JP6994243B2 - Tooth movement promoter and orthodontic treatment kit - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication in the 2017 Niigata Dental Society Second Regular Meeting Program (Abstracts of Lectures) on October 10, 2017

Application of Article 30, Paragraph 2 of the Patent Act Announced at the 2017 Niigata Dental Society Second Regular Meeting held at Niigata University on November 11, 2017.

The present invention relates to a tooth movement promoter and an orthodontic treatment kit.

Orthodontic treatment is a highly specialized dental treatment that moves teeth by applying mechanical and physical forces to the teeth and improves tooth alignment and meshing. The number of patients who receive orthodontic treatment is increasing year by year, and in recent years, the number of adults who wish to have orthodontic treatment has increased remarkably in addition to the conventional young patients who desire orthodontic treatment. However, in adult patients, compared to younger patients, delayed remodeling of periodontal tissues such as alveolar bone and periodontal ligament caused by physical force slows tooth movement, resulting in longer treatment. Tend to do. Prolonged treatment means a longer period of wearing the device, and there is a concern that the risk of gingival inflammation and periodontitis due to a decrease in tooth sweepability may increase.

Current main devices for orthodontic treatment include multi-bracket devices and mouthpiece type devices. These devices apply a continuous force to the teeth by the return force of a bent wire or a deformed polymer elastic material. However, in orthodontic treatment using this continuous force, it is necessary to always attach the device to the dentition during the treatment period of several years, and the physical and psychological burden on the patient is great.
On the other hand, as a conventional technique for promoting the movement of teeth by orthodontic treatment, there is corticotomy (alveolar bone cortical bone resection) in which a surgical operation is performed on the alveolar bone (see, for example, Non-Patent Document 1).

On the other hand, until now, ROCK inhibitors have been used as anticancer agents, therapeutic agents for bone-related diseases such as arthritis (see, for example, Patent Document 1), therapeutic agents for pain (see, for example, Patent Document 2) and the like. rice field.

Japanese Patent Publication No. 2013-510902 International Publication No. 2006/088088

Hassan A H et al., “Corticotomy-Assisted Orthodontic Treatment: Review.”, Open Dent J., Vol. 4, p159-164, 2010.

However, corticotomy requires a high degree of therapeutic technique and surgically invades, which imposes a heavy burden on the patient and has not reached a general method. In addition, ROCK inhibitors have been used as therapeutic agents for bone-related diseases, but have not been used for orthodontic treatment so far.

The present invention has been made in view of the above circumstances, and provides a tooth movement promoter and an orthodontic treatment kit that are minimally invasive and can shorten the period of orthodontic treatment.

That is, the present invention includes the following aspects.
The tooth movement promoter according to the first aspect of the present invention contains HA-1077, Y-27632 or a pharmaceutically acceptable salt thereof as an active ingredient .

The orthodontic treatment kit according to the second aspect of the present invention includes the tooth movement promoting agent according to the first aspect and the orthodontic appliance.

The tooth movement promoter and the orthodontic treatment kit of the above-described embodiment are minimally invasive and can shorten the orthodontic treatment period.

Arizarin Red S staining with mouse-derived precursor osteoblasts (MC3T3-E1 cells) cultured for 3 weeks in a medium containing two ROCK inhibitors (HA-1077 and Y-27632) having different concentrations in Example 2. It is an image which shows the evaluation result of the osteoblast calcification ability used. Mouse-derived macrophage-like cells (RAW264.7 cells) containing two ROCK inhibitors (HA-1077 and Y-27632) in Example 3 and cultured for 4 days in a ROCK inhibitor-free (control) medium. It is an image showing the evaluation result of the osteoclast differentiation promoting ability using Tartrate-Resistant Acid Phosphatase (TRAP) staining of Tartrate-Resistant Acid Phosphatase (TRAP). Stained by TRAP staining in RAW264.7 cells cultured for 4 days in ROCK inhibitor-free (control) medium containing two ROCK inhibitors (HA-1077 and Y-27632) in Example 3. It is a graph comparing the number of osteoclasts. For TRAP staining in mouse femoral osteoblasts cultured in a medium containing two ROCK inhibitors (HA-1077 and Y-27632) in Example 3 and free of ROCK inhibitors (control) for 4 days. It is an image which shows the evaluation result of the osteoclast differentiation promotion ability. Staining by TRAP staining in mouse femoral osteoblasts cultured for 4 days in a medium containing two ROCK inhibitors (HA-1077 and Y-27632) in Example 3 and free of ROCK inhibitors (control). It is a graph comparing the number of osteoclasts. MC3T3-E1 cells and mouse femoral osteoblasts cultured in a medium containing two ROCK inhibitors (HA-1077 and Y-27632) in Example 3 and free of ROCK inhibitors (control) for 3 weeks. It is an image showing the evaluation result of the osteoblast differentiation promoting ability using Alkaline phosphatase (ALP) staining and Alkaline phosphatase (ALP) staining. It is an angiogenesis marker in rat femoral osteoblasts cultured for 7 days in an osteoblast differentiation-inducing medium containing a ROCK inhibitor (Y-27632) and 0.1% DMSO (control) in Example 4. It is a graph which compared the expression level of the mRNA of the ANGPT1 gene. It is an angiogenesis marker in rat femoral osteoblasts cultured for 7 days in an osteoblast differentiation-inducing medium containing a ROCK inhibitor (Y-27632) and 0.1% DMSO (control) in Example 4. It is a graph which compared the expression level of the mRNA of the VEGFA gene. It is a schematic process diagram which shows the protocol of the influence confirmation test on the bone formation in the rat skull defect part using the ROCK inhibitor in Reference Example 1. FIG. It is an image comparing the state of bone formation in a rat skull defect portion filled with an atelocollagen sponge containing a ROCK inhibitor (HA-1077 and Y-27632) or PBS 4 weeks after the start of the test in Reference Example 1. The left side is a μCT image, the middle is a TRAP-stained image, and the right side is an immunostained image with an anti-RUNX2 antibody. Black arrows indicate bone formation sites, respectively. Newborn analyzed by ImageJ using μCT images of rat skull defects loaded with atelocollagen sponge containing ROCK inhibitors (HA-1077 and Y-27632) or PBS 4 weeks after the start of the test in Reference Example 1. It is a graph comparing the area of the bone part. It is a schematic process diagram which shows the protocol of the influence confirmation test on the tooth movement of a rat using the ROCK inhibitor in Example 5. FIG. ΜCT of maxillary right first molars, second molars and third molars of rats fitted with coils 7 days from the start of the test in Example 5 and administered with ROCK inhibitors (HA-1077 and Y-27632) or PBS. It is a statue. It is a graph comparing the movement distance of the maxillary right first molar of a rat to which a coil was attached and a ROCK inhibitor (HA-1077 and Y-27632) or PBS was administered on the 7th day from the start of the test in Example 5.

≪Tooth movement promoter≫
The tooth movement promoter of the present embodiment contains a ROCK inhibitor as an active ingredient.

Orthodontic treatment utilizes tissue remodeling of the alveolar bone and periodontal ligament, which are the supporting tissues of the tooth, by giving a mechanical stimulus to the tooth. When a mechanical stimulus is applied to the tooth, the periodontal ligament in the direction of movement is compressed, and bone resorption by bone-breaking cells is performed on the surface of the alveolar bone facing it, and the periodontal ligament is stretched on the opposite side of the periodontal ligament to the surface of the alveolar bone. Bone addition by osteoblasts occurs in. Therefore, the present inventors are searching for a drug capable of activating both osteoclasts and osteoblasts and accelerating the turnover of bone, thereby promoting the bone remodeling phenomenon and increasing the speed of tooth movement. Was done. As a result, it was found that the ROCK inhibitor promotes the differentiation of both osteoclasts and osteoblasts in the tooth, thereby increasing the moving speed of the tooth, and completed the present invention.

According to the tooth movement promoting agent of the present embodiment, by including the ROCK inhibitor, differentiation of both osteoclasts and osteoblasts can be promoted as described in Examples described later, and further, angiogenesis can be promoted. Be promoted. As a result, rather than orthodontic dentistry using only conventional orthodontic appliances such as a multi-bracket device and a mouthpiece type device, these conventional orthodontic appliances and the tooth movement promoting agent of the present embodiment can be used in combination. , The period of orthodontic treatment can be shortened.
In addition, the orthodontic treatment using the tooth movement promoting agent of the present embodiment does not require surgical invasion and can be performed with minimal invasion, so that the burden on the patient can be reduced.

<ROCK inhibitor>
The ROCK inhibitor contained in the tooth movement promoting agent of the present embodiment may be any as long as it inhibits Rho-associated protein kinase (ROCK), and is not particularly limited. Specifically, as a ROCK inhibitor, for example, HA-1077, Y-27632, Thiazovivin, GSK492286A, RKI-1447, GSK180736A, HA-1100, Y-39983, AR-13324, GSK269962, AT13148, K-115, KD025. , ZINC00881524, and pharmaceutically acceptable salts thereof. Among them, as the ROCK inhibitor contained in the tooth movement promoting agent of the present embodiment, HA-1077, Y-27632, or a pharmaceutically acceptable salt thereof is preferable, and Y-27632 or a pharmaceutically acceptable salt thereof is preferable. Is more preferable.

As used herein, the term "pharmaceutically acceptable" generally means a degree that does not harm the human body when properly administered to a test animal.

As the salt, a pharmaceutically acceptable acid addition salt or a basic salt is preferable.
The acid addition salt may be a salt with an inorganic acid or a salt with an organic acid. Examples of the inorganic acid include hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid and the like. Examples of the organic acid include acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartrate acid, citric acid, malic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid and the like.
The basic salt may be a salt with an inorganic base or a salt with an organic base. Examples of the inorganic base include sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide and the like. Examples of the organic base include caffeine, piperidine, trimethylamine, pyridine and the like.

<Other ingredients>
The tooth movement promoter of the present embodiment may contain other components in addition to the ROCK inhibitor. Other ingredients include, for example, additives, pharmaceutically acceptable carriers or diluents.
Examples of the additive include a buffer solution such as PBS and Tris-HCl, sodium azide, glycerol and the like.
Pharmaceutically acceptable carriers or diluents include, for example, excipients, diluting agents, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, viscous agents. Examples thereof include agents, flavoring agents, solubilizing agents, additives and the like. By using one or more of these carriers, a tooth movement-promoting agent in the form of a liquid, a suspension, an emulsion or the like can be prepared.
Further, a colloidal dispersion system can also be used as the carrier. The colloidal dispersion system has the effect of enhancing the in vivo stability of the ROCK inhibitor and the transfer of the ROCK inhibitor to specific organs, tissues, or cells (particularly osteoclasts, osteoblasts and their progenitor cells). Expected to have the effect of enhancing sex. Examples of the colloidal dispersion system include polyethylene glycols, polymer complexes, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. .. Among them, liposomes and artificial membrane vesicles having an effect of efficiently transporting a ROCK inhibitor to a specific organ, tissue, or cell are preferable.

Examples of the formulation of the tooth movement-promoting agent of the present embodiment are a sterile solution with water or other pharmaceutically acceptable liquid, a suspension, an injection or an oral cleaning agent (mouth wash), or an oral cleaning agent (mouth wash). , Gel-like, paste-like oral ointment or oral jelly, which is used parenterally.

Further, as a pharmacologically acceptable carrier or diluent, specifically, for example, sterile water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient. , Vehicles, preservatives, binders and the like are appropriately combined and formulated by mixing in a generally accepted unit dose form required for pharmaceutical practice.

When the tooth movement-promoting agent of the present embodiment is a sterile solution or suspension, for example, by impregnating the gingival sulcus or exclusion paste with the tooth movement-promoting agent of the present embodiment and wrapping it around the cervical region. The tooth movement-promoting agent of the present embodiment can be administered to the affected area through the gingival sulcus. Examples of the material of the gingival retraction thread include cotton and silk. Further, as the exclusion paste, for example, ESPE (registered trademark) exclusion paste manufactured by 3M Healthcare Co., Ltd. can be used.
When the tooth movement promoter of the present embodiment is a sterile solution or suspension, for example, the periodontal pack is impregnated with the tooth movement promoter of the present embodiment to pack the periodontal tissue. The tooth movement promoting agent of the present embodiment can be administered to the affected area. The periodontal tissue may be covered with a water-absorbent sponge impregnated with the tooth movement-promoting agent of the present embodiment, and the periodontal tissue may be covered with a periodontal pack. The periodontal pack is obtained by kneading and hardening the accelerator (catalyst) and the base material (base material). Examples of the accelerator (catalyst) in the periodontal pack include zinc oxide, vegetable oil, mineral oil, magnesium oxide and the like. Examples of the base material (base material) in the periodontal pack include rosin, lauric acid, natural rubber, lanolin, ethyl cellulose and the like.

When the tooth movement-promoting agent of the present embodiment is an injection, the sterile composition can be formulated according to the usual formulation practice using a vehicle such as distilled water for injection. In addition, the aqueous solution for injection may be used in combination with, for example, physiological saline, an isotonic solution containing glucose or other auxiliary agents, an appropriate solubilizing agent, or a nonionic surfactant.
Examples of the other adjuvant include D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like.
Examples of the suitable dissolution aid include alcohols such as ethanol and polyalcohols such as propylene glycol and polyethylene glycol.
Examples of the nonionic surfactant include polysorbate 80 and HCO-50.

Examples of the oily liquid include sesame oil and soybean oil, and examples of the solubilizing agent may be used in combination with, for example, benzyl benzoate, benzyl alcohol and the like. Further, a buffering agent, a soothing agent, a stabilizer, or an antioxidant may be further added.
Examples of the buffer include a phosphate buffer solution, a sodium acetate buffer solution and the like.
Examples of the pain-relieving agent include procaine hydrochloride and the like.
Examples of the stabilizer include benzyl alcohol, phenol and the like.
The prepared injection solution is usually filled in a suitable ampoule.

The injection can also be prepared as a non-aqueous diluent (for example, vegetable oil such as polyethylene glycol and olive oil, alcohols such as ethanol, etc.), a suspension agent, or an emulsion. Such sterilization of the injection can be performed by filtration sterilization with a filter, compounding of a bactericidal agent, or the like. Injections can be produced in the form of errand preparation. That is, it can be prepared into a sterile solid composition by a freeze-drying method or the like, and can be used by dissolving it in distilled water for injection or another solvent before use.

When the tooth movement-promoting agent of the present embodiment is an oral cleansing agent (mouthwash), for example, the tooth movement-promoting agent of the present embodiment and a base material or the like can be mixed to prepare an oral cleansing agent. By rinsing the oral cavity with the obtained oral cleanser, the tooth movement promoting agent of the present embodiment can be administered to the affected area. Examples of the base material in the oral detergent (mouthwash) include water, polyethylene glycol, dextrin and the like.

When the tooth movement-promoting agent of the present embodiment is an oral ointment or an oral jelly, a foaming agent, a foaming aid, an abrasive, a wetting agent, and a sweetness are used as long as the effects of the tooth movement-promoting agent of the present embodiment are not impaired. Agents, preservatives, fluorine ion supply compounds such as sodium fluoride, pigments, dyes, fragrances and the like can be appropriately contained.

When the tooth movement-promoting agent of the present embodiment is an oral ointment or an oral jelly, the tooth movement-promoting agent of the present embodiment and a base material are mixed to prepare an oral ointment or an oral jelly. Can be done. By applying the obtained oral ointment or oral jelly to the mouthpiece and attaching it to the teeth, the tooth movement promoting agent of the present embodiment can be administered to the affected area. At this time, examples of the base material in the oral ointment or the oral jelly include petrolatum, paraffin, polyethylene resin, beeswax, polyethylene glycol, cellulose and the like.

<Dose and administration method>
The tooth movement promoter of the present embodiment can be used for orthodontic treatment.
The dose of the tooth movement promoting agent of the present embodiment is appropriately adjusted in consideration of the age, sex, body weight, administration method, treatment time, etc. of the test animal.
Mammals are preferable as test animals, and specific examples thereof include humans, monkeys, mice, rats, rabbits, pigs, dogs, cats, horses, cows, goats, and sheep. Among them, humans are preferable as the test animal.

When the tooth movement-promoting agent of the present embodiment is administered parenterally, the single dose thereof varies depending on the symptoms and the administration method, but for example, in the form of an injection, it is usually in an adult (assuming a body weight of 60 kg). Is usually administered by local injection so that the amount of the ROCK inhibitor is 0.5 ng or more and 1 mg or less, preferably 1 ng or more and 0.5 mg or less, and more preferably 5 ng or more and 0.1 mg or less per day. Suitable.

As the number of administrations, it is preferable to administer once to several times per week on average.
The administration time may be any time during orthodontic treatment, for example, it may be at the time when orthodontic treatment with orthodontic appliances is started, or it may be while wearing orthodontic appliances. ..
As the administration form, it is preferable to directly administer to the tissue (periodontal tissue) around the tooth to be moved. For example, a method known to those skilled in the art such as injection into the oral mucosa or transmucosal administration by application of an oral ointment can be mentioned.

<Treatment method>
In the present embodiment, the above-mentioned tooth movement promoting agent for orthodontic treatment is provided.
Further, in the present embodiment, a therapeutically effective amount of the above-mentioned ROCK inhibitor and a tooth movement-promoting agent containing a pharmaceutically acceptable carrier or diluent are provided.
Further, in the present embodiment, there is provided an orthodontic treatment method comprising administering an effective amount of the ROCK inhibitor to a patient in need of orthodontic treatment.

≪Kit for orthodontic treatment≫
The orthodontic treatment kit of the present embodiment includes the tooth movement promoting agent and the orthodontic appliance according to the above embodiment.

According to the orthodontic treatment kit of the present embodiment, the period of orthodontic treatment can be shortened as compared with the conventional orthodontic dentistry using only the orthodontic appliance by including the tooth movement promoting agent.
Further, the orthodontic treatment using the orthodontic treatment kit of the present embodiment does not require surgical invasion and can be performed with minimal invasion, so that the burden on the patient can be reduced.

<Orthodontic appliances>
The orthodontic appliance included in the orthodontic treatment kit of the present embodiment is not particularly limited, and examples thereof include known ones such as a multi-bracket device and a mouthpiece type device.

As a method of using the orthodontic treatment kit of the present embodiment, first, an orthodontic appliance is attached to the patient. Then, the above-mentioned tooth movement promoting agent is administered to the tissue around the moved tooth. Specifically, when the tooth movement promoting agent is an injection, the injection may be subcutaneously injected (particularly into the oral mucosa) into the tissue surrounding the tooth to which the injection is to be moved. When the tooth movement promoting agent is an oral ointment, the oral ointment may be applied to the tissue around the tooth to be moved and administered transmucosally. When the tooth movement promoting agent is an oral ointment, the patient can administer it to the affected area as a prescription drug according to the prescription.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Screening of compounds having the ability to promote osteoclast differentiation First, screening of low molecular weight compounds that are candidates for tooth migration promoting agents was performed.

(1) Screening of compounds having the ability to promote osteolytic cell differentiation Standard inhibition containing 378 types of low molecular weight compounds as screening target compounds, existing anticancer agents, and inclusion of an inhibitor whose target molecule is obvious. A drug kit (provided from "Ministry of Education, Science and Technology Grants, Comprehensive Support Activities Based on Cancer Characteristics, Chemotherapy Infrastructure Support Activities") was used.
Pre-cultured mouse-derived macrophage-like cells (RAW264.7 cells) were subjected to each low-molecular-weight compound contained in the above kit and the cytokine RANKL (receptor activator of NF-κB ligand), which is an osteoclast differentiation factor, as a control. The cells were replaced with a medium containing 100 ng / mL each and cultured for 4 days.
Then, on the 5th day of culture, differentiation into osteoclasts was evaluated using a Tartrate-Resistant Acid Phosphatase (TRAP) staining solution (prepared by oneself). The results are shown in Table 1 below. In Table 1, the relative ratio (double) of the number of cells differentiated into osteoclasts in the cells to which each compound was added to the case where the number of cells differentiated into osteoclasts in the cells to which RANKL was added was 1. Is described as "osteoclast differentiation promoting effect". In addition, only 15 typical compounds having an promoting effect (8 types) or an inhibitory effect (7 types) on osteoclasts are described. The meaning of each abbreviation in Table 1 is as follows.
GSK: Abbreviation for Glycogen synthase kinase, which means glycogen synthase kinase.
CDK: Abbreviation for Cyclin-dependent kinase, which means cyclin-dependent kinase.
ROCK: An abbreviation for Rho-associated coiled-coil-connecting protein kinase, which means a serine / threonine protein kinase activated by active Rho.
CK: An abbreviation for Casein kinase, which means casein kinase.
PKC: An abbreviation for Protein kinase C, which means protein kinase C.
PKA: An abbreviation for Protein kinase A, which means cyclic adenosine monophosphate (cAMP) -dependent protein kinase (protein kinase A).
Wnt: A secretory glycoprotein. 7-transmembrane receptor Frizzled (Fz), 1-transmembrane receptor LRP5 / 6 (low-density lipoprotein receptor-related protein 5/6) that functions as a conjugated receptor, single membrane with tyrosine kinase activity It binds to the transmembrane receptors Ror and RYK and activates three pathways: the β-catenin pathway, the planar cell protein (PCP) pathway, and the calcium pathway.
P38: A signal molecule that connects extracellular stimuli to the transcriptional regulatory mechanism in the nucleus. It is a member of the MAPK (mitogen-activated protein kinase) family and is particularly activated by stress and inflammatory cytokines. For this reason, it is also called stress-activated protein kinase (SAPK) together with JNK (c-Jun N-terminal kinase) and ERK5.
CAMKII: Abbreviation for Ca ²⁺ / calmodulin-dependent protein kinase II, which means Ca ²⁺ calmodulin-dependent protein kinase.
Multi-kinases: means that multiple kinases are the targets of inhibition.
EGFR: Abbreviation for Epidermal growth factor receptor, which means epidermal growth factor receptor.
Retinoids: A general term for all-trans-retinoic acid (ATRA, retinoic acid), which is the active body of vitamin A, and a group of compounds showing equivalent activity.
G9a: Human histone methyltransferase.

From Table 1, we focused on ROCK inhibitors (HA-1077 and Y-27632) among the eight compounds that were found to have the ability to promote osteoclast differentiation, and their concentrations in the subsequent osteoblast calcification ability. It was used to examine the dependency.

[Example 2] Examination of concentration dependence of ROCK inhibitor on osteoblast calcification ability Next, osteoblasts of two types of ROCK inhibitors (HA-1077 and Y-27632) identified in Example 1. The concentration dependence on calcification ability was investigated. Specifically, for the two types of ROCK inhibitors (HA-1077 and Y-27632), except that the concentration of each compound contained in the medium was 0.2, 1 and 5 μM, the (1) of Example 1 ( Using the same method as in 2), MC3T3-E1 cells were cultured for 3 weeks, and the calcification ability of osteoblasts was evaluated using Alizarin Red S stain (prepared by themselves). The results are shown in FIG. In FIG. 1, "control" is MC3T3-E1 cells cultured for 3 weeks without the addition of a ROCK inhibitor.

From FIG. 1, in any of the compounds HA-1077 and Y-27632, activation of the calcification ability of osteoblasts was uniformly observed at a concentration of 0.2 μM to 5 μM. In particular, in Y-27632, the enhancement of calcification ability was remarkable.
From the above, the ROCK inhibitors HA-1077 and Y-27632 were identified as compounds that promote the differentiation into both osteoclasts and osteoblasts.

[Example 3] Osteoclast and osteoblast differentiation promoting ability confirmation test of ROCK inhibitor Next, osteoclast of two types of ROCK inhibitors (HA-1077 and Y-27632) identified in Example 1. And the ability to promote the differentiation of osteoblasts was reconfirmed.

(1) Test for confirming osteoclast differentiation-promoting ability of ROCK inhibitor Two types of ROCK inhibitors (HA-1077 and Y-27632) were used for RAW264.7 cells and mouse femoral bone marrow cells that had been cultured in advance. The cells were replaced with a medium containing 100 ng / mL each and cultured for 4 days. In addition, as a control, the cells were similarly cultured in a medium containing no ROCK inhibitor for 4 days.
Then, on the 5th day of culture, differentiation into osteoclasts was evaluated using a TRAP staining solution. The results with RAW264.7 cells are shown in FIGS. 2A and 2B, and the results with mouse femoral bone marrow cells are shown in FIGS. 3A and 3B. 2A and 3A are images of each TRAP-stained cell. 2B and 3B are graphs showing the number of cells differentiated into TRAP-stained cells, that is, osteoclasts, under each culture condition.

From FIGS. 2A to 3B, it was confirmed that the use of the ROCK inhibitor promoted the differentiation into osteoclasts.

(2) ROCK inhibitor osteoblast differentiation promoting ability confirmation test Next, pre-cultured MC3T3-E1 cells and mouse femoral bone marrow cells were subjected to two types of ROCK inhibitors (HA-1077 and Y-27632). ) Was replaced with a medium containing 5 μM each, and the cells were cultured for 3 weeks. In addition, as a control, the cells were similarly cultured in a medium containing no ROCK inhibitor for 3 weeks.
Then, the alizarin red S stain was used to evaluate the calcification ability of osteoblasts. In addition, for MC3T3-E1 cells, the ALP activity in osteoblasts was also evaluated using an alkaline phosphatase (ALP) staining solution (prepared by themselves). The results are shown in FIG.

From FIG. 4, it was confirmed that the use of the ROCK inhibitor promoted calcification, increased ALP activity, and promoted differentiation into osteoblasts.

[Example 4] Test for confirming the effect of a ROCK inhibitor on the expression of angiogenesis markers 5 μM Y-27632-containing osteoblast differentiation-inducing medium or control of pre-cultured rat femoral bone bone bone-derived stromal cells As (control), the cells were replaced with a medium containing 0.1% DMSO and cultured for 7 days. The composition of the osteoblast differentiation induction medium is α-MEM, 10% fetal bovine serum, 1% penicillin / streptomycin, 10 mM β-glycerophosphate, 50 μg / mL ascorbic acid and 10 nM dexamethasone.
Seven days after the start of culture, total RNA was recovered from the cells. Then, using a TaqMan® probe, the expression of mRNA of the angiogenesis markers ANGPT1 gene and VEGFA gene was analyzed by real-time PCR. The results are shown in FIG. 5A (ANGPT1 gene) and FIG. 5B (VEGFA gene).

From FIGS. 5A and 5B, in the presence of Y-27632, there was a significant increase in mRNA expression of about 5.6 times for the ANGPT1 gene and about 2.7 times for the VEGFA gene as compared with the control.
Therefore, by using the ROCK inhibitor, not only the differentiation of osteoclasts and osteoblasts is promoted, but also angiogenesis is promoted and nutrients are supplied, so that the movement of teeth is promoted more effectively. It was inferred.

[Reference Example 1] Test for confirming the effect of a ROCK inhibitor on bone formation Next, the effect of a ROCK inhibitor on bone formation was confirmed using rats. A specific protocol is shown in FIG. 6A.
First, a defect (diameter 5 mm) was formed in the skull of an 8-week-old SD rat (male), and an atelocollagen sponge containing two types of ROCK inhibitors (HA-1077 and Y-27632) or PBS was implanted therein. did. Then, atelocollagen sponge containing two kinds of ROCK inhibitors (HA-1077 and Y-27632) or PBS was charged twice a week and bred for 4 weeks from the start of the test. After 4 weeks, μCT analysis and tissue image analysis were performed. The results are shown in FIGS. 6B (image) and 6C (graph). In FIG. 6B, the left side is a μCT image, the center is a TRAP stained image, and the right side is an immunostained image with an anti-RUNX2 antibody. In general, "RUNX2" is a transcription factor essential for the differentiation of osteoblasts. Black arrows indicate bone formation sites, respectively. Further, FIG. 6C is obtained by analyzing the area of the new bone portion by ImageJ using each μCT image.

From FIGS. 6B and 6C, it was confirmed that the use of the ROCK inhibitor promoted the promotion of differentiation of both osteoclasts and osteoblasts, and promoted bone formation.

[Example 5] Test for confirming the effect of the ROCK inhibitor on tooth movement Next, the effect of the ROCK inhibitor on tooth movement was confirmed using rats. A specific protocol is shown in FIG. 7A.
First, a coil with a continuous traction force of 25 g was attached to the maxillary incisor and the maxillary right first molar of an 8-week-old SD rat (male). The rats were then divided into a control group and a ROCK inhibitor administration group, and 0, 3 and 5 days after the start of the experiment, PBS was added to the control group and 0.5 μM ROCK inhibitor (HA) was added to the ROCK inhibitor administration group. -1077 and Y-27632) -containing PBS were administered by 10 μL each to the palatal mucosa of the maxillary right first molar using a syringe. On the 7th day after the start, the maxilla was fixed with 4% paraformaldehyde and observed by μCT. The results are shown in FIG. 7B. Next, the moving distance of the maxillary right first molar was measured. As the moving distance, the distance between the maxillary right first molar and the second molar was measured. The results are shown in FIG. 7C and Table 3. FIG. 7C is an average of the moving distances (n = 5) shown in Table 3.

From FIGS. 7B, 7C and Table 3, it was revealed that the administration of ROCK inhibitors (HA-1077 and Y-27632) significantly promoted tooth movement.

The tooth movement promoter and the orthodontic treatment kit of the present embodiment are minimally invasive and can shorten the orthodontic treatment period.

Claims (2)

A tooth migration promoter containing HA-1077, Y-27632 or a pharmaceutically acceptable salt thereof as an active ingredient. The tooth movement promoter according to claim 1 and
Orthodontic appliances and
Orthodontic treatment kit equipped with.

Images