2.2. Liver S9 Metabolic Stability Assay

Both human (gender pooled, 10 individuals) and rat (Sprague Dawley; male; pooled) liver S9 fractions were purchased from BD Biosciences (San Jose, CA., USA). It has been demonstrated by Otwell et al., [10] that several Phase I and Phase II enzymes retain their activity, when stored at -70^oC for up to 10 years and up to 10 freeze/thaw cycles. Furthermore, our internal data from several different S9 lots has also demonstrated comparable Phase I and Phase II activities (as evaluated using 7-EC as a control) for the lifetime of the lot. The incubation conditions were developed by using four commercial compounds, 7-EC, diclofenac, 4-nitrocatecal, and phenolphthalein with known metabolic profiles (both Phase I and II metabolism). The rat and human S9 protein concentration and the cofactor concentrations were optimized to match the results of hepatocyte stability, quantitatively. The Phase I and Phase II metabolites; 7-hydroxycoumarin (7-HC), 7-HC sulfate, 7-HC glucuronide, 4-hydroxydiclofenac (4-HD), 4-HD glucuronide, diclofenac acyl glucuronide, 4-nitrocatechol sulfate (4-NC sulfate), and phenolphthalein glucuronide were also monitored. The identity of each of the metabolites was assessed by comparison of their retention time and mass spectra with those of authentic standards. Each new lot of S9 goes through this optimization process before being used for high throughput compound screening in the discovery DMPK flowchart. A cocktail of four activating cofactors was used in order to stimulate Phase I (NADPH), and Phase II (UDPGA, PAPS, GSH) metabolism. The final concentrations of NADPH, UDPGA, and GSH were 1, 0.5 and 2.5 mM, respectively while that of PAPS was 0.05 mg/mL.

Tris buffer was prepared as a 200 mM solution containing 2mM magnesium chloride (included MgCl₂ as a source for Mg⁺² ions to stimulate CYP activity) in deionized water and adjusted with 1 M NaOH to pH 7.4. Stock reference solutions (7-EC as the positive control) and test compounds were prepared at 5 mM concentration in DMSO, and then diluted to 0.3 mM with ACN prior to use. NADPH, UDPGA, and GSH solutions were prepared at 40, 20 and 2 mM, respectively while PAPS was prepared at 2 mg/mL, all in tris buffer prior to mixing together in a 1:1:1:1 ratio for use. S9’s were preincubated with test compound for 5 minutes at 37°C in tris buffer, pH 7.4, and then the reactions were initiated by adding the cofactor mixture. At two time points, zero and sixty minutes, aliquots of the sample mixture were removed and quenched by addition of two volumes of ice cold 50:50 ACN:MeOH. The plate of quenched samples was then centrifuged at 4000g for 10 minutes to sediment the precipitated proteins before injection onto LC-MS/MS for analysis of parent compound remaining. Percent of the parent compound remaining is calculated by comparing peak areas.