WO2013133650A1 - Pharmaceutical composition for preventing, alleviating or treating diseases associated with cranial nerves or spinal nerves, containing stem cell-conditioned culture medium powder - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing, alleviating, or treating diseases associated with the cranial nerves or the spinal nerves, containing a stem cell-conditioned culture medium powder. More specifically, the stem cell-conditioned culture medium powder is remarkably effective in alleviating ischemic strokes, and thus can be used for preventing, alleviating, or treating diseases associated with the cranial nerves or the spinal nerves. In addition, the present invention provides a novel, non-invasive drug delivery method through the nasal cavity, showing high pharmaceutical effects.

Description

A pharmaceutical composition for preventing, ameliorating or treating a cerebral or spinal nerve-related disease comprising stem cell conditioned medium powder

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating a cerebral or spinal nerve-related disease comprising a stem cell conditioned medium powder which uses a serum-free culture of stem cells to improve a brain or spinal nerve-related disease.

Cerebral infarction is the third-largest mortality disease in the United States, and most people who survive after it have to live life with irreversible neurological sequelae. Recombinant tissue plasminogen activator (rt-PA), the only thrombolytic drug approved by the US FDA, must be administered within 3 hours of cerebral infarction, and the actual success rate is only 30%, and the actual risk of complications such as cerebral hemorrhage is high. The drug can be used in a very limited number of subjects. Based on this reality, new treatments for the treatment of cerebral infarction have been developed until recently, and the most popular field is cell therapy.

There are various cell types that can be used as cell therapy products, but they are largely divided into embryonic stem cells and adult stem cells. Embryonic stem cells are differentiated into any cell of the human body and are expected to have a good effect due to their primitiveness. However, since human embryos should be used, there are major disadvantages of moral and ethical problems. Adult stem cells are typically bone marrow stem cells and fat stem cells. Cells, umbilical cord blood stem cells and the like, there is an advantage that can use the patient's own cells, there is a limitation in differentiation, there is a disadvantage that must be expanded in vitro. However, there are no ethical and moral limitations, and it has been in the spotlight as a cell therapy with a great potential for clinical application.

Tooth stem cells, which are a kind of adult stem cells, are mesenchymal stem cells that exist around nerves and blood vessels of pulp, which are a part of teeth, and the present inventors have recently developed a method of separating stem cells from teeth and multiplying them in large quantities. In addition, in vitro experiments, differentiation ability, such as proliferation is similar to other adult stem cells, and the ability to secrete various proteins was also reported to be excellent. In particular, conditioned media (CM) cultured dental pulp stem cells revealed that various proteins secreted by pulp stem cells exist, and vascular endothelial growth factor (VEGF) is excellent for neuroprotective substances and neovascular regeneration. It is known that it contains a large amount. These substances are expected to be excellent efficacy for various ischemic diseases including ischemic brain disease.

Stem cell therapy can be administered by direct intra-lesion graft and systemic administration. Intra-lesion direct transplantation has the advantage of preventing stem cell loss by directly transplanting stem cells to the lesions and acting directly on the lesions. However, invasive methods require general anesthesia for cerebral infarction and risk of secondary damage to the brain. have. Systemic administration is a method of transplanting stem cells through veins or arteries, which has a much more non-invasive advantage than direct transplantation.However, it is possible to administer stem cells to capillaries of other organs in the body (lungs, spleen, liver, etc.) Is trapped and stem cell loss is inevitable, and there are many questions about its efficacy due to limitations in moving to the brain through the brain-vascular barrier. Fortunately, many studies have been reported in which the neurological improvement effect is clearly observed even if there are no cells expected for brain lesions by remotely administering stem cells. Even though stem cells do not directly differentiate into damaged neurons, paracrine of various nutrients The theory that neurological function is improved by the effect is gradually gaining strength.

Therefore, a non-invasive and effective drug delivery method is required.

It is an object of the present invention to verify the various pharmaceutical efficacy of conditioned medium powder obtained from stem cell culture medium, and to use it for the prevention, improvement or therapeutic use of diseases related to brain or spinal nerve.

Another object of the present invention to provide a kit for the prevention, improvement or treatment of cerebral nerve or spinal nerve-related diseases using the conditioned medium powder.

In order to achieve the above object, the present invention comprises a conditioned medium powder, the conditioned medium powder is a brain or spinal nerve-related disease prevention, which is obtained by lyophilizing a culture obtained by culturing stem cells in a serum-free medium, Provided is a pharmaceutical composition for improvement or treatment.

The present invention also provides a kit for nasal administration for use in the prevention, amelioration or treatment of a brain or spinal nerve-related disease comprising a container containing the pharmaceutical composition.

According to the present invention, the conditioned medium powder of stem cells has an excellent effect on improving cerebral infarction, and thus can be used for preventing, ameliorating or treating cerebral nerve or spinal nerve-related diseases.

In addition, the present invention has the effect of providing a new drug delivery method through the nasal cavity, non-invasive and high pharmaceutical effect.

Figure 1 illustrates the MCAo experimental design for verifying the effect of improving the cerebral infarction of the stem cell conditioned medium powder of the present invention.

Figure 2 is a diagram for the MCAo model for verifying the effect of improving the cerebral infarction of the stem cell conditioned medium powder of the present invention, (a) indicates that the blood vessels are blocked when the MCA is blocked in the brain, the path through the suture (B) shows the location of the suture and the path of blood vessel progression.

Figure 3 shows the results of confirming the degree of cerebral infarction before cutting the brain tissue.

Figure 4 is an MRI photograph showing the cerebral infarction volume measurement results before and after treatment with the stem cell conditioned medium powder of the present invention.

5 is a result of confirming the part where the cerebral infarction appeared in the MRI picture of FIG.

Figure 6 shows the results of TTC staining in cerebral infarct animal model treated with the stem cell conditioned medium powder of the present invention.

7 is a graph measuring the size of the cerebral infarction portion using the image J program of the TTC staining result of FIG.

Figure 8 shows the EBST results of the cerebral infarction animal model treated with the stem cell conditioned medium powder of the present invention.

Figure 9 shows the step test results of the cerebral infarction animal model treated with the stem cell conditioned medium powder of the present invention.

Figure 10 is the result of measuring the weight of the cerebral infarct animal model treated with the stem cell conditioned medium powder of the present invention.

Figure 11 shows the degree of apoptosis after treatment with the stem cell conditioned medium powder of the present invention to the irradiated brain cortical neurons.

Figure 12 shows the changes in the cells after the treatment of the stem cell conditioned medium powder of the present invention after irradiating the brain cortical neurons.

Figure 13 shows the results of Western blot analysis using cells after treatment of the stem cell conditioned medium powder of the present invention to the irradiated brain cortical neurons.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present inventors concentrated and freeze-dried the conditioned medium obtained by culturing stem cells derived from the pulp of the child's teeth (teething) to prepare a powder form, and then tested whether the neurological function is improved in the cerebral infarction model. To this end, cerebral infarction induced a cerebral infarction in the white paper and dissolved conditional medium powder through the nasal cavity for a certain period of time and observed for 4 weeks, the area of cerebral infarction was reduced and excellent neurological improvement effect was confirmed. It was. Through this, various types of test substances were delivered to the animal model of cerebral infarction (white paper). for example,

Powder A is a freeze-dried conditioned medium obtained after culturing pulp-derived stem cells in DMEM, a basic medium, with 10% bovine serum, and incubating in serum-free DMEM when the confluency reaches 70-80%. ,

B powder was obtained by incubating pulp-derived stem cells with alphaMEM, a basic medium, by adding 10% bovine serum, growth factor and ascorbic acid, and then incubating in serum-free alphaMEM when confluency reached 70-80%. The conditioned medium is lyophilized,

C powder was cultured by adding 10% bovine serum, growth factor and ascorbic acid to pulp-derived stem cells in alphaMEM, which is the basic medium, and then 10% NSCM in serum-free alphaMEM when confluency reached 70-80%. After culturing by adding powder (B powder), the conditioned medium obtained is lyophilized.

After inducing a cerebral infarction model in white paper, the A, B, and C powders were delivered to the cranial nerve through the nasal mucosa.

EBST (elevated body swing test), step test (step test), and other weight measurements are carried out to perform a movement test.The results are analyzed by RI imaging and TTC staining before and after infarction volume (infarction volume) by image J Confirmation was made using the program.

In vitro experiments used brain cortical neurons to induce brain injury through radiation to reach apoptosis and then process A, B, and C powders for photo analysis, cell counting and Western blot. Quantitative analysis was performed (see FIG. 1).

As a result, MRI and TTC staining in the cerebral infarction model showed that the primary pulp stem cell conditioned medium powder had an excellent effect on the improvement of cerebral infarction and improved neurological behavior in the neurorobehavior test. It was found that the same effect was seen in brain cells.

In addition, direct infusion, I.P., I.V. treatment through the nasal cavity was found to be much more non-invasive and more effective.

In addition, in the A, B, C conditioned medium powder, C than A, B powder than C was more effective in improving the brain or spinal nerve-related disease function. The C powder is added to the B powder when cultured in the serum-free medium of the stem cells, provided an environment that offsets the starvation state in the medium according to the addition of the B powder to the active ingredients such as growth factors, cytokines into the medium It is thought to be less than B powder. Therefore, the improvement effect in the cerebral infarction model is considered to be low compared to the B powder.

Accordingly, the present invention comprises a conditioned medium powder, wherein the conditioned medium powder is a drug for preventing, improving or treating cerebral or spinal nerve-related diseases, which is obtained by lyophilizing a culture obtained by culturing stem cells in a serum-free medium. To provide a composition.

Conditioned medium powder of the present invention is characterized in that after culturing the stem cells in the appropriate medium and conditions, the conditioned medium (serum-free medium culture solution) containing the active ingredient is used by freeze drying.

According to one embodiment, the conditioned medium powder of the present invention contains a large amount of the active ingredient in the conditioned medium powder, a serum-free medium culture of stem cells, the powder has an excellent effect on preventing, improving or treating diseases related to the brain or spinal nerves. It can be to have.

The cerebral nerve or spinal nerve-related disease may be cerebral infarction, stroke, inflammatory disease, brain injury, brain trauma, Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis or mania, but is not particularly limited thereto.

The method for producing a conditioned medium powder of the present invention may be obtained by freeze-drying a culture medium, ie, a conditioned medium, obtained by culturing stem cells in a serum-free medium.

In the present specification, "conditioned medium" refers to a medium in which only a culture medium is collected after exchanging stem cells with a serum-free medium when the stem cells reach an algebraic growth stage, which is a cell division peak. This means using an unknown growth factor, cytokine, which is extracted from the stem cells in dividing cells in the medium. According to one embodiment, the conditioned medium is a growth factor derived from the pulp-derived mesenchymal stem cells, comprising a solution in which the pulp-derived mesenchymal stem cells are removed after culturing the pulp-derived mesenchymal stem cells in a serum-free medium. It may contain abundant substances such as cytokines.

The stem cells are human backpack, placenta, umbilical cord, umbilical cord blood, skin, peripheral blood, bone marrow, fat, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, omnilateral ligament, joint It may be at least one selected from the group consisting of mesenchymal stem cells isolated and / or cultured from tissues in which mesenchymal stem cells, including chondrocytes, teeth, perivascular cells, striatum, subpatellar fat mass, spleen and thymus, are present.

More specifically, the stem cells may be mesenchymal stem cells derived from pulp exhibiting the following characteristics:

(a) all show positive immunological characteristics for CD90, CD105, CD44, and CD73, (b) adhere to and grow in cell culture dishes, and exhibit morphological properties of the spine-shape; (c) It is characterized by having the ability to differentiate into mesodermal derived cells.

In the present specification, "mesenchymal stem cells (MSCs)" are cells that assist in making cartilage, bone, fat, myeloid epilepsy, muscle, nerves, etc., in adults, although they generally stay in the bone marrow, the cord blood It also exists in the peripheral blood, other tissues, etc., means a cell that can be obtained from them. For the purposes of the present invention, mesenchymal stem cells of the present invention means mesenchymal stem cells derived from pulp.

In the present invention, "differentiation" refers to a phenomenon in which a cell's structure or function is specialized during cell division and proliferation. Pluripotent mesenchymal stem cells can differentiate into lineage-defining progenitors (eg, mesodermal cells), and then further differentiate into other forms of progenitors (eg, osteoblasts, etc.), followed by specific tissues (eg, May be differentiated into terminal differentiated cells (eg, adipocytes, bone cells, chondrocytes, etc.) that play a characteristic role in bones, etc.).

In addition, the serum-free medium may be used without limitation as long as it is a medium for the purpose of maintaining and storing the cell type as a medium for initial cell culture without serum. For example, DMEM (Dulbecco's Modified Eagle's Medium), aMEM (alpha Minimal essential Medium), RPMI (Roswell Park Memorial Institute medium) and the like can be used.

The serum-free medium may further include growth factors or cytokines in the medium.

IGF, bFGF, TGF, HGF, EGF, VEGF or PDGF, etc. may be used alone or two or more as the growth factor, but is not particularly limited thereto.

As the cytokine, IL-1, IL-4, IL-6, IFN-γ, IL-10 or IL-17 may be used alone or in combination of two or more, but is not particularly limited thereto.

The conditioned medium can be prepared by culturing the cells in serum-free medium for 1 to 3 days at 25 to 40 ℃, but is not particularly limited thereto.

According to one embodiment, the step of preparing a conditioned medium using stem cells

Subcultured cells derived from primary teeth, pulp, periodontal ligament, alveolar bone, root tooth or gum from serum containing medium to obtain mesenchymal stem cells derived from pulp; And

The mesenchymal stem cells may comprise the step of culturing in serum-free medium.

The mesenchymal stem cells can be obtained through the purification process among the cells present in the teeth, teeth, pulp, periodontal ligament, alveolar bone, tooth root or gum, the process of extracting stem cells according to the characteristics of each tissue is known It is well known to those skilled in the art that the method can be used, and currently, cell lines which can use human-derived mesenchymal stem cells for research purposes can be easily and easily obtained mesenchymal stem cells.

Processes essential for the culturing of mesenchymal stem cells have been published in the literature, and biopsies extracted from sources through known methods prior to cell culture, ie teeth, pulp, periodontal ligament, alveolar bone, Tooth roots or gums can be reduced to a minimum in the subsequent culture by repeated washing process using a common cleaning medium containing antibiotics.

The medium for initial cell culture, including serum, is preferably a medium for the purpose of maintaining and storing cell types such as mesenchymal stem cells. In the present invention, DMEM (Dulbecco's Modified Eagle's Medium) and aMEM (alpha Minimal Essential Medium), which are generally used for cell culture, are used and include serum commonly used in cell culture.

Serum should preferably contain 0.1-20% fetal bovine serum (FBS) and antibiotics, antifungals and reagents to prevent the growth of mycoplasma causing contamination.

As antibiotics, antibiotics commonly used in cell culture such as penicillin, streptomycin, or fungizone can be used. It is preferable to use amphotericin B as an antifungal agent and tyrosine as a mycoplasma inhibitor, and may prevent mycoplasma contamination with gentamicin, ciprofloxacin, azithromycin, and the like.

If necessary, oxidative nutrients such as glutamine and energy metabolites such as sodium pyruvate can be further added.

General culture conditions of the initial culture is cultured in a humidity 90 to 95%, temperature 25 to 40 ℃, 5 to 10% CO ₂ incubator by applying the conditions most suitable for cell culture, the final concentration in 5 to 10% CO ₂ culture It is possible to add a carbon control source such as sodium bicarbonate such that is 0.17 to 0.22% by weight.

During the initial culture step, the tissue fragments are preferably attached to the bottom of the culture vessel and can promote growth with short stimulation due to trypsin-EDTA treatment according to standard techniques of cell culture.

The cumulative population doubling time is maintained until 70-80% confluence of the cells in culture in the flask is preferably subcultured by collecting the cells at 75% confluence.

Conditioned medium is prepared by culturing stem cells derived from pulp-derived pulp in the step-free medium at 25 to 40 ° C. for 1 to 3 days.

The conditioned medium may be lyophilized for 6 to 10 hours to obtain the conditioned medium powder of the present invention.

Conditioned medium powder of the present invention may be present in a highly concentrated active ingredient, for example, Decorin, MCP-1, DKK-3, TIMP-2, Angiogenin, Follistatin, Osteoprotegerin, betaIG-H3, MMP-10, MMP-7, LYVE-1, XEDAR, IL-22, RANK, IGFBP-6, Axl, FLRG, NRG1-beta1, PAI-1, Ferritin, Galectin-7, Resistin, Bate2 M, TSLP, VEGF-C, CD40, sTNT R1, IL-6, TRAIL R2, Osteopontin, TIMP-1, Growth Hormon, Thromboppoietin, CXCL-16, DPPIV, HVEM, IL-17B, IL-3, bFGF, IFN-γ, TGF, HGF or PDGF .

The pharmaceutical composition for preventing, ameliorating or treating a brain or spinal nerve-related disease of the present invention contains an active protein complex secreted by stem cells, which may result in immunological problems, safety problems, pharmaceutical problems, Individual deviation can be minimized. In addition, it can fundamentally prevent side effects that may occur due to cell injection in the human body.

The pharmaceutical composition for preventing, ameliorating or treating a brain or spinal nerve-related disease of the present invention may be intimately mixed with various forms of pharmaceutically acceptable carriers depending on the type of preparation required for administration.

The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may preferably be in a unit dosage form, and may be in a form that can be diluted and used so that the dosage can be adjusted and used according to a doctor's judgment.

The composition of the present invention is preferably for nasal injection. However, in embodiments of the present invention, the composition may be administered in a conventional manner also via intravenous, intraarterial, intramuscular, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. May be administered.

Injectable buffers and other adjuvant components used to formulate the compositions of the invention into injectable preparations are known in the art. Injectable formulations for the compositions of the present invention may include, in addition to injectable buffers, other adjuvants such as, for example, dissolution aids, pH adjusters, suspending agents. For example, physiological saline may be used as the buffer for injection.

The present invention also relates to a nasal administration kit for use in the prevention, amelioration or treatment of cerebral or spinal nerve-related diseases comprising a container containing the pharmaceutical composition.

Nasal dosing kits of the present invention may further comprise a container containing an injection buffer.

Nasal mucosa is connected to olfactory nerve cells and olfactory nerve cells are directly instructed by the brain. The method of administering the pharmaceutical composition of the present invention through the nasal cavity is noninvasive, but can be administered more drugs to brain lesions, and cranial nerves. Or spinal nerve-related diseases are characterized by high therapeutic effect. This can be done with conventional direct injection, I.P or I.V. Compared to the administration method, it is much non-invasive and has a high therapeutic effect on diseases related to the brain or spinal nerve.

The container used in the kit may be, for example, a glass or plastic container containing the pharmaceutical composition of the present invention and sealable. It is sufficient to contain the pharmaceutical composition of the present invention, but does not require a special form.

The present invention also provides a method of treating a brain or spinal nerve-related disease comprising administering to a mammal a pharmaceutical composition containing a therapeutically effective amount of stem cell conditioned medium powder.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “therapeutically effective amount” means the amount of a pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, doctor or other clinician, which is the disease being treated. Or an amount that induces alleviation of the symptoms of the disorder.

The method for treating a brain or spinal nerve-related disease described in the present invention can be performed using the pharmaceutical composition described above.

The dosage of the pharmaceutical composition of the present invention can determine the dosage of the composition according to the severity of the symptoms, the age, the weight of the patient and the like. The dosage of the pharmaceutical composition of the present invention may be administered once or several times a day from 0.1 to 100 mg / kg on an adult basis in the case of a nasal dosage form, and 1.0 to per day on an adult basis for an external preparation. Apply 1-5 times a day in an amount of 3.0 mL and continue for at least 1 month. However, the dosage does not limit the scope of the present invention. The pharmaceutical composition of the present invention may preferably be used by dissolving in 0.5 to 2 mL of injectable buffer in a single dose. Preferably, the pharmaceutical composition of the present invention and other auxiliary ingredients may be provided in a sealed package so that they can be dissolved and used in a buffer for injection in a separate vial, and the doctor determines the dosage according to the condition of the patient before administration. Can be dissolved in dosages.

It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the pharmaceutical compositions of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered may be readily determined and may vary depending on the particular active ingredient used, the mode of administration, the effect of the formulation and the development of the disease state. In addition, dosage adjustment according to the appropriate therapeutic level will be needed depending on factors of the individual being treated, including the patient's age, weight, diet and time of administration.

These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

<Example 1> Obtaining primary pulp stem cells

The pulp stem cells of childish teeth (ear teeth) were isolated from the donated teeth at tooth extraction. The pulp was recovered from the teeth, and collagenase (3 mg / mL) and disparate (4 mg / mL) were added to 10 mL of a-MEM medium supplemented with 1% penicillin-streptomycin or amphotericin B as an antifungal agent. Treatment was performed for 1 hour in a 5% CO ₂ incubator under about 90% humidity and about 37 ° C temperature. After the reaction, the tissues were passed through a 70 μm strainer to obtain cells.

To prepare the conditioned medium powder to be used in the experiment,

NSCM-DMEM (A powder) was obtained by incubating the stem cells derived from the pulp-derived stem cells in DMEM, 10% bovine serum and then 48 hours of incubation in DMEM when confluency reached 70-80%. The medium was lyophilized for 8 hours to obtain conditioned medium powder.

NSCM-alphaMEM (B powder) was obtained after incubating the stem cell derived from the pulp-derived stem cells in a medium containing 10ng of bFGF, 50㎍ of ascorbic acid and 10% bovine serum in alphaMEM. When it became -80%, the conditioned medium obtained after 48 hours incubation in serum-free medium without bovine serum was lyophilized for 8 hours to obtain conditioned medium powder.

NSCM-CM (C powder) was obtained by incubating the stem cell derived from the pulp-derived stem cells in a medium containing 10 ng of bFGF, 50 ㎍ of ascorbic acid, and 10% bovine serum in alphaMEM. When the medium reached -80%, 10% NSCM conditioned medium powder (B powder) was added to the serum-free medium without bovine serum, and the conditioned medium obtained after 48 hours of cultivation was lyophilized for 8 hours. Got.

Cultures of pulp stem cells were cultured in a 5% CO ₂ incubator under about 90% humidity and about 37 ° C. temperature conditions.

After culturing in serum-free medium of pulp stem cells, wash the flask from which the stem cell serum solution was removed with phosphate buffer solution, drop the cells with 0.25% Trypsin-EDTA (GIBCO), centrifuge the cell suspension, and measure the cell number. And viability was examined.

The medium used for subculture was 10% FBS, 1% penicillin-streptomycin or a-MEM medium supplemented with amphotericin B with an antifungal agent.

When the confluency of the stem cells cultured in serum-free medium reached about 70 to 80%, the culture medium was removed from the culture culture using a pipette, and then washed twice with phosphate buffer solution.

Experimental Example 1 Pharmaceutical Efficacy Test of Conditioned Medium Powder in Cerebral Infarction Model (MCAo)

300-320g rats were ordered and stabilized for one week in the animal breeding room, and the weight was measured and the MCAo surgery was performed at 320g.

After inhalation anesthesia, a 2 cm incision is made along the midline of the neck, and the left common carotid artery, the external carotid artery, the internal carotid artery, and the posterior carotid artery are carefully exposed so as not to damage the vagus nerve. The distal end of the posterior carotid artery was observed and the distal end of the internal carotid artery was ligated with a micro clip, and then a small hole was made using scissors in the vessel wall of the external carotid artery. The 4-0 silk suture was slowly pushed through the internal carotid artery from the small punctured external carotid artery while removing the clip. Surgery is divided into two types, one is general MCAo surgery, in which a suture is added and taken out after 60-90 minutes to make a cerebral infarction model and a suture is fixed to make a cerebral infarction model (pMCAo model of the present invention). Although the size of the inflammation is larger and more obvious than the general MCAo model, the disadvantage is that the experimental group may die well. The experiment was carried out by adding a suture, which is pMCAo surgery. Confirmed.

Figure 2 is a diagram of the MCAo model (a) shows the blockage of the blood vessels when blocking the MCA in the brain, it shows the path to enter the suture, (b) shows the location to put the suture and lying down the animal When pressed, the figure shows which side the blood vessel enters.

The suture was put in the form as above and the suture was made of silicone and rods made of silicon. As a sham model, one animal model without MCAo surgery, a control model with MCAo surgery and no test materials, A, B, and C were the test groups to which each test substance was administered, and the test materials were applied to PBS. Since it was dissolved and injected, it was divided into six groups, which were treated with PBS instead of experimental materials.

(Intranasal delivery of conditioned medium powder)

After induction of cerebral infarction, stabilization was performed for 48 to 72 hours, and the animals were checked while being protected. After 72 hours, neurological tests and movement tests were performed, and then powders were added to each group. When the powder was injected, the inhalation anesthesia was very weak, followed by injection of 10 μl through the nasal cavity of the cerebral infarction, followed by inhalation anesthesia for 2 minutes. The powder was added daily for 10 days, and the administration method was divided into 10 μl 10 times using a Hamilton micro syringe through the nasal cavity. The movement test was performed 7 times with 1, 5, 10, 17, 24, 31, and 38 days. After 38 days, the brains were extracted and sliced with TTC stained with slices.

(Infarction volume)

For the confirmation and volumetric cerebral ischemia, PBS was perfused into the brains of animals older than 38 days, and only brains were extracted. The extracted brain was immediately fixed to a rodent brain matrix, and then coronal section (2 mm thick) was immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC. T-8887, Sigma chemical) solution 37 It was immersed in the water bath of 30 degreeC about 30 minutes. It was then fixed in 10% formalin and the tissues were placed on slides by size and scanned using a scanner. The scanned image was calculated using the image J program to calculate the volume of cerebral infarction.

As a second experimental method, pMACo cerebral infarction animal model was made and 72 hours later, MRI 9.4T for animals was taken at the Korea Basic Science Institute, Ochang Branch Office (KBSI). At this time, the animals were taken MRI while checking the temperature and pulse rate under inhalation anesthesia. Each test substance was administered according to a predetermined protocol, and 38 days later, MRI was taken once more to confirm cerebral infarction volume. MRI images taken before and after the administration of the test material were used to calculate the volume of the brain where cerebral infarction occurred using the image J program.

Neurobehavior test

1. EBST

In the case of a normal experimental animal, it is about 15cm from the ground to twist the body to both sides and try to lift the head.In the case of the cerebral ischemic model, it is twisted in the opposite direction to the area where the brain ischemia is induced (C -shped lateral bending of the body. It is a test that sees the difference by dividing it into left and right and seeing how many times 20 moves to the right or left.

2. STEP test

In this test, when the normal group grabs the tail and moves the tail to the side with the forefoot attached to the ground, both feet are used to walk sideways.In the case of the cerebral ischemia model, the other side of the cerebral ischemia is not used well. If you move sideways, you will not move well and will turn off. This is a test to see the difference by observing how many times the right forefoot and the left forefoot move during the 20 foot rolls when moving sideways.

(In vitro study using radiation irradiation damaged cell model)

To determine what changes the pulp stem cell conditioned medium powder has at the cellular level, the cells used cortical neurons. After seeding the cerebral cortical neurons, the cells were stabilized and the number of cells was determined, followed by irradiation (3Gy) to give the cells the same effect as MCAo. After 6 hours, the buffer solution was placed in DMEM and pulp stem conditioned medium powder. After 24 hours, the number of cell death and the morphology and expression of BCL-2 in each cell were observed. To see the expression of BCL-2 is to see the effect of powder treatment after killing. Cortical neuronal seeding followed the protocol of the ordered company (neuromix) to order and use. Irradiation was tested in six-wells to allow radiation to spread evenly throughout the entire cell.

(Western Blot)

The cells (1 × 10 ⁴ ) were placed in 50 mL of Lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% deoxycholic acid sodium salt, deoxicholic acid sodium, 1% Triton X-100). After incubation at room temperature for 30 minutes, the cells were disrupted with a sonicator, and then centrifuged (1,2000 g, 3 minutes) to obtain a supernatant. The supernatant was quantified by BCA with an IntRON protein assay kit. Western blot analysis of Aliquotsed protein (50 μg) on Bcl-2 was 12% SDS-PAGE. At this time, β-tubulin was analyzed as a house keeping gene. After gel transfer to the PDVF membrane, blocking was performed at room temperature for 1 hour with 5% skim milk. After blocking, the primary antibody against rabbit polyclonal antibody (1: 1,000) against Bcl-2 and rabbit polyclonal antibody against 1: -tubulin (1: 4000), respectively, was reacted for 4 days at 4 ° C. After attaching the primary antibody and washed with TBST 1X, 1 hour. Goat polyclonal secondary antibodies against rabbit IgG against Bcl-2 and β-tubulin (1: 10,000 for Bcl-2 and 1: 20,000 for β-tubulin) were reacted by dilution. Thereafter, the cells were washed with TBST 1X for 1 hour and then developed by exposure to a film (Agfa) after a detection reaction using an ECL + detection kit.

(Image J program protocol)

The NIH program was downloaded from Wayne Rasband, National Institutes of Health, USA ( http://imageJ.nih.gov/ij ).

As shown in FIG. 3, 38 days after the powder was treated in the cerebral infarction model, the test was completed, and when the brain was extracted by perfusion with PBS, the cerebral infarction occurred severely. Could see. That is, when the brain was extracted, it was possible to see how large the external portion of the cerebral infarction occurred.

As shown in Figure 4, the results of the powder treatment before and after the powder treatment using the Korea Institute of Basic Science Animal MRI 9.4T as a result, the group treated with the powder compared to the control group infarction range (infarction volume, white) This excellent decrease was confirmed.

As a result of measuring the range in which cerebral infarction occurred using the image J program, as shown in FIG. 5, it was confirmed that the cerebral infarction range was clearly reduced in the group treated with C powder as compared to the untreated group.

Next, TTC staining was performed on each experimental animal. As a result, the sham group was only a cosmetic model without suture and no blood vessels, and the control model was 38 days after MCAo surgery. A, B, C test group was a model that showed the effect by administering each powder. PBS is an animal that was injected with only PBS instead of powder, and sham naturally performed MCAo surgery. Because it was not an experimental group, it was confirmed that the staining was dark, and that there was no white cerebral infarction, and the control group was found to have almost half the inflammation. In groups A, B, and C, inflammation was reduced or the color became stronger.

As a result of confirming the size of the cerebral infarction using the image J program based on the results, as shown in FIG. 7, the A, B and C powders showed better results than the PBS (Vehicle) group. Better results were obtained with B and C powders, especially with B powder.

Next, it was confirmed from the above results that the stem cell conditioned medium powder was effective for cerebral infarction, and it was confirmed whether the neurological function was improved through the neurological behavioral test.

As shown in Figure 8, it can be seen from the graph on the left that the powder treatment effect appeared on the 17th day, it was found that the statistical significance in the B, C powder treatment group on the 31st and 38th day.

As can be seen from the neurological behavioral test, the results of EBST indicate that the effect occurs one week after the powder treatment is finished.

Step test results for each experimental group are shown in Figure 9, it can be seen that significantly reduced on the 24th day. The statistical significance of the A, B, C powder treatment group was significantly higher than that of the control group at 24 days.

In the step test, the neurological behavior was improved two weeks after the powder treatment.

Figure 10 was to investigate the weight of each experimental group, the cerebral infarction model was confirmed that the weight is reduced from the day 1 was made normally. The sham model, which did not undergo MCAo surgery, did not lose weight unlike other groups, indicating that the MCAo model was successfully created.

Next, after confirming the effect of the pulp stem cell-derived conditioned medium powder in the MCAo animal test results, it was confirmed in vitro whether this effect also appears in brain cells. Irradiation may have a similar effect to cerebral infarction. After applying such damage to the cells, only one of them changed the buffer solution of the cell called DMEM, and one was treated with powder, and the control group was not irradiated.

FIG. 11 shows the death of cells 24 hours after treatment of each powder 6 hours after irradiation with cerebral cortical neurons, and treated with DMEM and C powder when the control group was viewed as 100%. The difference was found in the number of cells after irradiation.

From the above results, it can be seen that compared with the control group in the radiation damage, the cells treated with pulp stem cell conditioned medium powder did not die and were very effective.

In the case of brain tumor patients, especially pediatric brain tumor patients, causing the various side effects such as cognitive impairment due to the death of existing normal cells during radiation therapy, it is thought that the powder of the present invention can prevent this.

Figure 12 shows the change of cells after treatment of each powder after the radiation damage, the dendrite of the cells after the irradiation proceeds to the apoptosis stage, after processing the powder cell death is reduced, We could see the dendrites stretching very well. The black arrows show that the C-treated cells are much more likely to clump cells or dendrites than DMEM-treated cells.

Western blots were performed for quantitative analysis of these effects. The use of Bcl2 is intended to show anti-apoptotic effects. As shown in FIG. 13, the control group did not express a lot of Bcl2 (25kD) because apoptosis did not occur, but Bcl2 was expressed in the group treated with DMEM and C powder after radiation damage.

In addition, it was confirmed that a lot of dendrites in the group treated with C powder, and shown in the graph using the image J program is shown in Figure 13b.

As a result, the cell test results showed that the therapeutic effect on cerebral infarction was high in the group treated with the powder using pulp stem cells as in the animal test, and it is thought that the cell death of the brain can be treated therefrom.

The present invention can be used as an agent for preventing, ameliorating or treating a brain or spinal nerve-related disease.

Claims (10)

1. A medicinal composition for preventing, ameliorating or treating cerebral or spinal nerve-related diseases, comprising a conditioned medium powder, wherein the conditioned medium powder is obtained by freeze-drying a culture obtained by culturing stem cells in a serum-free medium.
2. The method of claim 1,

   Cerebral nerve or spinal nerve-related diseases include, but are not limited to, cerebral infarction, stroke, encephalitis, cerebral injuries, brain trauma, Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis, or manic cerebral or spinal nerve-related diseases. .
3. The method of claim 1,

   Stem cells include human backpack, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, fat, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, precross ligament, articular cartilage Cranial nerve or spinal nerve associated with at least one selected from the group consisting of mesenchymal stem cells isolated and / or cultured from tissues containing cells, teeth, perivascular cells, striatum, subpatellar masses, spleen and thymus Pharmaceutical composition for preventing, ameliorating or treating a disease.
4. The method of claim 3,

   Stem cell is a pharmaceutical composition for preventing, ameliorating or treating a cerebral nerve or spinal nerve-related disease, which is a pulp-derived mesenchymal stem cell having the following characteristics:

   (a) all show positive immunological characteristics for CD90, CD105, CD44 and CD73,

   (b) adheres to and grows in cell culture dishes and exhibits morphological characteristics of the spindle-shape;

   (c) has the ability to differentiate into mesodermal derived cells.
5. The method of claim 1,

   A serum-free medium is a pharmaceutical composition for preventing, ameliorating or treating a brain or spinal nerve-related disease further comprising a growth factor or cytokine.
6. The method of claim 5,

   A growth factor is a pharmaceutical composition for preventing, ameliorating or treating a cerebral nerve or spinal nerve-related disease which is at least one selected from the group consisting of IGF, bFGF, TGF, HGF, EGF, VEGF and PDGF.
7. The method of claim 5,

   A cytokine is at least one selected from the group consisting of IL-1, IL-4, IL-6, IFN-γ, IL-10, and IL-17. A pharmaceutical composition for preventing, ameliorating or treating a brain or spinal nerve-related disease.
8. The method of claim 1,

   Cultivation is carried out for 1 to 3 days at 25 to 40 ℃ brain or spinal nerve-related diseases for preventing, improving or treating a pharmaceutical composition.
9. Nasal dosing kit for use in the prevention, amelioration or treatment of cerebral or spinal nerve-related diseases comprising a container containing the pharmaceutical composition of any one of claims 1 to 8.
10. The method of claim 9,

    A kit further comprising a container containing an injection buffer.

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