norlevorphanol and 3-methoxymorphinan

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.

Topics: Animals; Body Remains; Bone and Bones; Chromatography, Liquid; Dextromethorphan; Dextrorphan; Mass Spectrometry; Rats

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.

Topics: Chromatography, Liquid; Dextromethorphan; Dextrorphan; Humans; Mass Spectrometry; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Time Factors

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.

Topics: Antitussive Agents; Calibration; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2D6 Inhibitors; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dextromethorphan; Dextrorphan; Gas Chromatography-Mass Spectrometry; Humans; Reproducibility of Results; Sensitivity and Specificity

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.

Topics: Chromatography, High Pressure Liquid; Dextromethorphan; Dextrorphan; Humans; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Time Factors

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Chromatography, Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Dextromethorphan; Dextrorphan; In Vitro Techniques; Intestinal Mucosa; Intestine, Small; Male; Mass Spectrometry; Oxidoreductases, N-Demethylating; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity

Interest in dextromethorphan (DM) has been renewed because of its anticonvulsant and neuroprotective properties. However, DM at supra-antitussive doses can produce psychotomimetic effects in humans. Recently, we demonstrated that DM exerts psychotropic effects in mice [Neurosci. Lett. 288 (2000) 76, Life Sci. 69 (2001) 615]. We synthesized a series of compounds with a modified morphinan ring system, with the intention of developing compounds that retain the anticonvulsant activity with weak psychotropic effects [Bioorg. Med. Chem. Lett. 11 (2001) 1651]. In order to extend our understanding of the pharmacological intervention of these morphinans, we assessed their behavioral effects, and then examined whether they exert protective effects on maximal electroshock convulsions (MES) in mice. Repeated treatment (20 or 40 mg/kg, i.p./day x 7) with DM or dextrorphan (a major metabolite of DM; DX) significantly enhanced locomotor activity in a dose-related manner. This locomotor stimulation was accentuated more in the animals treated with DX, and might be comparable to that of phencyclidine (PCP). By contrast, treatment with a metabolite of DM [3-methoxymorphinan (3MM) or 3-hydroxymorphinan (3HM)], 3-allyloxy-17-methylmorphinan (CPK-5), or 3-cyclopropylmethoxy-17-methylmorphinan (CPK-6) did not significantly alter locomotor activity or patterns. The behavioral effects mediated by these morphinans and PCP paralleled the effects of conditioned place preference. DM, DX, CPK-5, and CPK-6 had anticonvulsant effects against MES, while 3MM and 3HM did not show any anticonvulsant effects. We found that DM, DX, CPK-5 and CPK-6 were high-affinity ligands at sigma(1) receptors, while they all had low affinity at sigma(2) receptors. DX had relatively higher affinity for the PCP sites than DM. By contrast, CPK-5 and CPK-6 had very low affinities for PCP sites, suggesting that PCP sites are not requisites for their anticonvulsant actions. Our results suggest that the new morphinan analogs are promising anticonvulsants that are devoid of PCP-like behavioral side effects, and their anticonvulsant actions may be, in part, mediated via sigma(1) receptors.

Topics: Animals; Anticonvulsants; Dextromethorphan; Dose-Response Relationship, Drug; Electroshock; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Morphinans; Radioligand Assay; Receptors, sigma; Seizures

An HPLC method has been developed and validated for the determination of dextromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in urine samples. Deconjugated compounds were extracted on silica cartridges using dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separation was accomplished on a Phenyl analytical column serially connected with a Nitrile analytical column. The mobile phase consisted of a mixture of an aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence detector. Calibration curves were linear over the range investigated (0.2-8.0 microM) with correlation coefficients >0.999. The method was reproducible and precise. Coefficients of variation and deviations from nominal values were both below 10%. For all the analytes, recoveries exceeded 77% and the limits of detection were 0.01 microM. The validated assay proved to be suitable for the determination of DEM metabolic indexes reported to reflect the enzymatic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients, and in vitro, when applied to samples from the incubation of liver microsomes with dextromethorphan.

Topics: Aryl Hydrocarbon Hydroxylases; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dextromethorphan; Dextrorphan; HIV Infections; Humans; Malaria; Microsomes, Liver; Models, Chemical; Oxidoreductases, N-Demethylating; Reproducibility of Results; Sensitivity and Specificity

Dextromethorphan (DXM) is a widely used probe drug for human CYP2D6 activity both in vitro and in vivo. In humans, DXM is metabolized to dextrorphan (DXO), as well as 3-methoxymorphinan (MEM) and 3-hydroxymorphinan (HYM). The formation of MEM has been attributed primarily to CYP3A4, and the use of DXM has been debated as a simultaneous probe for CYP3A4 and CYP2D6 activities. Recently, we found that highly purified CYP2D6 has significant DXM N-demethylase activity in addition to its well known DXM O-demethylase activity. Therefore, we desired to further compare the contribution to DXM metabolism by individual human cDNA-expressed cytochromes P450, including 2C8, 2C9, 2C18, 2C19, 2D6, 2B6, and 3A4. Metabolites were quantified following separation by high-pressure liquid chromatography and apparent Michaelis-Menten constants determined for the appearance of DXO and MEM. Intrinsic clearance values were estimated for each P450 and normalized using the average percentage content and relative activity factor approaches for comparison. Simplified kinetic models (when [S] << K(m), V(max)/K(m) = V(o)/[S]) were used at fixed DXM concentrations of 20 (for DXM N-demethylation) and 0.2 microM (for DXM O-demethylation), as well as 2 microM to mimic plasma DXM concentrations in human extensive metabolizers. The results confirm that CYP2D6 contributes at least 80% to the formation of DXO, and CYP3A4 contributes more than 90% to the formation of MEM. All of our in vitro results are consistent and indicate that DXM as a marker for monitoring both CYP2D6 and CYP3A activities is practical in an average human or human liver microsomal preparation.

Topics: Antitussive Agents; Aryl Hydrocarbon Hydroxylases; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dextromethorphan; Dextrorphan; Humans; Isoenzymes; Microsomes, Liver; Oxidoreductases, N-Demethylating

A sensitive capillary electrophoretic method was developed and validated for the simultaneous determination of dextromethorphan and its metabolites, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan, in human plasma. After cleavage of conjugates by enzymatic hydrolysis with beta-glucuronidase, dextromethorphan and its metabolites were extracted from 1.5 ml of plasma by a liquid liquid extraction procedure using heptane-ethylacetate (50:50, v/v) and re-extracted to aqueous phase. The compounds were separated within 8 min on a fused silica capillary, 75 microm internal diameter using sodium borate (pH 9.4; 50 mM) as running buffer, and measured by UV-detection at 195 nm using a detection cell with a path length of 1.2 mm. The method was accurate and precise. Linear relationships were observed between the peak response and the concentration in the range of 1-400 ng ml(-1) plasma with correlation coefficients above 0.998. The limit of detection was 0.5-1 ng ml(-1) plasma for all compounds. The method was used for determination of plasma levels of dextromethorphan and its metabolites after transdermal and oral administration of dextromethorphan.

Topics: Administration, Cutaneous; Administration, Oral; Dextromethorphan; Dextrorphan; Electrophoresis, Capillary; Excitatory Amino Acid Antagonists; Humans; Models, Chemical; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxymorphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of CYP2d6 (quinidine), CYP1A1/2 (alpha-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.

Topics: Animals; Antitussive Agents; Benzoflavones; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dextromethorphan; Ditiocarb; Enzyme Inhibitors; Humans; In Vitro Techniques; Methylation; Microsomes, Liver; Mixed Function Oxygenases; Quinidine; Rats; Sulfaphenazole; Tissue Donors

1. The enzyme kinetics of dextromethorphan O-demethylation in liver microsomes from three extensive metabolisers (EM) with respect to CYP2D6 indicated high (Km1 2.2-9.4 microM) and low (Km2 55.5-307.3 microM) affinity sites whereas microsomes from two poor metabolisers (PM) indicated a single site (Km 560 and 157 microM). Similar differences were shown for 3-methoxymorphinan O-demethylation to 3-hydroxymorphinan (Km 6.9-9.6 microM in EM subjects; Km 307 and 213 microM in PM subjects). 2. Dextromethorphan O-demethylation was inhibited competitively by quinidine (Ki 0.1 microM), rac-perhexiline (Ki 0.4 microM), dextropropoxyphene (Ki 6 microM), rac-methadone (Ki 8 microM), and 3-methoxymorphinan (Ki 15 microM). These compounds were also potent inhibitors of 3-methoxymorphinan O-demethylation with IC50 values ranging from 0.02-12 microM. Anti-LKM1 serum inhibited both dextromethorphan and 3-methoxymorphinan O-demethylations in a titre-dependent manner. 3. The Michaelis-Menten constant for dextromethorphan N-demethylation to 3-methoxymorphinan (Km 632-977 microM) and dextrorphan N-demethylation to 3-hydroxymorphinan (Km 1571-4286 microM) did not differ between EM and PM microsomes. These N-demethylation reactions were not inhibited by quinidine and rac-methadone or LKM1 antibodies. 4. Dextromethorphan and 3-methoxymorphinan are metabolised by the same P450 isoform, CYP2D6, whereas the N-demethylation reactions are not carried out by CYP2D6.

Topics: Autoantibodies; Binding, Competitive; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System; Dextromethorphan; Dextropropoxyphene; Dextrorphan; Humans; In Vitro Techniques; Methadone; Methylation; Microsomes, Liver; Mixed Function Oxygenases; Oxidation-Reduction; Perhexiline; Phenotype; Quinidine

A simple, sensitive, and reproducible high-performance liquid chromatrography assay is described for the simultaneous determination of dextromethophan, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan in plasma and urine. A conventional solvent-solvent extraction procedure was used for the isolation of the analytes from plasma and urine samples. The compounds were separated on a cyano column (150 x 4.6 mm, 5-micron particle size) using a mobile phase of acetonitrile/triethylamine/distilled water (17:0.06:82.94, vol/vol), pH 3.0, and then were measured by fluorescence detection. Calibration curves in the range 2-200 ng/ml for plasma and 0.05-10 micrograms/ml for urine were linear and passed through the origin. The precision and accuracy were greater than 90% and the lowest detectable concentrations were 0.5 ng/ml for 3-hydroxymorphinan and 3-methoxymorphinan and 1 ng/ml for dextromethorphan and dextrophan in plasma. The utility of this method is demonstrated in a preliminary study of dextromethorphan metabolism and pharmacokinetics in man.

Topics: Chromatography, High Pressure Liquid; Dextromethorphan; Dextrorphan; Glucuronidase; Humans; Levorphanol