Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-in ammatory drugs

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1 British Journl of Phrmcology (1999) 126, 1824 ± 1830 ã 1999 Stockton Press All rights reserved 0007 ± 1188/99 $ Ex vivo ssy to determine the cyclooxygense selectivity of non-steroidl nti-in mmtory drugs 1 Frncesco Giulino & *,1 Timothy D. Wrner 1 Vsculr In mmtion, The Willim Hrvey Reserch Institute, St. Brtholomew's nd The Royl School of Medicine nd Dentistry, Chrterhouse Squre, London EC1M 6BQ 1 In this study we descrie experiments to estlish ex vivo the selectivity of non-steroidl ntiin mmtory drugs (NSAIDs) given in vivo. 2 Anesthetised (Inctin, 120 mg kg 71 ) mle Wistr rts (220 ± 250 g) received n i.v. dose of one of the following compounds (dose mg kg 71 ): spirin (20), diclofenc (3), L-745,337 (30), nimesulide (15), slicylte (20), sulindc (10). Blood smples were tken efore nd up to 6 h fter dosing nd the plsm otined from it ws tested for its ility to inhiit prostnoid formtion in IL-1treted A549 cells (COX-2 system) nd humn wshed pltelets (COX-1 system). For control the sme compounds were lso dded directly to the ssy systems. 3 All drugs, except sodium slicylte, inhiited COX-1 nd COX-2 when dded directly to the test systems. Plsm from spirin-treted rts ws without e ect on either COX-1 or COX-2, consistent with the rpid in vivo metolism to slicylte. Conversely, plsm from sulindc-treted rts inhiited COX-1 nd COX-2 with potencies ccording with in vivo metolism to sulindc sulphide. Diclofenc ws COX-1/2 non-selective when tested in vitro, ut slightly preferentil inhiitor of COX-2 when tested ex vivo. Nimesulide ws con rmed s preferentil inhiitor of COX-2 oth in vitro nd ex vivo. L-745,337 ws selective COX-2 inhiitor when tested in vitro or ex vivo. 4 In conclusion, our experiments show clerly () NSAIDs inctivtion, () ctivtion of prodrugs, nd (c) NSAIDs selectivity. Our ssy provides useful informtion out the selectivity of NSAIDs tht could e extended y the nlysis of plsm smples tken from humns similrly treted with test drugs. Keywords: COX-1; COX-2; NSAIDs selectivity; NSAIDs inctivtion; NSAIDs ctivtion; ex vivo evlution Arevitions: COX, cyclooxygense; DMEM, Dulecco's modi ed Egle medium; FBS, foetl ovine serum; IL-1, interleukin-1; NSAIDs, non-steroidl nti-in mmtory drugs; PG, prostglndin; PRP, pltelet rich plsm; TX, thromoxne Introduction Prostnoids re formed initilly y the conversion of rchidonic cid to common intermedite, prostglndin (PG) H 2, y the enzyme prostglndin endoperoxide synthse (PGHS) lso known s cyclooxygense (COX) (see Vne et l., 1998). This enzyme crries out two distinct rections. The cyclooxygense ctivity forms PGG 2 from rchidonic cid, while the peroxidse ctivity of PGHS converts PGG 2 to PGH 2. The susequent formtion of prticulr prostnoids depends on the ctions of speci c isomerses cting upon PGH 2 (see Hershmn, 1996). Following the discovery in the erly 1990s of n inducile isoform of COX (Xie et l., 1991; Kujuu et l., 1991; Wong et l., 1991; O'Bnion et l., 1991) it is now known tht COX exists in t lest two isoforms. A constitutive `house-keeping' enzyme, COX-1, which is responsile for homeosttic functions (Moncd et l., 1976; Whittle et l., 1980) nd n inducile isoform, COX-2, ssocited with mitogenic events nd in mmtory sttes (Rz et l., 1988; Fu et l., 1990; Msferr et l., 1990; O'Bnion et l., 1992; Lee et l., 1992; Xie et l., 1992). As re nement of the mechnism proposed y Vne in 1971 it hs therefore een suggested tht inhiition of COX-2 underlies the therpeutic e ccy of nonsteroidl nti-in mmtory drugs (NSAIDs) whilst inhiition of COX-1 underlies their side e ects (Mitchell et l., 1993). The *Author for correspondence; E-mil: t.d.wrner@mds.qmw.c.uk potentil therpeutic ene t of COX-2 selective inhiitors hs encourged the development of numerous in vitro test systems in which to de ne nd develop oth clssicl nd novel NSAIDs. The numerous in vitro test systems my e clssed into three min groups, puri ed/recominnt enzymes (Mede et l., 1993), cultures of intct cells (Cromlish et l., 1996) nd humn whole lood (Ptrignni et l., 1994; 1997). Although ll of these test systems hve provided informtion out the ilities of NSAIDs to inhiit COX-1 nd -2, they lso hve shortcomings tht limit their ppliction to the preliminry testing of drug. For exmple, ssys employing roken cells cnnot ccount for cell penetrtion nd intrcellulr drug distriution. Mny cell-sed ssy systems use non-humn cells nd fil to ccount for plsm inding. Humn loodsed ssys, therefore, hve cler dvntges. They do, however, still hve drwcks. In prticulr, di erent incution times for ssys of COX-1 nd COX-2 ctivity reduce the ility of these systems to predict with ccurcy the in vivo e ccy nd selectivity of NSAIDs. Here, therefore, we hve investigted the possiility of estlishing n ssy to ssess ex vivo the ctivity of NSAIDs given in vivo. To chieve this gol we injected rts intrvenously with NSAIDs nd then withdrew lood t di erent time points. The NSAID ctivity in the plsm ws determined y its ility to inhiit COX-1 nd COX-2 in, respectively, humn wshed pltelets nd IL-1-treted A549 cells. The NSAIDs used in this study for the in vivo experiments were chosen to represent the four functionlly

2 de ned clsses of NSAIDs: COX-1 selective (spirin, slicylte nd sulindc), COX-1/-2 non-selective (diclofenc), COX-2 preferentil inhiitors (nimesulide) nd COX-2 selective compounds (L-745,337). From this ssy system we hve otined good predictive dt with regrd to the ctivities of NSAIDs nd novel COX-2 selective compounds. Methods In vivo Mle Wistr rts (220 ± 250 g; Tuck, Ryleigh, U.K.) were nesthetised y intrperitonel injection of thioutrtil sodium (Inctin; 120 mg kg 71, i.p.; RBI, Ntick, U.S.A.). Body temperture ws mintined t 378C y mens of homeothermic lnket connected to rectl proe. The trche ws cnnulted to fcilitte ventiltion. The right crotid rtery ws cnnulted nd connected to pressure trnsducer (type , Trnsmeric Instruments) for the monitoring of systemic lood pressure displyed on Grphtec Linercorder. The jugulr vein ws lso cnnulted to llow injection of drugs nd infusion of sline. Animls were left for 30 min following surgery to stilise fter which time control plsm smple ws withdrwn (t=760). One hour lter (t=0) the rts received n i.v. olus of spirin (20 mg kg 71 ; n=4), diclofenc (3 mg kg 71 ; n=4), L-745,337 (30 mg kg 71 ; n=5), nimesulide (15 mg kg 71 ; n=4), sodium slicylte (20 mg kg 71 ; n=4) or sulindc (10 mg kg 71 ; n=5). Blood smples of %300 ml were tken from the crotid rtery t t=760, 5, 60, 120, 180, 240, 300 nd 360 min. The smples were centrifuged g for 2 min t 48C nd the plsm removed supplemented with heprin (15 U ml 71, Ntionl Veterinry Supplies, Stoke on Trent) nd snp frozen in liquid N 2. Three hundred ml of wrm sline ws injected i.v. fter ech withdrwl of lood. At the end of the time course, nimls were killed y n overdose of nesthetic. Individul drugs were dministered to rts on t lest 4 di erent study dys. Cell culture A549, humn epithelil crcinom cell line (ECACC Ref. No ), expresses COX-2 when exposed to IL-1 (Mitchell et l., 1994). Production of PGE 2 y this cell line cn therefore e used s n index of COX-2 ctivity. A549 cells were mintined in humidi ed tmosphere of 5% CO 2-95% ir t 378C nd grown in Dulecco's Modi ed Egle Medium (DMEM; Sigm, Poole, U.K.) supplemented with 10% foetl ovine serum (FBS; Sigm, Poole, U.K.). For the experimentl procedures, cells were seeded into 96-well pltes nd grown to con uence efore use. In order to induce COX-2 expression, cells were incuted for 24 h in fresh DMEM supplemented with 10% FBS nd IL-1 10 ng/ml. Before the experimentl procedure, the medium ws replced with fresh DMEM: C 2+ - free modi ed Kres-Ringer solution (see elow) (4 : 1, v/v) t 378C. Wshed pltelets F. Giulino & T.D. Wrner The production of thromoxne (TX) B 2 y pltelets ws used s index of COX-1 ctivity. Blood from helthy volunteers, who hd not tken NSAIDs for t lest two weeks, ws collected y venepuncture into geltine-coted (0.1% porcine geltine in H 2 O, 1±3 h t 378C) plstic tues contining trisodium citrte 3.15% (1 : 9, v/v). The lood ws centrifuged t Ex vivo determintion of NSAIDs selectivity g for 7 min to produce pltelet rich plsm (PRP). Prostcyclin (300 ng ml 71 ) ws then dded to the PRP followed y centrifugtion t 10006g for 15 min to sediment the pltelets. The resulting superntnt ws removed nd replced with n equl volume of C 2+ -free modi ed Kres- Ringer solution t 378C (10 mm HEPES, 20 mm NHCO 3, 120 mm NCl, 4 mm KCl, 2 mm N 2 SO % glucose, 0.1% ovine serum lumin). The pellet ws gently resuspended nd further prostcyclin (300 ng ml 71 ) dded. The pltelets were pelleted gin nd resuspended in C 2+ -free modi ed Kres- Ringer u er t 378C to mtch hlf of the initil plsm volume. Thirty minutes lter the pltelet suspension ws diluted 1 : 5 in DMEM supplemented with 10% FBS nd plted into geltine-coted 96-well pltes (100 ml well 71 ). Evlution of NSAIDs ctivity on COX-1 nd COX-2 To ssy NSAIDs ctivity in plsm collected from the rts, 10 ml of plsm ws dded to medium thing either preinduced A549 cells or wshed pltelets. Concentrtion response curves to spirin (n=4) or slicylte (n=4) (0.1 nm to 1 mm), diclofenc (n=4), L-745,337 (n=5), nimesulide (n=4), sulindc (n=4) or sulindc sulphide (n=5) (0.01 nm to 0.1 mm) were lso constructed on the sme culture pltes on which the corresponding plsm series were tested. After incution for 30 min t 378C, clcium ionophore A23187 (50 mm) ws dded nd the cells or pltelets incuted for further 15 min t 378C. At the end of the incution, pltes contining the pltelet suspension were centrifuged for 5 min t 15006g (48C) nd the superntnt removed nd snp frozen until nlysis y rdioimmunossy. Medium from A549 pltes ws lso removed nd frozen. Ech plte of cells nd pltelets included two sets of control wells, one incuted with vehicle (0.1% dimethyl sulfoxide) the other receiving control plsm withdrwn t t=760. Inhiition of COX-1 nd COX- 2 y test compounds or plsm smples ws clculted s percentge of the ctivity mesured in the corresponding control wells. The e ects of vehicle, plsm series nd NSAIDs on COX ctivity were mesured in t lest three seprte determintions (wells) on t lest four di erent experimentl dys. The sme set of plsm smples or drug dilutions were not used on more thn one experimentl dy. Mterils For the in vivo procedure, ll the drugs were dissolved in 5% icronte - 2.5% glucose u er in H 2 O nd sonicted until cler solution or very ne suspension were otined. The required mount of drug ws dissolved in 1 ml nd injected over period of 100 s. For the in vitro experiments, ll the drugs were dissolved in DMSO to form stock solutions of 0.1 to 1 M efore eing further diluted in DMEM. All compounds used were otined from Sigm (Poole, U.K.) unless otherwise stted. L-745,337 ws gift from Merck Frosst, Cnd. Sulindc sulphide ws purchsed from A niti (Exeter, U.K.). For the rdioimmunossy, ntiser to PGE 2 nd TXB 2 were otined from Sigm (Poole, U.K.); [ 3 H]-PGE 2 nd [ 3 H]-TXB 2 were purchsed from Amershm (Little Chlfont, U.K.). IL-1 ws otined from Genzyme (Kings Hill, U.K.). Dt nlysis Results re expressed s men+s.e.m. Concentrtion response curves were tted using sigmoidl regression with vrile slope.

3 1826 F. Giulino & T.D. Wrner Two-wy nlysis of the vrince (ANOVA) ws used to evlute sttisticl di erences etween dose response curves. IC 50 vlues were compred using the t-test for unpired oservtions. A one-smple t-test ws performed to determine signi cnt di erences from normlized controls. A P vlue less thn 0.05 ws considered sttisticlly signi cnt. All nlysis nd regressions were performed using GrphPd Prism (GrphPd Softwre, Sn Diego, CA, U.S.A.). Results In vivo The men rteril lood pressure of the rts t t=770 nd t=360 min ws, respectively, mmhg nd 75+2 mmhg with no signi cnt di erences etween tretment groups (P40.05, one wy ANOVA; n=25). A trnsient increse in lood pressure of 15+5 mmhg, lsting 5 ± 20 min, ws recorded fter the i.v. injection of ech drug. Agin, no signi cnt di erence etween drugs ws noted. Prostnoid production The stimulted relese of PGE 2 y A549 cells in the presence of drug vehicle or control plsm ws ng ml 71 (n=30) nd ng ml 71 (n=25), respectively. Thromoxne B 2 production y wshed pltelets ws 25+2 ngml 71 in the presence of drug vehicle (n=30) nd ng ml 71 in the presence of control plsm (n=25). Ex vivo determintion of NSAIDs selectivity thn COX-2 (IC mm) (Tle 1, Figure 3). However plsm tken from spirin-treted rts ws without ny signi cnt e ect (P40.05, one smple t-test) on the ctivities of either COX-1 or COX-2 s erly s 5 min fter dministrtion in vivo (Figure 3). Similrly slicylte, the metolite of spirin, ws without ctivity when dded directly to the ssy systems in concentrtions of up to 1 mm (Tle 1, Figure 4), or when plsm from slicylte-treted nimls ws tested (Figure 4). Sulindc nd sulindc sulphide Plsm tken from sulindctreted nimls inhiited COX-1 to mrkedly greter extent thn COX-2 throughout the smpling period (Figure 5). In similr fshion, when dded directly to the ssy systems, sulindc nd in prticulr its ctive metolite, sulindc sulphide, inhiited oth isoforms of COX with selectivity towrds COX-1 (Tle 1, Figure 5 nd c). Importntly, it should e noted tht sulindc sulphide inhiited COX times more potently thn the prent drug. L-745,337 The novel COX-2 selective drug L-745,337 clerly showed the gretest selectivity for COX-2 over COX-1 oth in vitro (1100 fold) (Tle 1, Figure 6) nd in the ex vivo ssy (Figure 6). In this ltter ssy, L-745,337 (30 mg kg 71 ) ws Evlution of NSAIDs ctivity on COX-1 nd COX-2 Diclofenc Of the compounds tested, diclofenc ws the most potent inhiitor of COX-1 nd COX-2, lthough it ws nonselective. Plsm tken from diclofenc-treted rts inhiited oth COX-1 nd COX-2 with smll ut signi cnt selectivity for COX-2 (P50.05, two-wy ANOVA) (Tle 1, Figure 1). Notly, the inhiitory ctivity of the plsm from diclofenctreted rts decresed throughout the in vivo experimentl period (Figure 1). Nimesulide In vitro, nimesulide showed preferentil inhiition of COX-2 (IC mm) over COX-1 (IC mm) (Tle 1, Figure 2). Consistently, in the ex vivo ssy, nimesulide showed signi cnt selectivity (P50.05, two-wy ANOVA) towrds the inhiition of COX-2. Plsm smples from nimesulide-treted rts lso demonstrted little chnge in ctivity over the 6 h smpling period (Tle 1, Figure 2). Aspirin nd slicylte In vitro, spirin ws pproximtely six times more potent s n inhiitor of COX-1 (IC mm) Tle 1 IC 50 vlues for NSAIDs on COX-1 nd COX-2 ctivity in wshed humn pltelets nd A549 cells. I=inctive IC 50 (mm) NSAIDs COX-1 COX-2 COX-2/COX-1 Aspirin Diclofenc L-745,337 Nimesulide Sodium slicylte Sulindc Sulindc sulphide I I ± Figure 1 E ect of diclofenc () nd plsm smples from diclofenc-treted rts () on COX-1 nd COX-2 ctivity. Pltelets (COX-1 system) nd A549 (COX-2 system) cells were exposed to diclofenc or plsm smples for 30 min nd then chllenged with clcium ionophore A23187 (50 mm) for 15 min. When dded directly to the ssy system, diclofenc ws COX-1/-2 non-selective wheres plsm smples from diclofenc-treted nimls showed smll ut signi cnt selectivity towrds COX-2. Dt re expressed s men+ s.e.m. from three determintions from four seprte experimentl dys.

4 F. Giulino & T.D. Wrner Ex vivo determintion of NSAIDs selectivity 1827 Figure 2 E ect of nimesulide () nd plsm smples from nimesulide-treted rts () on COX-1 nd COX-2 ctivity. Nimesulide showed preferentil inhiition of COX-2 oth when dded directly to the ssy system nd when plsm smples from nimesulide-treted rts were used. Dt re expressed s men+ s.e.m. from three determintions from four seprte experimentl Figure 3 E ect of spirin () nd plsm smples from spirintreted rts () on COX-1 nd COX-2 ctivity. Aspirin dded directly to the ssy system inhiited COX-1 more potently thn COX-2 wheres plsm smples from spirin-treted nimls filed to show ny signi cnt ctivity on oth isoforms. Dt re expressed s men+s.e.m. from three determintions from four seprte experimentl dys. without ny signi cnt e ect on COX-1 (P40.05, one smple t-test) wheres it cused strong inhiition of COX-2. Discussion Here we report novel pproch to ssessing the ctivity of NSAIDs ginst COX-1 nd COX-2. Our dt demonstrte selectivity nd metolism of oth clssicl NSAIDs nd, more importntly, for novel COX-2 selective compound. NSAIDs produce their ene cil e ects y inhiiting COX-2 nd their deleterious e ects y inhiiting COX-1 (see Vne et l., 1998). Thus, predictive in vitro nd in vivo ssys of NSAIDs selectivity for COX-1 vs COX-2 re essentil oth to rtionlize the use of existing NSAIDs nd to led to the production of the next genertion of NSAIDs. The most widely used test system for the evlution of NSAIDs e ccy nd selectivity is currently the whole humn lood ssy. In this system, the clotting of whole humn lood nd the mesurement of the TXB 2 formed is the sis for mesuring COX-1 ctivity wheres the formtion of PGE 2 or TXB 2 in lood incuted with cteril lipopolyscchride for 5 ± 24 h is the sis for mesuring COX-2 ctivity (Brideu et l., 1996; Ptrignni et l., 1994; 1997; Young et l., 1996). This ssy hs the cler dvntge of using esily ccessile humn COX-producing cells nd of tking into ccount drug inding to plsm proteins. However, the need to induce COX-2 ex vivo introduces n importnt vrile which in prticulr leds to considerle time discrepncy etween the COX-1 nd COX-2 ssys. Here we hve estlished test system tht voids this prolem. Signi cntly, it lso llows the nlysis of not only COX selectivity ut lso NSAIDs ctivtion nd inctivtion. Drugs were dministered intrvenously to limit vritions cused y di erences in sorption. However, the ssy could e pplied following orl dministrtion of NSAIDs. Aspirin dded directly to the test systems cused inhiition of COX-1 nd COX-2 with IC 50 vlues of, respectively, 1.88 nd mm. Conversely, plsm tken from spirin-treted rts hd no e ect on the ctivity of either COX isoform. This oservtion is in ccordnce with spirin eing rpidly hydrolysed in the lood strem to slicylte (Higgs et l., 1987) which is wek inhiitor of COX. Under our ssy conditions, where high levels of free rchidonic cid re chieved, these concentrtions of slicylte were without e ect on COX ctivity (Mitchell et l., 1997). In contrst to spirin, plsm from sulindc-treted nimls inhiited oth COX-1 nd COX-2 with selectivity towrds COX-1. Anlysis of the control concentrtion response curves indictes tht sulindc could only produce such n inhiition of COX-1 nd COX-2 t concentrtions greter thn 0.1 mm. This is greter thn tht chievle following the dose given

5 1828 F. Giulino & T.D. Wrner Ex vivo determintion of NSAIDs selectivity Figure 4 E ect of slicylte () nd plsm smples from slicyltetreted rts () on COX-1 nd COX-2 ctivity. Slicylte filed to show ny ctivity either when dded directly to the ssy system or when plsm from slicylte-treted nimls ws used. Dt re expressed s men+s.e.m from three determintions from four seprte experimentl dys. c nd indictes metolic ctivtion of sulindc, proly to sulindc sulphide (Kwn & Duggn, 1977). Indeed, our control curves indicted sulindc sulphide to produce inhiition of COX-1 nd COX-2 t concentrtions tht ccord much more closely with the dose given. Thus, this test system e ectively llows the testing nd chrcteriztion of pro-drugs. Moreover, the selectivity towrds COX-1 shown y sulindc sulphide in vitro ws clerly reproducile ex vivo. To sustntite the hypothesis tht our test system would e suitle for evluting ex vivo the selectivity of NSAIDs dministered in vivo, we tested three more drugs. Hving lredy tested COX-1 selective drugs, we chose diclofenc s representtive of COX-1/-2 non-selective NSAIDs (Mitchell, 1993), nimesulide s representtive of preferentil COX-2 inhiitors (see Fmey, 1997), nd L-745,337, COX-2 selective compound (Chn et l., 1995). When tested in vitro, diclofenc produced pproximtely equipotent inhiition of COX-1 nd COX-2. In the ex vivo ssy, diclofenc produced slightly preferentil inhiition of COX-2. It is tempting to propose tht the mild selectivity of diclofenc towrds the inhiition of COX-2 could ccount in prt for the low reltive risk of upper gstrointestinl toxicity reported y severl uthors for this drug (Henry et l., 1996; Grci Rodriguez et l., 1998). The ex vivo ssy my, therefore, provide etter indiction of gstrointestinl risk. In the ex vivo ssy diclofenc lso showed decrese in the Figure 5 E ect of sulindc sulphide (), plsm smples from sulindc-treted rts () nd sulindc (c) on COX-1 nd COX-2 ctivity. Sulindc sulphide nd sulindc oth showed selectivity towrds the inhiition of COX-1. Plsm smples from sulindctreted rts lso selectively inhiited COX-1 with potency ccording to the in vivo metolism of sulindc to sulindc sulphide. Dt re expressed s men+s.e.m. from three determintions from t lest four seprte experimentl dys. inhiitory ctivity throughout the in vivo experimentl period in ccordnce with its short hlf-life (Willis et l., 1979). Conversely, nimesulide ctivity in the plsm smples from nimesulide-treted rts ws pproximtely stle throughout the in vivo experimentl period. However the lck of relile intrvenous phrmcokinetic studies following intrvenous dministrtion of this drug (Bernreggi, 1993) does not llow ny vlidtion of our ndings. The selectivity of nimesulide s n inhiitor of COX-2 ws con rmed oth in vitro nd ex vivo. Nimesulide's preferentil inhiition of COX-2 over COX-1

6 F. Giulino & T.D. Wrner Figure 6 E ect of L-745,337 () nd plsm smples from L- 745,337-treted rts () on COX-1 nd COX-2 ctivity. When tested directly, L-745,337 inhiited COX fold more potently thn COX-1. Plsm smples form L-745,337-treted rts lso selectively inhiited COX-2 ctivity without ny signi cnt e ect on COX-1. Dt re expressed s men+s.e.m. from three determintions from ve seprte experimentl dys. well correltes with the high gstric tolerility oserved t low dosge (100 or 200 mg twice dily) for short tretment periods (Mrini & Spotti, 1993). However, s the dose nd/or dministrtion period increse, the gstric tolerility of nimesulide is reduced (Wrrington et l., 1993; Grci Rodriguez et l., 1998). The sfety mrgins shown y COX-2 preferentil inhiitors re lower thn those for COX-2 selective inhiitors. Here, in fct, the novel COX-2 selective drug L- 745,337 when tested in vitro ws 1100 fold more ctive ginst COX-2 thn COX-1. In the ex vivo ssy, L-745,337 ws lso con rmed to e highly selective COX-2 inhiitor, for plsm from L-745,337 treted rts ws without ny e ect on COX-1. This oservtion is consistent with the nti-in mmtory nd gstric spring pro le of L-745,337 (Chn et l., 1995). The ex vivo ssy lso showed tht L-745,337 ctivity ws very stle throughout the in vivo experimentl period. The dt ove suggest tht the ex vivo ssy system we hve developed hs very promising utility oth for pro ling Ex vivo determintion of NSAIDs selectivity 1829 the ctions of existing NSAIDs nd for the chrcteriztion of new compounds. It cn e used to study selectivity nd metolism under de ned conditions for COX-1 nd COX-2. It does, however, hve two disdvntges. Firstly the use of rt plsm in comintion with humn cells nd pltelets produces iologicl heterogeneity tht limits the potentil for predictivity in humns. Secondly, ecuse of the limited vilility of plsm from drug-treted rts it ws possile to test ex vivo only smll volumes (10 ml) of plsm. The resulting 10 fold dilution produces n underestimtion of the rel drug ctivity in the plsm. Nevertheless, it is cler tht this ssy provides informtion vlule in predicting the in vivo e ccy nd selectivity of old nd new NSAIDs. From the studies we hve performed, however, it is not possile to drw conclusions out the reltive e ects of NSAIDs ginst COX-1 nd COX-2 when used, for instnce, s nti-in mmtories. In prticulr, s we were interested in demonstrting in vivo ctivtion nd/or inctivtion, NSAIDs were dministered s i.v. oluses. Cliniclly, of course, these drugs re generlly dministered orlly. Further studies in which lood smples re tken from nimls receiving NSAIDs in orlly ctive nti-in mmtory nd nlgesic doses would of course clrify this issue. More importntly, this ssy could e of much usefulness in the nlysis of plsm smples tken from humns treted with NSAIDs. The use of pure plsm ex vivo in ssocition with humn cells or pltelets would provide high qulity stndrd from which to predict NSAIDs' in vivo e ccy nd selectivity. This test system my lso represent new pproch for the study of NSAID phrmcokinetic interctions with other drugs. Generlly, in most of the investigtions deling with phrmcokinetic interctions the totl lood concentrtion of the drug is determined. However the clinicl vlue of di erences in plsm concentrtions is questionle (Brouwers & de Smet, 1994). The evlution of the drug ctivity in plsm rther thn its totl lood concentrtion could represent much etter clinicl index. In conclusion, we hve estlished test system tht is cple of ssessing ex vivo the ctivity of NSAIDs dministered in vivo. In prticulr, using spirin nd sulindc, we hve shown tht our ssy cn unveil phenomen of drug inctivtion s well s drug ctivtion. More importntly, using diclofenc, nimesulide nd the novel COX-2 inhiitor L- 745,337, we hve demonstrted the possiility of studying the di erentil inhiition of COX isoforms with improved ccurcy in predicting NSAIDs selectivity in vivo. Therefore, the ppliction of this system to the nlysis of humn-derived smples my help to etter understnd the correltion etween NSAIDs di erentil inhiition of COX nd their reported gstrointestinl toxicity index. Studies on the phrmcokinetics interctions of NSAIDs with other drugs my lso ene t from the ppliction of this test system. TDW holds British Hert Foundtion Lectureship (BS/95003). This work ws lso supported y grnt from Boehringer Ingelheim Phrm KG. The uthors would like to thnk Miss E.G. Wood for her ssistnce with the cell culture.

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