Angiotensin 2

The structure of SDM‐878 is shown in Figure 1. First, based on π‐stacked horseshoe‐like conformation observed in the crystal structure of human OX₂ receptor bound to suvorexant,¹⁴ we found a lead compound with 3,8‐diazabicyclo[3.2.1]octane using ligand‐based drug design. Then, SDM‐878 was identified in the course of the extensive chemical optimization of the lead compound to find a highly selective OX₂ receptor antagonist with short half‐life in plasma. SDM‐878 was synthesized in‐house and used as a free base in all experiments. SDM‐878 was dissolved in dimethylsulfoxide and stored at −20°C to perform in vitro assays. SDM‐878 was suspended in 0.5% methylcellulose solution (Wako Pure Chemical Industries, Ltd.) and orally administered at a volume of 5 mL/kg. For the pharmacokinetic study, SDM‐878 was dissolved in 40% (v/v) N, N‐dimethylacetamide (Wako Pure Chemical Industries), 40% (v/v) polyethylene glycol 400 (Wako Pure Chemical Industries), and 20% (v/v) distilled water before being administered intravenously at a volume of 1 mL/kg. ADL‐OXB ([Ala¹¹, d‐Leu¹⁵]‐orexin‐B, Tocris Bioscience), an OX₂ receptor agonist, and angiotensin II (Peptide Institute, Inc) were dissolved in physiological saline and administered intracerebroventricularly at a volume of 5 μL/rat. Based on our previous studies, we selected doses of 3 nmol/rat for ADL‐OXB and 100 ng/rat for angiotensin II.¹⁵

Twelve‐month‐old male 5XFAD (n=24) and control C57BL/6J wild‐type mice (n=24) were randomly divided into 2 groups and received continuous intracerebroventricular (ICV) infusion of (1) vehicle (saline) or (2) angiotensin II (20 μg/(kg·h) (Peptide Institute, Inc, Osaka, Japan) for 4 weeks (n=12, in each group). The dosage of angiotensin II was determined according to previous reports.22, 23, 24 The detailed experimental protocol is shown in Figure 1.