Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and relationship to other cytochrome P-450 dependent xenobiotic oxidations.
Tolbutamide hydroxylation has been investigated in human liver microsomes. Anti-human liver NADPH-cytochrome P-450 reductase IgG inhibited hydroxytolbutamide formation and this metabolite was not formed when NADPH-generating system was omitted from microsomal incubations. Tolbutamide hydroxylation followed Michaelis-Menten kinetics, consistent with the involvement of a single form of cytochrome P-450 in this reaction. Mean apparent Km and Vmax values for hydroxytolbutamide formation were 120 +/- 41 microM and 0.273 +/- 0.066 nmol min-1 mg-1, respectively. A range of clinically used drugs and xenobiotics used as probes for cytochrome P-450 activity in laboratory animals was screened for inhibitory effects on hydroxytolbutamide formation. Caffeine, paraxanthine, theophylline, theobromine, debrisoquine, erythromycin, phenacetin, propranolol, aminopyrine, benzo(a)pyrene and 7-ethoxycoumarin were all found not to inhibit tolbutamide hydroxylation. In contrast, sulphaphenazole, phenylbutazone, nifedipine, verapamil, cimetidine, aniline, dextropropoxyphene and mephenytoin were competitive inhibitors of tolbutamide hydroxylation. The respective apparent Ki values for these compounds were 0.12 microM, 11 microM, 15 microM, 118 microM, 140 microM, 182 microM, 225 microM and 375 microM. Sulphinpyrazone inhibited tolbutamide hydroxylation with atypical kinetics. The in vitro data is in good agreement with in vivo drug interactions with tolbutamide. The data also confirm that tolbutamide hydroxylation is not associated with the cytochromes P-450 responsible for methylxanthine metabolism or with the form responsible for the polymorphic oxidation of debrisoquine.
Miners
J O
JO
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, Adelaide, South Australia.
Smith
K J
KJ
Robson
R A
RA
McManus
M E
ME
Veronese
M E
ME
Birkett
D J
DJ
D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D008617:Mephenytoin / Q000494:pharmacology; D008862:Microsomes, Liver / Q000378:metabolism*; D013806:Theophylline / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism*
D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D013806:Theophylline; D008617:Mephenytoin