S9 Stability Assay

S9 fraction Stability Assay is very well suited to obtain an early knowledge about the metabolism of a new chemical entity. This is valuable in the drug development process by providing 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 the therapeutic efficacy of the selected compounds.

The so called S9 fraction of liver homogenate contains the vast majority of both membrane-bound and soluble phase 1 (i.e. oxidative, reductive and hydrolytic) and phase 2 (i.e. conjugative) enzymes involved in drug metabolism. Thus, the S9 stability assay, compared to our microsomal stability assay, more broadly assesses the overall hepatic metabolism of the test compound.

Our standard S9 stability assay examines the disappearance over time of a test compound in incubations mixtures containing the liver S9 fraction, both in the presence and absence of the cofactors needed for CYP-mediated drug oxidations (i.e., nicotinamide adenine dinucleotide phosphate; NADPH) and glucuronidations (i.e., uridine diphosphoglucuronic acid; UDPGA). S9 fractions from a variety of animal species can be used to predict interspecies differences in the rate of metabolic elimination of the studied compound.

To download the protocol example, please click here.

The diagram shows the time-dependent depletion of an alcoholic group-containing test compound by NADH*-supplemented human, rat and mouse liver S9 fraction. The data are expressed as percentage of compound remaining at each time compared with time 0 min, and represent the mean ± SD (n=3). Error bars smaller than the symbols are not visible.
*NADH is a cofactor for both alcohol dehydrogenase- and aldehyde dehydrogenase-dependent oxidations.