Application of leucine in preparation of anti-cancer drugs and chemotherapy sensitizers

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of leucine in preparation of anti-cancer drugs and chemotherapy sensitizers. According to the application provided by the invention, the leucine is applied to products of anti-cancer drugs and chemotherapy sensitizers, and the result shows that the IC50 of the chemotherapy drugs to cancer cells is remarkably reduced by adding the leucine, the chemotherapy sensitivity of the cancer cells to the chemotherapy drugs such as gemcitabine and oxaliplatin is improved, the resistance of the cancer cells to chemotherapy is reduced, and the curative effect of the cancer cells is improved. The improvement of the chemotherapy curative effect is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of leucine in the preparation of anticancer drugs and chemotherapy sensitizers. Background Art

[0002] Cancer has always been a major problem that has plagued human health. It progresses rapidly and is difficult to treat. Although chemotherapy is often used in clinical treatment, some cancer cells have serious chemotherapy resistance, which often leads to poor chemotherapy effects or even chemotherapy failure.

[0003] Intrahepatic cholangiocarcinoma (ICC) is an adenocarcinoma originating from the epithelium of the secondary bile duct and its branches, accounting for approximately 10% to 15% of primary malignant liver tumors. It is the second most common primary malignant liver tumor after hepatocellular carcinoma in incidence. Both the incidence and mortality rates have been on the rise in recent years, and more than 70% of patients are already in the advanced stage when they first visit the doctor.

[0004] Since 2010, chemotherapy combined with platinum and gemcitabine has been used clinically for patients with advanced ICC. Starting in 2023, many international guidelines recommended that chemotherapy be combined with immunotherapy to improve prognosis. However, the objective response rate of both chemotherapy and chemotherapy combined with immunotherapy is still very low. Studies have shown that the median survival time of ICC patients receiving chemotherapy is only 11.7-16.8 months, and the objective response rate of chemotherapy is only about 17%.

[0005] The main reason for the above phenomenon is that ICC patients lack effective and safe adjuvants to enhance the chemotherapy effect during chemotherapy. Therefore, the safe chemotherapy dose usually used during chemotherapy is small, and the killing effect on tumors is limited. Tumors are prone to acquire drug resistance and thus relapse. So far, there is no effective treatment or method in clinical practice to improve ICC chemotherapy resistance. Summary of the invention

[0006] In order to solve the above technical problems, the present invention explores the mechanism of cancer chemotherapy resistance and provides a chemotherapy sensitizer product and corresponding applications that can improve the chemotherapy response rate of advanced cancer and improve the current status of cancer cell chemotherapy resistance.

[0007] In the process of exploring the chemotherapy resistance of cancer cells, the inventors unexpectedly found that adding leucine to the culture medium reduced the half inhibitory concentration (IC 50 ), improves the chemotherapy resistance of cancer cells and reduces the sensitivity of cancer cells to chemotherapy drugs. Based on this, the present invention is proposed.

[0008] The first aspect of the present invention is to provide the use of leucine in the preparation of anticancer drugs, wherein the cancers include but are not limited to the following: liver cancer, colon cancer, lung cancer, gastric cancer, colorectal cancer, and breast cancer.

[0009] Among the above-mentioned cancers, the liver cancer is intrahepatic bile duct carcinoma.

[0010] Another aspect of the present invention is to provide the use of leucine in the preparation of a chemosensitizer, wherein the chemosensitizer is a sensitizer that increases the sensitivity of at least one of the chemotherapeutic drugs gemcitabine and oxaliplatin.

[0011] Furthermore, in the above application provided by the present invention, the concentration of leucine in the anticancer drug or chemotherapy sensitizer is 0.1-50.0 mM.

[0012] Preferably, the concentration of leucine in the anticancer drug or chemosensitizer is 0.15-20.0 mM.

[0013] More preferably, the concentration of leucine in the anticancer drug or chemosensitizer is 0.5-15.0 mM.

[0014] As further preferred, the concentration of leucine in the anticancer drug or chemosensitizer is 0.5-2.0 mM.

[0015] As further preferred, the concentration of leucine in the anticancer drug or chemosensitizer is 1.6-2.0 mM.

[0016] The third aspect of the present invention is to provide a product for treating intrahepatic bile duct cancer, wherein the product comprises leucine and a chemotherapeutic drug, wherein the chemotherapeutic drug is at least one of gemcitabine and oxaliplatin.

[0017] Preferably, in the product, the concentration of leucine is 0.1-50.0 mM, the concentration of gemcitabine is 0.1-500.0 μM, and the concentration of oxaliplatin is 0.1-500.0 μM.

[0018] More preferably, in the product, the concentration of leucine is 0.5-15.0 mM, the concentration of gemcitabine is 0.1-400.0 μM, and the concentration of oxaliplatin is 0.1-400.0 μM.

[0019] Further preferably, in the product, the concentration of leucine is 1.0-2.0 mM, the concentration of gemcitabine is 0.1-200.0 μM, and the concentration of oxaliplatin is 0.1-200.0 μM.

[0020] In addition, when the above product is used for cancer treatment, leucine is taken simultaneously with at least one of the chemotherapy drugs gemcitabine and oxaliplatin.

[0021] The beneficial effects of the present invention are:

[0022] A new application of leucine is provided, that is, leucine is used in the preparation of anticancer drugs and chemotherapy sensitizers to improve the resistance of cancer patients to chemotherapy drugs, such as gemcitabine, oxaliplatin, etc., and to improve the efficacy of chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the results of the dose-dependent sensitization experiment of leucine on RBE cells in Example 1 of the present invention;

[0024] Figure 2 This is a graph showing the results of the dose-dependent sensitization experiment of leucine on HuCCT1 cells in Example 1 of the present invention;

[0025] Figure 3 is the IC of leucine on gemcitabine or oxaliplatin in RBE cells in Example 1 of the present invention 50 curve;

[0026] Figure 4 is the IC of leucine on gemcitabine or oxaliplatin in HuCCT1 cells in Example 1 of the present invention 50 curve;

[0027] Figure 5 This is a result diagram of leucine enhancing the chemotherapy sensitivity of the YAP / AKT mouse ICC model in Example 2 of the present invention;

[0028] Figure 6 This is a graph showing the results of leucine enhancing the chemotherapy sensitivity of the NICD / AKT mouse ICC model in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] Here, the inventors only use intrahepatic cholangiocarcinoma as an example for explanation.

[0032] Example 1

[0033] Experimental method: The revived hepatobiliary carcinoma cells (RBE cells) or cholangiocarcinoma adherent cells (HuCCT1 cells) were cultured in a 37°C incubator until the cell density was about 90%, and then digested and plated on a 96-well plate with 5×10 cells per well. 3 After the cells adhered for 24 hours, the supernatant was aspirated and culture medium containing leucine and gemcitabine / oxaliplatin was added for two days. CCK8 was then added to detect the absorbance at 450 nm to evaluate the killing effect.

[0034] in, Figure 1 and Figure 2 The graphs show the results of the dose-dependent sensitization experiment of leucine on RBE cells and HuCCT1 cells.

[0035] The experiment was divided into 5 groups, with leucine concentrations of 0 mM (i.e., control group), 1.0 mM, 1.3 mM, 1.6 mM, and 2.0 mM, respectively, and each leucine concentration had 6 replicate wells.

[0036] For RBE cells, the concentrations of gemcitabine were 0.1 μM, 1 μM, and 10 μM, and the concentrations of oxaliplatin were 2 μM, 10 μM, and 50 μM, respectively.

[0037] For HuCCT1 cells, the concentrations of gemcitabine were 1 μM, 10 μM, and 100 μM, and the concentrations of oxaliplatin were 10 μM, 20 μM, and 40 μM, respectively.

[0038] Figure 3 and Figure 4 are IC of RBE cells and HuCCT1 cells respectively 50 The curve was measured and divided into 3 groups, with leucine concentrations of 0 mM (i.e. control group), 0.4 mM, and 1.6 mM, respectively.

[0039] For RBE cells, the concentrations of gemcitabine were 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, and the concentrations of oxaliplatin were 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM.

[0040] For HuCCT1 cells, the concentrations of gemcitabine and oxaliplatin were 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM.

[0041] IC of groups treated with different concentrations of leucine 50 See Table 1 below.

[0042] Table 1 IC values ​​of different leucine concentrations in the treatment groups for chemotherapeutic drugs 50

[0043]

[0044]

[0045] The above results show that 1.6 mM leucine treatment can greatly reduce the IC of chemotherapeutic drugs on ICC cells. 50 , improving the chemotherapy resistance of ICC cells.

[0046] Example 2 Animal Experiment

[0047] Two different mouse ICC models were used in this example, as follows:

[0048] The first ICC model was formed by high-hydrodynamic injection of YAPS127A and myrAKT plasmids into the tail vein;

[0049] The second ICC model was generated by hydrodynamic tail vein injection of NICD and myrAKT plasmids.

[0050] The mice were divided into 4 groups, including normal drinking water group, gemcitabine + oxaliplatin (GEMOX) treatment + normal drinking water group, 114mM leucine (Leu) drinking water group, GEMOX treatment + 114mMLeu drinking water group, with 6 mice in each group. Treatment began 1.5 weeks after injection, and the mice were killed at 4 weeks. The mice drank about 5mL of water per day, which is equivalent to 0.66mmol of leucine intake.

[0051] The experimental results of two different mouse models are shown in the attached Figure 5 , Attachment Figure 6 shown.

[0052] from Figure 5 and Figure 6 From the gross observation of the two ICC model mice, it can be seen that the liver size of the GEMOX group was significantly smaller than that of the control group, while the liver of the mice in the GEMOX+Leu drinking water group was almost close to the normal liver size; from the data, compared with the control group, the liver weight to body weight ratio of the GEMOX group mice decreased by 20% to 30%, while the liver weight to body weight ratio of the GEMOX+Leu drinking water group decreased by 49% to 63%.

[0053] In addition, from the perspective of tissue pathology, the proportion of CK19+ tumor area in the GEMOX+Leu drinking water group decreased from 7% to about 1%, and the number of KI67+ proliferating tumor cells also decreased from about 200 to about 30.

[0054] The above data all indicate that the combined use of GEMOX+Leu drinking water can greatly reduce the ICC tumor burden.

[0055] In addition, further long-term survival experiment results also gave a consistent conclusion that compared with the control group and the GEMOX group alone, the combination of GEMOX+Leu drinking water can significantly improve the prognosis of ICC mice.

Claims

1. The use of leucine in the preparation of anticancer drugs, characterized in that: The cancers include but are not limited to the following: liver cancer, colon cancer, lung cancer, stomach cancer, colorectal cancer, and breast cancer.

2. The use according to claim 1, characterized in that The liver cancer is intrahepatic bile duct carcinoma.

3. The use of leucine in the preparation of a chemosensitizer, characterized in that: The chemotherapy sensitizer is a sensitizer that increases the sensitivity of at least one of the chemotherapy drugs gemcitabine and oxaliplatin.

4. The use according to claim 1 or 3, characterized in that: The concentration of leucine in the anticancer drug or chemotherapy sensitizer is 0.1-50.0 mM.

5. A product for treating intrahepatic bile duct cancer, characterized in that: The product comprises leucine and a chemotherapy drug, wherein the chemotherapy drug is at least one of gemcitabine and oxaliplatin.

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