WO2009136752A9 - Novel peptide for augmenting brain-derived neutrotrophic factor (bdnf) protein expression in hippocampal neurons, hippocampal tissue and cerebral-cortex tissue - Google Patents

Abstract

The present invention relates to a novel peptide for augmenting brain-derived neutrotrophic factor (BDNF) protein expression. More specifically, the present invention provides a novel peptide which can be used to prevent or treat neurological disorders such as Alzheimer's disease, Parkinson's disease, depression, stroke, Huntington's chorea, ischaemic disorders of the cerebrum, degenerative neurological disorders or diabetic neural disorders, by augmenting BDNF mRNA and protein expression in hippocampal and cerebral cortical tissue. A Methionine-Valine-Glycine (MVG) peptide comprising three different amino acids of the present invention augmented BDNF mRNA and protein expression in hippocampal neurons, hippocampal tissue and cerebral-cortex tissue; BDNF being an important functional substance in learning and memory, dopamine neuron protection and depression prevention.

Description

Novel peptides increase protein expression of WNDF in hippocampal neurons, hippocampal tissues, and cerebral cortical tissues

The present invention relates to novel peptides that increase protein expression of BDNF with various effects such as neuronal growth and differentiation and learning, memory, antidepression and the like.

Neurotrophic factor regulates the survival and differentiation of neurons during development (Davies, 1994), and maintains neural structure and activity of ion channels, neurotransmitter release, and axon pathways during the life of the individual (Davies, 1994). axon path-finding) (Schnell et al., 1994; Song and Poo, 1999; Schinder and Poo, 2000). Examples of such neurotrophic factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophs. Pin-4 / 5 (Neurotrophin-4 / 5, NT-4 / 5) is present.

Among the neurotrophic factors, BDNF is expressed in the hippocampus, cortex and basal brain which are responsible for learning, memory, and higher thinking in the central nervous system (CNS). BDNF expressed in this position is a major regulator of synaptic plasticity and recognition processes, which is the neurobiochemical basis for synaptic transmission and learning and memory (Lu B, 2003). Not only does it promote long-term potentiation (LTP), a cellular phenomenon in learning and memory processes, but it also inhibits long-term depression, LTD (opposite) (Ikegaya, Y. et al., 2002; Huber, K. et al., 1998). Among neurotrophic factors, this unique role is consistent with the fact that BDNF and its receptor, TrkB (tropomyosin-related kinase B), are widely distributed in glutamate synapses.

BDNF, an important regulator of synaptic transmission and synaptic plasticity, is associated with a variety of diseases. Typical examples include degenerative brain diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), depression from stress, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative diseases or diabetic neuropathy Etc. can be mentioned.

Alzheimer's disease causes deterioration of cognitive ability by the degeneration of cholinergic neurons in the whole brain and a decrease in the release of the neurotransmitter acetylcholine (Murer MG et al., 2001). BDNF is known to promote the survival and differentiation of basal cholinergic neurons as well as to stimulate the release of acetylcholine in these neurons (Knipper M et al., 1994). Therefore, it is suggested that deterioration of homeostasis of cells due to lack of synthesis of BDNF will cause Alzheimer's disease. It is also known that neuroplastic deficiency, ie reduction in synaptic contact, presents neuropathological and clinical signs (Mesulam MM., 1999). Clinical results showed that post-mortem brain necropsy in Alzheimer's disease significantly reduced the expression of BDNF and its receptor, TrkB, in the hippocampus and cortex, which are responsible for learning and memory (Phillips HS et al., 1991; Holsinger RM). et al., 2000; Allen SJ et al., 1999). BDNF is known to regulate long-term potentiation (LTP), a phenomenon in cellular processes of learning and memory through synaptic plasticity at these sites (Figurov, A. et al., 1996). Therefore, it is also thought that a decrease in the function of the recognition and memory process by reducing the expression of BDNF will cause Alzheimer's disease.

Parkinson's disease results in a decrease in the number of substantia nigral dopaminergic neurons, leading to depletion of striatal dopamine in the striatum, leading to symptoms such as motor dysfunction. Another characteristic of Parkinson's disease patients is that the cognitive function is deteriorated. As a protein associated with the cause of Parkinson's disease, neurotrophic factors are expected to play an important role in the protection of dopaminergic neurons (Siegel GJ and Chauhan NB., 2000). Among neurotrophic factors, the interaction of BDNF with dopaminergic neurons is well known. Dopaminergic neurons in the ventral midbrain, melanoma and ventral tegmental areas express BDNF (Seroogy KB et al., 1994), resulting in decreased BDNF expression in the melanoma region. The number of is significantly reduced (Porritt MJ et al., 2005). In addition, we report that BDNF is necessary to maintain the appropriate number of dopamine neurons in the vaginal cells (Baquet ZC et al., 2005) and that in the case of Parkinson's patients, the BDNF expression of the stratified dopamine neurons is significantly reduced (MogiM et. al., 1999; Howells DW et al., 2000) reported that the reduction of dopaminergic neurons in Parkinson's disease is associated with a deficiency of BDNF biosynthesis.

The number of depressed patients is increasing rapidly in the modern society, which is heavily stressed. Stress is known to reduce the volume of various brain regions, including nervous breakdown and hippocampus (Duman, RS and Monteggia, LM 2006), as well as reducing BDNF mRNA expression in the hippocampus (Duman, RS and Monteggia, LM 2006 Smith, MA et al., 1995). In postmortem brain necropsy of depressed patients, the expression of BDNF was significantly reduced compared to normal subjects, and the volume of hippocampus was significantly reduced in brain imaging (Sheline, Y.I et al., 2003). Various chemical antidepressants have been used to treat these depressions. Commonly, the expression of BDNF mRNA is significantly increased in the hippocampus, anterior cerebral cortex or both regions (Duman, R.S. and Monteggia, L.M. 2006). These results suggest the hypothesis of 'neurotrophin hypothesis of depression'. In other words, the treatment of mental disorders such as depression by restoring neuronal breakdown and cell number reduction by re-increasing the expression of reduced BDNF in the hippocampus and the anterior cerebral cortex by stress (Duman, R.S. and Monteggia, L.M. 2006).

As described above, BDNF exhibits various functions such as neuronal cell growth and differentiation, learning and memory, and antidepressant as well as maintenance of neural structure, activation of ion channels, and release of neurotransmitters. Along with these diverse functions, BDNF can be used for Alzheimer's disease, Parkinson's disease, depression, stroke (Schabitz et al., Stroke, 38: 2165-2172, 2007), and Huntington's chorea (Zuccato C et al., Science 293, 20). , July 2001), cerebral ischemic disease (Han BH et al., The Journal of neuroscience, 2000, 20 (15): 5775-5781, August 1, 2000), neurodegenerative diseases (Tsuzaka K et al., Muscle Nerve. 24 (4): 474-80, April 2001) or diabetic neuropathy (Nitta A et al., Neurotoxicology and Teratology, 24: 695-701, 2002). However, BDNF itself is a 14kDa macromolecular protein that does not cross the blood-brain barrier (BBB) and has problems such as administration method. Therefore, it is easy to cross the blood brain barrier and has very low cytotoxicity, and by discovering a substance that increases the expression of BDNF by administering a small amount, it is possible to detect neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, stroke, which are chronic stress diseases. It can be used for prevention and treatment.

 The present inventors prepared an MVG peptide consisting of three amino acids by selecting the amino acids with the highest expression of BDNF at each position through a peptide library technology called PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library). The present invention was completed by confirming that BDNF mRNA and protein expression is increased in hippocampal neurons, hippocampal tissues and cerebral cortical tissues by MVG peptide.

It is an object of the present invention to provide peptides that increase mRNA expression of Brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissues and cerebral cortical tissues.

Another object of the present invention is to provide a peptide for increasing protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissues and cerebral cortical tissues.

 Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of neurological diseases comprising a therapeutically effective amount of the peptide.

In order to achieve the above object, the present invention provides a peptide consisting of the amino acid (MVG; Methionine-Valine-Glycine) described in SEQ ID NO: 1 (hereinafter referred to as 'MVG peptide').

 The peptide of the present invention increases the expression of Brain-derived neurotrophic factor (BDNF) mRNA and / or the protein expression of Brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampus and cerebral cortex, and expresses protein expression of TrkB and May increase phosphorylation.

 The present invention also provides a polynucleotide consisting of a DNA sequence encoding the peptide. The polynucleotide comprises an equivalent nucleic acid sequence, that is, a codon degeneracy sequence that encodes MVG peptides of different but identical sequences.

 Specifically, the present inventors selected the amino acid with the highest expression of BDNF at each position through a peptide library technology called PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library) (MVG; Methionine-Valine-Glycine) Afterwards, it was again confirmed by RT-PCR and Western blotting that expression increased substantially at the cellular level. In addition, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test was performed to determine whether MVG peptide is toxic at the cellular level. To confirm the expression of BDNF at the actual animal level, 0.1 μg / kg of peptide was injected through the tail vein, and the hippocampus and cerebral cortex were removed and Western blotting was performed.

 As a result, the present inventors confirmed that the MVG peptide consisting of three amino acids induced an increase in BDNF expression not only in hippocampal neuronal cell line but also in hippocampal tissue and cerebral cortical tissue, and showed no cytotoxicity up to 1 mM at the cellular level. It was. Hereinafter, the present invention will be described in more detail.

In one embodiment, the present inventors incubated HT22 cells, a mouse hippocampal neuronal cell line, in a 24 well plate at 5 × 10 ⁴ per well for 12 hours, and then prepared PS-SPCL stock (stock). Was treated to each well. After 12 hours, the protein was obtained using RIPA (Radio-immunoprecipitation Assay) buffer to which the protease inhibitor was added, and the amount of BDNF was measured through Enzyme-Linked ImmunoSorbent Assay (ELISA). As a result, as shown in FIG. 1, M (Methionine) at the first position, V (Valine) at the second position, and G (Glycine) at the third position expressed the most BDNF.

 In another embodiment, the results show that the peptide composed of the amino acid (MVG) having the highest expression of BDNF at each position is resynthesized and treated to H19-7 cells of rat hippocampal neuronal cell line to increase the expression of BDNF mRNA. It was confirmed by RT-PCR. As a result, BDNF mRNA expression was increased in a dose-dependent manner in the experimental group treated with 0.001, 0.01, 0.1 μM MVG peptide as shown in FIG.

 In another embodiment, the BDNF protein expression was confirmed by Western blotting by treating the hippocampal neuronal H19-7 cells with MVG peptide. As a result, as shown in FIG. 3, BDNF protein expression was increased in a dose-dependent manner in the experimental group treated with 0.001, 0.01, and 0.1 μM MVG peptide as compared to the control group.

 In another embodiment, male SD (Sprague-Dawely) rats (4 weeks of age) are purchased and subjected to stabilization for 1 week, after which 1X PBS (control) and the MVG peptides determined above are 0.1 μg / Kg injections and hippocampal and cerebral cortical tissues were performed and Western blotting was performed. As a result, it was confirmed that the expression of BDNF and proBDNF protein in the experimental group increased as shown in Figure 4, 5 compared with the control group.

 In another embodiment, H19-7 cells were treated with MVG peptides from 10nM to 1mM to confirm cytotoxicity of the MVG peptides, and then the survival rate of the cells was confirmed by MTT solution. It was confirmed (FIG. 6).

In another embodiment, one of the spatial memory tests, the Y-maze test and the Morris water maze test, was performed to determine whether the MVG peptides promote spatial learning and memory at the experimental animal level. Enhancement of memory was confirmed through animal experiments (FIG. 8, FIG. 10), and increased BDNF expression by MVG peptides in hippocampus (FIG. 11A) and cerebral cortex (FIG. 11B). In addition, TrkB phosphorylation and TrkB expression were increased in the group injected with MVG peptides in the hippocampus and cerebral cortex, so that spatial learning and memory enhancement by MVG peptide increased the expression of its receptor (TrkB) as the hippocampus increased BDNF expression. It can be seen that the activation and the expression of the receptor is enhanced (Fig. 12a, 12b).

The present invention provides a pharmaceutical composition for the prevention and treatment of neurological diseases comprising a therapeutically effective amount of the MVG peptide of the present invention.

The present invention also provides a gene carrier comprising a polynucleotide consisting of a DNA sequence encoding the MVG peptide of the present invention, the gene carrier of the present invention can act as a pharmaceutical composition for the prevention and treatment of neurological diseases.

Examples of the neurological diseases include Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative disease or diabetic neuropathy, and the like. In general, the pharmaceutical composition is prepared in various forms by mixing a pharmaceutically acceptable non-toxic carrier that is commercially available according to the method of administration with the MVG peptide of the present invention, the prepared pharmaceutical composition is a neurological disease described above It can be administered as a prophylactic and therapeutic agent of.

In one embodiment of the present invention, to determine whether the MVG peptide of the present invention has an anti-parkinson effect, MVG peptide was treated with 0.001, 0.01, 0.1, 1 nM for 24 hours in SH-SY5Y cell line, a human dopaminergic cell line, and BDNF. Western blot expression of the anti-apoptotic protein, Bcl-2, was found to increase BDNF and Bcl-2 expression in the presence of 0.1 nM MVG peptide for 24h. In addition, MVG peptide was pretreated with 0.1 nM for 24 h in a SH-SY5Y cell line and treated with 2.5 mM MPP ⁺ for 24 h, and MVG peptide was measured by MTT assay 12 h and 24 h after MPP ⁺ treatment. By confirming that the cell viability was significantly high in the group pretreated, it was proved that the MVG peptide of the present invention can be usefully used as a composition for preventing and treating Parkinson's disease (FIG. 13).

The relationship between BDNF and Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative disease or diabetic neuropathy is fully disclosed in the documents listed below and the following references are incorporated herein by reference. Integrated, therefore, the use of MVG peptides to increase mRNA and protein expression of the brain-derived neurotrophic factor (BDNF) of the present invention as a pharmaceutical composition for the prevention and treatment of the disease is one technical idea in the present invention.

BDNF and Alzheimer's disease (AD)

(1) Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer s disease.

Alan H Nagahara, David A Merrill, Giovanni Coppola, Shingo Tsukada, Brock E Schroeder, Gideon M Shaked, Ling Wang, Armin Blesch, Albert Kim, James M Conner, Edward Rockenstein, Moses V Chao, Edward H Koo, Daniel Geschwind, Eliezer Masliah, Andrea A Chiba,

Mark H Tuszynski. NATURE MEDICINE LETTERS. 2009 Mar; 15 (3): 331-7.

(2) The expanding role of BDNF: a therapeutic target for Alzheimer's disease? Fumagalli, F., Racagni, G. and Riva, MA (2006) Pharmacogenomics J, 6, 8-15.

(3) New insights into brain BDNF function in normal aging and Alzheimer disease.

Tapia-Arancibia L, Aliaga E, Silhol M, Arancibia S. Brain Res Rev. 2008 Nov; 59 (1): 201-20. Epub 2008 Aug 3. Review.

(4) Protective effect of BDNF against beta-amyloid induced neurotoxicity in vitro and in vivo in rats.

Arancibia S, Silhol M, Mouli re F, Meffre J, H llinger I, Maurice T, Tapia-Arancibia L.

Neurobiol Dis. 2008 Sep; 31 (3): 316-26. Epub 2008 Jul 15.

(5) Association study of the decreased serum BDNF concentrations in amnestic mild cognitive impairment and the Val66Met polymorphism in Chinese Han.

Yu H, Zhang Z, Shi Y, Bai F, Xie C, Qian Y, Yuan Y, Deng L.

J Clin Psychiatry. 2008 Jul; 69 (7): 1104-11.

(6) Increase of BDNF serum concentration during donepezil treatment of patients with early Alzheimer's disease.

Leyhe T, Stransky E, Eschweiler GW, Buchkremer G, Laske C.

Eur Arch Psychiatry Clin Neurosci. 2008 Mar; 258 (2): 124-8. Epub 2007 Nov 7.

(7) [Protective effect of adeno-associated viral vector-mediated expression of human brain-derived neurotrophic factor in rat neurons against beta-amyloid-induced Alzheimer's disease in vitro]

Liu ZH, Ma DL, Jin H, Ma YB, Hu HT.

Nan Fang Yi Ke Da Xue Xue Bao. 2006 Oct; 26 (10): 1388-93. Chinese.

(8) The brain-derived neurotrophic factor gene as a possible susceptibility candidate for Alzheimer's disease in a chinese population.

Tsai SJ, Hong CJ, Liu HC, Liu TY, Liou YJ.

Dement Geriatr Cogn Disord. 2006; 21 (3): 139-43. Epub 2006 Jan 2.

(9) Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer's disease.

Peng S, Wuu J, Mufson EJ, Fahnestock M.

J Neurochem. 2005 Jun; 93 (6): 1412-21.

10 Decreased levels of BDNF protein in Alzheimer temporal cortex are independent of BDNF polymorphisms.

Lee J, Fukumoto H, Orne J, Klucken J, Raju S, Vanderburg CR, Irizarry MC, Hyman BT, Ingelsson M.

Exp Neurol. 2005 Jul; 194 (1): 91-6.

BDNF and Depression and anxiety

(1) New insights into BDNF function in depression and anxiety.

Keri Martinowich, Husseini Manji, Bai Lu. NATURE NEUROSCIENCE. VOLUME 10 NUMBER 9 SEPTEMBER 2007.

(2) Brain-derived neurotrophic factor, stress and depression: a minireview.

Yulu B, Ozan E, Gonul AS, Kilic E.

Brain Res Bull. 2009 Mar 30; 78 (6): 267-9. Epub 2008 Dec 26. Review.

(3) Brain-derived neurotrophic factor and antidepressant action: another piece of evidence from pharmacogenetics.

Tsai SJ, Hong CJ, Liou YJ.

Pharmacogenomics. 2008 Sep; 9 (9): 1353-8. Review.

(4) Acute hippocampal brain-derived neurotrophic factor restores motivational and forced swim performance after corticosterone.

Gourley SL, Kiraly DD, Howell JL, Olausson P, Taylor JR.

Biol Psychiatry. 2008 Nov 15; 64 (10): 884-90. Epub 2008 Aug 3.

(5) Targeting TrkB neurotrophin receptor to treat depression.

Rantam ki T, Castr n E.

Expert Opin Ther Targets. 2008 Jun; 12 (6): 705-15. Review.

BDNF and Parkinson's disease (PD)

(1) Shedding light into the role of BDNF in the pharmacotherapy of Parkinson's disease. Fumagalli, F., Racagni, G. and Riva, MA (2006) Pharmacogenomics J, 6, 95-104.

(2) Neurotrophic factors as a therapeutic target for Parkinson's disease.

Evans JR, Barker RA.

Expert Opin Ther Targets. 2008 Apr; 12 (4): 437-47. Review.

(3) Rasagiline promotes regeneration of substantia nigra dopaminergic neurons in post-MPTP-induced Parkinsonism via activation of tyrosine kinase receptor signaling pathway.

Mandel SA, Sagi Y, Amit T.

Neurochem Res. 2007 Oct; 32 (10): 1694-9. Epub 2007 Aug 16. Review.

(4) Neuroprotective effect of BDNF in young and aged 6-OHDA treated rat model of Parkinson disease.

Singh S, Ahmad R, Mathur D, Sagar RK, Krishana B.

Indian J Exp Biol. 2006 Sep; 44 (9): 699-704.

(5) Valproate protects dopaminergic neurons in midbrain neuron / glia cultures by stimulating the release of neurotrophic factors from astrocytes.

Chen PS, Peng GS, Li G, Yang S, Wu X, Wang CC, Wilson B, Lu RB, Gean PW, Chuang DM, Hong JS.

Mol Psychiatry. 2006 Dec; 11 (12): 1116-25. Epub 2006 Sep 12.

(6) Neurotrophic factors stabilize microtubules and protect against rotenone toxicity on dopaminergic neurons.

Jiang Q, Yan Z, Feng J.

J Biol Chem. 2006 Sep 29; 281 (39): 29391-400. Epub 2006 Aug 3.

(7) Repeated l-DOPA treatment increases c-fos and BDNF mRNAs in the subthalamic nucleus in the 6-OHDA rat model of Parkinson's disease.

Zhang X, Andren PE, Svenningsson P.

Brain Res. 2006 Jun 20; 1095 (1): 207-10. Epub 2006 May 24.

(8) Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death.

Onyango IG, Tuttle JB, Bennett JP Jr.

Neurobiol Dis. 2005 Oct; 20 (1): 141-54.

BDNF and Hungtington's disease

(1 ) Expression profiling of Huntington's disease models suggests that brain-derived neurotrophic factor depletion plays a major role in striatal degeneration.

Strand AD, Baquet ZC, Aragaki AK, et al (2007)J Neurosci                     27 (43): 11758 68

(2) Up-regulating BDNF with an ampakine rescues synaptic plasticity and memory in Huntington's disease knockin mice.

Simmons DA, Rex CS, Palmer L, Pandyarajan V, Fedulov V, Gall CM, Lynch G.

Proc Natl Acad Sci U S A. 2009 Mar 24; 106 (12): 4906-11. Epub 2009 Mar 5.

(3) Decreased BDNF levels are a major contributor to the embryonic phenotype of huntingtin knockdown zebrafish.

Diekmann H, Anichtchik O, Fleming A, Futter M, Goldsmith P, Roach A, Rubinsztein DC.

J Neurosci. 2009 Feb 4; 29 (5): 1343-9.

(4) Blood level of brain-derived neurotrophic factor mRNA is progressively reduced in rodent models of Huntington's disease: restoration by the neuroprotective compound CEP-1347.

Conforti P, Ramos C, Apostol BL, Simmons DA, Nguyen HP, Riess O, Thompson LM, Zuccato C, Cattaneo E.

Mol Cell Neurosci. 2008 Sep; 39 (1): 1-7. Epub 2008 May 10.

(5) Brain-derived neurotrophic factor over-expression in the forebrain ameliorates Huntington's disease phenotypes in mice.

Gharami K, Xie Y, An JJ, Tonegawa S, Xu B.

J Neurochem. 2008 Apr; 105 (2): 369-79. Epub 2007 Dec 12.

 Pharmaceutically acceptable carriers compatible with various formulations include all forms of diluents or solvents, fillers, fillers, binders, dispersants, disintegrants, surfactants, lubricants, excipients, and wetting agents. Furthermore, if necessary, commercially available dissolution aids, buffers, preservatives, colorants, flavors, sweeteners, and the like may also be added to the drug.

 The dosage unit form of the pharmaceutical composition according to the present invention is not particularly limited and may be widely selected according to various therapeutic purposes, for example, tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions. Oral dosages such as and the like, injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) and parenteral administration such as suppositories. Among these formulations, oral dosage formulations are preferred.

In addition, microinjection, electroporation using electroshock (electorporation), delivery using cations or liposomes, intracellular delivery methods of peptides known in the art, such as PTD (protein transduction domain) can be used.

 The various types of medicaments described above can be prepared by conventional methods, for example in the preparation of oral dosage forms such as tablets, capsules, granules and pills, they are known as sucrose, lactose, glucose, starch, mannitol Excipients; Binders such as syrup, gum arabic, sorbitol, trigacanth rubber, methylcellulose, polyvinylpyrrolidone and the like; Disintegrants such as starch, carboxymethyl cellulose and its calcium salts, microcrystalline cellulose, polyethylene glycol and the like; Lubricants such as talc, magnesium stearate, calcium stearate, silica and the like; It may be prepared by conventional methods using wetting agents such as sodium laurate, glycerol and the like.

 In the preparation of injectables, solutions, emulsions, suspensions and syrups, they are prepared by conventional methods, such as ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, castor oil and the like. Solvent for component dissolution; Surfactants such as sorbitol fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene ester, polyoxyethylene ether of hydrogenated castor oil, lecithin and the like; Manufactured using appropriate preservatives such as cellulose derivatives such as sodium carboxymethyl cellulose, methyl cellulose, suspending agents such as natural rubbers such as trigacanth rubber, gum arabic, esters of paraoxybenzoic acid, benzalkonium chloride, sorbitan fatty acid salts, etc. Can be.

 In preparing suppositories, they can be prepared using excipients such as polyethylene glycol, lanolin, coconut oil and the like using conventional methods.

 The dose of the pharmaceutical composition for the prevention and treatment of neurological diseases, including the therapeutically effective amount of the MVG peptide according to the present invention, depends on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease. It may be appropriately selected and is not specifically limited.

  The therapeutically effective amount of the MVG peptide included in the pharmaceutical composition of the present invention may be administered at 0.1 μg / kg / day to 10 μg / kg / day. Of course, the therapeutically effective amount may be in an amount outside the ranges described above.

 The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be envisaged, for example, by oral, subcutaneous, intravenous, intramuscular, nasal, intraperitoneal, rectal, intrauterine dural or intracerebroventricular injection. The MVG peptide of the present invention has little toxicity and side effects, and therefore can be used with confidence even for prolonged administration for prophylactic purposes.

In addition, the pharmaceutical composition of the present invention comprises a polynucleotide or equivalent nucleic acid sequence consisting of a DNA sequence encoding an MVG peptide, that is, a codon degeneracy sequence encoding a MVG peptide having a different sequence but the same DNA sequence. It may be a gene carrier, and the gene delivery method of the nucleotides is also included in the technical scope of the present invention. The oligonucleotides can be delivered intracellularly in a known form. For example, retroviral vectors, adenovirus vectors, and adeno-associated viral vectors that have been used as conventional gene carriers. Viral vector comprising a rule, liposomes, polylysine, polyethylenimine (PEI), protamine, histone, polyesteramine and their respective variants In addition to cationic polymers, non-viral vectors such as micelles, emulsions, nanoparticles, and the like can be used. In addition to the vector system, it can be efficiently delivered intracellularly using known intracellular substance transfer peptides.

 MVG peptide consisting of three amino acids according to the present invention not only increases the expression of BDNF without cytotoxicity in the hippocampal neuronal cell line but also increases the expression of BDNF in hippocampal and cortical tissues. Therefore, the small MVG peptides, such as the present invention, have a low molecular weight, easily cross the Blood Brain Barrier (BBB), and do not have cytotoxicity. Therefore, Alzheimer's disease, Parkinson's disease, depression, stroke, and Huntington's chorea It is very effective as a preventive and therapeutic agent for neurological diseases such as cerebral ischemic disease, neurodegenerative disease and diabetic neuropathy.

Figure 1 shows the results of finding the amino acid with the highest BDNF expression at each position of the peptide consisting of three amino acids (Methionine-Valine-Glycine) through the PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library) .

Figure 2 shows the results of mRNA expression of BDNF after treatment of MVG synthetic peptide of the present invention in rats (Hat), a hippocampal nerve cell line of rat (Rat) by concentration.

Figure 3 shows the results of protein expression of BDNF after treatment of the MVG synthetic peptide of the present invention to the rat hippocampal neuronal cell line H19-7 by concentration.

Figure 4 shows the results of BDNF protein expression by extracting the hippocampal tissue site after the rat intravenous injection of 0.1g / kg MVG synthetic peptide of the present invention.

Figure 5 shows the results of BDNF protein expression by extracting the cerebral cortical tissue site after the tail vein injection of 0.1V / kg MVG synthetic peptide of the present invention.

Figure 6 shows the cytotoxicity results of MVG synthetic peptides by MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test.

FIG. 7 illustrates a method of performing a Y-maze test, and FIG. 8 illustrates the number of times of entering the novel arm and the moving distance from the novel arm.

9 illustrates a method of performing a Morris water maze test.

Figure 10 shows the time spent on finding the platform and the test stayed on the platform.

FIG. 11 shows BDNF expression in the hippocampus (FIG. 11 (a)) and the cerebral cortex (FIG. 11 (b)) of mice that underwent the Y-maze test and Morris water maze test.

FIG. 12 shows phosphorylation and TrkB expression of TrkB in hippocampus (FIG. 12 (a)) and cortex (FIG. 12 (b)) of mice that underwent Y-maze test and Morris water maze test. It is.

FIG. 13 shows the results of in vitro experiments confirming the anti-parkinson effect of MVG peptides that regulate BDNF expression.

Example 1 Initial Screening with PS-SPCL

 PS-SPCL trimmer package stock was supported by the Peptide library support facility at Pohang University of Science and Technology for the discovery of peptides that increase BDNF expression. PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library), a peptide library technology used in the present invention, is a peptide consisting of three amino acids, the first position of which is substituted with tyrosine in alanine except cysteine. And the remaining second and third positions are fixed in such a way as to identify the most effective amino acid sequence at each position, thereby finding the amino acid with the highest BNDF expression at each position.

First, 57 mouse hippocampal neuronal cell lines, HT22 cells, were placed in a 24-well plate (SPL) at a total of 57 by ⁵ × 10 ⁴ per well for 12 hours at 37 ° C. in a 5% CO ₂ incubator (Incubator, VISION). Incubated in the. After spin down of the PS-SPCL trimmer package stock, the tube was opened, tapped with 15 μl of distilled water (DW) for cell culture, and then spun down again. 15 μl of PS-SPCL trimmer package stock prepared on a 24-well plate in which HT22 cells are incubated is placed in each well and incubated in an incubator for 12 hours. After 12 hours, the medium in the well was discarded and washed twice with 1 ml of 1X PBS (Potassium persulfate). 10 μl of protease inhibitor (Amersham) per 1 mL of RIPA was prepared, placed in each well, and the cell contents were placed in a 1.5 mL tube using a cell lifter (Cell Lifter, Corning). Pelletize the cell membrane by centrifugation (Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) for 5 minutes at 4 ° C., 9000 rpm, and transfer the supernatant containing protein to a new 1.5 ml tube. The concentration of was quantified using the Bradford assay.

 In order to measure the amount of BDNF expression, ELISA (Enzyme-linked immunosorbent assay, BDNF ELISA Kit, Promega) was performed according to the protocol. As shown in FIG. 1, M (Methionine) in the first position, V (Valine) in the second position, and G (Glycine) in the third position showed the highest BDNF expression.

Example 2: Increase of BDNF mRNA Expression by MVG Peptide in Hippocampal Neuronal Cell Line H19-7

As a result of Example 1, an MVG synthetic peptide consisting of three amino acids was purchased from Peptron Co., Ltd. H19-7 cells, the hippocampal neuronal cell lines of rats purchased from the American Type Culture Collection (ATCC), were placed in 6 well plates (NUNC) at 10 ⁶ per well and incubated in a 34 ° C., 5% CO ₂ incubator for 12 hours. , MVG synthetic peptides were treated with 0.001, 0.01, 0.1, 1μM and further incubated for 12 hours. RNA was isolated from each sample according to the protocol of the Easy-blue Total RNA extraction kit (Intron), and then 1 ug total RNA, 1 ol oligo (dT) ₁₈ , and the rest were processed using DW treated with DEPC (Diethylpyrocarbonate). The volume was added to the tube for PCR to 20 mu l and heated at 70 ℃ for 5 minutes. Complementary DNA (cDNA) was prepared by transferring 20 μl of the tube heated with RT premix (Reverse transcriptase premix, Bioneer) tube and performing reverse transcription at 42 ° C. for 1 hour. 5 μl of the prepared cDNA, 2 μl of BDNF primer and 13 μl of DDW (Double Distilled Water) PCR were placed in a PCR premix (Bioneer) tube for 5 minutes at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 60 ° C., 45 at 72 ° C. The cycle was turned 5 minutes and 29 cycles at 72 ° C. As a result, as shown in Figure 2 it can be seen that the amount of BDNF mRNA expression increases by concentration at 0.001, 0.01, 0.1μM. This demonstrates that MVG synthetic peptides increase BDNF mRNA expression in hippocampal neuronal cell lines of rats, and that peptide selection through Example 1 was achieved.

Example 3: Increase of BDNF Protein Expression by MVG Peptide in Hippocampal Neuronal Cell Line H19-7

As a result of Example 1, an MVG synthetic peptide consisting of three amino acids was purchased from Peptron Co., Ltd. H19-7 cells, rat hippocampal neuronal cell lines purchased from the American Type Culture Collection (ATCC), were placed in a 6 well plate (NUNC) at 10 ⁶ per well and incubated in a 34 ° C., 5% CO ₂ incubator for 12 hours. , MVG synthetic peptides were treated with 0.001, 0.01, 0.1, 1μM and further incubated for 12 hours. Obtain protein in the same manner as in Example 1, make 15% acrylamide gel through 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and load 30 μg sample 2 hours 30 minutes at 100V with an electrophoresis device (Electrophoresis Power supply, EPS 601, Amersham). Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in Figure 3 it can be seen that the amount of BDNF protein expression increased by concentration at 0.001, 0.01, 0.1μM. In addition, this shows the same increase pattern as the BDNF mRNA expression results shown in FIG.

Example 4 Increasing BDNF Protein Expression by MVG Peptides in Hippocampus

 Four-week-old male SD (Sprague-Dawley) rats were purchased from Orient Co., Ltd. and separated into three groups of experimental groups (MVG-administered group) and control group (1X PBS-administered group). Adaptation and stabilization were made. After one week, the experimental group was tail vein injection of 0.1 μg / kg of MVG and the control group of 0.1 μg / kg of 1 × PBS. After 12 hours, anesthesia was temporarily used in ether, and blood was collected from the heart after laparotomy. The brains of the rats were quickly extracted, rapidly cooled in liquid nitrogen, and stored at -80 ° C. After separating only the hippocampus from the brain, the RIPA buffer and protease inhibitor were prepared in the same ratio as in Example 1, and the hippocampal tissue was completely ground using a Homogenizer (RZR2020M, Heidolph). Centrifuge at 4 ° C and 2,000rpm for 30 minutes (Centrifuge, UNION 32R, Hanil Science Industrial Co., Ltd), transfer only the supernatant to 1.5ml tube, and then centrifuge for 30 minutes at 4 ° C, 9,000rpm ( Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) was used to quantify the protein by Bradford assay once again transfer only the supernatant to 1.5ml tube.

A 15% acrylamide gel was made through a 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and 30 μg of sample was loaded to obtain an electrophoresis power supply. EPS 601, Amersham) for 2 hours 30 minutes at 100V. Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in FIG. 4, the amount of BDNF and its precursor (precursor) form proBDNF in the experimental group compared to the control group was confirmed to increase compared to β-actin.

Example 5: Increased BDNF Protein Expression by MVG Peptides in Cerebral Cortical Tissues

 Four-week-old male SD (Sprague-Dawley) rats were purchased from Orient Co., Ltd. and separated into three groups of experimental groups (MVG-administered group) and control group (1X PBS-administered group). Adaptation and stabilization were made. After one week, the experimental group was tail vein injection of 0.1 μg / kg of MVG and the control group of 0.1 μg / kg of 1 × PBS. After 12 hours, anesthesia was temporarily used in ether, and blood was collected from the heart after laparotomy. The brains of the rats were quickly extracted, rapidly cooled in liquid nitrogen, and stored at -80 ° C. After only the cerebral cortex was separated from the extracted brain, RIPA buffer and protease inhibitor were prepared in the same ratio as in Example 1, and the cortical tissue was completely ground using a Homogenizer (RZR2020M, Heidolph). Centrifuge at 4 ° C and 2,000rpm for 30 minutes (Centrifuge, UNION 32R, Hanil Science Industrial Co., Ltd), transfer only the supernatant to 1.5ml tube, and then centrifuge for 30 minutes at 4 ° C, 9,000rpm ( Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) was used to quantify the protein by Bradford assay once again transfer only the supernatant to 1.5ml tube.

A 15% acrylamide gel was made through a 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and 30 μg of sample was loaded to obtain an electrophoresis power supply. EPS 601, Amersham) for 2 hours 30 minutes at 100V. Polyvinylidene Difluoride (PVDF) was used to transfer gels (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in FIG. 5, the amount of BDNF and its precursor (precursor) form proBDNF in the experimental group was higher than that of the control group, compared to β-actin.

Example 6: Determination of Cytotoxic Effect of MVG Peptides

H19-7 cells were incubated in 96 well plates (FALCON) at 2 × ¹⁰ 3/200 μl and incubated in 34 ° C., 5% CO ₂ incubator for 12 hours, and then treated with MVG synthetic peptide from 10 nM to 1 mM and 3 days Incubated for After 3 days, 20 μl of MTT (50 mg / ml) solution was treated, wrapped in aluminum foil, and incubated again in an incubator. After 4 hours, the medium and the MTT solution were removed, treated with 200 μl of DMSO (Dimetyl sulfoxide) solution, wrapped in foil, and placed at room temperature for 4 hours. The absorbance was measured at 570 nm using a microplate reader (Molecular device) to convert the control to 100% to calculate the value. As a result, as shown in Figure 6 it did not show cytotoxicity at a concentration from 10nM to 1mM compared to the control. Therefore, MVG synthetic peptide according to the present invention increases the protein expression of BDNF in hippocampal neuronal cell line, hippocampal tissue and cerebral cortical tissue, and because there is no cytotoxicity, Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, It can be effectively used as a pharmaceutical composition for the prevention and treatment of cerebral ischemic diseases, neurodegenerative diseases, diabetic neuropathy and the like.

Example 7 Measurement of Learning and Memory Enhancing Effects of MVG Peptides

7-1. Y-maze TEST

The Y-maze test, one of the spatial memory tests, was performed to see if MVG peptides enhance spatial learning and memory at the experimental animal level. Y-maze TEST (FIG. 7A) shows that the first arm blocked the novel arm and started the rat for 15 minutes to explore the other arm, and then moved to the novel arm for 5 minutes and moved on the novel arm in the second trial. This was done by examining the distance to see if memory was improved.

Four weeks old male rats were purchased from Orient Co., Ltd. and stabilized for one week. After injection of 0.1 μg / kg of MVG peptide through the tail vein, the first trial was performed 6 hours later, and the second trial was passed 6 hours later. It was. (FIG. 7B)

As a result of comparing the number of entry into the novel arm from the second trial and the distance from the novel arm to the control group, the MVG peptide-injected group entered the novel arm more significantly and the movement distance increased more significantly. Groups injected with peptides show enhanced activity in the learning and memory portions (FIG. 8)

7-2. Morris Underwater Maze TEST

The Morris water maze test (Fig. 9a), which measures spatial learning and memory, was performed. The mice were dropped in a tank containing water, and the visual cues installed outside the tank were recognized and the location of the platform was known. We measured spatial learning and memory by measuring how quickly we arrived at the platform.

Four weeks old male rats were purchased from Orient Co., Ltd., and then stabilized for one week, followed by a four-day Morris underwater maze test. Four tests were performed 6 hours after the injection of 0.1 μg / kg MVG peptide through the tail vein, and after 48 hours of Morris water maze testing, the platform was removed and the time spent on the platform was measured 48 hours later. It was. (FIG. 9B)

The experimental results showed that there was no significant difference between the two groups in the trial of four trials on the first day of the Morris Maze test, but in the first, third and fourth trials on the second day, the group injected with MVG peptide was significantly faster than the control group. In the first trial on the third day, the MVG group was found to be significantly faster than the control group. In addition, after 48 hours after the last trial on the third day, a probe test (measuring how long the platform was present) showed that the group injected with the MVG peptide was significantly longer in the target quadrant where the platform was compared to the control group. The time remaining in the quadrant other than where the platform resided was significantly reduced in the group injected with the MVG peptide.

 Thus, animal experiments confirmed that MVG peptides enhance spatial learning and memory. (Figure 10)

7-3. Increase of BDNF Protein Expression by MVG Peptide in Hippocampus and Cerebral Cortex of Rats Performing Y-maze and Morris Underwater Maze TEST

  The hippocampus and cerebral cortex of mice subjected to the Y-maze and Morris underwater maze test were extracted, and BDNF expression was compared using Western blotting. Experimental methods were the same as in Examples 4 and 5. As a result, it was confirmed that the expression of BDNF increased in the group injected with the MVG peptide compared to the control group, therefore, the learning and memory enhancement confirmed in the Y-maze and Morris underwater maze TEST was found in the hippocampus (Fig. 11a) and the cerebral cortex (Fig. It can be seen that the increase in BDNF expression by the MVG peptide in 11b).

Example 8: Determination of TrkB Expression Enhancement Effect of MVG Peptides

In order to compare the phosphorylation of TrkB and TrkB expression of BDNF receptor by MVG peptide, only the hippocampus and cerebral cortex sections were isolated from the brains of the Y-maze and Morris water maze TEST mice of Example 7, respectively. RIPA buffer and protease inhibitors were prepared in the same ratio as in Example 1, and only the hippocampus and the cerebral cortex portion were completely ground using a Homogenizer (RZR2020M, Heidolph). Centrifuge at 4 ° C and 2,000rpm for 30 minutes (Centrifuge, UNION 32R, Hanil Science Industrial Co., Ltd), transfer only the supernatant to 1.5ml tube, and then centrifuge for 30 minutes at 4 ° C, 9,000rpm ( Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) was used to quantify the protein by Bradford assay once again transfer only the supernatant to 1.5ml tube.

A 15% acrylamide gel was made through a 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and 30 μg of sample was loaded to obtain an electrophoresis power supply. EPS 601, Amersham) for 2 hours 30 minutes at 100V. Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and rabbit anti TrkB monoclonal antibody (Cell signaling) at a ratio of 1: 1000 Reacted overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, it was confirmed that TrkB phosphorylation and TrkB expression were increased in the group injected with MVG peptide in the hippocampus and cerebral cortex (FIG. 12A and FIG. 12B, respectively) and enhance spatial learning and memory by MVG peptide. The increase in BDNF expression in the hippocampus may be attributed to the activation of the receptor and enhanced expression of the receptor.

Example 9: Determination of anti-parkinson effect of MVG peptide

In vitro experiments were conducted to determine the anti-parkinson effect of MVG peptides that regulate BDNF expression. MVG peptides were treated in SH-SY5Y cell line, a human dopaminergic cell line, at concentrations of 0.001, 0.01, 0.1, and 1 nM for 24 hours, and the expression of BDNF, an anti-apoptotic protein, Bcl-2, was confirmed by Western blotting. Obtain protein in the same manner as in Example 1, make 15% acrylamide gel through 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and load 20 μg sample ) Was run for 2 hours and 30 minutes at 100V with an electrophoresis device (Electrophoresis Power supply, EPS 601, Amersham). Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD), rabbit anti BDNF polyclonal antibody (AB1534, chemicon), mouse anti Bcl-2 Monoclonal antibody (Mouse anti Bcl-2 antibody, sc-7382, Santa Cruz) was reacted overnight at a ratio of 1: 1000. Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents, after washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times 20 min) Goat anti-mouse IgG: Horseradish Peroxidase Conjugate, sc-2005, Santa Cruz was incubated at a ratio of 1: 3000, 1: 1000 for 50 minutes. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore).

As a result, it was confirmed that the expression of BDNF and Bcl-2 was increased compared to the control group in the state treated with 0.1 nM MVG peptide for 24 h (FIG. 13A).

In addition, in order to confirm the anti-parkinson effect of the BDNF regulatory peptide using MPP ⁺ , 2.5 mM MPP ⁺ (D048, Sigma) was pretreated after 0.1 h MMGG peptide in SH-SY5Y cell line for 24 h. Cell viability was measured by MTT assay in the same manner as in Example 6 after 12 hours and 24 hours after MPP ⁺ treatment. After 12 hours and 24 hours of MPP ⁺ treatment, 20 μl of MTT (50 mg / ml) solution was treated, wrapped in aluminum foil, and incubated again in an incubator. After 4 hours, the medium and the MTT solution were removed, treated with 200 μl of DMSO (Dimetyl sulfoxide) solution, wrapped in foil, and placed at room temperature for 4 hours. The absorbance was measured at 570 nm using a microplate reader (Molecular device) to convert the control to 100% to calculate the value. As a result, in the group pretreated with peptide, cell viability of 9.39% and 25.6% increased after 24 hours compared to the group treated with MPP ⁺ only after 12 hours.

By confirming that the cell viability is significantly high in the group pretreated with the MVG peptide (FIG. 13B), it can be seen that the MVG peptide according to the present invention has an anti-parkinson effect, and thus may function as a preventive and therapeutic agent for Parkinson's disease.

While the invention has been described above with reference to specific embodiments, various modifications, changes or modifications are possible in the art within the spirit and scope of the appended claims, and thus the foregoing description and drawings illustrate the invention. It should not be construed as limiting the technical spirit of the present invention but as illustrating the present invention. All documents cited in the present invention are incorporated herein by reference.

Claims (11)

1. Peptides consisting of the amino acids set forth in SEQ ID NO: 1
2. The peptide of claim 1, wherein the peptide increases mRNA expression or protein expression of a brain-derived neurotrophic factor (BDNF).
3.  The peptide according to claim 2, wherein the peptide increases the mRNA expression or protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissue and cerebral cortex.
4.  The peptide according to claim 2 or 3, wherein the hippocampal neurons are HT22 cell lines of mice or H19-7 cell lines of rats.
5.   The method of claim 1, wherein the peptide is TrkB Peptides characterized by increasing protein expression.
6.  A polynucleotide consisting of a DNA sequence encoding the peptide of claim 1.
7.  Gene delivery system comprising the polynucleotide of claim 6.
8.  Prevention of Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative disease, or diabetic neuropathy comprising a therapeutically effective amount of the peptide of claim 1 or the gene carrier of claim 7 And therapeutic pharmaceutical compositions.
9.  The pharmaceutical composition of claim 8, wherein the therapeutically effective amount of the peptide is 0.1 μg / kg / day to 10 μg / kg / day.
10.  The pharmaceutical composition of claim 8, which is administered orally, subcutaneously, intravenously, intramuscularly, intranasally, intraperitoneally, rectally, intrauterinely or intraventricularly.
11. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is prepared in the form of tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions, injections or suppositories.

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