Stabilized liquid composition of peptide

Abstract

The present specification discloses a stabilized liquid composition of a peptide comprising an amino acid sequence represented by SEQ ID NO: 1. In particular, the present invention provides a composition that exhibits improved storage stability and handleability through adjustment of a pH range so that purity and the content of related substances meet the standards even under refrigerated and room-temperature conditions in a liquid state. Such a liquid composition is applied as a cosmetic, a quasi-drug, or a pharmaceutical product, and thus can provide a long-term shelf life while maintaining the original functionality of peptides for treating dentin hypersensitivity.

Description

Stabilized peptide liquid composition

[0001] The present disclosure discloses a stabilized liquid composition of a peptide. In particular, the composition provides improved storage stability and handling by controlling the pH range so that the purity and content of flexible substances meet standards even under refrigerated and room temperature conditions. This liquid composition can be applied as a cosmetic, quasi-drug, or pharmaceutical product, maintaining the functionality of the original peptide while providing a long shelf life.

[0002] In this context, peptides for promoting dentin or pulp tissue regeneration and treating dentin hypersensitivity are attracting significant attention in dentistry. These peptides have the potential to modulate the inflammatory response of periodontal tissues or promote periodontal tissue regeneration. The development of peptides for promoting dentin or pulp tissue regeneration and treating dentin hypersensitivity could lead to novel approaches that provide non-surgical treatments for periodontal disease or enhance the effectiveness of existing treatments. The application of these peptides is fostering the development of novel therapeutic strategies in dentistry, particularly their potential as a means of modulating inflammation and promoting tissue regeneration.

[0003] The present inventors have continuously studied peptides for promoting regeneration of dentin or pulp tissue and treating dentin hypersensitivity, and have discovered that a peptide having the amino acid sequence of KYKQKRRSYK (SEQ ID NO: 1) has a mechanism for reactivating dental odontoblasts, sealing exposed dentinal tubules and regenerating damaged dentin, thereby promoting regeneration of dentin or pulp tissue and treating dentin hypersensitivity. (WO2024 / 122889, etc.)

[0004] Peptides are generally composed of fewer amino acids than proteins, resulting in smaller molecular weights and shorter amino acid chains. These structural characteristics make peptides more unstable and prone to degradation than proteins. They are particularly prone to degradation in water, making ensuring peptide stability a critical challenge when manufacturing finished products, such as pharmaceuticals, that contain peptides as their main ingredient.

[0005] Due to these issues, peptides are typically manufactured as finished products in powder form, with moisture removed by freeze-drying. However, this freeze-drying process, compared to conventional manufacturing processes, has higher production costs and requires demanding processing conditions, leading to increased costs and process issues.

[0006] Peptides with the amino acid sequence of SEQ ID NO: 1 also have storage stability and handling issues. Because these peptides are highly hygroscopic, they readily absorb moisture during storage, reducing their physical and chemical stability and making long-term storage difficult. This can lead to decreased peptide activity and quality, significantly hindering their clinical applications. Therefore, attempts have been made to develop peptides into lyophilized formulations. While lyophilized formulations have improved peptide stability to some extent, the problem of moisture absorption during lyophilized storage, which reduces the shelf life of pharmaceuticals and cosmetics, remains.

[0007] Therefore, in order to solve these problems and to promote the application of peptides having the above amino acid sequence, the present invention discloses a liquid composition of peptides with improved storage stability and handling properties.

[0008] The technical problems of this specification are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0009] The peptide-containing liquid composition of the present disclosure comprises a peptide having an amino acid sequence (KYKQKRRSYK) represented by SEQ ID NO: 1 and a buffer and has a pH in the range of about 2.5 to about 5.0.

[0010] In one embodiment, the pH of the peptide-containing liquid composition may range from about 2.5 to about 4.5.

[0011] In one embodiment, the peptide-containing liquid composition may further comprise a pH adjusting agent.

[0012] In one embodiment, the pH adjusting agent may be selected from the group consisting of acetic acid, sodium acetate, citric acid, sodium citrate, sulfuric acid, hydrochloric acid, tartaric acid, boric acid, or a combination thereof.

[0013] In one embodiment, the concentration of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 may range from about 0.2 to about 20 mg / mL.

[0014] In one embodiment, the concentration of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 may range from about 0.2 to about 10 mg / mL.

[0015] In one embodiment, the peptide-containing liquid composition may maintain a purity of about 95% or more when stored at room temperature for 3 months, as analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia.

[0016] In one embodiment, the peptide-containing liquid composition may maintain a purity of about 97% or more when stored at 2 to 8°C for 3 months, as analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia.

[0017] In one embodiment, a peptide-containing liquid composition may be one in which about 3% or less of a flexible substance is detected when stored at room temperature for 4 weeks, when analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia.

[0018] In one embodiment, the peptide-containing liquid composition may maintain a peptide content comprising the amino acid sequence represented by SEQ ID NO: 1 of about 95% or more based on the content before storage when stored at room temperature for 3 months.

[0019] In one embodiment, the peptide-containing liquid composition may further comprise a preservative.

[0020] In one embodiment, the preservative may be selected from a benzoate-based preservative, a sorbate-based preservative, or a paraben-based preservative.

[0021] In one embodiment, the peptide-containing liquid composition may be in a dosage form selected from among solutions, gels, elixirs, suspensions, emulsions, lemonades, perfumes, emulsions and infusions, liniments, tinctures, syrups, gargles, sprays, semi-solids, injections, eye drops, eye ointments, ear drops, nasal drops, liniments, lotions, aerosols, ointments, creams, and pastes.

[0022] In one embodiment, the peptide-containing liquid composition may further comprise a solvating agent.

[0023] In one embodiment, the peptide-containing liquid composition may further comprise one or more selected from a wetting agent, a viscosity agent, a surfactant, a carrier, or a diluent.

[0024] In one embodiment, the peptide-containing liquid composition may additionally comprise a fragrance.

[0025] A quasi-drug comprising a peptide-containing liquid composition of the present specification is disclosed.

[0026] A pharmaceutical composition for improving or treating tooth damage, comprising the peptide-containing liquid composition of the present specification as an active ingredient, is disclosed.

[0027] Specific details of other embodiments are included in the detailed description and drawings.

[0028] The composition of the present disclosure is a liquid composition comprising a peptide having an amino acid sequence represented by SEQ ID NO: 1 (KYKQKRRSYK). By fundamentally blocking hygroscopicity, the physical and chemical stability of the peptide is maintained, enabling long-term storage. In addition, the chemical stability of the peptide is maintained in the liquid formulation, minimizing degradation or deterioration during storage, and enabling the user to easily handle the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and use it conveniently in clinical applications.

[0029] Thus, the development of a stable liquid composition with a long shelf life may increase the clinical applicability of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and bring about positive changes in dental treatment.

[0030] Figure 1 is a graph showing the results of a moisture absorption and release characteristic measurement experiment (DVS; Dynamic Vapor Sorption / desorption) that confirmed the hygroscopicity of a peptide including an amino acid sequence represented by sequence number 1.

[0031] Figure 2 is a graph showing the results of an experiment confirming the stability of a peptide containing an amino acid sequence represented by sequence number 1 in artificial saliva.

[0032] Figure 3 is a graph showing the results of an experiment confirming the stability of a peptide containing an amino acid sequence represented by sequence number 1 in artificial gastric fluid.

[0033] Figure 4 is a graph showing the results of an experiment confirming the stability of a peptide containing an amino acid sequence represented by sequence number 1 in an artificial intestinal fluid.

[0034] Figure 5 is a graph showing the results of a 3-month stability test of a peptide-containing liquid composition, in which the purity change of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 at 1 mg / mL in a sodium acetate buffer solution at pH 4.5 was tested under various temperature conditions.

[0035] Figure 6 is a graph showing the results of a 3-month stability test of a peptide-containing liquid composition, in which the purity change of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 at 1 mg / mL in a sodium acetate buffer solution at pH 5.5 was tested under various temperature conditions.

[0036] Figure 7 is a graph showing the results of a 3-month stability test of a peptide-containing liquid composition, in which the purity change of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 at 1 mg / mL in a sodium acetate buffer solution at pH 6.0 was tested under various temperature conditions.

[0037] Figure 8 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the peptide purity change including the amino acid sequence represented by SEQ ID NO: 1 was tested under various temperature conditions in phosphate buffered saline at pH 7.5.

[0038] Figure 9 is a graph showing the results of a 3-month stability test of a peptide-containing liquid composition, in which the purity change of the peptide including the amino acid sequence represented by SEQ ID NO: 1 at 1 mg / mL in a Tris HCl buffer solution at pH 8.0 was tested under various temperature conditions.

[0039] Figure 10 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the purity change of the peptide including the amino acid sequence represented by SEQ ID NO: 1 at 1 mg / mL in a Tris HCl buffer solution at pH 9.5 was tested under various temperature conditions.

[0040] Figure 11 is a graph showing the degradation rate (%) of a peptide comprising an amino acid sequence represented by sequence number 1 according to pH in the range of pH 4.5 to 9.5.

[0041] Figure 12 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the purity change of the peptide including the amino acid sequence represented by SEQ ID NO: 1 was tested under various temperature conditions in a sodium acetate buffer solution at pH 2.5.

[0042] Figure 13 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the purity change of the peptide including the amino acid sequence represented by SEQ ID NO: 1 was tested under various temperature conditions in a sodium acetate buffer solution at pH 3.0.

[0043] Figure 14 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the change in peptide purity including the amino acid sequence represented by SEQ ID NO: 1 was tested under various temperature conditions in a sodium acetate buffer solution at pH 3.5.

[0044] Figure 15 is a graph showing the results of a 3-month stability test of a liquid composition containing a peptide, wherein the purity change of the peptide including the amino acid sequence represented by SEQ ID NO: 1 was tested under various temperature conditions in a sodium acetate buffer solution at pH 4.0.

[0045] Figure 16 is a graph showing the degradation rate (%) of a peptide comprising an amino acid sequence represented by sequence number 1 according to pH in the range of pH 2.5 to 9.5.

[0046] Figure 17 shows the results of an 8-week stability test confirmed by changes in peptide concentration of a liquid composition containing a peptide including an amino acid sequence represented by SEQ ID NO: 1 including a preservative.

[0047] Figure 18 shows the results of an 8-week stability test confirmed by changes in peptide purity of a liquid composition containing an amino acid sequence represented by sequence number 1 including a preservative.

[0048] FIG. 19 is HPLC result data confirming the purity of a peptide containing an amino acid sequence represented by SEQ ID NO: 1 after storing a peptide-containing liquid composition (5A) containing an amino acid sequence represented by SEQ ID NO: 1 according to one embodiment at various temperatures for 8 weeks.

[0049] FIG. 20 is HPLC result data confirming the purity of a peptide including an amino acid sequence represented by SEQ ID NO: 1 after storing a peptide-containing liquid composition (5B) including an amino acid sequence represented by SEQ ID NO: 1 according to one embodiment at various temperatures for 8 weeks.

[0050] FIG. 21 is HPLC result data confirming the purity of a peptide containing an amino acid sequence represented by SEQ ID NO: 1 after storing a peptide-containing liquid composition (5C) containing an amino acid sequence represented by SEQ ID NO: 1 according to one embodiment at various temperatures for 8 weeks.

[0051] FIG. 22 is HPLC result data confirming the purity of a peptide containing an amino acid sequence represented by SEQ ID NO: 1 after storing the peptide-containing liquid composition (5D) containing an amino acid sequence represented by SEQ ID NO: 1 at various temperatures for 8 weeks according to one embodiment.

[0052] Below, embodiments of the present disclosure are described in detail with reference to the attached drawings to enable those skilled in the art to easily implement the present disclosure. However, the present disclosure may be implemented in various different forms and is not limited to the embodiments described herein.

[0053] Throughout the specification and claims, whenever a part is said to "include" a certain component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0055] The present inventors have made efforts to develop a stable liquid composition to solve the problem of reduced storage stability due to hygroscopicity of a peptide comprising an amino acid sequence represented by SEQ ID NO: 1. As a result, they have confirmed that stability at room temperature and under refrigerated conditions is significantly improved when the peptide-containing liquid composition has a pH in the range of 2.5 to 5.0 or less, preferably 2.5 to 4.5 or less. In addition, they have confirmed that a long-term shelf life can be secured based on purity and active ingredient content during storage, and have completed the present invention.

[0056] In the present specification, a liquid composition containing a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 and a buffer, and having a pH range of 2.5 or more and 5.0 or less is provided.

[0057] The peptide of the present specification includes an amino acid sequence represented by SEQ ID NO: 1, and encompasses variants and salts thereof that contain the sequence and maintain the function. The peptide may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0058] Sequence number 1: KYKQKRRSYK

[0059] The above peptide may include a mutant peptide having a different sequence by deletion, insertion, substitution or a combination thereof of amino acid residues, as long as it does not affect the activity of the peptide, or may be in the form of a protein fragment having the same function. Amino acid modifications at the protein and peptide levels that do not entirely alter the activity due to including the amino acid sequence of SEQ ID NO: 1 are known in the art of the present invention, and may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., as needed. Accordingly, the peptide includes not only the amino acid sequence of SEQ ID NO: 1, but also a peptide or a mutant thereof having substantially the same amino acid sequence and the same activity. The peptide including the amino acid sequence represented by SEQ ID NO: 1 prevents or alleviates hypersensitivity by closing the defects of exposed dentinal tubules through physiological remineralization of dentin, and may be used for promoting regeneration of dentin or pulp tissue and / or treating dentin hypersensitivity.

[0060] The peptide-containing liquid composition of the present specification comprises a peptide having an amino acid sequence represented by SEQ ID NO: 1.

[0061] The content of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the peptide-containing liquid composition of the present specification is from about 0.2 mg / ml to about 20 mg / ml, from about 0.2 mg / ml to about 15 mg / ml, from about 0.2 mg / ml to about 10 mg / ml, from about 0.2 mg / ml to about 5 mg / ml, from about 0.2 mg / ml to about 1 mg / ml, from about 0.3 mg / ml to about 20 mg / ml, from about 0.3 mg / ml to about 15 mg / ml, from about 0.3 mg / ml to about 10 mg / ml, from about 0.3 mg / ml to about 5 mg / ml, from about 0.3 mg / ml to about 1 mg / ml, from about 0.5 mg / ml to about 20 mg / ml, from about 0.5 mg / ml to about 15 mg / ml, from about 0.5 mg / ml to about 10 mg / ml, about 0.5 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 1 mg / ml, for example, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, or about 20 mg / ml.

[0062] The pH of the peptide-containing liquid composition of the present disclosure is from about 2.5 to about 5.5, for example, from about 2.5 to about 5.5, from about 2.5 to about 5.4, from about 2.5 to about 5.3, from about 2.5 to about 5.2, from about 2.5 to about 5.1, from about 2.5 to about 5.0, from about 2.5 to about 4.9, from about 2.5 to about 4.8, from about 2.5 to about 4.7, from about 2.5 to about 4.6, from about 2.5 to about 4.5, from about 3.0 to about 5.5, from about 3.0 to about 5.4, from about 3.0 to about 5.3, from about 3.0 to about 5.2, from about 3.0 to about 5.1, from about 3.0 to about 5.0, pH about 3.0 to about 4.9, pH about 3.0 to about 4.8, pH about 3.0 to about 4.7, pH about 3.0 to about 4.6, pH about 3.0 to about 4.5, pH about 3.5 to about 5.5, pH about 3.5 to about 5.4, pH about 3.5 to about 5.3, pH about 3.5 to about 5.2, pH about 3.5 to about 5.1, pH about 3.5 to about 5.0, pH about 3.5 to about 4.9, pH about 3.5 to about 4.8, pH about 3.5 to about 4.7, pH about 3.5 to about 4.6, pH about 3.5 to about 4.5, pH about 4.5, or pH about 3.0.

[0063] In one embodiment, the buffering agent is phosphoric acid, acetic acid, citric acid, succinic acid and pharmaceutically acceptable salts of these acids (e.g., sodium salt, potassium salt), trisaminomethane and hydrochloric acid, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), tris(hydroxymethyl)aminomethane / sodium hydroxide, MOPS ((3-(N-Morpholino)propanesulfonic acid) / sodium hydroxide, MES (2-(N-morpholino) ethanesulfonic acid) / sodium hydroxide, glycine / hydrochloric acid, glycine / sodium hydroxide, boric acid / sodium tetraborate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid) / sodium hydroxide, Bicine / sodium hydroxide, PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid)) / sodium hydroxide, It may include at least one selected from sodium bicarbonate. In one example, the buffer may include sodium acetate, sodium citrate, sodium monophosphate, sodium diphosphate, or a combination thereof. Such a buffer may contribute to maintaining the pH of a peptide solution comprising the amino acid sequence represented by SEQ ID NO: 1. The type of buffer may be appropriately selected by a person skilled in the art depending on the purpose.

[0064] The buffer, based on the total liquid composition, is about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about It may be included at a concentration of 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, or 10 mM, 50 mM, 60 mM, or 80 mM.

[0065] In one embodiment, the pH of the buffer is from about 2.5 to about 9.5, for example, from about 2.5 to about 9.0, from about 2.5 to about 8.5, from about 2.5 to about 8.0, from about 2.5 to about 7.5, from about 2.5 to about 7.0, from about 2.5 to about 6.5, from about 2.5 to about 6.0, from about 2.5 to about 5.5, from about 2.5 to about 5.0, from about 2.5 to about 4.5, from about 2.5 to about 4.0, from about 3.0 to about 9.0, from about 3.0 to about 8.5, from about 3.0 to about 8.0, from about 3.0 to about 7.5, from about 3.0 to about 7.0, from about 3.0 to about 6.5, pH about 3.0 to about 6.0, pH about 3.0 to about 5.5, pH about 3.0 to about 5.0, pH about 3.0 to about 4.5, pH about 3.0 to about 4.0.

[0066] In one embodiment, the peptide-containing liquid composition of the present disclosure may further comprise a pH adjuster. The pH adjuster is selected to maintain stability by adjusting the pH of the liquid composition within a desired range. Possible pH adjusters include acids, bases, buffers, or buffer solutions. Examples of acidic pH adjusters include, but are not limited to, acetic acid, citric acid, hydrochloric acid, lactic acid, tartaric acid, boric acid, sulfuric acid, phosphoric acid, and the like. Examples of basic pH adjusters include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, and the like. Examples of buffers or buffer solutions include sodium acetate, sodium citrate, sodium monophosphate, sodium diphosphate, trisaminomethane and hydrochloric acid, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris(hydroxymethyl)aminomethane / sodium hydroxide, MOPS ((3-(N-Morpholino)propanesulfonic acid) / sodium hydroxide, MES (2-(N-morpholino) ethanesulfonic acid) / sodium hydroxide, glycine / hydrochloric acid, glycine / sodium hydroxide, boric acid / sodium tetraborate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid) / sodium hydroxide, Bicine / sodium hydroxide, PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid)) / sodium hydroxide, Including, but not limited to, sodium bicarbonate, etc. When the liquid composition containing a peptide including the amino acid sequence represented by SEQ ID NO: 1 has a pH range of 2.5 to 5.0, preferred pH adjusting agents that can be used include, but are not limited to, acetic acid, sodium acetate, citric acid, sodium citrate, sulfuric acid, hydrochloric acid, tartaric acid, boric acid, or combinations thereof.

[0067] In one embodiment, the peptide-containing liquid composition of the present disclosure comprises about 0.2 to about 20.0 mg / mL, about 0.2 to about 15.0 mg / mL, about 0.2 to about 10.0 mg / mL, about 0.2 to about 5.0 mg / mL, about 0.2 to about 2.0 mg / mL, about 0.2 to about 1.0 mg / mL, about 0.5 to about 20.0 mg / mL, about 0.5 to about 15.0 mg / mL, about 0.5 to about 10.0 mg / mL, about 0.5 to about 5.0 mg / mL, about 0.5 to about 2.0 mg / mL, about 0.5 to about 1.0 mg / mL, about 1.0 to about 20.0 mg / mL, about 1.0 to about 15.0 mg / mL, about 1.0 to It may be included at a concentration of about 10.0 mg / mL, about 1.0 to about 5.0 mg / mL, or about 1.0 to about 2.0 mg / mL.

[0068] The peptide-containing liquid composition of the present disclosure maintains high purity and content of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 even when stored under refrigeration and at room temperature.

[0069] In one embodiment, the peptide-containing liquid composition maintains a high purity even after storage at room temperature of 25°C for 3 months. For example, a peptide purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% may be maintained.

[0070] In one embodiment, the peptide-containing liquid composition maintains a high purity even after storage for 3 months under refrigerated conditions at about 2 to about 8° C. For example, a purity of about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, or about 99% or greater may be maintained.

[0071] In one embodiment, the peptide-containing liquid composition exhibits a small amount of volatile compounds when stored at room temperature for 4 weeks. For example, less than or equal to about 1%, less than or equal to about 1.5%, less than or equal to about 2%, less than or equal to about 2.5%, less than or equal to about 3%, or less than or equal to about 3.5% of volatile compounds may be detected.

[0072] In one embodiment, the peptide-containing liquid composition maintains a high level of peptide content for treating dentin hypersensitivity after 3 months of storage at room temperature, relative to the content prior to storage. For example, a content of at least about 95%, at least about 96%, at least about 97%, or at least about 98% may be maintained.

[0073] In one embodiment, the peptide-containing liquid composition may further comprise a preservative. The preservative may be added to improve the safety and effectiveness of the liquid composition, which is relatively susceptible to microbial growth. Examples of preservatives that may be used include, but are not limited to, benzoate-based preservatives, sorbate-based preservatives, and ester-based preservatives. Benzoate-based preservatives may include, but are not limited to, sodium benzoate and potassium benzoate. Sorbate-based preservatives may include, but are not limited to, sorbic acid and potassium sorbate. Ester-based preservatives may include, but are not limited to, methylparaben, ethylparaben, propylparaben, butylparaben, and isobutylparaben. In one embodiment, sodium benzoate preservative may be preferred. Preservatives should be carefully selected as they may affect the stability of the peptide-containing liquid composition. Other preservatives may be used as long as they do not impair the purpose of the present invention.

[0074] In one embodiment, the peptide-containing liquid composition can be prepared in various liquid dosage forms. For example, the liquid composition can be selected from solutions, gels, elixirs, suspensions, emulsions, lemonades, fragrances, infusions and infusions, liniments, tinctures, syrups, gargles, sprays, semi-solids, injections, eye drops, eye ointments, ear drops, nasal drops, liniments, lotions, aerosols, ointments, creams, and pastes. These dosage forms can be appropriately selected depending on the specific use and application site, and each dosage form can be optimized to maximize the stability and effectiveness of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1.

[0075] In one embodiment, the peptide-containing liquid composition may further comprise a solvating agent. The solvating agent may be used to enhance the solubility of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and to increase the homogeneity of the composition. Such solvating agents may be used in various forms, and may include, for example, polysorbate, propylene glycol, polyethylene glycol, sorbitan ester, and the like. The solvating agent may be appropriately selected by a person skilled in the art, taking into account the desired physical and chemical properties of the composition while maintaining the stability of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1.

[0076] In one embodiment, the peptide-containing liquid composition may further comprise a wetting agent, viscosity agent, surfactant, carrier, or diluent. These components can be appropriately selected by those skilled in the art to optimize the physical and chemical properties of the peptide-containing liquid composition comprising the amino acid sequence represented by SEQ ID NO: 1 and to enhance the convenience of use.

[0077] For example, humectants include, but are not limited to, glycerol, sorbitol, propylene glycol, butylene glycol, polyethylene glycol, trehalose, pyridoxine, allantoin, lactic acid, hyaluronic acid, urea, ceramides, diglycerin, trimethylglycine, aloe vera extract, panthenol, betaine, polyglycerin, arginine, amino acid complexes such as glutamine, alginate, PEG derivatives, acetyl glucosamine, phytosterols, trehalose derivatives, mannitol, glyceryl glucoside, propylene carbonate, butylene carbonate, erythritol, fructan, and glucose.

[0078] For example, viscosity agents include, but are not limited to, carbomer, hydroxypropylmethylcellulose, xanthan gum, guar gum, xanthan gum, carrageenan, acrylate / alkyl acrylate crosspolymer, sodium alginate, polyacrylamide, methylcellulose, hydroxyethylcellulose, (locust bean gum), tragacanth gum, sodium polyacrylate, pullulan, agarose, gelatin, dextran, polyvinyl alcohol, polyacrylic acid, polyquaternium, natural rubber, sulfated alginic acid, pluronic F127, algae extract, and sclerotium gum.

[0079] For example, surfactants include Tween 80, sodium lauryl sulfate (SLS), sodium cocoyl isethionate, sodium laureth sulfate (SLES), polysorbate 20, lauryl amphoacetate, cetyl trimethyl ammonium bromide, sodium methyl cocoyl taurate, cocamidopropyl betaine, disodium dimonium chloride, cetyl alcohol, stearyl alcohol, glyceryl stearate, Including, but not limited to, poloxamer, polyoxyethylene lauryl ether, sodium lauroyl sarcosinate, sodium coco sulfate, diethanolamine coconut amido propionate, methyl glucose sesquistearate, polyoxyethylene sorbitan monolaurate, PEG-40 hydrogenated castor oil.

[0080] In one embodiment, the peptide-containing liquid composition may include a carrier. The carrier can aid in the delivery of the main ingredient and control the physical properties of the liquid composition. For example, carriers include, but are not limited to, water, ethanol, butylene glycol, propylene glycol, dimethyl sulfoxide, glycerin, mineral oil, vegetable oils such as olive oil, coconut oil, avocado oil, and jojoba oil, silicone oil, triethanolamine, ethylene glycol, polyethylene glycol 400, polyethylene glycol 600, and sodium chloride.

[0081] In one embodiment, the peptide-containing liquid composition may further comprise a diluent. Examples of diluents include, but are not limited to, water, ethanol, butylene glycol, propylene glycol, dimethyl sulfoxide, glycerin, phenol, triclosan, vitamin E, vitamin C, panthenol, lecithin, sodium bicarbonate, polysorbate 80, sodium laureth sulfate, and the like.

[0082] In one embodiment, the peptide-containing liquid composition may further comprise a fragrance. Fragrances that may be used include natural essential oils and synthetic fragrances. Examples of natural essential oils include, but are not limited to, lavender oil, rosemary oil, tea tree oil, eucalyptus oil, peppermint oil, citrus oil, jasmine oil, ylang-ylang oil, sandalwood oil, vanilla extract, cinnamon oil, clove oil, geranium oil, patchouli oil, bergamot oil, lemongrass oil, nutmeg oil, mint oil, melissa oil, frankincense oil, chamomile oil, cedarwood oil, mandarin oil, grapefruit oil, anise oil, sweet orange oil, and the like. Examples of synthetic fragrances include, but are not limited to, citronellal, linalool, limonene, hexyl cinnamal, alpha-isomethyl ionone, butylphenyl methylpropional, hydroxycitronellal, methyl dihydrojasmonate, benzyl salicylate, hexyl cinnamaldehyde, and the like.

[0083] In one embodiment, the peptide-containing liquid composition of the present disclosure may further comprise physiologically acceptable additives, such as excipients, disintegrants, sweeteners, binders, coating agents, bulking agents, lubricants, diluents, dispersants, binders, and lubricants. Those skilled in the art can appropriately select and use these additives.

[0084] A quasi-drug comprising a liquid composition containing a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 of the present specification is provided. Quasi-drugs refer to products used for the diagnosis, treatment, improvement, alleviation, management, or prevention of diseases, which have a milder effect than or do not directly act on a pharmaceutical product. Such quasi-drugs include, but are not limited to, insect repellents, oral hygiene products, disinfectants, hygiene products, topical treatments, soaps, shampoos, and external cosmetics.

[0085] Disclosed is a pharmaceutical composition for improving or treating tooth damage, comprising a pharmaceutically effective amount of a liquid composition containing a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 of the present specification. The peptide comprising the amino acid sequence represented by SEQ ID NO: 1 exhibits preventive, ameliorating, and therapeutic effects on pulp diseases and periodontal diseases, including dentin hypersensitivity. The peptide comprising the amino acid sequence represented by SEQ ID NO: 1, which acts on dentin and periodontium, promotes regeneration of pulp and periodontal tissues through a cell activation mechanism. In one embodiment, the liquid composition containing a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 can be included in a pharmaceutical composition for improving or treating tooth damage in a pharmaceutically effective amount based on the peptide component for treating dentin hypersensitivity.

[0086] A pharmaceutically effective dose is a dose that is sufficient and safe to treat a disease or alleviate symptoms. This amount is determined by various factors, including the type and severity of the patient's disease, the drug's activity and sensitivity, the time of administration, the route of administration, the rate of excretion, the duration of treatment, and concurrent medications.

[0087] Pharmaceutical compositions can be prepared in various dosage forms. Examples include, but are not limited to, oral dosage forms such as tablets, capsules, granules, pills, liquids, suspensions, emulsions, troches, and oral gels; topical dosage forms such as ointments, creams, gels, lotions, patches, and suppositories; injectable dosage forms such as aqueous solutions, suspensions, emulsions, intramuscular injections, subcutaneous injections, and intravenous injections; eye drops, inhalants, and transdermal absorption agents. Each dosage form can be appropriately selected and formulated according to specific therapeutic purposes and patient needs.

[0088] Hereinafter, the present specification will be described in detail by way of examples.

[0089] However, the following examples are only intended to illustrate the present specification, and the contents of the present specification are not limited to the following examples.

[0090] <Example 1> Hygroscopicity of a peptide for promoting regeneration of dentin or pulp tissue and treating dentin hypersensitivity

[0091] The hygroscopicity of a peptide containing the amino acid sequence represented by SEQ ID NO: 1 was measured through a dynamic vapor sorption / desorption (DVS) experiment.

[0092] The results are shown in Fig. 1. The peptide containing the amino acid sequence represented by the used sequence number 1 was a linear peptide having the structure H-Lys-Tyr-Lys-Gln-Lys-Arg-Arg-Ser-Tyr-Lys-OH. The hygroscopicity of the peptide was confirmed according to the European Pharmacopoeia (EP) 5.11 characters section in monographs 'HYGROSCOPICTY'.

[0093] The peptide's absorbed moisture content, as determined from the change in peptide mass, did not increase up to a relative humidity of 5%. However, when the relative humidity exceeded 5%, the mass steadily increased up to 75%, indicating a significant moisture absorption rate. Furthermore, in the range of 75% to 95% relative humidity, moisture absorption rapidly increased, showing a maximum mass increase of 63.2%. This indicates that the peptide containing the amino acid sequence represented by SEQ ID NO: 1 is vulnerable to humidity and may be difficult to preserve when stored in a solid or lyophilized state.

[0094] <Example 2> Confirmation of the stability of peptide formulations in the gastrointestinal tract

[0095] Artificial saliva, artificial gastric juice, and artificial intestinal juice were prepared, and peptides of the structure H-Lys-Tyr-Lys-Gln-Lys-Arg-Arg-Ser-Tyr-Lys-OH were dissolved in these solutions, and the degree of decomposition was observed, confirming that different decomposition patterns were observed depending on the digestive tract.

[0096] The test conditions were as follows: HPLC test equipment (Shinadzu) was used, a column (250 x 4.6 mm, 5 um) was used, and the conditions were as follows: column temperature of approximately 30 ℃, sample temperature of approximately 30 ℃, flow rate of 1.0 mL / min, and injection volume of 10 uL. The mobile phase A was prepared by adding 0.5 mL of trifluoroacetic acid to purified water and making it 1,000 mL, and the mobile phase B was prepared by adding 0.5 mL of trifluoroacetic acid to acetonitrile and making it 1,000 mL. If the relative standard deviation of the peptide peak area was 2.0% or less after 6 injections of the standard solution, the system was judged to be suitable. The test was performed according to the liquid chromatography method in the general test methods of the Korean Pharmacopoeia. The injection order was 1 blank solution, 6 standard solution solutions, and 3 test solutions, and the test solutions were prepared anew each time.

[0097] A peptide containing the amino acid sequence represented by sequence number 1 was used after being frozen and stored at -20°C for approximately 6 months.

[0098] Artificial saliva was prepared at a pH of 6.8 with 0.1 mg / mL amylase in 20 mM phosphate buffer. The peptide was added at 1 mg / mL to the prepared artificial saliva, reacted at 37°C, and 200 μL was taken as a test solution. 70 μL of 1N HCl was added to inhibit the enzyme reaction. 1 mg / mL of the sample (peptide) was added to water, 200 μL was taken, and 70 μL of 1N HCl was added to this solution to prepare a standard solution. The sample reaction times of the test solution were measured at 0, 1, 2, 3, and 5 minutes. The test results for artificial saliva are shown in Table 1 below, and the line graph is shown in Fig. 2.

[0099] Content Time 123 Average Standard Deviation 0 min 100.000% 100.000% 100.000% 100.0% 0.01 min 100.363% 100.797% 99.083% 100.1% 0.92 min 100.187% 100.112% 98.877% 99.7% 0.73 min 99.838% 99.798% 99.393% 99.7% 0.25 min 99.865% 99.857% 97.830% 99.2% 1.2

[0100] An artificial gastric fluid was prepared in SGF (Simulated Gastric Fluid) buffer with a pH of 1.2 and a pepsin concentration of 3.2 mg / ml (2000 IU). The peptide was added to the prepared artificial gastric fluid at 1 mg / ml, reacted at 37°C, and 200 μl was taken as a test solution. 70 μl of 0.2 M Na2Co3 was added to inhibit the enzymatic reaction. The sample (peptide) was added to water at 1 mg / mL, 200 μL was taken, and the solution to which 70 μl of 0.2 M Na2Co3 was added was used as a standard solution. The sample reaction times of the test solution were measured at 0, 2, 3, 5, 10, 15, 30, and 60 minutes. The test results in the artificial gastric fluid are shown in Table 2 below, and the line graph is shown in Fig. 3.

[0101] Content Time 123 Mean Standard Deviation 0 min 100.000% 100.000% 100.000% 100.0% 0.00.25 min 98.403% 100.307% 100.914% 99.9% 1.30.5 min 98.076% 99.941% 101.750% 99.9% 1.81 min 98.500% 99.785% 101.510% 99.9% 1.52 min 98.307% 98.944% 102.607% 100.0% 2.33 min 97.249% 99.913% 101.778 % 99.6% 2.35 Minutes 98.495% 100.439% 101.593% 100.2% 1.610 Minutes 98.594% 100.078% 101.466% 100.0% 1.415 Minutes 96.821% 100.129% 101.903% 99.6% 2.630 Minutes 98.426% 99.910% 104.837% 101.1% 3.460 Minutes 98.305% 100.445% 103.299% 100.7% 2.5

[0102] An artificial intestinal fluid was prepared at a pH of 6.8 with 10 mg / ml (2000 IU) of pancreatin in SIF (Simulated Intestinal Fluid) buffer. Before adding the peptide to the prepared artificial intestinal fluid, the artificial intestinal fluid was reacted at 37°C for 5 minutes. The peptide was added at 1 mg / ml, reacted at 37°C, and 200 μl was taken as a test solution. 70 μl of 0.5 M citric acid was added to inhibit the enzyme reaction. The sample (peptide including the amino acid sequence represented by SEQ ID NO: 1) was added to water at a concentration of 1 mg / ml, and 200 μl was taken. The solution to which 70 μL of 0.5 M citric acid was added was used as a standard solution. The sample reaction times of the test solution were measured at 0.25, 0.5, 1, 2, 3, 5, 10, 15, 30, 60, and 120 minutes. The test results in the artificial intestinal fluid are shown in Table 3 below, and the broken line graph is shown in Figure 4.

[0103] Content Time 123 Mean Standard Deviation 0 min 4.122% 4.180% 4.294% 4.2% 0.10.25 min 4.429% 4.561% 4.607% 4.5% 0.10.5 min 4.452% 4.586% 4.945% 4.7% 0.31 min 4.325% 4.443% 4.926% 4.6% 0.32 min 4.048 % 4.111% 4.208 % 4.1% 0.13 min 3.459% 3.457% 2.492% 3.1% 0.65 min 1.802% 2.031% 1.949% 1.9% 0.110 min N / DN / DN / DN / DN / D

[0104] The test was stopped when the peptide containing the amino acid sequence represented by sequence number 1 was no longer detected. The test results showed a tendency to be stable in gastric juice with low pH conditions and a degradation pattern that was easily decomposed in intestinal juice with high pH conditions.

[0105] <Example 3>

[0106] Based on the experimental results of Example 2, the stability of a liquid composition containing a peptide including an amino acid sequence represented by sequence number 1 was confirmed by setting various conditions in the range of pH 2.5 to 9.5 in a liquid state.

[0107] A linear peptide consisting of 10 amino acids with the sequence H-Lys-Tyr-Lys-Gln-Lys-Arg-Arg-Ser-Tyr-Lys-OH was stored in a freezer at -20°C and then analyzed. The purity of the raw peptide was 99%, and the pure peptide content was 90.4%.

[0108] Preparation and incubation of peptide compositions

[0109] Peptide-containing liquid compositions containing the previously prepared peptides at concentrations of 0.2 mg / mL and 10 mg / mL were prepared in sodium acetate buffer, phosphate buffered saline, and Tris-HCl buffer. Table 4 below shows the composition information prepared under various pH conditions. Approximately 5 mL of each composition was prepared and placed in 2 cc screw-cap glass vials with a final volume of 0.2 mL to 10 mL. These vials were cultured and stored under freezing conditions at -20 °C, refrigerated conditions at 2 to 8 °C, and then incubated at 25 °C, 40 °C, and 60 °C. The accelerated temperature conditions in the incubator were 25 °C, 40 °C, and 60 °C. At each time point, 50 μL to 100 μL of sample was withdrawn from the vials and transferred to sample vials for RP-HPLC analysis. At the start of the measurement, all preparations were transparent and colorless in appearance.

[0110] Composition Number Buffer and Concentration Peptide Concentration (mg / mL) pH Last Measurement Time 3A 50 mM sodium acetate 104.53 months 3B 50 mM sodium acetate 105.53 months 3C 50 mM sodium acetate 106.03 months 3D Phosphate Buffered Saline 107.53 months 3E 50 mM Tris HCl 108.03 months 3F 50 mM Tris HCl 109.53 months 3G 50 mM sodium citrate 0.24.011 days 3H 50 mM sodium citrate 0.23.511 days 3I 50 mM sodium citrate 0.23.011 days 3J 50 mM sodium citrate 0.22.511 days 3K 50 mM sodium acetate 0.24.03 months 3L 50 mM sodium acetate 0.23.53 months 3M 50 mM acetic acid Sodium 0.23.01 months 3N50 mM sodium acetate 0.22.51 months

[0111] At the start of the stability study and until the end of one month of storage and incubation, samples were analyzed by the RP-HPLC method shown in Table 5 below.

[0112] Item Details Column YMC-Pack Pro C18, S-3 μm, 12 nm, 110Å, 150 x 4.6 mm, P / N: AS12S03-1546-WT Buffer A 0.1% Trifluoroacetic Acid in Water Buffer B 100% Acetonitrile Gradient 5% B to 25% B in 28 min Column Temperature 40° C ℃ Flow Rate 0.5 mL / min Injection Volume 25 uLUV Detection Wavelength 215 nm

[0113] After one month, i.e., the samples extracted during the 1-month, 2-month and 3-month incubation periods were analyzed using the RP-HPLC analysis method (TM 0055 HPLC) shown in the table below.

[0114] Item Details Column Phenomenex Luna C18, 3 μm, 100A, 4.6 x 150, P / N: 00F-4114-E0 Buffer A 0.1% Perchloric Acid in Water Buffer B 100% Methanol Gradient 0% B to 9% B in 25 min Column Temperature 55 ℃ Flow Rate 1.0 mL / min Injection Volume 10 uLUV Detection Wavelength 215 nm

[0115] Chemical stability results at pH 4.5

[0116] Composition 3A was prepared at a peptide concentration of 10 mg / mL in a sodium acetate buffer at pH 4.5. The composition was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. The accelerated aging results indicated that the peptide decomposed over time and temperature in the liquid formulation. The purity (%) measurement results are shown in Table 7 below, and the short-term chemical stability results are shown in Figure 5.

[0117] Mon-20 ℃2-8 ℃25 ℃40 ℃60 ℃098.998.998.998.998.91 97.796.191.9 2 97.29381.9 2.898.898.196.1 398.898.195.5

[0118] The shelf life of Composition 3A was estimated to be 2.4 years at 2-8°C and over 10 years when stored frozen at -20°C. This estimate was calculated using the Q10 rule and experimental aging results at 25°C, allowing for 8% degradation over the shelf life. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 8 below.

[0119] Temperature Experimental decomposition rate (% / month) Q10 factor value Calculated decomposition rate (% / month) Calculated shelf life (months) Calculated shelf life (years) -20 ℃ -0.010 16 -0.06 9 11 6 9.72 -8 ℃ -0.274 -0.28 29.12 4 25 ℃ -1.11 -1.17 30.640 ℃ -2.9 -2.9 2.8 0.260 ℃ -8.2 -8.2 10.1

[0120] Chemical stability results at pH=5.5

[0121] Composition 3B was prepared at a peptide concentration of 10 mg / mL in a sodium acetate buffer at pH 5.5. It was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. The accelerated aging results indicated that the peptide decomposed in a time- and temperature-dependent manner in the liquid formulation. The purity (%) measurement results are shown in Table 9 below, and the short-term chemical stability results are shown in Figure 6.

[0122] Mon-20 ℃2-8 ℃25 ℃40 ℃60 ℃098.998.998.998.998.91 97.195.188.82 94.59078.82.898.89893.3 398.997.992.9

[0123] The shelf life of Composition 3B was estimated to be 1.3 years at 2-8°C and more than 5 years when stored frozen at -20°C. This estimate was calculated using the Q10 rule and experimental aging results at 25°C, allowing for 8% degradation over the shelf life. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 10 below.

[0124] Temperature Experimental Decomposition Rate (% / month) Q10 Factor Calculated Decomposition Rate (% / month) Calculated Shelf Life (months) Calculated Shelf Life (years) -20 ℃ -0.01 216 -0.12 56 4.0 5.3 32 -8 ℃ -0.3 14 -0.5 0 16 0 1 3 3 2 5 ℃ -2.0 1 -2.0 4.0 0.3 3 40 ℃ -4.3 -4.3 1.9 0.1 6 60 ℃ -10 -10.0 0.8 0.07

[0125] Chemical stability results at pH=6.0

[0126] Composition 3C was prepared at a concentration of 10 mg / mL in a sodium acetate buffer solution at pH 6.0. The composition was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. The accelerated aging results indicated that the peptide decomposed in a time- and temperature-dependent manner in the liquid formulation. The purity (%) measurement results are shown in Table 11 below, and the short-term chemical stability results are shown in Figure 7.

[0127] Mon-20℃2-8℃25℃40℃60℃098.8798.8798.8798.8798.8798.871 96.0988.7481.382 92.12 64.482.898.8297.4489.12 398.8597.3788.68

[0128] The shelf life of Composition 3C was estimated to be 0.8 years at 2-8°C and more than 3 years when stored frozen at -20°C. This estimate was calculated using the Q10 rule and experimental aging results at 2-8°C, allowing for 8% degradation over the shelf life. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 12 below.

[0129] Temperature Experimental Decomposition Rate (% / month)Q10 FactorCalculated Decomposition Rate (% / month)Calculated Shelf Life (months)Calculated Shelf Life (years)-20 ℃-0.01216-0.213383.12-8 ℃-0.54-0.8590.825 ℃-3.41-3.420.240 ℃-10 -10.010.160 ℃-17 -17.000.0

[0130] Chemical stability results at pH=7.5

[0131] Composition 3D was prepared at a peptide concentration of 10 mg / mL in phosphate-buffered saline (pH 7.5). The composition was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. Accelerated aging results indicated that the peptide degraded in a time- and temperature-dependent manner in the liquid formulation. The purity (%) measurements are shown in Table 13 below, and the short-term chemical stability results are shown in Figure 8.

[0132] Mon-20 ℃2-8 ℃25 ℃40 ℃60 ℃098.8798.8798.8798.8798.871 38.972 2.898.7519.12 398.7615.775.73

[0133] The shelf life of Composition 3D was estimated to be 0.02 years at 2-8°C based on real-time measurements, and more than several years when stored frozen at -20°C. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 14 below.

[0134] Temperature Experimental decomposition rate (% / month) Q10 factor value Calculated decomposition rate (% / month) Calculated shelf life (months) Calculated shelf life (years) -20 ℃ -0.0404 -7.025 1.10.09 2 -8 ℃ -28.01 -28.10 0.30.02 25 ℃ -31.060 ℃ -60

[0135] Chemical stability results at pH=8.0

[0136] Composition 3E was prepared at a peptide concentration of 10 mg / mL in a 50 mM Tris-HCl solution at pH 8.0. The composition was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. The accelerated aging results indicated that the peptide decomposed with time and temperature in the liquid formulation. The purity (%) measurement results are shown in Table 15 below, and the short-term chemical stability results are shown in Figure 9.

[0137] Mon-20 ℃2-8 ℃25 ℃40 ℃60 ℃098.998.998.998.998.91 96.989.488.8 2 96.1 60.4 2.898.895.1 398.794.9

[0138] The shelf life of Composition 3E was estimated to be 0.5 years at 2 to 8°C and approximately 2 years when stored frozen at -20°C. This estimate was calculated using the Q10 rule and experimental aging results at 2 to 8°C, allowing for 8% degradation over the shelf life. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 16 below.

[0139] Temperature Experimental Decomposition Rate (% / month) Q10 Factor Calculated Decomposition Rate (% / month) Calculated Shelf Life (months) Calculated Shelf Life (years) -20 ℃ -0.04 44 -0.35 0 23 1.9 2 -8 ℃ -1.4 1 -1.4 0 5.7 0.5 25 ℃ -9.5 -9.5 0.8 0.140 ℃ -17 -17.0 0.5 0.06 0 ℃

[0140] Chemical stability results at pH=9.5

[0141] Composition 3F was prepared at a peptide concentration of 10 mg / mL in a 50 mM Tris-HCl solution at pH 9.5. The composition was incubated at -20°C, 2-8°C, 25°C, 40°C, and 60°C. Samples were collected and analyzed at baseline, 1 month, 2 months, 2.8 months, and 3 months. The accelerated aging results indicated that the peptide decomposed over time and temperature in the liquid formulation. The purity (%) measurement results are shown in Table 17 below, and the short-term chemical stability results are shown in Figure 10.

[0142] Mon-20℃2-8℃25℃40℃60℃098.998.998.998.998.998.9198.78463.927.6 298.581.547.318.4 2.898.780.739.8 398.680.138.8

[0143] The shelf life of Composition 3F was estimated to be 0.1 year at 2 to 8°C and approximately 0.4 years when stored frozen at -20°C. These estimates were calculated using the Q10 rule and experimental aging results at 2 to 8°C. The shelf life estimates based on the purity degradation rate of the peptide formulation are shown in Table 18 below.

[0144] Temperature Experimental Decomposition Rate (% / month)Q10 Factor Calculated Decomposition Rate (% / month) Calculated Shelf Life (months) Calculated Shelf Life (years) -20 ℃ -0.14 -1.80 04.40 37 2 -8 ℃ -7.21 -7.20 1.10 09 25 ℃ -22 -22.0 0.40 340 ℃ -47 -47.0 0.20 160 ℃

[0145] Experimental results showed that the initial degradation rate of the peptide measured at 60℃ and 40℃ was most stable in a sodium acetate buffer solution at pH approximately 4.5, and most unstable in a Tris-HCl buffer solution at pH 9.5. The tendency of increasing stability under acidic pH conditions was also observed at 25℃ and low temperatures of 2 to 8℃. The degradation rates at pH approximately 7.0 and pH approximately 7.5 were significantly higher than expected. The experimental results are shown in Fig. 11. It was confirmed that the degradation rate tended to increase as the pH of the composition increased. The experimental results confirmed that the most stable pH condition for the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in a liquid formulation was pH 4.5. Under this pH condition, the degradation rate at low temperatures, for example, the degradation rate at a refrigerated temperature of 2 to 8℃, which is estimated based on accelerated aging at high temperatures of 25℃ and 40℃, can be well predicted using the Q10 rule or the Arrhenius relationship. The real-time degradation rate at refrigerated temperatures of 2 to 8 °C was measured by incubation for up to 3 months, and the shelf life of the drug product was estimated under these storage conditions. The shelf life was accepted as the conditions of less than 8% degradation and ≥98% purity at the time of release. Although the degradation rate is an inherent characteristic of the drug product, the shelf life is affected by the initial purity of the drug product at release and the purity release specification. Assuming 8% degradation in a pH 4.5, sodium acetate environment in the liquid dosage form, and ≥98% purity at release and ≥90% purity specification, the estimated shelf life was a maximum of 2.4 years when refrigerated at 2 to 8 °C and a minimum of 10 years when stored frozen at -20 °C.

[0146] Experimental results showed that the peptide containing the amino acid sequence represented by SEQ ID NO: 1 in a liquid formulation in sodium acetate at pH 4.5 was the most stable, resulting in the longest calculated shelf life, which was estimated to be 2.4 years when refrigerated at 2 to 8°C and more than 10 years when frozen at -20°C. These estimates are based on the Q10 rule or Arrhenius relationship under pH conditions and accelerated aging tests at 25°C and 40°C. This allows for estimating the shelf life of a drug product under the given storage conditions. This value allows for an 8% decrease in purity during the shelf life.

[0147] Additionally, all 1 mg / mL peptide formulations containing the amino acid sequence represented by SEQ ID NO: 1 exhibited satisfactory physical stability by remaining clear and colorless before and after freeze-thaw cycles at all time points, pH, and temperature conditions.

[0148] From the above experiment, it was confirmed that the peptide can maintain stable properties even at low temperatures, and the recommended storage pH of the peptide liquid formulation was confirmed to be optimal at 4.5 or lower.

[0149] Table 19 below shows the estimated shelf life of a peptide-containing liquid composition calculated using the above-described method under various pH and temperature conditions.

[0150] pHTemperatureExperimental Decomposition Rate (% / month)Expiration Date (months)Expiration Date (years)Standard Conditions4.5-20 ℃0.071169.725 ℃4.52-8 ℃0.28292.425 ℃5.5-20 ℃0.13645.325 ℃5.52-8 ℃0.5161.325 ℃6-20 ℃0.43383.225 ℃62-8 ℃1.72100.825 ℃8-20 ℃0.35231.92-8 ℃82-8 ℃1.460.52-8 ℃9.5-20 ℃1.8040.42-8 ℃9.52-8 ℃7.210.12-8 ℃

[0151] A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 exhibited improved stability as the pH condition decreased. Accordingly, the short-term stability of peptide-containing compositions was measured at various temperature conditions at pHs ranging from 2.5 to 4.0 using sodium acetate and sodium citrate buffers. Additional tests were conducted in the same manner at various temperature conditions at pHs ranging from 2.5 to 4.0, and based on these, estimated shelf life values ​​were derived. The results are presented in Table 20 below.

[0152] Composition pH Experimental Decomposition Rate (% / month) Q10 Factor Expiration Date (months) Expiration Date (years) Storage Conditions 3N2.5-0.046 256 86 7.2-20 ℃ 3N2.5-0.264 221.82-8 ℃ 3N2.5-0.71 65.40.424 ℃ 3M3.0-0.012 256 329 27.4-20 ℃ 3M3.0-0.056 48 26.92-8 ℃ 3M3.0-0.216 211.724 ℃ 3L3.5-0.007 256 59549.6-20 ℃ 3L3.5-0.036 4149 12.42-8 ℃ 3L3.5-0.116 373.124 ℃3K4.0-0.00725653944.9-20 ℃3K4.0-0.036413511.22-8 ℃3K4.0-0.116342.824 ℃3J2.5-0.1---24 ℃3I3.0-0.1---24 ℃3H3.5-0.1---24℃3G4.0-0.1---24℃

[0153] The short-term chemical stability results by measuring the purity (%) at various temperature conditions of peptide-containing compositions of pH 2.5, pH 3.0, pH 3.5, and pH 4.0 are shown in Figs. 12 to 15, respectively. In addition, the degradation rate (%) of peptides for treating dentin hypersensitivity according to pH in the range of pH 2.5 to 9.5 is integrated and shown in Fig. 16.

[0154] <Example 4>

[0155] Since a tendency for stability to improve as pH conditions were lowered in Example 3 was observed, the stability of the liquid composition of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 at low pH was further confirmed.

[0156] According to the Ministry of Food and Drug Safety Notice No. 2019-132, "Stability Test Standards for Pharmaceuticals, etc.", the stability of a peptide composition containing the amino acid sequence represented by SEQ ID NO. 1 was evaluated for 4 weeks under refrigerated storage conditions, room temperature storage conditions, and accelerated storage conditions. The storage conditions were 40±2℃ and 75±5% relative humidity for the accelerated storage test, 25±2℃ and 60±5% relative humidity for the room temperature storage test, and 5±3℃ for the refrigerated storage test. To this end, a liquid composition containing the peptide was prepared by mixing it according to the prescription shown in Table 21 below.

[0157] This approximately 1g, the main ingredient, peptide (KYKQKRRSYK), 0.50 milligrams, viscosity regulator, glycerin 300.0 milligrams, sweetener, hydroxyethyl cellulose 15.0 milligrams, pH regulator, xylitol 30.0 milligrams, preservative, acetic anhydride 11.6 milligrams, solvent, methyl paraoxybenzoate 0.02 milligrams, solvent, ethanol, appropriate amount, purified water appropriate amount

[0158] For the stability evaluation, the appearance, identification test, pH, purity, and content were confirmed. The evaluation criteria were as follows: the appearance should be a colorless and transparent color maintained; the retention period of the main peak in the sample solution and the standard solution should be the same when tested according to the HPLC content test method; the pH range should be maintained in the range of 2.0 to 4.0; the viscosity range should be maintained in the range of 5,800 to 7,100 cP; each unknown reactive substance should be 1.0% or less, and the total reactive substance should be 3.0% or less; and the peptide content should be maintained in the range of 90.0 to 110.0%. The stability criteria were satisfied when the conditions were satisfied. The appearance was observed with the naked eye. The pH was measured according to the pH measurement method in the General Test Methods of the Korean Pharmacopoeia. The viscosity was tested according to the rotational viscometer method in the Viscosity Measurement Methods of the General Test Methods of the Korean Pharmacopoeia.

[0159] Purity evaluation was performed in the following manner.

[0160] About 10.0 mg of the peptide with sequence number 1 of CS (Chemical Standard) grade was precisely weighed as a standard and placed in a 100 mL volumetric flask. The diluent used was a mixture of trifluoroacetic acid, water, and acetonitrile in a ratio of 1:900:100 (v / v). This diluent was added, shaken ultrasonically, and filled to the mark on the flask with the diluent. The solution filtered through a 1 μm glass fiber filter was used as a system suitability solution. About 10 g (equivalent to 5.0 mg of peptide) of the prepared peptide-containing liquid composition was placed in a 50 mL volumetric flask, the same diluent was added, shaken and sonicated, cooled to room temperature, filled to the mark with the diluent, and the solution filtered through a 1 μm glass fiber filter was used as a test solution. Approximately 10 g of additives excluding the main ingredient were taken and processed in the same manner, and the filtered solution was used as the additive solution. The blank test solution, system suitability solution, additive solution, and test solution were tested using the liquid chromatography method in the general test methods of the Korean Pharmacopoeia according to the equipment operating conditions of the content test. However, the peaks detected in the blank test solution and additive solution were excluded from the calculation formula. When the standard solution is injected 6 times for system suitability, the relative standard deviation of the peptide peak area must be 2.0% or less.

[0161] The amount (%) of each unknown flexible substance is calculated by dividing the peak area (A) of each flexible substance in the test solution by the T ) and the purity (P) (%) of the CS grade peptide standard used was multiplied to obtain the peak area (A) of the peptide in the test solution. S ) was divided into . The total amount of flexible substances (%) was calculated as the sum of the amounts of flexible substances in each individual ant.

[0162] Peptide content evaluation was performed as follows. Approximately 10.0 mg of CS grade peptide standard was precisely weighed and placed in a 100 mL volumetric flask, the same diluent used in the purity test was added, the solution was shaken ultrasonically, and the solution was filled to the mark with the same diluent. The solution was then filtered through a 1 μm glass fiber filter and used as the standard solution. Approximately 10 g (equivalent to 5.0 mg of peptide) of the liquid composition of the peptide prepared above (equivalent to 5.0 mg of peptide) was placed in a 50 mL volumetric flask, the diluent was added, the solution was shaken, and then ultrasonically treated. After treatment, it was cooled to room temperature, filled to the mark with the diluent, and the solution filtered through a 1 μm glass fiber filter was used as the test solution. The instrument operating conditions are as follows: an ultraviolet spectrophotometer is used, the measurement wavelength is 215 nm, the column used is Poroshell Bonus-RP or an equivalent column, the column temperature is 13 ℃, the sample temperature is 5 ℃, the flow rate is 0.65 mL / min, and the injection volume is 100 μL. The mobile phase A was prepared by adding 3 mL of trifluoroacetic acid to purified water and making it 1,000 mL, and the mobile phase B was prepared by adding 3 mL of trifluoroacetic acid to acetonitrile and making it 1,000 mL. The ratio of the mobile phases was adjusted over time. For system suitability, when the standard solution is injected 6 times, the relative standard deviation of the peptide peak area containing the amino acid sequence represented by SEQ ID NO: 1 must be 2.0% or less. The test solution and standard solution were analyzed according to the liquid chromatography method in the general test methods of the Korean Pharmacopoeia under the above instrument operating conditions.

[0163] The peptide content for treating dentin hypersensitivity was calculated according to the following formula: Peptide content (%) = (AT × WS × P) / (AS × WT × 2 × D), where AT is the peak area of ​​the peptide in the test solution, AS is the average of the peak areas of the peptide in the standard solution, WS is the amount of the peptide standard sampled (mg), WT is the amount of the sample sampled (g), D is the indicated amount of peptide in about 1 g (mg / g), and P is the purity of the standard sample (%).

[0164] The results of the 4-week stability test under refrigerated storage conditions are shown in Table 22 below.

[0165] Test Item Initial 2 weeks 4 weeks Appearance Colorless transparent gel Colorless transparent gel Colorless transparent gel Identification Test The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same pH 3.0 2.9 2.9 Purity test (unknown related matter) 0.2% 0.2% 0.2% Purity test (total related matter) 0.2% 0.6% 0.8% Content test 97.7% 98.2% 98.5%

[0166] The results of the 4-week stability test under room temperature storage conditions are shown in Table 23 below.

[0167] Test Item Initial 2 weeks 4 weeks Appearance Colorless transparent gel Colorless transparent gel Colorless transparent gel Identification Test The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same pH 3.0 2.9 3.0 Purity test (unknown related matter) 0.2% 0.4% 0.5% Purity test (total related matter) 0.2% 1.1% 1.5% Content test 97.7% 97.7% 97.8%

[0168] The results of the 4-week stability test under accelerated test conditions are shown in Table 24 below.

[0169] Test Item Initial 2 weeks 4 weeks Appearance Colorless transparent gel Colorless transparent gel Colorless transparent gel Identification Test The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same The retention time of the main peak obtained from the test solution and the standard solution is the same pH 3.0 3.0 3.0 Purity Test (Unknown Related Matter) 0.2% 1.0% 1.8% Purity Test (Total Related Matter) 0.2% 2.8% 4.3% Content Test 97.7% 94.7% 93.3%

[0170] The test results showed that the lower the storage temperature, the more stable it was. Under refrigerated and room temperature storage conditions, the product met the standards for appearance, identification, pH, purity, and content for four weeks. Meanwhile, under accelerated testing conditions, the amount of flexible substances increased beyond the standard at relative retention times (RRTs) of 0.65, 0.98, 1.05, and 1.12, while the content showed a tendency to decrease somewhat. Therefore, the purity and content were re-checked after three months under refrigerated and room temperature storage conditions. The results are shown in Table 25 below.

[0171] Refrigerated condition (5±3℃) Room temperature storage condition (25±2℃) Purity 1.1% 2.9% Content 98 / 2% 97.2%

[0172] <Example 5> Peptide composition additionally containing a preservative

[0173] To prepare a liquid composition containing a peptide having an amino acid sequence represented by SEQ ID NO: 1 containing a preservative, a buffer containing a preservative was first prepared. Then, the peptide having an amino acid sequence represented by SEQ ID NO: 1 was added to prepare a volume of approximately 90% of the desired volume. The types of preservatives and buffers used are shown in Table 26 below. Here, a pH adjuster was added to adjust the pH to the desired level, and then the buffer was added again to obtain a formulation having a concentration of 0.2 mg / mL and pH 4.0. In this manner, the following compositions 5A to 5D were prepared.

[0174] No. Composition pH Final Volume 5A 0.2 mg / mL Peptide 80 mM Acetate Buffer 0.5% Sodium Benzoate 4.05 mL 5B 0.2 mg / mL Peptide 60 mM Acetate Buffer 0.2% Potassium Sorbate 4.05 mL 5C 0.2 mg / mL Peptide 10 mM Acetate Buffer 10 w / v% Ethanol 0.05 w / v% Methyl Paraben 0.0025 w / v% Propyl Paraben 4.05 mL 5D 0.2 mg / mL Peptide 10 mM Acetate Buffer, 0.2% Methyl Paraben 4.05 mL

[0175] As shown in Table 26 above, liquid compositions 5A, 5B, 5C, and 5D containing peptides having an amino acid sequence represented by SEQ ID NO: 1 were subjected to an 8-week stability test at 5°C and 25°C according to changes in peptide concentration and purity. The results are shown in FIGS. 17 and 18 . As shown in FIGS. 17 and 18 , it was confirmed that compositions 5A, 5B, and 5D were stable for more than 2 months at 5°C and 25°C, maintaining the peptide concentration. On the other hand, composition 5C showed a decrease in peptide concentration over time at both 5°C and 25°C. In addition, the results of measuring the change in purity of compositions 5A to 5D using an HPLC analysis method are shown in FIGS. 19 to 22 , respectively. To minimize interference from preservatives, chromatograms were integrated to determine the purity of the peptide having the amino acid sequence of SEQ ID NO: 1. For Composition 5C, two peaks were observed, with only the smaller peak matching the peptide's RT (Retention Time) (Fig. 21). Meanwhile, the peptide stability of Compositions 5A, 5B, and 5D was not affected by the presence of preservatives (Figs. 19, 20, and 22).

[0176] From the test results, it can be seen that a benzoate-based preservative or a paraben-based preservative may be preferable, as the preservative peak did not co-elute with the peptide peak or the peptide-related degradation impurities containing the amino acid sequence represented by the main sequence number 1.

[0177] Although the embodiments and examples of the present specification have been described with reference to the attached drawings, the present specification is not limited to the embodiments and examples described above, and can be manufactured in various different forms. Those skilled in the art to which the present specification pertains will understand that the present specification can be implemented in other specific forms without changing the technical spirit or essential characteristics of the present specification. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A peptide comprising the amino acid sequence KYKQKRRSYK represented by sequence number 1; and Contains buffers A liquid composition containing a peptide having a pH in the range of 2.5 to 5.

0.

2. A peptide-containing liquid composition according to claim 1, characterized in that the pH is in the range of 2.5 or more and 4.5 or less.

3. A peptide-containing liquid composition further comprising a pH adjuster according to claim 1.

4. A peptide-containing liquid composition in the second paragraph, wherein the pH adjusting agent is selected from the group consisting of acetic acid, sodium acetate, citric acid, sodium citrate, sulfuric acid, hydrochloric acid, tartaric acid, boric acid, or a combination thereof.

5. A liquid composition containing a peptide, wherein the concentration of the peptide including the amino acid sequence represented by sequence number 1 in the first paragraph is in the range of 0.2 to 20 mg / mL.

6. A peptide-containing liquid composition in the fifth paragraph, wherein the concentration of the peptide including the amino acid sequence represented by the sequence number 1 is in the range of 0.2 to 10 mg / mL.

7. A liquid composition containing a peptide, which maintains a purity of 95% or more when stored at room temperature for 3 months when analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia in paragraph 1.

8. A liquid composition containing a peptide, which maintains a purity of 97% or more when stored at 2 to 8°C for 3 months when analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia in paragraph 1.

9. A liquid composition containing a peptide in which, when analyzed according to the liquid chromatography method among the general test methods of the Korean Pharmacopoeia, less than 3% of flexible substances are detected when stored at room temperature for 4 weeks in accordance with paragraph 1.

10. A liquid composition containing a peptide, wherein the content of the peptide including the amino acid sequence represented by sequence number 1 is maintained at 95% or more based on the content before storage when stored at room temperature for 3 months in accordance with paragraph 1.

11. A peptide-containing liquid composition further comprising a preservative according to claim 1.

12. A peptide-containing liquid composition according to claim 11, wherein the preservative is selected from a benzoate-based preservative, a sorbate-based preservative, or a paraben-based preservative.

13. In the first paragraph, the peptide-containing liquid composition is a formulation selected from among solutions, gels, elixirs, suspensions, emulsions, lemonades, fragrances, emulsions and infusions, alcohols, tinctures, syrups, gargles, sprays, semi-solids, injections, eye drops, eye ointments, ear drops, nasal drops, liniments, lotions, aerosols, ointments, creams, and pastes.

14. A peptide-containing liquid composition further comprising a solvating agent according to claim 1.

15. A peptide-containing liquid composition according to claim 1, further comprising at least one selected from a wetting agent, a viscosity agent, a surfactant, a carrier, or a diluent.

16. A peptide-containing liquid composition further comprising a fragrance according to claim 1.

17. A quasi-drug comprising a liquid composition containing a peptide according to Article 1.

18. A pharmaceutical composition for improving or treating tooth damage, comprising a liquid composition containing a peptide according to Article 1 as an active ingredient.

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