Vol. 25, Issue 12, 1354-1358, 1997

Cytochrome P450 Isozymes Involved in Lisofylline Metabolism to Pentoxifylline in Human Liver Microsomes

Sun H. Lee¹ and John T. Slattery

Department of Pharmaceutics, University of Washington

We describe the kinetics of pentoxifylline formation from lisofylline in human liver microsomes using selective inhibitors of cytochrome P450 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 µM, V_max1 = 0.003 ± 0.001 nmol/min/mg protein, K_m2 = 158 ± 42.6 µM and V_max2 =0.928 ± 0.308 nmol/min/mg (N = 4). Pentoxifylline formation by the low K_m isoform (200 µM lisofylline) required NADPH, was not inhibited by any isozyme-specific P450 inhibitor, and was inhibited only 10% and 20%, respectively, by aminobenzotriazole and N-octamylamine. We concluded that the low K_m enzyme was not a cytochrome P450. At 5 µM of lisofylline the CYP1A2 inhibitor, furafylline, inhibited pentoxifylline formation by 58.8%, and the nonspecific CYP2E1 inhibitor, diethyldithiocarbamate, inhibited pentoxifylline formation by 21.7%. When preincubated with furafylline plus diethyldithiocarbamate, inhibition of pentoxifylline formation was increased 71.4%. Microsomal CYP1A2 activity correlated with pentoxifylline formation (r² = 0.870, p < 0.001). However, CYP2E1 activity did not correlate with pentoxifylline formation (r² = 0.143, p = 0.181). Baculovirus insect cell expressed human CYP1A2 formed pentoxifylline at 0.987 nmol/min/nmol cytochrome P450 at 5 µM lisofylline. cDNA expressed CYP2E1 did not catalyze formation of pentoxifylline. Diethyldithiocarbamate inhibited pentoxifylline formation by 85.7% in cDNA expressed CYP1A2. We conclude that CYP1A2 is the high affinity enzyme catalyzing pentoxifylline formation from lisofylline.