JP2021106551A - JAK3 inhibitor - Google Patents

Abstract

To provide a JAK3 inhibitor in which a highly safe material is used.SOLUTION: Tamarind extract (1), grape stalk extract (2), meadow sweet extract (3), gallnut extract (4), combination (5) of α-lipoic acid and/or a salt thereof and L-ornithine, pomegranate extract (6), and guava extract (7) can be used as an active ingredient for JAK3 inhibitor.SELECTED DRAWING: None

Description

The present invention relates to a JAK3 (Janus kinase 3) inhibitor using a highly safe material.

Janus kinase (JAKs) is one of the non-receptor tyrosine kinases, and four types of JAK1, JAK2, JAK3, and TYK2 are known. JAKs phosphorylate cytokine receptors and are involved in signal transduction of various cytokines via a transcription factor called Stat. While JAK1, JAK2 and TYK2 are widely expressed, JAK3 expression is mainly localized to lymphocytes such as T cells, B cells and natural killer cells.

Inhibition of JAKs inhibits the signaling of 6 cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) and plays an important role in the immune system. Since it results in specific suppression of the functions of lymphocytes such as T cells and B cells, JAKs inhibitors minimized the occurrence of side effects for diseases related to the activation of these lymphocytes. It is expected to be an effective therapeutic agent (see Non-Patent Documents 1 and 2).

For example, diseases that can be treated with JAK3 inhibitors include autoimmune diseases, allergic diseases, nervous system diseases, inflammatory bowel diseases, psoriasis, contact dermatitis, diabetes, celiac disease, acute respiratory urgency syndrome (ARDS), and transplantation. It has been reported that it can be a therapeutic agent for one-sided host disease (GVHD), transplant rejection, blood malignant tumors (lymphoma, leukemia), and other malignant tumors (Non-Patent Documents 3 to 5).

Conventionally, various components having various JAKs inhibitory actions have been reported. For example, tofacitinib, a JAK3 inhibitor, has been clinically put into practical use as a therapeutic agent for rheumatoid arthritis. It has also been reported that a specific azaindole derivative having a cyclic substituent at the 4-position and a specific azaindole derivative having a cyclic substituent at the 3-position and the 5-position are also effective as JAK inhibitors. (Patent Documents 1 and 2). However, since all of these JAK inhibitors are non-natural synthetic compounds, there are concerns about their safety in long-term ingestion. Therefore, it is desired to develop a component capable of inhibiting JAKs by using a highly safe material having eating experience.

Immunol. Rev. , 2009, Vol. 228, No. 1, p. 273-287 Int. J. Biochem Cell Biol. , 2009, Vol. 41, No. 12, p. 2376-2379 Trends Pharmacol. Sci. , 2004, Vol. 25, No. 11, p. 558-562 PLoS One. , 2012, Volume 7, Issue 2, e31721 Cancer Discov. , 2012, Vol. 2, No. 7, p. 591-597

International Publication No. 2006/127587 International Publication No. 2006/004984

An object of the present invention is to provide a JAK3 inhibitor using a highly safe material.

As a result of diligent studies to solve the above problems, the present inventor conducted various studies on materials having eating experience and confirmed safety, and (1) tamarind extract and (2) grape stalk extraction. , (3) Meadow sweet extract, (4) Five-fold extract, (5) α-lipoic acid and / or its salt in combination with L-ornithine, (6) Pomegranate extract, and (7) Guava extract It was found that the product has excellent JAK3 inhibitory activity. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the inventions of the following aspects.
Item 1. (1) Tamarind extract, (2) Grape stalk extract, (3) Meadow sweet extract, (4) Five-fold extract, (5) Α-lipoic acid and / or its salt in combination with L-ornithine, A JAK3 inhibitor containing at least one selected from the group consisting of (6) pomegranate extract and (7) guava extract.
Item 2. Item 2. The JAK3 inhibitor according to Item 1, which is a food or drink or a drug for internal use.

According to the present invention, since JAK3 can be inhibited by using a naturally derived ingredient having eating experience, a product having high safety and exhibiting a JAK3 inhibitory effect in various forms such as foods and drinks and oral medicines. Can be provided.

It is a figure which shows the result of having measured the serum IFN-γ concentration of the mouse of each group in Test Example 2.

The JAK3 inhibitor of the present invention is (1) tamarind extract, (2) grape stalk extract, (3) meadow sweet extract, (4) pentagonal extract, (5) α-lipoic acid and / or a salt thereof. It is characterized by containing at least one selected from the group consisting of (6) pomegranate extract and (7) guava extract as an active ingredient. Hereinafter, the JAK3 inhibitor of the present invention will be described in detail.

[Active ingredient]
In the JAK3 inhibitor of the present invention, as active ingredients, (1) tamarind extract, (2) grape stalk extract, (3) meadow sweet extract, (4) quintuplet extract, (5) α-lipoic acid and / Or at least one selected from the group consisting of a combination of a salt thereof and L-ornithine, (6) pomegranate extract, and (7) guava extract is used. Hereinafter, each active ingredient will be described.

(1) Tamarind Extract The tamarind extract is an extract obtained by extracting tamarind (Tamarindus indica L.).

The extraction target site of tamarind is not particularly limited, but seeds and seed coats are preferable. The extraction target site of tamarind may be in a raw state as it is, but may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, and roasting, if necessary.

The extraction treatment may be any general extraction method used for producing plant extracts, and examples thereof include solvent extraction treatment, supercritical extraction treatment, and steam distillation treatment. Among these, solvent extraction treatment is preferable.

The extraction solvent used in the solvent extraction treatment includes water; lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, n-propanol, isopropanol and n-butanol; and many alcohols such as propylene glycol and 1,3-butylene glycol. Valuable alcohols; ketones such as acetone; chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; and mixtures thereof. Among these extraction solvents, water, lower alcohol, and a mixture thereof, more preferably water, ethanol, and hydrous ethanol can be mentioned. Examples of the ethanol content in the hydrous ethanol include 30 to 90% by volume, preferably 40 to 90% by volume, and more preferably 50 to 80% by volume.

The solvent extraction treatment may be carried out by immersing or refluxing the extraction target site of tamarind in the extraction solvent. A tamarind extract is obtained by removing the solid matter by solid-liquid separation after the extraction treatment. The obtained extract may be used as a liquid extract as it is, or may be used as a concentrated extract or a dry extract by removing a part or all of the solvent, if necessary.

(2) Grape stem extract The grape stem extract is an extract obtained by extracting the stems of plants belonging to the genus Vitis. The grape stalk used as an extraction raw material may be any one containing resveratrol, and the variety is not particularly limited. The grape stalks used as the extraction raw material may be in the raw state as they are, or may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, roasting, etc., if necessary. good. The method of extraction treatment for obtaining the grape stem extract, the type of extraction solvent used for the solvent extraction treatment, and the like are the same as in the case of the tamarind extract. As the grape stalk extract, the grape stalk extract from which solids have been removed after the extraction treatment may be used as it is as a liquid extract, or it may be used as a concentrated extract or a dry extract by removing a part or all of the solvent. good.

(3) Meadowsweet Extract The Meadowsweet Extract is an extract obtained by extracting Meadowsweet (Filipendula ulmaria). The extraction target site of the meadow sweet is not particularly limited, but the whole plant and the above-ground part are preferable. The extraction target portion of the meadow sweet may be in a raw state as it is, but may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, and roasting, if necessary. The method of extraction treatment for obtaining the Meadow Sweet Extract, the type of extraction solvent used for the solvent extraction treatment, and the like are the same as in the case of the tamarind extract. As the Meadow Sweet Extract, the Meadow Sweet Extract from which solids have been removed after the extraction treatment may be used as it is as a liquid extract, or it may be used as a concentrated extract or a dry extract by removing a part or all of the solvent. good.

(4) Five-fold extract Five-fold is a sac-like insect that is produced on the leaves by the stimulation of Rhus chinensis aphid parasitizing the young shoots and leaves of Rhus chinensis of the family Rhus chinensis. It is an extract obtained by treatment. The extraction target site of Rhus chinensis may be in a raw state as it is, but may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, and roasting, if necessary. The method of extraction treatment for obtaining Rhus chinensis extract, the type of extraction solvent used in the solvent extraction treatment, and the like are the same as in the case of the tamarind extract. As the quintuplet extract, the quintuplet extract from which solids have been removed after the extraction treatment may be used as it is as a liquid extract, or may be used as a concentrated extract or a dry extract by removing a part or all of the solvent.

(5) α-lipoic acid and / or a salt thereof and L-ornithine α-lipoic acid are also called thioctic acids and are known compounds existing in plants and human bodies. Examples of the salt of α-lipoic acid include alkali metal salts such as sodium salt and potassium salt. In the present invention, one of α-lipoic acid and a salt thereof may be used alone, or these may be used in combination.

L-ornithine, also called 2,5-diaminopentanoic acid, is a known compound present in clams and the human body.

In the present invention, the ratio of α-lipoic acid and / or a salt thereof to L-ornithine is not particularly limited, but for example, L-ornithine is used per 100 parts by weight of the total amount of α-lipoic acid and / or a salt thereof. Examples thereof include 40 to 90 parts by weight, preferably 50 to 80 parts by weight, and more preferably 60 to 70 parts by weight.

(6) Pomegranate extract The pomegranate extract is an extract obtained by extracting pomegranate (Punica granatum L.). The extraction target site of pomegranate is not particularly limited, but fruit and / or pericarp is preferable. The pomegranate extraction target portion may be in a raw state as it is, but may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, and roasting, if necessary. The method of the extraction treatment for obtaining the pomegranate extract, the type of the extraction solvent used for the solvent extraction treatment, and the like are the same as in the case of the tamarind extract. As the pomegranate extract, the pomegranate extract from which solids have been removed after the extraction treatment may be used as it is as a liquid extract, or may be used as a concentrated extract or a dry extract by removing a part or all of the solvent.

(7) Guava extract The guava extract is an extract obtained by extracting guava (Psidium guava). The extraction target site of guava is not particularly limited, but leaves are preferable. The extraction target portion of guava may be in a raw state as it is, but may be subjected to pretreatment such as crushing, cutting, steaming, kneading, drying, and roasting, if necessary. The method of the extraction process for obtaining the guava extract, the type of the extraction solvent used for the solvent extraction process, and the like are the same as in the case of the tamarind extract. As the guar extract, the guar extract from which solids have been removed after the extraction treatment may be used as it is as a liquid extract, or it may be used as a concentrated extract or a dry extract by removing a part or all of the solvent.

[Dosage form / form]
The dosage form of the JAK3 inhibitor of the present invention is not particularly limited and may be solid, semi-solid, or liquid.

The JAK3 inhibitor of the present invention is preferably in the form of oral ingestion or oral administration, but may be in the form of enteral administration or intravenous administration.

Specific examples of the form of the JAK3 inhibitor of the present invention include foods and drinks and medicines for internal use (including quasi-drugs for internal use).

When the JAK3 inhibitor of the present invention is formed into a food or drink form (that is, when it is provided as a food or drink for inhibiting JAK3), the active ingredient can be used as it is or in combination with other food materials or additive components to obtain a desired form. It may be prepared. Examples of such foods and drinks include general foods and drinks, health functional foods (including foods for specified health use, nutritional functional foods, supplements, etc.), foods for the sick, and the like. The form of these foods and drinks is not particularly limited, but specifically, beverages such as tea beverages, energy drinks, fruit juice beverages, carbonated beverages, and lactic acid beverages; capsules (soft capsules, hard capsules), tablets, granules. , Powders, jellies, supplements such as liposome preparations; luxury drinks such as gummy, candy, jelly and the like. Among these foods and drinks, supplements are preferable.

When the JAK3 inhibitor of the present invention is made into a pharmaceutical form for internal use (including quasi-drugs for internal use), the active ingredient may be prepared as it is or in combination with other additives to form a desired form. good. Specific examples of such an internal medicine include drinks, tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), troches, chewables, and extracts (extracts). (Including soft extracts, dry extracts, etc.), jelly agents, syrup agents, alcoholic agents, elixir agents, liposome preparations, and the like.

In the JAK3 inhibitor of the present invention, the content of the active ingredient may be appropriately set in consideration of the body shape, age, symptomatology, morphology, etc. of the subject and the daily intake / dose of the active ingredient. .. Examples of the daily intake / dose of the active ingredient include about 1 to 3000 mg, preferably about 250 to 3000 mg. Here, the daily intake / dose of the active ingredient is a value in terms of dry weight if the active ingredient is an extract, and the active ingredient is α-lipoic acid and / or a salt thereof and L-ornithine. In the case of a combination with, it is the value of these total amounts.

The JAK3 inhibitor of the present invention may be ingested or administered once a day or in a plurality of times a day, preferably 1 to 3 times a day.

More specifically, in the case where the JAK3 inhibitor of the present invention is formed into a liquid pharmaceutical form such as a beverage, the content of the active ingredient in the JAK3 inhibitor of the present invention is, for example, 1 to 90% by weight. , Preferably 10 to 60% by weight, more preferably 10 to 40% by weight. Here, the content of the active ingredient is a value in terms of dry weight when the active ingredient is an extract, and when the active ingredient is a combination of α-lipoic acid and / or a salt thereof and L-ornithine. If, it is the value of the total amount of these.

[Use]
The JAK3 inhibitor of the present invention is used for the purpose of inhibiting JAK3. Inhibition of JAK3 results in specific suppression of the functions of lymphocytes such as T cells and B cells, and suppressively regulates the immune response. Therefore, the JAK3 inhibitor of the present invention has excessive activity of these lymphocytes. It is effective for the prevention or treatment of related diseases and symptoms, as well as diseases and symptoms that require suppression of the activity of these lymphocytes. The JAK3 inhibitor of the present invention is also effective for the prevention or treatment of diseases and symptoms associated with overexpression of JAK3.

Specific examples of diseases and symptoms targeted for prevention or treatment of the JAK3 inhibitor of the present invention include autoimmune diseases (rheumatoid arthritis, systemic erythematosus, scleroderma, polymyositis / dermatomyositis, Schegren's syndrome, Bechett. Diseases), allergic diseases (bronchial asthma, allergic rhinitis / pollinosis, atopic dermatomyositis, food allergies, anaphylaxis, drug allergies, urticaria, conjunctivitis, etc.), nervous system diseases (multiple sclerosis, Alzheimer's disease, etc.) , Inflammatory bowel disease (ulcerative colitis, Crohn's disease), psoriasis, leukoplakia, osteoarthritis, alopecia alopecia, contact dermatomyositis, diabetes, celiac disease, acute respiratory urgency syndrome (ARDS), transplant pair Examples include host disease (GVHD), transplant rejection, blood malignant tumors (lymphoma, leukemia), non-hematological malignant tumors (solid cancer), and the like. Among these, suitable targets for the prevention or treatment of the JAK3 inhibitor of the present invention include rheumatoid arthritis, osteoarthritis, atopic dermatitis, psoriasis, psoriasis, and alopecia areata.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

The sources of the ingredients used in the following tests are as follows.
-Tamarind extract : A water extract of tamarind seeds under the trade name "Cocoa Brown TR-RS" (manufactured by Yaegaki Fermentation Giken Co., Ltd.).
Grape stalk extract: trade name "grape stalk extract" (manufactured by Agurokemi), hydrous ethanol grape stalk (ethanol 70% by weight) of the extract
-Meadow Sweet Extract : An extract obtained by extracting the whole plant of Meadow Sweet (trade name "Meadow Sweet Crushed Product", manufactured by Basin Holdings Co., Ltd.) with water.
-Rhus chinensis extract : Product name "Rhus chinensis" (manufactured by Tochimoto Tenkaido Co., Ltd.), water extract of Rhus chinensis.
-Mixture of α-lipoic acid and L-ornithine : trade name "Lalon" (manufactured by Omnica Co., Ltd.), α-lipoic acid: mixture of L-ornithine with a weight ratio of 6: 4.
- pomegranate extract: trade name "pomegranate extract" (manufactured by Agurokemi), water extract of the fruit and rind of pomegranate.
-Guava extract : Brand name "guava leaf extract powder" (manufactured by Kinshu Bio Co., Ltd.), guava leaf water extract.

Test Example 1: Measurement of JAK3 inhibitory activity (IC ₅₀₎
The JAK3 inhibitory activity of each component was measured using the Human JAK3 ELIZA measurement kit (Carna Biosciences Co., Ltd.). The specific measurement procedure is as follows.

To each well of the streptavidin-coated 96-well plate (PerkinElmer 4009-0010), 20 μL of the substrate solution included in the measurement kit was added. Then, to each well, 20 μL of a solution containing 0.96 to 120 μg / mL of each test substance shown in Table 1 was added. Further, as a reaction negative control and a reaction positive control, 20 μL of an aqueous solution containing 0.4% by weight of dimethyl sulfoxide was added instead of the solution containing the test substance. Then, 40 μL of 1 × AB (assay buffer) diluted according to the manual was added to the wells of the reaction negative control, and JAK3 attached to the measurement kit was manually added to the other wells. 40 μL of the enzyme solution diluted according to the above was added and incubated at 37 ° C. for 60 minutes. The solution in the wells was then completely removed to stop the enzymatic reaction and each well was washed twice with 200 μL PBS (containing 0.1 wt% Tween 20) and twice with 200 μL PBS. Then, 200 μL of the blocking buffer attached to the measurement kit was added to each well, and the plate was incubated for 30 minutes at room temperature with stirring. The solution in the wells was then removed, 100 μL of an antibody solution obtained by manually diluting the HRP-labeled antiphosphotyrosine antibody included in the measurement kit was added to each well, and the plate was incubated for 30 minutes at room temperature with stirring. Then, the solution in the wells is removed, and each well is washed twice with 200 μL PBS (containing 0.1 wt% Tween 20) and twice with 200 μL PBS, and then 100 μL of the color-developing solution attached to the measurement kit is added to each. It was added to the wells and the plates were incubated for 5 minutes at room temperature with stirring. Next, 100 μL of a color stop solution (97 wt% sulfuric acid aqueous solution) was added to each well, and the absorbance of each well was measured with a microplate reader (450 nm).

According to the following formula, the JAK3 inhibition rate of the test substance at each concentration was calculated, and the IC ₅₀ was determined.

The results obtained are shown in Table 1. As a result, JAK3 was added to tamarind extract, grape stalk extract, meadow sweet extract, quintuplet extract, α-lipoic acid and / or a combination of a salt thereof and L-ornithine, pomegranate extract, and guava extract. It was revealed that it has excellent inhibitory activity and is effective as a JAK3 inhibitor.

Test Example 2: Verification of JAK3 inhibitory effect using inflammation model mice DBA / 1J mice (7 weeks old, male) were bred for 6 days in an environment where water and food were freely ingested, and then fasted for 24 hours. I let you. Mice were divided into 4 groups (5 mice in 1 group) so that the weights of each group were even, and treatment was performed under the conditions shown in Table 2. Serum was prepared from the blood obtained in each group, and the IFN-γ concentration in the serum was measured using the INF-γ Mouse In Vivo Capture Assay (BD Biosciences # 558491).

The obtained results are shown in FIG. When IL-2, which is an inflammation-inducing substance, and an anti-IFN-γ antibody were administered to the mice of Comparative Example 1 (distilled water administration group), the serum IFN-γ concentration increased and inflammation was induced. On the other hand, in the mice of Example 3 (Meadow sweet extract administration group), even if IL-2 and anti-IFN-γ antibody were administered, the serum was comparable to that of Reference Example 1 (tofacitinib administration group). The increase in IFN-γ concentration was suppressed, and inflammation could be suppressed. That is, this result suggests that administration of Meadowsweet extract inhibited JAK3 and suppressed the activation of the JAK-STAT pathway.

Claims (2)

(1) Tamarind extract, (2) Grape stalk extract, (3) Meadow sweet extract, (4) Five-fold extract, (5) Α-lipoic acid and / or its salt in combination with L-ornithine, A JAK3 inhibitor containing at least one selected from the group consisting of (6) pomegranate extract and (7) guava extract. The JAK3 inhibitor according to claim 1, which is a food or drink or a drug for internal use.

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