From street to lab: in vitro hepatotoxicity of buphedrone, butylone and 3,4-DMMC.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

New designer phenethylamines 2C-C and 2C-P have abuse potential and induce neurotoxicity in rodents.

2C (2C-x) is the general name for the family of phenethylamines containing two methoxy groups at the 2 and 5 positions of the benzene ring. The abuse of 2C family drugs has grown rapidly, although the abuse potential and neurotoxic properties of 2C drugs have not yet been fully investigated. In this study, we investigated the abuse potential and neurotoxicity of 4-chloro-2,5-dimethoxyphenethylamine (2C-C) and 2,5-dimethoxy-4-propylphenethylamine (2C-P). We found that 2C-C and 2C-P produced conditioned place preference in a dose-dependent manner in mice, and increased self-administration in rats, suggesting that 2C-C and 2C-P have abuse potential. To investigate the neurotoxicity of 2C-C and 2C-P, we examined motor performance and memory impairment after high doses of 2C-C and 2C-P. High doses of 2C-C and 2C-P decreased locomotor activity, rota-rod performance, and lower Y-maze test, novel objective recognition test, and passive avoidance test scores. We also observed that 2C-C and 2C-P affected expression levels of the D1 dopamine receptor, D2 dopamine receptor, dopamine transporter, and phospho-dopamine transporter in the nucleus accumbens and the medial prefrontal cortex, and increased c-Fos immuno-positive cells in the nucleus accumbens. Moreover, high doses of 2C-C and 2C-P induced microglial activation, which is involved in the inflammatory reaction in the striatum. These results suggest that 2C-C and 2C-P have abuse potential by affecting dopaminergic signaling and induce neurotoxicity via initiating neuroinflammation at high doses.

Designers Drugs-A New Challenge to Emergency Departments-An Observational Study in Poland.

Background and Objective: In the last decade, the phenomenon of using new psychoactive substances (NPS), called designer drugs, has been on rise. Though their production and marketing in Poland is prohibited, reports of the Supreme Audit Office noted that young people are increasingly reaching for new intoxication agents in the form of designer drugs. There is a significant increase in the number of patients with NPS abuse admitted to the emergency departments. As NPS cannot be detected by standard tests for the presence of psychoactive substances, it is difficult to choose the appropriate therapeutic intervention. Therefore, the aim of the present study was to evaluate the patient characteristics in the population of adults and children suspected of using NPS and formulate the protocol for diagnosis and treatment. Materials and Method: The paper is based on a retrospective analysis of medical records of hospitalized patients in the Clinical Emergency Department of The Regional Specialist Hospital in Olsztyn (SKOR WSS, emergency department (ED)) and the Pediatric Emergency Department of the Provincial Specialist Children's Hospital in Olsztyn (SORD WSSD, pediatric emergency department (PED)) between years 2013 to 2018. The patient records related to their general symptoms at admission, mental state and laboratory diagnostic tests were evaluated. Results: The majority of patients hospitalized due to the suspected use of NPS were adolescents in 2013-2016 and a reversal of this trend was observed in 2017-2018 when number of adults admitted to the emergency department (ED) due to NPS use was higher. The NPS abuse was significantly higher among male patients, alcoholics, people using other psychoactive substances, patients suffering from mental disorders and teenagers in difficult socio-economic family situations. Whereas, the most common symptoms among pediatric patients were co-ordination disorder and aggression, in adults mainly tachycardia and aggression was observed. The laboratory tests in significant number of adult patients showed leukocytosis and ketonuria. Conclusions: In the present study, no unambiguous toxidrome or biochemical pattern characteristic for using NPS was observed. However, evaluation of blood morphology, coagulation parameters, liver and kidney function can be helpful in the diagnostic and therapeutic process. Symptomatic treatment of patients, fluid therapy and sedation was sufficient in most cases to resolve the patient symptoms in 48 h.

The new designer drug buphedrone produces rewarding properties via dopamine D1 receptor activation.

Substituted cathinones are synthetic analogs of the active components of natural products and are widely abused worldwide. However, the rewarding properties of these agents have not yet been evaluated. In this study, we investigated the abuse potential of buphedrone [2-(methylamino)-1-phenylbutan-1-one, α-methylamino-butyrophenone] and its effects on the mesolimbic dopaminergic system in mice using conditioned place preference (CPP) analysis, a self-administration test, a locomotor activity test, a behavioral sensitization test and Western blot analysis. Treatment with buphedrone supported CPP and self-administration, enhanced locomotor activity and produced behavioral sensitization when mice were challenged with methamphetamine. SCH23390, a D1 dopamine antagonist, prevented buphedrone-induced CPP, whereas raclopride, a D2 dopamine antagonist, had no effect. SCH23390 also blocked locomotor activity increase by buphedrone, while raclopride partially attenuated locomotor activation. Western blot analysis revealed that repeated buphedrone treatment increased D1 dopamine receptor expression in the dorsal striatum and nucleus accumbens in mice. Collectively, these findings suggest the abuse potential of buphedrone and demonstrate the involvement of the dopaminergic system in the establishment of its rewarding properties.

Promoter-Specific Effects of DREADD Modulation on Hippocampal Synaptic Plasticity and Memory Formation.

Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn-HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn-HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn-HM3D impaired LTP and hSyn-HM4D facilitated LTP. Follow-up experiments indicated that the hSyn-HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn-HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα-HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα-HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα-HM3D hippocampal slices, whereas slices from CaMKIIα-HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. SIGNIFICANCE STATEMENT: This study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally modulating the hippocampus in not only a hippocampus-dependent task but also in hippocampal synaptic plasticity. This is the first study to evaluate the effects of DREADD-mediated inhibition and excitation in hippocampal long-term potentiation. More specifically, this study evaluates the effect of promoter-specific expression of DREADD viruses in a heterogenic cell population, which revealed surprising effects of different promoters. With chemogenetics becoming a more ubiquitous tool throughout studies investigating circuit-specific function, these data are of broad interest to the neuroscientific community because we have shown that promoter-specific effects can drastically alter synaptic function within a specific region, without parallel changes at the level of behavior.

Chemogenetic activation of the lateral hypothalamus reverses early life stress-induced deficits in motivational drive.

Altered motivated behaviour is a cardinal feature of several neuropsychiatric conditions including mood disorders. One well-characterized antecedent to the development of mood disorders is exposure to early life stress (ELS). A key brain substrate controlling motivated behaviour is the lateral hypothalamus (LH). Here, we examined the effect of ELS on LH activation and the motivation to self-administer sucrose. We tested whether chemogenetic activation of LH circuits could modify sucrose responding in ELS rats and examined the impact on LH cell populations. Male rat pups were maternally separated for 0 or 3 h on postnatal days 2-14. During adolescence, rats received bilateral injections of hM3D(Gq), the excitatory designer receptor exclusively activated by designer drugs, into LH. In adulthood, rats were trained to self-administer sucrose and tested under a progressive ratio schedule to determine their motivation for reward following injection with either vehicle or 5 mg/kg clozapine-N-oxide. Brains were processed for Fos-protein immunohistochemistry. ELS significantly suppressed lever responding for sucrose, indicating a long-lasting impact of ELS on motivation circuits. hM3D(Gq) activation of LH increased responding, normalizing deficits in ELS rats, and increased Fos-positive orexin and MCH cell numbers within LH. Our findings indicate that despite being susceptible to environmental stressors, LH circuits retain the capacity to overcome ELS-induced deficits in motivated behaviour.

DREADD-induced silencing of the medial amygdala reduces the preference for male pheromones and the expression of lordosis in estrous female mice.

Sexually naïve estrous female mice seek out male urinary pheromones; however, they initially display little receptive (lordosis) behavior in response to male mounts. Vomeronasal-accessory olfactory bulb inputs to the medial amygdala (Me) regulate courtship in female rodents. We used a reversible inhibitory chemogenetic technique (Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) to assess the contribution of Me signaling to females' preference for male pheromones and improvement in receptivity normally seen with repeated testing. Sexually naïve females received bilateral Me injections of an adeno-associated virus carrying an inhibitory DREADD. Females were later ovariectomized, treated with ovarian hormones, and given behavioral tests following intraperitoneal injections of saline or clozapine-N-oxide (CNO; which hyperpolarizes infected Me neurons). CNO attenuated females' preference to investigate male vs. female urinary odors. Repeated CNO treatment also slowed the increase in lordosis otherwise seen in females given saline. However, when saline was given to females previously treated with CNO, their lordosis quotients were as high as other females repeatedly given saline. No disruptive behavioral effects of CNO were seen in estrous females lacking DREADD infections of the Me. Finally, CNO attenuated the ability of male pheromones to stimulate Fos expression in the Me of DREADD-infected mice but not in non-infected females. Our results affirm the importance of Me signaling in females' chemosensory preferences and in the acute expression of lordosis. However, they provide no indication that Me signaling is required for the increase in receptivity normally seen after repeated hormone priming and testing with a male.

Individual Differences in the Relative Reinforcing Effects of 3,4-Methylenedioxypyrovalerone under Fixed and Progressive Ratio Schedules of Reinforcement in Rats.

The recreational use of designer drugs, including synthetic cathinones (bath salts), is associated with high levels of abuse and toxicity, and represents a growing threat to public health. 3,4-Methylenedioxypyrovalerone (MDPV) is a cocaine-like monoamine uptake inhibitor, and one of the most widely available and abused synthetic cathinones. The present study used male Sprague-Dawley rats to directly compare: (1) the acquisition of responding for MDPV and cocaine under a fixed ratio (FR) 1 schedule of reinforcement; (2) full dose-response curves for MDPV and cocaine under a FR5 schedule; and (3) progressive ratio (PR) schedules of reinforcement. Self-administration of MDPV and cocaine was acquired at comparable rates, and by a similar percentage of rats. Compared with cocaine, MDPV was ∼10-fold more potent and ∼3-fold more effective at maintaining responding (PR; final ratio completed). Unlike cocaine, for which little variability was observed among rats, the FR5 dose-response curve for MDPV was shifted ∼3-fold upward for a subset of rats (high-responders) relative to other rats with identical histories (low-responders). Compared with low-responding rats, high responders also self-administered more cocaine under the FR5 schedule, and earned significantly more MDPV, cocaine, and methamphetamine under a PR schedule of reinforcement. In addition to functioning as a significantly more effective reinforcer than either cocaine or methamphetamine, MDPV also appears to be unique in its capacity to establish an enduring phenotype in rats, characterized by unusually high levels of drug intake. Although the factors underlying this high-responder phenotype are unclear, they might be related to individual differences in human drug-taking behavior.

Injection of new psychoactive substance snow blow associated with recently acquired HIV infections among homeless people who inject drugs in Dublin, Ireland, 2015.

In February 2015, an outbreak of recently acquired HIV infections among people who inject drugs (PWID) was identified in Dublin, following similar outbreaks in Greece and Romania in 2011. We compared drug and risk behaviours among 15 HIV cases and 39 controls. Injecting a synthetic cathinone, snow blow, was associated with recent HIV infection (AOR: 49; p=0.003). Prevention and control efforts are underway among PWID in Dublin, but may also be needed elsewhere in Europe.

Potent rewarding and reinforcing effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV).

Reports of abuse and toxic effects of synthetic cathinones, frequently sold as 'bath salts' or 'legal highs', have increased dramatically in recent years. One of the most widely used synthetic cathinones is 3,4-methylenedioxypyrovalerone (MDPV). The current study evaluated the abuse potential of MDPV by assessing its ability to support intravenous self-administration and to lower thresholds for intracranial self-stimulation (ICSS) in rats. In the first experiment, the rats were trained to intravenously self-administer MDPV in daily 2-hour sessions for 10 days at doses of 0.05, 0.1 or 0.2 mg/kg per infusion. The rats were then allowed to self-administer MDPV under a progressive ratio (PR) schedule of reinforcement. Next, the rats self-administered MDPV for an additional 10 days under short access (ShA; 2 hours/day) or long access (LgA; 6 hours/day) conditions to assess escalation of intake. A separate group of rats underwent the same procedures, with the exception of self-administering methamphetamine (0.05 mg/kg per infusion) instead of MDPV. In the second experiment, the effects of MDPV on ICSS thresholds following acute administration (0.1, 0.5, 1 and 2 mg/kg, i.p.) were assessed. MDPV maintained self-administration across all doses tested. A positive relationship between MDPV dose and breakpoints for reinforcement under PR conditions was observed. LgA conditions led to escalation of drug intake at 0.1 and 0.2 mg/kg doses, and rats self-administering methamphetamine showed similar patterns of escalation. Finally, MDPV significantly lowered ICSS thresholds at all doses tested. Together, these findings indicate that MDPV has reinforcing properties and activates brain reward circuitry, suggesting a potential for abuse and addiction in humans.

An Internet Study of User's Experiences of the Synthetic Cathinone 4-Methylethcathinone (4-MEC).

Abstract A synthetic cathinone called 4-methylethcathinone (4-MEC) emerged online in 2010, and was cyber-marketed to be a replacement for mephedrone. The study aimed to present user experiences of 4-MEC as reported on the Internet, with a focus on user profiles, sourcing and product characteristics, routes of administration, dosage, positive and undesirable effects, and comparisons to mephedrone. Twenty-three individual, anonymous trip reports of the sole use of 4-MEC, and 112 screenshots of general 4-MEC user discussion boards, were taken from a purposeful sample of public drug-related sites. A content textual analysis was conducted on extracted qualitative information and produced 41 categories compiled into five general themes: "Type of 4-MEC user"; "Sourcing, informed decision making, product characteristics, and quality assurance"; "Routes of administration, gauging of dosage, and consumption of other drugs"; "Time course effects and outcomes"; and "Comparisons with mephedrone." 4-MEC is sold as white beads, crystalline shards, or green balls. User motives centered on curiosity, pricing, and ease of web sourcing. Oral, nasal, injecting, eyeball, and rectal routes of administration were described. Testing for purity, "allergy testing," and gauging of dosage were common. Users described euphoric but short-lived effects, with little comedown. Continued research is vital to inform harm reduction.

Current "legal highs".

BACKGROUND: A growing number of novel substances have been abused as recreational drugs by young people in the United States (US), Europe, and Australia. Called "legal highs," these substances range from plant-based to completely synthetic compounds. Spice, Salvia, mephedrone, methylenedioxypyrovalerone (MDPV), and other cathinone derivatives have psychotropic effects and are marketed for recreational use through exploitation of inadequacies in existing controlled substance laws. OBJECTIVES: This article reviews available literature on the most common "legal highs" as well as discussing the scientific basis for the legal difficulties in controlling trafficking in these novel substances. CONCLUSIONS: "Legal highs" continue to increase in use in the US, Europe, and Australia. These substances are powerful, can mimic effects of more traditional drugs of abuse, and are intentionally manufactured to circumvent existing controlled substance laws. As controlled substance legislation may be inadequate in the face of the quickly evolving legal highs, physicians are likely to see an increase in the prevalence of legal highs.

[Prevalence, reasons, and forms of use of legal highs by internet-based survey participants]. / Ocena rozpowszechnienia, powodów i form uiycia tak zwanych "dopalaczy" przez uczestników ankiety internetowej.

AIM: The aim of this study was to assess the prevalence, causes and forms of the use of legal highs in Poland. METHODS: The study was based on an original questionnaire and was carried out through the Internet before banning of the legal highs in Poland. RESULTS: The study covered 3013 people, aged 24.7 +/- 6.9, of which 9.3% admitted to use legal highs (6.5% ofwomen, and 14.7% ofmen, P < 0.01). The highest percentage of legal highs users was in the age groups 19-25 years (10.6%). Most people taking legal highs did so less than 1 time per month (71.8%). The most common reasons for taking legal highs were curiosity (46.4%), the desire to have fun (26.8%), and to improve the well-being (10.7%). Legal highs were most often used during meetings with friends (68.9% of cases), and social events (52.1%). The majority of their users adopted other risky behaviors: 56.4% took them with alcohol, 86.4% tried narcotics at least once, and 13.9% decided to try narcotics after trying legal highs. CONCLUSIONS: 1. Among the respondents of the online survey 9.3% admitted to use legal highs. 2. A typical profile of a legal high user is a young man, using them out of curiosity, to improve the well-being or just for fun, usually in social situations. 3. Initiatives undertaken to reduce the use of legal highs have to consider the social context.

[Clinical characteristics of 5 hospitalized 3,4-methylenedioxypyrovalerone (MDPV) users]. / A 3,4-methylenedioxypyrovaleron (MDPV) használatával kapcsolatos klinikai tapasztalatok öt hospitalizált paciens esete kapcsán.

A new illegal psychotropic substance appeared in Hungary during the first months of 2011. Acutely hospitalized patients with psychosis disclosed using a new type of designer drug, previously unknown to clinicians. As the new drug became better known, the cases with acute intoxication were often also transported to toxicology departments. In this study we summarize 5 short case studies that demonstrate the heterogeneous symptoms associated with MDPV abuse, and draw attention to the frequently occurring delusions, and the extended risk of intravenous substance use. Present case studies include patients with and without psychiatric history, regular and occasional users, intravenous and other routes of administration. In the short run antipsychotic therapy reduced the symptoms in all cases, but there is no clear therapeutic guideline for the treatment of patients having psychiatric problems associated with these drugs at the present time. The laboratory examination is unresolved as well. Mephedrone, MDPV and recent drugs have drawn attention to the problem of designer drugs again. This article examines the interaction of drug consumption trends and changes in law, apropos of the presented cases. This information can be helpful for the future treatment of MDPV or other designer drug users. Possible research and therapeutic implications are also discussed.

Determination of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in mouse blood and tissue after inhalation exposure to 'buzz' smoke by HPLC/MS/MS.

The disposition of the cannabimimetic naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in mice following inhalation of the smoke of the herbal incense product (HIP) 'Buzz' is presented. A high-pressure liquid chromatography with electrospray ionization triple quadrupole mass spectrometer (HPLC/MS/MS) method was validated for the analysis of JWH-018 in the specimens using deuterated Δ(9) -tetrahydrocannabinol (d(3) -THC) as the internal standard. JWH-018 was isolated by cold acetonitrile liquid-liquid extraction. Chromatographic separation was performed on a Zorbaz eclipse XDB-C(18) column. The assay was linear from 1 to 1000 ng/mL. Six C57BL6 mice were sacrificed 20 min after exposure to the smoke of 200 mg 'Buzz' containing 5.4% JWH-018. Specimen concentrations of JWH-018 were: blood, 54-166 ng/mL (mean 82 ± 42 ng/mL); brain, 316-708 ng/g (mean 510 ± 166 ng/g); and liver, 1370-3220 ng/mL (mean 1990 ± 752 ng/mL). The mean blood to brain ratio for JWH-018 was 6.8 and ranged from 4.2 to 10.9. After exposure, the responses of the mice were consistent with cannabinoid receptor type 1 activity: body temperatures dropped 7.3 ± 1.1 °C, and catalepsy, hyperreflexia, straub tail and ptosis were observed. The brain concentrations and physiological responses are consistent with the hypothesis that the behavioral effects of 'Buzz' are attributable to JWH-018.

Synthetic cathinone exposures reported to Texas poison centers.

BACKGROUND: Among the novel classes of synthetic "designer" drugs that have become increasingly popular among recreational drug users are synthetic cathinones. There is limited information on exposures to these substances. OBJECTIVE: The objective of this investigation was to describe the pattern of synthetic cathinone exposures reported to a statewide poison center network. METHODS: Synthetic cathinone exposures reported to Texas poison centers during 2010-2011 were identified and the distribution by various demographic and clinical factors determined. RESULTS: Of 362 total calls, 84.5% of the patients were 20 years or older and 74.0% male. The route of exposure was 47.8% by inhalation alone and 28.7% by ingestion alone. Other substances were involved in 19.3% of the exposures. The patient was already at or en route to a health-care facility in 75.1% of the exposures. The outcome was serious (moderate, major, potentially toxic, or death) in 74.0% of the exposures. The most frequently reported clinical effects were tachycardia (45.9%), agitation (39.2%), hypertension (21.0%), hallucinations (17.7%), and confusion (13.0%). The most common treatments were IV fluids (53.6%), benzodiazepines (40.9%), oxygen (11.0%), and other sedatives (7.5%). CONCLUSION: Synthetic cathinone exposures reported to Texas poison centers tended to occur through inhalation or ingestion, involve adult and male patients, be managed at health-care facilities, and involve potentially serious outcomes. SCIENTIFIC SIGNIFICANCE: This study adds to the limited information currently available on synthetic cathinone exposures.

[Designer drug induced psychosis]. / Dizájner drog indukálta pszichózis.

3,4-methylene-dioxy-pyrovalerone (MDPV) is a popular designer drug in Hungary, known as MP4. We present a case of a 34-year-old man, whose first psychotic episode was observed in the presence of MP4 use. The paranoid ideas of reference and the dereistic thinking could be the consequence of drug-induced psychosis. Within 24 hours after the intoxication was over delirium set in. The patient's history included only the use of MP4, use of other kinds of drugs was negated. The drug tests were negative, amphetamine derivates were not detectable in the urine sample. It is most likely that the MP4 pill contained an amount of MDPV less than detectable. In conclusion we suggest that the clinical picture could be the consequence of regular MDPV use.