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T. Kraemer, M. Bossmann, F.T. Peters, L.D. Paul, D. Springer, R.F. Staack and H.H. Maurer
Department of Experimental and Clinical Toxicology, Institute of Pharmacology and Toxicology, University of Saarland, Homburg, GERMANY
Aims: There is still no general consensus on which cytochrome P450 (CYP) isoenzymes are involved in the metabolism of amphetamine precursor drugs. Previous Studies on Wistar and Dark Agouti rats indicated that the polymorphically expressed human isoenzyme CYP2D6 might play a major role in metabolism of such drugs. The aim of our present study was to confirm this assumption by identifying the human hepatic CYP isoenzymes involved in N-dealkylation as well as in hydroxylation of the amphetamine precursors clobenzorex and fenproporex.
Methods: Activity screenings with nine individual cDNA expressed CYPs for general involvement were performed. Kinetic profiles were established using recombinant baculovirus infected insect cell microsomes and pooled human liver microsomes as enzyme sources. Experiments with chemical inhibitors completed the studies. Metabolites were quantified by NICI-GC-MS (HP5-MS column) after aqueous derivatization of the amine group with S-(-)-heptafluorobtuyrylprolyl chloride, liquid-liquid extraction and subsequent ethylation or heptafluorobutyrylation of the hydroxy groups.
Results: Besides CYP3A4, the most abundant human CYP isoform, CYP2B6 was involved in the N-dealkylation of clobenzorex to amphetamine. N-dealkylation of fenproporex was catalyzed by CYP3A4, CYP1A2, CYP2B6 and by CYP2D6. Hydroxylation to different hydroxy metabolites of clobenzorex was catalyzed by CYP1A2, CYP2B6 and also by CYP2C19. Hydroxylation to hydroxy fenproporex was catalyzed by CYP1A2, CYP2C19, CYP3A4 and also by CYP2D6. The kinetic constants, Km and Vmax, were determined for both enzyme sources.
Conclusions: As CYP3A4 is responsible for N dealkylation of both precursors, interactions with inhibitors or inducers of this isoenzyme are possible. Since CYP3A4 is the most abundant CYP isoenzyme in human liver, a major influence of polymorphisms and/or induction of other isoforms involved seems not likely. Concerning hydroxylation, different pharmacokinetic behaviours might be possible in vivo, because CYP2C19 and CYP2B6 are expressed polymorphically in humans and CYP1A2 is known to be inducible e.g. by smoking. However, additional involvement of CYP3A4 in fenproporex hydroxylation reduces the probability of different pharmacokinetic behaviour for this precursor caused e.g. by CYP2D6 polymorphism. The results of the study raise the question, why the Dark Agouti rat model overestimated the role of polymorphic CYP2D isoenzymes in amphetamine precursor metabolism.