ABSTRACT:The objective of the present study was to examine the accuracy of using unbound brain concentration determined by a brain homogenate method (C ub ), cerebral spinal fluid concentration (C CSF ), and unbound plasma concentration (C up ) as a surrogate for brain interstitial fluid concentration determined by brain microdialysis (C m ). Nine compounds-carbamazepine, citalopram, ganciclovir, metoclopramide, N-desmethylclozapine, quinidine, risperidone, 9-hydroxyrisperidone, and thiopental-were selected, and each was administered as an intravenous bolus (up to 5 mg/kg) followed by a constant intravenous infusion (1-9 mg/kg/h) for 6 h in rats. For eight of the nine compounds, the C ub s were within 3-fold of their C m ; thiopental had a C m 4-fold of its C ub . The C CSF s of eight of the nine compounds were within 3-fold of their corresponding C m ; 9-hydroxyrisperidone showed a C CSF 5-fold of its C m . The C up s of five of the nine compounds were within 3-fold of their C m ; four compounds (ganciclovir, metoclopramide, quinidine, and 9-hydroxyrisperidone) had C up s 6-to 14-fold of their C m . In conclusion, the C ub and C CSF were within 3-fold of the C m for the majority of the compounds tested. The C up s were within 3-fold of C m for lipophilic non-P-glycoprotein (؊P-gp) substrates and greater than 3-fold of C m for hydrophilic or P-gp substrates. The present study indicates that the brain homogenate and cerebral spinal fluid methods may be used as surrogate methods to predict brain interstitial fluid concentrations within 3-fold of error in drug discovery and development settings.For drugs with an intended action in the central nervous system it is assumed that unbound drug in brain interstitial fluid is in direct contact or in equilibrium with the site of action (de Lange and Danhof, 2002). Therefore, in preclinical and clinical pharmacokinetic/pharmacodynamic studies, it is critical to determine the concentration in the interstitial fluid for brain-targeted compounds. Unbound plasma concentration (C up ) has been used to represent the unbound concentration in tissue (Wilkinson, 2001). Because the brain is separated from the plasma by the blood-brain barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB), C up may not represent the interstitial fluid concentration (Davson and Segal, 1995;Hammarlund-Udenaes et al., 2008;Liu et al., 2008).Microdialysis has been considered as a standard approach to measure interstitial fluid concentration (C m ) (Joukhadar and Müller, 2005;Chaurasia et al., 2007). Although this technique has been developed for more than 2 decades, it is primarily used for determination of neurotransmitters and not drug concentrations in the brain. The main limitations of this technique include high resource requirements, low throughput, and special surgical skills to set up the experiment. In addition, many compounds in the discovery stage are often very lipophilic, and it is difficult to apply microdialysis technique to study these compounds because of high nonspecific bindi...

Our results demonstrate the selectivity of CPI and CPIII towards the OATP1B/MRP pathway, and the herein reported data further underline the potential of CPI and CPIII as selective and sensitive clinical biomarkers to quantify OATP1B-mediated DDIs.

Drug-induced mitochondrial dysfunction has been hypothesized to be an important determining factor in the onset of drug-induced liver injury. It is essential to develop robust screens with which to identify drug-induced mitochondrial toxicity and to dissect its role in hepatotoxicity. In this study we have characterised a mechanistically refined HepG2 model, using a panel of selected hepatotoxicants and non-hepatotoxicants. We have demonstrated that acute metabolic modification, via glucose-deprivation over a 4 h period immediately prior to compound addition, is sufficient to allow the identification of drugs which induce mitochondrial dysfunction, in the absence of cell death over a short exposure (2-8 h) using a plate-based screen to measure cellular ATP content and cytotoxicity. These effects were verified by measuring changes in cellular respiration, via oxygen consumption and extracellular acidification rates. Overall, these studies demonstrate the utility of HepG2 cells for the identification of mitochondrial toxins which act directly on the electron transport chain and that the dual assessment of ATP content alongside cytotoxicity provides an enhanced mechanistic understanding of the causes of toxicity.

With the rather easily-performed combinatory mapping approach, it was possible to provide quantitative information supporting the decision making in the drug discovery setting.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.