Screening method for periodontal tissue regeneration components

Abstract

This invention provides a method for screening new periodontal tissue regeneration components or bone tissue regeneration components. [Solution] A method for screening periodontal tissue regeneration components, comprising the step of evaluating the IκBα promoting effect of the component; and a method for evaluating the effect of the component on periodontal tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the component. Furthermore, a method for screening bone tissue regeneration components, comprising the step of evaluating the IκBα promoting effect of the component; and a method for evaluating the effect of the component on bone tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the component.

Description

Technical Field

[0001] The present invention relates to a method for screening periodontal tissue regeneration components. The present invention also relates to a method for screening bone tissue regeneration components.

Background Art

[0002] Periodontal disease is a disease in which the periodontal tissue supporting the teeth is invaded, and it is an extremely important disease that ultimately leads to tooth loss.

[0003] Periodontal disease is caused by bacteria such as Porphyromonas gingivalis, Porphyromonas gulae, and Actinobacillus actinomyceremcomitans, and antibiotics and the like are used to suppress the growth or kill these bacteria. In addition, methods for removing plaque such as brushing and scaling are used for the prevention of periodontal disease. However, with these therapies and methods, even if the progression of periodontal disease is stopped, it is difficult to regenerate or repair the destroyed periodontal tissue.

[0004] Various therapeutic agents have been proposed for promoting the regeneration, healing, and reattachment of these periodontal tissues. Non-Patent Document 1 describes the treatment results of periodontal tissue regeneration therapy using a preparation of basic fibroblast growth factor (also referred to as FGF-2 or bFGF).

[0005] However, Non-Patent Document 2 describes that a decrease in collagen I and an increase in MMP-1, which is a collagen-degrading enzyme, were confirmed by the excessive administration of bFGF. In addition, Non-Patent Document 3 describes that a dose-dependent increase in the percutaneous infection rate of Staphylococcus aureus was observed in the group locally administered with bFGF compared to the group not administered with bFGF, and it is considered that bFGF causes a decrease in immunity.

Prior Art Documents

Non-Patent Documents

[0006] [Non-Patent Document 1] Japanese Journal of Conservative Dentistry, Vol. 63, No. 3, pp. 219-227 [Non-Patent Document 2] Journal of Periodontology, Volume 71, Issue 6 (2000), 974-980 [Non-Patent Document 3] Acta Orthopedica, Volume 78, Issue 1 (2007), 63-73 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a method for screening new periodontal tissue regeneration components or bone tissue regeneration components. [Means for solving the problem]

[0008] To achieve the above objective, the inventors constructed a mathematical model that focuses on the complex signaling pathway mechanism as a new method for evaluating periodontal tissue regeneration. Through studies using this mathematical model, they found that suppressing pathways that promote bone resorption improves the bone metabolic balance of the alveolar bone and is effective in promoting periodontal tissue regeneration. More specifically, the inventors found that enhancing the activity of IκBα is the most effective in promoting periodontal tissue regeneration. This invention is completed based on these findings.

[0009] In other words, the present invention provides the following: [1] A method for screening periodontal tissue regeneration components, comprising the step of evaluating the IκBα promoting effect of the test component. [2] The screening method of [1], characterized in that the periodontal tissue regeneration component is for use in mammals. [3] The screening method according to [1] or [2], characterized in that the periodontal tissue regeneration component is used on periodontal tissue affected by periodontal disease. [4] A method for evaluating the effect of a test component on periodontal tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component. [5] A method for screening bone tissue regeneration components, comprising the step of evaluating the IκBα promoting effect of the test component. [6] A method for evaluating the effect of a test component on bone tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component. [7] A method for screening components for the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease, comprising the step of evaluating the IκBα-promoting effect of the component under test. [8] A method for evaluating the effects of a test component on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component. [Effects of the Invention]

[0010] The present invention provides a method for screening periodontal tissue regeneration components or bone tissue regeneration components. The present invention also provides a method for screening components for the treatment, improvement, alleviation, progression inhibition, healing promotion, and / or prevention of periodontal disease or bone disease. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the method for creating a mouse periodontal disease regeneration model in Example 1. [Figure 2] Figure 2 shows the test schedule for Example 1. [Figure 3] Figure 3 shows periodontal μCT images of the Test group and Control group in Example 1 during a 12-day periodontal tissue regeneration period. [Figure 4] Figure 4 shows the changes in the μCT values ​​converted to alveolar bone levels for the Test group and Control group in Example 1. [Figure 5] Figure 5 shows the RNA-seq data and analysis results from Example 2. [Figure 6]FIG. 6 is a diagram showing the bone regeneration mathematical model obtained in Example 3. [Figure 7] FIG. 7 is a diagram showing the results of the sensitivity analysis in Example 4. [Figure 8] FIG. 8 is a diagram showing the mechanism of action of Bay11-7082. [Figure 9] FIG. 9 is a diagram showing the test plan in Example 5. [Figure 10] FIG. 10 is a diagram showing the transition of the values obtained by converting the μCT values of the Bay group and the Control group in Example 5 to the alveolar bone level. [Figure 11] FIG. 11 is a diagram comparing the bone regeneration amounts of the Bay group and the Control group on the 6th day of administration in Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The inventors of the present invention constructed a mathematical model as a new method for evaluating periodontal tissue regeneration, and found that IκBα is related to periodontal tissue regeneration by sensitivity analysis. The present invention has been completed by finding that it is possible to evaluate or screen whether a test component can promote or inhibit IκBα and thereby regenerate periodontal tissue or bone tissue. IκBα exists in the pathway related to bone resorption, and the enhancement of IκBα can lead to the inhibition of bone resorption. In addition, the enhancement of IκBα is considered to be optimal for the prevention or treatment of periodontal disease in that it gently suppresses inflammation while maintaining the immunity to suppress periodontal disease-related bacterial cells and promotes bone regeneration to promote periodontal tissue regeneration.

[0013] (Definition) In the present specification, the periodontal tissue refers to the tissue that plays a role in supporting the tooth, and includes the gingiva (gum) covering the tooth, the periodontal ligament that serves as a cushion between the tooth and the alveolar bone, the alveolar bone that supports the tooth, and cementum that covers the root of the tooth.

[0014] In this specification, "periodontal disease" can be broadly divided into gingivitis and periodontitis based on its progression, with other known causes including occlusal trauma. Gingivitis is the initial stage of periodontal disease, characterized by swelling and easy bleeding of the gums due to plaque and tartar buildup around the teeth and between them. Periodontitis can be broadly classified into mild, moderate, and severe based on its symptoms. Mild periodontitis refers to a state where periodontal disease pathogens and toxins they produce enter the swollen gums, beginning to dissolve the alveolar bone. Moderate periodontitis refers to a state where the alveolar bone has dissolved to half the length of the tooth root due to periodontal disease. Severe periodontitis refers to a state where more than half the length of the tooth root has dissolved the alveolar bone. When the alveolar bone supporting the teeth decreases, the teeth become loose and eventually fall out. In this specification, "periodontal disease" also includes peri-implantitis. Peri-implantitis refers to the resorption of alveolar bone around an implant after implant placement. The effects of this invention include improving bone resorption and inflammation (bone grafting and anti-inflammatory action) caused by peri-implantitis.

[0015] In this specification, "test component" refers to, for example, proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances may be novel or known. Compound libraries prepared using combinatorial chemistry technology, random peptide libraries prepared by solid-phase synthesis or phage display methods, etc., can also be cited as test components.

[0016] In the screening or evaluation methods described herein, the promotion or suppression of IκBα may refer to the promotion or suppression of IκBα in cells. Here, the cells are not limited to periodontal ligament fibroblasts, cementocytes, osteoblasts, and osteoclasts, but may also include monocytes, macrophages, lymphocytes, neutrophils, etc. Of these, osteoclasts are preferred.

[0017] Examples of osteoclasts include cells obtained by differentiation induction from osteoclast progenitor cells, or osteoclasts isolated from tissues. Osteoclast progenitor cells include monocyte-macrophage cells capable of differentiating into osteoclasts, and any osteoclast progenitor cell capable of differentiating into osteoclasts can be used regardless of species or cell line. Tissues containing osteoclast progenitor cells, such as bone tissue, can also be used. For example, primary cultured cells prepared from an individual or established cell lines can be used.

[0018] In this specification, "for periodontal tissue regeneration" or "for bone tissue regeneration" is not particularly limited as long as it is intended for animals. That is, the subjects to whom the periodontal tissue regeneration composition or bone tissue regeneration composition is administered or ingested include mammals, including or excluding humans, such as eutherians. Carnivorous animals, including Canidae, Felidae, Ursidae, etc., and rodents, such as mice, squirrels, rats, guinea pigs, and hamsters, may be preferred subjects. Domesticated animals, pets, companion animals, etc., are also subject to this specification. More specifically, one or more animals selected from humans, monkeys, dogs, cats, bears, sea lions, mice, rats, squirrels, guinea pigs, hamsters, cattle, sheep, goats, pigs, deer, horses, or rabbits are also included.

[0019] [Screening of periodontal tissue regeneration components, evaluation of periodontal tissue regeneration] In one embodiment, the present invention provides a screening method for periodontal tissue regeneration components, comprising the step of evaluating the IκBα promoting or inhibiting effect of a test component. In another embodiment, the present invention provides a method for evaluating the effect of a component on periodontal tissue regeneration, comprising the step of evaluating the IκBα promoting or inhibiting effect of a test component.

[0020] In some embodiments, the screening or evaluation method of the present invention includes culturing cells in vitro in the presence of a test component and quantifying the change in IκBα levels over a certain period of time. More specifically, if there is an increase in IκBα levels after a certain period of time compared to the initial level, it can be determined that the test component has an IκBα-promoting effect, or if there is a decrease, it can be determined that the test component has an IκBα-inhibiting effect. In other embodiments, the screening or evaluation method of the present invention includes culturing cells in vitro in the presence and absence of a test component and quantifying and comparing the IκBα levels in the cells under both conditions. More specifically, if there is an increase in IκBα levels in the presence of a test component compared to the IκBα level in the absence of the test component, it can be determined that the test component has an IκBα-promoting effect, or if there is a decrease, it can be determined that the test component has an IκBα-inhibiting effect. More specifically, the screening method of the present invention screens for components that have the above-mentioned IκBα-promoting effect. A more specific method is described in Example 6 below.

[0021] In this specification, components that promote IκBα are referred to as periodontal tissue regeneration components, as they have the effect of promoting periodontal tissue regeneration. Components that inhibit IκBα are referred to as periodontal tissue regeneration inhibitory components.

[0022] IκBα levels can be measured by evaluating the amount of gene products that reflect the expression status of the gene encoding IκBα, for example. The gene products are not particularly limited and include, for example, mRNA, proteins, and peptides; one or more selected from these can be used. Such values ​​can be measured by known methods, such as Northern blotting, microarrays, RNA sequencing, PCR and other gene quantification methods, immunohistochemistry, Western blotting, in situ hybridization (ISH), next-generation sequencing (NGS), immunoblotting, staining, fluorescence, antibody methods, aptamer methods, ELISA, immunoprecipitation, and immunoturbidimetry. One or more selected from these methods may be used, or they may be used in appropriate combinations for detection or measurement. In such measurements, primers or antibodies specific to IκBα may be used.

[0023] By using the screening method or evaluation method of the present invention, it is possible to easily and efficiently screen for periodontal tissue regeneration components or periodontal tissue regeneration inhibiting components from among a large number of test components.

[0024] In one embodiment, the evaluation method of the present invention can be used to evaluate the effects of a test component on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease. Specifically, in the method described above, a component determined to have an IκBα-promoting effect can be determined to have an effect on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease.

[0025] (Composition for periodontal tissue regeneration) In one embodiment, components screened by the screening method of the present invention, or components determined to have an effect of promoting periodontal tissue regeneration by the evaluation method, are selected as periodontal tissue regeneration components and can be used more specifically as a composition for the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease. That is, in one embodiment, the screening method of the present invention is a screening method for components for the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease, comprising the step of evaluating the IκBα promoting effect of the test component. In this specification, the above composition and the composition containing the above component are referred to as a periodontal tissue regeneration composition. The periodontal tissue regeneration composition can be used as is, or added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, animal feed, or pet food, etc. In one embodiment, the periodontal tissue regeneration composition is a pharmaceutical composition or a food composition.

[0026] When periodontal tissue regeneration compositions are used as is, added to, or mixed with foods, beverages, animal feed, animal cosmetics, quasi-drugs for animals, or pet food, etc., they can be used as foods or beverages that explicitly or implicitly state that they maintain good condition of teeth and gums, or maintain the health of teeth and gums, as functional claims, i.e., health foods, functional foods, foods for the sick, and foods for specified health uses. Foods and pet foods include supplements. Furthermore, even without explicitly or implicitly stating the above functional claims, they can be used as so-called doctor's supplements recommended or presented by physicians in hospitals and / or clinics, such as internal medicine, orthopedics, and veterinary hospitals.

[0027] If the above-mentioned functionality is not explicitly stated, it is also possible to use the periodontal tissue regeneration composition by making a statement such as, for example, "for people concerned about dental health," "for people concerned about gum health," or "for people who want to eat a healthy diet," or a similar statement.

[0028] Health foods, functional foods, foods for the sick, foods for specified health uses, and pet foods can be used in various formulations, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candies, chewing gum, chewing candy, etc.), liquid preparations (syrups, suspensions), and liquid foods. Food formulations can be manufactured in the same way as known pharmaceutical preparations, by mixing the active ingredient with a food-acceptable carrier, such as a suitable excipient, and then manufacturing them using conventional methods. While the formulation form is not limited, orally disintegrating tablets, chewable tablets, candies, granules, powders, or liquid preparations are preferred from the viewpoint of significantly achieving the effects of the present invention.

[0029] (Embodiments as a pharmaceutical composition) The periodontal diseases targeted by the periodontal tissue regeneration composition include those described above, and can particularly target gingivitis and periodontitis caused by periodontal disease-causing bacteria. More specifically, it is desirable to target periodontal diseases in which alveolar bone has been absorbed and regeneration of periodontal tissue, including the alveolar bone, is necessary. In one embodiment, the periodontal tissue regeneration composition is a composition for treating periodontal disease in which alveolar bone has been absorbed.

[0030] When administering a periodontal tissue regeneration composition to a target, the administration route and dosage form are not particularly limited and can be appropriately selected and implemented by a person skilled in the art depending on the purpose of use of the composition, the target of administration, and the product's intended use. Preferably, the periodontal tissue regeneration composition is administered locally to periodontal tissues such as teeth, gums, and alveolar bone affected by periodontal disease. Methods of administration include injection, application, or spraying to the affected area. The periodontal tissue regeneration composition may be administered to the affected area in combination with flap surgery, or it may be administered to the periodontal tissue and periodontal pocket without the use of flap surgery.

[0031] The dosage form of the periodontal tissue regeneration composition can be any dosage form, such as liquids (solutions, emulsions, suspensions, injections, etc.), slurries, pastes, gels, creams, ointments, sprays, powders, tablets, gums, etc.

[0032] The administration schedule for the periodontal tissue regeneration composition is not particularly limited and can be appropriately determined by those skilled in the art, taking into account the gender, age, weight, and condition of the recipient. For example, administration schedules such as once every 3 to 5 days, once every 1 to 2 weeks, or once every 1 to 2 months are possible.

[0033] The dosage of the periodontal tissue regeneration composition is not particularly limited and can be appropriately determined by those skilled in the art, taking into account the condition of the periodontal tissues such as the teeth, gums, and alveolar bone affected by periodontal disease, the route of administration, the gender, age, and weight of the subject. For example, dosages of 0.1 to 10 ml per tooth or 0.1 to 1 ml per dose are possible.

[0034] In one embodiment, the periodontal tissue regeneration composition can be used in combination with treatment methods aimed at regenerating periodontal tissue, such as GTR (Guided Tissue Regeneration), bone graft material, collagen sponge, or bFGF.

[0035] In this specification, "bone graft material" refers to autogenous bone or artificial bone that can be used in combination for the regeneration of periodontal tissue damaged by periodontal disease, and refers to artificial bone composed of, for example, hydroxyapatite (HA), tricalcium phosphate (TCP), or β-calcium phosphate (β-TCP) activated glass. Recombinant human bFGF may also be administered in combination, and commercially available products (such as Regrowth, recombinant human bFGF preparation, Kaken Pharmaceutical Co., Ltd.) can be used in combination.

[0036] These treatment methods aimed at regenerating periodontal tissue can be used in conjunction with the administration of an effective therapeutic amount of the periodontal tissue regeneration composition to the affected area. While not limited to the following, for example, after applying the periodontal tissue regeneration composition and performing antibacterial treatment to the affected area, a membrane, bone graft material, or collagen sponge used in the GTR method can be applied to the affected area.

[0037] (Methods of treatment, prevention, or use) In one embodiment, the present invention provides a method for treating or preventing periodontal disease in a subject, including or excluding humans, using a component screened by the screening method of the present invention, or a component determined to have an effect of promoting periodontal tissue regeneration by the evaluation method. In another embodiment, the present invention provides the use of a component screened by the screening method of the present invention, or a component determined to have an effect of promoting periodontal tissue regeneration by the evaluation method, for producing a drug for treating or preventing periodontal disease.

[0038] (kit) In another embodiment, the present invention provides a kit for screening periodontal tissue regeneration components, comprising a primer or antibody for detecting the presence of IκBα, and optionally cells, a culture medium, and instructions.

[0039] [Screening of bone tissue regeneration components, evaluation of bone tissue regeneration] In one embodiment, the present invention provides a method for screening bone tissue regeneration components, comprising the step of evaluating the IκBα promoting effect of a test component. In another embodiment, the present invention provides a method for evaluating the effect of a component on bone tissue regeneration, comprising the step of evaluating the IκBα promoting or inhibiting effect by applying the test component.

[0040] In some embodiments, the screening or evaluation method of the present invention includes culturing cells in vitro in the presence of a test component and quantifying the change in IκBα levels over a certain period of time. More specifically, if there is an increase in IκBα levels after a certain period of time compared to the initial level, it can be determined that the test component has an IκBα-promoting effect, or if there is a decrease, it can be determined that the test component has an IκBα-inhibiting effect. In other embodiments, the screening or evaluation method of the present invention includes culturing cells in vitro in the presence and absence of a test component and quantifying and comparing the IκBα levels in the cells under both conditions. More specifically, if there is an increase in IκBα levels in the presence of a test component compared to the IκBα level in the absence of the test component, it can be determined that the test component has an IκBα-promoting effect, or if there is a decrease, it can be determined that the test component has an IκBα-inhibiting effect. More specifically, the screening method of the present invention screens for components that have the above-mentioned IκBα-promoting effect. A more specific method is described in Example 6 below.

[0041] In this specification, components that have an effect of promoting IκBα are selected as components that have an effect of promoting bone tissue regeneration and are referred to as bone tissue regeneration components. Components that have an effect of inhibiting IκBα are referred to as bone tissue regeneration inhibitory components.

[0042] IκBα levels can be measured by evaluating the amount of gene products that reflect the expression status of the gene encoding IκBα, for example. The gene products are not particularly limited and include, for example, mRNA, proteins, and peptides; one or more selected from these can be used. Such values ​​can be measured by known methods, such as Northern blotting, microarrays, RNA sequencing, PCR and other gene quantification methods, immunohistochemistry, Western blotting, in situ hybridization (ISH), next-generation sequencing (NGS), immunoblotting, staining, fluorescence, antibody methods, aptamer methods, ELISA, immunoprecipitation, and immunoturbidimetry. One or more selected from these methods may be used, or they may be used in appropriate combinations for detection or measurement. In such measurements, primers or antibodies specific to IκBα may be used.

[0043] By using the screening method or evaluation method of the present invention, it is possible to easily and efficiently screen for bone tissue regeneration components or bone tissue regeneration inhibiting components from among a large number of test components.

[0044] In one embodiment, the evaluation method of the present invention can be used to evaluate the effects of a test component on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of bone diseases. Specifically, in the method described above, a component determined to have an IκBα-promoting effect can be determined to have an effect on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of bone diseases.

[0045] In this specification, "bone disease" refers to a disorder characterized by a decrease in bone mass. Specifically, bone diseases include osteoporosis (e.g., postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of therapeutic drugs such as steroids and immunosuppressants, and osteoporosis associated with rheumatoid arthritis), bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases of cancer, giant cell tumor, osteopenia, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta. In some embodiments, the bone disease is a bone disease involving osteoclasts.

[0046] (Composition for bone tissue regeneration) In one embodiment, a component screened by the screening method of the present invention, or a component determined to have an effect of promoting bone tissue regeneration by the evaluation method, can be used as a bone tissue regeneration component, more specifically, as a composition for the treatment, improvement, alleviation, progression inhibition, and / or prevention of bone diseases. That is, in one embodiment, the screening method of the present invention is a screening method for components for the treatment, improvement, alleviation, progression inhibition, and / or prevention of bone diseases, comprising a step of evaluating the IκBα promoting effect of the test component. In this specification, the above composition and the composition containing the above component are referred to as a bone tissue regeneration composition. The bone tissue regeneration composition can be used as is, or added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, animal feed, or pet food. In one embodiment, the bone tissue regeneration composition is a pharmaceutical composition or a food composition.

[0047] When a bone tissue regeneration composition is used as is, added to, or mixed with food, beverages, animal feed, or pet food, it can be used as a food or beverage that explicitly or implicitly states that it has the functionality of maintaining good bone condition or bone health, i.e., as a health food, functional food, food for the sick, or food for specified health uses. Food and pet food include supplements. Furthermore, even without explicitly or implicitly stating the above functionality, it can be used as a so-called doctor's supplement recommended or presented by a physician in hospitals and / or clinics, such as internal medicine, orthopedics, or veterinary hospitals.

[0048] If the above-mentioned functionality is not explicitly stated, the composition of the present invention can be used with a label such as, for example, "for people concerned about bone health," "for people who want to maintain their athletic function," or a similar label.

[0049] Health foods, functional foods, foods for the sick, foods for specified health uses, and pet foods can be used in various formulations, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candies, chewing gum, chewing candy, etc.), liquid preparations (syrups, suspensions), and liquid foods. Food formulations can be manufactured in the same way as known pharmaceutical preparations, by mixing the active ingredient with a food-acceptable carrier, such as a suitable excipient, and then manufacturing them using conventional methods. While the formulation form is not limited, orally disintegrating tablets, chewable tablets, candies, granules, powders, or liquid preparations are preferred from the viewpoint of significantly achieving the effects of the present invention.

[0050] (Embodiments as a pharmaceutical composition) When administering a bone tissue regeneration composition to a target, the route of administration and dosage form are not particularly limited and can be appropriately selected and implemented by a person skilled in the art depending on the intended use of the composition, the target of administration, and the product's intended use. For example, parenteral or oral administration is possible. Parenteral preparations include, but are not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, and topical administration preparations. Topical administration includes direct administration to the injured, fractured, or impaired bone area, such as the skull, femur, sternum, vertebrae, or ribs, and may be administered as a transplant preparation containing the active ingredient in an artificial bone component such as hydroxyapatite.

[0051] The composition for bone tissue regeneration can be in any dosage form, such as liquids (solutions, emulsions, suspensions, injections, etc.), slurries, pastes, gels, creams, ointments, sprays, powders, suppositories, suppositories, tablets, pills, granules, capsules, etc.

[0052] The administration schedule for the bone tissue regeneration composition is not particularly limited and can be appropriately determined by those skilled in the art, taking into account the gender, age, weight, and condition of the recipient. For example, administration schedules such as once every 3 to 5 days, once every 1 to 2 weeks, or once every 1 to 2 months are possible.

[0053] The dosage of the bone tissue regeneration composition is not particularly limited and can be appropriately determined by those skilled in the art, taking into account the condition of the bone tissue, the route of administration, the sex, age, and weight of the subject. For example, dosages of approximately 0.1 μg / kg to 100 mg / kg and approximately 1 μg / kg to 10 mg / kg per dose are possible.

[0054] (Methods of treatment, prevention, or use) In one embodiment, the present invention provides a method for treating or preventing a bone disease in a subject, including or excluding humans, using a component screened by the screening method of the present invention, or a component determined to have an effect of promoting bone tissue regeneration by the evaluation method. In another embodiment, the present invention provides the use of a component screened by the screening method of the present invention, or a component determined to have an effect of promoting bone tissue regeneration by the evaluation method, for producing a drug for treating or preventing a bone disease.

[0055] (kit) In another embodiment, the present invention provides a kit for screening bone tissue regeneration components, comprising a primer or antibody for detecting the presence of IκBα, and optionally cells, a culture medium, and instructions. [Examples]

[0056] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0057] Example 1: Creation of a periodontal tissue regeneration model and acquisition of RNA-seq data. An artificial periodontal disease model was created by wrapping silk thread around the teeth of mice for 10 days, making the periodontal tissue more susceptible to periodontal disease-related bacteria (Figure 1).

[0058] Mice in which periodontal disease was induced using the method described above were designated as the Test group, and mice in which periodontal disease was not induced by not wrapping them with silk sutures were designated as the Control group. In both groups, periodontal tissue regeneration was induced by normal feeding management for 12 days after the removal of the silk sutures, and μCT images of the periodontal tissue were taken at 0, 3, 6, 9, and 12 days after the removal of the silk sutures (Figure 2).

[0059] μCT images confirmed that periodontal tissue regeneration occurred in the circled area of ​​the Test group in Figure 3. Furthermore, when the images were converted to alveolar bone level, an upward trend in alveolar bone level was observed from 0 to 12 days later (Figures 3 and 4).

[0060] Example 2: Analysis of RNA-seq data In this study, to investigate how the behavior of tissue cells changes during periodontal tissue regeneration, RNA was extracted from periodontal tissue at five points: Day 0, 3, 6, 9, and 12, and RNA-seq data was obtained (Figure 2).

[0061] RNA-seq is a method for comprehensively and quantitatively examining gene expression in a cell population. Genes whose expression levels changed significantly at five points from Day 0 to 12 were extracted, and clustering was performed based on gene expression levels (Figure 5). As a result, the mice were classified into four clusters: Cluster 1, which contained genes that were highly expressed on Day 0; Cluster 2, which contained genes that were highly expressed from Day 3 to 6; Cluster 3, which contained genes that were highly expressed on Day 9; and Cluster 4, which contained genes that were highly expressed on Day 12. GO (Gene Ontology) analysis was performed on the genes in each cluster. GO analysis is an analytical method that extracts gene functions that are observed to be significantly more abundant in a list of genes compared to the entire gene population. As a result, as shown in Figure 5, Cluster 1 showed increased expression of inflammatory genes such as those related to leukocyte migration; Cluster 2 showed increased expression of genes related to muscle cell and skin cell development; Cluster 3 showed increased expression of genes related to cell proliferation and immune responses; and finally, Cluster 4 showed increased expression of genes related to bone formation. From these findings, it became clear that periodontal tissue regeneration occurs in stages from Day 0 to 12 in periodontal disease-induced mice.

[0062] Example 3: Construction of a mathematical model for bone regeneration using RNA-seq data Based on the RNA-seq results performed above, we constructed a mathematical model that reflects the spatiotemporal molecular regulatory network in periodontal tissue regeneration, and used simulations to search for molecular targets that effectively enhance periodontal tissue regeneration function.

[0063] Teeth, like bones, are maintained by a balance between bone resorption and bone formation. However, in periodontal disease, excessive bone resorption occurs in the alveolar bone, leading to the dissolution and destruction of the alveolar bone, and ultimately tooth loss. Therefore, in order to reproduce the changes in the balance between bone resorption and bone regeneration in periodontal tissue regeneration using a mathematical model, this model describes the molecular networks important for bone resorption (left side) and the molecular networks important for bone formation (right side) (Figure 6). Furthermore, regarding the detailed molecular mechanisms, the main regulator of bone resorption is described as the RANKL signal-dependent NFκB signaling pathway, and the main regulator of bone formation is described as the BMP signal-dependent SMAD signaling pathway.

[0064] Then, by incorporating the results of gene expression changes obtained from RNA-seq into this model, the parameters of this mathematical model were predicted, and the time-series changes in the balance between these two in periodontal tissue regeneration were reproduced in the computer. Parameters refer to values ​​that determine the characteristics of a model. For example, the "reaction rate constant," which indicates how fast a certain chemical reaction proceeds, and the "threshold," which indicates how much of a certain substance is present before a reaction begins, are examples of parameters. The behavior of the model changes greatly depending on the parameter values. For example, if the reaction rate constant is large, the chemical reaction proceeds quickly, and if it is small, it proceeds slowly. In other words, parameters are important elements that determine the "properties" of the phenomenon that the model represents. Parameter values ​​are usually estimated from experimental data. In this study, the parameters of the entire mathematical model were estimated by using gene expression changes in the mRNA amounts of cFos, NFATc1, A20, Ctsk, and RUNX2 within the model as training data. As a result, a set of 30 parameters was predicted.

[0065] Example 4: Identification of molecular control mechanisms that activate bone regeneration. The final output of the above model is reflected as the bone regeneration score, which is shown at the bottom of Figure 6. To increase this bone regeneration score, we explored which molecules in the model should be targeted most effectively. Sensitivity analysis was used to investigate this. Intracellular signaling pathways are composed of complex response networks, but not all of their components contribute equally to the cellular response; there is variability in the influence of each molecule. Sensitivity analysis can quantify such influences, and the sensitivity coefficient corresponding to each molecule is calculated from the change in output when that element is perturbed. If the output changes significantly, that molecule is highly sensitive, and it is thought that controlling the expression level of that molecule may significantly change the dynamics of the output, meaning that bone regeneration may be effectively induced.

[0066] Figure 7 shows the results of this sensitivity analysis performed on the constructed bone regeneration model. In this study, all parameters in the model were perturbed to investigate which parameters affected the bone regeneration score in the lowest layer of the model. In Figure 7, the horizontal axis represents the parameters, and the vertical axis represents the set of 30 parameters. Red indicates positive sensitivity (increasing the value of the parameter increases the bone regeneration score), and blue indicates negative sensitivity (decreasing the value of the parameter increases the bone regeneration score). The left side shows the model parameters for the bone resorption region, and the right side shows the model parameters for the bone formation region. The darker colors indicate nuclear regulation, i.e., transcriptional activity of genes in the nucleus. In this study, we focused on parameters other than those for nuclear regulation in relation to molecular regulation in the cytoplasm. From the results of this sensitivity analysis, it became clear that the parameters related to IκBα production, enclosed in black frames, showed high sensitivity.

[0067] It was hypothesized that IκBα is present in the pathway involved in bone resorption, and that increased IκBα activity leads to inhibition of bone resorption.

[0068] Example 5: Evaluation of periodontal tissue regeneration function by IκBα activation Mathematical model simulations suggested that IκBα activation may be effective in enhancing bone regeneration. Therefore, we investigated whether there was a change in bone regeneration function by comparing cases where IκBα was activated with and without it in a mouse periodontal tissue regeneration model.

[0069] In this study, Bay11-7082 was locally administered orally to mice in the group exhibiting enhanced IκBα activation. IκBα is broken down into proteases via phosphorylation and ubiquitination, but Bay11-7082 inhibits the breakdown of IκBα by inhibiting the ubiquitin-specific proteases USP7 and USP21 (Figure 8).

[0070] Periodontal disease was induced in young mice by ligating silk threads to their second molars, and the threads were removed after 10 days. Periodontal tissue regeneration levels up to 9 days were evaluated by μCT imaging in the group that received local administration of Bay11-7082 every two days (Bay) and the control group (no administration) (Study plan: Figure 9, Results: Figures 10, 11). As a result, the amount of bone regeneration was significantly increased in the Bay group compared to the Control group. This indicates that treatment with Bay11-7082, i.e., activation of IκBα, promotes periodontal tissue regeneration.

[0071] Example 6: Screening of IκBα activating or inhibiting components Screening for IκBα activating or inhibiting components is performed according to the following protocol. 1. Bone-clast precursor cells: 200,000-800,000 cells / cm² 2 Sow the seeds. As a culture medium, you can use one containing 20-30 ng / ml RANKL and 20-30 ng / ml M-CSF. 2. Change the culture medium every other day until large osteoclasts appear. Large osteoclasts will appear after 3-5 days. 3. Once the appearance of large osteoclasts is confirmed, add the culture medium containing the target component for screening. 4. After culturing for a certain period of time, the change in IκBα levels in the cells is quantified using gene quantification methods, gene amplification methods, immunoassays, etc. Specifically, quantification is performed using PCR with IκBα-specific primers, Western blotting with IκBα-specific antibodies, or ELISA. This protocol allows for the determination of whether the test component is an activator, inhibitor, or ineffective component of IκBα. If the test component is an activator of IκBα, it can be identified as a periodontal tissue regeneration component or a bone tissue regeneration component. [Industrial applicability]

[0072] The present invention provides a method for screening periodontal tissue regeneration components or bone tissue regeneration components. Furthermore, the present invention enables the provision of novel periodontal tissue regeneration compositions or bone tissue regeneration compositions. Additionally, the present invention provides a method for screening components for the treatment, improvement, alleviation, progression inhibition, and / or prevention of periodontal disease. Moreover, the present invention enables the provision of novel compositions for the treatment, improvement, alleviation, progression inhibition, and / or prevention of periodontal disease. Therefore, the present invention can be used in the medical, veterinary, food, and cosmetic fields, among others.

Claims

1. A method for screening periodontal tissue regeneration components, comprising a step of evaluating the IκBα promoting effect of the test component.

2. The screening method according to claim 1, characterized in that the periodontal tissue regeneration component is for use in mammals.

3. The screening method according to claim 1 or 2, characterized in that the periodontal tissue regeneration component is used on periodontal tissue affected by periodontal disease.

4. A method for evaluating the effect of a test component on periodontal tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component.

5. A method for screening bone tissue regeneration components, comprising a step of evaluating the IκBα-promoting effect of the test component.

6. A method for evaluating the effect of a test component on bone tissue regeneration, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component.

7. A method for screening components for the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease, comprising the step of evaluating the IκBα-promoting effect of the test component.

8. A method for evaluating the effects of a test component on the treatment, improvement, alleviation, inhibition of progression, and / or prevention of periodontal disease, comprising the step of evaluating the IκBα promoting and / or IκBα inhibiting effect of the test component.

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