JP6320861B2 - Cholesterol absorption inhibitor - Google Patents
Cholesterol absorption inhibitor Download PDFInfo
- Publication number
- JP6320861B2 JP6320861B2 JP2014142050A JP2014142050A JP6320861B2 JP 6320861 B2 JP6320861 B2 JP 6320861B2 JP 2014142050 A JP2014142050 A JP 2014142050A JP 2014142050 A JP2014142050 A JP 2014142050A JP 6320861 B2 JP6320861 B2 JP 6320861B2
- Authority
- JP
- Japan
- Prior art keywords
- cholesterol
- cells
- ezetimibe
- test
- rnpc1l1
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、コレステロール吸収阻害剤に関する。 The present invention relates to a cholesterol absorption inhibitor.
血中コレステロールの上昇は冠状動脈性心疾患の主要な危険因子である。血中コレステロールを低下させるためには、基本的に、低コレステロールの食物、胆汁酸性の金属イオン封鎖剤（コレスチラミンおよびコレスチポール）、ニコチン酸（ナイアシン）、フィブラート類およびプロブコールなどに限られていた。しかしこれらの薬剤や療法には治療の限界があった。
近年、ロバスタチン、シンバスタチンおよびプラバスタチン（ＨＭＧ−ＣｏＡレダクターゼ阻害剤）が、冠動脈および頸動脈におけるアテローム性病変の進行を遅延させることが確認された。さらにシンバスタチンおよびプラバスタチンが冠状動脈性心臓病のリスクを低下させることが明らかとなった。
Increased blood cholesterol is a major risk factor for coronary heart disease. In order to lower blood cholesterol, it was basically limited to low cholesterol foods, bile acidic sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), fibrates and probucol. However, these drugs and therapies have limited treatment.
Recently, lovastatin, simvastatin and pravastatin (HMG-CoA reductase inhibitors) have been shown to delay the progression of atheromatous lesions in the coronary and carotid arteries. Furthermore, simvastatin and pravastatin were found to reduce the risk of coronary heart disease.
動脈硬化疾患において、コレステリルエステルは、アテローム性病変の主要な構成成分であり、動脈壁細胞におけるコレステロールの主要な貯蔵形態である。このコレステリルエステルの形成は、食事性コレステロールの腸管吸収における重要なステップでもある。
小腸上皮細胞に取り込まれたコレステロールは、小腸上皮細胞内の小胞体にてコレステリルエステルに変換された後、カイロミクロンを形成して体内に吸収されることが知られている。従って、コレステリルエステルの形成阻害が、食事性コレステロールの腸管吸収を阻止することが推測され、これに伴う血中コレステロールの減少が、アテローム性病変形成の進行を抑制し、動脈硬化の予防や改善につながるものと考えられている。
In arteriosclerotic diseases, cholesteryl esters are a major component of atherosclerotic lesions and a major storage form of cholesterol in arterial wall cells. The formation of this cholesteryl ester is also an important step in the intestinal absorption of dietary cholesterol.
It is known that cholesterol taken into small intestinal epithelial cells is converted into cholesteryl ester in the endoplasmic reticulum in small intestinal epithelial cells, and then formed into chylomicron and absorbed into the body. Therefore, it is speculated that the inhibition of cholesteryl ester formation prevents intestinal absorption of dietary cholesterol, and the concomitant reduction in blood cholesterol suppresses the progression of atheromatous lesion formation, preventing or improving arteriosclerosis. It is thought to be connected.
またエゼチミブ（Ｅｚｅｔｉｍｉｂｅ）などの特定のヒドロキシ置換アゼチジノン（特許文献１：米国特許第５，７６７，１１５号明細書）は、アテローム性動脈硬化症の治療および予防におけるコレステロール低下剤として有用であることが知られている。エゼチミブのコレステロール低下作用は、ノックアウトマウスやトランスジェニックマウスの研究から、小腸のコレステロール吸収を担う膜蛋白質であるＮｉｅｍａｎｎ Ｐｉｃｋ Ｔｙｐｅ Ｃ １ ｌｉｋｅ １（以下ＮＰＣ１Ｌ１という）とエゼチミブが結合することによって阻害されることが明らかとなった（特許文献２：特開２０１０−２５２７９８号公報）。
従って、エゼチミブとＮＰＣ１Ｌ１の結合を阻害する物質は、消化管内のコレステロール吸収を阻害し、アテローム性動脈硬化を改善することが期待されている。
Certain hydroxy-substituted azetidinones such as ezetimibe (US Pat. No. 5,767,115) may also be useful as cholesterol lowering agents in the treatment and prevention of atherosclerosis. Are known. The cholesterol-lowering action of ezetimibe is inhibited by binding of ezetimibe with Niemann Pick Type C 1 like 1 (hereinafter referred to as NPC1L1), which is a membrane protein responsible for cholesterol absorption in the small intestine, from studies on knockout mice and transgenic mice. Became clear (Patent Document 2: JP 2010-252798 A).
Therefore, a substance that inhibits the binding between ezetimibe and NPC1L1 is expected to inhibit cholesterol absorption in the gastrointestinal tract and improve atherosclerosis.
近年キノコの子実体から血中コレステロール低下効果のある物質や、動脈硬化の予防効果を有するものが得られている。特許文献３にはプレウロツス・エリンギ（Ｐｌｅｕｒｏｔｕｓ ｅｒｙｎｇｉ）ｖａｒ． ｆｅｒｕｌａｅやプロウロツス・オストレアツス（Ｐｌｅｕｒｏｔｕｓ ｏｓｕｔｒｅａｔｕｓｕ）などプレウロツス属のキノコが高コレステロール血症の治療剤であるロバスタチンを産生することが開示されている（特許文献３：特表２００３−５２０５７６号公報）。また特許文献４には担子菌類のシイタケ、キクラゲ、マイタケ、レイシがアディポネクチンを分泌促進しコレステロール低下に効果を示すことが開示されている（特許文献４：特開２００８−２９７２５６号公報）。このようにキノコ類の特定の種の子実体がコレステロールを低下させ、動脈硬化を軽減する作用を示すことは従前から知られていた。 In recent years, substances having a blood cholesterol lowering effect and those having an effect of preventing arteriosclerosis have been obtained from fruit bodies of mushrooms. In Patent Document 3, Pleurotus eringi var. It has been disclosed that mushrooms belonging to the genus Pleurotus such as ferulae and Pleurotus ostreatus produce lovastatin, which is a therapeutic agent for hypercholesterolemia (Patent Document 3: JP 2003-520576 A). Further, Patent Document 4 discloses that basidiomycetous fungi, shiitake, jellyfish, maitake, and litchi promote secretion of adiponectin and show an effect on lowering cholesterol (Patent Document 4: Japanese Patent Application Laid-Open No. 2008-297256). Thus, it has been known for a long time that the fruiting bodies of specific species of mushrooms have the effect of lowering cholesterol and reducing arteriosclerosis.
本発明者らは、担子菌の一種であるクロサルノコシカケ抽出物中に、ＮＰＣ１Ｌ１蛋白質と結合し、コレステロールの吸収を抑制する成分が存在し、コレステロール低下剤として有用であることを見出してすでに特許出願している（特願２０１３−０３３０８９号）。 The present inventors have already found that the extract of Kurosarokoshikatake, a kind of basidiomycete, binds to the NPC1L1 protein and suppresses absorption of cholesterol and is useful as a cholesterol lowering agent. (Japanese Patent Application No. 2013-033089).
本発明は、新規な高コレステロール吸収阻害剤、高コレステロール血症の改善剤、高脂血症の改善剤を提供することを課題とする。 An object of the present invention is to provide a novel high cholesterol absorption inhibitor, an improving agent for hypercholesterolemia, and an improving agent for hyperlipidemia.
本発明者らはクロサルノコシカケの示すコレステロール吸収阻害剤について研究を行い、コレステロール吸収阻害を示す物質を単離し、化学構造を決定した。 The present inventors conducted research on cholesterol absorption inhibitors exhibited by black salmon mushrooms, isolated substances showing inhibition of cholesterol absorption, and determined their chemical structures.
本発明の構成は次の通りである。
（１）次の化学式（Ｉ）で表される化合物を有効成分とするコレステロール吸収阻害剤。
（３）（１）記載のコレステロール吸収阻害剤を含む高脂血症改善剤。
The configuration of the present invention is as follows.
(1) A cholesterol absorption inhibitor comprising a compound represented by the following chemical formula (I) as an active ingredient.
(3) A hyperlipidemia improving agent comprising the cholesterol absorption inhibitor according to (1).
本発明により、新規なコレステロール吸収阻害剤及び高コレステロール血症改善剤が提供される。また高脂血症改善剤が提供される。 According to the present invention, a novel cholesterol absorption inhibitor and a hypercholesterolemia improving agent are provided. An agent for improving hyperlipidemia is also provided.
本発明は下記の化学式（Ｉ）で表される化合物を有効成分とするコレステロール吸収阻害剤及び高コレステロール血症改善剤である。 The present invention is a cholesterol absorption inhibitor and a hypercholesterolemia ameliorating agent comprising a compound represented by the following chemical formula (I) as an active ingredient.
本発明のコレステロール吸収阻害剤に用いる化合物は、ＩＵＰＡＣ名称 １５−Ｈｙｄｒｏｘｙ−１７−（１−ｈｙｄｒｏｘｙｍｅｔｈｙｌ−５−methyl−hex−4−enyl）−４，４，１０，１３，１４−ｐｅｎｔａｍｅｔｈｙｌ−１，２，４，５，６，７，１０, １１，１２，１３，１４，１５，１６，１７−tetraｄｅｃａｈｙｄｒｏ−ｃｙｃｌｏｐｅｎｔａ［ａ］ｐｈｅｎａｎｔｈｒｅｎ−３−ｏｎｅである。この化合物は化学合成することもできる。また、クロサルノコシカケ（Ｆｏｍｉｔｏｐｓｉｓ ｎｉｇｒａ）の子実体若しくは菌糸体から抽出しても良い。抽出は、水、低級脂肪族アルコール（メタノール、エタノール、イソプロピルアルコールなど）、低級脂肪族ケトン（アセトンなど）、ヘキサンなどの溶媒を用いる。抽出物を含む溶媒はあるいは、これらを含む溶媒により当業者が通常行う抽出処理により抽出する。抽出した溶液は容易に製造することができる。製造したエキスは、加熱処理、凍結乾燥あるいは減圧乾燥等の処理により、濃縮エキスや乾燥エキスにすることができる。 The compound used for the cholesterol absorption inhibitor of the present invention is IUPAC name 15-Hydroxy-17- (1-hydroxymethyl-5-methyl-hex-4-enyl) -4,4,10,13,14-pentamethyl-1, 2, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-cyclopenta [a] phenanthren-3-one. This compound can also be chemically synthesized. Alternatively, it may be extracted from the fruit body or mycelium of Fomitopsis nigra. The extraction uses a solvent such as water, a lower aliphatic alcohol (such as methanol, ethanol, isopropyl alcohol), a lower aliphatic ketone (such as acetone), or hexane. Alternatively, the solvent containing the extract is extracted by an extraction process usually performed by those skilled in the art using the solvent containing these. The extracted solution can be easily produced. The produced extract can be made into a concentrated extract or a dried extract by a treatment such as heat treatment, freeze-drying or reduced-pressure drying.
本発明の阻害剤または予防もしくは改善剤は、通常使用される任意成分を含有する錠剤、顆粒剤、散剤、カプセル剤、液剤などの剤形で経口により投与することができる。任意成分としては、例えば、賦形剤、結合剤、被覆剤、滑沢剤、糖衣剤、崩壊剤、増量剤、矯味矯臭剤、乳化・可溶化・分散剤、安定剤、ｐＨ調整剤、等張剤などが挙げられる。これらを常法に従って処理することにより、本発明の阻害剤等を製造することができる。 The inhibitor or the preventive or ameliorating agent of the present invention can be administered orally in a dosage form such as a tablet, granule, powder, capsule, or liquid containing an optional ingredient usually used. Examples of optional components include excipients, binders, coating agents, lubricants, sugar coatings, disintegrating agents, bulking agents, flavoring agents, emulsifying / solubilizing / dispersing agents, stabilizers, pH adjusting agents, and the like. Examples include tonicity agents. The inhibitor of this invention etc. can be manufactured by processing these according to a conventional method.
コレステロール吸収抑制剤又は高コレステロール血症改善剤として使用する場合の投与量は、年齢、性別、体重などによって異なるが、通常、原抽出物換算で成人１日あたり約１ｍｇ〜１ｇ、好ましくは５ｍｇ〜５００ｍｇである。
なお、剤には、医薬品のみならず、医薬部外品、健康食品なども包含される。
The dose for use as a cholesterol absorption inhibitor or hypercholesterolemia-improving agent varies depending on age, sex, body weight, etc., but is usually about 1 mg to 1 g, preferably 5 mg 500 mg.
The agent includes not only pharmaceutical products but also quasi drugs and health foods.
以下に実施例および試験例を挙げ、本発明をさらに具体的に説明する。
（コレステロール吸収阻害剤の製造例）
天然より採集したクロサルノコシカケ（Ｆｏｍｉｔｏｐｓｉｓ ｎｉｇｒａ）の未乾燥の子実体４２ｋｇを約４倍量のエタノール（Ｗａｋｏ）及び１倍量のアセトン（Ｗａｋｏ）で室温下、順次浸漬抽出後、エタノール及びアセトンの抽出液を混合し、ロータリーエバポレーターを用いて不溶物が析出しないよう溶媒を減圧濃縮した。次に、濃縮液５００ｍｌにヘキサン（Ｗａｋｏ）１Ｌを加え、分液漏斗を用いて液々分配した。この操作を複数回繰返し、回収したヘキサン層はロータリーエバポレーターを用いて濃縮乾固させ、４０．３ｇのヘキサン抽出部を得た。この内、ヘキサン抽出部２８．３ｇを１００ｍｌメタノール（Ｗａｋｏ）に溶解させ、９５０ｍｌヘキサン、９５０ｍｌの８０％アセトニトリル水を加えて液々分配し、ロータリーエバポレーターを用いて濃縮乾固させた。その結果、ヘキサン層６．８ｇ、中間層５．２ｇ、８０％アセトニトリル層１３．６ｇを得た。この液々分配による分画物は、後述する動物試験に供した。
Hereinafter, the present invention will be described more specifically with reference to examples and test examples.
(Production example of cholesterol absorption inhibitor)
Extraction of ethanol and acetone after sequential immersion extraction at room temperature of 42 kg of dried fruit bodies of black monkey (Fomitopsis nigra) collected from nature with about 4 times the amount of ethanol (Wako) and 1 time of acetone (Wako) The liquids were mixed, and the solvent was concentrated under reduced pressure using a rotary evaporator so that insolubles did not precipitate. Next, 1 L of hexane (Wako) was added to 500 ml of the concentrated liquid, and the liquid was partitioned using a separatory funnel. This operation was repeated a plurality of times, and the recovered hexane layer was concentrated and dried using a rotary evaporator to obtain 40.3 g of a hexane extraction part. Among these, 28.3 g of the hexane extraction part was dissolved in 100 ml methanol (Wako), 950 ml hexane and 950 ml of 80% acetonitrile water were added, liquid-divided and concentrated to dryness using a rotary evaporator. As a result, 6.8 g of a hexane layer, 5.2 g of an intermediate layer, and 13.6 g of an 80% acetonitrile layer were obtained. The fraction obtained by liquid-liquid distribution was subjected to the animal test described later.
次に８０％アセトニトリル層１１．６ｇをＯＤＳ（ＹＭＣ製）オープンクロマトグラフィー（桐山製作所製φ４×３０ ｃｍ）に供した。７５％アセトニトリル水にて溶出させ８画分に分離し、減圧下溶媒除去し、Ｆｒ２ ７２１．８ｍｇを得た。
更にＳｅｐｈａｄｅｘ ＬＨ２０（ＧＥヘルスジャパン製）及びメタノールを用いたオープンクロマトグラフィー（桐山製作所製φ２．２×１００ ｃｍ）を行い、Ｆｒ２全量を２２画分に分離し減圧下溶媒除去後、分画物を得た。この２２画分の内６画分を合わせ、ＯＤＳ（ＹＭＣ製）オープンクロマトグラフィー（桐山製作所製φ１．１×１４５ ｃｍ）に供した。７０％アセトニトリル水にて溶出させ８画分に分離し、更にＤｅｖｅｌｏｓｉｌＸＧ Ｃ３０（Ｎｏｍｕｒａ Ｃｈｅｍｉｃａｌ製）、アセトニトリル：０．１％ギ酸水（６：４）にてピーク分取を行い、最後に、ＤｅｖｅｌｏｓｉｌＸＧ Ｃ３０（Ｎｏｍｕｒａ Ｃｈｅｍｉｃａｌ製）、アセトニトリル：水（６：４）にてピーク分取を行い、減圧下溶媒除去後、化学式（Ｉ）の化合物 ４．０７ｍｇを単離した。
Next, 11.6 g of 80% acetonitrile layer was subjected to ODS (YMC) open chromatography (Kiriyama Seisakusho φ4 × 30 cm). Elution with 75% acetonitrile in water separated into 8 fractions, and the solvent was removed under reduced pressure to obtain 721.8 mg of Fr2.
Furthermore, Sephadex LH20 (manufactured by GE Health Japan) and open chromatography (φ2.2 × 100 cm, manufactured by Kiriyama Seisakusho) using methanol were performed, and the total amount of Fr2 was separated into 22 fractions, and the solvent was removed under reduced pressure. Obtained. Six of the 22 fractions were combined and subjected to ODS (YMC) open chromatography (Kiriyama Seisakusho φ1.1 × 145 cm). Elution with 70% acetonitrile water and separation into 8 fractions, followed by peak fractionation with Develosil XG C30 (manufactured by Nomura Chemical) and acetonitrile: 0.1% aqueous formic acid (6: 4), and finally Develosil XG C30 (Nomura Chemical), acetonitrile: water (6: 4) was used for peak fractionation, and after removing the solvent under reduced pressure, 4.07 mg of the compound of formula (I) was isolated.
（クロサルノコシカケ抽出物中のコレステロール阻害剤の構造決定）
単離した画分をＬＣ／ＭＳ、ＮＭＲにより構造を決定し、化学式（Ｉ）の化合物であることを確認した。
(Determining the structure of a cholesterol inhibitor in the extract of Black Sarcophagus)
The structure of the isolated fraction was determined by LC / MS and NMR, and confirmed to be a compound of the formula (I).
（試験１：エゼチミブの結合阻害試験）
製造例で得られた化学式（Ｉ）の化合物を、下記の試験法でＮＰＣ１Ｌ１とエゼチミブの結合阻害活性を評価する試験を行った。
（１）ラットＮＰＣ１Ｌ１遺伝子のクローニング
ラット空腸由来１本鎖ｃＤＮＡ（ジェノスタッフ株式会社）を鋳型にｉＰｒｏｏｆ Ｈｉｇｈ−Ｆｉｄｅｌｉｔｙ ＤＮＡ ポリメラーゼ（Ｂｉｏｒａｄ）を用いたＰＣＲ法により、５’端にＨｉｎｄ ＩＩＩ、３’端にＸｂａ Ｉを付加したｒＮＰＣ１Ｌ１全長を増幅し、Ｚｅｒｏ Ｂｌｕｎｔ ＴＯＰＯ ＰＣＲクローニング キット（Ｉｎｖｉｔｒｏｇｅｎ）を用いてｐＣＲ４ Ｂｌｕｎｔ ＴＯＰＯベクターにクローニングした。クローニングしたＤＮＡ配列を配列表配列番号１に示す。
なお、ｒＮＰＣ１Ｌ１全長増幅に使用したプライマー及びＰＣＲ条件は次のとおりである。
Ｓｅｎｓｅ： ５’− ａａｇｃｔｔａｃｃａｔｇｇｃａｇｃｔｇｃｃｔｇｇｃｔｇｇｇａｔｇｇｃ−３’（配列番号２）
Ａｎｔｉｓｅｎｓｅ： ５’− ｔｃｔａｇａｔｔａｇａａｃｔｔｔｔｇｇｔｃａｃｔｔｔｔｇｇｇｃ−３’ （配列番号３）
(Test 1: Ezetimibe binding inhibition test)
The compound of the formula (I) obtained in the production example was subjected to a test for evaluating the binding inhibitory activity between NPC1L1 and ezetimibe by the following test method.
(1) Cloning of rat NPC1L1 gene By PCR method using iProof High-Fidelity DNA polymerase (Biorad) using rat jejunum-derived single-stranded cDNA (Geno Staff Co., Ltd.) as a template, Hind III at 3 'end, 3' end The full length of rNPC1L1 with Xba I added thereto was amplified and cloned into the pCR4 Blunt TOPO vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen). The cloned DNA sequence is shown in SEQ ID NO: 1 in the sequence listing.
The primers and PCR conditions used for rNPC1L1 full-length amplification are as follows.
Sense: 5'- aagcttacatgggcaggctgcctggctgggaggg-3 '(SEQ ID NO: 2)
Antisense: 5'-tctagattagaactttttgggtactttgggg-3 '(SEQ ID NO: 3)
ＰＣＲ条件
１．９８℃ ３０秒、２．９８℃ １０秒、３．６８℃ ３０秒、４．７２℃ １．５分、５．７２℃ １０分、２〜４を３５サイクル実施する。
PCR conditions 1.98 ° C. for 30 seconds, 2.98 ° C. for 10 seconds, 3.68 ° C. for 30 seconds, 4.72 ° C. for 1.5 minutes, 5.72 ° C. for 10 minutes, 2 to 4 cycles are performed.
（２）ラットＮＰＣ１Ｌ１安定発現細胞の作製
ｐＣＲ４ Ｂｌｕｎｔ ＴＯＰＯ ｒＮＰＣ１Ｌ１ベクターをＨｉｎｄ ＩＩＩ及びＸｂａ Ｉ処理し、アガロース電気泳動後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ）を用いてｒＮＰＣ１Ｌ１を抽出し、発現用ベクターｐｃＤＮＡ３．１（Ｉｎｖｉｔｒｏｇｅｎ）のＨｉｎｄ ＩＩＩ、Ｘｂａ Ｉサイトにサブクローニングした。作製したｐｃＤＮＡ３．１ ｒＮＰＣ１Ｌ１の配列はプレミックスシーケンス解析サービス（タカラバイオ）を利用して確認を行った。
シーケンス用ｐｒｉｍｅｒ
Ｓｅｎｓｅ：５’−ｃａｃｔｇｃｔｔａｃｔｇｇｃｔｔａｔｃｇ−３’（配列番号４）
Ｓｅｎｓｅ：５’−ｇｃｃｔｔｔｔａｔｃａｇｃｇｃａｇｃｔｔｔ−３’（配列番号５）
Ｓｅｎｓｅ：５’−ａａｃｔｃｔｃａｃｃｃｃａｔａｃｃａｔｃ−３’（配列番号６）
Ｓｅｎｓｅ：５’−ｃａｇｔａｃｔｔｃｃａｇａａｃａａｃｃｇ−３’（配列番号７）
Ｓｅｎｓｅ：５’−ｔｔｃｔａｃｔｃｃｔａｃｃｔｇｇｇｔｇｔ−３’（配列番号８）
Ｓｅｎｓｅ：５’−ｃｔｔｃｔｔｃｃｇｃａａｇａｔａｔａｃｇｃ−３’（配列番号９）
Ｓｅｎｓｅ：５’−ｃｃａｃａｇｃｇｇａａｃａｇｔｔｔｃａｔ−３’（配列番号１０）
Ｓｅｎｓｅ：５’−ｔｃｃｃｔｃａｔｃａａｃｃｔｔｇｔｃａｃ−３’（配列番号１１）
Ｓｅｎｓｅ：５’−ｇｔｃｃａｔｇｃｔｔｃｔｔｇｃｔｇａｔｇ−３’（配列番号１２）
(2) Preparation of rat NPC1L1 stably expressing cells The pCR4 Blunt TOPO rNPC1L1 vector was treated with Hind III and Xba I, and after agarose electrophoresis, the rNPC1L1 vector was extracted using QIAquick Gel Extraction Kit (QIAGEN). (Invitrogen) Hind III, Xba I sites were subcloned. The sequence of the prepared pcDNA3.1 rNPC1L1 was confirmed using a premix sequence analysis service (Takara Bio).
Sequencer primer
Sense: 5'-cactgcttactgggcttatcg-3 '(SEQ ID NO: 4)
Sense: 5'-gccttttattcaggcgcacttt-3 '(SEQ ID NO: 5)
Sense: 5′-aactctaccaccataccatc-3 ′ (SEQ ID NO: 6)
Sense: 5′-cagtacttccagaacaaccg-3 ′ (SEQ ID NO: 7)
Sense: 5'-ttctactcctacctggggtgt-3 '(SEQ ID NO: 8)
Sense: 5'-ctttttccgcaagatatacgc-3 '(SEQ ID NO: 9)
Sense: 5′-ccacagcggagaacattttcat-3 ′ (SEQ ID NO: 10)
Sense: 5′-tcccccatcaactttgtcac-3 ′ (SEQ ID NO: 11)
Sense: 5'-gtccatgctttctgtctgatg-3 '(SEQ ID NO: 12)
次いで、ヒト腎臓由来ＨＥＫ２９３細胞（ＡＴＣＣ）に、Ｆｕｇｅｎｅ ＨＤ（Ｒｏｃｈｅ）を用いてｐｃＤＮＡ３．１ ｒＮＰＣ１Ｌ１をトランスフェクションし、最終濃度１ｍｇ／ｍｌ Ｇ４１８−ｓｕｌｆａｔｅ（Ｗａｋｏ）にてセレクションを行った。更に限界希釈法によりセレクションを行い、ＨＥＫ／ｒＮＰＣ１Ｌ１安定発現細胞の単一クローンを得た。 Subsequently, pcDNA3.1 rNPC1L1 was transfected into human kidney-derived HEK293 cells (ATCC) using Fugene HD (Roche), and selection was performed at a final concentration of 1 mg / ml G418-sulfate (Wako). Further, selection was performed by a limiting dilution method to obtain a single clone of HEK / rNPC1L1 stably expressing cells.
（３）膜画分の調製
φ１５０ｍｍｄｉｓｈ（Ｎｕｎｃ）にＨＥＫ２９３／ｒＮＰＣ１Ｌ１安定発現細胞を播種し、ＤＭＥＭ（Ｎａｃａｌａｉ ｔｅｓｑｕｅ）＋１０％ＦＢＳ（Ｇｉｂｃｏ）にてコンフルエントになるまで培養し、最終濃度４ｍＭ Ｓｏｄｉｕｍ Ｂｕｔｙｒａｔｅ（Ｓｉｇｍａ Ａｌｄｒｉｃｈ）２４時間処理後の細胞をＣｅｌｌ Ｄｉｓｓｏｃｉａｔｉｏｎ Ｂｕｆｆｅｒ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて回収した。８００ｒｐｍ、３分の遠心後、残渣の細胞を８％Ｓｕｃｒｏｓｅ含有２０ｍＭ ＨＥＰＥＳ／Ｔｒｉｓ Ｂｕｆｆｅｒ（ｐＨ７．４）＋ｐｒｏｔｅｉｎａｓｅ ｉｎｈｉｂｉｔｏｒ（Ｒｏｃｈｅ）に懸濁し、超音波を用いて破砕した。４０００ｒｐｍ、４℃、１０分の遠心後、上清を４８０００ｒｐｍ（１２５０００Ｇ）、４℃、３時間、超遠心し、残渣を膜画分として回収した。膜画分は１６０ｍＭ ＮａＣｌ、５％Ｇｌｙｃｅｒｏｌ含有２０ｍＭ ＨＥＰＥＳ／Ｔｒｉｓ Ｂｕｆｆｅｒに再懸濁し、ＢＣＡ ａｓｓａｙ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ Ｐｉｅｒｃｅ）にて蛋白質濃度を定量後、使用するまで−８０℃に保存した。
(3) Preparation of membrane fraction HEK293 / rNPC1L1 stably expressing cells were seeded on φ150 mm dish (Nunc), cultured until confluent in DMEM (Nacalai tesque) + 10% FBS (Gibco), and final concentration of 4 mM Sodium Butyrate (Sigma) Aldrich) Cells treated for 24 hours were collected using Cell Dissociation Buffer (Invitrogen). After centrifugation at 800 rpm for 3 minutes, the remaining cells were suspended in 20 mM HEPES / Tris Buffer (pH 7.4) + proteinase inhibitor (Roche) containing 8% sucrose, and disrupted using ultrasonic waves. After centrifugation at 4000 rpm, 4 ° C. for 10 minutes, the supernatant was ultracentrifuged at 48000 rpm (125000 G), 4 ° C. for 3 hours, and the residue was collected as a membrane fraction. The membrane fraction was resuspended in 20 mM HEPES / Tris Buffer containing 160 mM NaCl and 5% Glycerol, and the protein concentration was quantified with BCA assay (Thermo Scientific Pierce) and stored at −80 ° C. until use.
（４）結合阻害実験
９６ｗｅｌｌ ｐｌａｔｅ（Ｎｕｎｃ）に最終濃度２５ｎＭ ［３Ｈ］Ｅｚｅｔｉｍｉｂｅ−ｇｌｕｃｕｒｏｎｉｄｅ（Ａｍｅｒｉｃａｎ Ｒａｄｉｏｌａｂｅｌｅｄ Ｃｈｅｍｉｃａｌｓ， Ｉｎｃ．）、１．２５ｍｇ／ｍｌ ＨＥＫ／ｒＮＰＣ１Ｌ１由来膜蛋白質、各濃度の被験物質を緩衝液Ａ（２６ｍＭ ＮａＨＣＯ３、０．９６ｍＭ ＮａＨ２ＰＯ４、５ｍＭ ＨＥＰＥＳ、５．５ｍＭ Ｇｌｕｃｏｓｅ、１１７ｍＭ ＮａＣｌ、５．４ｍＭ ＫＣｌ、０．０３％ Ｓｏｄｉｕｍ ｔａｕｒｏｃｈｏｌａｔｅ、０．０５％ Ｄｉｇｉｔｏｎｉｎ、ｐＨ７．４）に加えＴｏｔａｌ ｖｏｌｕｍｅ ３０μｌとなるよう調製し、室温、１時間反応させた。反応液はＨａｒｖｅｓｔｅｒ（ＰｅｒｋｉｎＥｌｍｅｒ）を用いてＵｎｉＦｉｌｔｅｒ−９６ ＧＦ／Ｃ（ＰｅｒｋｉｎＥｌｍｅｒ）にトラップし、ミリＱ水（ミリポア）を用いて遊離［３Ｈ］Ｅｚｅｔｉｍｉｂｅ−ｇｌｕｃｕｒｏｎｉｄｅを洗浄した。乾燥後、Ｍｉｃｒｏｓｃｉｎｔ−２０（ＰｅｒｋｉｎＥｌｍｅｒ）を１５μｌ／ｗｅｌｌ添加し、Ｔｏｐｃｏｕｎｔ（ＰｅｒｋｉｎＥｌｍｅｒ）によりｒＮＰＣ１Ｌ１と結合した［３Ｈ］Ｅｚｅｔｉｍｉｂｅ−ｇｌｕｃｕｒｏｎｉｄｅの放射活性を測定した。
(4) Binding inhibition experiment 96 well plate (Nunc) with final concentration of 25 nM [ 3 H] Ezetimibe-glucuronide (American Radiolabeled Chemicals, Inc.), 1.25 mg / ml HEK / rNPC1L1-derived membrane protein at each concentration, In addition to solution A (26 mM NaHCO3, 0.96 mM NaH2PO4, 5 mM HEPES, 5.5 mM Glucose, 117 mM NaCl, 5.4 mM KCl, 0.03% Sodium taurocholate, 0.05% Digitonin, pH 7.4) and Total volume 30 μl And reacted at room temperature for 1 hour. The reaction solution was trapped in UniFilter-96 GF / C (PerkinElmer) using Harvester (PerkinElmer), and free [ 3 H] Ezetimibe-glucuronide was washed using MilliQ water (Millipore). After drying, 15 μl / well of Microscint-20 (PerkinElmer) was added, and the radioactivity of [ 3 H] Ezetimibe-glucuronide bound to rNPC1L1 was measured by Topcount (PerkinElmer).
（５）結果
クロサルノコシカケから抽出し、単離された化学式（Ｉ）の化合物は、ジメチルスルホキシド（ＤＭＳＯ）に溶解し、最終濃度１ｍｇ／ｍｌから８段階の３倍希釈系列を作製し、上記の手順で放射活性を測定し、その結果からＩＣ５０を算出した。ＩＣ５０は１５．３μｇ／ｍｌと低濃度でＮＰＣ１Ｌ１とエゼチミブの結合を阻害した。
(5) Results The compound of the formula (I) extracted and isolated from kurosarokoshikake is dissolved in dimethyl sulfoxide (DMSO) to prepare a 3-fold dilution series of 8 steps from the final concentration of 1 mg / ml. Radioactivity was measured by the procedure, and IC50 was calculated from the result. IC50 inhibited the binding of NPC1L1 and ezetimibe at a low concentration of 15.3 μg / ml.
ＩＣ５０を算出した希釈系列の阻害を図１に示す。
クロサルノコシカケから単離した化合物は、エゼチミブがＮＰＣ１Ｌ１に結合する反応を強く抑制した。従ってエゼチミブと同様に、ＮＰＣ１Ｌ１の機能を阻害し、コレステロールの腸管吸収を阻害する。
The inhibition of the dilution series for which the IC50 was calculated is shown in FIG.
The compound isolated from kurosarokoshikake strongly suppressed the reaction of ezetimibe binding to NPC1L1. Therefore, like ezetimibe, it inhibits the function of NPC1L1 and inhibits intestinal absorption of cholesterol.
（試験例２：高コレステロール血症ラットを用いたコレステロール低下試験）
本発明の化合物を含む抽出物（製造例に記載したヘキサン層、中間層、８０％アセトニトリル層）を用いて、高コレステロール含有食餌を摂取した場合に発生する高コレステロール血症に対する治療効果を確認した。
(Test Example 2: Cholesterol lowering test using hypercholesterolemic rats)
Using the extract containing the compound of the present invention (hexane layer, intermediate layer, 80% acetonitrile layer described in Production Examples), the therapeutic effect on hypercholesterolemia that occurs when a high cholesterol-containing diet is ingested was confirmed. .
１．試験動物
１週間の馴化後、体重測定を行い、層別連続無作為化法により群分けした８週齢のＷｉｓｔａｒ雄ラット（日本ＳＬＣ）を実験に供した。
高コレステロール血症は、高コレステロール食（ＣＲＦ−１を元に１％コレステロール、０．５％コール酸を添加；オリエンタル酵母工業株式会社）を与え、誘導した。高コレステロール血症を確認するため、通常食（ＣＲＦ−１；オリエンタル酵母工業株式会社）を与えたノーマル群を設定した。
群設定は、通常食を与えたノーマル群に加え、高コレステロール食を与えて、高コレステロール血症を誘導した状態にコーン油を投与したコントロール群、エゼチミブ（コスモバイオ株式会社）０．３ｍｇ／ｋｇを投与した陽性対照のエゼチミブ群、上記方法で製造したクロサルノコシカケヘキサン抽出部（分離前）、ヘキサン層、中間層、８０％アセトニトリル層を各５００ｍｇ／ｋｇ投与した（各群６匹の設定）。
1. Test animals After acclimatization for 1 week, body weights were measured, and 8-week-old Wistar male rats (Japan SLC) grouped by stratified continuous randomization were used for the experiment.
Hypercholesterolemia was induced by giving a high cholesterol diet (1% cholesterol and 0.5% cholic acid added based on CRF-1; Oriental Yeast Co., Ltd.). In order to confirm hypercholesterolemia, a normal group fed with a normal diet (CRF-1; Oriental Yeast Co., Ltd.) was set up.
The group setting is a control group in which corn oil is administered in a state in which hypercholesterolemia is induced in addition to a normal group given a normal diet, 0.3 mg / kg ezetimibe (Cosmo Bio Inc.) The ezetimibe group of the positive control to which was administered, the black sarcoma mushroom hexane extraction part (before separation), the hexane layer, the intermediate layer, and the 80% acetonitrile layer produced by the above method were each administered at 500 mg / kg (setting of 6 animals in each group).
２．試験方法
通常食、高コレステロール食、及び水は３日間自由摂取させ、被験物質はコーン油に溶解させ、試験開始の２日目から１日１回、２日間（初日は投与なし）、胃ゾンデを用いて強制経口投与した（２ｍｌ／ｋｇ）。ノーマル群及びコントロール群はコーン油を投与した。飼育は個別飼育（１匹／ケージ）とし、３日間の摂餌量を記録した。また、被験物質投与開始の２日目に床敷（パルソフト；オリエンタル酵母工業株式会社）を交換した。血液は、被験物質の最終投与から２時間絶食させた後（水は自由摂取）、ジエチルエーテル麻酔下、腹部大動脈からヘパリン処理した真空採血管（テルモ株式会社）を用いて回収した。回収した血液は、直ちに３，０００ｒｐｍ、１５分、４℃の条件下で遠心分離し、得られた血漿を分析するまで−８０℃に保存した。血漿中コレステロール量は、コレステロールＥテストワコー（和光純薬工業株式会社）、中性脂肪はトリグリセライドＥテストワコー（和光純薬工業株式会社）を用いて測定した。
2. Test method Regular diet, high-cholesterol diet, and water are allowed to freely take for 3 days, the test substance is dissolved in corn oil, once a day for 2 days (no administration on the first day) from the second day of the test, stomach tube Forcibly orally (2 ml / kg). The normal group and the control group were administered corn oil. The breeding was individual breeding (1 animal / cage), and food intake for 3 days was recorded. In addition, the floor (Palsoft; Oriental Yeast Co., Ltd.) was replaced on the second day after the start of test substance administration. The blood was fasted for 2 hours after the final administration of the test substance (water was freely ingested) and then collected using a vacuum blood collection tube (Terumo Corporation) heparinized from the abdominal aorta under diethyl ether anesthesia. The collected blood was immediately centrifuged at 3,000 rpm for 15 minutes at 4 ° C., and the obtained plasma was stored at −80 ° C. until analysis. The amount of cholesterol in plasma was measured using Cholesterol E Test Wako (Wako Pure Chemical Industries, Ltd.), and the neutral fat was measured using Triglyceride E Test Wako (Wako Pure Chemical Industries, Ltd.).
３．試験結果
結果は全て平均値±Ｓ．Ｅで表記した。統計解析はエクセル統計２０１０を使用し、ノーマル群を除いた高コレステロール食摂取の５群で行った。各測定データは一次元配置分散分析及びＤｕｎｎｅｔｔの多重検定を行った。有意水準はいずれの場合も両側５％未満とした。
血中総コレステロールの測定結果を図２、血中トリグリセライドの測定結果を図３に示す。
試験の結果、高コレステロール食を摂取させた５群間の摂餌量に差はなく、また体重に変動がなかった。このことから、高コレステロール血症の誘導は群間差なく行われたものと評価した。
血中コレステロールは、陽性対照のエゼチミブ投与群（Ｅｚｅ）、分離前群及び８０％アセトニトリル層群（ＡＣＮ層）においてコントロール群の血漿中コレステロールと比較して有意差が認められ、コレステロール低下作用が明らかとなった。ヘキサン層（ＨＥＸ層）、中間層は有意なコレステロール低下が認められなかった。
また、中性脂肪を測定した結果、コレステロールの測定結果と同様に、分離前群及び８０％アセトニトリル群において有意差が認められた。
したがって、クロサルノコシカケから単離した化学式（Ｉ）の化合物を含む層（ＡＣＮ層）は血漿中のコレステロールの低下作用と中性脂肪の低下作用を有していることが明らかとなった。
以上の試験結果から、ＮＰＣ１Ｌ１の阻害作用を有する化学式（Ｉ）の化合物は、食餌性高コレステロール血症及び高脂血症の改善剤として有用であることが明らかとなった。
3. Test results All results are mean ± S. Indicated by E. Statistical analysis was performed on 5 groups with high cholesterol diet intake, excluding the normal group, using Excel Statistics 2010. Each measurement data was subjected to a one-dimensional analysis of variance and Dunnett's multiple test. The significance level was less than 5% on both sides in all cases.
The measurement result of blood total cholesterol is shown in FIG. 2, and the measurement result of blood triglyceride is shown in FIG.
As a result of the test, there was no difference in food intake among the five groups fed with a high cholesterol diet, and there was no change in body weight. From this, it was evaluated that induction of hypercholesterolemia was performed without any difference between groups.
Blood cholesterol was significantly different from the plasma cholesterol of the control group in the positive control ezetimibe administration group (Eze), the pre-separation group and the 80% acetonitrile layer group (ACN layer), and the cholesterol lowering effect was clear It became. No significant cholesterol lowering was observed in the hexane layer (HEX layer) and the intermediate layer.
Moreover, as a result of measuring neutral fat, a significant difference was observed between the pre-separation group and the 80% acetonitrile group, similar to the cholesterol measurement result.
Therefore, it was revealed that the layer (ACN layer) containing the compound of the formula (I) isolated from black Sarcophagus has a cholesterol lowering action and a triglyceride lowering action in plasma.
From the above test results, it was revealed that the compound of the chemical formula (I) having an inhibitory action on NPC1L1 is useful as an agent for improving dietary hypercholesterolemia and hyperlipidemia.
（試験３：Ｃａｃｏ−２細胞を用いたコレステロールの細胞内取り込み阻害試験）
ヒト大腸がん由来細胞株Ｃａｃｏ−２細胞を用いて試験を行った。
１．レンチウイルス溶液及びCaco2/mock、Caco2/rNPC1L1安定発現細胞の作製
レンチウイルス用ベクターpCDH-CMV-MCS-EF1-Puro（System Biosciences）のSwaＩ、SgfＩサイトを利用して、同制限酵素サイトを付加したrNPC1L1全長をPCR法にて増幅し、サブクローニングした（以下、pCDH-rNPC1L1とする）。pCDH-rNPC1L1、エンベロープベクターのpMD2.G（Addgene）、パッキングベクターのpsPAX2（Addgene）の3種のベクターをリポフェクション用試薬LTX（invitrogen）を用いて293T（ATCC）細胞にトランスフェクションし、96時間後に培地を回収した。2500rpm、10分の遠心を2回行い、得られた上清を0.45μmのフィルター（ミリポア）を通し、レンチウイルス溶液を作製した。
ヒト大腸癌由来Caco2細胞（ATCC）にレンチウイルス溶液を添加して感染させ、更に限界希釈法を用いて最終濃度5μg/mlのpuromycin（Sigma Aldrich）にてセレクションを行いCaco2/rNPC1L1安定発現細胞の単一クローンを作製した。また、同様の方法よりインサートDNAを含まないpCDH-CMV-MCS-EF1-Puroベクターを用いて単一クローンを作製し、Caco2/mockとした。
(Test 3: Inhibition test of cholesterol uptake using Caco-2 cells)
The test was performed using human colon cancer-derived cell line Caco-2 cells.
1. Preparation of Lentiviral Solution and Caco2 / mock, Caco2 / rNPC1L1 Stable Expression Cells Using the SwaI and SgfI sites of lentiviral vector pCDH-CMV-MCS-EF1-Puro (System Biosciences), the same restriction enzyme sites were added. The full length of rNPC1L1 was amplified by PCR and subcloned (hereinafter referred to as pCDH-rNPC1L1). Three vectors, pCDH-rNPC1L1, envelope vector pMD2.G (Addgene) and packing vector psPAX2 (Addgene) were transfected into 293T (ATCC) cells using lipofection reagent LTX (invitrogen), and 96 hours later The medium was collected. Centrifugation was performed twice at 2500 rpm for 10 minutes, and the obtained supernatant was passed through a 0.45 μm filter (Millipore) to prepare a lentivirus solution.
Infection of human colon cancer-derived Caco2 cells (ATCC) with the addition of lentivirus solution, and further selection using puromycin (Sigma Aldrich) with a final concentration of 5 μg / ml using limiting dilution method, stable expression of Caco2 / rNPC1L1 cells A single clone was made. In addition, a single clone was prepared using the pCDH-CMV-MCS-EF1-Puro vector containing no insert DNA by the same method, and designated as Caco2 / mock.
２−１．[3H］ラベルコレステロールを用いた評価試験
Caco2/mock及びCaco2/rNPC1L1安定発現細胞のそれぞれを12well plate（Falcon）に5×105 cells/wellとなるよう播種し、2日置きに培地DMEM+10%FBSを交換して、オーバーコンフルエントの状態を14日間維持し、小腸様細胞に分化させた。DMEMにて細胞を洗浄後、最終濃度2mMタウロコール酸ナトリウム（Wako）、50μMフォスファチジルコリン（Sigma Aldrich）、1μMコレステロール（Sigma Aldrich）、1μCi/ml[3H]コレステロール（PerkinElmer）、各濃度のEzetimibeあるいは化合物Ｉを含有したDMEMミセル溶液を1ml/well添加し、37℃、1時間、処理した。1ml/well PBS+にて細胞を2回洗浄後、0.1%ドデシル硫酸リチウム（WAKO）、0.2N水酸化ナトリウム（Nacalai tesque）含有蒸留水を1ml/well添加し、室温48時間処理後、BCA assayにて蛋白質濃度を測定した。また、その細胞溶解液100μlを、2mlのクリアゾルII（Nacalai tesque）に加え、液体シンチレーションカウンター（ALOKA）にて総[3H]コレステロールの放射活性を測定し、蛋白質当りの量に換算して取り込み量とした。
2-1. Evaluation test using [ 3 H] labeled cholesterol
Each of the Caco2 / mock and Caco2 / rNPC1L1 stably expressing cells is seeded on a 12-well plate (Falcon) at 5 × 10 5 cells / well, and the medium DMEM + 10% FBS is changed every 2 days. The state was maintained for 14 days and differentiated into small intestine-like cells. After washing the cells with DMEM, final concentrations of 2 mM sodium taurocholate (Wako), 50 μM phosphatidylcholine (Sigma Aldrich), 1 μM cholesterol (Sigma Aldrich), 1 μCi / ml [ 3 H] cholesterol (PerkinElmer), 1 ml / well of DMEM micelle solution containing Ezetimibe or Compound I was added and treated at 37 ° C. for 1 hour. After washing the cells twice with 1 ml / well PBS +, add 1 ml / well of 0.1% lithium dodecyl sulfate (WAKO) and 0.2N sodium hydroxide (Nacalai tesque) in distilled water and treat for 48 hours at room temperature. The protein concentration was measured. In addition, add 100 μl of the cell lysate to 2 ml of Clearsol II (Nacalai tesque), measure the radioactivity of total [ 3 H] cholesterol using a liquid scintillation counter (ALOKA), and convert it into an amount per protein. The amount.
２−２．結果
Caco2/mock及びCaco2/rNPC1L1細胞それぞれに対し、溶媒を添加したContorol群、最終濃度3、10、30μM添加したEzetimibe群、最終濃度1、3、10、30μM添加した化合物Ｉ群の総[3H]コレステロールの測定結果を下記図４に示す。
Ezetimibe及び化合物Ｉ添加群は、mock及びrNPC1L1細胞のそれぞれのコントロールと比較し、いずれも取り込まれた[3H]コレステロール量が有意に減少しており、化合物Ｉはコレステロールの細胞内取り込みを顕著に抑制した。
2-2. result
For each of the Caco2 / mock and Caco2 / rNPC1L1 cells, the total of the Contorol group to which the solvent was added, the Ezetimibe group to which the final concentrations of 3, 10, and 30 μM were added, and the compound I group to which the final concentrations of 1, 3, 10, and 30 μM were added [ 3 H The measurement results of cholesterol are shown in FIG. 4 below.
The Ezetimibe and Compound I addition groups showed a significant decrease in the amount of [ 3 H] cholesterol incorporated, as compared with the respective controls of mock and rNPC1L1 cells. Suppressed.
３−１．［3H］ラベルコレステロールエステルを用いた評価試験
Caco2/mock及びCaco2/rNPC1L1安定発現細胞を12well plate（Falcon）に5×105 cells/wellとなるよう播種し、2日置きに培地DMEM+10%FBSを交換して、オーバーコンフルエントの状態を14日間維持し、小腸様細胞に分化させた。DMEMにて細胞を洗浄後、最終濃度5mMタウロコール酸ナトリウム（Wako）、500μMオレイン酸（Sigma Aldrich）、10μMコレステロール（Sigma Aldrich）、1μCi/ml[3H]コレステロール（PerkinElmer）、各濃度の被験物質を含有したDMEMミセル溶液を1ml/well添加し、37℃、1時間、処理した。DMEMにて洗浄後、無血清用カクテルITS（Thermo Fisher Scientific）含有DMEMに交換し、更に37℃、8時間、インキュベートし、エステル化を誘導した。PBS+で細胞を洗浄後、ヘキサン：イソプロパノール（3:2）（Nacalai tesque）混合液を用いて細胞から脂質を抽出し、クロロホルム：メタノール（2:1）（Nacalai tesque）に置換後、全量をTLC（Roche）にスポットし、ヘキサン：ジエチルエーテル：酢酸（60:40:1）（Nacalai tesque）を用いて展開した。展開したTLCはヨウ素（Nacalai tesque）を用いて発色させ、分離した[3H]コレステロール、[3H]コレステロールエステルをかきとり、クリアゾルII（Nacalai tesque）に浸し、液体シンチレーションカウンター（ALOKA）を用いて放射活性を測定した。
また、脂質抽出後の細胞は、0.1%ドデシル硫酸リチウム（WAKO）、0.2N水酸化ナトリウム（Nacalai tesque）含有蒸留水を添加し、室温48時間処理後、BCA assayにて蛋白質濃度を測定した。細胞中に取り込まれ、エステル化されたコレステロールを蛋白質当りの量に換算して取り込み量とした。
3-1. Evaluation test using [ 3 H] labeled cholesterol ester
Caco2 / mock and Caco2 / rNPC1L1 stably expressing cells are seeded on a 12-well plate (Falcon) at 5 × 10 5 cells / well, and the medium DMEM + 10% FBS is changed every 2 days to ensure overconfluence. Maintained for 14 days and differentiated into small intestine-like cells. After washing cells with DMEM, final concentrations of 5 mM sodium taurocholate (Wako), 500 μM oleic acid (Sigma Aldrich), 10 μM cholesterol (Sigma Aldrich), 1 μCi / ml [ 3 H] cholesterol (PerkinElmer), test substances at each concentration 1 ml / well of DMEM micelle solution containing was added and treated at 37 ° C. for 1 hour. After washing with DMEM, the sample was replaced with serum-free cocktail ITS (Thermo Fisher Scientific) -containing DMEM, and further incubated at 37 ° C. for 8 hours to induce esterification. After washing the cells with PBS +, lipids were extracted from the cells using a mixture of hexane: isopropanol (3: 2) (Nacalai tesque) and replaced with chloroform: methanol (2: 1) (Nacalai tesque). Spotted on (Roche) and developed using hexane: diethyl ether: acetic acid (60: 40: 1) (Nacalai tesque). The developed TLC is colored with iodine (Nacalai tesque), scraped the separated [ 3 H] cholesterol and [ 3 H] cholesterol ester, soaked in Clearsol II (Nacalai tesque), and using a liquid scintillation counter (ALOKA) Radioactivity was measured.
In addition, 0.1% lithium dodecyl sulfate (WAKO) and 0.2N sodium hydroxide (Nacalai tesque) -containing distilled water were added to the cells after lipid extraction, and after treatment for 48 hours at room temperature, the protein concentration was measured by BCA assay. Cholesterol taken up into cells and esterified was converted to the amount per protein and taken as the amount taken up.
３−２．結果
Caco2/mock及びCaco2/rNPC1L1細胞それぞれに対し、溶媒を添加したContorol群、最終濃度3、10、30μM添加したEzetimibe群、最終濃度1、3、10、30μM添加した化合物Ｉ群の [3H]コレステロールエステル量の測定結果を下記図５に示す。
Ezetimibe及び化合物Ｉ添加群は、mock及びrNPC1L1細胞のそれぞれのコントロールと比較し、いずれもコレステロールを細胞内に取り込んだ後に生成される [3H]コレステロールエステル量を濃度依存的に抑制し、細胞内へのコレステロール取り込み量を顕著に減少させた。
以上2つの試験から、化合物Ｉは、コレステロールの吸収部位である腸管壁の細胞であるCaco２へのコレステロールの取り込みを阻害していることが明らかとなった。
3-2. result
[ 3 H] of the Contorol group to which the solvent was added, the Ezetimibe group to which the final concentrations of 3, 10, and 30 μM were added, and the compound I group to which the final concentrations of 1, 3, 10, and 30 μM were added to the Caco2 / mock and Caco2 / rNPC1L1 cells, respectively. The measurement result of the amount of cholesterol ester is shown in FIG.
The Ezetimibe and Compound I addition groups compared the respective controls of mock and rNPC1L1 cells, and both inhibited the amount of [ 3 H] cholesterol ester produced after uptake of cholesterol into the cells in a concentration-dependent manner. Significantly reduced cholesterol uptake.
From the above two tests, it has been clarified that Compound I inhibits the uptake of cholesterol into Caco2, which is a cell of the intestinal tract wall which is the absorption site of cholesterol.
Claims (3)
A hyperlipidemia-improving agent comprising the cholesterol absorption inhibitor according to claim 1.
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