Application of small-molecule compound DPR-104 in preparation of medicine for treating uveal melanoma

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of a small molecule compound DRP (Dalicyclic Polymorphism)-104 in preparation of a medicine for treating uveal melanoma. The molecular formula of the DPR-104 is CHNO; an in-vitro experiment proves that the small molecular compound DPR-104 can effectively kill uveal melanoma cells, specifically, proliferation of the uveal melanoma cells is inhibited in an in-vitro concentration-dependent manner, and apoptosis of tumor cells is promoted; animal experiments prove that DPR-104 can effectively kill uveal melanoma in vivo and enhance the effect of uveal melanoma radiotherapy. On the basis, the invention provides a brand-new treatment medicine for uveal melanoma, the treatment effect can be effectively improved, and the life of a patient can be prolonged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a small molecule compound DRP-104 in preparation of a drug for treating uveal melanoma. BACKGROUND

[0002] Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with a high metastatic tendency. Due to the limited treatment methods, its mortality rate is high. More than 90% of uveal melanoma cases have activating mutations in GNAQ or GNA11 genes, leading to continuous activation of the Gαq / 11 signaling pathway and driving abnormal activation of downstream oncogenic pathways such as PKC-MAPK and FAK-YAP.

[0003] The existing targeted therapy drugs for uveal melanoma mainly focus on the classic oncogenic signaling pathways. For example, Gαq / 11 inhibitors YM-254890 and FR900359 exhibit significant tumor growth inhibition in preclinical studies, but due to their inability to distinguish between mutant and wild-type G proteins, they cause widespread inhibition of normal tissues, leading to serious systemic toxicity. Although inhibitors targeting downstream effectors such as PKC-MAPK and FAK-YAP signaling pathways show efficacy in cell and xenograft models, their clinical translation has been slow, and so far no drug has made a breakthrough in clinical trials. The current drug development dilemma may be due to the following reasons: ① compensatory activation of downstream pathways; ② heterogeneity of uveal melanoma's dependence on specific pathways; ③ the existing targets may not be the real fatal vulnerability of uveal melanoma.

[0004] In summary, the current targeted therapy for uveal melanoma faces the dual challenges of insufficient selectivity and gaps in mechanism understanding, and there is an urgent need to discover new targets or intervention drugs that specifically act on the core dependent mechanisms of tumors.

[0005] DRP-104 is a glutamine structural analog that can be activated and hydrolyzed to 6-diazo-5-oxo-L-norleucine (DON) in the tumor microenvironment by serine proteases, which then irreversibly binds to the active site of glutamine-utilizing enzymes (such as glutaminase GLS, phosphoribosyl pyrophosphate amidotransferase PPAT, cytidine triphosphate synthetase CTPS, and asparagine synthetase ASNS, etc.), blocking the glutamine metabolism of tumor cells. This mechanism can disrupt key metabolic processes such as intracellular TCA cycle, purine / pyrimidine synthesis, and non-essential amino acid synthesis, while inducing an anti-tumor immune response. Studies have shown that in a pancreatic cancer model, DRP-104 can inhibit tumor growth and synergize with the extracellular regulated protein kinase (ERK) inhibitor trametinib; in head and neck squamous cell carcinoma, it can enhance the sensitivity of tumor cells to ferroptosis. In addition, DRP-104 has a positive regulatory effect on immune cells, can activate CD8+ T cells and enhance the efficacy of PD-1 inhibitors. For example, in KEAP1 mutant lung cancer, DRP-104 can inhibit nucleotide metabolism and improve the response of tumors to immune checkpoint inhibitors.

[0006] Although DRP-104 exhibits significant anti-tumor activity in a variety of tumors, its efficacy and mechanism of action in uveal melanoma have not been reported. SUMMARY

[0007] To overcome the above-mentioned deficiencies, the present application aims to provide the application of small molecule compound DRP-104 in the preparation of uveal melanoma drugs, providing a new drug for the treatment of uveal melanoma.

[0008] To achieve the above-mentioned purposes, the specific technical solutions adopted by the present application are as follows:

[0009] In a first aspect, the present application provides the application of DRP-104 in the preparation of a drug for treating uveal melanoma, wherein the drug takes DRP-104 as the active ingredient, and the molecular formula of DRP-104 is C 22 H 27 N5O5, and the molecular structure is as shown below:

[0010]

[0011] Further, the DRP-104 can inhibit the proliferation of uveal melanoma cells, promote the apoptosis of uveal melanoma cells, and inhibit the tumor formation of uveal melanoma in vivo.

[0012] Further, the drug is applied alone or in combination with radiotherapy when administered to an individual.

[0013] Further, the administration mode of the drug includes intraperitoneal injection, oral administration, intravenous injection, subcutaneous injection, intravitreal injection, etc.

[0014] Further, the radiotherapy is eye in situ ray radiotherapy.

[0015] In a second aspect, the present application provides an application of DRP-104 in preparing a drug for enhancing the radiotherapy effect of uveal melanoma.

[0016] Further, the administration of DRP-104 is performed before radiotherapy.

[0017] In a third aspect, the present application provides a pharmaceutical composition for treating uveal melanoma, which comprises DRP-104 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0018] “Pharmaceutically acceptable” means that when the substances are properly administered to animals or humans, they do not produce adverse, allergic or other adverse reactions. The pharmaceutically acceptable excipient can be selected from one or more of diluents, fillers, surfactants, absorption promoters, disintegrants, wetting agents, dispersants, etc. In a preferred embodiment, the pharmaceutically acceptable excipient comprises the following components: DMSO, PEG300, Tween 80, ddH2O.

[0019] The present application has the following beneficial effects:

[0020] The present application provides an application of a small molecule in preparing a drug for treating uveal melanoma. The small molecule in the present application is DRP-104, which can effectively kill uveal melanoma cells, specifically showing that it inhibits the proliferation of uveal melanoma cells in a concentration-dependent manner in vitro, and promotes the apoptosis of tumor cells. Further, DRP-104 can effectively kill uveal melanoma in vivo and enhance the effect of radiotherapy of uveal melanoma. The present application provides a brand-new therapeutic drug for uveal melanoma, which can effectively improve the therapeutic effect and prolong the life of patients. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The experimental results in Example 1 of the present application demonstrate that DRP-104 can effectively inhibit the proliferation of uveal melanoma cells in vitro. In the figure: the left is the CCK-8 cell proliferation curve of human uveal melanoma cell line 92.1 treated with different concentrations of DRP-104, and the right is the CCK-8 cell proliferation curve of human uveal melanoma cell line OMM1 treated with different concentrations of DRP-104.

[0022] Figure 2The experimental results of Example 2 of the present application demonstrate that DRP-104 can effectively promote apoptosis of uveal melanoma cells in vitro.

[0023] Figure 3 The experimental results of Example 3 of the present application demonstrate that DRP-104 can inhibit uveal melanoma tumorigenesis and enhance the effect of radiotherapy in vivo. In the figure: A is the result of measuring tumor activity using the IVIS Spectrum small animal live optical imaging system, and B is the result of dissecting and sizing the tumor. DETAILED DESCRIPTION

[0024] The present application is further described below in conjunction with the accompanying drawings and specific examples. In the following examples, the reagents involved are commercially available conventional reagents unless otherwise specified, and the experimental operations involved are conventional operations in the art unless otherwise specified.

[0025] Example 1: DRP-104 can effectively inhibit the proliferation ability of uveal melanoma cells in vitro

[0026] Experimental materials: human uveal melanoma cell lines 92.1 and OMM1, DMSO, DRP-104 purchased from Selleck (USA), CCK-8 purchased from Dojindo (Japan).

[0027] Experimental steps: Take the 92.1 and OMM1 cells in the logarithmic culture period, count and resuspend according to the concentration of 2 000 cells / 100 μl. Set up the DMSO control group (Vehicle) and the 0.5, 1, 2 μM DRP-104 treatment group (DRP-104 is dissolved in DMSO to obtain a 10 μM stock solution, and then diluted to the desired concentration with complete culture medium), and inoculate 92.1 and OMM1 into 96-well plates at a density of 2 000 cells / well. At 0, 24, 48, 72, 96 hours, add 10 μl of CCK-8 solution to the corresponding wells, continue to culture in the incubator for 2 hours, and use the enzyme labeler to detect the absorbance of the corresponding wells at 450 nm. According to the detection results, draw the cell proliferation curve.

[0028] Experimental results: CCK-8 proliferation experiment shows that ( Figure 1 ), 0.5, 1, 2 μM of DRP-104 can effectively inhibit the proliferation ability of uveal melanoma cells.

[0029] Example 2: DRP-104 can effectively promote apoptosis of uveal melanoma cells in vitro

[0030] Experimental materials: human uveal melanoma cell lines 92.1 and OMM1, DMSO, DRP-104 were purchased from Selleck (USA), Caspase-3 antibody was purchased from CST (USA), Cleaved Caspase-3 antibody was purchased from Abeam (USA).

[0031] Experimental steps: Take the logarithmic growth period of uveal melanoma cells, count and inoculate 92.1 and OMM2.3 into 6-well plates at a density of 500 000 cells per well, set up DMSO control group (Vehicle) and 0.5, 1 μM DRP-104 treatment group. After 48 hours of drug treatment, the culture was terminated, and the next experiment was carried out. Collect adherent and supernatant suspended cells for Western Blot experiment to detect the protein expression level of apoptosis related protein Caspase-3 and Cleaved Caspase-3.

[0032] Experimental results: As shown in Figure 2 , Western Blot experiment detected the effect of DRP-104 treatment on apoptosis marker protein, and found that DRP-104 could promote the cleavage of Caspase-3, and the expression level of activated form Cleaved Caspase-3 was up-regulated, which confirmed that the cell apoptosis occurred.

[0033] Example 3: DRP-104 inhibits uveal melanoma tumor formation in vivo and enhances the effect of radiotherapy

[0034] Experimental materials: human uveal melanoma cell lines 92.1 and OMM1, BALB / c nude immunodeficient mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. (China), DMSO, PEG300, Tween 80, DRP-104 were purchased from Selleck (USA).

[0035] Experimental steps: BALB / c nude immunodeficient mice were used to construct xenotransplant tumor model, i.e. 3×10 5 Uveal melanoma cells labeled with luciferase (Luciferase) were injected into the suprachoroidal space of mice to form tumors in situ in the eyeball. On the 14th day after injection, the mice were randomly divided into groups and treated:

[0036] 1) Control group (Vehicle), intraperitoneal injection of 100 ul solvent, solvent formula: 5% DMSO + 40% PEG300 + 5% Tween80 + 50% ddH2O.

[0037] 2) DRP-104 group, two cycles of DRP-104 treatment were performed on day 14 and day 21. DRP-104 was dissolved in the same solvent as in 1), and was intraperitoneally injected at a drug dose of 2 mg / kg / d, continuously for 5 days, and then stopped for 2 days.

[0038] 3) RT group, two cycles of 4 Gy dose of gamma-ray radiotherapy were performed on day 16 and day 23.

[0039] 4) Comb group, two cycles of DRP-104 treatment were performed on day 14 and day 21. And radiotherapy was performed on day 16 and day 23.

[0040] At the end of the observation on day 30, the tumor activity was measured using the IVIS Spectrum small animal live optical imaging system, and then the mice were euthanized, and the tumors were dissected and sized.

[0041] Experimental results: As shown in Figure 3 A-B, DRP-104 can significantly inhibit the proliferation of uveal melanoma in vivo, and the tumor activity and size of the combination therapy group are further reduced. Therefore, it is proved by animal experiments that DRP-104 can significantly inhibit the proliferation of uveal melanoma in vivo, and can further enhance the effect of radiotherapy, providing a potential new drug for the treatment of uveal melanoma.

[0042] This specific embodiment is only an explanation of the present application, and is not a limitation of the present application. Any changes made by those skilled in the art after reading the specification of the present application will be protected by the patent law as long as it is within the scope of the claims of the present application.

Claims

1. Use of DRP-104 in the manufacture of a medicament for the treatment of uveal melanoma, characterized in that, The medicine takes DRP-104 as active ingredient, the molecular formula of the DRP-104 is C 22 H 27 N5O5, and the molecular structure is shown as follows: 。 2. Use according to claim 1, characterized in that, The DRP-104 can inhibit the proliferation of uveal melanoma cells, promote the apoptosis of uveal melanoma cells, and inhibit the tumor formation of uveal melanoma in vivo.

3. Use according to claim 1, characterized in that, The drug is used alone when administered to the individual.

4. Use according to claim 1, characterized in that, The drug is used in combination with radiotherapy when administered to the individual.

5. Use according to claim 3 or 4, characterized in that, The administration mode of the drug includes intraperitoneal injection, oral administration, intravenous injection, subcutaneous injection, and intravitreal injection.

6. Use according to claim 4, characterized in that, The radiotherapy is eye in situ ray radiotherapy.

7. Use of DRP-104 in the preparation of a drug for enhancing the efficacy of radiotherapy for uveal melanoma.

8. Use according to claim 7, characterized in that, The administration of the DRP-104 is performed before radiotherapy.

9. A pharmaceutical composition for treating uveal melanoma, characterized by, The drug comprises DRP-104 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutically acceptable excipient comprises the following components: DMSO, PEG300, Tween 80, and ddH2O.

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