Fang Shen1, Ai-Ping Bai, Zong-Ru Guo, Gui-Fang Cheng. 1. Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Abstract
AIM: To develop a whole-cell assay based on murine peritoneal macrophages and evaluate the inhibitory effect of candidate compounds on cyclooxygenase-1 (COX-1) and COX-2. METHODS: Macrophages were stimulated with calcimycin or lipopolysaccharide (LPS) for various periods. Their abilities to convert endogenous arachidonic acid to 6-keto-PGF1alpha or PGE2 were examined by radioimmunoassay (RIA). RNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and COX-1/2 was detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers. RESULTS: Rofecoxib selectively inhibited LPS-induced, COX-2-derived PGE2 synthesis with an IC50 value of (4.7+/-0.5) nmol/L compared with maximum inhibitory ratio of 17.3 % for the inhibition of calcimycin induced, COX-1-derived 6-keto-PGF1alpha synthesis. Indomethacin exhibited dual inhibitory effects on COX-1 and COX-2 with IC50 of (4.7+/-1.1) nmol/L and (7.1+/-1.2) nmol/L, respectively. Two series of 17 compounds were tested. Most of compounds in series II showed comparable inhibitory activities to rofecoxib on COX-2. The relative position of the sulfonylphenyl group to the lactam carbonyl group has important effects on COX-2 inhibitory activity. CONCLUSION: The established whole cell assay is appropriate for drug-design oriented in vitro assay. 3,4-Diaryl-3-pyrrolin-2-one derivatives were proved to be prospective new type of COX-2 selective inhibitors.
AIM: To develop a whole-cell assay based on murine peritoneal macrophages and evaluate the inhibitory effect of candidate compounds on cyclooxygenase-1 (COX-1) and COX-2. METHODS: Macrophages were stimulated with calcimycin or lipopolysaccharide (LPS) for various periods. Their abilities to convert endogenous arachidonic acid to 6-keto-PGF1alpha or PGE2 were examined by radioimmunoassay (RIA). RNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and COX-1/2 was detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers. RESULTS:Rofecoxib selectively inhibited LPS-induced, COX-2-derived PGE2 synthesis with an IC50 value of (4.7+/-0.5) nmol/L compared with maximum inhibitory ratio of 17.3 % for the inhibition of calcimycin induced, COX-1-derived 6-keto-PGF1alpha synthesis. Indomethacin exhibited dual inhibitory effects on COX-1 and COX-2 with IC50 of (4.7+/-1.1) nmol/L and (7.1+/-1.2) nmol/L, respectively. Two series of 17 compounds were tested. Most of compounds in series II showed comparable inhibitory activities to rofecoxib on COX-2. The relative position of the sulfonylphenyl group to the lactam carbonyl group has important effects on COX-2 inhibitory activity. CONCLUSION: The established whole cell assay is appropriate for drug-design oriented in vitro assay. 3,4-Diaryl-3-pyrrolin-2-one derivatives were proved to be prospective new type of COX-2 selective inhibitors.