Canine atopic dermatitis animal model of and use thereof

Abstract

The present invention relates to a method for producing a canine atopic dermatitis animal model, a canine atopic dermatitis animal model produced thereby, and a use thereof. According to the present invention, a canine atopic dermatitis animal model is produced by skin stimulation on a dog through epicutaneous sensitization with ovalbumin at a concentration of 1 to 5 mg / ml once daily for 1 to 2 weeks and then with ovalbumin at a concentration of 1 to 5 mg / ml once daily for 2 to 8 weeks. The resulting animal model exhibits clinical symptoms of atopic dermatitis, changes in relevant factors such as increased expression levels of IgE and CCL17, and elevated expression levels of Th2-related cytokines IL-4, IL-31, and IL-13. Therefore, this invention enables the establishment of a standardized animal model for symptoms and Th2 immune responses appearing upon the onset of atopic dermatitis, and the model can be used in clinical studies for screening candidate agents for the prevention, alleviation, or treatment of atopic dermatitis.

Description

Animal model of canine atopic dermatitis and its use

[0001] The present invention relates to a method for producing a canine atopic dermatitis animal model, a canine atopic dermatitis animal model produced thereby, and uses thereof.

[0002] Atopic dermatitis is a chronic, long-lasting skin condition characterized by the development of severely itchy, eczema-like lesions on the skin. It is common in people with a history or family history of atopic dermatitis. A characteristic of atopic dermatitis is that when symptoms appear, the affected area tends to be scratched or rubbed, which can worsen the skin condition. According to the Korea Centers for Disease Control and Prevention, atopic dermatitis is a chronic allergic inflammatory skin disease. It has characteristics of both allergic and inflammatory diseases, and since each disease operates through different mechanisms, it is necessary to clearly elucidate its pathogenesis.

[0003] Accordingly, research is actively underway to develop customized preventive or therapeutic agents to more effectively prevent or treat atopic skin diseases, including atopic dermatitis, based on their underlying causes. These studies require clinical trials, which can present various risks and raise ethical concerns. To overcome these challenges, various animal models are being used.

[0004] Meanwhile, in modern society, where single-person households are on the rise and the population is rapidly aging, the number of people living with companion animals has reached approximately 10 million. The Ministry of Agriculture, Food and Rural Affairs estimated the size of the Korean companion animal market at 1.8 trillion won in 2015, showing steady, high growth every year. However, pets' diets are becoming more sophisticated, and the lack of exercise they receive at home is leading to declines in organ function and weakened immunity to disease. While prevention and treatment with chemical agents are possible at pet hospitals, veterinarians require nutritional supplements that can provide lasting effects, such as health supplements.

[0005] Furthermore, as the pet population increases, various pet diseases are emerging. Among them, atopy is a chronic, recurrent inflammatory skin disease caused by rapid environmental changes, genetic factors, immunological abnormalities, and abnormalities in the skin barrier. It is an intractable disease that is difficult to completely cure not only in humans but also in pets. In particular, atopic dermatitis mostly occurs in animals living indoors, and because house dust or house dust mites act as allergens that cause atopy, the incidence rate is gradually increasing in social environments where pets are primarily raised.

[0006] Currently, companion animals suffering from atopic dermatitis in Korea are managed with medications such as steroids, with no other fundamental treatment options. While steroids are effective and can lead to rapid improvement in clinical symptoms, long-term administration carries significant side effects, making long-term use of steroids and medications less than optimal for managing atopic patients. Therefore, research on products with fewer side effects and effective atopic management is constantly needed. Clinical trials are essential for such research. Therefore, to study atopic dermatitis and screen for treatments, the development of animal models that represent the immune response of atopic dermatitis in companion animals is essential.

[0007] The present invention aims to solve the above-mentioned problems and other problems related thereto.

[0008] The purpose of the present invention is to provide a method for producing an animal model of atopic dermatitis, comprising the steps of: transdermally administering 1 to 5 mg / ml of ovalbumin to a dog whose hair has been removed once a day for 1 to 2 weeks to cause skin sensitization (epicutaneous sensitization); and transdermally administering 1 to 5 mg / ml of ovalbumin once a day for 2 to 8 weeks to cause skin irritation.

[0009] Another object of the present invention is to provide an atopic dermatitis animal model manufactured according to the above manufacturing method.

[0010] Another object of the present invention is to provide a method for screening for an agent for preventing, improving, or treating an atopic skin disease, comprising the steps of: (a) administering a candidate substance for preventing, improving, or treating an atopic skin disease to the prepared animal model of an atopic skin disease; and (b) measuring the degree of symptoms or the level of indicators related to an atopic skin disease in the animal model to which the candidate substance has been administered.

[0011] Another object of the present invention is to provide a use of an atopic dermatitis animal model for screening for atopic dermatitis prevention, improvement, or treatment agent.

[0012]

[0013] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0014] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0015]

[0016] As one aspect to achieve the above object, the present invention provides a method for producing a canine atopic dermatitis animal model. Specifically, the present invention provides a method for producing an atopic dermatitis animal model, comprising: (a) a step of transdermally administering ovalbumin at a concentration of 1 to 5 mg / ml to a dog whose hair has been removed once a day for 1 to 2 weeks (epicutaneous sensitization); and (b) a step of transdermally administering ovalbumin at a concentration of 1 to 5 mg / ml once a day for 2 to 8 weeks to cause skin stimulation.

[0017] In the present invention, skin sensitization may be induced through the transdermal administration in step (a). In step (a), ovalbumin is transdermally administered to induce skin sensitization, and the skin sensitization may increase the sensitivity to the antigen in a normal dog by repeatedly exposing the dog to the antigen. Thereafter, in step (b), ovalbumin is transdermally administered to the dog where skin sensitization has occurred to induce skin stimulation, and the skin stimulation may induce an allergic reaction by re-exposing the same antigen to a normal dog with an immune system that has become sensitized to the antigen, thereby inducing an atopic skin disease.

[0018] As a specific example, the present invention provides a method for producing an animal model of atopic dermatitis, comprising: (a) a step of causing skin sensitization by transdermal administration of ovalbumin at a concentration of 1 to 5 mg / ml to a dog whose hair has been removed once a day for 1 to 2 weeks (epicutaneous sensitization); and (b) a step of causing skin stimulation by transdermal administration of ovalbumin at a concentration of 1 to 5 mg / ml once a day for 2 to 8 weeks.

[0019] In the present invention, the term "atopic skin disease" includes both atopic dermatitis and atopic skin disease without inflammation, and the term "atopic dermatitis" may refer to a chronically relapsing skin eczema disease accompanied by severe itching. The term "atopic skin disease" may include atopic skin disease that occurs not only in humans but also in companion animals such as dogs.

[0020] In the present invention, the 'ovalbumin' refers to a protein that constitutes more than half of egg white, is encoded by the OVAL gene (Gene ID: 396058), and is used as an allergen that sensitizes the immune response.

[0021] In the present invention, the 'dog' may be a beagle, and the beagle may be a 7-month-old beagle, and may be a healthy beagle stabilized for a week with a 12-hour / 12-hour light / dark cycle at a temperature of 22 to 26°C and a humidity of 40 to 60%, but is not limited thereto.

[0022] In the present invention, the concentration of the ovalbumin may be 1 mg / ml.

[0023] The steps of transdermally administering the above ovalbumin to cause skin sensitization and skin irritation may each include: (i) applying ovalbumin to a patch and allowing it to be absorbed; and (ii) attaching the patch in which ovalbumin has been absorbed to a dog. The patch is specifically Tegaderm. TM ) may be used as a pad, but the type is not limited as long as it absorbs ovalbumin and can cause skin sensitization or irritation when attached to the dog.

[0024] In the present invention, the skin sensitization and skin irritation through the transdermal administration of ovalbumin may be performed on the skin of the back of the dog, and preferably, may be performed on the skin of four back areas. When the transdermal administration of ovalbumin is performed on the back of the dog, it is possible to prevent secondary damage to the wounded area due to the nature of the dog or the possibility of inducing systemic tolerance by licking the ovalbumin and entering the mouth, and it is characterized in that an atopic skin disease can be induced only through the local transdermal administration of ovalbumin. The skin of the four back areas may be selected in the range of a width of 2.5 to 5 cm and a height of 4 to 7 cm, and may be spaced 2 to 3 cm apart from each other on each back area, but is not limited thereto.

[0025] In the present invention, the 1 to 5 mg / ml ovalbumin may be applied to each patch in an amount of 100 μl to 1 ml, preferably 100 μl to 500 μl, and absorbed, but is not limited thereto.

[0026] In the present invention, after the step (ii) of attaching the patch having absorbed ovalbumin to the dog, the method may further include a step of first fixing the attached patch with a roll of cotton and then secondarily fixing it with an elastic bandage. By performing these two steps of fixation, it is possible to prevent systemic tolerance (immune tolerance) that may occur when attaching the patch having absorbed ovalbumin to the skin of the dog's back, thereby causing skin sensitization or skin irritation. In addition, after the step of causing skin sensitization or skin irritation, the method may further include a step of placing a collar on the dog's neck.

[0027] In the present invention, in the step of causing skin sensitization and skin irritation, the dosage and method of administering ovalbumin can be applied equally.

[0028] In the present invention, skin sensitization can be induced by transdermal administration of ovalbumin for 1 to 2 weeks, and skin irritation can be induced by transdermal administration of ovalbumin for a subsequent 2 to 8 weeks. As a specific example, skin sensitization can be induced by transdermal administration of ovalbumin for 1 week, and skin irritation can be induced by transdermal administration of ovalbumin for a subsequent 3 weeks. In addition, skin sensitization can be induced by transdermal administration of ovalbumin for a subsequent 2 weeks, and skin irritation can be induced by transdermal administration of ovalbumin for a subsequent 2 weeks.

[0029] In addition, after the step of causing skin sensitization, the step of causing skin irritation may be performed after a rest period of 1 to 3 weeks, and preferably, the step of causing skin irritation may be performed after a rest period of 2 weeks, but is not limited thereto.

[0030] In the present invention, the resting period may be used to ensure that the immune system, which responds through skin sensitization, has a period of reduced and minimal exposure to antigens. During this resting period, the dog's immune system returns to a normal state, and may then elicit a stronger immune response when stimulated.

[0031] As another aspect for achieving the above purpose, the present invention provides an atopic skin disease animal model manufactured according to the above manufacturing method.

[0032] The above ‘manufacturing method’ and ‘atopic skin disease’ are as described above.

[0033] In the present invention, the animal model may exhibit clinical symptoms including erythema and eschar formation and edema formation, and may exhibit symptoms of increased epidermal thickness and immune cell infiltration, compared to an animal that did not experience skin sensitization and irritation by percutaneous administration of ovalbumin.

[0034] In the present invention, the animal model may have an increased expression level of immunoglobulin E (IgE) and CCL17 chemokine in the blood compared to an animal that did not experience skin sensitization or irritation by percutaneous administration of ovalbumin. Preferably, when skin sensitization is induced by percutaneous administration of 1 mg / ml of ovalbumin, the expression level of IgE and CCL17 chemokine may be increased at a more significant level compared to an animal that did not experience skin sensitization or irritation by percutaneous administration of ovalbumin and an animal that did experience skin sensitization or irritation by percutaneous administration of 5 mg / ml of ovalbumin.

[0035] In the present invention, the animal model may exhibit an immune response of T-helper type 2 (Th2) cells in atopic dermatitis.

[0036] In the present invention, the animal model may be one in which the expression level of Th2-related cytokines in skin tissue is increased compared to an animal that does not cause skin sensitization and irritation by percutaneous administration of ovalbumin. Specifically, the relative expression levels of IL-4, IL-13, and IL-31 cytokines may be increased, and preferably, when skin sensitization and irritation are caused by percutaneous administration of 1 mg / ml of ovalbumin, the expression levels of IL-4, IL-13, and IL-31 cytokines may be increased at a more significant level compared to an animal that does not cause skin sensitization and irritation by percutaneous administration of ovalbumin and an animal that does cause skin sensitization and irritation by percutaneous administration of 5 mg / ml of ovalbumin.

[0037] In another aspect for achieving the above object, the present invention provides a method for screening for an atopic skin disease prevention, improvement or treatment agent, comprising the steps of (a) administering a candidate substance for preventing, improving or treating an atopic skin disease to the prepared atopic skin disease animal model; and (b) measuring the degree of symptoms or the level of an indicator related to an atopic skin disease in the animal model to which the candidate substance has been administered.

[0038] The above ‘atopic skin disease’ and ‘animal model’ are as described above.

[0039] In the present invention, administration of the candidate substance includes, but is not limited to, parenteral or oral administration, and a person skilled in the art can select an appropriate method to test the candidate substance on animals.

[0040] The above "candidate substance" refers to an unknown substance used in screening to determine whether it has an effect in preventing, improving, or treating atopic dermatitis, or whether it affects the severity of symptoms or indicator levels associated with atopic dermatitis. The candidate substance may include, but is not limited to, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, or plasma.

[0041] In the present invention, the symptoms related to atopic skin disease in step (b) may be at least one selected from the group consisting of erythema formation, scab formation, and edema formation, but are not limited thereto as long as they correspond to symptoms that may appear due to atopic skin disease. If the degree of the symptoms related to atopic skin disease measured in step (b) is reduced compared to the control group that was not administered the candidate substance, the candidate substance may be determined to be a preventive, improving, or therapeutic agent for atopic skin disease.

[0042] In the present invention, the indicator related to atopic skin disease in step (b) may be at least one selected from the group consisting of IgE, CCL17, IL-4, IL-13, and IL-31, but is not limited thereto as long as it corresponds to an indicator that may change due to atopic skin disease.

[0043] In the present invention, the step of measuring the level of an indicator related to atopic skin disease in an animal model administered with the candidate substance of step (b) means measuring the expression level of a gene or protein of the indicator.

[0044] The above gene expression level measurement method can be any of the conventional expression level measurement methods used in the art, and examples of analysis methods include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chips, etc.

[0045] In addition, any method commonly used in the art for measuring protein expression levels can be used, including, but not limited to, Western Blotting, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, oakteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation, complement fixation assay, flow cytometry (FACS), or protein chip methods.

[0046] In addition, the above method can use a preparation capable of confirming the expression level of one or more genes or proteins selected from the group consisting of IgE, CCL17, IL-4, IL-13 and IL-31 of the present invention, and the preparation preferably means an antibody, primer or probe specific for each gene or protein.

[0047] In the present invention, after the step (b), a step may be included of selecting a candidate substance that significantly restores the degree of the symptom or the level of the indicator in an animal model administered the candidate substance compared to a control group that was not administered the candidate substance, and determining the candidate substance as a preventive, improving, or treating agent for atopic dermatitis.

[0048] Specifically, if the expression level of one or more selected from the group consisting of IgE, CCL17, IL-4, IL-13, and IL-31 measured in step (b) above is lower than that of a control group that was not administered the candidate substance, the candidate substance can be determined as an agent for preventing, improving, or treating atopic dermatitis.

[0049] As another aspect for achieving the above object, the present invention provides the use of an atopic skin disease animal model for screening for an agent for preventing, improving, or treating atopic skin disease.

[0050] The above ‘atopic skin disease’ and ‘animal model’ are as described above.

[0051]

[0052] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0053] The present invention produces an animal model of atopic dermatitis by inducing skin sensitization and skin irritation by transdermally administering 1 to 5 mg / ml of ovalbumin to a dog once a day for 14 days, and the produced animal model not only exhibits clinical symptoms of atopic dermatitis and changes in related factors such as an increase in the expression levels of IgE and CCL17, but also exhibits an increase in the expression levels of Th2-related cytokines IL-4, IL-31, and IL-13, thereby establishing a standardized animal model for symptoms and Th2 immune responses that appear when atopic dermatitis occurs, and can also be utilized in clinical research for screening candidate substances for preventing, improving, or treating atopic dermatitis using the same.

[0054] Figure 1 shows the overall schedule and process of the experiment for producing a canine atopic dermatitis animal model of the present invention.

[0055] Figure 2 shows the clinical symptoms of atopic dermatitis observed according to the administered concentration of ovalbumin (T1: 1 mg / ml, T2: 5 mg / ml) on day 0 (D0) and day 14 (D14) after skin stimulation through transdermal administration of ovalbumin in a canine atopic dermatitis animal model of the present invention.

[0056] Figure 3 shows the severity of symptoms according to the administration concentration of ovalbumin (T1: 1 mg / ml, T2: 5 mg / ml) and the number of days after sensitization through administration, scored using the Draize dermal irritation scoring system (DDISS), in a canine atopic dermatitis animal model of the present invention.

[0057] Figure 4 shows the results of staining the skin tissue of an animal model of atopic dermatitis of the present invention with hematoxylin & eosin (H&E) according to the administered concentration of ovalbumin (1 mg / ml, 5 mg / ml). (Black arrow: lymphocyte, yellow arrow: lymphocytic dermal inflammation)

[0058] Figure 5 shows the results of staining the skin tissue of an animal model of atopic dermatitis of the present invention with Masson Trichrome (MT) according to the administered concentration of ovalbumin (1 mg / ml, 5 mg / ml).

[0059] Figure 6 shows the changes in epidermal thickness and degree of immune cell infiltration according to the administered concentration of ovalbumin (1 mg / ml, 5 mg / ml) in a canine atopic dermatitis animal model of the present invention.

[0060] Figure 7 shows the relative gene expression levels of COX-2 and PGES measured according to the administration concentration of ovalbumin (1 mg / ml, 5 mg / ml) in a canine atopic dermatitis animal model of the present invention.

[0061] Figure 8 shows the level of immunoglobulin E (IgE) antibody expression measured according to the administration concentration (1 mg / ml, 5 mg / ml) of ovalbumin in a canine atopic dermatitis animal model of the present invention.

[0062] Figure 9 shows the relative gene expression level of CCL17 chemokine measured according to the administration concentration of ovalbumin (1 mg / ml, 5 mg / ml) in a canine atopic dermatitis animal model of the present invention.

[0063] Figure 10 shows the relative gene expression levels of IL-4, IL-31, and IL-13 measured according to the administration concentration of ovalbumin (1 mg / ml, 5 mg / ml) in a canine atopic dermatitis animal model of the present invention.

[0064] Figure 11 shows the results of measuring the expression levels of IL-4 and IL-31 according to the administration concentration of ovalbumin (1 mg / ml, 5 mg / ml) in a canine atopic dermatitis animal model of the present invention.

[0065] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0066]

[0067] Example 1. Preparation of an animal model of atopic dermatitis disease

[0068] Six 7-month-old male beagles purchased from Raon Bio were used. The beagles were stabilized for one week in the laboratory animal breeding room of the Huvet Research Institute (temperature 24°C (±2°C), humidity 40–60%, 12 hr / 12 hr dark-light cycle). Afterwards, the hair on the dorsal and ventral areas of the beagles was completely removed using an electric razor for animals for transdermal administration. The administration (application) sites were marked with a marking pen at 5–7 cm intervals on four areas on the dorsal and ventral areas.

[0069] Sensitization was performed once a day for 14 days, and after a 2-week rest, stimulation was performed once a day for 14 days. Ovalbumin suitable for each dose was applied using TecAderm. Sensitization and stimulation were performed using the same ovalbumin administration concentration and method, and the overall experimental schedule and the appearance of the applied animal model are shown in Figures 1A and 1B, respectively.

[0070] Specifically, first, ovalbumin solutions were prepared by pipetting the final administration concentrations to be 1 mg / ml (T1) and 5 mg / ml (T2) (stock: 10 mg / mL, T1: 100 uL, T2: 500 uL), and these were stirred by resuspension or tapping frequently before and during administration. The prepared ovalbumin solutions of T1 and T2 were applied to the dorsal administration sites (4 sites) of the beagle once a day for a total of 2 weeks.

[0071] Tegaderm TM) After applying the prescribed dosage to the pad, attach it to the dorsal injection area, secure it with a roll of cotton for the first time to completely fix the patch, and secure it once more using an elastic bandage. Then, a neck collar was placed to prevent sensitization of areas other than the dermatitis modeling area. Since the patch does not have strong adhesiveness, the patch was replaced daily using the above method.

[0072] For the vehicle control (VC), the same beagles were used, administered transdermally using the same method, and sterile distilled water was applied daily. Afterwards, clinical symptoms and atopic dermatitis-related indicators of the beagles were analyzed before and after the stimulation period.

[0073]

[0074] Example 2. Analysis of clinical symptoms in an animal model of atopic dermatitis.

[0075] In the atopic dermatitis animal model prepared in Example 1, clinical symptoms of atopic dermatitis caused by ovalbumin were observed on days 0 and 14 after stimulation through ovalbumin administration, and scored using the Draize dermal irritation scoring system (DDISS) with reference to J. Pharmacol. Exp. Ther., 82, 377-390. The results of the observed clinical symptoms are as shown in Fig. 2, and the results of the DDISS scoring are as shown in Fig. 3.

[0076] In addition, each successive administration site and the sites of atopic symptom onset, such as around the mouth, ears, and soles, were individually evaluated and scored. As a result, as shown in Table 1 below, clinical symptoms of atopic dermatitis, such as erythema and eschar formation, and edema formation, were observed in the atopic dermatitis animal model of the present invention treated with ovalbumin.

[0077]

[0078] Control (VC) Oralbumin 1 mg / ml (T1) 5 mg / ml (T2) Erythema and crusting Number of beagles 222 Number of symptomatic beagles 022 Not observed---Minimum--1 Mild---21 Moderate---Severe---Score-21.5 Edema formation Number of symptomatic beagles 011 Not observed---Minimum--11 Mild---Moderate---Severe---Score-11

[0079]

[0080] Example 3. Histopathological analysis of an animal model of atopic dermatitis

[0081] In the animal model of atopic dermatitis prepared in Example 1, approximately 24 hours after the last stimulation with ovalbumin administration, the skin tissue of the beagle to which ovalbumin had been applied was biopsied for histopathological examination. The obtained skin tissue was fixed in formalin and then subjected to the Formalin Fixed Paraffin Embedding (FFPE) process to produce tissue slides, which were then deparaffinized with xylene and stained with hematoxylin & eosin (H&E) and Masson's Trichrome (MT). The staining results are shown in Figures 4 and 5, respectively.

[0082] The stained areas were analyzed at 200x magnification using an optical microscope (LECIA DM3000 LED). Each consecutive tissue was individually evaluated to determine changes in epidermal thickness and the extent of inflammatory cell infiltration, thereby conducting histological verification.

[0083] As a result, as shown in Fig. 6, increased epidermal thickness and immune cell infiltration were observed in the 1 mg / ml and 5 mg / ml ovalbumin treatment groups in the H&E and MT stained samples, and the epidermal thickness was greater in the 1 mg / ml ovalbumin administration group.

[0084] This suggests that continuous exposure to ovalbumin treatment above a certain concentration may cause skin inflammatory disease similar to atopic clinical symptoms, and this can also be observed in an atopic skin disease animal model using a beagle.

[0085]

[0086] Example 4. Analysis of inflammatory factor expression in skin tissue of an animal model of atopic dermatitis.

[0087] mRNA was extracted from the skin tissue of a beagle dog coated with ovalbumin, i.e., an animal model of atopic skin disease prepared in Example 1, and the mRNA expression levels of inflammatory factors such as COX-2 (cyclooxygenase-2) and PGES (prostaglandin E synthase) were confirmed using a quantitative real-time PCR test.

[0088] As a result, as shown in Fig. 7, it was confirmed that the gene expression level increased in a concentration-dependent manner depending on the administered ovalbumin, and it was determined that the infiltrated immune cells of Example 3 also increased in a trend similar to the inflammatory response.

[0089]

[0090] Example 5. Analysis of blood IgE and CCL17 expression levels following repeated administration of ovalbumin to an animal model of atopic dermatitis.

[0091] When an antigen penetrates from the outside due to a deficiency in the skin barrier function, chemokines such as CCL17 are released from immune cells and keratinocytes in the early stage of the immune response. Based on this, in the atopic skin disease animal model prepared in Example 1, mRNA was extracted from the blood of the ovalbumin-treated group and the mRNA level of CCL17 was confirmed using a quantitative real-time PCR test. In addition, immunoglobulin E (IgE) in the blood produced by the immune system is an antibody produced by the immune system in B cells in response to external allergens. In order to confirm whether atopic dermatitis was induced in the ovalbumin-treated group, the expression of the corresponding antibody and chemokine in the blood was analyzed using an ELISA test.

[0092] As a result, as shown in Fig. 8, in the control group (Vehicle control), the expression of IgE antibodies decreased compared to the baseline (BL), but in the ovalbumin treatment group, the expression of IgE antibodies relatively increased compared to the baseline, and in particular, it was confirmed that the expression amount increased significantly in the ovalbumin 1 mg / ml treatment group.

[0093] In addition, as shown in Figure 9, the mRNA expression level of CCL17 increased in all ovalbumin treatment groups compared to the control group (VC), and it was confirmed that it increased significantly in the 1 mg / ml group in particular.

[0094]

[0095] Example 6. Analysis of the expression level of Th2-related cytokines in skin tissue following repeated administration of ovalbumin to an animal model of atopic dermatitis.

[0096] Atopic dermatitis is known to be a Th2-dominant immune response, with Th2 cells secreting cytokines such as IL4, IL13, and IL31. To determine whether an ovalbumin-induced animal model of atopic dermatitis exhibits responses similar to atopic dermatitis, indicators related to T helper cells were examined.

[0097]

[0098] 6.1. Confirming mRNA expression level

[0099] mRNA was extracted from the skin tissue of a beagle dog coated with ovalbumin, i.e., an animal model of atopic skin disease prepared in Example 1, and the mRNA expression levels of Th2-related cytokines such as IL-4, IL-31, and IL-13 were confirmed using a quantitative real-time PCR test.

[0100] As a result, as shown in Fig. 10, it was confirmed that the expression of IL-4, IL-31, and IL-13 increased in the ovalbumin treatment group compared to the control group (VC), and in particular, it was confirmed that it was higher in the low concentration (1 mg / mL) treatment group compared to the high concentration (5 mg / mL).

[0101] This appears to be consistent with the results of inflammatory factor expression of COX-2 and PGES confirmed in Example 4, and it is judged that the overexpression of inflammatory factors in the group treated at high concentrations rather causes deviation in the skin inflammatory response and reduces the expression of Th2-related cytokines.

[0102] Therefore, based on the above results, it was determined that the optimal concentration of ovalbumin administration for producing an animal model of the present invention was 1 mg / ml.

[0103]

[0104] 6.2. Confirming protein expression levels

[0105] The expression of Th2-related cytokines was confirmed using an ELISA test method in the plasma of the atopic dermatitis animal model prepared in Example 1.

[0106] As a result, as shown in Fig. 11, the control group (Vehicle control) showed a similar or decreased relative IL-4 and IL-31 expression level compared to the baseline (BL), but the ovalbumin treatment group showed a relative increase in the expression of IL-4 and IL-31 compared to the baseline.

[0107] This suggests that Th2-related immune cells were activated by ovalbumin treatment, and this is supported by the increased levels of cytokines secreted from the activated immune cells.

[0108]

[0109] Based on the above results, it was confirmed that the canine atopic dermatitis animal model produced in the present invention can be established as an animal model in which a Th2-dominant immune response occurs.

[0110]

[0111] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. (a) a step of transdermal administration (epicutaneous sensitization) of ovalbumin at a concentration of 1 to 5 mg / ml to a dog once a day for 1 to 2 weeks; and (b) a step of causing skin irritation by percutaneously administering ovalbumin at a concentration of 1 to 5 mg / ml once a day for 2 to 8 weeks to a dog to which ovalbumin has been percutaneously administered according to step (a); a method for producing an animal model of atopic skin disease, comprising:

2. In paragraph 1, A manufacturing method wherein the concentration of the above ovalbumin is 1 mg / ml.

3. In paragraph 1, The step of transdermally administering ovalbumin of the above (a) is as follows: (i) a step of applying ovalbumin to a patch and allowing it to be absorbed; and (ii) A manufacturing method comprising a step of attaching a patch in which the above ovalbumin has been absorbed to a dog.

4. In paragraph 1, The step of causing skin irritation by transdermal administration of ovalbumin of the above (b) is as follows: (i) a step of applying ovalbumin to a patch and allowing it to be absorbed; and (ii) A manufacturing method comprising a step of attaching a patch in which the above ovalbumin has been absorbed to a dog.

5. In paragraph 1, A manufacturing method wherein the transdermal administration of ovalbumin of the above (a) and (b) is performed on the skin of the back of the dog.

6. In paragraph 5, A manufacturing method wherein the above-mentioned back portions are four back portions.

7. In paragraph 3, After the step of attaching the patch of step (ii) above to the dog, A manufacturing method further comprising the step of first fixing the attached patch with a roll of cotton, and then secondarily fixing it using an elastic bandage.

8. In paragraph 4, After the step of attaching the patch of step (ii) above to the dog, A manufacturing method further comprising the step of first fixing the attached patch with a roll of cotton, and then secondarily fixing it using an elastic bandage.

9. In paragraph 1, A manufacturing method for causing skin sensitization through the transdermal administration of step (a) above.

10. In paragraph 1, A manufacturing method further comprising, after step (a) above, a step of taking a rest period of 1 to 3 weeks.

11. An animal model of atopic skin disease manufactured according to any one of the manufacturing methods of clauses 1 to 10.

12. In paragraph 11, The above animal model is an atopic skin disease animal model that shows the immune response of T helper type 2 cells in atopic skin disease. 13.(a) A step of administering a candidate substance for preventing, improving or treating atopic skin disease to an animal model of atopic skin disease in Article 11; and (b) a step of measuring the degree of symptoms or expression level of indicators related to atopic skin disease in an animal model administered with the candidate substance; A method for screening for an agent for preventing, improving or treating atopic dermatitis, comprising:

14. In paragraph 13, A screening method, wherein the symptoms associated with the atopic skin disease of step (b) above are at least one selected from the group consisting of erythema formation, scab formation, and edema formation.

15. In paragraph 13, A screening method, wherein the indicator related to atopic skin disease in the step (b) is at least one selected from the group consisting of IgE, CCL17, IL-4, IL-13, and IL-31.

16. In paragraph 15, A screening method comprising a step of determining the candidate substance as a preventive, improving or treating agent for an atopic skin disease, when the expression level of at least one selected from the group consisting of IgE, CCL17, IL-4, IL-13 and IL-31 measured in the step (b) above shows a lower level compared to a control group that was not administered the candidate substance.

17. Use of an atopic skin disease animal model manufactured according to any one of the manufacturing methods of claims 1 to 10 for screening for an agent for preventing, improving, or treating atopic skin disease.

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