WO2023210882A1 - Composition for boosting immunity and preventing, ameliorating, or treating parkinson's disease, comprising o-cyclic phytosphingosine-1-phosphate derivative as active ingredient - Google Patents

Abstract

The present invention relates to a composition for boosting immunity and preventing, ameliorating, and treating Parkinson's disease, the composition comprising O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative as an active ingredient. More specifically, administration of cPS1P of the present invention significantly increased motor function of an animal model of Parkinson's disease, and in an MPTP-induced mouse and an NSE-hαSyn transgenic mouse, administration of cPS1P not only significantly increased the expression of the S1P1 receptor (S1PR1), but also significantly decreased the expression of αSyn. In addition, since there is an effect of a decrease in expression levels of inflammatory cytokines, which have increased in glial cells of an MPTP-induced mouse and an NSE-hαSyn transgenic mouse, by administration of cPS1P of the present invention, the active ingredient of the present invention can be used as a medicine for Parkinson's disease.

Description

Composition for improving immunity and preventing, improving or treating Parkinson's disease containing O-cyclic phytosphingosine-1-phosphate derivative as an active ingredient

The present invention relates to a composition for improving immunity and preventing or treating Parkinson's disease, comprising an O-cyclic phytosphingosine-1-phosphate derivative as an active ingredient.

Parkinson's disease is a progressive neurodegenerative disease characterized by parkinsonian symptoms such as slow movements, tremors at rest, muscle stiffness, shuffling walking, and hunched posture. It received its current name after James Parkinson, a British doctor who first reported the disease in the late 19th century. It is known to occur in about 1 in 1,000 people, but it is more frequent in the elderly population over 60 years of age. Uniquely, Koreans have the gene for Parkinson's disease, so the risk of developing it is higher than that of other races.

It develops when an abnormal protein called alpha-synuclein accumulates in brain cells. Recently, statistics have shown that the use of oral antibiotics increases the risk of Parkinson's disease 5 to 10 years later. This is likely to be related to specific anaerobic intestinal microorganisms. It is also known as a disease that occurs when dopamine secretion decreases due to damage and changes in nerve cells that secrete dopamine, a neurotransmitter in the brain.

In relation to Parkinson's disease, alpha synuclein first begins to accumulate in the brain stem, which connects the brain and torso, and gradually expands its distribution. It begins to accumulate at the bottom of the brainstem and reaches the substantia nigra of the midbrain tegmentum. When more than 50 to 70% of the brain cells in the substantia nigra are destroyed, externally observable symptoms occur. Acute Parkinson's disease also occurs when the substantia nigra is destroyed due to drug toxicity. The substantia nigra is like a factory in the brain that produces dopamine. Dopamine stimulates the brain to make precise movements and is involved in reward functions such as a sense of accomplishment. In Parkinson's disease, brain cells mainly in the areas that control movement are damaged, causing hand tremors, slow movements, and stiffness. It does not end here; over time, alpha synuclein spreads to all areas of the brain. It is thought that if it spreads to the cerebral cortex, dementia due to Parkinson's disease may occur.

Alpha synuclein aggregates within brain cells to form Lewy bodies, which are hyaline inclusions. Lewy body dementia is when brain cells die and leads to dementia symptoms.

When alpha-synuclein deposition is still only in the lower part of the brain, there are often no symptoms, as described above. Damage to the locus ceruleus and its surrounding areas may result in REM sleep behavior disorder, and damage to the olfactory cortex may result in incorrect smells. When REM sleep disorder occurs, the patient actually moves his or her arms and legs while performing dream actions while sleeping, so the patient's family thinks that the patient is talking excessively in his sleep. However, although it is true that these symptoms often appear in Parkinson's disease, the presence of these alone does not mean that one has Parkinson's disease. This is because you may be born with a bad sense of smell, and there are many other causes that can cause REM sleep disorders.

When Parkinson's disease reaches the substantia nigra and dopamine production falls below 70%, motor symptoms called Parkinson's symptoms occur. In the beginning, movements that require precision, movements using small tools such as chopsticks, writing, or fastening buttons do not work well. Dopamine in the substantia nigra is secreted by the basal ganglia, and the basal ganglia is responsible for performing tasks that do not require coordination from the cerebral cortex, that is, mindless actions. Therefore, movements made without much thought gradually decrease, and representative early symptoms include a decrease in walking speed (bradykinesia) and difficulty shaking the arms while walking.

When you think of Parkinson's disease, you usually think of resting tremor, but there are many cases where there is no hand tremor. The symptom that almost always appears is slow, narrow movements (bradykinesia). For reference, patients in whom hand tremor is the dominant symptom of the disease are said to have a better prognosis compared to patients in whom bradykinesia is the dominant symptom. Because the motor cortex is not properly stimulated due to a lack of dopamine, the range of movements of Parkinson's disease patients is mostly reduced. When you walk, your stride length decreases, and as you continue to write, you can see that the size decreases compared to the beginning (micrographia). The muscles in the face also move less and the facial expression becomes expressionless. For this reason, many Parkinson's disease patients receive treatment for depression first. As facial expressions decrease, the mouth opens naturally, which may cause drool to flow without realizing it. The volume of the voice also decreases. Some people find sleeping uncomfortable because they are unable to toss and turn while sleeping.

As neurodegeneration caused by alpha-synuclein continues, all symptoms worsen over time. At first, only one hand has symptoms, but then the other hand also develops symptoms, and walking goes from simply being slow to having difficulty maintaining balance. When you walk, your body keeps moving forward, but your feet can't keep up, so you often end up falling while walking. In the final stage, extreme motor impairment may lead to bed rest. In addition to motor symptoms, when the cerebral cortex is damaged, symptoms such as cognitive decline, visual hallucinations, and autonomic nervous system disorders also occur. In particular, the frequency of cognitive dysfunction is significantly higher than that of the general population, and even if the scope is limited to patients with dementia, the incidence rate is more than twice that of the normal population.

Meanwhile, as a technology related to phytosphingosine-1-phosphate, the use of phytosphingosine-1-phosphate or its derivatives as an immune enhancer is disclosed in Korean Patent No. 1865155, and phytosphingosine-1-phosphate is disclosed in Korean Patent No. 1842438. Although the use of gosine-1-phosphate or its derivatives for the prevention or treatment of dementia has been disclosed, there is still a method for improving immunity and treating Parkinson's disease containing the O-cyclic phytosphingosine-1-phosphate derivative of the present invention as an active ingredient. No composition has been disclosed for prevention, improvement or treatment.

The present invention was developed in response to the above-mentioned needs, and the present invention provides a composition for improving immunity and preventing, improving or treating Parkinson's disease, comprising an O-cyclic phytosphingosine-1-phosphate derivative as an active ingredient. In addition, the active ingredient significantly increased the motor function of the Parkinson's disease animal model by administering O-cyclic phytosphingosine-1-phosphate (cPS1P) of the present invention, and induced MPTP. And in NSE-hαSyn transgenic mice, not only the expression of S1P1 receptor (S1PR1) was significantly increased by cPS1P administration, but also the expression of αSyn was significantly decreased.

In addition, the present invention was completed by confirming that the increased expression of inflammatory cytokines in glial cells of MPTP-induced and NSE-hαSyn transgenic mice was reduced by administration of cPS1P of the present invention.

In order to achieve the above object, the present invention provides an immunizing agent comprising an O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for promoting and preventing or treating Parkinson's disease is provided.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a food-acceptable salt thereof as an active ingredient. Provides a health functional food composition for prevention or improvement.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent comprising O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. A feed composition for prevention or improvement is provided.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent comprising O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. A veterinary composition for prevention or treatment is provided.

The present invention relates to a composition for improving immunity and preventing, improving or treating Parkinson's disease, comprising an O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative as an active ingredient. , More specifically, the administration of cPS1P of the present invention significantly increased the motor function of an animal model of Parkinson's disease, and the expression of S1P1 receptor (S1PR1) was significantly increased by induction of MPTP and administration of cPS1P in NSE-hαSyn transgenic mice. Not only did it increase, but the expression of αSyn significantly decreased.

In addition, the amount of inflammatory cytokines increased in glial cells of MPTP-induced and NSE-hαSyn transgenic mice is reduced by administration of cPS1P of the present invention.

Figure 1 shows the results confirming the effect of cPS1P (O-cyclic phytosphingosine-1-phosphate) on motor dysfunction in MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mice, showing the total moving distance (A) in an open space box. , I); deadtime(B, J); number of square intersections(C, K); number of dependent behaviors (D, L); hanging time in wire hanging (E, M); Descent time in pole test (F, N); Hang time (G, O) and descent time (H, P) in the rotarod test; This is the result of checking. A to H are the results from the MPTP injection mouse model, and I to P are the results from the NSE-hαSyn transgenic mouse model. * indicates a statistically significant difference between the control group (Control) and the PD induction group (MPTP injection and NSE-hαSyn transgenic mice), p<0.05, and # indicates the PD induction group (MPTP injection and NSE-hαSyn transgenic mice). There is a statistically significant difference between the mouse) and the cPS1P administration group of the present invention, p<0.05.

Figure 2 shows the expression levels of S1PR1 and α-synuclein in the striatum and substantia nigra of MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mice. This is a result confirming the effect of cPS1P on regulation. A is a Western blot image of an MPTP-injected mouse and its quantification, B is a Western blot image of an NSE-hαSyn transgenic mouse and its quantification, * is a control group; and an MPTP-injected or NSE-hαSyn transgenic mouse; When comparing the expression levels of S1PR1 and β-synuclein, there is a statistically significant difference, p<0.05, # is MPTP-injected or NSE-hαSyn transgenic mouse; and S1PR1 and β-synuclein of the cPS1P administration group of the present invention. When comparing the expression levels, there is a statistically significant difference, p<0.05. The bands in A and B were quantified using Image J software, and β-actin was used as a loading control. C is the result of immunofluorescence analysis confirming the expression levels of S1PR1 and TLR4 in the striatum of wild type (WT), α-Syn Tag (NSE-hαSyn transgenic mouse), α-Syn Tag + cPS1P, and cPS1P groups. * indicates a statistically significant difference when comparing the expression levels of S1PR1 and TLR4 in control and MPTP-injected or NSE-hαSyn transgenic mice; p<0.05;# indicates MPTP-injected or NSE-hαSyn transgenic mice; When comparing the expression levels of S1PR1 and TLR4 in the cPS1P administration group of the present invention, there is a statistically significant difference, p<0.05.

Figure 3 shows the expression level of TH (tyrosine hydroxylase) in the substantia nigra and striatum of MPTP injection and NSE-hαSyn transgenic (αSyn-Tg) mice following cPS1P administration of the present invention. These are the results of immunohistochemistry (A-D) and Western blot (E, F) confirming the changes. * means there is a significant difference between the control group and the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group, # means the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group and the cPS1P administered group. It means that there is a significant difference between them, p<0.05.

Figure 4 shows the expression levels of VMAT-2 (Vesicular monoamine transporter 2) and DAT (dopamine transporter) in the substantia nigra and striatum of MPTP injection and NSE-hαSyn transgenic (αSyn-Tg) mice following cPS1P administration of the present invention. These are Western blot (A, B) results confirming the changes and immunofluorescence (C, D) results confirming the level of TH expression. * means there is a significant difference between the control group and the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group, # means the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group and the cPS1P administered group. It means that there is a significant difference between them, p<0.05.

Figure 5 shows GFAP (Glial fibrillary acidic protein) and Iba-1 (Ionized calcium-binding adapter protein-1) in the substantia nigra and striatum of MPTP injection and NSE-hαSyn transgenic (αSyn-Tg) mice following cPS1P administration of the present invention. expression level of Western blot (A, B) results confirming changes and expression levels of GFAP and Iba-1 in substantia nigra and striatum This is the immunofluorescence (C) result confirming the change. * means there is a significant difference between the control group and the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group, # means the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group and the cPS1P administered group. It means that there is a significant difference between them, p<0.05.

Figure 6 shows the expression levels of p-JNK, p-NFκB, TNF-α, and IL-1β in the substantia nigra and striatum of MPTP injection and NSE-hαSyn transgenic (αSyn-Tg) mice following cPS1P administration of the present invention. This is the Western blot result confirming the change. * means there is a significant difference between the control group and the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group, # means the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group and the cPS1P administered group. It means that there is a significant difference between them, p<0.05.

Figure 7 shows the results of immunohistochemical analysis confirming changes in dopaminergic neuron death in the substantia nigra and striatum of MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mice following cPS1P administration of the present invention. * means there is a significant difference between the control group and the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group, # means the MPTP-injected and NSE-hαSyn transgenic (αSyn-Tg) mouse group and the cPS1P administered group. It means that there is a significant difference between them, p<0.05.

Figure 8 shows the results of MTT analysis according to cPS1P treatment in SH-SY5Y human neuroblast cells. A confirms the cell viability of SH-SY5Y human neuroblast cells according to MPP ⁺ treatment, and B shows the results of MPP ⁺ and cPS1P of the present invention. Cell survival rate according to treatment was confirmed, C confirmed cytotoxicity, and D confirmed changes in caspase-3/7 activity. * means there is a significant difference between the control group and the MPP ⁺ treatment group, # means there is a significant difference between the MPP ⁺ treatment group and the cPS1P treatment group, p < 0.05.

The present invention provides a method for improving immunity and preventing Parkinson's disease, comprising an O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a pharmaceutical composition for therapeutic use.

In the present invention, 'pharmaceutically acceptable salt' refers to the relatively non-toxicity to patients and the beneficial effects of the O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative. It refers to any organic or inorganic addition salt that does not have side effects that deteriorate. For example, inorganic acid, organic acid, or non-toxic salts can be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, or tartaric acid can be used as the preferred inorganic acid. Preferred organic acids include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, and propionic acid ( propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid ), aspartic acid, ascorbic acid, carbonic acid, vanillic acid, or hydroiodic acid can be used.

Acid addition salts such as the above inorganic acids or organic acids can be prepared by conventional methods, such as dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. there is. Equimolar amounts of the compound and acid or alcohol in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be suction filtered.

The non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, and fluoride. , acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, Fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate phthalate , terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, Tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate can be preferably used.

In the present invention, the term 'prevention' refers to all actions that inhibit or delay the onset of Parkinson's disease by administering the pharmaceutical composition according to the present invention, and 'treatment' refers to the suspicion of Parkinson's disease and the diagnosis and treatment of Parkinson's disease by administering the pharmaceutical composition. It refers to any action that improves or changes the symptoms of an affected individual in a beneficial way.

The administration route for effective administration of the pharmaceutical composition according to the present invention is not particularly limited, and the administration route may be appropriately adopted for use in patients. For example, oral, rectal, transdermal, parenteral (subcutaneous, intramuscular, vascular), intrathecal, topical, inhalation and other administration methods can be used.

The pharmaceutical composition according to the present invention can be administered through various dosage forms according to common methods known in the pharmaceutical field. Preferred examples include powders, granules, tablets, troches, dispersions, suspensions, and solutions. , oral dosage forms such as capsules, emulsions, syrups, aerosols, external preparations, suppositories, injections, patches, and other suitable dosage forms can be used.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch or calcium carbonate, in the strain or endoplasmic reticulum derived from the strain. It is prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term 'pharmaceutically effective amount' in the present invention means sufficient to prevent or treat a disease with a reasonable benefit/risk ratio applicable to medical prevention or treatment. Means the amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and excretion rate, and the treatment. Factors including the duration of time, drugs used in combination or concurrently with the compositions of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

The frequency of administration of the composition of the present invention is not particularly limited, but can be administered once a day or divided into multiple doses, and the dosage depends on the patient's age, weight, gender, dosage form, health condition, and disease level. It may vary depending on the condition, and as a preferred example, based on an adult patient weighing 70 kg, the dosage is 0.1 to 300 mg/day, more preferably 0.5 to 100 mg/day, and even more preferably 1 It may be in the range of ~60 mg/day, but does not limit the scope of the present invention in any way.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a food-acceptable salt thereof as an active ingredient. It relates to a health functional food composition for prevention or improvement.

The active ingredient is preferably manufactured in a dosage form selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When the O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof is used as a health functional food composition, the O-cyclic phytosine-1-phosphate (cPS1P) derivative is used as a health functional food composition. Phingosine-1-phosphate (O-cyclic phytosphingosine-1-phosphate; cPS1P) derivatives or pharmaceutically acceptable salts thereof can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. You can. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

The health functional food composition of the present invention is commonly added during food production and may further include foodologically acceptable ingredients. For example, when manufactured as a beverage, in addition to the O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative of the present invention or a pharmaceutically acceptable salt thereof, citric acid, high fructose corn syrup, It may additionally contain one or more ingredients such as sugar, glucose, acetic acid, malic acid, fruit juice, etc.

The amount that can be included as an active ingredient in the health functional food composition according to the present invention can be appropriately selected depending on the age, gender, weight, condition, and symptoms of the disease of the person who wants a health functional food for preventing or improving Parkinson's disease, and is preferred. Preferably, it is contained in an amount of about 0.01 to 10.0 g per day for adults, and the effect of preventing or improving Parkinson's disease can be obtained by consuming foods with this content.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent comprising O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a feed composition for prevention or improvement.

The active ingredient can be added to the feed composition for the purpose of preventing or improving Parkinson's disease. The composition for feed may include feed additives. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act.

In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or an ingredient of the meal, for or suitable for eating, ingestion, and digestion by animals. The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

In addition, the present invention provides an immunostimulating and Parkinson's disease treatment agent comprising O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a veterinary composition for prevention or treatment.

The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate. , polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anti-aggregants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention provides rapid and sustained release of the active ingredient after administration to an animal. or may be formulated using methods well known in the art to provide sustained release, the dosage form being powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, It may be in the form of a suppository, sterile injectable solution, or sterile topical medication.

The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. Severity of the disease or condition, sensitivity to the active ingredient of the present invention depending on the individual's age, weight, health condition or gender, administration route, administration period, factors including other compositions mixed or used simultaneously with the composition, and other physiological or It can be determined based on factors well known in the veterinary field.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

1. Cell culture

Human neuroblastoma cells (SH-SY5Y) were purchased from the Korea Cell Bank (KCLB), and cultured for 5 days in DMEM (Dulbecco's modified Eagle's medium) containing 1% antibiotic-antimycotic solution and 10% fetal bovine serum. SH-SY5Y cells were treated with MPP ^{+ and} cultured under % CO ₂ and temperature conditions of 37°C.

The EGFP-alpha-synuclein-A53T plasmid was received from David Rubinsztein, and the cells were transformed using Lipofectamine 3000 (Invitrogen, CA, USA) according to the instructions provided by the manufacturer.

2. Animal model

As an animal model for Parkinson's disease, male wild-type C57BL/6J mice (approximately 25-28 g, 8 weeks old) administered MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) were transgenic. C57BL/6-Tg (NSE-hαSyn)(αSyn-Tg) Korl mice were used.

The male wild-type C57BL/6J mouse (approximately 25-28 g, 8 weeks old) was purchased from Samtako Biolabs (Ulsan), and transgenic C57BL/6-Tg (NSE-hαSyn) (αSyn-Tg) Korl Mice were purchased and used from the National Institute for Food Safety (SynG).

Samples obtained from mouse tails were used to confirm genotypes according to the PCR protocol of NIFDS. The animal model, the mouse, was provided with free access to water and food, and was acclimated for one week under a 12-hour light/dark cycle at a humidity of 60±10% and a temperature of 20±32°C.

MPTP (Sigma-Aldrich, MO, USA) was dissolved in distilled water and administered intraperitoneally (i.p.) to the mice (30 mg/kg) for 5 consecutive days.

Wild-type C57BL/6J mice were randomly divided into three groups (control group (vehicle treatment), MPTP group (MPTP treatment), and MPTP+cPS1P group).

Meanwhile, C57BL/6-Tg(NSE-hαSyn) Korl mice were classified into four groups (wild type (WT), α-Syn Tg(NSE-hαSyn), α-Syn Tg+cPS1P, and wild type (WT)+cPS1P). did. The cPS1P was provided by Axcesobiopharma (Anyang, Korea) and was used by dissolving in 0.01N NaOH solution.

3. Behavior analysis

(1) Open space test

The device used for the open space test consisted of an open space box (40 × 40 × 40 cm) divided into 16 equal-sized squares. Each mouse was placed in the center of an open space box and allowed to explore freely. The movements of the mice were recorded with a SMART video tracking system (Panlab, MA, USA). The experiments were conducted in a soundproof room under low lighting to prevent obstructions and unintentional freeze-thaw behavior.

(2) Pole test

The pole was a wooden stick with a length of 40 cm and a diameter of 10 mm. Mice were trained three times a day for two consecutive days to help them adapt to the behavior room. Each mouse was placed on a vertical wooden pole with its head facing upward. The total time taken to reach the bottom of the pole and place the front foot on the floor (T-LA) was recorded.

(3) Wire row test

The wire hang test apparatus consisted of a wire stretched horizontally to a height of 20 cm between two jars. Each mouse was placed on a wire held by its forelimbs, and the time each mouse stayed on the wire was recorded. The experiment was repeated 8 times, and the mice were allowed to rest sufficiently between experiments.

(4) Rotarod test

The rotarod test device consists of four adjacent rods with a height of 40 cm and a width of 10 cm. Mice were placed on a stick rotating at increasingly faster speeds, and the time the mouse remained on the stick was recorded. The experiment was repeated three times.

4. Preparation of tissue specimens for morphological analysis

After analyzing the mouse's behavior, saline (0.9%) and 4% paraformaldehyde were perfused into the mouse's carotid artery, the mouse's brain was fixed with paraformaldehyde for 72 hours at 4°C, and then sucrose phosphate buffer solution (20% ) and stored for 72 hours.

The brain was frozen using O.C.T compound, and brain slices were obtained using a CM3050C cryostat (Leica Germany). The obtained brain slices were thawed and mounted on gelatin-coated slides.

5. Protein extraction and Western blot analysis

Proteins were extracted using Pro-Prep protein extract, and the extracted protein concentration was measured using a spectrometer and Bio-Rad assay kit. Proteins were separated by 8% SDS-PAGE and transferred to PVDF (polyvinylidene-difluoride) membrane. Transfer was then blocked with 5% skim milk and incubated with each primary antibody listed in Table 1 overnight at 4°C (for 16 hours). After incubation, the primary antibody was reacted with the secondary antibody, and detected using ECL detection reagent (EzWestLumiOne). The detected images were scanned and quantified by ImageJ software.

Antibody Information

Antibody	Host	Application	manufacturing company	Catalog Number	Concentration
S1PR1	Rabbit	WB/IF	Invitrogen	PAI 1040	1:1000/1:100
α-Syn	Mouse	WB/IF	Santa Cruz Biotechnology	SC 58480	1:1000
TH	Rabbit	WB/IF	Merck	AB152	1:1000/1:100
VMAT-2	Rabbit	WB	Abcam	AB70808	1:1000
DAT	Rat	WB	Santa Cruz Biotechnology	SC 32259	1:1000
GFAP	Mouse	WB/IF	Santa Cruz Biotechnology	SC 33673	1:1000/1:100
Iba-1	Rabbit	WB	Thermo Fisher	PA527436	1:1000
P-JNK	Mouse	WB	Santa Cruz Biotechnology	SC 6254	1:1000
p-NFKB	Mouse	WB	Santa Cruz Biotechnology	SC 136548	1:1000
TNF-α	Mouse	WB	Santa Cruz Biotechnology	SC 52746	1:1000
IL-1β	Mouse	WB	Santa Cruz Biotechnology	SC 293072	1:1000

6. Immunochemical tissue staining

After washing the slides with phosphate buffer solution (PBS), the slides were washed with 10% H ₂ O ₂ It was treated with methanol for 10 minutes, and then proteinase K was applied for 10 minutes. After proteinase K treatment, the cells were blocked for 90 minutes with a blocking solution containing 5% BSA, normal goat serum, and tryptone X-100. Afterwards, each slide was treated with diluted primary antibodies (each antibody mixed at a ratio of 1:100 to 1:200 in blocking solution) and incubated at 4°C for 16 hours. After overnight incubation with the primary antibodies listed in Table 1, each slide was washed with PBS, treated with the corresponding secondary antibody (1:100 in PBS), incubated for 2 hours at room temperature, and then incubated with ABC at room temperature. It was treated with reagent (Vector Laboratory, CA, USA) for 1 hour and then washed with PBS. Finally, each slide was treated with 3,3-diaminobenzidine tetrahydrochloride hydrated (DAB) solution containing H ₂ O ₂ (Sigma-Aldrich, St. Louise, USA). After dehydration with ethanol (50% → 70% → 95% → 100%), it was treated with xylene for 5 minutes. Each slide was covered with a glass cover slip (Sigma-Aldrich, St. Louise, USA) using DPX mounting medium. Images were taken with an Axioscope 2 Plus microscope (Zeiss, Stuttgart, Germany).

7. Immunofluorescence Analysis

For immunofluorescence analysis, slides were washed with 1% PBS for 10 minutes and then incubated with proteinase K for 5 minutes at room temperature. Slides were washed and incubated for 1 hour with blocking solution containing 2% normal serum and 0.3% Triton X-100. The slides were then incubated and washed overnight at 4°C with the primary antibodies listed in Table 1, and then reacted with fluorescent secondary antibodies (Alexa Fluor 488 or 594, Invitrogen, California, USA) for 2 hours.

Each slide was washed with PBS and treated with DAPI (4',6-diamidino-2-phenylindole) for 10 minutes. Finally, the slides were covered with glass coverslips using fluorescent mounting medium. Images were captured with a confocal laser scanning microscope.

8. Nissl/Cresil Violet dyeing

For Nissl staining, slides were stained with 0.1% cresyl violet reagent (Sigma-Aldrich, St. Louis, USA) for 10 minutes and then washed with 1% PBS. After staining, the slides were washed with distilled water and dehydrated with ethanol (70% → 100%). Slides were immersed in differentiation solution (ethanol 90% + acetic acid), treated with xylene for 5 min, and then covered with glass coverslips using D.P.X mounting medium (Sigma-Aldrich, St. Louise, USA).

[Statistical analysis]

Western blot bands, immunohistology and immunofluorescence images of the present invention were quantified through ImageJ software. Density was expressed as mean ± standard error of the mean (SEM).

The data obtained in the present invention were statistically processed using one-way analysis of variance (ANOVA) and independent Student's t-test using GraphPad Prism 6, and the data were expressed as the mean ± SEM of three independent repeated experiments, p < 0.05 When , it was judged to be statistically significant.

Example 1. Effect of improving motor function impairment following administration of cPS1P of the present invention in a Parkinson's disease model

To analyze the effect of cPS1P on motor dysfunction, the open space test, pole test, wire hanging pole test, and rotarod test were performed.

In the open space test results, MPTP administration compared to the control group; and NSE-hαSyn transgenic; the total moving distance of the Parkinson's disease (PD) mouse group induced was significantly decreased, and the total moving distance of the Parkinson's disease-induced and cPS1P-administered group of the present invention was statistically significantly increased (Figures 1A and 1I).

In addition, the effect of cPS1P on immobility time was confirmed, and the results showed that MPTP administration compared to the control group; and NSE-hαSyn transgenic; immobility time increased in the Parkinson's disease (PD) mouse group, and in contrast, immobility time in the cPS1P administered group significantly decreased (Figures 1B and 1J).

Additionally, in the open field test, the number of square crossings and the number of rearing behaviors (the total number of times the mouse took an upright posture for the purpose of exploration during the test period) in the PD-induced group decreased compared to the control group, and the number of rearing behaviors decreased in the PD-induced group compared to the PD-induced group. The number of square crossings (Figures 1C and 1K) and the number of flapping behaviors (Figures 1D and 1L) in the cPS1P administered group of the invention significantly increased. It was confirmed that the hanging time in the wire hanging test (Figures 1E and 1M) and the descending time in the pole test (Figures 1F and 1N) were significantly changed.

Meanwhile, the motor coordination and balance of the experimental animals were evaluated through the rotarod test. As a result, compared to the control group, the time for the PD mouse group to fall from the rotarod rod was shorter. In contrast, the MPTP administration of the present invention; And the experimental group administered cPS1P to NSE-hαSyn transgenic Parkinson's disease (PD) mice had significantly improved hanging time (Figures 1G and 1H) and longer falling times (Figures 1H and 1P). was confirmed.

Example 2. Effect of controlling protein expression level by administration of cPS1P of the present invention in Parkinson's disease model

cPS1P administered MPTP; and NSE-hαSyn transgenic; expression of S1PR1 and β-synuclein was regulated in the striatum and substantia nigra of Parkinson's disease (PD) mice induced.

Through Western blot, it was confirmed whether cPS1P, the active ingredient of the present invention, can regulate the expression levels of S1PR1 and α-Syn (α-synuclein).

As a result, compared to the control group, MPTP administration; And NSE-hαSyn transgenic; in the brain (Substantia Nigra) and striatum of the Parkinson's disease (PD) mouse group induced by In contrast, it was confirmed that S1PR1 protein expression increased and β-synuclein (β-Sync) protein expression decreased in the cPS1P administration group of the present invention (Figures 2A and 2B).

In addition, the immune response of S1PR1 and TLR4 was confirmed by confocal microscopy, and the results showed that the expression level of S1PR1 was similar to the Western blot results, and the expression level of TLR4 increased in the PD-induced group compared to the control group. Therefore, the cPS1P administration group of the present invention was found to have a reduction effect to a level similar to that of the control group (Figure 2C).

Example 3. MPTP administration; Effect of cPS1P on TH (Tyrosine hydroxylase) expression in Parkinson's disease (PD) mouse model induced by NSE-hαSyn transgenic;

Tyrosine hydroxylase (TH) plays an important role in the synthesis of dopaminergic neurons. In this Example 3, MPTP administration; Tyrosine hydroxylase (TH) was used as a marker indicating the decrease in dopaminergic neurons in a Parkinson's disease (PD) mouse model induced by NSE-hαSyn transfection.

Immunohistochemistry analysis of TH expression in MPTP-injected mice and NSE-hαSyn transgenic mice showed that MPTP administration; And the expression of TH in the substantia nigra and striatum of the Parkinson's disease (PD) mouse group induced by NSE-hαSyn transgenics was significantly reduced compared to the control group. In contrast, the amount of TH expression in the cPS1P administered group was significantly higher than that of the PD induced group. was upregulated (Figures 3A-3D).

In addition, the Western blot results showed that the amount of TH expression was decreased in the substantia nigra and striatum of the PD model, and in contrast, the amount of TH expression in the substantia nigra and striatum was significantly increased in the group administered cPS1P of the present invention (Figure 3E and 3F).

Example 4. MPTP administration; and the effect of cPS1P on VMAT2 and DAT expression in a Parkinson's disease (PD) mouse model induced by NSE-hαSyn transgenic;

MPTP administration; and cPS1P on the expression of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) in the substantia nigra and striatum of a Parkinson's disease (PD) mouse model induced by NSE-hαSyn transgenic; The impact was evaluated.

The vesicular monoamine transporter 2 (VMAT2) is associated with the risk of PD, and VMAT2-deficient mice show progressive loss of dopaminergic neurons, dopamine deficiency, and accumulation of β-Syn, and the dopamine transporter (DAT) affects extracellular dopamine content. As one of the important influencing factors, DAT expression has been reported to be reduced in PD.

As a result of analyzing the expression levels of VMAT2 and DAT, which play a major role in the regulation of dopamine synthesis and transmission, by Western blot, MPTP administration compared to the control group; and NSE-hαSyn transgenic; expression levels of VMAT2 and DAT were decreased in the brains of the Parkinson's disease (PD) mouse group, and expression levels of VMAT2 and DAT were increased in the cPS1P administration group (Figures 4A and 4B).

Additionally, as a result of immunofluorescence analysis, it was confirmed that TH immunoreactivity in the substantia nigra and striatum of the PD mouse model brain was increased by cPS1P administration (Figures 4C and 4D).

Example 5. MPTP administration following administration of cPS1P; and NSE-hαSyn transgenic effect on improving gliosis in the brain of a Parkinson's disease (PD) mouse model.

The progressive loss of dopaminergic neurons in PD results in the production of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter protein-1, which are associated with activated glial cells that play important roles in neuroinflammation. binding adapter protein-1; Iba-1) are two specific markers of activated astrocytes and microglia, respectively.

To reveal the basic mechanism responsible for the anti-inflammatory effect of cPS1P, changes in the expression levels of activated astrocytes and microglia were confirmed.

Western blot results, MPTP administration; The expression of GFAP and Iba-1 was significantly up-regulated in the brain of the Parkinson's disease (PD) mouse group induced by NSE-hαSyn transfection, and the expression of GFAP and Iba-1 was significantly decreased in the brain of the cPS1P administration group. (Figures 5A and 5B).

In addition, in the immunofluorescence analysis, the immunofluorescence of GFAP increased in the substantia nigra of the MPTP-injected mouse brain, and the immunofluorescence of GFAP decreased in the substantia nigra of the brain of the cPS1P-administered group (Figure 5C).

Example 6. MPTP administration; and the effect of cPS1P on the expression of inflammatory factors in a Parkinson's disease (PD) mouse model induced by NSE-hαSyn transfection.

Excessive activation of c-Jun N-terminal kinases (JNK), phosphorylated nuclear factor κB (p-NFκB), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) contributes to neuroinflammation or neurodegeneration. It is known that it can have an effect.

MPTP administration, a PD induction animal model; and NSE-hαSyn transgenic; JNK (c-Jun N-terminal kinases), p-NFκB (phosphorylated nuclear factor-κB), tumor necrosis factor-α (TNF) in the brain of the Parkinson's disease (PD) mouse group. As a result of checking the expression levels of -α) and interleukin 1β (IL-1β), the expression levels of the above markers increased, and in contrast, the expression levels of the above markers significantly decreased in the cPS1P administration group of the present invention (Figures 6A and 6B).

Example 7. MPTP administration; And a decrease in neuronal death was confirmed by administering cPS1P to a Parkinson's disease (PD) mouse model induced by NSE-hαSyn transfection.

Dopaminergic neuron death appears in the substantia nigra and striatum of brains suffering from Parkinson's disease, and such neuron death is a major consequence of neuroinflammation and neurodegeneration. MPTP administration; To analyze the protective effect of cPS1P on neuronal death in a group of Parkinson's disease (PD) mice induced by NSE-hαSyn transfection, Nissl staining was performed on brain sections of mice.

As a result, the brain sections stained with cresyl violet received MPTP compared to the control group; and NSE-hαSyn transgenic; Nissl-stained neurons were reduced in the brains of the Parkinson's disease (PD) mouse group induced by MPTP co-administered with cPS1P; and NSE-hαSyn transgenic; Nissl-stained neurons increased in the brains of the Parkinson's disease (PD) mouse group (FIG. 7).

Example 8. Confirmation of cell viability, cytotoxicity and apoptosis in SH-SY5Y human neuroblasts following cPS1P treatment

Cytotoxicity following treatment with MPP ⁺ and cPS1P was confirmed in SHSY-5Y human neuroblasts through MTT analysis. As a result of analyzing MPP ⁺ at concentrations of 0.125, 0.25, 0.5, 1, 2, and 4mM, SHSY- When 5Y cells were treated with concentrations of 0.5, 1, 2, and 4mM, cell viability was significantly reduced (Figure 8A).

Therefore, to analyze cell viability, cytotoxicity, and neuronal death, 2mM of MPP ⁺ was treated, and in contrast, 1, 10, 50, or 100 μM of cPS1P of the present invention was treated to confirm changes in cell viability. Changes in cytotoxicity and caspase 3/7 activity were confirmed by treatment with 1, 10, or 50 μM.

As a result, the cPS1P treatment of the present invention increased cell survival (Figure 8B), decreased cytotoxicity (Figure 8C), and increased caspase 3/7 activity (Figure 8D).

Claims (7)

1. Pharmaceuticals for immune enhancement and prevention or treatment of Parkinson's disease containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivatives or pharmaceutically acceptable salts thereof as active ingredients Composition.
2. According to claim 1, wherein the pharmaceutically acceptable salt is any one inorganic acid selected from hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and tartaric acid; Methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid ( citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid. Any one organic acid selected from acid, carbonic acid, vanillic acid and hydroiodic acid; or sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, pro Cypionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, mali. ate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate phthalate, terephthalate, Benzenesulfonate, toluene sulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methane A pharmaceutical composition for enhancing immunity and preventing or treating Parkinson's disease, characterized in that it is a non-toxic salt selected from sulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.
3. The pharmaceutical composition for enhancing immunity and preventing or treating Parkinson's disease according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.
4. Health products for improving immunity and preventing or improving Parkinson's disease, containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivatives or food-acceptable salts thereof as active ingredients. Nutraceutical composition.
5. The health functional food according to claim 4, wherein the active ingredient is manufactured in a formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage, and is used to enhance immunity and prevent or improve Parkinson's disease. Composition.
6. Feed for improving immunity and preventing or improving Parkinson's disease containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivatives or pharmaceutically acceptable salts thereof as an active ingredient Composition.
7. Water for immune enhancement and prevention or treatment of Parkinson's disease containing O-cyclic phytosphingosine-1-phosphate (cPS1P) derivative or a pharmaceutically acceptable salt thereof as an active ingredient Medical composition.

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