| Genes encoding human cyclooxygenase 1 (hCOX-1) and cyclooxygenase 2 (hCOX-2) were cloned and introduced into Chinese hamster ovary (CHO) cell line to obtain CHO[hCOX-1] and CHO[hCOX-2] cell lines stably and highly expressing hCOX-1 and hCOX-2, respectively. Based on CHO[hCOX-1] and CHO[hCOX-2], a sensitive and specific model for screening of COX-1/COX-2 inhibitors was established to evaluate the inhibitory effects of compounds on human derived COX-1 and COX-2 under the same genetic background. In this COX-2 screening model, rofecoxib of 1×10-5 mol/L has no inhibitory effect on COX-1 activity; The inhibitory effect of indomethacin and SC-560 on COX-1 activity were dose-dependent at the concentrations of 1×10-51×10-8 mol/L, with the IC50 values of 1.12×10-8 mol/L and 6.20×10-9 mol/L, respectively; The inhibitory effect of imrecoxib at the concentration of 1×10-7 mol/L on COX-1 activity was about 50%. The inhibitory effect of rofecoxib on COX-2 activity was dose-dependent at the concentrations of 1×10-61×10-9 mol/L, with the IC50 value of 1.44×10-8 mol/L. The inhibitory effect of indomethacin, imrecoxib and SC-560 on COX-2 activity was dose-dependent at the concentrations of 1×10-51×10-8 mol/L, with the IC50 values of 2.97×10-8 mol/L, 4.78×10-8 mol/L, and 8.42×10-7 mol/L, respectively. In addition, primary functional studies were conducted on the cDNA encoding human COX-1 splice variant deleted of exon 9. No significant PGE2 production was detected in CHO cell transfected with COX-1sv cDNA. RT-PCR result suggested that the mRNA level of which is 1/3 of the mRNA level of full-length COX-1. The mRNA level of hCOX-1sv in the U937 cells was not affected by treatment of PMA and drugs including resveratrol, indomethacin, dexamethasone. While COX-2 selective inhibitor rofecoxib increased the mRNA level of COX-1sv to 1.5 fold of that of normal control.To develop new anti-inflammatory drugs with independent intellectual properties, more than 110 compounds were screened using an in vitro COX-1 and COX-2 assay system based on mouse peritoneal macrophages utilizing endogenous arachidonic acid. Four nimesulide analogs including GCB-0328, GCB-0329, GCB-0339B and GCB-0344, as well as a ditertbutyl-benzamide analog XZB-0702, were identified as COX-2 inhibitors. XZB-0702 was shown to inhibit COX-2 activity with the IC50 value between 1×10-7-1×10-6 mol/L. GCB-0328, GCB-0329, GCB-0339B, and...
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