Immunity checkpoint inhibitor supplement

Abstract

[Problem] To provide a supplement that blends Agaricus blazei Murrill subcritical water extract, Coprinus comatus powder, and Crispatus fungus, said supplement having an immune checkpoint inhibitory function. [Solution] An immune checkpoint inhibitor supplement containing at least subcritical water extract of Agaricus blazei Murrill, said supplement being characterized by containing 5-80 mass% Agaricus blazei Murrill subcritical water extract, 1-10 mass% Coprinus comatus powder, and 0.1-10 mass% Crispatus fungus.

Description

Immune Checkpoint Inhibitor Supplements

[0001] The present invention relates to an immune checkpoint inhibitor supplement containing a subcritical water extract of Agaricus blazei Murill.

[0002] In recent years, cancer immunotherapy has attracted attention as a new cancer treatment. It has been reported that, in addition to the signaling generated when T cell receptors recognize major histocompatibility complexes presenting cancer antigens between cancer cells and T cells and other cells that recognize and attack them, there is also a mechanism of auxiliary signals that act in an immunostimulatory or suppressive manner, and interest is growing in the possibility of treatments that focus on this mechanism. Molecules that can induce immunosuppressive signals in this mechanism are called "immune checkpoint molecules," and known examples include the PD-1 receptor on T cells, which is involved in the PD-1 ligand pathway, and the ligands PD-L1 and PD-L2 expressed on cancer cells, and the CTLA4 receptor on T cells, which is involved in the CTLA4 pathway (Non-Patent Documents 1 and 2).

[0003] The PD-1 molecule is an immunosuppressive coreceptor signal receptor belonging to the CD28 family. It is expressed on activated T cells, B cells, and myeloid cells, and has been shown to antigen-specifically suppress T cell activity by binding to its ligand (Non-Patent Document 1). PD-1 ligands include PD-L1 and PD-L2, which belong to the B7 family. The PD-1 pathway is believed to be involved in suppressing immunity against cancer cells, and anti-PD-1 antibodies and anti-PD-L1 antibodies targeting this pathway are expected to be candidates for cancer treatment, and antibody drugs are being developed (Patent Documents 1 to 3). Examples of known anti-PD-1 antibodies include nivolumab (Opdivo®) and pembrolizumab.

[0004] CTLA4 was identified by Brunet et al. in 1987 and has been shown to be a molecule involved in suppressing T cell immune responses by acting competitively with CD28 on T cells activated by the stimulatory co-signal B7 / CD28 pathway (Non-Patent Document 2). Ipilimumab is known as an example of an anti-CTLA4 antibody.

[0005] Recent studies have revealed that each cancer has its own unique genetic mutations. Lung cancer exhibits unique genetic mutations, such as ALK fusion genes and EGFR gene mutations, making it increasingly possible to perform personalized therapy targeting these mutations. Recently, it has been reported that activation of AXL kinase is a novel mechanism of EGFR-TKI resistance. Furthermore, a link between AXL and epithelial-mesenchymal transition (EMT) has been identified, contributing to poor prognosis, tumor growth, and metastasis. AXL is overexpressed in various cancers and is involved in their proliferation and progression. Furthermore, AXL activation is known to be involved in the acquisition of resistance to various anticancer drugs, including EGFR-TKIs, the anticancer drug cisplatin, and the HER2-targeted drug lapatinib.

[0006] Mushrooms have been used in traditional Chinese medicine since ancient times and are believed to be beneficial for the prevention and treatment of malignant tumors through immune enhancement and apoptosis induction. Among these, Agaricus blazei murrill (Himematsutake) in particular has strong antitumor activity, and the polysaccharide β-(1→6)-D-glucan protein complex (FIII2b fraction) in Agaricus blazei activates macrophages and NK cells, inhibiting cancer proliferation. Furthermore, a new immune mechanism, the activation of dendritic cells, has also been elucidated (Patent Document 4). However, the inhibitory effects of AXL and immune checkpoint inhibitors mentioned above have not been reported with mushroom extracts.

[0007] Drugs that act to inhibit the immune checkpoint molecules mentioned above, so-called "immune checkpoint inhibitors," are beginning to show clinical effectiveness. However, treatment with antibody drugs is very expensive, and the burden on patients can be excessive.

[0008] Patent Document 5 discloses naturally occurring components that inhibit the expression and / or function of proteins that act suppressively in the immune system, such as immune checkpoint molecules, and can improve immunity against cancer. Specifically, they are as follows: 1. An anticancer drug resistance suppressor containing an alkaline extract or subcritical water extract of Agaricus blazei Murill as an active ingredient. 2. The anticancer drug resistance suppressor according to above 1, which is an immune checkpoint inhibitor that inhibits the expression and / or function of an immune checkpoint molecule. 3. The anticancer drug resistance suppressor according to above 2, wherein the immune checkpoint molecule is PD-1, PD-L1, PD-L2 and / or CTLA4 molecule. 4. The anticancer drug resistance suppressor according to any one of above 1 to 3, which inhibits the expression and / or function of AXL, ADAM17 and / or HMGB1. 5. 5. The anticancer drug resistance inhibitor according to any one of above 1 to 4, wherein the Agaricus blazei alkaline extract or subcritical water extract contains an active ingredient with a molecular weight of less than 100,000. 6. The anticancer drug resistance inhibitor according to any one of above 1 to 5, wherein the active ingredient comprises one or more proteins detected at 17 kD, 26 kD, or 36 kD by SDS-polyacrylamide electrophoresis. 7. The anticancer drug resistance inhibitor according to any one of above 1 to 6, wherein the active ingredient comprises a component recovered after elution with a NaCl concentration of 0.3 M to 3.0 M. 8. A cancer therapeutic agent comprising the anticancer drug resistance inhibitor according to any one of above 1 to 7. 9. 8. A method for producing an anticancer drug resistance suppressor according to any one of items 1 to 7 above, comprising the following steps: (a) an extraction step of extracting Agaricus blazei fruiting bodies with hot water at 80-100°C, and (b) an extraction step of extracting the residue obtained in step (a) with a 3-10% NaOH aqueous solution, wherein the filtrate obtained in step (b) is obtained as an Agaricus blazei alkaline extract. 9. A method for producing an anticancer drug resistance suppressor according to any one of items 1 to 7 above, comprising the following steps: (a) an extraction step of extracting Agaricus blazei fruiting bodies with hot water at 80-100°C, and (b) an extraction step of extracting the residue obtained in step (a) with subcritical water at a pressure of 2-5 MPa and a temperature of 120-200°C, wherein the extract obtained in step (b) is obtained as an Agaricus blazei subcritical water extract.

[0009] The technology described in Patent Document 5 makes it possible to provide naturally derived drugs that act to inhibit the expression and / or function of proteins that act suppressively in the immune system, such as immune checkpoint molecules, and can promote immune responses against cancer.

[0010] Japanese Patent No. 4409430 Japanese Patent No. 5159730 Japanese Patent No. 5885764 Japanese Patent Publication No. 2015-78164 Japanese Patent No. 7141630

[0011] Annu Rev Immunol, 26: 677-704, 2008Nature 328: 267-270, 1987

[0012] Coprinus blazei (also known as Coprinus) is a type of mushroom with a cylindrical shape and a rounded cap tip. Crispatus bacteria are intestinal bacteria that are passed on to the intestines of infants via the mother's birth canal. Although the alkaline extract or subcritical water extract of Agaricus blazei Murill mentioned above is clearly effective as an immune checkpoint inhibitor, the synergistic effect of combining it with Coprinus blazei and Crispatus bacteria has not been verified.

[0013] The present invention was made in consideration of these circumstances, and aims to provide a supplement that has immune checkpoint inhibitory function by mixing Agaricus blazei (himematsutake) subcritical water extract, Coprinus blazei (coprinus comatus powder), and Crispatus fungus.

[0014] (1) In order to achieve the above object, the present application has taken the following measures: That is, an immune checkpoint inhibitor supplement according to one embodiment of the present invention is an immune checkpoint inhibitor supplement containing at least a subcritical water extract of Agaricus blazei Murill, and is characterized by containing 5 to 80 wt % of a subcritical water extract of Agaricus blazei Murill, 1 to 10 wt % of an extract of Coprinus comatus, and 0.1 to 10 wt % of crispatus fungus.

[0015] (2) Furthermore, the immune checkpoint inhibitor supplement of the present invention is an immune checkpoint inhibitor supplement containing at least a subcritical water extract of Agaricus blazei Murill, and is characterized by containing 5 to 60% by weight of a subcritical water extract of Agaricus blazei Murill, 1 to 20% by weight of an extract of Coprinus comatus, and 1% by weight of crispatus fungus.

[0016] According to one aspect of the present invention, immune cells are provided with both the ability to "protect against cancer cells" and the ability to "attack cancer cells," and a significant synergistic effect is expected.

[0017] FIG. 1 is a schematic diagram showing how cancer cells put the brakes on T cell attacks. FIG. 2 is a schematic diagram showing how T cells' inherent ability to attack cancer cells is exerted by inhibiting immune checkpoints. FIG. 3 is a schematic diagram showing how a supplement with immune checkpoint inhibitory effects is produced using subcritical water extraction. FIG. 4 is a schematic diagram showing how immunity is exerted in effect simultaneously with immune checkpoint inhibition. FIG. 5 is a graph showing test results. FIG. 6 is a graph showing other test results. FIG. 7 is a graph showing other test results.

[0018] [About immune checkpoint inhibitors] It is known that the human body's immune cells not only have the ability to attack cancer cells, but also have a "brake" function that suppresses that attack. Figure 1 is a schematic diagram showing how cancer cells put the brakes on T cell attacks. As shown in Figure 1, T cells, one type of immune cell, have a receptor called "PD-1" that acts as a brake. In order to survive, cancer cells under attack counter the attack by T cells by releasing a molecule called "PD-L1," which binds to the PD-1 receptor on T cells and suppresses the T cell attack. This mechanism was elucidated by Professor Honjo of Kyoto University, who won the 2018 Nobel Prize in Physiology or Medicine.

[0019] The PD-L1 molecule, which puts the brakes on the immune system in this way, is called an "immune checkpoint molecule." Based on the elucidation of this mechanism, drugs such as nivolumab and dostarlimab were developed that inhibit the action of immune checkpoint molecules, blocking the immune cells' ability to attack cancer cells. Figure 2 is a schematic diagram showing how inhibiting immune checkpoints allows T cells to exert their natural ability to attack cancer cells. In this way, the attacking function of T cells is not braked, allowing immune cells to exert their natural ability to attack cancer cells. Such cancer treatment drugs are called "immune checkpoint inhibitors."

[0020] [About immune checkpoint inhibitors made from naturally derived ingredients] The supplement industry has also been working on the development of immune checkpoint inhibitors made from naturally derived ingredients. Through its own research and exploration, the Iwade Mycology Institute discovered a naturally derived active ingredient in the cell wall of the mushroom species Agaricus blazei Murill. In Brazil, there is a mushroom called Agaricus blazei Murill (tentatively registered by Dr. Iwade), which does not grow naturally in Japan. The basidiomycete fungus of Agaricus blazei Murill was imported from Brazil to Japan, and after 10 years of work, in 1975, Iwade Inosuke, director of the Iwade Mycology Institute, became the world's first successful artificially cultivated Agaricus blazei Murill.

[0021] The Iwade Mycology Institute has partnered with Mie University and other institutions to conduct collaborative research on this "himematsutake" for approximately 50 years. Institute director Iwade Inosuke believed that in order to consistently cultivate the safest, highest-quality himematsutake, rich in active ingredients, it was necessary to cultivate a basic "seed fungus." After years of repeated testing, he developed the most suitable seed fungus for cultivating high-quality himematsutake, naming it "Iwade 101 strain." Currently, himematsutake grown using Iwade 101 strain as seed is cultivated organically without pesticides in Paraguay and Brazil in South America. Himematsutake grown using Iwade 101 strain as seed fungus, rich in immune checkpoint inhibitors, is a groundbreaking new mushroom species that Japan can be proud of.

[0022] Even if Agaricus blazei mushroom contains active ingredients with immune checkpoint inhibitory effects, simply consuming Agaricus blazei mushrooms alone will not provide sufficient immune checkpoint inhibitory effects. This is because the active ingredients are contained in Agaricus blazei mushroom's extremely tough cell walls, and humans lack the digestive enzymes required to break down these cell walls, preventing their full absorption. Therefore, to obtain the immune checkpoint inhibitory effects of Agaricus blazei mushrooms, it is desirable to extract the active ingredients from the Agaricus blazei mushroom cell walls and process them into a supplement before ingesting them. Currently, methods for extracting Agaricus blazei mushroom extracts used in supplements include hot water extraction and cell wall disruption. However, it is known that it is difficult to extract the active ingredients with immune checkpoint inhibitory effects using hot water extraction and cell wall disruption methods.

[0023] As a result of joint research, Iwade Mycology Research Institute, Mie University, and others have succeeded in efficiently extracting active ingredients with immune checkpoint inhibitory effects from the cell walls of Agaricus blazei Murrill using an extraction technology known as "subcritical water extraction." Figure 3 is a schematic diagram showing the process of producing a supplement with immune checkpoint inhibitory effects using subcritical water extraction. This subcritical water extraction method uses only water, temperature, and pressure, without using organic solvents, enzymes, acids, or bases. This method makes it possible to efficiently extract active ingredients with immune checkpoint inhibitory effects from the cell walls of Agaricus blazei Murrill without destroying their composition or activity (Patent No. 7141630).

[0024] The extract produced by this method is called "Apricot subcritical water extract (commonly known as CP-101)." "CP-101" is a supplement that can be described as a "naturally derived immune checkpoint inhibitor." It uses Agaricus blazei mushrooms grown from the Iwade 101 strain, which contains many active ingredients that have immune checkpoint inhibitory effects, and is said to have no side effects.

[0025] [About Coprinus comatus] Coprinus comatus (also known as Coprinus) is a type of mushroom with a cylindrical shape and a rounded cap tip. It has recently attracted attention in various industries due to its sulfur-containing amino acid, ergothioneine. Ergothioneine (L-Ergothioneine) has extremely high antioxidant activity, demonstrating stronger activity than other antioxidants, such as L-cysteine ​​and ascorbic acid (vitamin C). It is also known for its cosmetic benefits, including inhibiting elastase activity to prevent wrinkles and sagging, and tyrosinase activity to prevent age spots. Ergothioneine is also said to have various benefits, including anti-inflammatory, cellular energy-boosting, and anti-stress properties. It has also been shown to recognize and capture superoxide radicals, a type of reactive oxygen species.

[0026] [About Crispatus bacteria] Crispatus bacteria are lactic acid bacteria that are originally found in the female vagina and are passed on to the intestines of infants via the mother's birth canal during childbirth. Crispatus bacteria are said to reduce the risk of developing allergies and infectious diseases in the intestines. It is believed that the amount of crispatus bacteria in the intestines decreases with age, and the risk of developing these diseases increases accordingly. Therefore, supplementing with crispatus bacteria by taking it as a supplement is gaining attention.

[0027] [Mixture of Coprinus comatus extract and crispatus fungus] As mentioned above, CP-101 inhibits immune checkpoints. Meanwhile, the mixture of Coprinus comatus extract and crispatus fungus significantly strengthens the immune system. Figure 4 is a schematic diagram illustrating the simultaneous immune checkpoint inhibition and immune system enhancement. As shown in Figure 4, ingesting CP-101, Coprinus comatus extract, and crispatus fungus enhances immune cells' ability to both defend against and attack cancer cells, which is expected to have a significant synergistic effect.

[0028] With the cooperation of Company A, the inventors measured the immune activity (immunostimulatory effect measured by the induction of interleukin-12 production) of a "mixture of Coprinus comatus extract and Agaricus blazei subcritical water extract" alone and in combination with Bacillus crispatus.

[0029] [Test Overview] The samples used were a mixture of Coprinus formosanus extract and Agaricus blazei subcritical water extract and Crispatus fungus (10%, 1%, 0.1%).

[0030] [Test design] The test was conducted to evaluate the ability to induce interleukin 12 (IL-12) production using animal immune cells. Here, "IL-12" is a protein called a cytokine that is produced by immune cells and induces immunostimulatory and anti-allergic effects. "IL-12" has the function of activating "NK cells," which control the body's immune power, and enhancing immunity. Furthermore, "IL-12" has been reported to activate "Th1 cells," which induce anti-allergic effects, and contribute to the alleviation of allergic symptoms.

[0031] [Test Method] Macrophage-like cell line (immune cells) was used as the "cells." Cultured cells were suspended as the "medium" and dispensed into a microplate. A test substance was added to the medium to a final concentration, and the amount of "IL-12" produced in the culture supernatant was measured by ELISA.

[0032] [Results and Discussion] Figure 5 is a graph showing the results of the above test, from which the following can be read: - Immunoactivity (IL-12 pg / ml) of the control...3 - Immunoactivity (IL-12 pg / ml) of the "mixture of Coprinus pulcherrima extract and Agaricus blazei subcritical water extract"...6 - Immunoactivity (IL-12 pg / ml) of the "mixture of Coprinus pulcherrima extract and Agaricus blazei subcritical water extract" and "10% Crispatus fungus"...159 - Immunoactivity (IL-12 pg / ml) of "10% Crispatus fungus"...100 Figure 6 is a graph showing other results of the above test, from which the following can be read: - Immune activity of the control (IL-12pg / ml)...3 - Immune activity of the ``mixture of Coprinus pulcherrima extract and Agaricus blazei subcritical water extract'' (IL-12pg / ml)...6 - Immune activity of the ``mixture of Coprinus pulcherrima extract and Agaricus blazei subcritical water extract'' and ``1% Crispatus bacteria'' (IL-12pg / ml)...136 - Immune activity of ``1% Crispatus bacteria'' (IL-12pg / ml)...65 Figure 7 is a graph showing other results of the above test, from which the following can be read. - Immune activity of the control (IL-12pg / ml)...3 - Immune activity of the ``mixture of Coprinus glabra extract and Agaricus blazei subcritical water extract'' (IL-12pg / ml)...6 - Immune activity of the ``mixture of Coprinus glabra extract and Agaricus blazei subcritical water extract'' and ``0.1% Crispatus bacteria'' (IL-12pg / ml)...98 - Immune activity of ``0.1% Crispatus bacteria'' (IL-12pg / ml)...35 In other words, when 10%, 1%, and 0.1% Crispatus bacteria were added to the ``mixture of Coprinus glabra extract and Agaricus blazei subcritical water extract,'' the immune activity was higher than when each was used alone.

[0033] [About the supplement] The "immune checkpoint inhibitor supplement" according to this embodiment is made from the main ingredients "subcritical water extract of Agaricus blazei Murill (CP-101), Coprinus comatus extract, and crispatus fungus," and is recommended to be in tablet or hard capsule form. The recommended daily amount (as CP-101) is as follows: - For cancer prevention in healthy individuals: 50 mg / day, - For treatment in cancer patients: 100 mg to 200 mg / day

Claims

1. An immune checkpoint inhibitor supplement containing at least a subcritical water extract of Agaricus blazei Murill, characterized in that it contains 5 to 80% by weight of subcritical water extract of Agaricus blazei Murill, 1 to 10% by weight of an extract of Coprinus commune, and 0.1 to 10% by weight of Mycobacterium crispatus.

2. An immune checkpoint inhibitor supplement containing at least a subcritical water extract of Agaricus blazei Murill, characterized in that it contains 5 to 60% by weight of subcritical water extract of Agaricus blazei Murill, 1 to 20% by weight of extract of Coprinus commune, and 1% by weight of crispatus fungus.

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