| Description |
The predominant pathway for amphetamine metabolism in the rat is 4-hydroxylation. Limited in vitro data on the inhibition of debrisoquine hydroxylation by amphetamine suggest cytochrome P-450 (P450) 2D1 has a role in this reaction. In a preliminary experiment in which Lewis rats were pretreated with the P450 2D inhibitor, quinidine, reduced excretion of 4'-hydroxyamphetamine in the urine was found. As amphetamine and its metabolites are primarily excreted in the urine, measurement of urinary 4'-hydroxyamphetamine and amphetamine is a reasonable approach to study amphetamine's in vivo metabolism. An HPLC-UV method was developed in order to quantify 4'-hydroxyamphetamine and amphetamine in rat urine samples. In the initial study using P450 2D1-deficient Dark Agouti (DA) rats, a decrease in the excretion of 4'-hydroxyamphetamine (expressed as percentage of administrated dose) was found in the DA male (7.1 ± 2.3) and DA female (10.0 ± 2.8), as compared to the Sprague Dawley (SD) male (67.9 ± 6.3) and SD female (53.0 ± 8.3) rats. P450 2D's involvement in amphetamine 4'-hydroxylation was also established by enzyme induction and inhibition studies in SD rats. In vivo induction of P450s by 3-methylcholanthrene, phenobarbital, isosafrole, ethanol, pregnenolone-16/alpha-carbonitrile, clofibrate, and di-(2-ethylhexyl) phthalate produced minimal, if any, increase in the urinary excretion of 4'-hydroxyamphetamine. However, in vivo inhibition of P450s resulted in reduced excretion of 4'-hydroxyamphetamine (expressed as percentage of controls) in SD male rats pretreated with: 1-aminobenzotriazole, 7.2 ± 2.2; quinidine, 17.3 ± 1.6; quinine, 30.1 ± 5.9; and primaquine, 17.6 ± 7.4%. Neither diallyl sulfide, a P450 2E inhibitor, nor troleandomycin, a P450 3A inhibitor, reduced 4'-hydroxyamphetamine excretion (81.9 ± 13.4 and 76.4 ± 16.2% of control, respectively). The reduction in the urinary excretion of 4'-hydroxyamphetamine in the rats correlated positively p = 0.80) with decreases in a microsomal P450 2D1-dependent monooxygenase activity in rats which received the same inhibition treatments. This correlation, together with the findings of decreased 4-hydroxylation of amphetamine in female DA rats and in rats where P450 2D1-dependent monooxygenase activities have been inhibited, has now provided 2 independent lines of evidence to link P450 2D to amphetamine 4-hydroxylation. |
