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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 10, 2006 as doi:10.1096/fj.05-4434fje. |
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* The William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK; and
Department of Cardiothoracic Pharmacology, Unit of Critical Care Medicine, Imperial College London, Royal Brompton Hospital, London, UK
1Correspondence: The William Harvey Research Institute, Barts and the London, Charterhouse Square, London EC1M 6BQ, UK. E-mail: t.d.warner{at}qmul.ac.uk
SPECIFIC AIMS
In recent years the mechanisms of action, efficacies, and side effects of nonsteroid anti-inflammatory drugs (NSAIDs) have been the subjects of intense investigation. In these investigations it has been taken for granted that the activities of the NSAIDs are affected similarly by the presence of extracellular proteins. Of particular relevance to the NSAIDs, the protein concentrations in synovial fluid and the central nervous system are different from those in plasma. Here, therefore, we simply compared the potencies of cyclooxygenase inhibitors in the presence of the differing concentrations of protein present in blood, synovial fluid and cerebrospinal fluid, by modifying previously reported whole blood assays of cyclooxygenase activity.
PRINCIPAL FINDINGS
1. Extracellular protein does not affect the potency of aspirin, celecoxib, indomethacin, lumiracoxib, meloxicam, or SC560 as inhibitors of COX-1 or COX-2
Aspirin, celecoxib, indomethacin, lumiracoxib, meloxicam, and SC560 inhibited COX-1 in platelets, indicated by changes in the production of TxA2, with the different concentrations of protein having no effect on the IC50 values (Table 1
). The potencies of the same agents as inhibitors of COX-2 activity were also unaffected by the presence of differing concentrations of protein (Table 2
).
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2. Extracellular protein influences the potencies of diclofenac, naproxen, and rofecoxib against COX-1 but not against COX-2
The potencies of diclofenac, naproxen, and rofecoxib against COX-1 increased as the concentration of protein was decreased (Table 1)
. For diclofenac and naproxen, the IC50 values in 0.35 mg/mL bovine serum albumin (BSA), equivalent to cerebrospinal fluid, were > 10-fold lower than IC50 values derived from experiments conducted in whole blood. No statistical differences were found between inhibitor curves for the three compounds against production of PGE2 dependent on COX-2 in A549 cells, when experiments were performed in whole blood vs. media plus 35 mg/mL or 0.35 mg/mL BSA (Table 2)
.
3. Extracellular protein influences the potency of sodium salicylate against COX-1 and COX-2
The potency of sodium salicylate against COX-1 increased as the concentration of protein was decreased (Table 1)
such that the IC50 value in 0.35 mg/mL BSA was > 10-fold lower than the IC50 value derived from experiments conducted in whole blood. Sodium salicylate was also the only drug tested that demonstrated an increase in potency against COX-2 when the concentration of protein was decreased (Table 2)
such that the IC50 value in 0.35 mg/mL BSA was almost 10-fold lower than that derived from experiments conducted in whole blood.
4. Relative effects of differing protein concentrations on COX-1 vs. COX-2 selectivity
Calculated IC50 ratios for the inhibitors tested demonstrated that varying protein concentrations influenced the relative potencies of the drugs tested. Diclofenac was the most affected, showing a 34-fold increase in COX-1 selectivity between 0.35 mg/mL BSA and whole blood.
CONCLUSIONS AND SIGNIFICANCE
Here we show in two simple assay systems that the potencies of a range of NSAIDs are differently influenced by the presence of protein, with the strongest effects being shown against COX-1. This has important ramifications since it means the selectivity of NSAIDs for COX-1 vs. COX-2 will vary depending on their different chemical structures and their presence in different body compartments.
The protein concentration of plasma is 
70 mg/mL, whereas that in synovial fluid is some 50–80% lower, depending on disease state, and cerebrospinal fluid is an ultrafiltrate with very low protein levels, < 1% of that in plasma. The concentrations of BSA we have chosen therefore offer simple model systems equating to the environment within the blood compartment, joints and the central nervous system sites, at which NSAIDs are believed to be active. The potency of aspirin was unaffected by the concentrations of protein present in the incubation media. This may well be because aspirin is a noncompetitive inhibitor of both COX-1 and COX-2 acting to irreversibly acetylate serine residue 530. Thus, as there is not competition between free drug and substrate, protein binding of drug may have little impact. Sodium salicylate makes a good test of this hypothesis, since it is chemically very similar to aspirin but acts on COX as a weak competitive inhibitor, as shown in in vitro assay systems very similar to that used here. Here we found sodium salicylate to increase in potency as protein levels in the incubation medium were reduced, consistent with an increased concentration of free drug competing with substrate. The other drugs tested lie between these two simple models. Some are understood to be competitive inhibitors of COX-1 and so increases in free drug, i.e., reductions in protein binding, may well produce increased activity and leftwards shifts of the inhibitor curves as substrate is more readily competed away from the active site of the enzyme, as was seen for diclofenac and naproxen. Indomethacin binds tightly to both COX-1 and COX-2 producing inhibition which is largely independent of assay conditions consistent with the lack of effect of protein concentrations we found here. On COX-2, many drugs, such as naproxen, bind in a weak, pseudo-irreversible manner consistent with our observation of a lack of effect of protein on naproxen inhibition of COX-2.
In conclusion, our studies demonstrate that protein binding differently affects the activities of a range of NSAIDs against COX-1 and COX-2. This may well be explained by different kinetics of interaction between the inhibitors and the two COX isoforms. If a drug is an irreversible or slowly reversible COX inhibitor then protein binding seems to have little effect. This may be explained if the dissociation of the drug from the COX enzyme is slower than that of the drug from the binding proteins; i.e., under these conditions inhibitor molecules will always be present to bind to the COX enzyme. Competitive inhibitors of COX are affected by the presence of protein as their dissociation from the COX enzyme is much more rapid. Clearly, our findings show that the selectivities of inhibitors for COX-1 and COX-2, which are taken to be linked to their efficacy and side effects, change in different extracellular fluid conditions. In particular selectivity in one body compartment cannot be taken as evidence of selectivity in another body compartment and whole body safety or toxicity cannot be linked to one definitive measure of COX selectivity (Fig. 1
). Thus, these observations are extremely relevant to ongoing discussions regarding the safety of NSAIDs.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4434fje;
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