Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-inflammatory drugs

Abstract

1. In this study we describe experiments to establish ex vivo the selectivity of non-steroidal anti-inflammatory drugs (NSAIDs) given in vivo. 2. Anaesthetised (Inactin, 120 mg kg(-1)) male Wistar rats (220-250 g) received an i.v. dose of one of the following compounds (dose mg kg(-1)): aspirin (20), diclofenac (3), L-745,337 (30), nimesulide (15), salicylate (20), sulindac (10). Blood samples were taken before and up to 6 h after dosing and the plasma obtained from it was tested for its ability to inhibit prostanoid formation in IL-1beta-treated A549 cells (COX-2 system) and human washed platelets (COX-1 system). For control the same compounds were also added directly to the assay systems. 3. All drugs, except sodium salicylate, inhibited COX-1 and COX-2 when added directly to the test systems. Plasma from aspirin-treated rats was without effect on either COX-1 or COX-2, consistent with the rapid in vivo metabolism to salicylate. Conversely, plasma from sulindac-treated rats inhibited COX-1 and COX-2 with potencies according with in vivo metabolism to sulindac sulphide. Diclofenac was COX-1/2 non-selective when tested in vitro, but a slightly preferential inhibitor of COX-2 when tested ex vivo. Nimesulide was confirmed as preferential inhibitor of COX-2 both in vitro and ex vivo. L-745,337 was a selective COX-2 inhibitor when tested in vitro or ex vivo. 4. In conclusion, our experiments show clearly (a) NSAIDs inactivation, (b) activation of prodrugs, and (c) NSAIDs selectivity. Our assay provides useful information about the selectivity of NSAIDs that could be extended by the analysis of plasma samples taken from humans similarly treated with test drugs.

Figure 1

Effect of diclofenac (a) and plasma samples from diclofenac-treated rats (b) on COX-1 and COX-2 activity. Platelets (COX-1 system) and A549 (COX-2 system) cells were exposed to diclofenac or plasma samples for 30 min and then challenged with calcium ionophore A23187 (50 μM) for 15 min. When added directly to the assay system, diclofenac was COX-1/-2 non-selective whereas plasma samples from diclofenac-treated animals showed a small but significant selectivity towards COX-2. Data are expressed as mean±s.e.m. from three determinations from four separate experimental days.

Figure 2

Effect of nimesulide (a) and plasma samples from nimesulide-treated rats (b) on COX-1 and COX-2 activity. Nimesulide showed preferential inhibition of COX-2 both when added directly to the assay system and when plasma samples from nimesulide-treated rats were used. Data are expressed as mean±s.e.m. from three determinations from four separate experimental days.

Figure 3

Effect of aspirin (a) and plasma samples from aspirin-treated rats (b) on COX-1 and COX-2 activity. Aspirin added directly to the assay system inhibited COX-1 more potently than COX-2 whereas plasma samples from aspirin-treated animals failed to show any significant activity on both isoforms. Data are expressed as mean±s.e.m. from three determinations from four separate experimental days.

Figure 4

Effect of salicylate (a) and plasma samples from salicylate-treated rats (b) on COX-1 and COX-2 activity. Salicylate failed to show any activity either when added directly to the assay system or when plasma from salicylate-treated animals was used. Data are expressed as mean±s.e.m from three determinations from four separate experimental days.

Figure 5

Effect of sulindac sulphide (a), plasma samples from sulindac-treated rats (b) and sulindac (c) on COX-1 and COX-2 activity. Sulindac sulphide and sulindac both showed selectivity towards the inhibition of COX-1. Plasma samples from sulindac-treated rats also selectively inhibited COX-1 with potency according to the in vivo metabolism of sulindac to sulindac sulphide. Data are expressed as mean±s.e.m. from three determinations from at least four separate experimental days.

Figure 6

Effect of L-745,337 (a) and plasma samples from L-745,337-treated rats (b) on COX-1 and COX-2 activity. When tested directly, L-745,337 inhibited COX-2 1100 fold more potently than COX-1. Plasma samples form L-745,337-treated rats also selectively inhibited COX-2 activity without any significant effect on COX-1. Data are expressed as mean±s.e.m. from three determinations from five separate experimental days.