Anticonvulsant effects of dextrorphan in rats: possible involvement in dextromethorphan-induced seizure protection

The major metabolite of the non-opioid anticonvulsant/antitussive dextromethorphan is dextrorphan. In the present study, the effects of dextrorphan were determined in an experimental model of seizure activity (maximal electroshock convulsions) (MES). Subcutaneous administration of dextrorphan produced dose-related blockade of tonic hindlimb extension (THE) and a decrease in the duration of tonic forelimb extension (TFE). The anticonvulsant effect of dextrorphan was linear and maximally efficacious. Compared to the prototypical anticonvulsant drug diphenylhydantoin, dextrorphan was 2.5 times more potent (ED50's = 30 mumol/kg and 12 mumol/kg, respectively). Pretreatment with naloxone failed to antagonize dextrorphan-induced blockade of THE. Moreover, pretreatment with dextrophan failed to significantly enhance the anticonvulsant potency of diphenylhydantoin. It is likely that the anticonvulsant effects of dextrorphan are related to its actions at the phencyclidine/N-methyl-D-aspartate receptor complex, whereas the anticonvulsant effects of dextromethorphan have been attributed to binding to a specific dextromethorphan site in the brain. Therefore, we suggest that while metabolism to dextrorphan could possibly contribute to the anticonvulsant effects of dextromethorphan, it is probably through an unrelated receptor mechanism.     

Interaction of dextrorotatory opioids with phencyclidine recognition sites in rat brain membranes

The potencies of several dextrorotatory opioids, including four pairs of enantiomers, as inhibitors of specific [³H]PCP binding to rat brain synaptic membranes has been determined. Of the compounds tested unlabeled phencyclidine (PCP) was the most potent followed by (?)? cyclazocine > dextrorphan > (+) ketamine > (+) cyclazocine > (+)? SKF10,047 > levorphanol > dextromethorphan > (?) SKF10,047 > (?)? ketamine > (±) pentazocine and > (±) ethylketocyclazocine. The opiate mu receptor ligands, morphine, naloxone and naltrexone were virtually inactive as competitors of specific [³H]PCP binding. Unlike the stereostructural requirements for opiate mu receptors where activity resides predominantly in the levorotatory enantiomers, the present results support the contention that binding to the [³H]PCP labeled recognition site may reside in either the levorotatory or the dextrorotatory enantiomer. The specific binding of [³H]PCP which was defined as total binding minus that occurring in the presence of 10μM dextrorphan was found to be of a high affinity, saturable, reversible and sensitive to thermal degradation. These results suggest that certain dextrorotatory morphian derivatives may prove to be useful probes in further investigations of the molecular characteristics of the [³H]PCP binding site in brain membrane preparations.     

Comparative effects of opiate agonists methadone, levorphanol, and their isomers on the release of cortical ACh in vivo and in vitro.

The effect of analgesically active opiate agonists dl-methadone, levorphanol, and their less active forms d-methadone and dextrorphan, respectively, were tested on, (a) the spontaneous release of cortical acetylcholine (ACh) in vivo; (b) the spontaneous and K+-evoked release of cortical ACh in vitro. The injections of dl-methadone, but not d-methadone, inhibited the output of ACh in vivo. Naloxone completely reversed this effect of methadone. Levorphanol in small doses inhibited, and in larger doses stimulated, the in vivo release of ACh. Both effects were antagonized by naloxone. Its dextroisomer dextrorphan was completely inactive. The in vitro release of ACh from cortical slices was inhibited by all four agents. The effects of analgesically active opiates dl-methadone and levorphanol on the in vitro release were not clearly separable from the effects of their inactive forms d-methadone and dextrorphan.     

The blockade of serotonin uptake into synaptosomes:relationship to an interaction with monoamine oxidase inhibitors.

To test the hypothesis that the hyperpyrexia produced by meperidine and detromethorphan in rabbits pretreated with a monoamine oxidase inhibitor is related to inhibition of neuronal uptake of serotonin (5-hydroxytryptamine (5-HT)), fluoxetine (Lilly 110140) was studied. This potent and specific 5-HT neuronal uptake blocker was administered to phenelzine-pretreated rabbits and found to produce a lethal hyperpyrexia in doses equal to or greater than 2.5 mg/kg. The order of potency in blocking 5-[14C]HT uptake into synaptosomes prepared from rabbits was: fluoxetine greater than meperidine = dextromethorphan = levorphanol greater than anileridine greater than alphaprodine greater than morphine. Since fluoxetine, meperidine, and dextromethorphan produce hyperpyrexia in phenelzine-pretreated rabbits, whereas anileridine, alphaprodine, and morphine do not, there appears to be some correlation between the hyperpyrexic response and inhibition of 5-HT uptake. The exception is levorphanol, which is not hyperpyrexic despite being equipotent with meperidine and dextromethorphan in inhibiting 5-HT uptake. The ineffectiveness of levorphanol in producing hyperpyrexia may be due to its marked depressant properties, since the addition of another depressant drug (pentobarbital) antagonized the hyperpyrexic effect of meperidine.     

Determination of dextromethorphan and its metabolites in rat serum by liquid-liquid extraction and liquid chromatography with fluorescence detection

Dextromethorphan is an effective and safe antitussive, but has liabilities with respect to its abuse potential at doses above the therapeutic dose. At these higher doses, people report phencyclidine-like effects from the drug. A number of animal models have suggested that dextrorphan, an active metabolite of dextromethorphan, is responsible for the abuse liability of the parent compound when dextromethorphan is taken at high doses. Full pharmacokinetic profiles in single animals have not been demonstrated in these studies due to a lack of analytical sensitivity and/or selectivity for dextromethorphan and its metabolites. We have developed a low-cost liquid chromatographic method capable of characterizing the concentration-time profile for dextromethorphan and dextrorphan for 8 h in rats following an 18 mg/kg i.p. dose of dextromethorphan. Limits of quantitation (S/N=10) in 100 microL of serum were 0.25, 0.19, 0.27, and 0.22 nmol/mL for 3-hydroxymorphinan, dextrorphan, 3-methoxymorphinan, and dextromethorphan, respectively. Inter-day precision was better than 11% across the dynamic range of the method.     

Is Metaphit a phencyclidine antagonist? Studies with ketamine, phencyclidine and N-methylaspartate

The dissociative anaesthetics, phencyclidine and ketamine, block excitation of central neurones by N-methylaspartate. Using the technique of microelectrophoresis on rat spinal neurones in vivo Metaphit, a phencyclidine receptor acylating agent, was tested to see whether it would antagonise this effect of dissociative anaesthetics. The predominant effect of Metaphit was, however, to reduce N-methylaspartate induced excitation. It is concluded that Metaphit has mixed agonist/antagonist effects at the phencyclidine receptor.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Modified high-performance liquid chromatographic method to measure both dextromethorphan and proguanil for oxidative phenotyping

The activities of the polymorphic enzymes cytochromes P450 2D6 and 2C19 can be assessed by administering the probe drugs, dextromethorphan and proguanil, respectively. An existing high-performance liquid chromatographic technique, which measures dextromethorphan and its metabolites, has been modified to also measure proguanil and its polymorphic metabolite, cycloguanil in urine. Proguanil and cycloguanil are assayed in separate aliquots of urine to that used for dextromethorphan/dextrorphan as pretreatment with β-glucuronidase is required for the analysis of dextrorphan. To assay all four compounds a common extraction procedure is used and a single reversed-phase column and isocratic mobile phase with UV and fluorescence detectors connected in series are required. This technique is specific and sensitive for each analyte (limits of detection, dextrorphan/dextromethorphan/proguanil: 0.1 μg/ml, cycloguanil: 0.2 μg/ml). All assays are linear over the concentration ranges investigated (dextromethorphan/dextrorphan: 0.5–10 μg/ml, proguanil/cycloguanil: 1–20 μg/ml). The method described therefore uses laboratory resources very efficiently for all the assays required for hydroxylation phenotyping using proguanil and dextromethorphan.     

Identification of a Mr 58 000 glycoprotein subunit of the opiate receptor

A Mr 58 000 subunit of the opiate receptor has been identified using tritiated fentanyl isothiocyanate, a potent opiate alkylating reagent with specificity for the delta-opiate receptor subclass. The subunit is alkylated in the presence of dextrorphan but not levorphanol. The specifically labelled protein was retained on columns of immobilized wheat germ agglutinin and is therefore presumably a glycoprotein. Partial purification of the Mr 58 000 opiate receptor subunit from neuroblastoma X glioma NG108-15 hybrid cell membranes is described.     

Effect of morphine analogues on chemotaxis in Escherichia coli.

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1.1 mM-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.     

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs

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.     

Structure–activity relationships for ketamine esters as short-acting anaesthetics

A series of aliphatic esters of the non-opioid anaesthetic/analgesic ketamine were prepared and their properties as shorter-acting analogues of ketamine itself were explored in an infused rat model, measuring the time after infusion to recover from both the anaesthetic (righting reflex) and analgesic (response to stimulus) effects. The potency of the esters as sedatives was not significantly related to chain length, but Me, Et and i-Pr esters were the more dose potent (up to twofold less than ketamine), whereas n-Pr esters were less potent (from 2- to 6-fold less than ketamine). For the Me, Et and i-Pr esters recovery from anaesthesia was 10–15-fold faster than from ketamine itself, and for the n-Pr esters it was 20–25-fold faster than from ketamine. A new dimethylamino ketamine derivative (homoketamine) had ketamine-like sedative effects but was slightly less potent than, but ester analogues of homoketamine had very weak sedative effects.     

Simultaneous analysis of cytochrome P450 probes-dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A "cocktail" approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4'-hydroxy-flurbiprofen and 1'-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify cartridges) extraction procedure from 200 microl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm Lx3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4'-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1'-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: -9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Dextromethorphan attenuates trimethyltin-induced neurotoxicity via sigma1 receptor activation in rats

We showed that dextromethorphan (DM) provides neuroprotective/anticonvulsant effects and that DM and its major metabolite, dextrorphan, have a high-affinity for sigma(1) receptors, but a low affinity for sigma(2) receptors. In addition, we found that DM has a higher affinity than DX for sigma(1) sites, whereas DX has a higher affinity than DM for PCP sites. We extend our earlier findings by showing that DM attenuated trimethyltin (TMT)-induced neurotoxicity (convulsions, hippocampal degeneration and spatial memory impairment) in rats. This attenuation was reversed by the sigma(1) receptor antagonist BD 1047, but not by the sigma(2) receptor antagonist ifenprodil. DM attenuates TMT-induced reduction in the sigma(1) receptor-like immunoreactivity of the rat hippocampus, this attenuation was blocked by the treatment with BD 1047, but not by ifenprodil. These results suggest that DM prevents TMT-induced neurotoxicity, at least in part, via sigma(1) receptor stimulation.     

Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO(4) and methanol. The analysis was performed on a 3 microm, C(18) Tracer Excel 15 cm x 0.4 cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH = 3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10-500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.     

Metabolism to dextrorphan is not essential for dextromethorphan's anticonvulsant activity against kainate in mice

The effects of dextromethorphan (DM), and its major metabolite dextrorphan (DX) on kainic acid-induced seizures in mice were examined. Intracerebroventricular DM or DX (5 or 10 microg/0.5 microl) pretreatment significantly attenuated seizures induced by kainic acid (0.07 microg/0.07 microl) in a dose-related manner. DM or DX pretreatment significantly attenuated kainic acid-induced increases in AP-1 DNA-binding activity and fos-related antigen-immunoreactivity as well as neuronal loss in the hippocampus. DM appears to be a more potent neuroprotectant than DX. Since the high-affinity DM binding sites are recognized as being identical to the sigma-1 site, we examined the role of the sigma-1 receptor on the pharmacological action mediated by DM or DX. Pretreatment with the sigma-1 receptor antagonist BD1047 (2.5 or 5 mg/kg, i.p.) blocked the neuroprotection by DM in a dose-related manner. This effect of BD 1047 was more pronounced in the animals treated with DM than in those treated with DX. Combined, our results suggest that metabolism of DM to DX is not essential for DM to exert its effect. They also suggest that DM provides neuroprotection from kainic acid via sigma-1 receptor modulation.