Abstract:
Provided is a skin external composition, which includes tranexamic acid or a salt thereof and a skin penetration enhancer, thereby showing remarkably increased skin permeability and improved sense of use, skin irritation, and storage stability.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a skin external composition, which includes tranexamic acid or a salt thereof and a transdermal permeation enhancer, thereby showing remarkably increased skin permeability and improved sense of use, skin irritation, and storage stability. 
       BACKGROUND ART 
       [0002]    In general, tranexamic acid functions as a hemostatic agent when orally taken, and also functions as an anticoagulant agent, an anti-allergic agent, or an anti-inflammatory agent when topically applied. Tranexamic acid is used as ethical drugs and is also blended in OTC drugs. Owing to its effects on hyperpigmentation such as melasma, tranexamic acid is used for the treatment of hyperpigmentation and for whitening. 
         [0003]    Meanwhile, it has been noted that tranexamic acid has many problems in terms of sense of use, skin irritation, storage stability, and skin permeation, when used as an agent for external use. For example, Korean Patent No. 1087602 indicates a problem that a low-viscosity liquid composition blended with tranexamic acid leaves sticky or thick feeling when applied to the skin, and in particular, the feeling is remarkably increased when more than 0.5% by weight of tranexamic acid is used. Further, Korean Patent No. 1159574 indicates a problem that due to high crystallinity of tranexamic acid, crystals are precipitated in a tranexamic acid-blended agent for external use with evaporation over time. Furthermore, Japanese Patent Publication No. 2010-229100 indicates a problem that tranexamic acid shows very low skin permeability when applied to the skin. 
         [0004]    Moreover, the present inventors have analyzed various commercial agents for external use including tranexamic acid as an active ingredient, and as a result, they found that tranexamic acid included in the commercial products does not permeate the human skin in an in-vitro transdermal permeability test (see Experimental Example 1). 
       DISCLOSURE 
     Technical Problem 
       [0005]    Accordingly, the present inventors have developed a skin external composition which allows skin permeation of tranexamic acid without skin irritation and exhibits excellent sense of use and no precipitation of tranexamic acid in storage, thereby completing the present invention. 
       Technical Solution 
       [0006]    The present invention provides a skin external composition which allows skin permeation of tranexamic acid without skin irritation and exhibits excellent sense of use and storage stability. 
         [0007]    To this end, an object of the present invention is to provide a skin external composition including tranexamic acid or a salt thereof and a skin penetration enhancer. 
         [0008]    Another object of the present invention is to provide a method for enhancing skin permeability of tranexamic acid or a salt thereof, including the step of applying the composition including tranexamic acid or a salt thereof and a skin penetration enhancer to the skin. 
       Effect of the Invention 
       [0009]    The present invention provides a stable skin external composition which allows high skin permeation of tranexamic acid without skin irritation and exhibits excellent sense of use and no precipitation of tranexamic acid crystals in storage, and therefore, it may be applied to a variety of drugs and cosmetics employing tranexamic acid as an active ingredient. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0010]      FIG. 1  shows results of measuring transdermal permeability (%) of formulations of Examples 4, 8, and 10˜15 and Comparative Example 1 over time. 
           [0011]      FIG. 2  shows stability of the formulation of Example 4, which was observed under a microscope. 
           [0012]      FIG. 3  shows results of measuring skin melasma index of a negative control group (vehicle) and the formulations of Examples 4, 8, and 10˜12. 
       
    
    
     BEST MODE FOR CARRYING OUT THE INVENTION 
       [0013]    In an aspect, the present invention relates to a skin external composition including tranexamic acid or a salt thereof and a skin penetration enhancer. 
         [0014]    In another aspect, the present invention relates to a method for enhancing skin permeability of tranexamic acid or a salt thereof, including the step of applying the composition including tranexamic acid or a salt thereof and a skin penetration enhancer to the skin. 
         [0015]    In the present invention, tranexamic acid (TA) has a chemical name of 4-(aminomethyl)cyclohexanecarboxylic acid, and is a lysine derivative which acts by reversibly blocking lysine binding sites on plasmin or plasminogen. Therefore, tranexamic acid is an antiplasmin agent, and it has anticoagulant, anti-allergic, and anti-inflammatory effects and inhibitory effects on hyperpigmentation, such as melasma, blemish, freckle, or skin tone or skin texture improvement. 
         [0016]    In the present invention, tranexamic acid may be used in the form of a pharmaceutically or cosmetically acceptable salt, and the salt may include salts derived from inorganic acids, organic acids, or bases. For example, an alkali metal salt such as a gallium salt or a magnesium salt, an alkaline earth metal salt, an inorganic acid salt such as sulfate may be used. Further, tranexamic acid or a salt thereof may be synthesized by a method known in the art, or may be purchased from a commercially available source. 
         [0017]    In addition to the tranexamic acid of the present invention or a salt thereof, a derivative thereof may be applied, and known derivatives of tranexamic acid may be exemplified by dimers of tranexamic acid [trans-4-(trans-4-aminomethylcyclohexanecarbonyl) aminomethylcyclohexane carboxylate hydrochloride], esters of tranexamic acid and hydroxyquinone(trans-4-aminomethylcyclohexane carboxylate-4′-hydroxyphenylester), esters of tranexamic acid and gentisic acid [2-(trans-4-aminomethylcyclohexylcarbonyloxy)-5-hydroxybenzoic acid and its salts], amides of tranexamic acid [trans-4-aminomethylcyclohexanecarboxylate methylamides and their salts, trans-4-acetylaminomethylcyclohexane carboxylic acids and their salts, trans-4-(p-methoxybenzoyl)aminomethylcyclohexane carboxylic acids and their salts, trans-4-guanidinomethylcyclohexane carboxylic acids and their salts etc.] etc. 
         [0018]    In the composition for skin external use of the present invention, tranexamic acid or a salt thereof may be included in an amount of 0.01 to 10% by weight, based on the total weight of the composition. If the amount is less than the above range, it is difficult to expect a noticeable effect. If the amount exceeds the above range, skin permeability may be increased, but solubility of the composition base may be decreased to reduce stability or dispersion of the main ingredient. 
         [0019]    In the present invention, tranexamic acid is blended with a skin penetration enhancer, thereby providing a skin external composition showing remarkably enhanced skin absorption of tranexamic acid. 
         [0020]    Referring to Experimental Example 1 of the present invention, it can be seen that a commercially available tranexamic acid-containing composition for external use and a tranexamic acid-containing composition for external use disclosed in the prior art show no permeation of tranexamic acid through the human skin, regardless of time. 
         [0021]    Therefore, the present invention provides a skin external composition including tranexamic acid, which shows skin permeability of 10% or higher, 15% or higher, or 25% or higher after the composition is applied to the skin for 24 hrs, 48 hrs, or 72 hrs, respectively. 
         [0022]    Further, the composition of the present invention may include a skin penetration enhancer in an amount of 1 to 10% by weight, and more preferably, 2 to 5% by weight, based on the total weight of the composition, thereby showing excellent sense of use without skin irritation. If the content is less than the above range, it is difficult to achieve the transdermal absorption rate as intended in the present invention. If the content exceeds the above range, skin irritation may be caused and sense of use may be deteriorated. 
         [0023]    The skin penetration enhancer applied in the present invention may be, but is not limited thereto, one or more selected from the group consisting of sorbitol, isopropyl myristate, concentrated glycerin, propylene glycol monolaurate, polysorbate, butylene glycol, diethylene glycol monoethyl ether, glyceryl monooleate, polyglyceryl-6 dioleate, oleoyl polyoxyl-6 glycerides, caprylocaproyl polyoxyl glycerides, linoleoyl polyoxylglycerides, triglycerides, propylene glycol dicaprylocaprate, caprylic capric triglycerides, glycerol caprylate, and polyoxyethylene caprylic/capric glycerides (PEG-6 caprylic/capric glycerides). Preferably, one or more selected from the group consisting of sorbitol, isopropyl myristate, concentrated glycerin, propylene glycol monolaurate, and polysorbate may be used, but is not limited thereto. 
         [0024]    Further, the composition for skin external use of the present invention may be blended with a variety of known components, which are blended in a composition applied to the skin or mucous membrane. These components may be exemplified by a moisturizer, a UV absorber, a UV dispersing agent, vitamins, plant extracts, a skin astringent, an anti-inflammatory agent, a whitening agent, a cell stimulator, a vasodilator, a blood circulation stimulating agent, and a skin function enhancer, in addition to various additives such as a surfactant, a pH adjuster, a pigment, a flavoring agent, a preservative, a sterilizer, a thickener, an antioxidant, a metal ion blocker, a refreshing agent, a deodorizer, etc. A known base or carrier may be also used depending on the formulation. 
         [0025]    More preferably, the composition for skin external use of the present invention may further include one or more selected from the group consisting of a surfactant, a pH adjusting agent, and a thickener, thereby further improving sense of use and stability and minimizing skin irritation. 
         [0026]    If a surfactant is included, it is included in an amount of 1 to 10% by weight, and more preferably, 3 to 6% by weight, based on the total weight of the composition, thereby reducing the surface tension to help mixing of a water phase with an oil phase. 
         [0027]    The surfactant may be exemplified by, but is not limited to, anionic surfactants such as higher fatty acid soaps, alkyl sulfate ester salts, polyoxyethylene alkyl ether sulfates, alkyl ether phosphate ester salts, N-acylamino acid salts, and acryl-N-methyl taurine salts; cationic surfactants such as alkyl trimethyl ammonium chloride and dialkyl dimethyl ammonium chloride; amphoteric surfactants such as alkyl dimethyl aminoacetic acid betaine, alkyl amido dimethyl amino acetic acid betaine, and 2-alkyl-N-carboxy-N-hydroxy imidazolinium betaine; and non-ionic surfactants such as polyoxyethylene, polyol ester, and ethylene oxide-propylene oxide block copolymers. Surfactants belonging to polymeric surfactants or natural surfactants may be also used without particular limitation. 
         [0028]    If a pH adjusting agent is included, it is included in an amount of 0.01 to 2% by weight, and more preferably, 0.1 to 0.5% by weight, based on the total weight of the composition, thereby maintaining stability of the composition for external use. 
         [0029]    The pH adjusting agent may be exemplified by, but is not limited to, sodium hydroxide, boric acid, citric acid, alkanolamide, triethanolamine, acetic acid, sodium hydrogen carbonate, phosphoric acid, ammonia water, sodium sulfite, sodium hexametaphosphate, glucono-delta-lactone, adipic acid, and tetrasodium phosphate. 
         [0030]    If a thickener is included, it is included in an amount of 0.01 to 3% by weight, and more preferably, 0.1 to 1.5% by weight, based on the total weight of the composition, thereby providing a proper viscosity. 
         [0031]    The thickener may be exemplified by, but is not limited to, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyvinyl polymer, alkyl modified carboxyvinyl polymer, polyacrylamide, sodium alginate, propylene glycol alginate, agar, sodium polyacrylate, succinoglucan, dextran, mannan, marmelo, algae colloid, pectin, gellan gum, carrageenan, hyaluronic acid, polyvinyl alcohol, high polymerization degree polyethylene glycol, bentonite, magnesium aluminum silicate, Laponite, hectorite, and silicic anhydride. The metal ion blocker may be exemplified by, but is not limited to, a sodium salt of ethylene diamine tetra-acetic acid, phosphoric acid, and citric acid. 
         [0032]    The preservative may be exemplified by ethyl para-hydroxybenzoate, salicylic acid, and sorbic acid. 
         [0033]    If the formulation is a paste, a cream, or a gel, a carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tracant, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, or zinc oxide. 
         [0034]    If the formulation is a powder or a spray, a carrier component may be lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder, and in particular, in the case of spray, a propellent agent, such as chlorofluorohydrocarbon, propane/butane, or dimethyl ether, may be additionally included. 
         [0035]    If the formulation is a solution or an emulsion, a carrier component may be a solvent, a solubilizing agent, or an emulsifying agent, and it may be exemplified by water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan. 
         [0036]    If the formulation is a suspension, a carrier component may be a liquid diluent, such as water, ethanol, or propylene; a suspension, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminium metahydroxide, bentonite, agar, or tracant. 
         [0037]    The composition for skin external use of the present invention may be directly applied or sprayed onto the skin depending on its type. The amount and frequency of use of the composition per day may be properly determined according to a user&#39;s age, gender, use, and degree of symptoms, and for example, a proper amount of the composition may be applied to the skin at a frequency of once a day to 5 or 6 times a day. 
         [0038]    The composition for skin external use of the present invention may be applied to a variety of pharmaceutical compositions or cosmetic compositions employing tranexamic acid or a salt thereof as an active ingredient. If the composition may be used as a pharmaceutical composition, it may be used as an anti-plasmin agent, an anticoagulant agent, an anti-allergic agent, an anti-inflammatory agent, or a therapeutic agent for hyperpigmentation. If the composition may be used as a cosmetic composition, it may be used as a functional cosmetic composition for improving melasma, blemish, freckle, skin tone, or skin texture and for whitening the skin. 
         [0039]    In a preferred embodiment, the present invention relates to a pharmaceutical composition for external use, including tranexamic acid or a salt thereof and a skin penetration enhancer. 
         [0040]    Herein, a medical composition for external use includes a drug and a quasi-drug for external use. The formulation of the medical composition for external use is not particularly limited, as long as it can be applied to the skin or mucous membrane. For example, it may be any formulation such as an aqueous solution system, a soluble system, an emulsion system, a powder dispersion system, and a water/oil two layer system. Specifically, a liquid, an emulsion, a lotion, a liniment, an emulsion, a suspension, a cream, or an ointment may be exemplified. 
         [0041]    When the composition may be used for treating and improving hyperpigmentation, a known whitening component may be further blended, and exemplified by pantothenic acid or a salt thereof, hydroquinones, glucosamines, hinokitiols, azelaic acid or a salt thereof, tocopherols, pyridoxine or a salt thereof, ubiquinones, carotenes, flavones, iso flavones, flavanones, catechins, flavonols, glycinates, kojic acid or a salt thereof, glutathione or a salt thereof, other natural extracts having a whitening activity, but is not limited thereto. 
         [0042]    In another preferred embodiment, the present invention relates to a cosmetic composition including tranexamic acid or a salt thereof and a skin penetration enhancer. The cosmetic composition according to the present invention is characterized in that it includes tranexamic acid or a salt thereof as an active ingredient, thereby showing the effects of improving and whitening melasma, blemish, freckle, skin tone, skin texture, and inflammatory hyperpigmentation. 
         [0043]    The cosmetic composition according to the present invention may include tranexamic acid or a salt thereof in an amount of 0.01 to 10% by weight, based on the total weight of the composition. If the amount is less than the above range, it is difficult to expect a noticeable effect. If the amount exceeds the above range, skin permeability may be increased, but solubility of the composition base may be decreased to reduce stability or dispersion of the main ingredient. 
         [0044]    The composition according to the present invention may be formulated into a cosmetic composition in an embodiment of the present invention, which may be exemplified by cosmetics. In this case, the cosmetic composition according to the present invention includes a cosmetically or dermatologically acceptable medium or base. Such composition includes any formulations suitable for local applications, for example, a solution, a gel, a solid, anhydrous paste products, oil in water emulsion, a suspension, a microemulsion, microcapsules, microgranules or ionic (liposome) and non-ionic vesicular dispersion, or may be provided in the form of a cream, a skin, a lotion, a powder, an ointment, a spray, or a conceal stick. Such compositions may be obtained in a manner generally known in the art. Further, the composition according to the present invention may be used in the form of a foam or an aerosol composition further including a pressurized propellant. 
         [0045]    Further, the cosmetic composition according to the present invention may include an adjuvant currently used in the field of cosmetics and dermatology, such as fat, an organic solvent, a dissolving agent, a concentrating agent, a gelling agent, a softener, an anti-oxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or non-ionic emulsifier, a filler, a metal ion blocker, a chelating agent, a preservative, vitamins, a blocking agent, a wetting agent, essential oil, a dye, a pigment, a hydrophilic or hydrophobic activating agent, lipid vesicles or other ingredients currently used in cosmetic products. Such adjuvant may be used in an amount currently used in the field of cosmetics or dermatology. 
         [0046]    The cosmetic composition according to the present invention may be properly prepared in any desired formulation with no particular limitation. For example, the cosmetic composition may be prepared into a formulation such as a skin softener (skin lotion and milk lotion), a nutrient tonic, an essence, a nutrient cream, a massage cream, an eye cream, an eye essence, a pack, a patch, a gel, a stick, a spray, a cleansing cream, a cleansing foam, a cleansing water, a pack, a powder, a body lotion, a body cream, a body oil, or a body essence, but is not limited thereto. 
         [0047]    In an embodiment of the present invention, the composition may be applied to the face, in particular, eyes, mouth, cheeks, forehead, neck, hands or feet, but is not limited thereto. 
       DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0048]    Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are for illustrative purposes only, and the present invention is not limited to the following Examples. 
       COMPARATIVE EXAMPLE 1 
     White Lucent (Shiseido) 
       [0049]    White Lucent (Shiseido, Japan) was purchased and prepared. The main raw materials of White Lucent are summarized in Table 1. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Name of raw material 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 2-0-Ethyl ascorbic acid 
               
               
                   
                 Glucosyl hesperidin 
               
               
                   
                 Glycerin 
               
               
                   
                 Dimethicone 
               
               
                   
                 Disodium EDTA 
               
               
                   
                 Lauly betaine 
               
               
                   
                 Limonene 
               
               
                   
                 Benzyl benzoate 
               
               
                   
                 Butylene glycol 
               
               
                   
                 Serine 
               
               
                   
                 Cetyl ethyl hexanoate 
               
               
                   
                 Sodium metabisulfite 
               
               
                   
                 Sodium benzoate 
               
               
                   
                 Sodium hyaluronate 
               
               
                   
                 Aminomethyl propanediol 
               
               
                   
                 Acrylate/C10-30 alkyl acrylate copolymer 
               
               
                   
                 Isostearic acid 
               
               
                   
                 Xanthan gum 
               
               
                   
                 Purified water 
               
               
                   
                 Carbomer 
               
               
                   
                 Tocopherol acetate 
               
               
                   
                 Tranexamic acid 
               
               
                   
                 Phenoxyethanol 
               
               
                   
                 Potassium methoxysalicylate 
               
               
                   
                 Polyquaternium-51 
               
               
                   
                 PEG/PPG-14/dimethyl ester 
               
               
                   
                 PEG-150 
               
               
                   
                 Fragrance (natural) 
               
               
                   
                   
               
             
          
         
       
     
       COMPARATIVE EXAMPLE 2 
     Aestura RegeDerm Rx (Amore Pacific) 
       [0050]    Aestura RegeDerm Rx (Amore Pacific, Korea) was purchased and prepared. The main raw materials of Aestura RegeDerm Rx are summarized in Table 2. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Name of raw material 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Tranexamic acid 
               
               
                   
                 Glycolic acid 
               
               
                   
                 Glycerin 
               
               
                   
                 Glyceryl stearate 
               
               
                   
                 Glyceryl caprylate 
               
               
                   
                 Niacinamide 
               
               
                   
                 Disodium EDTA 
               
               
                   
                 Beta-glucan 
               
               
                   
                 Butylene glycol 
               
               
                   
                 Cyclopentasiloxane 
               
               
                   
                 Cyclohexasiloxane 
               
               
                   
                 Salicylic acid 
               
               
                   
                 Cetyl alcohol 
               
               
                   
                 Ceteth-20 
               
               
                   
                 Cetearyl alcohol 
               
               
                   
                 Sodium lactate 
               
               
                   
                 Sodium polyacrylate 
               
               
                   
                 Sodium hyaluronate 
               
               
                   
                 Steareth-20 
               
               
                   
                 Stearic acid 
               
               
                   
                 Ethanol 
               
               
                   
                 Ethoxydiglycol 
               
               
                   
                 Inulin lauryl carbamate 
               
               
                   
                 Tocophersolan 
               
               
                   
                 Tromethamine 
               
               
                   
                 Phenoxyethanol 
               
               
                   
                 Poloxamer 235 
               
               
                   
                 Poloxamer 338 
               
               
                   
                 Polyglyceryl-3 methylglucose distearate 
               
               
                   
                 Propanediol 
               
               
                   
                 PEG-75 stearate 
               
               
                   
                 Hydrogenated lecithin 
               
               
                   
                 Hydrogenatedpoly 
               
               
                   
                 Fragrance (natural) 
               
               
                   
                 Hexapeptide-9 Epigallocatechin gallate 
               
               
                   
                   
               
             
          
         
       
     
       COMPARATIVE EXAMPLE 3 
     Cream Formulation of Example 52 of Korean Patent No. 10-0251813 
       [0051]    A cream formulation of Example 52 of Korean Patent No. 10-0251813 was prepared. The main raw materials and content thereof are summarized in Table 3. 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                 Content 
               
               
                 Name of raw material 
                 (% by weight) 
               
               
                   
               
             
             
               
                 Tranexamic acid 
                 2.00 
               
               
                 Stearyl alcohol 
                 1.50 
               
               
                 Squalene 
                 2.00 
               
               
                 Vaseline 
                 2.50 
               
               
                 Deodorized liquid lanolin 
                 1.50 
               
               
                 Evening primrose oil 
                 2.00 
               
               
                 Isopropyl myristate 
                 5.00 
               
               
                 Glycerin monooleate 
                 2.00 
               
               
                 Polyoxyethylene (60 mol)hydrogenated castor oil 
                 2.00 
               
               
                 Tocopherol acetate 
                 0.05 
               
               
                 Ethyl parahydroxybenzoate 
                 0.20 
               
               
                 Butyl parahydroxybenzoate 
                 1.00 
               
               
                 Trans-4- ureidomethylcyclohexanecarboxylic acid 
                 1.00 
               
               
                 Trans-4-(N′- 
                 1.00 
               
               
                 ethylureidomethyl)cyclohexanecarboxylic acid 
               
               
                 Fragrance 
                 q.s. 
               
               
                 Sodium hydrogensulfite 
                 0.01 
               
               
                 Glycerin 
                 5.00 
               
               
                 Sodium hyaluronate 
                 0.01 
               
               
                 Carboxyl vinyl polymer 
                 0.20 
               
               
                 Potassium hydroxide 
                 0.20 
               
               
                 Purified water 
                 balance 
               
               
                   
               
             
          
         
       
     
       EXAMPLES 1-15 
     Formulations Containing Tranexamic Acid and Transdermal Absorber 
       [0052]    Formulations of Examples 1 to 10 were prepared according to the compositions given in the following Table 4, and Formulations of Examples 11 to 15 were prepared according to the compositions given in the following Table 5. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
             
             
               
                   
                   
               
               
                   
                 Section 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Example 
               
               
                 Name of raw 
                 Example 1 
                 Example 2 
                 Example 3 
                 Example 4 
                 Example 5 
                 Example 6 
                 Example 7 
                 Example 8 
                 Example 9 
                 10 
               
             
          
           
               
                 material 
                 Content (% by weight) 
               
               
                   
               
             
          
           
               
                 Tranexamic acid 
                 10.00  
                 10.00  
                 10.00  
                 10.00  
                 10.00  
                 10.00  
                 5.00 
                 5.00 
                 2.00 
                 2.00 
               
               
                 Sorbitol 
                 2.50 
                 — 
                 — 
                 5.00 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Isopropyl 
                 — 
                 2.50 
                 — 
                 — 
                 5.00 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 myristate 
               
               
                 Concentrated 
                 — 
                 — 
                 2.50 
                 — 
                 — 
                 5.00 
                 2.50 
                 — 
                 — 
                 — 
               
               
                 glycerin 
               
               
                 Propylene glycol 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 5.00 
                 2.50 
                 — 
               
               
                 monolaurate 
               
               
                 Polysorbate 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 5.00 
               
               
                 Glycol stearate 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
               
               
                 diethylene 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
               
               
                 glycol 
               
               
                 monoethyl ether 
               
               
                 Butylated 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 hydroxy toluene 
               
               
                 Cetyl alcohol 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
               
               
                 Olive oil 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
               
               
                 Liquid paraffin 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
               
               
                 Purified water 
                 75.15  
                 75.15  
                 75.15  
                 72.65  
                 72.65  
                 72.65  
                 80.15  
                 77.65  
                 83.15  
                 80.65  
               
               
                 Carbomer 940 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 Tocopherol 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
               
               
                 acetate 
               
               
                 Triethanolamine 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 Fragrance 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
             
             
               
                   
                   
               
               
                   
                 Section 
               
             
          
           
               
                   
                 Example 11 
                 Example 12 
                 Example 13 
                 Example 14 
                 Example 15 
               
             
          
           
               
                 Name of raw material 
                 Weight (%) 
               
               
                   
               
             
          
           
               
                 Tranexamic acid 
                 1.00 
                 0.50 
                 0.30 
                 0.10 
                 0.05 
               
               
                 Sorbitol 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
               
               
                 Isopropyl myristate 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Concentrated glycerin 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Propylene glycol monolaurate 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Polysorbate 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Glycol stearate 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
                 2.90 
               
               
                 diethylene glycol monoethyl ether 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
                 5.00 
               
               
                 Butylated hydroxy toluene 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 Cetyl alcohol 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
               
               
                 Olive oil 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
                 1.00 
               
               
                 Liquid paraffin 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
                 2.00 
               
               
                 Purified water 
                 81.65  
                 82.15  
                 82.35  
                 82.55  
                 82.60  
               
               
                 Carbomer 940 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 Tocopherol acetate 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
                 0.05 
               
               
                 Triethanolamine 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                 Fragrance 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
                 0.10 
               
               
                   
               
             
          
         
       
     
         [0053]    Preparation methods of the formulations of Examples 1 to 15 are summarized in the following Table 6. 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Process 
                 Operation 
               
               
                   
               
             
             
               
                 Preparation 
                 Purified water was added to a preparation tank and 
               
               
                 of aqueous 
                 then tranexamic acid was agitated at room 
               
               
                 phase 
                 temperature for 10 minutes. When tranexamic acid 
               
               
                   
                 was completely dissolved, carbomer 940 was slowly 
               
               
                   
                 added and completely dissolved. 
               
               
                   
                 Temperature: room temperature 
               
               
                   
                 Agitation speed: homomixer: 3,000 rpm 
               
               
                 Preparation 
                 An assistant preparation tank was heated, and then 
               
               
                 of oil phase 
                 liquid paraffin, sorbitol (or isopropyl myristate, 
               
               
                   
                 propylene glycol monolaurate, polysorbate, 
               
               
                   
                 concentrated glycerin), olive oil, cetyl alcohol, 
               
               
                   
                 glycol stearate, and butylated hydroxytoluene were 
               
               
                   
                 sequentially added and completely dissolved. 
               
               
                   
                 Temperature: 65~75° C. 
               
               
                   
                 Agitation speed: homomixer: 3,000 rpm 
               
               
                 Emulsification 
                 While agitating the preparation tank, the oil phase 
               
               
                   
                 liquid was slowly added to the aqueous phase liquid 
               
               
                   
                 of the preparation tank, and then tocopherol acetate 
               
               
                   
                 and diethylene glycol monoethyl ether were added and 
               
               
                   
                 agitated with heating. 
               
               
                   
                 Temperature: 60~70° C. 
               
               
                   
                 Agitation speed: homomixer: 3,000 rpm 
               
               
                 Cooling 
                 While cooling the tank, triethanolamine and fragrance 
               
               
                   
                 were added and agitated, and then bubbles were 
               
               
                   
                 removed under reduced pressure. 
               
               
                   
                 Temperature: 40° C. 
               
               
                   
                 Agitation speed: homomixer: 3,000 rpm 
               
               
                   
                 Reduced pressure: 70 mmHg 
               
               
                 Filtration 
                 Filtration was performed using a 80 mesh, and then 
               
               
                   
                 the product was transferred to a semi-finished 
               
               
                   
                 container. 
               
               
                   
               
             
          
         
       
     
       EXPERIMENTAL EXAMPLE 1 
     Transdermal Permeability Test 
       [0054]    1-1. Conditions for Transdermal Permeability Test
       Receptor: PBS (0.1% sodium azide)   Skin area contacting with receptor phase: 0.636 cm 2      Volume of receptor phase: 4.7 ml   Skin: a man aged 50 years   Temperature: 32° C.   Agitation speed: 600 rpm   Amount of sample collected: 1 ml per hr       
 
         [0062]    1-2. Test Method 
         [0063]    Skin permeation of tranexamic acid from the composition for external use through the human cadaver skin was measured using Franz diffusion cells. Franz diffusion cells were filled with receptors and agitated at 600 rpm while maintaining the temperature at 32° C. 
         [0064]    The human cadaver skin was placed between donor and receptor compartments of the Franz diffusion cells. The test drug was measured and loaded on the skin. As test solutions, 1 ml of receptor phase was collected at 6, 24, 48 and 72 hrs from a sampling port using a syringe, and quantification was performed by HPLC. The receptor at 32° C. was immediately added at an amount equal to the amount collected. 
         [0065]    1-3. Analysis Method 
         [0066]    The contents of tranexamic acid in the standard solution and the test solution were analyzed by the following liquid chromatography, and then a peak area was determined to prepare a calibration curve of the standard solution, and skin permeability was calculated therefrom. 
         [0067]    1-4. Test Equipment 
         [0068]    Equipment: HPLC 
         [0069]    Model: Agilent 1100 Series 
         [0070]    1-5. Analysis Conditions 
         [0071]    &lt;Conditions for Liquid Chromatography&gt;
       Column: Capcell pak (150 mm×4.6 mm, 5 μm)   Column temperature: 30° C.   Flow rate: 1 ml/min   Detection: UV 220 nm   Injection amount: 100 μl   Mobile phase: 11.0 g of anhydrous sodium dihydrogen phosphate was dissolved in 500 ml of water, and 5 ml of triethylamine and 1.4 g of sodium lauryl sulfate were added thereto. pH was adjusted to 4.0 with phosphate, and water was added to bring the volume to 600 ml. 400 ml of methanol was added to this solution.       
 
         [0078]    &lt;Preparation of Standard Solution&gt;
       Tranexamic acid standard solution: 10 mg of tranexamic acid was dissolved in a receptor solution, and then diluted with the receptor solution to prepare a standard solution (5, 20, 35, 50, 65 μg/ml).       
 
         [0080]    1-6. Equation 
         [0081]    C (concentration, μg/ml)=(peak area of test solution−intercept of calibration curve)/slope of calibration curve 
         [0082]    Cumulative permeability (Amount, μg/cm 2 )=((Cn×4.7)+(C1+C2+ . . . +Cn)×1)/0.636 
         [0083]    n=time point of collection of test solution 
         [0084]    Transdermal permeation rate (Flux, μg/cm 2 /hr)=((Cn×4.7)+(C1+C2+ . . . +Cn)×1)/0.636/T n=time point of collection of test solution, T=time of collection of test solution 
         [0085]    1-7. Test Result 
         [0086]    Transdermal permeabilities (%) of the formulations of Examples 4, 8, and 10-15, and the formulations of Comparative Examples 1, 2, and 3 were measured over time and summarized in the following Tables 7 and 8 and  FIG. 1 . The formulations of Comparative Examples 1, 2, and 3 showed no transdermal permeation, regardless of time, whereas the formulations of Examples showed high transdermal permeability and their transdermal permeabilities continuously increased over time. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
             
             
               
                   
                   
               
               
                   
                 Average permeability (%) 
               
             
          
           
               
                   
                   
                 Comparative 
                 Comparative 
                 Comparative 
               
               
                   
                 Time (h) 
                 Example 1 
                 Example 2 
                 Example 3 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 6 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 24 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 48 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 72 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 8 
               
             
             
               
                   
                   
               
               
                   
                 Average permeability (%) 
               
             
          
           
               
                 Time 
                   
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                 (h) 
                 Example 4 
                 Example 8 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
               
               
                   
               
             
          
           
               
                 0 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
                 0.00 
               
               
                 6 
                 3.31 
                 4.22 
                 3.58 
                 3.01 
                 2.84 
                 3.09 
                 2.69 
                 2.12 
               
               
                 24 
                 16.66 
                 16.22 
                 15.65 
                 15.22 
                 14.87 
                 16.00 
                 12.54 
                 10.50 
               
               
                 48 
                 33.54 
                 30.38 
                 30.74 
                 28.64 
                 28.17 
                 27.82 
                 21.38 
                 19.18 
               
               
                 72 
                 44.04 
                 40.07 
                 39.46 
                 39.34 
                 37.36 
                 35.59 
                 31.08 
                 28.24 
               
               
                   
               
             
          
         
       
     
       EXPERIMENTAL EXAMPLE 2 
     Skin Irritation Test 
       [0087]    In accordance with the guidelines for drug toxicity test, a skin irritation test was performed by applying the test material to the skin, and then 24 hrs later, briefly washing the skin with a solvent such as physiological saline which does not influence the test result in order to completely remove the test material, and observing changes such as erythema, edema, bleeding, and eschar formation after 24, 48, and 72 hrs. 
         [0088]    Eschar formation was evaluated in accordance with the criteria of the following Table 9, and edema was evaluated in accordance with the criteria of the following Table 10. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Grading scale 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 No erythema 0 
               
               
                   
                 Very slight erythema (barely perceptible) 1 
               
               
                   
                 Well-defined erythema 2 
               
               
                   
                 Moderate to severe erythema 3 
               
               
                   
                 Severe erythema(beet redness) and slight eschar formation 4 
               
               
                   
                 Possible total erythema score 4 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 Grading scale 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 No edema 0 
               
               
                   
                 Very slight edema(barely perceptible) 1 
               
               
                   
                 Slight edema (edges of area well defined by definite raising) 2 
               
               
                   
                 Moderate edema (raised approximately 1 mm) 3 
               
               
                   
                 Severe edema (raised more than 1 mm and extending beyond the 
               
               
                   
                 area of exposure) 4 
               
               
                   
                 Possible total edema score 4 
               
               
                   
                   
               
             
          
         
       
     
         [0089]    In accordance with the above criteria, skin irritation of the formulations of Examples 4, 8, and 10-15 and the formulations of Comparative Examples 1, 2, and 3 was tested, and the results are summarized in the following Table 11. All the formulations showed no skin irritation. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                 Section 
                 Degree of skin irritation 
               
               
                   
                   
               
             
             
               
                   
                 Example 4 
                 0 
               
               
                   
                 Example 8 
                 0 
               
               
                   
                 Example 10 
                 0 
               
               
                   
                 Example 11 
                 0 
               
               
                   
                 Example 12 
                 0 
               
               
                   
                 Example 13 
                 0 
               
               
                   
                 Example 14 
                 0 
               
               
                   
                 Example 15 
                 0 
               
               
                   
                 Comparative Example 1 
                 0 
               
               
                   
                 Comparative Example 2 
                 0 
               
               
                   
                 Comparative Example 3 
                 0 
               
               
                   
                   
               
             
          
         
       
     
       EXPERIMENTAL EXAMPLE 3 
     Test of Sense of Use 
       [0090]    To test sense of use, the test material was applied, and 10 minutes later, changes in three items of stickiness, glossiness, and thickness were examined Evaluation criteria are summarized in Table 12. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Evaluation criteria for sense of use 
               
             
          
           
               
                   
                 ⋆ 
                 ⊚ 
                 ◯ 
                 Δ 
                 X 
               
               
                   
                   
               
               
                   
                 Very good 
                 Good 
                 Moderate 
                 Slightly bad 
                 Bad 
               
               
                   
                   
               
             
          
         
       
     
         [0091]    The results of evaluating the sense of use of the formulations of Examples 4, 8, and 10-15 and the formulations of Comparative Examples 1, 2, and 3 in accordance with the above criteria are summarized in the following Table 13. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 Section 
                 Stickiness 
                 Glossiness 
                 Thickness 
               
               
                   
               
             
             
               
                 Comparative Example 1 
                 ⋆ 
                 ⊚ 
                 ⊚ 
               
               
                 Comparative Example 2 
                 ⊚ 
                 ◯ 
                 ⊚ 
               
               
                 Comparative Example 3 
                 ⊚ 
                 ⊚ 
                 ⊚ 
               
               
                 Example 4 
                 ◯ 
                 ⋆ 
                 ◯ 
               
               
                 Example 8 
                 ◯ 
                 ⋆ 
                 ◯ 
               
               
                 Example 10 
                 ⊚ 
                 ⊚ 
                 ⊚ 
               
               
                 Example 11 
                 ⊚ 
                 ⊚ 
                 ⊚ 
               
               
                 Example 12 
                 ⊚ 
                 ⊚ 
                 ⋆ 
               
               
                 Example 13 
                 ⋆ 
                 ◯ 
                 ⋆ 
               
               
                 Example 14 
                 ⋆ 
                 ◯ 
                 ⋆ 
               
               
                 Example 15 
                 ⋆ 
                 ◯ 
                 ⋆ 
               
               
                   
               
             
          
         
       
     
       EXPERIMENTAL EXAMPLE 4 
     Stability Test (Accelerated Test) 
       [0092]    In accordance with the guidelines for drug stability test, the test was performed at 40±2° C./relative humidity 75±5%. According to the above evaluation criteria, stability of the formulations of Examples 4, 6, and 8 was tested. As a result, as shown in  FIG. 2 , no crystal precipitation was observed under a microscope. 
         [0093]    As shown in the following Table 14, the formulations of Examples 4, 8, and 10˜15 showed stability in both content and appearance. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Stability test results of formulations of Examples 4, 8, and 10~15 
               
               
                 Accelerated stability test (storage conditions: 40 ± 2° C., 75 ± 5% RH) 
               
             
          
           
               
                   
                 Test results 
               
             
          
           
               
                 Section 
                 Test item 
                 Criteria 
                 Initial 
                 2 months 
                 4 months 
                 6 months 
               
               
                   
               
               
                 Example 4 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                   
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.89 
                 100.62 
                 100.37 
                 100.05 
               
               
                 Example 8 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                   
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 101.03 
                 100.55 
                 100.63 
                 100.29 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 10 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.39 
                 100.89 
                  99.72 
                  99.84 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 11 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.22 
                 100.51 
                 100.33 
                  99.91 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 12 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.75 
                 100.83 
                 100.79 
                  99.81 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 13 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.11 
                  99.87 
                 100.07 
                 100.18 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 14 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.27 
                 100.29 
                 100.03 
                 100.07 
               
               
                 Example 
                 Appearance 
                 White cream 
                 Suitable 
                 Suitable 
                 Suitable 
                 Suitable 
               
               
                 15 
                   
                 formulation 
               
               
                   
                 Content 
                 90.0~110.0% 
                 100.57 
                 100.27 
                 100.07 
                 100.08 
               
               
                   
               
             
          
         
       
     
       EXPERIMENTAL EXAMPLE 5 
     Efficacy Test 
       [0094]    5-1. Experimental Animal
       Species: Brown Guinea Pig (Al)   Sex and weight: male, 500˜550 g   Breeding environment:       
 
         [0098]    Temperature 23±3° C., 
         [0099]    Relative humidity 50±10%, 
         [0100]    Lighting 12 hr (08:0020:00), 
         [0101]    Number of air circulation 10˜15 times/hr, 
         [0102]    Illumination 150˜300 Lux. 
         [0103]    5-2. Pretreatment (UV Tanning) 
         [0104]    Experimental animals were brought and acclimated, and the hair of the animal&#39;s back was shaved. The animals were anesthetized with Zoletil, and the shaved area was covered with a perforated leather, followed by UV irradiation once a week for 3 weeks (500 mJ/cm 2 *3 times=1,500 mJ/cm 2 ) for induction of melanin pigmentation. A mexameter was used to measure pigmentation (melasma index). 
         [0105]    5-3. Treatment of Test Material 
         [0106]    Each 30 μl of the test materials (vehicle, Examples 4, 8, and 10-12) was applied to the induced pigment spot once a day for 5 days a week for 8 weeks. After treatment of the test material, a mexameter was used to measure skin melasma index once a week (measurement of the index for the same area was repeated three times, and the mean value was used). 
         [0107]    5-4. Test Results 
         [0108]    The test results of melasma index are shown in Table 15 and  FIG. 3 . 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                   
                   
                   
                   
                 Example 
                 Example 
                 Example 
               
               
                 Time 
                 vehicle 
                 Example 4 
                 Example 8 
                 10 
                 11 
                 12 
               
             
          
           
               
                 (day) 
                 Mean 
                 SD 
                 Mean 
                 SD 
                 Mean 
                 SD 
                 Mean 
                 SD 
                 Mean 
                 SD 
                 Mean 
                 SD 
               
               
                   
               
             
          
           
               
                 0 
                 497.2 
                 60.5 
                 496.6 
                 44.6 
                 500.7 
                 48.8 
                 494.0 
                 73.6 
                 495.6 
                 50.5 
                 495.7 
                 51.8 
               
               
                 7 
                 479.1 
                 55.6 
                 454.2 
                 55.2 
                 468.0 
                 56.1 
                 453.8 
                 27.6 
                 455.6 
                 47.7 
                 466.4 
                 71.2 
               
               
                 14 
                 476.8 
                 65.3 
                 411.9 
                 50.3 
                 425.1 
                 54.3 
                 414.6 
                 35.9 
                 408.9 
                 51.8 
                 433.7 
                 49.0 
               
               
                 21 
                 454.9 
                 59.3 
                 377.6 
                 42.9 
                 384.8 
                 44.5 
                 380.1 
                 41.8 
                 385.9 
                 50.9 
                 406.9 
                 49.7 
               
               
                 28 
                 457.1 
                 54.7 
                 353.0 
                 37.7 
                 360.1 
                 42.8 
                 350.0 
                 45.9 
                 363.8 
                 42.8 
                 381.7 
                 53.9 
               
               
                 35 
                 461.6 
                 54.5 
                 340.4 
                 39.4 
                 348.0 
                 43.0 
                 336.9 
                 40.7 
                 349.8 
                 41.6 
                 369.1 
                 47.7 
               
               
                 42 
                 460.7 
                 46.1 
                 321.2 
                 37.6 
                 328.6 
                 36.9 
                 312.7 
                 37.6 
                 325.4 
                 44.7 
                 352.7 
                 46.8 
               
               
                 49 
                 462.8 
                 42.4 
                 300.7 
                 43.1 
                 311.3 
                 32.7 
                 294.9 
                 36.9 
                 308.3 
                 41.7 
                 337.8 
                 43.1 
               
               
                 56 
                 457.9 
                 40.4 
                 279.1 
                 34.7 
                 284.3 
                 26.2 
                 271.1 
                 32.3 
                 286.2 
                 40.1 
                 327.2 
                 42.8