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-in¯ammatory 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 B 2 (TXB 2 ) during clotting and of prostaglandin E 2 (PGE 2 ) 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 26t18% and 18t13% respectively, and intersubject variation at 39% and 36%, respectively. The ratios of IC 50 s and, at best, of IC 80 s 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 ([x0.18, 0.21] for x0.03] for COX-2) but this was not the case for ibuprofen on COX-2 ([x0.14, 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.

Oral administration of tepoxalin in the horse: A PK/PD study

“…This method has the advantage of taking into account the clinically relevant target cells and the variable degree of protein binding of NSAIDs (Blain et al, 2002). In previous in vivo studies in other species, tepoxalin has been shown to undergo rapid and extensive conversion to its carboxylic acid hydrolysis product (RWJ-20142).…”

Pharmacokinetic-pharmacodynamic modeling of diclofenac in normal and Freund's complete adjuvant-induced arthritic rats

“…The PGE 2 released by LPS in the blood samples was documented according to a previously described method [20] . A 200-µL blood sample was added to a tube containing different levels of diclofenac sodium, heparin (0.3%) and aspirin (10 µg/mL).…”

Serum thromboxane B2 (TXB2) Determination is Influenced by Sample Incubation Temperature in Healthy Beagle Dogs

“…While searching for the literature on normal values of TXB 2 in canine serum and the measurement of serum TXB 2 after NSAID administration, we found inconsistency in the published data, which might influence the results obtained. In some studies (Blain et al 2002;Gilmer et al 2002;Jones et al 2002;McKellar et al 1990;Rinder et al 2002;Streppa et al 2002) blood samples for serum TXB 2 determination were incubated during blood clotting at 37 C for one hour as recommended (Brideau et al 1996;Patrignani et al 1997), whereas other studies (Frelinger et al 2006;Hashimoto et al 2002;Morchon et al 2006;Sunose et el. 2001a;Sunose et al 2001b;Tanus-Santos et al 2000) did not mention the incubation of samples.…”

“…The measurement of serum TXB 2 production by platelets following blood coagulation is a specific and most frequently used test for evaluation of platelet COX-1 activity in humans and other species (Brideau et al 1996;Blain et al 2002;Gilmer et al 2003;Jones et al 2002;Patrignani et al 1997;Rinder et al 2002;Sessions et al 2005;Van Kraaij et al 2002). Whole blood TXB 2 production is measured from clotted blood samples obtained from the patient after administration of NSAID or after collection of blood into tubes preloaded with NSAID.…”