Renal organic anion transporter-mediated drug-drug interaction between gemcabene and quinapril.

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a K(m) value of 13.4 microM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC(50) values of 35 and 48 microM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC(50) values of 197 and 133 microM, respectively. High plasma concentrations of gemcabene (>100 microM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

Evaluation of in vitro models for screening alkaline phosphatase-mediated bioconversion of phosphate ester prodrugs.

Generating a phosphate prodrug is one of the common approaches for circumventing poor solubility issues of a parent drug. Alkaline phosphatase (ALP) level was determined in rat intestine mucosa scraps, human colon carcinoma (Caco-2) cells, and Madin-Darby canine kidney (MDCK) cells to characterize in vitro models for ALP-mediated phosphate prodrug conversion. In addition, fosphenytoin and fosfluconazole were used as probe prodrugs to evaluate the models. The highest amount of ALP was detected in rat intestinal mucosa scraps, whereas ALP in 5-day cultured MDCK cells was minimal. As anticipated, ALP levels correlated with the parent drug conversion; the shortest cleavage half-life (t(1/2)) was observed in rat mucosa scraps; and MDCK cells showed the slowest conversion. Furthermore, the polarized conversion for the prodrugs was observed in Caco-2 monolayer cells, suggesting the polarized localization of alkaline in differentiated Caco-2 cells. The rate of ALP-mediated conversion was prodrug concentration-dependent with Michaelis-Menten constants of 1160 and 351 microM for fosphenytoin and fosfluconazole, respectively, determined in Caco-2 cells. The results revealed that whereas the intestinal mucosa scraps reserved the highest ALP activities and were shown as a promising in vitro tool for screening the bioconversion of phosphate prodrug, Caco-2 monolayers could provide the predictive information of bioconversion and further offer the capability in characterizing the permeability of prodrug and parent drug.

Torcetrapib induces aldosterone and cortisol production by an intracellular calcium-mediated mechanism independently of cholesteryl ester transfer protein inhibition.

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib's effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human.

The large interspecies differences of hepatobiliary transport present a challenge for the allometric prediction of human biliary excretion for drug candidates primarily cleared via hepatobiliary secretion. In the present study, we determined the metabolic stabilities of common fluorescent substrates of hepatobiliary efflux transporters and developed a rapid efflux assay to determine the functional activities of MRP/Mrp, BCRP/Bcrp and P-gp in hepatocytes of four species. The specificities of transporter-mediated dye efflux were confirmed by selective transporter inhibitors. Among tested species, transporter-specific dye efflux kinetics was consistent between freshly isolated and cryopreserved hepatocytes. Hepatocyte elimination half-lives of MRP/Mrp substrates GS-MF and calcein were observed in the rank order of human>monkey>dog>rat. The fourfold higher MRP/Mrp substrate efflux rate of rat hepatocytes compared to human is likely due to the species-specific functional differences of MRP2/Mrp2 expressed on the canalicular membrane. We also observed efficient BCRP-mediated pheophorbide A (PhA) efflux by human and dog hepatocytes, while PhA extrusion in monkey and rat hepatocytes appeared limited. P-gp function measured by DiOC2(3) efflux was minimal in hepatocytes of all origins and no significant species differences were detected. Our results demonstrated marked differences in hepatocyte MRP/Mrp and BCRP/Bcrp activities across species, indicating that they may contribute to the species differences of in vivo hepatobiliary excretion. These results also suggest the potential utility of primary hepatocytes, either fresh or cryopreserved, as an in vitro model to predict interspecies differences in the biliary transport of MRP/Mrp and BCRP/Bcrp substrates.

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

Species-specific, P450- and sulfotransferase-mediated novel ring contraction of a naphthyridine-N-oxide compound in cynomolgus monkey.

BMS-A78277 (1) is a 5,10-dihydrobenzo[beta][1,8]naphthyridine-N-oxide compound that resides in a class of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs), displaying improved activity against clinically relevant mutants of HIV-1 and possessing pharmacokinetic profiles amenable to once-daily dosing. In the course of investigating the nonclinical metabolism of 1, a circulating metabolite specific to the cynomolgus monkey was identified and subsequently characterized as the carboxyindole metabolite 2. The present investigation describes the biotransformation of this NNRTI in cynomolgus monkey, one which results in a net ring contraction of 1. The use of mass spectrometry and high field NMR analysis aided in the structural characterization of metabolite 2, the source of which originated from the urine and bile of cynomolgus monkeys receiving oral doses of 1. Preparation of a synthetic standard of 2 not only provided ultimate structural confirmation but also afforded ample material for biological testing. The metabolism of 1 was investigated in monkey hepatocytes and hepatic subcellular fractions. While microsomes were incapable of generating metabolite 2, incubation of 1 in monkey S9 fractions as well as hepatocytes resulted in measurable levels of the carboxyindole metabolite. Consequently, incubation of 1 in monkey hepatocytes, which were suspended in media containing (18)O-labeled water, resulted in the incorporation of (18)O into the carboxyindole metabolite, 2. These data implicate a mechanism involving the bioactivation of 1 to an electrophilic intermediate that upon hydrolysis undergoes a concerted ring contraction, resulting in the formation of 2. Previously confined to discussions regarding the metabolism of natural products and select aliphatic heterocycles, the present investigation extends the discussion of metabolism-mediated ring contraction to aromatics such as the present naphthyridine compound, 1.