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WO2017014545A1 - Composition pharmaceutique pour soigner la maladie de parkinson et inhiber les effets secondaires de la lévodopa, contenant une hormone de concentration de la mélanine en tant que principe actif - Google Patents

Composition pharmaceutique pour soigner la maladie de parkinson et inhiber les effets secondaires de la lévodopa, contenant une hormone de concentration de la mélanine en tant que principe actif Download PDF

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WO2017014545A1
WO2017014545A1 PCT/KR2016/007884 KR2016007884W WO2017014545A1 WO 2017014545 A1 WO2017014545 A1 WO 2017014545A1 KR 2016007884 W KR2016007884 W KR 2016007884W WO 2017014545 A1 WO2017014545 A1 WO 2017014545A1
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mch
levodopa
disease
parkinson
dopamine
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PCT/KR2016/007884
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Korean (ko)
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박히준
전송희
김종필
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동국대학교 산학협력단
경희대학교 산학협력단
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Publication of WO2017014545A1 publication Critical patent/WO2017014545A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones

Definitions

  • the present invention relates to a pharmaceutical composition for treating Parkinson's disease and inhibiting levodopa side effects and a health functional food comprising melanin concentrating hormone as an active ingredient.
  • Parkinson's disease is characterized by neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which gradually leads to depletion of dopamine in the striatum. Parkinson's disease breaks down the balance of the basal ganglia and impairs the function of motor neurons, causing rigidity, tremor, and ainesia. One to five percent of people over 50 are the second most common neurodegenerative diseases.
  • Parkinson's disease The ultimate cause of Parkinson's disease is unknown, but several symptoms appear to be due to a deficiency of dopamine, a neurotransmitter in the brain made from nerve cells in the substantia nigra area of the normal brain. .
  • Dopamine produced by neurons distributed in the black matter of the brain is linked to the basal ganglia of the brain, including the corpus striatum.
  • the basal ganglia are intricately linked to the motor cortex and many other areas of the brain, making it an important part of the body's movements, allowing them to perform smooth, harmonious and accurate movements.
  • Dopamine deficiency which is free from the end of basal ganglia dopaminergic nerves caused by such dopaminergic nerve damage, is a major cause of motility disorders in Parkinson's disease.
  • Parkinson's disease namely, the destruction of the neural cells of the black matter, but active research is being conducted to find out.
  • Infection theory such as viral encephalitis, immune theory that immune mechanisms are involved, genetic theory that innately inherits its substrate, theory that free radicals destroy nerve cells, poisoning theory of neurotoxic substances, and dopamine production and metabolism
  • Infection theory such as viral encephalitis, immune theory that immune mechanisms are involved, genetic theory that innately inherits its substrate, theory that free radicals destroy nerve cells, poisoning theory of neurotoxic substances, and dopamine production and metabolism
  • the neurotoxin 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4- 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 dopamine neurons in humans and nonhuman primates, causes astrocytes and activates microglia in cerebrospinal densities rather than specifically targeting dopaminergic neurons associated with Parkinson's disease (Nat Med, 1999, 5). (12), 1403-1409), causing typical biochemical or pathological symptoms of Parkinson's disease (Neurosci, 2000, 16 (2), 135-142).
  • L-dopa preparations As drugs for the treatment of Parkinson's disease, L-dopa preparations, dopamine receptor agonists, anticholinergic drugs, eldefril and the like are known.
  • the use of the dopamine precursor, L-dopa (levodopa), is an absolute criterion for treating Parkinson's disease, as motor neurons function dramatically in the early stages of Parkinson's disease.
  • levodopa causes side effects such as hallucination, insomnia, nausea and dyskinesia.
  • levodopa-induced dyskinesia LID is the most serious side effect in patients with Parkinson's disease who are chronically using levodopa.
  • Anticholinergic drugs may show autonomic nervous system abnormalities or mental function abnormalities, and thus they are limited to continuous administration to older patients.
  • surgical treatment such as high frequency nerve stimulation, that is, high frequency destruction or deep brain stimulation, is also performed, but there is a problem that requires invasive surgery and consumes a lot of money.
  • MCH melanin-concentrating hormone
  • One object of the present invention to provide a pharmaceutical composition for the treatment or prevention of Parkinson's disease comprising melanin concentrating hormone (melanin concentrating hormone) as an active ingredient.
  • Another object of the present invention 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, including MCH and levodopa.
  • Another object of the present invention to provide a pharmaceutical composition for inhibiting levodopa side effects, including melanin aggregation hormone and levodopa.
  • MCH is applied to dopamine neurons.
  • the present invention was completed by confirming that it has a protective effect and alleviates a motor disorder caused by levodopa.
  • composition comprising the melanin aggregation hormone of the present invention as an active ingredient restores the loss of dopamine neurons, and thus has an excellent effect on the treatment and prevention of Parkinson's disease, and improves dyskinesia caused by levodopa, a treatment for Parkinson's disease. There is.
  • FIG. 1 is a diagram showing the change in the number of dopamine neurons after the treatment of MCH, MCH + anti-MCH to the brain brain dopamine neurons.
  • Figure 2 is a diagram showing the change in the number of dopamine neurons through the immunofluorescence staining of Tuj1 and MAP2 by treating MCH, MCH + anti-MCH to the brain brain dopamine neurons.
  • Figure 3 is a diagram showing the qRT-PCR results of the dopamine neuron marker genes of MAP2, DAT, TH, AADC after the treatment of MCH in the brain brain dopamine neurons.
  • Figure 4 shows the number of dopamine neurons different in the treatment of MCH by concentration in the midbrain dopamine neurons.
  • 5 is a diagram showing the expression level of the marker gene.
  • Figure 6 is a diagram showing the change in the number of dopamine neurons after MCH treatment to 6-hydroxy dopamine or MPP + -treated midbrain dopamine neurons.
  • Figure 7 shows the number of dopamine neurons through immunofluorescence staining for TH, MAP2 after MCH treatment of 6-hydroxydopamine or MPP + -treated midbrain dopamine neurons and untreated midbrain dopamine neurons.
  • Figure 8 is a diagram showing the expression level of the gene through qRT-PCR for TH, DAT and synapsin after MCH treatment in 6-hydroxy dopamine or MPP + -treated midbrain dopamine neurons.
  • Figure 9 is a diagram analyzing the overall gene expression after the treatment of MCH in the brain brain dopamine neurons.
  • 10 is a diagram showing the expression of pCREB, pGSK-3 ⁇ , pAkt, and pERK proteins through Western blot after treatment with MCH in mesenchymal dopamine neurons.
  • 11 is a diagram showing the results of measuring the phosphorylation level of pCREB, pGSK-3 ⁇ , pAkt and pERK using phosphate-specific antibodies after MCH treatment in midbrain dopamine neurons.
  • FIG. 12 is a diagram showing that neuroprotective effect is suppressed through immunofluorescence staining of Tuj1 and MAP2 after treatment with PI3K and PKA signaling specific inhibitors to MCH-treated midbrain dopamine neurons.
  • 13 is a diagram showing the results of FIG. 12 numerically.
  • Figure 14 after treatment with MCH in MPP + -induced midbrain dopamine neuron killing environment, after treatment with PI3K and PKA signaling inhibitors, the neuronal protective effect against neuronal cell death through immunofluorescence staining of Tuj1 and MAP2 Is a diagram showing suppression. 15 is a diagram showing the results of FIG. 14 numerically.
  • FIG. 16 is a diagram showing that apoptosis is inhibited by MCH treatment of 0, 50 and 100 nM MCH in 6-OHDA induced human neuroblastoma SY-SY5Y cells.
  • FIG. 17 is a diagram illustrating phosphorylation levels of Ikb- ⁇ and p38 through Western blot after treatment of 6-OHDA-induced human neuroblastoma SY-SY5Y cells with MCH.
  • 18 is a diagram showing the results of FIG. 17 numerically.
  • 19 is a diagram showing the change of TH-positive neurons in the black matter after MCH injection into the MPTP-treated mice.
  • 20 is a diagram showing the density change of TH-positive fibers in the striatum after MCH injection into the MPTP-treated mice.
  • 21 is a diagram showing that the motor function is improved after injecting MCH to the MPTP-treated mice.
  • FIG. 22 is a diagram showing the expression of TH and synaptophysin proteins in black matter through Western blot after injection of MCH into mice treated with MPTP.
  • FIG. 23 is a diagram showing the results of FIG. 22 in TH and synaptophysin / ⁇ -actin (%).
  • FIG. 24 and 25 show the results of measuring the phosphorylation level of pAkt and pCREB through Western blot after injection of MCH into MPTP treated mice.
  • Figure 26 is a diagram showing the result of measuring the motor function through the rotarod test at 2 and 4 weeks after MCH injection into A53T alpha-synuclein transgenic mice.
  • FIG. 27 shows the results of measuring phosphorylation levels of pGKS-3 ⁇ and pAMPK through Western blot after MCH injection into A53T alpha-synuclein transgenic mice.
  • FIG. 28 is a diagram quantifying the results of FIG. 27.
  • FIG. 29 shows protein expression of synuclein, BDNF after MCH injection into A53T alpha-synuclein transgenic mice.
  • FIG. 30 is a diagram showing the results of FIG. 29 using synuclein and BDNF / ⁇ -actin.
  • FIG. 31 shows abnormal involuntary motility (AIM) at 1, 4 and 7 days after injection of levodopa and MCH at concentrations of 0.1 ⁇ g, 0.5 ⁇ g and 2.5 ⁇ g in 6-hydroxydopamine-induced Parkinson's disease model animals. The figure which shows the result of measuring.
  • AIM abnormal involuntary motility
  • the present invention provides a pharmaceutical composition for the treatment or prevention of Parkinson's disease comprising melanin concentrating hormone (melanin concentrating hormone) as an active ingredient.
  • 'melanin aggregation hormone' used in the present invention is a kind of neuropeptide, which is distributed in the central nervous system, and is known to be involved in dietary control and weight control of foods. It is known to have a function of softening and inhibiting melanin production. However, melanin aggregation hormone has a neuroprotective effect in the dopamine neurons, through which it has been identified for the first time through the Parkinson's disease treatment effect and dyskinesia induced effect from levodopa through the present invention.
  • the amino acid sequence of the melanin aggregation hormone and the nucleotide sequence information of the gene encoding the same are known to the NCBI (Genbank: NP_002665, NM_002674).
  • the term 'Parkinson' disease used in the present invention is caused by the progressive loss of dopamine neurons distributed in the substantia nigra of the brain, chronic progressive degenerative of the nervous system, which is characterized by stability, stiffness, locomotor movement and postural instability. Means disease.
  • the term 'prevention' refers to any action of inhibiting or delaying Parkinson's disease by administration of a composition containing the melanin aggregation hormone as an active ingredient.
  • treatment refers to any action in which the symptoms of the disease are improved or advantageously changed by administration of the composition containing the melanin aggregation hormone as an active ingredient.
  • the treatment of Parkinson's disease may be due to neuronal cell protection, motor function or neuronal cell number increase.
  • the neuron is a dopamine neuron, which may mean a neuron whose main neurotransmitter is dopamine, ventral tegmental area (VTA), substantia nigra pars compacta, hypothalamus arch of the middle brain It may be located in the nucleus (arcuate nucleus of the hypothalamus) or striatum.
  • VTA ventral tegmental area
  • substantia nigra pars compacta hypothalamus arch of the middle brain It may be located in the nucleus (arcuate nucleus of the hypothalamus) or striatum.
  • the melanin aggregation hormone of the present invention is treated to dopamine neurons, and then immunofluorescence staining is performed on neuron-related genes such as Tuj1 (class III beta-tubulin) and MAP2 (microtubule associated protein 2). It was confirmed that the number of dopamine neurons is increased, the number of dopamine neurons is effectively suppressed by the treatment of anti-melanin aggregation hormone (Fig. 1 to 2).
  • Tuj1 class III beta-tubulin
  • MAP2 microtubule associated protein 2
  • melanin aggregation hormone is MPP + or 6-OHDA It was confirmed to restore the death of neurons induced by (Figs. 6 to 8).
  • composition of the present invention may be to suppress the side effects of levodopa which is a therapeutic agent for Parkinson's disease.
  • levodopa is the most widely used drug in the treatment of Parkinson's disease, and it is known that administered levodopa freely crosses the blood brain barrier to artificially promote dopamine secretion.
  • side effects of levodopa include hallucination (hallucination), insomnia, nausea and dyskinesia.
  • exercise disorders include movement impairment, for example, slow and non-cooperative involuntary movements, shaking (the appearance of shaking, stiffness and gait disturbances. Patients treated with levodopa often have reduced Parkinson's disease symptoms, but they experience increasingly difficult to keep standing or even sitting.
  • composition of the present invention is tyrosine hydroxylase (TH), dopamine transporter (DAT), aromatic l-amino acid decarboxylase (AADC), MAP2 (microtuble associated protein 2) and increase the expression of one or more genes selected from the group consisting of synapsin, brain-derived neurotrophic factor protein, synaptophysin or tyrosine hydroxylase (tyrosine) It may be to increase the expression of hydroxylase (TH) protein or to reduce the expression of synuclin (synucelin) protein.
  • the MAP2 belongs to a microtubule associated protein and is involved in microtubule assembly. It is known that microtubules are primarily involved in microtubule assembly by combining with other microtubules and intermediate fibers.
  • composition of the present invention may be to increase the activity of pCREB, pGSK, pAkt or pAMPK, to inhibit the activity of Ikb ⁇ or p38, or to increase the activity of PI3K or PKA.
  • Akt signaling-related genes was increased through analysis of global gene expression in dopamine neurons treated with melanin aggregation hormone, and pCREB, pGSK and pAkt through Western blots for pCREB, pGSK and pAkt. It was confirmed that the phosphorylation of the Akt signaling-related protein including the (Fig. 9 to Fig. 11).
  • A53T alpha-synuclein transgenic mouse used in the present invention is a transformant in which the alanine, which is the 53rd amino acid, is transformed into threonine, and the mutant aggregates alpha-synuclein, a dopamine neurodegenerative protein, Causes degeneration of dopamine neurons.
  • the neuronal protective effect of melanin aggregation hormone was confirmed using A53T alpha-synuclein transgenic mice.
  • the alpha-synuclein is part of a large protein family including beta- and gamma-synuclein and synoretin. Dual alpha-synuclein is known to be expressed in the normal state associated with synapses and play a role in neuroplasticity, learning and memory. Alpha-synuclein can be an important protein in Parkinson's and related diseases because it can induce neuronal death and neuroinflammatory and can be a mediator of aggregate spreading in the brain (Proc Natl Acad Sci USA , 2009; 106: 13010-5; J Biol Chem, 2010a; 285: 9262-72).
  • the A53T alpha-synuclein transgenic mice injected with melanin coagulation hormone was longer in the rod than the non-injecting group, the melanin coagulation hormone through the exercise improvement effect It can be seen that (Fig. 26).
  • composition of the present invention may be administered in a pharmaceutically effective amount.
  • the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity, age, sex, disease of the individual. It can be determined according to the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including the drug used concurrently and other factors well known in the medical field.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations.
  • compositions of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and the suitable total daily dosage can be determined by the practitioner within the scope of good medical judgment, It may generally be in an amount of 0.001 to 1000 mg / kg.
  • the composition is not particularly limited as long as it is an individual for the purpose of preventing or treating Parkinson's disease, and any individual may be applied.
  • any individual such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cows, sheep, pigs, goats, and the like, and to humans, and the mode of administration is conventional in the art. If not included.
  • transdermal administration through topical application may be used, but is not limited thereto.
  • compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, 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 include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient described above.
  • the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • compositions of the present invention may be used in the form of oral dosage forms, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods.
  • it may be prepared by using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants which are commonly used.
  • Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like.
  • Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like.
  • excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like.
  • lubricants such as magnesium stearate, talc can also be used.
  • It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.
  • base of the suppository utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the route of administration of the pharmaceutical composition of the present invention may be one selected from the group consisting of oral, subcutaneous, intraperitoneal, pulmonary, intranasal, intramuscular, intravenous and arterial.
  • the melanin aggregation hormone solution was injected into the nasal cavity of the mouse injected with MPTP or A53T alpha-synuclein transgenic mice after dissolving melanin aggregation hormone in the saline solution.
  • the dosage depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the time of day, and may be appropriately selected by those skilled in the art.
  • the present invention 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 be to suppress the side effect of levodopa Parkinson's disease improver. Specifically, it may be to suppress the movement disorder caused by the side effects of levodopa, a Parkinson's disease improving agent.
  • Parkinson's disease The definition of the melanin aggregation hormone, Parkinson's disease is as described above.
  • 'health functional food' used in the present invention means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No.6775 of the Health Functional Food Act. This means that it is ingested for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on the structure and function of the human body.
  • the health functional food includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.
  • dietary supplement may be in the form of any one of meat, sausage, bread, chocolate, candy, confectionary, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcoholic beverage and vitamin complex. have.
  • the health functional food may further include food additives, and the suitability as a 'food additive' is related to the relevant items according to the General Regulations of the Food Additives Code and General Test Act, etc., unless otherwise specified. Judging by the standards and standards.
  • the chemical additives such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as navy dye, licorice extract, crystalline cellulose, guar gum, and sodium L-glutamate Mixed preparations, such as a preparation, an alkali addition agent, a preservative preparation, and a tar pigment preparation, are mentioned.
  • composition of the present invention provides a pharmaceutical composition for treating or preventing Parkinson's disease, including melanin aggregation hormone and levodopa.
  • composition of the present invention may be that levodopa side effects are suppressed compared to the levodopa alone administration group. Specifically, it may be that the movement disorder caused by levodopa compared to the levodopa alone administration group is suppressed.
  • the composition of the present invention exhibits a dopamine neuron protective effect, it may be administered alone to treat Parkinson's disease, or by administering the pharmaceutical composition of the present invention and levodopa in parallel, the Parkinson's disease may be treated.
  • the pharmaceutical composition of the present invention and levodopa a Parkinson's disease therapeutic agent
  • the activity of the conventional Parkinson's disease therapeutic agent and the therapeutic activity represented by the pharmaceutical composition of the present invention can be duplicated to more effectively treat Parkinson's disease.
  • the side effects of levodopa which is a side effect of movement disorders also have an effect.
  • the combination dosage agent which can be used at this time is not specifically limited as long as it shows the therapeutic activity of Parkinson's disease as well as levodopa.
  • dyskinesiasis in a group administered with levodopa and melanin aggregation hormone in combination with levodopa alone in a Parkinson's disease model animal induced with 6-hydroxydopamine (6-OHDA). was less induced (FIG. 31).
  • the present invention also provides a pharmaceutical composition for inhibiting levodopa side effects, including melanin aggregation hormone and levodopa.
  • composition of the present invention may be that levodopa side effects are suppressed compared to the levodopa alone administration group. Specifically, it may be that the movement disorder caused by levodopa compared to the levodopa alone administration group is suppressed.
  • composition of the present invention suppresses the side effects of levodopa
  • levodopa side effects can be suppressed.
  • the pharmaceutical composition of the present invention and levodopa which is a Parkinson's disease therapeutic agent
  • the activity of the conventional Parkinson's disease therapeutic agent and the therapeutic activity represented by the pharmaceutical composition of the present invention may overlap, thereby more effectively treating Parkinson's disease.
  • Exercise disorders, a side effect of levodopa also have an inhibitory effect.
  • Example 1 in primary dopamine neurons and human neuroblastoma cells MCH Functional Roles and Neuronal Protection Mechanisms
  • midbrain containing SNC and VTA was easily cut by treatment with papain at 37 ° C. for 30 minutes (5 U / ml; Worthington Biochemicals). The cut tissue was made into single cells through chewing using a small pipette tip. Cells were then centrifuged at 250 xg for 5 minutes and 5% fetal bovine serum, 1x B27 (Invitrogen), 1x GlutaMAX (Invitrogen), 0.4% D-glucose (Sigma-Aldrich), 10 U / ml penicillin (Invitrogen) , And 10 ⁇ g / ml streptomycin (invitrogen) were suspended in plating medium.
  • the cells isolated from each midbrain were divided equally and dispensed onto 4 poly-D-lysine (Sigma) and laminin (BD Bioscience) precoated 12-mm-round coverslips, followed by carbon dioxide It was kept at 37 ° C. in an incubator humidified with. At 24 hours after dispensing, the medium was changed to serum-free medium supplemented with 5 uM cytosine ⁇ -D-arabinofuranoside ( ⁇ -D-arabinofuranoside, Sigma Aldrich). glia) was used to inhibit the proliferation. Three days after plating, the medium was treated with MCH or other drugs.
  • Human neuroblastoma SH-SY5Y cells were purchased from American Type Culture Collection (Rockville, MD, USA). Cells were 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 unit / ml penicillin, and 100 ⁇ g / ml streptoceat at 37 ° C., 95% air, and 5% CO 2 atmosphere. DMEM (Dulbecco's modified Eagle's minimum essential medium, Invitrogen, Carlsbad, CA, USA with the addition of mycin (Gibco-BRL, Rockville, MD, USA) and additionally trypsin-EDTA (Gibco-BRL, Rockville, MD, USA) ).
  • MTT Thiazolyl Blue Tetrazolium Bromide
  • DMSO dimethyl sulfoxide
  • Cells were fixed with PBS containing 4% paraformaldehyde and immunostaining was performed using the following primary antibodies according to standard protocols: Tuj1 (mouse monoclonal, Covance); TH (rabbit polyclonal, Pelfreez); Appropriate Molecular Probes Secondary antibodies linked with Alexa Fluor® stains were used. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (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.
  • RNase H-mediated second-strand cDNA RNase H-treated cDNA
  • the cRNA produced through in vitro transcription was combined with a biotin-coupled nucleotide analog during cDNA synthesis. Samples were prepared by placing 15 ⁇ g of biotin-bound cDNA into a 1 ⁇ hybridization cocktail according to the Affymetrix Hybridization Manual.
  • GeneChip arrays (GeneChip array, mouse genome 430A 2.0 arrays) were hybridized in a gene chip hybridization oven at 45 °C for 16 hours at 60 RPM. Washing was performed in GeneChip Fluidics Station 450 with buffer provided by 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.
  • Cells were 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- Homogenized in lysis buffer containing 40 and protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 ⁇ g / ml aprotinin, and 5 ⁇ g / ml leupeptin).
  • protease inhibitor 0.1 mM phenylmethylsulfonylfluoride, 5 ⁇ g / ml aprotinin, and 5 ⁇ g / ml leupeptin.
  • qRT-PCR was performed using the dopamine neuron medium treated with MCH.
  • TH tyrosine hydroxylase
  • DAT dopamine transporter
  • AADC aromatic l-amino acid decarboxylase
  • fine particles were treated by the MCH treatment. It was confirmed that the expression of dopamine (dopamine, DA) marker gene including microtubule associated protein 2 (MAP2) increased (FIG. 3).
  • MCH plays an important role in the maintenance and survival of midbrain dopamine neurons.
  • MCH changes the overall gene expression, among which the expression of Akt signaling-related genes increased after the treatment of MCH (Fig. 9).
  • Akt signaling is involved in the neuronal cell protection mechanism of MCH, and CREB, Akt and GSK-3 ⁇ are involved in Akt signaling, but not ERK.
  • MPP + induced dopamine neurons were treated with MCH together with PI3K (LY294002) and PKA (H89) signaling specific inhibitors.
  • PI3K LY294002
  • PKA H89
  • Akt and CREB are involved in the protective effect of dopamine neurons induced by MCH using PI3K and PKA signaling inhibitors.
  • MCH exhibited a strong protective effect in neurons by inhibiting the activation of Ikb- ⁇ or p38.
  • the 12-week-old male C57BL / 6 mouse (Central Laboratories Animal Inc, Republic of Korea), weighing 23-25 g, was kept for 12 hours in light and 12 hours in dark, and free access to water and food. Room temperature was maintained at 23 ⁇ 1 ° C. All experiments were approved by Kyung Hee University Animal Protection Committee for Animal Welfare.
  • MCH was injected by intranasal injection, a noninvasive method that bypasses the blood brain barrier to deliver drugs from the nasal cavity to the brain.
  • intracerebroventricular injection is widely used for brain specific delivery of certain drugs, it is an invasive method and needs to anesthetize the experimental animal. Therefore, intranasal injection was chosen as the next best method for delivering 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. Prior to injection, MCH was dissolved in saline solution (saline, 0.5 mg / 30 ⁇ L) and stored at 4 ° C.
  • saline solution saline, 0.5 mg / 30 ⁇ L
  • mice All mice were held in a standing position to hold their heads and necks intact, and 15 ⁇ l of MCH solution was slowly injected with a pipette for about 15 seconds into one nasal cavity. After 2 to 3 minutes, the same injection was repeated in the other nostril. Another group of mice received a salt solution (i.n.).
  • Tissue sections were reacted with biotin-conjugated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour at room temperature, and for 1 hour with ABC reagent (Vector Laboratories Inc., Burlingame, CA, USA).
  • the reaction was carried out at room temperature and for 2 minutes in 1 M Tris-buffered saline (pH 7.5) containing 0.02% diaminobenzidine and 0.003% hydrogen peroxide.
  • the tissue sections were fixed and dried on gelatin-coated slides, water was removed and the cover was covered. Photographs of striatum and black matter were taken using a brightfield microscope (BX51; Olympus, Tokyo, Japan). Three independent observers who did not know the expected results counted TH-positive neurons in the black matter. Counted cells were checked three times for validity of the data.
  • Substantia nigra contains 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 (Na-pyrophosphate). ), 1% NP-40 and a protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 ⁇ g / ml aprotinin, and 5 ⁇ g / ml leupeptin) Homogenized.
  • phosphorylation levels of Akt and CREB were confirmed in black matter isolated from mice treated with MPTP.
  • phosphorylation levels of Akt and CREB decreased in the black matter of MPTP-treated mice, but recovered in the black matter of MCH-infused or acupuncture mice (FIGS. 24 and 25). ).
  • Heterozygous breeder of A53T alpha-synuclein (B6; C3-Tg-Prnp / SNCA * A53T / 83Vle / J) variant was purchased from Jackson Laboratories (Bar Harbor, ME, USA), and A53T transgenic and non-transformant Breeding was obtained to obtain litters.
  • mice Genotyped mice were reared free of water and food in a temperature and humidity controlled room at 12 hour light / dark cycles.
  • MCH Tocris biosciences, Bristol, UK
  • salt solution 0.5 mg / 30 ⁇ L
  • the control group CON
  • mice were pre-trained for 2 minutes in accelerated road speed mode one hour before the experiment.
  • the time on the road was up to 480 seconds at continuous load speed. Latency time was also analyzed.
  • cells and black matter were treated with 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 (Na dissolution including -pyrophosphate), 1% NP-40 and protease inhibitor (0.1 mM phenylmethylsulfonylfluoride, 5 ⁇ g / ml aprotinin, and 5 ⁇ g / ml leupeptin) Homogenized in solution.
  • protease inhibitor 0.1 mM phenylmethylsulfonylfluoride, 5 ⁇ g / ml aprotinin, and 5 ⁇ g / ml leupeptin
  • MCH phosphorylation of GSK3 ⁇ , phosphorylation of AMPK, brain-derived neurotrophic factor (BDNF) expression, and down-ampling of AMPK as targets of Akt in the black matter Expression of synuclein was confirmed as a stream target.
  • MCH increased phosphorylation expression of GSK3- ⁇ and AMPK in black matter of A53T alpha-synuclein transgenic mice, increased expression of BDNF protein, and decreased expression of synuclein protein (FIGS. 27-30). ).
  • mice Nine week old male C57BL / 6 mice (Central Lab. Animal Inc., Seoul, Republic of Korea) weighing 24-26 g each were used. All experiments were approved by the Kyung Hee University Animal Protection Committee for Animal Welfare and were strictly maintained according to the guidelines of the NIH and the Korean Medical Association. Stereotaxic surgery was performed to create unilateral 6-OHDA lesions in striata of mice.
  • mice were treated with tiletamine + zolazepam (30 mg / kg; Zoletil 50, Virbac, France) and xylazine (10 mg / kg; Rompun, Bayer Korea, Republic of Korea ) Was anesthetized with physiological saline and fixed in a stereotactic frame with a mouse adapter (Stoelting Co., USA). 6-OHDA-HCl mixture (3.0 mg / ml; Sigma Aldrich, USA) was dissolved in 0.02% ascorbic acid-based saline.
  • mice were injected with either saline solution or 6-OHDA in the right striatum, unilaterally according to the brain atlas of the mouse: +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 flow rate is 0.5 ⁇ l / min.
  • LID levodopa induced dyskinesia
  • AIM axial AIM
  • limb AIM repetitive and regular spasm or dystonia of the forelimbs
  • AIM orolingual AIM
  • dyskinesia was quantified on a 4-point scale that reflects frequency (0, none; 1, sometimes; 2, frequent; 3, interrupted by an external stimulus but continued; 4, external Continuous and serious without being interrupted by the stimulus).
  • three subtypes of AIM values were integrated.
  • AIM levels were evaluated after L-dopa injection.
  • the group administered with L-dopa (20 mg / kg) induced more dyskinesia (p ⁇ 0.05) compared with the 6-OHDA group and the control group.
  • the group injected with MCH (0.5 mg / kg) with L-dopa induced less dyskinesia than the group that received only L-dopa (p ⁇ 0.05) (FIG. 31).
  • Sub-analysis of AIM levels showed that all subcategories of AIM levels also had this tendency.

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Abstract

La présente invention concerne une composition pharmaceutique et un aliment fonctionnel diététique pour soigner la maladie de Parkinson ou inhiber les effets secondaires de la lévodopa, contenant une hormone de concentration de la mélanine en tant que principe actif. La composition selon la présente invention, contenant une hormone de concentration de la mélanine en tant que principe actif, permet de récupérer les cellules nerveuses dopaminergiques perdues, ce qui lui permet de soigner et prévenir la maladie de Parkinson de manière performante, et a pour effet de soulager la dyskinésie induite par la lévodopa.
PCT/KR2016/007884 2015-07-20 2016-07-20 Composition pharmaceutique pour soigner la maladie de parkinson et inhiber les effets secondaires de la lévodopa, contenant une hormone de concentration de la mélanine en tant que principe actif WO2017014545A1 (fr)

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KR102001338B1 (ko) 2018-02-08 2019-07-17 동국대학교 경주캠퍼스 산학협력단 혼합 생약 추출물을 유효성분으로 함유하는 파킨슨병의 예방 또는 치료용 약학적 조성물
WO2025080080A1 (fr) * 2023-10-10 2025-04-17 한양대학교 산학협력단 Composition pharmaceutique pour prévenir ou traiter la maladie de parkinson, contenant un inhibiteur de pdk en tant que principe actif

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5210076A (en) * 1988-09-13 1993-05-11 Berliner David L Methods of treating Parkinson's disease using melanin
US20030031707A1 (en) * 1997-04-08 2003-02-13 Alan A. Rubin Treatment of parkinson's disease and related disorders by novel formulations of the combination carbidopa-levodopa
KR20100088136A (ko) * 2007-10-12 2010-08-06 노파르티스 아게 파킨슨병의 치료를 위한 대사성 글루타메이트 수용체 조절제
KR20150017763A (ko) * 2012-06-11 2015-02-17 싸이코제닉스 아이엔씨. 파킨슨병 치료와 관련된 운동 장애 부작용의 치료

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5210076A (en) * 1988-09-13 1993-05-11 Berliner David L Methods of treating Parkinson's disease using melanin
US20030031707A1 (en) * 1997-04-08 2003-02-13 Alan A. Rubin Treatment of parkinson's disease and related disorders by novel formulations of the combination carbidopa-levodopa
KR20100088136A (ko) * 2007-10-12 2010-08-06 노파르티스 아게 파킨슨병의 치료를 위한 대사성 글루타메이트 수용체 조절제
KR20150017763A (ko) * 2012-06-11 2015-02-17 싸이코제닉스 아이엔씨. 파킨슨병 치료와 관련된 운동 장애 부작용의 치료

Non-Patent Citations (2)

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Title
LEE: "A Novel Target for L-dopa Induced Dyskinesia in Parkinson's Disease: the Role of Melanin Concentrating Hormone", MASTER'S THESIS OF KYUNGHEE UNIVERSITY, February 2016 (2016-02-01) *
THANVI ET AL.: "Levodopa-induced Dyskinesia in Parkinson's Disease: Clinical Features, Pathogenesis, Prevention and Treatment", POSTGRADUATE MEDICAL JOURNAL, vol. 83, 2007, pages 384 - 388, XP055348991 *

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