CN117323334A

CN117323334A - Application of NHE1 inhibitor in preparation of medicine for preventing and treating multiple myeloma

Info

Publication number: CN117323334A
Application number: CN202311274999.4A
Authority: CN
Inventors: 曾辉; 杨年会; 杜鹃; 萧伟豪; 邓素琪
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-01-02

Abstract

The invention relates to application of an NHE1 inhibitor in preparation of a medicament for preventing and treating multiple myeloma. The invention creatively discovers that the NHE1 inhibitor has obvious prevention and treatment effects on multiple myeloma, based on the MM cell strain and the experimental mice as research objects, verifies that the NHE1 inhibitor can inhibit proliferation of cells and promote apoptosis of cells in the MM cell strain, and verifies that the NHE1 inhibitor also has the effect of relieving tumor load on the MM mice in vivo by establishing a CDX mouse model, thereby providing a new strategy for preventing and treating the multiple myeloma.

Description

Application of NHE1 inhibitor in preparation of medicine for preventing and treating multiple myeloma

Technical Field

The invention belongs to the technical field of biological medicines, relates to a novel strategy for preventing and treating multiple myeloma, and in particular relates to application of an NHE1 inhibitor in preparation of a medicament for preventing and treating multiple myeloma.

Background

Multiple Myeloma (MM) is a disease of the blood system characterized by clonal proliferation of malignant plasma cells in the bone marrow, accounting for about 10-15% of the tumors of the blood system. Because of the advent of drugs such as proteasome inhibitors and immunomodulators, the median survival of MM patients has been significantly prolonged. However, MM is still an incurable disease and there are still many patients with recurrent drug resistance. Because of the infiltration of plasma cells and the secretion of monoclonal immunoglobulin, patients can have clinical symptoms such as hypercalcemia, renal insufficiency, anemia, bone destruction, infection and the like, the life quality of the patients is seriously influenced, and finding new targets for treating multiple myeloma is still crucial for improving the life quality and prognosis of the patients.

CN115414355a discloses the use of prodigiosin in the preparation of a medicament for the treatment of multiple myeloma. The first finding shows that the prodigiosin has selective killing effect on multiple myeloma cells and normal B cells, can inhibit proliferation of the multiple myeloma cells, induce apoptosis of the multiple myeloma cells, inhibit the colonization and growth of the human multiple myeloma cells in the bone marrow of mice, and prove that the prodigiosin has the effect of inhibiting the occurrence and development of the multiple myeloma cells.

CN115698065a provides a method for treating multiple myeloma (e.g., refractory multiple myeloma or relapsed refractory multiple myeloma) in an individual who has received one to three previous therapies for multiple myeloma, the method comprising administering to the individual an anti-CD 38 antibody, carfilzomib, and dexamethasone, which method is capable of extending the survival of a patient with multiple myeloma.

At present, specific drugs for multiple myeloma are still lacking; there are also limited strategies in the prior art for treating multiple myeloma, and it is of great interest to develop more strategies for treating multiple myeloma.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a novel strategy for preventing and treating multiple myeloma, and particularly provides application of an NHE1 inhibitor in preparing medicines for preventing and treating multiple myeloma.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides the use of an NHE1 inhibitor in the manufacture of a medicament for the prevention and treatment of multiple myeloma.

The invention creatively discovers that an NHE1 inhibitor has obvious prevention and treatment effects on multiple myeloma based on MM cell lines and experimental mice as research objects, verifies that the NHE1 inhibitor can inhibit proliferation and promote apoptosis of cells in the MM cell lines, and verifies that the NHE1 inhibitor also has the effect of relieving tumor burden on MM mice in vivo by establishing a CDX mouse model. The term control in the present invention means prevention or treatment.

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

The earliest widely used NHE1 inhibitor was amiloride, with pharmaceutically acceptable salts thereof (e.g., amiloride hydrochloride) and pharmaceutically acceptable derivatives thereof (e.g., 5- (N, N-hexamethylene) amiloride) also being common NHE1 inhibitors.

Preferably, the pharmaceutically acceptable derivatives of amiloride include 5- (N, N-hexamethylene) amiloride, the chemical structure of which is shown below:

the invention relocates the drug function of 5- (N, N-hexamethylene) amiloride (HMA), further expands the new application in preventing and treating Multiple Myeloma (MM), and provides potential treatment thought and reference for clinic.

Preferably, the medicament promotes apoptosis and/or inhibits proliferation of multiple myeloma cells.

Preferably, the medicament also contains pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifying agents, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.

Preferably, the dosage form of the medicament is any pharmaceutically acceptable dosage form.

In a second aspect, the invention provides the use of an NHE1 inhibitor in the preparation of a multiple myeloma cell apoptosis promoter.

According to the research result of the invention, the NHE1 inhibitor can promote apoptosis of multiple myeloma cells at a cellular level (in vitro level), namely the NHE1 inhibitor can be prepared into a simple preparation for test, which is used for exploring physiological metabolic processes of the multiple myeloma cells, and the apoptosis promoter claimed by the invention is not used for eliminating etiology or focus, namely, the application of the apoptosis promoter in preparing the multiple myeloma cell apoptosis promoter with non-treatment as a destination.

Preferably, the pharmaceutically acceptable derivative of amiloride comprises 5- (N, N-hexamethylene) amiloride.

In a third aspect, the invention provides the use of an NHE1 inhibitor in the preparation of an inhibitor of multiple myeloma cell proliferation.

According to the research results of the invention, the NHE1 inhibitor can inhibit proliferation of multiple myeloma cells at a cellular level (in vitro level), namely, the NHE1 inhibitor can be prepared into a simple preparation for test, which is used for exploring physiological metabolic processes of the multiple myeloma cells, and the claimed cell proliferation inhibitor is not used for eliminating etiology or focus, namely, the application of the claimed cell proliferation inhibitor in preparing the multiple myeloma cell proliferation inhibitor with non-therapeutic purposes.

In a fourth aspect, the present invention provides a method of promoting apoptosis and/or inhibiting proliferation of multiple myeloma cells, for non-therapeutic purposes, comprising: multiple myeloma cells are incubated in admixture with an effective dose of an NHE1 inhibitor.

The method claimed by the invention does not take living human or animal bodies as direct implementation objects, but takes pure in-vitro tumor cells as direct implementation objects, and only protects the tumor cells (cell level) against the growth inhibition mode; meanwhile, the method is not used for eliminating the etiology or focus, is not directly used for improving the health condition of the human body or the animal body, but is used for theoretical research on physiological metabolism behaviors of the multiple myeloma cells and screening more medicines for treating the multiple myeloma cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers that the NHE1 inhibitor has obvious prevention and treatment effects on multiple myeloma, based on the MM cell strain and the experimental mice as research objects, verifies that the NHE1 inhibitor can inhibit proliferation of cells and promote apoptosis of cells in the MM cell strain, and verifies that the NHE1 inhibitor also has the effect of relieving tumor load on the MM mice in vivo by establishing a CDX mouse model, thereby providing a new strategy for preventing and treating the multiple myeloma.

Drawings

FIG. 1 is a graph of the effect of HMA on pH in an RPMI-8226 cell line;

FIG. 2 is a graph of the effect of HMA on pH in the U266 cell line;

FIG. 3 is a graph of the effect of HMA on cell viability of the RPMI-8226 cell line;

FIG. 4 is a graph of the effect of HMA on cell viability of the U266 cell line;

FIG. 5 is a graph of the results of HMA affecting the proliferative capacity of the RPMI-8226 cell line;

FIG. 6 is a graph of the results of HMA affecting the proliferation potency of the U266 cell line;

FIG. 7 is a graph of the results of HMA induction of apoptosis in the RPMI-8226 cell line;

FIG. 8 is a graph of the results of HMA induction of apoptosis in the U266 cell line;

FIG. 9 is a graph showing the effect of HMA on Caspase-3/7 activity of the RPMI-8226 cell line;

FIG. 10 is a graph showing the effect of HMA on Caspase-3/7 activity of the U266 cell line;

FIG. 11 is a graph of experimental results of the effect of HMA on mouse body weight;

FIG. 12 is a graph of the effect of HMA on tumor volume in mice;

FIG. 13 is a graph of the effect of HMA on tumor weight in mice;

FIG. 14 is a graph of the results of a Ki67 immunohistochemical experiment of HMA on tumors in mice.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The NHE1 inhibitor HMA referred to in the following examples was purchased from Shanghai Haoyuan Biotechnology Co., ltd (MCE) and is model HY-128067; BALB/c-Nude mice referred to in the following examples were purchased from Guangdong Collection of Kangkang Biotech Co., ltd, and were aged for 4-6 weeks.

Example 1

This example explores the proliferation inhibition and apoptosis promoting effects of the NHE1 inhibitor HMA on multiple myeloma:

(1) Intracellular pH detection:

the RPMI-8226 and U266 cell lines were given HMA 10. Mu.M or corresponding volumes of DMSO treatment, respectively, for 48 hours. The medium was removed by centrifugation at 1000rpm for 5min. The PBS was washed once and then centrifuged to remove the PBS. To each sample, 100. Mu.L of BCECF-AM probe was added at a final concentration of 5. Mu.M, diluted with PBS. Followed by incubation at 37℃for 30 minutes in the absence of light. 1000rpm,5min centrifugation, and removal of BCECF-AM probe solution. The PBS was washed one pass, the following was performed:

preparing a pH standard curve sample: a portion of the control cell fraction 4 tubes incubated with the BCECF-AM probe was resuspended in 100. Mu.L of each of the standard solutions having pH values of 4.5, 5.5, 6.5 and 7.5, and then Nigericin (Nigericin) was added to a concentration of 10. Mu.M, and after mixing, incubated at 37℃for 10 minutes in the absence of light. Detection on a microplate reader, excitation wavelength ratio: ex=490 nm/440nm, em=535 nm. And (5) preparing a standard curve formula according to the fluorescence ratio of the standard sample.

Intracellular pH assay: and (3) detecting the sample cells dyed by the BCECF-AM probe by using an enzyme-labeled instrument, wherein the excitation light is 490nm and 440nm respectively, the emitted light is 535nm, the fluorescence intensity ratio of the excitation light is 490nm/440nm respectively is calculated, and the pH value in the sample cells is calculated through a standard curve. As shown in FIG. 1 (RPMI-8226 cell line) and FIG. 2 (U266 cell line), respectively, HMA decreased the intracellular pH of the RPMI-8226 and U266 cell lines.

(2) Cell viability was measured by CCK8 method:

RPMI-8226 cells or U266 cell suspensions (100. Mu.L/well) were seeded in 96-well plates at about 5X 10 per well ⁴ Cells were then incubated in culture for 48 hours with DMSO and different volumes of HMA, respectively, in suspension, and complete medium without drug and cells as a blank. After 48 hours, 10. Mu.L of CCK-8 solution was added to each well in the dark and incubation was continued for 3 hours in the cell incubator, after which the absorbance at 450nm was measured on a microplate reader.

The calculation formula is as follows: cell viability= (HMA absorbance-placebo absorbance)/(DMSO absorbance-placebo absorbance) ×100%. The results are shown in FIG. 3 (RPMI-8226 cell line) and FIG. 4 (U266 cell line), respectively, as can be seen, HMA significantly inhibited the viability of the RPMI-8226 and U266 cell lines and was concentration dependent.

(3) EdU cell proliferation assay:

RPMI-8226 and U266 cell lines were incubated in six well plates for 48 hours after 10 μm HMA or equivalent volumes of DMSO treatment. 2 XEdU working solution was prepared, and the mixture was added to the above-mentioned well plate in an equal volume so that the EdU concentration was 10. Mu.M, and the mixture was placed in an incubator for further culturing for 2 hours. After the EdU labeling of cells was completed, the culture broth was removed by centrifugation to collect cells in 1.5ml EP tube, and 1ml of 4% paraformaldehyde fixing solution was added thereto for fixation at 20℃for 15min. The fixative was removed and cells were washed 3 times for 3 minutes with 1ml PBS per tube. PBS was removed and each well was incubated with 1ml of permeate (PBS containing 0.3% Triton X-100) at 20℃for 15 minutes. The permeant was removed and cells were washed 2 times with 1ml PBS per well for 3 minutes. And preparing a Click reaction solution according to instructions. 0.5ml of Click reaction solution is added into each hole, and the mixture is uniformly mixed, and incubated for 30 minutes at 20 ℃ in a dark place. The Click reaction was aspirated and washed 3 times for 3 minutes with PBS. Hoechst33342 was diluted 1:1000 in PBS. After the washing solution was removed by pipetting, 1ml of diluted Hoechst33342 solution was added to each tube and incubated at 20℃for 10min in the dark. The Hoechst33342 solution was pipetted off. The PBS was washed 3 times for 3 minutes each. The PBS was removed, the cells were resuspended in 20. Mu.L of PBS, then spread evenly on a glass slide, and after the PBS was air-dried in the dark, the glass slide was dropped with a drop of anti-fluorescence quencher, then covered with a cover slip, during which air bubbles were avoided, and the cover slip was blocked with colorless nail polish. Photography was then performed under a fluorescence inverted microscope.

Hoechst33342 is blue fluorescence, the maximum excitation wavelength is 346nm, and the maximum emission wavelength is 460nm. The results are shown in FIG. 5 (RPMI-8226 cell line) and FIG. 6 (U266 cell line), respectively, which shows that the experimental group EdU positive ratio is significantly lower than the control group, indicating the ability of HMA to inhibit proliferation of the RPMI-8226 and U266 cell lines.

(4) Annexin V/PI apoptosis assay:

the RPMI-8226 and U266 cell lines were incubated for 48 hours after 10. Mu.M, 20. Mu.M HMA or equal volumes of DMSO treatment, respectively. The medium was removed by centrifugation at 1000rpm for 5min. The PBS was washed once and centrifuged to remove the PBS. Diluting 5×binding buffer to 1×with double distilled water to obtain working solution, adding 300 μl of working solution to each sample, adding 5 μl of Annexin V and 10 μl of PI to each sample, mixing, and dyeing at 20deg.C for 10min in dark place. Detecting apoptosis proportion by a flow meter: FITC channel detects Annexin V and PerCP-Cy5.5 channel detects PI. The results are shown in FIG. 7 (RPMI-8226 cell line) and FIG. 8 (U266 cell line), respectively, which shows that HMA induced an increase in the proportion of apoptosis in the RPMI-8226 and U266 cell lines and an increase in the proportion of apoptosis with increasing concentration.

(5) Caspase-3/7 Activity assay:

100 μl of RPMI-8226 cell fluid and U266 cell fluid were added to 96 well plates and incubated for 48 hours after 10 μM HMA or equivalent volumes of DMSO treatment. A Caspase 3/7 substrate working solution was prepared in a ratio of 50. Mu.l Caspase 3/7 substrate to 10ml assay buffer, and thoroughly mixed. 100. Mu.L/well of Caspase 3/7 substrate working solution was added and incubated in the incubator protected from light for 1h. Fluorescence intensity was detected with a microplate reader under excitation/emission wavelength=350/450 nm (cut-off=420 nm). As shown in FIG. 9 (RPMI-8226 cell line) and FIG. 10 (U266 cell line), respectively, the Caspase-3/7 activity of both the RPMI-8226 and U266 cell lines was activated after HMA treatment.

Example 2

This example explores the therapeutic effect of the NHE1 inhibitor HMA on multiple myeloma model mice:

(1) Construction of Multiple Myeloma (MM) mouse model:

and (3) culturing the RPMI-8226 cell line to a sufficient quantity to ensure that the cell growth state is good. Female BALB/c-Nude (Nude mice) of 6 weeks old were kept in SPF-class animal facilities at the laboratory animal center of Nanjing university and Nanjing university for one week of quarantine. RPMI-8226 cells were collected and stored in PBS in ice for resuspension. Skin at inoculation site of nude mice under right anterior axilla subcutaneous alcohol sterilization, 100. Mu.L of 6X 10 was aspirated by 1ml sterile syringe ⁶ RPMI-8226 cells were injected subcutaneously with a needle, and slowly. Modeling was indicated to be successful when nude mouse tumors were palpable.

(2) Mouse tumor burden detection:

nude mice failed to form tumor are knocked out, becomeThe number of nude mice which become tumor is 12. There was no significant difference in tumor volume size in 12 nude mice. The tumor-bearing nude mice were then randomly divided into two groups: experimental group 6, control group 6. Dosage of administration: the experimental group was intraperitoneally injected with HMA at a dose of 10mg/kg/d, and the control group was given the corresponding solvent. The body weight of the mice was monitored every 2 days, and the statistical result is shown in fig. 11, and the body weight change of the mice is not obvious, which suggests that the drug safety is good. Mice tumor volume was monitored every 2 days (tumor volume= (tumor long diameter x tumor short diameter) ² ) And/2), the statistical result is shown in fig. 12, and the tumor volume of the mice in the experimental group is significantly smaller than that of the mice in the control group.

Mice were sacrificed as experimental endpoints for 2 weeks of dosing or when tumors reached 1.5cm in diameter in either direction or the mice were unable to crawl or feed water, tumors were removed and weighed, and the statistical results are shown in fig. 13, with the tumor weights of the experimental mice significantly reduced from the control group. A portion of tumor paraformaldehyde was then immobilized for immunohistochemical detection, with the results shown in FIG. 14.

The applicant states that the use of the NHE1 inhibitors of the invention in the manufacture of a medicament for the treatment of multiple myeloma is illustrated by the examples above, but the invention is not limited to, i.e. it is not meant that the invention must be practiced in dependence upon the examples above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

Use of nhe1 inhibitors for the preparation of a medicament for the prevention and treatment of multiple myeloma.
2. The use according to claim 1, wherein the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof.
3. The use according to claim 2, wherein the pharmaceutically acceptable derivative comprises 5- (N, N-hexamethylene) amiloride.
4. The use according to any one of claims 1 to 3, wherein the medicament promotes apoptosis and/or inhibits proliferation of multiple myeloma cells.
5. The use according to any one of claims 1 to 4, wherein the medicament further comprises pharmaceutically acceptable excipients;

preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifying agents, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers;

preferably, the dosage form of the medicament is any pharmaceutically acceptable dosage form.
Use of nhe1 inhibitors in the preparation of a multiple myeloma apoptosis promoter.
7. The use according to claim 6, wherein the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof;

preferably, the pharmaceutically acceptable derivative comprises 5- (N, N-hexamethylene) amiloride.
Use of an nhe1 inhibitor in the preparation of an inhibitor of multiple myeloma cell proliferation.
9. The use according to claim 8, wherein the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof;

preferably, the pharmaceutically acceptable derivative comprises 5- (N, N-hexamethylene) amiloride.
10. A method of promoting apoptosis and/or inhibiting proliferation of multiple myeloma cells for a non-therapeutic purpose, the method comprising: incubating multiple myeloma cells in combination with an effective dose of an NHE1 inhibitor;

preferably, the NHE1 inhibitor comprises amiloride, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof;

preferably, the pharmaceutically acceptable derivative comprises 5- (N, N-hexamethylene) amiloride.