1521-0103/356/3/635–644$25.00 http://dx.doi.org/10.1124/jpet.115.228932THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:635–644, March 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Identification and Characterization of Novel MicrosomalProstaglandin E Synthase-1 Inhibitors for Analgesia

Srinivasan Chandrasekhar, Anita K. Harvey, Xiao-Peng Yu, Mark G. Chambers,Jennifer L. Oskins, Chaohua Lin, Thomas W. Seng, Stefan J. Thibodeaux,Bryan H. Norman, Norman E. Hughes, Matthew A. Schiffler, and Matthew J. FisherLilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana

Received August 25, 2015; accepted January 5, 2016

ABSTRACTProstaglandin (PG) E2 plays a critical role in eliciting inflamma-tion. Nonsteroidal anti-inflammatory drugs and selective inhib-itors of cyclooxygenase, which block PGE2 production, havebeen used as key agents in treating inflammation and painassociated with arthritis and other conditions. However, theseagents have significant side effects such as gastrointestinalbleeding and myocardial infarction, since they also block theproduction of prostanoids that are critical for other normalphysiologic functions. Microsomal prostaglandin E2 synthase-1is a membrane-bound terminal enzyme in the prostanoid

pathway, which acts downstream of cyclooxygenase 2 and isresponsible for PGE2 production during inflammation. Thus,inhibition of this enzyme would be expected to block PGE2production without inhibiting other prostanoids and would pro-vide analgesic efficacy without the side effects. In this report, wedescribe novel microsomal prostaglandin E2 synthase-1 inhib-itors that are potent in blocking PGE2 production and areefficacious in a guinea pig monoiodoacetate model of arthralgia.These molecules may be useful in treating the signs andsymptoms associated with arthritis.

IntroductionProstaglandins (PGs) play critical physiologic roles in a

variety of organ functions and serve as key mediators ofinflammation, pain, and fever (Smith, 1989; Funk, 2001;Smyth et al., 2009). PGE2, the most prominent prostanoid, isproduced by sequential enzymatic reactions starting with the re-lease of arachidonic acid from membrane glycerophospholipidsby phospholipase A2, followed by conversion to endoper-oxide PGH2 by either cyclooxygenase 1 or 2 (COX-1 or COX-2,respectively), and finally the isomerization of PGH2 to PGE2

by terminal prostaglandin E2 synthases (PGESs). The in-termediate PGH2 also serves as a substrate for othersynthases/isomerases leading to the production of throm-boxane Tx A2, PGI2, PGD2, and PGF2a (Funk, 2001). The COX-1isoform is constitutively expressed and is responsible for theproduction of PGs that preserve the gastric mucosa, whereasthe COX-2 isoform is induced in response to cytokines ininflammatory conditions such as rheumatoid arthritis andosteoarthritis (Sugimoto and Narumiya, 2007; FitzGerald,2004; Smyth et al., 2009). Nonsteroidal anti-inflammatorydrugs (NSAIDs) and COX-2 selective drugs provide symptom-atic relief by blocking the production of PGE2 through in-hibition of both COX-1 and COX-2 or COX-2 alone (Rainsford,2007). However, NSAIDs are associated with gastrointestinal

bleeding as a result of the inhibition of constitutively producedPGE2, whereas COX-2 selective inhibitors have been associ-ated with increased thrombotic risk and myocardial infarc-tion, which may be due to the inhibition of other inducibleprostanoids such as PGI2, which is required for propercardiovascular function (FitzGerald and Patrono, 2001;Mukherjee et al., 2001; Cheng et al., 2002, 2006; Fries andGrosser. 2005; Wang et al., 2005, 2006, 2011; Grosser et al.,2006). Thus, a selective inhibition of PGE2 without adverselyaffecting other prostanoids would be expected to provide anti-inflammatory and analgesic effects without the negative sideeffects.Microsomal prostaglandin E synthases 1 and 2 (mPGES-1

and mPGES-2, respectively) are terminal enzymes in convert-ing PGH2 to PGE2. mPGES-1 is expressed at low levels and isupregulated in a variety of inflammatory conditions, whereasmPGES-2 is constitutively expressed in a variety of tissues(Jakobsson et al., 1999; Tanikawa et al., 2002;Westmamet al.,2004; Samuelsson et al., 2007). Inhibition of mPGES-1 eitherby gene deletion or by pharmacological inhibition of activitydemonstrates selective blockage of PGE2 production andanalgesic and anti-inflammatory activity (Trebino et al., 2003;Kamei et al., 2004; Xu et al., 2008; Mbalaviele et al., 2010;Abdul-Malik et al., 2013; Bahia et al., 2014; Korotkova andJakobsson, 2014). These observations suggest that mPGES-1plays a critical role in eliciting PGE2–mediated inflammatoryresponse and that blocking the enzyme activity is likely todx.doi.org/10.1124/jpet.115.228932.

ABBREVIATIONS: COX, cyclooxygenase; EIA, enzyme immunoassay; FBS, fetal bovine serum; HEK-293, human embryonic kidney 293; IL,interleukin; LPS, lipopolysaccharide; MF-63, 2-(6-chloro-1H-phenanthro-[9,10-d]imidazol-2-yl)isophthalonitrile; MIA, monoiodoacetate; mPGES,microsomal prostaglandin E synthase; NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin; PGES, prostaglandin E synthase; TNFa,tumor necrosis factor a; Tx, thromboxane.

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provide analgesic and anti-inflammatory relief. We recentlyidentified and described novel mPGES-1 inhibitors that arehighly selective, potent, and orally available (Schiffler et al.,2016). In this report, we show that thesemolecules are selectivein blocking PGE2 production, while exhibiting no inhibition ofother prostanoids, such as PGI2, at the concentrations tested.We also show that the compounds are effective in reducing painin a guinea pig model of knee joint pain.

Materials and MethodsmPGES-1 Inhibitors (Compounds 1 and 2 and Reference

mPGES-1 Inhibitor MF-63). The synthesis of reference mPGES-1inhibitor MF-63 [2-(6-chloro-1H-phenanthro-[9,10-d]imidazol-2-yl)isophthalonitrile] (Xu et al., 2008) and compounds 1 and 2 weredescribed previously (Schiffler et al., 2016)

Human mPGES-1. Human mPGES-1 was purchased from Invi-trogen (catalog no. 97002RG, clone ID 6314722; Invitrogen, GrandIsland, NY) and was subcloned into pcDNA3.1 and transientlyexpressed in human embryonic kidney 293 (HEK-293) cells. Micro-somes were prepared from cell pellets based on published methods(Ouellet et al., 2002; Thorén et al., 2003). In brief, cell pellets weresonicated in a buffer of 15 mM TRIS-HCl, pH 8.0, 0.25 mM sucrose,0.1 mM EDTA, and 1 mM glutathione. The suspension was centrifugedat 5000� g for 10 minutes at 4°C. The supernatant fraction was loadedinto Beckman Quickseal tubes (342413; Beckman Coulter, Brea, CA)and centrifuged at 185,000 � g for 90 minutes at 4°C using a 70.1Ti rotor. Pellets were resuspended in a buffer of 10 mM sodiumphosphate, pH 7.0, 10% glycerol, 2.5 mM glutathione, and Completeprotease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN).Final concentrations were 4.4 mg/ml microsomes and 1.69 mM PGH2.All dilutions were made using the above buffer. After a 2.5-minuteincubation at room temperature, 2.5 ml/well SnCl2 in 0.5 N HCI wasadded to stop the reaction. PGE2 was quantitated by standard liquidchromatography/mass spectrometry analysis.

Guinea Pig mPGES-1. Guinea pig mPGES-1 was cloned frominterleukin (IL)-1–stimulated 104C1 (CRL-1405; American TypeCulture Collection, Manassas, VA) by 59-RACE and was subclonedinto pQCXIN. HEK-293 cells were infected with the plasmid for 24hours and then expanded under selection in Dulbecco’s ModifiedEagle Medium:Nutrient Mixture F-12 (DMEM/F12) 3:1 (Invitrogen),10% fetal bovine serum (FBS), and 1 mg/ml G418. Cell pellets wereprocessed into microsomes as described above. Activity was assessedas above with final concentrations of 15 mg/mL guinea pig mPGES-1microsomes and 2 mM PGH2. PGE2 was measured by enzymeimmunoassay (EIA) (500141; Cayman Chemical, Ann Arbor, MI) ata dilution of 1:1000.

Rat mPGES-1. Rat mPGES-1 cDNA was purchased from OpenBiosystems (catalog no.MRN1768- 99238049, clone ID 7456259; OpenBiosystems, Huntsville, AL) and was subcloned into pQCXIN. HEK-293 cells were infected with the plasmid for 24 hours and thenexpanded under selection in DMEM/F12 3:1 (Invitrogen), 10% FBS,and 1 mg/ml G418. Cell pellets were processed into microsomes asdescribed above. Activity was assessed as above with final concentra-tions of 2 mg/ml rat microsomes and 2 mMPGH2. PGE2 was measuredby EIA (500141; Cayman Chemical) at a dilution of 1:1000.

Human mPGES-2. Human mPGES-2 was obtained from OpenBiosystems (catalog no. MHS101 L-14465, clone ID 3946495) and wassubcloned into pET21d base vector. It was expressed in BL21CDE3cells. The enzyme was purified by nickel affinity and size exclusionchromatography. Activity was assessed in a buffer of 100 mM KPO4,pH 7.0, with 1 mM dithiothreitol. Final concentrations were 10 mg/mlhuman mPGES-2 and 2 mM PGH2. PGE2 was measured by EIA(500141; Cayman Chemical) at a dilution of 1:1000.

COX-1 and COX-2 Activity Assay. COX activity was assessedusing a commercially available kit utilizing ovine COX-1 and humanCOX-2 (560131; Cayman Chemical).

Enzyme-Inhibitor Reversibility Studies by a Rapid DilutionAssay. A rapid dilution assay was performed as previously describedto determine whether the inhibitor binding to the enzyme wasreversible (Copeland, 2005). Briefly, human mPGES-1 was dilutedinto buffer at 100� its usual assay concentration. Compounds (ordimethylsulfoxide) were added to the enzyme at 10� their respectiveIC50 values and incubated for 30 minutes at room temperature. PGH2

was diluted into buffer to give a 2 mM final concentration and a volumeequal to 100� the enzyme plus inhibitor volume was added to initiatethe reaction. At 20-second intervals, SnCl2 was added to stop thereactions. PGE2 wasmeasured by EIA (500141; CaymanChemical) at adilution of 1:1000.

A549 Epithelial Carcinoma Cell Assay. Human epithelial lungcarcinoma cell line A549 was purchased from American Type CultureCollection (CCL-185) and was maintained in Kaighn’s F12 plus 10%FBS in 5% CO2. For assay, cells were plated at 40,000/well in 96-wellFalcon plates (353072, Corning Incorporated, Corning, NY), 24 hoursprior to treatment. Compounds were diluted in dimethylsulfoxide andwere added at 1 ml/well (n 5 2), to give seven concentrations each.Cells were pretreated for 30 minutes at 37°C, 5% CO2. Recombinanthuman IL-1b (R&D Systems, Minneapolis, MN) was added to give0.2 ng/ml final. The treatment period was 18 hours. The conditionedmedium was assayed for levels of PGE2, PGD2, TxB2, and 6-ketoPGF1a by EIA (Cayman Chemical). The IC50 values were calculatedusing GraphPad Prism nonlinear regression sigmoidal dose responsecurve fitting (GraphPad Software Inc., La Jolla, CA). Data are themeans 6 S.D. of the indicated number of determinations.

Human Whole Blood Assay. Blood was collected from normalvolunteer donors into sodium heparin vacutainer tubes (BD, FranklinLakes, NJ). Donors had not taken NSAIDs, aspirin, celecoxib, orglucocorticoids within 2 weeks of the donation. Blood was distributedinto deep-well polypropylene plates and compounds were added. Theblood was pretreated at 37°C, 5% CO2, in a humidified atmosphere,loosely covered, for 30minutes; lipopolysaccharide (LPS) (serotype0111:B4; Sigma-Aldrich, St. Louis, MO) was then added to give a finalconcentration of 100 mg/ml. The plates were incubated for 20–24 hours,loosely covered, at 37°C, 5% CO2, in a humidified atmosphere, on anorbital shaker at 100 rpm.Theplateswere sealed tightlywith silicone capmats and were chilled on ice for 1 hour, then centrifuged at 1800 � g,10minutes,4°C, inanEppendorf5810Rcentrifuge (Eppendorf,Hauppauge,NY). Plasma was removed from the cell layer and transferred tov-bottom polypropylene plates. One hundred microliters was quanti-tatively transferred to Costar cluster tube blocks (4411, CorningIncorporated, Corning, NY) and 400 ml/well methanol/internal stan-dard was added. Solid phase extraction was performed using WatersHLB 30-mg/bed 96-well plates (Waters, Milford, MA), which weresubjected to liquid chromatography–tandemmass spectrometry analysis.Calibration curves were obtained by plotting the peak area ratio PGE2,PGF2a, TxB2, andrespective internal standardagainst the concentration.Aweighted (l/concentration) least-squares regression analysis was used toobtain a linear equation over the range of the calibration. The IC50 valueswerecalculatedusingGraphPadPrismnonlinear regressionsigmoidaldoseresponse (variable slope), with a fitted top of less than 1.5� the LPS controlanda fitted bottombetween zero and1.5� the reference standard.Dataarethe geometric means6 S.D. of determinations from six donors.

Intra-Articular Injection of LPS plus Tumor NecrosisFactor a. To assess the ability of mPGES-1 inhibitors to inhibitprostanoid production in the knee, a guinea pig model was used. Maleguinea pigs weighing around 300 g were first dosed subcutaneouslywith either vehicle (95% captex and 5% N-methyl-2-pyrrolidine(NMP), 50 mg/kg MF-63, or 30 mg/kg diclofenac (an NSAID used asa positive control). One hour postdose, animals were injected witheither 50 ml saline into both right and left knees or with 100 mg LPS(L2630 strain 0111:B4; Sigma) plus 50 ng tumor necrosis factor a

(TNFa) (5053-TG-025/CF; R&D Systems) in 50 ml saline into bothknees. Six hours after intra-articular injection, knee joints werelavaged to collect synovial fluid, and the fluid was measured forPGE2, PGI2, and PGF2a levels using EIA kits from Cayman Chemical.

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Monoiodoacetate (MIA) Pain Model. To assess pain efficacy,male Hartley guinea pigs (Charles River Laboratories, Burlington,MA) weighing approximately 200–250 g were used. To induce pain,the right knee of each guinea pigwas injectedwith 0.3mgMIA in 50mlsaline and the left knee with 50 ml saline. To test the efficacy ofcompounds, guinea pigs were either dosed 5 days (compound 1) or9 days (compound 2) after MIA injection with vehicle (10%Cremaphor

EL in saline), 30 mg/kg diclofenac (NSAID-positive control), two dosesof compound 1 (50 or 75 mg/kg), or two doses of compound 2 (10 or50 mg/kg). All dosing was subcutaneous at a dose volume of 5 ml/kgand the group size was six. Dose group was randomly assigned to eachanimal and dosing staggered by 10 minutes for each guinea pig. Painwas measured 4 hours postdosing via incapacitance testing. This testmeasures the difference in hind paw weight bearing between the

Fig. 1. Characterization of mPGES-1. (A) HEK-293 cells were transfected with cDNA for humanmPGES1; the microsomal and cytosolic fractionswere separated as described in the Materials andMethods. An aliquot was subjected to gel electro-phoresis, followed by immunoblot analysis withanti–mPGES-1 antibody (Cayman Chemical).Lane 1, starting material-cell homogenate; lane 2,low-speed supernatant fraction; lane 3, cytosolicfraction; and lane 4, microsomal fraction. (B) Themicrosomal fraction was assayed for mPGES-1activity using various concentrations of substrate(PGH2) and the PGE2 was quantitated by liquidchromatography–tandemmass spectrometry analysis.

Fig. 2. Chemical structure of mPGES-1inhibitors. Compounds 1 and 2 are newlydescribed mPGES-1 inhibitors. Celecoxib,rofecoxib (COX-2 inhibitors), and MF-63(a referencemPGES-1 inhibitor) were usedin some experiments.

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MIA- and saline-injected knees. For the following studies, each valuerepresents the average of three separate measurements, each ac-quired over a 1-second period for each animal, with the values thenaveraged for each treatment group. Data are presented as means withS.E.M. Data were evaluated by one-way analysis of variance. Groupswere compared with vehicle by Dunnett’s test with a Bonferronicorrection for comparison between groups. All statistical analyseswere performed using the JMP statistical analysis program (version 8;SAS Institute Inc., Cary, NC). Differences were considered to besignificant if the P value was less than 0.05.

ResultsPreparation and Characterization of mPGES-1 Enzyme.

Todevelop a reproduciblemPGES-1activity assay,HEK-293 cellswere transiently transfected with human mPGES-1 cDNA andmicrosomal and cytosolic fractions were evaluated for mPGES-1expression using immunoblot analysis. The results (Fig. 1A)demonstrate that the microsomal fraction contained mPGES-1.Theuntransfected cells containednobasalmPGES-1protein. Themicrosomal fraction was used to determine enzyme activity.The synthase activity was determined based on the ability of

mPGES-1 (microsomal preparation diluted in phosphatebuffer, pH 7) to convert PGH2 (substrate) to PGE2. A represen-tative example of the effects of various substrate concentra-tions on PGE2 production is shown in Fig. 1B. The Km value(13.2 mM) of the enzyme activity is comparable to the value of14 mM reported elsewhere (Ouellet et al., 2002).Identification of Novel and Selective mPGES-1 In-

hibitors. The in vitro enzyme activity assay was used toidentify and optimize novel chemical scaffolds as inhibitors.The structures of two optimizedmolecules (compounds 1 and 2)representing two different scaffolds, MF-63 (a reference

mPGES-1 inhibitor) and celecoxib, are shown in Fig. 2. Bothcompounds 1 and 2 (Fig. 3) demonstrate full efficacy (100%inhibition) and concentration-dependent inhibitory activityagainstmPGES-1 enzymewith IC50 values of 0.2416 0.085mMand0.0009460.0059mM, respectively. For comparison, the IC50

value of the reference mPGES-1 inhibitor was 0.0056 0.003 mM(data not shown). Compounds 1 and 2 show very little activityagainst isolated mPGES-2, COX-1, or COX-2. Furthermore,the COX-2 selective inhibitor (celecoxib) and nonselectiveNSAIDs (ibuprofen and diclofenac) show no activity againstmPGES-1 at the concentration tested. To determine relevantanimal species for evaluation as a diseasemodel, the compoundswere also tested against mPGES-1 from guinea pig and rat. Theinhibitors showed potent activity versus guinea pig mPGES-1but very poor activity versus rat mPGES-1 at the concentrationtested. A summary of enzyme activities of the mPGES-1inhibitors as well as celecoxib is shown in Table 1. Theseresults establish that the molecules are potent and selective.We also assessed whether the compounds bound to the

enzyme in a reversible fashion. A rapid dilution methodwas used to assess the nature of binding. mPGES-1 wasincubated with concentrations of the inhibitors that were10-fold higher than their respective IC50 values. Thesesolutions were then diluted 100-fold with the substratesolution resulting in inhibitor concentrations of one-tenth oftheir IC50 values. As shown in Fig. 4, the dilution resulted ina recovery of the enzyme activity over time. The resultsindicate that both compounds were reversible inhibitors.

Selective Inhibition of mPGES-1 in IL-1b–StimulatedA549 Cells. We next evaluated whether the mPGES-1inhibitors were effective in blocking PGE2 production in cellsin response to an inflammatory stimuli. The A549 human

Fig. 3. Concentration-dependent inhibition ofmPGES-1 activity by mPGES-1 inhibitors. Var-ious concentrations of compound 1 (A) or com-pound 2 (B) were first mixed with mPGES-1enzyme (microsomal preparation) followed theaddition of substrate PGH2. After a 2.5-minuteincubation at room temperature, the reactionwas stopped by the addition of SnCl2. Theproduct PGE2 was measured by liquid chroma-tography–tandem mass spectrometry.

TABLE 1In vitro characterization of mPGEs-1 inhibitorsData are presented as means 6 S.D.

Enzyme ActivityIC50 Value

Compound 1 Compound 2 Celecoxib

mMHuman mPGES-1 0.241 6 0.085 (n = 6) 0.000944 6 0.0059 (n = 10) .10Guinea pig mPGES-1 0.511 (n = l) 0.0044 6 0.0097 (n = 2) .10Rat mPGES-1 .100 54.5 6 16.3 (n = 2) .10Human mPGES-2 .100 1.33 6 0.92 (n = 2) n.d.COX-1 9% inhibition at 100 mM 21% inhibition at 10 mM 31% inhibition at 100 mMCOX-2 16% inhibition at 30 mM 21% inhibition at 30 mM 78% inhibition at 30 mM

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epithelial carcinoma cell line produces a variety of prostanoids(PGE2, PGI2, PGF2a, and PGD2) in response to IL-1 (Thorénand Jakobsson, 2000). Initially, we compared the effects of thereference mPGES-1 inhibitor (MF-63) and a COX-2 selectiveinhibitor (rofecoxib) on various prostanoids produced by IL-1treatedwithA549 cells. A549 cellswere pretreatedwith variousconcentrations of compounds for 30 minutes, followed by IL-1btreatment for an additional 18 hours, and the conditionedmedia were analyzed for various prostanoids using EIA. TheCOX-2 selective inhibitor rofecoxib blocked the production ofall prostanoids (PGE2, PGI2, PGF2a, and PGD2) in a dose-dependentmanner. The referencemPGES-1 inhibitor (MF-63)blocked PGE2 production (Fig. 5), but demonstrated varyinglevels of increase in other prostanoids, suggesting shuntingtoward these molecules. We next evaluated effects of

compounds 1 and 2 on IL-1b–treated A549 cells on PGE2 andPGI2 under similar conditions. The results (Fig. 6) demonstratethatmPGES1 inhibitors and celecoxibblockedPGE2production ina concentration-dependent manner. Compounds 1 and 2 demon-strated IC50 values of 0.87 6 0.423 mM and 0.012 6 0.006 mM,respectively. The mPGES-1 inhibitors also caused a 2- to3-fold increase in PGI2 levels, demonstrating a shuntingtoward other prostanoids. In comparison, celecoxib, a COX-2selective inhibitor, blocked the production of both prostanoids.These results established that mPGES-1 inhibitors wereeffective in blocking IL-1b–stimulated PGE2 production andthat the inhibitory effects were selective to PGE2 synthesis.Selective Inhibition of PGE2 Production in LPS-

Stimulated Human Whole Blood In Vitro. Previousstudies with NSAIDs and coxibs have shown a correlation

Fig. 4. Reversible inhibition of mPGES-1 activ-ity by the inhibitors. A 10-fold IC50 concentrationof each inhibitor was incubated with mPGES-1,followed by a 100-fold dilution with substrate,leaving a one-tenth–fold IC50 concentration ofinhibitor. This resulted in a recovery of enzymeactivity over time, indicating that these arereversible inhibitors. (A) Compound 1. (B) Com-pound 2.

Fig. 5. Characterization of prostanoid production in A549 cells. A549 cells were first treated for 30 minutes with various concentrations of either MF-63(reference mPGES-1 inhibitor) or rofecoxib (COX-2 inhibitor), followed by treatment with human IL-1b for 18 hours at 37°C in a humidified atmosphereof 95% air plus 5% CO2. The conditioned media were analyzed for PGE2 (A), PGI2 (B), TxB2 (C), PGF2a (D), and PGD2 (E) using respective EIA kits fromCayman Chemical. The results are expressed as the percent inhibition of respective prostanoids relative to IL-1b–treated control levels. The negativeinhibition indicates higher levels of production relative to IL-1b control values.

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between the in vitro human whole blood IC80 value and theplasma concentration achieved in vivo at clinically efficaciousdoses, thereby providing a basis for using biochemical potencyto predict analgesic efficacy (Huntjens et al., 2005). Therefore,we compared the activity of mPGES-1 inhibitors against thestandard of care, celecoxib, in human whole blood in vitro.Compounds were added to freshly collected human bloodobtained from normal volunteers who had not consumed anyanti-inflammatory drugs during the past 2 weeks. LPS(100 mg/ml) was added 30 minutes after the addition of thecompounds; after a 24-hour incubation at 37°C, the prosta-noids secreted into the plasma were quantified using liquidchromatography–tandem mass spectrometry. The results(Fig. 7) show that compound 1, compound 2, and celecoxib

blocked PGE2 production in a concentration-dependent man-ner. The IC50 values were 0.7926 0.267 mM, 0.0156 0.009 mM,and 0.551 6 0.490 mM, respectively. Once again, clear differ-ences were observed between mPGES-1 inhibitors andcelecoxib on other prostanoids. Whereas celecoxib blockedthe production of both PGF2a and TxB2, mPGES-1 inhibitorsshowed no inhibitory activity against either of these prosta-noids at the concentration tested. These results furtherdemonstrate that the two mPGES-1 inhibitors selectivelyblocked PGE2 production in whole human blood cells, withcompound 2 being more potent than celecoxib.mPGES-1 Inhibitors Were Efficacious in a Guinea

Pig MIA Model of Pain. We next wanted to evaluatewhether the mPGES-1 inhibitors were efficacious in an

Fig. 6. Selective inhibition of PGE2 activity in A549 cells by mPGES-1 inhibitors. A549 cells were pretreated for 30 minutes with various concentrationsof either compound 1 (A), compound 2 (B), or celecoxib (C), followed by treatment with human IL-1b for 18 hours at 37°C in a humidified atmosphere of95% air plus 5% CO2. The conditioned media were analyzed for PGE2 or PGI2 using respective EIA kits from Cayman Chemical. The results areexpressed as the percent inhibition of PGE2 or PGI2 relative to IL-1b–treated control levels. The negative inhibition indicates higher levels of productionrelative to IL-1b control values.

Fig. 7. Selective inhibition of PGE2 activity in human whole blood cells. (A–F) Freshly collected human blood obtained from normal volunteers, who hadnot consumed any anti-inflammatory medication for 2 weeks, was treated with various concentrations of compound 1, compound 2, or celecoxib for30 minutes, followed by LPS (100 mg/ml) for 24 hours. The plasma was separated from cells and the prostanoids in the plasma were assayed by liquidchromatography–tandem mass spectrometry after a solid phase extraction (refer to the Materials and Methods).

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animal model of pain that is known to be at least partiallymediated though PGE2 (Park et al., 2014). Previous studieshave suggested that a reference mPGES-1 inhibitor waseffective in a guinea pig MIA model of pain (Xu et al., 2008).Since compounds 1 and 2 did not inhibit rat mPGES-1 butwere effective against guinea pigmPGES-1 (Table 1), as a firststep we evaluated whether the reference mPGES-1 inhibitor(MF-63) was effective in blocking PGE2 production in the kneejoints of guinea pigs injected with TNFa plus LPS. Pre-liminary studies established that the optimal inflammationwas achieved by a combination of TNFa and LPS (data notshown). The guinea pigs were given either diclofenac (30 mg/kg)or MF-63 (50 mg/kg) by subcutaneous injection 1 hour priorto intra-articular injection of LPS plus TNFa. The jointfluid was collected by lavage with saline 6 hours after TNFaplus LPS injection and the lavage fluids were analyzedfor PGE2, PGI2, and PGF2a levels. The results demon-strate that the LPS plus TNFa stimulation of PGE2 wasblocked in animals dosed with the reference mPGES-1inhibitor or diclofenac (Fig. 8). Whereas diclofenac-treatedanimals showed suppression of other prostanoids (PGI2and PGF2a), the reference mPGES-1 inhibitor treatmentinhibited only PGE2. These results demonstrate that theselectivity of mPGES-1 was also observed in vivo at least forthe reference inhibitor.We next evaluated the ability of mPGES-1 inhibitors to

block the pain resulting from joint injury caused by the intra-articular injection of MIA. Previous studies have establishedthat the injection of MIA into the knee joint of rats and guineapigs produces an acute inflammatory insult, joint degenera-tion, and pain (Schwartz et al., 1981; Williams and Thonar,1989; Pomonis et al., 2005; Malfait et al., 2013). The painresulting from the joint injury can bemeasured via differentialweight bearing of the hind legs using an incapacitance tester.To evaluate the analgesic efficacy, MIA-injected guinea pigswere dosed with vehicle, mPGES-1 inhibitors at the indi-cated doses, or 30 mg/kg of the NSAID diclofenac (vehiclesaline). The pain was measured using incapacitance testing4 hours postdosing. The mPGES-1 inhibitors (compounds1 and 2) and diclofenac significantly inhibited pain versusvehicle, with the 75-mg/kg dose of compound 1 and the 50-mg/kgdose of compound 2 being significantly different from boththe low doses of the respective compounds and diclofenac(P , 0.05, Dunnett’s test with Bonferroni correction forcomparison between groups; Fig. 9). These results establishthat the mPGES-1 inhibitors were effective in a guinea pigmodel of pain.

DiscussionmPGES-1 is a terminal enzyme induced during inflam-

mation, is responsible for the production of PGE2, andis a potential target for effective analgesic and anti-inflammatory activity without causing side effects (Trebinoet al., 2003; Xu et al., 2008; Mbalaviele et al., 2010; Abdul-Malik et al., 2013; Bahia et al., 2014; Korotkova and Jakobsson2014). Here, we describe and characterize novel mPGES-1inhibitors that are potent, selective, and effective in a guineapig model of pain. The two molecules exemplified in this studyare potent against human, dog, and guinea pig mPGES-1enzymes and bind to the human enzyme in a reversiblemanner. They are highly selective and show no discernible

activity versus mPGES-2, COX-1, and COX-2 enzymes. Bothmolecules are effective in blocking PGE2 production in IL-1–stimulated A549 cells, as well as in LPS-stimulated human

Fig. 8. PGE2 inhibition after intra-articular injection of LPS plus TNFain guinea pigs. Male guinea pigs were first dosed subcutaneously witheither vehicle (95% captex and 5% NMP), 50 mg/kg MF-63, or 30 mg/kgdiclofenac. One hour postdosing, animals were injected with either 50 mlsaline into both right and left knees or with 100 mg LPS plus 50 ng TNFain 50 ml saline into both knees. The synovial fluid was collected from theknee joints by lavage 6 hours after intra-articular injection, and PGE2,PGI2, and PGF2a levels were determined using EIA kits from CaymanChemical.

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whole blood. Finally, they demonstrate efficacy in a guinea pigMIA model of pain.NSAIDs and COX-2 inhibitors have been extensively used

to treat the inflammation and pain associated with rheuma-toid arthritis and osteoarthritis but show significant sideeffects. Specifically, the cardiovascular side effects have beensuggested to be due to a general blockage of all prostanoids(FitzGerald and Patrono, 2001; Mukherjee et al., 2001; Friesand Grosser, 2005; Wang et al., 2005; Grosser et al., 2006).TxA2, a COX-1–mediated product produced in platelets, iscritical in vasoconstriction and platelet aggregation. Con-versely, COX-2–derived PGI2, produced in vascular smoothmuscle cells and endothelial cells, is a vasodilator and in-hibits platelet activation. Coxibs modulate the prothromboticTxA2 production only marginally, decrease the production ofantithrombotic PGI2, and create an alteration in the TxA2/PGI2ratio that favors the prothrombotic status (FitzGerald andPatrono, 2001; Mukherjee et al., 2001; Fries and Grosser,2005; Wang et al., 2005; Grosser et al., 2006). Because of thiscardiovascular liability, some coxibs have been withdrawnfrom the market (FitzGerald and Patrono, 2001; FitzGerald,2003). Thus, a significant need exists in developing saferalternatives to coxibs and NSAIDs.mPGES-1 is an inducible integral membrane protein and

acts as the terminal enzyme downstream of COX enzymes inproducing PGE2 from the intermediate PGH2. This enzyme is

normally coexpressed with COX-2 at very low levels in mosttissues, is induced by various inflammatory signals (e.g., IL-1and TNFa), and is upregulated in synovial tissue, cartilage,and chondrocytes of patients with osteoarthritis and rheuma-toid arthritis (Tanioka et al., 2000; Stichtenoth et al., 2001;Yamagata et al., 2001; Kojima et al., 2002, 2004; Lazarus et al.,2002; Claveau et al., 2003; Li et al., 2005). Two other enzymes,mPGES-2 and cytosolic PGES, also have been suggested to beinvolved in PGE2 production. mPGES-2 is expressed constitu-tively in several tissues along with COX-1 and is believed toplay a housekeeping function (Murakami et al., 2003). CytosolicPGES is present in the cytoplasm but its function in PGE2

production is poorly understood (Lovgren et al., 2007). There-fore, we have focused on identifying mPGES-1 inhibitors thatare highly potent, selective, and orally active in relevantpreclinical models.Traditionally, NSAIDs and COX-2 selective inhibitors have

been identified using in vitro enzyme activity, selectivityassays, human whole blood assays, and a variety of animalmodels that measure either pharmacodynamic end points suchas PGE2 levels or behavioral response such as nociception orhyperalgesia. Because the mPGES-1 inhibitors identified heredid not inhibit rat or mouse mPGES-l enzymes (Table 1), wewere unable to use traditional rodent animalmodels for efficacystudies. A meta-analysis of marketed NSAIDs and coxibssuggested that clinically efficacious doses of a variety of these

Fig. 9. Assessment of pain efficacy of mPGES-1 inhibitors in a guinea pig MIA model. Male Hartley guinea pigs were injected with 0.3 mg MIA in 50 mlsaline (right knee) or saline (left knee) and were dosed subcutaneously with compounds 5–9 days post-MIA injection. The compound doses were asfollows: vehicle (10% Cremaphor EL in saline), compound 1 (50 or 75 mg/kg), compound 2 (10 or 50 mg/kg), or diclofenac (30 mg/kg). The dose volume was5 ml/kg and the group size was six. Pain was measured 4 hours postdosing via incapacitance testing, as described in the Materials and Methods. Datawere evaluated by one-way analysis of variance and are presented as means (S.E.M.). Groups were compared with vehicle by Dunnett’s test with aBonferroni correction for comparison between groups. All statistical analyses were performed using the JMP statistical analysis program (version 8; SASInstitute Inc.). Differences were considered to be significant if the P value was less than 0.05 (*compared to vehicle/**compared to vehicle and diclofenac).

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drugs effectively blocked PGE2 production in LPS-stimulatedhuman whole blood at their respective IC80 concentrations(Huntjens et al., 2005).On this basis of observation,we initiateda biomarker-driven approach, which used IC50 and IC80 valuesfrom a human whole blood assay to determine compoundefficacy (Werner et al., 2002). Our goal was to first identifymolecules exhibiting high intrinsic potency in blocking PGE2

production in LPS-stimulated human whole blood. Further-more, a single oral dose of 200mg celecoxib in humans providesblood levels of drug that, at Cmax, reach the in vitro humanwhole blood IC80 value and exceed the IC50 value for duration of6–8 hours (Werner et al., 2002). Using these data, we soughtcompounds with high intrinsic potency that afforded exposurein rats approaching their IC80 values in the humanwhole bloodassay and remained above the human whole blood IC50 for6 hours or more after oral dosing (Schiffler et al., 2016).The initial assessment of enzyme inhibitory activity was

done by testing the ability of compounds to block mPGES-1activity of microsomal preparation of HEK-293 cells trans-fected with a human mPGES-1 cDNA. The immunoblot anal-ysis demonstrated the purity of the preparation and alsoshowed that the enzyme was present in the microsomalfraction, with very little being present in the cytosolic fraction(Fig. 1). The Km value of the enzyme preparation (13.2 nM) isin the range of reported activity for a similar preparation(Ouellet et al., 2002). The compounds bound to the enzyme in areversible manner (Fig. 4).PGE2 inhibition and selectivity was demonstrated in two

cell-based assays: 1) in IL-1b treated human epithelial cellcarcinoma cells (A549) and 2) in human whole blood treatedwith LPS. In both assays, the mPGES-1 inhibitors demon-strated selective inhibition of PGE2. The A549 cell line iscapable of synthesizing various prostanoids (PGE2, PGI2, andTxA2) in response to IL-1. Whereas NSAIDs and COX-2inhibitors blocked all prostanoids, mPGES-1 inhibitorsinhibited only the PGE2 production. Actives from A549 cellswere evaluated in human whole blood stimulated with LPS.Both compounds 1 and 2 demonstrate shunting toward

PGI2. As shown previously, shunting toward other prosta-noids is a mechanistic consequence of selective mPGES-1inhibition (Trebino et al., 2003).The biologic consequence ofshunting to PGI2 is unknown. Inhibition of PGI2 production orfunction is associated with adverse cardiovascular function(Flavahan, 2007; Arehart et al., 2008). However, it is impor-tant to consider that PGI2 shows paradoxical activities thatinclude both cardiovascular protective function as well asproinflammatory activity in arthritic models and conditions(Stitham et al., 2011). Prostacyclin-deficient mice are resis-tant to an inflammatory and arthritic challenge and prosta-cyclin inhibitors were efficacious in models of inflammationand arthritis (Honda et al., 2006; Pulichino et al., 2006). Bycontrast, COX-2, but not mPGES-1 deletion in mice, affordsprotection against thrombosis and hypertension (Yu et al.,2012; Chen et al., 2013). Thus far, cardiovascular protectionhas not yet been demonstrated using mPGES-1 inhibitors inanimal models because of the species selectivity issuesassociated with the compounds. Irrespective of the potentialdual role of PGI2, our results indicate that the mPGES-1inhibitors are effective in reducing pain in guinea pig modelsto a level comparable to an NSAID (diclofenac) at the testeddoses, although we do not know whether there is any PGI2produced in this model.

Although we did not use an animal model for compoundoptimization, we wanted to ensure that the key moleculeswere able to block a behavioral response that is known to be atleast partially mediated through PGE2. Since our compoundsdid not inhibit rat mPGES-1 at the concentration tested butwere effective against the guinea pig enzyme, we developed aguinea pig MIA model of pain. Initially we established that areference mPGES-1 inhibitor (MF-63) selectively inhibitedPGE2 production in guinea pig knee joints injected with LPSplus TNFa. by contrast, diclofenac, a traditional NSAID,blocked all prostanoids, further demonstrating in vivo evi-dence for the mechanism of action for this class of compounds.The two mPGES-1 inhibitors (compounds 1 and 2) wereefficacious in the guinea pig MIA model of pain.We do not know whether the therapeutic potential of

mPGES-1 inhibitors as anti-inflammatory/analgesic drugsalong with their potential safety features (cardiovascularand gastrointestinal) can be demonstrated in the clinic.Current options are restricted to NSAIDs and celecoxib, whichdisplay serious side effects. The availability of selective PGE2

inhibitors that do not alter the other prostanoids will facilitatethe evaluation of these molecules as safer alternatives toNSAIDs and coxibs.

Authorship Contributions

Participated in research design: Chandrasekhar, Harvey, Yu,Chambers, Fisher.

Conducted experiments: Harvey, Yu, Oskins, Lin, Seng,Thibodeaux.

Contributed new reagents or analytic tools: Harvey, Yu, Norman,Hughes, Schiffler, Fisher.

Performed data analysis: Chandrasekhar, Harvey, Yu, Chambers,Seng, Thibodeaux.

Wrote or contributed to the writing of the manuscript: Chandrase-khar, Harvey, Yu, Chambers, Schiffler, Fisher.

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Address correspondence to: Dr. Mark G. Chambers, Lilly ResearchLaboratories, Eli Lilly and Company, Lilly Corporate Center, Building 98CBasement, Drop Code 0403, Indianapolis, IN 46285. E-mail markgchambers@lilly.com

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