Promega (www.promega.com) developed a bioluminescent approach to in vitro ADME/Tox studies with its P450 Glo® assay, based on firefly luciferase. “We can use this in three ways,” according to James Cali, Ph.D., senior scientist. “We can measure levels of luciferase with a reagent that combines nonlimiting ATP and luciferin; limit ATP using a reagent with nonlimiting luciferase and luciferin; and limit luciferin with a reagent that combines nonlimiting APT and luciferase.” Consequently, the assay is used to screen P450 inhibitors, determine P450 activity or induction in cells, and measure P450 activity in enzyme assays.
The family of automated assays uses a type of luciferase that glows, with a half life of four to five hours, as opposed to technologies that use a wild-type luciferase that offers flash luminescence. P450-Glo is basically a two-step protocol, Dr. Cali said.
Luminescence has an advantage over fluorescence, Dr. Cali said, in that the former has low backgrounds and thus high sensitivity and wide dynamic range, and a water-soluble substrate. Fluorescent assays, in contrast, have high background noise because of their excitation range.
Tests showed that signal strength, using CYP2C9 and luciferin-H, had only marginal decay from zero to two hours when he left the lab for the night, and decayed gradually to 16 hours, after which the decay became dramatic. Luminescence was proportional to the concentration of luciferin. IC50 results show good correlation with the literature, he says.
Putting in vitro ADME/Tox research into practice at Amgen, Dr. Xu designed a program for in vitro ADME-screening assays for early drug discovery. “We have a conservative approach,” she said. The program includes a series of first-tier high-throughput assays, followed by more detailed second-tier assays.
The first tier of profiling assays is designed for lead selection and includes assays for microsomal metabolic stability, CYP3A4 and 2D6 inhibition, CYP3A4 TDI, parallel artificial membrane permeability assay (PAMPA), and ultrafiltration protein binding at a single concentration.
At this stage, Dr. Xu said, “Chemists love their compounds. So, if there is great potency, the chemists just don’t want to give up. They insist on seeing pharmacokinetic data.” The data generated from the first-tier assays will give chemists some ideas of what in vitro ADME properties of compounds to see; therefore, they can improve their SAR based on the information. Dr. Xu and her team developed time-dependent inhibition assays (TDI) in a high-throughput manner.
Second-tier, low-throughput pharmaceutical profiling assays offer more detail and include metabolic stability assays for microsomes and hepatocytes, a plasma protein-binding assay (using ultracentrifugation at 120,000 rpms), CYP3A4, 2D6, 1A2, 2C9, and 2C19 reversible and TDI inhibition assays in both human liver microsomal and hepatocyte preparations.
For the reactive drug-metabolism assay, she said, “we do protein covalent binding assay to address the bio-activation issue because we don’t want to see that compounds are metabolically reactive, which may be associated with their hepatoxicity. Covalent binding is done at a late stage,” she continued. “At that point, everything looks good. We do it for conservative reasons, so down the road if there is a problem, we have the data to support toxicity related to the bioactivation liability.”
The second-tier tests are run using triplicate wells with five points for time, concentration, and species. “We calculated half life, intrinsic clearance, IC50 for competitive CYP inhibition, free fractions, fold CYP induction,” and other parameters, she said.
Amgen also compares various species, evaluating, for example, rat liver microsome clearance with in vivo clearance for a rough idea of what to expect. Dr. Xu found that rat hepatocyte metabolic stability correlated well with rats’ in vivo data.