Limitation of the in vitro whole blood assay for predicting the COX selectivity of NSAIDs in clinical use

Abstract

Aims: To assess if the inhibitory potency of nonsteroidal anti-inflammatory drugs (NSAIDs) on cyclooxygenase (COX) isoenzymes, when given therapeutically in humans, can be predicted from their in vitro concentration-response curves using the whole blood assay.

Methods: Twenty-four healthy male volunteers aged 20--27 years were recruited. Inhibition of blood COX isoenzymes was determined in vitro before any drug intake and ex vivo after single and repeated intake of either 7.5 mg meloxicam once, 400 mg ibuprofen three times daily or 75 mg diclofenac SR once, taken in a randomized cross-over design. Production of thromboxane B2 (TXB2) during clotting and of prostaglandin E2 (PGE2) during endotoxin exposure served as indicators of platelet COX-1 and monocyte COX-2 activity, respectively. Drugs were determined in plasma by h.p.l.c., with a chiral separation of ibuprofen and free fractions after equilibrium dialysis.

Results: Intra-subject variation for COX-1 and COX-2 at baseline was at 26 +/- 18% and 18 +/- 13% respectively, and intersubject variation at 39% and 36%, respectively. The ratios of IC50s and, at best, of IC80s revealed diclofenac and meloxicam as selective COX-2 inhibitors and ibuprofen as a preferential COX-1 inhibitor in vitro. However, after oral intake, ibuprofen inhibited ex vivo COX-2 by 80% whereas diclofenac inhibited COX-1 by 70%. Meloxicam inhibited COX-1 from 30 to 55% depending on the repetition of the dose and increase in plasma concentrations. Using in vitro dose--response curves, the in vivo inhibitory potency of diclofenac was estimated adequately from its circulating concentration ([-0.18, 0.21] for COX-1 and [-0.13, -0.03] for COX-2) but this was not the case for ibuprofen on COX-2 ([-0.14, 0.27]) and meloxicam on COX-1 ([0.31, 1.05]). The limited predictability of the system was not improved through considering the unbound fraction of the drugs or the variable chiral inversion of ibuprofen.

Conclusions: Assessment of COX-2 selectivity based on in vitro studies and pharmacological modelling has a limited clinical relevance. There is a need to investigate COX selectivity at therapeutic plasma concentrations of NSAIDs using the ex vivo whole blood assay.

Figure 1

Comparison of NSAID concentrations and inhibition of COX activities in human blood assessed in vitro and ex vivo. The in vitro dose–response curves of platelet COX-1 (grey hatched line) and monocyte COX-2 (black line) activities were done for diclofenac (a), ibuprofen (b) and meloxicam (c). Increasing concentrations of NSAIDs were incubated with 1 ml whole blood samples allowed to clot for 1 h, and serum TXB₂ levels were measured as a reflection of thrombin-induced platelet thromboxane A₂ production. NSAIDs were also incubated with 1 ml heparinized whole blood samples in the presence of aspirin (10 µg ml⁻¹) and LPS (10 µg ml⁻¹) for 24 h, and plasma PGE₂ levels were assayed as a reflection of endotoxin-induced monocyte COX-2 activity. In vitro whole blood concentrations of NSAIDs were corrected into plasma concentrations using a dividing factor of 1.34 for diclofenac, 1.65 for ibuprofen and 1.49 for meloxicam. Results, depicted as percentage inhibition, were obtained from triplicates of the 24 volunteers and concentration-response curves were fitted according to the Hill function as described in the method section. In the ex vivo study, plasma NSAIDs concentrations measured after single and repeated intake of diclofenac 75 mg once, ibuprofen 400 mg three times daily or meloxicam 7.5 mg once were plotted on the same graph vs the percentage of inhibition of COX-1 (▵: single; ×: repeated) and COX-2 (▴: single; +: repeated) activities measured ex vivo at the same time.

Figure 2

Discrepancy between inhibition of platelet COX-1 activity by free meloxicam in vitro and percentage of COX-1 inhibition observed ex vivo after an oral intake of 7.5 mg day⁻¹ (▵: single intake; ×: repeated intake).

Figure 3

Discrepancy between inhibition of monocyte COX-2 activity by free ibuprofen racemate in vitro and percentage of COX-2 inhibition observed ex vivo after an oral intake of 400 mg three times daily (▴: single intake; +: repeated intake).

Figure 4

Influence of enantioselectivity on the inhibitory potency of ibuprofen towards COX activities in human blood assessed in vitro and ex vivo. The in vitro dose–response curves of platelet COX-1 (grey hatched line) and monocyte COX-2 (black line) activities were done considering that the S-(+)-enantiomer represented half of the racemate concentration. Results, depicted as percentage inhibition, were obtained from triplicates of the 24 volunteers and concentration-response curves were fitted according to the Hill function as described in the method section. In the ex vivo study, plasma concentrations of S-(+)-ibuprofen measured after single and repeated intake of 400 mg three times daily of ibuprofen racemate were plotted on the same graph vs the percentage of inhibition of COX-1 (▵: single; ×: repeated) and COX-2 (▴: single; +: repeated) activities measured ex vivo at the same time.