Determination of tolbutamide hydroxylation in rat liver microsomes by high-performance liquid chromatography: effect of psychoactive drugs on in vitro activity.
A simplified HPLC method for tolbutamide metabolism to hydroxytolbutamide has been used to screen sixty psychoactive drugs for their ability to inhibit rat liver microsomal tolbutamide hydroxylation. One-step extraction with diethyl ether was followed by reconstitution and isocratic HPLC analysis with a binary mobile phase (ammonium phosphate:methanol, 45:55, v/v). Nanogram amounts of hydroxytolbutamide formation were estimated with UV detection at 240 nm. Hydroxytolbutamide formation was linear with incubation times of 40-120 min, but specific activity increased with increases in microsomal protein (0.15-1.10 mg). A differential inhibitory response was demonstrated for tolbutamide and debrisoquine hydroxylation to 5 psychoactive drugs, suggesting that tolbutamide hydroxylation is not dependent on P4502D1. Sixty psychoactive drugs, or drug metabolites, (at 33 microM) were then co-incubated with tolbutamide (at 2.5 and 10.2 microM). Tolbutamide hydroxylation was refractory (< 25% inhibition) to twenty-four of the drugs and only mildly inhibited (25-50% inhibition) by twenty-eight. Two compounds, trans-3-methylfentanyl and flurazepam, produced > 50% inhibition that was independent of tolbutamide concentration. Five of the drugs (methadone, chlorpheniramine, meperidine, 6-monoacetylmorphine and methylphenidate), however, caused greater than 50% inhibition in a competitive manner which suggests these drugs may share an affinity for the substrate binding site for tolbutamide.
Ho
J W
JW
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84108.
Moody
D E
DE
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006900:Hydroxylation / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D011619:Psychotropic Drugs / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014044:Tolbutamide / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D000096926:Benzenesulfonamides
D011619:Psychotropic Drugs; D014044:Tolbutamide; C031014:hydroxymethyltolbutamide; D000096926:Benzenesulfonamides