Use of uap1 as a target in the preparation of a medicament for treating alopecia

Abstract

This invention relates to the field of biomedicine, disclosing the application of UAP1 as a target in the preparation of drugs for treating hair loss. This invention discovers that UAP1 is highly expressed in hair follicles in the bald areas of AGA patients, and can serve as a therapeutic target for hair loss. Simultaneously, this invention applies agents that reduce UAP1 expression to hair follicles, finding that downregulating UAP1 expression increases the diameter of the hair bulb, enhances follicle activity, prolongs the anagen phase, and delays the catagen phase. This invention is the first to discover that UAP1 can serve as a therapeutic target for hair loss, and designs corresponding nucleic acid molecules capable of reducing UAP1 expression, providing new targets and directions for the development of drugs for hair loss.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of UAP1 as a target in the preparation of drugs for treating hair loss. Background Technology

[0002] Alopecia is a common dermatological condition characterized by abnormal hair loss or thinning. There are several types of alopecia, such as androgenetic alopecia (AGA), alopecia areata, hereditary alopecia, and cicatricial alopecia, with AGA being the most common non-cicatricial form.

[0003] Hair follicles continuously undergo cycles of growth and decay throughout a person's life, making them an ideal model for studying organ regeneration. The hair follicle cycle can be divided into three phases: the anagen (growth) phase, the catagen (transitional) phase, and the telogen (resting) phase. The vitality and cyclical changes of hair follicles directly affect the health of hair. Taking AGA (Advanced Growth Alopecia) as an example, its main characteristics are a shortened anagen phase, a prolonged telogen phase, and gradual miniaturization of the hair follicle. The mechanisms of hair loss are not fully understood, but they involve the joint regulation of multiple genes and related signaling pathways, such as the Wnt / β-catenin, BMP, FGF, and SHH pathways. These pathways play a crucial role in regulating hair growth and hair follicle activity.

[0004] Currently, the only oral medications approved by my country's National Medical Products Administration for the treatment of alopecia include minoxidil and finasteride. Minoxidil has vasodilatory and potassium-channel-opening effects; finasteride is a type II 5α-reductase inhibitor that inhibits the conversion of testosterone to the more active dihydrotestosterone, thereby reducing the inhibitory effect of androgens on hair follicles. However, despite the widespread clinical use of these two drugs, some patients do not respond ideally to their treatments and may experience adverse reactions. Therefore, a deeper understanding of the pathogenesis of alopecia areata (AGA) and the further development of effective treatments and drugs are of significant research value and practical importance.

[0005] In recent years, factors involved in the pathogenesis of acute glycemic allergy (AGA), such as androgen receptor (AR), microinflammation, oxidative stress, and hypoxia-inducible factor, have been found to be related to protein N-glycosylation. N-glycosylation refers to the process by which N-glycans are linked to the amino acid side chains of proteins through glycosidic bonds to form glycoproteins. It is an important post-translational modification of proteins, playing a key role in cell adhesion and migration, interaction with the extracellular matrix, and signal transduction. Urate diphosphate-N-acetylglucosamine pyrophosphorylase 1 (UAP1) is crucial for N-glycosylation modification, catalyzing the conversion of uridine triphosphate and GlcNAc-1-phosphate to UDP-GlcNAc. UDP-GlcNAc is the most primitive substrate for the synthesis of N-glycan precursors, and its availability is the rate-limiting step in N-glycosylation, affecting its efficiency.

[0006] Therefore, studying UAP1 in AGA patients and developing a new target therapy for hair loss in AGA patients is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide an application of UAP1 as a target in the preparation of drugs for treating hair loss, based on the current state of research in the field. This invention utilizes an in vitro human hair follicle culture system to discover that UAP1 is highly expressed in hair follicles in the hair loss areas of AGA patients. Furthermore, reducing UAP1 expression can increase the diameter of the hair bulb and improve hair follicle activity in vitro, while also prolonging the anagen phase and delaying the entry of hair follicles into the catagen phase, thus serving as a new and effective target for hair loss treatment.

[0008] In a first aspect, the present invention provides the application of a reagent that targets UAP1 to reduce UAP1 expression or its protein activity in the preparation of a drug for treating hair loss.

[0009] Optionally, downregulation of UAP1 expression can increase the diameter of the hair bulb, improve hair follicle activity, or prolong the anagen phase of the hair follicle and delay the entry of the hair follicle into the catagen phase.

[0010] Optionally, the reagent for reducing UAP1 expression or its protein activity is selected from: nucleic acid molecules, gene editing vectors, anti-UAP1 antibodies, or homologous recombination vectors containing nucleotide sequences encoding inactive or weakened UAP1 with mutations; further, the gene editing vector is a CRISPR-CAS9 gene editing vector or a TALEN gene editing vector.

[0011] Optionally, the nucleic acid molecule includes siRNA, antisense RNA, or ribozyme.

[0012] Optionally, the sense strand sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand sequence of the siRNA is shown in SEQ ID NO.2.

[0013] Optionally, the hair loss condition is androgenetic alopecia.

[0014] Secondly, the present invention provides a pharmaceutical composition for treating hair loss, employing the following technical solution: A pharmaceutical composition for treating hair loss, the pharmaceutical composition comprising an agent that reduces UAP1 expression or its protein activity, wherein the agent that reduces UAP1 expression or its protein activity is the sole active ingredient or one of the active ingredients of the pharmaceutical composition.

[0015] Optionally, the reagent for reducing UAP1 expression or its protein activity is a siRNA targeting UAP1, the sense strand sequence of which is shown in SEQ ID NO.1, and the antisense strand sequence of which is shown in SEQ ID NO.2.

[0016] Optionally, the pharmaceutical composition may be in any pharmaceutically acceptable dosage form.

[0017] Thirdly, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating hair loss.

[0018] This invention includes at least one of the following beneficial technical effects: 1. This invention found that UAP1 is highly expressed in hair follicles in the alopecia area of ​​AGA patients, proving that it can serve as a target for the treatment of hair loss; 2. This invention applies reagents that reduce UAP1 expression to hair follicles and finds that downregulation of UAP1 expression can increase the diameter of the hair bulb, improve hair follicle activity, prolong the hair follicle growth phase, and delay the entry of hair follicles into the regression phase.

[0019] In summary, this invention provides the UAP1 target and discovers that downregulating UAP1 expression can treat AGA disease, specifically increasing the diameter of the hair bulb, improving hair follicle activity, prolonging the hair follicle growth phase, and delaying the entry of hair follicles into the regression phase. This has great clinical application and economic value. Attached Figure Description

[0020] Figure 1 This is a graph showing the expression levels of UAP1 protein in hair follicles on the top (bald area) and back (non-bald area) of AGA patients in Example 1; Figure 2 This is a graph showing the results of UAP1 protein expression levels in different groups of hair follicles in Example 2; Figure 3These are the results of hair follicle proliferation in different groups in Example 2; Figure 4 The images show HE-stained images (left) and statistical charts (right) of hair bulb diameters in different groups of hair follicles in Example 2. Figure 5 This is a graph showing the proportion of hair follicles in different groups during the hair follicle cycle in Example 2. Figure 5 A shows the morphological structure of hair follicles in the Si-UAP1 group and the NC group on days 0, 2, 4, and 6. Figure 5 B represents the count statistics of each hair follicle in each group on day 6, indicating the stage (retrograde / anagen phase) of the hair cycle. Figure 5 C represents the score for each hair follicle cycle stage. Detailed Implementation

[0021] The following is in conjunction with the appendix Figure 1 -Appendix Figure 5 The present invention will be further described in detail below. The specific embodiments of the present invention are intended to fully demonstrate the technical solution and effects of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent changes or modifications made by those skilled in the art based on the essence of the present invention should be covered within the scope of protection of the present invention.

[0022] Unless otherwise specified, the materials and equipment used in the various embodiments of the present invention are all commercially available products in the art.

[0023] Subjects: The hair follicle samples used in this study were obtained from the top or back of the head of patients who underwent hair transplantation surgery. After obtaining approval from the ethics committee and informed consent from the patients, they were used for subsequent in vitro experiments.

[0024] Example 1 UAP1 is highly expressed in hair follicles in the bald areas of AGA patients. Hair follicles were collected from the top (balding area) and the back (non-balding area) of AGA patients, respectively. The hair follicles were placed in 24-well cell culture plates, with one hair follicle in each well and 500 μL of serum-free William's medium added. The plates were then placed in a cell culture incubator (37°C, 5% CO2) for culture.

[0025] Hair follicles were collected and the expression level of UAP1 protein was confirmed by immunofluorescence staining; the UAP1 antibody used in the immunofluorescence staining was catalog number 67545-1-lg, manufactured by Wuhan Sanying Biotechnology Co., Ltd.

[0026] The results are as follows Figure 1 As shown, the results revealed that the expression of UAP1 protein in the hair follicles of the bald area on the top of AGA patients was higher than that in the hair follicles of the non-bald area on the back of the head.

[0027] Example 2 Effects of downregulating UAP1 protein expression levels on hair follicles 1. In vitro hair follicle tissue culture: Place hair follicles in 24-well cell culture plates, with one hair follicle in each well and add 500 μL of serum-free William's medium. Incubate in a cell culture incubator (37℃, 5% CO2) for culture.

[0028] 2. siRNA sequence design UAP1, Gene ID: 6675; UAP1 siRNA Chain of Justice: 5'-CAAUGAUGUACCAAUCCAA-3' (SEQ ID NO.1); Antonym: 5'-UUGGAUUGGUACAUCAUUG-3' (SEQ ID NO.2); negative control siRNA Chain of Justice: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.3); Antonym: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.4); The above siRNA sequences were all synthesized by Jiman Biotechnology Co., Ltd.

[0029] 3. Hair follicle grouping The in vitro hair follicles were divided into two groups: the control group (NC) and the UAP1 siRNA group (Si-UAP1), with 10 hair follicles in each group.

[0030] 4. siRNA transfection and culture Cell transfection was performed using Lipofectamine RNAiMAX (catalog number: 13778150, Thermo Fisher Scientific Inc.) to achieve an siRNA concentration of 80 nM; the NC group was transfected with negative control siRNA, and the Si-UAP1 group was transfected with UAP1 siRNA.

[0031] The hair follicles were photographed on the day of culture; the culture medium containing the transfection reagent was replaced every two days, and the culture was continued for 6 days.

[0032] 5. Immunofluorescence detection Hair follicles were collected and UAP1 protein expression levels were confirmed by immunofluorescence. The results are as follows: Figure 2As shown in the figure, the results showed that the expression level of UAP1 protein in the Si-UAP1 group was significantly lower than that in the NC group, indicating that transfecting UAP1 siRNA into hair follicles can effectively reduce the expression of UAP1 protein in hair follicles.

[0033] 6. Hair follicle bulb diameter and proliferation detection Hair follicles were cultured in vitro, transfected with siRNA, and fixed 48 hours later. After slide preparation, IF and HE staining were performed, and the diameter of the hair bulb was measured. Results are as follows: Figure 3 and Figure 4 As shown; Figure 3 It can be seen that the proliferative activity of hair follicles in the Si-UAP1 group was higher than that in the NC group, indicating that reducing UAP1 expression can enhance the proliferative capacity of hair follicles. Figure 4 It can be seen that the diameter of the hairball in the Si-UAP1 group was higher than that in the NC group, indicating that reducing UAP1 expression can increase the diameter of the hairball.

[0034] 7. Hair follicle cycle detection and evaluation After 6 days of continuous in vitro culture, the hair follicle's stage of the growth cycle was determined based on its morphology and structure. The number of hair follicles in the anagen and catagen phases was counted, and the differences in hair follicle stages among different groups were analyzed. The results are as follows: Figure 5 As shown in A-5C; Figure 5 A shows the morphological structure of hair follicles in the Si-UAP1 group and the NC group on days 0, 2, 4 and 6; Figure 5 B represents the count statistics of each hair follicle cycle (regression phase / growth phase) in each group on day 6; Figure 5 C represents the score for each hair follicle cycle stage. Follicles in the growth phase are scored as 1.0 points, those in the early regression phase are scored as 1.5 points, those in the middle regression phase are scored as 2.0 points, and those that have fully entered the regression phase are scored as 3.0 points.

[0035] The results showed that more hairs entered the anagen phase in the Si-UAP1 group compared to the NC group, indicating that UAP1 knockdown significantly altered the phase distribution of the hair cell cycle, causing more hair follicles to remain in the anagen phase and fewer hair follicles to be in the catagen phase.

[0036] In summary, this invention provides the UAP1 target and discovers that downregulating UAP1 expression can treat AGA disease, specifically increasing the diameter of the hair bulb, improving hair follicle activity, prolonging the hair follicle growth phase, and delaying the entry of hair follicles into the regression phase. This has great clinical application and economic value.

[0037] It should be noted that this embodiment only uses the siRNA method described above as an example. In fact, other agents that can inhibit UAP1 expression and / or its activity, such as other siRNAs, antisense RNAs, ribozymes and gene editing vectors (such as CRISPR-CAS9 gene editing vectors or TALEN gene editing vectors); anti-UAP1 antibodies; homologous recombination vectors containing nucleotide sequences encoding inactive or weakened UAP1 with mutations, can also achieve the above effects.

[0038] The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

Claims

1. The application of reagents that target UAP1 to reduce UAP1 expression or reduce its protein activity in the preparation of drugs for treating hair loss.

2. The application as described in claim 1, characterized in that, Downregulation of UAP1 expression increases the diameter of the hair bulb, enhances hair follicle activity, prolongs the anagen phase of the hair follicle, and delays the entry of the hair follicle into the catagen phase.

3. The application as described in claim 1, characterized in that, The reagents for reducing UAP1 expression or its protein activity are selected from: nucleic acid molecules, gene editing vectors, anti-UAP1 antibodies, or homologous recombination vectors containing nucleotide sequences encoding inactive or weakened UAP1 with mutations; further, the gene editing vector is a CRISPR-CAS9 gene editing vector or a TALEN gene editing vector.

4. The application as described in claim 3, characterized in that, The nucleic acid molecules include siRNA, antisense RNA, or ribozymes.

5. The application as described in claim 4, characterized in that, The sense strand sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand sequence of the siRNA is shown in SEQ ID NO.

2.

6. Use according to any one of claims 1 to 5, wherein The hair loss condition described is androgenetic alopecia.

7. A pharmaceutical composition for treating alopecia, characterized by, The pharmaceutical composition includes an agent that reduces UAP1 expression or its protein activity, wherein the agent that reduces UAP1 expression or its protein activity is the sole active ingredient or one of the active ingredients of the pharmaceutical composition.

8. The pharmaceutical composition of claim 7, wherein, The reagent for reducing UAP1 expression or its protein activity is a siRNA targeting UAP1, the sense strand sequence of which is shown in SEQ ID NO.1, and the antisense strand sequence of which is shown in SEQ ID NO.

2.

9. The pharmaceutical composition of claim 7, wherein The pharmaceutical composition is in any pharmaceutically acceptable dosage form.

10. Use of a pharmaceutical composition according to any one of claims 7-9 in the preparation of a medicament for treating hair loss.

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