Low-Affinity/High-Selectivity Dopamine Transport Inhibition Sufficient to Rescue Cognitive Functions in the Aging Rat

The worldwide increase in cognitive decline, both in aging and with psychiatric disorders, warrants a search for pharmacological treatment. Although dopaminergic treatment approaches represent a major step forward, current dopamine transporter (DAT) inhibitors are not sufficiently specific as they also target other transporters and receptors, thus showing unwanted side effects. Herein, we describe an enantiomerically pure, highly specific DAT inhibitor, S-CE-123, synthetized in our laboratory. Following binding studies to DAT, NET and SERT, GPCR and kinome screening, pharmacokinetics and a basic neurotoxic screen, S-CE-123 was tested for its potential to enhance and/or rescue cognitive functions in young and in aged rats in the non-invasive reward-motivated paradigm of a hole-board test for spatial learning. In addition, an open field study with young rats was carried out. We demonstrated that S-CE-123 is a low-affinity but highly selective dopamine reuptake inhibitor with good bioavailability. S-CE-123 did not induce hyperlocomotion or anxiogenic or stereotypic behaviour in young rats. Our compound improved the performance of aged but not young rats in a reward-motivated task. The well-described impairment of the dopaminergic system in aging may underlie the age-specific effect. We propose S-CE-123 as a possible candidate for developing a tentative therapeutic strategy for age-related cognitive decline and cognitive dysfunction in psychiatric disorders

Differential effects of novel dopamine reuptake inhibitors on interference with long-term social memory in mice

In the laboratory, long-term social recognition memory (SRM) in mice is highly susceptible to proactive and retroactive interference. Here, we investigate the ability of novel designed dopamine (DA) re-uptake inhibitors (rac-CE-123 and S-CE-123) to block retroactive and proactive interference, respectively. Our data show that administration of rac-CE-123 30 min before learning blocks retroactive interference that has been experimentally induced at 3 h, but not at 6 h, post-learning. In contrast, S-CE-123 treatment 30 min before learning blocked the induction of retroactive interference at 6 h, but not 3 h, post-learning. Administration of S-CE-123 failed to interfere with proactive interference at both 3 h and 6 h. Analysis of additional behavioral parameters collected during the memory task implies that the effects of the new DA re-uptake inhibitors on retroactive and proactive interference cannot easily be explained by non-specific effects on the animals’ general social behavior. Furthermore, we assessed the mechanisms of action of drugs using intracerebral in vivo-microdialysis technique. The results revealed that administration of rac-CE-123 and S-CE-123 dose-dependently increased DA release within the nucleus accumbens of freely behaving mice. Thus, the data from the present study suggests that the DA re-uptake inhibitors tested protect the consolidation of long-term social memory against interference for defined durations after learning. In addition, the data implies that DA signaling in distinct brain areas including the nucleus accumbens is involved in the consolidation of SRM in laboratory mice

R-Modafinil exerts weak effects on spatial memory acquisition and dentate gyrus synaptic plasticity

<div><p>Modafinil is a wake promoting drug approved for clinical use and also has cognitive enhancing properties. Its enantiomer R-Modafinil (R-MO) is not well studied in regard to cognitive enhancing properties. Hence we studied its effect in a spatial memory paradigm and its possible effects on dentate gyrus long-term potentiation (DG-LTP). Clinically relevant doses of R-MO, vehicle dimethyl sulfoxide (DMSO) or saline were administered for three days during the hole-board test and in <i>in vivo</i> DG-LTP. Synaptic levels of dopamine receptors D1R, D2R, dopamine transporter (DAT), and its phosphorylated form (ph-DAT) in DG tissue 4 h after LTP induction were quantified by western blot analysis. Monoamine reuptake and release assays were performed by using transfected HEK-293 cells. Possible neurotoxic side effects on general behaviour were also studied. R-MO at both doses significantly enhanced spatial reference memory during the last training session and during memory retrieval compared to DMSO vehicle but not when compared to saline treated rats. Similarly, R-MO rescues DG-LTP from impairing effects of DMSO. DMSO reduced memory performance and LTP magnitude when compared to saline treated groups. The synaptic DR1 levels in R-MO groups were significantly decreased compared to DMSO group but were comparable with saline treated animals. We found no effect of R-MO in neurotoxicity tests. Thus, our results support the notion that LTP-like synaptic plasticity processes could be one of the factors contributing to the cognitive enhancing effects of spatial memory traces. D1R may play an important regulatory role in these processes.</p></div

Effect of treatment on field potential measurements in DG: Stimulation with weak tetanus.

<p>Data was analyzed with two-way repeated measure ANOVA. Multiple comparison with Dunnett t (2-sided) test reveals difference between groups in fEPSP (A) and in PSA (B) measurements. * dmso vs 10 mg RMO (p<0.05), # saline vs dmso (p<0.05). Graphs shows mean ± SD. (C) Electrophysiological fEPSP (left panel) and PSA (right panel) analogue traces of baseline and 3h post LTP induction are given for DMSO, RMO and saline. (D) Analogue traces of PSA and fEPSP with marks of points of measurement.</p

Spatial learning and memory in the Hole-board.

<p>The performance was significantly better with improved RMI during day 2 and during retrieval (trial 10) on day 3 of the hole-board testing (A). (B) detailed data presentation for trial 10. Lower panels show the mean ± SEM and the upper panels the individual data points. Two-way repeated measure ANOVA for day 1 & 2 and univariate ANOVA on day 3 were used. * p < 0.05, DMSO vs R-MO 1 & 10 mg/kg. # p < 0.05, DMSO vs saline.</p

Experimental design of the hole-board and LTP experiments.

<p>For <i>in vivo</i> LTP and paired pulse facilitation test, treatment frequency and procedure was designed that matches with the hole-board test protocol. The term ‘treatment’ implies that the animals either administered with R-MO or DMSO or saline 30 min before the start of the actual experiment.</p

Monoamine neurotransmitter reuptake inhibition and release assay.

<p>(A) Inhibition of [³H]DA, [³H]MPP⁺ and [³H]5-HT reuptake by increasing concentrations of R-modafinil in HEK293 cells stably expressing human isoforms of DAT, NET and SERT. Experiments were performed as described in the methods section. Unspecific uptake was determined by using 10 μM mazindole for HEK-DAT and HEK- NET and 10 μM paroxetine for HEK-SERT. The percentage of maximum uptake was determined by using 1% DMSO in KHB. Graph shows mean values ± SD. (B) The release assay was performed in HEK-DAT cells. Cells were grown on PDL-coated coverslips, treated with 0,05 μM [³H]MPP⁺ at 37°C for 20 min and washed with KHB for 40 min in superfusion chambers. First three time points (baseline) and next four points were recorded without any compounds and with 10 μM Monensin, respectively. Final five points were with either 10 μM R modafinil or 10 μM D-amphetamine. Non-linear regression analysis was carried out. Values are presented as mean ± SD.</p

DMSO and R-MO diametrically modulate D1 receptor levels in the dentate gyrus: Synaptosome fraction of the dentate gyrus tissue was analyzed in western blots post LTP induction, probed for D1 (100 kDa), D2 (50 kDa), DAT (115 kDa) and DAT-phospho (75 kDa).

<p>D1 levels were significantly increased DMSO treated rats compared to the saline and R-MO (dissolved in DMSO) treated rats. There were no differences in other receptor or transporter levels analyzed. Student’s T-test statistical analysis was performed. Graph indicated mean ± SD. ** p < 0.005, ***p < 0.001.</p

Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine Transporter Inhibitors

Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogues have been reported so far, but more target-specific analogues remain to be discovered. It was the aim of this study to synthesize and characterize such analogues and, indeed, a series of compounds were showing higher activities on the DAT and a higher selectivity toward DAT versus serotonin and norepinephrine transporters than modafinil. This was achieved by substituting the amide moiety by five- and six-membered aromatic heterocycles. In vitro studies indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding different from that of cocaine. Moreover, no release of dopamine was observed, ruling out amphetamine-like effects. The absence of neurotoxicity of a representative analogue may encourage further preclinical studies of the above-mentioned compounds