JP2013199468A - Drug for prevention or inhibition of skin inflammation or injury - Google Patents
Drug for prevention or inhibition of skin inflammation or injury Download PDFInfo
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- JP2013199468A JP2013199468A JP2012258520A JP2012258520A JP2013199468A JP 2013199468 A JP2013199468 A JP 2013199468A JP 2012258520 A JP2012258520 A JP 2012258520A JP 2012258520 A JP2012258520 A JP 2012258520A JP 2013199468 A JP2013199468 A JP 2013199468A
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Abstract
Description
本発明は、皮膚の炎症または傷害、特に、紫外線照射に起因する皮膚の炎症または傷害の予防または抑制剤、および該予防または抑制剤を含み、化粧品または医薬として有用な組成物に関する。 The present invention relates to a preventive or suppressive agent for skin inflammation or injury, in particular, skin inflammation or injury caused by ultraviolet irradiation, and a composition useful as a cosmetic or pharmaceutical comprising the prophylactic or suppressive agent.
紫外線は、皮膚を構成する線維芽細胞や角化細胞のＤＮＡを損傷し、遺伝子変異による直接的な発がんの原因となる（直接的効果）。さらに、紫外線により細胞内の水が電離されることにより生じた活性酸素が、タンパク質や脂質を変性ないし失活させ、皮膚組織に損傷をもたらす（間接的効果）。紫外線（近紫外線）は、波長によりＡ波（ＵＶＡ：３２０〜４００ｎｍ）、Ｂ波（ＵＶＢ：３２０〜２８０ｎｍ）、およびＣ波（ＵＶＣ：＜２８０ｎｍ）に細分され、生体に対する影響がそれぞれ異なっている。 Ultraviolet rays damage the DNA of fibroblasts and keratinocytes that make up the skin and cause direct carcinogenesis due to genetic mutation (direct effects). Furthermore, active oxygen generated by ionizing intracellular water by ultraviolet rays denatures or inactivates proteins and lipids and causes damage to skin tissue (indirect effect). Ultraviolet rays (near ultraviolet rays) are subdivided into A waves (UVA: 320 to 400 nm), B waves (UVB: 320 to 280 nm), and C waves (UVC: <280 nm) depending on the wavelength, and their influence on the living body is different. .
ＵＶＡの皮膚に対する効果は、間接的効果が主であり、コラーゲンなど真皮の細胞外マトリックスを構成するタンパク質が活性酸素により変性され、皮膚のしわ、たるみ、弾力低下等の原因となる。これらは皮膚の光老化と呼ばれ、加齢に伴う生理的な皮膚の老化とは区別される。 The effect of UVA on the skin is mainly an indirect effect, and proteins that constitute the extracellular matrix of the dermis such as collagen are denatured by active oxygen, causing skin wrinkles, sagging, reduced elasticity, and the like. These are called skin photoaging and are distinguished from physiological skin aging with aging.
一方、ＵＶＢは、ＵＶＡよりも大きなエネルギーを持ち、その波長が、ＤＮＡが極大吸収を示す２６０ｎｍに近いため、ＤＮＡに直接的に損傷を与える。また、角化細胞にＵＶＢが照射されると、炎症性サイトカインであるインターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）、腫瘍壊死因子α（ＴＮＦ−α）等の発現が誘導され、炎症反応が起こる。この反応は、本来、組織侵襲に対する生体防御の一環として行われるものであり、生体の恒常性の維持に必要なものである。しかし、過剰な炎症反応は、炎症担当細胞（好中球やマクロファージ等）の過度の集中による活性酸素の過剰産生をもたらし、皮膚組織を傷害する結果となる。 On the other hand, UVB has a larger energy than UVA, and its wavelength is close to 260 nm at which DNA exhibits maximum absorption, and thus directly damages DNA. In addition, when keratinocytes are irradiated with UVB, expression of inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), etc. Is induced and an inflammatory reaction occurs. This reaction is originally performed as part of biological defense against tissue invasion, and is necessary for maintaining the homeostasis of the living body. However, an excessive inflammatory reaction results in excessive production of active oxygen due to excessive concentration of inflammatory cells (such as neutrophils and macrophages), resulting in injury to skin tissue.
ＵＶＣは、ＵＶＢよりもさらに大きなエネルギーを持ち、ＤＮＡに損傷を与える。また、培養角化細胞にアポトーシスを誘導することが報告されているが、生体の皮膚に照射された場合、表皮の角質層までしか到達しないため、細胞分裂が行われる基底部角化細胞を損傷するリスクは低いと考えられている。 UVC has even more energy than UVB and damages DNA. It has also been reported to induce apoptosis in cultured keratinocytes, but when irradiated to the skin of the living body, it only reaches the stratum corneum of the epidermis, thus damaging the basal keratinocytes where cell division occurs The risk of doing so is considered low.
このように、皮膚組織は紫外線照射により損傷を受けるが、太陽光に含まれるＵＶＢの９９％以上は成層圏に存在するオゾン層に吸収されて地表には到達しない。また、ＵＶＣは完全にオゾン層に吸収され、地表には全く到達しない。しかし、冷却器機の冷媒や電子基板の清浄剤として近年まで用いられてきたフロンガスは、分解されないまま成層圏に到達し、オゾンを分解する。その結果、１９８０年代以降、高緯度地域において経年的な成層圏オゾン量の減少が観測されてきた。かかるオゾン層の破壊により、地表におけるＵＶＢの量が増加すると共に、ＵＶＣが地表に到達することとなり、紫外線による皮膚傷害や光老化のリスクの増大が問題視されている。２０００年代以降、世界各国でフロンガス使用に対する規制が本格化した結果、新規に生産されるフロンガス量は激減し、極地におけるオゾンホールは漸減傾向にある。しかしながら、現在のペースでは、フロンガスの危険性が認識されるようになった１９８０年代のレベルまでオゾン層が回復するのは今世紀半ば以降になると予測されている。したがって、高緯度地域を中心に、紫外線による皮膚傷害や光老化を抑制する物質に対する社会的ニーズは、今後も高いまま持続するものと考えられる。 Thus, the skin tissue is damaged by ultraviolet irradiation, but 99% or more of UVB contained in sunlight is absorbed by the ozone layer present in the stratosphere and does not reach the ground surface. Moreover, UVC is completely absorbed by the ozone layer and does not reach the ground surface at all. However, chlorofluorocarbon gas, which has been used until recently as a refrigerant for a cooler machine and a cleaning agent for an electronic substrate, reaches the stratosphere without being decomposed and decomposes ozone. As a result, since the 1980s, a decrease in stratospheric ozone has been observed over time in high latitude areas. Due to the destruction of the ozone layer, the amount of UVB on the surface of the earth increases, and UVC reaches the surface of the earth, and there is a problem of increasing the risk of skin damage and photoaging due to ultraviolet rays. As a result of the full-scale regulation on the use of CFCs around the world since the 2000s, the amount of CFCs newly produced has been drastically reduced, and ozone holes in the polar regions are gradually decreasing. However, at the current pace, it is predicted that the ozone layer will recover after the mid-century to the level of 1980s when the dangers of chlorofluorocarbons were recognized. Therefore, it is considered that social needs for substances that suppress skin damage and photoaging due to ultraviolet rays will continue to be high, especially in high latitude areas.
紫外線照射による皮膚の傷害を防止する方法の一つは、活性酸素の発生を抑制するか、または発生した活性酸素を除去することである。活性酸素の産生を抑制する既知の生体物質としては、カタラーゼ、グルタチオンパーオキシダーゼ、グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ）、スーパーオキシドディスムターゼ（ＳＯＤ）等の酵素群、トランスフェリン、ラクトフェリン等の鉄イオンを捕捉するタンパク質、カロチノイド等の天然高分子（色素）が知られている。一例として、ＳＯＤを皮膚に投与すると、紫外線照射による光老化が抑制されることが知られている（特許文献１）。しかしながら、ＧＳＴやＳＯＤ等の酵素はタンパク質であるため、常温では分解ないし失活し易く、皮膚外用剤としての長期保存が困難であるという問題を有している。また、グルタチオン誘導体は悪臭があり、皮膚外用剤としての使用には適さない。 One of the methods for preventing skin damage caused by ultraviolet irradiation is to suppress the generation of active oxygen or to remove the generated active oxygen. Known biological substances that suppress the production of active oxygen include catalase, glutathione peroxidase, glutathione-S-transferase (GST), superoxide dismutase (SOD) and other enzyme groups, and iron ions such as transferrin and lactoferrin. Natural polymers (pigments) such as proteins and carotenoids are known. As an example, it is known that when SOD is administered to the skin, photoaging due to ultraviolet irradiation is suppressed (Patent Document 1). However, since enzymes such as GST and SOD are proteins, they are easily decomposed or inactivated at room temperature, and have a problem that long-term storage as a skin external preparation is difficult. Further, glutathione derivatives have a bad odor and are not suitable for use as a skin external preparation.
一方、発生した活性酸素が標的分子を攻撃する前にこれを捕捉して安定化または除去する作用を有する、いわゆる抗酸化物質としては、アスコルビン酸（ビタミンＣ）、アルブミン、トコフェロール（ビタミンＥ）等の天然物質がよく知られている。これらのうち、最も広範に用いられる抗酸化物質はアスコルビン酸およびその誘導体である。さらに、植物由来の成分であるポリフェノール類も、抗酸化作用を有することが知られている。 On the other hand, as a so-called antioxidant having an action of capturing and stabilizing or removing the active oxygen before attacking the target molecule, ascorbic acid (vitamin C), albumin, tocopherol (vitamin E), etc. The natural substances are well known. Of these, the most widely used antioxidant is ascorbic acid and its derivatives. Furthermore, polyphenols, which are plant-derived components, are also known to have an antioxidant effect.
紫外線照射による皮膚の傷害を防止する別の方法としては、紫外線照射により誘導される皮膚の炎症反応を抑制することが挙げられる。炎症反応の抑制により、活性酸素の過剰産生が回避され、皮膚傷害の発生が防止される。 Another method for preventing skin damage caused by ultraviolet irradiation is to suppress the inflammatory reaction of the skin induced by ultraviolet irradiation. By suppressing the inflammatory reaction, excessive production of active oxygen is avoided and the occurrence of skin injury is prevented.
皮膚の炎症反応を抑制する有効な方法の一つは、炎症性サイトカインの産生を抑制するか、またはこれを不活性化することである。前者の例としては、メルク（カルビオケム）社から発売されているｐ３８ＭＡＰキナーゼの阻害剤ＳＢ２０３５８０およびＳＢ２２００２５が挙げられる。これらの阻害剤は、角化細胞の細胞内情報伝達経路において、炎症性サイトカインの上流に位置するｐ３８ＭＡＰキナーゼの活性を抑制するため、炎症性患者の治療薬として有用である。後者の例としては、炎症性サイトカインの一つであるＴＮＦ−αの拮抗体を含む組成物を皮膚等に適用する方法が開発されている（特許文献２）。 One effective way to suppress the inflammatory response of the skin is to suppress or inactivate the production of inflammatory cytokines. Examples of the former include p38MAP kinase inhibitors SB203580 and SB220025 sold by Merck (Calbiochem). These inhibitors are useful as therapeutic agents for inflammatory patients because they suppress the activity of p38 MAP kinase located upstream of inflammatory cytokines in the intracellular signal transduction pathway of keratinocytes. As an example of the latter, a method of applying a composition containing an antagonist of TNF-α, which is one of inflammatory cytokines, to the skin or the like has been developed (Patent Document 2).
また、皮膚用の抗炎症剤として、様々なステロイド系または非ステロイド系抗炎症剤が開発されている。しかし、効果が顕著なステロイド系抗炎症剤は、長期間の投与により皮膚の劣化、副腎皮質機能の抑制等の副作用を示すことが報告されている。 In addition, various steroidal or non-steroidal anti-inflammatory agents have been developed as skin anti-inflammatory agents. However, it has been reported that steroidal anti-inflammatory agents with remarkable effects exhibit side effects such as skin deterioration and suppression of adrenal cortex function after long-term administration.
乳酸菌の菌体や細胞壁、および特定の条件で培養した乳酸菌培養物には、紫外線照射に起因する皮膚の炎症や炎症性サイトカインの産生を抑制する作用があることが知られている。例えば、特許文献３には、乳酸菌の菌体または細胞壁を実験動物の皮膚に塗布することで、紫外線照射による紅斑の発生が抑制されたことが記載されている。また、特許文献４には、ヤエナリ抽出物およびグルタミン酸モノナトリウムを含む培地中で乳酸菌を培養することにより得られる、ヤエナリ抽出物及びギャバを含有することを特徴とする乳酸菌培養物が、ヒト由来の角化細胞において、ＵＶＢ照射により発現誘導されるＩＬ−６の産生を抑制したことが記載されている。しかしながら、これら乳酸菌の菌体や細胞壁、および特定の乳酸菌培養物に関しては、紅斑の発生やＩＬ−６の産生を抑制する活性成分が特定されていない。そのため、皮膚への適用に際しては、乳酸菌の菌体もしくは細胞壁または乳酸菌培養物をそのまま用いるほかなく、皮膚外用剤として製剤化することは困難である。 Lactic acid bacteria and cell walls, and lactic acid bacteria cultures cultured under specific conditions are known to have an action of suppressing skin inflammation and production of inflammatory cytokines caused by ultraviolet irradiation. For example, Patent Document 3 describes that the occurrence of erythema caused by ultraviolet irradiation is suppressed by applying lactic acid bacteria or cell walls to the skin of a laboratory animal. Patent Document 4 discloses a human-derived lactic acid bacterium culture characterized by containing a peanut extract and a gab obtained by culturing a lactic acid bacterium in a medium containing a peanut extract and monosodium glutamate. It is described that IL-6 production induced by UVB irradiation was suppressed in keratinocytes. However, no active ingredient that suppresses the occurrence of erythema or IL-6 production has been identified for these lactic acid bacteria cells and cell walls, and specific lactic acid bacteria cultures. For this reason, when applied to the skin, it is difficult to formulate it as an external preparation for skin, except that lactic acid bacteria or cell walls or lactic acid bacteria cultures are used as they are.
本発明の目的は、紫外線照射等に起因する皮膚の炎症または傷害を予防または抑制することができる化合物を含み、副作用が少なく、かつ、外用剤としての利用に適した、皮膚の炎症または傷害の予防または抑制剤を提供することにある。 An object of the present invention is to contain a compound that can prevent or suppress skin inflammation or injury caused by ultraviolet irradiation or the like, has few side effects, and is suitable for use as an external preparation. It is to provide a preventive or suppressive agent.
本発明者らは、特定の構造を有するカルボン酸が、紫外線照射により誘導される炎症メディエーターの産生を抑制する作用、ＭＡＰキナーゼの活性化を抑制する作用、およびシクロオキシゲナーゼ（Ｃｏｘ）の産生を抑制する作用を有し、紫外線照射に起因する皮膚の炎症または傷害を予防または抑制するのに有効な化合物であることを見出した。 The present inventors have demonstrated that a carboxylic acid having a specific structure suppresses the production of inflammatory mediators induced by ultraviolet irradiation, suppresses the activation of MAP kinase, and suppresses the production of cyclooxygenase (Cox). It has been found that the compound has an action and is effective in preventing or suppressing skin inflammation or injury caused by ultraviolet irradiation.
すなわち、本発明は、式（Ｉ）の化合物を含む、皮膚の炎症または傷害の予防または抑制剤を提供する：
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。
That is, the present invention provides a preventive or suppressant for skin inflammation or injury comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
本発明はまた、式（Ｉ）の化合物を含む、炎症メディエーター産生抑制剤を提供する：
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。
The present invention also provides an inflammatory mediator production inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
本発明はまた、式（Ｉ）の化合物を含む、ＭＡＰキナーゼ活性化抑制剤を提供する：
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。
The present invention also provides a MAP kinase activation inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
本発明はまた、式（Ｉ）の化合物を含む、シクロオキシゲナーゼ産生抑制剤を提供する：
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。
The present invention also provides a cyclooxygenase production inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
さらに、本発明は、上記皮膚の炎症または傷害の予防または抑制剤、上記炎症メディエーター産生抑制剤、上記ＭＡＰキナーゼ活性化抑制剤、または上記シクロオキシゲナーゼ産生抑制剤を含む化粧品または医薬組成物を提供する。また、本発明は、上記皮膚の炎症または傷害の予防または抑制剤、上記炎症メディエーター産生抑制剤、上記ＭＡＰキナーゼ活性化抑制剤、または上記シクロオキシゲナーゼ産生抑制剤が配合された機能性飲食品を提供する。 Furthermore, the present invention provides a cosmetic or pharmaceutical composition comprising the above-mentioned skin inflammation or injury prevention or suppression agent, the above-mentioned inflammation mediator production inhibitor, the above MAP kinase activation inhibitor, or the above cyclooxygenase production inhibitor. The present invention also provides a functional food or drink containing the above-mentioned skin inflammation or injury prevention or suppression agent, the above-mentioned inflammation mediator production inhibitor, the above MAP kinase activation inhibitor, or the above cyclooxygenase production inhibitor. .
本発明の皮膚の炎症または傷害の予防または抑制剤（以下、単に「本発明の炎症/傷害の予防/抑制剤」とも称する）は、これを皮膚に塗布することにより、紫外線照射等に起因する皮膚の炎症反応を抑制し、炎症メディエーター（活性酸素、炎症性サイトカイン、プロテアーゼ、プロスタグランジン等）の過剰産生を防止すること、ＭＡＰキナーゼの過剰活性化を防止すること、およびシクロオキシゲナーゼの過剰産生を防止することができる。その結果、本発明の炎症/傷害の予防/抑制剤は、紫外線照射等に起因する皮膚傷害の発生を予防ないし軽減することができる。また、本発明の炎症/傷害の予防/抑制剤の有効成分である式（Ｉ）の化合物は、常温で長期保存することが可能であり、かつ、不快な臭気も有さない。したがって、本発明の炎症/傷害の予防/抑制剤は、紫外線等から皮膚を保護する化粧品や医薬品として利用することができ、特に外用剤の形態に適するものである。 The preventive or inhibitor of skin inflammation or injury of the present invention (hereinafter also simply referred to as “inflammation / injury preventive / suppressor of the present invention”) is caused by ultraviolet irradiation or the like by applying it to the skin. Inhibits skin inflammatory response, prevents overproduction of inflammatory mediators (reactive oxygen, inflammatory cytokines, proteases, prostaglandins, etc.), prevents overactivation of MAP kinase, and overproduction of cyclooxygenase Can be prevented. As a result, the inflammation / injury prevention / suppression agent of the present invention can prevent or reduce the occurrence of skin injury caused by ultraviolet irradiation or the like. Further, the compound of the formula (I) which is an active ingredient of the inflammation / injury prevention / suppression agent of the present invention can be stored for a long period of time at room temperature and does not have an unpleasant odor. Therefore, the inflammation / injury prevention / suppression agent of the present invention can be used as a cosmetic or pharmaceutical product for protecting the skin from ultraviolet rays or the like, and is particularly suitable for the form of an external preparation.
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤またはシクロオキシゲナーゼ産生抑制剤の有効成分である式（Ｉ）（Ｚ−Ａ−ＣＯＯＨ）の化合物において、Ｚは、水素であるかまたは置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、Ａは、置換基を有してもよい飽和または不飽和炭化水素基である。以下、式（Ｉ）の化合物の構造について、より詳細に説明する。 In the compound of formula (I) (ZA-COOH) which is an active ingredient of the inflammation / injury preventive / inhibitor, inflammatory mediator production inhibitor, MAP kinase activation inhibitor or cyclooxygenase production inhibitor of the present invention, Z Is an aromatic hydrocarbon group or a heterocyclic group which may be hydrogen or may have a substituent, and A is a saturated or unsaturated hydrocarbon group which may have a substituent. Hereinafter, the structure of the compound of the formula (I) will be described in more detail.
式（Ｉ）のＺに相当する複素環基としては、インドリル基、イソインドリル基、インドリニル基、イソインドリニル基等が挙げられ、インドリル基が好ましい。該複素環基が有してもよい置換基としては、ヒドロキシ基、アルキル基、アルケニル基、アルコキシ基、アミノ基等が挙げられる。 Examples of the heterocyclic group corresponding to Z in formula (I) include an indolyl group, an isoindolyl group, an indolinyl group, an isoindolinyl group, and the like, and an indolyl group is preferable. Examples of the substituent that the heterocyclic group may have include a hydroxy group, an alkyl group, an alkenyl group, an alkoxy group, and an amino group.
好ましくは、式（Ｉ）のＺに相当する複素環基は、下記式（Ｉａ）で表される基である：
（Ｉａ）
[式中、Ｒ１、Ｒ２およびＲ３は同一でも異なってもよく、各々独立に、水素、ヒドロキシ、Ｃ１−４アルキル、Ｃ１−４アルケニル、Ｃ１−４アルコキシおよびアミノからなる群より選択される基である]。また、式（Ｉａ）において、Ｒ１、Ｒ２およびＲ３は全て水素であることが好ましい。
Preferably, the heterocyclic group corresponding to Z in the formula (I) is a group represented by the following formula (Ia):
(Ia)
[Wherein R 1 , R 2 and R 3 may be the same or different and each independently represents a group consisting of hydrogen, hydroxy, C 1-4 alkyl, C 1-4 alkenyl, C 1-4 alkoxy and amino; Is a more selected group]. In the formula (Ia), R 1 , R 2 and R 3 are preferably all hydrogen.
式（Ｉ）のＺに相当する芳香族炭化水素基としては、フェニル基、ナフチル基等が挙げられ、フェニル基が好ましい。該芳香族炭化水素基が有してもよい置換基としては、ヒドロキシ基、アルキル基、アルケニル基、アルコキシ基、アミノ基等が挙げられる。 Examples of the aromatic hydrocarbon group corresponding to Z in the formula (I) include a phenyl group and a naphthyl group, and a phenyl group is preferable. Examples of the substituent that the aromatic hydrocarbon group may have include a hydroxy group, an alkyl group, an alkenyl group, an alkoxy group, and an amino group.
好ましくは、式（Ｉ）のＺに相当する芳香族炭化水素基は、下記式（Ｉｂ）で表される基である：
（Ｉｂ）
[式中、Ｒ４は水素、ヒドロキシ、Ｃ１−４アルキル、Ｃ１−４アルケニル、Ｃ１−４アルコキシおよびアミノからなる群より選択される基である]。また、式（Ｉｂ）において、Ｒ４はヒドロキシ基であることが好ましく、４−ヒドロキシ基であることがより好ましい。
Preferably, the aromatic hydrocarbon group corresponding to Z in the formula (I) is a group represented by the following formula (Ib):
(Ib)
[Wherein R 4 is a group selected from the group consisting of hydrogen, hydroxy, C 1-4 alkyl, C 1-4 alkenyl, C 1-4 alkoxy and amino]. In the formula (Ib), R 4 is preferably a hydroxy group, more preferably a 4-hydroxy group.
式（Ｉ）のＡに相当する炭化水素基としては、アルキレン基、アルケニレン基等が挙げられ、直鎖状、分枝状または環状のいずれであってもよい。また、該炭化水素基は、炭素数が１〜８であるものが好ましく、炭素数が１〜４であるものがより好ましく、炭素数が２であるものがさらに好ましい。式（Ｉ）のＡに相当する炭化水素基が有してもよい置換基としては、ヒドロキシ基、オキソ基、アミノ基等が挙げられる。 Examples of the hydrocarbon group corresponding to A in the formula (I) include an alkylene group and an alkenylene group, and may be any of linear, branched, or cyclic. In addition, the hydrocarbon group preferably has 1 to 8 carbon atoms, more preferably 1 to 4 carbon atoms, and still more preferably 2 carbon atoms. Examples of the substituent that the hydrocarbon group corresponding to A in formula (I) may have include a hydroxy group, an oxo group, and an amino group.
好ましくは、式（Ｉ）のＡに相当する炭化水素基は、ヒドロキシ基またはオキソ基を有する炭素数１〜４のアルキレン基であり、より好ましくは −ＣＨ２ＣＨ（ＯＨ）− または −ＣＨ２Ｃ（＝Ｏ）− である。 Preferably, the hydrocarbon group corresponding to A in formula (I) is a C 1-4 alkylene group having a hydroxy group or an oxo group, more preferably —CH 2 CH (OH) — or —CH 2. C (= O)-.
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤またはシクロオキシゲナーゼ産生抑制剤の有効成分として好ましい式（Ｉ）の化合物の例は、３−インドール乳酸、４−ヒドロキシフェニル乳酸、ピルビン酸および４−ヒドロキシフェニルピルビン酸である。 Examples of compounds of formula (I) that are preferred as active ingredients of the inflammation / injury preventive / inhibitor, inflammatory mediator production inhibitor, MAP kinase activation inhibitor or cyclooxygenase production inhibitor of the present invention include 3-indole lactic acid, 4 -Hydroxyphenyl lactic acid, pyruvic acid and 4-hydroxyphenyl pyruvic acid.
本発明の炎症/傷害の予防/抑制剤の適用対象となる炎症または傷害としては、紫外線照射、外傷、細菌または真菌の感染などの様々な要因によって引き起こされるものが挙げられ、好ましくは紫外線照射に起因するものである。該炎症または傷害の部位は特に限定されないが、好ましくは皮膚であり、より好ましくは表皮である。即ち、本発明の炎症/傷害の予防/抑制剤の適用対象は、好ましくは皮膚の炎症または傷害であり、より好ましくは、紫外線照射に起因する表皮の炎症または傷害である。 Examples of the inflammation or injury to which the agent for preventing / suppressing inflammation / injury of the present invention is applied include those caused by various factors such as ultraviolet irradiation, trauma, bacterial or fungal infection, and preferably ultraviolet irradiation. It is due. The site of the inflammation or injury is not particularly limited, but is preferably skin, more preferably epidermis. That is, the application target of the inflammation / injury prevention / inhibition agent of the present invention is preferably skin inflammation or injury, and more preferably epidermal inflammation or injury caused by ultraviolet irradiation.
また、炎症性サイトカインや活性酸素等の炎症メディエーターを介して生じる炎症または傷害に対しては、その発生原因や部位に関わらず、本発明の炎症/傷害の予防/抑制剤を好適に使用することができる。 In addition, for inflammation or injury caused by inflammatory mediators such as inflammatory cytokines and active oxygen, the prophylactic / inhibitory agent for inflammation / injury of the present invention is preferably used regardless of the cause or site of the occurrence. Can do.
本発明の炎症メディエーター産生抑制剤は、特に、炎症性サイトカインおよびプロスタグランジンの産生を抑制するものである。本発明の炎症メディエーター産生抑制剤を用いて産生を抑制することができる炎症性サイトカインとしては、ＩＬ−６、ＩＬ−１β、ＴＮＦ−α、インターロイキン８（ＩＬ−８）、インターフェロンγ（ＩＦＮ−γ）等が挙げられ、好ましくはＩＬ−６および／またはＩＬ−１βである。また、本発明の炎症メディエーター産生抑制剤を用いて産生を抑制することができるプロスタグランジンとしては、プロスタグランジンＥ２（ＰＧＥ２）およびプロスタグランジンＩ２（ＰＧＩ２）等が挙げられ、好ましくはプロスタグランジンＥ２である。これらの炎症性サイトカインおよびプロスタグランジンは、紫外線照射、外傷、細菌または真菌の感染などの様々な要因によって産生が誘導されるものである。本発明の炎症メディエーター産生抑制剤の適用対象は、好ましくは、紫外線照射によって誘導される炎症メディエーターの産生であり、より好ましくは、紫外線照射によって誘導されるＩＬ−６、ＩＬ−１βおよびプロスタグランジンＥ２からなる群より選択される１種以上の物質の産生である。 The inflammatory mediator production inhibitor of the present invention particularly suppresses the production of inflammatory cytokines and prostaglandins. Examples of inflammatory cytokines whose production can be suppressed using the inflammatory mediator production inhibitor of the present invention include IL-6, IL-1β, TNF-α, interleukin 8 (IL-8), interferon γ (IFN- (gamma)) etc. are mentioned, Preferably it is IL-6 and / or IL-1 (beta). In addition, examples of prostaglandins whose production can be suppressed using the inflammatory mediator production inhibitor of the present invention include prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), preferably prostaglandin. Gin E2. Production of these inflammatory cytokines and prostaglandins is induced by various factors such as ultraviolet irradiation, trauma, bacterial or fungal infection. The application target of the inflammatory mediator production inhibitor of the present invention is preferably production of inflammatory mediators induced by ultraviolet irradiation, more preferably IL-6, IL-1β and prostaglandins induced by ultraviolet irradiation. The production of one or more substances selected from the group consisting of E2.
本発明のＭＡＰキナーゼ活性化抑制剤によって活性化を抑制し得るＭＡＰキナーゼは、好ましくはｐ３８ＭＡＰキナーゼである。 The MAP kinase whose activation can be suppressed by the MAP kinase activation inhibitor of the present invention is preferably p38 MAP kinase.
本発明のシクロオキシゲナーゼ産生抑制剤を用いて産生を抑制することができるシクロオキシゲナーゼは、好ましくはシクロオキシゲナーゼ２（Ｃｏｘ−２）である。 The cyclooxygenase whose production can be suppressed using the cyclooxygenase production inhibitor of the present invention is preferably cyclooxygenase 2 (Cox-2).
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤またはシクロオキシゲナーゼ産生抑制剤の有効成分である式（Ｉ）の化合物は、当該分野において公知の合成方法を用いて合成することができる。あるいは、式（Ｉ）の化合物は、医農薬中間体や試薬等として市販されているものを使用してもよい。また、インドール乳酸やヒドロキシフェニル乳酸等の乳酸誘導体については、乳酸菌培養物から抽出することによって得ることもできる。 The compound of formula (I) which is an active ingredient of the inflammation / injury preventive / inhibitor, inflammatory mediator production inhibitor, MAP kinase activation inhibitor or cyclooxygenase production inhibitor of the present invention is synthesized by a synthesis method known in the art. Can be synthesized. Alternatively, as the compound of the formula (I), those commercially available as intermediates or reagents for medicines and agricultural chemicals may be used. In addition, lactic acid derivatives such as indole lactic acid and hydroxyphenyl lactic acid can also be obtained by extraction from a lactic acid bacteria culture.
本発明の炎症/傷害の予防/抑制剤は、式（Ｉ）の化合物を有効成分として含むものであればよく、皮膚の炎症または傷害を予防または抑制する作用を阻害しない限り、他の成分をさらに含むものであってもよい。例えば、本発明の炎症/傷害の予防/抑制剤は、式（Ｉ）の化合物を０．１重量％以上、または０．５重量％以上、または１．０重量％以上含むものであり得る。あるいは、本発明の炎症/傷害の予防/抑制剤は、式（Ｉ）の化合物のみからなるものであってもよい。 The prophylactic / inhibitory agent for inflammation / injury of the present invention is not limited as long as it contains the compound of formula (I) as an active ingredient, and other ingredients can be used as long as they do not inhibit the action of preventing or inhibiting skin inflammation or injury. Further, it may be included. For example, the inflammation / injury prevention / suppression agent of the present invention may contain 0.1% by weight or more, 0.5% by weight or more, or 1.0% by weight or more of the compound of formula (I). Alternatively, the prophylactic / inhibitory agent for inflammation / injury of the present invention may consist only of the compound of formula (I).
本発明の炎症メディエーター産生抑制剤は、式（Ｉ）の化合物を有効成分として含むものであればよく、炎症メディエーターの産生を抑制する作用を阻害しない限り、他の成分をさらに含むものであってもよい。例えば、本発明の炎症メディエーター産生抑制剤は、式（Ｉ）の化合物を０．１重量％以上、または０．５重量％以上、または１．０重量％以上含むものであり得る。あるいは、本発明の炎症メディエーター産生抑制剤は、式（Ｉ）の化合物のみからなるものであってもよい。 The inflammatory mediator production inhibitor of the present invention only needs to contain the compound of formula (I) as an active ingredient, and may further contain other ingredients as long as the action of inhibiting the production of inflammatory mediator is not inhibited. Also good. For example, the inflammation mediator production inhibitor of the present invention may contain 0.1% by weight or more, 0.5% by weight or more, or 1.0% by weight or more of the compound of formula (I). Alternatively, the inflammatory mediator production inhibitor of the present invention may consist only of the compound of formula (I).
本発明のＭＡＰキナーゼ活性化抑制剤は、式（Ｉ）の化合物を有効成分として含むものであればよく、ＭＡＰキナーゼの活性化を抑制する作用を阻害しない限り、他の成分をさらに含むものであってもよい。例えば、本発明のＭＡＰキナーゼ活性化抑制剤は、式（Ｉ）の化合物を０．１重量％以上、または０．５重量％以上、または１．０重量％以上含むものであり得る。あるいは、本発明のＭＡＰキナーゼ活性化抑制剤は、式（Ｉ）の化合物のみからなるものであってもよい。 The MAP kinase activation inhibitor of the present invention only needs to contain the compound of formula (I) as an active ingredient, and may further contain other ingredients as long as the action of inhibiting the activation of MAP kinase is not inhibited. There may be. For example, the MAP kinase activation inhibitor of the present invention may contain 0.1% by weight or more, 0.5% by weight or more, or 1.0% by weight or more of the compound of formula (I). Alternatively, the MAP kinase activation inhibitor of the present invention may consist only of the compound of formula (I).
本発明のシクロオキシゲナーゼ産生抑制剤は、式（Ｉ）の化合物を有効成分として含むものであればよく、シクロオキシゲナーゼの産生を抑制する作用を阻害しない限り、他の成分をさらに含むものであってもよい。例えば、本発明のシクロオキシゲナーゼ産生抑制剤は、式（Ｉ）の化合物を０．１重量％以上、または０．５重量％以上、または１．０重量％以上含むものであり得る。あるいは、本発明のシクロオキシゲナーゼ産生抑制剤は、式（Ｉ）の化合物のみからなるものであってもよい。 The cyclooxygenase production inhibitor of the present invention only needs to contain the compound of formula (I) as an active ingredient, and may further contain other ingredients as long as the action of inhibiting the production of cyclooxygenase is not inhibited. . For example, the cyclooxygenase production inhibitor of the present invention may contain 0.1% by weight or more, 0.5% by weight or more, or 1.0% by weight or more of the compound of formula (I). Or the cyclooxygenase production inhibitor of this invention may consist only of a compound of a formula (I).
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤およびシクロオキシゲナーゼ産生抑制剤は、化粧品または医薬組成物の成分として、あるいは食品添加剤として用いることができる。 The inflammation / injury prevention / inhibition agent, inflammation mediator production inhibitor, MAP kinase activation inhibitor and cyclooxygenase production inhibitor of the present invention can be used as a component of a cosmetic or pharmaceutical composition or as a food additive.
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤またはシクロオキシゲナーゼ産生抑制剤を含む化粧品（以下、「本発明の化粧品」とも称する）には、必要に応じ、抗酸化剤、紫外線吸収剤、溶剤、増粘剤、保湿剤、殺菌剤、防腐剤、ｐＨ調整剤、色素、香料等の化粧品に通常配合され得る他の成分を含有させることができる。 For the cosmetics (hereinafter also referred to as “the cosmetics of the present invention”) containing the inflammation / injury preventive / inhibitor, inflammatory mediator production inhibitor, MAP kinase activation inhibitor or cyclooxygenase production inhibitor of the present invention, as necessary , Antioxidants, ultraviolet absorbers, solvents, thickeners, moisturizers, bactericides, preservatives, pH adjusters, pigments, fragrances and other components that can be usually blended in cosmetics.
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤またはシクロオキシゲナーゼ産生抑制剤を含む医薬組成物（以下、「本発明の医薬組成物」とも称する）は、必要に応じ、医薬上許容される担体、賦形剤、希釈剤、結合剤、湿潤剤、溶剤、緩衝剤、懸濁化剤、増粘剤、着色剤、安定剤、乳化剤、分散剤、防腐剤等を含むものであり得る。 The pharmaceutical composition containing the inflammation / injury prevention / inhibition agent, inflammation mediator production inhibitor, MAP kinase activation inhibitor or cyclooxygenase production inhibitor of the present invention (hereinafter also referred to as “the pharmaceutical composition of the present invention”), If necessary, pharmaceutically acceptable carriers, excipients, diluents, binders, wetting agents, solvents, buffers, suspending agents, thickeners, colorants, stabilizers, emulsifiers, dispersants, preservatives An agent etc. may be included.
本発明の化粧品または医薬組成物の形態（剤形）は特に限定されないが、皮膚外用剤の形態であることが好ましい。皮膚外用剤の例としては、軟膏、クリーム、乳液、ゲル、貼付剤等が挙げられるが、これらに限定されるものではない。 The form (dosage form) of the cosmetic or pharmaceutical composition of the present invention is not particularly limited, but is preferably in the form of a skin external preparation. Examples of the external preparation for skin include, but are not limited to, ointments, creams, emulsions, gels, patches and the like.
本発明の化粧品または医薬組成物における式（Ｉ）の化合物の濃度は特に限定されないが、例えば、３−インドール乳酸であれば１０μＭ〜１００ｍＭ、４−ヒドロキシフェニル乳酸であれば１０μＭ〜１００ｍＭ、ピルビン酸であれば１０μＭ〜１００ｍＭ、４−ヒドロキシフェニルピルビン酸であれば１０μＭ〜１００ｍＭの濃度で皮膚に適用することができる。 The concentration of the compound of formula (I) in the cosmetic or pharmaceutical composition of the present invention is not particularly limited, but for example, 10 μM to 100 mM for 3-indole lactic acid, 10 μM to 100 mM for 4-hydroxyphenyl lactic acid, pyruvic acid Can be applied to the skin at a concentration of 10 μM to 100 mM, and 4-hydroxyphenylpyruvic acid at a concentration of 10 μM to 100 mM.
また、本発明の化粧品または医薬組成物の適用量および適用回数は特に限定されないが、例えば、３−インドール乳酸を有効成分として含む本発明の医薬組成物（皮膚外用剤）であれば１回あたり３−インドール乳酸１００〜５００μｇ／ｃｍ２（皮膚面積）の量で１日に１〜２回、４−ヒドロキシフェニルピルビン酸を有効成分として含む本発明の医薬組成物（皮膚外用剤）であれば１回あたり４−ヒドロキシフェニルピルビン酸１００〜５００μｇ／ｃｍ２（皮膚面積）の量で１日に１〜２回、皮膚に適用することができる。ピルビン酸を有効成分として含む本発明の医薬組成物、および４−ヒドロキシフェニル乳酸を有効成分として含む本発明の医薬組成物等についても同様の条件で皮膚に適用することができる。 Further, the application amount and the application frequency of the cosmetic or pharmaceutical composition of the present invention are not particularly limited. For example, the pharmaceutical composition (external preparation for skin) of the present invention containing 3-indolelactic acid as an active ingredient may be used once. If it is the pharmaceutical composition (skin external preparation) of this invention which contains 4-hydroxyphenyl pyruvic acid as an active ingredient once or twice a day in the quantity of 3-indole lactic acid 100-500 microgram / cm < 2 > (skin area). It can be applied to the skin once or twice a day in an amount of 100 to 500 μg / cm 2 (skin area) of 4-hydroxyphenylpyruvic acid per time. The pharmaceutical composition of the present invention containing pyruvic acid as an active ingredient and the pharmaceutical composition of the present invention containing 4-hydroxyphenyllactic acid as an active ingredient can be applied to the skin under the same conditions.
本発明の炎症/傷害の予防/抑制剤、本発明の炎症メディエーター産生抑制剤、本発明のＭＡＰキナーゼ活性化抑制剤、本発明のシクロオキシゲナーゼ産生抑制剤、または本発明の化粧品または医薬組成物は、紫外線への曝露の前もしくは後、または紫外線への曝露中のいずれの時点で皮膚に適用してもよい。 The prophylactic / inhibitory agent for inflammation / injury of the present invention, the inflammatory mediator production inhibitor of the present invention, the MAP kinase activation inhibitor of the present invention, the cyclooxygenase production inhibitor of the present invention, or the cosmetic or pharmaceutical composition of the present invention, It may be applied to the skin either before or after exposure to ultraviolet light, or at any point during exposure to ultraviolet light.
また、本発明の炎症/傷害の予防/抑制剤、本発明の炎症メディエーター産生抑制剤、本発明のＭＡＰキナーゼ活性化抑制剤、本発明のシクロオキシゲナーゼ産生抑制剤、または本発明の医薬組成物は、錠剤、顆粒剤、散剤、カプセル、シロップ等の形態において経口投与することも可能である。これにより、皮膚以外の部位における炎症もしくは傷害を予防または抑制すること、炎症メディエーターの産生を抑制すること、ＭＡＰキナーゼの活性化を抑制すること、またはシクロオキシゲナーゼ産生を抑制することができる。 Further, the prevention / suppression agent of inflammation / injury of the present invention, the inflammation mediator production inhibitor of the present invention, the MAP kinase activation inhibitor of the present invention, the cyclooxygenase production inhibitor of the present invention, or the pharmaceutical composition of the present invention, Oral administration in the form of tablets, granules, powders, capsules, syrups and the like is also possible. As a result, it is possible to prevent or suppress inflammation or injury at sites other than the skin, suppress the production of inflammatory mediators, suppress activation of MAP kinase, or suppress cyclooxygenase production.
さらに、本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、本発明のＭＡＰキナーゼ活性化抑制剤、または本発明のシクロオキシゲナーゼ産生抑制剤を、食品、飲料、サプリメント等に配合することにより、皮膚の炎症または傷害を予防または抑制する機能性飲食品、炎症メディエーターの産生を抑制する機能性飲食品、ＭＡＰキナーゼの活性化を抑制する機能性飲食品、あるいはシクロオキシゲナーゼの産生を抑制する機能性飲食品を提供することができる。 Furthermore, the inflammation / injury preventive / inhibitor of the present invention, the inflammation mediator production inhibitor, the MAP kinase activation inhibitor of the present invention, or the cyclooxygenase production inhibitor of the present invention is added to foods, beverages, supplements and the like. Functional foods and beverages that prevent or suppress skin inflammation or injury, functional foods or foods that suppress the production of inflammation mediators, functional foods or foods that suppress the activation of MAP kinase, or functions that suppress the production of cyclooxygenase Sexual foods and drinks can be provided.
以下、実施例によって本発明をより詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
実施例１
３−インドール乳酸の紫外線照射によるＩＬ−６産生抑制効果
ヒト由来株化角化細胞であるＨａＣａＴ細胞を１２ウェル細胞培養プレート（ＢＤバイオサイエンス）に播種し、１０％ウシ胎児血清を添加した高グルコースダルベッコ改変イーグル培地（ＤＭＥＭ）を培養液に用いて、３７℃、５％ＣＯ２の条件で、飽和密度になるまで培養した。メタノールに溶解した３−インドール乳酸（シグマ）を最終濃度１μＭから１００μＭの範囲で培養液に添加し、６０分後に、紫外線ランプ（三共電気：Ｇ８Ｔ５Ｅ、８Ｗ）を装着したＣＬ−１０００型ＵＶクロスリンカー（ＵＶＰ社）を用いて、紫外線Ｂ波を６０［ｍＪ／ｃｍ２］の強度で照射し、２４時間後に培養上清中のインターロイキン６（ＩＬ−６）濃度を、ＥＬＩＳＡ法を用いて測定した（ヒトＩＬ−６ＥＬＩＳＡキットを使用。ＢＤバイオサイエンス社製、製品番号５５５２２０）。結果を図１に示す。３−インドール乳酸は、最終濃度１００μＭにおいて、紫外線照射により誘導されるＩＬ−６の産生を抑制した。
Example 1
IL-6 production inhibitory effect by UV irradiation of 3-indole lactic acid High-glucose seeded with HaCaT cells, a human-derived keratinocyte cell line, in a 12-well cell culture plate (BD Bioscience) and supplemented with 10% fetal bovine serum Dulbecco's modified Eagle medium (DMEM) was used as a culture solution and cultured at 37 ° C. and 5% CO 2 until saturation density was reached. CL-1000 type UV crosslinker equipped with UV lamp (Sankyo Electric: G8T5E, 8W) 60 minutes later, 3-indole lactic acid (Sigma) dissolved in methanol was added to the culture solution at a final concentration of 1 μM to 100 μM. (UVP) was used to irradiate ultraviolet B waves at an intensity of 60 [mJ / cm 2 ], and after 24 hours, the interleukin 6 (IL-6) concentration in the culture supernatant was measured using an ELISA method. (Using human IL-6 ELISA kit. BD Biosciences, product number 555220). The results are shown in FIG. 3-indole lactic acid suppressed IL-6 production induced by UV irradiation at a final concentration of 100 μM.
実施例２
４−ヒドロキシフェニル乳酸の紫外線照射によるＩＬ−６産生抑制効果
３−インドール乳酸に代えて４−ヒドロキシフェニル乳酸（シグマ）を用いたこと以外は上記実施例１と同様の方法および条件において、紫外線照射後のＩＬ−６濃度を測定した。結果を図２に示す。４−ヒドロキシフェニル乳酸は、１、１０および１００μＭのいずれの濃度においても、紫外線照射により誘導されるＩＬ−６の産生を抑制した。
Example 2
IL-6 production inhibitory effect by ultraviolet irradiation of 4-hydroxyphenyl lactic acid UV irradiation in the same method and conditions as in Example 1 except that 4-hydroxyphenyl lactic acid (Sigma) was used instead of 3-indole lactic acid Later IL-6 concentrations were measured. The results are shown in FIG. 4-Hydroxyphenyl lactic acid suppressed the production of IL-6 induced by ultraviolet irradiation at any concentration of 1, 10 and 100 μM.
実施例３
ピルビン酸および４−ヒドロキシフェニルピルビン酸の紫外線照射によるＩＬ−６産生抑制効果
ＨａＣａＴ細胞を６ウェル細胞培養プレート（ＢＤバイオサイエンス）に播種し、１０％ウシ胎児血清を添加した高グルコースダルベッコ改変イーグル培地（ＤＭＥＭ）を培養液に用いて、３７℃、５％ＣＯ２の条件で、飽和密度になるまで培養した。培地をカルシウム・マグネシウム含有ハンクス平衡塩液（インビトロジェン）に置換し、紫外線ランプ（三共電気：Ｇ８Ｔ５Ｅ、８Ｗ）を装着したＣＬ−１０００型ＵＶクロスリンカー（ＵＶＰ社）を用いて、紫外線Ｂ波を６０［ｍＪ／ｃｍ２］の強度で照射した。照射後、ハンクス平衡塩液を、ピルビン酸（和光純薬）または４−ヒドロキシフェニルピルビン酸（シグマ）を１ｍＭから１００ｍＭの最終濃度で添加したＤＭＥＭ培地に置換した（化合物を添加していないＤＭＥＭ培地に置換したものを対照とした）。２４時間後に、培養上清中のインターロイキン６（ＩＬ−６）濃度を、ＥＬＩＳＡ法を用いて測定した（ヒトＩＬ−６ＥＬＩＳＡキットを使用。ＢＤバイオサイエンス社製、製品番号５５５２２０）。結果を図３に示す。ピルビン酸および４−ヒドロキシフェニルピルビン酸はいずれも、紫外線照射により誘導されるＩＬ−６の産生を抑制した。
Example 3
IL-6 production inhibitory effect of pyruvate and 4-hydroxyphenylpyruvate by UV irradiation High glucose Dulbecco's modified Eagle medium supplemented with 6% fetal bovine serum after seeding HaCaT cells in a 6-well cell culture plate (BD Bioscience) (DMEM) was used as a culture solution and cultured at 37 ° C. and 5% CO 2 until saturation density was reached. Using a CL-1000 type UV crosslinker (UVP) equipped with an ultraviolet lamp (Sankyo Electric: G8T5E, 8W), replacing the medium with calcium / magnesium-containing Hank's balanced salt solution (Invitrogen), 60 ultraviolet B waves were applied. Irradiation was performed at an intensity of [mJ / cm 2 ]. After irradiation, Hank's balanced salt solution was replaced with DMEM medium supplemented with pyruvic acid (Wako Pure Chemical) or 4-hydroxyphenylpyruvic acid (Sigma) at a final concentration of 1 mM to 100 mM (DMEM medium without compound added). As a control). After 24 hours, the interleukin 6 (IL-6) concentration in the culture supernatant was measured using an ELISA method (using a human IL-6 ELISA kit, manufactured by BD Biosciences, product number 555220). The results are shown in FIG. Both pyruvic acid and 4-hydroxyphenylpyruvic acid suppressed the production of IL-6 induced by ultraviolet irradiation.
実施例４
ピルビン酸の紫外線照射によるＩＬ−１β産生抑制効果
ＨａＣａＴ細胞を６ウェル細胞培養プレート（ＢＤバイオサイエンス）に播種し、１０％ウシ胎児血清を添加した高グルコースダルベッコ改変イーグル培地（ＤＭＥＭ）を培養液に用いて、３７℃、５％ＣＯ２の条件で、飽和密度になるまで培養した。培地をカルシウム・マグネシウム含有ハンクス平衡塩液（インビトロジェン）に置換し、紫外線ランプ（三共電気：Ｇ８Ｔ５Ｅ、８Ｗ）を装着したＣＬ−１０００型ＵＶクロスリンカー（ＵＶＰ社）を用いて、紫外線Ｂ波を６０［ｍＪ／ｃｍ２］の強度で照射した。照射後、ハンクス平衡塩液を、ピルビン酸（和光純薬）を１０ｍＭから１００ｍＭの最終濃度で添加したＤＭＥＭ培地に置換した（化合物を添加していないＤＭＥＭ培地に置換したものを対照とした）。２４時間後に、培養上清中のインターロイキン１β（ＩＬ−１β）濃度を、ＥＬＩＳＡ法を用いて測定した（ヒトＩＬ−１βＥＬＩＳＡキットを使用。ＢＤバイオサイエンス社製、製品番号５５８８４８）。結果を図４に示す。ピルビン酸は、紫外線照射により誘導されるＩＬ−１βの産生を顕著に抑制した。
Example 4
IL-1β production suppression effect of pyruvate by UV irradiation HaCaT cells were seeded in 6-well cell culture plates (BD Bioscience), and high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum was used as the culture solution. And cultured at 37 ° C. under 5% CO 2 until saturation density was reached. Using a CL-1000 type UV crosslinker (UVP) equipped with an ultraviolet lamp (Sankyo Electric: G8T5E, 8W), replacing the medium with calcium / magnesium-containing Hank's balanced salt solution (Invitrogen), 60 ultraviolet B waves were applied. Irradiation was performed at an intensity of [mJ / cm 2 ]. After irradiation, the Hank's balanced salt solution was replaced with DMEM medium to which pyruvic acid (Wako Pure Chemical Industries) was added at a final concentration of 10 mM to 100 mM (substitute with DMEM medium without compound added as a control). After 24 hours, the interleukin 1β (IL-1β) concentration in the culture supernatant was measured using an ELISA method (using a human IL-1β ELISA kit, manufactured by BD Biosciences, product number 558848). The results are shown in FIG. Pyruvate significantly suppressed IL-1β production induced by UV irradiation.
実施例５
４−ヒドロキシフェニルピルビン酸および３−インドール乳酸の紫外線照射によるヘアレスマウスの経皮水分蒸散抑制効果
８週齢の雄ヘアレスマウス（１群８匹）を１週間馴致した後、３０％プロピレングリコール溶液（和光純薬）を含む０．１Ｍリン酸緩衝液に４−ヒドロキシフェニルピルビン酸（シグマ）または３−インドール乳酸（シグマ）を各々最終濃度１００ｍＭで添加した液を調製し、１日に１００μＬずつ、４日間連続してコンラージ棒を用いて該マウスの背中に塗布した（塗布面積は約４ｃｍ２）。３０％プロピレングリコール溶液を含む０．１Ｍリン酸緩衝液のみを塗布した群を対照とした。実験開始１日目と３日目に、ハンディ型紫外線ランプ（ＵＶＰ社製：ＵＶＬＭ−２８）を用いて、紫外線Ｂ波を、１［Ｊ／ｃｍ２］の強度で照射した。実験開始５日目に、各群のマウスの経皮水分蒸散量を、水分蒸散量測定装置テヴァメーターＴＭ３００（Ｃｏｕｒａｇｅ−Ｋｈａｚａｋａ社製）を用いて測定した。ｔ検定を用い、群間の水分蒸散量の有意差を評価した。結果を図５に示す。
Example 5
Inhibitory effect of 4-hydroxyphenylpyruvic acid and 3-indolelactic acid on the transdermal water transpiration of hairless mice by UV irradiation After acclimatization of 8 weeks old male hairless mice (8 per group) for 1 week, 30% propylene glycol solution ( Prepared by adding 4-hydroxyphenylpyruvic acid (Sigma) or 3-indolelactic acid (Sigma) at a final concentration of 100 mM to 0.1 M phosphate buffer containing Wako Pure Chemical), 100 μL each day, It was applied to the back of the mouse using a congeal bar for 4 consecutive days (application area was about 4 cm 2 ). A group applied with only 0.1 M phosphate buffer containing 30% propylene glycol solution was used as a control. On the first and third days from the start of the experiment, an ultraviolet B wave was irradiated at an intensity of 1 [J / cm 2 ] using a handy ultraviolet lamp (UVLM: UVLM-28). On the fifth day from the start of the experiment, the transdermal water transpiration of each group of mice was measured using a water transpiration measuring device Tevameter TM300 (Courage-Khazaka). A t-test was used to evaluate the significant difference in water transpiration between groups. The results are shown in FIG.
化合物を塗布しなかったマウスでは、紫外線照射によって経皮水分蒸散量が増大した。これは、紫外線照射によって皮膚の角質層に炎症が生じ、皮膚のバリア機能が低下したためである。一方、３−インドール乳酸または４−ヒドロキシフェニルピルビン酸を塗布したマウスでは、これを塗布しなかったマウスと比較して経皮水分蒸散量の増大が抑制された。かかる結果は、３−インドール乳酸または４−ヒドロキシフェニルピルビン酸によって、紫外線照射による皮膚バリア機能の低下が抑制された事を示す。 In mice that did not receive the compound, transdermal water transpiration increased with UV irradiation. This is because the skin stratum corneum is inflamed by ultraviolet irradiation, and the barrier function of the skin is lowered. On the other hand, in the mice to which 3-indole lactic acid or 4-hydroxyphenylpyruvic acid was applied, the increase in the amount of transdermal water transpiration was suppressed as compared to the mice to which this was not applied. This result shows that 3-indole lactic acid or 4-hydroxyphenylpyruvic acid suppressed the reduction of the skin barrier function due to ultraviolet irradiation.
実施例６
ピルビン酸の紫外線照射による細胞死抑制効果
ＨａＣａＴ細胞を６ウェル細胞培養プレート（ＢＤバイオサイエンス）に播種し、１０％ウシ胎児血清を添加した高グルコースダルベッコ改変イーグル培地（ＤＭＥＭ）を培養液に用いて、３７℃、５％ＣＯ２の条件で、飽和密度になるまで培養した。培地をカルシウム・マグネシウム含有ハンクス平衡塩液（インビトロジェン）に置換し、紫外線ランプ（三共電気：Ｇ８Ｔ５Ｅ、８Ｗ）を装着したＣＬ−１０００型ＵＶクロスリンカー（ＵＶＰ社）を用いて、紫外線Ｂ波を６０［ｍＪ／ｃｍ２］の強度で照射した。照射後、ハンクス平衡塩液を、ピルビン酸（和光純薬）を最終濃度１００ｍＭで添加したＤＭＥＭ培地に置換した（化合物を添加していないＤＭＥＭ培地に置換したものを対照とした）。照射後２４時間後に細胞を、３．５％ホルムアルデヒド（和光純薬）を含むリン酸緩衝生理食塩水で１０分間固定した後、リン酸緩衝生理食塩水で２回洗浄した。この時点で細胞像を位相差顕微鏡により撮影した。細胞の写真を図６のＡ〜Ｄに示す。写真中のスケールの大きさは１００μｍである。撮影後、プレートに残存する細胞を半定量的に評価するため、０．１％クリスタルバイオレットを含むリン酸緩衝生理食塩水で細胞を１０分間染色し、リン酸緩衝生理食塩水で２回洗浄後、２ｍＬの１０％酢酸溶液でクリスタルバイオレットを抽出し、６００ｎｍの吸光度を測定した。この測定結果を示すグラフを図６Ｅに示す。紫外線照射により、対照区で認められる細胞数の減少（図６Ｃ）が、ピルビン酸添加区では軽減されている（図６Ｄ）。細胞数定量の結果も、位相差顕微鏡観察の結果を支持する。
Example 6
Cell death inhibitory effect of pyruvate by ultraviolet irradiation HaCaT cells were seeded in 6-well cell culture plates (BD Bioscience), and high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum was used as the culture solution. The cells were cultured at 37 ° C. and 5% CO 2 until saturation density was reached. Using a CL-1000 type UV crosslinker (UVP) equipped with an ultraviolet lamp (Sankyo Electric: G8T5E, 8W), replacing the medium with calcium / magnesium-containing Hank's balanced salt solution (Invitrogen), 60 ultraviolet B waves were applied. Irradiation was performed at an intensity of [mJ / cm 2 ]. After irradiation, Hank's balanced salt solution was replaced with DMEM medium to which pyruvic acid (Wako Pure Chemical Industries) was added at a final concentration of 100 mM (replaced with DMEM medium to which no compound was added was used as a control). 24 hours after irradiation, the cells were fixed with phosphate buffered saline containing 3.5% formaldehyde (Wako Pure Chemical Industries) for 10 minutes, and then washed twice with phosphate buffered saline. At this point, cell images were taken with a phase contrast microscope. Photographs of the cells are shown in FIGS. The scale size in the photograph is 100 μm. After imaging, in order to semi-quantitatively evaluate the cells remaining on the plate, the cells were stained with phosphate buffered saline containing 0.1% crystal violet for 10 minutes and washed twice with phosphate buffered saline. Crystal violet was extracted with 2 mL of 10% acetic acid solution, and absorbance at 600 nm was measured. A graph showing the measurement results is shown in FIG. 6E. The decrease in the number of cells observed in the control group (FIG. 6C) was reduced in the pyruvic acid-added group (FIG. 6D). The results of cell number quantification also support the results of phase contrast microscopy.
実施例７
上記の実施例６と同一の方法で、ＨａＣａＴ細胞に紫外線を照射し、ピルビン酸（最終濃度１００ｍＭ：和光純薬）またはアスコルビン酸（最終濃度１０ｍＭ：和光純薬）を添加したＤＭＥＭ培地に置換した。紫外線照射２４時間後に、１％ノニデットＰ４０（ナカライ）、５ｍＭエチレンジアミン四酢酸二ナトリウム（第一化学薬品）、１％プロテアーゼ阻害剤カクテル（シグマ）を添加した２０ｍＭトリス・塩酸緩衝液（ｐＨ７．４）を用いて細胞を可溶化し、レムリ法によるポリアクリルアミド電気泳動により蛋白質を分離した。分離された蛋白質は、セミドライブロッティング装置（バイオ・ラド社）によりニトロセルロース膜（ＧＥヘルスケアバイオサイエンス）に転写した。ニトロセルトース膜は、５％スキムミルク（和光純薬）、０．１％ポリオキシエチレンソルビタンモノラウレート（ツイン２０・ナカライ）を含むトリス緩衝生理食塩水（ｐＨ７．４）を用い、室温で２時間ブロッキング処理した後、抗シクロオキシゲナーゼ２（Ｃｏｘ−２）モノクローナル抗体（ＢＤバイオサイエンス：製品番号６１０２０３）を用いた一次抗体反応、西洋ワサビペルオキシダーゼ標識抗マウス抗体（ジャクソン社）を用いた二次抗体反応を行ったのち、ＥＣＬウェスタンブロッティング試薬（ＧＥヘルスケアバイオサイエンス）により、膜上の抗原量を化学発光法により検出した。内部標準としては、グリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）の発現量を用いた。検出結果を図７Ａに示す。
Example 7
In the same manner as in Example 6 above, the HaCaT cells were irradiated with ultraviolet rays and replaced with DMEM medium supplemented with pyruvic acid (final concentration 100 mM: Wako Pure Chemical Industries) or ascorbic acid (final concentration 10 mM: Wako Pure Chemical Industries). . 24 hours after UV irradiation, 20 mM Tris / HCl buffer (pH 7.4) supplemented with 1% Nonidet P40 (Nacalai), 5 mM disodium ethylenediaminetetraacetate (first chemical), 1% protease inhibitor cocktail (Sigma) The cells were solubilized using, and the proteins were separated by polyacrylamide electrophoresis using the Remli method. The separated protein was transferred to a nitrocellulose membrane (GE Healthcare Bioscience) using a semi-drive blotting device (Bio-Rad). The nitrocertose membrane uses Tris-buffered saline (pH 7.4) containing 5% skim milk (Wako Pure Chemicals) and 0.1% polyoxyethylene sorbitan monolaurate (Twin 20 / Nacalai), and 2 at room temperature. After time blocking treatment, primary antibody reaction using anti-cyclooxygenase 2 (Cox-2) monoclonal antibody (BD Bioscience: product number 610203), secondary antibody reaction using horseradish peroxidase-labeled anti-mouse antibody (Jackson) Then, the amount of antigen on the membrane was detected by a chemiluminescence method using an ECL western blotting reagent (GE Healthcare Bioscience). As an internal standard, the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. The detection result is shown in FIG. 7A.
また、同時点で細胞培養上清を回収し、その中のプロスタグランジンＥ２の濃度をＥＬＩＳＡにより測定した（プロスタグランジンＥ２キットを使用。カイマン・ケミカル社製・製品番号５１４５３１）。結果を図７Ｂに示す。ピルビン酸は紫外線照射によるシクロオキシゲナーゼ２の発現誘導を抑制し、その産物であるプロスタグランジンＥ２の産生を抑制した。 At the same time, the cell culture supernatant was collected, and the concentration of prostaglandin E2 in the supernatant was measured by ELISA (Prostaglandin E2 kit was used. Cayman Chemical Co., product number 514551). The result is shown in FIG. 7B. Pyruvate suppressed the induction of cyclooxygenase 2 expression by UV irradiation and the production of its product prostaglandin E2.
実施例８
実施例６と同様の方法で、ＨａＣａＴ細胞に紫外線を照射し、ピルビン酸（和光純薬）を最終濃度１００ｍＭで添加したＤＭＥＭ培地に置換した。紫外線照射後、３０分、６０分、および１２０分後に、実施例７と同様の方法で、ＨａＣａＴ細胞から蛋白質を回収し、１８０番目のスレオニンおよび１８２番目のチロシン残基がリン酸化されたｐ３８ストレス応答性ＭＡＰキナーゼを認識するポリクローナル抗体（ＣＳＴ社：製品番号９２１１）を一次抗体に、西洋ワサビペルオキシダーゼ標識抗ウサギ抗体（ＣＳＴ社：製品番号７０７４）を二次抗体に用いたウェスタンブロッティング法により、ｐ３８ストレス応答性ＭＡＰキナーゼのリン酸化（活性化）の程度を評価した。内部標準として、ｐ３８ストレス応答性ＭＡＰキナーゼに対するポリクローナル抗体（ＣＳＴ社：製品番号９２１２）を用いた。結果を図８に示す。ピルビン酸添加により、紫外線照射によるｐ３８ストレス応答性ＭＡＰキナーゼのリン酸化（活性化）が、全期間にわたって抑制された。
Example 8
In the same manner as in Example 6, the HaCaT cells were irradiated with ultraviolet rays and replaced with DMEM medium supplemented with pyruvic acid (Wako Pure Chemical Industries) at a final concentration of 100 mM. 30 minutes, 60 minutes, and 120 minutes after UV irradiation, the protein was recovered from the HaCaT cells in the same manner as in Example 7, and the p38 stress in which the 180th threonine and 182th tyrosine residues were phosphorylated By Western blotting using a polyclonal antibody that recognizes responsive MAP kinase (CST: product number 9211) as a primary antibody and a horseradish peroxidase-labeled anti-rabbit antibody (CST: product number 7074) as a secondary antibody, p38 The degree of phosphorylation (activation) of stress-responsive MAP kinase was evaluated. As an internal standard, a polyclonal antibody (CST: product number 9212) against p38 stress-responsive MAP kinase was used. The results are shown in FIG. By adding pyruvic acid, phosphorylation (activation) of p38 stress-responsive MAP kinase by ultraviolet irradiation was suppressed over the entire period.
本発明の炎症/傷害の予防/抑制剤、炎症メディエーター産生抑制剤、ＭＡＰキナーゼ活性化抑制剤およびシクロオキシゲナーゼ産生抑制剤は、抗炎症剤、日焼け防止剤、肌の光老化防止剤等として、化粧品および医薬品の分野において利用することができる。 Inflammation / injury preventive / inhibitor, inflammatory mediator production inhibitor, MAP kinase activation inhibitor and cyclooxygenase production inhibitor of the present invention include anti-inflammatory agents, sunscreen agents, skin photoaging inhibitors, etc. It can be used in the pharmaceutical field.
Claims (19)
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。 An agent for preventing or inhibiting skin inflammation or injury comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
（Ｉａ）
[式中、Ｒ１、Ｒ２およびＲ３は同一でも異なってもよく、各々独立に、水素、ヒドロキシ、Ｃ１−４アルキル、Ｃ１−４アルケニル、Ｃ１−４アルコキシおよびアミノからなる群より選択される基である]。 The prophylactic or suppressive agent according to claim 1, wherein Z is a group represented by the formula (Ia):
(Ia)
[Wherein R 1 , R 2 and R 3 may be the same or different and each independently represents a group consisting of hydrogen, hydroxy, C 1-4 alkyl, C 1-4 alkenyl, C 1-4 alkoxy and amino; Is a more selected group].
（Ｉｂ）
[式中、Ｒ４は水素、ヒドロキシ、Ｃ１−４アルキル、Ｃ１−４アルケニル、Ｃ１−４アルコキシおよびアミノからなる群より選択される基である]。 The preventive or suppressive agent according to claim 1, wherein Z is a group represented by the formula (Ib):
(Ib)
[Wherein R 4 is a group selected from the group consisting of hydrogen, hydroxy, C 1-4 alkyl, C 1-4 alkenyl, C 1-4 alkoxy and amino].
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。 Inflammatory mediator production inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。 MAP kinase activation inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
Ｚ−Ａ−ＣＯＯＨ
（Ｉ）
[式中、
Ｚは水素であるか、または置換基を有してもよい芳香族炭化水素基もしくは複素環基であり、
Ａは置換基を有してもよい飽和または不飽和炭化水素基である]。 Cyclooxygenase (Cox) production inhibitor comprising a compound of formula (I):
ZA-COOH
(I)
[Where
Z is hydrogen, or an aromatic hydrocarbon group or a heterocyclic group which may have a substituent,
A is a saturated or unsaturated hydrocarbon group which may have a substituent].
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