Use of targeting mif in the preparation of a medicament for the treatment of atopic dermatitis

Abstract

The application discloses one aspect of a kind of drug for treating atopic dermatitis in the preparation of targeting MIF application, another aspect discloses a kind of drug for treating atopic dermatitis.The drug for treating the atopic dermatitis is MIF small molecule inhibitor.The MIF small molecule inhibitor is MIF antagonist ISO-1.The application confirms that MIF has good therapeutic effect in HDM allergic mouse AD model by studying the therapeutic effect of phagocyte migration inhibitory factor MIF in HDM allergic mouse AD model.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a target phage migration inhibitory factor MIF in the preparation of a drug for treating atopic dermatitis, as well as a drug for targeted treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a relapsing, itchy, inflammatory skin disease, considered the first step in the atopic process. Many AD patients exhibit increased reactivity to airborne and food allergens. House dust mites (HDM) are the most common airborne allergen and are considered a significant factor in triggering and exacerbating AD. Serum HDM-specific IgE levels are elevated in AD patients and are positively correlated with disease severity; HDM desensitization therapy can effectively improve the clinical symptoms of AD. Furthermore, HDM stimulates keratinocytes in AD patients to release Th2-related cytokines, such as interleukin-25 and IL-33, thereby exacerbating the immune response.

[0003] Macrophage migration inhibitor factor (MIF) acts as a pleiotropic cytokine by participating in inflammatory and immune response processes, thereby contributing to the immune regulation and development of allergic diseases. Summary of the Invention

[0004] The purpose of this invention is to provide an application of targeting the phage migration inhibitory factor MIF in the preparation of a drug for treating atopic dermatitis, as well as a drug for targeted treatment of atopic dermatitis.

[0005] According to a first aspect of the present invention, a targeting MIF is used in the preparation of a medicament for treating atopic dermatitis, wherein the medicament for treating said atopic dermatitis is a small molecule inhibitor of MIF.

[0006] Preferably, the MIF small molecule inhibitor is an injectable formulation.

[0007] Preferably, the MIF small molecule inhibitor is an intraperitoneal injection.

[0008] Preferably, the MIF small molecule inhibitor is the MIF antagonist ISO-1.

[0009] Preferably, the injection dose of the MIF antagonist ISO-1 is 35 mg × (±10%) / kg.

[0010] According to a second aspect of the present invention, a medicament for targeted treatment of atopic dermatitis is provided, wherein the medicament for treating said atopic dermatitis is a small molecule inhibitor of MIF.

[0011] Preferably, the MIF small molecule inhibitor is an injectable formulation.

[0012] Preferably, the MIF small molecule inhibitor is an intraperitoneal injection.

[0013] Preferably, the MIF small molecule inhibitor is the MIF antagonist ISO-1.

[0014] Preferably, the injection dose of the MIF antagonist ISO-1 is 35 mg × (±10%) / kg.

[0015] This invention demonstrates the therapeutic effect of phage migration inhibitory factor MIF in an HDM allergic mouse AD model by studying the therapeutic effect of MIF in the model. Attached Figure Description

[0016] Figure 1 A schematic diagram of the induction protocol for HDM-sensitized AD mouse model;

[0017] Figure 2 A schematic diagram of the ISO-1 treatment regimen in an HDM-sensitized AD mouse model;

[0018] Figure 3 A schematic diagram illustrating the performance of an HDM-induced mouse AD model and the therapeutic effect of the MIF antagonist ISO-1 in this model;

[0019] Figure 4 A schematic diagram of mouse skin MIF immunofluorescence and RT-PCR results;

[0020] Figure 5 A schematic diagram illustrating changes in macrophage activation indicators. Detailed Implementation

[0021] This invention provides an application of targeting MIF in the preparation of a medicament for treating atopic dermatitis, wherein the medicament for treating atopic dermatitis is a small molecule inhibitor of MIF.

[0022] The present invention also provides a drug for targeted treatment of atopic dermatitis, wherein the drug for treating atopic dermatitis is a small molecule inhibitor of MIF.

[0023] The aforementioned MIF small molecule inhibitors are injectable formulations. In particular, the aforementioned MIF small molecule inhibitors are intraperitoneal injection formulations.

[0024] Specifically, the aforementioned MIF small molecule inhibitor is the MIF antagonist ISO-1. The injection dose of the aforementioned MIF antagonist ISO-1 is 35 mg × (±10%) / kg.

[0025] The present invention relates to the test methods, test procedures, test process control conditions, and test results.

[0026] Construction of HDM-induced AD mouse model:

[0027] This study used adult male (8-10 weeks old) wild-type C57BL / 6J mice (purchased from Beijing Spefolk). Mice were kept under specific pathogen-free conditions of 23°C, a 12:12 diurnal cycle, and 50±10% humidity, with free access to food and water. All experimental procedures were approved by the Animal Ethics Committee of Peking University First Hospital. To induce an HDM-allergic AD mouse model, calcipotriol dissolved in ethanol (MC903; 6 nmol / 60 μl; Tocris Bioscience, UK) was applied topically to a shaved area (2.5 cm * 2.5 cm) on the neck of mice daily for 7 days. Then, 100 mg of HDM ointment (Biostir AD, Japan) was applied to the same site three times a week after the MC903 application. After six HDM stimulations, the mice were euthanized, and skin specimens from the back were collected for analysis. See [link to relevant documentation]. Figure 1 .

[0028] ISO-1 treatment regimen for HDM-induced AD mouse model:

[0029] During the first week, MC903 was applied topically once daily to the shaved skin area (2.5cm x 2.5cm) on the neck of the mice. After one week, HDM ointment was applied every other day after MC903, for a total of 6 times. Throughout the modeling process, ISO-1 (35mg / kg) was injected intraperitoneally every other day.

[0030] Assessment of itching severity: Mice were placed in transparent observation boxes and videotaped for 60 minutes. Each instance of a mouse raising its hind legs to scratch the skin behind its ears and neck was recorded as one scratch. The number of scratches was observed and recorded over 60 minutes. AD Severity Index (Clinical Score): The intensity of skin erythema, hemorrhage, exudation, and desquamation was assessed based on gross photographs of the mouse's skin. Each sign was graded on a scale of four: 0 (none); 1 (mild); 2 (moderate); 3 (severe). The final score was the sum of the scores for all four signs. This index was assessed weekly throughout the experiment. See [link to relevant documentation]. Figure 2 .

[0031] Immunofluorescence staining:

[0032] Mouse dorsal skin tissue sections were dewaxed, dehydrated, and antigen-retrieved. They were then permeabilized with 0.2% Triton X100 at room temperature for 10 minutes, blocked with 0.5% BSA at room temperature for 30 minutes, and incubated overnight at 4°C with anti-MIF antibody (1:100, Cell Signaling Technology) as primary antibody. The tissue was then incubated with secondary antibody Alexa Fluor. TM488 goat anti-rabbit IgG (1:200, Invitrogen) was incubated at room temperature for 2 hours. After counterstaining with DAPI, the slides were mounted and observed and photographed under a confocal microscope (Leica).

[0033] Quantitative real-time polymerase chain reaction (qRT-PCR)

[0034] Total RNA was extracted using TRIzol (Invitrogen, CA, USA) and then subjected to full-scale gold extraction. The One-Step gDNA Removal and cDNA Synthesis SuperMix kit reverse-transcribed the DNA into cDNA. qRT-PCR was then performed. The total volume was 20 µL, including 10 µL PowerUp SYBR Green (Applied Biosystems, USA), 1 µL forward substrate, 1 µL reverse primer, and 2 µL cDNA. Cycles were performed at 95 °C for 30 seconds, followed by 5 seconds at 95 °C, then 34 seconds at 58–65 °C, for a total of 40 cycles. GAPDH was used as an internal control for relative expression.

[0035] This invention, through the construction of animal and cell models of Alzheimer's disease (AD) sensitized by dust mites, clarifies that the increased expression of MIF mediated by dust mite stimulation in keratinocytes plays an important role in the development and progression of atopic dermatitis in mice. The small molecule inhibitor of MIF has good therapeutic effects in the HDM allergic mouse AD model, providing a new potential target for drug development and clinical diagnosis and treatment of exogenous allergic AD.

[0036] This invention has the following characteristics:

[0037] 1) HDM stimulation of mice resulted in AD phenotype, and the MIF antagonist ISO-1 significantly improved the symptoms of HDM-induced AD.

[0038] HDM-derived allergens are a significant contributing factor to the development of Alzheimer's disease (AD). To mimic the skin barrier dysfunction in AD patients and facilitate the entry of external allergens such as HDM proteins into the skin, we established an HDM-induced AD model in C57BL / 6J mice using MC903 as an inducer. Compared to the control group, the HDM group exhibited severe redness, swelling, and exudation. Histopathological staining revealed a significantly increased epidermal thickness and inflammatory cell infiltration in the dermis in the HDM group. Furthermore, the number of scratches in HDM-induced AD mice was significantly increased. Figure 3 ).

[0039] Immunofluorescence results showed that, compared with the control group, MIF expression was increased in the skin of the HDM-induced AD mouse model, mainly in the epidermal basal layer; qRT-PCR results showed that the relative mRNA expression of MIF in the skin of the model mice was significantly increased compared with the control group. Figure 4 ).

[0040] To evaluate the therapeutic effect of the MIF antagonist ISO-1 on HDM-induced allergic atopic dermatitis (AD), a mouse model was established using MC903 and HDM. As previously described, ISO-1 (35 mg / kg) was administered intraperitoneally every other day. Following ISO-1 treatment, skin erythema, swelling, and exudation in mice were significantly reduced. H&E staining showed that ISO-1 significantly inhibited epidermal hyperkeratosis and dermal inflammatory cell infiltration. Compared with the HDM group, ISO-1 significantly improved pruritus. Furthermore, after ISO-1 treatment, the expression of type 2 inflammatory cytokines (such as IL-4, IL-13, IL-33, and TSLP) in the skin lesions was significantly reduced. Figure 3 ).

[0041] 2) MIF stimulates the activation of mouse macrophages, which can be inhibited by ISO-1.

[0042] Furthermore, MIF stimulated activation of the mouse macrophage cell line Raw264.7, increased the mRNA expression of inflammatory cytokines such as IL-1β, IL-6, and IL-10, and these expression levels were inhibited by ISO-1. Therefore, we believe that MIF induces macrophage activation, and its antagonist ISO-1 can alleviate symptoms of HDM-related allergic AD and regulate macrophage function. Figure 5 ).

[0043] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A use of a MIF small molecule inhibitor in the manufacture of a medicament for targeted treatment of atopic dermatitis, characterized in that, The MIF small molecule inhibitor is MIF antagonist ISO-1.

2. Use of a MIF small-molecule inhibitor according to claim 1 for the manufacture of a medicament for the targeted treatment of atopic dermatitis, characterized in that, The MIF antagonist ISO-1 is an injection.

3. Use of a MIF small-molecule inhibitor according to claim 2 for the manufacture of a medicament for the treatment of atopic dermatitis, characterized in that, The MIF antagonist ISO-1 is an intraperitoneal injection.

4. Use of a MIF small-molecule inhibitor according to claim 2 or 3 for the manufacture of a medicament for the targeted treatment of atopic dermatitis, characterized in that, One injection dose of the MIF antagonist ISO-1 injection or intraperitoneal injection is 35 mg / kg.

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