名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水谷武史氏の博士論文として提出された

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京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18550号 / 薬博第812号 / 新制||薬||238(附属図書館) / 31450 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 佐治 英郎, 教授 橋田 充, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Numerous natural products are widely used as first-line/alternative therapeutics and dietary supplements in both western and eastern society. However, the safety and efficacy profiles for herbal products are still limited. Organic anion transporters (OATs; SLC22 family) are expressed in many barrier organs and mediate in vivo body disposition of a broad array of endogenous substances and clinically important drugs. As some dietary flavonoids and phenolic acids were previously demonstrated to interact with OATs, it is necessary to explore the potential interaction of such components found in natural products in order to avoid potential OAT-mediated drug-drug interactions (DDIs). The inhibitory effects of 23 natural products were assessed on the function of human (h) OATs, hOAT1 (SLC22A6), hOAT3 (SLC22A7), and hOAT4 (SLC22A11) and/or the murine (m) orthologs mOat1 and mOat3. For compounds exhibiting marked inhibition at initial screening, dose-response curves (IC50 values) and DDI indices were determined. At the initial screening concentrations, 14, 19, and 2 test compounds exhibited significant inhibition on hOAT1, hOAT3, and hOAT4, respectively. Additionally, all test Danshen (a Chinese herbal medicine) hydrophilic components significantly reduced mOat1- and mOat3-mediated substrate uptake at 1 mM. For selected compounds, the IC50 and Ki values were estimated to be in the micromolar or even nanomolar range. Considering the clinical plasma concentration and unbound fraction in plasma, DDI indices for gallic acid, gentisic acid, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid B, and tanshinol indicated DDIs may occur in vivo in situations of co-administration of these compounds and clinical therapeutics known to be OAT substrates. Finally, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify gallic acid and gentisic acid in cell lysates in order to measure cellular uptake of these compounds in mOat1- or mOat3-expressing cells. Significant cellular uptake of gallic acid was observed in mOat1-expressing cells, compared with background control cells. The absorptive uptake was completely blocked by probenecid (known OAT inhibitor) at 1 mM. These results indicate that gallic acid is a substrate for mOat1 and suggest that human OAT1 might be involved in the active renal secretion of gallic acid.

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Organische Anionen werden aus dem Blut in die proximalen Tubuluszellen durch Organische-Anionen-Transporter 1 und 3 (OAT1 und OAT3) aufgenommen. Die Aufnahme erfolgt im Austausch gegen Dicarboxylate. In dieser Dissertation wurde die Affinität von Dicarboxylaten gegenüber humanen OAT1 und OAT3 untersucht mit dem Ziel mehr Informationen über die Struktur der Transporter zu erhalten. Es sollten Unterschiede zwischen dem OAT1 und OAT3 ermittelt werden, besonders bezüglich deren Substratspezifität. Alle Transporter wurden stabil in HEK293-Zellen exprimiert. Extrazellulär wurden Dicarboxylate als Inhibitoren gegen die 3H-p-Aminohippurat-Aufnahme (OAT1) oder 3H-Östronsulfat-Aufnahme (OAT3) zugefügt. OAT1 zeigt die höchste Affinität gegenüber Glutarat (IC50 3,3 µM), α-Ketoglutarat (IC50 4,7 µM) und Adipat (IC50 6,2 µM), gefolgt von Pimelat (IC50 18,6 µM) und Suberat (IC50 19,3 µM). Die Affinität von OAT1 gegenüber Succinat und Fumarat war gering. Der OAT3 zeigte dieselbe Dicarboxylat-Selektivität mit etwa 13-mal höheren IC50-Werten verglichen mit dem OAT1. Die Daten charakterisieren α-Ketoglutarat als hochaffines Substrat für den OAT1 und den OAT3. Die Ergebnisse deuten auf eine ähnliche Molekülstruktur der Bindungsstellen von OAT1 und OAT3 hin.