Anti-inflammatory lipid mediators for use in the treatment of neurodegenerative and / or autoimmune diseases - Patents.com

Abstract

The present invention relates to anti-inflammatory lipid mediators, including maresins, D-series resolvins, E-series resolvins, protectins, or lipoxins, or combinations thereof, for use in the treatment of neurodegenerative and / or autoimmune diseases.

Description

[Technical field]

[0001] Technical Field The present invention relates to the field of neurodegenerative and / or autoimmune diseases. More particularly, the present invention relates to a specialized pro-resolving lipid mediator, preferably maresin, and compositions comprising same for use in the treatment of neurodegenerative and / or autoimmune diseases, preferably multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). [Background technology]

[0002] Background technology Neurodegenerative diseases are a collective term for a series of conditions that primarily affect neurons in the human brain. Neurons are the basic units of the nervous system, including the brain and spinal cord. Neurons do not normally regenerate or replenish themselves, and therefore, when neurons are damaged or die, they cannot be replaced by the body. Examples of neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. Neurodegenerative diseases are incurable, debilitating conditions that result in the progressive degeneration and / or death of nerve cells. This causes problems with movement (called ataxia) or intellectual function (called dementia).

[0003] Similarly, immune system disorders or autoimmune diseases cause abnormally low or excessive activity of the immune system. Immunodeficiency reduces the body's ability to fight invaders and causes vulnerability to infections. In cases of immune system overactivity, the body attacks and damages its own tissues. This is the case of autoimmune diseases, where the immune system produces antibodies that attack the body's own tissue components rather than fighting infections. Treatment of autoimmune diseases generally focuses on reducing the activity of the immune system. Examples of autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and vasculitis.

[0004] Although neurodegenerative and autoimmune diseases have distinct etiologies, it is well known that localized inflammatory responses in the central nervous system have an essential role in the pathogenesis of these diseases.

[0005] Today, numerous research groups around the world are searching for complete or palliative solutions to many of these diseases, especially as the population affected by these diseases continues to grow due to current increased life expectancies.

[0006] Aiming at this goal, the inventors have surprisingly found that several molecules, termed "anti-inflammatory lipid mediators" or "SPMs", are useful in the treatment of such diseases.

[0007] "Anti-inflammatory lipid mediators" (SPMs, also called specialized pro-resolving mediators) are a large and growing class of cell signaling molecules formed in cells by the metabolism of polyunsaturated fatty acids (PUFAs) by one or a combination of enzymes, such as lipoxygenase, cyclooxygenase, and cytochrome P450 monooxygenase. Preclinical studies, primarily in animal models and human tissues, implicate SPMs in regulating the resolution of inflammation. These studies suggest that synthetic SPMs that are resistant to being metabolically inactivated may be promising clinically useful pharmacological tools to prevent and resolve a variety of pathological inflammatory responses, along with the tissue destruction and morbidity that these responses cause. These molecules include maresins, D-series resolvins, E-series resolvins, protectins, and lipoxins. Lipoxins are derived from arachidonic acid, E-series resolvins are derived from the long-chain n-3 fatty acid eicosapentaenoic acid (EPA), and D-series resolvins, protectins / neuroprotectins, and maresins are all derived from the n-3 fatty acid docosahexaenoic acid (DHA). There is increasing evidence for the role of these compounds in inflammatory processes.

[0008] For example, WO 2012 / 170791 discloses mono- and dihydroxy analogs of docosahexaenoic acid (DHA) for use in the treatment or prevention of inflammatory processes. WO 2010 / 033509 discloses 14-hydroxy analogs of docosahexaenoic acid (DHA) for use in the treatment or prevention of inflammatory processes. WO 2012 / 135032 discloses particles that are at least partially produced from cell-derived microparticles, have anti-inflammatory properties, and can be used as drug delivery systems, for example to treat inflammation, wounds, or pain, among others, resolvins, lipoxins, maresins, and protectins. Serhan et al., “Pro-resolving lipid mediators are leads for resolution physiology”, Nature, Vol. 10, p. 92-101, 5 June 2014 discloses SPMs and their involvement in inflammatory processes. WO 2013 / 170006 discloses anti-inflammatory mediators (SPMs) and SPM precursors obtained from natural sources (oils) and their use in nutritional supplements and pharmaceutical and cosmetic formulations to ameliorate inflammation and diseases having an inflammatory component.

[0009] As shown above, the use of SPMs as anti-inflammatory agents is well known, but the inventors have surprisingly found that these molecules, particularly maresins, are also useful in the treatment of neurodegenerative and / or autoimmune diseases, particularly multiple sclerosis and amyotrophic lateral sclerosis. This is highly advantageous, since the drugs currently used as anti-inflammatory drugs are not useful or even have adverse effects on patients suffering from neurodegenerative and / or autoimmune diseases, particularly multiple sclerosis and amyotrophic lateral sclerosis. Examples are Enbrel® (etanercept) or Celebrex® (celecoxib), further evidence can also be found in the articles: “TNF neutralization in MS Results of a randomized, placebo-controlled multicenter study” developed by The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS / MRI Analysis Group and published in Neurology (1999, pages 457-507); and “Trial of Celecoxib in Amyotrophic Lateral Sclerosis” by Cudkowicz et al., Ann. Neurol. 2006; 60: 22-31.

[0010] Therefore, there is no mention in the literature of using these compounds directly for the purposes disclosed herein. [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]FIG. 1 shows the therapeutic effect of maresin in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. (A) Assessment of locomotor loss in EAE mice over time. Note that treatment with maresin at disease onset was associated with a significant recovery of locomotor deficits (*p<0.05 vs. vehicle; two-way repeated measures ANOVA with Bonferroni post-hoc test). (B) Histological assessment of myelin loss in the spinal cord is assessed from Luxol Fast Blue stained tissue sections. Maresin provided significant protection from demyelination (*p<0.037 vs. vehicle; t-test). [Diagram 2] Figure 2: Administration of maresin significantly protects against functional losses in ALS. (A) Electrophysiological studies showing preservation of compound muscle action potential (CMAP) in tibialis anterior (TA) muscle. Note that maresin delayed the loss of CMAP amplitude by 4 weeks (*p<0.05 vs. vehicle; two-way repeated measures ANOVA with Bonferroni post-hoc test). (B) Treatment with maresin is associated with a significant preservation in functional outcome assessed by the rotarod test (*p=0.017; Mantel-Cox test). [Diagram 3] Figure 3 shows the therapeutic effect of oral maresin in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. (A) Assessment of locomotor loss in EAE mice over time. Note that treatment with maresin at disease onset was associated with a significant reversal of locomotor deficits (*p<0.05 vs. vehicle; two-way repeated measures ANOVA with Bonferroni post-hoc test). Summary of the Invention [Means for solving the problem]

[0012] Summary of the Invention The present invention relates to anti-inflammatory lipid mediators including maresins, D-series resolvins, E-series resolvins, protectins, or lipoxins, or combinations thereof, for use in the treatment of neurodegenerative and / or autoimmune diseases.

[0013] The present invention further relates to a composition comprising an anti-inflammatory lipid mediator selected from the group consisting of maresins, D-series resolvins, E-series resolvins, protectins, and lipoxins or combinations thereof for use in the treatment of neurodegenerative and / or autoimmune diseases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention The present invention relates to compounds which are anti-inflammatory lipid mediators as defined herein, including maresins, D series resolvins, E series resolvins, protectins, or lipoxins, or combinations thereof, for use in the treatment of neurodegenerative and / or autoimmune diseases.

[0015] The present invention further relates to a compound which is an anti-inflammatory lipid mediator as defined herein selected from the group consisting of maresins, D-series resolvins, E-series resolvins, protectins, and lipoxins or combinations thereof, for use in the treatment of neurodegenerative and / or autoimmune diseases.

[0016] In a preferred embodiment, the maresin is maresin-1 or maresin-2, the D series resolvin is resolvin D1, D2 D3 or D4, the E series resolvin is resolvin E1 or E2, the protectin is protectin D1 or neuroprotectin D1 and the lipoxin is lipoxin A4 or aspirin-triggered lipoxin. In a more preferred embodiment, the anti-inflammatory lipid mediator for use in the treatment of neurodegenerative and / or autoimmune diseases is maresin-1 or maresin-2. In the context of the present application, the term "maresin" includes maresin-1 and / or maresin-2.

[0017] Maresin 1 (7(R)-MaR1) is 7,14-dihydroxyDHA formed from 14(S)-hydroperoxyDHA exogenously supplied to resident peritoneal mouse macrophages activated with zymosan A. Maresin 2 (MaR2) is 13R,14S-dihydroxyDHA formed by recombinant human macrophage 12-lipoxygenase and soluble epoxide hydrolase co-incubated with DHA. Resolvin E1 (RvE1) is 5S,12R,18R-trihydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic acid). Resolvin E2 (RvE2) is 5S,18-dihydroxy-eicosa-6E,8Z,11Z,14Z,16E-pentaenoic acid. Protectin D1 (PD1) is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Resolvin D1 (RvD1) is 7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid. Resolvin D2 (RvD2) is 7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid. Resolvin D3 (RvD3) is 4S,11R,17S-trihydroxy-docosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid. Resolvin D4 (RvD4) is 4S,5,17S-trihydroxy-docosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid. Lipoxin A4 (LXA4) is 5S,6R,15S-trihydroxy-eicosa-7E,9E,11Z,13E-tetraenoic acid.

[0018] As used herein and defined in the Background section, "anti-inflammatory lipid mediators" (SPMs, also referred to as anti-inflammatory mediators) are a large and growing class of cell signaling molecules that are formed in cells by the metabolism of polyunsaturated fatty acids (PUFAs) by one or a combination of enzymes, such as lipoxygenase, cyclooxygenase, and cytochrome P450 monooxygenase.

[0019] In certain embodiments, the anti-inflammatory lipid mediators are in the form of a tautomer, solvate, hydrate, or a pharma- ceutically acceptable salt thereof, provided that the chemical structures of these compounds allow them to exist in these forms.

[0020] As used herein, "pharmaceutical acceptable salt" means that the salt derived from the corresponding compound is suitable for administration to a subject to achieve the treatment described herein without undue adverse side effects, taking into account the severity of the disease and the need for treatment. However, salts of non-pharmaceutical acids and bases may also find use, for example, in the preparation or purification of pharmaceutical acceptable compounds. All salts, whether pharmaceutical acceptable or not, are included within the scope of the present invention. The pharmaceutical acceptable acid and base addition salts mentioned above are meant to include therapeutically active non-toxic acid and base addition salt forms that the compounds disclosed herein can form. Pharmaceutically acceptable acid addition salts can be conveniently obtained by treating the base form with such appropriate acid. Suitable acids include inorganic acids such as, for example, hydrohalic acids, e.g., hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or organic acids such as, for example, acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid (i.e., ethanedioic acid), malonic acid, succinic acid (i.e., butanedioic acid), maleic acid, fumaric acid, malic acid (i.e., hydroxybutanedioic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid, and the like.

[0021] Conversely, said salt forms can be converted into the free base form by treatment with an appropriate base.

[0022] Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium salts, and the like, salts with organic bases, such as benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids, such as arginine, lysine, and the like.

[0023] In another preferred embodiment, the neurodegenerative and / or autoimmune disease is selected from the group consisting of multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, HIV dementia, epilepsy, schizophrenia, depression, bipolar disorder, neurodevelopmental disorders, autism, seizures, Huntington's disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. In a more preferred embodiment, the disease is multiple sclerosis (MS). In another more preferred embodiment, the disease is amyotrophic lateral sclerosis (ALS).

[0024] In another embodiment, the anti-inflammatory lipid mediator for use according to any of the above embodiments is comprised in a composition. In a preferred embodiment, the composition is formulated as a cosmetic composition, a pharmaceutical composition, a food formula, a food ingredient or supplement, a functional food, a dietary supplement, a nutraceutical composition, or is in a natural product extract. In a more preferred embodiment, the composition is a pharmaceutical composition. In another more preferred embodiment, the composition is a food product.

[0025] The "food" or "food ingredient or supplement", "functional food" or "nutraceutical" compositions described above may in principle be in any form suitable for human or animal consumption.

[0026] Moreover, the composition containing SPM may contain other ingredients. For example, the composition containing SPM may be mixed, dissolved, emulsified (e.g., in oil / water, water / oil, or double emulsion) or suspended in a matrix or base. The matrix or base may be, for example, an edible oil, such as an omega-3 PUFA-containing oil, an omega-3 PUFA concentrate containing high levels of EPA or DELA or a mixture of EPA and DELA, or another edible oil suitable for ingestion or administration. The matrix or base may also be water or an aqueous buffer. The composition containing SPM may also be prepared in a liposome, nanoparticle, or microparticle.

[0027] To extend shelf life, the composition may also contain one or more stabilizers, including antioxidants such as one or more tocopherols, ascorbic acid, and ascorbyl-fatty acid derivatives, as well as other antioxidants commonly used to stabilize edible oils, such as rosemary extract. The composition may further be packaged in a container that minimizes exposure to oxygen, heat, and incident light. These conditions will particularly increase the stability of SPM by preventing or limiting the oxidation and isomerization of double bonds. Since SPM is dissolved in oils that have significant levels of PUFAs that are sensitive to oxidation, the stability of bulk oils or formulated oils will also benefit from these conditions.

[0028] The compositions may also include one or more active ingredients such as aspirin, other nonsteroidal anti-inflammatory drugs, vitamins, antioxidants, flavonoids, minerals, trace elements, fatty acids, bioactive proteins and peptides such as lycopene, S-adenosylmethionine, oleocanthal, resveratrol, pterostilbene, bromelain, oligosaccharides, glucosinolates, and plant extracts such as Boswellia serrata, mangosteen, capsicum, turmeric, ginger, tea, neem, and / or willow bark extract, etc. The ingredients are not limited to the examples set forth herein.

[0029] Specific nutritional supplements containing fish oil, krill oil, or long chain omega-3 PUFA concentrates supplemented with compositions containing SPMs along with glucosamine and chondroitin for arthritis, or zinc, lutein, and zeaxanthin for eye health can be made to support specific health conditions.

[0030] Other nutritional supplements which contain SPMs are multivitamin preparations, sports nutrients, fortified fish oil capsules, oral health care products such as toothpastes and mouthwashes, certain oils used as food products such as spreads, dressings, cooking oils, snacks, health drinks, softgels, chewing gums and the like and in infant formulas.

[0031] A nutraceutical may be defined as a natural product used to supplement the diet by increasing the total dietary intake of important nutrients. This definition includes dietary supplements such as vitamins, minerals, herbal extracts, antioxidants, amino acids, and protein supplements. Dietary supplements fall under the newly created product category of "Dietary Supplements" established by the FDA in the Dietary Supplement Act of 1994. This act specifically defined dietary supplements to include: vitamins, minerals, herbs or other botanicals, antioxidants, amino acids, or other dietary substances used to supplement the diet by increasing the total daily intake. A "nutraceutical composition" is defined herein as a food composition that is fortified with ingredients that can provide health benefits. Such compositions in the context of the present invention may also be indicated as foods for use in special diets; medical foods; and dietary supplements. For example, a foodstuff or supplement may help prevent or reduce symptoms associated with inflammatory conditions such as allergies (e.g., hay fever) and the like. As with pharmaceutical compositions, the amount of active ingredient in a food or food additive will depend on several factors. Food products will generally contain concentrations sufficient to provide an effective amount of the active ingredient to the consumer upon ingestion of a normal (e.g., daily) portion of food. It will be recognized by those skilled in the art that optimal quantities and intervals for individual dosing to achieve the therapeutic effect of the pharmaceutical compositions, foodstuffs, or dietary supplements described herein may be readily determined by those skilled in the art.

[0032] The dosage range of the pharmaceutical composition can be adjusted according to the specific condition being treated, as required for the treatment of an individual patient.Any of a number of suitable pharmaceutical preparations may be utilized as a vehicle for administration of the composition of the present invention, and possibly a variety of administration routes are available.The specific mode selected will of course depend on the specific preparation selected, the severity of the disease, disorder, or condition being treated, and the dosage required for therapeutic efficacy.

[0033] Compositions containing anti-inflammatory lipid mediators for use according to any of the above embodiments should be administered by, but not limited to, oral, rectal, topical, vaginal, parenteral (e.g. subcutaneous, intramuscular, intradermal, inhalation, or intravenous), intrathecal, transdermal, intraperitoneal, and intrapulmonary and intranasal routes. Preferably, the compositions should be administered by oral or parenteral routes, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular active product being used.

[0034] Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets, lozenges, drops, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of combining the active compound and a suitable carrier (which may contain one or more accessory ingredients, as mentioned above).

[0035] In general, the formulations of the present invention are prepared by uniformly and thoroughly mixing the active compound with a liquid or finely divided solid carrier or both, and then molding the resulting mixture if necessary. For example, tablets may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form, such as a powder or granules, optionally mixed with a binder, a lubricant, an inert diluent, and / or a surfactant / dispersant. Molded tablets may be made by molding in a suitable machine the powdered compound moistened with an inert liquid binder.

[0036] The formulations of the present invention suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound, which preparation is preferably isotonic with the blood of the intended recipient. These preparations may be administered by subcutaneous, intravenous, intramuscular, inhalation, or intradermal injection. Such preparations may conveniently be prepared by mixing the compound with water or a glycine buffer and making the resulting solution sterile and isotonic with blood.

[0037] The formulations of the present invention are particularly suitable for topical application to the skin and preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that may be used include petrolatum, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0038] Formulations suitable for transdermal administration may also be presented as medicated bandages or discrete patches adapted to remain in intimate contact with the epidermis of the recipient for an extended period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (the passage of a small electrical current to "inject" electrically charged ions into the skin; also called electromotive drug administration (EMDA)) through the skin.

[0039] The present disclosure also relates to a method of treating a neurodegenerative disease and / or an autoimmune disease in a subject, comprising administering to said subject a therapeutically effective amount of an anti-inflammatory lipid mediator as defined above. Preferably, the subject is a human subject. Moreover, the present invention also relates to a method of treating a neurodegenerative disease and / or an autoimmune disease in a subject, comprising administering to said subject a therapeutically effective amount of a composition comprising an anti-inflammatory lipid mediator as defined above. The phrase "therapeutically effective amount" refers to an amount of such a substance that produces some desired local or systemic effect at a suitable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of such a substance will vary depending on the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the mode of administration, and the like, which can be easily determined by one skilled in the art. For example, a composition of the present disclosure may be administered in an amount sufficient to produce a suitable benefit / risk ratio applicable to such treatment.

[0040] It should be noted that all of the preceding embodiments can be implemented independently of each other or in combination with any other embodiment disclosed herein.

[0041] The invention will now be further illustrated by reference to the following examples, which are not intended to limit the scope of the invention. EXAMPLES

[0042] Working Example Example 1: Experimental Autoimmune Encephalomyelitis (EAE), Multiple Sclerosis (MS) Mouse Model EAE Induction and Functional Assessment: Active immunization was performed in adult female (8 weeks old) C57Bl / 6 mice. Briefly, 4 mg of M. tuberculosis ( M300 μg of myelin oligodendrocyte glycoprotein (MOG) emulsified in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA (DIFCO Laboratories) 35~55 Peptide (MEVGWYRSPFSRVVHLYRNGK , SEQ ID NO:1 ) was injected subcutaneously. On days 0 and 2, mice were further injected intraperitoneally (ip) with 500 ng of pertussis toxin (List Biological Laboratories). Animals were monitored daily for signs of EAE, and the scoring system was as follows: 0=no clinical signs; 0.5=partially lethargic tail, 1=lethargic tail; 2=mild hind limb weakness (fast righting reflex); 3=severe hind limb weakness (slow righting reflex); 3.5=hind limb weakness or paralysis of one hind limb; 4=paralysis of both hind limbs, 4.5=forelimb weakness; 5=forelimb paralysis; 6=moribund. Once animals showed the first signs, mice received daily intraperitoneal injections of maresin (1 μg in 200 μl saline) until the end of the experiment (day 21 after immunization). Control mice received an aqueous solution containing 200 μl saline following the same treatment protocol. On day 21 after immunization, mice were perfused with 4% paraformaldehyde solution, spinal cords were removed, and serial cryostat cross sections were cut (15 μm thick). Histological sections were then stained with Luxol Fast Blue to assess the area of ​​demyelination.

[0043] result: The inventors found that mice showed the first signs of disease between days 10 and 13 after immunization (Figure 1A). Functional deficits gradually progressed in saline-injected mice, reaching a plateau by day 18 after immunization. At this time point, saline-injected mice had a clinical score of about 4.5, indicating paralysis of both hind limbs and weakness of the forelimbs (Figure 1A). This score did not fluctuate until the end of follow-up. Interestingly, administration of maresin led to a significant reversal of functional impairment, showing a clinical score of about 2.5 at the peak of the disease (Figure 1A). This score indicates that the mice did not show paralysis of the hind limbs, but displayed slight or severe weakness in the hind limbs but not in the forelimbs. Histopathological sections of the spinal cord also revealed that treatment with maresin reduced the area of ​​demyelination by about 50% compared to saline-treated mice (Figure 1B). This data provides clear evidence that treatment with maresin protects against functional and myelin loss in a preclinical model of multiple sclerosis.

[0044] Example 2: Amyotrophic lateral sclerosis animal: Experiments were performed in female transgenic mice carrying the G93A human SOD1 mutation (B6-Tg[SOD1-G93A]1Gur) purchased from Jackson Laboratory (Bar Harbor, ME, USA) and provided by a colony maintained at the University of Zaragoza. Hemizygous transgenic mice were identified by PCR amplification of DNA extracted from tail samples and then maintained in a local facility. Mice were provided with food and water ad libitum at room temperature of 22±2°C under a 12:12-h light-dark cycle. Animals were considered to have reached the disease endpoint when the loss of righting reflex was longer than 30 s. At 8 weeks of age (before treatment began), animals were electrophysiologically tested to obtain baseline levels. Animals were then divided into different experimental groups, maresin-treated mice or saline SOD1 mice, according to mouse ancestry, body weight, and electrophysiological baseline values, keeping the groups balanced. G93AMice were administered maresin intraperitoneally (1 μg in 200 μl saline) on Mondays, Wednesdays, and Fridays starting at 8 weeks of age.

[0045] Functional testing: The animals' motor coordination, balance, and strength were assessed using the rotarod test. All mice were trained three times a week on a rod rotating at a constant speed of 14 rpm (rotating cylinder diameter 3.4 cm) for up to 180 s to reach a baseline level of performance. Animals were then tested weekly from 8 to 16 weeks of age at the same speed to measure the time each animal could remain on the rotating rod. The maximum time remaining on the rotating rod was conveniently set at 180 s.

[0046] Motor nerve conduction tests were performed every 2 weeks from 8 to 20 weeks of age (n=12 SOD1 G93A Vehicle, n=13 SOD treated with maresin G93A ). The sciatic nerve was stimulated percutaneously through paired needle electrodes placed at the sciatic notch with a single pulse (Grass S88) of 0.02 ms duration. Compound muscle action potentials (CMAP, M-wave) were recorded from the tibialis anterior (TA) muscle. All potentials were amplified and represented on a digital oscilloscope (Tektronix 450S) with appropriate settings to measure amplitude from baseline to maximum negative peak. The recording needle was placed using a microscope and indexed to anatomical landmarks to ensure reproducibility of needle position on all animals. During testing, mouse skin temperature was maintained between 34 and 36 °C using a thermostatically controlled heating pad. All assessors were blinded to the experimental groups.

[0047] result: The inventors demonstrated that the nerve-muscle integrity of the tibialis anterior muscle, assessed electrophysiologically by measuring the compound muscle action potential (CMAP), was significantly increased by SOD1 in mice aged from 8 to 20 weeks. G93AWe found that the electrophysiological loss gradually decreased in the ALS mice (Fig. 2A). Nevertheless, the first signs of overall motor loss assessed during the rotarod test were observed at 14 weeks, whereas the median disease onset in saline-treated ALS mice was 16 weeks (Fig. 2B). Interestingly, treatment with maresin delayed the electrophysiological loss by about 4 weeks (Fig. 2A). Consistent with the electrophysiological data, we also observed that maresin treatment also delayed disease onset based on the rotarod test by 3 weeks (median disease onset was 19 weeks) (Fig. 2B). These data provide clear evidence that maresin provides a significant therapeutic effect in a mouse model of ALS.

[0048] In the specification and claims, the terms "including" and "comprising" are open ended terms and should be construed to mean "including, but not limited to." These terms encompass the more restrictive terms "consisting essentially of" and "consisting of." It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0049] Example 3: Experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS) mouse models in oral treatment EAE induction and functional assessment: Active immunization was performed in adult female (8 weeks old) C57Bl / 6 mice. Briefly, 4 mg of M. tuberculosis ( M 300 μg of myelin oligodendrocyte glycoprotein (MOG) emulsified in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA (DIFCO Laboratories) 35~55 Peptide (MEVGWYRSPFSRVVHLYRNGK , SEQ ID NO:1) was injected subcutaneously. On days 0 and 2, mice were further injected intraperitoneally (ip) with 500 ng of pertussis toxin (List Biological Laboratories). Animals were monitored daily for signs of EAE, and the scoring system was as follows: 0=no clinical signs; 0.5=partially lethargic tail, 1=lethargic tail; 2=mild hind limb weakness (fast righting reflex); 3=severe hind limb weakness (slow righting reflex); 3.5=hind limb weakness or paralysis of one hind limb; 4=paralysis of both hind limbs, 4.5=forelimb weakness; 5=forelimb paralysis; 6=moribund. Once animals showed the first signs of disease, mice received daily oral treatments of maresin (1 μg in 200 μl saline) until the end of the experiment (day 21 after immunization). Control mice received 200 μl saline following the same treatment protocol.

[0050] result: We found that mice showed the first signs of disease between days 9 and 12 after immunization (Figure 3). Functional deficits gradually progressed in saline-injected mice, reaching a plateau by day 20 after immunization. At this time point, saline-injected mice had a clinical score of about 4, indicating paralysis of both hind limbs (Figure 3). This score did not fluctuate until the end of follow-up. Interestingly, oral administration of maresin led to a significant reversal of functional deficits, showing a clinical score of about 2.5 at the peak of the disease (Figure 3). This score indicates that the mice did not show hind limb paralysis, but did display slight or severe weakness in the hind legs.

Claims

1. A pharmaceutical composition comprising maresin for use in delaying motor loss in patients suffering from amyotrophic lateral sclerosis or for use in protecting against or reducing demyelination in patients suffering from multiple sclerosis.

2. 2. The pharmaceutical composition of claim 1, wherein the maresin is maresin-1 or maresin-2.

3. 3. The pharmaceutical composition of claim 2, wherein the maresin is maresin-1.

4. The pharmaceutical composition according to any one of claims 1 to 3, which is administered by oral, intravenous, subcutaneous, intramuscular, rectal, topical, vaginal, parenteral, transdermal, intraperitoneal, intrapulmonary, intrathecal, or intranasal route.

5. The pharmaceutical composition of claim 4, which is administered by oral or parenteral route.

6. A pharmaceutical composition comprising maresin for use in protecting against or reducing demyelination in patients suffering from multiple sclerosis.

7. A pharmaceutical composition comprising maresin for use in delaying motor loss in patients suffering from amyotrophic lateral sclerosis.

8. The pharmaceutical composition according to claim 6 or 7, wherein the maresin is maresin-1 or maresin-2.

9. The pharmaceutical composition of claim 8, wherein the maresin is maresin-1.

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