Editorial

COX-3: Fact or fancy?

Keywords: Cyclooxygenase; NSAID; Acetaminophen; Paracetamol

The identification of cyclooxygenase 2 (COX-2) in theearly 1990s was a turning point in the quest for explanationsto the effects of prostaglandins and nonsteroidal antiinflam-matory drugs (NSAIDs) [1]. Cyclooxygenases convertarachidonic acid released by membrane phospholipids intoprostaglandins. Whereas the first known isoform, COX-1, isconstitutively expressed and found throughout the body,COX-2 is induced during the inflammatory process. Thesedifferences suggested that the ability of NSAIDs and aspirinto induce gastric ulcers was related to decreased productionof gastroprotective prostaglandins due to COX-1 inhibition.This hypothesis was confirmed when newly developed drugsthat specifically inhibited COX-2 were shown to have the rateof gastrointestinal bleeding as compared to conventionalNSAIDs [2,3]. However, the “good” COX-1 versus “bad”COX-2 dichotomy has proved overly simplistic. COX-2 hasbeen shown to exert many physiological effects, for instancein regulating vascular and renal blood flows [4]. The identi-fication of COX-2 was not expected to increase the effective-ness of NSAID therapy, as conventional NSAIDs inhibitedboth COX-1 and COX-2.

The hypothesis that a third isoform, COX-3, may contrib-ute to resolve the inflammatory process was put forward inthe early 2000s [5]. In a mouse model of acute inflammation(carrageen injection into a subcutaneous air pouch), the pros-taglandin types produced during inflammation changed overtime. Initially, PGE2 was released in large amounts. Admin-istration of a specific COX-2 inhibitor stopped PGE2 produc-tion, and the inflammation resolved. In contrast, later on,when the inflammatory process was subsiding spontane-ously, PGD2 production increased and PGE2 productionstopped [6]. This led to the suggestion that a COX-3 isoformmay cause the late production of PGD2. However, no soundevidence supporting this hypothesis has been reported todate. Attention is turning to enzymes downstream from thecyclooxygenases that isomerize PGH2 (a COX product) intoeffector prostaglandins (prostaglandin E synthetase, prostag-landin D synthetase, etc...). The resolvins are recently iden-tified lipidic mediators derived from omega-3 fatty acids viaa COX-dependent process. Resolvins are produced in thepresence of aspirin and contribute to resolve the inflamma-

tory process [7]. It has been suggested that resolvins may beproduced by a COX-3 [8].

The COX-3 issue was also raised by researchers studyingacetaminophen (paracetamol). The mechanisms by whichthis agent exerts its potent analgesic and antipyretic effectsremain enigmatic [9]. In several experimental models ofinflammation, acetaminophen has virtually no antiinflamma-tory effects. Furthermore, in vitro, acetaminophen fails toinhibit COX in the usual concentrations, with the likelyexception of cerebral COX [10]. This raises the possibilitythat brain tissue may contain a COX-3 enzyme sensitive toacetaminophen and involved in the genesis of fever and in thecentral mechanisms responsible for pain. Supporting thishypothesis are several recent studies indicating a crucial rolefor COX in pain. Early studies established that prostaglan-dins produced at sites of inflammation sensitized the nocice-ptive fibers, thereby exacerbating pain sensations, while con-tributing to the classic triad of redness, warmth, and swelling.More recently, COX activity was identified in the spinal cord;although constitutively expressed, this enzyme was overpro-duced at sites of inflammation, where it exacerbated painsensations [11]. These data are consistent with the existenceof a COX-3 enzyme in the spinal cord. However, this spinalcord COX activity is related, not to an additional COXisoform, but to an acetaminophen-sensitive form of COX-2[12]. The analgesic effect of acetaminophen in inflammatoryconditions may be ascribable, in part at least, to inhibition ofthis spinal cord COX-2.

However, the acetaminophen-sensitive COX-3 hypothesishas gained new impetus from the discovery of a COX-1variant (vCOX-1) in dog cerebral cortex [13]. The authorsused the name “COX-3” to designate this variant. This nameis inappropriate, as the variant is not produced by a distinctgene but by alternate splicing of the COX-1 gene. The onlydifference is retention in COX-3 of intron 1 of the COX-1gene. COX-3, which contributes about 5% of total COX-1, isa 65-kDa membrane protein whose cyclooxygenase activityis about 80% lower than that of COX-1. This suggests thatintron 1 retention may modify the conformation of the activesite. Preferential expression of COX-3 in the brain and hearthas been reported [13,14]. In addition to COX-3, two shortervariants without cyclooxygenase activity have been identi-

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fied, PCOX-1a and PCOX-1b (Fig. 1). The function of thesetwo inactive, truncated COX-1 variants is unknown. Thedistinctive characteristic of COX-3 as compared to COX-1and COX-2 is its greater sensitivity to acetaminophen. TheCI50 (enzyme concentration associated with 50% inhibitionof substrate production) is 460 µM for COX-3 but far exceeds1000 µM for COX-1 and COX-2. This corroborates thehypothesis that an acetaminophen-sensitive COX differentfrom COX-1 and COX-2 exists, mainly within the brain, andmay explain the central effects of acetaminophen.

However, many unanswered questions remain. The weaklevel of COX-3 inhibition induced by acetaminophen prob-ably does not occur with standard oral dosages of acetami-

nophen (0.5–1 g/day) [15]. In addition, COX-3 is found inthe brain cortex, whereas body temperature regulation occursin the hypothalamus, which is therefore the likely site ofaction of antipyretic agents (including acetaminophen).Thus, there is room for other hypotheses on the mechanismsof action of acetaminophen. Finally, whether COX-3 exists inhumans remains unproven. A recent sequencing study of thehuman COX-1 gene found that the first intron contained anadditional nucleotide, as compared to canine COX-3. Thisdifference may lead to a frame shift precluding translationinto a functional protein [16,17]. Thus, the results obtained indogs may not apply to humans.

In sum, there is little support for existence of an additionalcyclooxygenase isoform encoded by its own gene. However,evidence that the sensitivity of COX-1 and COX-2 to ac-etaminophen varies across body sites suggests new explana-tions to the pain associated with inflammatory processes[18]. Thus, the constitutive COX-1 and inducible COX-2dichotomy is giving way to a more complex picture thatincludes several COX-1 and COX-2 variants, each actingwithin a specialized pathway, thereby producing a broadarray of substrates with multiple and, at times, oppositeeffects according to the site and time of production (Fig. 2).In addition, the sensitivity of these variants to NSAIDs andacetaminophen may vary according to the body site andenvironment. This new concept should help researchers totease apart the antiinflammatory and analgesic properties ofacetaminophen and NSAIDs, both in the conventional and inthe COX-2 inhibitor categories. This may lead eventually tothe development of new drugs that target the desired path-way, thereby providing more precise and more effectivetreatments.

Fig. 1. Structure of the COX-1 variants produced by alternate splicing. Notethe absence of the catalytic domain in PCOX1a and PCOX-1b (derived from[19]).

Fig. 2. Hypotheses on the mechanisms of action of acetaminophen. In addition to the ubiquitous constitutively expressed COX-1 and to the inducible COX-2,a COX-1 variant and a new catalytic COX-2 variant have been found in the central nervous system. These two enzymes may be the main targets ofacetaminophen. The production of components with antiinflammatory properties under the effect of COX activities, either spontaneously or with aspirin, maycontribute to the resolution of inflammation. vCOX-1: COX-1 variant; COX-2b: COX-2 with a different catalytic activity from conventional COX-2; COX-1rand COX-2-r: COX involved in the resolution of inflammation.

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References

[1] Berenbaum F. Cyclooxygenases and cyclooxygenase inhibitors: is anew class of drugs in the making? Rev Rhum Engl Ed 1998;65:634–40.

[2] Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R,Davis B, et al. Comparison of upper gastrointestinal toxicity of rofe-coxib and naproxen in patients with rheumatoid arthritis. N Engl JMed 2000;343:1520–8.

[3] Silverstein FE, Faich G, Goldstein JL, Simon LS, Pincus T, Whel-ton A, et al. Gastrointestinal toxicity with celecoxib vs. nonsteroidalanti-inflammatory drugs for osteoarthritis and rheumatoid arthritis:the CLASS study: a randomized controlled trial. JAMA 2000;284:1247–55.

[4] Crofford LJ. Is there a place for non-selective NSAIDs in the treat-ment of arthritis? Joint Bone Spine 2002;69:4–7.

[5] Willoughby DA, Moore AR, Colville-Nash PR. COX-1, COX-2, andCOX-3 and the future treatment of chronic inflammatory disease.Lancet 2000;355:646–8.

[6] Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ,Willoughby DA. Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med 1999;5:698–701.

[7] Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mir-ick G, et al. Resolvins: a family of bioactive products of omega-3 fattyacid transformation circuits initiated by aspirin treatment that counterproinflammation signals. J Exp Med 2002;196:1025–37.

[8] Schwab JM, Schluesener HJ, Meyermann R, Serhan CN. COX-3 theenzyme and the concept: steps towards highly specialized pathwaysand precision therapeutics? Prostaglandins Leukot Essent Fatty Acids2003;69:339–43.

[9] Botting RM. Mechanism of action of acetaminophen: is there acyclooxygenase 3? Clin Infect Dis 2000;31(Suppl 5):S202–10.

[10] Flower RJ, Vane JR. Inhibition of prostaglandin synthetase in brainexplains the anti-pyretic activity of paracetamol (4-acetamidophenol).Nature 1972;240:410–1.

[11] Samad TA, Moore KA, Sapirstein A, Billet S, Allchorne A,Poole S, et al. Interleukin-1beta-mediated induction of Cox-2 in theCNS contributes to inflammatory pain hypersensitivity. Nature 2001;410:471–5.

[12] Simmons DL, Botting RM, Robertson PM, Madsen ML, Vane JR.Induction of an acetaminophen-sensitive cyclooxygenase withreduced sensitivity to nonsteroid antiinflammatory drugs. Proc NatlAcad Sci USA 1999;96:3275–80.

[13] Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J,Elton TS, et al. COX-3, a cyclooxygenase-1 variant inhibited byacetaminophen and other analgesic/antipyretic drugs: cloning, struc-ture, and expression. Proc Natl Acad Sci USA 2002;99:13926–31.

[14] Shaftel SS, Olschowka JA, Hurley SD, Moore AH, O’Banion MK.COX-3: a splice variant of cyclooxygenase-1 in mouse neural tissueand cells. Brain Res Mol Brain Res 2003;119:213–5.

[15] Schwab JM, Schluesener HJ, Laufer S. COX-3: just another COX orthe solitary elusive target of paracetamol? Lancet 2003;361:981–2.

[16] Schwab JM, Beiter T, Linder JU, Laufer S, Schulz JE, Meyer-mann R, et al. COX-3—a virtual pain target in humans? FASEB J2003;1715:2174–5.

[17] Dinchuk JE, Liu RQ, Trzaskos JM. COX-3: in the wrong frame inmind. Immunol Lett 2003;86(1):121.

[18] Warner TD, Mitchell JA. Cyclooxygenase-3 (COX-3): filling in thegaps toward a COX continuum? Proc Natl Acad Sci USA 2002;99:13371–3.

[19] Simmons D. Variants of cyclooxygenase-1 and their roles in medicine.Thromb Res 2003;110:265–8.

Francis BerenbaumRheumatology department, Hôpital Saint Antoine et UMR

CNRS/Paris 6 Pierre et Marie Curie, 184, rue du faubourgSaint-Antoine, 75012 Paris, France

E-mail address: francis.berenbaum@sat.ap-hop-paris.fr(F. Berenbaum).

Received 5 January 2004; accepted 4 February 2004

Available online 22 March 2004

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