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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11693-11697, December 1994 Pharmacology Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible (cytokines/arachidonic add///mitogen/-like drugs) JANE A. MITCHELL*, PRAVIT AKARASEREENONT, CHRISTOPH THIEMERMANN, RODERICK J. FLOWER, AND JOHN R. VANE The William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London EClM 6BQ, United Kingdom Contributed by John R. Vane, September 9, 1993

ABSTRACT Constitutive cyclooxygenase (COX-1; pros- COX-2 may well explain their therapeutic utility as antiin- taglandin-endoperoxide synthase, EC 1.14.99.1) is present in flammatory drugs, whereas inhibition of COX-1 may explain cells under physiological conditions, whereas COX-2 is induced their unwanted side effects, such as gastric and renal damage. by some cytokines, mitogens, and endotoxin presumably in After establishing that bovine aortic endothelial cells in pathological conditions, such as inflammation. Therefore, we culture contain COX-1 and that endotoxin-activated J774.2 have assessed the relative inhibitory effects of some nonsteroi- macrophages contain COX-2, we have investigated the in- dal antiinflammatory drugs on the activities of COX-1 (in hibitory effects of some NSAIDs on the activity of COX-1 bovine aortic endothelial cells) and COX-2 (in endotoxin- and COX-2 in whole cells and broken cells and in purified activated J774.2 macrophages) in intact ceils, broken cells, and preparations. Our results show that NSAIDs have purified enzyme preparations (COX-1 in sheep seminal vesi- different profiles ofinhibition ofCOX-1 and COX-2 in a range cles; COX-2 in sheep placenta). Similar potencies of aspfrin, of models. indomethacin, and against the broken cell and purified enzyme preparations indicated no influence ofspecies. Aspirin, indomethacin, and ibuprofen were more potent in- METHODS hibitors of COX-1 than COX-2 in all models used. The relative Cell Culture. Murine macrophages (J774.2; The European potencies of aspirin and indomethacin varied only slightly Collection of Animal Cell Culture, Salisbury, U.K.) were between models, although the IC50 values were different. grown in 96-well culture plates with Dulbecco's modified Ibuprofen was more potent as an inhibitor of COX-2 in intact Eagle's medium supplemented with 10% fetal calf serum and cells than in either broken cells or purified . 4 mM L-glutamine. Bovine aortic endothelial cells (BAEC) salicylate was a weak inhibitor of both COX isoforms in intact were cultured from fresh bovine aortae as described (15) and cells and was inactive against COX in either broken cells or seeded onto 96-well culture plates. purifiedenzymepreparations. , BW755C, acetamin- Intact Cells. COX-L. BAEC were incubated for 30 min with ophen, and were approximately equipotent inhibi- aspirin (0.1 ng/ml to 1 mg/ml), indomethacin (0.1 ng/ml to 1 tors of COX-1 and COX-2 in intact cells. BF 389, an experi- mg/ml), ibuprofen (0.1 to 1 mg/ml), (0.1 to mental drug currently being tested in humans, was the most 100 Ag/ml), diclofenac (0.1 ng/ml to 100 pg/ml), naproxen potent and most selective inhibitor of COX-2 in intact cells. (0.1 ng/ml to 1 mg/ml), acetaminophen (0.1 ng/ml to 100 Thus, there are clear pharmacological differences between the ug/ml), BW 755C (0.1 ng/ml to 1 mg/ml), or a nonacidic two enzymes. The use of such models of COX-1 and COX-2 NSAID, BF 389 (0.1 ng/ml to 1 mg/ml; ref. 37). Arachidonic activity will lead to the identification of selective inhibitors of (30 ,uM) was then added, and the cells were incubated for COX-2 with presumably less side effects than present thera- a further 15 min at 37°C. The medium was then removed, and pies. Some inhibitors had higher activity in intact cells than radioimmunoassay (16) was used to measure the formation of against purified enzymes, suggesting that pure enzyme prep- 6-keto-PGF,a, PGE2, B2, or PGF2a. Antibodies arations may not be predictive of therapeutic action. to 6-keto-PGFia, PGE2, thromboxane B2, and PGF2a were obtained from Sigma. Tritiated 6-keto-PGF1,,, PGE2, throm- Cyclo-oxygenase (COX; -endoperoxide syn- boxane B2 or PGF2a were obtained from Amersham. thase, EC 1.14.99.1) converts to prostaglan- COX-2. Cultured J774.2 macrophages were treated with din (PG) H2, which is then further metabolized by other endotoxin at 1 gg/ml for 12 hr to induce COX-2. Culture enzymes to various PGs, , and medium was then changed, and one of the NSAIDs was (1). COX exists in at least two isoforms with similar molec- added (see above) for 30 min at 37°C. Arachidonic acid (30 ular weights ("'70 kDa). COX-1 is expressed constitutively ,uM) was then added, and the cells were incubated for a and was first characterized, purified, and cloned from sheep further 15 min at 37°C. The medium was removed and vesicular glands (2-7). Activation of COX-1 leads, for in- analyzed by radioimmunoassay as above. The inhibitory stance, to the production of prostacyclin, which when re- effects of NSAIDs on COX were measured in at least nine leased by the endothelium is antithrombogenic (8) and by the separate determinations (wellsj on at least 3 different exper- gastric mucosa is cytoprotective (9). COX-2 is induced in imental days. cells exposed to proinflammatory agents, including cytokines Cell Viability. Cell respiration, an indicator ofcell viability, (10), mitogens (11) and endotoxin (12, 13). Nonsteroidal was assessed by the mitochondrial-dependent reduction of antiinflammatory drugs (NSAIDs) inhibit the activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- COX, a property that accounts for their shared therapeutic mide to formazan as described (17). Treatment of J774.2 and side effects (14). Thus, the ability of NSAIDs to inhibit Abbreviations: BAEC, bovine aortic endothelial cells; COX, cyclo- The publication costs ofthis article were defrayed in part by page charge oxygenase; NSAIDs, nonsteroidal antiinflammatory drugs; PG, payment. This article must therefore be hereby marked "advertisement" prostaglandin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11693 Downloaded by guest on October 4, 2021 11694 Pharmacology: Mitchell et al. Proc. Natl. Acad Sci. USA 90 (1993) macrophages with lipopolysaccharide at 1 ,ug/ml for 12 hr did anti-rabbit IgG developed in sheep and linked to alkaline not significantly inhibit cell viability. phosphatase conjugate, and the blot was developed with Broken Cell Preparation. COX-L. BAEC were cultured in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetra- T175 flasks until confluent. The cells were washed and zolium. scraped into ice-cold phosphate-buffered saline (pH 7.4). The Materials and Statistical Analysis. All compounds used cells were then centrifuged at 1000 x g for 10 min, and the cell were obtained from Sigma unless otherwise stated. BF 389 pellet was homogenized with a glass Teflon homogenizer in was a gift from Biofor and BW 755C was a gift from Wellcome 50 mM Tris buffer (pH 7.4) containing 1 mM phenylmethyl- Research Laboratories. Data were analyzed by using Stu- sulfonyl fluoride, 50 ,M pepstatin A, and 0.2 mM leupeptin. dent's unpaired t test and taking a P value of <0.05 as The broken cells (150 ,ug of protein) were incubated at 37°C significant. in the presence of one of the NSAIDs (see above) for 30 min. Arachidonic acid (30 AM) was added, and the incubation was continued for a further 15 min. The reaction was then stopped RESULTS by boiling. The samples were centrifuged at 10,000 x g for 30 Characterization ofCOX-1 and COX-2 Activities. The basal min, and 6-keto-PGFi, was measured in the supematant as an release of eicosanoids from untreated J774.2 macrophages indicator of COX-1 activity. was below the limits of detection, which were 0.3 ng/ml for COX-2. J774.2 macrophages were cultured in T175 flasks 6-keto-PGF1,, PGE2, thromboxane B2, and PGF2a (n = until confluent. Endotoxin at 1 ,ug/ml was added for 12 hr, 6-15). After exposure to endotoxin at 1 JLg/ml for 12 hr, the after which time the cells were washed and homogenized; the cells released significantly higher amounts (P < 0.05, for all) effects of NSAIDs on COX-2 activity were assayed as for of 6-keto-PGFi, (1.55 ± 0.19 ng/ml), PGE2 (2.60 ± 0.08 COX-1 in the broken-cell preparations of BAEC. ng/ml), thromboxane B2 (0.87 ± 0.03 ng/ml), and PGF2a Purified Enzymes. Purified COX-1 and COX-2 were ob- (3.46 ± 0.10 ng/ml), (n = 9 for each). BAEC released tained from Cayman Chemicals (Ann Arbor, MI) and were 6-keto-PGF1, (2.30 ± 0.31 ng/ml; n = 9) and thromboxane B2 gifts from Biofor (Waverly, PA). Enzyme activity was mea- (0.43 ± 0.01 ng/ml) but <0.3 ng/ml of PGE2 or PGF2a (n = sured by the conversion of [14C]arachidonic acid to PGE2 9, for each). Therefore, the formation of 6-keto-PGFia was after separation by thin layer chromatography (TLC). A used as a common indicator of COX activity in BAEC or solution (100 ,u) of aspirin, indomethacin, ibuprofen, or endotoxin-activated J774.2 macrophages. sodium salicylate was added to a reaction mixture containing Characterization of COX Isoforms Present in BAEC or 6.6 uM arachidonic acid (saturating substrate concentration), Endotoxin-Activated J774.2 Macrophages. Antibodies to together with [14C]arachidonic acid (100,000 disintegrations COX-1 recognized a band of approximately 70 kDa in the per minute), and was made up to a final volume of 1 ml with extracts of BAEC (Fig. 1A, lane 1) that was not recognized 50 mM Tris buffer (pH 8 at 37°C) containing the cofactors by antibodies to COX-2 (Fig. 1B, lane 1). In contrast, COX-2 glutathione (5 mM), epinephrine (5 mM), and hematin (1 JIM). protein was present in extracts ofendotoxin-activated J774.2 The reaction was initiated by the addition of 10 units of macrophages (Fig. 1B, lane 3) with low levels ofCOX-1 (Fig. enzyme. Samples were incubated in a shaking water bath at 1A, lane 3). Untreated J774.2 macrophages contained no 37°C for 10 min (during which period the reaction was linear), COX-2 (Fig. 1B, lane 2) and low levels of COX-1 (Fig. 1A, after which the reaction was stopped by adding 30 ,ul of 1 M lane 2). HCl. One milliliter of saturated NaCl solution was added to Effects of NSAIDs on the Activity of Purified COX-1 and each sample followed by 1.5 ml of ethyl acetate. All samples COX-2. In these assays, there was no preincubation of the were mixed in a Vortex and centrifuged at 1500 rpm for 10 min NSAIDs with the enzyme before adding the substrate. As- (575 x g). The ethyl acetate layer (1 ml) was removed to a pirin, indomethacin, or ibuprofen were much more potent as separate tube and concentrated under a stream of nitrogen. inhibitors of purified COX-1 than of purified COX-2 (Table PGs were separated by TLC. Each sample was redissolved 1). Sodium salicylate was inactive against purified COX-1 at in 30 .ul ofchloroform/, 2:1 (vol/vol), and 20 ,ul was concentrations up to 1 mg/ml and produced only a 20% applied onto a glass-backed silica gel plate. The TLC plate was developed for =90 min at room temperature in a solvent A B consisting of the upper phase of ethyl acetate/trimethylpen- 1 2 3 1 2 3 tane//water, 110:50:20:100 (vol/vol). For detection of 14C-labeled PGs, autoradiography was performed by placing the plate in contact with x-ray film for 12 hr. The film was then developed and fixed. The PGE2 band was located on the plate and scraped off, and the absolute radioactivity was estimated by scintillation counting. <70 kDa> Immunoblot (Western Blot) Analysis. BAEC or endotoxin- activated (1 pg/ml; 12 hr) J774.2 macrophages cultured in T175 flasks were washed with phosphate-buffered saline (pH 7.4) and incubated for 10 min with 2-3 ml of extraction buffer FIG. 1. BAEC contain COX-1 and endotoxin-activated J774.2 [50mM Tris/10 mM EDTA/1% (vol/vol) Triton X-100/1 mM macrophages contain COX-2, as shown by Western blots with phenylmethylsulfonyl fluoride/50 ,M pepstatin A/0.2 mM polyclonal antibodies to COX-1 (A) or COX-2 (B). Cell extracts of leupeptin] while being gently shaken. The cell extract was BAEC (lanes 1) contained a protein (=70-kDa molecular mass) that then boiled for 10 min with gel-loading buffer [50 mM was recognized by antibodies to COX-1 (A) but not by antibodies to Tris/10% (wt/vol) SDS/10% (vol/vol) /10% 2-mer- COX-2 (B). Low levels of COX-1 or COX-2 protein were detected in captoethanol/2 mg of bromophenol blue per ml] in a ratio of untreated J774.2 macrophages (lanes 2). Endotoxin-activated J774.2 1:1 (vol/vol). The samples were loaded onto gradient gels macrophages contained a protein (=70-kDa molecular mass) that was (4-12% Tris recognized by antibodies to COX-2 (lanes 3). A band of =70 kDa was glycine; Novex, British Biotechnology, Oxford) recognized by COX-1 antibodies in cell extracts from endotoxin- and separated by electrophoresis. After transfer to nitrocel- activated J774.2 macrophages. As the COX-1 antibody used cross- lulose, the blot was primed with a selective antibody raised reacts with COX-2 (10%), this band is at least in part attributed to to ovine COX-1 developed in rabbits (a gift from K. Wu, COX-2 protein (lane 3). Equal amounts of protein were loaded in all Houston) or a rabbit antibody raised to murine COX-2 lanes (2-3 ,ug per lane). Similar results were obtained with cell (Cayman Chemical). The blot was then incubated with an extracts from two separate batches of cells. Downloaded by guest on October 4, 2021 Pharmacology: Mitchefl et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11695 Table 1. ICso values for aspirin, indomethacin, ibuprofen, sodium salicylate (salicylate), and BF 389 as inhibitors of COX activity in broken-cell preparations and of purified COX IC50, ,ug/ml Broken cells Purified enzyme NSAID COX-1 COX-2 Ratio COX-1 COX-2 Ratio Aspirin 8 200 25 5 210 42 Indomethacin 0.01 0.4 40 0.1 5 50 Ibuprofen 3 160 53 1 46 46 Salicylate Inactive >1 mg/ml Inactive Inactive BF 389 ND ND ND 4 8 2 The data on purified enzymes represent the mean oftwo determinations. Similar results were seen in separate experiments with different batches of COX-1 and COX-2. ND, not determined. inhibition of COX-2 at the maximum concentration used (1 COX-2 (ratio 0.2). It was substantially more potent in the mg/ml). The ratios ofactivities in favor ofCOX-1 varied from intact cells than against the purified enzymes. 42 to 50. In contrast, BF 389 with IC50 values of 4 ,ug/ml for COX-1 and 8 ,g/ml for COX-2 gave a ratio of 2. Effects of NSAIDs on Broken-Cell Preparations of BAEC DISCUSSION and Endotoxin-Activated J774.2 Macrophages. In these ex- The discovery of an inducible isoform (COX-2) of cyclooxy- periments, the NSAID was preincubated with the enzyme for genase (13) allows a reinterpretation and refinement of the 30 min. Aspirin, indomethacin, or ibuprofen inhibited the general theory that inhibition of COX activity explains the activity of COX-1 more potently than COX-2 in broken cells therapeutic and side effects of the aspirin-like drugs (14). (Table 1). The activity of aspirin was similar to that found COX-2 is induced in migratory and other cells by inflam- with purified enzymes, as was that of ibuprofen. However, matory stimuli (18) presumably through cytokine production. indomethacin showed IC50 values lower in the broken cells by Therefore, it is attractive to suggest that the antiinflammatory a factor of 10 than in the purified enzymes. COX-1 activity in actions of the NSAIDs are due to inhibition of COX-2, broken cells was unaffected by sodium salicylate. COX-2 whereas the unwanted side effects, such as gastric toxicity activity in broken cells was only slightly inhibited (20%o) by and , are due to inhibition of the constitutive the highest concentration ofsodium salicylate used (1 mg/ml; enzyme (COX-1), the products of which help to protect the Table 1). stomach and the kidney against damage. Here, we explore Effects of NSAIDs on COX Actity in Whole Cells. Indo- this hypothesis by testing some NSAIDs on different prep- methacin was the most potent inhibitor of COX-1 and was arations of COX-1 and COX-2. We show that BAEC contain some 60 times more potent than against COX-2 (Table 2; Fig. predominately COX-1 and that endotoxin-activated J774.2 2B). Aspirin was 166 times more active against COX-1 but macrophages contain predominately COX-2. Using the COX was less potent than indomethacin on either isoform. Ibu- activity in these cells as models for COX-1 and COX-2 or profen was 15 times more active against COX-1 than COX-2. using purified enzymes, we demonstrate that NSAIDs have Interestingly, the activity of ibuprofen against COX-2 in the different relative potencies as inhibitors of COX-1 and intact cells was some 10-fold greater than on the broken cells. COX-2. Sodium salicylate showed weak activity in both cell prepa- There is little evidence to suggest that the potencies of rations but was only 3 times more potent against COX-1 than NSAIDs as inhibitors of COX-1 and COX-2 vary from COX-2. Acetaminophen was also an inhibitor of COX-1 (IC30, 2.7 ± 2.0 ug/ml) and COX-2 (IC30, 20.0 ± 12.0 /g/ml) activity in intact cells. Diclofenac, naproxen, and BW 755C loo0 were approximately equipotent inhibitors of COX-1 or A 10C COX-2 in intact cells (Table 2). The nonacidic NSAID BF 389 o0 ., was the most potent inhibitor tested against COX-2 (IC50; o 0.03 jg/ml) and also showed the greatest selectivity for 0 e 0 1000 °1000 Table 2. IC50 values for NSAIDs on COX-1 and COX-2 activity in intact cells ._1 100 B 100 D IC50, ug/ml ct NSAID COX-1 COX-2 Ratio 0 Q Aspirin 0.3 ± 0.2 50 ± 10 166 Indomethacin 0.01 ± 0.001 0.6 ± 0.08 60 0 0 Ibuprofen 1.0 ± 0.07 15 ± 5.3 15 0 1000 0 1000 Acetaminophen* 2.7 ± 2.0 20 ± 12 7.4 Inhibitor, ,ug/ml Sodium salicylate 35 + 11 100 + 16 2.8 BW 755C 0.65 + 0.26 1.2 ± 0.8 1.8 FIG. 2. The effects of aspirin (A), indomethacin (B), ibuprofen Diclofenac 0.5 + 0.2 0.35 ± 0.15 0.7 (C), and sodium salicylate (D) on COX activity in BAEC (o) or J774.2 ± 1.3 ± 0.8 0.6 macrophages treated with endotoxin at 1 pg/ml for 12 hr (m). COX Naproxen 2.2 0.9 activity was measured by the formation of 6-keto-PGFia after BF 389 0.15 ± 0.01 0.03 ± 0.01 0.2 exposure to exogenous 30 ,tM arachidonic acid for 10 min. Aspirin The data show the mean ± the SEM for three to five determina- and indomethacin (Left) were more potent inhibitors of6-keto-PGF1i tions calculated from the means of triplicate determinations. The formation from BAEC (COX-1) than from endotoxin-activated ratio of the IC50 values for the NSAIDs on COX-2 relative to COX-1 J774.2 macrophages (COX-2). Sodium salicylate and ibuprofen is given in the final column of the table. (Right) were equipotent inhibitors. Data are expressed as mean ± *For acetaminophen, IC30 values are shown because 50% inhibition SEM from 9-15 determinations from at least three separate exper- of COX-2 was not achieved at concentrations up to 1 mg/ml. imental days. Downloaded by guest on October 4, 2021 116% Pharmacology: Mitchell et al. Proc. Natl. Acad Sci. USA 90 (1993) species to species. Nevertheless, because the two main cell Vane (14), using broken-cell preparations ofguinea pig lung types we have used are from ox (BAEC) and mouse (J774.2), (COX-1), found salicylate to be much weaker as an inhibitor we have compared the effects of four NSAIDs on crude of COX than aspirin. Here we confirm this observation by broken-cell preparations with those on purified enzyme prep- showing that aspirin is more potent than salicylate as an arations from the sheep. On the broken-cell preparations of inhibitor of COX-1 or COX-2. However, salicylate and guinea pig lung (presumably COX-1), aspirin had an IC50 of aspirin are said to be equally effective antiinflammatory 7 p,g/ml (14). Here we find the IC50 on broken BAEC cells to agents leading to the criticism (29) of the theory that inhibi- be 8 ,ug/ml and on purified sheep COX-1 to be 5 ,ug/ml. There tion of PG synthesis alone explains the actions of NSAIDs. is, then, good consistency for aspirin as an inhibitor ofCOX-1 Do the present results with salicylate help to explain this from several different species. anomaly? Here we show that aspirin is =100 times more In both crude and purified enzyme preparations (Table 1), potent than salicylate as an inhibitor of COX-1 in intact cells aspirin, indomethacin, and ibuprofen were much less active but only twice as potent as an inhibitor of COX-2. These against COX-2 than against COX-1, and the ratios of activity observations may explain those of Higgs et al. (30) showing remained remarkably consistent (25-53) between the two that salicylate and aspirin were approximately equipotent assay systems used. Indomethacin was about 10 times more inhibitors of COX activity in explants of acutely inflamed active as an inhibitor of COX activity in broken cell prepa- tissue (COX-2?). However, aspirin was found to be consid- rations than as an inhibitor ofpurified COX-1 or COX-2. This erably more potent than salicylate as an inhibitor of PGs in may be due to the different experimental protocols used, for the serum (COX-1?). Salicylate had negligible inhibitory indomethacin was preincubated for 30 min with the broken actions on COX activities in broken cell preparations or purified enzyme. The mechanism ofaction of salicylate as an cell preparations but was added simultaneously with arachi- inhibitor of COX activity in intact cells may be compounded donic acid substrate in the purified enzymes. by its suppression of the induction of COX (31). Our results with purified COX-1 and COX-2 to assess the Acetaminophen also posed a problem for the original inhibitory effects of indomethacin (50 times more potent theory of the mechanism of action of NSAIDs, for at thera- against COX-1 than COX-2) are very similar to those of peutic doses acetaminophen has weak antiinflammatory ac- Meade et al. (ref. 19; indomethacin was 32 times more potent tivity but is a stronger and (32). Flower against COX-1 than COX-2). They also found that aspirin was and Vane (21) found that COX preparations from the brain a selective inhibitor ofCOX-1 using membrane fractions from were more sensitive to acetaminophen than those from the transfected cells. spleen and suggested that there may be different isoforms of Can the effects of NSAIDs on COX-1 explain their un- COX. We found that acetaminophen inhibited both COX-1 wanted effects? The two strongest inhibitors of COX-1 in all and COX-2 activity in intact cells. However, acetaminophen assay systems used, aspirin and indomethacin, are the two was less potent as an inhibitor ofCOX-2 activity than all other NSAIDs that cause the most gastric damage (20). Even NSAIDs tested, with the exception of aspirin and sodium though indomethacin is more potent than aspirin as an salicylate. The weak inhibitory actions of acetaminophen on inhibitor of COX activity in a variety of systems (14, 21) and COX-2 may explain why this drug has only weak antiinflam- as an antiinflammatory drug (21, 22), it is also more potent matory actions. Perhaps there are other isoforms of COX than aspirin as an ulcerogenic agent (see ref. 23). What is ("COX-3") in the endothelial cells in the brain that initiate more important than the relative potencies of NSAIDs by the release of PGs (33). against each other on COX is the ratio of activities on COX-1 There is a third group of NSAIDs including BW 755C, and COX-2. Thus, assuming that COX-2 is relevant to diclofenac, and naproxen in which the ratio (IC50 against inflammation, an antiinflammatory dose of indomethacin or COX-2/IC5o against COX-1) in intact cells is about 1. The dual aspirin will suppress COX-1 activity in the stomach and COX and lipoxygenase inhibitor BW 755C (34) is an experi- kidney with a 50-fold greater potency. mental antiinflammatory agent with little or no ulcerogenic Aspirin irreversibly inhibits COX-1 by acetylation of a activity (9), which fits well with our general hypothesis. single serine residue on the enzyme (24). Indomethacin Diclofenac and naproxen are less ulcerogenic than aspirin or inhibits COX-1 activity by binding to its active site and indomethacin at antiinflammatory doses (ref. 35; see ref. 23). subsequently rendering it inactive (25), possibly by producing Interestingly, of all the NSAIDs tested, BF 389, an exper- a conformational change in an essential protein radical (26). imental drug now being tested in man, was the most potent Although the mechanisms by which aspirin and indomethacin agent against COX-2 in intact cells and also displayed a ratio inhibit COX are different, the relative potencies of these (0.2) with the greatest selectivity for COX-2. This fits well with drugs were similar in all of the assays used in this study and toxicological reports on this experimental drug, for BF 389 has in those using microsomal preparations from COS-1 trans- little or no gastric ulcerogenicity (36). The fact that BF 389 is fected cells for assay of COX-1 and COX-2 activity (19). more potent in intact cells than on purified enzymes may well Ibuprofen, which inhibits COX by substrate competition be due to the fact that it is concentrated in tissues (37). The with arachidonic acid (27), produces less side effects than results with BF 389 highlight the differences that can be either aspirin or indomethacin (28). This would not be pre- obtained between intact cells and purified enzymes. Clearly dicted from the ratios obtained on the enzyme preparations, the inhibition of purified enzymes as a screening method for but in intact cells ibuprofen was at least 5 times more potent antiinflammatory activity is a logical starting point, but results than aspirin as an inhibitor of COX-2, whereas the two from intact cell preparations may well correlate better with NSAIDs were equipotent at inhibiting COX-1 activity. These biological activity in animals and humans. results on intact cells may explain why at equiactive antiin- Thus, our results support the hypothesis that the side flammatory doses, ibuprofen produces less ulcerogenic side effects of NSAIDs correlate with their ability to inhibit effects than aspirin. However, Meade et al. (19) find that COX-1, while the antiinflammatory (therapeutic) effects of ibuprofen is equipotent as an inhibitor of COX-1 and COX-2 these agents are due to their ability to inhibit COX-2. Inhi- activity in microsomes from transfected COS-1 cells. As the bition of COX-2 activity may be achieved at several levels in mechanism of action of ibuprofen is based on substrate the cascade of events leading to induction of enzyme activity. competition, these differences in the potency of ibuprofen First, the actions of proinflammatory mitogens can be may be due to variations in the accessibility to the enzyme blocked with antagonists or antibodies. Second, and in the relative concentration of arachidonic acid present once the cell is activated, the synthesis of COX-2 may be in the different assay systems. blocked with agents such as glucocorticosteroids (38) or Downloaded by guest on October 4, 2021 Pharmacology: MitcheU et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11697

0 MACROPHAGES/OTHER CELLS 0g) COX-1 COX-2 Q CONSTITUTIVE INDUCED 0

TXA2 PGI2 PGE2 PROIrEASES PGs OTHER INFLAMMATORY 4 + MEDIATORS endothefium kidney stomach mucosa INFLAMMATION FIG. 3. Relationships between the pathways leading to the generation of eicosanoids by COX-1 or COX-2. Under physiological conditions, activation of COX-1 for instance in platelets, endothelium, stomach mucosa, or kidney results in the release of (TXA2), prostacyclin (PG12), or (PGE2). The release of these eicosanoids is selectively inhibited by drugs such as aspirin (1). Inflammatory stimuli release cytokines, such as interleukin 1, that induce the synthesis of COX-2 in cells, such as macrophages, resulting in the release of (PGs). The release of PGs together with proteases and other inflammatory mediators (such as reactive oxygen radicals) results in inflammation. The COX-2 pathway can be interrupted at several levels by antagonists or antibodies to cytokines and mitogens (2), inhibitors of the induction of COX-2 (e.g., glucocorticoids) (3), or selective inhibitors of COX-2 (4). salicylates (31). Finally, once COX-2 has been synthesized, 14. Vane, J. R. (1971) Nature (London) 231, 232-235. selective inhibitors of COX-2 would inhibit the production of 15. de Nucci, G., Gryglewski, R. J., Warner, T. D. & Vane, J. R. (1988) Proc. Natl. Acad. Sci. USA 85, 2334-2338. proinflammatory prostanoids without affecting, for instance, 16. Salmon, J. A. (1978) Prostaglandins 15, 383-397. prostacycin production by the endothelium (COX-1; Fig. 3). 17. Gross, S., Jaffe, E. A., Levi, R. & Kilbourn, R. G. (1991) The identification of selective inhibitors of COX-1 and Biochem. Biophys. Res. Commun. 187, 823-829. COX-2 will not only provide an opportunity to test this 18. Sano, H., Hla, T., Maier, J. M., Crofford, L. J., Case, J. P., hypothesis, but also lead to advances in the therapy of Maciag, T. & Wilder, R. L. (1992) J. Clin. Invest. 89, 97-108. inflammation. As new compounds become available to in- 19. Meade, E. A., Smith, W. L. & DeWitt, D. L. (1993) J. Biol. Chem. 268, 6610-6614. hibit COX-2, the use ofaspirin will diminish in inflammation, 20. Lanza, F. L. (1989) Scand. J. Gastroenterol. 24, 24-31. but expand as an inhibitor of COX-1 in platelets for the 21. Flower, R. J. & Vane, J. R. (1972) Nature (London) 240, prevention of thrombosis. 410-411. 22. Whittle, B. J. R. (1977) Br. J. Pharmacol. 60, 455-460. We are indebted to Ms. Elizabeth Wood for supplying the cultured 23. Rainsford, K. D. (1982) Rheumatol. Int. 2, 1-10. cells and Mr. David Bishop-Bailey and Ms. Kamlesh Garewal for 24. Roth, G. J., Stanford, N. & Majerus, P. W. (1975) Proc. Natl. technical assistance. We would also like to thank Drs. Timothy D. Acad. Sci. USA 72, 3073-3076. 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