Intrinsically disordered region of talin's FERM domain functions as an initial PIP2 recognition site.

Focal adhesions (FAs) mediate the interaction of the cytoskeleton with the extracellular matrix in a highly dynamic fashion. Talin is a central regulator, adaptor protein, and mechano-sensor of FA complexes. For recruitment and firm attachment at FAs, talin's N-terminal FERM domain binds to phosphatidylinositol 4,5-bisphosphate (PIP2)-enriched membranes. A newly published autoinhibitory structure of talin-1, where the known PIP2 interaction sites are covered up, lead us to hypothesize that a hitherto less examined loop insertion of the FERM domain acts as an additional and initial site of contact. We evaluated direct interactions of talin-1 with a PIP2 membrane by means of atomistic molecular dynamics simulations. We show that this unstructured, 33-residue-long loop strongly interacts with PIP2 and can facilitate further membrane contacts, including the canonical PIP2 interactions, by serving as a flexible membrane anchor. Under force as present at FAs, the extensible FERM loop ensures talin maintains membrane contacts when pulled away from the membrane by up to 7 nm. We identify key basic residues of the anchor mediating the highly dynamic talin-membrane interaction. Our results put forward an intrinsically disordered loop as a key and highly adaptable PIP2 recognition site of talin and potentially other PIP2-binding mechano-proteins.

Longitudinal strand ordering leads to shear thinning in Nafion.

Proton-exchange membrane fuel cells (PEMFC) offer a promising energy generation alternative for a wide range of technologies thanks to their ecological friendliness and unparalleled efficiency. At the heart of these electrochemical cells lies the membrane electrode assembly with its most important energy conversion components, the Proton Exchange Membrane. This component is created through the use of printing techniques and Nafion inks. The physicochemical properties of the ink, such as its viscosity under shear, are critical for the finished product. In this work we present non-equilibrium Molecular Dynamics simulations using a MARTINI based coarse-grained model for Nafion to understand the mechanism governing the shear viscosity of Nafion solutions. By simulating a Couette flow and calculating density maps of the Nafion chains in these simulations we shed light on the process that leads to the experimentally observed shear thinning effects of Nafion solutions under flow. We observe rod-shaped Nafion microstructures, 3 nm in size on average, when shear flow is absent or low. Higher shear rates instead break these structures and align Nafion strands along the direction of the flow, resulting in lower shear viscosities. Our work paves the way for a deeper understanding of the dynamic and mechanical properties of Nafion including studies of more complex CL and PEM inks.

Phase I metabolic profiling of the synthetic cannabinoids THJ-018 and THJ-2201 in human urine in comparison to human liver microsome and cytochrome P450 isoenzyme incubation.

Despite the increasing relevance of synthetic cannabinoids as one of the most important classes within "New Psychoactive Substances", there is still a lack of knowledge concerning their metabolism in humans. Due to the extensive metabolism of synthetic cannabinoids, metabolites are necessarily the best target analytes in urine, posing additional challenges to forensic analysis. The aims of this study were to identify appropriate urinary targets indicating intake of THJ-018 or THJ-2201 as well as to elucidate the most important cytochrome P450 isoenzymes within the metabolism of THJ-018 and THJ-2201 in vitro. For this purpose, the in vitro metabolism of THJ-018 and THJ-2201 was initially established using pooled human liver microsomes. The results obtained were compared to previously published in vitro results as well as to the results of the metabolic profiles from selected recombinant cytochrome P450 isoenzymes and from 23 urine samples from forensic cases. LC-HRMS was used to conduct product ion scans and to examine the metabolite spectra. For THJ-018, 17 different metabolite groups containing 33 different metabolites and isomers were detected after microsomal incubation, with the major metabolic pathways being monohydroxylation at the pentyl chain and of the naphthyl moiety as well as dihydroxylation of both residues. For THJ-2201, 19 different metabolite groups and 46 different metabolites and isomers were observed. The major metabolic pathways were monohydroxylation at the naphthyl moiety and oxidative defluorination. Significant contribution to the in vitro metabolism of THJ-018 and THJ-2201 originated from CYP2B6, CYP2C19, CYP3A4, and CYP3A5. As several cytochrome P450 isoenzymes are involved in the metabolism of these synthetic cannabinoids, a co-consumption with other drugs is unlikely to have an impact on their metabolism.

Activity-Based Detection of Consumption of Synthetic Cannabinoids in Authentic Urine Samples Using a Stable Cannabinoid Reporter System.

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first-line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation.

Immunoassay screening in urine for synthetic cannabinoids - an evaluation of the diagnostic efficiency.

BACKGROUND: The abuse of synthetic cannabinoids (SCs) as presumed legal alternative to cannabis poses a great risk to public health. For economic reasons many laboratories use immunoassays (IAs) to screen for these substances in urine. However, the structural diversity and high potency of these designer drugs places high demands on IAs regarding cross-reactivity of the antibodies used and detection limits. METHODS: Two retrospective studies were carried out in order to evaluate the capability of two homogenous enzyme IAs for the detection of currently prevalent SCs in authentic urine samples. Urine samples were analyzed utilizing a 'JWH-018' kit and a 'UR-144' kit. The IA results were confirmed by an up-to-date liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) screening method covering metabolites of 45 SCs. RESULTS: The first study (n=549) showed an 8% prevalence of SCs use (LC-MS/MS analysis) among inpatients of forensic-psychiatric clinics, whereas all samples were tested negative by the IAs. In a second study (n=200) the combined application of both IAs led to a sensitivity of 2% and a diagnostic accuracy of 51% when applying the recommended IA cut-offs. Overall, 10 different currently prevalent SCs were detected in this population. The results can be explained by an insufficient cross-reactivity of the antibodies towards current SCs in combination with relatively high detection limits of the IAs. CONCLUSIONS: In light of the presented study data it is strongly recommended not to rely on the evaluated IA tests for SCs in clinical or forensic settings. For IA kits of other providers similar results can be expected.

Detection and Activity Profiling of Synthetic Cannabinoids and Their Metabolites with a Newly Developed Bioassay.

Synthetic cannabinoids (SCs) are the largest group of compounds currently monitored in Europe by the EU Early Warning System on new psychoactive substances. Emerging recreational use of these products has led to multiple cases of adverse health effects and even death. In contrast to marijuana, where Δ9-tetrahydrocannabinol (Δ9THC) is metabolized to only one major active metabolite, it has been reported that several major phase I metabolites of SCs remain biologically active, exerting cannabinoid (CB) receptor affinity, potency, and efficacy greater than those of Δ9THC. It is therefore reasonable that more SCs can also be biotransformed into molecules with various levels of CB activity. Here, we developed and applied a new G-protein coupled receptor (GPCR) activation assay based on NanoLuc binary technology (Promega). More specifically, by demonstrating CB1 and CB2 receptor activation by JWH-018 and a selection of its metabolites, we are the first to show the suitability of the newly developed bioassay for monitoring GPCR-mediated activity. We also successfully applied this reporter system to evaluate the in vitro activity of JWH-122, JWH-210, and PB-22, their 5-fluoro analogues (MAM-2201, EAM-2201, and 5F-PB-22, respectively), and their main phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at cannabinoid receptors. All of these active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to its toxicity profile. We also demonstrate a proof of concept of the applicability of the newly developed bioassay for screening urine for CB receptor activity exerted by SCs.

Metabolites of synthetic cannabinoids in hair--proof of consumption or false friends for interpretation?

The detection of drug metabolites in hair is widely accepted as a proof for systemic uptake of the drug, unless the metabolites can be formed as artefacts. However, regarding synthetic cannabinoids, not much is known about mechanisms of incorporation into hair. For a correct interpretation concerning hair findings of these compounds and their metabolites, it is necessary to identify the different routes of incorporation and to assess their contribution to analytical findings. This study presents the results of the LC-ESI-MS/MS analysis of an authentic hair sample taken from a patient with a known history of heavy consumption of synthetic cannabinoids. In the authentic hair sample, 5F-PB-22 and AB-CHMINACA as well as their main metabolites 5F-PB-22 3-carboxyindole, PB-22 5-OH-pentyl, and AB-CHMINACA valine were detected in all segments, comprising segments grown in a time period where the substances had not been distributed on the 'legal high' market. To enable interpretation of the results regarding the distribution of the detected analytes along the hair shaft, the stability of 5F-PB-22 and AB-CHMINACA in hair matrix and under thermal stress was assessed. The stability tests revealed that the three 'metabolites' are also formed in externally contaminated hair after storage of the samples under different conditions. In addition, 5F-PB-22 3-carboxyindole and AB-CHMINACA valine were identified as degradation products in smoke condensate. Therefore, interpretation of 'metabolite' findings of compounds comprising chemically labile amide/ester bonds or 5-fluoro-pentyl side chains should be carried out with utmost care, taking into account the different mechanisms of formation and incorporation into hair.

Influence of adhesive point dimension and splint type on splint rigidity--evaluation by the dynamic Periotest method.

AIM: To evaluate the influence of adhesive point dimension and splint type on the rigidity of wire-composite splints in vitro. MATERIALS AND METHODS: A custom-made artificial model was used. The two central incisors served as injured teeth (degrees of loosening III and II) and the two lateral incisors as non-injured teeth (physiological mobility). Horizontal and vertical tooth mobilities were investigated before and after splinting with the Periotest(®) method; the percent change was taken as the relative splint effect. Teeth were splinted with three types of wire-composite splints: Dentaflex (0.45 mm), Strengtheners (0.8 × 1.8 mm), and Dentaflex completely covered with composite. Four adhesive point dimensions (2, 3, 4, and 5 mm) were evaluated. Normal distribution was tested with the Kolmogorov-Smirnov test. Differences were evaluated with the anova and post hoc tests for pair-wise comparisons. Significance level was set at 0.05. RESULTS: The adhesive point dimension did not influence splint rigidity, in general ( P  = 0.288). Significant effects were found in non-injured teeth with the Dentaflex ( P  < 0.001) and in injured teeth with the Strengtheners ( P  < 0.001). The Strengtheners splint rigidity increased significantly with increasing adhesive point dimensions. The three splints showed significantly different effects at 5-mm adhesive point dimension ( P  < 0.001). CONCLUSION: Splint rigidity for injured teeth was influenced by adhesive point dimension only when splinting with Strengtheners. We recommend adapting splint rigidity by selecting different wires and reducing the adhesive point dimension to a minimum. Dentaflex can be used for flexible splinting, Strengtheners, and composite covered Dentaflex for rigid splinting.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

Acute psychotropic, autonomic, and endocrine effects of 5,6-methylenedioxy-2-aminoindane (MDAI) compared with 3,4-methylenedioxymethamphetamine (MDMA) in human volunteers: A self-administration study.

The acute psychoactive, autonomic, and endocrine effects of the new psychoactive substance (NPS) 5,6-methylenedioxy-2-aminoindane (MDAI; 3.0 mg/kg, range 180-228 mg) were investigated in six healthy volunteers (four males, two females) in a non-blinded fashion without placebo. Subjective, cardiovascular, and endocrine responses were compared with two different doses of 3,4-methylenedioxymethamphetamine (MDMA) (75 mg and 125 mg) described in previously published placebo-controlled studies, which used identical outcome measures including Visual Analogue Scales (VAS), the Adjective Mood Rating Scale (AMRS), and the 5 Dimensions of Altered States of Consciousness (5D-ASC) scale. MDAI was well tolerated and produced subjective effects comparable with those of 125 mg MDMA. MDAI increased blood pressure similar to 125 mg MDMA but did not increase heart rate or body temperature. MDAI increased cortisol and prolactin levels and could be detected in serum about 20 min post ingestion and remained detectable at least for 4 days. In urine, MDAI was detectable over a period of at least 6 days. Further clinical investigations are warranted to assess whether MDAI could serve as drug with medicinal properties.

Advances in molecular simulations of protein mechanical properties and function.

Single-molecule force spectroscopy and classical molecular dynamics are natural allies. Recent advances in both experiments and simulations have increasingly facilitated a direct comparison of SMFS and MD data, most importantly by closing the gap between time scales, which has been traditionally at least 5 orders of magnitudes wide. In this review, we will explore these advances chiefly on the computational side. We focus on protein dynamics under force and highlight recent studies that showcase how lower loading rates and more statistics help to better interpret previous experiments and to also motivate new ones. At the same time, steadily increasing system sizes are used to mimic more closely the mechanical environment in the biological context. We showcase some of these advances on atomistic and coarse-grained scale, from asymmetric membrane tension to larger (multidomain/multimeric) protein assemblies under force.

Extraordinary long detection window of a synthetic cannabinoid metabolite in human urine - Potential impact on therapeutic decisions.

Synthetic cannabinoids (SCs) have become established drugs of abuse. They play an increasing role in drug therapy, where abstinence control testing is required. Differentiation between recent drug uptake and uptake in the distant past is important for drug therapy. This study aimed to evaluate the detection window of a metabolite commonly used as a consumption marker for AB-FUBINACA and AMB-FUBINACA (synonym: FUB-AMB) in urine analysis. The acidic hydrolysis metabolite was quantified in urine samples of a drug user by applying a validated analytical method. The concentration profile of the metabolite was correlated with usage data of the subject. Pharmacokinetic properties of AB-FUBINACA were collected by analysis of serum and urine samples from a controlled administration study (single oral ingestion of AB-FUBINACA). Thirteen urine samples were taken without advance notice over 2 years. The metabolite was detected in the first urine sample at 0.77 ng/mg creatinine and subsequently in concentrations ranging from 0.06 to 0.29 ng/mg creatinine. Usage data showed credible abstinence from SCs during this period. The pharmacokinetic properties observed within the controlled self-administration study supported the hypothesis of distribution into deeper compartments and long-lasting elimination (serum concentration-time curve showing biphasic kinetics). An elimination phase of over 1 year after the last drug uptake seems plausible in cases of extensive consumption. To avoid misinterpretation of positive findings, we recommend testing patients with known SC use at the beginning of the abstinence program and to re-test continuously at short time intervals. These data enable the correct interpretation of analytical findings.

Detection and phase I metabolism of the 7-azaindole-derived synthetic cannabinoid 5F-AB-P7AICA including a preliminary pharmacokinetic evaluation.

In June 2018, a 'research chemica'l labeled 'AB-FUB7AICA' was purchased online and analytically identified as 5F-AB-P7AICA, the 7-azaindole analog of 5F-AB-PINACA. Here we present data on structural characterization, suitable urinary consumption markers, and preliminary pharmacokinetic data. Structure characterization was performed by nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, infrared and Raman spectroscopy. Phase I metabolites were generated by applying a pooled human liver microsome assay (pHLM) to confirm the analysis results of authentic urine samples collected after oral self-administration of 2.5 mg 5F-AB-P7AICA. Analyses of pHLM and urine samples were performed by liquid chromatography-time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). An LC-MS/MS method for the quantification of 5F-AB-P7AICA in serum was validated. Ten phase I metabolites were detected in human urine samples and confirmed in vitro. The main metabolites were formed by hydroxylation, amide hydrolysis, and hydrolytic defluorination, though - in contrast with most other synthetic cannabinoids - the parent compound showed the highest signals in most urine samples. The compound detection window was more than 45 hours in serum. The concentration-time profile was best explained by a two-phase pharmacokinetic model. 5F-AB-P7AICA was detected in urine samples until 65 hours post ingestion. Monitoring of metabolite M07, hydroxylated at the alkyl chain, next to parent 5F-AB-P7AICA, is recommended to confirm the uptake of 5F-AB-P7AICA in urinalysis. It seems plausible that the shift of the nitrogen atom from position 2 to 7 (e.g. 5F-AB-PINACA to 5F-AB-P7AICA) leads to a lower metabolic reactivity, which might be of general interest in medicinal chemistry.

Human phase I metabolism of the novel synthetic cannabinoid 5F-CUMYL-PEGACLONE.

PURPOSE: 5F-CUMYL-PEGACLONE is a recently emerged γ-carbolinone derived synthetic cannabinoid. The present study aimed to identify phase I metabolites to reliably prove consumption of the substance by urine analysis and to differentiate from the uptake of the non-fluorinated analog CUMYL-PEGACLONE. METHODS: For metabolite characterization, phase I metabolites were analyzed by liquid chromatography-high resolution mass spectrometry after incubation with pooled human liver microsomes. Reliability of the biomarkers was evaluated by analysis of human urine samples (n = 20) by liquid chromatography-triple quadrupole tandem mass spectrometry. Sample preparation included ß-glucuronidase treatment followed by liquid-liquid extraction. RESULTS: In total, 15 metabolites were detected in vivo and characterized. Metabolic reactions were primarily observed at the γ-carbolinone core and the 5-fluoropentyl chain, and included N-dealkylation, hydroxylation, hydrolytic defluorination, formation of a dihydrodiol, oxidation to the pentanoic acid metabolite and formation of the propionic acid metabolite. Six of these metabolites were identical with phase I metabolites of CUMYL-PEGACLONE, which must be considered for interpretation of analytical findings in urine samples. CONCLUSIONS: 5F-CUMYL-PEGACLONE was subject to extensive metabolism in humans. The propionic acid metabolite was the most abundant metabolite in all urine samples and should be targeted when maximum sensitivity is needed (e.g., drug abstinence control). However, this metabolite also occurs in the biotransformation of the non-fluorinated analog and is, therefore, not a compound-specific marker. For differentiation, a metabolite hydroxylated at the γ-carbolinone core showed to be the most reliable marker and should be used as an additional target analyte.

5F-Cumyl-PINACA in 'e-liquids' for electronic cigarettes: comprehensive characterization of a new type of synthetic cannabinoid in a trendy product including investigations on the in vitro and in vivo phase I metabolism of 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA.

PURPOSE: In recent years e-liquids used in electronic cigarettes have become an attractive alternative to smoking tobacco. A new trend is the use of e-liquids containing synthetic cannabinoids (SCs) instead of smoking cannabis or herbal mixtures laced with SCs. In the frame of a systematic monitoring of the online market of 'legal high' products, e-liquids from online retailers who also sell herbal blends were bought. METHODS: The products were analyzed by gas chromatography-mass spectrometry. In some of the e-liquids an unknown compound was detected which was identified as the SC 5F-Cumyl-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) by nuclear magnetic resonance analysis. To investigate the phase I metabolism of this new class of compounds, 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA were incubated with pooled human liver microsomes (pHLM). Cumyl-PINACA was additionally ingested orally (0.6 mg) by a volunteer in a controlled self-experiment. To assess the relative potency of Cumyl-PINACA a set of SCs were characterized using a cAMP assay. RESULTS: Metabolism of 5F-Cumyl-PINACA and Cumyl-PINACA showed similarities with AM-2201 and JWH-018. The main metabolites were formed by hydroxylation at the N-pentyl side chain. The main metabolites detected in the volunteer's urine sample were the same as in the pHLM assay. All SCs tested with the cAMP assay were full agonists at the CB1 receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC50 value of 0.06 nM. CONCLUSIONS: The increasing popularity of e-liquids particularly among young people, and the extreme potency of the added SCs, pose a serious threat to public health. To our knowledge, this is the first report describing the tentative identification of human in vivo metabolites of Cumyl-PINACA and 5F-Cumyl-PINACA.

Stability of Biological Membranes upon Mechanical Indentation.

Mechanical perturbations are ubiquitous in living cells, and many biological functions are dependent on the mechanical response of lipid membranes. Recent force-spectroscopy studies have captured the stepwise fracture of stacks of bilayers, avoiding substrate effects. However, the effect of stacking bilayers, as well as the exact molecular mechanism of the fracture process, is unknown. Here, we use atomistic and coarse-grained force-clamp molecular dynamics simulation to assess the effects of mechanical indentation on stacked and single bilayers. Our simulations show that the rupture process obeys the laws of force-activated barrier crossing, and stacking multiple membranes stabilizes them. The rupture times follow a log-normal distribution which allows the interpretation of membrane rupture as a pore-growth process. Indenter hydrophobicity determines the type of pore formation, the preferred dwelling region, and the resistance of the bilayer against rupture. Our results provide a better understanding of the nanomechanics underlying the plastic rupture of lipid membranes.

Synthetic cannabinoids in hair - Pragmatic approach for method updates, compound prevalences and concentration ranges in authentic hair samples.

Since the first detection of synthetic cannabinoids (SCs) in so-called 'legal high' products (e.g. 'Spice') sold as legal alternatives to marihuana, the rapid development of this class of designer drugs poses a great challenge for analytical laboratories. The aim of this study was the comprehensive validation of an up-to-date LC-MS/MS method for detection of SCs in human hair for the purpose of drug abstinence testing and evaluation of a pragmatic re-validation approach for frequent method adaption. The validation demonstrated low quantification limits (0.5-5.0 pg mg-1) and acceptable selectivity, linearity, accuracy, and precision for 72 SCs. High matrix effects have been taken into consideration as a major limitation of the method. The partial re-validation approach proved to be an appropriate compromise between reduced validation effort and sufficient control of the method performance enabling analysts to keep pace with the dynamics of the drug market. The analysis of 294 authentic samples resulted in 163 positive samples and showed a broad concentration range (<1.0-5,700 pg mg-1) for 52 SCs in hair with up to 17 different compounds detected in a single hair sample. Periods of detection between one and 58 months were observed for single compounds in hair. Regarding the interpretation of analytical findings semi-quantitative concentrations were considered sufficient for a rough classification of the intensity of drug exposure in (i) passive exposure or exposure in the distant past (lower pg mg-1 range), (ii) more intense exposure (elevated concentration range, >20 pg mg-1 (upper 25th-percentile)), and (iii) heavy/recent exposure (>150 pg mg-1).

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL-PEGACLONE and detection in human urine samples.

Indole-, indazole-, or azaindole-based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl-derivative, CUMYL-PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ-carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography-tandem mass spectrometry and liquid chromatography-high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL-PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono-hydroxylated at the γ-carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL-PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross-reactivity.

Metabolism of Nine Synthetic Cannabinoid Receptor Agonists Encountered in Clinical Casework: Major in vivo Phase I Metabolites of AM-694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144 and XLR-11 in Human Urine Using LC-MS/MS.

BACKGROUND: `Herbal mixtures` containing synthetic cannabinoid receptor agonists (SCRAs) are promoted as legal alternative to marihuana and are easily available via the Internet. Keeping analytical methods for the detection of these SCRAs up-to-date is a continuous challenge for clinicians and toxicologists due to the high diversity of the chemical structures and the frequent emergence of new compounds. Since many SCRAs are extensively metabolized, analytical methods used for urine testing require previous identification of the major metabolites of each compound. OBJECTIVE: The aim of this study was to identify the in vivo major metabolites of nine SCRAs (AM- 694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144, XLR-11) for unambiguous detection of a drug uptake by analysis of urine samples. METHOD: Positive urine samples from patients of hospitals, detoxification and therapy centers as well as forensic-psychiatric clinics were analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LCqToF- MS) for investigation of the major in vivo metabolites. RESULTS: For all investigated SCRAs, monohydroxylation, dihydroxylation and/or formation of the Nhexanoic/ pentanoic acid metabolites were among the most abundant metabolites detected in human urine samples. Substitution of the fluorine atom was observed to be an important metabolic reaction for compounds carrying an N-(5-fluoropentyl) side chain. N-Dealkylated metabolites were not detected in vivo. CONCLUSION: The investigated metabolites facilitate the reliable detection of drug uptake by analysis of urine samples. For distinction between uptake of the fluorinated and the non-fluorinated analogs, the N-(4-hydroxypentyl) metabolite of the non-fluorinated analog was identified as a useful analytical target and consumption marker.

Phase I metabolism of the carbazole-derived synthetic cannabinoids EG-018, EG-2201, and MDMB-CHMCZCA and detection in human urine samples.

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG-018 (naphthalen-1-yl(9-pentyl-9H-carbazol-3-yl)methanone), EG-2201 ((9-(5-fluoropentyl)-9H-carbazol-3-yl)(naphthalen-1-yl)methanone), and MDMB-CHMCZCA (methyl 2-(9-(cyclohexylmethyl)-9H-carbazole-3-carboxamido)-3,3-dimethylbutanoate) are 3 representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-QToF-MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG-018 (n = 8). A hydroxypentyl metabolite, most probably the 4-hydroxypentyl isomer, and an N-dealkylated metabolite mono-hydroxylated at the carbazole core system were most abundant. In vitro studies of EG-018 and EG-2201 indicated that oxidative defluorination of the 5-fluoropentyl side chain of EG-2201 as well as dealkylation led to common metabolites with EG-018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG-018 and EG-2201 (n = 1) uptake is possible by the detection of compound-specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB-CHMCZCA users (n = 20), a metabolite mono-hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound-specific consumption marker while a biotransformation product of mono-hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance.