KR101749675B1 - Pharmaceutical composition for inhibition of levodopa induced side effect and treatment of Parkinson's disease comprising melanin concentrating hormone - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of Parkinson's disease or a levodopa side effect containing melanin concentrating hormone as an active ingredient and a health functional food. The composition comprising melanocyte hormone as an active ingredient of the present invention restores the loss of dopaminergic neurons and thus has an excellent effect in the treatment and prevention of Parkinson's disease and has the effect of improving the abnormal motility induced by levodopa.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for inhibiting Parkinson's disease and levodopa side effects comprising melanin cohesive hormone as an active ingredient and a pharmaceutical composition for inhibiting levodopa adverse effect of Parkinson's disease comprising melanin concentrating hormone

The present invention relates to a pharmaceutical composition for the treatment of Parkinson's disease comprising the melanin concentrating hormone (melanin concentrating hormone) as an active ingredient and a pharmaceutical composition for suppressing side effects of levodopa, and a health functional food.

Parkinson 's disease is characterized by neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which causes gradual depletion of dopamine in the striatum. Parkinson's features break down the balance of the basal ganglia and disrupt the functioning of motor neurons, causing rigidity, tremor, and akinesia. 1-5% of the population over 50 is the second most common neurodegenerative disease.

The ultimate cause of Parkinson's disease is unknown, but it is known that many symptoms occur due to dopamine deficiency, which is a neurotransmitter in the brain of neurons in the substantia nigra of the normal brain .

Dopamine produced in neurons distributed in the black matter of the brain is connected to the basal ganglia of the brain, including the corpus striatum. The basal ganglia is a complex part of the motor cortex and many other parts of the brain, making it a very important part of the body that enables smooth, harmonious and accurate exercise. The consequence of this dopaminergic neuronal injury is the deficiency of dopamine, which is consequently liberated from the basal ganglionic dopaminergic nerve endings, which is a major cause of motor impairment in Parkinson's disease.

There is no definite answer to the cause of Parkinson's disease, namely, what causes black nerve cells to be destroyed, but active research is under way to identify them. Infectious diseases such as viral encephalitis, paradoxical paradox that immune mechanism is involved, genetics that inherits its temperament, hypothesis that free radicals are generated and destroy nerve cells, toxic substance addiction, and the production and metabolism of dopamine But there is still a lack of explanation for Parkinson's disease.

As a model of Parkinson's disease, the neurotoxin 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine phenyl-1,2,3,6-tetrahydropyridine, MPTP) are mainly used (Nat Rev Neurosci, 2001, 2 (5), 325-334). 6-OHDA is absorbed by the dopamine transporter (DAT) and produces free radicals. MPTP reduces dopaminergic neurons in humans and non-human primates, causes astrocytomas, and activates microglial cells in the cerebral substantia nigra (Nat Med, 1999, 5 (12), 1403-1409), leading to typical biochemical or pathological symptoms of Parkinson's disease (Neurosci, 2000, 16 (2), 135-142).

As drugs for the treatment of Parkinson's disease, there are known L-dopa preparations, dopamine receptor agonists, anticholinergics, and elderpril. The use of L-dopa (levodopa), a dopamine precursor, is an absolute criterion for the treatment of Parkinson's disease, since the function of motor neurons is dramatically improved early in Parkinson's disease. However, levodopa causes side effects such as hallucination, insomnia, nausea and dyskinesia. Among these side effects, levodopa-induced movement disorder (LID) is the most serious side effect of Parkinson's disease, which is chronic use of levodopa. More than 50% of Parkinson's patients who are chronically using levodopa experienced these side effects within five years. Anticholinergic drugs may have autonomic abnormalities or mental dysfunctions, which limits their continued use in older patients. Other surgical treatments such as high frequency nerve stimulation, such as high frequency waves or deep brain stimulation, have also been performed, but they require invasive surgery and are costly.

Melanin-concentrating hormone (MCH), on the other hand, is a hypothalamic peptide consisting of the first 19 annular amino acids separated from the pituitary gland. The dominant expression of MCH in the brain was found in the lateral hypothalamus and protruded all over the brain. MCH is known to regulate energy homeostasis by consuming energy and stimulating feeding behavior, but no function associated with loss of MCH has been found for dopaminergic neurons.

Accordingly, the present inventors have made intensive efforts to develop a composition capable of inhibiting levodopa-induced adverse effects by treating Parkinson's disease using MCH. As a result, it has been found that MCH plays a role in dopaminergic neurons in primary dopaminergic neurons and Parkinson's disease mouse models The present inventors completed the present invention by confirming that they have a protective effect against levodopa and alleviate movement disorders caused by levodopa.

It is an object of the present invention to provide a pharmaceutical composition for treating or preventing Parkinson's disease comprising melanin concentrating hormone as an active ingredient.

Another object of the present invention is to provide a health functional food for improving or preventing Parkinson's disease comprising melanin aggregation hormone as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing Parkinson's disease comprising MCH and levodopa.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting levodopa side effects comprising melanin aggregated hormone and levodopa.

In order to solve the above problems, the present invention provides a pharmaceutical composition for treating or preventing Parkinson's disease comprising melanin concentrating hormone as an active ingredient.

The term 'melanin aggregation hormone' used in the present invention is a kind of neuropeptide that is distributed in the central nervous system and is known to be involved in dietary regulation and weight control of food, And is known to have a function of inhibiting melanin production. However, it has been clarified through the present invention that melanin aggregating hormone has a neuroprotective effect on dopaminergic neurons, and thus has an effect of treating Parkinson's disease and improving dyskinesia induced by levodopa. The amino acid sequence of the melanin aggregating hormone and the nucleotide sequence information of the gene encoding the melanin aggregating hormone are known from NCBI (Genbank: NP_002665, NM_002674).

As used herein, the term " Parkinson's disease " refers to a disease characterized by progressive loss of dopaminergic neurons in the substantia nigra of the brain, chronic progressive degeneration of the nervous system characterized by stable tremor, stiffness, ≪ / RTI >

As used herein, the term " prevention " means any action that inhibits or delays Parkinson's disease by administration of a composition comprising the melanin cohesive hormone as an active ingredient.

The term " treatment " used in the present invention means all the actions that improve or ameliorate symptoms of a disease by administration of a composition containing the melanin cohesive hormone as an active ingredient.

In the present invention, the treatment of Parkinson's disease may be by nerve cell protection, improvement of motor function, or increase of the number of nerve cells.

The neurons are dopaminergic neurons, which may be neurons whose main neurotransmitter is dopamine, and include the ventral tegmental area (VTA), the substantia nigra pars compacta, the hypothalamus It can be located in the nucleus (arcuate nucleus of the hypothalamus) or striatum.

In one specific embodiment, the melanocyte-mediated hormone of the present invention is treated to dopaminergic neurons, followed by immunofluorescence staining for neuron-related genes such as Tuj1 (class III beta-tubulin) and MAP2 (microtubule associated protein 2) And that the number of dopamine neurons was effectively suppressed by treating the antihyper melanin agglutination hormone (FIG. 1).

Further, in a specific embodiment, melanin aggregating hormone is treated with MPP ⁺ or 6-OHDA-treated dopaminergic neurons, and then, through immunofluorescence staining for TH and MAP2, melanin aggregating hormone is added to MPP ⁺ or 6-OHDA Induced neuronal death (Fig. 3).

The composition of the present invention may be to inhibit the levodopa side effect, which is a therapeutic agent for Parkinson's disease.

As used herein, the term "levodopa" is the most widely used drug currently used in the treatment of Parkinson's disease. It is known that administered levodopa freely passes through the blood brain barrier and artificially stimulates dopamine secretion. Although levodopa is the most widely used for the treatment of Parkinson's disease, it is known to have side effects. Side effects of levodopa include hallucination, insomnia, nausea, and dyskinesia. Of these side effects, exercise disorders include motor impairments such as slow and uncooperative involuntary movements, shaking, stiffness, and gait disturbances. Patients treated with levodopa often have symptoms of reduced Parkinsonism, but they experience an increasingly difficulty standing or even maintaining sitting.

The composition of the present invention can be used in combination with a tyrosine hydroxylase (TH), a dopamine transporter (DAT), an aromatic l-amino acid decarboxylase (AADC), a microtuble associated protein 2) and synapsin, or to increase the expression of one or more genes selected from the group consisting of brain-derived neurotrophic factor protein, synaptophysin or tyrosine hydroxylase hydroxylase, TH) protein, or decrease the expression of synuclein protein.

The MAP2 belongs to a microtubule associated protein and is involved in microtubule assembly. It is known that microtubules are mainly involved in microtubule assembly by binding to other microtubules and intermediate fibers.

In a specific embodiment, the melanocyte hormone of the present invention is treated to dopaminergic neurons, followed by qRT-PCR analysis to detect tyrosine hydroxylase (TH), dopamine transporter (DAT), aromatic 1- It was confirmed that expression of dopamine neuron-related genes including amino acid decarboxylase (AADC) and MAP2 (microtuble associated protein 2) was increased (FIGS. 1 and 2) .

Also, in a specific example, qRT-PCR was performed on melanin aggregating hormone in MPP ⁺ or 6-OHDA-treated dopaminergic neurons, resulting in expression of TH, DAT, synapsin gene by melanin aggregation hormone (Fig. 3).

In another specific example, the melanocyte-mediated hormone of the present invention was injected intranasally into MPTP-injected mice, and then it was confirmed by immunochemical staining that TH-positive neurons were increased in the black blood, and TH- It was confirmed that the fibers were present at a high density (Fig. 8A). In addition, when the melanin agglutination hormone was treated with the black substance of the MPTP-treated mouse as described above, the expression of TH protein and synaptopathic protein was restored (FIG. 9A).

In another specific embodiment, Western blot confirmed that melanin aggregating hormone increases the expression of brain-derived neurotrophin protein in the substantia nigra of A53T alpha-synuclein transgenic mice and reduces the expression of the synuclein protein C and D in Fig. 11).

The composition of the present invention may be one that increases the activity of pCREB, pGSK, pAkt or pAMPK, inhibits the activity of Ikb? Or p38, or increases the activity of PI3K or PKA.

In a specific example, the expression of the Akt signaling-related gene was found to be increased in the whole gene expression analysis in melanocyte-treated dopaminergic neurons, and pCREB, pGSK, and pAkt were obtained through western blotting for pCREB, pGSK and pAkt (Fig. 4). ≪ tb > < / TABLE >

In addition, it was confirmed that the phosphorylation level of Akt and CREB was restored when the melanin agglutination hormone was treated with the black substance of the MPTP-treated mouse (FIG. 9C, 9D).

In addition, in experiments using inhibitors for PI3K and PKA signaling, it was confirmed that PI3K and PKA were involved in the neuronal protection effect by melanin aggregation hormone (Fig. 6).

In another specific example, it was confirmed that cell death was inhibited by melanocyte-mediated hormone treatment in 6-OHDA-induced human neuroblastoma SH-SY5Y cells, and 6-OHDA-induced human neuroblastoma SH-SY5Y cells It was confirmed that the activity of Ikb? Or p38 was inhibited by the melanin cohesive hormone treatment (Fig. 7).

In addition, it was confirmed through Western blotting that the melanin aggregation hormone increased the expression of BDNF protein and the expression of the synuclein protein in the black body of the A53T alpha-synuclein transgenic mouse (Fig. 11)

The term "A53T alpha-synuclein transgenic mouse" used in the present invention is a transformant in which alanine, which is the 53rd amino acid, is mutated to threonine. The mutant has aggregated alpha-synuclein, a dopamine neurodegeneration inducing protein Resulting in degeneration of dopaminergic neurons. In the present invention, the neuroprotective effect of melanin aggregation hormone was confirmed by using A53T alpha-synuclein transgenic mouse.

The alpha-synuclein is part of a larger protein family including beta-and gamma-synuclein and synoretin. Double alpha-synuclein is expressed in synapse-related steady state and is known to play a role in neurite plasticity, learning and memory. Alpha-synuclein can be an important protein in Parkinson's disease and related diseases because it can cause neuronal cell death and neuroinflammation and can mediate aggregate spreading in the brain (Proc Natl Acad Sci USA , 2009; 106: 13010-5; J Biol Chem, 2010a; 285: 9262-72).

In one embodiment of the present invention, it was found that the group injected with melanin aggregation hormone into the A53T alpha-synuclein transgenic mice had a longer time on the rod than the group not injected with the melanin agglutination hormone, (Fig. 10).

The composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels are determined by well known factors in the species and severity, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, Can be determined accordingly.

The 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 can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition for treating or preventing Parkinson's disease of the present invention may contain, in addition to the above-described effective ingredient, a pharmaceutically acceptable carrier, excipient or diluent.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method . In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The administration route of the pharmaceutical composition of the present invention may be one selected from the group consisting of oral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, intramuscular, intravenous and intraarterial routes. In the present invention, in order to deliver the melanin aggregated hormone to the brain, melanocyte aggregation hormone solution was injected into the nasal cavity of mouse or A53T alpha-synuclein transgenic mouse injected with MPTP after dissolving melanin aggregation hormone in a salt solution.

The dose varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and time, but can be appropriately selected by those skilled in the art.

The present invention also provides a health functional food for improving or preventing Parkinson's disease comprising melanin aggregation hormone as an active ingredient.

The health functional food of the present invention may inhibit the levodopa side effect which is a Parkinson disease remedy. Specifically, it may be to inhibit movement disorders caused by side effects of levodopa, a Parkinson disease remedy.

The definitions of melanin aggregation hormone and Parkinson's disease are as described above.

The term " health functional food " used in the present invention means a food prepared and processed using raw materials or ingredients having useful functions in accordance with the Health Functional Food No. 6727, Means that the structure and function of the human body are ingested for the purpose of obtaining nutritional control effects or physiological effects and health effects.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The health functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, have.

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a 'food additive' is not limited to those mentioned in the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the above-mentioned 'food additives' include natural compounds such as ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid and the like, sensory coloring matter, licorice extract, crystalline cellulose and guar gum, sodium L- A mixed preparation such as a preparation, a noodle-added alkali agent, a preservative preparation, a tar coloring agent, and the like.

In addition, the composition of the present invention provides a pharmaceutical composition for treating or preventing Parkinson's disease comprising melanin aggregation hormone and levodopa.

The composition of the present invention may be more effective in suppressing side effects of levodopa than levodopa alone. Specifically, it may be that the movement disorder caused by levodopa is suppressed as compared with the levodopa alone administration group.

Since the composition of the present invention exhibits a protective effect on dopamine neurons, it can be administered alone to treat Parkinson's disease. Alternatively, the pharmaceutical composition of the present invention and levodopa can be administered in combination to treat Parkinson's disease. In particular, when the pharmaceutical composition of the present invention is administered in combination with Levodopa, which is a therapeutic agent for Parkinson's disease, the activity exhibited by the conventional Parkinson's disease agent and the therapeutic activity of the pharmaceutical composition of the present invention are overlapped with each other, It also has the effect of inhibiting movement disorder, which is a side effect of levodopa. In addition, the co-administration agent that can be used at this time is not limited to levodopa, but is not particularly limited thereto as long as it exhibits therapeutic activity of Parkinson's disease.

In one specific example, in a Parkinson's disease model animal induced with 6-hydroxydopamine (6-OHDA), levodopa and melanin cohesive hormone were administered in combination with levodopa alone, (Fig. 12).

The present invention also provides a pharmaceutical composition for inhibiting levodopa side effects comprising melanin aggregated hormone and levodopa.

The composition of the present invention may be more effective in suppressing side effects of levodopa than levodopa alone. Specifically, it may be that the movement disorder caused by levodopa is suppressed as compared with the levodopa alone administration group.

Since the composition of the present invention suppresses adverse effects of levodopa, levodopa side effects can be suppressed by administering the pharmaceutical composition of the present invention and levodopa in parallel. In particular, when the pharmaceutical composition of the present invention is administered in combination with Levodopa, which is a therapeutic agent for Parkinson's disease, the activity exhibited by the conventional Parkinson's disease agent and the therapeutic activity of the pharmaceutical composition of the present invention are overlapped, It also has the effect of suppressing movement disorder, which is a side effect of levodopa.

The composition comprising the melanin agglutinating hormone of the present invention as an active ingredient is excellent in the treatment and prevention of Parkinson's disease because it restores the loss of dopaminergic neurons and has the effect of improving the abnormal motility induced by Levodopa, .

FIG. 1 (A) is a graph showing changes in the number of dopamine neurons after treatment of MCH and MCH + anti-MCH on midbrain dopamine neurons. FIG. 1B is a graph showing changes in the number of dopamine neurons through immunofluorescence staining of Tuj1 and MAP2 by treating MCH, MCH + anti-MCH on midbrain dopaminergic neurons.
FIG. 2 (A) shows qRT-PCR results of dopamine neuron marker genes of MAP2, DAT, TH, and AADC after treatment of MCH in midbrain dopamine neurons. FIG. 2B shows the number of different dopamine neurons in the treatment of MCH by concentration in the middle cerebral dopamine neuron, and C in FIG. 2 shows the expression level of the marker gene.
FIG. 3A is a graph showing changes in the number of dopamine neurons after treatment with 6-hydroxydopamine or MPP ⁺ on MCH-doped neuron cells. FIG. 3B shows MCH-treated midbrain dopamine neurons treated with 6-hydroxydopamine or MPP ⁺ , and midbrain dopaminergic neurons not treated with the drug, and immunohistochemical staining for dopamine neurons Fig. FIG. 3C is a graph showing the expression level of the gene through qRT-PCR for TH, DAT, and synapsin after treating MCH with 6-hydroxydopamine or MPP ⁺ on mesenchymal dopaminergic neurons.
FIG. 4A is an analysis of overall gene expression after treatment of MCH in midbrain dopamine neurons. FIG. FIG. 4B shows the expression of proteins of pCREB, pGSK-3β, pAkt and pERK through Western blotting after treatment of MCH in midbrain dopaminergic neurons. FIG. 4C is a graph showing the results of measuring phosphorylation levels of pCREB, pGSK-3β, pAkt, and pERK using a phosphoric acid-specific antibody after treating MCH in midbrain dopamine neuron cells.
FIG. 5A shows that neuroprotective effect of Tuj1 and MAP2 is inhibited by immunofluorescence staining of MCH-treated midbrain dopamine neurons after treatment with PI3K and PKA signaling specific inhibitors. FIG. 5B is a graph showing numerical results of A of 5. FIG.
FIG. 6A shows the results of treatment of MCH with MPP ⁺ -induced midbrain dopamine neuronal cell death, followed by treatment with PI3K and PKA signaling inhibitor, followed by immunofluorescence staining of Tuj1 and MAP2 to inhibit neuronal cell death The effect is suppressed. Fig. 6B is a numerical value showing the result of A in 6. Fig.
FIG. 7A is a graph showing that cell death is inhibited by MCH treatment of MCH at 0, 50 and 100 nM in 6-OHDA-induced human neuroblastoma SY-SY5Y cells. FIG. 7B is a graph showing the levels of phosphorylation of Ikb-a and p38 by Western blotting after treating 6-OHDA-induced human neuroblastoma SY-SY5Y cells with MCH. 7C is a graph showing the results of FIG. 7B in numerical values.
FIG. 8A is a graph showing changes in TH-positive neurons in the black blood after MCH was injected into MPTP-treated mice. FIG. 8B is a graph showing changes in the density of TH-positive fibers in the striatum after MCH is injected into MPTP-treated mice. FIG. 8C shows that the motor function is improved after injecting MCH into MPTP-treated mice.
FIG. 9A shows the expression of TH and synaptophysin protein in the black body through Western blotting after MCH was injected into MPTP-treated mouse. FIG. 9B is a graph showing the results of A in FIG. 9 as TH and synaptophysin /? -Actin (%). FIG. 9C shows the result of measuring the phosphorylation level of pAkt and pCREB through Western blotting after injecting MCH into MPTP-treated mouse.
FIG. 10 is a graph showing the results of measurement of the motor function through the Rotarod test at 2 and 4 weeks after injection of MCH into A53T alpha-synuclein transgenic mice. FIG.
FIG. 11A shows the results of measurement of phosphorylation levels of pGKS-3? And pAMPK via Western blotting after injecting MCH into A53T alpha-synuclein transgenic mice. FIG. 11B is a graph obtained by digitizing the result of FIG. 11A. FIG. 11C shows protein expression of the synuclein and BDNF after injection of MCH into the A53T alpha-synuclein transgenic mouse. FIG. 11D is a diagram showing the results of FIG. 11C as a synuclein, BDNF /? -Actin.
FIG. 12 shows the results of abnormal unstimulated kinetic (AIM) values at days 1, 4, and 7 after levodopa and MCH were injected at a concentration of 0.1, 0.5, and 2.5 에 in an animal model of Parkinson's disease induced with 6-hydroxydodamine, Fig.

Hereinafter, the structure and effects of the present invention will be described in more detail with reference to examples. These examples are intended to be illustrative of the present invention only, and the scope of the present invention is not limited by these examples.

Example  1: Primary dopaminergic neurons and human neuroblastoma cells Of MCH  Functional Role and Neuronal Protection Mechanism

1-1: Primary dopamine neuron culture

Primary cells isolated from the midbrain of the born mice were cultured. Briefly, midbrain containing SNC and VTA was easily cut by treating papain (5 U / ml; Worthington Biochemicals) at 37 ° C for 30 minutes. The cut tissue was made into a single cell through mastication using a small pipette tip. Cells were then centrifuged at 250 xg for 5 minutes and resuspended in 5% fetal bovine serum, 1x B27 (Invitrogen), 1x GlutaMAX (Invitrogen), 0.4% D- glucose (Sigma- Aldrich), 10 U / ml penicillin (Invitrogen) , And 10 [mu] g / ml streptomycin (Invitrogen). Cells isolated from each midbrain were divided equally and placed on a 12-mm-circular cover slip precoated with four poly-D-lysine (Sigma) and laminin (BD Bioscience) RTI ID = 0.0 > 37 C < / RTI > in a humidified incubator. The medium was changed to a serum-free medium supplemented with 5 μM cytosine β-D-arabinofuranoside (Sigma Aldrich) for 24 hours after the injection, glia). On the third day after plating, the medium was treated with MCH or other drugs.

1-2. Human neuroblastoma SH-SY5Y cell culture

Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were 10% in the atmosphere consisting of CO ₂ in the temperature and 95% air and 5% of the 37 ℃ fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 unit / ml penicillin, and 100 μg / ml streptomycin (Dulbecco's modified Eagle's minimum essential medium, Invitrogen, Carlsbad, CA, USA) supplemented with trypsin-EDTA (Gibco-BRL, Rockville, MD, USA) ).

Cell viability was determined using Thiazolyl Blue Tetrazolium Bromide (MTT). Briefly, cells were plated in 12-well plates (Corning Incorporated, USA) at a density of 5 X 10 ³ cells / well, stabilized at 37 ° C for 16 hours and then differentiated into 10 uM retinoic acid for 24 hours. The differentiated cells were either treated with 100 uM of 6-OHDA (Sigma-Aldrich, St. Louis, Mo., USA) for 24 hours, UK). Cells were incubated with 2 mg / mL MTT solution. After incubation at 37 ° C in 5% CO ₂ for 4 hours, the supernatant was removed and dimethyl sulfoxide (DMSO, Sigma, USA) was added. The optical density (OD) of the reaction was measured at 580 nm using a microplate reader (iMARK, Bio-Rad, USA). Optical density was converted to percent using the following equation: Cell viability (%) = (OD sample / OD negative control) X 100. Negative control cells were completely treated with DMEM alone.

1-3: Immunocytochemistry

Cells were fixed with PBS containing 4% paraformaldehyde and immunostained using the following primary antibodies according to standard protocols: Tuj1 (mouse monoclonal, Covance); TH (rabbit polyclone, Pelfreez); A secondary antibody to which the appropriate molecular probe (Molecular Probes) Alexa Fluor® stain was attached was used. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). Cells were imaged with Nikon Eclipse Ti, and images were processed and analyzed with Adobe Photoshop software. Immunoblotting was performed according to standard protocols.

1-4: Whole genome expression analysis

Total RNA (total RNA) was isolated from neurons cultured using RNeasy Mini Kit (QIAGEN). 2 μg of total RNA (total RNA) was used to prepare biotin-binding cDNA (Affymetrix One Cycle cDNA Synthesis Kit). Briefly, this method used SuperScript II-directed reverse transcription using a T7-Oligo (dT) promoter primer to generate first strand cDNA. The RNase H-treated RNase H-mediated second-strand cDNA was synthesized by T7 RNA polymerase-directed in vitro transcription (T7 RNA polymerase-directed in vitro transcription). The cRNA produced by in vitro transcription was combined with biotin-linked nucleotide analogs during cDNA synthesis. Samples were prepared by adding 15 μg of biotin-conjugated cDNA to a 1X hybridization cocktail according to the Affymetrix hybridization manual.

Gene chip arrays (mouse genome 430A 2.0 arrays) were hybridized in a gene chip hybridization oven at 45 ° C for 16 hours at 60 RPM. Washes were performed on GeneChip Fluidics Station 450 with buffers from Affymetrix GeneChip Hybridization, Wash, and Stain Kit. Analysis results were scanned with GeneChip Scanner 3000, and images were extracted and analyzed with GeneChip Operating Software v1.4. For statistical analysis, CEL files were processed using GeneSpring 7.0 and normalized using robust multi-array analysis.

1-5. Western blot

The cells were washed twice with 50 mM Tris-base (pH 7.5), 150 mM sodium chloride, 2 mM EDTA, 1% glycerol, 10 mM NaF, 10 mM Na- pyrophosphate, 40 and a protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 μg / ml aprotinin, and 5 μg / ml leupeptin). 30 μg of lysate was electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and incubated for 16 hours at 4 ° C with anti-phospho-CREB (pCREB), anti- (PGSK-3?), Anti-GSK-3?, Anti-phospho-Akt (pAkt), anti- Akt, anti- phospho-ERK, anti-ERK (Cell signaling technology, Beverly, MA, USA), anti-phospho-p38, anti-p38, anti-phospho-Ikbα and anti-Icba (Cell signaling technology, Beverly, MA, USA) antibodies. After washing with TBS-T (0.05%), the blot was reacted with horseradish peroxidase-linked anti-rabbit antibody and the band was detected by ECL system (Thermo Fisher Scientific, USA) . Band images were obtained using Molecular Imager ChemiDoc XRS + (Bio-Rad, Hercules, Calif., USA) and the intensity of the bands was analyzed using Image Lab ^TM software version 2.0.1 (Bio-Rad).

1-6. The function of MCH in the survival of midbrain dopamine neurons

To investigate the function of MCH in maintenance and survival of midbrain dopamine neuronal cells, the following experiment was conducted.

After birth, primary dopaminergic neurons were isolated from day 1 midbrain, which was treated with MCH for 5 days and cultured for 24 hours. Dopaminergic neurons were observed in media containing dopamine neurons treated with the cultured MCH and immunofluorescent staining was performed on neuron-related genes such as Tuj1 and MAP2 to confirm the number of dopamine neurons. As a result, it was confirmed that the number of dopamine neurons was increased by MCH treatment. In addition, it was confirmed that the number of dopamine neurons was effectively inhibited when the MCH-treated neural cell medium was treated with anti-MCH (FIGS. 1A and 1B).

Next, qRT-PCR was performed using dopamine neuronal cell medium treated with MCH. As a result, the MCH treatment resulted in the production of tyrosine hydroxylase (TH), a dopamine transporter (DAT), an aromatic l-amino acid decarboxylase (AADC) It was confirmed that expression of a dopamine (DA) marker gene including microtubule associated protein 2 (MAP2) was increased (FIG. 2A).

In addition, qRT-PCR was performed on dopamine neuronal cell markers such as TH, DAT, AADC, and MAP2 after treatment with various concentrations of MCH in dopamine neuronal cell culture media. As a result, when the concentration of MCH was 2 uM, the number of dopamine neurons and the expression of the markers showed the greatest increase (Fig. 2B and C)

1-7. 6-hydroxydopamine or MPP ⁺  After treatment, midbrain dopamine neurons Of MCH  function

Next, in order to examine the protective effect of MCH on neuronal cells after treatment with drugs inducing Parkinson's disease model in midbrain dopaminergic neurons, at day 5 after plating, dopamine neurons exposed to MPP ⁺ or 6-OHDA MCH was processed.

Observation of the number of dopamine neurons and immunological staining for TH and MAP2 revealed that MCH restored neuronal death induced by MPP ⁺ or 6-OHDA in dopaminergic neurons (FIG. 3 A and B). In addition, qRT-PCR of TH, DAT and synapsin confirmed that MCH also increased the expression of TH, DAT and synapsin gene, thereby confirming that it has protective effect on neurons (see Fig. 3 C).

Thus, the above results indicate that MCH plays an important role in maintenance and survival of midbrain dopamine neuron.

1-8. Neuroprotective mechanisms of MCH in dopaminergic neurons

To investigate the neuronal protective effect mechanism of MCH in dopamine neurons, global gene expression in dopamine neurons treated with MCH was analyzed. After dopamine neuron cells were treated with MCH 24 hours later, the transcriptome of this dopamine neuron was analyzed and the transcriptional profile was compared between the control and the control. As a result, it was confirmed that MCH changes overall gene expression, and it was confirmed that the expression of Akt signaling-related gene increased after treatment with MCH (FIG. 4A).

Western blotting was also performed on the Akt signaling proteins pCREB, pGSK-3 [beta], pAkt and pERK. As a result, it was confirmed that the expression of Akt signaling protein including pCREB, pGSK-3? And pAkt was significantly increased in MCH-treated primary dopamine neurons compared to the untreated culture 4, B).

To examine the signaling pathway of MCH in dopaminergic neurons, we examined the activity of CREB, Akt, GSK-3β or ERK by treating phospho-specific antibody in MCH-treated primary dopamine neurons Respectively. At 3 hours after treatment with 2 mM MCH, cells were obtained and immunoblots were performed using each antibody. As a result, as shown in FIG. 4, phosphorylation levels of CREB, GSK-3? And Akt were increased by MCH treatment, but the phosphorylation level of ERK did not increase (FIG.

Therefore, it can be seen from the above results that Akt signaling is involved in the neuronal protection mechanism of MCH, and CREB, Akt and GSK-3β are involved in Akt signaling, and ERK is not involved.

1-9. Of MCH  In the nerve cell protection effect Phosphatidylinositol  3-kinase (phosphatidylinositol 3- kinase ) And Protein  Kinase ( protein kinase  A) involvement

PI3K (LY294002) and PKA (H89) signaling-specific inhibitors were applied to MCH-treated primary dopamine neurons in order to investigate whether PI3K / Akt and PKA / CREB were involved in the protective effect of MCH on neuronal cells in dopaminergic neurons To perform immunofluorescence staining for Tuj1 and MAP2. As a result, PI3K / Akt and PKA / CREB signaling specific inhibitors LY294002 and H89 inhibited the neuronal protective effects exhibited by MCH (FIG. 5).

In addition, MPP ⁺ - induced dopamine neurons were treated with MCH and PI3K (LY294002) and PKA (H89) signaling specific inhibitors. As a result, it was confirmed that the neuronal protective effect of MCH on MPP ⁺ -induced dopaminergic neuronal death was inhibited by inhibitors of PI3K / Akt and PKA / CREB (FIG. 6).

Thus, Akt and CREB were involved in the protective effect of MCH-induced dopamine neurons using PI3K and PKA signaling inhibitors.

1-10. 6- By OHDA  Induced human neuroblastoma SH - SY5Y  In a cell Of MCH  Nerve cell protection effect

To examine the effect of 6-OHDA-induced human neuroblastoma SH-SY5Y cells on neuronal protection of MCH, cells were differentiated with 10 μM retinoic acid for 24 hours, and then 100 μM of 6-OHDA 50 or 100 nM of MCH or treated without MCH. As a result, it was confirmed that 6-OHDA-induced apoptosis in SH-SY5Y cells was significantly inhibited by 100 nM MCH treatment (Fig. 7A).

Next, in order to examine the signaling pathway involved in the neuronal protection effect of MCH, phosphorylation level of Ikb-a or p38 was confirmed by Western blotting after 6-OHDA treatment in SH-SY5Y cells. As a result, it was confirmed that MCH inhibited phosphorylation of Ikb-a or p38 induced by 6-OHDA (Fig. 7B and Fig. 7C).

Therefore, it was found from the above results that MCH exhibited a strong protective effect on neurons by inhibiting the activation of Ikb-a or p38.

Example 2: Effect of MCH in MPTP-induced Parkinson's disease model animals

2-1. MPTP-induced Parkinson's disease model animal preparation

A 12-week-old male C57BL / 6 mouse (Central Laboratories Animal Inc, Republic of Korea) was weighed for 12 hours at light and 12 hours at dark for 23 to 25 g body weight and free access to water and food The room temperature was maintained at 23 ± 1 ° C. All experiments were approved by Kyung Hee University Animal Protection Committee for animal welfare.

 First, mice were randomly divided into 6 groups: CON (control, n = 5), MPTP (MPTP only, n = 5), AP (MPTP + acupuncture acupuncture, n = 5) (N = 5), MCH (MPTP + MCH, in, n = 5), and aMR1 (MPTP + AP + MCH-R 1 antagonist, ip). All groups were intraperitoneally injected with MPTP (30 mg / kg, dissolved in saline solution; Sigma-Aldrich, MO, USA) or saline solution for 5 days.

2-2. MCH injection

In order to deliver the drug from the nasal cavity to the brain, MCH was injected into the nasal cavity as a noninvasive method of bypassing the blood-brain barrier. Although intracerebroventricular infusion has been extensively used in the brain-specific delivery of certain drugs, this is an invasive procedure and requires the animals to be anesthetized. Therefore, intranasal injection was chosen as a second-line method to deliver MCH to the brain. Intranasal infusion was performed as described in De Rosa et al., 2005. MCH (Tocris biosciences, Bristol, UK) was injected intranasally into mice 2 hours after MPTP injection. Before injection, MCH was dissolved in saline (0.5 mg / 30 μL) and stored at 4 ° C. All mice were held in a straight posture so that they did not move with their head and neck, and 15 μl of MCH solution was slowly injected into a single nasal cavity with a pipette for about 15 seconds. After 2 to 3 minutes, the same injection was repeated in the other nostrils. Other groups of mice injected saline solution (i.n.).

2-3. Immunochemical staining

Animals were sacrificed on day 12 after the first MPTP or saline solution injection and perfused with gentle perfusion using 0.2 M phosphate buffer containing 4% paraformaldehyde. The brain was removed, fixed postfixed, and cryoprotected. Immunochemical staining was performed by Hirsch & Faucheux, 1998; As described in Hirsch et al., 1998, substantia nigra and striatum (levels between AP -3.08 to -3.28 mm from the midbrain bragg gemma along the atlas of the mouse brain and AP +0.38 to +0.98 from the striatum bremsma Free cross-sectional cryomicrotome-cut sections (Keith & Franklin, 2008).

Next, to stain the black substance, Hirsch & Faucheux, 1998; Immunohistochemical staining was performed as described in Hirsch et al., 1998. After reacting in 0.05 M phosphate-buffered saline containing 3% H ₂ O ₂ , the sections were blocked with 1% BSA and normal goat serum. The tissue sections were then reacted with primary antibody overnight at room temperature. The rabbit TH (1: 1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the primary antibody. The tissue sections were reacted with biotin-conjugated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour at room temperature and incubated with ABC reagent (Vector Laboratories Inc., Burlingame, Reacted at room temperature and reacted for 2 minutes in 1 M Tris-buffered saline (pH 7.5) containing 0.02% diaminobenzidine and 0.003% hydrogen peroxide. After the reaction, the tissue section was fixed on a gelatin-coated slide and dried, water was removed and the cover was covered. Using a brightfield microscope (BX51; Olympus, Tokyo, Japan), photographs of striations and black matter were taken. Three independent observers who did not know the expected result counted TH-positive neurons in the black matter. The counted cells were checked 3 times for data validity.

2-4. Western blot

The substantia nigra (SN) was obtained from a mixture of 50 mM Tris-base (pH 7.5), 150 mM sodium chloride, 2 mM EDTA, 1% glycerol, 10 mM sodium fluoride (NAF), 10 mM sodium pyrophosphate ), 1% NP-40 and a protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 μg / ml aprotinin, and 5 μg / ml leupeptin) Homogenized. 30 μg of lysate was electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and incubated for 16 hours at 4 ° C with anti-synaptotypic and anti-β-actin , Beverly, MA, USA) and anti-TH (Santa Cruz Biotechnology, CA, USA) antibodies. After washing with TBS-T (0.05%), the blot was reacted with horseradish peroxidase-linked anti-rabbit antibody and the band was detected using an ECL system (Thermo Fisher Scientific, USA) Respectively. Band images were obtained using Molecular Imager ChemiDoc XRS + (Bio-Rad, Hercules, Calif., USA) and the intensity of the bands was analyzed using Image Lab ^TM software version 2.0.1 (Bio-Rad).

2-5. Effect of MCH in MPTP-induced Parkinson's disease model animals

To investigate whether MCH plays an important role in protecting dopamine neurons in MPTP-treated mice, the effects of MCH in MPTP-induced Parkinson's disease models were confirmed by the following experiments.

As a result, injecting MCH into the nasal cavity of mice treated with MPTP was definitely effective. The MCH-injected group had significantly more TH-positive neurons (P <0.001) (Fig. 8A) than the MPTP-treated group and also had a higher density of TH-positive fibers in the striatum 8, B). Similar to the above experimental results, it was confirmed that MCH improves the motor function in the result of performing the rotarod test (FIG. 8C).

2-6. MPTP  In the treated mice, tyrosine hydroxylase ( tyrosine hydroxylase ), Synaptophysin, effect of MCH on pAkt and pCREB

In order to confirm the protective effect of MCH on neurons, the expression of TH and synaptophysin in the black body was confirmed. As a result, the expression of TH and synaptopathy was decreased in the MPTP-induced black body, but the expression of TH and the synaptopian protein in the black body of MCH-injected mice was significantly restored (FIGS. 9A and 9B).

Next, the phosphorylation levels of Akt and CREB in black blood cells isolated from mice treated with MPTP were examined to confirm the signaling pathway by MCH injection or acupuncture in the brain. As a result, as shown in FIG. 9, the levels of phosphorylation of Akt and CREB were reduced in the black of MPTP-treated mice but recovered in the black of MCH injected or acupunctured mice (FIGS. 9C and D).

Example 3. Effect of MCH on A53T alpha-synuclein transgenic mice

3-1. Transgenic mice

The heterozygous breeder of the A53T alpha-synuclein (B6; C3-Tg-Prnp / SNCA ^* A53T / 83Vle / J) was purchased from Jackson Laboratories (Bar Harbor, ME, USA) and the A53T transgenic and non- They were bred to obtain a transgenic chick. (5'-TGT AGG CTC CAA AAC CAA GG -3 ', SEQ ID NO: 1) and antisense (5'-TGT CAG GAT CCA CAG GCA TA -3', SEQ ID NO: 2) primers , And analyzed by electrophoresis on 1.5% agarose gel. The genetically analyzed mice were housed in a room where temperature and humidity were controlled in a 12-hour light / dark cycle to freely eat water and food. MCH (Tocris biosciences, Bristol, UK) was dissolved in saline solution (0.5 mg / 30 μL) and stored at 4 ° C. The mouse neck was then fixed and 15 μL of MCH drug was slowly pipetted. The procedure was repeated after 5 minutes. The control (CON) group was treated with saline solution. Male transgenic mice between 10 and 12 months of age were randomly divided into two groups: a control group (n = 5) and a group injected with MCH (n = 5) Lt; / RTI &gt; Weight gain was measured weekly to check for nutritional status and food intake was recorded weekly for 4 weeks at the same time.

3-2. Behavioral experiment

At 4 weeks, the RotaRoad experiment was performed to determine the behavior of the mice. The mouse was pre-programmed for 2 minutes in an accelerating load-speed mode one hour before the experiment.

The time on the load was up to 480 seconds at continuous load speeds. The latency time was also analyzed.

3-3. Western blot

For Western blotting, the cells and the supernatant were resuspended in 50 mM Tris-base (pH 7.5), 150 mM sodium chloride, 2 mM EDTA, 1% glycerol, 10 mM sodium fluoride (NAF), 10 mM sodium pyrophosphate -pyrophosphate), 1% NP-40 and a protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 μg / ml aprotinin, and 5 μg / ml leupeptin) Lt; / RTI &gt; 30 μg of lysate was electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane and incubated for 16 hours at 4 ° C with anti-phospho-Thr ¹⁷² AMPKa, anti-AMPKa, anti-BDNF Anti-TH (Santa Cruz Biotechnology, CA, USA) antibodies were reacted with anti-S-actin antibody, anti-sinuclene, and anti-beta-actin (Beverly, MA, USA) After washing with TBS-T (0.05%), the blot was reacted with horseradish peroxidase-linked anti-rabbit antibody and the band was detected using an ECL system (Thermo Fisher Scientific, USA) Respectively. Band images were obtained using Molecular Imager ChemiDoc XRS + (Bio-Rad, Hercules, Calif., USA) and band intensities were analyzed using Image Lab ^TM software version 2.0.1 (Bio-Rad).

3-4. A53T  Alpha- Sina Klein  In the transgenic mice, pGSK - 3β , pAMPK, synuclein and brain-derived neurofilament protein expression

In order to confirm the effect of MCH on A53T alpha-synuclein transgenic mice, experiments were conducted by dividing into two groups, MCH-injected group and non-injected group.

There was no difference in latency time between the two groups before injecting MCH. At week 4, the MCH injected group was significantly longer on the rod than in the group not injected with MCH (p <0.01) (Fig. 10). There was no difference in body weight and intake between the two groups.

To investigate the mechanism of action of MCH in A53T alpha-synuclein transgenic mice, phosphorylation of GSK3 [beta], phosphorylation of AMPK, expression of brain-derived neurotrophic factor (BDNF), reduction of AMPK The expression of synuclein was confirmed as a stream target. As a result, MCH increased the phosphorylation of GSK3-? And AMPK in the black body of A53T alpha-synuclein transgenic mice, increased the expression of BDNF protein, and decreased the expression of the synuclein protein (FIG. 11).

4. 6- With hydroxy dopamine  In a derived Parkinson's model Levodopa  Effect of MCH on induced side effects

4-1. Animal model preparation

A 9-week-old male C57BL / 6 mouse (Central Lab. Animal Inc., Seoul, Republic of Korea) having a weight of 24-26 g was used. All experiments were approved by the Animal Protection Committee of Kyung Hee University for animal welfare and strictly maintained in accordance with the guidelines of the NIH and the Korean Medical Association. Stereotaxic surgery was performed by creating unilateral 6-OHDA lesions in mouse striata.

4-2. Stereoscopic surgery for unilateral lesions

For stereotaxic surgery, mice were treated with tililetamine plus zolazepam (30 mg / kg; Zoletil 50, Virbac, France) and xylazine (10 mg / kg; Rompun, Bayer Korea, Republic of Korea ) And fixed with a mouse adapter (Stoelting Co., USA) in a stereotactic frame. 6-OHDA-HCl mixture (3.0 mg / ml; Sigma Aldrich, USA) was dissolved in 0.02% ascorbic acid-based physiological saline. Mice were injected saline solution or 6-OHDA into the right striatum at the side of the mouse atlas of the brain: +1.0 mm of AP, -2.1 mm of ML, -3.2 mm of DV; And +0.3 mm of AP, -2.3 mm of ML, -3.2 mm of DV (BJ and GP, 2008). Each injection rate (flow rate) is 0.5 l / min.

4-3. Abnormal involuntary movement (AIM) values

To quantify levodopa induced dyskinesia (LID), each mouse was placed in a separate cage so that the blinded observer could assess the abnormal behavior. Levodopa or vehicle (vehicle, Cenci et al., 1998) was injected every 20 minutes from 20 minutes to 140 minutes. (1) improved motion after levodopa injection, (2) movement toward the opposite side of the lesion, and (3) repetitive, unintentional, and undeniably unnatural movements.

Then, the subtypes of the AIM values were separated: (1) Axial AIM (axial AIM), stiffening body and neck toward the opposite side; (2) limb AIM, repetitive and regular seizures or dorsal postural dystocia; And (3) AIM (orolingual AIM) of the mouth and tongue, abnormal chewing movements and sometimes exaggerated movement of the tongue. To quantify these subtypes, anthropomorphic activity was quantified on a 4-point scale (0, no; 1, occasionally; 2, often; 3, Continuous and serious without being interrupted by the stimulation of. After the quantification, three subtypes of AIM values (axis, limb, ororiginal AIM) were integrated for comparison of each group.

4-4. Effect of MCH on abnormal unsteady movements induced by levodopa

To assess the effect of MCH on levodopa-induced side effects, AIM values were evaluated after L-dopa infusion.

As a result, the group to which L-dopa (20 mg / kg) was administered induced more aripipin (p <0.05) than the 6-OHDA group and the control group. In addition, the group injected with MCH (0.5 mg / kg) together with L-dopa induced less dyskinesia (p <0.05) than the group treated with L-dopa (Fig. 12). A sub-analysis of AIM figures showed that all subcategories of AIM figures also had this tendency.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Pharmaceutical composition for inhibition of levodopa induced          side effect and treatment of Parkinson's disease          melanin concentrating hormone <130> KPA150561-KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense_primer <400> 1 tgtaggctcc aaaaccaagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense_primer <400> 2 tgtcaggatc cacaggcata 20

Claims (10)

A pharmaceutical composition for suppressing side effects of levodopa comprising melanin concentrating hormone as an active ingredient.
delete The method according to claim 1,
Wherein the levodopa side effect is motor impairment.
A pharmaceutical composition for the treatment or prevention of Parkinson's disease comprising melanin concentrating hormone as an active ingredient,
Wherein the composition is administered in combination with levodopa in the treatment of Parkinson's disease.
The method according to claim 1,
Wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
A health functional food for suppressing side effects of levodopa containing melanin cohesive hormone as an active ingredient.
delete The method according to claim 6,
Wherein said levodopa side effect is a movement disorder.
A melanin aggregation hormone and levodopa.
A melanin cohesive hormone and levodopa.

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