CN112121152B

CN112121152B - Application of linatide in preparation of antitumor drugs

Info

Publication number: CN112121152B
Application number: CN202011028623.1A
Authority: CN
Inventors: 吴俊华; 张姗; 江春平; 刘淑雯; 马丁; 魏继武; 石维维; 王忠夏; 张广; 曹胤
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2021-06-22
Anticipated expiration: 2040-09-25
Also published as: CN112121152A

Abstract

The invention relates to the field of tumor treatment, in particular to application of linatide in preparation of antitumor drugs. The invention discloses the effect of the linatide on immune activation and the effect on anti-tumor immune activation; also discloses the function of the linatide in the aspect of anti-tumor immunotherapy. The results of the invention show that the linatide can significantly stimulate the proliferation of immune cells, and can significantly stimulate the release of interferon gamma (IFN-gamma); the linatide can obviously inhibit the growth of tumors and obviously prolong the life cycle of tumor-bearing mice on a plurality of in vivo animal tumor-bearing models; the linatide can obviously prolong the survival period of mice after metastatic recurrent tumor operations, and the safety of the linatide is basically ensured because the linatide is a drug on the market. In conclusion, the linatide can be used for preparing antitumor drugs.

Description

Application of linatide in preparation of antitumor drugs

Technical Field

The invention relates to the field of tumor treatment, in particular to an immune activation effect and an anti-tumor application of linatide.

Background

Tumors are a serious disease threatening the health of humans. The traditional schemes of surgery, radiotherapy, chemotherapy and the like inhibit the progress of tumors to a certain extent, but the relapse and the metastasis of the tumors are difficult to control. With the ever-rising exciting clinical findings, anti-tumor immunotherapy holds promise for tumor patients. The immune system has the capacity of identifying and eliminating heterosis, and tumors can inhibit the innate immune response of the immune system through various ways in the occurrence and development process, and can paralyze infiltrated immune effector cells by limiting the display of self novel antigens, starting immune check points and other methods, thereby achieving the purpose of avoiding immune identification and immune elimination. How to effectively activate anti-tumor immune response and relieve immune evasion so as to achieve the effect of immune elimination is always a goal pursued in the field of tumor immunotherapy.

Glucagon-like peptide-1 (GLP-1) is an endogenous polypeptide secreted by human body, and is a peptide hormone of 30 amino acids obtained by tissue-specific post-translational processing of glucagon gene. GLP-1 can stimulate insulin secretion by binding with its receptor GLP-1R, and has blood glucose lowering effect. GLP-1 can also produce weight loss effects through a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and feeding, delay of gastric emptying, and the like. In addition, GLP-1 acts on the central nervous system (especially the hypothalamus), thereby causing satiety and reducing appetite in humans. GLP-1 also has many physiological functions, such as lowering blood lipid, lowering blood pressure, protecting the cardiovascular system, enhancing learning and memory functions, protecting nerves, etc., by acting on the center. So far, no report of immune activation or anti-tumor immune activation exists.

GLP-1 has extremely short half-life of only 1.5-2 minutes, and cannot be clinically used for treating diabetes and other diseases. Therefore, the pharmaceutical field is trying to develop synthetic agonists of glucagon-like peptide-1 receptor (GLP-1R) with long half-life, and it is expected that GLP-1 similar activity and function will be obtained while prolonging half-life.

Linatide (rivastigmine) is a novel glucagon-like peptide-1 (GLP-1) receptor agonist. It is an artificially synthesized agonist of glucagon-like peptide-1 receptor (GLP-1R) developed by the company Sonofuran.

There has been no report of the use of risperidone for tumor therapy or anti-tumor, nor data or results of its immune activation or anti-tumor immune activation. When a polypeptide drug with an immune activation function is screened, the polypeptide drug is found to have an unexpected function, namely immune activation effect, so that the polypeptide drug is tried to be applied to anti-tumor immune activation and anti-tumor immunotherapy.

Disclosure of Invention

The invention aims to disclose the immune activation activity of the linatide and the application of the linatide in preparing an activated anti-tumor immune medicament through in vitro research; the immune activation effect of the linatide and the application of the linatide in preparing anti-tumor immunotherapy medicaments are confirmed through a plurality of in vivo animal models.

The invention confirms the application of the linatide in preparing the immune activation medicine in vitro. Furthermore, the invention confirms the application of the linatide in preparing anti-tumor immune activation medicaments on a plurality of in-vivo models, and confirms the application of the linatide in preparing anti-tumor immune treatment medicaments in vivo. At the same time, the basic safety of the linatide was also confirmed.

The invention is summarized as follows:

(1) the linatide can obviously stimulate the proliferation of immune cells in vitro and the release of interferon gamma (IFN-gamma), has definite immune activation effect, and discloses the application of the linatide in preparing immune activation medicaments.

(2) The linatide can remarkably stimulate the release of interferon gamma (IFN-gamma) in vivo, has definite immune activation effect, and discloses the application of the linatide in preparing anti-tumor immune activation medicaments.

(3) The linatide can obviously inhibit the growth of tumors in vivo, obviously prolong the life cycle of tumor-bearing mice, has definite anti-tumor immunotherapy effect, and discloses the application of the linatide in preparing anti-tumor immunotherapy medicaments.

(4) The linatide can obviously inhibit the growth of tumors and obviously prolong the survival period of tumor-bearing mice on a plurality of in vivo animal tumor models, wherein the tumors comprise: the breast cancer, the colon cancer and the melanoma have wide action range, have definite broad-spectrum anti-tumor immunotherapy action, and disclose the wide application value of the linatide in preparing the anti-tumor immunotherapy medicament.

(5) The linatide has the specific effects of inhibiting or delaying metastasis and recurrence in vivo and prolonging the survival period after tumor operation.

(6) Furthermore, it is important that the function of linatide is independent of its existing function, hypoglycemic, because it is tumor-bearing in immunocompromised, normoglycemic mice.

In general, the linatide can be used for preparing a medicine for stimulating immune cell proliferation, the linatide can be used for preparing a medicine for enhancing interferon gamma (IFN-gamma) expression, the linatide can be used for preparing an immune activation medicine, the linatide can be used for preparing an anti-tumor immunotherapy medicine, and the linatide can be used for preparing an anti-tumor medicine. The linatide can be used for preparing medicaments for inhibiting or delaying metastasis recurrence and prolonging the survival period after tumor operation. Moreover, because the tumor is carried on the mice with healthy immunity and normal blood sugar, the function of the linatide is not dependent on the existing function of reducing the blood sugar.

Drawings

FIG. 1 proliferation-stimulating effects of linatide on Peripheral Blood Mononuclear Cells (PBMCs)

FIG. 2 stimulation of IFN- γ secretion by Peripheral Blood Mononuclear Cells (PBMCs) by linatide

FIG. 3 anti-tumor immunotherapeutic Effect of linatide on mouse melanoma model (A) Elispot results (B) Change in tumor volume with time after different treatments (C) Change in survival in mice of different treatment groups

FIG. 4 anti-tumor immunotherapeutic Effect of linatide on mouse Breast cancer model (A) Elispot results (B) Change in tumor volume with time after different treatments (C) Change in survival in mice of different treatment groups

FIG. 5 anti-tumor immunotherapeutic Effect of linatide on mouse Colon cancer model (A) Elispot results (B) Change in tumor volume with time after different treatments (C) Change in survival of mice from different treatment groups

FIG. 6 survival Change in mice of different treatment groups post-operatively in mouse metastatic recurrent Breast cancer model

Detailed Description

Example 1 detection of immune activation function of polypeptide drug on peripheral blood mononuclear cells

First, experiment method

1. Determination of objects to be screened

We searched the drug library of polypeptides using drug bank to exclude FDA-approved polypeptides for infectious disease treatment, hematological disease treatment, tumor treatment and immunomodulation, leaving these polypeptides with approved functions unrelated to tumor treatment and immunomodulation. The functions are focused on the treatment of hormonal imbalance, the treatment of sexual dysfunction, the treatment of diabetes, the treatment of gastrointestinal digestive tract diseases, the treatment of genetic diseases, the treatment of cardiovascular diseases, the cure of bone diseases, the treatment of nervous system diseases, the treatment of respiratory disorders, the treatment of eye disorders and the treatment of malabsorption. This includes polypeptide drugs for the treatment of diabetes. In addition, the specific polypeptide drugs for diabetes treatment on the market in China were also searched. After summarizing FDA and chinese SFDA approved polypeptide drugs for diabetes treatment, we purchased these polypeptides for in vitro and in vivo activity detection and validation of the present invention by various routes. The immune activation function of the polypeptides is screened on peripheral blood mononuclear cells, and the screening finds that the peptide-risperidone for treating the diabetes has stronger immune activation function. The lisinopeptide was obtained from Xenoffy pharmaceutical Co, and was purchased from Nanjing pharmaceutical Co., Ltd. (production lot: 8F347A) and used as it was in a dose form or diluted with PBS.

2. Detection of proliferation-stimulating effect of linatide on Peripheral Blood Mononuclear Cells (PBMC)

Collecting peripheral blood of healthy volunteers under aseptic condition, anticoagulating with heparin, separating Peripheral Blood Mononuclear Cells (PBMC) with lymphocyte separation solution, suspending the cells in RPMI-1640 cell culture medium containing 10% fetal calf serum, and adjusting PBMC cell concentration to 3 × 10⁵Perml, PBMC were plated into 96 well cell plates at 200. mu.l per well, and linatide was added to a final concentration of 1. mu. mol/L and incubated with PBS as a control at 37 ℃ for 72h in 5% CO 2. After 72h, the proliferation of the cells was detected by using Biyun enhanced CCK-8 kit (cat No. C0042).

3. Detection of IFN-gamma stimulated secretion of Peripheral Blood Mononuclear Cells (PBMC) by linatide

Collecting peripheral blood of healthy volunteers under aseptic condition, anticoagulating with heparin, separating Peripheral Blood Mononuclear Cells (PBMC) with lymphocyte separation solution, suspending the cells in RPMI-1640 cell culture medium containing 10% fetal calf serum, and adjusting PBMC cell concentration to 3 × 10⁵Perml, PBMC were plated into 96 well cell plates at 200. mu.l per well, and linatide was added to a final concentration of 1. mu. mol/L and incubated with PBS as a control at 37 ℃ for 72h in 5% CO 2. After 72h, the supernatant was centrifuged and the cells were assayed for IFN-. gamma.stimulated secretion using an ELISA kit from Invitrogen (cat. No. 88-7316-76).

Second, experimental results

The linatide can remarkably stimulate the proliferation of Peripheral Blood Mononuclear Cells (PBMCs). The results in figure 1 show that the CCK-8 kit detects mean absorbance values a450 at 450nm of 0.4908 and 0.7568, respectively, of PBMCs after 72h incubation with PBS control and risperidone treatment, which is approximately 1.6-fold higher than the PBS control, and there is a significant difference between the two groups (P <0.05), and that risperidone is able to significantly stimulate PBMC proliferation. The results of this preliminary demonstration that the linatide has a certain immune activation effect are also quite unexpected, and suggest that the linatide may have potential therapeutic effects for anti-tumor or anti-virus.

The linatide can remarkably stimulate the secretion of IFN-gamma of Peripheral Blood Mononuclear Cells (PBMCs). The results in figure 2 show that the ELISA kit for IFN- γ detects the concentration of IFN- γ in the extracellular supernatant of PBMC after 72h of culture with PBS control and linatide treatments, and that the extracellular IFN- γ concentrations in PBS control and linatide treated groups were 72 and 115pg/ml, respectively, with significant difference between the two groups (P <0.05), indicating that linatide can significantly stimulate PBMC to secrete IFN- γ. The result confirms the previous proliferation stimulation experiment and also indicates that the linatide has a determined immune activation effect, and the result is also greatly unexpected, and the result suggests that the linatide has a potential therapeutic effect for resisting tumors or viruses.

Third, experiment summary

The above results of this example are very interesting, the first time it was detected that linatide had a definite immune activation in vitro, and since the cells were selected from peripheral blood PBMCs of normal healthy volunteers, the immune activation in vitro was not dependent on whether the PBMC source was a type 2 diabetic, i.e. the activity and function of linatide was not dependent on, nor independent of, its existing function. Based on the confirmation of definite immune activation, we will next test the anti-tumor immunotherapy effect on multiple tumor models, and the immune activation effect on in vivo animal models.

Example 2 Observation of the therapeutic Effect of Linasatide on Normal mouse melanoma model

First, experiment method

A normal healthy mouse C57BL/6 is selected, the mouse is immune-competent and is euglycemic, and mainly tumors (melanoma models) are wanted to be born on the immune-competent and euglycemic mouse, and then the treatment effect and the immune activation effect are observed. Because of the tumor bearing capacity of immunocompromised, normoglycemic mice, such function is not dependent on its existing function-hypoglycemic if the linatide is effective.

C57BL/6 mice 6-8 weeks old are selected, and a subcutaneous melanoma model is established in the right axilla. One side of each mouse was inoculated with 5X 10⁶A tumor size of about 200mm was measured after about 6 days for each B16/F10 melanoma cell³Mice were randomly divided into 2 groupsEach group of 12, respectively: PBS control group (100. mu.l PBS) and the risperidone subcutaneous injection treatment group (the dose of risperidone is 10. mu.g/kg, the injection volume is 100. mu.l) were injected 1 time per day subcutaneously. The day of initiation of the linatide treatment was recorded as day 0 of dosing, and tumor volumes and mice were weighed prior to linatide and PBS treatment on day 0 of dosing. According to the grouping, the inoculated mice are raised in a clean-grade animal room. In each group, 6 tumors were randomly selected on day 7 of administration, and tumor tissues were subjected to ELISpot assay using mouse Interferon gamma ELISPOT kit (cat # ab64029) from Abcam to detect IFN-. gamma.secretion in tumor tissues. The remaining 6 mice in each group were followed to measure tumor volume and body weight, and the survival time of the mice was recorded (day of survival was the day of tumor bearing). The tumor volume formula is V ═ 0.5 × Length × Width²。

Second, experimental results

IFN-gamma secretion was measured using the Interferon gamma ELISPOT kit after PBS control and rissin subcutaneous injection treatment in a normal mouse melanoma model, and the results in FIG. 3A show that IFN-gamma ELISPOT in PBS control and rissin treatment groups was 171 spots and 414 spots/2X 10⁵Cell, there was a significant difference between the two groups (P)<0.05), compared with the PBS control group, the risperidone treatment group is more than 2 times of the control group and can remarkably stimulate the secretion of IFN-gamma. The result not only sufficiently confirms the result of the in vitro immune activation of the linatide, but also can directly indicate that the linatide has a definite immune activation effect in vivo.

The results in fig. 3B show that on the normal mouse melanoma model, the PBS control and the risperidone treatment were injected subcutaneously, and the results at day 6 and day 9 after injection treatment showed that the risperidone-treated group was able to significantly inhibit the increase in tumor subvolume (P < 0.05). The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned lixisenatide, and can also directly indicate that the lixisenatide has a definite anti-tumor immunotherapy effect in vivo.

The results in fig. 3C show that in the normal mouse melanoma model, the PBS control group died beginning on day 27 after tumor loading and all died by day 34. However, mice died not beginning until day 31 in the rissin-treated group until all mice died at day 41. Subcutaneous injection of linatide can significantly prolong the survival of melanoma-bearing mice. The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned risperidone and the results of the obvious tumor growth inhibition, and can directly indicate that the risperidone has a definite anti-tumor immunotherapy effect in vivo.

Third, experiment summary

The in vivo result of a normal mouse melanoma model shows that the linatide has a definite immune activation effect and an anti-tumor immunotherapy effect, and the anti-tumor effect of the linatide is realized by activating immunity, namely the linatide has a remarkable effect of activating anti-tumor immunity. Moreover, because the tumor is carried on the mice with healthy immunity and normal blood sugar, the function of the linatide is not dependent on the existing function of reducing the blood sugar.

Example 3 observation of the therapeutic Effect of subcutaneous injection of Risinapine on a Normal mouse Breast cancer model

First, experiment method

The female mouse is a normal healthy BALB/c female mouse, the mouse is immune-competent and is euglycemic, the tumor-bearing mouse (breast cancer model) is wanted to be arranged on the immune-competent and euglycemic mouse, and then the treatment effect and the immune activation effect are observed. Because of the tumor bearing capacity of immunocompromised, normoglycemic mice, such function is not dependent on its existing function-hypoglycemic if the linatide is effective.

A BALB/c female mouse with the age of 6-8 weeks is selected to establish a subcutaneous breast cancer model at the right armpit fat pad. One side of each mouse was inoculated with 5X 10⁶Individual mouse 4T1 breast cancer cells, approximately 8 days later, were measured for tumor size to approximately 200mm³The mice were randomly divided into 2 groups of 12 mice each, which were: PBS control group (100. mu.l PBS) and the risperidone subcutaneous injection treatment group (the dose of risperidone is 10. mu.g/kg, the injection volume is 100. mu.l) were injected 1 time per day subcutaneously. The day of initiation of the risperidone treatment was recorded as day 0 of administrationTumor volumes, mice were weighed before risperidone and PBS treatment on day 0 of dosing. According to the grouping, the inoculated mice are raised in a clean-grade animal room. In which 6 tumors were randomly selected from each group on day 7, and tumor tissues were subjected to ELISpot assay using Abcam's mouse Interferon gamma ELISPOT kit (cat # ab64029) to detect IFN-. gamma.secretion in tumor tissues. The remaining 6 mice in each group were followed to measure tumor volume and body weight, and the survival time of the mice was recorded (day of survival was the day of tumor bearing). The tumor volume formula is V ═ 0.5 × Length × Width²。

Second, experimental results

The results in FIG. 4A show that IFN-. gamma.secretion was detected using the Interferon gamma ELISPOT kit after PBS control and rissin treatment by subcutaneous injection in a normal mouse breast cancer model, and that IFN-. gamma.ELISPOT was 181 and 314 spots/2X 10 in PBS control and rissin treatment groups, respectively⁵Cell, there was a significant difference between the two groups (P)<0.05), compared with the PBS control group, the risperidone-treated group is more than 1.7 times of the control group and can remarkably stimulate the secretion of IFN-gamma. The result confirms the result of the in vitro immune activation of the linatide and can directly indicate that the linatide has definite immune activation in vivo.

The results in fig. 4B show that the results at day 9, compared to the control group, after PBS control and risperidone subcutaneous injection treatment on a normal mouse breast cancer model, show that the risperidone-treated group was able to significantly inhibit the increase in tumor volume (P < 0.05). The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned lixisenatide, and can also directly indicate that the lixisenatide has a definite anti-tumor immunotherapy effect in vivo.

The results in figure 4C show that in the normal mouse breast cancer model, the PBS control group, mice died beginning on day 25 after tumor loading and all died by day 34. However, mice died not beginning until day 33 in the rissin-treated group until all mice died at day 49. Subcutaneous injection of linatide can significantly prolong the survival of melanoma-bearing mice. The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned risperidone and the results of the obvious tumor growth inhibition, and can directly indicate that the risperidone has a definite anti-tumor immunotherapy effect in vivo.

Third, experiment summary

The in vivo result of a normal mouse breast cancer model shows that the linatide has a definite immune activation effect and an anti-tumor immunotherapy effect, and the anti-tumor effect of the linatide is realized by activating immunity, namely the linatide has a remarkable effect of activating anti-tumor immunity. Moreover, because the tumor is carried on the mice with healthy immunity and normal blood sugar, the function of the linatide is not dependent on the existing function of reducing the blood sugar.

Example 4 Observation of the therapeutic Effect of Linfenacin on Normal mouse Colon cancer models

First, experiment method

The female mouse is a normal healthy BALB/c female mouse, the mouse is immune-competent and is euglycemic, the tumor-bearing mouse (colon cancer model) is wanted to be arranged on the immune-competent and euglycemic mouse, and then the treatment effect and the immune activation effect are observed. Because of the tumor bearing capacity of immunocompromised, normoglycemic mice, such function is not dependent on its existing function-hypoglycemic if the linatide is effective.

A BALB/c female mouse with the age of 6-8 weeks is selected to establish a subcutaneous colon cancer model at the right axillary fat pad. One side of each mouse was inoculated with 5X 10⁵Mouse CT26 colon cancer cells, tumor size was measured to about 200mm after about 10 days³The mice were randomly divided into 2 groups of 12 mice each, which were: PBS control group (100. mu.l PBS) and the risperidone subcutaneous injection treatment group (the dose of risperidone is 10. mu.g/kg, the injection volume is 100. mu.l) were injected 1 time per day subcutaneously. According to the grouping, the inoculated mice are raised in a clean-grade animal room. On day 7, 6 of each group were bled and tumor tissues were subjected to ELISpot assay using Abcam's mouse Interferon gamma ELISPOT kit (cat # ab64029) to detect IFN-. gamma.secretion in tumor tissues. The remaining 6 of each group were followed to measure tumorsVolume, body weight, mice survival recorded, day of starting treatment with linatide recorded as day 0, tumor volume measured before linatide and PBS treatment on day 0, and weighed. The tumor volume formula is V ═ 0.5 × Length × Width²。

Second, experimental results

The results in FIG. 5A show that IFN-. gamma.secretion was detected using the Interferon gamma ELISPOT kit after the PBS control and the rissin peptide were subcutaneously injected in the normal mouse colon cancer model, and that the IFN-. gamma.ELISPOT in the PBS control and the rissin peptide-treated groups was 197 and 305 spots/2X 10, respectively⁵Cell, there was a significant difference between the two groups (P)<0.05), compared with the PBS control group, the risperidone-treated group is more than 1.5 times of the control group and can remarkably stimulate the secretion of IFN-gamma. The result confirms the result of the in vitro immune activation of the linatide and can directly indicate that the linatide has definite immune activation in vivo.

The results in fig. 5B show that the results at day 6 show that the risperidone-treated group can inhibit the increase in tumor volume and that the tumor volume can be significantly inhibited at day 9 (P <0.05) compared to the control group after PBS control and risperidone subcutaneous injection treatment on a normal mouse colon cancer model. The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned lixisenatide, and can also directly indicate that the lixisenatide has a definite anti-tumor immunotherapy effect in vivo.

The results in figure 5C show that on the normal mouse colon cancer model, the PBS control group, mice died beginning on day 23 after tumor loading and all died by day 34. However, mice died not beginning until day 30 in the rissin-treated group until all mice died at day 42. Subcutaneous injection of linatide can significantly prolong the survival of colon cancer tumor-bearing mice. The results are consistent with the results of the in vitro and in vivo immune activation function of the aforementioned risperidone and the results of the obvious tumor growth inhibition, and can directly indicate that the risperidone has a definite anti-tumor immunotherapy effect in vivo.

Third, experiment summary

The in vivo result of a normal mouse colon cancer model shows that the linatide has definite immune activation effect and anti-tumor effect, and the anti-tumor effect of the linatide is realized by activated immunity, namely the linatide has obvious effect of activating anti-tumor immunity. Moreover, because the tumor is carried on the mice with healthy immunity and normal blood sugar, the function of the linatide is not dependent on the existing function of reducing the blood sugar.

Example 5 Observation of the Effect of linatide on postoperative survival in a model of metastatic recurrent Breast cancer in mice

First, experimental methods and materials

1. Experimental materials:

2. 4T1 cell culture

The culture medium of mouse breast cancer 4T1 cell is RPMI-1640 complete culture medium containing 10% fetal calf serum, 100U/ml penicillin and 100 μ g/ml streptomycin, and the cell is cultured at 37 deg.C and CO₂The culture was continued in a 5% strength incubator.

Human HCC cell lines BEL-7402, HuH-7, PLC/PRF/5 and SMMC-7721 were purchased from Shanghai national academy of sciences cell banks. Cell line MHCC97H was given by professor Jiafan, a secondary Zhongshan hospital, university of Compound Dan. The culture media of the five HCC cells were DMEM complete medium supplemented with 10% fetal bovine serum. The cells were continuously cultured in a constant temperature incubator at 37 ℃ and a CO2 concentration of 5%.

3. Mouse feeding

The experimental animals are: female SPF-grade BALB/c mice, 6 weeks old, weigh about 20 g. The animal is bred in an SPF animal house, the environment temperature is 22-25 ℃, the relative humidity is 40-60%, the day and night photoperiod is 12:12 hours, and the animal is bred by using autoclaved water and commercial mouse feed.

4. Construction and operation of mammary in-situ metastatic tumor model

Taking 4T1 cells in logarithmic growth phase, digesting with pancreatin, and preparing into cell suspension with concentration of 2 × 106/ml by using PBS; 0.1ml of 4T1 cell suspension is inoculated to the fourth mammary fat pad on the left side of the mouse to construct a mouse mammary orthotopic tumor model. After inoculation is finished, observing the state of the mouse, the infection of the inoculated part and the growth condition of the tumor day by day; the long diameter and the short diameter of the tumor are measured by a vernier caliper, the tumor volume is calculated according to the formula (length multiplied by width 2)/2 and recorded, and a curve is drawn. The mouse body weight was also weighed and recorded.

11 days after 4T1 cells are inoculated, the diameter of the breast in-situ tumor reaches about 5 mm. The mammary tumor tissue of the mice was completely excised under isoflurane gas anesthesia, the adherent mammary fat pad was removed, and the incised skin was sutured. After the surgery was completed, the state of the mice, whether the breast carcinoma had recurred, and the survival time of the mice were observed day by day.

The administration mode comprises the following steps: mice were randomly divided into 2 groups of 6 mice each, which were: PBS control group (100. mu.l PBS) and the risperidone subcutaneous injection treatment group (the dose of risperidone is 10. mu.g/kg, the injection volume is 100. mu.l) were injected 1 time per day subcutaneously. According to the grouping, the inoculated mice are raised in a clean-grade animal room. The first dose was given on day 9 (also 2 days prior to surgery) after tumor loading, followed by 1 subcutaneous injection per day for a total of 20 injections.

Second, experimental results

The results in fig. 6 show that breast cancer removal surgery was performed on day 11 after tumor bearing, followed by subcutaneous injections 1 per day for a total of 20 injections on day 9 after tumor bearing (also 2 days before surgery) on a mouse metastatic recurrent breast cancer model. In the PBS control group, mice died beginning on day 33 after tumor bearing and all mice died by day 44. However, mice died from the rissin-treated group until day 38, and all mice died until day 54. The subcutaneous injection of the risperidone can obviously prolong the postoperative life span of the metastatic recurrent breast cancer tumor-bearing mice.

Third, experiment summary

The result is consistent with the result of the in vitro and in vivo immune activation function of the risperidone and the result of the obvious tumor growth inhibition, and can directly indicate that the risperidone has a definite effect of prolonging the survival period of the metastatic recurrent tumor in vivo.

Summary of the detailed description section:

(1) the linatide can remarkably stimulate the proliferation of immune cells in vitro and remarkably stimulate the secretion and release of interferon gamma (IFN-gamma), has a definite immune activation effect, and discloses the application of the linatide in preparing immune activation medicaments.

(2) The linatide can remarkably stimulate the release of interferon gamma (IFN-gamma) in vivo, has definite immune activation effect, discloses the application of the linatide in preparing immune activation medicaments, and further discloses the application of the linatide in preparing anti-tumor immune activation medicaments.

(3) The linatide can obviously inhibit the growth of tumors in vivo and obviously prolong the life cycle of tumor-bearing mice, has definite anti-tumor immunotherapy effect and discloses the application of the linatide in preparing anti-tumor immunotherapy medicaments.

(5) It is important that the function of linatide is independent of its existing function, hypoglycemic, because it is tumor-bearing in immunocompromised, normoglycemic mice.

(6) The linatide has a definite function of prolonging the survival period of the metastatic recurrent tumor after operation in vivo.

In general, the linatide can be used for preparing a medicine for stimulating immune cell proliferation, the linatide can be used for preparing a medicine for enhancing interferon gamma (IFN-gamma) expression, the linatide can be used for preparing an immune activation medicine, the linatide can be used for preparing an anti-tumor immunotherapy medicine, the linatide can be used for preparing an anti-tumor medicine, and the linatide can be used for preparing a medicine for prolonging the survival period after metastatic recurrent tumor operation. Moreover, because the tumor is carried on the mice with healthy immunity and normal blood sugar, the function of the linatide is not dependent on the existing function of reducing the blood sugar.

Claims

1. The application of the linatide in preparing the anti-tumor medicament is characterized in that the tumor is melanoma.

2. The application of the linatide in the preparation of tumor immunotherapy medicaments is characterized in that the tumor is melanoma.