JP2010261871A - Labeled bile acid - Google Patents
Labeled bile acid Download PDFInfo
- Publication number
- JP2010261871A JP2010261871A JP2009114022A JP2009114022A JP2010261871A JP 2010261871 A JP2010261871 A JP 2010261871A JP 2009114022 A JP2009114022 A JP 2009114022A JP 2009114022 A JP2009114022 A JP 2009114022A JP 2010261871 A JP2010261871 A JP 2010261871A
- Authority
- JP
- Japan
- Prior art keywords
- bile acid
- labeled
- bsep
- conjugate
- bile
- 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.)
- Pending
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- 239000003613 bile acid Substances 0.000 title claims abstract description 171
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 title claims abstract description 125
- 230000000694 effects Effects 0.000 claims abstract description 97
- 108010093662 Member 11 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 claims abstract description 91
- 238000000034 method Methods 0.000 claims abstract description 62
- 108091022863 bile acid binding Proteins 0.000 claims abstract description 59
- 102000030904 bile acid binding Human genes 0.000 claims abstract description 59
- 238000012216 screening Methods 0.000 claims abstract description 13
- SLLFVLKNXABYGI-UHFFFAOYSA-N 1,2,3-benzoxadiazole Chemical class C1=CC=C2ON=NC2=C1 SLLFVLKNXABYGI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 8
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 5
- 102000011339 Bile salt export pump Human genes 0.000 claims abstract 10
- 239000000126 substance Substances 0.000 claims description 24
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- -1 N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzooxadiazolyl Chemical group 0.000 claims description 19
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- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 7
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- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims description 5
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- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical class C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 7
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
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- 108010078791 Carrier Proteins Proteins 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 239000004471 Glycine Substances 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
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- 150000003431 steroids Chemical class 0.000 description 5
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
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Abstract
Description
本発明は、側鎖が発色団で標識された胆汁酸又はその抱合体や、側鎖が発色団で標識された胆汁酸又はその抱合体を用いる胆汁酸トランスポーター活性の測定方法、胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニング方法等に関する。 The present invention relates to a method for measuring bile acid transporter activity using a bile acid having a side chain labeled with a chromophore or a conjugate thereof, a bile acid having a side chain labeled with a chromophore or a conjugate thereof, bile acid trans The present invention relates to a screening method for a porter activity promoter or inhibitor.
細胞は形質膜や小胞体，ミトコンドリアなどの細胞内小器官膜を介してイオンや栄養素の輸送ならびに老廃物や毒素の排出を行うことにより細胞機能を維持している。これらの物質輸送は、チャネル，トランスポーターと呼ばれる膜輸送タンパク質により選択的に行われている。その一つとしてＡＢＣトランスポーターが知られている。ＡＢＣタンパク質は、トランスポーター，チャネル，受容体（レギュレーター）という多様な機能に分化し、それぞれの生物で重要な生理機能を果たしており、細胞内ＡＴＰ、ＡＤＰによって駆動あるいは制御されている。ＡＴＰ加水分解のエネルギーをタンパク質分子の構造変化に変換し、それとカップリングすることによって薬剤を細胞の中から外へ輸送することから、薬物の体内動態プロファイル（吸収、分布、代謝、排泄、ターゲット部位での薬剤実効濃度）を規定し、ひいては薬物の全体的な薬理効果をも左右するといわれている。 Cells maintain their cell functions by transporting ions and nutrients and excreting waste products and toxins through plasma membranes, endoplasmic reticulum, and organelle membranes such as mitochondria. These substance transports are selectively carried out by membrane transport proteins called channels and transporters. As one of them, ABC transporter is known. ABC proteins differentiate into various functions such as transporters, channels, and receptors (regulators), play important physiological functions in each organism, and are driven or controlled by intracellular ATP and ADP. By converting the energy of ATP hydrolysis into structural changes in protein molecules and coupling it to the drug, the drug is transported from inside the cell to the outside, so that the pharmacokinetic profile of the drug (absorption, distribution, metabolism, excretion, target site) It is said that the drug's effective concentration) is determined, and in turn, affects the overall pharmacological effect of the drug.
例えば、ＡＢＣトランスポーターの一種であるＢＳＥＰ（bile salt export pump、ＡＢＣＢ１１）は、肝臓におけるタウロコール酸（Ｔ−ＣＡ）等の胆汁酸排泄を司る排出型ＡＢＣトランスポーターであり（例えば、非特許文献１参照）、ＢＳＥＰの機能障害が胆汁うっ滞等の肝疾患を誘起する一因と考えられている。さらに、ＢＳＥＰの胆汁酸輸送活性は、副作用として肝毒性を示す各種薬剤の投与により阻害を受けることが明らかにされており、臨床のみならず、医薬品開発の初期スクリーニングにおける肝毒性マーカーとして極めて有用とされていることから、ＢＳＥＰの胆汁酸輸送活性を測定することは、極めて重要である。 For example, BSEP (bile salt export pump, ABCB11), which is a type of ABC transporter, is an excretory ABC transporter that controls excretion of bile acids such as taurocholic acid (T-CA) in the liver (for example, Non-Patent Document 1). ), BSEP dysfunction is considered to be one of the causes of liver diseases such as cholestasis. Furthermore, it has been clarified that the bile acid transport activity of BSEP is inhibited by administration of various drugs showing hepatotoxicity as a side effect, and it is extremely useful as a hepatotoxicity marker not only in clinical but also in the initial screening of drug development. Therefore, it is extremely important to measure the bile acid transport activity of BSEP.
従来、ＢＳＥＰを含むＡＢＣトランスポーターの輸送活性の測定方法として、ＡＢＣトランスポーターが発現している膜に基質を添加した際のＡＴＰアーゼ活性を測定する方法が知られている。この方法は、輸送されるべき基質の存在によりＡＴＰがＡＢＣトランスポーターに結合し、ＡＤＰに分解されることから、ＡＢＣトランスポーターに結合した状態のＡＤＰを測定するものであるが、膜自体の持つ内因性ＡＴＰアーゼ活性によりバックグラウンドが高くなる場合がある、リン酸濃度の分解量を測定するため感度が比較低いという問題があった。 Conventionally, as a method for measuring transport activity of an ABC transporter containing BSEP, a method for measuring ATPase activity when a substrate is added to a membrane expressing ABC transporter is known. In this method, ATP binds to the ABC transporter due to the presence of the substrate to be transported and is decomposed into ADP. Therefore, ADP in the state bound to the ABC transporter is measured, but the membrane itself has. There was a problem that the background was high due to endogenous ATPase activity, and the sensitivity was relatively low because the amount of degradation of the phosphate concentration was measured.
これに代わる測定方法として、ＡＢＣトランスポーターを発現させた反転膜小胞を利用する薬物輸送実験法（ベシクル法）が開発されている。この方法では、ベシクル内に取り込まれたリガンド量から輸送活性を求めるため、トランスポーターの輸送能を直接評価することが可能である。ＢＳＥＰ活性測定にも同様の方法が適用されており、トレーサーとして放射性同位元素（ＲＩ）標識化胆汁酸（３Ｈ−標識Ｔ−ＣＡなど）を利用し、その放射活性よりＢＳＥＰの輸送活性を求めることができる。現在、このような測定系はキット化されて市販されており、各種薬剤によるＢＳＥＰ阻害活性評価のスクリーニング法として臨床並びに医薬品開発分野において広く利用されている。また、これまでに、３Ｈ−標識Ｔ−ＣＡをトレーサーとするＢＳＥＰ活性測定法により各種薬剤によるＢＳＥＰ阻害作用について検討が行われており、シクロスポリンＡ、グリベンクラミド、プラバスタチン、リファンピシン及びＴ−ＬＣＡなどの薬剤がＢＳＥＰの輸送活性を阻害することが明らかにされている。 As an alternative measurement method, a drug transport experiment method (vesicle method) using an inverted membrane vesicle expressing ABC transporter has been developed. In this method, since the transport activity is determined from the amount of ligand incorporated into the vesicle, the transport ability of the transporter can be directly evaluated. A similar method is applied to the measurement of BSEP activity, and a radioisotope (RI) -labeled bile acid (such as 3 H-labeled T-CA) is used as a tracer, and the transport activity of BSEP is determined from the radioactivity. be able to. Currently, such measurement systems are made into kits and are commercially available, and are widely used in clinical and pharmaceutical development fields as screening methods for evaluating BSEP inhibitory activity with various drugs. In addition, the BSEP inhibitory action by various drugs has been studied by the BSEP activity measurement method using 3 H-labeled T-CA as a tracer so far, such as cyclosporin A, glibenclamide, pravastatin, rifampicin and T-LCA. Drugs have been shown to inhibit the transport activity of BSEP.
また、ＢＳＥＰの胆汁酸輸送活性については、胆汁酸常成分に対する活性を解析した報告があり、胆汁酸のＢＳＥＰに対する親和性に関しては、タウロケノデオキシコール酸（Ｔ−ＣＤＣＡ）が最も高く、タウロコール酸（Ｔ−ＣＡ）及びタウロデオキシコール酸（Ｔ−ＤＣＡ）は同程度であり、側鎖抱合形式に関しては、グリシン抱合体に比し、タウリン抱合体が有意にＢＳＥＰにより輸送されることが報告され、ＢＳＥＰの輸送能は胆汁酸の分子構造により影響を受けることが示唆されている。 In addition, regarding the bile acid transport activity of BSEP, there is a report analyzing the activity of bile acid against normal components, and regarding the affinity of bile acid for BSEP, taurochenodeoxycholic acid (T-CDCA) is the highest, and taurocholic acid (T -CA) and taurodeoxycholic acid (T-DCA) are comparable, with respect to the side chain conjugation format, it has been reported that taurine conjugates are significantly transported by BSEP compared to glycine conjugates. It has been suggested that the transport ability of is influenced by the molecular structure of bile acids.
しかしながら、これまでに、ＢＳＥＰに対する胎児性胆汁酸並びに異常胆汁酸の親和性・反応性等の諸性質についての報告例はなく、ＢＳＥＰの基質特異性ついては未だ明らかにされていない。ＢＳＥＰをはじめとする胆汁酸トランスポーターの諸性質の解析において、従来の放射性同位元素標識化胆汁酸を利用することは、放射性同位元素使用に基づく様々な制約から、一般の実験室で実施することは不可能である。そこで、より汎用性に優れる胆汁酸トランスポーター輸送活性の測定方法、輸送活性の促進物質や阻害物質のスクリーニング法の開発が切望されている。 However, there have been no reports on various properties such as affinity and reactivity of fetal bile acids and abnormal bile acids to BSEP, and the substrate specificity of BSEP has not yet been clarified. In the analysis of various properties of bile acid transporters including BSEP, the use of conventional radioisotope-labeled bile acids should be carried out in a general laboratory due to various restrictions based on the use of radioisotopes. Is impossible. Therefore, development of a method for measuring bile acid transporter transport activity, which is more versatile, and a screening method for a transport activity promoting substance or inhibitor are strongly desired.
本発明の課題は、放射性同位元素標識を用いることなく、胆汁酸トランスポーターに対して非標識の胆汁酸と同等の親和性、反応性を有し、汎用性に優れた標識化胆汁酸や、胆汁酸トランスポーター活性の測定方法、胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニング方法等を提供することにある。 The subject of the present invention is a labeled bile acid having an affinity and reactivity equivalent to that of an unlabeled bile acid with respect to a bile acid transporter without using a radioisotope labeling, and excellent versatility, It is intended to provide a method for measuring bile acid transporter activity, a method for screening a substance for promoting or inhibiting bile acid transporter activity, and the like.
本発明者は、胆汁酸の側鎖部分に蛍光団を導入した新規の蛍光標識化胆汁酸を合成することにより、放射性標識を用いることなく、胆汁酸トランスポーターに対して非標識の胆汁酸と同等の親和性、反応性を有する標識化胆汁酸を開発し、さらに、該標識化胆汁酸が、胆汁酸トランスポーターの活性測定、胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニングに有用であることを見い出し、本発明を完成するに至った。 By synthesizing a novel fluorescently labeled bile acid in which a fluorophore is introduced into the side chain portion of the bile acid, the present inventor can use an unlabeled bile acid and a bile acid transporter without using a radiolabel. Developed labeled bile acids with equivalent affinity and reactivity, and the labeled bile acids are useful for measuring bile acid transporter activity and screening for substances that promote or inhibit bile acid transporter activity. I found something and came to complete the present invention.
すなわち本発明は、（１）ＢＳＥＰ（bile salt export pump）による輸送活性があり、２２位、２３位又は２４位の側鎖炭素原子を介して発色団が結合されていることを特徴とする標識化胆汁酸又はその抱合体や、（２）下記式で表されることを特徴とする上記（１）記載の標識化胆汁酸又はその抱合体 That is, the present invention provides (1) a label having a transport activity by BSEP (bile salt export pump) and having a chromophore bonded via a side chain carbon atom at position 22, 23 or 24. A labeled bile acid or a conjugate thereof, wherein (2) the labeled bile acid or the conjugate thereof is represented by the following formula:
［式中、Ｒ１〜Ｒ７は、それぞれ同一又は異なって、水素原子、ヒドロキシ基、置換若しくは非置換のＣ１−Ｃ６アルキル基、置換若しくは非置換のＣ１−Ｃ６アルコキシ基、置換若しくは非置換のＣ１−Ｃ８アシルオキシ基を表し、Ｘは、発色団を表し、ｎは、０〜２のいずれかの整数を表す。］や、（３）発色団が、フルオレセイン誘導体、アンスロイルニトリル誘導体、ベンゾオキサジアゾール誘導体からなる群より選択される上記（１）又は（２）記載の標識化胆汁酸又はその抱合体や、（４）ベンゾオキサジアゾール誘導体が、４−Ｎ，Ｎ−ジメチルアミノスルホニル−７−フルオロ−２，１，３−ベンゾオキサジアゾリル（ＤＢＤ−）である上記（３）記載の標識化胆汁酸又はその抱合体や、（５）タウリン抱合体であることを特徴とする上記（１）〜（４）のいずれかに記載の標識化胆汁酸又はその抱合体や、（６）Ｎ−（２４−［７−（４−Ｎ，Ｎ−ジメチルアミノスルホニル−２，１，３−ベンゾオキサジアゾール）］−アミノ−３α，７α，１２α−トリヒドロキシ−２７−ノル−５β−コレスタン−２６−オイル）−２’−アミノエタンスルホナートや、（７）Ｎ−（２３−［７−（４−Ｎ，Ｎ−ジメチルアミノスルホニル−２，１，３−ベンゾオキサジアゾール）］−アミノ−３α，７α，１２α−トリヒドロキシ−２４−ホモ−５β−コラン−２５−オイル）−２’−アミノエタンスルホナートに関する。 Wherein, R 1 to R 7 are the same or different and each is hydrogen atom, hydroxy group, substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted Represents a C1-C8 acyloxy group, X represents a chromophore, and n represents an integer of 0-2. (3) The labeled bile acid or conjugate thereof according to (1) or (2), wherein (3) the chromophore is selected from the group consisting of a fluorescein derivative, an anthroylnitrile derivative, and a benzooxadiazole derivative, (4) The labeled bile acid according to the above (3), wherein the benzooxadiazole derivative is 4-N, N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzooxadiazolyl (DBD-) Or a conjugate thereof, (5) a labeled bile acid or conjugate thereof according to any one of (1) to (4) above, which is a taurine conjugate, or (6) N- (24 -[7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzooxadiazole)]-amino-3α, 7α, 12α-trihydroxy-27-nor-5β-cholestan-26-oil ) 2'-aminoethanesulfonate, (7) N- (23- [7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzooxadiazole)]-amino-3α, 7α, 12α-Trihydroxy-24-homo-5β-cholan-25-oil) -2′-aminoethanesulfonate.
また本発明は、（８）胆汁酸の２２位、２３位又は２４位の側鎖炭素原子に活性基を導入する工程、導入した活性基に発色団を結合させる工程を含むことを特徴とする上記（１）〜（７）のいずれかに記載の標識化胆汁酸又はその抱合体の製造方法や、（９）上記（１）〜（７）のいずれかに記載の標識化胆汁酸又はその抱合体を用いることを特徴とする胆汁酸トランスポーター活性の測定方法や、（１０）上記（１）〜（７）のいずれかに記載の標識化胆汁酸又はその抱合体を用いることを特徴とする胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニング方法や、（１１）胆汁酸トランスポーターが、ＢＳＥＰ（bile salt export pump）であることを特徴とする上記（９）又は（１０）記載の方法や、（１２）上記（１）〜（７）のいずれかに記載の標識化胆汁酸又はその抱合体を含む、胆汁酸トランスポーター活性の測定用キットや、（１３）胆汁酸トランスポーターが、ＢＳＥＰ（bile salt export pump）であることを特徴とする上記（１２）記載のキットや、（１４）上記（１）〜（７）のいずれかに記載の標識化胆汁酸又はその抱合体を、胆汁酸トランスポーター活性の測定に使用する方法や、（１５）胆汁酸トランスポーターが、ＢＳＥＰ（bile salt export pump）であることを特徴とする上記（１４）記載の方法に関する。 The present invention also includes (8) a step of introducing an active group into a side chain carbon atom at the 22nd, 23rd or 24th position of bile acid, and a step of bonding a chromophore to the introduced active group. A method for producing a labeled bile acid or conjugate thereof according to any one of (1) to (7) above, or (9) a labeled bile acid according to any one of (1) to (7) above or a method thereof. A method for measuring a bile acid transporter activity characterized by using a conjugate, and (10) a labeled bile acid or a conjugate thereof according to any one of (1) to (7) above. Or (11) the bile acid transporter is BSEP (bile salt export pump), or the screening method for a bile acid transporter activity promoter or inhibitor (12) (1) to (7) A kit for measuring bile acid transporter activity comprising the labeled bile acid according to any one of the above or a conjugate thereof, and (13) the bile acid transporter is BSEP (bile salt export pump) Or (14) a method of using the labeled bile acid or conjugate thereof according to any one of (1) to (7) for measurement of bile acid transporter activity, (15) The method according to (14), wherein the bile acid transporter is BSEP (bile salt export pump).
本発明の標識化胆汁酸又はその抱合体は、胆汁酸トランスポーターに対して非標識の胆汁酸と同等の親和性及び反応性を有し、放射性同位元素を用いない汎用性の胆汁酸トレーサーとして利用することができる。また、本発明の標識化胆汁酸又はその抱合体によれば、胆汁酸トランスポーター活性の測定や、胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニングを正確かつ簡便に行うことができる。 The labeled bile acid of the present invention or a conjugate thereof has a similar affinity and reactivity to a bile acid transporter as an unlabeled bile acid, and is a versatile bile acid tracer that does not use a radioisotope. Can be used. In addition, according to the labeled bile acid or conjugate thereof of the present invention, measurement of bile acid transporter activity and screening for a substance that promotes or inhibits bile acid transporter activity can be performed accurately and simply.
本発明の標識化胆汁酸又はその抱合体としては、ＢＳＥＰ（bile salt export pump）による輸送活性があり、２２位、２３位又は２４位の側鎖炭素原子を介して発色団が結合されている、発色団で標識した胆汁酸、胆汁酸誘導体、又は、これらの抱合体であれば特に制限されず、本発明において胆汁酸とは、胆汁やその排泄物に含まれるコール酸誘導体を総称していう。健常成人における体液中胆汁酸はコール酸（ＣＡ）、ケノデオキシコール酸（ＣＤＣＡ）の一次胆汁酸、並びにデオキシコール酸（ＤＣＡ）、リトコール酸（ＬＣＡ）の二次胆汁酸が主要構成成分となっており、これら胆汁酸は、側鎖カルボキシル基あるいは母核水酸基を介して各種アミノ酸（グリシン、タウリン等）抱合体、並びに硫酸抱合体として体液中に存在することが知られている。以下に、胆汁酸常成分の構造（タウリン抱合体）の構造を示す。 The labeled bile acid of the present invention or a conjugate thereof has a transport activity by BSEP (bile salt export pump), and a chromophore is bonded via a side chain carbon atom at position 22, 23 or 24. Any bile acid labeled with a chromophore, a bile acid derivative, or a conjugate thereof is not particularly limited. In the present invention, bile acid is a general term for cholic acid derivatives contained in bile and its excreta. . Bile acids in body fluids of healthy adults are mainly composed of the primary bile acids of cholic acid (CA) and chenodeoxycholic acid (CDCA), and secondary bile acids of deoxycholic acid (DCA) and lithocholic acid (LCA). These bile acids are known to exist in body fluids as various amino acid (glycine, taurine, etc.) conjugates and sulfate conjugates via side chain carboxyl groups or mother nucleus hydroxyl groups. The structure of the bile acid ordinary component (taurine conjugate) is shown below.
また、胎児−新生児期には、前記の常成分の他に、１β−水酸化胆汁酸、６α−水酸化胆汁酸等のステロイド母核構造が異なる特異な胆汁酸（胎児性胆汁酸）が体液中に出現し、主要成分となることが知られている。 Further, during the fetal-neonatal period, in addition to the above-mentioned normal components, a specific bile acid (fetal bile acid) having a different steroid nucleus structure such as 1β-hydroxy bile acid, 6α-hydroxy bile acid, etc. is a body fluid. It is known to appear in and become a major component.
さらに、各種肝疾患時には、胆汁酸常成分の他に、ステロイド母核構造が不飽和であるΔ５−３β−ｏｌ、ＣＡ−Δ４−３−ｏｎｅなどの不飽和胆汁酸や、側鎖長が通常とは異なるホモＣＡ、ＴＨＣＡ、ノルＴＨＣＡ等の特異な胆汁酸（高級胆汁酸）が体液中に出現することが知られている。 Further, when various liver diseases, in addition to bile acids normally components, steroidal nucleus structure is unsaturated Δ 5 -3β-ol, and unsaturated bile acids, such as CA-Δ 4 -3-one, side chain length It is known that peculiar bile acids (higher bile acids) such as homo CA, THCA, nor THCA, which are different from normal, appear in body fluids.
したがって、本発明における胆汁酸には、胆汁酸常成分であるコール酸（ＣＡ）、ケノデオキシコール酸（ＣＤＣＡ）、デオキシコール酸（ＤＣＡ）、リトコール酸（ＬＣＡ）のほか、水酸化胆汁酸あるいは胎児性胆汁酸、不飽和胆汁酸、高級胆汁酸が含まれる。また、本発明の胆汁酸は、前記胆汁酸の誘導体、光学異性体、立体異性体であってもよく、遊離の胆汁酸、胆汁酸塩、あるいはこれらの塩であってもよい。 Therefore, the bile acids in the present invention include cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA), which are common components of bile acids, as well as hydroxylated bile acids or fetal. Bile acids, unsaturated bile acids, higher bile acids are included. Moreover, the bile acid of the present invention may be a derivative, optical isomer, or stereoisomer of the bile acid, or may be a free bile acid, a bile salt, or a salt thereof.
また、本発明の標識化胆汁酸又はその抱合体は、下記式
［式中、
Ｒ１−Ｒ7は、それぞれ同一又は異なって、水素原子、ヒドロキシ基、置換若しくは非置換のＣ１−Ｃ６アルキル基、置換若しくは非置換のＣ１−Ｃ６アルコキシ基、置換若しくは非置換のＣ１−Ｃ８アシルオキシ基を表し、
Ｘは、発色団を表し、
ｎは、０〜２のいずれかの整数を表す］
で表される。
[Where:
R 1 to R 7 are the same or different and each represents a hydrogen atom, a hydroxy group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C8 acyloxy. Represents a group,
X represents a chromophore,
n represents an integer of 0 to 2]
It is represented by
Ｃ１−Ｃ６アルキル基としては、具体的には、メチル基、エチル基、ｎ−プロピル基、ｉ−プロピル基、ｎ−ブチル基、ｉ−ブチル基、ｓ−ブチル基、ｔ−ブチル基、ｎ−ペンチル基、ｎ−ヘキシル基等が挙げられる。 Specific examples of the C1-C6 alkyl group include methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, s-butyl group, t-butyl group, n -Pentyl group, n-hexyl group, etc. are mentioned.
Ｃ１−Ｃ６アルコキシ基としては、具体的には、メトキシ基、エトキシ基、ｎ−プロポキシ基、ｉ−プロポキシ基、ｎ−ブトキシ基、ｉ−ブトキシ基、ｓ−ブトキシ基、ｔ−ブトキシ基、ｎ−ヘキシルオキシ基等が挙げられる。 Specific examples of the C1-C6 alkoxy group include methoxy group, ethoxy group, n-propoxy group, i-propoxy group, n-butoxy group, i-butoxy group, s-butoxy group, t-butoxy group, n -A hexyloxy group etc. are mentioned.
Ｃ１−Ｃ８アシルオキシ基としては、具体的には、ホルミルオキシ基、アセトキシ基、プロピオニルオキシ基、ブチリルオキシ基、イソブチリルオキシ基、バレリルオキシ基、イソバレリルオキシ基またはピバロイル基等のアルカノイルオキシ基や、ベンゾイルオキシ基等のアロイルオキシ基が挙げられる。 Specific examples of the C1-C8 acyloxy group include an alkanoyloxy group such as formyloxy group, acetoxy group, propionyloxy group, butyryloxy group, isobutyryloxy group, valeryloxy group, isovaleryloxy group or pivaloyl group; And aroyloxy group such as benzoyloxy group.
Ｃ１−Ｃ６アルキル基、Ｃ１−Ｃ６アルコキシ基、Ｃ１−Ｃ８アシルオキシ基の置換基としては、具体的には、Ｃ１−Ｃ６アルキル基、ヒドロキシ基、シアノ基、ニトロ基、アミノ基、カルボキシル基、ハロゲン原子等が挙げられる。Ｃ１−Ｃ６アルキル基は、前記と同義であり、ハロゲン原子としては、フッ素、塩素、臭素、ヨウ素等の各原子が挙げられる。 Specific examples of the substituent for the C1-C6 alkyl group, the C1-C6 alkoxy group, and the C1-C8 acyloxy group include a C1-C6 alkyl group, a hydroxy group, a cyano group, a nitro group, an amino group, a carboxyl group, and a halogen atom. An atom etc. are mentioned. The C1-C6 alkyl group has the same meaning as described above, and examples of the halogen atom include each atom such as fluorine, chlorine, bromine and iodine.
前記式において、Ｒ１、Ｒ３、Ｒ５及びＲ７は、好ましくはヒドロキシ基であり、Ｒ１、Ｒ３、及びＲ５は、好ましくはα配置であるヒドロキシ基である。また、Ｒ２、Ｒ４及びＲ６は、好ましくは水素原子であり、ｎは、好ましくは１又は２である。 In the above formula, R 1 , R 3 , R 5 and R 7 are preferably hydroxy groups, and R 1 , R 3 and R 5 are preferably hydroxy groups in the α configuration. R 2 , R 4 and R 6 are preferably hydrogen atoms, and n is preferably 1 or 2.
本発明の標識に用いる発色団としては、ベンゾフラザン誘導体、フルオレセイン誘導体、アンスロイルニトリル誘導体、ベンゾオキサジアゾール誘導体、スチルベン誘導体、ローダミン誘導体等の公知の蛍光発色団を用いることができる。発色団は、分子の大きさが小さく、蛍光波長が夾雑物の影響を受けにくい領域にあり、導入の容易さからアミノ基と反応性があるものが好適に用いられる。好ましくは、ベンゾフラザン誘導体であり、さらに好ましくは、ニトロベンゾオキサジアゾール（ＮＢＤ）誘導体、又はジメチルアミノスルホニルベンゾオキサジアゾール（ＤＢＤ）誘導体であり、特に、４−Ｎ，Ｎ−ジメチルアミノスルホニル−７−フルオロ−２，１，３−ベンゾオキサジアゾリル（ＤＢＤ−）が好ましく用いられる。 As the chromophore used for the labeling of the present invention, known fluorescent chromophores such as benzofurazan derivatives, fluorescein derivatives, anthroylnitrile derivatives, benzooxadiazole derivatives, stilbene derivatives, rhodamine derivatives and the like can be used. As the chromophore, those having a small molecular size and having a fluorescence wavelength that is hardly affected by impurities, and having reactivity with an amino group are preferably used because of easy introduction. Preferred is a benzofurazan derivative, more preferred is a nitrobenzooxadiazole (NBD) derivative or a dimethylaminosulfonylbenzooxadiazole (DBD) derivative, particularly 4-N, N-dimethylaminosulfonyl-7. -Fluoro-2,1,3-benzooxadiazolyl (DBD-) is preferably used.
本発明の標識化胆汁酸は、グリシン、タウリン、グルクロン酸、グルタチオン又は硫酸等と結合させた抱合体とすることができる。本発明の標識化胆汁酸又はその抱合体胆汁酸は、側鎖末端のカルボキシル基を介して発色団が標識されていないため、抱合形式は、ステロイド母核部分の抱合であっても、側鎖部分の抱合であってもよい。本発明の胆汁酸の抱合体としては、抱合形式が胆汁酸の胆汁酸トランスポーターへの親和性等の生理活性に影響を与えることから、胆汁酸側鎖のタウリン抱合体が好ましい。 The labeled bile acid of the present invention can be a conjugate conjugated with glycine, taurine, glucuronic acid, glutathione, sulfuric acid or the like. Since the labeled bile acid of the present invention or its conjugated bile acid is not labeled with a chromophore via a carboxyl group at the end of the side chain, the conjugation format is conjugated to a steroid mother nucleus part, but the side chain Partial conjugation may also be used. The bile acid conjugate of the present invention is preferably a taurine conjugate of a bile acid side chain because the conjugation format affects physiological activities such as the affinity of bile acids for bile acid transporters.
本発明の標識化胆汁酸又はその抱合体胆汁酸又はその抱合体としては、特に、ＢＳＥＰに対して非標識のタウロコール酸と同等の親和性、反応性を有する、Ｎ−（２４−［７−（４−Ｎ，Ｎ−ジメチルアミノスルホニル−２，１，３−ベンゾオキサジアゾール）］−アミノ−３α，７α，１２α−トリヒドロキシ−２７−ノル−５β−コレスタン−２６−オイル）−２’−アミノエタンスルホナート（Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ）、又は、Ｎ−（２３−［７−（４−Ｎ，Ｎ−ジメチルアミノスルホニル−２，１，３−ベンゾオキサジアゾール）］−アミノ−３α，７α，１２α−トリヒドロキシ−２４−ホモ−５β−コラン−２５−オイル）−２’−アミノエタンスルホナート（Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ）が好ましい。 As the labeled bile acid or its conjugate bile acid or its conjugate of the present invention, in particular, N- (24- [7-] having an affinity and reactivity equivalent to those of unlabeled taurocholic acid for BSEP. (4-N, N-dimethylaminosulfonyl-2,1,3-benzooxadiazole)]-amino-3α, 7α, 12α-trihydroxy-27-nor-5β-cholestan-26-oil) -2 ′ -Aminoethanesulfonate (Tauro-nor-THCA-24-DBD) or N- (23- [7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzooxadiazole)] -Amino-3 [alpha], 7 [alpha], 12 [alpha] -trihydroxy-24-homo-5 [beta] -cholan-25-oil) -2'-aminoethanesulfonate (Tauro-homo-CA-23-DBD) preferable.
本発明の標識化胆汁酸又はその抱合体の製造方法は、胆汁酸側鎖の２２位、２３位又は２４位の炭素原子を発色団で標識するため、胆汁酸の側鎖の２２位、２３位又は２４位の炭素原子に活性基を導入する工程と、導入した活性基に発色団を結合させる工程とを含む方法であれば特に制限されるものではないが、発色団と反応する活性基としては、反応の選択性・容易性等の観点からアミノ基を用いることが好ましい。 In the method for producing a labeled bile acid or a conjugate thereof according to the present invention, the carbon atom at position 22, 23 or 24 of the bile acid side chain is labeled with a chromophore, so that the position 22 or 23 of the bile acid side chain is The active group reacting with the chromophore is not particularly limited as long as the method includes a step of introducing an active group into the carbon atom at position 24 or 24 and a step of bonding a chromophore to the introduced active group. Is preferably an amino group from the viewpoint of selectivity and ease of reaction.
本発明の標識化胆汁酸又はその抱合体は、例えば、以下の工程に従い製造することができる。
ｉ）3α,7α,12α-Triformyroxy-5β-cholan-24-alや3α,7α,12α-Triformyroxy-24-nor-5β-cholan-23-al等のアルデヒド体（１）を出発原料として、ブロモ酢酸エチル等の保護基となりうるハロエステルを用いたＲｅｆｏｒｍａｔｓｋｙ反応により側鎖を伸長する。
ii）得られたβケトエステル体をＪｏｎｅ’ｓ酸化によりオキソ体（２）とする。
iii）酢酸アンモニウム、シアノ水素化ホウ素ナトリウム等を用いて、Ｂｏｒｃｈ反応等の還元的アミノ化反応を行い、アミン体（３）とする。
iv）続いて、例えばＤＢＤ−Ｆ等のアミノ基用蛍光誘導体化試薬とアミン体（３）をカップリングさせる。
ｖ）アルカリ加水分解により、エステルの脱保護を行い、蛍光標識誘導体（４）を得る。
さらに、vi）クロロギ酸エステル等を用い混合酸無水物法により側鎖カルボキシル基とタウリン、グリシン等のアミノ酸を縮合させ、あるいは酵素反応を用いて、蛍光標識誘導体（４）のタウリン抱合体を得ることができる。
The labeled bile acid or conjugate thereof of the present invention can be produced, for example, according to the following steps.
i) Bromo starting from aldehydes (1) such as 3α, 7α, 12α-Triformyroxy-5β-cholan-24-al and 3α, 7α, 12α-Triformyroxy-24-nor-5β-cholan-23-al The side chain is extended by a Reformatsky reaction using a haloester that can be a protecting group such as ethyl acetate.
ii) The obtained β-ketoester is converted to the oxo form (2) by Jones's oxidation.
iii) Ammonium acetate, sodium cyanoborohydride or the like is used to perform a reductive amination reaction such as a Borch reaction to obtain an amine body (3).
iv) Subsequently, an amino group fluorescent derivatization reagent such as DBD-F and an amine (3) are coupled.
v) Deprotection of the ester by alkaline hydrolysis to obtain the fluorescently labeled derivative (4).
Furthermore, vi) condensing side chain carboxyl groups with amino acids such as taurine and glycine using a mixed acid anhydride method using chloroformate or the like, or using enzyme reaction to obtain a taurine conjugate of fluorescently labeled derivative (4) be able to.
本発明の標識化胆汁酸又はその抱合体は、胆汁酸トランスポーター活性の測定に使用することができ、本発明の胆汁酸トランスポーター活性の測定に使用する方法には、胆汁酸トランスポーター活性の測定方法の他、胆汁酸トランスポーター活性の促進物質や阻害物質のスクリーニング方法が含まれる。 The labeled bile acid or conjugate thereof of the present invention can be used for measurement of bile acid transporter activity, and the method used for measurement of bile acid transporter activity of the present invention includes bile acid transporter activity. In addition to measurement methods, screening methods for substances that promote or inhibit bile acid transporter activity are included.
本発明の胆汁酸トランスポーター活性の測定方法は、側鎖が発色団で標識されている本発明の標識化胆汁酸又はその抱合体と、所望のタンパクあるいは胆汁酸トランスポーターとを接触させ、標識化胆汁酸又はその抱合体のタンパクあるいは胆汁酸トランスポーターによる輸送又は結合の程度を、標識された発色団による発色を指標として測定する方法であれば特に制限されない。胆汁酸トランスポーターは、動物等の生体内で発現しているものでも、遺伝子工学的・生化学的手段を用いて生体膜等に人為的に発現させたものでもよい。生体膜としては細胞膜や小胞体、ミトコンドリアなどの細胞内小器官膜を挙げることができ、具体的には、大腸菌等の細菌原核細胞や、酵母、アスペルギルス等の真核細胞や、ドロソフィラＳ２、スポドプテラＳｆ９等の昆虫細胞や、Ｌ細胞、ＣＨＯ細胞、ＣＯＳ細胞、ＨｅＬａ細胞、Ｃ１２７細胞、ＢＡＬＢ／ｃ３Ｔ３細胞、ＢＨＫ２１細胞、ＨＥＫ２９３細胞、ＶＥＲＯ細胞、ＣＶ−１細胞、ＭＤＣＫ細胞、Ｂｏｗｅｓメラノーマ細胞、アフリカツメガエル等の卵母細胞等の動植物細胞などを挙げることができる。 The method for measuring the bile acid transporter activity of the present invention comprises contacting the labeled bile acid of the present invention, whose side chain is labeled with a chromophore, or a conjugate thereof with the desired protein or bile acid transporter, There is no particular limitation as long as it is a method for measuring the degree of transport or binding of a conjugated bile acid or its conjugate by a protein or bile acid transporter using color development by a labeled chromophore as an index. The bile acid transporter may be expressed in a living body such as an animal or artificially expressed on a biological membrane or the like using genetic engineering / biochemical means. Examples of biological membranes include cell membranes, endoplasmic reticulum, and membranes of intracellular organelles such as mitochondria. Specifically, bacterial prokaryotic cells such as Escherichia coli, eukaryotic cells such as yeast and Aspergillus, Drosophila S2, and Spodoptera. Insect cells such as Sf9, L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB / c3T3 cells, BHK21 cells, HEK293 cells, VERO cells, CV-1 cells, MDCK cells, Bowes melanoma cells, Xenopus laevis And animal and plant cells such as oocytes.
本発明の胆汁酸トランスポーター活性の測定方法には、所望のタンパクあるいは胆汁酸トランスポーターを発現させた生体膜の反転ベシクルが、特に好適に用いられる。ベシクルを用いることにより、タンパクあるいは胆汁酸トランスポーターに輸送された標識化胆汁酸が小胞内に取り込まれるため、正確な活性測定が可能である。特に好ましいベシクルは、昆虫由来Ｓｆ９細胞の細胞膜反転ベシクルである。また、胆汁酸トランスポーター活性の測定方法においては、標識化胆汁酸と胆汁酸トランスポーターを接触させた後、輸送された標識化胆汁酸をグラスファイバーフィルター等に吸着し、吸着した標識化胆汁酸をエタノール等の溶媒で溶出後、溶媒を留去して、標識化胆汁酸を測定することができるが、特に、溶媒による抽出に代えて、輸送された標識化胆汁酸をＳＤＳ水溶液で直接捕集し、標識された発色団による発色を測定することにより、正確性を維持しながら簡便に胆汁酸トランスポーターの活性測定を行うことができる。 In the method for measuring bile acid transporter activity of the present invention, an inverted vesicle of a biological membrane in which a desired protein or bile acid transporter is expressed is particularly preferably used. By using vesicles, labeled bile acids transported to proteins or bile acid transporters are incorporated into vesicles, so that accurate activity measurement is possible. Particularly preferred vesicles are the cell membrane reversal vesicles of insect-derived Sf9 cells. In the method for measuring bile acid transporter activity, after the labeled bile acid and bile acid transporter are brought into contact, the transported labeled bile acid is adsorbed on a glass fiber filter or the like, and the adsorbed labeled bile acid is adsorbed. After elution with a solvent such as ethanol, the solvent can be distilled off to measure the labeled bile acid. In particular, instead of extraction with a solvent, the transported labeled bile acid can be directly captured with an aqueous SDS solution. By collecting and measuring the color developed by the labeled chromophore, the activity of the bile acid transporter can be easily measured while maintaining accuracy.
本発明の胆汁酸トランスポーター活性の測定用キットとしては、上記本発明の標識化胆汁酸又はその抱合体を含むものであれば特に制限されないが、通常、上記本発明のＢＳＥＰ等の胆汁酸トランスポーター活性の測定方法において用いられる胆汁酸トランスポーターを発現させた生体膜の反転ベシクル等を含むものが好ましい。すなわち、本発明の標識化胆汁酸又はその抱合体は、胆汁酸トランスポーターが発現した生体膜と組み合わせ、前記胆汁酸トランスポーター活性の測定方法に用いるためのキットとすることができる。さらにキットには、標識化胆汁酸捕集用の所定濃度のＳＤＳ溶液を組み合わせることができる。 The kit for measuring bile acid transporter activity of the present invention is not particularly limited as long as it contains the labeled bile acid of the present invention or a conjugate thereof, but usually a bile acid transport such as BSEP of the present invention. It is preferable to include an inverted vesicle of a biological membrane in which a bile acid transporter used in the method for measuring porter activity is expressed. That is, the labeled bile acid or conjugate thereof of the present invention can be combined with a biological membrane in which a bile acid transporter is expressed and used as a kit for use in the method for measuring bile acid transporter activity. Furthermore, the kit can be combined with an SDS solution having a predetermined concentration for collecting labeled bile acids.
本発明の胆汁酸トランスポーター活性の促進物質又は阻害物質のスクリーニング方法は、被験物質の存在下、及び非存在下に、本発明の胆汁酸トランスポーター活性の測定を行う、すなわち、本発明の標識化胆汁酸と胆汁酸トランスポーターを接触させ、標識化胆汁酸の胆汁酸トランスポーターによる輸送の程度を、標識された発色団による発色を指標として測定し、被験物質の存在下における測定結果と被験物質の非存在下における測定結果を比較評価する方法であれば特に制限されない。例えば、胆汁酸トランスポーターにより輸送された標識化胆汁酸による発色が、被験物質の存在により上昇する場合は、その物質が胆汁酸トランスポーター活性の促進物質であると判断され、利胆剤等の有効成分となる胆汁酸排泄促進物質をスクリーニングすることができる。あるいは、胆汁酸トランスポーターにより輸送された標識化胆汁酸による発色が、被験物質の存在により低下する場合は、その物質が胆汁酸トランスポーター活性の阻害物質であると判断され、被験物質として用いる薬剤の候補物質等から、肝毒性を有する薬剤をスクリーニングすることができる。 The method for screening a substance for promoting or inhibiting bile acid transporter activity of the present invention comprises measuring the bile acid transporter activity of the present invention in the presence and absence of a test substance, that is, the label of the present invention. The bile acid and bile acid transporter are brought into contact, and the degree of transport of the labeled bile acid by the bile acid transporter is measured using the color development by the labeled chromophore as an index, and the measurement result and test in the presence of the test substance The method is not particularly limited as long as it is a method for comparative evaluation of measurement results in the absence of a substance. For example, when the color development due to the labeled bile acid transported by the bile acid transporter increases due to the presence of the test substance, the substance is determined to be a substance that promotes bile acid transporter activity, and a bile acid etc. A bile acid excretion promoting substance that is an active ingredient can be screened. Alternatively, if the color development due to the labeled bile acid transported by the bile acid transporter decreases due to the presence of the test substance, the substance is judged to be an inhibitor of bile acid transporter activity and used as the test substance Drugs having hepatotoxicity can be screened from the candidate substances.
本発明において用いる胆汁酸トランスポーターとしては、例えば、ＮＴＣＰ、ＯＡＴＰ、ｃＭＯＡＴ、Ｐ−糖タンパク質（ＭＤＲ１）、ＭＤＲ２、ＭＲＰ２、ＢＳＥＰ等の公知のトランスポーターを挙げることができるが、これらに制限されるものではなく、トランスポーターがヒトあるいはその他の動物のいずれに由来するものであるかは問わない。本発明において用いる胆汁酸トランスポーターは、組み合わせて用いる標識化胆汁酸との親和性、反応性が、非標識の胆汁酸と同程度であることが好ましく、ＢＳＥＰ（bile salt export pump）、特にヒトＢＳＥＰ（ｈ−ＢＳＥＰ）を好適に例示することができる。 Examples of the bile acid transporter used in the present invention include known transporters such as NTCP, OATP, cMOAT, P-glycoprotein (MDR1), MDR2, MRP2, and BSEP, but are not limited thereto. It does not matter whether the transporter is derived from humans or other animals. The bile acid transporter used in the present invention preferably has the same affinity and reactivity with the labeled bile acid used in combination as that of the unlabeled bile acid. BSEP (bile salt export pump), particularly human BSEP (h-BSEP) can be preferably exemplified.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
１．ＢＳＥＰの基質特異性
（蛍光団の導入位置の決定）
ＢＳＥＰの胆汁酸輸送活性を測定する際にトレーサーとして用いる蛍光標識化胆汁酸を調製した。トレーサーとして用いる蛍光標識化胆汁酸は、ＢＳＥＰに対して、非標識のタウロコール酸（Ｔ−ＣＡ）と同等の親和性及び反応性を有することが必要となる。そこで、胆汁酸への蛍光団の導入位置を決定するために、ＢＳＥＰの基質特異性を解析した。
1. Substrate specificity of BSEP (determination of fluorophore introduction position)
Fluorescently labeled bile acid used as a tracer when measuring the bile acid transport activity of BSEP was prepared. The fluorescently labeled bile acid used as a tracer is required to have the same affinity and reactivity for BSEP as unlabeled taurocholic acid (T-CA). Therefore, in order to determine the position of introduction of the fluorophore into bile acids, the substrate specificity of BSEP was analyzed.
ＢＳＥＰの胆汁酸輸送活性の測定を、Ｓｆ９細胞にヒトＢＳＥＰを発現させたＢＳＥＰベシクル（ｈ−ＢＳＥＰベシクル）（株式会社ジェノメンブレン製）を用いて行った。胆汁酸としては、Ｔ−ＣＡ（シグマ社製）、Ｔ−ＣＤＣＡ（シグマ社製）、Ｔ−ＤＣＡ（シグマ社製）、Ｔ−ＬＣＡ（シグマ社製）、並びに、合成した側鎖長の異なる胆汁酸、胎児性胆汁酸、不飽和胆汁酸を用いた（表１）。各種胆汁酸及びｈ−ＢＳＥＰベシクル懸濁液（タンパク量５０μｇ）を含む輸送用緩衝液（１００ｍＭ ＫＮＯ３、１０ｍＭ Ｍｇ（ＮＯ３）２及び５０ｍＭ ｓｕｃｒｏｓｅを含む１０ｍＭ Ｈｅｐｅｓ−Ｔｒｉｓ緩衝液、ｐＨ７．４、３０μＬ）を３７℃で５分間プレインキュベートした後、１０ｍＭ ＡＴＰ（オリエンタル酵母社製）（２０μＬ）又は１０ｍＭ ＡＭＰ（オリエンタル酵母社製）（２０μＬ）を添加し、反応を開始した。２分間インキュベートした後、氷冷した反応停止用緩衝液（５０ｍＭ ｓｕｃｒｏｓｅ、１００ｍＭ ＫＮＯ３を含む１０ｍＭ Ｈｅｐｅｓ−Ｔｒｉｓ、ｐＨ７．４、０．２ｍＬ）を添加し、反応を停止した。次いで、反応液をグラスファイバーフィルター（マイクロプレートフィルター、孔計１．０μｍ）（ミリポア社製）を用いて吸引ろ過し、引き続き、反応停止用緩衝液（０．２ｍＬ×５回）で洗浄した。フィルター上に吸着した胆汁酸をエタノール（１．４ｍＬ）で溶出し、溶出液を減圧留去した。 The bile acid transport activity of BSEP was measured using a BSEP vesicle (h-BSEP vesicle) (manufactured by Genomembrane Co., Ltd.) in which human BSEP was expressed in Sf9 cells. As bile acids, T-CA (manufactured by Sigma), T-CDCA (manufactured by Sigma), T-DCA (manufactured by Sigma), T-LCA (manufactured by Sigma), and the synthesized side chain lengths are different. Bile acids, fetal bile acids and unsaturated bile acids were used (Table 1). Transport buffer containing various bile acids and h-BSEP vesicle suspension (protein amount 50 μg) (10 mM Hepes-Tris buffer containing 100 mM KNO 3 , 10 mM Mg (NO 3 ) 2 and 50 mM sucrose, pH 7.4, 30 μL) was preincubated at 37 ° C. for 5 minutes, and then 10 mM ATP (Oriental Yeast) (20 μL) or 10 mM AMP (Oriental Yeast) (20 μL) was added to initiate the reaction. After incubating for 2 minutes, an ice-cold buffer for stopping the reaction (50 mM sucrose, 10 mM Hepes-Tris containing 100 mM KNO 3 , pH 7.4, 0.2 mL) was added to stop the reaction. Next, the reaction solution was subjected to suction filtration using a glass fiber filter (microplate filter, pore meter: 1.0 μm) (manufactured by Millipore), and subsequently washed with a reaction stopping buffer solution (0.2 mL × 5 times). The bile acid adsorbed on the filter was eluted with ethanol (1.4 mL), and the eluate was distilled off under reduced pressure.
抽出した胆汁酸は、液体クロマトグラフィーエレクトロスプレーイオン化質量分析（ＬＣ−ＥＳＩ−ＭＳ）法により定量した。前記減圧留去した残渣を水（０．２ｍＬ）に溶解し、内標準物質を添加した後、Oasis HLB 96ウェルプレートカートリッジに通導した。カートリッジを水（０．２ｍＬ）で洗浄後、エタノール（０．４ｍＬ）で胆汁酸を溶出した。減圧下、溶出液を留去した後、残渣をＬＣ−ＭＳ用移動相（２０μＬ）に溶解し、その５μＬをＬＣ−ＭＳに注入した。液体クロマトグラフィーエレクトロスプレーイオン化質量分析（ＬＣ−ＥＳＩ−ＭＳ）は、島津QP-8000型液体クロマトグラフ/質量分析計を用いて、以下の方法で行った。分離カラムにはMightysil RP-18（１５０ｍｍ×２．０ｍｍ Ｉ．Ｄ．、５μｍ）（関東化学社製）を使用した。溶離液としては、２５〜４５％アセトニトリル／１０ｍＭ酢酸アンモニウム溶液を用い、流速は、０．２ｍＬ／ｍｉｎに設定してアイソクラティック溶出を行った。イオン化モードはエレクトロスプレーモード（ネガティブイオン検出モード）を使用した。ネブライザーガスには、窒素（４．５Ｌ／ｍｉｎ）を用い、デフレクター電圧は−４５Ｖに設定した。定量分析には、各胆汁酸の擬似分子イオン［Ｍ−１］−をモニターイオンとするSelected ion monitoring法を用い、内標準法により定量した。得られたクロマトグラムから、内標準法により胆汁酸の定量を行い活性値（ｐｍｏｌ／ｍｉｎ／ｍｇ ｐｒｏｔｅｉｎ）に換算した。なお、ＡＴＰ依存活性値はＡＴＰ共存下における活性値からＡＭＰ共存下における活性値を差し引いて求めた。 The extracted bile acid was quantified by the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method. The residue distilled off under reduced pressure was dissolved in water (0.2 mL), an internal standard substance was added, and the mixture was introduced into an Oasis HLB 96 well plate cartridge. After washing the cartridge with water (0.2 mL), bile acids were eluted with ethanol (0.4 mL). After evaporating the eluate under reduced pressure, the residue was dissolved in a mobile phase for LC-MS (20 μL), and 5 μL thereof was injected into LC-MS. Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) was performed by the following method using Shimadzu QP-8000 type liquid chromatograph / mass spectrometer. Mightysil RP-18 (150 mm × 2.0 mm ID, 5 μm) (manufactured by Kanto Chemical Co., Inc.) was used for the separation column. As the eluent, 25 to 45% acetonitrile / 10 mM ammonium acetate solution was used, and the flow rate was set to 0.2 mL / min to perform isocratic elution. As the ionization mode, an electrospray mode (negative ion detection mode) was used. Nitrogen (4.5 L / min) was used as the nebulizer gas, and the deflector voltage was set to -45V. The quantitative analysis, a pseudo molecular ion of each bile acid [M-1] - using the Selected ion monitoring method to monitor ions was quantified by internal standard method. From the obtained chromatogram, bile acid was quantified by an internal standard method and converted into an activity value (pmol / min / mg protein). The ATP-dependent activity value was determined by subtracting the activity value in the presence of AMP from the activity value in the presence of ATP.
定量した胆汁酸の活性値を求め、各胆汁酸に対するｈ−ＢＳＥＰ輸送活性の速度論的パラメーター（Ｋｍ値及びＶｍａｘ値）を以下に示すミカエリス−メンテン式から算出した。
ｖ ＝ Ｖｍａｘ × ｓ ／（Ｋｍ ＋ ｓ）
ｖ：輸送の初速度 （ｐｍｏｌ／ｍｉｎ／ｍｇ ｐｒｏｔｅｉｎ）
ｓ：胆汁酸濃度 （μＭ）
Ｋｍ：the Michaelis-Menten定数（μＭ）
Ｖｍａｘ：輸送の最大速度 （ｐｍｏｌ／ｍｉｎ／ｍｇ ｐｒｏｔｅｉｎ）
Ｋｍ値及びＶｍａｘ値は、KaleidaGraph （Ｓｙｎｅｒｇｙ Ｓｏｆｔｗａｒｅ、Ｒｅａｄｉｎｇ、ＰＡ）を利用し、非線形回帰によって求めた。その結果を表１に示した。
The quantified bile acid activity value was determined, and the kinetic parameters (Km value and Vmax value) of h-BSEP transport activity for each bile acid were calculated from the Michaelis-Menten equation shown below.
v = V max × s / ( K m + s)
v: Initial speed of transport (pmol / min / mg protein)
s: Bile acid concentration (μM)
K m : the Michaelis-Menten constant (μM)
V max : Maximum speed of transport (pmol / min / mg protein)
The K m value and the V max value were obtained by nonlinear regression using KaleidaGraph (Synergy Software, Reading, PA). The results are shown in Table 1.
胆汁酸常成分については、Ｋｍ値は、Ｔ−ＬＣＡ＜Ｔ−ＣＤＣＡ＜Ｔ−ＣＡ＜Ｔ−ＤＣＡの順に低い値を示し、ＢＳＥＰに対する親和性が、ステロイド母核上の水酸基の数及び位置により影響を受けることが推測された。また、側鎖構造の異なるＴ−ｈｏｍｏ−ＣＡ、Ｔ−ｎｏｒ−ＴＨＣＡ、及びＴ−ＴＨＣＡについては、いずれもＢＳＥＰによる輸送活性が認められ、Ｋｍ値はＴ−ＣＡと同程度の値を示した。一方、胎児性胆汁酸については、６α−水酸化胆汁酸（Ｔ−ＣＡ−６α−ｏｌ及びＴ−ＣＤＣＡ−６α−ｏｌ）のＢＳＥＰによる輸送活性は認められたが、他の胆汁酸では輸送活性がほとんど認められず、６α−水酸化胆汁酸を除いた胎児性胆汁酸はＢＳＥＰの基質とならないものと推測された。さらに、ステロイド母核構造の異なる不飽和胆汁酸についても同様に輸送活性は認められず、ＢＳＥＰの基質にはならないものと推測された。以上の結果と胆汁酸常成分に対するＢＳＥＰの胆汁酸輸送活性を解析した報告とを勘案し、ＢＳＥＰと胆汁酸との親和性は、胆汁酸の母核構造により大きく影響を受けるが、側鎖長の相違による影響は母核構造に比し少ないとの予測をした。また、グリシン抱合体又はタウリン抱合体等に例示される胆汁酸の側鎖抱合形式が親和性に影響を与えることも知られている。以上の知見に基づき、胆汁酸への蛍光団の導入位置としては、ＢＳＥＰに対する親和性への影響が小さい胆汁酸側鎖部分が最適であるとの結論を得た。 For the bile acid normal component, the Km value shows a low value in the order of T-LCA <T-CDCA <T-CA <T-DCA, and the affinity for BSEP depends on the number and position of hydroxyl groups on the steroid mother nucleus. It was speculated to be affected. Moreover, T-homo-CA, T-nor-THCA, and T-THCA having different side chain structures all showed transport activity by BSEP, and the Km value showed the same level as T-CA. . On the other hand, with regard to fetal bile acids, the transport activity of 6α-hydroxy bile acids (T-CA-6α-ol and T-CDCA-6α-ol) by BSEP was observed, but other bile acids were transported. The fetal bile acid excluding 6α-hydroxy bile acid was presumed not to be a substrate for BSEP. Furthermore, no transport activity was observed for unsaturated bile acids with different steroid matrix structures, and it was speculated that they would not be substrates for BSEP. Considering the above results and a report analyzing the bile acid transport activity of BSEP for bile acid normal components, the affinity between BSEP and bile acid is greatly influenced by the mother nucleus structure of bile acid. It was predicted that the effect of the difference in the amount of the difference was less than that of the mother nucleus structure. It is also known that the bile acid side chain conjugation format exemplified by glycine conjugates or taurine conjugates affects affinity. Based on the above findings, it was concluded that the bile acid side chain portion, which has a small influence on the affinity for BSEP, is the optimal position for introducing a fluorophore into bile acids.
２．蛍光標識化胆汁酸の合成
前記の結果に基づき、胆汁酸の側鎖部分への蛍光団導入を行った。胆汁酸分析に利用可能な蛍光標識化試薬としては、フルオレセイン誘導体、アンスロイルニトリル誘導体、ベンゾオキサジアゾール誘導体等の多種類の試薬が市販されている。これらを用いることにより、ｆｍｏｌからｐｍｏｌの胆汁酸の検出が可能であり、十分な感度のＢＳＥＰ輸送活性測定ができると考えられた。そこで、胆汁酸への蛍光標識化試薬としては、分子サイズが比較的小さく、蛍光波長が比較的長波長側である５７０ｎｍ付近にあるため夾雑物の影響を受けにくく、さらに、検出感度にも優れたベンゾオキサジアゾール誘導体を以下の標識化化合物の合成に用いた。
2. Synthesis of Fluorescently Labeled Bile Acid Based on the above results, a fluorophore was introduced into the side chain portion of bile acid. As fluorescent labeling reagents that can be used for bile acid analysis, many types of reagents such as fluorescein derivatives, anthroylnitrile derivatives, and benzooxadiazole derivatives are commercially available. By using these, it was considered that bmol acids from fmol to pmol could be detected, and BSEP transport activity measurement with sufficient sensitivity could be performed. Therefore, as a fluorescent labeling reagent for bile acids, the molecular size is relatively small, and the fluorescence wavelength is near 570 nm, which is a relatively long wavelength side, so that it is not easily affected by impurities, and also has excellent detection sensitivity. Benzoxadiazole derivatives were used for the synthesis of the following labeled compounds.
一般に、胆汁酸の蛍光標識化にはステロイド母核に存在する水酸基又は側鎖カルボキシル基を介して蛍光団を導入する方法が採用されている。しかしながら、胆汁酸の側鎖部分には、蛍光団を導入できる活性官能基は存在していないため、まず側鎖部分に活性基としてアミノ基を導入し、その後蛍光標識化試薬として4-(N,N-Dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole（ＤＢＤ−Ｆ）を用いて、蛍光標識化胆汁酸を合成した。 In general, a method of introducing a fluorophore through a hydroxyl group or a side chain carboxyl group present in a steroid mother nucleus is employed for fluorescent labeling of bile acids. However, since there is no active functional group capable of introducing a fluorophore in the side chain portion of bile acid, an amino group is first introduced as an active group into the side chain portion, and then 4- (N , N-Dimethylaminosulfonyl) -7-fluoro-2,1,3-benzoxadiazole (DBD-F) was used to synthesize fluorescently labeled bile acids.
標識化胆汁酸として、N-(24-[7-(4-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino- 3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2’-aminoethanesulfonate（Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ）を以下の方法に従い合成した。合成した各化合物の物性解析には、以下の手段を用いた。融点は、三田村融点測定装置を用いて測定し、未補正の値を示した。赤外吸収スペクトルは、JASCO FT / IR-300赤外分光光度計を用いて測定し、吸収波長はｃｍ−１で示した。核磁気共鳴スペクトルは、日本電子 JNM-ECA500 （５００ＭＨｚ）核磁気共鳴装置、内標準物質としてテトラメチルシラン（ＴＭＳ）を使用して測定し、化学シフト及び結合定数は、それぞれδ（ｐｐｍ）及びＨｚで示した（略号：ｓ=singlet、ｂｓ=broad singlet、ｄ=doublet、ｔ=triplet、ｑ=qualtet、ｍ=multiplet）。質量スペクトルは、マイクロマスAuto Spec 3000型質量分析装置を用いた。 As labeled bile acids, N- (24- [7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino-3α, 7α, 12α-trihydroxy-27-nor-5β- cholestan-26-oyl) -2′-aminoethanesulfonate (Tauro-nor-THCA-24-DBD) was synthesized according to the following method. The following means were used for the physical property analysis of each synthesized compound. The melting point was measured using a Mitamura melting point measuring apparatus and indicated an uncorrected value. The infrared absorption spectrum was measured using a JASCO FT / IR-300 infrared spectrophotometer, and the absorption wavelength was shown in cm- 1 . The nuclear magnetic resonance spectrum was measured using JEOL JNM-ECA500 (500 MHz) nuclear magnetic resonance apparatus and tetramethylsilane (TMS) as the internal standard substance. The chemical shift and the coupling constant were δ (ppm) and Hz, respectively. (Abbreviations: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = qualtet, m = multiplet). For the mass spectrum, a Micromass Auto Spec 3000 mass spectrometer was used.
［Ethyl 3α,7α,12α-triformyroxy-24-oxo-27-nor-5β-cholestan-26-oate （２）の合成］
合成したアルデヒド体、3α,7α,12α-Triformyroxy-5β-cholan-24-al（１） （３．１ｇ、６．５ｍｍｏｌ）を無水ベンゼン（１０ｍＬ）に溶解し、亜鉛（２．３ｇ、３６ｍｍｏｌ）、ブロモ酢酸エチル（２．７ｇ、１６ｍｍｏｌ）及び触媒量のヨウ素を加えた後、２０分間加熱還流した。反応液を氷冷し、ジエチルエーテルを加え、１５％硫酸で酸性とした後、ジエチルエーテルで抽出した。有機層を２ｍｏｌ／Ｌ塩酸、飽和炭酸水素ナトリウム溶液、飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥した。溶媒を減圧下留去し、得られた残渣をシリカゲルカラムクロマトグラフィー（ヘキサン／酢酸エチル＝３／２）により精製し、エチルエステル体（１．５ｇ、２．７ｍｍｏｌ）を無色結晶として得た。エチルエステル体（１．９ｇ、３．４ｍｍｏｌ）をアセトン（８ｍＬ）に溶解し、氷冷下、Jone’s試薬（２．５ｍＬ）を加え撹拌した。反応液に水を加え、析出した結晶をろ取し、酢酸エチルに溶解した。有機層を水及び飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥した。溶媒を減圧下留去し、得られた残渣をシリカゲルカラムクロマトグラフィー（ヘキサン／酢酸エチル＝２／１）により精製し、エステル体（２）（１．３ｇ、２．３ｍｍｏｌ）を無晶形粉末として得た。
[Synthesis of Ethyl 3α, 7α, 12α-triformyroxy-24-oxo-27-nor-5β-cholestan-26-oate (2)]
The synthesized aldehyde form, 3α, 7α, 12α-Triformyroxy-5β-cholan-24-al (1) (3.1 g, 6.5 mmol) was dissolved in anhydrous benzene (10 mL), and zinc (2.3 g, 36 mmol) was dissolved. , Ethyl bromoacetate (2.7 g, 16 mmol) and a catalytic amount of iodine were added, followed by heating to reflux for 20 minutes. The reaction solution was ice-cooled, diethyl ether was added, acidified with 15% sulfuric acid, and extracted with diethyl ether. The organic layer was washed with 2 mol / L hydrochloric acid, saturated sodium hydrogen carbonate solution and saturated brine, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3/2) to obtain an ethyl ester (1.5 g, 2.7 mmol) as colorless crystals. The ethyl ester form (1.9 g, 3.4 mmol) was dissolved in acetone (8 mL), and Jone's reagent (2.5 mL) was added and stirred under ice cooling. Water was added to the reaction solution, and the precipitated crystals were collected by filtration and dissolved in ethyl acetate. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 2/1) to obtain the ester (2) (1.3 g, 2.3 mmol) as an amorphous powder. Obtained.
mp: 142．5-144．0℃．1H-NMR (CDCl3):0．75(3H、s、18-CH3)、0．82(3H、d、J=6．2 Hz、 21-CH3)、 0．95 (3H、 s、 19-CH3)、 1．28 (3H、 t、 J=7．2 Hz、 -COOCH2 CH 3 )、 3．43 (2H、 s、 25-CH2)、 4．17 (2H、 q、 J=7．2 Hz、 -COOCH 2 CH3)、 4．72 (1H、 m、 3-H)、 5．07 (1H、 bs、 7-H)、 5．26 (1H、 bs、 12-H)、8．02、 8．11、 8．16 (each 1H、 s、 -CHO)． IR (nujol): 1720、 1750 cm-1、 HR-EI-MS m/z: 562．3144 (Calcdfor C31H46O9: 562．3142)． EI-MS m/z: 562 (M+、 3 %)、 470 (100 %)、 340 (70 %)、 299 (83 %)、 253 (61 %)． mp: 142.5-144.0 ° C. 1 H-NMR (CDCl 3 ): 0.75 (3H, s, 18-CH 3 ), 0.82 (3H, d, J = 6.2 Hz, 21-CH 3 ), 0.95 (3H, s, 19-CH 3 ), 1.28 (3H, t, J = 7.2 Hz, -COOCH 2 CH 3 ), 3.43 (2H, s, 25-CH 2 ), 4.17 (2H, q, J = 7.2 Hz, -COO CH 2 CH 3 ), 4.72 (1H, m, 3-H), 5.07 (1H, bs, 7-H), 5.26 (1H, bs 12-H), 8.02, 8.11, 8.16 (each 1H, s, -CHO). IR (nujol): 1720, 1750 cm -1, HR-EI-MS m / z: 562.3144 (Calcdfor C 31 H 46 O 9: 562.3142). EI-MS m / z: 562 (M + , 3%), 470 (100%), 340 (70%), 299 (83%), 253 (61%).
［Ethyl 24-amino-3α,7α,12α-triformyroxy-27-nor-5β-cholestan-26-oate（３）の合成］
エステル体（２）（１５０ｍｇ、０．２７ｍｍｏｌ）をエタノール（２ｍＬ）に溶解し、酢酸アンモニウム（２０８ｍｇ、２．７ｍｍｏｌ）及びモレキュラシーブスＡ４（２００ｍｇ）を加え、４５℃で５時間撹拌した。反応液に飽和炭酸水素ナトリウム溶液（５ｍＬ）を加え、酢酸エチルで抽出した。有機層を水、飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥した後、溶媒を減圧下留去した。得られた残渣をエタノールに溶解し、シアノ水素化ホウ素ナトリウム（２０ｍｇ、０．２７ｍｍｏｌ）を加え、室温で１時間撹拌した。反応液に飽和炭酸水素ナトリウム溶液（５ｍＬ）を加え、酢酸エチルで抽出した。有機層を水及び飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥した。溶媒を減圧下留去し、得られた残渣をシリカゲルカラムクロマトグラフィー（酢酸エチル）により精製し、アミノ体（３）（８０ｍｇ、０．１４ｍｍｏｌ）を無色無晶形粉末として得た。
[Synthesis of Ethyl 24-amino-3α, 7α, 12α-triformyroxy-27-nor-5β-cholestan-26-oate (3)]
The ester body (2) (150 mg, 0.27 mmol) was dissolved in ethanol (2 mL), ammonium acetate (208 mg, 2.7 mmol) and molecular sieves A4 (200 mg) were added, and the mixture was stirred at 45 ° C. for 5 hours. Saturated sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in ethanol, sodium cyanoborohydride (20 mg, 0.27 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Saturated sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (ethyl acetate) to obtain the amino compound (3) (80 mg, 0.14 mmol) as a colorless amorphous powder.
1H-NMR (CDCl3) : 0．73 (3H、s、 18-CH3)、 0．84 (3H、 d、 J=6．2 Hz、21-CH3)、 0．93 (3H、 s、 19-CH3)、 1．25 (3H、 t、 J=7．4 Hz、 -COOCH2 CH 3 )、 3．09 (1H、 m、 24-CH)、 4．13 (2H、 q、 J=7．2 Hz、 -COOCH 2 CH3)、 4．71 (1H、 m、 3-H)、 5．06 (1H、 bs、 7-H)、 5．26 (1H、 bs、 12-H)、 8．01、 8．10、 8．15 (each 1H、 s、-CHO)． IR (nujol): 1720、 3400 cm-1、 HR-EI-MS m/z: 563．3441 (Calcd for C31H46O9: 563．3458)． EI-MS m/z: 563 (M+、 3 %)、 518 (13 %)、476 (10 %)、 253 (4 %)、 116 (100 %)． ESI-MS m/z: 564 ([M+1]+、 100 %) 1 H-NMR (CDCl 3 ): 0.73 (3H, s, 18-CH 3 ), 0.84 (3H, d, J = 6.2 Hz, 21-CH 3 ), 0.93 (3H, s, 19-CH 3 ), 1.25 (3H, t, J = 7.4 Hz, -COOCH 2 CH 3 ), 3.09 (1H, m, 24-CH), 4.13 (2H, q , J = 7.2 Hz, -COO CH 2 CH 3 ), 4.71 (1H, m, 3-H), 5.06 (1H, bs, 7-H), 5.26 (1H, bs, 12-H), 8.01, 8.10, 8.15 (each 1H, s, -CHO). IR (nujol): 1720, 3400 cm −1 , HR-EI-MS m / z: 563.3441 (Calcd for C 31 H 46 O 9 : 563.3458). EI-MS m / z: 563 (M + , 3%), 518 (13%), 476 (10%), 253 (4%), 116 (100%). ESI-MS m / z: 564 ([M + 1] + , 100%)
［24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino- 3α,7α,12α-trihydroxy-27-nor-5β-cholestanoic acid（４）の合成］
アミノ体（３）（６０ｍｇ、０．１１ｍｍｏｌ）をアセトニトリル（２ｍＬ）に溶解し、トリエチルアミン（１１ｍｇ、０．１１ｍｍｏｌ）、及び4-(N,N-Dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole（ＤＢＤ−Ｆ）（東京化成工業社製）（２９ｍｇ、０．１２ｍｍｏｌ）を加え、５０℃で１時間撹拌した。減圧下、溶媒を留去した後、残渣をシリカゲルカラムクロマトグラフィー（ヘキサン／酢酸エチル＝３：２）により精製し、ＤＢＤ誘導体（５８ｍｇ、０．０７ｍｍｏｌ）を黄色無晶形粉末として得た。
[Synthesis of 24- [7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino-3α, 7α, 12α-trihydroxy-27-nor-5β-cholestanoic acid (4)]
Amino compound (3) (60 mg, 0.11 mmol) was dissolved in acetonitrile (2 mL), triethylamine (11 mg, 0.11 mmol), and 4- (N, N-dimethylaminosulfonyl) -7-fluoro-2,1,3 -Benzoxadiazole (DBD-F) (Tokyo Chemical Industry Co., Ltd.) (29 mg, 0.12 mmol) was added, and the mixture was stirred at 50 ° C. for 1 hour. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3: 2) to obtain a DBD derivative (58 mg, 0.07 mmol) as a yellow amorphous powder.
1H-NMR (CDCl3) : 0．73 (3H、s、 18-CH3)、 0．86 (3H、 d、 J=6．2 Hz、 21-CH3)、 0．93 (3H、 s、 19-CH3)、 1．25 (3H、 t、 J=7．2 Hz、 -COOCH2 CH 3 )、 2．65 (2H、 m、 25-CH2)、 4．02 (1H、 m、 24-CH)、 4．15 (2H、 q、 J=7．2 Hz、 -COOCH 2 CH3)、 4．70 (1H、 m、 3-H)、 5．05 (1H、 bs、 7-H)、 5．25 (1H、 bs、 12-H)、6．18 (1H、 d、 J=8．0、 CH=CNH)、 7．88 (1H、 d、 J=8．0、 CH=C-SO2(CH3)2、 8．01、 8．10、 8．14 (each 1H、 s、 -CHO)． EI-MS m/z: 788 (M+、 100 %)、 651 (9 %)、 563 (12 %)、 341 (77 %)、 253 (21 %)、 116 (100 %)． 1 H-NMR (CDCl 3 ): 0.73 (3H, s, 18-CH 3 ), 0.86 (3H, d, J = 6.2 Hz, 21-CH 3 ), 0.93 (3H, s, 19-CH 3 ), 1.25 (3H, t, J = 7.2 Hz, -COOCH 2 CH 3 ), 2.65 (2H, m, 25-CH 2 ), 4.02 (1H, m, 24-CH), 4.15 (2H, q, J = 7.2 Hz, -COO CH 2 CH 3), 4.70 (1H, m, 3-H), 5.05 (1H, bs 7-H), 5.25 (1H, bs, 12-H), 6.18 (1H, d, J = 8.0, CH = CNH), 7.88 (1H, d, J = 8. 0, CH = C-SO 2 (CH 3) 2, 8.01, 8.10, 8.14 (each 1H, s, -CHO) EI-MS m / z:. 788 (M +, 100%) , 651 (9%), 563 (12%), 341 (77%), 253 (21%), 116 (100%).
前記ＤＢＤ誘導体（５８ｍｇ、０．０７ｍｍｏｌ）をメタノールに溶解し１ｍｏｌ／Ｌ ＫＯＨ（０．４ｍＬ）を加え、３０分間加熱還流した。減圧下、溶媒を留去した。残渣を水に溶解し２ｍｏｌ／Ｌ塩酸で酸性とした後、クロロホルムで抽出した。有機層を水で洗浄後、無水硫酸ナトリウムで乾燥した。溶媒を減圧下留去し、カルボン酸（４）（４４ｍｇ、０．０６ｍｍｏｌ）を黄色無晶形粉末として得た。 The DBD derivative (58 mg, 0.07 mmol) was dissolved in methanol, 1 mol / L KOH (0.4 mL) was added, and the mixture was heated to reflux for 30 minutes. The solvent was distilled off under reduced pressure. The residue was dissolved in water, acidified with 2 mol / L hydrochloric acid, and extracted with chloroform. The organic layer was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain carboxylic acid (4) (44 mg, 0.06 mmol) as a yellow amorphous powder.
EI-MS m/z: 676(M+、 2 %)、 616 (3 %)、 253 (13 %)、 147 (100 %) ．
ESI-MS m/z: 675 ([M-1]−、 100 %)
EI-MS m / z: 676 (M + , 2%), 616 (3%), 253 (13%), 147 (100%).
ESI-MS m / z: 675 ([M-1] − , 100%)
［N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino- 3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2’-aminoethanesulfonate（Tauro-nor-THCA-24-DBD）（５）の合成］
カルボン酸（４）（４４ｍｇ、０．０６ｍｍｏｌ）を無水テトラヒドロフラン（２ｍＬ）に溶解し、氷冷した後、トリエチルアミン（９ｍｇ、０．０９ｍｍｏｌ）及びクロルギ酸エチル（９ｍｇ、０．０９ｍｍｏｌ）を加え、２０分間撹拌した。次いで、タウリン（２０ｍｇ、０．１６ｍｍｏｌ）の水溶液を加え３０分間撹拌した。溶媒を減圧下留去した後、残渣をＯＤＳカラムクロマトグラフィー（４８％エタノール）により精製し、Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ（５）（４５ｍｇ、０．０５ｍｍｏｌ）を黄色無晶形粉末として得た。
[N- (24- [7- (4-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)-]-amino-3α, 7α, 12α-trihydroxy-27-nor-5β-cholestan-26-oyl ) -2'-aminoethanesulfonate (Tauro-nor-THCA-24-DBD) (5)]
Carboxylic acid (4) (44 mg, 0.06 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL), ice-cooled, triethylamine (9 mg, 0.09 mmol) and ethyl chloroformate (9 mg, 0.09 mmol) were added, and 20 Stir for minutes. Then, an aqueous solution of taurine (20 mg, 0.16 mmol) was added and stirred for 30 minutes. After the solvent was distilled off under reduced pressure, the residue was purified by ODS column chromatography (48% ethanol) to obtain Tauro-nor-THCA-24-DBD (5) (45 mg, 0.05 mmol) as a yellow amorphous powder. It was.
ESI-MS m/z: 782 ([M-1]−、 100 %) ESI-MS m / z: 782 ([M-1] − , 100%)
合成したＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤの構造は、各種機器データにより確認した。また、励起スペクトル、蛍光スペクトルを測定したところ、励起極大波長は４４０ｎｍ、蛍光極大波長５７０ｎｍであった。さらに、ｎｏｒ−ＣＡを出発原料として、側鎖長がＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤよりメチレン基が１個短いＴａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤを同様の方法で合成した。蛍光検出プレートリーダーを用いて、これら蛍光標識体の検出限界を求めたところ、６０ｆｍｏｌ／ｗｅｌｌ（０．３ｐｍｏｌ／ｍｌ）と極めて高感度であり、ＢＳＥＰ活性測定に十分な測定感度を示した。これらの合成した蛍光標識化胆汁酸２種類を以下の試験に供した。 The structure of the synthesized Tauro-nor-THCA-24-DBD was confirmed by various instrument data. Moreover, when the excitation spectrum and the fluorescence spectrum were measured, the excitation maximum wavelength was 440 nm and the fluorescence maximum wavelength was 570 nm. Further, using nor-CA as a starting material, Tauro-homo-CA-23-DBD having a side chain length shorter than that of Tauro-nor-THCA-24-DBD by one methylene group was synthesized in the same manner. When the detection limit of these fluorescent labels was determined using a fluorescence detection plate reader, it was extremely high sensitivity of 60 fmol / well (0.3 pmol / ml), indicating sufficient measurement sensitivity for measuring BSEP activity. Two types of these fluorescently labeled bile acids synthesized were subjected to the following test.
３．蛍光標識化胆汁酸のＢＳＥＰベシクルによる輸送活性
ｈ−ＢＳＥＰベシクルを用いて、合成した蛍光標識化胆汁酸２種類の輸送活性を解析した。
3. Transport activity of fluorescently labeled bile acids by BSEP vesicles Using h-BSEP vesicles, the transport activities of two types of fluorescently labeled bile acids were analyzed.
合成した蛍光標識化胆汁酸（Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ及びＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ）を基質とするｈ−ＢＳＥＰ輸送活性の経時変化を検討した。ＡＴＰ依存輸送活性は、ＢＳＥＰベシクル又は対照のコントロールベシクルと各蛍光標識化胆汁酸をＡＴＰ又はＡＭＰ存在下、０〜５分間インキュベートし、ベシクル内に取り込まれた胆汁酸をエタノールで抽出して、胆汁酸量をＬＣ−ＥＳＩ−ＭＳ法により定量して求めた。蛍光標識化胆汁酸濃度は、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤについては、２．５μＭとし、Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤについては、４．７μＭとした。その結果を図１に示した。いずれの蛍光標識化胆汁酸を基質とした場合でも、ＢＳＥＰベシクルによるＡＴＰ依存輸送活性は、コントロールベシクルによる場合に比べて明らかに高い値を示した。また、ＢＳＥＰベシクルによるＡＴＰ依存輸送活性は、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ及びＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤのいずれにおいても経時的に上昇した。 The time course of h-BSEP transport activity using the synthesized fluorescently labeled bile acids (Tauro-homo-CA-23-DBD and Tauro-nor-THCA-24-DBD) as a substrate was examined. The ATP-dependent transport activity is determined by incubating BSEP vesicles or control control vesicles with each fluorescently labeled bile acid in the presence of ATP or AMP for 0 to 5 minutes, and extracting the bile acids incorporated into the vesicles with ethanol. The acid amount was quantified and determined by LC-ESI-MS method. The fluorescently labeled bile acid concentration was 2.5 μM for Tauro-homo-CA-23-DBD and 4.7 μM for Tauro-nor-THCA-24-DBD. The results are shown in FIG. Even when any fluorescently labeled bile acid was used as a substrate, the ATP-dependent transport activity by BSEP vesicles was clearly higher than that by control vesicles. In addition, the ATP-dependent transport activity by BSEP vesicles increased with time in both Tauro-homo-CA-23-DBD and Tauro-nor-THCA-24-DBD.
次に、合成した蛍光標識化胆汁酸（Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ及びＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ）を基質とするｈ−ＢＳＥＰ輸送活性の蛍光標識化胆汁酸に対する濃度依存性を検討した。その結果を図２に示した。いずれの蛍光標識化胆汁酸を基質とした場合でも、ＡＴＰ依存輸送活性は基質濃度依存的に上昇するとともに飽和性を示した。 Next, the concentration dependence of h-BSEP transport activity on the fluorescently labeled bile acids using the synthesized fluorescently labeled bile acids (Tauro-homo-CA-23-DBD and Tauro-nor-THCA-24-DBD) as substrates. It was investigated. The results are shown in FIG. Regardless of which fluorescently labeled bile acid was used as a substrate, the ATP-dependent transport activity increased depending on the substrate concentration and showed saturation.
ｈ−ＢＳＥＰ輸送活性の基質濃度依存性の測定結果から、各蛍光標識化胆汁酸に対するｈ−ＢＳＥＰベシクルのＫｍ値、及びＶｍａｘ値をミカエリス−メンテン式を用いて算出した。その結果を表２に示した。 From the measurement result of the substrate concentration dependence of h-BSEP transport activity, the Km value and Vmax value of the h-BSEP vesicle for each fluorescently labeled bile acid were calculated using the Michaelis-Menten equation. The results are shown in Table 2.
Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ、Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤに対するｈ−ＢＳＥＰのＫｍ値は、それぞれ２１．６μＭ、２３．１μＭを示し、ｈ−ＢＳＥＰに対する親和性は、いずれもＴ−ＣＡ（Ｋｍ値＝１９．０μＭ）に対してと同程度であった。さらに、基質の利用効率を示すパラメーターとしてＶｍａｘ／Ｋｍ値を算出した。Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤでは、２７．８を示し、Ｔ−ＣＡ（Ｖｍａｘ／Ｋｍ値＝２２．９）と同程度であった。一方、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤでは、３６．３を示し、Ｔ−ＣＡの約１．６倍の高値であった。 The K-values of h-BSEP for Tauro-homo-CA-23-DBD and Tauro-nor-THCA-24-DBD were 21.6 μM and 23.1 μM, respectively, and the affinity for h-BSEP was T -Similar to CA (Km value = 19.0 μM). Furthermore, Vmax / Km value was calculated as a parameter indicating the utilization efficiency of the substrate. Tauro-nor-THCA-24-DBD showed 27.8, comparable to T-CA (Vmax / Km value = 22.9). On the other hand, Tauro-homo-CA-23-DBD showed 36.3, which was about 1.6 times higher than T-CA.
以上の結果より、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ及びＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤは、いずれもＢＳＥＰベシクルの基質となることが明らかとなった。また、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤでは、基質利用率（Ｖｍａｘ／Ｋｍ値）が、Ｔ−ＣＡに比し高値を示すものの、ＢＳＥＰに対する親和性については、いずれの蛍光標識化胆汁酸も、非標識のＴ−ＣＡとほぼ一致しており、側鎖部分への蛍光団導入によるＢＳＥＰベシクルの親和性への影響は認められなかった。すなわち、胆汁酸とＢＳＥＰとの親和性に影響を与えることがない、胆汁酸への蛍光団の導入位置として、胆汁酸側鎖部分を選択することの有効性が示され、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ及びＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤの２種の合成した蛍光標識化胆汁酸が、いずれもＢＳＥＰの輸送能評価の指標として有用であることが示された。 From the above results, it was revealed that Tauro-homo-CA-23-DBD and Tauro-nor-THCA-24-DBD are both substrates of BSEP vesicles. Moreover, in Tauro-homo-CA-23-DBD, although the substrate utilization rate (Vmax / Km value) shows a high value compared with T-CA, as for the affinity for BSEP, any fluorescently labeled bile acids This was almost the same as unlabeled T-CA, and no influence on the affinity of BSEP vesicles due to the introduction of a fluorophore into the side chain portion was observed. That is, the effectiveness of selecting the bile acid side chain moiety as the introduction position of the fluorophore into the bile acid without affecting the affinity between the bile acid and BSEP is shown, and the Tauro-homo-CA It was shown that two kinds of synthesized fluorescently labeled bile acids, −23-DBD and Tauro-nor-THCA-24-DBD, are useful as indexes for evaluating the transport ability of BSEP.
４．蛍光標識化胆汁酸をトレーサーとするＢＳＥＰ輸送活性測定
汎用性に優れるＢＳＥＰ阻害活性評価法を確立するため、合成した蛍光標識化胆汁酸をトレーサーとして用いて、ＢＳＥＰ阻害活性を有する各種薬剤の存在下、ＢＳＥＰ輸送活性を測定した。
4). Measurement of BSEP transport activity using fluorescently labeled bile acid as tracer To establish BSEP inhibitory activity evaluation method with excellent versatility, using synthesized fluorescently labeled bile acid as tracer, in the presence of various drugs having BSEP inhibitory activity BSEP transport activity was measured.
４−１．蛍光標識化胆汁酸のトレーサーとして有用性
合成した蛍光標識化胆汁酸のトレーサーとしての有用性を確認するために、公知のＢＳＥＰ阻害剤によるＢＳＥＰ輸送阻害活性を測定・評価した。トレーサーとしては、合成したＴａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ（１．４μＭ）を用い、対照として、従来用いられている放射性同位元素標識化胆汁酸である３Ｈ−標識タウロコール酸（３Ｈ−標識Ｔ−ＣＡ）（１．４μＭ）を用いた。また、ＢＳＥＰ阻害剤としては、シクロスポリンＡ（シグマ社製）（１００μＭ）、グリベンクラミド（シグマ社製）（１００μＭ）、プラバスタチン（シグマ社製）（５００μＭ）、リファンピシン（シグマ社製）（５００μＭ）、及びタウロリトコール酸（１００μＭ）を、陰性対照としてテトラエチルアンモニウム（シグマ社製）（５００μＭ）を各濃度で用いた。Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤ（１．４μＭ）及びｈ−ＢＳＥＰベシクル懸濁液（蛋白量５０μｇ）を、各阻害剤の共存下又は非共存下、２分間インキュベートした後、氷冷した反応停止用緩衝液（５０ｍＭ ｓｕｃｒｏｓｅ、１００ｍＭ ＫＮＯ３及び１００ｍＭ Ｔ−ＣＡを含む１０ｍＭ Ｈｅｐｅｓ−Ｔｒｉｓ、ｐＨ７．４、０．２ｍＬ）を添加し反応を停止した。次いで、反応液をグラスファイバーフィルター（マイクロプレートフィルター、孔計１．０μｍ）を用い吸引ろ過し、引き続き、反応停止用緩衝液（０．２ｍＬ×５回）で洗浄した。フィルター上に吸着した胆汁酸をエタノール（１．４ｍＬ）で溶出し、溶出液を減圧留去した。得られた残渣をジメチルスルホキシド（０．４ｍＬ）に溶解し、その０．２ｍＬを９６穴マイクロプレートのウェルに分注し、プレートリーダーで蛍光強度を測定し、検量線から胆汁酸量を求め活性値（ｐｍｏｌ／ｍｉｎ／ｍｇ ｐｒｏｔｅｉｎ）に換算した。ＡＴＰ共存下における活性値からＡＭＰ共存下における活性値を差し引いて得られた値をＡＴＰ依存輸送活性値とした。ＢＳＥＰベシクル輸送活性への各阻害剤の阻害効果は、阻害剤非共存下でのＴａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤのＡＴＰ依存輸送活性値を１００％（コントロール）とする相対値（輸送率）として求めた。その結果を表３に示した。
4-1. Usefulness as tracer of fluorescently labeled bile acids To confirm the usefulness of synthesized fluorescently labeled bile acids as tracers, the BSEP transport inhibitory activity of known BSEP inhibitors was measured and evaluated. As a tracer, synthesized Tauro-homo-CA-23-DBD (1.4 μM) was used, and as a control, 3 H-labeled taurocholic acid ( 3 H--), a conventionally used radioisotope-labeled bile acid, was used. Labeled T-CA) (1.4 μM) was used. Further, as BSEP inhibitors, cyclosporin A (manufactured by Sigma) (100 μM), glibenclamide (manufactured by Sigma) (100 μM), pravastatin (manufactured by Sigma) (500 μM), rifampicin (manufactured by Sigma) (500 μM), and Taurolithocholic acid (100 μM) was used as a negative control and tetraethylammonium (manufactured by Sigma) (500 μM) was used at each concentration. Tauro-homo-CA-23-DBD (1.4 μM) and h-BSEP vesicle suspension (protein amount 50 μg) were incubated for 2 minutes in the presence or absence of each inhibitor, and then ice-cooled reaction Stopping buffer (10 mM Hepes-Tris, pH 7.4, 0.2 mL containing 50 mM sucrose, 100 mM KNO 3 and 100 mM T-CA) was added to stop the reaction. Subsequently, the reaction solution was subjected to suction filtration using a glass fiber filter (microplate filter, pore size: 1.0 μm), and subsequently washed with a reaction stopping buffer solution (0.2 mL × 5 times). The bile acid adsorbed on the filter was eluted with ethanol (1.4 mL), and the eluate was distilled off under reduced pressure. Dissolve the resulting residue in dimethyl sulfoxide (0.4 mL), dispense 0.2 mL of it into a well of a 96-well microplate, measure the fluorescence intensity with a plate reader, and determine the amount of bile acid from the calibration curve. It converted into a value (pmol / min / mg protein). The value obtained by subtracting the activity value in the presence of AMP from the activity value in the presence of ATP was defined as the ATP-dependent transport activity value. The inhibitory effect of each inhibitor on the BSEP vesicle transport activity is a relative value (transport rate) with the ATP-dependent transport activity value of Tauro-homo-CA-23-DBD being 100% (control) in the absence of the inhibitor. As sought. The results are shown in Table 3.
Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤを用いた場合のＡＴＰ依存輸送活性値は、テトラエチルアンモニウムを除く各阻害剤の共存下において、コントロールに比し低値を示した。また、各阻害剤共存下における輸送率は、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤを用いた場合と、３Ｈ−標識Ｔ−ＣＡを用いた場合とでほぼ同様の値を示した。これらの結果より、Ｔａｕｒｏ−ｈｏｍｏ−ＣＡ−２３−ＤＢＤは、ＢＳＥＰ阻害活性評価のトレーサーとして十分に機能することが判明し、本発明の蛍光標識化胆汁酸を用いた活性測定方法が、各種薬剤のＢＳＥＰ阻害活性を評価する方法へ適用可能であることが確認された。 The ATP-dependent transport activity value in the case of using Tauro-homo-CA-23-DBD was lower than that of the control in the presence of each inhibitor except tetraethylammonium. Moreover, the transport rate in the presence of each inhibitor showed almost the same value when Tauro-homo-CA-23-DBD was used and when 3 H-labeled T-CA was used. From these results, it was proved that Tauro-homo-CA-23-DBD functions sufficiently as a tracer for BSEP inhibitory activity evaluation, and the activity measurement method using the fluorescently labeled bile acid of the present invention is used for various drugs. It was confirmed that the method can be applied to a method for evaluating the BSEP inhibitory activity.
４−２．ＢＳＥＰ阻害活性評価法の開発
上記のＢＳＥＰ活性測定方法では、輸送されたトレーサーの測定は、ＢＳＥＰベシクル内に取り込まれたトレーサーをエタノールで抽出した後、減圧下溶媒を留去して行っているため、前処理操作が煩雑であり、抽出溶媒の留去に長時間を要している。そこで、より簡便に用いることができる簡略化された測定系からなるＢＳＥＰ阻害活性の評価方法を検討した。
4-2. Development of BSEP inhibitory activity evaluation method In the above BSEP activity measurement method, the transported tracer is measured by extracting the tracer incorporated in the BSEP vesicle with ethanol and then distilling off the solvent under reduced pressure. The pretreatment operation is complicated, and it takes a long time to evaporate the extraction solvent. Therefore, a method for evaluating BSEP inhibitory activity comprising a simplified measurement system that can be used more simply was studied.
トレーサーとしては、Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−ＤＢＤを用いて測定を行った。５．０μＭのＴａｕｒｏ−ｎｏｒ−ＴＨＣＡ−ＤＢＤとＢＳＥＰベシクルとをＡＴＰ又はＡＭＰ共存下インキュベートした後、ベシクル内に取り込まれたトレーサーを抽出した。抽出以降の工程としては、トレーサーをエタノールで抽出し、減圧下溶媒留去した後、ＤＭＳＯに溶解して、蛍光強度を測定した方法（Ｍｅｔｈｏｄ１）、トレーサーを１０％ＳＤＳ水溶液（１００μＬ）で抽出し、溶出液をマイクロプレートに直接捕集した後、そのままプレートリーダーを用いて蛍光強度を測定した方法（Ｍｅｔｈｏｄ２）、のそれぞれを行い、各ＢＳＥＰの輸送活性を測定した。その結果を表４に示した。 As a tracer, measurement was performed using Tauro-nor-THCA-DBD. After incubating 5.0 μM Tauro-nor-THCA-DBD and BSEP vesicles in the presence of ATP or AMP, the tracer incorporated in the vesicles was extracted. As the steps after extraction, the tracer was extracted with ethanol, the solvent was distilled off under reduced pressure, dissolved in DMSO, and the fluorescence intensity was measured (Method 1). The tracer was extracted with 10% SDS aqueous solution (100 μL). The eluate was directly collected on a microplate, and each of the methods (Method 2) in which the fluorescence intensity was directly measured using a plate reader was performed, and the transport activity of each BSEP was measured. The results are shown in Table 4.
ＡＴＰ共存下及びＡＭＰ共存下での活性値、並びにＡＴＰ依存輸送活性値のいずれについても、トレーサーの抽出方法を簡略化した方法（Ｍｅｔｈｏｄ２）と、エタノールで抽出し減圧下溶媒留去した方法（Ｍｅｔｈｏｄ１）との間では、よく符号する測定結果を得ることができた。トレーサーの抽出方法を簡略化した方法（Ｍｅｔｈｏｄ２）では、測定値のバラツキが若干大きく認められるものの、前処理操作の煩雑さが著しく改善され、より簡便にＢＳＥＰ輸送活性を測定することが可能であることが判明した。 For both the activity value in the presence of ATP and AMP, and the ATP-dependent transport activity value, a method (Method 2) in which the extraction method of the tracer is simplified, and a method (Method 1) in which the solvent is distilled off under reduced pressure after extraction with ethanol. ), We were able to obtain well-measured measurement results. In the method (Method 2) in which the tracer extraction method is simplified, the measurement value variation is slightly large, but the complexity of the pretreatment operation is remarkably improved, and the BSEP transport activity can be measured more easily. It has been found.
トレーサーの抽出方法を簡略化した方法（Ｍｅｔｈｏｄ２）を用いて、４−１と同様にして、公知のＢＳＥＰ阻害剤によるＢＳＥＰ輸送阻害活性を測定・評価した。トレーサーとしては、Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ（１．４μＭ）を用いた。ＢＳＥＰ阻害剤としては、シクロスポリンＡ（１００μＭ）、グリベンクラミド（１００μＭ）、プラバスタチン（５００μＭ）、リファンピシン（５００μＭ）、及びタウロリトコール酸（１００μＭ）を、陰性対照としてテトラエチルアンモニウム（５００μＭ）を各濃度で用いた。Ｔａｕｒｏ−ｎｏｒ−ＴＨＣＡ−２４−ＤＢＤ（１．４μＭ）及びｈ−ＢＳＥＰベシクル懸濁液（蛋白量５０μｇ）を５分間インキュベートした後、氷冷した反応停止用緩衝液（５０ｍＭ ｓｕｃｒｏｓｅ、１００ｍＭ ＫＮＯ３及び１００ｍＭ Ｔ−ＣＡを含む１０ｍＭ Ｈｅｐｅｓ−Ｔｒｉｓ、ｐＨ７．４、０．２ｍＬ）を添加し反応を停止した。次いで、反応液をグラスファイバーフィルター（マイクロプレートフィルター、孔計１．０μｍ）を用いて吸引ろ過し、引き続き、反応停止用緩衝液（０．２ｍＬ×５回）で洗浄した。フィルター上に吸着した胆汁酸を１０％ＳＤＳ溶液（５０μＬ×２回）で溶出し、溶出液を９６穴マイクロプレートに直接捕集し、プレートリーダーで蛍光強度を測定し、検量線から胆汁酸量を求め活性値（ｐｍｏｌ／ｍｉｎ／ｍｇ ｐｒｏｔｅｉｎ）に換算した。なお、はＡＴＰ共存下における活性値からＡＭＰ共存下における活性値を差し引いて得られた値をＡＴＰ依存活性値とした。その結果を表５に示した。 Using a method (Method 2) obtained by simplifying the tracer extraction method, BSEP transport inhibitory activity by a known BSEP inhibitor was measured and evaluated in the same manner as in 4-1. As a tracer, Tauro-nor-THCA-24-DBD (1.4 μM) was used. As BSEP inhibitors, cyclosporin A (100 μM), glibenclamide (100 μM), pravastatin (500 μM), rifampicin (500 μM), and taurolithocholic acid (100 μM) were used as negative controls at tetraethylammonium (500 μM) at each concentration. . Tauro-nor-THCA-24-DBD (1.4 μM) and h-BSEP vesicle suspension (protein amount 50 μg) were incubated for 5 minutes, and then ice-cooled reaction buffer (50 mM sucrose, 100 mM KNO 3 and 10 mM Hepes-Tris, pH 7.4, 0.2 mL) containing 100 mM T-CA was added to stop the reaction. Next, the reaction solution was subjected to suction filtration using a glass fiber filter (microplate filter, pore size 1.0 μm), and subsequently washed with a reaction stopping buffer solution (0.2 mL × 5 times). Bile acid adsorbed on the filter is eluted with 10% SDS solution (50 μL x 2), the eluate is directly collected on a 96-well microplate, the fluorescence intensity is measured with a plate reader, and the amount of bile acid is determined from the calibration curve. Was converted into an activity value (pmol / min / mg protein). In addition, the value obtained by subtracting the activity value in the presence of AMP from the activity value in the presence of ATP was defined as the ATP-dependent activity value. The results are shown in Table 5.
各阻害剤共存下における輸送率は、トレーサーの抽出方法を簡略化した方法（Ｍｅｔｈｏｄ２）を用いた場合であっても、従来法の３Ｈ−標識Ｔ−ＣＡを用いた場合とほぼ同様の値を示した。以上の結果から、本発明の活性測定方法は、簡便性に優れ、ＢＳＥＰ阻害活性を正しく評価することができる方法として適用できることが明らかとなった。さらに、本発明の測定方法は、従来不可能であった一般実験室での阻害物質のスクリーニングの実施を可能とするものであり、汎用性に優れることから、臨床、医薬品開発のみならず基礎研究分野においてもその利用価値は高く、ＢＳＥＰの機能解明の一助として極めて有用な知見を提供するものといえる。 Transport rates at each inhibitor presence, even when using the method (Method2) a simplified extraction process of the tracer, almost the same value as the case of using the 3 H- labeled T-CA of the prior art showed that. From the above results, it was revealed that the activity measurement method of the present invention is excellent in simplicity and can be applied as a method capable of correctly evaluating BSEP inhibitory activity. Furthermore, the measurement method of the present invention enables screening of inhibitory substances in a general laboratory, which has been impossible in the past, and is excellent in versatility. The utility value is high also in the field, and it can be said that it provides extremely useful knowledge as an aid in elucidating the function of BSEP.
Claims (15)
［式中、
Ｒ１−Ｒ７は、それぞれ同一又は異なって、水素原子、ヒドロキシ基、置換若しくは非置換のＣ１−Ｃ６アルキル基、置換若しくは非置換のＣ１−Ｃ６アルコキシ基、置換若しくは非置換のＣ１−Ｃ８アシルオキシ基を表し、
Ｘは、発色団を表し、
ｎは、０〜２のいずれかの整数を表す。］ The labeled bile acid or conjugate thereof according to claim 1, which is represented by the following formula.
[Where:
R 1 to R 7 are the same or different and each represents a hydrogen atom, a hydroxy group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C8 acyloxy. Represents a group,
X represents a chromophore,
n represents an integer of 0 to 2. ]
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| WO2014200816A1 (en) * | 2013-06-14 | 2014-12-18 | Biotranex, Llc | Method for measuring bile salt export transport and/or formation activity |
| US9772325B2 (en) | 2013-06-14 | 2017-09-26 | Biotranex, Llc | Method for measuring bile salt export transport and/or formation activity |
| US10481149B2 (en) | 2013-06-14 | 2019-11-19 | Biotranex, Llc | Method for measuring bile salt export transport and/or formation activity |
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