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Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum Toxicokinetics and Read-Across to provide an overview on research activities to develop in vitro toxicokinetics methods and physiologically-based kinetic (PBK) models and to find synergies to enhance use of toxicokinetic data to strengthen read-across. Currently, lacking toxicokinetic data often prevent the application of read-across. Preferably, toxicokinetic data should be generated using in vitro and in silico tools and anchored towards human relevance. In certain sectors, PBK modelling is being used for risk assessment, but less so in others. Specific activities were identified to facilitate the use of in vitro and in silico toxicokinetic data to support read-across: The collation of available tools indicating the parameters and applicability domains covered; endpoint-specific guidance on toxicokinetics parameters required for read-across; case studies exemplifying how toxicokinetic data help support read-across. Activities to enhance the scientific robustness of read-across include the further user-friendly combination of read-across tools and formal guidance by the authorities specifying the minimum information requirements to justify read-across for a given toxicity endpoint. The EPAA was invited to continue dissemination activities and to explore possibilities to collate a contemporaneous list of open toxicokinetics tools that assist risk assessment.

01 september 2012, Contains fulltext : 108286.pdf (publisher's version ) (Closed access), Na(+)-dependent taurocholate cotransporting polypeptide (NTCP, SLC10A1) is the main transporter facilitating the hepatic uptake of bile acids from the circulation. Consequently, the interaction of xenobiotics, including therapeutic drugs, with the bile acid binding pocket of NTCP could lead to impairment of hepatic bile acid uptake. We pursued a 3D-pharmacophore approach to model the NTCP substrate and inhibitor specificity and investigated whether it is possible to identify compounds with intrinsic NTCP inhibitory properties. Based on known endogenous NTCP substrates, a 3D-pharmacophore model was built, which was subsequently used to screen two virtual libraries together containing the structures of 10 million compounds. Studies with Chinese hamster ovary cells overexpressing human NTCP, human hepatocytes, ex vivo perfused rat livers, and bile duct-cannulated rats were conducted to validate the activity of the virtual screening hits. Modeling yielded a 3D-pharmacophore, consisting of two hydrogen bond acceptors and three hydrophobic features. Six out of 10 structurally diverse compounds selected in the first virtual screening procedure significantly inhibited taurocholate uptake in the NTCP overexpressing cells. For the most potent inhibitor identified, an anthraquinone derivative, this finding was confirmed in human hepatocytes and perfused rat livers. Subsequent structure and activity relationship studies with analogs of this derivative indicated that an appropriate distance between hydrogen bond acceptor features and presence of one or two negative charges appear critical for a successful NTCP interaction. In conclusion, pharmacophore modeling was successfully used to identify compounds that inhibit NTCP. Our approach represents an important first step toward the in silico flagging of potential cholestasis-inducing molecules.

Contains fulltext : 91944.pdf (publisher's version ) (Closed access)