Pharmaceutical composition and cosmetic composition

Abstract

To provide pharmaceutical compositions and cosmetic compositions that effectively utilize MANF, APLN, GDF11, TIMP2, THBS4, and / or CXCL4.SOLUTION: Provided is a lipolytic agent, a promoter for inducing differentiation from myoblasts to myotubes, a prostacyclin production enhancer, a promoter for hyaluronic acid production by chondrocytes, an anti-aging agent, a therapeutic or prophylactic agent for lifestyle-related diseases, or a therapeutic or prophylactic agent for adult diseases, comprising MANF. An amyloid β inhibitor, a therapeutic or prophylactic agent for neurological diseases, or a therapeutic or prophylactic agent for dementia is provided, comprising MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to pharmaceutical compositions and cosmetic compositions.

Background Art

[0002] Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein identified from the culture medium of rat mesencephalic type 1 astrocyte cells (see, for example, Non-Patent Document 1). The MANF protein is conserved among other vertebrates and invertebrates. For example, the human MANF protein has about 98% amino acid sequence homology with the mouse MANF protein, and it has been reported that the MANF proteins of Drosophila and C. elegans have about 50% amino acid sequence homology (see, for example, Non-Patent Documents 2 and 3).

[0003] It is known that the expression level of MANF decreases with aging, and among proteins other than neurotrophic factors, there are also those whose expression levels decrease with aging. For example, Growth differentiation factor 11 (GDF11), Tissue inhibitor of metalloproteinases 2 (TIMP2), Thrombospondin 4 (THBS4), platelet factor 4 (PF4; chemokine C-X-C motif ligand 4 (CXCL4)), Apelin (APLN), etc. also have decreasing expression levels with aging (see, for example, Non-Patent Documents 4, 5, 6, and 7). Therefore, the relationship with aging and anti-aging effects have been suggested for multiple factors such as MANF and these proteins (see, for example, Non-Patent Documents 4, 5, 6, and 7).

Prior Art Documents

Non-Patent Documents

[0004] [Non-Patent Document 1] Petrova, et al., MANF:a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons, Journal of Molecular Neuroscience, 20:173-187 (2003). [Non-Patent Document 2] Lindholm et al., Novel CDNF / MANF family of neurotrophic factors., Dev. Neurobiol., 70、361-371(2010). [Non-Patent Document 3] Lindholm et al.,MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain, Molecular and Cellular Neuroscience, Vol. 39, Issue 3, 356-371 (2008). [Non-Patent Document 4] Hsiao et al., Role of circulating molecules in age-related cardiovascular and metabolic disorders, Inflammation and Regeneration, Vol. 42, Issue 2 (2022). [Non-Patent Document 5] Kang et al., Circulating plasma factors involved in rejuvenation, Aging, Vol. 12, No. 22,23394-23408(2020). [Non-Patent Document 6] Schroer et al., Platelet factors attenuate inflammation and rescue cognition in ageing, Nature, Vol. 620, 1071-1079 (2023).

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0005] One object of the present invention is to provide a pharmaceutical composition and a cosmetic composition that effectively utilize MANF, APLN, GDF11, TIMP2, THBS4, and / or CXCL4.

Means for Solving the Problems

[0006] According to an aspect of the present invention, there is provided a lipolytic agent, an anti-obesity agent, an obesity-improving agent, a lipolysis promoter, a diet agent, and / or a therapeutic or prophylactic agent for diabetes, which contains MANF.

[0007] According to an aspect of the present invention, there is provided an agent for promoting differentiation of myoblasts into myotube cells, a muscle formation promoter, an agent for maintaining muscle mass, and / or an agent for increasing muscle mass, which contains MANF.

[0008] According to an aspect of the present invention, there is provided a prostacyclin production promoter, an adenylate cyclase activator, a cyclic adenosine monophosphate production promoter, a platelet aggregation inhibitor, a vasodilator, a therapeutic or prophylactic agent for hypertension, a therapeutic or prophylactic agent for arteriosclerosis, and / or an inhibitor of vascular endothelial cell proliferation, which contains MANF.

[0009] According to an aspect of the present invention, there is provided an agent for promoting hyaluronic acid production by chondrocytes and / or a therapeutic or prophylactic agent for joint diseases, which contains MANF.

[0010] According to an aspect of the present invention, an anti-aging agent containing MANF is provided.

[0011] According to an aspect of the present invention, a medicine for treating or preventing lifestyle-related diseases containing MANF is provided.

[0012] According to an aspect of the present invention, a medicine for treating or preventing adult diseases containing MANF is provided.

[0013] According to an aspect of the present invention, the use of MANF in the manufacture of a lipolytic agent, an anti-obesity agent, an obesity-improving agent, a lipolysis promoter, a diet agent, and / or a medicine for treating or preventing diabetes is provided.

[0014] According to an aspect of the present invention, the use of MANF in the manufacture of an agent for promoting differentiation induction from myoblasts to myotube cells, an agent for promoting muscle formation, an agent for maintaining muscle mass, and / or an agent for increasing muscle mass is provided.

[0015] According to an aspect of the present invention, the use of MANF in the manufacture of a prostacyclin production promoter, an adenylate cyclase activator, a cyclic adenosine monophosphate production promoter, a platelet aggregation inhibitor, a vasodilator, a medicine for treating or preventing hypertension, a medicine for treating or preventing arteriosclerosis, and / or a vascular endothelial cell growth inhibitor is provided.

[0016] According to an aspect of the present invention, the use of MANF in the manufacture of an agent for promoting hyaluronic acid production by chondrocytes and / or a medicine for treating or preventing joint diseases is provided.

[0017] According to an aspect of the present invention, the use of MANF in the manufacture of an anti-aging agent is provided.

[0018] According to an aspect of the present invention, the use of MANF in the manufacture of a medicine for treating or preventing lifestyle-related diseases is provided.

[0019] According to an aspect of the present invention, there is provided the use of MANF in the manufacture of a medicament for treating or preventing adult diseases.

[0020] According to an aspect of the present invention, there is provided the use of MANF for lipolysis treatment, anti-obesity treatment, obesity improvement treatment, lipolysis promotion treatment, diet treatment and / or treatment or prevention of diabetes.

[0021] According to an aspect of the present invention, there is provided the use of MANF for promoting the induction of differentiation from myoblasts to myotube cells, promoting muscle formation, maintaining muscle mass and / or increasing muscle mass.

[0022] According to an aspect of the present invention, there is provided the use of MANF for promoting prostacyclin production treatment, adenylate cyclase activity treatment, promoting cyclic adenosine monophosphate production treatment, platelet aggregation inhibition treatment, vasodilation treatment, treatment or prevention of hypertension, treatment or prevention of arteriosclerosis and / or treatment for inhibiting vascular endothelial cell proliferation.

[0023] According to an aspect of the present invention, there is provided the use of MANF for promoting hyaluronic acid production in chondrocytes and / or treating or preventing joint diseases.

[0024] According to an aspect of the present invention, there is provided the use of MANF for anti-aging treatment.

[0025] According to an aspect of the present invention, there is provided the use of MANF for treating or preventing lifestyle-related diseases.

[0026] According to an aspect of the present invention, there is provided the use of MANF for treating or preventing adult diseases.

[0027] According to an aspect of the present invention, there is provided a lipolysis treatment method, an obesity treatment method, an obesity improvement treatment method, a lipolysis promotion treatment method, a diet treatment method and / or a treatment or prevention method for diabetes, which comprises administering a composition containing MANF to a subject. The subject is a human, a non-human animal or their cells.

[0028] According to an aspect of the present invention, there is provided a method for promoting the differentiation of myoblasts into myotube cells, a method for promoting muscle formation, a method for maintaining muscle mass, and / or a method for increasing muscle mass, which comprises administering a composition containing MANF to a subject.

[0029] According to an aspect of the present invention, there is provided a method for promoting prostacyclin production, a method for activating adenylate cyclase, a method for promoting the production of cyclic adenosine monophosphate, a method for inhibiting platelet aggregation, a method for vasodilation, a method for treating or preventing hypertension, a method for treating or preventing arteriosclerosis, and / or a method for inhibiting the proliferation of vascular endothelial cells, which comprises administering a composition containing MANF to a subject.

[0030] According to an aspect of the present invention, there is provided a method for promoting the production of hyaluronic acid by chondrocytes and / or a method for treating or preventing joint diseases, which comprises administering a composition containing MANF to a subject.

[0031] According to an aspect of the present invention, there is provided an anti-aging treatment method, which comprises administering a composition containing MANF to a subject.

[0032] According to an aspect of the present invention, there is provided a method for treating lifestyle-related diseases, which comprises administering a composition containing MANF to a subject.

[0033] According to an aspect of the present invention, there is provided a method for treating or preventing adult diseases, which comprises administering a composition containing MANF to a subject.

[0034] According to an aspect of the present invention, there is provided an inhibitor for the accumulation, aggregation, or mislocalization of proteins, preferably an amyloid-β inhibitor, which contains MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

[0035] According to an aspect of the present invention, there is provided an inhibitor for suppressing the cell death of nerve cells, which contains MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

[0036] According to an aspect of the present invention, there is provided a therapeutic or prophylactic agent for a neurological disease, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. The neurological disease of this aspect is a neurological disease associated with the accumulation, aggregation, or mislocalization of a protein such as amyloid β, preferably a neurological disease caused by the accumulation or aggregation of amyloid β. The neurological disease of this aspect may be a neurofibrillary tangle (NFT).

[0037] According to an aspect of the present invention, there is provided a therapeutic or prophylactic agent for a disease caused by the accumulation, aggregation, or mislocalization of a protein, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. The protein of this aspect includes, for example, amyloid β, and the disease of this aspect includes, for example, amyloidosis.

[0038] According to an aspect of the present invention, there is provided a therapeutic or prophylactic agent for dementia, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. The dementia of this aspect may be Alzheimer's disease and / or Lewy body dementia, or may be dementia in diabetic patients and / or dementia in Down syndrome patients.

[0039] According to an aspect of the present invention, there is provided a therapeutic or prophylactic agent for a disease caused by nerve cell death, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. The disease of this aspect may be Alzheimer's disease, Huntington's disease, Parkinson's disease, and / or amyotrophic lateral sclerosis (ALS).

[0040] According to an aspect of the present invention, there is provided a therapeutic or prophylactic agent for a decrease in the number of nerve cells or nerve cell death, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

[0041] According to an aspect of the present invention, there is provided a rejuvenating or anti-aging agent for nerve cells, which comprises MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

[0042] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of an inhibitor of protein accumulation, aggregation, or mislocalization, preferably an amyloid-β inhibitor.

[0043] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a neuronal cell death inhibitor.

[0044] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a therapeutic or prophylactic agent for a neurological disease. The neurological disease of this aspect is a neurological disease associated with protein accumulation, aggregation, or mislocalization such as amyloid-β, preferably a neurological disease caused by amyloid-β accumulation or aggregation. The neurological disease of this aspect may be neurofibrillary tangles (NFT).

[0045] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a therapeutic or prophylactic agent for a disease caused by protein accumulation, aggregation, or mislocalization. The protein of this aspect includes, for example, amyloid-β, and the disease of this aspect includes, for example, amyloidosis.

[0046] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a therapeutic or prophylactic agent for dementia. The dementia of this aspect may be Alzheimer's disease and / or Lewy body dementia, or may be dementia in diabetic patients and / or dementia in Down syndrome patients.

[0047] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a medicament for treating or preventing a disease caused by neuronal cell death. The disease of this aspect may be Alzheimer's disease, Huntington's disease, Parkinson's disease and / or amyotrophic lateral sclerosis (ALS).

[0048] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a medicament for treating or preventing a decrease in the number of neuronal cells or neuronal cell death.

[0049] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a medicament for rejuvenating neuronal cells or preventing neuronal cell aging.

[0050] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for inhibiting protein accumulation, aggregation, or mislocalization, preferably for inhibiting amyloid-β.

[0051] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for inhibiting neuronal cell death.

[0052] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for treating or preventing a neurological disease. The neurological disease of this aspect is a neurological disease accompanied by protein accumulation, aggregation, or mislocalization such as amyloid-β, preferably a neurological disease caused by amyloid-β accumulation or aggregation. The neurological disease of this aspect may be neurofibrillary tangles (NFT).

[0053] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for the treatment or prevention of diseases caused by protein accumulation, aggregation, or mislocalization. The protein of this aspect includes, for example, amyloid β, and the disease of this aspect includes, for example, amyloidosis.

[0054] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for the treatment or prevention of dementia. The dementia of this aspect may include Alzheimer's disease and / or Lewy body dementia, and may also be dementia in diabetic patients and / or dementia in Down syndrome patients.

[0055] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for the treatment or prevention of diseases caused by nerve cell death. The disease of this aspect may be Alzheimer's disease, Huntington's disease, Parkinson's disease and / or amyotrophic lateral sclerosis (ALS).

[0056] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for the treatment or prevention of nerve cell number reduction or nerve cell death.

[0057] According to an aspect of the present invention, there is provided the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 for the rejuvenation or prevention of aging of nerve cells.

[0058] According to an aspect of the present invention, there is provided a method for inhibiting protein accumulation, aggregation, or mislocalization, preferably a method for inhibiting amyloid β, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 to a subject.

[0059] According to an aspect of the present invention, there is provided a method for suppressing cell death of nerve cells, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject.

[0060] According to an aspect of the present invention, there is provided a method for treating or preventing a neurological disease, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject. The neurological disease in this aspect is a neurological disease associated with the accumulation, aggregation, or mislocalization of proteins such as amyloid-β, preferably a neurological disease caused by the accumulation or aggregation of amyloid-β. The neurological disease in this aspect may be neurofibrillary tangles (NFT).

[0061] According to an aspect of the present invention, there is provided a method for treating or preventing a disease caused by the accumulation, aggregation, or mislocalization of a protein, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject. The protein in this aspect includes, for example, amyloid-β, and the disease in this aspect includes, for example, amyloidosis.

[0062] According to an aspect of the present invention, there is provided a method for treating or preventing dementia, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject. The dementia in this aspect may be Alzheimer's disease and / or Lewy body dementia, or may be dementia in diabetic patients and / or dementia in Down syndrome patients.

[0063] According to an aspect of the present invention, there is provided a method for treating or preventing a disease caused by nerve cell death, which includes administering a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject. The disease in this aspect may be Alzheimer's disease, Huntington's disease, Parkinson's disease, and / or amyotrophic lateral sclerosis (ALS).

[0064] According to an aspect of the present invention, there is provided a method for treating or preventing a decrease in the number of nerve cells or nerve cell death, which includes administering to a subject a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

[0065] According to an aspect of the present invention, there is provided a method for rejuvenating nerve cells or preventing nerve cell aging, which includes administering to a subject a composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2.

Advantages of the Invention

[0066] According to the present invention, it is possible to provide pharmaceutical compositions and cosmetic compositions that effectively utilize MANF, APLN, GDF11, TIMP2, THBS4, and / or CXCL4.

Brief Description of the Drawings

[0067]

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Mode for Carrying Out the Invention

[0068] Hereinafter, embodiments of the present invention will be described in detail. The embodiments shown below illustrate devices and methods for embodying the technical idea of this invention, and the technical idea of this invention does not specify combinations of constituent members, etc. as the following. Various changes can be made to the technical idea of this invention within the scope of the claims.

[0069] (First Embodiment) The pharmaceutical composition and / or cosmetic composition according to the first embodiment contains MANF.

[0070] The MANF contained in the pharmaceutical composition and / or cosmetic composition according to the first embodiment is, for example, an isolated protein or a recombinant protein, preferably a recombinant human MANF protein. The MANF protein may have, for example, the amino acid sequence of SEQ ID NO:1, or may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:1. The MANF protein may have the amino acid sequence from position 25 to position 182 (SEQ ID NO:2) in the amino acid sequence of SEQ ID NO:1, or may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:2.

[0071] The pharmaceutical composition and / or cosmetic composition containing MANF according to the first embodiment is, for example, a lipolytic agent, an anti-obesity drug, an obesity-improving drug, a lipolysis promoter, a diet drug, a therapeutic or preventive drug for diabetes, a promoter for inducing differentiation from myoblasts to myotube cells, a muscle formation promoter, a muscle mass maintenance agent, a muscle mass increasing agent, a prostacyclin production promoter, an adenylate cyclase activator, a cyclic adenosine monophosphate (cAMP) production promoter, a platelet aggregation inhibitor, a vasodilator, a therapeutic or preventive drug for hypertension, a therapeutic or preventive drug for arteriosclerosis, a vascular endothelial cell growth inhibitor, a promoter for promoting hyaluronic acid production in chondrocytes, a therapeutic or preventive drug for joint diseases, an anti-aging agent, a therapeutic or preventive drug for lifestyle-related diseases, and / or a therapeutic or preventive drug for adult diseases.

[0072] The inventor has first discovered that MANF decomposes fat. Therefore, MANF can be used as a lipolytic agent, an anti-obesity drug, an obesity-improving drug, a lipolysis promoter, and a diet drug. Also, in diabetes, it is known that the fat in the blood increases. Therefore, MANF can be used as a therapeutic or prophylactic drug for diabetes. The fat is, for example, neutral fat. The diabetes may be any of type 1 diabetes, type 2 diabetes, diabetes due to other specific mechanisms or diseases, and gestational diabetes.

[0073] The inventor has first found that MANF promotes the induction of differentiation from myoblasts to myotube cells. Therefore, MANF can be used as an agent for promoting the induction of differentiation from myoblasts to myotube cells. Also, myotube cells form muscle fibers and become muscles. Therefore, MANF can be used as a muscle formation promoter, an agent for maintaining muscle mass, and an agent for increasing muscle mass.

[0074] The inventor has first discovered that MANF promotes the production of prostacyclin by endothelial cells. Therefore, MANF can be used as a prostacyclin production promoter. Prostacyclin is also called prostaglandin I2 (PGI2). Prostacyclin activates adenylate cyclase and promotes the production of cyclic adenosine monophosphate (cAMP). Therefore, MANF can be used as an adenylate cyclase activator and a cyclic adenosine monophosphate (cAMP) production promoter.

[0075] Also, prostacyclin has an inhibitory effect on platelet aggregation and a vasodilatory effect on vascular smooth muscle. Therefore, MANF can be used as an antiplatelet aggregant, a vasodilator, a therapeutic or prophylactic drug for hypertension, and a therapeutic or prophylactic drug for arteriosclerosis. Furthermore, prostacyclin inhibits the proliferation of vascular endothelial cells. Therefore, MANF can be used as an inhibitor of vascular endothelial cell proliferation.

[0076] The present inventors have first found that MANF promotes chondrocytes to produce hyaluronic acid. Therefore, MANF can be used as an agent for promoting hyaluronic acid production in chondrocytes. Further, hyaluronic acid protects cartilage and has a lubricating effect, a buffering effect, and an anti-inflammatory effect on joints. Therefore, MANF can be used as a therapeutic or prophylactic agent for joint diseases. Joint diseases include, for example, arthritis, osteoarthritis, osteoarthritis of the knee, osteoarthritis of the hip, degenerative lumbar spondylosis, meniscus injury, rheumatoid arthritis, knee ligament injury, jumper's knee (patellar tendonitis), runner's knee (tibial collateral ligamentitis), pes anserine bursitis, Baker's cyst, osteochondritis dissecans of the knee, patellofemoral joint disease, prepatellar bursitis, etc. Joint diseases may be joint diseases caused by a decrease in chondrocytes, joint diseases caused by insufficient collagen production, joint diseases caused by insufficient hyaluronic acid production, or joint diseases caused by pain. Further, MANF can be used as a therapeutic or prophylactic agent for joint pain associated with these joint diseases.

[0077] In addition, anti-obesity effects, muscle mass-increasing effects, vasodilating effects, cartilage-protecting effects, etc. can suppress aging by protecting cells from cytotoxic stress and endoplasmic reticulum (ER) stress, leading to the treatment and prevention of lifestyle-related diseases and adult diseases. Therefore, MANF can be used as an anti-aging agent, a therapeutic or prophylactic agent for lifestyle-related diseases, and / or a therapeutic or prophylactic agent for adult diseases.

[0078] The pharmaceutical composition and the cosmetic composition according to the first embodiment contain an effective amount of MANF. Here, the effective amount refers to an amount capable of exerting an effect as a pharmaceutical composition or a cosmetic composition. The effective amount is appropriately set according to the patient's age, the target disease, the presence or absence of other active ingredients, and the amount of other formulations.

[0079] The pharmaceutical composition and / or cosmetic composition according to the first embodiment may contain pharmaceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline, etc. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrants include carboxymethyl cellulose and calcium carbonate. Examples of buffers include phosphates, citrates, and acetates. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth.

[0080] Examples of suspending agents include glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, and sodium lauryl sulfate. Examples of soothing agents include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics include benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.

[0081] In addition, the pharmaceutical composition and / or cosmetic composition according to the first embodiment contains water, alcohol, surfactants (cationic, anionic, nonionic, and amphoteric surfactants, etc.), humectants (glycerin, 1,3-butylene glycol, propylene glycol, propanediol, pentanediol, polyquaternium, amino acids, urea, pyrrolidone carboxylate, nucleic acids, monosaccharides, oligosaccharides, etc., and derivatives thereof, etc.), thickeners (polysaccharides, polyacrylates, carboxyvinyl polymer, polyvinylpyrrolidone, polyvinyl alcohol, chitin, chitosan, alginic acid, carrageenan, xanthan gum, methylcellulose, etc., and derivatives thereof, etc.), waxes, petrolatum, saturated fatty acids of hydrocarbons, unsaturated fatty acids, and silicone oils, etc., and derivatives thereof, triglycerides such as glyceryl tri(caprylate / caprate) and glyceryl trioctanoate, ester oils such as isopropyl stearate, natural fats and oils (olive oil, camellia oil, avocado oil, almond oil, cocoa butter, evening primrose oil, grape seed oil, macadamia nut oil, eucalyptus oil, rose hip oil, squalane, orange raffia oil, lanolin, and ceramide, etc.), preservatives (oxybenzoic acid derivatives, dehydroacetate, photosensitizers, sorbic acid, and phenoxyethanol, etc., and derivatives thereof, etc.), bactericides (sulfur, trichlorocarbanilide, salicylic acid, zinc pyrithione, and hinokitiol, etc., and derivatives thereof, etc.), ultraviolet absorbers (para-aminobenzoic acid, methoxycinnamic acid, etc., and derivatives thereof, etc.), anti-inflammatory agents (allantoin, bisabolol, ε-aminocaproic acid, acetylfarnesyl cysteine, and glycyrrhizic acid, etc., and derivatives thereof, etc.), antioxidants (tocopherol, BHA, BHT, and astaxanthin, etc., and derivatives thereof, etc.), chelating agents (ethylenediaminetetraacetic acid, hydroxyethanediphosphonic acid, etc., and derivatives thereof, etc.), extracts of animals and plants (ashitaba, aloe, ginseng, saffron, cinnamon, seaweed, quince, chamomile, licorice, kiwi, cucumber, mulberry, birch, rhodiola, garlic, button mushroom, hops, elm, lavender, rosemary, eucalyptus, milk, various peptides, placenta, royal jelly, Euglena extract, hydrolyzed Euglena extract, and Euglena oil, etc.,and purified products or fermented products of these contained components, etc., pH adjusters (inorganic acids, inorganic acid salts, organic acids, and organic acid salts, etc., and their derivatives, etc.), vitamins (vitamin A, vitamin B, vitamin C, vitamin D, ubiquinone, and nicotinamide, etc., and their derivatives, etc.), fermentation broths of yeast, Aspergillus, and lactic acid bacteria, Galactomyces culture broth, whitening agents (tranexamic acid, cetyl tranexamic acid hydrochloride, 4-n-butylresorcinol, arbutin, kojic acid, ellagic acid, glycyrrhizin flavonoid, niacinamide, and vitamin C derivatives, etc.), ceramide and ceramide derivatives, anti-wrinkle agents (retinol and retinal, and their derivatives, nicotinamide, and oligopeptides, etc., and their derivatives, etc., natural and synthetic components with neutrophil elastase inhibitory and MMP-1 and MMP-2 inhibitory effects, etc.), titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powdered materials processed from these, etc. can be blended within the scope of achieving the objectives of the pharmaceutical composition and cosmetic composition according to the first embodiment.

[0082] It should be noted that the components that can be added to the pharmaceutical composition and / or cosmetic composition according to the first embodiment are not limited to the above, and any components that can be used in the pharmaceutical composition and / or cosmetic composition can be freely selected. When the pharmaceutical composition and / or cosmetic composition according to the embodiment is used as a plaster, in addition to the above components, a base (kaolin, bentonite, etc.) and a gelling agent (polyacrylate, polyvinyl alcohol, etc.) can be blended within the scope of achieving the objective. When the pharmaceutical composition and / or cosmetic composition according to the first embodiment is used as a bath agent, sulfates, bicarbonates, borates, pigments, and moisturizing agents can be appropriately blended within the scope of achieving the objective and prepared into a powder type or a liquid type. The pharmaceutical composition and / or cosmetic composition according to the first embodiment may be a skin-applied composition.

[0083] Another aspect of the first embodiment is the use of MANF in the manufacture of pharmaceutical compositions and / or cosmetic compositions. The pharmaceutical compositions and / or cosmetic compositions are, for example, lipolytic agents, anti-obesity drugs, obesity-improving drugs, lipolysis-promoting agents, diet drugs, drugs for the treatment or prevention of diabetes, agents for promoting the differentiation induction from myoblasts to myotube cells, muscle formation-promoting agents, agents for maintaining muscle mass, agents for increasing muscle mass, prostacyclin production-promoting agents, adenylate cyclase activators, cyclic adenosine monophosphate (cAMP) production-promoting agents, platelet aggregation inhibitors, vasodilators, drugs for the treatment or prevention of hypertension, drugs for the treatment or prevention of arteriosclerosis, vascular endothelial cell growth inhibitors, chondrocyte hyaluronic acid production-promoting agents, drugs for the treatment or prevention of joint diseases, anti-aging agents, drugs for the treatment or prevention of lifestyle-related diseases, and / or drugs for the treatment or prevention of adult diseases.

[0084] In this aspect, it may include the step of adding MANF in the manufacture of pharmaceutical compositions and / or cosmetic compositions, and the timing and method of adding MANF are not limited. This aspect may, for example, include the step of mixing MANF with the above pharmaceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline, etc., and may also include the step of mixing MANF with the above water, alcohol, surfactants, humectants, thickeners, waxes, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids, and silicone oils, etc., and their derivatives, triglycerides, ester oils, natural fats and oils, antiseptics, bactericides, ultraviolet absorbers, anti-inflammatory agents, antioxidants, chelating agents, animal and plant extracts, pH adjusters, vitamins, fermentation broths of yeast, Aspergillus and lactic acid bacteria, Galactomyces culture broth, whitening agents, ceramide-ceramide derivatives, anti-wrinkle agents, titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders obtained by processing these, etc.

[0085] Another aspect of the first embodiment is a method of administering a pharmaceutical composition and / or a cosmetic composition containing the above-mentioned MANF. This aspect is, for example, a method for treating lipolysis, a method for treating obesity, a method for improving obesity, a method for promoting lipolysis, a method for diet treatment, a method for treating or preventing diabetes, a method for promoting the induction of differentiation from myoblasts to myotube cells, a method for promoting muscle formation, a method for maintaining muscle mass, a method for increasing muscle mass, a method for promoting prostacyclin production, a method for activating adenylate cyclase, a method for promoting the production of cyclic adenosine monophosphate, a method for inhibiting platelet aggregation, a method for treating vasodilation, a method for treating or preventing hypertension, a method for treating or preventing arteriosclerosis, a method for treating the inhibition of vascular endothelial cell proliferation, a method for promoting the production of hyaluronic acid by chondrocytes, a method for treating or preventing joint diseases, an anti-aging treatment method, a method for treating lifestyle-related diseases and / or a method for treating or preventing adult diseases.

[0086] In the method according to the first embodiment, a pharmaceutical composition and / or a cosmetic composition containing the above-mentioned MANF is administered to the subject. The subject is a human or non-human animal or their cells.

[0087] As used herein, "administration" includes oral administration, topical contact administration, intravascular administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, etc. of a pharmaceutical composition and / or a cosmetic composition so that an active ingredient is provided to a subject. The administration method is not limited to these, and a plurality of administration methods may be combined. For example, when a pharmaceutical composition and / or a cosmetic composition is prepared in a powder type or a liquid type, the powder type or liquid type composition can be orally administered, topically contacted, intravascularly administered, intravenously administered, intraperitoneally administered, intramuscularly administered, or subcutaneously administered. Also, for example, when a pharmaceutical composition and / or a cosmetic composition is a suppository or a skin application composition, the suppository or skin application composition can be transdermally administered by topical contact. Further, when the subject is a cell of a human or non-human animal, "administration" as used herein includes contacting the subject with a medium containing a pharmaceutical composition and / or a cosmetic composition. That is, the administration may be in vivo administration or in vitro administration.

[0088] In the method according to the first embodiment, a pharmaceutical composition and / or a cosmetic composition containing MANF is administered to a subject so that an effective amount of MANF is provided to the subject. Here, the effective amount refers to an amount capable of exerting an effect as a pharmaceutical composition or a cosmetic composition. The effective amount is appropriately set according to the age of the subject, the disease of the subject, the purpose of administration, the presence or absence of other active ingredients, and the amount of other formulations.

[0089] In the method according to the first embodiment, the frequency, number of times, period, etc. of administering the pharmaceutical composition and / or the cosmetic composition containing MANF to the subject are not limited, and these are appropriately adjusted so that an effective amount of MANF is provided to the subject. The frequency of administering the pharmaceutical composition and / or the cosmetic composition containing MANF to the subject is, for example, twice a day, once a day, once every two days, or once every three days.

[0090] (Second Embodiment) The pharmaceutical composition and / or the cosmetic composition according to the second embodiment contains MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. The pharmaceutical composition and / or the cosmetic composition may contain one selected from CXCL4, GDF11, THBS4, and TIMP2, or may contain two or more.

[0091] The CXCL4 contained in the pharmaceutical composition and / or cosmetic composition according to the second embodiment is, for example, an isolated protein or a recombinant protein, preferably a recombinant human CXCL4 protein. The CXCL4 protein may have, for example, the amino acid sequence of SEQ ID NO:3, or may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:3. The CXCL4 protein may have the amino acid sequence from position 32 to position 101 in the amino acid sequence of SEQ ID NO:3 (SEQ ID NO:4), or may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:4.

[0092] The GDF11 contained in the pharmaceutical composition and / or cosmetic composition according to the second embodiment is, for example, an isolated protein or a recombinant protein, preferably a recombinant human GDF11 protein. The GDF11 protein may have, for example, the amino acid sequence of SEQ ID NO:5, or may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:5. The GDF11 protein may have the amino acid sequence from position 299 to position 407 in the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:6), or may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:6.

[0093] The TIMP2 contained in the pharmaceutical composition and / or cosmetic composition according to the second embodiment is, for example, an isolated protein or a recombinant protein, preferably a recombinant human TIMP2 protein. The TIMP2 protein may have, for example, the amino acid sequence of SEQ ID NO:7, and may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:7. The TIMP2 protein may have the amino acid sequence from position 27 to position 220 (SEQ ID NO:8) in the amino acid sequence of SEQ ID NO:7, and may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:8.

[0094] The THBS4 contained in the pharmaceutical composition and / or cosmetic composition according to the second embodiment is, for example, an isolated protein or a recombinant protein, preferably a recombinant human THBS4 protein. The THBS4 protein may have, for example, the amino acid sequence of SEQ ID NO:9, and may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:9.

[0095] The APLN contained in the pharmaceutical composition and / or cosmetic composition according to the second embodiment is preferably Apelin-13. Apelin is a protein precursor consisting of 77 amino acid residues, which is cleaved into Apelin-36 consisting of 36 amino acid residues, Apelin-17 consisting of 17 amino acid residues, and Apelin-13 consisting of 13 amino acid residues. Among these, Apelin-13 has the highest activity. APLN, preferably Apelin-13, may have, for example, the amino acid sequence of SEQ ID NO:10 (XRPRLSHKGPMPF, where X is pyroglutamic acid.), or may have an amino acid sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO:10.

[0096] Amyloid beta (Aβ) is a relatively small protein (peptide) consisting of about 40 amino acid residues, and is known to accumulate in the brains of Alzheimer's disease patients to form amyloid plaques. When amyloid beta accumulates outside nerve cells in the brain, the accumulation of amyloid beta induces the accumulation of tau protein inside nerve cells. When the tau protein accumulated inside nerve cells aggregates, neurofibrillary tangles (NFT) are formed. It is considered that the toxicity of the accumulated amyloid beta and the accumulation of NFT cause nerve cell death and lead to the onset of Alzheimer's type dementia.

[0097] In addition to Alzheimer's type dementia, Lewy body dementia, vascular dementia, and frontotemporal dementia account for about 90% of all dementia, so these are called the four major dementias. Although Lewy body dementia, vascular dementia, and frontotemporal dementia are thought to develop by different mechanisms from Alzheimer's type dementia, there are also reports that the accumulation of amyloid beta is related to brain atrophy in Lewy body dementia.

[0098] Diabetes is known to increase the risk of developing Alzheimer's disease. In diabetic patients with increased insulin resistance, insulin secretion is promoted to lower blood glucose levels. The secreted insulin is decomposed by insulin-degrading enzyme (IED), and IED also plays a role in decomposing amyloid-β. When a large amount of insulin is present, IED is consumed for insulin decomposition, and the decomposition effect of amyloid-β decreases. As a result, it is thought that amyloid-β that remains undegraded accumulates.

[0099] Also, it is known that the risk of developing early-onset Alzheimer's disease is relatively high in people with Down syndrome. Down syndrome is trisomy of chromosome 21, and the gene encoding amyloid precursor protein (APP) is present on chromosome 21. Therefore, it is thought that the production of amyloid-β is higher in people with Down syndrome than in healthy individuals, which increases the risk of developing early-onset Alzheimer's disease in people with Down syndrome.

[0100] Diseases caused by the accumulation of amyloid proteins are also called amyloidosis. Amyloidosis can be classified into systemic amyloidosis in which amyloid accumulates in multiple organs and causes disorders, and localized amyloidosis in which amyloid accumulates in one organ and causes disorders. Alzheimer's disease, which is thought to be caused by the accumulation of amyloid-β, is a type of localized amyloidosis.

[0101] The present inventors confirmed that when amyloid-β is added to nerve cells, the nerve cells die, and confirmed the toxic effect of amyloid-β on nerve cells. On the other hand, the inventors have first found that when MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 are added to nerve cells simultaneously with amyloid-β, respectively, the death of nerve cells is suppressed. Therefore, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as an inhibitor of protein accumulation, aggregation, or mislocalization, preferably as an amyloid-β inhibitor. Here, the inhibition of amyloid-β includes the inhibition of amyloid-β accumulation, aggregation, or mislocalization, and includes the inhibition of the action of amyloid-β, particularly the action of amyloid-β on nerve cells.

[0102] As described above, it is considered that Alzheimer's disease develops due to the accumulation and aggregation of amyloid-β. Therefore, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a therapeutic or preventive agent for dementia. Dementia may include Alzheimer's disease and / or Lewy body dementia. Dementia may also be dementia in diabetic patients or dementia in Down syndrome patients.

[0103] In addition, since the death of nerve cells is suppressed in the presence of each of MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a nerve cell death inhibitor, or a therapeutic or preventive agent for nerve diseases. The nerve disease may be a nerve disease associated with the accumulation, aggregation, or mislocalization of proteins such as amyloid-β, preferably a nerve disease caused by amyloid-β accumulation or aggregation, for example, neurofibrillary tangles (NFT).

[0104] MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a therapeutic or prophylactic agent for amyloidosis because they each inhibit amyloid β. The amyloidosis may be systemic amyloidosis or localized amyloidosis. Amyloidosis includes, for example, Alzheimer's type dementia.

[0105] The inventor has first found that when GDF11, TIMP2, THBS4, and CXCL4 are added to nerve cells simultaneously with amyloid β, the number of surviving nerve cells increases as compared with the case where neither amyloid β nor GDF11, TIMP2, THBS4, and CXCL4 is added. Therefore, GDF11, TIMP2, THBS4, and CXCL4 can each be used as an agent for improving the survival rate of nerve cells or a protective agent for nerve cells.

[0106] The inventor has first found that when MANF and APLN are added to nerve cells simultaneously with amyloid β, the number of surviving nerve cells increases as compared with the case where only amyloid β is added. Therefore, MANF and APLN can each be used as an agent for improving the survival rate of nerve cells or a protective agent for nerve cells.

[0107] MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 each suppress nerve cell death or improve the survival rate of nerve cells. Therefore, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a therapeutic or prophylactic agent for diseases caused by nerve cell death. Nerve cell death includes apoptosis. Diseases caused by nerve cell death may include, for example, Alzheimer's disease, Huntington's disease, Parkinson's disease, and / or amyotrophic lateral sclerosis (ALS). Also, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a therapeutic or prophylactic agent for a decrease in the number of nerve cells or nerve cell death.

[0108] Furthermore, the suppression of nerve cell death, the improvement of the survival rate of nerve cells, or the protection of nerve cells leads to the rejuvenation of nerve cells or the prevention of aging. Therefore, MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 can each be used as a medicine for the rejuvenation of nerve cells or the prevention of aging.

[0109] The nerve cells according to the second embodiment are not limited, and include, for example, cerebral nerve cells, excitatory nerve cells, inhibitory nerve cells, motor nerve cells, peripheral nerve cells, and interneurons. The nerve cells may be collected nerve cells or nerve cells differentiated from stem cells. Stem cells include, for example, iPS cells (induced pluripotent stem cells). The collected or differentiated nerve cells may be positive for proteins such as vGlu (vesicular glutamate transporter), satb2 (Special AT-rich sequence-binding protein 2), CTIP (CtBP (carboxy-terminal binding protein) interacting protein), and / or Reelin.

[0110] The pharmaceutical composition and / or cosmetic composition according to the second embodiment contains an effective amount of MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. Here, the effective amount refers to an amount capable of exerting an effect as a pharmaceutical composition or a cosmetic composition. The effective amount is appropriately set according to the age of the patient, the target disease, the presence or absence of other active ingredients, and the amount of other formulations.

[0111] The pharmaceutical composition and / or cosmetic composition according to the second embodiment may contain the above pharmaceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline, etc., which can be included in the pharmaceutical composition and / or cosmetic composition according to the first embodiment, and the above water, alcohol, surfactant, humectant, thickener, wax, petrolatum, saturated fatty acid of hydrocarbon, unsaturated fatty acid, and silicone oil, etc., and their derivatives, triglycerides, ester oils, natural fats and oils, antiseptics, bactericides, ultraviolet absorbers, anti-inflammatory agents, antioxidants, chelating agents, animal and plant extracts, pH adjusters, vitamins, fermentation broth of yeast, Aspergillus and lactic acid bacteria, galactomyces culture solution, whitening agents, ceramide - ceramide derivatives, anti-wrinkle agents, titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders obtained by processing these, etc.

[0112] Another aspect of the second embodiment is the use of MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 in the manufacture of a pharmaceutical composition and / or cosmetic composition. The pharmaceutical composition and / or cosmetic composition is, for example, an inhibitor of protein accumulation, aggregation, or mislocalization, preferably an amyloid-β inhibitor; a therapeutic or prophylactic agent for neurological diseases; a therapeutic or prophylactic agent for dementia; a therapeutic or prophylactic agent for diseases caused by neuronal death; a therapeutic or prophylactic agent for a decrease in the number of neurons or neuronal death; a therapeutic or prophylactic agent for amyloidosis; and / or a rejuvenating or anti-aging agent for neurons.

[0113] In this aspect, the process of adding MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 in the manufacture of pharmaceutical compositions and / or cosmetic compositions may be included, and the timing and method of adding MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 are not limited. This aspect may include, for example, the step of mixing the above pharmaceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline, etc. with MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2, and may also include the step of mixing the above water, alcohol, surfactants, humectants, thickeners, waxes, petrolatum, saturated fatty acids of hydrocarbons, unsaturated fatty acids, and silicone oils, etc., and their derivatives, triglycerides, ester oils, natural fats and oils, preservatives, bactericides, ultraviolet absorbers, anti-inflammatory agents, antioxidants, chelating agents, extracts of animals and plants, pH adjusters, vitamins, fermentation broths of yeast, Aspergillus, and lactic acid bacteria, galactomyces culture broths, whitening agents, ceramide-ceramide derivatives, anti-wrinkle agents, titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders obtained by processing these, etc. with MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2. One selected from MANF, APLN, CXCL4, GDF11, THBS4, and TIMP2 may be added, or two or more may be added. When two or more selected from MANF, APLN, CXCL4, GDF11, THBS4, and TIMP2 are added, the order of addition is not limited.

[0114] Another aspect of the second embodiment is a method of administering to a pharmaceutical composition and / or a cosmetic composition containing the above-mentioned MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2. This aspect is, for example, a method of inhibiting protein accumulation, aggregation, or mislocalization, preferably a method of inhibiting amyloid β; a method of suppressing neuronal cell death; a method of treating or preventing a neurological disease; a method of treating or preventing dementia; a method of treating or preventing a disease caused by neuronal cell death; a method of treating or preventing a decrease in the number of neurons or neuronal cell death; a method of treating or preventing amyloidosis; and / or a method of rejuvenating or preventing aging of neurons. The neurological diseases targeted by this aspect are neurological diseases associated with the accumulation, aggregation, or mislocalization of proteins such as amyloid β, preferably neurological diseases caused by amyloid β accumulation. The neurological diseases targeted by this aspect may include neurofibrillary tangles (NFT). The dementia targeted by this aspect may include Alzheimer's disease and / or Lewy body dementia. The diseases caused by neuronal cell death targeted by this aspect may include Alzheimer's disease, Huntington's disease, Parkinson's disease, and / or amyotrophic lateral sclerosis (ALS). The amyloidosis targeted by this aspect may be systemic amyloidosis or may be localized amyloidosis.

[0115] In the method according to the second embodiment, a pharmaceutical composition and / or a cosmetic composition containing the above-mentioned MANF, APLN, CXCL4, GDF11, THBS4 and / or TIMP2 is administered to the subject. The subject is a human or non-human animal or their cells. The subject may be a human diabetic patient or a human Down syndrome patient.

[0116] In the method according to the second embodiment, "administration" refers to the same act as the administration in the method according to the first embodiment.

[0117] In the method according to the second embodiment, a pharmaceutical composition and / or a cosmetic composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 is administered to a subject so that an effective amount of MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 is provided to the subject. Here, the effective amount refers to an amount capable of exerting an effect as a pharmaceutical composition or a cosmetic composition. The effective amount is appropriately set according to the age of the subject, the disease of the subject, the purpose of administration, the presence or absence of other active ingredients, and the amount of other formulations.

[0118] In the method according to the second embodiment, the frequency, number of times, period, etc. of administering a pharmaceutical composition and / or a cosmetic composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject are not limited, and these conditions are appropriately adjusted so that an effective amount of MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 is provided to the subject. The frequency of administering a pharmaceutical composition and / or a cosmetic composition containing MANF, APLN, CXCL4, GDF11, THBS4, and / or TIMP2 to a subject is, for example, twice a day, once a day, once every two days, or once every three days.

Example

[0119] (Example 1: Preparation of Test Substance) As a test substance used in Examples 2 to 5 and 8 described below, recombinant human MANF protein (PEPROTECH) was prepared.

[0120] In addition, as the positive control substance for Example 2, isoproterenol (Cayman), which is known to have a lipolytic effect, was prepared. As the positive control substance for Example 3, insulin (Thermo Fisher Scientific), which is known to induce differentiation from myoblasts to myotubes, was prepared. As the positive control substance for Example 4, arachidonic acid (Sigma-Aldrich, A3611), which is known to promote the production of prostacyclin, was prepared. As the positive control substance for Example 5, N-acetylglucosamine (Sigma-Aldrich, A8625), which is known to promote the production of hyaluronic acid, was prepared.

[0121] As the test substances used in Example 7, as GDF11, recombinant human / mouse / rat GDF11 protein (PEPROTECH) was prepared. As TIMP2, recombinant human TIMP2 protein (PEPROTECH) was prepared. As THBS4, recombinant mouse THBS4 protein (PEPROTECH) was prepared. As CXCL4, recombinant human CXCL4 protein (PEPROTECH) was prepared. As APLN used in Example 8, [Pyr 1 -Apelin-13 with CAS number 217082-60-5 was prepared. As amyloid-β used in Examples 7 and 8, human recombinant amyloid-β protein (CAS number 107761-42-2) was prepared.

[0122] (Example 2: Lipolysis Test) Using a growth medium (PBM supplemented with PGM-2, 10% FBS, 2 mM L-glutamine, 50 μg / mL gentamicin, and 37 ng / mL amphotericin-B, Lonza, PT-8002), normal human subcutaneous preadipocytes (Lonza, PT-5020) were revived in a T-75 flask and cultured in a CO2 incubator (37°C, 5% CO2 humidified, the same hereinafter) until the normal human subcutaneous preadipocytes reached 60 to 80% confluence. Next, the normal human subcutaneous preadipocytes were detached from the T-75 flask using 0.25% trypsin-EDTA, and the normal human subcutaneous preadipocytes were centrifuged. Thereafter, using the growth medium, the normal human subcutaneous preadipocytes were seeded in a 96-well plate at 10,000 cells / 100 μL / well and cultured in a CO2 incubator.

[0123] The next day, the medium was replaced with 100 μL of a differentiation medium (PGM-2 supplemented with insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine, Lonza, PT-8002), and the cells were cultured in a CO2 incubator for 7 days to induce differentiation of the normal human subcutaneous preadipocytes into adipocytes. Next, the medium was replaced with 100 μL of a differentiation medium supplemented with MANF at a final volume concentration of 1 / 2000 and 1 / 500 and a positive control substance at a final concentration of 10 μmol / L, or 100 μL of a differentiation medium without MANF and the positive control substance, and the cells were cultured in a CO2 incubator for 24 hours to treat the adipocytes with either MANF or the positive control substance. A photograph of the treated cells is shown in Figure 1.

[0124] Subsequently, the culture supernatant was collected, and the amount of free glycerol in the supernatant was measured using a lipolysis analysis kit (Cayman, 10009381). The amount of free glycerol corresponds to the amount of lipolysis. The measurement method followed the protocol attached to the kit. Specifically, a multi-spectrum microplate reader (Varioskan Flash, Thermo Fisher Scientific) was used to measure the absorbance of the supernatant at a wavelength of 540 nm, and the relative free glycerol concentration was calculated. Using the group without MANF addition as a control, the control group was compared with the groups added with MANF and the positive control substance by two-sided test using Dunnett's test. When P < 0.05, a significant difference was considered to exist.

[0125] When the final volume concentration of MANF was 1 / 2000, as shown in Figure 2, the group added with MANF had a significantly higher amount of lipolysis compared to the control. When the final volume concentration of MANF was 1 / 500, the amount of lipolysis was also higher compared to the control. The group added with isoproterenol, which is known to have a lipolytic effect, had a significantly higher amount of lipolysis compared to the control, indicating that the experimental system was correct. These results indicate that MANF decomposes fat.

[0126] (Example 3: Muscle tube differentiation promotion test) Normal human skeletal muscle myoblasts (Lonza, CC-2580) were revived in a T-75 flask using a growth medium (SkGM-2, Lonza, CC-3245), and the normal human skeletal muscle myoblasts were cultured in a CO2 incubator until they reached approximately 60% confluence. Next, the normal human skeletal muscle myoblasts were detached from the T-75 flask using 0.05% trypsin-EDTA and phenol red. Subsequently, using the growth medium, the normal human skeletal muscle myoblasts were seeded in a 96-well plate at 7,500 cells / 100 μL / well and cultured in a CO2 incubator.

[0127] On the next day, the medium was replaced with 100 μL of differentiation medium (DMEM:F12 supplemented with 2% horse serum) supplemented with MANF at a final volume concentration of 1 / 2000 and 1 / 500, respectively, and a positive control substance at a final concentration of 50 nmol / L, or 100 μL of differentiation medium without MANF and the positive control substance, and the cells were cultured in a CO2 incubator for 3 days to induce differentiation of normal human skeletal muscle myoblasts into myotube cells.

[0128] Thereafter, the medium was removed from the wells, 100 μL of 4% paraformaldehyde at 4 °C was added to the wells, and the cells were fixed by incubating at 4 °C for 15 minutes. Next, the wells were washed 3 times with 100 μL of Dulbecco's PBS, and 100 μL of blocking / membrane permeabilization solution (Dulbecco's PBS supplemented with 3% bovine serum albumin and 0.3% Triton-X 100) was added to the wells and incubated at room temperature for 30 minutes.

[0129] Next, the solution in the wells was replaced with a solution containing an anti-myosin heavy chain (MHC) antibody (Affymetrix) and incubated overnight at 4 °C. Myosin heavy chain is a differentiation marker of myotube cells. After washing the wells 3 times with a buffer, a secondary antibody (goat anti-mouse IgG2b antibody, Alexa Fluor 555, Thermo Fisher Scientific) and a nuclear stain (Hoechst 33342, Thermo Fisher Scientific) were added to the wells and incubated at room temperature for 2 hours. Thereafter, after washing the wells 3 times with a buffer, buffer was added to the wells, and 9 fields in the center of each well were photographed using a 10× objective lens with a confocal imaging system (Operetta CLS, Perkin Elmer).

[0130] The images obtained by photographing are shown in Figure 3. From the photographed images, the number of cell nuclei and the number of myosin heavy chain-positive nuclei were counted, and the differentiation index I (%) given by the following formula (1) was calculated. The higher the differentiation index I, the more promoted the myotube differentiation. I = N M / N T × 100 (1) (1) In the formula, N M represents the number of myosin heavy chain-positive nuclei, and N T represents the total number of nuclei.

[0131] When the final concentration of MANF was 1 / 2000 by volume, as shown in Fig. 4, the group to which MANF was added had a higher differentiation index compared to the control. When the final volume concentration of MANF was 1 / 500, the group to which MANF was added also had a higher differentiation index compared to the control. The group to which insulin, which is known to induce differentiation from myoblasts to myotubes, was added had a significantly higher differentiation index compared to the control, indicating that the experimental system was correct. These results show that MANF promotes the induction of differentiation from normal human skeletal muscle myoblasts to myotube cells, promotes the proliferation of myotube cells, and / or increases the survival rate of myotube cells.

[0132] (Example 4: Prostacyclin production test) Using a medium (EGM-2, Lonza, CC-3162), normal human umbilical vein endothelial cells (Lonza, CC2519AS) were revived in a T-75 flask, and the normal human umbilical vein endothelial cells were cultured in a CO2 incubator until they reached 60 to 80% confluence. Next, the normal human umbilical vein endothelial cells were detached from the T-75 flask using 0.05% trypsin-EDTA and phenol red. Thereafter, using the medium, the normal human umbilical vein endothelial cells were seeded in a 96-well plate at 10,000 cells / 100 μL / well and cultured in a CO2 incubator.

[0133] The next day, the medium was replaced with 100 μL of medium to which each of MANF at a final volume concentration of 1 / 2000 and 1 / 500 and a positive control substance at a final concentration of 10 μmol / L was added, or 100 μL of medium to which neither MANF nor the positive control substance was added, and the cells were cultured in a CO2 incubator for 24 hours.

[0134] The culture supernatant was collected, and the amount of 6-ketoprostaglandin F1α, a metabolite of prostacyclin, was measured using an ELISA kit (abcam, ab133023) and a multi-spectro microplate reader (Varioskan Flash, Thermo Fisher Scientific).

[0135] When the final volume concentration of MANF was 1 / 2000, as shown in Figure 5, the group to which MANF was added had a significantly higher amount of 6-ketoprostaglandin F1α compared to the control without MANF addition. When the final volume concentration of MANF was 1 / 500, the group to which MANF was added had a higher amount of 6-ketoprostaglandin F1α compared to the control without MANF addition. The group to which arachidonic acid, which is known to promote the production of prostacyclin, was added had a significantly higher amount of 6-ketoprostaglandin F1α compared to the control, indicating that the experimental system was correct. These results show that MANF promotes the production of prostacyclin by normal human umbilical vein endothelial cells.

[0136] (Example 5: Hyaluronic acid production test) Using a growth medium (DMEM / F-12 supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution), a human chondrocyte cell line (SW1353, ATCC, HTB-94) was revived in a T-75 flask, and the human chondrocytes were cultured in a CO2 incubator until they reached 60 to 80% confluence. Next, the human chondrocytes were detached from the T-75 flask using 0.25% trypsin-EDTA and phenol red. Then, using the growth medium, the human chondrocytes were seeded in a 96-well plate at 10,000 cells / 100 μL / well and cultured in a CO2 incubator.

[0137] The next day, the medium was replaced with 100 μL of assay medium (DMEM / F-12 supplemented with 1% FBS and 1% penicillin-streptomycin mixed solution) with the addition of MANF at a final volume concentration of 1 / 2000 and 1 / 500 and a positive control substance at a final concentration of 10 mmol / L, or 100 μL of assay medium without the addition of MANF and the positive control substance, and human chondrocytes were cultured in a CO2 incubator for 24 hours or 72 hours.

[0138] The culture supernatant was collected, and the amount of hyaluronic acid was measured using an ELISA kit (Hyaluronan Quantikine ELISA Kit, R&D Systems) and a multi-spectro microplate reader (Varioskan Flash, Thermo Fisher Scientific).

[0139] When human chondrocytes were cultured for 24 hours and the final volume concentration of MANF was 1 / 2000, as shown in Figure 6, the group with MANF addition had a higher production of hyaluronic acid compared to the control without MANF addition. When human chondrocytes were cultured for 24 hours and the final volume concentration of MANF was 1 / 500, the group with MANF addition had a higher production of hyaluronic acid compared to the control without MANF addition. The group with the addition of N-acetylglucosamine, which is known to promote the production of hyaluronic acid, had a significantly higher production of hyaluronic acid compared to the control, indicating that the experimental system was correct. These results indicate that MANF promotes the production of hyaluronic acid by human chondrocytes.

[0140] Human chondrocytes were cultured for 72 hours. When the final volume concentration of MANF was 1 / 2000, as shown in Figure 7, the group to which MANF was added had a lower production amount of hyaluronic acid compared to the control without MANF addition. When human chondrocytes were cultured for 72 hours and the final volume concentration of MANF was 1 / 500, as shown in Figure 7, the group to which MANF was added had a lower production amount of hyaluronic acid compared to the control without MANF addition. The group to which N-acetylglucosamine, which is known to promote the production of hyaluronic acid, was added had a significantly higher production amount of hyaluronic acid compared to the control, indicating that the experimental system was correct.

[0141] (Example 6: Preparation of Cerebral Nerve Cells) A 12-well plate coated with a solubilized basement membrane preparation (Matrigel, Corning) was prepared, and a feeder-free medium (mTeSR (registered trademark) 1, Stemcell Technologies) containing a Rock (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor (Selleck) at a concentration of 10 nmol / mL was placed in each well.

[0142] iPS cells were dispersed with a tissue / culture cell detachment / separation / dispersion solution (Accutase, Innovative Cell Technologies) and seeded at a density of 1×10 5 cells / well. Then, the iPS cells were cultured in the feeder-free medium under gas conditions of 5% carbon dioxide concentration and 20% oxygen concentration for 24 hours.

[0143] On the next day (Day0), RNA of NGN2 (neurogenin2) connected to RNA corresponding to the puromycin (Puro) resistance gene was introduced into iPS cells. On the second day after the introduction of the NGN2-Puro resistance gene (Day2), the medium in the well was replaced with a neural induction medium (N3 medium) containing puromycin at a concentration of 2 μg / mL and cultured for 3 days to kill iPS cells other than those into which NGN2 was introduced and select iPS cells into which NGN2 was introduced. The N3 medium was prepared by adding 10 mL of B27, 5 mL of N2, and 1.6 mL of insulin with a concentration of 6.25 mg / mL to 500 mL of DMEM / F12.

[0144] On the seventh day after the introduction of the NGN2-Puro resistance gene (Day7), it was confirmed that vGlu-positive, satb2-positive, CTIP-positive, and REELIN-positive cerebral neurons were induced from iPS cells.

[0145] (Example 7: Survival test of cerebral neurons using GDF11, TIMP2, THBS4, CXCL4) After confirming that cerebral neurons were induced from iPS cells on the seventh day after the introduction of the NGN2-Puro resistance gene (Day7), a recombinant protein of amyloid β (concentration 10 μM) and any one of GDF11, TIMP2, THBS4, and CXCL4 were simultaneously added to the neural induction medium to culture cerebral neurons. As Comparative Control 1, a group was prepared in which cerebral neurons were cultured without adding any of amyloid β, GDF11, TIMP2, THBS4, and CXCL4 (No factor w / o Ab). As Comparative Control 2, a group was prepared in which cerebral neurons were cultured by adding only amyloid β (concentration 10 μM) (No factor w / Ab). The medium was changed once every 5 days. On the 17th day after the introduction of the NGN2-Puro resistance gene (Day17), the number of surviving cerebral neurons per field of view was measured in 5 fields of view and averaged. An example of a photograph of the field of view used for the measurement is shown in Fig. 8. Also, the averaged number of surviving cerebral neurons per field of view is shown in Fig. 9.

[0146] As shown in FIGS. 8 and 9, the number of surviving cerebral neurons per field of view was smaller in the group of comparison target 2 (No factor w / Ab) than in the group of comparison target 1 (No factor w / o Ab). These results indicate that amyloid-β induces death of cerebral neurons.

[0147] As shown in FIGS. 8 and 9, in the group in which amyloid-β and any one of GDF11, TIMP2, THBS4, and CXCL4 were simultaneously added to cerebral neurons, the number of surviving cerebral neurons per field of view was larger than in the group of comparison target 2 (No factor w / Ab). These results indicate that GDF11, TIMP2, THBS4, and CXCL4 each inhibit the effect of amyloid-β and suppress the death of cerebral neurons induced by amyloid-β. Furthermore, in the group in which amyloid-β and any one of GDF11, TIMP2, THBS4, and CXCL4 were simultaneously added to cerebral neurons, the number of surviving cerebral neurons per field of view was increased compared to the group of comparison target 1 (No factor w / o Ab).

[0148] (Example 8: Survival test of cerebral neurons using MANF and APLN) A test was conducted in the same manner as in Example 7, except that MANF and APLN were used instead of GDF11, TIMP2, THBS4, and CXCL. FIG. 10 shows an example of a photograph of the field of view used for measuring the number of surviving cerebral neurons. FIG. 11 shows the averaged number of surviving cerebral neurons per field of view.

[0149] As shown in FIGS. 10 and 11, the number of axons was decreased and the number of surviving cerebral neurons was small in the group of comparison target 2 (No factor w / Ab) in which cerebral neurons were cultured with only amyloid-β (concentration 10 μM), compared to the group of comparison target 1 (No factor w / o Ab) in which cerebral neurons were cultured without adding amyloid-β, MANF, or APLN. This result indicates that amyloid-β induces death of cerebral neurons, which is consistent with the result obtained in Example 7.

[0150] As shown in FIGS. 10 and 11, in the group in which amyloid β and either MANF or APLN were simultaneously added to the cerebral neurons, the number of surviving cerebral neurons per visual field was larger than that in the group of Comparative Example 2 (No factor w / Ab). These results indicate that MANF and APLN each inhibit the effect of amyloid β and suppress the death of cerebral neurons induced by amyloid β.

[0151] (Example 9: Apoptosis Marker Test) An anti-Cleaved Caspase-3 antibody (Abcam), which is an apoptosis marker, was added to the cerebral neurons cultured in Examples 7 and 8 at a concentration of 1 / 100 to stain the cerebral neurons. After staining, the number of Cleaved Caspase-3 positive cerebral neurons per visual field was measured in 5 visual fields and averaged. The averaged number of Cleaved Caspase-3 positive cerebral neurons per visual field is shown in FIG. 12.

[0152] As shown in FIG. 12, in the group in which amyloid β and any one of MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 were simultaneously added to the cerebral neurons, the number of Cleaved Caspase-3 positive cerebral neurons per visual field decreased compared to the group of Comparative Example 1 (No factor w / o Ab).

[0153] The results of Examples 7 to 9 indicate that MANF, APLN, GDF11, TIMP2, THBS4, and CXCL4 each suppress the cell death of neurons and improve the survival rate. In particular, in the group in which amyloid β and GDF11 were simultaneously added to the cerebral neurons, the number of surviving cerebral neurons per visual field increased by more than 4 times compared to the group of Comparative Example 1 (No factor w / o Ab). These results indicate that the effect of GDF11 in improving the survival rate of neurons or protecting neurons from cell death stress and ER stress is particularly large compared to MANF, APLN, TIMP2, THBS4, or CXCL4.

Claims

1. A lipolytic agent or lipolysis promoter containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

2. An anti-obesity agent or obesity preventive agent containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

3. A therapeutic or preventive agent for diabetes containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

4. A promoter for the production of prostacyclin or cyclic adenosine monophosphate containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

5. An antiplatelet aggregating agent containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

6. A vasodilator containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

7. A therapeutic or preventive agent for hypertension or arteriosclerosis containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

8. An inhibitor of vascular endothelial cell proliferation containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

9. A promoter for the production of hyaluronic acid containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

10. A therapeutic or preventive agent for joint diseases or joint pain containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

11. A promoter for inducing the differentiation of myoblasts into myotube cells containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

12. A muscle formation promoter containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

13. An agent for maintaining or increasing muscle mass, containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

14. A therapeutic or prophylactic agent for lifestyle-related diseases, containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

15. A therapeutic or prophylactic agent for adult diseases, containing MANF (Mesencephalic astrocyte-derived neurotrophic factor) protein.

16. An inhibitor of amyloid β, containing at least one protein selected from the group consisting of MANF (Mesencephalic astrocyte-derived neurotrophic factor), APLN (Apelin), CXCL4 (Chemokine C-X-C-Motif Ligand 4), GDF11 (Growth differentiation factor 11), THBS4 (Thrombospondin-4), and TIMP2 (Tissue inhibitor of metalloproteinases 2).

17. A therapeutic or prophylactic agent for nerve diseases, containing at least one protein selected from the group consisting of MANF (Mesencephalic astrocyte-derived neurotrophic factor), APLN (Apelin), CXCL4 (Chemokine C-X-C-Motif Ligand 4), GDF11 (Growth differentiation factor 11), THBS4 (Thrombospondin-4), and TIMP2 (Tissue inhibitor of metalloproteinases 2).

18. A therapeutic or prophylactic agent for dementia, comprising at least one protein selected from the group consisting of MANF (Mesencephalic astrocyte-derived neurotrophic factor), APLN (Apelin), CXCL4 (Chemokine C-X-C-Motif Ligand 4), GDF11 (Growth differentiation factor 11), THBS4 (Thrombospondin-4), and TIMP2 (Tissue inhibitor of metalloproteinases 2).

19. A cell death inhibitor for nerve cells, comprising at least one protein selected from the group consisting of MANF (Mesencephalic astrocyte-derived neurotrophic factor), APLN (Apelin), CXCL4 (Chemokine C-X-C-Motif Ligand 4), GDF11 (Growth differentiation factor 11), THBS4 (Thrombospondin-4), and TIMP2 (Tissue inhibitor of metalloproteinases 2).

20. A therapeutic or prophylactic agent for the treatment of or prevention of a decrease in the number of nerve cells, comprising at least one protein selected from the group consisting of MANF (Mesencephalic astrocyte-derived neurotrophic factor), APLN (Apelin), CXCL4 (Chemokine C-X-C-Motif Ligand 4), GDF11 (Growth differentiation factor 11), THBS4 (Thrombospondin-4), and TIMP2 (Tissue inhibitor of metalloproteinases 2).

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