Application of combination of vitamin C and tyrosine kinase inhibitor in preparation of medicine for resisting renal clear cell carcinoma and related medicine

Abstract

The invention belongs to the technical field of biological medicines, and provides application of combination of vitamin C (VC) and a tyrosine kinase inhibitor in resisting renal clear cell carcinoma. The invention relates to application of combination of VC and a tyrosine kinase inhibitor in preparation of a medicine for resisting renal clear cell carcinoma. The tyrosine kinase inhibitor comprises sunitinib and axitinib. Under the conditions that the concentration of VC is 0.039 mM to 0.625 mM and the concentration of sunitinib is 0.78 mu M to 6.25 mu M, the VC and the sunitinib have a synergistic effect to inhibit tumor cell proliferation. Under the conditions that the concentration of VC is 0.039 mM to 0.625 mM and the concentration of axitinib is 0.39 mu M to 3.13 mu M, the VC and the axitinib have a synergistic effect to inhibit tumor cell proliferation. In the application of the invention, the combination of VC and tyrosine kinase inhibitor can resist renal clear cell carcinoma and effectively inhibit the formation of tumor cell clone. The novel drug combination mode provides a new basis for clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of the combination of vitamin C and tyrosine kinase inhibitors in the preparation of drugs for treating renal clear cell carcinoma and related drugs. Background Art

[0002] The incidence of renal cancer has been increasing year by year. Among them, renal clear cell carcinoma (ccRCC) accounts for about 80% of renal cancer and is the most common pathological type. At present, the main treatment method is surgical treatment, but about 20%-50% of patients will develop metastases within 3 years after surgery, and the survival rate will be greatly reduced. Clinically, tyrosine kinase inhibitors (TKIs, such as Sunitinib, etc.) are the first-line drugs for treating ccRCC, but drug resistance is extremely likely to occur. Improving the drug resistance of Sunitinib and enhancing its therapeutic effect are difficult problems in the treatment of ccRCC.

[0003] Compared with the single drug treatment method, combination drug use is more inclined in clinical treatment. However, the existing combination methods, such as the combination of Atezolizumab and Axitinib, cannot completely relieve the disease process. Therefore, how to optimize the combination drug use plan with TKIs is a huge challenge faced by the clinical treatment of advanced ccRCC patients.

[0004] Vitamin C (VC), also known as ascorbic acid, is a very important vitamin for the human body and can be obtained from foods such as citrus fruits, strawberries, and tomatoes. Early studies found that VC is a powerful antioxidant that helps scavenge free radicals and reduce the damage of oxidative stress to cells. In addition, VC also participates in various biological processes, such as the synthesis of collagen and the release of antihistamines, and plays an important role in skin health and immune system function. Past research has mainly focused on the antioxidant and anti-inflammatory properties of VC and its direct effects on the growth of tumor cells. Compared with previous studies, we not only focused on the direct effects of VC on renal cancer, but also explored its drug resistance to TKIs and the feasibility of combined treatment with TKIs. It is suggested that VC is expected to provide new ideas for future cancer treatment research, especially in the combined treatment of integrating VC with first-line clinical drugs. Summary of the Invention

[0005] Based on this, the present invention provides the application of the combination of vitamin C and tyrosine kinase inhibitors in the preparation of drugs for treating renal clear cell carcinoma and related drugs, which solves the problems of drug resistance of tyrosine kinase inhibitor monotherapy and insufficient anti-renal cancer effect of combined drug use.

[0006] The first aspect of the present invention provides the application of the combination of vitamin C and tyrosine kinase inhibitors in the preparation of drugs for treating renal clear cell carcinoma, and the tyrosine kinase inhibitors include Sunitinib and Axitinib.

[0007] Specifically, under the conditions that the vitamin C concentration is 0.039 mM to 0.625 mM and the sunitinib concentration is 0.78 μM to 6.25 μM, the two act synergistically to inhibit the proliferation of tumor cells.

[0008] Specifically, under the conditions that the vitamin C concentration is 0.039 mM to 0.625 mM and the axitinib concentration is 0.39 μM to 3.13 μM, the two act synergistically to inhibit the proliferation of the tumor cells.

[0009] The second aspect of the present invention provides a combined drug for treating renal clear cell carcinoma, which comprises the vitamin C and the tyrosine kinase inhibitor in any of the above applications, and the tyrosine kinase inhibitor includes sunitinib and axitinib.

[0010] Specifically, the vitamin C concentration is 0.039 mM to 0.625 mM, and the sunitinib concentration is 0.78 μM to 6.25 μM.

[0011] Specifically, the vitamin C concentration is 0.039 mM to 0.625 mM, and the axitinib concentration is 0.39 μM to 3.13 μM.

[0012] Specifically, the combined drug further comprises a pharmaceutically acceptable excipient.

[0013] Specifically, the administration stage includes the early treatment stage after tumor discovery, the intermediate treatment stage after tumor progression, and the advanced treatment stage after tumor metastasis.

[0014] Specifically, the administration method includes oral administration.

[0015] In the application of the present invention, the combination of VC and the tyrosine kinase inhibitor can treat ccRCC and effectively inhibit the colony formation of tumor cells. The novel combined drug administration method of the present invention overcomes the problems of low response rate, drug resistance, and slow progress of curative effect existing in the existing treatment methods, and provides a new basis for clinical treatment.

[0016] At present, as a standard treatment drug for renal cancer, the single drug application of the tyrosine kinase inhibitor has defects, including a high incidence of drug resistance, and most patients develop secondary drug resistance within 6 to 15 months; about 30% to 40% of patients are ineffective in the initial treatment. In the application of the present invention, it is clarified that VC can reverse sunitinib resistance.

[0017] The combined drug of vitamin C and the tyrosine kinase inhibitor of the present invention can effectively inhibit the colony formation of tumor cells, and the effect is remarkable. Description of the Drawings

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art.

[0019] Figure 1 For the effect of VC on the proliferation of 786 - O and Caki - 1 cells, Figure 1 In which A shows the effect of different concentrations of VC on the activity of 786 - O cells, Figure 1 In which B shows the effect of different concentrations of VC on the activity of Caki - 1 cells.

[0020] Figure 2 For the effect of VC on Sunitinib resistance, Figure 2 In which A shows the effect of administering Sunitinib on the cell activities of 786 - O parental strain and resistant strain, Figure 2 In which B shows the effect of administering VC on the cell activity of 786 - O resistant strain, Figure 2 In which C shows the effect of administering Sunitinib on the cell activities of Caki - 1 parental strain and resistant strain, Figure 2 In which D shows the effect of administering VC on the cell activity of Caki - 1 resistant strain.

[0021] Figure 3 For the synergistic anti - tumor effect of combined use of VC and Sunitinib, Figure 3 In which A is a schematic diagram of the combined use of VC and Sunitinib, Figure 3 In which B shows the effect on the proliferation of 786 - O cells after 48 - hour administration of the VC group, Sunitinib group, and the combined - use group of VC and Sunitinib, Figure 3 In which C shows the effect on the proliferation of Caki - 1 cells after 48 - hour administration of the VC group, Sunitinib group, and the combined - use group of VC and Sunitinib.

[0022] Figure 4 For the effect of the combined use of VC and Sunitinib on the colony formation of tumor cells, Figure 4 In which A shows the effect of the combined use of VC and Sunitinib on the colony formation of 786 - O cells, Figure 4 In which B shows the effect of the combined use of VC and Sunitinib on the colony formation of Caki - 1 cells.

[0023] Figure 5 For the mechanism of the synergistic anti - tumor proliferation effect of the combined use of VC and Sunitinib, Figure 5 In which A is a flow cytometry diagram of the effect on cell apoptosis after treatment with VC and Sunitinib for 24 hours, Figure 5In it, B is a statistical chart of the flow cytometry results, showing the proportions of early apoptotic cells and late apoptotic cells after treatment with different administration methods. Figure 5 In it, C shows the effects of VC and Sunitinib treatments on the contents of proteins related to the apoptosis pathway (Bcl2, Caspase3, Bak, and P53) detected by the Western blot method.

[0024] Figure 6 is the synergistic anti-tumor effect of the combined use of VC and Axitinib. Figure 6 In A, the effects on the proliferation of 786-O cells after 48 hours of administration in the VC group, Axitinib group, and the combined use group of VC and Axitinib are shown. Figure 6 In B, the effects on the proliferation of Caki-1 cells after 48 hours of administration in the VC group, Axitinib group, and the combined use group of VC and Axitinib are shown. Specific implementation manners

[0025] Next, the technical solutions of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] A combined drug for treating clear cell renal cell carcinoma, which includes vitamin C and tyrosine kinase inhibitors. The tyrosine kinase inhibitors can be Sunitinib, Axitinib, Sorafenib, etc., preferably Sunitinib and Axitinib.

[0027] When vitamin C is combined with Sunitinib, the concentration of vitamin C is 0.039 mM to 0.625 mM, which can be 0.039 mM, 0.078 mM, 0.156 mM, 0.31 mM, 0.625 mM, etc., and the concentration of Sunitinib is 0.78 μM to 6.25 μM, which can be 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, etc.

[0028] When vitamin C is combined with Axitinib, the concentration of vitamin C is 0.039 mM to 0.625 mM, which can be 0.039 mM, 0.078 mM, 0.156 mM, 0.31 mM, 0.625 mM, etc., and the concentration of Axitinib is 0.39 μM to 3.13 μM, which can be 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, etc.

[0029] The combined drug also includes pharmaceutically acceptable excipients. The administration stages include the early treatment stage after tumor discovery, the intermediate treatment stage after tumor progression, and the advanced treatment stage after tumor metastasis. At present, especially the prevention and treatment of advanced ccRCC still face great challenges. The present invention can significantly improve the anti-kidney cancer effect, can be applied to each treatment stage, and can effectively solve the current problems.

[0030] In the following examples, 786-O (VHL mutant) and Caki-1 (VHL wild type) were purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection / Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0031] Example 1 In this example, the effect of VC against ccRCC was detected by using the representative ccRCC cell lines 786-O (VHL mutant) and Caki-1 (VHL wild type). The specific scheme is as follows: 1. The experiment detected the effect of VC at different concentrations on the cell proliferation of two cell lines, 786-O (VHL mutant) and Caki-1 (VHL wild type), by the MTT method. The detection results are as Figure 1 shown, Figure 1 in which A shows the effect of VC at different concentrations on the activity of 786-O cells, Figure 1 and B shows the effect of VC at different concentrations on the activity of Caki-1 cells.

[0032] The experimental results showed that in two different ccRCC tumor cell lines, VC had a concentration-dependent anti-proliferation effect.

[0033] 2. 786-O (VHL mutant) and Caki-1 (VHL wild type) cell lines resistant to sunitinib were constructed. Parallel control groups, VC group, and sunitinib group were set up. The MTT method was used to detect the effect of different treatment groups on the cell proliferation of the two cell lines to clarify whether VC inhibited the proliferation of the drug-resistant cell lines. The detection results are as Figure 2 shown, Figure 2 in which A shows the effect of administering sunitinib on the activity of 786-O parental and drug-resistant cell lines, Figure 2 B shows the effect of administering VC on the activity of 786-O drug-resistant cell lines, Figure 2 C shows the effect of administering sunitinib on the activity of Caki-1 parental and drug-resistant cell lines, Figure 2 and D shows the effect of administering VC on the activity of Caki-1 drug-resistant cell lines.

[0034] The experimental results showed that Figure 2 A in which is compared withFigure 2 C in it respectively shows that in 786 - O cells and Caki - 1 cells, compared with the parental cell line, the drug - resistant cell lines are insensitive to Sunitinib, indicating that the construction of the drug - resistant model is successful. Figure 2 B in Figure 2 D in it respectively shows that after treating different drug - resistant strains with VC, compared with the drug - resistant strains, VC significantly inhibits the proliferation of drug - resistant cell lines, indicating that VC has the effect of resisting Sunitinib resistance.

[0035] 3. Detect whether VC and Sunitinib in combination can have a synergistic effect by the MTT method. Select the renal cancer cell lines 786 - O and Caki - 1, and give different concentrations of VC and Sunitinib respectively. The experiment sets up parallel control groups, VC groups, Sunitinib groups, and the combined drug - using group of VC and Sunitinib. The detection results are as Figure 3 shown. Figure 3 A in it is the schematic diagram of the combined use of VC and Sunitinib. Figure 3 B in it is the effect on the proliferation of 786 - O cells after 48 - hour administration of the VC group, Sunitinib group, and the combined drug - using group of VC and Sunitinib. Figure 3 C in it is the effect on the proliferation of Caki - 1 cells after 48 - hour administration of the VC group, Sunitinib group, and the combined drug - using group of VC and Sunitinib.

[0036] The experimental results show that in 786 - O and Caki - 1 cells, when VC (0.039 mM - 0.625 mM) and Sunitinib (0.78 μM - 6.25 μM) are administered simultaneously, there is a trend of synergistic proliferation inhibition, clarifying the synergistic anti - ccRCC effect of VC and Sunitinib.

[0037] 4. Explore the mechanism of the synergistic anti - renal cancer proliferation effect of VC and Sunitinib, and use flow cytometry to detect the effect of different drug - using methods on cell apoptosis in different groups. The detection results are as Figure 4 shown. Figure 4 A in it is the effect of the combined use of VC and Sunitinib on the colony formation of 786 - O cells. Figure 4 B in it is the effect of the combined use of VC and Sunitinib on the colony formation of Caki - 1 cells.

[0038] The experimental results show that Figure 4 A in Figure 4In the B experiment results, 14 days after drug administration, compared with using VC or Sunitinib alone, the number of tumor cells treated with the combination of VC and Sunitinib was significantly reduced. The combination of VC and Sunitinib had a synergistic effect on inhibiting cell clone formation, achieving an effect of 1 + 1 > 2.

[0039] To explore the mechanism of the synergistic anti - renal cancer cell proliferation effect of VC and Sunitinib, flow cytometry was used to detect the effect on cell apoptosis after different drug treatments; Western blot was used to detect the effect on the content of proteins related to the cell apoptosis pathway (Bcl2, Caspase3, Bak, and P53) after different drug treatment methods. The detection results are as Figure 5 shown, Figure 5 In A of [reference], it is a flow cytometry diagram of the effect on cell apoptosis after treating with VC and Sunitinib for 24 hours, Figure 5 In B of [reference], it is a statistical chart of the flow cytometry results, showing the proportions of early - apoptotic and late - apoptotic cells after different drug treatment methods, Figure 5 In C of [reference], it is the effect on the content of proteins related to the cell apoptosis pathway (Bcl2, Caspase3, Bak, and P53) detected by Western blot after treating with VC and Sunitinib.

[0040] Figure 5 The experimental results of A in [reference] and Figure 5 B in [reference] showed that compared with single drugs, the combination of VC and Sunitinib in treating cells had a synergistic effect on promoting cell apoptosis. Further detecting the expression of proteins related to the cell apoptosis pathway (Bcl2, Caspase3, Bak, and P53), Figure 5 The experimental results of C in [reference] showed that compared with single drugs, the combined use of VC and Sunitinib significantly inhibited the expression of the apoptosis - related protein Bcl2 and increased the expression of the pro - apoptosis - related proteins Caspase3, Bak, and P53. These results proved that the combination of VC and Sunitinib inhibited the proliferation of ccRCC tumor cells by promoting apoptosis.

[0041] Example 2 The MTT method was used to detect whether the combination of VC and Axitinib could have a synergistic effect. The renal cancer cell lines 786 - O and Caki - 1 were selected, and different concentrations of VC and Axitinib were given respectively. In the experiment, parallel control groups, VC group, Axitinib group, and the combined - drug group of VC and Axitinib were set up. The detection results are as Figure 6 shown, Figure 6A shows the effect on the proliferation of 786-O cells after administration of the VC group, Axitinib group, and the combined administration of VC and Axitinib for 48 hours. Figure 6 B shows the effect on the proliferation of Caki-1 cells after administration of the VC group, Axitinib group, and the combined administration of VC and Axitinib for 48 hours.

[0042] The experimental results show that in 786-O and Caki-1 cells, the simultaneous administration of VC (0.039 mM - 0.625 mM) and Axitinib (0.39 μM - 3.13 μM) presents a trend of synergistic proliferation inhibition, clarifying the synergistic anti-renal cancer effect of VC and Axitinib.

[0043] The above are only the preferred embodiments of the present invention. Those skilled in the art should understand that without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the combination of vitamin C and tyrosine kinase inhibitor in the preparation of a drug for treating clear cell renal carcinoma, characterized in that, The tyrosine kinase inhibitors include sunitinib and axitinib.

2. The application according to claim 1, wherein Under the conditions that the vitamin C concentration is 0.039 mM to 0.625 mM and the sunitinib concentration is 0.78 μM to 6.25 μM, the two act synergistically to inhibit the proliferation of tumor cells.

3. The application according to claim 1, characterized in that, Under the conditions that the vitamin C concentration is 0.039 mM to 0.625 mM and the axitinib concentration is 0.39 μM to 3.13 μM, the two act synergistically to inhibit the proliferation of tumor cells.

4. A combined drug for treating clear cell renal cell carcinoma, characterized in that, It includes the vitamin C and the tyrosine kinase inhibitor in any one of the applications recited in claims 1 to 3, and the tyrosine kinase inhibitor includes sunitinib and axitinib.

5. The combined medicine according to claim 4, wherein, The vitamin C concentration is 0.039 mM to 0.625 mM, and the sunitinib concentration is 0.78 μM to 6.25 μM.

6. The combined medicine according to claim 4, characterized in that The vitamin C concentration is 0.039 mM to 0.625 mM, and the axitinib concentration is 0.39 μM to 3.13 μM.

7. The combined medicine according to claim 4, wherein It also includes pharmaceutically acceptable excipients.

8. The combined medicine according to claim 4, wherein The administration stage includes the early treatment stage after tumor discovery, the intermediate treatment stage after tumor progression, and the late treatment stage after tumor metastasis.

9. The combined medicine according to claim 4, wherein The administration method includes oral administration.

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