Fraction unbound () of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. and are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for and of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat Therefore, we recommend using rat as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of for liver tissues, hepatocytes, and other cell types. In addition, under hepatocyte stability incubation conditions should not be confused with , as one is a diluted and the other is an undiluted Cell density also plays a critical role in the accurate measurement of.

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ABSTRACT:Prediction of the metabolic sites for new compounds, synthesized or virtual, is important in the rational design of compounds with increased resistance to metabolism. The aim of the present investigation was to use rational design together with MetaSite, an in silico tool for predicting metabolic soft spots, to synthesize compounds that retain their pharmacological effects but are metabolically more stable in the presence of cytochrome P450 (P450) enzymes. The model compound for these studies was the phenethyl amide (1) derivative of the nonsteroidal anti-inflammatory drug (NSAID) indomethacin. Unlike the parent NSAID, 1 is a potent and selective cyclooxygenase-2 (COX-2) inhibitor and nonulcerogenic anti-inflammatory agent in the rat. This pharmacological benefit is offset by the finding that 1 is very unstable in rat and human microsomes because of extensive P4503 A4/2D6-mediated metabolism on the phenethyl group, experimental observations that were accurately predicted by MetaSite. The information was used to design analogs with polar (glycinyl) and/or electron-deficient (fluorophenyl, fluoropyridinyl) amide substituents to reduce metabolism in 1. MetaSite correctly predicted the metabolic shift from oxidation on the amide substituent to O-demethylation for these compounds, whereas rat and human microsomal stability studies and pharmacokinetic assessments in the rat confirmed that the design tactics for improving pharmacokinetic attributes of 1 had worked in our favor. In addition, the fluorophenyl and pyridinyl amide derivatives retained the potent and selective COX-2 inhibition demonstrated with 1. Overall, the predictions from MetaSite gave useful information leading to the design of new compounds with improved metabolic properties.In the early drug discovery phase, many compounds have high oxidative metabolic instability mediated by cytochrome P450 (P450) enzymes, which often results in poor oral pharmacokinetics in preclinical species. Whereas high-throughput in vitro microsomal assays that monitor metabolic stability provide a convenient means for "rankordering" large numbers of compounds, information pertaining to identification of "soft spots" for unstable compounds cannot be discerned from such analyses and requires separate metabolite identification studies. Unlike microsomal stability assays, metabolite identification experiments are low-throughput and cannot keep pace with high-speed chemistry efforts. In addition, there are scenarios in which metabolism studies will limit the labile site(s) to a certain region within the molecule. To guide chemists in the right direction, information on the exact metabolic site is often preferred. Therefore, in addition to existing in vitro assays, a value added proposition would be the use of in silico tools to precisely predict regiochemistry of metabolism for compounds (synthesized or only virtual), information that could be used for rational design of pharmacologically active compounds with improved metabolic properties.MetaSite is an in silico tool t...

As part of a strategy to deliver short-acting calcium-sensing receptor (CaSR) antagonists, the metabolically labile thiomethyl functionality was incorporated into the zwitterionic amino alcohol derivative 3 with the hope of increasing human clearance through oxidative metabolism, while delivering a pharmacologically inactive sulfoxide metabolite. The effort led to the identification of thioanisoles 22 and 23 as potent and orally active CaSR antagonists with a rapid onset of action and short pharmacokinetic half-lives, which led to a rapid and transient stimulation of parathyroid hormone in a dose-dependent fashion following oral administration to rats. On the basis of the balance between target pharmacology, safety, and human disposition profiles, 22 and 23 were advanced as clinical candidates for the treatment of osteoporosis.