CYP2D6 Activity Is Correlated with Changes in Plasma Concentrations of Taurocholic Acid during Pregnancy and Postpartum in CYP2D6 Extensive Metabolizers.

Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of >20% of marketed drugs. CYP2D6 expression and activity exhibit high interindividual variability and is induced during pregnancy. The farnesoid X receptor (FXR) is a transcriptional regulator of CYP2D6 that is activated by bile acids. In pregnancy, elevated plasma bile acid concentrations are associated with maternal and fetal risks. However, modest changes in bile acid concentrations may occur during healthy pregnancy, thereby altering FXR signaling. A previous study demonstrated that hepatic tissue concentrations of bile acids positively correlated with the hepatic mRNA expression of CYP2D6. This study sought to characterize the plasma bile acid metabolome in healthy women (n = 47) during midpregnancy (25-28 weeks gestation) and ≥3 months postpartum and to determine if plasma bile acids correlate with CYP2D6 activity. It is hypothesized that during pregnancy, plasma bile acids would favor less hydrophobic bile acids (cholic acid vs. chenodeoxycholic acid) and that plasma concentrations of cholic acid and its conjugates would positively correlate with the urinary ratio of dextrorphan/dextromethorphan. At 25-28 weeks gestation, taurine-conjugated bile acids comprised 23% of the quantified serum bile acids compared with 7% ≥3 months postpartum. Taurocholic acid positively associated with the urinary ratio of dextrorphan/dextromethorphan, a biomarker of CYP2D6 activity. Collectively, these results confirm that the bile acid plasma metabolome differs between pregnancy and postpartum and provide evidence that taurocholic acid may impact CYP2D6 activity during pregnancy. SIGNIFICANCE STATEMENT: Bile acid homeostasis is altered in pregnancy, and plasma concentrations of taurocholic acid positively correlate with CYP2D6 activity. Differences between plasma and/or tissue concentrations of farnesoid X receptor ligands such as bile acids may contribute to the high interindividual variability in CYP2D6 expression and activity.

A clinical pharmacokinetic drug-drug interaction study between dextromethorphan and emvododstat, a potent anti-SARS-CoV-2 dihydroorotate dehydrogenase inhibitor.

PURPOSE: A therapeutic agent that targets both viral replication and the hyper-reactive immune response would offer a highly desirable treatment for severe acute respiratory syndrome corona virus 2 (SARS-CoV-2, coronavirus disease 2019, COVID-19) management. Emvododstat (PTC299; 4-chlorophenyl 6-chloro-1-[4-methoxyphenyl]-1,3, 4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate) was found to be a potent inhibitor of immunomodulatory and inflammation-related processes by inhibition of dihydroorotate dehydrogenase to reduce the severity of SARS-CoV-2 infections This drug interaction study was performed to determine if emvododstat was an inhibitor of CYP2D6. METHODS: Potential drug-drug interactions between emvododstat and a CYP2D6 probe substrate (dextromethorphan) were investigated by measuring plasma dextromethorphan and metabolite (dextrorphan) concentrations before and after emvododstat administration. On day 1, 18 healthy subjects received an oral dose of 30 mg dextromethorphan followed by a 4-day washout period. On day 5, subjects received an oral dose of 250 mg emvododstat with food. Two hours later, 30 mg dextromethorphan was administered. RESULTS: When given with emvododstat, plasma dextromethorphan concentrations increased substantially, while metabolite levels (dextrorphan) remained essentially the same. Maximum plasma dextromethorphan concentration (Cmax) increased from 2006 to 5847 pg/mL. Dextromethorphan exposure (AUC) increased from 18,829 to 157,400 h·pg/mL for AUC0-last and from 21,585 to 362,107 h·pg/mL for AUC0-inf following administration of emvododstat. When dextromethorphan parameters were compared before and after emvododstat, least squares mean ratios (90% confidence interval) were found to be 2.9 (2.2, 3.8), 8.4 (6.1, 11.5), and 14.9 (10.0, 22.1) for Cmax, AUC0-last, and AUC0-inf, respectively. CONCLUSION: Emvododstat appears to be a strong CYP2D6 inhibitor. No drug-related treatment emergent adverse effects (TEAEs) were considered to be severe or serious. TRIAL REGISTRATION: EudraCT 2021-004626-29, 11 May 2021.

Evaluation of Drug-Drug Interaction Potential of Enarodustat (JTZ-951) Using a Cytochrome P450 Probe Cocktail.

The drug interaction potential of enarodustat (doses: 25, 50 mg) on the activity of cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 was evaluated after once-daily administration for 15 days in a phase 1 multiple-ascending-dose study in healthy subjects. Probe substrates specific for the enzymes, i.e., caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4), were administered orally as a cocktail with (day 15) and without (day -3) enarodustat. Drug interaction was based on geometric mean maximum plasma concentration (Cmax ) and area under the plasma concentration-time curve from the time of dosing to infinity (AUCinf ) ratios (day 15/day -3) for CYP1A2, 2C9, 2C19, 2D6, 3A4, and urinary excretion of dextromethorphan metabolite dextrorphan for CYP2D6. At the 2 enarodustat doses, for caffeine, the geometric mean ratios (range) for Cmax and AUCinf were 0.99-1.06 and 1.61-1.63, respectively. The ratios for peak concentrations and total exposures were 0.98-1.07 and 0.71-1.78 for tolbutamide and omeprazole, respectively. For dextrorphan the Cmax and AUCinf ratios were 0.83-0.90 and 1.02-1.04, respectively. The mean dextrorphan cumulative amount excreted into the urine from the time of dosing to 24 hours values on day -3 and day 15 were 8.25 mg and 8.20 mg at the lower dose, and 9.40 mg and 9.51 mg at the higher dose. The ratios for midazolam Cmax and AUCinf were 1.42-1.63. Overall, there was a lack of enarodustat dose dependency regarding the geometric mean ratios and 90% confidence intervals and urinary excretion of dextrorphan. There were some cases where the 90% confidence intervals at the 2 enarodustat doses were outside the 0.80-1.25 range, but changes in the geometric mean ratios were all <2-fold.

Analysis, occurrence, and consumption of substances with abuse potential in Xinjiang, China, from 2021 to 2022.

Understanding the consumption patterns of substances with abuse potential in the population is critical in combating drug crimes in the region. In recent years, wastewater-based drug monitoring has become a complementary tool worldwide. This study aimed to use this approach to understand the long-term consumption patterns of abuse potential substances in Xinjiang, China (2021-2022) and to provide more detailed and practical information on the current system. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to quantify the levels of abuse potential substances in wastewater. Subsequently, the detection rate and contribution rate of the drug concentrations were evaluated through analysis. Eleven of abuse potential substances were detected in this study. The influent concentrations ranged from (0.48 ng/L) to 133.41 ng/L, with dextrorphan having the highest concentration. The highest detection frequency rates were for morphine (82 %), dextrorphan (59 %), 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (43 %), methamphetamine (36 %), and tramadol (24 %). According to a study on wastewater treatment plants (WWTPs) removal efficiency, compared to the total removal efficiency in 2021, the total removal efficiency of WWTP1, WWTP3, and WWTP4 increased in 2022, while WWTP2 decreased slightly, and WWTP5 did not change significantly. Upon examination of the use of 18 selected analytes, it was determined that methadone, 3,4-methylenedioxy methamphetamine, ketamine, and cocaine were the primary substances of abuse in the Xinjiang region. This study identified significant abuse substances in Xinjiang and identified research priorities. Future studies should consider expanding the study site to obtain a comprehensive understanding of the consumption patterns of these substances in Xinjiang.

Development of an enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of methorphan and its O-demethylated metabolite in human blood and its application to post-mortem samples.

In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both.

Low-volume plasma sampling for determination of dextromethorphan and dextrorphan in rat plasma: LC-MS/MS method and its application in pharmacokinetic study.

Dextromethorphan (DM) and its metabolite dextrorphan (DX) continue to draw the attention of researchers owing to their diverse pharmacodynamics. Thus, there are possibilities for repurposing DM. Most of the pharmacodynamics of DM needs further validation in different preclinical models. Also, it is necessary to correlate the pharmacodynamics with relevant pharmacokinetics data. Multiple bioanalytical techniques developed for this purpose primarily use a high sample processing volume. Since sample volume is a limiting factor for many preclinical models, an effort was taken to develop an alternative method suitable for handling low sample processing volumes. An efficient solid-phase extraction technique, robust liquid chromatographic (LC) separation and highly sensitive tandem mass spectrometric detection (MS/MS) showed suitability for use of a 30 µl sample processing volume. This led to the development of a highly specific, selective, accurate and precise-bio-analytical method for simultaneous quantification of DM and DX in rat plasma. The validated method was linear in the range of 0.196-403.356 ng/ml for DM and 0.102-209.017 ng/ml for DX. The application of the method was demonstrated through the estimation of pharmacokinetic parameters that showed good congruence with earlier studies.

A longitudinal study of cytochrome P450 2D6 (CYP2D6) activity during adolescence.

CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7-15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4-h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention-deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model-estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype-based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.

Physiologically-based pharmacokinetic modeling of dextromethorphan to investigate interindividual variability within CYP2D6 activity score groups.

This study provides a whole-body physiologically-based pharmacokinetic (PBPK) model of dextromethorphan and its metabolites dextrorphan and dextrorphan O-glucuronide for predicting the effects of cytochrome P450 2D6 (CYP2D6) drug-gene interactions (DGIs) on dextromethorphan pharmacokinetics (PK). Moreover, the effect of interindividual variability (IIV) within CYP2D6 activity score groups on the PK of dextromethorphan and its metabolites was investigated. A parent-metabolite-metabolite PBPK model of dextromethorphan, dextrorphan, and dextrorphan O-glucuronide was developed in PK-Sim and MoBi. Drug-dependent parameters were obtained from the literature or optimized. Plasma concentration-time profiles of all three analytes were gathered from published studies and used for model development and model evaluation. The model was evaluated comparing simulated plasma concentration-time profiles, area under the concentration-time curve from the time of the first measurement to the time of the last measurement (AUClast ) and maximum concentration (Cmax ) values to observed study data. The final PBPK model accurately describes 28 population plasma concentration-time profiles and plasma concentration-time profiles of 72 individuals from four cocktail studies. Moreover, the model predicts CYP2D6 DGI scenarios with six of seven DGI AUClast and seven of seven DGI Cmax ratios within the acceptance criteria. The high IIV in plasma concentrations was analyzed by characterizing the distribution of individually optimized CYP2D6 kcat values stratified by activity score group. Population simulations with sampling from the resulting distributions with calculated log-normal dispersion and mean parameters could explain a large extent of the observed IIV. The model is publicly available alongside comprehensive documentation of model building and model evaluation.

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.

Ultrahigh-Throughput ESI-MS: Sampling Pushed to Six Samples per Second by Acoustic Ejection Mass Spectrometry.

We present an acoustic ejection mass spectrometry (AEMS) setup for contactless electrospray ionization mass spectrometry (ESI-MS)-based sample injection at a sampling rate faster than current ESI and matrix-assisted laser desorption ionization (MALDI) techniques. For the direct transfer of samples out of 384-well plates into a modified ESI source, an open port interface (OPI) was combined with a modified acoustic droplet ejection (ADE) system. AEMS has the potential to eliminate bottlenecks known from classical MS approaches, such as speed, reproducibility, carryover, ion suppression, as well as sample preparation and consumption. This setup provided a drastically reduced transfer distance between OPI and ESI electrode for optimum throughput performance and broadens the scope of applications for this emerging technique. To simulate label-free applications of drug metabolism and pharmacokinetics (DMPK) analysis and high-throughput screening (HTS) campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. The maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at full width at half-maximum (FWHM) of 105 ms and a relative standard deviation (%RSD) of 7.7/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51/73% at %RSD of 5.7/6.7% (n = 120). An HTS endurance buffer was used for >25 000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10 557). The optimized hardware and method setup resulted in high-throughput performance and enables further implementation in a fully automated platform for ESI-MS-based high-throughput screening.

Dextromethorphan and Dextrorphan Influence Insulin Secretion by Interacting with KATP and L-type Ca2+ Channels in Pancreatic ß-Cells.

The NMDA receptor antagonist dextromethorphan (DXM) and its metabolite dextrorphan (DXO) have been recommended for treatment of type 2 diabetes mellitus because of their beneficial effects on insulin secretion. This study investigates how different key points of the stimulus-secretion coupling in mouse islets and ß-cells are influenced by DXM or DXO. Both compounds elevated insulin secretion, electrical activity, and [Ca2+]c in islets at a concentration of 100 µM along with a stimulating glucose concentration. DXO and DXM increased insulin secretion approximately 30-fold at a substimulatory glucose concentration (3 mM). Patch-clamp experiments revealed that 100 µM DXM directly inhibited KATP channels by about 70%. Of note, DXM decreased the current through L-type Ca2+ channels about 25%, leading to a transient reduction in Ca2+ action potentials. This interaction might explain why elevating DXM to 500 µM drastically decreased insulin release. DXO inhibited KATP channels almost equally. In islets of KATP channel-deficient sulfonylurea receptor 1 knockout mice, the elevating effects of 100 µM DXM on [Ca2+]c and insulin release were completely lost. By contrast, 100 µM DXO still increased glucose-stimulated insulin release around 60%. In summary, DXM-induced alterations in stimulus-secretion coupling of wild-type islets result from a direct block of KATP channels and are partly counteracted by inhibition of L-type Ca2+ channels. The stimulatory effect of DXO seems to be based on a combined antagonism on KATP channels and NMDA receptors and already occurs under resting conditions. Consequently, both compounds seem not to be suitable candidates for treatment of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: This study shows that the use of dextromethorphan as an antidiabetic drug can cause unpredictable alterations in insulin secretion by direct interaction with KATP and L-type Ca2+ channels besides its actual target, the NMDA receptor.

Determination of dextromethorphan and dextrorphan solar photo-transformation products by LC/Q-TOF-MS: Laboratory scale experiments and real water samples analysis.

This work discusses the identification of the transformation products (TPs) generated during the photolytic degradation of dextromethorphan (DXM) and its metabolite dextrorphan (DXO), under simulated solar radiation in aqueous solutions (Milli-Q water and river water) in order to determinate its behavior into the aquatic environment. Tentative identification of the TPs was performed by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS), following a suspect screening approach. The use of high resolution-mass spectrometry (HRMS) allowed the tentative identification of DXM and DXO photoproducts based on the structure proposed by an in silico software, the accurate mass measurement, the MS/MS fragmentation pattern and the molecular formula finding. A total of 19 TPs were found to match some of the accurate masses included in a suspect list, and they were all tentatively identified by their characteristic MS-MS fragments. Most of the TPs identified showed a minor modified molecular structure like the introduction of hydroxyl groups, or demethylation. The time-evolution of precursors and TPs were monitored throughout the experiments, and degradation kinetics were presented for each analyte. Finally, the occurrence of DXM, DXO, and their tentatively proposed photodegradation TPs was evaluated in both surface and wastewater. In all real matrices, the results showed that the highest concentration was detected for DXO, followed by TP-244 (N-desmethyldextrorphan) and DXM.

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.

Analysis of Dextromethorphan and Three Metabolites in Decomposed Skeletal Tissues by UPLC-QToF-MS: Comparison of Acute and Repeated Drug Exposures.

Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) analysis of dextromethorphan (DXM) and its metabolites-dextrorphan, 3-methoxymorphinan (3-MEM) and 3-hydroxymorphinan-in skeletal remains of rats exposed to DXM under different dosing patterns is described. Rats (n = 20) received DXM in one of four dosing patterns: acute (ACU1 or ACU2-100 or 200 mg/kg, i.p.; n = 5, respectively) or repeated (REP1 or REP2-3 doses of 25 or 50 mg/kg, i.p., 30 min apart; n = 5, respectively). Drug-free animals (n = 5) served as negative controls. Following euthanasia, the animals decomposed to skeleton outdoors. Bones were sorted by animal and skeletal element (vertebra, femur, pelvis, tibia, rib and skull), washed, air-dried and pulverized prior to dynamic methanolic drug extraction, filtration/pass-through extraction and analysis by UPLC-QToF-MS in positive electrospray ionization mode. Analyte levels (expressed as mass-normalized response ratios, RR/m) differed significantly between ACU1 and ACU2 (Mann-Whitney (MW), P < 0.05) in all skeletal elements for all analytes investigated, and between REP1 and REP2 in most skeletal elements for 3-MEM and 3-HOM, but in all skeletal elements for DXM. Between ACU1 and ACU2, and between REP1 and REP2, analyte level ratios (RRi/RRj) differed significantly (MW, P < 0.05) in 3/6 to 6/6 skeletal elements, depending on the ratios concerned, with no analyte level ratio differing significantly between both ACU1 vs ACU2 and REP1 vs REP2. Kruskal-Wallis (KW) analysis showed skeletal element to be a main effect for all analyte levels and analyte level ratios in all ACU and REP groups examined (P < 0.05). For data pooled only according to exposure pattern, KW analysis showed dose pattern to be a main effect for both analyte levels and analyte level ratios (P < 0.05). These data illustrate a dependence of these measures on dose, dose pattern and skeletal element, suggesting that some exposure patterns may be distinguished by toxicological analysis of bone.

A novel and simple LC-MS/MS quantitative method for dextromethorphan and dextrorphan in oral fluid.

Aim: To develop and validate a simple method using LC-MS/MS for determination of dextromethorphan (DXM) and dextrorphan (DT) in human oral fluid. Results: Following protein precipitation, chromatographic separation used a phenyl column with isocratic elution (1 ml/min) of 10 mM ammonium-formate buffer and acetonitrile (65:35; v/v) with 0.1% formic acid. Retention times were 2.6 min for DT and 5 min for DXM. Total run time was 7 min. The intra- and inter-assay deviations (accuracy) for DT (1-100 ng/ml) and DXM (5-1000 ng/ml) ranged from -13.6 to 8.8% and -9.6 to 5.7%, respectively. Precision variations were ≤7.5%. Matrix effect was ≤11.8%. Conclusion: This method may prove helpful for quantification of DT and DXM in oral fluid for either clinical or toxicological purposes.

Dopamine enhancement of dextrorphan-induced skin antinociception in response to needle pinpricks in rats.

BACKGROUND: Dextrorphan with long-acting local anesthetic effects did not cause system toxicity as fast as bupivacaine, while catecholamines (i.e., epinephrine) with the vasoconstrictive characteristics enhanced the effects of local anesthetic drugs. The objective of the experiment was to examine the synergistic effect of local dopamine (a catecholamine) injection on cutaneous antinociception of dextrorphan. METHODS: The panniculus reflex in response to skin stimulation with a needle was used as the primary endpoint when dextrorphan (1.50, 2.61, 5.46, 10.20 and 20.40 µmol) alone, dopamine (16.20, 32.40, 51.60, 60.00 and 81.60 µmol) alone, or dopamine + dextrorphan (a ratio of ED50vs. ED50) was injected subcutaneously on the rat's back. We used an isobolographic modelling approach to determine whether a synergistic effect would be observed. RESULTS: We showed that dextrorphan, dopamine, or the mixture of dopamine and dextrorphan produced dose-related skin antinociception. The potency (ED50, 50% effective dose) for cutaneous antinociception was dextrorphan [6.02 (5.93-6.14) µmol] greater than dopamine [48.91 (48.80-49.06) µmol] (p < 0.01). The duration of nociceptive inhibition induced by dopamine was longer than that induced by dextrorphan (p < 0.01) based on their equipotent doses (ED25, ED50, and ED75). Enhancement and prolongation of skin antinociception occurred after co-administration of dopamine with dextrorphan. CONCLUSIONS: When compared to dopamine, dextrorphan was more potent and had a shorter duration of skin nociceptive block. Dopamine produced a synergistic effect on dextrorphan-mediated antinociception, and prolonged dextrorphan's antinociceptive duration.

Does In Vitro Cytochrome P450 Downregulation Translate to In Vivo Drug-Drug Interactions? Preclinical and Clinical Studies With 13-cis-Retinoic Acid.

All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted ~ 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.

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.

Identifying Levorphanol Ingestion Using Urine Biomarkers in Health Care Patients.

BACKGROUND: Levorphanol is a long-acting opioid analgesic that is an optical isomer of dextrorphan, a metabolite of the over-the-counter cough suppressant dextromethorphan. Providers prescribing levorphanol for pain management may need to assess compliance through urine drug testing, as this agent is subject to abuse. Therefore, it is important to differentiate between dextromethorphan and levorphanol ingestion. OBJECTIVES: This article is the first to report urine concentrations of levorphanol/dextrorphan and 3-hydroxymorphinan in human urine and assesses the need for an enantiomeric analysis to distinguish between dextromethorphan and levorphanol ingestion. STUDY DESIGN: Retrospective data review. METHODS: Medication compliance test results were reviewed for 521 urine samples submitted to Aegis Sciences Corporation between July 2014 and July 2016. Samples were included in this analysis if dextromethorphan or levorphanol testing was requested by the ordering provider. Urine samples were hydrolyzed with beta-glucuronidase and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An enantiomeric analysis to distinguish levorphanol from dextrorphan and (-)-3-hydroxymorphinan (norlevorphanol) from (+)-3-hydroxymorphinan was not performed. RESULTS: Nineteen urine samples with levorphanol listed as prescribed had median levorphanol/dextrorphan and 3-hydroxymorphinan concentrations of 1,881 ng/mL and 141 ng/mL, respectively. One-quarter of the urine samples with dextromethorphan listed as prescribed did not have any detectable dextromethorphan or 3-methoxymorphinan. LIMITATIONS: An enantiomeric analysis was not utilized with the LC-MS/MS testing method; therefore, levorphanol could not be differentiated from dextrorphan, and (-)-3-hydroxymorphinan could not be differentiated from (+)-3-hydroxymorphinan. The hepatic and renal function for these patients was unknown; however, both could impact the metabolism, distribution, and excretion of levorphanol biomarkers in urine. The dextromethorphan and/or levorphanol dose and timing of last ingestion was also not assessed. CONCLUSIONS: It may be impossible to distinguish between levorphanol and dextromethorphan ingestion based on urine biomarkers, unless dextromethorphan or 3-methoxymorphinan is present or an enantiomeric analysis is performed. Therefore, the potential exists for patients prescribed levorphanol to ingest dextromethorphan and appear compliant with levorphanol therapy. This should prompt clinicians to consider the parameters of their laboratory's testing method when interpreting levorphanol drug test results. KEY WORDS: Levorphanol, dextrorphan, dextromethorphan, 3-hydroxymorphinan, urine testing, urine concentration, drug testing, medication compliance testing.