Carboxyamidotriazole external ointment preparation and preparation method thereof

Abstract

The invention provides a carboxamide triazole external ointment preparation and a preparation method thereof, and relates to the technical field of medicines. The composition comprises the following components in parts by weight: 0.5-4 parts of carboxamide triazole, 60-90 parts of a matrix, 5-10 parts of a thickening agent, 2-9 parts of an emulsifier, 1-7 parts of a humectant, 2-8 parts of a penetration enhancer and 0.0005-0.05 part of an antioxidant. According to the carboxamidotriazole external ointment preparation provided by the invention, the physical and chemical properties of an obtained product are stable by optimizing prescription components and proportions and optimizing a preparation process: the ointment is regular in liquid drops and uniform in particle size when being observed under a microscope; under long-term test conditions, the chemical stability is good, the content is uniform, and related substances are not generated.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a carboxyamide triazole external ointment preparation and a preparation method thereof. Background Art

[0002] Carboxyamidotriazole (CAI) is a synthetic small molecule compound. Many studies have confirmed that it is a non-cytotoxic calcium ion antagonist. It has shown the effects of inhibiting the proliferation and metastasis of various tumors in both in vivo and in vitro models, and can inhibit angiogenesis. The results of previous studies have shown that carboxyamidotriazole also has significant anti-acute and chronic inflammatory effects.

[0003] Patent CN101553225B experimentally confirmed that carboxyamide triazole compounds can be used as inhibitors of inflammatory cytokines TNF-α and IL-1, and proposed the use of carboxyamide triazole compounds in the treatment of pain caused by any reason in mammals, inflammation involving inflammatory cytokines, and other immunological-based related diseases. Patent CN111249277A discloses the role of carboxyamide triazole compounds in the treatment of a series of autoinflammatory diseases, such as psoriasis and other common skin diseases.

[0004] Psoriasis is a chronic inflammatory skin disease with a high incidence, stubborn condition and easy recurrence. It is actually an immune disease, and the course of the disease mostly lasts a lifetime, which is extremely harmful to people's physical and mental health. Psoriasis is characterized by erythema, papules and scales. Its main histopathological changes are: excessive proliferation of keratinocytes (KC), infiltration of inflammatory cells in the dermis, and dilation of blood vessels in the dermal papilla; among them, excessive proliferation of KC is its most important feature, which is manifested as accelerated proliferation and metabolism of keratinocytes, shortened epidermal cell conversion cycle, thickening of the epidermis accompanied by abnormal keratinization such as hyperkeratosis and parakeratosis, and disappearance of the granular cell layer.

[0005] The etiology and pathogenesis of psoriasis are extremely complex and have not yet been fully understood. In recent years, it is believed that psoriasis is an immune disease caused by the complex interaction between highly proliferating keratinocytes and immune cells such as T cells, neutrophils, dendritic cells and macrophages, which is caused by the combined effects of genetic gene regulation and environmental factors, resulting in dysfunction of the patient's nervous system, biochemical substances, and immune system. The interaction between KC and these immune cells ultimately leads to excessive proliferation of KC and dermal inflammation, showing the unique clinical and pathological characteristics of psoriasis. A large number of cytokines are involved in its pathogenesis, among which cytokines such as TNF-α, IL-17, IL-12 and IL-23 are very important immune-mediated factors that have been identified. At present, there is no specific treatment for psoriasis, and the main treatment methods are symptomatic treatment of topical and oral drugs. Most of these drugs are hormones or antibiotics. Appropriate symptomatic treatment can temporarily control symptoms, but it only treats the symptoms and not the root cause. Long-term use will also cause repeated illness, incurable, and cause many adverse reactions.

[0006] As inhibitors of inflammatory factors such as inflammatory cytokines TNF-α, carboxyamide triazole compounds are further explored for their effects in the treatment of psoriasis, providing new ideas for the treatment of such diseases. At present, only carboxyamide triazole soft capsules have completed phase III clinical trials for the treatment of non-small cell lung cancer. Patent CN112353778B discloses a carboxyamide triazole soft capsule and a preparation method thereof. The soft capsule has good stability, and the drug content does not decrease during long-term storage, and the increase of related substances is not obvious. However, the amount of drug absorbed by the soft capsule after oral administration through the blood circulation to the skin is limited. Therefore, it is particularly important to develop local external preparations for the treatment of skin diseases. External preparations are administered at the site of lesions on the skin, requiring the drug to be released from the preparation and penetrate the stratum corneum of the skin to enter the skin, and to be able to stay in the skin for a long time to play a role.

[0007] In view of this, the present invention aims to provide a carboxyamide triazole topical ointment preparation and a preparation method thereof. The ointment provided has good physical and chemical stability, and the drug can be released from the preparation, penetrate the skin stratum corneum, and infiltrate and remain in the skin. Summary of the invention

[0008] In view of the above problems, the present invention provides a carboxyamide triazole external ointment preparation and a preparation method thereof. By screening the ingredients and proportions of the prescription and optimizing the preparation process, the prepared ointment has good physical and chemical stability.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] On the one hand, the present invention provides a carboxyamide triazole external ointment preparation, which comprises the following ingredients in proportion by weight: 0.5-4 parts of carboxyamide triazole, 60-90 parts of a base, 5-10 parts of a thickener, 2-9 parts of an emulsifier, 1-7 parts of a moisturizer, 2-8 parts of a penetration enhancer, and 0.0005-0.5 parts of an antioxidant.

[0011] Preferably, the matrix is ​​selected from at least one of white vaseline, yellow vaseline, liquid paraffin, paraffin, white beeswax, lanolin, lanolin alcohol, and silicone oil; further preferably, the matrix is ​​selected from at least one of white vaseline, yellow vaseline, liquid paraffin, lanolin alcohol, beeswax, and silicone oil; more preferably, the matrix is ​​selected from at least one of white vaseline and liquid paraffin.

[0012] Preferably, the thickener is selected from at least one of hydrogenated vegetable oil, palmitic acid, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, and paraffin; further preferably, the thickener is selected from at least one of hydrogenated vegetable oil, myristyl alcohol, and cetyl alcohol; more preferably, the thickener is cetyl alcohol.

[0013] Preferably, the emulsifier is selected from at least one of cetyl alcohol, lanolin, lanolin alcohol, Span 80, stearyl alcohol polyether, and Span 60; further preferably, the emulsifier is selected from at least one of lanolin, stearyl alcohol polyether, and Span 60; more preferably, the emulsifier is lanolin.

[0014] Preferably, the moisturizer is selected from at least one of isopropyl myristate, cyclomethicone, and triacetin; further preferably, the moisturizer is selected from isopropyl myristate.

[0015] Preferably, the penetration enhancer is selected from at least one of dimethyl sulfoxide, isopropyl myristate, azone, menthol, diethylene glycol monoethyl ether, and oleic acid; further preferably, the penetration enhancer is selected from at least one of dimethyl sulfoxide, azone, and menthol; more preferably, the penetration enhancer is dimethyl sulfoxide.

[0016] Preferably, the antioxidant is selected from at least one of butylated hydroxyanisole, dibutyl hydroxytoluene, and dl-α-tocopherol; further preferably, the antioxidant is selected from at least one of butylated hydroxyanisole and dl-α-tocopherol; more preferably, the antioxidant is butylated hydroxyanisole.

[0017] Preferably, the pH of the carboxyamide triazole external ointment preparation is 4-9.

[0018] Preferably, the carboxyamide triazole external ointment preparation comprises the following ingredients in proportion by weight: 0.5-4 parts of carboxyamide triazole, 55-80 parts of white vaseline, 2-10 parts of liquid paraffin, 5-10 parts of hexadecanol, 2-9 parts of lanolin, 1-7 parts of isopropyl myristate, 2-8 parts of a permeation enhancer, and 0.0005-0.1 parts of an antioxidant.

[0019] Further preferably, the following ingredients are included in proportion by weight: 2 parts of carboxyamide triazole, 71-75 parts of white vaseline, 4-6 parts of liquid paraffin, 6-7 parts of hexadecanol, 5-6 parts of lanolin, 3-5 parts of isopropyl myristate, 4-5 parts of permeation enhancer, and 0.001-0.01 parts of antioxidant.

[0020] More preferably, the following ingredients are included in proportion by weight: 2 parts of carboxyamide triazole, 71.473 parts of white vaseline, 6 parts of liquid paraffin, 7 parts of hexadecanol, 5 parts of lanolin, 3 parts of isopropyl myristate, 5 parts of penetration enhancer, and 0.001 parts of antioxidant.

[0021] On the other hand, the present invention provides a method for preparing a carboxyamide triazole external ointment preparation, comprising the following steps:

[0022] (1) white vaseline, cetyl alcohol, lanolin, liquid paraffin, isopropyl myristate and butylated hydroxyanisole are heated to melt the matrix;

[0023] (2) stirring and cooling the substrate in step (1) while it is in a molten state;

[0024] (3) preparing a buffer solution, adding the buffer solution to step (2), and stirring to homogenize the mixture;

[0025] (4) preparing a bulk drug dispersion, adding the bulk drug dispersion to step (3), and stirring to homogenize the mixture;

[0026] (5) Finally cool to 36°C-40°C.

[0027] Preferably, the specific parameters of the matrix melting described in step (1) are: temperature 70-80°C, stirring speed: 30rpm±5rpm; the stirring speed described in step (2) is 30rpm±5rpm, and the material temperature after cooling is 50-65°C; the stirring speed described in step (3) is 30rpm±5rpm, the homogenizing speed is 2000-2500rpm, and the mixing temperature is 50-65°C; the stirring speed described in step (4) is 30rpm±5rpm, the homogenizing speed is 2000-2500rpm, and the mixing temperature is 50-65°C; the specific parameters of the final cooling described in step (5) are: cooling end point temperature 36-40°C, stirring speed 15-25rpm.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The carboxyamide triazole external ointment preparation provided by the present invention optimizes the prescription components and the ratio and optimizes the preparation process, so that the obtained product has stable physical and chemical properties: the ointment has regular droplets and uniform particle sizes when observed under a microscope; it has good chemical stability and uniform content under long-term test conditions, and does not produce related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a microscopic image of the droplet structure of the ointment prepared in Example 1.

[0031] Figure 2 The in vitro skin permeation graphs of the ointments prepared in Examples 1, 15-18 and Comparative Examples 3, 14-15.

[0032] Figure 3 The figure shows the body weight changes of animals at different time points (n=5 or 10).

[0033] Figure 4 Animals were scored for changes in erythema at different time points (n=5 or 10).

[0034] Figure 5 Animals were scored for changes in scaling at different time points (n=5 or 10).

[0035] Figure 6 Animals were scored for changes in skin thickness at different time points (n=5 or 10).

[0036] Figure 7 The cumulative PASI score changes of animals at different time points (n=5 or 10).

[0037] Figure 8 Representative photos of animal modeling sites at different time points.

[0038] Fig. 9 These are the IL-12 concentrations of the mouse skin tissue model group, positive control group 1, positive control group 2, CAI ointment dose group 1, and CAI ointment dose group 2.

[0039] Fig.10 These are the IL-17 concentrations of the mouse skin tissue model group, positive control group 1, positive control group 2, CAI ointment dose group 1, and CAI ointment dose group 2.

[0040] Fig.11 These are the IL-23 concentrations of the mouse skin tissue model group, positive control group 1, positive control group 2, CAI ointment dose group 1, and CAI ointment dose group 2.

[0041] Fig.12These are the TNF-α concentrations of the mouse skin tissue model group, positive control group 1, positive control group 2, CAI ointment dose group 1, and CAI ointment dose group 2.

[0042] Fig.13 The following are 40x and 200x HE staining images of skin tissue sections of mouse 1 in the model group.

[0043] Fig.14 The following are 40x and 200x HE staining images of skin tissue sections of mouse 2 in the model group.

[0044] Fig.15 The following are 40x and 200x HE staining images of skin tissue sections of mouse 3 in the model group.

[0045] Fig.16 The following are 40x and 200x HE staining images of skin tissue sections of 4 mice in the model group.

[0046] Fig.17 These are 40x and 200x HE staining images of the skin tissue sections of mouse 1 in CAI ointment dose 1 group.

[0047] Fig.18 These are 40x and 200x HE staining images of skin tissue sections of mice in group 2 of CAI ointment dose 1.

[0048] Fig.19 These are 40x and 200x HE staining images of skin tissue sections of 3 mice in CAI ointment dose 1 group.

[0049] Fig. 20 These are 40x and 200x HE staining images of skin tissue sections of 4 mice in CAI ointment dose 1 group.

[0050] Fig.21 These are 40x and 200x HE staining images of the skin tissue sections of mouse 1 in CAI ointment dose 1 group.

[0051] Fig. 22 These are 40x and 200x HE staining images of skin tissue sections of mice in group 2 of CAI ointment dose 1.

[0052] Fig.23 These are 40x and 200x HE staining images of skin tissue sections of 3 mice in CAI ointment dose 1 group.

[0053] Fig.24 These are 40x and 200x HE staining images of skin tissue sections of 4 mice in CAI ointment dose 1 group.

[0054] Fig.25 These are 40x and 200x HE staining images of the skin tissue section of mouse 1 in the positive group 1.

[0055] Fig.26These are 40x and 200x HE staining images of skin tissue sections of mouse 2 in the positive group 1.

[0056] Fig. 27 These are 40x and 200x HE staining images of skin tissue sections of mouse 3 in the positive group 1.

[0057] Fig.28 These are 40x and 200x HE staining images of skin tissue sections of mouse 1 in the positive group 2.

[0058] Fig.29 These are 40x and 200x HE staining images of skin tissue sections of mice in the positive group 2.

[0059] Fig.30 These are 40x and 200x HE staining images of skin tissue sections of mice 3 in the positive group 2.

[0060] Fig.31 Statistical chart of pathological scores of mice. DETAILED DESCRIPTION

[0061] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0062] Examples 1-5

[0063] The prescription of the carboxyamide triazole external ointment preparation of Example 1-5 comprises the following components in parts by weight:

[0064]

[0065] Example 1-5 A method for preparing a carboxyamide triazole topical ointment preparation comprises the following steps:

[0066] (1) Take white vaseline, cetyl alcohol, lanolin, liquid paraffin, isopropyl myristate and butylated hydroxyanisole and heat and stir to melt at 70-80°C, and continue stirring while cooling to 50-65°C.

[0067] (2) stirring and cooling the matrix from step (1) (keeping the matrix molten);

[0068] (3) preparing a buffer solution, slowly adding the buffer solution to step (2), and stirring to homogenize the mixture;

[0069] (4) preparing a bulk drug dispersion, slowly adding the bulk drug dispersion to step (3), and stirring to homogenize the mixture;

[0070] (5) Stir and cool to a temperature of 36-38°C.

[0071] Embodiment 6-8

[0072] Compared with Example 1, the bases in Examples 6-8 are different, and the other auxiliary materials are the same.

[0073] Example Name Matrix 1 Matrix 2 Example 6 White Vaseline Lanolin alcohol Example 7 Yellow Vaseline White Beeswax Example 8 White Vaseline Silicone oil

[0074] Examples 9-18

[0075] Compared with Example 1, Examples 9-18 are prescriptions of different types of thickeners, emulsifiers and moisturizers, and the other auxiliary materials are the same.

[0076]

[0077] Examples 19-22

[0078] The prescription of the carboxyamide triazole external ointment preparation of Examples 19-22 comprises the following components in parts by weight:

[0079]

[0080] Comparative Example 1

[0081] Compared with Example 1, hexadecanol is not added in the prescription, and white vaseline is added in the same proportion, and the preparation method is the same.

[0082] Comparative Example 2

[0083] Compared with Example 1, lanolin is not added in the prescription, and white vaseline is added in the same proportion, and the preparation method is the same.

[0084] Comparative Example 3

[0085] Compared with Example 1, isopropyl myristate is not added in the prescription, and white vaseline is added in an equal proportion, and the preparation method is the same.

[0086] Comparative Example 4

[0087] Compared with Example 1, a pH adjuster, sodium dihydrogen phosphate monohydrate, was added to the formulation to adjust the pH to 10.

[0088] Comparative Examples 5-7

[0089] Compared with Example 1, the bases in the prescriptions are respectively aqueous lanolin (Comparative Example 5), stearic acid (Comparative Example 6), and vegetable oil (Comparative Example 7).

[0090] Comparative Examples 8-9

[0091] Compared with Example 1, the thickeners in the prescription are anionic emulsified wax (Comparative Example 8) and white beeswax (Comparative Example 9).

[0092] Comparative Examples 10-11

[0093] Compared with Example 1, the emulsifiers in the prescription are polysorbate 80 (Comparative Example 10) and glyceryl monostearate (Comparative Example 11).

[0094] Comparative Examples 12-13

[0095] Compared with Example 1, the moisturizers in the prescription are propylene glycol (Comparative Example 12) and glycerin (Comparative Example 13).

[0096] Comparative Examples 14-15

[0097] Compared with Example 1, the penetration enhancers in the prescriptions are respectively triolein (Comparative Example 14) and polyoxyethylene glycol tetrahydrofuran (Comparative Example 15).

[0098] Comparative Examples 16-17

[0099] Compared with Example 1, the antioxidants in the prescriptions are gallic acid (Comparative Example 16) and ascorbic acid (Comparative Example 17).

[0100] Test Case

[0101] 1. Physical and chemical stability test

[0102] The ointments prepared in Examples 1-22 showed regular droplets and uniform particle sizes under a microscope, and no stratification under a high-speed centrifugation test; the consistency was moderate and the spreadability was good. There was no stratification at 30°C and the stability was good. Compared with Example 1, the properties, microstructures, and centrifugal stability of Comparative Examples 1, 2, 3, 5, 6, 7, and 9 were worse.

[0103] Table 1 Physical stability test results of Examples 1-22 and Comparative Examples 1-17

[0104]

[0105]

[0106]

[0107] Table 2 Accelerated stability results (30℃±2℃ / 65RH±5RH)

[0108]

[0109]

[0110] Table 3 Long-term stability results of Examples 1, 5, and 7 (20°C ± 2°C / 60% RH ± 5% RH)

[0111]

[0112] The above results show that the ointments prepared in Examples 1-22 have good chemical stability under accelerated test conditions, the content does not change significantly, and the related substances increase slowly. The pH of Comparative Example 4 is higher, and Comparative Examples 10 and 11 replace the emulsifier, and Comparative Examples 16 and 17 replace the antioxidant, and the content has a significant downward trend, and the impurities increase significantly.

[0113] 2. In vitro release and in vitro skin penetration

[0114] In vitro skin penetration is as follows Figure 2 The results show that the ointment has permeation within 24 hours, the permeation rate is fast in the first 2 hours, and then slows down. The cumulative permeation amount of Example 1 and Examples 15-18 is higher than that of Comparative Examples 3, 14, and 15, indicating that the permeation enhancer used in Examples 1 and Examples 15-18 can promote the skin permeation of the drug.

[0115] 3. Pharmacological and efficacy experiments

[0116] Purpose of the test:

[0117] The therapeutic effect of the test substance on psoriasis was investigated using a psoriasis mouse model induced by imiquimod cream.

[0118] Test method:

[0119] After the quarantine period, 40 quarantine qualified animals were selected and randomly divided into a model group (n=10), a positive control group 1 (n=5), a positive control group 2 (n=5), a CAI ointment dosage group 1 (n=10), and a CAI ointment dosage group 2 (n=10) according to their weight. Ensure that the weight level of animals between groups is balanced. Modeling was performed after grouping. The area of ​​about 5cm×3cm on the back of all mice was depilated and prepared for skin. Imiquimod cream was applied to the back of the animals twice a day for 5 consecutive days, and the drug was administered on the same day after the modeling was completed. The administration period was 3 days, and the corresponding test substance was applied to the modeling site twice a day. During the test period, the animals were photographed, weighed, and scored for PASI every day. One day after the last administration, the animals were euthanized, and the full-thickness skin of the modeling site was separated for ELISA detection and pathological detection. CAI ointment was prepared according to the method of Example 1.

[0120] Table 4 Information of test substances, reference substances and modeling agents

[0121]

[0122] Modeling method:

[0123] 40 SPF BALB / c male mice were adaptively raised in a barrier facility for 7 days and observed daily. Modeling was performed after confirming that the mice were healthy and normal. The back of all mice was depilated and skinned in an area of ​​about 5 cm × 3 cm. Imiquimod cream was applied to the back of the animals twice a day for 7 consecutive days. After confirming that the animals were in normal condition, they were returned to the cage and raised normally. After modeling, the skin lesions at the modeling site were closely monitored.

[0124] Grouping and dosing:

[0125] After the quarantine period, 40 quarantine qualified animals were randomly divided into 7 groups according to body weight, with 5 or 10 animals in each group. The weight level of animals in each group was ensured to be balanced. Modeling was carried out immediately after grouping, and medication was carried out on the same day after modeling. Details of grouping and medication are shown in Table 5.

[0126] Table 5 Group and dosage design list

[0127]

[0128] Note: bid*3: administer twice a day for 3 consecutive days.

[0129] Observation and indicator monitoring

[0130] General Observations

[0131] Modeling period: After the animal modeling, the animals were observed and recorded every day. The observation contents included: whether they were dead, dying, feed and water intake, trauma, feces, appearance and coat, mental state, activity status, etc.

[0132] Dosing period: The animals in this group were observed and recorded within 15 minutes to 1 hour after daily dosing. The observation contents were the same as above.

[0133] Weight: Quarantine period: The animals are weighed and recorded upon receipt and after the 7-day quarantine period.

[0134] Modeling and drug administration period: The animals were weighed and recorded daily after the modeling began, and were weighed and recorded daily during the drug administration period.

[0135] PASI score

[0136] After modeling, the animals were scored and recorded daily according to Table 6.

[0137] Table 6 PASI scoring criteria for psoriasis model

[0138] score erythema Scaly thickness 0 No erythema No scale on the surface The lesions are flush with normal skin 1 Reddish red erythema Some lesions have scales on the surface The lesions are slightly higher than normal skin 2 Red erythema Most lesions have scaling Moderately raised lesions 3 Dark red erythema The lesion is almost covered with scales Skin lesions are thickened and raised 4 Very deep erythema The lesions are completely covered with scales The lesions are highly thickened and prominent

[0139] Animal handling

[0140] After the last administration, the mice were killed by cervical dislocation, and the full-thickness skin of the modeling site of each animal was separated, fixed with 4% paraformaldehyde and frozen at -80℃.

[0141] Pathological examination and cytokine level detection

[0142] The sample tissues fixed with 4% paraformaldehyde were dehydrated, paraffin-embedded and sectioned, and stained with HE. The pathological changes of the skin at the modeling sites of each animal were observed under a microscope.

[0143] Elisa was used to detect changes in IL-12, IL-23, IL-17, and TNF-α in tissue homogenates of samples frozen at -80℃.

[0144] Data analysis and processing

[0145] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Before comparing the experimental groups, the inter-group variance analysis (F-test) was performed. When the inter-group variances were equal, the inter-group comparison was performed using the Student-T test (unpaired T test). When the inter-group variances were unequal, the corrected Student-T test was used for statistical analysis. The data were statistically analyzed and statistical graphs were drawn using Graphpad prism 8 software.

[0146] Test results

[0147] Gross observation: The mental state and activity of animals in each group were normal after administration, and no abnormalities were observed.

[0148] Body weight: The body weight changes of animals in each group at different time points are as follows Figure 3 As shown in Table 7. The changes in the body weight levels of animals in each drug-administered group were consistent, with a certain degree of decrease in the modeling period (d0-d5), and a stable body weight level from the end of the late modeling period to the start of drug administration (d5-d10). There was no significant difference in the body weight levels of animals in each group (P>0.05).

[0149] Table 7 Body weight of animals in each group at different time points (n=5 or 10)

[0150]

[0151] PASI score

[0152] Erythema score: The erythema scores of the modeling sites of each group of animals at different time points are as follows Figure 4As shown in Table 8, compared with the model group, the erythema scores of mice in CAI ointment dose group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the erythema scores of mice in CAI ointment dose group 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01); the erythema scores of mice in the positive control group 1 were significantly reduced on d9-d10 (2-3 days after administration) (P < 0.01). There was no statistical difference in the erythema scores of mice in the positive control group 2 compared with the control group (P > 0.05).

[0153] Table 8 Erythema scores of animals in each group at different time points (n=5 or 10)

[0154]

[0155] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01.

[0156] Scaly score

[0157] The scale scores of the modeling sites of each group of animals at different time points are as follows Figure 5 As shown in Table 9, compared with the model group, the squama scores of mice in CAI ointment dose groups 1 and 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the squama scores of mice in the positive control group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01). The squama scores of mice in the positive control group 2 were significantly reduced on d10 (3 days after administration) (P < 0.01).

[0158] Table 9 Squamous scores of animals in each group at different time points (n=5 or 10)

[0159]

[0160] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01

[0161] Skin thickness score

[0162] The skin thickness scores of the modeling sites of each group of animals at different time points are as follows Figure 6 As shown in Table 10, compared with the model, the skin thickness scores of mice in CAI ointment dose groups 1 and 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01); the skin thickness scores of mice in the positive control group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05). The skin thickness scores of mice in the positive control group 2 were significantly reduced on d10 (3 days after administration) (P < 0.05).

[0163] Table 10 Skin thickness scores of animals in each group at different time points (n=5 or 10)

[0164]

[0165] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01.

[0166] Cumulative rating

[0167] The PASI cumulative scores of the modeling sites of each group of animals at different time points are as follows Figure 7 As shown in Table 11, compared with the model group, the PASI cumulative scores of mice in CAI ointment dose groups 1 and 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the PASI cumulative scores of mice in the positive control group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the PASI cumulative scores of mice in the positive control group 2 were significantly reduced on d10 (3 days after administration) (P < 0.01).

[0168] Table 11 PASI cumulative scores of animals in each group at different time points (n=5 or 10)

[0169]

[0170]

[0171] Note: Compared with the model group, ** indicates P < 0.01.

[0172] Animal psoriasis skin lesions

[0173] The skin lesions of the animals in each group at different times are as follows Figure 8 As shown, it can be clearly seen that compared with the model group, the skin lesions at the modeling site of mice in CAI ointment dose groups 1 and 2 were significantly improved on d8-d10 (1-3 days after administration); the skin lesions at the modeling site of mice in B drug dose group 1 were significantly improved on d8 (1 day after administration) and significantly improved on d9-d10 (2-3 days after administration); the skin lesions at the modeling site of mice in B drug dose group 2 were significantly improved on d9 (2 days after administration) and significantly improved on d10 (3 days after administration).

[0174] 4. Biochemical index test results

[0175] Biochemical index detection: The ELISA method was used to detect the concentrations of IL-12, IL-17, IL-23 and TNF-α in the mouse skin tissue model group, positive control group 1, positive control group 2, CAI ointment dose group 1, and CAI ointment dose group 2.

[0176] Experimental operation

[0177] 1. After centrifugation of mouse skin tissue samples, take the supernatant for ELISA experiment;

[0178] 2.ELISA experiment:

[0179] (1) Standard sample addition: set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well;

[0180] (2) Sample addition: Add 50 μL of the sample to be tested to the sample well; do not add any sample to the blank well;

[0181] (3) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well except the blank well;

[0182] (4) Incubation: Seal the reaction wells with a sealing film and incubate in a 37°C water bath or incubator for 60 min;

[0183] (5) Washing: Tear off the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times;

[0184] Color development: Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min;

[0185] Stop: Add 50 μL of stop solution to each well;

[0186] Determination: Within 15 minutes, use an enzyme-labeled instrument to measure the OD value of each well at a wavelength of 450 nm.

[0187] V. Experimental Results

[0188] 1. Results Statistics

[0189] Table 12 IL-12 experimental results statistics

[0190]

[0191] Table 13 IL-17 experimental results statistics

[0192]

[0193]

[0194] Table 14 IL-23 experimental results statistics

[0195]

[0196] Table 15 TNF-a experimental results statistics

[0197]

[0198]

[0199] 2. Histogram of experimental results

[0200] The experimental results are as follows Figure 9-12 shown.

[0201] 3. Conclusion:

[0202] From the above results, it can be seen that compared with the model group, the carboxyamide triazole ointment treatment group can reduce the concentrations of IL-12 and IL-23 to a certain extent. Compared with the model group, the IL-17 level in the skin of the CAI ointment dose 1 group and the CAI ointment dose 2 group was significantly reduced (P<0.05). Compared with the model group, the TNF-a level in the skin of the CAI ointment dose 1 group was significantly reduced (P<0.05).

[0203] summary:

[0204] General observations: The mental state and activity of animals in each group were normal after administration, and no abnormalities were observed.

[0205] Body weight: The changes in body weight levels of animals in each drug-dosing group were consistent, with a certain degree of decrease during the modeling period (d0-d5), and the body weight levels remained stable from the end of the late modeling period to the start of drug administration (d5-d10). There was no significant difference in the body weight levels of animals in each group (P>0.05).

[0206] Erythema score: Compared with the model group, the erythema score of mice in CAI ointment dose group 1 was significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the erythema score of mice in CAI ointment dose group 2 was significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01); the erythema score of mice in the positive control group 1 was significantly reduced on d9-d10 (2-3 days after administration) (P < 0.01). There was no statistical difference in the erythema score of mice in the positive control group 2 compared with the control group (P > 0.05).

[0207] Squamous scale score: Compared with the model group, the squamous scale score of mice in CAI ointment dose groups 1 and 2 was significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the squamous scale score of mice in the positive control group 1 was significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01). The squamous scale score of mice in the positive control group 2 was significantly reduced on d10 (3 days after administration) (P < 0.01).

[0208] Skin thickness score: Compared with the model, the skin thickness scores of mice in CAI ointment dose groups 1 and 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05 or P < 0.01); the skin thickness scores of mice in the positive control group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.05). The skin thickness scores of mice in the positive control group 2 were significantly reduced on d10 (3 days after administration) (P < 0.05).

[0209] PASI cumulative score: Compared with the model group, the PASI cumulative scores of mice in CAI ointment dose groups 1 and 2 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the PASI cumulative scores of mice in the positive control group 1 were significantly reduced on d8-d10 (1-3 days after administration) (P < 0.01); the PASI cumulative scores of mice in the positive control group 2 were significantly reduced on d10 (3 days after administration) (P < 0.01).

[0210] Animal psoriasis skin lesions: It can be clearly seen that compared with the model group, the skin lesions at the modeling site of mice in CAI ointment dose groups 1 and 2 were significantly improved on d8-d10 (1-3 days after administration).

[0211] Pathology report

[0212] 1. Materials and Methods

[0213] 1.1 Samples and Film Reading

[0214] The samples sent for inspection are fixed with paraformaldehyde. After the fixation is in good condition, they are trimmed, dehydrated, embedded, sliced, stained, and sealed in strict accordance with the SOP procedures of the pathological laboratory detection of this unit. Finally, qualified samples are examined under the microscope. (For different clients, the corresponding preparation laboratory reports are detailed on the company's official website, which is briefly described here).

[0215] Browse the slices under a microscope or browse digital slices, observe the tissue slices in detail at different magnifications, and describe in writing the basic pathological changes in the slices, such as congestion, congestion, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, inflammatory changes, etc., and reflect the differences between the slices.

[0216] 1.2 Image acquisition instrument

[0217] name factory model Upright white light microscope Nikon(JaPan) EcliPse Ci-L

[0218] 1.3 Evaluation Criteria

[0219]

[0220] Evaluation criteria reference: [US] Peter. Mann et al. International Standards of Terminology and Diagnostic Criteria for Pathological Changes in Rats and Mice (INHAND) [M]. Translated by Yang Lifeng, Zhou Xiangmei, and Zhao Deming. Beijing: China Agriculture Press, 2019.

[0221] 2. Check the results

[0222] 2.1 Lesion description

[0223] Fig.13 It can be seen that in the model group mouse 1 (#22), small focal necrosis and nuclear fragmentation were occasionally seen in the epidermis and superficial dermis of the skin tissue, accompanied by a small amount of basophilic inflammatory exudate, local epidermis was thick, local collagen fibers were irregularly arranged, and a large number of scattered lymphocytes and granulocytes infiltrated the dermis.

[0224] Fig.14 It can be seen that the dermis of the skin tissue of model group mouse 2 (#24) is thin, and the number of accessory organs such as hair follicles and sebaceous glands is small; more epidermal necrosis and shedding can be seen, the cell nucleus is fragmented, and it is eosinophilic and homogeneous, accompanied by more inflammatory exudates, and the local dermal collagen fibers are irregularly arranged, and more lymphocytes and granulocytes can be seen scattered infiltrates.

[0225] Fig.15 It can be seen that a small amount of epidermal necrosis and shedding can be seen in the skin tissue of mouse 3 (#28) in the model group, the cell nucleus is fragmented, and it is eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The dermal collagen fibers are arranged more regularly, and the number of accessory organs such as hair follicles and sebaceous glands is abundant. A small amount of lymphocytes can be seen scattered infiltration.

[0226] Fig.16 It can be seen that in the skin tissue of mouse 4 (#60) in the model group, small focal necrosis and shedding of the epidermis were occasionally observed, the nuclei were fragmented, and the epidermis was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. A small number of prickle cells with loose cytoplasm and irregular cell arrangement were observed, and the local dermal collagen fibers were irregularly arranged. A small number of scattered lymphocytes infiltration were observed.

[0227] Fig.17 It can be seen that in the skin tissue of mouse 1 (#29) in group 1 of CAI ointment, small focal necrosis of the epidermis was occasionally observed, the nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local dermal collagen fibers were irregularly arranged, and a small amount of lymphocytes and granulocytes were scattered infiltrates.

[0228] Fig.18 It can be seen that in the skin tissue of mouse 2 (#3) in the CAI ointment dose 1 group, small focal necrosis of the epidermis was occasionally observed, the cell nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local dermal collagen fibers were irregularly arranged, and a small amount of lymphocytes and granulocytes were scattered infiltrates.

[0229] Fig.19It can be seen that in the skin tissue of mouse 3 (#30) in the CAI ointment dose 1 group, small focal necrosis of the epidermis was occasionally observed, the cell nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local dermal collagen fibers were irregularly arranged, and a small amount of lymphocytes and granulocytes were scattered infiltrates.

[0230] Fig. 20 It can be seen that in the epidermis and superficial dermis of the skin tissue of mouse 4 (#32) in the CAI ointment dose 1 group, small focal necrosis was occasionally observed, the cell nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local dermal collagen fibers were irregularly arranged, and a small amount of lymphocytes were scattered infiltration.

[0231] Fig.21 It can be seen that in the skin tissue of mouse 1 (#16) in the CAI ointment dose 2 group, small focal necrosis and shedding of the epidermis were occasionally observed, with cell nuclei fragmentation, accompanied by a small amount of inflammatory exudate, and the dermal collagen fibers were arranged more regularly, with a small amount of scattered lymphocyte infiltration.

[0232] Fig. 22 It can be seen that the epidermis of the skin tissue of mouse 2 (#19) in the CAI ointment dose 2 group was intact, with a clear structure and a visible keratinized layer; the dermis was rich in collagen fibers and arranged regularly, with visible accessory organs such as hair follicles and sebaceous glands, and occasionally a small amount of scattered lymphocytes infiltration.

[0233] Fig.23 It can be seen that in the skin tissue of mouse 3 (#21) in the CAI ointment dose 2 group, small focal necrosis and shedding of the epidermis were occasionally observed, with cell nuclei fragmented, accompanied by a small amount of inflammatory exudate, irregular arrangement of local dermal collagen fibers, and a small amount of scattered infiltration of lymphocytes and granulocytes.

[0234] Fig.24 It can be seen that in the epidermis and superficial dermis of the skin tissue of mouse 4 (#50) in the CAI ointment dose 2 group, small focal necrosis was occasionally observed, the cell nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local dermal collagen fibers were irregularly arranged, and a small amount of lymphocytes were scattered infiltration.

[0235] Fig.25 It can be seen that in the skin tissue of the positive group 1 mouse 1 (#18), small focal necrosis of the epidermis and nuclear fragmentation were occasionally observed, and the collagen fibers in the local epidermis and dermis were arranged irregularly, and a small amount of lymphocytes and granulocytes were scattered infiltrates.

[0236] Fig.26 It can be seen that the epidermis of the skin tissue of mouse 2 (#54) in the positive group 1 is intact, with a clear structure and a visible keratinized layer; the dermis is rich in collagen fibers and arranged regularly, and accessory organs such as hair follicles and sebaceous glands can be seen, and a small amount of scattered lymphocytes can be seen occasionally.

[0237] Fig. 27It can be seen that in the skin tissue of mouse 3 (#7) in the positive group 1, small focal necrosis of the epidermis was occasionally observed, the cell nucleus was fragmented, the epidermis was locally thick, the dermal collagen fibers were arranged more regularly, and a small amount of scattered lymphocyte infiltration was occasionally observed.

[0238] Fig.28 It can be seen that in the skin tissue of the positive group 2 mouse 1 (#38), small focal necrosis was occasionally seen in the epidermis and superficial dermis, the cell nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The dermal collagen fibers were arranged relatively regularly, and a small amount of lymphocytes were occasionally scattered and infiltrated.

[0239] Fig.29 It can be seen that the epidermis of the skin tissue of mouse 2 (#44) in the positive group 2 is intact, with a clear structure, a visible keratinized layer, and a thick epidermis in some areas; the dermis is rich in collagen fibers and arranged regularly, and accessory organs such as hair follicles and sebaceous glands can be seen, and occasionally a small number of lymphocytes are scattered in the infiltration.

[0240] Fig.30 It can be seen that in the skin tissue of mouse 3 (#52) in the positive group 2, small focal necrosis of the epidermis was occasionally observed, the nucleus was fragmented, and it was eosinophilic and homogeneous, accompanied by a small amount of inflammatory exudate. The local epidermis was thick, the dermal collagen fibers were arranged more regularly, and a small amount of lymphocytes and granulocytes were occasionally scattered infiltrates.

[0241] 2.2 Pathological scoring

[0242] The pathological scores are shown in Table 16. Fig.31 As shown in the figure, compared with the model group, the pathological score of animals in the CAI ointment dose 1 group was significantly decreased (P < 0.01).

[0243] Table 16 Pathological scoring table

[0244]

[0245]

[0246] in conclusion:

[0247] Under the experimental conditions, the test substance had a significant therapeutic effect on imiquimod-induced psoriasis mice. The changes in erythema, scaling, thickness of the skin lesions in the model mice, as well as the onset time of the drug effect were significantly better than those in other groups of mice. The test substance was set up in two dose groups, and the low dose drug had better efficacy.

[0248] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A carboxyamide triazole external ointment preparation, characterized in that The invention comprises the following ingredients in proportion by weight: 0.5-4 parts of carboxyamide triazole, 60-90 parts of base, 5-10 parts of thickener, 2-9 parts of emulsifier, 1-7 parts of moisturizer, 2-8 parts of penetration enhancer and 0.0005-0.5 parts of antioxidant.

2. The carboxyamide triazole external ointment preparation according to claim 1, characterized in that The matrix is ​​selected from at least one of white vaseline, yellow vaseline, liquid paraffin, paraffin, white beeswax, lanolin, lanolin alcohol, and silicone oil; the thickener is selected from at least one of hydrogenated vegetable oil, palmitic acid, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, and paraffin; the emulsifier is selected from at least one of stearyl alcohol polyether, Span 60, cetyl alcohol, lanolin, lanolin alcohol, and Span 80; the moisturizer is selected from at least one of isopropyl myristate, cyclomethicone, and triacetin; the penetration enhancer is selected from at least one of dimethyl sulfoxide, isopropyl myristate, azone, menthol, diethylene glycol monoethyl ether, and oleic acid; the antioxidant is selected from at least one of butylated hydroxyanisole, dibutylhydroxytoluene, and dl-α-tocopherol.

3. The carboxyamide triazole external ointment preparation according to claim 1, characterized in that The pH of the carboxyamide triazole external ointment preparation is 4-9.

4. The carboxyamide triazole external ointment preparation according to claim 2, characterized in that The invention comprises the following ingredients in proportion by weight: 0.5-4 parts of carboxyamide triazole, 55-80 parts of white vaseline, 2-10 parts of liquid paraffin, 5-10 parts of hexadecanol, 2-9 parts of lanolin, 1-7 parts of isopropyl myristate, 2-8 parts of a penetration enhancer and 0.0005-0.1 parts of an antioxidant.

5. The carboxyamide triazole external ointment preparation according to claim 4, characterized in that The invention comprises the following ingredients in proportion by weight: 2 parts of carboxyamide triazole, 71-75 parts of white vaseline, 4-6 parts of liquid paraffin, 6-7 parts of hexadecanol, 5-6 parts of lanolin, 3-5 parts of isopropyl myristate, 4-5 parts of a penetration enhancer and 0.001-0.01 parts of an antioxidant.

6. The carboxyamide triazole external ointment preparation according to claim 5, characterized in that: The composition comprises the following ingredients in proportion by weight: 2 parts of carboxyamide triazole, 71.473 parts of white vaseline, 6 parts of liquid paraffin, 7 parts of hexadecanol, 5 parts of lanolin, 3 parts of isopropyl myristate, 5 parts of a penetration enhancer, and 0.001 parts of an antioxidant.

7. The carboxyamide triazole external ointment preparation according to claim 6, characterized in that The penetration enhancer is dimethyl sulfoxide; the antioxidant is butylated hydroxyanisole.

8. A method for preparing a carboxyamide triazole external ointment preparation, characterized in that: The following steps are involved: (1) white vaseline, cetyl alcohol, lanolin, liquid paraffin, isopropyl myristate and butylated hydroxyanisole are heated to melt the matrix; (2) stirring and cooling the substrate in step (1) while it is in a molten state; (3) preparing a buffer solution, adding the buffer solution to step (2), and stirring to homogenize the mixture; (4) preparing a bulk drug dispersion, adding the bulk drug dispersion to step (3), and stirring to homogenize the mixture; (5) Finally cool to 36°C-40°C.

9. The preparation method according to claim 8, characterized in that: The specific parameters of the matrix melting in step (1) are: The temperature is 70-80°C, and the stirring speed is 30rpm±5rpm; the stirring speed in step (2) is 30rpm±5rpm, and the material temperature after cooling is 50-65°C; the stirring speed in step (3) is 30rpm±5rpm, the homogenizing speed is 2000-2500rpm, and the mixing temperature is 50-65°C; the stirring speed in step (4) is 30rpm±5rpm, the homogenizing speed is 2000-2500rpm, and the mixing temperature is 50-65°C; the final cooling in step (5) has the following specific parameters: the cooling end point temperature is 36-40°C, and the stirring speed is 15-25rpm.

Images