RESUMEN
BACKGROUND: Parenchymal accumulation of beta-amyloid (Aß) characterizes Alzheimer's disease (AD). Aß homeostasis is maintained by two ATP-binding cassette (ABC) transporters (ABCC1 and ABCB1) mediating efflux, and the receptor for advanced glycation end products (RAGE) mediating influx across the blood-brain barrier (BBB). Altered transporter levels and disruption of tight junctions (TJ) were linked to AD. However, Aß transport and the activity of ABCC1, ABCB1 and RAGE as well as the functionality of TJ in AD are unclear. METHODS: ISMICAP, a BBB model involving microperfusion of capillaries, was used to assess BBB properties in acute cortical brain slices from Tg2576 mice compared to wild-type (WT) controls using two-photon microscopy. TJ integrity was tested by vascularly perfusing biocytin-tetramethylrhodamine (TMR) and quantifying its extravascular diffusion as well as the diffusion of FM1-43 from luminal to abluminal membranes of endothelial cells (ECs). To assess ABCC1 and ABCB1 activity, calcein-AM was perfused, which is converted to fluorescent calcein in ECs and gets actively extruded by both transporters. To probe which transporter is involved, probenecid or Elacridar were applied, individually or combined, to block ABCC1 and ABCB1, respectively. To assess RAGE activity, the binding of 5-FAM-tagged Aß by ECs was quantified with or without applying FPS-ZM1, a RAGE antagonist. RESULTS: In Tg2576 mouse brain, extravascular TMR was 1.8-fold that in WT mice, indicating increased paracellular leakage. FM1-43 staining of abluminal membranes in Tg2576 capillaries was 1.7-fold that in WT mice, indicating reduced TJ integrity in AD. While calcein was undetectable in WT mice, its accumulation was significant in Tg2576 mice, suggesting lower calcein extrusion in AD. Incubation with probenecid or Elacridar in WT mice resulted in a marked calcein accumulation, yet probenecid alone had no effect in Tg2576 mice, implying the absence of probenecid-sensitive ABC transporters. In WT mice, Aß accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aß fluorescence was observed in Tg2576 vessels, and FPS-ZM1 was without effect, suggesting reduced RAGE binding activity. CONCLUSIONS: Disrupted TJ integrity, reduced ABCC1 functionality and decreased RAGE binding were identified as BBB alterations in Tg2576 mice, with the latter finding challenging the current concepts. Our results suggest to manage AD by including modulation of TJ proteins and Aß-RAGE binding.
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Enfermedad de Alzheimer , Barrera Hematoencefálica , Ratones , Animales , Barrera Hematoencefálica/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Capilares/metabolismo , Células Endoteliales/metabolismo , Probenecid/metabolismo , Homeostasis , PerfusiónRESUMEN
Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.
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Transportadores de Anión Orgánico , Ratas , Animales , Transportadores de Anión Orgánico/metabolismo , Probenecid/farmacología , Probenecid/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Eliminación Renal , Furosemida/farmacología , Furosemida/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Ácido Quinurénico/metabolismo , Ácido Quinurénico/farmacología , Ácido Piridóxico/metabolismo , Ácido Piridóxico/farmacología , Interacciones Farmacológicas , Biomarcadores/metabolismo , Riñón/metabolismoRESUMEN
Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1RC/RC mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1RC/RC and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1RC/RC mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCDcl4 cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCDcl4 epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology.
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Quistes , Riñón Poliquístico Autosómico Dominante , Ratones , Masculino , Femenino , Humanos , Animales , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Probenecid/farmacología , Probenecid/metabolismo , Probenecid/uso terapéutico , Modelos Animales de Enfermedad , Riñón/metabolismo , Progresión de la Enfermedad , Adenosina Trifosfato/metabolismo , Quistes/metabolismo , Quistes/patología , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Canales Catiónicos TRPP/farmacologíaRESUMEN
Organic anion transporters 1 and 3 (OAT1/3) occupy a key role in mediating renal elimination. Kynurenic acid (KYNA) was previously discovered as an effective endogenous biomarker to assess drug-drug interaction (DDI) for OAT inhibitors. Here, further in vitro and in vivo investigation was performed to characterize the elimination routes and feasibility of KYNA, along with other reported endogenous metabolites, as biomarkers of Oat1/3 inhibition in bile duct-cannulated (BDC) cynomolgus monkeys. Our results suggested that KYNA is a substrate of OAT1/3 and OAT2, but not OCT2, MATE1/2K, or NTCP, and that it shares comparable affinities between OAT1 and OAT3. Renal and biliary excretions and plasma concentration-time profiles of KYNA, pyridoxic acid (PDA), homovanillic acid (HVA), and coproporphyrin I (CP-I) were assessed in BDC monkeys dosed with either probenecid (PROB) at 100 mg/kg or the control vehicle. Renal excretion of KYNA, PDA, and HVA was determined to be the major elimination route. The maximum concentration and the area under the plasma concentration-time curve (Cmax and AUC0-24h) of KYNA were about 11.6- and 3.7-fold higher in the PROB group than in the vehicle group. Renal clearance of KYNA decreased by 3.2-fold, but biliary clearance (CLbile) was not altered after PROB administration. A similar trend was observed for PDA and HVA. Interestingly, an elevation of plasma concentration and reduction of CP-I CLbile were observed after PROB treatment, which suggested inhibition of the CP-I Oatp-Mrp2 transport axis by PROB. Overall, our results indicated that KYNA could potentially facilitate early and reliable assessment of DDI liabilities of Oat inhibition in monkeys. SIGNIFICANCE STATEMENT: This work reported renal excretion as the major elimination pathway for kynurenic acid, pyridoxic acid, and homovanillic acid. Administration of probenecid reduced renal clearance and increased plasma exposure of these biomarkers in monkeys, consistent with the observation in humans. These endogenous biomarkers discovered in monkeys could be potentially used to evaluate the clinical drug-drug interactions in the early phase of drug development.
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Transportadores de Anión Orgánico , Probenecid , Humanos , Animales , Macaca fascicularis/metabolismo , Probenecid/farmacología , Probenecid/metabolismo , Ácido Piridóxico , Ácido Homovanílico , Estudios de Factibilidad , Ácido Quinurénico , Transportadores de Anión Orgánico/metabolismo , Biomarcadores/metabolismo , Interacciones Farmacológicas , Riñón/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismoRESUMEN
OBJECTIVES: To evaluate the effect of tartaric acid (TTA) on Madin-Darby canine kidney (MDCK) cells compared to human kidney (HK)-2 cells. Secondarily, to evaluate the effects of probenecid, an organic anion transporter (OAT)-1 inhibitor, as well as human (h)OAT-4 transfection into MDCK cells to prevent TTA-induced cytotoxicity through decreasing accumulation via OAT-1 uptake inhibition or increasing OAT-4-mediated TTA efflux. DESIGN: Seventy-two-hour TTA concentration response and inhibitor studies in immortalized cell lines. SETTING: School of Pharmacy biomedical research laboratory and tissue culture facility. ANIMALS/SAMPLES: MDCK and HK-2 immortalized cell lines. INTERVENTIONS: Both cell lines were treated with increasing concentrations of TTA for 72 hours. Additionally, MDCK cells were co-incubated with TTA and increasing concentrations of probenecid or had been transfected with hOAT-4 and subsequently treated with TTA for 72 hours. MEASUREMENTS AND MAIN RESULTS: Media and samples were collected and lactate dehydrogenase (LDH) release was measured. LDH release was measured to assess TTA-induced cytotoxicity after 72 hours. LDH was not significantly increased in the HK-2 cells at any concentration but was significantly increased in the MDCK cells from 10 to 100 mM. LDH concentrations were significantly decreased (61%) in MDCK cells incubated with 50 mM TTA and probenecid when compared to TTA alone. hOAT-4 MDCK cell transfection also significantly reduced LDH release (57%) when comparing the transfected MDCK cells to the nontransfected MDCK cells treated with 50 mM TTA. CONCLUSIONS: TTA is a species-specific nephrotoxicant in dogs due to an interspecies difference in OAT-4 expression. Inhibiting TTA uptake in MDCK cells in vitro using the OAT-specific inhibitor, probenecid, prevents TTA-induced cytotoxicity.
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Transportadores de Anión Orgánico , Humanos , Animales , Perros , Células de Riñón Canino Madin Darby , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Avena , Probenecid/farmacología , Probenecid/metabolismo , Riñón/metabolismo , Transfección/veterinariaRESUMEN
The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non-hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility.
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Anticonceptivos , Dinoprostona , Humanos , Femenino , Ratones , Animales , Dinoprostona/metabolismo , Anticonceptivos/metabolismo , Anticonceptivos/farmacología , Probenecid/metabolismo , Probenecid/farmacología , Folículo Ovárico/metabolismo , Ovulación/fisiología , Proteínas de Transporte de Membrana/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.
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Transportadores de Anión Orgánico , Uricosúricos , Ratas , Animales , Uricosúricos/farmacología , Benzbromarona , Probenecid/farmacología , Probenecid/metabolismo , Ácido Úrico , Ratas Sprague-Dawley , Riñón/metabolismo , Transportadores de Anión Orgánico/metabolismoRESUMEN
Bentysrepinine (Y101) is a novel phenylalanine dipeptide for the treatment of hepatitis B virus. Renal excretion played an important role in the elimination of Y101 and its metabolites, M8 and M9, in healthy Chinese subjects, although the molecular mechanisms of renal excretion and potential drug-drug interactions (DDIs) remain unclear. The present study aimed to determine the organic anion transporters (OATs) involved in the renal disposition of Y101 and to predict the potential DDI between Y101 and entecavir, the first-line agent against HBV and a substrate of OAT1/3. Pharmacokinetic studies and uptake assays using rat kidney slices, as well as hOAT1/3-HEK293 cells, were performed to evaluate potential DDI. The co-administration of probenecid (an inhibitor of OATs) significantly increased the plasma concentrations and area under the plasma concentration-time curves of M8 and M9 but not Y101, while reduced renal clearance and the cumulative urinary excretion of M8 were observed in rats. The time course of Y101 and M8 uptake via rat kidney slices was temperature-dependent. Moreover, the uptake of M8 was inhibited significantly by probenecid and benzylpenicillin, but not by p-aminohippurate or tetraethyl ammonium. M8 was found to be a substrate of hOAT3, but Y101 is not a substrate of either hOAT1 or hOAT3. Additionally, the entecavir inhibited the uptake of M8 in the hOAT3-transfected cells and rat kidney slices in vitro. Interestingly, no significant changes were observed in the pharmacokinetic parameters of Y101, M8 or entecavir, regardless of intravenous or oral co-administration of Y101 and entecavir in rats. In conclusion, M8 is a substrate of OAT3 in rats and humans. Furthermore, M8 also mediates the DDI between Y101 and entecavir in vitro, mediated by OAT3. We speculate that it would be safe to use Y101 with entecavir in clinical practice. Our results provide useful information with which to predict the DDIs between Y101 and other drugs that act as substrates of OAT3.
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Proteína 1 de Transporte de Anión Orgánico , Transportadores de Anión Orgánico Sodio-Independiente , Humanos , Ratas , Animales , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Probenecid/metabolismo , Probenecid/farmacología , Ratas Wistar , Células HEK293 , Dipéptidos/metabolismo , Interacciones Farmacológicas , Riñón/metabolismoRESUMEN
Tight control of skeletal muscle contractile activation is secured by the excitation-contraction (EC) coupling protein complex, a molecular machinery allowing the plasma membrane voltage to control the activity of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. This machinery has been shown to be intimately linked to the plasma membrane protein pannexin-1 (Panx1). We investigated whether the prescription drug probenecid, a widely used Panx1 blocker, affects Ca2+ signaling, EC coupling, and muscle force. The effect of probenecid was tested on membrane current, resting Ca2+, and SR Ca2+ release in isolated mouse muscle fibers, using a combination of whole-cell voltage-clamp and Ca2+ imaging, and on electrically triggered contraction of isolated muscles. Probenecid (1 mM) induces SR Ca2+ leak at rest and reduces peak voltage-activated SR Ca2+ release and contractile force by 40%. Carbenoxolone, another Panx1 blocker, also reduces Ca2+ release, but neither a Panx1 channel inhibitory peptide nor a purinergic antagonist affected Ca2+ release, suggesting that probenecid and carbenoxolone do not act through inhibition of Panx1-mediated ATP release and consequently altered purinergic signaling. Probenecid may act by altering Panx1 interaction with the EC coupling machinery, yet the implication of another molecular target cannot be excluded. Since probenecid has been used both in the clinic and as a masking agent for doping in sports, these results should encourage evaluation of possible effects on muscle function in treated individuals. In addition, they also raise the question of whether probenecid-induced altered Ca2+ homeostasis may be shared by other tissues.
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Calcio , Probenecid , Ratones , Animales , Probenecid/metabolismo , Probenecid/farmacología , Calcio/metabolismo , Carbenoxolona/metabolismo , Carbenoxolona/farmacología , Fibras Musculares Esqueléticas/metabolismo , Contracción Muscular , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Conexinas/metabolismoRESUMEN
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 (10Panx) 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.
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Carbenoxolona , Neuralgia , Adenosina Trifosfato/farmacología , Animales , Carbenoxolona/farmacología , Carbenoxolona/uso terapéutico , Conexinas/genética , Conexinas/metabolismo , Citocinas/metabolismo , Etidio/metabolismo , Etidio/farmacología , Etidio/uso terapéutico , Hiperalgesia/metabolismo , Lipopolisacáridos/farmacología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Probenecid/metabolismo , Probenecid/farmacología , Probenecid/uso terapéutico , ARN Interferente Pequeño/metabolismo , Células de SchwannRESUMEN
Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.
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Anemia de Células Falciformes , Reticulocitos , Adenosina Trifosfato/metabolismo , Anemia de Células Falciformes/metabolismo , Apirasa/metabolismo , Cationes/metabolismo , Células Cultivadas , Eritrocitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxia/metabolismo , Probenecid/metabolismo , Reticulocitos/metabolismo , Suramina/metabolismo , Suramina/farmacologíaRESUMEN
OBJECTIVE: To investigate whether probenecid (PROB) could improve the proliferation and migration ability of rats' pulmonary artery smooth muscle cells induced by platelet-derived growth factor-BB (PDGF-BB). METHODS: Primary pulmonary artery smooth muscle cells (PASMCs) of SD rats were cultured in vitro, and were randomly divided into control group (CON group), PDGF-BB group (10 ng/ml PDGF-BB treatment for 24 h) and PDGF-BB+PROB group (10 ng/ml PDGF-BB and 200 µmol/L PROB treatment for 24 h, PROB is a specific blocker of pannexin-1). CCK-8 method was used to select the suitable intervention concentrations of PROB and PDGF-BB, and to detect the proliferation of PASMCs in each group. The migration ability of PASMCs was detected by TranswellTM assay and cell scratch test. Immunofluorescence cytochemistry and Western blot were used to detect the protein expressions and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in PASMCs. RESULTS: Compared with CON group, the migration and proliferation ability of PASMCs in PDGF-BB group were enhanced (Pï¼0.05). After treated with PROB, the migration and proliferation ability of PASMCs in PDGF-BB+PROB group were decreased significantly (Pï¼0.05). Compared with CON group, the expression and protein levels of OPN and PCNA in PDGF-BB group were increased significantly (Pï¼0.05), while the expression and protein levels of OPN and PCNA in PDGF-BB+PROB were decreased significantly (Pï¼0.05). CONCLUSION: Probenecid inhibits the migration and proliferation of PDGF-BB-induced PASMCs by blocking Pannexin-1.
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Probenecid , Arteria Pulmonar , Ratas , Animales , Becaplermina/metabolismo , Becaplermina/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Probenecid/farmacología , Probenecid/metabolismo , Proteínas Proto-Oncogénicas c-sis/farmacología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Proliferación Celular , Ratas Sprague-Dawley , Miocitos del Músculo Liso , Células CultivadasRESUMEN
A naphthoquinone molecule known as plumbagin (PL), which has a wide range of pharmacological properties including antitumor, antioxidation, anti-inflammation, and neuroprotective effects, is extracted from the roots of the medicinal herb Plumbago zeylanica L. Plumbagin has been studied for its potential to treat Parkinson's disease (PD). However, its effectiveness and mechanism are still unknown. This study intends to evaluate plumbagin's effectiveness against PD in vitro and in vivo. Plumbagin partially repaired the loss of dopaminergic neurons in the nigral substantia nigra and the resulting behavioural impairment caused by MPTP or MPTP/probenecid in mice. Furthermore, plumbagin treatment significantly inhibited the TLR/NF-κB pathways. It reduced the TNF-α, IL-6, and IL-1ß mRNA expression in PD mice induced by MPTP or MPTP/probenecid, which was consistent with the findings in the inflammatory model of BV2 cells induced by MPP+ or LPS. In addition, plumbagin treatment enhanced the microtubule-associated protein 1 light chain 3 beta (LC3) LC3-II/LC3-I levels while decreasing the p-mTOR and p62 protein accumulation in PD mice induced by MPTP or MPTP/probenecid, which was similar to the results obtained from the experiments in SH-SY5Y and PC12 cells induced by MPP+. Consequently, our results support the hypothesis that plumbagin, by promoting autophagy and inhibiting the activation of the TLR/NF-κB signaling pathway, is a promising treatment agent for treating Parkinson's disease (PD). However, to confirm plumbagin's anti-PD action more thoroughly, other animal and cell PD models must be used in future studies.
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Naftoquinonas , Neuroblastoma , Fármacos Neuroprotectores , Enfermedad de Parkinson , Ratas , Humanos , Ratones , Animales , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , FN-kappa B/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/metabolismo , Probenecid/metabolismo , Probenecid/farmacología , Neuroblastoma/metabolismo , Transducción de Señal , Naftoquinonas/farmacología , Naftoquinonas/uso terapéutico , Naftoquinonas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Autofagia , Ratones Endogámicos C57BL , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , Modelos Animales de EnfermedadRESUMEN
PURPOSE: To build a physiologically based pharmacokinetic (PBPK) model of the clinical OATP1B1/OATP1B3/BCRP victim drug rosuvastatin for the investigation and prediction of its transporter-mediated drug-drug interactions (DDIs). METHODS: The Rosuvastatin model was developed using the open-source PBPK software PK-Sim®, following a middle-out approach. 42 clinical studies (dosing range 0.002-80.0 mg), providing rosuvastatin plasma, urine and feces data, positron emission tomography (PET) measurements of tissue concentrations and 7 different rosuvastatin DDI studies with rifampicin, gemfibrozil and probenecid as the perpetrator drugs, were included to build and qualify the model. RESULTS: The carefully developed and thoroughly evaluated model adequately describes the analyzed clinical data, including blood, liver, feces and urine measurements. The processes implemented to describe the rosuvastatin pharmacokinetics and DDIs are active uptake by OATP2B1, OATP1B1/OATP1B3 and OAT3, active efflux by BCRP and Pgp, metabolism by CYP2C9 and passive glomerular filtration. The available clinical rifampicin, gemfibrozil and probenecid DDI studies were modeled using in vitro inhibition constants without adjustments. The good prediction of DDIs was demonstrated by simulated rosuvastatin plasma profiles, DDI AUClast ratios (AUClast during DDI/AUClast without co-administration) and DDI Cmax ratios (Cmax during DDI/Cmax without co-administration), with all simulated DDI ratios within 1.6-fold of the observed values. CONCLUSIONS: A whole-body PBPK model of rosuvastatin was built and qualified for the prediction of rosuvastatin pharmacokinetics and transporter-mediated DDIs. The model is freely available in the Open Systems Pharmacology model repository, to support future investigations of rosuvastatin pharmacokinetics, rosuvastatin therapy and DDI studies during model-informed drug discovery and development (MID3).
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Interacciones Farmacológicas , Modelos Biológicos , Rosuvastatina Cálcica/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Adulto , Factores de Edad , Área Bajo la Curva , Transporte Biológico , Estatura , Peso Corporal , Etnicidad , Heces/química , Gemfibrozilo/metabolismo , Humanos , Hígado , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Masculino , Proteínas de Neoplasias/metabolismo , Probenecid/metabolismo , Rifampin/metabolismo , Rosuvastatina Cálcica/sangre , Rosuvastatina Cálcica/orina , Factores Sexuales , Programas Informáticos , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismoRESUMEN
Fevipiprant, an oral, nonsteroidal, highly selective, reversible, and competitive prostaglandin D2 receptor 2 antagonist, is eliminated by glucuronidation and by direct renal excretion predominantly via organic anion transporter (OAT) 3. This study aimed to assess the effect of simultaneous UDP-glucuronosyltransferase (UGT) and OAT3 inhibition by probenecid on the pharmacokinetics of fevipiprant and its acyl glucuronide (AG) metabolite to support the dosing recommendation of fevipiprant in the presence of drugs inhibiting these pathways; however, phase III clinical trial results did not support its submission. This was a single-center, open-label, single-sequence, two-period crossover study in healthy subjects. Liquid chromatography with tandem mass spectrometry was used to measure concentrations of fevipiprant and its AG metabolite in plasma and urine. In the presence of probenecid, the mean maximum concentrations of fevipiprant increased approximately 1.7-fold, and the area under the concentration-time curve in plasma increased approximately 2.5-fold, whereas the mean apparent volume of distribution and the AG metabolite:fevipiprant ratio decreased. The apparent systemic clearance decreased by approximately 60% and the renal clearance decreased by approximately 88% in the presence of probenecid. Using these data and those from previous studies, the relative contribution of OAT and UGT inhibition to the overall effect of probenecid was estimated. Furthermore, a general disposition scheme for fevipiprant was developed, showing how a perpetrator drug such as probenecid, which interferes with two key elimination pathways of fevipiprant, causes only a moderate increase in exposure and allows estimation of the drug-drug inhibition when only one of the two pathways is inhibited. SIGNIFICANCE STATEMENT: In this drug-drug interaction (DDI) study, probenecid was used as a tool to inhibit both glucuronidation and active renal secretion of fevipiprant. The combination of plasma and urine pharmacokinetic data from this study with available data allowed the development of a quantitative scheme to describe the fate of fevipiprant in the body, illustrating why the DDI effect on fevipiprant is weak-to-moderate even if a perpetrator drug inhibits several elimination pathways.
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Adyuvantes Farmacéuticos/metabolismo , Ácidos Indolacéticos/metabolismo , Riñón/metabolismo , Tasa de Depuración Metabólica/fisiología , Probenecid/metabolismo , Piridinas/metabolismo , Eliminación Renal/fisiología , Adyuvantes Farmacéuticos/farmacología , Adulto , Estudios Cruzados , Interacciones Farmacológicas/fisiología , Femenino , Humanos , Ácidos Indolacéticos/farmacología , Riñón/efectos de los fármacos , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Persona de Mediana Edad , Probenecid/farmacología , Piridinas/farmacología , Eliminación Renal/efectos de los fármacos , Adulto JovenRESUMEN
Organic anion transporter 3 (OAT3) plays a critical role in the renal excretion of many xenobiotics. Because steviol acyl glucuronide (SVAG), an OAT3 substrate, is the major circulating metabolite after oral ingestion of steviol glycosides and is excreted into the urine, inhibition of OAT3 activity may alter pharmacokinetic profiles of SVAG. The present study showed that drugs such as probenecid and glimepiride displayed potent inhibition toward the OAT3-mediated SVAG transport, with IC50 values of 4.9 and 0.8 µM, respectively. No species differences were observed. Probenecid and glimepiride could significantly elevate plasma concentrations of SVAG after oral administration of rebaudioside A, with significant increases in plasma maximum (Cmax) and area under the plasma time-concentration curve values. The inhibitory effect on the OAT3-mediated SVAG transport exemplified a unique case between drugs and the metabolite of a food additive. Our data suggest that caution should be exercised when giving steviol glycoside products to human subjects with compromised renal function.
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Diterpenos de Tipo Kaurano/metabolismo , Glucósidos/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Probenecid/metabolismo , Compuestos de Sulfonilurea/metabolismo , Animales , Transporte Biológico , Diterpenos de Tipo Kaurano/química , Glucósidos/química , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Transportadores de Anión Orgánico Sodio-Independiente/genética , Probenecid/administración & dosificación , Probenecid/química , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfonilurea/administración & dosificación , Compuestos de Sulfonilurea/químicaRESUMEN
Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.
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Aciclovir/farmacocinética , Riñón/metabolismo , Leflunamida/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteína 1 de Transporte de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Aciclovir/administración & dosificación , Aciclovir/metabolismo , Administración Intravenosa , Animales , Células Cultivadas , Crotonatos/administración & dosificación , Crotonatos/metabolismo , Crotonatos/farmacología , Perros , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Hidroxibutiratos , Leflunamida/administración & dosificación , Leflunamida/metabolismo , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/metabolismo , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Nitrilos , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Probenecid/administración & dosificación , Probenecid/metabolismo , Probenecid/farmacología , Propionatos/administración & dosificación , Propionatos/metabolismo , Propionatos/farmacología , Quinolinas/administración & dosificación , Quinolinas/metabolismo , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Distribución Tisular , Toluidinas/administración & dosificación , Toluidinas/metabolismo , Toluidinas/farmacologíaRESUMEN
Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted.
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Biomarcadores/sangre , Ácido Homovanílico/sangre , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ácido Piridóxico/sangre , Animales , Transporte Biológico/fisiología , Línea Celular , Células HEK293 , Humanos , Macaca fascicularis , Metabolómica/métodos , Probenecid/metabolismoRESUMEN
This study aimed to demonstrate that organic anion transporters (OATs) mediate the drug-drug interaction (DDI) between piperacillin and tazobactam. After co-administration with piperacillin in rats, the AUC of tazobactam in plasma was significantly increased, and t1/2ß was prolonged with significant reduction in plasma clearance, renal clearance and cumulative urinary excretion. In rat and human kidney slices, probenecid, p-aminohippurate and benzylpenicillin inhibited the uptake of piperacillin and tazobactam. Piperacillin significantly inhibited the uptake of tazobactam. Moreover, the uptakes of piperacillin, tazobactam and sulbactam in hOAT1/3-HEK293 cells were significantly higher compared with mock-HEK293 cells, respectively. Piperacillin significantly inhibited the uptake of tazobactam in hOAT1/3-HEK293 cells. The Km values of tazobactam (431⯱â¯67⯵M for hOAT1, 377⯱â¯63⯵M for hOAT3) were significantly higher than those of piperacillin (37⯱â¯5⯵M for hOAT1, 172⯱â¯28⯵M for hOAT3). This suggested that piperacillin has a stronger affinity to hOAT1/3 than tazobactam. Meanwhile, the Km values of tazobactam were increased in the presence of piperacillin with unchanged Vmax. This indicated that piperacillin inhibited the uptake of tazobactam in a competitive manner. In conclusion, piperacillin and tazobactam are the substrates of hOAT1/3, and OAT1/3 mediate the DDI between piperacillin and tazobactam.
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Interacciones Farmacológicas/fisiología , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ácido Penicilánico/análogos & derivados , Piperacilina/farmacología , Animales , Línea Celular , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ácido Penicilánico/farmacología , Penicilina G/metabolismo , Probenecid/metabolismo , Ratas , Ratas Wistar , Sulbactam/farmacología , Tazobactam , Ácido p-Aminohipúrico/metabolismoRESUMEN
A PBPK modelling approach was used to predict organic anion transporter (OAT) mediated drug-drug interactions involving S44121, a substrate and an inhibitor of OAT1 and OAT3. Model predictions were then compared to the results of a clinical DDI study which was carried out to investigate the interaction of S44121 with probenecid, tenofovir and ciprofloxacin. PBPK models were developed and qualified using existing clinical data, and inhibition constants were determined in vitro. The model predictions for S44121 as an OAT inhibitor were similar to the results obtained from the clinical DDI study, with no interaction observed for tenofovir or ciprofloxacin in the presence of S44121. An observed AUC ratio of 2.2 was obtained for S44121 in the presence of probenecid, which was slightly higher than the model predicted AUC ratio of 1.6. A DDI study in the monkey was also carried out for the interaction between S44121 and probenecid, since the monkey has previously been reported to be a good preclinical model for OAT-mediated DDI. However, this study highlighted a species difference in the major route of S44121 elimination between monkey (mainly hepatic metabolism) and human (mainly renal excretion of unchanged drug), rendering a comparison between the two DDI studies difficult. Overall, for S44121 the PBPK modelling approach gave a better prediction of the extent of DDI than the static predictions based on inhibitor Cmax and IC50, therefore this can be considered a potentially valuable tool within drug development.