NSAIDs and cardiovascular disease: transducing human pharmacology results into clinical read-outs in the general population

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1 Pharmacological Reports 2010, 62, ISSN Copyright 2010 by Institute of Pharmacology Polish Academy of Sciences Review NSAIDs and cardiovascular disease: transducing human pharmacology results into clinical read-outs in the general population Marta L. Capone 1, Stefania Tacconelli 1, Luis Garcia Rodriguez 2, Paola Patrignani 1 Department of Medicine and Center of Excellence on Aging and G. d Annunzio University Foundation, Ce.S.I., Via dei Vestini 31, Chieti, Italy Centro Español de Investigación Farmacoepidemiológica (CEIFE), Almirante 28 (2º), Madrid 28004, Spain Correspondence: Paola Patrignani, ppatrignani@unich.it Abstract: Traditional (t) non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase (COX)-2 inhibitors (coxibs) are important and efficacious drugs for the management of musculoskeletal symptoms. These drugs have both beneficial and adverse effects due to the inhibition of prostanoids. Although the tnsaid and coxib inhibition of COX-2-dependent prostaglandin (PG)E production is effective in ameliorating symptoms of inflammation and pain, a small but consistent increased risk of myocardial infarction has been detected in association with their use. Convincing evidence suggests that cardiovascular toxicity associated with the administration of these compounds occurs through a common mechanism involving inhibition of COX-2-dependent prostacyclin. The development of biomarkers that predict the impact of NSAIDs on COX-1 and COX-2 activities in vitro, ex vivo and in vivo has been essential to read-out the clinical consequences of the varying degrees of inhibition of the two COX-isozymes in humans. Whole blood assays for COX-1 and COX-2 might be candidates as surrogate end-points of toxicity and efficacy of NSAIDs. Using a biomarker strategy, we have shown that the degree of inhibition of COX-2 and the functional selectivity with which it is achieved are relevant to the level of cardiovascular hazard from NSAIDs and relate to drug potency (exposure). We propose that the assessment of COX-2 in whole blood ex vivo, either alone or in combination with urinary levels of 2,3-dinor-6-keto-PGF = a biomarker of prostacyclin biosynthesis in vivo, may represent a valid surrogate end-point to predict cardiovascular risk for functionally selective COX-2 inhibitors. Key words: NSAIDs, selectivity, prostacyclin, thromboxane Abbreviations: AA arachidonic acid, COX cyclooxygenase, cpges cytosolic PGE synthase, GI gastrointestinal, HO-1 heme-oxygenase-1, HUVECs human umbilical vein endothelial cells, IL interleukin, IP prostacyclin receptor, LPS bacterial endotoxin, LSS laminar shear stress, MI myocardial infarction, PG prostaglandin, PGI prostacyclin, PMA phorbol myristate acetate, RCTs randomized clinical trials, RR relative risk, Ser serine, SNPs single-nucleotide polymorphisms, THIN The Health Improvement Network, TNF tumor necrosis factor, tnsaids traditional nonsteroidal anti-inflammatory drugs, TX thromboxane Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are an important and efficacious class of drugs for the management of musculoskeletal symptoms. These compounds cause both beneficial and adverse effects due to the inhibition of prostanoids [12, 37]. Prostanoids are biologically active derivatives of ara- 530 Pharmacological Reports, 2010, 62,

2 NSAIDs and cardiovascular disease Marta L. Capone et al. chidonic acid (AA) released from membrane phospholipids by phospholipases [14]. AA is transformed into prostaglandin (PG)H 2, through the activity of cyclooxygenase (COX) enzymes (i.e., COX-1 and COX-2). PGH 2 is subsequently metabolized by terminal synthases into the biologically active prostanoids, such as prostacyclin (PGI 2 ), PGD 2, PGF 2, PGE 2 and thromboxane (TX)A 2 [14, 17]. In particular, the isomerization of PGH 2 to PGE 2 is catalyzed by three different isomerases: a cytosolic PGE synthase (cpges) and two membrane-bound PGESs, mpges-1 and mpges-2 [22]. Of these isomerases, cpges and mpges-2 are constitutive enzymes whereas mpges-1 is mainly an induced isoform. COX-1 and COX-2 have the same catalytic activities: cyclooxygenase and peroxidase [34, 38]. However, the two isoforms of COX are the products of different genes. The COX-1 gene is considered a housekeeping gene by virtue of the constitutively low levels of expression in most cell types. In contrast, the gene for COX-2 is a primary response gene with multiple regulatory sites. COX-2 expression can be rapidly induced by bacterial endotoxin (LPS), cytokines (such as interleukin (IL)-1 and tumor necrosis factor (TNF)- ), growth factors, and the tumor promoter phorbol myristate acetate (PMA) [19]. COX-1- dependent prostanoids play an essential homeostatic role in gastrointestinal (GI) cytoprotection [20, 30, 39], while COX-2-dependent prostanoids play dominant roles in pathophysiologic processes, such as inflammation [6, 9]. Several lines of evidence suggest that the main mechanism of action for traditional (t)nsaids and NSAIDs selective for COX-2 (named coxibs), used for the treatment of pain and inflammatory joint disease, is the inhibition of COX-2- dependent PGE 2 [11]. Inhibition of constitutively expressed COX-1 in the GI tract and presumably in platelets by tnsaids seems to play a role in the increased risk of upper GI bleeding/perforation [30]. Proof of concept of this hypothesis came from the finding that reduced incidence of serious GI adverse effects has been shown for two COX-2 inhibitors, such as rofecoxib and lumiracoxib, when compared to tnsaids in large randomized clinical trials (RCTs) [3, 33]. Although the inhibition of COX-2-dependent PGE 2 production by tnsaids and coxibs is effective in ameliorating symptoms of inflammation and pain, a small but consistent increased risk of myocardial infarction (MI) has been detected in NSAID users [15, 16, 26]. Increased incidences of thrombotic events have been detected in placebo-controlled trials involving the COX-2 inhibitors celecoxib, rofecoxib and valdecoxib [4, 24, 25, 35]. However, the results of observational studies and a meta-analysis of data derived from trials with coxibs have shown that the cardiovascular hazard is also related to some tnsaids, such as diclofenac [15, 18, 21]. Convincing evidence suggests that cardiovascular toxicity associated with the administration of NSAIDs selective for COX-2 and some tnsaids occurs through a common mechanism involving the inhibition of COX-2-dependent prostacyclin. Pharmacology of NSAIDs The biochemical selectivity of COX inhibitors for COX-2 has been assessed in the past using different in vitro assays. These experiments yielded variable results depending on the assay used. In fact, several variables may affect the heterogeneity of the results, including the concentrations of exogenous AA, the lack of plasma proteins, and different types of isolated enzyme [5]. To overcome the variability associated with these assays, the human whole blood assays were developed. They are considered the gold standard method to assess the biochemical COX-2 selectivity of the drugs. These assays are based on the measurement of PGE 2 production in response to a 24-h incubation of LPS with heparinized blood samples, which reflects the time-dependent induction of COX-2 in circulating monocytes [27]. A parallel measurement of TXB 2 production during whole blood clotting is used as an index of platelet COX-1 activity [29]. The human whole blood assay allows the evaluation of the effects of drugs on platelet COX-1 and monocyte COX-2 both in vitro and, more importantly, ex vivo, after the administration of the drugs in humans. In vitro it is possible to define the experimental COX-2 selectivity obtained based on the assessment of IC 50 values, the concentration of the drug required to inhibit COX activity by 50%, for COX-1 and COX-2 and the calculation of COX-1/COX-2 IC 50 ratios. However, this describes only a chemical feature of NSAIDs. The same assays can be used to evaluate the achieved COX-2 selectivity of the drug at plasma concentrations. This information is critical for interpreting and possibly Pharmacological Reports, 2010, 62,

3 predicting the clinical consequences of NSAID administration because the degree of inhibition of COX-1 and COX-2 in vivo is dose-dependent. Cardiovascular hazard of NSAIDs It has recently been shown that patients taking NSAIDs, both coxibs and tnsaids, had a 35% increased risk of non-fatal MI [relative risk 1.35, (95% CI), 1.23 to 1.48] [15]. This elevation of risk increased with increasing treatment duration and daily dose. Using a translational medicine approach, from proof of concept in cells and experimental animals to studies in humans, it was possible to show that the inhibition of COX-2-dependent prostacyclin is the most plausible mechanism for the increase in cardiovascular hazard by NSAIDs. COX-2 is one of the endothelial genes upregulated by uniform laminar shear stress (LSS), which is characteristically associated with atherosclerotic lesionprotected areas [36]. We have recently shown that COX-2 protein was significantly induced in human umbilical vein endothelial cells (HUVECs) exposed to steady LSS [10]. This was associated with a significant increase of 6-keto-PGF 1, the hydrolysis product of prostacyclin. In contrast, the TNF-, a proatherogenic cytokine, released in the medium or detected in cell lysates was significantly reduced compared to the static condition. This was associated with a coincident induction of heme oxygenase (HO)-1, an antioxidant enzyme. The LSS-dependent reduction of TNF- production and the induction of HO-1 were both inhibited by the selective COX-2 inhibitor NS-398, the nonselective COX inhibitor aspirin, and the specific prostacyclin receptor (IP) antagonist RO This illustrates the central role played by LSS-induced COX-2-dependent prostacyclin in the modulation of endothelial inflammation [10]. Thus, inhibition of COX-2-dependent prostacyclin might contribute to the acceleration of atherogenesis in patients taking tnsaids and NSAIDs selective for COX-2 through the down-regulation of HO-1 and the resulting increase of TNF- production in human endothelial cells [10]. In mice, it has been demonstrated that endothelial COX-2-dependent prostacyclin modulates thrombosis induced in vivo by photochemical injury to the vasculature [8] using multiple approaches: the administration of the COX-2 selective inhibitor DFU, deletion of IP and specific deletion of COX-2 in endothelial cells. Single-nucleotide polymorphisms (SNPs) within the IP receptor gene have been identified. Interestingly, the R212C variant is associated with defective prostacyclin signaling. Arehart et al. [2] have shown that patients who are carriers of the SNP R212C show an increased propensity towards cardiovascular disease and events, analogous to the effects of COX-2 inhibition. The increased incidence of thrombotic events associated with the profound inhibition of COX-2- dependent prostacyclin might be mitigated, by a complete suppression of platelet COX-1 activity [16, 26]. This complete and permanent suppression of platelet COX-1 activity is mandatory to obtain the cardioprotective effects because a nonlinear relationship exists between the inhibition of platelet TXA 2 production and the inhibition of TXA 2 -mediated platelet aggregation. As a result, an inhibition of over 95% of COX-1 activity is required to influence platelet function [32]. In fact, even very small concentrations of TXA 2 have been shown to cause platelet activation [23, 31]. Aspirin is the only NSAID that has been shown to reduce non-fatal cardiovascular disease events (25 to 30%) and fatal events (10 to 15%) in RCTs of secondary prevention [1]. This is because aspirin, but not other NSAIDs, is an irreversible inhibitor of platelet COX-1 activity. This inhibition occurs through acetylation of a specific serine residue (Ser529) of human COX-1, which translates into an almost complete suppression (> 95%) of platelet capacity to produce TXA 2 at low doses throughout the 24-hour dosing interval. In contrast, tnsaids, which are reversible inhibitors of platelet COX-1, generally cause an incomplete and intermittent inhibition of platelet TXA 2. Clinical studies have shown that this reversible inhibition may be inadequate to prevent cardiovascular events [28]. These findings led us to introduce the concept of functional COX-2 selectivity, with respect to platelets, by NSAIDs [15]. This is a feature of drugs that suppress COX-2 in the presence of an insufficient reduction of platelet COX-1 activity (< 95%) to translate into inhibition of platelet function. The vast majority of NSAIDs are functionally selective for COX-2 at therapeutic doses [15]. With the exception 532 Pharmacological Reports, 2010, 62,

4 NSAIDs and cardiovascular disease Marta L. Capone et al. of aspirin, the only NSAID that lacks functional COX-2 selectivity in platelets is naproxen, and this is only seen at high doses in some individuals [7]. Due to the fact that most tnsaids are functionally selective for COX-2, they may confer cardiovascular hazard. We hypothesized that within this group the degree of inhibition of COX-2, due to either potency or the length of drug exposure, might be an important determinant. Thus, to address this important issue, we performed a population-based retrospective cohort study with nested case-control analysis using data from The Health Improvement Network (THIN) database in the United Kingdom [15]. First, we established the cardiovascular safety profile for specific individual NSAIDs. Then, we addressed the hypothesis that the degree of inhibition of whole blood COX-2 in vitro, as measured by plasma concentrations, corresponding to the average NSAID therapeutic dose in patients, an index of drug potency/exposure, could be used to predict the relative risk (RR) of MI observed in the general population for each individual NSAID that was functionally selective for COX-2. We found that the extent of COX-2-dependent prostacyclin inhibition may represent an independent key determinant of the increased risk of MI among NSAIDs with nonfunctional suppression of platelet COX-1, a property shared by coxibs and most tnsaids. We also found that the assessment of whole blood COX-2 may represent a surrogate end-points to predict the cardiovascular risk of these drugs. Interestingly, individual NSAIDs with a degree of COX-2 inhibition < 90% at therapeutic concentrations presented a RR of 1.18 (95% CI, ), while those with a greater COX-2 inhibition had a RR of 1.60 (95% CI, ) [15]. Perspectives The development of biomarkers capable of predicting the impact of NSAIDs on COX-1 and COX-2 activities in vitro, ex vivo and in vivo has been essential to understanding the clinical consequences of varying degree of inhibition of COX-isozymes in humans. Whole blood assays for COX-1 and COX-2 might be candidates for monitoring levels of toxicity and efficacy of NSAIDs. Using a biomarker strategy, we have shown that, within the group of functionally COX-2 selective NSAIDs with respect to platelets (coxibs and most tnsaids), the degree of inhibition of COX-2 is relevant to the cardiovascular hazard from NSAIDs and relates to drug potency (exposure). Thus, a reduction of the dose is recommended and will presumably limit the number of patients exposed to a cardiovascular hazard by NSAIDs. It will not, however, eliminate the risk on an individual level due to the marked variability in how different people react to these drugs, based on their genetic background [13]. Further studies should be performed to verify whether the assessment of whole blood COX-2 ex vivo, either alone or in combination with the measurement of urinary levels of 2,3-dinor-6-keto-PGF 1 (a biomarker of prostacyclin biosynthesis in vivo), in association with genetic biomarkers, such as polymorphisms in the IP receptor, may represent valid surrogate end-points to predict cardiovascular risk for functionally selective COX-2 inhibitors. Acknowledgments: We would like to thank Dr. Licia Totani, Dr. Virgilio Evangelista, Dr. Luigia Di Francesco and Dr. Annalisa Bruno for their invaluable contributions to the results discussed in this review. References: 1. Antithrombotic Trialists Collaboration: Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ, 2002, 324, Arehart E, Stitham J, Asselbergs FW, Douville K, MacKenzie T, Fetalvero KM, Gleim S et al: Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition. Circ Res, 2008, 102, Bombardier C, Laine L, Reicin A, Shapiro D, Burgos- Vargas R, Davis B, Day R et al.: Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med, 2000, 343, Bresalier RS, Sandler RS, Quan H, Bolognese JA, Oxenius B, Horgan K, Lines C et al.: Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med, 2005, 352, Brooks P, Emery P, Evans JF, Fenner H, Hawkey CJ, Patrono C, Smolen J et al.: Interpreting the clinical significance of the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2. Rheumatology, 1999, 38, Cao Y, Prescott SM: Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol, 2002, 190, Capone ML, Tacconelli S, Sciulli MG, Grana M, Ricciotti E, Minuz P, Di Gregorio P et al.: Clinical pharmacol- Pharmacological Reports, 2010, 62,

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