Timeframe Analysis of Novel Synthetic Cannabinoids Effects: A Study on Behavioral Response and Endogenous Cannabinoids Disruption.

This study investigates the impact of SCs consumption by assessing the effects of three novel synthetic cannabinoids (SCs); MDMB-CHMINACA, 5F-ADB-PINACA, and APICA post-drug treatment. SCs are known for their rapid onset (<1 min) and prolonged duration (≥5 h). Therefore, this research aimed to assess behavioral responses and their correlation with endocannabinoids (ECs) accumulation in the hippocampus, and EC's metabolic enzymes alteration at different timeframes (1-3-5-h) following drug administration. Different extents of locomotive disruption and sustained anxiety-like symptoms were observed throughout all-encompassing timeframes of drug administration. Notably, MDMB-CHMINACA induced significant memory impairment at 1 and 3 h. Elevated levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were detected 1 h post-MDMB-CHMINACA and 5F-ADB-PINACA administration. Reduced mRNA expression levels of fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL) (AEA and 2-AG degrading enzymes, respectively), and brain-derived neurotrophic factor (BDNF) occurred at 1 h, with FAAH levels remaining reduced at 3 h. These findings suggest a connection between increased EC content and decreased BDNF expression following SC exposure. Cognitive disruption, particularly motor coordination decline and progressive loss manifested in a time-dependent manner across all the analyzed SCs. Our study highlights the importance of adopting a temporal framework when assessing the effects of SCs.

Clinical Profiles of Japanese Patients with Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Collected by a Nationwide System from 2006 to 2023.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are potentially life-threatening severe cutaneous adverse drug reactions. These diseases are rare, and their onset is difficult to predict because of their idiosyncratic reactivity. The Japan Severe Adverse Reactions Research Group, led by the National Institute of Health Sciences, has operated a nationwide to collect clinical information and genomic samples from patients with SJS/TEN since 2006. This study evaluated the associations of clinical symptoms with sequelae and specific causative drugs/drug groups in Japanese patients with SJS/TEN to identify clinical clues for SJS/TEN treatment and prognosis. Acetaminophen, antibiotics, and carbocisteine were linked to high frequencies of severe ocular symptoms and ocular sequelae (p < 0.05). For erythema and erosion areas, antipyretic analgesics had higher rates of skin symptom affecting <10% of the skin than the other drugs, suggesting narrower lesions (p < 0.004). Hepatic dysfunction, was common in both SJS and TEN, and antiepileptic drugs carried higher risks of hepatic dysfunction than the other drug groups (p = 0.0032). This study revealed that the clinical manifestations of SJS/TEN vary according to the causative drugs.

Identification of hexahydrocannabinol (HHC), dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC) and hexahydrocannabiphorol (HHCP) in electronic cigarette cartridge products.

PURPOSE: Since 2021, products claiming to contain hexahydrocannabinol (HHC) and hexahydrocannabiphorol (HHCP), which are tetrahydrocannabinol (THC) analogs, have been distributed via the Internet. Owing to the presence of three asymmetric carbons in their structure, HHC and HHCP have multiple stereoisomers. This study aimed to identify the actual stereoisomers of HHC and HHCP isolated from electronic cigarette cartridge products using nuclear magnetic resonance (NMR) spectroscopy. METHODS: Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) were used for the analyses of two major peaks and one minor peak in product A and two major peaks in product B. These five compounds were isolated by silica gel column chromatography, and their structures were analyzed by 1H, 13C-NMR and various two-dimensional NMR techniques, i.e., H-H correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy. RESULTS: Three compounds isolated from product A were identified as rel-(6aR,9R,10aR)-hexahydrocannabinol (11ß-hexahydrocannabinol; 11ß-HHC), rel-(6aR,9S,10aR)-hexahydrocannabinol (11α-hexahydrocannabinol, 11α-HHC), and a minor compound (2R,5S,6R)-dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC). Meanwhile, the structural isomers of the major compound isolated from product B were identified as rel-(6aR, 9R, 10aR)-hexahydrocannabiphorol (11ß-hexahydrocannabiphorol; 11ß-HHCP) and rel-(6aR, 9S, 10aR)-hexahydrocannabiphorol (11α-hexahydrocannabiphorol; 11α-HHCP). CONCLUSIONS: The presence of both 11ß-HHC and 11α-HHC in the HHC products analyzed in this study suggests that they were most likely synthesized via the reduction reaction of Δ8-THC or Δ9-THC. Dihydro-iso-THC was probably obtained as a byproduct of the synthesis of Δ8-THC or Δ9-THC from cannabidiol. Similarly, 11ß-HHCP and 11α-HHCP in the HHCP product could stem from Δ9-tetrahydrocannabiphorol.

Structural analysis of an lysergic acid diethylamide (LSD) analogue N-methyl-N-isopropyllysergamide (MiPLA): Insights from Rotamers in NMR spectra.

Lysergic acid diethylamide (LSD) is a hallucinogenic compound that binds to and activates the serotonin 2A receptor and is classified as a controlled narcotic in Japan. Recently, MiPLA, an N-methyl-N-isopropyl derivative of LSD, has been detected in paper-sheet products in several countries. This study focuses on the synthesis of MiPLA and includes a comprehensive analysis involving structural and liquid chromatography-mass spectrometry (LC-MS). Particularly, MiPLA was synthesized in three-steps starting from ergometrine maleate, which resulted in the formation of (8S)-isomer, iso-MiPLA, as a by-product. The LC-MS results showed that LSD, MiPLA, and iso-MiPLA exhibited different retention times. Their chemical structures were determined using nuclear magnetic resonance spectroscopy, which revealed the presence of rotamers involving the N-methyl-N-isopropyl groups of tertiary amides in MiPLA and iso-MiPLA.

Characterization of the lysergic acid diethylamide analog, 1-(thiophene-2-carbonyl)-N,N-diethyllysergamide (1T-LSD) from a blotter product.

Recently, lysergic acid diethylamide (LSD) analogs have appeared worldwide as designer drugs. In this study, we identified a distributed LSD analog from a paper-sheet product. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS), and liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were used to analyze the sheet product. The sheet product claimed to contain 1-(1,2-dimethylcyclobutanoyl)-N,N-diethyllysergamide (1D-LSD). However, an unknown compound was detected in the product together with tryptamine and L-tryptophan methyl ester. This compound was isolated from the sheets and identified as 1-(thiophene-2-carbonyl)-N,N-diethyl-6-methyl-9,10-didehydroergoline-8ß-carboxamide (1-thiophenoyl LSD; 1-(2-thienoyl)-LSD, 1T-LSD), using 1 H, 13 C nuclear magnetic resonance (NMR) spectroscopy and various two-dimensional NMR techniques. 1T-LSD was shown to have the thiophene-2-carbonyl group at the N1 position instead of the 1,2-dimethylcyclobutane-carbonyl group as claimed. The amount of 1T-LSD (free base) in three individual unit from one sheet was determined to be 87-100 µg per unit using a proton-specific quantitative NMR (1 H-qNMR) method. Deacylation of 1T-LSD to LSD was also observed to occur in methanol-d4 during NMR analysis. The UV spectrum of 1T-LSD differed from that of other LSD analogs, and the fluorescence sensitivity was much lower. Because of concerns about the future distribution of products containing new LSD analogs, continued monitoring of newly detected compounds in sheet products is encouraged.

[Determination of 11 Cannabinoids in Cannabis sativa L. by Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-TOF-MS)].

Eleven major cannabinoids from each subdivided tissue of drug-type and fiber-type cannabis plants were determined by means of a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The cannabinoids analyzed in this study were tetrahydrocannabinol acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol acid (CBDA), cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), cannabinol (CBN), cannabichromene (CBC), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG) and tetrahydrocannabivarin (THCV). As a result, THCA was detected in the bracts at 28.4 µg/mg, in the buds at 24.8 µg/mg, and in the leaves at 5.1 to 10.5 µg/mg in the drug-type cannabis plant. In addition, Δ9-THC, CBGA, CBN, CBG, CBC, and THCV were mainly detected in bracts, buds, and leaves. On the other hand, as for the fiber-type cannabis plant, CBDA was detected in the bracts at 27.5 µg/mg, in the buds at 10.6 µg/mg, and in the leaves at 1.5-3.3 µg/mg. In addition, Δ9-THCA, CBD, Δ9-THC, CBC, and CBG were mainly detected in bracts, buds, and leaves.

Identification of LSD analogs, 1cP-AL-LAD, 1cP-MIPLA, 1V-LSD and LSZ in sheet products.

PURPOSE: Many analogs of lysergic acid diethylamide (LSD) have recently appeared as designer drugs around the world. These compounds are mainly distributed as sheet products. In this study, we identified three more newly distributed LSD analogs from paper sheet products. METHODS: The structures of the compounds were determined by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS), liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: From the NMR analysis, the compounds in the four products were identified as 4-(cyclopropanecarbonyl)-N,N-diethyl-7-(prop-2-en-1-yl)-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1cP-AL-LAD), 4-(cyclopropanecarbonyl)-N-methyl-N-isopropyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo-[4,3-fg]quinoline-9-carboxamide (1cP-MIPLA), N,N-diethyl-7-methyl-4-pentanoyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1V-LSD) and (2'S,4'S)-lysergic acid 2,4-dimethylazetidide (LSZ). In comparison with the structure of LSD, 1cP-AL-LAD was converted at the positions at N1 and N6, and 1cP-MIPLA was converted at the positions at N1 and N18. The metabolic pathways and biological activities of 1cP-AL-LAD and 1cP-MIPLA have not been reported. CONCLUSIONS: This is the first report showing that LSD analogs that were converted at multiple positions have been detected in sheet products in Japan. There are concerns about the future distribution of sheet drug products containing new LSD analogs. Therefore, the continuous monitoring for newly detected compounds in sheet products is important.

Derivatives of methoxetamine and major methoxetamine metabolites potently block NMDA receptors.

N-Methyl-D-aspartate receptors (NMDARs) in the brain are influenced by psychoactive drugs such as 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine) and its analog 2-(ethylamino)-2-(3-methoxyphenyl)-cyclohexanone (methoxetamine). The recreational methoxetamine use can cause several toxicities and methoxetamine-related deaths have also been reported. Therefore, it has been banned in many countries. Since 2020, methoxetamine derivatives, 2-(ethylamino)-2-(m-tolyl)cyclohexan-1-one (deoxymethoxetamine) and 2-(isopropylamino)-2-(3-methoxyphenyl)cyclohexan-1-one (methoxisopropamine), have been sold online as designer drugs. However, how deoxymethoxetamine and methoxisopropamine act on NMDARs remains unknown. In this study, we first performed in silico docking studies of NMDARs, and deoxymethoxetamine and methoxisopropamine in addition to the major methoxetamine metabolites, 2-amino-2-(3-methoxyphenyl)-cyclohexanone (N-desethyl methoxetamine) and 2-(ethylamino)-2-(3-hydroxyphenyl)-cyclohexanone (O-desmethyl methoxetamine). The docking study suggested each compound interacts with NMDARs. We also determined the half-maximal inhibitory concentration (IC50s) of the methoxetamine-related compounds for NMDARs using NMDAR-expressing cartwheel interneurons of mice and patch-clamp recordings. We found that the IC50s of methoxetamine, deoxymethoxetamine, methoxisopropamine, N-desethyl methoxetamine, and O-desmethyl methoxetamine for NMDARs were 0.524, 0.679, 0.661, 1.649, and 0.227 µM, respectively. These results indicate that the methoxetamine-related compounds act as potent NMDAR blockers. Thus, deoxymethoxetamine and methoxisopropamine, both of which may cause damage by blocking NMDARs, are serious concerns. N-Desethyl methoxetamine and O-desmethyl methoxetamine may cause several adverse effects when methoxetamine is metabolized.

Derivatization-assisted immunoassays: application for group-specific detection of potent methamphetamine and amphetamine enantiomers.

Reliable and feasible tools for detecting (S)-methamphetamine [(S)-MAP] and (S)-amphetamine [(S)-AP] are required for regulating their illicit circulation. Antibodies that react equally to these stimulants are desirable for this purpose, but have been difficult to generate because of the crucial difference between their characteristic structures: i.e., N-methylamino (MAP) and amino (AP) groups. Furthermore, their small molecular masses (Mr < 150) have hampered the generation of high-affinity antibodies. To overcome these problems, we converted (S)-MAP and -AP into their 2-(trimethylsilyl)ethyl carbamate forms, Teoc-(S)-MAP and -AP, respectively, as surrogate analytes. The Teoc-derivatization not only increases their molecular masses, but also masks their structural differences. We generated a novel monoclonal antibody that showed a satisfactory affinity to Teoc-(S)-MAP residues (Kd = 13 nM as the IgG form) and developed a competitive enzyme-linked immunosorbent assay (ELISA) using microplates containing immobilized Teoc-(S)-MAP residues. Almost overlapping dose-response curves were obtained for Teoc-(S)-MAP and -AP, with the limit of detection of 0.078 and 0.10 ng per assay, respectively. A fixed amount of test powder sample (1 mg) was derivatized with Teoc-O-succinimidyl for 5 min, and subjected to ELISA using Teoc-(S)-MAP as the calibration standard. Under this protocol, (S)-MAP and -AP were converted to their Teoc derivatives with 30% and 34% yield, respectively, determined using ELISA as "Teoc-(S)-MAP equivalent," being distinguished from the derivatization products of (R)-MAP, (R)-AP, ephedrine, (S)-methylenedioxymethamphetamine, tyramine, dopamine, and ß-alanine. This ELISA detected as little as 10 µg of (S)-MAP and -AP, and (S)-MAP in urine obtained from (S)-MAP-administered rats. Immunochromatography devices were also developed using gold nanoparticles coated with the monoclonal antibody, with which 0.10 mg of (S)-MAP and -AP was detected by the naked eye. We conclude that the present derivatization-assisted immunoassays may be useful for the detection of (S)-MAP and/or -AP in early stage screening of suspicious substances.

[Identification of Three Arylcyclohexylamines (MXPr, MXiPr, and DMXE) in Illegal Products].

Arylcyclohexylamines are a category of substances to which the anesthetic ketamine belongs. The arylcyclohexylamines have been reported to act as antagonists of the N-methyl-d-aspartate (NMDA) receptor. An analog of ketamine, 2-(ethylamino)-2-(3-methoxyphenyl)-cyclohexanone (methoxetamine; MXE), has been controlled as a narcotic in Japan and overdoses of MXE have been reported to cause health problems. In recent years, MXE derivatives have beendetected in illegal products in Japan. In this study, we describe the identification of three MXE derivatives, 2-(3-methoxyphenyl)-2-(propylamino)cyclohexan-1-one (methoxpropamine; MXPr), 2-(isopropylamino)-2-(3-methoxyphenyl)cyclohexan-1-one (methoxisopropamine; MXiPr) and 2-(3-methoxyphenyl)-2-(propylamino)cyclohexan-1-one (deoxymethoxetamine; DMXE), from illegal products.

Derivatization-assisted enzyme-linked immunosorbent assay for identifying hallucinogenic mushrooms with enhanced sensitivity.

A sensitive immunochemical method for identifying hallucinogenic mushrooms (magic mushrooms) is required for regulating their illicit use. We have previously generated a monoclonal antibody (mAb) that targets psilocin (Psi), the major psychoactive compound in hallucinogenic mushrooms, and developed an enzyme-linked immunosorbent assay (ELISA). However, this ELISA failed to achieve the expected low-picomole-range sensitivity, as a result of insufficient affinity of the mAb to Psi. It is recognized that haptenic antigens with a larger molecular mass tend to induce antibodies with higher affinities. Thus, we herein report a "derivatization-assisted ELISA," in which the "real analyte" Psi was determined as a "surrogate analyte," the tert-butyldimethylsilyl ether analog thereof (TBS/Psi) having a 1.6-fold greater molecular mass (Mr 318.53) than Psi. A novel mAb against TBS/Psi, prepared by immunizing mice with a TBS/Psi-albumin conjugate showed a 69-fold higher affinity to TBS/Psi residues (Ka = 3.6 × 107 M-1 as IgG) than that of our previous mAb against Psi. This mAb consequently enabled a competitive ELISA for measuring TBS/Psi with the desired sensitivity: the dose-response curve midpoint (12.1 pmol per assay) was >100-fold lower than that of the previous ELISA for determining Psi. Extracts of dried mushroom powders were mixed with TBS triflate for 30 min at room temperature, converting Psi into TBS/Psi in approximately 50% yield. The reaction mixture was then subjected to an ELISA using the anti-TBS/Psi mAb to determine TBS/Psi. Psilocybe cubensis, a species of hallucinogenic mushrooms, gave rise to positive signals, indicating the presence of Psi therein in the expected quantity, while no detectable response was observed for four kinds of edible mushrooms available in the markets.

[NMR Study of the Discrimination of Enantiomers of Methamphetamine and Its Raw Materials Using Chiral Solvating Agents].

Some controlled substances, such as stimulants and narcotics, have asymmetric carbons in their molecules. Because the enantiomers do not always show the same pharmacological effects, and there are substances with different controls due to differences in their stereochemistry, a simple and unambiguous method for assessment of the composition of enantiomers is necessary. In this study, to develop a simple and rapid stereoscopic identification method for methamphetamine and its raw materials (ephedrine and pseudoephedrine), the 1H-NMR method was studied using three commercially available chiral solvating agents (CSAs); 1,1'-bi(2-naphthol)(BINOL), 2,2,2-trifluoro-1-(9-anthryl)ethanol (TFAE) and α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA). In addition, the accuracy of the optical purity, which was measured using samples mixed with enantiomers in various ratios, was investigated. The NMR peaks of the enantiomers were separated by adding (R)- or (S)-form of BINOL, TFAE or MTPA to the chloroform-d solution of methamphetamine, ephedrine or pseudoephedrine. A sufficient discrimination of enantiomers was obtained by adding about 10 equal amounts of each CSA to the solutions. With regard to the optical purity, it was possible to determine accurately the mixing of small amounts of enantiomers of about 5% even if the NMR peaks did not reach the baseline separation, when impurity peaks do not overlap. This method will be one of the useful techniques for the rapid and simple discrimination of enantiomers of illegal methamphetamine and its raw materials.

[Discrimination of Kratom Products by an Improved PCR-RFLP Method].

In Japan, mitragynine, 7-hydroxymitragynine and Mitragyna speciosa Korth. (M. speciosa, "Kratom") were controlled as Designated Substances under the Pharmaceutical and Medical Device Act from March 2016. In this study, the origins of 16 Kratom products obtained from the illegal drug market in Japan were investigated by DNA analyses and LC-MS analyses. When the PCR-restriction fragment length polymorphism (RFLP) was performed using the restriction enzyme XmaI (as reported by Sukrong et al. to be able to distinguish M. speciosa), the same DNA fragment patterns were obtained from all 16 products. On the other hand, as a result of the identification of the plant species of each product by nucleotide sequence analyses, the sequences of M. speciosa were detected in only 14 products. Despite the facts that mitragynine and 7-hydroxymitragynine were detected also in the other two products by the LC-MS analyses, M. speciosa DNAs were not amplified from these products by the PCR. Moreover, the DNA amplicons of the other psychotropic plant (Mesembryanthemum sp., e.g. "Kanna") were detected. This plant PCR amplicon has the restriction site for the XmaI at the same position of the M. speciosa PCR amplicon and it is difficult to distinguish "Kratom" and "Kanna" by the conventional PCR-RFLP. When the restriction enzyme XhoI was used simultaneously with the Xmal, the specific DNA fragment was only observed from the M. speciosa amplicon and it was possible to distinguish both species using this improved PCR-RFLP method. This method is useful to identify the origin of Kratom products distributed in the illegal drug market.

[Identification of LSD Derivatives, 1cP-LSD, MIPLA and 1B-LSD in Illegal Products as Paper Sheet].

Lysergic acid diethylamide (LSD) is a hallucinogen, synthesized from ergot alkaloid, and controlled as a narcotic in Japan. Recently, LSD derivatives have appeared as designer drugs, all over the world. In previous study, we reported identification and analysis of four LSD derivatives in four paper sheet products. In this study, we detected three additional LSD derivatives from three paper sheet products, which were obtained from September 2019 to March 2020 in Japan. We extracted the compounds from paper sheet products with methanol for LC-MS, high-resolution MS and GC-MS analyses. The compounds were identified as 4-cyclopropionyl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1cP-LSD), N-methyl-N-isopropyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo-[4,3-fg]quinoline-9-carboxamide (MIPLA), 4-butyryl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1B-LSD), by GC-MS, LC-MS, LC-Q-TOF-MS and NMR analyses. As well as other N1-acylated LSD derivatives, 1cP-LSD and 1B-LSD were easily deacylated to LSD during GC-MS analysis, we have to be careful to analyze these compounds.

Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms.

Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20-20 or 0.040-2.0 µg/assay (limit of detection was 0.14 or 0.029 µg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

F-phenibut (ß-(4-Fluorophenyl)-GABA), a potent GABAB receptor agonist, activates an outward-rectifying K+ current and suppresses the generation of action potentials in mouse cerebellar Purkinje cells.

The GABA analog phenibut (ß-Phenyl-GABA) is a GABAB receptor agonist that has been licensed for various uses in Russia. Phenibut is also available as a dietary supplement from online vendors worldwide, and previous studies have indicated that phenibut overdose results in intoxication, withdrawal symptoms, and addiction. F-phenibut (ß-(4-Fluorophenyl)-GABA), a derivative of phenibut, has not been approved for clinical use. However, it is also available as a nootropic supplement from online suppliers. F-phenibut binds to GABAB with a higher affinity than phenibut; therefore, F-phenibut may lead to more serious intoxication than phenibut. However, the mechanisms by which F-phenibut acts on GABAB receptors and influences neuronal function remain unknown. In the present study, we compared the potency of F-phenibut, phenibut, and the GABAB agonist (±)-baclofen (baclofen) using in vitro patch-clamp recordings obtained from mouse cerebellar Purkinje cells slice preparations Our findings indicate that F-phenibut acted as a potent GABAB agonist. EC50 of outward current density evoked by the three GABAB agonists decreased in the following order: phenibut (1362 µM) > F-phenibut (23.3 µM) > baclofen (6.0 µM). The outward current induced by GABAB agonists was an outward-rectifying K+ current, in contrast to the previous finding that GABAB agonists activates an inward-rectifying K+ current. The K+ current recorded in the present study was insensitive to extracellular Ba2+, intra- or extracellular Cs+, and intra- or extracellular tetraethylammonium-Cl. Moreover, F-phenibut suppressed action potential generation in Purkinje cells. Thus, abuse of F-phenibut may lead to severe damage by inhibiting the excitability of GABAB-expressing neurons.

[Identification and Analysis of LSD Derivatives in Illegal Products as Paper Sheet].

To prevent the abuse of new psychoactive substances (NPS), a total of 2372 substances and two plants are controlled as "Designated Substances" in Japan as of September 2019. Although the distribution of these substances has decreased for the past three years, newly-emerged NPS are still being found. In this study, we detected four lysergic acid diethylamide (LSD) derivatives as designer drugs from four paper sheet products, which were obtained from 2014 to 2017 in Japan. The compounds were identified as 4-Acetyl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ALD-52), N,N,7-triethyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ETH-LAD), 7-Allyl-N,N-diethyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (AL-LAD), N,N-diethyl-7-methyl-4-propionyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1P-LSD), by GC-MS, LC-MS, LC-Q-TOF-MS and NMR analyses. Further, we studied the extraction methods of LSD derivatives from paper sheet, and the analytical conditions of GC-MS, LC-MS and LC-FL(fluorescence). Among LSD derivatives, 1P-LSD have been controlled as designated substances (Shitei Yakubutsu) under the Pharmaceutical and Medical Device Act in Japan since April 2016. For the legislation of the other derivatives identified in this study, the evaluation of their pharmacological properties are now in progress.

Elevation of endocannabinoids in the brain by synthetic cannabinoid JWH-018: mechanism and effect on learning and memory.

The impairment of learning and memory is a well-documented effect of both natural and synthetic cannabinoids. In the present study, we aimed to investigate the effect of acute administration of JWH-018, a synthetic cannabinoid, on the hippocampal metabolome to assess biochemical changes in vivo. JWH-018 elevated levels of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The increase of endocannabinoid levels in response to JWH-018 could be inhibited by co-administration of AM251, a CB1 receptor antagonist. Biochemical analyses revealed that this was the result of suppression of two hydrolases involved in endocannabinoid degradation (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL]). Additionally, we showed that JWH-018 causes a reduction in the levels of brain-derived neurotrophic factor (BDNF), which is known to modulate synaptic plasticity and adaptive processes underlying learning and memory. The decrease of BDNF following JWH-018 treatment was also rescued by co-administration of AM251. As both endocannabinoids and BDNF have been shown to modulate learning and memory in the hippocampus, the alteration of their levels in response to JWH-018 may explain the contribution of synthetic cannabinoids to impairment of memory.

Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay.

INTRODUCTION: In recent years, new psychoactive substances (NPS) have been widely distributed for abuse purposes. Effective measures to counter the spread of NPS are to promptly legislate them through the risk assessment. Phencyclidine analogues having inhibitory effects toward NMDA receptor (NMDAR) have recently emerged in Japan. Therefore, it is important to establish a high-throughput system for efficiently detecting NPS that can inhibit NMDAR activity. METHODS: Hippocampal neurons prepared from embryonic rats were incubated in 96-well microplates. After 3 weeks in vitro, cultured neurons were preincubated with phencyclidine (PCP) or PCP-analogues, including 3-methoxyphencyclidine (3-MeO-PCP) and 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo), and then treated with 100 µM glutamate for 10 min. After fixation, cultured neurons were immunostained with anti-drebrin and anti-MAP2 antibodies. The linear cluster density of drebrin along the dendrites was automatically quantified using a protocol that was originally developed by us. RESULTS: The high-throughput immunocytochemical assay, measuring drebrin cluster density of cultured neurons, demonstrated that glutamate-induced reduction of drebrin cluster density in 96-well plates is competitively inhibited by NMDAR antagonist, APV. The reduction was also antagonized by PCP, 3-MeO-PCP and 3-MeO-PCMo. The inhibitory activity of 3-MeO-PCMo was lower than that of PCP or 3-MeO-PCP, with IC50 values of 26.67 µM (3-MeO-PCMo), 2.02 µM (PCP) and 1.51 µM (3-MeO-PCP). DISCUSSION: The relative efficacy among PCP, 3-MeO-PCP and 3-MeO-PCMo calculated from IC50 are similar to those from Ki values. This suggests that the high-throughput imaging analysis is useful to speculate the Ki values of new PCP analogues without performing the kinetic studies.