Immunity checkpoint inhibiting supplement

Abstract

To blend Agaricus blazei Murrill subcritical water extract, Coprinus comatus powder, and Lactobacillus crispatus and provide a supplement having an immune checkpoint inhibitory function.SOLUTION: An immune checkpoint inhibiting supplement contains at least subcritical water extract of Agaricus blazei Murrill, the supplement being characterized by containing 5-80 wt.% of Agaricus blazei Murrill subcritical water extract, 1-10 wt.% of Coprinus comatus extract, and 0.1-10 wt.% of Lactobacillus crispatus.SELECTED DRAWING: Figure 3

Description

[Technical field]

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

[0002] In recent years, cancer immunotherapy has been attracting attention as a new cancer treatment method. In addition to the signal generated by the T cell receptor recognizing the major histocompatibility complex presenting the cancer antigen between cancer cells and T cells that recognize and attack them, it has been reported that there is a mechanism of auxiliary signals that act in an immunostimulatory or inhibitory manner, and interest in the possibility of treatment focusing on this mechanism is increasing. 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 involved in the PD-1 ligand pathway, the ligands PD-L1 and PD-L2 expressed on cancer cells, and the CTLA4 receptor on T cells involved in the CTLA4 pathway (Non-Patent Documents 1 and 2).

[0003] The PD-1 molecule is an immunosuppressive co-signal receptor belonging to the CD28 family, and is expressed in activated T cells, B cells, and myeloid cells. It has been shown that the PD-1 molecule inhibits T cell activity in an antigen-specific manner by binding to its ligand (Non-Patent Document 1). PD-L1 and PD-L2, which belong to the B7 family, are known as PD-1 ligands. 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 therapeutic drugs, and antibody drugs are being developed (Patent Documents 1 to 3). Nivolumab (Opdivo (registered trademark)) and Pembrolizumab are known as examples of anti-PD-1 antibodies.

[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 there are genetic mutations specific to each cancer. In lung cancer, specific genetic mutations such as "ALK fusion gene" and "EGFR gene mutation" are seen, and it is becoming possible to perform "individualized treatment" targeting these genetic mutations. Recently, it has been reported that activation of AXL kinase is a new mechanism of EGFR-TKI resistance. Furthermore, it has been reported that AXL is associated with epithelial-mesenchymal transition (EMT), and is involved in poor prognosis, tumor growth, and metastasis. AXL is overexpressed in various cancers and is involved in their proliferation and progression. It is also known that activation of AXL is involved in the acquisition of resistance to various anticancer drugs, including EGFR-TKI, anticancer drug cisplatin, and HER2-targeted drug lapatinib.

[0006] On the other hand, mushrooms have been used in traditional oriental medicine since ancient times, and are considered to be beneficial for the prevention and treatment of malignant tumors by strengthening the immune system and inducing apoptosis. Among them, Agaricus blazei murrill in particular has high antitumor activity, and the polysaccharide β-(1→6)-D-glucan protein complex (FIII2b fraction) in Agaricus blazei activates macrophages and NK cells and has a cancer proliferation inhibitory effect. Furthermore, a new immune mechanism, the activation of dendritic cells, has also been elucidated (Patent Document 4). However, there have been no reports so far on the inhibitory effect of AXL or immune checkpoint inhibition effects of mushroom extracts.

[0007] The effectiveness of the so-called "immune checkpoint inhibitors," which act to inhibit the immune checkpoint molecules described above, is gradually being recognized clinically. However, treatment with antibody drugs is very expensive, and the burden on patients may be excessive.

[0008] Patent Document 5 discloses naturally derived components 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 improve immunity against cancer. Specifically, the components are as follows. 1. An agent for suppressing resistance to anticancer drugs, comprising an alkaline extract or a subcritical water extract of Agarigus blazei Murill as an active ingredient. 2. The anticancer drug resistance suppressant described in 1 above, 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 2 above, wherein the immune checkpoint molecule is PD-1, PD-L1, PD-L2 and / or CTLA4 molecule. 4. The anticancer agent resistance suppressor according to any one of 1 to 3 above, which inhibits the expression and / or function of AXL, ADAM17 and / or HMGB1. 5. The anticancer drug resistance suppressant according to any one of 1 to 4 above, wherein the alkaline extract or subcritical water extract of Agaricus blazei contains an active ingredient having a molecular weight of less than 100,000. 6. The anticancer drug resistance suppressor according to any one of 1 to 5 above, 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 suppressor according to any one of 1 to 6 above, comprising a component recovered after the active ingredient is eluted at a NaCl concentration of 0.3 M to 3.0 M. 8. A cancer therapeutic agent comprising the anticancer drug resistance suppressant according to any one of 1 to 7 above. 9. The following steps: (a) an extraction step using hot water at 80-100°C on Agaricus blazei fruiting bodies; and (b) A method for producing an anticancer drug resistance inhibitor according to any one of 1 to 7 above, comprising an extraction step of using a 3-10% aqueous NaOH solution on the residue obtained in step (a), and obtaining the filtrate obtained in step (b) as an alkaline extract of Agaricus blazei. 10. The following steps: (a) an extraction step using hot water at 80-100°C on Agaricus blazei fruiting bodies; and (b) A method for producing an anticancer drug resistance inhibitor according to any one of 1 to 7 above, comprising an extraction step of using subcritical water at a pressure of 2-5 MPa and a temperature of 120-200°C on the residue obtained in step (a), and obtaining the extract obtained in step (b) as a Agaricus blazei subcritical water extract.

[0009] The technology described in Patent Document 5 makes it possible to provide naturally derived drugs that act inhibitoryly on 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. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 4409430 specification [Patent Document 2] Patent No. 5159730 specification [Patent Document 3] Patent No. 5885764 specification [Patent Document 4] JP 2015-78164 A [Patent Document 5] Patent No. 7141630 specification [Non-patent literature]

[0011] [Non-Patent Document 1] Annu Rev Immunol, 26: 677-704, 2008 [Non-Patent Document 2] Nature 328: 267-270, 1987 Summary of the Invention [Problem to be solved by the invention]

[0012] "Coprinus blazei" is a type of mushroom with a cylindrical shape and a rounded cap tip. "Crispatus" is an intestinal bacterium that is passed on to the intestines of infants via the mother's birth canal during childbirth. Although it is clear that the above-mentioned "alkaline extract or subcritical water extract of Agarigus blazei Murill" is effective as an immune checkpoint inhibitor, it has not been verified that a synergistic effect can be obtained by mixing "Coprinus blazei" and "Crispatus".

[0013] The present invention has been made in consideration of these circumstances, and aims to provide a supplement that has immune checkpoint inhibitory function by mixing subcritical water extract of Agaricus blazei (Agaricus), Coprinus blazei (powder of Coprinus formosum), and Porphyromonas crispatus. [Means for solving the problem]

[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, and is characterized in that it contains 5 to 80% by weight of a subcritical water extract of Agaricus blazei, 1 to 10% by weight of an extract of Coprinus sativus, and 0.1 to 10% by weight 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 weight % subcritical water extract of Agaricus blazei Murill, 1 to 20 weight % extract of Coprinus sativus, and 0.1 to 20 weight % crispatus fungus. Effect of the Invention

[0016] According to one embodiment of the present invention, immune cells are provided with both the "power to defend against cancer cells" and the "power to attack cancer cells," and a significant synergistic effect is expected. [Brief description of the drawings]

[0017] [Figure 1] This is a schematic diagram showing how cancer cells put the brakes on T cell attacks. [Diagram 2] This is a schematic diagram showing how inhibition of immune checkpoints allows T cells to exert their inherent ability to attack cancer cells. [Diagram 3] FIG. 1 is a schematic diagram showing the process of producing a supplement with immune checkpoint inhibitory effects using subcritical water extraction. [Figure 4] FIG. 1 is a schematic diagram showing immune checkpoint inhibition and immune activation at the same time. [Diagram 5] 1 is a graph showing test results. [Figure 6] 13 is a graph showing other results of the test. [Figure 7] 13 is a graph showing other results of the test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] [About immune checkpoint inhibitors] It is known that the immune cells of the human body have the function of attacking cancer cells, as well as the function of acting as a brake to suppress such attacks. 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 release a molecule called "PD-L1" to counter the attack of T cells, and this PD-L1 binds to the PD-1 receptor on T cells to suppress the attack of T cells. This mechanism was elucidated by Professor Honjo of Kyoto University, who was awarded the 2018 Nobel Prize in Physiology or Medicine.

[0019] The PD-L1 molecule, which puts the brakes on immunity in this way, is called an "immune checkpoint molecule." Based on the elucidation of this mechanism, drugs 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 inherent ability to attack cancer cells. In this way, the attacking function of T cells is not put under strain, allowing immune cells to exert their inherent ability to attack cancer cells. Such cancer treatment drugs are called "immune checkpoint inhibitors."

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

[0021] Iwade Mycology Research Institute has been conducting joint research on this "Himematsutake" for about 50 years in partnership with Mie University and other institutions. In order to steadily cultivate high-quality Himematsutake that are safe and contain the most effective ingredients, Iwade Inosuke, the director of the institute, felt that it was necessary to create a base "seed fungus". After repeated testing over the years, the most suitable seed fungus for cultivating high-quality Himematsutake was created and named "Iwade 101 strain". Currently, Himematsutake using Iwade 101 strain as seed fungus is cultivated organically without pesticides in Paraguay and Brazil in South America. Himematsutake using Iwade 101 strain as seed fungus, which contains many ingredients that have immune checkpoint inhibitory effects, can be said to be a groundbreaking new species of mushroom that Japan can be proud of.

[0022] Now, even if Agaricus blazei contains active ingredients with immune checkpoint inhibitory effects, simply eating Agaricus blazei as is will not provide sufficient immune checkpoint inhibitory effects. This is because the active ingredients with immune checkpoint inhibitory effects are present in Agaricus blazei's very sturdy cell walls, and humans do not have the digestive enzymes to break down these cell walls, so they cannot be fully absorbed. Therefore, in order to obtain immune checkpoint inhibitory effects from Agaricus blazei, it is desirable to extract the active ingredients in the Agaricus blazei cell walls and process them into supplements before taking them. Here, the methods of extracting Agaricus blazei extracts currently used in supplements include hot water extraction and cell wall destruction. However, it is known that it is difficult to extract active ingredients with immune checkpoint inhibitory effects using hot water extraction and cell wall destruction 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 Agaricus blazei cell walls using an extraction technology called "subcritical water extraction method." Figure 3 is a schematic diagram showing how to manufacture a supplement with immune checkpoint inhibitory effects using subcritical water extraction method. This subcritical water extraction method does not use organic solvents, enzymes, acids, bases, etc., but extracts using only water, temperature, and pressure. This method makes it possible to efficiently extract active ingredients with immune checkpoint inhibitory effects from Agaricus blazei cell walls without destroying their composition or activity (Patent No. 7141630).

[0024] The extract produced by this method is called "Phimematsutake 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 grown from the Iwade 101 strain, which contains many active ingredients that have immune checkpoint inhibitory effects, and is said to have no risk of side effects.

[0025] [About Coprinus comatus] Coprinus comatus (also known as Coprinus) is a type of mushroom with a rounded cylindrical shape at the tip of the cap. It has attracted attention in various industries in recent years because it contains a sulfur-containing amino acid called ergothioneine. This ergothioneine (L-Ergothioneine) has very high antioxidant activity, and is recognized to have stronger activity than other antioxidant ingredients such as L-cysteine ​​and ascorbic acid (vitamin C). It is also known to have beauty effects, and ergothioneine is said to have elastase activity inhibitory effects that prevent wrinkles and sagging, and tyrosinase activity inhibitory effects that prevent age spots. In addition, ergothioneine is said to have various benefits such as anti-inflammatory effects, cellular energy promotion effects, and anti-stress effects, and it is known that ergothioneine has the ability to recognize and capture superoxide radicals, a type of active oxygen.

[0026] [About Cristapus bacteria] Lactobacillus crispatus is a lactic acid bacterium that originally exists in the vagina of women and is passed on to the intestines of infants via the birth canal of the mother during childbirth. It is said that Lactobacillus crispatus in the intestines reduces the risk of developing allergies and infectious diseases. It is believed that the amount of Lactobacillus crispatus in the intestines decreases with age, and the risk of developing these diseases increases accordingly. Therefore, a method of supplementing with Lactobacillus crispatus by taking it as a supplement has been attracting attention.

[0027] [Mixture of Coprinus comatus extract and crispatus fungus] As mentioned above, CP-101 inhibits immune checkpoints. On the other hand, the mixture of Coprinus comatus extract and crispatus fungus significantly strengthens immunity. Figure 4 is a schematic diagram showing how immunity is strengthened simultaneously with immune checkpoint inhibition. As shown in Figure 4, by taking CP-101, Coprinus comatus extract, and crispatus fungus, immune cells are given both the ability to "protect against cancer cells" and the ability to "attack cancer cells," and it is believed that a significant synergistic effect can be expected. EXAMPLES

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

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

[0030] [Study 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 leads to immune activation and anti-allergic effects. "IL-12" has the function of activating "NK cells," which control the body's immune power, and enhancing immunity. Furthermore, it has been reported that "IL-12" activates "Th1 cells," which lead to anti-allergic effects, and contributes 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. The test substance was added to the medium so as to reach a final concentration, and the amount of "IL-12" produced in the culture supernatant was measured by ELISA.

[0032] [Results and Discussion] FIG. 5 is a graph showing the results of the above test, from which the following can be read. · Control immune activity (IL-12pg / ml)…3 · Immune activity (IL-12pg / ml) of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract"...6 · Immune activity (IL-12pg / ml) of a mixture of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract" and "10% Bacillus crispatus"...159 · Immune activity of "10% Crispatus bacteria" (IL-12pg / ml)...100 FIG. 6 is a graph showing other results of the above test, from which the following can be read. · Control immune activity (IL-12pg / ml)…3 · Immune activity (IL-12pg / ml) of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract"...6 · Immune activity (IL-12pg / ml) of a mixture of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract" and "1% Bacillus crispatus"...136 · Immune activity of "1% Crispatus bacteria" (IL-12pg / ml)…65 FIG. 7 is a graph showing other results of the above test, from which the following can be read. · Control immune activity (IL-12pg / ml)…3 · Immune activity (IL-12pg / ml) of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract"...6 · Immune activity (IL-12pg / ml) of a mixture of "a mixture of Coprinus fasciatus extract and Agaricus blazei subcritical water extract" and "0.1% Bacillus crispatus"…98 · Immune activity of "0.1% Crispatus bacteria" (IL-12pg / ml)…35 In other words, when 10%, 1%, or 0.1% of crispatus bacteria was added to a mixture of Coprinus commune extract and Agaricus blazei subcritical water extract, immune activity was increased more than when either was used alone.

[0033] [About supplements] The "immune checkpoint inhibitor supplement" of this embodiment has as its main ingredients "subcritical water extract of Agaricus blazei (CP-101), Coprinus gracilis extract, and crispatus fungus" and is in the form of "tablets or hard capsules recommended," with the recommended daily amount (as CP-101) determined as follows: For cancer prevention in healthy individuals: 50 mg / day For cancer treatment: 100mg~ 200mg / day

Claims

1. An immune checkpoint inhibitor supplement comprising at least a subcritical water extract of Agaricus blazei Murill, 5 to 80% by weight of a subcritical water extract of Agaricus blazei Murill; 1 to 10% by weight of a Coprinus comatus extract, An immune checkpoint inhibitor supplement containing 0.1 to 10% by weight of crispatus bacteria.

2. An immune checkpoint inhibitor supplement comprising at least a subcritical water extract of Agaricus blazei Murill, 5 to 60% by weight of a subcritical water extract of Agaricus blazei Murill; 1 to 20% by weight of a Coprinus comatus extract, An immune checkpoint inhibitor supplement containing 0.1 to 20% by weight of crispatus bacteria.

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