JP2020143008A - Novel brown adipocyte differentiation inducing agent - Google Patents

Abstract

To provide means that is useful for inducing the differentiation of a brown adipocyte and has excellent practical utility.SOLUTION: There is provided a brown adipocyte differentiation inducing agent containing vitamin D or a derivative thereof as an active ingredient. The brown adipocyte differentiation inducing agent is used as, for example, an active ingredient of a pharmaceutical, a quasi drug, food or cosmetics.SELECTED DRAWING: None

Description

The present invention relates to an agent that promotes differentiation into brown adipocytes and its use (for example, prevention / treatment of obesity).

There are two types of adipose tissue, white adipose tissue and brown adipose tissue. While white adipose tissue is a storage organ that stores energy, brown adipose tissue consumes the stored energy and releases it as heat. It is a burning organ. Although brown adipose tissue is abundant in humans in newborns, it has since been significantly reduced and has been thought to be absent in physiologically meaningful amounts in adults. However, in recent years, it has become clear that brown adipose tissue not only functions physiologically even in adults, but its decrease causes obesity and diabetes (Non-Patent Document 1). Therefore, activation and weight increase of brown adipose tissue and brown adipose tissue formation of white adipose tissue are expected as epoch-making measures against obesity and metabolic syndrome (Non-Patent Document 2). To date, PRDM16, BMP7, PGC1α and the like have been reported as factors that control the differentiation of brown adipocytes (Non-Patent Documents 3 and 4), but none of these methods have been put into practical use. In addition, it has been clarified that sympathetic nerve stimulation is effective as a method for activating brown adipose tissue, but development of an activator using a sympathomimetic agent has been discontinued due to side effects. In addition, some means for inducing differentiation into brown adipocytes have been proposed (see, for example, Patent Documents 1 to 3).

International Publication No. 2014/027608 Pamphlet Japanese Unexamined Patent Publication No. 2016-199481 JP-A-2017-193508

N Engl J Med 360: 15091-1517,2009 Science and Technology Trends June 2009 (Science and Technology Trends Research Center, Science and Technology Policy Research Institute, Ministry of Education, Culture, Sports, Science and Technology) Cell Metab 1: 361-370,2005 Nature 454: 961-967,2008 Eur J Nutr. 2015 Sep; 54 (6): 1001-12.

Various measures have been proposed for activating brown adipocytes and inducing differentiation of brown adipocytes, but at present, there are no practical measures. In view of such a situation, an object of the present invention is to provide an effective means for inducing differentiation of brown adipocytes and to contribute to the establishment of a new preventive / therapeutic method for obesity, metabolic syndrome and the like. Is the subject.

In the course of studies to solve the above problems, the present inventors focused on vitamin D, which plays an important role in maintaining blood calcium concentration. Vitamin D is used for the prevention and treatment of osteoporosis and psoriasis. It is also being considered for use in cancer prevention. In relation to brown adipocytes, it has been reported that active vitamin D ₃ suppresses differentiation into brown adipocytes (Non-Patent Document 5). However, the experiments in this report used active vitamin D ₃ at non-physiological concentrations, and it cannot be said that it was intended for use in living organisms. Therefore, it does not suggest the usefulness of vitamin D clinically or practically.

The present inventors constructed an experimental system assuming use in a living body, and investigated the action of active vitamin D ₃ at physiological concentrations. As a result, active vitamin D ₃ promoted brown fatification. That is, the surprising fact that active vitamin D ₃ exhibits the opposite activity or action to the previously reported activity at physiological concentrations has been revealed. In addition, as a result of further studies, similar effects were observed for the precursor of active vitamin D ₃ (25 (OH) D ₃ ) and vitamin D derivatives. The results of this experiment support that the bioactivity of promoting brown adipose tissue is not unique to active vitamin D ₃ , but is a highly common or general property found in precursors and analogs. Vitamin D is originally a natural product and also a component existing in the living body, and it can be said that its safety is high. This feature is important in utilizing the new usefulness found in vitamin D and its derivatives, and shows that the practical value and significance of the present invention are high.
Based on the above results and considerations, the following inventions are provided.
[1] A brown adipocyte differentiation inducer containing vitamin D or a derivative thereof as an active ingredient.
[2] The brown adipocyte differentiation inducer according to [1], wherein the vitamin D is vitamin D ₂ or vitamin D ₃ .
[3] Vitamin D ₂ is 1 α, 25 (OH) ₂ D ₂ or 25 (OH) D ₂ α, and vitamin D ₃ is 1 α, 25 (OH) ₂ D ₃ or 25 (OH) D ₃ . The brown adipocyte differentiation inducer according to [2].
[4] The brown adipocyte differentiation inducer according to [1], wherein the derivative is eldecalcitol or phalecalcitriol.
[5] A composition for treating or preventing metabolic disorders or related diseases, which comprises the brown adipocyte differentiation inducer according to any one of [1] to [4].
[6] The composition according to [5], wherein the metabolic disorder or a related disease is obesity, diabetes, dyslipidemia, arteriosclerosis or metabolic syndrome.
[7] The composition according to [5] or [6], which is a medicine, a quasi drug, a food or a cosmetic.

Promotion of brown fatification by active vitamin D ₃ (1α, 25 (OH) ₂ D ₃ ). Left figure: The results of fat staining of cells after induction of differentiation (left) (black part is lipid) and the relative expression level of the fat marker gene FABP4 (right). Promotion of brown fatification by active vitamin D ₃ (1α, 25 (OH) ₂ D ₃ ). The relative expression level of the brown-like adipocyte marker gene UCP1 is shown. Promotion of brown adipose formation by 25 (OH) D ₃ . The relative expression level of the brown-like adipocyte marker gene UCP1 is shown. Promotion of brown fatification by erdecalcitol. The relative expression level of the brown-like adipocyte marker gene UCP1 is shown.

1. 1. Brown adipocyte differentiation inducer The first aspect of the present invention relates to a brown adipocyte differentiation inducer (hereinafter, also referred to as “differentiation inducer” for convenience of explanation). The "brown adipocyte differentiation inducer" is a brown adipocyte that acts on cells having the ability to differentiate into brown adipocytes (that is, progenitor cells or stem cells, typically muscle / brown adipocyte or adipose precursor cells). A drug that promotes differentiation into cells. The use of a brown adipocyte differentiation inducer promotes the differentiation into brown adipocytes, resulting in an increase in the number of brown adipocytes.

The active ingredient of the differentiation inducer of the present invention is vitamin D or a vitamin D derivative. Vitamins D _{2 to} D ₇ are present due to the difference in side chain structure at position 17. Widely distributed in nature and physiologically important are vitamin D ₂ (ergocalciferol) and vitamin D ₃ (cholecalciferol). Vitamin D ₂ or vitamin D ₃ is preferably used as the active ingredient of the present invention. Vitamin D ₂ and vitamin D ₃ are similarly metabolized in the human body and are considered to have the same activity.

In a preferred embodiment, the active form of vitamin D ₂ (1α, 25 (OH) ₂ D ₂ ) or vitamin D ₃ (1 α, 25 (OH) ₂ D ₃ ), or a precursor thereof, 25 (OH) D ₂ Alternatively, 25 (OH) D ₃ is the active ingredient.

Many vitamin D derivatives have been synthesized. Examples of existing vitamin D derivatives are alfacalcidol, maxacalcitol, eldecalcitol, and farecalcitriol. The vitamin D derivative which is the active ingredient of the present invention is not particularly limited as long as the expected effect, that is, the effect of inducing differentiation into brown adipocytes is observed. Therefore, vitamin D derivatives to be developed in the future can also be adopted as the active ingredient of the present invention. Eldecalcitol can be mentioned as an example of a preferred vitamin D derivative.

The differentiation inducer of the present invention may contain two or more kinds of active ingredients. In other words, a plurality of types of vitamin D or vitamin D derivatives may be used in combination to constitute the differentiation inducer of the present invention. In that case, the combination, the number of active ingredients, etc. are arbitrary.

2. 2. Compositions Containing Brown Adipocyte Differentiation Inducers Brown adipocytes are special cells that burn triglycerides and consume excess energy, and it has become clear that their decrease causes obesity and the development of metabolic syndrome. It was. Therefore, increasing brown adipocytes is an effective means for preventing and treating metabolic disorders and related diseases such as obesity and metabolic syndrome. Therefore, the second aspect of the present invention provides a composition for treating or preventing metabolic disorders or related diseases.

The compositions of the present invention are preferably provided in the form of pharmaceuticals, quasi-drugs, foods and cosmetics. That is, as a preferred embodiment, the present invention provides a pharmaceutical composition, a quasi-drug composition, a food composition and a cosmetic composition containing the differentiation inducer of the present invention as an active ingredient. Specific examples of "metabolic disorders or related diseases" (hereinafter referred to as "target diseases" for convenience of explanation) to be treated or prevented by the composition of the present invention include obesity, diabetes, dyslipidemia, and atherosclerosis. , Metabolic syndrome.

"Obesity" generally refers to a state in which adipose tissue is excessively accumulated in the body. In the present specification, the term "obesity" shall be construed in a broad sense, and the concept includes obesity. "Obesity" refers to a condition in which obesity causes or is associated with a health disorder (complication) or is expected to occur in the future and medically requires weight loss. As a method for determining obesity, for example, there is a method based on BMI (body mass index), which is widely used internationally. BMI is a value obtained by dividing weight (kg) by the square of height (m) (BMI = weight (kg) / height (m) ² ). BMI <18.5 is underweight, 18.5 ≤ BMI <25 is normal range, 25 ≤ BMI <30 is preobese, 30 ≤ BMI <35 is obese class I , 35 ≤ BMI <40 is determined to be obese class II, and 40 ≤ BMI is determined to be obese class III (WHO). In addition, using BMI, the standard weight (ideal weight) of a Japanese adult is calculated from the following formula, standard weight (kg) = height (m) ² x 22, and the measured weight is the standard weight (calculated value). There is also a method of determining that a condition exceeding 120% of the body weight is obese. However, since the standard body weight (ideal body weight) differs for each individual due to differences in gender, age, lifestyle, etc., it is considered inappropriate to uniformly determine obesity by this method.

"Metabolic syndrome" refers to a condition in which insulin resistance, arteriosclerosis-induced lipoprotein abnormality, and hypertension are complicated based on obesity and accumulation of visceral fat, and the risk of contracting cardiovascular disease is increased. Regarding the diagnostic criteria for metabolic syndrome, in April 2005, eight internal medicine societies (Japan Society for Atherosclerosis, Japan Society for the Study of Obesity, Japan Society for the Study of Diabetes, Japan Society for Hypertension, Japan Cardiovascular Society, Japan Society for Internal Medicine, Japan Society for Kidney Disease, Japan The Thrombosis and Hematology Society) has jointly created the following diagnostic criteria.
<Diagnostic criteria by 8 academic societies such as the Japanese Society of Internal Medicine>
In addition to (a) below, two or more of (b) to (d) are satisfied.
(a) Abdominal circumference: 85 cm or more for men and 90 cm or more for women
(b) Neutral fat is 150 mg / dl or more and / or HDL-C is less than 40 mg / dl
(c) Blood pressure is 130/85 mmHg or higher
(d) Fasting blood glucose is 110 mg / dl or higher

The International Federation of Diabetes (IDF) has created the following universal diagnostic criteria.
<International Diabetes Federation (IDF) diagnostic criteria>
In addition to (a) below, two or more of (b) to (e) are satisfied.
(a) Abdominal circumference: 90 cm or more for men and 80 cm or more for women
(b) Neutral fat is 150mg / dl or more
(c) Fasting blood glucose is 100 mg / dl or higher
(d) HDL-C: less than 40 mg / dl for men and less than 45 mg / dl for women
(e) Blood pressure is 130/85 mmHg or higher

(1) Pharmaceutical composition / quasi-drug composition The "pharmaceutical" and "quasi-drug" in the present invention are drug compositions that exhibit a therapeutic or prophylactic effect on a target disease. Therapeutic effects include alleviating the symptoms or associated symptoms characteristic of the target disease (mitigation), preventing or delaying the exacerbation of the symptoms, and the like. The latter can be regarded as one of the preventive effects in terms of preventing aggravation. Thus, therapeutic and prophylactic effects are partly overlapping concepts, difficult to distinguish clearly, and the practical benefits of doing so are small. A typical preventive effect is to prevent or delay the onset (onset) or recurrence of symptoms and pathological conditions characteristic of the target disease. As long as it shows some therapeutic effect or preventive effect on the target disease, or both, it corresponds to the pharmaceutical composition or the quasi-drug composition of the present invention.

The pharmaceutical composition and the quasi-drug composition of the present invention can be formulated according to a conventional method. When formulated, other formulationally acceptable ingredients (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiology (Saline, etc.) can be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. As the buffer, phosphate, citrate, acetate and the like can be used. As the emulsifier, gum arabic, sodium alginate, tragant and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the pain-relieving agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. Propylene glycol, ascorbic acid and the like can be used as the stabilizer. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited, and for example, the pharmaceutical composition or quasi-drug of the present invention as tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, suppositories and the like. A quasi-drug composition can be provided.

The pharmaceutical composition of the present invention contains an amount of an active ingredient necessary for obtaining the expected therapeutic effect or preventive effect (that is, a therapeutically effective amount). Similarly, the quasi-drug composition of the present invention contains an amount of the active ingredient necessary for obtaining the expected improving effect, preventive effect and the like. The amount of the active ingredient contained in the pharmaceutical composition or the quasi-drug composition of the present invention generally varies depending on the dosage form and form, but the amount of the active ingredient is, for example, about 0.1% by weight to about 99 so as to achieve a desired dose. Set within the range of% by weight.

The pharmaceutical composition and quasi-drug composition of the present invention are oral or parenteral (intravenous, intraarterial, subcutaneous, muscular, or intraperitoneal injection, transdermal, nasal, transmucosal, depending on the dosage form and form. , Apply, etc.) to the subject. The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, livestock, and experimental animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quail, etc.). In a preferred embodiment, the subject of application is human.

The dose and amount of the pharmaceutical composition and the quasi-drug composition of the present invention are set so as to obtain the expected effects. In setting an effective dose, the symptom, age, sex, weight, etc. of the target are generally taken into consideration. Those skilled in the art can set an appropriate dose in consideration of these matters. Considering that the effect peculiar to the physiological concentration of vitamin D (induction of differentiation into brown adipocytes) was observed (see the examples below), it is possible to set the dose so that an excessive amount is not administered. preferable. The "excess amount" is an amount that greatly exceeds the normal concentration (that is, physiological concentration) in the living body, and is used when active vitamin D ₃ (1α, 25 (OH) ₂ D ₃ ) is administered. If there is, the dose is excessive so that the maximum blood concentration after administration is, for example, 1000 pM or more (preferably 750 pM or more, more preferably 500 pM or more, even more preferably 250 pM or more, even more preferably 150 pM or more). It can be used as a guide for the amount. Therefore, the dose should be adjusted so that the maximum blood concentration after administration is, for example, less than 1000 pM, preferably less than 750 pM, more preferably less than 500 pM, even more preferably less than 250 pM, and even more preferably less than 150 pM. It is good to set. The optimal dose may be determined by preliminary experiments. Regarding the dose of vitamin D other than active vitamin D ₃ or vitamin D derivative, if it is present in the living body to be administered (for example, 25 (OH) D ₃ ), its physiological concentration is taken into consideration. However, if it can be set according to the above dose of active vitamin D ₃ and does not exist in the living body to be administered (for example, a synthetic compound such as eldecalcitol or phalecalcitriol), its activity. The activity of active vitamin D ₃ may be compared with that of active vitamin D ₃ , and the dose may be set according to the above dose of active vitamin D ₃ .

As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the symptoms to be applied and the duration of effect of the active ingredient can be taken into consideration.

As is clear from the above description, the present application states that a therapeutically effective amount of the pharmaceutical composition of the present invention is administered to a patient with a metabolic disorder or a related disease or a potential patient (a person who may be affected in the future). It also provides characteristic preventive and therapeutic methods.

(2) Food Composition As described above, one aspect of the present invention is a food composition containing the differentiation inducer of the present invention. Examples of the "food composition" in the present invention include rice, bread, processed vegetable products, processed meat products, noodles, miso juice, soup, confectionery (for example, biscuits, cookies, chocolate, candy, cakes, ice cream). , Chewing gum, tablets), tea drinks, soft drinks, carbonated drinks, fruit juice drinks, dairy drinks, lactic acid bacteria drinks, fermented milk drinks, alcoholic drinks, spreads / jams (margarine, fast spreads, jams, etc.), sprinkles, mayonnaise / Dressings, sauces / sauces, nutritional supplements or health functional foods (nutrition functional foods, functional foods, foods for specified health use), food additives, pet animal foods, pet animal nutritional supplements it can. In the case of dietary supplements or health functional foods, food additives, pet animal foods, pet animal dietary supplements, they can be provided in the form of powders, granule powders, tablets (tablets), pastes, liquids and the like. By providing in the form of a food composition, it becomes easy to take the active ingredient of the present invention on a daily basis or continuously.

The food composition of the present invention preferably contains an amount of the active ingredient that can be expected to have a therapeutic or prophylactic effect. The content (addition amount) can be determined in consideration of the medical condition, health condition, age, sex, weight, etc. of the person to whom it is used.

(3) Cosmetic composition As described above, one aspect of the present invention is a cosmetic composition containing the differentiation inducer of the present invention as an active ingredient. The cosmetic composition of the present invention comprises the differentiation inducer of the present invention and the components / base materials (for example, various fats and oils, mineral oil, petrolatum, squalane, lanolin, honeybee, modified alcohol, palmitic acid) usually used in cosmetics. Contains dextrin, glycerin, glycerin fatty acid ester, ethylene glycol, paraben, camphor, menthol, various vitamins, zinc oxide, titanium oxide, benzoic acid, edetic acid, chamomile oil, carrageenan, chitin powder, chitosan, fragrance, colorant, etc.) Can be obtained by doing.

Examples of the form of the cosmetic composition include emulsions for faces or bodies, lotions, creams, lotions, essences, oils, facial masks, sheets, and cleansers. The amount of the differentiation inducer added to the cosmetic composition is not particularly limited. For example, the differentiation inducer may be added so as to be 0.001% by weight to 60% by weight.

Focusing on vitamin D, which plays an important physiological function such as maintaining blood calcium concentration, the following experiments were conducted with the expectation that vitamin D exhibits the ability to induce brown adipose differentiation.

1. 1. Method In order to evaluate the ability to induce brown adipose differentiation, C3H10T1 / 2 cells derived from mouse embryos, which have the ability to differentiate into brown adipocytes, were used. Dulbecco's modified Eagle's Medium containing 10% FBS was used for normal culture, and the cells were cultured in a CO ₂ incubator (37 ° C, CO ₂ concentration 5%). For differentiation into brown fat, when the cells became confluent, 1 μM Dexamethasone, 10 μg / ml Insulin, 0.5 μM 3-isobutyl-1-methyl-xanthine, 125 μM Indomethacin, and 3 nM Triiodothyronine were added to the above medium. In addition, vitamin D (1α, 25 (OH) ₂ D ₃ or 25 (OH) D ₃ ) with a final concentration of 0-1000 pM (for 1α, 25 (OH) ₂ D ₃ ), 0-2000 pM (25 (25) In the case of OH) D _3, the cells were replaced with a differentiation-inducing medium added so as to be (OH) D ₃ ) and treated for 48 hours. After 48 hours, change to a medium containing 10 μg / ml Insulin, 3 nM Triiodothyronine, and vitamin D (1α, 25 (OH) ₂ D ₃ or 25 (OH) D ₃ ), and change the medium every 2 days. The cells were cultured for a total of 7 days. After completion of the culture, RNA was extracted from the cells, and the expression levels of FABP4, which is a fat marker, and UCP1, which is a brown fat marker, were measured and evaluated by real-time PCR. On the other hand, the cultured cells were subjected to oil red O staining.

2. 2. Results The active vitamin D ₃ , 1α, 25 (OH) ₂ D ₃ , suppressed the differentiation of C3H10T1 / 2 cells into adipocytes in a dose-dependent manner (Fig. 1). Looking at the expression of the brown adipocyte UCP1, low concentrations of active vitamin D ₃ promoted differentiation into brown adipocytes (Fig. 2). Human blood levels of active vitamin D ₃ from it is about 10 to 150 pM, active vitamin D ₃ at physiological concentrations can be said to have a brown fat reduction effect.

Similarly, 25 (OH) D ₃ , a precursor of active vitamin D ₃ , also suppressed the differentiation into brown adipocytes at high concentrations, but promoted the expression of brown adipocytes at low concentrations (Fig. 3). .. Since the concentration of 25 (OH) D _{3 in} human blood is about 30 to 250 nM, it is shown that the physiological concentration of 25 (OH) D ₃ has a brown adipose effect similar to that of active vitamin D _3. It was.

Based on the above experimental results, we decided to investigate whether vitamin D derivatives also have a brown fattening effect. Eldecalcitol was used as the test compound. As shown in FIG. 4, eldecalcitol was also required to have a brown adipose action, and it was found that the brown adipose action is a characteristic having high commonality and generality in vitamin D and its derivatives.

The brown adipose cell differentiation inducer of the present invention can be used for prevention and treatment of diabetes, dyslipidemia, arteriosclerosis and the like as well as obesity and metabolic syndrome. The present invention containing vitamin D or a derivative thereof as an active ingredient is expected to have high safety, and can be said to be applicable to a wide range of uses and applications such as pharmaceuticals, foods, and food materials. In particular, it is suitable for daily or continuous ingestion (administration).

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (7)

A brown adipocyte differentiation inducer containing vitamin D or a derivative thereof as an active ingredient. The brown adipocyte differentiation inducer according to claim 1, wherein the vitamin D is vitamin D ₂ or vitamin D ₃ . Claim ₂ where vitamin D ₂ is 1 α, 25 (OH) ₂ D ₂ or 25 (OH) D ₂ α and vitamin D ₃ is 1 α, 25 (OH) ₂ D ₃ or 25 (OH) D _3. The brown adipocyte differentiation inducer according to. The brown adipocyte differentiation inducer according to claim 1, wherein the derivative is eldecalcitol or phalecalcitriol. A composition for treating or preventing metabolic disorders or related diseases, which comprises the brown adipocyte differentiation inducer according to any one of claims 1 to 4. The composition according to claim 5, wherein the metabolic disorder or a related disease is obesity, diabetes, dyslipidemia, arteriosclerosis or metabolic syndrome. The composition according to claim 5 or 6, which is a medicine, a quasi drug, a food or a cosmetic.

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