Baicalein topical formulation and use thereof in treatment of atopic dermatitis

Abstract

Disclosed in the present invention are a baicalein topical formulation and use thereof in the treatment of atopic dermatitis. The active ingredient of the formulation is baicalein, which is combined with a pharmaceutical excipient to prepare a dosage form that can be applied externally to the skin and mucous membranes. The topical formulation can be used in the treatment of atopic dermatitis and is characterized by convenience in use and significant efficacy, thereby offering broad application prospects.

Description

A baicalein external preparation and its application in treating atopic dermatitis Technical Field

[0001] The present invention relates to pharmaceutical preparations and applications, and in particular to a baicalein external preparation and its application in treating atopic dermatitis. Background Art

[0002] Atopic dermatitis is a chronic, relapsing inflammatory skin disease characterized by dry skin, chronic eczematous lesions, and significant itching. Over the past few decades, the prevalence and incidence of atopic dermatitis have steadily increased. Data indicate that the global prevalence among children ranges from 15% to 20%, and among adults as high as 10%. Atopic dermatitis has become the 15th most common non-fatal disease and the skin disease with the highest disease burden. According to the medication recommendations in the "Guidelines for the Diagnosis and Treatment of Atopic Dermatitis in China" (2020 edition), drug treatment is categorized into topical and systemic medications. Topical medications are first-line treatments, but long-term, large-area use can cause cutaneous and systemic adverse reactions. Maintenance therapy for topical medications involves topical anti-inflammatory drugs, such as moderate-to-low-potency glucocorticoids or calcineurin inhibitors, to minimize recurrences. Adverse reactions primarily include local burning and irritation. Systemic medications are primarily used as adjunctive therapy for atopic dermatitis and include oral antihistamines, immunosuppressants, and glucocorticoids. Based on this, a safe and effective topical drug preparation that can be used to treat atopic dermatitis is urgently needed by society.

[0003] Baicalein is a monomer compound extracted and separated from the traditional Chinese medicine Scutellaria baicalensis. Its chemical name is 5,6,7-trihydroxyflavone (5,6,7-trihydroxyflavone) and its molecular formula is C 15 H 10 O5 (MW.270.24). Baicalein has multiple pharmacological effects, including antibacterial, antiviral, antiallergic, and immunomodulatory. It is one of the flavonoids with the highest content in Scutellaria baicalensis.

[0004] Currently, there is no patent reporting on the application of baicalein external preparations in the treatment of atopic dermatitis. The present invention designs and uses an external preparation in the form of an external preparation to explore the application of baicalein in the treatment of atopic dermatitis.

[0005] Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a baicalein external preparation and prove its good application effect in the treatment of atopic dermatitis.

[0007] The compound baicalein described in the present invention has a structural formula as shown below:

[0008] Technical solution: The external preparation of baicalein of the present invention is a pharmaceutical liquid preparation prepared by dissolving the raw material powder of baicalein or its pharmaceutically acceptable salt in a clear solution obtained by mixing any one solvent selected from PEG300, PEG400, propylene glycol, and ethanol or a combination of multiple solvents.

[0009] The external preparation of baicalein and the liquid pharmaceutical preparation are sprays.

[0010] The topical preparation of baicalein is a liquid drug preparation poured and sealed in a container with a single opening. The liquid drug preparation is released from the container into the atmosphere through accessories of the open container at atmospheric pressure. After entering the atmosphere, the liquid drug preparation can form liquid particles with a particle size of microns. The liquid particles can adhere to the outer layer of keratinocytes or mucosal epithelial cells of an organism.

[0011] The baicalein external preparation or the liquid pharmaceutical preparation can be further prepared into an ointment or gel by adding pharmaceutical excipients to increase the viscosity of the preparation. Preferably, the viscosity of the preparation is further modified so that the preparation can adhere to the keratinocytes and mucosal cell layers without using a specific container or device based on the atmospheric pressure principle.

[0012] The external preparation of baicalein, the pharmaceutical excipients include one or more of a solvent, an oil phase matrix, an emulsifier, a thickener, a pH regulator, and an antibacterial agent; wherein the solvent includes glycerol and water, the oil phase matrix includes liquid paraffin, vaseline, stearic acid, glyceryl monostearate, and white beeswax, the emulsifier includes Tween 80 and polyoxyethylene hydrogenated castor oil RH40, the thickener includes sodium carboxymethyl cellulose, xanthan gum, sodium hydroxypropyl methylcellulose, an aqueous gel matrix carbomer, chitosan, sodium alginate, sodium carboxymethyl cellulose, and sodium hydroxypropyl methylcellulose, the pH regulator includes triethanolamine, acetic acid, and hydrochloric acid, and the antibacterial agent includes benzalkonium chloride.

[0013] The external-use preparation of baicalein and the spray are clear and transparent solutions.

[0014] The external preparation of baicalein, the clear and transparent solution is a solution that is colorless, light yellow, bright yellow, or dark yellow to the naked eye.

[0015] Application of the external preparation of baicalein in atopic dermatitis medicine.

[0016] The application, the atopic dermatitis is a pathological phenomenon in which an organism is exposed to unknown substances or causes an inflammatory reaction and manifests on the surface of the skin or mucous membrane.

[0017] The application exerts an inhibitory effect on inflammation after contacting the keratinocytes or mucosal cell layer of an organism having atopic dermatitis.

[0018] In the external preparation of baicalein, the mass volume ratio of baicalein to other solvents or excipients is 0.005-0.015 g / mL, preferably 0.01 g / mL.

[0019] Baicalein is prepared into an external preparation that can be used for atopic dermatitis. The anti-inflammatory and antibacterial pharmacological effects of the external preparation of baicalein are beneficial to the recovery of chronic eczema-like skin damage. At the same time, the anti-allergic and immunomodulatory effects of baicalein are also beneficial to alleviate the clinical symptoms of itching of atopic dermatitis.

[0020] Beneficial effects: This topical preparation can significantly improve the pathological signs of atopic dermatitis skin tissue, reduce the transcriptional levels of inflammatory factors in the lesions, significantly reduce the degree of transepidermal water loss in the lesions of atopic dermatitis skin tissue in mice, and repair the skin barrier. Its effect is better than levocetirizine, a commonly used drug for atopic dermatitis. Compared with the existing technology, the topical preparation of baicalein has a more significant therapeutic effect on atopic dermatitis by the commonly used route of administration of baicalein (oral). The present invention has demonstrated through experiments that the topical preparation of baicalein has a significant therapeutic effect on atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a picture of the appearance and properties of the baicalein external preparation in Example 4;

[0022] FIG2 shows the effect of the baicalein external preparation on the skin of atopic dermatitis-like mice in Example 5;

[0023] FIG3 shows the effect of the baicalein external preparation on the pathological changes of the skin tissues of atopic dermatitis-like mice in Example 5;

[0024] FIG4 shows the effects of the baicalein external preparation in Example 5 and Comparative Example 1 on mast cell infiltration in the skin of atopic dermatitis-like mice;

[0025] Figure 5 shows the effects of baicalein gavage and topical preparation on involucrin and filaggrin in the epidermis of eczema-like mice in Comparative Example 1; DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] Preparation method of baicalein spray external preparation

[0031] Baicalein is highly hydrophobic and poorly water soluble. This example systematically studies the solubility of baicalein in different excipients. Research methods:

[0032] Method 1: An excess of the raw material baicalein (Batch No. HR228W3) is placed in an appropriate amount of excipient or excipient solution and observed for dissolution by ultrasound. After centrifugation at 10,000 rpm for 10 minutes, the supernatant is filtered through a 0.45 μm PES membrane. The filtrate is diluted or undiluted and assayed using HPLC or UV spectrophotometry.

[0033] Method 2: Weigh the raw material baicalein (batch number: HR228W3), add it to the excipients, and observe the dissolution phenomenon by ultrasound. If it is clearly dissolved, continue to add and record the amount of raw material added.

[0034] Table 1 Saturated solubility of baicalein in different excipients or excipient solutions

[0035] Note [1]: Except for this auxiliary material, which adopts method 2, the rest of the auxiliary materials adopt method 1.

[0036] The above results (Table 1) show that baicalein has a high solubility in PEG300, PEG400, propylene glycol, and 75% ethanol. Therefore, the present invention uses PEG300, propylene glycol, and ethanol as solvents to prepare baicalein spray.

[0037] Prepared according to the following recipe process:

[0038] Table 2 Baicalein spray prescription (300mL)

[0039] Preparation method:

[0040] 1. Measure the prescribed amount of PEG300, propylene glycol (if included in the prescription), and ethanol (if included in the prescription) and mix them evenly to obtain mixture 1;

[0041] 2. Weigh the prescribed amount of baicalein and add it to mixture 1. Stir well and ultrasonicate for 5-30 minutes to dissolve it to obtain mixture 2.

[0042] 3. Measure the prescribed amount of purified water, add it to mixture 2, stir evenly, and obtain a yellow-brown clear solution.

[0043] Table 3 Application feeling of baicalin spray

[0044] The above results (Table 3) show that increasing the amount of propylene glycol results in a more sticky feel upon application, while increasing the amount of ethanol results in a lighter feel upon application. Considering that the primary skin symptoms of atopic dermatitis patients are dry skin and chronic eczema, clinical medications require careful attention to moisturizing properties. Propylene glycol has properties similar to those of glycerin and offers excellent moisturizing properties, so Spray 1 was selected as the optimal formulation.

[0045] Example 2

[0046] Stability study of a baicalein external preparation using an atmospheric pressure device

[0047] The topical preparation was prepared according to the optimal prescription in Example 1 and placed under accelerated conditions (40°C ± 2°C, RH 75% ± 5%) and long-term conditions (25°C ± 2°C, RH 60% ± 5%). Samples were taken at 0, 5, 9, and 15 days to detect the content and related substances. The statistical method for related substances was the area normalization method.

[0048] Table 4 Baicalein atmospheric pressure device spray external preparation 6 prescription (according to 300ml package)

[0049] Preparation process: Measure the prescribed amount of PEG300 and propylene glycol and mix them evenly to obtain mixture 1; weigh the prescribed amount of baicalein, add it to mixture 1, stir evenly, and then ultrasonicate for 5 to 30 minutes to dissolve it, to obtain mixture 2; measure the prescribed amount of purified water, add it to mixture 2, stir evenly, and obtain a yellow-brown clear solution.

[0050] Table 5 Stability test results of baicalein spray external preparation 6 in atmospheric pressure device

[0051] The above results (Table 5) show that the content and related substances of the topical preparation are relatively stable under accelerated conditions (40°C ± 2°C, RH 75% ± 5%) and long-term conditions (25°C ± 2°C, RH 60% ± 5%) for 15 days.

[0052] Example 3

[0053] Method for preparing an external preparation of baicalein after further adding pharmaceutical excipients

[0054] The baicalein raw material is dark in color, and the liquid particles released by atmospheric pressure easily stain clothing, making it difficult to clean and having low adhesion. Considering the compliance and ease of use of clinical patients, this example attempts to further add suitable pharmaceutical excipients to the preparation to improve the viscosity of the preparation, increase the adhesion of the drug, and enable the drug to be released without atmospheric pressure. The following table shows the formula and process for preparation:

[0055] Table 6 Prescription of external preparation of baicalein after further adding pharmaceutical excipients

[0056] Preparation method:

[0057] 1. Preparation of oil phase: Weigh the prescribed amount of oil phase, heat to 75℃ and melt for later use;

[0058] 2. Preparation of drug-containing solution: Weigh the prescribed amount of API, PEG300, propylene glycol, and ethanol, mix well, and heat at 75°C to dissolve clearly. This is the drug-containing solution and set aside.

[0059] 3. Preparation of aqueous phase: Weigh the prescribed amount of purified water, heat it to 75°C, add sodium carboxymethyl cellulose, dissolve it, add polyoxyethylene hydrogenated castor oil RH40, and set aside;

[0060] 4. Preparation: Maintaining a temperature of 75°C, slowly add the oil phase to the aqueous phase while high shearing at 3000-6000 rpm for 5-30 minutes. Once a uniform preparation is obtained, slowly add the drug-containing solution and continue high shearing at 3000-6000 rpm for 5-30 minutes. Cool to complete.

[0061] Table 7 Appearance and properties of the preparation

[0062] The above results (Table 7) show that formulations 2 to 4 can all be prepared with moderate viscosity after cooling and uniformity without stratification.

[0063] Example 4

[0064] Method 2 for preparing an external preparation of baicalein cream further adding pharmaceutical excipients

[0065] In order to increase the advantages of the preparation such as easy spread, comfortable feeling, non-greasy, easy to wash off, and no interference with the normal physiological functions of the skin, based on this, this embodiment provides a preparation method of a new preparation and explores its feasibility.

[0066] Prepared according to the following prescription and process:

[0067] Table 8 Prescription of external preparation of baicalein after further adding pharmaceutical excipients

[0068] Preparation method:

[0069] 1. Weigh the prescribed amount of carbomer and add it to the prescribed amount of water. Leave it overnight until it swells naturally. Then add the prescribed amount of triethanolamine while stirring until a transparent colloidal liquid is formed.

[0070] 2. Preparation of drug-containing solution: Weigh the prescribed amount of baicalein, PEG300, propylene glycol (if included in the prescription), glycerin (if included in the prescription), and ethanol (if included in the prescription), stir well, and sonicate for 5-30 minutes. After dissolving, add the prescribed amount of 50% benzalkonium chloride ethanol solution, mix well, and set aside.

[0071] 3. Add the drug-containing solution to the colloidal liquid in "Step 1", stirring while adding, mix evenly, and form a yellow transparent gel.

[0072] Table 9 Preparation application feeling

[0073] Comparison of the appearance and properties of the preparations with different formulations (Figure 1) and the application feeling (Table 9) showed that Preparation 8 can achieve better appearance and more comfortable application feeling.

[0074] Example 5

[0075] Study on the main pharmacodynamic effects of baicalin topical preparation in the treatment of atopic dermatitis

[0076] 1. Experimental Materials

[0077] (1) Drugs

[0078] Baicalein external preparation (10 mg / ml) prepared in Example 1

[0079] (2) Experimental animals

[0080] C57BL / 6J mice, female, 8 weeks old, were housed in an SPF environment at a temperature of 20-25°C, a relative humidity of 30-70%, and a 12:12 h light-dark cycle; they had free access to water and food.

[0081] (3) Reagents

[0082] 1) Calcipotriol (MC903): MCE Company, Product No.: HY-10001.

[0083] 2) Positive drug: Levocetirizine: MCE Company, product number: HY-B0814.

[0084] 3) PBS solution: Dissolve 8.0 g of NaCl, 2.0 g of KH2PO4, 1.56 g of Na2HPO4·H2O, and 0.20 g of KCl in 1000 mL of ultrapure water, sterilize with high-pressure steam, and store at 4°C.

[0085] 2. Experimental methods:

[0086] 19 model mice were randomly divided into 5 groups according to body weight, namely blank control group, model + solvent control group, positive drug control group, baicalein low-dose group and baicalein high-dose group, with 6 mice in each group. The grouping is shown in the table below:

[0087] Table 10 Animal grouping

[0088] Except for the blank group, 10 nmol of MC903 was applied to the left ear skin of mice in the other groups to establish a mouse atopic dermatitis model. After modeling, different doses of the test samples were given according to the grouping for treatment. The test sample group was administered by applying the drug to the affected skin of the mice, once a day for 13 consecutive days, and the positive drug control group was gavage-administered once a day for 13 consecutive days, with a dosage volume of 0.1 mL / 10 g. The ear margin thickness of the mice was measured every three days for seven days before the experiment, and every two days for seven days after the experiment. On the last day of the experiment, the mice were killed by cervical dislocation, and the ear tissue of the same area was taken and weighed.

[0089] HE staining was used to analyze the pathological changes of mouse ear tissues, and the epidermal thickness was calculated and statistically analyzed.

[0090] The transcription levels of IL-4 and TSLP in the mouse model skin tissue were detected by real-time fluorescence quantitative PCR.

[0091] 3. Statistical methods:

[0092] All measurement data are The comparison among multiple groups was performed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

[0093] 4. Experimental results:

[0094] Table 11 Effect of external application of baicalein on the thickness of the ear margin of mice ( n=6)

[0095] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0096] Table 12 Effect of external application of baicalein on the thickness of the ear margin of mice ( n=6)

[0097] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0098] Table 13 Effect of external application of baicalein on epidermal thickness in atopic dermatitis-like mice

[0099] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0100] Table 14 Effects of external application of baicalein on the expression of specific cytokines in mice with atopic dermatitis ( n=6)

[0101] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0102] Table 15 Effect of external application of baicalein on mast cell infiltration in the skin of atopic dermatitis-like mice

[0103] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0104] The effect of topical baicalein on MC903-induced atopic dermatitis-like symptoms ( Figure 2 ) showed that topical baicalein could significantly alleviate atopic dermatitis-like symptoms.

[0105] The measurement results of the ear margin thickness of mice treated with external application of baicalein (Table 11 and Table 12) showed that the ear margin thickness of the control group mice remained within a certain range during the experiment and did not increase significantly; the ear margin thickness of the model group mice increased significantly with the passage of modeling time; the external application of baicalein inhibited the increase in the ear margin thickness of mice to a certain extent, and the effect was comparable to that of the positive drug.

[0106] The pathological changes of mouse ear tissue after external application of baicalein (Figure 3 and Table 13) showed that external application of baicalein could significantly inhibit the thickening of the mouse epidermis.

[0107] The effect of topical baicalein on the transcriptional levels of IL-4 and TSLP (Table 14) showed that the transcriptional levels of IL-4 and TSLP in the model group were significantly increased compared to the blank control group. In the group treated with topical baicalein, baicalein significantly reduced the transcriptional levels of IL-4 and TSLP, achieving the effect of reducing the levels of specific cytokines and improving the symptoms of atopic dermatitis.

[0108] The results of the effect of topical baicalein on mast cell infiltration in the skin of atopic dermatitis-like mice (Table 15, Figure 4) showed that compared with the blank control group, the level of mast cells in the model group was significantly increased. In the group treated with topical baicalein, baicalein significantly reduced the level of mast cells, and the effect was equivalent to that of positive drugs.

[0109] The above results indicate that baicalein topical preparations can play a role in the treatment of atopic dermatitis.

[0110] Comparative Example 1

[0111] Comparative testing of the commonly used oral (po) route of baicalein and the baicalein topical preparation of the present invention (Example 1). Animal experimental studies showed that both oral and topical baicalein administration had significant inhibitory effects on atopic dermatitis, but the topical baicalein preparation was more effective. Its inhibitory effect on mouse skin thickness (Table 16), its effect on involucrin and filaggrin in the mouse epidermis (Figure 5), and its ability to inhibit mast cell infiltration in mouse skin (Figure 4) were all superior to those of baicalein administered via the commonly used oral route.

[0112] Table 16 Comparison of the efficacy of baicalein administered orally and skin preparations in AD-like mouse models

[0113] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model control group.

[0114] Comparative Example 2

[0115] Published technology has previously disclosed the use of a baicalein topical preparation (hereinafter referred to as "Preparation BE") for atopic dermatitis [Therapeutic effects of Baicalein on atopic dermatitis-like skin lesions of NC / Nga mice induced by dermatophagoides pteronyssinus, Int Immunopharmacol. 2010 Sep; 10(9): 1142-8. doi: 10.1016 / j.intimp.2010.06.020. Epub 2010 Jul 9.]. The data from this technology showed that Preparation BE reduced the level of IL-4, an inflammatory indicator in atopic dermatitis, by 31.16%, which was lower than the 92.61% reduction achieved by this technology. This indicates that the therapeutic effect of the baicalein topical preparation prepared by this technology is significantly superior to that of the existing technology.

Claims

1. A baicalein external preparation, characterized in that: The liquid drug preparation is prepared by dissolving raw material powder of baicalein or its pharmaceutically acceptable salt in a clear solution obtained by mixing any one solvent of PEG300, PEG400, propylene glycol and ethanol or a plurality of solvents.

2. The external preparation of baicalein according to claim 1, characterized in that: The liquid drug preparation is a spray.

3. The external preparation of baicalein according to claim 2, characterized in that: The liquid drug preparation is poured and sealed in a container with a single opening. The liquid drug preparation is released from the container into the atmosphere through the accessories of the open container in the form of atmospheric pressure. After entering the atmosphere, the liquid drug preparation can form liquid particles with a particle size of micrometer level. The liquid particles can adhere to the outer layer of keratinocytes or mucosal epithelial cells of the organism.

4. The external preparation of baicalin according to claim 1, characterized in that: The liquid drug preparation can be made into an ointment or a gel after further adding pharmaceutical excipients to improve the viscosity of the preparation.

5. The external preparation of baicalein according to claim 4, characterized in that: The pharmaceutical excipients include one or more of a solvent, an oil phase matrix, an emulsifier, a thickener, a pH regulator, and an antibacterial agent; wherein the solvent includes glycerol and water; the oil phase matrix includes liquid paraffin, vaseline, stearic acid, glyceryl monostearate, and white beeswax; the emulsifier includes Tween 80 and polyoxyethylene hydrogenated castor oil RH40; the thickener includes sodium carboxymethyl cellulose, xanthan gum, sodium hydroxypropyl methylcellulose, an aqueous gel matrix carbomer, chitosan, sodium alginate, sodium carboxymethyl cellulose, and sodium hydroxypropyl methylcellulose; the pH regulator includes triethanolamine, acetic acid, and hydrochloric acid; and the antibacterial agent includes benzalkonium chloride.

6. The external preparation of baicalin according to claim 2, characterized in that: The spray is a clear and transparent solution.

7. The external preparation of baicalin according to claim 6, characterized in that: The clear and transparent solution is a solution that is colorless, light yellow, bright yellow or dark yellow to the naked eye.

8. Use of the external preparation of baicalin according to any one of claims 1 to 7 in atopic dermatitis medicine.

9. The use according to claim 8, characterized in that: Atopic dermatitis is a pathological phenomenon in which an organism is exposed to unknown substances or causes an inflammatory reaction and manifests itself on the surface of the skin or mucous membranes.

10. The use according to claim 8, characterized in that: It exerts an inhibitory effect on inflammation after contact with the keratinocytes or mucosal cell layer of organisms with atopic dermatitis.

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