ORAL RINSE COMPOSITIONS FOR ALLEVIATING XEROSTOMIA COMPRISING POLYETHYLENE GLYCOL DERIVATIVES

Abstract

An oral rinse composition for alleviating xerostomia includes a polyethylene glycol derivative as an active ingredient, which allows not only covalent bonding of the composition to the oral mucosa without irritation to increase the oral moisturizing capacity, but also allows easy manufacture, storage, use and packaging by having a granule formulation.

Claims

1. An oral rinse composition for alleviating xerostomia, the composition comprises a polyethylene glycol derivative of the following Chemical Formula (1) as an active ingredient, an acidity adjusting agent and a flavoring agent, the polyethylene glycol derivative has a molecular weight of 8,000 to 15,000 Da, the composition is in the form of a granule formulation: ##STR00005##

2. The oral rinse composition of claim 1, wherein the granules have a particle size of 200 to 1500 μm.

3. The oral rinse composition of claim 1, wherein the composition is dissolved in water and reconstituted in the form of a solution for use.

4. The oral rinse composition of claim 1, wherein the acidity adjusting agent is comprised in an amount of 1 to 10 parts by weight based on 100 parts by weight of the composition.

5. The oral rinse composition of claim 3, wherein the composition exhibits a pH of 7 to 8 when dissolved in water and reconstituted in a solution form for use.

6. The oral rinse composition of claim 3, wherein the solution exhibits a viscosity of 0.001 to 0.01 Pa.Math.s.

7. A method of alleviating xerostomia, comprising applying the oral rinse composition for alleviating xerostomia of claim 1 to a subject in need thereof.

8. The method of claim 7, wherein the application comprises administering and rinsing an oral rinse composition for alleviating xerostomia reconstituted in solution form.

9. The method of claim 7, wherein the application is carried out 1 to 3 times a day.

10. A method of preparing an oral rinse composition for alleviating xerostomia of claim 1, comprising: preparing a polyethylene glycol derivative powder of Chemical Formula 1 having a molecular weight of 8,000 to 15,000 Da; mixing an acidity adjusting agent and a flavoring agent with the polyethylene glycol derivative powder, then transferring it to an airtight container and liquefying it at a high temperature; when the liquefaction is completed, transferring the airtight container to a low temperature to solidify; and when the solidification is completed, pulverizing to prepare granules; ##STR00006##

11. The method of claim 10, comprising adding an acidity adjusting agent to bring the pH to 7 to 8.

12. The method of claim 10, wherein the liquefying is carried out at 50 to 70° C. for 1 hour.

13. The method of claim 10, wherein the solidifying comprises: primary hardening for 1 hour at room temperature; and secondary hardening at −20° C.

14. A product for relieving xerostomia, comprising a container capable of measuring 15 to 30 ml of water per use and a packet packaged with an oral rinse composition for alleviating xerostomia of 1 to 1.5 g of the granule formulation of claim 1 per use.

15. The product of claim 14, wherein the amount of water per use is 20 ml, and the oral rinse composition per use is 1 g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIGS. 1A and 1B show the results of nuclear magnetic resonance (NMR) analysis of the powder formulation and the granule formulation of the oral rinse composition for alleviating xerostomia according to the present invention.

[0048] FIG. 2 is a comparison result of surface static electricity according to the formulation of the oral rinse composition for alleviating xerostomia according to the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0049] Hereinafter, the present invention will be described in detail by way of Examples. The following Examples merely illustrate the present invention and do not limit the scope of the present invention.

Experimental Example 1. Moisture Absorption Confirmation Test of PEG Derivatives for Oral Moisture Absorption

[0050] In order to identify and determine the PEG-OH structure having high oral moisture absorption, the moisture absorption rates of PEG derivatives having different structures were tested.

[0051] Test materials: As a PEG derivative for the moisture absorption test, a dry powder having a molecular weight of 3,000 Da or more rather than liquid (wax) PEG was used, and the test was carried out using powders of a linear structure of PEG (Chemical Formula A) having molecular weights of 3,400 Da and 10,000 Da, and a branched structure of 4arm PEG (Chemical Formula B) and 6arm PEG (Chemical Formula C) having molecular weights of 10,000 Da as described below.

##STR00002##

[0052] The test materials were purchased from Daechang Chemical, and for the test, the powder particles were uniformly pretreated by extraction and powdering processes using an organic solvent, and then the test was carried out.

[0053] Method for Measuring Moisture Absorption: The moisture absorption was confirmed by the weight of water vapor absorbed per mass of polyethylene glycol (PEG). After pretreatment by drying PEG in a desiccator at a temperature of 25±1° C., it was left for 24 hours under a condition of 60±2% relative humidity at a temperature of 25±1° C., which is a moisture absorption condition, and the weight increase (mg) per 1 g of PEG was defined as the moisture absorption of the PEG. The moisture absorption of each PEG was determined by comparing the weight difference before and after leaving the moisture absorption condition to be calculated as a percentage.

[0054] The results are shown in Table 2 below.

TABLE-US-00001 TABLE 1 Moisture absorption (%) Molecular Molecular Numerical Structure Weight (Da) Value(%) Mean(%) Comparative Di-PEG-OH; 3,400 10.00 10.14 Example 1 P2OH 10.74 9.66 Comparative Di-PEG-OH; 10,000 13.65 12.31 Example 2 P2OH 11.99 11.29 Example 1 4-arm PEG-OH; 10,000 16.46 15.48 P4OH 13.85 16.14 Comparative 6-arm PEG-OH; 10,000 14.09 12.59 Example 3 P6OH 11.95 11.72

[0055] As shown above, it was confirmed that the linear Di-PEG-OH having a molecular weight of 3,400 Da (Comparative Example 1) showed the lowest moisture absorption rate of 10.14%, and 10,000 Da (Comparative Example 2) showed a rate of 12.31%, and the branched 4-arm PEG-OH having a molecular weight of 10,000 Da (Example 1) showed the highest moisture absorption rate of 15.48%.

[0056] As a result of confirming the moisture absorption of PEG-OH of linear and branched structures, the higher the molecular weight of the linear PEG-OH of the solid powder, the higher the moisture absorption (that is, the moisture absorption of the molecular weight of 10,000 Da (Comparative Example 2) is higher than that of 3,400 Da (Comparative Example 1)), and the moisture absorption of the branched PEG was higher when comparing the moisture absorption between the linear and branched PEG structures based on the same molecular weight of 10,000 Da. In particular, compared to the 6-arm PEG-OH (Comparative Example 3), the 4-arm PEG-OH (Example 1) structure exhibited the highest moisture absorption, so the 4 arm-PEG-OH (Example 1) was selected as the main ingredient of the present invention.

[0057] In addition to the branched 4arm-PEG-OH, 6arm-PEG-OH and 8arm-PEG-OH can be used for oral moisturizing, but no superiority to 4arm-PEG-OH could be confirmed in the synthesis/manufacturing process and moisturizing effect for PEG derivatives.

Experimental Example 2. Moisture Content Confirmation Test by 4Arm-Peg-OH Molecular Weight for Oral Moisture Absorption

[0058] According to the results of Experimental Example 1, it was confirmed that the branched 4arm-PEG-OH structure had high moisture absorption, and in order to confirm the moisture absorption according to the molecular weight of the same structure, a moisture content confirmation test according to 4arm PEG-OH of 10,000 Da, 20,000 Da, and 30,000 Da molecular weights was carried out. The test method was carried out by exposing 4arm-PEG-OH test materials for each molecular weight to a temperature of 25±1° C. and a relative humidity of 60±2% under moisture absorption conditions for 4 hours, and then analyzing the moisture content contained in each test material using a moisture meter (MS-70; METTLER TOLEDO, 400 W straight halogen lamp heating system with SRA filter and SHS weighting technology). That is, moisture absorption was confirmed using the difference in moisture content before and after exposure to moisture absorption conditions.

[0059] As shown in the table below, the moisture content and moisture absorption were measured according to the 4arm-PEG-OH structure having the molecular weights of 10,000 Da, 20,000 Da, and 30,000 Da. 4arm-PEG-OH PEG derivatives with a molecular weight of 5,000 Da or less (for example, 2,000 Da and 5,000 Da) were excluded from this test material because powdering was not easy in the production/synthesis process. 4arm-PEG-OH of each molecular weight was purchased from Daechang Chemical, and for the test, the powder particles were uniformly pretreated by extraction and powdering processes using an organic solvent, and then the test was carried out. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Moisture Content (%) Molecular Before After Molecular Weight exposure exposure A − Structure (Da) (B) (A) B AVR Example 1 4-arm 10,000 0.707 1.638 0.931 0.750 PEG-OH; 0.753 1.400 0.647 P4OH 0.807 1.480 0.673 Compar- 4-arm 20,000 0.783 1.398 0.615 0.558 ative PEG-OH; 0.724 1.368 0.644 Example 4 P4OH 0.764 1.180 0.416 Compar- 4-arm 30,000 0.708 0.857 0.149 0.136 ative PEG-OH; 0.691 0.811 0.12 Example 5 P4OH 0.695 0.834 0.139

[0060] As shown in the above results, the smaller the molecular weight, the greater the difference in moisture content before and after 4 hours of exposure at 60% relative humidity. That is, it was found that the higher the molecular weight of 4arm-PEG-OH, the lower the moisture absorption.

[0061] That is, 4arm-PEG-OH (Example 1) of 10,000 Da among molecular weights 10,000 Da, 20,000 Da, and 30,000 Da of 4arm-PEG-OH showed the greatest difference (A−B) in moisture content before and after exposure to 60% relative humidity and was found to absorb a lot of moisture.

[0062] As described above, a 4arm-PEG-OH molecular weight 2,000 Da or 5,000 Da was excluded due to the difficulty of the final powder manufacturing process for the PEG derivative and the resulting low production yield. In the overall reaction process for PEG derivatization, the crystallization and powder manufacturing process affect the production yield in the chemical reaction process and the crystallization and recrystallization process using an organic solvent after filtration/concentration. In addition, it is an essential consideration because the production yield is lowered in the washing/recrystallization process to remove impurities with an organic solvent and the powdering process of the final even particles after drying. According to the results of Experimental Example 2, of 4arm-PEG-OH molecular weights 20,000 Da and 30,000 Da were not suitable for oral moisturizing because the moisturizing effect according to the increase in molecular weight was low.

[0063] For this reason, (Example 1) a 4arm-PEG-OH structure having a molecular weight of 10,000 Da was used as a main raw material for the synthesis and preparation of the PEG derivative, which is the main ingredient for oral moisturizing of the present invention.

Experimental Example 3. Selection of PEG Derivative Reactive Group

[0064] In order to select a reactive group of a PEG derivative that forms a covalent bond with an amine group present in the oral mucosa, the half-life according to the reactive group of the PEG derivative and the number of production processes (number of steps) required for the synthesis of the reactive group were comparatively analyzed. When the half-life is short, the covalent bond with the amine group of the oral mucosa cannot occur because hydrolysis occurs quickly during dissolution of the composition in water. Therefore, it is difficult to use a reactive group with a short half-life for preparing an oral rinse composition for alleviating xerostomia.

[0065] The half-life was measured based on the UV absorbance of the N-hydroxy succinimide (NHS) group hydrolyzed in a buffer condition of 25° C. and pH 8. The results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Number of Production Reaction Half-life Processes Molecular Structure Reactive Group (minutes)* (Steps) Comparative PEG- Succinimidyl 33.6 4 Example 6 CH2CH2CH2CH2—CO2—NHS Valerate (SVA) Comparative PEG-O—CO2—NHS Succinimidyl 20.4 1 Example 7 Carbonate (SC) Example 2 PEG-O2C—CH2CH2CH2—CO2—NHS Succinimidyl 17.6 2 Glutarate (SG) Comparative PEG-O2C—CH2CH2—CO2—NHS Succinimidyl 9.8 2 Example 8 Succinate (SS) Comparative PEG-O—CH2—CO2—NHS Succinimidyl 0.75 3 Example 9 Carboxymethylated (SCM) Comparative PEG-O—CH2 CH2—CO2—NHS Succinimidyl 23.3 4 Example 10 Butanoate (SBA) Comparative PEG-O—CH2 CH2—CO2—NHS Succinimidyl 16.5 5 Example 11 Propionate (SPA)

[0066] As shown in the above results, succinimidyl carbonate (SC) ester (Comparative Example 7) had a simple production process of 1 step and a relatively long half-life, but there was risks and environmental risks of using phosgene gas in the manufacturing process. On the other hand, in the case of the derivative (Example 2) modified with succinimidyl glutarate (SG) according to the present invention, the amide reaction rate in oral application is fast, there are no reagents required before and after the reaction, and no by-products, and there is no problem with discomfort and safety in oral application. In addition, compared with other reactive groups, the half-life of Example 2 is relatively long, the number of production processes is small and simple, and the risk in the production process is low. Accordingly, a derivative (Chemical Formula 1) modified with succinimidyl glutarate (SG) was prepared through Preparation Example 1 below and used in subsequent experiments.

Preparation Example 1. Preparation of Polyethylene Glycol Derivatives of Chemical Formula 1

[0067] ##STR00003## ##STR00004##

[0068] After dissolving the compound of Chemical Formula 2 in methylene chloride at room temperature, triethylamine was added. After adding glutaric anhydride to the reaction solution, stirring was performed at room temperature for 20 to 24 hours. The reaction solution was washed with a 14% ammonium chloride aqueous solution, and when the layers were separated, the lower organic solution layer was collected. The aqueous solution layer was extracted using methylene chloride. The combined organic solution layer was precipitated in diethyl ether after moisture was removed using magnesium sulfate and the solvent was concentrated. The precipitate was filtered and dried under vacuum at room temperature for 24 hours to obtain a compound of Chemical Formula 3.

[0069] The compound of Chemical Formula 3 was dissolved in methylene chloride, and N-hydroxy succinimide (NHS) and dicyclohexyl carbodiimide (DCC) were added thereto. The reaction solution was stirred at room temperature for 15 to 20 hours. After the reaction, dicyclohexyl urea (DCU) as a by-product was filtered using a glass filter, and the filtered solution was precipitated in diethyl ether after concentrating the solvent. After filtering the precipitate, it was dissolved in ethyl acetate at 55±5° C. and recrystallized at 0-5° C. for 15-17 hours. The recrystallized product was filtered, washed 3 times with diethyl ether, and dried under vacuum at room temperature for 24 hours to obtain a compound (n=57) of Chemical Formula 1 having a weight average molecular weight of 10,000.

Experimental Example 4: Composition Test Using a pH Adjusting Agent

[0070] As a result of research by the present inventors, it was found that the conditions for forming a covalent bond between the amine group of the oral mucosa and the reactive group of the PEG-SG derivative of Chemical Formula 1 were pH 6.0 to 8.0.

[0071] However, when the PEG-SG derivative of Chemical Formula 1 used as a main ingredient for oral moisturizing and the flavoring agent were dissolved in water, weak acidity (pH4.5), which was not suitable for the above pH conditions, was exhibited.

[0072] In order to adjust the composition of the present invention to a pH condition suitable for covalent bonding in the oral cavity, an acidity adjusting agent (sodium bicarbonate) used as a food additive was added to determine the amount of the acidity adjusting agent so that the pH was 6.0 to 8.0″.

[0073] Test Materials: As shown in Table 4 below, the acidity adjusting agent (sodium bicarbonate) was prepared by adding 0 (control), 1, 5, and 10% of the product content (1.5 g). A pH test was carried out using three 1 g samples for each composition.

TABLE-US-00004 TABLE 4 Polyethylene Glycol Derivative Acidity of Chemical Flavoring Adjusting Formula 1 Agent Agent Total Comparative 93.3% 6.7% 0% 100% Example 12  (1.4 g) (0.1 g) (0.000 g) (1.5 g) Example 3 92.3% 6.7% 1% 100% (1.385 g) (0.1 g) (0.015 g) (1.5 g) Example 4 88.3% 6.7% 5% 100% (1.325 g) (0.1 g) (0.075 g) (1.5 g) Example 5 83.3% 6.7% 10%  100%  (1.25 g) (0.1 g) (0.150 g) (1.5 g)

[0074] Test Methods: To accurately measure the pH of the prepared samples, pH meter calibration was carried out in 3 sections (pH 4.0, 7.0, 10.0 standard buffer), and the allowable parameter pH gradient was 90 to 105% and 0±30 mV (0.5 at 25° C. pH units) were checked. After completely dissolving 1 g of each test material in 20 ml of distilled water, the pH was checked.

[0075] The results are shown in Table 5 below.

TABLE-US-00005 TABLE 5 pH Mean pH Comparative 4.5 4.4 4.5 4.5 Example 12 Example 3 7.0 7.1 7.0 7.0 Example 4 7.4 7.5 7.5 7.5 Example 5 7.8 7.8 7.9 7.8

[0076] As shown in the above results, the mean pH of each sample to which 1, 5, and 10% of the acidity adjusting agent was added was 7.0, 7.5, and 7.8. As a result, it was confirmed that 1 to 10% of the addition amount of the acidity adjusting agent was in an amount range suitable for covalent pH 6.0 to 8.0 conditions.

[0077] However, as a result of sensory evaluation, the composition in which the acidity adjusting agent was added in an amount of 10% or more (Example 5) was excluded because it caused a bitter taste and irritation to the tongue when applied orally. In subsequent experiments, a composition (Example 4) having an acidity adjusting agent addition amount of 5% was prepared and used according to Preparation Example 2 below.

Preparation Example 2. Preparation of Oral Rinse Composition for Alleviating Xerostomia

[0078] An oral rinse composition for alleviating xerostomia, including the polyethylene glycol derivative of Chemical Formula 1, an acidity adjusting agent and a flavoring agent, was prepared in various formulations, and cytotoxicity (human safety), human applicability such as moisturizing effect (functionality), and ease of handling in a commercialization process, product stability, etc. were evaluated through the following Experimental Examples.

Preparation Example 2-1. Preparation of Oral Rinse Composition for Alleviating Xerostomia in Powder Formation

[0079] Each of 89 parts by weight of a polyethylene glycol derivative, 5 parts by weight of sodium hydrogen carbonate as an acidity adjusting agent, and 6 parts by weight of mint as a flavoring agent was added to a V-type mixer, and mixed at 20 to rpm for 15 minutes to prepare a finished composition in powder form.

Preparation Example 2-2. Preparation of Oral Rinse Composition for Alleviating Xerostomia in Liquid Formation

[0080] 1.5 g of the powder composition of Preparation Example 2-1 was dissolved in 30 ml of water to prepare a liquid composition.

Preparation Example 2-3. Preparation of Oral Rinse Composition for Alleviating Xerostomia in Granular Formation

[0081] An airtight container accommodating the powder composition prepared in Preparation Example 2-1 was liquefied at 60° C. for 1 hour. When the liquefaction was completed, the airtight container was transferred to room temperature and primarily hardened for 1 hour, and then secondarily hardened at −20° C. to solidify. When the solidification was completed, the solidified product was pulverized to a particle size of 355 to 1400 μm.

Experimental Example 5. Cytotoxicity Test

[0082] The cytotoxicity test is a test to confirm the safety of the composition according to the present invention, and the presence and degree of a cytotoxic reaction in cultured cells (L-929) was evaluated. A good laboratory practice (GLP) institution was commissioned to perform the test according to each test method presented in ISO 10993.

[0083] Test Materials:

[0084] As shown in Table 6 below, each test solution was prepared using a powder formulation (A) without an acidity adjusting agent, a powder formulation (B, Preparation Example 2-1) containing an acidity adjusting agent, and a liquid formulation test material (C, Preparation Example 2-2) in which a powder formulation (B) was dissolved in 30 ml of water.

TABLE-US-00006 TABLE 6 Powder formulation (A) Powder Comparative formulation (B) Example 13 Comparative (Same as Example 14 Liquid Comparative (Same as formulation (C) Classification Example 12) Example 4) Example 6 Polyethylene 1.4 g 1.325 g 1.325 g Glycol Derivative (4arm-PEG-SG) Flavoring Agent 0.1 g  0.1 g  0.1 g Acidity — 0.075 g 0.075 g Adjusting Agent Total 1.5 g  1.5 g 1.5 g in 1 Oz(30 ml) DW

[0085] Cytotoxicity Test Method

[0086] 5-1. Test Method of Powder Sample: In the extract (E-MEM +5% FBS), the powder test material and the control were dissolved at a concentration of 0.2 g/ml, and dissolved for 24±2 hours to prepare a test solution. The culture medium of cell L-929 was removed from the plate well, replaced with 1 mL of test solution and control medium (extract solution), and then the cells were cultured. During culturing, microscopic examinations were carried out every 24, 48, 72±4 hours to evaluate cytotoxicity. Positive and negative controls were also used in the test.

[0087] 5-2. Test Method of Liquid Sample: A liquid test solution was prepared in which a dilution solution (2× E-MEM+10% FBS) and the liquid test material were combined/diluted in a 1:1 ratio. The culture medium of cell L-929 was removed from the plate well, replaced with 1 mL of test solution and control medium (diluent), and then the cells were cultured. During culturing, microscopic examinations were carried out every 24, 48, 72±4 hours to evaluate cytotoxicity. Cadmium chloride (100 uM CdCl2) as a positive control and E-MEM+5% FBS as a negative control were also used in the test.

[0088] As the evaluation criteria of the cytotoxicity test, a score of 0 to 4 was recorded by evaluating the morphological change of cells and the degree of change in viable cells according to lysis or separation. Scores 3 and 4 were judged to be cytotoxic, and scores of 1 and 2 were judged to be non-cytotoxic.

[0089] The results are shown in Tables 7 and 8 below.

TABLE-US-00007 TABLE 7 Cytotoxicity Score Comparative Comparative Example 13 Example 14 Example 6 Classification 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr Test Material 4/4/4 4/4/4 4/4/4 4/4/4 4/4/4 4/4/4 0/0/0 0/0/0 0/0/0 Positive Control 4/4/4 4/4/4 4/4/4 4/4/4 4/4/4 4/4/4 0/0/0 0/0/0 0/0/0 Negative Control 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 Cell Control 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0

[0090] As shown in the above results, as a result of confirming the cytotoxicity test for each composition and both formulations, for Comparative Examples 13 and 14, a cytotoxicity score of ‘4’ was recorded and strong cytotoxicity was exhibited, and Example 6 did not exhibit any cytotoxicity with a cytotoxicity score of ‘0’. Example 6 includes an acidity adjusting agent, and by dissolving the composition in 30 ml of water and applying it to the cells as a liquid, it was confirmed as a safe composition and formulation that did not affect the morphological change and survival of L-929 cells.

TABLE-US-00008 TABLE 8 Powder Powder Formulation (a) Formulation (b) Liquid Comparative Comparative Formulation (c) Classification Example 13 Example 14 Example 6 Amount of 1.5 g 1.5 g 0.05 g/ml test material (1.5 g/30 ml in DW) Cytotoxic Test Material Extraction Type Test Material sample Dilution Type* concentration 0.2 g/ml in 0.2 g/ml in 0.025 g/ml in media media media Cytotoxicity Cytotoxicity. Cytotoxicity. No Cytotoxicity. result

[0091] As shown in the above results, since the powder compositions (Comparative Examples 13 and 14) exhibit cytotoxicity, it is not suitable to directly apply the dry powder formulation to a xerostomia patient with a small amount of saliva secretion.

[0092] Therefore, after changing to a liquid formulation by adding water to the powder formulation of the present invention, no toxicity was observed as the concentration of the test material for the cytotoxicity test was reduced from 0.2 g/ml to 0.025 g/ml (1.5 g/30 ml×½). In addition, unlike applying a powder composition to the dry oral mucosa, which gives irritation to the sensitive mucous membrane, when the powder was reconstituted into a liquid formulation, moisture that the oral cavity lacked was additionally supplied, and the expected effect of increasing the moisturizing effect was exhibited by allowing the patient to easily apply the product in the oral cavity.

Experimental Example 6. Moisturizing Effect Confirmation Test According to Cell Viability

[0093] By applying the composition of the present invention to human epithelial cells, a moisturizing layer is formed by covalent bonding of the main ingredient PEG derivative to the cell surface, and an in vitro test was carried out to confirm cell viability by exposing the cells to a dry environment.

[0094] Test Materials:

[0095] Example 6 of the present invention, in which cytotoxicity safety was confirmed in Experimental Example 5, and Hydris Oral Rinse, a product for relieving xerostomia manufactured by Colgate, were used as test materials. Both test materials are liquids in the same formulation. As a negative control, PBS (phosphate-buffered saline; Gibco) was used.

TABLE-US-00009 TABLE 9 Hydris ™ Oral Rinse Example 6 Main Ingredient Glycerin, Polyethylene Glycol PEG Moisturizer Derivative (Main Ingredient) (4arm-PEG- SG) Other Sorbitol, Sodium Saccharin, None (Sweetener, Thickener, Sucralose/Cellulose Gum, Xanthan Surfactant, Gum, Carbomer/Poloxamer Preservative) 407/Cetylpyridinium Chloride, Sodium Benzoate Buffer Disodium Phosphate, Sodium Sodium Phosphate Bicarbonate Coloring Agent, FD&C Blue 1, Mint Mint Flavoring Agent

[0096] Test Method:

[0097] Using human pharynx epithelial cells, the composition of the present invention, Hydris™ Oral Rinse of Colgate, and phosphate-buffered saline (PBS) were applied to the cells at 37° C. for 15 minutes and cultured, then the test material was removed and washed with PBS, the cells were exposed to dry conditions of 30% humidity at 25° C., and cell viability was measured. The effect of the treated test materials to protect cells by reducing apoptosis due to drying was confirmed by cell viability. Cells were treated with PBS as a positive control, and the viability of cells not exposed to dry conditions was set to 1 (100%), and cell viability according to treatment with a test material was expressed as a percentage. Cell viability was analyzed using Cell Counting Kit 8 (CCK8; Dojindo Molecular Technologies, Inc., Rockville, Md., USA). This kit is used to quantify the number of living cells by generating orange formazan dye during bioreduction in the presence of electron carriers using a water-soluble tetrazolium salt.

[0098] The results are shown in Table 10 below.

TABLE-US-00010 TABLE 10 Non-Dry Condition (Positive Control) Dry Condition PBS PBS Hydris ™ Example 6 Classification (N = 8) (N = 16) (N = 8) (N = 8) Mean 100 11.6 12.0 71.9 Median 101.9 4.6 11.5 81.5 SD 7.34 17.06 3.38 47.26

[0099] As shown above, the mean value of the cell viability of Example 6 of the present invention based on the cell viability (%) of the positive control was 71.9% (median: 81.5%), showing the highest cell viability, and the Hydris product of GSK showed a relatively low viability with a mean value of 12.0% (median: 11.5%).

[0100] Because the moisturizing ingredient remained in the cells even after cell washing by covalent bonding of the main ingredient PEG derivative of Example 6 and oral cells to maintain the effect of the moisturizing coating, cells are washed and then exposed to a dry environment to minimize cell death due to drying to maintain cell viability. In other words, the composition including the existing moisturizer is washed off by talking or ingestion of drinks/food after oral application, so it has a limited role as a moisturizer, but the moisturizing effect is temporary, and therefore, it must be frequently applied intraorally. However, the main ingredient of the PEG derivative of the present invention is for maintaining a moisturizing effect for a long time without being washed away by physical stimulation by covalent bonding with cells.

Experimental Example 7. Reactive Group Activity and Electrostatic Test of the Main Ingredient 4Arm-Peg-Sg Derivative

[0101] Common PEG derivatives are prepared in the form of final powder (powder type) through chemical reaction, synthesis, a manufacturing process, and a recrystallization process after crystallization and washing. These powdered PEG derivatives are mixed with additives, and the recovered mixture is filled into the hopper of the equipment for fixed quantity (1 g) packet packaging, and then the powder is supplied to the scale (load cell) from the vibrating plate and the weight is measured, and the fixed quantity is filled in aluminum packets. However, in the case of a powder formulation such as Preparation Example 2-1, a loss due to static electricity occurs during the weighing, mixing and recovery of the mixture at the beginning of the production process, and for the same reason, it was found that the recovery rate was lowered.

[0102] Therefore, changes in reactive group activity and whether static electricity was generated were analyzed by changing the physical form of the granule formulation of Preparation Example 2-3 to solve the static electricity problem in the manufacturing process caused by the physical form, that is, powder, of 4arm-PEG-SG, which is the main ingredient of the PEG derivative of the present invention.

[0103] Test materials: As in the previous experimental examples, 4arm-PEG-SG, a PEG derivative in powder form, and a sample in the form of granules were prepared.

[0104] Test Method:

[0105] Prior to the electrostatic test, it was confirmed by nuclear magnetic resonance (NMR) analysis that there was no problem in the reactive group activity and stability of the PEG derivative even after the physical state change of melting and solidifying 4arm-PEG-SG in powder form. FIGS. 1A and 1B are the results of nuclear magnetic resonance (NMR) analysis of the powder formulation and the granule formulation of the oral rinse composition for alleviating xerostomia according to the present invention, confirming that there is no problem in the activity and stability of the reactive group regardless of the change in physical state.

[0106] The electrostatic test was measured using an electrostatic meter (EYE-02) capable of measuring the surface electrostatic force of the main ingredient PEG derivatives of the two formulations. 50 g and 100 g of each of the PEG derivatives, the main ingredient of the powders and granules, were placed in a PET storage pack (vinyl pack) used for weighing in the manufacturing process, and the surface electrostatic force was measured and compared.

[0107] As shown in FIG. 2, as a result of confirming and comparing the surface electrostatic force generated depending on the physical form, that is, powders and granules of the PEG derivative 4arm-PEG-SG, it was confirmed that the surface static electricity of the powder decreases by about 50% or more when the morphology changes into granules.

[0108] As a result, in the case of the granule formulation of Preparation Example 2-3, the loss in the hopper due to static electricity was reduced and packet filling was facilitated, unlike the powder formulation of Preparation Example 2-1.

Experimental Example 8. Stability Confirmation Test According to the Formulation of the Final Finished Composition

[0109] A test to confirm product stability according to the formulation was carried out as follows.

[0110] Test Materials: An oral rinse composition of the powder formulation of Preparation Example 2-1 (Comparative Example 15) and the granule formulation of Preparation Example 2-3 (Example 7) of the present invention was prepared, and 1 g of each was packaged in an aluminum packet.

[0111] Stability Test Method: 1 g of the finished composition in powder and granule form was filled in an aluminum packet (9 cm×2 cm), sealed with a heat adhesive, and stored in a thermo-hygrostat chamber with a relative humidity of 60%±5% at 25±2° C. Stability test analysis was performed on days 0, 1, 2, 3, 4, 5, 6, 7, 8 and 10 of storing the sample, 10 mg of the analyzed sample was weighed, put into an NMR tube and dissolved in 0.62 mL of CDCl.sub.3, and analyzed by 1H-NMR (400 MHz). The analysis method confirmed the reactive group activity of the main ingredient 4arm-PEG-SG derivative by the succinimidyl group peak of 2.80-2.90 ppm (parts per million; proton) of the NMR spectrum. That is, since 4arm-PEG-SG is the main ingredient that binds to the oral mucosa and provides a moisturizing effect, the functional group (succinimidyl group) activity of 4arm-PEG-SG, which covalently bonds with the amine (NH.sub.2) group of the oral mucosa, was analyzed by 1H-NMR, and the stability of the finished composition was confirmed. The stability of the finished composition was judged as “Stable” when the NMR activity value of the main ingredient 4arm-PEG-SG was 90% or more, and “Not stable” when it was less than 90%.

[0112] The results are shown in Table 11 below.

TABLE-US-00011 TABLE 11 Result of NMR (%) Analysis According to the Test Period Test Day Day Day Day Day Day Day Day Day Day Material 0 1 2 3 4 5 6 7 8 10 Comparative 96.03 95.97 95.44 92.15 88.14 N/A N/A N/A N/A N/A Example 15 96.07 95.95 95.00 92.18 87.01 N/A N/A N/A N/A N/A (Powder) 96.04 95.96 95.34 92.25 87.04 N/A N/A N/A N/A N/A Stable Not stable Example 7 96.51 96.36 96.21 95.50 93.96 93.63 90.26 *90.21 89.25 86.88 (Granule) 96.41 96.40 95.81 95.48 94.08 92.28 90.29 88.81 86.94 80.84 96.33 96.32 95.97 94.55 93.85 92.66 92.68 88.22 87.95 83.94 Stable Not stable *On day 7, one batch among the three batches satisfies the standard (90% or more), but the mean value of the three batches is below the standard, so it is judged as Not stable.

[0113] As shown in the above results, as a result of confirming the stability test under severe conditions (25±2° C., relative humidity 60%±5%) according to the formulation of the finished composition, the reactive group activity of the main ingredient 4arm-PEG-SG derivative showed stable results of 92.15%, 92.19%, and 92.25% on day 3 for composition in the form of a powder (Comparative Example 15), and 90.26%, 90.29%, and 92.68% on day 6 for composition in the form of a granule (Example 7).

[0114] When the formulation of the finished composition was in the form of powder (Comparative Example 15), the granule finished composition (Example 7) showed stable results for 6 days compared to the results stable for 3 days under severe conditions, and it was confirmed that the stability was improved. The formulation of the composition is changed to a granule form, and compared to a powder form, the exposure area to the air is reduced, and it appears that the absorption of moisture in the air according to the decrease in exposure is reduced and the stability is increased.

[0115] Therefore, the formulation of the final finished composition was determined to be granule. A product for alleviating xerostomia including a container capable of measuring the amount of water per use and a packet packaged with a composition for alleviating xerostomia in the form of granules per use was developed as a final product.