Uses of oligopeptides in the treatment of gingivitis and gingival recession and in the repair of oral mucosa

Abstract

The present invention discloses the use of oligopeptides in the treatment of gingivitis and gingival recession and in the repair of oral mucosa. The oligopeptide has a structure represented by formula (I), i.e., X1X2X3X4X5X6(I). X1 is glycine (Gly, G) or lysine (Lys, K). X2 is glutamic acid (Glu, E) or threonine (Thr, T). X3 is K or T. X4 is G or K. X5 is serine (Ser, S), phenylalanine (Phe, F) or absent. And, X6 is F or absent.

Description

Technical Field

[0001] The present invention relates to the use of oligopeptides in the treatment of gingivitis and gingival recession and the repair of oral mucosa. The present invention belongs to the technical field of medicine.

Background Art

[0002] Patent Document 1 discloses an oral care composition containing a free or salt form of basic amino acid and a fluoride ion source.

[0003] Patent Document 2 discloses an oral composition containing a mixture selected from extracts of at least three of pomegranate, prickly ash, ginger and Zingiber zerumbet.

[0004] Patent Document 3 discloses a method for treating gingival injury by administering cells containing isolated non - hair bulb dermal sheath (NDBS) into the oral cavity.

[0005] Patent Document 4 discloses the use of an oligopeptide containing six amino acids with substituted N - terminus and C - terminus in the treatment of oral mucosa.

[0006] Patent Document 5 discloses an anti - inflammatory and analgesic agent for oral soft tissues containing adrenocorticotropic hormone or an anti - inflammatory compound.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

[0008] Conventional technology has neither taught nor proposed that unsubstituted oligopeptides containing four, five, or six amino acids can be applied to the treatment of gingivitis and gingival recession and the repair of oral mucosa. [Means for solving the problem]

[0009] This invention discloses the use of an oligopeptide (or peptide) of the following formula (I) in the preparation of agents for the treatment of gingivitis and gingival recession and for the repair of oral mucosa.

[0010] X1X2X3X4X5X6(I)

[0011] X1 is either glycine (Gly, G) or lysine (Lys, K).

[0012] X2 is either glutamic acid (Glu,E) or threonine (Thr,T).

[0013] X3 is either K or T.

[0014] X4 is either G or K.

[0015] X5 is either serine (Ser,S), phenylalanine (Phe,F), or absent.

[0016] X6 is either F or does not exist.

[0017] In preferred embodiments, the oligopeptide of formula (I) is GEKGF (SEQ ID NO.2) and / or GEKG (SEQ ID NO.1).

[0018] In another preferred embodiment, the oligopeptide of formula (I) is GEKG.

[0019] In a preferred embodiment, the oligopeptide of formula (I) is KTTKS (SEQ ID NO.3) and / or KTTKSF (SEQ ID NO.4).

[0020] In another preferred embodiment, the oligopeptide of formula (I) is KTTKS.

[0021] In a preferred embodiment, the oligopeptide of formula (I) is a combination of GEKG and KTTKS.

[0022] In a preferred embodiment, the agent comprises 0.1 to 10% by weight of GEKG, 0.1 to 10% by weight of KTTKS, and one or more pharmaceutically acceptable carriers. The agent preferably comprises 0.2 to 2% by weight of GEKG and 0.2 to 2% by weight of KTTKS.

[0023] In another preferred embodiment, the one or more pharmaceutically acceptable carriers include solid carriers, semi-solid carriers and liquid carriers, and the types and amounts used of these solid carriers, semi-solid carriers and liquid carriers are well-known in the art.

[0024] In a preferred embodiment, the agent is in the form of a powder, aerosol, ointment, gel, paste or solution. The solution may be in the form of a water-soluble buffer. The water-soluble buffer includes, but is not limited to, an aqueous solution of phosphate or citrate.

[0025] In a preferred embodiment, the agent is administered orally. The oral administration includes, but is not limited to, transmucosal absorption and oral absorption.

[0026] In a preferred embodiment, the agent is a sustained-release type, particularly a sustained-release type for transmucosal absorption and oral absorption.

[0027] In preferred embodiments, GEKG and GEKGF, and KTTKS and KTTKSF can be prepared by methods described in the prior art (for example, Taiwan Patent Application Publication No. 201129368A1 and Taiwan Patent Application Publication No. 201333045A1).

[0028] To enable those skilled in the art to further understand the structure, features and other objectives of the present invention, several preferred embodiments of the present invention are given below and described in detail in conjunction with the drawings, so that those skilled in the art can implement them concretely. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows the results of an analysis of tumor necrosis factor-alpha (TNFα) suppression and cytotoxicity in L929 cells at different dilution ratios of KTTKS. [Figure 2] Figure 2 shows the analysis of TNFα suppression rates and cytotoxicity for L929 cells at different GEKG dilution ratios. [Figure 3] Figure 3 shows that KTTKS suppresses nitric oxide (NO) production by macrophages induced by inflammatory response inducers (LPS). [Figure 4] Figure 4 shows that GEKG suppresses macrophage NO production induced by inflammatory response inducers (LPS). [Figure 5] Figure 5 shows that GEKG and KTTKS suppress the production of hydrogen peroxide (H2O2) by macrophages induced by inflammatory response-inducing substances (LPS). [Figure 6] Figure 6 shows that different concentrations of GEKG and KTTKS promote the proliferation of early oral epithelial cells. [Figure 7] Figure 7 shows that different concentrations of GEKG and KTTKS reduce oral epithelial cell death caused by H2O2 free radicals in vitro. [Figure 8A]Figure 8A shows the effects of different concentrations of GEKG and KTTKS on the proliferation of in vitro human gingival fibroblasts (HGF). [Figure 8B] Figure 8B shows the effects of different concentrations of GEKG and KTTKS on the production of type I collagen in vitro. [Figure 9A] Figure 9A shows the gel electrophoresis of the RT-PCR products TGF-β1 and GAPDH. [Figure 9B] Figure 9B shows that different concentrations of GEKG and KTTKS promote TGF-β1 expression, and that a dose-dependent relationship exists. [Figure 10] Figure 10 shows that TGF-β1 and KTTKS or GEKG induce extracellular matrix (ECM) synthesis in human gingival fibroblasts (HGF). [Figure 11] Figure 11 shows a photograph of gingival growth after using a gingival restorative solution containing KTTKS or GEKG. [Modes for carrying out the invention]

[0030] The present invention relates to the use of oligopeptides in the treatment of gingivitis and gingival recession and in the repair of oral mucosa. The technical aspects of the present invention will be described below with reference to specific embodiments so that those skilled in the art can easily understand the advantages and effects of the present invention from what is disclosed herein. However, the present invention can also be implemented or applied by other different specific embodiments.

[0031] Example 1: Evaluation of TNFα inhibitory activity and cytotoxicity of GEKG and KTTKS by proliferation / cytotoxicity analysis of L929 cells in vitro.

[0032] In vitro proliferation / cytotoxicity analysis of L929 cells was performed on microtiter plates. L929 cells were cultured in Eagle's Minimal Essential Medium (EMEM) containing 10% bovine serum, 1% P / S, and 1% non-essential amino acids. Next, the aggregated L929 cells were washed with 2 ml of PBS solution, triedpsinized, and resuspended in EMEM. Subsequently, 200 μl of the cell suspension was aspirated and the cell density was counted, and the remaining suspension was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and EMEM was added to the diluted cells to obtain a final concentration of 1.5 x 10⁶ cells. 5 Cells / ml were prepared. Then, 100 μl of the cell suspension was added to each well of a 96-well flat-bottom microtiter plate and incubated at 37°C in a 5% CO2 atmosphere for 24 hours (h).

[0033] TNFα inhibitory activity

[0034] First, GEKG and KTTKS were dissolved in 1×PBS to concentrations equivalent to 1 mg GEKG / ml and 1 mg KTTKS / ml, respectively. Next, GEKG and KTTKS were diluted in 1×PBS solution to concentrations of 500 μg / ml, 125 μg / ml, 31.25 μg / ml, and 7.81 μg / ml, respectively. The diluted solutions were then mixed with an equal volume of TNFα solution and incubated for 1 hour. Subsequently, 50 μl of each of these four mixed solutions was taken and added to a 96-well flat-bottom microtiter plate in which the above-mentioned L929 cells had been cultured for 24 hours. The final concentration of actinomycin D (ActD) in the culture medium was adjusted to 2 μg / ml, and the final concentration of TNFα was adjusted to 0.1 ng / ml. Test wells containing only 2 μg / ml ActD and test wells containing only 0.1 ng / ml TNFα were designated as the positive control group and the blank control group, respectively. The above 96-well flat-bottom microtiter plates were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. Then, the values ​​were measured using an enzyme-linked immunoassay analyzer, with readings taken at OD490 / 630nm. The formula for calculating the TNFα inhibitory effect was as follows.

[0035]

number

[0036] cytotoxicity

[0037] First, GEKG and KTTKS were dissolved in 1×PBS to concentrations equivalent to 1 mg GEKG / ml and 1 mg KTTKS / ml, respectively. Next, GEKG and KTTKS were diluted in 1×PBS solution to concentrations of 500 μg / ml, 125 μg / ml, 31.25 μg / ml, and 7.81 μg / ml, respectively. Subsequently, 50 μl of each of these four diluted solutions was taken and added to a 96-well flat-bottom microtiter plate in which the above L929 cells had been cultured for 24 hours, and the final concentration of ActD in the culture medium was adjusted to 2 μg / ml. The above 96-well flat-bottom microtiter plate was then cultured at 37°C in a 5% CO2 atmosphere for 24 hours. After that, 50 μl of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) was added to each well and cultured for 4 hours. The cytotoxicity was then measured using an enzyme-linked immunosorbent assay (ISO) analyzer, with values ​​read at OD490 / 630nm. The formula for calculating cytotoxicity was as follows.

[0038]

number

[0039] result

[0040] Table 1 shows the analysis results for L929 cells at different dilution ratios of KTTKS. The TNFα inhibitory rate of KTTKS was 179.4%. As shown in Figure 1, this indicates that KTTKS exhibits excellent TNFα inhibitory activity without cytotoxicity.

[0041] [Table 1]

[0042] Table 2 shows the analysis results for L929 cells at different GEKG dilution ratios. The TNFα inhibitory rate of GEKG was 182.3%. Therefore, as shown in Figure 2, GEKG exhibits excellent TNFα inhibitory activity without cytotoxicity.

[0043] [Table 2]

[0044] Example 2: Evaluated the NO free radical scavenging ability (anti-inflammatory effect) of GEKG and KTTKS by suppressing nitric oxide (NO) induced by an inflammation-inducing substance (LPS) (macrophage experimental model) in vitro.

[0045] The anti-inflammatory capabilities of GEKG and KTTKS were evaluated in vitro using an experimental model in which macrophage NO production is induced with a known inflammatory response-inducing substance (LPS).

[0046] The experimental results for KTTKS and GEKG are shown in Figures 3 and 4, respectively. These results demonstrate that both KTTKS and GEKG can suppress NO production caused by LPS (i.e., exhibit anti-inflammatory effects), and that a dose-dependent relationship exists between them.

[0047] Example 3: Evaluating the H2O2 scavenging ability (anti-inflammatory oxidative stress effect) of GEKG and KTTKS from hydrogen peroxide (H2O2) induced and produced in vitro by an inflammation-inducing substance (LPS) (macrophage experimental model).

[0048] The anti-inflammatory oxidative stress capacity of GEKG and KTTKS was evaluated in vitro using an experimental model in which macrophage H2O2 production is induced with a known inflammatory response inducer (LPS).

[0049] Dichlorofluorescin diacetate (DCFH-DA), which is permeable to cell membranes, was added to the test samples. Upon entering cells, DCFH-DA underwent cleavage and oxidization of intracellular H2O2, forming a DCF product with green fluorescence. This revealed that peptides GEKG and KTTKS can suppress free radical H2O2 induced by LPS at wavelengths of 488-530 nm. Furthermore, increasing the peptide concentration significantly reduced the concentration of free radicals (H2O2) released by cells in response to LPS stimulation. The experimental results for KTTKS and GEKG are shown in Figure 5. From these results, it was demonstrated that both KTTKS and GEKG can suppress H2O2 production caused by LPS (i.e., exhibit anti-inflammatory oxidative stress effects), and that a dose-dependent relationship exists between the two.

[0050] Example 4: Evaluation of the promotion of proliferation of early oral epithelial cells

[0051] Figure 6 shows that different concentrations of GEKG and KTTKS can promote the proliferation of early oral epithelial cells in vitro, and that a dose-dependent relationship exists. Therefore, it has been demonstrated that GEKG and KTTKS can be applied to the healing of oral mucosal wounds.

[0052] Example 5: Evaluation of early oral epithelial cell death induced by anti-free radical / inflammatory response

[0053] Figure 7 shows that different concentrations of GEKG and KTTKS can reduce oral epithelial cell death caused by H2O2 free radicals in vitro, and that a dose-dependent relationship exists. Therefore, it has been demonstrated that GEKG and KTTKS can be applied to prevent oral epithelial cell death, and furthermore, that they can enable the protection of oral epithelial cells, prevention of mucosal lesions, prevention of oral cancer, and treatment of oral ulcers.

[0054] Example 6: Evaluation of the promotion of human gingival fibroblast (HGF) proliferation and type I collagen production.

[0055] Figure 8A shows that different concentrations of GEKG and KTTKS do not significantly affect the proliferation of HGF in vitro.

[0056] In Figure 8B, the gel electrophoresis of the RT-PCR product shows that the DNA bands for GAPDH and type I collagen appeared at positions 467 bp and 409 bp, respectively. This indicates that GEKG and KTTKS stimulated the expression of type I collagen genes during transcription. Furthermore, the formation of type I collagen was observed in human gingival fibroblasts. In addition, a dose-dependent relationship was observed between the expression level of type I collagen and the concentrations of GEKG and KTTKS in the range of 0 to 40 μl.

[0057] Based on the above, it has been demonstrated that the effect of GEKG and KTTKS on increasing type I collagen in the gingiva is not due to an increase in the number of human gingival fibroblasts, but rather that the proliferation of type I collagen is achieved through stimulation or activation of human gingival fibroblasts.

[0058] Example 7: Evaluation of TGF-β1 gene expression enhancement in human gingival fibroblasts (HGF).

[0059] TGF-β1 is known to regulate the proliferation of gingival fibroblasts and osteoblasts, as well as to promote the synthesis of the extracellular matrix (ECM) and type I and type III collagen.

[0060] In vitro enhancement of TGF-β1 gene expression in human HGF

[0061] In Figure 9A, the gel electrophoresis of the RT-PCR product shows that the GAPDH and TGF-β1 DNA bands appeared at positions 467 bp and 161 bp, respectively. This indicates that GEKG and KTTKS stimulated the expression of the TGF-β1 gene during transcription.

[0062] Figure 9B shows that there is a dose-dependent relationship between TGF-β1 expression levels and the concentrations of GEKG and KTTKS.

[0063] Based on the above, it has been demonstrated that GEKG and KTTKS can promote TGF-β1 expression by stimulating human HGF.

[0064] TGF-β1 promotes extracellular matrix synthesis in human HGF.

[0065] We investigated whether human HGF has a synergistic effect on extracellular matrix synthesis by treating it with TGF-β1 (0.5 ng / ml or 5 ng / ml) alone or in combination with KTTKS or GEKG (50 μM).

[0066] Human HGF was pre-cultured for 24 hours in Dulbecco's Modified Eagle Medium (DMEM) containing 0.2% serum. Then, it was treated for 24 hours at three different TGFβ1 concentrations (0 ng / ml, 0.5 ng / ml, and 5 ng / ml), and cultured for another 24 hours either in the absence of KTTKS or GEKG (solid line) or in the presence of 50 μM (dotted line).

[0067] Figure 10 shows the amount of collagen and fibrin synthesized when KTTKS or GEKG is not present (solid line) or when 50 μM is present (dotted line), respectively.

[0068] This demonstrates that TGFβ1, KTTKS, and GEKG enhance ECM biosynthesis through independent mechanisms, and that KTTKS and GEKG are more capable of inducing ECM synthesis in human HGF than TGFβ1. Furthermore, another possible mechanism suggests that TGFβ1 and KTTKS or GEKG work together to increase ECM biosynthesis.

[0069] Example 8: Preparation of a gingival repair solution containing KTTKS or GEKG and its effect on gingival growth.

[0070] A gingival repair solution was prepared using the ingredients and proportions listed in Table 3 below.

[0071] [Table 3]

[0072] Deionized water was weighed and placed in an emulsifying kettle. While maintaining the temperature of the kettle at 20°C, weighed EDTA-2Na, sodium benzoate, and citric acid were added in order. Next, a stirrer was started to dissolve the ingredients uniformly, and then glycerol and sorbitol were added in order and continued to stir uniformly. After mixing peppermint oil and PEG-40 hydrogenated castor oil, the mixture was added to the emulsifying kettle and stirred uniformly. Finally, KTTKS and GEKG were added and stirred uniformly.

[0073] The gingival repair solution was administered twice daily. Approximately 5-6 ml was given each time, and the patient was instructed to hold it in their mouth for about 10-15 minutes before spitting it out.

[0074] As shown in Figure 11, the gingiva of tooth A was clearly receding before treatment, but one month after using the gingival repair solution, the gingiva of tooth A grew without further recession. Furthermore, six months after using the gingival repair solution, the gingiva of tooth A had clearly grown, and the gap between the teeth had narrowed.

[0075] This demonstrates that the gingival repair solution has the function of stimulating the growth of receding gums.

[0076] Based on the above, the present invention not only effectively solves the problems present in the prior art, but also significantly enhances its effects. Furthermore, no identical or similar products have been created, disclosed, used, or enhanced in the same art.

Claims

1. The use of oligopeptides in the preparation of drugs for the treatment of gingivitis and gingival recession and for the repair of oral mucosa, The oligopeptides are oligopeptides (GEKG) consisting of the amino acid sequence described in SEQ ID NO: 1, and oligopeptides (KTTKS) consisting of the amino acid sequence described in SEQ ID NO: 3, The aforementioned agent is characterized by containing 0.2 to 2% by weight of GEKG and 0.2 to 2% by weight of KTTKS.

2. The use according to claim 1, wherein the agent further comprises one or more pharmaceutically acceptable carriers.

3. The use according to claim 1 or 2, characterized in that the drug is in the form of a powder, aerosol, ointment, gel, paste, or solution.

4. The use according to claim 3, characterized in that the drug is administered orally.

5. The use according to claim 4, characterized in that the aforementioned drug is of the sustained-release type.

6. The use according to claim 5, characterized in that the sustained-release type is a sustained-release type absorbed via mucosal or oral absorption.

7. The use according to claim 1, wherein the agent further comprises glycerol, sorbitol, EDTA, sodium benzoate, citric acid, peppermint oil, and PEG-40 hydrogenated castor oil.

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