Infant Dextromethorphan and Dextrorphan Exposure via Breast Milk From Mothers Who Are CYP2D6 Extensive Metabolizers.

The risk of infant exposure to dextromethorphan (DM) and its active metabolite, dextrorphan (DX), through breast milk has not been evaluated. In this study, bound and unbound DM and DX concentrations in breast milk and plasma at 2 hours post-dose were measured in 20 lactating women (n = 20) following a single 30 mg oral dose of DM. The DM and DX concentrations in breast milk were positively correlated with their respective plasma concentrations. The breast milk-to-plasma (M/P) ratios of 1.0 and 1.6 and the unbound M/P ratios of 1.1 and 2.0 for DM and DX, respectively, suggested that DM and DX are extensively distributed into breast milk. The infant exposure following a single dose of 30 mg DM was estimated using breast milk concentrations of 0.33 ± 0.32 and 1.8 ± 1.0 µg/kg/day for DM and DX, respectively. The steady-state infant exposure was estimated using the M/P ratios and previously reported area under the concentration-time curve (AUC) of DM and DX following repeated dosing of DM 60 mg orally, twice daily, to be 0.64 ± 0.22 and 1.23 ± 0.38 µg/kg/day, respectively. Based on these estimated infant doses, the relative infant doses (RIDs) were estimated to be <1%, suggesting the infant is only exposed to a minor fraction of adult dose through breast milk; however, one nursing infant developed an erythematous rash during this study, which warrants additional research to fully elucidate the risks of infant exposure to DM and DX through breast milk.

N-Substituted-3-alkoxy-derivatives of dextromethorphan are functional NMDA receptor antagonists in vivo: Evidence from an NMDA-induced seizure model in rats.

Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = -0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = -0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6-Mediated Metabolic Activity.

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype-phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next-generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele-specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next-generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes.

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 µL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 µL), microsomal protein amount (5 µg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ±â€¯SD, n = 4), which showed a maximum velocity of 2.24 ±â€¯0.42 pmol/min/mg and a MM constant of 282 ±â€¯62 µM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.

Pharmacokinetics of dextromethorphan and its metabolites in horses following a single oral administration.

Dextromethorphan is an N-methyl-D-aspartate (NMDA) non-competitive antagonist commonly used in human medicine as an antitussive. Dextromethorphan is metabolized in humans by cytochrome P450 2D6 into dextrorphan, which is reported to be more potent than the parent compound. The goal of this study is to describe the metabolism of and determine the pharmacokinetics of dextromethorphan and its major metabolites following oral administration to horses. A total of 23 horses received a single oral dose of 2 mg/kg. Blood samples were collected at time 0 and at various times up to 96 h post drug administration. Urine samples were collected from 12 horses up to 120 h post administration. Plasma and urine samples were analyzed using liquid chromatography-mass spectrometry, and the resulting data analyzed using non-compartmental analysis. The Cmax , Tmax , and the t1/2 of dextromethorphan were 519.4 ng/mL, 0.55 h, and 12.4 h respectively. The area under the curve of dextromethorphan, free dextrorphan, and conjugated dextrorphan were 563.8, 2.19, and 6,691 h*ng/mL respectively. In addition to free and glucuronidated dextrorphan, several additional glucuronide metabolites were identified in plasma, including hydroxyl-desmethyl dextrorphan, desmethyl dextrorphan, and three forms of hydroxylated dextrorphan. Dextromethorphan was found to be eliminated from the urine predominately as the O-demethylated metabolite, dextrorphan. Several additional metabolites including several novel hydroxy-dextrorphan metabolites were also detected in the urine in both free and glucuronidated forms. No significant undesirable behavioural effects were noted throughout the duration of the study. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacogenetic comparison of CYP2D6 predictive and measured phenotypes in a South African cohort.

The relationship between genetic variation in CYP2D6 and variable drug response represents a potentially powerful pharmacogenetic tool. However, little is known regarding this relationship in the genetically diverse South African population. The aim was therefore to evaluate the relationship between predicted and measured CYP2D6 phenotype. An XL-PCR+Sequencing approach was used to determine CYP2D6 genotype in 100 healthy volunteers and phenotype was predicted using activity scores. With dextromethorphan as the probe drug, metabolic ratios served as a surrogate measure of in vivo CYP2D6 activity. Three-hour plasma metabolic ratios of dextrorphan/dextromethorphan were measured simultaneously using semi-automated online solid phase extraction coupled with tandem mass spectrometry. Partial adaptation of the activity score system demonstrated a strong association between genotype and phenotype, as illustrated by a kappa value of 0.792, inter-rater discrepancy of 0.051 and sensitivity of 72.7%. Predicted phenotype frequencies using the modified activity score were 1.3% for poor metabolisers (PM), 7.6% for intermediate metabolisers (IM) and 87.3% for extensive metabolisers (EM). Measured phenotype frequencies were 1.3% for PM, 13.9% for IM and 84.8% for EM. Comprehensive CYP2D6 genotyping reliably predicts CYP2D6 activity in this South African cohort and can be utilised as a valuable pharmacogenetic tool.

Correlation of genotype, phenotype, and mRNA expression of CYP2D6 and CYP2C19 in peripheral blood leukocytes (PBLs).

INTRODUCTION: The genetic polymorphism of drug metabolizing enzymes of the cytochrome P450 (CYP) families, especially CYP2D6 and CYP2C19, is the most important cause of variable responses of many drugs. Enzyme activity ranges from complete deficiency, so called poor metabolizers (PMs), to an ultrafast metabolism. While PMs and extensive metabolizers (EMs) can be well distinguished by genotyping, phenotyping is necessary to subdivide EMs from intermediate metabolizers (IMs). The aim of the study was to evaluate if messenger RNA (mRNA) concentration for CYP-enzymes in peripheral blood leukocytes (PBLs) will be predictive of systemic enzyme activity, allowing an easy and safe determination of metabolic activity. METHODS: The genotype, phenotype, and mRNA-expression in PBLs were evaluated in 124 healthy Caucasian volunteers (males and females, age range 23 - 59 years) on three occasions (every 4 weeks). Genotyping was performed by Taqman allelic discrimination on the most common null alleles for CYP2D6 (*3, *4, *6, *7, and *8) and CYP2C19 (*2 and *3). For phenotyping CYP2D6, dextromethorphan/dextrorphan metabolic ratios were determined in collected urine (8 hours) after administration of 30 mg dextromethorphan. For phenotyping CYP2C19, we used the plasma concentration ratio of omeprazole/hydroxyomeprazole 4 hours after ingestion of 40 mg omeprazole. mRNA-expression in PBLs for CYP2D6 and CYP2C19 was measured by Taqman real-time PCR before medication and 4 hours afterwards. RESULTS: Genotyping for CYP2D6 and CYP2C19 showed a regular distribution of EMs and PMs compared to studies of a comparable population. The median dextromethorphan/dextrorphan metabolic ratio was 0.47 in EMs/IMs and 2.29 in PMs. The median omeprazole/hydroxyomeprazole metabolic ratio was 3.06 in EMs/IMs and 35.29 in PMs. CYP2D6 and CYP2C19 mRNA expression was detected without evidence of correlation to the respective metabolic ratio. CONCLUSION: The results do not support the concept of using mRNA expression profiles for CYP2D6 and CYP2C19 enzymes in PBLs for prediction of systemic enzyme activity.

Hepatic Cyp2d and Cyp26a1 mRNAs and activities are increased during mouse pregnancy.

There is considerable evidence that drug disposition is altered during human pregnancy and based on probe drug studies, CYP2D6 activity increases during human pregnancy. The aim of this study was to determine whether the changes of CYP2D6 activity observed during human pregnancy could be replicated in the mouse, and explore possible mechanisms of increased CYP2D6 activity during pregnancy. Cyp2d11, Cyp2d22, Cyp2d26 and Cyp2d40 mRNA was increased (P < 0.05) on gestational days (GD) 15 and 19 compared with the non-pregnant controls. There was no change (P > 0.05) in Cyp2d9 and Cyp2d10 mRNA. In agreement with the increased Cyp2d mRNA, Cyp2d-mediated dextrorphan formation from dextromethorphan was increased 2.7-fold (P < 0.05) on GD19 (56.8±39.4 pmol/min/mg protein) when compared with the non-pregnant controls (20.8±11.2 pmol/min/mg protein). An increase in Cyp26a1 mRNA (10-fold) and retinoic acid receptor (Rar)ß mRNA (2.8-fold) was also observed during pregnancy. The increase in Cyp26a1 and Rarß mRNA during pregnancy indicates increased retinoic acid signaling in the liver during pregnancy. A putative retinoic acid response element was identified within the Cyp2d40 promoter and the mRNA of Cyp2d40 correlated (P < 0.05) with Cyp26a1 and Rarß. These results show that Cyp2d mRNA is increased during mouse pregnancy the and mouse may provide a suitable model to investigate the mechanisms underlying the increased clearance of CYP2D6 probes observed during human pregnancy. Our findings also suggest that retinoic acid signaling in the liver is increased during pregnancy, which may have broader implications to energy homeostasis in the liver during pregnancy.

Liver kidney microsomal type 1 antibodies reduce the CYP2D6 activity in patients with chronic hepatitis C virus infection.

Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies.

In vitro characterization of the drug-drug interaction potential of catabolites of antibody-maytansinoid conjugates.

The in vitro characterization of the inhibition potential of four representative maytansinoid species observed upon hepatic and/or tumor in vivo processing of antibody-maytansine conjugates (AMCs) with cleavable and noncleavable linkers is reported. We investigated the free maytansinoid species N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1), (S)-methyl-DM1, and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) as representative cleavable linker catabolites and Lysine-N(ε)-N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate-DM1 (Lys-MCC-DM1) as the representative noncleavable linker catabolite. Studies with recombinant human cytochromes P450 (P450s) indicate CYP2D6, CYP3A4, and CYP3A5 are the primary isoforms responsible for the oxidative metabolism of DM1, (S)-methyl-DM1, and DM4. Lys-MCC-DM1 was not metabolized by any of the P450 isoforms studied. DM1 was shown to be a reversible inhibitor of CYP2C8 (K(i) = 11 ± 3 µM) and CYP2D6 (K(i) = 14 ± 2 µM). Lys-MCC-DM1 and (S)-methyl-DM1 showed no reversible or time-dependent inactivation of any of the P450s studied. DM1 and DM4 inactivated CYP3A from human liver microsomes with K(i)/k(inact) values of 4.8 ± 0.9 µM/0.035 ± 0.002 min(-1) and 3.3 ± 0.2 µM/0.114 ± 0.002 min(-1), respectively. DM1 and DM4 inactivated recombinant CYP3A4 with K(i)/k(inact) values of 3.4 ± 1.0 µM/0.058 ± 0.005 min(-1) and 1.4 ± 0.3 µM/0.117 ± 0.006 min(-1), respectively. Because of instability in plasma, further characterization of the DM1 and DM4 intramolecular and intermolecular disulfide conjugates observed in vivo is required before an accurate drug-drug interaction (DDI) prediction can be made. AMCs with noncleavable thioether-linked DM1 as the cytotoxic agent are predicted to have no potential for a DDI with any of the major human P450s studied.

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes.

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 µg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 µg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 µg/ml) also decreased CYP2D6 activity (IC(50)=265.8 µg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 µM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 µM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 µM, 16.5 µM and 61.4 µM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 µg/ml and 2.53 µM, respectively, compared to quinidine, K(i)=0.41 µM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.

An extension of hypotheses regarding rapid-acting, treatment-refractory, and conventional antidepressant activity of dextromethorphan and dextrorphan.

It was previously hypothesized that dextromethorphan (DM) and dextrorphan (DX) may possess antidepressant properties, including rapid and conventional onsets of action and utility in treatment-refractory depression, based on pharmacodynamic similarities to ketamine. These similarities included sigma-1 (σ(1)) agonist and NMDA antagonist properties, calcium channel blockade, muscarinic binding, serotonin transporter (5HTT) inhibition, and µ receptor potentiation. Here, six specific hypotheses are developed in light of additional mechanisms and evidence. Comparable potencies to ketamine for DM and DX are detailed for σ(1) (DX>DM>ketamine), NMDA PCP site (DX>ketamine>DM), and muscarinic (DX>ketamine>>>>DM) receptors, 5HTT (DM>DX≫ketamine), and NMDA antagonist potentiation of µ receptor stimulation (DM>ketamine). Rapid acting antidepressant properties of DM include NMDA high-affinity site, NMDR-2A, and functional NMDR-2B receptor antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation, µ potentiation, and 5HTT inhibition), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, µ potentiation, and 5HTT inhibition), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Those for dextrorphan include NMDA high-affinity site and NMDR-2A antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation and ß adrenoreceptor stimulation), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, ß stimulation, and µ antagonism), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Conventional antidepressant properties for dextromethorphan and dextrorphan include 5HTT and norepinephrine transporter inhibition, σ(1) stimulation, NMDA and PCP antagonism, and possible serotonin 5HT1b/d receptor stimulation. Additional properties for dextromethorphan include possible presynaptic α(2) adrenoreceptor antagonism or postsynaptic α(2) stimulation and, for dextrorphan, ß stimulation and possible muscarinic and µ antagonism. Treatment-refractory depression properties include increased serotonin and norepinephrine availability, PCP, NMDR-2B, presynaptic alpha-2 antagonism, and the multiplicity of other antidepressant receptor mechanisms. Suggestions for clinical trials are provided for oral high-dose dextromethorphan and Nuedexta (dextromethorphan combined with quinidine to block metabolism to dextrorphan, thereby increasing dextromethorphan plasma concentrations). Suggestions include exclusionary criteria, oral dosing, observation periods, dose-response approaches, and safety and tolerability are considered. Although oral dextromethorphan may be somewhat more likely to show efficacy through complementary antidepressant mechanisms of dextrorphan, a clinical trial will be more logistically complex than one of Nuedexta due to high doses and plasma level variability. Clinical trials may increase our therapeutic armamentarium and our pharmacological understanding of treatment-refractory depression and antidepressant onset of action.

Simultaneous determination of dextromethorphan and its metabolite dextrorphan in plasma samples using second-order calibration coupled with excitation-emission matrix fluorescence.

Dextromethorphan (DEX) is an antitussive agent used in many cough and cold medications, and dextrorphan (DOR) is its metabolite. Owing to their similar structures, optimization of the condition for the chromatography approach, which is in common use for determination, is both demanding and time-consuming. This paper describes a methodology that combines excitation-emission matrix fluorescence spectra with second-order calibration, and was applied to simultaneously and directly determine DEX and DOR contents in plasma samples.

Effect of black seed on dextromethorphan O- and N-demethylation in human liver microsomes and healthy human subjects.

OBJECTIVE: To investigate the effects of black seed on the metabolic activities of CYP3A4 and CYP2D6 in human liver microsomes and in human subjects using dextromethorphan as a probe drug. METHODS: CYP2D6-mediated O-demethylation and CYP3A4-mediated N-demethylation of dextromethorphan (DEX) to dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively, were utilized to assess the metabolic activities of the two enzymatic pathways. In the in vitro experiments, DEX was incubated with microsomes and NADPH in absence or presence of black seed extract (10-100 microg/ml) and the formation of the metabolites were measured by HPLC. In the clinical study, four healthy volunteers received a single oral dose of DEX 30 mg alone in phase I, and along with last dose of black seed (2.5 g twice daily for seven days) in phase II. Activities of the two enzymes were evaluated based on the urinary metabolic ratios (MRs), which were calculated from eight-hour urine collections. DEX and its metabolites were assayed in urine samples by HPLC following a liquid-liquid extraction. RESULTS: Black seed extracts significantly inhibited the formation of both metabolites in microsomes. The maximum inhibition was observed at the highest extract concentration (i.e., 100 microg/ml), which was about 80% and 60% for DOR and 3-MM, respectively. In the clinical study, the urinary MRs of DEX/DOR and DEX/3-MM increased by factors of 127 and 1.6-fold, respectively, after consumption of black seed. CONCLUSION: Black seed significantly inhibited CYP2D6 and CYP3A4 mediated metabolism of DEX in human liver microsomes and healthy human volunteers indicating that it has the potential to interact with CYP2D6 and CYP3A4 substrates.

Intersexual differences in inhibitory influence of trans-resveratrol on activity of cytochrome P450 2D2 in rats.

OBJECTIVES: Differences in the metabolism between males and females have been seen over time. Hormonal regulation of cytochrome P450 activity is understood to be involved. Trans-resveratrol (RES) is an estrogenically active plant polyphenol with many protective biological activities including neuroprotection. The present report studied the influence of sex and RES on variances in rat's cytochrome P450 2D2 hepatic metabolic activity. METHODS AND DESIGN: Isolated perfused rat liver was used for determination of cytochrome P450 2D2 activity. Wistar albino rats of both sexes were treated with RES at the dose of 5 mg/kg/day for 10 days prior to liver isolation. Levels of marker substance dextromethorphan (DEM) and its 2D2 specific metabolite dextrorphan (DEX) were measured during perfusion. The metabolic ratios (DEM/DEX) and the levels of DEM and DEX in perfusate were compared. RESULTS: In the controls, the activity of CYP2D2 was found to be higher in male rats compared to females. RES produced inhibition of CYP2D2, expressed by significant changes of both DEM and DEX levels in males and significant increase of only DEM levels in females. There were no gender changes in DEX levels in RES treated animals whilst DEM levels were significantly increased during the whole perfusion in females. CONCLUSION: The results confirmed gender differences in the metabolic activity of CYP450 2D2 with a higher rate in male rats. RES acted as an inhibitor, however again with greater impact in males than in females. This metabolic divergence could be a cause for different sensitivity or even toxicity of drugs metabolized by the CYP450 2D2.

CYP2D6-CYP2C9 protein-protein interactions and isoform-selective effects on substrate binding and catalysis.

Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.

O-demethylation of dextromethorphan using microbial cultures.

The potential of various microbes to metabolize the dextromethorphan (DXM) , a CYP 2D6 substrate was studied to investigate similarities between microbial and mammalian metabolism. Eight microbes were screened for their ability to metabolize DXM in a manner comparable to humans with a view to develop alternative systems to study human drug metabolism. The major metabolite of DXM produced by Saccharomyces cerevisiae (NCIM 3090) was characterized by HPLC and Liquid Chromatography Mass Spectrometry (LC/MS). DXM was biotransformed by Saccharomyces cerevisiae into a major metabolite dextrorphan which is a known , active metabolite of DXM in human. The microbial metabolism of DXM was similar to the metabolism in mammals. The Saccharomyces cerevisiae could be used as a suitable model strain in vitro to mimic CYP 2D6 metabolism and to produce dextrorphan an active metabolite of DXM for further pharmacological, toxicological studies.

Metabolism of dextrorphan by CYP2D6 in different recombinantly expressed systems and its implications for the in vitro assessment of dextromethorphan metabolism.

Cytochrome P450 2D6 (CYP2D6) mediated formation of dextrorphan (DOR) from dextromethorphan (DEX) is widely used as a marker to assess the activity of this enzyme both in vitro and in vivo. The sequential metabolism of DOR during in vitro studies, particularly using recombinant systems (rCYPs) expressing human CYP2D6, is assumed to be negligible. The extent of metabolism was investigated for a range of DEX and DOR concentrations in microsomal preparations from three different rCYPs expressing human CYP2D6 (yeast, Supersomes and Bactosomes) containing 10 pmol of the enzyme. Bactosomes and Supersomes, but not yeast rCYP microsomes, were capable of metabolising DOR to 3-hydroxymorphinan (HYM). Two novel CYP2D6 related metabolites were identified in Bactosomes, and assigned as single hydroxylations in the phenyl rings of DOR and HYM using ion-trap mass spectrometry. Therefore, in rCYP systems with high turn over rate (e.g. Bactosomes) DOR may not be considered as an end product particularly at low concentrations of DEX; leading to an underestimation of true metabolic rate. The results also put further emphasis on the necessity of optimising study conditions when switching between rCYP sources.

Development and validation of a chemical hydrolysis method for dextromethorphan and dextrophan determination in urine samples: application to the assessment of CYP2D6 activity in fibromyalgia patients.

Dextromethorphan (DEM) is a widely used probe drug for human cytochrome P450 2D6 isozyme activity assessment by measuring the ratio between DEM and its N-demethylated metabolite dextrorphan (DOR). DOR is excreted in urine mainly conjugated to glucuronic acid. Prior to quantification, DOR must be deconjugated to avoid variability caused by the polymorphic glucuronosyltransferase enzyme. A chemical hydrolysis method was optimized using a chemometric approach. Three factors (acid concentration, hydrolysis time and temperature) were selected and simultaneously varied to study their effect on conjugated DOR hydrolysis. Hydrolysis conditions that maximize DOR release without significant degradation of both DEM and DOR were chosen and results were compared to those obtained by enzymatic method using beta-glucuronidase. An HPLC method with fluorescence detection was developed for the simultaneous quantitation of DEM, DOR and levallorphan, used as an internal standard. Separation was performed on a phenyl analytical column (150 mmx4.6 mm i.d., 5 microm) with a mobile phase consisting of 18% acetonitrile and 50 mM phosphoric acid (pH 3). The overall analytical procedure was validated and showed good performances in terms of selectivity, linearity, sensitivity, precision and accuracy. Finally, this assay was used to determine DEM/DOR molar ratios in fibromyalgia patients for the purpose of determining phenotype status for the CYP2D6.

Effect of sodium ozagrel on the activity of rat CYP2D6.

The aim of the study was to investigate the influence of sodium ozagrel on CYP2D6 (cytochromeP450 2D6) activity. The studies were performed with rat urine and liver microsomes and chemical inhibitors. The metabolism of dextromethorphan (dextrophan/dextromethorphan, dextrophan is a metabolite of dextromethorphan) and phenacetin (paracetamol/phenacetin, paracetamol is a metabolites of phenacetin) was used as probe to measure CYP2D6 and CYP1A2 (cytochromeP450 1A2) activity, respectively, determined by high-performance liquid chromatography (HPLC). The results showed that the metabolism of dextrophan/dextromethorphan in the sodium ozagrel-treated group (37 mg/kg) was higher than that of the control (P<0.05/6) in both in vivo and in vitro studies (r=0.9811). The rate of dextromethorphan metabolism was inhibited by sodium ozagrel and cimetidine in rat liver microsomes prepared from sodium ozagrel-treated rats and control rats group (sodium ozagrel IC(50)=26.5 microM, cimetidine IC(50)=86.3 microM in sodium ozagrel-treated group; sodium ozagrel IC(50)=13.9 microM, cimetidine IC(50)=24.8 microM in control group). The inhibitory effect of sodium ozagrel on CYP2D6 activity was noncompetitive with dextromethorphan with a K(i) of 324.94 microM. Kinetic parameters of the reactions were established by using Lineweaver-Burk with K(m)=0.67 mM and V(max)=2.13 pm/min/mg protein for the sodium ozagrel-treated group and K(m)=0.29 mM, and V(max)=0.91 pm/min/mg protein for the control group, respectively. The expression of CYP2D6 protein in the treated group was higher than that of the control group, as determined by Western blotting. The activity and expression of CYP1A2 did not show obvious differences in the control group and sodium ozagrel treated group. In conclusion, sodium ozagrel metabolism in rats is mediated primarily through CYP2D6, and sodium ozagrel can induce CYP2D6 activity.