Assessment Under Initial Rate Conditions

Assessment Under Initial Rate Conditions

Published:  23 October 2014

Assessment Under Initial Rate Conditions of the Selectivity and Time Course of Cytochrome P450 Inactivation in Pooled Human Liver Microsomes and Hepatocytes: Optimization of Inhibitor Conditions Used for Reaction Phenotyping Studies

The identification of the metabolic pathways (reaction phenotyping) for new drug candidates is an essential component of drug development, as these studies increase the understanding of how a drug is cleared and the potential for the drug to be subject to drug-drug interactions with co-administrated medications. One approach used to identify which cytochrome P450 (CYP) enzymes are responsible for the metabolism of a drug is the use of specific chemical inhibitors in test systems such as pooled human liver microsomes (HLM) or pooled cryopreserved human hepatocytes (CHH). The design of these studies is critical because factors such as metabolic depletion of the inhibitor, protein binding, and insufficient enzyme inactivation can produce convoluted and misleading results. In the present study, we examined the selectivity and duration of CYP inactivation, with a range of protein concentrations and incubation times, for a variety of commonly used CYP inhibitors all under initial rate probe substrate conditions. Briefly, NADPH-fortified pooled HLM (n = 200) at 0.1, 0.5 and 1 mg/mL or pooled CHH (n = 100) at 1 million cells/mL, were pre-incubated for 30 min at 37°C with various CYP inhibitors, namely furafylline (10 μM), phencyclidine (up to 30 μM), gemfibrozil glucuronide (100 μM), tienilic acid (20 μM), esomeprazole (10 μM), quinidine (5 μM), paroxetine (1 or 5 μM), ketoconazole (1 or 4 μM), CYP3cide (2.5 μM), mibefradil (1 μM), and troleandomycin (50 μM). Following the pre-incubation step, marker substrate (» Km) incubations were performed for up to 5 min (10 min for CHH) at three post pre-incubation time points (0, 30, 120 min; simulating the time course of an unknown drug) to determine CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (amodiaquine), CYP2C9 (diclofenac), CYP2C19 (S‑mephenytoin), CYP2D6 (dextromethorphan), CYP2E1 (chlorzoxazone) and CYP3A4/5 (midazolam) residual activities. Metabolite formation was determined by LC-MS/MS analysis. Results indicated that, in both HLM and CHH, inhibition of specific CYP enzymes can be accomplished over the duration of 120 minutes after pre-incubation with an inhibitor.