Flakka Drug: Symptoms, Side Effects & Complications

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).

Both synthetic cathinones lowered NE levels in hippocampus and altered GLU levels in some brain regions compared to saline, but the effects on GLU occurred primarily for males (Table 2 and Fig. 4). Many studies measure the neurochemical effects of stimulants of abuse during direct drug effects (Ahmed et al., 2004; Mantsch et al., 2004), which does not provide information on lasting brain changes. 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). 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 maximal cytotoxic effect increases with the elongation of the α-aliphatic side-chain, which can cause major health problems, as longer-chain substances produce less pronounced stimulatory effects and hence are used in higher doses. Additionally, the presented findings implicate the presence of disturbances in the plasma membrane fluidity as another important factor underlying the cytotoxicity of α-pyrrolidinophenones. There were two scenarios in which neurotransmitter data were excluded from graphs and analyses. First, there were a few cases in which amygdala samples had very elevated levels of DA, DOPAC, and HVA. Because of the closeness of amygdala and striatum in the brain, it is likely that a small amount of striatum was inadvertently included as part of the amygdala sample.

• Some brand names of synthetic cathinones include Bliss, Vanilla Sky, Lunar Wave, Cloud Nine, and White Lightning.
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• There were sex differences in 5-HT levels for ShA rats, with both synthetic cathinones producing greater 5-HT levels for females than males in amygdala and PFC (Table 4 and Fig. 4).
• The following day, we performed an extensive neurochemical profiling across the striatum and prefrontal cortex for monoamine neurochemistry.
• Exposure to 4-F-PVP for 24 h led to a marked, concentration-dependent reduction in the survival of SH-SY5Y (10–300 μM), Hep G2 (10–300 μM), RPMI 2650 (10–300 μM), and H9c2(2-1) (25–300 μM) cell lines.
• There were also large sex differences in GLU levels in PFC, and 5-HIAA levels in striatum and thalamus for ShA groups (Fig. 4).

Effects of PVP, 4-F-PVP, and 4-MeO-PVP on the Survival of SH-SY5Y, Hep G2, RPMI 2650, and H9c2(2- Cells

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). Neurotransmitter and metabolite concentrations for all brain regions and all LgA and naïve rat groups are shown in Fig. There were differences between condition (LgA vs naïve), between males and females, and there were interactions between condition and sex.

In most instances, data from naïve and LgA groups (present study) were statistically analyzed separately from ShA groups (Marusich et al., 2019a; Marusich et al., 2019b) because the neurotransmitter assays were conducted at different times for these studies. Additionally, one male 4MMC ShA and saline ShA rat were exposed to the incorrect infusion volume. All self-administration and neurotransmitter data for these three rats were excluded from graphs and analyses. Some complications of flakka drug abuse can happen while the drug user is acutely agitated if they were to harm themselves or others; however, medically, the severe consequences of the agitation caused by the drug appear later. Agitated patients can go into a state called “excited delirium,” which is a medical emergency. In the excited delirium state, restrained patients struggle to free themselves, scream, flail, and can even have seizures.

5. Drug Self-Administration

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. Significant reduction of cell viability was observed in RPMI 2650 cells at concentrations of 10 to 300 μM, in SH-SY5Y cells between 50 and 300 μM, in Hep G2 cells between 100 and 300 μM, and in H9c2(2-1) cells at 200 and 300 μM after 24-h incubation. Significant reductions were also seen in H9c2(2-1) cells between 10 and 300 μM, and in SH-SY5Y, Hep G2, and RPMI 2650 cells between 25 and 300 μM after 72-h incubation (Fig. 6b).

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 example, binge-like self-administration of MDPV resulted in neurodegeneration and deficits in the novel object recognition (NOR) test (Sewalia et al. 2018). Likewise, adolescent rats exposed to methylone exhibited persistent depletions of serotonin and deficits in reference memory (Lopez-Arnau et al. 2014).

Figure 2.

Body weight and rectal temperature were recorded at baseline, immediately prior to each injection, and two hours after the last injection. Temperature was measured by inserting a lubricated probe 1.5 cm into the rectum and recording the readout from a connected TH-8 Thermalert temperature monitor (Physitemp Instruments; Clifton, NJ, USA) after the signal reached steady state. For the GLU analysis, an additional 10 μl aliquot of supernatant was transferred to a 700 μl deep, 96 well plate, and diluted with 490 μl of 5 mM ammonium acetate and 0.1% formic acid, aqueous (LC-MS/MS mobile phase A).

It is an ongoing challenge, as each time one type of bath salt is made illegal, the drug labs change the chemical structure slightly and a new drug that is technically not illegal is created. In the case of flakka, the new chemical is called alpha-pyrrolidinopentiophenone or alpha-PVP. Drug users take flakka to get a feeling of euphoria, a heightened sense of awareness, stimulation, and energy. Word on the street is that flakka (also called gravel or flocka) is a combination of heroin and crack cocaine, or heroin and methamphetamines, but in reality, flakka is just a newer-generation version of a type of synthetic drug called bath salts (MDPV). “Baths salts” is a term used to refer to synthetic cathinones as a whole, some of which may cause far more intense hyperstimulation than others.

The rise in popularity of synthetic cathinones is linked to their ease of procurement over the internet and in gas stations, smoke shops, and novelty stores (Spiller et al., 2011). Synthetic cathinones have been falsely marketed as numerous products, the most recognizable being “bath salts,” and are typically labeled “not for human consumption” or “for research purposes only” to alpha-pyrrolidinopentiophenone function evade drug laws (United States Department of Justice, 2011c). Although many synthetic cathinone derivatives exist, 3,4-methylenedioxypyrovalerone (MDPV), mephedrone, or methylone, initially comprised approximately 98% of all synthetic cathinones encountered in US drug seizures (United States Department of Justice, 2011a). Citing imminent threats to public health and safety, the US Drug Enforcement Administration (DEA) used their emergency scheduling authority to temporarily classify these 3 drugs (now often referred to as first-generation bath salts) as Schedule I substances in October of 2011 (United States Department of Justice, 2011b).

Cell Lines

Male and female Sprague-Dawley rats were trained to self-administer α-PVP (0.1 mg/kg/infusion) or 4MMC (0.5 mg/kg/infusion) through autoshaping, and then self-administered for 21 days during 1 h (short access; ShA) or 6 h (long access; LgA) sessions. Amygdala, hippocampus, hypothalamus, prefrontal cortex (PFC), striatum, and thalamus were extracted, and tissue was analyzed with electrochemical detection and liquid chromatography mass spectrometry. Rats acquired self-administration of α-PVP and 4MMC, and LgA rats showed more escalation of self-administration than ShA rats. 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.

Next, slopes for the log-transformed drug doses on the linear portion of the descending slopes (ED50 values in mg/kg) and maximal ICSS threshold decreases for METH, α-PVP, and 4-MEC were compared by a 1-way between-subjects analysis of variance with Bonferroni posthoc tests. Slope and ED50 values from previously published data (Watterson et al., 2012, 2014) were also calculated (Table 1) but were not compared statistically because of the possibility of cohort effects. However, there are currently no published behavioral studies that have directly assessed the potential abuse liability of α-PVP and 4-MEC.

Body Weight and Rectal Temperature

Furthermore, the dorsal striatum is involved in escalation of drug taking and compulsive behaviors (Clark et al., 2013; Willuhn et al., 2012). LgA, but not ShA, synthetic cathinone self-administration elevated DOPAC and HVA levels in striatum compared to saline and synthetic cathinone ShA levels (Fig. 5), encompassing the binge and intoxication stage of Koob and Volkow’s model. Interestingly, while the metabolites were altered, DA levels only changed in striatum and thalamus for 4MMC and α-PVP LgA groups, respectively, compared to saline ShA. These findings are consistent with a past study showing that ShA 4MMC self-administration did not alter DA, DOPAC, or HVA in striatum (Motbey et al., 2013). Thus, LgA synthetic cathinone self-administration for 21 days models most neurochemical changes predicted by the Koob and Volkow hypothesis for the binge and intoxication stage. The Koob and Volkow hypothesis of drug abuse posits that treatment needs may differ based on the amount of drug consumed over time, and that stimulant-induced neurochemical changes may occur at different times for different brain regions or neurotransmitter systems (Koob and Volkow, 2010).

For 5-HIAA/5-HT ratios, LgA self-administration of both synthetic cathinones lowered 5-HIAA/5-HT compared to ShA groups. Furthermore, α-PVP LgA, but not 4MMC LgA, elevated 5-HIAA/5-HT in PFC compared to α-PVP ShA and saline ShA. While an in-depth discussion of the significance of these ratios is beyond the scope of this manuscript, it is notable that the duration of synthetic cathinone self-administration not only alters levels of neurotransmitters but possibly affects the rate of neurotransmitter metabolism or clearance.

At a concentration of 300 μM, PVP produced a decrease in the viability of SH-SY5Y, Hep G2, RPMI 2650, and H9c2(2-1) cells by, respectively, 25, 51, 44, and 21% of control values. Exposure of cells to 300 μM of PVP reduced their viability by 48% (SH-SY5Y), 55% (Hep G2), 61% (RPMI 2650), and 44% (H9c2(2-1)) of the respective control values (Fig. 2a). 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. 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.

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In the current study, we assessed the persistent behavioral and neurochemical effects of exposure to the second-generation synthetic cathinone α-pyrrolidinopropiophenone (α-PPP). Both 4MMC and α-PVP produce robust functional changes in the DA system, albeit by different mechanisms of action, releasing DA, or blocking DA transport, respectively (Baumann et al., 2012; Cameron et al., 2013; Glennon and Young, 2016; Marusich et al., 2014; Simmler et al., 2013). Overall, there were few neurochemical differences following self-administration of a DA uptake inhibitor versus a DA releaser, and the magnitude of the differences between effects of these synthetic cathinones was small (Fig. 3 and 4). This suggests that synthetic cathinones may lead to similar neuroplasticity, regardless of the mechanism of action. In contrast, the amount of drug access during self-administration (ShA vs. LgA) is a pivotal factor in the neurochemical effects of synthetic cathinones.

The strongest effect was observed at 300 μM of 4-F-PVP and 4-MeO-PVP, being 43 and 45% of positive control cytotoxicity, respectively (Fig. 3). Exposure to 4-F-PVP for 24 h led to a marked, concentration-dependent reduction in the survival of SH-SY5Y (10–300 μM), Hep G2 (10–300 μM), RPMI 2650 (10–300 μM), and H9c2(2-1) (25–300 μM) cell lines. The greatest reduction in the viability, i.e., by 53% of the control value, was observed in RPMI 2650 cells, while reductions by 45, 47, and 45% of control values were observed in SH-SY5Y, Hep G2, and H9c2(2-1) cells, respectively (Fig. 2b). Incubation of cells with 4-F-PVP for 72 h resulted in a marked, concentration-dependent decline in the survival of all cell lines, with the maximal effect at the similar level in SH-SY5Y (reduction by 60%), Hep G2 (reduction by 54%), RPMI 2650 (reduction by 59%), and H9c2(2-1) (reduction by 46%) (Fig. 2b). 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.

6 hours after familiarization (Leger et al. 2013), each mouse was tested in the same box with a novel object replacing one of the familiar objects.Familiarization and testing sessions lasted 10 minutes, were video recorded, and were assessed offline. Exploration was defined as sniffing the object or orienting the head towards the object while the subject was within 1 cm of distance from the object. 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).