As for safe profiles, we tested single-dose toxicity, repeat-dose toxicity, hERG inhibition, and mutagenicity of JX01. Both single-dose toxicity study and repeat-dose toxicity study were conducted in mice. No mortality, signs of morbidity, or alterations in relative organ weight were observed in mice within 14 days after single administration of JX01 (2000 mg/kg, 200-fold greater than the therapeutic dose, p.o.), comparable with either vehicle- or EMPA-treated mice (Supporting Information Fig. S7). In repeat-dose toxicity study, no deleterious changes in body weight or relative organ weight were observed after 14 days of repeated administration of JX01 (100 mg/kg, 10-fold greater than that therapeutic dose, p.o.) (Fig. 7A and B). Analysis of biochemical parameters, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), and urea, and histomorphological analysis by hematoxylin-eosin staining to assess potential effects on liver and kidney function further demonstrated that JX01 had no deleterious effects on the liver or kidney (Fig. 7C−G). JX01 did not significantly inhibit hERG at a concentration of 30 μmol/L (IC50 > 30 μmol/L, Table 2). In addition, to examine mutagenicity potential of JX01, mini-Ames reverse mutation test was performed in Salmonella typhimurium strains TA98 and TA100 with and without S9 metabolic activation. The results showed that JX01 did not induce a ≥ 2-fold increase in colony formation in both strains, regardless of S9 metabolic activation, indicating that JX01 exhibits no genotoxic effect (Table 2). Together, these preliminary data verified that JX01 has good pharmaceutical properties and safety, which strongly supports the further development of JX01 as a novel anti-HF drug.This work was supported by the National key R&D Program of China (2021YFA0804904), the National Natural Science Foundation of China (22107030), the Chinese Postdoctoral Science Foundation (2020M681211), the Shanghai Morning Light Program (20CG36, China), the Shanghai Frontier Science Center of Optogenetic Techniques for Cell Metabolism (2021 Sci & Tech 03-28, China), the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZDCX20212702, China), and the Chinese Special Fund for State Key Laboratory of Bioreactor Engineering (2060204).
