A sensitive method for determination of cytochrome P4502D6 activity in vitro using bupranolol as substrate

Previous studies have shown that bupranolol, a beta-adrenoceptor blocker, is a substrate of cytochrome P4502D6 (CYP2D6). A sensitive in vitro assay was developed to quantify the formation of hydroxybupranolol using HPLC. A TLC method, using radiolabeled bupranolol, was also developed to test the reproducibility of the two methods. Both of them gave virtually identical results; however, the HPLC method was sensitive to 20 pmol and the TLC with radiolabeled substrate to <1 pmol of hydroxybupranolol. The KM value for bupranolol was lower than that reported for any other substrate of CYP2D6. The KM value in microsomes of a typical human liver (L-1) was 0.272 +/- 0.02 (SE) mu M and the Vmax was 360 +/- 10 (SE) pmol/mg/min (0.83 +/- 0.02 pmol/pmol cytochrome P450/min). The KM value for the CYP2D6 expressed in yeast was 0.076 +/- 0.003 (SE) mu M, and the Vmax was 43 +/- 1 (SE) pmol/mg/min (0.64 +/- 0.01 pmol/pmol cytochrome P450/min). Quinidine competitively inhibited the formation of hydroxybupranolol, with Ki values of 5 nM in expressed CYP2D6 and 14.05 nM in human liver (L-1).