Disappearance of a parent compound or the appearance of metabolites is measured following incubation with hepatocytes, liver microsomes or plasma, enabling calculations of half-life and intrinsic clearance. Biotransformation of test article and metabolite profiles can then be elucidated (see Metabolite ID studies).
Regulatory considerations for metabolic stability studies
These studies are used to measure compound half-life, to calculate intrinsic clearance and to predict human pharmacokinetic profiles in anticipation of first-in-human trials.
Method for Metabolic Stability
- Test System: Pooled cryopreserved hepatocytes, hepatic microsomes or plasma
- Test Species: Mouse, rat, dog, rabbit, minipig, monkey and human; additional species upon request
- Test Article Concentrations: Two concentrations tested per species (1 and 10 µM) in triplicates
Metabolic Stability in Hepatocytes
The test article is incubated at two concentrations with approximately 1 x 106 hepatocytes/mL suspended in William’s Medium E for up to 5 time points and stopped by addition of organic solvent.
Control incubations are performed in the incubation medium only to determine the stability of the test article under incubation conditions. The metabolic integrity of each hepatocyte lot is assessed with 7-ethoxycoumarin or other suitable marker.
Metabolic Stability in Microsomes
The test article is incubated at two concentrations with microsomes (0.5 mg/ml protein) in the presence of NADPH for up to 5 time points and stopped by addition of an organic solvent. Control incubations are performed in the absence of NADPH to determine the stability of the test article under incubation conditions.
The metabolic integrity of each microsome lot is assessed for Phase 1 enzymes using 7-ethoxycoumarin or other suitable marker.
Metabolic Stability in Plasma
The test article is incubated at two concentrations in plasma for up to 5 time points and stopped by addition of an organic solvent. Positive control incubations include procaine (hydrolyzed by carboxylesterase) or other suitable metabolic marker.
Incubation samples are analyzed for the remaining test article using a liquid chromatography-mass spectrometry (LC-MS) analytical method. The percentage of remaining test article in samples is then determined as compared to the initial concentration. Upon approval, selected samples may be further analyzed to characterize or identify metabolites by LC-MS.
These assays provide drug stability information (calculated at the half-life and intrinsic clearance) utilizing cellular or subcellular fractions. Further analysis of incubations for metabolite profiling and identification can elucidate biotransformation pathways and allow for a better comparison between preclinical test species and human metabolites.