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Nature Communications Dec 2022TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron...
TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.
Topics: Rats; Humans; Animals; Cannabidiol; TRPV Cation Channels; Cannabinoids; Probenecid; Ligands; Cryoelectron Microscopy
PubMed: 36470868
DOI: 10.1038/s41467-022-35163-y -
Scientific Reports Nov 2022Solid contact ion selective electrodes are extensively utilized owing to their marvelous performance over traditional liquid contact ones. The main drawback of those...
Solid contact ion selective electrodes are extensively utilized owing to their marvelous performance over traditional liquid contact ones. The main drawback of those solid contact electrodes is aqueous layer formation which affects their constancy. Herein and to overcome this common drawback, a carbon paste electrode containing poly(3,4-ethylenedioxythiophene) was constructed and used for determination of probenecid at variant pH values. This modification decreased the potential drift down to 0.8 mV/h and improved its stability over 30 days. A Nernstian slope of - 57.8 mV/decade associated with a linear range of 1.0 × 10-1.0 × 10 mol/L was obtained. The modified carbon paste electrode successfully detected up to 8.0 × 10 mol/L probenecid. Results of this modified carbon paste electrode were also compared to unmodified one.
Topics: Humans; Probenecid; Carbon; Potentiometry; Ion-Selective Electrodes; Poly A
PubMed: 36443448
DOI: 10.1038/s41598-022-24920-0 -
Frontiers in Cell and Developmental... 2022An exaggerated inflammatory response is the hallmark of a plethora of disorders. ATP is a central signaling molecule that orchestrates the initiation and resolution of... (Review)
Review
An exaggerated inflammatory response is the hallmark of a plethora of disorders. ATP is a central signaling molecule that orchestrates the initiation and resolution of the inflammatory response by enhancing activation of the inflammasome, leukocyte recruitment and activation of T cells. ATP can be released from cells through pannexin (Panx) channels, a family of glycoproteins consisting of three members, Panx1, Panx2, and Panx3. Panx1 is ubiquitously expressed and forms heptameric channels in the plasma membrane mediating paracrine and autocrine signaling. Besides their involvement in the inflammatory response, Panx1 channels have been shown to contribute to different modes of cell death (i.e., pyroptosis, necrosis and apoptosis). Both genetic ablation and pharmacological inhibition of Panx1 channels decrease inflammation and contribute to a better outcome in several animal models of inflammatory disease involving various organs, including the brain, lung, kidney and heart. Up to date, several molecules have been identified to inhibit Panx1 channels, for instance probenecid (Pbn), mefloquine (Mfq), flufenamic acid (FFA), carbenoxolone (Cbx) or mimetic peptides like Panx1. Unfortunately, the vast majority of these compounds lack specificity and/or serum stability, which limits their application. The recent availability of detailed structural information on the Panx1 channel from cryo-electron microscopy studies may open up innovative approaches to acquire new classes of synthetic Panx1 channel blockers with high target specificity. Selective inhibition of Panx1 channels may not only limit acute inflammatory responses but may also prove useful in chronic inflammatory diseases, thereby improving human health. Here, we reviewed the current knowledge on the role of Panx1 in the initiation and resolution of the inflammatory response, we summarized the effects of Panx1 inhibition in inflammatory pathologies and recapitulate current Panx1 channel pharmacology with an outlook towards future approaches.
PubMed: 36438559
DOI: 10.3389/fcell.2022.1020826 -
Molecules (Basel, Switzerland) Nov 2022Accumulating evidence has shown that Parkinson's disease (PD) is a systemic disease other than a mere central nervous system (CNS) disorder. One of the most important...
Accumulating evidence has shown that Parkinson's disease (PD) is a systemic disease other than a mere central nervous system (CNS) disorder. One of the most important peripheral symptoms is gastrointestinal dysfunction. The enteric nervous system (ENS) is regarded as an essential gateway to the environment. The discovery of the prion-like behavior of α-synuclein makes it possible for the neurodegenerative process to start in the ENS and spread via the gut-brain axis to the CNS. We first confirmed that synucleinopathies existed in the stomachs of chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid (MPTP/p)-induced PD mice, as indicated by the significant increase in abnormal aggregated and nitrated α-synuclein in the TH-positive neurons and enteric glial cells (EGCs) of the gastric myenteric plexus. Next, we attempted to clarify the mechanisms in single MPTP-injected mice. The stomach naturally possesses high monoamine oxidase-B (MAO-B) activity and low superoxide dismutase (SOD) activity, making the stomach susceptible to MPTP-induced oxidative stress, as indicated by the significant increase in reactive oxygen species (ROS) in the stomach and elevated 4-hydroxynonenal (4-HNE) in the EGCs after MPTP exposure for 3 h. Additionally, stomach synucleinopathies appear before those of the nigrostriatal system, as determined by Western blotting 12 h after MPTP injection. Notably, nitrated α-synuclein was considerably increased in the EGCs after 3 h and 12 h of MPTP exposure. Taken together, our work demonstrated that the EGCs could be new contributors to synucleinopathies in the stomach. The early-initiated synucleinopathies might further influence neighboring neurons in the myenteric plexus and the CNS. Our results offer a new experimental clue for interpreting the etiology of PD.
Topics: Mice; Animals; alpha-Synuclein; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Synucleinopathies; MPTP Poisoning; Mice, Inbred C57BL; Disease Models, Animal; Parkinsonian Disorders; Parkinson Disease; Neuroglia; Stomach
PubMed: 36364248
DOI: 10.3390/molecules27217414 -
Molecular Vision 2022Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the...
PURPOSE
Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release.
METHODS
Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space.
RESULTS
The incubation of the rat lenses in the AAH that lacked Ca induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid.
CONCLUSIONS
Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.
Topics: Rats; Animals; Probenecid; Carbenoxolone; Flufenamic Acid; Dextrans; Connexins; Adenosine Triphosphate
PubMed: 36284672
DOI: No ID Found -
Clinical Pharmacokinetics Nov 2022Pexidartinib is a novel oral small-molecule inhibitor that selectively targets colony-stimulating factor 1 receptor, KIT proto-oncogene receptor tyrosine kinase, and...
BACKGROUND AND OBJECTIVE
Pexidartinib is a novel oral small-molecule inhibitor that selectively targets colony-stimulating factor 1 receptor, KIT proto-oncogene receptor tyrosine kinase, and FMS-like tyrosine kinase 3 harboring an internal tandem duplication mutation. It is approved in the United States for the treatment of adult patients with symptomatic tenosynovial giant cell tumor (TGCT) associated with severe morbidity or functional limitations and not amenable to improvement with surgery. Pexidartinib in vitro data indicate the potential for absorption- and metabolism-related drug-drug interactions (DDIs). The objective was to present a comprehensive DDI risk assessment of agents that can impact pexidartinib exposure by altering its absorption and metabolism potentially affecting efficacy and safety of pexidartinib.
METHODS
Four open-label crossover studies were performed to assess the effects of a pH modifier (esomeprazole), a strong cytochrome P450 (CYP) 3A4 inhibitor (itraconazole), a strong CYP3A/5'-diphospho-glucuronosyltransferase (UGT) inducer (rifampin), and a UGT inhibitor (probenecid) on the single-dose pharmacokinetics of pexidartinib. In addition, a physiologically based pharmacokinetic model was developed to predict the effect of a moderate CYP3A4 inhibitor (fluconazole) and a moderate CYP3A inducer (efavirenz) on the pharmacokinetics of pexidartinib.
RESULTS
Co-administration of pexidartinib with esomeprazole modestly decreased pexidartinib exposure (maximum plasma concentration [C], ng/mL: geometric mean ratio [90% confidence interval (CI)], 45.4% [36.8-55.9]; area under the drug plasma concentration-time curve from time 0 to infinity [AUC], ng•h/mL: geometric mean ratio [90% CI], 53.1% [47.4-59.3]), likely related to decreased solubility of pexidartinib at increased pH levels. As expected, the strong CYP3A4 inhibitor itraconazole increased pexidartinib exposure (C, ng/mL: geometric mean ratio [90% CI], 148.3% [127.8-172.0]; AUC, ng•h/mL: geometric mean ratio [90% CI], 173.0% [160.7-186.3]) while the strong CYP3A/UGT inducer rifampin decreased exposure (C, ng/mL: geometric mean ratio [90% CI], 67.1% [53.1-84.8]; AUC, ng•h/mL: geometric mean ratio [90% CI], 37.0% [30.6-44.8]). In addition, UGT inhibition increased pexidartinib exposure (C, ng/mL: geometric mean ratio [90% CI], 105.8% [92.4-121.0]; AUC, ng•h/mL: geometric mean ratio [90% CI], 159.8% [143.4-178.0]), consistent with the fact that pexidartinib is a substrate of the UGT1A4 enzyme, which is responsible for the generation of the major metabolite, ZAAD-1006a.
CONCLUSIONS
The physiologically based pharmacokinetic model predicted that a moderate CYP3A4 inhibitor and a moderate CYP3A inducer would produce modest increases and decreases, respectively, in pexidartinib exposure. These results provide a basis for pexidartinib dosing recommendations when administered concomitantly with drugs with drug-drug interaction potential, including dose adjustments when concomitant administration cannot be avoided.
CLINICAL TRIAL REGISTRATION
Probenecid: phase I trial, NCT03138759, 3 May, 2017; esomeprazole, itraconazole, rifampin: phase I trials, not registered with ClinicalTrials.gov.
Topics: Adult; Humans; Area Under Curve; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inducers; Cytochrome P-450 CYP3A Inhibitors; Drug Interactions; Esomeprazole; Healthy Volunteers; Itraconazole; Probenecid; Rifampin
PubMed: 36264536
DOI: 10.1007/s40262-022-01172-9 -
CNS Neuroscience & Therapeutics Jan 2023The aim of this study was to assess the influence of the major transporters at blood-brain barrier and blood-cerebrospinal fluid barrier on levofloxacin (LVFX)...
AIMS
The aim of this study was to assess the influence of the major transporters at blood-brain barrier and blood-cerebrospinal fluid barrier on levofloxacin (LVFX) pharmacokinetics in rat. To explore the different effects of transporters on drug concentrations in cerebrospinal fluid (CSF) and brain extracellular fluid (ECF).
METHODS
High-performance liquid chromatography coupled with microdialysis was used to continuously and synchronously measure unbound concentrations of LVFX in rat blood, hippocampal ECF, and lateral ventricle CSF for comprehensive characterization of brain pharmacokinetics. The role of transporters in the brain efflux mechanism of LVFX was analyzed in the absence and presence of various transporter inhibitors.
RESULTS
Following LVFX (50 mg/kg) administration, the unbound partition coefficient of LVFX in brain ECF and CSF (K and K ) were 34.0 ± 1.7% and 41.2 ± 2.4%, respectively. When probenecid was coadministered with LVFX, the AUC and the mean residence time (MRT) in rat blood increased significantly (p < 0.05). After MK571 intervention, 1.35-fold and 1.16-fold increases in K and K were observed, respectively (p < 0.05). Treatment with Ko143 increased the levels of LVFX in brain ECF. The difference in LVFX concentration in brain ECF and CSF was <3-fold with or without treatment with transporter inhibitors.
CONCLUSION
Efflux of LVFX from the central nervous system (CNS) involves multidrug resistance-associated proteins (MRPs), breast cancer resistance protein (BCRP), and organic anion transporters (OATs). MRPs play an important role in mediating the brain/CSF-to-blood efflux of LVFX. LVFX concentrations in CSF can be used as a surrogate to predict the concentrations inside brain parenchyma.
Topics: Animals; Rats; ATP Binding Cassette Transporter, Subfamily G, Member 2; Blood-Brain Barrier; Brain; Levofloxacin; Multidrug Resistance-Associated Proteins
PubMed: 36253925
DOI: 10.1111/cns.13989 -
Journal of Neuroinflammation Oct 2022Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of...
BACKGROUND
Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain.
METHODS
A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake.
RESULTS
CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression.
CONCLUSION
In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.
Topics: Adenosine Triphosphate; Animals; Carbenoxolone; Connexins; Cytokines; Ethidium; Hyperalgesia; Lipopolysaccharides; Mice; Nerve Tissue Proteins; Neuralgia; Probenecid; RNA, Small Interfering; Schwann Cells
PubMed: 36195881
DOI: 10.1186/s12974-022-02603-x -
Artificial Intelligence in Medicine Sep 2022In health-care, there is a need to quantify medical errors. Among these errors, we observe wrong dose prescriptions. Drug dose titration (DT) is the process by which...
In health-care, there is a need to quantify medical errors. Among these errors, we observe wrong dose prescriptions. Drug dose titration (DT) is the process by which dosage is progressively adjusted to the patient till a steady dose is reached. Depending on the clinical disease, drug, and patient condition, dose titration can follow different procedures. Once modeled, these procedures can serve for clinical homogenization, standardization, decision support and retrospective analysis. Here, we propose a language to model dose titration procedures. The language was used to formalize one- and two-drug titration of chronic and acute cases, and to perform retrospective analysis of the drug titration processes on 253 patients diagnosed of diabetes mellitus type 2 and treated with metformin, 321 patients treated of chonic heart failure with furosemide, 155 patients with hyperuricemia treated with allopurinol as initial drug and febuxostat as alternative drug, and 187 hyperuricemia patients with primary drug allopurinol and supplementary drug probenecid, in order to identify different types of drug titration deviations from standard DT methods.
Topics: Allopurinol; Gout; Gout Suppressants; Humans; Hyperuricemia; Retrospective Studies; Uric Acid
PubMed: 36100337
DOI: 10.1016/j.artmed.2022.102343 -
International Journal of Molecular... Sep 2022Erythrocytes' aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells' (RBCs) behavior and function. The aging pattern can be...
Erythrocytes' aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells' (RBCs) behavior and function. The aging pattern can be influenced, in morphological, biochemical, and metabolic terms by the environmental conditions. In this paper, we studied the effect of a moderate mechanical stimulation applied through external shaking during the RBCs aging and revealed a strong acceleration of the aging pattern induced by such stimulation. Moreover, we evaluated the behavior of the main cellular effectors and resources in the presence of drugs (diamide) or of specific inhibitors of the mechano-transduction (probenecid, carbenoxolone, and glibenclamide). This approach provided the first evidence of a direct cross-correlation between aging and mechano-transduction and permitted an evaluation of the overall metabolic regulation and of the insurgence of specific morphological features, such as micro-vesicles and roughness alterations. Overall, for the first time the present data provided a schematic to understand the integration of distinct complex patterns in a comprehensive view of the cell and of its interactions with the environment. Mechano-transduction produces structural effects that are correlated with the stimulation and the strength of the environmental stimulation is paramount to effectively activate and trigger the biological cascades initiated by the mechano-sensing.
Topics: Cellular Senescence; Erythrocytes; Humans
PubMed: 36077573
DOI: 10.3390/ijms231710180