region-specific stability of dendritic extent in normal human aging and regression in Alzheimer’s disease. II. Subiculum. Brain Research, 540(1-2), 83-95. https: / / doi.Org / 10.1016 / 0006-8993(91 )90494-g

[0168] 34. Golovyashkina, N., Penazzi, L., Ballatore, C., Smith, A. B., Bakota, L., i Brandt, R. (2015). Region-specific dendritic simplification induced by A , mediated by tau via dysregulation of microtubule dynamics: A mechanistic distinct event from other neurodegenerative processes. Molecular Neurodegeneration, 10(1 ), 60. https: / / doi.Org / 10.1186 / s13024-015- 0049-0

[0169] 35. Hoover, B. R., Reed, M. N., Su, J., Penrod, R. D., Kotilinek, L. A., Grant, M. K., Pitstick, R., Carlson, G. A., Lanier, L. M., Yuan, L.-L., Ashe, K. H., i Liao, D. (2010). Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron, 68(6), 1067. https: / / d0i.0rg / l 0.1016 / j. neuron.2010.11 .030

[0170] 36. Ittner, L. M., Ke, Y. D., Delerue, F., Bi, M., Gladbach, A., Eersel, J. van, Wolfing, H., Chieng, B. C., Christie, M. J., Napier, I. A., Eckert, A., Staufenbiel, M., Hardeman, E., i Gotz, J. (2010). Dendritic Function of Tau Mediates Amyloid-p Toxicity in Alzheimer’s Disease Mouse Models. Cell, 142(3), 387-397. https: / / doi.Org / 10.1016 / j.cell.2010.06.036

[0171] 37. Jaworski, T., Lechat, B., Demedts, D., Gielis, L., Devijver, H., Borghgraef, P., Duimel, H., Verheyen, F., Kugler, S., i Van Leuven, F. (2011 ). Dendritic degeneration, neurovascular defects, and inflammation precede neuronal loss in a mouse model fortau-mediated neurodegeneration. The American Journal of Pathology, 179(4), 2001-2015. https: / / d0i.0rg / l 0.1016 / j.ajpath.2011 .06.025

[0172] 38. Moyer, C. E., Shelton, M. A., i Sweet, R. A. (2015). Dendritic Spine Alterations in Schizophrenia. Neuroscience letters, 601 , 46-53. https: / / d0i.0rg / l 0.1016 / j.neulet.2014.11 .042

[0173] 39. Immadisetty, K., Geffert, L. M., Surratt, C. K., i Madura, J. D. (2013). New design strategies for antidepressant drugs. Expert Opinion on Drug Discovery, 8(11 ), 1399-1414. https: / / doi.Org / 10.1517 / 17460441 .2013.830102

[0174] 40. Wong, D. T., Horng, J. S., Bymaster, F. P., Hauser, K. L., i Molloy, B. B. (1974). A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-N-methyl-3- phenylpropylamine. Life Sciences, 15(3), 471-479. https: / / doi.org / 10.1016 / 0024- 3205(74)90345-2

[0175] 41 . Nolan, T. L., Lapinsky, D. J., Talbot, J. N., Indarte, M., Liu, Y., Manepalli, S., Geffert, L. M., Amos, M. E., Taylor, P. N., Madura, J. D., i Surratt, C. K. (2011 ). Identification of a novel selective serotonin reuptake inhibitor by coupling monoamine transporter-based virtual screening and rational molecular hybridization. ACS Chemical Neuroscience, 2(9), 544- 552. https: / / d0i.0rg / l 0.1021 / cn200044x

[0176] 42. Viegas-Junior O’Brien, R. J., Kamboj, S., Ehlers, M. D., Rosen, K. R., Fischbach, G. D., i Huganir, R. L. (1998). Activity-dependent modulation of synaptic AMPA receptor accumulation. Neuron, 21 (5), 1067-1078. https: / / doi.org / 10.1016 / s0896-6273(00)80624-8

[0177] 43. Viegas-Junior O’Brien, R. J., Kamboj, S., Ehlers, M. D., Rosen, K. R., Fischbach, G. D., i Huganir, R. L. (1998). Activity-dependent modulation of synaptic AMPA receptor accumulation. Neuron, 21 (5), 1067-1078. https: / / doi.org / 10.1016 / s0896-6273(00)80624-8

[0178] 44. Leelananda, S. P., i Lindert, S. (2016). Computational methods in drug discovery. Beilstein Journal of Organic Chemistry, 12, 2694-2718. https: / / doi.org / 10.3762 / bjoc.12.267

[0179] 45. Sadybekov, A. V., i Katritch, V. (2023). Computational approaches streamlining drug discovery. Nature, 616(7958), Article 7958. https: / / d0i.0rg / l 0.1038 / s41586-023-05905-z

[0180] 46. Sheridan, R. P. (2008). Alternative global goodness metrics and sensitivity analysis: Heuristics to check the robustness of conclusions from studies comparing virtual screening methods. Journal of Chemical Information and Modeling, 48(2), 426-433. https: / / d0i.0rg / l 0.1021 / ci700380x

[0181] 47. Willett, P. (2003). Similarity-based approaches to virtual screening. Biochemical Society transactions, 31 , 603-606. https: / / doi.org / 10.1042 / BST0310603 Zhang, Q., i Muegge, I. (2006). Scaffold hopping through virtual screening using 2D and 3D similarity descriptors: Ranking, voting, and consensus scoring. Journal of Medicinal Chemistry, 49(5), 1536-1548. https: / / doi.org / 10.1021 / jm050468i Mayr, L. M., i Fuerst, P. (2008). The future of high-throughput screening. Journal of Biomolecular Screening, 13(6), 443-448. https: / / doi.Org / 10.1177 / 1087057108319644 Bajorath, J. (2002). Integration of virtual and high-throughput screening. Nature Reviews.

[0182] Drug Discovery, 1 (11 ), 882-894. https: / / doi.org / 10.1038 / nrd941

[0183] Inglese, J., Johnson, R. L., Simeonov, A., Xia, M., Zheng, W., Austin, C. P., i Auld, D. S. (2007). High-throughput screening assays for the identification of chemical probes. Nature Chemical Biology, 3(8), Article 8. https: / / d0i.0rg / l 0.1038 / nchembio.2007.17 Mayr, L. M., i Fuerst, P. (2008). The future of high-throughput screening. Journal of Biomolecular Screening, 13(6), 443-448. https: / / d0i.0rg / l 0.1177 / 1087057108319644 Szymanski, P., Markowicz, M., i Mikiciuk-Olasik, E. (2011). Adaptation of High-Throughput Screening in Drug Discovery — Toxicological Screening Tests. International Journal of Molecular Sciences, 13(1 ), 427-452. https: / / d0i.0rg / l 0.3390 / ijms13010427 Delvecchio, C., Tiefenbach, J., i Krause, H. M. (2011 ). The zebrafish: A powerful platform for in vivo, HTS drug discovery. Assay and Drug Development Technologies, 9(4), 354-361 . https: / / doi.org / 10.1089 / adt.2010.0346 Caban, A., Pisarczyk, K., Kopacz, K., Kapusniak, A., Toumi, M., Remuzat, C., i Kornfeld, A. (2017). Filling the gap in CNS drug development: Evaluation of the role of drug repurposing. Journal of Market Access & Health Policy, 5(1 ), 1299833. https: / / doi.Org / 10.1080 / 20016689.2017.1299833 Padhy, B. M., i Gupta, Y. K. (2011 ). Drug repositioning: Re-investigating existing drugs for new therapeutic indications. Journal of Postgraduate Medicine, 57(2), 153-160. https: / / doi.Org / 10.4103 / 0022-3859.81870 Rao, N., Poojari, T., Poojary, C., Sande, R., i Sawant, S. (2022). Drug Repurposing: A Shortcut to New Biological Entities. Pharmaceutical Chemistry Journal, 56(9), 1203-1214. https: / / doi.Org / 10.1007 / sl 1094-022-02778-w

Claims

Claims:1 . A method to screen for compounds which increase the synapse stability of neurons cultured in vitro comprising the following steps: a. adding a synaptic destabiliser and at least one compound to a portion of a neuron culture, b. adding a synaptic destabiliser without said at least one compound to an other portion of a same neuron culture as in step a, c. monitoring the postsynaptic compartment of said neuron cultures in steps a) and b), and d. obtaining a compound which induces a stabilization of the synapses at least at the level of neurons treated with no destabiliser.

2. Dilazep having the SMILES code: COC1=CC(=CC(=C1OC)OC)C(=O)OCCCN2CCCN(CC2)CCCOC(=O)C3=CC(=C(C( =C3)OC)OC)OC and salts thereof for use to prevent or to treat depressive disorders.

3. Penbutolol having the SMILES code:CC(C)(C)NCC(COC1=CC=CC=C1C2CCCC2)O and salts thereof for use to prevent or to treat depressive disorders.

4. The use according to claims 2-3, where depressive disorders comprise major depressive disorder, single episode depressive disorder, dysthymic disorder, mixed depressive and anxiety disorder, premenstrual dysphoric disorder, persistent depressive disorder, premenstrual dysphoric disorder, substance / medication-induced depressive disorder, depressive disorder due to another medical condition and treatment-resistant depressive disorder.

5. The use according to claims 2-4, wherein said compounds are administered in fixed- dose combination formulations or in separate formulations for concurrent administration for treating depressive disorders.

6. A composition for use according to claims 2-5, further comprising one or more additional active ingredients selected from the group consisting of antidepressants, anxiolytics, mood stabilizers, antipsychotics, and combinations thereof.