External preparation of microsphere-loaded medicine for treating chloasma and preparation method of external preparation

Abstract

The invention discloses an external preparation of a microsphere-loaded medicine for treating chloasma and a preparation method of the external preparation. The preparation method comprises the following steps: preparing polyvinyl alcohol acrylate microspheres, loading a tranexamic acid medicine and adding a matrix to prepare the external preparation of the microsphere-loaded medicine. By means of oleophylic and hydrophilic segments on the polyvinyl alcohol acrylate microspheres, fusion with skin lipid components is increased, permeability of the medicine is improved, meanwhile, due to the large specific surface area of the microspheres, the medicine can be more evenly loaded on the microspheres, and due to the hydrophilicity of the microspheres, moisture can be kept for a longer time; and the problem of crystallization of tranexamic acid caused by moisture volatilization is well solved. The ointment or gel external preparation prepared by the invention is relatively good in character and stability, good in in-vitro permeation and in-vitro release performance and remarkable in treatment effect on chloasma of mice.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and relates to an external preparation of microsphere-loaded drugs for treating chloasma and a preparation method thereof. Background Art

[0002] Tranexamic acid (TA), also known as tranexamic acid, is approved by the US FDA for the treatment of menorrhagia and prevention of bleeding. It is the only classic hemostatic drug that has been widely used in clinical practice for half a century. In 1979, scholars first discovered that TA can significantly lighten patients' melasma, and thus TA has attracted people's attention as a new drug for the treatment of melasma. As a plasmin inhibitor, TA may mainly work through: (1) inhibiting plasmin-plasminogen. Arachidonic acid (AA) and α-melanocyte stimulating hormone (α-MSH) increase with the increase of plasmin in keratinocytes. Prostaglandins (PG) as metabolites of arachidonic acid can stimulate melanin synthesis and the production of melanocyte stimulating hormone. Therefore, by inhibiting the plasmin pathway, tranexamic acid will reduce the production of melanin, thereby achieving the effect of lightening spots. (2) Tyrosinase is a key enzyme in the formation of melanin. The chemical structure of tranexamic acid is similar to that of tyrosine. Therefore, early studies have reported that tranexamic acid may also be used to treat melasma by competing with tyrosine, thereby interfering with the catalytic effect of tyrosinase on tyrosine and inhibiting the formation of melanin.

[0003] Clinically, tranexamic acid is mainly used to treat melasma in three ways: oral administration, subcutaneous injection and topical administration. Among them, oral preparations for the treatment of melasma have been widely studied and have shown good freckle removal effects. However, the oral dosage is large and may cause some gastrointestinal side effects. In addition, the oral first-pass effect is obvious, the body metabolism is fast, and the half-life is short. Compared with oral and injection administration, topical preparations are a more attractive alternative treatment option that can improve drug bioavailability and eliminate potential systemic side effects.

[0004] However, in order to achieve the therapeutic effect, tranexamic acid must penetrate the stratum corneum to reach the dermis where pigmentation occurs, but tranexamic acid is highly hydrophilic and has limited affinity with skin lipid components, so the permeability and retention effect of tranexamic acid are not ideal. At present, many improvements have been made in the method of promoting transdermal absorption of tranexamic acid, including microemulsion, solid lipid nano, liposome, etc. Although this method improves the fusion of the drug with the stratum corneum to a certain extent, due to the very high crystallinity of tranexamic acid, it is easy to crystallize, and solids will be precipitated during the water release process, thereby affecting the therapeutic effect. Summary of the invention

[0005] In view of the above problems, the present invention provides an external preparation for treating chloasma with a microsphere loaded drug and a preparation method thereof, which comprises the preparation of polyvinyl alcohol acrylate microspheres, loading of tranexamic acid drugs and adding a matrix to prepare an external preparation for microsphere loaded drugs. The present invention not only increases the fusion with the lipid components of the skin and increases the permeability of the drug through the lipophilic and hydrophilic segments on the polyvinyl alcohol acrylate microspheres, but also increases the permeability of the drug due to the large specific surface area of ​​the microspheres, so that the drug can be more evenly loaded on the microspheres, and the hydrophilicity of the microspheres allows the moisture to be retained for a longer time, which well solves the crystallization problem caused by the volatilization of tranexamic acid.

[0006] The technical solution of the present invention is: a method for preparing an external preparation of microsphere-loaded drugs for treating chloasma, characterized in that it comprises the following steps:

[0007] 1) Preparation of polyvinyl alcohol acrylate microspheres

[0008] Add sodium dodecyl diphenyl ether disulfonate and polyvinyl alcohol into purified water and stir to dissolve to prepare the aqueous phase;

[0009] Take monomers ethyl acrylate, hexadecane and azobisisobutyronitrile and stir and mix them evenly to form the oil phase;

[0010] The aqueous phase is slowly added to the oil phase under stirring, and stirred evenly to prepare a milky white pre-emulsion; then emulsified to obtain a polyvinyl alcohol acrylate microsphere emulsion;

[0011] 2) Drug loading

[0012] Adding tranexamic acid to the polyvinyl alcohol acrylate microsphere emulsion and stirring and dispersing the mixture, and then performing ultrasound, stirring and reacting the mixture after the ultrasound is completed, filtering the mixture, and naturally air-drying the mixture after the filtrate is concentrated under reduced pressure to obtain drug-loaded microspheres;

[0013] 3) Preparation of ointment or gel external preparation

[0014] In the step 1), the mass ratio of sodium dodecyl diphenyl oxide disulfonate, polyvinyl alcohol, monomer ethyl acrylate, hexadecane and azobisisobutyronitrile is 1:18-22:28-32:0.8-1.2:0.4-0.6;

[0015] The drug loading amount of tranexamic acid is 12-15%.

[0016] The ointment in step 3) has a formula of 28-32 parts by weight of stearic acid, 3-5 parts of castor oil, 8-12 parts of glyceryl monostearate, 18-22 parts of glycerol, 24-28 parts of triethanolamine, 20-25 parts of drug-loaded microspheres, and 40-60 parts of distilled water;

[0017] The preparation method is as follows: heat stearic acid, glyceryl monostearate, and castor oil to 50-60°C until they are completely dissolved, keep warm, prepare an oil phase, and set aside; heat glycerol, drug-loaded microspheres, and distilled water to 50-60°C, slowly add triethanolamine under stirring until the drug-loaded microspheres are completely dissolved, keep warm, prepare an aqueous phase, and set aside; add the oil phase to the aqueous phase under stirring, stir and disperse after the addition, cool to 30-40°C for filling and sealing.

[0018] The formula of the gel external preparation in step 3) is: 100 parts gelatin, 28-32 parts glycerol, 40-50 parts carbomer, 12-18 parts xanthan gum, 30-35 parts drug-loaded microspheres, and 8-12 parts deionized water.

[0019] The preparation method is as follows: drug-loaded microspheres, deionized water and glycerol are added to an oil phase tank, and then heated to 50-60°C to dissolve, to prepare a mixed material 1 for standby use; gelatin, xanthan gum and carbomer are added to another oil phase tank, and then heated to 60-90°C until completely melted, to prepare a mixed material 2 for standby use; mixed material 2 and mixed material 1 are transferred to an emulsification tank in turn and vacuum stirred evenly; then the temperature is lowered to 40-50°C and high-speed stirring is started to complete dispersion, the temperature is further lowered to 35-40°C, filling is completed, and sealing is performed.

[0020] The ointment or gel external preparation prepared by the above method has good properties and stability, good in vitro permeability and in vitro release performance, and has a significant therapeutic effect on mouse chloasma.

[0021] At present, there are PLGA microspheres loaded with clobetasol propionate for local administration, but clobetasol propionate is a poorly soluble drug, and PLGA microspheres are mostly hydrophilic segments, which increase the solubility of the drug, while tranexamic acid is a water-soluble drug, and a lipophilic and hydrophilic microsphere is needed to increase the lipid solubility of the drug while avoiding the solvent from volatilizing and precipitating crystals. Therefore, the present invention provides a polyvinyl alcohol acrylate microsphere with a lipophilic and hydrophilic segment, which can prolong the release time of the drug after loading the drug, increase the affinity with the lipid components of the skin, thereby increasing the permeability of the drug, minimizing systemic drug absorption, and reducing the possible side effects of the drug. At the same time, the hydrophilic segment carried by the microsphere avoids the problem of drug crystallization caused by solvent volatilization.

[0022] Tests have proven that:

[0023] 1. Under the preferred ratio, the cream external preparation prepared by the present invention has uniform color, no granular feeling when applied, relatively fine, no stratification, and uniform color; the amount of emulsifier triethanolamine has a relatively large influence on the stability of the cream. The prepared gel is easy to apply, has good elasticity, is comfortable on the skin, has no stratification, and has uniform color; the matrix adopts a compound of carbomer, gelatin and xanthan gum for the best effect.

[0024] 2. The ointment or gel external preparation prepared by the present invention has good properties and stability, a relatively consistent release rate in vitro, and a duration of up to 6 hours; the in vitro permeation flux can be maintained for 24 hours.

[0025] 3. The ointment or gel external preparation prepared by the present invention has a significant therapeutic effect on mouse chloasma; the intensity of the effect is gel>ointment>positive control hydroquinone cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a picture of polyvinyl alcohol acrylate microsphere emulsion dispersed in water and observed under a microscope;

[0027] Figure 2 It is a linear curve of the cumulative drug release amount of SE-2024-006 in vitro and the square root of time;

[0028] Figure 3 It is the linear curve of the cumulative drug release amount of SE-2024-012 in vitro and the square root of time;

[0029] Figure 4 The in vitro permeation flux and cumulative permeation amount graph of SE-2024-006;

[0030] Figure 5 The in vitro permeation flux and cumulative permeation amount graph of SE-2024-012;

[0031] Figure 6 Representative images of HE staining of the normal control group (Figure A), model control group (Figure B), positive control group (Figure C), SE-2024-006 ointment group (Figure D), and SE-2024-012 gel group (Figure E) after 60 days of drug administration;

[0032] Figure 7 Representative images of MF staining of the normal control group (Figure A), model control group (Figure B), positive control group (Figure C), SE-2024-006 ointment group (Figure D) and SE-2024-012 gel group (Figure E) after 60 days of drug administration. DETAILED DESCRIPTION

[0033] Example 1: Preparation of polyvinyl alcohol acrylate microspheres

[0034] Under stirring, add 1.0 g of sodium dodecyl diphenyl ether disulfonate and 20 g of polyvinyl alcohol to 120 g of purified water, stir and dissolve, as the aqueous phase;

[0035] Take 30g of monomer ethyl acrylate, 1g of hexadecane and 0.5g of azobisisobutyronitrile and stir and mix them evenly to form the oil phase;

[0036] The aqueous phase was slowly added to the oil phase under stirring and stirred for 20 minutes to prepare a milky white pre-emulsion; then a dispersion emulsifier (model FM200) was used for high-speed emulsification at a speed greater than 15000 rpm for 5 minutes to obtain a polyvinyl alcohol acrylate microsphere emulsion.

[0037] The emulsion was dispersed with water and observed under a microscope. Figure 1 The microsphere structure shown has a diameter of approximately 243.33 μm.

[0038] Example 2: Drug loading

[0039] 20 g of tranexamic acid was added to the polyvinyl alcohol acrylate microsphere emulsion prepared above and stirred and dispersed, and then placed under ultrasonic conditions (40KHz) for 30 minutes. After the ultrasonication was completed, the reaction was stirred for 24 hours, filtered (the microspheres will dissolve after loading the drug, the undissolved part will be filtered out, the solid will be discarded, and the filtrate will be obtained), and washed with purified water, the filtrate was collected, and the material was naturally air-dried after reduced pressure concentration to obtain drug-loaded microspheres.

[0040] The encapsulation efficiency and drug loading of the drug in the microspheres are calculated as follows:

[0041] Encapsulation efficiency = ((mass of drug delivered - mass of drug in supernatant) / mass of drug delivered) × 100%

[0042] Drug loading = ((weight of drug delivered - weight of drug in supernatant) / total weight of drug-loaded microspheres) × 100%

[0043] The drug encapsulation efficiency obtained in this example is 80.2%, and the drug loading is 14.6%.

[0044] Example 3: Preparation of external preparations

[0045] Table 1: Preparation formula of cream

[0046]

[0047] The process descriptions of Prescription 1 to Prescription 6 are as follows:

[0048] 1) heating the prescribed amount of stearic acid, glyceryl monostearate and castor oil to 50-60° C. until they are completely dissolved, keeping the temperature to prepare an oil phase for later use;

[0049] 2) Heat the prescribed amount of glycerol, loaded TA microspheres, and distilled water to 50-60° C., slowly add triethanolamine under stirring until the loaded TA microspheres are completely dissolved, keep warm, and prepare an aqueous phase for later use;

[0050] 3) Add the oil phase to the water phase under stirring. After the addition is completed, turn on high-speed stirring, control the stirring rate to 1000 rpm, stir for 30 minutes (stir for 10 minutes, stop for 5 minutes, and cycle once). After dispersion is completed, cool to 30-40°C;

[0051] 4) Filling and maintaining the temperature at 30-40°C. After filling, seal the tubes. The average filling volume of each tube is 5g. The batch numbers are recorded as: SE-2024-001 to SE-2024-006.

[0052] Table 2: Gel preparation recipe

[0053] prescription Prescription 7 Prescription 8 Prescription 9 Prescription 10 Prescription 11 Prescription 12 Deionized water / g 10 10 10 10 10 10 Glycerol / g 30 30 30 30 30 30 Carbomer / g 160 / / 50 60 45 Gelatin / g / 160 40 / 100 100 Xanthan gum / g / / 120 110 / 15 Loading TA microspheres / g 32 32 32 32 32 32

[0054] The process descriptions of Prescription 7 to Prescription 12 are as follows:

[0055] 1) Add the prescribed amount of loaded TA microspheres, deionized water and glycerol to the oil phase tank, and then heat to 50-60° C. to dissolve to prepare a mixed material 1 for standby use;

[0056] 2) Add the prescribed amount of gelatin, xanthan gum and carbomer into another oil phase tank, and then heat to 60-90° C. until completely melted to prepare mixed material 2 for standby use;

[0057] 3) Transfer the mixed material 2 into the emulsification tank and stir it under vacuum. Then transfer the mixed material 1 into the emulsification tank and continue to stir it at 20-30 rpm while maintaining the vacuum state.

[0058] 4) Cool down to 40-50°C and start high-speed stirring, control the stirring rate to 1000 rpm, stir for 30 minutes (stir for 10 minutes, stop for 5 minutes, cycle once), and after dispersion is completed, continue to cool down to 35-40°C;

[0059] 5) Filling and maintaining the temperature at 35-40°C. After filling, seal the tubes. The average filling volume of each tube is 5g. The batch numbers are recorded as SE-2024-007 to SE-2024-012.

[0060] Product Testing

[0061] 1. Product properties testing

[0062] The property test results of Prescription 1 to Prescription 6 are shown in Table 3.

[0063] Table 3: Property test results of prescription 1 to prescription 6

[0064]

[0065]

[0066] It can be seen from the results in Table 3 that this prescription is an O / W emulsion matrix cream. The amount of emulsifier triethanolamine has a relatively large influence on the stability of the cream. According to the test results of creams with different prescriptions, the results obtained with batch number SE-2024-006 (prescription 6) are better.

[0067] The results of the property tests of prescriptions 7 to 12 are shown in Table 4:

[0068] Table 4: Property test results of prescriptions 7 to 12

[0069]

[0070] It can be seen from the results in Table 4 that different gel matrices have a relatively large influence on the stability of the prepared gel. According to the selection of the gel matrix, the compound of carbomer, gelatin and xanthan gum with batch number SE-2024-012 (prescription 12) obtained better results.

[0071] 2. In vitro release test

[0072] The in vitro release of external preparations with batch numbers SE-2024-006 and SE-2024-012 was tested. The specific test process and results are as follows:

[0073] Device: vertical diffusion cell; temperature: 32°C; rotation speed: 600rpm;

[0074] Release medium: pH 6.8 phosphate buffer solution;

[0075] Sample volume: 300 mg (2 mm quantitative loop);

[0076] Artificial membrane: PES polyethersulfone (Defilter), pore size: 0.45 μm;

[0077] Medium volume: 10 ml; Sampling time: 1h, 2h, 3h, 4h, 5h, 6h;

[0078] Sampling volume: 1ml; Refill volume: 1ml; Diffusion cell orifice diameter: 15mm; Diffusion cell orifice area: 1.77cm 2 ;

[0079] The HPLC chromatographic conditions used an Agela C18 (4.6×250 mm, 5 μm) column; pH 2.5 phosphate buffer-methanol (60:40) as the mobile phase; a flow rate of 1.0 ml per minute; a detection wavelength of 220 nm; a column temperature of 30°C; and an injection volume of 20 μl.

[0080] The results are shown in Table 5 and Figure 2-3As shown: SE-2024-006 and SE-2024-012 have good properties and stability, and the release rates of the two batches in vitro are relatively consistent, lasting up to 6 hours.

[0081] Table 5: In vitro release test results

[0082]

[0083] 3. In vitro penetration test

[0084] In vitro permeation testing was performed on external preparations with batch numbers SE-2024-006 and SE-2024-012. The specific testing process and results are as follows:

[0085] Device: vertical diffusion cell; temperature: 32°C; rotation speed: 600rpm;

[0086] Sample volume: 75 mg (0.5 mm quantitative loop); Release medium: pH 6.8 phosphate buffer solution;

[0087] Skin: pig skin (abdomen);

[0088] Medium volume: 10ml; Sampling volume: 1.5ml; Rehydration volume: 1.5ml;

[0089] Diffusion cell orifice diameter: 15mm; Diffusion cell orifice area: 1.77cm 2 ;

[0090] Sampling time: 1h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, 24h.

[0091] The HPLC chromatographic conditions used an Agela C18 (4.6×250 mm, 5 μm) column; pH 2.5 phosphate buffer-methanol (60:40) as the mobile phase; a flow rate of 1.0 ml per minute; a detection wavelength of 220 nm; a column temperature of 30°C; and an injection volume of 20 μl.

[0092] The results are as follows Figure 6 and Figure 4 , 5 As shown: The in vitro permeation flux of SE-2024-006 and SE-2024-012 can be maintained for 24 hours.

[0093] Table 6: In vitro permeation test results

[0094]

[0095]

[0096] 4. Study on the effect of topical preparations with batch numbers SE-2024-006 and SE-2024-012 on the mouse chloasma model 1. Animal selection and identification

[0097] C57BL / 6J mice (SFP) were selected as experimental subjects and provided by Jinan Mingyue Experimental Animal Breeding Co., Ltd. The mice lived in a well-ventilated environment with 12 hours of normal light per day, room temperature of 20-26°C, relative humidity of 40-70%, and indoor ventilation. The mice needed to be fed for a week to adapt to the laboratory environment before the experiment. Five groups were designed, with 10 animals in the normal control group and 12 animals in each of the other groups, a total of 58 animals were required to be enrolled, so this experiment expected 60 animals to be screened.

[0098] Each group of animals was numbered from 1 to n. Picric acid was used to rub the fur of different parts of the animals for marking.

[0099] Table 7: Staining sites of animals in each group

[0100]

[0101] 2. Animal Model Preparation

[0102] Under anesthesia with a 10:1 mixture of Shutai and Su Mianxin diluted 1 time, the back was shaved to fully expose the skin, the shaved area was 2cm×2cm, and the hair was shaved once a week; each mouse was injected with progesterone injection solution in the hind limb muscle, 20mg / kg, and the two hind limbs were injected alternately, once a day, for 39 consecutive days. 30 minutes after the injection, UVB with a wavelength of 275-320nm was selected, the distance between the mouse skin and the light source was 20cm, and the skin was locally irradiated once a day, each time for 45 minutes, and the group medication was carried out after 39 consecutive days.

[0103] 3. Animal Grouping

[0104] Group design: A total of 5 groups were set up, namely normal control group, model control group, SE-2024-006 (ointment) group, SE-2024-012 (gel) group, and hydroquinone cream positive control group;

[0105] Number of animals: 10 animals in the normal control group, 11 animals in the model control group and the positive control group, 12 animals in the SE-2024-006 (ointment) group and the SE-2024-012 (gel) group, a total of 56 animals;

[0106] Gender ratio: All females;

[0107] Grouping method: The groups were balanced according to the severity scores of modeling.

[0108] See Table 8 for specific grouping information.

[0109] Table 8: Dosage regimen and group information

[0110]

[0111]

[0112] Note: The first digit of the animal number represents the group, and the last two digits represent the animal serial number.

[0113] Each medication group took an appropriate amount of the corresponding ointment and applied it evenly on the shaved skin of the back, while the normal control group and the model control group did not apply any medicine. Apply twice a day for 60 consecutive days.

[0114] 4. Index detection

[0115] The classification of apparent indicators is shown in Table 9.

[0116] Table 9: Classification of apparent indicators

[0117]

[0118] 1) Determination of tyrosine content and MDA and SOD activity in skin tissue

[0119] Each time, an appropriate amount of skin from the ultraviolet irradiated area on the back of the mouse was taken and made into 10% tissue homogenate in an ice bath. The activities of tyrosinase (TYR) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the skin tissue were determined by ELISA strictly according to the method provided by the kit.

[0120] 2) Histopathological examination

[0121] After 30 days of drug administration, 5 mice were selected from the normal control group, 5 mice from the model control group, 5 mice from the positive control group, and 6 mice from each of the two test groups, for a total of 27 mice; after 60 days of drug administration, 5 mice were selected from the normal control group and 6 mice from each of the other groups, for a total of 29 mice. After euthanasia, the skin tissue of the lesion area was fixed with formaldehyde, and HE staining was used to observe skin pathological changes. The Masson-Fontana staining method was used to observe the number, distribution and deposition of melanocytes.

[0122] V. Experimental Results and Analysis

[0123] 1) Symptom observation and scoring at the back modeling site

[0124] After modeling, the mice had severe skin damage, with obvious spots, larger spots, dull skin, and fur loss. Seven days after successful modeling, the model group began to show mild symptoms on the back that alleviated on their own, and the score decreased slightly.

[0125] As shown in Table 10, compared with the model group, the symptom scores of SE-2024-006 (ointment) and SE-2024-012 (gel) were significantly reduced starting from 7 days after administration (p < 0.05 or p < 0.01), and continued until the end of 60 days of administration, with the scores getting lower and lower, indicating that the skin symptoms of melasma are getting milder.

[0126] Compared with the model group, the symptom score of the hydroquinone cream control group began to decrease significantly from the 21st day of administration (p < 0.05 or p < 0.01), indicating that the onset of action was later than SE-2024-006 (ointment) and SE-2024-012 (gel). As the administration time prolonged, the symptom score gradually decreased, with statistical differences on the 21st, 28th, and 35th days (p < 0.05 or p < 0.01), but after 42 days, as the model recovered on its own, there was no statistical difference in the skin symptom score between the hydroquinone cream control group and the model group (p > 0.05).

[0127] Compared with hydroquinone cream, the skin symptom scores of mice in the SE-2024-006 (ointment) and SE-2024-012 (gel) groups were significantly reduced since 21 days after administration, among which statistical differences were shown on the 42nd, 56th and 60th days after administration of SE-2024-006 (ointment) (p < 0.05 or p < 0.01); statistical differences were shown on the 21st, 30th, 35th, 42nd, 49th, 56th and 60th days after administration of SE-2024-012 (gel) (p < 0.05 or p < 0.01); this indicates that the effects of SE-2024-006 (ointment) and SE-2024-012 (gel) on chloasma are significantly better than those of hydroquinone cream.

[0128] Table 10: Observation scores of the back of chloasma mice

[0129]

[0130]

[0131] Note: * is p<0.05, ** is p<0.01, compared with the model group. # is p<0.05, ## is p<0.01, compared with the hydroquinone cream group. 0-28d: blank group n=10, model group and positive control group n=11, SE-2024-006 and SE-2024-012 groups n=12; 30d: each group n=5; 35-60d: blank group n=5, other groups n=6.

[0132] 2) TYR, SOD activity and MDA content in skin tissue

[0133] Compared with the normal group, the TYR activity in the skin tissue of the mice in the model group was significantly increased, the SOD activity was significantly decreased, and the MDA content was significantly increased (p < 0.01), indicating that the membrane-forming factors had a significant effect on the TYR, SOD activity and MDA content of the mice, and the model was successfully prepared.

[0134] Compared with the model group, after 30 days of administration, the TYR activity in the skin tissue of mice in the hydroquinone cream group, SE-2024-006 (ointment) and SE-2024-012 (gel) groups was slightly decreased, the SOD activity was slightly increased, and the MDA content was slightly decreased, but there was no statistical difference (p>0.05), indicating that the effect of 30 days of administration was not obvious. After 60 days of administration, the TYR activity in the skin tissue of mice in the hydroquinone cream group, SE-2024-006 (ointment) and SE-2024-012 (gel) groups was decreased, the SOD activity was increased, and the MDA content was decreased, but only the TYR activity in the SE-2024-012 (gel) group showed a statistical difference (p<0.01), indicating that SE-2024-012 (gel) had a significant inhibitory effect on the TYR activity in the skin tissue of model mice after 60 days of administration, and the other groups also had a certain inhibitory effect.

[0135] Table 11-1 TYR, SOD activities and MDA content in skin tissue of mice treated for 30 days ( )

[0136]

[0137] Note: ## means p<0.01, compared with the normal control group.

[0138] Table 11-2 TYR, SOD activity and MDA content in skin tissue of mice treated for 60 days ( )

[0139]

[0140] Note: **p<0.01, compared with the model group; ##p<0.01, compared with the normal control group.

[0141] 3) Pathological examination results

[0142] Pathological results after 60 days of drug administration Figure 6 and Figure 7 As shown, Figure 6 Representative images of HE staining of the normal control group (Figure A), model control group (Figure B), positive control group (Figure C), SE-2024-006 ointment group (Figure D), and SE-2024-012 gel group (Figure E) after 60 days of drug administration; Figure 7Representative images of MF staining of the normal control group (Figure A), model control group (Figure B), positive control group (Figure C), SE-2024-006 ointment group (Figure D) and 10% SE-2024-012 gel group (Figure E) after 60 days of drug administration.

[0143] VI. Conclusion

[0144] (1) Back symptom observation score

[0145] SE-2024-006 (ointment), SE-2024-002 (gel), and hydroquinone cream have significant improvements on the skin appearance of melasma model mice, including spot color, area, fur loss, and skin color. The improvement effects of SE-2024-006 (ointment) and SE-2024-002 (gel) are stronger than those of hydroquinone cream.

[0146] (2) TYR, SOD activity and MDA content in skin tissue

[0147] SE-2024-006 (ointment), SE-2024-002 (gel), and hydroquinone cream had certain effects on TYR activity, increasing SOD activity, and reducing MDA content in the skin of melasma model mice, but only SE-2024-012 (gel) had a significant inhibitory effect on TYR activity after 60 days of administration.

[0148] (3) Pathological examination results

[0149] Administration of SE-2024-006 (ointment), SE-2024-012 (gel), and hydroquinone cream for 60 days significantly improved epidermal thickening and dermal collagen fiber disorder in chloasma model mice, significantly inhibited inflammatory cell infiltration, significantly improved the number and distribution of pigment cells, and significantly reduced melanocyte deposition. The improvement effect of SE-2024-012 (gel) was better than that of the hydroquinone cream positive control. Administration for 30 days only slightly improved epidermal thickening and inflammatory cell infiltration in model mice, but had no significant improvement on the number and distribution of pigment cells.

[0150] (4) Conclusion:

[0151] Under the experimental conditions: SE-2024-006 (ointment), SE-2024-012 (gel) and hydroquinone cream have significant therapeutic effects on mouse melasma; the intensity of their effects is SE-2024-012 (gel) > SE-2024-006 (ointment) > hydroquinone cream.

Claims

1. A method for preparing a topical preparation of microsphere-loaded drugs for treating chloasma, characterized in that: The loaded drug is tranexamic acid; It includes the following steps: 1) Preparation of polyvinyl alcohol acrylate microspheres Add sodium dodecyl diphenyl ether disulfonate and polyvinyl alcohol into water and stir to dissolve to form the aqueous phase; Take monomers ethyl acrylate, hexadecane and azobisisobutyronitrile and stir and mix them evenly to form the oil phase; The aqueous phase is slowly added to the oil phase under stirring, and stirred evenly to prepare a milky white pre-emulsion; then emulsified to obtain a polyvinyl alcohol acrylate microsphere emulsion; 2) Drug loading Adding tranexamic acid to the polyvinyl alcohol acrylate microsphere emulsion and stirring and dispersing the mixture, and then performing ultrasound, stirring and reacting the mixture after the ultrasound is completed, filtering the mixture, and naturally air-drying the mixture after the filtrate is concentrated under reduced pressure to obtain drug-loaded microspheres; 3) further preparing an ointment or gel external preparation.

2. The preparation method according to claim 1, characterized in that: In the step 1), the mass ratio of sodium dodecyl diphenyl oxide disulfonate, polyvinyl alcohol, monomer ethyl acrylate, hexadecane and azobisisobutyronitrile is 1:18-22:28-32:0.8-1.2:0.4-0.

6.

3. The preparation method according to claim 1, characterized in that: The drug loading amount of tranexamic acid is 12-15%.

4. The preparation method according to claim 1, characterized in that: The components and weight proportions of the ointment in step 3) are: 28-32 of stearic acid, 3-5 of castor oil, 8-12 of glyceryl monostearate, 18-22 of glycerol, 24-28 of triethanolamine, 20-25 of drug-loaded microspheres, and 40-60 of distilled water.

5. The preparation method according to claim 4, characterized in that: The preparation method of the ointment in step 3) is as follows: heating stearic acid, glyceryl monostearate, and castor oil to 50-60° C. until they are completely dissolved, keeping the temperature, preparing an oil phase for standby use; heating glycerol, drug-loaded microspheres, and distilled water to 50-60° C., slowly adding triethanolamine under stirring until the drug-loaded microspheres are completely dissolved, keeping the temperature, preparing an aqueous phase for standby use; adding the oil phase to the aqueous phase under stirring, stirring and dispersing after the addition is completed, cooling to 30-40° C. for filling and sealing.

6. The preparation method according to claim 1, characterized in that: The components of the gel external preparation in step 3) are: 100 parts gelatin, 28-32 parts glycerol, 40-50 parts carbomer, 12-18 parts xanthan gum, 30-35 parts drug-loaded microspheres, and 8-12 parts deionized water.

7. The preparation method according to claim 6, characterized in that: The preparation method of the gel external preparation in step 3) is as follows: adding drug-loaded microspheres, deionized water and glycerol to an oil phase tank, and then heating to 50-60° C. to dissolve, to prepare a mixed material 1 for use; adding gelatin, xanthan gum and carbomer to another oil phase tank, and then heating to 60-90° C. to completely melt, to prepare a mixed material 2 for use; transferring the mixed material 2 and the mixed material 1 to an emulsifying tank in turn and vacuum stirring them evenly; then cooling to 40-50° C., starting high-speed stirring to complete dispersion, and continuing to cool to 35-40° C., filling is completed, and sealing is performed.

8. An external preparation for treating chloasma prepared by the method according to any one of claims 1 to 7, comprising microspheres loaded with drugs.

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