Application of probiotics and metabolite indole thereof in antitumor drugs
By using probiotics and their metabolite indole, the MDSCs in the tumor microenvironment are eliminated, and the problem of limited efficacy of PD-1 monoclonal antibody is solved, which significantly enhances the anti-tumor efficacy, prolongs the patient's survival, and provides safe and economical Treatment plan.
Patent Information
- Application Number
- CN202411335870.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-17
AI Technical Summary
The efficacy of the existing anti-tumor drug PD-1 monoclonal antibody is limited by the existence of myelogenic inhibitory cells (MDSCs) in the tumor microenvironment, and patients have poor response to their treatment, and there is a lack of strategies to effectively clear MDSCs to improve the efficacy.
By using specific probiotics and their metabolite indole, the tumor immune environment is optimized and MDSCs in the tumor microenvironment is eliminated, thereby enhancing the anti-tumor efficacy of PD-1 monoclonal antibody. Specifically, it uses probiotics that can metabolize indoles in the body after oral administration, such as Lactobacillus acidophilus Lactobacillus acidophilus BNCC336636, which directly act on MDSCs or regulate their microenvironment to reduce their number and function.
Significantly inhibit tumor growth, reduce the number and function of MDSCs, enhance the anti-tumor efficacy of PD-1 monoclonal antibody, improve clinical treatment effect, and prolong patient survival. Lactobacillus acidophilus and its metabolite indole have no toxic side effects, and the cost is low , has good clinical application prospects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of medicine, and in particular to the application of probiotics and their metabolite indole in anti-tumor drugs. Background Art
[0002] Malignant tumors are diseases with high morbidity and mortality worldwide, and their treatment has always been a major challenge in the medical community. In recent years, immune checkpoint inhibitors represented by PD-1 monoclonal antibodies have shown certain efficacy in clinical applications, but unfortunately, some patients do not respond well to their treatment. Studies have shown that the presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment is one of the main reasons for the limited efficacy of PD-1 monoclonal antibodies. Therefore, exploring effective strategies to eliminate MDSCs is of great significance for improving the efficacy of PD-1 monoclonal antibodies.
[0003] Intestinal flora and its metabolites play a key role in regulating the tumor immune microenvironment, but the specific relationship between these factors and MDSCs function and the efficacy of PD-1 monoclonal antibodies has not yet been clarified. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies in the prior art, provide the use of probiotics and their metabolites indole in anti-tumor drugs, optimize the tumor immune environment through specific probiotics and their metabolites, and then effectively eliminate MDSCs in the tumor microenvironment, so as to enhance the anti-tumor efficacy of PD-1 monoclonal antibodies.
[0005] The present invention is achieved through the following technical solutions:
[0006] First, the application of indole in anti-tumor and PD-1 monoclonal antibody enhancing drugs is provided.
[0007] Then, the invention provides the use of indole-producing probiotics in anti-tumor and PD-1 monoclonal antibody enhancing drugs, wherein the indole-producing probiotics are probiotics that can metabolize and produce indole in vivo after oral administration.
[0008] Through the above technical solutions, there are currently no suitable drugs for clinical anti-tumor and enhancing the efficacy of PD-1 monoclonal antibodies. Once patients develop PD-1 monoclonal antibody resistance, the clinical treatment effect and patient survival will be significantly reduced; myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are one of the main reasons for the poor efficacy of PD-1 monoclonal antibodies. Intestinal flora and its metabolites play an important role in regulating the tumor immune microenvironment. In order to reshape intestinal microorganisms and increase the colonization of probiotics, indole and / or indole-producing probiotics are used to remove MDSCs and sensitize the anti-tumor efficacy of PD-1 monoclonal antibodies, which helps to improve clinical treatment effects and prolong patient survival.
[0009] Furthermore, the probiotics that can metabolize and produce indole in vivo after oral administration are Lactobacillus acidophilus BNCC336636. The Lactobacillus acidophilus used is an edible bacterium with no toxic side effects and has good application prospects in clinical transformation.
[0010] Furthermore, the drug directly acts on MDSCs or regulates their microenvironment to reduce their number and inhibit their function.
[0011] Preferably, the subject of administration of the drug is a human or a mammal.
[0012] Through the above technical scheme, the medicine contains an effective dose of indole or an effective dose of indole-producing probiotics. The effective dose is in the form of a unit dosage (such as the content in a tablet of medicine) or a unit dose of the patient to be treated (such as a unit body weight dose). In the present invention, the drug is administered to mammals, including humans, mice, etc. According to the isodose conversion relationship between humans and experimental animals, the dose per unit body weight of humans can be inferred using the dose of animals.
[0013] Finally, a drug for anti-tumor and PD-1 monoclonal antibody enhancement is provided, wherein the active ingredient includes indole and / or indole-producing probiotics.
[0014] Through the above technical solution, indole and / or indole-producing probiotics can be used not only as one of the effective ingredients, but also as the only effective ingredient. Specifically, when preparing the drug, a carrier material can also be added; the carrier includes but is not limited to a diluent, a suspending agent, a buffer, a granule, an emulsion, an excipient, an encapsulating agent spray, an adhesive, a filler, a disintegrant, a wetting agent, a transdermal absorbent, an absorption accelerator, a surfactant, a flavoring agent, a colorant or an adsorption carrier.
[0015] The drug is prepared into tablets, capsules, solutions or injections according to conventional methods known to those skilled in the art; preferably, it is prepared into oral preparations.
[0016] Furthermore, the indole-producing probiotics are probiotics that can metabolize and produce indole in vivo after oral administration.
[0017] Furthermore, the probiotic bacteria that can metabolize and produce indole in vivo after oral administration is Lactobacillus acidophilus BNCC336636.
[0018] Furthermore, the drug reduces the number of MDSCs and inhibits their function by acting on MDSCs or regulating the microenvironment of MDSCs.
[0019] Beneficial Effects
[0020] The probiotics of the present invention are Lactobacillus acidophilus and its metabolite indole. Treatment with Lactobacillus acidophilus or its product indole can significantly inhibit tumor growth, reduce the abundance of Lactobacillus acidophilus in the intestines of tumor-bearing mice, and further reduce the number and function of myeloid-derived suppressor cells (MDSCs); drug-resistant patients can clear MDSCs and sensitize the anti-tumor efficacy of PD-1 monoclonal antibodies by oral administration of Lactobacillus acidophilus or its metabolite indole, significantly enhance the anti-tumor function of PD-1 monoclonal antibodies, improve clinical treatment effects, and prolong patient survival.
[0021] The present invention has strong effects in inhibiting tumor growth and increasing the efficacy of PD-1 monoclonal antibody. Lactobacillus acidophilus is an edible bacterium with no toxic side effects. Lactobacillus acidophilus or its product indole is safe to eat, has no toxic side effects, and has low cost, and has good application prospects in clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The difference in the proportion of MDSCs between tumor-bearing and non-tumor-bearing mice, where the horizontal axis is the group and the vertical axis is the proportion of MDSCs. The proportion of MDSCs in tumor-bearing mice is higher than that in non-tumor-bearing mice (****p<0.0001);
[0023] Figure 2 This is a bar graph showing the LDA value distribution of intestinal microorganisms in tumor-bearing and non-tumor-bearing mice. The blue color represents the non-tumor-bearing mouse group, the pink color represents the tumor-bearing mouse group, and the red box marks Lactobacillus, which is enriched in non-tumor-bearing mice.
[0024] Figure 3 The flow cytometric detection of the changes in the number of MDSCs after in vitro treatment with Lactobacillus acidophilus, where the horizontal axis is the group and the vertical axis is the proportion of MDSCs. Direct treatment with Lactobacillus does not affect the number of MDSCs (p>0.05);
[0025] Figure 4 The flow cytometry test for the change in the number of MDSCs after indole treatment in vitro, where the horizontal axis is the group and the vertical axis is the proportion of MDSCs. Only treatment with indole, a metabolite of Lactobacillus, can reduce the number of MDSCs (****p<0.0001);
[0026] Figure 5 In vitro indole treatment reduces the expression of genes related to the immunosuppressive function of MDSCs. The horizontal axis is the group, and the vertical axis is the differentially expressed genes. The genes related to the immunosuppression of MDSCs in the indole treatment group are downregulated;
[0027] Figure 6The statistical results of tumor size in LLC tumor-bearing mice treated with Lactobacillus acidophilus or indole alone or in combination with PD-1 monoclonal antibody. The horizontal axis is the number of days of tumor growth (the horizontal axis day 0 represents the first day of tumor measurement), and the vertical axis is the tumor size. Indole and lactobacillus can inhibit tumor growth when used alone, and the effect is better when used in combination with PD-1 monoclonal antibody.
[0028] Figure 7 The statistical results of the MDSCs proportions in the peripheral blood, tumor and spleen of LLC tumor-bearing mice treated with Lactobacillus acidophilus or indole alone or in combination with PD-1 monoclonal antibody are shown, where the horizontal axis represents the group and the vertical axis represents the MDSCs proportion. After the use of Lactobacillus and indole, the MDSCs proportion in the mice was reduced. DETAILED DESCRIPTION
[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0030] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.
[0031] Unless otherwise specified, the reagents and raw materials used in the examples and comparative examples of the present invention can be obtained through commercial channels.
[0032] Example 1: Analysis of the proportion of MDSCs in tumor-bearing mice
[0033] The LLC tumor-bearing mouse model was established with the approval of the Ethics Committee of Zhengzhou University, and non-tumor-bearing mice were used as controls. The experimental group and the control group were set up with 5 mice each. The control group was tumor-free, and each mouse in the experimental group was subcutaneously loaded with 1×10 6 cells, and when the tumor grows to 1000mm 3 When the size is about 1500 mm (ethical requirements require that the volume of mouse tumors should not exceed 1500 mm 3 ), peripheral blood of mice was obtained by sampling blood from the eye sockets, spleen tissue was obtained after the mice were killed, red blood cells were lysed by red blood cell lysis buffer, red blood cell lysis buffer was added to the spleen tissue after grinding to lyse the red blood cells, and finally a single cell suspension was obtained and stained with flow cytometry antibodies (CD11b and Gr1) that marked mouse MDSCs cells, the proportion of MDSCs in peripheral blood and spleen was analyzed, and finally the proportion of MDSCs in the two groups was compared.
[0034] The results are as follows Figure 1 As shown, from left to right are the proportions of MDSCs in spleen and peripheral blood (****p<0.0001).
[0035] Example 2: Expression characteristics of mouse intestinal microorganisms and analysis of transfer-related bacterial flora
[0036] The LLC tumor-bearing mouse model was established with the approval of the Ethics Committee of Zhengzhou University, and non-tumor-bearing mice were used as controls. The control group did not bear tumors, and each mouse in the experimental group was subcutaneously loaded with 1×10 6 LLC cells, and when the tumor grows to 1000mm 3 When the size is about 1500 mm (ethical requirements require that the volume of mouse tumors should not exceed 1500 mm 3 ), the feces of the mice were collected under sterile conditions into sterile EP tubes. The mouse feces obtained above were transported to Wuhan Kangce Technology Co., Ltd. for 16S rRNA gene sequencing using dry ice. The differences in microbiota between the two groups were further analyzed.
[0037] Bone marrow cells of wild-type mice were obtained, G-CSF and GM-CSF were added, and induced in vitro for 48 hours to obtain MDSCs. The harvested MDSCs were re-plated in a 24-well plate with 1×106 cells per well. Inactivated Lactobacillus acidophilus was added to the experimental group. After 12 hours of co-culture, the cells were collected and stained with flow cytometry antibodies (CD11b and Gr1) that mark mouse MDSCs cells, and stained with PI and Annexin V apoptosis dyes to detect MDSCs apoptosis.
[0038] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 The results of 16S rRNA gene sequencing of fecal specimens from tumor-bearing mice and non-tumor-bearing mice are shown. Figure 3 The apoptotic rate of MDSCs after Lactobacillus acidophilus treatment in vitro.
[0039] Example 3: Lactobacillus regulates the proportion and function of MDSCs through its metabolite indole
[0040] Wild-type mouse bone marrow cells were taken, G-CSF and GM-CSF were added, and induced in vitro for 48 hours to obtain MDSCs. The harvested MDSCs were re-plated in 24-well plates, with 1×106g cells per well. The experimental group added the lactobacillus metabolite indole, and the cells were collected after 12 hours of co-culture. They were stained with flow cytometry antibodies (CD11b and Gr1) that marked mouse MDSCs cells, and stained with PI and Annexin V apoptosis dyes to detect MDSCs apoptosis. At the same time, the treated centrifuged and removed the culture supernatant, resuspended with PBS, centrifuged again and discarded the supernatant, and the cell pellet was quickly frozen in liquid nitrogen and transported to Wuhan Kangce Technology Co., Ltd. on dry ice for RNA-seq sequencing to analyze the expression of genes related to the inhibitory function of MDSCs.
[0041] The results are as follows Figure 4 and Figure 5 As shown, Figure 4 To detect apoptosis of MDSCs treated with indole in vitro; Figure 5 RNA-seq results of MDSCs treated with indole in vitro.
[0042] Example 4: Mouse model verification shows that Lactobacillus acidophilus and its metabolite indole can inhibit tumor growth and enhance the efficacy of PD-1 monoclonal antibody
[0043] The LLC tumor-bearing mouse model was established with the approval of the Ethics Committee of Zhengzhou University. A total of six groups were set up, namely control group (Control), PD-1 monoclonal antibody alone group (aPD-1), indole alone group (Indole), Lactobacillus acidophilus alone group (LSC), indole and PD-1 monoclonal antibody combination group (Indole+aPD-1) and Lactobacillus acidophilus and PD-1 monoclonal antibody combination group (LSC+aPD-1), with 5 mice in each group. Each mouse was subcutaneously tumor-bearing 1×106 LLC cells. On the fifth day, the tumor was visible to the naked eye. The tumor was measured and the tumor size was approximately 70mm3. The tumor was then measured every other day. On the sixth day, indole and Lactobacillus acidophilus were gavaged every day and PD-1 monoclonal antibody treatment was given every two days. After a total of five treatments, the mice were killed on the eighteenth day, and peripheral blood, tumor and spleen tissues were obtained, respectively. Single cell suspensions were obtained after grinding the tissues and lysing red blood cells, and stained with flow cytometry antibodies (CD11b and Gr1) that mark mouse MDSCs cells, and the proportion of MDSCs was detected by flow cytometry.
[0044] Figure 6 The tumor growth curves of the experimental group and the treatment group are shown (the horizontal axis day 0 represents the first day of tumor measurement). After the use of indole and Lactobacillus acidophilus, tumor growth was inhibited, and the inhibitory effect on tumors was better when combined with PD-1 monoclonal antibody; Figure 7The difference in the number of MDSCs in each group is shown. After oral administration of indole and Lactobacillus acidophilus, the number of MDSCs in various parts of the mice was significantly reduced. It can be seen that supplementing Lactobacillus acidophilus and its metabolite indole in tumor-bearing mice can inhibit tumor growth and enhance the anti-tumor function of PD-1 monoclonal antibodies.
[0045] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. Application of indole in anti-tumor and PD-1 monoclonal antibody enhancing drugs.
2. Application of indole-producing probiotics in anti-tumor and PD-1 antibody-enhancing drugs.
3. The use according to claim 2, It is characterized in that The indole-producing probiotics are probiotics that can metabolize and produce indole in vivo after oral administration.
4. The use according to claim 3, It is characterized in that The probiotic bacteria that can metabolize and produce indole in vivo after oral administration is Lactobacillus acidophilus BNCC336636.
5. The use according to claim 1 or 2, It is characterized in that The drug directly acts on MDSCs or regulates their microenvironment to reduce their number and inhibit their function.
6. The use according to any one of claims 1 to 4, It is characterized in that The drug is administered to humans or mammals.
7. A drug for anti-tumor and enhancing PD-1 monoclonal antibody, It is characterized in that The active ingredients are indole and / or indole-producing probiotics.
8. The drug according to claim 7, It is characterized in that The indole-producing probiotics are probiotics that can metabolize and produce indole in vivo after oral administration.
9. The drug according to claim 8, It is characterized in that The probiotic bacteria that can metabolize and produce indole in vivo after oral administration is Lactobacillus acidophilus BNCC336636.
10. The drug according to claim 7, It is characterized in that The drug reduces the number of MDSCs and inhibits their function by acting on MDSCs or regulating the microenvironment of MDSCs.