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Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy

Vascular Inflammation and

^a Experimental Pathology, The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, U.K.; and

^b Unit of Critical Care Medicine, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, U.K.

	ABSTRACT

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Prostanoids produced via the action of cyclo-oxygenase-2 (COX-2) appear central to many inflammatory conditions. Here we show in LPS-treated rats, however, that COX-2 induction alone does not greatly increase prostanoid production in vivo. For this, a second, arachidonic acid liberating stimulus is also required. Thus, only after intravenous injection of bradykinin or exogenous arachidonic acid was a marked increase in prostanoid formation seen. There is, therefore, synergy between proinflammatory mediators: both induction of COX-2 protein and an increase in the supply of arachidonic acid are required to greatly enhance prostanoid production. Second, we show that supplying arachidonic acid to increase prostanoid production reduces the effectiveness of both currently used nonsteroidal antiinflammatory drugs (NSAIDs) (diclofenac) and novel COX-2-selective inhibitors (NS-398, celecoxib) as inhibitors of COX-2 activity. Our data lead to two important conclusions. First, increased prostanoid production in inflammation is a two-component response: increased COX-2 expression and increased arachidonic acid supply. Second, the supply of arachidonic acid to COX-2 determines the effectiveness of NSAIDs. NSAIDs and selective COX-2 inhibitors, therefore, will generally be less effective at more inflamed sites, providing a rationale for the very high doses of NSAIDs required in human conditions such as rheumatoid arthritis.—Hamilton, L. C., Tomlinson, A. M., Mitchell, J. A., Warner, T. D. Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy.

Key Words: antiinflammatory agents • cyclooxygenase inhibitors • disease models • prostaglandins • prostaglandin endoperoxide synthase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PROSTANOIDS ARE physiologically important as locally acting autacoids and pathologically important as proinflammatory mediators. They are produced via a complex enzyme cascade regulated by two principle enzymes, phospholipase A₂ (PLA₂)² and cyclo-oxygenase (COX). PLA₂ mobilizes arachidonic acid from cellular phospholipids, which is then metabolized by COX to prostaglandin H₂ (PGH₂) (1), the common substrate for a number of synthetases and isomerases essential to the formation of a variety of prostanoids.

PLA₂ exists in both calcium-dependent and -independent isoforms. The extracellular or secretory PLA₂ (sPLA₂) requires millimolar levels of calcium for activation whereas the intracellular or cytosolic PLA₂ (cPLA₂) requires only nanomolar levels of calcium (for review, see ref 2 ). Thus, sPLA₂ is activated continuously by the levels of calcium found in the extracellular environment, whereas cPLA₂ is activated via increases in intracellular calcium elicited by inflammatory mediators such as bradykinin (3–5).

Similar to PLA₂, COX exists in multiple isoforms. A constitutive isoform (COX-1) is thought to be responsible for the `housekeeping' functions of the enzyme whereas the inducible (COX-2) isoform (6, 7) is thought to mediate inflammatory events. COX-2 is expressed in vitro in response to a number of proinflammatory mediators (8–11) and in vivo at the site of inflammation (12, 13). As traditional nonsteroidal antiinflammatory drugs (NSAIDs) appear to produce at least some of their beneficial effects by inhibiting COX-2 and their deleterious side effects by inhibiting COX-1 (7), the development of selective COX-2 inhibitors has been an area of intense pharmaceutical research. Increasing the supply of arachidonic acid markedly reduces the potency of both traditional NSAIDs and newer selective COX-2 inhibitors as inhibitors of COX-2 activity in vitro (14, 15). As the activity of phospholipase A₂ and hence free arachidonic acid levels are elevated at inflammatory sites, this implies that in vivo application of an NSAID such as sodium salicylate will produce lesser reductions in prostanoid production at inflammatory sites than at noninflammatory sites.

Therefore we have addressed for the first time in vivo two important and related questions. First, what is the effect of increasing the supply of arachidonic acid from either exogenous or endogenous sources on the activity of COX-2? Second, what effect does increasing the levels of free arachidonic acid have on the COX-2-inhibiting effects of traditional NSAIDs and novel COX-2-selective compounds?

	MATERIALS AND METHODS

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Materials
[³H] 6-Keto PGF₁ was bought from Amersham International (Little Chalfont, Bucks). NS 398 was obtained from Cayman Chemicals (Ann Arbor, Mich.). Celecoxib was prepared by Boehringer Ingleheim (Germany). All other compounds were purchased from Sigma Chemical Company (Poole, Dorset).

Surgical procedure
Male Wistar rats (220–250 g; Tuck, U.K.) were anesthetized with thiobutabarbital sodium (Inactin; 120 mg kg ^-1, i.p.). The trachea was cannulated to facilitate respiration. The right carotid artery was cannulated and connected to a pressure transducer (Elcomatic Type 750) for the measurement of systemic blood pressure, which was recorded on a Graphtec Linearcorder (Type WR 3101). Mean arterial pressure (MAP) was calculated as the diastolic pressure plus one third of the pulse pressure. Hemodynamic parameters were measured throughout the protocol. A cannula was also introduced into the left jugular vein for the administration of drugs. Body temperature was maintained at 37°C via a homeothermic blanket regulated by a rectal thermometer (Biosciences, Shernedd, Kent).

Upon completion of the surgical procedure, animals were left for 15 min to allow stabilization of the cardiovascular parameters. Animals were treated (t=-1 h) with one of the following: NS 398 (0.3 to 10 mg kg^-1 i.p.) (16), celecoxib (0.01 to 3 mg kg^-1 i.p.) (17), diclofenac (5 or 10 mg kg^-1 i.p.), or drug vehicle (10% w/v DMSO or saline, as appropriate). Commencing 1 h later (t=0), animals received a 6 h continuous infusion of lipopolysaccharide [LPS, serotype 0127:B8 (12,500,000 endotoxin units/mg) 0.2 mg kg^-1 h^-1] or LPS vehicle (saline). At the end of the infusion period with LPS or vehicle (t=6 h), animals were challenged with a final bolus of arachidonic acid (3 mg kg ^-1 i.v.) or bradykinin (10 µmol kg^-1, i.v.). Blood samples (500 µl) were taken via the carotid artery cannula at t = 0, 2, 4, 6 h, and 1 min after administration of the final bolus of arachidonic acid or bradykinin. The animals were then killed by overdose of anesthetic and organs were snap-frozen, under hexane, for immunohistochemical analysis of COX-2 expression.

Measurement of plasma 6 keto-PGF₁
The plasma concentration of 6 keto-prostaglandin (PG) F₁, the stable hydrolysis product of prostacyclin (PGI₂), was measured by specific radioimmunoassay as a determinant of COX activity.

Data analysis
Significant differences were determined by one-way analysis of variance, followed by Dunnett's test. P <0.05 was considered to be statistically significant.

	RESULTS

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Effects of LPS on COX-2 expression and plasma levels of 6 keto-PGF₁
Infusion of LPS to the rats caused a low (but significant), time-dependent increase in the plasma concentration of 6 keto-PGF₁ such that at t = 6 h levels were elevated more than 30-fold (Fig. 1 ).Moreover, after 6 h of LPS infusion, COX-2 immunoreactivity was expressed in the endothelium and smooth muscle of all organs studied (heart, spleen, aorta, and kidney; data not shown) and was particularly prevalent in the vasculature of the lung (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. The effect of infusion for 6 h of LPS (0.2 mg kg-1 h-1) or LPS vehicle (saline) on the concentration of 6 keto-PGF1 in the plasma of anesthetized rats (n=5–8). Results are expressed as mean ±SEM.

Synergy between supply of arachidonic acid and induction of COX-2
In naive rats, a bolus injection of arachidonic acid (3 mg kg^-1, i.v.) caused a small increase in the circulating levels of 6 keto-PGF₁ (Fig. 2A ).However, infusion of LPS caused a dramatic elevation in the acute formation of 6 keto-PGF₁ such that at 4 and 6 h it was increased by more than 120-fold (Fig. 2A ). Similarly, at 6 h there was a clear synergy between bradykinin and LPS (Fig. 2B ). For example, in LPS-treated animals, 10 µmol kg^-1 bradykinin caused a far greater increase in plasma 6 keto-PGF₁ levels than did 100 µmol kg^-1 bradykinin in time-matched controls (Fig. 2B ).

View larger version (24K): [in this window] [in a new window]   Figure 2. A) Effect of LPS infusion on the production of 6 keto-PGF1 after bolus injection of arachidonic acid (AA, 3 mg kg-1, i.v.) in rats receiving vehicle or LPS for 2–6 h (n=5–8). B) Effect of LPS infusion or vehicle for 6 h on the production of 6 keto-PGF1 after injection of a bolus of bradykinin (BK) (1–100 µmol kg-1) (n=4–8). Results are expressed as mean ±SEM.

Effect of a `classical NSAID', diclofenac
Diclofenac (5 mg kg^-1) inhibited by more than 90% the increase in plasma 6 keto-PGF₁ concentration caused by infusion of LPS for 6 h, but was without effect against the large increase in plasma 6 keto-PGF₁ levels recorded 1 min after bolus injection of arachidonic acid (Table 1 ).At a dose of 10 mg kg^-1, however, diclofenac inhibited by more than 95% the rise in plasma 6 keto-PGF₁ caused by arachidonic acid injection (Table 1) . In naive animals, diclofenac (10 mg kg^-1) potently inhibited 6 keto-PGF₁ levels after arachidonic acid (Table 2 ).

View this table: [in this window] [in a new window]   Table 1. Effect of the classical NSAID, diclofenac, on the accumulation and acute formation of plasma 6 keto-PGF1 in LPS-treated rats

View this table: [in this window] [in a new window]   Table 2. Effect of diclofenac and the COX-2-selective inhibitors NS 398 and celecoxib on the formation of 6-keto-PGF1 (ng ml-1) in naive animalsa

Effects of COX-2-selective inhibitors in LPS-treated animals
NS 398 at all doses tested (0.3 to 10 mg kg^-1) reduced by more than 90% the increase in plasma 6 keto-PGF₁ concentration caused by infusion of LPS for 6 h (Fig. 3A ).However, in LPS-treated rats NS 398 inhibited the arachidonic acid-induced rise in 6 keto-PGF₁ only at doses greater than 3 mg kg^-1 (Fig. 3B ).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of NS 398 on the concentration of 6 keto-PGF1 in the plasma of anesthetized rats treated with LPS (0.2 mg kg-1 h-1) for 6 h. A) In rats receiving LPS alone (n=8). B) In rats 1 min after injection of a bolus of arachidonic acid (3 mg kg-1, i.v.). Results are expressed as mean ±SEM. of 5–8 observations. *P <0.05, NS 398-treated vs. vehicle-treated.

Celecoxib at doses between 0.01 mg kg^-1 and 3 mg kg^-1 inhibited by more than 80% the increases in plasma 6 keto-PGF₁ levels caused by LPS infusion for 6 h (Fig. 4A ).At the lowest dose tested, however, celecoxib was without effect against the rapid elevations in plasma 6 keto-PGF₁ caused by bolus injection of arachidonic acid (Fig. 4B ).

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of celecoxib on the concentration of 6 keto-PGF1 in the plasma of anesthetized rats treated with LPS (0.2 mg kg-1 h-1) at t = 6 h. A) In rats receiving LPS alone (n=8). B) In rats 1 min after injection of a bolus of arachidonic acid (3 mg kg-1, i.v.). Results are expressed as mean ±SEM of 5–6 observations. *P <0.05 celecoxib-treated vs. vehicle-treated.

Effects of COX-2-selective inhibitors in naive animals
NS 398 at 3 and 10 mg kg^-1 and celecoxib at doses between 0.3 mg kg^-1 and 3 mg kg^-1 were without significant effect on the plasma 6-keto-PGF₁ concentration after arachidonic acid bolus in naive animals (Table 2) .

Cardiovascular parameters
LPS infusion (0.2 mg kg^-1 h^-1) for 6 h produced a significant fall in MAP between 2 h and 6 h, in comparison with time-matched controls (Table 3 ).Treatment with diclofenac or the COX-2-selective inhibitors were without effect on the fall in MAP between 2 and 6 h (Table 3) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Here we demonstrate that the COX-2-dependent production of prostanoids in vivo is powerfully regulated by the supply of arachidonic acid from either exogenous or endogenous sources. Thus, there is a profound synergy between proinflammatory mediators: 1 µmol bradykinin induces the release of more 6 keto-PGF₁ in a COX-2-induced rat than does 100 µmol bradykinin in a naive animal. Our second important observation is that increasing the supply of arachidonic acid in vivo reduces the effectiveness of NSAIDs as inhibitors of prostanoid production. This clearly indicates that at sites of inflammation, where phospholipase A₂ activity and hence free arachidonic acid levels are highest, competitively acting NSAIDs must be present in higher concentrations than other systemic sites to produce equivalent reductions in prostanoid formation.

It seems clear that an increase in the expression of COX-2 was responsible for the exaggerated production of prostanoids accumulating in the plasma and after arachidonic acid administration in LPS-treated rats. First, the time course of the increase in basal and arachidonic acid-stimulated 6 keto-PGF₁ formation was consistent with the time course of COX-2 induction (18–21). Second, the selective COX-2 inhibitors NS-398 (16) and celecoxib (17) reduced both basal and arachidonic acid-stimulated 6 keto-PGF₁ formation in LPS-treated rats but not in naive animals. Third, immunohistochemical analysis showed that treatment with LPS greatly increased the expression of COX-2 in blood vessels throughout the rats. In LPS-treated animals, the formation of 6 keto-PGF₁ after arachidonic acid bolus did not increase after 4 h, whereas plasma levels of 6 keto-PGF₁ increased the most between 4 and 6 h. These observations are consistent with induction of COX-2 protein being maximal within 4 h, after which time the activity of this COX-2 was reflected by the increase in plasma 6 keto-PGF₁ levels. However, at both 4 and 6 h the basal formation of 6 keto-PGF₁ represented only a fraction of the potential synthetic capacity of COX-2 (20). Supplying arachidonic acid revealed this synthetic capacity had increased by more than 140-fold. Clearly, arachidonic acid supplied as a bolus is not a direct model for the release of arachidonic acid from endogenous stores. To this end, we treated animals with bradykinin, which can release arachidonic acid from endogenous stores through the activation of PLA₂ (22). As with exogenous arachidonic acid, the formation of 6 keto-PGF₁ after bolus injection of bradykinin was greatly increased (>120-fold) after infusion of LPS.

These observations lead to the inevitable conclusion that COX-2 induction alone, irrespective of its degree, does not automatically result in an exaggerated production of prostanoids. An increase in the supply of arachidonic acid is also needed. This increased supply can obviously follow the stimulation of phospholipase A₂ by local mediators such as bradykinin, as has been demonstrated in the isolated kidney (23). Similarly, arachidonic acid can be supplied exogenously, as has been demonstrated also in the isolated kidney (19) and in vivo (20). The release of prostanoids dependent on the activity of COX-2 must then be seen as a two-component event: induction of COX-2, event one, and supply of arachidonic acid, event two. Both events are essential. Indeed, it has recently been demonstrated (24) that in essential fatty acid-deficient rats, a reduction in arachidonic acid levels was associated with a reduction in paw swelling after injection of Mycobacterium butyricum.

Having established that the supply of arachidonic acid is a key regulator of the activity of COX-2 in vivo, we investigated the impact of this on the potencies of NSAID drugs. As the supply of arachidonic acid influences the potencies of NSAIDs in vitro (15, 25), we reasoned it would similarly influence their potencies in vivo. This was indeed the case, as typified by the `classical' NSAID, diclofenac. Thus, though diclofenac at lower doses effectively inhibited the increase in circulating 6-keto-PGF₁ caused by LPS infusion, it was without effect against the formation of 6 keto-PGF₁ that followed the bolus injection of arachidonic acid. Higher doses were required to inhibit this latter response. This effect of COX-2 substrate was not limited to the diclofenac. The selective COX-2 inhibitors celecoxib (17), and especially NS-398 (26), were less able to reduce 6 keto-PGF₁ production when free arachidonic acid levels were high.

Our observations have clear implications for the therapeutic use of NSAIDs. In local sites of inflammation (e.g., in arthritic joints), cellular levels of free arachidonic acid will be higher than those found systemically. This increased supply of arachidonic acid will blunt the ability of NSAIDs to inhibit prostanoid formation at these inflamed sites. To achieve good inhibition at inflammatory sites, NSAIDs must be applied at doses that will produce at all other sites containing lower levels of free arachidonic acid even greater inhibition of prostanoid production. As many of these other sites contain COX-1, which is responsible for the generation of physiologically important prostanoids, the application of high doses of non-COX-1/2-selective or poorly COX-2-selective NSAIDs frequently produces unwanted side effects. What about COX-2-selective compounds? At doses of 0.1 mg kg^-1 or greater, celecoxib effectively inhibited both the LPS-induced increases in circulating 6 keto-PGF₁ levels and the large increase in plasma 6 keto-PGF₁ that followed bolus injection of arachidonic acid. This agrees with the efficacy of celecoxib as an inhibitor of the chronic inflammation associated with adjuvant arthritis in the rat (ED₅₀ 0.37 mg^-1 kg^-1 day) (17). Celecoxib is much less effective as an analgesic agent in the Hargreaves hyperalgesia model (ED₅₀ 34.5 mg^-1 kg^-1) (17). Indeed, the more than 100-fold difference between the doses of celecoxib effective in producing analgesia and in inhibiting 6 keto-PGF₁ formation (even in the presence of high free arachidonic acid levels) suggests that there may be roles for COX-1 products as mediators of hyperalgesia.

In summary, our data lead to two important conclusions. First, the exaggerated production of prostanoids that often follows COX-2 induction is regulated by the supply of arachidonic acid. Increasing prostanoid production in inflammation therefore requires a two-component response: increased COX-2 expression and increased arachidonic acid supply. Second, the supply of arachidonic acid to COX-2 determines the effectiveness of NSAIDs. Particularly at sites of inflammation, NSAIDs will inhibit prostanoid production in a manner that is inversely related to arachidonic acid supply. This observation was true both for `classical NSAIDs' and for the newer COX-2-selective inhibitors NS398 and celecoxib. NSAIDs and COX-2-selective inhibitors will, therefore, generally be less effective at more inflamed sites where phospholipase A₂ activity is increased (27). This provides a rationale for the very high doses of NSAIDs required in human conditions such as rheumatoid arthritis and for the discrepancies between in vitro tests of COX selectivity and in vivo efficacy.

	ACKNOWLEDGMENTS

 
L.C.H. is the recipient of a British Heart Foundation Ph.D. studentship (FS/97013), J.A.M. is a Wellcome Career Development Fellow, and T.D.W. holds a British Heart Foundation Lectureship (BS/95003). This work was also supported by a grant from Boehringer Ingelheim, Germany. The authors would like to thank Mr. Lorne Dye for his assistance with the immunohistochemistry.

	FOOTNOTES

 
¹ Correspondence: Vascular Inflammation, The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, U.K. E-mail: t.d.warner{at}mds.qmw.ac.uk

² Abbreviations: COX, cyclo-oxygenase; COX-1, constitutive isoform; COX-2, inducible isoform; cPLA₂, cytosolic phospholipase A₂; LPS, lipopolysaccharide; MAP, mean arterial pressure; NSAIDs, nonsteroidal antiinflammatory drugs; PG, prostaglandin; sPLA₂, secretory PLA₂;.

Received for publication August 20, 1998. Revision received October 14, 1998.

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