CN115227715A

CN115227715A - Combined medicine and pharmaceutical composition for treating cancer

Info

Publication number: CN115227715A
Application number: CN202211021673.6A
Authority: CN
Inventors: 赵行; 宋绍涓; 吉宁; 夏鑫; 陈谦明
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-10-28
Filing date: 2022-08-24
Publication date: 2022-10-25

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a combined medicine and a pharmaceutical composition for treating cancer. The combination of the invention is CX-4945 and cisplatin administered separately or simultaneously. The pharmaceutical composition is prepared by taking CX-4945 and cisplatin as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients. CX-4945 can induce cancer cell megakaryosis and increase the intracellular content of DDP, thereby enhancing the apoptosis of cancer cells. Therefore, the combination drug and the pharmaceutical composition can enhance the treatment effect of DDP on cancer under the condition that the application dose of DDP is limited, and have good application prospect.

Description

Combined medicine and pharmaceutical composition for treating cancer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a combined medicine and a pharmaceutical composition for treating cancer.

Background

Cisplatin (DDP) has become the first line treatment for most malignancies since its approval in 1979. However, the significant systemic toxicity of DDP (including nephrotoxicity, ototoxicity, neurotoxicity, etc.) causes its dose range to be limited, requiring increased dosing frequency. In addition, the therapeutic efficacy of DDP is also greatly affected by the limited dosage. After palliative treatment with drugs such as DDP, median survival time and efficacy remain unsatisfactory for patients with recurrent and metastatic cancer.

Therefore, how to improve the anticancer effect of DDP under the condition of limited dosage is a very important research topic.

The anticancer efficacy of DDP is positively correlated with its intracellular uptake. Passive diffusion and transporter uptake are the major mechanistic pathways by which cells take up DDP. Increasing the accumulation of DDP in cancer cells will help promote tumor suppression when the dose range of administration is limited. It follows that finding innovative strategies for DDP uptake in cancer cells to increase intracellular DDP concentrations would likely be an effective solution to increase the efficacy of DDP treatment.

CX-4945 is a potent, orally administrable, highly selective CK2 inhibitor. Megakaryosis has been shown to be induced in biliary tract cancer, non-small cell lung cancer and colorectal cancer cells. Macrobiosis is an endocytic mechanism of cellular uptake of extracellular material, resulting from intracellular and extracellular stimuli, in the formation of cell membrane folds and megasomes, which has been shown to contribute to intracellular uptake of anticancer drugs (Biomaterials 2020: 119795.Mol Ther 20126. However, macrobiosis is only a mechanism for helping cells to take in substances, and whether the macrobiosis can exert a synergistic effect to improve the curative effect of the anticancer drugs or not is not reported in relevant researches at present.

In order to allow more options for clinical treatment of cancer, there is a need to develop more studies of cancer cells to increase DDP uptake, thereby providing new cancer treatment drug regimens.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a combined medicine and a pharmaceutical composition, aiming at enhancing the uptake of a chemotherapy drug cisplatin by cells, thereby enhancing the anticancer effect of the cisplatin.

A combination of CX-4945 and cisplatin for use in the treatment of cancer, for separate or simultaneous administration.

Preferably, the combination is for use in the treatment of oral squamous cell carcinoma.

Preferably, the dosage ratio of CX-4945 to cisplatin is as follows by mass ratio: 7:4-7:1.

Preferably, the dosage ratio of CX-4945 to cisplatin is as the mass ratio: 7:4.

Preferably, the combination is a solution containing 10-20. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

Preferably, the combination is a solution containing 10. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

The invention also provides the use of CX-4945 and cisplatin in the manufacture of a combination medicament for the treatment of cancer.

Preferably, the dosage ratio of CX-4945 to cisplatin is as the mass ratio: 7:4.

The invention also provides a pharmaceutical composition, which is prepared by taking CX-4945 and cisplatin as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Preferably, the dosage ratio of CX-4945 to cisplatin is as follows by mass ratio: 7:4.

Preferably, the pharmaceutical composition is a solution containing 10-20. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

Preferably, the pharmaceutical composition is a solution containing 10. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

Preferably, the adjuvant or auxiliary ingredient is selected from at least one of diluents, fillers, colorants, glidants, lubricants, binders, stabilizers, suspending agents and buffering agents.

Experiments show that CX-4945 can induce Oral Squamous Cell Carcinoma (OSCC) macropinocytosis and increase the intracellular content of DDP, thereby enhancing the apoptosis of OSCC cells. Therefore, the combined use of CX-4945 and cisplatin or the preparation of pharmaceutical composition thereof can effectively improve the treatment effect of cisplatin on OSCC and other cancers on the premise of limiting the using dose of cisplatin.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a graph showing the results of experiment of inducing OSCC megacytopenia by CX-4945 in Experimental example 1;

FIG. 2 is a graph showing the result of an experiment of increasing the intracellular content of DDP in OSCC cellular megalobin according to example 2 of the present invention;

FIG. 3 shows the results of the screening of the optimal dose ratio of CX-4945 in combination with DDP in Experimental example 3 of the present invention.

FIG. 4 is a graph showing the results of experiments in which CX-4945 in combination with DDP enhances OSCC apoptosis in vitro in Experimental example 4 of the present invention.

Detailed Description

The reagents and materials used in the present invention are commercially available.

EXAMPLE 1 combination drug for treating oral squamous cell carcinoma CX-4945 and cisplatin

This example provides a combination of CX-4945 and cisplatin, which can be used simultaneously or separately, wherein the ratio of CX-4945 to cisplatin is as follows: 7:4.

As a preferred embodiment, the combination is 1mg/mL DDP in water and 10mM CX-4945 in DMSO, which are used independently.

Example 2 pharmaceutical composition for the treatment of oral squamous cell carcinoma CX-4945 and cisplatin

The embodiment provides a pharmaceutical composition of CX-4945 and cisplatin, which comprises CX-4945 and cisplatin, wherein the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:1.

In order to further illustrate the technical solution of the present invention, the following experimental examples demonstrate the technical effects of the present invention.

Experimental example 1 CX-4945 can induce OSCC megacytopathogenesis:

1. experimental methods

After treating four OSCC cells (Cal-27, HSC-3, HSC-4, UM 1) with 10-40. Mu.M CX-4945 for 12h, the change in cell morphology was observed under a phase contrast microscope. Cal-27 cells were selected, labeled with lysosome probe (Lyso-tracker), endoplasmic reticulum probe (ER-tracker) and mitochondrial probe (Mito-tracker), respectively, in combination with a fluorescent marker Dextran AF488 specific for macropinocytosis, and then observed with a confocal microscope.

2. Results of the experiment

The results of the experiment are shown in FIG. 1. FIG. 1a shows microscope pictures of Cal-27, HSC-4 cells treated with 20. Mu.M CX-4945 for 12h and HSC-3, UM1 cells treated with 40. Mu.M CX-4945 for 12 h. As can be seen, vacuoles were produced in all four OSCC cells after CX-4945 treatment. FIG. 1b shows confocal microscopy pictures obtained after co-culturing Cal-27 cells for 12h with 0.5mg/mL Dextran AF488 and 10. Mu.M CX-4945. It can be seen that most vacuoles had taken up a significant amount of Dextran AF488, which was found to be positive for Dextran AF488, indicating the presence of macrocells. FIG. 1c shows confocal microscopy pictures obtained after co-culturing Cal-27 cells with 0.5mg/mL Dextran AF488, 10. Mu.M CX-4945 and endoplasmic reticulum probe for 12 h. Red arrows indicate Dextran AF488 positive vacuoles that do not co-localize with the endoplasmic reticulum, indicating that Dextran AF488 positive vacuoles do not co-localize with the endoplasmic reticulum. FIG. 1d shows confocal microscopy pictures obtained after co-culturing Cal-27 cells with 0.5mg/mL Dextran AF488, 10. Mu.M CX-4945 and lysosomal probe for 12 h. The red arrows indicate Dextran AF 488-positive vacuoles that did not co-localize with lysosomes, and the green arrows indicate Dextran AF 488-positive vacuoles that co-localize with lysosomes, indicating that a portion of the Dextran AF 488-positive vacuoles co-localize with lysosomes. FIG. 1e shows confocal microscopy pictures obtained after co-culturing Cal-27 cells with 0.5mg/mL Dextran AF488, 10. Mu.M CX-4945 and mitochondrial probe for 12 h. Red arrows indicate Dextran AF488 positive vacuoles that did not co-localize with mitochondria, indicating that Dextran AF488 positive vacuoles did not co-localize with mitochondria.

These manifestations are consistent with the characteristics of megastigmatosis, confirming that the vacuole induced by CX-4945 is derived from megastigmatosis. Namely CX-4945 can induce OSCC megacytopenia.

Experimental example 2 CX-4945 induced megalocytosis can increase the intracellular content of DDP

1. Experimental methods

OSCC cells (Cal-27) were treated with a solution containing 1mg/mL DDP as DPP experimental group.

OSCC cells (Cal-27) were treated with a solution containing 100. Mu.M CX-4945 (Silmitasertib) and 1mg/mL DDP as a Silmitasertib + DDP experimental group.

OSCC cells (Cal-27) were treated with a solution containing 100. Mu.M CX-4945 (silmitastiib) and 1mg/mL DDP, and megacaryotic pharmacological inhibitor BAF1 (bafilomycin A1) was introduced to inhibit the megacaryotic drink induced by CX-4945, as a silmitastiib + DDP + BAF1 experimental group.

The intracellular DDP content of the above experimental groups was quantified by HPLC. Wherein the specific conditions of the high performance liquid chromatography are as follows: the cells treated accordingly were collected in 1.5mL EP tubes and counted to ensure a cell count of 1X 10 ⁷ After washing the cells twice with PBS, the cells were centrifuged at 2000rpm for 5 minutes, and the cell pellet was collected. To each EP tube was added 400 μ L RIPA buffer, and then immediately lysed cells with an ultrasonic lysis cell disruptor (Diagenode, belgium) (4 ℃,20 cycles, 1 minute each). After cell lysis, the cells were centrifuged at 10,000rpm for 10 minutes, and the supernatant was collected. The protein was precipitated by adding 4 volumes of methanol, centrifuged at 10,000rpm for 10min, and the supernatant was subjected to HPLC (Shimadzu corporation, japan). A Diamonsil C-18 column (4.6 mm. Times.250mm, 5 μm) was used, the mobile phase was methanol-water (10, 90), the flow rate was 0.5mL/min, the detection wavelength was 254nm, and the column temperature was 25 ℃.

2. Results of the experiment

The results are shown in FIG. 2. FIG. 2a shows the microscope pictures obtained after the DPP experiment group, the Silmitasertib + DDP experiment group, and the Silmitasertib + DDP + BAF1 experiment group all treated Cal-27 cells for 4 h. FIG. 2b shows a peak of DDP obtained by HPLC test at 254 nm. FIG. 2c shows a standard graph of DDP obtained by HPLC test, with the formula f (x) =147.279 x +730.147, rr ² =0.9997887. FIG. 2d shows a histogram of the intracellular concentration of DDP and a comparison of differences in the cells of each experimental group obtained by HPLC testing after each experimental group treated Cal-27 cells for 4 h. It can be seen that the macromers drink induced by silmitasterib significantly increased the intracellular content of DDP{(*)P<0.05,(**)P<0.01,(***)P<0.001}. By calculation, the intracellular content of the simmitasertib + DDP group was increased by 127.06% + -7.25% compared to the DDP group. Whereas the addition of BAF1 reversed this phenomenon.

The above results indicate that CX-4945-induced OSCC macrobiosis can increase the intracellular content of DDP.

Experimental example 3 CX-4945 in combination with optimum dose ratio screening of DDP

1. Experimental method

OSCC cells (Cal-27) were treated with a solution containing 10. Mu.M CX-4945 (Silmitasertib) and 1. Mu.g/mL DDP as Silmitasertib + DDP experimental group 1.

OSCC cells (Cal-27) were treated with a solution containing 10. Mu.M CX-4945 (Silmitasertib) and 2. Mu.g/mL DDP as Silmitasertib + DDP experimental group 2.

OSCC cells (Cal-27) were treated with a solution containing 20. Mu.M CX-4945 (Silmitasertib) and 1. Mu.g/mL DDP as Silmitasertib + DDP experimental group 3.

OSCC cells (Cal-27) were treated with a solution containing 20. Mu.M CX-4945 (Silmitasertib) and 2. Mu.g/mL DDP as Silmitasertib + DDP experimental group 4.

For the above experimental group, the cell activity was tested by CCK8, and the Theoretical Value (TV) of the combined inhibition rate of cell activity of the two drugs was calculated by a bliss independent model calculation formula, and compared with the Actual Value (AV) of the combined inhibition rate of cell activity. AV (audio video)<TV, the two drugs have antagonistic action; AV = TV, the two drugs acting independently; AV (Audio video)>TV, the two drugs act synergistically. CCK8 conditions were such that cells were applied at 1X 10 per well ⁴ The density of the/well is inoculated in a 96-well plate for 24h, and then the cells are treated correspondingly by the medicine. Then 10. Mu.L of CCK8 solution was added to each well. After incubation at 37 ℃ for 1 hour, absorbance at a wavelength of 450nm was measured using Varioskan Flash (Thermo Scientific, USA).

2. Results of the experiment

The results are shown in FIG. 3 and Table 1.

TABLE 1 Combined inhibition of cellular Activity

FIGS. 3a-3d show histograms of cell activity using CCK8 after 24h and 48h treatment of Cal-27 cells in each experimental group. The cell activity of the drug combination group is remarkably reduced. Table 1 shows that the TV values of both drugs (CX-4945 and DDP) are smaller than the AV value in each treatment group, and both show synergistic effects. The difference between the TV value and the AV value of the silmitasertib + DDP experimental group 2 and the silmitasertib + DDP experimental group 4 is the largest, and the combined effect is the best. To achieve a better synergy with a lower drug dose, the amount of silmitasertib + DDP panel 2 (equivalent to CX-4945 and DDP mass ratio 7:4) is the more preferred amount. Subsequent experimental examples therefore selected drug concentrations of simmitasertib + DDP experimental group 2 for subsequent experiments.

Experimental example 4 CX-4945 in combination with DDP enhance OSCC apoptosis in vitro

1. Experimental method

OSCC cells (Cal-27) were treated with a solution containing 2. Mu.g/mL DDP as DDP experimental groups.

OSCC cells (Cal-27) were treated with a solution containing 10. Mu.M CX-4945 (Silmitasertib) as a Silmitasertib experimental group.

OSCC cells (Cal-27) were treated with a solution containing 10. Mu.M CX-4945 (Silmitasertib) and 2. Mu.g/mL DDP as a Silmitasertib + DDP experimental group.

For the above experimental groups, the cell activity was tested with CCK8, the apoptosis rate was quantified with flow cytometry, and the level of apoptosis-related proteins was tested with WB. The specific conditions of CCK8, flow cytometry and WB test are as follows: CCK8 conditions were such that cells were applied at 1X 10 per well ⁴ The density of the/well is inoculated in a 96-well plate for 24h, and then the cells are treated correspondingly by the medicine. After which 10 μ LCCK8 solution was added to each well. After incubation at 37 ℃ for 1 hour, absorbance at a wavelength of 450nm was measured using Varioskan Flash (Thermo Scientific, USA).

The conditions of flow cytometry are that the cells are arranged at 1X 10 ⁵ The density of each well is inoculated in a 6-well plate for 24h, and then the cells are treated correspondingly by the medicine. The medium was then removed and the cells were harvested and washed twice with PBS. Centrifuging at 4000rpm for 5min, and collecting cell precipitate. Then is atAdding 500 μ L LBinding Buffer,5 μ L Annexin V-FITC and 5 μ L Propidium Iodide into the cell sediment to suspend the cells, and reacting for 15-60min at room temperature in a dark place. Flow cytometry (Beckman FC500, break, CA, USA) detected the apoptosis rate with excitation wavelength Ex =488nm and emission wavelength Em =530nm.

WB was performed under conditions in which the cells were lysed with RIPA buffer, and then centrifuged at 14000rpm for 15min to collect the supernatant. Then using Pierce ^TM BCA (bicinchoninic acid) protein detection kit (Thermo Scientific) ^TM ) The protein was quantified. The samples were subjected to electrophoresis (SDS-PAGE) using 12% sodium dodecyl sulfate polyacrylamide hydrogel under conditions of 80V for 30 minutes and 120V for 1 hour, and then transferred onto a 0.22 μm polyvinylidene difluoride membrane (PVDF, MA, USA) by wet transfer (300mA, 75 minutes). The membranes were blocked with 5% skim milk in TBST for 1 hour at room temperature, then covered with primary antibodies (anti-cleared Caspase 3, anti-cleared Caspase 8, anti-ERK, anti-p 38, anti-JNK, anti-p-JNK and anti- β -actin, dilution ratio 1/1000, abcam) and left overnight at 4 ℃. The following day, membranes were washed with TBST for 15 minutes and incubated with secondary antibody (goat anti mouse/rabbit) for 1 hour at room temperature. Finally, the results are shown by BM chemiluminescence Western Blotting kit (Roche).

2. Results of the experiment

The results are shown in FIG. 4. FIG. 4a shows microscope pictures obtained after Cal-27 cells were treated for 24h in the DPP, silmitartertib, and Silmitartertib + DDP experimental groups. It can be seen that the number of dead cells in the simmitasertib + DDP experimental group was visually increased compared to the other experimental groups. FIG. 4b shows a bar graph of the activity of cells tested with CCK8 after 24h and 48h treatment of Cal-27 cells for each experimental group. The cellular activities of the simmitasertib + DDP experimental group were decreased time-dependently compared to the other experimental groups. FIGS. 4c and d show the apoptosis scatter plot and apoptosis rate histogram obtained by flow cytometry after Cal-27 cells were treated for 24h and 48h in each experimental group, respectively. The apoptosis rate of the simmitartertib + DDP experimental group was significantly increased from the other experimental groups, { () P <0.05, () P <0.01, (×) P <0.001}. In addition, as can be seen from fig. 4d, the apoptosis rate of the simmitasertib + DDP experimental group is significantly higher than the sum of the apoptosis rates of the DPP experimental group and the simmitasertib experimental group, which indicates that DPP and simmitasertib generate a synergistic effect in the apoptosis experiment. FIG. 4e shows the banding patterns of the resulting apoptotic proteins (clear Caspase 3, clear Caspase 8, ERK, p-ERK, p38, JNKs, p-JNKs) tested by WB after each experimental group treated Cal-27 cells for 24h and 48 h. The level of apoptotic proteins (cleared Caspase 3, cleared Caspase 8, p-ERK, p-JNKs) was elevated in the simmitasertib + DDP experimental group compared to the other experimental groups.

The results show that CX-4945 can enhance the apoptosis of OSCC cells after being used in combination with DDP, and the CX-4945 and the DDP have synergistic effect in the treatment of OSCC.

As can be seen from the above examples and experimental examples, CX-4945 can induce megacytopenia of OSCC cells and increase intracellular content of DDP, thereby enhancing apoptosis of OSCC cells. Therefore, the combination drug and the drug composition provided by the invention can enhance the treatment effect of DDP on OSCC and other cancers under the condition that the use dose of DDP is limited. Therefore, the invention has good application prospect.

Claims

1. A combination for the treatment of cancer, which comprises: the combination is CX-4945 and cisplatin administered separately or simultaneously.

2. The combination as claimed in claim 1, wherein: the combination is useful for the treatment of oral squamous cell carcinoma.

3. The combination as claimed in claim 1, wherein: the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:4-7:1.

4. The combination as claimed in claim 3, wherein: the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:4.

5. The combination as claimed in claim 1, wherein: the combination drug is a solution containing 10-20 mu M CX-4945 and 2 mu g/mL cisplatin.

6. The combination as claimed in claim 5, wherein: the combination was a solution containing 10. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

Use of cx-4945 and cisplatin in the manufacture of a combination medicament for the treatment of cancer.

8. Use according to claim 7, characterized in that: the combination is useful for the treatment of oral squamous cell carcinoma.

9. Use according to claim 7, characterized in that: the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:4-7:1.

10. Use according to claim 9, characterized in that: the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:4.

11. Use according to claim 7, characterized in that: the combination drug is a solution containing 10-20 mu M CX-4945 and 2 mu g/mL cisplatin.

12. Use according to claim 11, characterized in that: the combination was a solution containing 10. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

13. A pharmaceutical composition characterized by: it is prepared by taking CX-4945 and cisplatin as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

14. The pharmaceutical composition according to claim 13, wherein: the dosage proportion of CX-4945 and cisplatin is as the mass ratio: 7:4-7:1.

15. The pharmaceutical composition according to claim 14, wherein: the dosage ratio of CX-4945 to cisplatin is as follows by mass: 7:4.

16. The pharmaceutical composition according to claim 13, wherein: the pharmaceutical composition is a solution containing 10-20 mu M CX-4945 and 2 mu g/mL cisplatin.

17. The combination as claimed in claim 16, wherein: the pharmaceutical composition is a solution containing 10. Mu.M CX-4945 and 2. Mu.g/mL cisplatin.

18. The pharmaceutical composition according to claim 13, wherein: the adjuvant or auxiliary component is selected from at least one of diluent, filler, colorant, glidant, lubricant, binder, stabilizer, suspending agent and buffer.