CN117899094A

CN117899094A - Application of acarbose combined with PD-1 monoclonal antibody in cancer treatment

Info

Publication number: CN117899094A
Application number: CN202410058677.4A
Authority: CN
Inventors: 张世龙; 王志明; 王妍; 张争艳
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2024-01-15
Filing date: 2024-01-15
Publication date: 2024-04-19

Abstract

The invention discloses an application of acarbose combined with PD-1 monoclonal antibody in cancer treatment. The invention verifies that acarbose combined with PD-1 monoclonal antibody has the effect of promoting the immunotherapy of malignant solid tumors in a tumor mouse model by establishing various tumor mouse models including melanoma, colon cancer and AOM/DSS induced colon adenocarcinoma mouse models: acarbose can increase the proportion of CD8 ⁺ T cells in the tumor microenvironment, and activate the anti-tumor immune effect of CD8 ⁺ T cells; can regulate intestinal flora, reduce harmful bacteria, increase beneficial bacteria, and further enhance the curative effect of PD-1 monoclonal antibody; in mice, the use of PD-1 mab alone inhibited tumor cell growth compared to control or single drug groups, but the combination of acarbose and PD-1 mab significantly improved the effect of PD-1 mab treatment. The invention provides a new thought for improving the curative effect of PD-1 monoclonal antibody, and has important value and good clinical application prospect.

Description

Application of acarbose combined with PD-1 monoclonal antibody in cancer treatment

Technical Field

The invention relates to an application of acarbose combined with PD-1 monoclonal antibody in cancer treatment, belonging to the technical field of biological medicine.

Background

Tumor immunotherapy is one of the important breakthroughs in the field of tumor therapy in recent years, and has received a great deal of attention with its unique therapeutic mechanism. Monoclonal antibody (PD-1 monoclonal antibody) of the apoptosis protein 1 is used as an important immune checkpoint inhibitor, and can activate the immune system of a patient to attack tumor cells by blocking the combination between PD-1 and a ligand PD-L1 thereof, thereby achieving remarkable curative effect. However, there are significant differences in the efficacy of PD-1 mab from individual to individual, and a fraction of patients exhibit resistance. This suggests that the effect of immunotherapy is affected not only by the characteristics of the tumor itself, but also by the host immune status and the immune microenvironment. In recent years, intestinal flora has attracted considerable interest to researchers as an important component of the immune microenvironment. The intestinal flora can influence the immune response of a host, regulate the activity of immune cells and further influence the occurrence, development and therapeutic effect of tumors. The intestinal flora is involved in many basic physiological activities of the human body, such as promoting the developmental maturation of the intestinal tract, promoting the digestion and absorption of food, synthesizing vitamins, resisting the invasion of pathogens, maintaining the intestinal barrier, maintaining the homeostasis of the immune system, etc. Changes in intestinal flora are closely related to the occurrence and development of tumors. For example, tumor patients have reduced flora diversity and markedly disturbed composition compared to healthy persons. Recent researches find that the intestinal flora disturbance of tumor patients is one of the important reasons for the treatment resistance of PD-1 monoclonal antibodies, and a new idea is provided for overcoming the treatment of cancers by the PD-1 monoclonal antibodies.

Acarbose has also been found in recent years to have an effect of modulating the immune system and intestinal flora as a commonly used antidiabetic agent. The discovery not only expands the application field of acarbose, but also provides an example for fully exerting the versatility of the medicine. First, with the intensive research, scientists find that acarbose is not only a hypoglycemic drug, but also has a regulating effect on the immune system, can enhance the activity of immune cells and improve immune response. The discovery lays a foundation for the application of acarbose in tumor immunotherapy. Secondly, the effect of acarbose on the intestinal flora is also of increasing concern. Acarbose can influence the metabolic activity and metabolic products of the flora by adjusting the structure and abundance of the flora in the intestinal tract, thereby changing the environment in the intestinal tract and further regulating the functions of the immune system. The indirect immunoregulation effect provides a theoretical basis for potential application of acarbose in tumor treatment. In addition, the low cost, safety and good tolerability of acarbose also make it a widely used drug, further increasing its attractiveness in tumor therapy. In the field of immunotherapy, low-cost drugs may greatly reduce the treatment cost of patients, and promote popularization of immunotherapy.

In combination, acarbose has been shown to have various potential applications as a commonly used antidiabetic agent, particularly in the modulation of the immune system and in the modulation of the intestinal flora. However, no research on the immunotherapeutic effect and application value of acarbose on tumors is currently seen, and the immunotherapeutic effect of acarbose on tumor models and the regulatory effect of acarbose on intestinal flora of tumor models after oral administration are not clear.

Disclosure of Invention

The purpose of the invention is that: aiming at the problems of poor curative effect, drug resistance and the like of PD-1 monoclonal antibodies in cancer treatment, the invention provides the application of combined acarbose and PD-1 monoclonal antibodies in cancer treatment, and the invention takes various mouse tumor models as research objects and adopts PD-1 monoclonal antibodies for treatment; meanwhile, through the intervention of acarbose, the acarbose is found to have sensitization effect on the PD-1 monoclonal antibody treatment of a tumor model, and the treatment effect of the PD-1 monoclonal antibody can be improved.

In order to achieve the aim, the invention provides application of acarbose combined immune checkpoint inhibitor in preparing anti-tumor drugs.

Preferably, the tumor is a malignant solid tumor that is resistant to immunotherapy.

Preferably, the tumor comprises melanoma, colorectal cancer or colon adenocarcinoma.

Preferably, the immune checkpoint inhibitor is at least one of PD-1 monoclonal antibody, PD-L1 monoclonal antibody and CTLA4 monoclonal antibody.

Preferably, the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is acarbose and PD-1 monoclonal antibody.

Preferably, the dosage form of the medicament is a tablet, a capsule, a powder, a granule or an oral liquid preparation.

Compared with the prior art, the invention has the beneficial effects that:

The result of the invention shows that acarbose has promotion effect on PD-1 monoclonal antibody treatment effect in a mouse tumor model, PD-1 monoclonal antibody is an important means of tumor immunotherapy, but the treatment effect has individual difference, acarbose can increase CD8 ⁺ T cell proportion, and activate CD8 ⁺ T cell anti-tumor immune effect; the invention can regulate intestinal flora, reduce harmful bacteria, increase beneficial bacteria and further enhance the curative effect of PD-1 monoclonal antibody, thus providing a new thought for improving the curative effect of PD-1 monoclonal antibody; the invention combines the new application of acarbose with tumor immunotherapy, fully plays the potential of new application of old drugs, and expands a new way for clinical application of acarbose.

Drawings

Figure 1 demonstrates the effect of acarbose to increase the efficacy of PD-1 mab in the treatment of melanoma models: A. tumor growth curves in isotype control, PD-1 mab group, acarbose group, PD-1 mab+acarbose group; B. tumor photographs of each group of mice at the end of the experiment;

Figure 2 shows the effect of acarbose on increasing PD-1 mab in the treatment of colon cancer model: A. tumor growth curves in isotype control, PD-1 mab group, acarbose group, PD-1 mab+acarbose group; B. tumor weight of each group of tumor-bearing mice at the end of the experiment; C. tumor photographs of each group of mice at the end of the experiment; D. weight change of each group of mice during the experiment;

FIG. 3 shows the effect of acarbose on increasing PD-1 mab in the treatment of AOM/DSS induced colon adenocarcinoma model: A. number of tumors in isotype control, PD-1 mab group, acarbose group, PD-1 mab+acarbose group; B. at the end of the experiment, tumor pictures of each group of tumor-bearing mice; C. h & E staining pictures of tumor tissue of each group of mice at the end of the experiment;

Figure 4 demonstrates that acarbose can significantly improve tumor immune microenvironment, increasing T cell infiltration in tumor microenvironment: kegg enrichment analysis suggested a pathway for PD-1 mab + acarbose group enrichment; GSEA enrichment analysis suggests T cell activation channels in PD-1 monoclonal antibody+acarbose group are enriched; gsea enrichment analysis suggested positive regulation of chemokine production pathway enrichment in PD-1 mab + acarbose group; the relative abundance of 25 immune cells in PD-1 mab group and PD-1 mab+acarbose group tumors, the results are shown in bar graphs; comparison of the relative abundance of different types of immune cells between the PD-1 mab group and the PD-1 mab+acarbose group;

Figure 5 shows that acarbose can promote CXCL10 secretion and thereby enhance CD8 ⁺ T cell infiltration: A. immunohistochemical staining of CD8 ⁺ T cells and CXCL10 in tumor tissue of each group of mice at the end of the experiment; B. at the end of the experiment, immunohistochemical staining quantitative analysis of CD8 ⁺ T cells and CXCL10 in tumor tissues of each group of mice; CXCl10 expression and the Spearman correlation of immunohistochemical scoring of CD8 ⁺ T cells; E. ELISA detection results of CXCL10 levels in peripheral serum of various groups (n=4);

Figure 6 shows the intestinal flora richness and diversity of acarbose-altered tumor-bearing mice: A. PCoA analysis based on Bray-Curtis dissimilarity coefficient evaluates the beta diversity of intestinal flora species between the PD-1 mab group and the PD-1 mab+acarbose group; B. comparing the α diversity of the PD-1 mab group and the PD-1 mab+acarbose group, e.g., shannon index;

Figure 7 shows the effect of acarbose on intestinal flora composition: the composition ratio of the PD-1 monoclonal antibody group to the PD-1 monoclonal antibody+acarbose group intestinal flora at the portal level; the composition ratio of the PD-1 monoclonal antibody group to the PD-1 monoclonal antibody+acarbose group intestinal flora at the genus level;

Figure 8 shows a differential analysis of acarbose versus gut flora composition at the genus level: A. comparing community composition differences of the PD-1 monoclonal antibody group and the PD-1 monoclonal antibody+acarbose group on a genus level by drawing a differential heat map; B. in random forest analysis of unsupervised learning, importance ranking is performed on microorganisms at genus level, and the higher the average Accuracy drop (MEAN DECREASE IN Accuracy, MDA) is, the more important the genus is;

Figure 9 shows the effect of acarbose on gut microbiota on mouse serum metabonomics: A. PCoA analysis is carried out on the serum metabonomics characteristics of the PD-1 monoclonal antibody group and the PD-1 monoclonal antibody+acarbose group mice by calculating the difference coefficient of Bray-Curtis; B. comparing the structural differences of the serometabonomics characteristics between the PD-1 monoclonal antibody group and the PD-1 monoclonal antibody+acarbose group by drawing a differential heat map;

In the above figures, ns indicates no significant difference, P is less than 0.05, P is less than 0.01, P is less than 0.001, P is less than 0.0001.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Example 1 therapeutic Effect of acarbose on tumor models with PD-1 mab treatment resistance

To demonstrate the potential effect of acarbose in overcoming immunotherapeutic resistant tumors, tumor models with some immunotherapeutic resistance were selected in this example, including tumor models that mimic human melanoma, colon cancer, and AOM/DSS induction. These models are clinically significant representatives, by which the potential impact of acarbose on tumor therapy can be more fully studied.

Melanoma model: melanoma is a highly malignant skin tumor characterized by high invasiveness and metastasis and often exhibits some resistance to immunotherapy. A melanoma mouse model was selected as a subject to mimic the characteristics of human melanoma. The mouse model of melanoma is formed by inoculating melanoma cells into the skin of the mouse. Then, treatment was performed by administration of acarbose, and its effect on melanoma was observed. The specific experimental steps are as follows:

B16F10 melanoma cells were cultured to log phase and then the cells were collected from the flask. The cell suspension was diluted with PBS buffer, ensuring that each mouse was inoculated with approximately 5.0X10- ⁵ B16F10 cells in vivo. The cell suspension was injected subcutaneously into mice by means of ventral injection, and tumor growth was observed periodically after inoculation. Tumor diameters (long and short diameters) were measured 1 time every 3 days after tumor formation, and the calculation formula of tumor volume in mice: v=0.5×long diameter×short diameter.

Acarbose administration: acarbose is administered orally at a dose of 500mg/kg. The oral administration mode is selected, and the clinical common administration way is simulated, so that the absorption and the action of the medicine are ensured. Treatment was started on day 1 of tumor inoculation and dosing was continued for 4 weeks.

PD-1 mab administration: PD-1 mab (InVivoMAb anti-mouse PD-1,Bio X cellCo.Ltd.USA, # BE 0146) was administered as an immunotherapeutic agent by intraperitoneal injection at a dose of 10mg/kg at a frequency of 1 every three days for a total of 3 times.

Subcutaneous model of colon cancer: colon cancer is a common malignancy with high heterogeneity and resistance to immunotherapy. In order to study the immunotherapeutic effect of acarbose on colon cancer, a subcutaneous tumor-bearing model of colon cancer was selected. Colon cancer cells were implanted into mice to form tumors subcutaneously and then acarbose was administered for treatment to evaluate their inhibitory effect on colon cancer. The specific experimental steps are as follows:

MC38 colon cancer cells were cultured to log phase and then the cells were collected from the flask. The cell suspension was diluted with PBS buffer, ensuring that each mouse was inoculated with approximately 5.0X10- ⁵ MC38 cells in vivo. The cell suspension was injected subcutaneously into mice by means of ventral injection, and tumor growth was observed periodically after inoculation. Tumor diameters (long and short diameters) were measured 1 time every 3 days after tumor formation, and the calculation formula of tumor volume in mice: v=0.5×long diameter×short diameter.

PD-1 mab administration: PD-1 monoclonal antibody is used as an immunotherapeutic medicine and is administrated by intraperitoneal injection at a dosage of 10mg/kg, wherein the administration frequency is 1 time every three days, and the total dosage is 3 times.

Model of AOM/DSS (Azoxymethane/Dextran Sulfate Sodium) induced colon adenocarcinoma: the AOM/DSS model is a commonly used model of colon cancer, which can be induced to produce colitis and colon cancer in mice by intraperitoneal injection of azomethane (Azoxymethane, AOM) and sodium dextran sulfate (Dextran Sulfate Sodium Salt, DSS). The model has the characteristics of intestinal inflammation and canceration, and can simulate the development process of human colon cancer. The effect of AOM/DSS induced mouse model on inflammation and tumor is studied through acarbose intervention treatment so as to show the therapeutic potential of the mouse model on immune therapy resistant tumor model. The specific experimental steps are as follows:

Experimental results:

1. Melanoma mouse model

In the melanoma mouse model, we observed that acarbose alone had no significant inhibitory effect on tumor growth. However, when acarbose was used in combination with PD-1 mab, melanoma growth was significantly inhibited. This result suggests that acarbose may increase the effectiveness of PD-1 mab in treating melanoma (fig. 1).

2. Subcutaneous tumor-bearing model for colon cancer

In a subcutaneous tumor-bearing model of colon cancer, we also observed that acarbose alone had no significant inhibitory effect on tumor growth. However, when acarbose is used in combination with PD-1 mab, the therapeutic effect is significantly enhanced, far superior to that of PD-1 mab alone. This further demonstrates that acarbose can enhance the therapeutic effect of PD-1 mab, particularly in colon cancer treatment (fig. 2A-C). In addition, the body weight of each group of mice remained steadily increasing throughout the experiment, with no significant differences between groups of body weights (fig. 2C). In conclusion, acarbose can promote the sensitivity of PD-1 monoclonal antibody to treat tumor-bearing mice, and has good safety.

AOM/DSS induced tumor model

In the AOM/DSS induced tumor model, as in the first two models, acarbose alone did not produce significant inhibition of tumor growth. However, the combined use of acarbose and PD-1 mab significantly inhibited tumor growth, and the therapeutic effect was significantly better than that of PD-1 mab alone (fig. 3). This result underscores the potential of acarbose as an adjuvant immunotherapeutic agent.

Example 2 acarbose can significantly increase the number of CD8 ⁺ T cells in a tumor, stimulate the immune response, and promote the anti-tumor immune effect of PD-1 mAb

To further elucidate the molecular and cellular mechanisms involved in the treatment of PD-1 mab, RNA-seq sequencing analysis was performed on mouse tumor tissue in this example to explore the effect of acarbose on tumor immune microenvironment. First, KEGG analysis identified some high-level functional modules, such as the activation of the immune system(such as T cell receptor signaling pathway,Th1 and Th2 cell differentiation,Antigen processing and presentation,NF-kappa B signaling pathway and chemokine signaling pathway, etc., that were abundantly expressed in tumor tissue of acarbose-treated groups (fig. 4A). GSEA enrichment analysis further confirmed this result. Of particular note, this group showed significant enrichment of T cell activation and positive regulation of chemokine production (fig. 4B-C), which are key regulators of the tumor immune microenvironment. The RNA-seq data is then used to estimate immune cells that are tumor infiltrated. We found that in tumour tissue treated with acarbose, the proportion of T cells is higher (FIG. 4D), in particular the proportion of CD8 ⁺ T cells in total cells is increased (FIG. 4E), and that such cells play a critical role in the anti-tumour immune response of the body.

Further analysis showed that the use of acarbose not only increased the proportion of CD8 ⁺ T cells (FIGS. 5A-B), but also stimulated an increase in the expression level of the chemokine CXCL10 in tumor tissue (FIGS. 5A-B) and in peripheral serum (FIG. 5D) (FIGS. 5A-B). CXCL10 plays an important regulatory role in the migration of CD8 ⁺ T cells into the tumor microenvironment. These findings indicate that acarbose successfully recruits and increases the infiltration and activation levels of killing CD8 ⁺ T lymphocytes in tumor tissue by promoting the expression of CXCL10 chemokines by tumor cells. This effect effectively promotes the therapeutic effect of the PD-1 antibody, and significantly inhibits tumor growth. The above results suggest that acarbose-induced increased infiltration of CD8 ⁺ T lymphocytes is one of the very promising strategies for converting "cold" tumors into "hot" tumors, which can be applied in tumor therapy or as immunomodulators to enhance the immunotherapeutic effect of PD-1 antibodies.

EXAMPLE 3 acarbose reversal of intestinal microbiota imbalance in tumor-bearing mice

To determine whether and how acarbose affects the intestinal flora of tumor-bearing mice, bacterial 16s rRNA (V3-V4 region) in the faeces of each group of mice was collected on day 27 post tumor inoculation for MiSeq-based sequencing. To investigate the degree of difference between microflora we calculated β -diversity using principal coordinate analysis (PCOA), the analysis results indicated that significant separation of the intestinal microflora occurred for each group (fig. 6A). At the same time, acarbose intervention significantly increased intestinal microbial alpha diversity (Shannon index, fig. 6B). These results suggest that acarbose significantly increases the richness and diversity of the intestinal flora of tumor-bearing mice.

Studies have shown that the mouse microbiome is predominantly Firmicutes (firmicutes) and Bacteroidetes (bacteroides). The Firmicutes/Bacteroidetes ratio could be significantly reduced after acarbose intervention compared to the PD-1 mab group (fig. 7A). Of the first 20 genera, there were significant changes in relative abundance for a total of 15 genera, belonging to Firmicutes and Bacteroidetes. In PD-1 mab group mice, part of the genus (Faecalibaculum、Streptococcus、Rhizobium、Cupriavidus、Gordonibacter、Peptococcus、Oscillibacter、Enterorhabdus、Lachnoclostridium) was significantly increased, while others (Faecalibaculum and bifidobacteria) were significantly decreased (fig. 7B). Furthermore, acarbose significantly increased Lactobacillus, parabacteroides, intestinimonas and bifidobacteria et al (fig. 8A), which are the major microorganisms producing short chain fatty acids, closely related to the balance of the intestinal micro-ecosystem. Further, random forest analysis based on unsupervised learning showed that Enterorhabdus, parabacteroides, rhizobium, oscillibacter, butyricicoccus and bifidobacteria were more regulated by acarbose in these abundantly differing genera (fig. 8B).

EXAMPLE 4 acarbose has a modulating effect on the metabolites associated with the intestinal microbiota

Intestinal microorganisms in humans produce large quantities of metabolites at once, some of which, in particular short chain fatty acids, can profoundly influence the immune response in the host's intestine. Thus, we studied the effect of acarbose on serum metabolic characteristics of tumor-bearing mice using liquid chromatography-mass spectrometry (LC-MS) non-targeting method. PCoA analysis showed that the metabolic characteristics of PD-1 mab and PD-1 mab+acarbose group mice were significantly different (FIG. 9A), 42 metabolites with significant differences in abundance between PD-1 mab and PD-1 mab+acarbose group were identified. Acarbose administration significantly increased serum levels of 2-dhahma[dmed-fahfa]、6-Phospho-D-gluconate、6-Aminocaproic acid、Kynurenic acid、N-oleoylethanolamine、2-epahpa[dmed-fahfa]、N-(3-oxooctanoyl)-l-homoserine、lactone、1-Oleoyl-sn-glycero-3-phosphocholine、Permethrin and Procyanidin B other metabolites in tumor-bearing mice (fig. 9B).

Claims

1. The application of acarbose combined immune checkpoint inhibitor in preparing antitumor medicine is provided.

2. The use of claim 1, wherein the tumor is a malignant solid tumor that is resistant to immunotherapy.

3. The use according to claim 2, wherein the tumour comprises melanoma, colorectal cancer or colon adenocarcinoma.

4. The use according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is at least one of PD-1 mab, PD-L1 mab and CTLA4 mab.

5. The use according to claim 4, wherein the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, the active ingredient being acarbose and PD-1 mab.

6. The use according to claim 5, wherein the pharmaceutical is in the form of a tablet, capsule, powder, granule or oral liquid.