In vitro activity: PRX-08066 inhibits 5-HT-induced mitogen-activated protein kinase activation with IC50 of 12 nM and markedly reduces thymidine incorporation with IC50 of 3 nM in Chinese hamster ovary cells expressing the human 5-HT2BR, which suggests that PRX-08066 can potentially inhibit the pathologic 5-HT-induced vascular muscularization associated with PAH. PRX-08066 inhibits cell proliferation with IC50 of 0.46 nM and with a maximum inhibition of 20% and 5-HT secretion with IC50 of 6.9 nM with a maximum inhibition of 30% in the 5-HT(2B) expressing SI-NET cell line, KRJ-I. PRX-08066 inhibits isoproterenol-stimulated 5-HT release with IC50 of 1.25 nM and a maximum inhibition of 60% in NCI-H720 cells. PRX-08066 (0.5 nM) significantly inhibits ERK phosphorylation in KRJ-I cells. PRX-08066 inhibits TGFβ1, CTGF and FGF2 transcription and secretion in KRJ-I cells. PRX-08066 decreases level of transcripts for Ki67 (84%) as well as Ki67 protein (36.8%) associated with an increase in caspase 3 transcript levels in KRJ-I cells. PRX-08066 decreases level of transcripts of TGFβ1, FGF2 and TPH1 in KRJ-I cells. PRX-08066 significantly increases the number of dead cells (34%) compared with untreated controls in KRJ-I cells. PRX-08066 causes a significant increase in dead/caspase 3 positive cells (76%) and caspase 3 activity (52%) in HEK293 cells.

Kinase Assay:

Cell Assay: 5×103 cells/mL, seeds in 96-well plates at 100 μL (4 plates/experimental condition) are stimulated with PRX-08066 (0.1 μM to 100 nM: n = 6 wells/concentration). After 24 hours, mitochondrial activity is measured after adding MTT (3-[4,5-dimethylthiazol-2-ly]-2.5-diphenyltetrazolium bromide: 0.5mg/mL per well) for 3 hours. The optical density is read photospectrometrically at 595 nm using a microplate reader. 29 Results are normalized to control (unstimulated cells) and the effective half-maximal concentrations calculated.

J Pharmacol Exp Ther. 2010 Aug;334(2):364-72; Cancer. 2010 Jun 15;116(12):2902-12