Use of mTOR kinase inhibitors with pdk inhibitors and medicaments

Abstract

This invention relates to the application of a composition of an mTOR kinase inhibitor and a PDKs inhibitor in the synergistic inhibition of tumor growth. The composition is a combination of intraperitoneal injection of compound I and oral administration of compound II via drinking water. Compound I has immunosuppressive effects, thus alleviating rejection reactions after organ transplantation, and possesses an anti-tumor mechanism; compound II is a PDKs inhibitor drug that controls cancer metabolism; the combined use of both significantly inhibits tumor growth, therefore this composition can be used to prepare drugs for treating the progression of malignant tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the combination of mTOR kinase inhibitors and PDKs inhibitors drugs described in this application and their pharmaceutically acceptable salt compounds to change the tumor growth microenvironment, induce tumor cell apoptosis and change cancer metabolism to reverse cancer growth, belonging to the field of medicine. BACKGROUND

[0002] Cancer has become the primary killer of global health at this stage, a large number of studies have shown that malignant tumor cells have the characteristics of being unaffected by the apoptosis mechanism; unrestricted growth; insensitive to growth limiting factors; and accelerated cell division rate. A large number of studies have shown that the mammalian target of rapamycin (mTOR) controls cell anabolism, including protein translation, DNA and RNA synthesis. The mTOR signaling pathway is highly activated in most cancer cells, so inhibiting mTOR can achieve the purpose of limiting the growth of cancer cells; but compared with chemotherapy drugs, inhibiting the mTOR pathway has less effect on the growth of cancer cells. One of the reasons is that autophagy induced by inhibiting the mTOR pathway can help cancer cells resist the pressure of nutrient deficiency. Secondly, inhibiting the mTOR signaling pathway limits the growth rate of cancer cells rather than inducing apoptosis. Therefore, the combination of inhibiting the mTOR signaling pathway with other therapies shows considerable therapeutic effect. Previous studies have found that DCA treatment has limited inhibitory effect on the growth of cancer cells in vivo and in vitro. Compared with the use of DCA or rapamycin alone, we found that DCA combined with rapamycin treatment showed better inhibition of tumor growth, which also showed that DCA combined with other anticancer drugs may have better therapeutic effect. SUMMARY

[0003] The present application relates to the discovery of drugs that synergistically inhibit tumor progression, one of the purposes is to provide a combination of mTOR kinase inhibitors and PDKs inhibitors for inhibiting tumor growth; the second purpose is to establish a new treatment method for synergistically inhibiting tumor progression.

[0004] The compound I is the mTOR kinase inhibitor Rapamycin, which is shown in formula (I); the compound II is the PDKs inhibitor Dichloroacetic acid (DCA), which is shown in formula (II); the combination of the compound I (Rapamycin) intravenous injection and the compound II (Dichloroacetic acid) oral administration in drinking water.

[0005] The present application relates to the discovery of drugs that synergistically inhibit tumor progression, one of the purposes of the present application is to provide a combination of mTOR kinase inhibitors and PDKs inhibitors for inhibiting tumor progression.

[0006] Including but not limited to: the use of the composition in the preparation of a pharmaceutically acceptable carrier or excipient for synergistically inhibiting tumor progression.

[0007] Experiments show that the composition described in the present application can inhibit the proliferation of tumor cells relative to the use of compound I or compound II alone, and thus can be used synergistically to prepare a drug for inhibiting tumor progression.

[0008] Compound I has an immunosuppressive effect to reduce rejection after organ transplantation and has an anti-tumor mechanism; compound II is a PDKs inhibitor drug that controls cancer metabolism; the combination of the two significantly inhibits tumor growth, and thus the composition can be used to prepare a drug for treating the progression of malignant tumors. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Figure 1 is a graph showing that DCA combined with Rapamycin treatment and enhanced Rapamycin-induced cell growth inhibition;

[0010] Figure 2 Figure 2 is a graph showing that DCA and Rapamycin have a higher growth inhibition effect than Rapamycin; DCA and Rapamycin combined treatment significantly reduces the number of cell clusters;

[0011] Figure 3 Figure 3 is a graph showing that DCA makes cancer cells sensitive to Rapamycin in vivo. DETAILED DESCRIPTION

[0012] The present application will be further described in conjunction with examples. The examples are only limited to illustrate the present application, and are not a limitation on the present application.

[0013] Example 1

[0014] DCA (purchased from Shanghai Macklin) combined with Rapamycin (purchased from Shanghai Macklin) treatment and enhanced Rapamycin-induced cell growth inhibition.

[0015] (a) 4T1 cells (from ATCC cell bank) were treated with different final concentrations of Rapamycin (0, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100; nM) for 96 hours, and cell viability was tested using the SRB (Rhodamine, purchased from Beijing Bailingwei) assay, with three wells for each treatment.

[0016] (b) and (c) CT-26 and MDA-MB-231 (from ATCC cell bank) cells were treated with different concentrations of Rapamycin (0, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20; nM) for 72 hours, respectively. Cell viability was tested using the SRB assay, with three wells for each treatment.

[0017] Experimental procedure: The following were respectively... Figure 1 The cell lines shown (4T1, CT-26, MDA-MB-231) were passaged and seeded into 96-well plates, with 1000 cells per well. 4T1 and CT-26 were cultured in RPM-1640 medium, and MDA-MB-231 was cultured in DMEM medium. 200 μL of medium was added to each well. After 12 hours of adherent culture in an incubator with 5% CO2 at 37°C, the cells were treated with different concentrations of Rapamycin as shown above. Each treatment (including a control group and an experimental group; the control group was treated with water (DCA solvent), and the experimental group was treated with DCA) corresponded to three replicate wells. The DCA group was treated with DCA (1M stock solution dissolved in water) to a final concentration of 2 mM, while the control group was treated with an equal volume of water. Cell viability was assessed after 96 hours of culture for 4T1 cells, and after 72 hours of culture for CT-26 and MDA-MB-231 cells.

[0018] The method used to detect cell viability was the SRB assay. The procedure was briefly described as follows: Trichloroacetic acid (final concentration 10%, g / ml) was added to the wells of a 96-well plate containing cells, and the plate was fixed at 4°C for 1 hour. The supernatant was then discarded, and the cells were washed three times with water and air-dried. 50 μL of staining solution (0.04% SRB (rhodamine) dissolved in 1% acetic acid aqueous solution) was added to each well, and staining was performed for half an hour. The supernatant was then discarded, and the cells were washed three times with 1% acetic acid aqueous solution and air-dried. Finally, the dye was dissolved in 50 μL of a 10 mM Tris (pH = 10.5) solution, and the absorbance at 510 nm was read to characterize cell viability.

[0019] The results are as follows Figure 1 As shown in the figure, treatment with 2mM DCA can increase the inhibitory effect of rapamycin on cell proliferation in all three cell lines, indicating that rapamycin and DCA have a synergistic effect in inhibiting tumor cell proliferation.

[0020] Example 2

[0021] DCA combined with Rapamycin showed higher growth inhibition efficacy than Rapamycin alone; the combination of DCA and Rapamycin significantly reduced the number of cell colonies.

[0022] (a) and (b) MDA-MB-231 and 4T1 cells were seeded into 12-well plates, respectively, and treated with... Figure 2The drug treatment was performed for 6 days. Cell clones were stained with SRB (using the same staining method as the SRB assay in Example 1 above; after washing three times with 1% acetic acid solution and air-drying, they were photographed and statistically analyzed). Images and quantification were then performed. Each group consisted of four wells (one well as the control group, one well as the DCA group, one well as the rapamycin group, and one well as the DCA + rapamycin group). The final concentration of DCA (stock solution concentration of 1M, solvent: water) was 2mM; the final concentration of rapamycin (stock solution concentration of 10μM, solvent: DMSO) was 10nM.

[0023] Experimental procedure: 200 MDA-MB-231 and 4T1 cells were seeded in each well of a 12-well plate. MDA-MB-231 cells were cultured in DMEM medium, and 4T1 cells were cultured in RPMI-1640 medium. The plates were incubated at 37°C with 5% CO2 for 12 hours. Compound I (rapamycin group, with water added to make up the amount of solvent less than that in the DCA+rapamycin group), Compound II (DCA group, with DMSO added to make up the amount of solvent less than that in the DCA+rapamycin group), Compound I and Compound II (DCA+rapamycin group), and the control group were added with the same volume of solvent water and DMSO as the other groups (DCA+rapamycin group). After 6 days, the cells were stained and statistically analyzed using the SRB assay method mentioned in Example 1.

[0024] The results are as follows Figure 2 As shown in the figure, DCA alone did not significantly alter cell proliferation in MDA-MB-231 cells, while rapamycin alone significantly inhibited cell proliferation. Importantly, compared with rapamycin alone, the combination of DCA and rapamycin significantly reduced the number of cell clones. In 4T1 cells, compared with the two compounds used alone, the combination of DCA and rapamycin not only reduced the number of cell clones but also reduced the size of the clones, further demonstrating that DCA and rapamycin synergistically inhibit tumor cell proliferation.

[0025] Example 3

[0026] DCA sensitizes cancer cells to rapamycin in the body.

[0027] (a) Schematic diagram of DCA and rapamycin treatment in mice injected with 4T1 cells (100,000 cells per mouse). DCA: dissolved in drinking water at 0.75 g / L, orally administered with water (average 3-4 ml per mouse per day); rapamycin (solvent: DMSO / PEG300 / water, volume ratio 20 / 40 / 40, concentration 0.5 mg / ml): 2 mg / kg intraperitoneally, once daily.

[0028] (b) Measure the tumor volume every two days using calipers (direct measurement with electronic vernier calipers, measuring the longest end of the tumor as L and the shortest end as W), and calculate the volume using the standard formula 0.5 × L × W. 2 Calculate the tumor volume, where L is the longest diameter and W is the shortest diameter.

[0029] (c) Tumors were removed and photographed 14 days after treatment with the method shown in (a), with 5 mice in each group.

[0030] (d) Mouse tumor weight derived from (c).

[0031] (e) After 14 days of treatment using the method shown in (a), the spleens of the mice were removed and photographed, with 5 mice in each group.

[0032] (f) Mouse spleen weight derived from (e)

[0033] Composition and experimental procedure of the experimental and control groups: The mice were 6-8 week old female BALB / c mice. 100,000 4T1 cells were injected subcutaneously into the thigh of each mouse. After 4 days of tumor growth, a solid tumor protrusion under the thigh skin was visible to the naked eye. The mice were then divided into four groups (5 mice in each group) as shown in (a). The control group and DCA group were injected with water (containing no rapamycin) to dissolve rapamycin, while the other two groups received rapamycin via intraperitoneal injection once a day, based on body weight (2 mg / kg). The DCA group and DCA+Rapamycin group drank water containing DCA, while the other two groups drank water without DCA. The tumor volume was measured every two days. Finally, on the eighteenth day, the mice were sacrificed, the solid tumors were removed, and the spleen weight of each mouse was measured and photographed.

[0034] The results are as follows Figure 3 As shown in the figure, DCA alone had no significant effect on the proliferation of subcutaneous tumors formed by 4T1 cells, but it reduced the weight of the spleen in mice, indicating a decrease in tumor cell infiltration in the spleen. Rapamycin alone significantly inhibited the proliferation of 4T1 subcutaneous tumors and significantly reduced spleen weight. The combined use of DCA and rapamycin resulted in the smallest tumor weight and volume, and also the smallest spleen weight, compared to the use of either compound alone. These results indicate that rapamycin and DCA synergistically inhibit tumor cell proliferation in vivo.

[0035] The in vivo and in vitro experiments described above demonstrate that the combined use of the mTOR inhibitors rapamycin and DCA effectively inhibits tumor cell proliferation, tumor cell colony formation, and cell survival. Therefore, the combined use of these two inhibitors provides a candidate approach for the development of anticancer drugs and holds great promise for clinical application in oncology.

Claims

1. Application of mTOR kinase inhibitors and PDK inhibitors in the preparation of drugs for treating tumors; The mTOR kinase inhibitor is compound I, rapamycin, which has the structural formula (I); the PDK inhibitor is compound II, dichloroacetic acid (DCA), which has the structural formula (II). ， The dosages of the mTOR kinase inhibitor and PDKs inhibitor are 0.5-2 mg rapamycin and 0.25-1 g DCA per day, respectively. The tumor is a tumor with highly activated mTOR pathway, specifically one or both of breast cancer and colon cancer.

2. Application of mTOR kinase inhibitors in the preparation of drugs for the combined treatment of tumors with PDK inhibitors; The mTOR kinase inhibitor is compound I, rapamycin, which has the structural formula (I); the PDK inhibitor is compound II, dichloroacetic acid (DCA), which has the structural formula (II). , The dosages of the mTOR kinase inhibitor and PDKs inhibitor are 0.5-2 mg rapamycin and 0.25-1 g DCA per day, respectively. The tumor is a tumor with highly activated mTOR pathway, specifically one or both of breast cancer and colon cancer.

3. Use according to claim 1 or 2, characterized in that: Compound I is an intravenous injection formulation, and compound II is an oral formulation.

4. Use according to claim 3, characterized in that: The intravenous or oral formulations also include a pharmaceutically acceptable carrier.

5. Use according to claim 1 or 2, characterized in that: The dosages of the mTOR kinase inhibitor and PDKs inhibitor are 0.8-1.2 mg rapamycin and 0.4-0.6 g DCA per day, respectively.

6. A drug for treating tumors and / or inhibiting tumor growth, comprising an mTOR kinase inhibitor and a PDKs inhibitor; The mTOR kinase inhibitor is compound I, rapamycin, which has the structural formula (I); the PDK inhibitor is compound II, dichloroacetic acid (DCA), which has the structural formula (II). ， The dosages of the mTOR kinase inhibitor and PDKs inhibitor are 0.5-2 mg rapamycin and 0.25-1 g DCA, respectively. The tumor is a tumor with highly activated mTOR pathway, specifically one or both of breast cancer and colon cancer.

7. The medicament according to claim 6, characterized in that: Compound I is an intravenous injection formulation, and compound II is an oral formulation.

8. The medicament according to claim 7, characterized in that: The intravenous or oral formulations also include a pharmaceutically acceptable carrier.

9. The medicament according to claim 6, characterized in that: The dosages of the mTOR kinase inhibitor and PDKs inhibitor are 0.8-1.2 mg of rapamycin and 0.4-0.6 g of DCA, respectively.

Images