JP2022177817A - Composition containing ketone donor - Google Patents

Abstract

To provide a composition containing poly(D-3-hydroxybutyric acid) at an effective dose.SOLUTION: The present invention discloses: a composition containing poly(D-3-hydroxybutyric acid) of 0.02 wt.% or more; or a pharmaceutical composition, a feed composition, and a food or beverage composition, each containing poly(D-3-hydroxybutyric acid) of 0.2 wt.% or more and less than 5.0 wt.% or 0.2 wt.% or more and less than 2.0 wt.%. The composition is used such that the daily intake of poly(D-3-hydroxybutyric acid) is 90 mg/m2 body surface area or 900 mg/m2 body surface area or more.SELECTED DRAWING: None

Description

There is an application for exception to loss of novelty

The present invention relates to compositions containing ketone donors, in particular poly(D-3-hydroxybutyric acid), which have anti-tumor and anti-intestinal inflammatory effects.

A ketone body is a general term for compounds in which a carbonyl group and two hydrocarbons are bonded. 3-hydroxybutyric acid (hereinafter also referred to as "3HB"), acetoacetic acid, acetone, etc. exist in the body and exert various physiological actions. For example, for 3-hydroxybutyric acid, various diseases that appear with aging, such as cancer and heart disease (Non-Patent Document 1), neurological diseases such as Alzheimer's disease (Non-Patent Document 2) A therapeutic effect on diseases has been reported. It has also been reported that it has a therapeutic effect on diseases including epileptic seizures in dogs used as pets (Non-Patent Document 3).

Ketone bodies are degraded by hydrolytic enzymes in the small intestine and bacterial flora present in the large intestine, are rapidly absorbed from the intestinal epithelium, and are then released into the blood. However, it is difficult to maintain the concentration of ketone bodies in the blood for a long period of time. In this regard, it is possible to administer a ketone donor to the body to maintain blood ketone body levels. Ketone donors have also been reported to be effective in treating various diseases. For example, oral administration of poly(D-3-hydroxybutyric acid), a ketone donor, exerts an inhibitory effect on cancer growth (Patent Document 1); It has been reported that it exerts preventive and therapeutic effects on diseases (Patent Document 2), exerts an effect of suppressing blood sugar level (Patent Document 3), and the like.

As described above, ketone bodies or ketone donors exert preventive and/or therapeutic effects on various diseases, and thus are expected to be applied to pharmaceuticals and the like. However, at present, there is no report on the lower limit of the effective dose, etc., in order to avoid side effects caused by administration of ketone bodies and ketone donors.

WO2020/250980 publication WO2005/021013 publication WO2019/035486 publication

Han et al., Exp Mol Med., 52:548-555 2020. Avgerionos et al., Int Rev Neurobiol., 154:79-110 2020. Berk et al., J Vet Intern Med., 34:1248-1259 2020

In view of the above circumstances, the present invention provides a composition containing a ketone donor poly(D-3-hydroxybutyric acid) (hereinafter also referred to as "PHB"), The problem to be solved is the effective amount of PHB contained in the composition (the lower limit of the amount contained in the composition, etc.).

The inventors fed breast cancer tumor model mice with diets containing 0.02% by weight, 0.20% by weight, and 2.0% by weight of PHB by free feeding, and found that the diet containing PHB at all concentrations showed no tumor growth. In addition, the extension of survival period was confirmed in 0.20 wt% and 2.0 wt% PHB-containing feeds. In addition, the inventors have found that breast cancer tumor model mice, malignant melanoma model mice, and lymphoma model mice were fed with a diet containing PHB and administered with an anticancer agent, and found that tumor growth inhibitory effects and survival A synergistic effect of PHB and anticancer drugs was confirmed for the effect of prolonging the period.

Furthermore, mice were pre-fed with 2.0 wt% PHB-supplemented diet ad libitum, and then dextran sulfate sodium (DSS) was administered to the mice to induce colitis. . As a result, mice fed 2.0 wt% PHB-supplemented diet had longer survival times and better clinical scores and body weight than control mice (mice not fed 2.0 wt% PHB-supplemented diet). good results were obtained. Previously, it was reported that inflammatory bowel disease model mice were prevented and treated with a feed containing 5.0% by weight of PHB (Patent Document 2). It was demonstrated for the first time by the present invention that the administration of PHB can also prevent and treat inflammatory effects. Furthermore, the present inventors also clarified that the anti-inflammatory action of PHB is mediated by promoting the expression of anti-inflammatory cytokines and enhancing the action of regulatory T cells.
Therefore, the present inventors suggested that PHB exhibits broad therapeutic effects on autoimmune diseases such as inflammatory bowel disease.

The present invention has been completed based on the above findings. That is, the present invention is the following (1) to (17).
(1) A composition containing 0.02% by weight or more of poly(D-3-hydroxybutyric acid).
(2) The composition according to (1) above, containing 0.2% by weight or more of poly(D-3-hydroxybutyric acid).
(3) The composition according to (1) or (2) above, containing less than 5.0% by weight of poly(D-3-hydroxybutyric acid).
(4) The composition according to (3) above, containing less than 2.0% by weight of poly(D-3-hydroxybutyric acid).
(5) A composition containing poly(D-3-hydroxybutyric acid), which is used so that 90 mg/m ² body surface area or more of poly(D-3-hydroxybutyric acid) is ingested per day. The composition according to (1) above, characterized in that
(6) The composition according to (5) above, which is used such that 900 mg/m ² body surface area or more of poly(D-3-hydroxybutyric acid) is ingested per day.
(7) The composition according to (1) above, which is used for disease treatment.
(8) The composition according to (7) above, wherein the disease is cancer.
(9) A combination composition comprising the composition according to (8) above and an anticancer agent, wherein the composition and the anticancer agent have a synergistic effect. thing.
(10) The composition according to (7) above, wherein the disease is an autoimmune disease.
(11) The composition according to (10) above, wherein the autoimmune disease is inflammatory bowel disease.
(12) The composition according to (1) above, wherein the composition is a pharmaceutical composition, a feed composition, or a food composition.
(13) The composition according to (12) above, wherein the feed composition is a dietary food.
(14) A composition for suppressing an excessive immune reaction, the composition containing poly(D-3-hydroxybutyric acid) (PHB).
(15) The composition according to (14) above, wherein the composition is a pharmaceutical composition, a feed composition, or a food composition.
(16) The composition according to (15) above, wherein the feed composition is a dietary food.
(17) The composition according to (15) above, wherein the pharmaceutical composition is for treatment of autoimmune disease or allergic disease.
(18) The composition according to (14) above, which suppresses an excessive immune response by promoting the expression of anti-inflammatory cytokines.
(19) The composition according to (14) above, which suppresses excessive immune reactions by increasing the number of regulatory T cells.
In this specification, the sign "-" indicates a numerical range including the values on the left and right of it.

INDUSTRIAL APPLICABILITY According to the present invention, the lower limit of the effective content contained in a PHB composition or the lower limit of the effective intake of PHB per day becomes clear, and it becomes possible to provide a composition containing an appropriate amount of PHB. . Where the composition is a medicament or pharmaceutical composition, the present invention is also advantageous in that it provides a low dose medication of PHB and avoids possible side effects.

Investigation of the degradation process of PHB in vivo. A shows a PHB degradation model in the intestine. B shows time course of 3HB concentration in mouse feces after administration of PHB. C shows time course of ketone body (3HB) concentration in mouse blood after administration of PHB. For C, n=5, ^* P<0.05, values are shown as mean±s.e.m. Examination of the antitumor effect of PHB using a transplanted tumor (breast cancer) model. After implanting mammary adenocarcinoma E0771 cells into mice, the mice were fed diets containing various concentrations of PHB by ad libitum. Tumor volume (A), survival rate (B) and body weight change (C) after feeding are shown. The table summarizes the median survival times and P-values for groups of mice fed various concentrations of the PHB-mixed diet. Investigation of anti-tumor effects of combined use of PHB and anti-cancer drugs using transplanted tumor (breast cancer, malignant melanoma and malignant lymphoma) models. A shows changes in tumor volume over time (left) and survival rate (right) when 2.0% by weight of PHB and Carboplatin (CBDCA) were administered in combination to a breast cancer model implanted with E0771 cells. B shows changes in tumor volume over time (left) and survival rate (right) when 2.0% by weight of PHB and Carboplatin (CBDCA) were administered in combination to a malignant melanoma model implanted with B16F10 cells. C shows changes in tumor volume over time (left) and survival rate (right) when 2.0% by weight of PHB and Vincristine (VCR) were administered in combination to a lymphoma model implanted with EL4 cells. n=8, ^* P<0.05, ^** P<0.01, ^*** P<0.001, values are shown as mean ± standard error. Investigation of combined effects of ketone ester addition and radiation irradiation using breast cancer cells. Fig. 2 shows the relationship between irradiation intensity and cell viability when E0771 cells cultured in a medium supplemented with 0.5 mM ketone ester (3HB dimer) were irradiated with X-rays. CTRL is the result of culturing in a medium containing no ketone ester, and KE is the result of culturing in a medium containing a ketone ester. Investigation of the antitumor effect of PHB using a mouse model implanted with human breast cancer. After transplanting human breast cancer MDAMB231 cells into mice, mice were fed diets containing various concentrations of PHB by ad libitum. Post-feeding tumor volume (A) and survival (B) are shown. Investigation of the effect of prior administration of PHB monotherapy on inflammatory bowel disease. The figure above shows the outline of the experiment. Mice were fed with a 2.0% by weight PHB mixed diet, and two weeks after the feeding, 3% dextran sulfate sodium (DSS) aqueous solution was given freely to the mice to induce colitis. The mouse survival rate (A), clinical score (Disease activity index: DAI) (B) and body weight (C) calculated or measured over time after administration of 3% DSS are shown. Investigation of the effect of post-administration of PHB monotherapy on inflammatory bowel disease (1). The figure above shows the outline of the experiment. Mice were given a 2% dextran sulfate sodium (DSS) aqueous solution ad libitum to induce colitis, and then fed a diet containing 2.0% by weight of PHB on day 4. The mouse survival rate (A), clinical score (Disease activity index: DAI) (B) and body weight (C) calculated or measured over time after administration of 2% DSS are shown. Investigation of the effect of post-administration of PHB monotherapy on inflammatory bowel disease (2). Mice were given a 2% dextran sulfate sodium (DSS) aqueous solution ad libitum to induce colitis, and then fed a diet containing 2.0% by weight of PHB on day 4. Then, mouse large intestine tissue sections were prepared, stained with HA (A), and evaluated for histological score (HS) (B). Furthermore, A shows HE-stained images of mice, and B shows the results of evaluation of histological scores in large intestine tissues collected from mice. Furthermore, gene expression levels of cytokines (IL-2 and IL-10) and Foxp3 were measured (C). Investigation of the effect of post-administration of PHB monotherapy on inflammatory bowel disease (2). Mice were given a 2% dextran sulfate sodium (DSS) aqueous solution ad libitum to induce colitis, and then fed a diet containing 2.0% by weight of PHB on day 4. Thereafter, mouse large intestine tissue sections were prepared, immunostained using an anti-Foxp3 antibody (A), and the number of Foxp3-positive cells was counted (B). Arrowheads in A indicate Foxp3-positive cells.

式（１）において、ｎは整数であって、特に限定はしないが、例えば、100以上3000以下、好ましくは500以上2500以下、より好ましくは2000程度である。本実施形態にかかるPHBは、当業者であれば公知の方法により容易に合成することができ、例えば、WO2020/250980に開示された合成方法により合成してもよい。または、市販品を購入してもよい。 A first embodiment of the present invention is a composition containing poly(D-3-hydroxybutyric acid) (PHB).
The PHB according to the present embodiment is a compound represented by the following formula 1, and is decomposed in vivo to form D-3-hydroxybutyric acid (3HB) represented by the following formula 2. becomes.

In formula (1), n is an integer and is not particularly limited. The PHB according to this embodiment can be easily synthesized by a method known to those skilled in the art, and may be synthesized by the synthesis method disclosed in WO2020/250980, for example. Or you may purchase a commercial item.

The lower limit amount of PHB contained in the composition according to the present embodiment is, for example, 0.02% by weight or more, 0.03% by weight or more, 0.04% by weight or more, 0.05% by weight or more, 0.06% by weight or more, 0.07% by weight or more, and 0.08% by weight. 0.09 wt% or more, 0.10 wt% or more, 0.11 wt% or more, 0.12 wt% or more, 0.13 wt% or more, 0.14 wt% or more, 0.15 wt% or more, preferably 0.02 wt% or more, 0.05 wt% or more % or more and 0.1% by weight or more. There is no particular upper limit to the amount of PHB contained in the composition. The upper limit of the amount of PHB contained in the product is, for example, less than 5.0% by weight, preferably 4.0% by weight or less, 3.0% by weight or less, 2.0% by weight or less, 1.0% by weight or less, 0.5% by weight or less, and 0.4% by weight. 0.3% by weight or less, 0.2% by weight or less, and more preferably 2.0% by weight or less, 1.0% by weight or less, and 0.2% by weight or less.
The PHB content can also be specified by combining the above-mentioned lower limit and upper limit of PHB.
The effective intake of the composition according to the present embodiment (the intake required to improve the disease or pathological condition) is not particularly limited, but for example, 90 mg or more of PHB per m ² of body surface area per day. , More preferably, the amount is enough to ingest 900 mg of PHB, and the sufficient amount for ingestion of PHB is 9,000 mg.
The effective intake may be recalculated per kg body weight for each animal type. The coefficient for converting the intake per 1 m ² of body surface area to 1 kg of body weight is, for example, but not limited to, the US Food and Drug Administration (FDA; Food and Drug Administration) Guidance for setting the initial dose (Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, July 2005: FDA Guidance) may be used. Specifically, although not particularly limited, for humans, it may be divided by the above factor, for example, 37, and converted to 2.4 mg or more of PHB, more preferably 24.3 mg or more of PHB per 1 kg of body weight per day. , for dogs, it may be divided by the above factor, for example, 20, and converted to PHB of 4.5 mg or more, more preferably PHB of 45.0 mg or more per kg of body weight per day, and the above factor for rabbits, for example, It may be divided by 12 and converted to 7.5 mg or more of PHB, more preferably 75.0 mg or more of PHB per 1 kg of body weight per day. PHB may be converted to 11.2 mg or more, more preferably PHB 112.5 mg or more per day, and divided by the above coefficient for ferrets, for example, 7, and PHB 12.8 mg or more per 1 kg of body weight per day. Preferably, PHB may be converted to 128.5 mg or more, and divided by the above factor, for example, 6 for rats, and PHB is converted to 15 mg or more, more preferably PHB to 150 mg or more per 1 kg of body weight per day. It may be divided by the above factor, for example, 5 for hamsters, and converted to 18 mg or more of PHB, more preferably 180 mg or more of PHB per 1 kg of body weight per day. By dividing the above factor by 3, for example, PHB may be converted to 30 mg or more, more preferably 300 mg or more, of PHB per 1 kg of body weight per day.

The composition according to this embodiment has a preventive and / or therapeutic effect on diseases such as cancer and autoimmune diseases, and may be used as a pharmaceutical composition, food and drink composition, and It can be provided as a feed composition (including dietetic food) and the like, but is not limited to these compositions. In addition, even compositions other than pharmaceutical compositions, including feed compositions and food and drink compositions, can be used to treat various diseases of humans and non-human animals (e.g., cancer, autoimmune diseases, etc.), and to treat pathological conditions. may be used for the improvement, remission and cure of

When the composition according to the present embodiment is a composition for treating a disease (a composition used for the purpose of treating a disease, etc.), the disease to be treated by the composition is not particularly limited, but cancer and It is an autoimmune disease. Examples of the cancer include hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, and myxosarcoma. , liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, malignant meningioma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, brain tumor, epithelial Cell-derived neoplasms (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, lip cancer, oral cancer, esophageal cancer, gastrointestinal cancer such as small and gastric cancer, colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer cancer, cervical cancer, lung cancer, breast cancer, skin cancer such as squamous cell carcinoma and basal cell carcinoma, prostate cancer and renal cell carcinoma, and others that affect epithelial, mesenchymal or blood cells throughout the body. Known cancers, etc.

Autoimmune disease is a general term for diseases that develop when the immune system fails to function normally and attacks self-organizations and cells.
Examples of autoimmune diseases according to this embodiment include inflammatory bowel diseases (controlled ulcerative colitis, Crohn's disease, etc.), type 1 diabetes, which are caused by decreased or lost function of regulatory T cells, etc. Rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, and the like. In particular, the therapeutic composition according to this embodiment is expected to have therapeutic effects on inflammatory bowel disease.

When the composition according to this embodiment is a pharmaceutical composition, the subject of administration may be not only humans but also non-human animals. The pharmaceutical composition may be in an oral or parenteral dosage form, and is not particularly limited. Examples include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, Examples include creams, gels, patches, inhalants, injections, and the like. These formulations are prepared according to a conventional method. Liquid formulations may be dissolved or suspended in water or other suitable solvents at the time of use. Moreover, tablets and granules may be coated by a well-known method. Injections are prepared by dissolving PHB in water, but if necessary, they may be dissolved in physiological saline or glucose solution, and buffers and preservatives may be added.

The type of pharmaceutical additive used in the production of the pharmaceutical composition according to the present embodiment, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical composition are appropriately selected by those skilled in the art according to the form. It is possible to As additives for formulations, inorganic or organic substances, or solid or liquid substances can be used. It can be blended at 95.0% by weight, or between 1% and 90.0% by weight. Specifically, examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, and calcium carboxymethylcellulose. , ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, refined lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol or water.

In order to produce a solid preparation for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate or silicic anhydride to form a powder, or if necessary A binder such as sucrose, hydroxypropyl cellulose or polyvinylpyrrolidone, and a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium are added and wet or dry granulated to form granules. To produce tablets, these powders and granules may be compressed directly or after adding a lubricant such as magnesium stearate or talc. These granules or tablets are coated with an enteric base such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate polymer to form an enteric preparation, or ethylcellulose, carnauba wax or hardened oil to form a sustained release preparation. You can also To manufacture capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with gelatin to form soft capsules. be able to.

For the production of injections, the active ingredient may be mixed with hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, pH adjusters such as sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, if necessary. Dissolve in distilled water for injection with a tonicity agent, filter aseptically and fill in an ampoule, or further add mannitol, dextrin, cyclodextrin or gelatin and lyophilize in a vacuum to prepare a dissolution type injection for use. good too. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, or the like may be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

For the preparation of rectal formulations, the active ingredient is dissolved by moistening with a suppository base such as cocoa butter, tri-, di- and monoglycerides of fatty acids or polyethylene glycol, poured into molds and allowed to cool, or the active ingredient is extruded. After dissolving in polyethylene glycol, soybean oil or the like, it may be coated with a gelatin film or the like.

The dosage and frequency of administration of the pharmaceutical composition according to the present embodiment are not particularly limited, and depending on the conditions such as prevention of deterioration and progression of the disease to be treated and / or purpose of treatment, type of disease, patient's weight and age, etc. Physicians, pharmacists, and veterinarians in the case of veterinary medicines can make appropriate selections based on their judgment.
In general, the daily dose for oral administration is about 90 mg or more, preferably about 900 mg, per ^square meter of body surface area, and can be administered once or several times a day. . When converted to 1 kg body weight, humans are about 2.4 mg or more, preferably about 24.3 mg, dogs are about 4.5 mg or more, preferably about 45.0 mg, and rabbits are about 7.5 mg or more, preferably about 75.0 mg. becomes.

When the composition according to the present embodiment is a food and drink composition, its form is not particularly limited, and examples include beverages such as soft drinks and nutritional drinks; Milk beverages, fermented milk, yogurt drinks, dairy products such as butter, and other supplements may also be used.

The composition according to this embodiment may be a feed composition that can be ingested by non-human animals. Feed as used herein refers to so-called feeds that non-human animals need to take as a source of nutrients, but is not particularly limited, but for example, livestock such as pigs and cattle, feed additives used for breeding farmed fish, dogs and cats. , and pet food (dietary food) given for the purpose of dietary therapy for pets such as rabbits.
"Pet food given for the purpose of diet therapy" is generally defined as a food in which the amount and ratio of nutritional components are adjusted, and which is used by veterinarians in veterinary medicine for the purpose of nutritional support for pets with specific diseases or health conditions. Means intended to be used for dietary management under the guidance of , and is sold at veterinary hospitals, but is not limited to this.
"Pet food given for the purpose of diet therapy" is not particularly limited, but may be, for example, solid pet food, liquid pet food, or pet supplements given for the purpose of supplementing nutritional sources. Well, in the case of supplements for pets, although not particularly limited, it may be in the form of tablets, capsules, granules, powders, syrups, liquids, and the like.
A person skilled in the art can appropriately select the raw materials of the "pet food given for the purpose of dietary therapy" according to its form. Specifically, grains, potatoes, starches, sugars, nuts, beans, vegetables, fruits, mushrooms, algae, seafood, meat, eggs, milk, oils, vitamins, minerals , amino acids, and other additives may be used.
A person skilled in the art can appropriately select the manufacturing method of the “pet food given for the purpose of diet therapy” according to its form. Specifically, it may be produced using a mixer, steamer, molding machine, dryer, heat sterilizer, freezer or the like, or produced by a simple method such as drying in the sun.

A second embodiment of the present invention is a composition for treating and/or treating cancer, characterized in that the composition according to the first embodiment and an anticancer agent are used in combination (the above-mentioned A combination composition of the composition and an anticancer agent), and a combination composition having a synergistic action of the composition and the anticancer agent. The anticancer agent in the combination composition according to this embodiment is not particularly limited, and can be selected according to the cancer to be prevented or treated. Examples include, but are not limited to, aclarubicin, doxorubicin, evirubicin, biralubicin, nimtine, prednisolone, anastrozole, irinotecan, carboplatin, cisplatin, nedaplatin, gemcitabine, cytarabine, cyclophosphamide, tamoxifen, dexamethasone, methotrexate, trastuzumab, nivolumab , ibilimumab, lavatinib, paclitaxel, docetaxel, vincristine, etoposide, fluorouracil, mitomycin C, and other anticancer agents.

In the administration method of the combination composition according to the present embodiment, the order of administration of the composition according to the first embodiment and the anticancer agent is not particularly limited. administration, or administration after the anticancer agent. In addition, the administration interval when the composition according to the first embodiment and the anticancer agent are administered at different times is not particularly limited, but is, for example, 1 to 12 hours, 12 to 24 hours, 24 hours to 48 hours, etc., can be appropriately selected by doctors, veterinarians, and other experts according to the condition of the target disease.

In addition, in the combination composition according to this embodiment, the dosage of the anticancer drug can be appropriately determined by doctors, veterinarians, and other experts according to the anticancer drug used. For example, when the cancer to be treated is breast cancer and malignant melanoma, and the anticancer agent is carboplatin, it may be administered in a unit dose of 50 mg/kg to 200 mg/kg. When the cancer is lymphoma and the anticancer agent is vincristine, it may be administered at a unit dose of 0.1 mg/kg to 1.0 mg/kg.

A third embodiment of the present invention provides a method for preventing and/or preventing a disease, comprising administering the composition according to the first embodiment or the combination composition according to the second embodiment to a subject for prevention and/or treatment. or a method of treatment. Furthermore, when the treatment target is cancer, the administration of the composition according to the first embodiment or the combination composition according to the second embodiment to the treatment target may be combined with irradiation. Here, the order of administration of the composition according to the first or second embodiment and radiation exposure is preferably that the composition according to the first or second embodiment is administered first, but is limited to this order. not a thing
Diseases according to this embodiment are not particularly limited, but examples include cancer (e.g., breast cancer, malignant melanoma, lymphoma, etc.) and inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, etc.). be able to.
As used herein, "treatment" means preventing or alleviating the progression and worsening of the condition in a subject (human and non-human animal) already afflicted with the disease, thereby preventing or slowing down the progression and worsening of the disease. A treatment intended to provide relief.
In addition, "prevention" means to prevent the onset of a disease in subjects (humans and non-human animals) that may develop a disease requiring treatment, thereby preventing the onset of the disease in advance. It is a procedure intended to
In addition, the subject of treatment and prevention is not limited to humans, non-human mammals such as mice, rats, dogs, cats, small animals such as rabbits, ferrets, guinea pigs, hamsters, chinchillas, hedgehogs, degus, cows, Livestock such as horses, sheep and pigs, monkeys, primates such as chimpanzees and gorillas, and the like may also be used.

A fourth embodiment is a composition for suppressing an excessive immune response, comprising poly(D-3-hydroxybutyric acid) (PHB), Alternatively, it is the use of a composition containing poly(D-3-hydroxybutyric acid) (PHB) for suppressing an excessive immune reaction.
Since PHB has the effect of increasing regulatory T cells, it exerts the function of suppressing excessive immune reactions in vivo that occur in autoimmune diseases, allergic diseases, and the like. Therefore, the composition according to the fourth embodiment can also be used for the purpose of treating autoimmune diseases, allergic diseases, etc., or alleviating pathological conditions.
For the composition containing poly(D-3-hydroxybutyric acid) (PHB) according to the fourth embodiment, see also the descriptions of other embodiments.

Where this specification is translated into English and contains the words "a", "an" and "the" in the singular, the plural as well as the singular unless the context clearly indicates otherwise. It shall also include things.
EXAMPLES The present invention will be further described below with reference to Examples, but these Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Administration method of PHB to mice
PHB was purchased from Beyond Science Co., Ltd. with a weight average molecular weight of 590,000 or less derived from the genus Halomonas produced according to a previous report (Patent Document 1). Mice (C57BL6/N) used in the experiments were purchased from Oriental Yeast Co., Ltd. For administration to mice, add PHB to rodent diet (CE-2, Clea) to 0.02% by weight, 0.2% by weight or 2.0% by weight, mix well and feed by free feeding method. (oral administration). In addition, when mice were fed with a diet containing 0.2% by weight of PHB, the average PHB mixed diet intake of mice was 3,000 mg, and the daily intake of PHB was calculated. A certain amount of PHB was ingested. Therefore, when mice were fed diets containing 0.02 wt% PHB, 0.2 wt% PHB, and 2.0 wt% PHB, the mice received 30 mg/kg body weight and 300 mg/kg body weight, respectively, per day. And about 3,000 mg/kg body weight of PHB was ingested.
To convert the intake per kg of body weight to the intake per m ² of body surface area, see Table 1 of the FDA guidance (Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, July 2005). The indicated coefficients were used. Therefore, multiplied by a factor of 3 in mice, they ingested about 90 mg/m ² body surface area, 900 mg/m ² body surface area and 9,000 mg/m ² body surface area of PHB, respectively. Furthermore, when the intake per 1 m ² of body surface area is converted to 1 kg of body weight for each animal type, divide by the coefficient 37 described in the FDA guidance for humans, and each day is 2.4 mg/kg body weight. , 24.3 mg/kg body weight, and 243.2 mg/kg body weight, divided by the factor of 20 described in the FDA guidance for dogs, resulting in 4.5 mg/kg body weight, 45.0 mg/kg body weight, and 450 mg/kg body weight per day, respectively. mg/kg body weight, divided by the factor of 12 given in the FDA guidance for rabbits, resulting in 7.5 mg/kg body weight, 75.0 mg/kg body weight and 750 mg/kg body weight per day, respectively.

2. Investigation of PHB degradation process in vivo We investigated the process through which PHB is degraded into 3HB in vivo.
Eight-week-old male mice were fed diets supplemented with 0.2% or 2.0% by weight of PHB ad libitum.
2-1. Method (1) Measurement of 3HB content in feces
On days 0, 2, 4, 6 and 8 after PHB administration, 10 mg of a sample (stool) was collected from the mouse and dissolved in 90 μL of MilliQ water. 50 μL of the fecal suspension was collected, and 5 μL of the internal standard substance 2-isopropylmalic acid (1 mg/mL) and methanol/water/chloroform (5:2:2) solution were added to the collected suspension. 250 μL was added, vortexed for 30 seconds, and then incubated at 37° C. for 30 minutes. After incubation, the solution was centrifuged (16,000×g, 5 min, 4° C.), 225 μL of the resulting supernatant was taken, 200 μL of MilliQ water was added, and vortexed for 30 seconds. After vortexing, the solution was further centrifuged (16,000×g, 5 min, 4° C.), and 250 μL of the resulting supernatant was taken and concentrated by centrifugation at room temperature for 20 minutes. The concentrate was frozen at -80°C and lyophilized to complete dryness.
40 μL of methoxyamine hydrochloride (40 mg/ml, pyridine solution) was added to the dry matter, vortexed for 30 seconds, and incubated at 30° C. for 90 minutes. 40 μL of MSTFA (N-Methyl-N-trimethylsilyltrifluoroacetamide)+1%TMCS (trimethylchlorosilane) was added to the solution after incubation, vortexed for 30 seconds, and incubated at 37° C. for 45 minutes. The obtained reaction solution was transferred to a vial and measured by GC-MS. The measurement conditions of GC-MS are as follows.
Injection volume: 1 μl
Injection method: split (10:1)
Inlet temperature: 250°C
Column: DB-5ms Duraguard (30m, 250μm, 0.25μm)
Temperature rising conditions: 60℃(1min)-R10℃/min→240℃-R20℃/min→325℃(0min)
Flow rate: 1.0ml/min (constant pressure) 57kPa
Transfer line temperature: 290℃
Ionization mode: ESI

(2) Measurement of 3HB level in blood
Ketone bodies (3HB) in the blood collected using Niprostat strips and ketone body measurement chips, blood was collected from the tail vein of mice on days 0, 2, 4, 6 and 8 after PHB administration. amount was measured.

2-2. result
Fig. 1B shows the time course of 3HB level in feces of PHB-administered mice, and Fig. 1C shows the time course of 3HB level in blood.
The amount of 3HB in the feces peaked on the 1st day after the start of PHB administration and then decreased. On the other hand, 3HB in the blood peaked 3 days after the start of PHB administration and then decreased. From the above results, it is considered that orally administered PHB is first degraded into 3HB by bacteria in the large intestine, absorbed in the large intestine, and then released into the blood (Fig. 1A).

3. Examination of the effect of PHB monotherapy on mouse breast cancer 3-1. Methods The effect of PHB monotherapy on tumor volume increase and mouse survival was evaluated in a transplanted tumor model. 2 × 10 ⁶ mouse mammary adenocarcinoma E0771 cells and 100 µL of Dulbecco's modified Eagle (DMEM) medium containing 30% Matrigel were implanted into the dorsum of C57BL/6N mice, and the tumor size was measured over time to determine the tumor volume. was calculated. Tumor volume was calculated by the following formula.
Tumor volume = major axis x minor axis x minor axis x 0.52
Eight days after tumor transplantation, PHB mixed feed containing 0% by weight (control: CTRL), 0.02% by weight, 0.2% by weight and 2.0% by weight of PHB was fed ad libitum. Tumor volume was measured over time, death, tumor volume exceeding 1500 mm ³ , obvious respiratory abnormalities, body weight loss of 10% or more, macroscopically clear metastasis. Survival rate was analyzed with the case of death as the endpoint.

3-2. Results Figure 2A shows the tumor volume of mice implanted with mammary adenocarcinoma, Figure 2B shows the survival rate, and Figure 2C shows the change in body weight. In addition, the median survival rate and P value of the group of mice fed each PHB mixed diet were summarized in the table. A tumor growth-inhibiting effect and a prolonged survival period were observed in the mouse group fed with the PHB-mixed diet containing 0.2% by weight and 2.0% by weight of PHB (FIGS. 2A and B). On the other hand, in the group of mice fed with a PHB mixed diet containing 0.02% by weight of PHB, although a tumor growth inhibitory effect was observed, no extension of the survival period was observed (Figs. 2A and B). In addition, no significant change in body weight was observed in any of the PHB mixed feed fed groups (Fig. 2C).
From the above results, it was suggested that PHB should be administered (orally) in an amount of 0.2% by weight or more for the treatment of cancer under the administration conditions in this example.

4. Examination of effect of combined administration of PHB and anticancer agent on mouse breast cancer, mouse malignant melanoma and mouse malignant lymphoma 4-1. METHODS: The effects of combined administration of PHB and anticancer agents on tumor volume increase and mouse survival were evaluated in an implanted tumor model. 100 μL containing mouse mammary adenocarcinoma E0771 cells (2×10 ⁶ ), mouse malignant melanoma B16F10 cells (5×10 ⁵ ) or mouse malignant lymphoma EL4 cells (2×10 ⁶ ) and 30% Matrigel of Dulbecco's Modified Eagle (DMEM) medium was implanted into the dorsum of C57BL/6N mice, and tumor size was measured over time. The tumor volume was calculated by the formula described in 3-1 above.
A PHB-mixed diet containing 2.0% by weight of PHB was fed 6 or 8 days after tumor transplantation. Carboplatin (100 mg/kg, i.p.) was administered to the breast cancer and melanoma models, and vincristine (0.5 mg/kg, i.v.) was administered to the malignant lymphoma model 24 h after feeding with the PHB diet. . Tumor volume was measured over time, death, tumor volume exceeding 1500 mm ³ , obvious respiratory abnormalities, body weight loss of 20% or more, macroscopically clear metastasis. Survival rate was analyzed with the case of death as the endpoint.

4-2. result
Fig. 3A shows changes in tumor volume over time and the survival rate when 2.0% by weight of PHB and Carboplatin (CBDCA) were administered in combination to a breast cancer model implanted with E0771 cells. Fig. 3B shows changes in tumor volume over time and survival rate when weight% PHB and carboplatin were administered in combination, and 2.0% by weight PHB and vincristine (VCR) were administered in combination to a lymphoma model implanted with EL4 cells. Figure 3C shows the time course of tumor volume and survival rate. Table 1 shows the results of fitting the curves of the tumor volume graphs of FIGS.

マウス乳腺がんE0771細胞腫瘍を例にすると、PHB処理および抗がん剤処理のいずれも行わなかった場合、腫瘍の体積が3.656日で2倍になる。PHBを含む飼料を与えた場合の腫瘍倍化日数は4.171日（無処理と比較して、腫瘍倍化日数が1.14倍延長された）、カルボプラチン単独投与の場合腫瘍倍化日数は4.576日（無処理と比較して、腫瘍倍化日数が1.25倍延長された）延長された。これに対し、PHB含有飼料とカルボプラチンを併用投与した場合の腫瘍倍化日数は、5.346日（無処理と比較して、腫瘍倍化日数が1.462倍延長された）であり、併用投与の効果はPHB含有飼料の効果とカルボプラチンの効果の相加のはずであるが、実際の併用群では、1.46倍程度となり、PHB含有飼料とカルボプラチンの併用効果は、相乗的であることが認められた（表１）。B16F10細胞腫瘍およびEL4細胞腫瘍においては、PHB含有飼料とカルボプラチンの併用による相乗効果がより顕著であった（表１）。
以上、図３の結果および表１に示す解析結果から、全ての腫瘍モデルにおいて、PHBと抗がん剤の併用投与に相乗効果があることが示された。

Taking the mouse mammary adenocarcinoma E0771 cell tumor as an example, the volume of the tumor doubled in 3.656 days when neither PHB treatment nor anticancer drug treatment was performed. The number of days for tumor doubling was 4.171 days with PHB-containing diet (1.14 times longer than no treatment), and 4.576 days with carboplatin alone (no treatment). Tumor doubling days were prolonged 1.25-fold compared to treatment). In contrast, when PHB-containing feed and carboplatin were administered in combination, the number of days for tumor doubling was 5.346 days (the number of days for tumor doubling was prolonged by 1.462 times compared to no treatment). The effect of the PHB-containing feed and carboplatin should be additive, but in the actual combination group, the effect was about 1.46 times higher, and it was confirmed that the combined effect of the PHB-containing feed and carboplatin was synergistic (Table 1). 1). In B16F10 cell tumors and EL4 cell tumors, the synergistic effect of the combination of PHB-containing diet and carboplatin was more pronounced (Table 1).
As described above, from the results of FIG. 3 and the analysis results shown in Table 1, it was shown that the combined administration of PHB and anticancer drugs has a synergistic effect in all tumor models.

5. Investigation of combined effect of ketone ester addition and irradiation on breast cancer cells 5-1. Methods Analysis was performed using a colony formation test. Mouse mammary adenocarcinoma E0771 cells were trypsinized to a cell density of 20 to 200 cells/ml, and 5 ml each was plated on a 60 mm dish. After culturing in a CO ₂ incubator for 6 hours, the medium was replaced with 2 ml of medium containing 0.5 mM of ketone ester (KE), a dimer of 3HB. After 24 hours, the cells were irradiated with X-rays and cultured in a CO ₂ incubator for 7 to 10 days. After culturing, the medium was removed and the cells were fixed using methanol. After drying for 10 minutes, 2 ml of 0.03% crystal violet was added to each petri dish to stain the colonies. After standing still for 20 minutes, the staining solution was discarded and dried. The number of colonies formed with 50 or more cells was counted, and the survival rate was calculated using the following formula.

5-2. result
FIG. 4 shows the relationship between the Surviving Fraction and the intensity of the irradiated radiation. In FIG. 4, the curve was fitted to the LQ model, and the X-ray dose at which 50% cell death was obtained was CTRL = 2.27 Gy and KE = 1.91 Gy. This result indicated that the ketone ester had a synergistic effect that enhanced the radiation effect by 1.19 times.

6. Examination of the effect of PHB monotherapy on human breast cancer 6-1. Method 3×10 ⁶ human breast cancer MDAMB231 cells were transplanted into mouse mammary glands, and 0.2% by weight PHB diet was fed from 4 weeks later.
6-2. Results Figure 5A shows the tumor volume of mice implanted with human mammary adenocarcinoma, and Figure 5B shows the survival rate. In mice fed with a PHB diet containing 2.0% by weight of PHB, an inhibitory effect on tumor growth and prolongation of survival were observed.

7. Examination of the effect of pre-administration of PHB monotherapy on inflammatory bowel disease 7-1. Methods Mice were fed with a 2.0% by weight PHB mixed diet, and 2 weeks after the feeding, 3% dextran sulfate sodium (DSS) aqueous solution was given freely to the mice to induce colitis. After that, body weight and diarrhea/bleeding clinical score (Disease activity index: DAI) were analyzed. Survival rates were analyzed with endpoints of death, obvious respiratory abnormalities, and weight loss of 20% or more.

7-2. Results The changes in survival rate, DAI and body weight of colitis-induced mice over time are shown in Figures 6A, 6B and 6C, respectively. All values of survival rate, DAI and body weight were improved in the group of mice pretreated with 2.0% by weight of PHB mixed diet compared to the control group (the group of mice not pretreated with 2.0% by weight of PHB mixed diet). was Based on the above results, pre-administration of at least 2.0% by weight of PHB was found to improve the pathology of inflammatory bowel disease.

8. Examination of the effect of post-administration of PHB monotherapy on inflammatory bowel disease 8-1. Method
Mice were given a 2% dextran sulfate sodium (DSS) aqueous solution ad libitum to induce colitis, and then fed a diet containing 2.0% by weight of PHB on day 4. After that, body weight and diarrhea/bleeding clinical score (Disease activity index; DAI) were analyzed. Survival rates were analyzed with endpoints of death, obvious respiratory abnormalities, and weight loss of 20% or more.

8-2. Results The changes in survival rate, DAI and body weight of colitis-induced mice over time are shown in Figures 7A, 7B and 7C, respectively. All values of survival rate, DAI and body weight were higher in the group of mice administered 2.0% by weight of PHB mixed diet after colitis induction than in the control group (the group of mice not administered with 2.0% by weight of PHB mixed diet). was improving. Based on the above results, administration of at least 2.0% by weight of PHB to mice that had already developed inflammatory bowel disease improved the pathology of the disease.

9. Examination of the effects of PHB on anti-inflammatory cytokines and regulatory T cells 9-1. Method
A 2% dextran sulfate sodium (DSS) aqueous solution was administered to mice ad libitum to induce colitis, and on the 4th day, feeding with a diet containing 2.0% by weight of PHB was started. Eight days after DSS drinking, colon tissues were collected, the sections were HE-stained, and histological scores (HS) were evaluated. In addition, mRNA expression levels of cytokines (IL-2 and IL-10) and Foxp3 (regulatory T cell marker) in the collected colon tissues were analyzed by RT-qPCR.
Furthermore, the collected large intestine tissue was immunostained using an anti-Foxp3 antibody, and the number of Foxp3-positive cells was counted.

9-2. result
Compared with mice not fed with PHB (CTL in FIG. 8), mice fed with PHB (PHB in FIG. 8) showed a decrease in HS score in colon tissue (FIG. 8B). Gene expression of IL-10, an inflammatory cytokine, was found to be elevated (Fig. 8C). These results suggest that PHB promotes the expression of anti-inflammatory cytokines and exhibits anti-inflammatory effects.
In addition, the expression of Foxp3 mRNA in the large intestine tissue is elevated (Fig. 8C), and the number of Foxp3-positive cells in the PHB-fed mice was higher than that in the PHB-fed mice (CTL in Fig. 9). (PHB in FIG. 9), it was found that the number of Foxp3-positive cells increased (FIG. 9B). This result indicates that PHB has the effect of increasing the number of regulatory T cells, and suggests that it functions effectively to suppress excessive immune reactions.

INDUSTRIAL APPLICABILITY The present invention provides a composition containing an amount of PHB that is effective in treating or preventing cancer and inflammatory growth diseases, and is expected to be used in the fields of food and drink production as well as the fields of medicine and veterinary medicine. be.

Claims (19)

A composition containing 0.02% by weight or more of poly(D-3-hydroxybutyric acid). 2. The composition of claim 1, containing 0.2% or more by weight of poly(D-3-hydroxybutyric acid). 3. A composition according to claim 1 or 2, containing less than 5.0% by weight of poly(D-3-hydroxybutyric acid). 4. The composition of claim 3, containing less than 2.0% by weight of poly(D-3-hydroxybutyric acid). A composition containing poly(D-3-hydroxybutyrate), which is used such that 90 mg/m ² body surface area or more of poly(D-3-hydroxybutyrate) is ingested per day. 2. The composition of claim 1, characterized in that: 6. Composition according to claim 5, characterized in that it is used to ingest more than 900 mg/ ^m2 body surface area of poly(D-3-hydroxybutyric acid) per day. The composition according to claim 1, characterized in that it is used for disease treatment. 8. The composition of claim 7, wherein said disease is cancer. 9. A combination composition comprising the composition according to claim 8 and an anticancer agent, wherein the composition and the anticancer agent have a synergistic action. 8. The composition of claim 7, wherein said disease is an autoimmune disease. 11. The composition of claim 10, wherein said autoimmune disease is inflammatory bowel disease. The composition according to claim 1, wherein the composition is a pharmaceutical composition, a feed composition, or a food composition. 13. The composition of claim 12, wherein said feed composition is a diet. A composition for suppressing an excessive immune response, the composition comprising poly(D-3-hydroxybutyric acid) (PHB). 15. The composition according to claim 14, wherein said composition is a pharmaceutical composition, a feed composition or a food composition. 16. The composition of claim 15, wherein said feed composition is a diet. 16. The composition according to claim 15, wherein said pharmaceutical composition is for treating autoimmune or allergic diseases. 15. The composition according to claim 14, which suppresses an excessive immune response by promoting the expression of anti-inflammatory cytokines. 15. The composition of claim 14, which suppresses excessive immune responses by increasing the number of regulatory T cells.

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