REVIEWS
`
`THE DEVELOPMENT OF COX2
`
`INHIBITORS
`
`Ron‘ I. Flower
`
`Aspirin, arguably the worlds favourite drug. has been around since the late nineteenth century.
`but it wasn't until the late 1970s that its ability to inhibit prostaglandin production by the
`cyolooxygonasa enzyme was identified as the basis of its therapeutic action. Early hints of a
`second form of the cyclooxygenase that was ditiarentially sensitive to other aspirin-like drugs
`ultimately ushered in an exciting era of drug disoovery. culminating In the introduction of an
`entirely new generation of anti-inflamrnatories. This article reviews the story of this discovery and
`looks at the future of cyolooxygenaso pharmacoiogy.
`
`ISL)Z)'.\l.E t1.50Ii.‘lZYhiEi
`Urn: of several forms «if an
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`umr: l‘E:lL‘1i||1\lll.II dilfer from
`each nlher in such properties.
`as substrate aflinitv and
`nnucintuni rain of
`enz_\'n1c- s1.Ibtilt'JlL‘ reanioii.
`ANTIPYRETIC
`IJi.-.~cr1iu.-»- ihe rciwcr-ieupprmiuwc
`:n.lirit!' ui'ai.irzqt.
`
`Dcp:tr'trtte:iI trffliixiteirrirai
`Piianrmcoiaggir.
`Tire Wiifinrrl Hitnrey
`RL-sm rcir Iirsritiiia,
`Queen ."l‘Inry Uriiversiry
`aftoririori.
`Charierirauaefiuiiriret
`London ECIM 630. UK.
`9-.-mu'i:
`i‘.j.fl01tTT{'PtIldS.qF!illl.fl€.it-it
`tini-.|IJ.it:I.Wnrtl|t1_1-I
`
`Ti?
`The league table listing the ‘top 20' drugs includes
`rofecox ih I \-’imtx,i and cell.-rcnt ilt {Celehrex 1,
`two
`iJt.hjl:Ii[ot‘s of the pruslaglrlridiri -l'on'nJ'ng Cycluuxygenase
`(COX) ii-"Io. 1:, which between them cornnianded sales
`in excess of US 54 billion in the year 2000 met. 1|. This
`statistic ntight seem surprising —— after all. the thera-
`peutic use of COX irihihitors has a venerable history
`dating back to the introduction of aspirin in 1898 (or
`even earlier it‘ lite use of saliqrlatc-containing plant
`extracts is included} and, since then, the field has a
`record of almost continuous clevcloprneri l. The iflrltis
`saw the introduction of pl'|ei1)'llJIJl£i2Dl‘lE. the feiianiates
`appeared in the 19505. indomethacin in the 19t5i.is. the
`proprionatcsiri the i9i'iis and the coricams in Lhe i9iitis.
`With such a long reconl ofdrug discovery in the area
`and such a vast range of drugs to choose from. one
`might be forgiven for thinking that the wellspring of
`chemical innovation that nurtured the field so effi—
`cicntlv over the past CEfltI.1r_\'l'l‘il.l§l have long since run
`dry — or at least begun to falter—and that the current
`Inttrkel would liave little room for new versions of what
`would seem to be at rather tired and \vell—mor11 formula.
`However. the discovery in the early 1990s of ii second
`COX isozviitr revitalized the field and stimulated a hunt
`for new and selective isoforra inhibitors. This culmi-
`nated in the introduction ofViox.\ and Celehrett in
`Europe and North A|1\e'r'ir;:I within a mere ten years. and.
`at the same time, brought a Fresh perspective on the
`
`unusual therapeutic profile ofseveral existing non-
`steroidal anti-ii1ii‘3Tl'Ii'l‘Iu1lt‘|l'}‘d1'l1g5iNSr\lDSil. But we are
`getting ahead oiuurselves —how did this idea ofihe
`second isoform come ubotil in the firs: place and what
`are the therapeutic advalitages of these new inhibitors
`over the [1'li1l1)t older drugs which. after all. have seen
`valiant clinical service over the decades? To answer these
`questions, we must return to the 19?0s when much of
`the seminal work on COX inhibition was publislied.
`
`Earif work
`Although the NSAID5 do not reverse the course oi"
`systemic diseases such as arthritis. they form the n1ain-
`slay Syrnptonwtic treatment‘ ofrnany inflamniatory
`t.li50]'Lll’1'!5ill‘l(.l soit—tissue injuries. Approximately Si}
`NSAID preparations are listed in Mcriiliiy iridcx of
`i't«!.-_=n‘i'mi Speriaiitiesand. as a class. these are among the
`most commonly prescribed drugs.
`in the United
`liingdoni. for cxarnplc. recent data [ 1999.1 indicate that
`[35 million prcscriptioits are written for NSA].Ds each
`yearnl it cool of Elm rtiillioti —ancl Ll']l3 figure doeslfl
`take into ncco tint the over-the-counter sales. which are
`considerable. Aspirin itself is still oorisumed in prodi-
`gious amounts around the \\01'l(l and new tises an: con-
`tinually being found for this drug.
`NBA IDs are sometimes lcnown as the aspirin-like
`drugs hecause they liave an activity profile that is
`broadly similar to that of tispit-in. That is,tl1ey all possess
`
`DRUG DlS€U‘i'EfiV
`Ni\'i'i.JIi.E llE\'|I:'\\".\'
`\'LILL~M1-;_? Maittillliiiifl 11'!
`
`
`Page 1 of 13
`
`® 2053 Nature Publishing Group
`
`Patent Owner Est. 2030
`CFAD v. POZBI1
`IF'R2Ui5-O1?‘l8
`
`

`
`
`
`Mitogens. oytotones and many other
`slirnus acting mrough the MAPK oathway
`
`HgLno1tAnovenriewo!pI'ustag|and]n syI11ha5lsandrneIabo|Isn'I.tnlha0n,t treetattyacicts
`sutth asaracltidonale can be totmad from several sotiroes. although phospholrptd-botnzl
`aI'anhrdcmateBpI'Gl13lJ’9l"‘3"‘05l5lEIf1lfir:aI1l pool F'hosptvuhoeees.est:ecislvvcnosctnc
`tttxvstattttpiase A, [GpLKi}.atElllQl5y-r8gtJ‘al9tIEl12flTES{b)'ll1E MAPktnase IMAPKJ pamwayl that
`lbemeamdfidm-atowhid1ottmtramfonndbyuncydowogerosetCO>Qommex.fle
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`by a I‘poxy-gaaase racttm. followed by a COX I'EtflCIiCX‘|. This generates PGGz.a15—h5dro—p£roxi:B
`prtstag1mdhuatenodmo1mPGFg.dnDorrespomngItydrmqoomct.Bomoftese
`irttoltnacflalas are shmtvecl but mayhave indeparncient btologlcaiactivity. However. in Hunt (mes
`itelxatyetotearvmotttertnisrxtepeetiawtadnityissiga-ificartmwio.Amntabottayofarinmes
`transforrr-PGI-1,:moavanetyofuoo1ncts.SoTooffl'eseeru)mes.suehastt1u*nbo:enefi>¢
`synthaseand tart:-eltaqottlln [F’Gl;J8yl'|‘lh85B'. §1ow rranrocl tissue Iorrallzalion t‘lorexan1pte.TJ(
`wrlthasae in otatelelsanct PGL_, 5yrrfl1amI'n altar endolhelml. Other en23.nIwzt.L-‘u<;l1a5lI\tt
`mdDua0fldBmdudaso5afiBonEraaBs.afiquflemddydnnbmed.aHug1haxmmsaemw
`ca1nenmcoufwmW\grmanwmoysflrnm.mamateast.Iheydq3aomdesPGG:mdPGFh
`can aim decay E}.lZl'll3'lm\t§y (53 to PGFW PGEE aid PGDE The 1l prnrtuuts. TKA. and
`PGII. decavspontarteotnty to that respective rxetaive metabolites. TXB2 and B-toato PGI-',_. ‘fine
`taotogncaiacfivrtyotu-ueoaterorostaglat-reins:seurtaI|edfanowi1gun1akentoce!s,byase4'iasor
`mefibctic enzymes H\atarEnre5ar1tin5Dmetts5t.Ies{f0tenarrIr)le.lt1téhJItgialtugtitxJr\Denlratit:|1s.
`tnaotivte metahontasottt-ueseorostat1octstndo@ocaa'tmc;t1ain st1oner~i1'igtespeo'a1|yfntheHvan
`priottosecIetaottinfl'Ieurine,tm~hIt:l1e'stiT1ateSoltotalbadyorc:stagLarIdintuT\ovtarcat'IbeI'oade
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`ammgetDmacfimarflpasshemEmWpHflg2§a.mdTXAfibe(2LEeui|BImpGfla1lffieIn
`ptatetalaggregation. Colour coda led. precursors: orange. trrlennedates: yellow.
`rsrootaglancinsthatmedatemostotfl1ebbtogicatac1h+tyofmiss3otern:gmen.troofinewhrgetv
`inacfiiemetabolfles:bruM1.HormtaUfltesmatarea:ooetcdpnnanhm#ernne
`
`analgesic. anti-inflammatory and aorneraenc properties
`to some degree. and produce characteristic side effects,
`including gastric intolerance and depression of blood
`clotting through inhibitory action on platelet fiinction.
`A5 a group. the NSAIDs are structurally diverse, with
`most lbut not all} being carboxylic acids ti’-IG-.21. The
`main question. front the phar1'nacologist's point of view.
`was how these apparclttly disparate therapeutic and side
`cfi’tcLs were mecltnnisl ically linked.
`There were several early sttggestinns titers 2.3]. but
`the real breakdmough came in 19?}. Vane tells us‘ that
`the idea that the aspir-in—like drugs blocked the conver-
`sion of substrate arachidon ic acid to prostaglandins
`came to him while reviewing experiments in which
`aspirin blocked the release of‘rabbit aorta contracting
`substance’ {RC5} from guinea—pig and dog lu ng.
`Believing that RC5 was an interrnr.-dian: in prostaglandin
`synthesis. he wrote “. . .a logical corollary was that
`aspirin might well be blocking the synthesis of
`proataglandin5".r\ quartet of papers appeared that year
`from Value and I‘rte11'Ihers of h is group. showing that
`aspirin itself. indonterhacin and (less effectively) sodium
`salicylarc blocked prostaglandin synthesis in a cell-free
`system-" and in isolated perfused spleen of dogs“. in
`humans. therapeutic doses of aspirin taken by volun-
`teers reduced prostagiandin generation by aggregating
`platelets ex viva’ or in seminal plasma samples collected
`during the course of the treatment‘. The overall message
`was clear — at least some NS:\IDs were able to prevent
`the generation of prostaglandins bvdirect action on the
`CDX el1z‘y'[1'IE.:I nd did so in humans in clinical doses.
`But how was this linked to their therapeutic actions?
`The Tale waits and early l97tJs had seelt an explosion
`of interest in the biology ofthc Pt'05[agl£ll'tdl..I'lti. It had
`already been shown that prostaglandins a re generated
`during platelet agregation to produce fever. rn’PEtw.GE£ta
`and inflammation (reviewed by \'\'ilJjs‘J.Pmstap,landi11s
`had also been detected in gastric mucosa and been
`shown to inhibit ulcer formation in rodents“. In other
`\-vords,the ability of NSAlDs' to block COX provided the
`much sought-after link between the therapeutic and side
`effects ofthcsc drugs.
`Over the next coupleofyears, awcral other important
`Fmdin 35 em erged. Significantly. it was Shown that the
`entire garnut ofNSAlDs inhibit ODX at concentrations
`well within their therapeutic plasma range and that the
`overall order of potency corresponded to their thera-
`peutic activity". Other types ofanti- inflarnmalories,
`such as the glucocorticoids and thc 5‘I:I—cal.lccl disease-
`ntodifying drugs, were inactive in these cell-free assays.
`providing further evidence for the specificity of the efi‘ect.
`Using Enrmrlousnxc pairs of NSMD5. such as n:tprmre11'1.
`an exquisite correlation was observed between the anti-
`inflatnmatorvand anti—COX activity. and many more
`studies confirmed the notion that this is a fundamental
`rncchanisin of this class of drug {reviewed in
`:5}.
`By 19.74. \iane's concept was firmly cstahl ished.
`Rt.‘:ie‘dI'I:l‘lt‘rt~' were qulcl: to seize upon the Fact that
`NSMDS could he used to probe the functions of
`prostanoids in physiology and pathology. thereby
`opening an entirely new chapter in cicosanoid research.
`
`um I MJ\ttL‘1I Ions I Vt‘ILUME 1 j' ’
`
`tnwtmrlalI.lr2.|:|:m|,fre\r|e'n|'!.it‘lI'l.Igtil5:
`
`Page 2 °f 13
`
`E ZIIOS Nature Publishing Group
`
`Patent Owner EH. 2030
`CFAD V. Pozen
`lPR2D15-D1T1B
`
`

`
`5”“
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`REVIEWS
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`Figure 2 | chemical structures of NSAIDS and related compounds. Structures of some 'cia.ssicai' NSAIDS. including
`repreaeiitalive salioylalas. pyrazoiones. lonarnales, proprionates. oxicarns and indomethacin. Note the general presence ol
`a calboxyfic-acid moiety.
`
`Parenthetically. one might add that the phannaceutical
`industry now possessed, probably for the first time.a
`simple and robust in wire teclinique to screen com-
`potmds for putative anti- inflammatory activity. This
`in itself was a significant advance, and the number of
`chemical abstracts dealing with potential inhibitors of
`the COX enzyme rose markedly. with more than 2.500
`per year recorded within a decade of these ideas
`taking hold.
`But right from the beginning ofthe story, several
`anomalies were noted. The most relevant concerns
`paracetamol {acetarninophen} — another hugely popu-
`lar drug that was also introduced in the l89l}s(a]tl1ou@
`it was not in common use until some 40 years later}. As
`for all other aspirin-like drugs. paracetamol possessed
`antipyretic and analgesic activity but. unlike most, had
`little anti-inilamntalory activity and caused virtually no
`gastric or platelet side effects. Apparently in accord with
`its therapeutic profile, paracetamol was found to ltavt: a
`diliercnt pattern of inhibitory activity. being more effec-
`tive against brain COX than enzyrne prepared from
`peripheral Iissues such as the spleen “. A1 Ihc time, th is
`was put forward as at putative explanation for the selec-
`tivity of its therapeutic action. and the idea that there are
`several forms of the enzyme was formulated. Wide
`
`variations in the inhibitory potency of indontethacin
`against COX enzymes prepared from a range of tissues
`was subsequently reported "’ and the isoenzyme idea
`was further elaborated in several 1'ev'iews"'”’.
`
`The development of the field
`Despite the early indications for alternative forins of
`COX. little concrete evidence emerged to support this
`idea for several years. The structural features of COX.
`which ltas dual ltydro-peroxidase and cyclooxygenast:
`activity", were not well understood. \-\"ith the Imtable
`exception of bovine or ovine seminal vesicles. COX is
`ttsu:tlly expressed in tissues in low abundance. As a
`dirneric inc-mbrane—bound protein, it posed many
`challenges to purification and was not sequenced until
`I98-S I RE:-‘S 1s.i9J. Other factors contpiicating the inter-
`pretation of potency differences included the wide
`variations in assay conditions that were used by difi'er—
`enl groups — which is still .1 problent today —- as well
`as differences in the kinetics ofthe COX inltibitors.
`some of which produced ‘competitive reversible‘ inhi-
`lsitinn. wltereas others exltibiled unustinl inhibitory
`effects such as the ‘contpetitive non—1'eversibIe' mecha-
`nism that is observed in the case of l]1ElOl'l’l£'tl'Ii]Cll'I2°.
`The diseotieryi”-’ that. alone among the group. aspirin
`
`HYPERALGESIA
`An alvnornnl state ofitwreami
`sc:n.i1irlty to painful sllmuli.
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`moleu.I.1a|'.\trur.lure.~ are minor
`imagn ul c:|t‘h min.-r.
`
`l\'.-\'i'|.lRl-I RE\-'li_’\\'S I DRUG DISFOVERY
`
`voi.L-'.\iE 1|:\1i-\at:H zero [151
`
`Page 3 of 13
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`
`REVIEWS
`
`CONTRALATERA.L
`On or affecting the opposite side.
`HOUSEKEEPING GENE
`A gene that is usually expressed
`at a fairly constant rate in cells
`as it subserves some constant
`physiological requirement. By
`comparison, an inducible gene is
`one that normally appears at a
`very specific lime and in
`response to a specific stimulus.
`
`irreversibly acetylated a serine residue (SerS30) within
`the COX active site to produce its effects, reinforced
`the opinion held by many workers at that time that the
`NSAlDs were a chemically heterogeneous group of
`drugs with widely differing modes of inhibitory action.
`ln short, it seemed at least possible that some of these
`differences in inhibitor potency could be the result of
`factors other than the presence of isozymes.
`Meanwhile, other apparently unrelated investigations
`were to provide the backdrop for future dramatic revela-
`tions. Using a model of rabbit kidney inflammation
`induced by ligation of the urethra, Needleman’s group
`observed” that the affected, but not the CONTRALATERAL,
`organ unexpectedly developed an enormous capacity to
`generate prostaglandins. 1n the following year“, the
`group showed that this increase was due to de novo
`synthesis of fresh enzyme, although no suggestion was
`made at this time that the new enzyme was a variant
`form. Nevertheless, this theme was pursued by the
`Needleman group over the next few years and, in 1982, a
`paper appeared that suggested the presence of two dis-
`tinct forms of COX in brain tissue with differing sensitivi-
`ties to indomethacin”. Other studies in gastrointestinal
`tissue were also supportive of the selectivity of action of
`NSAIDS in dilferent tissues“.
`The problem was taken up again by several labora-
`tories towards the end of the 19805. Treatment of vas-
`cular smooth muscle cells with epidermal growth factor
`(EGF)27 or fibroblasts, and monocytes with pro-
`inflammatory stimuli such as interleukin-11”’ or
`lipopolysaccharide”, induced COXmRNA and de novo
`synthesis of enzyme which, when partially sequenced,
`seemed to be identical to the bovine seminal vesicle
`COX. In one paper“, Needleman’s group wrote “Clearly,
`those putative enzyme pools may arise as different gene
`products, possibly through the expression of different
`COX genes. . . ”. Interestingly, glucocorticoids inhibited
`the induction of this new COX protein without altering
`the amount of enzyme that was constitutively present
`in cells”. Elsewhere, further evidence emerged indi-
`cating different forms of the enzyme. For example,
`Wong and Richards, using immunological techniques,
`reported two isoforms in the rat ovary, one of which
`was regulated by hormones”.
`
`The discovery of COX2
`Paradoxically, the next significant step forward came in
`1991 from workers in an entirely diflerent field. While
`investigating the expression of early-response genes in
`fibroblasts transformed with Rous sarcoma virus,
`Simmons and his colleagues" identified a novel mRNA
`transcript that coded for a protein that had a high
`sequence similarity, but was not identical, to the seminal
`vesicle COX enzyme. The suggestion was that a COX
`isozyme had been discovered. Independent and simulta-
`neous confirmation of this exciting finding came from
`the laboratory of Herschman and colleagues, who dis-
`covered a novel cDNA species encoding a protein with a
`predicted structure similar to COX1 while studying
`phorbol—ester-induced genes in Swiss 3T3 cells”. The
`same laboratory subsequently showed that this gene
`
`product was indeed a novel COX” and that its induction
`was inhibited by’de.\'amethasone37. Similarly indicative
`findings were also reported in mouse fibroblasts”, cul-
`tured rat mesangial cells”, RAW 264.7 cells”, rat alveolar
`macrophages‘“"”, the ovary"-“ and other cell types“.
`But was this enzyme physiologically relevant?
`Needleman’s group conclusively identified their
`inflammation-inducible form of COX as the species
`that both Simmons and l-lerschman had cloned". As
`the evidence for the relevance of the two enzymes
`mounted, they were renamed COX1, referring to the
`original enzyme isolated from seminal vesicles and sub-
`sequently found to be distributed almost ubiquitously,
`and COX2, denoting the ‘inducible’ form of the enzyme
`(although it was expressed basally in the brain“). The
`two genes had a different chromosomal organization in
`rodents“ and humans“, and COXIICOX2 mRNA was
`diflerentially expressed” in human tissues. Promoter
`analysis confirmed a fundamental difference between
`the two isozymes, with the COX2 promoter containing
`elements strongly reminiscent of those genes that are
`switched on during cellular stress and switched off by
`glucocorficoids”, whereas COX1 had the appearance of
`a ‘HOUSElCE.EPING’GENE. Histological and other studies con-
`firmed this apparent division of labour between the
`two enzymes, and COX1 seemed to be the predomi-
`nant isoform in healthy gastrointestinal tissue from rat,
`dog and monkeys‘.
`These facts begged a key question — if COX2 was
`the predominant inflammatory species, shouldn’t this
`be the target for NSAlDs? If this was so, the corollary
`was surely that COX2 inhibition was the true thera-
`peutic modality of NSAIDs, whereas COX1 inhibition
`might be the cause of side effects such as gastric irrita-
`tion and depression of platelet aggregation. This was
`the notion put forward by more than one group5“5‘,
`which came to be known in a rather Orwellian way as
`the ‘COX2-bad:COX1-good’ hypothesis. If these
`propositions were true,
`then a selective COX2
`inhibitor would be an ideal drug, possessing the anti-
`inflammatory action but lacking the gastric and other
`side effects. But would it be possible to find or design
`such a drug?
`Some encouragement for this idea could be
`derived from publications of the day”-5’. Most of the
`non-steroidal drugs available at that time actually
`inhibited both enzymes to a greater or lesser degree,
`but some selectivity of action was seen with experi-
`mental drugs such as 6-MNA and BF389. But other
`data that subsequently emerged from transgenic
`approaches were less encouraging. Cox] —null (REF. 55)
`and Cox2-null(RI—:1=. 56) mice did not behave exactly as
`expected — Cox1—deficient mice did not have sponta-
`neous stomach ulcers and, whereas the ulceration
`caused by indomethacin in these animals was less than
`in the wild types, it was still very much in evidence.
`Again contrary to theoretical predictions, homo-_
`zygous mutant mice lacking Cox2 showed a normal
`(albeit acute) inflammatory response when treated
`with several agonists, apparently contradicting the
`idea that Cox2 was the predominant enzyme in
`
`132 MARCH 2003 VOLUME 2
`
`_
`
`www.nature.com/reviews/drugdisc
`
`Page 4 of 13
`
`© 2003 Nature Publishing Group
`
`Patent Owner Ex. 2030
`CFAD v. Pozen
`lPR2015-01718
`
`

`
`REVIEWS
`
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`cit;
`celecoxib
`
`°\~S
`WC’ “O
`Rofecoxib
`
`N02
`
`NS398
`
`0“
`\ CONH
`,N.
`o"S“o cm
`Meloxicam
`
`NHCOCH3
`
`NHCOCH3
`
`OH
`Paracetamol
`
`OCHZH5
`Phenacetin
`
`I
`
`0
`N
`N,
`,
`Hac
`Phenazone
`(antipyrine)
`
`CH3
`
`.
`CH3
`I o\ ,0
`N\/S‘ ,Na
`°
`
`0
`\
` N l
`N
`Haé
`Dipyrone
`(Methampyrone)
`
`CH3
`
`0H3
`1
`N\
`
`0
`‘
`QM |
`,N
`Hac
`Amidopyrine
`(aminopyrine)
`
`CH
`
`CH;
`
`3
`
`I)
`
`1:
`
`Figure 3 I Chemical structures of NSAIDs and related compounds. a | Structures of DuP697, NS398 and other similar
`compounds. b l Selective COX2 inhibitors that were discovered as a result of a search for selective lsoform inhibitors (ceiecoxib and
`rofecoxib) or that were ‘revealed’ as being COX2 selective (meloxicam, etodolac and nimesulide). c I Structures of some
`compounds that are more effective inhibitors of COX3 according to Slmmonsl“.
`
`inflammation“. From the medicinal chemist’s point
`of view, there was also another depressing fact —— the
`sequence of the catalytic domains of the two isozymes
`was so similar that the prospect of finding a specific
`inhibitor seemed remote. Fortunately, this perception
`was soon to be changed in a rather dramatic way.
`
`‘sleepers’
`DuP697 was a drug reported in 1990 by the Dupont
`Company to be an effective anti-inflammatory agent
`with reduced ulcerogenic properties”. Significantly,
`DuP697 showed only feeble activity in in vitro assays
`of COX using seminal vesicle or rat kidney prepara-
`tions (known to predominantly contain COX1), but
`was more effective against rat brain prostanoid syn-
`thesis. The authors originally attributed the gastro-
`intestinal safety of this compound to its chemical
`structure (a non-acidic thiophene) (1=IG.3a),which was
`presumed to have different pharmacokinetic proper-
`ties to other COX inhibitors, most of which were car-
`boxylic acids. Reports of other compounds with simi-
`lar properties were published later, including some
`experimental compounds such as NS398, fiosulide
`and CGP28238 (REF. 53) (FIG. 3a). However, in light of
`the discoveryofCOX2, it was not long before alert phar-
`macologists realized that these drugs might have this
`unusual behaviour because they acted predominately on
`the COX2 isozyme.
`
`SYNOVIAL TISSUE
`The tissues that surround
`the joints.
`
`NATURE REVIEWS l DRUG DISCOVERY
`
`Page 5 of 13
`
`© 2003 Nature Publishing Group
`
`The moment of this Damascene revelation cannot
`be pinpointed precisely but, in the context of future
`drug development, two ‘prostaglandin’ meetings in
`1992 (in Keystone, Colorado, in January, and Montreal
`in July) seem to have been of particular significance. At
`the first meeting, the Dupont Group presented evi-
`dence that gastric tolerance to DuP697 might be
`accounted for by differential inhibition of COX
`enzymes. At the second meeting, workers from Taisho
`described similardata with another structurally unre-
`lated compound, NS398 (REFS 59,50). These meetings
`were attended by many industrial scientists, and it
`seems clear from published accounts that these events
`initiated — or at least greatly accelerated — many in-
`house ‘COX2’ programmes. It is also clear that the
`structures of DuP697 and NS398 were crucial starting
`points in the hunt for new selective inhibitors. Two
`companies who capitalized on these leads were Searle
`Monsanto (now Pharmacia) and Merck. The former
`focused on sulphonamide-substituted l,5—diaryl pyra-
`zole compounds, whereas Merck scientists settled on a
`series of methylsulphonylphenyl compounds.
`While medicinal chemistry programmes were being
`developed, the field continued to produce data that were,
`on the whole, supportive to the COX1/COX2 concept.
`For example, COX2 was found in human SYNOVIALTISSUE
`taken from patients with rheumatoid arthritis, and
`the ‘inducibility’ of this enzyme by cytokines was
`
`‘
`
`VOLUME2 MARCH 2003 18:!
`
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`REVIEWS
`
`CARRAGEENAN
`A sulphated cell-wall
`polysaccharide that is found in
`certain red algae, which
`contains repeating sulphated
`disaccharidcs ofgalactose, and
`sometimes anhydrogalactose,
`and is used to induce an
`inflammatory lesion when
`injected into experimental
`animals.
`
`DYSMENORRHOEA
`Painful menstruation, often
`associated with nausea,
`vomiting. headache and
`faintness. It is thought to be
`related to excessive
`prostaglandin production.
`
`confirmed“-‘Z. Support for the COX1/COX2 concept
`also came from pharmacological studies. Masferrer at
`al.“ showed that the onset of inflammation in the rat air
`pouch correlated with the appearance of COX2 in the
`lesion, and that NS398 blocked the production of
`prostaglandins at this site without causing intestinal
`lesions or affecting gastric prostaglandin synthesis.
`Similar results were seen in various inflammatory models
`with an early Searle Monsanto selective inhibitor
`SC58 I25 (REFS 64,65). A number of other useful, selective
`COX2 inhibitors were soon described in the literature —
`SC558 (a celecoxib prototype) showed good efficacyin
`rodent models of inflammation, fever and pain, whereas
`the closely related SC560, a selective COXI inhibitor, was
`ineffective“. Celecoxib itself (then known as SC58635)
`reversed CARRAGEENAN-ll'ldL1C€d hyperalgesia and local
`prostaglandin production“ in rats, and a related com-
`pound was active intrathecally“. Recombinant COXI
`and COX2 had by now been prepared, and the selectivity
`of DUP697 and NS398 had been confirmed with in vitro
`assay systems using these enzymes"-7°.
`The solution of the crystal structures ofCOXI in
`1994 (REF. 71) and COX2 in 1996 (REF. 72) made a sub-
`stantial impact on the field. In the former paper, atten-
`tion was drawn to the crucial roles of arginine 120
`(Arg120, which interacted with the carboxyl group of
`both substrate and inhibitors) and tyrosine 355
`(Tyr355) in determining the stereospecificity of NSAID
`binding (FIGS 4, 5). In the latter paper, the structure of
`COX2 was determined in the presence of several
`inhibitors, enabling the authors to deduce the confor-
`mational and other changes required for selective inhi-
`bition. Despitethe great similarity in the sequence data,
`detailed examination of the structure of the catalytic
`sites revealed the substrate binding ‘channel’ in the two
`enzymes to be quite different. A single amino-acid
`change, from the comparatively bulky isoleucine (Ile)
`in COX1 to valine at position 523 in COX2 (equivalent
`to position 509 in COX1), and the conformational
`changes that this produced resulted in enhanced access
`to a ‘side pocket’ that allowed the binding of COX2—
`specific inhibitors by providing a docking site for the
`bulky phenylsulphonamide residue ofdrugs such as
`SC558 (FIG.4). In an elegant demonstration ofhow
`crucial this single change was, Gierse etul.” showed
`that mutation of this residue in COX2 back to He
`largely prevented selective inhibitors such as SC58 1 25,
`SC236 and NS398 from working. Further work on the
`structural basis for inhibition of both isoforms delin-
`eated the role ofArgI06 in the binding of substrate and
`certain NSAIDs", as well as the role of Tyr35S located
`at the entrance of the active site ofCOX2 (REF. 75).
`These structural data also helped explain differences
`in the inhibitory kinetics of COX] and COX2 with
`drugs such as DuP697 and NS398 (REF. 76). There are, as
`stated above, several distinct mechanisms by which
`COX1 inhibitors can inhibit the enzyme, but many are
`of the competitive reversible type. By contrast, the
`COX2 inhibitors are irreversible, time-dependent (in
`the context of enzyme kinetics) inhibitors, partly as a
`result of the binding of the sulphonamide (or related)
`
`moiety into the enzyme‘side pocket’. In an analysis of
`the kinetic behaviour of several COX inhibitors, Gierse
`etal.” subsequently discerned four separate modes of
`enzyme inhibition, ranging from the simple competi-
`tive inhibition of drugs such as ibuprofen, through the
`‘weak binding,
`time-dependent’ mechanism of
`naproxen and the oxicams and the ‘tight binding, time-
`dependent’ inhibition of indomethacin , to the covalent
`modification produced by aspirin.
`
`The development of the ‘coxibs’
`Encouraged by the ‘concept testing’ experiments with
`selective inhibitors, and armed with several solid leads
`and a clear idea ofthe nature of the binding site, devel-
`opment of this field was rapid. Celecoxib arose from the
`Searle Monsanto programme and showed marked selec-
`tivity for COX2 in vitro”. Preclinical studies with this
`compound revealed that the drug had good efficacy in
`rodent models of inflammation, fever and pain. Early
`human studies confirmed its effectiveness in the treat-
`ment of osteoarthritis, rheumatoid arthritis and post-
`surgical pain when tested in comparison with a placebo
`or with comparator NSAlDs such as naproxen, ibupro-
`fen and diclofenac. Crucially, there was also confirmation
`ofthe reduced incidence of platelet ‘and gastrointestinal
`side effects (reviewed by Lefltowith”), and celecoxib was
`subsequently licensed in the United Kingdom for
`osteoarthritis and rheumatoid arthritis in 2000.
`From Merck’s methylsulphonylphenyl series came
`MK0966, later named rofecoxib. Once again, a series of
`preclinical studies confirmed the efficacy and gastro-
`intestinal safety ofthis compound in rodent models of
`inflammation, and early human studies stablished its
`clinical utility in fever and pain (dental, DYSMENORRHOEA
`and post-operative models) when compared with stan-
`dard NSAlD_s such as ibuprofen or naproxen (reviewed
`by Morrison“). In osteoarthritis and rheumatoid arthri-
`tis, rofecoxib proved superior to placebo and comparable
`in efficacy to standard doses of other NSAIDs such as
`diclofenac. Rofecoxib was also found to have greater
`gastrointestinal safety and not to affect platelet aggrega-
`tion — it was licensed in the United Kingdom for
`osteoarthritis in 1999 and rheumatoid arthritis in 2001.
`But it was not just the qust for new drugs that was
`stimulated by the discovery of COX2. The enolcarbox—
`amide meloxicam (Boehringer Ingleheim), as well as the
`tetrahydropyranoindole etodolac (Wyeth/Shire), were
`drugs already under development at the time that the
`COX2 story ‘broke’, whereas a sulphonanilide drug,
`nimesulide (Helsinn), had been marketed in Europe
`since 1985 (FIG. 3|»). In each case, clinical and experimen-
`tal evidence already indicated that these agents were dif-
`ferent from the other NSAIDs, especially in terms of
`their good gastrointestinal tolerance. Pharmacologists
`could now_view these anomalous data with a vision
`greatly sharpened by the emerging COX2 concept, and
`these drugs turned out
`to be effective COX2
`inhibitors‘“‘”’. In an authoritative survey, Warner et :11.“
`found, for example, that meloxicam and etodolac
`showed almost the same order of selectivity for
`COX1/COX2 as some of the newer agents.
`
`134 | MARCH 2003 |\’OLUME 2
`
`www.nature.com/reviews/drugdisc
`
`Page 6 of 13
`
`© 2003 Nature Publishing Group
`
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`lPR2015-01718
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`
`REVIEWS
`
`
`
`COX1 inhibitor
`Flurbiprofen
`F-gLI'e4|Oomparisonoftl1eNSAlDbh1dirtgsibesofGO)l1 andGO)t2afberBrowrtenSchetrati:;cafloorIirmdfiedlmu11 REF. H2).
`showing It-nedrtlartanoesintl-teNSAlD birndirtgshesofcmtl andODX2. hlote1t'althaOOX2binctrrg sltelsrnoreacoornmodatingancl
`Lschatacierized oya 'sldeomket'matcmaowmwmatemI<ygro.psa:d1asuememwoJphony|wolewm DuP69? OOK
`cyclooxygenasa; hk‘iA1D. non-steroidal anti-tnflamrrratcnr drug.
`
`COX2 nnhibilor
`DuPE9T
`
`The GOXHGDX2 colloopt today
`I-lowdoes thisyery influential idea stack up today in the
`light of the results that have been published on the
`actions of the coicibs and olherselcctive inhibitors of the
`COX2 isoform? Like all ideas. it has seen some modifi-
`cations over the passage of time. At the