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Pharmaceutics Apr 2024Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most prescribed drugs to treat pain or fever. However, oral administration of NSAIDs is frequently...
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most prescribed drugs to treat pain or fever. However, oral administration of NSAIDs is frequently associated with adverse effects due to their inhibitory effect on the constitutively expressed cyclooxygenase enzyme 1 (COX-1) in, for instance, the gastrointestinal tract. A systemic delivery, such as a buccal delivery, of NSAIDs would be beneficial and additionally has the advantage of a non-invasive administration route, especially favourable for children or the elderly. To investigate the transport of NSAIDs across the buccal mucosa and determine their potential for buccal therapeutic usage, celecoxib, diclofenac, ibuprofen and piroxicam were tested using an established oral mucosa Transwell model based on human cell line TR146. Carboxyfluorescein and diazepam were applied as internal paracellular and transcellular marker molecule, respectively. Calculated permeability coefficients revealed a transport ranking of ibuprofen > piroxicam > diclofenac > celecoxib. Transporter protein inhibitor verapamil increased the permeability for ibuprofen, piroxicam and celecoxib, whereas probenecid increased the permeability for all tested NSAIDs. Furthermore, influence of local inflammation of the buccal mucosa on the transport of NSAIDs was mimicked by treating cells with a cytokine mixture of TNF-α, IL-1ß and IFN-γ followed by transport studies with ibuprofen (+ probenecid). Cellular response to pro-inflammatory stimuli was confirmed by upregulation of cytokine targets at the mRNA level, increased secreted cytokine levels and a significant decrease in the paracellular barrier. Permeability of ibuprofen was increased across cell layers treated with cytokines, while addition of probenecid increased permeability of ibuprofen in controls, but not across cell layers treated with cytokines. In summary, the suitability of the in vitro oral mucosa model to measure NSAID transport rankings was demonstrated, and the involvement of transporter proteins was confirmed; an inflammation model was established, and increased NSAID transport upon inflammation was measured.
PubMed: 38675204
DOI: 10.3390/pharmaceutics16040543 -
International Journal of Molecular... Apr 2024Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the...
Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120/α7nAChR mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.
Topics: Animals; Astrocytes; Glutamic Acid; Mice; Kynurenine; Kynurenic Acid; alpha7 Nicotinic Acetylcholine Receptor; HIV Envelope Protein gp120; Signal Transduction; Mice, Knockout; Probenecid; Mice, Inbred C57BL; Male; Brain; NF-kappa B
PubMed: 38673879
DOI: 10.3390/ijms25084286 -
JCI Insight Apr 2024Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the...
Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.
Topics: Animals; Receptors, Aryl Hydrocarbon; Mice; Male; Renal Insufficiency, Chronic; Tryptophan; Muscle, Skeletal; Humans; Oxidative Phosphorylation; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Mice, Inbred C57BL; Uremia; Mitochondria, Muscle; Basic Helix-Loop-Helix Transcription Factors; Female; Mice, Knockout; Cytochrome P-450 CYP1B1; Cytochrome P-450 CYP1A1; Middle Aged; Energy Metabolism; Disease Models, Animal
PubMed: 38652558
DOI: 10.1172/jci.insight.178372 -
BMC Chemistry Apr 2024Therapeutic drug monitoring is an important clinical testing of the drugs to monitor their concentrations in plasma in order to guarantee their optimal impact, and to...
Therapeutic drug monitoring is an important clinical testing of the drugs to monitor their concentrations in plasma in order to guarantee their optimal impact, and to avoid any side effects resulting from drug-drug interactions. A green reversed-phase high-performance liquid chromatographic method using a photodiode array detector (RP-HPLC-PDA) was developed for the simultaneous determination of three carbapenem antibiotics (Imipenem, ertapenem, and meropenem) with the co-formulated drug (cilastatin) and contraindicated drugs (probenecid and warfarin) in spiked human plasma. The separation was achieved at 25 °C using a gradient elution of a mixture of mobile phase A: methanol and mobile phase B: phosphate buffer (pH 3.0). The photodiode array detector was adjusted at 220 nm. Bioanalytical method validation was carried out as per the FDA guidelines, and the method showed good linearity ranges for the six drugs that included their Cmax levels along with low limits of quantification. Based on the results, the method was found to be accurate and precise; with high % recovery and good % RSD, respectively. The method was successfully applied to spiked human plasma, signifying a good potential to be implemented in future TDM studies of these drugs when co-administered together.
PubMed: 38581021
DOI: 10.1186/s13065-024-01161-y -
Open Forum Infectious Diseases Mar 2024To gauge the safety and utility of extended tecovirimat/cidofovir for severe mpox, here we report our experience caring for 4 patients with mpox and advanced human...
To gauge the safety and utility of extended tecovirimat/cidofovir for severe mpox, here we report our experience caring for 4 patients with mpox and advanced human immunodeficiency virus (HIV) at the Hospitals of the University of Pennsylvania during the 2022 global outbreak. Three patients had recurrent courses complicated by superinfections, coinfections and insufficient nutrition/housing, requiring extended tecovirimat (5-16 weeks) and cidofovir (1-12 doses) with probenecid and fluids. At follow-up, patients had undetectable HIV RNA on antiretrovirals, improved ulcers and stable renal function on antivirals. Serology guided cessation for one 7-month cidofovir course. Overall findings support a comprehensive approach of prolonged tecovirimat/cidofovir with antiretrovirals for severe mpox, while addressing social factors.
PubMed: 38486814
DOI: 10.1093/ofid/ofae110 -
Nature Communications Mar 2024Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2...
Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.
Topics: Humans; Animals; Rats; Phosphorylation; Probenecid; Binding Sites; Biological Assay; Biological Transport; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2
PubMed: 38438394
DOI: 10.1038/s41467-024-46392-8 -
Journal of Integrative Neuroscience Feb 2024Mitochondrial dysfunction is one of the major hallmarks of Parkinson's disease (PD). Recently, angiotensin II type 1 and type 2 receptors (AT1R, AT2R) were reported to...
Telmisartan Protects Mitochondrial Function, Gait, and Neuronal Apoptosis by Activating the Akt/GSK3β/PGC1α Pathway in an MPTP-Induced Mouse Model of Parkinson's Disease.
BACKGROUND
Mitochondrial dysfunction is one of the major hallmarks of Parkinson's disease (PD). Recently, angiotensin II type 1 and type 2 receptors (AT1R, AT2R) were reported to be present on the mitochondrial membrane. Both are crucial players in the brain renin-angiotensin system (RAS). Current evidence indicates that blockade of brain AT1R protects dopaminergic neurons in PD.
METHODS
Thus, the current study was aimed to explore the effects of Telmisartan (Tel), a selective AT1R blocker, on mitochondrial function and a mouse model by exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [250 mg/kg body weight (10 divided i.p. injections, each 25 mg/kg body weight at 3.5 days interval) + Probenecid 250 mg/kg]. Gait function was assessed by beam walk, and mice were euthanized on the 35th day and their brain tissues isolated for Western blot analysis.
RESULTS
Pretreatment with Tel significantly protected motor functions during the beam walk in MPTP-treated mice. Tel attenuated the increased levels of AT1R, α-syn, and inflammatory markers such as inducible nitric oxide synthase (iNOS) and ionized calcium-binding adaptor molecule 1 (IBA1) in MPTP-treated mice. In addition, Tel preserved the expression of AT2R, tyrosine hydroxylase (TH), p-Akt/Akt, and p-GSK3β (Ser-9)/GSK3β, as well as protecting mitofusin protein 1 (MFN1) and Peroxisome proliferator-activated receptor-gamma coactivator-α (PGC1α), a critical activator of mitochondrial biogenesis.
CONCLUSION
These results indicate that Tel protects mitochondrial function and gait in a mouse model of PD by modulating the Akt/GSK3β/PGC1α pathway.
Topics: Animals; Mice; Telmisartan; Parkinson Disease; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Proto-Oncogene Proteins c-akt; Glycogen Synthase Kinase 3 beta; Gait; Apoptosis; Mitochondria; Body Weight; Mice, Inbred C57BL; Disease Models, Animal
PubMed: 38419447
DOI: 10.31083/j.jin2302029 -
Experimental Neurology Apr 2024The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment...
The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment approaches. Biomarkers to identify the early stages of PD are therefore of paramount importance for the advancement of the therapy of PD. In the present study, by using a mouse model of PD obtained by subchronic treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the clearance inhibitor probenecid (MPTPp), we identified prodromal markers of PD by combining in vivo positron emission tomography (PET) imaging and ex vivo immunohistochemistry. Longitudinal PET imaging of the dopamine transporter (DAT) by [F]-N-(3-fluoropropyl)-2β-carboxymethoxy-3β-(4-iodophenyl) nortropane ([F]-FP-CIT), and brain glucose metabolism by 2-deoxy-2-[F]-fluoroglucose ([F]-FDG) were performed before MPTPp treatment and after 1, 3, and 10 MPTPp administrations, in order to assess relation between dopamine neuron integrity and brain connectivity. The results show that in vivo [F]-FP-CIT in the dorsal striatum was not modified after the first administration of MPTPp, tended to decrease after 3 administrations, and significantly decreased after 10 MPTPp administrations. Post-mortem immunohistochemical analyses of DAT and tyrosine hydroxylase (TH) in the striatum showed a positive correlation with [F]-FP-CIT, confirming the validity of repeated MPTPp-treated mice as a model that can reproduce the progressive pathological changes in the early phases of PD. Analysis of [F]-FDG uptake in several brain areas connected to the striatum showed that metabolic connectivity was progressively disrupted, starting from the first MPTPp administration, and that significant connections between cortical and subcortical regions were lost after 10 MPTPp administrations, suggesting an association between dopamine neuron degeneration and connectivity disruption in this PD model. The results of this study provide a relevant model, where new drugs that can alleviate neurodegeneration in PD could be evaluated preclinically.
Topics: Humans; Parkinson Disease; Dopamine; Probenecid; Dopaminergic Neurons; Fluorodeoxyglucose F18; Dopamine Plasma Membrane Transport Proteins; Corpus Striatum; Nerve Degeneration; Tropanes
PubMed: 38281587
DOI: 10.1016/j.expneurol.2024.114704 -
Viruses Jan 2024Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly...
Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.
Topics: Animals; Humans; Mice; Influenza, Human; Influenza in Birds; Influenza A Virus, H7N9 Subtype; Influenza A Virus, H5N1 Subtype; Probenecid; Birds; Mammals
PubMed: 38275962
DOI: 10.3390/v16010152 -
Computational and Structural... Dec 2024TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is...
TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in using a novel medium-throughput Ca influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.
PubMed: 38261868
DOI: 10.1016/j.csbj.2023.12.028