Evaluation of P-450 in metabolism of EFC and delavirdine. Possible reaction metabolite of delavirdine.
P-450 isoforms catalyzes numerous Phase I drug metabolizing reactions. Previously, a P-450 dependent reaction demonstrated 7-ethoxy-4trifluoromethylcoumarin was metabolized to a fluorescent product 7-hydroxy-4trifluoromethylcoumarin (Buters 1993). The present investigation assessed the ability of the fluorescence assay to monitor cytochrome P-450 activity. A kinetic study on pooled rat liver microsomes and human liver microsomes demonstrated a single phase indicating a simple enzymatic process. Km and Vmax, determined using non-linear regression analysis were 266.2 ±8.0 and 10.8 ±1.1 for pooled rat liver microsomes and were 204.4 ±4.0 and 0.35 ±0.004 for human liver microsomes, respectively. Delavirdine, a specific non-nucleoside reverse transcriptase inhibitor of HIV-1, is known to be metabolized by cytochromes 3A4 and 2D6 and to inactivate cytochrome 3A4. Inactivation caused by delavirdine was not detected spectrophotometrically in rat and human liver microsomes. However, delavirdine was found to irreversibly bind to pooled rat and human liver microsomal protein. Initial studies using recombinant cytochrome isoforms 3A4 and 2D6 established evidence of metabolism by means of radio-assay and HPLC-radiochemical analysis. HPLC-radiochemical analysis and mass spectrometry, on samples containing nucleophilic trapping agents, suggested generation of a reactive metabolite in delavirdine metabolism.