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Jeffrey R Idle

Publications and source records attributed to Jeffrey R Idle.

18 recordsLinked to original sources
PubMed2006

The PREgnane X receptor gene-humanized mouse: a model for investigating drug-drug interactions mediated by cytochromes P450 3A.

The most common clinical implication for the activation of the human pregnane X receptor (PXR) is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in Pxr-null mice using a BAC clone containing the complete human PXR gene and 5'- and 3'-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an approximately 60% decrease was observed for both the maximal midazolam serum concentration (C(max)) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1'-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs.

Animals↗
PubMed2006

The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases.

The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

Acetylcysteine↗
PubMed2006

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but <1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P = 0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered. (237 words).

Animals↗
PubMed2006

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio.

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

Adult↗
PubMed2006

Comparative aromatic hydroxylation and N-demethylation of MPTP neurotoxin and its analogs, N-methylated beta-carboline and isoquinoline alkaloids, by human cytochrome P450 2D6.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin is a chemical inducer of Parkinson's disease (PD) whereas N-methylated beta-carbolines and isoquinolines are naturally occurring analogues of MPTP involved in PD. This research has studied the oxidation of MPTP by human CYP2D6 (CYP2D6*1 and CYP2D6*10 allelic variants) as well as by a mixture of cytochrome P450s-resembling HLM, and the products generated compared with those afforded by human monoamine oxidase (MAO-B). MPTP was efficiently oxidized by CYP2D6 to two main products: MPTP-OH (p-hydroxylation) and PTP (N-demethylation), with turnover numbers of 10.09 min-1 and Km of 79.36+/-3 microM (formation of MPTP-OH) and 18.95 min-1 and Km 69.6+/-2.2 microM (PTP). Small amounts of dehydrogenated toxins MPDP+ and MPP+ were also detected. CYP2D6 competed with MAO-B for the oxidation of MPTP. MPTP oxidation by MAO-B to MPDP+ and MPP+ toxins (bioactivation) was up to 3-fold higher than CYP2D6 detoxification to PTP and MPTP-OH. Several N-methylated beta-carbolines and isoquinolines were screened for N-demethylation (detoxification) that was not significantly catalyzed by CYP2D6 or the P450s mixture. In contrast, various beta-carbolines were efficiently hydroxylated to hydroxy-beta-carbolines by CYP2D6. Thus, N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline (a close MPTP analog) was highly hydroxylated to 6-hydroxy-N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline and a corresponding 7-hydroxy-derivative. Thus, CYP2D6 could participate in the bioactivation and/or detoxification of these neuroactive compounds by an active hydroxylation pathway. The CYP2D6*1 enzymatic variant exhibited much higher metabolism of both MPTP and N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline than the CYP2D6*10 variant, highlighting the importance of CYP2D6 polymorphism in the oxidation of these toxins. Altogether, these results suggest that CYP2D6 can play an important role in the metabolic outcome of both MPTP and beta-carbolines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2006

Urinary metabolite profiling reveals CYP1A2-mediated metabolism of NSC686288 (aminoflavone).

NSC686288 [aminoflavone (AF)], a candidate chemotherapeutic agent, possesses a unique antiproliferative profile against tumor cells. Metabolic bioactivation of AF by drug-metabolizing enzymes, especially CYP1A monooxygenases, has been implicated as an underlying mechanism for its selective cytotoxicity in several cell culture-based studies. However, in vivo metabolism of AF has not been investigated in detail. In this study, the structural identities of 13 AF metabolites (12 of which are novel) in mouse urine or from microsomal incubations, including three monohydroxy-AFs, two dihydroxy-AFs and their sulfate and glucuronide conjugates, as well as one N-glucuronide, were determined by accurate mass measurements and liquid chromatography-tandem mass spectrometry fragmentation patterns, and a comprehensive map of the AF metabolic pathways was constructed. Significant differences between wild-type and Cyp1a2-null mice, within the relative composition of urinary metabolites of AF, demonstrated that CYP1A2-mediated regioselective oxidation was a major contributor to the metabolism of AF. Comparisons between wild-type and CYP1A2-humanized mice further revealed interspecies differences in CYP1A2-mediated catalytic activity. Incubation of AF with liver microsomes from all three mouse lines and with pooled human liver microsomes confirmed the observations from urinary metabolite profiling. Results from enzyme kinetic analysis further indicated that in addition to CYP1A P450s, CYP2C P450s may also play some role in the metabolism of AF.

Animals↗
PubMed2006

A metabolomic approach to the metabolism of the areca nut alkaloids arecoline and arecaidine in the mouse.

The areca alkaloids comprise arecoline, arecaidine, guvacoline, and guvacine. Approximately 600 million users of areca nut products, for example, betel quid chewers, are exposed to these alkaloids, principally arecoline and arecaidine. Metabolism of arecoline (20 mg/kg p.o. and i.p.) and arecaidine (20 mg/kg p.o. and i.p.) was investigated in the mouse using a metabolomic approach employing ultra-performance liquid chromatography-time-of-flight mass spectrometric analysis of urines. Eleven metabolites of arecoline were identified, including arecaidine, arecoline N-oxide, arecaidine N-oxide, N-methylnipecotic acid, N-methylnipecotylglycine, arecaidinylglycine, arecaidinylglycerol, arecaidine mercapturic acid, arecoline mercapturic acid, and arecoline N-oxide mercapturic acid, together with nine unidentified metabolites. Arecaidine shared six of these metabolites with arecoline. Unchanged arecoline comprised 0.3-0.4%, arecaidine 7.1-13.1%, arecoline N-oxide 7.4-19.0%, and N-methylnipecotic acid 13.5-30.3% of the dose excreted in 0-12 h urine after arecoline administration. Unchanged arecaidine comprised 15.1-23.0%, and N-methylnipecotic acid 14.8%-37.7% of the dose excreted in 0-12 h urine after arecaidine administration. The major metabolite of both arecoline and arecaidine, N-methylnipecotic acid, is a novel metabolite arising from carbon-carbon double-bond reduction. Another unusual metabolite found was the monoacylglyceride of arecaidine. What role, if any, that is played by these uncommon metabolites in the toxicology of arecoline and arecaidine is not known. However, the enhanced understanding of the metabolic transformation of arecoline and arecaidine should contribute to further research into the clinical toxicology of the areca alkaloids.

Animals↗
PubMed2006

Urinary metabolites and antioxidant products of exogenous melatonin in the mouse.

Exogenous melatonin is widely used for sleep disorders and has potential value in neuroprotection, cardioprotection and as an antioxidant. Here, a novel method is described for the determination of melatonin and six metabolites in mouse urine by use of LC-MS/MS and GC-MS. LC-MS/MS is used for the measurement of melatonin, N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin (6-HMEL), while GC/MS is used for the determination of N-[2-(5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (2-OMEL) and cyclic 3-hydroxymelatonin (3-HMEL) with detection limits on column of 0.02-0.5 pmol, depending on the metabolite. Following oral administration of melatonin to mice, a 0-24 hr urine collection revealed the presence of melatonin (0.2% dose), 6-HMEL (37.1%) and NAS (3.1%) comprising >90% of the total metabolites; AMK and AFMK were also detected at 0.01% each; 2-OMEL was found at 2.2% of the dose, which is >100 times more than the AMK/AFMK pathway, and comprises >5% of the melatonin-related material detected in mouse urine. 3-HMEL was largely found as a sulfate conjugate. These studies establish sensitive assays for determination of six melatonin metabolites in mouse urine and confirm the potential for antioxidant activity of melatonin through the identification in vivo of AMK and AFMK, ring-opened metabolites with a high capacity for scavenging reactive oxygen species.

Animals↗
PubMed2005

Cerebral formation in situ of S-carboxymethylcysteine after ifosfamide administration to mice: a further clue to the mechanism of ifosfamide encephalopathy.

The clinical use of the alkylating oxazaphosphorine ifosfamide is hampered by a potentially severe encephalopathy. S-carboxymethylcysteine (SCMC), a metabolite of ifosfamide (IF), activates the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor, causes neuronal acidification, and could thus be responsible for the encephalopathy. Since the presence of SCMC in brain has not been documented following administration of IF, SCMC was measured in the brain of mice following both the individual i.p. administration of IF and SCMC. SCMC was found in a concentration of 108.2 +/- 29.7 nmol/g following IF, but was detectable at much lower levels following the administration of SCMC (21.1 +/- 21.2 nmol/g). Together with the observation that the concentration of SCMC was 10-fold higher in liver than in brain 1h after administration of SCMC, these findings suggest that the SCMC found after IF was formed in the brain in situ. The concentration of glutamic acid was similar in IF and SCMC treated animals. Methylene blue, which is used clinically to treat and to prevent IF encephalopathy, did not decrease the formation of SCMC in brain. By inhibiting monoamine oxidase activity it did, however, markedly increase the concentration of serotonin in brain which could modulate the effects of SCMC on AMPA/kainate receptors. Thus, SCMC is present in brain following the administration of IF and could contribute to the IF-associated encephalopathy by activation of AMPA/kainate receptors.

Animals↗
PubMed2005

Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice humanized for CYP1A1 and CYP1A2.

The procarcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine formed during the cooking of foods. Metabolism of PhIP by CYP1A2 differs substantially between humans and rodents, with more N2-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Therefore, the human response to PhIP and other heterocyclic amine exposure may not be accurately reflected in the laboratory rodent. By generating mouse models expressing the human genes, species differences in heterocyclic amine metabolism can be addressed. Two transgenic mouse lines were developed, one expressing the human CYP1A1 CYP1A2 transgene in a mouse Cyp1a1-null background (hCYP1A1) and another expressing human CYP1A1 CYP1A2 in a mouse Cyp1a2-null background (hCYP1A2). Expression of human CYP1A2 protein was detected in the liver and also at considerably lower levels in extrahepatic tissues such as lung, kidney, colon, and heart. In the hCYP1A1 and hCYP1A2 mice, 3-methylcholanthrene (3-MC) induced both human CYP1A1 and CYP1A2 protein in the liver. Differences in the metabolism of the heterocyclic amine PhIP were observed between wild-type and hCYP1A2 mice. PhIP was preferentially metabolized by N2-hydroxylation in hCYP1A2 mice, whereas in wild-type mice, 4'-hydroxylation was the predominant pathway. Since the N2-hydroxylation pathway for PhIP metabolism has been reported to be predominant in humans, these results illustrate the potential effectiveness of using these transgenic, humanized mice as models for determining human health risks to PhIP and other heterocyclic amines instead of wild-type mice.

Animals↗
PubMed2004

Metabolism of melatonin by human cytochromes p450.

In humans, the pineal hormone melatonin (MEL) is principally metabolized to 6-hydroxymelatonin (6-HMEL), which is further conjugated with sulfate and excreted in urine. MEL O-demethylation represents a minor reaction. The exact role of individual human cytochromes P450 (P450s) in these pathways has not been established. We used a panel of 11 recombinant human P450 isozymes to investigate for the first time the 6-hydroxylation and O-demethylation of MEL. CYP1A1, CYP1A2, and CYP1B1 all 6-hydroxylated MEL, with CYP2C19 playing a minor role. These reactions were NADPH-dependent. CYP2C19 and, to some extent CYP1A2, O-demethylated MEL. The K(m) (microM) and V(max) (k(cat), pmol min(-1) pmol(-1) P450) for 6-hydroxylation were estimated as 19.2 +/- 2.01 and 6.46 +/- 0.22 (CYP1A1), 25.9 +/- 2.47 and 10.6 +/- 0.32 (CYP1A2), and 30.9 +/- 3.76 and 5.31 +/- 0.21 (CYP1B1). These findings confirm the suggestion of others that CYP1A2 is probably the foremost hepatic P450 in the 6-hydroxylation of MEL and a single report that CYP1A1 is also able to mediate this reaction. However, this is the first time that CYP1B1 has been shown to 6-hydroxylate MEL. The IC50 for the CYP1B1-selective inhibitor (E)-2,4,3',5'-tetramethoxystilbene was estimated to be 30 nM for MEL 6-hydroxylation by recombinant human CYP1B1. Comparison of brain homogenates from wild-type and cyp1b1-null mice revealed that MEL 6-hydroxylation was clearly mediated to a significant degree by CYP1B1. CYP1B1 is not expressed in the liver but has a ubiquitous extrahepatic distribution, and is found at high levels in tissues that also accumulate either MEL or 6-HMEL, such as intestine and cerebral cortex, where it may assist in regulating levels of MEL and 6-HMEL.

Animals↗
PubMed2004

Polymorphic cytochrome P450 2D6: humanized mouse model and endogenous substrates.

Cytochrome P450 2D6 (CYP2D6) is the first well-characterized polymorphic phase I drug-metabolizing enzyme, and more than 80 allelic variants have been identified for the CYP2D6 gene, located on human chromosome 22q13.1. Human debrisoquine and sparteine metabolism is subdivided into two principal phenotypes--extensive metabolizer and poor metabolizer--that arise from variant CYP2D6 genotypes. It has been estimated that CYP2D6 is involved in the metabolism and disposition of more than 20% of prescribed drugs, and most of them act in the central nervous system or on the heart. These drug substrates are characterized as organic bases containing one nitrogen atom with a distance about 5, 7, or 10 A from the oxidation site. Aspartic acid 301 and glutamic acid 216 were determined as the key acidic residues for substrate-enzyme binding through electrostatic interactions. CYP2D6 transgenic mice, generated using a lambda phage clone containing the complete wild-type CYP2D6 gene, exhibits enhanced metabolism and disposition of debrisoquine. This transgenic mouse line and its wild-type control are models for human extensive metabolizers and poor metabolizers, respectively, and would have broad application in the study of CYP2D6 polymorphism in drug discovery and development, and in clinical practice toward individualized drug therapy. Endogenous 5-methoxyindole- thylamines derived from 5-hydroxytryptamine were identified as high-affinity substrates of CYP2D6 that catalyzes their O-demethylations with high enzymatic capacity and specificity. Thus, polymorphic CYP2D6 may play an important role in the interconversions of these psychoactive tryptamines, including a crucial step in a serotonin-melatonin cycle.

Animals↗
PubMed2003

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.

5-Methoxytryptamine↗
PubMed2003

Screening for endogenous substrates reveals that CYP2D6 is a 5-methoxyindolethylamine O-demethylase.

The objective of this investigation was to screen for potential endogenous substrates for CYP2D6. Using recombinant CYP2D6, together with hepatic microsomes from CYP2D6-transgenic mice, human liver microsomes, and a specific anti-CYP2D6 monoclonal antibody, it was ascertained that CYP2D6 does not significantly metabolize the endogenous phenylethylamines 2-phenylethylamine, octopamine, synephrine, 3-methoxy-p-tyramine, 4-methoxy-m-tyramine, metanephrine, and normetanephrine, nor the indolethylamines tryptamine, serotonin, 6-methoxytryptamine, and melatonin, nor the beta-carbolines harman, norharman and tryptoline. However, the indolethylamines 5-methoxy-N,N-dimethyltryptamine (5-MDMT) and pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) showed relatively high affinity for CYP2D6 in a spectral binding assay (K(s) 28 +/- 5, and 0.5 +/- 0.3 microm (mean +/- SEM), respectively) and were O-demethylated only by CYP2D6 in a panel of 15 recombinant common human P450s. Pinoline and 5-MDMT O-demethylase activities were 35- and 11-fold greater in liver microsomes from CYP2D6-humanized mice, respectively, than those in liver microsomes from control mice. Moreover, the increased activities were completely inhibited by an anti-CYP2D6 monoclonal antibody. Kinetic analysis with recombinant CYP2D6 gave K(m) and k(cat) values for 5-MDMT and pinoline O-demethylations of 12 +/- 1 microm and 65 +/- 1 min(-1) and 1.8 +/- 0.3 microm and 26 +/- 1 min(-1), respectively. These two substrates can be added to 5-methoxytryptamine, which we have recently reported to be an endogenous CYP2D6 substrate. CYP2D6 is therefore a relatively highly specific, high-affinity, high-capacity 5-methoxyindolethylamine O-demethylase. Polymorphic cytochrome CYP2D6 may therefore exert an influence on mood and behavior by the O-demethylation of these 5-methoxyindolethylamines found in the brain and pineal gland. These processes may also impact on mental and neurological health. The findings may open new vistas for the determination of CYP2D6 phenotype.

Animals↗
PubMed2003

The relative contribution of monoamine oxidase and cytochrome p450 isozymes to the metabolic deamination of the trace amine tryptamine.

Tryptamine is a trace amine in mammalian central nervous system that interacts with the trace amine TA(2) receptor and is now thought to function as a neurotransmitter or neuromodulator. It had been reported that deamination of tryptamine to tryptophol was mediated by CYP2D6, a cytochrome P450 that is expressed in human brain, suggesting that tryptamine may be an endogenous substrate for this polymorphic enzyme. We were unable to confirm this report and have reinvestigated tryptamine metabolism in human liver microsomes (HLM) and in microsomes expressing recombinant human cytochrome P450 and monoamine oxidase (MAO) isozymes. Tryptamine was oxidized to indole-3-acetaldehyde by HLM and recombinant human MAO-A in the absence of NADPH, and indole-3-acetaldehyde was further reduced to tryptophol by aldehyde reductase in HLM in the presence of NADPH. Steady-state kinetic parameters were estimated for each reaction step by HLM and MAO-A. The CYP2D6 substrates bufuralol and debrisoquine showed strong inhibition of both tryptophol production from tryptamine in HLM and the formation of indole-3-acetaldehyde from tryptamine catalyzed by recombinant MAO-A. Anti-CYP2D6 monoclonal antibody did not inhibit these reactions. Pargyline, a nonselective MAO inhibitor, did not show cross inhibition to debrisoquine 4-hydroxylation and dextromethorphan O-demethylation by HLM and recombinant CYP2D6 enzyme. This is the first unequivocal report of the selective conversion of tryptamine to tryptophol by MAO-A. CYP2D6 does not contribute to this reaction.

Cytochrome P-450 CYP2D6↗
PubMed2003

Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine.

The psychotropic beta-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for the O-demethylations mediated by each isozyme and by pooled HLMs. K(cat) (min(-1)) and K(m) Awake M) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline.

Animals↗
PubMed2002

4-Hydroxylation of debrisoquine by human CYP1A1 and its inhibition by quinidine and quinine.

A panel of 15 recombinant cytochromes P450 expressed in human B-lymphoblastoid cells was used to study debrisoquine 4-hydroxylation. Both CYP2D6 and CYP1A1 carried out the reaction. The apparent K(m) (micromolar) and V(max) (picomoles per minute per picomole of P450) for CYP2D6 were 12.1 and 18.2 and for CYP1A1 were 23.1 and 15.2, respectively. CYP1A1 debrisoquine 4-hydroxylase was inhibited by the CYP1A1 inhibitor alpha-naphthoflavone and the CYP1A1 substrate 7-ethoxyresorufin. Additionally and surprisingly, this reaction was also inhibited by quinidine and quinine, with respective IC(50) values of 1.38 +/- 0.10 and 3.31 +/- 0.14 microM, compared with those for CYP2D6 debrisoquine 4-hydroxylase of 0.018 +/- 0.05 and 3.75 +/- 2.07 microM, respectively. Anti-CYP1A1 monoclonal antibody (mAb) 1-7-1 abolished CYP1A1 debrisoquine hydroxylase and anti-CYP2D6 mAb 50-1-3 eradicated CYP2D6 debrisoquine 4-hydroxylase. Three further CYP2D6-specific reactions were tested: dextromethorphan O-demethylation, bufuralol 1'-hydroxylation, and sparteine dehydrogenation. The CYP2D6 specificity, judged by the CYP2D6/CYP1A1 activity ratios was 18.5, 7.0, 6.0, and 1.6 for dextromethorphan, bufuralol, sparteine, and debrisoquine, respectively. Thus, debrisoquine is not a specific CYP2D6 substrate and quinidine is not a specific CYP2D6 inhibitor. These findings have significant implications for the conduct of in vitro drug metabolism inhibition studies and underscore the fallacy of "specific chemical inhibitors" of a supergene family of enzymes that have overlapping substrate specificities. The use of highly specific mAbs in such studies is mandated. It is unclear as yet whether these findings have implications for the relationship between CYP2D6 genotype and in vivo debrisoquine 4-hydroxylase activity.

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