Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters

AbstractPsychostimulants such as amphetamine and cocaine are illicitly used drugs that act on neurotransmitter transporters for dopamine, serotonin or norepinephrine. These drugs can by themselves already cause severe neurotoxicity. However, an additional health threat arises from adulterant substances which are added to the illicit compound without declaration. One of the most frequently added adulterants in street drugs sold as cocaine is the anthelmintic drug levamisole. We tested the effects of levamisole on neurotransmitter transporters heterologously expressed in HEK293 cells. Levamisole was 100 and 300-fold less potent than cocaine in blocking norepinephrine and dopamine uptake, and had only very low affinity for the serotonin transporter. In addition, levamisole did not trigger any appreciable substrate efflux. Because levamisole and cocaine are frequently co-administered, we searched for possible allosteric effects; at 30μM, a concentration at which levamisole displayed already mild effects on norepinephrine transport it did not enhance the inhibitory action of cocaine. Levamisole is metabolized to aminorex, a formerly marketed anorectic drug, which is classified as an amphetamine-like substance. We examined the uptake-inhibitory and efflux-eliciting properties of aminorex and found it to exert strong effects on all three neurotransmitter transporters in a manner similar to amphetamine. We therefore conclude that while the adulterant levamisole itself has only moderate effects on neurotransmitter transporters, its metabolite aminorex may exert distinct psychostimulant effects by itself. Given that the half-time of levamisole and aminorex exceeds that of cocaine, it may be safe to conclude that after the cocaine effect “fades out” the levamisole/aminorex effect “kicks in”

On the role of Levamisole as Cocaine adulterant

Kokain ist das am häufigsten eingenommene illegale Stimulanz der westlichen Gesellschaft. Untersuchungen des österreichischen gemeinnützigen Projekts Checkit! ergaben, dass 69% aller gestreckten Kokainproben aus Wien, das Anthelminthikum Levamisol enthielten. Levamisole, der aktive Wirkstoff des Medikaments Ergamisol®, welches 1999 vom Markt genommen wurde, ist seitdem nur mehr in der Veterinärmedizin zugelassen. Als 2004 eine gehäufte Anzahl von Kokainkonsumenten an Agranulozytose erkrankten, ein Krankheitsbild, das bereits als eine der Nebenwirkungen von Levamisole bekannt war, wurde erstmals die Verwendung von Levamisol als Kokainstreckmittel verifiziert. Über die genauen Ursachen der Verwendung von Levamisol wurde viel spekuliert, jedoch fehlten experimentelle Beweise. In dieser Arbeit wurde erstmals die pharmakologische Wirkung von Levamisol an humanen monoaminer-gen Neurotransmitter Transportern untersucht, welche unter anderem von Kokain inhibiert werden. Dabei handelte es sich um den Dopamin- (DAT), Noradrenalin- (NET), sowie Serotonin-Transporter (SERT). Unter Zuhilfenahme pharmakologischer Versuche, konnte gezeigt werden, dass Levamisol nur eine schwache Inhibierung am NET jedoch keine am DAT oder SERT erzeugen konnte. Unter der Annahme, Levamisol könnte, Amphetaminen gleich, eine Umkehr der Transportrichtung erzwingen, wurde auch das Freisetzungpotential gemessen. Jedoch führte Levamisol zu keiner Freisetzung in einem der getesteten Transportern. Weiters war zu diesem Zeitpunkt bekannt, dass Levamisol im menschlichen Körper zu unter anderem Aminorex metabolisiert wird. Aminorex war bereits aufgrund seiner dem Amphetamin ähnlichen Eigenschaften bekannt. Unter Berücksichtigung dieses Wissens wurden Aminorex ebenfalls an DAT, NET sowie SERT getestet. Dabei konnte gezeigt werden, dass Aminorex ein dem Kokain sehr ähnliches Inhibierungspotential besitzt. Experimente zur Messung der Neurotransmitterfreisetzung konnten zeigen, dass Aminorex einen starken Effekt am SERT, einen geringen am NET und keinen Effekt am DAT hat. Dabei zeigte Aminorex einen Kokaineffekt am DAT und NET sowie einen Amphetamineffekt am SERT, welches einen extraneuronalen Anstieg der Neurotransmitter Dopamin, Noradrenalin sowie Serotonin zufolge hätte. Demnach würde der Effekt von Aminorex jenem von Kokain zeitlich verzögert folgen und damit zu einer Verlängerung der psychoaktiven Wirkung von Kokain führen.Cocaine is one of the most widely used illicit stimulant drugs in western society. Although cocaine adulteration is common practice amongst the dealers, the choice of adulterant often remains elusive. Since 2003, multiple cases of agranulocytosis amongst cocaine users has pointed towards a new adulterant - levamisole. An investigation of “street cocaine” samples in the year 2012 by the Austrian drug prevention program “Checkit!” revealed that 69% of analysed adulterated cocaine samples contained levamisole. Although it was speculated that levamisole may itself exert a pharmacological effect in humans there was no experimental evidence to substantiate this claim. During this doctoral thesis, the effect of levamisole on three human neurotransmitter transporters, the dopamine transporter (DAT), the norepinephrine transporter (NET), as well as the serotonin transporter (SERT) was investigated, since all three are targets of cocaine. It was found that levamisole exerted a weak inhibitory effect on NET, but not on DAT or SERT. Since substances like amphetamines cause release of these monoamines, the possibility of levamisole as a releaser had also been tested. No releasing property of levamisole could be detected. Previous publications have demonstrated that levamisole is heavily metabolized in the human body. The amphetamine-like substance aminorex was found to be one of the major levamisole metabolites. Testing aminorex on DAT, NET and SERT showed that it displayed a similar strength of inhibition on all three monoamine transporters as cocaine. Furthermore, aminorex exerted a strong releasing property on SERT, whereas releasing effects on NET were weak and absent on DAT, which was distinctive to the action of cocaine. This work provides the first evidence that it is not levamisole, but its metabolite aminorex, that exerts pharmacological effects similar to those of cocaine on the human monoamine transporters. Furthermore, these data could give a possible explanation for the use of levamisole as a cocaine adulterant: as the effect of cocaine starts to wear off, the metabolized aminorex starts to “kick in”, prolonging the psychoactive action of cocaine.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)331650

Direct PIP2 binding mediates stable oligomer

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.(VLID)459696

Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by αCaMKII.

Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT). Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anxiety disorders. In addition, SERT is a major molecular target for psychostimulants such as cocaine and amphetamines. Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. The interaction between SERT and αCaMKII was verified using biochemical assays and FRET analysis and colocalization of the two molecules was confirmed in primary serotonergic neurons in culture. Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to 3,4-methylenedioxymethamphetamine (also known as "ecstasy") and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII. This finding supports the concept that uptake and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction

Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

<div><p>Background</p><p>The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene <i>SLC6A4</i> and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence.</p><p>Methods</p><p>A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (V_max) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet V_max.</p><p>Results</p><p>The present study demonstrates that increases in platelet V_max significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within <i>SLC6A4</i>. Furthermore, functional connectivity analyses indicate that platelet V_max is related to global DMN activation and not intrinsic DMN connectivity.</p><p>Conclusion</p><p>This study provides evidence that platelet V_max predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal V_max coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation.</p></div

Correlation analysis between maximal platelet 5-HT uptake velocity (V_max) and neuronal activation (n = 48).

<p>Medial prefrontal cortex, mPFC; anterior cingulate cortex, ACC; motor cortex, MOC; premotor cortex, PMC; posterior cingulate cortex, PCC; precuneus, PRE; middle temporal gyrus, MTG; inferior temporal gyrus, ITG; p, uncorrected p value; significance level after correction for multiple comparisons based on recent recommendations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092543#pone.0092543-Johnson1" target="_blank">[63]</a>: ***p<0.001; **p<0.005; *p<0.05; x, y, z are coordinates in Talairach space; L, left hemisphere; R, right hemisphere; cluster size expressed as number of voxels.</p

Functional brain correlates of platelet 5-HT uptake velocity.

<p>(<b>A–B</b>) Figures display right-hemispheric surface mappings of a whole-brain correlation analysis between platelet V_max and BOLD activity (n = 48). Significant brain areas showed positive and negative correlations. Negatively correlated clusters comprised areas of the DMN such as regions within the mPFC/ACC as well as the PCC, MTG, and ITG. Positive correlations were found in the fronto-parietal control system encompassing the CEN and SN with a significant cluster located in the right MOC and PMC. The corresponding left-hemispheric mapping is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092543#pone.0092543.s002" target="_blank">Figure S2</a>. Colorbar represents t-values. (<b>C</b>) Scatter plot shows the negative relationship between platelet V_max and BOLD activity averaged across the mPFC cluster (peak at [−7.7, 44.1, 27.5]). (<b>D</b>) Scatter plot shows the positive relationship between platelet V_max and BOLD activity averaged across the MOC cluster (peak at [20.8, −21.6, 69.1]). All analyses are controlled for age, gender and 5-HTTLPR. Serotonin, 5-HT; maximal 5-HT uptake velocity, V_max; default mode network, DMN; medial prefrontal cortex, mPFC; anterior cingulate cortex, ACC; posterior cingulate cortex, PCC; middle temporal gyrus, MTG; inferior temporal gyrus, ITG; central executive network, CEN; salience network, SN; motor cortex, MOC; premotor cortex, PMC; blood-oxygen-level dependent, BOLD; a.u., arbitrary units.</p