Application of anti-TCRγδ antibody, anti-CSF1R antibody and / or anti-TNF-α antibody in the preparation of drugs for treating androgenetic alopecia

Abstract

This invention discloses the application of anti-TCRγδ antibodies, anti-CSF1R antibodies, and / or anti-TNF-α antibodies in the preparation of drugs for treating androgenetic alopecia, belonging to the field of biomedical technology. By applying anti-TCRγδ antibodies, anti-CSF1R antibodies, and anti-TNF-α antibodies to DHT-induced AGA model mice, it was found that all three antibodies promoted hair growth and effectively alleviated the development of AGA. This result indicates that specifically deleting skin γδT cells and macrophages or specifically blocking TNF-α in the skin can clear pathogenic γδT cells, macrophages, or TNF-α in the pathogenesis area of ​​androgenetic alopecia, promoting hair growth and providing a new treatment option for androgenetic alopecia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of anti-TCRγδ antibody, anti-CSF1R antibody and / or anti-TNF-α antibody in the preparation of drugs for treating androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss in humans. It is characterized by a shortened anagen (growth) phase in the normal hair growth cycle, leading to an increase in the number of telogen (resting) phase hair follicles, follicle miniaturization, and the transformation of terminal hairs into vellus hairs, ultimately preventing the hair from penetrating the epidermis. In men, it mainly manifests as a receding hairline and / or progressive thinning and reduction of hair on the crown, while in women, it mainly manifests as diffuse thinning and fineness of hair on the crown. AGA significantly impacts patients' mental health and quality of life.

[0003] Abnormal androgen metabolism throughout the body and on the scalp, as well as excessive follicular response to androgens, are important aspects of AGA pathogenesis. In men, the main androgen is testosterone, which is converted into the more active 5α-dihydrotestosterone (DHT) by 5α-reductase. DHT levels are generally elevated in the scalp of AGA patients and are an important molecule leading to AGA. Therefore, finasteride (a 5α-reductase inhibitor) is currently an important drug for the treatment of AGA. At the same time, topical minoxidil solution (local vasodilator, increasing blood and oxygen supply) is used to promote hair growth and improve symptoms.

[0004] Although the aforementioned medications are widely used to treat AGA, significant limitations remain: approximately 39.5% of Japanese male AGA patients experienced only minimal hair growth after daily oral administration of 1 mg finasteride, indicating poor efficacy. Furthermore, some finasteride users have reported persistent side effects such as decreased libido, erectile dysfunction, breast discomfort, depression, and skin allergies. The efficacy of topical minoxidil is highly dependent on patient adherence; patients must use it 1-2 times daily as prescribed and persist for 4-8 months to observe any effects. Therefore, new treatment methods are urgently needed to improve the treatment outcomes for AGA.

[0005] γδT cells are a type of innate T lymphocytes whose T cell receptor (TCR) is composed of γ and δ chains. γδT cells are highly prevalent in the skin. Currently, the research and application of anti-TCRγδ antibodies as direct therapeutic agents are not yet mature. In clinical studies, anti-TCRγδ antibodies are mainly used to assist, activate, or construct immunotherapies centered on γδT cells (especially cancer immunotherapy). CSF1R is a receptor tyrosine kinase on the surface of macrophages, which mainly participates in regulating macrophage survival, proliferation, differentiation, and function. Anti-CSF1R antibodies can directly target and eliminate pathogenic macrophages in the body and are currently mainly used in clinical research on tumors and chronic graft-versus-host disease. TNF-α is a pleiotropic cytokine that can be produced by various immune cells, including γδT cells and macrophages, and participates in coordinating inflammatory responses, cell proliferation, and death. Anti-TNF-α antibodies can precisely block TNF-α, and are currently approved for use in diseases such as rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, and psoriasis. In summary, there are currently no reports of anti-TCRγδ antibodies, anti-CSF1R antibodies, or anti-TNF-α antibodies being able to treat AGA. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide the application of anti-TCRγδ antibody, anti-CSF1R antibody and / or anti-TNF-α antibody in the preparation of drugs for treating androgenetic alopecia, so as to solve the technical problems of existing AGA treatments having many side effects, slow onset of action and poor efficacy.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the invention discloses the use of anti-TCRγδ antibody, anti-CSF1R antibody and / or anti-TNF-α antibody in the preparation of a drug for treating androgenetic alopecia.

[0008] Preferably, the dosage of the anti-TCRγδ antibody is 10 mg / Kg, and the dosing frequency is once every other day after 3 consecutive days of dosing.

[0009] Preferably, the dosage of the anti-CSF1R antibody is 25 mg / kg, and the dosing frequency is once every other day.

[0010] Preferably, the dosage of the anti-TNF-α antibody is 20 mg / kg, and the dosing frequency is once every other day.

[0011] Preferably, the drug is an intradermal injection.

[0012] More preferably, the intradermal injection uses phosphate buffer as a solvent.

[0013] Preferably, the drug further includes a pharmaceutically acceptable carrier and / or excipients.

[0014] More preferably, the carrier comprises one or more of a solvent, a polymer, and liposomes; the excipients comprise one or more of a diluent, an excipient, and a stabilizer.

[0015] Preferably, the drug also includes other drugs for treating androgenetic alopecia.

[0016] A second aspect of the present invention discloses a composition for treating androgenetic alopecia, comprising any combination of anti-TCRγδ antibody, anti-CSF1R antibody, anti-TNF-α antibody, and other drugs for treating androgenetic alopecia.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The application of anti-TCRγδ antibody, anti-CSF1R antibody, and / or anti-TNF-α antibody provided by this invention in the preparation of drugs for treating androgenetic alopecia (AGA) was demonstrated by applying these antibodies to DHT-induced AGA model mice. It was found that all three antibodies promoted hair growth and effectively alleviated the development of AGA. This result indicates that specifically deleting skin γδT cells or macrophages, or specifically blocking TNF-α in the skin, can clear pathogenic γδT cells, macrophages, or TNF-α in the AGA lesion area, promoting hair growth and providing a new treatment option for androgenetic alopecia. Attached Figure Description

[0018] Figure 1 is a diagram of the AGA model construction of the present invention; wherein, A is a flowchart of the AGA mouse model construction process, and B is a diagram of the hair growth on the back of mice in the control group and the AGA group. Figure 2Figure 1 shows the results of detecting changes in γδT cells and macrophages in the skin of AGA mice according to the present invention. A represents the flow cytometry plot of the proportion of γδT cells in the skin of AGA and control mice; B represents the statistical results of the proportion and number of γδT cells in the skin of AGA and control mice; C represents the flow cytometry plot of the proportion of γδT cells expressing TNF-α in the skin of AGA and control mice; D represents the statistical results of the proportion of γδT cells expressing TNF-α in the skin of AGA and control mice; E represents the flow cytometry plot of the proportion of macrophages in the skin of AGA and control mice; F represents the statistical results of the proportion and number of macrophages in the skin of AGA and control mice; G represents the flow cytometry plot of the proportion of macrophages expressing TNF-α in the skin of AGA and control mice; H represents the statistical results of the proportion of macrophages expressing TNF-α in the skin of AGA and control mice; *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001; Figure 3 shows the results of the anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody of the present invention effectively alleviating AGA symptoms; where A is a record of hair growth on the back of mice in the control group, AGA negative control group, and treatment groups of anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody; B is the HE staining result of the back skin of mice in the control group, AGA group, and treatment groups of anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody; C is the statistical results of hair follicle density, the proportion of hair follicles in the growth phase, and hair bulb diameter of mice in the control group, AGA group, and treatment groups of anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody; *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Detailed Implementation

[0019] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0020] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0021] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0022] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0023] The reagents used in this article include: Deoxyribonuclease I (DNase I), Fetal Bovine Serum (FBS), and Phosphate Buffered Saline (PBS).

[0024] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0025] This invention provides the application of anti-TCRγδ antibody, anti-CSF1R antibody, and / or anti-TNF-α antibody in the preparation of drugs for treating androgenetic alopecia. Intradermal injection of anti-TCRγδ antibody into the skin of DHT-induced AGA model mice showed that the anti-TCRγδ antibody promoted hair growth and alleviated AGA symptoms. Intradermal injection of anti-CSF1R antibody into the skin of DHT-induced AGA model mice showed that the anti-CSF1R antibody promoted hair growth and alleviated AGA symptoms. Intradermal injection of anti-TNF-α antibody into the skin of DHT-induced AGA model mice showed that the anti-TNF-α antibody promoted hair growth and alleviated AGA symptoms.

[0026] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0027] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0028] I. Constructing the AGA Model As shown in Figure 1A, 7-week-old male C57BL / 6J wild-type mice were randomly divided into an AGA model group and a control group. Mice in the AGA model group received daily subcutaneous injections of DHT (0.1 mg / 100 μL / mouse / day) into their backs to establish the AGA model mouse group. Mice in the control group received daily subcutaneous injections of corn oil (100 μL / mouse / day) into their backs to establish the control group mouse group. These injections were performed for 21 consecutive days. On day 11, the hair on the backs of the mice in each group was removed using a shaver and depilatory cream. Hair growth on the backs was recorded by photograph on days 11 and 21.

[0029] Hair growth is shown in Figure 1B. The skin on the backs of mice in the control group turned completely black, indicating that the hair follicles entered the growth phase. The skin on the backs of mice in the AGA group was pink, indicating that the hair follicles were in the resting phase, indicating that the AGA model was successfully constructed.

[0030] II. Preparation of Mouse Skin Single-Cell Suspension On day 21 of AGA model construction, mice in each group were sacrificed by cervical dislocation, and the skin on the back was separated and cut into 1 mm pieces. 3 Tissue blocks of various sizes were added to 10 mL of digestion solution (RPMI 1640 containing 1 mg / mL collagenase IV and 0.2 mg / mL DNase I) and placed in a shaker at 37°C and 80 rpm for 90 min. The digestion supernatant was filtered through a 70 μm filter and centrifuged at 4°C and 2000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in 500 μL of FACS (1×PBS containing 2% FBS) to obtain single-cell suspensions of skin cells from AGA mice and control mice.

[0031] III. Detection of changes in γδT cells and macrophages in AGA mice 1. Flow cytometry analysis of changes in the proportion, number, and function of γδT cells in mouse skin. 100 μL of AGA mouse skin single-cell suspension and control mouse skin single-cell suspension were taken, and FITC-labeled anti-mouse CD45.2 antibody (purchased from Biolegend, Cat:109806, Clone:104, Mouse (SJL) IgG2a, κ, 1:400), Pacific Blue (PB)-labeled anti-mouse CD3 antibody (purchased from Biolegend, Cat:100214, Clone:17A2, Rat IgG2b, κ, 1:1600), were added to the suspension. PECY7-labeled anti-mouse TCRγδ antibody (purchased from Biolegend, Cat:118124, Clone:GL3, Armenian Hamster) was also added to the suspension. IgG (1:1600) was stained at 4°C in the dark for 30 min; 1 mL of FACS (PBS containing 2% FBS) was added to stop the staining, and the cells were centrifuged at 4°C and 1700 rpm for 5 min; the supernatant was discarded, and 100 μL of FACS was added to resuspend the cells. The changes in the proportion and number of γδT cells were detected by flow cytometry. After surface staining, 100 μL of cell permeabilization fixative (Biolegend, Cat: 420801) was added, and the cells were fixed at 4°C in the dark for 30 min. 500 μL of intracellular staining permeability wash buffer (Biolegend, Cat: 421002) was added to each tube to terminate fixation, and the cells were centrifuged at 4°C, 1700 rpm for 5 min. The supernatant was discarded, and APCY7-labeled anti-mouse TNF-α antibody (Biolegend, Cat: 506343, Clone: ​​MP6-XT22, Rat IgG1, κ, 1:800) was added. The cells were stained at 4°C in the dark for 30 min. 500 μL of intracellular staining permeability wash buffer was added to terminate staining, and the cells were centrifuged at 4°C, 1700 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 100 μL of FACS. Flow cytometry was used to detect the expression level of TNF-α in γδT cells.

[0032] Changes in the proportion and number of γδT cells are as follows: Figure 2 As shown in Figures A and B, although the proportion of γδT cells in the skin of AGA mice did not increase, their number did. The results of TNF-α expression level detection in γδT cells are as follows... Figure 2 As shown in C and D, the expression level of TNF-α in γδT cells in the skin of AGA mice is increased.

[0033] 2. Flow cytometry analysis of changes in the proportion, number, and function of macrophages in mouse skin. 100 μL of AGA mouse skin single-cell suspension and control mouse skin single-cell suspension were taken respectively, and FITC-labeled anti-mouse CD45.2 antibody (purchased from Biolegend, Cat:109806, Clone:104, Mouse (SJL) IgG2a, κ, 1:400), PECY7-labeled anti-mouse CD11b antibody (purchased from Biolegend, Cat:101216, Clone:M1 / 70, Rat IgG2b, κ, 1:400), and phycoerythrin (PE)-labeled anti-mouse F4 / 80 antibody (purchased from Biolegend, Cat:123110, Clone:BM8, Rat IgG2a, κ, 1:400) were added. Staining was performed at 4℃ in the dark for 30 min. Staining was terminated by adding 1 mL of FACS (PBS containing 2% FBS), and centrifuged at 1700 rpm for 5 minutes at 4℃. After surface staining, add 100 μL of cell permeabilization fixative and fix at 4°C in the dark for 30 min. Discard the supernatant, resuspend in 100 μL of FACS, and detect changes in macrophage proportion and number by flow cytometry. After surface staining, add 100 μL of cell permeabilization fixative and fix at 4°C in the dark for 30 min. Add 500 μL of intracellular staining permeabilization wash buffer to each tube to terminate fixation, centrifuge at 1700 rpm for 5 min at 4°C. Discard the supernatant, add allophycocyanin (APC)-labeled anti-mouse TNF-α antibody (purchased from Biolegend, Cat:506308, Clone:MP6-XT22, Rat IgG1, κ, 1:800), and stain at 4°C in the dark for 30 min. Add 500 μL of intracellular staining permeabilization wash buffer to terminate staining, centrifuge at 1700 rpm for 5 min at 4°C. Discard the supernatant, resuspend in 100 μL of FACS, and detect the expression level of TNF-α in macrophages by flow cytometry.

[0034] Changes in the proportion and number of macrophages are as follows Figure 2 As shown in Figures E and F, the proportion and number of macrophages in the skin of AGA mice were increased. The expression level of TNF-α in macrophages was detected as follows: Figure 2 As shown in G and H, macrophages in the skin of AGA mice exhibit elevated levels of TNF-α expression.

[0035] IV. Application of Targeted Antibodies in the Preparation of Drugs for the Treatment of AGA 1. Intradermal injection of anti-TCRγδ antibody / anti-CSF1R antibody / anti-TNF-α antibody for the treatment of AGA 1) Intradermal injection of anti-TCRγδ antibody for the treatment of AGA On days 2, 1, and 1 of the AGA model mouse model, mice were intradermally injected with anti-TCRγδ antibody (purchased from BioXcell, Cat:BE0070, Clone:UC7-13D5, Armenian Hamster IgG,κ) in their backs for three consecutive days. The antibody was then administered every other day until day 21, at a dose of 10 mg / kg, for a total of 13 administrations. Simultaneously, AGA model mice injected with an equal volume of PBS served as the AGA negative control (AGA+PBS), and male C57BL / 6J wild-type mice injected with an equal volume of PBS served as the blank control (control group+PBS).

[0036] As shown in Figure 3A, after treatment with anti-TCRγδ antibody, nearly 60% of the skin on the back of AGA model mice turned black, suggesting that intradermal injection of anti-TCRγδ antibody can significantly alleviate AGA symptoms.

[0037] 2) Intradermal injection of anti-CSF1R antibody for the treatment of AGA On days 2, 1, and 1 of the AGA model mouse model, mice were intradermally injected with anti-CSF1R antibody (purchased from BioXcell, Cat:BE0213, Clone:AFS98, Rat IgG2a, κ) in their backs for three consecutive days. The antibody was then administered every other day until day 21, at a dose of 25 mg / kg, for a total of 13 administrations. Simultaneously, AGA model mice injected with an equal volume of PBS served as the AGA negative control (AGA+PBS), and male C57BL / 6J wild-type mice injected with an equal volume of PBS served as the blank control (control group+PBS).

[0038] As shown in Figure 3A, after treatment with anti-CSF1R antibody, nearly 50% of the skin on the back of AGA model mice turned black, suggesting that intradermal injection of anti-CSF1R antibody can effectively alleviate AGA symptoms.

[0039] 3) Intradermal injection of anti-TNF-α antibody for the treatment of AGA From day 1 of AGA model mouse establishment, anti-TNF-α antibody (purchased from BioXcell, Cat:BE0058, Clone:XT3.11, Rat IgG1) was injected intradermally into the back of AGA model mice, once every other day until day 21, with each dose being 20 mg / Kg, for a total of 11 administrations. Simultaneously, AGA model mice injected with an equal volume of PBS served as AGA negative controls (AGA+PBS), and male C57BL / 6J wild-type mice injected with an equal volume of PBS served as blank controls (control group+PBS).

[0040] As shown in Figure 3A, after treatment with anti-TNF-α antibody, nearly 55% of the skin on the back of AGA model mice turned black, suggesting that intradermal injection of anti-TNF-α antibody can effectively alleviate AGA symptoms.

[0041] 2. Hematoxylin-eosin (HE) staining of mouse skin tissue On day 21 of AGA model establishment, mice in each group were sacrificed by cervical dislocation, and 1 cm of skin was harvested from the midline of the back. 0.5 cm samples were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. 4 μm sections were cut and baked in a 60°C oven, then treated in xylene for 15 min to remove paraffin, followed by a second soaking to ensure complete dewaxing. Subsequently, the sections were stained in hematoxylin aqueous solution for 5 min and rinsed with running water for 15 min. After staining, the sections were dehydrated stepwise in 75% and 90% ethanol, 10 min each time. Finally, the sections were immersed in 0.5% ethanol. Stain with eosin for 2 min and rinse with running water for 1 min. After staining, the sections are dehydrated with anhydrous ethanol and then cleared again in xylene for 3 min, repeated once. Then, mount with neutral resin. The mounted sections are observed and photographed under a microscope. Hair growth is assessed using the color of the mouse dorsal skin (pink: hair follicles in the resting phase; black: hair follicles in the anagen phase) combined with HE staining (HE statistical indicators: hair follicle density, percentage of anagen-phase hair follicles, and hair bulb diameter).

[0042] As shown in Figures 3B and 3C, in the control group mice, melanocytes in the skin began to produce melanin, and all hair follicles entered the anagen phase. In the AGA negative control group mice, no melanin was produced, and all hair follicles were in the telogen phase. However, in the three groups of AGA mice treated with anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody, HE staining of the skin showed that melanocytes began to produce melanin, most hair follicles entered the anagen phase, and a small number of hair follicles were in the telogen phase (Figure 3B). Compared with the control group mice, the AGA negative control group mice had significantly lower hair follicle density, a significantly lower proportion of hair follicles in the anagen phase, and a significantly smaller hair bulb diameter. However, after treatment with anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody, compared with the AGA group, the hair follicle density in the anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody treatment groups was significantly higher, the proportion of hair follicles in the anagen phase was significantly higher, and the hair bulb diameter was also significantly larger (Figure 3C). These results suggest that intradermal injection of anti-TCRγδ antibody, anti-CSF1R antibody, and anti-TNF-α antibody can effectively alleviate AGA.

[0043] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of anti-TCRγδ antibody, anti-CSF1R antibody and / or anti-TNF-α antibody in the preparation of drugs for treating androgenetic alopecia.

2. The application according to claim 1, characterized in that, The dosage of the anti-TCRγδ antibody is 10 mg / Kg, and the dosing frequency is once every other day after 3 consecutive days of administration.

3. The application according to claim 1, characterized in that, The dosage of the anti-CSF1R antibody is 25 mg / kg, and the dosing frequency is once every other day.

4. The application according to claim 1, characterized in that, The dosage of the anti-TNF-α antibody is 20 mg / kg, and the dosing frequency is once every other day.

5. The application according to claim 1, characterized in that, The drug is an intradermal injection.

6. The application according to claim 5, characterized in that, The intradermal injection solution uses phosphate buffer as a solvent.

7. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

8. The application according to claim 7, characterized in that, The carrier includes one or more of solvents, polymers, and liposomes; the excipients include one or more of diluents, excipients, and stabilizers.

9. The application according to claim 1, characterized in that, The medications also include other medications for treating androgenetic alopecia.

10. A composition for treating androgenetic alopecia, characterized in that, This includes any combination of anti-TCRγδ antibodies, anti-CSF1R antibodies, anti-TNF-α antibodies, and other medications used to treat androgenetic alopecia.

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