Abstract
levo-α-Acetylmethadol (LAAM) is a long-acting opioid agonist prodrug used for preventing opiate withdrawal. LAAM undergoes bioactivation via sequential N-demethylation to nor-LAAM and dinor-LAAM, which are more potent and longer-acting than LAAM. This study examined LAAM and nor-LAAM metabolism using human liver microsomes, cDNA-expressed CYP, CYP isoform-selective chemical inhibitors, and monoclonal antibody to determine kinetic parameters for predicting in vivo drug interactions, involvement of constitutive CYP isoforms, and mechanistic aspects of sequentialN-demethylation. N-Demethylation of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model,Km values were 19 and 600 μM for nor-LAAM and 4 and 450 μM for dinor-LAAM formation, respectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selective inhibitors troleandomycin, erythromycin, ketoconazole, and midazolam. Of the cDNA-expressed isoforms examined, CYP2B6 and 3A4 had the highest activity toward LAAM and nor-LAAM at both low (2 μM) and high (250 μM) substrate concentrations. N-Demethylation of LAAM and nor-LAAM by expressed CYP3A4 was unusual, with hyperbolic velocity curves and Eadie-Hofstee plots and without evidence of positive cooperativity. Using a two-site model, Kmvalues were 6 and 0.2 μM, 1250 and 530 μM, respectively. Monoclonal antibody against CYP2B6 inhibited CYP2B6-catalyzed but not microsomal LAAM or nor-LAAM metabolism, whereas troleandomycin inhibited metabolism in all microsomes studied. The ratio [dinor-LAAM/(nor-LAAM plus dinor-LAAM)] with microsomes and CYP3A4 decreased with increasing LAAM concentration, suggesting most dinor-LAAM is formed from released nor-LAAM that subsequently reassociates with CYP3A4. Based on these results, we conclude that LAAM and nor-LAAM are predominantly metabolized by CYP3A4 in human liver microsomes, and CYP3A4 exhibits unusual multisite kinetics.
Footnotes
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Send reprint requests to: Dr. Evan D. Kharasch, Department of Anesthesiology, Box 356540, University of Washington, Seattle, WA 98195-6540. E-mail: kharasch{at}u.washington.edu
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This study was supported by a Merit Review Award from the Veterans Affairs Medical Research Bureau and National Institutes of Health Grant K24DA00417.
- Abbreviations:
- LAAM
- levo-α-acetylmethadol
- CYP
- cytochrome P450
- cDNA
- complementary deoxyribonucleic acid
- DDC
- diethyldithiocarbamate
- TAO
- troleandomycin
- HPLC
- high performance liquid chromatography
- HLM
- human liver microsome
- AIC
- Akaike's information criterion
- E
- enzyme
- S
- substrate
- CL
- clearance
- Received October 13, 2000.
- Accepted January 2, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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