Depilation compound, animal depilation model and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an unhairing compound, an animal unhairing model and a preparation method and application of the unhairing compound. Wherein the unhairing compound is a conjugate, and the conjugate comprises interferon IFN gamma and an antibody targeting hair follicle stem cells or hair bulb / hair papilla tissues. According to the unhairing compound, the interferon IFN gamma and the antibody targeting the hair follicle stem cells or the hair bulbs / hair papilla tissues are coupled to form a conjugate, the interferon IFN gamma is delivered to a designated area by means of the targeting effect of the antibody targeting the hair follicle stem cells or the hair bulbs / hair papilla tissues, and hot compress is carried out on the skin, so that the unhairing effect of the unhairing compound is improved. According to the method, the immune tolerance of hair follicle stem cells or hair bulb / hair papilla tissues is broken, temporary unhairing or permanent unhairing is further achieved, simulation of human hair disease alopecia areata and scar alopecia is achieved, and a corresponding animal model is obtained and can be used for screening of therapeutic drugs and drug efficacy testing.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a depilatory compound, an animal depilatory model, and a preparation method and application thereof. Background Art

[0002] There are many different methods of hair removal, which are mainly divided into temporary hair removal and permanent hair removal. At present, temporary hair removal methods include shaving, plucking, depilatory cream depilatory, hot wax depilatory, beeswax depilatory, etc. Among them, shaving may cause damage to the surface of the skin, and because it cannot directly contact the roots of the hair, there are also residual hairs, resulting in poor depilatory effects; depilatory creams contain more chemical ingredients, which may irritate the skin; plucking, hot wax depilatory and beeswax depilatory are very painful and can easily cause skin redness, swelling and allergic reactions. Permanent hair removal is mainly performed by laser or electrolysis, which requires repeated operations, and each depilation requires a certain time interval to finally achieve permanent hair removal. The depilatory cycle is long and may cause skin inflammation or discomfort.

[0003] Alopecia areata and (primary) scarring alopecia are naturally occurring human hair diseases. Their mechanism of occurrence is the attack of the autoimmune system on different parts of the hair follicles: the attack on the hair bulb / pillary tissue is reversible, and the hair follicles can recover, becoming alopecia areata, involving about 0.2-2% of the population; the attack on the hair follicle stem cells is irreversible, and the hair follicles cannot recover, becoming (primary) scarring alopecia, involving even fewer people. At present, there is still a lack of animal models that simulate alopecia areata and (primary) scarring alopecia, which is not convenient for carrying out corresponding drug screening or efficacy verification.

[0004] Therefore the prior art still needs to be improved. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a hair removal compound, aiming to solve the problems of physical discomfort or long cycle in the existing hair removal methods.

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

[0007] In a first aspect of the present application, a depilatory compound is provided, which is a conjugate comprising interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue, wherein the interferon IFNγ is coupled to the antibody targeting hair follicle stem cells or hair bulb / dermal papilla tissue via a linker.

[0008] Optionally, the antibodies targeting hair follicle stem cells include 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 antibodies targeting hair bulb / hair 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.

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

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

[0011] The third aspect of the present application provides a use of the depilatory compound of the first aspect of the present application in preparing an animal depilatory model.

[0012] Optionally, the animal comprises a mouse.

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

[0014] The fifth aspect of the present application provides an application of the mouse hair loss model of the fifth aspect of the present application in screening drugs for treating alopecia areata and cicatricial alopecia.

[0015] Optionally, the screening of drugs for treating alopecia areata and cicatricial alopecia includes performing drug rescue treatment on 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] The sixth aspect of the present application provides a use of the depilatory compound of the first aspect of the present application in the preparation of a depilatory product.

[0017] Beneficial effects of the present application: The depilatory compound in the present application forms a conjugate by coupling interferon IFNγ and antibodies targeting hair follicle stem cells or hair bulb / hair papilla tissue. By means of the targeting effect of antibodies targeting hair follicle stem cells or hair bulb / hair papilla tissue, interferon IFNγ is delivered to the designated area, and combined with hot compresses on the skin, the immune tolerance of hair follicle stem cells or hair bulb / hair papilla tissue is destroyed, thereby achieving the depilatory effect. This method can target different parts of the hair follicles through the selection of antibodies. When the antibodies target hair follicle stem cells, permanent hair removal can be achieved, and when the antibodies target hair bulb / hair papilla tissue, temporary hair removal can be achieved. Compared with laser hair removal or electrolysis hair removal in the prior art, the number of hair removal times is significantly reduced, shortening the time required for permanent hair removal, and at the same time, it can avoid skin inflammation or discomfort caused by laser hair removal or electrolysis hair removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 The preparation method of the depilatory composition provided in the examples of the present application and the coupling efficiency detection diagram:

[0020] a is a schematic diagram of the process of preparing the depilatory composition; b is a diagram of the synthesis analysis of the depilatory composition; c is an analysis of the coupling efficiency of the depilatory composition;

[0021] Figure 2 This is an analysis chart of the effects of different hot compress treatment temperatures on the hair removal effect on mice provided in the examples of this application:

[0022] a is a schematic diagram of the experimental process; b is an observation diagram of the mouse depilatory area; c is a pathological analysis diagram of the skin tissue in the depilatory area;

[0023] Figure 3 This is an analysis diagram of the effects of the depilatory compounds anti-NCAM1-IFNγ and anti-CD200-IFNγ on the depilatory effect of mice provided in the examples of this application:

[0024] a is a schematic diagram of the experimental process; b is an observation diagram of the mouse depilatory area; c is a pathological analysis diagram of the skin tissue in the depilatory area;

[0025] Figure 4 This is a diagram analyzing the drug rescue effect of the mouse hair loss model provided in the examples of this application:

[0026] a is a schematic diagram of the experimental process; b is an observation diagram of the mouse hair loss area; c is an analysis diagram of the alopecia areata area in the mouse hair loss area;

[0027] Figure 5 This is an analysis diagram of the drug rescue effect of different traditional Chinese medicine components provided in the examples of the present application on the mouse hair loss model;

[0028] Figure 6 This is an analysis diagram of the effects of the depilatory compounds anti-COL17A1-IFNγ and anti-LGR5-IFNγ on the depilatory effects of mice provided in the examples of this application:

[0029] a is the observation picture of the mouse depilatory area; b is the analysis picture of the alopecia areata area in the mouse depilatory area; c is the pathological analysis picture of the skin tissue in the depilatory area. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0031] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0032] The embodiment of the present application provides a depilatory compound. Specifically, the depilatory compound is a conjugate, including interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue.

[0033] The conjugate is an antibody-drug conjugate (ADC), which is a complex molecule that combines a monoclonal antibody with a cytotoxic drug. It is mainly composed of a monoclonal antibody, a linker, and a cytotoxic substance. The linker is a bridge connecting the antibody and the drug. It can be divided into cleavable linkers and non-cleavable linkers. Cleavable linkers can break under specific conditions in the cell (such as acidic environment, enzymatic environment, etc.) to release the drug. Non-cleavable linkers are when the antibody is degraded, the drug is still connected to the antibody fragment, and then exerts its effect through other mechanisms. Researchers familiar with this field can also achieve the coupling of antibodies and interferon IFNγ through other similar methods, such as making fusion proteins through genetic engineering to achieve the expression and production of antibodies and interferon IFNγ in the same molecule.

[0034] Interferon IFNγ affects the growth and function of hair follicles through multiple mechanisms. IFNγ can induce hair follicles to enter the catagen phase, which is a stage in the hair growth cycle. In this process, IFNγ inhibits the expression of some key genes in epidermal papilla cells (hDPCs), such as ALP, Axin2, IGF-1, FGF 7, and FGF10, by inhibiting the expression of β-catenin, activating GSK-3β, and enhancing the expression of Wnt inhibitors such as DKK-1 and TGF-β2. The inhibition of these genes causes hair follicles to enter the catagen phase, which in turn affects hair growth.

[0035] Previous work has shown that intravenous or subcutaneous injection of interferon IFNγ alone in mice does not cause hair loss, indicating that the body has further protective mechanisms. We found that applying heat to the skin can promote the function of interferon and lead to skin ulcer reactions. Therefore, by combining heat and the effects of interferon IFNγ, it is expected to achieve a more effective attack on hair follicles. Antibodies targeting hair follicle stem cells and antibodies targeting hair bulb / hair papilla tissue can deliver the conjugate to hair follicle stem cells or hair bulb / hair papilla tissue, combined with 50°C heat application to the skin, to promote the function of interferon IFNγ and inhibit hair growth.

[0036] When interferon IFNγ and antibodies targeting hair follicle stem cells are included in the depilatory compound, the immune immunity of the hair follicle stem cells is destroyed, causing the body's immune system to attack the hair follicle stem cells, thereby achieving permanent hair removal, which is also the pathogenesis of the human hair disease (primary) cicatricial alopecia. When interferon IFNγ and antibodies targeting hair bulb / hair papilla tissue are included in the depilatory compound, the immune immunity of the hair bulb is destroyed, causing the body's immune system to attack the hair bulb / hair papilla tissue, thereby achieving reversible hair removal, which is also the pathogenesis of the human hair disease alopecia areata.

[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] The antibodies targeting the 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 antibodies targeting the hair bulb / dermal papilla tissue are selected from NCAM1 antibody.

[0039] The present application also provides a method for preparing the depilatory compound as described above, wherein interferon IFNγ and an antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue are coupled by click chemistry reaction. The mass ratio of interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue is 1:0.1-10, for example, the mass ratio of interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / hair 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 embodiments, see Figure 1 In A, the method for preparing the depilatory compound comprises the steps of:

[0041] S101, reacting an antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue with tetrazine succinimide ester to obtain a first reaction product.

[0042] Specifically, the antibody and tetrazine succinimide ester (NHS-TZ) are reacted in a carbonate buffer at pH 9.0 at room temperature for 3 hours. The molar ratio of the antibody to tetrazine succinimide ester is 1:50 or about 1:50. After the reaction, it is diluted with a phosphate buffer at pH 7.4, and ultrafiltration centrifuged three times to remove excess impurities.

[0043] S102, reacting interferon IFNγ with trans-cyclooctene-N-hydroxysuccinimide ester to obtain a second reaction product.

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

[0045] S103, mixing the first reaction product and the second reaction product, and reacting them to obtain a depilatory compound.

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

[0047] It should be noted that the preparation method of the depilatory compound in the embodiment of the present application is not limited to click chemistry reaction or Figure 1 The preparation method shown in A can prepare a depilatory composition by coupling interferon IFNγ with an antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue, including fusing the antibody gene with the interferon IFNγ gene by genetic engineering and producing it in the form of a fusion protein to obtain a depilatory composition.

[0048] The present application also provides a depilatory composition, comprising the depilatory compound described above in the present application.

[0049] In some embodiments, the depilatory composition further comprises ingredients having synergistic effects.

[0050] The present application also provides a use of the depilatory compound as described above in preparing an animal depilatory model. Preferably, the animal depilatory model includes mice.

[0051] The present application also provides a method for preparing a mouse hair loss model, comprising the steps of:

[0052] S201, applying heat to the part of the mouse to be depilated, and then subcutaneously injecting the depilatory compound as described above into the part of the mouse to be depilated, and repeating this process three times to obtain a mouse depilatory model.

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

[0054] The effect of interferon IFNγ in the depilatory composition was activated by hot compress treatment and injection of the depilatory composition, and a mouse depilatory model was constructed.

[0055] In some embodiments, before applying heat to the area to be depilated, the area to be depilated may be plucked. Plucking can induce hair follicle regeneration, which is beneficial to the establishment of a mouse depilation model.

[0056] In some embodiments, the examples of the present application also provide a use of the mouse hair loss model as described above in the screening of drugs for treating alopecia areata and scarring alopecia.

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

[0058] The mouse depilation model in the embodiment of the present application can be subjected to drug rescue treatment. For example, the mouse depilation model is orally administered with drugs, and the hair follicle damage can be observed for five consecutive days. The oral drugs include baricitinib or a Chinese medicine composition. The depilation condition (temporary depilation or permanent depilation) of the mouse depilation model can be verified by drug rescue treatment, and it 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 depilation model with potential drugs for the treatment of alopecia areata and scarring alopecia.

[0059] In some embodiments, the Chinese medicine composition includes licorice, astragalus, and honeysuckle. The mass ratio of licorice, astragalus, and honeysuckle is 5:5:3. Specifically, 5g of licorice, 5g of astragalus, and 3g of honeysuckle can be added to 250mL of water, boiled for 15min, then maintained at 85°C for 30min, filtered to remove solid components, and then fed orally to a mouse depilatory model after cooling for 5 consecutive days, and the hair condition of the depilatory part of the mouse depilatory model was observed.

[0060] The present application also provides an application of the depilatory compound as described above in the preparation of a depilatory product.

[0061] The following is further described by means of specific examples.

[0062] The reagents or instruments in the following examples without indicating the manufacturer are all conventional products that can be obtained commercially.

[0063] Example 1

[0064] (1) 30 μg of anti-CD200 antibody and 6 μg of tetrazine succinimide ester (NHS-TZ) were added to 200 μL of pH 9.0 carbonate buffer and reacted at room temperature for 3 h. The mixture was then diluted with pH 7.4 phosphate buffer (total volume of each time was 500 μL) and ultrafiltrated and centrifuged three times to obtain a first reaction product.

[0065] (2) 30 μg of interferon IFNγ and 6 μg of trans-cyclooctene-N-hydroxysuccinimide ester (NHS-TCO) were added to 200 μL of pH 9.0 carbonate buffer, and reacted at room temperature for 3 h. The mixture was then diluted with pH 7.4 phosphate buffer (total volume of 500 μL each time), and ultrafiltration and centrifugation were performed three times to obtain a second reaction product.

[0066] (3) The first reaction product and the second reaction product were mixed, placed at 4° C. for overnight reaction, and purified by ultrafiltration and centrifugation to obtain the depilatory compound anti-CD200-IFNγ.

[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 molecular weight of the uncoupled interferon IFNγ is small, while the molecular weight after coupling is large. Through this method, it was determined that the coupling efficiency of interferon IFNγ was about 65%.

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

[0069] Example 2

[0070] 30 μg of anti-NCAM1 antibody and 30 μg of interferon IFNγ were taken and the same method as in Example 1 was used to prepare the hair removal compound anti-NCAM1-IFNγ.

[0071] Example 3

[0072] like Figure 2As shown in a, 2-3 month old C57BL / 6 mice were selected and the hair removal area was plucked to induce hair follicle regeneration; then on the 8th, 9th and 10th days of feeding, the hair removal area of ​​the mice was subjected to hot compress treatment at 48°C, 50°C and 52°C for 5 minutes respectively, and the mice were continued to be fed after treatment.

[0073] The hot compress-treated areas were photographed on the 8th, 11th, 15th, 18th, 22nd, and 25th days of feeding. Figure 2 As shown in b. Figure 2 As can be seen in Figure b, after 5 minutes of hot compress treatment at 48°C, the hair of the mice had obviously resumed growing on the 15th day. After 5 minutes of hot compress treatment at 52°C, the mice's skin was damaged and ulcerated. After 5 minutes of hot compress treatment at 50°C, the hair of the mice still did not grow significantly on the 25th day. This further indicates that hot compress treatment between 48-52°C has a good inhibitory effect on hair regeneration. Figure 2 As can be seen from Figure c, 48°C hot compress treatment has no significant effect on hair follicles; while 50°C hot compress treatment significantly inhibits hair follicle growth, but cannot eradicate hair follicle stem cells and hair follicle regeneration.

[0074] Example 4

[0075] like Figure 3 As shown in a, 2-3 month old C57BL / 6 mice were taken and the hair removal area was plucked to induce hair follicle regeneration. On the 8th, 10th and 12th days of feeding, the hair removal compound anti-CD200-IFNγ prepared in Example 1 and the hair removal compound anti-NCAM1-IFNγ prepared in Example 2 were used for treatment, respectively. The specific steps included first applying a 50°C hot compress to the skin of the hair removal area on the back of the mouse for 5 minutes, and then subcutaneously injecting a hair removal compound anti-CD200-IFNγ or a hair removal compound anti-NCAM1-IFNγ containing 2 μg of interferon IFNγ after 1 hour of hot compress treatment. After treatment, the mice continued to be raised.

[0076] The treated hair loss areas were photographed on the 8th, 12th, 16th, 20th and 25th days of feeding. Figure 3 As shown in b, from Figure 3As can be seen in b, after the hair plucking treatment on the 8th day, the back area of ​​the mouse was hairless. The mouse group treated with the hair removal compound anti-CD200-IFNγ gradually grew hair over time, but the hair in the subcutaneous injection area had not grown on the 25th day, indicating that the mice treated with the hair removal compound anti-CD200-IFNγ almost achieved permanent hair removal. In the mouse group treated with the hair removal compound anti-NCAM1-IFNγ, the hair gradually grew over time, and the subcutaneous injection area had basically grown completely on the 20th day, and had grown completely on the 25th day, indicating that the mice treated with the hair removal compound anti-NCAM1-IFNγ achieved the effect of temporary hair removal.

[0077] On the 16th, 20th and 35th days of feeding, the treated hair loss areas were subjected to histopathological analysis. The specific steps included routine pathological section staining, including sampling, formalin fixation, dehydration, paraffin embedding, pathological sectioning and hematoxylin-eosin staining. The results were as follows: Figure 3 As shown in c, from Figure 3 As can be seen in middle c, the skin tissue of mice treated with the depilatory compound anti-NCAM1-IFNγ had regrown hair follicles on the 35th day, while the skin tissue of mice treated with the depilatory compound anti-CD200-IFNγ had no hair follicle growth on the 35th day.

[0078] It should be noted that although Example 2 proves that only using 50°C hot compress for 5 minutes has the effect of inhibiting hair growth, Figure 3 As can be seen from b and c, after 25 days of feeding, the mice treated in the manner of Example 2 were subcutaneously injected with the hair removal compound anit-CD200-IFNγ and obvious alopecia were observed with the naked eye ( Figure 3 b), histopathological analysis also shows that the hair follicles have not regenerated ( Figure 3 c), which further indicates that the depilatory compound has a significant effect of inhibiting hair growth; and infiltration of inflammatory cells and immune attack on hair follicles are clearly visible in the tissue sections on the 25th day.

[0079] Example 5

[0080] The mouse hair loss model with temporary hair loss prepared in Example 4 was used for drug rescue test. Figure 4As shown in a, after the mouse was treated with the compound anti-NCAM1-IFNγ to obtain a mouse hair loss model, the mouse hair loss model was treated with baricitinib and a Chinese medicine composition, respectively. An untreated control group (control), 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 and then treated with baricitinib, and a Chinese medicine composition experimental group (GHR) treated with the compound anti-NCAM1-IFNγ and then treated with a Chinese medicine composition were set up.

[0081] The steps of baricitinib treatment are: orally administer baricitinib to mice at a dose of 50 mg / kg / day on the 13th, 14th, 15th, 16th, and 17th days of feeding. The steps of the Chinese medicine composition treatment are: add 5g licorice, 5g astragalus, and 3g honeysuckle to 250mL water, boil for 15 minutes, then maintain at 85°C for 30 minutes, filter to remove solid components, and use the obtained Chinese medicine composition liquid as drinking water for the mouse hair loss model on the 13th, 14th, 15th, 16th, and 17th days of feeding, and continue feeding. On the 18th day of feeding, the hair loss area was photographed and observed, and the results are as follows Figure 4 As shown in b and c.

[0082] from Figure 4 As can be seen from b and c, after treatment with baricitinib and the Chinese medicine composition GHR, the hair loss areas have recovered to a certain extent, and the hairless areas have been significantly reduced, which further indicates that the mouse hair loss model obtained by treating mice with the compound anti-NCAM1-IFNγ is temporary hair loss and can be rescued by drugs; it also indicates that the Chinese medicine composition provided in the example has the effect of promoting hair regeneration.

[0083] Furthermore, in order to verify the effects of different components in the Chinese medicine composition, licorice, astragalus, and honeysuckle were used respectively, and the same method as above was used for drug rescue treatment, and compared with the group treated with the Chinese medicine composition and baricitinib. The results are as follows Figure 5 As shown. Figure 5 It can be seen that the area of ​​the hairless area is reduced in the groups treated with licorice, astragalus or honeysuckle alone, but the effect is not as good as the group treated with the Chinese medicine composition GHR for drug rescue. This further shows that the Chinese medicine composition in the embodiment of the present application has a good effect of promoting hair regeneration and can be used as a potential Chinese medicine composition for the treatment of alopecia areata.

[0084] Example 6

[0085] The depilatory composition anti-LGR5-IFNγ of LGR5 antibody and the depilatory composition anti-COL17A1-IFNγ of COL17A1 antibody were prepared in the same manner as in Example 1, and the depilatory composition anti-LGR5-IFNγ and the depilatory composition anti-COL17A1-IFNγ were used to treat mice in the same manner as in Example 2. The depilatory area was observed and photographed on the 25th day of feeding, and pathological analysis was performed on the 16th and 20th days of feeding. The results are as follows: Figure 6 As shown in a, b, and c. Figure 6 As can be seen in a and b, after the mice were treated with the hair removal composition anti-LGR5-IFNγ and the hair removal composition anti-COL17A1-IFNγ, the hair in the hair removal area did not grow significantly, and the area of ​​alopecia areata was large. Figure 6 As can be seen in Figure c, after the mice were treated with the hair removal composition anti-LGR5-IFNγ and the hair removal composition anti-COL17A1-IFNγ, an obvious immune attack on the hair follicles was observed on the 16th day of feeding, and no obvious hair follicles grew in the skin tissue on the 35th day of feeding. The above results indicate that the hair removal composition anti-LGR5-IFNγ and the hair removal composition anti-COL17A1-IFNγ have the effect of inhibiting hair growth and can achieve permanent hair removal.

[0086] In summary, the depilatory compound in the present application forms a conjugate by coupling interferon IFNγ and antibodies targeting hair follicle stem cells or hair bulb / hair papilla tissue, and delivers interferon IFNγ to a designated area by means of the targeting effect of antibodies targeting hair follicle stem cells or hair bulb / hair papilla tissue, and combines hot compresses on the skin to destroy the immune tolerance of hair follicle stem cells or hair bulb / hair papilla tissue, thereby achieving a depilatory effect. In addition, by simulating the occurrence mechanism of alopecia areata and (primary) cicatricial alopecia, targeted antibodies are designed to target different areas of hair follicles, coupled with interferon IFNγ, and hot compresses are applied to the skin to achieve reversible and irreversible depilation, and an animal model (mouse model) simulating these two diseases is constructed, and finally a Chinese medicine composition that can effectively rescue the mouse depilatory model is obtained, providing new ideas for the development of drugs for the treatment of alopecia areata.

[0087] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A depilatory compound, characterized in that The depilatory compound is a conjugate, which includes interferon IFNγ and an antibody targeting hair follicle stem cells or an antibody targeting hair bulb / hair papilla tissue, wherein the interferon IFNγ is coupled to the antibody targeting hair follicle stem cells or the antibody targeting hair bulb / hair papilla tissue via a linker.

2. The depilatory compound according to claim 1, characterized in that 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 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.

3. A method for preparing a depilatory compound according to claim 1 or 2, characterized in that: The interferon IFNγ and the antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue are coupled by click chemistry reaction.

4. The method for preparing a depilatory compound according to claim 3, characterized in that: The method for preparing the depilatory compound comprises the steps of: reacting the antibody targeting hair follicle stem cells or hair bulb / hair papilla tissue with tetrazine succinimide ester to obtain a first reaction product; reacting the interferon IFNγ with trans-cyclooctene-N-hydroxysuccinimide ester to obtain a second reaction product; The first reaction product and the second reaction product are mixed and reacted to obtain a depilatory compound.

5. Use of the depilatory compound according to claim 1 or 2 in preparing an animal depilatory model.

6. The use according to claim 5, characterized in that: The animals include mice.

7. A method for preparing a mouse hair loss model, characterized in that: Includes steps: The mouse hair removal part is subjected to hot compress treatment, and then the hair removal compound according to claim 1 or 2 is subcutaneously injected into the mouse hair removal part, and this is repeated three times to obtain a mouse hair removal model.

8. Use of the mouse hair loss model according to claim 7 in screening drugs for treating alopecia areata and cicatricial alopecia.

9. The use according to claim 8, characterized in that: The screening of drugs for treating alopecia areata and cicatricial alopecia includes performing drug rescue treatment on the mouse hair loss model; The drug used in the drug rescue treatment includes baricitinib or a traditional Chinese medicine composition; The traditional Chinese medicine composition comprises liquorice, astragalus and honeysuckle, and the mass ratio of the liquorice, astragalus and honeysuckle is 5:5:

3.

10. Use of the depilatory compound according to claim 1 or 2 in the preparation of depilatory products.

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