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Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.
Mol Pharmacol. 1989 May; 35(5):736-43.MP

Abstract

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)

Authors+Show Affiliations

Department of Medicine, Westmead Hospital, NSW, Australia.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

2725477

Citation

Reidy, G F., et al. "Inhibition of Oxidative Drug Metabolism By Orphenadrine: in Vitro and in Vivo Evidence for Isozyme-specific Complexation of Cytochrome P-450 and Inhibition Kinetics." Molecular Pharmacology, vol. 35, no. 5, 1989, pp. 736-43.
Reidy GF, Mehta I, Murray M. Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics. Mol Pharmacol. 1989;35(5):736-43.
Reidy, G. F., Mehta, I., & Murray, M. (1989). Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics. Molecular Pharmacology, 35(5), 736-43.
Reidy GF, Mehta I, Murray M. Inhibition of Oxidative Drug Metabolism By Orphenadrine: in Vitro and in Vivo Evidence for Isozyme-specific Complexation of Cytochrome P-450 and Inhibition Kinetics. Mol Pharmacol. 1989;35(5):736-43. PubMed PMID: 2725477.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics. AU - Reidy,G F, AU - Mehta,I, AU - Murray,M, PY - 1989/5/1/pubmed PY - 1989/5/1/medline PY - 1989/5/1/entrez SP - 736 EP - 43 JF - Molecular pharmacology JO - Mol Pharmacol VL - 35 IS - 5 N2 - 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) SN - 0026-895X UR - https://www.unboundmedicine.com/medline/citation/2725477/Inhibition_of_oxidative_drug_metabolism_by_orphenadrine:_in_vitro_and_in_vivo_evidence_for_isozyme_specific_complexation_of_cytochrome_P_450_and_inhibition_kinetics_ DB - PRIME DP - Unbound Medicine ER -