Drug combination of purinostat mesylate and analog thereof for preventing and treating multiple myeloma, and use thereof

Abstract

A drug combination of purinostat mesylate and an analog thereof for preventing and treating multiple myeloma, and the use thereof. Specifically, provided is a three-drug or four-drug combination of purinostat mesylate and an analog thereof, dexamethasone and other drug, which drug combination has significantly enhanced synergistic anti-tumor activity in vitro and in vivo, synergistically inhibits the expression of the key survival protein in multiple myeloma, is superior to existing clinical combination regimens, and has no significant toxic side effects. In addition, the drug combination significantly down-regulates the expression of CDK6, and overcomes the drug resistance to an immunomodulator. In particular, the three-drug combination of puisostat mesylate, a glucocorticoid and an immunomodulator has an excellent prevention and treatment effect on multiple myeloma, especially relapsed / refractory multiple myeloma.

Description

Combined drug of prilocastat mesylate and its analogs for preventing and treating multiple myeloma and use thereof Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a combined drug of prilocastat mesylate and its analogs for preventing and treating multiple myeloma and uses thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant plasma cell hematologic malignancy originating in the bone marrow. Among hematologic malignancies, MM ranks second in incidence, accounting for 10%-13%, with an increasing incidence rate annually, making it one of the most common hematologic malignancies worldwide. Although existing drugs can significantly alleviate disease progression in MM patients, most patients eventually relapse and become insensitive to these drugs. To date, MM remains incurable and progresses to relapsed / refractory multiple myeloma (R / R MM).

[0003] Literature 1 (Landgren O, etc.: Modern multiple myeloma therapy: deep, sustained treatment response and good clinical outcomes. Journal of Internal Medicine 2017, 281: 365-82.) reported that in modern therapy, three-drug or four-drug combination regimens containing new drugs are used as much as possible for MM or R / R MM, which have higher overall survival and progression-free survival compared with patients treated with traditional two-drug combination regimens. Literature 2 (Rodríguez-Lobato LG, etc.: CAR T-cells in multiple myeloma: state of the art and future directions. Frontiers in Oncology 2020, 10: 1243.) reported that among the many current treatment methods and means, chimeric antigen receptor T-cell immunotherapy (CAR-T) has the best efficacy, but due to its high cost and poor accessibility at this stage, it cannot meet the needs of the majority of R / R MM patients. In addition, proteasome inhibitors and immunomodulators have been widely used in clinical practice, resulting in an increasing rate of drug resistance. There is an urgent need to develop drugs with new or different mechanisms of action or more potent drugs with greater accessibility.

[0004] Histone deacetylase inhibitors (HDACi) are promising drugs for the treatment of multiple myeloma. For example, panobinostat (LBH589) significantly prolonged progression-free survival (PFS) in patients with relapsed / relapsed (R / R) multiple myeloma. However, its toxic side effects have limited its clinical use and have led to the withdrawal of its approval by the FDA. Therefore, the development of highly selective HDAC inhibitors with enhanced activity and reduced toxicity could further address the clinical needs of patients with relapsed / relapsed (R / R) multiple myeloma. Furthermore, with the increasing understanding of the mechanisms of HDACi tumor inhibition and its demonstrated potential in the treatment of hematologic malignancies, combination therapy has become an important approach to improving the therapeutic efficacy of HDACi. Combining HDACi with other drugs that enhance their activity can not only further enhance the anti-tumor effect but also reduce the toxicity or drug resistance associated with the drug dose. Therefore, combination therapy is another important approach to improving the efficacy of HDAC inhibitors in the treatment of multiple myeloma.

[0005] Purinostat Mesylate (PM) is a highly selective HDACi for types I and IIb developed independently by our company. Its inhibitory activity against HDAC class I / IIb enzymes is superior to all currently marketed HDAC inhibitors, and its cardiotoxicity is significantly lower than that of LBH589. Therefore, preclinical pharmacodynamic evaluation of PM in combination with other drugs for the treatment of multiple myeloma has important clinical significance and scientific value, laying a preclinical pharmacodynamic foundation for clinical trials in the treatment of MM patients. Furthermore, developing a combination therapy with PM and its analogs that has the advantages of high efficacy, low side effects, low toxicity, and the ability to overcome drug resistance can further improve patients' quality of life. Summary of the Invention

[0006] The present invention explores the mechanism of action of PM in treating MM and explores the combination of PM (including PM analogs with similar activity) with other drugs to study the feasibility of PM combination therapy for MM. Through various experiments such as in vitro cell lines and various model mice, the in vivo anti-MM activity, efficacy and toxicity of PM in combination with immunomodulators (such as pomalidomide, lenalidomide, etc.), glucocorticoids (such as dexamethasone, etc.), and PM with other drugs (CD38 monoclonal antibodies such as daratumumab, proteasome inhibitors such as bortezomib, and selective inhibitors of nuclear export such as selinexor) are compared to obtain a combination regimen with excellent results.

[0007] The present invention first provides a combination drug of prilocastat mesylate and its analogs for preventing and treating multiple myeloma. The combination drug is a three-drug combination drug of prilocastat mesylate and its analogs, a glucocorticoid, and a third drug, which are administered separately or simultaneously; or, the combination drug is a four-drug combination drug of prilocastat mesylate and its analogs, a glucocorticoid, a fourth drug, and a fifth drug, which are administered separately or simultaneously; wherein, in the three-drug combination drug, the third drug is selected from an immunomodulator, a monoclonal antibody, or a proteasome inhibitor; and in the three-drug combination drug, the fourth drug is a monoclonal antibody, and the fifth drug is a proteasome inhibitor.

[0008] Among them, in the above-mentioned combination drug, the prilocastat mesylate and its analogs are selected from compounds having the following structures:

[0009] Preferably, in the above-mentioned combination drug, the prilocastat mesylate and its analogs are selected from compounds having the following structures:

[0010] More preferably, in the above-mentioned combination drug, the prilocastat mesylate and its analogs are prilocastat mesylate for injection.

[0011] Wherein, in the above-mentioned combined drug, the glucocorticoid is selected from dexamethasone or prednisone.

[0012] Preferably, in the above-mentioned combination drug, the glucocorticoid is dexamethasone.

[0013] Among them, in the above-mentioned combination drugs, in the three-drug combination drugs, the third drug is selected from immunomodulators or monoclonal antibodies.

[0014] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, the third drug is an immunomodulator.

[0015] Among them, in the above-mentioned combination drugs, in the three-drug combination drugs, the immunomodulator is selected from pomalidomide, lenalidomide or thalidomide.

[0016] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, the immunomodulator is selected from pomalidomide or lenalidomide.

[0017] More preferably, in the above combination drug, in the three-drug combination drug, the immunomodulator is pomalidomide.

[0018] Among them, in the above-mentioned combination drug, in the three-drug combination drug, the monoclonal antibody is selected from daratumumab, elotuzumab or isatuximab.

[0019] Preferably, in the above-mentioned combination drug, in the triple-drug combination drug, the monoclonal antibody is daratumumab.

[0020] Among them, in the above-mentioned combination drug, in the three-drug combination drug, the proteasome inhibitor is selected from bortezomib, ixazomib or carfilzomib.

[0021] Preferably, in the above-mentioned combination drug, in the triple-drug combination drug, the proteasome inhibitor is bortezomib.

[0022] Among them, in the above-mentioned combination drug, in the four-drug combination drug, the monoclonal antibody is selected from daratumumab, elotuzumab or isatuximab.

[0023] Preferably, in the above-mentioned combination drug, in the four-drug combination drug, the monoclonal antibody is daratumumab.

[0024] Among them, in the above-mentioned combination drug, in the four-drug combination drug, the proteasome inhibitor is selected from bortezomib, ixazomib or carfilzomib.

[0025] Preferably, in the above-mentioned combination drug, in the four-drug combination drug, the proteasome inhibitor is bortezomib.

[0026] Among them, in the above-mentioned combination drugs, in the three-drug combination drugs, when the third drug is an immunomodulator, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulators is 1.25-5:1:0.5-30.

[0027] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the immunomodulator is pomalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulator is 1.25-5:1:0.5-2.5.

[0028] More preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the immunomodulator is pomalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulator is 5:1:0.5-2.5.

[0029] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the immunomodulator is lenalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulators is 1.25-5:1:5-30.

[0030] More preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the immunomodulator is lenalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulator is 5:1:10.

[0031] Among them, in the above-mentioned combination drugs, in the three-drug combination drugs, when the third drug is a monoclonal antibody, the mass ratio of the main drugs of the prilocastat mesylate and its analogs, glucocorticoids and monoclonal antibodies is 1.25-5:1:2.5-25.

[0032] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the third drug is a monoclonal antibody, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and monoclonal antibodies is 5:1:5;

[0033] Among them, in the above-mentioned combination drugs, in the three-drug combination drugs, when the third drug is a proteasome inhibitor, the mass ratio of the main drugs of prilocastat mesylate, glucocorticoid and proteasome inhibitor is 1.25-5:1:0.1-1.

[0034] Preferably, in the above-mentioned combination drug, in the three-drug combination drug, when the third drug is a proteasome inhibitor, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and proteasome inhibitors is 5:1:0.1.

[0035] Among them, in the above-mentioned combination drugs, in the four-drug combination drugs, the mass ratio of the main drugs of the prilocastat mesylate and its analogs, glucocorticoids, monoclonal antibodies and proteasome inhibitors is 1.25-5:1:2.5-25:0.1-1.

[0036] Preferably, in the above-mentioned combination drug, in the four-drug combination drug, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids, monoclonal antibodies and proteasome inhibitors is 5:1:5:0.1.

[0037] Among them, in the above-mentioned combination drug, the combination drug is a preparation prepared by using prilocastat mesylate and its analogs, glucocorticoids and a third drug as active ingredients at the same time, and adding pharmaceutically acceptable excipients; or the combination drug is a preparation prepared by using prilocastat mesylate and its analogs, glucocorticoids and a fourth drug and a fifth drug as active ingredients at the same time, and adding pharmaceutically acceptable excipients.

[0038] Preferably, in the above-mentioned combination drug, the preparation of the combination drug is an injectable preparation.

[0039] More preferably, in the above combination drug, the injection route is at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection or intramyocardial injection.

[0040] Wherein, in the above-mentioned combination drug, the multiple myeloma is relapsed and refractory multiple myeloma.

[0041] The present invention also provides the use of the above-mentioned prilocastat mesylate and its analogues combined with a drug for preventing and treating multiple myeloma in the preparation of a drug for treating and / or preventing multiple myeloma.

[0042] Wherein, in the above use, the multiple myeloma is relapsed refractory multiple myeloma.

[0043] Beneficial effects of the present invention:

[0044] In the early stage of this invention, PM was evaluated for its efficacy in treating MM through preclinical studies, demonstrating that PM, a highly selective and active inhibitor of HDACI / IIb, is a promising drug for the treatment of MM. It is speculated that PM may have strong synergistic anti-MM activity when used in combination with other drugs to treat MM. Based on this, this invention combines bulk RNA-seq and molecular cell experimental studies to explore the efficacy of PM in the treatment of R / R Further investigation of potential MM combination therapies involved experiments in sensitive and resistant MM cell lines and MM mouse models. The in vitro and in vivo antitumor activity of PM in combination with pomalidomide and dexamethasone, existing three-drug combinations, and PM with other three-drug and four-drug combinations, such as daratumumab and bortezomib, was investigated. A superior combination was identified. In particular, the combination of PM with pomalidomide and dexamethasone exhibited significantly enhanced synergistic in vitro and in vivo antitumor activity, synergistically inhibiting the expression of key MM survival proteins. This combination was superior to several existing clinical three-drug combinations and PM with other three-drug and four-drug combinations, with no significant side effects. The combination significantly downregulated CDK6 expression, overcoming Pom resistance. This study provides a preclinical pharmacodynamic foundation for a Phase Ib / IIa clinical trial of PM combination therapy for R / R MM, which is of great clinical value and academic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a graph showing that PM treatment upregulates myeloma cell-cell adhesion and downregulates cellular CDK6 protein expression in Example 1; wherein, A shows that MM1S cells were treated with control and PM 5nM for 24 hours, and cells were collected for bulk RNA-seq and GSEA plasma cell-cell adhesion signaling pathway enrichment analysis (n=3); B shows that after the MM1S mouse model was treated with vehicle and PM 10mg / kg for 24 hours, tumor tissue was subjected to bulk RNA-seq and GSEA plasma cell-cell adhesion signaling pathway enrichment analysis; C shows that dexamethasone-sensitive and -resistant MM1S and MM1R cells were treated with different concentrations of PM and LBH589 for 24 hours, and CDK6 protein expression in the cells was determined by western blot; D shows that bortezomib-sensitive and -resistant RPMI-8226 and RPMI-8226R cells were treated with different concentrations of PM and LBH589 for 24 hours, and CDK6 protein expression in the cells was determined by western blot.

[0046] Figure 2 is a correlation graph of the anti-proliferative activity of RPMI-8226 cells treated with the combination of PM, Pom and DXM in Example 2 and the CI index; wherein, A represents the combined treatment of PM and Pom for 48 h; B represents the combined treatment of PM and Pom for 72 h; C represents the combined treatment of PM, Pom and DXM for 48 h; D represents the combined treatment of PM, Pom and DXM for 72 h; compared with the blank control group, ns indicates P>0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001; compared with the corresponding concentrations of Pom+DXM in combination, ## indicates P<0.01, ### indicates P<0.001, and #### indicates P<0.0001, using t-test.

[0047] Figure 3 is a correlation graph of the anti-proliferative activity of MM1S cells treated with the combination of PM, Pom and DXM in Example 2 and the CI index; wherein, A represents the combined treatment of PM and Pom for 48 h; B represents the combined treatment of PM and Pom for 72 h; C represents the combined treatment of PM, Pom and DXM for 48 h; D represents the combined treatment of PM, Pom and DXM for 72 h; compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001; compared with the corresponding concentrations of Pom + DXM in combination, ns indicates P > 0.05, ## indicates P < 0.01, ### indicates P < 0.001, and #### indicates P < 0.0001, using t-test.

[0048] Figure 4 is a correlation graph of the anti-proliferative activity of MM1R cells treated with the combination of PM, Pom and DXM in Example 2 and the CI index; wherein, A represents the combined treatment of PM and Pom for 48 h; B represents the combined treatment of PM and Pom for 72 h; C represents the combined treatment of PM, Pom and DXM for 48 h; D represents the combined treatment of PM, Pom and DXM for 72 h; compared with the blank control group, ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001; compared with the corresponding concentrations of Pom+DXM in combination, ### indicates P<0.001, and #### indicates P<0.0001, using t-test.

[0049] Figure 5 shows the synergistic inhibition of the expression of key survival-related proteins in MM1S and MM1R cells by PM in combination with Pom and DXM using β-ACTIN as the internal reference in Example 3; wherein A is the protein expression level in MM1S cells; B is the protein expression level in MM1R cells.

[0050] FIG6 is a graph showing the concentration-dependent upregulation of CD38 expression in MM1S cells treated with PM for 24 hours in Example 5; wherein A is a waveform graph; and B is a bar graph.

[0051] Figure 7 shows the synergistic inhibition of RPMI-8226 tumor growth by PM in combination with various existing multiple myeloma drugs in Example 6; wherein A is the tumor growth curve; B is the tumor weight; C is the percentage change in body weight; compared with the blank control group, *** indicates P < 0.001; compared with the combination groups, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; #### indicates P < 0.0001, t-test.

[0052] FIG8 is a diagram showing the effects of PM combined with Pom and DXM on the heart, liver, spleen, lung, kidney, stomach and intestine of mice treated with the RPMI-8226 model in Example 6 using HE staining.

[0053] Figure 9 is a graph showing the effects of PM combined with Pom and DXM on the levels of key proteins in RPMI-8226 model mouse tumors detected by immunohistochemistry in Example 6; wherein A is the immunohistochemistry graph; B is the immunohistochemistry quantification; compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and NS indicates P > 0.05, t-test.

[0054] Figure 10 is a diagram showing the effects of PM in combination with Pom and DXM on key proteins in tumor tissue of RPMI-8226 model mice with β-ACTIN as the internal reference in Example 6; wherein A is the protein expression level; B is a bar graph; compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; # indicates P < 0.05, ## indicates P < 0.01, t-test.

[0055] Figure 11 is a graph showing that the combination of PM with Pom and DXM for the treatment of MM1R in Example 7 is superior to the combination of PM with other drugs and multiple clinical three-drug combination regimens; wherein A is the tumor growth curve; B is the tumor weight; C is the percentage change in body weight; compared with the blank control group, * indicates P < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001; compared with the combination groups, NS indicates P > 0.05, # indicates P < 0.05, and ### indicates P < 0.001, using t-test. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0057] 1. The reagents and materials used in the examples are shown in Table 1.

[0058] Table 1 Reagent and material information

[0059] Prilostat mesylate structural formula, molecular formula: C 23 H 26 N 10 O3·CH4O3S, molecular weight: 586.62.

[0060] 2. The information of antibodies used in the examples is shown in Table 2.

[0061] Table 2 Antibody information

[0062] 3. The information of experimental animals used in the examples is as follows:

[0063] All animal experiments and procedures involved in the present invention were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China). The experimental endpoints of all multiple myeloma tumor-bearing mouse models were approved by the Animal Ethics Committee of Sichuan University, and the tumor diameter (d) did not exceed 20 mm.

[0064] (1) MM1S mouse model: NOD-SCID female mice, weighing 16-18 g, 5-6 weeks old, were purchased from Spefox (Beijing) Biotechnology Co., Ltd. All animals were housed in the SPF animal room of the State Key Laboratory of Biotherapy, Sichuan University.

[0065] (2) MM1R mouse model: NTG female mice, weighing 18-20 g, 6-7 weeks old, were purchased from Spefox (Beijing) Biotechnology Co., Ltd. and maintained in the SPF animal room of the Animal Experiment Center, West China Hospital, Sichuan University.

[0066] (3) RPMI-8226 mouse model: NOD-SCID female mice, weighing 16–18 g, 5–6 weeks old. They were purchased from Beijing Huafukang Biotechnology Co., Ltd. with a production license number of SCXK(Beijing)2019-0008. They were housed in the SPF animal room of the State Key Laboratory of Biotherapy, Sichuan University.

[0067] 4. The main software and data processing systems used in the examples are as follows:

[0068] Microsoft Excel 2010 version, Microsoft Corporation, Microsoft Excel is used for data record keeping, calculation, conversion, and organization and formatting.

[0069] Adobe illustrator 2021 version, Adobe Systems Incorporated, Adobe illustrator is used to organize and layout image data.

[0070] Graphpad Prism, version 9.0, Graphpad Software Co., Ltd., was used to organize and analyze dose-effect fitting curves and to organize quantitative data to produce bar graphs.

[0071] Image-Pro Plus, version 6.0, MEDIA CYBERNETICS, USA. Image-Pro Plus software was used for quantitative statistical analysis of immunohistochemical images.

[0072] 5. The preparation methods of the reagents and common solutions used in the experiments in the examples are as follows:

[0073] (1) Solutions of compounds such as Prilosec mesylate (PM), pomalidomide (Pom), dexamethasone (DXM), daratumumab (Dara), bortezomib (Bort), lenalidomide (Len), selinexor, and panobinostat lactate (LBH589) for cell culture: The volume of DMSO required to prepare a 10 mmol / L stock solution (μL) = (m / M) × 10 5 , where m is the mass of the drug and M is the molar mass of the drug. Precisely weigh PM, Pom, DXM, and other drugs on an analytical balance. After centrifugation, transfer the mixture to a biosafety cabinet. Prepare all compounds into 10 mmol / L stock solutions using sterile DMSO and store in a -20°C refrigerator.

[0074] (2) RPMI-1640 complete medium: Prepared in a biosafety cabinet containing 10% fetal bovine serum, 100 U mL -1 Penicillin and 100 mg·L -1 RPMI-1640 complete medium containing streptomycin. Shake gently to mix, seal the bottle with sealing film, and store in a refrigerator at 4°C until ready for use.

[0075] (3) PBS phosphate buffer: Add an appropriate amount of UP water to PBS phosphate buffer powder, shake to dissolve, then dilute to 2 L with UP water and store at room temperature until used.

[0076] (4) PBST buffer: Prepare PBS buffer (2 L) in advance according to the steps shown in (3), then add 2 mL of Tween 20, shake well, dissolve and store at room temperature for later use.

[0077] (5) SDS-PAGE electrophoresis buffer (2 L system): Weigh 6.06 g Tris base, 37.5 g glycine, and 2 g SDS powder on a balance. Add appropriate amount of UP water and shake until completely dissolved. Then, dilute to 2 L and store at room temperature until ready to use.

[0078] (6) Transfer buffer (2 L system): Weigh 5.8 g of Tris base and 29 g of glycine powder on a balance. Add an appropriate amount of UP water and shake until completely dissolved. Then add 400 mL of methanol solution and continue to add UP water to make up to 2 L. Let stand at room temperature until ready to use.

[0079] (7) 5% skim milk: Weigh 2.5 g of skim milk powder on a balance, add an appropriate amount of PBST solution, shake to mix, and then dilute to 50 mL with PBST solution. Store at 4°C until use.

[0080] (8) Primary antibody incubation solution: Dilute the antibody with the primary antibody diluent according to the dilution ratio recommended in the antibody instructions for Western blot experiments, vortex mix, and store at -20°C until use.

[0081] (9) Secondary antibody incubation solution: Prepare 5% skim milk in advance according to the steps and methods shown in (7). Select the required secondary antibody according to the species type of the primary antibody and add it to the skim milk at a ratio of 1:5000 for dilution. Vortex mix and store at 4°C until ready for use.

[0082] 6. The cell line information used in the examples is as follows:

[0083] (1) MM1S: purchased from the American Type Culture Collection (ATCC), a 3rd-4th generation cell line, and stored in liquid nitrogen tank A in the Quality Control Room of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0084] (2) MM1R: From the American Type Culture Collection (ATCC), it is a 3rd-4th generation cell line and is stored in the liquid nitrogen tank No. A in the Quality Control Room of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0085] (3) RPMI-8226: purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, it is a 3rd-4th generation cell line and stored in the liquid nitrogen tank No. A in the Quality Control Room of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0086] (4) RPMI-8226R: Originated from Nanjing University of Chinese Medicine, the cells were authenticated and stored in liquid nitrogen tank A in the Quality Control Room of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0087] The above cells were well maintained and were authenticated before use.

[0088] 7. Experimental methods in the embodiments:

[0089] 7.1 Cell Culture

[0090] Multiple myeloma cell lines MM1S, MM1R (DXM-resistant), RPMI-8226, and RPMI-8226R (Bort-resistant) were routinely cultured in a 10% fetal bovine serum (FBS) medium containing 100 U mL -1 Penicillin and 100 mg·L -1The cells were cultured in RPMI-1640 complete medium containing streptomycin in a humidified, 5% CO2 cell culture incubator at 37°C.

[0091] 7.2. CCK8 Assay for Synergistic Antiproliferative Activity of PM Combined with Pom and DXM on MM Cells in Vitro

[0092] Cells were grown to the logarithmic phase and counted. MM1S, MM1R, or RPMI-8226 cells were seeded into 96-well plates at 20,000-40,000 cells / well (100 μL volume / well) and cultured in a CO2 incubator overnight. The experimental groups were supplemented with 100 μL of culture medium containing different concentrations of PM, Pom, DXM alone, and different concentrations of the combined agents. The blank control group was supplemented with 100 μL of fresh culture medium per well, while the solvent control group was supplemented with an equal volume of fresh culture medium containing DMSO equal to the highest experimental concentration of the drug. Three parallel wells were set up in each group and cultured in an incubator.

[0093] After 48 and 72 hours of drug exposure, 20 μL of CCK8 working solution was added to each well and the cells were incubated in a 37°C incubator for 1–4 hours before terminating the culture. Absorbance at 450 nm was measured on a microplate reader. Inhibition rates were calculated based on absorbance values ​​at different concentrations using the formula: Inhibition rate = {1-[(average OD value of the experimental group - average OD value of the blank control group) / (average OD value of the solvent control group - average OD value of the blank control group)]} × 100%. Calcusyn 2.0 software was used to fit the CI values ​​to the corresponding compound treatment concentrations.

[0094] 7.3 Western blot analysis of MM survival-related protein expression

[0095] (1) Cell plating: MM1S and MM1R (dexamethasone-resistant) cells were plated at 5×10 6 The cells were plated at a concentration of 100 μg / 10 mL in a 10 cm culture dish.

[0096] (2) Drug treatment: Observe under a microscope to confirm the cell status and density again, place the cells in an incubator and culture for about 4 hours before adding drugs. Add different concentrations of PM, Pom, DXM alone, and different concentrations of combined culture media for 24 hours.

[0097] (3) Protein extraction: After the drug treatment, semi-adherent cells were gently scraped off in one direction with a cell scraper and then gently pipetted to collect the cell suspension. Suspended cells were directly pipetted to collect the cell suspension. Centrifuge at 320g for 5 minutes, discard the supernatant, and the pellet was the cell sample required for the experiment. Wash the cell sample twice with pre-chilled PBS and discard the supernatant.

[0098] Prepare the lysis buffer by adding 10μl PMSF (100mM) and 10μl phosphatase inhibitor cocktail to every 1ml RIPA lysis buffer, and add 100-200uL of lysis buffer to each sample. Resuspend and lyse the cell samples with this strong lysis buffer. Vortex the sample for 1min, place it on ice for 5min, and repeat this operation for a total of 30min. To ensure more complete cell lysis, use a cell ultrasonic disruptor to sonicate 5 times, each time for 3s, with a rest period of 9s, and operate on ice. Centrifuge the sonicated sample at 13000rpm and 4℃ for 15min. Collect the supernatant in a 1.5mL EP tube and store it on ice.

[0099] Prepare a 96-well plate with Bradford reagent and UP water. Prepare protein standards and add them to the 96-well plate at varying concentrations. Add Bradford reagent and measure absorbance at 595 nm using a microplate reader. Calculate a standard curve using Excel, plotting protein content (μg) on ​​the horizontal axis against absorbance on the vertical axis. The standard curve is: y = 0.5789x + 0.535, R = 0.9984.

[0100] Use a 96-well plate, take 2 μL of the supernatant of the sample to be tested, add 18 μL of up water, and then add 250 μL of Bradford reagent. Set up 3 replicate wells for each sample, mix well, and measure the absorbance at 595 nm on a microplate reader.

[0101] Calculate protein concentration based on the standard curve and adjust the sample to the same concentration using the prepared RIPA lysis buffer. Add 5× SDS-PAGE Loading Buffer, vortex to mix, and heat in a metal bath at 100°C for 10 minutes to denature the protein. This completes the protein sample preparation. After cooling to room temperature, the sample can be stored at -20°C for extended periods.

[0102] (4) SDS-PAGE electrophoresis: Prepare a 10% SDS-PAGE gel based on the molecular weight of the protein involved in this experiment. Place the solidified gel on an electrophoresis clamp and place it in the electrophoresis tank. Add enough electrophoresis solution and start preparing for sample loading. Take out the protein sample frozen at -20℃ and slowly thaw it at room temperature. Mix it again and then load it. Adjust the voltage to 80V for electrophoresis. When the protein sample to be tested is electrophoresed to the lower layer of gel, adjust the voltage to 120V. Stop electrophoresis when the sample is electrophoresed to the bottom of the PAGE gel.

[0103] (5) Transfer: Soak the transfer clip and sponge in pre-cooled transfer buffer in advance. Take out the gel glass plate after electrophoresis, and cut the excess gel outside the area where the target protein is located according to the molecular weight indicated by the protein marker band. Activate the PVDF membrane with methanol and place the membrane on the gel, remove the bubbles between the PVDF membrane and the gel, and cover with the transfer clip. Place the transfer clip in the transfer tank, put it in the ice box, add enough pre-cooled transfer buffer, and then cover the transfer tank with ice. Adjust the current to 250-300mA for transfer, and the transfer time is about 1-2 hours (adjust according to the molecular weight of the protein).

[0104] (6) Blocking: Prepare 5% milk with PBST. Remove the PVDF membrane and wash it twice with PBST. Place it in the pre-prepared milk blocking buffer and incubate it slowly at room temperature for 1-2 hours.

[0105] (7) Primary antibody incubation: Dilute the required antibody in proportion with primary antibody diluent, then pour it into the antibody incubation box. Wash the blocked PVDF membrane with PBST and place it in the primary antibody, and incubate it on a vertical shaker at 4°C overnight.

[0106] (8) Washing the membrane: Place the PVDF membrane incubated with the primary antibody in PBST and wash repeatedly on a shaker, replacing the PBST every 5 minutes for a total of 30 minutes.

[0107] (9) Secondary antibody incubation: Prepare secondary antibody with 5% milk in a certain proportion. Pour the prepared secondary antibody into the antibody incubation box and incubate the PVDF membrane at room temperature with shaking for 1 hour.

[0108] (10) Washing the membrane: Place the PVDF membrane incubated with the secondary antibody in PBST and wash repeatedly on a shaker, changing the PBST every 5 minutes for a total of 30 minutes.

[0109] (11) Development: Prepare and mix the developer solution A:B in a ratio of 1:1 and store in a dark place. After washing, add the developer solution dropwise onto the PVDF membrane and take photos using ECL (Rio-Rad) chemiluminescence imaging.

[0110] (12) Image data processing: Organize image data and use Adobe illustrator software to complete the organization and layout of images.

[0111] 7.4 Detection of CD38 Expression in MM1S Cells by Flow Cytometry

[0112] 1.0×10 MM1S cells in logarithmic growth phase 6Cells were seeded in each well of a 6-well plate. Different concentrations of PM (0, 0.25, 0.5, and 1 nM) were added for 24 hours. Cells were harvested, washed with pre-chilled PBS, and incubated with a human CD38 flow cytometry antibody according to the manufacturer's instructions. CD38 expression in MM1S cells was determined by flow cytometry.

[0113] 7.5 Establishment of Human Multiple Myeloma RPMI-8226 and MM1R Mouse Models

[0114] 1.0×10 RPMI-8226 cells in logarithmic growth phase 7 Cells / mouse / 100 μl were inoculated into 6-7 week old female NOD-SCID mice to establish RPMI-8226 subcutaneous tumor mouse model. 3 The mice were randomly divided into groups. The specific dosing regimen and grouping are shown in Table 3. A total of 8 groups, 6 mice each, were included. During the experiment, changes in mouse weight were recorded and their condition was observed. The long and short diameters of the tumors were measured with a vernier caliper every 2 days to dynamically observe the anti-tumor effects of the test drug.

[0115] Table 3 RPMI-8226 mouse model grouping and dosing regimen

[0116] After treatment termination, mice were anesthetized with 2.5% avertin solution and sacrificed. Tumor tissue and heart, liver, spleen, lung, kidney, stomach, and intestinal tissue were dissected using dissecting tools. Tumor tissue was weighed and photographed. Visceral and partial tumor tissue was fixed with paraformaldehyde and sent to Wuhan Sewell Biotechnology Co., Ltd. for hematoxylin and eosin staining, immunohistochemical sectioning, and pathological analysis. The remaining tumor tissue was quickly frozen in liquid nitrogen for subsequent Western blot analysis. Immunohistochemistry sections were scanned using a full-information tissue slide scanner and subjected to quantitative optical density analysis using Image-Pro Plus 6.0 software to compare protein expression levels in the samples.

[0117] MM1R cells in the logarithmic growth phase were adjusted to a cell density of 1 × 10 7 After the cells were collected, the cell suspension was stored on ice. 100 μL of MM1S cell suspension was drawn up with a 1 mL disposable sterile syringe and injected subcutaneously into the right armpit of NTG mice to establish the MM1R xenograft mouse model. When the average tumor volume of MM1R tumor-bearing mice grew to 200 mm 3 About 30 tumor-bearing mice were selected and randomly divided into groups. The specific dosing regimen and grouping are shown in Table 4. There were 8 groups in total, with 6 mice in each group. During the treatment period, the mice were observed, their body weight was weighed every 2 days, and the long and short diameters of the tumors were measured with a vernier caliper.

[0118] Table 4 MM1R mouse model grouping and dosing regimen

[0119] The calculation formula of tumor volume (TV) is: TV (mm 3 )=a×b 2 ×0.5; where a and b represent the major diameter and minor diameter, respectively.

[0120] The calculation formula of tumor inhibition rate (%) is: tumor inhibition rate (%) = (average tumor weight of negative control group (g) - average tumor weight of drug-treated group (g)) / average tumor weight of negative control group (g) × 100%.

[0121] Effective judgment criteria: tumor inhibition rate (ie, tumor growth inhibition rate) < 40% is ineffective; tumor inhibition rate ≥ 40% and P < 0.05 after statistical analysis is effective.

[0122] 7.6. Western blot experiments on tumor tissue

[0123] Weigh about 50 mg of tumor tissue stored in liquid nitrogen and gently rinse it in pre-cooled saline or PBS. Place the tissue in a petri dish, mince it thoroughly with ophthalmic scissors, and transfer it to a grinding tube. Use protease inhibitor PMSF solution and phosphatase inhibitor Cocktail to mix with RIPA lysis buffer (strong) in proportion (Cocktail / PMSF:RIPA=1:100) to prepare a strong lysis buffer, and add this strong lysis buffer to the grinding tube, 600 μL / tube. Add two steel beads to each grinding tube and then place it in a freezer grinder for grinding. After grinding, perform vortexing and other protein extraction operations. The specific steps are the same as the Western blot experiment in 7.3.

[0124] Example 1: PM upregulates malignant plasma cell intermolecular adhesion and significantly downregulates CDK6 expression in MM cells, suggesting that PM combined with immunomodulators may have synergistic anti-MM activity

[0125] Currently, PM has completed Phase I clinical trials for the treatment of relapsed and refractory B-cell hematological malignancies. 2 Eleven patients with R / R MM were enrolled in the 3+3 dose-escalation phase of the PM Phase I clinical trial. The disease control rate (DCR) was 72.7%. Although the majority of patients with R / R MM treated in the PM Phase I clinical trial were treated at a low-dose escalation stage, they still achieved a moderate efficacy and a favorable safety window. Unlike LBH589, no significant QTc prolongation was observed in the PM Phase I clinical trial. Therefore, PM is expected to achieve even better clinical efficacy in combination therapy in Phase Ib and Phase IIa treatments.

[0126] Initially, the present inventors conducted in-depth preclinical studies on the pharmacodynamics and mechanism of action of PM, including bulk RNA-seq analysis of MM1S cell lines and tumor tissues in MM1S model mice. To explore combination therapy strategies for PM, the present inventors further analyzed the sequencing data. Bulk RNA-seq and GSEA enrichment analysis revealed that PM treatment significantly upregulated the "GOBP_CELL_CELL_ADHESION_VIA_PLASMA_MEMBRANE_ADHESION_MOLE ECULES" gene set in MM1S cell lines and tumor tissues compared to the control and vehicle treatment groups (Figure 1, A and B). IMiDs used in MM treatment can downregulate the molecular adhesion of malignant plasma cells. Furthermore, recent studies have shown that upregulation of CDK6 is a key factor in the development of drug resistance to IMiDs such as Len and Pom in MM. Research conducted by the present invention has found that PM can concentration-dependently downregulate CDK6 protein expression in various MM cell lines, including dexamethasone-sensitive and -resistant MM1S and MM1R cells, as well as bortezomib-sensitive and -resistant RPMI-8226 and RPMI-8226R cells. Furthermore, activation of the MEK / ERK pathway is implicated in the resistance of MM cells to IMiDs, and previous studies conducted by the present invention have shown that PM treatment significantly inhibits the RAF / MEK / ERK / AKT signaling pathway in tumor cells. In summary, the present invention hypothesizes that the combination of PM and immunomodulators for the treatment of MM may have potent synergistic anti-MM activity.

[0127] Example 2: PM combined with Pom and DXM synergistically inhibits MM cell proliferation

[0128] Previous studies on the mechanism of PM in treating MM have suggested that PM combined with immunomodulators may have synergistic anti-MM activity. Based on this, the present invention first tested whether PM combined with the immunomodulator pomalidomide (Pomalidomide) exhibits synergistic effects in MM cell lines RPMI-8226, MM1S, and dexamethasone-resistant MM1R cells.

[0129] After 48 and 72 hours of treatment with various concentrations of PM and Pom alone, or in combination with PM and Pom, the antiproliferative activity of MM cell lines was determined using CCK8 assay, and the combination index (CI) was calculated using Calcusyn 2.0 software. As shown in Figures 2A and B, 3A and B, and 4A and B, the minimum combination indexes for the combination of PM and Pom in the RPMI-8226, MM1S, and dexamethasone-resistant MM1R cell lines were 0.225, 0.382, and 0.270, respectively. These results demonstrate that the combination of PM and Pom exhibits a synergistic antiproliferative effect in RPMI-8226, MM1S, and dexamethasone-resistant MM1R cell lines.

[0130] Furthermore, low-dose dexamethasone can enhance the efficacy of combined therapy and reduce toxic side effects. Therefore, the present invention further explored the in vitro synergistic antiproliferative effect of the triple combination of PM, Pom, and DXM in the treatment of multiple myeloma. As shown in Figures 2, C and D, the minimum combination indexes of PM, Pom, and DXM for the RPMI-8226 cell line at 48 hours and 72 hours were 0.092 and 0.135, respectively. As shown in Figures 3, C and D, the minimum combination indexes for the MM1S cell line at 48 hours and 72 hours were 0.272 and 0.121, respectively. As shown in Figures 4, C and D, the minimum combination indexes for the dexamethasone-resistant MM1R cell line at 48 hours and 72 hours were 0.535 and 0.437, respectively. These results demonstrate that the triple combination of PM, Pom, and DXM exhibits synergistic antiproliferative activity against all MM cell lines tested.

[0131] Example 3: PM combined with Pom and DXM synergistically downregulates the expression of MM survival-related proteins

[0132] In vitro studies on MM cell lines have demonstrated that PM combined with Pom and DXM exhibits synergistic anti-tumor cell proliferation activity, significantly outperforming either drug alone or the combination of Pom and DXM. Based on this, the present invention further investigated whether this triple-drug combination exhibits synergistic inhibitory activity against the expression of key proteins associated with MM survival in MM cell lines.

[0133] The expression levels of CDK6, c-MYC, IKZF1, IKZF3, and EZH2 proteins are closely associated with the development and poor prognosis of multiple cellular membranes (MM). CDK6 is a key factor in resistance to immunomodulatory agents in MM, and downregulating CDK6 can resensitize MM to immunomodulatory agents. As shown in Figure 5, PM alone and in combination with Pom and DXM significantly downregulated CDK6 expression in MM1S and MM1R cell lines, whereas Pom combined with DXM upregulated or had no effect on CDK6. Furthermore, PM combined with Pom and DXM at concentrations of 1 nM and 3 nM significantly downregulated the expression levels of these proteins in MM1S and MM1R cells compared to Pom combined with DXM. Furthermore, the combination of PM with Pom and DXM significantly downregulated the expression of these proteins compared to PM alone at the same or higher concentrations. These results suggest that PM combined with Pom and DXM synergistically inhibits the expression of MM survival-related proteins, resulting in synergistic anti-MM activity.

[0134] Example 4: PM concentration-dependent upregulation of CD38 expression in MM1S cells

[0135] Literature reports indicate that HDAC inhibitors can upregulate multiple antigens on tumor cells. The HDAC6 inhibitors ricolinostat and ACY-241 significantly upregulate CD38 expression in MM cells. Combining them with the CD38 monoclonal antibody daratumumab significantly enhances the anti-MM activity of daratumumab. Therefore, the present invention treated MM1S cells with varying concentrations of PM for 24 hours. Flow cytometry was used to measure the effect of PM treatment on CD38 expression in multiple myeloma cells to explore the potential for combined use of PM and CD38 monoclonal antibodies. As shown in Figure 6, PM, at low concentrations that do not cause MM1S cell death, upregulates CD38 expression in MM1S cells in a concentration-dependent manner. Therefore, the present invention will further investigate whether the combination of PM and daratumumab has synergistic anti-MM activity in vivo in model mice.

[0136] Example 5: PM combined with multiple drugs synergistically inhibits tumor growth in the RPMI-8226 mouse model

[0137] TP53 is a key tumor suppressor gene located on the short arm of human chromosome 17. It inhibits tumor cell proliferation and promotes apoptosis. Mutations in the tumor suppressor TP53 are common in patients with high-risk or advanced multiple myeloma (MM). These mutations can lead to increased tumor invasiveness and increased resistance to treatment, resulting in a poorer prognosis. The MM cell line RPMI-8226 carries this mutation, making the RPMI-8226 mouse model a common preclinical model for evaluating the efficacy of drug treatments for multiple myeloma.

[0138] Previous research results have shown that PM combined with Pom and DXM can synergistically inhibit the proliferation of multiple MM cells and the protein expression of key oncogenes in vitro, with superior efficacy compared to PM alone and the combination of Pom and DXM. To further investigate the efficacy of PM combined with pomalidomide and dexamethasone in a multiple myeloma mouse model, the present invention established an RPMI-8226 mouse model to explore the efficacy of this combination. Furthermore, the present invention also found that PM treatment can concentration-dependently upregulate CD38 expression in MM1S cells, suggesting that PM combined with daratumumab may also have synergistic anti-MM activity. Bortezomib is a commonly used drug for the treatment of MM and is also used in combination with various myeloma drugs. Therefore, in the RPMI-8226 mouse model, the present invention simultaneously explored the efficacy of various PM combination regimens, comparing the PM combination with pomalidomide and dexamethasone with other combination regimens (daratumumab and bortezomib).

[0139] 1. PM combined with pomalidomide and dexamethasone synergistically inhibited tumor growth in the RPMI-8226 mouse model, which was superior to other triple-drug combinations such as PM combined with daratumumab, bortezomib, and dexamethasone.

[0140] Establish RPMI-8226 model mice. When the tumor volume of tumor-bearing mice grows to 100-200mm 3 The patients were divided into groups and treated as shown in Table 3. As shown in Figure 7A, compared with the vehicle treatment group, the PM (5 mg / kg), Dara+Bort+DXM (5+0.1+1 mg / kg), Pom+DXM (Pd, 2.5+1 mg / kg), PM+Dara+Bort+DXM (5+5+0.1+1 mg / kg), PM+Dara+DXM (5+5+1 mg / kg), PM+Pd (5+2.5+1 mg / kg), and PM+Bort+DXM (5+0.1+1 mg / kg) administration groups had significant inhibitory effects on tumor growth in the NOD-SCID mouse subcutaneous tumor model of human multiple myeloma RPMI-8226 cell line. After 28 days of continuous treatment, the tumor inhibition rates of each treatment group calculated based on tumor weight were 90.64%, 87.12%, 95.37%, 97.58%, 97.50%, 99.65% and 95.52%, respectively; the relative tumor proliferation rate T / C% values ​​were 14.04%, 17.11%, 5.99%, 3.87%, 6.14%, 1.55% and 6.49%, respectively. The PM + Pd (5 + 2.5 + 1 mg / kg) combination group showed the greatest tumor suppression effect, with tumor regression in the model mice. This was superior to the PM alone (P < 0.0001), PM + daratumumab + dexamethasone (P = 0.0110), PM + daratumumab + bortezomib + dexamethasone (P = 0.0264), and PM + bortezomib + dexamethasone (P = 0.0003) groups, and significantly superior to the positive control groups of daratumumab + bortezomib + dexamethasone (P = 0.0057) and pomalidomide + dexamethasone (P = 0.0019) (Figure 7B). During the dosing period, mice in any group showed no significant weight loss (Figure 8C), had normal eating and activity, had no abnormal secretions or lesions around the eyes, mouth, nose, or ears, and were not observed to be agitated, aggressive, or depressed. No dose-related abnormal animal activities were found in the PM single or combination groups and the positive control group.

[0141] In summary, the combination of PM with pomalidomide and dexamethasone has strong synergistic inhibitory activity for tumor growth, and the tumors in the model mice basically regressed. The efficacy of this combination is better than that of PM with other three-drug or four-drug combinations such as daratumumab, bortezomib, and dexamethasone, and is also significantly better than the positive control of the three-drug combination of daratumumab, bortezomib, and dexamethasone.

[0142] 2. PM combined with Pom and DXM has no obvious toxicity to mouse organ tissues

[0143] In the RPMI-8226 mouse model, the combination of PM with Pom and DXM demonstrated the most effective treatment, with no significant weight loss, compared to the other PM combination groups and the positive control group. Therefore, the present invention performed HE staining on organ tissues of mice in the vehicle group, PM alone, Pom + DXM combination group, and PM + Pom + DXM combination group to investigate the effects of these combinations on various mouse organs.

[0144] The results are shown in Figure 8. The heart, liver, spleen, lung, kidney, stomach and intestinal tissue cells of mice in each treatment group had normal morphology, clear tissue structure and no obvious abnormalities. A small amount of myocardial fiber vacuolar degeneration was observed in the ventricular septum of the heart tissue of each treatment group, and no obvious inflammatory cell infiltration was observed. The liver tissue structure of the pomalidomide + dexamethasone combination group and the PM + pomalidomide + dexamethasone combination group was clear, except for a small amount of eosinophilic granules in the cytoplasm, no obvious inflammatory cell infiltration was observed. A slight increase in multinucleated giant cells was observed in the spleen tissue of each treatment group, including the vehicle, which may be related to the breeding environment and not caused by drug toxicity. No other obvious abnormalities were observed. Lung tissue in the pomalidomide + dexamethasone and PM + pomalidomide + dexamethasone groups showed focal alveolar dilatation, with no significant abnormalities in other structures. The lung tissue surface was covered with a smooth serous membrane, showing no significant abnormalities. The lung parenchyma consisted of numerous alveoli at the various levels of bronchial branches and their terminals. The bronchial structures at all levels were normal, and the alveolar walls were composed of a single layer of epithelium with a clear structure. The interstitium, including the connective tissue and blood vessels within the lungs, showed no significant abnormalities. No significant inflammatory cell infiltration was observed. In kidney tissue, glomeruli were evenly distributed, with uniform cell number and matrix within the glomeruli. The renal tubular epithelial cells were round and plump, with no significant inflammatory cell infiltration. The gastric tissue had clear structural layers. The gastric mucosal epithelium was a single layer of columnar epithelium with normal, tightly packed cells. The gastric glands were abundant, with normal, regularly packed cells. The muscularis was evenly stained, with normal muscle fiber morphology and structure, and no significant inflammatory cell infiltration was observed. The intestinal tissue layers were clearly structured and distinctly demarcated, the mucosal epithelium was intact, the cell morphology was normal, and the intestinal glands were abundant and regularly arranged. No significant inflammatory cell infiltration was observed. In summary, PM combined with Pom and DXM in the treatment of RPMI-8226 model mice had no significant toxic side effects.

[0145] 3. PM combined with Pom and DXM synergistically downregulates the expression of key proteins in MM tumor tissues

[0146] Further immunohistochemistry and Western blot analysis were performed on the tumor tissues of mice in the blank control group, PM alone, Pom+DXM combination group, and PM+Pom+DXM combination group to explore the effects of each treatment group on key proteins for tumor survival.

[0147] Immunohistochemistry results, as shown in Figure 9, show that Ac-H3 protein levels were significantly upregulated in tumor tissues of RPMI-8226 mouse models treated with PM alone and in combination with Pom and DXM. Ki67, a cell proliferation marker, was significantly downregulated compared to the vehicle group. Furthermore, levels of key proteins involved in MM cell survival, such as c-MYC and IRF4, were significantly downregulated, with the PM, Pom, and DXM combination group showing the greatest efficacy.

[0148] Consistent with the immunohistochemistry results, Western blot analysis of tumor tissues also showed that compared to the vehicle group, all treatment groups significantly downregulated the levels of c-MYC, IRF4, EZH2, and IKZF1, key myeloma cell survival factors. Furthermore, PM, in combination with Pom and DXM, exhibited significant synergistic activity. Furthermore, while literature reports indicate that long-term treatment with Pom and DXM for myeloma upregulates CDK6 levels, the combination of PM with Pom and DXM in the present invention significantly downregulated CDK6 expression (Figure 10), thereby overcoming Pom resistance.

[0149] In summary, consistent with the in vitro results, the combination of PM with Pom and DXM can synergistically inhibit key proteins for the survival of RPMI-8226 tumor cells, thereby inhibiting tumor progression in the RPMI-8226 mouse model, and the therapeutic effect is significantly better than that of PM alone and the combination of Pom and DXM.

[0150] Example 6: PM combined with pomalidomide and dexamethasone synergistically inhibited tumor growth in the MM1R mouse model, outperforming other triple-drug combinations of PM with daratumumab, lenalidomide, and dexamethasone.

[0151] Dexamethasone (DXM) is a widely used glucocorticoid in clinical practice and plays a crucial role in various chemotherapy regimens for multiple myeloma. Most patients with relapsed or refractory multiple myeloma have previously been exposed to dexamethasone. To establish a dexamethasone-resistant MM1R mouse model of multiple myeloma, we further investigated the in vivo efficacy of PM in combination with pomalidomide and dexamethasone. We also investigated the efficacy and toxicity of this combination compared with commonly used clinical triplet regimens, PM alone, and PM in combination with other drugs.

[0152] Dexamethasone-resistant MM1R cells were inoculated into NTG mice. When the tumor volume grew to 200 mm 3As shown in Figure 11A and B, each group of tumor-bearing mice was continuously administered for 16 days (Day 0-16). Compared with the blank control group, PM (5 mg / kg), Len+Bort+DXM (10+0.1+1 mg / kg), Selinexor+Pd (10+0.5+1 mg / kg), Dara+Pd (5+0.5+1 mg / kg), PM+Pd (5+0.5+1 mg / kg), PM+Len+DXM (5+10+1 mg / kg), and PM+Dara+DXM (5+5+1 mg / kg) had a significant inhibitory effect on the growth of human multiple myeloma cell MM1R subcutaneous tumor model. Statistical analysis of tumor weights in each group of mice revealed tumor inhibition rates of 80.56%, 51.85%, 72.22%, 37.96%, 88.89%, 84.26%, and 82.41% for each treatment group, respectively. The relative tumor growth rates (T / C%) were 36.72%, 48.50%, 28.88%, 60.66%, 27.04%, 27.33%, and 33.79%, respectively. Among the treatment groups, PM 5 mg / kg alone significantly outperformed the first-line combination therapy for multiple myeloma, Len+Bort+DXM (P=0.0006), the positive control, the Dara+Pd combination (P=0.0005), and slightly outperformed the Selinexor+Pd combination (P=0.0782). Among them, the PM+Pd (5+0.5+1 mg / kg) combined administration group had the best tumor inhibition effect, which was better than PM 5 mg / kg alone (P=0.0420).

[0153] These results demonstrate that PM monotherapy has a promising therapeutic effect in the dexamethasone-resistant MM1R mouse model. Furthermore, consistent with previous in vitro results and in vivo results in RPMI-8226 and MM1S mouse models, the combination of PM, pomalidomide, and dexamethasone demonstrated optimal efficacy, exhibiting significant combined activity and superior tumor inhibition to multiple clinical triplet combinations and other PM combination regimens. Furthermore, with the exception of the Selinexor + Pd combination group, which exhibited significant weight loss and diarrhea, no other treatment groups exhibited significant weight loss (Figure 11C). PM monotherapy and combination treatments had no significant effect on the diet, activity, or body weight of tumor-bearing mice.

[0154] Based on the above experiments, the present invention initially demonstrated, through preclinical efficacy evaluation of PM in the treatment of MM, that PM, a highly selective and active HDACI / IIb inhibitor, is a promising drug for the treatment of MM. Based on this, combined with bulk RNA-seq and molecular and cellular studies, potential combinations of PM for the treatment of R / R MM were explored. Further, using sensitive and resistant MM cell lines and MM mouse models, PM in combination with pomalidomide and dexamethasone demonstrated synergistic in vitro and in vivo antitumor activity, synergistically inhibiting the expression of key MM survival proteins. This combination demonstrated superiority to multiple existing clinical three-drug regimens, as well as PM with other three- and four-drug combinations, such as daratumumab and bortezomib, without significant toxic side effects. This preclinical pharmacodynamic evidence provides a foundation for Phase Ib / IIa clinical trials of PM in combination with R / R MM, which is of great clinical value and academic significance.

Claims

1. A combination drug of prilocastat mesylate and its analogs for preventing and treating multiple myeloma, characterized in that: The combination drug is a three-drug combination drug of prilocastat mesylate and its analogs, glucocorticoids and a third drug, which are administered separately or simultaneously; or, the combination drug is a four-drug combination drug of prilocastat mesylate and its analogs, glucocorticoids and a fourth drug and a fifth drug, which are administered separately or simultaneously; wherein, in the three-drug combination drug, the third drug is selected from an immunomodulator, a monoclonal antibody or a proteasome inhibitor; and in the three-drug combination drug, the fourth drug is a monoclonal antibody and the fifth drug is a proteasome inhibitor.

2. The combined drug according to claim 1, characterized in that: The prilocastat mesylate and its analogs are selected from compounds having the following structures: Preferably, the prilocastat mesylate and its analogs are selected from compounds having the following structures: More preferably, the prilocastat mesylate and its analogs are prilocastat mesylate for injection.

3. The combined drug according to claim 1, characterized in that: The glucocorticoid is selected from dexamethasone or prednisone; preferably, the glucocorticoid is dexamethasone.

4. The combined drug according to any one of claims 1 to 3, characterized in that: In the triple-drug combination, the third drug is selected from an immunomodulator or a monoclonal antibody; preferably, the third drug is an immunomodulator.

5. The combined drug according to any one of claims 1 to 3, characterized in that: The triple-drug combination drug must meet at least one of the following requirements: The immunomodulator is selected from pomalidomide, lenalidomide or thalidomide; preferably, the immunomodulator is selected from pomalidomide or lenalidomide; more preferably, the immunomodulator is pomalidomide; The monoclonal antibody is selected from daratumumab, elotuzumab or isatuximab; preferably, the monoclonal antibody is daratumumab; The proteasome inhibitor is selected from bortezomib, ixazomib or carfilzomib; preferably, the proteasome inhibitor is bortezomib.

6. The combined drug according to any one of claims 1 to 3, characterized in that: The four-drug combination drug must meet at least one of the following requirements: The monoclonal antibody is selected from daratumumab, elotuzumab or isatuximab; preferably, the monoclonal antibody is daratumumab; The proteasome inhibitor is selected from bortezomib, ixazomib or carfilzomib; preferably, the proteasome inhibitor is bortezomib.

7. The combined drug according to any one of claims 1 to 3, characterized in that: The triple-drug combination drug must meet at least one of the following requirements: When the third drug is an immunomodulator, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulators is 1.25-5:1:0.5-30; preferably, when the immunomodulator is pomalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulators is 1.25-5:1:0.5-2.5; preferably, when the immunomodulator is lenalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids and immunomodulators is 1.25-5:1:0.5-2.

5. The mass ratio of the main drugs of the analogue, glucocorticoid and immunomodulator is 1.25-5:1:5-30; more preferably, when the immunomodulator is pomalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogues, glucocorticoid and immunomodulator is 5:1:0.5-2.5; more preferably, when the immunomodulator is lenalidomide, the mass ratio of the main drugs of prilocastat mesylate and its analogues, glucocorticoid and immunomodulator is 5:1:10; When the third drug is a monoclonal antibody, the mass ratio of the main drug of prilocastat mesylate and its analogs, glucocorticoids and monoclonal antibodies is 1.25-5:1:2.5-25; preferably 5:1:5; When the third drug is a proteasome inhibitor, the main drug mass ratio of prilocastat mesylate and its analogs, glucocorticoids and proteasome inhibitors is 1.25-5:1:0.1-1; preferably 5:1:0.

1.

8. The combined drug according to any one of claims 1 to 3, characterized in that: In the four-drug combination drug, the mass ratio of the main drugs of prilocastat mesylate and its analogs, glucocorticoids, monoclonal antibodies and proteasome inhibitors is 1.25-5:1:2.5-25:0.1-1; preferably 5:1:5:0.

1.

9. The combined drug according to any one of claims 1 to 3, characterized in that: The combination drug is a preparation prepared by using prilocastat mesylate and its analogs, glucocorticoids and a third drug as active ingredients, and adding pharmaceutically acceptable excipients; or the combination drug is a preparation prepared by using prilocastat mesylate and its analogs, glucocorticoids and a fourth drug and a fifth drug as active ingredients, and adding pharmaceutically acceptable excipients; preferably, the preparation is an injectable preparation; more preferably, the injection route is at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection or intramyocardial injection.

10. The combined drug according to any one of claims 1 to 9, characterized in that: The multiple myeloma is relapsed or refractory multiple myeloma.

11. Use of the combined medicament of prilocastat mesylate and its analogs for preventing and treating multiple myeloma according to any one of claims 1 to 10 in the preparation of a drug for treating and / or preventing multiple myeloma.

12. The use according to claim 11, characterized in that: The multiple myeloma is relapsed or refractory multiple myeloma.

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