Application of carbidopa in preparation of medicine for treating melanoma

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of carbidopa in preparation of a medicine for treating melanoma. Carbidopa can target CD147 and has an inhibition effect on CD147, so that invasion, migration and growth of melanoma cells are inhibited, Carbidopa can be used for preparing drugs for treating melanoma, and new safe and effective drugs are provided for the field of melanoma treatment.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to an application of carbidopa in the preparation of a drug for treating melanoma. Background Art

[0002] Melanoma is a malignant tumor originating from melanocytes in the skin, mucous membranes and uveal tract. It has a high invasive ability and can rapidly metastasize to other organs. Metastatic melanoma is one of the most deadly cancers with a very poor prognosis. In the visceral metastasis stage, the median survival is only 6 months. Therefore, although melanoma only accounts for 5% of all skin cancers, it is the cause of 80% of skin cancer deaths. The occurrence and development of melanoma is associated with a variety of somatic mutations, the most common mutations include BRAF, NRAS, and NF1, among which BRAF V600E The mutation is present in approximately 57% of melanoma patients. Surgery remains the main treatment for early-stage melanoma, but the cure rate for patients with advanced melanoma is extremely low.

[0003] In addition to traditional chemotherapy drugs, targeted therapy has become a new method for treating melanoma. Since 2011, the U.S. Food and Drug Administration (FDA) has approved BRAF inhibitors such as vemurafenib and dabrafenib for BRAF V600E Treatment of unresectable or metastatic melanoma with BRAF mutations. Although targeted therapies are effective and can significantly inhibit melanoma progression, nearly all patients treated with BRAF inhibitors develop resistance within one year of treatment. Therefore, finding new, safe and effective drugs is an urgent issue in the field of melanoma treatment. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a use of carbidopa in the preparation of a drug for treating melanoma, aiming to solve the problem of drug resistance in existing melanoma treatment drugs.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a method for preparing a drug for treating melanoma is provided.

[0007] Optionally, the carbidopa inhibits invasion, migration and growth of melanoma cells.

[0008] Optionally, the inhibiting the growth of melanoma cells includes inhibiting the viability of melanoma cells, inducing senescence or apoptosis of melanoma cells.

[0009] Optionally, the carbidopa has an inhibitory effect on CD147.

[0010] In a second aspect of the present application, a pharmaceutical composition for treating melanoma is provided, wherein the pharmaceutical composition comprises carbidopa or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.

[0011] Optionally, the pharmaceutical composition includes a pharmaceutically acceptable excipient of carbidopa.

[0012] Optionally, the auxiliary material includes at least one of a pharmaceutical carrier, a diluent, an adjuvant, and an excipient.

[0013] Optionally, the pharmaceutical composition is in the form of a capsule, tablet, powder, granule or injection.

[0014] In a third aspect of the present application, a CD147 inhibitor is provided, wherein the CD147 inhibitor includes carbidopa.

[0015] Compared with the existing technology, this application has the following advantages:

[0016] Carbidopa in the present application can target CD147 and has an inhibitory effect on CD147, thereby hindering the cellular pathway based on CD147-induced expression of cell matrix metalloproteinases to enhance the invasion and migration ability of melanoma cells and the drug resistance of melanoma cells; at the same time, it activates the protein Caspase-8 to induce melanoma cell apoptosis, thereby inhibiting the invasion, migration and growth of melanoma cells, making up for the defects of existing BRAF inhibitors vemurafenib and dabrafenib in causing drug resistance due to reactivation of the MAPK cell pathway or compensation of bypass signals, and has great application potential in the preparation of drugs for the treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 The CD147 inhibitor screening results provided in the examples of this application are:

[0019] A is the specific molecular structure of carbidopa and the Hex Score of its binding to CD147; B is the binding model of carbidopa and CD147; C is the verification of the binding effect of carbidopa on CD147;

[0020] Figure 2 The examples provided in this application provide an analysis of the inhibitory effects of different concentrations of carbidopa on the proliferation of melanoma cells A375, SK-MEL-5, and SK-MEL-28:

[0021] A is melanoma cell A375; B is melanoma cell SK-MEL-5; C is melanoma cell SK-MEL-28;

[0022] Figure 3 Analysis of the inhibitory effect of carbidopa on the cell proliferation ability of melanoma cells resistant to vemurafenib provided in the examples of this application:

[0023] A is an analysis of the inhibitory effect of vemurafenib on the proliferation of melanoma cells A375; B is an analysis of the inhibitory effect of vemurafenib on the proliferation of melanoma cells resistant to vemurafenib; C is an analysis of the inhibitory effect of carbidopa on the proliferation of melanoma cells resistant to vemurafenib;

[0024] Figure 4 Analysis of the inhibitory effect of carbidopa on the migration ability of melanoma cells A375, SK-MEL-5, and SK-MEL-28 provided in the examples of this application:

[0025] A is melanoma cell A375; B is melanoma cell SK-MEL-5; C is melanoma cell SK-MEL-28;

[0026] Figure 5 Analysis of the inhibitory effect of carbidopa on the invasion ability of melanoma cells A375 and SK-MEL-28 provided in the examples of this application:

[0027] A is melanoma cell A375; B is melanoma cell SK-MEL-28;

[0028] Figure 6 Analysis of the inhibitory effect of carbidopa on the cloning ability of melanoma cells A375 and melanoma vemurafenib-resistant RA provided in the examples of this application:

[0029] A is melanoma cell A375; B is melanoma vemurafenib-resistant strain RA;

[0030] Figure 7 Western blotting analysis of MMP2 protein and Caspase-8 protein provided in the examples of this application;

[0031] Figure 8 This is a flow cytometric analysis diagram of melanoma cells A375 provided in the examples of this application. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings and embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. In the absence of conflict, the features in the following embodiments and embodiments can be combined with each other.

[0033] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions such as "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 the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such 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 this field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0034] The present invention provides an application of carbidopa in a drug for treating melanoma. Specifically, carbidopa can inhibit the invasion, migration, and growth of melanoma cells.

[0035] Carbidopa is a peripheral decarboxylase inhibitor, commonly used clinically in combination with levodopa to treat Parkinson's disease. While it has no therapeutic use in Parkinson's disease on its own, when used with levodopa, it can inhibit the activity of aromatic amino acid decarboxylase, preventing the peripheral conversion of levodopa to dopamine. However, carbidopa does not cross the blood-brain barrier and therefore does not affect the conversion of levodopa to dopamine in the brain. The present invention demonstrates through experiments that carbidopa can inhibit the invasion, migration, and growth of melanoma cells, and thus can be used in the preparation of drugs for treating melanoma.

[0036] In some embodiments, carbidopa has an inhibitory effect on CD147.

[0037] CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN) or baserin, is a transmembrane glycoprotein with two immunoglobulin domains and a member of the immunoglobulin superfamily. The inventors previously established a nude mouse xenograft model of malignant melanoma and discovered that the CD147 gene may act as an oncogene in malignant melanoma. Targeting CD147 can inhibit tumor cell viability, proliferation, and invasion, while inducing melanoma cell senescence and apoptosis.

[0038] Based on this, a structure-based virtual ligand approach was used to screen the FDA-approved drug library, docking small molecules into the structure of large molecular targets and scoring their potential complementarity, ultimately identifying carbidopa as a potential inhibitor of CD147.

[0039] Virtual screening (VS) is a method that searches compound databases for compounds that bind to a target protein or conform to a quantitative structure-activity relationship (QSAR) model based on its three-dimensional structure or quantitative structure-activity relationship (QSAR) model. FDA-approved marketed drugs and compounds that have passed Phase I clinical trials are particularly well-suited for drug repurposing due to their excellent biological activity, pharmacokinetic properties, and safety profile. This can significantly accelerate drug development and reduce risk. Furthermore, drug repositioning offers numerous advantages over new drug development. It reduces the risk of drug development failure, shortens the development cycle, and lowers development costs.

[0040] Furthermore, an in vitro protein binding experiment (pull down assay) confirmed that carbidopa could effectively bind to the CD147 protein gene. A formazan test (MTS assay) then confirmed that carbidopa could inhibit the proliferation of different melanoma cell lines A375, SK-MEL-5, and SK-MEL-28. A scratch test and a Transwell assay also demonstrated that carbidopa could fully bind to CD147, was an inhibitor of CD147, and could inhibit the invasion, migration, and growth of melanoma cells.

[0041] The present application also provides a pharmaceutical composition for treating melanoma, comprising carbidopa or its pharmaceutically acceptable salts, stereoisomers, solvates, and polymorphs. Specifically, the composition may include carbidopa and derivatives obtained by salt modification, esterification, amidation, aminomethylation, etherification, ring opening, and cyclization of carbidopa. These derivatives are structurally similar to carbidopa and may exhibit specific pharmacological effects similar to those of carbidopa, and are also within the scope of protection of this application.

[0042] The term "pharmaceutically acceptable" refers to a substance (such as a carrier or excipient) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, that is, the substance can be administered to a subject without causing adverse biological reactions or interacting in an adverse manner with any components contained in the composition.

[0043] In some embodiments, the pharmaceutical composition for treating melanoma further comprises a pharmaceutically acceptable excipient for carbidopa. Further, the excipient may comprise at least one of a pharmaceutically acceptable carrier, a diluent, an adjuvant, and an excipient.

[0044] Carriers, diluents, adjuvants or excipients may include carbohydrates, water-soluble or swellable polymers, hydrophilic or hydrophobic materials, waxes, gelatin, oils, solvents, water, etc. Specifically, the carriers, diluents or excipients include, for example, water, starch, lactose, dextrose, fructose, sucrose, polyethylene glycol, propylene glycol, sorbitol, mannitol, polyvinyl alcohol, rubber, gelatin, alginate, calcium silicate, calcium phosphate, cellulose, sugar water, methylcellulose, polyvinyl pyrrolidone, alkyl parahydroxyphenyl sorbate, talc, magnesium stearate, stearic acid, glycerin, sesame oil, olive oil, soybean oil, etc.

[0045] In some embodiments, the pharmaceutical composition for treating melanoma is in the form of a solid, semi-solid, liquid, or gaseous preparation, such as a tablet, pill, capsule, powder, granule, ointment, suspension, solution, injection, inhalant, gel, or aerosol. Preferably, the pharmaceutical composition for treating melanoma is in the form of a capsule, tablet, powder, granule, or injection.

[0046] The present invention also provides a CD147 inhibitor, including carbidopa. Using a structure-based virtual ligand approach to screen an FDA-approved drug library, small molecules were docked into the structure of a macromolecular target and their potential complementarity was scored. Ultimately, carbidopa was identified as a potential CD147 inhibitor. Furthermore, in vitro protein binding experiments confirmed that carbidopa effectively binds to the CD147 protein gene.

[0047] The following is further described with reference to specific embodiments.

[0048] Unless otherwise specified, the methods and reagents used in the examples are conventional experimental methods in the art.

[0049] Example 1

[0050] By using a computer-based structure-based virtual ligand screening method, we screened the clinically approved drug database to search for potential CD147 inhibitors. Virtual screening of FDA-approved drugs was performed in XP (Extra Precision) mode, and several potential inhibitors targeting CD147 were identified. Carbidopa was found to be the preferred CD147 inhibitor by Hex scoring ( Figure 1 Then, the hierarchical docking algorithm Glide docking experiment was used to evaluate the possible binding modes between CD147 and inhibitors ( Figure 1 Carbidopa was identified as a potential inhibitor of CD147. Finally, a pulldown assay was performed to confirm that carbidopa could effectively bind to CD147 ( Figure 1 (as shown in C in the figure).

[0051] Example 2

[0052] Melanoma cell lines (A375, SK-MEL-5, SK-MEL-28, G361, RA) were plated in 96-well plates (3×10 3 Cells were plated at 400 μM for 24 h, 48 h, and 72 h, respectively. After cell attachment, carbidopa was added in a gradient of concentrations (0, 20, 40, and 80 μM). Cell proliferation was assessed using MTS. A375, SK-MEL-5, SK-MEL-28, and G361 are all melanoma cell lines, and RA is a vemurafenib-resistant melanoma cell line.

[0053] (1) Melanoma cells A375, SK-MEL-5, and SK-MEL-28 were treated with different concentrations (0, 20, 40, and 80 μM) of carbidopa for different time periods (0, 24, 48, and 72 h) and then subjected to MTS assay to investigate cell proliferation. Figure 2 As shown in the table, the data are statistically expressed as mean (n=5) ± SD, and asterisks (*) indicate statistically significant differences, *p<0.05, **p<0.01, ***p<0.001. Figure 2 As can be seen from Figures A, B, and C, carbidopa has an inhibitory effect on the proliferation of A375, SK-MEL-5, and SK-MEL-28 cells. When the concentration of carbidopa is 80 μM, the inhibitory effect is obvious.

[0054] (2) The melanoma vemurafenib-resistant strain RA was successfully constructed using 2 μM vemurafenib. Melanoma cells A375 were treated with different concentrations (0, 20, 40, 80 μM) and RA for different time periods (0, 24, 48, 72 h) and then the cell proliferation ability was tested by MTS assay. The results are shown in the figure. Figure 3 As shown in A and B. Figure 3 As shown in Figures A and B, vemurafenib can inhibit the proliferation of melanoma cells A375, but not RA cells, indicating that the melanoma resistant strain RA was successfully constructed. Then, RA was treated with different concentrations (0, 20, 40, 80 μM) of carbidopa for different time periods (0, 24, 48, 72 h) and then the cell proliferation capacity was tested by MTS assay. The results are shown in Figure 3. Figure 3 As shown in C. Figure 3 As shown in Figure C, carbidopa also inhibits cell proliferation in the vemurafenib-resistant melanoma cell line RA. This further demonstrates that carbidopa can still inhibit the proliferation of melanoma cells that have already developed resistance to vemurafenib, resolving the issue of developing resistance to the existing BRAF inhibitor vemurafenib after a period of use.

[0055] Example 3

[0056] (1) Melanoma cells A375, SK-MEL-5, and SK-MEL-28 were plated in 96-well plates. After the cells adhered, they were treated with 20 μM carbidopa. The effect of carbidopa on the migration ability of different melanoma cell lines A375, SK-MEL-5, and SK-MEL-28 was detected by scratch test. The results are shown in the figure. Figure 4 As shown. Figure 4 As shown in Figures A, B, and C, compared with the control group (not treated with carbidopa), carbidopa can inhibit the migration ability of various melanoma cells, especially significantly inhibit the migration ability of A375 and SK-MEL-28 cells.

[0057] (2) Melanoma cells A375 and SK-MEL-28 were plated in 96-well plates, and after the cells adhered, they were treated with 20 μM carbidopa for 24 h. The effect of carbidopa on the invasion ability of melanoma cells A375 and SK-MEL-28 was verified by Transwell assay. The results are as follows: Figure 5 As shown. Figure 5 As can be seen from Figures A and B, carbidopa can significantly inhibit the invasion ability of melanoma cells A375 and SK-MEL-28.

[0058] (3) Melanoma cells A375 and melanoma RA cells were plated in 96-well plates. After the cells adhered, they were treated with 20 μM carbidopa for 24 h. After culturing for 2 weeks, the clone formation was observed. The results are as follows: Figure 6 As shown, from Figure 6 As shown in Figures A and B, carbidopa can inhibit the cloning ability of melanoma cells A375 and melanoma RA cells (resistant to velofilin).

[0059] Example 4

[0060] (1) To further explore the mechanism by which carbidopa targets CD147 to inhibit melanoma invasion and metastasis, melanoma A375 cells were treated with 20 μM carbidopa for different time periods and the protein expression changes of MMP2 and Caspase-8 in the apoptosis pathway were verified by Western blot. The results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that carbidopa can inhibit the expression of MMP2 in a time-dependent manner and induce the cleavage of apoptosis-related protein Caspase-8.

[0061] (2) To further explore the effect of carbidopa on apoptosis of melanoma cells, melanoma A375 cells were plated in 96-well plates and treated with 80 μM carbidopa for 0, 24, and 48 h after cell attachment. Flow cytometry was performed on cells treated at different times. The results are shown in Table 2. Figure 8 As shown. Figure 8 As can be seen from the figure, 80 μM carbidopa can significantly induce apoptosis in melanoma A375 cells.

[0062] In summary, this application screened an FDA-approved drug library through a structure-based virtual ligand method, and screened out carbidopa as a potential inhibitor of CD147. Experimental verification also demonstrated that carbidopa not only blocked the cellular pathway based on CD147-mediated expression of matrix metalloproteinases that enhances the invasion and migration ability of melanoma cells and the drug resistance of melanoma cells, but also activated the protein Caspase-8 to induce apoptosis of melanoma cells, thereby inhibiting the invasion, migration and growth of melanoma cells. This method compensates for the defects of existing BRAF inhibitors such as vemurafenib and dabrafenib, which lead to drug resistance due to reactivation of the MAPK cell pathway or compensation of bypass signals, and has great application potential in the preparation of drugs for the treatment of melanoma.

[0063] It should be understood that the application of this application is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes 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. Application of carbidopa in the preparation of drugs for treating melanoma.

2. The use according to claim 1, characterized in that The carbidopa inhibits the invasion, migration and growth of melanoma cells.

3. The use according to claim 2, characterized in that The inhibition of melanoma cell growth includes inhibiting the activity of melanoma cells and inducing melanoma cell senescence or apoptosis.

4. The use according to claim 1, characterized in that Carbidopa has an inhibitory effect on CD147.

5. A pharmaceutical composition for treating melanoma, characterized in that: The pharmaceutical composition comprises carbidopa or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.

6. The pharmaceutical composition for treating melanoma according to claim 5, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient of carbidopa.

7. The pharmaceutical composition for treating melanoma according to claim 6, characterized in that The auxiliary materials include at least one of a pharmaceutical carrier, a diluent, an adjuvant, and an excipient.

8. The pharmaceutical composition for treating melanoma according to claim 5, characterized in that The pharmaceutical composition is one of capsules, tablets, powders, granules and injections.

9. A CD147 inhibitor, characterized in that The CD147 inhibitors include carbidopa.

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