Application of vitamin C combined with tyrosine kinase inhibitors in the preparation of anti-renal clear cell carcinoma drugs and related drugs

Abstract

This invention belongs to the field of biopharmaceutical technology and provides the use of vitamin C (VC) in combination with a tyrosine kinase inhibitor for the treatment of clear cell renal cell carcinoma. This combination of VC and a tyrosine kinase inhibitor is used to prepare a drug for treating clear cell renal cell carcinoma. Tyrosine kinase inhibitors include sunitinib and axitinib. When VC concentrations range from 0.039 mM to 0.625 mM and sunitinib concentrations range from 0.78 μM to 6.25 μM, the two agents synergistically inhibit tumor cell proliferation. When VC concentrations range from 0.039 mM to 0.625 mM and axitinib concentrations range from 0.39 μM to 3.13 μM, the two agents synergistically inhibit tumor cell proliferation. In this application, the combination of VC and a tyrosine kinase inhibitor can treat clear cell renal cell carcinoma and effectively inhibit tumor cell colony formation. This novel combination therapy provides new insights for clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of vitamin C in combination with a tyrosine kinase inhibitor in the preparation of an anti-renal clear cell carcinoma drug and related drugs. Background Art

[0002] The incidence of kidney cancer is increasing annually. Clear cell renal cell carcinoma (ccRCC) is the most common pathological type, accounting for approximately 80% of renal cancer cases. Surgery is currently the primary treatment, but approximately 20%-50% of patients will develop metastases within three years of surgery, significantly reducing survival. Clinically, tyrosine kinase inhibitors (TKIs, such as sunitinib) are the first-line treatment for ccRCC, but resistance is highly prevalent. Improving sunitinib resistance and enhancing its therapeutic efficacy are challenging challenges in treating ccRCC.

[0003] Combination therapy is increasingly being used in clinical treatment, rather than single-drug therapies. However, existing combinations, such as atezolizumab plus axitinib, fail to completely alleviate disease progression. Therefore, optimizing combination therapy with TKIs presents a significant challenge in the clinical treatment of patients with advanced ccRCC.

[0004] Vitamin C (VC), also known as ascorbic acid, is a vital vitamin for the human body and can be obtained from foods such as citrus fruits, strawberries, and tomatoes. Early studies have shown that VC is a powerful antioxidant that helps scavenge free radicals and mitigate cellular damage caused by oxidative stress. VC is also involved in various biological processes, such as collagen synthesis and the release of antihistamines, playing an important role in skin health and immune system function. Previous studies have primarily focused on VC's antioxidant and anti-inflammatory properties and its direct effects on tumor cell growth. Compared to previous studies, our study not only focused on VC's direct effects on renal cancer but also explored its resistance to TKIs and the feasibility of combination therapy with TKIs. This suggests that VC may provide new insights for future cancer treatment research, particularly when combined with first-line clinical agents. Summary of the Invention

[0005] Based on this, the present invention provides the use of vitamin C in combination with tyrosine kinase inhibitors in the preparation of anti-renal clear cell carcinoma drugs and related drugs, which solve 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 use of vitamin C in combination with a tyrosine kinase inhibitor in the preparation of an anti-renal clear cell carcinoma drug, wherein the tyrosine kinase inhibitor includes 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 tumor cell proliferation.

[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 combination drug for treating renal clear cell carcinoma, comprising the vitamin C and the tyrosine kinase inhibitor in any of the above applications, wherein 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 combination drug also includes pharmaceutically acceptable excipients.

[0013] Specifically, the administration stage includes an early treatment stage after tumor discovery, a mid-term treatment stage after tumor progression, and a late treatment stage after tumor metastasis.

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

[0015] In the application of this invention, VC combined with a tyrosine kinase inhibitor can fight ccRCC and effectively inhibit tumor cell clone formation. This novel combination therapy overcomes the problems of low response rate, drug resistance, and slow progress in efficacy of existing treatments, providing a new basis for clinical treatment.

[0016] Currently, tyrosine kinase inhibitors, the standard treatment for renal cancer, have drawbacks when used alone, including a high incidence of drug resistance, with most patients developing secondary resistance within 6-15 months; and approximately 30-40% of patients failing initial treatment. The present invention demonstrates that VC can reverse sunitinib resistance.

[0017] The vitamin C combined with the tyrosine kinase inhibitor of the present invention can effectively inhibit the formation of tumor cell clones with remarkable effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0019] Figure 1 The effect of VC on the proliferation of 786-O and Caki-1 cells. Figure 1 A in the figure shows the effect of different concentrations of VC on the activity of 786-O cells. Figure 1 B in the figure shows the effect of different concentrations of VC on the activity of Caki-1 cells.

[0020] Figure 2 It is the VC's resistance to Sunitinib. Figure 2 A in the figure represents the effect of Sunitinib on the cell activity of 786-O parent strain and drug-resistant strain. Figure 2 B in the figure is the effect of VC on the cell activity of 786-O drug-resistant strain. Figure 2 C in the figure represents the effect of Sunitinib on the cell activity of the parental and drug-resistant strains of Caki-1. Figure 2 D in the figure is the effect of VC on the cell activity of Caki-1 drug-resistant strain.

[0021] Figure 3 The combined use of VC and Sunitinib has a synergistic anti-tumor effect. Figure 3 A in the figure is a schematic diagram of the combined use of VC and Sunitinib. Figure 3 B in the figure shows the effect of VC group, Sunitinib group and VC and Sunitinib combination group on the proliferation of 786-O cells after 48 hours of administration. Figure 3 C in the figure represents the effect of VC group, Sunitinib group and VC and Sunitinib combination group on the proliferation of Caki-1 cells 48 hours after administration.

[0022] Figure 4 To investigate the effect of VC combined with Sunitinib on the clone formation of tumor cells. Figure 4 A in the figure represents the effect of VC combined with Sunitinib on the clone formation of 786-O cells. Figure 4 B in the figure shows the effect of the combination of VC and Sunitinib on the clone formation of Caki-1 cells.

[0023] Figure 5 The mechanism of synergistic anti-tumor proliferation effect of VC combined with Sunitinib is Figure 5 A in the figure is the flow cytometry graph showing the effect of VC and Sunitinib on cell apoptosis after 24 hours of treatment. Figure 5B is a statistical graph of flow cytometry results, showing the proportion of cells undergoing early and late apoptosis after treatment with different drug administration methods. Figure 5 C in the figure is the effect of VC and Sunitinib treatment on the levels of apoptosis pathway-related proteins (Bcl2, Caspase3, Bak and P53) detected by Western blot.

[0024] Figure 6 The combined use of VC and Axitinib has a synergistic anti-tumor effect. Figure 6 A in the figure represents the effect of VC group, Axitinib group and VC and Axitinib combination group on the proliferation of 786-O cells after 48 hours of administration. Figure 6 B in the figure shows the effect of VC group, Axitinib group and VC and Axitinib combination group on the proliferation of Caki-1 cells after 48 hours of administration. DETAILED DESCRIPTION

[0025] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0026] A combination drug for treating renal clear cell carcinoma, comprising vitamin C and a tyrosine kinase inhibitor. The tyrosine kinase inhibitor may be sunitinib, axitinib, sorafenib, etc., with sunitinib and axitinib being preferred.

[0027] When vitamin C is used in combination with sunitinib, the concentration of vitamin C is 0.039mM~0.625mM, which can be 0.039mM, 0.078mM, 0.156mM, 0.31mM, 0.625mM, etc., and the concentration of sunitinib is 0.78μM~6.25μM, which can be 0.78μM, 1.56μM, 3.13μM, 6.25μM, etc.

[0028] When vitamin C is used in combination with axitinib, the concentration of vitamin C is 0.039mM~0.625mM, which can be 0.039mM, 0.078mM, 0.156mM, 0.31mM, 0.625mM, etc., and the concentration of axitinib is 0.39μM~3.13μM, which can be 0.39μM, 0.78μM, 1.56μM, 3.13μM, etc.

[0029] The combination drug also includes a pharmaceutically acceptable excipient. Administration phases include early treatment following tumor discovery, mid-stage treatment following tumor progression, and late-stage treatment following tumor metastasis. Currently, the prevention and treatment of advanced ccRCC, in particular, remain significant challenges. This invention significantly enhances the effectiveness of its treatment for renal cancer and can be applied at all stages of treatment, effectively addressing current challenges.

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

[0031] Example 1

[0032] This example uses representative ccRCC cell lines 786-O (VHL mutant) and Caki-1 (VHL wild-type) to test the anti-ccRCC effect of VC. The specific protocol is as follows:

[0033] 1. The experiment used MTT method to detect the effect of different concentrations of VC on the cell proliferation of 786-O (VHL mutant) and Caki-1 (VHL wild type) cells. The test results are as follows: Figure 1 As shown, Figure 1 A in the figure shows the effect of different concentrations of VC on the activity of 786-O cells. Figure 1 B in the figure shows the effect of different concentrations of VC on the activity of Caki-1 cells.

[0034] The experimental results showed that VC had a concentration-dependent anti-proliferative effect in two different ccRCC tumor cell lines.

[0035] 2. Construct sunitinib-resistant 786-O (VHL mutant) and Caki-1 (VHL wild-type) cell lines, set up parallel control group, VC group, and sunitinib group (Sunitinib), use MTT method to detect the effect of different treatment groups on the cell proliferation of the two cell lines, to determine whether VC inhibits the proliferation of drug-resistant cell lines, the test results are as follows Figure 2 As shown, Figure 2 A in the figure represents the effect of Sunitinib on the cell activity of 786-O parent strain and drug-resistant strain. Figure 2 B in the figure is the effect of VC on the cell activity of 786-O drug-resistant strain. Figure 2 C in the figure represents the effect of Sunitinib on the cell activity of the parental and drug-resistant strains of Caki-1. Figure 2 D in the figure is the effect of VC on the cell activity of Caki-1 drug-resistant strain.

[0036] The experimental results show that Figure 2 A in Figure 2 C in the figure shows that in 786-O cells and Caki-1 cells, compared with the parental cell lines, the resistant cell lines are insensitive to Sunitinib, indicating that the resistance model was successfully constructed. Figure 2 B in Figure 2 D in the figure shows that when different drug-resistant strains were treated with VC, VC significantly inhibited the proliferation of drug-resistant cell lines compared with the resistant strains, indicating that VC has the effect of resisting Sunitinib resistance.

[0037] 3. The MTT assay was used to test whether the combination of VC and Sunitinib could have a synergistic effect. Renal cancer cell lines 786-O and Caki-1 were selected and given different concentrations of VC and Sunitinib respectively. The experiment was conducted by setting up parallel control group, VC group, Sunitinib group and VC and Sunitinib combination group. The test results are as follows: Figure 3 As shown, Figure 3 A in the figure is a schematic diagram of the combined use of VC and Sunitinib. Figure 3 B in the figure shows the effect of VC group, Sunitinib group and VC and Sunitinib combination group on the proliferation of 786-O cells after 48 hours of administration. Figure 3 C in the figure represents the effect of VC group, Sunitinib group and VC and Sunitinib combination group on the proliferation of Caki-1 cells 48 hours after administration.

[0038] The experimental results showed that in 786-O and Caki-1 cells, simultaneous administration of VC (0.039mM~0.625mM) and Sunitinib (0.78μM~6.25μM) showed a synergistic proliferation inhibition trend, clarifying the synergistic anti-ccRCC effect of VC and Sunitinib.

[0039] 4. To explore the mechanism of synergistic anti-proliferation effect of VC and Sunitinib in renal cancer, flow cytometry was used to detect the effects of different medication methods on cell apoptosis. Figure 4 As shown, Figure 4 A in the figure represents the effect of VC combined with Sunitinib on the clone formation of 786-O cells. Figure 4 B in the figure shows the effect of the combination of VC and Sunitinib on the clone formation of Caki-1 cells.

[0040] The experimental results show that Figure 4 A and Figure 4The results of experiment B showed that 14 days after administration, the number of tumor cells treated with the combination of VC and Sunitinib was significantly reduced compared with those treated with VC or Sunitinib alone. The combination of VC and Sunitinib had a synergistic inhibitory effect on cell clone formation, achieving a 1+1>2 effect.

[0041] To explore the mechanism of synergistic anti-proliferation effect of VC and Sunitinib on renal cancer, flow cytometry was used to detect the effect of different drug treatments on cell apoptosis; Western blot was used to detect the effect of different drug treatments on the levels of apoptosis pathway-related proteins (Bcl2, Caspase3, Bak and P53). Figure 5 As shown, Figure 5 A in the figure is the flow cytometry graph showing the effect of VC and Sunitinib on cell apoptosis after 24 hours of treatment. Figure 5 B is a statistical graph of flow cytometry results, showing the proportion of cells undergoing early and late apoptosis after treatment with different drug administration methods. Figure 5 C in the figure is the effect of VC and Sunitinib treatment on the levels of apoptosis pathway-related proteins (Bcl2, Caspase3, Bak and P53) detected by Western blot.

[0042] Figure 5 A and Figure 5 The results of experiment B in Figure 3 showed that compared with single drug, the combined treatment of VC and Sunitinib played a synergistic role in promoting cell apoptosis. The expression of proteins related to the cell apoptosis pathway (Bcl2, Caspase3, Bak and P53) was further detected. Figure 5 The experimental results in Figure C show that compared with either drug alone, the combination of VC and Sunitinib significantly inhibited the expression of the apoptosis-related protein Bcl2 and increased the expression of the pro-apoptotic proteins Caspase3, Bak, and P53. These results demonstrate that the combination of VC and Sunitinib inhibits the proliferation of ccRCC tumor cells by promoting apoptosis.

[0043] Example 2

[0044] The MTT assay was used to test whether the combination of VC and Axitinib could have a synergistic effect. Renal cancer cell lines 786-O and Caki-1 were selected and given different concentrations of VC and Axitinib, respectively. The experiment was conducted by setting up a parallel control group, VC group, Axitinib group, and VC and Axitinib combination group. The test results are shown in Figure 2. Figure 6 As shown, Figure 6A in the figure represents the effect of VC group, Axitinib group and VC and Axitinib combination group on the proliferation of 786-O cells after 48 hours of administration. Figure 6 B in the figure shows the effect of VC group, Axitinib group and VC and Axitinib combination group on the proliferation of Caki-1 cells after 48 hours of administration.

[0045] The experimental results showed that in 786-O and Caki-1 cells, the simultaneous administration of VC (0.039mM~0.625mM) and Axitinib (0.39μM~3.13μM) showed a synergistic proliferation inhibition trend, clarifying the synergistic anti-renal cancer effect of VC and Axitinib.

[0046] The above is only a preferred embodiment of the present invention. Those skilled in the art should understand that several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of protection of the present invention.

Claims

1. The use of vitamin C in combination with a tyrosine kinase inhibitor in the preparation of an anti-renal clear cell carcinoma drug, characterized in that: The tyrosine kinase inhibitor is sunitinib or axitinib, The vitamin C concentration is 0.039 mM to 0.625 mM, the sunitinib concentration is 0.78 μM to 6.25 μM, and the axitinib concentration is 0.39 μM to 3.13 μM.

2. A combination drug for treating renal clear cell carcinoma, characterized in that: The vitamin C and the tyrosine kinase inhibitor in the use according to claim 1 are active ingredients, and the tyrosine kinase inhibitor is sunitinib or axitinib. The vitamin C concentration is 0.039 mM to 0.625 mM, the sunitinib concentration is 0.78 μM to 6.25 μM, and the axitinib concentration is 0.39 μM to 3.13 μM.

3. The combination drug according to claim 2, characterized in that Pharmaceutically acceptable excipients are also included.

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