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 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, Km1 = 0.282 +/- 0.135 microM, Vmax1 = 0.003 +/- 0.001 nmol/min/mg protein, Km2 = 158 +/- 42.6 microM and Vmax2 =0.928 +/- 0.308 nmol/min/mg (N = 4). Pentoxifylline formation by the low Km isoform (200 microM 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 Km enzyme was not a cytochrome P450. At 5 microM 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 microM 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.
Lee
S H
SH
Department of Pharmaceutics, University of Washington, Seattle, WA 98195-7610, USA.
Slattery
J T
JT
D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010431:Pentoxifylline / Q000031:analogs & derivatives* / Q000378:metabolism*; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D019388:Cytochrome P-450 CYP1A2; C025189:lisofylline; D010431:Pentoxifylline