Recombinant Cytochrome P450 2D6 Research Articles

Adult and infant pharmacokinetic profiling of dihydrocodeine using physiologically based pharmacokinetic modeling.

We previously analysed the serum concentrations of dihydrocodeine in a 1-month-old infant with respiratory depression after being prescribed dihydrocodeine phosphate 2.0 mg/day divided t.i.d. for 2 days. The purpose was to develop a full physiologically based pharmacokinetic (PBPK) model that could account for these and other drug monitoring results. Based on experiments in Caco-2 cell monolayers, the effective permeability of dihydrocodeine in human jejunum was established as 1.28 × 10-4 cm/s. The in vitro Vmax /Km values for dihydrocodeine demethylation mediated by recombinant cytochrome P450 2D6 and 3A4 were 0.19 and 0.066 μl/min/pmol, respectively, and for dihydrocodeine 6-O-glucuronidation mediated by recombinant UGT2B4 and 2B7, the Vmax /Km values were 0.14 and 0.22 μl/min/mg protein, respectively. Renal clearance was calculated as 5.37 L/h on the total clearance value multiplied by the fraction recovered in urine. The reported plasma concentration-time profiles of dihydrocodeine after intravenous administration in healthy volunteers were used to adjust the tissue partitioning ratios. The developed model simulated the pharmacokinetic profiles of dihydrocodeine after single and multiple oral administrations reasonably well in the same population. Subsequently, the validated model was used to simulate pharmacokinetic profiles for five pediatric cases, including the 1-month-old Japanese boy and a 14-year-old Japanese girl who took an overdose of dihydrocodeine phosphate (37 mg). The simulated pharmacokinetic profiles for five virtual pediatric subjects matching the age, gender, and P450 2D6 phenotype of each case approximately reflected the observed values. These results suggested that our dihydrocodeine PBPK model reproduced the results of clinical cases reasonably well for subjects.

A combinatorial approach for the discovery of cytochrome P450 2D6 inhibitors from nature

The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism and metabolizes at least 20% of all clinically relevant drugs. Therefore, it is an important target for drug-drug interaction (DDI) studies. High-throughput screening (HTS) assays are commonly used tools to examine DDI, but show certain drawbacks with regard to their applicability to natural products. We propose an in silico – in vitro workflow for the reliable identification of natural products with CYP2D6 inhibitory potential. In order to identify candidates from natural product-based databases that share similar structural features with established inhibitors, a pharmacophore model was applied. The virtual hits were tested for the inhibition of recombinant human CYP2D6 in a bioluminescence-based assay. By controlling for unspecific interferences of the test compounds with the detection reaction, the number of false positives were reduced. The success rate of the reported workflow was 76%, as most of the candidates identified in the in silico approach were able to inhibit CYP2D6 activity. In summary, the workflow presented here is a suitable and cost-efficient strategy for the discovery of new CYP2D6 inhibitors with natural product libraries.

Effect of 24 Cytochrome P450 2D6 Variants Found in the Chinese Population on Atomoxetine Metabolism in vitro

Objective: The aim of this article was to assess the catalytic activities of 24 cytochrome P450 2D6 (CYP2D6) variants found in the Chinese population toward atomoxetine in vitro as well as CYP2D6.1. Methods: In this study, the co-expression enzyme of human recombinant CYPOR, CYPb5, and CYP2D6.1 or other CYP2D6 variants with the baculovirus-mediated insect cells (Sf21) was used to study the catalytic activities of 24 CYP2D6 variants toward atomoxetine metabolism. The metabolite of atomoxetine (4-hydroxyatomoxetine) was detected by ultra-high performance liquid chromatography-mass spectrometry method. Results: The intrinsic clearance (Vmax/Km) values of most variants were significantly altered when compared with CYP2D6.1. CYP2D6.94, CYP2D6.D336N, CYP2D6.R440C exhibited marked increased values 172, 126, 121% respectively. CYP2D6.89 and CYP2D6.98 exhibited similar catalytic activity as the wild type, whereas 17 variants exhibited significantly decreased values (from 5 to 87%) due to increase Km and/or decrease Vmax values. However, CYP2D6.92 and CYP2D6.96 showed no or few activity because of producing nothing. Conclusions: Our results suggest that most of these newly found variants exhibit significantly changed catalytic activities compared with the wild type. And these findings provide valuable information for the growth and development of personalized medicine in China.

Regeneration of serotonin from 5-methoxytryptamine by polymorphic human CYP2D6.

Polymorphic cytochrome P450 2D6 (CYP2D6) is expressed in several types of central neurons but its function in human brain is currently unknown. Using recombinant enzymes and CYP2D6-transgenic mice, we established that 5-methoxytryptamine (5-MT), a metabolite and precursor of melatonin, is a specific and high-turnover endogenous substrate of CYP2D6. This potent serotonergic neuromodulator in numerous physiological systems binds tightly to recombinant CYP2D6 enzyme with an equilibrium dissociation constant (K(s)) of 23.4 micromol/l, and is O-demethylated to serotonin (5-hydroxytryptamine, 5-HT) with a high turnover of 51.7 min(-1) and low Michaelis-Menten constant of 19.5 micromol/l. The production of 5-HT from 5-MT catalyzed by CYP2D6 was inhibited by selective serotonin reuptake inhibitors, and their inhibition potency (K(i), micromol/l) decreased in the order of fluoxetine (0.411) > norfluoxetine (1.38) > fluvoxamine (10.1) > citalopram (10.9). Liver microsomes prepared from CYP2D6-transgenic mice showed about 16-fold higher 5-MT O-demethylase activity than that from wild-type mice. After the intravenous co-administration of 5-MT (10 mg/kg) and pargyline (20 mg/kg), serum 5-HT level was about 3-fold higher in CYP2D6-transgenic mice than wild-type mice. When dosed with alpha,alpha,beta,beta-d -5-MT, alpha,alpha,beta,beta-d4-5-HT was detected in transgenic mouse serum, and its content was much higher than wild-type mouse. alpha,alpha,beta,beta-d4-5-HT was not produced in CYP2D6-transgenic mice pretreated with quinidine. The regeneration of 5-HT from 5-MT provides the missing link in the serotonin-melatonin cycle. Up to 10% of the population lacks this enzyme. It is proposed that this common inborn error in 5-MT O-demethylation to serotonin influences a range of neurophysiologic and pathophysiologic events.

Studies on the metabolism of the α‐pyrrolidinophenone designer drug methylenedioxy‐pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC–MS and LC–high‐resolution MS and its detectability in urine by GC–MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy- N, N-dimethyltryptamine metabolism and pharmacokinetics

5-Methoxy- N, N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency ( V max/ K m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities ( R 2 = 0.98; P < 0.0001) and CYP2D6 contents ( R 2 = 0.77; P = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg- CYP2D6) mouse models showed that Tg- CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg- CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.

The endocannabinoid anandamide is a substrate of cytochrome P450 2D6

The endocannabinoid anandamide, an arachidonic acid derivative, activates the cannabinoid receptors CB1 and CB2. These receptors and the enzymes involved in the synthesis and inactivation of their endogenous ligands represent novel targets for the treatment of many nervous system and peripheral disorders. We recently demonstrated that anandamide is a substrate for the liver and kidney cytochrome P450 (CYP) enzymes 3A4 and 4F2. Several CYP isoforms are present in the brain and have important roles in the oxidation of endogenous substrates. The objective of the current studies was to determine if anandamide is a substrate for the human polymorphic CYP 2D6, one of the major CYP isoforms in human brain. We found that recombinant CYP 2D6 converted anandamide to 20‐hydroxyeicosatetraenoic acid ethanolamide (20‐HETE‐EA) and 5,6‐, 8,9‐, 11,12‐, and 14,15‐epoxyeicosatrienoic acid ethanolamides (EET‐EAs) with apparent Km values of 1–2 μM. CYP 2D6 further metabolized the epoxides of anandamide to form several di‐oxygenated derivatives. To our knowledge, anandamide and its epoxides are the first eicosanoid molecules to be identified as CYP2D6 substrates, raising the possibility that this enzyme could be involved in the metabolism of other endogenous mediators with similar structural properties. Supported in part by the National Institutes of Health (Grants CA‐16954 and T32 GM007767).

P.1.g.008 Maternal deprivation and reactivity to different stress in male and female adult rats according to their estrous cycle

4-Hydroxylation is an important pathway of tamoxifen metabolism because the product of this reaction is intrinsically 100 times more potent as an oestrogen receptor antagonist than is the parent drug. Although tamoxifen 4-hydroxylation is catalysed by human cytochrome P450 (CYP), data conflict on the specific isoforms responsible. The aim of this study was to define unequivocally the role of individual CYPs in the 4-hydroxylation of tamoxifen by human liver microsomes. Microsomes from each of 10 human livers catalysed the reaction [range = 0.6–2.9 pmol/mg protein/min (1 μM substrate concentration) and 6–25 pmol/mg protein/min (18 μM)]. Three of the livers with the lowest tamoxifen 4-hydroxylation activity were from genetically poor metabolisers with respect to CYP2D6. Inhibition of activity by quinidine (1μM), sulphaphenazole (20 μM) and ketoconazole (2 μM), selective inhibitors of CYPs 2D6, 2C9 and 3A4, respectively, was 0–80%, 0–80% and 12–57%. The proportion of activity inhibited by quinidine correlated positively with total microsomal tamoxifen 4-hydroxylation activity (rs = 0.89, P < 0.01), indicating a major involvement of CYP2D6 in this reaction. Recombinant human CYPs 2D6, 2C9 and 3A4 but not CYPs 1A1, 1A2, 2C19 and 2E1 displayed significant 4-hydroxylation activity. Similar inhibition and correlation experiments confirmed that tamoxifen N-demethylation is catalysed predominantly by CYP3A4. These findings indicate that the 4-hydroxylation of tamoxifen is catalysed almost exclusively by CYPs 2D6, 2C9 and 3A4 in human liver microsomes. However, the marked between-subject variation in the contribution of these isoforms underlines the need to study metabolic reactions in a sufficient number of livers that are characterised with respect to a range of cytochrome P450 activities.

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes.

Anti-liver-kidney microsome antibodies (anti-LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti-LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti-LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti-LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti-LKM-positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254-339 (254-273, 257-269, 270-294, 291-310, 307-324, 321-339, 373-389) as well as the novel peptide 196-218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6-dependent bufuralol 1'-hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257-269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196-218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254-273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti-LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti-LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257-269 a further immunodominant epitope exists on CYP2D6 which is recognized by sera from patients with AIH II but hardly by sera from patients with hepatitis C.

The Potential for CYP2D6 Inhibition Screening Using a Novel Scintillation Proximity Assay-Based Approach

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

Coumarin Substrates for Cytochrome P450 2D6 Fluorescence Assays

A set of nine 4-aminomethyl-7-alkoxycoumarin derivatives was synthesized and characterized as substrates for O-dealkylation by recombinant cytochrome P450 2D6, a major human enzyme involved in drug metabolism. Enzymatic O-dealkylation yields 7-hydroxycoumarins, which have useful fluorescence properties. The substrates, which differed in substitution at the amino and 7-hydroxy positions, varied in terms of catalytic efficiency of O-dealkylation and in their selectivity as substrates for cytochrome P450 2D6 in human liver microsomes. Several of the compounds are useful as cytochrome P450 2D6 substrates in single-phase, rapid-throughput assays.

Role of cytochrome P450 2D6 (CYP2D6) in the stereospecific metabolism of E- and Z-doxepin.

The tricyclic antidepressant, doxepin, is formulated as an irrational mixture of E (trans) and Z (cis) stereoisomers (85%: 15%). We examined the stereoselective metabolism of doxepin in vitro, with the use of human liver microsomes, recombinant CYP2D6 and gas chromatography-mass spectrometry. In human liver microsomes over the concentration range 5-1500 microM, the rate of Z-doxepin N-demethylation exceeded that of E-doxepin above 100 microM in two of three livers. Eadie-Hofstee plots were curvilinear indicating the involvement of several enzymes in N-demethylation. Coincubation of doxepin with 7,8-naphthoflavone and ketoconazole reduced the rates of N-demethylation of E- and Z-doxepin by 30-50% and 40-60%, respectively, suggesting the involvement of CYP1A and CYP3A4, whilst quinidine had little effect on N-demethylation. In contrast, doxepin hydroxylation was exclusively stereo-specific; E-doxepin and E-N-desmethyldoxepin were hydroxylated with high affinity in liver microsomes and by recombinant CYP2D6 (Km in the range of 5-8 microM), but there was no evidence of Z-doxepin hydroxylation. In 'metabolic consumption' experiments with liver microsomes (having measurable CYP2D6 activity) and initial substrate concentration of 1 microM, the consumption of E-doxepin was greater (P < 0.05, n = 5) than that of Z-doxepin. Quinidine inhibited the consumption of E-doxepin but did not affect the consumption of Z-doxepin. With N-desmethyldoxepin, quinidine inhibited the consumption of E-N-desmethyl-doxepin whereas Z-N-desmethyldoxepin appeared to be a terminal oxidative metabolite. In summary, CYP2D6 is a major oxidative enzyme in doxepin metabolism; predominantly catalysing hydroxylation with an exclusive preference for the E-isomers. The relatively more rapid metabolism of E-isomeric forms, and the limited metabolic pathways for the Z-isomers may explain the apparent enrichment of Z-N-desmethyldoxepin that is observed in vivo.

Establishment of a novel radioligand assay using eukaryotically expressed cytchrome P4502D6 for the measurement of liver kidney microsomal type 1 antibdy in patients with autoimmune hepatitis and hepatitis C virus infection

Background/Aims: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. Methods: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection ), 128 LKM1-negative patients and 57 normal controls were tested. Results: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls ( p<0.0001), and were correlated with the immunofluorescence titres of LKM1 ( r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively.). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. Conclusions: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Variable contribution of cytochromes p450 2d6, 2c9 and 3a4 to the 4-hydroxylation of tamoxifen by human liver microsomes

4-Hydroxylation is an important pathway of tamoxifen metabolism because the product of this reaction is intrinsically 100 times more potent as an oestrogen receptor antagonist than is the parent drug. Although tamoxifen 4-hydroxylation is catalysed by human cytochrome P450 (CYP), data conflict on the specific isoforms responsible. The aim of this study was to define unequivocally the role of individual CYPs in the 4-hydroxylation of tamoxifen by human liver microsomes. Microsomes from each of 10 human livers catalysed the reaction [range = 0.6–2.9 pmol/mg protein/min (1 μM substrate concentration) and 6–25 pmol/mg protein/min (18 μM)]. Three of the livers with the lowest tamoxifen 4-hydroxylation activity were from genetically poor metabolisers with respect to CYP2D6. Inhibition of activity by quinidine (1μM), sulphaphenazole (20 μM) and ketoconazole (2 μM), selective inhibitors of CYPs 2D6, 2C9 and 3A4, respectively, was 0–80%, 0–80% and 12–57%. The proportion of activity inhibited by quinidine correlated positively with total microsomal tamoxifen 4-hydroxylation activity (r s = 0.89, P < 0.01), indicating a major involvement of CYP2D6 in this reaction. Recombinant human CYPs 2D6, 2C9 and 3A4 but not CYPs 1A1, 1A2, 2C19 and 2E1 displayed significant 4-hydroxylation activity. Similar inhibition and correlation experiments confirmed that tamoxifen N-demethylation is catalysed predominantly by CYP3A4. These findings indicate that the 4-hydroxylation of tamoxifen is catalysed almost exclusively by CYPs 2D6, 2C9 and 3A4 in human liver microsomes. However, the marked between-subject variation in the contribution of these isoforms underlines the need to study metabolic reactions in a sufficient number of livers that are characterised with respect to a range of cytochrome P450 activities.

Atypical metabolism of deprenyl and its enantiomer, (S)-(+)-N,alpha-dimethyl-N-propynylphenethylamine, by cytochrome P450 2D6.

Debrisoquine 4-hydroxylase is a unique cytochrome P450 that effects oxidation of protonated substrates at sites distal from the basic nitrogen. A basic tenet of the several models that have been proposed for the active site of P450 2D6 is that oxidation occurs at distances of approximately 5 or approximately 7 A from the protonated site. In this study, the metabolism of both stereoisomers of deprenyl, a therapeutically valuable monoamine oxidase B inhibitor, was shown to produce N-demethylation and N-depropargylation of the sole basic nitrogen in the molecule by recombinant cytochrome P450 2D6. N-Demethylation of L-(-)-deprenyl leading to nordeprenyl was favored by approximately 13:1 over N-depropargylation which produced methamphetamine. The Km and kcat values for formation of methamphetamine, the minor metabolite, were 56 +/- 5 microM and 0.63 +/- 0.063 nmol of methamphetamine min-1 (nmol of P450)-1, respectively; the kcat for nordeprenyl formation was approximately 8.2 nmol of nordeprenyl min-1 (nmol of P450)-1. Although these pathways would be the anticipated processes for monoamine oxidases and most cytochrome P450s, this mode of biotransformation is not predicted by current active site models and represents a novel pathway for P450 2D6. Statistical analysis indicates that the therapeutically important L-(-)-isomer was preferentially metabolized [kcat/Km (-)/(+) ratio = 2.66]. Competitive inhibition of deprenyl metabolism by both quinidine and quinine with an approximate 10(3) differential confirms that this metabolic pathway is P450 2D6 mediated.(ABSTRACT TRUNCATED AT 250 WORDS)