Subacute administration of both methcathinone and manganese causes basal ganglia damage in mice resembling that in methcathinone abusers

Abuse of a ‘homemade’ version of the psychostimulant drug methcathinone (Mcat) causes a persistent and disabling extrapyramidal syndrome (Sikk et al. 2011, 2013; Stepens et al. 2014).This shares some common features with Parkinson’s disease, but has distinct symptoms including early falls, dystonia, severe dysarthria, and a poor response to l-DOPA treatment (Sikk et al. 2011). The precise cause of the neurological symptoms remains unknown. However, the recipe for producing ‘homemade’ Mcat involves the oxidation of pseudoephedrine using potassium permanganate. This produces a solution containing high levels of both Mcat and manganese (Mn) which is then injected intravenously on multiple occasions each day (Sikk et al. 2007). The neurological syndrome that appears in man is accompanied by Mn deposition in the basal ganglia and altered basal ganglia function as assessed by abnormal fluorodeoxyglucose (FDG) uptake (Sikk et al. 2013; Stepens et al. 2014) and decreased striatal D2 receptor binding density (Kessler et al. 2003) shown by MRI and PET investigations.

To date the development of neurological syndrome produced by ‘homemade’ Mcat abuse has been associated with Mn toxicity based on the known effects of exposure to the metal in man (Taba 2013). Mn impairs the release of dopamine without causing a significant loss of nigro-striatal dopaminergic neurons (Guilarte 2013). This would fit with the fact that ‘homemade’ Mcat produced using sodium dichromate as the oxidizing agent, does not appear to have the same toxic effects. However, Mcat itself also causes striatal dopamine release and inhibits its reuptake after both acute and chronic administration (Cozzi et al. 2013) and its potential toxicity on longer term administration is unclear. This raises the possibility that effects of ‘homemade’ Mcat seen in man are either due to Mn toxicity or to the actions of Mcat or to a synergistic interaction. This has never previously been investigated.

In this investigation, we have studied the effects of long-term administration of Mcat or Mn to mice. Importantly, we have compared the effects to those produced by ‘homemade’ Mcat produced according to the recipe used by drug addicts in their kitchens and that contains the same ratio of Mcat to Mn found in the illegal drug mixture. We investigated alterations in motor performance and the effects on dopaminergic neurons in basal ganglia, and changes in postsynaptic dopamine D2 receptor binding SPECT analysis with ¹²³I-epidepride.

Throughout the drug treatment period both Mcat and Mcat/Mn stimulated motor activity, with activity increasing compared to control immediately after dosing. In the motility box, distance covered, time in motion and time spent on rear feet were significantly decreased compared to the control group (Table 1). After 4 weeks of drug treatment a clear suppression in overall motor activity of animals treated with Mcat, Mn, or Mcat/Mn was found.

Weight of the animals did not change significantly during the 4 weeks. The mean weight by treatment groups were control 22.8 g (95% CI 21.9–23.7), Mcat/Mn 23.3 g (95% CI 22.4–24.2), Mcat 22.7 g (95% CI 21.5–23.9) and Mn 22.1 g (95% CI 21.2–23.1) at baseline and control 25.2 g (95% CI 24.5–25.9), Mcat/Mn 25.2 g (95% CI 23.8–26.7), Mcat 24.3 g (95% CI 23.1–25.5) and Mn 22.2 g (95% CI 21.4–23.1), respectively, by the end of the 4 week treatment period.

SPECT imaging demonstrated a significant decrease in specific to nonspecific epidepride binding ratio in Mcat (1.85; 95% CI 0.9–2.7), Mn (1.61; 95% CI 0.7–2.5) and Mcat/Mn (2.06; 95% CI 1.5–2.7) groups compared to the controls (mean 7.22; 95% CI 3.7–10.8; details shown in Fig. 1, left). Representative SPECT images of the anatomical regions are shown in Fig. 1 (right panels).

Results of the immunohistochemical staining studies are summarized on Figs. 2 and 3. TH positive cell counting showed a significant decrease of overall cell density in the SN in Mcat, Mn and Mcat/Mn treated animals (Fig. 2). There was a decrease in DAT expression in Mcat-treated animals as compared to the control group (Fig. 3). GAD65/67 staining was reduced by the administration of Mn alone. There was no change in VMAT2 expression following any of the treatments.

We analyzed the effects of two potentially toxic compounds, Mcat and Mn, in a homemade drug based on pseudoephedrine conversion. While Mcat is the desired oxidation product, Mn is generated in a free form from a major ingredient, potassium permanganate. The onset of a parkinsonian syndrome in individuals abusing this cocktail has previously been attributed to Mn toxicity since occupational exposure to the metal is associated with the occurrence of similar motor symptoms (Lucchini et al. 2009; Sikk et al. 2007). In addition, a similarly acting Mcat mixture prepared using sodium dichromate, instead of potassium permanganate does not lead to evident neurological sequelae based on short term exposure (Emerson and Cisek 1993). However, neither the conclusion that Mn alone is responsible for the syndrome occurring in man nor the potential role played by Mcat has been tested. We now show in mice that both components of this drug cocktail, Mn and Mcat, contribute to its toxicity.

The abovementioned syndrome in itself, although devastating for the affected population, is less known throughout the world. At the same time, the use of methcathinone is part of a larger epidemiological trend of synthetic cathinone abuse. The mixture of manganese and methcathinone, also termed ephedrone in literature, is mainly reported as case series or case reports and larger studies are lacking (Sikk and Taba 2015).

Administration of Mcat, Mn and a “home-made” mixture containing the two led to similar and consistent changes in motor function. As expected, there was an immediate acute response to each injection of Mcat alone or in combination with Mn over the 4-week period, in the form of stereotypical movements lasting 30–60 min. This effect has been described in relation to Mcat as well as other psychostimulants (Anneken et al. 2017). But importantly, both components (Mcat and Mn), alone or in combination, equally suppressed overall motor activity by the end of the 4-week treatment period as shown in the motility box. An interesting observation was that this effect was, if anything, more evident with Mcat alone than with Mn alone or Mn in combination with Mcat. Overall, the changes observed in motor function reflect the development of the extrapyramidal syndrome as seen in man. The described locomotor changes are in concordance with previously published literature (see the excellent review by Angoa-Perez et al. (2017).

The alterations in motor function and a marked decrease in the number of TH immunoreactive cells in substantia nigra is in good correlation. The decrease in cell number occurred after the administration of Mn or Mcat or a combination of the two. A similar change following Mn administration was described previously in mice (Stanwood et al. 2009) but this is the first time that this has been shown following Mcat treatment. The decrease presumably reflects a loss of dopaminergic cell bodies although a reduction in TH expression cannot be excluded. Psychostimulants are known to be directly toxic to nigral dopaminergic cells. For example, Ares-Santos et al. (2014) demonstrated a marked reduction in the number of nigral dopaminergic neurons together with changes in locomotor activity in response to methamphetamine administration. The overall conclusion from the examination of nigral tissue, would be that both Mn and Mcat exert toxicity and that this effect is reflected in the combination of these drugs and explains the persistence of the motor deficits seen in man.

More puzzling are the changes in the markers of presynaptic function of dopaminergic neurons in the striatum. It might have been expected that there would be reductions in VMAT2 and DAT that reflected the loss of nigral dopaminergic cells, but this was not the case. No changes in VMAT2 occurred in any treatment group and only Mcat given alone decreased DAT expression. These findings suggest that presynaptic dopaminergic function was occurring relatively normally despite substantial nigral cell loss. The explanation may be that, at the timepoints studied, there was on-going nigral TH positive cell degeneration that had yet to be reflected in changes in striatal markers. Alternatively, it may be that remaining dopaminergic terminals in the striatum have upregulated levels of VMAT2 and DAT to compensate for the losses that had already occurred. Why DAT should only be decreased by Mcat treatment while there is no effect of a combination of Mcat and Mn is a subject for further investigation. If we hypothesize that the toxicity of Mn is preferentially postsynaptic (e.g. GABA neuron) and that of Mcat presynaptic (dopamine neurons), the dose differences may also become meaningful. Doses in the home-made mixture may simply be too low to cause measurable changes. Mn alone has been shown to alter DAT functioning. A study by Roth et al. (2013) for example suggests that Mn induces DAT internalization and thus leads to disruption of dopamine signaling. However, this situation reflects the results of SPECT imaging studies in patients abusing the Mcat and Mn cocktail where presynaptic markers were unaltered despite the presence of marked neurological symptoms (Sikk et al. 2010).

Post-synaptic changes in I¹²³epidepride binding to post-synaptic D2 receptors in the striatum on SPECT imaging were marked in animals treated with Mcat alone, Mn alone or a combination of the two. We have assumed that the changes in I¹²³-epidepride binding represent a decrease in receptor number and not competition with Mcat for receptor occupancy. Mcat should be cleared from the body by the time of SPECT scanning based on its plasma half-life. The psychomotor effects of another closely related derivative of Mcat, mephedrone, can be measured for 2 h after administration but its plasma concentrations become undetectable 5 h after intravenous administration (Martinez-Clemente et al. 2013). In addition, ¹²³I-epidepride has such a high affinity for D2 receptors (Km about 50 pM) that Mcat, having a much lower affinity (micromolar), would be unlikely to displace it (Tibbo et al. 1997; Kornhuber et al. 1995). Presuming this to be the case, there are two potential explanations for these findings. The first would be that there is downregulation of post-synaptic D2 receptors reflected in decreased numbers of epidepride binding sites as a result of receptor internalization. Such a decrease would lead to decreased motor function as previously observed in mice after D2 receptor block or absence (Klinker et al. 2013). The alternative explanation is that there is a loss of striatal cells bearing D2 receptors as a result of the toxicity of Mcat and Mn. This would be consistent with previous studies that have specifically investigated the toxic actions of Mn (Stanwood et al. 2009). D2 receptor bearing interneurons are mostly GABAergic but also cholinergic and glutamatergic neurons are important. We found decreased GAD, a marker of GABA neurons, only in the Mn group. The striatal cells that have D2 receptors on their cell bodies are largely making up the indirect output pathway to the external segment of the globus pallidus. Cell loss would disrupt the striato-thalamo-cortical loop controlling voluntary movement and explain the changes seen in motor function in both man and animals. A strong argument for the importance of postsynaptic damage can be derived from the clinical poor l-DOPA response of the patients. If there is a substantial damage of the cells bearing D2 receptors, the target of l-DOPA therapy is reduced.

One of the main limitations of the present study includes the timing of experiments (locomotor effects, immunohistochemical and SPECT studies). It could be argued that a longer experiment duration could have resulted in more significant changes. While this is true, it was our goal to demonstrate the presence of abovementioned alterations rather than quantify them in detail. In addition, originating from the clinical practice, there are reports of patients suffering from markedly severe symptoms after only a short period of using the drug.

Based on our results, we conclude that both components of the Mcat and Mn cocktail can exert toxic actions that disrupt dopaminergic function and striatal output that leads to the generation of an extrapyramidal syndrome. While both lead to altered function of the basal ganglia, it is in all probability the addictive properties of Mcat that drive the frequent daily abuse of the cocktail that then inflicts cellular damage. These findings are of importance as despite the best efforts, there has been no detailed examination of post-mortem brain material from the addict population that would have allowed the uncovering of pathophysiological mechanisms responsible for their neurological symptoms to be uncovered.