CN115089601B

CN115089601B - Use of ivermectin in the preparation of a medicament for the treatment of multiple myeloma

Info

Publication number: CN115089601B
Application number: CN202210822340.7A
Authority: CN
Inventors: 郑宇欢; 罗红梅
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2023-06-09
Anticipated expiration: 2042-07-13
Also published as: CN115089601A

Abstract

The invention provides the use of ivermectin (abbreviated as IVM) as the sole active ingredient in the manufacture of a medicament for the treatment of multiple myeloma (abbreviated as MM) and the use of ivermectin in combination with an anti-myeloma medicament in the manufacture of a medicament for the treatment of multiple myeloma. The invention discovers that IVM has killing effect on MM cells for the first time. Based on the hypothesis of the mechanism of action of IVM on MM cells, the combination of IVM and bortezomib (abbreviated as BTZ) shows that the dual-drug combination anti-MM effect is significantly better than that of BTZ or IVM single drug. Because IVM is a mature medicine used clinically for years, clinical practice shows that the medicine has higher safety in use in patients, and the concept of 'IVM combined with BTZ for treating MM' has a great clinical transformation prospect.

Description

Use of ivermectin in the preparation of a medicament for the treatment of multiple myeloma

Technical Field

The invention particularly relates to application of ivermectin in preparing medicines for treating multiple myeloma.

Background

Multiple Myela (MM) is a plasma cell malignancy that can secrete large amounts of misfolded or non-functioning monoclonal immunoglobulins. The ubiquitin-proteasome system (UPS) directs its degradation by proteasomes to maintain intracellular homeostasis by ubiquitin-labeling unfolded/misfolded proteins. Therefore, UPS plays an important role in the development and progress of MM. The 26S structural proteasome is a multi-subunit complex, consisting of a central 20S Core (CP) and two terminal 19S regulatory parts (regulatory particles, RP) (FIG. 1A). The 20S core consists of 2 alpha and 2 beta loops, each comprising 7 corresponding subunits. The proteasome has 3 active catalytic centers, all on the beta ring, β1 (PSMB 6 encoded, with caspase-like catalytic activity), β2 (PSMB 7 encoded, with trypsin-like catalytic activity) and β5 (PSMB 5 encoded, with chymotrypsin-like catalytic activity) (fig. 1B). Proteasome inhibitors (Proteasome inhibitors, PIs) are a class of anti-MM drugs currently in common use. PSMB5 is a major target for PIs. MM cells rely more on UPS systems to maintain intracellular homeostasis than normal cells, and when PIs inhibit key steps of protein degradation, they lead to endoplasmic reticulum stress (Endoplasmic reticulum stress, ER stress), activation of unfolded protein responses (Unfolded Protein Response, UPR), ultimately leading to cell death. MM cells were therefore sensitive to PIs-like drugs (fig. 1C).

Ivermectin (IVM) is a macrolide antibiotic with good efficacy against a variety of nematode and arthropod parasitic diseases, and was originally widely used for the treatment of animal parasitic diseases. With the intensive research, new roles of IVM have been gradually explored. Researchers found that IVM blocked viral protein entry into the nucleus by inhibiting the import alpha/beta pathway of importin, thereby achieving anti-HIV-1 and dengue virus effects. Also, it has been shown that IVM has therapeutic effects on various viruses such as SARS-CoV-2.

In recent years, it has been found that IVM may also have an anti-tumour effect. In the research of leukemia and a plurality of solid tumors (such as breast cancer, liver cancer, gastric cancer and the like), IVM has strong anti-tumor effect. The relevant mechanism is summarized as follows: 1) IVM induces apoptosis in cells: apoptosis, autophagy, and inflammatory necrosis; 2) Inhibiting tumor stem cells; 3) Reversing tumor multidrug resistance; 4) Enhancing the curative effect of targeted therapy. Numerous signaling pathways/molecules, such as Wnt pathway, akt/mTOR pathway, MAPK pathway, HSP27, KPNB1, etc., may be affected by IVM. The continuous progress in mechanism exploration has driven the expansion of clinical applications of IVM, and related clinical experiments have been conducted in various solid tumors and leukemias. However, the literature search suggests that the role of IVM in MM is not reported.

Disclosure of Invention

The present invention provides the use of ivermectin as the sole active ingredient in the manufacture of a medicament for the treatment of multiple myeloma.

Further, the drug is a drug that inhibits myeloma cell proliferation, inhibits myeloma cell proteasome activity, and/or causes DNA damage to myeloma cells.

Further, the medicine is an oral preparation or an injection.

The invention also provides application of ivermectin and an anti-myeloma drug in preparing a combined drug for treating multiple myeloma.

Further, the anti-myeloma drug is any one or more of a proteasome inhibitor, a glucocorticoid, an immunomodulator, an XOP1 inhibitor, CD38 monoclonal antibody, marflange and doxorubicin.

Still further, the proteasome inhibitor comprises bortezomib, carfilzomib, ifenprodil Sha Zuo meters; the glucocorticoid comprises dexamethasone and prednisone; the immunomodulator comprises thalidomide, lenalidomide and pomalidomide; the XPO1 inhibitor includes plug Li Nisuo; the CD38 mab includes up to Lei Tuoyou mab.

Further, the anti-myeloma drug is bortezomib.

Further, the mass ratio of bortezomib to ivermectin in the combination is 1-5: 25 to 30, preferably 3:28.

the invention finally provides a combination for the treatment of multiple myeloma comprising ivermectin and an anti-myeloma agent for simultaneous or separate administration.

Further, the anti-myeloma drug is bortezomib.

Further, the mass ratio of bortezomib to ivermectin is 1-5: 25 to 30, preferably 3:28.

further, the combination drug is an injection and/or an oral preparation.

The application of ivermectin in preparing medicaments for treating multiple myeloma is disclosed, and IVM has killing effect on MM cells for the first time. Based on the hypothesis of the action mechanism of IVM on MM cells, the combination application of IVM and BTZ shows that the dual-drug combination anti-MM effect is obviously superior to that of BTZ or IVM single drug, and the dual-drug combination anti-MM has a synergistic effect. Because IVM is a mature drug used clinically for many years, clinical practice shows that the drug has higher safety in use in patients. From the aspects of curative effect and safety, the combination of IVM and BTZ can prolong the service life of patients with multiple myeloma and improve the life quality of the patients, and the concept of treating MM by the combination of IVM and BTZ has a great clinical transformation prospect.

In light of the foregoing, many modifications, substitutions, and variations can be made in accordance with the ordinary skill in the art without departing from the basic spirit and scope thereof.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 schematic diagram of the structure of the proteasome

FIG. 2Ivermectin inhibits proliferation of myeloma cells

FIG. 3Ivermectin inhibits proteasome activity of myeloma cells

FIG. 4Ivermectin can cause DNA damage to myeloma cells

FIG. 5 synergistic effect of Ivermectin and bortezomib

Detailed Description

Example 1

1. Main reagent and consumable

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Second, the main instrument

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3. Experimental method

1. Detection of proteasome Activity by fluorogenic substrates

1.1 preparation of related Agents

(1) Proteasome buffer preparation: 50mM Tris (pH 7.5), 5mM MgCl ₂ 10% glycerol, 1mM ATP,1mM DTT, all of which are the final concentrations of the various reagents in solution, were stored at-20deg.C after packaging.

(2) 2mM Suc-LLVY-AMC substrate preparation: 5mg of substrate was centrifuged at 4℃at 10000rpm/min for 1min. DMSO (3.273 ml) was then added to prepare 2mM Suc-LLVY-AMC, which was dispensed in 100 ul/tube and stored at-80 ℃.

1.2 specific steps

(1) After the cells are treated, protein is extracted and BCA is quantified (see later for specific procedures).

(2) Protein lysate collected in the previous step, proteasome buffer, and fluorogenic substrate formulated with DMSO were added sequentially to a 96-well plate (white). Overall 200ul: 100ul of fluorogenic substrate, protein lysate and proteasome buffer total 100ul.

(3) Mix well, act gently.

(4) Incubate for 1h at 37 ℃.

(5) Detecting and exciting by a multifunctional fluorescent enzyme-labeled instrument: 345nm, emission: 445nm.

2.Western blotting

2.1 preparation of related Agents

(1) Preparation of 10×running buffer: taking 24.16g of Tris base, 150.4g of glycine and 8g of SDS, adding double distilled water to a volume of 800mL, fully and uniformly mixing, and preserving at normal temperature for standby.

(2) Preparation of 1×running buffer: 100mL of 10×running buffer is taken, double distilled water is added to a constant volume of 1L, and the mixture is fully and uniformly mixed for preparation.

(3) Preparation of 10×trans buffer: taking 30.3g of Tris base and 144g of glycine, adding double distilled water to a volume of 1L, fully and uniformly mixing, and preserving at normal temperature for standby.

(4) Preparation of 1×trans buffer: 100mL of 10×running buffer,150mL of methanol are taken, double distilled water is added to a constant volume of 1L, and the mixture is fully and uniformly mixed, and precooled in a refrigerator at 4 ℃ for standby.

(5) Preparation of 10×tbs: taking 24.2g of Tris base and 80g of NaCl, adding double distilled water to a volume of 1L, adjusting the pH value to 7.6, and preserving at normal temperature for standby.

(6) Preparation of 1×tbst (as-prepared): taking 100mL of 10 XTBS, 1mL of Tween-20, adding double distilled water to a volume of 1L, fully mixing, and preserving at normal temperature for standby.

2.2 extraction of Total protein

(1) The cells were collected into a 15mL centrifuge tube and centrifuged at 800rpm for 5min at room temperature.

(2) The supernatant was discarded, resuspended in an appropriate amount of PBS and centrifuged at 800rpm for 5min at room temperature.

(3) The supernatant was discarded, 50-100. Mu.l of whole cell lysate (RIPA 10ml, cocktail 1 piece) was added according to the number of cells, and the cell suspension was transferred to an EP tube by pipetting and mixing.

(4) On a horizontal shaker, the cells were lysed thoroughly by ice bath for 20-40 min.

(5) Centrifuge at 4℃at full speed (> 10000 rpm), centrifuge for 20min.

(6) The supernatant was aspirated to clean EP tubes and the pellet was discarded.

2.3BCA reagent protein quantification

(1) BCA standard substances with different concentration gradients of A-G are prepared according to the specification, 50 mu L of standard substance stock solution is added into a tube A, 50 mu L of PBS is added into a tube B-G in advance, 50 mu L of PBS is added into a tube B in sequence after uniform mixing, and the protein concentration gradient is gradually reduced in multiple from 2 mg/ml. 8 wells of a 96-well plate were added sequentially.

(2) 20 mu L of PBS is added into each well of the 96-well plate, 5 mu L of protein samples to be detected are respectively added into corresponding wells, and corresponding marks are made to avoid confusion.

(3) And (3) preparing a proper amount of color reagent (A solution: B solution=50:1), uniformly mixing, and adding 200 mu L of color reagent into each hole of the standard substance hole and the detection protein sample hole.

(4)CO ₂ Incubate in a constant temperature incubator at 37℃for 30min.

(5) The absorbance (OD) of each well at 562nm was measured with a microplate reader, a standard curve was drawn, and the protein concentration of each experimental sample was calculated with reference to the standard curve.

(6) And (3) preparing a proper amount of protein liquid, 2×loading buffer and RIPA into a sample liquid according to an experimental plan, heating the sample liquid at the temperature of 100 ℃ for 10min in a metal bath for denaturation, and preserving the sample liquid at the temperature of-80 ℃ for later use.

2.4Western blotting specific procedure

(1) And (3) glue preparation: the glass plate is cleaned, aligned and placed in a clamp for clamping, and then vertically placed on a bracket. Preparing a separation gel with a required concentration according to the molecular weight of the detected protein (TEMED is added last), then slowly adding double-distilled water pressure gel, and standing at room temperature. After the separation gel is solidified, the double distilled water covered on the upper layer of the separation gel is poured out. Preparing 5% concentrated gelatin (TEMED is added last), then inserting into clean comb, standing at room temperature until the concentrated gelatin is solidified. The formula of the separating gel and the concentrated gel is as follows:

(2) Sample adding: after the gel was prepared, the gel was mounted on an electrophoresis tank together with a glass plate, and about 1000mL of 1 Xrunningbuffer was added, and the comb in the gel plate was gently taken out. And calculating the required loading volume, mass and volume balancing of an equivalent protein sample (30-50 mug) according to the protein concentration, sequentially adding each loading hole, paying attention to the loading sequence, avoiding confusion, and simultaneously adding a protein Marker as a reference of the size of a protein molecule and the loading sequence.

(3) Electrophoresis: constant pressure, initial 80V, after bromophenol blue strip enters into the separation gel, adjusting the voltage to 100V, continuing electrophoresis for 1-2h, and taking the condition that the target protein with the minimum molecular weight does not electrophoresis out the gel.

(4) Transferring: and (3) cutting the adhesive properly to remove the excessive adhesive blocks, wherein all the target strips are still on the adhesive. Placing the glue on one side of the cathode of the film-transferring clamping plate, covering a foam cushion and filter paper between the clamping plate and the glue in advance to ensure that the glue is completely covered by a1×trans buffer, cutting a PVDF film slightly larger than the glue, soaking in methanol for 30 seconds, slightly transferring the PVDF film to the glue by holding the clamp, and taking care of removing bubbles between the PVDF film and the glue. The anode side clamping plate which is covered with the foam cushion and the filter paper in advance is closed to form a sandwich-like transfer film clamping plate, the sandwich-like transfer film clamping plate is placed into a transfer tank, and the positive and negative poles (black to black and red to red) of the transfer tank are determined. Sufficient 1×trans buffer was added to completely soak it. The membrane is transferred for 1-2h at a constant pressure of 100V and a low temperature (the specific time is determined according to the molecular weight of the detected protein).

(5) Closing: the PVDF membrane is taken out from the membrane transferring clamping plate and placed in a container with enough sealing liquid (1 XTBST with 5% skimmed milk), the PVDF membrane is required to be completely covered by the sealing liquid, and the container is placed on a shaking table to shake at low speed and incubated for 1h at room temperature.

(6) Incubation resistance: after the end of the sealing, the PVDF film is cut according to the molecular weight of the detected protein by comparing with a Marker strip. The primary antibody of the protein to be tested (diluted according to the required proportion, the primary antibody dilution is configured according to the instruction) prepared in advance is placed in a hybridization bag together with a PVDF film (no bubbles are generated), a rotary table is rotated at a low speed after sealing, and the protein to be tested is incubated overnight at 4 ℃.

(7) Secondary antibody incubation: the PVDF membrane in the hybridization bag was taken out the next day while recovering the primary antibody, the PVDF membrane was washed 3 times with a sufficient amount of 1 XTBE, and placed on a horizontal shaker (150-200 rpm/min) for 5-10min each time. The secondary antibody (diluted according to the required proportion, the secondary antibody diluent is 1 XTBST containing 5% skimmed milk) prepared in advance and PVDF membrane are placed in a hybridization bag, a shaking table (60-80 rpm/min) is placed after sealing, and the mixture is incubated for 1h at room temperature.

(8) Developing: after the secondary antibody incubation is completed, the secondary antibody incubation liquid is removed, and the membrane is washed in the manner described above. The washed PVDF film was placed in freshly prepared ECL developer (reagent a: reagent b=1:1), film exposure time was adjusted according to the banding conditions, developed using a developer, and archived after scanning.

3. Apoptosis experiments

(1) The cells were collected in a flow tube and centrifuged at 1000rpm for 5min at room temperature.

(2) The supernatant was discarded, and the cells were resuspended in 3mL of PBS (pre-chilled at 4 ℃), centrifuged at 1000rpm for 5min at room temperature.

(3) The cells were resuspended in Binding buffer 50-100uL, and then Annexin V-APC 2uL was added and mixed well and stained at room temperature in the dark for 5min.

(4) Adding PI (propidine iodide) uL, mixing, and dyeing at room temperature for 5min.

(5) 300 μl of PBS was added and the flow cytometer was run on-line.

CCK8 experiment

(1) 100uL of cell suspension per well was added to a 96-well plate, 2X 10 cells per well ³ . Complete medium 1640 is added in a circle nearest the edge to prevent evaporation.

(2) The cells were returned to the incubator and cultured for 24 hours, 48 hours and 72 hours.

(3) After the detection time, the 96-well plate was removed and 10uLCCK8 solution was added to each well.

(4) After the addition of CCK8, the culture plate is put back into the cell incubator for further culture for 4 hours.

(5) OD at 490nm was measured with a microplate reader.

(6) The calculation method comprises the following steps: cell actual OD = cell OD-blank OD

5. Cell cycle experiments

(1) Collecting cells: the cells were pelleted by centrifugation at 1500rpm for 5min at room temperature.

(2) The supernatant was discarded and a small amount of liquid remained.

(3) The cells were resuspended with 2mL of pre-chilled PBS and the cell suspension transferred into a flow tube.

(4) The cells were pelleted by centrifugation at 1500rpm for 5min at room temperature.

(5) The supernatant was discarded and a small amount of liquid remained.

(6) Cells were resuspended with 1mL of pre-chilled PBS.

(7) The cell suspension was added dropwise to pre-chilled 95% ethanol while shaking at low speed, and after mixing, fixed overnight at 4 ℃.

(8) The cells were pelleted by centrifugation at 1500rpm for 5min at room temperature.

(9) The supernatant was discarded and a small amount of liquid remained.

(10) Cells were resuspended with 5mL of pre-chilled PBS.

(11) The cells were pelleted by centrifugation at 1500rpm for 5min at room temperature.

(12) The supernatant was discarded, about 50uL of liquid was left, and the bottom of the tube was flicked with a finger to avoid cell clumping.

(13) And adding 0.4mL of PI staining solution into each tube of cell sample, blowing and mixing uniformly by using a gun tip, and carrying out light-shielding water bath at 37 ℃ for 30min.

(14) The cell filter is filtered and then is detected by a machine.

6. Immunofluorescence

(1) Cells were collected into a 15mL centrifuge tube and centrifuged at 1000rpm for 5min at room temperature.

(2) Cells were washed with PBS, the supernatant was discarded, and a small amount of liquid was left to re-suspend the cells. Subsequent experiments were performed in EP tubes, 4X 10 ⁵ Tube.

(3) Cells were fixed with 3% -4% paraformaldehyde at room temperature for 15min.

(4) Cells were washed 2 times with PBS for 5 min/time. After each washing, the mixture was centrifuged at 2500rpm for 5min at room temperature, and the supernatant was discarded.

(5) Closing: cells were incubated with PBST containing 1% bsa for 30min at room temperature.

(6) Incubating primary antibody: primary antibodies were diluted with PBST containing 1% bsa (specific dilution ratio is based on antibody instructions), 4 ℃, and the shaker was spun overnight.

(7) Centrifuge at 2500rpm for 5min at room temperature and remove supernatant.

(8) Cells were washed 2 times with PBST, 1 time with PBS, 5 min/time. After each washing, the mixture was centrifuged at 2500rpm at room temperature for 5min, and the supernatant was discarded.

(9) Incubating a fluorescent secondary antibody: the secondary antibody was diluted with PBST containing 1% bsa and incubated at room temperature for 1h in the dark.

(10) Centrifuge at 2500rpm for 5min at room temperature and remove supernatant.

(11) Cells were washed 2 times with PBST, 1 time with PBS, 5 min/time.

(12) Sealing piece: the 20uL caplets (containing DAPI) and the cell suspension from the previous step were mixed well and then dropped onto the center of the slide, covered with a cover slip, and left overnight at 4 ℃.

(13) And storing at-20deg.C in dark place.

B-NSG immunodeficient murine in vivo nodulation

(1) And (3) raising B-NSG female mice under SPF conditions, so as to ensure that the health condition of the mice is suitable for the next experiment.

(2) ARD cells (ARD-LUC) stably expressing luciferase (luciferases) were cultured in sufficient amounts for use to ensure a good cell status.

(3) Cells were inoculated via the tail vein 2X 10 when mice grew to 6-8 weeks ⁶ /only.

(4) Mice were examined for neoplasia weekly by bioluminescence In Vivo Imaging (IVIS) of the mice after cell inoculation.

8. Living animal imaging

(1) Fluorescein salt was formulated with PBS to a final concentration of 15 mg/mL.

(2) Each mouse was intraperitoneally injected with 200uL.

(3) Mice were placed on an IVIS test table for detection within 15min after injection of the potassium salt of fluorescein to monitor tumor size, location in the mice.

(4) The fluorescence intensity reflects the tumor load in mice, and the fluorescence intensity can be quantitatively analyzed to monitor the disease progression of each group of mice.

9.B-NSG immunodeficiency murine model in vivo treatment experiment

(1) Mice were tumorigened about day 14 post-inoculation, and mice were divided into four groups of 5 mice each, each treated with PBS, bortezomib, IVM, bortezomib in combination with IVM.

(2) The specific administration method comprises the following steps: bortezomib 0.75ug/g was administered twice a week (intraperitoneally) for a total of 4 treatments. IVM 2ug/g, once daily (intraperitoneal administration). The combination of the two drugs is prepared by simultaneously carrying out bortezomib and IVM according to the method. I.e. the bortezomib dose was 0.75ug/g with 4=3 ug/g and the IVM dose was 2×14=28 ug/g over two weeks. The CTR group was administered PBS intraperitoneally as a treatment control.

(3) After modeling was successful, the fluorescein intensity of the experimental mice was measured weekly with IVIS.

4. Experimental results

Ivermectin inhibits proliferation of myeloma cells

Multiple myeloma tumor cell lines were affected with different concentrations of Ivermectin (IVM), with significant apoptosis of myeloma cells being induced as the drug concentration increased (fig. 2A). The effect of IVM on myeloma cell proliferation was examined with CCK8, and the results suggest that: IVM inhibited myeloma cell proliferation, and the higher the drug concentration, the stronger the inhibition (fig. 2B). Lower concentrations of IVM (7.5 uM) acted on ARP1 and ARD cells, and flow-detected cell cycle found: after drug treatment, the proportion of cells arrested in S phase increased (fig. 2c, d).

Taken together, the data indicate that IVM can effectively inhibit proliferation of myeloma cells and has a certain killing effect on tumor cells.

Ivermectin inhibits proteasome Activity of myeloma cells

In the subsequent research, the invention adopts a fluorogenic substrate method to directly examine the proteasome activity of the cells. The fluorescent substrate (Suc-LLVY-AMC) can act with PSMB5 subunit of the proteasome to release the fluorescent group, and the higher the activity of the proteasome can be detected by combining the fluorescence intensity with a multifunctional fluorescence microplate reader, the stronger the fluorescence. MM cells (IM 9, ARP 1) were treated with IVM and cellular proteasome activity was detected by fluorogenic substrate method. The discovery is as follows: after IVM treatment, the proteasome activity of MM cells was significantly reduced (fig. 3A). In addition, western blotting detection suggests that: IVM can cause elevated levels of ubiquitination of MM cell proteins, activation of UPR pathways and apoptotic pathways (fig. 3b, c). These data indicate that IVM has some killing effect on MM cells by inhibiting proteasome activity.

Ivermectin causes DNA damage to myeloma cells

More and more studies have shown that the genomic stability of MM decreases and most patients have aneuploidy chromosomal abnormalities. The specific mechanisms leading to reduced chromosomal stability are currently unknown and may be associated with DNA double strand break repair (double-strand DNA breaks repair) disorders or excessive homologous recombination repair (homologous recombination repair, HRR) caused by non-homologous end joining repair abnormalities. Thus, MM cells may rely more on "surviving" DNA repair pathways than normal cells to maintain good survival. Thus, targeting certain DNA repair pathways in MM cells may be a new therapeutic direction in the future.

The literature has reported that IVM can cause DNA damage to HeLa cells. The invention sends out by western blottingNow: 1) ATM pathway activation of MM cells after IVM treatment, p-ATM, gamma H ₂ AX and pCHK1/2 expression is increased-DNA damage is caused by medicine; 2) IVM inhibited the HRR pathway of MM cells, and DNA damage repair-related factors RAD51, FANCD2, BRCA1 were all significantly reduced (FIG. 4A). Gamma H ₂ AX is a marker of DNA double strand breaks, and immunofluorescence data indicate that gamma H in MM nuclei increases with IVM concentration ₂ AX expression was elevated, further confirming that IVM could cause DNA damage to MM cells (FIG. 4B).

In summary, IVM can inhibit DNA damage repair while causing DNA damage to MM cells.

Ivermectin and bortezomib have a synergistic effect

Clinical use of Proteasome Inhibitors (PIs) has led to significant advances in the treatment of MM, with PIs, represented by BTZ, causing killing of MM by inhibiting proteasome activity to disrupt MM intracellular protein homeostasis. In addition to the well-known effects of BTZ in inhibiting protein degradation, inhibiting NF- κb signaling, etc., other novel downstream effects have been increasingly explored. There are studies showing that BTZ can hinder DNA damage repair by depleting free ubiquitin in the nucleus of MM cells. The invention discovers that IVM has a certain killing effect on MM cells. The possible mechanism is as follows: 1) Inhibiting proteasome activity; 2) Causes MM cell DNA damage and inhibits DNA damage repair. In combination with the analysis of the mechanism of action of IVM on MM cells, IVM and BTZ are suspected to be effective against myeloma. The experimental result of the combined application also proves that the combination of the two medicaments has more obvious killing effect on the MM cells.

CCK8 results suggest that even with BTZ in combination with low concentrations of IVM (7.5 uM), proliferation inhibition on MM cells was significantly higher than in the single drug group (fig. 5A). The MM cells were treated in combination with IVM and BTZ at different concentrations and apoptosis was detected by flow-through, which indicated that: the killing effect of the double-drug combination group on MM cells is obviously better than that of the single-drug combination group under different drug concentrations. This result was verified in different cell lines (fig. 5B). In terms of inhibition of proteasome activity, the experimental results were consistent with CCK8 and apoptosis, with more pronounced dual drug combination (fig. 5C). In addition, western blotting examined the related molecular mechanisms revealed that the UPR and apoptotic pathways of the combination of the two drugs activated to a higher extent (fig. 5D). The in vitro experiments described above all demonstrate that the combined use of IVM and BTZ has significantly higher killing effect on MM cells than either BTZ or IVM alone.

In vivo validation was performed in a B-NSG immunodeficient murine model, ARD cells (ARD-LUC) stably expressing luciferase (luciferase) were injected into 7-8 week old female B-NSG immunodeficient mice via the tail vein and tumors developed in the mice around day 14 (D14) after injection. Mice were divided into 4 groups and treated with PBS, IVM, BTZ, IVM +btz, respectively. The specific administration method comprises the following steps: BTZ 0.75ug/g, twice weekly (intraperitoneal administration), total treatment 4 times; IVM 2ug/g, once daily (intraperitoneal administration); a combination of two drugs, BTZ and IVM simultaneously in accordance with the method described above; the CTR group was administered PBS intraperitoneally as a treatment control. Tumor burden in D14, D21, D28 mice after MM cell inoculation was monitored with a small animal In Vivo Imaging System (IVIS) (see experimental methods section for details). There was no significant difference in fluorescence intensity in the 4 groups of mice at D14, indicating that the tumor burden was approximately the same for each group of mice at the onset of treatment. During subsequent treatment, each group of mice had an increased tumor burden over time, but the fluorescence intensity was still reduced compared to the PBS treated group, i.e. both BTZ and IVM treatments were effective. In addition, the fluorescence intensity of the double-drug combination group is lower, and the curative effect is better (fig. 5E and 5F).

From the results, in vivo and in vitro experiments prove that the anti-MM effect of the dual-drug combination group of IVM and BTZ is stronger than that of any single-drug group.

In summary, the present invention first discovered that IVM has killing effect on MM cells, and first proposed that IVM can inhibit proteasome activity. This finding may extend the awareness of IVM. Based on the hypothesis of the mechanism of action of IVM on MM cells, the combination of IVM and BTZ showed that the anti-MM effect was significantly better than either BTZ or IVM single drug. Because IVM is a mature medicine used clinically for years, clinical practice shows that the medicine has higher safety in use in patients, and the concept of 'IVM combined with BTZ for treating MM' has a great clinical transformation prospect.

Claims

1. Use of ivermectin in combination with bortezomib for the manufacture of a medicament for the treatment of multiple myeloma; the mass ratio of bortezomib to ivermectin in the combined medicament is 3:28.

2. a combination for the treatment of multiple myeloma, characterized by: it contains ivermectin and bortezomib for simultaneous administration; the mass ratio of bortezomib to ivermectin is 3:28.