identification and characterization of novel microsomal...
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JPET#228932
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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
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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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