fda approved a2a ar agonist  (Gilead Sciences)

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 2. Activation of A2A AR downmodulates P-gp expres- sion and function in human brain endothelial cells. (A) Western blot analysis of P-gp expression in HCMEC-D3 cells treated with Lexiscan (LEX) (1 μM) for up to 72 hours. GAPDH was used as loading control. (B) For densitometric analysis, the intensity of P-gp upon Lexiscan treatment was divided by that of DMSO control and graphed. Densitometric analysis of short-time point (dotted box) is depicted as an inset. (C) Rho123 uptake assay of HCMEC-D3 cells treated with Lexiscan (1 μM) was performed. Concentrations of Rho123 accumulation in brain endothelial cells were analyzed by fluorometry (Synergy, Biotek), with excitation at 488 nm and emission at 523 nm. *P < 0.05; **P < 0.01 (n = 4, 2-tailed Student’s t test, 1 representative result of 3 different experi- ments). (D) Western blot analy- sis depicting P-gp expression in human primary brain endothelial cells treated with Lexiscan (1 μM) for up to 72 hours. GAPDH was used as loading control. (E) Densitometric analysis was used to measure the intensity of P-gp expression with Lexiscan or NECA treatment and was divided by the DMSO control and graphed. Densitometric analysis of short-time point (dotted box) is depicted as an inset. (F) Rho123 uptake assay of human primary brain endothelial cells treated with Lexiscan (1 μM). The concentration of Rho123 accumulation in endothelial cells was quantified by fluorometry (Synergy, Biotek), with excitation at 488 nm and emission at 523 nm. *P < 0.05; **P < 0.01 (n = 4, 2-tailed Student’s t test, 1 representative result of 3 different experiments). (G) Sche- matic diagram of Rho123 accu- mulation assay from C and F.

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Activation Assay, Western Blot, Expressing, Control, Concentration Assay

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 3. Activation of A2A AR by Lexiscan induces rapid transmigration of Rho123 across an in vitro human BBB and accumulation of Rho123 in primary brain endothelial cell. (A) Diagram depicting methodology of transmigration assay of Rho123 using an in vitro BBB model. (B) Results of in vitro BBB model using primary human brain endothelial cell monolayers cultured on porous membranes to determine Rho123 migration across the BBB. Endothelial cell monolayers cultured on porous membranes were treated with Lexiscan (0.25 μM) or NECA (0.25 μM) concomitantly with 2.5 μM of Rho123, and the concentration of Rho123 at the bottom chambers was analyzed by fluorimetry, with excitation at 488 nm and emission at 523 nm. *P < 0.05 (n = 4, 2-tailed Student’s t test, 1 representative result of 3 different experiments). (C–F) Human primary brain endothelial cells were cultured on coverslips and treated with 2.5 μM of Rho123 with or without 1 μM of Lexiscan or NECA at different time points (15 minutes, 1, 4, 24 hours). Cells were fixed with 4% PFA and costained with P-gp and visualized with fluorescent microscope. Scale bar: 25 μm.

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Activation Assay, Transmigration Assay, In Vitro, Cell Culture, Migration, Concentration Assay, Microscopy

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 5. P-gp downmodulation by Lexiscan is also mediated by ubiquitination. (A) Immunoprecipitation analysis of ubiquitination of P-gp in human pri- mary brain endothelial cells upon activation with Lexiscan for 15 minutes. P-gp was pulled down with an anti–P-gp antibody and immunoblotted with an antiubiquitin antibody. (B) IFA depicting ubiquitination of P-gp in human primary brain endothelial cells upon activation with Lexiscan for 15 minutes. Cells were fixed, permeabilized, and stained with an anti–P-gp (green) and antiubiquitin (red) antibody. Nucleus was counterstained with DAPI (blue). Scale bar: 25 μm. (C) Schematic diagram depicting potential mechanisms of P-gp downregulation by Lexiscan. Upon activation of A2A AR, P-gp is cleaved by MMP9 and secreted to the extracellular space (a), or ubiquitylated and digested by proteasome (b), or can relocate from soluble fraction to the insoluble fraction (c). These mechanisms may occur independently or in combination.

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Activation Assay, Staining

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 6. Ablation of CD73 or ARs increased P-gp expression and decreased P-gp substrate accumulation in the brain. (A) Western blot analysis of P-gp from primary brain endothelial cells of brains of WT, A1, A2A, and CD73 KO mice. β-Actin was used as a loading control. Intensity of bands was normalized with that of β-actin. *P < 0.05 (n = 3 from 3 different Western blots, 2-tailed Student’s t test). (B) Rho123 uptake assay was performed using primary brain endothelial cells from brains of WT, A1, A2A, and CD73 KO mice. Cells were grown to confluence and treated with 2.5 μM of Rho123 at 5, 15, 30, and 60 min- utes. Cells were lysed with lysis buffer and were analyzed by fluorometry, with excitation at 488 nm and emission at 523 nm. *P < 0.05; **P < 0.01 (n = 4, 2-tailed Student’s t test, 1 representative result of 2 different experiments). (C) Western blot analysis of P-gp expression levels from brains of WT, A1 KO, A2A KO, and CD73 KO mice. β-Actin was used as loading control. Band intensity was normalized to that of β-actin and graphed. *P < 0.05 (n = 3, 2-tailed Student’s t test). (D) IFA on the brain of WT, A1 KO, A2A KO, and CD73 KO mice. Frozen brain sections were stained with GLUT1 (an endothelial marker, red) and P-gp (green) and counterstained with DAPI (blue). Nucleus was counterstained with DAPI (blue). Scale bar: 100 μm.

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Expressing, Western Blot, Control, Lysis, Staining, Marker

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 7. A2A receptor activation by Lexiscan induces rapid and reversible downmodulation of P-gp expression and function in brain vascular endothelial cells in WT mice. (A) Immunofluorescence images of brains from Lexiscan-treated mice of WT brain at 15 and 30 minutes after Lexiscan treatment. Frozen brain sections were stained with GLUT1 (red), or P-gp (green) and counterstained with DAPI (blue). (B) Enlarged image of A. (C) Brain epirubicin accumula- tion assay in the Lexiscan- and vehicle-treated (control) mice. 10 mg/kg of epirubicin was injected intravenously with or without 0.05 mg/kg of Lexiscan. Mice were perfused with ice-cold PBS and sacrificed at different time points. The accumulation of epirubicin in the brain was measured using fluorometric excitation at 488 nm and emission at 590 nm. *P < 0.05 (n = 4, 2-tailed Student’s t test). (D) Fluorescent microscopic analysis of epirubicin accumulation in the brains of mice treated with Lexiscan or PSC833 (a functional P-gp inhibitor) compared with vehicle (control). 10 mg/kg of epirubicin was injected intravenously with or without 0.05 mg/kg of Lexiscan or 50 mg/kg of PSC833 for 15 minutes. Mice were perfused with ice-cold PBS and sacrificed. Brain was sectioned for microscopic analysis for full-brain image, and focal zoomed image from cortex was laid as an inset. Epirubicin is in red, and nucleus was counterstained with DAPI (blue). (E) Intensity of epirubicin from different regions of cortex (CTX), cerebellum (CRBL), and hippocampus (HPC) from brains of control, Lexiscan-, or PSC833-injected animals was quantified and depicted as graphs. Scale bars: 100 μm (A); 50 μm (B) 5 mm (D). ***P < 0.001 (n = 50, 2-tailed Student’s t test).

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Control, Injection, Functional Assay

Journal: Journal of Clinical Investigation

Article Title: A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

doi: 10.1172/jci76207

Figure Lengend Snippet: Figure 9. Mechanism of regulation of transcellular permeability by A2A AR signaling. (a) In basal status, single layer of brain endothelial cells highly express transporters including P-gp. (b) When AR is acti- vated by adenosine or Lexiscan, (c) it downregulates P-gp on brain endo- thelial cells (d), which increases transcellular permeability and delivery of P-gp substrate into the brain. When P-gp substrate crosses the brain endothelial cells, it is still not clear which transporters subsequently aid the final delivery of molecules into the brain side.

Article Snippet: To test this, we used the FDA-approved A2A AR agonist Lexiscan or the broad-spectrum AR agonist NECA to determine the impact of A2A AR activation on P-gp expression and function.

Techniques: Permeability