In vitro activity: Fasudil (1-10 μM) and hydroxyfasudil (0.3-10 μM) significantly prevented endothelin-induced cardiomyocyte hypertrophy. Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses.

Kinase Assay: Hydroxyfasudil HCl (also called HA1100 HCl), a metabolite of Fasudil, is a potent Rho-kinase inhibitor and vasodilator. It acts as a ROCK inhibitor, with IC50s of 0.73 and 0.72 μM for ROCK1 and ROCK2, respectively. Hydroxyfasudil prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. In a concentration-dependent manner, hydroxyfasudil increases eNOS mRNA and protein expression, resulting in a 1.9- and 1.6-fold increase, respectively, at 10 μmol/L. This correlates with a 1.5- and 2.3-fold increase in eNOS activity and NO production, respectively.

Cell Assay: Human vascular endothelial cells are treated with increasing concentrations of hydroxyfasudil (0.1 to 100 μmol/L) and eNOS expression and activity are measured.