Seite - (000094) - in Biomedical Chemistry: Current Trends and Developments

the lower chamber and the total drug concentration prior to ultrafiltration are determined and then used to determine the extent of protein binding. Commercially available 96- well ultrafiltration apparatus. HPLC is frequently employed. Equilibrium Dialysis Equilibrium dialysis is based on the principle of the diffusion of solutes along a concentration gradient across a semi-permeable membrane. The protein solution containing drug is placed in one chamber while buffer is placed in the opposing chamber. The unbound drug passes through the membrane and at equilibrium, the unbound drug will be at equal concentrations on both sides of the membrane while the bound drug will remain in the protein chamber. Equilibration times are long (typically 12– 48 h). HPLC is used to quantify the free and total drug concentrations. Frequently used in drug discovery in order to determine the percentage of drug binding to plasma proteins maintaining a true equilibrium during the whole experiment. Metabolic in vitro models Expressed enzymes Expressed enzymes are concentrated enzymes with high activity toward a specific substrate, they are often used as ‘‘bioreactors’’ to generate metabolites. Using the generated metabolites that are collected as chromatographic fractions, it is possible to test for their pharmacological activity. Conduct reaction-phenotyping for drug candidates and identify non-CYP microsomal or cytosolic enzymes often involved in drug metabolism. Identify the relative contribution of each CYP isoform to the total hepatic clearance. Identify the involvement of CYPs in metabolic pathways.