A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat.

The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A -demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A -demethylation yielded and values of 39.7 pmol/min per milligram protein and 43.2 M for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 M for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A -demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A -demethylase activity of HLMs was significantly correlated with lauric acid -hydroxylase activity, a marker of CYP4A11 activity ( = 0.904, < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC = 1.82 M) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC = 0.0137-0.0182 M); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC = 5.70-17.7 M). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC = 0.913 M) and HRMs (IC = 0.659 M). These results indicated that luciferin-4A -demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.