JP2022115260A - Test reagent for measuring function of organic anion transporter - Google Patents
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Abstract
Description
本発明は、有機アニオントランスポーターの機能を測定するための検査薬に関する。 The present invention relates to test reagents for measuring the function of organic anion transporters.
治療薬を経口投与した場合、小腸腸管から血液内に吸収される。その取り込みを担うトランスポーターが有機アニオントランスポーター（ＯＡＴＰ）、有機カチオントランスポーター（ＯＣＴ）などで、このＯＡＴＰトランスポーターは特に、複数の臨床的な薬物の取り込みに関係している。例えば、コレステロール低下のため処方されるスタチン系薬物は、ＯＡＴＰ１Ｂ１の輸送基質となる。このＯＡＴＰの取り込み能はヒトにおいて多様性があるので、化学療法等を行う場合も、創薬の場合も、事前に評価をしておくことが好ましい。 When administered orally, therapeutic agents are absorbed into the blood through the small intestine. Transporters responsible for the uptake include organic anion transporters (OATP), organic cation transporters (OCT), etc., and OATP transporters are particularly implicated in the uptake of multiple clinical drugs. For example, statin drugs prescribed for cholesterol lowering are transport substrates for OATP1B1. Since this OATP uptake ability varies among humans, it is preferable to evaluate in advance both in the case of chemotherapy and drug discovery.
しかしながら、経口薬の消化管吸収量の確認作業は、質量分析器等を用いた血液分析により行われており、患者への負荷が大きく、経口薬の消化管吸収に関与する個々の薬物トランスポーター機能は測定できない。 However, confirmation of gastrointestinal absorption of oral drugs is performed by blood analysis using mass spectrometers, etc., which imposes a large burden on patients. Function cannot be measured.
一方、１２５Ｉ－２－ヨードアセトアミノフェン（１２５Ｉ－ＩＡＰ）等の放射性核種で標識されたアセトアミノフェンは、特許文献１に薬物代謝機能を測定するための検査薬として有用であることが開示されているが、トランスポーターとの関連性についてはこれまで報告されていない。 On the other hand, acetaminophen labeled with a radionuclide such as 125 I-2-iodoacetaminophen ( 125 I-IAP) is described in Patent Document 1 as being useful as a test agent for measuring drug metabolism function. Although disclosed, no association with transporters has been previously reported.
本発明の課題は、簡便にＯＡＴＰの取り込み能を評価できる検査薬を提供することである。 An object of the present invention is to provide a test agent that can easily evaluate the OATP uptake ability.
本発明者らは、前記課題を解決すべく鋭意検討を重ねた結果、放射性核種で標識されたアセトアミノフェン又はその類縁体を用いることにより、簡便にＯＡＴＰの取り込み能を評価できることを見出し、本発明を完成するに至った。 The present inventors have made intensive studies to solve the above problems, and found that the use of radionuclide-labeled acetaminophen or an analogue thereof makes it possible to easily evaluate the OATP uptake ability. I have perfected my invention.
すなわち、本発明の要旨は以下のとおりである。
（１）次式（Ｉ）：
で示される化合物を含有する有機アニオントランスポーターの機能を測定するための検査薬。
（２）前記式（Ｉ）において、Ｘで表される放射性核種が１２３－ヨード（１２３Ｉ）、１２４－ヨード（１２４Ｉ）、１２５－ヨード（１２５Ｉ）、１３１－ヨード（１３１Ｉ）、１８－フッ素（１８Ｆ）、７６－臭素（７６Ｂｒ）、７７－臭素（７７Ｂｒ）又は２１１－アスタチン(２１１Ａｔ)である前記（１）に記載の検査薬。
（３）経口投与され、前記放射性核種の膀胱での集積量を測定することによって有機アニオントランスポーターの機能を測定するための前記（１）又は（２）に記載の検査薬。
（４）経口投与された対象者由来の尿試料の放射能を液体シンチレーションカウンター又はγカウンターで測定することによって有機アニオントランスポーターの機能を測定するための前記（１）又は（２）に記載の検査薬。
That is, the gist of the present invention is as follows.
(1) Formula (I):
A test agent for measuring the function of an organic anion transporter containing a compound represented by.
(2) In the above formula (I), the radionuclide represented by X is 123-iodine ( 123 I), 124-iodine ( 124 I), 125-iodine ( 125 I), 131-iodine ( 131 I), The test agent according to (1) above, which is 18-fluorine ( 18 F), 76-bromine ( 76 Br), 77-bromine ( 77 Br) or 211-astatin ( 211 At).
(3) The test agent according to (1) or (2) above, which is administered orally and used to measure the function of the organic anion transporter by measuring the amount of radionuclide accumulated in the bladder.
(4) The method according to (1) or (2) above for measuring the function of the organic anion transporter by measuring the radioactivity of a urine sample derived from an orally administered subject with a liquid scintillation counter or a γ counter. test drug.
本発明によれば、簡便にＯＡＴＰの取り込み能を評価できる。本発明の検査薬を用いて、経口薬の投与前に患者個々のトランスポーター機能を測定した上で経口薬の薬剤投与量を各患者で調整できるようになる。 According to the present invention, the OATP uptake ability can be easily evaluated. By using the test agent of the present invention, it becomes possible to measure the transporter function of each patient before administering the oral drug, and then adjust the dosage of the oral drug for each patient.
本発明に用いる前記式（Ｉ）で示される化合物は、解熱鎮痛剤アセトアミノフェン（４－（アセチルアミノ）フェノール）の２位を放射性核種で標識したものである。 The compound represented by the formula (I) used in the present invention is an antipyretic analgesic, acetaminophen (4-(acetylamino)phenol) labeled with a radionuclide at the 2-position.
前記式（Ｉ）において、Ｘで表される放射性核種としては、例えばトリチウム（３Ｈ）、１１－炭素（１１Ｃ）、１８－フッ素（１８Ｆ）、７６－臭素（７６Ｂｒ）、７７－臭素（７７Ｂｒ）、１２３－ヨード（１２３Ｉ）、１２４－ヨード（１２４Ｉ）、１２５－ヨード（１２５Ｉ）、１３１－ヨード（１３１Ｉ）及び２１１－アスタチン(２１１Ａｔ)、好ましくは１８－フッ素（１８Ｆ）、７６－臭素（７６Ｂｒ）、７７－臭素（７７Ｂｒ）、１２３－ヨード（１２３Ｉ）、１２４－ヨード（１２４Ｉ）、１２５－ヨード（１２５Ｉ）、１３１－ヨード（１３１Ｉ）及び２１１－アスタチン(２１１Ａｔ)等が挙げられる。 Radionuclides represented by X in the formula (I) include, for example, tritium ( 3 H), 11-carbon ( 11 C), 18-fluorine ( 18 F), 76-bromine ( 76 Br), 77- Bromine ( 77 Br), 123-iodo ( 123 I), 124-iodo ( 124 I), 125-iodo ( 125 I), 131-iodo ( 131 I) and 211-astatin ( 211 At), preferably 18- fluorine ( 18 F), 76-bromine ( 76 Br), 77-bromine ( 77 Br), 123-iodine ( 123 I), 124-iodine ( 124 I), 125-iodine ( 125 I), 131-iodine ( 131 I) and 211-astatin ( 211 At).
前記式（Ｉ）において、Ｒで表される炭素数１～５のアルキル基としては、例えばメチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ペンチル基が挙げられる。 In the above formula (I), examples of alkyl groups having 1 to 5 carbon atoms represented by R include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group and sec-butyl group. , tert-butyl group and pentyl group.
本発明に用いる前記式（Ｉ）で示される化合物は、次式（II）：
で示される化合物に放射性核種を導入することにより製造することができる。
The compound represented by the formula (I) used in the present invention has the following formula (II):
It can be produced by introducing a radionuclide into the compound represented by.
前記式（II）で示される化合物への放射性核種の導入法としては一般的な方法を用いることができ、例えば、放射性核種が１２３－ヨード（１２３Ｉ）、１２４－ヨード（１２４Ｉ）、１２５－ヨード（１２５Ｉ）又は１３１－ヨード（１３１Ｉ）である場合は、クロラミンＴ法を用いることができる。 As a method for introducing a radionuclide into the compound represented by the formula ( II ), a general method can be used. -iodine ( 125 I) or 131-iodine ( 131 I), the chloramine T method can be used.
本発明の検査薬の投与経路としては、経口投与が挙げられる。 An administration route of the test agent of the present invention includes oral administration.
本発明の検査薬の投与形態としては、投与経路に適した剤形であれば、液剤、錠剤、カプセル剤等から適宜選択すればよく、本発明の作用及び効果を損なわない限り、薬学的に許容される担体、又は剤形によって当該技術分野において一般的に使用される添加剤を更に含んでもよい。添加剤として、例えば、着色剤、保存剤、風味剤、香り改善剤、呈味改善剤、甘味剤、又は安定剤、その他薬学的に許容される添加剤を含有することができる。 The dosage form of the test agent of the present invention may be appropriately selected from liquids, tablets, capsules, etc., as long as it is a dosage form suitable for the administration route. It may further comprise acceptable carriers or additives commonly used in the art depending on the dosage form. Additives may include, for example, coloring agents, preservatives, flavoring agents, flavor improving agents, taste improving agents, sweetening agents, stabilizers, and other pharmaceutically acceptable additives.
本発明の検査薬の投与量は、投与方法、投与する化合物ならびに患者の年齢、性別及び体重によって、適宜決定すればよい。 The dosage of the test agent of the present invention may be appropriately determined depending on the administration method, the compound to be administered, and the patient's age, sex and weight.
本発明の検査薬を画像診断薬として、イメージングのために用いる場合は、体内の透過性を必要とするので、放射性核種として１２３Ｉ等の放射線エネルギーの高いものを用いることが好ましいが、本発明の検査薬を経口投与した対象者由来の尿試料を用いて検査する場合は、１２５Ｉ及び１２３Ｉのいずれを用いてもよい。 When the test agent of the present invention is used as a diagnostic imaging agent for imaging, it needs to be able to penetrate the body. Either 125 I or 123 I may be used in the case of testing using a urine sample derived from a subject who orally administered the test drug.
画像診断薬として、イメージングのために用いる場合は、経口投与後の対象者の１２３Ｉ等の放射性核種の膀胱での集積量を測定することによって有機アニオントランスポーター（ＯＡＴＰ）の機能を測定することができる。 When used as a diagnostic imaging agent for imaging, to measure the function of the organic anion transporter (OATP) by measuring the accumulation of radionuclides such as 123 I in the bladder of a subject after oral administration. can be done.
対象者由来の尿試料を用いて検査する場合は、例えば、１２５／１２３Ｉ－ＩＡＰを経口投与した後に、１０～３０分後の間に尿試料を採取して、γカウンターで測定することで、放射能を取得する。健常者とＯＡＴＰ低下患者において、その放射能を比較すると、ＯＡＴＰ低下患者の尿試料の放射能は健常者と比較して低下する。その結果、小腸を中心とする消化管のＯＡＴＰ発現量を推測することができる。 When testing using a urine sample derived from a subject, for example, after oral administration of 125/123 I-IAP, a urine sample is collected 10 to 30 minutes later and measured with a γ counter. , to get radioactivity. Comparing the radioactivity in healthy subjects and in OATP-depleted patients, the radioactivity in urine samples of OATP-depleted patients is decreased compared to healthy subjects. As a result, the OATP expression level in the gastrointestinal tract centering on the small intestine can be estimated.
以下、実施例を挙げて本発明を更に具体的に説明するが、本発明の範囲は以下の実施例に限定されるものではない。 EXAMPLES The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the following examples.
（実施例１）
Ｉ．１２５Ｉ－２－ヨードアセトアミノフェンの合成
(Example 1)
I. Synthesis of 125 I-2-iodoacetaminophen
クロラミンＴ法でアセトアミノフェンの１２５Ｉ標識を行った。１２５Ｉ－ＮａＩ（１．５ＭＢｑ）をリン酸緩衝化食塩水（ｐＨ７．４）で１０μＬに希釈し、１０ｍＭアセトアミノフェンのエタノール溶液１００μＬ及び４ｍＭクロラミンＴ水溶液（塩酸でｐＨ５．６に調整したｍｉｌｌｉＱに溶解）２５μＬを加えて十分に撹拌し、２５℃で３０分間反応させた後、ピロ亜硫酸ナトリウムの１／１０飽和溶液２５μＬを加え、反応を停止した。反応液を窒素気流下で濃縮し、高速液体クロマトグラフィー（ＨＰＬＣ）を用いて分離精製した。検出機器には、ＵＶ－ＶＩＳ検出器（ＳＰＤ－１０Ａ，Ｓｈｉｍａｄｚｕ）、ＲＩ検出器；ラジオアナライザー（ＲＬＣ－７０１，Ａｌｏｋａ）を使用し、下記の分析条件で行った。条件を最適化した結果、標識率は８０％以上、放射化学的純度は９９％以上で１２５Ｉ－２－ヨードアセトアミノフェン（１２５Ｉ－ＩＡＰ）が得られた。 125 I labeling of acetaminophen was performed by the chloramine T method. 125 I-NaI (1.5 MBq) was diluted to 10 μL with phosphate-buffered saline (pH 7.4) and mixed with 100 μL of ethanol solution of 10 mM acetaminophen and 4 mM chloramine T aqueous solution (milliQ adjusted to pH 5.6 with hydrochloric acid). ) was added and stirred thoroughly, and reacted at 25°C for 30 minutes, then 25 µL of a 1/10 saturated solution of sodium pyrosulfite was added to terminate the reaction. The reaction solution was concentrated under a nitrogen stream and separated and purified using high performance liquid chromatography (HPLC). UV-VIS detector (SPD-10A, Shimadzu), RI detector; Radioanalyzer (RLC-701, Aloka) was used as detection equipment, and analysis was performed under the following analysis conditions. As a result of optimizing the conditions, 125 I-2-iodoacetaminophen ( 125 I-IAP) was obtained with a labeling rate of over 80% and a radiochemical purity of over 99%.
（ＨＰＬＣ分析条件）
カラム 5C18-MS-II(Nacalai tesque)
移動相 20％メタノール：80％50mM KH2PO4(pH4.7), 0-10分
50％メタノール：50％50mM KH2PO4(pH4.7), 10-18分
流速 1.0mL/分
UV 225nm
RI 27keV±5%
(HPLC analysis conditions)
Column 5C 18 -MS-II (Nacalai tesque)
50% Methanol: 50% 50mM KH 2 PO 4 (pH4.7), 10-18min Flow rate 1.0mL/min
UV 225nm
RI 27keV±5%
II．１２５Ｉ－ＩＡＰの正常マウス体内分布実験
Ａ）実験方法と材料
１２５Ｉ－ＩＡＰを約４０ｋＢｑ／２００μＬとなるように生理食塩水で希釈し、経口ゾンデ（Ｆｕｃｈｉｇａｍｉ）を用いて予め６時間絶食しておいたｄｄＹマウス（雄、８週齢、日本ＳＬＣ）に１匹当たり約３７ｋＢｑ／２００μＬを経口投与法として胃内投与した。投与５分、１０分、３０分、６０分後（ｎ＝４）に、イソフルラン（Ｗａｋｏ）麻酔下で心臓採血を行い、頸椎脱臼によりマウスを屠殺した。その後、速やかに各臓器（血液、心臓、肺、肝臓、脾臓、膵臓、胆嚢、胃、小腸、大腸、腎臓、膀胱、脳、甲状腺）を摘出し、それぞれの重量と放射能を電子天秤（Ｓｈｉｍａｄｚｕ）及びオートウェルガンマカウンタで測定した。
II. 125 I-IAP normal mouse biodistribution study A) Experimental methods and materials
125 I-IAP was diluted with physiological saline to about 40 kBq/200 μL, and ddY mice (male, 8 weeks old, Japan SLC) that had been fasted for 6 hours in advance were given 1 dose using an oral probe (Fuchigami). About 37 kBq/200 μL per animal was administered intragastrically as an oral administration method. Five minutes, 10 minutes, 30 minutes, and 60 minutes after administration (n=4), cardiac blood collection was performed under isoflurane (Wako) anesthesia, and the mice were sacrificed by cervical dislocation. Thereafter, each organ (blood, heart, lung, liver, spleen, pancreas, gallbladder, stomach, small intestine, large intestine, kidney, bladder, brain, thyroid) was promptly removed, and the weight and radioactivity of each were measured using an electronic balance (Shimadzu). ) and an Autowell Gamma Counter.
また、１２５Ｉ－ＩＡＰを約２０ｋＢｑ／２００μＬとなるように生理食塩水で希釈し、マウスの尾静脈から静脈投与した。投与５分、１５分、３０分、６０分後に、心臓採血を行い、重量と放射能を測定した。 In addition, 125 I-IAP was diluted with physiological saline to about 20 kBq/200 μL and administered intravenously through the tail vein of mice. At 5, 15, 30 and 60 minutes after administration, heart blood was collected and weight and radioactivity were measured.
Ｂ）結果と考察
経口投与時と静脈投与時の血液の重量集積率を図１に示した。経口投与時の血液の時間放射能曲線は、静脈投与時の結果と同様に、最大放射能を示した後に放射能の低下が確認できた。したがって、１２５Ｉ－ＩＡＰは経口投与後、小腸から血液内に吸収されたと考えられた。
B) Results and Consideration FIG. 1 shows the weight accumulation rate of blood during oral administration and intravenous administration. Similar to the result of intravenous administration, the time radioactivity curve of blood after oral administration showed the maximum radioactivity and then decreased radioactivity. Therefore, it was considered that 125 I-IAP was absorbed into the blood from the small intestine after oral administration.
III．１２５Ｉ－ＩＡＰのＯＡＴＰ阻害マウス体内分布実験
Ａ）実験方法と材料
予め６時間絶食しておいたマウスにＯＡＴＰ特異的阻害薬bromosulfalein（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を経口ゾンデにより経口投与法として胃内投与した。１２５Ｉ－ＩＡＰを約２０ｋＢｑ／１００μＬとなるように生理食塩水で希釈し、bromosulfalein投与（１ｍＭ／１００μＬ）５分後に、１２５Ｉ－ＩＡＰを経口ゾンデによりマウスに１匹当たり２０ｋＢｑ／１００μＬを経口投与した。１２５Ｉ－ＩＡＰ投与５分、１０分、３０分、６０分後（ｎ＝４）に、イソフルラン麻酔下で心臓採血を行い、頸椎脱臼によりマウスを屠殺した。その後、速やかに各臓器を摘出し、それぞれの重量と放射能を電子天秤及びオートウェルガンマカウンタで測定した。
III. 125 I-IAP OATP Inhibition Mouse Body Distribution Experiment A) Experimental method and materials Bromosulfalein (Sigma-Aldrich), an OATP-specific inhibitor, was intragastricly administered to mice that had been fasted for 6 hours by an oral probe. . 125 I-IAP was diluted with physiological saline to about 20 kBq/100 μL, and 5 minutes after administration of bromosulfalein (1 mM/100 μL), 125 I-IAP was orally administered to each mouse at 20 kBq/100 μL using an oral probe. did. At 5, 10, 30 and 60 minutes (n=4) after administration of 125 I-IAP, cardiac blood collection was performed under isoflurane anesthesia, and the mice were sacrificed by cervical dislocation. Then, each organ was promptly extracted, and the weight and radioactivity of each were measured using an electronic balance and an Autowell gamma counter.
Ｂ）結果と考察
正常マウス群とＯＡＴＰ阻害群の経口投与時の血液と、高集積を示した主要臓器である膀胱、肝臓及び胆嚢の重量集積率を図２に示した。ＯＡＴＰ阻害群では血液集積率が、正常群に比べて１２５Ｉ－ＩＡＰ投与後５分で有意に低下したため、ＯＡＴＰ阻害薬の影響により、１２５Ｉ－ＩＡＰの消化管吸収機能が一時的に阻害され、小腸から血液内への取り込みが減少したと考えられた。したがって、１２５Ｉ－ＩＡＰはＯＡＴＰを介して血液内に吸収されていることが確認された。また、膀胱では、ＯＡＴＰ阻害群が正常群に比べて１２５Ｉ－ＩＡＰの集積率の遅延が見られた。特に、１２５Ｉ－ＩＡＰ投与後１０分と３０分において有意な集積率低下が確認された。一方、肝臓と胆嚢ではともにＯＡＴＰ阻害群は正常群と比べて、集積率の低下傾向が確認されたが、有意差は見られなかった。膀胱、肝臓、胆嚢以外の臓器において、正常群とＯＡＴＰ阻害群で集積率がほとんど変わらなかった。したがって、膀胱の集積率を測定することで、小腸を中心とする消化管のＯＡＴＰ機能を推測できる可能性が示された。
B) Results and Discussion FIG. 2 shows the weight accumulation rates of the blood of the normal mouse group and the OATP inhibition group after oral administration, and the major organs showing high accumulation, ie, the bladder, liver and gallbladder. In the OATP inhibition group, the blood accumulation rate was significantly decreased 5 minutes after administration of 125 I-IAP compared to the normal group . , it was thought that the uptake from the small intestine into the blood decreased. Therefore, it was confirmed that 125 I-IAP was absorbed into the blood via OATP. In the bladder, the OATP inhibition group showed a delay in the rate of accumulation of 125 I-IAP compared to the normal group. In particular, a significant decrease in accumulation rate was confirmed at 10 minutes and 30 minutes after administration of 125 I-IAP. On the other hand, in both the liver and gallbladder, the OATP-inhibited group tended to show a lower accumulation rate than the normal group, but no significant difference was observed. In organs other than the bladder, liver, and gallbladder, there was almost no difference in the accumulation rate between the normal group and the OATP inhibition group. Therefore, by measuring the accumulation rate in the bladder, it was possible to estimate the OATP function of the gastrointestinal tract centering on the small intestine.
IV．結語
１２５Ｉ－ＩＡＰは小腸刷子縁膜上のＯＡＴＰを介して血液内に吸収されることが確認された。更に、小腸に発現するＯＡＴＰの機能を阻害することで、１２５Ｉ－ＩＡＰの膀胱集積が有意に低下した。
IV. Conclusion
It was confirmed that 125 I-IAP is absorbed into the blood via OATP on the small intestinal brush border membrane. Furthermore, inhibition of the function of OATP expressed in the small intestine significantly reduced 125 I-IAP accumulation in the bladder.
以上の実施例では、１２３Ｉよりも放射線半減期が長い１２５Ｉを放射性核種として用いたが、画像診断薬として、イメージングのためには放射線エネルギーの高い１２３Ｉが用いられている。 Although 125 I, which has a longer radiation half-life than 123 I, was used as the radionuclide in the above examples, 123 I, which has high radiation energy, is used as a diagnostic imaging agent for imaging.
したがって、１２３Ｉ－ＩＡＰイメージングにおける膀胱の集積を測定することで、ＯＡＴＰ発現量を反映した消化管吸収機能を推測できる可能性が示された。
Therefore, by measuring the accumulation in the bladder in 123 I-IAP imaging, it was possible to estimate the gastrointestinal absorption function reflecting the OATP expression level.
Claims (4)
で示される化合物を含有する有機アニオントランスポーターの機能を測定するための検査薬。 Formula (I):
A test agent for measuring the function of an organic anion transporter containing a compound represented by.
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