Application of neutralizing antibody of targeted macrophage in preparation of medicine for treating atopic dermatitis

Abstract

The invention provides application of a neutralizing antibody of a targeted macrophage to preparation of a medicine for treating atopic dermatitis (AD), relates to the technical field of biomedicine, and is characterized in that the neutralizing antibody of the targeted macrophage is provided to deplete the macrophage so as to treat the AD. The method is characterized in that a neutralizing antibody capable of specifically recognizing and combining a key target spot-colony stimulating factor 1 receptor (CSF1R) on the surface of the macrophage is applied to block a CSF1 / CSF1R signal channel, so that the macrophage for driving the AD pathological process is eliminated. According to the application, the atopic dermatitis (AD) is treated by applying the neutralizing antibody targeting the key target spot of the macrophage, and remarkable and multi-aspect beneficial technical effects are generated. The effects are verified in an AD mouse model induced by MC903 and are specifically reflected in relieving of disease core symptoms, intervention of key pathological links and improvement of overall health conditions, and a foundation is laid for clinical treatment of atopic dermatitis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of neutralizing antibodies targeting macrophages in the preparation of drugs for the treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common, chronic, relapsing, inflammatory skin disease characterized by intense itching, impaired skin barrier function, and abnormal immune response. The pathogenesis of AD is complex, involving multiple factors including genetics, environment, skin barrier defects, and immune dysregulation. For a long time, research has focused on the role of type 2 helper T cells (Th2) and their produced cytokines (such as IL-4, IL-13, and IL-31), as well as mast cells / basophils. Biologics targeting these pathways (such as dupilumab and troroculumab) have achieved significant clinical efficacy, confirming the central role of the type 2 immune pathway in AD.

[0003] However, existing therapies still have limitations: some patients do not respond adequately (primary or secondary non-response) or experience adverse reactions. This suggests that the pathogenesis of Alzheimer's disease (AD) involves a more complex cellular network and immune redundancy. In recent years, the key role of innate immune cells, especially skin-resident and recruited macrophages, in the initiation, chronicity, and pruritus perception of AD has received increasing attention, providing novel targets for developing new therapeutic strategies.

[0004] The skin contains a complex network of antigen-presenting cells, including Langerhans cells, dendritic cells, and resident inflammatory macrophages. Macrophages exhibit high plasticity and can polarize into different functional subtypes based on microenvironmental signals. In the acute phase of Alzheimer's disease (AD), classically activated (M1-like) and alternatively activated (M2-like) macrophages mediating type 2 inflammation are predominant. They amplify local inflammatory responses and recruit more immune cells (such as Th2 cells and eosinophils) by producing large amounts of pro-inflammatory factors (e.g., TNF-α, IL-1β, IL-6) and chemokines. In the chronic phase of AD, the macrophage phenotype further transforms into subtypes with stronger tissue remodeling and fibrosis functions, participating in skin thickening (lichenification) and barrier repair.

[0005] Although the core role of macrophages in Alzheimer's disease (AD) has been preliminarily revealed, there are currently no specific antibody drugs designed to precisely target and regulate the function of macrophages in skin lesions, especially their key pathogenic cytokines or surface receptors. Summary of the Invention

[0006] This invention provides a method for treating Alzheimer's disease (AD) by targeting macrophages with a neutralizing antibody to deplete macrophages. The core of this method lies in the application of a neutralizing antibody (αCSF1R) that specifically recognizes and binds to a key target on the surface of macrophages—the colony-stimulating factor 1 receptor (CSF1R)—blocking the CSF1 / CSF1R signaling pathway and thereby eliminating the macrophages driving the pathological progression of AD.

[0007] This application provides the use of a neutralizing antibody targeting macrophages in the preparation of a drug for treating atopic dermatitis, wherein the neutralizing antibody is a neutralizing antibody against colony-stimulating factor 1 receptor.

[0008] Preferably, the αCSF1R eliminates macrophages that drive the pathological process of AD by blocking the CSF1 / CSF1R signaling pathway.

[0009] This application also provides a medicament for treating atopic dermatitis, the medicament comprising a neutralizing antibody against colony-stimulating factor 1 receptor.

[0010] Preferably, the drug includes other medically acceptable adjuvants.

[0011] Preferably, the dosage form of the drug is selected from at least one of injection, topical preparation or tablet.

[0012] Preferably, the injectable includes intraperitoneal injection, subcutaneous injection, and intravenous injection.

[0013] Preferably, the topical preparation includes creams, gels, and lotions.

[0014] Preferably, the drug can relieve the core symptoms of atopic dermatitis, including reducing the frequency of itching and scratching, slowing down skin thickening, and lowering the clinical dermatitis score.

[0015] Preferably, the drug can downregulate the mRNA expression levels of IL-1β, TNF-α, IL-23, and IL-33 in skin tissue.

[0016] Compared with the prior art, this application has at least the following beneficial effects:

[0017] Treatment of atopic dermatitis (AD) with a neutralizing antibody (αCSF1R) targeting key macrophage targets has yielded significant and multifaceted beneficial effects. These effects were systematically validated in an MC903-induced AD mouse model, specifically manifested in the relief of core disease symptoms, intervention in key pathological processes, and improvement in overall health status. This lays the foundation for the clinical treatment of atopic dermatitis and has significant application and promotion value. Attached Figure Description

[0018] Figure 1 Effects of intraperitoneal injection of neutralizing antibody αCSF1R on mouse skin macrophages.

[0019] Immunofluorescence results showed that the CD68 fluorescence signal of macrophages in the αCSF1R group on day 8 was significantly reduced compared with that in the IgG group, and the number of CD68-labeled macrophages in the skin lesions was significantly reduced (scale bar = 50 μm).

[0020] Figure 2 Intraperitoneal injection of the neutralizing antibody αCSF1R can alleviate the effects of palmitic acid-induced pruritus.

[0021] (A) Schematic diagram of intradermal (id) injection of PA, intraperitoneal (ip) injection of αCSF1R or IgG in normal mice; (B) Behavioral results showing that the number of scratches in the αCSF1R group was significantly lower than that in the IgG group, with a statistically significant difference at the 1-hour time point, suggesting that αCSF1R can alleviate itching behavior induced by intradermal PA injection. Behavioral statistical analysis was performed using two-way ANOVA, with P < 0.0001, compared with the IgG group at the corresponding time points.

[0022] Figure 3 Schematic diagram of the application of the neutralizing antibody αCSF1R in an MC903-induced specific dermatitis model by intraperitoneal injection to deplete macrophages.

[0023] Figure 4 In vivo experiments showed that the αCSF1R group had a significant reduction in atopic dermatitis symptoms, including changes in facial skin appearance, scratching behavior, mouse weight, clinical scores, and the thickness and weight of facial skin.

[0024] (A) Images showing facial skin changes after intraperitoneal injection of neutralizing antibodies αCSF1R or IgG. The images show a significant reduction in facial skin damage in the αCSF1R group. (B) Behavioral data showed a significant decrease in scratching frequency in the αCSF1R group. (C) Mouse weight changes showed that mice in the αCSF1R group lost weight more slowly. (D) Clinical scores showed that the αCSF1R group had lower clinical scores, indicating that the αCSF1R group could significantly alleviate the symptoms of atopic dermatitis induced by MC903. (E, F) The results indicate that the thickness and weight of facial skin in mice in the αCSF1R group were significantly reduced 8 days after MC903 application. Statistical analysis of behavioral data, weight changes, and clinical scores was performed using two-way ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001, compared with the IgG group at the corresponding time points. The thickness and weight of facial skin were measured using a t-test (Student's T-test), with **P < 0.01 and ***P < 0.001, compared with the corresponding IgG group.

[0025] Figure 5 HE staining and quantitative analysis showed that epidermal hyperplasia was significantly reduced in the αCSF1R group, and the number of epidermal infiltrating cells was significantly decreased.

[0026] (A) shows representative HE staining images; (B) shows the statistical results of image A. Statistical results were analyzed using a t-test (Student's T-test). ***P < 0.001, compared with the IgG group.

[0027] Figure 6 This invention verifies in vivo experiments the effect of α-CSF1R on the expression level of inflammatory factors in the skin of mice with MC903-induced specific dermatitis.

[0028] Real-time PCR results showed that, compared with the IgG group, the mRNA expression levels of inflammatory factors IL-1β (A), TNF-α (B), IL-23 (C), and IL-33 (D) in the skin of mice in the αCSF1R group were significantly reduced. Statistical results were analyzed using a t-test (Student's T-test). *P < 0.05, **P < 0.01, compared with the IgG group.

[0029] Figure 7 Western blot was used to detect the levels of p-ERK, p-NF-κb, and TLR4 in skin lesions of the αCSF1R and IgG groups.

[0030] (A) Western blot results show that, compared with the IgG group, the levels of p-ERK, p-NF-κb, and TLR4 in the skin of mice in the αCSF1R group were significantly reduced. (B) Statistical results of Figure A. The statistical results were analyzed using a t-test (Student's t-test). *P < 0.05, ****P < 0.0001, compared with the IgG group. Detailed Implementation

[0031] This application provides the use of a neutralizing antibody targeting macrophages in the preparation of a drug for treating atopic dermatitis. The neutralizing antibody is a colony-stimulating factor 1 receptor (CSF1R) neutralizing antibody (abbreviated as αCSF1R). The αCSF1R eliminates macrophages that drive the pathological process of AD by blocking the CSF1 / CSF1R signaling pathway.

[0032] This application also provides a medicament for treating atopic dermatitis, the medicament comprising a neutralizing antibody against colony-stimulating factor 1 receptor.

[0033] In one embodiment, the drug also includes other medically acceptable adjuvants.

[0034] The dosage form of the drug is selected from at least one of injections and topical preparations. In one embodiment, the injections include intraperitoneal injections, subcutaneous injections, and intravenous injections; the topical preparations include creams, gels, and lotions.

[0035] The drug can relieve the core symptoms of atopic dermatitis, including reducing the frequency of itching and scratching, slowing down skin thickening, and lowering clinical dermatitis scores, while downregulating the mRNA expression levels of IL-1β, TNF-α, IL-23, and IL-33 in skin tissue.

[0036] The above content will be elaborated below with specific verification experiments:

[0037] I. Synthesis of neutralizing antibody αCSF1R

[0038] The αCSF1R reagent was purchased from Selleck, catalog number A2159.

[0039] In this application, αCSF1R refers to the mouse colony-stimulating factor 1 receptor (CSF1R).

[0040] II. Changes in the expression of inflammatory factors in the skin of model mice

[0041] Real-time quantitative PCR results showed that on day 8 of atopic dermatitis modeling, the expression of IL-1β, TNF-α, IL-23, and IL-33 mRNA in αCSF1R skin was significantly increased. Figure 6 ).

[0042] 2.1 Preparation of specific dermatitis model mice

[0043] Mice (strain: C57BL / 6) were purchased from the Experimental Animal Center of Nantong University, 8 weeks old, weighing 25±2 grams, and acclimatized for 3 days. After anesthesia, hair removal cream was applied to the face of the mice, followed by 3 days of acclimatization. The mice were kept anesthetized using a Reward mask anesthesia machine, and MC903 (200 μM, 20 μL) was applied to the face once daily for 8 consecutive days.

[0044] 2.2 Animal tissue sample extraction, RNA extraction, reverse transcription, and Real-time PCR process

[0045] 2.2.1 Extraction of tissue samples

[0046] (1) After anesthetizing the mice, the thoracic cavity was opened and the heart was perfused with 0.9% sodium chloride solution;

[0047] (2) After the perfusion was completed, the skin of the mouse with atopic dermatitis was taken and placed in 200 μL of Trizol reagent and placed on ice;

[0048] 2.2.2 RNA Extraction

[0049] (1) After taking the sample, use micro-scissors to cut it into small pieces and homogenize it once every 30 seconds for a total of 4 times until no blocky tissue is visible. Then add 800 μL Trizol and let it stand for 5 min.

[0050] (2) Add 200 μL of chloroform to each tube, shake vigorously for 1 min, and let stand for 5 min;

[0051] (3) Centrifuge at 4℃, 12000 rpm for 15 min;

[0052] (4) Gently aspirate the supernatant into an RNAase-Free EP tube, add an equal volume of isopropanol, mix gently until filaments are no longer visible to the naked eye, and let stand for 10 min.

[0053] (5) Centrifuge at 4℃, 12000 rpm, for 15 min;

[0054] (6) Discard the supernatant, add 1 mL of anhydrous ethanol and mix gently, then centrifuge at 4℃, 12000 rpm for 15 min.

[0055] (7) Discard the supernatant, invert it onto filter paper, and let it air dry at room temperature until the precipitate is translucent;

[0056] (8) Add 20 μL of RNase-free H2O to each tube, heat at 60℃, and bathe in a water bath for 10 min to promote dissolution. After dissolution, place on ice.

[0057] (9) Measure RNA concentration using an OD tester.

[0058] 2.2.3 Total RNA was reverse transcribed into cDNA

[0059] (1) Removal of DNA impurities from the sample (10 μL system)

[0060]

[0061] (2) RNA was reverse transcribed into cDNA (20 μL system)

[0062]

[0063] After reverse transcription to cDNA, dilute 8 times with deionized water and store at -20℃ for later use.

[0064] (3) Primer sequence

[0065] For primer design, this patent uses mouse IL-1β, IL-6, TNF-α, IL-4, and IL-33 mRNA sequences from the NCBI database, designs primers on the NCBI website, and uses BLAST for specificity verification to predict the size of the amplification product.

[0066] The primer sequences required for the experiment are shown in the table below.

[0067]

[0068] 2.2.4 Real-time PCR experiment

[0069] (1) Prepare the Real-time PCR reaction system (10 μL) according to the table below:

[0070]

[0071] (2) PCR reaction using a Life Technology instrument, under the following conditions:

[0072]

[0073] III. Effects of intraperitoneal injection of αCSF1R on mouse skin macrophages

[0074] Immunofluorescence results showed that, compared with the IgG group, the αCSF1R group mice expressed less CD68, a marker of macrophages, in the skin. Figure 1 ).

[0075] 3.1 Immunofluorescence sample preparation, staining, and imaging

[0076] 3.1.1 Tissue section preparation

[0077] After anesthetizing mice, their hearts were perfused with physiological saline and 4% paraformaldehyde. The mice were then fixed on ice for 2 hours. Skin from the ruptured facial area was placed in a 15mL EP tube containing 4% paraformaldehyde, pre-chilled, and incubated overnight at 4°C. The skin was then dehydrated with 20% sucrose. Once the skin sank to the bottom of the tube, the sucrose solution was replaced with 30% sucrose. After the skin sank again, the skin was removed. The trimmed skin sections were grouped and placed in a model chamber, then rapidly frozen on a microtome. After solidification, the embedded blocks were attached to the freezing stage using OTC adhesive for cryosectioning. Skin tissue patches, 12μm thick, were mounted on adhesive slides and allowed to air dry.

[0078] 3.1.2 Immunofluorescence staining

[0079] (1) Place the slides in a container containing 0.01 mol / L PBS and wash 3 times, 6 min each time;

[0080] (2) After washing the slides, add immunofluorescence blocking solution and block at room temperature for 2 h.

[0081] (3) After the blocking is completed, add the primary antibody prepared with the blocking solution and incubate overnight at 4°C.

[0082] (4) Take it out on the second day, let it return to room temperature, and wash it three times with 0.01 mol / L PBS for 6 min each time.

[0083] (5) Dilute the secondary antibody with 0.01 mol / L PBS and incubate at room temperature in the dark for 2 h.

[0084] (6) Discard the secondary antibody and wash the slide three times with 0.01 mol / L PBS for 6 min each time.

[0085] (7) Place the slide at room temperature away from light. After the slide is half-dry, drop a sealing agent on the slide, cover it with a coverslip, and let it dry before taking pictures with a fluorescence microscope.

[0086] (8) Quantitative analysis was performed using ImageJ software.

[0087] IV. Detection of TLR4 signaling pathway protein expression in the skin of model mice

[0088] Western blot results showed that, compared with the IgG group, the levels of TLR4, p-NF-κb, and p-ERK were significantly reduced in the αCSF1R group. Figure 7 ).

[0089] 4.1 Sample preparation, protein extraction, electrophoresis, membrane transfer, antibody incubation, and chemiluminescent development for Western Blot

[0090] 4.1.1 Tissue Sample Preparation

[0091] Sampling was performed as in 2.2.1. Mice were anesthetized, their thoracic cavity was cut open, their hearts were perfused, and skin from the broken areas of their faces was taken and placed in EP tubes.

[0092] 4.1.2 Protein Extraction

[0093] (1) Prepare the lysis buffer according to the table below:

[0094]

[0095] (2) Add 200 μL of lysis buffer to each tube, homogenize, and continue until no sedimentation occurs;

[0096] (3) Ice bath for 30 min, then centrifuge at 4℃, 14000 rpm for 15 min;

[0097] (4) Aspirate the supernatant into a new EP tube; measure the protein concentration;

[0098] (5) Prepare the protein loading solution according to the reaction system, and boil it in a water bath at 100°C for 5 min to denature the protein;

[0099]

[0100] 4.1.3 Electrophoresis

[0101] (1) Prepare SDS-PAGE gels of appropriate concentrations according to the molecular weight of the target protein;

[0102] (2) Add the sample into the lane of the SDS-PAGE gel and maintain a constant voltage of 110V for 2 hours;

[0103] 4.1.4 Transfer of film

[0104] Transfer the protein from the SDS-PAGE gel to a membrane (such as a PVDF or nitrocellulose membrane) and hold at a constant current of 250 mA for 90 min.

[0105] 4.1.5 Antibody incubation

[0106] (1) Wash the membrane once with TBST (1X) on a decolorizing shaker for 8 min;

[0107] (2) 5% skim milk powder sealed with a membrane to prevent nonspecific binding, at room temperature for 2 hours;

[0108] (3) 5% skim milk powder was prepared with a specific primary antibody and incubated overnight at 4°C;

[0109] (4) The next day, after removing the membrane and allowing it to return to room temperature, wash it three times with TBST (1X) on a decolorizing shaker for 8 minutes each time;

[0110] (5) Prepare secondary antibody with TBST(1X) and incubate at room temperature for 2 hours; then wash three times with TBST(1X) every 8 minutes.

[0111] 4.1.6 Chemiluminescence Development

[0112] (1) Prepare the luminescent solution according to the proportions specified in the reagent manufacturer's instructions;

[0113] (2) Add the luminescent solution to the washed membrane and incubate for 1 min;

[0114] (3) The membrane was developed in a high-sensitivity chemiluminescence system to obtain the target protein band.

[0115] 4.1.7 ImageJ software analysis of target protein bands

[0116] (1) Import the target protein strip into ImageJ software.

[0117] (2) Calculate the grayscale value of the bar according to the Image J software operation guide.

[0118] (3) Plot the graph based on the grayscale values.

[0119] V. Effects of αCSF1R on itching behavior in mice with atopic dermatitis

[0120] This invention involved intraperitoneal injection of αCSF1R every other day, at a dose of 200 μg / 200 μL, with continuous monitoring of scratching behavior in mice from the start. Behavioral results showed that αCSF1R could alleviate scratching behavior induced by MC903-specific dermatitis. Figure 4 ).

[0121] 5.1 Intraperitoneal injection in mice

[0122] To establish the MC903-specific dermatitis model, αCSF1R was administered via intraperitoneal injection. (See section 2.1 for the MC903 model.) The intraperitoneal injection method for mice is as follows: Lift the mouse by its tail with your right hand and place it on the cage lid. Use your left thumb and forefinger to quickly and gently pinch the skin at the back of the neck near the scapula to prevent the head from turning backward. Simultaneously, use your little and ring fingers to press down on the base of the tail, straightening the mouse's body so that the abdomen is facing upward. Inject the medication in the area slightly to the left and below the midline of the abdomen, with the needle at a 15-30 degree angle to the mouse's body and the tip pointing towards the head. Quickly and steadily pierce the skin and abdominal wall to enter the peritoneal cavity. Aspirate to ensure no blood, urine, or intestinal contents are aspirated before slowly injecting the medication. After injection, quickly withdraw the needle. Finally, return the mouse to its cage and observe it briefly. Successful administration is confirmed if no abnormalities are observed.

[0123] 5.2 Behavioral testing of mice

[0124] Three days prior to the behavioral test, mice were allowed to acclimatize, with the same experimental personnel and environment maintained, and a double-blind method was used for the test.

[0125] For itch behavior detection, mice were placed in a transparent nine-square grid on a metal frame and allowed to acclimatize for 30 minutes, followed by 1 hour of video recording. The number of scratches was counted as one scratch: the hind paws scratched the face, then the mice licked or bit their paws, or were placed back on the base. This cycle was counted as one scratch. If the front paws rubbed the face, it was not counted as itch behavior.

[0126] Based on the above description, this application utilizes a neutralizing antibody (αCSF1R) targeting key macrophage targets to treat atopic dermatitis (AD), producing significant and multifaceted beneficial technical effects. These effects were systematically validated in an MC903-induced AD mouse model, specifically manifested in the relief of core disease symptoms, intervention in key pathological processes, and improvement in overall health status. This lays the foundation for the clinical treatment of atopic dermatitis and has significant application and promotion value.

Claims

1. The application of neutralizing antibodies targeting macrophages in the preparation of drugs for treating atopic dermatitis, characterized in that: The neutralizing antibody is a neutralizing antibody against colony-stimulating factor 1 receptor.

2. The application of the neutralizing antibody targeting macrophages according to claim 1 in the preparation of a drug for treating atopic dermatitis, characterized in that: The αCSF1R eliminates macrophages that drive the pathological process of AD by blocking the CSF1 / CSF1R signaling pathway.

3. A drug for treating atopic dermatitis, characterized in that: The drug includes a neutralizing antibody against the colony-stimulating factor 1 receptor.

4. The drug according to claim 3, characterized in that: The drug also includes other medically acceptable adjuvants.

5. A drug according to claim 3, characterized in that: The dosage form of the drug is selected from at least one of injection, topical preparation or tablet.

6. The drug according to claim 5, characterized in that: The injectables include intraperitoneal injections, subcutaneous injections, and intravenous injections.

7. The drug according to claim 5, characterized in that: The topical preparations include creams, gels, and lotions.

8. A drug according to claim 3, characterized in that: The medication can relieve the core symptoms of atopic dermatitis, including reducing the frequency of itching and scratching, slowing down skin thickening, and lowering clinical dermatitis scores.

9. A drug according to claim 3, characterized in that: The drug can downregulate the mRNA expression levels of IL-1β, TNF-α, IL-23, and IL-33 in skin tissue.

Images