Flakka is a highly potent drug that can be snorted, injected, eaten, smoked, or vaporized in e-cigarettes. When heated up, it gives off a foul-smelling smoke characterized as smelling like dirty socks. Frozen tissues were weighed, sonically disrupted in 100 µl of 0.3 N HClO4 and centrifuged for 10 minutes at 4ºC to remove cellular debris. A 100 µl aliquot of the supernatant was placed in an WPS-3000TBSL autosampler maintained at 10ºC, and 10 µl was injected onto a Thermo Scientific (Waltham, MA) Hypersil BDS C18 column (35ºC) with Thermo Scientific Dionex Test Phase running at a flow rate of 0.5 mL/min.
- Our findings are in line with those published by Matsunaga et al. (2017), demonstrating that cytotoxicity of pyrovalerones increases with the elongation of the α-carbon side-chain.
- GLU levels were similar to the original analyses (Supplementary Material Table S2), and the differences between LgA and ShA were still evident in the reanalysis.
- Meanwhile, synthetic cathinone-induced DA metabolism in striatum appears to occur later or has a longer duration of effect.
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). While effects on DA were largely absent in this study, self-administration of either synthetic cathinone altered 5-HT and 5-HIAA levels. 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).
Effects on Neurochemistry
Using a discrete trials current intensity threshold intracranial self-stimulation procedure, the present study assessed the effects of 2 common second-generation synthetic cathinones, α‐pyrrolidinopentiophenone (0.1–5mg/kg) and 4-methyl-N-ethcathinone (1–100mg/kg) on brain reward function. Our findings demonstrate that α-pyrrolidinophenones decrease the viability of cell lines derived from neuroblasts, hepatic epithelium, upper airway epithelium, and cardiomyoblasts in a concentration- and time-dependent manner. The length of the α-aliphatic side-chain seems to be the most important factor determining the maximal cytotoxic effect, i.e., neither α-PVP, with five carbon atoms, nor its substituted analogs lowered cell viability to considerably below 40% of the control values in all tested cell lines. On the other hand, the incubation of cells with PV8 and its analogs, containing an additional two carbon atoms, reduced cell viability by a maximum of 70–80% after 24 h, and by more than 90% after 72 h. Further elongation of the side-chain increased the cytotoxic activity, as observed in the tests on PV9 and its substituted analogs. Incubation for 72 h with the highest tested doses (200 and 300 μM) resulted in almost complete extinction of cell viability in all but the H9C2(2-1) cell lines.
Effects of PV8, 4-F-PV8, and 4-MeO-PV8 on the Survival of SH-SY5Y, Hep G2, RPMI 2650, and H9c2(2- Cells
Interestingly, ShA drug self-administration also led to escalation of intake, particularly for 4MMC. This finding suggests that dysregulated drug intake is not limited to LgA conditions for α-PVP and 4MMC, similar to a past study showing that LgA and ShA cocaine self-administration both led to escalation of intake (Beckmann et al., 2012). To our knowledge, this is the first study to statistically compare male and female rodents for 4MMC and α-PVP self-administration, and the resulting neurochemical changes. Sex differences for α-PVP during autoshaping (Fig. S2) did not lead to sex differences in α-PVP self-administration (Fig. 2).
LgA self-administration of α-PVP increased 5-HIAA levels in all brain regions, compared to control. In contrast, both LgA and ShA 4MMC self-administration decreased 5-HT and 5-HIAA levels in most brain regions. LgA exposure to both synthetic cathinones increased DOPAC levels in hypothalamus and striatum, and increased HVA levels in striatum compared to control. LgA self-administration of either synthetic cathinone produced region-specific increases in NE levels, whereas ShA self-administration lowered NE levels in select locations compared to control. These alterations in neurotransmitter levels indicate that synthetic cathinone use may produce differential neurochemical changes during the transition from use to abuse, and that 21 days of self-administration only models the beginning stages of dysregulated drug intake. To our knowledge, there are currently no published studies directly assessing the potential abuse liability of the second-generation synthetic cathinones α-PVP or 4-MEC.
8. Data Analysis
Drug concentrations used in this in vitro study, reaching 300 μM, exceed those normally found in the blood obtained from intoxicated patients and during autopsies (Kudo et al. 2015; Marinetti and Antonides 2013). However, as discussed in our previous work (Wojcieszak et al. 2016), organs such as the liver, brain, and upper airway epithelium can be exposed to significantly higher local drug concentrations than those measured in blood. 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).
- Consistent with previous studies of α-PPP (Gatch et al. 2017; Marusich et al. 2014), we report that it has robust locomotor-stimulant effects in our laboratory that are comparable to cocaine.
- The test subjects were male Swiss Webster mice (Charles River Laboratories; Wilmington, MA) that weighed 27–33 grams and ranged in age from 2–3 months.
- Results obtained using LDH assay further confirm the impact of the side-chain length on the cytotoxicity of pyrrolidinophenones.
- For the experiments examining the persistent effects of α-PPP on neurochemistry and behavior, 18 mice treated with α-PPP and 16 mice were treated with saline were included in the final analyses, divided between two groups.
- Group one underwent dosing with α-PPP (80 mg/kg, QID, q2h, IP) and was assessed in the Y-maze and elevated plus maze (EPM) four days later, with EPM assessments occurring at least three hours after the Y-maze assessments.
Adverse effects
Meanwhile, synthetic cathinone-induced DA metabolism in striatum appears to occur later or has a longer duration of effect. The levels of DA metabolites in striatum following ShA 4MMC or ShA saline self-administration were consistent with that of a previous study, however, the present study produced approximately 60% higher levels of DA in striatum compared to the previous study (Motbey et al., 2013). Due to methodological differences in the self-administration doses used across studies, it is unclear why the self-administration regimen used in the present study led to greater DA levels.
It is also notable that both α-PPP (Eshleman et al. 2017) and MDPV (Simmler et al. 2013) function as reuptake inhibitors rather than substrate-based releasers, and the mechanism of action of methylone appears to include both reuptake inhibition and substrate-based release (Simmler et al. 2013). Although α-PPP and MDPV are amphetamine derivatives, their pharmacological mechanism of action is closer to cocaine than to amphetamines. Most previous work with stimulant-induced neurotoxicity has focused on substrate-based releasers rather than reuptake inhibitors. Moreover, hyperthermic responses to reuptake inhibitors have been less forthcoming than with substrate-based releasers, and we did not observe a hyperthermic response to cocaine in the present study.
There were also sex differences in 5-HT levels for hippocampus, hypothalamus, and striatum for 4MMC ShA groups, but the direction of the effects was region dependent. 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.
The percentage of the total exploration time spent exploring the novel object was calculated to reflect recognition memory. Spontaneous alternation in the Y-maze has been proposed to measure hippocampus-dependent spatial working memory (Walker and Gold 1994). Performance in the Y-maze was assessed by both automated quantitation (Maze Engineers; Cambridge, MA) and manual observation.
The standard was transferred to a volumetric flask and diluted to volume with mobile phase A (5 mM ammonium acetate and 0.1% formic acid, aqueous) and labeled as stock solution. The stock solution containing approximately 2.5 mg/ml of GLU was then diluted to encompass a concentration range from 2500 to 10 ng/ml. Standards for ECD were prepared by weighing approximately 1 mg of analytes DA (Sigma-Aldrich, Buchs, Switzerland), DOPAC (Sigma-Aldrich, Buchs, Switzerland), HVA (Sigma-Aldrich, Buchs, Switzerland), 5-HT (Sigma-Aldrich, St. Louis, MO), 5-HIAA (Sigma-Aldrich, St. Louis, MO), and NE (Sigma-Aldrich, St. Louis, MO). Each standard was transferred to a volumetric flask and diluted to volume with tissue buffer to create stock solutions. A stock solution containing approximately 10 μg/ml of analyte was then diluted to encompass a concentration range from 1000 to 0.5 ng/ml.
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. Cellular membrane integrity was assessed by measuring the activity of lactate dehydrogenase released from damaged cells into the culture medium using LDH Cytotoxicity Assay (ScienCell Research Laboratories, Carlsbad, CA, USA) following 48-h exposure to the drugs, according to the manufacturer’s instruction.
Consistent with previous studies of α-PPP (Gatch et al. 2017; Marusich et al. 2014), we report that it has robust locomotor-stimulant effects in our laboratory that are comparable to cocaine. We have found that α-PPP persistently depletes the levels of monoamine neurotransmitters in the striatum and frontal cortex, and induces significant memory impairments, as measured in the Y-maze assay, and decreased exploratory activity in the NOR test. However, these deficits were not matched by similar changes in the EPM test or deficits in recognition memory in the NOR test. We operationally define “second-generation” cathinones as the compounds, such as pentylone and alpha-pyrrolidinopentiophenone (α-PVP), that have shown increased abuse more recently and were placed into the Controlled Substances Act in 2014 or remain unscheduled. In recent years, numerous second-generation synthetic cathinones have emerged that contain a pyrrolidine ring, similar to MDPV and α-PVP, in place of the secondary amine of methamphetamine-like synthetic cathinones.
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 alpha-pyrrolidinopentiophenone function 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). There were also large sex differences in GLU levels in PFC, and 5-HIAA levels in striatum and thalamus for ShA groups (Fig. 4). Sex differences in neurochemistry in LgA groups were largely confined to 5-HIAA levels (Fig. 3). Future studies are needed to determine why there were vast sex differences in the neurochemistry of ShA groups that seemed to dissipate in the LgA groups.
A prototypical second-generation pyrrolidine synthetic cathinone is the compound alpha-pyrrolidinopropiophenone (α-PPP). Its molecular pharmacology was recently characterized, demonstrating that α-PPP has high affinity for the dopamine and norepinephrine transporters (approximately 1–2 μM), where it functions as a reuptake inhibitor. In contrast, α-PPP did not have any substrate-based releasing properties and substantially lower affinity for the serotonin transporter (approximately 163 μM) (Eshleman et al. 2017). Furthermore, it was recently demonstrated that α-PPP is a locomotor stimulant and has cocaine-like and methamphetamine-like discriminative stimulus effects (Gatch et al. 2017), and its locomotor-stimulant effects can be blocked by the selective dopamine D1-like receptor antagonist SCH (Marusich et al. 2014).
Then 100 μl was transferred to a new 700 μl deep, 96 well plate and diluted with an additional 400 μl of LC-MS/MS mobile phase A (total dilution of 250 fold). The plate was sealed and mixed again at 1000 RPM for 4 min on an Eppendorf MixMate orbital shaker prior to analysis. RTI shares its evidence-based research – through peer-reviewed publications and media – to ensure that it is accessible for others to build on, in line with our mission and scientific standards.