(T1130-10-69) Identification of Drug Metabolizing Enzymes that Catalyze Formation of a Novel Oxidative Metabolite from 7-ethoxycoumarin

Purpose: In the development of new drugs, in vitro metabolic study of drug candidates should be conducted before starting the first clinical study. Drug candidates are metabolized to their metabolites by phase I and II reactions in many cases. Cryopreserved hepatocytes catalyze both phase I and II reactions, and are frequently utilized for assessing metabolism of drug candidates. In in vitro metabolic study of drug candidates, hepatocytes used should retain drug-metabolizing enzyme activities of phase I and II reactions. 7-Ethoxycoumarin (EC) is the most common substrate used for checking enzyme activities of phase I and II reactions. As reported, EC is metabolized to 7-hydroxycoumarin (HC) by phase I reaction and formed HC is metabolized to its glucuronosyl- (HCG) and sulfo- (HCS) conjugates by phase II reaction, and these substrate and metabolites are determined using their synthetic standards commercially available. We identified a novel oxidative metabolite, 4-ethoxy-2-hydroxyphenylacetic acid (EHP-acetic acid), as the oxidative metabolite in ¹⁴C-EC metabolism by human hepatocytes and liver microsomes. Furthermore, using human cytochrome P450 (CYP) expression system and human liver microsomes with CYP inhibitors, we reported that EHP-acetic acid formation was catalyzed by CYP1A2 (main isomer), and CYP2C19 (minor isomer). Metabolic pathway of EC to EHP-acetic acid is proposed to be metabolized via epoxide and 4-ethoxy-2-hydroxyphenylacetaldehyde (EHP-aldehyde). In addition, 4-ethoxy-2-hydroxyphenylethanol (EHP-alcohol) is thought to exist as a metabolite of EHP-aldehyde. The objective of this study is to estimate drug-metabolizing enzymes that catalyze EHP-acetic acid formation using human liver 9,000×g supernatants (S9) with semicarbazide (aldehyde trapping agent), cyanamide (inhibitor of aldehyde dehydrogenase), and pyrazole (inhibitor of alcohol dehydrogenase).

Methods: EC (100 mmol/L) was incubated with human S9 with 10000 μmol/L semicarbazide, 1000 μmol/L cyanamide, and 1000 μmol/L pyrazole in the presence of NADPH-generating system for 60 minutes at 37°C. The produced amount of EHP-acetic acid, EHP-aldehyde, and EHP-alcohol was determined by liquid chromatography / tandem mass spectrometry.

Results: The metabolism of EC to EHP-acetic acid and EHP-ethanol in human liver S9 was inhibited by semicarbazide and the produced amount of EHP-acetaldehyde was increased (Table 1). The metabolism of EC to EHP-acetic acid was inhibited by cyanamide and the produced amount of EHP-acetaldehyde and EHP-ethanol did not change. The metabolism of EC to EHP-ethanol was inhibited by pyrazole and the produced amount of EHP-acetaldehyde and EHP-acetic acid did not change.

Conclusion: Based on the results from this study and our previous report, EHP-acetic acid was formed mainly by CYP1A2 and partly by CYP2C19 in human and EHP-aldehyde was present as a metabolic intermediate in the metabolic reaction of EHP-acetic acid from EC (Figure 1). Aldehyde dehydrogenase contributed to the metabolic reaction of EHP-aldehyde to EHP-acetic acid in human hepatocytes.

Acknowledgements: We have no financial relationship to disclose for our presentation contents.

Figure 1. Proposed metabolic pathway of 7-ethoxycoumarin by human hepatocytes

Table 1. Effect of modifiers on metabolism of 7-ethoxycoumarin by human liver S9