KR20250082490A - Canine atopic skin disease animal model and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing a canine atopic skin disease animal model, a canine atopic skin disease animal model produced thereby, and uses thereof.
The present invention produces an atopic skin disease animal model by transdermally sensitizing a dog with 1 to 5 mg/ml of ovalbumin once a day for 1 to 2 weeks, and then transdermally sensitizing the dog with 1 to 5 mg/ml of ovalbumin once a day for 2 to 8 weeks to cause skin stimulation. The animal model produced as described above exhibits clinical symptoms of atopic skin disease and changes in related factors such as increases in the expression levels of IgE and CCL17, and also exhibits increases in the expression levels of Th2-related cytokines IL-4, IL-31, and IL-13. Accordingly, the present invention establishes a standardized animal model for symptoms and Th2 immune responses that occur when atopic skin disease occurs, and can also be utilized in clinical research for screening candidates for prevention, improvement, or treatment of atopic skin disease.

Description

{Canine atopic skin disease animal model and uses thereof}

The present invention relates to a method for producing a canine atopic skin disease animal model, a canine atopic skin disease animal model produced thereby, and uses thereof.

Atopic dermatitis is a chronic skin disease that lasts a long time and causes severe itching eczematous lesions on the skin. It is common in people with a history or family history of atopic disease. When symptoms appear, the area is scratched or rubbed, which worsens the skin symptoms as a result, which can be seen as a characteristic of atopic dermatitis. According to the definition of the Korea Centers for Disease Control and Prevention, atopic dermatitis is a chronic allergic inflammatory disease that occurs on the skin, and it has the characteristics of both allergic and inflammatory diseases, and since each disease acts through different mechanisms, it is necessary to clearly identify the pathogenesis.

Accordingly, research on customized prevention or treatment methods to more effectively prevent or treat atopic skin diseases, including atopic dermatitis, according to their causes is actively being conducted. In such research, clinical trials are essential, and in this process, there may be various risk factors and ethical issues may arise, so various animal models are being used as a means to overcome these.

Meanwhile, in modern society, where single-person households are on the rise and the population is rapidly entering an era of hyper-aging, the number of people living with companion animals is approximately 10 million. The Ministry of Agriculture, Food and Rural Affairs estimates that the size of the domestic companion animal market in Korea was 1.8 trillion won in 2015, and it is showing continuous high growth every year. However, as the diet of pets becomes more luxurious, and as they are raised at home, the function of all organs is deteriorating due to lack of exercise, and immunity to diseases is weakening. Of course, prevention or treatment using chemical drugs is possible at pet hospitals, but nutritional supplements in the form of health supplements that can have a continuous effect are needed.

In addition, as the pet population increases, various pet diseases are emerging, among which atopy is a chronic recurrent inflammatory skin disease caused by rapid changes in the environment, genetic factors, immunological abnormalities, and skin barrier abnormalities, and is an intractable disease that is difficult to completely cure not only for humans but also for pets. In particular, atopic dermatitis mostly occurs in animals that live 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 mainly raised.

Up to now, companion animals suffering from atopic dermatitis in Korea have been managed with drugs such as steroids without any other fundamental treatment methods. Drugs such as steroids are effective and can be expected to improve clinical symptoms quickly, but because the side effects of long-term administration are very significant, managing atopic patients through long-term administration of steroids and drugs is not the best method. Therefore, research on products with fewer side effects and effective in managing atopic dermatitis is constantly required, and since clinical trials are essential for such research, it is necessary to develop an animal model that represents the immune response of actual companion animal atopic dermatitis in order to study such atopic dermatitis and screen for prevention, improvement, or treatment of atopic dermatitis.

Republic of Korea Patent No. 10-0882505

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

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

Another object of the present invention is to provide an atopic skin disease animal model manufactured according to the above manufacturing method.

Another object of the present invention is to provide 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 indicators related to an atopic skin disease in the animal model to which the candidate substance has been administered.

The technical problems to be achieved according to the technical idea of the invention disclosed in this specification are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

In one aspect to achieve the above object, the present invention provides a method for producing a canine atopic skin disease animal model. Specifically, the present invention provides a method for producing an atopic skin disease animal model, comprising: (a) a step of transdermally administering ovalbumin at a concentration of 1 to 5 mg/ml to a dog 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.

In the present invention, skin sensitization may be induced through the transdermal administration in step (a). In step (a), skin sensitization may be induced by transdermal administration of ovalbumin, and the skin sensitization may be to increase the sensitivity to an 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 cause skin stimulation, and the skin stimulation may be to 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.

As a specific example, the present invention provides a method for producing an animal model of atopic skin disease, 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 from which 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.

In the present invention, the 'atopic skin disease' includes both atopic dermatitis and atopic skin disease without inflammation, and the 'atopic dermatitis' may mean a chronic relapsing skin eczema disease accompanied by severe itching. The atopic skin disease may include an atopic skin disease that appears not only in humans but also in companion animals such as dogs.

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 immune responses.

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.

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

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 with the ovalbumin absorbed to a dog. Specifically, the patch may use a Tegaderm ^TM pad, but its type is not limited as long as it can cause skin sensitization or irritation when ovalbumin is absorbed and attached to a dog.

In the present invention, skin sensitization and skin stimulation through the transdermal administration of ovalbumin may be performed on the skin of the back area 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 area of the dog, it is possible to prevent the possibility of secondary damage to a wound area due to the characteristics of the dog, or of inducing systemic tolerance by licking the ovalbumin and causing it to enter the mouth, and it is characterized in that an atopic skin disease can be induced only by 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, a height of 4 to 7 cm, and may be spaced apart from each other by a distance of 2 to 3 cm, but is not limited thereto.

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 allowed to be absorbed, but is not limited thereto.

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 using 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 back of the dog to cause skin sensitization or skin irritation. In addition, the method may further include a step of putting a neck collar on the neck of the dog after the step of causing skin sensitization or skin irritation.

In the present invention, in the step of causing skin sensitization and skin irritation, both the administration dosage and administration method of ovalbumin can be applied equally.

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 2 to 8 weeks thereafter. 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 3 weeks thereafter. In addition, skin sensitization can be induced by transdermal administration of ovalbumin for 2 weeks, and skin irritation can be induced by transdermal administration of ovalbumin for 2 weeks thereafter.

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.

In the present invention, the resting period may be provided so that the immune system that reacts through skin sensitization has a period in which exposure to the antigen is reduced and minimized. During the resting period, the dog's immune system returns to a normal state, and may cause a stronger immune response when stimulated thereafter.

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.

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

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 exhibit skin sensitization and irritation by transdermal administration of ovalbumin.

In the present invention, the animal model may be one in which the expression levels of immunoglobulin E (IgE) and CCL17 chemokines in the blood are increased compared to animals that did not cause skin sensitization and irritation by percutaneous administration of ovalbumin. Preferably, when skin sensitization is caused by percutaneous administration of 1 mg/ml of ovalbumin, the expression levels of IgE and CCL17 chemokines may be increased at a more significant level compared to animals that did not cause skin sensitization and irritation by percutaneous administration of ovalbumin and animals that did cause skin sensitization and irritation by percutaneous administration of 5 mg/ml of ovalbumin.

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

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

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: (a) a step of administering a candidate substance for preventing, improving or treating an atopic skin disease to the prepared atopic skin disease animal model; and (b) a step of measuring the degree of symptoms or the level of an indicator related to an atopic skin disease in the animal model administered with the candidate substance.

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

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.

The above 'candidate substance' refers to an unknown substance used in screening to examine whether it has an effect in preventing, improving or treating atopic skin disease, or whether it affects the degree of symptoms or the level of indicators related to atopic skin disease. The candidate substance may include, but is not limited to, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract or plasma.

In the present invention, the symptoms related to atopic skin disease in the 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 if 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 the step (b) is reduced compared to the control group that was not administered the candidate substance, the candidate substance may be determined to be an agent for preventing, improving, or treating atopic skin disease.

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 can change due to atopic skin disease.

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.

The above gene expression level measurement method can use 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.

In addition, any method commonly used in the art can be used for measuring the protein expression level, and examples thereof include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, orchite immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation, complement fixation assay, flow cytometry (FACS), or protein chip methods.

In addition, the method can utilize a formulation 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 formulation preferably means an antibody, primer or probe specific for each gene or protein.

In the present invention, after step (b), a step may be included for selecting a candidate substance that significantly recovers the degree of symptoms or the level of indicators 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 skin disease.

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 the control group that was not administered the candidate substance, the candidate substance can be determined as an agent for preventing, improving, or treating atopic skin disease.

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

The present invention produces an atopic skin disease animal model by transdermally sensitizing a dog with 1 to 5 mg/ml of ovalbumin once a day for 1 to 2 weeks, and then transdermally sensitizing the dog with 1 to 5 mg/ml of ovalbumin once a day for 2 to 8 weeks to cause skin stimulation. The produced animal model exhibits clinical symptoms of atopic skin disease and not only exhibits changes in related factors such as increases in the expression levels of IgE and CCL17, but also exhibits increases in the expression levels of Th2-related cytokines IL-4, IL-31, and IL-13. Accordingly, a standardized animal model is constructed for symptoms and Th2 immune responses that occur when atopic skin disease occurs, and can also be utilized in clinical research to screen candidates for prevention, improvement, or treatment of atopic skin disease using the same.

Figure 1 shows the overall schedule and process of the experiment for producing an animal model of canine atopic skin disease of the present invention.
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.
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, in a canine atopic dermatitis animal model of the present invention, as scored using the DDISS (Draize dermal irritation scoring system).
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 (OVA) (1 mg/ml, 5 mg/ml). (Black arrow: lymphocyte, yellow arrow: lymphocytic dermal inflammation)
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).
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.
Figure 7 shows the relative gene expression levels of COX-2 and PGES measured according to the administered concentration of ovalbumin (1 mg/ml, 5 mg/ml) in a canine atopic dermatitis animal model of the present invention.
Figure 8 shows the results of measuring the level of immunoglobulin E (IgE) antibody expression according to the administered concentration of ovalbumin (1 mg/ml, 5 mg/ml) in a canine atopic dermatitis animal model of the present invention.
Figure 9 shows the relative gene expression level of CCL17 chemokine according to the administration concentration (1 mg/ml, 5 mg/ml) of ovalbumin in a canine atopic dermatitis animal model of the present invention.
Figure 10 shows the relative gene expression levels of IL-4, IL-31, and IL-13 measured according to the administered concentration of ovalbumin (1 mg/ml, 5 mg/ml) in a canine atopic dermatitis animal model of the present invention.
Figure 11 shows the results of measuring the expression levels of IL-4 and IL-31 according to the administered concentration of ovalbumin (1 mg/ml, 5 mg/ml) in a canine atopic dermatitis animal model of the present invention.

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.

Example 1. Production of animal model of atopic skin disease

Beagles were used, six males, 7 months old, purchased from Raon Bio. The beagles were stabilized for one week in the laboratory animal breeding room of the Huvet Research Institute (temperature 24℃ (±2℃), humidity 40-60%, 12 hr/12 hr dark-light cycle). Afterwards, the hair on the back and abdomen of the beagles was completely removed using an electric razor for animals for percutaneous administration. The administration (application) sites were marked with a marking pen at intervals of 5-7 cm on four areas on the back and abdomen.

Sensitization was performed once a day for 14 days, and after 2 weeks of 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 concentration and method of ovalbumin administration, and the overall experimental schedule and the appearance of the applied animal model are as shown in Fig. 1A and Fig. 1B, respectively.

Specifically, first, ovalbumin solutions were prepared by quantitatively pipetting the final administration concentrations to 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 T1 and T2 ovalbumin solutions were applied to the dorsal administration sites (4 sites) of the beagle once a day for a total of 2 weeks.

After applying the prescribed dosage to the Tegaderm ^TM pad, attach it to the dorsal application 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 put on 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.

For the control group (Vehicle control, VC), the same beagles were used as above, and the same method was used to administer the drug percutaneously, and sterile distilled water was applied every day. Afterwards, the clinical symptoms and atopic skin disease-related indicators of the beagles were analyzed before and after the stimulation period.

Example 2. Analysis of clinical symptoms of an animal model of atopic dermatitis

In the atopic dermatitis animal model manufactured in Example 1, clinical symptoms of atopic dermatitis caused by ovalbumin were observed on day 0 (D0) and day 14 (D14) of stimulation through ovalbumin administration, and scored using the Draize dermal irritation scoring system (DDISS) with reference to J. Pharmacol. Exp. Ther., 82, 377-390, etc. 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.

In addition, each successive administration site and atopic symptom expression site 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.

Control group
(VC) Ovalbumin 1mg/ml (T1) 5 mg/ml (T2) Erythema and crusting Number of beagle dogs 2 2 2 Number of symptomatic beagles 0 2 2 Not observed - - - Minimum - - 1 Mild - 2 1 Moderate - - - Severe - - - score - 2 1.5 Edema formation Number of symptomatic beagles 0 1 1 Not observed - - - Minimum - 1 1 Mild - - - Moderate - - - Severe - - - score - 1 1

Example 3. Histopathological analysis of an animal model of atopic dermatitis

In Example 1, for the animal model of atopic dermatitis disease, about 24 hours after the last stimulation through ovalbumin administration, a biopsy was performed on the skin tissue of the beagle to which ovalbumin had been applied for histopathological examination. The obtained skin tissue was fixed in formalin and then went through the Formalin Fixed Paraffin Embedding (FFPE) process to create tissue slides, deparaffinized with xylene, and stained with hematoxylin & eosin (H&E) and Masson's Trichrome (MT). The staining results are as shown in Figs. 4 and 5, respectively.

The above 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 degree of inflammatory cell infiltration, thereby conducting histological verification.

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.

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

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

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.

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

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

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 initial stage of the immune response. Based on this, in the atopic skin disease animal model manufactured 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 chemokines in the blood was analyzed using an ELISA test.

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, the expression amount was confirmed to have increased significantly in the ovalbumin 1 mg/ml treatment group.

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 significantly increased in the 1 mg/ml group in particular.

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

Atopic dermatitis is known to be a Th2-dominant immune response, and Th2 cells secrete cytokines such as IL4, IL13, and IL31. To confirm whether the atopic skin disease animal model induced by ovalbumin shows a response similar to atopic dermatitis, indicators related to T helper cells were identified.

6.1. Confirming mRNA expression level

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.

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).

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.

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.

6.2. Check protein expression level

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

As a result, as shown in Fig. 11, the control group (Vehicle control) showed similar or decreased relative IL-4 and IL-31 expression levels 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.

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.

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.

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

Claims (16)

(a) a step of transdermal 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:
In the first paragraph,
A manufacturing method wherein the concentration of the above ovalbumin is 1 mg/ml.
In the first paragraph,
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.
In the first paragraph,
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.
In the first paragraph,
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.
In paragraph 5,
A manufacturing method wherein the above-mentioned back portions are four back portions.
In the third paragraph,
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.
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.
In the first paragraph,
A manufacturing method for causing skin sensitization through the transdermal administration of step (a) above.
In the first paragraph,
A manufacturing method further comprising, after step (a) above, a step of taking a rest period of 1 to 3 weeks.
An atopic dermatitis animal model manufactured according to any one of the manufacturing methods of claims 1 to 10.
In Article 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.
(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:
In Article 13,
A screening method, wherein the symptoms associated with the atopic skin disease of step (b) are at least one selected from the group consisting of erythema formation, scab formation, and edema formation.
In Article 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.
In Article 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.