Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (APOB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic depletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor-associated cholesterol deposits, and photoreceptor cell death, and loss of rod but not cone function. RPE-specific reduction in Mttp had no significant effect on plasma lipids and lipoproteins. While APOB was decreased in the RPE, most ocular retinoids remained unchanged, with the exception of the storage form of retinoid, retinyl ester. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but is not directly involved in plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

Prostaglandin production selectively in brain endothelial cells is both necessary and sufficient for eliciting fever.

Fever is known to be elicited by prostaglandin E2 acting on the brain, but its origin has remained disputed. We show in mice that selective deletion of prostaglandin synthesis in brain endothelial cells, but not in neural cells or myeloid cells, abolished fever induced by intravenous administration of lipopolysaccharide and that selective rescue of prostaglandin synthesis in brain endothelial cells reinstated fever. These data demonstrate that prostaglandin production in brain endothelial cells is both necessary and sufficient for eliciting fever.

Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (rs1051740) and Enos (rs1799983).

Recurrent pregnancy loss (RPL) affects around 2% of women of reproductive age. Primary RPL is defined by ≥2 pregnancy losses and no normal birth delivery. In secondary RPL, the losses are after a normal pregnancy and delivery. Most cases have no clear aetiology, although primary cases are the most complex. Several gene single nucleotide polymorphisms (SNPs) have been associated with RPL. The frequency of some SNPs is increased in women suffering from RLP from Asian or Caucasian races; however, in admixed populations, the information on possible genetic links is scarce and contradictory. This study aimed to assess the frequency of two SNPs present in two different enzymes involved in medical conditions observed during pregnancy. It is a case-control study. Microsomal epoxy hydrolase (mEPH) is involved in detoxifying xenobiotics, is present in the ovaries, and is hormonally regulated. The endothelial nitric oxide synthase (NOS3) that forms nitric is involved in vascular tone. Two SNPs, rs1051740 (mEPH) and rs1799983 (NOS3), were assessed. The study included 50 controls and 63 primary RPL patients. The frequency of mutated alleles in both SNPs was significantly higher in patients (p < 0.05). Double-mutated homozygotes were encountered only in RPL patients (p < 0.05). Genetic polymorphisms rs1051740 and rs1799983 may be involved in primary RPL in the Venezuelan admix population. Genetic studies could provide crucial information on the aetiology of primary RPL.

Adaptive Effects of Intermittent Hypoxia Training on Oxygen-Dependent Processes as a Potential Therapeutic Strategy Tool.

BACKGROUND/AIMS: Important benefits of intermittent hypoxic training (IHT) have emerged as an effective tool for enhancing adaptive potential in different pathological states, among which acute hypoxia dominates. Therefore, the aim of our study was to evaluate the mechanisms related to the effects of the nitric oxide system (nitrites, nitrates, carbamide, and total polyamine content) on ADP-stimulated oxygen consumption and oxidative phosphorylation in heart and liver mitochondria and biomarkers of oxidative stress in the blood, heart, and liver of rats exposed to the IHT method and acute hypoxia and treated with the amino acid L-arginine (600 mg/kg, 30 min) or the NO synthase inhibitor L-NNA (35 mg/kg, 30 min) prior to each IHT session. METHODS: We analysed the modulation of the system of oxygen-dependent processes (mitochondrial respiration with the oxygraphic method, microsomal oxidation, and lipoperoxidation processes using biochemical methods) in tissues during IHT in the formation of short-term and long-term effects (30, 60, and 180 days after the last IHT session) with simultaneous administration of L-arginine. In particular, we investigated how mitochondrial functions are modulated during intermittent hypoxia with the use of oxidation substrates (succinate or α-ketoglutarate) in bioenergetic mechanisms of cellular stability and adaptation. RESULTS: The IHT method is associated with a significant increase in the production of endogenous nitric oxide measured by the levels of its stable metabolite, nitrite anion, in both plasma (almost 7-fold) and erythrocytes (more than 7-fold) of rats. The intensification of nitric oxide-dependent pathways of metabolic transformations in the energy supply processes in the heart and liver, accompanied by oscillatory mechanisms of adaptation in the interval mode, causes a probable decrease in the production of urea and polyamines in plasma and liver, but not in erythrocytes. The administration of L-arginine prior to the IHT sessions increased the level of the nitrite-reducing component of the nitric oxide cycle, which persisted for up to 180 days of the experiment. CONCLUSION: Thus, the efficacy of IHT and its nitrite-dependent component shown in this study is associated with the formation of long-term adaptive responses by preventing the intensification of lipoperoxidation processes in tissues due to pronounced changes in the main enzymes of antioxidant defence and stabilisation of erythrocyte membranes, which has a pronounced protective effect on the system of regulation of oxygen-dependent processes as a whole.

Vaccine-induced neutralizing antibodies bind to the H protein of a historical measles virus.

Measles is a highly contagious airborne viral disease. It can lead to serious complications and death and is preventable by vaccination. The live-attenuated measles vaccine (LAMV) derived from a measles virus (MV) isolated in 1954 has been in use globally for six decades and protects effectively by providing a durable humoral and cell-mediated immunity. Our study addresses the temporal stability of epitopes on the viral surface glycoprotein hemagglutinin (H) which is the major target of MV-neutralizing antibodies. We investigated the binding of seven vaccine-induced MV-H-specific monoclonal antibodies (mAbs) to cell-free synthesized MV-H proteins derived from the H gene sequences obtained from a lung specimen of a fatal case of measles pneumonia in 1912 and an isolate from a current case. The binding of four out of seven mAbs to the H protein of both MV strains provides evidence of epitopes that are stable for more than 100 years. The binding of the universally neutralizing mAbs RKI-MV-12b and RKI-MV-34c to the H protein of the 1912 MV suggests the long-term stability of highly conserved epitopes on the MV surface.

A phase 2 trial investigating the efficacy and safety of the mPGES-1 inhibitor vipoglanstat in systemic sclerosis-related Raynaud's.

OBJECTIVE: Our objective was to test the hypothesis, in a double-blind, placebo-controlled study that vipoglanstat, an inhibitor of microsomal prostaglandin E synthase-1 (mPGES-1) which decreases prostaglandin E2 (PGE2) and increases prostacyclin biosynthesis, improves RP. METHODS: Patients with systemic sclerosis (SSc) and ≥7 RP attacks during the last screening week prior to a baseline visit were randomised to four weeks treatment with vipoglanstat 120 mg or placebo. A daily electronic diary captured RP attacks (duration and pain) and Raynaud's Condition Score, with change in RP attacks/week as primary end point. Cold challenge assessments were performed at baseline and end of treatment. Exploratory endpoints included patients' and physicians' global impression of change, Assessment of Scleroderma-associated Raynaud's Phenomenon questionnaire, mPGES-1 activity, and urinary excretion of arachidonic acid metabolites. RESULTS: Sixty-nine subjects received vipoglanstat (n = 33) or placebo (n = 36). Mean weekly number of RP attacks (baseline; vipoglanstat 14.4[SD 6.7], placebo 18.2[12.6]) decreased by 3.4[95% CI -5.8;-1.0] and 4.2[-6.5;-2.0] attacks per week (p= 0.628) respectively. All patient reported outcomes improved, with no difference between the groups. Mean change in recovery of peripheral blood flow after cold challenge did not differ between the study groups. Vipoglanstat fully inhibited mPGES-1, resulting in 57% reduction of PGE2 and 50% increase of prostacyclin metabolites in urine. Vipoglanstat was safe and well tolerated. CONCLUSION: Although vipoglanstat was safe, and well tolerated in a dose achieving full inhibition of mPGES-1, it was ineffective in SSc-related RP. Further development and evaluation of vipoglanstat will therefore be in other diseases where mPGES-1 plays a pathogenetic role.

Characterization of the 263K-derived microsomal fraction: a source of prions for nanofiltration validation studies.

BACKGROUND: The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain-derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. STUDY DESIGN AND METHODS: The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K-infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster-PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. RESULTS: A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22-528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. CONCLUSION: Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration.

Design, synthesis, and evaluation of formylpiperazine analogs of Ferrostatin-1 as novel improved ferroptosis inhibitors.

In this study, a series of new formylpiperazine-derived ferroptosis inhibitors were designed and synthesized based on the structure of a known ferroptosis inhibitor, ferrostatin-1 (Fer-1). The anti-ferroptosis activity of these synthetic compounds in human umbilical vein endothelial cells (HUVECs) induced by Erastin was evaluated. It was found that some of the new compounds, especially compound 26, showed potent anti-ferroptosis activity, as evidenced by its ability to restore cell viability, reduce iron accumulation, scavenge reactive oxygen species, maintain mitochondrial membrane potential, increase GSH levels, decrease LPO and MDA content, and upregulate GPX4 expression. Moreover, compound 26 exhibited superior microsomal stability than Fer-1. The present results suggest that compound 26 is a promising lead compound for the development of new ferroptosis inhibitors for the treatment of vascular diseases.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson's disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.

Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells.

High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases.

Do the Effects of Krebs Cycle Intermediates on Oxygen-Dependent Processes in Hypoxia Mediated by the Nitric Oxide System Have Reciprocal or Competitive Relationships?

BACKGROUND/AIMS: Currently, it is proven that the cellular metabolism of nitric oxide is necessary to maintain optimal health and adaptation of the organism to the impact of various environmental factors. The aim of this work was to reveal the biological role of nitric oxide, its metabolic changes, and its mechanism of action in tissues under hypoxia, as well as the possibility of tissue metabolism correction through NO-dependent systems under the influence of Krebs cycle intermediates. METHODS: A systematic assessment of the effect of succinate (SC, 50 mg/kg b.w.) and α-ketoglutarate (KGL, 50 mg/kg b.w.) in the regulation of oxygendependent processes in rats (mitochondrial oxidative phosphorylation, microsomal oxidation, intensity of lipid peroxidation processes, and the state of the antioxidant defense system) depending on functional changes in nitric oxide production during hypoxia was evaluated. The state of the nitric oxide system was estimated spectrophotometrically by determination of the concentration of its stable nitrite anion metabolite (NO2 -). The levels of catecholamines were estimated from the content of epinephrine and norepinephrine using the differentially fluorescent method. The activity of cytochrome P450-dependent aminopyrine-N-demethylase was determined with the Nash reagent. RESULTS: Tissue hypoxia and metabolic disorders caused by this condition through changes in the content of catecholamines (epinephrine, norepinephrine, dopamine, DOPA) as well as the cholinesterase-related system (acetylcholine content and acetylcholinesterase activity) were the studied experimental parameters under acute hypoxia (AH, 7% O2 in N2, 30 min). The activation of lipid peroxidation and oxidatively modified proteins and an increase in the epinephrine content in AH are associated with an increased role of SC and a decrease in KGL as substrates of oxidation in mitochondria. A more pronounced effect of exogenous KGL, compared to SC, on the content of nitrite anion as a stable metabolite of nitric oxide in the liver under acute hypoxia against the background of a decrease in the intensity of lipid peroxidation processes was revealed. The activation of SC-dependent mitochondrial oxidative processes caused by AH was found to decrease in animals after an intermittent hypoxia training (IHT) course. IHT (7% O2 in N2, 15-min, 5 times daily, 14 days) prevented the activation of oxidative stress in tissues and blood after the AH impact and increased the efficiency of energy-related reactions in the functioning of hepatic mitochondria through increased oxidation of KGL. CONCLUSION: The studied effects of adaptation are mediated by an increase in the role of NO-dependent mechanisms, as assessed by changes in the pool of nitrates, nitrites, carbamides, and total polyamines.

A Symbolic Regression Model for the Prediction of Drug Binding to Human Liver Microsomes.

It is common practice in the early drug discovery process to conduct in vitro screening experiments using liver microsomes in order to obtain an initial assessment of test compound metabolic stability. Compounds which bind to liver microsomes are unavailable for interaction with the drug metabolizing enzymes. As such, assessment of the unbound fraction of compound available for biotransformation is an important factor for interpretation of in vitro experimental results and to improve prediction of the in vivo metabolic clearance. Various in silico methods have been proposed for the prediction of test compound binding to microsomes, from various simple lipophilicity-based models with moderate performance to sophisticated machine learning models which demonstrate superior performance at the cost of increased complexity and higher data requirements. In this work, we attempt to strike a middle ground by developing easily implementable equations with improved predictive performance. We employ a symbolic regression approach based on a medium-size in-house data set of fraction unbound in human liver microsomes measurements allowing the identification of novel equations with improved performance. We validate the model performance on an in-house held-out test set and an external validation set.

Microsomal glutathione transferase 1 controls metastasis and therapeutic response in melanoma.

While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.

Neuroinflammatory mediators in acquired epilepsy: an update.

Epilepsy is a group of chronic neurological disorders that have diverse etiologies but are commonly characterized by spontaneous seizures and behavioral comorbidities. Although the mechanisms underlying the epileptic seizures mostly remain poorly understood and the causes often can be idiopathic, a considerable portion of cases are known as acquired epilepsy. This form of epilepsy is typically associated with prior neurological insults, which lead to the initiation and progression of epileptogenesis, eventually resulting in unprovoked seizures. A convergence of evidence in the past two decades suggests that inflammation within the brain may be a major contributing factor to acquired epileptogenesis. As evidenced in mounting preclinical and human studies, neuroinflammatory processes, such as activation and proliferation of microglia and astrocytes, elevated production of pro-inflammatory cytokines and chemokines, blood-brain barrier breakdown, and upregulation of inflammatory signaling pathways, are commonly observed after seizure-precipitating events. An increased knowledge of these neuroinflammatory processes in the epileptic brain has led to a growing list of inflammatory mediators that can be leveraged as potential targets for new therapies of epilepsy and/or biomarkers that may provide valued information for the diagnosis and prognosis of the otherwise unpredictable seizures. In this review, we mainly focus on the most recent progress in understanding the roles of these inflammatory molecules in acquired epilepsy and highlight the emerging evidence supporting their candidacy as novel molecular targets for new pharmacotherapies of acquired epilepsy and the associated behavioral deficits.

Structure-activity relationship studies of benzothiazole-phenyl analogs as multi-target ligands to alleviate pain without affecting normal behavior.

Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are potential targets for several diseases. Previous studies have reported that concomitant selective inhibition of sEH and FAAH produced antinociception effects in an animal model of pain. However, the co-administration of a selective sEH inhibitor and a selective FAAH inhibitor might produce serious side effects due to drug-drug interactions that could complicate drug development in the long term. Thus, discovering dual sEH/FAAH inhibitors, single small molecules that can simultaneously inhibit both sEH and FAAH, would be a significant accomplishment in the medicinal chemistry field. Herein, we report the synthesis and biological evaluation of benzothiazole-phenyl-based analogs as potential dual sEH/FAAH inhibitors. This work represents a follow-up structure-activity relationship (SAR) and metabolic-stability studies of our best dual sEH/FAAH inhibitor identified previously, as well as in vivo evaluation of its effects on voluntary locomotor behavior in rats. Our SAR study indicates that trifluoromethyl groups on the aromatic rings are well tolerated by the targeted enzymes when placed at the ortho and para positions; however, they, surprisingly, did not improve metabolic stability in liver microsomes. Our behavioral studies indicate that doses of dual sEH/FAAH inhibitors that alleviate pain do not depress voluntary behavior in naïve rats, which is a common side effect of currently available analgesic drugs (e.g., opioids). Thus, dual sEH/FAAH inhibitors may be a safe and effective approach to treat pain.

In vitro hepatic metabolism of the natural product quebecol.

Quebecol (2,3,3-tri-(3-methoxy-4-hydroxyphenyl)-1-propanol) is a polyphenolic compound, which is formed during maple syrup production from Acer spp. Quebecol bears structural similarities to the chemotherapy drug tamoxifen, which has led to synthesis of structural analogues and investigations into their pharmacological properties, however there are no reports on the hepatic metabolism of quebecol.This interest in therapeutic properties spurred us to investigate the in vitro microsomal Phase I and II metabolism of quebecol. We were unable to detect any P450 metabolites for quebecol in either human liver microsomes (HLM) or rat liver microsomes (RLM). In contrast we observed marked formation of three glucuronide metabolites in both RLM and HLM, suggesting that clearance via Phase II pathways is likely to predominate.To further understand the hepatic contribution to first-pass glucuronidation we have validated an HPLC method following FDA and EMA guidelines (selectivity, linearity, accuracy, and precision) to quantify quebecol in microsomes. In vitro enzyme kinetics were performed for quebecol glucuronidation by HLM including 8 concentrations from 5-30 µM. We determined a Michaelis-Menten constant (KM) of 5.1 µM, intrinsic clearance (Clint,u) of 0.038 ± 0.001 mL/min/mg, and maximum velocity (Vmax) of 0.22 ± 0.01 µmol/min/mg.

Investigation of pharmacokinetic properties of a PEGylated bilirubin nanoparticle in male Sprague-Dawley rats using liquid chromatography-quadrupole time-of-flight mass spectrometry.

Polyethylene glycol (PEG) was introduced into synthetic bilirubin 3α and a PEGylated bilirubin 3α nanoparticle (BX-001N, Brixelle®) was developed for the first time.An in vitro microsomal stability study, in vivo PK studies with intravenous bolus (IV) and subcutaneous injection (SC), and a semi-mass balance study of BX-001N were investigated to evaluate its pharmacokinetic (PK) properties in male Sprague-Dawley (SD) rats using developed liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF/MS).Following IV administration at 10 or 30 mg/kg, BX-001N showed very low clearance (0.33-0.67 mL/min/kg) with predominant distribution in the vascular system (Vd = 51.73-83.02 mL/kg). BX-001N was also very stable in vitro liver microsomal stability study.Following SC administration at 10 or 30 mg/kg, the bioavailability of BX-001N in plasma at 10 mg/kg was around 43% and showed the less dose-proportionality at 30 mg/kg dose.BX-001N was mainly excreted via the urinary pathway (86.59-92.99% of total amount of parent drug in excreta; urine and feces) not via the biliary one.

The Generation of a Nanobody-Based ELISA for Human Microsomal Epoxide Hydrolase.

A microsomal epoxide hydrolase (mEH) metabolizes in vivo in both xenobiotic and endogenous epoxides associated with signaling function. Findings in patients suggest that mEH might be a biomarker for several diseases, including metastatic cancer and viral hepatitis. To easily quantify mEH, nanobodies specific to the human mEH were isolated from a phage library of llama VHHs. Four unique clones were obtained and used for developing ELISAs. Three formats of double antibody sandwich assays were investigated using different detection strategies. Using PolyHRP, the signal was strongly amplified, yielding a 22-fold lower LOD (12 pg mL-1) than the 'conventional'. To further validate the performance of the immunoassays, human tissue samples were analyzed by nanobody-based ELISAs and compared to the enzyme activities (R2 > 0.95). The results demonstrate that these nanobodies are powerful tools for the quantification of human mEH and could eventually result in a bedside assay.

Microsomal Prostaglandin E Synthase-1 and -2: Emerging Targets in Non-Alcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) affects a substantial proportion of the general population and is even more prevalent in obese and diabetic patients. NAFLD, and particularly the more advanced manifestation of the disease, nonalcoholic steatohepatitis (NASH), increases the risk for both liver-related and cardiovascular morbidity. The pathogenesis of NAFLD is complex and multifactorial, with many molecular pathways implicated. Emerging data suggest that microsomal prostaglandin E synthase-1 and -2 might participate in the development and progression of NAFLD. It also appears that targeting these enzymes might represent a novel therapeutic approach for NAFLD. In the present review, we discuss the association between microsomal prostaglandin E synthase-1 and -2 and NAFLD.