Microsomal Stability Assay provides an early knowledge regarding the metabolism of a new chemical entity. This is valuable in the drug development process as it provides essential information for the selection of leads from among a number of compounds having a similar therapeutic potential. Early in vitro metabolism studies can be used to rank ordering drug hits/leads, and provide an insight into possible interspecies differences in toxicity and therapeutic efficacy of the selected compounds.
The liver is the main site of drug metabolism in mammals, and more than 60% of currently marketed drugs are cleared from the human body via oxidation catalyzed by microsomal enzymes belonging to the cytochrome P450 (CYP) family. In addition, mammalian liver microsomes contain other important drug metabolizing enzymes including flavin monooxygenases (FMO), glucuronosyltransferases and esterases (Parkinson et al. “Biotransformation of xenobiotics in Casarett & Doull’s Toxicology: The Basic Science of Poisons”. Klaassen CD ed pp 185–367, McGraw-Hill, Inc., New York, 2013).
Our standard microsomal stability assay examines the time dependent decrease of a test compound in the incubation mixtures containing liver microsomes in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), the cofactor for CYP- and FMO-dependent oxidations. Liver microsomes from a variety of animal species can be used to predict interspecies differences in the rate of metabolic elimination of the studied compound. All test compounds are tested in triplicate. The metabolic competency of the test system is validated by the inclusion of a positive control compound with a known stability under the assay conditions. The final report includes graphs showing the remaining test compound (in percentage) over time, as well as disappearance half-life and/or intrinsic clearance data.
By supplementing the cofactor uridine diphosphoglucuronic acid (UDPGA), liver microsomes can also be used to screen compounds for glucuronidation, a major phase II pathway in the metabolism of several drugs (Williams et al. “Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios.” Drug Metab Dispos. 32:1201-8, 2004).
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The diagram shows the time-dependent depletion of two different test compounds by NADPH-supplemented human liver microsomes. The data are expressed as a percentage of the compound remaining at each time compared to time 0 min, and represent the mean ± SD (n = 3). Error bars smaller than the symbols are not visible.