Method for rapidly establishing EGFRIs-induced skin toxicity animal model

Abstract

The invention relates to a method for rapidly establishing an EGFRIs-induced dermatotoxicity animal model. According to the method, an oral EGFRIs drug is subjected to solubilization and permeation promotion and then smeared on animal skin to induce EGFRIs dermatotoxicity reaction. The model only needs 7-10 days to have obvious skin toxicity reaction, skin histopathologic change, inflammatory and immune cell infiltration increase and inflammatory factor level increase, and accords with EGFRIs skin toxicity reaction characteristics. Compared with an intragastric erlotinib hydrochloride induction model, the model has the advantages that the occurrence and development speed of skin toxic reaction is higher, the occurrence rate and stability of the model are higher, animal death is avoided and the like. The construction method of the model is simple and easy to operate, the modeling time is shortened, the model is good in stability and repeatability, the model can be widely popularized and applied, and an animal model is provided for research and development of pathogenesis of EGFRIs-induced skin toxicity and drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of animal models, and in particular relates to a method for quickly establishing an animal model of skin toxicity caused by EGFRIs. Background Art

[0002] In cancer treatment, the use of epidermal growth factor receptor inhibitors (EGFRIs) has become a common treatment. However, 3 to 10 days after oral or injection of EGFRIs, patients develop skin adverse reactions such as papulopustular rash, dry and itchy skin, and abnormal hair regeneration, with an incidence rate as high as 92.4%. Skin toxicity usually affects the parts of the body that are rich in sebaceous glands and exposed to sunlight, including the face (forehead, under the nose, mandible, cheeks), upper chest, back, scalp, abdomen, arms, and waist. From a histological point of view, skin biopsies of patients treated with EGFRIs will reveal some typical characteristics, mainly including infiltration of mast cells in the basal layer and infiltration and aggregation of inflammatory cells such as macrophages and T lymphocytes, as well as infiltration of epidermal inflammatory cells and inflammation of hair follicles, and even loosening of the stratum spinosum. This type of skin toxicity not only seriously interferes with the patient's daily life, but also has a negative impact on the patient's mood. What is more serious is that this adverse reaction may lead to the need to reduce the dosage of EGFRIs or even be forced to stop taking the medicine. The interruption of treatment may further aggravate the cancer condition and greatly hinder the patient's treatment progress.

[0003] Although EGFRIs-induced skin adverse reactions have a high incidence and significant harmfulness, their pathogenesis has not been fully elucidated so far, and there is a lack of specific therapeutic drugs for this type of skin adverse reactions in clinical practice.

[0004] Animal models play a vital role in studying the pathogenesis of diseases and therapeutic drugs. There are two main methods for establishing animal models of skin toxicity reactions related to EGFRIs: one is gene knockout technology, and the other is the method of inducing EGFRIs drugs by oral gavage (CN201910220571, CN202210458564.4). However, both methods have many disadvantages, such as the high cost, short lifespan and low survival rate of knockout mice, long experimental cycle, and strict breeding conditions, which limit their application in large-scale experiments. Another method of inducing EGFRIs drugs by oral gavage requires long-term (at least 4 weeks) large-dose oral administration, which can easily cause animal death, thus failing to ensure the smooth completion of the entire experiment. In addition, when using the oral gavage induction method, there are large differences in the time and severity of skin toxicity reactions in mice of different individuals, which brings great difficulties to the evaluation of the efficacy of therapeutic drugs, and the sites of disease are usually concentrated on the head, face and claws of mice. When developing topical drugs, the efficacy evaluation of these sites will face the problem of difficulty in drug administration.

[0005] In summary, given the various limitations of existing animal models in studying skin toxicity reactions caused by EGFRIs, there is an urgent need to establish a rapid, stable, and widely promoted and applied animal model to provide a method for exploring the pathogenesis of EGFRIs-induced skin toxicity and developing therapeutic drugs. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for rapidly constructing EGFRIs-induced skin toxicity reactions, the method comprising: applying the oral EGFRIs drug to the animal skin after solubilization and penetration enhancement to induce EGFRIs-type skin toxicity reactions.

[0007] To achieve the above object, the present invention adopts the following technical solution: The EGFRIs drugs may be erlotinib hydrochloride, gefitinib, lapatinib, afatinib, etc., including but not limited to the above-mentioned anti-tumor molecular targeted drugs.

[0008] Preferably, the drug is erlotinib hydrochloride; the concentration of erlotinib hydrochloride is 0.2%.

[0009] The solubilizing agent of the EGFRIs drug can be PEG 400, Tween-80, propylene glycol, etc.; Preferably, the solubilizer for EGFRIs is PEG 400; Preferably, the concentration of PEG 400 is 5-15%, more preferably 10%.

[0010] The penetration enhancers of the EGFRIs drug are azone, propylene glycol, and menthol; Preferably, the penetration enhancer of the EGFRIs drug is azone; Preferably, the azone concentration is 0.5-2.5%, more preferably 1%.

[0011] The preparation method of the drug for constructing the EGFRIs-type skin toxicity reaction animal model is as follows: take 100 mg of erlotinib hydrochloride, add 10% PEG400 and 1% azone, add pure water to 50 mL, ultrasonicate for 30 minutes, mix well, and obtain the product.

[0012] Preferably, the EGFRIs drug is applied externally to the modeling site of the animal, and the modeling site is the back skin, ear skin or other skin of the animal after depilation, more preferably the back skin.

[0013] The dosage of the EGFRIs drug is 0.05-0.2 mg erlotinib hydrochloride / cm 2 The area of ​​the modeling site is preferably 0.1-0.15 mg erlotinib hydrochloride / cm 2 The area of ​​the modeling site; more preferably, the frequency of application is once a day, for a total of 7 to 10 days.

[0014] The model animal is a rat or a mouse, preferably a mouse; of course, those skilled in the art can use other types of experimental animals for modeling according to actual use requirements, including but not limited to the above-mentioned rats and mice.

[0015] Compared with the traditional gavage erlotinib hydrochloride induced model, the EGFRIs-type skin toxicity reaction model construction method provided by the present invention shows the following beneficial effects: the occurrence and development speed of the EGFRIs-type skin toxicity reaction model established by this method is faster, only 7 to 10 days, the incidence rate is as high as 100%, the animal mortality rate is 0%, and the time and severity of the skin toxic reaction are relatively uniform. The modeling site can be selected according to actual use needs to select the site where the therapeutic drug is applied to the appropriate part of the animal to construct the model. In addition, the skin tissue pathological changes, the infiltration of inflammatory and immune cells, the level of inflammatory factors, etc. all show that there is no significant difference with the skin toxicity model induced by gavage erlotinib hydrochloride. The construction method of the model of the present invention is simple and easy to operate, shortens the modeling time, has a high animal survival rate, and has good model stability and repeatability, and can be widely promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The toxic reactions of the mice dorsal skin after intragastric administration and application of erlotinib hydrochloride in Examples 1 and 2 are shown.

[0017] Figure 2This is the histopathological observation of the back skin of the mouse in Example 3 (HE, the scale bar in the figure is 100 μm).

[0018] Figure 3 Statistical results of epidermal thickness of mouse back skin in Example 3 (n=10, X±SD, *** P <0.001).

[0019] Figure 4 This is the histopathological observation of the back skin of mice in Example 4 (Oil Red, the scale bar in the figure is 100 μm).

[0020] Figure 5 The infiltration of mast cells, macrophages and T lymphocytes in the back skin of mice in Example 5 (the scale bar in the figure is 100 μm).

[0021] Figure 6 The expression levels of TNF-α, IL-6, and IL-17 inflammatory factors in the back skin of mice in Example 6 (n=10, X±SD, *** P < 0.001).

[0022] Figure 7 The water content of the back skin of mice in Example 7 (n=10, X±SD, *** P < 0.001). DETAILED DESCRIPTION

[0023] The sources of animals, drugs, reagents, and instruments involved in the examples are as follows: Experimental animals: SPF-grade BALB / c female mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with experimental animal license number (SCXK (Su) 2022-0009), and were raised in the Experimental Animal Center of Jiangsu Institute of Traditional Chinese Medicine under light / dark (12 h / 12 ​​h) and temperature (22±3)°C, with free access to food and water, and experimental animal use license (SYXK (Su) 2016-0018).

[0024] Drugs and reagents: Erlotinib hydrochloride (Shanghai Aladdin Biochemical Technology Co., Ltd.); PEG400, propylene glycol, Tween 80, (Xilong Scientific); sodium carboxymethyl cellulose, azone, menthol (McLean); TNF-α, IL-6, IL-17 Elisa kit (KeyGen Biotech Co., Ltd.).

[0025] Instruments: MS205DU 1 / 100,000 electronic balance (Mettler Toledo); ultrasonic cleaning machine (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.); water purifier (Millipore, TANKPE 060); others are of commercial origin unless otherwise specified.

[0026] Example 1: Establishment of an EGFRIs skin toxicity model induced by intragastric administration of erlotinib hydrochloride BALB / c mice (about 20 g, female) were fed for 7 days to adapt to the environment and then randomly divided into 2 groups according to body weight. They were randomly divided into a blank group and an erlotinib hydrochloride group, with 10 mice in the blank group and 10 mice in the erlotinib hydrochloride group. Preparation of erlotinib hydrochloride suspension: 500 mg of erlotinib hydrochloride was added with 0.25% sodium carboxymethyl cellulose, and then pure water was added to 50 mL, ultrasonicated for 30 min, and shaken well when administered. The day before administration, the back of BALB / c mice was depilated and erlotinib hydrochloride solution was administered by gavage at a dose of 150 mg / kg (i.e., 0.015 mL / g) once a day. Skin toxicity occurred after continuous administration (about 48 days). The toxic reactions of the mouse skin were observed and recorded daily.

[0027] Example 2: Establishment of an EGFRIs skin toxicity reaction model induced by applying erlotinib hydrochloride BALB / c mice (about 20 g, female) were fed for 7 days to adapt to the environment, and then erlotinib hydrochloride was applied to induce the EGFRIs skin toxicity reaction model. Preparation of erlotinib hydrochloride: Take 100 mg of erlotinib hydrochloride, add 10% PEG400 and 1% azone, add pure water to 50 mL, ultrasonicate for 30 min, mix well, and the result is obtained. The back of BALB / c mice was depilated one day before administration, and erlotinib hydrochloride solution was applied in the form of application, with a dose of 0.1 mg erlotinib hydrochloride / cm 2 The area of ​​the modeling site was 1 time per day for about 7-10 days. The weight of the mice was recorded daily, and the toxicity of the skin on the back of the mice was observed.

[0028] The results are as follows Figure 1 As shown in Table 1: It takes at least 48 days for oral administration of erlotinib hydrochloride to induce skin toxicity, and it is very easy to cause animal death (mortality rate of 50%). Compared with the model induced by oral administration of erlotinib hydrochloride, the smear method to establish the EGFRIs skin toxicity model has a faster occurrence and development speed, which only takes 7 to 10 days, with an incidence rate of up to 100%, an animal mortality rate of 0%, and the time and severity of skin toxicity are relatively uniform, indicating that the smear method to establish the EGFRIs skin toxicity model has a faster modeling time, a high animal survival rate, and a stable model.

[0029] Table 1 Skin toxicity and mortality of animals in two modeling methods

[0030] Example 3: HE staining of the back skin of animals in Examples 1 and 2 Dewax the paraffin sections in xylene for 10 min, replace with fresh xylene and dewax for another 10 min. Then soak the sections in 100%, 90%, 70% and pure water for 5 min each to rehydrate the tissue. After that, add hematoxylin stain to the slide so that the stain completely immerses the tissue, stain for 3 min, and then slowly rinse in tap water for 10 min to remove excess stain. Finally, add eosin stain to the slide so that the stain completely immerses the tissue, stain for 30 to 60 seconds, and slowly rinse with 70% ethanol for 3 min. Soak in 70%, 90%, and 100% ethanol for 10 seconds each for dehydration, then make it clear in xylene for 5 min, replace with fresh xylene, and make it clear for another 5 min. Seal the slide with neutral gum, observe and take pictures under a microscope or in a slide scanner.

[0031] The results are as follows Figure 2 As shown: Compared with the blank group, mice in the oral and topical administration of erlotinib hydrochloride groups showed parakeratosis (retention of stratum corneum cell nuclei), pus-like mast cell clusters outside the hair follicle ducts, follicular expansion, hair follicle obstruction, and significant epidermal thickening (such as Figure 3 As shown, P < 0.001), and there was no significant difference between the oral gavage group and the smear group, indicating that the parakeratosis, follicular expansion, and hair follicle inflammation and obstruction of the mouse back skin in the EGFRIs skin toxicity reaction model established by the smear administration of erlotinib hydrochloride group can achieve the same effect as the model established by traditional oral gavage.

[0032] Example 4: Oil Red staining of the back skin of animals in Examples 1 and 2 Take the skin tissue samples from the back of the animals in Examples 1 and 2, take the frozen sections out of the refrigerator, and air dry. Wash with PBS 3 times, 5 min each time. Fix with 4% paraformaldehyde for 5-10 min. Wash twice with distilled water, 5 min each time. Wash the sections with 60% isopropanol twice, 5 min each time. Stain the sections with oil red working solution for 15 min. Wash twice with 60% isopropanol, 5 min each time. Wash twice with distilled water, 5 min each time. Counterstain with hematoxylin for 2 min. Rinse thoroughly with water for 10 min, wipe dry the surrounding water. Cover with a coverslip, seal the sections with a sealing agent, and observe and film.

[0033] The results are as follows Figure 4As shown: Compared with the blank group, the number of sebaceous glands in the skin of mice in the groups given erlotinib hydrochloride by oral gavage and smear application was reduced, the dilated follicles ruptured, and the oil secretion was significantly reduced, and there was no significant difference between the oral gavage group and the smear application group. This shows that the sebaceous glands, follicle rupture and oil secretion of the back skin of mice in the EGFRIs skin toxicity reaction model established by the group given erlotinib hydrochloride by smear application can achieve the same effect as the model established by traditional gavage method.

[0034] Example 5: Infiltration of mast cells, macrophages and T lymphocytes in the back skin of animals in Examples 1 and 2 The paraffin-embedded skin tissue samples from the back of the animals in the experiments of Examples 1 and 2 were taken and the infiltration of mast cells, macrophages and T lymphocytes in the skin tissues was analyzed by immunohistochemical staining.

[0035] The results are as follows Figure 5 As shown: Compared with the blank group, there was obvious infiltration and large-scale aggregation of mast cells (benzylamine blue, purple), macrophages (CD68, brown) and T lymphocytes (CD3, brown) in the skin of mice in the groups given erlotinib hydrochloride by oral gavage and smear, and there was no significant difference between the oral gavage group and the smear group, indicating that the infiltration and aggregation of inflammatory cells in the back skin of mice in the EGFRIs skin toxicity reaction model established by smear can achieve the same effect as the model established by traditional gavage method.

[0036] Example 6: Expression levels of TNF-α, IL-6, and IL-17 inflammatory factors in the back skin of animals in Examples 1 and 2 The skin tissue samples from the back of the animals in the experiments of Examples 1 and 2 were taken, and the expression levels of inflammatory factors in the skin were determined according to the instructions of the Elisa kit.

[0037] The results are as follows Figure 6 As shown: Compared with the blank group, the expressions of IL-6, IL-17, and TNF-α in the skin of mice in the group given erlotinib hydrochloride by oral gavage and application were significantly increased ( P < 0.001), and there was no significant difference between the oral gavage group and the smear group, indicating that the inflammatory factors in the back skin of mice in the EGFRIs skin toxicity reaction model established by the erlotinib hydrochloride smear group can achieve the same effect as the model established by traditional oral gavage.

[0038] Example 7: Water content of the back skin of animals in Examples 1 and 2 Take the skin tissue samples from the back of the animals in Examples 1 and 2, wipe the mouse skin as dry as possible with filter paper, cut the depilated mouse skin of about 1 cm × 1 cm on the back of the neck, accurately weigh the wet weight of the skin, and then put it in a 60 ℃ oven and dry it for 2 hours. After the moisture is basically dissipated, take it out and accurately weigh the dry weight of the skin according to the following formula: Skin moisture percentage (%) = (wet weight - dry weight) / wet weight × 100%.

[0039] The results are as follows Figure 7 As shown: Compared with the blank group, the water content in the skin of mice in the group given erlotinib hydrochloride by oral gavage and application was significantly reduced ( P < 0.001), and there was no significant difference between the oral gavage group and the smear group, indicating that the EGFRIs skin toxicity reaction established by the smear administration of erlotinib hydrochloride group had the same effect as the model established by the traditional oral gavage method, causing damage to the skin barrier on the back of mice, weakening the moisturizing function, and reducing the water content of the skin.

[0040] In summary, the animal model of EGFRIs-like skin toxicity induced by applying oral EGFRIs drugs to animal skin after solubilization and penetration enhancement is consistent with the physiological and pathological characteristics of EGFRIs-like skin toxicity in terms of skin tissue pathological characteristics, infiltration of mast cells, macrophages, T lymphocytes, and expression of inflammatory cytokines. Compared with the oral administration of erlotinib hydrochloride group, this method forms skin toxicity faster, no animal deaths, more obvious pathological characteristics, higher incidence and stability of the model, and simple and easy operation, which can be widely promoted and applied to the exploration of pathogenesis and the development of therapeutic drugs.

Claims

1. A method for rapidly establishing an animal model of skin toxicity caused by EGFRIs, characterized in that The method comprises the following steps: applying the oral EGFRIs drug to the skin of animals after solubilization and penetration enhancement to induce EGFRIs-type skin toxicity reactions.

2. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The EGFRIs drug can be anti-tumor molecular targeted drugs such as erlotinib hydrochloride, gefitinib, lapatinib, afatinib, etc., preferably erlotinib hydrochloride; the concentration of erlotinib hydrochloride is 0.2%.

3. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The solubilizer of the EGFRIs drug can be PEG 400, Tween-80, propylene glycol, etc., preferably PEG 400; the concentration of the PEG 400 is 5-15%, preferably 10%.

4. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The penetration enhancer of the EGFRIs drug can be azone, propylene glycol, menthol, preferably azone; the concentration of azone is 0.5-2.5%, preferably 1%.

5. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The preparation method of the above-mentioned drug is as follows: take 100 mg of erlotinib hydrochloride, add 10% PEG400 and 1% azone, add pure water to 50 mL, ultrasonicate for 30 min, mix well, and obtain the drug.

6. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The EGFRIs drug is applied externally to the modeling site of the animal, which is the back skin, ear skin or other skin of the animal after depilation, preferably the back skin.

7. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The dosage of the EGFRIs drug is 0.05-0.2 mg erlotinib hydrochloride / cm 2 The area of ​​the modeling site is preferably 0.1-0.15 mg erlotinib hydrochloride / cm 2 The application frequency is once a day for a total of 7 to 10 days.

8. The method of a rapid EGFRIs-induced skin toxicity animal model according to claim 1, characterized in that: The model animal is a rat or a mouse, preferably a mouse.

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