identification and characterization of novel microsomal...

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Identification and Characterization of Novel Microsomal Prostaglandin 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, Matthew J. Fisher

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 6, 2016 as DOI: 10.1124/jpet.115.228932

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Running Title

a. Running Title: Novel mPGES-1 inhibitors for Analgesia.

b. Corresponding author: Dr. Mark G. Chambers, Eli Lilly and Company, Lilly Corporate Center,

Building 98C Basement, Drop code 0403, Indianapolis, IN 46285. Phone 317-655-0361. Fax 317-651-

6333. Email [email protected]

c . Number of pages 31

d. Number of Tables 1

Number of Figures 9

Number of words

Abstract 163

Introduction 486

Materials and Methods 1408

Results 1400

Discussion 1333

d. Abbreviations

COX-1 = cyclooxygenase-1; COX-2 = cyclooxygenase-2

mPGES-1 and mPGES-2 = Microsomal prostaglandin E synthase 1 and 2

cPGES = cytosolic prostaglandin E synthase; Monoiodoacetate (MIA)

Human whole blood (HWB)

e. Recommended section: Inflammation, Immuno-pharmacology, and Asthma


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Abstract

Prostaglandin E2 (PGE2) plays a critical role in eliciting inflammation. Non-steroidal anti-

inflammatory drugs and selective inhibitors of cyclooxygenase, which block PGE2 production, have been

used as key agents in treating inflammation and pain associated with arthritis and other conditions.

However, these agents have significant side effects such as gastro-intestinal bleeding and myocardial

infarction, since they also block the production of prostanoids that are critical for other normal

physiological functions. Microsomal prostaglandin E2 synthase (mPGES-1) is a membrane bound

terminal enzyme in the prostanoid pathway, which acts downstream of cyclooxygenase 2 and is

responsible for PGE2 production during inflammation. Thus inhibition of this enzyme would be expected

to block PGE2 production without inhibiting other prostanoids and would provide analgesic efficacy

without the side-effects. In this report, we describe novel mPGES-1 inhibitors that are potent in blocking

PGE2 production and are efficacious in a guinea pig monoiodoacetate model of arthralgia. These

molecules may be useful in treating the signs and symptoms associated with arthritis.


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Introduction

Prostaglandins play critical physiological roles in a variety of organ functions and serve as key

mediators of inflammation, pain, and fever (Funk 2001, Smith 1989, Smyth et al 2009). PGE2, the most

prominent prostanoid, is produced by sequential enzymatic reactions starting with the release of

arachidonic acid from membrane glycerophospholipids by phospholipase A2, followed by conversion to

endoperoxide PGH2 by either cyclooxygenase-1 or cyclooxygenase-2 (COX-1 and COX-2, respectively),

and finally the isomerization of PGH2 to PGE2 by terminal prostaglandin E2 synthases (PGES). The

intermediate PGH2 also serves as a substrate for other synthases/isomerases leading to the production of

TxA2, PGI2, PGD2, and PGF2α (Funk 2001). The COX-1 isoform is constitutively expressed and is

responsible for the production of prostaglandins that preserve the gastric mucosa, while the COX-2

isoform is induced in response to cytokines in inflammatory conditions such as rheumatoid arthritis and

osteoarthritis (FitzGerald 2009, Smyth et al 2009, Sujimoto and Narumiya, 2007). Nonsteroidal anti-

inflammatory drugs (NSAIDs) and COX-2 selective drugs provide symptomatic relief by blocking the

production of PGE2 through inhibition of both COX-1 and COX-2 or COX-2 alone (Rainsford 2007).

However, NSAIDs are associated with gastrointestinal bleeding due to the inhibition of constitutively

produced PGE2, while COX-2 selective inhibitors have been associated with increased thrombotic risk

and myocardial infarction, which may be due to the inhibition of other inducible prostanoids such as

PGI2, which is required for proper cardiovascular function (FitzGerald and Petrono 2001, Wang et al

2005, Mukerjhee at al 2001, Fries and Grosser 2005, Grosser et al 2006). Thus, a selective inhibition of

PGE2 without adversely affecting other prostanoids would be expected to provide anti-inflammatory and

analgesic effects without the negative side effects.

Microsomal prostaglandin E synthases 1and 2 (mPGES-1 and mPGES-2) are terminal enzymes in

converting PGH2 to PGE2. mPGES-1 is expressed at low levels and is up regulated in a variety of

inflammatory conditions, while mPGES-2 is constitutively expressed in a variety of tissues (Jakobsson et

al 1999, Samuelsson et al 2007, Tanikawa et al 2002). Inhibition of mPGES-1 either by gene deletion or


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by pharmacological inhibition of activity demonstrates selective blockage of PGE2 production and

analgesic and anti-inflammatory activity (Trebino et al, 2003, Xu et al. 2008; Mbalaviele et al. 2010,

Abdul-Malik et al, 2013; Bahia et al. 2014, Korotkova and Jakobsson 2014). These observations suggest

that mPGES-1 plays a critical role in eliciting PGE2 mediated inflammatory response and that blocking

the enzyme activity is likely to provide analgesic and anti-inflammatory relief. Recently, we have

identified and described novel mPGES-1 inhibitors that are highly selective, potent, and are orally

available (Schiffler et al. 2015 a; Schiffler et al 2015 b). In this report, we show that these molecules are

selective in blocking PGE2 production, while exhibiting no inhibition of other prostanoids, such as PGI2,

at the concentrations tested. We also show that the compounds are effective in reducing pain in a guinea

pig model of knee joint pain.


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Materials and Methods

mPGES-1 inhibitors (Compound 1 and 2, reference mPGES-1 inhibitor MF-63).

The synthesis of MF-63 (Xu et al. 2008) and compounds 1 and 2 have been described (Schiffler

et al. 2015 a; Schiffler et al 2015 b)

Human mPGES-1

Human mPGES-1 was purchased from Invitrogen (Cat# 97002RG, clone ID 6314722), was

subcloned into pcDNA3.1, and was transiently expressed in HEK 293 cells. Microsomes were prepared

from cell pellets based on published methods (Ouellet et al, 2002, Thoren 2003). In brief, cell pellets were

sonicated 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 centrifuged at 5,000 x g for 10 min at 4oC. The supernatant fraction was

loaded into Beckman Quickseal tubes (342413) and centrifuged at 185,000 x g for 90 min at 4oC using

70.1 Ti rotor. Pellets were resuspended in a buffer of 10 mM sodium phosphate, pH 7.0; 10 % glycerol,

2.5 mM glutathione; and Complete protease inhibitor cocktail (Roche). Final concentrations were 4.4

μg/mL microsomes and 1.69 μM PGH2. All dilutions were made using the above buffer. After a 2.5

minute incubation at room temperature, 2.5 μL/well SnCl2 in 0.5 N HCI was added to stop the reaction.

PGE2 was quantitated by standard LC/MS analysis.

Guinea pig mPGES-1

Guinea pig mPGES-1 was cloned from IL-1 stimulated 104C1 (ATCC: CRL- 1405) by 5'-RACE

and was subcloned into pQCXIN. HEK-293 cells were infected with the plasmid for 24 hours, and then

expanded under selection in DMF/F12 3:1 (Invitrogen) + 10% FBS + 1 mg/mL G418. Cell pellets were

processed into microsomes as described above. Activity was assessed as above with final concentrations

of 15 μg/mL guinea pig mPGES-1 microsomes and 2 μM PGH2. PGE2 was measured by enzyme

immunoassay (EIA, Cayman 500141) at a dilution of 1:1000.


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Rat mPGES-1

Rat mPGES-1 cDNA was purchased from Open Biosystems (Cat# MRN1768- 99238049, clone

ID 7456259) and was subcloned into pQCXIN. HEK-293 cells were infected with the plasmid for 24

hours, and then expanded under selection in DME/F12 3:1 (Invitrogen) + 10%FBS + 1 mg/mL G418.

Cell pellets were processed into microsomes as described above. Activity was assessed as above with

final concentrations of 2 μg/mL rat microsomes and 2 μM PGH2 in 50 μL of buffer. PGE2 was measured

by EIA (Cayman 500141) at a dilution of 1:1000.

Human mPGES-2

Human mPGES-2 was obtained from Open Biosystems (Cat# MHS101 L-14465, Clone ID

3946495) and was subcloned into pET21d base vector. It was expressed in BL21CDE3 cells. The enzyme

was purified by nickel affinity and size exclusion chromatography. Activity was assessed in a buffer of

100 mM KPO4, pH 7.0, with 1 mM dithiothreitol. Final concentrations were 10 μg/mL human mPGES-2

and 2 μM PGH2. PGE2 was measured by EIA (Cayman 500141) at a dilution of 1:1000.

COX-1 and -2 Activity Assay

COX activity was assessed using a commercially available kit utilizing ovine COX-1 and human

COX-2 (Cayman 560131).

Enzyme-Inhibitor Reversibility Studies by a Rapid Dilution Assay

A rapid dilution assay was performed as previously described in order to determine whether the

inhibitor binding to the enzyme was reversible (Copeland, 2005). Briefly, human mPGES-1 was diluted

into buffer at 100x its usual assay concentration. Compounds (or DMSO) were added to the enzyme at

10x their respective IC50s and incubated for 30 minutes at room temperature. PGH2 was diluted into

buffer to give a 2 μM final concentration and a volume equal to 100x the enzyme + inhibitor volume was


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added to initiate the reaction. At 20 second intervals, SnCl2 was added to stop the reactions. PGE2 was

measured by EIA (Cayman 500141) at a dilution of 1:1000.

A549 Epithelial Carcinoma Cell assay

Human epithelial lung carcinoma cell line A549 was purchased from ATCC (CCL-185) and was

maintained in Kaighn's F12 + 10% FBS, in 5% CO2. For assay, cells were plated at 40,000/well in 96 well

Falcon plates, 24 hours prior to treatment. Compounds were diluted in DMSO and were added at 1

μL/well, n=2, to give seven concentrations each. Cells were pretreated for 30 minutes at 37oC, 5%CO2.

rhIL-1β (R&D Systems) was added to give 0.2 ng/mL final. The treatment period was 18 hours. The

conditioned medium was assayed for levels of PGE2 PGD2, TxB2 and 6-keto PGF1α by EIA (Cayman).

The IC50 values were calculated using Graphpad Prism nonlinear regression sigmoidal dose response

curve fitting. Data are the mean ± sd of the indicated number of determinations.

Human Whole Blood Assay

Blood was collected from normal volunteer donors into sodium heparin vacutainer tubes (BD).

Donors had not taken NSAIDs, aspirin, Celebrex, or glucocorticoids within two weeks of the donation.

Blood was distributed into deep well polypropylene plates and compounds were added. The blood was

pretreated at 37oC, 5% CO2, in a humidified atmosphere, loosely covered, for 30 minutes, then LPS

(Sigma, Serotype 0111:B4) was added to give a final concentration of 100 μg/mL. The plates were

incubated for 20-24 hours, loosely covered, at 37oC, 5% CO2, in a humidified atmosphere, on an orbital

shaker at 100 rpm. The plates were sealed tightly with silicone cap mats and were chilled on ice for 1

hour, then centrifuged at 1800 x g, 10 minutes, 4oC, in an Eppendorf 5810R centrifuge. Plasma was

removed from the cell layer and transferred to v-bottom polypropylene plates. One hundred microliters

was quantitatively transferred to Costar cluster tube blocks and 400 μL/well methanol/internal standard

was added. Solid phase extraction was performed using Waters HLB 30mg/bed 96 well plates and

subjected to LC/MS/MS analysis. Calibration curves were obtained by plotting the peak area ratio PGE2,


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PGF2a, TxB2, and respective internal standard against the concentration. A weighted (l/concentration)

least squares regression analysis was used to obtain a linear equation over the range of the calibration.

The IC50 values were calculated using Graphpad Prism nonlinear regression sigmoidal dose response

(variable slope), with a fitted top of less than 1.5x the LPS control and a fitted bottom between zero and

1.5x the reference standard. Data are the geometric mean ± sd of determinations from six donors.

Intra-articular injection of LPS plus TNFα

To assess the ability of mPGES-1 inhibitors to inhibit prostanoid production in the knee a guinea

pig model was used. Male guinea pigs of around 300g were first dosed subcutaneously with either vehicle

(95% captex and 5% NMP), 50 mg/kg MF-63 or 30 mg/kg diclofenac (an NSAID used as a positive

control). One hour post dose animals were injected with either 50 μL of saline into both right and left

knees or with 100 µg lipopolysaccharide (LPS-Sigma L2630 strain 0111:B4) plus 50 ng TNFα (R&D

Systems 5053-TG-025/CF) in 50 μL saline into both knees. Six hours post intra-articular injection, knee

joints were lavaged to collect synovial fluid, and the fluid was measured for PGE2, PGI2, and PGF2α levels

using EIA kits from Cayman.

Monoiodoacetate (MIA) pain model

To assess pain efficacy, male Hartley guinea pigs (Charles River) of approximately 200-250

grams were used. To induce pain, the right knee of each guinea pig was injected with 0.3 mg MIA in 50

µL of saline and the left knee with 50 µL of saline. To test the efficacy of compounds, guinea pigs were

either dosed 5 days (compound 1) or 9 days (compound 2) post MIA injection with vehicle (10%

Cremaphor EL in saline), 30 mg/kg of diclofenac (NSAID-positive control), two doses of compound 1

(50 or 75 mg/kg) or 2 doses of compound 2 (10 or 50 mg/kg). All dosing was subcutaneous at a dose

volume of 5 mL/kg and the group size was n=6. Dose group was randomly assigned to each animal and

dosing staggered by 10 minutes for each guinea pig. Pain was measured 4 hours post dosing via

incapacitance testing. This test measures the difference in hind paw weight bearing between the MIA and


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saline injected knees, and for the following studies each value represents the average of 3 separate

measurements, each acquired over a 1 second period for each animal, with the values then averaged for

each treatment group. Data are presented as means with standard error of the means (SEM). Data were

evaluated by one way analysis of variance (ANOVA). Groups were compared to vehicle by Dunnett's test

with a Bonferroni correction for comparison between groups. All statistical analyses were performed

using JMP (version 8) statistical analysis program (SAS Institute Inc., NC). Differences were considered

to be significant if the P value was less than 0.05.


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Results

Preparation and Characterization of mPGES-1 Enzyme

In order to develop a reproducible mGES-1 activity assay, HEK293 cells were transiently

transfected with human mPGES-1 cDNA and microsomal and cytosolic fractions were evaluated for

mPGES-1 expression using immunoblot analysis. The results (Fig 1 A) demonstrate that the microsomal

fraction contained mPGES-1. The untransfected cells contained no basal mPGES-1 protein. The

microsomal fraction was used to determine enzyme activity.

The synthase activity was determined based on the ability of mPGES-1 (microsomal preparation

diluted in phosphate buffer, pH 7) to convert PGH2 (substrate) to PGE2. A representative example of the

effects of various substrate concentrations on PGE2 production is shown in (Figure 1 B). The Km value

(13.2 µM) of the enzyme activity is comparable to the value of 14 µM reported elsewhere (Ouellet et al

2002).

Identification of Novel and Selective mPGES-1 Inhibitors

The in vitro enzyme activity assay was utilized to identify and optimize novel chemical scaffolds

as inhibitors. The structures of two optimized molecules (Compound 1 and Compound 2) representing

two different scaffolds, MF-63 (a reference mPGES-1 inhibitor), and celecoxib are shown in Figure 2.

Both compounds 1 and 2 (Figure 3 A and B) demonstrate full efficacy (100% inhibition) and

concentration dependent inhibitory activity against mPGES-1 enzyme with IC50 values of 0.241 ± 0.085

μM and 0.00094 ± 0.0059 μM, respectively. For comparison, the IC50 value of the reference mPGES-1

inhibitor was 0.005 ± 0.003 µM (data not shown). Compounds 1 and 2 show very little activity against

isolated mPGES-2, COX-1, or COX-2. Further, the COX-2 selective inhibitor (celecoxib) and non-

selective NSAIDs (ibuprofen and diclofenac) show no activity against mPGES-1 at the concentration

tested. To determine relevant animal species for evaluation as a disease model, the compounds were also

tested against mPGES-1 from guinea pig and rat. The inhibitors showed potent activity versus guinea pig


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mPGES-1 but very poor activity versus rat mPGES-1 at the concentration tested. A summary of enzyme

activities of the mPGES-1 inhibitors as well as celecoxib are shown in Table 1. These results establish

that the molecules are potent and selective.

We also assessed whether or not the compounds bound to the enzyme in a reversible fashion. A

rapid dilution method was used to assess the nature of binding. mPGES-1 was incubated with

concentrations of the inhibitors that were 10 fold higher than their respective IC50 values. These solutions

were then diluted 100 fold with the substrate solution resulting in inhibitor concentrations of 1/10 their

IC50 values. As shown in Figures 4A and 4B, the dilution resulted in a recovery of the enzyme activity

over time. The results indicate that both compounds were reversible inhibitors.

Selective Inhibition of mPGES-1 in IL-1β Stimulated A549 Cells

We next evaluated whether the mPGES-1 inhibitors were effective in blocking PGE2 production

in cells in response to an inflammatory stimuli. The human epithelial carcinoma cell line A549 produces a

variety of prostanoids (PGE2, PGI2, PGF 2α, and PGD2) in response to IL-1 (Thoren et al 2000). Initially,

we compared the effects of the reference mPGES-1 inhibitor (MF-63) and a COX-2 selective inhibitor

(rofecoxib) on various prostanoids produced by IL-1 treated with A549 cells. A549 cells were pre-treated

with various concentrations of compounds for 30 min, followed by IL-1β treatment for an additional 18 h,

and the conditioned media were analyzed for various prostanoids using EIA. The COX-2 selective

inhibitor rofecoxib blocked the production of all prostanoids (PGE2, PGI2, PGF2α, and PGD2) in a dose-

dependent manner. The reference mPGES-1 inhibitor (MF-63) blocked PGE2 production (Fig 5 A-E), but

demonstrated varying levels of increase in other prostanoids suggesting shunting towards these

molecules. We next evaluated effects of compounds 1 and 2 on IL-1β treated A549 cells on PGE2 and

PGI2 under similar conditions. The results (Fig 6 A, B, and C) demonstrate that mPGES1 inhibitors and

celecoxib blocked PGE2 production in a concentration dependent manner. Compounds 1 and 2

demonstrated IC50 values of 0.87 ± 0.423 μM and 0.012 ± 0.006 μM, respectively. The mPGES-1


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inhibitors also caused a 2-3 fold increase in PGI2 levels, demonstrating a shunting towards other

prostanoids. In comparison, celecoxib, a COX-2 selective inhibitor blocked the production of both

prostanoids. These results established that mPGES-1 inhibitors were effective in blocking IL-1β

stimulated PGE2 production, and that the inhibitory effects were selective to PGE2 synthesis.

Selective Inhibition of PGE2 Production in LPS Stimulated Human Whole Blood in vitro

Previous studies with NSAIDs and coxibs have shown a correlation between the in vitro human

whole blood IC80 and the plasma concentration achieved in vivo at clinically efficacious doses, thereby

providing a basis for using biochemical potency to predict analgesic efficacy (Huntjens et al 2005).

Therefore, we compared the activity of mPGES-1 inhibitors against the standard of care, celecoxib, in

human whole blood in vitro. Compounds were added to freshly collected human blood obtained from

normal volunteers who had not consumed any anti-inflammatory during the past 2 weeks. LPS (100

μg/mL) was added 30 min after the addition of the compounds, and after a 24 h incubation at 37oC, the

prostanoids secreted into the plasma were quantified using LC/MS/MS. The results (Fig. 7) show that

compound 1, compound 2, and celecoxib blocked PGE2 production in a concentration dependent manner.

The IC50 values are 0.792 ± 0.267 μM; 0.015 ± 0.009 μM; and 0.551 ± 0.490 μM, respectively. Once

again, clear differences were observed between mPGES-1 inhibitors and celecoxib on other prostanoids.

While celecoxib blocked the production of both PGF2α and TxB2, mPGES-1 inhibitors showed no

inhibitory activity against either of these prostanoids at the concentration tested. These results further

demonstrate that the two mPGES-1 inhibitors selectively blocked PGE2 production in whole human blood

cells with compound 2 being more potent than celecoxib.

mPGES-1 Inhibitors were Efficacious in Guinea Pig Monoiodoacetate Model of Pain

We next wanted to evaluate whether or not the mPGES-1 inhibitors were efficacious in an animal

model of pain that is known to be at least partially mediated though PGE2 (Park et al 2014). Previous

studies have suggested that a reference mPGES-1 inhibitor was effective in a guinea pig monoiodoacetate


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model of pain (Xu et al. 2008). Since compounds 1 and 2 did not inhibit rat mPGES-1, but were effective

against guinea pig mPGES-1 (Table 1), as a first step we evaluated whether the reference mPGES-1

inhibitor (MF-63) was effective in blocking PGE2 production in the knee joints of guinea pigs injected

with TNFα + LPS. Preliminary studies established that the optimal inflammation was achieved by a

combination of TNFα and LPS (data not shown). The guinea pigs were given either diclofenac, (30

mg/kg) or MF-63 (50 mg/kg) by subcutaneous injection 1 hr prior to intra-articular injection of LPS +

TNFα. The joint fluid was collected by lavage with saline 6 h post TNFα+LPS injection and the lavage

fluids were analyzed for PGE2, PGI2, and PGF2α levels. The results demonstrate that the LPS+TNFα

stimulation of PGE2 was blocked in animals dosed with the reference mPGES-1 inhibitor or diclofenac

(Fig 8). While diclofenac treated animals showed suppression of other prostanoids (PGI2 and PGF2α), the

reference mPGES-1 inhibitor treatment inhibited only PGE2. These results demonstrate that the selectivity

of mPGES-1 was also observed in vivo at least for the 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 monoiodoacetate. Previous studies have established that the

injection of monoiodoacetic acid (MIA) into the knee joint of rats and guinea pigs produces an acute

inflammatory insult, joint degeneration, and pain (Schwartz et al, 1981, Williams and Thonar, 1989,

Pomonis et al 2005, Malfait et al, 2013). The pain resulting from the joint injury can be measured via

differential weight bearing of the hind legs using an incapacitance tester. In order to evaluate the

analgesic efficacy, MIA-injected guinea pigs were dosed with vehicle, mPGES-1 inhibitors at the

indicated doses, or 30 mg/kg of the NSAID diclofenac (vehicle saline). The pain was measured using

incapacitance testing 4 hours post dosing. The mPGES-1 inhibitors (compounds 1 and 2) and diclofenac

significantly inhibited pain versus vehicle, with the 75 mg/kg dose of compound 1 and 50mg/kg dose of

compound 2 being significantly different from both the low doses of the respective compounds and

diclofenac (p<0.05 Dunnett's test with Bonferroni correction for comparison between groups; Figure 9).

These results establish that the mPGES-1 inhibitors were effective in a guinea pig model of pain.


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Discussion

mPGES-1 is a terminal enzyme induced during inflammation, is responsible for the production of

PGE2 and is a potential target for effective analgesic and anti-inflammatory activity without causing side

effects (Trebino et al, 2003, Xu et al. 2008; Mbalaviele et al. 2010, Abdul-Malik et al, 2013; Bahia et al.

2014, Korotkova and Jakobsson 2014). In this paper we describe and characterize novel mPGES-1

inhibitors that are potent, selective, and are effective in a guinea pig model of pain. The two molecules

exemplified in this paper are potent against human, dog, and guinea pig mPGES-1 enzymes and bind to

the human enzyme in a reversible manner. They are highly selective and show no discernible activity

versus mPGES-2, COX-1, and COX-2 enzymes. Both molecules are effective in blocking PGE2

production in IL-1 stimulated A549 cells, as well as in LPS stimulated human whole blood. Finally, they

demonstrate efficacy in a guinea pig monoiodoacetate (MIA) model of pain.

NSAIDs and COX-2 inhibitors have been extensively used to treat the inflammation and pain

associated with rheumatoid arthritis and osteoarthritis but show significant side-effects. Specifically, the

cardiovascular side effects have been suggested to be due to a general blockage of all prostanoids

(FitzGerald and Petrono 2001; Wang et al.2005; Mukerjhee at al 2001; Fries and Grosser 2005; Grosser et

al, 2006). Thromboxane A2, a COX-1 mediated product produced in platelets, is critical in

vasoconstriction and platelet aggregation. Conversely, COX-2 derived PGI2, produced in vascular smooth

muscle cells and endothelial cells, is a vasodilator and inhibits platelet activation. Coxibs modulate the

prothrombotic TxA2 production only marginally, decrease the production of antithrombotic PGI2, and

create an alteration in the TxA2/PGI2 ratio that favors the prothrombotic status (FitzGerald and Petrono

2001, Wang et al 2005, Mukerjhee at al 2001, Fries and Grosser 2005, Grosser et al 2006). Because of

this cardiovascular liability, some coxibs have been withdrawn from the market (FitzGerald 2003,

FitzGerald and Patrono 2001). Thus a significant need exists in developing safer alternatives to coxibs

and NSAIDs.


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mPGES-1 is an inducible integral membrane protein and acts as the terminal enzyme downstream

of COX enzymes in producing PGE2 from the intermediate PGH2. This enzyme is normally co-expressed

with COX-2 at very low levels in most tissues, is induced by various inflammatory signals such as IL-1,

and TNFα, and is up regulated in synovial tissue, cartilage, and chondrocytes of osteoarthritis and

rheumatoid arthritis patients (Tanioka 2000; Stichtenoth 2001; Yamagata 2001; Kojima 2002; Lazarus,

2002, Claveau 2003; Kojima 2004; Li 2005). Two other enzymes, mPGES-2 and cPGES, also have been

suggested to be involved in PGE2 production. mPGES-2 is expressed constitutively in several tissues

along with COX-1 and is believed to play a house-keeping function (Murakami 2003). cPGES is present

in the cytoplasm but its function in PGE2 production is poorly understood (Lovgren et al 2007).

Therefore, we have focused on identifying mPGES-1 inhibitors that are highly potent, selective, and

orally active in relevant pre-clinical models.

Traditionally, NSAIDs and COX-2 selective inhibitors have been identified using in vitro enzyme

activity, selectivity assays, human whole blood assay, and a variety of animal models that measure either

pharmacodynamic end points such as PGE2 levels or behavioral response such as nociception or

hyperalgesia. Because the mPGES-1 inhibitors identified here did not inhibit rat or mouse mPGES-l

enzymes (Table 1), we were unable to utilize traditional rodent animal models for efficacy studies. A

meta analysis of marketed NSAIDs and coxibs suggested that clinically efficacious doses of a variety of

these drugs effectively blocked PGE2 production in LPS-stimulated human whole blood at their

respective IC80 concentrations (Huntjens et al. 2005). Based on this observation we initiated a biomarker-

driven approach which utilized IC50 and IC80 values from a human whole blood assay to determine

compound efficacy (Werner et al. 2002). Our goal was to first identify molecules exhibiting high intrinsic

potency in blocking PGE2 production in LPS-stimulated human whole blood. Further, a single oral dose

of 200 mg of celecoxib in humans provides blood levels of drug that, at Cmax, reach the in vitro human

whole blood IC80 value and exceed the lC50 value for duration of 6-8 hours (Werner et al. 2002). Using

these data, we sought compounds with high intrinsic potency that afforded exposure in rats approaching


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their IC80 values in the human whole blood assay and remained above the HWB IC50 for 6 hrs or more

after oral dosing (Schiffler et al. 2015 a; Schiffler et al. 2015 b).

The initial assessment of enzyme inhibitory activity was done by testing the ability of compounds

to block mPGES-1 activity of microsomal preparation of HEK293 cells transfected with a human

mPGES-1 cDNA. The immunoblot analysis demonstrated the purity of the preparation and also showed

that the enzyme was present in the microsomal fraction with very little being present in the cytosolic

fraction (Figure 1). The Km value of the enzyme preparation (13.2 nM) is in the range of reported activity

for similar preparation (Ouellet et al. 2002). The compounds bound to the enzyme in a reversible manner

(Fig 4).

PGE2 inhibition and selectivity was demonstrated in two cell-based assays: a) in an IL-1β treated

human epithelial cell carcinoma cells (A549) and b) human whole blood treated with LPS. In both the

assays, the mPGES-1 inhibitors demonstrate selective inhibition of PGE2. The A549 cell line is capable of

synthesizing various prostanoids (PGE2, PGI2, and TxA2) in response to IL-1. While NSAIDS and COX-2

inhibitors blocked all prostanoids, mPGES-1 inhibitors inhibited only the PGE2 production. Actives from

A549 cells were evaluated in human whole blood stimulated with LPS.

Both compounds 1 and 2 demonstrate shunting towards PGI2. As shown before, shunting towards

other prostanoids is a mechanistic consequence of selective mPGES-1 inhibition (Trebino et al. 2003).The

biological consequence of shunting to PGI2 is unknown. Inhibition of PGI2 production or function is

associated with adverse cardiovascular function (Flavahan 2001; Arehart et al, 2008). However, it is

important to consider that PGI2 shows paradoxical activities that include both cardiovascular protective

function as well as pro-inflammatory activity in arthritic models and conditions (Stitham et al. 2011).

Prostacyclin deficient mice are resistant to an inflammatory and arthritic challenge and prostacyclin

inhibitors were efficacious in models of inflammation and arthritis ((Honda et al. 2006; Pulichino et al.

2006). In contrast, COX-2, but not mPGES-1 deletion in mice, affords protection against thrombosis and


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hypertension (Yu et al, 2012, Chen et al, 2013). So far, cardiovascular protection has yet not been

demonstrated using mPGES-1 inhibitors in animal models because of the species selectivity issues

associated with the compounds. Irrespective of the potential dual role of PGI2, our results indicate that the

mPGES-1 inhibitors are effective in reducing pain in guinea pig models to a level comparable to an

NSAID, diclofenac at the tested doses, although we do not know whether there is any PGI2 produced in

this model.

Although we did not utilize an animal model for compound optimization, we wanted to ensure

that the key molecules were able to block a behavioral response that is known to be at least partially

mediated through PGE2. Since our compounds did not inhibit rat mPGES-1 at the concentration tested,

but was effective against the guinea pig enzyme, we developed a guinea pig monoiodoacetate model of

pain. Initially we established that a reference mPGES-1 inhibitor MF-63 selectively inhibited PGE2

production in guinea pig knee joints injected with LPS+TNFα. In contrast, diclofenac, a traditional

NSAID, blocked all prostanoids, further demonstrating in vivo evidence for the mechanism of action for

this class of compounds. The two mPGES-1 inhibitors (compound 1 and 2) were efficacious in the guinea

pig MIA model of pain.

We do not know whether the therapeutic potential of mPGES-1 inhibitors as anti-

inflammatory/analgesic drug along with its potential safety features (cardiovascular and GI) can be

demonstrated in the clinic. Current options are restricted to NSAIDs and celecoxib which display serious

side-effects. The availability of selective PGE2 inhibitors that do not alter the other prostanoids will

facilitate the evaluation of these molecules as safer alternatives to NSAIDs and Coxibs.


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Author Contribution

Participated in Research Design: Srinivasan Chandrasekhar, Anita K Harvey, Xiao-Peng Yu , Mark Chambers, Matthew J. Fisher

Conducted Experiments: Anita K. Harvey, Xiao-Peng Yu, Thomas W. Seng, Stefan J. Thibodeaux, Jennifer L. Oskins, Chaohua Lin

Contributed new reagents: Xiao-Peng Yu, Anita Harvey, Bryan H. Norman, Norman E. Hughes, Matthew A. Schiffler, Matthew J. Fisher

Performed data Analysis: Anita K. Harvey, Xiao-Peng Yu, Thomas W. Seng, Stefan J. Thibodeaux, Srinivasan Chandrasekhar, Anita K Harvey, Mark Chambers

Wrote or contributed to the writing: Srinivasan Chandrasekhar, Anita K Harvey, Xiao-Peng Yu, Mark Chambers, Matthew A. Schiffler, Matthew J. Fisher


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Footnote

Please address all correspondence to

Dr. Mark G. Chambers, Endocrine Division, Lilly Research Laboratories, DC 0403

Indianapolis, IN 46285. [email protected]


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Figure Legends

Figure 1. Characterization of mPGES-1

Panel A. HEK293 cells were transfected with cDNA for human mPGES1; the microsmal and cytosolic

fractions were separated as described in Materials and Methods. An aliquot was subjected to gel

electrophoresis followed by immunoblot analysis with anti mPGES-1 antibody (Cayman). Lanes 1=

starting material-cell homogenate; Lane 2 = low speed supernatant fraction; lane 3 = cytosolic fraction;

lane 4 = microsomal fraction

Panel B. The microsomal fraction was assayed for mPGES-1 activity using various concentrations of

substrate (PGH2) and the PGE2 was quantitated by LC/MS/MS analysis.

Figure 2. Chemical Structure of mPGES-1 Inhibitors

Compounds 1 and 2 are newly described mPGES-1 inhibitors. Celecoxib, Rofecoxib (Cox-2 inhibitors)

and MF-63, a reference mPGES-1 inhibitor, were used in some experiments.

Figure 3. Concentration-dependent Inhibition of mPGES-1 Activity by mPGES-1 Inhibitors

Various concentrations of compound 1 or compound 2 were first mixed with mPGES-1 enzyme

(microsomal preparation) followed the addition of substrate PGH2. After a 2.5 min incubation at room

temperature, the reaction was stopped by the addition of SnCl2. The product PGE2 was measured by

LC/MS/MS. Panel A = Compound 1; Panel B = Compound 2

Figure 4. Reversible Inhibition of mPGES-1 Activity by the inhibitors

A 10 fold IC50 concentration of each inhibitor was incubated with mPGES-1 followed by a 100 fold

dilution with substrate, leaving a 1/10 fold IC50 concentration of inhibitor. This resulted in a recovery of

enzyme activity over time, indicating that these are reversible inhibitors. Panel A = Compound 1; Panel B

= Compound 2.


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Figure 5. Characterization of Prostanoid Production in A549 Cells

A549 cells were first treated for 30 min with various concentrations of either MF-63 (reference mPGES-1

inhibitor) or Rofecoxib (COX-2 inhibitor), followed by treatment with hIL-1β for 18 h at 37oC in a

humidified atmosphere of 95% air+5% CO2. The conditioned media were analyzed for PGE2 (Panel A)

PGI2 (Panel B), TxB2 (Panel C), PGF2α (Panel D), and PGD2 (Panel E) using respective EIA kits from

Cayman. The results are expressed as % inhibition of respective prostanoids relative to IL-1 β treated

control levels. The negative inhibition indicates higher levels of production relative to IL-1 β control

values.

Figure 6. Selective Inhibition of PGE2 Activity in A549 Cells by mPGES-1 Inhibitors

A549 cells were pre-treated for 30 min with various concentrations of either compound 1 (Panel A),

compound 2 (Panel B), or celecoxib (Panel C) followed by treatment with hIL-1β for 18 h at 37oC in a

humidified atmosphere of 95% air+5% CO2. The conditioned media were analyzed for PGE2 or PGI2

using respective EIA kits from Cayman. The results are expressed as % inhibition of PGE2 or PGI2

relative to IL-1 β treated control levels. The negative inhibition indicates higher levels of production

relative to IL-1 β control values.

Figure 7. Selective Inhibition of PGE2 Activity in Human Whole Blood Cells

Freshly collected human blood obtained from normal volunteers, who had not consumed any anti-

inflammatory medication for two weeks, was treated with various concentrations of compounds 1, 2, or

celecoxib for 30 min, followed by LPS (100 μg/mL) for 24 hours. The plasma was separated from cells

and the prostanoids in the plasma were assayed by LC/MS/MS after a solid phase extraction (refer to

Materials and Methods).

Figure 8. PGE2 inhibition after Intra-articular injection of LPS plus TNFα in guinea pigs


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Male guinea pigs were first dosed subcutaneously with either vehicle (95% captex and 5% NMP), 50

mg/kg MF-63 or 30 mg/kg diclofenac. One hour post dosing, animals were injected with either 50 μL of

saline into both right and left knees or with 100 µg LPS + 50 ng TNFα in 50 μL saline into both knees.

The synovial fluid was collected from the knee joints by lavage 6 hours post intra-articular injection and

PGE2, PGI2 and PGF2α levels were determined using EIA kits from Cayman.

Figure 9. Assessment of Pain Efficacy of mPGES-1 Inhibitors in Guinea Pig MIA Model

Male Hartley guinea pigs were injected with 0.3 mg MIA in 50 μL of saline (right knee) or saline (left

knee), and were dosed subcutaneously with compounds 5-9 days post MIA injection. The compound

doses were as follows: 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 was 5 mL/kg and the group size was n=6.

Pain was measured 4 hours post dosing via an incapacitance testing, as described in Materials and

Methods. Data were evaluated by one way analysis of variance (ANOVA) and are presented as means

with standard error of the means (SEM). Groups were compared to vehicle by Dunnett's test with a

Bonferroni correction for comparison between groups. All statistical analyses were performed using JMP

(version 8) statistical analysis program (SAS Institute Inc., NC). Differences were considered to be

significant if the P value was less than 0.05 (*/**).


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Table 1

Compound-1 Compound-2 Celecoxib

Enzyme Activity

Human mPGES-1 0.241 ± 0.085, n=6 0.000944 ± 0.0059, n=10 >10

Guinea Pig mPGES-1 0.511, n=1 0.0044 ± 0.0097, n=2 >10

Rat mPGES-1 >100 54.5 ± 16.3, n=2 >10

Human mPGES-2 >100 1.33 ± 0.92, n=2 nd

COX-1 9 % Inhibition @ 100 μM -1 % Inhibition @ 10 μM 31 % Inhibition @ 100 μM

COX-2 16 % Inhibition @ 30 μM -1 % Inhibition @ 30 μM 78 % Inhibition @ 30 μM

IC50 (μM) ± sd


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