Methods of treating mood disorders

Abstract

A method of treating or preventing a mood disorder of a subject is disclosed. The method comprises orally administering to the subject a therapeutically effective amount of hyaluronic acid. Compositions comprising same are also disclosed.

Description

[0001] METHODS OF TREATING MOOD DISORDERS

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of IL Patent Application No. 311338, filed on March 07, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to methods of treating mood disorders including anxiety and depression by providing agents that alter gut permeability.

[0006] Social interactions are critical for the survival and development of animals and humans playing a fundamental role in everyday life, and greatly influencing well-being and quality of life. The impact of social interactions and their deficits are relevant for the development and manifestation of a wide spectrum of disorders, including anxiety and depression. Accumulating evidence demonstrates the role of genetic and environmental factors in the development and establishment of social behavior.

[0007] Numerous human and animal studies have demonstrated that pathways underlying inflammatory and stress responses may play an essential role in the etiopathology of social deficits, depression, and anxiety [Moieni M, Eisenberger NI. Ann N Y Acad Sci. 2018;1428:5-13]. Inflammation can directly or indirectly affect mental health, thus substantially increasing asocial behavior. For example, humans who received an endotoxin derived from Escherichia coli developed an immune reaction that resulted in emotions of depression and social disconnection [Eisenberger NI, et al Brain Behav Immun. 2010;24:558-63]. On the other hand, social defeat has been shown to lead to monocyte-mediated exacerbation of gut inflammation [Schneider KM. et al., Cell. 2023;186:2823-2838. e20].

[0008] Moreover, social interactions have been correlated with the gut microbiota composition [Vernier CL, et al., Sci Adv. 2020;6]. Fecal microbiota transplantation (FMT) from specific - pathogen-free to germ- free (GF) mice increased GF mice social behavior [Desbonnet L, et al., Mol Psychiatry. 2014;19:146-8]. Social defeat, which leads to anxiety- and depressive-like behaviors, has been associated with changes in the alpha-diversity of the mouse gut microbiota and reduced the relative abundance of various bacterial genera . [McGaughey KD et al. Sci Rep. 2019;9:3281] . Similarly, rat offspring from antibiotic-treated mothers demonstrated decreased social investigation [Degroote S, et al., Prog Neurop sychopharmacol Biol Psychiatry. 2016;71:76-82]. Background art includes Bairachnaya, M., et al., Aging (Albany NY) 11, 9901-9911 (2019) and Agranyoni, O. et al. NPJ Biofilms Microbiomes 7, 28 (2021). Additional art includes Chinese Patent No. CN112195128B.

[0009] SUMMARY OF THE INVENTION

[0010] According to an aspect of the present invention there is provided a method of treating or preventing a mood disorder of a subject in need thereof, comprising orally administering to the subject a therapeutically effective amount of hyaluronic acid (HA), thereby treating the mood disorder.

[0011] According to an aspect of the present invention there is provided a method of treating or preventing a mood disorder of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that decreases permeability of the gut, wherein the agent is not a bacteria, thereby treating the mood disorder.

[0012] According to another aspect of the invention there is provided a composition comprising HA, for use in treating a mood disorder.

[0013] According to still another aspect of the invention there is provided a pharmaceutical composition comprising HA and an anti-inflammatory agent, wherein the composition is formulated for oral delivery.

[0014] According to an embodiment of the present invention, the subject does not have a disease of the gastrointestinal tract (GIT).

[0015] According to an embodiment of the present invention, the disease of the GIT is selected from the group consisting of inflammatory bowel disease, metabolic syndrome, gut infection and gut autoinflammation.

[0016] According to an embodiment of the present invention, the agent which reduces permeability of the gut comprises hyaluronic acid (HA), or a derivative thereof.

[0017] According to an embodiment of the present invention, the HA comprises linear HA.

[0018] According to an embodiment of the present invention, the HA comprises crosslinked HA.

[0019] According to an embodiment of the present invention, the agent which reduces permeability of the gut comprises a histamine receptor antagonist.

[0020] According to an embodiment of the present invention, the histamine receptor antagonist is selected from the group consisting of Cimetidine, Famotidine, Lafutidine, Nizatidine, Ranitidine, Roxatidine and Tiotidine. According to an embodiment of the present invention, the agent which reduces permeability of the gut is a metabolite selected from the group consisting of taurine, tryptamine and L-homo serine.

[0021] According to an embodiment of the present invention, the administering comprises orally administering.

[0022] According to an embodiment of the present invention, the administering comprises administering at least once a day.

[0023] According to an embodiment of the present invention, the method further comprises administering to the subject an anti-inflammatory agent.

[0024] According to an embodiment of the present invention, the anti-inflammatory agent comprises a steroid agent.

[0025] According to an embodiment of the present invention, the anti-inflammatory agent comprises a non-steroidal anti-inflammatory agent.

[0026] According to an embodiment of the present invention, the anti-inflammatory agent comprises a COX-2 inhibitor.

[0027] According to an embodiment of the present invention, the COX-2 inhibitor comprises celecoxib.

[0028] According to an embodiment of the present invention, the anti-inflammatory agent comprises a biologic.

[0029] According to an embodiment of the present invention, the biologic comprises TNF inhibitor.

[0030] According to an embodiment of the present invention, the anti-inflammatory agent and said agent that decreases permeability of the gut are formulated in a single composition.

[0031] According to an embodiment of the present invention, the anti-inflammatory agent and said agent that decreases permeability of the gut are formulated in separate compositions.

[0032] According to an embodiment of the present invention, the mood disorder comprises anxiety and / or depression.

[0033] According to an aspect of the present invention there is provided a agent that decreases permeability of the gut, for use in treating a mood disorder, wherein the agent is not a bacteria.

[0034] According to an aspect of the present invention there is provided a pharmaceutical composition comprising an agent that decreases permeability of the gut and an anti-inflammatory agent, wherein the agent that decreases permeability of the gut is not a bacteria.

[0035] According to an embodiment of the present invention, the pharmaceutical composition is formulated for oral delivery or rectal delivery. According to an embodiment of the present invention, the agent that decreases permeability of the gut is HA or a derivative thereof, and the anti-inflammatory agent is a Cox-2 inhibitor.

[0036] Unless otherwise defined, all technical and / or 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0037] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0038] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0039] In the drawings:

[0040] FIGs. 1A-F. Dom and Sub mice differ in body weight, eWAT content, and colon development from early infancy to adulthood. (A) Body-weight follow-up of Dom (n=90; 45 males) and Sub (n=92; 42 males) mice. (B) eWAT mass (normalized to body weight) follow-up of Dom and Sub male mice. (C) Visualization of eight representative mouse intestines removed from Dom and Sub male mice. Colon length (in millimeters, mm) of Dom vs. Sub at 1-4 weeks of age; Means are 29 vs. 20, 38 vs. 35, 57 vs. 48, and 70 vs. 50 mm, respectively. (D) Complete intestine length follow-up. (E) Small intestine length follow-up. (F) Colon length follow-up. eWAT and colon tissue follow-up was performed on the same mice (Dom, n=53; Sub, n= 51). Statistical significance was determined using a student’s t-test, (*) p<0.05, (**) p<0.01, (***) p<0.001. Error bars show standard deviation.

[0041] FIGs. 2A-L. Sub mice compared to Dom mice possess age-dependent higher mucin expression and increased gut permeability. (A) PAS staining of Dom (n=32 males) and Sub mice (n=29 males), at ages 1, 2, 3, and 12 weeks old, and 12-weeks-old BS mice (n=4 males). (B) H&E staining of 12-week-old Dom (n=10 males) and Sub mice (n=10 males). (C) Quantification of the goblet cell number according to the mucin droplets per crypt from 10 fields of each mouse at ages 1, 2, 3, 4, and 12 weeks old. (D) Quantification of the goblet cell area (pm2) according to the mucin droplets from 10 fields of each mouse at ages 1, 2, 3, 4, and 12 weeks old. (E) Quantification of the goblet cell area (p m2) according to the mucin droplets from 10 fields of Dom, Sub, and BS mice at the age of 12 weeks. (F) Dom and Sub mouse colon mucin staining using lectin labeling. (G) Dom and Sub mouse colon bacteria staining using FISH probes. (H) Dom and Sub mouse colon nuclei staining using Hoechst labeling. (I) Merged staining. (J) Quantification of the distance between the lumen bacteria and the colon epithelium based on the FISH staining (panels F-I), using the Zen 3.4 software. White arrows define the distance measured. (K) MUC2 gene expression normalized to HPRT in Dom (n=30 males) and Sub mice (n=34 males), at the ages of 0, 1, 2, 3, 4, 8, and 12 weeks old. (L) Gut permeability of Dom and Sub mice (n=9 males in each group, in ng / ml), at the ages of 3 and 4 months-old, based on the detection of serum FITC-dextran levels, 5- 6 hr post FITC-dextran oral gavage administration. Statistical significance was determined using a student’s t-test; (*) p<0.05, (**) p<0.01, (***) p<0.001. Error bars show standard deviation.

[0042] FIGs. 3A-F. Adult Sub mice possess microbiota-induced lower fecal SCFA levels compared to Dom mice. (A) damate, (B) acetate and (C) butyrate concentrations (nmol / mg feces) in Dom (n=8) and Sub (n=7) stools. (D) propionate (E) acetate and (F) butyrate concentrations (nmol / mg feces) in 2.5 months transplanted GF mice. Transplants were of PBS (n=3), or stools from Dom (n=6) or Sub (n=7) mice resuspended in PBS. Statistical significance was determined using a student’s t-test and One-way ANOVA, (*) p<0.05. Error bars show standard deviation.

[0043] FIGs. 4A-I. Anti-inflammatory and gut permeability reduction treatments modulated Sub mouse behavior, colon length and inflammation, and gut permeability. (A) The study design of the treatment experiment performed on 40 mice (4 groups of 10 mice each - control (water-treated), HA-treated, Al-treated, and HA+AI- treated). (B) EPM test of Sub (n=40 males, 10 in each group) controls and mice treated with HA, Al, and HA+AI agents. Y-axis shows the frequency of entering the open arms normalized to the frequency of entering the open and close arms. (C) FST of Sub mice treated with HA, Al, and HA+AI agents and controls. Y-axis shows the time mice were immobile in the water. (D) TCST of Sub mice treated with HA, Al, and HA+AI agents and controls. Y-axis shows the nose-point frequency to enter the area around the stranger mouse. (E) Colon length of Sub mice treated with HA, Al, and HA+AI agents and Controls. (F) FITC-dextran assay of gut permeability, Y axis shows FITC-dextran concentration in the mouse serum (ng / ml) 5 hr after oral gavage at 4.5 months old. (G-I) A cytokine array comparison of pooled proteins extracted from the colons of Sub mice: controls (n=3) and those treated with (G) HA (n=3), (H) Al, and (I) HA+AI. Panels G, H, and I present only the colon cytokines with significantly different expression per treatment, compared to the Sub control group. Each bar represents the average duplicate cytokine expression normalized to the positive control. Control Sub mice demonstrated a significantly higher cytokine level than Al-treated Sub mice. Statistical significance was determined using a student's t-test, (*) p<0.05, (**) p<0.01, and (***) p<0.001. Error bars show standard deviation. Al- Anti-inflammatory, HA- Hyaluronic acid, EPM- elevated plus maze, TCT- Three chambers test, FST- Forced swim test.

[0044] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0045] The present invention, in some embodiments thereof, relates to methods of treating mood disorders including anxiety and depression by providing agents that alter gut permeability.

[0046] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0047] Individuals with psychiatric disorders, behavioral deficits and high stress vulnerability often exhibit symptoms of gastrointestinal inflammation together with anatomical and functional gut impairments. The present inventors previously developed a mouse model with strong features of dominance (Dom) and submissiveness (Sub), essential elements of social behavior. Using behavioral and pharmacological approaches, they demonstrated that Dom and Sub mice possess different cognitive and learning capabilities. Moreover, compared to Dom mice, Sub mice exhibit strong stress sensitivity, along with depressive-like and anti-social characteristics, and systematic inflammation, demonstrated by higher- IL-6 and IL- lb serum levels. It was further reported that adult Sub mice also exhibit an altered gut microbiota composition compared to Dom and Sabra mice.

[0048] The present inventors have now shown that Sub mice exhibited colon anatomical and physiological abnormalities including higher gut permeability (Figure 2L), reduced colon length (Figure IF), increased Goblet cell area (Figure 2E) and mucin hyperproduction associated with elevated MUC-2 mRNA expression (Figure 2K).

[0049] In order to investigate whether altered colon anatomical features and gut permeability are causatively connected with behavioral disorders, the present inventors treated Sub mice with a gut permeability modulator agent, hyaluronic acid (HA). Results showed that this treatment rescued Sub mouse social behavior. Without being bound to theory, the present inventors conceive that by modulating the gut permeability of the animals, inflammation in the colon was prevented, thereby reducing the amount of inflammatory mediators from reaching the bloodstream, which ultimately acted to increase sociability behaviors (Figures 4A-I).

[0050] Whilst further reducing the present invention to practice, the present inventors showed that treating mice with a combined therapy of HA and an anti-inflammatory agent (celecoxib) significantly improved Submissive mice behavior, as reflected by a reduction in depressive and anxiety-like behaviors (Figures 4A-I).

[0051] Thus, according to an aspect of the present invention, there is provided a method of treating or preventing a mood disorder of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that decreases permeability of the gut, wherein the agent is not a bacteria, thereby treating the mood disorder.

[0052] According to one embodiment, a “mood disorder” refers to disruption of feeling, tone or emotional state experienced by an individual for an extensive period of time. Mood disorders include, but are not limited to, depression (i.e., depressive disorders), anxiety (anxiety-related disorders) bipolar disorders, substance-induced mood disorders, social behavior deficits, alcohol- induced mood disorders, benzodiazepine-induced mood disorders, mood disorders due to general medical conditions, as well as many others. See, e.g., DSM-5 (www(dot)dsm5(dot)org), incorporated herein by reference.

[0053] The subject is typically a mammalian subject - e.g. human.

[0054] The subject may have an increased level of gut (e.g. intestinal) permeability (i.e. impaired intestinal barrier function). The term “Intestinal permeability” is a term describing the control of material passing from inside the gastrointestinal tract through the epithelial cells lining the mucosal surface of the gut wall, into the rest of the body, (i.e. through the gut epithelial barrier). The intestine normally exhibits some permeability, which allows nutrients to pass through the gut, while also maintaining a barrier function to keep potentially harmful substances (such as antigens) from leaving the intestine and migrating to the body more widely. In a healthy human intestine, small particles (< 4 A in radius) can migrate through tight junction claudin pore pathways, and particles up to 10-15 A (3.5 kDa) can transit through the paracellular space uptake route. The subject being treated may have an intestine, through which small particles greater than 4 A in radius (e.g. greater than 5 A, greater than 6 A, greater than 7 A , greater than 8 A, greater than 10 A may migrate through tight junction claudin pore pathways. The subject being treated may have an intestine, through which particles greater than 15 A (3.5 kDa), greater than 16 A greater than 17 A, greater than 18 A, greater than 19 A, greater than 20 A can transit through the paracellular space uptake route.

[0055] According to a particular embodiment, the subject is not suffering from a chronic inflammatory gastro-intestinal disorder (e.g. colitis or Crohn’s disease). According to another embodiment, the subject is not suffering from Celiac disease.

[0056] According to another embodiment, the subject has been diagnosed with a depression related disorder. For example, the subject may have been previously prescribed (for at least one month, 2 months, 3 months or longer) a selective serotonin reuptake inhibitor (SSRI) for treatment of a depression related disorder. Examples of SSRIs include, but are not limited to fluoxetine, citalopram, sertraline, paroxetine, and escitalopram.

[0057] Additionally, or alternatively, the subject has been diagnosed with an anxiety related disorder. For example, the subject may have been previously prescribed (for at least one month, 2 months, 3 months or longer) an SSRI or a selective serotonin norepinephrine reuptake inhibitor (SNRI) or a benzodiadapene. Examples of anxiolytic agents include, but are not limited to fluoxetine, citalopram, sertraline, paroxetine, escitalopram, duloxetine, venlafazine, clomipramine, pregabalin.

[0058] According to one embodiment, the mood disorder is a depression-related disorder.

[0059] The term “depression” or “depressive disorder” or “depression-related disorder” includes a mood disorder involving any of the following symptoms: persistent sad, anxious, and / or “empty” mood; feelings of hopelessness and / or pessimism; feelings of guilt, worthlessness, and / or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed; decreased energy, fatigue, and / or being “slowed down”; difficulty concentrating, remembering, and / or making decisions; insomnia, early-morning awakening, and / or oversleeping; loss of appetite and / or weight loss, overeating and / or weight gain; thoughts of death and / or suicide; suicide attempts; restlessness and / or irritability; persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and / or chronic pain; and combinations thereof. See, e.g., DSM-5 (described above).

[0060] Non-limiting examples of depression-related disorders include, but are not limited to, major depression disorder (MDD), atypical depression, melancholic depression, psychotic major depression or psychotic depression, catatonic depression, postpartum depression, seasonal affective disorder (SAD), chronic depression (dysthymia), severe depression, unipolar depression, double depression, depressive disorder not otherwise specified, depressive personality disorder (DPD), recurrent brief depression (RBD), minor depressive disorder (minor depression), premenstrual syndrome, premenstrual dysphoric disorder, depression caused by chronic medical conditions (e.g., cancer, chronic pain, chemotherapy, chronic stress), and combinations thereof. Various subtypes of depression are described in, e.g., DSM-5 (described above). In particular embodiments, the depression is major depression disorder (MDD). In certain instances, the methods of the present invention treat or alleviate one or more symptoms of depression. In certain other instances, the methods of the present invention treat depression. According to a specific embodiment, the depression-related disorder comprises a major depression disorder (MDD). According to one embodiment, the MDD is associated with suicidal ideation.

[0061] According to a specific embodiment, the depression-related disorder comprises a treatment-resistant depression (TRD). TRD typically refers to inadequate response to at least one antidepressant therapy of adequate doses and duration. Such an adequate dose and duration is well known to one of skill in the art.

[0062] According to one embodiment, the mood disorder is a bipolar disorder.

[0063] According to one embodiment, “bipolar disorder” refers to a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing from being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM-5 (described above). Bipolar disorder is also known as manic depression.

[0064] Non-limiting examples of bipolar disorders include, but are not limited to, mania, acute mania, severe mania, hypomania, depression, moderate depression, dysthymia, severe depression, episodes of mania and / or depression, psychosis / psychotic symptoms (e.g. hallucinations, delusions), mixed bipolar state, bipolar I disorder (mania with or without major depression), bipolar II disorder (hypomania with major depression), rapid-cycling bipolar disorder, Cyclothymia and / or Bipolar Disorder Not Otherwise Specified (BD-NOS). See, e.g., DSM-5 (described above).

[0065] Exemplary anxiety-related disorders include panic disorder agoraphobia, generalized anxiety disorder (GAD), social anxiety disorder, separation anxiety disorder of childhood and selective mutism.

[0066] The method described herein comprises administering to the subject a therapeutically effective amount of an agent that decreases the permeability of the gut (e.g. intestine).

[0067] Agents which decrease the permeability of the gut (i.e. improve the intestinal barrier function) can be selected on the basis of their ability to increase Tra sepithelial Electrical Resistance (TER) in an epithelial cell layer to > 110%, 120%, 130%, 140% or even 150% relative to TER at the beginning of the treatment. An epithelial cell tight junction disruptor agent may be included in this assay, as further described herein below.

[0068] Examples of epithelial (or epithelial-like) cell lines which may be used in this assay include, but are not limited to CaCo-2 cells, DLD-1 cells, HT-29 cells, T-84 cells and LoVo cells. The assay may be personalized using epithelial cells from a candidate subject. This may be particularly relevant in subjects who are known to have compromised epithelial intestinal barrier.

[0069] The epithelial cells are cultured under conditions that promote tight junction formation between the cells.

[0070] The term "tight junction (TJ)," as used herein, describes the closely associated apical areas of two cells whose membranes join together by specialized transmembrane proteins forming a virtually impermeable barrier to fluid.

[0071] Preferably, the epithelial cells are cultured as a monolayer. Examples of media that can be used to culture the epithelial cells include, but are not limited to DMEM, EMEM and RPMI.

[0072] In one embodiment, the cells are cultured directly on a solid surface (e.g. plastic, glass etc.). Alternatively, the solid surface is coated with an adhesive matrix, such as an extracellular matrix protein. Examples of contemplated extracellular matrix proteins include, but are not limited to collagen (e.g. type I collagen), MatrigelTMor fibronectin.

[0073] In one embodiment, the epithelial cells are cultured in an Ussing chamber.

[0074] Preferably, the cells are cultured for at least 12 hours, more preferably at least 24 hours so as to promote generation of coherent and uniform monolayers with highly organized tight junctions. When an extracellular matrix protein is used to coat the solid surface, the cells may be cultured such that stable focal adhesions are formed with the underlying ECM. The thickness of the underlying ECM coating may be adjusted for experiments aiming at the analysis of tight junctions and those used for testing focal adhesions.

[0075] Once the tight junctions are generated between the epithelial cells, a disrupting agent may be used which is capable of disrupting or destabilizing the tight junctions.

[0076] As used herein, the term “disrupting agent” refers to an agent that is capable of disrupting or destabilizing tight junctions of epithelial cells as assayed by light microscopy compared to a control (i.e. absence of the agent) under identical conditions in less than 48 hours, more preferably less than 24 hours. Exemplary disruptors include TNFa and LPS.

[0077] In one embodiment, the agent that decreases the permeability of the gut (i.e. restores the gut barrier) comprises hyaluronic acid (HA), or a derivative of HA.

[0078] Hyaluronic acid (HA) is biological polymer and is one of the main components of the extracellular matrix. It is an unsulfated glycosaminoglycan (GAG) and is composed of repeating disaccharide units composed of (P-l,4)-linked D-glucuronic acid and (P- 1,3) -linked N-acetyl-D- glucos amine. The term “HA” refers to low and high molecular weight hyaluronic acid, in its pure or salt form, but also all cross-linked, modified or hybrid forms of HA. Cross-linker agents for HA operating via the carboxylic groups or via the amine groups after HA deacetylation include: Glutaraldehyde and other aldehydes, Dialdehydes, Genipin, Cinnamic acid or derivatives of it, synthetic cross-linkers from the carbodiimide family (EDC), Divinylsulfone, BODE and Mannitol, Ribose and other sugars. The modified HA group include Polyvinylpyrrolidone-sodium hyaluronate, disulfide cross-linked hyaluronan hydrogels, Glycidyl trimethylammonium chloride (GTAC), Phenyl succinic acid derivatives HA, Sodium caproyl hyaluronate, Sodium tyramino hyaluronate, Sodium rhodaminylamino hyaluronate, Sodium fluoresceinylamino hyaluronate, DTPA - Hyaluronate, DTPA (Gd) - Hyaluronate, Sodium formyl hyaluronate, Sodium palmitoyl hyaluronate, Sodium propinylamino hyaluronate, Sodium azidopropylamino hyaluronate. Hybrid HA group include diphenylalanin HA, Albumin HA, Fibrinogen or fibrin HA, Chitosan HA and any other kind protein or carbohydrate polymers with HA.

[0079] In some embodiments, the hyaluronic acid (HA) has an average molecular weight (being high MW hyaluronic acid) of at least 100 kDa, preferably of at least 500 kDa, or at least 1,000 kDa or at least 2,000 kDa, e.g., of from 100 kDa to 100,000 kDa, or from 1,000 kDa to 100,000 kDa, for example, from 1,000 kDa to 50,000 kDa, or from 1,000 kDa to 20,000 kDa, or from 1,000 kDa to 10,000 kDa, or from 1,000 kDa to 5,000 kDa, or from 2,000 kDa to 5,000 kDa, including any intermediate values and subranges therebetween.

[0080] Other examples of agents that increase the permeability of the gut include histamine antagonists including but not limited to Cimetidine, Famotidine, Lafutidine, Nizatidine, Ranitidine, Roxatidine and Tiotidine.

[0081] Other exemplary small molecule agents known to reduce permeability of the gut are metabolites including, but not limited to taurine, tryptamine and L-homoserine.

[0082] Collagen (and collagen peptides) are also contemplated as they are known to ameliorate intestinal barrier dysfunction.

[0083] Other agents known to increase the permeability of the gut are disclosed in International Application No. WO2019 / 155465, the contents of which are incorporated herein by reference.

[0084] In one embodiment, the agent is not a bacterial agent (e.g. a probiotic agent).

[0085] The present inventors further contemplate treating the subject with an anti-inflammatory agent.

[0086] The agents which increase the permeability of the gut may be co-formulated with the antiinflammatory agent described herein, or may be provided as separate compositions to the subject. Thus, each agent included in the combination can be formulated separately for use in combination. The drugs are said to be used "in combination" when, in a recipient of both drugs, the effect of one drug enhances or at least influences the effect of the other drug.

[0087] The two agents in the combination cooperate to provide an effect on target cells that is greater than the effect of either drug alone. This benefit manifests as a statistically significant improvement in a given parameter of target cell effect. In embodiments, the improvement resulting from treatment with the drug combination can manifest as an effect that is at least additive and desirably synergistic, relative to results obtained when only a single agent is used.

[0088] In use, each drug in the combination can be formulated as it would be for monotherapy, in terms of dosage size and form and regimen. In this regard, the synergy resulting from their combined use may permit the use of somewhat reduced dosage sizes or frequencies, as would be revealed in an appropriately controlled clinical trial.

[0089] According to one embodiment, the agents known to increase the permeability of the gut and the anti-inflammatory agent are administered concomitantly.

[0090] According to another embodiment, the agent known to increase the permeability of the gut and the anti-inflammatory agent are administered sequentially, wherein the first agent is used, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, a week, a month or more after the second agent. Such a determination is well within the capacity of one of skill in the art. In another embodiment, the agent known to increase the permeability of the gut and the anti-inflammatory agent are administered sequentially, wherein the second agent is used, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, a week, a month or more after the first agent.

[0091] The phrase “anti-inflammatory agent” refers to an agent or compound that has antiinflammatory effects. These agents may remedy pain by reducing inflammation. Examples of antiinflammatory agents include, but are not limited to, a statin, sulindac, sulfasalazine, naroxyn, diclofenac, indomethacin, ibuprofen, flurbiprofen, ketoprofen, aclofenac, aloxiprin, aproxen, aspirin, diflunisal, fenoprofen, mefenamic acid, naproxen, phenylbutazone, piroxicam, meloxicam, salicylamide, salicylic acid, desoxysulindac, tenoxicam, ketoralac, clonidine, flufenisal, salsalate, triethanolamine salicylate, aminopyrine, antipyrine, oxyphenbutazone, apazone, cintazone, flufenamic acid, clonixeril, clonixin, meclofenamic acid, flunixin, colchicine, demecolcine, allopurinol, oxypurinol, benzydamine hydrochloride, dimefadane, indoxole, intrazole, numbane hydrochloride, paranylene hydrochloride, tetrydamine, benzindopyrine hydrochloride, fluprofen, ibufenac, naproxol, fenbufen, cinchophen, diflumidone sodium, fenamole, flutiazin, metazamide, letimide hydrochloride, nexeridine hydrochloride, octazamide, molinazole, neocinchophen, nimazole, proxazole citrate, tesicam, tesimide, tolmetin, triflumidate, fenamates (mefenamic acid, meclofenamic acid), nabumetone, celecoxib, etodolac, nimesulide, apazone, gold, tepoxalin; di thiocarbamate, or a combination thereof. Anti -inflammatory' agents also include other compounds such as steroids, such as for example, fluocinolone, cortisol, cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, beclomethasone, fluticasone interleukin- 1 receptor antagonists, thalidomide (a TNF-a release inhibitor), thalidomide analogues (which reduce TNF-a production by macrophages), bone morphogenetic protein (BMP) type 2 or BMP- 4 (inhibitors of caspase 8, a TNF-a activator), quinapril (an inhibitor of angiotensin II, which upregulates TNF-a), interferons such as IL-11 (which modulate TNF-a receptor expression), and aurin-tricarboxylic acid (which inhibits TNF-a), guanidinoethyldisulfide, or a combination thereof.

[0092] Exemplary anti-inflammatory agents include, for example, naproxen; diclofenac; celecoxib; sulindac; diflunisal; piroxicam; indomethacin; etodolac; meloxicam; ibuprofen; ketoprofen; r- flurbiprofen; mefenamic; nabumetone; tolmetin, and sodium salts of each of the foregoing; ketorolac bromethamine; ketorolac tromethamine: ketorolac acid; choline magnesium trisalicylate; rofecoxib; vakiecoxib; lumiracoxib: etoricoxib; aspirin; salicylic acid and its sodium salt; salicylate esters of alpha, beta, gamma-tocopherols and tocotrienols (and all their d, 1, and racemic isomers); methyl, ethyl, propyl, isopropyl, n -butyl, sec-butyl, t-butyl, esters of acetylsalicylic acid; ten oxicam; aceclofenac; nimesulide; nepafenac; amfenac; bromfenac; flufenamate; phenylbutazone, or a. combination thereof.

[0093] In some embodiments, the anti-inflammatory inchide, but are not limited to, salicylates, diflunisal, indomethacin, ibuprofen, naproxen, tolmetin, ketorolac, diclofenac, ketoprofen, fenamates (mefenamic acid, meclofenamic acid), enolic acids (piroxicam, meloxicam), nabumetone, celecoxib, etodolac. nimesulide, apazone, gold, sulindac or tepoxalin; antioxidants, such as dithiocarbamate, and other compounds such as sulfasalazine [2-hydroxy-5-[-4-[C2- pyridinylamino)sulfonyl]azo]benzoic acid], steroids, such as fluocinolone, cortisol, cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, beclomethasone, fluticasone, protein inhibitors of TNF, such as etanercept, Remicade, IL-1 , such as Kineret®, p38, RANK, RANKL or a combination thereof.

[0094] According to one embodiment, the anti -inflammatory' agent is an NSAID. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid. meclofenamic acid, flufenamic acid, tolfenamic acid, and COX -2 inhibitors.

[0095] According to a particular embodiment, the anti-inflammatory agent is a COX-2 inhibitor. Examples of COX-2 inhibitors include, but are not limited to celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib.

[0096] According to another embodiment, the anti-inflammatory agent is a tumor necrosis factor alpha (TNFa) blocker such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certoHzumab pegol (Cimzia), golimumab (Simponi),

[0097] The agent that decreases the permeability of the gut (and optionally the anti-inflammatory agent” may be used per se or as part of a pharmaceutical composition, where it is mixed with suitable carriers or excipients.

[0098] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0099] Herein the term "active ingredient" refers to the agent that decreases the permeability of the gut accountable for the biological effect.

[0100] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0101] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0102] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0103] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections. Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, spray drying, coating or lyophilizing processes.

[0104] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0105] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0106] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0107] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0108] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0109] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0110] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0111] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0112] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0113] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0114] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g. hyaluronic acid) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., mood disorder) or prolong the survival of the subject being treated.

[0115] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0116] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0117] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0118] Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0119] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0120] In one embodiment, the treatment regimen may be administration may be at least once a day, at least twice a day or at least three times a day. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0121] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0122] As used herein the term “about” refers to ± 10 %

[0123] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0124] The term “consisting of’ means “including and limited to”.

[0125] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0126] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0127] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0128] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0129] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0130] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0131] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0132] EXAMPLES

[0133] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0134] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes LIII Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

[0135] Materials and Methods

[0136] Animals

[0137] Mice were housed in groups of five males or 6 females in a temperature- (21 ± 2°C), humidity - (55 ± 5%), and light-controlled (from 7 AM to 7 PM) room. Standard laboratory chow and water were available ad libitum. Mice from generations 25-47 were used in this study. Each group of mice was grouped from at least two different cages to avoid the cage and litter effects.

[0138] Dominant-Submissive Relationship (DSR) Test

[0139] Dominant (Dom) and submissive (Sub) mice were selectively bred from outbred Sabra background strain mice based on their behavior in the DSR test. The DSR test is a food competition paradigm used to assess social interaction between pairs of mice. This test was performed at each generation once mice reached the age of 8 weeks old. Briefly, pairs of mice of the same sex and similar weights (average 43.7 ± 2.1 g) were paired and tested according to the DSR protocol in the DSR apparatus. Made up of Plexiglas, the DSR apparatus consisted of two identical chambers (12 cm x8.5 cm x 7 cm) placed on opposite sides of the apparatus and connected by a tunnel (2.5 cm x 2.5 cm x 27 cm). In the center of the tunnel was a feeder tube with a 0.5 cm diameter hole in its bottom that provides sweetened milk (3% fat, 10% sugar); only one animal has accessed the feeder tube at any given time. On the tunnel, at the entrance to each chamber, gates prevented the mice from reaching the milk until they were removed, creating an equal starting point at the beginning of each session. Fourteen hours before each session, the mice were deprived of food, but water was provided ad libitum. A pair of mice were placed in the two separate chambers behind the gates on the test day. Mice were left in the chamber for 30 seconds for habituation. The gates were then removed, and for 5 min the milk-drinking time was recorded manually for each mouse. DSR sessions were carried out for four consecutive days. The mice will initially fight for their turn in drinking the milk but during each day the Dom mouse will dominate the drinking area and the Sub mice will not attempt to drink the milk. A dominant-submissive relationship was determined if a significant difference (p<0.05) was observed between the two mice’s daily drinking durations and if the difference in drinking scores was at least 40%. In the DSR mouse model, more than 99% of the selectively bred Dom and Sub mice developed strong and stable DSRs inherited from their parents [Feder Y et al. J Affect Disord. 2010;126:214-22]. Therefore, pups, younger than 8 weeks included in this study, although they have not been characterized by the DSR test, most likely possess a distinct Dominant or Submissive feature.

[0140] Three-Chamber Sociability Test (TCST)

[0141] The TCST was employed to assess the motivation of mice to socially interact with a stranger mouse. A mouse was placed in the center chamber with free access to all chambers. On one of the two side chambers, an unfamiliar mouse of the same strain was caged inside a cylinder. An identical empty cylinder was placed in the opposite side chamber. The number of entries of the focal mouse into each of the side chambers was measured for 10 min. Social mice will prefer to spend significantly more time around the stranger mouse than in the empty chamber. Mouse movements were recorded using EthoVision 9.1 (Noldus, Netherlands).

[0142] Forced Swim Test (FST)

[0143] The FST was used for characterizing mice depressive-like behavior. Mice were placed individually into an inescapable transparent glass cylinder (30 cm in height, 10 cm in diameter) filled 25-cm high with water (25±2°C). All animals were forced to swim for 6 min, during which immobility (floating in the water with only minor movements to keep afloat) was recorded and compared. Animals that failed to stay afloat were removed immediately. Each animal underwent the FST only once, after which they were dried with paper towels and placed in cages under a warm lamp for 10 min before being returned to their home cages. Elevated Plus Maze (EPM)

[0144] The EPM was used to assess mice emotional behavior by measuring general exploratory performance and avoidance of the aversive open arms of the maze. Behavior in this task (i.e., activity in the open arms) reflects a conflict between rodent preference for protected areas (e.g., closed arms) and their innate motivation to explore novel environments. Arranged in a “+” shape, the EPM apparatus (54 cm in height and 66 cm in length) has two closed and two open arms, as well as an open center. A single mouse was placed in the center of the maze with its head directed toward a closed arm. The locomotory activity (distance traveled and speed) and exploratory behavior (the number of entries and the time spent in each arm) were recorded for 5 minutes using EthoVision 9.1 (Noldus, Netherlands). Anti-anxiety behavior was determined as increased time spent in the open arm and an increase in the ratio of open arm to closed arm entries.

[0145] Colon Histology Hematoxylin and Eosin (H&E) and Periodic Acid-Schiff (PAS) Staining

[0146] Colon specimens were collected from Dom (total n=32 males) and Sub mice (total n=29 males) at ages 1, 2, 3, and 12 weeks old, and from 12-week- old BS (n=4 males) mice and incubated in 4% formalin for 24 h, after which the specimens were transferred to a 70% ethanol. The tissues were embedded in paraffin and sectioned into 4 m sections. Slides were stained in H&E and PAS staining (Abeam, Cambridge, United Kingdom). PAS staining was done according to manufacture instruction. Analyses of the PAS staining were done using ImageJ software.

[0147] Fluorescent In-Situ Hybridization (FISH)

[0148] FISH analyses were used to quantify the distance of the colon bacteria from the epithelium and quantify the inner mucus layer length in the lumen. Tissue fixation (24-48 hrs.) was performed using Camoy fixative [60% (v / v) dry methanol, 30% (v / v) chloroform, 10% (v / v) glacial acetic acid]. After fixation, the fixed tissue was washed twice in dry methanol for 30 min each, followed by two times in absolute ethanol for 20 min each, and incubated in two baths of xylene for 15 min each before paraffin embedding. Before rehydrating the slides, the tissues were incubated in washing (20 mM Tris-HCl, 0.9% NaCl; pH 7.4) and hybridization (20 mM Tris-HCl, 0.1% SDS; pH 7.4) buffers at 56°C overnight. Then slides were rehydrated and dipped in washing buffer at 56°C for 10 min. Probes (EUB338-1 / 2 / 3, for total of 16S bacterial staining) diluted in the hybridization buffer covered the slides with parafilm at 56°C overnight. The slides were stained for mucin using lectin (1:200, Vector Laboratories) for 2 hrs. at room temperature in the dark. Hoechst (1:10, Vector Laboratories) was used to stain the nuclei. The results were viewed and analyzed with a fluorescent microscope using red, green, and blue channels (Zeiss). Gene Expression Quantification

[0149] RNA was extracted from the colon tissues of random Dom and Sub male mice (n = 5 from each group) longitudinally (0, 1, 2, 3, 4, 8, and 12 weeks old), using RNeasy Micro Kit (Qiagen, Germany). Reverse transcription was performed with a commercial cDNA Kit GoScript™ Reverse Transcriptase (Promega, Madison, WI, USA). RT-PCR was performed using a Fast SYBR® Green Master Mix (Applied Biosystems, MA, USA). The primers are presented in table 1 (Hylabs, Rehovot, Israel). Reactions were performed using the QuantStudiol 96 RT-PCR System (Applied Biosystems, MA, USA).

[0150] Fluorescein-5-Isothiocyanate (FITC)-Dextran Assay

[0151] To measure the gut permeability, a FITC-dextran assay was performed. Mice were fasted (no food or water) over night before the assay. Mice (weight 25-35 g) received an oral gavage of FITC-dextran (150 .L of 80 mg / ml FITC-dextran in PBS, Sigma Chemical), returned to their home cage, and provided food and water ad libitum. Mice were sacrificed 5-6 hours after gavage, and cardiac blood was collected with a 1 ml syringe. Blood was incubated at room temperature for 30 min (protected from light) and then centrifuged for 20 min at 1500 x g at 4°C. Simultaneously, we prepared standards of FITC-dextran in PBS by dilution series: 8000, 4000, 2000, 1000, 500, 250, 125, 0 ng / ml. Sera were diluted 1:1 in PBS. Mice sera and standards were dispensed in duplicates onto black 96-well plates, and fluorescence imaged: Excitation: 485 nm, Emission: 528 nm in a microplate reader (Tecan).

[0152] Fecal Sample Collection and Short-Chain Fatty Acid Quantification

[0153] Fresh fecal samples (-250 mg each) were collected from 3-month-old Dom (n=8) and Sub (n=7) mice. Samples were collected from 9:00 AM to 12:00 AM by placing mice individually in sterile cages and retrieving the feces using sterile forceps. Samples were placed in pre-weighed sterile tubes and immediately stored at -80°C. The targeted metabolomics was performed using LC-MS analysis to detect butyrate, propionate, and acetate SCFAs.

[0154] In Vivo Therapeutic Evaluation

[0155] The therapeutic effects of an anti-inflammatory (Al) agent and a gut permeability reducing agent were evaluated in Sub mice. Briefly, 40 Sub male mice were divided randomly into four groups (n=10) as follows: control group, that received normal drinking water daily (P / O); A Al group - mice were orally administered (16 mg / kg) with celecoxib (Cox-2 inhibitor) (Trima, Israel) daily for four weeks; A hyaluronic acid (HA) group - mice were orally administered (30 mg / kg) (low Mw HA, R&D Systems, Minneapolis, MN, United States), once a week for six weeks; and a combination treatment group that were orally administered with a combination of the two agents. To ensure accurate therapeutic dosing we used oral gavage administration. Behavioral assessment of all mice (using TCST, EPM and FST) was performed during 4 to 6 weeks of treatment, and gut permeability assessment using the FITC-dextran assay was performed after six weeks of treatment.

[0156] Cytokine Profiles in Colon Tissues

[0157] Cytokine differences in the colon tissue of control and treated Sub mice, at 4-month-old, were assessed by an antibody-based protein array [Proteome Profiler: Mouse Cytokine Array (R&D Systems, Minneapolis, MN, USA)], according to the manufacturer’s instructions. We used a pooled protein extract from colon tissues removed from three random mice from each group. The average signal of pixel density from duplicate cytokines was determined using ImageQuant TF software. The relative intensity of the reference values (three inside control duplicates in each membrane) was included with densitometry calculations ImageQuant TL software.

[0158] Statistical Analyses

[0159] All statistical analyses were performed using GraphPad Prism 6 unless otherwise noted. Quantitative results are expressed as means ± SD and were analyzed using a student's / -test for individual comparisons or one- or two-way ANOVA, followed by a Bonferroni means separation test for multiple comparisons. The statistical significance of differences between groups is presented graphically as (*) for p<0.05, (**) for p<0.01, and (***) for p<0.001.

[0160] RESULTS

[0161] Age-Dependent eWAT and Gut Tissue Growth Patterns Differ Between Dom and Sub Mice. Dom mice weighed significantly more than Sub mice from the second week of life (Figure 1A), in spite similar food intake of both mice phenotypes measured in adulthood. These body weight differences are presumably derived from early infancy gut microbiota differences, and smaller epididymis white adipose tissue (eWAT) mass. A longitudinal eWAT and gastrointestinal (GI) tract comparative development analysis of both mouse phenotypes revealed an earlier eWAT tissue development in male Dom mouse compared to Sub mice; (3.9-fold increase in eWAT tissue, already at the age of two weeks where at this age, the eWAT of Sub mice was barely detectable (Figure IB, p<0.001). Moreover, although Sub male mice started to develop high eWAT mass at the age of three weeks and thereafter, it was continually significantly lower than that of Dom mice (p<0.01).

[0162] Additional mouse tissue analyses of the spleen, liver, and GI revealed similar mass of the spleen and liver tissues in Dom and Sub mice, at the different ages tested. Length measurements of the GI showed that the entire gut length of Dom and Sub mice was similar and only differed at the age of two and four- weeks (Figures 1C-D). Detailed analyzes of the gut parts, revealed that although Dom and Sub mice small intestine lengths were similar (p>0.05, except at the age of two weeks, Figure IE), a dramatic sex-independent colon length reduction was observed in Sub mice compared to Dom mice, from the third week of life (Figure IF, p<0.001).

[0163] Sub mice exhibit higher mucin expression and age-dependent elevated gut permeability.

[0164] To characterize the differences in Dom and Sub mice colon physiology, the colon mucus layer was evaluated and gut permeability was determined. Total mucin analysis of colon tissues, performed by Periodic acid-Schiff (PAS) staining (Figure 2A), demonstrated that while the number of mucin-producing goblet cells per crypt was similar between Dom and Sub mice in all the tested ages (Figure 2C), the mucin vesicle area from early infancy, was significantly higher in Sub mice compared to Dom mice, and increased significantly with age (Figure 2D, p<0.01). To determine if the mucus layer thickness is increased in Sub mice, or decreased in Dom mice, these findings were compared to the mucin levels in adult Sabra background strain (BS) mice, at 12 weeks old, the age at which the highest difference in mucin levels was observed between Dom and Sub mice. Indeed, at this age, the colon mucin area of Dom and BS mice was similar, while Sub mice colon tissue exhibited a higher mucin area (Figure 2E, p<0.01), suggesting that Sub mice are mucin hyper-producers.

[0165] To further evaluate the mucin structure, using Fluorescence in Situ Hybridization (FISH), the distance from the inner mucus layer to outside the lumen epithelium was measured in adult Dom and Sub mice (Figure 2F - I). Quantification of the average distance between the bacteria and the epithelium indicated that in Sub mice colons, the bacteria are more distant from the mucin epithelium layer than in Dom mice (Figure 2J, mean ± SD, 50 pm ± 15.1 in Sub mice versus 40 ± 10.4 pm in Dom mice, p=0.09). Although this difference was statistically insignificant, this 10 pm difference in bacterial-mucus layer distance is in line with the heavier mucus layer that characterizes the Sub colons. It was highly noticeable that Sub mouse mucin was greater than that of Dom mice at 3 -month-old at the lumen area, which was not shown in the PAS staining due to the hydrophilic formalin that washed the lumen mucin away during the tissue fixation (Figure 2A).

[0166] To further confirm mucin hyper-production in Sub mouse, the expression levels of MUC2, the most abundant peptide in the colon mucin was determined by RT-PCR, and discovered significantly higher expression in Sub mouse colons than in Dom mice, from birth to adulthood, at all the ages tested (Figure 2K). Of note, at the age of three weeks, when Sub mice MUC2 expression was the highest, the differences in colon length between Dom and Sub mice also became significant.

[0167] These results reveal that Sub mice colons are significantly shorter than Dom mice colons, which possibly suggest an inflammatory process. Therefore, the present inventors sought to evaluate and compare the colon function in both mouse phenotypes by determining their gut permeability. Using a FITC-dextran assay, which measures the sera level of labeled dextran leaked from the gut due to enhanced gut permeability, they demonstrated that at the age of 3-months, Sub and Dom mice had similar gut permeability; however, at the age of 4-month, Sub mice possess age-dependent increased gut permeability (Figure 2L).

[0168] Sub Mice Possess Lower Gut Short-Chain Fatty Acids Levels.

[0169] Next, the present inventors performed targeted metabolomics on adult 8 week old Dom and Sub mice to quantify the three primary SCFAs which regulate Treg activity and tight junction expression in the colon: propionate, acetate and butyrate. Fecal metabolomic analysis revealed that Sub mice possessed a 52% and 53% decrease in propionate and acetate, respectively (Figure 3A and 3B, p<0.05), and showed a tendency toward lower butyrate levels (Figure 3C; 1.8-fold compared to Dom mice, p>0.05). A similar pattern was observed in fecal-transplanted GF mice, with a significant 32% reduction in propionate in Sub-transplanted GF mice compared to Dom- transplanted GF mice (Figure 3D), suggesting that the decreased propionate levels in Sub mice may result from their altered gut microbiota composition.

[0170] In-vivo Therapeutics with Hyaluronic Acid or Celecoxib Drug Improved Sub Mouse Social Behavior and Reduced the Inflamed Colon Features.

[0171] Considering the inflamed colons and dysregulated gut permeability, the present inventors sought to examine the effect of two agents on adult (2.5 months old) Sub mice. The treatment regimens included: (i) a gut permeability reduction agent hyaluronic acid (HA); (ii) an antiinflammatory (Al) agent - celecoxib (Cox-2 inhibitor); and (iii) a combined treatment of the two agents (Figure 4A). Following the indicated treatment modalities, EPM, TCST, and FST behavioral paradigms were carried out, to examine treatment-induced behavioral changes of Sub mice. The order of the sequential behavioral tests was set to minimize the stress effects, as EPM is the least stressful and FST is the most. Moreover, to reduce the stress between the tests, the mice rested for at least four days between each test. Sub mice treated with the celecoxib exhibited a significant decrease in their anxiety- and depressive-like behaviors (Figure 4B-C, 24% and 66%, respectively). Moreover, they were significantly more sociable than the control Sub mice treated with water (Figure 4D). In addition, treating mice with celecoxib significantly increased their colon length compared to the control group (Figure 4E). These results support the strong relation between Sub mice's inflammatory state and their behavioral deficits. To examine whether their inflammation state originated from their increased gut permeability, Sub mice were treated with HA, an agent known to increase colonic tight-j unction protein expression [Kim Y, et al. Matrix Biol. 2018;66:93-109]. HA-treated Sub mice demonstrated significantly decreased gut permeability (Figure 4F, 25%), 6% increase in colon length (Figure 4E), and improved social behavior (Figure 4D). Moreover, a colon cytokine array demonstrated a significant decrease in 15 pro-inflammatory cytokines after celecoxib treatment (Figure 4H), and a decrease in seven pro- inflammatory cytokines after a combination treatment of HA and Al (Figure 41). After HA treatment, Sub mice exhibited similar colon cytokine profiles with changes only in three cytokine levels (Figure 4G). Overall, intervention with both agents improved the inflammatory status of Sub mice colons and significantly promoted the social behavior deficits of Sub mice.

[0172] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0173] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing a mood disorder of a subject in need thereof, comprising orally administering to the subject a therapeutically effective amount of hyaluronic acid (HA), thereby treating the mood disorder.

2. The method of claim 1, wherein the subject does not have a disease of the gastrointestinal tract (GIT).

3. The method of claim 2, wherein said disease of the GIT is selected from the group consiting of inflammatory bowel disease, metabolic syndrome, gut infection and gut autoinflammation .

4. The method of claim 1, wherein said HA comprises linear HA.

5. The method of claim 1, wherein said HA comprises crosslinked HA.

6. The method of any one of claims 1-5, wherein said administering comprises administering at least once a day.

7. The method of any one of claims 1-6, wherein further comprising administering to the subject an anti-inflammatory agent.

8. The method of claim 7, wherein said anti-inflammatory agent comprises a steroid agent.

9. The method of claim 8, wherein said anti-inflammatory agent comprises a nonsteroidal anti-inflammatory agent.

10. The method of claim 7, wherein said anti-inflammatory agent comprises a COX-2 inhibitor.

11. The method of claim 10, wherein said COX-2 inhibitor comprises celecoxib.

12. The method of claim 7, wherein said anti-inflammatory agent comprises a biologic.

13. The method of claim 12, wherein said biologic comprises TNF inhibitor.

14. The method of any one of claims 7-13, wherein said anti-inflammatory agent and said agent that decreases permeability of the gut are formulated in a single composition.

15. The method of any one of claims 7-14, wherein said anti-inflammatory agent and said HA are formulated in separate compositions.

16. The method of any one of claims 1-15, wherein the mood disorder comprises anxiety and / or depression.

17. A composition comprising HA, for use in treating a mood disorder.

18. A pharmaceutical composition comprising HA and an anti-inflammatory agent, wherein the composition is formulated for oral delivery.

19. The pharmaceutical composition of claim 18, wherein the anti-inflammatory agent is a Cox-2 inhibitor.

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