Characterization of iso-LSD metabolism using human liver microsomes in comparison to LSD and its applicability as urinary biomarker for LSD consumption.

Urinalysis of lysergic acid diethylamide (LSD) poses a challenge due to its rapid metabolism, resulting in little to no LSD detectable in urine. Instead, its primary metabolite, 2-oxo-3-hydroxy-LSD, is predominantly detected. In this study, we observed several urine profiles with iso-LSD detected together with 2-oxo-3-hydroxy-LSD. Iso-LSD is derived from illicit preparation of LSD as a major contaminant, and it was detected at higher abundance than LSD and 2-oxo-3-hydroxy-LSD in certain urine samples. Therefore, the metabolism of iso-LSD and its potential as a viable urinary biomarker for confirming LSD consumption is of interest. For metabolism studies, LSD and iso-LSD were incubated in human liver microsomes (HLMs) at 0 min, 60 min and 120 min to characterize their metabolites using LC-QTOF-MS. For urinary analysis, 500 µL of urine samples underwent enzymatic hydrolysis and clean-up using supported-liquid extraction (SLE) prior to analysis by LC-QTOF-MS. From HLM incubation study of LSD, the metabolites detected were dihydroxy-LSD, 2-oxo-LSD, N-desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD with LSD levels decreasing significantly throughout all time points, consistent with the existing literatures. For HLM study of iso-LSD, metabolites eluting at retention times after the corresponding metabolites of LSD were detected, with iso-LSD levels showing only a slight decrease throughout all time points, due to a slower metabolism of iso-LSD compared to LSD. These findings corroborate with the urinalysis of 24 authentic urine samples, where iso-LSD with 2-oxo-3-hydroxy-LSD was detected in the absence of LSD. Based on our findings, iso-LSD is commonly detected in urine (18 out of 24 samples) sometimes with traces of possible 2-oxo-3-hydroxy-iso-LSD. The slower metabolism and high detection rate in urine make iso-LSD a viable urinary biomarker for confirming LSD consumption, especially in the absence of LSD and/or 2-oxo-3-hydroxy-LSD.

Lockdown! Learning public health of infectious diseases through gameplay.

The COVID-19 pandemic has demonstrated the detrimental effects of a lack of understanding of public health measures. During the pandemic, lockdowns, social distancing, and mask mandates introduced by governments were met with skepticism, doubt, and an unwillingness to comply, increasing the extent of negative outcomes as a result. Albeit devastating, the pandemic has offered an invaluable opportunity to observe the correlation between the prevalence of public health education and compliance with public health measures during critical times. In this article, we describe a card game that was developed during the COVID-19 pandemic to educate the public (including children) about how specific public health measures address the pandemic and how global cooperation is essential in addressing even one country's problems. The game can be used in primary, secondary, or tertiary education classrooms, initiating conversations about the topic and providing a basic understanding before more in-depth learning.

Qualitative Confirmation of 94 New Psychoactive Substances and Metabolites in Urine Using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry.

Numerous methods and techniques have been published for the identification of new psychoactive substances (NPS) and their metabolites in urine. However, there lacks a holistic approach to analyze different groups of NPS and their metabolites with decision points for reporting their use. In this study, data-dependent acquisition workflow using liquid chromatography--quadrupole time-of-flight mass spectrometry was developed and validated for the identification of a total of 94 NPS and metabolites in urine using the established decision points. The limit of identification for all analytes was determined at 25% below their respective decision points. The method was demonstrated to be accurate and precise at their respective decision points with extraction recoveries and ion suppression/enhancement ranging from 51.0% to 103.5% and -81.6% to 159.1%, respectively. There was no observed carryover up to 200 ng/mL for all analytes and no interferences from urine matrixes, internal standards and other common drugs of abuse. The extracted drug analytes were stable at 4 and 15°C for up to 3 days. The validated method was successfully evaluated and applied in the testing of urine samples from NPS users. In conclusion, this validated method can analyze a wide range of NPS and their metabolites with the use of decision points for consistency in reporting.

Transesterification of Indazole-3-carboxamide Synthetic Cannabinoids: Identification of Metabolite Biomarkers for Diagnosing Co-abuse of 5F-MDMB-PINACA and Alcohol.

Concurrent use of alcohol with synthetic cannabinoids (SCs) has been widely recorded among drug abusers. The susceptibilities of three indazole-3-carboxamide type SCs with methyl ester moiety, 5F-MDMB-PINACA, 5F-MMB-PINACA, and MMB-FUBINACA, to transesterification in the presence of ethanol warranted further investigation in view of probable augmented toxicity. In vitro metabolite identification experiments were first performed using human liver microsomes (HLMs) to characterize the novel metabolites of the three parent SCs in the presence of ethanol. Formation of transesterified metabolite, hydrolyzed metabolite, and several oxidative metabolites in HLM in the presence of alcohol was further determined for each parent SC and the respective ethyl ester analog, 5F-EDMB-PINACA, 5F-EMB-PINACA, and EMB-FUBINACA, to quantitatively elucidate transesterification and hydrolysis activities. Our results suggested that all three SCs undergo carboxylesterase-mediated transesterification to their respective ethyl ester analog in the presence of ethanol, which was incubation time- and ethanol concentration-dependent. Each ethyl ester metabolite was sequentially and readily metabolized to novel oxidative metabolites with the intact ethyl ester moiety and the same hydrolyzed metabolite as derived from its parent SC. A smaller extent of transesterification was non-enzymatically driven. Notably, we proposed 5F-EDMB-PINACA oxidative defluorination metabolite as the biomarker for diagnosing the potential co-abuse of 5F-MDMB-PINACA and alcohol. Due to the comparable pharmacological activities between each SC and its ethyl ester metabolite, augmented toxicity associated with co-abuse of SCs and alcohol is probable and deserves further investigation.

Identification of Optimal Urinary Biomarkers of Synthetic Cannabinoids BZO-HEXOXIZID, BZO-POXIZID, 5F-BZO-POXIZID, and BZO-CHMOXIZID for Illicit Abuse Monitoring.

BACKGROUND: The continuous introduction of new synthetic cannabinoid (SC) subtypes and analogues remains a major problem worldwide. Recently, a new "OXIZID" generation of SCs surfaced in seized materials across various countries. Hence, there is an impetus to identify urinary biomarkers of the OXIZIDs to detect their abuse. METHODS: We adapted our previously reported two-pronged approach to investigate the metabolite profiles and disposition kinetics of 4 OXIZID analogues, namely, BZO-HEXOXIZID (MDA-19), BZO-POXIZID (5C-MDA-19), 5F-BZO-POXIZID (5F-MDA-19), and BZO-CHMOXIZID (CHM-MDA-19). First, bottom-up in vitro incubation experiments comprising metabolite identification, metabolic stability, and reaction phenotyping were performed using human liver microsomes and recombinant human cytochrome P450 enzymes. Second, top-down analysis of authentic urine samples from drug abusers was performed to corroborate the in vitro findings and establish a panel of urinary biomarkers. RESULTS: A total of 42 to 51 metabolites were detected for each OXIZID, and their major metabolic pathways included N-alkyl and phenyl hydroxylation, oxidative defluorination (for 5F-BZO-POXIZID), oxidation to ketone and carboxylate, amide hydrolysis, and N-dealkylation. The OXIZIDs were metabolically unstable, mainly metabolized by cytochromes P3A4, P3A5, and P2C9, and demonstrated mechanism-based inactivation of cytochrome P3A4. Integrating with the results of 4 authentic urine samples, the parent drug and both N-alkyl and phenyl mono-hydroxylated metabolites of each OXIZID were determined as suitable urinary biomarkers. CONCLUSIONS: Drug enforcement agencies worldwide may apply these biomarkers in routine monitoring procedures to identify abusers and counter the escalation of OXIZID abuse.

Urinary Metabolite Biomarkers for the Detection of Synthetic Cannabinoid ADB-BUTINACA Abuse.

BACKGROUND: (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3carboxamide (ADB-BUTINACA) is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene in January 2020. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. METHODS: We characterized the metabolites of ADB-BUTINACA in human liver microsomes using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. We further used recombinant enzymes to generate a pool of key metabolites in situ and determined their metabolic stability. By coupling in vitro metabolism and authentic urine analyses, a panel of urinary metabolite biomarkers of ADB-BUTINACA was curated. RESULTS: Fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. Reaction phenotyping established that ADB-BUTINACA was rapidly eliminated via CYP2C19-, CYP3A4-, and CYP3A5-mediated metabolism. Three major monohydroxylated metabolites (M6, M12, and M14) were generated in situ, which demonstrated greater metabolic stability compared to ADB-BUTINACA. Coupling metabolite profiling with urinary analysis, we identified four urinary biomarker metabolites of ADB-BUTINACA: 3 hydroxylated metabolites (M6, M11, and M14) and 1 oxidative deaminated metabolite (M15). CONCLUSIONS: Our data support a panel of four urinary metabolite biomarkers for diagnosing the consumption of ADB-BUTINACA.

Diagnosing intake and rationalizing toxicities associated with 5F-MDMB-PINACA and 4F-MDMB-BINACA abuse.

5F-MDMB-PINACA and 4F-MDMB-BINACA are synthetic cannabinoids (SCs) that elicit cannabinoid psychoactive effects. Defining pharmacokinetic-pharmacodynamic (PK-PD) relationships governing SCs and their metabolites are paramount to investigating their in vivo toxicological outcomes. However, the disposition kinetics and cannabinoid receptor (CB) activities of the primary metabolites of SCs are largely unknown. Additionally, reasons underlying the selection of ester hydrolysis metabolites (EHMs) as urinary biomarkers are often unclear. Here, metabolic reaction phenotyping was performed to identify key metabolizing enzymes of the parent SCs. Hepatic clearances of parent SCs and their EHMs were estimated from microsomal metabolic stability studies. Renal clearances were simulated using a mechanistic kidney model incorporating in vitro permeability and organic anionic transporter 3 (OAT3)-mediated uptake data. Overall clearances were considered in tandem with estimated volumes of distribution for in vivo biological half-lives (t1/2) predictions. Interactions of the compounds with CB1 and CB2 were investigated using a G-protein coupled receptor activation assay. We demonstrated that similar enzymatic isoforms were implicated in the metabolism of 5F-MDMB-PINACA and 4F-MDMB-BINACA. Our in vivo t1/2 determinations verified the rapid elimination of parent SCs and suggest prolonged circulation of their EHMs. The pronounced attenuation of the potencies and efficacies of the metabolites against CB1 and CB2 further suggests how toxic manifestations of SC abuse are likely precipitated by augmented exposure to parent SCs. Notably, basolateral OAT3-mediated uptake of the EHMs substantiates their higher urinary abundance. These novel insights underscore the importance of mechanistic, quantitative and systematic characterization of PK-PD relationships in rationalizing the toxicities of SCs.

A rapid SPE-based analytical method for UPLC/MS/MS determination of aminoglycoside antibiotic residues in bovine milk, muscle, and kidney.

An SPE-based cleanup protocol was developed for ultra-performance LC (UPLC)/MS/MS determination of residues of the common aminoglycoside antibiotics streptomycin, dihydrostreptomycin, neomycin, and gentamicin in bovine milk, kidney, and muscle. Recoveries for all compounds except neomycin ranged from 80 to 104% for all matrixes studied; recoveries for neomycin ranged from 71 to 84%. Intraday and interday precision data were under 15% for all sample matrixes. Compared with other recently reported cleanup methods, less sample is required, the use of potentially dangerous reagents is minimized, and fewer manipulations are required by the analyst. A high throughput 96-well plate format was used for SPE cleanup and UPLC/MS analysis.

Preclinical metabolism and pharmacokinetics of SB1317 (TG02), a potent CDK/JAK2/FLT3 inhibitor.

SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed > 99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 µM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution ( > 0.6 L/kg), oral bioavailability of 24%, ∼ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate.

Preclinical metabolism and disposition of SB939 (Pracinostat), an orally active histone deacetylase inhibitor, and prediction of human pharmacokinetics.

The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino) ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 µM) but inhibited 2C19 (IC(50) = 5.8 µM). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between ∼84 and 94%. The blood-to-plasma ratio was ∼1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l · h(-1) · kg(-1) and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and ∼3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.

In vitro phase I cytochrome P450 metabolism, permeability and pharmacokinetics of SB639, a novel histone deacetylase inhibitor in preclinical species.

In vitro liver microsomal stability, permeability, pharmacokinetics (PK) and oral bioavailability of SB639, a novel HDACi (Histone Deacetylase inhibitor), were determined. The in vitro metabolism was examined in mouse, rat, dog and human liver microsomes. The permeability and efflux potential of SB639 were determined using Caco-2 cell monolayers. To determine pharmacokinetics and oral bioavailability, blood samples were drawn at pre-determined intervals up to 24 h post-dose after single intravenous (i.v.) or oral (p.o.) administration of SB639 to mouse or rat. The concentrations of SB639 in plasma samples were determined by a validated LC-MS/MS method. In vitro liver microsomal stability data revealed that SB639 was stable in human and dog liver microsomes, unstable in mouse and rat liver microsomes. The Caco-2 data has shown that SB639 is highly permeable with an apparent permeability of 3.01.10(-6) cm/s at 10 microM. After oral administration, maximum concentrations of SB639 were achieved within 0.5 h of post dose. Following i.v. administration, the concentration of SB639 declined in a bi-exponential fashion with terminal elimination half-life of 1.67 h for mice and 1.12 h for rats. The systemic clearance and volume of distribution of SB639 in mice were 15.8 l/h/kg and 38 l/kg, respectively, while the respective values in rats were 3.84 l/h/kg and 3.67 l/kg. Elimination half-life in rats ranged between 1.12-2.26 h. Absolute oral bioavailability of SB639 in mouse and rat was 13% and 10%, respectively. In conclusion, the superior potency, physicochemical and PK properties of SB639 compared to the recently FDA approved drug Zolinza (Suberoylanilide hydroxamic acid or Vorinostat) in the preclinical setting makes it a potential clinical candidate.

Development and validation of high-performance liquid chromatography-tandem mass spectrometry assay for 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide, a novel HDAC inhibitor, in mouse plasma for pharmacokinetic studies.

A liquid chromatography/tandem mass spectrometric method for the quantification of 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide (EX-2), a novel histone deacetylase (HDAC) inhibitor, in mouse plasma was developed to support in-house pharmacokinetic (PK) studies in the lead optimization stage. In order to determine the PK parameters for EX-2 in comparison to other HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), PXD-101 and LBH-589, which are currently in different stages of clinical trials, research-grade bio-analytical method validations were carried out for EX-2 and these reference HDAC inhibitors, which were synthesized by in-house medicinal chemists. The components of validation consisted of specificity, extraction efficiency, signal-response of calibration standards, lower limit of quantification, autosampler stability and accuracy and precision of quality control samples. The validated LC/MS/MS methods were accurate and precise. The calibration curve ranged from 1 to 1600 ng/mL for all the analytes. The methods developed were used to quantify EX-2 and other HDAC inhibitors in mouse plasma obtained from pharmacokinetic studies. The results suggest that EX-2 has better PK parameters compared with the reference drugs and is a promising drug development candidate.