Comparative affinity of duloxetine and venlafaxine for serotonin and norepinephrine transporters in vitro and in vivo, human serotonin receptor subtypes, and other neuronal receptors.

The blockade of serotonin (5-HT) and norepinephrine (NE) transporters in vitro and in vivo by the dual 5-HT/NE reuptake inhibitors duloxetine and venlafaxine was compared. Duloxetine inhibited binding to the human NE and 5-HT transporters with K(i) values of 7.5 and 0.8 nM, respectively, and with a K(i) ratio of 9. Venlafaxine inhibited binding to the human NE and 5-HT transporters with K(i) values of 2480 and 82 nM, respectively, and with a K(i) ratio of 30. Duloxetine inhibited ex vivo binding to rat 5-HT transporters and NE transporters with ED(50) values of 0.03 and 0.7 mg/kg, respectively, whereas venlafaxine had ED(50) values of 2 and 54 mg/kg, respectively. The depletion of rat brain 5-HT by p-chloramphetamine and depletion of rat hypothalamic NE by 6-hydroxydopamine was blocked by duloxetine with ED(50) values of 2.3 and 12 mg/kg, respectively. Venlafaxine had ED(50) values of 5.9 and 94 mg/kg for blocking p-chloramphetamine- and 6-hydroxydopamine-induced monoamine depletion, respectively. Thus, duloxetine more potently blocks 5-HT and NE transporters in vitro and in vivo than venlafaxine.

S33005, a novel ligand at both serotonin and norepinephrine transporters: I. Receptor binding, electrophysiological, and neurochemical profile in comparison with venlafaxine, reboxetine, citalopram, and clomipramine.

S33005 displayed marked affinity for native, rat, and cloned human serotonin (5-HT) transporters (SERT) and less pronounced affinity for norepinephrine (NE) transporters (NET), while its affinity at dopamine (DA) transporters and >50 other sites was negligible. Reuptake of 5-HT and (less potently) NE into cerebral synaptosomes was inhibited by S33005, whereas DA reuptake was little affected. In vivo, S33005 prevented depletion of cerebral pools of 5-HT by parachloroamphetamine. Furthermore, it decreased electrical activity of raphe-localized serotonergic neurones, an action abolished by the 5-HT1A antagonist WAY100,635. At higher doses, S33005 blocked firing of locus ceruleus-localized adrenergic neurones, an action abolished by the alpha2-adrenergic antagonist idazoxan. In contrast, S33005 did not inhibit ventrotegmental dopaminergic neurones. In frontal cortex of freely moving rats, S33005 dose dependently elevated dialysate levels of 5-HT, NE, and DA. In hippocampus, levels of 5-HT and NE were similarly elevated, while in nucleus accumbens and striatum, levels of 5-HT were increased whereas DA was unaffected. Upon chronic (2 weeks) administration, basal levels of NE were elevated in frontal cortex and, therein, 5-HT2A receptor density was decreased. Comparative studies with clinically used antidepressants showed that venlafaxine possessed a profile similar to S33005 but was less potent. Clomipramine likewise interacted with SERTs and NETs but also with several other receptors types, while citalopram and reboxetine were preferential ligands of SERTs and NETs, respectively. In conclusion, S33005 interacts potently with SERTs and, less markedly, with NETs. It enhances extracellular levels of 5-HT and NE throughout corticolimbic structures and selectively elevates dialysis levels of DA in frontal cortex versus subcortical regions.

Lack of neuroprotective effect of sigma receptor ligands in the neurotoxicity of p-chloroamphetamine in rat brain.

We studied the mechanism of antagonism of p-chloroamphetamine-induced neurotoxicity by dextromethorphan. The pretreatment with potent and selective sigma receptor ligands, 4-phenyl-4-(1-phenylbutyl)piperidine (4-PPBP) and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100), did not alter the reduction of 5-hydroxytryptamine and 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the cerebral cortex by repeated administration of p-chloroamphetamine. These results suggest that sigma receptors might not play a significant role in the antagonism of p-chloroamphetamine-induced neurotoxicity by dextromethorphan.

Effects of duloxetine, an antidepressant drug candidate, on concentrations of monoamines and their metabolites in rats and mice.

Duloxetine, (+)-N-methyl-3-(1-naphthalenyloxy)-2-thiophenepropanamine, is an inhibitor of the serotonin and norepinephrine neuronal transporters (Wong et al., 1993). In mice, duloxetine antagonized the depletion of brain serotonin by p-chloroamphetamine (ED50 = 2.5 mg/kg, i.p.) and the depletion of heart norepinephrine by 6-hydroxydopamine (ED50 = 1.1 mg/kg, i.p.). Brain concentrations of 5-hydroxyindoleacetic acid were decreased by duloxetine at 2 hr after doses of 1, 3 and 10 mg/kg and at 1 to 8 hr (but not 24 hr) after a 10 mg/kg i.p. dose of duloxetine. Duloxetine antagonized norepinephrine depletion in frontal cortex, but not dopamine depletion in striatum, after treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In rats, duloxetine decreased brain 5-hydroxyindoleacetic acid dose-dependently for up to 8 hr and decreased serotonin turnover measured by the accumulation of 5-hydroxytryptophan in rat hypothalamus after decarboxylase inhibition. In rats, duloxetine antagonized the depletion of brain serotonin by p-chloramphetamine and the depletion of norepinephrine and epinephrine in hypothalamus after i.c.v. injection of 6-hydroxydopamine. In vitro, duloxetine had little effect on either type A (serotonin as substrate) or type B (phenylethylamine as substrate) monoamine oxidase, IC50 concentrations being above 10(-5) M. These data extend evidence that duloxetine inhibits serotonin and norepinephrine transporters in vivo, actions that may lead to therapeutic efficacy in mental depression.

Evaluation of nefopam as a monoamine uptake inhibitor in vivo in mice.

Nefopam antagonized 6-hydroxydopamine-induced depletion of heart norepinephrine in mice with an ED50 value of 12 mg/kg. Nefopam was ineffective in antagonizing p-chloroamphetamine-induced depletion of brain serotonin in our standard assay in mice, apparently due to a short duration of action. Brain concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were decreased after a 32 mg/kg, i.p., dose of nefopam at 1 and 2 hr but not at 4 hr. When nefopam was injected simultaneously with p-chloroamphetamine, it prevented brain serotonin depletion initially, but by 6 hr the protective effect was essentially lost, suggesting that p-chloroamphetamine persisted in mouse brain longer than did nefopam. Nefopam caused a dose-related antagonism of brain serotonin depletion at 2 hr after injection of a low dose of p-chloroamphetamine hydrochloride (10 mg/kg, i.p.), with a calculated ED50 value of 11 mg/kg. The lowering of brain 5-HIAA concentration 2 hr after nefopam injection occurred after a 32 mg/kg dose but not after a 3 or 10 mg/kg dose. These data suggest that nefopam is effective as an inhibitor of norepinephrine and serotonin uptake at doses previously shown to be analgesic in mice, consistent with uptake inhibition being a postulated mechanism important in its analgesic effect. Nonetheless, nefopam is a relatively weak inhibitor of monoamine uptake with a short duration of action in mice.

Drug concentrations in mouse brain at pharmacologically active doses of fluoxetine enantiomers.

The i.p. injection of R-fluoxetine into mice at doses of 1-10 mg/kg led to higher concentrations of the desmethyl metabolite, R-norfluoxetine, in whole brain than was true for S-fluoxetine. R-Norfluoxetine, but not S-norfluoxetine, concentrations predominated over those of the parent drug at 7-24 hr after injection of the corresponding fluoxetine enantiomer. The more rapid N-demethylation of R-fluoxetine, and the relative inactivity of R-norfluoxetine as a serotonin uptake inhibitor compared with S-norfluoxetine, may explain the earlier report that R-fluoxetine is less potent than S-fluoxetine in antagonizing p-chloroamphetamine depletion of brain serotonin in mice. In the present study, a 10 mg/kg, i.p., dose of S-fluoxetine completely prevented p-chloroamphetamine given 24 hr later from depleting brain serotonin, whereas R-fluoxetine offered no protection at this time.

5-HT loss in rat brain following 3,4-methylenedioxymethamphetamine (MDMA), p-chloroamphetamine and fenfluramine administration and effects of chlormethiazole and dizocilpine.

1. The present study has investigated whether the neurotoxic effects of the relatively selective 5-hydroxytryptamine (5-HT) neurotoxins, 3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy'), p-chloroamphetamine (PCA) and fenfluramine on hippocampal and cortical 5-HT terminals in rat brain could be prevented by administration of either chlormethiazole or dizocilpine. 2. Administration of MDMA (20 mg kg-1, i.p.) resulted in an approximate 30% loss of cortical and hippocampal 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content 4 days later. Injection of chlormethiazole (50 mg kg-1) 5 min before and 55 min after the MDMA provided complete protection in both regions, while dizocilpine (1 mg kg-1, i.p.) protected only the hippocampus. 3. Administration of a single dose of chlormethiazole (100 mg kg-1) 20 min after the MDMA also provided complete protection to the hippocampus but not the cortex. This regime also attenuated the sustained hyperthermia (approx +2.5 degrees C) induced by the MDMA injection. 4. Injection of PCA (5 mg kg-1, i.p.) resulted in a 70% loss of 5-HT and 5-HIAA content in hippocampus and cortex 4 days later. Injection of chlormethiazole (100 mg kg-1, i.p.) or dizocilpine (1 mg kg-1, i.p.) 5 min before and 55 min after the PCA failed to protect against the neurotoxicity, nor was protection afforded by chlormethiazole when a lower dose of PCA (2.5 mg kg-1, i.p.) was given which produced only a 30% loss of 5-HT content. Chlormethiazole did prevent the hyperthermia induced by PCA (5 mg kg-1), while the lower dose of PCA (2.5 mg kg-1) did not produce a change in body temperature.5. Neither chlormethiazole nor dizocilpine prevented the neurotoxic loss of hippocampal or cortical 5-HT neurones measured 4 days following administration of fenfluramine (25 mg kg-1, i.p.).6. In general, chlormethiazole and dizocilpine were effective antagonists of the 5-HT-mediated behaviours of head weaving and forepaw treading which appeared following injection of all three neurotoxins.7. Both chlormethiazole and dizocilpine have previously been shown to prevent the neurotoxic effects ofa high dose of methamphetamine on cerebral 5-HT and dopamine pathways. These drugs also prevent MDMA-induced neurotoxicity of 5-HT pathways, but not that induced by injection of PCA or fenfluramine. This suggests that the mechanisms of neurotoxic damage to 5-HT pathways produced by substituted amphetamines cannot be identical. The monoamine loss does not appear to result from the hyperthermia produced by the neurotoxic compounds.

The N-methyl-D-aspartate antagonist MK-801 protects against serotonin depletions induced by methamphetamine, 3,4-methylenedioxymethamphetamine and p-chloroamphetamine.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocks the ability of D-methamphetamine (MA) to deplete striatal dopamine (DA). We now report that MK-801 attenuates decreases in serotonin (5-HT) concentration induced by MA and two other amphetamine analogues, 3,4-methylenedioxymethamphetamine (MDMA) and p-chloroamphetamine (PCA). Rats were injected with saline (1.0 ml/kg) or MK-801 (0.5, 1.0 or 2.5 mg/kg) followed by either saline (1.0 mg/kg), MA (4, 2 or 1 injection(s); 10.0, 20.0 or 40.0 mg/kg), MDMA (20.0 or 40.0 mg/kg) or PCA (5.0 or 10.0 mg/kg). In some experiments, two injections of MK-801 or saline were used. Seventy-two hours after the last injection rats were sacrificed and concentrations of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and DA were determined in hippocampus and striatum. MA caused a depletion of 5-HT to 33% of control in hippocampus and to 50% of control in striatum after the 4 x 10.0 mg/kg dose regimen. When MK-801 (2.5 mg/kg) was co-administered with MA, concentrations of 5-HT did not differ from control levels in either brain region. MDMA depleted 5-HT to approximately 58% of control in hippocampus and 66% of control in striatum at the 40 mg/kg dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Dextromethorphan antagonizes the acute depletion of brain serotonin by p-chloroamphetamine and H75/12 in rats.

A role for calcium in p-chloroamphetamine-induced neurotoxicity has been inferred previously from protective effects of dextromethorphan. We found that dextromethorphan reduces rat brain concentrations of 5-hydroxyindoleacetic acid and blocks the acute, non-neurotoxic depletion of brain serotonin by p-chloroamphetamine and by H75/12. Inhibition of the membrane transporter on brain serotonin neurons by dextromethorphan in vivo might explain its protective effect against p-chloroamphetamine neurotoxicity.

Dextromethorphan protects against the neurotoxic effects of p-chloroamphetamine in rats.

Dextromethorphan, an agent that blocks the neuronal-damaging effects of hypoxemia in vitro, was tested for its ability to prevent the neurotoxic effects of p-chloroamphetamine (PCA). Rats were treated with either saline, PCA, dextromethorphan, or the combination of PCA and increasing doses of dextromethorphan. Dextromethorphan provided a dose-related protection against the serotonin (5-HT)-depleting effects of PCA. These observations may offer a clue as to the mechanism responsible for PCA-induced neurotoxicity.

Biochemical and functional studies on EMD 49,980: a potent, selectively presynaptic D-2 dopamine agonist with actions on serotonin systems.

EMD 49,980 proved to be a potent and selectively presynaptic D-2 dopamine receptor agonist in biochemical studies with rats. Thus, the gamma-butyrolactone-induced accumulation of dihydroxyphenylalanine, used as a presynaptic model, was antagonized with ED50 values of 0.29 and 0.09 mumol/kg in striatum and t. olfactorium, respectively, with high maximal effects. In contrast, striatal acetylcholine concentrations, reflecting actions at normosensitive postsynaptic D-2 receptors, were only moderately increased by about 30% over a dose range of 2.3-68 mumol/kg. In rats with unilateral nigrostriatal lesions, EMD 49,980 induced long-lasting contralateral turning, indicative of agonistic actions at denervated postsynaptic D-2 receptors. In addition, EMD 49,980 potently inhibited serotonin (5-HT) uptake in vitro and in vivo. Binding studies confirmed D-2 activity in the nM range but, similarly potent effects were observed at 5-HT1A binding sites. Measurement of 5-hydroxytryptophan (5-HTP) accumulation in the n. raphe revealed that, in vivo, the net effect of EMD 49,980 on 5-HT systems is an agonistic one. Control experiments indicate that inhibition of 5-HTP accumulation by EMD 49,980 is induced mainly via direct activation of 5-HT1A receptors, although some contribution due to 5-HT uptake inhibition is likely. Furthermore, results with various reference compounds make it unlikely that there are indirect effects, also via alpha 2-receptors in the models used and support the view that D-2 agonistic, 5-HT uptake inhibiting and 5-HT1A agonistic actions are independent properties of EMD 49,980.

Synthesis and antidepressant properties of novel 2-substituted 4,5-dihydro-1H-imidazole derivatives.

A unique combination of alpha 2-adrenoreceptor antagonist and serotonin-selective reuptake inhibitory activities has been identified in a series of 2-substituted 4,5-dihydro-1H-imidazole derivatives. This combination of blocking activities has provided one of these derivatives, napamezole hydrochloride (2), with potential as an antidepressant. A discussion of the syntheses of these compounds includes a convenient method for the conversion of nitriles to imidazolines with ethylenediamine and trimethylaluminum.

Effect of cysteine on the persistent depletion of brain monoamines by amphetamine, p-chloroamphetamine and MPTP.

The administration of L-cysteine (500 mg/kg i.p.) 30 min before and 5 h after the administration of (+)-amphetamine sulfate markedly attenuated the persistent decreases in striatal dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rats one week after the administration of a single dose of amphetamine (9.2 mg/kg i.p.) to iprindole-treated animals and in mice one week after the last of four daily injections of amphetamine (30 mg/kg i.p.). Cysteine prevented the persistent decreases in striatal serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) one week after the administration of p-chloroamphetamine to rats, but failed to alter the persistent decreases in striatal DA, DOPAC and HVA in mice one week after the last of four daily doses of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 30 mg/kg s.c.). The results suggest that the mechanisms by which amphetamine and p-chloroamphetamine, but not MPTP, produce persistent depletions of striatal monoamines involve the generation of neurotoxic electrophilic intermediates which can be inactivated by the administration of cysteine.

Cyproheptadine antagonism of p-chloroamphetamine hypothermia in mice.

Serotonin and 5-hydroxyindoleacetic acid levels were measured in the brains of mice following intraperitoneal administration of 10 mg/kg p-chloramphetamine (PCA). Indolyl levels were decreased at the 60 min interval, but were unchanged 30 min following PCA injection. PCA-induced hypothermia was found to be dependent upon dose and ambient temperature. Hypothermia was attenuated by cyproheptadine and slightly enhanced in the presence of fluoxetine. Mechanisms other than intraneuronal release and serotonin depletion appear to mediate PCA-induced hypothermia.

Fluoxetine enantiomers as antagonists of p-chloroamphetamine effects in rats.

The dextrorotatory enantiomer of fluoxetine was slightly more potent than the levorotatory enantiomer in antagonizing the depletion of brain serotonin by p-chloroamphetamine in rats. The time course of the depletion of brain serotonin at times out to 24 hr after the injection of p-chloroamphetamine was determined with or without simultaneous administration of one of the fluoxetine enantiomers. The dextrorotatory enantiomer prevented the depletion of brain serotonin at any time after p-chloroamphetamine. The levorotatory enantiomer prevented the initial depletion of brain serotonin at 2 and 4 hr, but by 8 hr brain serotonin concentration was decreased and by 24 hr the depletion of serotonin was almost as great as in rats treated with p-chloroamphetamine alone. The elevation of serum corticosterone that occurred acutely after injection of a low dose of p-chloroamphetamine was significantly antagonized by both enantiomers of fluoxetine, the dextrorotatory enantiomer being slightly more potent. In contrast, the lowering of DOPAC (3,4-dihydroxyphenylacetic acid) concentration in rat brain by p-chloroamphetamine was not antagonized by either enantiomer of fluoxetine, indicating this effect is not secondary to serotonin release by p-chloroamphetamine. The results are consistent with other evidence that both enantiomers of fluoxetine are potent inhibitors of serotonin uptake, the dextrorotatory enantiomer being longer-acting than the levorotatory enantiomer in rats.

Serotonergic involvement in pharmacological action of the anxiolytic-sedatives thalidomide and supidimide.

The anxiolytic-sedative drugs thalidomide and supidimide inhibited spontaneous motor activity in rats. Both compounds inhibited the serotonin (5-HT) behavioural syndrome induced by tranylcypromine (TCP) plus L-tryptophan (TRP) or clorgyline plus the selective 5-HT uptake blocker, LM 5008 (4-[2-(3-indolyl)ethyl]piperidine) and delayed the behavioural effects of p-chloro-amphetamine, a releaser of 5-HT. The behavioural syndrome induced by the 5-HT agonist, 5-methoxy-N,N'-dimethyltryptamine (5-MeODMT) was unaffected by supidimide pretreatment. Thus supidimide does not possess 5-HT receptor antagonistic properties. This was further substantiated by the unaltered 5-HT-induced platelet aggregation in the presence of supidimide (10(-7)-10(-4) M). A decrease of 5-HT release into the synaptic cleft will lead to a diminished behavioural response to drugs that act presynaptically. Supidimide induced a greater increase in accumulation of brain 5-HT in TCP (5 mg/kg) plus TRP (100 mg/kg)-treated animals as compared to that in the corresponding controls. These data indicate that the behavioural and pharmacological actions of supidimide may be related to its inhibition of 5-HT release.

Monoaminergic involvement in the pharmacological actions of buspirone.

Buspirone, MJ-13805 and MJ-13653 did not produce a '5-hydroxytryptamine (5-HT) syndrome' in rats at doses up to 20 mg kg-1. These drugs were very weak 5-HT uptake blockers (IC50 much greater than 10 microM) compared to drugs such as chlorimipramine. These drugs did not inhibit either monoamine oxidase (MAO)-A or MAO-B. The Ki values for these agents as inhibitors of [3H]-5-HT and [3H]-ketanserin binding to rat frontal cortex or hippocampal membranes were in the microM range, well above the brain concentrations achieved after an oral dose of 25 mg kg-1. Parenterally administered buspirone blocked apomorphine-induced stereotypy, inhibited the 5-HT syndrome elicited by 5-methoxy-N,N-dimethyltryptamine, and delayed the onset of p-chloroamphetamine induced behaviours.

Ejaculations induced by p-chloroamphetamine in the rat.

The ejaculatory response following acute injections of p-chloroamphetamine (PCA) and several other drugs was measured by weighing the compact seminal material accumulated over 2 hr. p-Chloroamphetamine caused a dose-dependent ejaculatory response that was inhibited by the inhibitor of the synthesis of 5-hydroxytryptamine (5-HT), p-chlorophenylalanine (PCPA), neurotoxic doses of PCA, reserpine, DSP 4 a selective noradrenergic neurotoxin given 48 hr before PCA, the inhibitor of synthesis of noradrenaline (NA) FLA 63, the specific inhibitors of uptake of 5-HT, alaproclate, fluoxetine and norzimeldine and the selective inhibitor of the uptake of NA, CPP 199, the E form of norzimeldine. The doses of several receptor antagonists producing a 50% decrease in the weight of seminal material were determined. The non-selective 5-HT receptor antagonists, metitepine and methergoline, the selective alpha 1-adrenoreceptor antagonists, prazosin and phenoxybenzamine and the non-selective alpha-adrenoreceptor antagonist, phentolamine, had strong effects, followed by the selective 5-HT2 antagonists, ketanserin and pirenperone. Yohimbine, an alpha 2-adrenoreceptor antagonist and atropine, a muscarinic receptor antagonist, only produced a partial blockade. The rank order of potency for some dopamine (DA) receptor antagonists was chlorpromazine, domperidone, haloperidol, pimozide. Remoxipride, a selective DA2 receptor antagonist and the selective DA1 antagonist, Sch 23390, had no effect. The following drugs had no effect: propranolol, naloxone, picrotoxin, cimetidine and mepyramine. The 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MeODMT 3 mg/kg, i.p.) produced a small effect on the weight of seminal material, although 72% of the rats ejaculated. d-Amphetamine did not induce ejaculation at 5 mg/kg but had a marked effect at 15 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism by which uptake inhibitors antagonize p-chloroamphetamine-induced depletion of brain serotonin.

The mechanism by which uptake inhibitors antagonize p-chloroamphetamine (PCA)-induced depletion of brain serotonin has been suggested to involve: (1) blockade of PCA uptake into the serotonin neuron by means of the membrane carrier, or (2) blockade of the efflux of released serotonin from the neuron, this efflux also requiring the membrane carrier. According to either mechanism, antagonism of serotonin depletion by PCA would provide a valid index of inhibition of the amine uptake system on serotonin neurons in vivo.