Rho-Kinase-IN-2 (Compound 23) is an orally active and selective inhibitor of Rho Kinase (ROCK) with CNS penetration, exhibiting a high affinity for ROCK2 with an IC50 of 3 nM. This compound is of potential interest for further investigations in Huntington's disease research [1].

ROCK2:3 nM

Treatment with Rho-Kinase-IN-2 (0-10 mM) for 1 hour led to concentration-dependent alterations in phosphorylation levels, specifically an increase in AKT phosphorylation and a decrease in MYPT1 phosphorylation, observed across A7r5 and PANC1 cell lines. Western Blot Analysis confirmed these effects, quantifying the potency of Rho-Kinase-IN-2 in modulating AKT and MYPT1 phosphorylation with EC50 and IC50 values of 28 nM and 14 nM, respectively.

Administering Rho-Kinase-IN-2 orally at 10 mg/kg six times at intervals of 0.5, 1, 2, 4, 8, and 12 hours demonstrated a dose- and time-dependent engagement with ROCK1 and ROCK2 targets. Similar administration at doses of 10 or 20 mg/kg, either once daily (QD) or twice daily (BID) for two weeks, exhibited excellent tolerability. A single dose ranging from 1-20 mg/kg revealed a direct dose- and time-dependent correlation between brain exposure and MYPT1 phosphorylation status. Moreover, a single administration at 10 or 20 mg/kg resulted in decreased mean arterial, systolic, diastolic blood pressures, and heart rate. Continuous treatment at 10 mg/kg twice daily for 90 days led to lower-than-expected brain concentrations. These findings were consistent across various animal models, including male C57BL/6 mice, 3-4-month-old heterozygote Q175DN KI and wild-type littermate mice, heterozygote HTT zQ175DN knock-in mice, and CD1 mice, demonstrating the compound’s pharmacodynamics and pharmacokinetics profiles in preclinical settings.