WO2020020343A1

WO2020020343A1 - Use of total glucosides of paeony as potentiating agent for immunological checkpoint inhibitor for tumor treatment

Info

Publication number: WO2020020343A1
Application number: PCT/CN2019/097898
Authority: WO
Inventors: 张作光; 田晖; 李克峰
Original assignee: 张作光
Priority date: 2018-07-26
Filing date: 2019-07-26
Publication date: 2020-01-30

Abstract

The present invention provides a use of total glucosides of paeony in the preparation of a potentiating agent for an immunological checkpoint inhibitor for tumor treatment, wherein the immunological checkpoint inhibitor is a PD-1/L1 inhibitor, and wherein the total glucosides of paeony include albiflorin and paeoniflorin. The present invention also provides a method for tumor treatment, comprising using total glucosides of paeony as a potentiating agent for an immunological checkpoint inhibitor, wherein the total glucosides of paeony enhances an efficacy of the immunological checkpoint inhibitor by improving intestinal flora balance of a cancer patient by means of stress resistance, wherein improving intestinal flora balance involves increasing amounts of gut probiotic bacteria, such as Akk bacteria, ruminococci, lactic acid bacteria, bifidobacteria, Bifidobacterium longum, etc.

Description

Use of total glucosides of paeony as synergist of immune checkpoint inhibitor for treating tumor

Technical field

The invention belongs to the field of medicine and relates to the use of total glucosides of paeony as a synergist for an immune checkpoint inhibitor for treating tumors. Specifically, the present invention relates to the use of total glucosides of paeony and its main components paeoniflorin and paeoniflorin to improve the efficacy of immune checkpoint inhibitors to treat tumors by improving the intestinal flora balance, and more specifically, the applicant has found that The total glucosides of paeony can increase the anti-cancer function of immune checkpoint inhibitors such as PD-1 / L1 by increasing the intestinal probiotics, especially the contents of microorganisms such as Akk bacteria, rumen cocci, bifidobacterium, bifidobacterium longum, and lactic acid bacteria. Response rate and efficacy, so that it can be prepared as a synergist of immune anti-cancer drugs such as PD-1 / L1 inhibitors, or it can be used to prepare tumor health products, food nutrients, and food additives.

Background technique

Intestinal microorganisms are the largest and most important micro-ecosystem of the human body. The total weight of intestinal bacteria in a healthy adult can reach 1-1.5kg. Under normal circumstances, the intestinal flora in the human body and the host cell form a symbiotic relationship, in a dynamic and balanced ecology. The role of intestinal flora and its impact on human health have attracted much attention from the medical community. The intestinal flora and the host cell continuously communicate information, evolve with the host, regulate multiple metabolic pathways of the host, affect the immune-anti-inflammatory axis, and are closely related to the host's physiological and biochemical activities, affecting the host's health. More and more studies show that the composition of the intestinal flora is closely related to the occurrence and treatment of cancer, especially in the immunotherapy of tumors.

As early as November 27, 2015, Science magazine published two heavy papers that disclosed the composition of intestinal microorganisms in cancer patients, which may determine immune checkpoint inhibitors (CTLA-4, PD-1 / L1) Represents the effectiveness of cancer immunotherapy.

On November 2, 2017, two years later, "Science" magazine published two more heavy articles. Through clinical case analysis of cancer patients, it was proved that intestinal microorganisms directly affect the effect of immunotherapy for cancer patients. What exactly is an immune checkpoint inhibitor for cancer treatment "effective? Ineffective? It depends on the gut microbe complexion."

Dr. Laurence Zivogel from the Gustave Roussy Cancer Institute in France analyzed 249 lung cancer, kidney cancer and other cancer patients treated with PD-1 / L1 inhibitors. It was found that among these 249 patients, 69 patients had taken antibiotics due to dental treatment or urinary tract infections just before and after treatment. The flora is normal. Cancer patients with antibiotics that have a disordered intestinal flora have an overall survival that is nearly 45% shorter than the normal group. For further verification, the researchers transplanted the patient's intestinal microbes into a sterile mouse model, and found that transplantation of mice that treated patients with intestinal microbes would also be effective with PD-1 / L1 inhibitor treatment. Mice that transplanted intestinal microorganisms from patients who failed to respond did not respond.

Another paper published by the same journal is the research result of the Jennifer Wargo team of the University of Texas Anderson Cancer Center. They performed oral and intestinal tract on 112 melanoma patients treated with PD-1 / L1 inhibitors. Microbiological analysis to determine the effect of microbes on tumor immunotherapy.

The similarities between the results of the two papers have confirmed that there is a significant difference between the intestinal microbes of the two groups of patients with effective and ineffective immunotherapy; the difference lies in the specific bacteria involved in response to immunotherapy, the former being prominently Akk Bacteria (Akkermansia muciniphila), Enterococcus sheila; the latter is mainly Ruminococaceae.

On January 5, 2018, Thomas Gajewski's team who revealed the relationship between microbes and immunotherapy two years ago, published their latest research results in the journal Science, and analyzed the composition of 42 patients with metastatic melanoma The results proved that there was a significant correlation between the composition of the intestinal faecal flora of the patients and the effect of the PD-1 inhibitor treatment, and in the intestinal flora of patients who responded significantly to the treatment, long abundance was identified Bifidobacterium Iongum, Collinsella aerofaciens and Enterococcus faecium. Similarly, when researchers transplanted gut microbes from these patients into sterile mice, they could significantly enhance tumor immune response and tumor control.

Five consecutive heavy papers have been published in the journal Science for two years, confirming the significant impact of the intestinal flora that coexists with the human body on cancer immunotherapy. The results of this study revealed that by regulating the composition of the intestinal flora, such as increasing intestinal The abundance of microorganisms such as Akk bacteria, Enterococcus shela, rumenococcus, Bifidobacterium longum can not only enhance the effect of tumor immunotherapy, but also determine whether this treatment is effective to a certain extent.

Summary of the Invention

The purpose of the present invention is to provide the use of total glucosides of paeony and its main functional components, paeoniflorin (Albiflorin) and paeoniflorin, for the preparation of a synergist for an immune checkpoint inhibitor. The inventors of the present application have found that the total glucosides of paeony can improve the antitumor response rate and efficacy of PD-1 / L1 inhibitors by improving the composition and balance of intestinal flora.

The inventors discovered that slow stress (CUMS) damages the intestine of model rats in the process of using the targeted metabolomics method to study the main functional components of total glucosides of paeonia lactiflorin and paeoniflorin in the treatment of depression by regulating the intestinal brain axis. The bacterial flora is balanced in metabolism, and the peiolide can quickly restore the normal metabolism of the intestinal flora. Then, the inventors performed metabolomics and 16SrDNA metagenome tests on rat stool samples, and found that paeoniflorin can significantly improve the composition and metabolic characteristics of intestinal flora in depression rats, and make them into the normal rat intestines. The flora pattern is approaching. Among them, it is particularly important that the inventors found that the abundance of microorganisms such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestinal flora of stress-depression rats was significantly reduced, especially CUMS The Akk bacteria in the intestinal tract of model rats is almost zero, and taking paeoniflorin can increase the abundance of Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestine of CUMS rats. In addition, the total glucosides of paeony and its main functional components paeoniflorin and paeoniflorin can also prevent colon cancer by regulating the function of the endocannabinoid system.

Based on the above, the object of the present invention is achieved by the following technical solutions:

In one aspect, the present invention provides the use of total glucosides of paeony in the preparation of a synergist for an immune checkpoint inhibitor for the treatment of tumors.

Preferably, the immune checkpoint inhibitor is a PD-1 / L1 inhibitor.

Preferably, the total glucosides of paeony comprises paeoniflorin and paeoniflorin.

In another aspect, the present invention provides the use of total glucosides of paeony in the preparation of a medicament for improving the balance of the intestinal flora through anti-stress treatment of tumors or enhancing the efficacy of checkpoint inhibitors.

Preferably, the function of improving the intestinal flora balance is to increase the content of probiotic bacteria such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestine.

Preferably, the tumor is a tumor associated with depression and treated with an immune checkpoint inhibitor.

The total glucosides of paeony provided by the invention have the application of antidepressant and improvement of intestinal flora disorder, and are suitable as a synergist for PD-1 / L1 inhibitors to treat tumors in cancer patients with depression.

The tumor is a tumor associated with depression or associated intestinal flora and treated with an immune checkpoint inhibitor.

Preferably, the tumor is colon cancer or pancreatic cancer.

The total glucosides of paeony of the present invention increase the intestinal flora lactic acid bacteria, thereby promoting the expression of cannabinoid receptor CB2 in intestinal epithelial cells, and can be used to synergize PD-1 / L1 inhibitors to treat colon cancer.

The total glucosides of paeony of the present invention can improve the efficacy of immune anti-cancer drugs such as PD-1 / L1 inhibitors by improving the intestinal flora disorder of tumor patients, and can be used as drugs, health products, nutritional supplements or food for preventing and treating tumors. additive.

In another aspect, the present invention provides a method for treating a tumor, the method comprising using the total paeony glycosides of the present invention as a potentiator of an immune checkpoint inhibitor.

Preferably, the immune checkpoint inhibitor is a PD-1 / L1 inhibitor.

Wherein the tumor is a tumor that is associated with depression or associated intestinal flora and is treated with an immune checkpoint inhibitor.

Preferably, the tumor is colon cancer or pancreatic cancer.

The total glucosides of paeony enhance the efficacy of checkpoint inhibitors through anti-stress to improve the intestinal flora balance of cancer patients.

The total glucosides of paeony improve the intestinal flora balance through anti-stress, which can increase the microorganisms such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestine, thereby strengthening immunity such as PD-1 / L1 inhibitors. Antitumor response rate and efficacy of antitumor drugs.

Preferably, the present invention provides a method for improving immunologic anticancer drugs such as PD-1 / L1 inhibitors through antidepression to improve intestinal flora disturbance, and this method is particularly suitable for treating cancer patients associated with depression.

Preferably, the present invention provides a method for treating patients with cancer associated with depression using drugs such as PD-1 / L1 inhibitors. This method is characterized by the use of antidepressants capable of improving the intestinal flora balance. It is understood that the total glycosides, paeoniflorin, and paeoniflorin belong to natural preparations with this function.

The inventors of the present invention have found that by restoring and improving the composition of the intestinal flora, the efficacy of immune anticancer drugs such as PD-1 / L1 inhibitors can be maintained and increased; drugs or health care that can restore and improve the composition of the intestinal flora Foods can be used as synergists for immunotherapy cancer drugs such as PD-1 / L1 inhibitors to ensure and improve the efficacy of the drugs; drugs or health foods that can restore and improve the composition of the intestinal flora may become immune anti-cancer Standard for drug treatment of tumors.

The present invention relates to a huge drug market: Some experts have analyzed that in the development of new anti-cancer drugs, PD-1 / L1 inhibitors are considered to be promising star species in tumor treatment drugs and can be widely used in the treatment of various tumors. According to market statistics, the global sales revenue of monoclonal antibodies in 2017 was 103.8 billion U.S. dollars. Based on this calculation, sales of intestinal flora regulators, which are potentiators of immune anti-cancer drugs such as PD-1 / L1 inhibitors, will surely increase. It's amazing.

The research results of the present invention suggest:

1. Total glucosides of paeony and its main functional components paeoniflorin and paeoniflorin, as a natural product that rapidly improves the function of the intestinal flora system, can significantly increase the intestinal Akk bacteria by adjusting the intestinal flora balance, The abundance of microorganisms such as rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium positively affect and enhance the efficacy of immunological anticancer drugs such as PD-1 / L1 inhibitors.

2. Cancer patients co-morbid with depression will have intestinal flora disorder, which is manifested by a significant decrease in the abundance of microorganisms such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium longum. Therefore, the use of PD -1 / L1 inhibitors and other immunological anticancer drugs for tumor treatment, it is necessary to simultaneously treat depression, and be sure to choose those antidepressant drugs that can regulate and restore the intestinal flora balance. By increasing the abundance of microorganisms such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium long in the intestinal flora, immunological anticancer drugs such as PD-1 / L1 inhibitors have the desired effect.

In addition, the research of the present invention also proves that the total glucosides of paeony and its main functional components paeoniflorin and paeoniflorin can improve the function of the intestinal flora system by regulating the intestinal endocannabinoid system: paeoniflorin is a cannabinoid Receptor CB2 agonist, by promoting the upregulation of intestinal lactic acid bacteria, increasing the expression of cannabinoid receptor CB2 in intestinal epithelial cells, treating inflammatory bowel disease (IBD) and preventing and treating colon cancer.

The invention discloses total glucosides of paeony and its main functional components, paeoniflorin and paeoniflorin, by increasing the content of bile acids, amino acids and vitamins in the intestinal tract of patients with depression and tumors, significantly increasing the beneficial metabolism of the intestinal flora, making it normal. The function of the intestinal flora is restored. This improvement in the function of the intestinal flora is manifested in the restoration of the probiotic bacteria's firmicutes, among which, in particular, the Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and long bifidus in the intestinal flora have been increased. The content of microorganisms such as Bacillus provides an intestinal flora environment for immune anti-cancer drugs such as PD-1 inhibitors, and enhances the function of immune anti-cancer drugs such as PD-1 / L1 inhibitors. .

Compared with the prior art, the present invention has the following advantages:

1. The present invention restores and improves the metabolic balance of the intestinal flora by increasing the content of bile acids, vitamins and amino acids in the intestinal flora, increasing the Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, Abundance of microorganisms such as Bifidobacterium longum. Experiments have shown that the total glucosides of paeony, paeoniflorin, and paeoniflorin can rapidly improve the intestinal tract caused by various stresses (including stress caused by diseases such as depression and cancer) within one week of administration. Flora disorder.

2. The invention restores the intestinal flora balance in the human body, increases the abundance of microorganisms such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, bifidobacterium longum, etc., improves the function of intestinal flora, and improves PD-1 / The efficacy of drugs such as L1 inhibitors in treating tumors. This directly promotes the growth of endogenous probiotics, and has advantages in maintaining the activity, stability and richness of probiotics compared with the existing technology of supplementing exogenous probiotics.

3. The present invention finds that antidepressants such as SSRIs such as fluoxetine, will further aggravate the intestinal flora of patients after taking it, and will have a negative impact on the performance of immune anticancer drugs such as PD-1 / L1 inhibitors. Paeony total glycosides, paeoniflorin, and paeoniflorin can rapidly improve the intestinal flora balance while antidepressant, restore and enhance the intestinal Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, bifidobacterium longum, etc. Therefore, they are suitable for treating depression coexisting with cancer, and are synergistic agents for PD-1 / L1 inhibitors to treat tumors.

Brief description of the drawings

The drawings are used to illustrate the integration of next-generation metabolomics and high-throughput metagenomic sequencing, which show that paeoniflorin has rapid recovery and improved function of regulating intestinal flora.

Figure 1.Multiple Comparative Analysis of Targeted Metabolomics (PLS-DA)

Figure 2. VIP analysis of intestinal flora metabolism in rats in the depression model group and the blank group (Top20 metabolites, VIP> 1.5 is a significant difference)

Figure 3. Structural analysis of intestinal flora of high-throughput sequencing stress rat model group (depression group) and blank group (Control: blank group; CUMS: stress rat model)

Figure 4. Cluster analysis of intestinal population structure of high-throughput metagenomic sequencing after administration of paeoniflorin. Control: blank group; Alb: peiolide group, CUMS: rat model.

Figure 5: The peony lactone group corrects the disturbance of intestinal microbial metabolism in the model group rats (metabolites with significant changes after Top25 administration)

Figure 6: PLS-DA shows that the fluoxetine group increased the intestinal flora of the model group rats.

Figure 7. Relative abundance of flora species at the level of intestinal tract (Family) in each group of CUMS rats

Figure 8. Ratio of Bacteroides / Thick Bacillus (B / F ratio) in feces of peiolide group and CUMS group

The best way to implement the invention

The present invention will be further explained with reference to specific implementation cases. However, the following implementation examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

In previous studies, we integrated targeted metabolomics and high-throughput sequencing technology and found that peony lactone glycosides and total peony glycosides containing peony lactone glycosides can rapidly improve the intestinal flora disorder in rats and restore Normal function of the intestinal flora.

Example 1: "Sub-healthy" state of internal and external environmental stress causes disturbance of intestinal flora

We have established a chronic unpredictable mild stress rat model (chronic unpredictable mild stress (CUMS). Targeted metabolomics was used to study the metabolic function of the intestinal flora of stressed rats. PLS-DA multivariate analysis showed that the intestinal flora of the depression model group (depression group) rats was blank and blank. There are significant differences between them, see Figure 1.

By VIP analysis, the metabolism of intestinal flora of rats in the depression group was significantly reduced compared with the blank group. Among the two groups of significant Top20 metabolites, 16 (80%) metabolites were significantly reduced in the depression model (VIP> 1.5). The reduced metabolites were mainly amino acids and vitamins and their derivatives, see attached figure 2.

High-throughput 16sRNA sequencing showed that the intestinal flora structure and type of intestinal flora of the stress rat model had changed significantly compared with the blank group, as shown in FIG. 3.

Studies have shown that the composition of the intestinal flora, the disruption of the abundance and balance of various flora, and the disturbance of function are all closely related to the type and degree of stress in the internal and external environment.

Example 2: Paeonolide administration for 7 days can quickly restore the structure and function of intestinal flora of slow stress rats

We further administered peony lactone glycosides in the stress model rats, continuously, each treatment dose was 7mg / kg / d. After 7 days, we used metabonomics and 16sDNA high-throughput metagenomic sequencing technology to re-evaluate the function and structure of rat intestinal flora. Through multiple comparative analysis of metabolomics data (PLS-DA), it was found that after administration of paeoniflorin, the intestinal flora of rats moved toward the normal group, almost completely overlapping with the normal group, indicating that paeonilactone Glycosides help the intestinal flora of stress rats return to normal, see Figure 1 below. This is consistent with the results of the flora structure analysis.

According to the VIP analysis, the overall metabolism of the intestinal flora was significantly increased compared with the depression group after 7 days of paeoniflorin administration. The main manifestations were increased bile acid content and increased amino acid and vitamin content, see Figure 4 below.

Fig. 5 is a clustering analysis diagram of intestinal flora structure correlation. The structure of the peony lactone administration group (Alb) is similar to that of the normal group (Control), and they are clustered together without statistical difference (P> 0.05). Compared with the stress rat model group, the beneficial bacteria Firmicutes, especially lactic acid bacteria, have increased significantly.

Example 3: Administration of fluoxetine for 7 days exacerbates intestinal flora disturbance in stressed rats

The positive drug fluoxetine was administered continuously for 7 days at a dose of 10 mg / kg / d. Multivariate PLS-DA analysis showed that the effect of fluoxetine was opposite to that of paeoniflorin, which caused the metabolism of the rat intestinal flora to deviate more. Normal state, indicating that SSRI antidepressants further exacerbated the intestinal flora disorder, see Figure 6.

Example 4: Effect of Paeoniflorin on 16S rDNA Expression in Feces of Slow Stress Rats

Experimental results

1.OTU analysis and species annotation

Relative abundance (%) of the top 10 species with the largest abundance at the Family level and Akk bacteria

↑: the relative abundance of the bacteria increased in the model group compared with the normal control group; ↓: the relative abundance of the bacteria decreased in the model group compared with the normal control group; ↑: the relative abundance of the bacteria increased in the paelide group compared with the model group; ↓: The relative abundance of bacteria in the peony lactone group was reduced compared with the model group.

Based on the abundance data of Top10 species and Akk bacteria that are the most abundant species at the family level shown in Figure 7 and the attached table, CUMS model rat intestinal Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacterium, and bifidobacterium can be found. And other microorganisms were significantly reduced, in which the intestinal Akk bacteria was nearly 0; the peony lactone administration group, for 7 days, could reverse the intestinal flora disorder caused by CUMS, and increase rumen cocci, lactic acid bacteria, bifidobacteria, and long double The abundance of probiotics such as Bifidobacterium and Akk bacteria, and the increase and recovery of the flora expression of these species can enhance the effectiveness of immune anticancer drugs such as PD-1 / L1 inhibitors.

2.The ratio of Bacillus and Pachybacteria in feces of peony lactone group and CUMS group

Bacteroides and pachybacteria are the main components of the intestinal flora of rats, and the change in the ratio of Bacteroides / thick-wall bacteria (B / F) is a key indicator of the ecological imbalance of the intestinal flora. Compared with the normal group, the abundance of Bacteroides and the B / F ratio in the feces of the CUMS group (Figure 8, P <0,05) indicate that CUMS disrupted the balance of the intestinal flora. After 7 days of administration of paeoniflorin, the B / F ratio was lower than that of CUMS, indicating that paeoniflorin relieved the intestinal flora disturbance induced by CUMS in rats. See Figure 8.

In summary, the integrated study of metabolomics and high-throughput sequencing of 16sRNA showed that paeoniflorin and paeoniflorin containing paeoniflorin and paeoniflorin can rapidly regulate and improve the intestinal flora of CUMS rats Function, can increase the abundance of microorganisms such as rumen cocci, lactic acid bacteria, bifidobacteria, bifidobacterium longum, Akk bacteria in the intestinal flora, and the increase and recovery of these probiotics can enhance PD-1 / L1 inhibition Efficacy of anti-cancer drugs such as agents.

Paeoniflorin, paeoniflorin, paeoniflorin, or paeony extract can be used to rapidly improve the intestinal flora disturbance, increase the content of intestinal probiotics, and increase the proliferation of immune anticancer drugs such as PD-1 / L1 inhibitor As an effective agent and a therapeutic partner, a composition containing these ingredients can also be prepared as a health food, a food additive, or a nutritional supplement to improve the intestinal flora balance and prevent tumors.

These products have extremely high application value and huge market potential.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention, as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims of the present invention.

Claims

Use of total glucosides of paeony for the preparation of a synergist of an immune checkpoint inhibitor for treating tumors.
The use according to claim 1, wherein the immune checkpoint inhibitor is a PD-1 / L1 inhibitor.
The use according to claim 1, wherein the total glucosides of paeony comprises paeoniflorin and paeoniflorin.
The use according to claim 1, wherein the total glucosides of paeony enhance the efficacy of an epidemic checkpoint inhibitor through anti-stress to improve the intestinal flora balance of cancer patients;

The function of improving the intestinal flora balance is to increase the content of probiotics such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestine.
The use according to claim 1, wherein the tumor is a tumor associated with depression or associated intestinal flora and treated with an immune checkpoint inhibitor;

The tumor is colon cancer or pancreatic cancer.
The use according to claim 1, wherein the synergist is a medicine, a health food or a nutritional supplement.
A method for treating a tumor, the method comprising using total glucosides of paeony as a synergist for an immune checkpoint inhibitor.
The method according to claim 7, wherein the immune checkpoint inhibitor is a PD-1 / L1 inhibitor.
The method according to claim 7, wherein the total glucosides of paeony comprises paeoniflorin and paeoniflorin.
The method according to claim 7, wherein the tumor is a tumor associated with depression or associated intestinal flora and treated with an immune checkpoint inhibitor; the tumor is colon cancer or pancreatic cancer.
The method according to claim 7, wherein the total glucosides of paeony enhance the efficacy of checkpoint inhibitors by anti-stress to improve the intestinal flora balance of cancer patients;

The function of improving the intestinal flora balance is to increase the content of probiotics such as Akk bacteria, rumen cocci, lactic acid bacteria, bifidobacteria, and bifidobacterium in the intestine.

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