Different development patterns of reward behaviors induced by ketamine and JWH-018 in striatal GAD67 knockdown mice.

IMPORTANCE: Glutamic acid decarboxylase 67 (GAD67) is a gamma-aminobutyric acid (GABA) synthesis enzyme associated with the function of other neurotransmitter receptors, such as the N-methyl-D-aspartate (NMDA) receptor and cannabinoid receptor 1. However, the role of GAD67 in the development of different abused drug-induced reward behaviors remains unknown. In order to elucidate the mechanisms of substance use disorder, it is crucial to study changes in biomarkers within the brain's reward circuit induced by drug use. OBJECTIVE: The study was designed to examine the effects of the downregulation of GAD67 expression in the dorsal striatum on reward behavior development. METHODS: We evaluated the effects of GAD67 knockdown on depression-like behavior and anxiety using the forced swim test and elevated plus maze test in a mouse model. We further determined the effects of GAD67 knockdown on ketamine- and JWH-018-induced conditioned place preference (CPP). RESULTS: Knockdown of GAD67 in the dorsal striatum of mice increased depression-like behavior, but it decreased anxiety. Moreover, the CPP score on the NMDA receptor antagonist ketamine was increased by GAD67 knockdown, whereas the administration of JWH-018, a cannabinoid receptor agonist, did not affect the CPP score in the GAD67 knockdown mice group compared with the control group. CONCLUSIONS AND RELEVANCE: These results suggest that striatal GAD67 reduces GABAergic neuronal activity and may cause ketamine-induced NMDA receptor inhibition. Consequently, GAD67 downregulation induces vulnerability to the drug reward behavior of ketamine.

Assessment of illicit drug use in Seoul, the capital of South Korea for 21 days by wastewater-based epidemiology.

22 illicit drugs were monitored in Seoul, the capital of South Korea for 21 days using wastewater-based epidemiology to assess the drug usage patterns for the first time by region and social status. Among the targeted compounds, 10 were detected, with consistent detection of methamphetamine in samples over the entire sampling period. In addition, ketamine had the highest estimated consumption rate at 47.5 mg/day/1000 people, followed by methamphetamine at 12.5 mg/day/1000 people. Methamphetamine and MDMA(3,4-methylenedioxymethamphetamine) exhibited relatively stable daily patterns, with coefficients of variation of 7.03 %, and 13.4 % respectively. Furthermore, no statistically significant differences were observed between weekends and weekdays for all compounds (Mann-Whitney Rank Sum test, p > 0.05). Statistically significant regional differences in drug consumption were observed for methamphetamine, MDMA, and ketamine (Mann-Whitney Rank Sum test, p < 0.05). These differences were found to be related to average annual income and educational levels.

Tracking methamphetamine and amphetamine consumption patterns in South Korea via enantiomeric analysis of wastewater.

Wastewater-based epidemiology (WBE) has emerged as an effective method for monitoring a community's health status and lifestyle. In recent years, enantiomeric profiling has shown promise as a tool for tracing the sources of abused drugs through WBE. This study investigated amphetamine (AMP) and methamphetamine (METH) consumption in South Korea using enantiomeric analysis of untreated wastewater samples collected from 27 wastewater-treatment plants (WWTPs). Both AMP and METH were detected, with the predominant detection of S-(+)-METH indicating widespread illegal use of METH, which is primarily produced by a clandestine synthesis procedure that involves the reduction of ephedrine/pseudoephedrine. Most AMP/METH ratios in the samples were consistent with the expected METH excretion profile, indicating that the presence of AMP was primarily due to METH metabolism. However, R-(-) AMP was detected in 18.5 % and 25.9 % of wastewater samples in winter and spring, respectively, and the high AMP/METH ratio (>0.27) indicated potential AMP abuse. By differentiating between the sources of AMP and METH in wastewater, enantiomeric analysis could help authorities to target and address specific drug-abuse issues affecting the population more effectively.

Deletion of Cryab increases the vulnerability of mice to the addiction-like effects of the cannabinoid JWH-018 via upregulation of striatal NF-κB expression.

Synthetic cannabinoids have exhibited unpredictable abuse liabilities, especially self-administration (SA) responses in normal rodent models, despite seemingly inducing addiction-like effects in humans. Thus, an efficient pre-clinical model must be developed to determine cannabinoid abuse potential in animals and describe the mechanism that may mediate cannabinoid sensitivity. The Cryab knockout (KO) mice were recently discovered to be potentially sensitive to the addictive effects of psychoactive drugs. Herein, we examined the responses of Cryab KO mice to JWH-018 using SA, conditioned place preference, and electroencephalography. Additionally, the effects of repeated JWH-018 exposure on endocannabinoid- and dopamine-related genes in various addiction-associated brain regions were examined, along with protein expressions involving neuroinflammation and synaptic plasticity. Cryab KO mice exhibited greater cannabinoid-induced SA responses and place preference, along with divergent gamma wave alterations, compared to wild-type (WT) mice, implying their higher sensitivity to cannabinoids. Endocannabinoid- or dopamine-related mRNA expressions and accumbal dopamine concentrations after repeated JWH-018 exposure were not significantly different between the WT and Cryab KO mice. Further analyses revealed that repeated JWH-018 administration led to possibly greater neuroinflammation in Cryab KO mice, which may arise from upregulated NF-κB, accompanied by higher expressions of synaptic plasticity markers, which might have contributed to the development of cannabinoid addiction-related behavior in Cryab KO mice. These findings signify that increased neuroinflammation via NF-κB may mediate the enhanced addiction-like responses of Cryab KO mice to cannabinoids. Altogether, Cryab KO mice may be a potential model for cannabinoid abuse susceptibility.

Prenatal ketamine exposure impairs prepulse inhibition via arginine vasopressin receptor 1A-mediated GABAergic neuronal dysfunction in the striatum.

Ketamine is a widely used anesthetic with N-methyl-D-aspartate (NMDA) receptor antagonism. Exposure to ketamine and NMDA receptor antagonists may induce psychosis. However, the mechanism underlying the effects of ketamine on the immature brain remains unclear. In this study, NMDA receptor antagonists, ketamine and methoxetamine, were administered to pregnant F344 rats (E17). These regimens induce psychosis-like behaviors in the offspring, such as hyperlocomotion induced by MK-801, a non-competitive NMDA receptor antagonist. We also observed that prepulse inhibition (PPI) was significantly reduced. Interestingly, ketamine administration increased the arginine vasopressin receptor 1A (Avpr1a) expression levels in the striatum of offspring with abnormal behaviors. Methoxetamine, another NMDA receptor antagonist, also showed similar results. In addition, we demonstrated a viral vector-induced Avpr1a overexpression in the striatum-inhibited PPI. In the striatum of offspring, ketamine or methoxetamine treatment increased glutamate decarboxylase 67 (GAD67) and δ-aminobutyric acid (GABA) levels. These results show that prenatal NMDA receptor antagonist treatment induces GABAergic neuronal dysfunction and abnormalities in sensorimotor gating via regulating Avpr1a expression in the striatum.

The designer benzodiazepine, flubromazepam, induces reward-enhancing and cardiotoxic effects in rodents.

The use of many benzodiazepines is controlled worldwide due to their high likelihood of abuse and potential adverse effects. Flubromazepam-a designer benzodiazepine-is a long-acting gamma-aminobutyric acid subtype A receptor agonist. There is currently a lack of scientific evidence regarding the potential for flubromazepam dependence or other adverse effects. This study aimed to evaluate the dependence potential, and cardiotoxicity via confirmation of the QT and RR intervals which are the factors on the electrical properties of the heart of flubromazepam in rodents. Using a conditioned place preference test, we discovered that mice treated intraperitoneally with flubromazepam (0.1 mg/kg) exhibited a significant preference for the flubromazepam-paired compartment, suggesting a potential for flubromazepam dependence. In addition, we observed several cardiotoxic effects of flubromazepam; 100-µM flubromazepam reduced cell viability, increased RR intervals but not QT intervals in the electrocardiography measurements, and considerably inhibited potassium channels in a human ether-à-go-go-related gene assay. Collectively, these findings suggest that flubromazepam may have adverse effects on psychological and cardiovascular health, laying the foundation for further efforts to list flubromazepam as a controlled substance at both national and international levels.

25I-NBOMe, a phenethylamine derivative, induces adverse cardiovascular effects in rodents: possible involvement of p21 (CDC42/RAC)-activated kinase 1.

Abuse of new psychoactive substances is an emerging social problem. Several phenethylamines are internationally controlled substances as they are likely to be abused and have adverse effects. Phenethylamine analog 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25I-NBOMe) has been reported as one of the most commonly abused psychoactive substance. However, the cardiotoxicity of this compound has not been extensively evaluated. Thus, in this study, we investigated the adverse cardiovascular effects of 25I-NBOMe, related to p21 (CDC42/RAC)-activated kinase 1 (PAK1). The cardiotoxicity of 25I-NBOMe was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, live/dead cytotoxicity assay, PAK1/CDC42 kinase assay, and in vivo electrocardiography (ECG). Also, we analyzed the expression level of PAK1, which is known to play key roles in the cardiovascular system. In the MTT assay, cell viability of 25I-NBOMe-treated H9c2 cells or primary cardiomyocytes of ICR mice decreased in a concentration-dependent manner. Results from the in vitro cytotoxicity assay in cardiomyocytes showed that 25I-NBOMe decreased the viability of H9c2 rat cardiomyocytes, and TC50 of 25I-NBOMe was found to be 70.4 µM. We also observed that 25I-NBOMe reduced PAK1 activity in vitro. Surface ECG measurement revealed that intravenous injection of 25I-NBOMe (doses of 1.0 and 3.0 mg/kg, corresponding to serum concentrations of 18.1 and 28.6 ng/mL, respectively) prolonged the QTc interval in SD rats. Furthermore, treatment with 25I-NBOMe downregulated the expression of PAK1 in the hearts of SD rats and H9c2 cells. In summary, our findings indicate that PAK1-related adverse effects of 25I-NBOMe can cause toxicity to cardiomyocytes and induce an abnormal ECG pattern in animals.

2-((2-(4-Iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH) and 2-(((2-(4-chloro-2,5-dimethoxyphenyl)ethyl)amino)methyl)phenol (25C-NBOH) induce adverse effects on the cardiovascular system.

Two new psychoactive substances (NPSs) classified as phenethylamines, namely 2-((2-(4-Iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH) and 2-(((2-(4-chloro-2,5-dimethoxyphenyl)ethyl)amino)methyl)phenol (25C-NBOH), are being abused by people seeking recreational hallucinogens. These NPSs may cause serious health problems as their adverse effects are not known in most cases. Therefore, in the present study, we evaluated the cardiotoxicity of 25I-NBOH and 25C-NBOH using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), Langendorff test, and human ether-a-go-go-related gene (hERG) assay. Furthermore, we analyzed the expression levels of p21 CDC42/RAC1-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, treatment with 25I-NBOH or 25C-NBOH dramatically decreased viability of H9c2 cardiomyocytes. Meanwhile, these two compounds significantly increased QT intervals and RR intervals in the rat ECG measurement. 25I-NBOH down-regulated the PAK1 protein expression in rat primary cardiomyocytes as well as H9c2 cells. However, 25C-NBOH had no effect on the PAK1 expression in H9c2 cells. In an in-depth study, 25I-NBOH inhibited potassium channels in the hERG assay, but in ex vivo test, the substance did not affect the left ventricular developed pressure (LVDP) and heart rate of the isolated rat hearts. Taken together, these results suggest that both 25I-NBOH and 25C-NBOH may have adverse cardiovascular effect. Further investigation would be needed to determine which factors mainly influence the relationship between PAK1 expression and cardiotoxicity.

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription.

Interaction of cannabinoid receptor type 1 (CB1) and GABAergic neuronal activity is involved in drug abuse-related behavior. However, its role in drug-dependent Pavlovian conditioning is not well understood. In this study, we aimed to evaluate the effects of a CB1 agonist, JWH-210, on the development of conditioned place preference (CPP)-induced by methamphetamine (METH). Pretreatment with a synthetic cannabinoid, JWH-210 (CB1 agonist), increased METH-induced CPP score and METH-induced dopamine release in acute striatal slices. Interestingly, CB1 was expressed in glutamate decarboxylase 67 (GAD67) positive cells, and overexpression of CB1 increased GAD67 expression, while CB1 knockdown reduced GAD67 expression in vivo and in vitro. GAD67 is known as an enzyme involved in the synthesis of GABA. CB1 knockdown in the mice striatum increased METH-induced CPP. When GAD67 decreased in the mice striatum, mRNA level of CB1 did not change, suggesting that CB1 can regulate GAD67 expression. GAD67 knockdown in the mouse striatum augmented apomorphine (dopamine receptor D2 agonist)-induced climbing behavior and METH-induced CPP score. Moreover, in the human brain, mRNA level of GAD67 was found to be decreased in drug users. Therefore, we suggest that CB1 potentiates METH-induced CPP through inhibitory GABAergic regulation of dopaminergic neuronal activity.

Pharmacology and adverse effects of new psychoactive substances: synthetic cannabinoid receptor agonists.

Over the last decade, new psychoactive substances (NPS) have continuously been the focus of the international society since their emergence on the illicit drug market. NPS can be classified into six groups including; synthetic cannabinoid receptor agonists (SCRAs), stimulants, opioids, dissociatives, sedatives/hypnotics, and classic hallucinogens with psychoactive effects. These are sold as "herbal incense," "bath salts," "legal highs," and "research chemicals". They can be synthesized easily with slight changes in the chemical moieties of known psychoactive substances. NPS are sold worldwide via on- and off-line markets without proper scientific evaluation regarding their safety or harmfulness. Abuse of NPS poses a serious public health issue, and systematic studies on their adverse effects are lacking. Therefore, it would be meaningful to collect currently available data in order to understand NPS and to establish viable solutions to cope with the various health issues related to them. In this article, we reviewed the general pharmacological characteristics, recent findings, and adverse effects of representative NPS; SCRAs. SCRAs are known as the most commonly abused NPS. Most SCRAs, cannabinoid receptor 1 and cannabinoid receptor 2 agonists, are often associated with severe toxicities, including cardiotoxicity, immunotoxicity, and even death, unlike natural cannabinoid Δ9-Tetrahydrocannabinol.

Limonene has anti-anxiety activity via adenosine A2A receptor-mediated regulation of dopaminergic and GABAergic neuronal function in the striatum.

BACKGROUND: Limonene, a common terpene found in citrus fruits, is assumed to reduce stress and mood disorders. Dopamine and γ-aminobutyric acid (GABA) have been reported to play an important role in modulating anxiety in different parts of the brain. HYPOTHESIS/PURPOSE: Herein, we report the anxiolytic activity of limonene. In addition, we identified a possible mechanism underlying the effect of limonene on DAergic and GABAergic neurotransmission. STUDY DESIGN: In this study, mice were injected with saline in the control group and limonene in the test group before behavioral analysis. We performed immunoblotting and high-performance liquid chromatography (HPLC) analysis after the behavioral study. RESULTS: The limonene treated group showed increased locomotor activity and open-arm preference in the elevated plus maze experiment. Limonene treatment increased the expression of both tyrosine hydroxylase and GAD-67 proteins and significantly upregulated dopamine levels in the striatum. Furthermore, tissue dopamine levels were increased in the striatum of mice following limonene treatment, and depolarization-induced GABA release was enhanced by limonene pre-treatment in PC-12 cells. Interestingly, limonene-induced anxiolytic activity and GABA release augmentation were blocked by an adenosine A2A receptor (A2AR) antagonist. CONCLUSION: Our results suggest that limonene inhibits anxiety-related behavior through A2A receptor-mediated regulation of DAergic and GABAergic neuronal activity.

D-limonene Inhibits Pentylenetetrazole-Induced Seizure via Adenosine A2A Receptor Modulation on GABAergic Neuronal Activity.

BACKGROUND: Epilepsy is a chronic neurological disorder characterized by the recurrence of seizures. One-third of patients with epilepsy may not respond to antiseizure drugs. PURPOSE: We aimed to examine whether D-limonene, a cyclic monoterpene, exhibited any antiseizure activity in the pentylenetetrazole (PTZ)-induced kindling mouse model and in vitro. METHODS: PTZ kindling mouse model was established by administering PTZ (30 mg/kg) intraperitoneally to mice once every 48 h. We performed immunoblot blots, immunohistochemistry (IHC), and high-performance liquid chromatography (HPLC) analysis after the behavioral study. RESULTS: An acute injection of PTZ (60 mg/kg) induced seizure in mice, while pretreatment with D-limonene inhibited PTZ-induced seizure. Repeated administration of PTZ (30 mg/kg) increased the seizure score gradually in mice, which was reduced in D-limonene (10 mg/kg)-pretreated group. In addition, D-limonene treatment increased glutamate decarboxylase-67 (GAD-67) expression in the hippocampus. Axonal sprouting of hippocampal neurons after kindling was inhibited by D-limonene pretreatment. Moreover, D-limonene reduced the expression levels of Neuronal PAS Domain Protein 4 (Npas4)-induced by PTZ. Furthermore, the adenosine A2A antagonist SCH58261 and ZM241385 inhibited anticonvulsant activity and gamma-aminobutyric acid (GABA)ergic neurotransmission-induced by D-limonene. CONCLUSION: These results suggest that D-limonene exhibits anticonvulsant activity through modulation of adenosine A2A receptors on GABAergic neuronal function.

Dopamine D1 receptor antagonist reduces stimulant-induced conditioned place preferences and dopamine receptor supersensitivity.

Repeated administration of stimulants induces conditioned place preference (CPP). Dopamine receptor supersensitivity is developed in stimulant-induced CPP animals; however, dopamine receptor subtypes associated with the development of supersensitivity in CPP animals are largely unknown. The present preclinical study aimed to examine whether dopamine D1 or D2 receptor antagonists exert inhibitory effects on stimulant-induced psychological behaviors. Additionally, the authors aimed to elucidate the role of dopamine receptor supersensitivity on the development of reward-related behavior. Sprague Dawley rats subjected to methamphetamine- and cocaine-induced CPP tests were treated with dopamine D1 (SCH23390) or D2 (sulpiride) receptor antagonists. Following the CPP experiment, rats were challenged with apomorphine (dopamine receptor agonist), and locomotor activity was measured. Methamphetamine- and cocaine-induced CPP was reduced with the administration of SCH23390, but not sulpiride. In addition, the apomorphine challenge evoked an increase in locomotor activity in stimulant-pre-treated rats, reflecting dopamine receptor supersensitivity. SCH23390 pre-treatment inhibited the development of dopamine receptor supersensitivity, while sulpiride demonstrated no inhibitory effects. These results suggest that the dopamine D1 receptor antagonist SCH23390 inhibits the development of dopamine receptor supersensitivity which is associated with the development of CPP.

Correction to: Dopamine D1 receptor antagonist reduces stimulant-induced conditioned place preferences and dopamine receptor supersensitivity.

In the originally published article, the name of the first author was incorrectly presented as Su Mi Gu. The correct name is Sun Mi Gu, which is also given above.

Cardiotoxic effects of [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol.

Two synthetic tryptamines, namely [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate (4-AcO-DET) and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol (4-HO-MET), are abused by individuals seeking recreational hallucinogens. These new psychoactive substances (NPSs) can cause serious health problems because their adverse effects are mostly unknown. In the present study, we evaluated the cardiotoxicity of 4-AcO-DET and 4-HO-MET using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, electrocardiography (ECG), and the human ether-a-go-go-related gene (hERG) assay. In addition, we analyzed the expression level of p21 (CDC42/RAC)-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, 4-AcO-DET- and 4-HO-MET-treated H9c2 cells proliferated in a concentration-dependent manner. Moreover, both substances increased QT intervals (as determined using ECG) in Sprague-Dawley rats and inhibited potassium channels (as verified by the hERG assay) in Chinese hamster ovary cells. However, there was no change in PAK1 expression. Collectively, the results indicated that 4-AcO-DET and 4-HO-MET might cause adverse effects on the cardiovascular system. Further studies are required to confirm the relationship between PAK1 expression and cardiotoxicity. The findings of the present study would provide science-based evidence for scheduling the two NPSs.

Rewarding effects of 2-desoxypipradrol in mice.

Desoxypipradrol (2-DPMP), a new psychoactive substance (NPS), acts as a norepinephrine-dopamine reuptake inhibitor (NDRI). NDRIs can be addictive due to their action mechanisms similar to cocaine and methamphetamine. However, there is a lack of scientific information regarding the exact dependency of 2-DPMP. Thus, the purpose of this study was to evaluate rewarding and reinforcing effects of 2-DPMP in rodents. The effective dose range of 2-DPMP was determined by climbing behavior test. To evaluate rewarding effects of 2-DPMP, conditioned place preference (CPP) test was performed at selected doses in mice. Self-administration (SA) test was then undertaken at two doses that caused the highest effects in the CPP test. Dopamine level changes were analyzed using synaptosomes in order to investigate effects of 2-DPMP on the central nervous system (CNS). Significant responses were observed in the climbing behavior test at doses of 0.1, 0.5, and 1 mg/kg by intraperitoneal injection (i.p.). In the CPP test, mice i.p. administered 2-DPMP at 1 mg/kg showed a significant preference in drug-paired compartment. In the SA test, mice intravenously given 0.1 mg/kg/infusion showed significantly higher active lever responses. Further, dopamine was increased in a dose-dependent manner. Taken together, these results suggest that 2-DPMP may act on the CNS and induce rewarding and reinforcing effects, indicating its dependence liability.

Abuse potential of 2-(4-iodo-2, 5-dimethoxyphenyl)N-(2-methoxybenzyl)ethanamine (25INBOMe); in vivo and ex vivo approaches.

25INBOMe ("25-I", "N-Bomb"), one of new psychoactive substances (NPSs), is being abused for recreational purpose. However, the liability for abuse or dependence has not been systematically studied yet. The objective of the present study was to evaluate rewarding and reinforcing effects of 25INBOMe using conditioned place preference (CPP) and self-administration (SA) paradigms. In addition, ultrasonic vocalizations (USVs) were measured to investigate relationships between USVs and emotional state regarding dependence on psychoactive substances. To understand molecular mechanism involved in its action, dopamine (DA) level changes were analyzed using synaptosomes extracted from the striatal region of the brain. Expression level changes of SGK1 (serum/glucocorticoid regulated kinase 1) and PER2 (period circadian protein homolog 2), two putative biomarkers for drug dependence, were also analyzed. Results showed that 25INBOMe increased both CPP (0.3 mg/kg) and SA (0.03 mg/kg/infusion) and produced higher frequencies in USVs analysis. It also increased DA levels in the striatal region and changed expression levels of SGK1 and PER2. Results of the present study suggest that 25INBOMe might produce rewarding and reinforcing effects, indicating its dependence liability. In addition, frequencies of USV might be associated with emotional state of mice induced by psychoactive substances regarding substance dependence. This is the first systemic preclinical report on the dependence liability of 25INBOMe and the first attempt to introduce a possible relationship between USVs and emotional state of mice regarding substance dependency. Further studies are needed to clarify the mechanism involved in 25INBOMe dependency and determine the usefulness of USV measurement as a method for evaluating dependence liability.

Receptor Binding Affinities of Synthetic Cannabinoids Determined by Non-Isotopic Receptor Binding Assay.

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor (CB1) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified CB1 were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid Δ9-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to CB1 were determined to be (from highest to lowest) 9.52 × 10-13 M (JWH-210), 6.54 × 10-12 M (JWH-250), 1.56 × 10-11 M (Δ9-tetrahydrocannabinol), 2.75 × 10-11 M (RCS-4), and 6.80 ×10-11 M (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

2-(2,5-Dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe) induce adverse cardiac effects in vitro and in vivo.

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 µM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.

Methoxetamine Induces Cytotoxicity in H9c2 Cells: Possible Role of p21 Protein (Cdc42/Rac)-Activated Kinase 1.

The abuse of new psychoactive substances (NPS) is an emerging social problem. Methoxetamine, one of the NPS, was designed as an alternative to ketamine and it was considered an NPS candidate owing to its high addictive potential. However, cardiotoxicity of the phencyclidine analogue, methoxetamine, has not been extensively evaluated. P21 protein (Cdc42/Rac)-activated kinase 1 (PAK-1) is associated with the drug-induced cardiotoxicity and hypertrophy of cardiomyocytes. In the present study, we investigated the effects of methoxetamine on rat cardiomyocytes and PAK-1. Methoxetamine (at 10 µM) reduced cell viability and PAK-1 mRNA levels in H9c2 cells. Methoxetamine treatment (100 µM) decreased the beating rate of primary cardiomyocytes. However, 100 µM methoxetamine-induced heart rate decline was less than 100 µM PCP- or ketamine-induced heart rate decline. Meanwhile, fingolimod hydrochloride (FTY720, 1 µM), a PAK-1 activator, increased cell viability and inhibited hypertrophy induced by methoxetamine in H9c2 cells. These results suggest that methoxetamine may have harmful effects on the cardiovascular system through the regulation of the expression and function of PAK-1.