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Postmortem metabolomics holds promise for identifying crucial biological markers relevant to death investigations and clinical scenarios. We aimed to assess its applicability in diagnosing hypothermia, a condition lacking definitive biomarkers. Our retrospective analysis involved 1095 postmortem femoral blood samples, including 150 hypothermia cases, 278 matched controls, and 667 randomly selected test cases, analyzed using UHPLC-QTOF mass spectrometry. The model demonstrated robustness with an R2 and Q2 value of 0.73 and 0.68, achieving 94% classification accuracy, 92% sensitivity, and 96% specificity. Discriminative metabolite patterns, including acylcarnitines, stress hormones, and NAD metabolites, along with identified pathways, suggest that metabolomics analysis can be helpful to diagnose fatal hypothermia. Exposure to cold seems to trigger a stress response in the body, increasing cortisol production to maintain core temperature, possibly explaining the observed upregulation of cortisol levels and alterations in metabolic markers related to renal function. In addition, thermogenesis seems to increase metabolism in brown adipose tissue, contributing to changes in nicotinamide metabolism and elevated levels of ketone bodies and acylcarnitines, these findings highlight the effectiveness of UHPLC-QTOF mass spectrometry, multivariate analysis, and pathway identification of postmortem samples in identifying metabolite markers with forensic and clinical significance. The discovered patterns may offer valuable clinical insights and diagnostic markers, emphasizing the broader potential of postmortem metabolomics in understanding critical states or diseases.
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Biomarcadores , Hipotermia , Metabolómica , Humanos , Metabolómica/métodos , Biomarcadores/sangre , Masculino , Hipotermia/metabolismo , Hipotermia/diagnóstico , Femenino , Persona de Mediana Edad , Adulto , Anciano , Autopsia , Estudios Retrospectivos , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina/sangre , Cromatografía Líquida de Alta Presión , Espectrometría de Masas/métodosRESUMEN
Synthetic cannabinoids (SCs) remain a major public health concern, as they continuously are linked to severe intoxications and drug-related deaths worldwide. As new SCs continue to emerge on the illicit drug market, an understanding of SC metabolism is needed to identify formed metabolites that may serve as biomarkers in forensic toxicology screening and for understanding the pharmacokinetics of the drugs. In this work, the metabolism of ADB-4en-P-5Br-INACA and ADB-P-5Br-INACA ((S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-(pent-4-en-1-yl)-1H-indazole-3-carboxamide, (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-pentyl-1H-indazole-3-carboxamide respectively) were investigated using human hepatocytes in vitro and in-house synthesized references. Both SCs were incubated with pooled human hepatocytes over 3 h, with the aim to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total nine metabolites were identified for ADB-4en-P-5Br-INACA and 10 metabolites for ADB-P-5Br-INACA. The observed biotransformations included dihydrodiol formation, terminal amide hydrolysis, hydroxylation, dehydrogenation, carbonyl formation, glucuronidation, and combinations thereof. The major metabolites were confirmed by in-house synthesized references. Recommended biomarkers for ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA are the terminal hydroxy and dihydrodiol metabolite respectively.
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The mechanisms contributing to alcohol use disorder (AUD) are complex and the orexigenic peptide ghrelin, which enhances alcohol reward, is implied as a crucial modulator. The major proportion of circulating ghrelin is however the non-octanoylated form of ghrelin, des-acyl ghrelin (DAG), whose role in reward processes is unknown. As recent studies show that DAG decreases food intake, we hypothesize that DAG attenuates alcohol-related responses in animal models. Acute and repeated DAG treatment dose-dependently decreased alcohol drinking in male and female rats. In these alcohol-consuming male rats, repeated DAG treatment causes higher levels of dopamine metabolites in the ventral tegmental area, an area central to reward processing. The role of DAG in reward processing is further supported as DAG prevents alcohol-induced locomotor stimulation, reward in the conditioned place preference paradigm, and dopamine release in the nucleus accumbens in male rodents. On the contrary, DAG does not alter the memory of alcohol reward or affect neurotransmission in the hippocampus, an area central to memory. Further, circulating DAG levels are positively correlated with alcohol drinking in female but not male rats. Studies were conducted in attempts to identify tentative targets of DAG, which currently are unknown. Data from these recombinant cell system revealed that DAG does not bind to either of the monoamine transporters, 5HT2A, CB1, or µ-opioid receptors. Collectively, our data show that DAG attenuates alcohol-related responses in rodents, an effect opposite to that of ghrelin, and contributes towards a deeper insight into behaviors regulated by the ghrelinergic signaling pathway.
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Consumo de Bebidas Alcohólicas , Dopamina , Ghrelina , Núcleo Accumbens , Recompensa , Área Tegmental Ventral , Animales , Ghrelina/farmacología , Ghrelina/metabolismo , Masculino , Ratas , Femenino , Dopamina/metabolismo , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efectos de los fármacos , Etanol/farmacología , Etanol/administración & dosificación , Humanos , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
Semi-synthetic cannabinoids (SSCs) including hexahydrocannabinol (HHC) are emerging on the drug market and sold openly as purportedly legal replacements for cannabis and Δ9-THC. By the beginning of 2024, 24 European countries had identified HHC, often sold openly in edibles (foods/candy), vapes and low-THC cannabis flowers and resins. The SSC market is developing rapidly, with HHC acetate (HHC-O), hexahydrocannabiphorol (HHC-P) and others recently identified. These developments may mark the first major change in the market for 'legal' replacements to cannabis since 'Spice' containing synthetic cannabinoids, such as JWH-018, emerged in 2008. Currently, there are some data available on the pharmacology of SSCs, which is crucial for understanding their effects, evaluating health risks and informing public health responses. This study focused on characterizing the in vitro activation of the human CB1 receptor by the (R)- and (S)-epimers of HHC, HHC-P and HHC-O. Using recombinant CHO-K1 cells expressing the human CB1 receptor, the potency (EC50) and efficacy were determined. It was established that (9R)-HHC and (9R)-HHC-P activated the CB1 receptor as partial agonists and with five and two times lower potency compared to JWH-018, respectively, while the (S)-epimers exhibited even lower potency. The (R)-epimer of HHC-O activate the CB1 receptor to even lesser extent and the (S)-epimer showed no activation. For HHC and HHC-P, all epimers exhibited similar level of efficacy. This available evidence suggests cannabimimetic effects of the tested SSC with the exception for the acetates that likely function as pro-drugs in vivo.
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Persistent taxane-induced peripheral neuropathy (TIPN) is highly prevalent among early-stage breast cancer survivors (ESBCS) and has detrimental effect on quality of life. We leveraged logistic regression models to develop and validate polygenic prediction models to estimate the risk of persistent PN symptoms in a training cohort and validation cohort taking clinical risk factors into account. Based on 337 whole-exome sequenced ESBCS two of five prediction models for individual PN symptoms obtained AUC results above 60% when validated. Using the model for numbness in feet (35 SNVs) in the test cohort, 73% survivors were correctly predicted. For tingling in feet (55 SNVs) 70% were correctly predicted. Both models included SNVs from the ADAMTS20, APT6V0A2, CCDC88C, CYP2C8, EPHA5, NR1H3, PSKH2/APTV0D2, and SCN10A genes. For cramps in feet, difficulty climbing stairs and difficulty opening a jar the validation was unsuccessful. Polygenic prediction models including clinical risk factors can estimate the risk of persistent taxane-induced numbness in feet and tingling in feet in ESBCS.
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Autopsy rates are declining globally, impacting cause-of-death (CoD) diagnoses and quality control. Postmortem metabolomics was evaluated for CoD screening using 4,282 human cases, encompassing CoD groups: acidosis, drug intoxication, hanging, ischemic heart disease (IHD), and pneumonia. Cases were split 3:1 into training and test sets. High-resolution mass spectrometry data from femoral blood were analyzed via orthogonal-partial least squares discriminant analysis (OPLS-DA) to discriminate CoD groups. OPLS-DA achieved an R2 = 0.52 and Q2 = 0.30, with true-positive prediction rates of 68% and 65% for training and test sets, respectively, across all groups. Specificity-optimized thresholds predicted 56% of test cases with a unique CoD, average 45% sensitivity, and average 96% specificity. Prediction accuracies varied: 98.7% for acidosis, 80.5% for drug intoxication, 81.6% for hanging, 73.1% for IHD, and 93.6% for pneumonia. This study demonstrates the potential of large-scale postmortem metabolomics for CoD screening, offering high specificity and enhancing throughput and decision-making in human death investigations.
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Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC-O and HHCP-O, respectively, are emerging in Europe as alternatives to tetrahydrocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi-synthetic cannabinoids HHC, HHCP, HHC-O and HHCP-O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC-O and HHCP-O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non-hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra-high performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight mass spectrometer (QToF-MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC-O and HHCP-O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuronidated. 11-OH-HHC, 5'OH-HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non-acetate analogues.
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Background: Any difference in biomarkers between genotype-positive individuals with overt hypertrophic cardiomyopathy (HCM), and genotype-positive but phenotype-negative individuals (G+P-) in HCM-associated pathways might shed light on pathophysiological mechanisms. We studied this in young HCM patients. Methods: 29 HCM patients, 17 G+P--individuals, and age- and sex-matched controls were prospectively included. We analyzed 184 cardiovascular disease-associated proteins by two proximity extension assays, categorized into biological pathways, and analyzed with multivariate logistic regression analysis. Significant proteins were dichotomized into groups above/below median concentration in control group. Results: Dichotomized values of significant proteins showed high odds ratio (OR) in overt HCMphenotype for Fibroblast growth factor-21 (FGF-21) 10 (p = 0.001), P-selectin glycoprotein ligand-1 (PSGL-1) OR 8.6 (p = 0.005), and Galectin-9 (Gal-9) OR 5.91 (p = 0.004). For G+P-, however, angiopoietin-1 receptor (TIE2) was notably raised, OR 65.5 (p = 0.004), whereas metalloproteinase inhibitor 4 (TIMP4) involved in proteolysis, in contrast, had reduced OR 0.06 (p = 0.013). Conclusions: This study is one of the first in young HCM patients and G+P- individuals. We found significantly increased OR for HCM in FGF-21 involved in RAS-MAPK pathway, associated with cardiomyocyte hypertrophy. Upregulation of FGF-21 indicates involvement of the RAS-MAPK pathway in HCM regardless of genetic background, which is a novel finding.
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Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The aim of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross-reactivity. Blood samples from driving under the influence of drugs (DUID) cases screened positive for cannabis using enzyme-linked immunoadsorbent assay (ELISA) and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS-MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias and autosampler stability. Cross-reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5 and 200 ng/mL. The cross-reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LC-MS-MS method, 9R-HHC-COOH, 9S-HHC-COOH and 11-OH-9R-HHC showed matrix effects <25% at both concentrations, while 8-OH-9R-HHC, 9R-HHC and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and intra day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper LOQ was 20 and 200 ng/mL, respectively. Reanalysis of cases (n = 145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.
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Dronabinol , Detección de Abuso de Sustancias , Espectrometría de Masas en Tándem , Humanos , Dronabinol/sangre , Dronabinol/análogos & derivados , Detección de Abuso de Sustancias/métodos , Conducir bajo la Influencia , Ensayo de Inmunoadsorción Enzimática , Reproducibilidad de los Resultados , Límite de Detección , Cromatografía LiquidaRESUMEN
A genetic predisposition to central nervous system (CNS) toxicity induced by antimicrobial drugs (antibiotics, antivirals, antifungals, and antiparasitic drugs) has been suspected. Whole genome sequencing of 66 cases and 833 controls was performed to investigate whether antimicrobial drug-induced CNS toxicity was associated with genetic variation. The primary objective was to test whether antimicrobial-induced CNS toxicity was associated with seventeen efflux transporters at the blood-brain barrier. In this study, variants or structural elements in efflux transporters were not significantly associated with CNS toxicity. Secondary objectives were to test whether antimicrobial-induced CNS toxicity was associated with genes over the whole genome, with HLA, or with structural genetic variation. Uncommon variants in and close to three genes were significantly associated with CNS toxicity according to a sequence kernel association test combined with an optimal unified test (SKAT-O). These genes were LCP1 (q = 0.013), RETSAT (q = 0.013) and SFMBT2 (q = 0.035). Two variants were driving the LCP1 association: rs6561297 (p = 1.15x10-6, OR: 4.60 [95% CI: 2.51-8.46]) and the regulatory variant rs10492451 (p = 1.15x10-6, OR: 4.60 [95% CI: 2.51-8.46]). No common genetic variant, HLA-type or structural variation was associated with CNS toxicity. In conclusion, CNS toxicity due to antimicrobial drugs was associated with uncommon variants in LCP1, RETSAT and SFMBT2.
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Antiinfecciosos , Estudios de Casos y Controles , Antiinfecciosos/toxicidad , Sistema Nervioso Central , Antibacterianos , Antifúngicos , Proteínas de Transporte de MembranaRESUMEN
Non-small cell lung cancer is often diagnosed at advanced stages, and many patients are still treated with classical chemotherapy. The unselective nature of chemotherapy often results in severe myelosuppression. Previous studies showed that protein-coding mutations could not fully explain the predisposition to myelosuppression. Here, we investigate the possible role of enhancer mutations in myelosuppression susceptibility. We produced transcriptome and promoter-interaction maps (using HiCap) of three blood stem-like cell lines treated with carboplatin or gemcitabine. Taking advantage of publicly available enhancer datasets, we validated HiCap results in silico and in living cells using epigenetic CRISPR technology. We also developed a network approach for interactome analysis and detection of differentially interacting genes. Differential interaction analysis provided additional information on relevant genes and pathways for myelosuppression compared with differential gene expression analysis at the bulk level. Moreover, we showed that enhancers of differentially interacting genes are highly enriched for variants associated with differing levels of myelosuppression. Altogether, our work represents a prominent example of integrative transcriptome and gene regulatory datasets analysis for the functional annotation of noncoding mutations.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Carboplatino/efectos adversos , Antineoplásicos/efectos adversos , Mutación/genéticaRESUMEN
Objective: The primary aim of this study was to examine the association between the different predicted phenotypes of the polymorphic CYP2D6 gene and the prevalence of adverse drug reactions in patients suffering from depressive disorders. The secondary aim was to investigate if comedication with CYP2D6 inhibitors resulted in more adverse drug reactions due to phenoconversion.Methods: Between January 2012 and December 2021, 415 patients with a depressive disorder and insufficient treatment response in secondary psychiatric care were included in the naturalistic observational study Genes, Depression, and Suicidality (GEN-DS). The patients were subjected to a semistructured interview and diagnosed according to DSM-IV. Patients were also required to complete the self-rating version of the UKU Side Effect Rating Scale. All patients were genotyped for CYP2D6 and assigned a corresponding predicted CYP2D6 phenotype.Results: Out of the 415 patients, 147 patients with available genotyping and UKU scale results were also prescribed 1 or more drugs metabolized by CYP2D6. We did not find any evidence of an effect of the predicted CYP2D6 phenotype on the total burden of adverse drug reactions or in any of the specific symptom domains as measured with the UKU scale among these patients. We also investigated if comedication with 1 or more substances that inhibited the effect of the CYP2D6 enzyme resulted in more reported adverse drug reactions due to phenoconversion. Even though the rate of phenotypic PMs increased from 13 to 38 patients, we did not find any support for increased adverse drug reactions in this group.Conclusions: We did not find that CYP2D6 phenotype could predict the occurrence of adverse drug reactions in patients with depressive disorders in this naturalistic setting. However, information about CYP2D6 genotype may still be important in antidepressant treatment for the selection of appropriate drugs, for dosing recommendations of certain medications, or when the patient is suffering from severe adverse reactions.
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Antidepresivos , Citocromo P-450 CYP2D6 , Trastorno Depresivo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Antidepresivos/efectos adversos , Citocromo P-450 CYP2D6/genética , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/genética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , GenotipoRESUMEN
1-(2,3-Dihydro-1H-inden-5-yl)-2-(piperidin-1-yl)pentan-1-one (3,4-Pr-PipVP), a novel synthetic cathinone (SCat), was first identified in 2022 in Germany. The product was marketed as 1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one (3,4-EtPV), a substance not covered by the German New Psychoactive Substances Act (NpSG). Although originally intended to be an exploratory new synthetic cathinone containing the novel bicyclo[4.2.0]octatrienyl function, the compound was subsequently confirmed to contain an indanyl ring system scheduled under generic legislation like the NpSG. However, it is one of only a few marketed SCats carrying a piperidine ring. Inhibition experiments involving norepinephrine, dopamine, and serotonin transporters showed that 3,4-Pr-PipVP was a low potency blocker at all three monoamine transporters compared to related substances such as MDPV. Additionally, pharmacokinetic data were collected from pooled human liver microsomes incubations and from the analysis of authentic urine samples received after oral administration of 5 mg 3,4-Pr-PipVP hydrochloride. Phase I metabolites were tentatively identified in vitro and in vivo using liquid chromatography-time-of-flight mass spectrometry. Main metabolites were formed by metabolic reduction of the carbonyl function with and without additional hydroxylations at the propylene bridge of the molecule. Keto-reduced H2 -3,4-Pr-PipVP and H2 -piperidine-OH-3,4-Pr-PipVP as well as aryl-OH-3,4-Pr-PipVP, and indanyl-OH-piperidine-OH-3,4-Pr-PipVP are suggested as most suitable biomarkers for the detection of 3,4-Pr-PipVP since they were detected for much longer than the parent compound. 3,4-Pr-PipVP could be detected for up to 21 h whereas its metabolites were detectable for up to about 4 days.
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Líquidos Corporales , Cathinona Sintética , Humanos , Microsomas Hepáticos/metabolismo , Biomarcadores/metabolismo , Piperidinas/metabolismoRESUMEN
Many new psychoactive substances (NPS) are stimulants, and information about their potency and abuse potential is often lacking. To start addressing this need, a method measuring the inhibition of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, and NET) by stimulant drugs was developed. The use of a proprietary fluorescent dye mixture and three cell lines (CHO-K1, HEK 293, and MDCK), each expressing a single transporter, allowed for a semiautomated, one-pot determination of inhibition in a 384-well format. The method was validated using well characterized stimulants, including cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), α-PVP, and fluoxetine and performed similarly to other methods. Seven synthetic cathinones all showed highest potency for DAT inhibition, followed by NET and SERT. The rank potency for DAT inhibition IC50 (nM) was MPHP (4.53) > 4Cl-α-PVP (8.05) > 3F-α-PVP (12.7) > α-PiHP (13.4) > N-ethylpentylone (16.9) > N-ethylhexedrone (44.5) > 4-methylpentedrone (261). All but 4-methylpentedrone were more potent than amphetamine (257) and cocaine (111). The DAT/SERT inhibition ratio for the cathinones was in the range from 5.02 for 4-methylpentedrone to >3730 for α-PiHP, compared to 1.64 for cocaine and >4030 for α-PVP. All seven substances had inhibition profiles similar to those of potent stimulants with high abuse potential.
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Alcaloides , Estimulantes del Sistema Nervioso Central , Cocaína , Metilaminas , Pentanonas , Pirrolidinas , Humanos , Colorantes Fluorescentes , Células HEK293 , Estimulantes del Sistema Nervioso Central/farmacología , Anfetamina , Cocaína/farmacología , Neurotransmisores , Proteínas de Transporte de Dopamina a través de la Membrana PlasmáticaRESUMEN
Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after ß-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge.
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The emergence of new synthetic cannabinoid receptor agonists (SCRAs) onto the illicit drugs market continues to cause harm, and the overall availability of physicochemical and pharmacokinetic data for new psychoactive substances is lacking. The lipophilicity of 23 SCRAs and the plasma protein binding (PPB) of 11 SCRAs was determined. Lipophilicity was determined using a validated chromatographic hydrophobicity index (CHI) log D method; tested SCRAs showed moderate to high lipophilicity, with experimental log D7.4 ranging from 2.48 (AB-FUBINACA) to 4.95 (4F-ABUTINACA). These results were also compared to in silico predictions generated using seven commercially available software packages and online tools (Canvas; ChemDraw; Gastroplus; MoKa; PreADMET; SwissADME; and XlogP). Licenced, dedicated software packages provided more accurate lipophilicity predictions than those which were free or had prediction as a secondary function; however, the latter still provided competitive estimates in most cases. PPB of tested SCRAs, as determined by equilibrium dialysis, was in the upper range of the lipophilicity scale, ranging from 90.8% (ADB-BUTINACA) to 99.9% (BZO-HEXOXIZID). The high PPB of these drugs may contribute to reduced rate of clearance and extended durations of pharmacological effects compared to lesser-bound SCRAs. The presented data improve understanding of the behaviour of these drugs in the body. Ultimately, similar data and predictions may be used in the prediction of the structure and properties of drugs yet to emerge on the illicit market.
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Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB1 and CB2 ß-arrestin 2 recruitment assays and a CB1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB1 agonists. Also, at CB2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1 ; however, this effect was less prominent at CB2 . Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected.
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Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.
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Agonistas de Receptores de Cannabinoides , Cannabinoides , Humanos , Agonistas de Receptores de Cannabinoides/metabolismo , Cannabinoides/análisis , Espectrometría de Masas en Tándem/métodos , Metaboloma , Estándares de Referencia , Hepatocitos/metabolismo , Amidas/metabolismo , Cetonas/metabolismo , Microsomas Hepáticos/metabolismoRESUMEN
BACKGROUND: Pancreatic cancer remains a leading cause of cancer-related death. To individualise management and improve survival, more accurate prognostic models are needed. METHODS: All patients resected for pancreatic ductal adenocarcinoma in a tertiary Swedish centre during 2009-2019 were thoroughly analysed with regards to pathological and clinical parameters including tumour grade, resection margin status, para-aortic lymph node engagement (node station 16), and systemic treatment. RESULTS: The study cohort included 275 patients. Overall median survival was 21.2 months (95% CI 17.5-24.8). Year of resection, margin status (R1 subdivided into R11mm/R1ink), perineural invasion, differentiation grade, TNM stage, and adjuvant therapy were independent factors with significant impact on survival. Margin status also significantly affected recurrence-free survival and relapse patterns, with local and peritoneal relapses being associated with R1-status (p < 0.001 and p = 0.007). Presence of para-aortic lymph node metastases was associated with shorter recurrence-free survival as compared to N1 status only. CONCLUSION: Survival in resected pancreatic cancer is improving over time. Resection margin status is a key factor affecting recurrence patterns and prognosis. Given the poor recurrence-free survival in node station 16 metastasised patients, the rational for resection remains in doubt, and improved treatment strategies for this patient group is necessary.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Estudios de Cohortes , Márgenes de Escisión , Metástasis Linfática/patología , Estudios Retrospectivos , Recurrencia Local de Neoplasia/patología , Neoplasias Pancreáticas/patología , Pronóstico , Ganglios Linfáticos/cirugía , Ganglios Linfáticos/patología , Carcinoma Ductal Pancreático/patología , Neoplasias PancreáticasRESUMEN
Rilmazafone is a pro-drug that can be prescribed in Japan to treat insomnia. Rilmazafone metabolizes into active compounds by a ring closure resulting in a triazolo benzodiazepine structure similar to alprazolam. In mid-2022, the National Board of Forensic Medicine in Sweden were requested to investigate two separate deaths with the suspected use of pagoclone. Packages labeled "Pagoclone" were found at each scene that was suspected to contain rilmazafone based on website information. During screening by high resolution mass spectrometry, rilmazafone metabolites were presumptively identified. Due to the lack of reference material for the active metabolites, the metabolites were synthesized in house and quantification of the compounds identified in the two autopsy cases was prompted. In Case 1, femoral blood concentrations of 7.9, 65 and 170 ng/g of the metabolites rilmazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included the medications haloperidol, alimemazine, fluoxetine, olanzapine and acetaminophen. In Case 2, femoral blood concentrations of 1.7, 1.4 and 70 ng/g of rimazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included loperamide, alimemazine and pregabalin. The intake of rilmazafone was determined as the cause of death in Case 1 and contributed in the Case 2.