Assessing granisetron as a specific CYP1A1 substrate in primary human hepatocytes: A comprehensive evaluation for...

Cytochrome P450 (CYP) enzymes are essential in metabolic pathways and drug-drug interactions, making their investigation highly relevant during drug development. These studies are typically conducted in liver systems, where primary human hepatocytes (PHH) are considered the gold standard. Current regulatory guidelines focus primarily on CYP1A2 for drug interaction studies, neglecting CYP1A1, which is highly inducible and particularly relevant in populations exposed to pollutants like polycyclic aromatic hydrocarbons, commonly found in tobacco smoke. This study applied granisetron as a specific substrate for CYP1A1 in drug interaction research, establishing assay parameters for its use in PHH, with the aim of providing clear recommendations for measuring CYP1A1 enzyme activity in industry applications. It was confirmed that 7-OH-granisetron is representative of CYP1A1 enzyme activity in PHH. Furthermore, enzyme kinetics indicated biphasic Michaelis-Menten kinetics for granisetron-7-hydroxylation, with V_max = 0.3 pmol/(min× million cells) and K_m = 5.5 μM. Optimal incubation conditions for measurements under V_max conditions were determined to be 30-40 μM granisetron, with a minimum incubation time of 90 minutes. These conditions were validated in a CYP1A induction experiment, confirming the effectiveness of the parameters. CYP1A1 exhibited high inducibility, which is relevant in clinical settings for patients exposed to CYP1A1 inducers. In conclusion, this study developed an assay to investigate CYP1A1 enzyme activity in PHH during drug interaction studies such as enzyme induction or enzyme inhibition. This work highlights granisetron-7-hydroxylation as a marker reaction to uncover specific CYP1A1 reactions in vitro and enhance the understanding of metabolic variations in systems involving CYP1A1 induction. SIGNIFICANCE STATEMENT: This study applied granisetron as a specific substrate for CYP1A1 in drug interaction studies and determined the assay parameters for the use of granisetron in primary human hepatocytes. This work contributes to the field by yielding clear recommendations for the application of granisetron in primary human hepatocytes for enzymatic activity measurements under V_max conditions, providing guidance for industry applications on measuring specific CYP1A1 enzyme activity in induction studies.