Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 164-188).

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Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl− and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8−/− cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl− current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa. VRAC activity is fully reconstituted in LRRC8−/− human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.

Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for treatment of acute ischemia/reperfusion injury, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can "rescue" salvageable brain tissue and/or protect BBB integrity during cerebral hypoxia and subsequent reoxygenation stress (H/R). One approach that may enable neural tissue rescue following H/R is CNS delivery of drugs with brain protective effects such as HMG-CoA reductase inhibitors (i.e., statins). Our present in vivo data demonstrates that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain following H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter Oatp1a4 at the BBB under H/R conditions. In rat brain microvessels H/R (6% O₂, 60 min followed by 21% O₂, 10 min) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate, fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of TGF-β/ALK5 signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

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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

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21783号 / 工博第4600号 / 新制||工||1717(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 安部 武志, 教授 作花 哲夫, 教授 阿部 竜 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. </p><p>The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut^® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut^® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil.</p><p>Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.</p>

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