Supernatant was then transferred to a 96 well plate for analysis by electrochemical detection (ECD). A Thermo Scientific CoulArray Multi-Channel ECD Array system (model 5600A; Thermo Scientific, Waltham, MA, USA) was used to analyze neurotransmitter concentrations. The array detector contained 16 coulometric electrochemical cells that provided quantitation of multiple neurotransmitters and metabolites simultaneously. An Agilent 1100 HPLC System (Santa Clara, CA, USA) and an Applied Biosystems API 4000 Triple Quadrupole liquid chromatography mass spectrometer (LC-MS) with Turbo Ion Spray source (Foster City, CA, USA) were used for quantitation of GLU.
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).
Drugs and Chemicals
- Flakka is a dangerous drug that has many bad side effects, mostly including changes in behavior or mood.
- Group two underwent dosing with α-PPP (80 mg/kg, QID, q2h, IP) and was assessed in the NOR test four days later and sacrificed for neurochemical analysis the following day.
- 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).
Neurotransmitter levels were measured to investigate if neuronal signaling changed as a function of duration of synthetic cathinone exposure and modeled different stages of drug abuse (Koob and Volkow, 2010). 4MMC shows escalation of self-administration during LgA conditions (Nguyen et al., 2017b; Watterson et al., 2014). 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.
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). The most remarkable examples of α-pyrrolidinophenones include 3,4-methylenedioxypyrovalerone (3,4-MDPV) and α-pyrrolidinopentiophenone (α-PVP). The term “man on flakka” does not refer to a specific individual but rather describes the behavior typically exhibited by individuals under the influence of flakka (alpha-PVP), a synthetic cathinone known for inducing extreme and unpredictable actions. ED50 values represent the mean dose leading to 50% maximal response with upper 95% confidence limits (UL) and lower 95% confidence limits (LL). Maximal response values represent the mean maximum intracranial self-stimulation (ICSS) threshold reduction (independent of dose)±95% confidence intervals.
Muscle tissue begins to break down, releasing proteins and other cellular products into the bloodstream, in a process called rhabdomyolysis. The result of the cellular products and proteins released during rhabdomyolysis and dehydration can impair the filtering function of the kidneys, leading to renal failure and death. In addition, such agitation may trigger Taser use or other methods that have the potential to harm the individual when law enforcement personnel have to intervene. 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. Coulometric detection was accomplished with a Thermo Scientific Dionex 6011RS electrode cell, and the signal analyzed on a Thermo Scientific Dionex Chromeleon CDS processing platform.
“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. While terminology varies, and those who purchase illicit drugs can rarely be 100% sure of the product they purchase, Flakka generally refers to Alpha-PVP, a specific synthetic cathinone. The legal status of flakka remains a moving target as authorities try to keep pace with the evolving synthetic drug market. 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.
This caused people most at risk, poor desperate drug addicts and homeless people, to use it instead of more expensive drugs like cocaine or methamphetamines. Internationally, the legal status of flakka varies by country, but many countries have taken steps to control its use due to its dangerous effects. This was part of an effort to curb the abuse of synthetic drugs, which were becoming increasingly popular and causing significant health issues. Various instances of “Man on Flakka” have been captured in videos on news channels and social media platforms such as YouTube. These videos often depict alarming scenes of erratic behavior, such as individuals running naked through streets, attempting to break into homes, jumping from heights, exhibiting extreme aggression towards others, or displaying bizarre and dangerous acts. These instances highlight the profound and often alarming effects that flakka can have on behavior and mental state, emphasizing its dangers to both users and the community at large.
7. Neurotransmitter Quantitation
Flakka is the street name for the synthetic cathinone called alpha-pyrrolidinopentiophenone (Alpha-PVP). 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.
Likewise, adolescent rats exposed to methylone exhibited persistent depletions of serotonin and deficits in reference memory (Lopez-Arnau et al. 2014). However, mephedrone and methylone have also been reported to not persistently deplete brain monoamine content (Baumann et al. 2012). 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).
The plate was sealed and mixed again at 1000 RPM for 4 min on an Eppendorf MixMate orbital shaker prior to analysis. Ark Behavioral Health Is an accredited drug and alcohol rehabilitation program, that believes addiction treatment should not just address “how to stay sober” but needs to transform the life of the addict and empower him or her to create a more meaningful and positive life. We are dedicated to transforming the despair of addiction into a purposeful life of confidence, self-respect and happiness. We want to give recovering addicts the tools to return to the outside world completely substance-free and successful. Ark Behavioral Health offers 100% confidential substance abuse assessment and treatment placement tailored to your individual needs.
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. This study sought to examine effects of long access (LgA) to α-PVP and 4MMC, using procedures similar to those employed for ShA exposure (Marusich et al., 2019a; Marusich et al., 2019b).
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. 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. 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.
Adverse effects
Extending incubation time to 72 h increased the cytotoxicity at 300 μM, leading to the decrease of the viability by 91% for SH-SY5Y, 97% for Hep G2, 98% for RPMI 2650, and 63% for H9c2(2-1). Moreover, a broader concentration range was found to elicit a significant drop in the viability for the Hep G2 (25–300 μM) and H9c2(2-1) (10–300 μM) cell lines, compared to 24-h exposure (Fg. 4c). Cell viability and mitochondrial function were measured by assessment of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases after 24- and 72-h exposure to the drugs. A solution of MTT (0.5 mg/ml) was added to the cells, and the culture was incubated for a further 3-h at 37 °C. After aspiration of culture medium, formazan crystals were dissolved in DMSO, and its absorbance was measured at 570 nm using Bio-Rad microplate reader model 680; this value being proportional to the number of cells with intact mitochondria. The mean values for each group were obtained by subtracting the mean OD of the positive control (1% (v/v) Triton-X100, added 30 min before MTT) from the value.
In the United States, flakka is classified as a Schedule I controlled substance under the Controlled Substances Act. Schedule I drugs are defined as substances with a high potential for abuse, no currently accepted medical use in treatment, and a lack of accepted safety for use under medical supervision. On January 22, 2013, the US DEA published a request for information specifically regarding 8 additional synthetic cathinones, 2 of the most prominent being 4-methyl-N-ethcathinone (4-MEC) and α‐pyrrolidinopentiophenone (α-PVP) (DEA, 2013). Their similarity in chemical structure suggests that α-PVP and 4-MEC likely emerged as replacements for MDPV and mephedrone, respectively (Figure 1). Rats were surgically implanted with chronic indwelling jugular catheters under general anesthesia as previously described (Marusich et al., 2019a; Marusich et al., 2019b). Catheters were flushed daily with saline prior to the session, and with 0.2 ml of a solution containing 0.96% gentamicin, 2.88% heparin, and 96.2% saline after the session to maintain patency.
Treating Flakka Abuse
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. 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). In contrast, α-PVP inhibits uptake of DA and NE transport (Glennon and Young, 2016; Koob and Volkow, 2010; Marusich et al., 2014).
Statistics on Flakka drug:
However, neither PVP, PV8, or PV9 lowered the fluorescence anisotropy of the TMA-DPH probe, which reflects the fluidity of the polar head-group portion of the cell membrane (Fig. 8). Hep G2 and H9c2(2-1) cells were cultivated in DMEM, SH-SY5Y in DMEM/F12, and RPMI 2650 in MEM with Earle’s salts and 1× Non-Essential Amino Acids Solution media, supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)-streptomycin (100 μg/ml) alpha-pyrrolidinopentiophenone function at 37 °C in a humidified atmosphere enriched with 5% CO2. 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. Brains were sliced into 1 mm thick coronal sections, and these slices were placed flat on a cold plate over ice.
Absolute tissue concentrations (ng/mg) for the monoamine neurotransmitters dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin, and norepinephrine, were determined by comparison with external standard curves and corrected for tissue weight, as we have described previously (Murnane et al. 2012). Α-PPP exposure results in persistent changes in exploratory behavior, spatial working memory, and monoamine neurochemistry. This research highlights potential dangers of α-PPP, including potential neurotoxicity, and suggests that the mechanisms underlying the persistent untoward effects of the cathinones may be distinct from those of the amphetamines. Statistical analyses were conducted using NCSS (Number Cruncher Statistical Systems, Kaysville, Utah, USA). 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.
After extending incubation time to 72 h, PV9 caused almost complete loss of viable SH-SY5Y (max. reduction by 94%), Hep G2 (max. reduction by 95%), and RPMI 2650 cells (max. reduction by 99%) when applied at 200 and 300 μM, and a 91% reduction of H9c2(2-1) when applied at 300 μM. A significant reduction of viable cells was observed in concentrations of 10–300 μM in H9c2(2-1) cells, and at concentrations ranging from 25 to 300 μM in SH-SY5Y, Hep G2, and RPMI 2650 cells (Fig. 6a). All graphical data presentations were created using GraphPad Prism 7.0 (GraphPad Software Inc.; La Jolla, CA). The time courses of the telemetry data (locomotor activity and core temperature) comparing saline versus α-PPP were integrated by standard area under the curve analysis in GraphPad, and then analyzed by repeated measures one-way analysis of variance, with post-hoc comparisons by Tukey’s test. All other data comparing saline versus α-PPP were analyzed by unpaired t-test, and corrected for multiple comparisons by the Bonferroni method to maintain the probability of making a type 1 error at 5%. Use of synthetic cathinones, which are designer stimulants found in “bath salts,” has increased dramatically in recent years.