Tiplaxtinin (PAI-039)

The chromogenic assay is initiated by the addition of Tiplaxtinin (PAI-039) (10 – 100 µM final concentration, maximum DMSO concentration of 0.2%) to recombinant human PAI-1 (140 nM in pH 6.6 buffer). After a 15 minute incubation at 25°C, 70 nM of recombinant human t-PA is added, and the combination of this compound, PAI-1 and tPA are incubated for an additional 30 minutes. After the second incubation, Spectrozyme tPA, is added and absorbance read at 405 nm at 0 and 60 minutes. Relative PAI-1 inhibitory activity is equal to the residual tPA activity in the tiplaxtinin / PAI-1 treatment. Control treatments include the complete inhibition of tPA by PAI-1 at the molar ratio employed (2:1), and the absence of any effect of it on t-PA alone. The immunofunctional assay is based upon the non-SDS dissociable interaction between tPA and active PAI-1. Assay plates are coated with 100 µl of a solution of t-PA (10 µg/ml in TBS), and kept at 4 °C overnight. This compound is dissolved in DMSO and diluted to a final concentration of 1-100 µM as described above. It is then incubated with human PAI-1 (50 ng/ml) for 15 minutes, and an aliquot of this solution added to the t-PA-coated plate for 1 h. The solution is aspirated from the plate, which is then washed with a buffer consisting of 0.05% Tween 20 and 0.1% BSA in TBS. This assay detects only active inhibitory PAI-1 (not latent or substrate) bound to the plate, and is quantitated using a monoclonal antibody against human PAI-1 (MA33B8). A 1000X dilution of MA33B8 is added to the plate and incubated at for one hour, aspirated and washed. A secondary antibody consisting of goat anti-mouse IgG (H+L)-AP alkaline phosphatase conjugate is added, incubated for one hour, aspirated and washed. A 100 µl aliquot of alkaline phosphatase solution is added, followed by determination of absorbance at 405 nm 60 minutes later. The quantitation of residual active PAI-1 bound to t-PA at varying concentrations of it is used to determine the IC50 by fitting the results to a logistic dose-response program, with the IC50 defined as the concentration of compound required to achieve 50% inhibition of PAI-1 activity. The assay sensitivity is 5 ng/ml of human PAI-1 as determined from a standard curve ranging from 0-100 ng/ml of human PAI-1.

T24, UM-UC-14, UROtsa, and HeLa cells

~50 μM

24 h

Briefly, cell lines, T24, UM-UC-14, UROtsa, and HeLa cells are plated in 96-well dishes in triplicate at 1 ?10³ cells per well and allowed to adhere for 24 hours. Subsequently, tiplaxtinin (PAI-039) is added to the wells and allowed to incubate at the indicated concentrations. Cellular proliferation is determined by CellTiter-Glo Luminescent Cell Viability Assay according to manufacturer's instructions at 24 hours, and its IC50 is determined in Graphpad Prism. Luminescence was measured using a FLUOstar OPTIMA Reader.

Rat with carotid thrombosis

1 mg/kg

p.o.