Exploration of catalytic properties of CYP2D6 and CYP3A4 through metabolic studies of levorphanol and levallorphan.

CYP2D6 and CYP3A4, two members of the cytochrome P450 superfamily of monooxygenases, mediate the biotransformation of a variety of xenobiotics. The two enzymes differ in substrate specificity and size and characteristics of the active site cavity. The aim of this study was to determine whether the catalytic properties of these isoforms, reflected by the differences observed from crystal structures and homology models, could be confirmed with experimental data. Detailed metabolite identification, reversible inhibition, and time-dependent inhibition were examined for levorphanol and levallorphan with CYP2D6 and CYP3A4. The studies were designed to provide a comparison of the orientations of substrates, the catalytic sites of the two enzymes, and the subsequent outcomes on metabolism and inhibition. The metabolite identification revealed that CYP3A4 catalyzed the formation of a variety of metabolites as a result of presenting different parts of the substrates to the heme. CYP2D6 was a poorer catalyst that led to a more limited number of metabolites that were interpreted in terms to two orientations of the substrates. The inhibition studies showed evidence for strong reversible inhibition of CYP2D6 but not for CYP3A4. Levallorphan acted as a time-dependent inhibitor on CYP3A4, indicating a productive binding mode with this enzyme not observed with CYP2D6 that presumably resulted from close interactions of the N-allyl moiety oriented toward the heme. All the results are in agreement with the large and flexible active site of CYP3A4 and the more restricted active site of CYP2D6.

Synthesis and pharmacological evaluation of 6,7-indolo/thiazolo-morphinans--further SAR of levorphanol.

To further extend the structure-activity relationships of levorphanol, two series of novel morphinans were prepared by incorporation of an indole or aminothiazole fragment to the hexyl ring (ring C) in levorphanol. Such morphinans differed from previously reported ligands in that such indole- or aminothiazole-containing morphinans displayed enhanced binding affinity to the delta opioid receptor, while the affinity to kappa and micro receptors was slightly reduced.

Effects of dextromethorphan, a nonopioid antitussive, on development and expression of amygdaloid kindled seizures.

The effects of dextromethorphan (DM), a nonopioid antitussive and a functional N-methyl-D-aspartate (NMDA) antagonist, on expression and development of amygdaloid kindled seizures were examined. The maximum anticonvulsant effect of DM (30 mg/kg) on fully kindled seizures appeared within 30 min of administration and lasted for at least 2 h. DM decreased, in a dose-dependent manner [10-70 mg/kg, intraperitoneally (i.p.)], the severity of kindled seizures 30 min after injection, but the estimated ED50 was 3 times higher than the previously reported value for maximal electroshock convulsions. Furthermore, the high dose (70 mg/kg), while suppressing kindled seizures, produced myoclonus which coincided with EEG spike activity in the amygdala and the cortex. When tested on the development of kindling, 30 mg/kg DM retarded the growth of afterdischarge in the amygdala and the cortex, but had no effect on the development of behavioral seizures. DM 60 mg/kg accelerated development of kindling and produced spontaneous seizures. These results indicate that DM, unlike other NMDA antagonists, has a narrow therapeutic window as an anticonvulsant on kindled seizures and that higher doses may potentiate the kindling process.

Pharmacokinetic comparison of a dextromethorphan-salbutamol combination tablet and a plain dextromethorphan tablet.

We compared in this double-blind crossover study the bioavailability of dextromethorphan from a dextromethorphan-salbutamol combination tablet (Redol comp) and from a plain dextromethorphan tablet (Extuson) by determining dextrorphan concentrations after single-dose oral administration in 10 healthy volunteers. The absorption of salbutamol from the combined preparation was also determined. The absorption of dextromethorphan was slightly faster from the plain dextromethorphan preparation. The peak concentration of dextrorphan was achieved at 1.5 h after Extuson and at 2 h after Redol comp (1,053.0 +/- 366.5 ng/ml and 901.5 +/- 210.9 ng/ml, NS). AUC0-12 values of dextrorphan were 4,315.6 +/- 295.0 (ng/ml)h after Extuson and 3,983.8 +/- 205.6 (ng/ml)h after Redol comp (p less than 0.05). Salbutamol was well absorbed from the combined preparation and the peak concentration was achieved at 3 h (6.57 +/- 2.95 ng/ml). Four subjects reported side-effects typical for salbutamol after the combination tablet. No side-effects were reported after the plain dextromethorphan tablet. On the basis of the present study, we conclude that the absorption of dextromethorphan from the preparations tested is almost equal and the dextromethorphan-salbutamol combination can be administered in tablet form for the treatment of cough.

Suppression by ketamine and dextromethorphan of precipitated abstinence syndrome in rats.

The development of physical dependence on opiates appears to involve an inhibition by opiates of L-asparaginase and glutaminase, and the blockade by opiates of aspartatergic (ASPergic)/glutamatergic (GLUergic) receptors. Ketamine (K) (0.5 or 1 mg/kg) or dextromethorphan (DM) (1 or 2 mg/kg), both of which are known to decrease the responsiveness of ASPergic/GLUergic receptors, were administered to the three morphine (M)-containing pellets implanted rats prior to 2 mg/kg naloxone (NL) injection. Whereas 0.5 mg/kg K showed no significant effect on abstinence syndrome signs, 1 mg/kg K and 1 mg/kg DM significantly attenuated some of the signs. The attenuation or prevention of all the signs were observed after 2 mg/kg DM administration. Almost complete prevention was seen from the second minute on during the ten-minute observation period. As ASP and GLU antagonists K and DM have this antagonizing effect on the precipitated abstinence syndrome signs, the manifestation of abstinence syndrome may mainly result from the normalization of ASP and GLU production because of the disinhibition by NL of the enzymes and the stronger stimulation of ASPergic/GLUergic receptors which have no opiate blockade after NL injection.

Rapid screening for polymorphisms in dextromethorphan and mephenytoin metabolism.

1. The phenotyping parameters for dextromethorphan and mephenytoin were assessed in 48 normal male volunteers following administration of each metabolic probe drug on separate occasions and together according to a randomized 3-way crossover design. 2. Neither the urinary S-/R-mephenytoin ratio nor the dextromethorphan metabolic ratio were altered significantly by coadministration of the probe drugs. 3. Five-hundred and nineteen subjects were screened for expression of mephenytoin 4-hydroxylase and dextromethorphan O-demethylase activity following the coadministration of mephenytoin and dextromethorphan. The activity was determined in each case by methods not requiring any quantitative measurements. 4. Nineteen (3.7%) of the subjects were identified as poor metabolizers (PMs) of mephenytoin and 35 subjects (6.7%) as PMs of dextromethorphan. 5. All PMs of dextromethorphan were confirmed by more rigorous evaluation of the metabolic ratio.

The treatment of heroin addicts with dextromethorphan: a double-blind comparison of dextromethorphan with chlorpromazine.

According to the hypothesis that the development of physical dependence on and tolerance to opiates depends on the inhibition by opiates of L-asparaginase and L-glutaminase activities in the brain, and the blockade by opiates of the aspartatergic/glutamatergic receptors especially NMDA, four female and fourty-four male heroin addicts were included in a double-blind clinical trial. Four mg chlorpromazine (CPZ) was administered every hour and 10 mg diazepam (DIA) every 6 hours to a group consisting of two female and nineteen male inpatients. The remaining subjects received 15 mg non-opioid antitussive dextromethorphan (DM) instead of CPZ. The withdrawn addicts were controlled twice a day and yawning, lacrimation, rhinorrhoea, perspiration, goose flesh, muscle tremor, dilated pupils, anorexia, joint and muscle aches, restlessness, insomnia, emesis, diarrhea, craving and rejection of smoking as abstinence syndrome signs were observed and rated on a scale of 1, 2 and 3 points according to their intensity. All signs, except perspiration and emesis, were significantly less intense in the group given DM + DIA than CPZ + DIA. The other plus points included the immediate stop of craving and the early onset of smoking in DM + DIA group. The results are considered to be supporting evidence for the hypothesis emphasizing the blockade of NMDA receptors by opiates in opiate addiction. Furthermore, the decrease caused by non-opioid NMDA antagonists in the responsiveness of NMDA receptors appears very promising for the treatment of opiate addicts.

Inhibitory studies of mexiletine and dextromethorphan oxidation in human liver microsomes.

The cytochrome P-450dbl isozyme (P-450bdl) is responsible for the genetic sparteine-debrisoquine type polymorphism of drug oxidation in humans. To investigate the relationship between mexiletine oxidation and the activity of this isozyme, cross-inhibition studies were performed in human liver microsomes with mexiletine and dextromethorphan, a prototype substrate for P-450dbl. The formation of hydroxymethylmexiletine and p-hydroxymexiletine, two major mexiletine metabolites, was competitively inhibited by dextromethorphan. Mexiletine competitively inhibited the high affinity component of dextromethorphan O-demethylation. In addition, there was a good agreement between the apparent Km values for the formation of both mexiletine metabolites and the high affinity component of dextromethorphan O-demethylation and their respective apparent Ki values. Several drugs were tested for their ability to inhibit mexiletine oxidation. Quinidine, quinine, propafenone, oxprenolol, propranolol, ajmaline, desipramine, imipramine, chlorpromazine and amitryptiline were competitive inhibitors for the formation of hydroxymethylmexiletine and p-hydroxymexiletine as for prototype reactions of the sparteine-debrisoquine type polymorphism. Amobarbital, valproic acid, ethosuximide, caffeine, theophylline, disopyramide and phenytoin, known to be non-inhibitors of P-450dbl activity, were found not to inhibit the formation of these mexiletine metabolites. Moreover, the formation of both metabolites was strongly inhibited by an antiserum containing anti-liver/kidney microsomes antibodies type I (anti-LKMI) directed against P-450dbl. These data suggest that the formation of two major metabolites of mexiletine is predominantly catalysed by the genetically variable human liver P-450dbl.

Dextromethorphan for treatment of complex partial seizures.

We performed a double-blind, crossover, add-on study of the antitussive agent dextromethorphan (DM 120 mg/d) as therapy for seizures on 9 patients suffering from severe complex partial seizures. DM had no significant influence on key laboratory values, nor on anticonvulsant drug levels. Side effects were negligible. Complex partial seizure frequency increased 25% during the DM arm of the study, although this increase was not clinically significant.

Polymorphic formation of morphine from codeine in poor and extensive metabolizers of dextromethorphan: relationship to the presence of immunoidentified cytochrome P-450IID1.

We studied the oxidation capacity in liver biopsies of a series of extensive metabolizers (n = 10) and poor metabolizers (n = 2) as identified by in vivo phenotyping with dextromethorphan. Codeine and dextromethorphan were used as probe drugs in vitro. The data were compared with the contents of cytochrome P-450IID1 as quantitated by Western immunoblotting by use of a specific monoclonal antibody (MAb 114/2). The O-demethylation of codeine was highly correlated with the O-demethylation of dextromethorphan (r = 0.90). The N-demethylation of codeine was catalyzed at a considerably higher rate than the O-demethylation. The N-demethylation to O-demethylation ratio of codeine was 46 in the poor metabolizer and, on average, 6.2 (range, 2.6 to 11) in the extensive metabolizers, respectively. The band intensity in Western blots correlated with the rate of O-demethylation of codeine (r = 0.95) and of dextromethorphan (r = 0.88) in the extensive metabolizers. The comeasurement of the O-demethylation and N-demethylation of codeine may provide a tool with which to phenotype individuals in vitro with respect to the polymorphism of the cytochrome P-450IID1.

Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man.

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.

Ropizine concurrently enhances and inhibits [3H]dextromethorphan binding to different structures of the guinea pig brain: autoradiographic evidence for multiple binding sites.

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.

Dextromethorphan sites, sigma receptors, and the psychotomimetic effects of sigma opiates.

A review of the literature shows that the dysphoric and psychotomimetic side effects of opiate agonist-antagonist reside in the opiate levorotatory isomer. Thus, the current definition of the sigma receptor cannot explain the psychotomimetic effects of sigma opiates.

Dextromethorphan and neuromodulation: old drug coughs up new activities.

Dextromethorphan is one of the most widely used non-opioid cough suppressants, representing the active ingredient in several over-the-counter antitussive formulations. It does not possess the CNS pharmacology of other opiates in humans (i.e. analgesia, respiratory depression, abuse liability or psychotomimetic properties), but since the discovery in 1981 of high affinity recognition sites in brain for dextromethorphan a unique neuropharmacological profile has emerged for this relatively innocuous drug. Anticonvulsant and neuroprotective properties have been demonstrated, and treatment with dextromethorphan has been shown to improve the cerebrovascular and functional consequences of global cerebral ischemia. Frank Tortella and colleagues review the CNS pharmacology of dextromethorphan, its possible involvement with NMDA or sigma-receptors, and the potential clinical importance of this old 'new' drug.