JP2016210730A - Pharmaceutical compositions and production methods thereof as well as pharmaceuticals - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide pharmaceutical compositions for treating allergic rhinitis, pollinosis, and central nervous system disease which have high therapeutic effect and can be supplied stably, and to provide production methods thereof.SOLUTION: The invention provides a pharmaceutical composition for treating at least either one of allergic rhinitis and pollinosis containing an immortalization stem cell-culture supernatant containing a secretory component from immortalization stem cells obtained by culturing the immortalization stem cells which have been immortalized, or their secretory component, the stem cells being either one of tooth-pulp stem cells or myeloid stem cells.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition, a method for producing the same, and a pharmaceutical product.

As a general-purpose alternative technique for diseases difficult to treat with conventional medicine, a treatment method using stem cells has attracted attention.
The treatment method using stem cells can target many diseases, and so far various methods including embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and somatic stem cells. Of stem cells have been reported.

  The treatment method using human induced pluripotent stem (iPS) cells does not use fertilized eggs like ES cells, but can produce cells that are almost the same quality as embryonic stem cells using adult tissues. If iPS cells are used, there is no problem of rejection due to immune reaction.

  Furthermore, Ueda et al. Reported a method using various biological factors produced by stem cells, for example, various growth factors, instead of using the culture supernatant of dental pulp-derived stem cells, that is, stem cells themselves ( For example, Patent Document 1).

International Publication No. 2011/118795

In recent years, the number of people who develop allergic rhinitis and hay fever has been increasing year by year and is recognized as a major social problem. In allergic rhinitis and hay fever, patients are required to be treated with no pain or financial burden, and the number of patients is enormous. There must be.
In contrast, the usual treatments with various drugs and vaccines are less economical, but they still have a low therapeutic effect or require long-term administration. The reality is not definitive treatment.
In addition, examples of diseases in which a large number of patients are present include central nervous system diseases such as cerebral infarction and Alzheimer. These diseases are also required to be a low-cost treatment for patients. At the same time, since a long treatment period is required, it is necessary to overcome the supply-related problems in the drugs that are necessary as described above.

Treatment methods using ES cells and somatic stem cells for the above-mentioned diseases have problems with ethics and safety, and there is a problem in the supply of active ingredients in terms of using own cells. Have
In addition, human induced pluripotent stem (iPS) cells are necessary for use in therapy, such as the risk of malignant tumors (germ cell carcinoma) cannot be excluded and the establishment of iPS cells is troublesome. The fact is that the burden is great on the preparation and management of cells.

Patent Document 1 can be said to be an excellent technique in that it provides the above-described composition for treating an injured part that is effective in recovery of damage due to photoaging of the skin, bone regeneration, and the like. However, no investigation has been made as to whether or not these culture supernatants of stem cells are effective in treating allergic rhinitis and hay fever.
In addition, since the stem cells used are not cell lines, the desired culture supernatant can be obtained unless these stem cells are prepared carefully or if the cryopreserved sample is thawed to proliferate the cells. In addition, if the number of divisions as a cell is repeated, the culture supernatant of a certain composition secreted from the cell will not be secreted and the cell will die, resulting in large restrictions on use, resulting in the above-mentioned damaged part. There is a problem in the supply side of the active ingredient in the therapeutic composition.
On the other hand, the use of cancer cells, which are typical infinitely proliferating cells, can be considered, but the risk of producing biological factors harmful to the living body cannot be denied.
Thus, in the conventional method, when considering the treatment of hay fever, allergic rhinitis and central nervous system disease, not only is there no knowledge about the symptoms and the effect of improving the disease, but also the active ingredient is supplied stably. There was also a problem that it was difficult.

  In view of the above situation, an object of the present invention is to provide a pharmaceutical composition for treating allergic rhinitis, hay fever and central nervous system disease, which has a high therapeutic effect and can be stably supplied, and its production It is to provide a method.

The present invention includes the following aspects.
[1] An immortalized stem cell culture supernatant containing a secreted component from an immortalized stem cell obtained by culturing an immortalized immortalized stem cell or a secreted component thereof, wherein the immortalized stem cell is a dental pulp stem cell or bone marrow A pharmaceutical composition for treating at least one of allergic rhinitis and hay fever, which is one of stem cells.
[2] An immortalized stem cell culture supernatant containing a secreted component from an immortalized stem cell obtained by culturing an immortalized immortalized stem cell or a secreted component thereof, wherein the immortalized stem cell is a dental pulp stem cell or bone marrow A pharmaceutical composition for the treatment of a central nervous system disease, which is any one of stem cells.
[3] The pharmaceutical composition according to [1] or [2], comprising ultrapure water, wherein the secretory component is dissolved in ultrapure water.
[4] The pharmaceutical composition according to [3], wherein the secretory component is contained in an amount of 1 × 10 ⁻⁷ to 1% by mass with respect to ultrapure water.
[5] The pharmaceutical composition according to any one of [1] to [4], further comprising a reversible heat-thickening compound.
[6] A pharmaceutical having the pharmaceutical composition according to any one of [1] to [5] in the form of an injection, an oral preparation, a nasal drop, an eye drop or a coating.
[7] A method for producing the pharmaceutical composition according to any one of [1] to [5], wherein the stem cell selected from dental pulp stem cells or bone marrow stem cells is immortalized; A step of culturing an immortalized stem cell obtained by crystallization, and a step of recovering a culture supernatant containing a secretory component from the immortalized stem cell obtained by culturing the immortalized stem cell. Method.
[8] The production method according to [7], further comprising a step of extracting a secretory component in the collected culture supernatant.
[9] The method according to [7] or [8], further comprising a step of dissolving the secretory component in ultrapure water.

  According to the present invention, it is possible to provide a pharmaceutical composition for treating allergic rhinitis, hay fever and central nervous system disease, which has a high therapeutic effect and can be stably supplied, and a method for producing the same. it can.

It is a figure which shows the evaluation score of the nasal symptom before and behind treating the patient with pollinosis using the pharmaceutical composition which concerns on this invention. It is a figure which shows the evaluation score of the ocular symptom before and after treating the patient with hay fever using the pharmaceutical composition which concerns on this invention. It is a graph which shows the effect with respect to the improvement of the movement disorder after the pharmaceutical composition based on this invention in a cerebral infarction rat. It is a fluorescence microscope image which shows the magnitude | size of the cerebral infarction area | region after administration of the pharmaceutical composition concerning this invention in a cerebral infarction rat. It is the fluorescence image in the brain after administration of the pharmaceutical composition containing the fluorescent molecule and (A) purified water or (B) ultrapure water in the cerebral infarction rat.

≪Pharmaceutical composition≫
The pharmaceutical composition according to the first aspect of the present disclosure comprises an immortalized stem cell culture supernatant containing secreted components from immortalized stem cells or a secreted component thereof obtained by culturing immortalized immortalized stem cells. A pharmaceutical composition used for the treatment of at least one of allergic rhinitis and hay fever, wherein the immortalized stem cell is either a dental pulp stem cell or a bone marrow stem cell.
In addition, the pharmaceutical composition according to the second aspect of the present disclosure is an immortalized stem cell culture supernatant containing secreted components from an immortalized stem cell obtained by culturing an immortalized immortalized stem cell or its secretion. A pharmaceutical composition used for the treatment of a central nervous system disease comprising a component, wherein the immortalized stem cell is either a dental pulp stem cell or a bone marrow stem cell.
Hereinafter, both the pharmaceutical composition according to the first aspect and the second aspect described above are collectively referred to as “the pharmaceutical composition of the present disclosure”.

  In the present disclosure, “treatment” means that part or all of the tissue function lost due to the disease is maintained or restored compared to the function of the tissue before the disease occurs, In addition to the restoration of the function of the organization, it also broadly encompasses regeneration as a functional organization. The evaluation of whether the function is maintained or restored differs depending on the tissue in which the disease occurs, but may be performed based on an appearance or an assay usually used for evaluating the degree of the target function.

  In the present disclosure, the scope of “treatment” includes not only a treatment that cures a disease or tissue abnormality but also a treatment that stops the progression of the disease or tissue abnormality until it is not completely cured, or delays compared to the case where no treatment is performed. Is also included. In the present disclosure, the treatment start time may be after the onset is observed, or may be performed before the onset.

  In this specification, dental pulp stem cells and bone marrow stem cells refer to mammalian mesenchymal cells, and mesenchymal cells are cells belonging to the mesenchymal system such as osteoblasts, adipocytes, muscle cells, and chondrocytes. A cell that has the ability to differentiate.

  “Allergic rhinitis” refers to a type I allergic disease mainly having seizure recurrent sneezing, aqueous nasal discharge, and nasal congestion. Allergic rhinitis includes both perennial and seasonal. Seasonal things include hay fever.

  “Pollenosis” refers to a series of symptoms such as recurrent sneezing, runny nose, nasal congestion and itchy eyes caused by plant pollen coming into contact with mucous membranes such as the nose and eyes. And refers to type I allergic diseases. Although hay fever is generally accompanied by the onset of both nasal symptoms (nasal symptoms) and eye symptoms (eye symptoms), it also includes those with only one of nasal symptoms or eye symptoms. Moreover, the causative plant of pollinosis is not specifically limited.

  “Central nervous system disease” refers to a disease in a large area where many nerve cells gather in the nervous system. In the spinal torso, including humans, it refers to a disease in the brain or spinal cord. Say.

(Immortalized stem cell culture supernatant)
The pharmaceutical composition of the present disclosure comprises an immortalized stem cell culture supernatant containing secreted components from immortalized stem cells obtained by culturing immortalized immortalized stem cells or a secreted component thereof, allergic rhinitis, hay fever And as an active ingredient of a pharmaceutical composition for the treatment of central nervous system diseases. As used herein, the immortalized stem cell means a cell obtained by immortalizing either a dental pulp stem cell or a bone marrow stem cell. Further, when expressed as “immortalized stem cells”, unless otherwise specified, it means that the stem cells obtained by immortalizing one of the above two types of stem cells are collectively referred to as “immortalized stem cell culture supernatant” The culture supernatant containing the secretory component from the immortalized stem cell, obtained by culturing the immortalized immortalized stem cell. Further, for example, when expressed as “immortalized dental pulp stem cell culture supernatant”, it represents a culture supernatant obtained by culturing immortalized dental pulp stem cells, and cultured immortalized stem cells derived from other tissues. The culture supernatant obtained is not meant.
In addition, when simply expressed as “immortalized stem cell culture supernatant”, it means a culture supernatant itself obtained by culturing immortalized stem cells, or one that has been stored without altering or changing its volume, It may be referred to as “untreated immortalized stem cell culture supernatant”. Here, the treatment means that the untreated immortalized stem cell culture supernatant is subjected to treatment such as centrifugation, concentration, solvent replacement, dialysis, drying, freeze-drying, dilution, desalting and the like.

The “secretory component” refers to all components secreted into the culture medium by the above-described immortalized stem cells, and refers to various components including proteins as described later. That is, the remaining thing after removing the component derived from water or a solvent and a culture medium in said immortalized stem cell culture supernatant. In addition, since it is difficult to remove only the medium components from the immortalized stem cell culture supernatant, the immortalized stem cell culture supernatant is generally handled. In this case, solvent replacement or lyophilization of the immortalized stem cell culture supernatant is performed. The thing after processing etc. will also contain a secretory component.
The mass of the secretory component can be determined by subtracting the mass of the medium component from the total mass obtained by removing water and solvent from the immortalized stem cell culture supernatant.

  “Immortalized stem cell culture supernatant” is defined as a culture solution obtained by culturing either immortalized dental pulp stem cells or immortalized bone marrow stem cells and containing no cell components. The pharmaceutical composition contains an immortalized stem cell culture supernatant or a secreted component thereof as an active ingredient, but the composition as a whole may or may not contain cells (regardless of cell type). May be. However, the pharmaceutical composition of the present disclosure is clearly distinguished from a composition containing the above stem cells and / or immortalized stem cells themselves as active ingredients, or a composition using other cells as active ingredients. In addition, the immortalized stem cell culture supernatant can be obtained as a culture supernatant that can be used in the pharmaceutical composition of the present disclosure by, for example, separating and removing cell components after the culture. Specifically, a culture supernatant containing no cell component can be prepared by appropriately performing various treatments (for example, centrifugation, dialysis, membrane separation, etc.).

In the pharmaceutical composition of the present disclosure, the mechanism of action against allergic rhinitis, hay fever and central nervous system diseases is unclear, but the present inventors consider as follows.
The immortalized stem cell culture supernatant or a secretory component thereof contained in the pharmaceutical composition according to the first aspect acts to suppress the activity of inflamed cells such as allergic rhinitis or pollinosis, Since it acts to suppress the secretion of substances necessary for inflammation (for example, inflammatory cytokines) to resident cells, continuous administration of the immortalized stem cell culture supernatant results in This is considered to normalize the inflamed tissue. Here, since the immortalized stem cell culture supernatant or its secretory component contains many types and sufficient amount of components suitable for the treatment of inflammation, the immortalized stem cell culture supernatant or A sufficient therapeutic effect can be obtained by administering the secretory component for a short period of time.
Moreover, when the immortalized stem cell culture supernatant or its secretory component is administered for a certain period of time, it can not only suppress the inflammation but also shift to a state where the inflammation does not occur again, that is, an immune tolerance state.
Further, the immortalized stem cell culture supernatant or the secretory component thereof contained in the pharmaceutical composition according to the second aspect has various types and sufficient amounts necessary for repairing damaged nerve tissue or degenerated nerve tissue. Immediately repairs damaged or denatured parts by the action of components. Furthermore, with respect to endogenous stem cells and the like, when the component continues to act as a constant induction signal so that they form a normal tissue, the endogenous stem cells and the like can be quickly differentiated and proliferated. Can be inferred. As a result, it is considered that the tissue causing the central nervous system disease is repaired and the treatment is completed.

Furthermore, since the immortalized stem cells can continue to secrete the necessary factors with a certain quality no matter how many times they are passaged, the composition containing the supernatant has a stable therapeutic effect.
Further, in the preparation of the immortalized stem cell culture supernatant in the present disclosure, there is no limitation that the patient's own stem cells must be used, as described later. In addition, culture supernatant of immortalized stem cells obtained by immortalizing stem cells derived from dental pulp or bone marrow obtained from mammals other than humans (such as cattle, horses, pigs, monkeys, and sheep) (on non-human-derived immortalized stem cell cultures) Qing) has a sufficient therapeutic effect on humans and non-human mammals. Furthermore, a culture supernatant of immortalized stem cells obtained by immortalizing human dental pulp or bone marrow-derived stem cells (human-derived immortalized stem cell culture supernatant) has a sufficient therapeutic effect on the mammals other than humans.
Thus, the immortalized stem cell culture supernatant of the present disclosure has an advantage that the industrial productivity can be improved. Furthermore, since the immortalized stem cells used for the preparation of the immortalized stem cell culture supernatant are simply immortalized stem cells, unlike the tumor cells, the factors secreted from them are the same as the non-immortalized stem cells. Safety is guaranteed.
Further, for example, for the purpose of treating diseases of livestock and pets, there is an advantage that both the non-human-derived immortalized stem cell culture supernatant and the human-derived immortalized stem cell culture supernatant can be used.

Moreover, as will be described later, by using ultrapure water in the pharmaceutical composition, the therapeutic effect can be dramatically improved. This means that the penetration of low molecular weight compounds into the skin, subcutaneous, epithelial mucosal tissue and the underlying tissue with ultrapure water is possible even with large molecules such as polymers. This puts a burden on the patient with components necessary for treatment secreted by immortalized stem cells, that is, various cytokines such as VGF and TGF, and macromolecules such as extracellular matrix (ECM) such as collagen and hyaluronic acid. Without the need for efficient administration. In addition, for example, in a disease that targets the brain, by administering a pharmaceutical composition by nasal nose, etc., it is possible to efficiently reach a necessary component for treatment without considering the blood-brain barrier. It also has the advantage of being able to.
Furthermore, by using ultrapure water in a pharmaceutical composition containing an immortalized stem cell culture supernatant derived from non-human stem cells, the penetration effect thereof can be dramatically increased as in humans. For this reason, the pharmaceutical composition containing the ultrapure water of the present disclosure exhibits a sufficient therapeutic effect on animals other than humans.

  The stem cells used for the preparation of the immortalized stem cell culture supernatant are stem cells that are either mammalian dental pulp stem cells or bone marrow stem cells. Here, the mammal is selected from the group consisting of humans, pigs, cows, sheep, horses, and monkeys because of its high genetic similarity to human cells and low risk of infection. It is preferable.

  An immortalized stem cell was prepared by introducing four types of genes into an initial cultured cell obtained by initially culturing a stem cell, and selecting STRO-1 expression from the gene-transferred cell as an index. An immortalized stem cell. Here, STRO-1 serves as an index of cell immortalization, and the high expression rate also serves as an index indicating the same properties as primary cultured cells in stem cells.

The four types of genes are preferably four types selected from the group consisting of hTERT, bmi-1, E6, E7, Oct3 / 4, Sox2, Klf4, c-Myc, and p16INK4a. Here, the genes to be introduced are preferably hTERT, bmi-1, E6 and E7.
The above-described immortalized stem cell preferably has a telomere repair ability and at least a division ability capable of dividing 200 times or more in the number of individual doublings. Further, at the time when the number of individual doublings is 20, it is preferable that at least 40% or more of the cell population is STRO-1-expressing cells and has a regenerative ability equivalent to that of primary cultured cells.

The immortalized stem cell culture supernatant or the secretory component thereof in the present disclosure includes many types of low-molecular compounds, proteins, polysaccharides, and the like secreted from the above-described immortalized stem cells into the culture medium.
The immortalized stem cell in the present disclosure is a group consisting of insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and HGF which is a hepatocyte growth factor. At least two or more growth factors selected from are secreted into the culture supernatant. Here, the “growth factor” is a general term for polypeptides that promote cell division and cause morphological changes and hypertrophy. Factors vary depending on the type of cells that produce growth factors, and are roughly classified into epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), tumor growth factor (TGF), and the like.
Furthermore, receptors on the cell membrane of each cell have tyrosine kinase activity, and when a growth factor binds, the tyrosine residue of the protein is phosphorylated, causing cell proliferation and differentiation. Several examples are known in which growth factors are mesoderm inducers in ontogeny. In addition, some examples of lymphokines that regulate the immune system have become mesoderm inducers in ontogeny. Such growth factors can be quantified by a known ELISA method, microarray method or the like.

  IGF-1 is a polypeptide that is highly similar in sequence to insulin, and is known to cause reactions such as mitogenesis in cell culture as well as to influence the growth of nerve cells in the same manner as insulin. ing. VEGF is a group of glycoproteins involved in angiogenesis that forms new blood vessels in the absence of blood vessels and angiogenesis that branches from existing blood vessels to form blood vessels in the embryogenesis stage. is there. TGF-β is also a potent growth inhibitory factor for many cells and is closely involved in cell differentiation / migration / adhesion, ontogeny and tissue reconstruction, wound healing, inflammation / immunity, cancer invasion / It plays an important role in a wide range of areas such as metastasis. Furthermore, HGF is versatile not only for hepatocytes but also for various cells that promote cell proliferation, promote cell motility, induce anti-apoptosis (cell death), induce morphogenesis, and regenerate and protect other tissues and organs. It has various physiological activities.

The immortalized stem cells are vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β). It is considered that various cytokines such as -1 and -3, TGF-α, KGF, HBEGF, and SPARC can be continuously produced.
Thus, the immortalized stem cell culture supernatant or its secretory component contains a great many types of components including the above-mentioned proteins, and each of these components can be identified and quantified using known techniques. It can be carried out.

  In the pharmaceutical composition of the present disclosure, the immortalized stem cell culture supernatant or secretory component thereof preferably contains at least two cytokines, among which vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth. More preferably, it comprises a combination of two or more selected from the group consisting of factor (IGF), platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-β).

  The mixture containing cytokines in the immortalized stem cell culture supernatant contained in the pharmaceutical composition of the present disclosure is a mixture of cytokines isolated as part of the immortalized stem cell culture supernatant or from the whole immortalized stem cell culture supernatant. It can be used as a mixture. In a mixture of cytokines isolated from an immortalized stem cell culture supernatant, a portion of the cytokines may be replaced with one or more known corresponding cytokines.

(Dental pulp stem cells)
The “dental pulp stem cell” refers to a kind of stem cell contained in a dental nerve having regenerative ability. Because it is protected by a hard material called teeth, it has the property that it does not transmit ultraviolet rays or radiation, and genes are not easily damaged. The dental pulp stem cell is preferably a stem cell obtained from the dental pulp of any tooth selected from the group consisting of a dropped deciduous tooth, a dropped permanent tooth, a extracted deciduous tooth, and a extracted permanent tooth. Among these, stem cells obtained from deciduous teeth are particularly preferred from the viewpoint of therapeutic effects on allergic rhinitis, hay fever and central nervous system diseases.

  In the pharmaceutical composition according to the first aspect of the present disclosure, the pharmaceutical composition comprising an immortalized pulp stem cell culture supernatant (SHED-CM) obtained by immortalizing dental pulp stem cells or a secretory component thereof comprises allergic rhinitis and pollen It is preferably used for the treatment of hay fever, from the viewpoint of the therapeutic effect of SHED-CM.

In the pharmaceutical composition according to the second aspect of the present disclosure, the pharmaceutical composition containing an immortalized dental pulp stem cell culture supernatant (SHED-CM) or a secretory component thereof is used for the treatment of diseases of the central nervous system.
Examples of central nervous system diseases include spinal cord injury, acute, chronic and progressive neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Alzheimer's dementia, Parkinson's disease, progressive supranuclear palsy, Huntington Disease, multisystem atrophy, spinocerebellar degeneration, etc.), neuronal degeneration / dropout due to acute and chronic cerebral infarction associated with cerebral ischemia, intracerebral hemorrhage, etc., and retinal diseases with neuronal damage. Here, “cerebral infarction” refers to a cerebral ischemic condition caused by blockage of blood flow to or through the cerebrum.
Among the above-mentioned diseases, it is preferably used for the treatment of diseases or pathologies selected from the group consisting of neurological diseases and cerebral infarction.
Furthermore, it is preferably used for treatment of a disease or pathological condition selected from the group consisting of chronic cerebral infarction, amyotrophic lateral sclerosis (ALS), and Alzheimer type dementia.

As a method for preparing dental pulp stem cells, for example, it can be prepared from human fallen deciduous teeth as follows.
First, after the deciduous deciduous teeth are sterilized with, for example, chlorohexidine, isodine solution or other disinfectant, the crown portion is divided, and the pulp tissue is collected with a dental reamer.
The collected dental pulp tissue contains a basic medium such as 5% to 15% bovine serum (hereinafter sometimes referred to as “CS”) and 50 units / ml to 150 units / ml antibiotic. It is suspended in Dulbecco's Modified Eagle's Medium (hereinafter referred to as “DMEM”). Subsequently, the cells are treated with 1 mg / ml to 5 mg / ml collagenase and 1 mg / ml to 5 mg / ml dispase at 37 ° C. for 0.5 to 2 hours.

As the basal medium, in addition to DMEM, Iskov modified Dulbecco medium (IMDM) (manufactured by GIBCO, etc.), Ham F12 medium (HamF12) (manufactured by SIGMA or GIBCO, etc.), RPMI 1640 medium, and the like can be used. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (manufactured by GIBCO)) can be mentioned.
In addition, fetal bovine serum (fetal bovine serum, hereinafter referred to as “FBS” or “FCS”), human serum, sheep serum and other sera, serum substitutes (Knockout) serum replacement (KSR)), bovine serum albumin (hereinafter sometimes referred to as “BSA”), penicillin, streptomycin and other antibiotics, various vitamins, and various minerals.
The above basic medium can also be used for culturing cells to be described later and culturing cells after sorting.

After the enzyme treatment, centrifugation is performed for 3 to 10 minutes (3,000 rpm / 7,000 to 7,000 rpm) to collect dental pulp cells. If necessary, cell sorting is performed using a cell strainer. The selected cells are resuspended in, for example, 3 to 6 ml of the above basic medium, and seeded in an adherent cell culture dish having a diameter of 4 to 8 cm.
Next, after adding a culture solution, for example, DMEM containing 10% fetal calf serum (FCS), the cells are cultured at 37 ° C. for about 2 weeks in an incubator under a 5% CO ₂ atmosphere. After removing the culture solution, the cells are washed once or several times with phosphate buffered saline (PBS) or the like. Instead of removing the culture medium and washing the cells, the adhesive dental pulp stem cells that formed colonies can also be recovered. Adhesive dental pulp stem cells are treated with, for example, 0.025% to 0.1% (w / v) trypsin and 0.3 mM to 1 mM EDTA for several minutes at 37 ° C. and detached from the dish. Cells are then harvested.

Next, the adherent cells selected as described above are cultured. For example, the dental pulp stem cells obtained as described above are seeded in an adherent cell culture dish, and collected after culturing in an incubator under a 5% CO ₂ atmosphere at 37 ° C. Cultured cells (SHED-P) can be obtained and can be used for the preparation of immortalized stem cells (SHED-T).
For example, when subculture or subconfinence is reached by observation with the naked eye, subculture is performed by removing the cells from the culture vessel using trypsin and EDTA and collecting them again, as described above. Seed in a culture vessel containing Here, sub-confluent means a state in which cells adhere to about 70% of the cell attachment surface in the culture vessel. Next, for example, subculture is performed 1 to 8 times, and the selected cells are grown to the required number of cells. After culturing as described above, the cells are collected and stored in liquid nitrogen. Cells collected from various donors may be stored in the form of dental pulp stem cell banks.

(Bone marrow stem cells)
“Bone marrow stem cell” is a general term for cells obtained in a bone marrow aspirate, and includes leukocyte cells such as myeloblasts, erythroid cells, bone marrow megakaryocytes, and plasma cells. As the bone marrow stem cell, the cell type to be collected is not particularly limited, but it is preferably collected from the femur, tibia or pelvis (iliac bone) from the viewpoint that a large amount of cells can be collected and is easily collected. . In the case of mammals other than humans, it can also be collected from the iliac and tibia. Methods for collecting bone marrow-derived mesenchymal stem cells are known to those skilled in the art, and for example, ordinary methods used in medicine can be used.
To obtain autologous or allogeneic stem cells in bone marrow stem cells, for example, humans have mesenchymal cells of the human femur, iliac, and jawbone with informed consent to the patient Aseptically collect bone marrow and peripheral blood from tissues and organs using syringes, puncture needles, etc., and inoculate in culture containers and use them by separating floating cells and adherent cells, or flow By using a technique such as cytometry or density gradient centrifugation, mesenchymal cells can be collected and separated.
In addition, bone marrow-derived stem cells obtained from several tissues are commercially available and can be easily obtained.

  In the pharmaceutical composition according to the first aspect of the present disclosure, the pharmaceutical composition comprising an immortalized bone marrow stem cell culture supernatant (BM-CM) obtained by immortalizing bone marrow stem cells or a secretory component thereof is used for allergic rhinitis and pollen. It is preferably used for the treatment of hay fever from the viewpoint of the therapeutic effect of BM-CM.

  Moreover, in the pharmaceutical composition according to the second aspect of the present disclosure, the pharmaceutical composition containing an immortalized bone marrow stem cell culture supernatant (BM-CM) or a secretory component thereof is used for the treatment of the above-mentioned central nervous system disease. In this case, an example of a central nervous disease and a preferable treatment target are the same as described above.

When collecting mesenchymal stem cells from the bone marrow of mammals other than humans, for example, cut both ends of the bone (femur, tibia), wash the inside of the bone with a medium suitable for culturing mesenchymal stem cells, Mesenchymal stem cells can be obtained from the washed culture.
As described above, bone marrow-derived stem cells (BM-P) can be obtained and used for immortalization of stem cells.

[Preparation of immortalized stem cell culture supernatant]
To obtain an immortalized stem cell supernatant, first, either dental pulp stem cells or bone marrow stem cells are obtained from mammalian tissue as described above. The mammal is preferably selected from the group consisting of humans, cows, sheep, pigs, horses, and monkeys.

<Immortalization of stem cells>
In order to obtain immortalized stem cells, genes introduced into any of the stem cells obtained from the above pulp and bone marrow include hTERT, bmi-1, E6, E7, Oct3 / 4, Sox2, Klf4, c-Myc. And p16INK4a. hTERT is a gene for telomere repair enzyme, and bmi-1 is a gene for Bmi-1, which is one of the proteins constituting the polycomb complex. Here, Bmi-1 is necessary for maintenance of hematopoietic stem cells, and has an effect that hematopoietic stem cells can be increased by enhancing the activity.
E6 and E7 are early genes of HPV-16 or HPV-18. Oct3 / 4 is a gene that activates transcription of a target gene in cooperation with Sox2. Klf4 (Kruppel-type transcription factor 4) regulates genes involved in cell division and embryogenesis and is involved as a cancer suppressor of digestive system cancer.
Sox2 belongs to the SRY-related HMG box gene family, and is a gene known to be involved in maintaining undifferentiated (pluripotent) functions. c-Myc is an oncogene and is a gene that promotes both cell survival and death in tumors induced with c-Myc. p16INK4a is a gene that plays an important role in controlling the cell cycle of cancer cells.

  The specific immortalization of stem cells will be described below, but the immortalization of stem cells is not limited to this method.

Four types of genes are introduced into primary cultured cells obtained by initial culture of the stem cells to create gene-transferred cells. The genes introduced here are preferably four types selected from the group consisting of hTERT, bmi-1, E6, E7, Oct3 / 4, Sox2, Klf4, c-Myc, and p16INK4a. By introducing hTERT, bmi-1, E6, and E7, an immortalized stem cell having a larger number of individual doublings can be obtained. Here, hTERT is a gene for human telomerase reverse transcriptase, and bmi-1 is a polycomb group gene involved in stem cell self-renewal and differentiation control. E6 and E7 are genes present in an open reading frame (ORF) that encodes the early genes used by human papillomavirus for self-replication.
Such gene introduction can be performed as follows.

A plasmid for incorporating the above-described gene of interest is prepared, and this is incorporated into a shuttle vector such as pShuttle2 (Clontech) to clone the above gene. E. coli is transformed with this shuttle vector, and a kanamycin resistant transformant is selected. The plasmid DNA of the selected kanamycin resistant transformant is purified and the restriction enzyme site is analyzed to identify the recombinant.
Next, the expression cassette is excised from the above shuttle vector using restriction enzymes such as PI-SceI and I-CueI, and this is ligated to an adenovirus vector such as Adeno-X viral DNA (Clontech). . The resulting ligation product is cleaved with SwaI and used to transform E. coli.

From the obtained transformants, ampicillin resistant transformants are selected. The recombinant adenovirus DNA into which the above gene is incorporated is purified, and the restriction enzyme site is analyzed to identify the recombinant.
The recombinant adenovirus is then digested with PacI and transfected into HEK293 cells. Recombinant adenovirus is propagated and collected to determine the virus titer to confirm the presence of the virus. Virus is purified according to a conventional method, and the stem cells (for example, SHED-P) are infected.
The cell group after virus infection is stained with FITC according to a conventional method, and STRO-1-positive cells are detected using a flow cytometer. Here, STRO-1 is considered as one of markers of mesenchymal stem cells having pluripotency in bone marrow, and serves as an index of cell immortalization.
By the above procedure, immortalized stem cells (SHED-T or BM-T) can be obtained.

<Preparation of immortalized stem cell culture supernatant>
Next, the immortalized stem cells obtained by the above method are subjected to the above basic medium, for example, a medium (DMEM) supplemented with 10% FBS as serum under conditions of 37 ° C. in a 5% CO ₂ atmosphere. Incubate for 24 to 48 hours and centrifuge at 200 g to 500 g for 3 to 7 minutes, or use a separation membrane that does not allow immortalized stem cells to pass through, and immortalized stem cell culture supernatant (SHED-CM or BM- CM). For collection of the culture supernatant, for example, a cornagome pipette can be used. The collected culture supernatant may be used as it is as an active ingredient of the pharmaceutical composition of the present disclosure, and after the concentration, solvent replacement, dialysis, lyophilization, dilution and other treatments, the effective of the pharmaceutical composition of the present disclosure will be used. It may be used as an ingredient.

In the above case, the presence or absence, type, and added concentration of serum, medium, etc. may be appropriately set in consideration of the type of immortalized stem cell to be used. In addition, the safety | security of the pharmaceutical composition of this indication may be improved by not containing serum. Therefore, in order to obtain a serum-free pharmaceutical composition, for example, a culture supernatant without serum can be prepared by culturing immortalized stem cells in a serum-free medium (serum-free medium). . A serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.
In order to prepare “immortalized stem cell culture supernatant” that does not contain serum, a serum-free medium may be used throughout the entire process or for the last or several subcultures from the end. In addition to DMEM, Iskov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI 1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (manufactured by GIBCO)) can be mentioned. Examples of components that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

The culture time for obtaining the culture supernatant is, for example, 5 hours to 7 days, and may be 1 day to 6 days. The culture temperature is, for example, 36 ° C to 38 ° C, for example, 37 ° C, and the CO ₂ concentration is 4 to 6%, for example, 5%. Further, the culture may be performed, for example, by three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregation suspension culture, etc.).

  In addition, as described later, the culture supernatant of the immortalized stem cells obtained as described above contains various growth factors and exhibits various actions without high purification. That is, since a pharmaceutical composition that can be used for the treatment of various diseases can be produced by a simple process, it is possible to avoid a decrease in physiological activity of various growth factors accompanying high-level purification.

  The “immortalized stem cell culture supernatant” used in the pharmaceutical composition of the present disclosure refers to a culture supernatant containing various secreted biological factors obtained by culturing immortalized stem cells. A cell-free solution. Due to this characteristic, the pharmaceutical composition of the present disclosure is clearly distinguished from various compositions containing stem cells, as a matter of course. A typical example of this embodiment is a pharmaceutical composition that does not contain stem cells and is composed of only stem cell culture supernatants. Therefore, for example, by separating and removing cell components after culturing, a culture supernatant that can be used in the pharmaceutical composition of the present disclosure can be obtained. In addition, the above-described immortalized stem cell culture supernatant is obtained by appropriately treating the culture supernatant obtained after the immortalized stem cell culture with various treatments (eg, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying). , Diluted, desalted, preserved, etc.) and then used in the composition.

Immortalized stem cells selected and cultured by the above method are tissues and cells collected from a living body and have the same properties as the primary cultured cells seeded first. In general, primary cultured cells are important in that they have properties similar to the tissue from which they are sourced and are close to normal cells. However, the growth is slower than that of the established cell line, and de-differentiation may occur while the culture is continued, which is difficult to maintain while maintaining its properties. On the other hand, as described above, the immortalized stem cell in the present disclosure has a characteristic that it can retain the properties of the primary cultured cell even after many times of division.
Here, in the immortalized stem cells in the present disclosure, the expression rate of STRO-1, which is a marker of the degree of undifferentiation of cells, is more significant than that of stem cells that are not immortalized stem cells when the cell doubling number is 20 or 40 times. It is preferable that the ratio is as high as about 1.5 to 3 times.

The above immortalized stem cell culture supernatant can be used as a pharmaceutical composition after being diluted with an aqueous solution or other solvent. In this case, water and a buffer solution can be used as the aqueous solution. As other solvents, organic solvents can be used as long as the effects of the pharmaceutical composition of the present disclosure can be obtained.
Moreover, as water used for water and a buffer solution, ion-exchange water, purified water, ultrapure water, etc. can be used. These waters can be prepared with a general-purpose manufacturing apparatus or purchased.
When the immortalized stem cell culture supernatant is diluted with an aqueous solution (excluding ultrapure water) and used as a pharmaceutical composition, the culture conditions when the immortalized stem cells are cultured, the type and disease of the disease to be treated It is preferable to dilute the supernatant as appropriate in consideration of the degree of the above, but the concentration (%) in the pharmaceutical composition of the untreated immortalized stem cell culture supernatant (100 × untreated immortalized stem cell culture supernatant (Volume / volume of the pharmaceutical composition) is preferably used at a concentration of 0.1% to 300%. In addition, 300% means that the supernatant is concentrated. When it is 0.1% or more, the therapeutic effect of the immortalized stem cell culture supernatant is further enhanced, and by being 300% or less, the deposition of factors in the immortalized stem cell culture supernatant can be further prevented, It is more economical. Furthermore, it is more preferably 0.2% to 100%, and particularly preferably 0.5% to 50%.
Moreover, as a secretory component, it is preferable to be contained by 1 * 10 <^-6> mass%-10 mass% with respect to pharmaceutical composition whole quantity. By being 1 × 10 ⁻⁶ mass% or more, it is possible to secure a component necessary for a more sufficient therapeutic effect, and by being 10 mass% or less, excessive components are reduced and the economic efficiency in treatment is further increased. be able to. It is more preferably 1 × 10 ⁻⁶ mass% to 1 mass%, and further preferably 1 × 10 ⁻⁴ mass% to 1 mass%.

(Ultra pure water)
The pharmaceutical composition of the present disclosure may include ultrapure water, and the secretory component may be dissolved in ultrapure water. The pharmaceutical composition in which the above-mentioned immortalized stem cell culture supernatant or its secretory component is dissolved in ultrapure water not only penetrates these active ingredients into the skin and subcutaneous skin, but also applies to epithelial mucosal tissue and its underlying tissue. Penetration can be dramatically increased. Therefore, when the intended pharmaceutical composition is administered mainly to the nose, eyes, etc., as in the treatment of allergic rhinitis and hay fever, the effect can be dramatically improved. Moreover, the effect of such ultrapure water is the same for components in an immortalized stem cell culture supernatant prepared using stem cells other than human.
In the pharmaceutical composition containing ultrapure water in the present disclosure, not only low molecular weight compounds but also various cytokines such as VGF and TGF as described above, and extracellular matrix (ECM) such as collagen and hyaluronic acid are used. This is also the first method that targets components of high polymers.
Here, ultrapure water refers to water having an electrical resistivity of 17.5 MΩ · cm or more, and the organic substance is preferably 10 ppb or less. Such water can be obtained by purchasing a commercially available product, or can be prepared by a commercially available ultrapure water production apparatus. By containing in water having an electrical resistivity of 17.5 MΩ · cm or more, the penetration effect of the components in the immortalized stem cell culture supernatant can be further enhanced. More preferably, the electrical resistivity is 18.0 MΩ · cm or more. The electrical resistivity of water can be detected by a detector of electrical resistivity of water provided in the ultrapure water production apparatus, and can be easily detected by, for example, an electrical resistivity meter (manufactured by Horiba, Ltd.).
Moreover, the penetration effect of the said component can further be heightened because the organic substance in ultrapure water is water of 10 ppb or less.
The polymer is a molecule having a molecular weight of 2000 or more (more preferably 3000 or more) and includes proteins, nucleic acids, and polysaccharides.

Further, as a method for dissolving the immortalized stem cell culture supernatant in ultrapure water, it may be prepared by adding ultrapure water as it is to the immortalized stem cell culture supernatant. Ultrapure water may be added to those appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.).
When the untreated immortalized stem cell culture supernatant is dissolved in ultrapure water, the concentration of the culture supernatant in the pharmaceutical composition is the culture conditions when the immortalized stem cells are cultured, the components to be added, and the treatment target It is preferable to adjust appropriately in consideration of the type of disease and the degree of disease, etc., but the concentration (%) in the pharmaceutical composition when converted to the amount of the untreated immortalized stem cell culture supernatant ( 100 × untreated immortalized stem cell culture supernatant / volume of pharmaceutical composition) is preferably used at a concentration of 0.01% to 80%. If it is 0.01% or more, the therapeutic effect of the immortalized stem cell culture supernatant is further enhanced, and if it is 80% or less, it is possible to contain a certain amount of ultrapure water. Can be further enhanced. Further, it is preferably 0.01% to 50%, more preferably 0.01% to 10%, particularly preferably 0.05% to 3%, and 0.1% to 2%. Most preferably.
Moreover, it is preferable that 1 * 10 <^-7> mass%-1 mass% are contained as a secretory component in this case with respect to ultrapure water. By being 1 × 10 ⁻⁷ mass% or more, it is possible to secure components necessary for a more sufficient therapeutic effect, and by being 1 mass% or less, excessive components are reduced and the economic efficiency in treatment is further increased. be able to. It is more preferably 1 × 10 ⁻⁶ mass% to 0.1 mass%, and further preferably 1 × 10 ⁻⁵ mass% to 0.1 mass%.

  In addition, when ultrapure water is included in the pharmaceutical composition, the content ratio of ultrapure water (100 × volume of ultrapure water / volume of the pharmaceutical composition) is the culture condition and addition when immortalized stem cells are cultured. It is preferable to adjust appropriately considering the components to be treated, the type of disease to be treated, the degree of disease, and the like, but it is preferably 20% to 99.99%. By being 20% or more, the effect of ultrapure water can be further enhanced, and by being 99.99% or less, the therapeutic effect of the immortalized stem cell culture supernatant can be further enhanced. Further, it is preferably 50% to 99.99%, more preferably 90% to 99.9%, and particularly preferably 97% to 99.5%.

(Reversible heat-thickening compound)
The pharmaceutical composition of the present disclosure may further contain a reversible heat-thickening compound. A reversible heat-thickening compound is a compound that thickens by heating. When administered to a living body such as a human, it easily thickens and gels at its body temperature. By including such a compound in the pharmaceutical composition, it is highly usable in preparation and transportation before administration, and after administration, the thickened gel stays in the affected area. More sustainable. Such a reversible heat-thickening compound is described in detail in WO2009-001899.
The concentration of the reversible heat-thickening compound in the pharmaceutical composition (100 × mass of reversible heat-thickening compound / volume of the pharmaceutical composition, (w / v)%) is the therapeutic effect of the pharmaceutical composition of the present invention. However, it is preferably 0.01% to 10%. When it is 0.01% or more, it is easy to thicken with heat, and when it is 10% or less, it is easy to handle as the viscosity of the pharmaceutical composition.

(Liposome)
The pharmaceutical composition of the present disclosure may further contain a liposome. Liposomes are closed vesicles consisting of a monolayer or multiple layers of lipids obtained by mixing phospholipids and water as a basic skeleton, and various substances such as tissue-specific substances are contained in the inner water layer or lipid layer. It can also hold sensitive molecules. For this reason, by using this as a drug delivery system (DDS), the above-mentioned liposome is transferred to the target tissue, and the fusion of the lipid bilayer and the liposome with the cells on the tissue and the other methods are used. By gradually releasing the molecules, the therapeutic effect of the target tissue can be enhanced.
Known methods can be used for the kind of phospholipid in the liposome and the method for preparing the liposome in which the pharmaceutical composition is encapsulated. For example, it can be prepared by the method according to JP-A-2000-229815. Such a reagent for preparing liposomes for DDS is commercially available, and can be purchased from, for example, Yuka Sangyo Co., Ltd.

(Concentration method of immortalized stem cell culture supernatant)
The pharmaceutical composition of the present disclosure can include a concentrated immortalized stem cell culture supernatant. As a method for concentrating the immortalized stem cell culture supernatant, a conventional method can be applied. Examples of the concentration method include the following two methods.
1. Spin Column Concentration Method Immortalized stem cell culture supernatant is concentrated using Amicon Ultra Centrifugal Filter Units-10K (Millipore) (maximum 75-fold concentration). The specific operation procedure is as follows.
(I) The culture supernatant (up to 15 ml) is put into Amicon Ultra Centrifugal Filter Units-10K, centrifuged at 4000 g for about 60 minutes, and concentrated to 200 μl.
(Ii) The same amount of sterilized PBS as the culture supernatant is put into the tube, and centrifuged again at 4000 g for about 60 minutes to replace the base solution with PBS.
(Iii) Collect 200 μl of the obtained solution into a micro test tube to obtain a concentrated immortalized stem cell culture supernatant.

2. Ethanol precipitation concentration method Immortalized stem cell culture supernatant is concentrated using ethanol precipitation method (concentration up to 10 times). The specific operation procedure is as follows.
(I) 45 ml of 100% ethanol is added to 5 ml of the culture supernatant, mixed and left at −20 ° C. for 60 minutes.
(Ii) Centrifuge at 15000 g for 15 minutes at 4 ° C.
(Iii) Remove the supernatant, add 10 ml of 90% ethanol and stir well.
(Iv) Centrifuge at 15000 g for 5 minutes at 4 ° C.
(V) The supernatant is removed, and the resulting pellet is dissolved in 500 μl of sterilized water, collected in a microtest tube, and used as a concentrated immortalized stem cell culture supernatant.

(Additive ingredients)
Depending on the condition of the subject to be applied, other components other than those described above can be additionally used in the pharmaceutical composition, provided that the expected therapeutic effect is maintained. An example of ingredients that can be additionally used in a pharmaceutical composition is as follows.
(i) Bioabsorbable material As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) and the like can be used.

(ii) Gelling material As the gelling material, a material having high biocompatibility is preferably used, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (application tissue) of the pharmaceutical composition. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(iii) Others Pharmaceutically acceptable other components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc. ) Can also be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used. Antibiotics, pH adjusters, growth factors (for example, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)) and the like may be included.

The final form of the pharmaceutical composition is not particularly limited. Examples of forms are liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.).
The dosage of the pharmaceutical composition may be a therapeutically effective amount. Since the pharmaceutical composition contains an immortalized stem cell culture supernatant as an active ingredient, the dosage of the pharmaceutical composition may be appropriately adjusted when used for treatment. As described below, the active ingredient is concentrated. It may be used.

≪Pharmaceuticals≫
As another aspect of the invention according to the present disclosure, the pharmaceutical composition may be a pharmaceutical having the above-described pharmaceutical composition in the form of an injection, an oral preparation, a nasal drop, an eye drop, or a coating. Thus, the therapeutic effect can be further enhanced by limiting the place and method for administering the pharmaceutical composition. Among these, from the viewpoint of reducing the burden on the patient and the therapeutic effect, it is preferable to use a medicine having the form of a nose drop, an eye drop, or a coating agent, and more preferably a nose drop or an eye drop. . These pharmaceuticals may be prepared by including the above pharmaceutical composition as it is, or may be appropriately treated (concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage). It may be prepared. Furthermore, you may prepare by including an additive required as a pharmaceutical. Examples of additives include excipients, disintegrants, lubricants, binders, coating agents, colorants, anti-aggregation agents, absorption accelerators, solubilizers, stabilizers, health food ingredients, and nutritional supplements. Materials, vitamins, fragrances, sweeteners, preservatives, preservatives, antioxidants and the like can be mentioned.

≪Treatment method≫
As other aspects of the invention according to the present disclosure, there are a therapeutic method for treating allergic rhinitis or hay fever, and a therapeutic method for treating central nervous disease and the like. These methods include administering the above-described immortalized stem cell culture supernatant or a secreted component thereof, or a processed one of the immortalized stem cell culture supernatant into the body. Thereby, it can treat efficiently.

  The administration method and administration route of the pharmaceutical composition are not particularly limited. When the pharmaceutical composition is used for the treatment of allergic rhinitis or hay fever, parenteral administration is preferred, and parenteral administration is preferred. Examples of topical administration include injection into the target tissue, application or spraying. Examples of the method for administering the pharmaceutical composition include ophthalmic administration and intranasal administration. Among these administration methods, intranasal administration and the like are preferable from the viewpoint of therapeutic effect and minimal invasiveness.

Moreover, when using the said pharmaceutical composition for the treatment of a central nervous system disease, it is preferable that it is parenteral administration, and it may be systemic administration or local administration as parenteral administration. Examples of the administration method include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, ophthalmic administration, and intranasal administration. Of these, intranasal administration and the like are preferable because they are less invasive.
As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.
The dosage of the pharmaceutical composition is, for example, 0.1 mg / kg / day in terms of the amount of untreated immortalized stem cell culture supernatant when used for the treatment of allergic rhinitis or hay fever. It is -1000 mg / kg / day, and 1 mg / kg / day-100 mg / kg / day may be sufficient.
Furthermore, in the above, the dosage when the pharmaceutical composition is dissolved in ultrapure water is, for example, 0.01 mg / kg / day to 100 mg / kg / day, and 0.1 mg / kg / day. -10 mg / kg / day may be sufficient.

  The subject to which the pharmaceutical composition is administered is typically an affected human patient, but includes mammals other than humans (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs) Hamster, monkey, cow, pig, goat, sheep, dog, cat, etc.).

≪Method for producing pharmaceutical composition≫
In the present disclosure, (1) a step of immortalizing any one stem cell selected from dental pulp stem cells or bone marrow stem cells, (2) a step of culturing immortalized stem cells obtained by the immortalization, and (3) And a step of recovering a culture supernatant obtained by culturing the immortalized stem cells.
You may further include the process of extracting the secretory component in the collect | recovered culture supernatant in said process. By including this step, handling, storage and transportation of the pharmaceutical composition become easier. The manufacturing method may further include a step of dissolving the secretory component in ultrapure water. By including this step, it is possible to provide a method for producing a pharmaceutical composition aiming at high penetrating power of the secretory component to the affected area.
The production method further includes a step of performing at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, drying, dilution and desalting before and after any of the above steps. May be.
The production method may further include a step of adding an additional component to the collected culture supernatant. By adding such an additional component, it is possible to change the physical properties of the entire pharmaceutical composition and to improve its properties. About each process and an additional component, the matter described in description of the pharmaceutical composition which concerns on this indication is applied as it is. Subjecting the collected culture supernatant to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage; and the recovery When both steps of adding an additional component to the cultured supernatant are included, both of the steps may be performed first, and may be performed concurrently when possible. In addition, the step of dissolving in ultrapure water is performed after (3) the step of collecting the culture supernatant obtained by culturing the immortalized stem cells, and before and after the other steps. be able to.
The culture supernatant in the above step (3) can be collected by sucking the culture solution with a dropper or pipette, for example. The collected culture supernatant is used as an active ingredient of the pharmaceutical composition according to the present disclosure as it is or after one or more treatments. The culture supernatant of immortalized stem cells exhibits the desired action without complicated and sophisticated purification. For this reason, the pharmaceutical composition according to the present disclosure can be produced by a simple process. The fact that a complicated purification step is not required is also advantageous in that a decrease in activity associated with purification can be avoided. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., −80 ° C.).
Moreover, when said process (1) is a dental pulp stem cell, you may add the process of selecting an adhesive cell from a dental pulp cell as a previous process before a process (1). In addition, the matter described in description of the pharmaceutical composition which concerns on this indication applies as it is the said process to select.

<Method for Producing Lyophilized Product of Immortalized Stem Cell Culture Supernatant>
Moreover, in this indication, the method of manufacturing the freeze-dried material of the immortalized stem cell culture supernatant used for the pharmaceutical composition of this indication can also be provided. Thereby, good storage stability is obtained. As a lyophilization method of the immortalized stem cell culture supernatant, a method commonly used for this purpose can be applied. Examples of the method for producing a lyophilized product of immortalized stem cell culture supernatant include the following methods.
(I) The immortalized stem cell culture supernatant obtained by the above method or a concentrate thereof is frozen at −200 ° C. to −20 ° C. for 2 hours to half a day.
(Ii) After freezing, open the lid of the sample tube and set it in the freeze dryer.
(Iii) Lyophilize for 1-2 days.
(Iv) The obtained product is used as a lyophilized product of an immortalized stem cell culture supernatant (can be stored at -200 ° C to -20 ° C).

  Examples of the present invention will be described below, but the present invention is not limited thereto.

<Materials and methods>
≪Preparation of immortalized dental pulp stem cells≫
An immortalized dental pulp stem cell (SHED-T) is prepared by immortalization treatment after collecting a deciduous dental stem cell according to the method described in Paragraph 0046 to Paragraph 0077 of International Publication No. 2013-47082. Used for preparation of dental pulp stem cell culture supernatant (SHED-CM).

<< Preparation of bone marrow stem cells (BM) and immortalized bone marrow stem cells (BM-T) >>
Human bone marrow stem cells (BM) (bone marrow mesenchymal stem cells, Bone Marrow Mesenchymal stem cells) were purchased from Lonza and cultured according to the manufacturer's instructions. Next, immortalized bone marrow stem cells (BM-T) were produced from the cultured BM by the same method as described above, and used for the production of the next immortalized bone marrow stem cells (BM-CM).

≪Preparation of immortalized stem cell culture supernatant≫
Each of the immortalized stem cells (SHED-T or BM-T) obtained above is determined in a culture solution (DMEM) containing 15% fetal calf serum (FCS) at 37 ° C. in a 5% CO ₂ atmosphere. Cultured for a period of time, centrifuged at 300 g for 5 minutes to collect the supernatant, and filtered using a 0.22 mm syringe filter to obtain an immortalized stem cell culture supernatant stock solution (SHED-CM or BM-CM).

{Effects against hay fever}
[Example 1]
For 6 men and 5 women who are aware of hay fever, the SHED-CM or BM-CM prepared above is used in purified water (Japanese Pharmacopoeia) at a concentration of 1% to 10% (v / v). A pharmaceutical composition was prepared by diluting, and any of these was administered nasally every day for 2 weeks from 0.1 ml to 0.3 ml (average 0.3 ml). In addition, the content ratio (w / v)% with respect to the purified water of the secretory component in SHED-CM and BM-CM used in Example 1 is 5 × 10 ⁻⁶ % to 5 × 10 ⁻⁵ %.
As a clinical evaluation, as described below, subjective symptoms (nasal symptoms and eye symptoms) before and after treatment, that is, before treatment and after 2 weeks, were evaluated by scores as follows.

<Score evaluation of nasal symptoms>
As shown below, (1) the number of times of sneezing per day, (2) the number of times of runny nose treatment, and (3) the degree of stuffy nose, were scored, and the average of the scores in these three items was entered by four people The score in the evaluation of nasal symptoms.
(1) Number of sneezing: 21 points or more per day: 5 points, 20-11 times: 4 points, 10-6 times: 3 points, 5-1 times: 2 points, 0 times: 1 point.
(2) Number of times of runny nose treatment: 21 points or more per day: 5 points, 20-11 times: 4 points, 10-6 times: 3 points, 5-1 times: 2 points, 0 times: 1 point .
(3) Nose clogging: 5 points for complete nasal congestion, 4 points for strong nasal congestion, mostly mouth breathing, 3 points for strong nasal congestion, sometimes mouth breathing, nasal congestion , 2 points when there is no mouth breathing, 1 point when there is no stuffy nose and mouth breathing.
<Evaluation of eye symptom score>
Regarding the state before and after treatment, a score is provided as follows, and the score is used in the evaluation of eye symptoms.
・ Severe symptoms such as hyperemia, tears and itching: 4 points ・ The above symptoms are severe: 3 points ・ In the above symptoms, there is no problem: 2 points ・ In the above symptoms, there is no problem: 1 point <Effective days>
In the above-described clinical evaluation, the period from the day when the administration was started to the day when the effect can be judged, that is, the day when the score started to decrease, was defined as the number of days when the effect was manifested.

[Example 2]
For 3 men and 2 women who are aware of hay fever, the SHED-CM or BM-CM prepared above was added to ultrapure water (manufactured by Sanei Co., Ltd.) at a concentration of 0.1% to 1% ( The pharmaceutical composition was prepared by diluting with v / v), and 0.1 ml to 0.3 ml (average 0.3 ml) was administered nasally every day for 2 weeks. In addition, the content ratio (w / v)% of the secretory component to the ultrapure water in the SHED-CM and BM-CM used in Example 2 is 5 × 10 ⁻⁷ % to 5 × 10 ⁻⁶ %.
The clinical evaluation was performed by evaluating the score in the same manner as in Example 1.

  Details of data of each patient in the above examples, the type of culture supernatant used for each patient (S: SHED-CM, B; BM-CM), concentration of the culture supernatant, clinical evaluation, and number of days of effect expression Are shown in Tables 1 and 2.

From the results of Tables 1 and 2, in Example 1 using purified water, both nasal symptoms and ocular symptoms were improved, and particularly high in nasal symptoms. Moreover, in 11 patients in Example 1, no symptoms that seemed to be side effects were observed.
In Example 2 using ultrapure water, as shown in Table 2, the concentration of the immortalized stem cell culture supernatant was 1/10 of that in Example 1 using purified water. Both nasal symptoms and ocular symptoms showed significant improvement effects. Moreover, in all the five patients in Example 2, no symptoms that seemed to be side effects were observed. In addition, all these five patients did not have any subjective symptoms of hay fever even after 3 months.
FIG. 1 and FIG. 2 show changes in scores (average evaluation scores) before and after treatment with nasal symptoms and ocular symptoms in Example 1 and Example 2. FIG. Moreover, the black circle in a figure shows the evaluation score of Example 2, and the black triangle shows the evaluation score of Example 1. From FIG. 1 and FIG. 2, it can be clearly seen that in Example 2, the pharmaceutical composition using ultrapure water has a higher therapeutic effect than in the case of using purified water (Example 1). Indicated.
Moreover, it was shown that Example 2 using ultrapure water has an effect at an early stage clearly compared with Example 1 using purified water also in the number of days of effect expression.
Thus, it was shown that the pharmaceutical composition containing the immortalized stem cell culture supernatant in the present disclosure has a high therapeutic effect for allergic rhinitis and hay fever by short-term administration.

{Effect on cerebral infarction}
The effects of SHED-CM and BM-CM on animals serving as models of cerebral infarction were verified as follows.

Example 3
[Evaluation for movement disorders]
<Cerebral infarction rat>
As an animal serving as a model of cerebral infarction (cerebral ischemia model), Sprague-Dawley rats (SD rat) was purchased, and the method described in Inoue et al. [Tissue Eng Part A, Vol. 19, 24 (2013)]. Thus, the middle cerebral artery was occluded in the rat, and a rat having cerebral ischemia (cerebral infarction rat) was prepared.

<Administration of pharmaceutical composition to rats with cerebral infarction>
According to the method described by Inoue et al., 72 hours after the middle cerebral artery occlusion of the rat (day 3), the rat was anesthetized. And it divided into the administration group (A)-(C) of the pharmaceutical composition used for a comparative example. Thereafter, according to the method described in the same document, 100 μl / day of each corresponding pharmaceutical composition was introduced into the rats of each administration group using a microsyringe (manufactured by Hamilton). I went to my eyes every day.
<Administration group>
-Pharmaceutical composition administration group (A): This is a group to administer a pharmaceutical composition in which SHED-CM is mixed at 50% (v / v) with purified water (Japanese Pharmacopoeia). The total number of individuals is 3.
-Pharmaceutical composition administration group (B): It is a group which administers the pharmaceutical composition which mixed SHED-CM with the ultrapure water (made by Yamaei Co., Ltd.) by 50% (v / v). The total number of individuals is 3.
-Pharmaceutical composition administration group (C): A group to which only PBS is administered. The total number of individuals is 3.

<Evaluation method for movement disorders>
In the pharmaceutical composition administration groups (A), (B) and (C), the evaluation of the movement disorder at the time of each day from the 3rd day to the 12th day was scored according to the method described in the above-mentioned Inoue et al. I went by making it. In addition, evaluation of movement disorders was performed on all three animals in each of the above administration groups, and was scored. In addition, the evaluation score used by the evaluation in the said movement disorder shows that there are few movement disorders, so that a score is low.
Moreover, in the evaluation score obtained at the time of each day, between the pharmaceutical composition administration group (A) and the pharmaceutical composition administration group (C) and between the pharmaceutical composition administration group (B) and the pharmaceutical composition administration group ( P values (significance probabilities) with C) were determined by Student's T test.

In FIG. 3, the result of the evaluation score obtained at the time of each day progress of each administration group is shown. Moreover, the case where P value between those administration groups is 5% or less is shown simultaneously.
As shown in FIG. 3, in the pharmaceutical composition administration group (A) given SHED-CM mixed with purified water, the score gradually decreased with the passage of days, and the pharmaceutical composition administration group (C) on the 12th day. Significantly reduced to 1/3 of the evaluation score. Further, in the pharmaceutical composition administration group (B) given SHED-CM mixed with ultrapure water, the evaluation score decreased more significantly than in the pharmaceutical composition administration group (A). The evaluation score decreased to about one-half of the product administration group (A).
From the above results, it was shown that the pharmaceutical composition containing SHED-CM has an effect of recovering the symptoms of cerebral infarction in cerebral infarction rats at an early stage. Furthermore, it was shown that the SHED-CM containing pharmaceutical composition using ultrapure water has a higher effect on recovery from symptoms of cerebral infarction.

Example 4
[Evaluation in the area of cerebral infarction]
In order to evaluate the size of the cerebral infarction region after administration of the pharmaceutical composition in each of the pharmaceutical composition administration groups (A) to (C), the above-mentioned Inoue et al. And Ginsberg et al. [Stroke, Vol. 34, 214 (2013)], the rat brain of each administration group on day 13 was collected, and then incisions (coronal cross section) 12 having a thickness of 20 μm at intervals of 1 mm so as to cover the entire brain. After producing the sheet, hematoxylin-eosin staining was performed, and the cerebral infarction region was visualized by using a fluorescence microscope. In addition, cerebral infarction in each of two rats in each administration group based on the data obtained from each of these incisions in accordance with the method described in Leach et al. [Stroke, Vol. 24, 1063 (1993)]. The volume (mm ³ ) of the area was calculated.

FIG. 4 is an image obtained by photographing a coronal section of the central portion of the brain of each rat in each administration group under a fluorescence microscope. Further, the number at the right end of each coronal section indicates the calculated volume (mm ³ ) of the cerebral infarction region.
As shown in FIG. 4, cerebral infarct volume region (35 mm ³ and 33 mm ³⁾ of the pharmaceutical composition administered group gave SHED-CM was mixed with purified water (A), the pharmaceutical compositions administered to received PBS only Compared to the volume of group (C) (42 mm ³ and 45 mm ³ ), it was shown that the cerebral infarction area was clearly reduced. Furthermore, the volume (26 mm ³ and 19 mm ³ ) of the pharmaceutical composition administration group (B) mixed with ultrapure water has a more reduced cerebral infarction region than the pharmaceutical composition administration group (A). It has been shown.
Thus, it was shown that the pharmaceutical composition containing SHED-CM has an effect of reducing the cerebral infarction region in cerebral infarction rats. Furthermore, it was shown that the SHED-CM containing pharmaceutical composition using ultrapure water has a higher effect.

Example 5
[Evaluation of SHED-CM in the transition to the brain]
Fluorescence labeling of how the difference in the type of water (purified water or ultrapure water) contained in the pharmaceutical composition affects the difference in the amount transferred to the brain after nasal administration of SHED-CM The substance (Qdot (manufactured by Life Technology)) was used for verification.
Load SHED-CM into Amicon Ultra Centrifugal Filter Units-10K (Millipore), centrifuge at 4 ° C. and 4000 g for about 60 minutes to obtain a 25-fold concentrate, and then add the same amount of sterile PBS as it was loaded Centrifuge again under the same conditions. By repeating this three times, a solution (SHED-CMH) from which low molecules having a molecular weight of about 5000 or less were removed from SHED-CM was obtained. Next, a SHED-CMH-Qdot solution containing particles in which SHED-CMH was bonded onto Qdot nanocrystals was prepared according to the protocol of Qdot innovators Tool Kit ITKTM (product number Q21531MP, manufactured by Life Technology). Next, the obtained SHED-CMH-Qdot solution was added to 0.1% (v / v) in the pharmaceutical composition to obtain a solution (a) and a solution (b) having the following composition conditions. .
<Composition conditions>
(A) Solution / SHED-CMH-Qdot solution 0.1%
・ SHED-CM 49.9%
・ Purified water 50.0%
(B) Solution / SHED-CMH-Qdot solution 0.1%
・ SHED-CM 49.9%
・ Ultrapure water 50.0%
In the rats of the 13th day of the pharmaceutical composition administration groups (A) and (B), the rats of the pharmaceutical composition administration group (A) were (a) the solution, the rats of the pharmaceutical composition administration group (B) were (b) ) Each solution was administered intranasally, and 30 minutes later, the rat was fixed on a fluorescence imager and the head was photographed to perform fluorescence imaging of the rat brain.

FIG. 5 shows a fluorescence image of the rat brain 30 minutes after administration of the (a) solution or (b) solution, respectively.
As shown in FIGS. 5 (A) and 5 (B), it is shown that the high molecular weight component of SHED-CM (SHED-CMH) is transferred to the rat brain when any solution is used. (The elongated and shining part in the upper part of the figure). In addition, when the solution (b) mixed with ultrapure water was administered, the region with strong fluorescence intensity was wide compared with the solution (a) mixed with purified water. It was shown that the transfer of SHED-CMH to the brain occurred more strongly.

〔Comprehensive evaluation〕
From the results and evaluations of Examples 3 to 5, the pharmaceutical composition of the present disclosure migrates quickly and extensively to the brain suffering from cerebral infarction after its administration, and repairs damaged brain tissue to a normal state. It has been shown that it has an effect of being transferred and recovering from symptoms associated with cerebral infarction at an early stage.

Claims (9)

An immortalized stem cell culture supernatant containing a secretory component from an immortalized stem cell obtained by culturing an immortalized immortalized stem cell, or a secretory component thereof,
A pharmaceutical composition for the treatment of at least one of allergic rhinitis and hay fever, wherein the immortalized stem cells are either dental pulp stem cells or bone marrow stem cells. An immortalized stem cell culture supernatant containing a secretory component from an immortalized stem cell obtained by culturing an immortalized immortalized stem cell, or a secretory component thereof,
A pharmaceutical composition for treating a central nervous system disease, wherein the immortalized stem cell is either a dental pulp stem cell or a bone marrow stem cell.   The pharmaceutical composition according to claim 1 or 2, comprising ultrapure water, wherein the secretory component is dissolved in ultrapure water. The pharmaceutical composition according to claim 3, wherein the secretory component is contained in an amount of 1 x ^10-7 mass% to 1 mass% with respect to ultrapure water.   The pharmaceutical composition according to any one of claims 1 to 4, further comprising a reversible heat-thickening compound.   The pharmaceutical which has the pharmaceutical composition of any one of Claims 1-5 in the form of an injection, an oral preparation, a nasal drop, an eye drop, or a coating agent. A method for producing the pharmaceutical composition according to any one of claims 1 to 5,
Immortalizing any one stem cell selected from dental pulp stem cells or bone marrow stem cells;
Culturing the immortalized stem cells obtained by the immortalization;
Recovering the culture supernatant containing the secretory component from the immortalized stem cell, obtained by culturing the immortalized stem cell, and a method for producing a pharmaceutical composition.   Furthermore, the manufacturing method of Claim 7 including the process of extracting the secretory component in the collect | recovered culture supernatant.   The method according to claim 7 or 8, further comprising a step of dissolving the secretory component in ultrapure water.