Journal: Cancer research

Article Title: Interactions of abiraterone, eplerenone and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100

doi: 10.1158/0008-5472.CAN-11-3980

Figure Lengend Snippet: Sigmoidal dose response curves show activation of (A) WT-AR by R1881 and spironolactone and (B) T877A-AR by R1881, spironolactone, eplerenone, prednisolone, dexamethasone. Fold change from the DMSO control was plotted and EC50s calculated using non-linear regression (GraphPad). EC50 and 95% confidence intervals are given. C, LNCaP and VCaP prostate cancer cells in CSS were treated with eplerenone or spironolactone alone or in combination with 0.1, 1 or 5μM abiraterone, 10μM bicalutamide or 10μM MDV3100 or for 7 days and then analyzed for cell viability. Fold change from the DMSO control was then calculated and plotted. Significance is shown for stimulation by eplerenone or spironolactone compared to DMSO control and for inhibition by bicalutamide, MDV3100 or abiraterone when compared to stimulated levels. D, LNCaP and VCaP cells were treated with 0.1nM R1881 or 0.1 - 10μM eplerenone for 5 hours. RNA was extracted and cDNA synthesized for analysis by quantitative PCR to determine relative levels of PSA and TMPRSS2 mRNA expression. Significance compared to DMSO controls is shown. Data shown for all experiments are the mean (error bars, standard error of the mean, SEM) of 3 independent experiments of 16 replicates (A, B) or in duplicate (C, D). *, P<0.05; **, P<0.01; ***, P< 0.001, one-way ANOVA with Bonferroni correction.

Article Snippet: Bicalutamide, dexamethasone, prednisone and dihydrotestosterone (DHT) (Sigma-Aldrich, UK), tritiated [ 3 H]-R1881 (Perkin-Elmer), R1881 (Steraloids, RI), eplerenone and spironolactone (Tocris-Bioscience, Bristol, UK) were obtained from commercial sources.

Techniques: Activation Assay, Control, Inhibition, Synthesized, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer research

Article Title: Interactions of abiraterone, eplerenone and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100

doi: 10.1158/0008-5472.CAN-11-3980

Figure Lengend Snippet: A, PC-3 cells were co-transfected with ARE3-luciferase and WT or mutant AR (T877A, D879G, W741C, M749L, R629Q). Cells were treated with 0.1 - 25μM abiraterone, 10μM bicalutamide or 10μM MDV3100 in CSS medium containing 0.1nM R1881 for 16 hours and then analyzed for luciferase activity. Fold change from the DMSO control was calculated and then percentage change relative to the R1881-stimulated DMSO control was determined. Data shown are representative of 3 independent experiments and represent mean and SEM of 8 replicates. B, COS-7 cells were co-transfected with GRE2-TATA-Luc and the WT or mutant human expression plasmid pSVARo (T877A, G142V, P533S, T575A, H874Y, R629Q). Cells were treated with 0.1 - 5μM abiraterone or MDV3100 in CSS medium containing 10nM DHT for 24 hours. The luciferase activities were assayed in duplicate and normalized for the amounts of expressed AR determined immunologically by dot blot analysis and normalized for protein concentration. The change in normalized luciferase activity relative to cells incubated without any compound for each AR variant was determined. Data shown represent two or three independent experiments performed in quadruplicate. C, Dose-proportional inhibition of proliferation of LNCaP and VCaP cells by abiraterone, MDV3100 and bicalutamide. LNCaP and VCaP prostate cancer cells in CSS with 0.1nM R1881 were treated with 0.1, 1 or 5μM abiraterone, 10μM bicalutamide or 10μM MDV3100 for 7 days and then analyzed for cell viability. Fold change from the DMSO control was then calculated and plotted. Data shown are the mean (error bars, standard error of the mean, SEM) of 3 independent experiments in quadruplicate. D, LNCaP cells were treated with 0.1nM R1881 or 10μM eplerenone in combination with DMSO, 10μM bicalutamide, 10μM MDV3100 or 5μM abiraterone for 5 hours. RNA was extracted and cDNA synthesized for analysis by quantitative PCR to determine relative levels of PSA and TMPRSS2 mRNA expression. Data shown are the mean and SEM of 3 independent experiments in duplicate. Significance is shown for *, P<0.05; **, P<0.01; ***, P< 0.001; ****, P<0.0001 relative to DMSO control (one-way ANOVA with Bonferroni correction).

Article Snippet: Bicalutamide, dexamethasone, prednisone and dihydrotestosterone (DHT) (Sigma-Aldrich, UK), tritiated [ 3 H]-R1881 (Perkin-Elmer), R1881 (Steraloids, RI), eplerenone and spironolactone (Tocris-Bioscience, Bristol, UK) were obtained from commercial sources.

Techniques: Transfection, Luciferase, Mutagenesis, Activity Assay, Control, Expressing, Plasmid Preparation, Dot Blot, Protein Concentration, Incubation, Variant Assay, Inhibition, Synthesized, Real-time Polymerase Chain Reaction

Journal: Cancer research

Article Title: Interactions of abiraterone, eplerenone and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100

doi: 10.1158/0008-5472.CAN-11-3980

Figure Lengend Snippet: PC-3 cells were transfected with WT AR (A) or T877A mutant AR (B) and then treated with CSS media containing 5nM of [3H] R1881 in combination with cold R1881, DHT, eplerenone, abiraterone or bicalutamide at the concentrations shown for 2 hours. Abiraterone was insoluble in cell media at concentrations greater than 25μM. Cell-associated radioactivity was measured and the data analyzed by nonlinear regression to determine the EC50 for each test compound (GraphPad Prism). Data shown are the mean and SEM of three independent experiments in triplicate for percentage (%) [3H]-R1881 bound versus log10 of concentration (μM) of cold competitor. EC50 and 95% confidence intervals are given. C, Inhibition of eplerenone-stimulated AR activation by bicalutamide, MDV3100 and abiraterone. PC-3 cells were co-transfected with ARE3-luciferase and T877A mutant AR. Cells were treated with DMSO (control) or eplerenone in combination with DMSO, 10μM bicalutamide, 10μM MDV3100 or 5μM abiraterone for 16 hours and then analyzed for luciferase activity. Fold change from the DMSO control was calculated. Data shown are from three independent experiments and represent mean and SEM of 13 replicates. D, Increased hormone levels reduce AR inhibition by MDV3100. PC-3 cells were co-transfected with ARE3-luciferase and WT-AR. Cells were treated with R1881 in combination with DMSO or MDV3100 at the concentrations indicated for 16 hours and then analyzed for luciferase activity. Fold change from the DMSO control was calculated. Data shown are from three independent experiments and represent mean and SEM of 24 replicates. ***, P< 0.01 relative to R1881 or DHT control with DMSO (one-way ANOVA with Bonferroni correction).

Article Snippet: Bicalutamide, dexamethasone, prednisone and dihydrotestosterone (DHT) (Sigma-Aldrich, UK), tritiated [ 3 H]-R1881 (Perkin-Elmer), R1881 (Steraloids, RI), eplerenone and spironolactone (Tocris-Bioscience, Bristol, UK) were obtained from commercial sources.

Techniques: Transfection, Mutagenesis, Radioactivity, Concentration Assay, Inhibition, Activation Assay, Luciferase, Control, Activity Assay

Journal: BMJ Open Diabetes Research & Care

Article Title: miR-196b-5p-enriched extracellular vesicles from tubular epithelial cells mediated aldosterone-induced renal fibrosis in mice with diabetes

doi: 10.1136/bmjdrc-2019-001101

Figure Lengend Snippet: Aldosterone (Aldo) impaired renal function and aggravated renal fibrosis. (A) Blood urea nitrogen (BUN) and urine albumin-to-creatinine ratio (UACR) of db/db mice after exposure to Aldo for 6 weeks (n=10/group). (B) Representative image of H&E and periodic acid-Schiff (PAS) staining for tubulointerstitial lesions detection, Sirius red and Masson’s trichrome staining for interstitial fibrosis detection and immunofluorescence staining for fibronectin (FN, green), collagen type I alpha 1 (col1A1) (red) and Hoechst (blue) and quantification. Scale bar, 100 µm. (C) FN and alpha-smooth muscle actin (α-SMA) protein levels of db/db renal cortex with different treatments detected by western blot analysis and quantified. *P<0.05, **p<0.01 vs control, #p<0.05, ##p<0.01 vs Aldo. Numeric data are presented as the means±SEM. DAPI, 4,6-diamidino-2-phenyiindole 2 hci; Epl, eplerenone; MTS, Masson’s trichrome staining.

Article Snippet: Eplerenone (s1707, Selleck) 100 mg/kg/day results in a substantial reduction in albuminuria and the progression of DKD with a lesser risk for hyperkalemia.

Techniques: Staining, Immunofluorescence, Western Blot, Control

Journal: BMJ Open Diabetes Research & Care

Article Title: miR-196b-5p-enriched extracellular vesicles from tubular epithelial cells mediated aldosterone-induced renal fibrosis in mice with diabetes

doi: 10.1136/bmjdrc-2019-001101

Figure Lengend Snippet: Effect of aldosterone (Aldo) on tubular epithelial cells and fibroblasts. (A) Representative image of E-cadherin staining in mice. Scale bar, 100 µm. (B) NGAL protein levels in db/db renal cortex with different treatments detected by western blot analysis and quantified. (C) E-cadherin and Snail protein levels in proximal tubule epithelial cells (PTECs) with different treatments detected by western blot analysis and quantified. (D) Representative photomicrographs of PTEC images by transmission electron microscopy (TEM). Scale bar, 2 µm. (E) Fibronectin (FN), collagen type I alpha 1 (col1A1) and alpha-smooth muscle actin (α-SMA) protein levels in fibroblasts with different treatments for 72 hours detected by western blot analysis and quantified. (F) MR protein levels of PTECs and fibroblasts detected by western blot analysis. *P<0.05, **p<0.01 vs control, #p<0.05, ##p<0.01 vs Aldo. Numeric data are presented as the means±SEM. Epl, eplerenone; MR, mineralocorticoid receptor; NGAL, neutrophil gelatinase-associated lipocalin.

Article Snippet: Eplerenone (s1707, Selleck) 100 mg/kg/day results in a substantial reduction in albuminuria and the progression of DKD with a lesser risk for hyperkalemia.

Techniques: Staining, Western Blot, Transmission Assay, Electron Microscopy, Control

Journal: BMJ Open Diabetes Research & Care

Article Title: miR-196b-5p-enriched extracellular vesicles from tubular epithelial cells mediated aldosterone-induced renal fibrosis in mice with diabetes

doi: 10.1136/bmjdrc-2019-001101

Figure Lengend Snippet: Extracellular vesicles (EVs) from proximal tubule epithelial cells (PTECs) activated fibroblasts. (A) Electron microscopic image of whole-mounted EVs purified from PTECs (left, bar, 2 µm; right, bar, 100 nm). EV protein fractions were processed for western blot analysis with the indicated antibodies against EV markers CD9, CD63 and TSG101 and the negative marker GRP94. The size of the EVs was analyzed by a Zetasizer Nano particle analyzer. (B) An in vitro coculture system was used in which different treated PTECs were seeded in the top compartment, separated by a porous membrane from fibroblasts that were cultured in the bottom compartment. Representative images of EVs released from Dio-labeled PTECs and internalized by fibroblasts. GW4869 was used as an EV secretion inhibitor (three independent experiments; n=3). Scale bar, 100 µm. (C) EV uptake experiment. PTEC-derived EVs were labeled with red fluorescent dye and incubated with fibroblasts. Fibroblasts were incubated with PKH26-labeled EVs for 24 hours (four independent experiments; n=4). Scale bar, 100 µm. (D) MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay showing proliferation of fibroblasts after treatment with EVs that were isolated from different treated PTECs. (E) Migration analysis of fibroblasts 48 hours after treatment with EVs that were isolated from different treated PTECs and quantification of migrated cells. (F) Fibronectin (FN), collagen type I alpha 1 (col1A1) and alpha-smooth muscle actin (α-SMA) protein levels in fibroblasts with different treatments detected by western blot analysis for interstitial fibrosis and quantified. (G) Representative images of immunofluorescence staining for col1A1 (green), FN (green), α-SMA (green) and Hoechst (blue) in fibroblasts treated with EVs extracted from PTECs under different treatments and quantification. Scale bar, 100 µm. *P<0.05, **p<0.01 vs EVs, #p<0.05, ##p<0.01 vs Aldo-EVs. Numeric data are presented as the means±SEM. Aldo, aldosterone; DAPI, 4,6-diamidino-2-phenyiindole 2 hci; Epl, eplerenone.

Article Snippet: Eplerenone (s1707, Selleck) 100 mg/kg/day results in a substantial reduction in albuminuria and the progression of DKD with a lesser risk for hyperkalemia.

Techniques: Purification, Western Blot, Marker, In Vitro, Membrane, Cell Culture, Labeling, Derivative Assay, Incubation, Isolation, Migration, Immunofluorescence, Staining

Journal: BMJ Open Diabetes Research & Care

Article Title: miR-196b-5p-enriched extracellular vesicles from tubular epithelial cells mediated aldosterone-induced renal fibrosis in mice with diabetes

doi: 10.1136/bmjdrc-2019-001101

Figure Lengend Snippet: miRNA-196b-5p promoted fibroblast activation and proliferation. (A) Fibronectin (FN), collagen type I alpha 1 (col1A1) and alpha-smooth muscle actin (α-SMA) protein levels in fibroblasts with different treatments detected by western blot analysis for interstitial fibrosis and quantified. The control fraction contains both RNA and proteins; the ribonuclease (RNase)+proteinase fraction contains trace RNAs and trace proteins; the proteinase fraction contains RNAs and trace proteins and the RNase fraction contains proteins and trace RNAs. *P<0.05, **p<0.01 vs RNase or RNase+proteinase. Numeric data are presented as the means±SEM. (B) miR-196b-5p abundance in extracellular vesicles (EVs) secreted by proximal tubule epithelial cells (PTECs). (C) Expression level of miR-196b-5p in PTECs. (D) Expression levels of miR-196b-5p in fibroblasts. (E) Expression level of miR-196b-5p in the renal cortex of db/db mice. (F) miR-196b-5p abundance in EVs derived from the renal cortex of db/db mice. Data were normalized to levels of U6. *P<0.05, **p<0.01 vs EVs or control, #p<0.05, ##p<0.01 vs Aldo-EVs or Aldo. (G) The expression of miR-196b-5p in fibroblasts after transfection of miR-196b-5p mimic and inhibitor. (H) MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay of fibroblasts after overexpression or knockdown of miR-196b-5p. (I) The expression of the SOCS family, a miR-196b-5p target gene, after overexpression or knockdown of miR-196b-5p. (J) FN, col1A1, alpha-smooth muscle actin (α-SMA), SOCS2 and p-stat3 protein levels in fibroblasts. *P<0.05, **p<0.01 vs miR-con. Numeric data are presented as the means±SEM. Aldo, aldosterone; Epl, eplerenone; miRNA, microRNA.

Article Snippet: Eplerenone (s1707, Selleck) 100 mg/kg/day results in a substantial reduction in albuminuria and the progression of DKD with a lesser risk for hyperkalemia.

Techniques: Activation Assay, Western Blot, Control, Expressing, Derivative Assay, Transfection, Over Expression, Knockdown

Journal: BMJ Open Diabetes Research & Care

Article Title: miR-196b-5p-enriched extracellular vesicles from tubular epithelial cells mediated aldosterone-induced renal fibrosis in mice with diabetes

doi: 10.1136/bmjdrc-2019-001101

Figure Lengend Snippet: Extracellular vesicles (EVs) from the renal cortex of Aldo-treated mice caused kidney injury in recipient mice. (A) Electron microscopic image of whole-mounted EVs purified from the renal cortex of db/db mice. Scale bar: 100 nm. EV protein fractions were processed for western blot analysis with the indicated EV marker antibodies against CD9, CD63 and TSG101. The size of the EVs was analyzed using a Zetasizer Nano particle analyzer. (B) C57BL/6 mice were injected via the tail vein with PKH26-labeled EVs of approximately 30 mg/mouse generated in vitro by the renal cortex of db/db mice or phosphate buffered saline. Representative kidney sections after 16 hours of PKH26-labeled EV injection (three independent experiments; n=3). Scale bar, 100 µm. (C) Representative image of miR-196b-5p staining in C57BL/6 mice, representative image of Sirius Red staining and Masson’s trichrome staining for interstitial fibrosis detection and representative image of fibronectin (FN), collagen type I alpha 1 (col1A1) and alpha-smooth muscle actin (α-SMA) staining for renal injury in C57BL/6 mice after 72 hours of EV injection and quantification. Scale bar, 100 µm. (D) FN, col1A1 and α-SMA protein levels in the renal cortex of C57BL/6 mice with different treatments detected by western blot analysis for interstitial fibrosis and quantified. *P<0.05, **p<0.01 vs EVs, #p<0.05, ##p<0.01 vs Aldo-EVs. Numeric data are presented as the means±SEM. Aldo, aldosterone; DAPI, 4,6-diamidino-2-phenyiindole 2 hci; Epl, eplerenone; MTS, Masson’s trichrome staining; PBS, phosphate-buffered saline.

Article Snippet: Eplerenone (s1707, Selleck) 100 mg/kg/day results in a substantial reduction in albuminuria and the progression of DKD with a lesser risk for hyperkalemia.

Techniques: Purification, Western Blot, Marker, Injection, Labeling, Generated, In Vitro, Saline, Staining

Journal: Frontiers in Pharmacology

Article Title: Combination of ADAM17 knockdown with eplerenone is more effective than single therapy in ameliorating diabetic cardiomyopathy

doi: 10.3389/fphar.2024.1364827

Figure Lengend Snippet: Cardiac fibroblasts transformation and RAAS activation following ADAM17 knockdown, eplerenone administration and combined treatment in DCM mice. (A) Representative of fibroblast activation protein (FAP) staining (scale bar = 20 μm) in four groups of mice. (B) Quantification of FAP expression in four groups of mice. (C) Representative Western blotting images of Collagen III, Collagen I, FAP and α-SMA expression in four groups of mice. (D–G) Quantification of Collagen III, Collagen I, FAP and α-SMA expression in four groups of mice. (H) Representative Western blotting images of ADAM17, ACE2, AT1R and AT2R expression in four groups of mice. (I–L) Quantification of ADAM17, ACE2, AT1R and AT2R expression in four groups of mice. Values shown were mean and SEM ( n = 6 in each group). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Eplerenone was purchased from BOC Sciences (USA).

Techniques: Transformation Assay, Activation Assay, Knockdown, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Combination of ADAM17 knockdown with eplerenone is more effective than single therapy in ameliorating diabetic cardiomyopathy

doi: 10.3389/fphar.2024.1364827

Figure Lengend Snippet: Cardiac fibroblasts transformation and RAAS activation following ADAM17 knockdown, eplerenone intervention and combined-administration in primary cardiac fibroblasts. (A) Representative Western blotting images of ADAM17 expression in cardiac fibroblasts treated with different concentrations glucose. (B,C) Quantification analysis of ADAM17 protein expression and activity in cardiac fibroblasts treated with different concentrations glucose. (D) Representative Western blotting images of Collagen III, Collagen I, FAP and α-SMA expression in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. (E–H) Quantification of Collagen III, Collagen I, FAP and α-SMA expression in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. (I) Representative Western blotting images of ADAM17, ACE2, AT1R and AT2R expression in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. (J–M) Quantification of ADAM17, ACE2, AT1R and AT2R expression in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. Values shown were mean and SEM ( n = 6 in each group). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Eplerenone was purchased from BOC Sciences (USA).

Techniques: Transformation Assay, Activation Assay, Knockdown, Western Blot, Expressing, Activity Assay, Saline

Journal: Frontiers in Pharmacology

Article Title: Combination of ADAM17 knockdown with eplerenone is more effective than single therapy in ameliorating diabetic cardiomyopathy

doi: 10.3389/fphar.2024.1364827

Figure Lengend Snippet: The activation of TGF-β1/Smad3 pathway and mechanism of the roles of combination of ADAM17 knockdown and eplerenone treatment in DCM-induced cardiac fibrosis. (A) Representative Western blotting images of TGF-β1, Smad3 and phospho-Smad3 expression in six groups of mice. (B,C) Quantification of TGF-β1 and the ratio of pSmad3/Smad3 in six groups of mice. (D) Representative Western blotting images of TGF-β1, Smad3 and phospho-Smad3 expression in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. (E,F) Quantification of TGF-β1 and the ratio of pSmad3/Smad3 in cardiac fibroblasts treated with LG, HG, HG + siNC + saline, HG + siA17+saline, HG + siNC + Epl, HG + siA17+Epl, respectively. Values shown were mean and SEM ( n = 6 in each group). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Eplerenone was purchased from BOC Sciences (USA).

Techniques: Activation Assay, Knockdown, Western Blot, Expressing, Saline

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: Structures of AZD9977 ((S)-2-(7-Fluoro-4-(3-oxo-3,4-dihydro-2H-benzo[b][ , ]oxazine-6-carbonyl)-3,4-dihydro-2H-benzo[b][ , ]oxazin-3-yl)-N-methylacetamide) and eplerenone.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques:

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: AZD9977 and eplerenone activities on MR, GR, PR and AR in binding assays.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: Concentration response curves of AZD9977 and eplerenone tested in a reporter gene assay in (a) presence or (b) absence of 0.1 nM aldosterone. n = 4, average ± SD.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: Concentration Assay, Reporter Gene Assay

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques:

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: The structures of the mineralocorticoid receptor (MR) ligand binding domain (LBD) in complex with (a) eplerenone and (b) AZD9977. The ligands are shown in magenta surrounded by refined 2mFo-DFc electron density as indicated by blue mesh. Putative hydrogen bonds are marked as dashed lines. Near the interface in between helices 3 and 5, the AZD9977 amide extension is placed with the oxygen in a position within hydrogen bonding distance of Gln776, Arg817, Ser810 and an ordered water molecule. In contrast, the eplerenone 3-keto group is further removed from the helix 3–5 interface and only appeared to make a direct interaction with Arg817. In addition, the AZD9977 3,4-dihydro-2 H -1,4-benzoxazine oxygen is positioned to make a direct interaction to one of the observed rotamers of Ser811. As Ser811 is unique to MR within the steroid receptor family, this interaction may contribute to the selectivity profile of AZD9977. At the other end of the ligand binding pocket, the AZD9977 N -methyl-acetamide side chain is optimally placed to form a direct interaction to Thr945 and a bidentate interaction with Asn770. While the eplerenone γ-lactone is positioned to interact with Thr945 it is further away from Asn770 and only appears to make a weak interaction with the asparagine Nδ. (c) Reporter gene data for AZD9977 (solid) or eplerenone (open) tested on Met777Val MR mutation in presence (black) or absence (blue) of 0.1 nM aldosterone. n = 4, average ± SD. (d) Recruitment, relative to reference compound (dexamethasone), of MR-LBD to seven different co-regulator peptides for aldosterone (green), AZD9977 (red) and eplerenone (blue) at saturated binding conditions (50 μM). n = 2, average ± SD.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: Ligand Binding Assay, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: Uni-nephrectomised rats with an aldosterone mini-pump and on a high salt diet were treated for 4 weeks with AZD9977 or eplerenone as food admixtures. Doses in mg kg -1 d -1 are indicated under the bars. (a) UACR was measured in urine collected for 24h at day 28. (b) UACR vs free steady average plasma drug exposure normalized to rat in vitro IC 50 (C uss -average/ in vitro IC 50 ). Drug exposure levels correspond to the exposure levels achieved at the studied doses (a). c) Renal pathology scores (renal fibrosis and glomerular nephritis). d) Histological sections stained with PAS.Representative photomicrographs of glomeruli/tubular sections from rats treated with vehicle (V), AZD9977 at 10 (AZ 10), 30 (AZ 30) or 100 mg kg -1 d -1 (AZ 100) or eplerenone at 10 (EP 10) or 30 mg kg -1 d -1 (EP 30). The scale bar corresponds to 200 μm original size. Average +/- SEM; n = 7–8; *p<0.05 compared to vehicle.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: Clinical Proteomics, In Vitro, Staining

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: db/db mice uni-nephrectomised at 8 weeks of age were treated from age 18w to age 22w with 100 mg kg -1 d -1 AZD9977 or 100 mg kg -1 d -1 eplerenone alone or in combination with enalapril. (a) Total urine albumine secreted over 24 hours was assessed. (b) Glomerular ECM scores were quantitated as % of glomerular tuft staining. (c) Histological sections stained with PAS. Representative photomicrographs of glomeruli from lean control mice (L), diabetic db/db mice without treatment at 18 weeks of age (w18) or at 22 weeks of age (w22) and diabetic db/db mice treated with AZD9977 (AZ), eplerenone (EP), enalapril (EN), combination of AZD9977 and enalapril (EN+AZ) and eplerenone and enalapril (EN+EP). The scale bar corresponds to 50 μm original size. Average +/- SEM; n = 7–9; *p<0.05 compared to vehicle at 22 weeks of age. # p<0.05 compared to enalapril treatment.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: a) Salt deprived rats were treated with increasing doses of eplerenone (a) or AZD9977 (b), urine collection for 8 hours after dose. Average +/- SEM; n = 8; *p<0.05 compared to vehicle. c) Salt deprived rats were treated with 10 or 30 mg kg -1 eplerenone in absence or presence of 100 mg kg -1 AZD9977, urine collection for 8 hours after dose. Average +/- SEM; n = 8, *p<0.05 vs vehicle. # p<0.05 vs eplerenone treatment in absence of AZD9977.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques:

Journal: PLoS ONE

Article Title: Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

doi: 10.1371/journal.pone.0193380

Figure Lengend Snippet: Average drug exposure of AZD9977 and eplerenone after oral administration to salt deprived rats.

Article Snippet: Indivuals were randomised on body weight and orally gavaged with vehicle (0.5% HPMC, 0.1% Tween80, 5% Solutol HS15) and/or AZD9977 and/or eplerenone (Molekula Limited, UK) at 7:00 am at the day of the experiment.

Techniques: In Vitro

Journal: BioMed Research International

Article Title: The Influence of Ionizing Radiation, Temperature, and Light on Eplerenone in the Solid State

doi: 10.1155/2014/571376

Figure Lengend Snippet: Structural formulas of eplerenone and its potential impurities .

Article Snippet: Eplerenone and its impurities were obtained from Industriale Chimica s.r.l., Saronno, Italy.

Techniques:

Journal: BioMed Research International

Article Title: The Influence of Ionizing Radiation, Temperature, and Light on Eplerenone in the Solid State

doi: 10.1155/2014/571376

Figure Lengend Snippet: Chromatograms of eplerenone and its 7 potential impurities.

Article Snippet: Eplerenone and its impurities were obtained from Industriale Chimica s.r.l., Saronno, Italy.

Techniques:

Journal: BioMed Research International

Article Title: The Influence of Ionizing Radiation, Temperature, and Light on Eplerenone in the Solid State

doi: 10.1155/2014/571376

Figure Lengend Snippet: Eplerenone and impurities of known structure in degraded and nondegraded samples.

Article Snippet: Eplerenone and its impurities were obtained from Industriale Chimica s.r.l., Saronno, Italy.

Techniques:

Journal: BioMed Research International

Article Title: The Influence of Ionizing Radiation, Temperature, and Light on Eplerenone in the Solid State

doi: 10.1155/2014/571376

Figure Lengend Snippet: Eplerenone and impurities of unknown structure in degraded and nondegraded samples.

Article Snippet: Eplerenone and its impurities were obtained from Industriale Chimica s.r.l., Saronno, Italy.

Techniques:

Journal: American Journal of Translational Research

Article Title: An additive effect of eplerenone to ACE inhibitor on slowing the progression of diabetic nephropathy in the db/db mice

doi:

Figure Lengend Snippet: Effect of enalapril, elperenone and combination therapy on glomerular immunofluorescent staining for podocin and WT-1 in diabetic db/db mice from weeks 18 to 22. (A) Representative photomicrographs (at original magnification × 400) of glomeruli from normal control mice (db/m), diabetic db/db mice without treatment at week 18 (db/db-18 wk) or at week 22 (db/db-22 wk) and diabetic db/db mice treated with enalapril (Ena), eplerenone (Epl), combination of enalapril and eplerenone (Ena+Epl). Graphic representation of glomerular podocin (B) staining scores or glomerular number of WT-1 positive podocytes (C) was shown below. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18 wk. §P<0.05, vs. db/db-22 wk. ‡P<0.05, vs. db/db+Ena. ζP<0.05, vs. db/db+Epl.

Article Snippet: Eplerenone was provided by AstraZeneca (Molndal, Sweden).

Techniques: Staining, Control

Journal: American Journal of Translational Research

Article Title: An additive effect of eplerenone to ACE inhibitor on slowing the progression of diabetic nephropathy in the db/db mice

doi:

Figure Lengend Snippet: Effect of enalapril, elperenone and combination therapy on glomerular matrix protein accumulation in diabetic db/db mice from weeks 18 to 22. (A) The histological sections stained with PAS and glomerular immunofluorescent staining for fibronectin (FN) and type IV collagen (Col IV) are presented at 400 × magnification. Representative photomicrographs of glomeruli from normal control mice (db/m), diabetic db/db mice without treatment at week 18 (db/db-18 wk) or at week 22 (db/db-22 wk) and diabetic db/db mice treated with enalapril (Ena), eplerenone (Epl), combination of enalapril and eplerenone (Ena+Epl). Graphic representations of glomerular matrix score (B), FN staining score (C) and Col IV staining score (D) are shown below. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18 wk. §P<0.05, vs. db/db-22 wk. ‡P<0.05, vs. db/db+Ena. ζP<0.05, vs. db/db+Epl.

Article Snippet: Eplerenone was provided by AstraZeneca (Molndal, Sweden).

Techniques: Staining, Control

Journal: American Journal of Translational Research

Article Title: An additive effect of eplerenone to ACE inhibitor on slowing the progression of diabetic nephropathy in the db/db mice

doi:

Figure Lengend Snippet: Effect of enalapril, elperenone and combination therapy on protein production of TNFα in renal cortical tissue and urine TNFα and MCP-1 levels. (A) Representative western blots illustrating TNFα and β-actin protein expression. The lower respective graph summarizes the results of band density measurements. Urinary TNFα (B) MCP-1 (C) levels were measured by ELISA. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18 wk. §P<0.05, vs. db/db-22 wk. ‡P<0.05, vs. db/db+Ena. ζP<0.05, vs. db/db+Epl. Diabetic db/db mice were treated with enalapril (Ena), eplerenone (Epl), combination of enalapril and eplerenone (Ena+Epl) from weeks 18 to 22.

Article Snippet: Eplerenone was provided by AstraZeneca (Molndal, Sweden).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Translational Research

Article Title: An additive effect of eplerenone to ACE inhibitor on slowing the progression of diabetic nephropathy in the db/db mice

doi:

Figure Lengend Snippet: Effect of enalapril, elperenone and combination therapy on protein production of renal NAPDH oxidases and the TBARS levels in renal cortex tissue. Representative western blots illustrate Nox2, p47phox and β-actin protein expression and graphic representation of the mean band intensity of Nox2 (A) and p47phox (B) normalized against the band intensity of β-actin. (C) Renal TBARS levels were detected by a colorimetric assay. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18 wk. §P<0.05, vs. db/db-22 wk. ‡P<0.05, vs. db/db+Ena. ζP<0.05, vs. db/db+Epl. Diabetic db/db mice were treated with enalapril (Ena), eplerenone (Epl), combination of enalapril and eplerenone (Ena+Epl) from weeks 18 to 22.

Article Snippet: Eplerenone was provided by AstraZeneca (Molndal, Sweden).

Techniques: Western Blot, Expressing, Colorimetric Assay