Use of nhe1 inhibitors for the preparation of a medicament for the prevention and treatment of multiple myeloma

Abstract

The application relates to application of an NHE1 inhibitor in preparation of a medicine for preventing and treating multiple myeloma. The application creatively finds that the NHE1 inhibitor has obvious prevention and treatment effects on multiple myeloma, and based on MM cell strains and experimental mice as research objects, it is verified that the NHE1 inhibitor can inhibit the proliferation of cells and promote the apoptosis of cells in the MM cell strains, and through establishment of a CDX mouse model, it is verified that the NHE1 inhibitor also has the effect of reducing tumor load on the MM mice in the body, which provides a new strategy for prevention and treatment of multiple myeloma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new strategy for the prevention and treatment of multiple myeloma, specifically the application of NHE1 inhibitors in the preparation of drugs for the prevention and treatment of multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is a hematologic disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow, accounting for approximately 10-15% of all hematologic malignancies. The median survival of MM patients has been significantly prolonged due to the availability of drugs such as proteasome inhibitors and immunomodulators. However, MM remains incurable, and many patients still experience relapse and drug resistance. Due to plasma cell infiltration and monoclonal immunoglobulin secretion, patients experience clinical symptoms such as hypercalcemia, renal insufficiency, anemia, bone destruction, and infection, severely impacting their quality of life. Therefore, identifying new therapeutic targets for multiple myeloma remains crucial for improving patient survival and prognosis.

[0003] CN115414355A discloses the application of styraxin in the preparation of drugs for treating multiple myeloma. It is the first discovery that styraxin has a selective killing effect on multiple myeloma cells and normal B cells, inhibiting the proliferation of multiple myeloma cells, inducing apoptosis of multiple myeloma cells, and inhibiting the colonization and growth of human multiple myeloma cells in mouse bone marrow, thus confirming that styraxin has an inhibitory effect on the occurrence and development of multiple myeloma cells.

[0004] CN115698065A provides a method for treating multiple myeloma (such as refractory or relapsed refractory multiple myeloma) in individuals who have received one to three prior therapies for multiple myeloma, the method comprising administering an anti-CD38 antibody, carfilzomib, and dexamethasone to the individual, the method being able to prolong the survival of patients with multiple myeloma.

[0005] Currently, there is still a lack of specific drugs for multiple myeloma; existing strategies for treating multiple myeloma are still very limited, and it is very meaningful to develop more treatment strategies for multiple myeloma. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new strategy for the prevention and treatment of multiple myeloma, specifically the application of NHE1 inhibitors in the preparation of drugs for the prevention and treatment of multiple myeloma.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of NHE1 inhibitors in the preparation of drugs for the prevention and treatment of multiple myeloma.

[0009] Cellular pH balance maintenance depends on ion transporters such as sodium-hydrogen exchangers (NHEs). NHE1 is the most widely expressed member of the NHE family. This invention creatively discovers that NHE1 inhibitors have significant preventive and therapeutic effects against multiple myeloma. Based on MM cell lines and experimental mice, this invention verifies that NHE1 inhibitors can inhibit cell proliferation and promote apoptosis in MM cell lines. Furthermore, by establishing a CDX mouse model, it is confirmed that NHE1 inhibitors also reduce tumor burden in MM mice in vivo. This provides a new strategy for the prevention and treatment of multiple myeloma. In this invention, "prevention and treatment" refers to both prevention and treatment.

[0010] Preferably, the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof.

[0011] The earliest widely used NHE1 inhibitor was amiloride, and its pharmaceutically acceptable salts (such as amiloride hydrochloride) and its pharmaceutically acceptable derivatives (such as 5-(N,N-hexamethylene)amiloride) are also common NHE1 inhibitors.

[0012] Preferably, the pharmaceutically acceptable derivative of amiloride includes 5-(N,N-hexamethylene)amiloride, whose chemical structure is shown below:

[0013]

[0014] This invention repositions the pharmacological function of 5-(N,N-hexamethylene)amilolide (HMA), further expanding its new applications in the prevention and treatment of multiple myeloma (MM), and providing potential treatment ideas and references for clinical practice.

[0015] Preferably, the drug promotes apoptosis of multiple myeloma cells and / or inhibits the proliferation of multiple myeloma cells.

[0016] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0017] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0018] Preferably, the dosage form of the drug is any pharmaceutically acceptable dosage form.

[0019] Secondly, the present invention provides the application of NHE1 inhibitors in the preparation of multiple myeloma cell apoptosis promoters.

[0020] According to the research results of this invention, NHE1 inhibitors can promote apoptosis of multiple myeloma cells at the cellular level (in vitro level). That is, NHE1 inhibitors can be made into a simple experimental preparation for exploring the physiological metabolic process of multiple myeloma cells. The apoptosis promoter claimed in this invention is not for eliminating the cause or lesion, that is, it is an application in the preparation of apoptosis promoters for multiple myeloma cells for a non-therapeutic purpose.

[0021] Preferably, the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof.

[0022] Preferably, the pharmaceutically acceptable derivatives of amiloride include 5-(N,N-hexamethylene)amiloride.

[0023] Thirdly, this invention provides the application of NHE1 inhibitors in the preparation of inhibitors for the proliferation of multiple myeloma cells.

[0024] According to the research results of this invention, NHE1 inhibitors can inhibit the proliferation of multiple myeloma cells at the cellular level (in vitro level). That is, NHE1 inhibitors can be made into a simple experimental preparation for exploring the physiological metabolic process of multiple myeloma cells. The cell proliferation inhibitor claimed in this invention is not for eliminating the cause or lesion, that is, it is an application in the preparation of multiple myeloma cell proliferation inhibitors for non-therapeutic purposes.

[0025] Preferably, the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof.

[0026] Preferably, the pharmaceutically acceptable derivatives of amiloride include 5-(N,N-hexamethylene)amiloride.

[0027] Fourthly, the present invention provides a method for promoting apoptosis of multiple myeloma cells and / or inhibiting the proliferation of multiple myeloma cells for non-therapeutic purposes, the method comprising: incubating multiple myeloma cells with an effective dose of an NHE1 inhibitor.

[0028] The method claimed in this invention does not directly target living human or animal bodies, but rather isolated tumor cells. It only protects the method of inhibiting the growth of tumor cells (at the cellular level). Furthermore, this method is not a process of eliminating the cause or lesion, nor is it a treatment method directly used to improve the health of human or animal bodies. Rather, it is intended for theoretical research on the physiological and metabolic behavior of multiple myeloma cells and for screening more drugs to treat multiple myeloma cells.

[0029] Preferably, the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof.

[0030] Preferably, the pharmaceutically acceptable derivatives of amiloride include 5-(N,N-hexamethylene)amiloride.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention creatively discovers that NHE1 inhibitors have significant preventive and therapeutic effects on multiple myeloma. Based on MM cell lines and experimental mice, this invention verifies that NHE1 inhibitors can inhibit cell proliferation and promote apoptosis in MM cell lines. Furthermore, by establishing a CDX mouse model, it is confirmed that NHE1 inhibitors also reduce tumor burden in MM mice in vivo. This provides a new strategy for the prevention and treatment of multiple myeloma. Attached Figure Description

[0033] Figure 1 This is a graph showing the effect of HMA on pH in the RPMI-8226 cell line;

[0034] Figure 2 This is a graph showing the effect of HMA on pH in the U266 cell line;

[0035] Figure 3 This is a graph showing the effect of HMA on the cell viability of the RPMI-8226 cell line;

[0036] Figure 4 This is a graph showing the effect of HMA on the cell viability of the U266 cell line;

[0037] Figure 5 This is a graph showing the effect of HMA on the proliferation of the RPMI-8226 cell line;

[0038] Figure 6 This is a graph showing the effect of HMA on the proliferation ability of the U266 cell line;

[0039] Figure 7 This is a diagram showing the results of HMA-induced apoptosis in the RPMI-8226 cell line;

[0040] Figure 8 This is a diagram showing the results of HMA-induced apoptosis in the U266 cell line;

[0041] Figure 9 The figure shows the effect of HMA on the Caspase-3 / 7 activity of the RPMI-8226 cell line.

[0042] Figure 10 The figure shows the effect of HMA on the Caspase-3 / 7 activity of the U266 cell line.

[0043] Figure 11 This is a graph showing the experimental results of the effect of HMA on mouse body weight;

[0044] Figure 12 This is a graph showing the effect of HMA on tumor volume in mice;

[0045] Figure 13 This is a graph showing the effect of HMA on tumor weight in mice;

[0046] Figure 14 This is a diagram showing the results of the Ki67 immunohistochemical experiment on mouse tumors using HMA. Detailed Implementation

[0047] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0048] The NHE1 inhibitor HMA involved in the following examples was purchased from Shanghai Haoyuan Biotechnology Co., Ltd. (MCE), model number HY-128067; the BALB / c-Nude mice involved in the following examples were purchased from Guangdong Jicui Yaokang Biotechnology Co., Ltd., aged 4-6 weeks.

[0049] Example 1

[0050] This example investigates the inhibitory effect of the NHE1 inhibitor HMA on the proliferation inhibition and apoptosis promotion of multiple myeloma:

[0051] (1) Intracellular pH detection:

[0052] RPMI-8226 and U266 cell lines were treated with 10 μM HMA or the corresponding volume of DMSO for 48 hours. The culture medium was removed by centrifugation at 1000 rpm for 5 min. The cells were washed once with PBS, followed by centrifugation to remove the PBS. 100 μL of BCECF-AM probe was added to each sample, bringing the final probe concentration to 5 μM, and diluted with PBS. The cells were then incubated at 37°C in the dark for 30 minutes. The BCECF-AM probe solution was removed by centrifugation at 1000 rpm for 5 min. The cells were washed once with PBS, and the following steps were performed:

[0053] Preparation of pH standard curve samples: A portion of the control group cells incubated with the above BCECF-AM probe was divided into four tubes. Each tube was resuspended in 100 μL of standard solutions at pH 4.5, 5.5, 6.5, and 7.5. Nigericin was then added to a concentration of 10 μM, and the mixture was incubated at 37°C in the dark for 10 minutes. Detection was performed using a microplate reader with an excitation wavelength ratio of Ex = 490 nm / 440 nm and Em = 535 nm. A standard curve formula was constructed based on the fluorescence ratio of the standard samples.

[0054] Intracellular pH measurement: Cell samples stained with the BCECF-AM probe were analyzed using a microplate reader at excitation wavelengths of 490 nm and 440 nm and an emission wavelength of 535 nm. The fluorescence intensity ratio at excitation wavelengths of 490 nm and 440 nm was calculated, and the intracellular pH value was determined using a standard curve. The results are shown below. Figure 1 (RPMI-8226 cell line) and Figure 2 As shown in the figure (U266 cell line), HMA causes a decrease in intracellular pH in RPMI-8226 and U266 cell lines.

[0055] (2) CCK8 assay for cell viability:

[0056] Seed RPMI-8226 cells or U266 cell suspension (100 μL / well) into 96-well plates, approximately 5 × 10⁶ cells per well. 4 Cells were collected, and then DMSO and different volumes of HMA were added to the suspension, along with complete culture medium without drugs and cells as a blank control. The cells were incubated for 48 hours. After 48 hours, 10 μL of CCK-8 solution was added to each well in the dark, and the cells were incubated for another 3 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0057] Calculation formula: Cell viability = (HMA group absorbance - blank control group absorbance) / (DMSO group absorbance - blank control group absorbance) × 100%. The results are as follows: Figure 3 (RPMI-8226 cell line) and Figure 4 As shown in the figure (U266 cell line), HMA significantly inhibited the viability of RPMI-8226 and U266 cell lines in a concentration-dependent manner.

[0058] (3) EdU cell proliferation detection:

[0059] RPMI-8226 and U266 cell lines were treated with 10 μM HMA or an equal volume of DMSO in six-well plates and cultured for 48 hours. 2× EdU working solution was prepared and added to the wells to achieve an EdU concentration of 10 μM. The plates were then incubated for another 2 hours. After EdU labeling, the cells were centrifuged to remove the culture medium and collected into 1.5 ml EP tubes. 1 ml of 4% paraformaldehyde fixative was added, and the tubes were fixed at 20°C for 15 min. The fixative was removed, and each tube was washed three times with 1 ml PBS for 3 minutes each time. The PBS was removed, and each well was incubated at 20°C for 15 minutes with 1 ml permeabilization buffer (PBS containing 0.3% Triton X-100). The permeabilization buffer was removed, and each well was washed twice with 1 ml PBS for 3 minutes each time. Click reaction solution was prepared according to the manufacturer's instructions. 0.5 ml of Click reaction solution was added to each well, mixed well, and incubated at 20°C in the dark for 30 minutes. Aspirate the Click reaction solution and wash three times with PBS for 3 minutes each time. Dilute Hoechst 33342 with PBS at a ratio of 1:1000. After aspirating the washings, add 1 ml of the diluted Hoechst 33342 solution to each tube and incubate at 20°C in the dark for 10 minutes. Aspirate the Hoechst 33342 solution. Wash three times with PBS for 3 minutes each time. Aspirate the PBS, resuspend the cells in 20 μL of PBS, and then spread them evenly on a glass slide. Allow the PBS to air dry in the dark. Place a drop of anti-fluorescence quencher on the slide, then cover it with a coverslip, avoiding air bubbles. Seal the edges of the coverslip with colorless nail polish. Then, photograph under an inverted fluorescence microscope.

[0060] Hoechst 33342 is a blue fluorescent compound with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. The results are as follows: Figure 5 (RPMI-8226 cell line) and Figure 6 As shown in the figure (U266 cell line), the EdU positivity rate in the experimental group was significantly lower than that in the control group, indicating that HMA inhibited the proliferation of RPMI-8226 and U266 cell lines.

[0061] (4) Annexin V / PI apoptosis detection:

[0062] RPMI-8226 and U266 cell lines were treated with 10 μM, 20 μM HMA, or an equal volume of DMSO and cultured for 48 hours. The culture medium was removed by centrifugation at 1000 rpm for 5 min. The cells were washed once with PBS and centrifuged to remove the PBS. Working solutions were prepared by diluting 5× Binding buffer to 1× with double-distilled water. 300 μl of working solution was added to each sample, followed by 5 μl Annexin V and 10 μl PI. The mixture was incubated at 20°C in the dark for 10 min. Apoptosis rates were detected by flow cytometry: Annexin V was detected using the FITC channel, and PI was detected using the PerCP-Cy5.5 channel. The results are shown below. Figure 7 (RPMI-8226 cell line) and Figure 8 As shown in the figure (U266 cell line), HMA induces an increase in the apoptosis rate of RPMI-8226 and U266 cell lines, and the apoptosis rate increases with increasing concentration.

[0063] (5) Caspase-3 / 7 activity assay:

[0064] Add 100 μl of RPMI-8226 cell culture and U266 cell culture to 96-well plates, treat with 10 μM HMA or an equal volume of DMSO, and incubate for 48 hours. Prepare Caspase 3 / 7 substrate working solution by mixing 50 μl of Caspase 3 / 7 substrate with 10 ml of assay buffer. Add 100 μL / well of Caspase 3 / 7 substrate working solution and incubate in the dark for 1 hour. Detect fluorescence intensity using a microplate reader at excitation / emission wavelengths of 350 / 450 nm (cutoff value = 420 nm). The results are shown below. Figure 9 (RPMI-8226 cell line) and Figure 10 As shown in the figure (U266 cell line), the Caspase-3 / 7 activity of RPMI-8226 and U266 cell lines was activated after HMA treatment.

[0065] Example 2

[0066] This embodiment investigates the therapeutic effect of the NHE1 inhibitor HMA on a mouse model of multiple myeloma:

[0067] (1) Construction of a mouse model of multiple myeloma (MM):

[0068] RPMI-8226 cell lines were cultured to a sufficient quantity to ensure good cell growth. Six-week-old female BALB / c-Nude mice were quarantined and housed in the SPF-grade animal facility of the Experimental Animal Center at Jinan University after one week of isolation and control. RPMI-8226 cells were collected, resuspended in PBS, and stored on ice. The skin at the injection site was disinfected with alcohol under the right anterior axilla of the nude mice, and 100 μL of 6 × 10⁶ cells was drawn into a 1 ml sterile syringe. 6 One RPMI-8226 cell was slowly injected subcutaneously via an oblique needle. Successful modeling was indicated when the tumor was palpable in the nude mouse.

[0069] (2) Tumor burden detection in mice:

[0070] After excluding mice that failed to develop tumors, 12 mice successfully developed tumors. There was no significant difference in tumor size among the 12 mice. The mice were then randomly divided into two groups: an experimental group (n=6) and a control group (n=6). Dosage: The experimental group received intraperitoneal injection of HMA at a dose of 10 mg / kg / day, while the control group received the corresponding solvent. Mouse weight was monitored every two days, and the results are shown below. Figure 11 As shown, the mice's body weight did not change significantly, indicating good drug safety. Tumor volume in mice was monitored every two days (tumor volume = (tumor long axis × tumor short axis)). 2 ) / 2), the statistical results are as follows Figure 12 As shown, the tumor volume in the experimental group mice was significantly smaller than that in the control group.

[0071] Two weeks after administration, or when the tumor diameter in any direction reaches 1.5 cm, or when the mouse is unable to crawl, eat, or drink, the experiment is considered an endpoint. The mouse is then sacrificed, the tumor is removed, and weighed. The results are statistically analyzed as follows: Figure 13 As shown, the tumor weight in the experimental group mice was significantly lower than that in the control group. A portion of the tumor was then fixed with paraformaldehyde for immunohistochemical detection, and the results are as follows. Figure 14 As shown.

[0072] The applicant declares that this invention illustrates the application of the NHE1 inhibitor in the preparation of drugs for the prevention and treatment of multiple myeloma through the above embodiments. However, this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0073] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0074] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of NHE1 inhibitors in the preparation of drugs for the prevention and treatment of multiple myeloma. The NHE1 inhibitor is 5-(N,N-hexamethylene)amilolide.

2. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

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