Feasibility Study of Single-Photon Emission Computed Tomography with Iodine-123 Labeled Metaiodobenzylguanidine for Preclinical Evaluation of Labetalol as a ß-Adrenergic Receptor Blocker.

Among clinically used radiopharmaceuticals, iodine-123 labeled metaiodobenzylguanidine ([123I]mIBG) serves for diagnosing neuroendocrine tumors and obtaining images of myocardial sympathetic innervation. mIBG, a structural analogue of norepinephrine (NE), a neurotransmitter acting in peripheral and central nerves, follows a pathway similar to NE, transmitting signals through the NE transporter (NET) located at synaptic terminals. It moves through the body without decomposing, enabling noninvasive image evaluation. In this study, we aimed to quantify [123I]mIBG uptake in the adrenal glands using small animal single-photon emission computed tomography/computed tomography (SPECT/CT) images post [123I]mIBG administration. We investigated the possibility of assessing the effectiveness of ß-adrenergic receptor blockers by quantifying SPECT/CT images and biodistribution results to determine the degree of [123I]mIBG uptake in the adrenal glands treated with labetalol, a known ß-adrenergic receptor blocker. Upon intravenous administration of [123I]mIBG to mice, SPECT/CT images were acquired over time to confirm the in vivo distribution pattern, revealing a clear uptake in the adrenal glands. Labetalol inhibited the uptake of [123I]mIBG in cell lines expressing NET. A decrease in [123I]mIBG uptake in the adrenal glands was observed in the labetalol-treated group compared with the normal group through SPECT/CT imaging and biodistribution studies. These results demonstrate that SPECT/CT imaging with [123I]mIBG could be applicable for evaluating the preclinical efficacy of new antihypertensive drug candidates such as labetalol, a ß-adrenergic receptor blocker.

Proteochemometric Modeling Identifies Chemically Diverse Norepinephrine Transporter Inhibitors.

Solute carriers (SLCs) are relatively underexplored compared to other prominent protein families such as kinases and G protein-coupled receptors. However, proteins from the SLC family play an essential role in various diseases. One such SLC is the high-affinity norepinephrine transporter (NET/SLC6A2). In contrast to most other SLCs, the NET has been relatively well studied. However, the chemical space of known ligands has a low chemical diversity, making it challenging to identify chemically novel ligands. Here, a computational screening pipeline was developed to find new NET inhibitors. The approach increases the chemical space to model for NETs using the chemical space of related proteins that were selected utilizing similarity networks. Prior proteochemometric models added data from related proteins, but here we use a data-driven approach to select the optimal proteins to add to the modeled data set. After optimizing the data set, the proteochemometric model was optimized using stepwise feature selection. The final model was created using a two-step approach combining several proteochemometric machine learning models through stacking. This model was applied to the extensive virtual compound database of Enamine, from which the top predicted 22,000 of the 600 million virtual compounds were clustered to end up with 46 chemically diverse candidates. A subselection of 32 candidates was synthesized and subsequently tested using an impedance-based assay. There were five hit compounds identified (hit rate 16%) with sub-micromolar inhibitory potencies toward NET, which are promising for follow-up experimental research. This study demonstrates a data-driven approach to diversify known chemical space to identify novel ligands and is to our knowledge the first to select this set based on the sequence similarity of related targets.

The Pharmacological Profile of Second Generation Pyrovalerone Cathinones and Related Cathinone Derivative.

Pyrovalerone cathinones are potent psychoactive substances that possess a pyrrolidine moiety. Pyrovalerone-type novel psychoactive substances (NPS) are continuously detected but their pharmacology and toxicology are largely unknown. We assessed several pyrovalerone and related cathinone derivatives at the human norepinephrine (NET), dopamine (DAT), and serotonin (SERT) uptake transporters using HEK293 cells overexpressing each respective transporter. We examined the transporter-mediated monoamine efflux in preloaded cells. The receptor binding and activation potency was also assessed at the 5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C receptors. All pyrovalerone cathinones were potent DAT (IC50 = 0.02-8.7 µM) and NET inhibitors (IC50 = 0.03-4.6 µM), and exhibited no SERT activity at concentrations < 10 µM. None of the compounds induced monoamine efflux. NEH was a potent DAT/NET inhibitor (IC50 = 0.17-0.18 µM). 4F-PBP and NEH exhibited a high selectivity for the DAT (DAT/SERT ratio = 264-356). Extension of the alkyl chain enhanced NET and DAT inhibition potency, while presence of a 3,4-methylenedioxy moiety increased SERT inhibition potency. Most compounds did not exhibit any relevant activity at other monoamine receptors. In conclusion, 4F-PBP and NEH were selective DAT/NET inhibitors indicating that these substances likely produce strong psychostimulant effects and have a high abuse liability.

Design, synthesis, and biological evaluation of novel bifunctional thyrointegrin antagonists for neuroblastoma.

Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma membrane integrin αvß3. Dual targeting of the NET and integrin αvß3 receptors using a Drug-Drug Conjugate (DDC) might provide effective treatment strategy in the fight against neuroblastoma and other neuroendocrine tumors. In this work, we synthesized three dual-targeting BG-P400-TAT derivatives, dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400-PAT containing di-iodobenzene, di-methoxybenzene, and piperazine groups, respectively. These derivatives utilize to norepinephrine transporter (NET) and the integrin αvß3 receptor to simultaneously modulate both targets based on evaluation in a neuroblastoma animal model using the neuroblastoma SK-N-F1 cell line. Among the three synthesized agents, the piperazine substituted BG-P400-PAT exhibited potent integrin αvß3 antagonism and reduced neuroblastoma tumor growth and cancer cell viability by >90%. In conclusion, BG-P400-PAT and derivatives represent a potential therapeutic approach in the management of neuroblastoma.

Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.

The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z' = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

Inhibition of the norepinephrine transporter rescues vascular hyporeactivity to catecholamine in obstructive jaundice.

In patients with obstructive jaundice, the cardiovascular system exhibits hypotension and vascular hyporeactivity. Most norepinephrine is taken up through the neuronal norepinephrine transporter (NET), which is implicated in cardiovascular diseases. A previous study demonstrated that pharmacological NET inhibition could increase resting blood pressure. However, the role of NETs in vascular hyporeactivity induced by obstructive jaundice is poorly understood. This study used the NET inhibitor nisoxetine and a rat model of bile duct ligation (BDL) to investigate whether NET is associated with BDL-induced vascular hyporeactivity. Rats were injected with nisoxetine via the tail vein for 7 consecutive days after BDL. Samples of the superior cervical sympathetic ganglion (SCG) and thoracic aortic rings were processed for investigations. Our results showed that NET expression in the SCG was significantly increased after BDL. Nisoxetine prevented the augmentation of NET expression, increased α1-adrenoceptor activation, and enhanced the weakened contractile responses of thoracic aortic rings after BDL. Our study demonstrates that nisoxetine plays a protective role in BDL-induced vascular hyporeactivity through increased α1-adrenoceptor activation in rats.

A novel dopamine D3R agonist SK609 with norepinephrine transporter inhibition promotes improvement in cognitive task performance in rodent and non-human primate models of Parkinson's disease.

Mild cognitive impairment is present in a number of neurodegenerative disorders including Parkinson's disease (PD). Mild cognitive impairment in PD (PD-MCI) often manifests as deficits in executive functioning, attention, and spatial and working memory. Clinical studies have suggested that the development of mild cognitive impairment may be an early symptom of PD and may even precede the onset of motor impairment by several years. Dysfunction in several neurotransmitter systems, including dopamine (DA), norepinephrine (NE), may be involved in PD-MCI, making it difficult to treat pharmacologically. In addition, many agents used to treat motor impairment in PD may exacerbate cognitive impairment. Thus, there is a significant unmet need to develop therapeutics that can treat both motor and cognitive impairments in PD. We have recently developed SK609, a selective, G-protein biased signaling agonist of dopamine D3 receptors. SK609 was successfully used to treat motor impairment and reduce levodopa-induced dyskinesia in a rodent model of PD. Further characterization of SK609 suggested that it is a selective norepinephrine transporter (NET) inhibitor with the ability to increase both DA and NE levels in the prefrontal cortex. Pharmacokinetic analysis of SK609 under systemic administration demonstrated 98% oral bioavailability and high brain distribution in striatum, hippocampus and prefrontal cortex. To evaluate the effects of SK609 on cognitive deficits of potential relevance to PD-MCI, we used unilateral 6-hydroxydopamine (6-OHDA) lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus macaques, with deficits in performance in a sustained attention and an object retrieval task, respectively. SK609 dose dependently improved the performance of 6-OHDA-lesioned rats, with peak performance achieved using a 4 mg/kg dose. This improvement was predominantly due to a significant reduction in the number of misses and false alarm errors, contributing to an increase in sustained attention. In MPTP-lesioned monkeys, this same dose also improved performance in an object retrieval task, significantly reducing cognitive errors (barrier reaches) and motor errors (fine motor dexterity problems). These data demonstrate that SK609 with its unique pharmacological effects on modulating both DA and NE can ameliorate cognitive impairment in PD models and may provide a therapeutic option to treat both motor and cognitive impairment in PD patients.

Reticular Epithelial Edema: An Uncommon Side Effect of ROCK/NET Inhibitor Netarsudil.

A 66-year-old female with advanced primary open-angle glaucoma and Descemet's stripping endothelial keratoplasty OD with previously noted inferior stromal edema presented with a 1-month history of progressive decreased visual acuity after starting netarsudil twice daily. Her best-corrected visual acuity was 20/80 OD and no light perception OS. The right cornea was notable for inferior small epithelial bullae in a reticular pattern from 2 to 9 o'clock encroaching on the visual axis involving both sides of the graft-host junction. The reticular epithelial edema resolved upon discontinuation of netarsudil and best-corrected visual acuity improved to 20/50 but was limited by persistent stromal edema. We report a patient with a history of a partially decompensated Descemet's stripping endothelial keratoplasty who develops reticular epithelial corneal edema after starting netarsudil. This unique pattern of edema may present in the setting of preexisting endothelial cell dysfunction when netarsudil is used, a complication not noted in the Food and Drug Administration (FDA) trials.

Dual Targeting of Norepinephrine Transporter (NET) Function and Thyrointegrin αvß3 Receptors in the Treatment of Neuroblastoma.

Therapeutic targeting of the norepinephrine transporter (NET) function with benzylguanidine (BG), conjugated with the high-affinity thyrointegrin αvß3 antagonist triazole tetraiodothyroacetic acid, TAT, via noncleavable bonding to poly(ethylene glycol) (PEG400) (P) might allow for effective treatment options in neuroblastoma. BG-P-TAT is a dual-targeting agent, targeting the NET function and the thyrointegrin αvß3 receptors that are overexpressed in neuroblastoma and other neuroendocrine tumors. Various cancer cells and actively dividing tumor-endothelial cells express the thyrointegrin αvß3 receptors. In this work, the novel compound BG-P-TAT was synthesized and evaluated in the neuroblastoma SK-N-FI cell line for improved targeting and to offer a new strategy for patients with neuroblastoma. BG-P-TAT demonstrated significant suppression of neuroblastoma tumor progression, growth, and viability in a dose-dependent manner. In conclusion, BG-P-TAT represents a potential lead candidate for the treatment of neuroblastoma and other neuroendocrine tumors.

Pharmacological norepinephrine transporter inhibition for the prevention of vasovagal syncope in young and adult subjects: A systematic review and meta-analysis.

BACKGROUND: Vasovagal syncope (VVS) significantly reduces quality of life, yet lacks effective medical therapies. Pharmacological norepinephrine transporter (NET) inhibition increases synaptic norepinephrine reuptake, which may be able to prevent hypotension, bradycardia, and syncope. OBJECTIVE: The objective of this systematic review was to evaluate the ability of 3 NET inhibitors-reboxetine, sibutramine, and atomoxetine-to prevent head-up tilt-induced vasovagal outcomes in healthy participants and patients with VVS. METHODS: Relevant studies were identified from Medical Literature Analysis and Retrieval System Online, Excerpta Medica Database, Cochrane Central Register of Controlled Trials, and Cumulative Index to Nursing and Allied Health Literature without language restriction from database inception to August 2019. All randomized controlled trials comparing the benefit of a NET inhibitor vs placebo in adult populations were selected for review and meta-analysis. RESULTS: Four studies (101 participants) met inclusion criteria. The mean study size was 25 (range 11-56) participants. NET inhibition reduced the likelihood of vasovagal reactions marked by hypotension and bradycardia in healthy participants during head-up tilt testing (relative risk 0.15; 95% confidence interval 0.04-0.52; P = .003). This relative risk reduction also occurred in patients with VVS during head-up tilt when given atomoxetine (relative risk 0.49; 95% confidence interval 0.28-0.86; P = .01). This was achieved through heart rate compensation with NET inhibition toward the end of tilt testing (106 ± 32 beats/min vs 60 ± 22 beats/min; P < .001), which in turn preserved cardiac output and mean arterial pressure (71 ± 20 mm Hg vs 43 ± 13 mm Hg; P < .001) in the absence of significantly increased systemic vascular resistance. CONCLUSION: NET inhibition prevents severe vasovagal reactions and syncope induced by head-up tilt testing in both healthy participants and patients with VVS. Pharmacological NET inhibition is a promising potential treatment of recurrent syncope.

Dynamic monitoring of single-terminal norepinephrine transporter rate in the rodent cardiovascular system: A novel fluorescence imaging method.

Here, we validate the use of a novel fluorescent norepinephrine transporter (NET) substrate for dynamic measurements of transporter function in rodent cardiovascular tissue; this technique avoids the use of radiotracers and provides single-terminal resolution. Rodent (Wistar rats and C57BL/6 mice) hearts and mesenteric arteries (MA) were isolated, loaded with NET substrate Neurotransmitter Transporter Uptake Assay (NTUA) ex vivo and imaged with confocal microscopy. NTUA labelled noradrenergic nerve terminals in all four chambers of the heart and on the surface of MA. In all tissues, a temperature-dependent, stable linear increase in intra-terminal fluorescence upon NTUA exposure was observed; this was abolished by NET inhibitor desipramine (1 µM) and reversed by indirectly-acting sympathomimetic amine tyramine (10 µM). NET reuptake rates were similar across the mouse cardiac chambers. In both species, cardiac NET activity was significantly greater than in MA (by 62 ± 29% (mouse) and 21 ± 16% (rat)). We also show that mouse NET reuptake rate was twice as fast as that in the rat (for example, in the heart, by 94 ± 30%). Finally, NET reuptake rate in the mouse heart was attenuated with muscarinic agonist carbachol (10 µM) thus demonstrating the potential for parasympathetic regulation of norepinephrine clearance. Our data provide the first demonstration of monitoring intra-terminal NET function in rodent cardiovascular tissue. This straightforward method allows dynamic measurements of transporter rate in response to varying physiological conditions and drug treatments; this offers the potential to study new mechanisms of sympathetic dysfunction associated with cardiovascular disease.

Structure-Activity Relationships of Novel Thiazole-Based Modafinil Analogues Acting at Monoamine Transporters.

Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping disorders and investigated as potential therapeutics against cocaine addiction and for cognitive enhancement. Our continuous effort to find modafinil analogues with higher inhibitory activity on and selectivity toward the dopamine transporter (DAT) has previously led to the promising thiazole-containing derivatives CE-103, CE-111, CE-123, and CE-125. Here, we describe the synthesis and activity of a series of compounds based on these scaffolds, which resulted in several new selective DAT inhibitors and gave valuable insights into the structure-activity relationships. Introduction of the second chiral center and subsequent chiral separations provided all four stereoisomers, whereby the S-configuration on both generally exerted the highest activity and selectivity on DAT. The representative compound of this series was further characterized by in silico, in vitro, and in vivo studies that have demonstrated both safety and efficacy profile of this compound class.

Dopamine and norepinephrine transporter inhibition for long-term fear memory enhancement.

Psychostimulants are highly effective cognitive-enhancing therapeutics yet have a significant potential for abuse and addiction. While psychostimulants likely exert their rewarding and addictive properties through dopamine transporter (DAT) inhibition, the mechanisms of their procognitive effects are less certain. By one prevalent view, psychostimulants exert their procognitive effects exclusively through norepinephrine transporter (NET) inhibition, however increasing evidence suggests that DAT also plays a critical role in their cognitive-enhancing properties, including long-term memory enhancement. The present experiments test the hypothesis that combined strong NET and weak DAT inhibition will mimic the fear memory-enhancing but not the addiction-related effects of psychostimulants in mice. We examined the effects of the high affinity NET inhibitors atomoxetine or nisoxetine and the low affinity DAT inhibitor bupropion, either alone or in combination, on short- and long-term memory of Pavlovian fear conditioning. We also examined the addiction-related effects of combined strong NET and weak DAT inhibition using conditioned place preference and a locomotor activity test. While atomoxetine or nisoxetine alone enhanced short-term fear memory, the addition of bupropion was required to significantly enhance long-term fear memory. Additionally, combined atomoxetine and bupropion did not produce substantial motor stimulation or place preference. These findings suggest that combining strong NET and weak DAT inhibition could lead to the development of a highly effective cognitive enhancer that lacks the potential for addiction.

D-578, an orally active triple monoamine reuptake inhibitor, displays antidepressant and anti-PTSD like effects in rats.

Significant unmet needs exist for development of better pharmacotherapeutic agents for major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) as the current drugs are inadequate. Our goal in this study is to investigate behavioral pharmacological characterization of a novel triple reuptake inhibitor (TRI) D-578 which exhibits nanomolar potency at all three monoamine transporters (Ki; 16.2. 16.2, 3.23 nM, and 29.6, 20.6, 6.10 nM for the rat brain and cloned human dopamine, serotonin and norepinephrine transporters, respectively) and exhibited little to no affinity for other off-target CNS receptors. In a rat forced swim test, compound D-578 upon oral administration displayed high efficacy and not stimulating in locomotor behavior. The effects of D-578 and paroxetine were next evaluated in a rat model for traumatic stress exposure - the single prolonged stress (SPS) model - which has been shown to have construct, predictive, and behavioral validity in modeling aspects of PTSD. Our results show that SPS had no effect on the acquisition of conditioned fear, but impaired extinction learning and extinction retention of fear behavior compared to sham treatment. D-578, but not paroxetine, attenuated the extinction and extinction-retention deficit induced by SPS. These findings suggest that D-578 has greater efficacy in normalizing traumatic stress-induced extinction-retention learning in a model for PTSD compared to paroxetine. Overall these results suggest that D-578, in addition to producing a robust and efficacious antidepressant effect, may attenuate maladaptive retention of fearful memories and support further testing of this agent for the pharmacotherapy of depression and PTSD.

Novel Meta-iodobenzylguanidine-Based Copper Thiosemicarbazide-1-guanidinomethylbenzyl Anticancer Compounds Targeting Norepinephrine Transporter in Neuroblastoma.

Meta-iodobenzylguanidine (MIBG) is a ligand with high affinity against norepinephrine transporter (NET) that has been used for diagnostic imaging and radionuclide therapy of NET-expressing tumors, such as neuroblastoma. We hypothesize that MIBG can be used as a ligand for development of new anticancer drugs targeting NET-expressing neuroblastoma (NB). To test our hypothesis, we synthesized two MIBG-based anticancer copper complexes [Cu(m-TSBG)2 and Cu(p-TSBG)2] by conjugation of a thiosemicarbazone copper group onto MIBG ligand. Both Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds showed potent anticancer activity against NB cells (BE2C and SK-N-DZ cells). The NB-specific anticancer activity of Cu(m-TSBG)2 and Cu(p-TSBG)2 was further demonstrated by the reduced anticancer activities when nonconjugated MIBG ligand was used to competitively block binding of Cu(m-TSBG)2 or Cu(p-TSBG)2 onto NET-expressing NB cells. Both Cu(m-TSBG)2 or Cu(p-TSBG)2 compounds hold potential as promising new drugs for targeted therapy of neuroblastoma and other NET-expressing tumors.

'Messy' Processing of χ-conotoxin MrIA Generates Homologues with Reduced hNET Potency.

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

Pharmacological characterization of the aminorex analogs 4-MAR, 4,4'-DMAR, and 3,4-DMAR.

4,4'-Dimethylaminorex (4,4'-DMAR) is a novel psychoactive substance (NPS) that appeared on the illicit drug market in addition to the psychostimulant 4-methylaminorex (4-MAR). Both substances are methylated derivatives of aminorex, an amphetamine-like anorectic used in the 1960ies and withdrawn from the marked due to severe cardiovascular toxicity. The aim of the present study was to characterize the in vitro pharmacological profiles of 4-MAR, 4,4'-DMAR, and 3,4-dimethylaminorex (3,4-DMAR, direx). We assessed norepinephrine (NE), dopamine (DA), and serotonin (5-HT) transporter inhibition potencies and monoamine release in transporter-transfected human embryonic kidney (HEK) 293 cells. We also assessed monoamine receptor and transporter binding affinities. 4,4'-DMAR potently inhibited all monoamine transporters (IC50<1 µM) with greater potency than 3,4-methlyenedioxymethamphetaime (MDMA) and displayed a higher serotonergic over dopaminergic preference, relatively similar to MDMA (DA transporter / 5-HT transporter inhibition ratio of 0.4 and 0.08 for 4,4'-DMAR and MDMA, respectively). In contrast, 4-MAR preferentially inhibited the NE and DA transporter, exhibiting a pharmacological profile more similar to amphetamine. Both 4-MAR and 4,4'-DMAR were also substrate releasers at the DAT. 3,4-DMAR only weakly inhibited the NE transporter and showed no relevant activity at the DA and 5-HT transporter. Binding affinities of all three aminorex derivatives at various monoamine receptors were negligible (Ki values >2 µM). The in vitro pharmacological profiles indicate that 4,4'-DMAR has comparable psychoactive properties and serotonergic toxicity to MDMA and may be more potent. 4-MAR is a psychostimulant similar to amphetamine or methamphetamine. 3,4-DMAR likely has only weak psychostimulant properties.

Chronic methylphenidate preferentially alters catecholamine protein targets in the parietal cortex and ventral striatum.

The psychostimulant methylphenidate (MPH) is the primary drug treatment for attention deficit hyperactivity disorder (ADHD) in children. MPH is well known to acutely block the dopamine (DAT) and noradrenaline (NET) transporters. Its effect on additional catecholamine targets is however less known. This study was aimed at comparing the effects of acute (2 mg/kg, i.p.) and chronic (2 mg/kg twice daily for 2 weeks) MPH treatment to young rats on key catecholamine protein targets in brain regions implicated in the symptoms and treatment of ADHD. For this purpose, the density of DAT, NET, the vesicular monoamine transporter 2 (VMAT2), the rate limiting enzyme for catecholamine synthesis tyrosine hydroxylase (TH) and the dopamine D1 receptor were measured in frontal (FC), parietal cortex (PCx) and the dorsal (DS) and ventral (VS) striatum. The data demonstrate that the effects of MPH depend on duration of treatment and brain region investigated. With the exception of DAT in the VS our results indicate that chronic but not acute administration of MPH increases levels of DAT, NET, TH, VMAT2 and D1. These effects were further more prominent in the VS over DS and in the PCx compared to the FC. In addition, chronic MPH enhanced DAT levels in the left DS but not in right side. To summarize, this study shows new evidence that chronic MPH to young rats preferentially alters catecholamine targets in PCx and VS over DS and FC. The effect of chronic MPH to increase levels of DAT, NET and VMAT2 suggests that the drug might long-term loose some of its acute action to increase extracellular levels of dopamine and noradrenaline. In conclusion, these findings provide novel insights into the mechanism of action by MPH in the treatment of ADHD and further suggest that the long-term effectiveness of the stimulant drug could be limited.

Selective activation of Dopamine D3 receptors and norepinephrine transporter blockade enhances sustained attention.

Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of ∼500 nM and behaves as a NET substrate. Systemic dosing of SK609 (4 mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4 mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05 mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25 mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8 mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.

Analytical quantification, intoxication case series, and pharmacological mechanism of action for N-ethylnorpentylone (N-ethylpentylone or ephylone).

Synthetic cathinones continue to proliferate in clandestine drug markets worldwide. N-ethylnorpentylone (also known as N-ethylpentylone or ephylone) is a popular emergent cathinone, yet little information is available about its toxicology and pharmacology. Here we characterize the analytical quantification, clinical presentation, and pharmacological mechanism of action for N-ethylnorpentylone. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to quantify N-ethylnorpentylone in blood obtained from human cases. Clinical features exhibited by the intoxicated individuals are described. The activity of N-ethylnorpentylone at plasma membrane transporters for dopamine (DAT), norepinephrine (NET) and 5-HT (SERT) was assessed using in vitro assays measuring uptake inhibition and evoked release of [3 H] neurotransmitters in rat brain synaptosomes. Our LC-MS/MS method assayed N-ethylnorpentylone concentrations with limits of detection and quantification of 1 and 5 ng/mL, respectively. Quantitation was linear from 5 to 500 ng/mL, and the method displayed specificity and reproducibility. Circulating concentrations of N-ethylnorpentylone ranged from 7 to 170 ng/mL in clinical cases, and the associated symptoms included palpitations, tachycardia, agitation, hallucinations, coma and death. N-Ethylnorpentylone was a potent inhibitor at DAT (IC50  = 37 nM), NET (IC50  = 105 nM) and SERT (IC50  = 383 nM) but displayed no transporter releasing activity. We present a validated method for quantifying N-ethylnorpentylone in human case work. The drug is a psychomotor stimulant capable of inducing serious cardiovascular and neurological side-effects which can be fatal. In vitro findings indicate that N-ethylnorpentylone exerts its effects by potent blockade of DAT and NET, thereby elevating extracellular levels of dopamine and norepinephrine in the brain and periphery.