KR20150085578A - Pharmaceutical composition comprising adipose stem cell extract for treating or preventing neurologic disease - Google Patents

Abstract

The present invention provides fat stem cell extracts and a usage of the same. A pharmaceutical composition comprising adipose stem cell extracts for treating or preventing nervous system disease according to the present invention has a good effect on preventing or treating nervous system diseases and thus can be useful in preventing or treating acute, subacute or chronic neurodegeneration diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for treating or preventing a neurological disease including an adipose stem cell extract,

The present invention is in the field of pharmaceutical compositions related to neurological diseases including stem cell extracts.

As the lifespan of humans increases, the incidence of neurodegenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, Alzheimer's disease, diabetic retinopathy, multiple infarct dementia and disc macular degeneration is also increasing. The number of neurodegenerative disease patients is estimated to be 24 million worldwide. In addition, diseases caused by other types of neurodegenerative diseases such as strokes or other trauma or injuries are increasing year by year. In Korea, as of the end of 2003, the number of elderly people aged 65 or older, who need to receive medical treatment or care for their neurological diseases such as dementia and stroke, should reach 640,000, , And the proportion of elderly population arithmetically will reach 870,000 in 2015 and 1.64 million in 2030. The management costs for these will exceed 4.8 trillion won in 2015 and reach 9 trillion by 2030 .

There is a need for the development of therapeutic agents for neuroprotection that effectively treat these acute and chronic neurodegenerative diseases and / or prevent damage to neuronal cell death or neuronal cells.

One of the most recent treatments for neurodegenerative diseases is stem cell therapy. However, there is a problem in that it is difficult to obtain autologous stem cells or the rate of cell survival after transplantation is limited and the efficiency of differentiation into functional cells is low.

In particular, adipose stem cells are expected to be a source of stem cells, but there is no proper transplantation method to the nervous system and the problem that they are not completely differentiated into neurons is not solved. In addition, brain stem surgery is required to transplant adipose stem cells directly into the brain, and the survival rate of adipose stem cells is only 13% a month after transplantation, and it is difficult to survive after several months (Lee et al., Ann Neurol 2009 Nov; 66 (5): 671-81).

Therefore, it is necessary to develop a highly stable neuroprotective agent that is easy to administer and can be repeatedly treated as a substitute for stem cell transplantation.

International Patent Application Publication No. WO2011 / 054100 relates to a stem cell extract and its immunomodulating use, and particularly relates to the use thereof in the treatment of autoimmune diseases and arthritis using extracts derived from human embryonic stem cells.

U.S. Published Patent Application No. 2010/0184225 discloses the use of chromosome of germline cell extract such as ovary for remodeling cell division.

The present invention aims at providing a composition which is more easily administered and can be repeatedly treated, and which can prevent or treat a neurological disease more effectively than a therapeutic agent using cells themselves.

The present invention provides a pharmaceutical composition for preventing or treating a neurological disease, comprising an adipocyte stem cell extract. In one embodiment, the neurological disease prevention or treatment according to the present invention is used for activating the pCREB-PGC-1α pathway, It may be due to death suppression.

The adipocyte stem cell extract according to the present invention may be a self-derived, homogenous or heterogeneous derived stem cell extract derived from a soluble and non-soluble fraction or a soluble fraction, wherein the soluble fraction is a protein, a polypeptide or a peptide Component, RNA, DNA, or lipid. In one embodiment, the effect of the composition according to the invention is on a proteinaceous component, for example a protein, a polypeptide or a peptide, in the soluble fraction.

Neurological diseases in which the pharmaceutical compositions according to the present invention may be used include acute, subacute or chronic neurodegenerative diseases.

In one embodiment according to the present application, an adipose stem cell extract according to the present invention comprises a) providing an adipose stem cell; b) fusing, crushing or permeabilizing said adipose stem cells in a buffer to produce a cell suspension, said method comprising the step of fractionating the cell suspension into soluble and non-soluble substances . ≪ / RTI > In one embodiment, for the production of an extract according to the present invention, the adipose stem cells are fused, treated with collagenase prior to said fusing, washed with phosphate buffered solution, and then thawed by ultrasonication.

The adipose stem cell fusion according to the present invention and the pharmaceutical composition containing the same are effective for neuroprotective effect against acute, subacute and / or chronic neuropathic diseases requiring protection from damage of nerve cells such as damage or apoptosis . In addition, the extract or composition according to the present invention can be repetitively treated because it is easy to administer, unlike the case where the cell itself is used, and it is possible to prevent or treat diseases more effectively because the cells do not need to depend on survival or migration.

FIG. 1 shows the protective effect of hASC-E on the OGD-induced Neuro-2a cells in vitro. FIG. 1a shows the result of observing the morphology of cells in the treated and control cell groups. FIGS. 1B and 1C show that the cell viability is improved by pretreatment of hASC-E with WST-1 and LDH analysis, respectively. Specifically, the heat-treated hASC-E protein, DNA, RNA, In the case of extracting and processing only a single component of < RTI ID = 0.0 &gt; FIG. 1D shows that when hASC-E was treated simultaneously with OGD, cell viability was improved by WST-1 analysis. * p < 0.05 and ** p < 0.01, ANOVA. Cell viability was optimized for results that did not induce OGD.
FIG. 2 shows the neuroprotective effect of hASC-E on various ischemic stroke models of Invivo. FIG. 2a shows permanent ischemia, FIG. 2b shows 90 min temporary ischemia, FIG. 2c shows MCA (middle cerebral artery) As a result of TTC staining and ischemic volume measurement of hASC-E, fibroblast extract, and control brain after MCA electrocoagulation, hASC-E shows a neuroprotective effect as compared with the control. * p &lt; 0.05, ANOVA. # p < 0.05 (between fibroblast-E and media-treated controls), Student's t-test. The scale bar is 0.8 cm.
FIG. 3 shows functional and neuroprotective effects in the ICH model of hASC-E administration. FIG. 3A shows that brain water content in the ischemic hemisphere treated with hASC-E was significantly lower than that of the control (FIG. ** p <0.01, ANOVA). FIG. 3B is a photograph of brain slices of ischemic hemispheres showing the neuroprotective effect of hASC-E compared with the control group (** p <0.05, Student's t- test). The scale bar is 0.8 cm). Figure 3c shows that behavioral deficits in the hASC-E treated group on days 1 and 3 after ICH via the modified limb placing test (MLPT) were significantly reduced compared to the control group ** p < 0.01, ANOVA.
Figure 4 shows the experimental design and phenotype for R6 / 2 mice, 4a is a graphical representation of the time progression of the experiments performed in one embodiment of the invention, Figure 4b shows the 12 weeks of age FIG. 4C shows that 10, 11 and 12-week-old R6 / 2 mice treated with hASC-E were superior to the control group in the rotarod test This is a result indicating the motion performance.
FIG. 5 shows the results of striatel apha and mHtt agglutination. FIG. 5A shows the results of a decrease in mutant huntingtin aggregation in rat hMSC-E-treated R6 / 2 mice compared with the control group , Figure 5b shows the results of Western blot analysis of mutant huntingtin protein aggregates and Figure 5c shows the results of quantification of Western blots showing that in the brains of hASC-E treated R6 / 2 mice the mutant Huntingtin protein The results show that the degree of aggregation is low . FIG. 5d shows the result of staining of the R5 / 2 mice treated with hASC-E or medium with Nissle, and FIG. 5e shows the results of staining with striatum / marginal striatum peristriatum) is higher. Bar = 100 mm, * p < 0.01 in Figs. 5A to 5E.
FIG. 6 shows activation of p-Akt, CREB and PGC-1α pathways by hASC-E. FIG. 6a shows the expression of PGC-1a, p-CREB, and p-CREB in the brain of RAS mice treated with hASC-
( * P < 0.05), and Figure 6b shows the results of Western blotting (left) showing that Akt was up-regulated and beta-actin in the neuro-2a cells treated with hASC- (Left) and p-CREB (right) ( * p &lt; 0.05).
Figure 7 shows changes and statistical differences in the expression levels (N, normal; C, control; A, hASCs-E) in microarray analysis of hASC-E treated brain after permanent MCA occlusion in rats ( p < 0.05, Mann- Whitney U test), and C / N or A / C represents the ratio of C: N or A: C, respectively.

In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating an adipocyte stem cell extract or a neurological disease comprising the same.

To date, there has been no application of adipose stem cell fusion as a therapeutic agent for neurological diseases as described below.

As used herein, the term "adipose-derived stem cell" (ASC) refers to isolated, plastic-adherent pluripotent cells and includes adipose-derived stem cells (ADS), adipose-derived adult stromal cell, adipose-derived stromal cell (ADSC), adipose stromal cell (ASC), adipose mesenchymal stem cells (AdMSC), lipoblast, pericyte, preadipocyte and PLA cells (processed lipoaspirate). Adipose stem cells have fibroblast-like morphology and express many CD markers such as CD10, CD13, CD29, CD34, CD44, CD54, CD71, CD90, CD105, CD106 and CD117 and can be propagated in Invitro, Gt; cells. &Lt; / RTI &gt; The adipose stem cells according to the present invention are those directly extracted from the tissue of the subject, or cultured in Invitro or cells engineered to express a specific recombinant protein.

The adipose stem cells used in the preparation of an extract of adipose stem cells according to the present invention can be easily obtained from mammals through known methods. For example, it can be easily separated from the product of liposuction or subcutaneous adipose tissue, and the separated cells can be easily cultured and amplified in Invitro. See, for example, Rodriguez AM, et al., Biochimie 87 (1): 125-128, 2005; Zannettino AC, et al., J Cell Physiol 214 (2): 413-421, 2008; Traktuev DO, et al., Circ Res 102 (1): 77-85, 2008.

The term "extract ", as used herein, refers to an extract that lyses, ruptures and / or separates cells, including one or more of natural or recombinant biological material such as proteins, nucleic acids or lipids, Or permeabilization (poration), which is produced by using the entire cell or a part thereof.

The extract according to the present invention may comprise a soluble and / or insoluble fraction, and in one embodiment a soluble fraction is used. In one embodiment, the soluble fraction is a protein, polypeptide, or peptide, DNA, RNA component, and / or lipid component. In other embodiments, the soluble fraction comprises a protein, polypeptide, or peptide component. In another embodiment, the non-soluble fraction comprises a fat component.

Fusion, crushing or perforation of cells for preparing a cell extract can be accomplished by physical and chemical methods and include, for example, freezing and thawing, ultrasonication, hyaluronidase, dispase, collagenase, Enzymatic treatment, including, for example, enzymatic treatment, and the like, and chemical treatment such as DTT (Dithiothreitol). For example Taranger CK et al., Mol Biol Cell 16 (12): 5719-35, 2005; Cho HJ et al., Blood 116 (3): 386-95, 2010; Lee ST, et al., Plos One 6 (7): e21801, 2011; Jeon D, et al., Epilepsia. 52 (9): 1617-26, 2011.

The term "neurological disease or symptom" as used herein is a disease or symptom that affects the brain, spinal cord, or peripheral nerve, and includes structural, chemical, and electrical abnormalities of the nervous system, resulting in paralysis, Disorder, convulsive seizure, and the like.

The term "neuroprotection" as used herein means protecting nerve cells of the brain, central nervous system or peripheral nervous system (especially in the brain or spinal cord) from killing and / or damage. It also means to delay or prevent the treatment, the onset of a neurological disease affecting the nervous system due to the above protection.

In another aspect herein, "neuroprotection" includes neuroprotection after injury of the brain, central nervous system, or peripheral nervous system, where damage is due to chemical, toxic, infectious, radiation and / or traumatic injury, But is not limited thereto.

The extract according to the present invention or a composition containing the same exhibits an effect of delaying disease progression by controlling direct neuroprotective effect or pathological mechanism of disease.

Accordingly, the extract or composition according to the present invention can be used for the prevention or treatment of various diseases which are effective by direct neuroprotection or which slow down disease progression by controlling the disease pathology. Such diseases include, but are not limited to, acute, subacute, or chronic neurodegenerative diseases.

By way of example and not limitation, the extract or pharmaceutical composition according to the present invention is due to the activation of the pCREB-PGC-1 alpha pathway. Thus, in another aspect, it can be used in a variety of diseases that can be prevented or treated by activation of the pCREB-PGC-1 alpha pathway.

As used herein, "acute neurodegenerative disease" includes, but is not limited to, various types of diseases associated with the death or impairment of nerve cells, including cerebral blood flow deficit, local brain trauma, brain injury and spinal cord injury, , Reperfusion following embolic occlusion and thrombotic occlusion, acute ischemia, perinatal hypoxia-ischemic injury, cardiac arrest and any type of intracranial hemorrhage (e. G., Epidural, subdural, subarachnoid and intracerebral) Includes cerebral ischemia or cerebral infarction due to my lesions (including bruises, penetrating, compression, and lacerations). In one embodiment, the acute neurodegenerative disease is the result of stroke, cerebral infarction, cerebral hemorrhage, head injury, or spinal cord injury.

"Subacute neurodegenerative disease" as used herein means a neurological dysfunction disorder occurring over a period of several days or weeks, including, but not limited to, dehydrative diseases such as multiple sclerosis, neurogenic tumor syndrome, subacute combined degeneration, subacute necrotizing encephalitis, and subacute sclerosing encephalitis.

The term "chronic neurodegenerative disease" as used herein includes, but is not limited to, senile dementia, vascular dementia, diffuse white matter disease (Vince Disease), dementia of endocrine or metabolic origin, dementia caused by head trauma and diffuse brain injury, Alzheimer's disease, peptic disease, diffuse rheumatoid disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration (Schie-Drager's syndrome), neurodegeneration Amyotrophic lateral sclerosis, incompatibility, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's Diseases, Parkinson's disease, synucleinopathies, primary progressive aphasia, ancestral malformation, macado-Joseph disease / spinal cord cerebellum (Including atopic dermatitis), motor neuron diseases including olivary schwannomas, giardi &lt; RTI ID = 0.0 &gt; Lutret &lt; / RTI &gt; disease, training and false training paralysis, spinal cord and spinal cord training muscle atrophy (Kennedy disease), multiple sclerosis, Welfare-Kugelberg-Welander disease, ankylosing spondylitis, progressive multifocal &lt; RTI ID = 0.0 &gt; Prion diseases including cerebral palsy, hereditary autonomic ataxia (Lilly-Day Syndrome), Creutzfeldt-Jakob, Gerstmann-Straussler-Shinkler disease, Kurus disease and fatal hereditary insomnia, or cerebral palsy. In one embodiment, the chronic neurodegenerative disease includes Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis or cerebral palsy.

The composition according to the present invention provides a protective effect against nerve damage caused by the above-mentioned neurological diseases and is administered to a subject in need of prevention or treatment of a neurodegenerative disease in an effective amount in a preventive or therapeutic manner, Can be effectively used for prevention or treatment.

A therapeutically effective amount means an amount necessary to alleviate, ameliorate, or alleviate one or more symptoms of a neurodegenerative disease.

As used herein, the term "treatment" refers to any action that improves or alleviates the symptoms of obesity or a disease caused by the administration of a composition according to the invention. Those skilled in the art will be able to ascertain the precise standards of obesity and determine the degree of improvement, improvement, and cure based on the data presented by the Korean Medical Association.

As used herein, the term "prophylactic " means any act that inhibits or delays the onset of obesity or other diseases resulting therefrom upon administration of the composition herein. It will be apparent to those skilled in the art that compositions of the present invention having therapeutic effects on obesity can prevent such diseases when taken prior to the appearance of the initial symptoms or symptoms of obesity.

The composition of the present invention comprises the above-mentioned extract according to the present invention as an active ingredient, and additionally, one or more kinds of active ingredients exhibiting the same or similar functions or a compound which maintains / increases the solubility and / May be further contained.

The extract or composition according to the present invention may also be used alone or in combination with methods using surgery, drug therapy and biological response modifiers.

In addition to the above-mentioned active ingredients, the composition of the present invention may further comprise at least one pharmaceutically or physiologically acceptable carrier.

The carrier as used herein means a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. Examples of such carriers include buffers such as saline, Ringer's solution, buffered saline, phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, Or immunoglobulins; Other hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and glucose, or other carbohydrates including mannose, chelating agents such as EDTA, For example, mannitol or sorbitol, salt-forming counter-ions such as sodium, and / or non-ionic surfactants such as tweens, polyethylene glycol (PEG) and PLURONICS.

If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administering the extract or composition of the present invention is not particularly limited thereto, and the known method of administering the inhibitor can be applied. The method of the present invention can be applied to parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, Or parenteral administration. In the case of parenteral administration, it can be administered through a patch type nasal / respiratory patch applied to the skin. In order to obtain a rapid therapeutic effect, administration by intravenous injection is preferable.

The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. Particularly, parenteral administration may be preferred for protein preparations, Path and means are not excluded. For typical drugs, the dosage unit comprises, for example, from about 0.01 mg to 100 mg, but does not exclude the following ranges and ranges above. The daily dose may be about 1 g to 10 g, and may be administered once a day or divided into several times a day.

In another aspect, the disclosure also relates to a method of making an adipose stem cell extract. In one embodiment, the method comprises: a) providing an adipose stem cell; b) fusing, crushing or permeabilizing said adipose stem cells in a buffer to produce a cell suspension.

In one embodiment according to the present application, the method may further comprise fractionating the cell suspension into a soluble and a non-soluble material.

In one embodiment according to the present invention, the adipose stem cells are fused. In another embodiment, the adipose stem cells are treated with collagenase, washed with phosphate buffered saline, and then thawed by sonication.

The method according to the present invention further comprises the step of treating the adipose stem cells with collagenase and washing with phosphate buffered solution before step b).

In place of step b) in the method according to the invention, said adipose stem cells are treated with collagenase, washed with phosphate buffered solution and then melted by ultrasonication.

In another aspect, the present invention also provides an adipocyte stem cell extract prepared by the above method, and a pharmaceutical composition for preventing or treating a neurological disease, comprising the same, as described above.

Hereinafter, examples for the purpose of facilitating understanding of the present invention will be described. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental Method

Production of human adipose stem cells and production of adipose stem cell extract (hASC-E)

The adipose stem cells used in the examples of the present application were obtained from human adipose tissue with the approval of the Bioethics Review Committee of Seoul National University.

To obtain adipose stem cells, human adipose tissue was transfected and cultured in PBS containing 0.075% collagenase type I tissue solution (Invitrogen, USA) for 1 hour at 37 DEG C with gentle shaking. The treated tissue was centrifuged at 200 xg for 10 minutes, then the upper fat cell fraction was removed and the lower stromal fraction (pellet) was treated with a red blood cell fusion buffer (Sigma, USA) for 10 minutes at room temperature. Thereafter, the mixture was centrifuged at 200 xg for 10 minutes using a 100 μm pore nylon membrane, and then filtered. The filtrate was cultured in a suspension of endothelial growth medium, 2 MV (EGM2 MV; Lonza, USA).

For the production of human ASC-E, cultured ASCs were harvested with cold PBS and centrifuged at 200 xg for 5 minutes. ASC was suspended in extraction buffer (1 mM DTT, 1 mM EDTA, protease inhibitor cocktail P8340, 0.1% DEPC in PBS) and then thawed several times with a needle connected to a syringe. The melt was then centrifuged at 14,000 xg for 15 minutes and the supernatant was filtered through a syringe filter unit (0.45 mu m). DMEM (Dulbecco's modified Eagles medium, Welgene, Korea) was used as an extraction buffer for in vitro treatment. 100 ug of extract (hASC-E) was prepared using about 5x10 ⁶ cells.

Human fibroblast cultures and extracts were prepared as previously described (Jeon, D., et al. 2011. Epilepsia 52, 1617-1626).

Neuro-2a culture and hASC-E treatment

Neuro-2a (American Type Culture Collection, USA) was cultured in DMEM medium containing 10% fetal bovine serum, 100 U penicillin and 0.1 mg / mL of streptomycin at 37 ° C in 5% CO ₂ /95% Lt; / RTI &gt;

Two days after seeding, Neuro-2a cells were treated with 10 [mu] g / ml of hASC-E prepared in Example 1 for 24 hours. Cells were harvested and then thawed in RIPA (Radio Immuno Precipitation Assay) buffer containing protease inhibitor and phosphatase inhibitor (Roche, USA). The cell fusion residues were removed by centrifugation, and the protein concentration was measured using a BCA kit (Pierce, USA) according to the manufacturer's instructions.

Analysis of ischemia and cell viability in vitro

In vitro ischemia and cell viability assays were performed as previously described (Jung et al., 2006 Neurosci. Lett., 394, 168-173, Park et al., 2009 Neurosci. Lett. Briefly, mouse neuroblast (Neuro-2a, American Type Culture Collection, USA) was placed in each well of a 96-well plate at a density of 5000 cells per well and contained 10% fetal bovine serum, 100 U penicillin and 0.1 mg / mL streptomycin The cells were cultured in DMEM medium at 37 ° C, 5% CO ₂ /95% atmosphere for one day. Then, the medium was replaced with a culture medium containing 2 μM hASC-E (100 μg / well), and cultured in the same manner as above. Treated with PBS, heat-treated hASC-E, DNA, RNA, or lipid components as a control or comparison group. On the third day of culture, OGD (oixygen deprivation, no glucose and sodium pyruvate, O ₂ levels 1-1.5%) was induced for 16 hours using a humidified hypoxic chamber (Bactron 1.5, Sheldon Manufacturing, USA) . DNA, RNA, lipid, and heat-treated hASC-E (heated at 100 for 10 minutes) were obtained using the same volume of adipose stem cells as used for the preparation of 100 μg of hASC-E.

For simultaneous treatment, Neuro-2a cells were treated with hASC-E (100 μg / well) or PBS and immediately induced OGD conditions as above.

Cell viability and cytotoxicity were measured by WST-1 reagent (5 mmol / L, 1: 9, Roche, Mannheim, Germany) and lactate dehydrogenase (LDH, CytoTox 96, Promega, USA) Lt; / RTI &gt; All experiments were performed in triplicate and the results were titrated to the ratio of each non -OGD value.

Stroke model and administration of hASC-E

Stroke models including transient and permanent MCA occlusion and intracerebral hemorrhage (ICH) were performed using male Sprague-Dawley rats (weight, 240-260 g, Orient Bio, Korea) and male BALB / c (Chu, K. et al., 2007, Stroke 38, 177-182; Wang et al., 2002) using the mouse (weight, 22-25 g, Orient Bio). Lee, ST, 2008. Brain 131, 616629). One hour after stroke induction, hASC-E (40 mg / kg per abdominal administration per animal) and the same volume of the fusion buffer control medium and fibroblast-E (40 mg / kg per abdominal administration per animal) Permanent Tamura MCA electrocoagulation was used as an additional cerebral infarction model. In this model, hASC-E was administered intraperitoneally 3 hours after induction of cerebral infarction. Arterial blood pressure, blood gas and blood glucose were measured during all strokes and rectal temperature was maintained at 37 ± 0.5 ℃.

Ischemic volume measurement

After 24 hours of ischemic induction, the volume of the ischemic volume (infarct and border area) was performed as previously described using TTC (2,3,7-triphenyltetrazolium chloride) (Chu, K. 2007. Stroke 38, 177 -182). In summary, after brain extraction, the frontal tip was cut into 1 mm thickness and immersed in 2% TCC solution. After staining the sections with 4% paraformaldehyde in PBS, the ischemic area and hemispheric area of each section were traced and measured using an image analysis system (Image-Pro Plus, Media Cybernetics, USA). To correct for edema due to brain edema, the ischemic volume was calibrated in the following manner: Corrected ischemic volume value: measured ischemic area × 1 - {[(ipsilateral hemisphere area - contralateral hemisphere area) / contralateral hemisphere ]}. Ischemic volume was measured by two independent authors with no prior knowledge of the type of section, expressed as a percentage of the total hemispherical volume.

Measurement of ischemic volume and moisture content of brain in ICH model

In the ICH model, ischemic volume and brain water content were measured as previously described (Lee, S. T. 2008. Brain 131, 616-629). Briefly, after brain was harvested, it was divided into two hemispheres along the center line, immediately weighed, and dried in an oven at 100 ° C for 24 hours to determine the dry weight. The moisture content was calculated as ((wet weight dry weight) / wet weight) 100 (%).

Ischemic volume was measured using Drabkin's reagent (Sigma, USA) according to the manufacturer's instructions. The ICH hemispheric brain was separated, blood was removed, homogenized and sonicated. Followed by centrifugation at 12,000 rpm for 30 minutes. The supernatant (04 ml) was mixed with Drabkin's reagent and allowed to stand at room temperature for 20 minutes. The absorbance at 540 nm was measured. Absorbance was compared with the standard curves (0, 2, 4, 8, 16, 32, 50, and 100 μl) prepared using normal hemispheres and allogeneic blood.

Huntington's disease animal model and hASC-E administration

Transgenic HD mice and wild type (WT) female mice (Jackson Laboratories, USA) of R6 / 2 series (B6CBATg (HDexon1) 62Gpb / 3J, 111 CAGs) were used. R6 / 2 transgenic mice express exon 1 of human mHtt and are widely used as animal models of HD. Mice were randomly grouped and 12 h light / dark cycle, water and feed were taken freely. Genotypes were verified by PCR analysis.

Disease in R6 / 2 mice occurs at 8 weeks of age. In consideration of the protective effect of hASC-E, the extract of the present invention was administered twice per week (40 mg / kg) from 6 weeks of age and the control mice received only the buffer solution (medium) used for extracting the same volume. hASC-E was prepared just before use.

Rotaroad performance and weight measurement

Rotarod was measured using a Rotarod apparatus (Jungdo Instruments, Korea). The mouse was placed on a rod accelerating at a rotational speed of 4 to 40 rpm over a period of 3 minutes. There was a 15-minute break between each test. Four-week-old mice were subjected to a three-day rotarod test three times a day for three days to practice. Three tests were performed, and the average latency time to fall was used for data analysis. Rotarod evaluations and weight measurements were measured weekly between 5 and 12 weeks (see FIG. 1A).

MLPT  Test

MLPT testing was performed as described previously (Chu, K. et al. 2007. Stroke 38, 177-182) before, one day and three days after induction of ICH induction. In summary, the rats were placed 10 cm away from the table, and the degree of stretching of the forelegs to the table was evaluated. In the normal case, 0 point was given, and abnormal bending was given 1 point. The rats were placed along the edge of the table, and then the forelimbs were suspended through the edges to allow the forelegs to move freely. Each front leg (second experiment: front leg; third experiment: hind leg) was gently pulled down and the retrieval placement was assessed. Finally, the rats were placed on the edge of the table, and the forelimbs were evaluated for lateral positioning. Three evaluation results were scored as follows: normal, 0 point; Delay (at least 2 seconds) and / or incomplete performance, 1 point; Failed to perform, 2 points. A total of 7 points represent the greatest neurological deficit, and 0 points to normal.

Immunochemistry and volume analysis of striatum

For immunochemical analysis, 12-week old mice were anesthetized and perfused through 10% cold saline and 4% paraformaldehyde in 0.1 M PBS through the heart. The brains were harvested and frozen with 4% 30% sucrose solution and cut with 20 μm sections. The free floating sections were washed, blocked with normal goat serum and stained with EM48 mHtt antibody (1: 300; Millipore). The next day, the sections were washed 3 times with PBS and incubated with FITC conjugated anti-rabbit IgG (1:50; Jackson Immuno Research Laboratories) for 2 hours. In order to measure the striatal volume of R6 / 2 mice, a 200 μm coronal tissue section, which was continuously cut from the mouth to the anterior border, was used for Nissl staining. The area of the striatum and the area around the striatum were defined in each serial section, and then the total volume was measured using Image-Pro Plus (Media Cybernetics, USA).

Protein Extract and Western Blot Analysis

Brains of R6 / 2 mice were harvested, frozen immediately in liquid nitrogen and stored at -70 &lt; 0 &gt; C. The cultured Neuro-2a cells were washed and recovered in PBS. Protein extracts were prepared with RIPA (Radio Immuno Precipitation Assay) buffer containing protease inhibitor and phosphatase inhibitor (Roche, USA). 30 μg of total protein was electrophoresed on 10% SDS-PAGE and transferred to PVDF membrane. (1: 1000; Cell signaling), CREB (1: 1000; Cell signaling), EM48 (1: 200; Millipore ) And cultured with a horseradish peroxidase-conjugated anti-mouse or rabbit antibody (1: 5000; GE Healthcare) as a secondary antibody, and developed with an ECL kit. After color development, signals were quantified using ImageJ software.

Analysis of gene expression changes through microarray

24 hours after induction of ischemia, whole brain except cerebellum of rat subjected to permanent local cerebral ischemia was extracted and used. After the homogenization of the extracts, TRIzol (Gibco) was used in accordance with the manufacturer's instructions and RNA (N, normal, without any treatment, n = 3; C, control, occlusion and treated with lysate buffer, n = 3; A, hASCs-E , occlusion and treated with hASCs-E, n = 3). Genes with altered expression levels were classified according to their function. Statistical difference between C and N and statistical difference between A and C ( p <0.05, MannWhitney U test) were used as a data analysis criterion, showing more than double difference in C: N (C / N) ratio. Hybridization in situ Hybridization kit (Agilent, USA) was used for the hybridization of Agilent oligonucleotides. Data analysis was performed using a GenePix 4000B scanner (Axon Instruments, USA).

Microarray analysis was also performed for the Neuro-2a model, which was an in vitro co-processed OGD and hASC-E (or control medium). In order to minimize the effect of the mutation among the groups, three RNA samples per group were combined to form one microarray sample (No-OGD + control, No-OGD + hASC-E, OGD + And OGD + hASC-E).

RT-PCR (reverse transcription polymerase chain reaction) using brain and spleen in ICH model

To analyze the anti-inflammatory effect of hASC-E treatment in the ICH model, RT-PCR analysis was performed. ICH rats (n = 3 per group, PBS or hASC-E treated) were sacrificed at 24 hours after ICH induction and RNA was extracted from the brain and spleen using TRIzol (Gibco) according to the manufacturer's instructions. First strand cDNA was synthesized using the cDNA Synthesis Kit (Roche, USA) according to the manufacturer's instructions and amplified using the following primer sets and conditions: 25 cycles of 95, 58 and 72 ° C for 40 sec each : Tumor necrosis factor-α: 5'-TAC TGA ACT TCG GGG TGA TTG GTCC-3 '(sense) and 5'-CAG CCT TGT CCC TTG AAG AG ACC-3'(antisense); IL-6 (interleukin-6): 5'-CTT GGG ACT GAT GTT GTTGAC-3 '(sense) and 5'-TCT GAA TGA CTC TGG CTT TG-3' (antisense). After amplification products were separated from agarose gels, they were scanned and analyzed using ImageJ software NIH, Bethesda, MD, USA). mRNA expression levels were optimized for GAPDH concentrations.

Statistical analysis

All results are expressed as mean ± standard error. Rotarod performance and body weight were analyzed weekly using the Fishers post hoc test ANOVA. Western blot and tissue analysis were performed using Student's t-test. 0.05 was considered statistically significant. Data were analyzed using SPSS version 17.0 (SPSS Inc., USA).

Experimental Example 1. Improvement of cell viability by hASC-E

In order to investigate the protective effect of neurons, hASC-E was treated with Neuro-2a cells cultured as described in the experimental section. The results are shown in FIG. As shown in Fig. 1A, the cells treated with the extract according to the present invention were found to have long neurites. In contrast, the control (heat-treated hASC-E, DNA, RNA, and lipid) was smaller, denser, rounded, aggregated, and curled to have atrophied or reduced neurite outgrowths. As shown in Figs. 1B and 1C, hASC-E treated cells also showed improved results in terms of life compared to the control group. In addition, as shown in Fig. 1 (d), even when the extract was treated with OGD at the same time, the life was improved compared to the control group.

These results indicate that the extract according to the present invention has a neuroprotective effect on ischemia.

Experimental Example 2 Reduction of Ischemic Volume in a Local Cerebellar Ischemic Model by hASC-E Treatment

As described in the experimental section, the protective effect of hASC-E on hASC-E-induced ischemic nerve injury was investigated by treating hASC-E with a mouse model in which a temporary or permanent MCA (middle cerebral artery) occlusion was applied. The results are shown in FIG. In the case of permanent ischemia, as compared with the control PBS (n = 11, 261.24 ± 7.03 mm ³ ) in the hASC-E treated mice (n = 9, 174.02 ± 25.94 mm ³ ) Of the respondents.

In the case of temporary ischemia, as shown in FIG. 2B, the volume (n = 10, 107.44 ± 24.00 mm ³ ) of mice treated with hASC-E 1 hour after ischemia compared with the control group (n = 11, 182.48 ± 12.91 mm ³ ) 41%, respectively. When treated with fibroblast extract, the volume of ischemia was increased. (n = 10, 259.81 +/- 26.90, P < 0.01).

In addition, as shown in FIG. 2C, 43% ischemic volume decreased after 3 hours of induction of permanent electrocoagulation in the cortical (MCA electrocoagulation) (hASC-E, n = 9, 21.99 ± 6.62 mm ³ ; control group n = 8, 38.72 ± 2.43 mm ³ , p <0.05). When treated with fibroblast cell extract, there was no significant difference from control group.

Experimental Example 3: Inhibition of neural degeneration by hASC-E in the ICH model, decrease of brain moisture content and decrease of ischemic volume

To investigate the effect of hASC-E in the ICH model as described in the Experimental Methods section, it was administered to the ICH model. The results are shown in FIG. FIG. 3A shows the water content of the brain, and FIG. 3B shows the MLPT test result. As shown in FIG. 3A, when the hASC-E treatment was performed 1 hour after ICH induction, the moisture content of the ischemic brain hemisphere decreased compared to the control group (control group n = 11, 80.65 ± 0.14% 6, 80.04 ± 0.11%, fibroblast-E, n = 6, 80.96 ± 0.16%, p <0.01). On the other hand, there was no difference in the results between the treated group (n = 7, 12.52 ± 2.48) and the control group (n = 8, 21.70 ± 3.07, p <0.05) in the brain hemisphere at the non-ischemic site as shown in FIG. 3b. As shown in FIG. 3C, the hASC-E treated group showed a reduced score in the MLPT test as compared to the control group. On day 1, control = 5.78 + 0.15, hASC-E = 4.170.60, fibroblast-E = 6.00 + 0.29); 3, fibroblast-E (control), n = 9, hASC-E, n = 6, fibroblast-E , n = 6. Fibroblast extract and medium control showed similar results.

The results show the neuronal recovery effect by treatment of hASC-E according to the present invention.

Experimental Example 4. Improvement of R6 / 2 mouse behavior by hASC-E

To investigate the effect of hASC-E on the behavioral binding of R6 / 2 mice, hASC-E was injected into mice at 6 weeks of age as described in the experimental section. The untreated mice showed progressive weight loss between 10 and 12 weeks. On the other hand, mice treated with hASC-E showed a decrease in weight loss at 12 weeks of age. (22.1 ± 0.2 vs. 24 ± 0.9, p <0.01) (see FIG. 4). In the RotaRod test, hASC-E treated mice showed increased rotavirus run time compared to vehicle control group (Groups 10, 11, p < 0.05) at 12 weeks (p < 0.01) (see FIG.

These results indicate that hASC-E prevents weight loss and increases motor function in Huntington transgenic mice.

Experimental Example 5. Reduction of striatum and mutant Htt aggregation in R6 / 2 mice by hASC-E treatment

R6 / 2 mice exhibit mHtt aggregation of striatum and striatum and cortex during disease development. To investigate the histological changes of the brain, striate volume and mHtt agglutination were examined in 12-week-old mice. As described in the Experimental Methods section, mHtt agglutination studies stained brain tissue sections with EM48 antibody, which detects the aggregation of mutant hunting tones. The results are shown in FIG. 5A, in which mHtt aggregation in the striatum was reduced by hASC-E treatment, but not in the cortex. Proteins were then extracted from mouse brain and mHtt agglutination was measured using Western blot. The results are shown in Figures 5b and 5c. In the brain of hASC-E treated group, mHtt aggregation was decreased compared to the medium control group. After measuring the volume around the striatum and striatum, the ratio of the striatum to the surrounding striatum was calculated. The results are shown in Figures 5d and 5e. As shown in the figure, the ratio of striatum to striatum was decreased in hMSC-E treated R6 / 2 mouse brain compared with medium control brain (0.22 ± 0.01 vs. 0.25 ± 0.01, p <0.01, n = 7 ).

These results indicate that hASC-E modulates the pathology of Huntington's disease and inhibits the progression of the disease.

Experimental Example 6. Activation of CREB and PGC-1 and nerve cells in R6 / 2 mice by hASC-E

In HD progression, the failure of the cAMP-responsive element binding protein (pCREB) -PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1) Inhibition of CREB activity results in PGC-1 disorder, which results in the formation of the HD striatum &lt; RTI ID = 0.0 &gt; (&lt; / RTI &gt; (Cui L, et al. 2006. Cell 127: 59-69). Considering that PGC-1 alpha activation has cytotoxicity induced by mHtt and protective effects against mitochondrial dysfunction, activation of the pCREB-PGC-1 alpha pathway may slow the invention and / or progression of HD .

Thus, in order to investigate the effect of the extract according to the present invention on this pathway, 12-week-old R6 / 2 mice were treated with hASC-E or medium for 6 weeks and Western blotting was performed as described in the experimental method. The results are shown in FIG. 6A, and the expression of p-Akt, p-CREB and PGC-1α (p, 0.05 vs. R6 / 2 control) was increased by treatment with hASC-E, but decreased in the control group.

In vitro experiments using Neuro-2a cells also revealed that the expression of p-CREB and PGC1α was significantly increased as shown in FIG. 6b. These results indicate that the CREB-PGC-1α pathway is activated by hASC-E. These results indicate that the extract of the present invention has a therapeutic effect through neurotransmitter CREB-PGC-1α pathway activation.

Experimental Example 7. Gene Expression Change by hASC-E Treatment

After 1 hour of ischemic induction, hASC-E was treated, and cell and molecular changes were analyzed by microarray. As a result, as shown in Fig. 7 and the following Tables 1-1 to 1-6 and 2-1 to 2-6 Similarly, the expression of genes related to inflammation and immune responses was down-expressed in ischemic rats treated with hASC-E (see Tables 1-1 to 1-6). In addition, expression of genes associated with apoptosis or apoptosis was inhibited in the brain of ischemic rats treated with ASC-E (see Tables 2-1 to 2-6).

[Table 1-1]

[Table 1-2]

[Table 1-3]

[Table 1-4]

[Table 1-5]

[Table 1-6]

[Table 2-1]

[Table 2-2]

[Table 2-3]

[Table 2-4]

[Table 2-5]

[Table 2-6]

These results are consistent with the results of microarray experiments using Neuro-2a cells. Comparing the results of the OGD + hASC-E and OGD + control experiments, it has been shown that the genes involved in the glomerular or immune response constitute a significant portion of the genes that are more than two-fold altered by hASC-E treatment (results not shown).

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (13)

A pharmaceutical composition for preventing or treating a neurological disease, comprising an adipocyte stem cell extract.
The method according to claim 1,
Wherein the prevention or treatment of the neurological disease is caused by activation of the pCREB-PGC-1? Pathway, regulation of inflammatory reaction or inhibition of cell death.
The method according to claim 1,
Wherein said adipose stem cells are autologous, allogeneic, or heterologous.
The method according to claim 1,
Wherein the adipocyte stem cell extract comprises a soluble and non-soluble fraction, a non-soluble fraction or a soluble fraction.
5. The method of claim 4,
Wherein said soluble fraction comprises a protein, polypeptide or peptide component, RNA, DNA, or lipid.
6. The method of claim 5,
Wherein the soluble fraction comprises a protein, a polypeptide or a peptide.
The method according to claim 1,
Wherein the prevention or treatment of the nervous system disease is by a protein, a polypeptide or a peptide contained in the adipocyte extract.
The method according to claim 1,
Wherein the nervous system disease includes acute, subacute or chronic neurodegenerative diseases.
9. The method of claim 8,
The acute neurodegenerative diseases include stroke, cerebral infarction, cerebral hemorrhage, head injury, or spinal cord injury. The subacute neurodegenerative diseases include dehydration diseases, neurogenic tumor syndrome, subacute combined degeneration, subacute necrosis A subacute necrotizing encephalitis, or a subacute sclerosing encephalitis. &Lt; Desc / Clms Page number 24 &gt;
9. The method of claim 8,
Such chronic neurodegenerative diseases include memory associated with senile dementia, vascular dementia, diffuse white matter disease (vandal dementia), dementia due to endocrine or metabolic origin, head trauma and dementia due to diffuse brain injury, boxer dementia and frontal dementia Chronic epilepsy associated with neurodegeneration, amyotrophic lateral sclerosis (Alzheimer's disease, Alzheimer's disease, Pick disease, diffuse rheisome disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration Amyotrophic lateral sclerosis, incompatibility dysfunction, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, ancestral black degeneration, macado-Joseph disease / spinal cord cerebral ataxia, (Kennedy disease), multiple sclerosis, primary sidewalk sclerosis, hereditary fascioliasis, Werdnihi-Hoffman's disease, Kugelberg &apos; s disease, - Welland disease, Tay-Sachs disease, Sandhoff disease, hereditary ankylosing spondylitis, Wohlfart-Kugelberg-Welander disease, Ankylosing spondylitis, Progressive multifocal white matter encephalopathy, Hereditary autonomic ataxia Lary-Day syndrome), Creutzfeldt-Jakob disease, Gerstmann-Straussler-Shinker disease, Kurus disease and prion diseases including fatal hereditary insomnia, or cerebral palsy. A pharmaceutical composition.
11. The method according to any one of claims 1 to 10,
Wherein said adipocyte stem cell extract comprises: a) providing adipose stem cells; and b) producing the cell suspension by dissolving, crushing or permeabilizing the adipose stem cells in a buffer solution to thereby prepare a cell suspension.
12. The method of claim 11,
Wherein the method further comprises fractionating the cell suspension into a soluble and a non-soluble material.
12. The method of claim 11,
Wherein the adipose stem cells are fused, treated with collagenase before the fusion, washed with a phosphate buffer solution, and then melted by ultrasonic treatment.

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