Application of Lanisimine or pharmaceutically acceptable salt thereof in preparation of medicine for resisting colon cancer

Abstract

The invention relates to the field of medical application, in particular to application of Lanisimine or pharmaceutically acceptable salt thereof in preparation of drugs for resisting colon cancer. Researches show that the medicine as the NMDAR antagonist has an anti-depression effect; the research finds that the medicine has the effect of resisting colon cancer. The medicine is beneficial to treatment and rehabilitation of colon cancer patients, and the medicine with the colon cancer resisting and depression resisting effects is successfully developed and used for treating the patients with colon cancer accompanied by depression.

Description

Technical Field

[0001] The present invention relates to the field of medical applications, and in particular to applications of lanisamine or a pharmaceutically acceptable salt thereof in the preparation of anti-colon cancer drugs. Background Art

[0002] Colon cancer is a common digestive tract malignancy located in the colon. According to the latest global data, the number of new cases and mortality rates of colorectal cancer in my country are rapidly increasing. By 2022, colorectal cancer had become the third leading cause of malignant tumor mortality, posing a serious threat to human health. Furthermore, many colorectal cancer patients often experience symptoms such as depression, anxiety, pain, and cognitive impairment, which not only exacerbate their suffering but also significantly impact treatment outcomes and prognosis, posing a significant concern. Statistics show that cancer patients with concomitant depressive symptoms have a 25% increased mortality rate, and the prevalence of depression in colorectal cancer patients ranges from 1.6% to 57%. A clinical study of 1,732 colorectal cancer patients showed that concomitant depression increased the risk of death by 16%. These data suggest that depression can promote tumor development and adversely affect prognosis and treatment. Emphasizing the improvement of depressive symptoms in colorectal cancer patients has positive implications for the effectiveness of colorectal cancer treatment.

[0003] Rac-Lanicemine is a low-capture NMDA receptor antagonist with antidepressant effects. Its use in the treatment of tumors has not been reported. Summary of the Invention

[0004] Therefore, the first object of the present invention is to provide a new use of lanisamine or a pharmaceutically acceptable salt thereof, namely, use of lanisamine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colon cancer.

[0005] For patients with colorectal cancer and depression, a multimodal treatment strategy is often adopted clinically, with radical tumor resection and systemic chemotherapy as the core of anti-tumor therapy, combined with psychotropic medications to improve depressive symptoms. Although combined medications can significantly improve patient compliance and effectively alleviate cancer-related symptoms (such as pain and fatigue), this strategy still has significant clinical limitations. In cancer treatment, traditional treatments (such as surgery, radiotherapy, and chemotherapy) are associated with systemic toxicity and acquired drug resistance. Regarding antidepressant therapy, although selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) have been shown in multiple studies to effectively improve depression scores in cancer patients, their combination with anti-tumor drugs may lead to adverse consequences such as reduced drug efficacy, additive drug toxicity, and reduced anti-tumor immune responses. Therefore, the search for a dual-action therapeutic agent with both anti-colorectal and anti-depressant effects is becoming increasingly important. On the one hand, it can prevent the occurrence of depression in the early stages of cancer diagnosis; on the other hand, while the drug exerts its anti-colon cancer effect, it can also play a role in preventing and treating depression, which will be beneficial to the treatment and recovery of colon cancer patients.

[0006] To this end, the second object of the present invention is to provide a new use of lanisamine or a pharmaceutically acceptable salt thereof, namely, the use of lanisamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating colon cancer combined with depression.

[0007] Furthermore, the drug is prepared according to conventional processes in the art with lanisamine or a pharmaceutically acceptable salt thereof as an active ingredient, with or without the addition of pharmaceutically acceptable excipients.

[0008] Furthermore, the pharmaceutically acceptable excipients include one or more of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, and preservatives.

[0009] Furthermore, the dosage form of the drug is a liquid preparation or a solid preparation.

[0010] Furthermore, the solid preparation includes powders, tablets, capsules, granules or pills; the liquid preparation includes injections, mixtures, syrups or suspensions.

[0011] Furthermore, the drug can prevent or treat colon cancer whose pathogenic mechanism is the immune function of macrophages.

[0012] The technical solution of the present invention has the following advantages:

[0013] This invention, for the first time, uses lanisamine or a pharmaceutically acceptable salt thereof for the treatment of colon cancer. By establishing a mouse model of colon cancer-associated depression, the anti-cancer effects of lanisamine in these mice were investigated by comparing tumor size and morphological characteristics across groups. Transcriptomics analysis of tumor tissues was also performed to analyze differential expression of tumor-related genes and explore its anti-cancer mechanism. Studies have shown that the drug not only exerts its anti-cancer effects but also prevents and treats depression, potentially benefiting the treatment and recovery of colon cancer patients. This demonstrates the successful development of a drug with both anti-cancer and anti-depressant properties for the treatment of patients with colon cancer-associated depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a schematic diagram of the construction and drug administration of the colon cancer-combined depression mouse model in Experimental Example 1. In the figure, "Tumorgrowth stage" is translated as the tumor growth stage, "Administration of R-Lan" is translated as the lanisamine treatment stage, and "sacrifice" is translated as the sacrifice of experimental animals.

[0016] Figure 2 These are the behavioral test results of mice after 14 days of chronic restraint stress (CRS);

[0017] Figure 3 These are the behavioral test results of mice after 28 days of chronic restraint stress (CRS);

[0018] Figure 4 The effect of lanisimine on open field test and tail suspension test in mice with colon cancer and depression (n=8-12, mean ± SD); Figure 2-4 (A) Number of entries into the central zone in the open field test; (B) Total movement distance (m); (C) Representative diagram of movement trajectory; (D) Immobility time (s) in the tail suspension test; Compared with the control group, * p<0.05, ** p<0.01, *** p<0.001; compared with group D, * p<0.05, ** p<0.01, *** p<0.001; compared with T group, *p<0.05; compared with T+D group, # p<0.05, ## p < 0.01;

[0019] Figure 5 Effects of lanisimine on body weight, tumor volume, and tumor weight in mice with colon cancer and depression (n=8, mean ± SD); (A) Body weight change; (B) Photographs of tumor tissue; (C) Tumor volume; (D) Tumor weight; Compared with the T+D group, ** P < 0.01, *** P < 0.001;

[0020] Figure 6 Representative images of H&E staining (Scale Bar = 50 μm, 20 μm);

[0021] Figure 7 Effects of lanisamine on the proliferation of MC38 cells (n=3; mean ± SD); (A) Representative images of Ki-67 staining (Scale Bar = 20 μm); (B) Statistical graph of the average optical density of Ki-67-positive cells; Compared with the T+D group, ** p<0.01, *** p < 0.001;

[0022] Figure 8 Effects of lanisimine on apoptosis of MC38 cells (n=3; mean ± SD); (A) Representative images of TUNEL staining (Scale Bar = 50 μm); (B) Statistical graph of the average optical density of TUNEL-positive cells; Compared with the T+D group, *** p < 0.001;

[0023] Figure 9 RNA sequencing results of tumor tissues in the TD group and TD group (H); (A) Volcano plot of differential gene expression; (B) KEGG pathway analysis diagram; (C) up-KEGG pathway analysis diagram;

[0024] Figure 10 To investigate the effect of lanisimine on six immune infiltrates (B cells, CD4 + T cells, CD8 + The effects of the expression of cytokines on T cells, neutrophils, macrophages, and dendritic cells were observed (n=4, mean ± SD); A: Gene list that met the screening criteria for differentially expressed genes; B: P values ​​of up-regulated genes (Dcn and Aspn) and macrophages (0.646 and 0.625); C: P value of down-regulated genes (Edil3) and macrophages (0.632);

[0025] Figure 11 Lanisimide has no direct killing effect on tumor cells (n=4, mean ± SD); (A) Detection of R-Lan on MC38 activity; (B) Detection of R-Lan on CT26 activity; Compared with the Control group, * p<0.05, ** p<0.01, *** p < 0.001;

[0026] Figure 12 Effects of lanisimine on tumor volume and weight after immune cell depletion (n=6, mean ± SD); A: Representative images of tumor tissue; B: Changes in tumor volume after NK cell depletion; C: Changes in tumor weight after CD8 + T cell-depleted tumor volume changes; D: Macrophage-depleted tumor volume changes; E: Tumor weight statistics; Compared with the control group, ** p<0.01, *** p<0.001; Day after inoculation is translated as the number of days after inoculation; Tumor volume is translated as tumor volume;

[0027] Figure 13 macrophages in the TME and tumor-infiltrating immune cells (CD8 + Expression of NMDAR1 on T and NK cells (n=3, mean ± SD); A: Expression of NMDAR1 on macrophages; B: Expression of CD8 + NMDAR1 expression on T cells; C: NMDAR1 expression on NK cells. DETAILED DESCRIPTION

[0028] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0029] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0030] The behavioral tests of this application are as follows:

[0031] The open field test can detect the spontaneous activity of animals and evaluate their learning and cognitive functions. The fewer times an animal enters the central area, the more avoidance behavior it exhibits; the shorter the total movement distance, the more it exhibits a lack of pleasure, despair, and a higher level of depression. During the test, mice were placed in the field for 2 hours to adapt before the experiment began. The test field was a 50×50×40cm 3 The experimental parameters of the Smart3.0 animal behavior video analysis and acquisition system were set up, and the central 15×15 cm test area was divided into 2 The central zone is the central area, and the remaining area is the peripheral zone. Data acquisition takes 5 minutes, and evaluation indicators are the number of entries into the central zone and the total distance moved. At the beginning of the experiment, each mouse was gently placed in the test box facing any corner and acclimated to the open field box group 1. Then, spontaneous activity indicators were recorded for 5 minutes.

[0032] Document 1: AN,AC,G R.Cytokines in breast cancer[J].Cytokine Growth FactorRev,2006,17(5);

[0033] Document 2: RESEARCH DOC,IMMUNOLOGY GSWFOLS,TEL-AVIV UNIVERSITY,ISRAEL.AABB@POST.TAU.AC.IL.Host microenvironment in breast cancerdevelopment:inflammatory cells,cytokines and chemokines in breast cancerprogression:reciprocal tumor-microenvironment interactions[J].Breast cancerresearch:BCR,2003,5(1):31-6.

[0034] Tail suspension test: After being suspended by the tail, mice attempt to escape but are unable to do so, giving up the struggle and entering a unique state of depression and immobility. The longer the immobility period, the higher the level of depression. During the test, the experiment was carried out during the dark period of the circadian rhythm cycle. During the entire experimental process, no light source or noise that might stimulate the mice was ensured in the room. The mice were placed in the test room for 2 hours before the experiment began. The experimental apparatus consisted of a plastic box (50 cm, 20 cm long, 20 cm wide) with a hook hanging in the middle of the box. At the beginning of the experiment, the skin 1 cm away from the tip of the mouse's tail was fixed with pressure-sensitive tape and hung on the hook in the middle of the beam. The mouse's head was facing downward, with a distance of approximately 5 cm between its head and the ground. There were no objects around the mouse for it to grab. The Smart 3.0 Animal Behavior Video Analysis and Acquisition System was used to record the mouse's behavior for 6 minutes, and the mouse's immobility time was recorded for the next 4 minutes.

[0035] Reference 1: DANTZER R. Cytokine, Sickness Behavior, and Depression[J]. Neurological Clinics, 2006, 24(3): 441-60.

[0036] Example 1

[0037] 1. Test drugs and reagents

[0038] Lanisamine (R-Lan) solution: Lanisamine was dissolved in DMSO to prepare a 100 mg / ml lanisamine stock solution, which was then diluted with normal saline to prepare 0.125 mg / ml, 0.5 mg / ml, and 2 mg / ml lanisamine solutions, respectively.

[0039] 5-Fu (5-fluorouracil) solution: Dissolve 5-Fu in normal saline to prepare a 5-Fu solution with a concentration of 2 mg / ml.

[0040] 2. Animal modeling and drug administration

[0041] (1) Construction of a mouse model of colon cancer with depression

[0042] Male C57BL / 6J mice (6-8 weeks old, 18-24 g) were randomly divided into two groups: normal control group (Control group) and depression model group (CRS group). Figure 1As shown in Figure 2, chronic restraint stress (CRS) was used to establish a depression model in mice. The specific modeling method was as follows: mice in the model group were restrained in a 50 mL ventilated centrifuge tube for 4 hours at random times each day, deprived of food and water during this period. After 14 days, behavioral tests were performed using the open field test (OFT) and the tail suspension test (TST).

[0043] The mice in the normal control group were further randomly divided into two groups: a normal control group (Control group) and a tumor model group (T group). The mice in the depression model group were also randomly divided into two groups: a chronic restraint stress model group (D group) and a tumor-depression model group (T+D group). Subsequently, MC38 colon cancer cells were inoculated subcutaneously in the axilla of mice in the T and T+D groups. The tumors were then inoculated subcutaneously (in the axilla). Well-grown MC38 colon cancer cells were digested and centrifuged, counted, and the cell suspension concentration adjusted to 0.5×10 6 0.1 mL of the drug was inoculated into the right axilla of CRS-depressed mice. During this period, mice in the CRS and T+D groups continued to receive CRS stimulation for a total of 28 days. The establishment of a mouse model of colon cancer with depression was verified through behavioral testing and tumor growth status.

[0044] The results are as follows Figure 2-3 As shown in the figure, mice underwent behavioral testing after 14 days of chronic restraint stress (CRS). Compared with the control group, the CRS group had no significant difference in the number of explorations of the central area in the open field test (P>0.05), but the immobility time in the tail suspension test was prolonged (P<0.01), indicating that the mice exhibited depressive-like behavior. MC38 colon cancer cells were inoculated, and behavioral tests were performed 14 days later. Compared with the Control group, the D group explored the central area less frequently in the open field test (P < 0.01), and the immobility time in the tail suspension test was prolonged (P < 0.01); compared with the D group, the T group and the T+D group explored the central area less frequently in the open field test (P < 0.05, P < 0.01), and the immobility time in the tail suspension test was prolonged (P < 0.01, P < 0.001); compared with the T group, the T+D group explored the central area less frequently in the open field test (P < 0.01), and the immobility time in the tail suspension test was prolonged (P < 0.05), indicating that the colon cancer-associated depression mouse model was successfully established.

[0045] (2) Grouped administration

[0046] When the tumor volume reaches about 80 mm 3 After 14 days, the tumor-depression model group (T+D group) was randomly divided into groups (n=8) and drug administration began. The group administration method was as follows:

[0047] ① Tumor depression model group (T+D group): intraperitoneal injection of normal saline once a day;

[0048] ② Tumor depression model + low-dose lanisamine group (T+DLR-Lan group, also called LR-Lan group): Lanisamine solution was intraperitoneally injected at a dose of 1.25 mg / kg;

[0049] ③ Tumor depression model + medium-dose lanisamine group (T+DMR-Lan group, also called MR-Lan group): Lanisamine solution was intraperitoneally injected at a dose of 5 mg / kg;

[0050] ④ Tumor depression model + high-dose lanisamine group (T+DHR-Lan group, also called HR-Lan group): Lanisamine solution was intraperitoneally injected at a dose of 20 mg / kg;

[0051] ⑤ Tumor depression model + 5-fluorouracil group (T+D-5-Fu group, also called 5-Fu group): 5-Fu solution was intraperitoneally injected at a dose of 20 mg / kg;

[0052] The five groups were all administered intraperitoneally once daily for 14 consecutive days, with a dosing volume of 10 mL / kg in the morning.

[0053] 3. Test methods

[0054] (1) Behavioral testing

[0055] After 14 days of administration, behavioral tests were performed using open field test and tail suspension test to evaluate the antidepressant effect of lanisimine.

[0056] (2) Evaluation of the anti-colon cancer effect of lanisimine

[0057] After 14 days of drug administration and behavioral testing, the tumor tissue was removed and weighed, and the tumor tissue samples were analyzed from a morphological perspective. The specific experimental methods are as follows:

[0058] ① General status observation: During the experiment, the hair condition and activity behavior of the mice were recorded, and the body weight, tumor volume, and tumor weight changes were measured regularly.

[0059] ②H&E staining analysis: Hematoxylin-eosin (H&E) staining was used to observe the number of tumor cells, staining depth, changes in nuclear-cytoplasmic ratio, and nuclear division in tumor tissues.

[0060] ③ Immunohistochemistry: Immunohistochemistry is used to detect the expression level of the proliferation marker Ki67 in tumor tissue to evaluate the proliferation activity of tumor cells.

[0061] ④TUNEL staining analysis: TUNEL staining was used to detect cell apoptosis in tumor tissues to evaluate the apoptosis of tumor cells.

[0062] (3) Study on the mechanism of action of lanisimine against colon cancer

[0063] RNA sequencing analysis: RNA sequencing (RNA-Seq) was used to compare differential gene expression profiles in colon cancer tumor tissues between the T+D group and the T+DHR-Lan group. The anti-colon cancer effects of lanisamine were further analyzed using the TIMER database in the context of colon cancer.

[0064] 4. Experimental results

[0065] (1) Lanisimine has an antidepressant effect on colon cancer-associated depression model mice

[0066] See Figure 4 As shown in the results, compared with the D group, the T+D+HR-Lan (20 mg / kg) group had no significant difference in the number of explorations of the central area in the open field test (P>0.05), and the immobility time in the tail suspension test was prolonged (P<0.05). Compared with the T+D group, the T+D+HR-Lan (20 mg / kg) group had an increased number of explorations of the central area in the open field test (P<0.01), and a decreased immobility time in the tail suspension test (P<0.05), indicating that R-Lan can alleviate the depressive-like behavior of mice with colon cancer and depression.

[0067] (2) Lanisimine has an anti-colon cancer effect in colon cancer-associated depression model mice

[0068] During the experiment, the mice in the Control group were in good condition, with a steady increase in weight. The mice in the T group were in good condition in the early stages of the experiment, but as the tumor volume increased, they later developed weight fluctuations, decreased activity, sluggish reactions, and matted hair. Due to the stimulation of chronic restraint stress, the T+D group later exhibited a similar condition to the T group, accompanied by an increase in tumor volume. In contrast, after treatment with medium and high doses of R-Lan (5 mg / kg and 20 mg / kg), the mice showed a more stable weight gain trend, and their overall condition was significantly improved compared to the T and T+D groups.

[0069] like Figure 5As shown, compared with the T group, the tumor volume in the T+D group was significantly increased (P<0.001). Compared with the T+D group, treatment with medium and high doses of R-Lan (5 mg / kg and 20 mg / kg) significantly reduced tumor volume and weight (P<0.01, P<0.001). Treatment with the positive drug 5-Fu (20 mg / kg) also significantly reduced tumor volume and weight (P<0.01, P<0.001). These results indicate that R-Lan has an inhibitory effect on colon cancer growth.

[0070] (3) Effects of Ranisimide on MC38 cells in colon cancer tumor tissue

[0071] like Figure 6 As shown, in the T and T+D groups, tumor cells showed diffuse growth, dense arrangement, and a large number. Their nuclei were darkly stained, with a large nuclear-to-cytoplasmic ratio, abundant cytoplasm, prominent nucleoli, and typical pathological mitotic activity. Treatment with medium and high doses of R-Lan (5 mg / kg and 20 mg / kg) and 5-Fu (20 mg / kg) reduced the number of tumor cells, lightened the staining, decreased the nuclear-to-cytoplasmic ratio, and improved mitotic activity. These results demonstrate that R-Lan can inhibit the growth of MC38 cells.

[0072] (4) Effect of 1.5-lanisamine on the proliferation level of MC38 cells in colon cancer tumor tissue

[0073] like Figure 7 As shown in the data, compared with the T+D group, the number of Ki67-positive cells was significantly reduced after treatment with low, medium and high doses of R-Lan (1.25 mg / kg, 5 mg / kg, 20 mg / kg) and 5-Fu (20 mg / kg) (P < 0.05, P < 0.01, P < 0.001 and P < 0.001), indicating that R-Lan can inhibit the proliferation of MC38 cells.

[0074] (5) Effect of Ranisimide on the apoptosis level of MC38 cells in colon cancer tumor tissue

[0075] like Figure 8 As shown in the figure, compared with the T+D group, the number of TUNEL-positive cells was significantly reduced after treatment with low, medium and high R-Lan (1.25 mg / kg, 5 mg / kg, 20 mg / kg) and 5-Fu (20 mg / kg) (P < 0.001), indicating that R-Lan can promote the apoptosis of MC38 cells.

[0076] (6) RNA sequencing results of colon cancer tumor tissue

[0077] The sequencing results are as follows Figure 9Compared with the T+D group, a total of 417 genes were significantly up-regulated and 387 genes were significantly down-regulated in the T+DHR-Lan (20 mg / kg) group, and the differential gene expression was significant (pvalue<0.05). KEGG pathway analysis ( Figure 9 In panels B, C, and D, differentially expressed genes were significantly enriched in multiple immune and inflammation-related pathways, including the NOD-like receptor signaling pathway, the Jak-STAT signaling pathway, the cytokine-cytokine receptor interaction pathway, and the C-type lectin receptor signaling pathway. This suggests that the anti-colon cancer effect of R-Lan may be related to the regulation of immune cells.

[0078] (7) TIMER data estimation of immune cell infiltration in colon cancer tumor tissue

[0079] Differential genes that meet the screening conditions (log2 Fold Change>2, p value<0.05) (such as Figure 10 Compared with the T+D group, the up-regulated genes Dcn (p=0.646) and Aspn (p=0.625) and the down-regulated gene Edil3 (p=0.632) in the T+DHR-Lan (20 mg / kg) group were most correlated with macrophages (Fig. Figure 10 (As shown in B and C in Figure 5 ). This indicates that the anti-colon cancer effect of R-Lan may be related to the regulation of macrophages.

[0080] Example 2

[0081] The CCK-8 assay was used to detect the effect of lanisimine on the viability of MC38 and CT26 colon cancer cells to evaluate its direct inhibitory effect on colon cancer cells.

[0082] 1. Drugs and reagents

[0083] 1 mg of lanisamine (purchased from MCE, product number HY-108235B, 20 mg) was added to DMSO to prepare a 1 mM lanisamine stock solution. The lanisamine stock solution was then diluted with physiological saline to obtain lanisamine solutions with concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μM, respectively. Physiological saline was used as the 0 μM lanisamine solution.

[0084] The complete culture medium was prepared as follows: 5 mL of fetal bovine serum (FBS), 500 μL of penicillin-streptomycin solution (double antibody, 100×), 500 μL of HEPES buffer (1 M, pH 7.3), 500 μL of non-essential amino acid solution (NEAA, 100×), and 43.5 mL of DMEM medium were added to a 50 mL sterile centrifuge tube, mixed thoroughly, and stored in a 4°C refrigerator for later use.

[0085] 2. Cell culture

[0086] MC38 and CT26 colon cancer cells were used as the study subjects. A water bath was set to 37°C. Cryopreserved tubes of different cell lines were removed from the liquid nitrogen tank and quickly placed in a 37°C water bath until the culture medium dissolved. The tubes were transferred to 15ml centrifuge tubes, 5ml of fresh complete culture medium was added, pipetted to mix thoroughly, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, complete culture medium was added, and the tubes were gently pipetted. The tubes were transferred to cell culture flasks and incubated in a 37°C, 5% CO2 incubator. The cells were expanded and subcultured until the cell count reached approximately 90%. The tubes were trypsinized with 0.25% EDTA and expanded as needed.

[0087] MC38 colon cancer cell inoculation and administration: take the above MC38 colon cancer cells in the logarithmic growth phase and 3 / 100 μL was inoculated in a 96-well plate, and four groups of experiments were set up for each species of cells. The dosing scheme was as follows: Group A: blank culture medium group without cells; Group B: lanisamine treatment group (administered with lanisamine solution at concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg / mL respectively); Group C: negative control group (cell suspension not treated with drug); 6 replicate wells were set up in each group.

[0088] Inoculation and administration of CT26 colon cancer cells: Take the above CT26 colon cancer cells in the logarithmic growth phase and 3 / 100 μL was inoculated in a 96-well plate, and four groups of experiments were set up for each species of cells. The dosing scheme was as follows: Group A: blank culture medium group without cells; Group B: lanisamine treatment group (administered with lanisamine solution at concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg / mL respectively); Group C: negative control group (cell suspension not treated with drug); 6 replicate wells were set up in each group.

[0089] 3. Experimental results

[0090] The results are as follows Figure 11As shown in the figure, when the drug concentration of lanisamine is 64 μM, it has a certain inhibitory effect on MC38 and CT26 cells, indicating that R-Lan has no significant direct killing effect on colon cancer cells.

[0091] Example 3 Immune cell depletion experiment

[0092] 1. Test drugs and reagents

[0093] Lanisamine solution: Lanisamine was dissolved in DMSO to prepare a 100 mg / ml lanisamine stock solution, which was then diluted with physiological saline to prepare a 2 mg / ml lanisamine solution.

[0094] 2. Animal grouping, modeling and drug administration

[0095] 60 male C57BL / 6J mice, 6-8 weeks old, weighing 18-24 g, were first implanted with tumors and grouped, then divided into groups and given drug treatments.

[0096] (1) Tumor implantation and grouping: Normal C57BL / 6J mice were shaved and disinfected under the right armpit. MC38 colon cancer cells with good growth status were digested and centrifuged, and the cell suspension concentration was adjusted to 1.0×10 6 / mL, 0.1mL was inoculated into the right armpit of mice; the mice were divided into four groups, 12 mice in each group:

[0097] ①NK cell depletion group: NK cells in mice were depleted by intraperitoneal injection of Anti-NK-1.1 antibody (100 μg / mouse) on days 1, 3, and 7, respectively;

[0098] ②Depletion of CD8 + T cells (CD8 + T depletion) group: CD8 in mice was depleted by intraperitoneal injection of anti-CD8β antibody (100 μg / mouse) on days 1, 3, and 7, respectively. + T cells;

[0099] ③ Macrophage depletion group (also called clodronate group): Macrophages were eliminated from mice by intraperitoneal injection of clodronate disodium liposomes (1 mg / mouse) on days 1, 5, and 10, respectively;

[0100] ④Liposome control group (liposome): Ordinary blank liposomes (1 mg / mouse) were injected intraperitoneally on days 1, 5, and 10 as a control;

[0101] (2) Regrouping and drug administration: When the tumor grows to 80 (mm 3) After that, each group was divided into two groups and administered with drugs (n=6). The NK cell depletion group was randomly divided into NK depletion-Control group and NK depletion-R-Lan group. + The T cell group was randomly divided into CD8 + T depletion-Control group and CD8 + The depletion-R-Lan group, the depleted macrophage group was randomly divided into the clodronate-Control group and the clodronate-R-Lan group, and the liposome control group was randomly divided into the liposome-Control group and the liposome-R-Lan group, and the drugs were administered according to the following method.

[0102] ①NK depletion-Control group: intraperitoneal injection of normal saline, once a day;

[0103] ②NK depletion-R-Lan group: 20 mg / kg of Lanisamine solution (concentration 2 mg / ml) was intraperitoneally injected;

[0104] ③CD8 + T depletion-Control group: intraperitoneal injection of normal saline, once a day;

[0105] ④CD8 + T depletion-R-Lan group: lanisamine solution 20 mg / kg (concentration 2 mg / ml) was intraperitoneally injected;

[0106] ⑤Clodronate-Control group: intraperitoneal injection of normal saline, once a day;

[0107] ⑥Clodronate-R-Lan group: lanisamine solution 20 mg / kg (concentration 2 mg / ml) was intraperitoneally injected;

[0108] ⑦Liposome-Control group: intraperitoneal injection of normal saline, once a day;

[0109] ⑧liposome-R-Lan group: lanisamine solution 20 mg / kg (concentration 2 mg / ml) was intraperitoneally injected;

[0110] All eight groups received a single intraperitoneal injection of 10 mL / kg daily for seven consecutive days. During the treatment period, the mice were observed daily for activity, weight changes were recorded, and tumor volume changes in tumor-bearing mice were measured with a vernier caliper. Seven days after dosing, tumor tissue was removed from the mice and photographed.

[0111] 3. Detection indicators

[0112] (1) The status of the mice was observed during the experiment, and the changes in body weight and tumor volume were recorded regularly. After 14 days of tumor growth, the tumor tissue was removed and weighed to evaluate the anti-colon cancer effect of lanisimine under different immune cell depletion conditions.

[0113] (2) The colon cancer tumor tissues from the liposome-R-Lan group mice were prepared into tumor cell suspensions using the following method, and the CD8 + The expression levels of N-methyl-D-aspartate receptor (NMDAR) on T cells, NK cells and tumor-associated macrophages (TAMs) were investigated to explore the regulation of specific immune cells by the anti-colon cancer effect of lanisimine.

[0114] Preparation of tumor cell suspension (clean bench operation):

[0115] 1) Place the excised tumor tissue in sterile culture medium (process as quickly as possible to avoid cell death);

[0116] 2) Wash the tumor tissue with PBS, remove blood and necrotic tissue, and cut the tissue into 1-2 mm 3 small pieces;

[0117] 3) Place the tissue block in the digestion solution and digest in a 37°C incubator for 1 hour. Gently shake or pipette to promote digestion.

[0118] 4) After digestion, serum-containing culture medium (DMEM containing 5% FBS) was added to terminate the reaction;

[0119] 5) Pipette repeatedly to disperse the tissue fragments into a single-cell suspension. Filter the suspension through a 70 μm cell sieve to remove undigested tissue fragments and cell clumps.

[0120] 6) Centrifuge the filtered cell suspension at 1000 rpm for 5 minutes;

[0121] 7) Discard the supernatant and resuspend the cells in PBS to obtain a tumor cell suspension.

[0122] 4. Experimental results

[0123] (1) Effects of lanisimine on tumor volume and weight after immune cell depletion

[0124] The results are as follows Figure 12 As shown, compared with the Control group, the depletion of NK cells and CD8 +After depletion of T cells and macrophages, there was no significant difference in tumor volume after treatment with R-Lan (20 mg / kg, concentration 2 mg / ml) (P>0.05). Compared with the case of macrophage depletion, treatment with R-Lan (20 mg / kg, concentration 2 mg / ml) without macrophage depletion significantly reduced tumor volume (P<0.01) and tumor weight (P<0.001).

[0125] (2) Macrophages and tumor-infiltrating immune subsets in the TME (CD8 + Expression of NMDAR1 on T cells, NK cells

[0126] like Figure 13 As shown in Figure 2, NMDAR1 was highly expressed on TAMs, but not on CD8 + There is almost no NMDAR1 expression on T and NK cells.

[0127] In summary, the anti-colon cancer effect of R-Lan was significantly weakened in macrophage-depleted mice. Among the immune cell subsets infiltrating TME, only TAMs expressed NMDAR, indicating that R-Lan exerted its anti-colon cancer effect through macrophages, and CD8 + T cells and NK cells can synergistically participate in its anti-colon cancer effect.

[0128] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of lanisamine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colon cancer.

2. Use of lanisamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating colon cancer complicated with depression.

3. The use according to claim 1 or 2, characterized in that The drug uses lanisamine or a pharmaceutically acceptable salt thereof as an active ingredient, with or without the addition of pharmaceutically acceptable excipients, and is prepared according to conventional processes in the art.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable excipients include one or more of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, and preservatives.

5. The use according to claim 1 or 2, characterized in that The dosage form of the drug is a liquid preparation or a solid preparation.

6. The use according to claim 5, characterized in that The solid preparations include powders, tablets, capsules, granules or pills; the liquid preparations include injections, mixtures, syrups or suspensions.

7. The use according to claim 1 or 2, characterized in that The medicine can prevent or treat colon cancer whose pathogenic mechanism is the immune effect of macrophages.

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