Pharmaceutical composition for treating atopic dermatitis as well as preparation method and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating atopic dermatitis and a preparation method and application of the pharmaceutical composition for treating atopic dermatitis. The pharmaceutical composition comprises matrix vaseline, a humectant, a moistening agent, a repairing agent and an antioxidant. Two lipid components (gamma-linolenic acid and linoleic acid) are supplemented, so that eczema-like dermatitis can be obviously relieved, skin lesion inflammatory cell infiltration is reduced, the content of fatty acid in a skin barrier is recovered, treatment of atopic dermatitis is assisted, and the treatment effect on the atopic dermatitis is remarkable; the pharmaceutical composition disclosed by the invention has the advantages of quick effect on atopic dermatitis, small toxic and side effects, good safety and good medicinal prospect.

Description

Technical Field

[0001] The present invention relates to an external pharmaceutical composition for treating skin diseases and a preparation method thereof, and particularly relates to an external pharmaceutical composition for treating atopic dermatitis, a preparation method and an application thereof, belonging to the field of medicine. Background Art

[0002] Atopic dermatitis (AD) is a common chronic, recurrent, non-infectious inflammatory skin disease, also known as atopic eczema. According to whether other allergic diseases are combined, atopic dermatitis can be divided into two types: simplex type and mixed type. Among them, simplex atopic dermatitis only shows dermatitis, and can be further divided into endogenous type and exogenous type. At present, the cause of atopic dermatitis is not fully understood, and it may be related to multiple factors such as genetic factors, environmental factors, and immune system abnormalities.

[0003] Atopic dermatitis is characterized by repeated attacks, polymorphic lesions, dry skin, and severe itching. The occurrence of this disease is related to factors such as genetic susceptibility, immune imbalance, skin barrier function damage, and skin flora disorder. The treatment principle of atopic dermatitis is to restore the normal barrier function of the skin, relieve or eliminate clinical symptoms, find and eliminate inducing and / or aggravating factors, reduce and prevent recurrence, and improve the quality of life of patients.

[0004] At present, the treatment of atopic dermatitis mainly includes: using external emollients as a basic nursing method, applying glucocorticoids externally to the skin lesions and taking oral antihistamines. In severe cases, glucocorticoids can be used systemically or immunosuppressants, biological agents, and small molecule inhibitor drugs can be adopted. However, drug treatments all have problems of increased adverse reactions and weakened efficacy during long-term use.

[0005] The lipids on the skin surface are an important part of the skin barrier and play a crucial role in maintaining the integrity and function of the skin barrier. The lipids in the skin barrier include ceramides, cholesterol, and free fatty acids. These three components usually exist stably in the stratum corneum of the skin in a molar ratio of 1:1:1 to form a lipid barrier. Among them, free fatty acids (FFAs), most of which are non-branched long-chain saturated fatty acids (such as palmitic acid and stearic acid), a small number are monounsaturated long-chain fatty acids (such as palmitoleic acid and oleic acid) or polyunsaturated long-chain fatty acids (such as linoleic acid and eicosapentaenoic acid). FFAs account for about 10%-20% of the total lipid dry weight of the stratum corneum, and their content varies depending on the anatomical location. The content on the face (~20%) and abdomen (~19%) is higher than that on the lower limbs (~14%) and soles (~9%).

[0006] Although the currently commonly used topical ceramide drugs have a certain effect in repairing the skin barrier, a single component is difficult to fully meet the needs of patients with atopic dermatitis. Ceramide mainly supplements the ceramide component of the skin barrier, but the other main component in the skin barrier, medium-chain and long-chain fatty acids, also needs to be repaired, and the effect of a single ceramide on the overall skin metabolism regulation and improvement of inflammatory response is limited.

[0007] In patients with atopic dermatitis, the contents of both ceramide and medium-chain and long-chain free fatty acids are significantly decreased, resulting in dry skin and impaired barrier function. Microorganisms and allergens invade the epidermis through the damaged barrier, thereby inducing an inflammatory response and presenting clinical symptoms such as skin itching, erythema, and exudation. Previous studies have shown that unsaturated fatty acids can relieve eczema-like skin inflammatory responses by activating specific receptors to increase the level of regulatory T cells and the content of anti-inflammatory M2 macrophages. In current skin emollient products, ceramide is used as the main barrier repair component, while the clinical value of free fatty acids is ignored.

[0008] However, the applicant's research found that the contents of various long-chain fatty acids in the skin lesions of patients with atopic dermatitis are significantly decreased, mainly including two long-chain polyunsaturated fatty acids, γ-linolenic acid and linoleic acid. In an animal model, topical application of the above two lipid components can significantly relieve eczema-like skin inflammation and reduce the infiltration of inflammatory cells in the skin lesions.

[0009] The present invention supplements the deficiencies of the existing topical barrier repair drugs for atopic dermatitis and assists in the treatment of atopic dermatitis by restoring the fatty acid content in the skin barrier. Summary of the Invention

[0010] The object of the present invention is to address the limitations of existing topical repair drugs for treating atopic pruritus, such as single drug components, single repair effects, and poor effects on overall skin metabolism regulation and improvement of inflammatory responses. The present invention provides a pharmaceutical composition for treating specific dermatitis and its preparation method. The pharmaceutical composition of the present invention is topically applied to the skin lesions of specific dermatitis, supplementing two lipid components, significantly alleviating eczematous skin inflammation, reducing infiltration of inflammatory cells in the skin lesions, restoring the fatty acid content in the skin barrier, assisting in the treatment of atopic dermatitis, and having a significant therapeutic effect on specific dermatitis; the cream contains gamma-linolenic acid and linoleic acid, and these components can not only supplement the medium and long-chain fatty acids in the skin barrier, but also reduce the infiltration of inflammatory cells and inflammatory factors, and alleviate the inflammatory response. Gamma-linolenic acid and linoleic acid in the cream can regulate the oil balance of the skin and reduce transepidermal water loss. In addition, the main components gamma-linolenic acid and linoleic acid of the present invention are derived from natural plants, have good biocompatibility and low irritation, and are suitable for long-term use. The pharmaceutical composition of the present invention has a rapid onset of effect, low toxicity and side effects, and good safety for atopic dermatitis, and has good medicinal prospects.

[0011] To achieve the object of the present invention, on the one hand, the present invention provides a pharmaceutical composition for treating atopic dermatitis, comprising a matrix petrolatum, a humectant, a emollient, a repair agent, and an antioxidant.

[0012] Among them, in the pharmaceutical composition

[0013] The weight ratio of the humectant (glycerol) to the matrix petrolatum is (10 - 15):100, preferably (10 - 12.5):100, and more preferably 11.4:100;

[0014] The weight ratio of the emollient (gamma-linolenic acid) to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100;

[0015] The weight ratio of the repair agent (linoleic acid) to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100;

[0016] The weight ratio of the antioxidant (vitamin E, BHA, BHT, propyl gallate) to the matrix petrolatum is (0.5 - 3):100, preferably (1.26 - 1.69):100, and more preferably 1.26:100.

[0017] In particular, the weight ratio of antioxidant BHA to matrix petrolatum is (0.01 - 0.1):100, preferably (0.05 - 0.07):100, and more preferably 0.06:100. The weight ratio of antioxidant BHT to matrix petrolatum is (0.01 - 0.1):100, preferably (0.05 - 0.07):100, and more preferably 0.06:100. The weight ratio of antioxidant propyl gallate to matrix petrolatum is (0.01 - 0.1):100, preferably (0.05 - 0.07):100, and more preferably 0.06:100. The weight ratio of antioxidant vitamin E to matrix petrolatum is (0.5 - 2):100, preferably (0.8 - 1.5):100, and more preferably 1.08:100.

[0018] In particular, the moisturizer is selected from one or more of glycerol, hyaluronic acid, sorbitol, propylene glycol or urea, preferably glycerol;

[0019] The emollient is selected from one or more of γ-linolenic acid, olive oil, squalane, avocado oil or jojoba oil, preferably γ-linolenic acid;

[0020] The repair agent is selected from one or more of linoleic acid, ceramide, cholesterol, hyaluronic acid or squalene, preferably linoleic acid;

[0021] The antioxidant is a mixture of natural antioxidants and synthetic antioxidants, where the natural antioxidants are selected from one or more of vitamin E, tea polyphenols, astaxanthin, lycopene or coenzyme Q10; the synthetic antioxidants are a mixture of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene) and propyl gallate.

[0022] In particular, the weight ratio of natural antioxidant to synthetic antioxidant is (8 - 10):(1 - 2), preferably 9:1.5.

[0023] In particular, the weight ratio of the synthetic antioxidants BHA, BHT, and propyl gallate is (1 - 2):(1 - 1.5):(0.5 - 1), preferably 1:1:1.

[0024] On the other hand, the present invention provides a method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising the following steps carried out in sequence:

[0025] 1) Add a moisturizer to the heated and melted matrix petrolatum and stir evenly to form a first mixture;

[0026] 2) Add an emollient and a repair agent to the first mixture whose temperature has been reduced to 40 - 50°C in sequence, and stir evenly to form a second mixture;

[0027] 3) Add an antioxidant to the second mixture and stir evenly to obtain the product.

[0028] The purpose of homogenization is to make the internal components of the pharmaceutical composition more evenly distributed and reduce the impact of heterogeneity on product performance. Although natural antioxidants and synthetic antioxidants have been added and stirred evenly in step 3), the distribution of these components may still be somewhat uneven. Homogenization can further refine the particles through shear force, cavitation, and turbulence to ensure that all components are evenly dispersed in the matrix.

[0029] In particular, the antioxidant described in step 3) is a mixture of natural antioxidants and synthetic antioxidants, where the natural antioxidant is selected from one or more of vitamin E, tea polyphenols, astaxanthin, lycopene, or coenzyme Q10; the synthetic antioxidant is a mixture of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene), and propyl gallate.

[0030] In particular, adding the antioxidant in step 3) includes the following steps in sequence:

[0031] 3A) Add BHA (butylated hydroxyanisole) and BHT (dibutylhydroxytoluene) as synthetic antioxidants to the second mixture whose temperature has been reduced to 40 - 50 °C and stir evenly;

[0032] 3B) Add natural antioxidants and propyl gallate as a synthetic antioxidant to the second mixture whose temperature has been reduced to below 40 °C and stir evenly.

[0033] In particular, it further includes step 4), after cooling the mixture to which the antioxidant has been added and mixed evenly, perform homogenization treatment.

[0034] In particular, it further includes step 4A), measure the pH value of the mixture to which the antioxidant has been added and mixed evenly. If the pH value of the mixture is 5.5 - 6.5, then perform step 4), that is, perform homogenization treatment; if the pH value of the mixture is less than 5.5 or greater than 6.5, then use a pH regulator to adjust the pH of the mixture so that the pH value of the mixture is 5.5 - 6.5.

[0035] In particular, the pH regulator is selected from lactic acid and triethanolamine.

[0036] In particular, if the pH value of the mixture is less than 5.5, then add a triethanolamine solution to the mixture to adjust the pH value of the mixture to 5.5 - 6.5; if the pH value of the mixture is greater than 6.5, then add a lactic acid solution to the mixture to adjust the pH value of the mixture to 5.5 - 6.5.

[0037] In particular, the lactic acid solution is prepared as follows: 1 g of lactic acid is dissolved in 10 ml of sterile water; the triethanolamine solution is prepared as follows: 1 g of triethanolamine is dissolved in 10 ml of sterile water.

[0038] Among them, in step 1), the weight ratio of the humectant glycerol to the matrix petrolatum is (10 - 15):100, preferably (10 - 12.5):100, and more preferably 11.4:100.

[0039] In particular, in step 1), after heating the petrolatum to melt it into a liquid state, the humectant glycerol is added.

[0040] In particular, the melting temperature of the petrolatum is 60 - 70 °C, preferably 60 °C.

[0041] In particular, glycerol is heated to 60 - 70 °C, preferably 60 °C, and then added to the petrolatum and mixed with the petrolatum.

[0042] In particular, in step 2), the weight ratio of the emollient to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100; the weight ratio of the repair agent to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100.

[0043] In particular, in step 2), the weight ratio of the emollient γ-linolenic acid to the matrix petrolatum is (2 - 5):100, preferably 3.6:100; the weight ratio of the repair agent linoleic acid to the matrix petrolatum is (2 - 5):100, preferably 3.6:100.

[0044] In particular, in step 2), the weight ratio of the emollient to the repair agent is 1:(0.5 - 2), preferably 1:1.

[0045] In particular, in step 3), the weight ratio of the natural antioxidant to the matrix petrolatum is (0.5 - 2):100, preferably (0.8 - 1.5):100, and more preferably 1.08:100; the weight ratio of the synthetic antioxidant to the matrix petrolatum is (0.03 - 0.3):100, preferably (0.15 - 0.19):100, and more preferably 0.18:100.

[0046] In particular, in step 3), the weight ratio of the natural antioxidant to the matrix petrolatum is (0.4 - 1.8):100, preferably 0.96:100; the weight ratio of the synthetic antioxidant to the matrix petrolatum is (0.05 - 0.5):100, preferably 0.18:100.

[0047] In particular, the weight ratio of the natural antioxidant to the synthetic antioxidant is preferably (8 - 10) : (1 - 2), and more preferably 9 : 1.5.

[0048] In particular, in step 3), the natural antioxidant is selected from one or more of vitamin E, tea polyphenols, astaxanthin, lycopene or coenzyme Q10; the synthetic antioxidant is a mixture of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene) and propyl gallate.

[0049] In particular, in step 3), after adding the natural antioxidant, the synthetic antioxidant is added in the order of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene), and propyl gallate.

[0050] In particular, in step 3), the weight ratio of the natural antioxidant to the synthetic antioxidant is (8 - 10) : (1 - 2), preferably 9 : 1.5.

[0051] The pharmaceutical composition of the present invention is an external medicine capable of restoring the skin lipid barrier of atopic dermatitis, and is used for the treatment and daily care of patients with this disease. An external preparation containing unsaturated fatty acids γ-linolenic acid and linoleic acid is used for basic nursing treatment of skin barrier repair in patients with atopic dermatitis.

[0052] Use of the pharmaceutical composition for treating atopic dermatitis of the present invention: Apply the mixture externally to the lesions of atopic dermatitis without exudation or breakage and the dry skin without lesions, twice a day.

[0053] In the present invention, vaseline as a matrix also has lubricating and moisturizing effects, and synergistically with glycerol, increases the moisturizing effect of the drug and significantly reduces the trans-epidermal water loss of the skin; the moisturizer γ-linolenic acid also has an antioxidant effect and synergistically with the antioxidant, enhances the antioxidant capacity at the lesion site; the synthetic antioxidants BHA (butylated hydroxyanisole) and BHT (dibutylhydroxytoluene) can also improve the stability of the cream and improve the stability of the therapeutic effect of the cream.

[0054] Advantages and Benefits of the Present Invention

[0055] γ-Linolenic acid and linoleic acid in the pharmaceutical composition for treating atopic dermatitis of the present invention are important components of natural skin lipids and can promote the repair of the skin barrier. They can enhance the hydration of the skin, reduce water loss, and at the same time promote the normal metabolism of the stratum corneum.

[0056] Moreover, topical application of the polyunsaturated fatty acids γ-linolenic acid and linoleic acid can reduce the expression of pro-inflammatory factors such as IL-1b, and reduce the expression of type 2 inflammatory factors related to atopic dermatitis such as IL-4, IL-33, and TSLP, and has a significant anti-inflammatory effect;

[0057] γ-linolenic acid and linoleic acid are naturally occurring fatty acids, usually derived from vegetable oils (such as linseed oil, corn oil, etc.), and have good tolerance to human skin. The use of chemical additives is reduced, and the risks of allergy and irritation are lowered, making it particularly suitable for the sensitive skin of patients with atopic dermatitis.

[0058] The ointment of the pharmaceutical composition for treating atopic dermatitis containing long-chain unsaturated acids of the present invention can not only relieve the inflammatory reaction in the acute phase, but also prevent recurrence by repairing the skin barrier.

[0059] The pharmaceutical composition of the present invention is externally applied to the skin lesions, supplementing two lipid components (γ-linolenic acid, linoleic acid), significantly relieving the skin inflammatory reaction of atopic dermatitis, reducing the infiltration of inflammatory cells in the skin lesions, restoring the fatty acid content in the skin barrier, assisting in the treatment of atopic dermatitis, and having a significant therapeutic effect on atopic dermatitis; the pharmaceutical composition of the present invention has a rapid onset of action, low toxicity and side effects, and good safety for atopic dermatitis, and has good medicinal prospects. Description of the Drawings

[0060] Figure 1 For patients with mild localized atopic dermatitis, the changes in erythema and papules at the skin lesions after applying the cream.

[0061] Figure 2 For the changes in the severity of the disease before and after treatment.

[0062] Figure 3 For the changes in the severity of itching before and after treatment.

[0063] Figure 4 For the changes in the level of transepidermal water loss after applying the cream to patients with mild localized atopic dermatitis. *P<0.05, **P<0.001.

[0064] Figure 5 For the animal experiment process.

[0065] Figure 6A For the clinical manifestations at the end of the experiment of two groups of mice.

[0066] Figure 6B For the HE staining performance at the end of the experiment of two groups of mice.

[0067] Figure 6C For the immunohistochemical staining of IL-1β and IL-4 at the end of the experiment of two groups of mice.

[0068] Figure 7 For the infiltration levels of (A) IL-33, (B) IL-4, (C) IL-33, and (D) TSLP at the end of the experiment of two groups of mice, where MC903 is the control group of mice and MC903+GLA is the experimental group of mice. Detailed Description of the Invention

[0069] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and substitutions all fall within the protection scope of the present invention.

[0070] Example 1 Preparation of Ointment

[0071] (1) Melting of the matrix petrolatum

[0072] Heat the petrolatum to 65 ± 5 °C (usually 60 - 70 °C) until it is completely melted, and set aside;

[0073] The melting temperature of the auxiliary petrolatum should not be too high to avoid damaging the structure of the petrolatum. In this example, the amount of petrolatum used is 100 g as an illustration.

[0074] (2) Adding glycerol

[0075] 2 - 1) Preheat glycerol to 60 °C (usually consistent with the melting temperature of petrolatum, 50 - 70 °C);

[0076] 2 - 2) Slowly add the heated glycerol to the melted petrolatum and stir evenly to obtain the first mixture, where the mass ratio of glycerol to petrolatum is 11.4:100 (usually (10 - 15):100); the stirring rate is 200 rpm (usually 100 - 300 rpm).

[0077] Glycerol needs to be preheated to a temperature close to that of petrolatum (about 60 °C) to avoid stratification caused by temperature differences.

[0078] (3) Adding linolenic acid and linoleic acid

[0079] Let the first mixture stand at room temperature. When the temperature drops to 45 °C (usually 40 - 50 °C), add γ - linolenic acid and linoleic acid in sequence, and continue to stir until uniform to obtain the second mixture, where the mass ratio of γ - linolenic acid to petrolatum is 3.6:100 (usually (2 - 5):100); the mass ratio of linoleic acid to petrolatum is 3.6:100 (usually (2 - 5):100); the stirring rate is 40 rpm (usually 30 - 60 rpm); the mass ratio of γ - linolenic acid to linoleic acid is 1:1 (usually 1:(0.5 - 2)).

[0080] γ-linolenic acid is more easily oxidized than linoleic acid. Adding it first can reduce the time it is exposed to air during the mixing process. The addition of γ-linolenic acid can provide a more uniform matrix for the subsequent mixing of linoleic acid. Moreover, since linseed oil and linoleic acid are sensitive to heat, they need to be added at a lower temperature.

[0081] (4) Add antioxidants

[0082] When the temperature of the above-mentioned second mixture drops to 45 ± 5 °C (usually 40 - 50 °C), add BHA and BHT, and stir evenly; when the temperature drops below 40 °C, add vitamin E and propyl gallate in sequence, and stir evenly to obtain the ointment precursor mixture; among them, the mass ratio of BHA to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the mass ratio of BHT to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the mass ratio of vitamin E to petrolatum is 1.08:100 (usually (0.5 - 2):100); the mass ratio of propyl gallate to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the stirring rate is 40 rpm (usually 30 - 60 rpm).

[0083] Add BHA and BHT first: add them at a temperature of 40 - 60 °C to ensure complete dissolution and uniform distribution; add vitamin E when the temperature drops below 40 °C to reduce oxidation loss; finally, add propyl gallate when the temperature drops below 40 °C to avoid reacting with other components and reducing the antioxidant effect.

[0084] Vitamin E, BHA, BHT, and propyl gallate are all antioxidants and need to be added at a lower temperature to maintain their activity. The addition amounts of BHA, BHT, and propyl gallate need to be strictly controlled, usually not exceeding 0.1%.

[0085] (5) pH measurement

[0086] Use a surface pH electrode to detect the pH value of the ointment precursor mixture. The pH value suitable for skin use is 5.5 - 6.5 (preferably 6.0), that is, close to the physiological pH value of the skin;

[0087] If the measured pH value is too high (greater than 6.5), slowly add lactic acid solution while stirring until the pH value reaches 6.0 (usually the target range is 5.5 - 6.5);

[0088] If the pH value is too low (less than 5.5), slowly add triethanolamine solution while stirring until the pH value reaches 6.0 (usually the target range is 5.5 - 6.5);

[0089] After adjusting the pH value, continue to stir the cream until it reaches room temperature to ensure uniform distribution of the ingredients.

[0090] Preparation methods of lactic acid solution and triethanolamine solution:

[0091] Take an appropriate amount of lactic acid (such as 1 g), add a small amount of water (such as 10 mL), and stir until it is completely dissolved. Take an appropriate amount of triethanolamine (such as 1 g), add a small amount of water (such as 10 mL), and stir until it is completely dissolved.

[0092] (6) Cooling and homogenization

[0093] After measuring the pH value, stir and cool the ointment precursor mixture at room temperature until it completely solidifies; then use a homogenizer to further mix to ensure uniform distribution of all ingredients, and obtain the finished ointment.

[0094] Fill the finished ointment into an opaque container and seal it for storage.

[0095] Note: The filling environment should be kept clean to avoid contamination.

[0096] The finished ointment prepared by the method of the present invention is uniform, delicate, without particles and without stratification.

[0097] Use a surface pH electrode to detect the pH value of the finished ointment. The pH value is 6.0 (usually 5.5 - 6.5), which is close to the physiological pH value of the skin and is suitable for skin use.

[0098] During the preparation of the specific dermatitis ointment of the present invention, the temperature is strictly controlled: avoid destroying the activities of linseed oil, linoleic acid and antioxidants at too high temperature; control the stirring rate as follows: mixing glycerol and petrolatum: 100 - 300 rpm, adding γ-linolenic acid, linoleic acid, antioxidant: 30 - 60 rpm; avoid air mixing into the ointment, resulting in oxidation, deterioration of the ointment, shortened use time and shortened shelf life;

[0099] During the preparation of the ointment, it is necessary to ensure a clean production environment to avoid microbial contamination;

[0100] Choose an opaque container for the packaging of the ointment to avoid oxidation of antioxidants and oils caused by light;

[0101] The addition order of antioxidants during the preparation process is particularly important: first add BHA and BHT, and then add vitamin E and propyl gallate when the temperature is lowered below 40 °C to ensure the thermal stability and uniform dispersion of the ingredients.

[0102] Stirring speed: moderate, to prevent air mixing and causing oxidation.

[0103] Example 2

[0104] (1) Melting of the matrix petrolatum

[0105] Same as step (1) of Example 1.

[0106] (2) Add glycerol

[0107] Except that the mass ratio of glycerol to petrolatum is 12.5:100 (usually (10 - 15):100); the stirring rate is 150 rpm, the rest is the same as step (2) of Example 1.

[0108] (3) Add linolenic acid and linoleic acid

[0109] Except that the mass ratio of γ-linolenic acid to petrolatum is 4:100 (usually (2 - 5):100); the mass ratio of linoleic acid to petrolatum is 3:100 (usually (2 - 5):100); the stirring rate is 50 rpm; the mass ratio of γ-linolenic acid to linoleic acid is 1:0.75 (usually 1:(0.5 - 2)), the rest is the same as step (3) of Example 1.

[0110] (4) Add antioxidants

[0111] Except that the mass ratio of vitamin E to petrolatum is 1.5:100 (usually (0.5 - 2):100); the mass ratio of BHA to petrolatum is 0.07:100 (usually (0.01 - 0.1):100); the mass ratio of BHT to petrolatum is 0.05:100 (usually (0.01 - 0.1):100); the mass ratio of propyl gallate to petrolatum is 0.07:100 (usually (0.01 - 0.1):100); the stirring rate is 50 rpm, the rest is the same as step (4) of Example 1.

[0112] (5) pH measurement

[0113] Same as step (5) of Example 1.

[0114] (6) Cooling and homogenization

[0115] Same as step (6) of Example 1.

[0116] Example 3

[0117] (1) Melt the matrix petrolatum

[0118] Same as step (1) of Example 1.

[0119] (2) Add glycerol

[0120] Except that the mass ratio of glycerol to petrolatum is 10:100 (usually (10 - 15):100), the rest is the same as step (2) of Example 1.

[0121] (3) Add linseed oil and linoleic acid

[0122] Except that the mass ratio of γ-linolenic acid to petrolatum is 2.5:100 (usually (2-5):100); the mass ratio of linoleic acid to petrolatum is 2.5:100 (usually (2-5):100); and the stirring rate is 50 rpm, the rest is the same as step (3) of Example 1.

[0123] (4) Add antioxidant

[0124] Except that the mass ratio of vitamin E to petrolatum is 0.8:100 (usually (0.5-2):100); the mass ratio of BHA to petrolatum is 0.05:100 (usually (0.01-0.1):100); the mass ratio of BHT to petrolatum is 0.05:100 (usually (0.01-0.1):100); and the mass ratio of propyl gallate to petrolatum is 0.05:100 (usually (0.01-0.1):100), the rest is the same as step (4) of Example 1.

[0125] (5) pH measurement

[0126] The same as step (5) of Example 1.

[0127] (6) Cooling and homogenization

[0128] The same as step (6) of Example 1.

[0129] Control Example 1 Excipient preparation

[0130] (1) Melt the matrix petrolatum

[0131] Heat the petrolatum to 65±5°C (usually 60-70°C) until it is completely melted and set aside;

[0132] (2) Add glycerol

[0133] Preheat the glycerol to 60°C (usually consistent with the melting temperature of petrolatum, 50-70°C);

[0134] Slowly add the heated glycerol to the melted petrolatum and stir evenly to obtain the first mixture, wherein the mass ratio of glycerol to petrolatum is 11.4:100 (usually (10-15):100); the stirring rate is 200 rpm (usually 100-300 rpm).

[0135] (3) Add antioxidant

[0136] When the temperature of the first mixture drops to 45 ± 5 °C (usually 40 - 50 °C), add BHA and BHT, and stir evenly; when the temperature drops below 40 °C, add vitamin E and propyl gallate in sequence, and stir evenly to obtain the ointment precursor mixture; wherein, the mass ratio of BHA to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the mass ratio of BHT to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the mass ratio of vitamin E to petrolatum is 1.08:100 (usually (0.5 - 2):100); the mass ratio of propyl gallate to petrolatum is 0.06:100 (usually (0.01 - 0.1):100); the stirring rate is 40 rpm (usually 30 - 60 rpm).

[0137] Stir and cool the ointment precursor mixture at room temperature until it completely solidifies; then use a homogenizer to further mix to ensure that all components are evenly distributed to obtain the finished ointment.

[0138] Test Example 1 Clinical Effect Evaluation and Skin Barrier Function Evaluation

[0139] 1. Test drug:

[0140] The ointment prepared in Example 1 of the present invention.

[0141] 2. Research subjects:

[0142] Outpatients in the Dermatology Clinic of Peking University First Hospital from January 2015 to April 2025 were selected, who were clearly diagnosed as patients with mild localized atopic dermatitis, met the diagnostic and inclusion / exclusion criteria, aged 18 - 80 years old, and voluntarily accepted observation and treatment.

[0143] A: Patients with atopic dermatitis

[0144] (1) Inclusion criteria

[0145] ① Aged 18 - 80 years old

[0146] ② Diagnosed as a patient with atopic dermatitis according to the Haniffin diagnostic criteria and having the disease for at least 1 year

[0147] ③ Patients with mild AD (the investigator's global assessment (IGA) score is 1 or 2)

[0148] ④ Having skin lesions in the cubital fossa or posterior neck

[0149] ⑤ Having no other active skin diseases

[0150] (2) Exclusion criteria

[0151] ① Long-term use of glucocorticoids, immunosuppressants, or other drugs that may affect the immune system

[0152] ② Topical use of glucocorticoids or other immunosuppressive drugs in the sampling area in the past 4 weeks

[0153] ③ Suffering from severe systemic diseases

[0154] ④ Allergic to known drug components

[0155] B: The control group consisted of healthy subjects without atopic dermatitis:

[0156] (1) Inclusion criteria for the control group:

[0157] ① Aged 18 - 80 years old

[0158] ② Without active skin diseases

[0159] (2) Exclusion criteria

[0160] ① Long-term use of glucocorticoids, immunosuppressants, or other drugs that may affect the immune system

[0161] ② Topical use of glucocorticoids or other immunosuppressive drugs in the sampling area in the past 4 weeks

[0162] ③ Suffering from severe systemic diseases

[0163] 7 subjects with mild localized atopic dermatitis were treated with topical application of this cream product. After washing the affected area with warm water, an appropriate amount of this cream (about 2 - 5 mg per square centimeter) was taken and applied to the affected area 2 times a day for 2 consecutive weeks.

[0164] 2 - 1) Observation indicators for subjects with atopic dermatitis:

[0165] (1) Clinical manifestations: Observe the degree of remission of erythema and papules in patients.

[0166] (2) Evaluation by Investigator Global Assessment (IGA).

[0167] 2 - 2) Peak Pruritus Numerical Rating Scale (ppNRS)

[0168] ppNRS is a tool used to evaluate the severity of pruritus symptoms in patients, and is used to quantify the most severe pruritus experienced by patients in the past 24 hours. An 11 - point scale is used, with 0 points indicating "no pruritus" and 10 points indicating "the most severe pruritus imaginable", and patients score the most severe pruritus according to their own feelings.

[0169] 2 - 3) TEWL

[0170] TEWL is an important indicator for detecting the improvement of the skin barrier. AD patients have severe barrier function damage, and the TEWL value is significantly increased. Therefore, the TEWL level was measured before and after treatment to evaluate the treatment effect. The transepidermal water loss (TEWL) of the subjects was measured using a Tewameter (C - K Electronic, Germany) to evaluate the improvement of the skin barrier function. The average value was calculated for each group, and whether there were differences was analyzed.

[0171] 2 - 4) IGA score

[0172] The IGA score is a tool used to evaluate the severity of atopic dermatitis (AD). The evaluation is based on the morphological characteristics of skin lesions:

[0173] Erythema: Evaluate the severity of erythema, from hardly noticeable to significant erythema.

[0174] Infiltration / papules: Evaluate the obviousness of skin infiltration or papules.

[0175] Lichenification: Evaluate the degree of skin lichenification.

[0176] Exudation / crusting: Evaluate the presence of exudation or crusting.

[0177] Scoring criteria:

[0178] 0 points (Clear): No signs of inflammation, no erythema, infiltration / papules, lichenification, exudation or crusting. Post - inflammatory hyperpigmentation or hypopigmentation may be present.

[0179] 1 point (Almost clear): Hardly noticeable erythema, infiltration / papules and / or mild lichenification, no exudation or crusting.

[0180] 2 points (Mild): Slight but definite erythema (pink), infiltration / papules and / or lichenification, no exudation or crusting.

[0181] 3 points (Moderate): Clearly noticeable erythema (dark red), infiltration / papules and / or lichenification, exudation and crusting may be present.

[0182] 4 points (Severe): Significant erythema (dark red or bright red), significant infiltration / papules and / or significant lichenification, the disease is widely distributed, exudation or crusting may be present.

[0183] 3. Efficacy evaluation criteria

[0184] After applying the topical cream, the skin TEWL level decreased significantly, being significantly lower than the pre-treatment TEWL level.

[0185] 4. Experimental Results and Analysis

[0186] Data analysis was performed using the Prism 10 software package. Paired t-tests were used for the before-and-after control data. A P < 0.05 was considered to indicate a significant difference.

[0187] The results showed that the skin lesion symptoms of AD patients were significantly alleviated after 2 weeks of topical medication (such as Figure 1 ), the IGA score was significantly reduced (P < 0.001) (such as Figure 2 ), and at the same time, the severity of itching (ppNRS) was significantly alleviated (P < 0.0001 (such as Figure 3 )).

[0188] The TEWL level of AD patients at the baseline was significantly higher than that of the control group. After 2 weeks of topical medication, the TEWL decreased significantly and returned to a level similar to that of healthy controls, indicating that the skin barrier was significantly improved, such as Figure 4 .

[0189] Experimental Example 2: Animal Experiment

[0190] 1. Experimental Animals

[0191] Wild-type C57BL / 6 female mice at 6 - 8 weeks of age (purchased from Spiff (Beijing) Biotechnology Co., Ltd.).

[0192] 2. Experimental Drugs and Instruments

[0193] Calcipotriol (MC903, Beijing Solarbio Science & Technology Co., Ltd.); phosphate buffer saline (PBS); TRIzol (RNA extraction reagent); SYBR Green Master Mix (SYBRGreen premix, ThermoFisher Scientific, Waltham, MA, USA); Applied Biosystems TM ViiA TM 7 (Thermo Fisher Scientific, Waltham, MA, USA).

[0194] Long-chain unsaturated fatty acid solution, and its preparation method is as follows: Add gamma-linolenic acid (1 mg) and linoleic acid (1 mg) into 1 ml of absolute ethanol to prepare a storage solution with the concentrations of gamma-linolenic acid and linoleic acid being 1 mg / ml, and store it frozen at -20°C. On the day of the experiment, dilute each unsaturated fatty acid storage solution to 1 mM using phosphate buffer saline (PBS).

[0195] 3. Experimental methods

[0196] In the previous animal models, MC903 was used as the storage solution (450 μM). Among them, the preparation method of MC903: Dissolve 1 mg of calcipotriol in 5.386 ml of absolute ethanol, and store the storage solution frozen at -20°C.

[0197] On the day of the experiment, dilute this solution 10 times with absolute ethanol to prepare a working solution (45 μM).

[0198] Animal experiment protocol:

[0199] The animal experiment process is as Figure 5 .

[0200] 1) Establish an animal model of AD-like dermatitis

[0201] Depilate the back skin of the mice using a blade, aspirate 100 μl of the MC903 working solution with a pipette tip, and evenly apply it to the back skin of the mice for 7 consecutive days to establish an AD-like dermatitis mouse model. The clinical manifestations of the back skin of the model mice are as Figure 6A ;

[0202] 2) Administration

[0203] Randomly divide the model mice into 2 groups: experimental group (ointment group), control group (excipient group).

[0204] Among them: The experimental group (ointment group) externally applies the ointment prepared in Example 1 of the present invention; the control group (excipient group) externally applies an ointment without fatty acids; administer the drugs for 3 days. Sacrifice the mice on the 11th day and cut off the back skin. The clinical manifestations of the back skin of the mice on the 11th day are as Figure 6A .

[0205] Fix 1 / 4 of the skin tissues of each group of mice cut off with formalin, embed them in wax blocks and perform hematoxylin-eosin (HE) staining, as well as immunohistochemical staining of IL-1b and IL-4. The specific steps are as follows:

[0206] HE staining: Mouse skin tissues were fixed in 10% formalin solution at room temperature for 24 hours, and then paraffin sections were prepared. The sections were baked at 65 °C for 2 hours, and then immersed in xylene I and II for 15 minutes each. After dehydration with 100%, 95%, 85% and 75% ethanol, the sections were washed with distilled water. The sections were stained with Harris hematoxylin for 5 minutes, differentiated in hydrochloric acid ethanol for 5 seconds, and then immersed in ammonia water for 30 seconds. After staining with eosin for 2 minutes, the sections were dehydrated through gradient dehydration, cleared with xylene, and sealed with resin. Finally, the results were analyzed under an optical microscope, and the observed results were as Figure 6B .

[0207] Immunohistochemical staining: Paraffin sections were first dewaxed with a series of solutions, and then antigen retrieval was performed in citrate / EDTA buffer. After blocking the activity of endogenous peroxidase with 3% hydrogen peroxide solution, the tissues were blocked with 3% bovine serum albumin. The sections were then incubated with the primary antibody overnight at 4 °C, followed by incubation with the corresponding horseradish peroxidase-labeled secondary antibody. After washing, DAB solution was applied for color development, and the reaction was terminated with tap water. The sections were counterstained with hematoxylin, dehydrated, and sealed with resin. Finally, the results were analyzed under an optical microscope, as Figure 6C .

[0208] 4) Determination of the infiltration level of pro-inflammatory factors

[0209] Three-fourths of the remaining skin tissue was frozen at -80 °C, and then RNA was extracted. Specific primers were used to detect inflammatory factors such as TSLP and IL-33. The specific procedure is as follows:

[0210] RNA extraction: The mouse skin tissue samples cut were washed with PBS, 1 mL of TRIzol was added, and incubated on ice for 10 minutes. Subsequently, the solution was transferred to a 1.5 mL centrifuge tube.

[0211] The mouse skin tissue (20 mg) cut was washed with cold PBS, chopped and placed in a 1.5 mL centrifuge tube. 1 mL of TRIzol was added, and ground at a speed of 12,000 revolutions per minute for 10 minutes. The supernatant was collected, 200 μL of chloroform was added, incubated for 5 minutes, and then centrifuged at a speed of 12,000 revolutions per minute for 10 minutes. The supernatant was mixed with 600 μL of isopropanol, incubated at -20 °C for 15 minutes, and centrifuged again. After discarding the supernatant, the RNA was washed with 1 mL of 70% ethanol, dried, and dissolved in 20 μL of DEPC water for RNA quantification using NanoDrop.

[0212] Reverse transcription: Mix 2 μg of RNA with 5× gDNA Eraser Buffer and gDNA Eraser, and incubate at 42 °C for 2 minutes to remove genomic DNA. Subsequently, add the remaining components, incubate the mixture at 37 °C for 15 minutes, and then incubate at 85 °C for 5 seconds to synthesize cDNA. The cDNA is stored at -20 °C.

[0213] Real-time quantitative PCR: The reaction system consists of 1 μL of cDNA, 10 μL of SYBR Green Master Mix, 1 μL each of the forward and reverse primers, and 7 μL of RNase-free water. Use Applied Biosystems TM ViiA TM 7 system for PCR reaction, and the cycling conditions are as follows: 2 minutes at 50 °C, 2 minutes at 95 °C, 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C, and finally 15 seconds at 95 °C and 1 minute at 60 °C. The results of real-time quantitative PCR are as Figure 7 .

[0214] Real-time quantitative PCR was used to detect the expression levels of skin cytokines in animal experiments, and the primers used are shown in Table 1.

[0215] Table 1 Primers for real-time fluorescence PCR reaction

[0216]

[0217] 4. Experimental results and conclusions

[0218] In the experimental group, applying the ointment of the present invention (containing long-chain unsaturated fatty acids, namely γ-linolenic acid + linoleic acid) externally to mice can significantly reduce skin inflammation. The results of reverse transcription and real-time fluorescence quantitative PCR detection of tissue RNA extracted from skin tissues show that applying the fatty acid-containing cream externally reduces the levels of type 2 inflammation-related pro-inflammatory factors such as IL-1b, IL-4, TSLP, and IL-33, indicating that this solution has a therapeutic effect on atopic dermatitis.

[0219] From the experimental results, it can be seen that the ointment prepared by the method of the present invention can significantly inhibit skin inflammatory reactions.

[0220] Experimental conclusion: Applying the ointment containing γ-linolenic acid and linoleic acid externally can significantly improve the clinical manifestations of dermatitis in mice, reduce the infiltration of inflammatory factors such as IL-1β, IL-4, IL-33, and TSLP, and has the therapeutic value of relieving atopic dermatitis.

[0221] Examples should describe in detail and specifically all the necessary conditions required for those of ordinary skill in the art to implement and reproduce the present invention, such as parameters, materials, equipment, tools, etc., as well as the necessary specifications and models. If new substances or self-prepared materials are used therein, the manufacturing methods thereof should also be described.

Claims

1. A pharmaceutical composition for treating atopic dermatitis, characterized in that, It includes matrix petrolatum, humectant, emollient, repair agent and antioxidant.

2. The pharmaceutical composition according to claim 1, wherein, In the said pharmaceutical composition The weight ratio of the humectant to the matrix petrolatum is (10 - 15):100, preferably (10 - 12.5):100, and more preferably 11.4:100; The weight ratio of the emollient to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100; The weight ratio of the repair agent to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100; The weight ratio of the antioxidant to the matrix petrolatum is (0.5 - 3):100, preferably (1.26 - 1.69):100, and more preferably 1.26:

100.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The said humectant is selected from one or more of glycerol, hyaluronic acid, sorbitol, propylene glycol or urea, preferably glycerol; The said emollient is selected from one or more of γ-linolenic acid, olive oil, squalane, avocado oil or jojoba oil, preferably γ-linolenic acid; The said repair agent is selected from one or more of linoleic acid, ceramide, cholesterol, hyaluronic acid or squalene, preferably linoleic acid; The said antioxidant is a mixture of natural antioxidant and synthetic antioxidant, wherein the natural antioxidant is selected from one or more of vitamin E, tea polyphenols, astaxanthin, lycopene or coenzyme Q10; the synthetic antioxidant is a mixture of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene) and propyl gallate.

4. A method for preparing a pharmaceutical composition for treating atopic dermatitis, characterized in that, It includes the following steps carried out in sequence: 1) Add the humectant to the heated and melted matrix petrolatum and stir evenly to make the first mixture; 2) Add the emollient and the repair agent to the first mixture whose temperature has dropped to 40 - 50 °C in sequence and stir evenly to make the second mixture; 3) Add the antioxidant to the second mixture and stir evenly to obtain it.

5. The preparation method according to claim 4, characterized in that, In step 1), the weight ratio of the humectant glycerol to the matrix petrolatum is (10 - 15):100, preferably (10 - 12.5):100, and more preferably 11.4:

100.

6. The preparation method according to claim 4 or 5, characterized in that, In step 2), the weight ratio of the emollient to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:100; the weight ratio of the repair agent to the matrix petrolatum is (2 - 5):100, preferably (2.5 - 4):100, and more preferably 3.6:

100.

7. The preparation method according to claim 4 or 5, characterized in that, In step 3), the weight ratio of the natural antioxidant to the matrix petrolatum is (0.5 - 2):100, preferably (0.8 - 1.5):100, and more preferably 1.08:100; the weight ratio of the synthetic antioxidant to the matrix petrolatum is (0.03 - 0.3):100, preferably (0.15 - 0.19):100, and more preferably 0.18:

100.

8. The preparation method according to claim 4 or 5, characterized in that, The natural antioxidant described in step 3) is selected from one or more of vitamin E, tea polyphenols, astaxanthin, lycopene or coenzyme Q10, preferably vitamin E; the synthetic antioxidant is a mixture of BHA (butylated hydroxyanisole), BHT (dibutylhydroxytoluene) and propyl gallate.

9. The preparation method according to claim 8, characterized in that, In step 2), the weight ratio of the moisturizer to the repair agent is 1:(0.5 - 2), preferably 1:1.

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