Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.
The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition kinetics to noncompetitive. Treatment of rats with orphenadrine (75 mg/kg/day intraperitoneally for 3 days) was associated with a 2-fold induction of total hepatic P-450, a 5- and 2.4-fold induction of androstenedione 16 beta- and 6 beta-hydroxylase activity, respectively, and formation of an orphenadrine-P-450 MI complex. Western blots of orphenadrine-induced microsomes revealed a 20-fold increase in P-450 PB-B/D-immunoreactive protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Reidy
G F
GF
Department of Medicine, Westmead Hospital, NSW, Australia.
Mehta
I
I
Murray
M
M
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005292:Ferricyanides / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009966:Orphenadrine / Q000494:pharmacology*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000032:analysis
D065607:Cytochrome P-450 Enzyme Inhibitors; D005292:Ferricyanides; D007527:Isoenzymes; D000735:Androstenedione; D003577:Cytochrome P-450 Enzyme System; D009966:Orphenadrine; D013250:Steroid Hydroxylases; C028033:potassium ferricyanide