Use of tectorigenin in the preparation of a drug for treating multiple myeloma

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of iridoid glucoside in preparation of a medicine for treating multiple myeloma. The structure of the iridoid glucoside is shown in the following formula 1), and the application aims to provide a new application of the iridoid glucoside in treatment of multiple myeloma. Researches show that the iridoid glucoside can significantly inhibit the proliferation of multiple myeloma cells, reduce the cell viability and DNA synthesis level, and effectively inhibit the growth of tumors in vivo. Further mechanism researches show that the iridoid glucoside can inhibit the PI3K / AKT / mTOR / c-Myc pathway to resist cancer, and ensure that the medicine has no obvious tissue damage to the heart, liver, spleen, lung and kidney of mice at an effective dose. Formula 1).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of iris flavonoids in the preparation of drugs for treating multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is a malignant tumor caused by the abnormal proliferation of plasma cells, and its development is closely related to a complex signaling network. Signaling pathways and proliferation: Overactivation of the PI3K / AKT / mTOR pathway is a core factor driving MM cell proliferation and metabolism, while high expression of the downstream transcription factor c-Myc further promotes DNA replication and survival of cancer cells.

[0003] Although proteasome inhibitors (such as bortezomib, carfilzomib, and ixazomib), immunomodulatory agents (such as lenalidomide, thalidomide, and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and ixazomib), and bispecific antibodies are available that can significantly improve the prognosis of patients with relapsed or refractory multiple myeloma, these drugs eventually lead to drug resistance in most patients, resulting in disease relapse. Summary of the Invention

[0004] To develop a new approach for treating multiple myeloma and address the problem that existing drugs eventually develop resistance and lead to disease relapse in most patients, this invention provides the application of iris flavonoids in the preparation of drugs for treating multiple myeloma. This invention exerts a highly effective anti-tumor effect by inhibiting specific signaling pathways, and has no significant toxic side effects on vital organs, providing a new drug option for the clinical treatment of multiple myeloma. To achieve the above objectives, this invention adopts the following technical solution.

[0005] Iris flavonoids are natural active ingredients extracted from plants of the Iridaceae family (such as Belamcanda chinensis), possessing anti-inflammatory and antioxidant effects. However, their anticancer efficacy and complete mechanism of action in multiple myeloma, a specific malignant tumor, have not been fully elucidated. Therefore, this invention provides the application of iris flavonoids in the preparation of drugs for treating multiple myeloma. The structure of the iris flavonoids is shown in formula 1):

[0006] Formula 1).

[0007] Iris flavonoids inhibit the phosphorylation level of the PI3K / AKT / mTOR signaling pathway and downregulate the expression of downstream transcription factor c-Myc, thereby blocking the metabolic reprogramming and proliferation signals of tumor cells from the upstream. This differs from existing therapies such as proteasome inhibitors, immunomodulators, and monoclonal antibodies, which exert their effects through direct killing or immunomodulation, in terms of their target and molecular mechanism. Iris flavonoids act on the core metabolic-growth signaling axis of tumor cells, and their mechanism of action is distinct from existing drugs. For patients with relapsed or refractory multiple myeloma who have developed resistance to the three existing classes of drugs (proteasome inhibitors, immunomodulators, and anti-CD38 monoclonal antibodies), iris flavonoids hold promise as a new treatment option, effectively overcoming the drug resistance bottleneck of existing technologies and addressing the problem that many patients eventually develop drug resistance and relapse due to current technologies.

[0008] Furthermore, the drug uses iris flavonoids as its sole active ingredient.

[0009] Furthermore, the drug is a liquid preparation.

[0010] Furthermore, the drug is an injectable dosage form.

[0011] Furthermore, the drug also includes excipients.

[0012] Furthermore, the excipients are selected from any one or more of physiological saline, water for injection, glucose solution, propylene glycol, and polyethylene glycol.

[0013] Furthermore, the concentration of iris flavonoids in the drug is 0.08 μM to 500 μM.

[0014] Furthermore, the drug is used to inhibit the phosphorylation level of the PI3K / AKT / mTOR signaling pathway and downregulate the expression of downstream transcription factor c-Myc, thereby inhibiting the metabolism and growth of multiple myeloma cells.

[0015] Furthermore, the tumor cells are human multiple myeloma cell lines RPMI 8226 and NCI H929.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides the application of iris flavonoids in the preparation of drugs for treating multiple myeloma, offering a novel treatment strategy for multiple myeloma. The research results of this invention indicate that iris flavonoids, belonging to the natural flavonoid class, inhibit the proliferation of multiple myeloma cells, reduce cell viability and DNA synthesis levels. This can be achieved by inhibiting the phosphorylation level of key proteins in the PI3K / AKT / mTOR signaling pathway, blocking signal cascade transmission, and simultaneously downregulating the expression of downstream transcription factor c-Myc. Iris flavonoids, by inhibiting the phosphorylation level of the PI3K / AKT / mTOR signaling pathway and downregulating the expression of downstream transcription factor c-Myc, block the metabolic reprogramming and proliferation signals of tumor cells from the upstream. This differs in target and molecular mechanism from existing therapeutic approaches such as proteasome inhibitors, immunomodulators, and monoclonal antibodies, which exert their effects through direct killing or immunomodulation. Iris flavonoids act on the core metabolic-growth signaling axis of tumor cells, and its mechanism of action is distinct from existing drugs. For patients with relapsed or refractory multiple myeloma who have developed resistance to three existing classes of drugs (proteasome inhibitors, immunomodulators, and anti-CD38 monoclonal antibodies), iris flavonoids are expected to provide a new treatment option, thereby effectively overcoming the drug resistance bottleneck of existing technologies and solving the problem that most patients will eventually develop drug resistance and relapse due to existing treatment methods.

[0017] 2. This invention aims to provide a novel application of iris flavonoids in the treatment of multiple myeloma (MM), by inhibiting the PI3K / AKT / mTOR / c-Myc pathway to fight cancer, while ensuring that the drug, at an effective dose, does not cause significant tissue damage to the heart, liver, spleen, lungs, and kidneys. Specifically:

[0018] (1) Anti-proliferative efficacy: CCK8 assay confirmed that iris flavonoids can significantly reduce the viability of MM cells (such as RPMI 8226 and NCI H929).

[0019] (2) Verification of proliferation inhibition: EdU cell proliferation experiment (combined with fluorescence microscopy) confirmed that iris flavonoids can significantly downregulate the DNA synthesis level of MM cells and inhibit cell proliferation.

[0020] (3) Mechanism of action (metabolism and growth): Iris flavonoids inhibit the metabolism and growth of tumor cells by inhibiting the phosphorylation level of the PI3K / AKT / mTOR signaling pathway and downregulating the expression of downstream transcription factor c-Myc.

[0021] (4) In vivo efficacy: In the animal model of MM xenograft tumor, iris flavonoids significantly inhibited the growth of tumor volume and weight.

[0022] (5) Biosafety: HE staining of the heart, liver, spleen, lungs and kidneys of experimental animals confirmed that iris flavonoids did not cause histological damage to normal organs at effective doses. Attached Figure Description

[0023] Figure 1 To explain the antitumor activity and IC50 of iris flavonoids in this invention. 50 Values; where: (A) Cell viability of human multiple myeloma cell line RPMI 8226 after treatment with different concentrations of iris flavonoids for 24h, 48h, and 72h; (B) IC50 values ​​of human multiple myeloma cell line RPMI 8226 after 24 h of treatment with different concentrations of iris flavonoids; (C) Cell viability of human multiple myeloma cell line NCI H929 after treatment with different concentrations of iris flavonoids for 24h, 48h, and 72h; (D) represents the IC50 values ​​of human multiple myeloma cell line NCI H929 after 24 h of treatment with different concentrations of iris flavonoids.

[0024] Figure 2 This is the Edu cell proliferation experiment in this invention; wherein: (A) Representative fluorescence images of EdU in human multiple myeloma cell line RPMI 8226 after treatment with different concentrations of iris flavonoids for 24 h; (B) Representative fluorescence images of EdU in human multiple myeloma cell line NCI H929 after 24 h of treatment with different concentrations of iris flavonoids.

[0025] Figure 3 In this invention, iris flavonoids inhibit the PI3K / AKT / mTOR signaling pathway; wherein: (A) Effects of different concentrations of iris flavonoids on the expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, c-Myc, and other proteins in the human multiple myeloma cell line RPMI 8226 after 24 h of treatment. ACTIN was used as a loading control.

[0026] (B) Effects of different concentrations of iris flavonoids on the expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, and c-Myc proteins in human multiple myeloma cell line NCI H929 after 24 h of treatment. ACTIN was used as a loading control.

[0027] Figure 4 The present invention describes the inhibitory effect of iris flavonoids on tumor growth in mice; wherein: (A) is an image of a subcutaneous xenograft; (B) shows the change in subcutaneous xenograft volume over time; (C) is the weight of the subcutaneous xenograft at the end of treatment.

[0028] Figure 5 This invention demonstrates that iris flavonoids caused no histological damage to the heart, liver, spleen, lungs, and kidneys of mice; HE-stained sections of mouse heart, liver, spleen, lungs, and kidneys, scale bar 100 μm. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1: Cell viability assay. The CCK8 assay was used to evaluate the antitumor effect of iris flavonoids on MM cells (RPMI 8226, NCIH929). The specific steps are as follows: 1. 50,000 RPMI 8226 cells / well and NCI H929 cells were seeded into 96-well plates, with 3 replicates per well, as the experimental group. A control group (cells added but no drugs) and a blank group (no cells or drugs added, only culture medium) were also set up.

[0031] The RPMI 8226 cells were purchased from the Chinese Academy of Sciences and cultured under the following conditions: RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (P / S).

[0032] NCI H929 cells were purchased from Shanghai Jiayi Company. The culture conditions were as follows: RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (P / S).

[0033] All of the above cell lines have been identified by SPR.

[0034] 2. Drug Intervention: Different concentrations of iris flavonoid solution were added to the cells in the experimental group, with concentration gradients of 0, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM. Each well contained 100 μL of culture medium. Cells were incubated at 37°C with 5% CO2 for 24 h, 48 h, and 72 h. After 24 h, 48 h, and 72 h of iris flavonoid intervention, the 96-well plates were removed, and 10 μL of CCK8 solution was added to each well under dark conditions. After adding CCK8, the 96-well plates were vortexed on a cell culture shaker to mix, and then incubated in a cell culture incubator for another 2 h.

[0035] The iris flavonoid solution was prepared by dissolving iris flavonoid powder in DMSO to obtain a 160mM stock solution. The iris flavonoid powder was purchased from Shanghai Taoshu Biotechnology Co., Ltd., product number T3488.

[0036] 3. The OD value of each well is detected by a multi-functional microplate reader at a wavelength of 450nm.

[0037] 4. Calculate cell viability based on OD value readings and calculate IC50 using Grapad Prism 9. 50 .

[0038] The results showed that iris flavonoids significantly inhibited MM cell viability in a dose- and time-dependent manner. (See...) Figure 1 .

[0039] Depend on Figure 1 It was found that in RPMI 8226 and NCI H929 cells, CCK8 assay showed that iris flavonoids significantly reduced cell viability in a time- and dose-dependent manner, and its IC50 was calculated, indicating that iris flavonoids have significant time- and concentration-dependent cytotoxic effects on both MM cell types.

[0040] Example 2: Cell proliferation assay using EdU assay to evaluate the antitumor effect of iris flavonoids on MM cells (RPMI 8226 cells and NCI H929 cells). The specific steps are as follows: 1. Cell preparation and plating: Each well contains 100 μL of culture medium, containing 3 × 10⁶ cells / well. 4 RPMI 8226 cells and NCI H929 cells were seeded into 96-well plates and divided into control and drug intervention groups. The control group was the DMSO group, in which 100 μL of culture medium containing 0.1% (v / v) DMSO was added to each well. The drug intervention groups included iris flavonoid 80 μM group, iris flavonoid 120 μM group and iris flavonoid 160 μM group, in which 100 μL of culture medium containing iris flavonoid at final concentrations of 80 μM, 120 μM and 160 μM were added to each well respectively. Each group was configured with 3 replicates.

[0041] 2. Drug intervention: According to the experimental design, different concentrations of iris flavonoids were added to the drug intervention group, with concentration gradients of 80μM, 120μM and 160μM, and the cells were treated for 24h.

[0042] 3. EdU Incubation: Prepare the EdU working solution using culture medium according to the EdU kit instructions to a final concentration of 10 μM. Add 100 μL of EdU solution to each well and incubate the 96-well plate in a cell culture incubator for 2 hours.

[0043] 4. Fixation: After incubation, centrifuge the cells in the well plate and carefully remove the culture medium from the side wall of the well with a 1mL syringe. Add 100μL of 4% paraformaldehyde (m / v) to each well and incubate at room temperature for 15min to fix the cells.

[0044] 5. Washing: After fixation, wash the cells with PBS, repeating 3 times to completely remove the fixative. PBS was purchased from Sevier Biotechnology Co., Ltd., with a pH of 7.4.

[0045] 6. Permeabilization treatment: After centrifugation to remove the washing buffer, add 100 μL of strong immunostaining permeabilization buffer to each well and incubate at room temperature for 15 min to enhance cell membrane permeability. Then centrifuge to remove the permeabilization buffer and repeat the washing step twice.

[0046] 7. Click reaction: Prepare the Click reaction solution according to the kit instructions, and add 50 μL of the reaction mixture to each well. Incubate at room temperature in the dark for 30 min. After incubation, repeat the washing step 3 times to remove unbound reaction mixture.

[0047] 8. Nuclear staining: Dilute Hoechst 33342 staining solution with PBS at a ratio of 1:1000 (v / v), add 100 μL of Hoechst 33342 staining solution to each well, and incubate at room temperature in the dark for 10 min to stain the cell nuclei. Centrifuge to remove the staining solution, and repeat the washing step 3 times.

[0048] 9. High-content screening and instrumental observation: Finally, a fluorescence microscope was used to photograph cells and assess the effect of iris flavonoids on cell proliferation. For example... Figure 2 .

[0049] Depend on Figure 2 It was found that in RPMI 8226 and NCI H929 cells, compared with the control group, the proportion of EdU-positive cells gradually decreased with increasing iris flavonoid concentrations (80, 120, 160 μM). These results indicate that iris flavonoids can inhibit DNA synthesis in multiple myeloma cells in a concentration-dependent manner and further suppress cell proliferation.

[0050] Example 3: Mechanism of Action (Metabolic Relations and Growth) The specific steps are as follows: 1. Treatment groups were set up with 80μM, 120μM and 160μM of iris flavonoids, and a control group without drug treatment was set up.

[0051] The treatment groups refer to cells treated with different concentrations of iris flavonoids, with concentration gradients of 80 μM, 120 μM, and 160 μM, for 24 hours. The procedure was the same as the drug intervention group in Example 2.

[0052] 2. After 24 hours of cell intervention, cells were collected by centrifugation at 2000 rpm for 5 minutes and washed twice with pre-cooled PBS. The supernatant was discarded, and the cell pellet was placed on ice for later use.

[0053] 3. Based on the cell pellet volume in step 2, prepare freshly made protein lysis buffer, adding 100 μL of buffer per million cells. Mix RIPA lysis buffer: PMSF protease inhibitor: phosphorylase inhibitor at a ratio of 100:1:1 (v / v), vortex to mix, and incubate on ice for 30 min. Then, use an ultrasonic lysis apparatus for 5 min. Centrifuge at 12000g for 20 min at 4°C and collect the supernatant.

[0054] 4. Determine the protein concentration of each sample using the BCA protein quantification method to ensure consistent protein loading in each sample. Calculate the protein concentration based on absorbance (562 nm) using the standard curve method to ensure that the same amount of protein is used for subsequent sample loading.

[0055] 5. Protein denaturation treatment: Mix the protein sample with 5× protein loading buffer at a ratio of 4:1, mix thoroughly by pipetting, boil in a 100℃ metal bath for 10 minutes, then quickly transfer to ice to cool, and finally store in a -20℃ freezer for later use.

[0056] 6. Gel preparation: Select different concentrations of separating gel according to the target molecular weight, and prepare it using a one-step method. Insert a comb with the appropriate number of holes (observe carefully to prevent air bubbles from forming), let it stand at room temperature for 15 minutes, and follow the principle of preparing it immediately before use.

[0057] 7. Sample loading: Load 20 μg of sample, and add marker and pre-extracted protein samples in sequence according to the experimental group.

[0058] 8. Electrophoresis: 80V, 30min, 120V, 1h.

[0059] 9. Transfer: Use rapid transfer solution, constant current 400mA, for 30 minutes.

[0060] 10. Blocking, applying antibodies (p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, c-Myc), and exposure are the same as for ordinary WB.

[0061] The results showed that iris flavonoids inhibited tumor cell metabolism and growth by suppressing phosphorylation levels in the PI3K / AKT / mTOR signaling pathway and downregulating the expression of downstream transcription factor c-Myc. Figure 3 ).

[0062] Example 4: In vivo efficacy: In an animal model of MM xenograft tumors, iris flavonoids significantly inhibited the growth of tumor volume and weight.

[0063] The specific steps are as follows: 1. The concentration of RPMI 8226 cells was 3 × 10⁻⁶. 6 Tumor cell solutions were obtained by dissolving each cell in 100 μL of PBS.

[0064] 2. Subcutaneously inoculate 100 μL (3 × 10⁻⁶) of the above tumor cell solution into NVSG mice. 6 (Number of mice). Among them, the NVSG mice were obtained from Beijing Weishang Lide Biotechnology Co., Ltd.

[0065] 3. Once the tumor reaches a size of approximately 100 mm... 3 During the experiment, the experimental group received intraperitoneal injections of iris flavonoid solution at a dose of 50 mg / kg, administered every three days for a total of five injections. The control group received an equal volume of the solution.

[0066] 4. Observe the mice's mental state and tumor formation every three days. Measure the tumor size and end the experiment on day 32. Weigh the tumor, photograph the tumor, statistically analyze the tumor weight, and use GraphPad to plot the tumor growth curve.

[0067] The results showed that in the MM xenograft animal model, iris flavonoids significantly inhibited the growth of tumor volume and weight (e.g., Figure 4 ).

[0068] Example 5: Biosafety The specific steps are as follows: 1. Collect organ samples (including heart, liver, spleen, lung, and kidney) from NVSG mice, and then fix them in 4% (m / v) paraformaldehyde solution for 24 hours to ensure that the tissue morphology is preserved.

[0069] 2. After fixation, the tissue was dehydrated, cleared, and impregnated with paraffin before being embedded in paraffin. The embedded tissue was then cut into 3 μm sections using a microtome and placed on glass slides for staining.

[0070] 3. HE staining: First, hematoxylin is used to stain the cell nuclei, making them appear purple or blue; then eosin is used to stain the cytoplasm and other extracellular matrix. After staining, the tissue sections are dehydrated and cleared to make them suitable for microscopic observation.

[0071] The results showed that HE staining of the heart, liver, spleen, lungs, and kidneys of experimental animals confirmed that iris flavonoids, at effective doses, did not cause histological damage to normal organs (e.g., Figure 5 ).

[0072] As can be seen from the above, the iris flavonoids described in this invention exhibit significant anti-multiple myeloma activity in both in vitro cell experiments and in vivo animal experiments. Specifically, the CCK8 and EdU assays showed that iris flavonoids significantly inhibited the viability and DNA synthesis level of multiple myeloma cells, thereby effectively inhibiting tumor cell proliferation. Furthermore, Western blotting results confirmed that iris flavonoids reduced the phosphorylation levels of PI3K, AKT, and mTOR, and downregulated the expression of the downstream transcription factor c-Myc, indicating that it can inhibit tumor cell metabolism and growth at the signaling pathway level.

[0073] Furthermore, in a multiple myeloma xenograft model, iris flavonoids significantly inhibited the growth of tumor volume and weight, further validating its antitumor effect in vivo. Simultaneously, HE staining analysis of the major organs of the experimental animals revealed no significant histological damage, indicating that it exhibits good biocompatibility while exerting its antitumor effects.

[0074] Based on the above experimental results and mechanism of action analysis, it can be concluded that the technical solution of the present invention can block the abnormal proliferation and metabolic process of multiple myeloma cells by inhibiting the PI3K / AKT / mTOR / c-Myc signaling pathway, thereby effectively solving the problems of drug resistance and short-lasting efficacy of existing treatment methods, and reducing the toxic side effects on normal tissues while ensuring anti-tumor effects.

[0075] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The application of iris flavonoids in the preparation of drugs for treating multiple myeloma, characterized in that, The structure of the iris flavonoid is shown in formula 1): Formula 1).

2. The application according to claim 1, characterized in that, The drug uses iris flavonoids as its sole active ingredient.

3. The application according to claim 2, characterized in that, The drug is a liquid preparation.

4. The application according to claim 3, characterized in that, The drug is an injectable dosage form.

5. The application according to claim 4, characterized in that, The drug also includes excipients.

6. The application according to claim 5. Its characteristic is that, The excipients are selected from any one or more of physiological saline, water for injection, glucose solution, propylene glycol, and polyethylene glycol.

7. The application according to claim 6, characterized in that, The concentration of iris flavonoids in the drug is 0.08 μM to 500 μM.

8. The application according to claim 1, characterized in that, The drug is used to inhibit the phosphorylation level of the PI3K / AKT / mTOR signaling pathway and downregulate the expression of downstream transcription factor c-Myc, thereby inhibiting the metabolism and growth of multiple myeloma cells.

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