Construction method and application of psoriasis animal model

Abstract

The invention discloses a construction method and application of a psoriasis animal model. The construction method of the psoriasis animal model comprises the following step: locally smearing imiquimod ointment on a non-human primate animal to induce the animal to generate psoriasis-like skin lesion. And evaluating the formation of the psoriasis model by regularly observing and recording the severity of erythema, scales, thickening and other symptoms in the smeared area. Compared with the prior art, local inflammatory response of cynomolgus monkey skin is triggered by an immunomodulator imiquimod, and symptoms such as erythema, scales and inflammation presented by a human psoriasis patient are effectively simulated. More importantly, the modeling method has high controllability, the attack degree of psoriasis can be accurately controlled by adjusting the use amount and the smearing area of the imiquimod, medication can be stopped after an expected model state is achieved, further deterioration of the model is prevented, and therefore the stability and repeatability of the model are guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of disease animal models, and in particular to a method for constructing a psoriasis animal model and application thereof. Background Art

[0002] Psoriasis is a chronic, relapsing, immune-mediated skin disease characterized by erythema, scaling, and inflammation on the skin. Although the exact cause of psoriasis is not fully understood, abnormal responses of the immune system play a key role in its pathogenesis. Currently, one of the main challenges facing psoriasis research and new drug development is the lack of reliable animal models to simulate the pathological process of human psoriasis.

[0003] At present, Imiquimod is an immunomodulator widely used in the establishment of psoriasis models. Imiquimod induces local inflammatory reactions in the skin by activating Toll-like receptor 7 (TLR7), which leads to the formation of psoriasis-like lesions. Mouse models are the main research objects of imiquimod-induced psoriasis-like symptoms, but due to physiological differences between species, mouse models cannot completely simulate the pathological characteristics of human psoriasis. For example: CN114568386A discloses a method for establishing an animal model of chronic psoriasis inflammation, which effectively reduces the dehydration and weight loss of mice by simulating the chronic and repeated pathogenesis of psoriasis in the human body. The specific steps are as follows: First, the modeling mice are raised in a standard feeding method; secondly, two days before modeling, the back of the mouse is divided into at least two parts, the hair of the first part is removed first, and then the imiquimod cream is applied, which is used continuously for 5 days, and the use is stopped for 2 days as a cycle. At the end of the second cycle, when the hair of the first part of the mouse shows signs of growing again, the hair of the second part is removed, and the cream is continued to be applied. Through this method of intermittent topical application of imiquimod cream, the defect of long-term continuous topical application leading to the gradual stabilization or alleviation of psoriasis lesions was avoided, thus successfully establishing a chronic inflammatory animal model of psoriasis. Summary of the invention

[0004] As a primate with a physiological and immune system highly similar to that of humans, cynomolgus monkeys have unique advantages in drug evaluation and disease model research. However, there is currently no mature method for inducing psoriasis-like lesions in cynomolgus monkeys by imiquimod. Therefore, the present invention provides a method for constructing an animal model of psoriasis and its application. This is of great significance for in-depth understanding of the pathogenesis of psoriasis and screening of effective therapeutic drugs.

[0005] The method for constructing the psoriasis animal model is as follows: by topically applying imiquimod ointment to non-human primates, psoriasis-like skin lesions are induced in the animals. The formation of the psoriasis model is evaluated by regularly observing and recording the severity of symptoms such as erythema, scaling, and thickening in the application area. After stopping the application of imiquimod ointment, the changes in the skin symptoms of the model animals can be continued to be observed to evaluate the stability and reversibility of the model.

[0006] The non-human primates were cynomolgus monkeys, ranging in age from 5 to 15 years old.

[0007] Before applying imiquimod ointment, disinfect the application area with iodine and gently massage after application to promote absorption of the ointment.

[0008] For local application, use 5% imiquimod ointment, with each application area ranging from 2cm×2cm to 6cm×6cm and the application dosage ranging from 0.5g to 2g.

[0009] The topical application frequency is once daily for 7 to 14 days until obvious psoriasis-like lesions appear.

[0010] More specifically, the method for constructing the psoriasis animal model is:

[0011] 1. Animal anesthesia: General anesthesia was performed on the cynomolgus monkeys using conventional anesthesia methods to ensure that the animals remained pain-free during the entire operation. Appropriate anesthetics and doses can be selected according to the laboratory standard operating procedures.

[0012] 2. Preparation of the target area: Select the back or limbs of the cynomolgus monkey as the application area. Use a sterile cotton swab dipped in iodine solution to thoroughly clean and disinfect the skin in the selected area to ensure that there is no contamination.

[0013] 3. Imiquimod ointment application: Take 5% imiquimod ointment and apply the ointment evenly on the disinfected skin area using sterile gloves or an applicator. The application area and dosage can be adjusted according to the experimental design. Usually, the area applied each time is about 3cm×3cm.

[0014] 4. Ointment penetration and absorption: After application, gently massage the application area for about 2-3 minutes to promote the penetration and absorption of the ointment. Be careful to avoid hair in the application area that may hinder the absorption of the ointment.

[0015] 5. Repeated application: According to the experimental design, apply imiquimod ointment once a day for 1-2 weeks. Observe the skin changes in the application area until psoriasis-like erythema, scaling, skin thickening and other symptoms appear. The application dose is 0.5g / time or 1.3g / time. The control group applies the same dose of vaseline ointment.

[0016] 6. Observation of skin symptoms: During the entire application period, regularly check and record skin symptoms in the application area. A scoring system (such as the PASI score) can be used to assess the severity of symptoms such as erythema, scaling, and thickening. If necessary, a skin biopsy is performed to further analyze histological changes.

[0017] 7. Model maintenance and observation: When the skin symptoms of cynomolgus monkeys reach the expected model state, the application of imiquimod ointment can be stopped and subsequent observation and research can be started. During this period, attention should be paid to the cleaning and care of the application area to prevent infection or other complications.

[0018] 8. Animal awakening and recovery: After the operation is completed, monitor the animal's awakening process to ensure its safe recovery. If necessary, use saline or artificial tears to moisten the eyes to prevent dryness.

[0019] 9. Follow-up treatment: Observe the overall health status of the animals and provide appropriate treatment when necessary. Collect and analyze experimental data to evaluate the success rate and application value of the model.

[0020] In the method for constructing an animal model of psoriasis of the present invention, after applying imiquimod ointment, the model animal exhibits typical skin symptoms such as psoriasis-like erythema, scaling, and skin thickening.

[0021] The 5% imiquimod ointment mentioned in the present invention refers to the commercially available imiquimod ointment containing 5wt% imiquimod.

[0022] Beneficial effects of the present invention:

[0023] Existing psoriasis models mainly rely on induction in rodents such as mice. However, due to physiological differences between species, these models often cannot fully simulate the complex pathological characteristics of human psoriasis. The method of inducing a psoriasis model in cynomolgus monkeys using imiquimod ointment, in which the immunomodulator imiquimod induces a local inflammatory response in the skin of cynomolgus monkeys, effectively simulates the symptoms of erythema, scaling and inflammation exhibited by human psoriasis patients. More importantly, this modeling method is highly controllable. By adjusting the amount of imiquimod used and the application area, the severity of psoriasis can be precisely controlled, and the medication can be terminated after the expected model state is reached to prevent the model from further deteriorating, thereby ensuring the stability and repeatability of the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the technical roadmap of the present invention.

[0025] Figure 2 It is a skin lesion scoring chart.

[0026] Figure 3 It is a skin condition map. DETAILED DESCRIPTION

[0027] Parameters for specific chemical substances used, sources.

[0028] Example 1-2

[0029] 1. Animal anesthesia: General anesthesia was performed on the cynomolgus monkeys using conventional anesthesia methods to ensure that the animals remained pain-free during the entire operation. Appropriate anesthetics and doses can be selected according to the laboratory standard operating procedures.

[0030] 2. Preparation of the target area: Select the back or limbs of the cynomolgus monkey as the application area. Use a sterile cotton swab dipped in iodine solution to thoroughly clean and disinfect the skin in the selected area to ensure that there is no contamination.

[0031] 3. Imiquimod ointment application: Take 5% imiquimod ointment and apply the ointment evenly on the disinfected skin area using sterile gloves or an applicator. The application area and dosage can be adjusted according to the experimental design. Usually, the area applied each time is about 3cm×3cm.

[0032] 4. Ointment penetration and absorption: After application, gently massage the application area for about 2-3 minutes to promote the penetration and absorption of the ointment. Be careful to avoid hair in the application area that may hinder the absorption of the ointment.

[0033] 5. Repeated application: According to the experimental design, imiquimod ointment was applied repeatedly once a day for 1-2 weeks. The skin changes in the application area were observed until psoriasis-like erythema, scaling, skin thickening and other symptoms appeared. The application dose of Example 1 was 0.5 g / time, and the application dose of Example 2 was 1.3 g / time.

[0034] 6. Observation of skin symptoms: During the entire application period, regularly check and record skin symptoms in the application area. A scoring system (such as the PASI score) can be used to assess the severity of symptoms such as erythema, scaling, and thickening. If necessary, a skin biopsy is performed to further analyze histological changes.

[0035] 7. Model maintenance and observation: When the skin symptoms of cynomolgus monkeys reach the expected model state, the application of imiquimod ointment can be stopped and subsequent observation and research can be started. During this period, attention should be paid to the cleaning and care of the application area to prevent infection or other complications.

[0036] 8. Animal awakening and recovery: After the operation is completed, monitor the animal's awakening process to ensure its safe recovery. If necessary, use saline or artificial tears to moisten the eyes to prevent dryness.

[0037] 9. Follow-up treatment: Observe the overall health status of the animals and provide appropriate treatment when necessary. Collect and analyze experimental data to evaluate the success rate and application value of the model.

[0038] In animal models of the disease, the PASI (Psoriasis Area and Severity Index) score is mainly used to assess the severity of the psoriasis model, which includes the assessment of the severity of skin inflammation, skin thickness, crusting and erythema. The scoring system uses a 5-point system (0-4 points) to evaluate these symptoms separately.

[0039] Specific evaluation criteria for PASI scores

[0040] Skin thickness: Scored based on the degree of skin folds.

[0041] 0 points: The skin is smooth and wrinkle-free.

[0042] 1 point: Slight wrinkling of the skin at the edge of the application area.

[0043] 2 points: Slight wrinkles appear on the entire skin of the application area.

[0044] 3 points: The wrinkles in the application area are further deepened.

[0045] 4 points: Based on the score of 3, the mice showed weight loss or poor condition.

[0046] Crusting: Score based on the extent of skin scaling.

[0047] 0 points: The skin is smooth and without scaling.

[0048] 1 point: Slight scaling of the skin in the application area.

[0049] 2 points: The skin in the application area is completely covered with scales.

[0050] 3 points: The degree of scaling in the applied area deepens further.

[0051] 4 points: Based on the score of 3, the mice showed weight loss or poor condition.

[0052] Erythema: Score is based on the degree of redness of the skin.

[0053] 0 points: smooth skin.

[0054] 1 point: Slight redness appears on the skin in the application area.

[0055] 2 points: The entire skin in the application area turns red.

[0056] 3 points: The red color of the applied area deepens further.

[0057] 4 points: Based on the score of 3, the mice showed weight loss or poor condition.

[0058] Total score calculation

[0059] The total score is the sum of the scores of skin thickness, crusting and erythema. In this way, the severity of psoriasis in mice can be comprehensively evaluated.

[0060] The PASI score has important application value in the study of psoriasis animal models. It can better evaluate the severity of the disease and the effect of treatment, and provide strong support for the research and treatment of psoriasis.

[0061] In this experiment, different doses (0.5 g and 1.3 g) of imiquimod ointment were used to induce skin lesions in experimental animals. Figure 2 The results showed that the 1.3g group showed significantly higher inflammatory responses than the 0.5g group in terms of skin thickness, crusting and erythema scores, and the scores of various indicators continued to rise throughout the experiment, eventually reaching the highest point on the 14th day. This suggests that high doses of imiquimod ointment can more strongly and rapidly induce psoriasis-like lesions of the skin. In contrast, the inflammatory response of the 0.5g group was milder, the lesions developed more slowly, and the final total score was significantly lower than that of the 1.3g group. The experimental results clearly show the positive correlation between the dose of imiquimod ointment and the inflammatory response of the skin. High doses are more suitable for simulating pathological models of severe psoriasis, while low doses may be more suitable for studying mild or early psoriasis.

[0062] The effect of imiquimod ointment at different doses on inducing skin lesions in cynomolgus monkeys, the two groups of examples used ointment doses of 0.5g and 1.3g, respectively.

[0063] Day 0: Initial state, the skin surface is smooth and has no obvious lesions, and appears healthy overall.

[0064] Day 2: The skin begins to show slight changes, with mild redness or color changes, indicating that the skin is beginning to respond to some treatment or stimulation.

[0065] Day 4: The color change is more obvious and there may be slight signs of inflammation on the surface of the skin, but the overall changes are still relatively minor.

[0066] Day 6: The area of ​​erythema increases, the skin reaction becomes more obvious, and there may be some infiltration of inflammatory cells.

[0067] Day 8: The erythema continues to worsen, and scaling or slight desquamation may appear on the skin surface, indicating that the inflammatory response is increasing.

[0068] Day 10: The skin condition worsens further, the erythema expands, and there may be more obvious skin thickening or tissue reaction.

[0069] Day 12: The lesion area becomes more obvious and may have oozing or more severe scaling, indicating that the psoriasis-like lesions or other skin inflammation are getting worse.

[0070] Day 14: The most serious condition, skin lesions reach their peak, with obvious inflammation, erythema, scaling or other pathological features.

[0071] Example 3

[0072] The same as Example 2, the only difference is that the 5% imiquimod ointment is replaced by the improved imiquimod ointment.

[0073] The improved imiquimod ointment is prepared by mixing 100 g of 5% imiquimod ointment and 5 g of lipopolysaccharide at 200 rpm and 70° C. for 15 min to obtain the improved imiquimod ointment.

[0074] The lipopolysaccharide is derived from Escherichia coli 055:B5 and is composed of a lipid A portion connected to an antigenic O-polysaccharide. Product number: ST1470-10mg; source: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0075] Example 4

[0076] The same as Example 2, the only difference is that the 5% imiquimod ointment is replaced by the improved imiquimod ointment.

[0077] The improved imiquimod ointment is prepared by mixing 100 g of 5% imiquimod ointment and 5 g of phospholipid permeation enhancer at 200 rpm and 70° C. for 15 min to obtain the improved imiquimod ointment.

[0078] The preparation method of the phospholipid penetration enhancer is as follows: 210.2 g of isooctyl phosphate and 61.08 g of ethanolamine are mixed evenly, and 500 mL of acetone is added and stirred to mix evenly; the acetone is removed by rotary evaporation at 50° C.; and vacuum drying is performed to obtain the phospholipid penetration enhancer.

[0079] Example 5

[0080] The same as Example 2, the only difference is that the 5% imiquimod ointment is replaced by the improved imiquimod ointment.

[0081] The improved imiquimod ointment is prepared by mixing 100 g of 5% imiquimod ointment and 5 g of phospholipid permeation enhancer at 200 rpm and 70° C. for 15 min to obtain the improved imiquimod ointment.

[0082] The preparation method of the phospholipid penetration enhancer is as follows: 154.10g of mono(2-methylpropyl) phosphate and 61.08g of ethanolamine are mixed evenly, and 500mL of acetone is added and stirred to mix evenly; the acetone is removed by rotary evaporation at 50°C; and vacuum drying is performed to obtain the phospholipid penetration enhancer.

[0083] Example 6

[0084] The same as Example 2, the only difference is that the 5% imiquimod ointment is replaced by the improved imiquimod ointment.

[0085] The improved imiquimod ointment is prepared by mixing 100 g of 5% imiquimod ointment and 5 g of phospholipid permeation enhancer at 200 rpm and 70° C. for 15 min to obtain the improved imiquimod ointment.

[0086] The preparation method of the phospholipid penetration enhancer is as follows: 266.31 g of lauryl phosphate monoester and 61.08 g of ethanolamine are mixed evenly, and 500 mL of acetone is added and stirred to mix evenly; the acetone is removed by rotary evaporation at 50° C.; and vacuum drying is performed to obtain the phospholipid penetration enhancer.

[0087] Transdermal absorption test:

[0088] The 5% imiquimod ointment and the improved imiquimod ointment prepared in Examples 3-6 were subjected to penetration test.

[0089] Fresh pig skin was taken, subcutaneous fat was removed, and then the skin samples were cut into 5 cm × 5 cm for subsequent experiments.

[0090] The prepared pig skin sample was tightly clamped between the donor chamber and the receptor chamber of the transdermal diffusion instrument, ensuring that the stratum corneum of the skin was facing the donor chamber. 5 g of the sample to be tested was added to the donor chamber, and 4.0 mL of phosphate buffered saline (PBS, pH 7.4) was added to the receptor chamber.

[0091] During the entire experiment, the solution was kept stirring at 600 rpm in the receptor chamber and the temperature was controlled at 32 ± 0.5 °C to simulate the skin environment under physiological conditions and ensure the accuracy and reliability of the experimental results.

[0092] After 8 hours, 2.0 mL of sample was taken from the receptor chamber and the sample was centrifuged at 16,000 rpm and the imiquimod concentration was measured using a high performance liquid chromatography (HPLC) system.

[0093] Table 1

[0094] Cumulative permeation amount (mg) Example 2 (5% Imiquimod Ointment) 0.325 Example 3 0.322 Example 4 0.394 Example 5 0.488 Example 6 0.384

[0095] The animal model induced by imiquimod usually disappears within a few days, which may be due to the poor absorption of imiquimod by the skin and the lack of lasting efficacy. In view of this problem, the present invention improves the 5% imiquimod ointment by additionally adding a penetration enhancer to improve the transdermal absorption effect of imiquimod.

[0096] Compared with the 5% imiquimod ointment of Example 2, Examples 4-6 contain additional substances that promote penetration, such as lipopolysaccharide and phospholipid penetration enhancers, which can effectively promote the transdermal absorption effect of imiquimod ointment and further stabilize the effect of psoriasis animal model.

[0097] Phospholipid penetration enhancers diffuse more easily into the skin and disrupt the orderly arrangement of stratum corneum lipids, thereby enhancing the interaction between the drug and the skin and improving the skin penetration effect of imiquimod.

[0098] In Examples 4-6, the effect of preparing the phospholipid penetration enhancer using mono(2-methylpropyl) phosphate as the phosphate source is significantly better than isooctyl phosphate and lauryl phosphate monoester. The possible reason is that the structure of the phospholipid penetration enhancer obtained by using mono(2-methylpropyl) phosphate and ethanolamine achieves a more appropriate balance between lipophilicity and hydrophilicity, so that the penetration enhancer can better combine with imiquimod and more easily penetrate the stratum corneum and other barriers of the skin, thereby showing a better penetration effect.

[0099] Model persistence: After successful modeling, the animal model is continued to be raised according to conventional feeding methods, and subsequent changes in the animal model are observed.

[0100] Table 2

[0101] The number of days required for the animal model symptoms to completely disappear after successful modeling Example 1 4 days Example 2 7 days Example 3 14 days Example 4 16 days Example 5 24 days Example 6 18 days

[0102] As can be seen from the above table, the animal model constructed in Example 3 maintains the corresponding symptoms longer after the model is successfully established than in Examples 1-2. The lipopolysaccharide in Example 3 is different from the phospholipid permeation enhancer and does not significantly promote the transdermal absorption of imiquimod, but lipopolysaccharide is a component of the cell wall of Gram-negative bacteria and has a certain irritation itself, which can assist imiquimod in enhancing the inflammatory effect and improve the duration of symptoms of the successful animal model.

[0103] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for constructing an animal model of psoriasis, characterized in that: By topically applying imiquimod ointment to non-human primates, psoriasis-like skin lesions are induced in the animals; the formation of the psoriasis model is evaluated by regularly observing and recording the severity of symptoms such as erythema, scaling, and thickening in the application area; after stopping the application of imiquimod ointment, the changes in skin symptoms of the model animals can continue to be observed to evaluate the stability and reversibility of the model.

2. The method for constructing an animal model of psoriasis according to claim 1, characterized in that: The non-human primates were cynomolgus monkeys, ranging in age from 5 to 15 years old.

3. The method for constructing an animal model of psoriasis according to claim 1, characterized in that: Before applying imiquimod ointment, disinfect the application area with iodine and gently massage after application to promote absorption of the ointment.

4. The method for constructing an animal model of psoriasis according to claim 1, characterized in that: For local application, use 5% imiquimod ointment, with each application area ranging from 2cm×2cm to 6cm×6cm and the application dosage ranging from 0.5g to 2g.

5. The method for constructing an animal model of psoriasis according to claim 1, characterized in that: The topical application frequency is once daily for 7 to 14 days until obvious psoriasis-like lesions appear.

6. The method for constructing an animal model of psoriasis according to claim 1, wherein: The method for constructing the psoriasis animal model is as follows: (1) Animal anesthesia: General anesthesia was performed on the cynomolgus monkeys using conventional anesthesia methods to ensure that the animals remained pain-free during the entire operation. Appropriate anesthetics and doses were selected according to the laboratory standard operating procedures; (2) Preparation of the target area: The back or limbs of the cynomolgus monkey were selected as the application area. A sterile cotton swab was dipped in iodine solution to thoroughly clean and disinfect the skin of the selected area to ensure that there was no contamination. (3) Application of imiquimod ointment: Take 5% imiquimod ointment and apply it evenly on the disinfected skin area using sterile gloves or an applicator; the application area and dosage can be adjusted according to the experimental design; usually, the application area is about 3 cm × 3 cm each time; (4) Ointment penetration and absorption: After application, gently massage the applied area for about 2-3 minutes to promote the penetration and absorption of the ointment. Be careful to avoid hair in the application area that may hinder the absorption of the ointment. (5) Repeated application: According to the experimental design, apply imiquimod ointment once a day for 1-2 weeks; observe the skin changes in the application area until psoriasis-like erythema, scaling, skin thickening and other symptoms appear; the application dose is 0.5 g / time or 1.3 g / time; (6) Observation of skin symptoms: During the entire application period, skin symptoms in the application area should be checked and recorded regularly. Scoring systems (such as the PASI score) can be used to assess the severity of symptoms such as erythema, scaling, and thickening. If necessary, skin biopsy should be performed to further analyze histological changes. (7) Model maintenance and observation: When the skin symptoms of the cynomolgus monkeys reach the expected model state, the application of imiquimod ointment can be stopped and subsequent observation and research can be started; during this period, attention should be paid to the cleaning and care of the application area to prevent infection or other complications; (8) Animal awakening and recovery: After the operation is completed, monitor the animal's awakening process to ensure its safe recovery; if necessary, use saline or artificial tears to moisten the eyes to prevent dryness; (9) Follow-up treatment: observe the overall health status of the animals and provide appropriate treatment when necessary; collect and analyze experimental data to evaluate the success rate and application value of the model.

7. Use of the psoriasis animal model according to any one of claims 1 to 6 in psoriasis treatment drugs.

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