Pyrrolidinophenones are a class of stimulant recreational designer drugs including many substituted cathinones. The NOR test has been proposed to measure recognition memory by allowing mice to explore novel and familiar objects (Antunes and Biala 2012). Each animal had a familiarization (training) session with a pair of identical objects (~ 5 cm long x 5 cm wide x 10 cm high) placed 5 cm away from the walls but adjacent in the open field.
Cocaine and amphetamine, which have similar mechanisms of action as α-PVP and 4MMC, respectively, induce synaptic plasticity within the DA system and DA receptive neurons. This plasticity hijacks normal learning mechanisms to create habits that persist despite adverse consequences and contribute to the deterioration of cognitive performance (Koob and Volkow, 2010). Thus, it is possible that the same processes caused dysfunctional learning or deterioration of cognitive performance for rats self-administering α-PVP and 4MMC in the present study.
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. 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.
Materials and Methods:
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. LgA self-administration only affected DA levels in hypothalamus and thalamus compared to naïve, and in striatum and thalamus compared to saline ShA, and these changes were small in magnitude. Surprisingly, only LgA 4MMC affected total DA in striatum when compared to saline ShA, a region in which LgA exposure increased DA metabolites (Fig. 3 and 5).
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. Ultra-high pressure liquid chromatography (UPLC) coupled with ECD was used to simultaneously measure DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT, 5-hydroxy-3-acetic acid (5-HIAA), and NE.
As such, α-PPP is an emerging second-generation pyrrolidine derivative of MDPV with clear reinforcing and stimulant effects, and individuals who consume α-PPP may be at risk of persistent untoward behavioral and neurobiological changes consistent with neurotoxicity. 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.
“Flakka drug dance”
PV9 and its substituted analogs caused profound disruption of cell membranes in all assessed cell lines (Fig. 7). The effect was always significant at 200 and 300 μM for all drugs and cell lines; in addition, the membrane integrity of RPMI 2650 cells was also significantly affected by all drugs at 100 μM. Moreover, 4-MeO-PV9 also damaged the membranes of SH-SY5Y and Hep G2 cells when administered at 100 μM. All PV9 analogs produced a maximal effect of at least 70% of the positive control group in SH-SY5Y, Hep G2, and RPMI 2650 cells, while the maximal effect of PV9 derivatives in H9c2(2-1) cells always exceeded 65% of the positive control (Fig. 7). After 24-h incubation PVP caused significant reductions in the survival of SH-SY5Y (25–300 μM), Hep G2 (10–300 μM), RPMI 2650 (50–300 μM), and H9c2(2-1) (10–300 μM) cell lines.
8. Data Analysis
The present study suggests that 21 days of LgA self-administration models the neurochemistry of the beginning stages of the Koob and Volkow model of dysregulated drug intake (Koob and Le Moal, 2005, 2008; Koob and Volkow, 2010). Most past research using the escalation model of self-administration has used 21 sessions (Anker et al., 2010; Gipson et al., 2011; Kitamura et al., 2006; Marusich et al., 2010; Wee et al., 2007a), or fewer. The identified studies that used more than 21 sessions of self-administration (Ahmed and Koob, 1999; Belin et al., 2009; Greenwell et al., 2009a; Greenwell et al., 2009b) provided very little information on the neurochemical changes that occur beyond 21 sessions. Thus, future research should extend the duration of self-administration past 21 sessions to examine what neurochemical changes transpire and if increasing neurochemical dysregulation occurs with continued drug use as hypothesized. 4-Methylmethcathinone (4MMC; mephedrone) releases dopamine (DA), norepinephrine (NE), and serotonin (5-HT) (Baumann et al., 2012; Cameron et al., 2013; Simmler et al., 2013).
Drugs
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. Importantly, our results suggest that the risk of intoxication with pyrovalerones, resulting from their cytotoxic properties, could be positively related to the length of their aliphatic side-chain. Pyrovalerone derivatives (α-pyrrolidinophenones) constitute a branch of synthetic cathinones, a second most prominent group of novel psychoactive substances (NPS). Since 2008, each year has seen the introduction of a number of novel synthetic cathinone derivatives into the dynamic, clandestine NPS market, in an attempt to circumvent legal restrictions (EMCDDA 2017; Majchrzak et al. 2018; Zawilska and Wojcieszak 2017).
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. Rats were given 7 days to recover from surgery before commencement of ICSS procedures, during which they received daily injections of 2.5mg/mL meloxicam (0.15mL volume) to minimize postsurgical discomfort. Law enforcement agencies and health care providers have expressed significant concerns about the widespread use of flakka, particularly among younger demographics and in clubbing scenes where synthetic drugs are more prevalent. If you or someone you know needs assistance, consider reaching out to a professional or a helpline. Alpha-Pyrrolidinopentiophenone is a synthetic cathinone that acts as a stimulant similar to amphetamine, the active ingredient in certain ADHD medications.
In H9c2(2-1) cells, the viability was reduced by 76–78% at 200 μM and by approximately 96% at 300 μM, irrespective of the incubation time. 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).
Statistics on Flakka drug:
Rats were euthanized by rapid decapitation approximately 24 h after their last self-administration session in order to avoid direct drug effects (Marusich et al., 2019a; Marusich et al., 2019b; Wee et al., 2007b). 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.
As of 2013, these first-generation synthetic cathinones are now permanently classified as Schedule I substances in the United States (United States of America, 2012; United States Department of Justice, 2013a). 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.
- Notably, sex differences in neurochemistry were more abundant for ShA than LgA groups, and the cause of this is unknown.
- We hypothesized that exposure to α-PPP would deplete brain levels of dopamine and norepinephrine as well as induce memory deficits and increase anxiety.
- For 5-HIAA/5-HT ratios, LgA self-administration of both synthetic cathinones lowered 5-HIAA/5-HT compared to ShA groups.
- Rats were given 7 days to recover from surgery before commencement of ICSS procedures, during which they received daily injections of 2.5mg/mL meloxicam (0.15mL volume) to minimize postsurgical discomfort.
Given that the present study primarily observed early-stage neurochemical changes, experimental paradigms using greater than 21 sessions of self-administration may be necessary to reach the negative reinforcement and withdrawal stage of the Koob and Volkow model. It is also possible that Koob and Volkow’s hypothesized neurochemical changes are dependent on drug contingency, dose, or other methodological considerations. The present study sought to determine if stimulant-induced neurochemical changes spread as drug use persists (cf. Koob and Le Moal, 2005, 2008; Koob and Volkow, 2010). Neurotransmitter data from groups that self-administered under ShA or LgA conditions are compared in Fig. Changes in DA levels in striatum and thalamus are hypothesized to occur during the binge and intoxication stage because activation of the mesolimbic DA system produces acute reinforcing properties of psychostimulants (Koob and Volkow, 2010).
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). There were also large sex differences in GLU levels in alpha-pyrrolidinopentiophenone function PFC, and 5-HIAA levels in striatum and thalamus for ShA groups (Fig. 4).
When heated up, it gives off a foul-smelling smoke characterized as smelling like dirty socks. Results obtained using LDH assay further confirm the impact of the side-chain length on the cytotoxicity of pyrrolidinophenones. PVP and its analogs caused only benign disruption of SH-SY5Y and H9C2(2-1) cell membranes, while PV8 and PV9, and their substituted derivatives, evoked marked damages.