CGS 15435

Kinase experiment:

Thromboxane synthetase, cyclooxygenase, prostacyclin synthetase, and lipoxygenase enzymes are prepared. For the individual enzyme assays. [1-14C]Arachidonic acid (AA) is incubated with partially purified enzyme obtained from human platelets (thromboxane synthetase), sheep seminal vesicles (cyclooxygenase), bovine aorta (prostacyclin synthetase), and guinea-pig leukocytes (lipoxygenase). At the end of the incubation period, the products are extracted into ethyl acetate, the extracts are evaporated to dryness, the residues are redissolved in acetone, and these solutions are spotted on thin layer chromatography plates. The plates are developed in the appropriate solvents, scanned and radioactive spots corresponding to those of TxB2, PGE2, 6-keto PGFu, and 5-HETE, are scraped off and counted by a radiospectrometer. The IC50 values are determined by employing a range of concentrations of test compounds over the linear range of the assay and analyzed graphically. All determinations are done in duplicate and repeated once[1].

Animal experiment:

Rabbits[1]Adult male New Zealand rabbits (2.3-3.5 kg) are anesthetized with sodium pentobarbital (30 mg/kg i.v.). The animals are tracheotomized and the left femoral artery and vein are cannulated for the recording of mean arterial blood pressure (MABP) and the injection of either vehicle or drug, respectively. The animals are allowed to stabilize for at least 15 rain prior to drug or vehicle administration. CGS 15435 and DAZ are dissolved in 2 mL of 0.5 M Tris buffer (pH 8.4) and injected i.v. over a 15 s period. CGS 15435 (8.6 μmol/kg; 3.1 mg/kg) is administered at 15 min (n=4) or 24 h (n=6) prior to AA.

References:

[1]. Olson RW, et al. CGS 15435A, a thromboxane synthetase inhibitor with an extended duration of action: a comparison with dazoxiben. Eur J Pharmacol. 1987 Jan 20;133(3):265-73.