KR102317259B1 - A composition for accelerating orthodontic tooth movement comprising 4-hexylresorcinol - Google Patents

Abstract

The present invention relates to a composition for promoting orthodontic tooth movement comprising 4-hexylresorcinol as an active ingredient. By using the composition of the present invention, it is possible to effectively solve the time problem caused by slow tooth movement during orthodontic treatment without surgical operation.

Description

A composition for accelerating orthodontic tooth movement comprising 4-hexylresorcinol as an active ingredient {A composition for accelerating orthodontic tooth movement comprising 4-hexylresorcinol}

The present invention relates to a composition for promoting orthodontic tooth movement comprising 4-hexylresorcinol as an active ingredient.

Orthodontic treatment is usually required for more than a year. Maintaining oral hygiene due to orthodontic braces is difficult for the patient and can lead to tooth decay and periodontitis. A long treatment period is not satisfactory for the patient. In addition, orthodontic treatment of elderly patients may delay tooth movement due to an imbalance between bone formation and bone resorption.

Several attempts have been made to accelerate tooth movement. The application of parathyroid hormone accelerates orthodontic tooth movement by increasing the activity of osteoclasts. However, applying hormones can damage the endocrine system and lead to unexpected health problems. Surgical methods such as adrenal resection can lead to accelerated bone remodeling. However, adrenal resection can damage the root of the tooth and requires surgical operation.

4-Hexylresorcinol (4HR) is a resorcinolic lipid and has been commonly used in cosmetics and preservatives. 4-hexylresorcinol has been used in bone graft materials for the purpose of inhibiting foreign body giant cell formation and NF-κB pathway. In addition, 4-hexylresorcinol is known to increase angiogenesis through an HIF-independent pathway, but the detailed mechanism is unclear. Recently, in an unpublished study by this investigator, it was found that 4-hexylresorcinol increased the expression of TGF-β1. It is known that 4-hexylresorcinol is attached to bone graft materials to promote bone formation, but the fact that it inhibits the NF-κB pathway and the inhibition of the formation of foreign giant cells, which are multinucleated cells similar to osteoclasts morphologically Because of this, it is estimated that the function of osteoclasts will be inhibited, and it has been thought that it cannot be used for orthodontic procedures.

The present inventors confirmed that 4-hexylresorcinol increased bone resorption by physical pressure at the same time as bone formation, and confirmed that it can be utilized for orthodontic procedures requiring tooth movement.

Korean Patent Publication No. 10-2011-0050308

An object of the present invention is to reduce orthodontic tooth movement (orthodontic tooth movement) containing 4-hexylresorcinol as an active ingredient, which can reduce orthodontic time and cost by accelerating tooth movement through a simple procedure without surgical operation ) to provide a composition for promotion.

Orthodontic treatment is performed for the purpose of treating irregular dentition such as malocclusion. It is known that orthodontic tooth movement for orthodontic orthodontic tooth movement accompanies alveolar bone turnover, and physiological alveolar bone replacement is performed by fixing force. The alveolar bone replacement process during orthodontic tooth movement consists of repeating bone formation on the tension side and bone resorption on the compression side. It is known to occur throughout. In particular, bone resorption induced on the compression side by the orthodontic force of the orthodontic appliance lasts for 1-14 days, and osteoclasts derived from hepatocytes in the bone marrow are expressed on the compression side during tooth movement, and acid phosphatase (ACP) and tartrate- It is also known that tartrate-resistant acid phosphatase (TRAP) activity is high. It is also known that cells involved in bone formation exhibit high alkaline phosphatase (ALP) activity and persist for up to 21 days.

During the study on the efficacy of 4-hexylresorcinol, the present inventors not only promoted the expression of factors related to bone formation at the site treated with 4-hexylresorcinol, but also conversely, under the condition of applying physical pressure, bone resorption It was observed that the expression of factors related to

Accordingly, the present invention provides a composition for promoting orthodontic tooth movement comprising 4-hexylresorcinol as an active ingredient.

The present invention also provides a method of promoting orthodontic tooth movement comprising administering to a subject an effective amount of the composition.

The composition of the present invention can be administered to a variety of subjects in mammals such as rats, mice, livestock, and humans, for example, orally, or intraperitoneally, intrarectally, intravenously, intramuscularly, subcutaneously, intrauterine dura mater or intraventricular (Intracerebroventricular) can be administered in various formulations. Specifically, the composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, or injections, respectively, according to a conventional method. have. In an embodiment of the present invention, it was administered in the form of an injection to the subcutaneous tissue requiring tooth movement.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route of administration and the duration, but may be appropriately selected by those skilled in the art. For a desirable effect, a single dose of the composition of the present invention is 0.01 mg/kg to 100 mg/kg, preferably 0.1 mg, based on a 70 kg adult male based on the content of 4-hexylresorcinol as the active ingredient. It may be /kg to 50mg/kg. If it is less than 0.01 mg/kg, the effect is insignificant, and if it exceeds 100 mg/kg, side effects such as irritation of the mucous membrane or skin may occur.

The administration period may be administered in a single shot, or may be administered in several divided doses.

By using a composition containing 4-hexylresorcinol as an active ingredient, it is possible to effectively solve a time problem caused by slow tooth movement during orthodontic treatment without surgical operation.

1 shows the results of comparing the expression changes of bone formation markers by time and treatment concentration in Saos-2 cells treated with 4-hexylresorcinol.
Figure 2 shows the orthodontic braces applied to each rat of the control group, group A and group B.
Figure 3 shows the results of comparing the expression levels of bone turnover-related markers (osteocalcin, CTX and TRACP-5b) in the blood of each rat of the control group, negative control group, group A and group B.
4 shows the results obtained in the ratio of root length to microcomputerized tomography findings and distal alveolar level of the distal root of the mandibular first molar at the time when orthodontic treatment of the control group, group A and group B is completed.
5 shows the results of confirming the expression levels of BMP-2 (protein that promotes bone formation) and RANKL (marker of osteoclasts) by performing hematoxylin and eosin staining and immunochemical staining on mandibular samples.

Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the specific content.

<Experimental example> Experimental materials and methods

1. Cell Experiments

Saos-2 cells (Korea Cell Line Bank No.30085) were mixed with 10% fetal bovine serum (Euroclone, UK), 50 U/mL penicillin G, 50 µg/mL streptomycin sulfate, 2 g/L sodium carbonate, and 0.11 g Suspended in RPMI 1640 supplemented with /L sodium pyruvate. 4-Hexylresorcinol was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2. Western Blot Experiment

To analyze the protein expression level of alkaline phosphatase, osteocalcin, osteopontin, type I collagen and runx2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SAOS cells were treated with 1, 10 and 100 μM of 4-hexylresorcinol. As a control group, cells treated with solvent alone were used. Proteins were collected at 2, 8 and 24 hours after treatment. The collected proteins were separated by mixing with sodium dodecyl sulfate buffer and heating. The isolated proteins were electrophoresed on a 10% polyacrylamide gel. The gel was transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was irradiated with primary antibody (dilution ratio = 1:500). Blots were imaged and quantified using a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

3. Animal Experiments

Six-week-old Crl:CD (Sprague-Dawley)-specific pathogen-free (SPF)/viral antibody-free (VAF) mated female rats (Orientbio Inc., Sungnam, Korea) were used for the study. All procedures were performed in accordance with the guidelines for laboratory animal care approved by Gangneung-Wonju University Animal Research (GWNU-2019-17). 30 (2-3 per cage) rats were housed and fed. All rats were housed for one week for acclimatization before the experiment. Each rat underwent oophorectomy on both sides of the ovaries and rested for 2 weeks after surgery. The ovaries are the main producers of estrogen. Therefore, by examining blood samples 2 weeks after surgery, the changed level of estrogen in the blood was confirmed to confirm the success of ovariectomy.

The ovarian resected rats were divided into 3 groups of 10 rats each. Each rat was treated with an orthodontic appliance as shown in FIG. 2 . The rat group treated with solvent alone was grouped into two groups, and the rats treated with 4-hexylresorcinol at low and high doses were grouped as a control group. The subcutaneous injection site was established under the skin of the back. Rats in the low-dose group (Group A) were administered 1.28 mg/kg of 4-hexylresorcinol via weekly subcutaneous injection. The rats in the high-dose group (Group B) were administered 128 mg/kg of 4-hexylresorcinol via weekly subcutaneous injection. The distance between the mesial surface (M1) of the first molar and the distal surface (I) of the incisor was measured weekly using a vernier caliper. The degree of tooth movement was defined as the distance changed at each observation compared to day 0. The final distance between M2 and M3 was measured using radiography with a SigmaScan Pro 5 (SPSS Inc. Chicago, IL).

Two hours after the last injection, blood was drawn and all rats were sacrificed. To confirm that ovariectomy was successful, animals that had not undergone ovariectomy (n = 5) were included in the blood sample analysis and used as negative controls. Blood osteocalcin (CAT #: E-EL-R1456), CTX (CAT #: E-EL-R0243) and TRACP-5b (CAT #: E-EL-R0628) measurement kits were purchased from Elabscience. For ELISA or CLIA analysis, whole blood samples were centrifuged and plasma was collected. Plasma was mixed with protein lysis buffer in a 1:1 volume ratio. These mixtures were used for ELISA analysis. Subsequent procedures were performed according to the manufacturer's protocol. A biopsy of the mandible was performed to perform histological and molecular analysis.

4. Microcomputerized tomography and evaluation of alveolar bone level

The obtained mandibular sample was commissioned for microcomputed tomography, and a three-dimensional image was obtained using this. Since there may be loss of alveolar bone in case of rapid tooth movement, the length of the root of the distal root of the first molar and the length of the adjacent alveolar bone were measured respectively, and the ratio was calculated using the length of the root as the denominator and the length of the alveolar bone as the numerator.

5. Histological analysis and immunochemical staining analysis

After microcomputerized tomography, the specimens were decalcified and embedded in paraffin. After cutting the sample to a thickness of 10 μm, hematoxylin and eosin staining methods and immunochemical staining were performed. Antibodies used for immunochemical staining were BMP-2 and RANKL. BMP-2 is a protein that promotes bone formation and is found in osteoblasts and macrophages, and RANKL as a marker of osteoclasts is thought to be found where bone resorption is actively occurring.

<Example> Experimental results

1. Confirmation of increase in bone formation markers by application of 4-hexylresorcinol in Saos-2 cells

According to the above experimental example, the expression of bone-forming protein markers according to the treatment of 4-hexylresorcinol in Saos-2 cells was confirmed through Western blotting.

As a result, as shown in FIG. 1 , it was confirmed that the expression of bone formation markers was increased through the treatment of 4-hexylresorcinol in Saos-2 cells. Specifically, the expression levels of representative alkaline phosphatase (AP), osteocalcin (OC), osteopontin (OP), type I collagen and runx2 as markers related to bone formation were After administration of 4-hexylzolcinol, the expression level increased in a dose (1-100 μM) and time (2-24 h)-dependent manner. In particular, OC and OP were significantly increased by administration of 4-hexylzolcinol. did.

2. Confirmation of tooth movement promotion by application of 4-hexylresorcinol

As shown in FIG. 2 , when 4-hexylresorcinol was administered after dental braces were applied to the rats of each group, the actual tooth movement speed was confirmed by measuring the movement distance (refer to the animal experiment part of the experimental example).

As a result, the tooth movement distances at 1 week were 0.24±0.84 mm, 0.92±1.00 mm, and 0.89±0.61 mm in the control group, group A and group B, respectively. Differences between groups were not statistically significant. The tooth movement distance at 2 weeks was 1.98±1.12 mm, 2.63±0.68 mm, and 2.90±0.42 mm in the control group, group A and group B, respectively, indicating a statistically significant difference between groups (P = 0.043). In the post-hoc test, the difference between the control group and group B was also statistically significant (P = 0.046).

Comparison of tooth movement distance between groups group Day 7 Day 14 control 0.24 ± 0.84 mm 1.98 ± 1.12 mm group A 0.92 ± 1.00 mm 2.63 ± 0.68 mm group B 0.89 ± 0.61 mm 2.90 ± 0.42 mm

*P<0.05, comparison between the experimental groups and control group at each time point. The value was mean ± SD.

The gaps between M2 and M3 were 0.22±0.19 mm, 0.44±0.23 mm, and 0.51±0.21 mm in the control group, group A and group B, respectively, and the difference between groups was statistically significant (P = 0.011). In the post-hoc test, the difference between the control group and groups A and B was statistically significant (P = 0.012).

3. Check for changes in markers of bone turnover in the blood

The negative control (NC) group did not undergo ovariectomy. Ovariectomy showed significant changes in bone turnover markers. OC levels between groups showed statistically significant differences (P < 0.001). Compared with the NC groups, the control group, group A and group B showed significantly higher levels of OC (P=0.004, <0.001 and 0.001 for control group, group A and group B, respectively). Interestingly, group A had significantly higher levels of OC than the control group (P = 0.029).

CTX is a protein fragment produced when type 1 collagen is decomposed, and since the main component of bone matrix protein is type 1 collagen, it can be an indirect indicator of the degree of bone resorption. However, if osteogenesis is relatively dominant over resorption, the value may decrease despite active bone resorption. In contrast, TRACP-5p is an independent and direct indicator of osteoclast activity. Changes in blood expression levels of CTX and TRACP-5p, known as bone resorption markers, were confirmed through administration of 4-hexylresorcinol.

As a result, CTX and TRACP-5p levels between groups showed statistically significant differences (P = 0.010 and 0.002, respectively). In a post-hoc test, the CTX level of group B was significantly lower than that of the control group (P = 0.007). It can be seen that in the balance between bone formation and bone resorption, bone formation appears more predominantly in the group administered with 4-hexylresorcinol. However, this alone cannot move the teeth. In the case of TRACP-5p level, both group A and group B showed significantly higher values than the NC group (P = 0.003 and 0.009, respectively). (FIG. 3) Therefore, it can be seen that osteoclast activity is achieved in the group administered with 4-hexylresorcinol.

Accordingly, administration of 4-hexylresorcinol could be predicted to accelerate orthodontic tooth movement by simultaneously promoting bone formation and bone resorption.

4. Microcomputerized tomography findings and alveolar bone level evaluation

According to the 3-dimensionally reconstructed microcomputerized tomography findings, in the group administered with 4-hexylresorcinol, a clear gap due to tooth movement between the first and second molars was confirmed. (Fig. 4)

The ratio of alveolar bone length to root is 0.65 ± 0.17 for the control group, 0.64 ± 0.10 for group A, and 0.63 ± 0.15 for group B. There was no statistically significant difference (P>0.05). Therefore, it can be seen that the formation of alveolar bone is as fast as the control group with slow tooth movement even in Group B, which has a lot of tooth movement.

5. Histological findings

According to histological findings, root resorption due to rapid tooth movement was hardly observed in Groups A and B. (Fig. 5)

In addition, BMP-2 and RANKL were highly expressed in Groups A and B compared to the control group.

Claims (3)

A composition for promoting orthodontic tooth movement comprising 4-hexylresorcinol as an active ingredient.
The composition for promoting orthodontic tooth movement according to claim 1, wherein the 4-hexylresorcinol activates bone resorption by physical pressure.
The composition for promoting orthodontic movement according to claim 2, wherein the 4-hexylresorcinol simultaneously activates bone formation.

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