RELATIVE QUANTITIES OF CATALYTICALLY ACTIVE CYP 2C9 AND 2C19 IN HUMANLIVER-MICROSOMES - APPLICATION OF THE RELATIVE ACTIVITY FACTOR APPROACH

The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activit y factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4' -hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, r espectively. The kinetics of these reactions were studied in human liv er microsomes, in microsomes from human lymphoblastoid cells, and in i nsect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of V-max (reaction velocity at saturating substrate concentrati ons) in human liver microsomes of the isoform-specific index reaction divided by the V-max of the reaction catalyzed by the cDNA expressed i soform. RAFs were also determined for SUPERMIX, a commercially availab le mixture of cDNA expressed human drug metabolizing CYPs formulated t o achieve a balance of enzyme activities similar to that found in huma n liver microsomes. Lymphoblast RAF(2C9) in human liver microsomes ran ged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF (2C9) in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg pro tein (mean value: 49), while a value of 201 pmol CYP/mg protein was ob tained for SUPERMIX. Both lymphoblast and insect cell RAF(2C19) in hum an liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean valu es: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system us ed had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or oi CYP2C19 as a S-mephenytoin hydroxylase. Howev er insect cell expressed CYP2C19 (which includes oxidoreductase) had s ubstantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphobla st-determined RAF(2C9) to RAF(2C19) in human livers was 3.0 (range 1.6 -17.9; n = 10), while this ratio for SUPERMIX was 8.6. The ratio of me an insect cell-determined RAF(2C9) to RAF(2C19) in human livers was 1. 7 (range 0.04-16.2; n 10), while this ratio for SUPERMIX was 7.0. Neit her ratio is in agreement with the 20:1 ratio of immuno-quantified lev els of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels hi gher than those in human liver microsomes.