Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.cholesterol ͉ intestinal brush border membranes

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Diclofenac is eliminated predominantly (ϳ50%) as its 4Ј-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (ϳ50%) and dogs (Ͼ80 -90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4Ј-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL int ϭ V max /K m ) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4Ј-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ϳ75% of the estimated hepatic CL int of diclofenac is apparently inconsistent with the 4Ј-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4Ј-hydroxy diclofenac AG, was observed. The estimated hepatic CL int of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4Ј-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4Ј-hydroxylation of diclofenac. These findings may have general implications in the use of total (free ϩ conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4Ј-hydroxy diclofenac urinary excretion.In vitro drug metabolism systems, especially liver microsomes, offer tremendous promise as a tool in drug discovery and development to make human pharmacokinetic projections for potential drug candidates (Obach et al., 1997;Obach, 1999). These systems allow for lead selection based on metabolism data in human tissue that seem more relevant to the human in vivo situation than the in vivo animal models. The popularity of liver microsomes, in comparison with other in vitro systems such as hepatocytes and liver slices, stems from the ease of their preparation, use, and long-term storage and viability. However, the use of liver microsomes for extrapolation of in vivo clearance suffers from a number of limitations such as nonspecific binding of compounds to microsomal components, reduced rates of metabolism because of potential product inhibition kinetics, and the difficulties in examining conjugative metabolism (e.g., glucuronidation) in microsomal incubations. These limitations lead to frequent underprediction of in vivo clearance from microsomal metabolism data (Houston and Carlile, 1997;Obach, 1999). There have been only a few attempts to extrapolate in vivo clearance from microsomal metabolism data for compound...

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (K m ϭ 162 M), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively. Sitagliptin did not inhibit hOAT1-mediated cidofovir uptake, but it showed weak inhibition of hOAT3-mediated cimetidine uptake (IC 50 ϭ 160 M). hOAT3-mediated sitagliptin uptake was inhibited by probenecid, ibuprofen, furosemide, fenofibric acid, quinapril, indapamide, and cimetidine with IC 50 values of 5.6, 3.7, 1.7, 2.2, 6.2, 11, and 79 M, respectively. Sitagliptin did not inhibit Pgp-mediated transport of digoxin, verapamil, ritonavir, quinidine, and vinblastine. Cyclosporine A significantly inhibited Pgp-mediated transport of sitagliptin (IC 50 ϭ 1 M). Our data indicate that sitagliptin is unlikely to be a perpetrator of drug-drug interactions with Pgp, hOAT1, or hOAT3 substrates at clinically relevant concentrations. Renal secretion of sitagliptin could be inhibited if coadministered with OAT3 inhibitors such as probenecid. However, the magnitude of interactions should be low, and the effects may not be clinically meaningful, due to the high safety margin of sitagliptin.Sitagliptin [also known as MK-0431 ( Fig. 1)], is a selective, reversible inhibitor of dipeptidyl-peptidase 4 recently approved by the Food and Drug Administration for the treatment of type 2 diabetes (Deacon, 2005;Kim et al., 2005). Dipeptidyl-peptidase 4 inhibitors have a glucose-lowering effect by inhibiting the inactivation of incretin peptides, including glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are released upon nutrient ingestion, stimulate meal-induced insulin secretion, and contribute to glucose homeostasis (Kieffer and Habener, 1999). The pharmacokinetics, metabolism, and excretion of sitagliptin have been investigated in humans and animals Herman et al., 2005Herman et al., , 2006Beconi et al., 2007). After a single oral dose of sitagliptin (1.5-600 mg) in healthy male volunteers, sitagliptin was well absorbed with an apparent terminal half-life ranging from 8 to 14 h. Sitagliptin was primarily renally eliminated as unchanged drug (Herman et al., 2005), with metabolism playing only a minor role (Vincent et al., 2007). Renal clearance of sitagliptin averaged approximately 388 ml/min in humans (Herman et al., 2005). Given that sitagliptin is approximately Article, publication date, and citation information can be found at

In the current study, the identification of the rat and human UDP-glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of diclofenac was determined. Recombinant human UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate of 166-pmol/min/mg protein, while UGT1A6 and 2B15 catalyzed the glucuronidation of diclofenac at low rates (<20-pmol/min/mg protein). Conversely, human UGT2B7 displayed a high rate of diclofenac glucuronide formation (>500 pmol/min/mg protein). Recombinant rat UGT2B1 catalyzed the glucuronidation of diclofenac at a rate of 250-pmol/min/mg protein. Rat UGT2B1 and human UGT2B7 displayed a similar, low apparent Km value of <15 microM for both UGT isoforms and high Vmax values 0.3 and 2.8 nmol/min/mg, respectively. Using diclofenac as a substrate, enzyme kinetics in rat and human liver microsomes showed that the enzyme(s) involved in diclofenac glucuronidation had a low apparent Km value of <20 microM and a high Vmax value of 0.9 and 4.3 nmol/min/mg protein, respectively. Morphine is a known substrate for rat UGT2B1 and human UGT2B7 and both total morphine glucuronidation (3-O- and 6-O-glucuronides) and diclofenac glucuronidation reactions showed a strong correlation with one another in human liver microsome samples. In addition, diclofenac inhibited the glucuronidation of morphine in human liver microsomes. These data suggested that rat UGT2B1 and human UGT2B7 were the major UGT isoforms involved in the glucuronidation of diclofenac.