Whether amphetamine acts principally at the plasma membrane or at synaptic vesicles is controversial. We find that d-amphetamine injection into the Planorbis giant dopamine neuron causes robust dopamine release, demonstrating that specific amphetamine uptake is not required. Arguing for action at vesicles, whole-cell capillary electrophoresis of single Planorbis dopamine neurons shows that amphetamine reduces vesicular dopamine, while amphetamine reduces quantal dopamine release from PC12 cells by > 50% per vesicle. Intracellular injection of dopamine into the Planorbis dopamine neuron produces rapid nomifensine-sensitive release, showing that an increased substrate concentration gradient is sufficient to induce release. These experiments indicate that amphetamine acts at the vesicular level where it redistributes dopamine to the cytosol, promoting reverse transport, and dopamine release.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Sixty-four compounds with diverse structures were used in evaluation of intrinsic clearance by various hepatocyte preparations from rats, dogs, monkeys, and humans. Intrinsic clearance (CL int ) was calculated from the ratio of the initial amount of the test compound minus the amount remaining after 2 h of incubation and the corresponding area under the concentration versus time curve. The predictive potential of this in vitro model was tested by comparing the intrinsic clearance with in vivo clearance using linear regression analysis. In addition, the intrinsic clearance values obtained from three different types of hepatocytes (cryopreserved, fresh, and sandwich-cultured) from the same species were compared to determine the influence of preservation and culture conditions. It seems that intrinsic clearance determined from human cryopreserved hepatocyte (R 2 ‫؍‬ 0. In the early phase of drug discovery, animal pharmacokinetic data are used for advancing drug candidates to the clinical phase of drug development. According to a study by Prentis et al. (1988), approximately 40% of drug candidates failed because of poor human pharmacokinetic characteristics. For this reason, numerous attempts have been performed in recent years to find a reliable way of predicting in vivo hepatic clearance in humans. For example, Lavé et al. (1997) demonstrated a correlation between intrinsic clearance in human hepatocytes and hepatic extraction ratio in humans, and used the correlation to classify compounds into low, intermediate, or high hepatic extraction ratio in humans. Obach (1997a) used hepatic microsomal intrinsic clearance to predict human in vivo clearance and Houston and Carlile (1997) compared the use of microsomes, hepatocytes, and liver slices for prediction of hepatic clearance in rats. They showed that both microsomes and hepatocytes might be useful for obtaining the rank order of clearances. Schneider et al. (1999) used multivariate statistical models and artificial neural networks to predict the hepatic drug clearance in humans from in vitro hepatocyte data. These in vitro models yielded a wide range of in vivo-in vitro correlation indexes that, in general, suggest the potential for prediction of in vivo human clearance using in vitro clearance data.In addition to developing various in vitro physiological models for the evaluation of in vivo-in vitro correlations, considerable effort has been made toward methods for calculation of intrinsic clearance. In some studies, intrinsic clearance was obtained by determining the enzyme kinetic parameters (V max and K m ). The ratio of these two parameters was then equated with intrinsic clearance. In the second method, referred to as the "in vitro t 1/2 method", intrinsic clearance (CL int ) was calculated by measuring the first order rate constant for consumption of the substrate at low concentration. In a third method, intrinsic clearance was calculated from the ratio of the initial amount of the t...

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

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