Development and Validation of A GC-MS Method Based on Solid Phase Extraction and Derivatization for Analysis of Free and Glucuronide-Conjugated Bisphenol F in Biological Samples.

BACKGROUND: As one of the speculated bisphenols to replace bisphenol A (BPA), bisphenol F (BPF), naturally present in mustard, is structurally similar to BPA and may have similar estrogenic activity, but information on its toxicity is very limited compared to BPA. OBJECTIVE: In order to support the toxicology study of BPF at Heath Canada, a gas chromatography (GC) with mass spectrometry (MS) method based on solid phase extraction and derivatization was developed for analysis of BPF in liver samples. METHODS: Samples were treated with ß-glucuronidase to convert BPF glucuronide to free BPF for analysis of total BPF. RESULTS: The method was validated for free BPF at different spiking levels, and recoveries ranged from 90.0-97.5% with relative standard deviations from 0.11-5.54%. The method was also validated for glucuronide-conjugated BPF at different spiking levels of BPF mono-ß-D-glucuronide, recoveries ranged from 72.3-93.3% with relative standard deviations from 1.7-8.94%. The method was used to analyse 60 liver tissue samples from the rats dosed with BPF at different levels in a toxicology study. Free and glucuronide-conjugated BPF were not detected in any of the control samples which were not dosed with BPF (average method detection limit: 0.31 ng/g), but detected in all the other liver tissue samples with levels increasing at higher doses. The percent of glucuronide-conjugated BPF in total BPF varied among the liver samples, from as low as 9.8% to as high as 77.9%, indicating the importance of analysing biological samples for BPF in both free and conjugated forms for total exposure. CONCLUSION: A GC-MS method based on solid phase extraction and derivatization was developed for analysis of both free and glucuronide-conjugated BPF in liver samples. This method was validated not only for free BPF, but also for mono-ß-D-glucuronide conjugated BPF for the first time to confirm the efficiency of the deconjugation procedure with enzyme. HIGHLIGHTS: This method can be adapted and applied for analysis of free and glucuronide-conjugated BPF in other biological samples with appropriate validation in target sample matrices.

The role of drug efflux and uptake transporters in the plasma pharmacokinetics and tissue disposition of morphine and its main metabolites.

Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.

Unraveling the therapeutic potential of quercetin and quercetin-3-O-glucuronide in Alzheimer's disease through network pharmacology, molecular docking, and dynamic simulations.

Quercetin is a flavonoid with notable pharmacological effects and promising therapeutic potential. Quercetin plays a significant role in neuroinflammation, which helps reduce Alzheimer's disease (AD) severity. Quercetin (Q) and quercetin 3-O-glucuronide (Q3OG) are some of the most potent antioxidants available from natural sources. However, the natural form of quercetin converted into Q3OG when reacted with intestinal microbes. The study aims to ensure the therapeutic potential of Q and Q3OG. In this study, potential molecular targets of Q and Q3OG were first identified using the Swiss Target Prediction platform and pathogenic targets of AD were identified using the DisGeNET database. Followed by compound and disease target overlapping, 77 targets were placed in that AKT1, EGFR, MMP9, TNF, PTGS2, MMP2, IGF1R, MCL1, MET and PARP1 was the top-ranked target, which was estimated by CytoHubba plug-in. The Molecular docking was performed for Q and Q3OG towards the PDB:1UNQ target. The binding score of Q and Q3OG was - 6.2 kcal/mol and - 6.58 kcal/mol respectively. Molecular dynamics simulation was conducted for Q and Q3OG towards the PDB:1UNQ target at 200 ns. This study's results help identify the multiple target sites for the bioactive compounds. Thus, synthesizing new chemical entity-based quercetin on structural modification may aid in eradicating AD complications.

Naturalistic comparison of clomethiazole and Diazepam treatment in alcohol withdrawal: effects on oxidative stress, inflammatory cytokines and hepatic biomarkers.

Ethanol is metabolized by alcohol dehydrogenase to acetaldehyde and induces cytochrome P450 2E1 (CYP2E1), which generates reactive oxygen species that cause inflammatory liver damage. Clomethiazole, a drug approved for alcohol withdrawal treatment (AWT) in some European countries, inhibits CYP2E1. We hypothesized that clomethiazole would lead to a faster reduction in oxidative stress, inflammatory cytokines, and liver enzymes compared to diazepam treatment. We analysed respective biomarkers in 50 patients undergoing AWT and 25 healthy individuals but found no statistical difference between the two medication groups over 3-5 days. Hence, our hypothesis was not confirmed during this observation period.

Confirmation of ethanol administration in racing greyhounds by LC-MS-MS.

Ethanol is a prohibited substance in professional animal racing as its administration causes physiological effects such as depression of the central nervous system. Regulation of potential doping agents, including those that inhibit performance, is critical to ensure integrity and animal welfare in greyhound racing, but the detection of ethanol is complicated by dietary and/or environmental exposure. In response, a reliable analytical method capable of detecting recent ethanol administration in greyhound urine samples was validated and implemented. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used to investigate the variation in urinary ethanol metabolites; ethyl-ß-D glucuronide (EG; γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ = 1.0 µg/ml, s EG $$ {s}_{\mathrm{EG}} $$ = 3.3 µg/ml) and ethyl sulfate (ES; γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ = 0.9 µg/ml, s ES $$ {s}_{\mathrm{ES}} $$ = 1.9 µg/ml) levels from a reference population of 202 racing greyhounds. These were compared to urine samples collected following administration of ethanol to one male and one female greyhound. Results were used to establish a threshold within the national rules of greyhound racing: γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ and γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ > 20 µg/ml in urine are defensible criteria to confirm ethanol administration to greyhounds. Case studies of competition samples are provided to demonstrate the forensic translation of this work.

Evaluation of harmful drinking among professional drivers by direct ethanol biomarkers and its relation with psychological distress.

OBJECTIVE: This study aimed to evaluate the alcohol consumption among professional truck and bus drivers using direct ethanol biomarkers, and to explore its relationship with anxiety, depression, and stress. METHODS: The assessment of potential harmful drinking was conducted through the measurement of direct biomarkers: phosphatidylethanol (PEth), ethyl glucuronide (EtG), and ethyl sulfate (EtS), using dried blood spots (DBS). Additionally, self-reported data from the Alcohol Use Disorders Identification Test (AUDIT-C) were used. Emotional states, including depression, anxiety, and stress, were evaluated using the Depression, Anxiety, and Stress Scale (DASS-21). RESULTS: A total of 97 drivers participated in the study, with the majority being male (96%) and identified as truck drivers (75.3%). Among them, 43.3% reported working more than 10 h daily. The majority of volunteers exhibited normal levels of stress (81.4%), anxiety (83%), and depression (86.6%). According to the AUDIT-C assessment, 30.9% were categorized as having a moderate risk, while 11.3% were deemed to be at high risk for harmful alcohol consumption behavior. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) levels, indicating recent ethanol consumption, were detected in 14.4% of the drivers. In contrast, the long half-life metabolite PEth (16:0-18:1) was present in 88.7% of the volunteers. A moderate correlation (rs = 0.45, p < .01) was observed between PEth levels and AUDIT-C scores. The Receiver Operating Characteristic (ROC) curve, utilizing a PEth threshold of ≥ 59.0 ng ml-1, displayed 78% sensitivity and 73% specificity in effectively distinguishing high risk for alcohol intake. Notably, no significant associations were found between alcohol consumption and levels of stress, depression, and anxiety. CONCLUSIONS: The study findings indicate a noteworthy proportion of drivers engaging in regular alcohol consumption alongside a demanding workload. Notably, PEth measurements highlighted an underreporting within the AUDIT-C self-reports. These results lend robust support for the utilization of biomarkers in assessing alcohol consumption patterns among drivers.

Tauroursodeoxycholate prevents estradiol 17ß-d-glucuronide-induced cholestasis and endocytosis of canalicular transporters by switching off pro-cholestatic signaling pathways.

AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.

Luteolin-7-O-ß-d-Glucuronide Attenuated Cerebral Ischemia/Reperfusion Injury: Involvement of the Blood-Brain Barrier.

Ischemic stroke is a common cerebrovascular disease with high mortality, high morbidity, and high disability. Cerebral ischemia/reperfusion injury seriously affects the quality of life of patients. Luteolin-7-O-ß-d-glucuronide (LGU) is a major active flavonoid compound extracted from Ixeris sonchifolia (Bge.) Hance, a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the protective effect of LGU on cerebral ischemia/reperfusion injury was investigated in an oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal model and a transient middle cerebral artery occlusion (tMCAO) rat model. In in vitro experiments, LGU was found to improve the OGD/R-induced decrease in neuronal viability effectively by the MTT assay. In in vivo experiments, neurological deficit scores, infarction volume rates, and brain water content rates were improved after a single intravenous administration of LGU. These findings suggest that LGU has significant protective effects on cerebral ischemia/reperfusion injury in vitro and in vivo. To further explore the potential mechanism of LGU on cerebral ischemia/reperfusion injury, we performed a series of tests. The results showed that a single administration of LGU decreased the content of EB and S100B and ameliorated the abnormal expression of tight junction proteins ZO-1 and occludin and metalloproteinase MMP-9 in the ischemic cerebral cortex of the tMCAO 24-h injury model. In addition, LGU also improved the tight junction structure between endothelial cells and the degree of basement membrane degradation and reduced the content of TNF-α and IL-1ß in the brain tissue. Thereby, LGU attenuated cerebral ischemia/reperfusion injury by improving the permeability of the blood-brain barrier. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

Transacylation and hydrolysis of the acyl glucuronides of ibuprofen and its α-methyl-substituted analogues investigated by 1H NMR spectroscopy and computational chemistry: Implications for drug design.

Drugs and drug metabolites containing a carboxylic-acid moiety can undergo in vivo conjugation to form 1-ß-O-acyl-glucuronides (1-ß-O-AGs). In addition to hydrolysis, these conjugates can undergo spontaneous acyl migration, and anomerisation reactions, resulting in a range of positional isomers. Facile transacylation has been suggested as a mechanism contributing to the toxicity of acyl glucuronides, with the kinetics of these processes thought to be a factor. Previous 1H NMR spectroscopic and HPLC-MS studies have been conducted to measure the degradation rates of the 1-ß-O-AGs of three nonsteroidal anti-inflammatory drugs (ibufenac, R-ibuprofen, S-ibuprofen) and a dimethyl-analogue (termed here as "bibuprofen"). These studies have also determined the relative contributions of hydrolysis and acyl migration in both buffered aqueous solution, and human plasma. Here, a detailed kinetic analysis is reported, providing the individual rate constants for the acyl migration and hydrolysis reactions observed in buffer for each of the 4 AGs, together with the overall degradation rate constants of the parent 1-ß-O-AGs. Computational modelling of the reactants and transition states of the transacylation reaction using density functional theory indicated differences in the activation energies that reflected the influence of both substitution and stereochemistry on the rate of transacylation/hydrolysis.

Population pharmacokinetics of primaquine and its metabolites in African males.

BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites' pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

PURPOSE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. RESULTS: We found that the recombinant ß-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis.

The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.

Luteolin-7-O-ß-d-glucuronide Ameliorates Cerebral Ischemic Injury: Involvement of RIP3/MLKL Signaling Pathway.

Luteolin-7-O-ß-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca2+ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

Quercetin-3-O-ß-D-glucuronide attenuates osteoarthritis by inhibiting cartilage extracellular matrix degradation and inflammation.

Objective: Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage damage. In order to find a safer and more effective drug to treat OA, we investigated the role of quercetin-3-O-ß-D-glucuronide (Q3GA) in OA. Methods: We used qRT-PCR and western blots to detect the effects of Q3GA on extracellular matrix (ECM) and inflammation related genes and proteins in interleukin-1ß (IL-1ß) induced chondrocytes. We determined the effect of Q3GA on the NF-κB pathway using western blots and immunofluorescence. Moreover, the effect of Q3GA on the Nrf2 pathway was evaluated through molecular docking, western blots, and immunofluorescence experiments and further validated by transfection with Nrf2 siRNA. Subsequently, we established a rat model of OA and injected Q3GA into the joint cavity for treatment. After 5 weeks of Q3GA administration, samples were obtained for micro-computed tomography scanning and histopathological staining to determine the effects of Q3GA on OA rats. Results: We found that Q3GA reduced the degradation of ECM and the expression of inflammatory related proteins and genes in primary chondrocytes of rats induced by IL-1ß, as well as the expression of nitric oxide (NO) and reactive oxygen species (ROS). It inhibited the activation of the NF-κB pathway by increasing the expression of Nrf2 in the nucleus. In addition, Q3GA inhibited cartilage degradation in OA rats and promoted cartilage repair. Conclusion: Q3GA attenuates OA by inhibiting ECM degradation and inflammation via the Nrf2/NF-κB axis. The translational potential of this article: The results of our study demonstrate the promising potential of Q3GA as a candidate drug for the treatment of OA and reveal its key mechanisms.

Construction of a fused grid-based CYP2C8-Template system and the application.

A ligand-accessible space in the CYP2C8 active site was reconstituted as a fused grid-based Template∗ with the use of structural data of the ligands. An evaluation system of CYP2C8-mediated metabolism has been developed on Template with the introduction of the idea of Trigger-residue initiated ligand-movement and fastening. Reciprocal comparison of the data of simulation on Template with experimental results suggested a unified way of the interaction of CYP2C8 and its ligands through the simultaneous plural-contact with Rear-wall of Template. CYP2C8 was expected to have a room for ligands between vertically standing parallel walls termed Facial-wall and Rear-wall. Both the walls were separated by a distance corresponding to 1.5-Ring (grid) diameter size, which was termed Width-gauge. The ligand sittings were stabilized through contacts with Facial-wall and the left-side borders of Template including specific Position 29, left-side border of Rings I/J, or Left-end, after Trigger-residue initiated ligand-movement. Trigger-residue movement is suggested to force ligands to stay firmly in the active site and then to initiate CYP2C8 reactions. Simulation experiments for over 350 reactions of CYP2C8 ligands supported the system established.

An LC-MS/MS Method for Quantification of Lamotrigine and Its Main Metabolite in Dried Blood Spots.

BACKGROUND: The antiepileptic drug lamotrigine (LTG) shows high pharmacokinetic variability due to genotype influence and concomitant use of glucuronidation inducers and inhibitors, both of which may be frequently taken by elderly patients. Our goal was to develop a reliable quantification method for lamotrigine and its main glucuronide metabolite lamotrigine-N2-glucuronide (LTG-N2-GLU) in dried blood spots (DBS) to enable routine therapeutic drug monitoring and to identify altered metabolic activity for early detection of drug interactions possibly leading to suboptimal drug response. RESULTS: The analytical method was validated in terms of selectivity, accuracy, precision, matrix effects, haematocrit, blood spot volume influence, and stability. It was applied to a clinical study, and the DBS results were compared to the concentrations determined in plasma samples. A good correlation was established for both analytes in DBS and plasma samples, taking into account the haematocrit and blood cell-to-plasma partition coefficients. It was demonstrated that the method is suitable for the determination of the metabolite-to-parent ratio to reveal the metabolic status of individual patients. CONCLUSIONS: The clinical validation performed confirmed that the DBS technique is a reliable alternative for plasma lamotrigine and its glucuronide determination.

Non-linear blood-brain barrier transport and dosing strategies influence receptor occupancy ratios of morphine and its metabolites in pain matrix.

BACKGROUND AND PURPOSE: Morphine is important for treatment of acute and chronic pain. However, there is high interpatient variability and often inadequate pain relief and adverse effects. To better understand variability in the dose-effect relationships of morphine, we investigated the effects of its non-linear blood-brain barrier (BBB) transport on µ-receptor occupancy in different CNS locations, in conjunction with its main metabolites that bind to the same receptor. EXPERIMENTAL APPROACH: CNS exposure profiles for morphine, M3G and M6G for clinically relevant dosing regimens based on intravenous, oral immediate- and extended-release formulations were generated using a physiology-based pharmacokinetic model of the CNS, with non-linear BBB transport of morphine. The simulated CNS exposure profiles were then used to derive corresponding µ-receptor occupancies at multiple CNS pain matrix locations. KEY RESULTS: Simulated CNS exposure profiles for morphine, M3G and M6G, associated with non-linear BBB transport of morphine resulted in varying µ-receptor occupancies between different dose regimens, formulations and CNS locations. At lower doses, the µ-receptor occupancy of morphine was relatively higher than at higher doses of morphine, due to the relative contribution of M3G and M6G. At such higher doses, M6G showed higher occupancy than morphine, whereas M3G occupancy was low throughout the dose ranges. CONCLUSION AND IMPLICATIONS: Non-linear BBB transport of morphine affects the µ-receptor occupancy ratios of morphine with its metabolites, depending on dose and route of administration, and CNS location. These predictions need validation in animal or clinical experiments, to understand the clinical implications.

The Active Glucuronide Metabolite of the Brain Protectant IMM-H004 with Poor Blood-Brain Barrier Permeability Demonstrates a High Partition in the Rat Brain via Multiple Mechanisms.

BACKGROUND: Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. METHODS: First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood-brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. RESULTS: IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. CONCLUSIONS: The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood-brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood-brain barrier metabolism and transport of active glucuronide conjugates.

In vitro and in vivo protective potential of quercetin-3-glucuronide against lipopolysaccharide-induced pulmonary injury through dual activation of nuclear factor-erythroid 2 related factor 2 and autophagy.

In vitro and in vivo models of lipopolysaccharide (LPS)-induced pulmonary injury, quercetin-3-glucuronide (Q3G) has been previously revealed the lung-protective potential via downregulation of inflammation, pyroptotic, and apoptotic cell death. However, the upstream signals mediating anti-pulmonary injury of Q3G have not yet been clarified. It has been reported that concerted dual activation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and autophagy may prove to be a better treatment strategy in pulmonary injury. In this study, the effect of Q3G on antioxidant and autophagy were further investigated. Noncytotoxic doses of Q3G abolished the LPS-caused cell injury, and reactive oxygen species (ROS) generation with inductions in Nrf2-antioxidant signaling. Moreover, Q3G treatment repressed Nrf2 ubiquitination, and enhanced the association of Keap1 and p62 in the LPS-treated cells. Q3G also showed potential in inducing autophagy, as demonstrated by formation of acidic vesicular organelles (AVOs) and upregulation of autophagy factors. Next, the autolysosomes formation and cell survival were decreased by Q3G under pre-treatment with a lysosome inhibitor, chloroquine (CQ). Furthermore, mechanistic assays indicated that anti-pulmonary injury effects of Q3G might be mediated via Nrf2 signaling, as confirmed by the transfection of Nrf2 siRNA. Finally, Q3G significantly alleviated the development of pulmonary injury in vivo, which may result from inhibiting the LPS-induced lung dysfunction and edema. These findings emphasize a toxicological perspective, providing new insights into the mechanisms of Q3G's protective effects on LPS-induced pulmonary injury and highlighting its role in dual activating Nrf2 and autophagy pathways.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. AIM OF THE STUDY: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. MATERIALS AND METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-ß-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 µM, 9.05 µM, 3.87 µM, and 7.00 µM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. CONCLUSION: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.