JP2007315852A - Estimation method of lipid metabolism disorder - Google Patents
Estimation method of lipid metabolism disorder Download PDFInfo
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- JP2007315852A JP2007315852A JP2006144034A JP2006144034A JP2007315852A JP 2007315852 A JP2007315852 A JP 2007315852A JP 2006144034 A JP2006144034 A JP 2006144034A JP 2006144034 A JP2006144034 A JP 2006144034A JP 2007315852 A JP2007315852 A JP 2007315852A
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Abstract
Description
本発明は、被験物質が脂質代謝異常症を誘発する可能性があるか否かを予測又は診断する方法に関する。より具体的には、特定の生体内物質の変動を測定することにより、被験物質が脂質代謝異常症を誘発する可能性があるか否かを簡便かつ確実に予測又は診断する方法に関する。 The present invention relates to a method for predicting or diagnosing whether or not a test substance may induce dyslipidemia. More specifically, the present invention relates to a method for easily and reliably predicting or diagnosing whether or not a test substance may induce lipid metabolism abnormality by measuring a change in a specific in-vivo substance.
薬物投与により生体組織中にリン脂質蓄積が引き起こされる場合があり、この病態は薬物誘発性リン脂質症(drug-induced phospholipidosis)と呼ばれている。薬剤誘発性リン脂質症は、親水性と疎水性の両者の性質を有する種々の陽性イオン化合物の投与により発現し、そのメカニズムとしては、(1)薬物とリン脂質との結合物のphospholipaseによる消化不良、(2)薬物による直接的なphospholipase活性阻害、(3)ライソゾームへの薬物の取り込みの結果生じるライソゾーム内pH上昇によるphospholipase活性の低下,(4)薬物によるリン脂質の合成亢進が考えられている。また、蓄積する物質が中性脂肪及びスフィンゴミエリンの場合には、それぞれ脂肪症(steatosis)及びスフィンゴミエリン蓄積症(sphingolipidosis)と呼ばれ、リン脂質症を含め、これらの病態は脂質代謝異常症(lipidosis)に分類される。脂質代謝異常症のヒトでの発現リスクが低い医薬候補化合物を創製するためには、新薬の探索研究の段階で医薬候補化合物の脂質代謝異常症の誘発ポテンシャルを簡便かつ確実に予測することが極めて重要である。しかしながら、新薬開発の際の非臨床安全性評価における一般的な脂質代謝異常症の診断方法は病理組織学的検査であるが、脂質代謝異常症がある程度進行しないと病理組織学的変化として観察することは困難である。また、臨床推定使用量よりも極めて高い投与用量で医薬候補化合物を実験動物に投与した場合に脂質代謝異常症が認められるケース、あるいは観察された脂質代謝異常症の程度が軽微なケースなどでは、該医薬候補化合物がヒトに投薬された場合のリスクとベネフィットを考慮した上で、臨床試験段階に進む場合もあり,その際には外科的侵襲の少ない方法で採取された生体試料により、脂質代謝異常症を診断しながら、注意深く臨床試験を取り進める必要がある。このような理由から、脂質代謝異常症を簡便かつ確実に予測する、または外科的侵襲の少ない方法で採取された生体試料を用いて脂質代謝異常症を確実に診断する方法の開発が求められている。 Drug administration may cause phospholipid accumulation in living tissues, and this condition is called drug-induced phospholipidosis. Drug-induced phospholipidosis is manifested by administration of various positive ion compounds having both hydrophilic and hydrophobic properties. (1) Digestion of drug-phospholipid conjugate with phospholipase Poor, (2) Inhibition of direct phospholipase activity by drug, (3) Decrease in phospholipase activity due to increase in lysosomal pH resulting from incorporation of drug into lysosome, (4) Increase in phospholipid synthesis by drug Yes. In addition, when the accumulated substances are neutral fat and sphingomyelin, they are called steatosis and sphingolipidosis, respectively, and these pathologies including phospholipidosis are dyslipidemia ( lipidosis). In order to create a drug candidate compound with low risk of dyslipidemia in humans, it is extremely easy to reliably and reliably predict the induction potential of dyslipidemia of a drug candidate compound at the stage of exploratory research for new drugs. is important. However, histopathological examination is a general method for diagnosing dyslipidemia in non-clinical safety evaluation during the development of new drugs. However, if dyslipidemia does not progress to some extent, it is observed as histopathological changes. It is difficult. In addition, in cases where dyslipidemia is observed when the drug candidate compound is administered to an experimental animal at a dose much higher than the clinically estimated use amount, or in cases where the observed degree of dyslipidemia is minor, Considering the risks and benefits when the drug candidate compound is administered to humans, it may proceed to the clinical trial stage, in which case lipid metabolism is measured using a biological sample collected by a method with less surgical invasion. It is necessary to proceed with clinical trials carefully while diagnosing abnormalities. For these reasons, there is a need for the development of a method for predicting dyslipidemia easily and reliably, or for reliably diagnosing dyslipidemia using biological samples collected by a method with little surgical invasion. Yes.
そのような手段として、生体内物質をマーカーとして利用して脂質代謝異常症を誘発する可能性のある物質を予測又は診断する手段が提案されている。例えば、フェナセルツ酸(フェニルアセチルグリシン、PAG; Magn. Reson. Chem., 39, pp.559-565, 2001; Biomarkers, 5, pp.410-423, 2000)、リゾビスホスファチジン酸(Biochim. Biophys. Acta, 1631, pp.136-146, 2003)、白血球中脂肪滴(J. Appl. Toxicol., 26, pp.167-177, 2005)、白血球における遺伝子発現(米国毒性学会要旨集, pp.35, 2006)などはリン脂質症のバイオマーカーとして利用できることが知られており、被験物質のリン脂質代謝異常症誘発可能性を評価するためのバイオマーカーとして利用できることが期待できる。また、フェニルアラニン代謝経路のバランス変化を指標として、医薬の開発段階において候補化合物の脂質代謝異常症誘発可能性を予測する方法も提案されている(国際公開WO2005/64344)。
本発明の課題は、被験物質が薬剤誘発性リン脂質症などの脂質代謝異常症を誘発する可能性があるか否かを簡便かつ確実に予測する方法を提供することにある。より具体的には、特定の生体内物質の変動を検出することにより、被験物質について脂質代謝異常症の誘発可能性を簡便かつ確実に予測する方法を提供することが本発明の課題である。
また、本発明の別の課題は、特定の生体内物質の変動を検出することにより、薬剤誘発性リン脂質症などの脂質代謝異常症の発症の有無を簡便かつ確実に診断する方法及びそのための手段を提供することにある。
An object of the present invention is to provide a method for easily and reliably predicting whether or not a test substance has a possibility of inducing lipid metabolism disorders such as drug-induced phospholipidosis. More specifically, it is an object of the present invention to provide a method for easily and reliably predicting the possibility of inducing dyslipidemia for a test substance by detecting a change in a specific in-vivo substance.
Another object of the present invention is to provide a method for easily and reliably diagnosing the presence or absence of onset of lipid metabolism disorders such as drug-induced phospholipidosis by detecting changes in specific in-vivo substances, and for the same It is to provide means.
本発明者らは上記の課題を解決すべく鋭意研究を行った結果、脂質代謝異常症の一つであるリン脂質症を誘発することが知られている物質を動物に投与すると23種の特定の生体内物質の変動が生じることを見出した。この知見を基にして、被験物質を投与した後に該生体物質の変動を検出することによって、その被験物質が脂質代謝異常症を誘発する可能性を有するか否かを簡便かつ確実に予測することができること、及びこの特定の生体内物質をバイオマーカーとして利用することによりヒトを含む哺乳類動物において薬剤誘発性リン脂質症などの脂質代謝異常症の発症可能性を外科的侵襲の少ない方法で採取された生体試料を用いて簡便かつ確実に診断することができることを見出した。本発明はこれらの知見を基にして完成された。 As a result of intensive studies to solve the above problems, the present inventors have found that when a substance known to induce phospholipidosis, which is one of dyslipidemias, is administered to animals, 23 types of identification It has been found that fluctuations in in vivo substances occur. Based on this knowledge, predicting whether or not the test substance has the possibility of inducing lipid metabolism disorder by detecting a change in the biological substance after administering the test substance And by using this specific in vivo substance as a biomarker, the possibility of developing lipid metabolism disorders such as drug-induced phospholipidosis in mammals including humans was collected by a method with less surgical invasion. It has been found that simple and reliable diagnosis can be performed using a biological sample. The present invention has been completed based on these findings.
すなわち、本発明により、被験物質の脂質代謝異常症誘発可能性を予測又は診断する方法であって、下記の工程:
(1)被験物質をヒト以外の哺乳類動物に投与した後、該哺乳類動物から生体試料を採取する工程;
(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を被験物質を投与していない哺乳類動物から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該被験物質が脂質代謝異常症を誘発する可能性があると判定する工程
を含み、かつ該特定生体内物質がアミオダロン及び下記の式で表されるフェノキシプロピルアミン化合物(I):
(1) A step of collecting a biological sample from a mammal after administering the test substance to a mammal other than a human;
(2) Measure the specific biological substance contained in the biological sample, and the measurement result obtained is a measurement result of the specific biological substance contained in the biological sample collected from a mammal not administered the test substance And a step of determining that the test substance may induce dyslipidemia when a significant difference is found in these measurement results, and the specific biological substance is amiodarone and Phenoxypropylamine compound (I) represented by the formula:
上記の発明の好ましい態様によれば,特定生体内物質の測定をメタボローム解析により行う上記の方法;特定生体内物質がL-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4,6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸、トリメチルアミン N−オキシド、ヒポタウリン、フルクトシルリジン、ベタイン、L-アスパラギン、L-オルニチン、L-リジン、L-メチオニン、及びL-ヒスチジンからなる群から選ばれる1種又は2種以上の物質である上記の方法;特定生体内物質がL-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸からなる群から選ばれる1種又は2種以上の物質である上記の方法;特定生体内物質がL-カルニチンである上記の方法;生体試料が尿、血漿、血清、または組織ホモジネートである上記の方法;組織ホモジネートが肺ホモジネートまたは肝ホモジネートである上記の方法;及び被験物質が医薬候補化合物である上記の方法が提供される。 According to a preferred embodiment of the above invention, the method of measuring a specific in vivo substance by metabolome analysis; the specific in vivo substance is L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4,6- Dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, The above method, which is one or more substances selected from the group consisting of L-methionine and L-histidine; Internal substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxy Selected from the group consisting of phenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid The above method wherein the specific biological substance is L-carnitine; the biological sample is urine, plasma, serum, or tissue homogenate; the tissue homogenate is There is provided the above method which is a lung homogenate or a liver homogenate; and the above method wherein the test substance is a drug candidate compound.
また、本発明により、医薬候補化合物のスクリーニング方法であって、上記の予測方法により脂質代謝異常症を誘発する可能性があると判定された被験物質を被験物質群から除外して脂質代謝異常症の誘発可能性のない医薬候補化合物を選抜する工程を含む方法、及び該スクリーニング方法により選抜された医薬候補化合物を含むライブラリーが提供される。
さらに、被験物質の脂質代謝異常症誘発可能性を予測するためのバイオマーカーとしての上記の特定生体内物質の使用、及び被験物質の脂質代謝異常症誘発可能性を予測するためのバイオマーカーであって、上記の特定生体内物質を含むバイオマーカーが本発明により提供される。
Further, according to the present invention, there is provided a screening method for a drug candidate compound, wherein a test substance determined to have a possibility of inducing a lipid metabolism disorder by the above prediction method is excluded from the test substance group, and the lipid metabolism disorder is detected. There are provided a method comprising the step of selecting a drug candidate compound that is not inducible, and a library comprising the drug candidate compound selected by the screening method.
Furthermore, the biomarker is used for predicting the possibility of the test substance to induce lipid metabolism disorders and the use of the specific in vivo substance as a biomarker to predict the possibility of the test substance to induce lipid metabolism disorders. Thus, the present invention provides a biomarker containing the above specific biological substance.
別の観点からは、脂質代謝異常症の診断方法であって、下記の工程:
(1)ヒトを含む哺乳類動物個体から生体試料を採取する工程;
(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を健常者から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該個体は脂質代謝異常症の発症可能性を有すると判定する工程
を含み、かつ該特定生体内物質がアミオダロン及び上記の構造式で表されるフェノキシプロピルアミン化合物(I)のそれぞれの投与により有意に変動する1種又は2種以上の物質である方法が提供される。
From another viewpoint, a method for diagnosing dyslipidemia, comprising the following steps:
(1) a step of collecting a biological sample from a mammal individual including a human;
(2) Measure a specific biological substance contained in the biological sample, compare the obtained measurement results with the measurement result of the specific biological substance contained in a biological sample collected from a healthy person, and measure these When a significant difference is found in the result, the individual includes a step of determining that the individual has a possibility of developing dyslipidemia, and the specific biological substance is amiodarone and phenoxypropyl represented by the above structural formula Methods are provided that are one or more substances that vary significantly with each administration of amine compound (I).
上記の発明の好ましい態様によれば、特定生体内物質の測定をメタボローム解析により行う上記の方法;特定生体内物質がL-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4,6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸、トリメチルアミン N−オキシド、ヒポタウリン、フルクトシルリジン、ベタイン、L-アスパラギン、L-オルニチン、L-リジン、L-メチオニン、及びL-ヒスチジンからなる群から選ばれる1種又は2種以上の物質である上記の方法;特定生体内物質がL-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸からなる群から選ばれる1種又は2種以上の物質である上記の方法;特定生体内物質がL-カルニチンである上記の方法;生体試料が尿、血漿、血清、又は組織ホモジネートである上記の方法;組織ホモジネートが、肺ホモジネート又は肝臓ホモジネートである上記の方法;及び脂質代謝異常症が遺伝性リピドーシス、薬物起因性リピドーシス、又は脂肪酸代謝ホメオスタシス異常である上記の方法が提供される。 According to a preferred embodiment of the above invention, the method of measuring a specific in vivo substance by metabolome analysis; the specific in vivo substance is L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4,6- Dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, The above method, which is one or more substances selected from the group consisting of L-methionine and L-histidine; Internal substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxy Selected from the group consisting of phenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid The above method wherein the specific biological substance is L-carnitine; the biological sample is urine, plasma, serum or tissue homogenate; the tissue homogenate is A method as described above, wherein the disorder is pulmonary homogenate or liver homogenate; and dyslipidemia is hereditary lipidosis, drug-induced lipidosis, or The above methods are provided wherein the fatty acid metabolism homeostasis is abnormal.
また、脂質代謝異常症の診断用マーカーとしての上記の特定生体内物質の使用、及び脂質代謝異常症の診断用マーカーであって、上記の特定生体内物質を含む診断用マーカーが本発明により提供される。
さらに、上記のバイオマーカー又は上記の診断用マーカーを化学的又は生物学的に検出可能な測定用試薬;上記の特定生体内物質の変動を抑制する作用を有する物質を有効成分として含む脂質代謝異常症の予防及び/又は治療のための医薬;及び上記の特定生体内物質の発現を抑制する作用又は上記の特定生体内物質の発現を亢進する作用を有する物質を有効成分として含む脂質代謝異常症の予防及び/又は治療のための医薬も本発明により提供される。
Also, the use of the above specific biological substance as a diagnostic marker for dyslipidemia and a diagnostic marker for dyslipidemia, comprising the above specific biological substance, are provided by the present invention. Is done.
Furthermore, a reagent for measurement capable of chemically or biologically detecting the above biomarker or the above diagnostic marker; a lipid metabolism abnormality comprising as an active ingredient a substance having an action of suppressing the fluctuation of the above specific biological substance A lipid metabolism disorder comprising as an active ingredient a drug for the prevention and / or treatment of infectious diseases; and a substance having an action of suppressing the expression of the specific biological substance or an action of enhancing the expression of the specific biological substance A medicament for the prevention and / or treatment of is also provided by the present invention.
本発明の予測方法によれば、実験動物を用いた長期にわたる毒性試験を行うことなく、簡便かつ確実に被験物質が脂質代謝異常症誘発可能性を有するか否かを判定することができ、安全な医薬候補化合物を迅速にスクリーニングすることができる。また、本発明の診断方法は上記の診断用マーカーを用いることにより患者に苦痛を与えずに簡便かつ精度の高い診断を行うことができるという特徴がある。 According to the prediction method of the present invention, it is possible to easily and surely determine whether or not a test substance has a possibility of inducing dyslipidemia without performing a long-term toxicity test using an experimental animal. New drug candidate compounds can be rapidly screened. In addition, the diagnostic method of the present invention is characterized in that a simple and highly accurate diagnosis can be performed without causing pain to the patient by using the diagnostic marker.
本発明の方法は、被験物質の脂質代謝異常症誘発可能性を予測する方法であって、下記の工程:(1)被験物質をヒト以外の哺乳類動物に投与した後、該哺乳類動物から生体試料を採取する工程;及び(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を被験物質を投与していない哺乳類動物から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該被験物質が脂質代謝異常症を誘発する可能性があると判定する工程を含み、かつ該特定生体内物質がアミオダロン及び上記構造式で表されるフェノキシプロピルアミン化合物(I)のそれぞれの投与により有意に変動する1種又は2種以上の物質である方法ことを特徴としている。 The method of the present invention is a method for predicting the possibility of inducing dyslipidemia of a test substance, the following steps: (1) After administering the test substance to a mammal other than a human, a biological sample from the mammal And (2) measuring a specific biological substance contained in the biological sample, and specifying the measurement result obtained from the biological sample collected from a mammal not administered with the test substance. A step of determining that there is a possibility that the test substance induces dyslipidemia when there is a significant difference in the measurement results compared to the measurement results of the in vivo substance, and The method is characterized in that the in-vivo substance is one or more substances that vary significantly depending on the administration of amiodarone and the phenoxypropylamine compound (I) represented by the above structural formula.
バイオセンサーを用いて医薬候補化合物に起因する脂質代謝異常症を予測する方法及び脂質代謝異常症及びその関連疾患を診断する方法については国際公開WO 2005/64344に記載があり、その予測方法に使用される生体試料の種類(細胞又は組織など)、生体試料の採取方法、実験動物の種類、及び被験物質に実験動物を曝露させる手段、並びにその診断方法の適用対象となる脂質代謝異常症及びその関連疾患などが詳細かつ具体的に説明されている。これらの説明は本発明の予測方法及び診断方法を理解するために有用であり、当業者は上記刊行物を参照しつつ本発明を容易に理解することができる。上記刊行物の全ての開示を参照により本明細書の開示として含める。 A method for predicting dyslipidemia caused by a drug candidate compound using a biosensor and a method for diagnosing dyslipidemia and related diseases are described in International Publication WO 2005/64344, and used for the prediction method. Type of biological sample (cell or tissue, etc.) to be collected, biological sample collection method, type of experimental animal, means for exposing the experimental animal to the test substance, and lipid metabolism disorder to which the diagnostic method is applied and its Related diseases and the like are described in detail and specifically. These explanations are useful for understanding the prediction method and diagnosis method of the present invention, and those skilled in the art can easily understand the present invention with reference to the above-mentioned publications. The entire disclosures of the above publications are incorporated herein by reference.
本発明の予測方法において適用される被験物質の種類は特に限定されず、有機低分子化合物のほか、高分子化合物、無機化合物、核酸類(RNAiなどに用いる小分子RNAや非天然型塩基を含む核酸などを含む)、糖類、脂質類、又はペプチド類(オリゴペプチド又はポリペプチド類を含む)などの任意の物質が包含される。一般的には、本発明の予測方法を医薬候補化合物のスクリーニング手段として使用することが好ましく、被験物質は医薬候補化合物であることが好ましい。 The type of test substance applied in the prediction method of the present invention is not particularly limited. In addition to organic low molecular weight compounds, high molecular weight compounds, inorganic compounds, nucleic acids (including small molecule RNA and non-natural bases used for RNAi and the like) Any substance such as saccharides, lipids, or peptides (including oligopeptides or polypeptides) is included. In general, the prediction method of the present invention is preferably used as a screening means for drug candidate compounds, and the test substance is preferably a drug candidate compound.
本発明の予測方法において使用されるヒト以外の哺乳類動物の種類は特に限定されず、例えば、サル、ラット、マウス、モルモット、イヌ、ネコ、ウサギなどの通常の実験動物を使用することができる。被験物質を上記哺乳類動物に投与する方法は特に限定されず、被験物質の物理化学的性状や動物の種類などに応じて適宜の投与経路を選択することができる。例えば、経口投与、静脈内投与、又は腹腔内投与など適宜の投与経路を選択できる。 The kind of mammals other than humans used in the prediction method of the present invention is not particularly limited. For example, normal laboratory animals such as monkeys, rats, mice, guinea pigs, dogs, cats, and rabbits can be used. The method for administering the test substance to the mammal is not particularly limited, and an appropriate administration route can be selected according to the physicochemical properties of the test substance, the kind of animal, and the like. For example, an appropriate administration route such as oral administration, intravenous administration, or intraperitoneal administration can be selected.
生体試料の種類も特に限定されず、例えば、尿、血液、唾液、リンパ液、組織、又は細胞など任意の生体試料を使用することができ、それらの種類の応じて、適宜の分離手段を採用することができる。例えば、採血により採取した血液を生体試料として用いることができ、採取した血液から分離した血清を生体試料として用いてもよい。また、手術などの手段で摘出した臓器などから採取した組織のホモジネートを生体試料として用いることもでき、ホモジネート上清を生体試料として用いてもよい。例えば、肺や肝臓などの臓器から採取した組織のホモジネート又はホモジネート上清を好ましく用いることができる。 The type of biological sample is not particularly limited, and any biological sample such as urine, blood, saliva, lymph, tissue, or cell can be used, and an appropriate separation means is adopted depending on the type. be able to. For example, blood collected by blood collection can be used as the biological sample, and serum separated from the collected blood may be used as the biological sample. Further, a homogenate of a tissue collected from an organ or the like removed by means such as surgery can be used as a biological sample, and a homogenate supernatant may be used as a biological sample. For example, a tissue homogenate or a homogenate supernatant collected from an organ such as the lung or liver can be preferably used.
生体試料中に含まれる特定生体内物質の種類は特に限定されないが、該特定生体内物質は、脂質代謝異常症を誘発することが知られている2種の物質:アミオダロン及び上記の構造式で表されるフェノキシプロピルアミン化合物(I)のそれぞれの投与により変動する生体内物質を用いる必要がある。アミオダロンについてはToxicol. Appl. Pharmacol., 97, pp.124-133, 1987及びHepatology, 8, pp.1063-1068, 1988に脂質代謝異常症を発症することが記載されている。フェノキシプロピルアミン化合物(I)はWO 00/71517に開示された化合物である。また、アミオダロン投与により観察される白血球中脂肪滴は、フェノキシプロピルアミン化合物(I)の投与では観察されない。 The type of the specific biological substance contained in the biological sample is not particularly limited, but the specific biological substance includes two substances known to induce dyslipidemia: amiodarone and the above structural formula. It is necessary to use an in vivo substance that varies depending on each administration of the phenoxypropylamine compound (I) represented. About amiodarone, it is described in Toxicol. Appl. Pharmacol., 97, pp.124-133, 1987 and Hepatology, 8, pp.1063-1068, 1988 to develop dyslipidemia. The phenoxypropylamine compound (I) is a compound disclosed in WO 00/71517. Further, the lipid droplets in leukocytes observed by amiodarone administration are not observed by administration of phenoxypropylamine compound (I).
本発明の予測方法で測定の対象となる特定生体内物質は、生体内に本来的に存在する物質、好ましくは本来的に一定の濃度範囲内で存在する物質であってもよく、あるいは脂質代謝異常症において出現又は消失する物質であってもよい。特定生体内物質は任意の生体内物質又は被験物質の代謝物であってもよい。2種以上の特定生体内物質を測定することによって、より精度の高い予測が可能になる場合がある。特定生体内物質としては、例えば、L-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸、トリメチルアミン N-オキシド、ヒポタウリン、フルクトシルリジン、ベタイン、L-アスパラギン、L-オルニチン、L-リジン、L-メチオニン、及びL-ヒスチジンからなる群から選ばれる1種又は2種以上の物質を挙げることができ、好ましくはL-カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネート、グアニジドアセテート、4-グアニジノブタノエート、N-アミジノ-L-アスパラギン酸からなる群から選ばれる1種又は2種以上の物質を挙げることができる。これらのうち、L―カルニチン、4-ベータ-アセチルアミノエチルイミダゾール、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネートが更に好ましく、L-カルニチンが最も好ましい。もっとも、特定生体内物質はこれらに限定されることはない。 The specific biological substance to be measured by the prediction method of the present invention may be a substance that is inherently present in the living body, preferably a substance that is inherently present in a certain concentration range, or lipid metabolism. It may be a substance that appears or disappears in an abnormality. The specific in-vivo substance may be any in-vivo substance or metabolite of the test substance. By measuring two or more kinds of specific in vivo substances, more accurate prediction may be possible. Specific biological substances include, for example, L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinoline Carboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-asparagine One or more substances selected from the group consisting of acids, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, L-methionine, and L-histidine Preferably L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-hist , 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxy Mention may be made of one or more substances selected from the group consisting of picolinate, guanidide acetate, 4-guanidinobutanoate and N-amidino-L-aspartic acid. Of these, L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4- Hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4_6-dihydroxypicolinate are more preferred, and L-carnitine is most preferred. However, the specific in vivo substance is not limited to these.
本明細書において用いられる「測定」という用語は、定性及び定量又はそれらのどちらかを目的として特定生体内物質を検出することを意味しており、濃度測定のほか、同定などの概念を含めて最も広義に解釈しなければならず、いかなる意味においてもこの用語を限定的に解釈してはならない。「測定結果」という用語は濃度の測定値などのほか検出の有無などの指標も含めて最も広義に解釈する必要があり、必ずしも得られた数値自体を意味するものではない。例えば、ある特定生体内物質の濃度が検出限界以下の濃度に変動した場合、この結果も「測定結果」の概念に含まれる。 As used herein, the term “measurement” means to detect a specific in-vivo substance for the purpose of qualitative and / or quantitative, and includes concepts such as identification as well as concentration measurement. It should be interpreted in the broadest sense and the term should not be interpreted in any way restrictive. The term “measurement result” needs to be interpreted in the broadest sense including not only the measurement value of concentration but also an indicator such as the presence or absence of detection, and does not necessarily mean the obtained numerical value itself. For example, when the concentration of a specific in-vivo substance changes to a concentration below the detection limit, this result is also included in the concept of “measurement result”.
特定生体内物質の測定手段は特に限定されず、当業者に利用可能な任意の測定手段を1種又は2種以上組み合わせて用いることができるが、例えば、キャピラリー電気泳動と質量分析法とを組み合わせて測定を行うことが好ましい。例えば、Anal. Chem., 77, pp.78-84, 2005; Electrophoresis, 25, pp.1964-1972, 2004; Journal of Proteome Research., 2, pp488-494, 2003などを参照することにより当業者はこの測定を容易に行うことができる。この方法によれば、分子量及び電気泳動時間から特定生体内物質を高感度かつ精度よく同定し、かつ定量することができるという特徴がある。もっとも、特定生体内物質の種類に応じて適宜の測定手段を採用できることは当業者に自明である。 The measuring means for the specific in vivo substance is not particularly limited, and any measuring means available to those skilled in the art can be used singly or in combination of two or more. For example, capillary electrophoresis and mass spectrometry are combined. It is preferable to perform measurement. See, for example, Anal. Chem., 77, pp. 78-84, 2005; Electrophoresis, 25, pp. 1964-1972, 2004; Journal of Proteome Research., 2, pp 488-494, 2003, etc. Can make this measurement easily. This method is characterized in that a specific in-vivo substance can be identified and quantified with high sensitivity and accuracy from the molecular weight and electrophoresis time. However, it is obvious to those skilled in the art that an appropriate measurement means can be adopted according to the type of the specific biological substance.
メタボローム解析とは、生体内の代謝物質の総体(メタボローム)の網羅的な測定を意味する。メタボローム測定法としては、キャピラリー電気泳動・質量分析法、ガスクロマトグラフィー・質量分析法、高速液体クロマトグラフィー・質量分析法、フーリエ変換イオンサイクロトロン共鳴質量分析法、核磁気共鳴分析法(NMR)などが知られている(メタボローム研究の最前線、2003、シュプリンガー・フェアラーク東京株式会社(富田勝、西岡孝明編))。 Metabolome analysis means comprehensive measurement of the total amount of metabolites in the living body (metabolome). Metabolome measurement methods include capillary electrophoresis / mass spectrometry, gas chromatography / mass spectrometry, high performance liquid chromatography / mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry, nuclear magnetic resonance analysis (NMR), etc. Known (the forefront of metabolomic research, 2003, Springer Fairlark Tokyo Co., Ltd. (edited by Masaru Tomita, Takaaki Nishioka)).
また、特定生体内物質は、未処理あるいは適切な誘導体化処理の後、高速液体クロマトグラフィーやガスクロマトグラフィーなどによる分離手法と、紫外線、蛍光、電気化学的検出方法、Free Induction Decay(FID:自動誘導減衰)、質量分析などの機器分析手法を適切に組み合わせた測定方法、酵素免疫測定(ELISA)、放射性同位体による免疫測定(RIA)、化学発光酵素免疫測定(CLEIA)などの免疫反応を利用した測定方法、酵素反応と吸光度、蛍光あるいは発光検出などを組み合わせた生化学的手法でも測定可能である。 In addition, specific biological substances are untreated or appropriately derivatized, followed by separation techniques such as high-performance liquid chromatography and gas chromatography, UV, fluorescence, electrochemical detection methods, and free induction decay (FID: automatic). Induction decay), measurement methods that combine instrumental analysis methods such as mass spectrometry, enzyme immunoassay (ELISA), radioisotope immunoassay (RIA), chemiluminescent enzyme immunoassay (CLEIA), and other immune reactions The measurement can also be performed by a biochemical method that combines enzyme reaction and absorbance, fluorescence or luminescence detection.
生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を被験物質を投与していない哺乳類動物から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該被験物質が脂質代謝異常症を誘発する可能性があると判定することができる。本明細書において「有意差」とは一般的には周知かつ慣用の統計分析手法により2つの測定結果に有意な差が得られることを意味するが、必ずしも厳密な統計学的手法によらずにみかけ上の差を認めた場合にも有意差ありと判定してもよい。 Measuring a specific biological substance contained in a biological sample, and comparing the obtained measurement result with a measurement result of the specific biological substance contained in a biological sample collected from a mammal not administered with the test substance; When a significant difference is recognized in these measurement results, it can be determined that the test substance may induce dyslipidemia. In the present specification, “significant difference” generally means that a significant difference is obtained between two measurement results by a well-known and conventional statistical analysis method, but not necessarily by a strict statistical method. If an apparent difference is recognized, it may be determined that there is a significant difference.
被験物質が脂質代謝異常症の誘発性を示す場合には、特定生体内物質の濃度や組織分布などに変動が生じる。本明細書において「変動」という用語は、例えば、特定生体内物質の濃度上昇又は濃度減少、消失、出現、組織内若しくは細胞内又は生体中の分布変化、あるいは発現亢進などの1種又は2種以上を含む概念であり、いかなる意味においても限定的に解釈してはならない。この変動は、被験物質を投与した動物から採取した生体試料中に含まれる特定生体内物質の測定結果と被験物質を投与していない哺乳類動物から採取した生体試料中に含まれる該特定生体内物質の測定結果とを比較することにより、容易に確認することができる。一般的には、被験物質の非投与群をコントロール群とすることにより容易に変動を確認することができるが、健常な哺乳類動物からあらかじめ得た測定結果を標準結果とし用意しておき、その標準結果と比較して有意差の有無を判定してもよい。このような態様も本発明の方法に包含されることは言うまでもない。 When the test substance shows an inducibility of dyslipidemia, the concentration of the specific biological substance, tissue distribution, etc. vary. In this specification, the term “variation” refers to one or two types, for example, an increase or decrease in the concentration of a specific biological substance, disappearance, appearance, change in distribution within a tissue or cell or in a living body, or increased expression. It is a concept that includes the above, and should not be construed as limiting in any way. This fluctuation is caused by the measurement result of the specific biological substance contained in the biological sample collected from the animal administered with the test substance and the specific biological substance contained in the biological sample collected from the mammal not administered with the test substance. This can be easily confirmed by comparing with the measurement results. In general, fluctuations can be easily confirmed by setting the non-administration group of the test substance as the control group, but the measurement results obtained in advance from healthy mammals are prepared as standard results. You may determine the presence or absence of a significant difference compared with a result. It goes without saying that such an embodiment is also included in the method of the present invention.
上記の予測方法を用いて、脂質代謝異常症を誘発することのない安全な医薬候補化合物をスクリーニングすることができる。このスクリーニング方法は、スクリーニングの対象となる被験物質群のなかから上記の予測方法を用いて脂質代謝異常症を誘発する可能性のある被験物質を選択し、被験物質群からその被験物質を除外して、残りの被験物質群をスクリーニング結果物として得る工程を含んでいる。このようにして得られたスクリーニング結果物に含まれる物質は、脂質代謝異常症を誘発する可能性がないか、あるいはその可能性が極めて低い安全な物質であり、医薬として好適に使用可能な物質である。また、このようにして得た安全な物質群からなるライブラリーは、新規な医薬の開発のための物質ソース群として有用である。 Using the above prediction method, a safe drug candidate compound that does not induce dyslipidemia can be screened. In this screening method, a test substance that may induce dyslipidemia is selected from the test substance group to be screened using the prediction method described above, and the test substance is excluded from the test substance group. And a step of obtaining the remaining test substance group as a screening result. Substances contained in the screening results thus obtained are safe substances that have no possibility of inducing dyslipidemia or extremely low possibility, and can be suitably used as pharmaceuticals. It is. In addition, a library composed of safe substance groups obtained in this way is useful as a substance source group for the development of new medicines.
別の観点から提供される本発明の方法は、脂質代謝異常症の診断方法であって、下記の工程:(1)ヒトを含む哺乳類動物個体から生体試料を採取する工程;(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を健常者から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該個体は脂質代謝異常症の発症可能性を有すると判定する工程を含み、かつ該特定生体内物質がアミオダロン及び上記の構造式で表されるフェノキシプロピルアミン化合物(I)のそれぞれの投与により有意に変動する1種又は2種以上の物質であることを特徴としている。哺乳類動物としては、ヒトのほか、イヌ又はネコなどのペット類、あるいはウシやブタなどの家畜類が挙げられるが、これらに限定されることはない。好ましくはヒトを対象として診断を行うことができる。 The method of the present invention provided from another viewpoint is a method for diagnosing dyslipidemia, comprising the following steps: (1) collecting a biological sample from an individual mammal including human; (2) the living body The specific biological substance contained in the sample is measured, and the obtained measurement result is compared with the measurement result of the specific biological substance contained in the biological sample collected from a healthy person, and there is a significant difference between these measurement results. And the phenoxypropylamine compound (I) wherein the individual includes a step of determining that the individual has the possibility of developing dyslipidemia, and the specific in vivo substance is amiodarone and the structural formula above. It is characterized in that it is one or more substances that vary significantly depending on the administration of each of these. Examples of mammals include, but are not limited to, humans, pets such as dogs and cats, and livestock such as cows and pigs. Preferably, diagnosis can be performed on human subjects.
上記の診断方法において、脂質代謝異常症としては、例えば、遺伝性リピドーシス(例えばゴーシェ病、ニーマンピック病(A〜C型)、ファーブリ病、ウォルマン病、コレステロールエステル蓄積症、脳腱黄色腫病、フィトステロール血症、レフサム病、テイ-サックス病、全身性(GM1)ガングリオシドーシス、サルファチドリピドーシス(黒染性白質萎縮症)、ガラクトシルセラミドリピドーシスなど)、薬物起因性リピドーシス(例えばPLsis、脂肪症、スフィンゴミエリン蓄積症など)、脂肪酸代謝ホメオスタシス異常(例えば、脂肪酸β酸化異常など)などを挙げることができるが、これらに限定されることはない。本明細書において「脂質代謝異常症」という用語には、脂質代謝異常を伴う疾患、又は脂質代謝異常に起因する疾患なども包含される。このような疾患は脂質代謝異常症の関連疾患として知られており、例えば、高血圧症、粥状硬化症、動脈硬化、心筋梗塞、脂肪肝、肝炎、肝硬変、糖尿病、痴呆症、アルツハイマー症、心臓病、及び慢性疲労症候群などのほか、薬物起因性リピドーシス関連疾患として肺線維症、失明、及び脳症などが挙げられる。もっとも、本発明の診断方法の対象となる脂質代謝異常症はこれらに限定されることはない。 In the above diagnostic method, examples of dyslipidemia include hereditary lipidosis (eg, Gaucher disease, Niemann-Pick disease (types A to C), Fabry disease, Wolman disease, cholesterol ester storage disease, cerebral tendon xanthoma disease, Phytosterolemia, refsum disease, Tay-Sachs disease, systemic (GM1) gangliosidosis, sulfatid lipidosis (black-stained leukotrophy), galactosylceramide lipidosis, etc., drug-induced lipidosis (eg, PLsis, steatosis, Sphingomyelin accumulation disease, etc.), fatty acid metabolism homeostasis abnormality (for example, fatty acid β oxidation abnormality, etc.) can be mentioned, but are not limited thereto. In the present specification, the term “dyslipidemia” includes diseases associated with abnormal lipid metabolism, diseases caused by abnormal lipid metabolism, and the like. Such diseases are known as diseases related to dyslipidemia, such as hypertension, atherosclerosis, arteriosclerosis, myocardial infarction, fatty liver, hepatitis, cirrhosis, diabetes, dementia, Alzheimer's disease, heart In addition to diseases and chronic fatigue syndrome, pulmonary fibrosis, blindness, and encephalopathy are examples of drug-induced lipidosis-related diseases. However, the dyslipidemia which is the target of the diagnostic method of the present invention is not limited to these.
上記の診断方法は基本的に上記の予測方法と同様にして行うことができ、好ましくは個体から分離された生体試料として尿又は血液などを用いることができる。健常者から得た測定結果として、あらかじめ健常者から得た測定結果を標準結果として用意しておき、その結果を利用することもできる。被験個体から得た測定値と健常者から得た測定結果との間に有意差が認められた場合には、その被験個体は脂質代謝異常症をすでに発症しているか、あるいは脂質代謝異常症を発症する危険性がある。本明細書において「脂質代謝異常症の発症可能性を有する」という用語は、その個体が脂質代謝異常症をすでに発症していることを病理組織学的手段などにより明確に確認できる場合のほか、その個体が脂質代謝異常症に罹患していることを病理学的には証明できないものの他の臨床データーから脂質代謝異常症を発症していることが強く疑われる場合、あるいは脂質代謝異常症を将来発症する危険性を有している場合などを含めて、最も広義に解釈する必要がある。 The above diagnostic method can be performed basically in the same manner as the above prediction method. Preferably, urine or blood can be used as a biological sample separated from an individual. As a measurement result obtained from a healthy person, a measurement result obtained from a healthy person is prepared in advance as a standard result, and the result can be used. If there is a significant difference between the measured value obtained from the test individual and the measurement result obtained from the healthy subject, the test individual has already developed dyslipidemia or has dyslipidemia. Risk of developing. In the present specification, the term “having the possibility of developing dyslipidemia” means that the individual can clearly confirm that the individual has already developed dyslipidemia by histopathological means, Pathological evidence that the individual is suffering from dyslipidemia but other clinical data strongly suspected of developing dyslipidemia, or dyslipidemia in the future It should be interpreted in the broadest sense, including when there is a risk of developing it.
上記の特定生体内物質は脂質代謝異常症の診断用マーカーとして有用である。診断用マーカーとしては、1種の特定生体内物質を用いてもよいが、2種以上の特定生体内物質を組み合わせて用いてもよい。上記の診断方法の便宜のために、該診断用マーカーを化学的又は生物学的手段により特異的に検出できる測定用試薬を提供することも望ましく、2種以上の該診断用マーカーをそれぞれ特異的に検出できる測定用試薬を組み合わせてキットとして提供することも望ましい。特定生体内物質を特異的に検出可能な試薬としては、例えば、特定生体内物質に特異的に反応する標識化抗体などを挙げることができるが、この特定の試薬に限定されることはない。 The specific in-vivo substance is useful as a diagnostic marker for dyslipidemia. As a diagnostic marker, one kind of specific in vivo substance may be used, or two or more kinds of specific in vivo substances may be used in combination. For the convenience of the above diagnostic method, it is also desirable to provide a measuring reagent capable of specifically detecting the diagnostic marker by chemical or biological means, and each of the two or more types of diagnostic markers is specific. It is also desirable to provide a kit by combining the detection reagents that can be detected. Examples of the reagent that can specifically detect a specific in-vivo substance include labeled antibodies that specifically react with the specific in-vivo substance, but are not limited to this specific reagent.
以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。
例1
アミオダロン及びフェノキシプロピルアミン化合物(I)を試験物質として、リン脂質症の発現の有無を病理組織学検査及び肺組織中のホスファチジルコリン定量により調べた。また、尿及び血清中の中間・最終代謝産物をCE-TOFMSを用いて網羅的に分析した。
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
With amiodarone and phenoxypropylamine compound (I) as test substances, the presence or absence of phospholipidosis was examined by histopathological examination and phosphatidylcholine determination in lung tissue. In addition, intermediate and final metabolites in urine and serum were comprehensively analyzed using CE-TOFMS.
アミオダロンはSIGMA社より購入し、フェノキシプロピルアミン化合物(I)は三菱ウェルファーマ株式会社で合成し、実験に使用した。6週齢のCrl:CD(SD)ラットを日本チャールスリバーから購入し、5日間馴化飼育した後に実験に用いた。動物は1群4匹からなる3群に無作為に群分けし、溶媒対照群には0.5%ヒドロキシプロピルメチルセルロース溶液(HPMC)、アミオダロン投与群とフェノキシプロピルアミン化合物(I)投与群はそれぞれHPMCによる懸濁液を150mg/kgと100mg/kgの投与用量で14日間反復経口投与した。最終投与の直後にすべての動物を代謝ケージに移し、24時間にわたり尿を採取した。最終投与24時間後に動物をジエチルエーテルで麻酔した上で採血し、さらに放血後、肺を採取した。血液は定法にて血清を分離した後、メタノール・クロロホルムによる抽出液をCE-TOFMSに添加した。尿は遠心分離により夾雑物を取り除いた後、CE-TOFMSに添加した。CE-TOFMSによる解析は特願2005-258684号明細書に記載された方法に準じて実施した。肺は二分割し、一部の組織片については定法に従い病理組織標本を作製して病理組織変化を観察した。他方の組織片は採取後速やかに液体窒素にて凍結した上で、定法に従いホスファチジルコリンを定量した。 Amiodarone was purchased from SIGMA, and phenoxypropylamine compound (I) was synthesized by Mitsubishi Pharma Corporation and used in the experiment. Six-week-old Crl: CD (SD) rats were purchased from Japanese Charles River and used for experiments after habituation for 5 days. The animals were randomly divided into 3 groups of 4 animals. The solvent control group was treated with 0.5% hydroxypropylmethylcellulose solution (HPMC), the amiodarone-administered group and the phenoxypropylamine compound (I) -administered group with HPMC, respectively. The suspension was orally administered repeatedly for 14 days at doses of 150 mg / kg and 100 mg / kg. Immediately after the last dose, all animals were transferred to metabolic cages and urine collected for 24 hours. At 24 hours after the final administration, the animals were anesthetized with diethyl ether and blood was collected. After exsanguination, the lungs were collected. After separating serum from blood by a conventional method, an extract with methanol / chloroform was added to CE-TOFMS. Urine was added to CE-TOFMS after removing impurities by centrifugation. The analysis by CE-TOFMS was performed according to the method described in Japanese Patent Application No. 2005-258684. The lung was divided into two parts, and a part of the tissue piece was prepared according to a conventional method to observe a histopathological change. The other tissue piece was frozen in liquid nitrogen immediately after collection, and phosphatidylcholine was quantified according to a conventional method.
病理組織学検査では、アミオダロン投与群の全例でリン脂質症の特徴である泡沫肺胞マクロファージの集簇が認められた。一方、フェノキシプロピルアミン化合物(I)投与群及び溶媒対照群ではいずれの個体においてもリン脂質症の発現を疑わせる所見は観察されなかった。肺のリン脂質定量の結果を図1に示す。アミオダロン投与群及びフェノキシプロピルアミン化合物(I)投与群のホスファチジルコリン量は、いずれも溶媒対照群に比べて有意な増加が認められた。フェノキシプロピルアミン化合物(I)投与群の増加率は約1.5倍に対し、アミオダロン投与群は約3.5倍の増加率を示し、本実験条件においては、アミオダロン投与群の方がフェノキシプロピルアミン化合物(I)投与群よりリン脂質症の程度は重度であると考えられた。 Histopathological examination revealed foamy alveolar macrophage aggregation, a characteristic of phospholipidosis, in all patients in the amiodarone-administered group. On the other hand, in the phenoxypropylamine compound (I) -administered group and the solvent control group, there was no observation suspicious of phospholipidosis in any individual. The results of phospholipid quantification in the lung are shown in FIG. The amount of phosphatidylcholine in the amiodarone-administered group and the phenoxypropylamine compound (I) -administered group was significantly increased as compared with the solvent control group. The increase rate of the phenoxypropylamine compound (I) administration group was about 1.5 times, while the amiodarone administration group showed an increase rate of about 3.5 times. Under these experimental conditions, the amiodarone administration group was more phenoxypropylamine compound (I ) From the administration group, the degree of phospholipidosis was considered to be severe.
CE-TOFMSにより生体内物質の一斉分析を実施した後、メタボローム・ディファレンシャルディスプレイ法による各生体内物質量の群間比較をWilliamsの多重比較検定で行うとともに各個体毎に肺組織中のホスファチジルコリン量との間でピアソンとスピアマンの相関係数を求め、相関係数0.5以上又は-0.5以下のピークを抽出した。個々のピークについては精密質量及び電気移動度から候補物質の絞込み、質量分析装置(MS/MS)による物質の構造推定を行い、標準物質が入手可能な物質については比較し物質を同定した。その結果、表1に示すように、アミオダロン投与群とフェノキシプロピルアミン化合物(I)投与群においてともに変動する代謝物として、尿からはトリメチルアミン N−オキシド、ヒポタウリン、グアニジドアセテート、4-グアニジノブタノエート、4-ベータ-アセチルアミノエチルイミダゾール、L-カルニチン、N-アミジノ-L-アスパラギン酸、N-アセチル-L-ヒスチジン、1-リボシルイミダゾール-4-アセテート、フルクトシルリジン、L-グルタミン酸、4-ヒドロキシ-2-キノリンカルボン酸、フェニルアセチルグリシン、4-ヒドロキシフェニルアセチルグリシン、3-インドキシルスルフェート、及び5-2'-カルボキシエチル-4_6-ジヒドロキシピコリネートが見出され、血清からはトリメチルアミン N−オキシド、ベタイン、L-アスパラギン、L-オルニチン、L-リジン、L-メチオニン及びL-ヒスチジンが見出された。同定もしくは推定された物質は、ヒスチジンやトリプトファンなどのアミノ酸代謝経路、脂肪酸のβ酸化経路及び尿素回路などに由来する生体内代謝物質であった。この中のフェニルアセチルグリシンは薬物誘発性リン脂質症のバイオマーカーとしての有用性が報告されており(Biomarkers, 5, pp.410-423, 2000;Magn. Reson. Chem., 39, pp.559-565, 2000)、本発明の予測・診断方法が高い精度を有していることを示唆している。
Claims (12)
(1)被験物質をヒト以外の哺乳類動物に投与した後、該哺乳類動物から生体試料を採取する工程;及び
(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を被験物質を投与していない哺乳類動物から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該被験物質が脂質代謝異常症を誘発する可能性があると判定する工程
を含み、かつ該特定生体内物質がアミオダロン及び下記の式で表されるフェノキシプロピルアミン化合物(I):
(1) collecting a biological sample from a mammal after administering the test substance to a mammal other than a human; and
(2) Measure the specific biological substance contained in the biological sample, and the measurement result obtained is a measurement result of the specific biological substance contained in the biological sample collected from a mammal not administered the test substance And a step of determining that the test substance may induce dyslipidemia when a significant difference is found in these measurement results, and the specific biological substance is amiodarone and Phenoxypropylamine compound (I) represented by the formula:
(1)ヒトを含む哺乳類動物個体から生体試料を採取する工程;
(2)該生体試料中に含まれる特定生体内物質を測定し、得られた測定結果を健常者から採取した生体試料中に含まれる該特定生体内物質の測定結果と比較し、これらの測定結果に有意差が認められた場合には該個体は脂質代謝異常症の発症可能性を有すると判定する工程
を含み、かつ該特定生体内物質がアミオダロン及び請求項1に記載のフェノキシプロピルアミン化合物(I)のそれぞれの投与により有意に変動する1種又は2種以上の物質である方法。 A method for diagnosing dyslipidemia, comprising the following steps:
(1) a step of collecting a biological sample from a mammal individual including a human;
(2) Measure a specific biological substance contained in the biological sample, compare the obtained measurement results with the measurement result of the specific biological substance contained in a biological sample collected from a healthy person, and measure these The method includes a step of determining that the individual has a possibility of developing dyslipidemia when a significant difference is found in the results, and the specific in vivo substance is amiodarone and the phenoxypropylamine compound according to claim 1 The method which is 1 type, or 2 or more types of substance which changes significantly by each administration of (I).
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WO2005064344A1 (en) * | 2003-12-26 | 2005-07-14 | Takeda Pharmaceutical Company Limited | Method of predicting abnormality of lipid metabolism |
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JPH07196485A (en) * | 1993-12-28 | 1995-08-01 | Yakult Honsha Co Ltd | Lipid metabolism improver and food products improving lipid metabolism |
WO1998012170A1 (en) * | 1996-09-20 | 1998-03-26 | Yamanouchi Pharmaceutical Co., Ltd. | Novel tricyclic fused ring derivatives |
WO2005021779A1 (en) * | 2003-08-29 | 2005-03-10 | Perkinelmer Las, Inc. | Mass spectrometry methods for simultaneous detection of metabolic enzyme activity and metabolite levels |
WO2005064344A1 (en) * | 2003-12-26 | 2005-07-14 | Takeda Pharmaceutical Company Limited | Method of predicting abnormality of lipid metabolism |
Cited By (3)
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JP2013134129A (en) * | 2011-12-26 | 2013-07-08 | Lion Corp | Method for determining dyslipidemia |
CN110161256A (en) * | 2012-03-18 | 2019-08-23 | 株式会社资生堂 | Disease sample analytical equipment, analysis system and analysis method |
CN110161256B (en) * | 2012-03-18 | 2022-03-25 | 镜株式会社 | Disease sample analysis device, disease sample analysis system, and disease sample analysis method |
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