Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-11-22
2002-11-12
Rotman, Alan L. (Department: 1625)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C514S001000, C549S385000
Reexamination Certificate
active
06479231
ABSTRACT:
INTRODUCTION
Organic anion transporter (OAT) is an essential component of the renal tubular secretory pathway of small negatively charged molecules (1). OAT, which is located in the basolateral (anti-luminal) membrane of renal proximal convoluted tubules (2), mediates the active uptake of anionic substrates from the systemic blood circulation into the proximal tubular epithelium and functions in concert with luminal (apical) efflux carrier(s) or channel(s) (3). Biochemical studies using the renal cortical slices, intact tubules, or basolateral membrane vesicles isolated from the renal cortex have provided initial information on the function and substrate specificity of the OAT system (4, 5, 6). Recent cloning of OAT from several species including human (hOAT1) accelerated further characterization of this physiologically important membrane transport protein (7, 8, 9). Upon heterologous functional expression in
Xenopus laevis
oocytes, OAT variants from different species have shown the ability to transport p-aminohippuric acid (PAH), a prototypical organic anion substrate, in exchange for intracellular dicarboxylate (e.g. (&agr;-ketoglutarate, glutarate) (9, 10). Subsequent studies revealed a broad substrate specificity for OAT, which includes endogenous metabolites (urate), signal molecules (cyclic nucleotides, prostaglandins), toxins (ochratoxin A), as well as xenobiotics (2,4-dichlorophenoxyacetic acid) (11, 12, 13). In addition, recent studies indicated that the OAT system is involved in the tubular secretion of many important therapeutics such as &bgr;-lactam antibiotics (14), nonsteroidal anti-inflamatory drugs (15), antiviral nucleotide analogs (9), and non-peptidic angiotensin inhibitors (16). It has been postulated that in some cases, compounds transported by the OAT system may induce pharmacokinetic drug-drug interactions or cause nephrotoxicity (17, 18). In the past, the evaluation of potential OAT substrates and/or inhibitors has been restricted to inhibition assays using the isolated renal tubules or basolateral membrane vesicles and radioactive OAT substrates (most frequently PAH) (4, 19, 20). Although more recent approaches have relied on the transport assays in Xenopus oocytes transiently expressing rat OAT or hOAT1 (9, 14, 15), there was no straightforward, efficient, and reproducible assay available for OAT transport activity. In this study, we describe the development of a fluorescent cell-based assay, which allows for an efficient and reliable evaluation of the interaction between small molecules and hOAT1.
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Covington Raymond
Gilead Sciences, Inc.
Hensley Max D.
Rotman Alan L.
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