Effect of the Pleiotropic Drug CNB-001 on Tissue Plasminogen Activator (tPA) Protease Activity in vitro: Support for Combination Therapy to Treat Acute Ischemic Stroke

Current state-of-the-art acute ischemic stroke clinical trials are designed to study neuroprotectants when administered following thrombolysis; tissue plasminogen activator (tPA) is administered to patients within 3–4.5 hours of an ischemic event. Thus, in order to develop a novel neuroprotectant and move it forward to a clinical trial, it is important to assess the effects of the drug on tPA’s proteolytic activity in vitro, prior to extensive in vivo analysis. In this study, we determined if CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol)], would affect, either enhance or inhibit tPA activity in vitro. In this tPA-inhibitor (plasminogen activator inhibitor-1; PAI-1 and 2,7-Bis-(4-Amidinobenzylidene)-Cycloheptan-1-One Dihydrochloride; tPA stop) controlled study, we used a chromogenic substrate (CH3SO2-D-hexahydrotyrosine-Gly-Arg-p-nitroanilide•AcOH) to study drug interactions in vitro, spectrophotometrically measuring protease released p-Nitroaniline from the substrate. We found that PAI-1 (0.25 μM) and tPA stop (5 μM) significantly (p<0.0001) inhibited substrate release, by 98.6% and 83.4%, respectively, thus inhibiting tPA activity in vitro. In comparison, CNB-001 (0.7–7 μM) reduced tPA activity by 28–32%, with an extrapolated IC50 value of 65.2–704 μM. Thus, although high concentrations of CNB-001 does affects tPA activity in vitro, the study supports the use of CNB-001 in combination with tPA to treat stroke, However, CNB-001 should be administered following thrombolysis to promote neuroprotection and repair.