Table 1.
AZD9977 and eplerenone activities on MR, GR, PR and AR in binding assays.
Fig 1.
Structures of AZD9977 and eplerenone.
Structures of AZD9977 ((S)-2-(7-Fluoro-4-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carbonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-yl)-N-methylacetamide) and eplerenone.
Fig 2.
AZD9977 and eplerenone activities in reporter gene assays.
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.
Table 2.
AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.
Fig 3.
Crystal structure, reporter gene data on M777V mutation and co-peptide recruitment comparing AZD9977 and eplerenone.
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-2H-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.
Fig 4.
AZD9977 and eplerenone protect against aldosterone and high salt induced renal injury.
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 IC50 (Cuss-average/in vitro IC50). 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.
Fig 5.
AZD9977, eplerenone and co-treatment with enalapril reduce albuminuria in diabetic kidney disease.
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.
Fig 6.
AZD9977 does not change urinary Na+/K+ ratio in rat urine electrolyte secretion model.
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.
Table 3.
Average drug exposure of AZD9977 and eplerenone after oral administration to salt deprived rats.