Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases from XIAP inhibition is not clear. It is generally believed that binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse inhibition of the effector caspases. Surprisingly, we show here that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined the dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP. We also show that XIAP homotrimerizes via its C-terminal Ring domain, making its inhibitory activity toward caspase-3 more susceptible to Smac.

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca(2+)-mediated cell-cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca(2+)-dependent cell-cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

ABSTRACT:17␣-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K m (Michaelis constant) model (K m1 ‫؍‬ 220 nM; K m2 ‫؍‬ 15.5 M). Uptake was inhibited (ϳ90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na ؉ /taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K m values (87 nM and 141 M), whereas OATP2B1-and NTCP-mediated uptake into HEK-293 cells conformed to single K m kinetics (10.7 and 2.6 M, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression Pglycoprotein. Only vesicles that contained BCRP exhibited ATPdependent uptake of EE2-Sul (K m1 ‫؍‬ 2.9 and K m2 ‫؍‬ 307 M). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Co-administration of gemfibrozil significantly increases the exposure of repaglinide.• CYP3A4 and CYP2C8 are important in the metabolism of repaglinide.• OATP1B1 polymorphism is an independent predictor of repaglinide pharmacokinetics.• Gemfibrozil and its O-glucuronide are CYP2C8 and OATP1B1 inhibitors. WHAT THIS STUDY ADDS• Acyl glucuronidation is a major metabolic pathway of repaglinide in vitro.• Gemfibrozil and its glucuronide inhibit the glucuronidation of repaglinide. • UGT1A1 is a major enzyme responsible for the glucuronidation of repaglinide. AIMTo further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. METHODSRepaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. RESULTSThe results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [ 3 H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (~78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (~80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. CONCLUSIONSInhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.

ABSTRACT:17␣-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells A synthetic and potent estrogen receptor agonist, 17␣-ethinylestradiol (EE2) is a major estrogen component of oral contraceptive formulations (Zhang et al., 2007). Although EE2 is well absorbed, oral bioavailability is variable (e.g., 20 -65%) because of extensive first-pass metabolism in both the intestine and liver. Such metabolism involves sulfotransferase-catalyzed 3-O-sulfation, UDP-glucuronosyltransferase-catalyzed 3-O-glucuronidation, and cytochrome P450-mediated 2-hydroxylation. Of the three pathways, sulfation dominates and the resulting metabolite (EE2-Sul) circulates at concentrations that are at least one order of magnitude greater than the parent EE2 (Back et al., 1980). The results of various pharmacokinetic and radiolabeled studies have demonstrated that EE2 undergoes enterohepatic recirculation, with the various metabolites recovered in bile (ϳ40% of dose) and urine (ϳ30% of the dose) (Maggs et al., 1983).The presence of both EE2-Sul and EE2-Glu in the urine is thought to reflect active transport in kidneys (Maggs et al., 1983).In an accompanying manuscript (Han et al., 2010), EE2-Sul was shown to be a substrate of numerous liver-expressed transporters (OATP1B1, OATP2B1, NTCP, and BCRP). However, information related to the transporters involved in the renal excretion of EE2-Sul is lacking. In the human kidney, a variety of solute carrier (SLC) and ATP-binding cassette (ABC) transporters are expressed in proximal tubule cells and play a major role in the uptake and secretion of organic compounds (Lee and Kim, 2004;Robertson and Rankin, 2006). Namely, organic anion transporter (OAT)1 and OAT3 are SLCs expressed on the basolateral membrane, whereas OAT4, multidrug resistance-associated protein (MRP)2, MRP4, and BCRP are apical transporters known to actively transport organic anions. However, additional transporters, such as organic cation transporter (OCT)2 (e.g., prostaglandins) and MATE1 (e.g., estrone-3-sulfate, acyclovir, and ganciclovir), are also able to transport organic anions (Tanihara et al., 2007). Therefore, the purpose of the present study was to characterize the drug transporters that are responsible for renal Article, publication date, and citation information can be found at

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Despite a recent expansion in the recognition of the potential utility of coproporphyrin (CP) as an endogenous biomarker of organic anion-transporting polypeptide (OATP) 1B activity, there have been few detailed studies of CP's pharmacokinetic behavior and an overall poor understanding of its pharmacokinetic fate from tissues and excretion. Here, we describe the pharmacokinetics of octadeuterium-labeled coproporphyrin I (CPI-d8) in cynomolgus monkeys following oral and intravenous administration. CPI-d8 has a half-life and bioavailability of 7.6 hours and 3.2%, respectively. Cynomolgus monkeys received oral cyclosporin A (CsA) at 4, 20, and 100 mg/kg which yielded maximum blood concentrations (C max ) and area under the plasma concentration-time curve (AUC) values of 0.