Analysis of Synthetic and Natural Cannabinoids in the Forensic Field Applying High-Resolution Mass Spectrometry

Multidimensional challenges arise in the field of forensic chemistry and toxicology from the ongoing emergence of synthetic cannabinoids (SCs) as well as the increasing legalization and medicalization of Cannabis sativa (C. sativa). This work addresses these challenges from different angles under the application of state-of-the-art mass spectrometry. “Phase I In vitro Metabolic Profiling of the Synthetic Cannabinoid Receptor Agonists CUMYL-THPINACA and ADAMANTYL-THPINACA” (study I) investigated the in vitro metabolic fate of two SCs. As data on the metabolism of newly emerging SCs is typically scarce, in vitro metabolism studies are required for the identification of suitable screening targets. The implementation of an in silico assisted workflow aided identification and structure elucidation of metabolites. It was observed that both SCs are vastly metabolized. Suitable screening targets were proposed. Additionally, investigation of the involved cytochrome P450 (CYP) isoenzymes gave valuable information on potential metabolic drug-drug adverse reactions and the potential influence of CYP polymorphisms. “Adulteration of low-delta-9-tetrahydrocannabinol products with synthetic cannabinoids: Results from drug checking services” (study II) presents data gained on the phenomenon of low THC cannabis products adulterated with SCs. Since 2020, such products have been increasingly detected in Switzerland and various European countries. The drug user’s unawareness about the presence of SCs combined with the typically higher potencies of SCs when compared to Δ9-tetrahydrocannabinol (THC) raised public health concerns. Cannabis samples and data on the drugs’ effects obtained from three drug checking services were investigated. A comprehensive screening method for SCs applying high resolution mass spectrometry (HRMS) was developed and validated. The carrier material was characterized regarding its THC and cannabidiol (CBD) contents. Data obtained from drug checking services included user self-reports on adverse effects after consumption of the respective adulterated and non-adulterated cannabis products. Increased risks for adverse effects, in particular cardiovascular and psychologic adverse effects, were found for products containing SCs when compared to regular cannabis products. The role of drug checking services as market monitoring tool and as source on effects of newly emerging new psychoactive substances (NPS) was highlighted. “Beyond Δ9-tetrahydrocannabinol and cannabidiol: Chemical differentiation of cannabis varieties applying targeted and untargeted analysis” (study III) presents the development and validation of a comprehensive analytical method for the determination of major and minor cannabinoids in cannabis inflorescences. Minor cannabinoids are gaining interest for various applications, ranging from improved product characterization and differentiation of cannabis varieties to bioanalytical questions in the medico-legal field. Samples derived from 18 cannabis varieties grown and stored under standardized conditions were characterized, applying the targeted and untargeted analyses using HRMS. Multivariate statistics, e.g., principal component analysis, were conducted to investigate similarities and differences between varieties. The presented methods allowed for a refined representation of chemical differences, i.e., chemical fingerprints, between varieties, expanding traditionally applied classification systems based on THC and CBD alone

Phase I In Vitro Metabolic Profiling of the Synthetic Cannabinoid Receptor Agonists CUMYL-THPINACA and ADAMANTYL-THPINACA

Synthetic cannabinoid receptor agonists (SCRAs) remain popular drugs of abuse. As many SCRAs are known to be mostly metabolized, in vitro phase I metabolic profiling was conducted of the two indazole-3-carboxamide SCRAs: CUMYL-THPINACA and ADAMANTYL-THPINACA. Both compounds were incubated using pooled human liver microsomes. The sample clean-up consisted of solid phase extraction, followed by analysis using liquid chromatography coupled to a high resolution mass spectrometer. In silico-assisted metabolite identification and structure elucidation with the data-mining software Compound Discoverer was applied. Overall, 28 metabolites were detected for CUMYL-THPINACA and 13 metabolites for ADAMATYL-THPINACA. Various mono-, di-, and tri-hydroxylated metabolites were detected. For each SCRA, an abundant and characteristic di-hydroxylated metabolite was identified as a possible in vivo biomarker for screening methods. Metabolizing cytochrome P450 isoenzymes were investigated via incubation of relevant recombinant liver enzymes. The involvement of mainly CYP3A4 and CYP3A5 in the metabolism of both substances were noted, and for CUMYL-THPINACA the additional involvement (to a lesser extent) of CYP2C8, CYP2C9, and CYP2C19 was observed. The results suggest that ADAMANTYL-THPINACA might be more prone to metabolic drug−drug interactions than CUMYL-THPINACA, when co-administrated with strong CYP3A4 inhibitors

Production and Quality Control of [¹⁷⁷Lu]Lu-PSMA-I&T: Development of an Investigational Medicinal Product Dossier for Clinical Trials

Since prostate cancer is the most commonly diagnosed malignancy in men, the theranostic approach has become very attractive since the discovery of urea-based PSMA inhibitors. Different molecules have been synthesized starting from the Glu-urea-Lys scaffold as the pharmacophore and then optimizing the linker and the chelate to improve functional characteristics. This article aimed to highlight the quality aspects, which could have an impact on clinical practice, describing the development of an Investigational Medicinal Product Dossier (IMPD) for clinical trials with [177Lu]Lu-PSMA-I&T in prostate cancer and other solid tumors expressing PSMA. The results highlighted some important quality issues of the final preparation: radiolabeling of PSMA-I&T with lutetium-177 needs a considerably longer time compared with the radiolabeling of the well-known [177Lu]Lu-PSMA-617. When the final product was formulated in saline, the stability of [177Lu]Lu-PSMA-I&T was reduced by radiolysis, showing a decrease in radiochemical purity (<95% in 24 h). Different formulations of the final product with increasing concentrations of ascorbic acid have been tested to counteract radiolysis and extend stability. A solution of 20 mg/mL of ascorbic acid in saline prevents radiolysis and ensures stability over 30 h

[18F]F-PSMA-1007 Radiolabelling without an On-Site Cyclotron: A Quality Issue

Radiopharmaceuticals targeting the prostate-specific membrane antigen (PSMA) has become the gold standard for PET imaging of prostate cancer. [68Ga]Ga-PSMA-11 has been the forerunner but a [18F]F-PSMA ligand has been developed because of the intrinsic advantages of Fluorine-18. Fluorine-18 labelled compounds are usually prepared in centers with an on-site cyclotron. Since our center has not an on-site cyclotron, we decided to verify the feasibility of producing the experimental 18F-labelled radiopharmaceutical [18F]F-PSMA-1007 with [18F]F- from different external suppliers. A quality agreement has been signed with two different suppliers, and a well-established and correctly implemented quality assurance protocol has been followed. The [18F]F- was produced with cyclotrons, on Nb target, but with different beam energy and current. Extensive validation of the [18F]F-PSMA-1007 synthesis process has been performed. The aim of this paper was the description of all the quality documentation which allowed the submission and approval of the Investigational Medicinal Product Dossier (IMPD) to the Competent Authority, addressing the quality problems due to different external suppliers. The result indicates that no significant differences have been found between the [18F]F- from the two suppliers in terms of radionuclidic and radiochemical purity and [18F]F- impacted neither the radiochemical yield of the labelling reaction nor the quality control parameters of the IMP [18F]F-PSMA-1007. These results prove how a correct quality assurance system can overcome some Regulatory Authorities issue that may represent an obstacle to the clinical use of F-18-labelled radiopharmaceuticals without an on-site cyclotron

Additional file 1: of Ex vivo immunosuppressive effects of mesenchymal stem cells on Crohn’s disease mucosal T cells are largely dependent on indoleamine 2,3-dioxygenase activity and cell-cell contact

Mesenchymal stem cells with T cells from inflamed Crohn’s mucosa. Representative time-lapse imaging of a co-culture with bone marrow-derived mesenchymal stem cells of a healthy donor and T cells isolated from inflamed mucosa of a patient with Crohn’s disease. A dynamic and intense interaction is clearly evident mostly during the first 6 h, when the binding between these two cell populations is non-fixed but instead appears as a continuous attachment and detachment, after which a growing number of cells with morphological features of apoptosis (those with nuclear fragmentation and cellular swelling) and apoptotic bodies become evident. The images were serially recorded every 30 min for 12 h. Magnification: 100×. (ZIP 6780 kb

Annotated corpora and tools of the PARSEME Shared Task on Automatic Identification of Verbal Multiword Expressions (edition 1.0)

The PARSEME shared task aims at identifying verbal MWEs in running texts. Verbal MWEs include idioms (let the cat out of the bag), light verb constructions (make a decision), verb-particle constructions (give up), and inherently reflexive verbs (se suicider 'to suicide' in French). VMWEs were annotated according to the universal guidelines in 18 languages. The corpora are provided in the parsemetsv format, inspired by the CONLL-U format. For most languages, paired files in the CONLL-U format - not necessarily using UD tagsets - containing parts of speech, lemmas, morphological features and/or syntactic dependencies are also provided. Depending on the language, the information comes from treebanks (e.g., Universal Dependencies) or from automatic parsers trained on treebanks (e.g., UDPipe). This item contains training and test data, tools and the universal guidelines file

PARSEME corpora annotated for verbal multiword expressions (version 1.3)

This multilingual resource contains corpora in which verbal MWEs have been manually annotated. VMWEs include idioms (let the cat out of the bag), light-verb constructions (make a decision), verb-particle constructions (give up), inherently reflexive verbs (help oneself), and multi-verb constructions (make do). This is the first release of the corpora without an associated shared task. Previous version (1.2) was associated with the PARSEME Shared Task on semi-supervised Identification of Verbal MWEs (2020). The data covers 26 languages corresponding to the combination of the corpora for all previous three editions (1.0, 1.1 and 1.2) of the corpora. VMWEs were annotated according to the universal guidelines. The corpora are provided in the cupt format, inspired by the CONLL-U format. Morphological and syntactic information, ­­­­including parts of speech, lemmas, morphological features and/or syntactic dependencies, are also provided. Depending on the language, the information comes from treebanks (e.g., Universal Dependencies) or from automatic parsers trained on treebanks (e.g., UDPipe). All corpora are split into training, development and test data, following the splitting strategy adopted for the PARSEME Shared Task 1.2. The annotation guidelines are available online: https://parsemefr.lis-lab.fr/parseme-st-guidelines/1.3 The .cupt format is detailed here: https://multiword.sourceforge.net/cupt-format

Effects on the incidence of cardiovascular events of the addition of pioglitazone versus sulfonylureas in patients with type 2 diabetes inadequately controlled with metformin (TOSCA.IT): a randomised, multicentre trial

Background The best treatment option for patients with type 2 diabetes in whom treatment with metformin alone fails to achieve adequate glycaemic control is debated. We aimed to compare the long-term effects of pioglitazone versus sulfonylureas, given in addition to metformin, on cardiovascular events in patients with type 2 diabetes. Methods TOSCA.IT was a multicentre, randomised, pragmatic clinical trial, in which patients aged 50\ue2\u80\u9375 years with type 2 diabetes inadequately controlled with metformin monotherapy (2\ue2\u80\u933 g per day) were recruited from 57 diabetes clinics in Italy. Patients were randomly assigned (1:1), by permuted blocks randomisation (block size 10), stratified by site and previous cardiovascular events, to add-on pioglitazone (15\ue2\u80\u9345 mg) or a sulfonylurea (5\ue2\u80\u9315 mg glibenclamide, 2\ue2\u80\u936 mg glimepiride, or 30\ue2\u80\u93120 mg gliclazide, in accordance with local practice). The trial was unblinded, but event adjudicators were unaware of treatment assignment. The primary outcome, assessed with a Cox proportional-hazards model, was a composite of first occurrence of all-cause death, non-fatal myocardial infarction, non-fatal stroke, or urgent coronary revascularisation, assessed in the modified intention-to-treat population (all randomly assigned participants with baseline data available and without any protocol violations in relation to inclusion or exclusion criteria). This study is registered with ClinicalTrials.gov, number NCT00700856. Findings Between Sept 18, 2008, and Jan 15, 2014, 3028 patients were randomly assigned and included in the analyses. 1535 were assigned to pioglitazone and 1493 to sulfonylureas (glibenclamide 24 [2%], glimepiride 723 [48%], gliclazide 745 [50%]). At baseline, 335 (11%) participants had a previous cardiovascular event. The study was stopped early on the basis of a futility analysis after a median follow-up of 57\uc2\ub73 months. The primary outcome occurred in 105 patients (1\uc2\ub75 per 100 person-years) who were given pioglitazone and 108 (1\uc2\ub75 per 100 person-years) who were given sulfonylureas (hazard ratio 0\uc2\ub796, 95% CI 0\uc2\ub774\ue2\u80\u931\uc2\ub726, p=0\uc2\ub779). Fewer patients had hypoglycaemias in the pioglitazone group than in the sulfonylureas group (148 [10%] vs 508 [34%], p&lt;0\uc2\ub70001). Moderate weight gain (less than 2 kg, on average) occurred in both groups. Rates of heart failure, bladder cancer, and fractures were not significantly different between treatment groups. Interpretation In this long-term, pragmatic trial, incidence of cardiovascular events was similar with sulfonylureas (mostly glimepiride and gliclazide) and pioglitazone as add-on treatments to metformin. Both of these widely available and affordable treatments are suitable options with respect to efficacy and adverse events, although pioglitazone was associated with fewer hypoglycaemia events. Funding Italian Medicines Agency, Diabete Ricerca, and Italian Diabetes Society