Respective Metabolites Research Articles

Viloxazine Increases Cortical Serotonin Without Inhibiting Serotonin Reuptake at Doses Used to Treat ADHD

AbstractBackgroundMost FDA-approved ADHD treatments increase norepinephrine (NE) and dopamine (DA); however, our prior preclinical studies of the non-stimulant ADHD treatment viloxazine ER (Qelbree®) demonstrated that viloxazine also increases serotonin (5-HT). A prior microdialysis study showed increases in NE, DA, and 5-HT in the rat prefrontal cortex (PFC); however, the 50 mg/kg dose resulted in supratherapeutic plasma concentrations. Viloxazine is a moderate affinity selective NE reuptake inhibitor, structurally different than traditional SSRI antidepressants. Viloxazine has negligible activity at the serotonin reuptake transporter (SERT), suggesting viloxazine has a different mechanism of 5-HT PFC elevation than SSRIs. The current microdialysis study was undertaken to further characterize if viloxazine affects 5-HT and its 5-HIAA metabolite at therapeutically relevant plasma concentrations. Results are compared to similar microdialysis studies of SSRIs.MethodsRats were implanted with I-shaped microdialysis probes connected to a microperfusion pump, delivering artificial cerebrospinal fluid, in the PFC. After a 2-hour baseline period, viloxazine (1, 3, 10, or 30 mg/kg) was administered (ip). Dialysate samples were collected from the interstitial fluid (ISF) of the PFC before and after dosing. LC-MS/MS was used to determine the dialysate concentrations of viloxazine and viloxazine-induced changes in NE, 5-HT, and their respective metabolites, DHPG and 5-HIAA. Viloxazine plasma concentrations were also measured.Animal research was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.ResultsViloxazine administration resulted in significant dose-dependent increases in ISF NE levels and corresponding decreases in DHPG (NE metabolite) at all doses tested, reflecting viloxazine’s activity as a NET inhibitor. Viloxazine treatment also resulted in a dose-dependent elevation of ISF 5-HT levels in the PFC. Of the doses tested, 30 mg/kg was found to be clinically relevant as it induced ISF concentrations approximating unbound plasma concentrations in pediatric ADHD patients. At this dose, 5-HT levels were significantly increased over baseline and higher than vehicle levels. Coincident changes in 5-HIAA concentrations were not observed, reaffirming viloxazine’s lack of activity as a SERT inhibitor.ConclusionViloxazine induced dose-dependent increases in NE and 5-HT in the PFC, a critical target region for ADHD therapies. At clinically relevant viloxazine plasma concentrations, 5-HT was increased in the PFC. Unlike SSRIs, which correspondingly decrease the 5-HT metabolite in the PFC (indicating serotonin reuptake inhibition), viloxazine did not affect 5-HIAA levels. Thus, viloxazine increases cortical 5-HT levels by a different mechanism than SSRIs. Whether 5-HT effects aid in viloxazine therapeutic efficacy in ADHD is yet unknown.FundingSupernus Pharmaceuticals, Inc.

The fate of bisphenol A, bisphenol S, and their respective glucuronide metabolites in ovarian cells

Ovarian cells are critical for reproduction and steroidogenesis, which are functions that can be impacted by exposure to xenobiotics. As in other extra-hepatic tissues, biotransformation events may occur at the ovarian level. Such metabolic events deserve interest, notably as they may modulate the overall exposure and toxicity of xenobiotics. In this study, the comparative metabolic fate of two bisphenols was investigated in ovarian cells. Bisphenol A (BPA), a model endocrine disruptor, and its major substitute bisphenol S (BPS) were selected. Bovine granulosa cells (primary cultures) and theca explants (ex vivo tissue) were exposed for 24 hr to tritium-labeled BPA, BPS and their respective glucuronides (i.e. their major circulating forms), at concentrations consistent with low-dose exposure scenarios. Mass balance studies were performed, followed by radio-HPLC profiling. The capability of both cell compartments to biotransform BPA and BPS into their respective sulfo-conjugates was demonstrated, with sulfation being the predominant metabolic route. In theca, there was a significantly higher persistence of BPA (compared to BPS) residues over 24 hr. Moreover, only theca explants were able to deconjugate inactive BPA-glucuronide and BPS-glucuronide back into their biologically active aglycone forms. Deconjugation rates were demonstrated to be higher for BPS-G than for BPA-G. These findings raise concerns about the in situ direct release of bisphenols at the level of the ovary and demonstrate the relevance of exploring the biotransformation of bisphenols and their circulating metabolites in different ovarian cells with specific metabolic capabilities. This work also provides essential knowledge for the improved risk assessment of bisphenols.

Occupational Exposure and Health Impact Assessment of Diisocyanates in Finland.

Diisocyanates are a group of chemicals widely used in different industrial applications. The critical health effects related to diisocyanate exposure are isocyanate sensitisation, occupational asthma and bronchial hyperresponsiveness (BHR). Industrial air measurements and human biomonitoring (HBM) samples were gathered in specific occupational sectors to examine MDI, TDI, HDI and IPDI and the respective metabolites from Finnish screening studies. HBM data can give a more accurate picture of diisocyanate exposure, especially if workers have been exposed dermally or used respiratory protection. The HBM data were used for conducting a health impact assessment (HIA) in specific Finnish occupational sectors. For this purpose, exposure reconstruction was performed on the basis of HBM measurements of TDI and MDI exposures using a PBPK model, and a correlation equation was made for HDI exposure. Subsequently, the exposure estimates were compared to a previously published dose-response curve for excess BHR risk. The results showed that the mean and median diisocyanate exposure levels and HBM concentrations were low for all diisocyanates. In HIA, the excess risk of BHR from MDI exposure over a working life period was highest in the construction and motor and vehicle industries and repair sectors, resulting in estimated excess risks of BHR of 2.0% and 2.6%, and 113 and 244 extra BHR cases in Finland, respectively. Occupational exposure to diisocyanates must be monitored because a clear threshold for DI sensitisation cannot be established.

Improved Method for the Detection of Highly Polar Pesticides and Their Main Metabolites in Foods of Animal Origin: Method Validation and Application to Monitoring Programme

The application of polar pesticides in agricultural production has been of great interest due to their low costs and their high effectiveness. For this reason, the possibility of their transfer to foods of animal origin is of great concern for human health. The manuscript describes the implementation and validation of an analytical method to detect polar pesticides, at regulatory levels, in three foods of animal origin, including bovine fat, chicken eggs, and cow milk. The method was fully validated to detect glyphosate, glufosinate, and their respective metabolites in the above-mentioned foods obtaining fit-for-purpose sensitivity, recoveries (76–119%), repeatability (≤20%), within-laboratory reproducibility (≤20%), and experimental measurement uncertainty less than 50% as required by the SANTE/11312/2021 criteria. Given the satisfactory results, the applicability of the method to additional molecules belonging to the same category (AMPA, cyanuric acid, ethephon, fosetyl aluminum, HEPA, maleic hydrazide, and N-acetyl-glyphosate) was also evaluated in order to meet possible future requests. Finally, the implemented method was applied to analyse samples over the period of March 2021 to August 2022 from two Italian regions (Umbria and Marche) within the national monitoring programme. In agreement with previously available data, none of the samples analysed showed the presence of glyphosate and glufosinate at levels above the legal limit.

Smoked cannabis reduces peak cocaine plasma levels and subjective effects in a controlled drug administration study of polysubstance use in men

BackgroundDespite the high prevalence of polysubstance use, outcomes and potential risks associated with common drug combinations are not well characterized. Many individuals who use cocaine also use cannabis, yet little is known about how interactions between the two drugs might contribute to continued co-use. MethodsThe aim of this double-blind, placebo-controlled study was to determine the physiological and subjective effects of smoked cannabis with smoked cocaine, to identify variables that may contribute to the continued use of this drug combination. Healthy, non-treatment seeking volunteers who reported smoking both cocaine and cannabis (N = 9, all males) completed a 13-day inpatient protocol. On session days, cannabis [0.0 or 5.6 % tetrahydrocannabinol (THC)] was administered 28 min prior to cocaine (0, 12, or 25 mg). Dependent measures included pharmacokinetic assessment of THC and cocaine and their respective metabolites, in addition to subjective and cardiovascular effects. ResultsActive cannabis (5.6 % THC) increased plasma levels of THC and the metabolite 11-nor-9-carboxy-Δ9-THC (THCCOOH), as well as subjective ratings of cannabis effects and heart rate relative to inactive cannabis. Cocaine dose-dependently increased plasma cocaine and metabolites and subjective ratings of cocaine effects. Active cannabis pre-treatment decreased plasma levels of cocaine and metabolites. Furthermore, active cannabis attenuated cocaine-related reductions in ‘Hunger’ and ‘Calm.’ ConclusionsCannabis pre-treatment altered the subjective experience of smoked cocaine and reduced peak plasma levels of cocaine. Future studies should explore additional doses of each drug and whether these changes also impact cocaine’s reinforcing effects.

Knockout of ABCC1 in NCI-H441 cells reveals CF to be a suboptimal substrate to study MRP1 activity in organotypic in vitro models

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an efflux transporter responsible for the extrusion of endogenous substances as well as xenobiotics and their respective metabolites. Its high expression levels in lung tissue imply a key role in pulmonary drug disposition. Moreover, its association with inflammatory lung diseases underline MRP1’s relevance in drug development and precision-medicine. With the aim to develop a tool to better understand MRP1’s role in drug disposition and lung disease, we generated an ABCC1−/− clone based on the human distal lung epithelial cell line NCI-H441 via a targeted CRISPR/Cas9 approach. Successful knockout (KO) of MRP1 was confirmed by qPCR, immunoblot and Sanger sequencing. To assess potential compensatory upregulation of transporters with a similar substrate recognition pattern as MRP1, expression levels of MRP2–9 as well as OAT1–4, 6, 7 and 10 were measured. Functional transporter activity was determined via release studies with two prodrug/substrate pairs, i.e. 5(6)-carboxyfluorescein (CF; formed from its diacetate prodrug) and S-(6-(7-methylpurinyl))glutathione (MPG; formed from its prodrug 6-bromo-7-methylpurine, BMP), respectively. Lastly, transepithelial electrical resistance (TEER) of monolayers of the KO clone were compared with wildtype (WT) NCI-H441 cells. Of eight initially generated clones, the M2 titled clone showed complete absence of mRNA and protein in accordance with the designed genome edit. In transport studies using the substrate CF, however, no differences between the KO clone and WT NCI-H441 cells were observed, whilst no differences in expression of potential compensatory transporters was noted. On the other hand, when using BMP/MPG, the release of MPG was reduced to 11.5% in the KO clone. Based on these results, CF appears to be a suboptimal probe for the study of MRP1 function, particularly in organotypic in vitro and ex vivo models. Our ABCC1−/− NCI-H441 clone further retained the ability to form electrically tight barriers, making it a useful model to study MRP1 function in vitro.

MR-spectroscopy in metachromatic leukodystrophy: A model free approach and clinical correlation

Background and purposeMetachromatic leukodystrophy (MLD) is a lysosomal enzyme deficiency disorder leading to demyelination and subsequently to a progressive decline in cognitive and motor function. It affects mainly white matter where changes during the course of the disease can be visualized on T2-weighted MRI as hyperintense areas. Associated changes in brain metabolism can be quantified by MR spectroscopy (MRS) and may give complementary information as biomarkers for disease characterisation and progression. Our study aimed to further investigate the correlation of MRS with clinical parameters for motor and cognitive function by using a model free MRS analysis approach that would be precise and straightforward to implement. Materials and methods53 MRS datasets derived from 29 patients (10 late-infantile, 19 juvenile) and 12 controls were acquired using a semi-LASER CSI sequence covering a slice through the centrum semiovale above the corpus callosum. We defined four regions of interest in the white matter (frontal white matter [FWM] and the cortico-spinal tract [CST] area, each left and right) and one in cortical grey matter. Spectra were analysed using a model and fitting free approach by calculating the definite integral of 10 intervals which were distributed along the whole spectrum. These 10 intervals were orientated towards the main peaks of the metabolites N-acetylaspartate (NAA), creatine, myo-inositol, choline, glutamine/glutamate and aspartate to approximately attribute changes in the intervals to corresponding metabolites. Their ratios to the main creatine peak integral were correlated with clinical parameters assessing motor and cognitive abilities. Furthermore, in a post-hoc analysis, NAA levels of a subset of 21 MR datasets were correlated to NAA levels in urine measured by 1H (proton) nuclear magnetic resonance (NMR) spectroscopy.The applied interval integration method was validated in the control cohort against the standard approach, using spectral profile templates of known metabolites (LCModel). Both methods showed good agreement, with coefficients of variance being slightly lower for our approach compared to the related LCModel results. Moreover, the new approach was able to extract information out of the frequency range around the main peaks of aspartate and glutamine where LCModel showed only few usable values for the respective metabolites. ResultsMLD spectra clearly differed from controls. The most pronounced differences were found in white matter (much less in grey matter), with larger values corresponding to main peaks of myo-inositol, choline and aspartate, and smaller values associated with NAA and glutamine. Late-infantile patients had more severe changes compared to later-onset patients, especially in intervals corresponding to NAA, aspartate, myo-inositol, choline and glutamine.There was a high correlation of several intervals in the corticospinal tract region with motor function (with the most relevant interval corresponding to NAA peak with a correlation coefficient of −0.75; p < 0.001), while cognitive function, by means of IQ, was found to be most correlating in frontal white matter corresponding to the NAA peak (r = 0.84, p < 0.001).The post-hoc analysis showed that the main NAA peak interval correlated negatively with the NAA in urine (r = −0.584, p < 0.001). ConclusionThe applied model and fitting free interval integration approach to analyse MRS data of a semi-LASER sequence at 3T suits well to detect and quantify pathological changes in MLD patients through the different courses of the disease and correlates well with clinical symptoms while showing smaller dimensions of variation compared to the more sophisticated single metabolite analysis using LCModel. NAA seems the most clinically meaningful biomarker to use in this context. Its correlation with urine measurements further underlines its potential as a clinically and biologically useful parameter of disease progression in MLD.

Estrogen-DNA Adducts and Breast Cancer Risk in Premenopausal Asian Women.

This study provides evidence that endogenous estrogen-induced genotoxicity may contribute to the carcinogenesis of breast cancer in premenopausal Asian women. This work could have important preventive implication for the emerging disease in East Asia.

Recent advances in the detection of drugs of abuse by dried blood spots.

The purpose of this review study was to sum up the main recent advances reported in the scientific literature about the detection of common drugs of abuse in biological samples upon their collection by dried blood spot devices. The most recent, innovative and fully validated methods for the qualitative and/or quantitative detection of common drugs of abuse are reported, including alprazolam, clonazepam, diazepam, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methyl-enedioxyethylamphetamine, amphetamine, methamphetamine, cocaine, tetrahydrocannabinol, 6-monoacetylmorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, noroxycodone, tramadol, methadone, buprenorphine, fentanyl, ketamine and their respective metabolites and γ-hydroxybutyric acid. Dried blood spot proved to be extremely promising for routine analysis of forensic cases, although large-scale experiments on real samples need to be performed to confirm the emerging advantages of the technique and remove the potential limitations still affecting its widespread application.

Ecotoxicological risk assessment of 14 pesticides and corresponding metabolites to groundwater and soil organisms using China-PEARL model and RQ approach.

Global use of pesticides brings uncertain risks to human and nontarget species via environmental matrix. Currently, various models for exposure risk assessment are developed and widely used to forecast the impact of pesticides on environmental organisms. In this study, five commonly used insecticides, seven herbicides and three fungicides were chosen to analyze the subsequent risks in groundwater in simulated scenarios using China-PEARL (Pesticide Emission Assessment at Regional and Local Scales) model. In addition, their exposure risks to soil organisms were characterized based on risk quotient (RQ) approach. The results indicated that 23.3% of the total 528 predicted environmental concentrations (PECs) of pesticides and respective metabolites in groundwater from six Chinese simulated locations with ten crops were above 10μgL-1. Furthermore, acceptable human risks of pesticides in groundwater were observed for all simulation scenarios (RQ < 1). Based on the derived PECs in soil short-term and long-term exposure simulation scenarios, all compounds were evaluated to be with acceptable risks to soil organisms, except that imidacloprid was estimated to be with unacceptable chronic risk (RQ = 27.5) to earthworms. Overall, the present findings provide an opportunity for a more-comprehensive understanding of exposure toxicity risks of pesticides leaching into groundwater and soil.

Analysis of the Antioxidant Mechanism of Tamarix ramosissima Roots under NaCl Stress Based on Physiology, Transcriptomic and Metabolomic.

There is a serious problem with soil salinization that affects the growth and development of plants. Tamarix ramosissima Ledeb (T. ramosissima), as a halophyte, is widely used for afforestation in salinized soils. At present, there are few reports on the antioxidant mechanism of T. ramosissima under NaCl stress. In this study, we learned about the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and hydrogen peroxide (H2O2) and malondialdehyde (MDA) content changes in T. ramosissima. We also mined the relevant metabolic pathways in the antioxidant mechanism, candidate key genes, and their related differential metabolites and verified them using quantitative real-time PCR (qRT-PCR). The results show that the SOD, POD, and CAT activities, and the H2O2 and MDA content reached the highest values in the roots of T. ramosissima. Simultaneously, 92 differentially expressed genes (DEGs) related to antioxidant enzyme activities changed during 48 and 168 h of NaCl stress, and these DEGs were mainly upregulated in 168 h. Based on the association analysis of transcriptomic and metabolomic data, we found Unigene0089358 and Unigene0007782 as genes related to key enzymes in the flavonoid biosynthesis pathway. They were located in the upstream positive regulation at 48 and 168 h under NaCl stress, and their respective related metabolites (phloretin and pinocembrin) were involved in resistance to NaCl stress, and they were significantly correlated with their respective metabolites. In conclusion, at 48 and 168 h under NaCl stress, the roots of T. ramosissima resist NaCl stress by enhancing enzymatic and nonenzymatic antioxidant mechanisms, scavenging ROS generated by high-salt stress, alleviating NaCl toxicity, and maintaining the growth of T. ramosissima. This study provides genetic resources and a scientific theoretical basis for further breeding of salt-tolerant Tamarix plants and the molecular mechanism of antioxidants to alleviate NaCl toxicity.

Abstract 12910: Exercise-Induced Specialized Proresolving Mediators Stimulate AMPK Phosphorylation to Promote Mitochondrial Respiration in Macrophages

Emerging epidemiological data suggests that exercise imparts its CVD risk-reducing effects in part through improvements in systemic inflammation. The mechanisms underpinning the salutary effects of physical activity, however, remain incompletely understood. We recently reported that exercise enhances the production of specialized proresolving mediators (SPMs), which act as agonist of inflammation resolution through actions on macrophages (Mϕ). In this study, we find that wheel running accelerated the metabolism of proinflammatory lipid mediators LTB 4 and PGD 2 into their respective metabolites 20-OH-LTB 4 (Sed 27.14±5.88 vs Exe 70.40±10.82 pg/mL; P&lt;0.05; n =3-4) and PGJ 2 (Sed 92.63±16.08 vs Exe 227.90±30.23 pg/mL; P&lt;0.05; n =3-4), resulting in an increase in the ratio of proresolving-to-proinflammatory lipid mediator levels (RvE1+RvD1:leukotrienes; Sed 0.19±0.02 vs Exe 0.32±0.90; P&lt;0.05; n =3-4). These findings were associated with enhanced mitochondrial metabolism in Mϕ, as evidenced by increased basal (Sed 0.12±0.06 vs Exe 1.53±0.07; P&lt;0.05; n =3-4), maximum (Sed 9.20±0.30 vs Exe 12.02±0.82; P&lt;0.05; n =3-4), and ATP-linked oxygen consumption rate (Sed 1.25±0.06 vs Exe 1.47±0.09 pmol/min/μg protein; P&lt;0.05; n =3-4). We also find that SPMs RvD1, RvD2, RvE1 or MaR1 stimulate basal (Ctrl 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P&lt;0.05; n =3) and ATP-linked respiration (Ctrl 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P&lt;0.05; n =3). We find that an increase in basal (Ctrl 1.80±0.13 vs RvD1 2.43±0.19 pmol/min/μg protein; P&lt;0.05; n =3-5) and ATP-linked respiration (Ctrl 1.44±0.10 vs RvD1 1.70±0.15 pmol/min/μg protein; P&lt;0.05; n =3-5) with RvD1 treatment occurs concomitantly with AMPK phosphorylation (Ctrl 1.00±0.07 vs RvD1 1.40±0.02 pAMPK/AMPK fold change; P&lt;0.05; n =3) and inhibiting the kinase activity of AMPK with Compound C abrogates SPM-induced mitochondrial respiration. Taken together, these data suggest that exercise-enhanced SPM production stimulates mitochondrial metabolism in Mϕ via AMPK, which may contribute to exercise-induced amelioration of chronic inflammation.

Influence of Zhuanggu Guanjie Pill on Seven Cytochrome P450 Enzymes Based on Probe Cocktail and Pharmacokinetics Approaches.

The use of herbal medicines has tremendously increased over the past few decades. Case reports and controlled clinical investigations of herbal-drug interactions have been reported. Since Cytochrome P450 (CYP) enzymes play an important role in drug interactions. The evaluation of the influence of herbal medicines on the activities of CYPs is beneficial to promote scientific and rational clinical use of herbal medicines. Herein, we aimed to develop and validate a method to simultaneously quantify seven CYP cocktail probe drugs consisting of phenacetin (PNC), bupropion (BPP), losartan potassium (LK), omeprazole (OMP), dextromethorphan (DM), chlorzoxazone (CZZ) and midazolam (MDZ) and their respective metabolites in a single acquisition run and use this method to evaluate the influence of Zhuanggu Guanjie Pill (ZGGJP) on seven CYPs. A cost-effective and simple UHPLC-(±)ESI-MS/MS method for simultaneous determination of seven probe drugs and metabolites in rat plasma was developed and validated. Male and female rats were randomly divided into three groups and treated with 1.2 g/kg/d ZGGJP, 5 g/kg/d ZGGJP and 0.5% CMC-Na for 14 consecutive days. After 24 h of the last administration, all rats were administrated orally with probe drugs. The influence of ZGGJP on the CYPs was carried out by comparing the metabolic ratio (Cmax, AUC0-t) of metabolites/probe drugs in rats. The calibration curves were linear, with correlation coefficient > 0.99 for seven probe drugs and their corresponding metabolites. Intra- and inter-day precisions were not greater than 15% RSD and the accuracies were within ± 15% of nominal concentrations. The ZGGJP showed significant inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats. ZGGJP had inductive effects on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.

Cannabidiol, ∆9-tetrahydrocannabinol, and metabolites in human blood by volumetric absorptive microsampling and LC-MS/MS following controlled administration in epilepsy patients.

Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5-25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10-500 CBD and 7-OH-CBD; and 20-5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30µL blood.

Spatial-temporal occurrence of contaminants of emerging concern in urban rivers in southern Brazil

The widespread use and misuse of antibiotics and pesticides has been linked with several risks to the environment and human health. In the present report, the results of the monitoring of 64 pharmaceuticals and 134 pesticides occurrence in an urban river in Southern Brazil are presented and discussed. Sampling campaigns have covered the period 2016–2018. The identification and determination of the analytes were achieved by high-resolution mass spectrometry. The data were analyzed using chemometric tools to obtain spatial-temporal models. Toxicological evaluation was achieved using acute toxicity (zebrafish standardized protocol), and determination of risk quotient. Within the 198 analytes included in the targeted analysis method for surface water, 33 were identified in an urban river during 2 years of monitoring, being 20 pharmaceuticals and 13 pesticides. Using high-resolution mass spectrometry, a suspect screening approach was established in an un-target analysis. The evaluation was carried out using a data bank built from consumption data of drugs and pesticides, in the metropolitan region of Porto Alegre - RS and their respective metabolites. The suspect screening analysis done with a data bank with more than 1450 compounds results in 27 suspect findings. The target analysis results showed a continuous prevalence of non-steroidal anti-inflammatories, analgesics, antipyretics, beta-blockers, corticoids, and antibiotics. Regarding the pesticides, the main classes were fungicides, especially those from triazol and strobilurin classes.

Hepatic and renal toxicities and metabolism of fentanyl analogues in rats.

New synthetic opioids continue to emerge in the illicit market, and among them, fentanyl analogues pose a serious threat to the public health with their abuse and trafficking. We investigated the toxicity of fentanyl analogues on the liver and kidneys mediated by the µ-opioid receptor (MOR). Our study focused on 4-fluoro-isobutyrylfentanyl (4F-iBF), which is classified as a "narcotic" in Japan; structurally similar analogues 4-chloro-isobutyrylfentanyl (4Cl-iBF) and isobutyrylfentanyl (iBF) were also investigated. Rats that were intraperitoneally administered 4F-iBF (5mg/kg (12.3μmol/kg)) or iBF (12.3μmol/kg) displayed hepatic and renal ischemic-like damage, but 4Cl-iBF (12.3μmol/kg) did milder renal damage only. We found that the agonist activity of 4F-iBF, at MORs was approximately 7.2 times that of 4Cl-iBF, and that pretreatment with MOR antagonist naltrexone (0.8mg/kg) alleviated liver and kidney injuries caused by 4F-iBF. These results suggested that 4F-iBF might cause ischemic damage to the liver and kidneys, induced by respiratory depression mediated by MORs. Furthermore, to elucidate the metabolism of fentanyl analogues, we investigated the change over time in the amount of 4F-iBF, 4Cl-iBF, iBF (6.15μmol/kg, respectively), and their respective metabolites in serum after intraperitoneal administration to rats. The results showed that in 24-h post-dose serum, 4Cl-iBF and iBF were substantially eliminated while 4F-iBF remained at about 30% of the maximum level, and each of the N-dephenylethylated metabolites of 4F-iBF, 4Cl-iBF, and iBF was detected in 2-h post-dose serum. The results from this study revealed information on the hepatic and renal toxicities and metabolism related to fentanyl analogues.

High-altitude Hypoxia Influences the Activities of the Drug-Metabolizing Enzyme CYP3A1 and the Pharmacokinetics of Four Cardiovascular System Drugs.

(1) Background: High-altitude hypoxia has been shown to affect the pharmacokinetic properties of drugs. Although there is a high incidence of cardiovascular disease among individuals living in high-altitude areas, studies on the effect of high-altitude hypoxia on the pharmacokinetic properties of cardiovascular drugs are limited. (2) Methods: The aim of this study was to evaluate the pharmacokinetics of nifedipine, bosentan, simvastatin, sildenafil, and their respective main metabolites, dehydronifedipine, hydroxybosentan, simvastatin hydroxy acid, and N-desmethyl sildenafil, in rats exposed to high-altitude hypoxia. Additionally, the protein and mRNA expression of cytochrome P450 3A1 (CYP3A1), a drug-metabolizing enzyme, were examined. (3) Results: There were significant changes in the pharmacokinetic properties of the drugs in rats exposed to high-altitude hypoxia, as evidenced by an increase in the area under the curve (AUC) and the half-life (t1/2z) and a decrease in total plasma clearance (CLz/F). However, most of these changes were reversed when the rats returned to a normoxic environment. Additionally, there was a significant decrease in CYP3A1 expression in rats exposed to high-altitude hypoxia at both the protein and mRNA levels. (4) Conclusions: High-altitude hypoxia suppressed the metabolism of the drugs, indicating that the pharmacokinetics of the drugs should be re-examined, and the optimal dose should be reassessed in patients living in high-altitude areas.

LC-DAD-ESI-MS/MS and NMR Analysis of Conifer Wood Specialized Metabolites.

Many species from the Pinaceae family have been recognized as a rich source of lignans, flavonoids, and other polyphenolics. The great common occurrence of conifers in Europe, as well as their use in the wood industry, makes both plant material and industrial waste material easily accessible and inexpensive. This is a promising prognosis for both discovery of new active compounds as well as for finding new applications for wood and its industry waste products. This study aimed to analyze and phytochemically profile 13 wood extracts of the Pinaceae family species, endemic or introduced in Polish flora, using the LC-DAD–ESI-MS/MS method and compare their respective metabolite profiles. Branch wood methanolic extracts were phytochemically profiled. Lignans, stilbenes, flavonoids, diterpenes, procyanidins, and other compounds were detected, with a considerable variety of chemical content among distinct species. Norway spruce (Picea abies (L.) H.Karst.) branch wood was the most abundant source of stilbenes, European larch (Larix decidua Mill.) mostly contained flavonoids, while silver fir (Abies alba Mill.) was rich in lignans. Furthermore, 10 lignans were isolated from the studied material. Our findings confirm that wood industry waste materials, such as conifer branches, can be a potent source of different phytochemicals, with the plant matrix being relatively simple, facilitating future isolation of target compounds.

Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization

Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues. Herein, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI), an ambient liquid extraction-based ionization technique, as a viable alternative to other MSI techniques to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. MSI data acquired over a broad m/z range showed low signals of the drug and its metabolites resulting from the low ionization efficiency and substantial signal suppression on the tissue. Significant improvements in the signal-to-noise were obtained using selected ion monitoring (SIM) with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide and hydroxydiclofenac are localized to the inner medulla and cortex of the kidney, respectively, which is consistent with the previously reported localization of enzymes that process diclofenac into its respective metabolites. In contrast, a uniform distribution of diclofenac and its metabolites was observed in the liver tissue. Concentration ratios of diclofenac and hydroxydiclofenac calculated from nano-DESI MSI data are generally in agreement to those obtained using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collectively, our results demonstrate that nano-DESI MSI can be successfully used to image diclofenac and its primary metabolites and derive relative quantitative data from different tissue regions. Our approach will enable a better understanding of metabolic processes associated with diclofenac and other drugs that are difficult to analyze using commercially available MSI platforms.

Effects of social isolation on 50-kHz ultrasonic vocalizations, affective state, cognition, and neurotransmitter concentrations in the ventral tegmental and locus coeruleus of adult rats

Vocal communication, cognition, and affective state are key features of sustained health and wellness, and because vocalizations are often socially-motivated, social experience likely plays a role in these behaviors. The monoaminergic systems of the ventral tegmental area (VTA) and the locus coeruleus (LC) are associated with social and reward processing, vocalization production, and neurotransmitter changes in response to environmental stressors. The effect of social isolation on these complex behaviors and the underlying neural mechanisms is relatively unknown. To add to this body of literature, we randomized adult male Long-Evans rats to control (housed with a cagemate) or isolated (housed individually) conditions and assayed ultrasonic vocalizations, cognition (novel object recognition test), anxiety (elevated plus maze) and anhedonia (sucrose preference test) at 2, 4, 6, 8, and 10 months of age. At 10 months, VTA and LC samples were assayed for dopamine, norepinephrine, and serotonin using high performance liquid chromatography. We tested the hypotheses that isolation 1) diminishes vocalizations and cognition, 2) increases anxiety and depression, and 3) increases levels of dopamine, norepinephrine, and serotonin in the VTA and LC. Results showed isolation significantly reduced vocalization tonality (signal-to-noise ratio) and increased maximum frequency. There were no significant findings for cognition, anxiety, or anhedonia. Dopamine and serotonin and their respective metabolites were significantly increased in the VTA in isolated rats. These findings suggest chronic changes to social condition such as isolation affects vocalization production and levels of VTA neurotransmitters.