56 research outputs found
Metabolism and Pharmacological Characterization at μ-, κ-, and δ- Opioid Receptors Using a Novel Non-Radioactive Assay of Nitazenes and Methadone-like Synthetic Opioids
No full textI nuovi oppioidi sintetici (NSO), inclusi i nitazeni e i derivati della difenilmetilpiperidina, rappresentano sfide significative per la salute pubblica a causa della loro elevata potenza, rapida diffusione e limitata caratterizzazione. Questo studio mira a migliorare la comprensione del loro metabolismo e della loro attività farmacologica, fornendo strumenti essenziali per il rilevamento e la valutazione del rischio. Utilizzando epatociti primari umani e microsomi epatici umani e analisi LC-HRMS/MS, abbiamo caratterizzato il metabolismo in vitro e in vivo di etonitazepipne, dipianone, protonitazepine e metonitazepine. Un’analisi approfondita dei dati, supportata da software predittivi, ha permesso l’identificazione delle principali vie metaboliche e biomarcatori.
La caratterizzazione farmacologica di questi composti si è concentrata sulla loro attivazione dei recettori oppioidi μ-, κ- e δ- (MOR, KOR e DOR). È stato sviluppato e ottimizzato un saggio ad alta capacità basato su HTRF® per il legame del GTP Gi, per valutarne la potenza e l’efficacia. Come previsto, etonitazepipne, protonitazepine e metonitazepine hanno condiviso, come principali trasformazioni metaboliche, l’O-dealchilazione, l’idrossilazione e l’apertura del gruppo pirrolidinico o piperidinico in acido butanoico. Il dipianone ha mostrato un profilo metabolico unico, con metaboliti principali generati dopo l’apertura del pirrolidinico in butanolo e acido butanoico, seguita da ciclizzazione.
Grazie al saggio HTRF® basato sul legame GTP Gi, il protonitazepine è risultato il più potente ed efficace agonista del MOR, con una potenza circa 6 volte superiore al fentanyl, seguita da etonitazepipne, metonitazepine e dipianone. Questa ricerca fornisce informazioni fondamentali sul metabolismo e sulla farmacologia degli NSO, proponendo biomarcatori unici di consumo e un nuovo saggio HTRF® rapido e semplice per lo studio dell’attività dei recettori.Novel Synthetic Opioids (NSOs), including nitazenes and diphenylmethylpiperidine derivatives, pose significant challenges to public health due to their high potency, rapid emergence, and limited characterization. This study aims to advance the understanding of their metabolism and pharmacological activity, providing essential tools for detection and risk assessment. Using cryopreserved primary human hepatocytes, Pooled Human Liver Microsomes and LC-HRMS/MS analysis, we characterized the in vitro and in vivo metabolism of etonitazepipne, dipyanone, protonitazepyne, and metonitazepyne. Comprehensive data mining supported by predictive software enabled the identification of key metabolic pathways and biomarkers. The pharmacological characterization of these compounds focused on their activation of the μ-, κ-, and δ-opioid receptors (MOR, KOR, and DOR). A high-throughput HTRF®-based GTP Gi binding assay was developed and optimized to assess their potency and efficacy.
As expected, etonitazepipne, protonitazepyne and metonitazepyne shared as main transformation O-dealkylation, hydroxylation and pyrrolidine or piperidine opening to butanoic acid. Dipyanone presented a unique metabolic profile, and the main metabolites were generated after pyrrolidine opening to butanol and butanoic acid followed by cyclisation. As a result of the HTRF®-based GTP Gi binding assay, protonitazepyne was the most potent and efficacious agonist at MOR, resulting around 6 times more potent than fentanyl, followed by etonitazepipne, metonitazepyne and dipyanone. This research provides critical insights into the metabolism and pharmacology of NSOs, proposing unique biomarkers of consumption and a novel, fast and easy HTRF®-based GTP Gi binding assay for receptor activity study
Exploring the Metabolism of Flubrotizolam, a Potent Thieno-Triazolo Diazepine, Using Human Hepatocytes and High-Resolution Mass Spectrometry
No full textBackground: The abuse of psychoactive substances presents challenges in clinical and forensic toxicology. The emergence of novel and potent drugs that pose significant health risks, in particular towards frequent abusers and users unaware of the ingredients, further complicates the situation. Designer benzodiazepines have become a fast-growing subgroup of these new psychoactive substances (NPSs), and their overdose may potentially turn fatal, especially when combined with other central nervous system depressants. In 2021, flubrotizolam, a potent thieno-triazolo designer benzodiazepine, emerged on the illicit market, available online as a "research chemical". The identification of markers of consumption for this designer benzodiazepine is essential in analytical toxicology, especially in clinical and forensic cases. Methods: We therefore aimed to identify biomarkers of flubrotizolam uptake in ten-donor-pooled human hepatocytes, applying liquid chromatography high-resolution mass spectrometry and software-aided data mining supported by in silico prediction tools. Results: Prediction studies resulted in 10 and 13 first- and second-generation metabolites, respectively, mainly transformed through hydroxylation and sulfation, methylation, and glucuronidation reactions. We identified six metabolites after 3 h human hepatocyte incubation: two hydroxylated metabolites (α- and 6-hydroxy-flubrotizolam), two 6-hydroxy-glucuronides, a reduced-hydroxy-N-glucuronide, and an N-glucuronide. Conclusions: We suggest detecting flubrotizolam and its hydroxylated metabolites as markers of consumption after the glucuronide hydrolysis of biological samples. The results are consistent with the in vivo metabolism of brotizolam, a medically used benzodiazepine and a chloro-phenyl analog of flubrotizolam
In silico and in vitro human metabolism of IOX2, a performance-enhancing doping agent
No full textIOX2 is a potent inhibitor of prolyl hydroxylase 2, a key enzyme in the regulation of hypoxia-inducible factor (HIF) and oxygen homeostasis. As such, it can be used to enhance athletic performance and is currently banned by the World Anti-Doping Agency (WADA). Detection of metabolites is critical to demonstrate drug use in doping. However, there is currently little data on IOX2 human metabolism. Our aim was to identify relevant biomarkers of IOX2 use in humans. For this purpose, IOX2 was incubated with 10-donor-pooled human hepatocytes for 3 h, incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and LC-HRMS/MS data were screened with Compound Discoverer (Thermo Scientific) for a comprehensive identification of IOX2 metabolites. Additionally, IOX2 human metabolites were predicted with GLORYx open-access software (University of Hamburg, Germany) to assist in the LC-HRMS/MS analysis and data mining. Thirteen metabolites were identified, oxidation at the quinolinyl group, O-glucuronidation, and combinations being predominant biotransformations. The results were consistent with previous animal studies and a single case of oral microdose administration. We suggest hydroxyquinolinyl-IOX2 as major biomarker of IOX2 use in biological samples, glucuronide hydrolysis being critical to increase IOX2 and hydroxyquinolinyl-IOX2 detectability in urine
3F-α-pyrrolydinovalerophenone (3F-α-PVP) in vitro human metabolism: Multiple in silico predictions to assist in LC-HRMS/MS analysis and targeted/untargeted data mining
No full textSynthetic cathinones (SCs) constitute a heterogenous class of new psychoactive substances (NPS), structurally related to cathinone. SCs represent the widest NPS class, second to synthetic cannabinoids, accounting for approximately 160 different analogues with substitution at the phenyl group, the amine group, or the alkyl chain. In 2020, α-pyrrolidonophenone analogues were the most trafficked SCs, and were involved in many fatalities and intoxication cases. In particular, 3F-α-pyrrolidinovalerophenone (3F-α-PVP) was the cause of the highest number of SC-related fatal intoxications in Sweden in 2018. Minor structural modifications are used to avoid legal controls and analytical detection, but may also induce different toxicological profile. Therefore, the identification of specific markers of consumption is essential to discriminate SCs in clinical and forensic toxicology. In this study, we assessed 3F-α-PVP metabolic profile. 3F-α-PVP was incubated with 10-donor-pooled human hepatocytes, LC-HRMS/MS analysis, and software-assisted data mining. This well-established workflow was completed by in silico metabolite predictions using three different freeware. Ten metabolites were identified after 3 h incubation, including hydrogenated, hydroxylated, oxidated, and N-dealkylated metabolites. A total of 51 phase I and II metabolites were predicted, among which 7 were detected in the incubations. We suggest 3F-α-PVP N-butanoic acid, 3F-α-PVP pentanol, and 3F-α-PVP 2-ketopyrrolidinyl-pentanol as specific biomarkers of 3F-α-PVP consumption. This is the first time that an N-ethanoic acid is detected in the metabolic pathway of a pyrrolidine SC, demonstrating the importance of a dual targeted/untargeted data mining strategy
A possible deliberate Mahābhārata-echo in the imagery of the Buddhacarita compounded-rūpakas
No full textThe main focus of the paper is to tentatively document traces of hypertextuality between the Buddhacarita and the Mahābhārata, under the assumption that Aśvaghoṣa probably knew this latter work, albeit non-definitive version of it. The selected methodological approach is a comparison between Bc and MBh in-compound-rūpakas. Indeed, since it is plausible that he benefited from an erudite court audience, Aśvaghoṣa is here assumed to take for granted that even indirect hints at MBh passages would be promptly understood. Therefore, he sometimes re-uses Mahābhārata expressions, and merely changes the word-order or replaces a single constituent in the matching figurative phrases or compounds, and sometimes plays with the MBh rūpakas in a more complex way. On the basis of the survey and analysis of all the Bc’s rūpakas and their supposed inspirational MBh source, the present inquiry tries to show how the singled-out cross-references are not only aimed at building a generic sophisticated literary pattern for his mahākāvya and his learned audience, but they are also intentionally targeted at evoking Epic heroic imagery as clues for the kingly commitment the author attributes to Buddha
Per un’analisi preliminare della poiesi di Aśvaghoṣa: fra epica, retorica ed estetica
No full textAśvaghoṣa (I-II C.E.), the multifaceted buddhist author of the mahākāvyas Saundarananda and Buddhacarita, seems to have mastered the rhetorical devices of metaphora in absentia (rūpaka) and simile (upamā) in both of his works. These aspects will become systematised only until much later (VI-VII C.E.) and eventually investigated in contemporary cognitive linguistics studies (Black 1962; Levin 1977; Lakoff 1980). By means of a diachronic and synchronic approach, this article analyses the poetics and poiesis of the author through the lens of intertextuality and the dynamics of literary reuse in South-Asian and Buddhist literature. In this context, applying a synchronic approach means interpreting different types of rūpakas and upamās, foregrounding the author’s self-consciousness on rhetoric and stylistic forms. Similarly, the diachronic approach imposes a bidirectional criterion, namely a) the evaluation of a pre-systematised use of analogy forms in texts belonging to the epic genre (Itihāsa), and b) the assessment on already systematised analogy forms in later canonical alaṃkāraśāstras, from Bhāmaha’s Kāvyālaṃkāra (VII C.E.) to Mammaṭa’s Kāvyaprakāśa (XI C.E.)
Development of an enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of methorphan and its O-demethylated metabolite in human blood and its application to post-mortem samples
No full textIn the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both
Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances
No full textAdulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer’s profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations’ websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as “non-biological” (n = 35) or “biological” (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers’ health
Human metabolism and basic pharmacokinetic evaluation of AP‐238: A recently emerged acylpiperazine opioid
No full textAs a consequence of recently implemented legal restrictions on fentanyl analogs, a new generation of acylpiperazine opioids appeared on the illicit drug market. AP-238 was the latest opioid in this series to be notified by the European Early Warning System in 2020 and was involved in an increasing number of acute intoxications. AP-238 metabolism was investigated to provide useful markers of consumption. For the tentative identification of the main phase I metabolites, a pooled human liver microsome assay was performed. Further, four whole blood and two urine samples collected during post-mortem examinations and samples from a controlled oral self-administration study were screened for anticipated metabolites. In total, 12 AP-238 phase I metabolites were identified through liquid chromatography-quadrupole time-of-flight mass spectrometry in the in vitro assay. All of these were confirmed in vivo, and additionally, 15 phase I and five phase II metabolites were detected in the human urine samples, adding up to a total of 32 metabolites. Most of these metabolites were also detected in the blood samples, although mostly with lower abundances. The main in vivo metabolites were built by hydroxylation combined with further metabolic reactions such as O-methylation or N-deacylation. The controlled oral self-administration allowed us to confirm the usefulness of these metabolites as proof of intake in abstinence control. The detection of metabolites is often crucial to documenting consumption, especially when small traces of the parent drug can be found in real samples. The in vitro assay proved to be suitable for the prediction of valid biomarkers of novel synthetic opioid intake
- …
