Application of lacidipine in preparation of IDO inhibitor and / or antitumor drug

Abstract

The invention provides application of lacidipine in preparation of IDO inhibitors and / or antitumor drugs, and belongs to the technical field of chemical drugs. Research finds that lacidipine is an IDO inhibitor with double functions, can inhibit expression of IDO in tumors and can inhibit enzyme activity; the lacidipine plays a good anti-tumor role by inhibiting the difunction of IDO (indoleamine-2, 3-dioxygenase). The lacidipine can promote effector T cells, inhibit tumor infiltration and functions of Tregs cells and inhibit further metastasis of tumors. The lacidipine can also be combined with other anti-tumor drugs to play a synergistic anti-tumor effect. The invention also provides a new treatment strategy for enhancing antitumor immune response by clinically applying antihypertensive drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drugs, and specifically relates to use of lacidipine in preparing IDO inhibitors and / or anti-tumor drugs. Background Art

[0002] In the early stages of breast cancer, acute inflammation activates the immune system, initiating tumor-specific T cell responses to eliminate cancer cells. The adaptive immune system maintains surviving tumor cells in a dormant state. Once this balance is broken, tumor cells stimulate acute inflammation to turn into chronic inflammation, thereby establishing a complex tumor immunosuppressive microenvironment (TME) consisting of myeloid-derived suppressor cells (MDSCs), suppressive immune cells (regulatory T cells, Tregs) and B cells, allowing tumors to escape the surveillance and elimination of the immune system, thereby promoting the occurrence of breast cancer. The immune system is crucial to the disease outcome of triple-negative breast cancer (TNBC). Immune cells have been found to infiltrate in a variety of tumors, including breast cancer, and are becoming important biomarkers for predicting efficacy and treatment outcomes. The prognosis and efficacy of tumor patients are closely related to the infiltration of tumor immune cells. Tumors can be divided into "cold" tumors and "hot" tumors based on the level of tumor infiltrating lymphocytes (TILs). Cold tumors show T cell deficiency or immune rejection, while hot tumors are characterized by T cell infiltration and immune activation. Since hot tumors have a higher response rate to immunotherapy, most studies are aimed at how to convert cold tumors into hot tumors to obtain better immunotherapy responses. Among different breast cancer subtypes, TNBC and Her2-positive breast cancer show higher immunogenicity. More importantly, TILs are an independent prognostic biomarker in breast cancer, and in lymph node-positive TNBC, high TILs are associated with improved survival. Therefore, given the importance of tumor immune monitoring for cancer prevention and the high immunogenicity of TNBC, immunotherapy is becoming a promising innovative treatment for TNBC. Choosing to develop tumor immunotherapy strategies that convert cold tumors into hot tumors may produce more effective anti-tumor responses.

[0003] Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunomodulatory enzyme that leads to the depletion of tryptophan and the accumulation of its metabolite kynurenine, thereby inducing local immunosuppression in the tumor microenvironment. High IDO activity in local tissues leads to rapid depletion of tryptophan, increases the number of unloaded tRNAs, and activates the stress response kinase GCN2 and mTOR signaling pathways, leading to G1 phase arrest of effector T cell cell cycle, thereby inhibiting the activity and proliferation of effector T cells. In addition, accumulated kynurenine interacts with the aryl hydrocarbon receptor (AhR) and activates it, thereby inducing the activation of naive CD4 +T cells differentiate into regulatory T cells (Tregs). However, the differentiation of Tregs can in turn inhibit the activation of effector T cells. All these changes work synergistically in the tumor microenvironment, responsible for the survival and immune escape of cancer cells. The expression of IDO1 is usually induced by proinflammatory cytokines such as interferon γ (IFNγ) and tumor necrosis factor α (TNFα), and is regulated by the JAK / STAT and NF-κB signaling pathways (Bilir and Sarisozen, 2017). Studies have shown that tumors with high expression of IDO1 have high expression of CD8 + Reduced infiltration of effector T cells is closely associated with poor prognosis in patients with various tumors, revealing that IDO1 is an important immune regulator and may be an ideal target for cancer immunotherapy.

[0004] At present, a variety of IDO1 inhibitors such as epacadostat, 1-methyl-D-tryptophan (1-MT), and Indoximod have entered clinical trials. However, the treatment effect of IDO1 inhibitors alone is poor, and chemotherapy and other immunotherapy are required. The failure of the Phase III trial of epacadostat combined with pembrolizumab for the treatment of melanoma revealed the shortcomings of the existing IDO1 inhibitor treatment strategy. First, the epacadostat dose tested in ECHO-301 may not achieve sufficient drug exposure, and the degree of IDO1 inhibition in the tumor is unclear. Secondly, epacadostat and pembrolizumab may not be the best combination strategy. It has been shown that IDO inhibitors are more effective in combination with DNA damage-inducing drugs. Finally, epacadostat is a highly specific IDO1 inhibitor, and selective IDO1 blockade is not enough to continuously relieve tumor immunosuppression mediated by elevated kynurenine and tryptophan deficiency. Tumor cells achieve compensatory immune escape through the expression of TDO and activation of downstream effector pathways (such as aryl hydrocarbon receptors) (Wang et al., 2019). In addition, preclinical mouse tumor models have shown that IDO1-mediated immunosuppression is not only dependent on its enzymatic activity (Zhai et al., 2015), but IDO1 may also serve as a signaling molecule to mediate or maintain immune tolerance independent of its enzymatic activity. Therefore, while targeting IDO transcriptional levels, it may be necessary to inhibit its enzymatic activity in order to significantly restore IDO-mediated immunosuppression. Given the important immunomodulatory function of IDO1, the discovery of new IDO1 inhibitors and combination therapy strategies is of great significance.

[0005] Voltage-gated calcium channels (VGCCs) are the targets of dihydropyridines (DHPs) for the treatment of hypertension. DHPs interact with VGCCs, blocking the depolarization of vascular smooth muscle cells, cardiomyocytes, and cardiac lymph node tissues, inhibiting Ca 2+In addition, VGCCs are closely related to the initiation and progression of many types of cancer (such as colon cancer, prostate cancer, lung cancer, breast cancer and pancreatic cancer). Compared with healthy cells, cancer cells have a depolarized cell membrane potential, which may promote the abnormal activation of L-type calcium channels in cancer cells. Excessive activation of L-type calcium channels regulates intracellular Ca 2+ Changes in concentration induce the proliferation, invasion and metastasis of tumor cells (Martinez-Delgado and Felix, 2017). 1,4-dihydropyridine (1,4-DHP) drugs, such as lacidipine, nifedipine and amlodipine, are the most widely used and effective L-type calcium channel blockers (CCBs). Many studies have shown that a few drugs in 1,4-dihydropyridine have anti-tumor activity, such as nifedipine and amlodipine. Among them, nifedipine can inhibit the migration, proliferation and autophagy of prostate cancer, cervical cancer and colon cancer cells (Fourbon et al., 2017). Amlodipine can inhibit the proliferation of human HT-39 breast cancer cells and A431 human epidermoid carcinoma cells, and has been shown to overcome tumor resistance by inducing cell apoptosis (Li et al., 2006). However, none of these drugs work by blocking their known targets VGCCs. In addition, it is still unclear whether voltage-gated channel inhibitors can exert anti-tumor effects by inhibiting tumor immunity.

[0006] Lacidipine is a third-generation new dihydropyridine calcium channel blocker with stronger and more sustained antihypertensive effects and has been widely used in clinical practice. However, the calcium channel blocking activity of lacidipine has not been found to mediate antitumor effects.

[0007] Summary of the invention

[0008] The purpose of the present invention is to provide the use of lacidipine in preparing IDO inhibitors and / or anti-tumor drugs.

[0009] The present invention provides use of lacidipine in preparing an IDO inhibitor and / or a drug for preventing and / or treating a disease related to IDO.

[0010] Furthermore, the IDO inhibitor is an IDO1 inhibitor or an IDO2 inhibitor.

[0011] Furthermore, the drug for preventing and / or treating diseases related to IDO is a drug for preventing and / or treating tumors.

[0012] Furthermore, the drug is a drug that inhibits tumor growth and / or inhibits tumor metastasis.

[0013] Furthermore, the tumor is breast cancer, cervical cancer, lung cancer, liver cancer, gastric cancer, or colorectal cancer.

[0014] The present invention also provides the use of lacidipine in combination with a chemotherapeutic drug in the preparation of a drug for preventing and / or treating tumors;

[0015] Preferably, the drug is a drug that inhibits tumor growth and / or tumor metastasis;

[0016] More preferably, the tumor is breast cancer, cervical cancer, lung cancer, liver cancer, gastric cancer, or colorectal cancer.

[0017] Furthermore, the chemotherapy drug is doxorubicin, cisplatin, cyclophosphamide, 5-fluorouracil, methotrexate, paclitaxel, vinblastine or rapamycin.

[0018] The present invention also provides an IDO inhibitor and / or a pharmaceutical preparation for preventing and / or treating diseases related to IDO, characterized in that it is prepared by taking lacidipine as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0019] The present invention also provides a pharmaceutical composition for preventing and / or treating tumors, which comprises lacidipine and a chemotherapeutic drug.

[0020] The present invention also provides a pharmaceutical preparation for preventing and / or treating tumors, which is prepared by taking lacidipine and chemotherapy drugs as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention has found that lacidipine is a dual-function IDO inhibitor that can inhibit both the expression of IDO in tumors and the enzyme activity; lacidipine exerts a good anti-tumor effect through dual-function inhibition of IDO. Lacidipine can promote effector T cells, inhibit Treg cell tumor infiltration and function, and inhibit further tumor metastasis. Lacidipine can also be used in combination with other anti-tumor drugs to exert a synergistic anti-tumor effect. The present invention also provides a new treatment strategy for the clinical application of antihypertensive drugs to enhance anti-tumor immune response.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of different concentrations of lacidipine inhibiting IDO1 activity of breast cancer MDA-MB-231 and MCF-7 cells: MDA-MB-231 and MCF-7 cells were treated with different concentrations of lacidipine (5, 10, 20, 40, 80 μM) for 2 h, and then recombinant human IFNγ (100 ng / mL) was added for 24 h, and the concentration of L-kynurenine in the cell supernatant was measured.

[0026] Figure 2 The cytotoxicity results of different concentrations of lacidipine on breast cancer MDA-MB-231 and MCF-7 cells: A is MDA-MB-231 cells; B is MCF-7 cells; MDA-MB-231 and MCF-7 cells were treated with different concentrations of lacidipine (5, 10, 20, 40, 80 μM) for 2 h, and then treated with human recombinant IFNγ (100 ng / mL) for 24 h, and CCK-8 was used to detect cell viability.

[0027] Figure 3 The results of different concentrations of lacidipine inhibiting IDO1 activity of cervical cancer HeLa cells and their cytotoxicity to HeLa cells: A is the inhibition of IDO1 activity of cervical cancer HeLa cells; B is the cytotoxicity to HeLa cells; HeLa cells were treated with different concentrations of lacidipine (0.5, 1, 2.5, 5, 10, 20 μM) for 2 h, and then treated with human recombinant IFNγ (100 ng / mL) for 24 h, the concentration of L-kynurenine in the cell supernatant was determined, and the cell viability was determined by the CCK-8 method.

[0028] Figure 4 The results of lacidipine inhibiting the expression of IDO1 signaling pathway related proteins in different breast cancer cells: A is MDA-MB-231; B is MCF-7; MDA-MB-231 and MCF-7 cells were pretreated with lacidipine (10, 20, 40 μΜ) and JAK Inhibitor I (5 μM) for 2 h, and then stimulated with IFNγ (100 ng / mL) for 24 h, and the expression of IDO1, IDO2 and TDO2 was analyzed by Western blotting, with GAPDH as the internal reference.

[0029] Figure 5The results of lacidipine inhibiting the expression of IDO1 mRNA in breast cancer cells: MDA-MB-231 and MCF-7 cells were pretreated with lacidipine (10, 20, 40 μM) and JAK Inhibitor I (5 μM) for 2 h, and then stimulated with IFNγ (100 ng / mL) for 24 h. The expression of IDO1 mRNA was detected by qRT-PCR, and GAPDH was used as the internal reference gene; *p<0.05, **p<0.01, ***p<0.001 compared with the IFNγ treatment group.

[0030] Figure 6 Lacidipine inhibits the activities of IDO1, IDO2 and TDO2: A is IDO1; B is IDO2; C is TDO2; HEK293A cells were transfected with pCMV3-IDO1, pCMV3-IDO2 and pCMV3-TDO2 plasmids for 24 h, and then treated with different concentrations of lacidipine (10, 20, 40 μΜ) and epacadostat (25 nM) for 6 h. The activities of IDO1, IDO2 and TDO2 were detected using cell supernatants, *p<0.05, **p<0.01 and ***p<0.001 vs. pCMV3-IDO1 / 2 or pCMV3-TDO2 groups. The cell lysates were analyzed by western blotting using IDO1, IDO2 and TDO2 antibodies, with GAPDH as an internal reference.

[0031] Figure 7 The results of 1,4-dihydropyridine antihypertensive drugs inhibiting IDO1 activity of breast cancer cells and their cytotoxicity to breast cancer cells were as follows: A is isradipine; B is nifedipine; C is nitrendipine; MDA-MB-231 cells were treated with different concentrations of isradipine, nifedipine and nitrendipine (5, 10, 20, 40, 80 μM) for 2 h, and then human recombinant IFNγ (100 ng / mL) was added for 24 h. The concentration of L-kynurenine in the cell supernatant was determined and the cell viability was determined by CCK-8 method.

[0032] Figure 81,4-dihydropyridine antihypertensive drugs inhibit the activity of IDO1 and IDO2: AC is HEK293A cells transfected with pCMV3-IDO1 plasmid for 24 hours, and then treated with different concentrations of isradipine (A), nifedipine (B), nitrendipine (C) (10, 20 and 40μΜ) and epacadostat (25nM) for 6 hours. The activity of IDO1 was detected in the cell supernatant, **p<0.01 and ***p<0.001 compared with the pCMV3-IDO1 group. IDO1 antibody was used for western blot Blotting analysis, GAPDH was used as an internal reference; D was HEK293A cells transfected with pCMV3-IDO2 plasmid for 24 hours, and then treated with isradipine, nifedipine, nitrendipine (10μΜ) and epacadostat (25nM) for 6 hours. The activity of IDO2 was detected using the cell supernatant, *p<0.05 and **p<0.01 compared with the pCMV3-IDO2 group. Western blotting analysis was performed using IDO2 antibody, with GAPDH as an internal reference.

[0033] Fig. 9 Effect of 1,4-dihydropyridine antihypertensive drugs on TDO2 activity: HEK293A cells were transfected with pCMV3-TDO2 plasmid for 24 hours, and then treated with isradipine, nifedipine, nitrendipine (10μΜ) and epacadostat (25nM) for 6 hours. The activity of TDO2 was detected by cell supernatant, *p<0.05 compared with the pCMV3-TDO2 group. Western blotting analysis was performed using TDO2 antibody, and GAPDH was used as an internal reference.

[0034] Fig.10 Isradipine inhibits the expression of IDO1 signaling pathway-related proteins in breast cancer cells: A and B are MDA-MB-231 (A) and MCF-7 (B) cells pretreated with isradipine (10, 20, 40 μM) and JAK Inhibitor I (5 μM) for 2 h, and then stimulated with IFNγ (100 ng / mL) for 24 h. Western blotting was used to analyze the expression of IDO1, IDO2 and TDO2, with GAPDH as the internal reference.

[0035] Fig.11 In vivo safety evaluation of lacidipine: A and B are the body weight changes of mice in each group.

[0036] Fig.12The effect of lacidipine on the growth of 4T1-Luc orthotopic tumor in mice: A is the tumor volume of mice treated with doxorubicin and lacidipine alone or in combination at different times, and the mouse tumor volume is expressed as the mean ± standard error (SE); B is the tumor size and weight of mice in each group treated with doxorubicin and lacidipine alone or in combination, **p<0.01, ***p<0.001 and ****p<0.0001 compared with the control group; ####p<0.0001 compared with the doxorubicin group; C is the tumor volume of mice treated with cisplatin and lacidipine alone or in combination at different times, and the mouse tumor volume is expressed as the mean ± standard error (SE); D is the tumor size and weight of mice in each group treated with cisplatin and lacidipine alone or in combination, and the mouse tumor weight is expressed as the mean ± standard error (SE); ***p<0.001 and ****p<0.0001 compared with the control group; #p<0.05 compared with the cisplatin group.

[0037] Fig.13 Effect of lacidipine on 4T1-Luc orthotopic tumor growth in mice: Mouse tumor growth was monitored by in vivo imaging, and the luminescence intensity of tumors in mice carrying 4T1-Luc cells was detected on days 7, 16, and 28.

[0038] Fig.14 Effect of lacidipine on IDO activity in vivo: The Kyn / Try ratio in mouse serum was determined by HPLC, *p<0.05, **p<0.01 and ***p<0.001 compared with the control group.

[0039] Fig.15 Effects of lacidipine on tumor-infiltrating T cells: A and B are flow cytometry analysis of immune infiltrating CD8 T cells in tumor tissues at the end of administration. + CD45 + Effector T cells (A) and CD4 + CD25 + Foxp3 + Percentage of Trges (B) cells (n=6).

[0040] Fig.16 Effect of lacidipine on protein expression in tumor tissues: Immunohistochemistry and immunofluorescence were used to detect the expression of IDO1, CD8 and Foxp3 in mouse breast tumor tissues, and CD8 + and Foxp3 + T cell infiltration.

[0041] Fig.17 Effects of lacidipine on splenic T cells: A and B are flow cytometry analysis of CD8 + CD45 +Effector T cells (A) and CD4 + CD25 + Foxp3 + Percentage of Trges (B) cells (n=6).

[0042] Fig.18 Effects of lacidipine on T cell function: A and B are ELISA tests for the levels of cytokines IFNγ (A), IL-10 (B) and TGF-β (C) in tumor tissue homogenates; *p<0.05 and **p<0.01 compared with the control group, #p<0.05 compared with the doxorubicin / cisplatin group.

[0043] Fig.19 The results of lacidipine inhibiting the migration and invasion of breast cancer MDA-MB-231 cells: A is a scratch assay showing that lacidipine (10, 20 or 40 μM) treatment inhibits the scratch healing of MDA-MB-231 cells; B is a transwell migration assay to evaluate the effect of lacidipine (10, 20 or 40 μM) on the migration ability of MDA-MB-231 cells; C is a transwell invasion assay to evaluate the effect of lacidipine (10, 20 or 40 μM) on the invasion ability of MDA-MB-231. DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercial products. The main reagents and antibodies used in Western blotting are shown in Tables 1 and 2.

[0045] Table 1. Main reagents

[0046]

[0047] Table 2. Antibodies used in Western blotting

[0048]

[0049]

[0050] Example 1. Inhibitory effect of lacidipine on IDO1 in vitro and in vivo and its anti-tumor effect

[0051] 1. Experimental Methods

[0052] 1. In vitro experimental methods

[0053] 1.1 Cell culture

[0054] Human breast cancer cell line MDA-MB-231 was purchased from Wuhan Pucell Life Science Co., Ltd., China, human breast cancer cell line MCF-7, and human embryonic kidney cell HEK293A were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. MDA-MB-231 cells were cultured in Leibovitz's L15 medium (Solarbio), HEK293A cells were cultured in DMEM medium (Hyclone), and MCF-7 cells were cultured in MEM medium (Hyclone) containing 1% glutamine (Invitrogen), 1% 100× non-essential amino acids (Invitrogen), 1% sodium pyruvate (100mM) solution (Invitrogen) and 0.01mg / ml human recombinant insulin (Sigma). All culture media were supplemented with 10% (v / v) fetal bovine serum (Excell Bio) and 1% P / S (penicillin / streptomycin, Gibco) and placed in a cell culture incubator at 37°C and 5% CO2. All cells were analyzed by short tandem repeat analysis (STR) and tested for mycoplasma contamination by Suzhou Jianda Biotechnology Co., Ltd.

[0055] 1.2 Detection of intracellular IDO1 / 2 activity

[0056] 1.2.1 Detection of intracellular IDO1 activity

[0057] (1) When the confluence of MDA-MB-231, MCF-7 and HeLa cells inoculated in a 96-well plate reached about 70%, the culture medium was discarded and 200 μL of complete culture medium containing L-tryptophan (15 μg / mL) was added to each well.

[0058] (2) Subsequently, 0.1% DMSO solution was added to MDA-MB-231 and MCF-7 cells as negative controls and different concentrations (5, 10, 20, 40, 80 μM) of lacidipine, isradipine, nifedipine, and nitrendipine were treated for 2 h; 0.1% DMSO solution was added to HeLa cells as negative controls and different concentrations (0.5, 1, 2.5, 5, 10, 20 μM) of lacidipine were treated for 2 h. Three replicate wells of cells were set up in each group and implanted into a 96-well plate.

[0059] (3) Recombinant human IFNγ (100 ng / mL) was added to the cells to induce the expression of IDO1. The cells were placed in a CO2 incubator at 37°C and incubated for 24 h.

[0060] (4) Collect 100 μL of supernatant and add 25 μL of 30% trichloroacetic acid. Incubate the mixture at 50°C for 30 min. Centrifuge at 25°C, 2500 rpm for 10 min.

[0061] (5) Pipette 100 μL of the supernatant into a new 96-well culture plate, and add 100 μL of 2% (w / v) p-dimethylaminobenzaldehyde solution in glacial acetic acid.

[0062] (6) After mixing, the absorbance value was measured at 480 nm. The inhibition rate (%) = (A 药物 / A DMSO -1)×100%.

[0063] 1.2.2 Detection of intracellular IDO1 / 2 activity

[0064] (1) HEK293A cells were seeded into a 6-well plate, gently shaken to make them evenly distributed, and then placed in an incubator at 37° C. and 5% CO 2 for 24 h.

[0065] (2) When the cell density reaches about 70%-80%, use TransIntro TM EL transfection reagent was used to transfect pCMV3-IDO1 and pCMV3-IDO2 plasmids into HEK293A cells for 24 h.

[0066] (3) Different concentrations (10, 20, 40 μM) of compounds (lacidipine, isradipine, nifedipine, nitrendipine) and 25 nM Epacadostat were added to the cells and incubated for 6 h.

[0067] (4) Pipette 100 μL of cell culture medium into a 1.5 mL EP tube, then add 25 μL of 30% trichloroacetic acid and incubate at 50° C. for 30 min.

[0068] (5) Centrifuge at 13,000 rpm for 10 min at 25°C.

[0069] (6) Take 100 μL of the supernatant and add 100 μL of a 2% (w / v) solution of p-dimethylaminobenzaldehyde in glacial acetic acid.

[0070] (7) After mixing, measure the absorbance at 480 nm.

[0071] 1.3 TDO2 cell assay

[0072] (1) HEK293A cells were seeded into a 6-well plate, gently shaken to make them evenly distributed, and then placed in an incubator at 37° C. and 5% CO 2 for 24 h.

[0073] (2) When the cell density reaches about 70%-80%, use TransIntro TM HEK293A cells were transfected with pCMV3-TDO2 plasmid using EL transfection reagent for 24 h.

[0074] (3) Different concentrations (10, 20, 40 μM) of compounds (lacidipine, isradipine, nifedipine, nitrendipine) and 25 nM epacadostat) were added to the cells and incubated for 6 h.

[0075] (4) 300 μL of cell culture medium was pipetted into a 1.5 mL EP tube, followed by the addition of 90 μL of 30% trichloroacetic acid and incubation at 65° C. for 30 min.

[0076] (5) Centrifuge at 13,000 rpm for 10 min at 25°C.

[0077] (6) Take 100 μL of the supernatant and add 100 μL of a 2% (w / v) solution of p-dimethylaminobenzaldehyde in glacial acetic acid.

[0078] (7) After mixing, measure the absorbance at 480 nm.

[0079] 1.4CCK-8 detection of cell viability

[0080] (1) MDA-MB-231, MCF-7 and HeLa cells seeded in 96-well plates were cultured overnight. When the cell confluence reached about 70%-80%, different concentrations (5, 10, 20, 40, 80 μM) of drugs (lacidipine, isradipine, nifedipine, nitrendipine) were added and cultured for 2 h. Human recombinant IFNγ (100 ng / mL) was then added for treatment for 24 h. Three replicate wells were set for each drug addition group.

[0081] (2) Prepare CCK-8 working solution: Mix CCK-8 solution and serum-free culture medium in a ratio of 1:9.

[0082] (3) Add 100 μL of CCK-8 working solution to each well and place in a 37°C constant temperature incubator for 1-4 h.

[0083] (4) When the solution of the negative control group with DMSO turned orange-yellow, remove the cell culture plate and use ThermoScientific The Flash instrument detected the absorbance at a wavelength of 570 nm and calculated the survival rate (%).

[0084] 1.5 Western blotting

[0085] (1) MDA-MB-231 and MCF-7 cells seeded in 6-well plates were cultured overnight. When the cell confluence reached about 70%-80%, different concentrations (10, 20, 40 μM) of compounds (lacidipine, isradipine) and JAK inhibitor I (5 μM) were added and cultured for 2 h. Human recombinant IFNγ (100 ng / mL) was then added for 24 h, and total protein was extracted.

[0086] (2) Preparation of SDS-PAGE gel: Prepare separation gels of different concentrations according to the molecular weight of the target protein according to Table 3 below:

[0087] Table 3. SDS-PAGE separation gel formula (10mL)

[0088]

[0089] (3) Place the prepared gel in the electrophoresis tank and add 1× running buffer. Then add an appropriate amount of the protein sample to be tested and protein marker to the loading well. Perform electrophoresis at 75V constant voltage for about 40 minutes. After the protein sample is pressed into a straight line, change the voltage to 100V and stop the electrophoresis after bromophenol blue reaches the bottom of the gel. Carefully remove the gel and soak it in 1× Transbuffer transfer buffer.

[0090] (4) Before transfer, soak the filter paper, NC membrane, and sponge in 1×Transbuffer for about 10 minutes; then, place the sponge, 3 layers of filter paper, gel, NC membrane, 3 layers of filter paper, and sponge on the clamp in order, remove bubbles, and then place them in the transfer electrophoresis tank; according to the molecular weight of the target protein, transfer at a constant current of 260A for 60-90 minutes.

[0091] (5) After the transfer, the NC membrane was taken out and immersed in 5% BSA (1×TBST) solution and blocked at room temperature for 2 h.

[0092] (6) Dilute the primary antibody in proportion with 5% BSA according to the antibody instructions, then add the designated primary antibody onto the NC membrane and incubate at 4°C overnight.

[0093] (7) After the primary antibody incubation, wash the membrane five times with 1× TBST solution, each time for 10 min.

[0094] (8) Add the diluted secondary antibody onto the NC membrane and incubate at room temperature for 2 h.

[0095] (9) The membrane was then washed five times with 1× TBST solution, each time for 10 min.

[0096] (10) ECL reagent A and B were mixed in equal amounts, added dropwise and evenly covered on the NC film. After reacting in the dark for 30 seconds, the film was scanned and the image data was recorded.

[0097] 1.6qRT-PCR experiments

[0098] 1.6.1 Extraction of total RNA from cells

[0099] (1) MDA-MB-231 and MCF-7 cells seeded in 6-well plates were cultured overnight. When the cell confluence reached about 70%-80%, different concentrations of lacidipine (10, 20, 40 μM) and JAK Inhibitor I (5 μM) were added and cultured for 2 h. Human recombinant IFNγ (100 ng / mL) was then added for 24 h.

[0100] (2) Wash the cells twice with pre-cooled 1× PBS, add 500 μL Trizol reagent, incubate on ice for 5 minutes, blow the cells with a 1 mL pipette tip to completely lyse them, and transfer the lysate to a centrifuge tube.

[0101] (3) Add 100 μL of chloroform to each tube, shake for 30 seconds, and place on ice for 5 minutes.

[0102] (4) Centrifuge at 13,000 rpm for 15 min at 4°C, transfer the upper colorless aqueous phase to a new centrifuge tube, add 250 μL of isopropanol, mix well, and let stand at room temperature for 10 min.

[0103] (5) Centrifuge at 4°C, 13,000 rpm for 15 minutes, remove the supernatant, and wash the precipitate with freshly prepared 75% ethanol. Centrifuge at 4°C, 13,000 rpm for 10 minutes, remove the supernatant.

[0104] (6) Repeat washing the precipitate twice and add DEPC water to dissolve the extracted RNA.

[0105] 1.6.2qRT-PCR

[0106] (1) Use The All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Detection Kit reverse transcribes mRNA into cDNA.

[0107] Table 4. cDNA synthesis system

[0108]

[0109] (2) Gently tap the tube to mix, and incubate at 42°C for 15 min.

[0110] (3) Incubate at 85°C for 5 seconds.

[0111] (4) Then, use Tip Top Green qPCR SuperMix Assay performs qRT-PCR on cDNA in a 20 μL reaction mixture.

[0112] Table 5. qRT-PCR reaction system

[0113]

[0114]

[0115] The qPCR reaction was performed using a two-step method:

[0116] Table 6. qRT-PCR reaction program

[0117]

[0118] Table 7. Primer sequences used in qRT-PCR experiments

[0119]

[0120] (5) The relative expression of IDO1 and CYP1A1 mRNA was calculated using 2 -ΔΔCt Methods GAPDH was selected as the internal reference for normalization.

[0121] 2. In vivo experimental methods

[0122] Mouse breast cancer cells 4T1 transfected with luciferase (4T1-Luc) were purchased from Wuhan Pusail Life Science Co., Ltd., China. 4T1-Luc cells were cultured in RPMI-1640 medium (Hyclone). The medium was supplemented with 10% (v / v) fetal bovine serum (Excell Bio) and 1% P / S (penicillin / streptomycin, Gibco) and placed in a cell culture incubator at 37°C and 5% CO2. The cells were analyzed by short tandem repeat analysis (STR) and tested for mycoplasma contamination by Suzhou Jianda Biotechnology Co., Ltd.

[0123] 2.1 Mouse orthotopic breast tumor experiment

[0124] (1) Female BALB / c mice aged 6-8 weeks were orthotopically transplanted with 4T1-Luc cells (1×10 5 ) injected into the mammary fat pad.

[0125] (2) In vivo imaging of mice was performed on days 7, 16, and 28 after orthotopic implantation. Before in vivo imaging, mice were anesthetized with chlorofluoroethane using a small animal anesthesia machine, D-luciferin potassium salt was dissolved in DPBS without calcium and magnesium, filtered with a 0.2 μm filter membrane, and 200 μL of 15 mg / mL D-luciferin solution was injected intraperitoneally for imaging to observe the size of the tumor in the mouse.

[0126] (3) After the first in vivo imaging test, the mice were randomly divided into 9 groups (12 mice in each group) and given the following treatments: a. negative control (solvent group), b. doxorubicin (intravenous injection, ip, 1 mg / kg), c. 1-methyl-D-tryptophan (1-MT, oral, op, 1 mg / kg) + doxorubicin (intravenous injection, ip, 1 mg / kg), d. lacidipine (oral, op, 10 mg / kg), e. lacidipine (oral, op, 20 mg / kg), f. lacidipine (oral, op, 20 mg / kg) + doxorubicin (intravenous injection, ip, 1 mg / kg), g. cisplatin (intravenous injection, ip, 1 mg / kg), h. 1-MT (oral, op, 1 mg / kg) + cisplatin (intravenous injection, ip, 1 mg / kg), i. lacidipine (oral, op, 20 mg / kg) + cisplatin (intravenous injection, ip, 1 mg / kg). Adriamycin and cisplatin were administered every 3 days, and lacidipine and 1-MT were administered every 2 days. Tumor volume was measured with a vernier caliper every 3 days. The tumor volume was calculated using the following formula: 0.5236×L1×(L2) 2 , where L1 and L2 are the long and short axes of the tumor, respectively. The weight of the mice was monitored every three days. After the experiment, the model mice were killed by cervical dislocation, and blood, tumor tissues, and major organs such as heart, liver, spleen, lung, and kidney were collected for further research.

[0127] 2.2 High performance liquid chromatography (HPLC) analysis

[0128] Fresh blood was collected from mice, 100 μL of methanol was added to the serum to precipitate protein, the precipitate was removed by centrifugation, and 20 μL of supernatant was added to the column (250 mm × 4.6 mm; Eclipse XDB-C18, 5 μm). The mobile phase was phosphoric acid water (pH = 2.3). The content of kynurenine and tryptophan was detected at 235 nm and 280 nm, respectively.

[0129] 2.3 Flow cytometry analysis of tumor-infiltrating and spleen immune cells

[0130] Tumor tissues and spleen were collected for flow cytometric analysis.

[0131] 2.3.1 Extraction of cells from tumor tissue

[0132] (1) The freshly removed animal tumor tissue was digested with 1× HBSS buffer containing 1 mg / ml collagenase I, 1 mg / ml collagenase IV and 0.5 mg / ml DNase I at 37°C for 45 min to form a tumor tissue homogenate.

[0133] (2) After the tumor tissue homogenate was filtered through a 70 μM cell filter, an appropriate amount of red blood cell lysis buffer was added to fully remove red blood cells.

[0134] 2.3.2 Extraction of spleen cells

[0135] (1) Fresh spleen was removed from the mouse and immersed in PBS containing 1% P / S.

[0136] (2) The surface of the spleen was washed twice with PBS containing 1% P / S, and then placed on a 70 μM cell strainer. The spleen was crushed by pressing it with the piston of a syringe.

[0137] (3) Use a pipette to repeatedly blow up and down the suspension to break up the remaining tissue clumps.

[0138] (4) After the spleen tissue homogenate was filtered through a 70 μM cell filter, the cell pellet was collected and the red blood cells were removed using red blood cell lysis buffer.

[0139] 2.3.3 Flow cytometric analysis of T cells

[0140] (1) Centrifuge the liquid after removing red blood cells at 1500 rpm for 5 min, remove the supernatant, and resuspend the cells in PBS.

[0141] (2) The obtained tumor cells and spleen cells were stained with APC anti-CD4 antibody, PE anti-CD25 antibody, PE anti-CD45 and APC anti-CD8 antibody at 37°C for 30 min, respectively.

[0142] (3)Use True-Nuclear TM Transcription Factor Buffer is used to fix and disrupt the nuclei of cells: 1 mL of True-Nuclear TM 1×Fix concentrate solution, vortex to mix, and incubate at room temperature in the dark for 60 minutes.

[0143] (4) Without washing, add 2 mL of True-Nuclear to each tube. TM 1×Perm buffer, 2000rpm, centrifuge at 37℃ for 5min.

[0144] (5) Remove the supernatant and add 2 mL of True-Nuclear to each tube. TM 1×Perm buffer, 2000rpm, centrifuge at 37℃ for 5min, and remove the supernatant.

[0145] (6) Add 100 μL True-Nuclear TM Resuspend the cells in 1× Perm buffer. Add an appropriate amount of True-Nuclear TM Alexa Fluor 500 diluted in 1× Perm buffer 488Foxp3 antibody, incubate at room temperature in the dark for 1 h.

[0146] (7) Add 2 mL of True-Nuclear to each tube TM 1×Perm buffer, 2000rpm, centrifuge at 37℃ for 5min, remove the supernatant. Repeat the operation twice.

[0147] (8) Add PBS to resuspend the cells and analyze CD45 by flow cytometry. + CD8 + T cells and CD4 + CD25 + Foxp3 + Tregs cells.

[0148] 2.4 Enzyme-linked immunosorbent assay (ELISA) for cytokine determination

[0149] Prepare the working solution according to the following table:

[0150] Table 8. Preparation of working solution

[0151]

[0152] (1) Add 100 μL of sample to each well and use Dilution buffer R (1×) as a blank control.

[0153] (2) Add 50 μL of Biotinylated antibody working solution to each well, shake to mix evenly, cover with sealing film, and incubate at 37°C for 90 min.

[0154] (3) Discard the liquid in the wells and add 300 μL of 1× Washing buffer to each well. After leaving for 1 min, remove the supernatant and then dry the liquid in the wells on filter paper. Repeat this operation 4 times.

[0155] (4) Add 100 μL of Streptavidin-HRP working solution to each well and incubate at 37°C for 30 min.

[0156] (5) Repeat step (3), then add 100 μL TMB to each well and incubate at 37°C for approximately 20 min. The termination time is determined by the color in the well.

[0157] (6) Immediately add 100 μL Stop solution to each well to terminate the reaction.

[0158] (7) After 10 min, the levels of cytokines such as TGF-β, IFNγ, and IL-10 were analyzed at a wavelength of 450 nm.

[0159] 2.5 Immunohistochemical staining

[0160] (1) Place the tissue sections in a 60°C constant temperature oven for 1-2 h, and then soak them in xylene (twice, 10 min each), 100% alcohol (three times, 5 min each), 95% alcohol (twice, 5 min each), 90%, 85%, 80% alcohol (once, 5 min each), and 75% alcohol (once, 5 min).

[0161] (2) Wash with distilled water for 15 min, rinse twice with 1× PBS for 5 min each time, and then immerse in citric acid buffer for 3 min at high temperature and high pressure for antigen repair. Remove the tissue and allow it to cool naturally.

[0162] (3) Rinse twice with 1× PBS, 5 min each time, and then immerse the tissue in 3% H2O2 (methanol) solution and incubate at room temperature for 15 min.

[0163] (4) Rinse with distilled water, add 3% BSA to block at room temperature for 30 min, and rinse twice with 1× PBS, each time for 5 min.

[0164] (5) Add specific primary antibody to the tissue, incubate overnight at 4°C, and rinse twice with 1× PBS, each time for 5 min.

[0165] (6) Add secondary antibody and incubate at 37°C for 2 h. After rinsing with PBS, add the prepared DAB solution and react for 3-10 min (the reaction time is determined according to the depth of staining).

[0166] (7) Rinse the tissue with distilled water and counterstain with hematoxylin.

[0167] (8) Wash with distilled water for 15 min, place the tissue sections in a 1% hydrochloric acid-anhydrous ethanol solution to differentiate the stained hematoxylin for 15 s, and rinse the tissue with tap water to turn it blue again.

[0168] (9) Then, soak in 75% alcohol (once for 5 min), 80% and 85% alcohol (once for 1 min each), 90% and 95% alcohol (once for 3 min each), 100% alcohol (three times for 10 min each), and xylene (twice for 10 min each).

[0169] (10) Sealing: Add a drop of neutral resin to the tissue and then cover it with a coverslip. Observe the staining effect under a microscope and take photos.

[0170] 2. Experimental Results

[0171] 1. Inhibitory effect of lacidipine on IDO1

[0172] Figure 1 to Figure 3 The results showed that lacidipine could inhibit the IDO1 activity of breast cancer MDA-MB-231 and MCF-7 cells and cervical cancer Hela cells in a dose-dependent manner. 50 The values ​​were 12.4μM, 13.2μM and 18.9μM respectively; while lacidipine had no obvious cytotoxic effect on breast cancer cells and cervical cancer cells. This indicates that the inhibitory effect of lacidipine on IDO1 activity is not due to nonspecific cytotoxicity.

[0173] In addition, lacidipine significantly inhibited the protein expression of IDO1, IDO2 and TDO2 in breast cancer MDA-MB-231 and MCF-7 cells ( Figure 4 ) and inhibited the upregulation of IDO1 mRNA expression induced by IFNγ in MDA-MB-231 and MCF-7 cells ( Figure 5 ).

[0174] In order to further verify the inhibitory effect of lacidipine on the enzyme activities of IDO1, IDO2 and TDO2, the present invention overexpressed IDO1, IDO2 and TDO2 in HEK293A cells. The results showed that lacidipine inhibited the activities of IDO1 and IDO2 enzymes at the same time, but had no effect on the enzyme activity of TDO2 ( Figure 6 ).

[0175] 2. Effects of various 1,4-dihydropyridine antihypertensive drugs on IDO1 activity

[0176] Next, the present invention evaluated whether other 1,4-dihydropyridine antihypertensive drugs, isradipine, nifedipine and nitrendipine, have similar effects to lacidipine. The results showed that ( Figure 7 ), IC of isradipine, nifedipine and nitrendipine on the inhibition of L-kynurenine biosynthesis 50The values ​​were 23.23 μM, 27.23 μM and >80 μM, respectively, which were all higher than lacidipine. In addition, the inhibitory effect of nifedipine on IDO1 activity may be partly due to cytotoxicity. In HEK293A cells overexpressing IDO1 or IDO2, these drugs were able to inhibit the enzymatic activity of IDO1 and IDO2 ( Figure 8 ), except for nifedipine which had no inhibitory effect on TDO2 activity, isradipine and nitrendipine had weak inhibitory effects on TDO2 ( Fig. 9 In addition, isradipine can also inhibit the expression of IDO1 / IDO2 / TDO2 in MDA-MB-231 cells ( Fig.10 ), indicating that 1,4-dihydropyridine antihypertensive drugs have similar inhibitory effects on IDO, and lacidipine exhibits the strongest inhibitory activity. These results indicate that lacidipine can not only inhibit the expression of IDO1, but also directly target IDO1 to regulate IDO1 enzyme activity, and is a dual-functional IDO1 inhibitor.

[0177] 3. Lacidipine has potential anti-tumor effects in vivo

[0178] In order to evaluate the in vivo antitumor activity of lacidipine, the present invention injected 4T1-Luc cells into the fat pad of BALB / c mice to establish a mouse orthotopic breast cancer model. After 7 days, the mice were divided into 9 groups. The body weight of the mice was monitored every three days to evaluate the safety of the drug. Fig.11 As shown, lacidipine alone slightly reduced the weight of mice, while chemotherapeutic drugs doxorubicin and cisplatin alone significantly reduced the weight of mice. The weight of mice treated with lacidipine and IDO1 inhibitor 1-MT combined with doxorubicin and cisplatin, respectively, was consistent with that of mice treated with doxorubicin / cisplatin alone. This indicates that the systemic toxicity of doxorubicin / cisplatin combined with lacidipine and 1-MT is caused by the toxicity of doxorubicin / cisplatin, while lacidipine has less systemic toxicity to mice. The above results indicate that lacidipine has good safety in vivo.

[0179] In addition, lacidipine (10 or 20 mg / kg) can significantly inhibit the growth of tumors in mice, and combined with doxorubicin / cisplatin can play a synergistic role, further enhancing the inhibitory effect of these chemotherapy drugs on tumor growth. Its anti-tumor effect is significantly better than that of 1-MT combined with doxorubicin / cisplatin ( Fig.12 ). Among them, lacidipine combined with cisplatin almost completely inhibited tumor growth. Small animal in vivo imaging detected the luminescence intensity of tumor growth in mice carrying 4T1-Luc cells on days 7, 16, and 28 of modeling. The results showed that compared with the control group, the luminescence area of ​​each treatment group was significantly reduced, and the inhibitory effect of lacidipine combined with doxorubicin / cisplatin on the luminescence intensity of tumor growth was better than that of doxorubicin / cisplatin alone ( Fig.13). These results indicate that lacidipine alone can effectively inhibit tumor growth, and combined with the chemotherapy drugs doxorubicin and cisplatin, it can exert a synergistic effect and have a stronger anti-tumor effect.

[0180] 4. Effect of lacidipine on IDO activity in vivo

[0181] In order to verify the effect of lacidipine on IDO activity in mice, the ratio of kynurenine / tryptophan (Kyn / Try) in mouse serum was determined by high performance liquid chromatography (HPLC). The results showed that lacidipine alone significantly reduced the Kyn / Trp ratio in a dose-dependent manner. More importantly, the combined administration of lacidipine and doxorubicin / cisplatin further enhanced the inhibitory effect of doxorubicin / cisplatin alone on the conversion of tryptophan to kynurenine, exerting a synergistic effect ( Fig.14 ).

[0182] 5. Effect of lacidipine on tumor immune cell infiltration

[0183] To further study the effect of lacidipine on tumor immune cell infiltration, flow cytometry and immunofluorescence were used to detect the infiltration of immune cells in tumor tissues. The results showed that lacidipine increased the expression of CD45 + CD8 + Compared with the control group, lacidipine (20 mg / kg) combined with doxorubicin or cisplatin also promoted the expression of CD45 in tumor tissues. + CD8 + The proportion of effector T cells ( Fig.15 A). In addition, lacidipine further significantly inhibited tumor-infiltrating CD4 + CD25 + Foxp3 + However, compared with doxorubicin or cisplatin alone, lacidipine combined with doxorubicin or cisplatin had a significant effect on CD4 + CD25 + Foxp3 + There was no significant difference in the number of Trges cells ( Fig.15 B). Immunofluorescence analysis of tumor tissues also obtained consistent results ( Fig.16 ), compared with the control group, lacidipine alone or in combination with chemotherapy drugs could increase the expression of CD8 in tumor tissue cells and inhibit the expression of Foxp3, indicating that lacidipine alone or in combination with chemotherapy drugs could promote the infiltration of effector T cells and inhibit the infiltration of Tregs cells into tumor tissues.

[0184] In order to further verify that the regulatory effect of lacidipine on immune-infiltrating T cells in tumor tissue is caused by inhibiting IDO1, the expression of IDO1 protein in mouse breast tumor tissue was detected by immunohistochemistry (IHC). The results showed that lacidipine alone and in combination with doxorubicin or cisplatin could reduce the expression of IDO1, and its effect was better than that of 1-MT combined with chemotherapy drugs ( Fig.16 ). Next, the present invention detected the number of immune cells in the spleen of mice, and lacidipine was also able to increase the number of CD45 + CD8 + T cell population and suppress CD4 + CD25 + Foxp3 + Trges cells ( Fig.17 ). In order to evaluate the function of tumor tissue infiltrating effector T cells and Tregs cells, the present invention detected the levels of cytokines IFNγ, IL-10 and TGF-β in tumor tissue homogenate by ELISA. The results showed that lacidipine alone significantly increased IFNγ and reduced the content of IL-10 and TGF-β; combined with doxorubicin or cisplatin, it was also able to significantly increase IFNγ and reduce the level of TGF-β. Compared with the doxorubicin-treated group, the IFNγ level was significantly increased, and the IL-10 and TGF-β levels were significantly reduced when lacidipine was combined with it ( Fig.18 ).

[0185] The above results indicate that lacidipine can promote the infiltration of effector T cells in tumor tissues, improve their function, inactivate Tregs cells, thereby mediating anti-tumor immunity and further exerting tumor suppressive activity.

[0186] 6. Lacidipine has the effect of inhibiting breast cancer metastasis

[0187] The present invention studies whether lacidipine has a potential inhibitory effect on metastasis. Scratch and transwell migration experiments show that lacidipine alone or in combination with IFNγ inhibits the metastasis of MDA-MB-231 ( Fig.19 Transwell invasion assay showed that lacidipine alone or in combination with IFNγ could also significantly inhibit the migration of MDA-MB-231 ( Fig.19 C) Cell invasion.

[0188] 3. Conclusion:

[0189] (1) The present invention has found that lacidipine has a significant inhibitory effect on IDO, and it has an inhibitory effect on both IDO1 and IDO2 enzyme activities. Lacidipine is also a bifunctional IDO inhibitor that participates in IDO-mediated tumor immunity by inhibiting the expression and enzyme activity of IDO in tumors. Importantly, IDO1-mediated immunosuppression is not only related to its enzyme activity, but may also serve as a signaling molecule that maintains or mediates immune tolerance. Therefore, compared with IDO enzyme activity inhibitors, simultaneous inhibition of IDO1 expression and activity may have a dual anti-tumor effect. Compared with other similar 1,4-dihydropyridine antihypertensive drugs, the lacidipine of the present invention has the best inhibitory effect on IDO.

[0190] (2) The present invention has found that lacidipine has good safety in vivo and has a good effect in inhibiting tumor growth, and also has a good inhibitory effect on tumor metastasis in vitro.

[0191] (3) The present invention has found that the combination of lacidipine and other anti-tumor drugs can reduce the toxicity of other anti-tumor drugs and improve the safety of clinical use; moreover, it can exert a synergistic anti-tumor effect.

[0192] In summary, the present invention has found that lacidipine is a dual-function IDO inhibitor that can inhibit both the expression of IDO in tumors and the enzyme activity; lacidipine exerts a good anti-tumor effect through dual-function inhibition of IDO. Lacidipine can promote effector T cells, inhibit Tregs cell tumor infiltration and function, and inhibit further tumor metastasis. Lacidipine can also be used in combination with other anti-tumor drugs to exert a synergistic anti-tumor effect. The present invention also provides a new therapeutic strategy for the clinical application of antihypertensive drugs to enhance anti-tumor immune response.

Claims

1. Use of lacidipine in the preparation of an IDO inhibitor and / or a medicament for preventing and / or treating an IDO-related disease.

2. The use according to claim 1, characterized in that: The IDO inhibitor is an IDO1 inhibitor or an IDO2 inhibitor.

3. The use according to claim 1, characterized in that: The drug for preventing and / or treating diseases related to IDO is a drug for preventing and / or treating tumors.

4. The use according to claim 3, characterized in that: The drug is a drug for inhibiting tumor growth and / or inhibiting tumor metastasis.

5. The use according to claim 3 or 4, characterized in that: The tumor is breast cancer, cervical cancer, lung cancer, liver cancer, stomach cancer, and colorectal cancer.

6. Use of lacidipine in combination with chemotherapy drugs in the preparation of drugs for preventing and / or treating tumors; Preferably, the drug is a drug that inhibits tumor growth and / or tumor metastasis; More preferably, the tumor is breast cancer, cervical cancer, lung cancer, liver cancer, gastric cancer, or colorectal cancer.

7. The use according to claim 6, characterized in that: The chemotherapy drug is doxorubicin, cisplatin, cyclophosphamide, 5-fluorouracil, methotrexate, paclitaxel, vinblastine or rapamycin.

8. An IDO inhibitor and / or a pharmaceutical preparation for preventing and / or treating an IDO-related disease, characterized in that: The invention is prepared by taking lacidipine as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

9. A pharmaceutical composition for preventing and / or treating tumors, characterized in that: It includes lacidipine and chemotherapy drugs.

10. A pharmaceutical preparation for preventing and / or treating tumors, characterized in that: The drug is prepared by taking lacidipine and chemotherapy drugs as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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