Localization of rat brain binding sites for [3H]tomoxetine, an enantiomerically pure ligand for norepinephrine reuptake sites

doi:10.1016/0304-3940(93)90737-6

Neuroscience Letters

Volume 157, Issue 2, 23 July 1993, Pages 203-206

https://doi.org/10.1016/0304-3940(93)90737-6 Get rights and content

Abstract

The distribution of binding sites for the potent inhibitor of norepinephrine (NE) reuptake, [³H]tomoxetine, was examined in rat brain using quantitative autoradiography. Scatchard analysis of [³H]tomoxetine-binding to slide-mounted sections of rat forebrain indicated that the ligand bound to two sites, a high-affinity site with a K_d of 0.29 nM and a lower-affinity site with a K_d of 16 nM. Pharmacological characterization of this high-affinity site was consistent with labelling a NE-uptake site in brain. Autoradiographic localization of the binding sites for [³H]tomoxetine was performed at a ligand concentration of 1 nM representing the distribution of high-affinity sites. The radioligand bound with a distribution of binding sites that was consistent with the known distribution of NE-containing neurons. The highest levels of binding were seen in regions, such as the locus coeruleus, bed nucleus of the stria terminalis, anterior ventral nucleus of the thalamus and the paraventricular nucleus of the hypothalamus. Low levels were seen in regions such as the caudate-putamen, ventral tegmental area and zona reticulata of the substantia nigra, where NE-containing neurons have been reported to be low. Binding to all these sites was inhibited by 1 μM desipramine which produced autoradiograms with a uniform nonspecific binding. These results indicate that low concentrations of [³H]tomoxetine can be used to localize and characterize NE-binding sites. Further study will be necessary to determine the nature of the low-affinity binding site.

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Actually, we found that the potency of 071031B to NE uptake inhibition was greater in frontal cortex than hippocampus (data not shown). Gehlert et al (14) found similar brain region difference, and they reported that the potency to inhibit NE uptake by duloxetine was greater in hypothalamus. Like frontal cortex, hypothalamus also received extensive serotonergic and noradrenergic innervations.
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Atomoxetine, Placebo and Parent Management Training in Autism; http://clinicaltrials.gov/; NCT00844753.
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Prefrontal catecholamine neurotransmission plays a key role in the therapeutic actions of drugs for attention-deficit/hyperactivity disorder (ADHD). We have recently shown that serotonin/noradrenaline reuptake inhibitors and the noradrenaline reuptake inhibitor desipramine attenuated horizontal hyperactivity in spontaneously hypertensive rats, an animal model of ADHD, and that these drugs are potential pharmacotherapeutics for ADHD. In this study, we used in vivo microdialysis to study the effects of acute and chronic (once daily for 3 weeks) administration of the serotonin/noradrenaline reuptake inhibitor venlafaxine and the noradrenaline reuptake inhibitor desipramine on noradrenaline, dopamine, and serotonin levels, and the expression of the neuronal activity marker c-Fos in the mouse prefrontal cortex and striatum. Both acute and chronic venlafaxine administration increased prefrontal noradrenaline, dopamine, and serotonin levels and striatal noradrenaline and serotonin levels. Both acute and chronic desipramine administration increased prefrontal noradrenaline and dopamine levels and striatal noradrenaline levels, with chronic administration yielding stronger increase. Chronic desipramine did not affect striatal dopamine and serotonin levels. Both acute and chronic venlafaxine administration increased the expression of c-Fos in the prefrontal cortex, whereas chronic, but not acute, desipramine administration increased the expression of c-Fos in the prefrontal cortex. Both acute and chronic venlafaxine administration increased the striatal c-Fos expression to some degree, whereas desipramine administration did not. These results suggest that acute and chronic venlafaxine and chronic desipramine administration maximally activate the prefrontal adrenergic and dopaminergic systems without affecting striatal dopaminergic systems in mice.
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Catecholamine neurotransmission in the prefrontal cortex plays a key role in the therapeutic actions of drugs for attention-deficit/hyperactivity disorder (ADHD). Recent clinical studies show that several serotonin–norepinephrine reuptake inhibitors have potential for treating ADHD. In this study, we examined the effects of acute treatment with serotonin–norepinephrine reuptake inhibitors on locomotion and the extracellular levels of monoamines in the prefrontal cortex in spontaneously hypertensive rats (SHR), an animal model of ADHD. Adolescent male SHR exhibited greater horizontal locomotion in an open-field test than male WKY control rats. Psychostimulant methylphenidate (0.3 and 1 mg/kg), the selective norepinephrine reuptake inhibitor atomoxetine (1 and 3 mg/kg), and serotonin–norepinephrine reuptake inhibitors duloxetine (10 mg/kg), venlafaxine (10 and 30 mg/kg) and milnacipran (30 mg/kg) reduced the horizontal activity in SHR, but did not affect in WKY rats. The selective norepinephrine reuptake inhibitor reboxetine (10 mg/kg) and the tricyclic antidepressant desipramine (10 and 30 mg/kg) also reduced the horizontal activity in SHR, whereas the selective serotonin reuptake inhibitor citalopram (30 mg/kg) did not. Microdialysis studies showed that atomoxetine, methylphenidate, duloxetine, venlafaxine, milnacipran, and reboxetine increased the extracellular levels of norepinephrine and dopamine in the prefrontal cortex in SHR. Citalopram did not affect norepinephrine and dopamine levels in the prefrontal cortex, although it increased the serotonin levels. Neither duloxetine nor venlafaxine increased the dopamine levels in the striatum. These findings suggest that serotonin–norepinephrine reuptake inhibitors, similar to methylphenidate and atomoxetine, have potential for ameliorating motor abnormality in the SHR model.
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In 2002, atomoxetine (ATX), a non stimulant drug was approved for the treatment of ADHD in children, adolescents, and adults as an alternative to methylphenidate (MPH) [7,9,10]. ATX is a potent inhibitor of the noradrenaline neuronal reuptake membrane transporter with minimal affinity for other monoamine transporters or receptors [11,12]. Following oral administration, ATX is well absorbed and metabolized predominantly in the liver by cytochrome P-450 enzyme pathway.
Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate.
A procedure based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine – 4 hydroxyATX – and N-desmethylatomoxetine – des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2 ml 1 M NaOH at 45 °C. Then, analytes were extracted from alkaline solution with two different 2 ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water–60% 5 mM ammonium acetate, 50 mM formic acid, 4 mM trifluoroacetic acid in acetonitrile–water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring.
The method was linear over the concentration range 0.2–50 ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0 ng/mg hair and from 0.3 to 1.0 ng/mg, respectively.
Notwithstanding the absence of any dose–hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.

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¹: Present address: David W. Robertson, Ph.D., Ligand Pharmaceuticals, Inc., 9393 Town Centre Drive, Suite 100, San Diego, CA 92121.

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Localization of rat brain binding sites for [3H]tomoxetine, an enantiomerically pure ligand for norepinephrine reuptake sites

Abstract

Brain Res.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

[3H]-Desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons

J. Neurochem.

[3H]Mazindol binding associated with neuronal dopamine and norepinephrine uptake sites

Mol. Pharmacol.

Localization of rat brain binding sites for [³H]tomoxetine, an enantiomerically pure ligand for norepinephrine reuptake sites

[³H]-Desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons

[³H]Mazindol binding associated with neuronal dopamine and norepinephrine uptake sites