Kinase experiment:

Binding of [125I]-Ang-(1-7) is performed. Briefly, 100 μg of membranes from primary cultured bovine aortic endothelial cells (BAECs, passage 1) are incubated in a total volume of 200 μL for 45 minutes at 25℃ in HEPES-buffered saline (10 mM HEPES, 0.1 M NaCl, 5 mM MgCl2) containing 0.2% BSA and protease inhibitor cocktail Complete (Boehringer Mannheim). Saturable binding of [125I]-Ang-(1-7) is calculated by subtracting nonspecific binding (40% to 50%), determined in the presence of 10 μM unlabeled Ang-(1-7) from total binding. Competition experiments with increasing concentrations of AVE 0991 and unlabeled Ang-(1-7) are performed in the presence of 10 nM [125I]-Ang-(1-7). Assays are terminated by vacuum filtration (≤15 mm Hg) over Durapore filters (0.65 μm, Opak 96-well plates) presoaked with 1% BSA. The filters are washed 3 times with each 100 μL of PBS (50 mM, NaHPO4 and 0.15 M NaCl, pH 7.2). Radioactivity on dried filters is quantified with a gamma counter[1].

Animal experiment:

Mice[2] Swiss male mice, Mas-KO (Mas-/-) male mice on the pure genetic background C57BL/6, and WT C57BL/6 control mice (Mas+/+) are used. Water diuresis is induced by intraperitoneal water injection (0.05 mL/g of body weight [BW]) in conscious mice. Drugs are administered in the same injection with water load at prefixed volumes (0.01 mL/g BW). In the first set of experiments, WT mice (C57BL/6, control group) or Mas-KO mice are treated with: (1) 0.58 nmol/g AVE 0991 (n=9, control; n=11, Mas-KO mice); or (2) vehicle for AVE 0991 (10 μM KOH, 0.01 mL/g; n=9, control; n=9, Mas-KO). In the second set, Swiss mice are treated with: (1) vehicle (n=36); (2) 0.58 nmol/g AVE 0991 (n=16); (3) 46 pmol/g Ang-(1-7) antagonist A-779 (n=4); (4) 2 nmol/g losartan or valsartan (n=5); (5) 2 nmol/g AT2 receptor antagonists PD123319 or PD123177 (n=9); (6) AVE 0991 combined with A-779; (7) AVE 0991 combined with losartan or valsartan (n=4 for each); (8) or AVE 0991combined with PD123319 (n=5) or PD123177 (n=4). The urinary volume is measured for 60 minutes after water loading, and urine samples are obtained to determine the osmolality. The dose of AVE 0991is based in preliminary experiments performed in Swiss mice. Rats[3] Male Wistar rats weighting 250-300 g are used. Rats are treated either with AVE-0991 (1 mg/kg, n=9) or vehicle (0.9% NaCl, n=11) administered orally by gavage. At the end of the 7 day period of AVE-0991 treatment, the animals are decapitated 10-15 min after intraperitoneal injection of 400 IU of heparin. After the thorax is opened, the heart is carefully dissected, removed from the thoracic cavity, and placed in a plate containing ice-cold Krebs-Ringer solution (KRS) to attenuate any potential cardiac damage during dissection of aorta artery.

IC50: AVE 0991 and Ang-(1–7) competitively bind to bovine aortic endothelial cell membranes with IC50 values of 21 ± 35 and 220 ± 280 nM, respectively.

Angiotensin-(1–7) (Ang-[1–7]) acts as a crucial component of the renin-angiotensin system which can regulate and maintain the blood pressure by offsetting effects of Ang II, a typical vasoconstrictor in vivo. AVE 0991, a nonpeptide mimic of Ang-(1–7), performs as an agonist of angiotensin-(1-7) receptor. It plays an important role in exploring effects of Ang-(1-7) and evaluating the potential of Ang-(1-7) as a cardiovascular drug. [1]

In vitro: AVE 0991 was found to compete with Ang-(1-7) for bovine aortic endothelial cell membrane receptors with an IC50 of 21±35 nM. There was no significant difference in peak concentrations of NO and O2- released from AVE 0991 treated group and Ang-(1-7) treated group. However, the released amount of NO was approximately 5 times higher in AVE 0991 group than that in Ang-(1-7) group. Moreover, AVE 0091 significantly displaced the binding of I-Ang-(1-7) in both Mas-transfected monkey kidney cells (COS) and Mas-transfected Chinese hamster ovary (CHO) cells. [1]

In vivo: AVE 0091 at a dosage of 0.58 nmol/g resulted in a significant decrease in urinary volume, together with an increase in urinary osmolality in water-loaded C57BL/6 mice. [2] The antidiuretic effect of AVE was completely offset by the Ang II antagonists, which indicting a high specificity of AVE 0991. Since Ang II induced atherogenesis and ang-(1–7) offset Ang II action, it was proved that AVE 0991 blocked atherogenesis in apolipoprotein E (apoE)-knockout mice model. [3]

Clinical trial: So far, no clinical trial has been conducted.

References:
[1]Wiemer G, Dobrucki LW, Louka FR, Malinski T, Heitsch H.  AVE 0991, a nonpeptide mimic of the effects of angiotensin-(1–7) on the endothelium. Hypertension. 2002 Dec; 40: 847-52.
[2] Pinheiro SV, Silva AC, Sampaio WO, Paula RD, Mendes EP, Bontempo ED, Pesquero JB, Walther T, Alenina N, Bader M, Bleich M and Santos RA.  Nonpeptide AVE 0991 is an angiotensin-(1-7) receptor Mas agonist in the mouse kidney. Hypertension. 2004 Oct; 44(4):490-6.
[3]Toton-Zuranska J, Gajda M, Pyka-Fosciak G, Kus K, Pawlowska M, Niepsuj A, Wolkow P, Olszanecki R, Jawien J and Korbut R.  AVE 0991- angiotensin-(1-7) receptor agonist, inhibits atherogenesis in APOE-knockout mice. J Physiol Pharm. 2010, 61(2):181-183.