Specialized pro-resolving lipid mediators for treating depression, anxiety, and / or stress

Abstract

The invention described herein concerns the use of specialized pro-resolving mediators (SPMs) in the treatment of mood disorders and conditions associated with a serotonin imbalance, such as depression, anxiety, and / or stress. Such treatment may be carried out with the SPMs alone or in combination with existing anti-depressants and / or anxiolytics. The efficacy of the treatment is demonstrated in an animal model.

Description

[0001] SPECIALIZED PRO-RESOLVING LIPID MEDIATORS FOR TREATING DEPRESSION, ANXIETY, AND / OR STRESS Field of the invention

[0002] The present invention concerns the use of specialized pro-resolving mediators (SPMs) in the treatment of mood disorders and conditions associated with a serotonin imbalance, such as depression, anxiety, and / or stress. Such treatment may be carried out with the SPMs alone or in combination with existing medications, such as anti-depressants and / or anxiolytics.

[0003] Background of the invention

[0004] Mood disorders, depression, anxiety, and stress

[0005] A mood disorder also known as affective disorder is a type of mental health condition where there is a disconnect between actual life circumstances and the person's state of mind or feeling. A mood disorder can negatively affect your ability to function normally. It can have serious consequences in all aspects of life, from personal to professional. Children, teens, and adults can all have mood disorders. Therapy, medicines, support and self-care can help treat mood disorders.

[0006] Depression, anxiety, and stress are seriously debilitating conditions that each year cause significant disruptions in people’s lives and affect their ability to carry out their work. As such, the cost to society of these conditions is highly significant.

[0007] Depression affects an estimated one in 15 adults (6.7%) in any given year. And one in six people (16.6%) will experience depression at some time in their life. Depression can occur at any time but, on average, first appears during the late teens to mid-20s. Women are more likely than men to experience depression. Some studies show that one-third of women will experience a major depressive episode in their lifetime. There is a high degree of heritability (approximately 40%) when first-degree relatives (parents / children / siblings) have depression. Depression is not only a medical concern but also a social problem.

[0008] Depression is among the most treatable mental disorders. Between 80% and 90% percent of people with depression eventually respond well to treatment. Almost all patients gain some relief from their symptoms.

[0009] Before a diagnosis or treatment, a health professional should conduct a thorough diagnostic evaluation, including an interview and a physical examination. In some cases, a blood test might be done to make sure the depression is not due to a medical condition like a thyroid problem or a vitamin deficiency (reversing the medical cause would alleviate the depression-like symptoms). The evaluation will identify specific symptoms and explore medical and family histories as well as cultural and environmental factors with the goal of arriving at a diagnosis and planning a course of action. Psychological treatments are the first treatments for depression. They can be combined with antidepressant medications in moderate and severe depression. Antidepressant medications are not needed for mild depression. Brain chemistry may contribute to an individual’s depression and may factor into their treatment. For this reason, antidepressants might be prescribed to help modify one’s brain chemistry. These medications are not sedatives, “uppers” or tranquilizers. They are not habit-forming. Generally, antidepressant medications have no stimulating effect on people not experiencing depression.

[0010] Antidepressants may produce some improvement within the first week or two of use, yet full benefits may not be seen for two to three months. If a patient feels little or no improvement after several weeks, his or her psychiatrist can alter the dose of the medication or add or substitute another antidepressant. In some situations, other psychotropic medications may be helpful.

[0011] Psychiatrists usually recommend that patients continue to take medication for six or more months after the symptoms have improved. Longer-term maintenance treatment may be suggested to decrease the risk of future episodes for certain people at high risk.

[0012] Depression may also be caused by an underlying chronic medical condition, such as e.g. multiple sclerosis or other conditions affecting quality of life and currently not having positive treatment prospects.

[0013] Anxiety is a feeling of unease, such as worry or fear, that can be mild or severe. People can experience feelings of anxiety at some point in their life, but some people find it hard to control their worries. Their feelings of anxiety are more constant and can often affect their daily lives.

[0014] Anxiety is the main symptom of several conditions, including panic disorder, phobias (i.e. agoraphobia or claustrophobia), post-traumatic stress disorder (PTSD) and social anxiety disorder (social phobia).

[0015] In addition to psychological treatment, many different types of medications are used in the treatment of anxiety disorders, including traditional anti-anxiety drugs such as benzodiazepines (typically prescribed for short-term use) and newer options like SSRI antidepressants (often recommended as a long-term anxiety solution). These drugs can provide temporary relief, but they also come with side effects and safety concerns - some significant.

[0016] There’s a fine line between stress and anxiety. Both are emotional responses, but stress is typically caused by an external trigger. The trigger can be short-term, such as a work deadline or a fight with a loved one or long-term, such as being unable to work, discrimination, or chronic illness. People under stress experience mental and physical symptoms, such as irritability, anger, fatigue, muscle pain, digestive troubles, and difficulty sleeping. Anxiety, on the other hand, is defined by persistent, excessive worries that don’t go away even in the absence of a stressor. Anxiety leads to a nearly identical set of symptoms as stress: insomnia, difficulty concentrating, fatigue, muscle tension, and irritability.

[0017] SPMs are essential for resolution of inflammation.

[0018] Omega-3 fatty acids are found in resolving inflammatory exudates and serve as substrates for the synthesis of specific signalling molecules - the so-called specialized pro-resolving mediators (SPMs), which comprise resolvins, protectins, lipoxins and maresins. These SPMs are crucial for sufficient resolution of inflammatory processes because they promote critical pathways leading to resolution of the inflammatory response. Treatment with SPMs or their precursors have been found to resolve inflammation.

[0019] It has been described that patients with depression have elevated levels of pro-inflammatory markers and it has therefore been proposed that anti-inflammatory medication, such as NSAIDs or COX inhibitors, could aid in treating depression. However, results are inconclusive, and it has been suggested that any potential benefit on depression would be outweighed by potential side effects of the anti-inflammatory medication (Kohler et al., Current Neuropharmacology, 2016, 14, 732- 742).

[0020] Presently available anti-depressants and anxiolytics have narrow therapeutic windows and can have serious side effects, including addiction, resulting in non-compliance with the treatment regimen. It would therefore be desirable to provide compounds capable of treating mood disorders, depression, anxiety, and / or stress without the side effect profile of existing anti-depressants and anxiolytics.

[0021] Summary of the invention

[0022] In one aspect, the present invention concerns a composition for use in the treatment of mood disorders, major depressive disorder, generalized anxiety disorder, chronic stress, and / or acute stress disorder, wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and / or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

[0023] Figures

[0024] Figure 1 discloses the latency and grooming periods in the splash test (ST).

[0025] Figure 2 discloses the number of colony forming units (CFU) in spleen and liver induced during CMS and after treatment with SPMs.

[0026] Figure 3 discloses the amount of lipopolysaccharide binding proteins (LBP) in plasma, induced due to chronic moderate stress (CMS) and after treatment with SPMs. Figure 4 discloses plasma levels of Tryptophan, kynurenine and serotonin (5-HT) after chronic mild stress (CMS) and after treatment with SPMs.

[0027] Figure 5 discloses quinolinic acid (QUINA) and kynurenic acid (KYNA) plasma levels and the ratio between both levels after chronic moderate stress and after treatment with SPMs.

[0028] Figure 6 discloses levels of quinolinic acid (QUINA) and kynurenic acid (KYNA) in the frontal cortex and ratio between them (excitotoxicity risk) after chronic moderate stress (CMS) and treatment with SPMs.

[0029] Figure 7 discloses TNF-alpha levels in the frontal cortex, after chronic mild stress (CMS) and after treatment with the SPMs.

[0030] Detailed description of the invention

[0031] Definitions

[0032] In the present context, the term "SPM" or “specialized pro-resolving lipid mediator” is intended to mean a compound which is a resolvin, a protectin, a lipoxin, or a maresin.

[0033] In the context of the present invention, the term "resolvin" is intended to cover resolvins resulting from the cascade of EPA, also referred to as “E series resolvins”, and from the cascade of DHA, also referred to as “D series resolvins”. Examples of E series resolvins include the compounds RvE1 , RvE2, and RvE3. Examples of D series resolvins include the compounds RvD1 , RvD2, RvD3, RvD4, RvD5, and RvD6.

[0034] In the context of the present invention, the term "protectin'1is intended to cover protectins resulting from the cascade of DHA. Protectins include the compounds PD1 , and PDX.

[0035] In the context of the present invention, the term "maresin" is intended to cover maresins resulting from the cascade of DHA. Maresins include the compounds MaR1 , MaR2, MaR3, MCTR1 , MCTR2, and MCTR3.

[0036] In the context of the present invention, the term “SPM precursor” or “SPM precursors” refers to 18- hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), and / or 14-hydroxydocosahexaenoic acid (14-HDHA).

[0037] In the context of the present invention, the term “mood disorder” refers to any of a group of conditions of mental and behavioural disorder where a disturbance in the person's mood is the main underlying feature. Mood disorders fall into seven groups, including; abnormally elevated mood, such as mania or hypomania; depressed mood, of which the best-known and most researched is major depressive disorder (MDD) (alternatively known as clinical depression, unipolar depression, or major depression); and moods which cycle between mania and depression, known as bipolar disorder (BD) (formerly known as manic depression). In the context of the present invention, the term “major depressive disorder” refers to a mental disorder characterized by at least two weeks of pervasive low mood, low self-esteem, and loss of interest or pleasure in normally enjoyable activities, as diagnosed by a clinician. This is to be distinguished from a feeling of momentary sadness or grief, or any other feeling that may lead a person to express that he / she is “depressed”.

[0038] In the context of the present invention, the term “generalized anxiety disorder” is an anxiety disorder characterized by excessive, uncontrollable, and irrational worry about events or activities, as diagnosed by a clinician.

[0039] In the context of the present invention, the term “chronic stress” is intended to mean the physiological or psychological response induced by a long-term internal or external stressor, as diagnosed by a clinician. It is not to be confused with a feeling of being very busy when a person may otherwise state to feel “stressed”.

[0040] In the context of the present invention, the term “acute stress disorder” is intended to mean a psychological response to a terrifying, traumatic or surprising experience, as diagnosed by a clinician. If symptoms persist longer than a month, people are diagnosed as having posttraumatic stress disorder (PTSD).

[0041] Compositions for treatment of mood disorders, depression, anxiety, and / or stress.

[0042] In one aspect, the present invention concerns a composition for use in the treatment of mood disorders, major depressive disorder, generalized anxiety disorder, chronic stress, and / or acute stress disorder, wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and / or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

[0043] Accordingly, in one embodiment, the composition of the invention comprises an SPM precursor and / or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

[0044] In a further embodiment, the composition comprises one or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof.

[0045] In another embodiment, the composition comprises two or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof.

[0046] In still another embodiment, the composition comprises three or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof.

[0047] In still a further embodiment, the composition further comprises EPA and / or DHA.

[0048] In an alternative embodiment, the composition according to the invention comprises an SPM and / or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

[0049] In another embodiment, the composition comprises one or more compounds selected from the group consisting of 5S,6R,15S-trihydroxy-7E,9E,11 Z,13E-eicosatetraenoic acid (LxA4), 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid (LxB4), 5S,6R,15R-trihydroxy- 7E,9E,11 Z,13E-eicosatetraenoic acid (15-epi-LxA4), 5S,14R,15R-trihydroxy-6E,8Z,10E,12E- eicosatetraenoic acid (15-epi-LxB4), 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (RvE1), 5S,12R,18S-trihydroxy-6Z,8E,10E,14Z,16E'-eicosapentaenoic acid (18S-RvE1), 5S,18R-dihydroxy-6Z,8E,10E,14Z,16E'-eicosapentaenoic acid (RvE2), 17R,18RZS-dihydroxy- 5Z,8Z,11Z,13E,15H-eicosapentaenoic acid (RvE3), 7S,8R,17S-trihydroxy-4Z,9E,11 E,13Z,15E,19Z- docosahexaenoic acid (RvD1), 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (RvD2), 4S,11 R,17S-trihydroxy-5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid (RvD3), 4S,5R,17S-trihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD4), 7S,17S-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD5), 4S,17S-dihydroxy- 4Z,8H,10Z,13Z,15H,19Z-docosahexaenoic acid (RvD6), 7S, 8R,17R-tri hydroxy- 42, 9E,11 E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD1), 7S, 16R,17R-tri hydroxy- 42, 8E,10Z,12H,14E,19Z-docosahexaenoic acid (17R-RvD2), 4S, 11 R,17R-tri hydroxy- 52, 7E,9E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD3), 4S,5R,17R-trihydroxy- 6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD4), 7S,17R-dihydroxy- 4Z,8E,10Z,13Z,15H,19Z-docosahexaenoic acid (17R-RvD5), 4S,17R-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD6), 7,13R,20-trihydroxy- 8H,10Z,14E,16Z,18E-docosapentaenoic acid (RvT1), 7,8,13R-trihydroxy-9E,11 E,14E,16Z,19Z- docosapentaenoic acid (RvT2), 7,12,13R-trihydroxy-8Z,10E,14E,16Z,19Z-docosapentaenoic acid (RvT3), 7,13R-dihydroxy-8E,10Z,14E,16Z,19Z-docosapentaenoic acid (RvT4), 7,8,17-trihydroxy- 8,10,13,15,19-docosapentaenoic acid (RvD1n-3), 7, 16, 17-tri hydroxy-8,10, 12, 14, 19- docosapentaenoic acid (RvD2n-3), 7,17-dihydroxy-8,10,13,15,19-docosapentaenoic acid (RvD5n-3), 10R,17S-dihydroxy-4Z,7Z,11 E,13E,15Z,19Z-docosahexaenoic acid (PD1), 10S,17S-dihydroxy- 4Z,7Z,11 E,13E,15Z,19Z-docosahexaenoic acid (PDX), 10R,17S,22-trihydroxy- 4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (22-hydroxy-PD1), 10R,17R-dihydroxy- 4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (17-epi-PD1), 10S,17S-dihydroxy- 4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (10-epi-PD1), 10,17-dihydroxy-7,11 ,13,15,19- docosapentaenoic acid (PD1n-3), 16,17-dihydroxy-7,10,12,14,19-docosapentaenoic acid (PD2n-3), 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1), 13R,14S-dihydroxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (MaR2), 7S,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z- docosahexaenoic acid (7-epi-MaR1), 14S,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L1), 14R,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L2), and 7 S, 14S-dihydroxy-8E, 10E, 12Z, 16Z, 19Z-docosapentaenoic acid (MaR1 n-3) •

[0050] Preparation of SPMs and their active precursors is known in the art, including WO 2013 / 170006.

[0051] The composition for use according to the present invention may in one embodiment further comprise the administration of an anti-depressant and / or an anxiolytic. The anti-depressant and / or anxiolytic may be in a separate composition, or it may be part of the same composition. In one embodiment, the anti-depressant and / or anxiolytic is in a separate composition. In a further embodiment, the treatment may further comprise the administration of a compound selected from the group consisting of alprazolam (anxiety), amitriptyline (depression), amoxapine (depression), bupropion (anxiety), buspirone (anxiety), butriptyline (depression), citalopram (depression), clomipramine (anxiety and depression), desipramine (depression), dibenzepin (depression), dosulepin (depression), doxepin (anxiety and depression), duloxetine (anxiety), escitalopram (anxiety and depression), fluoxetine (depression), fluvoxamine (depression), gabapentin (anxiety), imipramine (depression), iprindole (depression), lofepramine (depression), mirtazapine (anxiety), nefazodone (depression), norfluoxetine (depression), opipramol (anxiety), paroxetine (depression), protriptyline (depression), reboxetine (depression), sertraline (anxiety and depression), sulpiride (anxiety), tandospirone (anxiety and depression), trimipramine (anxiety and depression), trazodone (anxiety) and venlafaxine (anxiety and depression), or pharmaceutically acceptable salts thereof. In another embodiment, the composition for use according to the present invention does not comprise an anti-depressant and / or an anxiolytic.

[0052] In another embodiment, the composition for use according to the present invention is for use in treating major depressive disorder. In yet another embodiment, the treatment further comprises the administration of an anti-depressant, such as amitriptyline, amoxapine, butriptyline, citalopram, clomipramine, desipramine, dibenzepin, dosulepin, doxepin, escitalopram, fluoxetine, fluvoxamine, imipramine, iprindole, lofepramine, nefazodone, norfluoxetine, paroxetine, protriptyline, reboxetine, sertraline, tandospirone, trimipramine, or venlafaxine, or pharmaceutically acceptable salts thereof.

[0053] In still another embodiment, the composition for use according to the present invention is for use in treating general anxiety disorder. In a further embodiment, the treatment further comprises the administration of an anxiolytic, such as alprazolam, bupropion, buspirone, clomipramine, doxepin, duloxetine, escitalopram, gabapentin, mirtazapine, opipramol, sertraline, sulpiride, tandospirone, trimipramine, trazodone, or venlafaxine, or pharmaceutically acceptable salts thereof.

[0054] In still a further embodiment, the composition for use according to the present in invention is for use in the treatment of chronic stress. In yet a further embodiment, the composition for use according to the present invention is for use in the treatment of acute stress disorder. In another embodiment the treatment of chronic stress or acute stress disorder further comprises the administration of an anti-depressant and / or an anxiolytic.

[0055] The treatment of mood disorders, major depressive disorder, generalized anxiety disorder, chronic stress, and / or acute stress disorder has a duration for as long as necessary to effectively relieve the patient of symptoms. Indeed, treatment may even continue after symptoms have disappeared. The SPMs and SPM precursors as defined herein have an advantage compared to traditional antiinflammatory agents, such as non-steroidal anti-inflammatory drugs (NSAIDs) and Cox inhibitors, in their favourable side effect profile, which allows them to be administered for longer periods of time than traditional inflammatory agents. In one embodiment, the treatment of mood disorders, major depressive disorder, generalized anxiety disorder, chronic stress, and / or acute stress disorder has a duration of at least 3 months, such as at least 6 months, e.g. at least 12 months. In a further embodiment, the treatment has a duration of between 3 months and 24 months. In yet a further embodiment, the treatment has a duration of between 6 months and 18 months. In still a further embodiment, the treatment has a duration of between 9 months and 15 months.

[0056] Examples

[0057] Example 1 - Behavioural assessment in an animal model + biochemical markers

[0058] Animal model

[0059] A chronic stress study (chronic mild stress model - CMS) was carried out as a behavioral test in vivo in rats - including the analysis of specific analytes in plasma and in the prefrontal cortex.

[0060] Young male adult rats Wistar Hannover (HsdRccHamWist), with initial weight between 200 and 225 grams and not consanguineous, were used throughout. Animals were housed in individual cages under standard humidity and temperature conditions, with a 12-hour light / dark cycle (switched on at 8 am) and with water and food ad libitum. They were kept under constant conditions for the 7 days prior to the onset of stress. All experimental protocols followed the guidelines of the Animal Experimentation Committee of the Complutense University of Madrid, Spain, in accordance with Spanish legislation (RD 53 / 2013, of 1 February) and European legislation in force (2010 / 63 / EU) and all necessary permits were obtained. In order to minimize animal suffering and the number of animals used, the three R principles (reduction, replacement and refinement) were applied wherever possible. The depression model is known as the "chronic mild stress" (CMS). It is a chronic stress of mixed physical-psychological type, which is unpredictable, inescapable and uncontrollable for animals, and which results in the induction of depressive type behavior, in addition to sharing biochemical and structural alterations related to this psychiatric disease. These characteristics give the model sufficient robustness for the experimental study of depression, as it is endorsed by numerous studies that support its validity of content, prediction, and construct:

[0061] 1 . Content validity: CMS induces in animals many of the typical symptoms of human depression, validated through corresponding behavioral tests, such as anhedonia, apathy and lack of motivation, anxiety or weight loss, even biochemical alterations such as the decrease in the bioavailability of serotonin, inflammatory markers in plasma and the increase of the main stress hormone (in humans cortisol, in rodents corticosterone).

[0062] 2. Predictive validity: the main pharmacological treatments used in humans against depression (tricyclic antidepressants, selective serotonin reuptake inhibitors, selective serotonin-noradrenaline reuptake inhibitors) are capable of reversing or attenuating the behavioral changes following CMS.

[0063] 3. Construct validity: chronic stress has been identified as one of the main triggers of depressive processes. The etiology of the disease is thus confirmed in this model.

[0064] The total duration of chronic stress was 21 days after having verified in previous studies of our research that it was sufficient to induce depressive behavior and biochemical alterations related to depression. Each animal was individually housed and subjected to two different stressors changed daily every 12 hours, with two mornings of rest a week, following an irregular pattern that was unpredictable for the animals. The application of stressors over each of the three weeks is summarized in Table 1 :

[0065] Table 1 : Chronic mild stress protocol.

[0066] The different stressors used constitute a stress of moderate intensity. The description is as follows: • Matching animals: represents the invader-host paradigm. They also pair the animals after a period of deprivation of food and drink, which is an additional stress for animals.

[0067] • Flash lighting: changing light / dark cycle every 2 hours.

[0068] • Wet sawdust: moisten sawdust with 350 mL of water. At the end of the stress, it was changed to new sawdust.

[0069] • Food and drink deprivation: withdrawal of free access to food and drink.

[0070] • Tilted cage: inclination of the cages at a 45 degrees angle.

[0071] • Strobe: placement of an LED spotlight with a lighting frequency of 150 flashes / minute.

[0072] SPM composition and treatment

[0073] The composition used in the experiments is an oil composition of marine origin, which has been enriched with SPMs and SPM precursors by fractionation using the procedure disclosed in WO 2013 / 170006. The specifications for the oil are the following:

[0074] SPM precursor content

[0075] • 17-HDHA as FFA (mg / kg) 80-400 (LC / MS)

[0076] • 18-HEPE as FFA (mg / kg) 50-400 (LC / MS)

[0077] • 14-HDHA as FFA (mg / kg) 40-200 (LC / MS)

[0078] Fatty acid profile

[0079] • EPA mg / g (as FFA) 100-300 (Eur.Ph. 2.4.29)

[0080] • DHA mg / g (as FFA) 200-450 (Eur.Ph. 2.4.29)

[0081] • Total Omega-3 mg / g (as FFA) 500-850 (Eur.Ph. 2.4.29)

[0082] • Triglycerides (% aera) Min. 60 (Eur.Ph. 2.2.30)

[0083] *FFA = free fatty acid

[0084] Each rat was administered 0.3 mL of the SPM composition or placebo (skimmed milk) daily by oral gavage for four weeks (1 week pre-treatment, 3 weeks of CMS). The rats were divided into four different groups: Control or CMS rats, each of these groups further divided into rats receiving placebo and rats receiving the SPM composition.

[0085] Protocol - behavioural studies

[0086] All behavioural studies were carried out under controlled conditions of humidity, temperature, lighting and acoustics and minimizing as much as possible the time of the researcher in the behavioral room during their performance. The different behaviors to be studied were recorded using a video camera, and the videos were later analyzed with the researcher, who was unaware of the research conditions.

[0087] The behavioral tests consisted of: • Splash test (ST) (anhedonia-related behaviors and degree of personal self-care)

[0088] The "splash" test is a behavioural test about parameters related to depressive type behaviour. The evaluation was carried out after day 21 of stress (day 22 of the experiment) through the grooming behavior of the animal after spraying a solution of 10% sucrose in the dorsal coat during the activity phase and under dark conditions. Grooming is considered a personal hygiene index, as well as an indirect measure of appetite for a sweet solution related to anhedonic characteristics. Behaviours were analyzed / quantified with the help of a camcorder for a period of 5 minutes: o Latency period (time until the start of grooming) o Grooming (duration of grooming)

[0089] Results - behavioural studies

[0090] The administered SPMs significantly reduce the latency period in the group of stressed rats. This is directly related to anhedonia and therefore a measure of the antidepressant effect of the SPMs. The duration of grooming was similar for both groups.

[0091] The SPMs decreased the latency time in the ST, as well as a decrease in the time spent grooming, so they showed ability to improve anhedonia and the degree of personal self-care.

[0092] Protocol - sample collection and processing

[0093] Biological sampling in rats was performed in a staggered and balanced manner between the different groups, to minimize possible interference by circadian variations of corticosterone levels. After an overdose of intraperitoneal anesthesia (320 mg / kg) with sodium pentobarbital (Vetoquinol®), the absence of reflexes, both in foot and in tail, was verified, the chest cavity was opened after sterilization with 70% ethanol (v / v) and the removal of tissues. a) Extraction process:

[0094] • Plasma: blood was extracted by cardiac puncture after the opening of the chest cavity. Using sterile syringes loaded with 0.1 ml of ethylenediaminetetra-acetic acid (EDTA) 0.5 M as an anticoagulant. The separation of the different blood fractions was performed by centrifugation at 1500 r.p.m. for 15 minutes, at room temperature, and following a separation protocol based on Ficoll. Plasma and peripheral blood mononuclear cells (PBMCs) were stored at -80 °C until further use for the various tests.

[0095] • Brain: brain was extracted from the skull separating and collecting the frontal cortex divided into 6 segments. They were immediately frozen in carbonic snow and stored at -80 °C for later use.

[0096] The choice of the frontal cortex is due to its involvement in depression as it plays a crucial role in the so-called executive function that includes the processing of emotions and decision making and planning. It is a structure with special sensitivity towards pro and anti-inflammatory mediators induced by stress, the etiological factor of depression that emulates the animal model. In addition, previous studies have shown that TLR-4, the innate immunity receptor is activated in the prefrontal cortex after CMS.

[0097] • Mesenteric lymph nodes, spleen and liver: under sterile conditions, the same amount of each tissue type was weighed and homogenized in 2 mL of sterile saline for the subsequent microbiological cultures to be performed within the study of colon bacterial translocation.

[0098] • Preparation of total tissue homogenate

[0099] In order to study intercellular mediators and global protein quantities within the tissue, a procedure was performed to obtain total tissue homogenate. To do this, the portion of brain was homogenized in 600 pl (TissueLyser, QIAGEN) to 50 oscillations / s and 4 °C for 4 minutes. Total homogenization was obtained by centrifuging at 12000 rpm for 10 minutes in cold and collecting the supernatant. Samples were stored at -80 °C until the corresponding tests were performed.

[0100] • Preparation for microbiology studies

[0101] Bacterial translocation was measured by the growth of colony-forming units (CFUs) in different organs. Mesenteric lymph nodes (GLMs), liver and spleen were selected, in addition to the blood itself. The entire procedure was performed under sterility conditions. Each tissue was weighed and homogenized in 2 mL of sterile saline 9% (v / v) by performing two serial dilutions of each sample and inoculating them in microbial culture media.

[0102] Biochemical protocol a) Microbiology studies The culture media used were Blood Agar and MacConkey Agar 5% for aerobic bacteria count and Blood Agar Brucella supplemented with vitamin K1 for anaerobic bacteria. After 24 hours of incubation at 37 °C for aerobic bacteria and 48 hours for anaerobics, CFUs were counted as CFUs / mg of tissue. The identification of the different bacterial species was done by Gram staining and standard biochemical tests. In the case of aerobic microorganisms the following assays were used: the oxidase assays (Mast Diagnostics®), antibiotic discs of optoquine, bacitracin and novobiocin (Mast®), coagulase (Beckton Dickinson®), catalase, and the biochemical identification system BBLTM Enterotubo IITM (Difco Laboratories, Detroit, Ml, USA). The API 20 NE system (Biomerieux®) was used for non fermenter Gram negative bacilli, and the API 20 Strep for enterococcus. For anaerobes, the identification system Rapid ID 32 A (Biomerieux®) was used. b) Determination of the lipopolysaccharide (LPS), Lipopolysaccharide binding protein (LBP), tryptophan (Trp), kynurenine (Kyn), serotonin (5-HT), quinolinic acid (QUINA) and kynurenic acid

[0103] Commercial ELISA kits were used: LAL Chromogenic Endpoint Assay and LBP ELISA kit from Hycult® Biotechnology, USA; Trp, Kyn, 5-HT ELISA kits from LDN; QUINA, KYNA ELISA kits from Cloud-Clone Corp., USA. Absorbance was measured with the Synergy 2 microplate reader (BioTek®, USA) and data processing was performed with the built-in Gen5 Data Analysis Software (BioTek®, USA). c) Statistical analysis

[0104] Data were expressed as the mean ± standard error of the mean (SEM). The ROUT test was performed for the detection of outliers, the Saphiro-Wilks test to evaluate the normality of distribution and the Brown-Forsythe test to check the homogeneity of variances. If the data presented a normal distribution and equality of variances, a one-way ANOVA was performed followed by a Tukey post-hoc for multiple comparisons between groups. If the distribution was normal but equal variances could not be assumed, a Brown-Forsythe ANOVA followed by a Tamhane T2 post-hoc was applied for multiple comparisons between groups. If the data did not show a normal distribution, a nonparametric Kruskal-Wallis test with a post-hoc Dunn was performed for multiple comparisons between groups. A value of p 0.05 was considered statistically significant. The data was analyzed using the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).

[0105] Biochemical results in plasma - bacterial translocation

[0106] The gut-brain axis (GBA) is a bilateral communication network between the gastrointestinal (Gl) tract and the central nervous system (CNS). The essential amino acid tryptophan contributes to the normal growth and health of both animals and humans and, importantly, exerts modulatory functions at multiple levels of the GBA. Tryptophan is used for protein synthesis, affecting the growth and health of animals and humans.

[0107] Changes in the gut microbiota composition affect the GBA by modulating the tryptophan metabolism. Most tryptophan from ingested protein is absorbed in the small intestine. The remaining tryptophan reaches the large intestine, where it is degraded by commensal microbes. Tryptophan is the sole precursor of serotonin [5-hydroxytryptamine (5-HT)], which is a key neurotransmitter involved in the modulation of emotional control, food intake, sleep and pain. Tryptophan can be metabolized into kynurenine, tryptamine and indolic compounds, thereby modulating neuroendocrine and intestinal immune responses and having profound effects on the interaction between gut microbiota and the GBA. Tryptamine, a monoamine that is structurally similar to serotonin, can be generated from the decarboxylation of tryptophan by tryptophan decarboxylases (TrpD) from commensal bacteria. Bacteria belonging to Clostridium, Ruminococcus, Blautia, and Lactobacillus have been identified as being able to convert tryptophan to tryptamine in a TrpD-dependent manner.

[0108] During a depressive process, there is an increase in intestinal permeability, which leads to the translocation of bacteria from the intestine to the blood, causing a generalized inflammatory process in the body. The inflammatory process can be communicated to the brain, producing neuro-inflammation and leading to further depressive processes.

[0109] When bacterial translocation occurs and bacteria pass into the blood, it is expected to find high levels of bacterial debris, such as lipopolysaccharides (LPSs), in plasma. LPSs are found in the plasma membrane of GRAM-negative bacteria. Many of these LPSs act as toxins and cause inflammation. It is expected to find LPSs in blood when there are bacteria in the body. Lipopolysaccharide binding proteins (LBPs) bind to LPSs and remove the LPSs from the body, cleaning the blood and reducing the level of inflammation.

[0110] SPMs decrease the amount of colony-forming units (CFUs) in the spleen and liver. No CFUs are detected in the CMS-Y group in the liver. This indicates that SPMs correct the intestinal permeability occurring during the depressed state.

[0111] LBP levels increased significantly after treatment with SPMs (p< 0.05). Without being bound by a particular theory, the increase in the LBP levels induced by the SPMs is considered a response to the increase in LPS levels mediated by the anti-inflammatory properties of SPMs.

[0112] Biochemical results in plasma and prefrontal cortex - kynurenine pathway

[0113] The metabolism of tryptophan and kynurenine has been studied both in plasma and in the prefrontal cortex.

[0114] More than 90% of total tryptophan is oxidized, via the kynurenine pathway, into kynurenine in the liver. This kynurenine pathway exerts a primary role in affecting tryptophan availability by the clearance of excess tryptophan. The tryptophan metabolism along the kynurenine pathway is mainly initialized by the induction of either of the rate limiting enzyme indoleamine-2,3-dioxygenase (IDO) or by tryptophan 2,3-dioxygenase (TDO).

[0115] While IDO exists in various organs, such as the brain, the gastrointestinal (Gl) tract, and liver, TDO is almost entirely expressed in the liver. IDO can be activated in response to immune stimuli, with interferon-gamma being the most efficient inducer. Among other inflammatory conditions, overexpression of IDO can be observed in the colonic mucosa of patients with inflammatory bowel disease. TDO activity is regulated by tryptophan’s availability, with its activity being relatively stable, while stress-induced changes in the expression of TDO in the liver are primarily influenced by the activation of the hypothalamic-pituitary-adrenal axis through the action of glucocorticoids.

[0116] In the case of CMS, the production of indolamine 2,3-dioxygenase (IDO) is induced. The production of IDO reduces the bioavailability of tryptophan, since it is consumed through the kynurenine pathway. In the absence of IDO, tryptophan is a precursor to serotonin (5-HT) thanks to tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (DDC).

[0117] When IDO is produced and tryptophan passes through the kynurenine pathway, the following kynurenine metabolites are produced:

[0118] 1) Kynurenic acid (KYNA), which is neuroprotective, since it is an antagonist of the glutamate N- methyl D-aspartate receptor (NMDA receptor antagonist).

[0119] 2) Quinolinic acid (QUINA), which is neurotoxic, since it is an agonist of the glutamate N-methyl D-aspartate receptor (NMDA agonist).

[0120] The ratio between the two metabolites (QUINA / KYNA) was measured. This ratio increases in the case of CMS, with levels returning to normal after the administration of antidepressants (AD).

[0121] In groups treated with SPMs, the QUINA:KYNA ratio obtained in plasma and in frontal cortex is neuroprotective:

[0122] KYNA is produced in a larger proportion than QUINA.

[0123] Both metabolites bind to the same brain receptor: NMDA receptor, which regulates brain arousal. As the ratio of QUINA / KYNA is reduced, it is concluded that excitotoxicity is reduced.

[0124] The results of kynurenine levels in the prefrontal cortex support what was observed in plasma.

[0125] Treatment with SPMs increased the levels of KYNA in the frontal cortex, with maintained QUINA levels. This decreases the risk of excitotoxicity (damage or death of nerve cells when levels of otherwise necessary and safe neurotransmitters become pathologically high).

[0126] Biochemical results in prefrontal cortex - inflammation

[0127] A statistically significant decrease in the TNF-a factor was seen in the prefrontal cortex, which confirms the anti-inflammatory nature of the SPMs. TNF-a levels in the frontal cortex were measured in the CMS situation. TNF-a is a key cytokine in inflammatory reactions. Its neutralization is important for the treatment of chronic inflammatory illnesses. The SPMs were able to decrease the TNF-alpha levels in the frontal cortex. CMS does not induce an increase in the TNF-a levels when compared with the controls.

[0128] Conclusions The behavioural study in rats demonstrated an anti-depressive effect of administering SPMs, more particularly manifested in the observation of shorter latency times in the splash test.

[0129] The behavioural results were corroborated by the observed biochemical changes as a result of the chronic mild stress and the subsequent SPM treatment. The results thus support the use of SPMs for treating mood disorders, depression, anxiety and / or stress.

Claims

1. Claims1. A composition for use in the treatment of generalized anxiety disorder, chronic stress, acute stress disorder, mood disorders, and / or major depressive disorder, wherein said composition comprises one or more specialized pro-resolving lipid mediator (SPM) precursors and / or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof, wherein said one or more precursors are selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18- HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), and 14-hydroxydocosahexaenoic acid (14- HDHA).

2. The composition for use according to claim 1 , wherein said composition further comprises EPA and / or DHA.

3. The composition for use according to any one of the preceding claims, wherein the composition is for use in the treatment of generalized anxiety disorder.

4. The composition for use according to claim 3, wherein the treatment further comprises the administration of an anxiolytic.

5. The composition for use according to any one of claims 1 to 2, wherein the composition is for use in the treatment of chronic stress.

6. The composition for use according to any one of claims 1 to 2, wherein the composition is for use in the treatment of acute stress disorder.

7. The composition for use according to claim 5 or 6, wherein the treatment further comprises the administration of an anti-depressant and / or an anxiolytic.

8. The composition for use according to any one of claims 1 to 2, wherein the composition is for use in the treatment of major depressive disorder.

9. The composition for use according to claim 7, wherein the treatment further comprises the administration of an anti-depressant.

10. The composition for use according to any one of the preceding claims, wherein the treatment has a duration of at least 3 months, such as at least 6 months, e.g. at least 12 months.11 . The composition for use according to any one of claims 1 to 9, wherein the treatment has a duration of between 3 months and 24 months, such as between 6 months and 18 months, e.g. between 9 months and 15 months.

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