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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., (© 2023 John Wiley & Sons Ltd.)

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-8β-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 N 1 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-d 4 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., (© 2023 John Wiley & Sons Ltd.)

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.

Since around 2021, products claiming to contain a Δ 9 -tetrahydrocannabinol (THC) analog with different lengths of alkyl chain at C-3 position have been sold on the internet in Japan. Δ 9 -THC has a pentyl group derived from the precursor olivetol at the C-3 position. These products include liquid cartridges for electronic cigarettes, herbal products, and gummy products. This study analyzed and determined the ingredients in five oil products distributed on the internet from 2022 to 2023 that claim to contain THC analogs. Samples of each product were used for GC-MS and LC-MS measurements. After isolating and purifying the unknown components from the products, structural analysis was performed by measuring 1 H, 13 C-NMR and various two-dimensional NMR [HH correlation spectroscopy (H-H COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY)]. The analysis identified Δ 8 -tetrahydrocannabivarin (THCV), Δ 9 -THCV, Δ 8 -tetrahydrocannabutol (THCB), Δ 9 -THCB, Δ 8 -tetrahydrocannabihexol (THCH), Δ 9 -THCH, Δ 8 -3-octyl-THC (THCjd) and Δ 9 -THCjd. These compounds were Δ 8 -THC or Δ 9 -THC analogs with different lengths of alkyl chain at C-3 position. Meanwhile, Δ 4(8) -iso-THCV and Δ 11 -THCB were identified as minor components of the product, and were considered to be the reaction byproducts of the synthesis of the Δ 8 -THC or Δ 9 -THC analogs. In the future, there are concerns about the distribution of products containing new THC analogs. Therefore, continuous provision monitoring of newly detected in the products is important.

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.

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 1 H, 13 C-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 (11β-hexahydrocannabinol; 11β-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 (11β-hexahydrocannabiphorol; 11β-HHCP) and rel-(6aR, 9S, 10aR)-hexahydrocannabiphorol (11α-hexahydrocannabiphorol; 11α-HHCP)., Conclusions: The presence of both 11β-HHC and 11α-HHC in the HHC products analyzed in this study suggests that they were most likely synthesized via the reduction reaction of Δ 8 -THC or Δ 9 -THC. Dihydro-iso-THC was probably obtained as a byproduct of the synthesis of Δ 8 -THC or Δ 9 -THC from cannabidiol. Similarly, 11β-HHCP and 11α-HHCP in the HHCP product could stem from Δ 9 -tetrahydrocannabiphorol., (© 2023. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

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., (© 2023. The Author(s).)

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), Δ 9 -tetrahydrocannabinol (Δ 9 -THC), cannabidiol acid (CBDA), cannabidiol (CBD), Δ 8 -tetrahydrocannabinol (Δ 8 -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, Δ 9 -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, Δ 9 -THCA, CBD, Δ 9 -THC, CBC, and CBG were mainly detected in bracts, buds, and leaves.

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 (IC 50 s) of the methoxetamine-related compounds for NMDARs using NMDAR-expressing cartwheel interneurons of mice and patch-clamp recordings. We found that the IC 50 s 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., Competing Interests: Declarations of interest None., (Copyright © 2022 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

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 ( M r < 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 ( K d = 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 β-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.

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.

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 ( M r 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 ( K a = 3.6 × 10 7 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.

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 1 H-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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

The GABA analog phenibut (β-Phenyl-GABA) is a GABA B 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 (β-(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 GABA B 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 GABA B receptors and influences neuronal function remain unknown. In the present study, we compared the potency of F-phenibut, phenibut, and the GABA B 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 GABA B agonist. EC 50 of outward current density evoked by the three GABA B agonists decreased in the following order: phenibut (1362 μM) > F-phenibut (23.3 μM) > baclofen (6.0 μM). The outward current induced by GABA B agonists was an outward-rectifying K + current, in contrast to the previous finding that GABA B agonists activates an inward-rectifying K + current. The K + current recorded in the present study was insensitive to extracellular Ba 2+ , 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 GABA B -expressing neurons., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

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.

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 IC 50 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 IC 50 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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Context: Despite widespread use of diverse synthetic cannabinoid (sCB) compounds, the pathophysiology associated with intoxication with many sCB compounds, including AB-CHMINACA, is poorly understood, as is their metabolism and distribution into blood and organs., Case Details: A young man died shortly after ingesting an herb product containing sCB compounds. Toxicological analyses of blood samples revealed high levels of AB-CHMINACA (7.61 ± 0.59 ng/mL) and its metabolites (M2, 56.73 ± 4.16 ng/mL; M4, 2.29 ± 0.14 ng/mL) and trace amounts of 5-fluoro-AMB, FUB-PB-22, and AB-FUBINACA. The autopsy revealed severe pulmonary edema, and histology showed air bubbles in the alveolar effusion, suggesting rapid progression of edema. Low blood levels of N-terminal pro-brain natriuretic peptide excluded cardiogenic pulmonary edema. Histological examination revealed diffuse neuronal (brain) and myocardial (sub-endocardial) hyper-eosinophilia, indicating hypoxic encephalopathy and systemic hypoxemia, respectively., Conclusions: The findings show that AB-CHMINACA induced rapid progression of pulmonary edema resulting in hypoxic encephalopathy and systemic hypoxemia, possibly through severe seizures. The high blood ratio of the M2 metabolite to the parent compound, AB-CHMINACA, demonstrates rapid metabolism. This highlights the usefulness of quantification of M2 in diagnosing AB-CHMINACA intoxication.

Natural cannabinoids and their synthetic substitutes are the most widely used recreational drugs. Numerous clinical cases describe acute toxic symptoms and neurological consequences following inhalation of the mixture of synthetic cannabinoids known as "Spice." Here we report that an intraperitoneal administration of the natural cannabinoid Δ 9 -tetrahydrocannabinol (10 mg/kg), one of the main constituent of marijuana, or the synthetic cannabinoid JWH-018 (2.5 mg/kg) triggered electrographic seizures in mice, recorded by electroencephalography and videography. Administration of JWH-018 (1.5, 2.5 and 5 mg/kg) increased seizure spikes dose-dependently. Pretreatment of mice with AM-251 (5 mg/kg), a cannabinoid receptor 1-selective antagonist, completely prevented cannabinoid-induced seizures. These data imply that abuse of cannabinoids can be dangerous and represents an emerging public health threat. Additionally, our data strongly suggest that AM-251 could be used as a crucial prophylactic therapy for cannabinoid-induced seizures or similar life-threatening conditions.

N -Benzyl-substituted 2C class phenethylamines (NBOMes) are psychoactive designer drugs, with strong hallucinogenic and stimulant effects, even at low doses. The designer drug, 2-(4-bromo-2, 5-dimethoxyphenyl)- N -(2-methoxybenzyl) ethanamine (25B-NBOMe) is considered to be one of the most potent agonists of the serotonin-2A (5-HT 2A ) receptor. Recently, we reported the first lethal case of 25B-NBOMe intoxication with severe rhabdomyolysis, concluded by clinical, pathological and toxicological analyses. There are currently no good animal models that closely recapitulate serotonin receptor-dependent rhabdomyolysis. In the present study, we created animal models of rhabdomyolysis using zebrafish larvae to study the pathomechanism of rhabdomyolysis, and demonstrated that 25B-NBOMe can simulate lethal rhabdomyolysis in this animal. Treatment of the larvae with 25B-NBOMe decreased their survival rate, locomotion, altered birefringence of the skeletal muscle and immunostainings for dystroglycan (a myoseptal protein) and myosin heavy chain (a myofibril protein), which were consistent with rhabdomyolysis. This 25B-NBOMe-induced rhabdomyolysis was inhibited by the 5-HT 2A receptor antagonists ritanserin and aripirazole, but not by the 5-HT 1A  + 5-HT 1B receptor antagonist propranolol and the 5-HT 3 receptor antagonist granisetron, indicating 5-HT 2A -dependent rhabdomyolysis. The 25B-NBOMe-treated zebrafish is, therefore, a highly useful model of rhabdomyolysis for studying the pathomechanism of rhabdomyolysis as well as for therapeutic drug screening.

Considering the pharmacological effects of chiral drugs, enantiopure drugs may differ from their racemic mixture formulation in efficacy, potency, or adverse effects. Levomethorphan (LVM) and Dextromethorphan (DXM) act on the central nervous system and exhibit different pharmacological features. LVM, the l-stereoisomer of methorphan, shows many similarities to opiates such as heroin, morphine and codeine, including the potential for addiction, while the d-stereoisomer, DXM, does not have the same opioid effect. In the present study, NMR-based metabolomics were performed on the urine of rats treated with these stereoisomers, and showed significant differences in metabolic profiles. In urine within 24 h after treatment of these samples, levels of citrate, 2-oxoglutarate, creatine, and dimethylglycine were higher in LVM-treated rats than in DXM-treated rats. While urinary levels of hippurate and creatinine gradually increased over 72 h in DXM-treated rats, these metabolites were decreased in the urine by 48-72 h after treatment with LVM. The levels of these changed metabolites may provide the first evidence for different cellular responses to the metabolism of stereoisomers.

A woman in her thirties was found dead on a bed. Considerable amounts of "aroma liquid" and "bath salt" products and hypnotic drug tablets were scattered beside the bed. Autopsy showed pulmonary congestion and edema. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses of "aroma liquid" and "bath salt" products showed the presence of new cathinone designer drugs, 4-methoxy PV8 (4-methoxy PHPP), PV9 (α-POP), and 4-methoxy PV9 (4-methoxy α-POP), and a dissociative agent, diphenidine. Drug screening in stomach contents, blood and hydrolyzed urine of the woman by GC-MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the presence of the above 4 types of drugs and 3 types of benzodiazepines, triazolam, flunitrazepam, and nitrazepam, and their metabolites. The above 7 drugs and 3 benzodiazepine metabolites were simultaneously determined by LC-MS/MS after modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction using diazepam-d5 as the internal standard. The concentrations of 4-methoxy PV8, PV9, 4-methoxy PV9, and diphenidine in the femoral blood were 2.69, 0.743, 0.261, and 1.38μg/ml, respectively, which were significantly higher than concentrations reported in previous cases. Alcohol concentration in the femoral blood was 1.52mg/ml. Based on the pathological and toxicological findings, the cause of death was determined to be 3 types of cathinone drugs, 4-methoxy PV8, PV9, and 4-methoxy PV9, and diphenidine poisoning under the influence of 3 benzodiazepines and alcohol., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Herbal products containing synthetic cannabinoids-initially sold as legal alternatives to marijuana-have become major drugs of abuse. Among the synthetic cannabinoids, [1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone (MAM-2201) has been recently detected in herbal products and has psychoactive and intoxicating effects in humans, suggesting that MAM-2201 alters brain function. Nevertheless, the pharmacological actions of MAM-2201 on cannabinoid receptor type 1 (CB1R) and neuronal functions have not been elucidated. We found that MAM-2201 acted as an agonist of human CB1Rs expressed in AtT-20 cells. In whole-cell patch-clamp recordings made from Purkinje cells (PCs) in slice preparations of the mouse cerebellum, we also found that MAM-2201 inhibited glutamate release at parallel fiber-PC synapses via activation of presynaptic CB1Rs. MAM-2201 inhibited neurotransmitter release with an inhibitory concentration 50% of 0.36 μM. MAM-2201 caused greater inhibition of neurotransmitter release than Δ(9)-tetrahydrocannabinol within the range of 0.1-30 μM and JWH-018, one of the most popular and potent synthetic cannabinoids detected in the herbal products, within the range of 0.03-3 μM. MAM-2201 caused a concentration-dependent suppression of GABA release onto PCs. Furthermore, MAM-2201 induced suppression of glutamate release at climbing fiber-PC synapses, leading to reduced dendritic Ca(2+) transients in PCs. These results suggest that MAM-2201 is likely to suppress neurotransmitter release at CB1R-expressing synapses in humans. The reduction of neurotransmitter release from CB1R-containing synapses could contribute to some of the symptoms of synthetic cannabinoid intoxication including impairments in cerebellum-dependent motor coordination and motor learning., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Methylone (2-methylamino-1-[3,4-methylenedioxy-phenyl]propan-1-one), an amphetamine analog, has emerged as a popular drug of abuse worldwide. Methylone induces hyperthermia, which is thought to contribute toward the lethal consequences of methylone overdose. Methylone has been assumed to induce hyperthermic effects through inhibition of serotonin and/or dopamine transporters (SERT and DAT, respectively). To examine the roles of each of these proteins in methylone-induced toxic effects, we used SERT and DAT knockout (KO) mice and assessed the hyperthermic and lethal effects caused by a single administration of methylone. Methylone produced higher rates of lethal toxicity compared with other amphetamine analogs in wild-type mice. Compared with wild-type mice, lethality was significantly lower in DAT KO mice, but not in SERT KO mice. By contrast, only a slight diminution in the hyperthermic effects of methylone was observed in DAT KO mice, whereas a slight enhancement of these effects was observed in SERT KO mice. Administration of the selective D1 receptor antagonist SCH 23390 and the D2 receptor antagonist raclopride reduced methylone-induced hyperthermia, but these drugs also had hypothermic effects in saline-treated mice, albeit to a smaller extent than the effects observed in methylone-treated mice. In contradistinction to 3,4-methylenedioxymethamphetamine, which induces its toxicity through SERT and DAT, these data indicate that DAT, but not SERT, is strongly associated with the lethal toxicity produced by methylone, which did not seem to be dependent on the hyperthermic effects of methylone. DAT is therefore a strong candidate molecule for interventions aimed at preventing acute neurotoxic and lethal effects of methylone.

Recently, illegal herbal or liquid products containing psychoactive compounds have been a serious problem damaging human health and causing numerous traffic accidents. Reports indicate that most of those herbal products contain various types of synthetic cannabinoids. There are many on-site drug-testing devices; however, synthetic cannabinoids are not targeted compounds for such devices. In this study, we evaluated the on-site drug-testing device "K2/Spice Test" for the detection of 12 different types of 38 synthetic cannabinoids (including 13 naphthoylindole-type synthetic cannabinoids) and a natural cannabinoid (Δ(9)-tetrahydrocannabinol). Although this device is primarily used for the detection of metabolites of naphthoylindole-type synthetic cannabinoids in urine samples, we applied it to detect synthetic cannabinoids in illegal herbal products for rapid screening analyses. As a result of the on-site examination of synthetic cannabinoids, 10 naphthoylindole-type synthetic cannabinoids [five narcotics (JWH-018, JWH-073, AM-2201, MAM-2201, and JWH-122); five designated substances (JWH-015, JWH-200, AM-1220, JWH-019, and JWH-020)], and two other types of synthetic cannabinoid [designated substances (a benzoylindole AM-694 and a naphthoylnaphthalene CB-13)] showed positive results (the limit of detection ranged from 50 to 250 μg/mL). Furthermore, MeOH extracts of illegal herbal products containing naphthoylindole-type synthetic cannabinoids also showed positive results (the limit of detection ranged from 2.5 to 10 mg herbal products/mL). Therefore, we found that this device may be useful for the on-site examination of some naphthoylindole-type synthetic cannabinoids not only in urine samples but also in illegal herbal products.

Six new psychoactive substances were identified together with two other substances (compounds 1 - 8 ) in illegal products by our ongoing survey in Japan between January and July 2014. A new synthetic cannabinoid, FDU-NNEI [1-(4-fluorobenzyl)- N -(naphthalen-1-yl)-1 H -indole-3-carboxamide, 2 ], was detected with the newly distributed synthetic cannabinoid FDU-PB-22 ( 1 ). Two 2 H -indazole isomers of synthetic cannabinoids, AB-CHMINACA 2 H -indazole analog ( 3 ) and NNEI 2 H -indazole analog ( 4 ), were newly identified with 1 H -indazoles [AB-CHMINACA and NNEI indazole analog (MN-18)]. In addition, 2-methylpropyl N -(naphthalen-1-yl) carbamate ( 5 ) and isobutyl 1-pentyl-1 H -indazole-3-carboxylate ( 6 ) were detected in illegal products. Compound 6 is considered to be a by-product of the preparation of NNEI indazole analog from compound 5 and 1-pentyl-1 H -indazole. A phenethylamine derivative, N -OH-EDMA [ N -hydroxy-3,4-ethylenedioxy- N -methylamphetamine, 7 ], and a cathinone derivative, dimethoxy-α-PHP (dimethoxy-α-pyrrolidinohexanophenone, 8 ), were newly identified in illegal products. Among them, compounds 1 and 8 have been controlled as designated substances (Shitei-Yakubutsu) under the Pharmaceutical Affairs Law in Japan since August and November 2014, respectively.

A reliable method using supercritical fluid chromatography with mass spectrometry (SFC-MS) was developed for cannabinoids using compressed carbon dioxide (CO2) and methanol as the mobile-phase. The cannabinoids, i.e., cannabicyclohexanol (CCH: cis-isomer), trans-CCH, 5-(1,1-dimethylheptyl)-2-[(1R,3S)-3-hydroxycyclohexyl]-phenol (CP-47497), 5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol (CP-55940), 3-(1,1'-dimethylheptyl)-6aR,7,10,10aR-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol (HU-210), 2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol (CBD), (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-018), (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073) and 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), were determined within 12 min using a conventional column (2-EP) for SFC. Furthermore, two optical isomers of CCH and trans-CCH were completely and rapidly separated by a chiral stationary phase column (AMY1). A highly sensitive detection (0.002-3.75 ppb) was also obtained by these methods using 2-EP and AMY1 columns. These methods were applied to the qualitative and quantitative determination of cannabinoids in dried plant products. Although the concentration and species were different in the products, JWH-018, JWH-073 and CCH, including the cis-isomer, trans-isomer and the optical isomers, were detected in the products. Therefore, the proposed SFC-MS method seems to be useful as an alternative method to GC-MS and LC-MS for illegal drugs, such as cannabinoids.

Our continuous survey of illegal products in Japan revealed the new distribution of 15 designer drugs. We identified four synthetic cannabinoids, i.e., NNEI (1), 5-fluoro-NNEI (2), 5-chloro-NNEI (3) and NNEI indazole analog (4), and seven cathinone derivatives, i.e., MPHP (5), α-PHPP (6), α-POP (7), 3,4-dimethoxy-α-PVP (8), 4-fluoro-α-PVP (9), α-ethylaminopentiophenone (10) and N-ethyl-4-methylpentedrone (11). We also determined LY-2183240 (12) and its 2'-isomer (13), which were reported to inhibit endocannabinoid uptake, a methylphenidate analog, 3,4-dichloromethylphenidate (14), and an MDA analog, 5-APDB (15). No chemical and pharmaceutical data for compounds 3, 4, 6 and 7 had been reported, making this the first report on these compounds., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

To counter the spread of the many analogues of psychoactive substances, the Pharmaceutical Affairs Law in Japan was amended in 2006 to establish a new category - Designated Substances - in order to more promptly control these drugs. As of March 2013, 106 substances (including one plant, Salvia divinorum) were listed in the category of Designated Substances, and 13 of them had had their category changed from Designated Substances into the much stricter category, Narcotics. However, new analogues of controlled substances, especially synthetic cannabinoids, appeared one-by-one since the new category was introduced. To avoid a cat-and-mouse game between regulators and illicit drug manufacturers, a comprehensive system (generic scheduling) for designating naphthoylindole-type synthetic cannabinoids, with particular substituents, was introduced into the Designated Substances in 2013. Since late 2012, the naphthoylindole-type compounds have been gradually replaced by other types of synthetic cannabinoids, such as cyclopropylmethanones, cannabimimetic carboxamide derivatives, adamanthoyl indoles, and cannabimimetic quinolinyl carboxylates. After the enforcement of the generic scheduling for designating naphthoylindoles in March 2013, these naphthoylindoles have been completely replaced by other types and have rarely been detected in the products. New types of psychoactive substances, including opioid receptor agonists (e.g. AH-7921, MT-45), hallucinogenic phenethylamines (e.g. NBOMe-type compounds), and thiophene derivatives (e.g. methiopropamine, α-PVT) have also appeared. The almost infinite possibilities of altered structures of chemicals make it difficult to carry out effective and exhaustive scheduling. To prevent the widespread distribution and abuse of these new psychoactive substances, continuous and dedicated monitoring for the emergence of these substances is necessary., (Copyright © 2013 John Wiley & Sons, Ltd.)

The metabolism by human liver microsomes of several new illicit drugs, that is, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3- carboxamide (ADB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1- (4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC), quinolin-8-yl 1-(5-fluoropentyl)-(1H-indole)-3-carboxylate (5 F-QUPIC) and α-pyrrolidinovalerothiophenone (α-PVT), which have indole, indazole, quinolinol ester and thiophene structures, was investigated using reversed-phase chromatography and mass spectrometry. The present method is based upon the oxidation by cytochrome p450 superfamily enzymes in the microsomes. The oxidation of ADB-FUBINACA and AB-FUBINACA mainly occurred on the N-(1-amino-alkyl-1-oxobutan) moiety. However, the oxidation of AB-PINACA seemed to occur on the 1-pentyl moiety. On the other hand, QUPIC and 5 F-QUPIC, which have a quinolinol ester structure, predominantly underwent a cleavage reaction to produce indoleacetic acid type metabolites. In contrast, the metabolism reaction of α-PVT was different from that of the other tested drugs, and various oxidation products were observed on the chromatograms. The obtained metabolites are not in conflict with the results predicted by MetaboLynx software. However, the exact structures of the metabolites, except for 1-pentyl-1H-indole-3-carboxylic acid (QUPIC metabolite) and 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid (5 F-QUPIC metabolite), are currently not proven, because we have no authentic compounds for comparison. The proposed approach using human liver microsome seems to provide a new technology for the prediction of possible metabolites occuring in humans., (Copyright © 2014 John Wiley & Sons, Ltd.)

In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.

In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

URB-754 (6-methyl-2-[(4-methylphenyl)amino]-1-benzoxazin-4-one) was identified as a new type of designer drug in illegal products. Though many of the synthetic cannabinoids detected in illegal products are known to have affinities for cannabinoid CB1/CB2 receptors, URB-754 was reported to inhibit an endocannabinoid deactivating enzyme. Furthermore, an unknown compound (N,5-dimethyl-N-(1-oxo-1-(p-tolyl)butan-2-yl)-2-(N'-(p-tolyl)ureido)benzamide), which is deduced to be the product of a reaction between URB-754 and a cathinone derivative 4-methylbuphedrone (4-Me-MABP), was identified along with URB-754 and 4-Me-MABP in the same product. It is of interest that the product of a reaction between two different types of designer drugs, namely, a cannabinoid-related designer drug and a cathinone-type designer drug, was found in one illegal product. In addition, 12 cannabimimetic compounds, 5-fluoropentyl-3-pyridinoylindole, JWH-307, JWH-030, UR-144, 5FUR-144 (synonym: XLR11), (4-methylnaphtyl)-JWH-022 [synonym: N-(5-fluoropentyl)-JWH-122], AM-2232, (4-methylnaphtyl)-AM-2201 (MAM-2201), N-(4-pentenyl)-JWH-122, JWH-213, (4-ethylnaphtyl)-AM-2201 (EAM-2201) and AB-001, were also detected herein as newly distributed designer drugs in Japan. Furthermore, a tryptamine derivative, 4-hydroxy-diethyltryptamine (4-OH-DET), was detected together with a synthetic cannabinoid, APINACA, in the same product., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In recent years, many analogs of narcotics have been widely distributed as easily available psychotropic substances and have become a serious problem in Japan. To counter the spread of these non-controlled substances, the Pharmaceutical Affairs Law in Japan was amended in 2006 to establish a new category; Designated Substances in order to more strictly control these substances. In April 2007, 31 compounds and 1 plant were first controlled as Designated Substances. Before 2007, the major compounds distributed in the Japanese illegal drug market were tryptamines, phenethylamines and piperazines. Alkyl nitrites, such as isobutyl nitrite and isopentyl nitrite, were also widely distributed. After they were listed as Narcotics or Designated Substances in 2007, these compounds, especially the tryptamines, quickly disappeared from the market. In their place, cathinone derivatives have been widely distributed, as well as different phenethylamines and piperazines. Additionally, in recent years, new herbal products containing synthetic cannabinoids have appeared globally. As at July 2012, 78 substances (including 1 plant; Salvia divinorum) were listed in the category of Designated Substances. They were 13 tryptamines, 17 phenethylamines, 11 cathinones, 4 piperazines, 23 synthetic cannabinoids, 6 alkyl nitrites, 3 other compounds and 1 plant. In this review, we show our survey of the spread of new designer drugs in Japan, focusing especially on synthetic cannabinoids and cathinone derivatives. Also, the prevalence and legal status of these substances in other countries will be presented.

A rapid enantiomeric separation and simultaneous determination method based on ultra high performance liquid chromatography (UHPLC) was developed for phenethylamine-type abused drugs using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(-)-DBD-Py-NCS) as the chiral fluorescent derivatization reagent. The derivatives were rapidly enantiomerically separated by reversed-phase UHPLC using a column of 2.3-µm octadecylsilica (ODS) particles by isocratic elution with water-methanol or water-acetonitrile systems as the mobile phase. The proposed method was applied to the analysis of products containing illicit drugs distributed in the Japanese market. Among the products, 1-(3,4-methylenedioxyphenyl)butan-2-amine (BDB) and 1-(2-methoxy4,5-methylenedioxyphenyl)propan-2-amine (MMDA-2) were detected in racemic form. Furthermore, the method was successfully applied to the analysis of hair specimens from rats that were continuously dosed with diphenyl(pyrrolidin-2-yl)methanol (D2PM). Using UHPLC-fluorescence (FL) detection, (R)- and (S)-D2PM from hair specimens were enantiomerically separated and detected with high sensitivity. The detection limits of (R)- and (S)-D2PM were 0.12 and 0.21 ng/mg hair, respectively (signal-to-noise ratio (S/N) = 3)., (Copyright © 2012 John Wiley & Sons, Ltd.)

Several synthetic cannabinoids have recently been distributed as psychoactive adulterants in many herbal products on the illegal drug market around the world. However, there is little information on pharmacology and toxicology of such compounds. Although Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a psychoactive cannabinoid of marijuana, was reported to affect electroencephalograms (EEG) of rats, the effects of synthetic cannabinoids are unknown. We examined the pharmacological activities of three synthetic cannabinoids; cannabicyclohexanol (CCH), CP-47,497 and JWH-018; by analyzing EEG power spectra and locomotor activity after intraperitoneal administration to rats and compared them with those of Δ(9)-THC. The three compounds significantly increased the EEG power in the frequency range of 5.0-6.0 Hz for the first 3h, while Δ(9)-THC decreased the power spectra in the wide range of 7.0-20.0 Hz during the first hour. These results indicate that the effect of the three compounds on EEG is different from that of Δ(9)-THC. Additionally, CCH, CP-47,497 and JWH-018 significantly decreased the locomotor activity for 11.5h, 11h and 4.5h, respectively, after administration which was longer than that of Δ(9)-THC (3.5h). Furthermore, all three compounds significantly reduced the total amounts of locomotor activity during a 3-h, 6-h and 12-h period after injection, whereas no statistical difference was observed for the Δ(9)-THC injection. Among the three compounds, CCH and CP-47,497 exerted a longer duration of the change in the EEG power spectra and suppression of the locomotor activity than JWH-018., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

An HPLC-fluorescence detection method for simultaneous determination of N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP) labeled with 4-(4,5-diphenyl-1 H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was described. DIB-BZP and -TFMPP were well separated within 13 min without interference of peaks from plasma components. The lower detection limits of BZP and TFMPP at a signal-to-noise ratio of 3 were 0.9 and 4.6 ng/mL, respectively. Precisions of the proposed method for intra- and inter-day assays were less than 4.8 and 9.1% as %RSD (n =5). Furthermore, the method could be successfully applied to monitor both compounds in plasma after their sole or co-administration to rats (each dose, 2 mg/kg). Clearance of TFMPP was significantly different under the conditions (P=0.047)., (Copyright © 2011 John Wiley & Sons, Ltd.)

In recent years, many analogs of narcotics have been widely distributed as easily available psychotropic substances and have become a serious problem in Japan. To counter the spread of these non-controlled substances, the Pharmaceutical Affairs Law in Japan was amended in 2006 to establish a new category, "designated substances", to more strictly control these psychotropic substances. Fifty-one substances have been listed in this category as of December 2010. However, many new analogs have appeared, one after the other. Although the distribution of tryptamine-type designer drugs has decreased since the amendment of the law, the distribution of cathinone derivatives, as well as of phenetylamine-type and piperazine-type designer drugs, has increased. Moreover, non-controlled psychotropic plants have become popular in place of chemical psychotropic substances, which are now subject to stricter controls. Additionally, since 2008, new herbal products containing synthetic cannabinoids (for example, a brand named "Spice") have appeared. Sixteen synthetic cannabinoids, classified into four groups, have been detected in products purchased up to December 2010 via Japanese-based websites. The distribution of products containing the psychoactive substances described above (so-called "legal highs" in European countries) is a worldwide problem. In this review, we survey current trends in the abuse of psychotropic substances and plants in Japan, focusing especially on synthetic cannabinoids, cathinone derivatives and psychotropic plants., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid-acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.

Three indole alkaloids, voacamine (1), 3,6-oxidovoacangine (2), and a new alkaloid, 5-hydroxy-3,6-oxidovoacangine (3), isolated from Voacanga africana were found to exhibit potent cannabinoid CB1 receptor antagonistic activity. This is the first example of CB1 antagonists derived from natural alkaloids., (Copyright © 2011 Elsevier Ltd. All rights reserved.)