ABSTRACT
BACKGROUND: There is a lack of information on CYP2D6, a major metabolizing enzyme, in Africa ethnic nationalities. The objective was to determine CYP2D6 phenotype in Yoruba Nigerians using dextromethorphan (DEX). METHOD: A total of 89 healthy volunteers received 30 mg of DEX orally followed by blood and urine sample collection at 3-hour and over 8 h post-dose, respectively. DEX and dextrorphan (DOR) concentrations were determined using High Performance Liquid Chromatography (HPLC). The metabolic ratio (MR, DEX/DOR) were plotted for the phenotype determination. RESULTS: The log MR that separated poor (PMs) from normal metabolizers (NMs) was 0.28 and 0.75 for urine and plasma, respectively. Two subjects (2.3%) identified as PMs had a mean MR of 17 and 3.2 in plasma and urine, significantly higher than that of NMs (p < .0001). A positive correlation between urine and plasma MR was noted. CONCLUSION: The prevalence of PMs in the Yoruba Nigerians was similar to that reported among blacks.
Subject(s)
Black People , Cytochrome P-450 CYP2D6/metabolism , Dextromethorphan/pharmacokinetics , Administration, Oral , Adult , Chromatography, High Pressure Liquid/methods , Dextromethorphan/administration & dosage , Dextrorphan/blood , Dextrorphan/urine , Female , Humans , Male , Middle Aged , Nigeria , Phenotype , Time FactorsABSTRACT
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.
Subject(s)
Dextromethorphan/blood , Dextromethorphan/urine , Excitatory Amino Acid Antagonists/blood , Excitatory Amino Acid Antagonists/urine , Horses/blood , Horses/urine , Administration, Oral , Animals , Antitussive Agents/administration & dosage , Antitussive Agents/blood , Antitussive Agents/metabolism , Antitussive Agents/urine , Chromatography, Liquid/methods , Dextromethorphan/administration & dosage , Dextromethorphan/metabolism , Dextrorphan/blood , Dextrorphan/metabolism , Dextrorphan/urine , Drug Monitoring/methods , Excitatory Amino Acid Antagonists/administration & dosage , Excitatory Amino Acid Antagonists/metabolism , Female , Glucuronides/blood , Glucuronides/metabolism , Glucuronides/urine , Horses/metabolism , Male , Mass Spectrometry/methodsABSTRACT
PURPOSE: Determining metabolic ratio from single-point plasma is potentially a good phenotyping method of CYP2D6 to reduce the required time interval and increase the reliability of data. It is difficult to conduct large sample size clinical trials to evaluate this phenotyping method for multiple plasma points. A physiologically based pharmacokinetic (PBPK) model can be developed to do simulations based on the large virtual Chinese population and evaluate single-point plasma phenotyping method of CYP2D6. METHODS: Pharmacokinetic data of dextromethorphan (DM) and its metabolite dextrorphan (DX) after oral administration were used for model development. The SimCYP® model incorporating Chinese demographic, physiological, and enzyme data was used to simulate DM and DX pharmacokinetics in different phenotype groups. RESULTS: The ratios of the simulated to the observed mean AUC and Cmax of DM were 1.01 and 0.81 for extensive metabolizers (EMs), 0.90 and 0.81 for intermediate metabolizers (IMs), and 1.12 and 0.84 for poor metabolizers (PMs). The ratios of the simulated to the observed mean AUC and Cmax of DX were 1.12 and 0.89 for EMs, 0.66 and 0.62 for IMs. All ratios were within the predefined criterion of 0.5-2. The simulations of DM and DX pharmacokinetic profiles in 1000 virtual Chinese subjects with reported frequencies of different phenotypes indicated that statistically significant correlations were found between metabolic ratio of DM to DX (MRDM/DX) from AUC and from single-point plasma from 1 to 30h (all p-values <0.001). CONCLUSION: MRDM/DX from single-point plasma from 1 to 30h after the administration of 30mg controlled-release DM could predict the MRDM/DX from AUC well and could be used as the phenotyping method of CYP2D6 for EMs, IMs, and PMs.
Subject(s)
Cytochrome P-450 CYP2D6/metabolism , Dextromethorphan/pharmacokinetics , Models, Biological , Adult , Antitussive Agents/blood , Antitussive Agents/pharmacokinetics , Area Under Curve , Asian People , Computer Simulation , Dextromethorphan/blood , Dextrorphan/blood , Female , Humans , Male , Middle Aged , Phenotype , Young AdultABSTRACT
The use of khat (Catha edulis) while on medication may alter treatment outcome. In particular, the influence of khat on the metabolic activities of drug-metabolizing enzymes is not known. We performed a comparative 1-way crossover study to evaluate the effect of khat on cytochrome P450 (CYP)2D6 and CYP3A4 enzyme activity. After 1 week of khat abstinence, baseline CYP2D6 and CYP3A4 metabolic activities were determined in 40 Ethiopian male volunteers using 30 mg dextromethorphan (DM) as a probe drug and then repeated after 1 week of daily use of 400 g fresh khat leaves. Urinary concentrations of cathinone and cathine were determined to monitor the subjects' compliance to the study protocol. Genotyping for CYP2D6*3 and CYP2D6*4 was done. Plasma DM, dextrorphan and 3-methoxymorphinan concentrations were quantified. CYP2D6 and CYP3A4 enzyme activities were assessed by comparing plasma log DM/dextrorphan and log DM/methoxymorphinan metabolic ratio (MR) respectively in the presence and absence of khat. Cytochrome 2D6 MR was significantly increased from baseline by concurrent khat use (paired t test, P = 0.003; geometric mean ratio, 1.38; 95% confidence interval [95% CI], 1.12-1.53). Moreover, the inhibition of CYP2D6 activity by khat was more pronounced in CYP2D6*1/*1 compared with CYP2D6*1/*4 genotypes (P = 0.01). A marginal inhibition of CYP3A4 activity in the presence of khat was observed (P = 0.24). The mean percentage increase of CYP2D6 and CYP3A4 MR from baseline by khat use was 46% (95% CI, 20-72) and 31% (95% CI, 8-54), respectively. This is the first report linking khat use with significant inhibition of CYP2D6 metabolic activity in humans.
Subject(s)
Catha , Cytochrome P-450 CYP2D6 Inhibitors/pharmacology , Cytochrome P-450 CYP2D6/drug effects , Cytochrome P-450 CYP3A/drug effects , Plant Leaves , Adult , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6 Inhibitors/administration & dosage , Cytochrome P-450 CYP3A/genetics , Dextromethorphan/administration & dosage , Dextromethorphan/analogs & derivatives , Dextromethorphan/blood , Dextrorphan/blood , Ethiopia , Excitatory Amino Acid Antagonists/administration & dosage , Excitatory Amino Acid Antagonists/blood , Humans , Male , Young AdultABSTRACT
BACKGROUND: Cytochrome P450 2D6 (CYP2D6) metabolizes around 25% of the drugs used in therapeutics and different polymorphisms have been identified in various populations. This study aimed at finding the prevalence of CYP2D6 polymorphisms using dextromethorphan as a probe drug. MATERIALS AND METHODS: Healthy participants were administered 60 mg dextromethorphan after an overnight fast and 5 ml of blood was collected 3 h postdose. A validated laboratory method was used to measure both dextromethorphan and its active metabolite, dextrorphan from plasma. Metabolic ratio (MR) of dextromethorphan to dextrorphan was calculated for each of the participants. Probit analysis was done and antimode was defined. Individuals with log MR equal to or higher than the antimode were classified as poor metabolizers (PMs) and those with values less than antimode were categorized as extensive metabolizers (EMs). RESULTS: Data from a total of 149 participants were evaluated and the median (range) of MR was 0.25 (0.03-3.01). The polynomial equation obtained in probit analysis gave an antimode for MR of 1.39. Five (3.36%) participants were PMs and 144 (96.64%) were found to be EMs. One participant had reported mild drowsiness 2 h postdose that subsided spontaneously without any intervention. CONCLUSION: The prevalence of CYP2D6 polymorphism in Western Indian population is low (3.36%) and is similar to other populations.
Subject(s)
Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Dextromethorphan/pharmacokinetics , White People/genetics , Adolescent , Adult , Dextromethorphan/blood , Dextrorphan/blood , Female , Humans , India , Male , Phenotype , Polymorphism, Genetic , Young AdultABSTRACT
A sensitive and accurate HPLC-MS/MS method was developed for the simultaneous determination of dextromethorphan, dextrorphan and chlorphenamine in human plasma. Three analytes were extracted from plasma by liquid-liquid extraction using ethyl acetate and separated on a Kromasil 60-5CN column (3 µm, 2.1 × 150 mm) with mobile phase of acetonitrile-water (containing 0.1% formic acid; 50:50, v/v) at a flow rate of 0.2 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The calibration curve was linear over the range of 0.01-5 ng/mL for dextromethorphan, 0.02-5 ng/mL for dextrorphan and 0.025-20 ng/mL for chlorphenamine. The lower limits of quantification for dextromethorphan, dextrorphan and chlorphenamine were 0.01, 0.02 and 0.025 ng/mL, respectively. The intra- and inter-day precisions were within 11% and accuracies were in the range of 92.9-102.5%. All analytes were proved to be stable during sample storage, preparation and analytic procedures. This method was first applied to the pharmacokinetic study in healthy Chinese volunteers after a single oral dose of the formulation containing dextromethorphan hydrobromide (18 mg) and chlorpheniramine malaeate (8 mg).
Subject(s)
Chlorpheniramine/blood , Chromatography, High Pressure Liquid/methods , Dextromethorphan/blood , Dextrorphan/blood , Tandem Mass Spectrometry/methods , Adult , Chlorpheniramine/chemistry , Chlorpheniramine/pharmacokinetics , Dextromethorphan/chemistry , Dextromethorphan/pharmacokinetics , Dextrorphan/chemistry , Dextrorphan/pharmacokinetics , Drug Stability , Female , Humans , Least-Squares Analysis , Male , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , Young AdultABSTRACT
A highly selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantitating dextromethorphan (DXM) and its metabolite dextrophan (DXO) in rat plasma using pirfenidone as an internal standard. Protein precipitation with acetonitrile was employed for the sample preparation. Chromatographic separation was achieved on a SB-C18 column at 25 degrees C, with a gradient elution programme of which acetonitrile-0.1% formic acid in water as mobile phase. The flow rate was 0.4 mL/min. Detection is carried out by multiple reaction monitoring (MRM) on a ion-trap LC-MS/MS system with an electrospray ionization interface. The assay is linear over the range 1-500 ng/mL for DXM and 1-250 ng/mL for DXO, with a lower limit of quantitation of 1 ng/mL for both. Intra- and inter-day precision of the assay were less than 9.80% and the accuracy were in the range 96.35-106.39%. The developed method was successfully applied to analyze the drug in samples of rat plasma for pharmacokinetic study.
Subject(s)
Dextromethorphan/blood , Dextromethorphan/pharmacokinetics , Dextrorphan/blood , Animals , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Indicators and Reagents , Male , Quality Control , Rats , Rats, Wistar , Reproducibility of Results , Specimen Handling , Spectrometry, Mass, Electrospray IonizationABSTRACT
BACKGROUND: An association between CYP2D6 variation and clinical outcomes among women with breast cancer treated with tamoxifen (TAM) has been demonstrated, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes. This association is mainly due to the CYP2D6-mediated hydroxylation of N-desmethyltamoxifen (NDT) to yield endoxifen (EDF), which because of its high antiestrogenic potency, is mainly responsible for the therapeutic efficacy of TAM. The aim of this study was to evaluate the relation of CYP2D6 genotyping and phenotyping with EDF levels and [NDT]/[EDF] metabolic ratio in breast cancer patients from South of Brazil under TAM therapy. METHODS: Trough blood samples were collected from 97 patients. CYP2D6 genotyping was performed with a luminex assay and calculation of genotypic activity scores. Tamoxifen and metabolites EDF, NDT, and 4-hydroxy-TAM were measured in plasma by high performance liquid chromatography with photo diode array detector. CYP2D6 phenotyping was performed by the determination of dextromethorphan (DMT) and dextrorphan (DTF) by high-performance liquid chromatography with fluorescence detection at plasma collected 3 hours after oral administration of 33 mg of DMF. Phenotypes were given according to [DMT]/[DTF] metabolic ratio. RESULTS: CYP2D6 genotyping indicated a prevalence of 4.1% poor metabolizer, 4.1% intermediate metabolizer, 49.5% extensive metabolizer slow activity, 39.2% extensive metabolizer fast activity, and 3.1% ultrarapid metabolizer. Genotype (genotypic activity scores) was significantly correlated with phenotype ([DMT]/[DTF]), with a moderate association (rs = -0.463; P < 0.001). Median plasma concentrations (nanograms per milliliter; N = 97) were TAM 57.17; 4-hydroxy-TAM 1.01; EDF 6.21; NDT 125.50. EDF levels were lower in poor metabolizers than that in extensive metabolizers (P < 0.05). Phenotype showed stronger, but still moderate, association with EDF and [NDT]/[EDF] than genotype (r = -0.507, r = 0.625, P < 0.001 versus r = 0.356, r = 0.516, P < 0.01). Phenotype accounted for 26% of the variability in EDF levels and 38% of [NDT]/[EDF], whereas genotype accounted for 12% and 27%, respectively. CONCLUSIONS: CYP2D6 genotyping and/or phenotyping could not fully predict EDF concentrations. Monitoring EDF itself could be considered during TAM therapy.
Subject(s)
Breast Neoplasms/blood , Breast Neoplasms/drug therapy , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacokinetics , Tamoxifen/therapeutic use , Adult , Aged, 80 and over , Brazil , Breast Neoplasms/genetics , Cytochrome P-450 CYP2D6 Inhibitors , Dextromethorphan/blood , Dextrorphan/blood , Female , Genotype , Humans , Hydroxylation , Middle Aged , Phenotype , Tamoxifen/bloodABSTRACT
In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0min at a flow-rate of 1.5 mL min⻹ into a Phenomenex Gemini® C18, 5 µm analytical column (150 × 4.6 mm i.d.). The calibration curve was linear over the range from 0.2 to 200 ng mL⻹ for dextromethorphan and doxylamine and 0.05 to 10 ng mL⻹ for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study.
Subject(s)
Chromatography, High Pressure Liquid/methods , Dextromethorphan/blood , Dextrorphan/blood , Doxylamine/blood , Spectrometry, Mass, Electrospray Ionization/methods , Adolescent , Adult , Dextromethorphan/pharmacokinetics , Dextrorphan/pharmacokinetics , Doxylamine/pharmacokinetics , Female , Humans , Male , Middle Aged , Sensitivity and Specificity , Tandem Mass Spectrometry/methods , Young AdultABSTRACT
PURPOSE: Cytochrome P450 2D6 (CYP2D6) genotypes and the dextromethorphan/dextrorphan (DXM/DXT) metabolic ratio (MR), which is a marker of CYP2D6 activity, were studied in 118 unrelated healthy Ecuadorians. METHODS: Genotyping of CYP2D6 was performed by amplification of entire CYP2D6 gene by XL-PCR for CYP2D6*5 and multiplication alleles and by real time-PCR for CYP2D6 *2, *3, *4, *6, *10, *17, *29, *35, *41, and copy number. The plasma levels of DXM and its metabolite DXT were determined on a high-performance liquid chromatography-UV system. RESULTS: The proportions of non-functional alleles were 0.4, 10.6, 0.8, 2.1, and 0% for CYP2D6*3, *4, *4 × N, *5, and *6, respectively. Genotypically, only one of the subjects (0.9%) was homozygous for two inactive alleles and phenotypically classified as a poor metabolizer (PM). The MRs (mean ± standard deviation) corresponding to "activity scores" of 0, 0.5, 1, 1.5, 2, and 2.5 were 10.57 (n = 1), 1.63 ± 0.35 (n = 2), 1.16 ± 0.74 (n = 29), 1.00 ± 0.47 (n = 8), 1.24 ± 0.82 (n = 76), and 1.30 ± 0.32 (n = 2), respectively. CONCLUSIONS: Our data suggest that only 1% of subjects of this Ecuadorian population were PMs and that none were phenotypically ultrarapid metabolizers, which is in agreement with previous findings in other Amerindian populations.
Subject(s)
Antitussive Agents/pharmacokinetics , Cytochrome P-450 CYP2D6/genetics , Dextromethorphan/pharmacokinetics , Polymorphism, Genetic , Adolescent , Adult , Alleles , Antitussive Agents/blood , Biotransformation , Cytochrome P-450 CYP2D6/metabolism , Dextromethorphan/blood , Dextrorphan/blood , Ecuador , Female , Gene Dosage , Gene Frequency , Genetic Association Studies , Humans , Hydroxylation , Male , Young AdultABSTRACT
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.
Subject(s)
Dextromethorphan/blood , Dextrorphan/blood , Calibration , Dextrorphan/metabolism , Humans , Spectrometry, FluorescenceABSTRACT
A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the simultaneous quantitative determination of dextromethorphan (DM) and its metabolites dextrorphan (DX), 3-methoxymorphinan (3MM) and 3-hydroxymorphinan (3HM), in human lithium heparinized plasma. The extraction involved a simple liquid-liquid extraction with 1 ml n-butylchloride from 200µl aliquots of plasma, after the addition of 20 µl 4% (v/v) ammonium hydroxide and 100 µl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC(®) BEH C(18) 1.7 µm 2.1 mm x 100mm column eluted at a flow-rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7 min, with elution times of 1.3min for DX and 3HM, 2.8 min for 3MM and 2.9min for DM. The multiple reaction monitoring transitions were set at 272>215 (m/z), at 258>133 (m/z), at 258>213 (m/z) and at 244>157 (m/z) for DM, DX, 3MM and 3HM, respectively. The calibration curves were linear (r²≥0.995) over the range of 0.500-100 nM with the lower limit of quantitation validated at 0.500 nM for all compounds, which is equivalent to 136, 129, 129 and 122 pg/ml for DM, DX, 3MM and 3HM, respectively. Extraction recoveries were constant, but ranged from 39% for DM to 83% for DX. The within-run and between-run precisions were within 11.6%, while the accuracy ranged from 92.7 to 110.6%. The applicability of the bioanalytical method was demonstrated and is currently implemented in a clinical trial to study DM as probe-drug for individualized tamoxifen treatment in breast cancer patients.
Subject(s)
Dextromethorphan/analogs & derivatives , Dextromethorphan/blood , Dextrorphan/blood , Mass Spectrometry/methods , Chromatography, Liquid , Dextromethorphan/administration & dosage , Humans , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
The pharmacokinetics of dextromethorphan (DM) is markedly influenced by cytochrome P450 2D6 (CYP2D6) enzyme polymorphisms. The aim of this study was to quantify the effects of the CYP2D6*1, *2, and *41 variants on DM metabolism in vivo and to identify other sources of pharmacokinetic variability. Concentrations of DM and dextrorphan (DO) in plasma and urine were evaluated in 36 healthy Caucasian men. These volunteers participated in three clinical studies and received a single oral dose of 30 mg DM-HBr. Data were modeled simultaneously using the population pharmacokinetics NONMEM software. A five-compartment model adequately described the data. The activity levels of the alleles assessed differed significantly. The clearance attributable to an individual CYP2D6*1 copy was 2.5-fold higher as compared with CYP2D6*2 (5,010 vs. 2,020 l/h), whereas the metabolic activity of CYP2D6*41 was very low (85 l/h). Urinary pH was confirmed as a significant covariate for DM renal clearance. These results refine genotype-based predictions of pharmacokinetics for DM and presumably for other CYP2D6 substrates as well.
Subject(s)
Antitussive Agents/pharmacokinetics , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Dextromethorphan/pharmacokinetics , Dextrorphan/pharmacokinetics , Models, Biological , Polymorphism, Genetic , Administration, Oral , Adult , Antitussive Agents/administration & dosage , Antitussive Agents/blood , Antitussive Agents/urine , Biotransformation , Clinical Trials as Topic , Dextromethorphan/administration & dosage , Dextromethorphan/blood , Dextromethorphan/urine , Dextrorphan/blood , Dextrorphan/urine , Gene Frequency , Genotype , Humans , Hydrogen-Ion Concentration , Male , Middle Aged , Phenotype , White People/genetics , Young AdultABSTRACT
For the first time, a highly sensitive and simple LC-MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C(18) Column (100 mm x 2.0 mm, 3 microm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10-5000 ng/mL for PA, 2-1000 ng/mL for PE, 0.05-25 ng/mL for DT and 0.1-50 ng/mL for CP. The accuracies as determined from quality control samples were in range of -8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.
Subject(s)
Acetaminophen/blood , Chromatography, Liquid/methods , Dextrorphan/blood , Pseudoephedrine/blood , Tandem Mass Spectrometry/methods , Drug Stability , Humans , Linear Models , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Dextromethorphan is used as a probe drug for assessing CYP2D6 and CYP3A4 activity in vivo and in vitro. A SIM GC/MS method without derivatization for the simultaneous determination of dextromethorphan and its metabolites, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan, in human plasma, urine and in vitro incubation matrix was developed and validated. Calibration curves indicated good linearity with a coefficient of variation (r) better than 0.995. The lower limit of quantitation was found to be 10 ng/mL for all analytes in all matrices. Intra-day and inter-day precision for dextromethorphan and its metabolites was better than 9.02 and 9.91%, respectively and accuracy ranged between 91.76 and 106.27%. Recovery for dextromethorphan, its metabolites and internal standard levallorphan was greater than 72.68%. The method has been successfully applied for the in vitro inhibition of metabolism of dextromethorphan by CYP2D6 and CYP3A4 using known inhibitors of CYPs such as quinidine and verapamil.
Subject(s)
Cytochrome P-450 CYP2D6 Inhibitors , Cytochrome P-450 CYP3A Inhibitors , Dextromethorphan/analogs & derivatives , Dextromethorphan/analysis , Dextromethorphan/metabolism , Dextrorphan/analysis , Antitussive Agents/blood , Antitussive Agents/metabolism , Antitussive Agents/urine , Calibration , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP3A/metabolism , Dextromethorphan/blood , Dextromethorphan/urine , Dextrorphan/blood , Dextrorphan/urine , Gas Chromatography-Mass Spectrometry , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC-MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2-200ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.
Subject(s)
Blood Specimen Collection/methods , Dextromethorphan/blood , Dextrorphan/blood , Chromatography, High Pressure Liquid , Humans , Tandem Mass SpectrometryABSTRACT
AIMS: To investigate the influence of different cytochrome P450 (CYP) activities and other potential covariates on the disposition of methadone in patients on methadone maintenance therapy (MMT). METHODS: Eighty-eight patients (58 male; 21-55 years; 84 White) on MMT were studied. CYP2D6 activity [3 h plasma metabolic ratio of dextromethorphan (DEX) to dextrorphan (DOR)] was determined in 44 patients (29 male; 24-55 years), CYP1A2 activity (salivary caffeine elimination half-life) in 44 patients (21 male; 24-55 years) and CYP3A activity (oral clearance of midazolam) in 49 patients (33 male; 23-55 years). Data on all three CYPs were obtained from 32 subjects. Total plasma concentrations of (RS)-methadone and total and unbound plasma concentrations of both enantiomers were measured by LC/MS. Population pharmacokinetics and subsequent multiple regression analysis were used to calculate methadone oral clearance and to identify its covariates. RESULTS: Between 61 and 68% of the overall variation in total plasma trough concentrations of (RS)-, (R)- and (S)-methadone was explained by methadone dose, duration of addiction before starting MMT, CYP3A activity and illicit morphine use. CYP3A activity explained 22, 16, 15 and 23% of the variation in unbound (R)-, unbound (S)-, total (RS)- and total (S)-methadone clearances, respectively. Neither CYP2D6 nor CYP1A2 activity was related to methadone disposition. CONCLUSIONS: CYP3A activity has a modest influence on methadone disposition. Inhibitors and inducers of this enzyme should be monitored in patients taking methadone.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Methadone/pharmacokinetics , Narcotics/pharmacokinetics , Opioid-Related Disorders/rehabilitation , Adult , Biomarkers/blood , Caffeine/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP3A/metabolism , Dextromethorphan/blood , Dextrorphan/blood , Female , Humans , Isomerism , Male , Methadone/blood , Methadone/therapeutic use , Midazolam/metabolism , Middle Aged , Narcotics/blood , Narcotics/therapeutic use , Opioid-Related Disorders/metabolism , Saliva/chemistry , Young AdultABSTRACT
A simple and improved HPLC method with fluorometric detection for simultaneous determination of dextromethorphan (DM) and its three metabolites (dextrorphan (DX), 3-methoxymorphinan (MM), 3-hydroxymorphinan (HM)) in human plasma was developed and validated. The method involved a simple and efficient extraction protocol using an n-heptane/ethyl acetate (1:1) solvent mixture that achieved recoveries of 70-90% with an insignificant interference from the plasma matrix. The analysis was performed on a phenyl column with isocratic elution, a mobile phase composed of 20% methanol, 30% acetonitrile, and 50% KH2PO4 buffer (10mM, with adding 0.02% of TEA; adjusted with phosphoric acid to pH 3.5), and a run time of only 15 min. Linear calibration curves were constructed in the concentration range of 1-200 nM for DM and its three metabolites. The lower limit of quantitation (LLOQ) in human plasma was 1 nM for each compound. The coefficient of variation and RSE% of the intraday and interday analyses for DM and its three metabolites all complied with USFDA requirements. This analytical method was preliminarily applied to determine the polymorphic functions of CYP2D6 and CYP3A4 in the metabolic pathway of DM to DX and then to HM.
Subject(s)
Chromatography, High Pressure Liquid/methods , Dextromethorphan/analogs & derivatives , Dextromethorphan/blood , Dextrorphan/blood , Spectrometry, Fluorescence , Chromatography, High Pressure Liquid/instrumentation , Dextromethorphan/administration & dosage , Humans , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
Honey is a common food supplement but not many studies have studied honey and drug interaction. This study investigates the influence of 7 days of honey administration on the activity of CYP3A4, CYP2D6 and CYP2C19 drug-metabolizing enzymes in healthy volunteers by using appropriate biomarker and probe drugs. A within-group pharmacokinetic study was done in 12 healthy volunteers. Urine samples (0-8 hr) were collected after administration of 30 mg of oral dextromethorphan (probe drug for CYP2D6) for analysis of dextromethorphan and dextrorphan. A plasma sample (4 hr) was collected after administration of 200 mg of oral proguanil (probe drug for CYP2C19) for the analysis of proguanil and cycloguanil. Urine samples (0-24 hr) were collected for the analysis of 6beta-hydroxycortisol (biomarker for CYP3A4). The volunteers were administered honey for 7 days. Subsequently blood and urine samples were collected after drug dosing as before. These samples were analysed for drug and metabolite concentrations in urine and plasma using high performance liquid chromatography method. Seven days of honey administration resulted in statistically significant increase in 24-hr urinary excretion of 6beta-hydroxycortisol. However, the metabolic ratios of dextromethorphan and proguanil were not significantly altered after 7 days of honey administration. Honey obtained from Western Ghats of southern India may induce CYP3A4 enzyme activity but not CYP2D6 and CYP2C19 enzyme activities.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 Enzyme System/metabolism , Honey , Mixed Function Oxygenases/metabolism , Administration, Oral , Adult , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP2C19 , Cytochrome P-450 CYP3A , Dextromethorphan/blood , Dextromethorphan/metabolism , Dextromethorphan/pharmacokinetics , Dextrorphan/blood , Humans , Hydrocortisone/analogs & derivatives , Hydrocortisone/urine , India , Male , Proguanil/metabolism , Proguanil/pharmacokinetics , Proguanil/urine , Triazines/urineABSTRACT
In vitro-in vivo extrapolation of clearance, embedded in a clinical trial simulation, was used to investigate differences in the pharmacokinetics and pharmacodynamics of dextromethorphan between CYP2D6 poor and extensive metabolizer phenotypes. Information on the genetic variation of CYP2D6, as well as the in vitro metabolism and pharmacodynamics of dextromethorphan and its active metabolite dextrorphan, was integrated to assess the power of studies to detect differences between phenotypes. Whereas 6 subjects of each phenotype were adequate to achieve 80% power in showing pharmacokinetic differences, the power required to detect a difference in antitussive response was less than 80% with 500 subjects in each study arm. Combining in vitro-in vivo extrapolation with a clinical trial simulation is useful in assessing different elements of study design and could be used a priori to avoid inconclusive pharmacogenetic studies.