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.

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.

Lotus leaf alkaloid fraction can strongly inhibit CYP2D6 isoenzyme activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal medicine He-Ye, the leaves of the lotus (Nelumbo nucifera) plant, is traditionally used in China for the treatment of sunstroke, thirst, diarrhea, and fever. Currently, the leaf is used not only as an herbal tea to reduce lipid level and control body weight, but also as a major ingredient in some lipid-lowering Chinese patented medicines. Our previous study demonstrated that the alkaloid fraction (AF) of the herb has a strong inhibitory effect on CYP2D6 isoenzyme activity in vitro. The present study aims to further verify this activity using the in vivo rat model and to explore the inhibitory mechanism on CYP2D6 using human liver microsomes (HLMs). MATERIALS AND METHODS: After a continuous 7-d oral dose of AF (50mg/kg) or a vehicle, Sprague Dawley rats received a single intravenous dose of dextromethorphan or metoprolol. Blood samples were collected at various time points, and the plasma concentrations of the relevant metabolites dextrorphan and hydroxymetoprolol were assayed by LC-MS/MS for evaluating the effect of AF on their pharmacokinetics and CYP2D6 activity. Dextromethorphan as a probe at different concentrations was incubated with HLMs in an incubation buffer system, in the presence or absence of AF at different concentrations. After incubation, the produced metabolite was assayed. RESULTS: After being pretreated with AF in rats, the plasma concentrations of dextrorphan and hydroxymetoprolol significantly decreased, with Cmax going from 79.44 to 29.96 and 151.18 to 83.39hng/mL (P<0.05), AUCall from 167.27 to 62.25 and 347.68 to 223.24hng/mL (P<0.05), and AUCinf from 183.39 to 84.76 and 350.59 to 234.57hng/mL (P<0.05), respectively, in comparison with those of untreated rats. The t1/2 of hydroxymetoprolol significantly increased from 1.14 to 1.99h (P<0.05). The in vitro incubation test showed that AF competitively inhibited the CYP2D6, with apparent Ki value of 0.64µg/mL. CONCLUSIONS: AF can strongly inhibit the activity of CYP2D6 enzyme, as confirmed by in vivo and in vitro models. Possible drug interactions may occur between AF and other medications metabolized by CYP2D6. Thus, caution should be paid when the lotus leaf and its preparations are concurrently administered with conventional medicines.

Human Cytosolic Sulfotransferase SULT1A3 Mediates the Sulfation of Dextrorphan.

Dextrorphan, an active metabolite of the antitussive dextromethorphan, has been shown to be subjected to sulfation by several zebrafish cytosolic sulfotransferases (SULTs). We were interested in finding out which of the human SULT(s) is(are) capable of catalyzing the sulfation of dextrorphan, and to verify whether sulfation of dextrorphan may occur in cultured human cells and human organ cytosols. Data from the enzymatic assays showed that, of all thirteen known human SULTs, SULT1A3 displayed the strongest dextrorphan-sulfating activity. Cell culture experiments using HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells incubated with [(35)S]sulfate together with varying concentrations of dextrorphan revealed indeed the production and release of [(35)S]sulfated dextrorphan in a concentration-dependent manner. Additionally, significant dextrorphan-sulfating activity was detected in human liver, small intestine and lung cytosols. Taken together, these results provided a biochemical basis for the sulfation of dextrorphan in humans.

CYP2D6 Is Inducible by Endogenous and Exogenous Corticosteroids.

Although cytochrome P450 (CYP) 2D6 has been widely considered to be noninducible on the basis of human hepatocyte studies, in vivo data suggests that it is inducible by endo- and xenobiotics. Therefore, we investigated if the experimental conditions routinely used in human hepatocyte studies may be a confounding factor in the lack of in vitro induction of CYP2D6. Sandwich cultured human hepatocytes (SCHH) were preincubated with or without dexamethasone (100 nM) for 72 hours before incubation with 1µM endogenous (cortisol or corticosterone) or exogenous (dexamethasone or prednisolone) corticosteroids. At 72 hours, CYP2D6 mRNA, protein, and activity were quantified by real-time quantitative polymerase chain reaction, quantitative proteomics, and formation of dextrorphan from dextromethorphan, respectively. In the absence of supplemental dexamethasone, CYP2D6 activity, mRNA, and protein were significantly and robustly (>10-fold) induced by all four corticosteroids. However, this CYP2D6 induction was abolished in cells preincubated with supplemental dexamethasone. These data show, for the first time, that CYP2D6 is inducible in vitro but the routine presence of 100 nM dexamethasone in the culture medium masks this induction. Our cortisol data are in agreement with the clinical observation that CYP2D6 is inducible during the third trimester of pregnancy when the plasma concentrations of cortisol increase to ∼1µM. These findings, if confirmed in vivo, have implications for predicting CYP2D6-mediated drug-drug interactions and call for re-evaluation of regulatory guidelines on screening for CYP2D6 induction by xenobiotics. Our findings also suggest that cortisol may be a causative factor in the in vivo induction of CYP2D6 during pregnancy.

The Origin, Expression, Function and Future Research Focus of a G Protein-coupled Receptor, Mas-related Gene X2 (MrgX2).

Mas-related genes (Mrgs) belong to a large family of G protein-coupled receptor genes found in rodents. Human MRGX proteins are G protein-coupled 7-transmembrane proteins sharing 41-52% amino acid identity with each other, but have no orthologs in rodents. MrgX2 is a member of the MrgX family. MRGX2 is expressed in the small neurons of sensory ganglia and mast cells. It can interact with a series of factors and genes such as the peptides substance P, vasoactive intestinal peptide, cortistatin (CST), proadrenomedullin N-terminal peptide (PAMP), LL-37, PMX-53 and ß-defensins. MRGX2 is related to nociception, adrenal gland secretion and mast cell degranulation. Recent research on MrgX2 provides insights into its role in nociception and anti-microbial activities. This article reviewed the origin, expression and function of MrgX2, and discussed possible future research focus.

Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 µM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 µM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 µM) and dog (Ki = 2.4 ± 1.3 µM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

Dynamics and persistence of CYP2D6 inhibition by paroxetine.

WHAT IS KNOWN AND OBJECTIVE: Paroxetine is both a substrate and an inhibitor of CYP2D6. The objective of the presented study was to determine the persistence of CYP2D6 inhibition after short term (6 weeks) and long term (18·7 ± 10·6 weeks) paroxetine treatment. METHODS: Two the studies consisted of 30 depressive/anxiety patients each. In the first study, patients were subdivided into three groups treated with paroxetine (A1), alprazolam (A2) and paroxetine + alprazolam (A3). After 6 weeks, all the patients (A1+A2+A3) were switched to alprazolam treatment; metabolic activity was evaluated at the beginning, after 6 weeks of paroxetine/alprazolam/alprazolam + paroxetine treatment (A1/A2/A3) and 4 weeks after the switch to alprazolam treatment (Week 0, 6, 10). In the second study patients on previous long term paroxetine treatment were subdivided into two groups treated with mirtazapine (B1) or paroxetine (B2); metabolic activity of CYP2D6 was evaluated at the beginning and after 6 weeks of therapy. RESULTS AND DISCUSSION: Metabolic ratio of dextromethorphan to dextrorphan has normalized in all subjects after 4 weeks of paroxetine wash out in the first study. In the second study, 6 weeks after paroxetine discontinuation, restoration of metabolic activity of CYP2D6 was observed in only five of eight originally poor metabolizers. WHAT IS NEW AND CONCLUSION: We conclude that a wash-out period of 4 weeks seems to be sufficient for CYP2D6 disinhibition after short-term paroxetine treatment (6 weeks). On the other hand, treatment with a CYP2D6 substrate less than 6 weeks after long-term paroxetine treatment (18·7 weeks on average) could result in elevated drug plasma levels and occasionally also in drug toxicity.

An extension of hypotheses regarding rapid-acting, treatment-refractory, and conventional antidepressant activity of dextromethorphan and dextrorphan.

It was previously hypothesized that dextromethorphan (DM) and dextrorphan (DX) may possess antidepressant properties, including rapid and conventional onsets of action and utility in treatment-refractory depression, based on pharmacodynamic similarities to ketamine. These similarities included sigma-1 (σ(1)) agonist and NMDA antagonist properties, calcium channel blockade, muscarinic binding, serotonin transporter (5HTT) inhibition, and µ receptor potentiation. Here, six specific hypotheses are developed in light of additional mechanisms and evidence. Comparable potencies to ketamine for DM and DX are detailed for σ(1) (DX>DM>ketamine), NMDA PCP site (DX>ketamine>DM), and muscarinic (DX>ketamine>>>>DM) receptors, 5HTT (DM>DX≫ketamine), and NMDA antagonist potentiation of µ receptor stimulation (DM>ketamine). Rapid acting antidepressant properties of DM include NMDA high-affinity site, NMDR-2A, and functional NMDR-2B receptor antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation, µ potentiation, and 5HTT inhibition), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, µ potentiation, and 5HTT inhibition), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Those for dextrorphan include NMDA high-affinity site and NMDR-2A antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation and ß adrenoreceptor stimulation), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, ß stimulation, and µ antagonism), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Conventional antidepressant properties for dextromethorphan and dextrorphan include 5HTT and norepinephrine transporter inhibition, σ(1) stimulation, NMDA and PCP antagonism, and possible serotonin 5HT1b/d receptor stimulation. Additional properties for dextromethorphan include possible presynaptic α(2) adrenoreceptor antagonism or postsynaptic α(2) stimulation and, for dextrorphan, ß stimulation and possible muscarinic and µ antagonism. Treatment-refractory depression properties include increased serotonin and norepinephrine availability, PCP, NMDR-2B, presynaptic alpha-2 antagonism, and the multiplicity of other antidepressant receptor mechanisms. Suggestions for clinical trials are provided for oral high-dose dextromethorphan and Nuedexta (dextromethorphan combined with quinidine to block metabolism to dextrorphan, thereby increasing dextromethorphan plasma concentrations). Suggestions include exclusionary criteria, oral dosing, observation periods, dose-response approaches, and safety and tolerability are considered. Although oral dextromethorphan may be somewhat more likely to show efficacy through complementary antidepressant mechanisms of dextrorphan, a clinical trial will be more logistically complex than one of Nuedexta due to high doses and plasma level variability. Clinical trials may increase our therapeutic armamentarium and our pharmacological understanding of treatment-refractory depression and antidepressant onset of action.

Clonidine as adjuvant for oxybuprocaine, bupivacaine or dextrorphan has a significant peripheral action in intensifying and prolonging analgesia in response to local dorsal cutaneous noxious pinprick in rats.

The aim of the study was to evaluate co-administration of clonidine with oxybuprocaine (ester type), bupivacaine (amide type) or dextrorphan (non-ester or non-amide type) and to see whether it could have a peripheral action in enhancing local anesthesia on infiltrative cutaneous analgesia in rats. Cutaneous analgesia was evaluated by a block of the cutaneous trunci muscle reflex (CTMR) in response to local dorsal cutaneous noxious pinprick in rats. The analgesic effect of the addition of clonidine with oxybuprocaine, bupivacaine or dextrorphan by subcutaneous injection was evaluated. On an ED(50) basis, the rank of drug potency was oxybuprocaine>bupivacaine>dextrorphan (P<0.01). Mixtures of clonidine (0.12µmol) with oxybuprocaine, bupivacaine or dextrorphan (ED(50) or ED(95)) extended the duration of action and increased the potency on infiltrative cutaneous analgesia. Among these drugs, the addition of clonidine to bupivacaine (amide type) elicits the most effective cutaneous analgesia. Clonidine at the dose of 0.12 and 0.24µmol did not produce cutaneous analgesia. Oxybuprocaine showed more potent cutaneous analgesia than bupivacaine or dextrorphan in rats. Co-administration of oxybuprocaine, bupivacaine or dextrorphan with clonidine increased the potency and duration on infiltrative cutaneous analgesia. The addition of clonidine to bupivacaine (amide type) elicits more effective cutaneous analgesia than oxybuprocaine (ester type) or dextrorphan (non-ester or non-amide type).

N-methyl-D-aspartate (NMDA) antagonists--S(+)-ketamine, dextrorphan, and dextromethorphan--act as calcium antagonists on bovine cerebral arteries.

Ketamine, an intravenous anesthetic and a major drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Ketamine's enantiomer, S(+)-ketamine, acts stereoselectively on neuronal NMDA receptors. The purpose of this in vitro study was to compare the direct effects of S(+)-ketamine, 2 other noncompetitive NMDA receptor antagonists (dextrorphan and dextromethorphan), and the calcium entry blocker nimodipine on the cerebral vasculature, using bovine middle cerebral arteries as an experimental model. Arterial rings were mounted in isolated tissue chambers equipped with isometric tension transducers to obtain pharmacologic dose-response curves. In the absence of exogenous vasoconstrictors, the NMDA antagonists or nimodipine had negligible effects on cerebral arterial tone. When rings were preconstricted with either potassium or the stable thromboxane A2 mimetic U46619, the NMDA antagonists and nimodipine each produced dose-dependent relaxation. Prior endothelial stripping had no effect on subsequent drug-induced relaxation of K+-constricted rings. In Ca2+-deficient media containing either potassium or U46619, the NMDA antagonists and nimodipine each produced competitive inhibition of subsequent Cainduced constriction. In additional experiments, arterial strips were mounted in isolated tissue chambers to directly measure calcium uptake, using 45calcium (45Ca) as a radioactive tracer. The NMDA antagonists and nimodipine each blocked potassium-stimulated or U46619-stimulated Ca2+ uptake into arterial strips. These results indicate that S(+)-ketamine, dextrorphan, and dextromethorphan, like nimodipine, directly dilate cerebral arteries by acting as calcium antagonists; they all inhibit 45Ca uptake through both potential-operated (potassium) and receptor-operated (U46619) channels in cerebrovascular smooth muscle.

Brain levels of dextromethorphan and the intensity of opioid withdrawal in mice.

Consistent with their antagonistic actions at N-methyl-D-aspartate type glutamate receptors, dextromethorphan (DXM) and its metabolite, dextrorphan (DXT) decrease the intensity of opioid withdrawal syndrome. Since quinidine (QND) affects CYP2D6-mediated metabolism and P-glycoprotein governed transport, we sought to determine whether co-treatment with QND would affect brain levels of DXM and DXT as well as the effect of these compounds on opioid withdrawal syndrome in mice. We found that DXM dose dependently inhibited the intensity of opioid withdrawal syndrome and that there was a tendency for a further decrease when QND was co-administered with DXM. Administration of 30 mg/kg of DXM resulted in higher brain levels of DXM and DXT than administration of 10 mg/kg of DXM, but much lower DXT levels than that produced by 30 mg/kg of DXT. Co-treatment with QND resulted in higher brain levels of DXM (but not DXT) suggesting that QND produces an increase in the brain availability of DXM. In summary, brain levels of DXM were inversely correlated with the intensity of opioid withdrawal syndrome. QND induced increased brain levels of DXM tend to attenuate the intensity of opioid withdrawal syndrome. We suggest that it is DXM, rather than DXT, that is responsible for the attenuating effect on the intensity of opioid withdrawal syndrome, and that the beneficial action of QND on the effect of DXM should be more pronounced in humans.

Isobolographic analysis of epinephrine with bupivacaine, dextromethorphan, 3-methoxymorphinan, or dextrorphan on infiltrative anesthesia in rats: dose-response studies.

BACKGROUND AND OBJECTIVES: The aims of this study were to establish the potencies of epinephrine, bupivacaine, dextromethorphan, 3-methoxymorphinan, and dextrorphan and evaluate interactions of epinephrine with bupivacaine, dextromethorphan, 3-methoxymorphinan, or dextrorphan as an infiltrative anesthetic. Bupivacaine, a common and long-acting local anesthetic, was used as control. METHODS: Dose-dependent responses of epinephrine, dextromethorphan, 3-methoxymorphinan, and dextrorphan on cutaneous analgesia were compared with bupivacaine in rats. The interactions of drugs were evaluated via an isobolographic analysis. RESULTS: We found that epinephrine, bupivacaine, dextromethorphan, 3-methoxymorphinan, and dextrorphan produced a dose-dependent local anesthetic effect as infiltrative cutaneous analgesia. Relative potencies were epinephrine > bupivacaine > dextromethorphan > 3-methoxymorphinan > dextrorphan (P < .01 for each comparison). Coadministration of bupivacaine with epinephrine produced a synergistic effect, and coadministration of dextromethorphan, 3-methoxymorphinan, or dextrorphan with epinephrine produced an additive effect. CONCLUSIONS: Epinephrine, dextromethorphan, 3-methoxymorphinan, and dextrorphan are known to have local anesthetic effects as infiltrative cutaneous analgesia in rats. Epinephrine increased the potency of bupivacaine, but not dextromethorphan, 3-methoxymorphinan, or dextrorphan as an infiltrative anesthetic. The cutaneous analgesic effects of adding epinephrine to dextromethorphan, 3-methoxymorphinan, or dextrorphan, are similar to combinations of 2 local anesthetics.

The spinal anaesthetic effect of dextromethorphan, dextrorphan, and 3-methoxymorphinan.

Dextromethorphan, an antitussive, has a complex pharmacologic profile and has not been well studied. Our aim was to evaluate whether dextromethorphan and its metabolites, dextrorphan and 3-methoxymorphinan, have a spinal anaesthetic effect. Using a method of spinal blockade in rats, we evaluated the potencies and durations of the effects of dextromethorphan and its metabolites on spinal blockades of motor function and nociception. Bupivacaine was the active control. We found that dextromethorphan and its metabolites produced a dose-related spinal blockade of motor function and nociception. On an ED(50) basis, the ranks of potencies were bupivacaine>dextrorphan>3-methoxymorphinan>dextromethorphan (p<0.05 for the differences). On an equipotent basis, dextrorphan and bupivacaine produced similarly longer nociceptive blockades than did dextromethorphan and 3-methoxymorphinan (p<0.05 for the differences). Co-administration of dextromethorphan or its metabolites with bupivacaine produced an additive effect. In conclusion, intrathecal injections of dextromethorphan or its metabolites, dextrorphan and 3-methoxymorphinan, produced dose-related spinal blockades of motor function and nociception. The suitability of these drugs as clinical spinal anaesthetics is worth further evaluation.

Effect of sodium ozagrel on the activity of rat CYP2D6.

The aim of the study was to investigate the influence of sodium ozagrel on CYP2D6 (cytochromeP450 2D6) activity. The studies were performed with rat urine and liver microsomes and chemical inhibitors. The metabolism of dextromethorphan (dextrophan/dextromethorphan, dextrophan is a metabolite of dextromethorphan) and phenacetin (paracetamol/phenacetin, paracetamol is a metabolites of phenacetin) was used as probe to measure CYP2D6 and CYP1A2 (cytochromeP450 1A2) activity, respectively, determined by high-performance liquid chromatography (HPLC). The results showed that the metabolism of dextrophan/dextromethorphan in the sodium ozagrel-treated group (37 mg/kg) was higher than that of the control (P<0.05/6) in both in vivo and in vitro studies (r=0.9811). The rate of dextromethorphan metabolism was inhibited by sodium ozagrel and cimetidine in rat liver microsomes prepared from sodium ozagrel-treated rats and control rats group (sodium ozagrel IC(50)=26.5 microM, cimetidine IC(50)=86.3 microM in sodium ozagrel-treated group; sodium ozagrel IC(50)=13.9 microM, cimetidine IC(50)=24.8 microM in control group). The inhibitory effect of sodium ozagrel on CYP2D6 activity was noncompetitive with dextromethorphan with a K(i) of 324.94 microM. Kinetic parameters of the reactions were established by using Lineweaver-Burk with K(m)=0.67 mM and V(max)=2.13 pm/min/mg protein for the sodium ozagrel-treated group and K(m)=0.29 mM, and V(max)=0.91 pm/min/mg protein for the control group, respectively. The expression of CYP2D6 protein in the treated group was higher than that of the control group, as determined by Western blotting. The activity and expression of CYP1A2 did not show obvious differences in the control group and sodium ozagrel treated group. In conclusion, sodium ozagrel metabolism in rats is mediated primarily through CYP2D6, and sodium ozagrel can induce CYP2D6 activity.

Inactivation of CYP2D6 by methylenedioxymethamphetamine in different recombinant expression systems.

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomestrade mark and Bactosomestrade mark) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20microM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50microM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI (k(inact), the maximum inactivation rate constant and K(I), the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater (p<0.05) for Bactosomestrade mark (0.95+/-0.33min(-1), 42.9+/-20.1microM) than those found using yeast microsomes (0.28+/-0.04min(-1), 2.86+/-1.18microM) and Supersomestrade mark (0.38+/-0.05min(-1), 3.66+/-0.10microM). After correction for depletion of MDMA during pre-incubation, k(inact) and K(I) values determined using Bactosomestrade mark decreased significantly but remained higher than for the other rCYP systems (p<0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomestrade mark and Bactosomestrade mark. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.

Comparative effects of dextromethorphan and dextrorphan on nicotine discrimination in rats.

While the role of dextrorphan and dextromethorphan as N-methyl-d-aspartate (NMDA) receptor antagonists has received considerable research attention, their effects on nicotinic acetylcholine receptors (nAChR) has been less well characterized. Recent in vitro and in vivo research has suggested that these drugs noncompetitively block alpha3beta4*, alpha4beta2, and alpha7 nAChR subtypes and antagonize nicotine's antinociceptive and reinforcing effects. Both drugs were most potent at blocking alpha3beta4* AChR. This study investigated the effects of dextrorphan and dextromethorphan on nicotine's discriminative stimulus effects. Three groups of rats were trained in a two-lever drug discrimination procedure to discriminate 0.4 mg/kg s.c. nicotine from saline. Nicotine dose-dependently substituted for itself in all three groups. In contrast, when dextrorphan (group 1) or dextromethorphan (group 2) were injected i.p., neither substitution for nor antagonism of nicotine was observed for either drug. Since i.p. administration allows substantial metabolism of dextromethorphan to its parent compound dextrorphan, the two drugs were also tested following s.c. administration (group 3). Discrimination results were similar across both routes of administration, in that neither substitution nor antagonism occurred, however, s.c. administration reduced response rates to a much greater extent than did i.p. administration. Previous work suggests that beta2 subunits are crucial for mediation of nicotine's discriminative stimulus effects and may play a role in its reinforcing effects, albeit other research suggests a role for alpha3beta4* nicotinic receptors in the latter. Our results suggest that alpha3beta4* nicotinic receptors do not play a major role in nicotine's discriminative stimulus effects. Further, they suggest that the role of cholinergic mediation of the behavioral effects of dextrorphan and dextromethorphan related to the abuse properties of nicotine may be minimal.

Potential interactions of methylphenidate and atomoxetine with dextromethorphan.

OBJECTIVE: To examine the potential for drug-drug interactions to influence drug metabolism between the attention-deficit/hyperactivity disorder (ADHD) dl-methylphenidate and atomoxetine with dextromethorphan, a probe for interactions involving cytochrome P450 (CYP) 2D6 isoenzyme. DESIGN: In vitro and ex vivo analysis of changes in metabolism of study drugs. SETTING: Laboratory. PATIENTS: Not applicable. INTERVENTIONS: Pooled human liver microsomal fractions prepared at CEDRA Corporation (now CellzDirect, Austin, Tex.) by the standard differential centrifugation method (lot 821-1). Human liver microsomes were pooled from 15 donors. Recombinant CYP 2D6-containing microsomes (Supersomes; lots 20 and 24 BD Gentest; Woburn, Mass.) were prepared from a baculovirus-infected insect cell line that expressed only the human CYP 2D6 isoform. Dextromethorphan, with and without effector, was incubated with pooled human liver and recombinant CYP 2D6-containing microsomes. Atomoxetine and dl-methylphenidate were tested at 0.1x, 1x, and 10x their reported therapeutic concentrations. Paroxetine, a known inhibitor of CYP 2D6, was used as a reference agent, and quinidine was used as a positive control inhibitor of CYP 2D6. MAIN OUTCOME MEASURES: Changes in substrate metabolism indicative of CYP 2D6-mediated interactions. RESULTS: Atomoxetine and paroxetine inhibited the formation of dextrorphan by about 50% in human liver microsomes and by more than 80% in recombinant microsomes; the profiles of atomoxetine and the known 2D6 inhibitor paroxetine were similar. High concentrations of dextromethorphan reversed the inhibition of its metabolism, indicating a competitive mechanism of the interaction. Conversely, dextromethorphan and dextrorphan only modestly inhibited atomoxetine and paroxetine metabolism. dl-Methylphenidate did not inhibit dextrorphan formation in either microsome preparation, and dl-methylphenidate metabolism was unaffected by dextromethorphan or dextrorphan. CONCLUSION: These results demonstrate the potential for in vivo interactions between dextromethorphan and atomoxetine in patients with ADHD. However, they do not support the plausibility of an in vivo interaction between dextromethorphan and dl-methylphenidate.

Dextromethorphan, 3-methoxymorphinan, and dextrorphan have local anaesthetic effect on sciatic nerve blockade in rats.

Dextromethorphan has been used as an antitussive for more than 40 years and is considered a drug with a good margin of safety. The aim of the study was to evaluate whether dextromethorphan and its metabolites--3-methoxymorphinan and dextrorphan--had local anaesthetic effects. Using a method of sciatic nerve blockade in rats, the potencies and durations of actions of dextromethorphan and its metabolites on sciatic nerve blockades of motor function, proprioception, and nociception were evaluated. Lidocaine was used as control. We found that dextromethorphan and its metabolites produced dose-related local anaesthetic effects on sciatic nerve blockades of motor function, proprioception, and nociception. The ranks of potencies were lidocaine>dextromethorphan>3-methoxymorphinan>dextrorphan (P<0.01 for each comparison). Under an equi-potent basis, dextrorphan and 3-methoxymorphinan had durations of actions longer than that of lidocaine (P<0.05 for each comparison). Co-administration of dextromethorphan or its metabolites with lidocaine produced an additive effect on sciatic nerve blockades. In conclusion, dextromethorphan and its metabolites - 3-methoxymorphinan and dextrorphan- had a local anaesthetic effect on sciatic nerve blockades of motor function, proprioception and nociception with durations of actions longer than that of lidocaine. Co-administration of dextromethorphan and its metabolites produced an additive effect on sciatic nerve blockades.

Opioid peptides and receptors in joint tissues: study in the rat.

Using immunohistochemical and biochemical techniques, the occurrence of endogenous opioid peptides and their receptors in normal rat bone and joint tissues was investigated. Opioid receptors were detected, quantified, and characterized in homogenates from capsule/synovium and periosteum using radioligand binding assays. Receptor binding of the nonselective opioid [3H]naloxone to tissue homogenates was stereospecific and saturable, showing similar characteristics to that of brain tissue, although with lower binding capacities. By immunohistochemistry, the neuronal occurrence of four different enkephalins was demonstrated in synovium, bone marrow, periosteum, and juxta-articular bone, whereas no neuronal dynorphin immunoreactivity was detected. Double-staining studies disclosed that enkephalins coexisted with substance P in primary afferent fibers. The applied techniques can be used to assess changes in the distribution of endogenous opioids and their receptors in joint tissues in conditions associated with pain and inflammation. The endogenous opioid system now demonstrated might be targeted and exploited therapeutically to obtain peripheral control of symptoms in joint disorders.