Depilatory compounds, animal depilation models and methods of making and using the same

Abstract

This application relates to the field of biomedical technology, specifically disclosing a hair removal compound, an animal hair removal model, its preparation method, and its applications. The hair removal compound is a conjugate comprising interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue. The hair removal compound in this application forms a conjugate by coupling interferon IFNγ with an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue. Utilizing the targeting effect of the antibody, interferon IFNγ is delivered to the designated area. Combined with hot compresses to the skin, the immune tolerance of hair follicle stem cells or hair bulb / dermal papilla tissue is disrupted, thereby achieving temporary or permanent hair removal. This also simulates human hair diseases such as alopecia areata and scarring alopecia, obtaining corresponding animal models that can be used for drug screening and efficacy testing.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a hair removal compound, an animal hair removal model, its preparation method and application. Background Technology

[0002] There are various methods for hair removal, mainly divided into temporary and permanent methods. Currently, temporary hair removal methods include shaving, plucking, using depilatory creams, hot waxing, and beeswaxing. Shaving may damage the skin's surface and, because it doesn't directly reach the hair follicle, may leave hair residue, resulting in poor removal. Depilatory creams contain many chemicals that may irritate the skin. Plucking, hot waxing, and beeswaxing are quite painful and can easily cause redness, swelling, and allergic reactions. Permanent hair removal mainly uses laser or electrolysis methods, requiring repeated treatments with sufficient time intervals between each session to achieve permanent hair removal. This process is lengthy and may cause skin inflammation or discomfort.

[0003] Alopecia areata and (primary) cicatricial alopecia areata are naturally occurring human hair disorders. Their pathogenesis involves the autoimmune system attacking different parts of the hair follicle: attacks on the hair bulb / dermal papilla tissue are reversible, allowing the follicle to recover, resulting in alopecia areata, affecting approximately 0.2-2% of the population; attacks on hair follicle stem cells are irreversible, preventing follicle recovery, resulting in (primary) cicatricial alopecia, affecting an even smaller population. Currently, there is a lack of animal models simulating alopecia areata and (primary) cicatricial alopecia, hindering corresponding drug screening or efficacy validation.

[0004] Therefore, existing technologies still need improvement. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this application is to provide a hair removal compound that aims to solve the problems of discomfort or long treatment time of existing hair removal methods.

[0006] The technical solution of this application is as follows:

[0007] A first aspect of this application provides a hair removal compound, the hair removal compound being a conjugate comprising interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue, wherein the interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue are conjugated via a linker.

[0008] Optionally, the antibody targeting hair follicle stem cells includes at least one of CD200 antibody, LGR5 antibody, COL17A1 antibody, CD34 antibody, ITGB6 antibody, TNC antibody, ANGPTL7 antibody, EDN2 antibody, CDH13 antibody, SLC1A4 antibody, SLC7A1 antibody, SLC7A11 antibody, LAMB3 antibody, LAMC2 antibody, JAG2 antibody, COMP antibody, and FGF18 antibody; the antibody targeting hair bulb / dermal papilla tissue includes at least one of NCAM1 antibody, CDH3 antibody, WNT5A antibody, RSPO3 antibody, RSPO4 antibody, VCAN antibody, LEFR antibody, SCARA5 antibody, CD133 antibody, FGFR1 antibody, FGFR2 antibody, and CORIN antibody.

[0009] A second aspect of this application provides a method for preparing the hair removal compound of the first aspect of this application, wherein the interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue are coupled by a click chemical reaction.

[0010] Optionally, the method for preparing the hair removal compound includes the following steps: reacting the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue with tetraazine succinimide ester to obtain a first reaction product; reacting the interferon IFNγ with trans-cyclooctene-N-hydroxysuccinimide ester to obtain a second reaction product; and mixing the first reaction product and the second reaction product to react and obtain the hair removal compound.

[0011] A third aspect of this application provides the use of the hair-removing compound of the first aspect of this application in the preparation of an animal hair-removal model.

[0012] Optionally, the animal includes a mouse.

[0013] The fourth aspect of this application provides a method for preparing a mouse hair removal model, comprising the steps of: applying a hot compress to the area of ​​the mouse to be hair removed, and then subcutaneously injecting the hair removal compound of the first aspect of this application into the area of ​​the mouse to be hair removed, repeating the process three times to obtain a mouse hair removal model.

[0014] The fifth aspect of this application provides the use of a mouse hair loss model of the fifth aspect of this application in the screening of drugs for treating alopecia areata and scarring alopecia.

[0015] Optionally, the screening of drugs for treating alopecia areata and scarring alopecia includes drug rescue treatment of the mouse hair loss model; the drugs used in the drug rescue treatment include licorice, astragalus, and honeysuckle, and the mass ratio of licorice, astragalus, and honeysuckle is 5:5:3.

[0016] A sixth aspect of this application provides the use of the hair removal compound of the first aspect of this application in the preparation of hair removal products.

[0017] The beneficial effects of this application: The hair removal compound in this application forms a conjugate by coupling interferon IFNγ with an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue. Utilizing the targeting effect of the antibody, interferon IFNγ is delivered to the designated area. Combined with heat application to the skin, the immune tolerance of hair follicle stem cells or hair bulb / dermal papilla tissue is disrupted, thereby achieving hair removal. This method can target different parts of the hair follicle through antibody selection. When the antibody targets hair follicle stem cells, permanent hair removal can be achieved; when the antibody targets hair bulb / dermal papilla tissue, temporary hair removal can be achieved. Compared with existing laser or electrolysis hair removal technologies, the number of hair removal sessions is significantly reduced, shortening the time required for permanent hair removal, while avoiding skin inflammation or discomfort caused by laser or electrolysis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0019] Figure 1 The preparation method of the hair removal composition provided in the embodiments of this application and the coupling efficiency test graph are as follows:

[0020] a is a schematic diagram of the preparation method of the hair removal composition; b is a synthesis analysis diagram of the hair removal composition; c is a coupling efficiency analysis of the hair removal composition.

[0021] Figure 2 A graph illustrating the effect of different heat treatment temperatures on hair removal in mice, provided in the embodiments of this application:

[0022] a is a schematic diagram of the experimental procedure; b is an observation diagram of the hair-removed area of ​​the mouse; c is a pathological analysis diagram of the skin tissue in the hair-removed area.

[0023] Figure 3 The following is a graph showing the effect of the hair removal compounds anti-NCAM1-IFNγ and anti-CD200-IFNγ provided in the embodiments of this application on the hair removal efficacy in mice:

[0024] a is a schematic diagram of the experimental procedure; b is an observation diagram of the hair-removed area of ​​the mouse; c is a pathological analysis diagram of the skin tissue in the hair-removed area;

[0025] Figure 4 A graph illustrating the drug rescue effect in a mouse hair loss model provided in this application embodiment:

[0026] a is a schematic diagram of the experimental procedure; b is an observation diagram of the hair-removed area in mice; c is an analysis diagram of the area of ​​patchy baldness in the hair-removed area of ​​mice.

[0027] Figure 5 This is a graph showing the drug rescue effect of different traditional Chinese medicine components on a mouse hair loss model, provided in the embodiments of this application.

[0028] Figure 6 The following is a graph showing the effect of the hair removal compounds anti-COL17A1-IFNγ and anti-LGR5-IFNγ provided in the embodiments of this application on the hair removal efficacy in mice:

[0029] a) Observation of the hair-removed area in mice; b) Analysis of the area of ​​patchy baldness in the hair-removed area of ​​mice; c) Pathological analysis of the skin tissue in the hair-removed area. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0031] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0032] This application provides a hair removal compound, specifically, the hair removal compound is a conjugate comprising interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue.

[0033] Antibody-drug conjugates (ADCs) are complex molecules that bind monoclonal antibodies to cytotoxic drugs, primarily composed of monoclonal antibodies, linkers, and cytotoxic substances. Linkers act as the bridge between the antibody and the drug. They can be classified as cleavable or non-cleavable linkers. Cleavable linkers break down under specific intracellular conditions (such as acidic or enzymatic environments), releasing the drug. Non-cleavable linkers, on the other hand, retain the drug linking to the antibody fragment even after antibody degradation, and then exert their effects through other mechanisms. Researchers skilled in this field can also achieve antibody-interferon IFNγ conjugation through similar methods, such as creating fusion proteins via genetic engineering, enabling the expression and production of both antibodies and IFNγ within the same molecule.

[0034] Interferon γ (IFNγ) affects hair follicle growth and function through multiple mechanisms. IFNγ induces hair follicles to enter the catagen phase, a stage in the hair growth cycle. During this process, IFNγ inhibits the expression of key genes in epidermal papillary cells (hDPCs), such as ALP, Axin2, IGF-1, FGF7, and FGF10, by suppressing β-catenin expression, activating GSK-3β, and enhancing the expression of Wnt repressors such as DKK-1 and TGF-β2. The inhibition of these genes leads to the hair follicle entering the catagen phase, thereby affecting hair growth.

[0035] Previous work has shown that intravenous or subcutaneous injection of interferon-IFNγ alone in mice does not induce hair loss, indicating the existence of further protective mechanisms in the body. We found that applying heat to the skin can promote the function of interferon, leading to a skin ulceration reaction. Therefore, combining heat application with the action of interferon-IFNγ may achieve a more effective attack on hair follicles. Antibodies targeting hair follicle stem cells and antibodies targeting hair bulb / dermal papilla tissue can deliver conjugates to hair follicle stem cells or hair bulb / dermal papilla tissue. Combined with 50°C heat application to the skin, this promotes the effect of interferon-IFNγ and inhibits hair growth.

[0036] When hair removal compounds include interferon IFNγ and antibodies targeting hair follicle stem cells, they disrupt the immune immunity of these stem cells, causing the body's immune system to attack them, thus achieving permanent hair removal. This is the pathogenesis of primary hair loss (alopecia areata). Conversely, when hair removal compounds include interferon IFNγ and antibodies targeting the hair bulb / dermal papilla, they disrupt the immune immunity of the hair bulb, causing the body's immune system to attack it, thus achieving reversible hair removal. This is the pathogenesis of alopecia areata (patchy hair loss).

[0037] In some embodiments, the antibody targeting hair follicle stem cells includes at least one of CD200 antibody, LGR5 antibody, COL17A1 antibody, CD34 antibody, ITGB6 antibody, TNC antibody, ANGPTL7 antibody, EDN2 antibody, CDH13 antibody, SLC1A4 antibody, SLC7A1 antibody, SLC7A11 antibody, LAMB3 antibody, LAMC2 antibody, JAG2 antibody, COMP antibody, and FGF18 antibody. Preferably, the antibody targeting hair follicle stem cells is selected from one of CD200 antibody, LGR5 antibody, and COL17A1 antibody.

[0038] Antibodies targeting hair bulb / dermal papilla tissue include at least one of NCAM1 antibody, CDH3 antibody, WNT5A antibody, RSPO3 antibody, RSPO4 antibody, VCAN antibody, LEFR antibody, SCARA5 antibody, CD133 antibody, FGFR1 antibody, FGFR2 antibody, and CORIN antibody. Preferably, the antibody targeting hair bulb / dermal papilla tissue is selected from NCAM1 antibody.

[0039] This application also provides a method for preparing the hair removal compound as described above, wherein interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue are coupled via a click chemical reaction. The mass ratio of interferon IFNγ to the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue is 1:0.1-10, for example, the mass ratio of interferon IFNγ to the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue is 1:0.1, 1:0.5, 1:1, 1:3, 1:5, 1:7, 1:8, or 1:10, etc.

[0040] In some implementations, please refer to Figure 1 The preparation method of the hair removal compound (A) includes the following steps:

[0041] S101. The antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue is reacted with tetraazine succinimide ester to obtain the first reaction product.

[0042] Specifically, the antibody was reacted with tetraazine succinimide ester (NHS-TZ) in carbonate buffer at pH 9.0 at room temperature for 3 hours. The molar ratio of antibody to tetraazine succinimide ester was 1:50 or approximately 1:50. After the reaction, the mixture was diluted with phosphate buffer at pH 7.4, and then subjected to ultrafiltration and centrifugation three times to remove excess impurities.

[0043] S102. Interferon IFNγ is reacted with trans-cyclooctene-N-hydroxysuccinimide ester to obtain the second reaction product.

[0044] Specifically, interferon IFNγ and trans-cyclooctene-N-hydroxysuccinimide ester (NHS-TCO) were reacted in carbonate buffer at pH 9.0 at room temperature for 3 hours. The molar ratio of interferon IFNγ to trans-cyclooctene-N-hydroxysuccinimide ester was 1:20 or approximately 1:20. After the reaction, the mixture was diluted with phosphate buffer at pH 7.4, and then subjected to ultrafiltration and centrifugation three times to remove excess impurities.

[0045] S103. The first reaction product and the second reaction product are mixed to obtain a hair removal compound.

[0046] Specifically, the first reaction product and the second reaction product are mixed and coupled overnight at 4°C. After the coupling reaction is completed, ultrafiltration and centrifugation are performed to purify the coupled compound.

[0047] It should be noted that the preparation method of the hair removal compound in the embodiments of this application is not limited to click chemical reaction or other methods. Figure 1 The preparation method shown in A can be used to prepare a hair removal composition by conjugating interferon IFNγ with an antibody that targets hair follicle stem cells or hair bulb / dermal papilla tissue. This includes fusing the antibody gene with the interferon IFNγ gene through genetic engineering to produce a fusion protein and obtain the hair removal composition.

[0048] This application also provides a hair removal composition, including the hair removal compound described above in this application.

[0049] In some embodiments, the hair removal composition also includes components with synergistic effects.

[0050] This application also provides an application of the hair-removing compound described above in the preparation of an animal hair-removal model. Preferably, the animal hair-removal model includes mice.

[0051] This application also provides a method for preparing a mouse hair loss model, including the following steps:

[0052] S201. Apply heat to the hair removal area of ​​the mouse, and then inject the hair removal compound as described above into the subcutaneous tissue of the hair removal area of ​​the mouse. Repeat this process three times to obtain a mouse hair removal model.

[0053] The temperature of the hot compress treatment is 48-52℃, preferably 50℃. The injection contains 0.1-10μg of interferon IFNγ, for example, the content of interferon IFNγ can be 0.1μg, 0.5μg, 1μg, 2μg, 5μg, 7μg, 8μg or 10μg, etc.

[0054] A mouse hair removal model was established by activating the effect of interferon IFNγ in the hair removal composition through hot compress treatment and injection of the hair removal composition.

[0055] In some implementations, before applying heat to the area to be hair-removed in mice, the area can be plucked. Plucking can induce hair follicle regeneration, which is beneficial for establishing a mouse hair removal model.

[0056] In some embodiments, this application also provides the application of the mouse hair loss model described above in the screening of drugs for treating alopecia areata and scarring alopecia.

[0057] Based on the aforementioned hair removal composition and the preparation method of the mouse hair removal model, a mouse hair removal model with temporary hair removal can be constructed, which can be used to screen drugs for treating alopecia areata; at the same time, a mouse hair removal model with permanent hair removal can also be constructed, which can be used to screen drugs for treating scarring alopecia.

[0058] The mouse hair loss model in this application embodiment can undergo drug rescue treatment. For example, the mouse hair loss model can be orally administered a drug for five consecutive days to observe hair follicle damage. The orally administered drug includes baricitinib or a traditional Chinese medicine composition. Drug rescue treatment can verify the hair loss status (temporary or permanent hair loss) of the mouse hair loss model, and can also be used as a positive control for drug screening experiments, so as to achieve the effect of drug screening by feeding the mouse hair loss model with potential drugs for treating alopecia areata and scarring alopecia.

[0059] In some embodiments, the traditional Chinese medicine composition includes licorice root, astragalus root, and honeysuckle flower. The mass ratio of licorice root, astragalus root, and honeysuckle flower is 5:5:3. Specifically, 5g of licorice root, 5g of astragalus root, and 3g of honeysuckle flower can be added to 250mL of water, boiled for 15 minutes, and then maintained at 85℃ for 30 minutes. The solid components are filtered out, and after cooling, the mixture is administered orally to a mouse hair loss model for 5 consecutive days, and the hair condition of the hair loss area in the mouse hair loss model is observed.

[0060] This application also provides an application of the hair removal compound described above in the preparation of hair removal products.

[0061] The following specific examples will provide further details.

[0062] Unless otherwise specified, all reagents or instruments mentioned in the following examples are commercially available products.

[0063] Example 1

[0064] (1) Take 30 μg of anti-CD200 antibody and 6 μg of tetrazine succinimide ester (NHS-TZ), add them to 200 μL of pH 9.0 carbonate buffer, and react at room temperature for 3 h. Then dilute with pH 7.4 phosphate buffer (total volume of 500 μL each time), ultrafilter and centrifuge three times to obtain the first reaction product.

[0065] (2) Take 30 μg of interferon IFNγ and 6 μg of trans-cyclooctene-N-hydroxysuccinimide ester (NHS-TCO) and add them to 200 μL of pH 9.0 carbonate buffer. Let it react at room temperature for 3 h. Then dilute it with pH 7.4 phosphate buffer (total volume of 500 μL each time), and ultrafilter and centrifuge three times to obtain the second reaction product.

[0066] (3) The first reaction product and the second reaction product were mixed and reacted overnight at 4°C. After purification by ultrafiltration and centrifugation, the hair removal compound anti-CD200-IFNγ was obtained.

[0067] (4) SDS gel electrophoresis was performed on the components before the reaction and the mixture after the coupling reaction to analyze the coupling efficiency. The uncoupled interferon IFNγ had a smaller molecular weight, while the coupled interferon IFNγ had a larger molecular weight. Using this method, the coupling efficiency of interferon IFNγ was determined to be approximately 65%.

[0068] (5) In another method, we first conjugated interferon IFNγ with the fluorescent small molecule FITC by mixing interferon IFNγ molecules and fluorescent small molecule FITC at a molecular ratio of 1:50 and reacting overnight in carbonate buffer at pH 9.0. Then, the interferon IFNγ-FITC conjugate product was further coupled with an anti-CD200 antibody molecule in the aforementioned click chemical conjugation reaction, and the conjugation efficiency was analyzed by SDS gel electrophoresis. The results of fluorescence quantification showed that the conjugation efficiency of interferon IFNγ was approximately 65%.

[0069] Example 2

[0070] Take 30 μg of anti-NCAM1 antibody and 30 μg of interferon IFNγ and prepare the hair removal compound anti-NCAM1-IFNγ using the same method as in Example 1.

[0071] Example 3

[0072] like Figure 2As shown in Figure a, 2-3 month old C57BL / 6 mice were used to induce hair follicle regeneration by plucking the hair in the area to be de-haired. Then, on the 8th, 9th and 10th days of feeding, the area to be de-haired of the mice was treated with heat at 48℃, 50℃ and 52℃ for 5 minutes, respectively. The mice were then fed again after treatment.

[0073] Photos were taken of the areas treated with heat therapy on days 8, 11, 15, 18, 22, and 25 of the rearing period. The results are as follows: Figure 2 As shown in b. From Figure 2 As shown in Figure b, after 5 minutes of heat treatment at 48℃, the mice's hair had significantly resumed growth by day 15. After 5 minutes of heat treatment at 52℃, the mice's skin was damaged and ulcerated. After 5 minutes of heat treatment at 50℃, hair growth was still not obvious by day 25. This indicates that heat treatment between 48-52℃ has a good inhibitory effect on hair regeneration. Figure 2 As can be seen from the data, the 48℃ heat treatment has no significant effect on hair follicles; while the 50℃ heat treatment significantly inhibits hair follicle growth, but cannot eradicate hair follicle stem cells or promote hair follicle regeneration.

[0074] Example 4

[0075] like Figure 3 As shown in Figure a, 2-3 month old C57BL / 6 mice were used, and hair follicle regeneration was induced by plucking the hair from the areas to be dehaired. On days 8, 10, and 12 of feeding, the mice were treated with the hair-removing compound anti-CD200-IFNγ prepared in Example 1 and the hair-removing compound anti-NCAM1-IFNγ prepared in Example 2, respectively. The specific steps included first applying a 50°C heat compress to the skin of the area to be dehaired on the back of the mouse for 5 minutes, and then subcutaneously injecting either the hair-removing compound anti-CD200-IFNγ or the hair-removing compound anti-NCAM1-IFNγ containing 2 μg of interferon IFNγ 1 hour later. The mice were then fed again after treatment.

[0076] The treated hair-removal areas were photographed on days 8, 12, 16, 20, and 25 of the rearing period. The results are as follows: Figure 3 As shown in b, from Figure 3As shown in Figure b, after plucking on day 8, the mice had no hair on their backs. In the group treated with the hair-removing compound anti-CD200-IFNγ, hair gradually grew back over time; however, hair in the subcutaneous injection area had not grown back by day 25, indicating that the mice treated with anti-CD200-IFNγ achieved almost permanent hair removal. In contrast, in the group treated with the hair-removing compound anti-NCAM1-IFNγ, hair gradually grew back over time, with the subcutaneous injection area showing almost complete hair growth by day 20 and complete hair growth by day 25, indicating that the mice treated with anti-NCAM1-IFNγ achieved temporary hair removal.

[0077] Histopathological analysis was performed on the treated hair-removed areas on days 16, 20, and 35. The specific steps included: routine pathological section staining, including sampling, formalin fixation, dehydration, paraffin embedding, pathological sectioning, and hematoxylin-eosin staining. The results are as follows: Figure 3 As shown in c, from Figure 3 As can be seen from Figure c, the skin tissue of mice treated with the hair removal compound anti-NCAM1-IFNγ showed that hair follicles had regrowth by day 35, while the skin tissue of mice treated with the hair removal compound anti-CD200-IFNγ showed no hair follicle growth by day 35.

[0078] It should be noted that although Example 2 demonstrated that applying a 50°C heat treatment for 5 minutes alone could inhibit hair growth, but combined with... Figure 3 As can be seen from Figures b and c, mice treated in the manner described in Example 2 showed obvious alopecia areata after subcutaneous injection of the hair-removing compound anit-CD200-IFNγ 25 days after feeding. Figure 3 (b) Histopathological analysis also shows that the hair follicles have stopped regenerating. Figure 3 (c) This further demonstrates that the hair removal compound has a significant effect on inhibiting hair growth; moreover, the infiltration of inflammatory cells and immune attack on hair follicles were clearly visible in the tissue sections on day 25.

[0079] Example 5

[0080] The mouse hair loss model with temporary hair loss prepared in Example 4 was used for drug rescue testing. Figure 4As shown in Figure a, after obtaining a mouse hair loss model by treating mice with the compound anti-NCAM1-IFNγ, the mouse hair loss models were treated with baricitinib and a traditional Chinese medicine composition, respectively. There were four experimental groups: a control group (no treatment), a positive control group (anti-NCAM1-IFNγ) treated only with the compound anti-NCAM1-IFNγ, a baricitinib experimental group (Baricitinib) treated with the compound anti-NCAM1-IFNγ followed by treatment with the traditional Chinese medicine composition, and a traditional Chinese medicine composition experimental group (GHR) treated with the compound anti-NCAM1-IFNγ followed by treatment with the traditional Chinese medicine composition.

[0081] The treatment with baricitinib involved administering it orally to mice at a dose of 50 mg / kg / day on days 13, 14, 15, 16, and 17. The treatment with the traditional Chinese medicine composition involved adding 5g of licorice root, 5g of astragalus root, and 3g of honeysuckle flower to 250mL of water, boiling for 15 minutes, then maintaining the temperature at 85℃ for 30 minutes. The solid components were then filtered out. The resulting liquid was used as drinking water for the mouse hair loss model on days 13, 14, 15, 16, and 17. On day 18, the hair-loss areas were photographed for observation, and the results were as follows: Figure 4 As shown in b and c.

[0082] from Figure 4 As can be seen from Figures b and c, after treatment with baricitinib and the traditional Chinese medicine composition GHR, the hair loss areas all showed some degree of recovery, and the hairless areas showed a significant reduction. This indicates that the mouse hair loss model obtained by treating mice with the compound anti-NCAM1-IFNγ is temporary hair loss and can be salvaged by medication. It also shows that the traditional Chinese medicine composition provided in the examples has the effect of promoting hair regeneration.

[0083] Furthermore, to verify the effects of different components in the traditional Chinese medicine composition, licorice, astragalus, and honeysuckle were used respectively, and drug rescue treatment was performed using the same method as described above. The results were compared with those of the groups treated with the traditional Chinese medicine composition and baricitinib. Figure 5 As shown. From Figure 5 As can be seen, the area of ​​hairless region was reduced in the groups treated with licorice, astragalus or honeysuckle alone, but the effect was not as good as the group treated with the traditional Chinese medicine composition GHR. This shows that the traditional Chinese medicine composition in the embodiments of this application has a good effect on promoting hair regeneration and can be used as a potential traditional Chinese medicine composition for treating alopecia areata.

[0084] Example 6

[0085] The hair removal compositions anti-LGR5-IFNγ (containing LGR5 antibody) and anti-COL17A1-IFNγ (containing COL17A1 antibody) were prepared using the same method as in Example 1. Mice were then treated with these two hair removal compositions using the same method as in Example 2. The hair-removed areas were observed and photographed on day 25 of feeding, and pathological analysis was performed on days 16 and 20 of feeding. The results are as follows: Figure 6 As shown in a, b, and c. From... Figure 6 As can be seen from Figures a and b, after treating mice with the hair removal compositions anti-LGR5-IFNγ and anti-COL17A1-IFNγ, no significant hair regrowth occurred in the areas to be treated, resulting in large areas of alopecia areata. From... Figure 6 As shown in Figure c, after treating mice with the hair removal compositions anti-LGR5-IFNγ and anti-COL17A1-IFNγ, a significant immune challenge targeting hair follicles was observed on day 16 of feeding, and no obvious hair follicle growth was observed in the skin tissue on day 35 of feeding. These results indicate that the hair removal compositions anti-LGR5-IFNγ and anti-COL17A1-IFNγ have an inhibitory effect on hair growth and can achieve permanent hair removal.

[0086] In summary, the hair removal compound in this application delivers interferon IFNγ to the designated area by conjugating it with an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue. Combined with heat application to the skin, this disrupts the immune tolerance of the hair follicle stem cells or hair bulb / dermal papilla tissue, thereby achieving hair removal. Furthermore, by simulating the mechanisms of alopecia areata and (primary) cicatricial alopecia, targeted antibodies were designed to target different areas of the hair follicle, conjugated with interferon IFNγ. Combined with heat application to the skin, reversible and irreversible hair removal was achieved. Animal models (mouse models) simulating these two diseases were constructed. Finally, a traditional Chinese medicine composition that can effectively rescue hair loss in mouse models was obtained, providing new insights for drug development for the treatment of alopecia areata.

[0087] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A depilatory compound, characterized in that, The hair removal compound is a conjugate comprising interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue. The interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue are conjugated via a click chemical reaction. The antibody targeting hair follicle stem cells is selected from at least one of CD200 antibody, LGR5 antibody, and COL17A1 antibody, and the antibody targeting hair bulb / dermal papilla tissue is selected from NCAM1 antibody. The method for preparing the hair removal compound includes the following steps: The antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue is reacted with tetraazine succinimide ester to obtain the first reaction product; The interferon IFN γ was reacted with trans-cyclooctene-N-hydroxysuccinimide ester to obtain the second reaction product; The first reaction product and the second reaction product are mixed to obtain a hair removal compound.

2. The use of the hair-removing compound of claim 1 in the preparation of an animal hair-removal model.

3. Use according to claim 2, characterized in that, The animals mentioned include mice.

4. A method for preparing a mouse alopecia model, characterized by, Including the following steps: The mouse hair removal model was obtained by applying a hot compress to the hair removal area and then subcutaneously injecting the hair removal compound of claim 1 into the hair removal area. This process was repeated three times.

5. The application of the mouse hair loss model of claim 4 in the screening of drugs for treating alopecia areata and scarring alopecia.

6. Use according to claim 5, characterized in that, The screening of drugs for treating alopecia areata and scarring alopecia includes drug rescue treatment of the mouse hair loss model; The drugs used in the drug rescue treatment include baricitinib or a traditional Chinese medicine composition; The traditional Chinese medicine composition includes licorice, astragalus, and honeysuckle, with the mass ratio of licorice, astragalus, and honeysuckle being 5:5:

3.

7. The use of the hair removal compound of claim 1 in the preparation of hair removal products.

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