Category: Preclinical Development
Purpose: Entacapone is used in Parkinson’s disease (PD) as an adjunct to levodopa and carbidopa. Entacapone is a selective and reversible inhibitor of catechol-o-methyl transferase (COMT) in peripheral tissues, an enzyme that is responsible for the metabolism of levodopa1. The maximum recommended daily dose is 1600 mg. The objective of the study is to evaluate the direct and time dependent inhibition (TDI) of P450 enzymes by Entacapone.
Methods: Inhibition potential towards major CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was determined using marker reactions in human liver microsomes. For measuring direct inhibition potential, entacapone was incubated with HLM and probe substrates in the presence of NADPH followed by termination of reactions with acetonitrile. Time dependent inhibition potential was evaluated in a single point assay at 10 µM by incubating entacapone with HLM and NADPH in the presence and absence of pre-incubation for 30min followed by dilution in to an activity assay containing probe substrate at 10x the Km. TDI was also evaluated by an IC50 shift assay where entacapone is incubated with HLM and NADPH with and without pre-incubation followed by dilution in to activity assay containing probe substrate at Km to measure the residual activity.
Results: Entacapone inhibited CYP2C9 mediated diclofenac hydroxylation and CYP2C8 mediated amodiaquine deethylation with IC50 values of 1.11 µM and 19.0 µM respectively. The IC50 values of entacapone values against CYP1A2, 2A6, 2B6, 2C19, 2E1, and 3A4 are greater than 100 µM, the highest tested concentration. Entacapone inhibited CYP2C8 mediated amodiaquine deethylation by 62 ± 3 % (Mean ± SEM of three experiments with triplicates run in each experiment) in human liver microsomes in pre-incubation mode. No significant TDI was observed with other enzymes. The IC50 values of entacapone in the absence and presence of NADPH with pre-incubation are 17.5 and 1.9 µM respectively with a ~ 9 fold shift in inhibition potential. The IC50 obtained in the absence of pre-incubation (19.0 µM) is comparable with that obtained in the absence pre-incubation without NADPH. The inactivation kinetics of entacapone against CYP2C8 is characterized by KI and Kinact values of 33.2 µM and 0.088 min-1 respectively.
Conclusion: The results indicate that entacapone is a time dependent inhibitor of CYP2C8. Entacapone can serve as a positive control in CYP2C8 TDI assay.