Application of darafenib in preparation of medicine for treating skin fibrosis diseases

Abstract

The invention provides application of darafenib or pharmaceutically acceptable salts, esters and hydrates thereof in preparation of drugs for treating skin fibrosis diseases. The application shows that tamsulosin has the effect of slowing down and treating skin fibrosis, has a good application prospect in the aspect of developing the medicine for resisting skin fibrosis diseases, provides a new medicine research and development direction for treating, slowing down and improving skin fibrosis diseases, and has the potential of becoming a new medicine for treating skin fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a new use of dabrafenib, and specifically to the use of dabrafenib in preparing a drug for treating skin fibrosis diseases. Background Art

[0002] Skin fibrosis is a connective tissue disease that results from abnormal repair of damaged deep dermis. It can occur in a variety of pathological processes, including the immune disease scleroderma (SSc), keloid scars caused by abnormal wound healing, and hypertrophic scars (HS). The appearance of skin fibrosis can cause psychological distress to patients, while the resulting pain and functional impairment can severely impact their quality of life.

[0003] The skin is composed of the epidermis, dermis, and subcutaneous tissue. The epidermis is composed of multiple layers of cells, while the dermis is primarily composed of a network of elastic microfibrils interwoven with fibrous proteins (collagen and elastin), adhesion molecules (fibronectin), and proteoglycans. Multiple cell types, including fibroblasts, endothelial cells, and mast cells, are interspersed within this network. Fibroblasts are considered the most important effector cells in the pathogenesis of skin fibrosis. Skin injury, inflammation, infection, and immune dysfunction can lead to dysregulated fibroblast activation, excessive secretion of extracellular matrix, and invasion of surrounding normal skin tissue. This is accompanied by infiltration of inflammatory factors and excessive production of cytokines, resulting in skin thickening. Hypertrophic scars and keloids occur after skin injuries (such as surgery, chemical burns, burns, scratches, and insect bites), and can also occur naturally after allergic reactions. Hypertrophic scars are confined to the original wound site and can resolve spontaneously. Numerous contractile myofibroblasts are attached to the extracellular matrix through focal adhesion-like structures. Keloids extend beyond the wound edge into the surrounding skin, resulting in a strong inflammatory response, active dermal fibroblast proliferation, and active angiogenesis. Epidemiological data show that approximately 100 million people develop scars after surgery each year, and burn wounds that fail to heal within 21 days have a 70% or higher risk of developing hypertrophic scars.

[0004] Skin fibrosis is a difficult-to-treat disease. Intraperitoneal steroid injections have been the gold standard of treatment since the mid-1960s. However, this treatment primarily relieves symptoms and is often associated with side effects, such as atrophy of surrounding healthy skin, fat, and muscle, as well as osteoporosis. Existing prevention and treatment strategies primarily focus on reducing inflammation, including scar revision surgery, pressure / cryotherapy, radiation therapy, and laser therapy. These strategies are associated with high recurrence rates and significant adverse reactions. In addition, emerging therapies, including mesenchymal stem cell therapy, autologous fat transfer, local interferon injections, and intralesional botulinum toxin injections, require further clinical investigation. Currently, due to the lack of clarity regarding the pathological mechanisms underlying different types of skin fibrosis, few satisfactory treatments have entered clinical practice. Therefore, exploring new potential drug targets and developing proven, relatively safe, and affordable drugs for skin fibrosis are of great social and medical significance.

[0005] Dabrafenib is a potent and selective BRAF kinase inhibitor used to treat BRAF-mutated melanoma and other related diseases. 50 The value is 5.2nM. In the treatment of cancers with BRAF V600 mutations, dabrafenib is often used in combination with MEK inhibitors (such as trametinib) to fully inhibit the MAPK signaling pathway. Common side effects of dabrafenib as an oral targeted drug include hair loss, skin thickening, headache, swelling or peeling of the hands and feet, and joint or muscle pain. To date, there are no reports that dabrafenib can slow or treat skin fibrosis caused by non-coronavirus diseases.

[0006] The structural formula of dabrafenib is as follows:

[0007] Summary of the Invention

[0008] In view of this, the present invention aims to propose a new use of dabrafenib, specifically the use of dabrafenib or its pharmaceutically acceptable salts, esters, and hydrates in the preparation of drugs for treating skin fibrosis diseases. The present invention shows that dabrafenib has the effect of slowing down and treating skin fibrosis, and has good application prospects in the development of drugs against skin fibrosis diseases, providing a new drug research and development direction for the treatment, relief, and improvement of skin fibrosis diseases.

[0009] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0010] In one aspect, the present invention provides the use of dabrafenib or a pharmaceutically acceptable salt, ester, or hydrate thereof in the preparation of a medicament for treating skin fibrosis.

[0011] In some preferred embodiments of the present invention for use in preparing a medicament for treating skin fibrosis, the skin fibrosis is bleomycin-induced skin fibrosis and / or keloid.

[0012] In some preferred embodiments of the present invention for use in preparing a drug for treating skin fibrosis, the dabrafenib improves physiological indicators caused by skin fibrosis, including at least one of reducing dermal thickness, reducing collagen deposition, reducing the degree of skin fibrosis, reducing the expression of fibrosis-related proteins, and reducing scar tissue weight.

[0013] In some preferred embodiments of the present invention for use in preparing a drug for treating skin fibrosis, the reduction in fibrosis-related protein expression is the reduction in the expression of α-SMA, Col1α1, Col3α1, and Fn in keloid tissue.

[0014] In some preferred embodiments of the present invention for use in preparing a drug for treating skin fibrosis, the drug comprises tamsulosin or a pharmaceutically acceptable salt, ester, or hydrate thereof as an active ingredient, and pharmaceutically acceptable excipients.

[0015] The pharmaceutically acceptable excipients in the present invention include one or more of a carrier, an excipient, and a diluent; some examples of suitable carriers, excipients, and diluents include: one or more of lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl paraben, talc, magnesium stearate, and liquid paraffin.

[0016] The pharmaceutically acceptable excipients of the present invention also include lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners or flavoring agents and other auxiliary agents.

[0017] In some preferred embodiments of the present invention for use in preparing a drug for treating skin fibrosis, the drug dosage form is one or more of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, wines, tinctures, lotions, and films.

[0018] The method of preparing the active ingredient into a medicine in the present invention can be prepared by methods known to those skilled in the art. For example, the active ingredient can be diluted with a carrier or encapsulated in a carrier so that it can be quickly released, slowly released, or delayed released after administration to a subject.

[0019] In some preferred embodiments of the present invention for use in preparing a medicament for treating skin fibrosis, the administration route is one or more of oral administration, injection, implantation, and external application.

[0020] In some preferred embodiments of the present invention for preparing a drug for treating skin fibrosis, the effective dosage of dabrafenib is 0.1-50 mg / kg / d.

[0021] In some preferred embodiments of the present invention for preparing a drug for treating skin fibrosis, the effective dosage of dabrafenib is 10-30 mg / kg / d.

[0022] Another aspect of the present invention provides a drug for treating skin fibrosis, comprising dabrafenib or a pharmaceutically acceptable salt, ester or hydrate thereof as an active ingredient.

[0023] The term "treatment" as used herein includes its generally accepted meaning, which includes preventing, preventing, inhibiting, improving, and slowing, stopping or reversing the development of the symptoms or expected pathology. As such, the present invention encompasses both therapeutic and prophylactic administration.

[0024] As used herein, the term "effective amount" refers to the amount or dosage of an active ingredient that provides the desired effect upon a single or multiple administration to a patient. The effective amount can be determined by the attending diagnostician as a skilled artisan using known techniques and observations obtained under similar circumstances. In determining the effective amount or dosage of the active ingredient to be administered, the attending diagnostician should consider a variety of factors, including, but not limited to: the species of the mammal; the size, age, and general health; the specific disease involved; the degree of involvement or severity of the disease; the response of the individual patient; the specific compound to be administered; the mode of administration; the bioavailability properties of the administered formulation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0025] Compared with the prior art, the use of dabrafenib in the preparation of a drug for treating skin fibrosis has the following advantages:

[0026] The present invention provides a new use of dabrafenib, namely, the use of dabrafenib or a pharmaceutically acceptable salt, ester, or hydrate thereof in the preparation of a medicament for treating skin fibrosis. Dabrafenib in the present invention has excellent efficacy in slowing and treating skin fibrosis, improving dermal thickness, reducing collagen deposition, and alleviating the degree of skin fibrosis in patients with skin fibrosis. This provides a new medicament for treating, alleviating, or ameliorating skin fibrosis and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1A Schematic diagram of hydroxyproline content in skin tissue of each group of mice in Example 1 of the present invention;

[0029] Figure 1B Schematic diagram of H&E staining of mouse skin tissue in each group in Example 1 of the present invention;

[0030] Figure 1C This is a statistical diagram of the thickness of the dermis layer in the skin tissue of each group of mice in Example 1 of the present invention;

[0031] Figure 2A This is a statistical chart of scar weight after drug administration in each group in the keloid xenograft model of Example 2 of the present invention;

[0032] Figure 2B This is a statistical graph showing the expression levels of fibrosis markers α-SMA, Col1α1, Col3α1, and Fn in the keloids after drug administration in each group in the keloid xenograft model of Example 2 of the present invention. DETAILED DESCRIPTION

[0033] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0034] The present invention will be described in detail below with reference to the embodiments and accompanying drawings.

[0035] Example 1

[0036] Effect of dabrafenib on bleomycin-induced skin fibrosis in mice

[0037] 1. Preparation of bleomycin-induced skin fibrosis animal model:

[0038] The bleomycin (BLM)-induced skin fibrosis model is a commonly used skin fibrosis model. Male C57BL / 6J wild-type mice (8-10 weeks old) were shaved of the hair on the same part and injected subcutaneously with 100 μL of 0.5U BLM solution. The specific implementation method is as follows: the mice were divided into a control group (normal saline group), a bleomycin model group and a dabrafenib hydrochloride treatment group. The hair on the same part of the back of all mice was shaved within a range of approximately 2.0 cm × 2.0 cm, and then 100 μL of normal saline (0.9% NaCl) solution was injected subcutaneously on the back of the mice in the NaCl group. Similarly, 100 μL of 0.5U BLM solution was injected subcutaneously on the back of the mice in the model group, and 100 μL of a mixture of 0.5U BLM and 10 μM dabrafenib was injected on the back of the mice in the experimental group, once a day, for continuous injection. On the 21st day, the mice in each group were anesthetized, and skin specimens from the injection site on the back were obtained to analyze the pathological changes in the skin of the mice.

[0039] 2. Mouse grouping:

[0040] The mice were divided into three groups: control group (normal saline group), bleomycin model group and dabrafenib treatment group, with 6 mice in each group.

[0041] 3. Grouping and drug administration of mice:

[0042] The control group (normal saline group) was injected subcutaneously on the back of mice every day with 100 μL normal saline (0.9% NaCl) solution, the same volume as that of the dabrafenib-treated mice;

[0043] Mice in the bleomycin model group were subcutaneously injected with 100 μL of 0.5 U BLM solution to establish the model;

[0044] The dabrafenib-treated group was injected with 100 μL of a mixture of 0.5 U BLM and 10 μM dabrafenib on the back of mice;

[0045] The injection is given once a day for 21 consecutive days. The corresponding experimental dose of dabrafenib is converted based on the optimal dose reported in clinical practice or literature.

[0046] 4. Detection method:

[0047] After 21 consecutive days of injection, skin tissue was collected to detect the level of skin fibrosis and collagen content in mice.

[0048] 1) Skin Collagen Content Assay: To determine hydroxyproline content, mouse skin tissue (10 mg) was collected 21 consecutive days after injection, placed in a 5 mL ampoule, and oven-dried at 120°C. The sample was hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5-8.0. The residue was filtered, and the total volume was adjusted to 10 mL with PBS. A 50 μL sample was taken, 350 μL of deionized water was added, and 200 μL of chloramine T solution was added, incubated at room temperature for 20 minutes. The sample was then incubated with 200 μL of perchloric acid and incubated at room temperature for 5 minutes. The sample was then incubated with 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) at 65°C for 20 minutes. A 200 μL aliquot was added to a 96-well plate, and the absorbance at 570 nm was measured. A standard curve was constructed using the readings from the standard sample, and the hydroxyproline concentration (Cs) of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline contained in the skin tissue was converted into W according to the following formula: W = Cs × 8 (dilution factor of the measured sample) × total sample volume.

[0049] 2) Fibrosis level detection: Skin tissue sections were pathologically stained and the dermis thickness and collagen deposition level were calculated.

[0050] Pathological staining: Skin samples were fixed with 10% formalin, dehydrated, embedded in paraffin, and cut into 5-μm-thick sections. After dewaxing in xylene and rehydration using an alcohol series, sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and Sirius red. Images were randomly captured using an upright transmission fluorescence microscope and analyzed using Image-Pro Plus version 6.0.

[0051] 5. Test results:

[0052] 1) Skin collagen content test results: The hydroxyproline content in the bleomycin model group was significantly increased, while the hydroxyproline content in the skin tissue of mice treated with dabrafenib was significantly decreased. This indicates that dabrafenib can inhibit the synthesis of collagen in the skin induced by bleomycin (such as Figure 1A shown).

[0053] 2) Pathological staining: H&E staining was performed on mouse skin tissue sections (scale: 100 μm), and the skin thickness of the skin tissue sections was quantitatively analyzed. It was found that the skin thickness of the mice in the dabrafenib treatment group was significantly lower than that of the mice in the bleomycin model group (such as Figure 1B 、 Figure 1C shown).

[0054] in, Figure 1A 、 1BIn Figure 1C, the control group represents the normal saline group, the bleomycin model group represents the mouse model group, and the dabrafenib treatment group represents the drug treatment group; * represents the significant difference (P value) between the drug treatment group and the model group, where *: P < 0.05, **: P < 0.01.

[0055] Example 2

[0056] Effects of dabrafenib on scar weight and fibrosis-related gene expression in a keloid xenograft mouse model

[0057] 1. Preparation of keloid xenograft mouse model:

[0058] Freshly removed skin tissue was stripped of excess fat in a clean bench, leaving only the epidermis and dermis. The keloid tissue was cut into slices of approximately 5×5×5 mm, with each tissue block weighing 0.08 to 0.1 g. BALB / c nude mice were anesthetized, and an approximately 0.5 cm incision was made on the back. The subcutaneous pouch was then formed by subcutaneous dissection, and the tissue block was implanted in the subcutaneous pouch. A keloid xenograft model was established approximately 14 days after surgery. The mouse grafts were divided into two groups and received in situ injections of saline (0.9% NaCl) or dabrafenib for two weeks, for a total of six treatments. After treatment, the grafts were collected, the weight of the transplanted scars was assessed, and gene expression analysis was further performed.

[0059] 2. Mouse grouping:

[0060] The patients were divided into 2 groups: control group (normal saline group) and dabrafenib treatment group, with 3 grafts in each group.

[0061] 3. Grouping and drug administration of mice:

[0062] In the control group (normal saline group), mice were injected orally with 50 μL normal saline (0.9% NaCl) solution as a control;

[0063] The dabrafenib-treated group was injected with a mixture of 50 μL of normal saline and 10 μM dabrafenib on the back of mice;

[0064] The treatment lasted for two weeks, with a total of 6 treatments. The corresponding experimental dose of dabrafenib was converted based on the optimal dose reported in clinical practice or literature.

[0065] 4. Detection method:

[0066] After 6 treatments, the excised transplanted keloid tissues were weighed and analyzed, and the expression of fibrosis-related genes was further analyzed.

[0067] 1) Keloid weight analysis: The excised and transplanted keloid tissue was weighed and analyzed, and the ratio of the keloid weight loss after drug administration to the initial weight was used as the reference value.

[0068] 2) Analysis of fibrosis-related gene expression: This study used real-time fluorescence quantitative PCR experiments to detect changes in the RNA expression levels of skin fibrosis markers in keloids.

[0069] 5. Test results:

[0070] 1) Keloid weight analysis results: The tissue weight of the dabrafenib-treated group decreased (e.g. Figure 2A shown).

[0071] 2) Fibrosis-related gene expression analysis results: qPCR analysis showed that dabrafenib reduced the expression of α-SMA, Col1α1, Col3α1 and Fn in keloid xenograft tissues (e.g. Figure 2B shown).

[0072] in, Figure 2A 、 2B In the table, the control group represents the saline group, and the dabrafenib-treated group represents the drug-treated group. * represents a significant difference (P value) between the drug-treated group and the control group, where *: P < 0.05, **: P < 0.01.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of dabrafenib or its pharmaceutically acceptable salts, esters, and hydrates in the preparation of a medicament for treating skin fibrosis.

2. The use according to claim 1, characterized in that: The skin fibrosis disease is bleomycin-induced skin fibrosis and / or keloid.

3. The use according to claim 1, characterized in that: The dabrafenib improves physiological indicators caused by skin fibrosis, including at least one of reducing dermal thickness, reducing collagen deposition, alleviating skin fibrosis, reducing fibrosis-related protein expression, and reducing scar tissue weight.

4. The use according to claim 3, characterized in that: The reduction of fibrosis-related protein expression is to reduce the expression of α-SMA, Col1α1, Col3α1 and Fn in keloid tissue.

5. The use according to any one of claims 1 to 4, characterized in that: The drug for treating skin fibrosis comprises tamsulosin or its pharmaceutically acceptable salt, ester, hydrate as an active ingredient, and pharmaceutically acceptable excipients.

6. The use according to any one of claims 1 to 4, characterized in that: The pharmaceutical dosage form is one or more of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, alcoholic beverages, tinctures, lotions, and films.

7. The use according to any one of claims 1 to 4, characterized in that: The administration route is one or more of oral administration, injection, implantation, and external application.

8. The use according to any one of claims 1 to 4, characterized in that: The effective dosage of dabrafenib is 0.1-50 mg / kg / d.

9. The use according to claim 8, characterized in that: The effective dosage of dabrafenib is 10-30 mg / kg / d.

10. A drug for treating skin fibrosis, characterized in that: The invention comprises dabrafenib or a pharmaceutically acceptable salt, ester or hydrate thereof as an active ingredient.

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