CYTOCHROME-P450 ISOZYMES INVOLVED IN LISOFYLLINE METABOLISM TO PENTOXIFYLLINE IN HUMAN LIVER-MICROSOMES

We describe the kinetics of pentoxifylline formation from lisofylline in human liver microsomes using selective inhibitors of cytochrome P45 0 isozymes, correlation studies with specific isozyme activities, and cDNA-expressed human CYP1A2 and 2E1. A biphasic model fitted the data best for the formation of pentoxifylline, K-m1, = 0.282 +/- 0.135 mu M , V-max1 = 0.003 +/- 0.001 nmol/min/mg protein, K-m2 = 158 +/- 42.6 mu M and V-max2 = 0.928 +/- 0.308 nmol/min/mg (N = 4), Pentoxifylline fo rmation by the low K-m isoform (200 mu M lisofylline) required NADPH, was not inhibited by any isozyme-specific P450 inhibitor, and was inhi bited only 10% and 20%, respectively, by aminobenzotriazole and N-octa mylamine. We concluded that the low K-m enzyme was not a cytochrome P4 50. At 5 mu M of lisofylline the GYP1A2 inhibitor, furafylline, inhibi ted pentoxifylline formation by 58.8%, and the nonspecific CYP2E1 inhi bitor, diethyldithiocarbamate, inhibited pentoxifylline formation by 2 1.7%, When preincubated with furafylline plus diethyldithiocarbamate, inhibition of pentoxifylline formation was increased 71.4%. Microsomal CYP1A2 activity correlated with pentoxifylline formation (r(2) = 0.87 0, p < 0.001). However, CYP2E1 activity did not correlate with pentoxi fylline formation (r(2) = 0.143, p = 0.181). Baculovirus insect cell e xpressed human CYP1A2 formed pentoxifylline at 0.987 nmol/min/nmol cyt ochrome P450 at 5 mu M lisofylline, cDNA expressed CYP2E1 did not cata lyze formation of pentoxifylline. Diethyldithiocarbamate inhibited pen toxifylline formation by 85.7% in cDNA expressed CYP1A2, We conclude t hat CYP1A2 is the high affinity enzyme catalyzing pentoxifylline forma tion from lisofylline.