SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists
SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists
SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists
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B4 <br />
REASONS FOR OVERESTIMATION OF THE ROLE OF CYP2D6 IN HUMAN METABOLISM<br />
OF AMPHETAMINE PRECURSOR DRUGS USING THE DARK AGOUTI RAT MODEL<br />
Thomas Kraemer', Denis Theobald and Hans H. Maurer: University Hospital, Homburg, Germany<br />
Background: Female Dark Agouti rats (fDA) have been proposed as a model <strong>of</strong> the human CYP2D6 poor<br />
metabolizer phenotype (PM) allowing a preliminary screening for CYP2D6 substrates. This rat model has<br />
successfully been used by the author's working group to investigate the role <strong>of</strong> CYP2D6 in human<br />
metabolism <strong>of</strong> the new designer drug TFMPP (1-(3-trifluoromethylphenyl)piperazine). However, the<br />
crucial role <strong>of</strong> CYP2D6 in N-dealkylation <strong>of</strong> amphetamine precursors (e.g. fenproporex) to amphetamine<br />
predicted by this rat model could not be confirmed using recombinant human CYPs and human liver<br />
microsomes (HLM). Four different CYP is<strong>of</strong>orms, namely CYPIA2, CYP2B6, CYP2D6 and CYP3A4,<br />
were involved in this metabolic step with CYP2D6 obviously not being <strong>of</strong> major importance. Using the<br />
data on intrinsic clearance for the particular is<strong>of</strong>orms the percentage <strong>of</strong> contribution <strong>of</strong> each is<strong>of</strong>orm to the<br />
entire microsomal clearance in vitro was calculated for fenproporex. The highest contribution was found<br />
for CYP2B6 (65 % for R(-) and 72 % for the S(+)-enantiomer) and the lowest values were found for<br />
CYP2D6 (7 % and 4 % for R(-) and S(+)-enantiomers, respectively). The aim <strong>of</strong> this study was to elucidate<br />
the reasons for the failure <strong>of</strong>the rat model. As a characteristic test reaction for CYP2B6 activity (CYP<br />
is<strong>of</strong>orm with the highest contribution), bupropion side chain hydroxylation was chosen.<br />
Methods: Data for calculation <strong>of</strong> intrinsic clearances for fenproporex N-dealkylation were taken from<br />
previous studies with recombinant CYPs. Bupropion hydrochloride (R,S-2-(tert-butylamino)-3'<br />
chloropropiophenone; amfebutamone; BU) was administered to Wistar (model <strong>of</strong> the human CYP2D6<br />
extensive metabolizer phenotype) and Dark Agouti rats for toxicological diagnostic reasons according to<br />
the corresponding German law. Urine was collected separately from the feces and analyzed directly after<br />
sampling. After enzymatic cleavage <strong>of</strong> conjugates and centrifugation, bupropion and hydroxy-bupropion<br />
were separated and quantified using an Agilent Technologies (AT, Waldbronn, Germany) AT 1100 series<br />
atmospheric pressure chemical ionisation (APCl) electrospray LC-MSD, SL version and an LC-MSD<br />
ChemStation using the A.OS.03 s<strong>of</strong>tware. Gradient elution was achieved on a Merck LiChroCART®<br />
column (125 x 2 mm LD.) with Superspher®60 RP Select B as stationary phase and a LiChroCART®1O-2<br />
Superspher®60 RP Select B guard column. The mobile phase consisted <strong>of</strong> ammonium formate (5 mM,<br />
adjusted to pH 3 with formic acid) (eluent A) and acetonitrile (eluent B). Human urine samples from<br />
volunteers after ingestion <strong>of</strong> BU were analyzed in the same way.<br />
Results and Discussion: BU and its metabolites could easily be separated using the applied LC-MS<br />
conditions. Characteristic fragments in the APCI-mass spectra allowed easy discrimination <strong>of</strong> the sidechain<br />
hydroxy from the ring hydroxy metabolites. Bupropion side chain hydroxylation was chosen as<br />
specific test reaction for CYP2B activity. Indeed, the side chain hydroxy bupropion (HO-BU) was the<br />
major BU metabolite in human urine samples, whereas it was almost completely absent in urine <strong>of</strong> the two<br />
rat strains. CYP2B6 seems to playa major role in human metabolism <strong>of</strong> amphetamine precursors. BU side<br />
chain hydroxylation, a characteristic test reaction for this is<strong>of</strong>orm, hardly occurs in either rat strain. As a<br />
consequence, it could be possible, that for lack <strong>of</strong> a corresponding CYP2B is<strong>of</strong>orm, CYP2D is<strong>of</strong>orms take<br />
over such metabolic reactions in rats leading to an overestimation <strong>of</strong> the role <strong>of</strong> CYP2D for human<br />
metabolism. In addition, it should also be kept in mind, that the lower extent <strong>of</strong> ring hydroxylation <strong>of</strong><br />
amphetamine precursors under in vitro conditions may also contribute to the differences found between<br />
animal in vivo models and human in vitro test systems.<br />
Keywords: Dark Agouti rats; cytochrome P450; amphetamine precursor drugs<br />
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