LB30057, an orally effective direct thrombin inhibitor, prevents arterial and venous thrombosis in rats and dogs.

The anti-thrombotic effects of LB30057, a direct thrombin inhibitor, were evaluated with in vivo rat and dog thrombosis models. In rats, 1 mg/kg of LB30057 inhibited half of the clot formations in the inferior vena cava at 5 minutes after intravenous application. When measured at 2 hours after oral application, 100 mg/kg prevented approximately half of the clot formations in the inferior vena cava and 50 mg/kg prolonged the mean occlusion time from 15.6 +/- 1.3 minutes to 47.2 +/- 8.3 minutes in the carotid artery. In dogs, the formation of thrombus in the jugular vein was reduced to half at a dose range of 20-30 mg/kg at 6 hours after oral application. In addition, the LB30057 dosage required to reduce venous clot formation by approximately 80-90% in dogs was only about 10% of that required for the same reduction in rats. This is probably due to the variation in its time-dependent blood concentration profiles in each species; for example, the plasma half-life of LB71350 in dogs was longer than that in rats (153.0 +/- 3.0 vs. 129.7 +/- 12.7 min at 30 mg/kg, i.v., respectively). AUC, T(max), C(max), and BA in dogs were 59, 8.9, 9.17, and 13.3 times higher than those in rats at oral 30 mg/kg, respectively. Taken together, these results suggest that LB30057 administered orally is effective in the prevention of arterial and venous thrombosis in rats and dogs. It therefore represents a good lead compound for investigations to discover a new, orally available, therapeutic agent for treating thrombotic diseases.

Cell-based assay for beta-secretase activity.

The cerebral deposition of amyloid beta-peptide (Abeta) is a major factor in the etiology of Alzheimer's disease. beta-Secretase (BACE) initiates the generation of Abeta by cleaving the amyloid precursor protein at the beta-site and is therefore a prime target for therapeutic intervention. Here we report a cell-based method suitable for monitoring BACE activity and the efficacy of protease inhibitors. A fusion protein containing the amino-terminal transmembrane domain of Golgi alpha-mannosidase II, a Drosophila Golgi integral membrane protein, linked to human alkaline phosphatase (AP) by a short beta-site sequence, was expressed in Drosophila S2 cells. While the uncleaved fusion protein was retained in the Golgi apparatus, cleavage of the beta-site by BACE resulted in the release of AP to the culture medium, where it was easily detected and quantified. Three peptidomimetic inhibitors (LB83190, LB83192, LB83202) were tested for their efficacy with this cell-based assay. While LB83190 and LB83192 effectively blocked BACE activity, LB83202, a carboxylated derivative of LB83192, did not. This is consistent with the inability of LB83202 to permeate the cell membrane. The present cell-based assay could provide a convenient tool for high-throughput screening of substances that can interfere with BACE in living cells.