Pharmacological and toxicological investigations of new psychoactive substances

Since the mid-2000s, an unprecedented amount of new psychoactive substances (NPSs) have emerged on the recreational drug market and have since then gained popularity as alternatives to traditional drugs of abuse. Besides clinical case reports, the pharmacological profiling and assessment of toxicity in vitro provide valuable information on the mechanism of action and possible risks associated with NPSs use.
Within the scope of the pharmacology part of this thesis, focus was laid on the potential of NPSs to inhibit norepinephrine (NE), dopamine (DA), and serotonin (5-HT) transporters (NET, DAT, and SERT, respectively) in transporter-transfected human embryonic kidney (HEK) 293 cells. In addition, monoamine transporter and receptor affinities were determined.
Analogs of the popular NPS 4-methylmethcathinone (mephedrone) potently inhibited NET and, with the exception of 3-methylmethcathinone (3-MMC), inhibited SERT more potently than DAT. Mephedrone and its analogs were substrate-type releasers of NE, DA, and 5-HT. The indole NPS 5-(2-aminopropyl)indole (5-IT) potently inhibited NE, DA, and 5-HT uptake and mediated an efflux of DA and 5-HT. Like amphetamine, its 4-methylated analog 4-methylamphetamine (4-MA) was a transporter substrate but in contrast to amphetamine, 4-MA had a higher selectivity for SERT vs. DAT. The two indane NPSs N-methyl-2-aminoindane (N-methyl-2-AI) and 5-methoxy-6-methyl-2-aminoindane (MMAI) were selective inhibitors of NE and 5-HT uptake, respectively, and selective releasers of the respective neurotransmitters. The predominant actions on SERT vs. DAT suggests that dimethylmethcathinones, 4-MA, and MMAI may mediate certain entactogenic effects similar to 3,4-methylenedioxymethamphetamine (MDMA), whereas 3-MMC, 5-IT, and N-methyl-2-AI are stimulants similar to amphetamine. Analogs of the prescription drug methylphenidate (MPH) mainly inhibited NET and DAT and showed only weak inhibition of SERT. Like MPH and cocaine, MPH-based NPSs did not elicit transporter-mediated efflux of any monoamines. The predominant actions on DAT vs. SERT indicates that theses compounds are associated with an increased abuse liability. Diclofensine, a NPS that was originally developed as antidepressant, was a triple monoamine inhibitor without releasing properties. The dissociative NPS diphenidine was an inhibitor of NET and DAT with moderate potency, and its methoxylated derivative methoxphenidine was a selective but weak NET inhibitor. Diphenidine and methoxphenidine were both devoid of any monoamine releasing properties.
2,5-Dimethoxy-4-substituted phenethylamines (2C drugs) and their N-2-methoxybenzyl (“NBOMe”) analogs potently interacted with serotonergic 5-HT2A, 5-HT2B, and 5-HT2C receptors. The N-2-methoxybenzyl substitution of 2C drugs increased the binding affinity at serotonergic 5-HT2A and 5-HT2C receptors and monoamine transporters but reduced binding to the 5-HT1A receptor. NBOMes and 2C drugs were mostly potent partial agonists at the 5-HT2A and 5-HT2B receptors. However, drugs with a bulky and lipophilic 4-substituent had a decreased or absent activation potential or efficacy at these receptors.
A correlation analysis revealed that the assessed in vitro data can help to predict human effective doses of stimulants and psychedelics. For stimulants, DAT and NET inhibition potency positively, and SERT inhibition potency inversely correlated with human doses reported on the Internet. For psychedelics, serotonin 5-HT2A and 5-HT2C but not 5-HT1A receptor affinity significantly correlated with human effective doses. Serotonin receptor activation data did not correlate with human doses. However, it is a necessity to determine whether a drug activates the 5-HT2A receptor in order to predict its potential to induce psychedelic effects in humans.
For the toxicological part of the thesis, focus was laid on hepatotoxic mechanisms of six synthetic cathinones in two hepatocellular cell lines. For bupropion, 3,4-methylenedioxypyrovalerone (MDPV), mephedrone, and naphyrone the depletion of cellular ATP content preceded cytotoxicity, suggesting mitochondrial toxicity. In contrast, methedrone and methylone depleted the cellular ATP pool and induced cytotoxicity at similar concentrations. Bupropion, MDPV, and naphyrone additionally decreased the mitochondrial membrane potential, confirming mitochondrial toxicity. Bupropion was the only compounds that uncoupled oxidative phosphorylation. Bupropion, MDPV, mephedrone, and naphyrone inhibited complex I and II of the electron transport chain, naphyrone also complex III. The cathinones associated with mitochondrial toxicity were shown to increase mitochondrial reactive oxygen species (ROS) and lactate production, and naphyrone and MDPV additionally depleted the cellular total glutathione (GSH) pool. Liver injury associated with these drugs is rare and affected persons likely have susceptibility factors rendering them more sensitive for the hepatotoxicity of these drugs.