Fentanyl-Type Antagonist of the µ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation.

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the "opioid crisis." To design safer therapeutic agents, we accidentally discovered µ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Synthetic cannabinoid CP-55,940 induces apoptosis in a human skeletal muscle model via regulation of CB1 receptors and L-type Ca2+ channels.

Rhabdomyolysis has been reported in patients who abuse synthetic cannabinoids. However, no studies have yet assessed whether these cases reflect the direct cytotoxicity of synthetic cannabinoids on skeletal muscle, a possibility that the present study sought to address. Specifically, this study investigated the cytotoxicity of the synthetic cannabinoid CP-55,940, a compound that acts equally on both types of cannabinoid receptors (CB1 and CB2), in a human embryonic rhabdomyosarcoma (RD) cell line. Exposure of these cells to CP-55,940 resulted in concentration-dependent decreases in cell viability. These effects were attenuated by pre-incubation with AM251 (30 µM), a selective CB1 receptor antagonist, but not by pre-incubation with AM630 (30 µM), a selective CB2 receptor antagonist. Following treatment with CP-55,940, RD cells exhibited apoptosis, as indicated by the accumulation of annexin-V, activation of caspase-3, and a loss of the mitochondrial membrane potential. Additionally, CP-55,940 treatment of RD cells led to increases in intracellular Ca2+ levels. CP-55,940-induced cell death was significantly attenuated in the absence of extracellular Ca2+, and was partially decreased by pre-incubation with verapamil (5 µM) or diltiazem (5 µM), compounds that block the L-type Ca2+ channel. Our results indicate that the cytotoxicity of CP-55,940 towards RD cells (skeletal muscle cells) is mediated by the CB1 receptor, but not by the CB2 receptor. Our results further suggest that calcium influx through the L-type channel may play an important role in the apoptosis induced by these compounds.

Synthetic cannabinoids enhanced ethanol-induced motor impairments through reduction of central glutamate neurotransmission.

Marijuana or synthetic cannabinoids and alcohol are often used together, with these combinations causing motor impairments that can subsequently lead to motor vehicle accidents. This study investigated the combined use of both synthetic cannabinoids and ethanol and their effect on motor coordination in mice in addition to examining the neurochemical changes in the cerebellum. Ethanol (2 g/kg, i.p.) significantly induced motor impairment in the accelerating rotarod test in mice. Furthermore, ethanol-induced motor impairments were further accentuated when combined with the synthetic cannabinoid, JWH-018 or AB-CHMINACA. The enhancement effects of the synthetic cannabinoids were completely antagonized by pretreatment with the selective CB1 receptor antagonist AM251, but not by the selective CB2 receptor antagonist AM630. Neurochemical study results showed that ethanol caused a reduction in the extracellular glutamate levels in the cerebellum during periods of ethanol-induced motor impairment. In addition to the enhanced motor impairment seen when ethanol was combined with JWH-018, these combinations also enhanced the reduction of the extracellular glutamate levels in the cerebellum. We additionally used microelectrode array recordings to examine the effects of ethanol and/or JWH-018 on the spontaneous network activity in primary cultures from mouse cerebellum. Results showed that ethanol combined with JWH-018 significantly reduced spontaneous neuronal network activity in the primary cerebellar culture. Our findings demonstrate that ethanol-induced motor impairments are enhanced by synthetic cannabinoids, with these effects potentially mediated by CB1 receptors. An accentuated reduction of neurotransmissions in the cerebellum may play an important role in motor impairments caused by ethanol combined with synthetic cannabinoids.

[Present Conditions of Marijuana Regulation in USA: Medical and Recreational Use].

In most countries marijuana is regulated by the Single Convention on Narcotic Drugs. In Japan marijuana use is illegal under the Marijuana Control Law. In USA, marijuana is also classified as a schedule I drug, which is the most stringent regulation category under federal law. On the other hand, California became the first state to legalize marijuana for medical uses in 1996. Since then, several other US states have approved marijuana for medical or recreational use. However, marijuana remains completely illegal in most states, while some allow only cannabidiol (CBD) extracted from marijuana for medical use. In June 2018, the US Food and Drug Administration approved Epidiolex, the first marijuana-derived drug, containing purified CBD, to treat certain rare childhood seizure syndromes. Therefore the situation surrounding control of marijuana in USA is complex. Recently, a definite trend toward reconsidering marijuana regulation has been seen around the world, which could have a major impact on marijuana policy in Japan. In this review, we investigated existing medical and recreational marijuana laws in various US states, with a focus on California, which approved recreational use in 2018. Here, we describe the current state of marijuana regulation in terms of both medical and recreational use. In addition, we discuss public safety issues associated with marijuana, including crime, traffic accidents, and emergency department visits from possible marijuana exposure, as well as generated tax revenues, from official marijuana-related reports in Colorado, which legalized marijuana use in 2012.

[Dependence and Cytotoxicity of Components of Cannabis].

Cannabis use among the younger population in Japan has been steadily increasing. The aim of the present review is to highlight recent knowledge regarding the molecular mechanisms of action and health risks associated with cannabis and synthetic cannabinoid consumption. We investigated the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoids on place conditioning in ICR mice. Both Δ9-THC and synthetic cannabinoids produce a significant conditioned place preference. These rewarding effects were completely suppressed by the cannabinoid CB1 receptor type antagonist AM251. The cytotoxicological effects of Δ9-THC and synthetic cannabinoids were also characterized in the limbic forebrain of mice in primary culture in vitro. Δ9-THC and synthetic cannabinoids caused cell death in a dose-dependent manner. The rank order of cytotoxicological potency was synthetic cannabinoids>Δ9-THC and related to the agonistic activities of the CB1 receptor. A recent review on the harmful effects of cannabis use in humans reported that behavioral impairments, especially in terms of attention, memory, and complex information-processing ability, can last for many weeks after cessation of cannabis use among heavy users. In addition, cannabis use could be a risk factor for drug dependence and later psychosis among adolescents. The results of animal and human studies suggest that CB1 receptors play an important role in the expression of harmful effects of cannabis and synthetic cannabinoid use. Moreover, concern regarding increasing concentrations of Δ9-THC in cannabis in many countries has been noted, because more potent cannabis may be associated with worse adverse effects.

Δ9-Tetrahydrocannabinol elicited 22-kHz ultrasonic vocalization changes after air puff stimulus through CB1 receptor in adult rats.

Δ9-Tetrahydrocannabinol (THC) is known to have various pharmacological effects mediated through activation of cannabinoid CB1 and CB2 receptors in rodents. In adult rats, 22- and 50-kHz ultrasonic vocalizations (USVs) serve as an effective communication system and as indicators of negative and positive states, respectively. The present study was performed to determine whether THC affects USVs in adult rats, and to determine the roles of cannabinoid receptors in these effects. THC (1, 3 mg/kg) was administered intraperitoneally to adult male Wistar rats 60 min before measurement of USVs. The CB1 antagonist, SR141716 (3, 6 mg/kg), or CB2 antagonist, AM630 (1, 10 mg/kg), was administered intraperitoneally 10 min before THC. USVs were measured during a 5-minute period without air puff stimulus or with air puff stimulus. THC did not affect 22- or 50-kHz USVs without air puff stimulus. On the other hand, THC significantly increased the number of 22-kHz USVs, but not 50-kHz USVs, after air puff stimulus. Moreover, SR141716 at 6 mg/kg, but not AM630 at either dose, inhibited the increase in number of 22-kHz USVs induced by THC after air puff stimulus. These results suggest that THC induced changes in sensitivity to aversive air puff stimuli through CB1 receptors, and as a result increased emission of 22-kHz USVs in rats.

Synthetic cannabinoid AM2201 induces seizures: Involvement of cannabinoid CB1 receptors and glutamatergic transmission.

Abuse of synthetic cannabinoids is a serious social problem worldwide. Intentional ingestion of synthetic cannabinoids can cause severe toxicity, including seizures. Here we investigated the effects of acute administration of synthetic cannabinoids on the induction of epileptic seizures by monitoring electroencephalographic activity in freely moving mice. The synthetic cannabinoid, AM2201, induced abnormal, high-amplitude (>2-fold baseline amplitude), sharp-wave activity. The abnormal spike-wave discharges were accompanied by epileptiform behavior: rigid posture, tail extension, rearing with forepaws extended, jumping, and intermittent tonic-clonic jerking movements. The abnormal spike-wave discharges and behavioral changes were suppressed by pretreatment with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630 or the vanilloid receptor antagonist, capsazepine. Furthermore, the group 1 metabotropic glutamate receptor antagonist SIB1757 eliminated AM2201-induced spike-wave discharges and episodes of epileptiform behavior. AM2201 markedly increased the extracellular glutamate concentration in the hippocampus during periods of AM2201-induced abnormal spike-wave discharges and behavioral changes. These findings are the first evidence that AM2201 induces epileptic seizures by enhancing glutamatergic transmission in the hippocampus. Our findings demonstrate that induction of epileptic seizures by synthetic cannabinoids is mediated by CB1 receptors, but not by CB2 receptors, and further suggest that rapid elevation of glutamatergic transmission may play an important role in the induction of seizures following intentional ingestion of synthetic cannabinoids.

Pyrolysis of UR-144, a synthetic cannabinoid, augments an affinity to human CB1 receptor and cannabimimetic effects in mice.

Drug abusers most often smoke 'herbal incense' as a cigarette or inhale it using a smoking tool. Smoking may cause pyrolysis of the drug and produce decomposed products of which biological effect has never been investigated. The synthetic cannabinoid UR-144 is known to undergo thermal degradation, giving a ring-opened isomer, so-called UR-144 degradant. The present study demonstrates by using UR-144 as a model drug that the smoke of burned UR-144 contains the UR-144 degradant. The UR-144 degradant showed approximately four fold higher agonist activity to human CB1 receptor and augmented hypothermic and akinetic actions in mice compared to UR-144. These results indicate that smoking behavior may increase psychological actions of the certain synthetic cannabinoids.

[Drug Dependence and Toxicity of Law-Evading Drugs: Their Mechanisms Explored from Basic Research].

The number of law-evading chemical substances that pose serious health risks to humans is increasing worldwide, including Japan and the Western world, and their abuse is becoming a serious social problem. Analysis of the chemical substances in these herbal products revealed the presence of synthetic cannabinoids. This review summarizes the pharmacological actions and dangers of chemical substances contained in law-evading herbal products by focusing on synthetic cannabinoids as a group of chemical substances contained in these products. We established a psychic-dependence liability and cytotoxicity screening system for synthetic cannabinoids by using animals and cell cultures. In a place-conditioning study, the synthetic cannabinoids produced a significant conditioned-place preference. The rewarding effects of synthetic cannabinoids were completely suppressed by a cannabinoid CB1 receptor antagonist. Administration of synthetic cannabinoids to primary striatal culture caused cell death in a dose-dependent manner. Our findings show that the CB1 receptor might be involved in the expression of synthetic cannabinoid-induced rewarding effect. These behavioral data indicate that synthetic cannabinoids have a psychic-dependence liability. Furthermore, our data from primary striatal culture indicate that synthetic cannabinoids have strong neurotoxicity. These behavioral and neurochemical data indicate that synthetic cannabinoids might have strong adverse effects and a psychic-dependence liability.

[Drug Dependence and Cytotoxicity of Law-evading Drugs: Their Identities Explored from Basic Research].

  Cases of people experiencing disturbed consciousness or dyspnea, causing traffic accidents, or requiring ambulance transport to hospital due to abuse of law-evading chemical substances have become a serious social problem in Japan. Most law-evading herbal products are marketed as incense or herbs and consist of finely chopped, dry vegetative matter mixed with chemical substances (drugs). Analysis of the chemical substances in these herbal products has demonstrated that they contain synthetic cannabinoids. Because there are many cannabinoid compounds, even if a particular drug is regulated, similar compounds that differ only slightly in structure may be added in their place. Therefore a cat-and-mouse game exists between regulations on chemical substances and their propagation. This paper summarizes the pharmacological actions and dangers of chemical substances contained in law-evading herbal products by focusing on synthetic cannabinoids, as a group of chemical substances contained in these products. Furthermore, comprehensive designations of synthetic cannabinoids have been introduced as a new method of regulation that emphasizes the similarity of chemical structures; this paper also outlines the comprehensive designations. We established a psychic-dependence liability and cytotoxicity screening system for synthetic cannabinoids using animals (behavioral analysis in vivo) and cell cultures (cytotoxicity analysis in vitro). With our drug-screening system, we were able rapidly to evaluate and quantify psychic-dependence liabilities and cytotoxicity of synthetic cannabinoids contained in law-evading herbal products. These scientific data using our screening system contributed to the establishment of legislation for comprehensive designations of synthetic cannabinoids.

[Pharmacological properties of law-evading chemical substances].

In recent years, frequent cases of people suffering disturbed consciousness, dyspnea, etc. due to abuse of synthetic cannabis and being transported by ambulance or causing traffic accidents are occurring and are becoming a serious social problem in Japan. Most law-evading herbal products have colorful illustrations and logos and are sold as incense or herbs. Law-evading herbal products consist of finely chopped dry vegetative matter mixed with chemical substances (drugs), and the drugs are injurious to health. Analysis of chemical substances in herbal products clarified that they contain synthetic cannabinoid, a chemical component that exhibits action similar to that of hemp. There are many affinity compounds of cannabinoid, so presently, even if a particular drug is regulated, similar compounds that partially differ in structure will propagate. There is thus a cat-and-mouse game between regulations on chemical substances and their propagation. This paper summarizes the pharmacological actions and danger of chemical substances contained in law-evading herbal products by focusing on synthetic cannabinoid or synthetic cathinone, a chemical substance it contains.

Splenectomy modifies hyperactive states of the dopaminergic system induced by morphine in C57BL/6J-bg(J)/bg(J) (beige-J) mice.

Genetic factors affect the locomotor activity induced by morphine, which mainly depends on the activation of dopaminergic systems, and morphine has distinct pharmacological activities in C57BL/6J-bg(J)bg(J) (beige-J) mice, which have genetic deficiencies in immunological function. We previously showed that beige-J mice exhibited greater locomotor activity and dopamine turnover, whereas splenectomy reduced this hyperlocomotion and dopamine turnover, which suggests that beige-J mice could be an experimental animal model for investigating hyperactivation of the dopaminergic system, and that the spleen may contribute to the susceptibility to activation of the dopaminergic system. Furthermore, morphine can induce hyperlocomotion mediated by activation of the dopaminergic system. Therefore, we examined the effects of splenectomy on the hyperlocomotion and regulation of the dopaminergic system induced by morphine in beige-J mice. Morphine induced hyperlocomotion, which was accompanied by activation of the dopaminergic system, in beige-J mice. Furthermore, splenectomy enhanced the hyperlocomotion and activation of the mesolimbic dopaminergic system induced by morphine in beige-J mice. Our findings indicate that substances originating from the spleen may regulate both spontaneous activation of the mesolimbic dopaminergic system and the µ-opioidergic system-mediated activation of the mesolimbic dopaminergic system by morphine through different modes of action. These results imply that beige-J mice could be a practical animal model for investigating the interactions between immune-modulation and the µ-opioidergic system and/or dopaminergic system.

Dopaminergic hyperactivity accompanied by hyperlocomotion in C57BL/6J-bg(J)/bg(J) (beige-J) mice.

Genetic factors affect locomotor activity, which mainly depends on the activation of dopaminergic systems. C57BL/6J-bg(J)bg(J) (beige-J) mice, which exhibit deficiencies in immunological function, show behavioral hyperactivity. The present study was designed to investigate the locomotor activity of beige-J mice accompanied by a change in the dopaminergic system. Beige-J mice showed higher locomotor activity and dopamine turnover, whereas splenectomy reduced this hyperlocomotion and dopamine turnover. These results suggest that beige-J mice could be suitable as an experimental animal model for investigating hyperactivation of the dopaminergic system, and the spleen may contribute to the susceptibility of dopaminergic systems to activation.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB1 receptors and apoptotic cell death.

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB1 receptor, but not by the CB2 receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain.

Ecstasy (3, 4-methylenedioxymethamphetamine) use among Japanese rave population.

AIMS: The aim of this study was to examine the prevalence of 3, 4-methylenedioxymethamphetamine (MDMA) use and to identify characteristics of MDMA users among rave attendees in Japan. This is the first rave-population study focusing on MDMA use in Japan. METHODS: The anonymous self-administrative questionnaire was conducted using laptop computers at four rave parties at three different venues in Tokyo, Japan. Participants were asked about lifetime use of MDMA and other club drug use, characteristics of rave attendance, and their demographics. RESULTS: Questionnaires were completed by 300 rave attendees (47.3% female), 68.3% of whom were aged 20-29 years, and 92.3% of whom had completed high school. Among the participants, 8.0% reported MDMA use. Compared with 'non-drug controls' (the participants who had never used any illicit drugs), MDMA users were significantly more likely to be 30-39-year-old men. In addition, compared with 'cannabis users' (non-MDMA users who had used cannabis), MDMA users were significantly more likely to use other drugs and reported more adverse health effects due to 'polydrug use'. Furthermore, MDMA users were significantly more likely to go to raves and preferred smaller venues. CONCLUSIONS: Our results clearly suggest that rave attendees have a higher lifetime prevalence of MDMA use than the Japanese general population (0.2% reported in 2007). MDMA users are deeply involved in rave parties, and MDMA use may have high potential to generate close relationships among rave attendees. Therefore, MDMA users may have more opportunities to access MDMA than cannabis users and non-drug controls.

Current status of substance abuse and HIV infection in Japan.

Japan has experienced an epidemic of methamphetamine (MAP) abuse three times: The first epidemic was from 1951 to 1957, the second epidemic was from 1970 to 1994, and the third epidemic started in 1995 and continues today. Fortunately, HIV infection is not as serious a problem in Japan as it is in other countries. The major route of HIV infection in Japan has been through male homosexual transmission. In cumulative number, homosexual transmission accounted for 63% of the 11,146 HIV-positive patients and 40% of 5,158 AIDS patients as of December 30, 2011. Intravenous drug use accounted for 0.3% and 0.4% of these cases, respectively. Drug abuse has changed during the past 20 years in Japan. The changes are summarized as follows: There has been (1) a remarkable decrease in solvent abuse, (2) a stabilization of MAP abuse, (3) a penetration of cannabis abuse, (4) an emergence of evasive drug abuse, and (5) a silent increase in medical drug dependence. This implies that (1) there has been a change from a "solvent dominant type" of use to a "cannabis dominant type," that is, from a "Japanese type" to a "Western type," (2) a shift to drugs which do not have a high potential to cause drug-induced psychosis, and (3) a shift from conduct that leads to arrest to conduct that does not lead to arrest. Regardless of whether the drug use is illicit or not, drug dependence is a mental disorder. Japan is urged to deal with drug abuse and dependence using not only the criminal model but also the medical model.