2.1 Animals
Dahl Salt Sensitive (DSS) male rats were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were free to access sterilized standard laboratory food and water. The animal room environment was controlled (target conditions: temperature 20 to 26°C, relative humidity 30 to 70%, 12 h of artificial light, and 12 h of dark). Temperature and relative humidity were monitored twice daily. The study was conducted in accordance with the Institutional Animal Care and Use Committee at WuXi AppTec.
2.2 Reagents
All the test drugs were supplied by Huizhou Salubris Co. Ltd (Storage condition: 25°C nitrogen cabinet free of light): S086 (Lot: ZXM18002, Purity: 95.51%), EXP3174 (Lot: SXMA18002, Purity: 91.47%), sacubitril calcium salt (Lot: SXMA18001, Purity: 95.36%), and LCZ696 (Lot: DYF17006, Purity: 94.37%).
The study used 0.5% CMC-Na as the vehicle for all the compounds. This vehicle was prepared by weighing and dissolving CMC-Na in deionized water at a concentration of 0.5 g CMC-Na/100 ml water. All the compounds were dissolved in this 0.5% CMC-Na solution at a concentration calculated based on their anhydrous-free acid weight. The theoretical weight of each compound was multiplied by a corresponding conversion factor (S086: 1.12; sacubitril calcium salt: 1.10; EXP3174: 1.09; and LCZ696: 1.14) to determine the actual weight to be used in the solution. The prepared solutions were stored at 2-8°C and were used within specific timeframes. S086 and EXP3174 solutions were prepared every 3 days, while sacubitril calcium salt and LCZ696 solutions were prepared daily. Prior to dosing, the solutions were allowed to reach room temperature for 10-15 minutes. The rats were each given a dosage of 5 ml/kg of body weight, which was well-mixed with their respective solutions.
Pentobarbital, supplier: Alfasan International B.V.; lot: 1709296-02; property: white powder; storage: light-free at room temperature. Na+test kit, supplier: MedicalSystem; lot: 180908101, 180921101; storage: 2-8℃. 8% salt forage (blue particles), supplier: Jiangsu Medicience; storage: 2-8℃. 0.3% salt forage (yellow particles), supplier: Jiangsu Medicience; storage: 2-8℃. Meloxicam injection, supplier: Qilu Animal Health Products CO., LTD. lot: 1805001. Gentamicin, supplier: Yichang Humanwell Pharma CO., LTD. lot: 20180139.