Application of CYP27A1 inhibitor in preparation of medicine for treating melanoma

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a CYP27A1 inhibitor in preparation of a medicine for treating melanoma. The CYP27A1 inhibitor is Dafadine A, and the CYP27A1 inhibitor is Dafadine-A. The invention proves that 27-hydroxycholesterol and cytochrome P450 family member 27A1 (CYP27A1) are potential new targets for treating melanoma, Dafadine-A can obviously enhance the killing ability of Vemrafenib to melanoma cells and effectively inhibit the formation of melanoma stem cell microspheres, so that the drug resistance of a Vemrafenib resistant cell strain A375-VR to Vemrafenib is improved to a great extent, and the Dafadine-A can be used for treating melanoma. It is prompted that the drug resistance problem generated in the process of treating melanoma through Vemurafenib is expected to be solved through the drug combination mode, and the good clinical transformation value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a CYP27A1 inhibitor in the preparation of a drug for treating melanoma. Background Art

[0002] Melanoma is a malignant tumor that occurs in skin melanocytes and is the most lethal among all skin cancers, currently accounting for more than 75% of the deaths related to skin cancers. Nearly half of melanomas carry a BRAF V600E mutation, that is, the 600th amino acid of the BRAF gene mutates from valine to glutamic acid, making the BRAF protein always in an activated state. Through the Mitogen-Activated Protein Kinase (MAPK) signaling pathway, it promotes the occurrence and development of melanoma.

[0003] Vemurafenib is a selective BRAF inhibitor that can bind to BRAF V600E and then inhibit its protein activity, block the mitogen-activated protein kinase signaling pathway, and relieve the occurrence and development of melanoma. Therefore, the US Food and Drug Administration (FDA) approved the listing of Vemurafenib in 2011 for the treatment of inoperable or metastatic melanoma with BRAF V600E mutation, which can effectively extend the survival period of patients. However, like other tumor treatment drugs, this targeted drug for BRAF V600E mutation generally shows drug resistance after patients take it for a period of time, resulting in a weakened effect of taking the drug later or even tumor recurrence.

[0004] Therefore, how to find new targets and effective drug regimens to solve melanoma drug resistance is the technical problem to be solved by the present invention. Summary of the Invention

[0005] Aiming at the problems existing in the above-mentioned prior art, the present invention aims to provide a new target for melanoma drug resistance and a new use of an inhibitor of this target.

[0006] The first aspect of the present invention lies in providing the use of a CYP27A1 inhibitor in the preparation of a drug for treating melanoma, and the CYP27A1 inhibitor is Dafadine-A.

[0007] Furthermore, the CYP27A1 inhibitor in the drug is used as the only active ingredient or one of the active ingredients.

[0008] Even further, when the CYP27A1 inhibitor in the drug is used as one of the active ingredients, the active ingredients further include Vemurafenib.

[0009] Furthermore, the CYP27A1 inhibitor and Vemurafenib are administered simultaneously or sequentially in any order.

[0010] The second aspect of the present invention is to provide a medicament for treating melanoma, comprising the CYP27A1 inhibitor.

[0011] Furthermore, in the medicament, the content of the CYP27A1 inhibitor is 0.1-99 wt%.

[0012] Furthermore, the medicament further comprises Vemurafenib.

[0013] Furthermore, the molar ratio of the CYP27A1 inhibitor to Vemurafenib is 5-50:0.1-20.

[0014] Furthermore, the medicament further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes conventional diluents (such as at least one of water for injection, microcrystalline cellulose, etc.), fillers (such as at least one of mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binders (such as at least one of carbomer, gum arabic, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrants (such as at least one of sodium carboxymethyl starch, cross-linked carboxymethyl cellulose sodium, hypromellose, low-substituted hydroxypropyl cellulose, etc.), lubricants (such as at least one of talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, colloidal silica, etc.), wetting agents (such as at least one of propylene glycol, glycerol, ethanol, etc.), stabilizers (such as at least one of disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, vitamin C, etc.), osmotic pressure regulators (such as at least one of sodium chloride, glucose, etc.), pH regulators (such as at least one of triethanolamine, sodium hydroxide, sodium citrate, etc.), and preservatives (such as at least one of chlorobutanol, parabens, ethylparaben, benzalkonium bromide, etc.). The above excipients can be in common doses and are mixed with the CYP27A1 inhibitor and Vemurafenib in common ratios. After the dosages of the CYP27A1 inhibitor and Vemurafenib are determined, the ratios between the various pharmaceutical excipients can be appropriately adjusted as needed.

[0015] Furthermore, the dosage form of the medicament is a granule, tablet, capsule, pill, dripping pill, oral liquid preparation, gavage preparation or injection dosage form.

[0016] The present invention has the following beneficial effects:

[0017] (1) The present invention increases cholesterol synthesis by molecular means, significantly enhancing the proliferation and metastasis ability of melanoma cells;

[0018] (2) The present invention reduces cholesterol synthesis by molecular and pharmaceutical means, thereby reducing the content of 27-hydroxycholesterol and significantly decreasing the proliferation and metastasis ability of melanoma cells;

[0019] (3) The present invention inhibits cytochrome P450 family member 27A1 by Dafadine-A, thereby reducing the content of 27-hydroxycholesterol and significantly improving the problem of Vemurafenib resistance in melanoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In which:

[0021] A is the chemical structure of the CYP27A1 inhibitor Dafadine-A.

[0022] B is the killing effect of Dafadine-A alone and Dafadine-A combined with Vemurafenib on melanoma cells.

[0023] C is the effect of Dafadine-A treatment on the formation and proliferation of melanoma tumor stem cell microspheres.

[0024] (D) is the effect of Dafadine-A on the Rap1 / AKT pathway.

[0025] (E) is the effect of simultaneously adding Dafadine-A and HJC0197 (RAP1 activator) on the formation and proliferation of melanoma tumor stem cell microspheres.

[0026] Figure 2 In which:

[0027] A is the property of the A375VR cell line resistant to Vemurafenib in terms of cell proliferation and morphological characterization.

[0028] B is the half-lethal concentration (IC50) of the A375VR cell line to Vemurafenib.

[0029] C is the expression of the CYP27A1 gene in A375VR.

[0030] D is the effect of Dafadine-A alone on the proliferation of wild-type melanoma cells.

[0031] E is the effect of Dafadine-A combined with Vemurafenib on the proliferation of the A375VR drug-resistant cell line. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0033] Currently, combination drug therapy is often adopted, which can greatly improve the effect of single drug use. In previous work, through the analysis of melanoma metabolomics, we discovered a potential new target for melanoma treatment, 27-hydroxycholesterol (27-HC). The synthesis of 27-hydroxycholesterol requires catalysis by cytochrome P450 family member 27A1 (CYP27A1). Therefore, by inhibiting cytochrome P450 family member 27A1, the production of 27-hydroxycholesterol in melanoma cells can be inhibited. We used the inhibitor of cytochrome P450 family member 27A1, Dafadine-A, to treat melanoma cells, which can greatly improve the drug sensitivity of melanoma cells to Vemurafenib, effectively reduce the number of melanoma stem-like cells, and inhibit the proliferation and migration of melanoma cells.

[0034] The following are specific embodiments. Unless otherwise specified, the raw materials used in the embodiments are commercially available products.

[0035] Example 1: Effect of combination drug therapy on A375 cells

[0036] 1 Experimental method

[0037] In this experiment, Dafadine-A (50 μm) (structural formula as shown in Figure 1 A) was used in combination with different concentrations of Vemurafenib to treat A375 melanoma cells, specifically including the following steps:

[0038] (1) Cell culture and treatment

[0039] Take A375 melanoma cells in the logarithmic growth phase and inoculate them in a 96-well culture plate at a concentration of 4×10 3 cells / well. The cells were cultured in DMEM medium supplemented with 10% FBS, 1% double antibody, 1% non-essential amino acids, and 0.1% mercapto reducing agent, and Dafadine-A (50 μm) and Vemurafenib (0.1, 0.2, 0.5, 1, 2 μm) were added simultaneously, and incubated in an incubator at 37 °C, 5% CO 2 concentration and saturated humidity conditions for 24 hours.

[0040] (2) Use an inverted microscope to observe the cell density and morphology and take pictures.

[0041] 2 Experimental Results

[0042] The results are as Figure 1 shown in B. Compared with the treatment with Vemurafenib alone, in the presence of Dafadine-A (50 μm), the higher the concentration of Vemurafenib in combination, the more A375 melanoma cells die.

[0043] Example 2: Effects of Dafadine-A on Two Kinds of Melanoma Cells, A375 and A2058

[0044] 1 Experimental Method

[0045] In the tumor sphere formation experiment, 1% l-glutamine, 1% penicillin / streptomycin double antibody, 2% B27, 20 ng / ml epidermal growth factor (EGF), and 20 ng / ml basic fibroblast growth factor (bFGF) were added to serum-free DMEM / F12 medium to prepare stem cell medium. The single-cell suspension was inoculated into an ultra-low attachment six-well plate at a density of 1000 cells / ml. After 10 days of seeding, the formed spheres were digested and re-inoculated into an ultra-low attachment six-well plate at a density of 1000 cells / ml. After continuing to culture for 10 days, spheres with a diameter > 30 μm were counted under an inverted microscope (×100), and the colony formation number was calculated. In this experiment, the solvent (DMSO) was used as a control, and Dafadine-A (5, 10 μm) and cholesterol (20 μm), either alone or added simultaneously, were added to the stem cell medium as the experimental group. The tumor stem cell microspheres formed for the first time after 10 days were digested, and after secondary sphere formation and continued culture for 10 days, the changes in the number and volume of the formed tumor stem cell microspheres were observed.

[0046] 2 Experimental Results

[0047] The results are as Figure 1 shown in C. Dafadine-A has an obvious inhibitory effect on the formation of tumor microspheres of both A375 and A2058 melanoma cells. Even when cholesterol is added to enhance the stem cell formation ability of these two melanoma cells, it can be effectively weakened by Dafadine-A.

[0048] Example 3: Western Blot Verification that 27-HC Plays a Role through the RAP1 / AKT Signaling Pathway

[0049] 1 Experimental Method

[0050] In this experiment, cells in good growth state were inoculated into six-well plates and cultured with DMEM medium supplemented with 10% FBS, 1% double antibiotics, 1% non-essential amino acids, and 0.1% mercapto reducing agent. At the same time as adding the cells, the solvent (DMSO) was added as the control group. The experimental groups were respectively added with 5 μm and 10 μm Dafadine-A. After culturing for 48 h, the cells were taken, lysed into proteins, and the protein expression was verified by western blot.

[0051] 2 Experimental results

[0052] As shown in Figure D of the results, Dafadine-A can reduce the phosphorylation levels of RAP1 and AKT in two melanoma cells, A375 and A2058, indicating that 27-HC promotes the formation of tumor stem cells by activating the phosphorylation of RAP1 and AKT.

[0053] Example 4: The RAP1 activator can alleviate the inhibition of Dafadine-A on the formation of tumor stem cells

[0054] 1 Experimental method

[0055] In this experiment, two melanoma cells, A375 and A2058, in good growth state were respectively inoculated into ultra-low attachment six-well plates and cultured in stem cell medium (prepared as in Example 2) at a density of 1000 cells / ml. At the same time as adding the cells, the solvent (DMSO), Dafadine-A, or Dafadine-A and HJC0197 (RAP1 activator) were added as the experimental groups. After sowing for 10 days, the formed spheres were digested and re-inoculated into ultra-low attachment six-well plates at a density of 1000 cells / ml. After continuing to culture for 10 days, the spheres with a diameter > 30 μm were counted under an inverted microscope (×100), and the colony formation number was calculated.

[0056] 2 Experimental results

[0057] The results are as Figure 1 shown in E. The number and volume of stem cells formed by adding Dafadine-A and HJC0197 (RAP1 activator) simultaneously are significantly more than those formed by adding Dafadine-A alone, verifying the ability of 27-HC to promote the formation of tumor stem cells through the RAP1 signaling pathway.

[0058] Example 5: Inhibition of vemurafenib on melanoma cells

[0059] 1 Experimental method

[0060] In this experiment, after the cells developed drug resistance by gradually increasing the concentration of Vemurafenib in the cell culture medium, the cells were inoculated into DMEM medium supplemented with 10% FBS, 1% double antibody, 1% non-essential amino acids, and 0.1% mercapto reducing agent for culture. At the same time, wild-type melanoma cells A375WT and drug-resistant melanoma cells A375VR (cells resistant to Vemurafenib) were stimulated with different concentrations of Vemurafenib (2, 4, 8, 15, 20 μM). After culturing for 24 hours, the effects of Vemurafenib on the cell number and morphology were observed under a microscope.

[0061] 2 Experimental results

[0062] The results are as Figure 2 shown in Figure A. After wild-type melanoma cells A375WT were treated with different concentrations of Vemurafenib, the cell number decreased significantly. However, for drug-resistant melanoma cells A375VR (cells resistant to Vemurafenib), with the increase in the concentration of Vemurafenib, the cell number did not change significantly, indicating that the construction of the drug-resistant cell line was successful.

[0063] Example 6: Determination of the half-inhibitory concentration of Vemurafenib

[0064] 1 Experimental method

[0065] In this experiment, wild-type cells and drug-resistant cells were simultaneously inoculated into a 96-well plate and cultured in DMEM medium supplemented with 10% FBS, 1% double antibody, 1% non-essential amino acids, and 0.1% mercapto reducing agent for 48 hours. After stimulating wild-type melanoma cells A375WT and drug-resistant melanoma cells A375VR with different concentrations of Vemurafenib, CCK-8 and DMEM complete medium were added to the 96-well plate in a ratio of 1:9 for reaction. The OD value at 450 nm was measured by an enzyme-labeled instrument to determine the drug concentration at which the cells were half-lethal.

[0066] 2 Experimental results

[0067] The results are as Figure 2 shown in Figure B. The half-inhibitory concentrations (IC50) of Vemurafenib for wild-type melanoma cells A375WT and drug-resistant melanoma cells A375VR were 0.12 μM and 8.953 μM, respectively. Melanoma cells A375VR showed resistance to Vemurafenib.

[0068] Example 7: mRNA expression levels of CYP27A1 in A375WT and A375VR

[0069] 1 Experimental methods

[0070] In this experiment, total RNA of A375WT and A375VR was extracted, 1 μg of RNA was reverse-transcribed into cDNA, and after real-time quantitative PCR, the mRNA expression levels of CYP27A1 in the two types of cells were compared. The Q-PCR reaction system had a total volume of 20 μL, including 0.5 μL each of upstream and downstream primers, 10 μL of PCR mix, 7 μL of ultrapure water, and 2 μL of reverse-transcribed cDNA.

[0071] 2 Experimental results

[0072] The results were as Figure 2 shown in Figure C. The expression of Cytochrome P450 family member 27A1 (CYP27A1) was significantly upregulated in the drug-resistant melanoma cell line A375VR.

[0073] Example 8: Effect of Dafadine-A on the survival of wild-type A375WT

[0074] 1 Experimental methods

[0075] In this experiment, drug-resistant cells were simultaneously seeded in 96-well plates and cultured in DMEM medium supplemented with 10% FBS, 1% double antibody, 1% non-essential amino acids, and 0.1% mercapto reducing agent for 48 h. Meanwhile, wild-type A375WT was stimulated with different concentrations of Dafadine-A. CCK-8 and DMEM complete medium were added to the 96-well plates at a ratio of 1:9 for reaction, and the OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0076] 2 Experimental results

[0077] The results were as Figure 2 shown in Figure D. Treatment of A375WT with different concentrations of Dafadine-A did not have a significant effect on cell survival.

[0078] Example 9: Inhibition of combined drug treatment on A375VR

[0079] 1 Experimental methods

[0080] In this experiment, drug-resistant cells were simultaneously seeded in 96-well plates and cultured in DMEM medium supplemented with 10% FBS, 1% double antibody, 1% non-essential amino acids, and 0.1% mercapto reducing agent. Two groups were added with Vemurafenib or a combination of Vemurafenib and Dafadine-A and cultured for 48 h. CCK-8 and DMEM complete medium were added to the 96-well plates at a ratio of 1:9 for reaction, and the OD value at 450 nm was measured using an ELISA reader.

[0081] 2 Experimental results

[0082] The results are as Figure 2 shown in E. Dafadine-A combined with Vemurafenib has a significant inhibitory effect on A375VR.

[0083] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0084] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a CYP27A1 inhibitor in the preparation of a medicament for treating melanoma, characterized in that, the CYP27A1 inhibitor is Dafadine-A.

2. The use according to claim 1, characterized in that, the CYP27A1 inhibitor in the medicament is the sole active ingredient or one of the active ingredients.

3. The use according to claim 2, characterized in that, when the CYP27A1 inhibitor in the medicament is one of the active ingredients, the active ingredients further include Vemurafenib.

4. The use according to claim 3, characterized in that, the CYP27A1 inhibitor and Vemurafenib are administered simultaneously or sequentially in any order.

5. The use according to claim 4, characterized in that, the medicament further includes a pharmaceutically acceptable carrier.

6. A medicament for treating melanoma, characterized in that, it includes the CYP27A1 inhibitor according to claim 1.

7. The medicament according to claim 6, characterized in that, in the medicament, the content of the CYP27A1 inhibitor is 0.1-99 wt%.

8. The medicament according to claim 6, characterized in that, the medicament further includes Vemurafenib.

9. The medicament according to claim 6, characterized in that, the molar ratio of the CYP27A1 inhibitor to Vemurafenib is 5-50:0.1-20.

10. The medicament according to claim 9, characterized in that, the dosage form of the medicament is a granule, tablet, capsule, pill, dripping pill, oral liquid preparation, gavage preparation or injection dosage form.

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