Application of JTC801 as macrovesicle-like cell death inducer in tumor resistance

Abstract

The invention provides an application of JTC801 as a macrovesicle-like cell death inducer in tumor resistance. Specifically, the invention relates to JTC801 as a small molecule kinase inhibitor drug, the JTC801 has a good death induction effect on tumor cells, especially uveal melanoma cells, and the induced cell death form is not classical apoptosis, pyroptosis, ferroptosis and the like, but is a giant vesicle-like death mode. The death mode is similar to giant vesicle type death, a large number of vacuoles are accumulated in cytoplasm, but different from the giant vesicle type death mode, the death mode does not have the phenomenon that fusion of lysosome and macropinosome is inhibited, and therefore the death mode is defined as the giant vesicle type death mode. Therefore, the JTC801 as a newly discovered macrovesicle-like cell death inducer has a good application prospect in tumor treatment, especially in treatment of uveal melanoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to cell death induction applications and related drugs. Specifically, it relates to the mechanism of action, death effect, and implementation method and use of a small molecule kinase inhibitor, JTC801, as an inducer of giant vesicular cell death in antitumor therapy. Background Technology

[0002] Cell death is a complex and interconnected process that plays a crucial role in maintaining tissue homeostasis and preventing disease. Targeting cell death processes can be used to inhibit tumor progression, making it a viable cancer treatment strategy. Most tumor cells exhibit resistance to the classic death mechanism—apoptosis—during treatment. Finding alternative cell death mechanisms as targets for cancer therapy could help improve treatment efficacy for cancer patients.

[0003] Uveal melanoma (UM) is a type of malignant tumor of the eye in adults, and is the second most common type of melanoma after cutaneous melanoma, with characteristics distinct from cutaneous melanoma. UM is highly aggressive and malignant, with over 50% of diagnosed patients developing hematogenous metastases, most commonly involving the liver (>90%). The median survival for patients with liver metastases is only 4-6 months, and the 2-year survival rate is less than 8%. Furthermore, compared to patients abroad, the age of onset for UM patients in my country is relatively low (47.3 vs. 60), requiring sufficient attention and concern. Although various strategies have achieved some success in recent years in treating UM patients, such as immune checkpoint therapy represented by PD-1 / PD-L1 antibodies, CAR-T therapy, DC vaccines, and TCR / anti-CD3 bispecific fusion protein targeting the HLA-0201-restricted tumor antigen peptide gp100 (Tebentafusp / IMCgp100), the overall survival rate has not significantly improved. Therefore, there is an urgent need to develop new therapeutic drugs to improve the treatment effect of UM, prolong the survival time of patients, and improve their quality of life.

[0004] The human kinase genome consists of approximately 560 protein kinases, which participate in physiological processes such as cell proliferation, apoptosis, and subcellular translocation by mediating protein phosphorylation and dephosphorylation, playing important roles in inflammation, metabolic diseases, and tumorigenesis. In 2001, the FDA approved the first kinase inhibitor (MKI), imatinib, for the treatment of chronic myeloid leukemia (CML), marking the rise of molecular targeted therapy and making MKIs a popular treatment option for cancer. Currently, 71 small molecule kinase inhibitors (SMKIs) have been approved by the FDA, mostly for cancer treatment. For example, kinase inhibitors targeting EGFR mutations have shown good efficacy in non-small cell lung cancer, significantly improving patient survival. However, novel kinase inhibitors for tumors such as umbilical leukemia (UM) are still in the research stage, and no clear therapeutic effects have yet been observed. A key characteristic of UM is that approximately 90% of patients carry activating mutations in the G protein subunits Q and 11 (GNAQ / 11) genes, activating the intracellular mitogen-activated protein kinase (MAPK) signaling pathway. Data from an ongoing Phase I / II clinical trial (NCT03947385) shows that the protein kinase C (PKC) inhibitor Darovasertib (IDE196) demonstrates promising therapeutic efficacy in patients with metastatic UM and has received FDA orphan drug designation. Therefore, SMKIs hold promise for UM treatment. Discovering SMKIs capable of inducing novel modes of death in UM is of significant scientific and clinical value for improving the treatment outcomes of malignant UM tumors. Summary of the Invention

[0005] This invention builds upon the aforementioned research, employing a small molecule kinase inhibitor library to screen for small molecule inhibitors with strong tumor cell killing toxicity against human UM cell lines, and exploring their mechanisms of inducing tumor cell death. This invention reveals that JTC801 exhibits excellent cell death induction activity in human UM cell lines. The induced cell death mechanism is not the classic apoptosis, pyroptosis, or ferroptosis, but rather a macrovesicular-like cell death. This mechanism resembles macrovesicular cell death, with the accumulation of numerous vacuoles in the cytoplasm. However, unlike classic macrovesicular cell death, this mechanism does not involve the inhibition of lysosomal and macropinocytosome fusion, and is accompanied by dysfunction and metabolic disorders of organelles such as lysosomes and mitochondria. Therefore, it is defined as a macrovesicular-like cell death mechanism. Thus, JTC801, as a newly discovered macrovesicular-like cell death inducer, shows promising application potential in tumor therapy, especially in the treatment of uveal melanoma.

[0006] The purpose of this invention is to provide the application, mechanism of action, death effect, and use of the small molecule kinase inhibitor JTC801 as a macrovesicular cell death inducer in antitumor therapy, especially in the treatment of uveal melanoma. Another purpose of this invention is to provide a pharmaceutical composition for inducing macrovesicular cell death in antitumor therapy.

[0007] In a first aspect, the invention provides a novel use of the small molecule kinase inhibitor JTC801 as an inducer of giant vesicle-like cell death.

[0008] In one embodiment of the present invention, the giant bubble-like death mode has the following characteristics:

[0009] (i) A large number of monomembrane vacuoles accumulate in the cytoplasm;

[0010] (ii) Cytoplasmic vacuoles are caused by macropinocytosis or interruption of endosomal lysosomal transport;

[0011] (iii) There is no phenomenon of inhibited fusion of intracellular lysosomes and macrocytic pinocytosis.

[0012] In one embodiment of the present invention, the induction mechanism of the giant bubble-like death mode has the following characteristics:

[0013] (i) The expression of glucose transporter solute carrier family 2 member 1 (SLC2A1) and amino acid transporter SLC7A5 was downregulated;

[0014] (ii) Mitochondrial membrane potential is impaired, the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) are significantly downregulated, ROS are increased, and lipid peroxidation accumulates;

[0015] (iii) Loss of lysosomal membrane integrity;

[0016] (iv) Macrocytic pinocytosis and lysosomes form fusion vacuoles.

[0017] Preferably, the cells are uveal melanoma cells derived from mammals such as humans, mice, rabbits, and sheep.

[0018] In one embodiment of the present invention, in a cell culture model, the recommended induction dose of JTC801 is 3-5 μM, and the induction time is 24-36 h.

[0019] In a second aspect, the present invention provides a giant vesicle-like cell death inducer, the active ingredient of which is JTC801, and further comprises pharmaceutically or immunologically usable excipients.

[0020] In a third aspect, the invention provides the application of JTC801 as a giant vesicle-like cell death inducer in tumor treatment, wherein the tumor is preferably uveal melanoma.

[0021] In terms of its mechanism of action, JTC801 inhibits tumor proliferation, promotes tumor-like vesicular cell death, and suppresses tumor metastasis. Specifically:

[0022] In one embodiment of the present invention, the JTC801 inhibits tumor cell proliferation. The concentration of JTC801 used to inhibit tumor cell proliferation is 3 μM, and the induction time is 24h-72h. The tumor cells include human uveal melanoma cells and mouse skin melanoma cells.

[0023] In one embodiment of the present invention, JTC801 exerts a cytotoxic effect by killing tumor cells. The concentration of JTC801 used to kill tumor cells is 3 μM, and the induction time is 24h-72h. The tumor cells include human uveal melanoma cells and mouse skin melanoma cells. The way JTC801 kills uveal melanoma cells and induces cell death is through giant vesicular cell death.

[0024] In one embodiment of the present invention, JTC801 can inhibit tumor progression and metastasis in vivo. The administration routes of JTC801 can be oral, intratumoral injection, intraocular injection, or intraperitoneal injection. The recommended dosage of JTC801 is 5-20 mg / kg, exhibiting good antitumor activity. The efficacy of JTC801 is closely related to the duration of administration, with a recommended dosing period of 7-14 consecutive days. The tumors mentioned are human uveal melanoma and mouse cutaneous melanoma, but the possibility of similar effects in other tumors cannot be ruled out. The dosage and duration of administration have good biocompatibility. This is also a method for treating tumors with JTC801.

[0025] A fourth aspect of the present invention provides an antitumor drug combination that induces macrovesicular cell death, characterized in that it comprises:

[0026] (i) a therapeutically effective dose of JTC801; and

[0027] (ii) Pharmaceutically or immunologically acceptable carriers or excipients.

[0028] Furthermore, the JTC801 component in the pharmaceutical composition accounts for 0.001 to 99.9 wt% of the total weight of the pharmaceutical composition, preferably 1 to 95 wt%, more preferably 5 to 90 wt%, and even more preferably 10 to 80 wt%. The balance consists of pharmaceutically acceptable carriers and other additives.

[0029] Furthermore, other antitumor active substances are administered before, simultaneously with, or after the pharmaceutical composition of the present invention. These other active substances have antitumor effects and may be chemotherapeutic drugs, targeted drugs, or other drugs with antitumor effects.

[0030] In another embodiment of the invention, the tumor is uveal melanoma, but its use in inducing giant vesicular cell death in other tumors is not excluded.

[0031] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this invention. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein.

[0032] Beneficial technical effects of the present invention:

[0033] This invention, through extensive research and animal model experiments, screened and obtained JTC801, a small molecule inhibitor with the most significant cytotoxic effect against uveal melanoma cytokines. Based on this, the mechanism of action of JTC801 against uveal melanoma was studied. This invention is the first to discover that JTC801 acts as a macrovesicle-like cell death inducer in tumor cells. This previously undiscovered inducer of an apoptosis-independent cell death type exhibits good anti-cancer effects and biosafety, and has significant clinical implications for improving the treatment efficacy of uveal melanoma patients with apoptosis resistance. It can also serve as a candidate for anti-cancer drug combinations, providing new exploration and technical support for tumor treatment, especially the treatment of uveal melanoma. Attached Figure Description

[0034] Figure 1 The results show that JTC801 inhibits the proliferation of uveal melanoma cells and promotes their death: A, JTC801 inhibits the proliferation of MUM2B and OMM2.5 uveal melanoma cells at a concentration of 3 μM. (Figure: CCK8 proliferation analysis); B, JTC801 promotes an increased proportion of MUM2B and OMM2.5 cell death at a concentration of 3 μM. (Figure: Flow cytometry analysis) (***, P < 0.001).

[0035] Figure 2 The study showed that JTC801 induced giant vesicle-like cell death in tumor cells: A, the overall morphological changes of MUM2B cells after JTC801 treatment (20x objective); B, cell morphology under electron microscopy after 16 h of treatment with JTC801 (3 μM).

[0036] Figure 3 The study showed that the expression of glucose transporter solute carrier family 2 member 1 (SLC2A1) and amino acid transporter SLC7A5 was downregulated in MUM2B cells after treatment with JTC801. The figure shows the Western blot.

[0037] Figure 4 The JTC801 treatment resulted in lysosomal membrane leakage in MUM2B cells. The figure shows the flow cytometry analysis (**, P < 0.01; ***, P < 0.001).

[0038] Figure 5 The results show that JTC801 impairs energy metabolism in MUM2B cells: A shows that JTC801 treatment of MUM2B cells reduces ATP production in mitochondria. The figure shows the analysis using a multi-mode microplate reader (*, P<0.05; **, P<0.01); B shows that JTC801 treatment of MUM2B cells increases intracellular reactive oxygen species (ROS). The figure shows the flow cytometry analysis (*, P<0.05).

[0039] Figure 6 The study showed that gavage treatment with JTC801 significantly inhibited the growth of subcutaneous tumors of MUM2B uveal melanoma. The figure shows the tumor growth curves in mice (**, P < 0.01; ***, P < 0.001).

[0040] Figure 7 Intratumoral injection of JTC801 significantly inhibited the growth and metastasis of MUM2B uveal melanoma in situ and prolonged the survival of mice. The figure shows the tumor growth curve and survival curve of mice (*, P<0.05; **, P<0.01).

[0041] Figure 8 Intratumoral injection of JTC801 significantly inhibited the growth of B16F10 cutaneous melanoma and prolonged the survival of mice. The figure shows the tumor growth curve and survival curve of mice (*, P<0.05; **, P<0.01).

[0042] Figure 9 The results show that JTC801 treatment did not cause any toxic side effects on the heart, liver, spleen, lungs, kidneys, or stomach. The image shows hematoxylin-eosin (HE) staining. Detailed Implementation

[0043] The following are specific embodiments of the present invention, intended to further illustrate the antitumor effect of JTC801 and the selection of administration methods. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.

[0044] JTC801, molecular formula C 26 H 26 ClN3O2, the structural formula is as follows:

[0045]

[0046] As used herein, the term "JTC801" includes the JTC801 compound itself and any modifications, variants, isomers, or derivatives thereof that use JTC801 as an active ingredient, as well as pharmaceutically acceptable salts or esters thereof. The JTC801 compound itself is a compound having the above-described chemical structure.

[0047] JTC801 can be structurally modified and optimized using conventional techniques in the art, such as improving its stability, bioavailability, and toxicity. The compounds of this invention can also be used in pharmaceutically or physiologically acceptable forms derived from acid or base salts. These salts include (but are not limited to) salts formed with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or hydroxyethanesulfonic acid. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, calcium, potassium, or magnesium), as well as esters, carbamates, or other conventional forms.

[0048] As used herein, the term "JTC801" also includes carriers for therapeutic administration, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (MackPub.Co., NJ 1991).

[0049] Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8.

[0050] The source of the JTC801 described in this invention is not particularly limited and can be any commercially available JTC801, such as one purchased from MCE, part number 244218-51-7.

[0051] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0052] Experimental methods not specifically described in the following examples may be performed using conventional methods in the art, such as referring to Molecular Cloning: A Laboratory Manual (3rd Edition, New York: Cold Spring Harbor Laboratory Press, 1989) or following the conditions recommended by the supplier.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0054] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0055] The compositions of the present invention can be prepared into dosage forms required for single-dose administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0056] It should be understood that the effective dose of JTC801 used may vary depending on the severity of the condition of the patient being treated. The specific dosage is determined based on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), within the judgment of a skilled physician.

[0057] The compositions of the present invention can be in solid form (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (e.g., oral liquid) or other suitable forms. The routes of administration can be: oral administration; intratumoral administration.

[0058] In addition, the compositions of the present invention may also contain substances that enhance the therapeutic effects of other active substances on tumors, the other active substances being selected from the group consisting of: commonly used chemotherapy drugs; targeted therapy drugs; and other tumor treatment strategies.

[0059] Example 1: JTC801 inhibits the proliferation of uveal melanoma cells and promotes the death of uveal melanoma cells.

[0060] Uveal melanoma cells MUM2B and OMM2.5 were seeded at 5000 cells / well in 96-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). JTC801 was added to the experimental group to a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. At time points of 0, 6, 12, 24, and 48 hours, 10 μL of CCK8 solution was added to each well for an additional 1 hour of incubation. OD values ​​at 450 nm were read using a BioTek Cytation 5 microplate reader. Cell growth was observed as follows: Figure 1 As shown in Figure A.

[0061] MUM2B and OMM2.5 cells were grown at 1×10⁻⁶. 5 Cells were seeded at a density of 1 / ml in 6-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). The experimental group received JTC801 at a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. At 24 and 48 h, the supernatant and digested cells were collected. The supernatant and cells were mixed and centrifuged. The cell pellet was stained with Annex V-FITC dye at room temperature in the dark for 20 min, followed by the addition of propidium iodide (PI) dye. Apoptosis was immediately detected by flow cytometry. The results are as follows: Figure 1 As shown in B.

[0062] The results showed that JTC801 treatment reduced the proliferation capacity of MUM2B and OMM2.5 cells and increased apoptosis.

[0063] The results indicate that JTC801 inhibits the proliferation of uveal melanoma cells and has the ability to kill uveal melanoma cells.

[0064] Example 2: JTC801 induces macrovesicle-like cell death in tumor cells

[0065] MUM2B cells were seeded at a rate of 5000 cells / well in 96-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). Then, JTC801 was added to the wells to a final concentration of 3 μM, and the cells were cultured for another 16 hours. During this time, images were taken using an Incucyte imaging system to observe intracellular vacuolation and cell death. Results are as follows: Figure 2 As shown in Figure A, the white arrows indicate the giant vacuoles formed within the cells. 3 μM JTC801 induces the formation of numerous vacuoles within tumor cells and induces giant vacuolar cell death.

[0066] Uveal melanoma cells MUM2B and OMM2.5 were mixed at a ratio of 2×10⁻⁶. 5Cells were seeded per well in 6-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). The experimental group received JTC801 at a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. After 16 hours, the culture medium was discarded, and each well was fixed with 2.5% glutaraldehyde at room temperature for 1 hour. Cells were then washed repeatedly with PBS (phosphate-buffered saline) 3-4 times, 5-10 minutes each time. The fixed cell samples were then sequentially placed in different concentrations of ethanol (50%, 70%, 80%, 90%, 100%) for 10-20 minutes each time, followed by multiple applications of 100% ethanol to ensure complete dehydration. The samples were then embedded in resin and placed in an oven (60°C) for 24 hours to complete the embedding process. After sectioning, the samples were placed on an electron microscope grid for transmission electron microscopy observation. Results are as follows: Figure 2 As shown in B, the white arrow indicates a giant vacuole formed inside the cell.

[0067] The results showed that 3 μM JTC801 could induce the formation of a large number of vacuoles in tumor cells and induce giant vesicle-like cell death.

[0068] The results indicate that JTC801 can induce giant bubble-like cell death in tumor cells.

[0069] Example 3: JTC801 downregulates the expression of MUM2B glucose transporter solute carrier family 2 member 1 (SLC2A1) and amino acid transporter SLC7A5.

[0070] MUM2B cells were fed at 1.5 × 10⁻⁶ 5 Cells were seeded at a density of 1 / ml in 6-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). The experimental group received JTC801 at a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. Cells were digested and collected at 0, 1, 2, 4, 8, and 12 hours, and lysed with whole-cell lysis buffer containing protease inhibitors. Proteins were obtained by centrifugation at 12,000 rpm for 15 minutes. Separation was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to a nitrocellulose blot membrane. After blocking with TBST solution containing 5% skim milk powder, the membrane was incubated overnight at 4°C with primary antibodies (including SLC2A1, SLC7A5, and GAPDH), followed by incubation with secondary antibodies. Immunoblotting was performed using a high-sensitivity chemiluminescent substrate and detected using a chemiluminescent protein blot scanner. GAPDH was used as an internal control.

[0071] The results showed that the expression levels of SLC2A1 and SLC7A5 proteins decreased with increasing JTC801 (3 μM) treatment time.

[0072] The results indicate that treatment with 3 μM JTC801 can inhibit the protein expression of glucose transporter SLC2A1 and amino acid transporter SLC7A5 in MUM2B cells.

[0073] Example 4: JTC801 increases intracellular lysosomal permeability in MUM2B cells

[0074] MUM2B cells were used at 1×10 5 Cells were seeded at a density of 1 / ml in 12-well plates and cultured overnight in a CO2 incubator (37℃, 5% CO2). JTC801 was added to the experimental group to a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. After 24 hours, the original cell culture medium was replaced with acridine orange solution (1 μg / mL), and the cells were incubated in a CO2 incubator for 30 minutes. Cells were washed 2-3 times with PBS to remove acridine orange that had not yet entered the cells. Cells were then digested, collected, and analyzed by flow cytometry. Results are shown below. Figure 4 As shown.

[0075] The results showed that treatment with 3 μM JTC801 increased the fluorescence intensity of FTIC, which represents the disruption of lysosomal membrane integrity, and decreased the fluorescence intensity of PE, which represents lysosomal membrane integrity.

[0076] The results indicate that JTC801 induces increased lysosomal permeability in MUM2B cells.

[0077] Example 5: JTC801 induced impaired intracellular energy metabolism in MUM2B cells

[0078] 5000 MUM2B cells / well were seeded into 96-well plates. The experimental group received JTC801 to a final concentration of 3 μM, while the control group received the corresponding volume of DMSO. The experiments were performed according to the manufacturer's instructions for the enhanced ATP assay kit (S0026, Beyotime). At 0h, 2h, 4h, and 8h, cells were lysed with 100 μL of lysis buffer per well, followed by centrifugation at 12,000×g for 15 minutes, and the cell supernatant was collected. 100 μL of freshly prepared ATP assay working solution was added to each well of the 96-well plate and incubated at room temperature for 5 minutes. Then, the cell lysis supernatant was added to the wells and mixed thoroughly. The plates were immediately read using a Synergy microplate reader. TM H1 (BioTek) measured the luminescence value. The results are as follows: Figure 5 As shown in Figure A.

[0079] MUM2B cells were used at 1×10 5Cells were seeded at a density of / ml in 12-well plates and cultured overnight in a CO2 incubator (37℃, 5% CO2). JTC801 was added to the experimental group to a final concentration of 3μM, while the control group received the corresponding volume of DMSO. After 16 hours, the original cell culture medium was replaced with 10μM working solution of DCFH-DA, and the cells were incubated in a CO2 incubator for 30 minutes. Cells were gently washed 2-3 times with PBS or culture medium to remove excess DCFH-DA that had not yet entered the cells; cells were then digested and collected, and analyzed by flow cytometry. Results are shown below. Figure 5 As shown in B.

[0080] The results showed that after treatment with 3 μM JTC801, ATP production gradually decreased over time, while ROS production increased.

[0081] The results indicate that JTC801 causes oxidative damage and energy metabolism disorder in MUM2B cells, suggesting impaired mitochondrial function.

[0082] Example 6: Gavage treatment with JTC801 significantly inhibited the growth of subcutaneous tumors of MUM2B uveal melanoma.

[0083] Select 6-8 week old male nude mice, using 5×10 6 MUM2B cells were administered subcutaneously to mice. Starting on day 7, mice were treated with JTC801 via gavage at a dose of 10 mg / kg once daily for 14 consecutive days. The control group received PBS solution instead of JTC801. Tumor size was measured every other day during the experiment, and mouse survival time was recorded until all mice in one group died. The longest diameter (a mm) and shortest diameter (b mm) of the tumor were used for measurement. Tumor size was calculated using the formula a × b × b / 2 (mm). 3 The result is as follows: Figure 6 As shown.

[0084] The results showed that gavage treatment with JTC801 (10 mg / kg) reduced the volume of MUM2B subcutaneous tumors.

[0085] The results indicate that JTC801 (10 mg / kg) can inhibit the growth of subcutaneous tumors of MUM2B uveal melanoma via gavage.

[0086] Example 7: Intratumoral injection of JTC801 significantly inhibited the growth and metastasis of MUM2B uveal melanoma in situ and prolonged the survival of mice.

[0087] Select 6-8 week old male nude mice, and use 5×10⁻⁶ mice with luciferase. 6MUM2B cells were administered via intraocular uveal injection to mice at a dose that was 10 mg / kg. Starting on day 7, mice were treated with JTC801 via intratumoral injection, once daily for 14 days. The control group received PBS solution instead of JTC801. Tumor fluorescence intensity was measured weekly using a small animal in vivo imaging system, and mouse survival time was recorded until all mice in one group died. Results are as follows: Figure 7 As shown.

[0088] The results showed that intratumoral treatment with JTC801 (10 mg / kg) inhibited the growth of MUM2B in situ tumors, reduced the metastasis rate, and increased the survival rate.

[0089] The results indicate that JTC801 (10 mg / kg) can inhibit the growth and metastasis of MUM2B uveal melanoma in situ and prolong the survival of mice through intratumoral therapy.

[0090] Example 8: Intratumoral injection of JTC801 significantly inhibited the growth of B16F10 cutaneous melanoma and prolonged the survival of mice.

[0091] Select 6-8 week old male immunocompetent mice (C57BL / 6) and use 3×10⁻⁶ mice with luciferase. 5 B16F10 cells / mouse were administered subcutaneously, followed by intratumoral injection of JTC801 starting on day 7 at a dose of 20 mg / kg, administered every other day for 14 days. The control group received PBS solution instead of JTC801. Tumor fluorescence intensity was measured weekly using a small animal in vivo imaging system, and mouse survival time was recorded until all mice in one group died. Results are as follows: Figure 8 As shown.

[0092] The results showed that intratumoral treatment with JTC801 (20 mg / kg) inhibited the growth of B16F10 melanoma and increased the survival rate of mice.

[0093] The results indicate that intratumoral treatment with JTC801 (20 mg / kg) also inhibits the growth of melanoma in immunocompetent mice.

[0094] Example 9: Intraperitoneal injection of JTC801 does not cause toxic side effects on the heart, liver, spleen, lungs, kidneys, and stomach.

[0095] Six- to eight-week-old male immunocompetent mice (C57BL / 6 and BALB / c) were selected and treated with JTC801 via intraperitoneal injection at a dose of 10 mg / kg, administered once daily for 21 days. The control group received PBS solution instead of JTC801. Mice were sacrificed on day 21, and heart, liver, spleen, lung, kidney, and stomach tissues were collected from both groups. These tissues were fixed with paraformaldehyde for 24 hours and then dehydrated by soaking in different concentrations of ethanol (70%, 80%, 90%, 95%, and 100%) for 1 hour at each concentration. The dehydrated tissue samples were then immersed in xylene for 30 minutes each time, three times, until the tissue samples were completely transparent. The transparent tissue samples were then immersed in molten paraffin for 4 hours to ensure complete paraffin infiltration. The paraffin-impregnated tissue samples were placed in embedding molds, molten paraffin was added, and after cooling and solidification, the tissue blocks were removed. The paraffin-embedded tissue blocks were cut into sections 4-6 micrometers thick using a microtome and placed on glass slides. The sections were dewaxed in xylene for 10 minutes each time, three times in total. The sections were then sequentially passed through 100%, 95%, 90%, 80%, and 70% ethanol for 10 minutes each time, and finally rinsed with distilled water to ensure complete hydration. The sections were then immersed in hematoxylin staining solution for 5 minutes. The sections were rinsed with tap water. The sections were then dehydrated sequentially by passing through 70%, 80%, 90%, 95%, and 100% ethanol for 5 minutes at each concentration. The dehydrated sections were then cleared in xylene for 5 minutes each time, three times in total. Mounting adhesive was added, and a coverslip was gently placed over the sections. After drying, the tissue morphology was observed under a microscope, and toxicity was evaluated. Results are as follows: Figure 9 As shown.

[0096] The results showed that tissue section analysis indicated that JTC801 did not affect the physiological state of the heart, liver, spleen, lungs, kidneys, and stomach in mice.

[0097] These results indicate that JTC801 has good biocompatibility at the dosage used.

[0098] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of JTC801 as a giant vesicle-like cell death inducer.

2. The application according to claim 1, characterized in that, The cells in question are uveal melanoma tumor cells.

3. A giant vesicle-like cell death inducer, characterized in that, Its active ingredient is JTC801, and it also contains pharmaceutically usable excipients.

4. The use of JTC801 as a giant vesicle-like cell death inducer in the preparation of antitumor drugs.

5. The use according to claim 4, characterized in that, The tumor is a uveal melanoma.

6. The use according to claim 5, characterized in that, JTC801 in combination with other anti-uveal melanoma drugs.

7. A pharmaceutical composition for inducing macrovesicular cell death in the treatment of tumors, characterized in that, Include: (i) Therapeutic effective dose of JTC801; (ii) Pharmaceutically or immunologically acceptable carriers or excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an oral formulation, an intratumoral injection formulation, or an intraocular injection formulation.

9. The pharmaceutical composition according to claim 7, characterized in that, The tumor is a uveal melanoma.

10. The pharmaceutical composition according to claim 7, characterized in that, The drug composition is used in combination with other drugs against uveal melanoma.

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