Relating specific neurochemical depletions to specific behavior impairments presents rich research opportunities. In this study, we document persistent effects of the second-generation pyrrolidine synthetic cathinone α-PPP on behavior and neurochemistry. We document that, consistent with previous studies of amphetamine derivatives (Murnane et al. 2012), α-PPP acutely decreases body weight over the course of a standard 6-hour dosing regimen.
The brain was then cut down the mid line and each cortical half was opened, and the hippocampus was removed (Spijker, 2011). Next, each half was cut into three coronal slices using midline anatomical markers moving rostral to caudal. The first cut was made at the beginning of the corpus callosum, the second at the fornix, and the third cut at the end of the corpus callosum. Finally, thalamus and amygdala were removed from the third section (Chiu et al., 2007; Honkanen, 1999). All tissue samples were placed in aluminum foil, inserted into a cryovial, flash frozen in liquid nitrogen, and then stored at −80°C.
Brain Dissection
It may, however, cause an excited delirium resulting in wild, unpredictable, and severely violent behavior in those under this drug’s influence. Flakka only arrived on the drug market in the mid-2000s and has since become a significant threat to public health in many areas of Florida, and Broward County in particular. Flakka, a street name for the inexpensive and dangerous “designer drug” Alpha-PVP, is one such newcomer.
Is Flakka illegal?
In rhesus macaques, provision of a tryptophan deficient diet resulted in significant reductions in cerebrospinal fluid biomarkers of serotonin tone, yet no significant change in recognition memory and significant improvement in spatial working memory (Taffe et al. 2003). The role of serotonin systems in working and recognition memory is an area ripe for additional research. ShA synthetic cathinone self-administration did not alter total DA in any measured brain region.
Fig. 3.
Moreover, it is noteworthy that immortalized cancer cell lines, which are a convenient model for in vitro studies, can be more resistant to cytotoxicity, and therefore, cell damage can be observed in concentrations higher than in normal cells in vivo (den Hollander et al. 2014; Wojcieszak et al. 2016). Long incubation times were applied in order to show whether the cytotoxicity of studied compounds increase with time, which is relevant since the common abuse pattern of synthetic cathinones includes long sessions during which multiple doses are administered (Zawilska and Wojcieszak 2013). The effects of α-PPP on serotonin levels are somewhat surprising, as it has reported selectivity for the dopamine and norepinephrine transporters over the serotonin transporter (Eshleman et al. 2017). However, we used a relatively high dose regimen to ensure near maximal levels of toxicity, and α-PPP likely loses some selectivity at such doses. Given the paucity of literature on the neurotoxic effects of synthetic cathinones, it is difficult to overly generalize or compare our findings to the literature.
- The combination of a high body temperature and extreme muscle overactivity can cause other metabolic problems to happen in the body.
- Amygdala, hippocampus, hypothalamus, prefrontal cortex (PFC), striatum, and thalamus were extracted, and tissue was analyzed with electrochemical detection and liquid chromatography mass spectrometry.
- Synthetic cathinones (“bath salts”) are β-ketone analogs of amphetamines, yet few studies have examined their potential neurotoxic effects.
- H9c2(2-1) (ATCC® CRL-1446™) cell line was purchased from the European Collection of Cell Cultures (ECACC, Porton Down, UK).
Although the doses were located at similar points on the dose-effect curves (Aarde et al., 2015; Gannon et al., 2017; Nguyen et al., 2016), it is unknown if the results of this study will generalize across doses. In contrast to the minor sex differences in self-administration behavior, sex differences in neurochemical changes were more widespread. Notably, sex differences in neurochemistry were more abundant for ShA than LgA groups, and the cause of this is unknown. Prominent sex differences emerged for NE levels in amygdala, hippocampus, PFC, and striatum for ShA groups (Fig. 4).
Synthetic cathinones are widely available throughout the U.S. (Baumann, 2014; DEA, 2017; Madras, 2017), and unintentional ingestion of synthetic cathinones is rising (Oliver et al., 2019). Identifications of synthetic cathinones in seized drug products (DEA, 2017, 2019; NDEWS, 2018b) and identification of new synthetic cathinones are rising in some parts of the U.S. (NDEWS, 2019). High school students continue to use α-pyrrolidinopentiophenone (α-PVP) (Palamar et al., 2019), and α-PVP has caused multiple medical emergencies and deaths (NDEWS, 2015, 2018a).
In the concentration range of 10–300 μM, significant cytotoxic effects were observed in SH-SY5Y, Hep G2, and RPMI 2650 cells after 24-h incubation, and in SH-SY5Y and RPMI 2650 cells after 72-h incubation. In H9c2(2-1) cells, significant effects were observed at 100–300 μM, irrespective of the incubation time (Fig. 6c). Substituted analogs differ from native PV8 as they affect H9c2(2-1) cell viability even after 24 h. 4-F-PV8 applied for 24 h markedly reduced the viability of SH-SY5Y (100–300 μM), Hep G2 (50–300 μM), RPMI 2650 (10–300 μM), and H9c2(2-1) (200 and 300 μM) cell lines, with the greatest reduction by 42% (SH-SY5Y), 77% (Hep G2), 79% (RPMI 2650), and 72% (H9c2(2-1)) (Fig. 4b). Cell viability was significantly decreased in SH-SY5Y (25–300 μM; maximal reduction by 83%), Hep G2 (50–300 μM; maximal reduction by 97%), RPMI 2650 (10–300 μM; maximal reduction by 97%), and H9c2(2-1) cells (10–300 μM; maximal reduction by 79%) (Fig. 4b). PVP and its fluoro- and methoxy-substituted derivatives produced moderate and concentration- and time-dependent decline in the viability of SH-SY5Y, Hep G2, RPMI 2650, and H9c2(2-1) cells measured as mitochondrial activity.
Effects on Neurochemistry
Each experiment included a positive control of 1% (v/v) Triton-X100, as recommended by the manufacturer. Results are expressed as a percentage value of the positive control group, considered as 100% cytotoxicity. The desired psychostimulatory effects include raised alertness and awareness, improved mood, impression of increased motivation, energy, and euphoria (Zawilska and Wojcieszak 2017).
What happened to the “Man on Flakka?”
Using a 1.5 mm diameter tissue biopsy-punch, regions of interest were taken from individual slices, as we have described previously (Murnane et al. 2012). Synthetic cathinones (“bath salts”) are β-ketone analogs of amphetamines, yet few studies have examined their potential neurotoxic effects. Taken together, we predict that α-PVP possesses a potential for compulsive abuse (ie, addiction) that is roughly similar to that of METH and MDPV but much greater than that of 4-MEC, methylone, and MDMA. Accordingly, we also predict that 4-MEC will have a relatively lower potential for compulsive use than that of MDPV and METH, would be most similar to methylone and MDMA (ie, episodic use), and may exert primarily entactogenic effects.
Side effects
5-HIAA levels were higher for females than males for rats in most self-administration conditions and in most brain regions, effects that were observed for both synthetic cathinones. In contrast, female ShA rats had lower DA levels than males in PFC, higher DOPAC levels than males in amygdala, hypothalamus, and striatum, and higher HVA levels than males in amygdala and striatum. LgA females showed lower NE levels than males in hypothalamus, whereas ShA rats showed sex differences in NE levels in the other brain regions. Females had lower NE levels than males in amygdala and thalamus for ShA rats, the latter of which only occurred for 4MMC groups, and higher NE levels than ShA males in hippocampus, PFC, and striatum. There were a few sex differences in GLU levels, but the affected brain region and direction of the effects varied by synthetic cathinone and by self-administration condition (Fig. 3–4). Overall, these data suggest that 21 days of LgA, but not ShA, self-administration of synthetic cathinones produces most of the early-stage neurochemical changes predicted by Koob and Volkow (Koob and Volkow, 2010).
It may be mixed with or sold as other illicit substances ranging from cocaine to ecstasy, LSD, methamphetamine, and more. SH-SY5Y (ATCC® CRL-2266™), Hep G2 (ATCC® HB-8065™), and RPMI 2650 (ATCC® CCL-30™) alpha-pyrrolidinopentiophenone function cell lines were purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). H9c2(2-1) (ATCC® CRL-1446™) cell line was purchased from the European Collection of Cell Cultures (ECACC, Porton Down, UK). Effective treatment typically includes a combination of medical detoxification, behavioral therapies, and ongoing support to manage cravings and prevent relapse. The epicenter of the flakka epidemic was Broward County, Florida, which includes the city of Fort Lauderdale. Despite the DEA’s efforts to ban flakka, the drug is still being manufactured and sold illegally, often through online vendors.
Male, Swiss-Webster mice were exposed to α-PPP (80mg/kg) using a binge-like dosing regimen (QID, q2h). Behavior was assessed 4–5 days after the dosing regimen, and neurochemistry was assessed the following day. Behavior was studied using the elevated plus maze, Y-maze, and novel object recognition tests. Regional levels of dopamine, serotonin, norepinephrine, and the major dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in the prefrontal cortex and striatum using high-pressure liquid chromatography.
Finally, it is also important to mention that both first- and second-generation synthetic cathinones are often sold as mixtures. Specifically, synthetic cathinone products have been shown to often contain more than one cathinone, as well as other adulterants, including illicit amphetamines, piperazines, cutting/binding agents, caffeine, and topical anesthetics (Brandt et al., 2010; German et al., 2014). Thus, although abuse liability assessment of these individual drugs is now emerging, assessment of the effects and abuse potential of combinations of these drugs will be more difficult yet should be a central focus of future research. Together, the results of the present study suggest that second-generation synthetic cathinones likely possess a similar potential for abuse as their first-generation predecessors as well as the illicit amphetamines they are designed to mimic. Furthermore, these findings have important implications for future research on synthetic cathinone abuse and dependence and legislative efforts to classify these drugs according to the proper controlled substance schedule.
In contrast, other studies measured neurochemical effects several days after the last drug exposure (Briand et al., 2008; Hadlock et al., 2011; Schwendt et al., 2009), which may be during withdrawal. Brain tissue was collected approximately 24 h after the last drug exposure in this study and the past companion studies (Marusich et al., 2019a; Marusich et al., 2019b). All statistically significant effects of drug (synthetic cathinone vs saline) on neurotransmitters in ShA groups, and interactions between drug and sex are shown in Table 2 and Fig.
The serotonergic effects of 4MMC were expected because it releases 5-HT (Baumann et al., 2012). Α-PVP does not have functional serotoninergic effects (Marusich et al., 2014), but α-PVP surprisingly increased 5-HIAA levels in several brain regions under LgA conditions. It is unclear if these unexpected serotonergic effects are a result of α-PVP binding to 5-HT1A (Rickli et al., 2015). Furthermore, it was noteworthy that 4MMC decreased 5-HT and 5-HIAA levels, whereas α-PVP increased 5-HIAA levels.