Demyelination inhibitor, myelination enhancer, pharmaceutical composition for preventing or treating diseases associated with demyelination or myelination, and food and drink for use in inhibiting demyelination or enhancing myelination

Abstract

This demyelination inhibitor contains a compound represented by general formula (I).

Description

Demyelination inhibitor, myelination enhancer, pharmaceutical composition for preventing or treating diseases associated with demyelination or myelination, and food and drink for use in inhibiting demyelination or enhancing myelination

[0001] The present invention relates to a demyelination inhibitor, a myelination enhancer, a pharmaceutical composition for preventing or treating diseases associated with demyelination or myelination, and a food or drink for use in inhibiting demyelination or enhancing myelination.

[0002] The phenomenon in which the myelin surrounding nerve axons is destroyed is called "demyelination." Multiple sclerosis (MS), one of the demyelinating diseases, is a designated intractable disease in which demyelination causes neuroinflammation, motor disorders, and other problems. Multiple sclerosis is prevalent in Europe and the United States, with an estimated 2.3 million patients worldwide, and the number of patients in Japan is rapidly increasing, approaching 20,000.

[0003] Currently, there are existing drugs that can suppress relapses in multiple sclerosis, but no new drugs have yet been developed that can halt progression. Therefore, there is a strong demand for a fundamental treatment that can suppress demyelination and regenerate destroyed myelin.

[0004] To date, it has been reported, for example, that cyclic phosphatidic acid suppresses cuprizone (hereinafter sometimes referred to as "CPZ")-induced demyelination and motor dysfunction in mice (see, for example, Non-Patent Document 1), that the flavonoid baicalein reduces CPZ-induced demyelination by suppressing neuroinflammation (see, for example, Non-Patent Document 2), and that ursolic acid suppresses CPZ-induced demyelination and motor dysfunction through IGF-1 upregulation (see, for example, Non-Patent Document 3).

[0005] Yamamoto S. et al. , Cyclic phosphatidic acid treatment suppression cuprizone-induced demyelination and motor dysfunction in mice, European Journal of Pharmacology, 741, 2014, 17-24 Hashimoto M. et al. Brain Research Bulletin, 135, 2017, 47-52 Yamamoto S. et al. , Ursolic acid treatment suppresses cuprizone-induced demyelination and motor disfunction via upregulation of IGF-1, Journal of Pharmacological Sciences, 144, 2020, 119-122

[0006] The present invention aims to solve the above-mentioned conventional problems and achieve the following objectives: Namely, the present invention aims to provide a demyelination inhibitor capable of suppressing demyelination, a myelination increaser capable of increasing myelin formation, and a pharmaceutical composition for preventing or treating diseases associated with demyelination or myelin formation, or a food or beverage product for use in suppressing demyelination or increasing myelination.

[0007] As a result of extensive research conducted by the present inventors to achieve the above object, they have found that the compound represented by general formula (I) has excellent demyelination inhibitory activity and myelination enhancing activity.

[0008] The present invention is based on the findings of the present inventors, and provides the following means for solving the above problems: <1> A demyelination inhibitor characterized by containing a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) <2> A myelination enhancer characterized by containing a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) <3> A pharmaceutical composition for preventing or treating a disease associated with demyelination or myelination, comprising at least one of the demyelination inhibitor described in <1> and the myelination enhancer described in <2>. <4> A food or drink for use in inhibiting demyelination or enhancing myelination, comprising a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.)

[0009] According to the present invention, the above-mentioned conventional problems can be solved, the above-mentioned objects can be achieved, and it is possible to provide a demyelination inhibitor that can suppress demyelination, a myelination formation enhancer that can increase myelin formation, and a pharmaceutical composition for preventing or treating diseases associated with demyelination or myelin formation, or a food or beverage product for use in suppressing demyelination or increasing myelin formation.

[0010] FIG. 1 is a diagram showing an example of the results of myelin staining of the control group in Test Example 1. FIG. 2 is a diagram showing an example of the results of myelin staining of the group in Test Example 1 that was fed a powdered feed for breeding containing 0.2% CPZ. FIG. 3 is a diagram showing an example of the results of myelin staining of the group in Test Example 1 that was fed a feed in which a test sample was blended with a powdered feed for breeding containing 0.2% CPZ. FIG. 4 is a graph showing the results of calculating the myelin score for each group in Test Example 1. FIG. 5 is a graph showing the results of measuring the maximum exercise duration for each group in Test Example 1. FIG. 6 is a graph showing the results of measuring the number of motor impairments for each group in Test Example 1. FIG. 7 is a diagram showing an example of the results of myelin staining of the control group in Test Example 2. FIG. 8 is a diagram showing an example of the results of myelin staining of the group in Test Example 2 that was fed a powdered feed for breeding containing 0.2% CPZ. FIG. 9 is a diagram showing an example of the results of myelin staining for a group fed a feed containing 0.2% CPZ powdered feed for breeding and a test sample "raw food" in Test Example 2. FIG. 10 is a diagram showing an example of the results of myelin staining for a group fed a feed containing 0.2% CPZ powdered feed for breeding and a test sample "heat-treated" in Test Example 2. FIG. 11 is a diagram showing an example of the results of myelin staining for a group fed a feed containing 0.2% CPZ powdered feed for breeding and a test sample "dried" in Test Example 2. FIG. 12 is a diagram showing an example of the results of myelin staining for a group fed a feed containing 0.2% CPZ powdered feed for breeding and a test sample "discarded part" in Test Example 2. FIG. 13 is a graph showing the results of calculating the myelin scores for each group in Test Example 2. FIG. 14 is a diagram showing an example of the results of myelin staining for the control group in Test Example 3. Fig. 15 is a diagram showing an example of the results of myelin staining of a group fed a powdered feed for breeding containing 0.2% CPZ in Test Example 3. Fig. 16 is a diagram showing an example of the results of myelin staining of a group fed a feed in which a test sample compound represented by structural formula (1) was blended with a powdered feed for breeding containing 0.2% CPZ in Test Example 3.Figure 17 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed in Test Example 3, and the compound represented by structural formula (2) of the test sample. Figure 18 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed in Test Example 3, and the compound represented by structural formula (3) of the test sample. Figure 19 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed in Test Example 3, and the compound represented by structural formula (4) of the test sample. Figure 20 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed in Test Example 3, and the compound represented by structural formula (5) of the test sample. Figure 21 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed and the compound represented by structural formula (6) of the test sample. Figure 22 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed and the compound represented by structural formula (7) of the test sample. Figure 23 shows an example of myelin staining results for a group fed with a feed containing 0.2% CPZ in a breeding powder feed and the compound represented by structural formula (8) of the test sample. Figure 24 is a graph showing the calculated myelin scores for each group in Test Example 3. Figure 25 shows the state after treatment with a chemiluminescent reagent in Test Example 4. Figure 26 is a graph showing the calculated expression levels of myelin basic protein in Test Example 4. Figure 27 shows the state after treatment with a chemiluminescent reagent in Test Example 5. FIG. 28 is a graph showing the results of calculating the expression level of myelin basic protein in Test Example 5.

[0011] (Demyelination inhibitor, myelination enhancer) The demyelination inhibitor of the present invention contains a compound represented by general formula (I) and, if necessary, further contains other ingredients. The myelination enhancer of the present invention contains a compound represented by general formula (I) and, if necessary, further contains other ingredients.

[0012] Demyelination refers to the phenomenon in which the myelin surrounding nerve axons is destroyed (the myelin sheath is destroyed). As used herein, inhibiting demyelination refers to preventing myelin from being destroyed. Inhibiting demyelination includes not only completely preventing myelin destruction, but also reducing the degree of myelin destruction compared to when a demyelination inhibitor is not used.

[0013] As used herein, "increasing myelin formation" refers to increasing the amount of myelin, more specifically, increasing axonal myelination. Increasing myelin formation also includes regenerating destroyed myelin.

[0014] <Compound Represented by General Formula (I)> The compound represented by the following general formula (I) is a compound having a structure in which a retinol skeleton and an acyl group (R—CO—) are bonded via an ester bond.

[0015] In the general formula (I) above, R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.

[0016] The aforementioned R is preferably an acyclic hydrocarbon group.

[0017] The aforementioned R is preferably a saturated hydrocarbon group.

[0018] The number of carbon atoms in R is not particularly limited as long as it is 1 to 22, and can be appropriately selected depending on the purpose. However, it is preferably 10 to 20, more preferably 12 to 18, even more preferably 14 to 16, and particularly preferably 15.

[0019] The aforementioned R is preferably a pentadecyl group.

[0020] -Compound Represented by Structural Formula (1)- The compound represented by the following structural formula (1) is a compound in which R is a pentadecyl group, and is called all-trans-retinyl palmitate (molecular formula: C ３６ H ６０ O ２ , molecular weight: 524.86).

[0021] The compound represented by the general formula (I) may be a commercially available product or may be prepared as appropriate.

[0022] The compound represented by the general formula (I) can also be used in the form of a material containing the compound. Examples of the material include crustaceans containing the compound represented by the general formula (I). Examples of crustaceans containing the compound represented by the general formula (I) include cherry shrimp.

[0023] The form of the material is not particularly limited as long as it contains the compound represented by the general formula (I) and can be appropriately selected depending on the purpose, and may be, for example, a raw form or a processed form that has been subjected to processing treatment such as heating, drying, etc. Furthermore, even parts that are normally discarded can be used as long as they contain the compound represented by the general formula (I).

[0024] The fact that the compound represented by the general formula (I) possesses both demyelination inhibitory activity and myelin formation increasing activity was discovered for the first time by the present inventors. Since the compound represented by the general formula (I) is contained in food ingredients, it is highly safe and extremely useful.

[0025] The content of the compound represented by general formula (I) in the demyelination inhibitor or the myelination enhancer is not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the demyelination inhibitor or the myelination enhancer may consist solely of the compound represented by general formula (I).

[0026] <Other Components> The other components in the demyelination inhibitor or the myelination enhancer are not particularly limited and can be selected appropriately depending on the purpose, as long as they do not impair the effects of the present invention, and examples thereof include pharmaceutically acceptable carriers. The carrier is also not particularly limited and can be selected appropriately depending on the dosage form, etc. The other components may be used alone, or two or more may be used in combination. The content of the other components in the demyelination inhibitor or the myelination enhancer is not particularly limited and can be selected appropriately depending on the purpose.

[0027] <Dosage Form> The dosage form of the demyelination inhibitor or the myelination enhancer is not particularly limited and can be appropriately selected depending on the desired administration method, etc. Examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations to be dissolved when used, etc.), ointments, patches, gels, creams, powders for external use, sprays, and inhalation powders.

[0028] The oral solid preparation can be prepared by a conventional method by adding, for example, a compound represented by general formula (I) (hereinafter sometimes referred to as the "active ingredient"), an excipient, and, if necessary, additives such as a binder, disintegrant, lubricant, colorant, and flavoring agent. Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrant include dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricant include purified talc, stearates, borax, and polyethylene glycol. Examples of the coloring agent include titanium oxide, iron oxide, etc. Examples of the flavoring agent include sucrose, orange peel, citric acid, tartaric acid, etc.

[0029] The oral liquid preparation can be produced by a conventional method by adding additives such as flavorings, buffers, and stabilizers to the active ingredient. Examples of flavorings include sucrose, orange peel, citric acid, and tartaric acid. Examples of buffers include sodium citrate. Examples of stabilizers include tragacanth, gum arabic, and gelatin.

[0030] The injection preparation can be prepared by adding, for example, a pH adjuster, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, etc. to the active ingredient, and then using a conventional method to prepare an injection preparation for subcutaneous, intramuscular, intravenous, etc. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Examples of the isotonicity agent include sodium chloride, glucose, etc. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride, etc.

[0031] The ointment can be prepared by, for example, blending the active ingredient with known bases, stabilizers, humectants, preservatives, etc., and mixing them in a conventional manner. Examples of the bases include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.

[0032] The patch can be produced, for example, by applying the ointment as a cream, gel, paste, etc. to a known support by a conventional method. Examples of the support include woven fabrics made of cotton, staple fiber, or chemical fiber, nonwoven fabrics, films made of soft vinyl chloride, polyethylene, polyurethane, etc., and foam sheets.

[0033] <Use> The administration method, dosage, number of administrations, administration interval, administration time, and administration target of the demyelination inhibitor or the myelination enhancer are not particularly limited and can be appropriately selected depending on the purpose.

[0034] The method of administration of the demyelination inhibitor or the myelination enhancer is not particularly limited, and either systemic administration or local administration can be selected depending on, for example, the dosage form of the demyelination inhibitor or the myelination enhancer, the age, weight, and presence or absence of disease of the individual to be administered. For example, in systemic administration (e.g., oral administration, intraperitoneal administration, administration into the blood, etc.), it is preferable to appropriately apply a conventionally known drug delivery technique so that the active ingredient is delivered stably and efficiently to the desired site. In addition, in local administration, the active ingredient can be administered by direct injection into the desired site. For the injection, a conventionally known method such as injection can be appropriately used. The demyelination inhibitor or the myelination enhancer can be suitably used for oral administration.

[0035] The dose, frequency of administration, and administration interval of the demyelination inhibitor or the myelination enhancer are not particularly limited and can be appropriately selected depending on the age, body weight, presence or absence of disease, etc. of the individual to be administered. Examples of the dose include an amount of the compound represented by general formula (I) selected within the range of 0.1 to 1,000 mg per kg of body weight per day of the individual to be administered. Examples of the frequency of administration include a daily dose administered once a day or administered in multiple divided doses. Examples of the administration interval include daily administration, administration every 1 to 3 days, etc.

[0036] The timing of administration of the demyelination inhibitor or the myelination enhancer is not particularly limited and can be appropriately selected depending on the purpose. For example, the administration may be preventive or therapeutic against demyelination.

[0037] The subjects to which the demyelination inhibitor or the myelination enhancer is administered are not particularly limited and can be appropriately selected depending on the purpose. Examples include humans, mice, rats, cows, pigs, monkeys, dogs, cats, sheep, goats, and birds.

[0038] The demyelination inhibitor or the myelin formation increasing agent may be used in combination with other drugs.

[0039] The demyelination inhibitor can effectively inhibit demyelination. Therefore, the present invention also relates to a method for inhibiting demyelination, which comprises administering the demyelination inhibitor to an individual.

[0040] The myelination enhancer can effectively enhance myelin formation. Therefore, the present invention also relates to a method for increasing myelination, which comprises administering the myelination enhancer to an individual.

[0041] (Pharmaceutical Composition) The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating a disease associated with demyelination or myelination. The pharmaceutical composition of the present invention contains at least one of a demyelination inhibitor and a myelination enhancer of the present invention, and further contains other ingredients as necessary.

[0042] The disease associated with demyelination or myelination is not particularly limited and can be appropriately selected depending on the purpose, but preferred examples include at least one selected from the group consisting of multiple sclerosis, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, neuromyelitis optica spectrum disorder (sometimes referred to as "neuromyelitis optica"), Krabbe disease, and Guillain-Barré syndrome. Multiple sclerosis includes progressive and relapsing-remitting forms.

[0043] As used herein, "prevention of a disease associated with demyelination or myelination" refers to preventing or delaying the onset of a disease associated with demyelination or myelination, and also includes preventing recurrence. Furthermore, as used herein, "treatment of a disease associated with demyelination or myelination" refers to delaying the progression of a disease associated with demyelination or myelination or alleviating symptoms of the disease associated with demyelination or myelination by suppressing demyelination or increasing myelin formation in an already occurring disease associated with demyelination or myelination.

[0044] Since the compound represented by the general formula (I) is contained in a food ingredient, it is possible to prevent or treat diseases associated with demyelination or myelination with high safety and high quality of life.

[0045] <At least one of a demyelination inhibitor and a myelination increaser> The demyelination inhibitor is the demyelination inhibitor of the present invention described above. The myelination increaser is the myelination increaser of the present invention described above. Either the demyelination inhibitor or the myelination increaser may be used alone, or both may be used in combination.

[0046] The total content of the demyelination inhibitor and the myelination enhancer in the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the purpose. Alternatively, the pharmaceutical composition may consist of at least one of the demyelination inhibitor and the myelination enhancer.

[0047] <Other Components> The other components in the pharmaceutical composition are not particularly limited and can be selected appropriately depending on the purpose, as long as they do not impair the effects of the present invention, and examples thereof include the same as the other components in the demyelination inhibitors or myelination enhancers described above. The content of the other components in the pharmaceutical composition is not particularly limited and can be selected appropriately depending on the purpose.

[0048] <Dosage Form> The dosage form of the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the desired administration method, etc. Examples include dosage forms similar to those of the demyelination inhibitors or myelination enhancers described above.

[0049] <Use> The administration method, dosage, number of administrations, administration interval, administration time, and administration target of the pharmaceutical composition are not particularly limited and can be selected appropriately depending on the purpose. For example, they can be similar to those described in the "Use" section of the above-mentioned demyelination inhibitor or myelination enhancer.

[0050] The pharmaceutical composition may be used in combination with other pharmaceutical compositions.

[0051] The pharmaceutical composition can inhibit demyelination and / or increase myelination, thereby effectively preventing or treating diseases associated with demyelination or myelination. Therefore, the present invention also relates to a method for preventing or treating diseases associated with demyelination or myelination, which comprises administering the pharmaceutical composition to an individual.

[0052] (Food and Beverage) The food and beverage of the present invention is a food and beverage used for inhibiting demyelination or increasing myelin formation. The food and beverage of the present invention contains a compound represented by general formula (I), and optionally contains other components.

[0053] <Compound represented by general formula (I)> The compound represented by general formula (I) is the same compound as the compound represented by general formula (I) in the demyelinating inhibitor of the present invention described above, and the preferred embodiment is also the same.

[0054] There are no particular restrictions on the content of the compound represented by general formula (I) in the food and beverage, and it can be appropriately selected depending on the purpose. Furthermore, the food and beverage may consist solely of the compound represented by general formula (I).

[0055] <Other Components> The other components in the food and drink are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected from components commonly used in foods and drinks depending on the purpose. The content of the other components in the food and drink is not particularly limited, and can be appropriately selected depending on the purpose.

[0056] There are no particular restrictions on the aforementioned food and beverages, and they can be appropriately selected according to the purpose. Examples include health foods, functional foods, foods for specified health uses, nutritional supplements, foods for sick people, and food additives.

[0057] Specific examples of the aforementioned foods and beverages include liquid foods such as drinks, soups, dairy beverages, soft drinks, tea beverages, alcoholic beverages, jelly-like beverages, and functional beverages; oil-containing products such as edible oils, dressings, mayonnaise, and margarine; carbohydrate-containing foods such as rice dishes, noodles, and bread; processed livestock products such as ham and sausages; processed marine products such as kamaboko, dried fish, and salted seafood; processed vegetable products such as pickles; semi-solid foods such as jelly and yogurt; fermented foods such as miso and fermented beverages; various confectionery products such as Western-style sweets, Japanese-style sweets, candies, gums, gummies, frozen desserts, and ice cream; retort products such as curry, thickened sauces, and Chinese soups; and instant foods such as instant soups and instant miso soups, as well as microwaveable foods. Health foods prepared in powder, granule, tablet, capsule, liquid, paste, and jelly form are also included.

[0058] The food and drink can be produced by a known production method except for using the compound represented by the general formula (I). The compound represented by the general formula (I) may be blended during the production of the food and drink, or may be added to the food and drink after production.

[0059] The food or drink may contain ingredients other than the compound represented by general formula (I) that are useful for inhibiting demyelination and / or increasing myelination.

[0060] There are no particular restrictions on the amount, frequency, and interval of intake of the aforementioned food and beverages, and they can be appropriately selected according to the age, weight, presence or absence of disease, etc., of the individual receiving the intake. As for the amount, for example, the amount of the compound represented by general formula (I) per day of the individual receiving the intake can be selected within the range of 0.1 to 1,000 mg per kg of body weight. As for the frequency of intake, for example, the daily intake amount may be taken once a day, or it may be taken in multiple doses. As for the interval of intake, for example, it may be taken every day, or every 1 to 3 days.

[0061] The timing of intake of the food or drink is not particularly limited and can be appropriately selected depending on the purpose. For example, the food or drink may be taken preventively or therapeutically for diseases related to demyelination or myelination.

[0062] There are no particular restrictions on the target animals for the aforementioned food and beverages, and they can be appropriately selected according to the purpose. Examples include humans, mice, rats, cattle, pigs, monkeys, dogs, cats, sheep, goats, and birds.

[0063] Test examples of the present invention will be explained below, but the present invention is not limited to the following test examples.

[0064] (Test Example 1) The demyelination inhibitory effect of the test sample was investigated using cuprizon (CPZ)-induced demyelinating model mice (experimental model mice for multiple sclerosis).

[0065] [Mice] Male C57BL / 6j mice (Tokyo Laboratory Animal Science) were delivered to a suitable animal facility at Saitama Medical University at 9 weeks of age and reared in a 12-hour light / 12-hour dark cycle. Mice that had been acclimatized for one week were used in the experiment. In addition, the mice's weight was measured weekly to monitor their condition.

[0066] <Preparation of CPZ-induced demyelinating model mice> Demyelinating model mice were prepared by freely feeding CE-2 (Nippon Crea Co., Ltd.), a breeding and rearing powder feed containing 0.2% CPZ (Merck KGaA), to 10-week-old male C57BL / 6j mice for 5 weeks (the peak period of CPZ-induced demyelinating). The breeding and rearing powder feed containing 0.2% CPZ (hereinafter sometimes referred to as "mixed feed") was mechanically mixed at a low speed (50 rpm) for 12 hours using a mixer (Mazemaze Man (Misugi Co., Ltd.)). The mixed feed was given to the mice ad libitum using a powder feeder (KN-675-4 (110 x 60 mm, Natsume Seisakusho)). Stock feed was prepared weekly and stored at 4°C, and replaced with fresh mixed feed twice a week. Control mice were given powder feed without CPZ. The mice in the test sample group were fed a feed prepared as follows, in which the test sample was mixed with powder feed for breeding containing 0.2% CPZ.

[0067] <Test Sample> Sakura shrimp (from the Yui Fishing Port direct sales store) were used as the test sample. The test sample was crushed in a mortar and pestle and mixed at a dose of 10% by weight when preparing powdered feed for rearing and breeding that contained 0.2% CPZ.

[0068] [Analysis] <Myelin Staining> Mice were intracardially perfused with 4% paraformaldehyde (PFA) / PBS solution to fix the brain. The brains were then removed and post-fixed overnight in 4% PFA / PBS solution, followed by cryoprotection overnight in 30% sucrose / PBS solution. The brains were then rapidly frozen in liquid nitrogen, and sections (25 μm thick) were cut using a cryostat (LEICA CM1950) and mounted on gelatin-coated slides. The sections were stained with 0.3% Black Gold II (Histo-Chem) solution at 60°C for 10 minutes, rinsed in PBS, fixed in 1% sodium thiosulfate, rinsed in PBS, and cover-slipped using Poly-Mount (Polysciences Inc.). Brain sections used for Black Gold II staining were selected between -0.22 mm and -0.58 mm from Bregma. Brain sections were photographed using a KEYENCE BZ-X700 microscope (Keyence). Mean optical density (OD) within the corpus callosum was measured using ImageJ 1.46r. The background was subtracted from the tissue OD value as a blank. The mean corpus callosum OD value for each experimental group, with the control set at 100%, was calculated as the myelin score. Statistical analysis was performed using the Newman-Keuls test.

[0069] Microscopic photographs of Black Gold II-stained brain sections are shown in Figures 1 to 3. Figure 1 shows an example of the results for the control group, Figure 2 shows an example of the results for the group fed a powdered feed for breeding containing 0.2% CPZ (hereinafter, sometimes referred to as the "CPZ group"), and Figure 3 shows an example of the results for the group fed a powdered feed for breeding containing 0.2% CPZ and a test sample (hereinafter, sometimes referred to as the "CPZ + shrimp group"). Figure 4 also shows graphs of the myelin scores for the control group ((A) in Figure 4), the CPZ group ((B) in Figure 4), and the CPZ + shrimp group ((I) in Figure 4). In Figure 4, "**" indicates a significant difference (p<0.01) compared to the control group, "***" indicates a significant difference (p<0.001) compared to the control group, and "###" indicates a significant difference (p<0.001) compared to the CPZ group.

[0070] <Behavioral Test> Motor dysfunction due to demyelination was evaluated as follows. Mice were placed on the rod of a rotarod apparatus (Mouse Rotarod, Ugo Basile) and rotated at a speed of 28 rpm. The time until the mouse fell off the cylinder (hereinafter sometimes referred to as "exercise duration") was counted. The maximum exercise time was set at 5 minutes, and even if the mouse fell off, it was allowed to immediately return to the cylinder to continue exercising. The total number of times the mouse fell off and rotated while clinging to the cylinder (hereinafter sometimes referred to as "number of motor impairments") was counted. Statistical analysis was performed using the Newman-Keuls test for exercise duration and the Kruskal-Wallis test for the number of motor impairments.

[0071] The results of measuring the time allowed for exercise are shown in Figure 5, and the results of measuring the number of motor impairments are shown in Figure 6. In Figures 5 and 6, (A) shows the results for the control group, (B) shows the results for the CPZ group, and (I) shows the results for the CPZ + shrimp group. In Figures 5 and 6, "*" indicates a significant difference (p<0.05) from the control group, "***" indicates a significant difference (p<0.001) from the control group, "#" indicates a significant difference (p<0.05) from the CPZ group, and "##" indicates a significant difference (p<0.01) from the CPZ group.

[0072] As shown in Figures 1 to 4, the group administered Sakura shrimp had a high myelin score (high myelin content) and significantly suppressed demyelination in the CPZ-induced demyelination model mice. Furthermore, as shown in Figures 5 and 6, the group administered Sakura shrimp also significantly suppressed motor dysfunction.

[0073] (Test Example 2) The demyelination inhibitory effect of the test sample was examined using a CPZ-induced demyelination model mouse.

[0074] [Mice] As in Test Example 1, male C57BL / 6j mice were used.

[0075] <Preparation of CPZ-induced demyelination model mice> Demyelination model mice were prepared in the same manner as in Test Example 1. Mice in the test sample group were fed a feed prepared as follows, in which the test sample was mixed with powdered feed for breeding containing 0.2% CPZ.

[0076] <Test Samples> The following were used as test samples. The test samples were mixed at a dosage of 10% by weight when preparing powdered feed for breeding containing 0.2% CPZ. - Raw food: Freshly caught sakura shrimp stored frozen. - Heat-treated: Boiled caught sakura shrimp. - Dried: Dried caught sakura shrimp. - Discarded parts: Hairs and small pieces of shell removed when processing caught sakura shrimp.

[0077] [Analysis] <Myelin staining> Myelin staining was performed in the same manner as in Test Example 1. Micrographs of brain sections stained with Black Gold II are shown in Figures 7-12. FIG. 7 shows an example of the results for the control group, FIG. 8 shows a group given a powdered feed for breeding containing 0.2% CPZ (hereinafter sometimes referred to as the "CPZ group"), FIG. 9 shows a group given a feed containing 0.2% CPZ powdered feed for breeding mixed with the test sample "raw" (hereinafter sometimes referred to as the "CPZ + raw group"), FIG. 10 shows a group given a feed containing 0.2% CPZ powdered feed for breeding mixed with the test sample "heat-treated" (hereinafter sometimes referred to as the "CPZ + heat-treated group"), FIG. 11 shows a group given a feed containing 0.2% CPZ powdered feed for breeding mixed with the test sample "dried" (hereinafter sometimes referred to as the "CPZ + dried group"), and FIG. 12 shows a group given a feed containing 0.2% CPZ powdered feed for breeding mixed with the test sample "discarded part" (hereinafter sometimes referred to as the "CPZ + discarded part group"). 13 shows graphs of the myelin scores for the control group ((A) in FIG. 13), CPZ group ((B) in FIG. 13), CPZ + saline group ((i) in FIG. 13), CPZ + heat-treated group ((ii) in FIG. 13), CPZ + desiccation group ((iii) in FIG. 13), and CPZ + discarded part group ((iv) in FIG. 13). In FIG. 13, "*" indicates a significant difference (p<0.05) compared to the control group, "***" indicates a significant difference (p<0.001) compared to the control group, and "###" indicates a significant difference (p<0.001) compared to the CPZ group.

[0078] As shown in Figures 7 to 13, even when the shrimp was processed, the effect of suppressing demyelination in the CPZ-induced demyelination model mice was obtained.

[0079] Test Example 3 Using a mouse model of CPZ-induced demyelination, the demyelination inhibitory effect of the test sample was examined.

[0080] [Mice] As in Test Example 1, male C57BL / 6j mice were used.

[0081] <Preparation of CPZ-induced demyelination model mice> Demyelination model mice were prepared in the same manner as in Test Example 1. Mice in the test sample group were fed a feed prepared as follows, in which the test sample was mixed with powdered feed for breeding containing 0.2% CPZ.

[0082] <Test Samples> As test samples, compounds represented by the following structural formulas (1) to (8), which are fatty acids contained in sakura shrimp or their metabolites, were used. The test samples were mixed at a dose of 10 mg / kg / day when preparing powdered feed for rearing and breeding that contained 0.2% CPZ.

[0083] - Compound represented by structural formula (1) (all-trans-Retinyl Palmitate) (Catalog number: R275450, Toronto Research)

[0084] Compound represented by structural formula (2) (13-cis retinoic acid) (catalog number: 181-02301, Wako)

[0085] Compound represented by structural formula (3) (Palmitoleyl Oleate) (Catalog number: WE-1393, Nu-Chek-Prep)

[0086] - Compound represented by structural formula (4) (Arachidonic Acid methyl ester) (Catalog number: 90014, Cayman)

[0087] Compound represented by structural formula (5) (α-Linolenic Acid ethyl ester) (Catalog number: 10008199, Cayman)

[0088] Compound represented by structural formula (6) (Palmitic acid) (Catalog number: 165-00102, Wako)

[0089] Compound represented by structural formula (7) (Vitamin A) (catalog number: 20241, Wako)

[0090] Compound represented by structural formula (8) (all-trans retinoic acid) (catalog number: 182-01111, Wako)

[0091] [Analysis] <Myelin staining> Myelin staining was performed in the same manner as in Test Example 1. Micrographs of brain sections stained with Black Gold II are shown in Figures 14-23. 14 shows the control group, FIG. 15 shows the group fed with a powdered feed for breeding containing 0.2% CPZ (hereinafter, sometimes referred to as the "CPZ group"), FIG. 16 shows the group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample compound represented by structural formula (1) (hereinafter, sometimes referred to as the "CPZ+ATRP group"), FIG. 17 shows the group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample compound represented by structural formula (2) (hereinafter, sometimes referred to as the "CPZ+13cisRA group"), FIG. 18 shows the group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample compound represented by structural formula (3) (hereinafter, sometimes referred to as the "CPZ+PO group"), and FIG. 19 shows the group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample compound represented by structural formula (4) 20 shows an example of the results for a group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample containing the compound represented by structural formula (5) (hereinafter, this may be referred to as the "CPZ+aLA group"); FIG. 21 shows a group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample containing the compound represented by structural formula (6) (hereinafter, this may be referred to as the "CPZ+PA group"); FIG. 22 shows a group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample containing the compound represented by structural formula (7) (hereinafter, this may be referred to as the "CPZ+VitA group"); and FIG. 23 shows an example of the results for a group fed with a powdered feed for breeding containing 0.2% CPZ and a test sample containing the compound represented by structural formula (8) (hereinafter, this may be referred to as the "CPZ+tRA group").

[0092] Figure 24 also shows graphs of myelin scores for the control group (Figure 24 (A)), CPZ group (Figure 24 (B)), CPZ + ATRP group (Figure 24 (1)), CPZ + 13cisRA group (Figure 24 (2)), CPZ + PO group (Figure 24 (3)), CPZ + AAME group (Figure 24 (4)), CPZ + aLA group (Figure 24 (5)), CPZ + PA group (Figure 24 (6)), CPZ + VitA group (Figure 24 (7)), and CPZ + tRA group (Figure 24 (8)). In Figure 24, "#" indicates a significant difference (p < 0.05) compared to the CPZ group, "##" indicates a significant difference (p < 0.01) compared to the CPZ group, "###" indicates a significant difference (p < 0.001) compared to the CPZ group, and "+++" indicates a significant difference (p < 0.001) compared to the CPZ + ATRP group.

[0093] As shown in Figures 14-24, the compound represented by structural formula (1), which is an extremely long-chain unsaturated fatty acid contained in sakura shrimp, showed a significant demyelination inhibitory effect compared to the compounds represented by structural formulas (2)-(8).

[0094] (Test Example 4) Using a cell line of oligodendrocytes that form myelin, the expression of myelin basic protein (MBP), a myelin marker, was analyzed, and the effect of the compound represented by structural formula (1) on increasing myelin formation was investigated.

[0095] [Tests and Analysis] MO3.13 cells, a cell line of oligodendrocytes that form myelin, were measured in 5.0 × 10⁻⁶ units. ５Cells were seeded individually in 6 wells. After 24 hours, the medium was changed to serum-free medium, and the compound represented by structural formula (1) (10 μM, 100 μM) was added and the cells were cultured for 5 days. The cells were homogenized in Tris-buffered saline (TBS) containing a cocktail of protease and phosphatase inhibitors, and centrifuged at 4°C and 15,000 × g for 10 minutes. Protein concentration was measured using a BCA protein assay kit (Nacalai Tesque). Protein (10 μg / lane) in the lysis solution was subjected to SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). After blocking with 5% skim milk (Megmilk Snow Brand Co., Ltd.) in PBS (PBS-T) containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate, Nacalai Tesque), the samples were infused overnight with a primary antibody (Anti-Myelin Basic Protein antibody ab218011 abcam). The samples were then infused for 1 hour with a secondary antibody (Cell Signaling Technology) and washed three times with PBS-T. The samples were immersed in a chemiluminescent reagent (Chemi-Lumi One Super, Nacalai Tesque), images were acquired using a C-DiGit blot scanner (LI-COR), and analysis was performed using ImageJ.

[0096] Figure 25 shows the state after treatment with a chemiluminescent reagent. Figure 26 shows a graph of MBP expression levels when the compound represented by structural formula (1) is administered, with the MBP expression level in the control set to 100%. In Figures 25-26, "C" indicates the control (no addition of the compound represented by structural formula (1)), "100 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 100 μM, and "10 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 10 μM. In Figure 26, "*" indicates a statistically significant difference (p < 0.05) compared to the control.

[0097] As shown in Figures 25 and 26, the compound represented by structural formula (1) increased the amount of myelin.

[0098] (Test Example 5) The effect of increasing myelin formation was investigated in the same manner as in Test Example 4, except that the amount of compound represented by structural formula (1) added was "none", "50 μM", "30 μM", "10 μM", or "5 μM".

[0099] Figure 27 shows the state after treatment with a chemiluminescent reagent. Figure 28 shows a graph of MBP expression levels when the compound represented by structural formula (1) is administered, with the MBP expression level in the control set to 100%. In Figures 27-28, "C" indicates the control (no addition of the compound represented by structural formula (1)), "50 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 50 μM, "30 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 30 μM, "10 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 10 μM, and "5 μM" indicates the result when the compound represented by structural formula (1) is added at a dose of 5 μM. In Figure 28, "*" and "**" indicate values ​​that showed a significant difference compared to the control (*: p < 0.05, **: p < 0.01).

[0100] As shown in Figures 27-28, the compound represented by structural formula (1) increased the amount of myelin.

[0101] Furthermore, it was confirmed that the amount of compound represented by structural formula (1) added is preferably 10 μM or 30 μM, with 30 μM being more preferable. From these results, it is considered that the appropriate dosage (intake) of the compound represented by general formula (I) for an individual is preferably 0.2 to 2.0 mg per kg of body weight per day for the target individual.

[0102] As shown above, the compound represented by general formula (I) was confirmed not only to suppress demyelination but also to increase myelin formation.

[0103] Examples of the present invention include the following: <1> A demyelination inhibitor characterized by containing a compound represented by the following general formula (I). (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) <2> The demyelination inhibitor described in <1> above, wherein R in general formula (I) is a pentadecyl group. <3> A myelin formation increasing agent characterized by containing a compound represented by the following general formula (I). (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) <4> The myelin formation increasing agent according to <3>, wherein R in general formula (I) is a pentadecyl group. <5> A pharmaceutical composition for the prevention or treatment of diseases related to demyelination or myelin formation, characterized by containing at least one of the demyelination inhibitor according to <1> or <2> and the myelin formation increasing agent according to <3> or <4>. <6> The pharmaceutical composition for the prevention or treatment of diseases related to demyelination or myelin formation according to <5>, wherein the disease related to demyelination or myelin formation is at least one selected from the group consisting of multiple sclerosis, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenal spinal neuropathy, Leber's hereditary optic neuropathy, neuromyelitis optica spectrum disorder, Krabbe disease, and Guillain-Barré syndrome. <7> A food or drink for use in inhibiting demyelination or increasing myelination, characterized by containing a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) <8> A food or beverage for use in suppressing demyelination or increasing myelin formation as described in <7>, wherein R in general formula (I) is a pentadecyl group. <9> A method for suppressing demyelination, characterized by administering the demyelination inhibitor described in <1> or <2> to an individual. <10> A method for increasing myelin formation, characterized by administering the myelin formation increasing agent described in <3> or <4> to an individual. <11> A method for preventing or treating diseases related to demyelination or myelin formation, characterized by administering the pharmaceutical composition for the prevention or treatment of diseases related to demyelination or myelin formation described in <5> or <6> to an individual.

[0104] This application claims priority based on Japanese Patent Application No. 2024-152714, filed on September 4, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A demyelination inhibitor characterized by containing a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) 2. The demyelination inhibitor according to claim 1, wherein R in the general formula (I) is a pentadecyl group.

3. A myelination enhancer comprising a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) 4. The myelination enhancer according to claim 3, wherein R in said general formula (I) is a pentadecyl group.

5. A pharmaceutical composition for the prevention or treatment of diseases associated with demyelination or myelination, comprising at least one of the demyelination inhibitor described in claim 1 or 2 and the myelination enhancer described in claim 3 or 4.

6. A pharmaceutical composition for the prevention or treatment of a disease associated with demyelination or myelination according to claim 5, wherein the disease associated with demyelination or myelination is at least one selected from the group consisting of multiple sclerosis, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, neuromyelitis optica spectrum disorder, Krabbe disease, and Guillain-Barré syndrome.

7. A food or drink for use in inhibiting demyelination or increasing myelination, characterized by containing a compound represented by the following general formula (I): (In general formula (I), R represents a saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms.) 8. A food or beverage for use in inhibiting demyelination or increasing myelination according to claim 7, wherein R in the general formula (I) is a pentadecyl group.

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