Biological characterization of new inhibitors of microsomal PGE synthase-1 in preclinical models of inflammation and vascular tone

Article abstract of DOI:10.1111/bph.14827

Background and Purpose: Microsomal PGE synthase-1 (mPGES-1), the inducible synthase that catalyses the terminal step in PGE₂ biosynthesis, is of high interest as therapeutic target to treat inflammation. Inhibition of mPGES-1 is suggested to be safer than traditional NSAIDs, and recent data demonstrate anti-constrictive effects on vascular tone, indicating new therapeutic opportunities. However, there is a lack of potent mPGES-1 inhibitors lacking interspecies differences for conducting in vivo studies in relevant preclinical disease models. Experimental Approach: Potency was determined based on the reduction of PGE₂ formation in recombinant enzyme assays, cellular assay, human whole blood assay, and air pouch mouse model. Anti-inflammatory properties were assessed by acute paw swelling in a paw oedema rat model. Effect on vascular tone was determined with human ex vivo wire myography. Key Results: We report five new mPGES-1 inhibitors (named 934, 117, 118, 322, and 323) that selectively inhibit recombinant human and rat mPGES-1 with IC₅₀ values of 10–29 and 67–250 nM respectively. The compounds inhibited PGE₂ production in a cellular assay (IC₅₀ values 0.15–0.82 μM) and in a human whole blood assay (IC₅₀ values 3.3–8.7 μM). Moreover, the compounds blocked PGE₂ formation in an air pouch mouse model and reduced acute paw swelling in a paw oedema rat model. Human ex vivo wire myography analysis showed reduced adrenergic vasoconstriction after incubation with the compounds. Conclusion and Implications: These mPGES-1 inhibitors can be used as refined tools in further investigations of the role of mPGES-1 in inflammation and microvascular disease.

Full text of DOI:10.1111/bph.14827

Bergqvist Filip (Orcid ID: 0000-0002-0075-6751)
Biological characterization of new mPGES-1 inhibitors in preclinical models of
inflammation and vascular tone
Karin Larsson1,#, Julia Steinmetz1,#, Filip Bergqvist1,#, Samsul Arefin2, Linda Spahiu3, Johan
Wannberg4, Sven-Christian Pawelzik1,5, Ralf Morgenstern3, Patric Stenberg6, Karolina
Kublickiene2, Marina Korotkova1, Per-Johan Jakobsson1,*
#These authors contributed equally to this work.
1Rheumatology Unit, Dep. of Medicine, Solna, Karolinska Institutet, Karolinska University
Hospital, SE-171 76 Stockholm, Sweden
2Division of Renal Medicine, Dep. of Clinical Science, Intervention and Technology,
Karolinska Institutet, Karolinska University Hospital, SE-141 86 Stockholm, Sweden
3Biochemical Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet,
SE-171 77 Stockholm, Sweden
4SciLifeLab Drug Discovery & Development Platform, Medicinal Chemistry - Lead
Identification, Dep. of Medicinal Chemistry, Uppsala University, SE-751 23 Uppsala,
Sweden
5Theme Heart and Vessels, Division of Valvular and Coronary Diseases, Karolinska
University Hospital, Stockholm, Sweden
6Gesynta Pharma AB, Fogdevreten 2a, SE-171 65 Solna, Sweden
*Corresponding author:
Prof. Per-Johan Jakobsson
Address: Dep. of Medicine, Solna, Karolinska Institutet, Rheumatology Unit, D2:01,
Karolinska University Hospital, Solna, SE-171 76 Stockholm, Sweden
Email: per-johan.jakobsson@ki.se
Telephone: +46 (0) 70-483 80 18
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/bph.14827
This article is protected by copyright. All rights reserved.

Running title: New mPGES-1 inhibitors for preclinical studies
Abstract
Background and Purpose
Microsomal prostaglandin E synthase-1 (mPGES-1), the inducible synthase that catalyses the
terminal step in PGE2 biosynthesis, is of high interest as therapeutic target to treat
inflammation. Inhibition of mPGES-1 is suggested to be safer than traditional NSAIDs and
recent data demonstrate anti-constrictive effects on vascular tone, indicating new therapeutic
opportunities. However, there is a lack of potent mPGES-1 inhibitors lacking interspecies
differences for conducting in vivo studies in relevant preclinical disease models.
Experimental Approach
Potency was determined based on the reduction of PGE2 formation in recombinant enzyme
assays, cellular assay, human whole blood assay, and air pouch mouse model. Anti-
inflammatory properties were assessed by acute paw swelling in a paw oedema rat model.
Effect on vascular tone was determined with human ex vivo wire-myography.
Key Results
We report five new mPGES-1 inhibitors (named 934, 117, 118, 322, and 323) that selectively
inhibit recombinant human and rat mPGES-1 with IC50 values of 10-29 nM and 67-250 nM,
respectively. The compounds inhibited PGE2 production in a cellular assay (IC50 values 0.15-
0.82 µM) and in a human whole blood assay (IC50 values 3.3-8.7 µM). Moreover, the
compounds blocked PGE2 formation in an air pouch mouse model and reduced acute paw
swelling in a paw oedema rat model. Human ex vivo wire-myography analysis showed
reduced adrenergic vasoconstriction after incubation with the compounds.
Conclusion and Implications
These mPGES-1 inhibitors can be used as refined tools in further investigations of the role of
mPGES-1 in inflammation and microvascular disease.
This article is protected by copyright. All rights reserved.

Abbreviations
12-HHT
AA
12-hydroxyheptadecatrienoic acid
Arachidonic acid
CA
Carrageenan
CIII
Compound III
COX
Cyclooxygenase
Cxb
Celecoxib
EIA
Enzyme immunoassay
H-PGDS
KPSS
LC-MS/MS
L-PGDS
MDA
mPGES-1
mPGES-2
NSAIDs
PG
Hematopoietic-type prostaglandin D synthase
High potassium physiological solution
Liquid chromatography tandem mass spectrometry
Lipocalin-type prostaglandin D synthase
Malondialdehyde
Microsomal prostaglandin E synthase-1
Microsomal prostaglandin E synthase-2
Nonsteroidal anti-inflammatory drugs
Prostaglandin
PGIS
Prostacyclin synthase
PSS
SPE
Physiological salt solution
Solid-phase extraction
TBA
2-thiobarbituric acid
What is already known about this subject
 NSAIDs targeting COXs are valuables tools to treat inflammation but can cause severe
side effects.
 Targeting downstream mPGES-1 constitutes a potentially safer therapeutic alternative to
treat inflammation and cancer.
What this study adds
 Five new mPGES-1 inhibitors with cross-species activity.
Clinical significance
 Cross-species mPGES-1 inhibitors enable preclinical investigations of mPGES-1 as drug
target necessary to drive clinical trials.
This article is protected by copyright. All rights reserved.

Introduction
Microsomal prostaglandin E synthase-1 (mPGES-1) is the key terminal enzyme in the
production of prostaglandin E2 (PGE2) from arachidonic acid in the cyclooxygenase (COX)
pathway. PGE2 is a potent bioactive mediator involved in both physiological homeostatic
functions e.g. regulation of blood flow (Kauffman, 1981), smooth muscle function (Ren et
al., 1995), and mucosal integrity (Takeuchi, 2012) as well as pathological processes in
autoimmune diseases and cancer (Fattahi & Mirshafiey, 2012; Korotkova & Jakobsson, 2010;
Wang & Dubois, 2010). Early upon inflammatory challenge, PGE2 induces local vasodilation
and vascular permeability, which promote leukocyte infiltration to the site of inflammation
(Morimoto et al., 2014). When the inflammatory stimuli are removed the inflammation can
recede and resolve. To limit non-specific inflammation, PGE2 also induces cytokines such as
IL-10, leading to an immunosuppressive state that if persistent is associated with chronic
inflammation and cancer (Nakanishi & Rosenberg, 2013; Stolina et al., 2000). In a chronic
inflammatory state, PGE2 mediates pain, inflammatory angiogenesis, and tissue destruction
(Kamei et al., 2004; Robinson et al., 1975). In cancer, PGE2 is associated with increased
proliferation and survival of tumour cells, increased angiogenesis, enhanced invasion, and
metastasis (Buchanan et al., 2003; Pai et al., 2001; Sheng et al., 1998; Sheng et al., 2001).
Non-steroidal anti-inflammatory drugs (NSAIDs) that reduce PGE2 production via COX
inhibition are widely used drugs for inflammation and pain management. Since COX
inhibition also blocks the production of the other prostanoids, i.e. PGD2, PGF2α, prostacyclin
(PGI2), and thromboxane (TXA2), which are important for normal cellular functions and
homeostasis, NSAIDs are associated with severe side effects. COX-1 selective inhibitors are
associated with bleeding and gastrointestinal side effects (Bombardier et al., 2000) whereas
COX-2 selective inhibitors increase the risk of cardiovascular adverse effects (Baron et al.,
2008; Bresalier et al., 2005), which has led to caution in the use of these drugs. Recent
studies have also indicated an increased risk of cardiovascular adverse effects even for non-
selective COX inhibitors (Nissen et al., 2016; Sondergaard et al., 2017). In contrast, selective
inhibition of downstream mPGES-1 has been suggested as a potential safer alternative to
NSAIDs (Samuelsson et al., 2007).
Despite promising results with genetic knock-out of mPGES-1 in numerous mouse models of
inflammation (Kojima et al., 2008; Trebino et al., 2003) and cancer (Howe et al., 2013;
Nakanishi et al., 2011; Nakanishi et al., 2008), there are no mPGES-1 inhibitors in the clinic
This article is protected by copyright. All rights reserved.

today. The first phase I trials with an mPGES-1 inhibitor, LY3023703, in healthy volunteers
showed inhibition of LPS-induced PGE2 production in ex vivo blood and increased levels of
systemic prostacyclin, as measured by stable urine metabolite PGIM. LY3023703, as well as
an additional mPGES-1 inhibitor from the same program, were discontinued due liver
toxicity (Jin et al., 2018; Jin et al., 2016), and the toxicity could subsequently be attributed to
a reactive metabolite of 2-aminoimidazole, which was a common feature to both compounds
(Norman et al., 2018). Aside from such compound-specific set-backs, there are at least two
likely reasons for the lack of mPGES-1 inhibitors on the market. First, the inhibitors
developed by pharmaceutical companies have been screened towards the human enzyme.
There are differences in amino acid sequences between human and murine mPGES-1 in the
active site rendering the murine catalytic cleft less accessible to compounds, thus published
inhibitors developed towards human mPGES-1 are generally ineffective against murine
mPGES-1 (Pawelzik et al., 2010; Sjögren et al., 2013). This has limited preclinical
investigations in commonly used animal models of several diseases and thus hampered the
exploration of novel mPGES-1 inhibitors for use in indications beyond those for which they
were initially developed, typically inflammatory pain.
We have previously characterized mPGES-1 inhibitors lacking interspecies differences in
murine models of inflammation (Leclerc et al., 2013a; Leclerc et al., 2013b) as well as in
cancer models (Kock et al., 2018; Olesch et al., 2015) and recently Ding and co-workers
described new cross-species inhibitors (Ding et al., 2018), but there is still a need for further
improved mPGES-1 inhibitors for preclinical investigations. New mPGES-1 inhibitors are
required to have superior affinity and improved bioavailability. Second, COX-2 inhibitors are
associated with cardiovascular side effects, and since mPGES-1 is functionally coupled to
COX-2, there has been a fear that also mPGES-1 inhibitors will present cardiovascular
adverse effects. However, recent studies have indicated that mPGES-1 depletion will not only
evade cardiovascular concerns associated with COX-2 inhibition (Raouf et al., 2016a; Raouf
et al., 2016b), but also that the mPGES-1 inhibitor Compound III (CIII) increases levels of
vasoprotective prostacyclin (Leclerc et al., 2013a) and reduces vasoconstriction in large
blood vessels, measured by wire-myography (Ozen et al., 2017). Collectively, the observed
anti-inflammatory (through reduction of PGE2) and vasoprotective (through increase of
prostacyclin) properties of mPGES-1 inhibitors suggests further studies in models of
resistance-sized arteries.
This article is protected by copyright. All rights reserved.

In this study, we have characterized new inhibitors of human and rodent mPGES-1,
demonstrating improved pharmacological properties, and tested them in vitro, in vivo, and ex
vivo models of inflammation and vascular tone in human small arteries.
METHODS
Compounds
For experimental descriptions of the synthesis of compounds 934, 117, 118, 322, and 323
used in this study (Figure 1) see Supporting Information. COX-1 inhibitor SC-560, COX-2
inhibitor celecoxib, mPGES-1 inhibitor MK-886, and dual COX-1/2 inhibitor diclofenac
were purchased from Sigma-Aldrich. COX-2 inhibitor NS-398 was purchased from Biomol,
Germany. Hematopoietic-type prostaglandin D synthase (H-PGDS) inhibitor HQL-79 and
prostacyclin synthase (PGIS) inhibitor U-51605 were purchased from Cayman Chemicals,
Ann Arbor, MI, USA. Selective mPGES-1 inhibitor CIII was produced by NovaSaid AB,
Stockholm, Sweden. All compounds used were diluted from DMSO stock solutions if not
stated otherwise.
Enzyme inhibition assay
In order to determine the ability of the test compounds to inhibit mPGES-1 enzyme activity,
PGH2 (Lipidox, Sweden) was incubated with recombinant mPGES-1 and remaining PGH2
was indirectly assessed by measuring its degradation product malondialdehyde (MDA) as
described previously (Basevich et al., 1983). In brief, recombinant human (30 µg/ml) and rat
(1 mg/ml) mPGES-1 membrane fraction produced in Escherichia coli was pre-incubated with
the test compounds at 4°C at concentrations ranging from 0.1 nM to 3.3 µM for human and 5
nM to 37 µM for rat, in duplicates. After 30 min, the substrate PGH2 (10 µM final
concentration) was added to the enzyme-compound mixture and incubated for 90 s at room
temperature. An excess of FeCl2 in the presence of citric acid, pH 3, stopped the reaction by
converting any remaining PGH2 into MDA and 12-hydroxyheptadecatrienoic acid (12-HHT).
Subsequently, 2-thiobarbituric acid (TBA, Sigma-Aldrich) was added and the samples were
heated at 80°C for 30 min. Any formed MDA-TBA conjugate was measured using
absorbance at 530 nm (and subtracting absorbance at 560 nm) or using fluorescence at
excitation 485 nm/emission 545 nm. Inhibition of mPGES-1 by the test compounds was
expressed as the percentage relative to the inhibition of mPGES-1 by a reference mPGES-1
inhibitor MK-886 or CIII, to reduce inter-assay variability, and calculated as follows:
퐼푛ℎꢀ푏ꢀ푡ꢀ표푛 = (푇푒푠푡 퐶표푚푝표푢푛푑 − 푃표푠ꢀ푡ꢀ푣푒) ÷ (푅푒푓푒푟푒푛푐푒 − 푃표푠ꢀ푡ꢀ푣푒) × 100
This article is protected by copyright. All rights reserved.

Inhibition is the percent inhibitory activity, Positive is the signal obtained after incubation of
PGH2 with mPGES-1, Test compound is the signal obtained after incubation of PGH2 with
mPGES-1 in the presence of test compound, and Reference is the signal obtained after
incubation of PGH2 with mPGES-1 in the presence of 10 µM MK-886 or 10 µM CIII. The
assay was performed without mPGES-1 enzyme or with denatured mPGES-1 enzyme (boiled
for 5 min) as negative controls. The inhibition assay was repeated three times for 118 and
four times for 934, 322 and 323.
The test compounds were also assayed for inhibition of microsomal prostaglandin E
synthase-2 (mPGES-2), PGDS, and PGIS activity using the same approach as the mPGES-1
inhibition assay. Recombinant simian mPGES-2 membrane fraction was used, and the
compounds were assayed in duplicates at 10 concentrations between 2.5 nM and 50 M or
100 M. The inhibition assay was performed four times for 934, 117, and 118 and twice for
322 and 323. For PGDS, human recombinant lipocalin-type PGDS (L-PGDS, #10006788)
and human recombinant hematopoietic-type PGDS (H-PGDS, #10006593, both from
Cayman Chemical, Ann Arbor, MI, USA) were used. Compounds were assayed in duplicates
with 9 concentrations between 7.5 nM to 50 M for L-PGDS and 10 concentrations between
5 nM to 100 M for H-PGDS. The L-PGDS inhibition assay was performed twice for all
compounds. The H-PGDS inhibition assay was performed twice for 934, 117, and 188 and
once for 322 and 323. The inhibitory activity of a test compound was calculated as %
inhibition using the same formula as for mPGES-1, with L-PGDS inhibition relative to
denatured protein and H-PGDS inhibition relative to the H-PGDS inhibitor HQL-79.
Human recombinant PGIS (membrane fraction) was used to assess the test compounds
inhibition on PGIS activity. Inhibition of PGIS was reported as relative to inhibition by PGIS
inhibitor U-51605 at 10 M. All compounds were tested once in duplicates between 2.5 nM
and 50 M.
COX inhibition assay
To screen for cross-reactivity against COX-1 and COX-2, the compounds were tested in a
COX inhibitor screening assay (#560131, Cayman Chemical) according to manufacturer’s
recommendations with a minor modification, and formed PGF was measured by EIA

2
(Cayman Chemical). In brief, compounds 934, 117, 118, 322 and 323 were assayed in
triplicates at 10 M and compared to reference compounds SC-560 and NS-398. Compounds
This article is protected by copyright. All rights reserved.

were incubated with ovine COX-1 and human recombinant COX-2 for 10 min at 37 C prior
to addition of arachidonic acid. After 3 min, the reaction was stopped with HCl, stannous
chloride was added, and the reaction mixture was incubated at room temperature for 1 h in
order to allow formed PGH to convert into PGF . The reaction volumes were reduced to

2
2
one fifth of the recommended volumes.
mPGES-1 inhibition in intact cells
A549 human lung carcinoma cells (ATCC, Cat# CCL-185, RRID:CVCL_0023) were
cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin,
100 µg/ml streptavidin and L-glutamine (all from Invitrogen AB, Sweden) at 37°C in a
humidified atmosphere containing 5% CO2. A549 cells were seeded in 96-well plates at a
density of 25.000 cells/well and incubated for 20 h in RPMI-1640 culture medium
supplemented with 2% FBS. After 20 h, cells were treated with 10 ng/ml interleukin-1β (IL-
1β, # I9401, Sigma-Aldrich) and various concentrations of test compounds or vehicle control
(1% DMSO) in culture medium supplemented with 2% FBS and incubated for 24 h. NS-398
at 10 µM was used as positive control. The reaction was stopped by aspiring the supernatants
and cell viability was assessed by MTT assay (Sigma-Aldrich) according to manufacturer’s
protocol. PGE2 concentration was determined by enzyme immunoassay (EIA, Cayman
chemicals) in cell culture supernatants according to manufacturer’s instructions.
Whole blood assay and prostanoid profiling using LC-MS/MS
Compounds, reference compounds (diclofenac and NS-398), and vehicle controls (DMSO) in
25 µl of PBS were prepared in a 96-well plate. 200 µl of freshly drawn heparin blood (Ethical
approval Dnr 02-196, Karolinska Institutet) was added to each well and the plate was
incubated at 37°C for 30 min. After incubation, 25 µl of 0.1 mg/ml LPS (Sigma-Aldrich) in
PBS was added (final concentration 10 µg/ml of LPS) followed by pipetting up and down 3
times. The plate was incubated at 37°C for 24 h and then centrifuged at 3000 g for 10 min at
4°C. Working on ice, 120 µl of plasma was recovered to a new plate that was sealed with
aluminium foil and stored at -80°C. For prostanoid profiling using LC-MS/MS, plasma
samples were thawed on ice and then transferred to a collection plate prepared with 50 µl of
deuterated internal standard mix containing 6-keto-PGF1α-d4, PGF2α-d4, PGE2-d4, PGD2-d4,
TxB2-d4, and 15-deoxy-Δ12,14PGJ2-d4 (Cayman Chemical, Ann Arbor, MI, USA) in 100%
MeOH. Proteins were precipitated by addition of 800 µl 100% MeOH, pipetting up and down
10 times, and the plate was incubated on ice for 20 min. The plate was then centrifuged at
This article is protected by copyright. All rights reserved.

3000 g for 10 min at 4°C. The supernatants were transferred to a new plate and evaporated
under vacuum for 4 h. The evaporated samples (about 200 µl) were diluted with 1 ml of
0.05% formic acid in water and then loaded onto Oasis HLB 1cc 30 mg plate (Waters,
Ireland) that had been pre-conditioned with 1 ml of 100% MeOH and 1 ml of 0.05% formic
acid in water. The plate was washed with 10% MeOH, 0.05% formic acid in water, and
analytes were eluted with 100% MeOH. The eluates were evaporated under vacuum to
complete dryness and then stored at -20°C until reconstituted in 50 µl of 20% acetonitrile in
water prior to LC-MS/MS analysis. Analytes were quantified in negative mode with multiple
reaction monitoring method, using an Acquity triple quadrupole detector mass spectrometer
equipped with an Acquity H-class UPLC (Waters, MA, USA). Separation was performed on
a 50 x 2.1 mm Acquity UPLC BEH C18 column 1.7 µm (Waters, Ireland) with a 12 min
stepwise linear gradient (20-95%) at a flowrate of 0.6 ml/min with 0.05% formic acid in
acetonitrile as mobile phase B and 0.05% formic acid in water as mobile phase A. Data were
analysed using MassLynx software, version 4.1, with internal standard calibration.
Air pouch model
The air pouch model is an established model for preclinical anti-inflammatory drug efficiency
studies. The air pouch mimics the synovial cavity and when challenged with carrageenan,
provides a localized sterile inflammatory environment suitable to study prostaglandins
(Duarte et al., 2012). Male C57BL/6JBomTac mice (Taconic, Denmark), weighing
approximately 20 g, were used for the air pouch experiments. The mice were housed in
groups of 4 to 8 animals in cages containing bedding and environmental enrichment.
Husbandry and care practices were based on veterinary guidance. The animals had access to
food and water ad libitum and were inspected at least twice a day so that any health issues
were immediately apparent and veterinary guidance could be obtained. Ethical approval for
this study was granted by the regional ethical committee of Stockholm, Sweden (N86/13).
To form the air pouches, 3 ml of sterile air were injected into the interscapular area of the
back of the mice under isoflurane anaesthesia (Univentor 400, 3%). To provide stable air
pouches, they were re-inflated with 1.5 ml sterile air after 5 days under light anaesthesia. 24 h
after the second air injection, animals were randomized into receiving 30 mg/kg or 100
mg/kg of mPGES-1 inhibitor (934, 117, 118, 322 or 323), 50 mg/kg celecoxib, or vehicle
control (1% Tween 80 and 0.5% carboxymethyl cellulose in MilliQ water) administered
through oral gavage (p.o.). One hour after administration of compounds or vehicle, 1 ml 1%
This article is protected by copyright. All rights reserved.

carrageenan (CA) was injected into the pouch under light isoflurane anaesthesia. Sterile
inflammation was allowed to develop for 6 h before sacrifice by an overdose of isoflurane
combined with cervical dislocation, and exudate was collected. Exudates were immediately
centrifuged at 1500 g for 3 min and then stored at -20°C until further analysis. PGE2 and 6-
keto-PGF concentrations were measured by EIA according to manufacturer’s protocol

1
(#514010, #515211, Cayman Chemical). Four of the unstimulated control samples and three
of the celecoxib treated mice were below the detection limit of the EIA analysis (15 pg/ml).
Therefore, these samples were assigned a value of 20 pg/pouch for the subsequent data
analysis. Due to non-successful pouch formation, 7 out of 48 mice were excluded from the
study. The remaining 41 mice were randomized among the different groups, ensuring a
minimum number of mice (n=3) for the saline control and equal distribution for each
treatment group with the aim of 10 per group based on earlier experiments. This resulted in
two treatment groups with a sample size of 9 (30 mg/kg and celecoxib) and two treatment
groups with 10 mice each (CA control and 100 mg/kg). For the following four experiments
the same number of mice was used in each experiment.
Carrageenan-induced paw oedema model
The CA-induced paw oedema model is a widely used model to study the anti-inflammatory
response of NSAIDs in vivo (Morris, 2003). Anthem Biosciences was assigned to perform
experiments to evaluate the anti-inflammatory effect of the compounds in the CA-induced
paw oedema model in rats. All experiments were performed according to protocols approved
by the Institutional Animal Ethics Committee (IAEC) under the supervision of the Committee
for the Purpose of Control and Supervision on Experiment on Animals (CPCSEA), Ministry
of Environment, Forest and Climate Change, Government of India. Male Wistar rats, 6 weeks
old and weighing approximately 150 g, kept on regular chow diet were used in the
experiment. Seven rats were randomized into treatment groups based on weight. The identity
of compounds was unknown to Anthem Biosciences. Sample size was based on earlier
experience and existing literature (Morris, 2003). Animals were fasted overnight before the
day of experiment. Compounds were administered at 1, 3, 10, 30 and 100 mg/kg and the
reference compound, celecoxib was administered at 10 mg/kg in a suspension containing a
final concentration of 1% Tween 80 and 0.5% carboxymethyl cellulose by oral gavage. One
hour later, inflammation was induced with 0.1 ml of 1% CA solution that was injected into
the subplantar region of the hind paw of the rats. Swelling of the paw was monitored using a
This article is protected by copyright. All rights reserved.

plethysmometer (Ugo Basile, Italy) before (baseline) and at 1, 2, 3, and 4 h after CA
injection.
In vivo pharmacokinetic studies in rat
Eurofins Cerep Laboratories was assigned to investigate the pharmacokinetic properties of
the new mPGES-1 inhibitors. The pharmacokinetic study was reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC). Each value represents the mean of
three animals (Supporting Information Table S1). Intravenous (i.v.) dose was 2 mg/kg and
oral dose was 10 mg/kg. Blood was drawn 3,10, 30, 60, 120, 240, 360, and 1440 min after i.v.
dosing and 10, 30, 60, 120, 240, 360, 480, and 1440 min after p.o. dosing. The
pharmacokinetic properties of the inhibitors were assessed in male CD IGS rats from Charles
River Laboratories, weighing between 180-250 g.
Vascular reactivity studies in resistance size arteries
Ethical approval for this study was granted by the Ethics Committee at Karolinska University
Hospital, Huddinge (273/94). Full informed consent according to the Declaration of Helsinki
was obtained from all subjects. Arterial segmentation and vascular reactivity studies were
carried out as previously described (Arefin et al., 2014). Briefly, the subcutaneous fat
biopsies were obtained from the lower abdomen from healthy donors and placed in cold
physiological salt solution (PSS: NaCl 119 mM, KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.17
mM, NaHCO3 25 mM, KH2PO4 1.18 mM, EDTA 0.026 mM, and glucose 5.5 mM). In ice-
cold PSS, resistance size arteries (Ø100-500µm) were dissected from subcutaneous fat
biopsies and cleaned from surrounding non-vascular tissue using a stereomicroscope. Vessel
tension was measured using a Mulvany’s type 4-channel Multi Myograph system (Danish
Myotechnology, Model 610), and isometric force was registered using Lab chart 8 software
(AD Instruments, New Zealand). Each organ bath contained warmed (37°C) PSS that was
continuously bubbled with 5% CO2 /95% O2. Every 30 min all solutions, including the
incubation solutions were refreshed. Viability and endothelial function was assessed by an
initial stretching protocol, followed by specific smooth muscle activation that included a first
stimulation with a mixture of high potassium physiological salt solution (KPSS, equimolar
substitution of 125 mM Na+ with K+). The arteries were then washed with PSS to return to
resting basal tone followed by treatment with norepinephrine at 10 µM. The arteries were
washed with PSS, and then relaxation was tested with 1 µM acetylcholine or 1 µM
This article is protected by copyright. All rights reserved.

bradykinin after preconstruction with 1 µM norepinephrine. Arteries that did not fulfil
viability criteria of >50% relaxation to acetylcholine or bradykinin were excluded.
To determine any effect of mPGES-1 inhibitors in respect to contractility, arteries were first
contracted with increasing concentrations of norepinephrine (0.001-3 µM) in a cumulative
manner until a stable plateau was reached. The arteries were then washed with PSS to return
to a resting basal tone followed by a 30 min treatment with CIII (10 µM), 934 (3 µM), 118 (3
µM), or vehicle control (DMSO). After the 30 min incubation, a second norepinephrine
(0.001-3 µM) concentration-response curve was recorded, and concentration response curves
were expressed as % of initial high potassium contraction. Thereafter, arterial viability was
assessed by treatment with norepinephrine at 1 µM. Due to paired analysis, randomization
was not needed.
Data and statistical analyses
Data are presented as mean ± SD or median with inter-quartile range (25th-75th percentile)
and individual data points. All IC50 values were calculated using nonlinear regression and
sigmoidal concentration-response curve fit. Statistical significance in the air pouch model and
in the paw swelling assay was calculated using one-way ANOVA (normally distributed data)
followed by Dunnett’s multiple comparison test, with a single pooled variance (the mean of
each treatment group was compared with the mean of the vehicle control group). Post-hoc
tests were only run if F achieved P<0.05. Statistical analysis of constriction of arteries was
performed by paired t-test of individual EC50 values (before and after inhibitor incubation).
Significance was set to P<0.05 (denoted by * in figures). Calculations and graphs were
prepared using GraphPad Prism 7.0e. The data and statistical analysis comply with the
recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018).
Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a; Alexander et al.,
2017b).
This article is protected by copyright. All rights reserved.

RESULTS
Compound potency and selectivity towards mPGES-1
The compounds profiled herein are the result of an optimization effort that started from a
screen of a compound library towards human mPGES-1. One of the original hit series
consisted of 1-(benzothiazol-2-yl)piperidine-4-carboxamides. This hit series evolved into a
series of 1-(1H-benzoimidazol-2-yl)piperidine-4-carboxamides with various substitutions on
the benzimidazole and amide parts with CIII (Leclerc et al., 2013a) as an early example.
Further optimizations focused on properties such as potency, aqueous solubility, and in vitro
metabolic stability. The improved compounds combined with the knowledge that earlier
bezimidazole compounds could reach submicromolar IC50 values in rat recombinant mPGES-
1 (e.g. CIII) provided the basis for further in vitro and in vivo profiling of these potentially
cross species human/murine mPGES-1 inhibitors.
The five compounds 934, 117, 118, 322, and 323 (Figure 1) were found to be potent
inhibitors of recombinant human and rat mPGES-1 (Figure 2, Supporting Information Figure
S1). The IC50 values were 10-29 nM and 67-250 nM towards human or rat enzyme,
respectively. The selectivity towards mPGES-1 was determined by screening other enzymes
in the prostanoid synthesis pathway. The compounds showed no inhibitory capacities towards
COX-1, PGIS, L-PGDS or H-PGDS at concentrations up to 10 µM (Table 1). All compounds
showed weak to moderate inhibition of mPGES-2 at 10 µM. Compound 323 showed weak
inhibition of COX-2.
Inhibition of PGE2 production in intact cells
Human lung cancer cell line A549 in culture medium supplemented with 2% FBS was used
to determine the potency of the compounds to inhibit PGE2 production in a cell assay. All
compounds inhibited the IL-1β induced PGE2 production in a concentration dependent
manner, showing IC50 values in the range of 0.15-0.82 µM (Figure 3). The COX-2 inhibitor
NS-398, used as positive control, completely reduced PGE2 production at 10 µM. The tested
compounds did not affect cell viability when used below 100 µM (Supporting Information
Figure S2).
Inhibition of PGE2 production in human whole blood
Human whole blood assay was used to assess the compounds’ capacity to inhibit LPS-
induced PGE2 production in a complex biological matrix. The compounds targeting mPGES-
This article is protected by copyright. All rights reserved.

1 displayed similar potency to block PGE2 production, with IC50 values in the range of 3.3-
8.7 µM (Figure 4). The positive control, diclofenac, fully inhibited PGE2 production at 10
µM, while the tested compounds showed 10-15% residual PGE2 production at the highest
tested concentration (20 µM). Reference compound NS-398 showed weak inhibition at 0.1
µM. Diclofenac fully inhibited the thromboxane production (as measured by the stable
metabolite TXB2), while NS-398 and the compounds targeting mPGES-1 showed no
consistent effect on TXB2 formation (Supporting Information Figure S3).
In vivo pharmacokinetic study in rats
All the compounds displayed similar properties, although 117 reached a higher maximum
concentration and displayed a higher bioavailability than the other compounds (Supporting
Information Table S1). The obtained exposure of the compounds after oral administration
supports an in vivo effect with the doses used in the animal studies.
Inhibition of PGE2 production in air pouch model
To assess the compounds’ ability to reduce PGE2 in vivo, the CA air pouch model was used
to induce inflammation and prostaglandin production in mice (Leclerc et al., 2013a).
Compounds or vehicle were administered via p.o. one hour before injection of CA in the
pouch. CA successfully induced PGE2 production in the air pouches, and celecoxib, used as a
positive control in all experiments, significantly reduced the formation of PGE2. All test
compounds reduced PGE2 production in the pouch exudates (Figure 5). Compound 322 and
323 significantly reduced PGE2 production at 30 mg/kg versus 100 mg/kg for the other
compounds. In contrast to celecoxib, the mPGES-1 inhibitors did not reduce prostacyclin
levels. For Compound 117 there was even an increase in prostacyclin formation (Supporting
Information Figure S4).
Efficacy on acute inflammation in vivo
To further investigate the compounds’ efficacy in vivo, a carrageenan-induced paw oedema
model in rats was used. Celecoxib was used as a reference compound and significantly
reduced paw swelling in all the individual experiments with 50% (one experiment per
compound). A significant reduction in swelling 1 h post CA induction was seen for all
compounds compared to vehicle control, with a maximum reduction in paw swelling of 45-
65%. 934, 322 and 323 reduced paw swelling at all tested doses (1-100 mg/kg) with a clear
trend to dose-dependency (Figure 6). A significant reduction in paw swelling was also seen
for 117 and 118 1 h post CA, but a dose-response trend was not observed for 117, and only a
This article is protected by copyright. All rights reserved.

weak dose-response was observed for 118 (Figure 6). At 4 h post CA induction, paw swelling
reached its maximum (Figure S5). When comparing the area under the curve (AUC) of the
different inhibitors to the vehicle treated CA induced control, a significant decrease in paw
swelling over 4 h was observed for 934 (10, 30 and 100 mg/kg), 117 (1, 3, 10 and 30 mg/kg),
118 (10 mg/kg), 322 (3 and 100 mg/kg), and 323 (100 mg/kg). Celecoxib (10 mg/kg)
significantly reduced paw swelling in all experiments.
Effect of mPGES-1 inhibition on vasoconstriction in small arteries
To study the effects of mPGES-1 inhibition on human peripheral resistance vasculature, we
assessed norepinephrine-induced vasoconstriction using wire-myography. The reference
mPGES-1 inhibitor CIII (Leclerc et al., 2013a; Ozen et al., 2017) reduced constriction at 10
µM; however, without reaching statistical significance. Based on structural differences and
performance in vitro and in vivo, 934 and 118 were selected for the wire-myography
experiments. Compound 118 showed better efficacy than CIII at 3-fold lower concentration
(3 µM), indicating an increased potency (Figure 7). Compound 934 showed a high
variability, possibly due to poor solubility in the assay buffer; however, a trend towards
reduced constriction was seen.
DISCUSSION
Inhibition of mPGES-1 was initially proposed as a promising alternative to traditional COX
inhibitors to manage pain and inflammation. Selective mPGES-1 inhibitors are envisioned to
present less side effects than COX inhibitors, as they target only inducible PGE2 production
and spare the production of other prostanoids that are important for physiological functions.
However, as recent data suggests that mPGES-1 inhibition is not only safe, but also may
elicit beneficial cardiovascular effects, we set out to first develop improved human/rodent
mPGES-1 inhibitors that can be used to study such effects in multiple preclinical disease
models and then to demonstrate the ability of such inhibitors to attenuate norepinephrine-
induced vasoconstriction in resistance-size human arteries.
The compounds 934, 117, 118, 322, and 323 selectively inhibited recombinant human and rat
mPGES-1 in vitro with IC50 values in the low nanomolar range. The compounds displayed
potent inhibition of PGE2 production in IL-1β treated human intact cells. The compounds
were further tested in an LPS-treated human whole blood assay (24 h), where the compounds
blocked PGE2 production with IC50 values in the low micromolar range without affecting
This article is protected by copyright. All rights reserved.

thromboxane synthesis. A summary of the compounds performance in in vitro assays is given
in Table 2.
To establish the compounds’ efficacy in rodent models in vivo, we used the CA-induced air
pouch mouse model (Leclerc et al., 2013a) and a CA-induced paw oedema rat model. All five
compounds dose-dependently reduced the concentration of PGE2 in the pouch exudates.
While the COX-inhibitor celecoxib almost completely blocked PGE2 formation, full
inhibition of PGE2 production was not reached with the mPGES-1 inhibitors. This difference
could be due to non-enzymatic degradation of PGH2 or because PGH2 is converted to PGE2
by the other PGE2 synthases mPGES-2 and cPGES (Murakami et al., 2003; Tanioka et al.,
2000). Inhibition of mPGES-1 in the air pouch did not affect prostacyclin concentration,
while celecoxib inhibited both PGE2 and prostacyclin production. Moreover, the five
compounds significantly reduced acute paw swelling.
In contrast to COX inhibitors, mPGES-1 inhibitors are likely to have cardioprotective
properties based on several studies in mPGES-1 knockout mice (Cheng et al., 2006; Leclerc
et al., 2013a; Tang et al., 2016; Wang et al., 2011; Wang et al., 2006). Also, it was recently
reported that treatment with mPGES-1 inhibitor CIII results in reduced contraction of larger
human blood vessels ex vivo (Ozen et al., 2017). We set out to prove that mPGES-1
inhibition results in reduced contraction in human small resistance size arteries. We conclude
that the new compounds replicate the effects of CIII regarding reduction of vasoconstriction.
However, further studies are warranted to elucidate the underlying mechanisms of mPGES-1
inhibition and cardiovascular protection. The established dogma behind the cardiovascular
side effects imposed by NSAIDs is a reduction in vasodilating prostacyclin, while platelet
derived thromboxane (platelet activator and vasoconstrictor) biosynthesis remains (Grosser et
al., 2006). The proposed cardioprotective effect of mPGES-1 inhibition is mediated by
shunting of PGH2 from PGE2 to prostacyclin (Cheng et al., 2006; Jin et al., 2016; Ozen et al.,
2017; Tang et al., 2016).
Recently, the results from the first clinical phase I trial with an mPGES-1 inhibitor were
published (Jin et al., 2016). The Eli Lilly compound LY3023703 showed very potent
inhibition of PGE2, and in contrast to a COX-2 inhibitor celecoxib, there was an increased
release of urinary prostacyclin metabolite, suggesting a systemic increase of cardioprotective
prostacyclin during mPGES-1 inhibition in man. Mechanistic data in mice showed that
mPGES-1 derived PGE2 drives vascular remodelling, stiffness, and endothelial dysfunction in
This article is protected by copyright. All rights reserved.

hypertension (Avendano et al., 2018). This potentially protective side effect of mPGES-1
inhibition opens up the possibility to tackle the production of pro-inflammatory and
immunosuppressive PGE2, while increasing the production of cardioprotective prostacyclin.
In the present study, we have characterized five new cross-species mPGES-1 inhibitors
suitable for oral delivery with improved potency and selectivity compared to published
inhibitors lacking interspecies differences (Ding et al., 2018; Leclerc et al., 2013a; Leclerc et
al., 2013b). All five compounds presented comparable selectivity and potency. Our results
indicate a class effect of mPGES-1 inhibition in reduction of inflammation and protection
against cardiovascular events. We envision that these compounds will be valuable tools in
preclinical research to evaluate mPGES-1 as a therapeutic target in inflammation, cancer, and
microvascular disease.
ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Research Council (grant no: 2015-
03955, 2017-01391, and 2017-02577), the Swedish Rheumatism Association (grant no: R-
755861), King Gustaf V 80 Year Foundation (grant no: n/a), the Swedish Childhood Cancer
Foundation (grant no: TJ2017-0040), the Swedish Cancer Society (grant no: CAN 2016/739),
the Cancer Society in Stockholm (grant no: 171073), Innovative Medicines Initiative
(EU/EFPIA, ULTRA-DD, grant no: 115766), Stockholm County Council (ALF, grant no:
20160378), VINNOVA (Innovativa Start-ups, grant no: 2017-04031), and Karolinska
Institutet Foundation (grant no: n/a).
CONFLICTS OF INTEREST
R.M., P.S., and P-J.J. are engaged in Gesynta Pharma AB, a company that develops mPGES-
1 inhibitors. All other authors have no conflicts of interests to declare.
AUTHOR CONTRIBUTIONS
K.L., J.S., F.B., L.S., J.W., R.M., P.S., K.K., M.K, and P-J.J. contributed to study conception
and design. J.S., F.B., S.A., L.S., J.W., and P.S. performed experiments. K.L., J.S., F.B.,
S.A., L.S., J.W., and P.S. analysed data. K.L., J.S., F.B, and J.W. drafted the manuscript.
R.M, P.S., K.K., M.K, and P-J.J. critically revised the manuscript. S-C.P. contributed with
new reagents. All authors read and approved the final manuscript.
This article is protected by copyright. All rights reserved.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research as stated in the BJP guidelines for
Design & Analysis, and Animal Experimentation, and as recommended by funding agencies,
publishers and other organisations engaged with supporting research.
REFERENCES
Alexander, S. P., Christopoulos, A., Davenport, A. P., Kelly, E., Marrion, N. V., Peters, J. A.,
et al. (2017a). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G protein-
coupled receptors. Br J Pharmacol, 174 Suppl 1, S17-S129. doi:10.1111/bph.13878
Alexander, S. P., Fabbro, D., Kelly, E., Marrion, N. V., Peters, J. A., Faccenda, E., et al.
(2017b). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Enzymes. Br J
Pharmacol, 174 Suppl 1, S272-S359. doi:10.1111/bph.13877
Arefin, S., Simoncini, T., Wieland, R., Hammarqvist, F., Spina, S., Goglia, L., et al. (2014).
Vasodilatory effects of the selective GPER agonist G-1 is maximal in arteries of
postmenopausal women. Maturitas, 78(2), 123-130.
doi:10.1016/j.maturitas.2014.04.002
Avendano, M. S., Garcia-Redondo, A. B., Zalba, G., Gonzalez-Amor, M., Aguado, A.,
Martinez-Revelles, S., et al. (2018). mPGES-1 (Microsomal Prostaglandin E
Synthase-1) Mediates Vascular Dysfunction in Hypertension Through Oxidative
Stress. Hypertension, 72(2), 492-502. doi:10.1161/HYPERTENSIONAHA.118.10833
Baron, J. A., Sandler, R. S., Bresalier, R. S., Lanas, A., Morton, D. G., Riddell, R., et al.
(2008). Cardiovascular events associated with rofecoxib: final analysis of the
APPROVe trial. Lancet, 372(9651), 1756-1764. doi:10.1016/S0140-6736(08)61490-7
Basevich, V. V., Mevkh, A. T., & Varfolomeev, S. D. (1983). [Kinetics of prostaglandin H2
degradation. A method of determining prostaglandin H-convertase activity]. Bioorg
Khim, 9(5), 658-665.
This article is protected by copyright. All rights reserved.

Bombardier, C., Laine, L., Reicin, A., Shapiro, D., Burgos-Vargas, R., Davis, B., et al.
(2000). Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in
patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med, 343(21),
1520-1528, 1522 p following 1528. doi:10.1056/NEJM200011233432103
Bresalier, R. S., Sandler, R. S., Quan, H., Bolognese, J. A., Oxenius, B., Horgan, K., et al.
(2005). Cardiovascular events associated with rofecoxib in a colorectal adenoma
chemoprevention trial. N Engl J Med, 352(11), 1092-1102.
doi:10.1056/NEJMoa050493
Buchanan, F. G., Wang, D., Bargiacchi, F., & DuBois, R. N. (2003). Prostaglandin E2
regulates cell migration via the intracellular activation of the epidermal growth factor
receptor. J Biol Chem, 278(37), 35451-35457. doi:10.1074/jbc.M302474200
Cheng, Y., Wang, M., Yu, Y., Lawson, J., Funk, C. D., & Fitzgerald, G. A. (2006).
Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular
function. J Clin Invest, 116(5), 1391-1399. doi:10.1172/JCI27540
Curtis, M. J., Alexander, S., Cirino, G., Docherty, J. R., George, C. H., Giembycz, M. A., et
al. (2018). Experimental design and analysis and their reporting II: updated and
simplified guidance for authors and peer reviewers. Br J Pharmacol, 175(7), 987-993.
doi:10.1111/bph.14153
Ding, K., Zhou, Z. Y., Hou, S. R., Yuan, Y. X., Zhou, S., Zheng, X. R., et al. (2018).
Structure-based discovery of mPGES-1 inhibitors suitable for preclinical testing in
wild-type mice as a new generation of anti-inflammatory drugs. Scientific Reports, 8.
doi:10.1038/s41598-018-23482-4
Duarte, D. B., Vasko, M. R., & Fehrenbacher, J. C. (2012). Models of inflammation:
carrageenan air pouch. Curr Protoc Pharmacol, Chapter 5, Unit5 6.
doi:10.1002/0471141755.ph0506s56
This article is protected by copyright. All rights reserved.

Fattahi, M. J., & Mirshafiey, A. (2012). Prostaglandins and rheumatoid arthritis. Arthritis,
2012, 239310. doi:10.1155/2012/239310
Grosser, T., Fries, S., & FitzGerald, G. A. (2006). Biological basis for the cardiovascular
consequences of COX-2 inhibition: therapeutic challenges and opportunities. Journal
of Clinical Investigation, 116(1), 4-15. doi:10.1172/JCI27291
Harding, S. D., Sharman, J. L., Faccenda, E., Southan, C., Pawson, A. J., Ireland, S., et al.
(2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and
expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucleic
Acids Res, 46(D1), D1091-D1106. doi:10.1093/nar/gkx1121
Howe, L. R., Subbaramaiah, K., Kent, C. V., Zhou, X. K., Chang, S. H., Hla, T., et al. (2013).
Genetic deletion of microsomal prostaglandin E synthase-1 suppresses mouse
mammary tumor growth and angiogenesis. Prostaglandins Other Lipid Mediat, 106,
99-105. doi:10.1016/j.prostaglandins.2013.04.002
Jin, Y., Regev, A., Kam, J., Phipps, K., Smith, C., Henck, J., et al. (2018). Dose-dependent
acute liver injury with hypersensitivity features in humans due to a novel microsomal
prostaglandin E synthase 1 inhibitor. British Journal of Clinical Pharmacology, 84(1),
179-188. doi:10.1111/bcp.13423
Jin, Y., Smith, C. L., Hu, L., Campanale, K. M., Stoltz, R., Huffman, L. G., Jr., et al. (2016).
Pharmacodynamic comparison of LY3023703, a novel microsomal prostaglandin e
synthase 1 inhibitor, with celecoxib. Clin Pharmacol Ther, 99(3), 274-284.
doi:10.1002/cpt.260
Kamei, D., Yamakawa, K., Takegoshi, Y., Mikami-Nakanishi, M., Nakatani, Y., Oh-Ishi, S.,
et al. (2004). Reduced pain hypersensitivity and inflammation in mice lacking
microsomal prostaglandin e synthase-1. J Biol Chem, 279(32), 33684-33695.
doi:10.1074/jbc.M400199200
This article is protected by copyright. All rights reserved.

Kauffman, G. L., Jr. (1981). The role of prostaglandins in the regulation of gastric mucosal
blood flow. Prostaglandins, 21 Suppl, 33-38.
Kock, A., Larsson, K., Bergqvist, F., Eissler, N., Elfman, L. H. M., Raouf, J., et al. (2018).
Inhibition of Microsomal Prostaglandin E Synthase-1 in Cancer-Associated
Fibroblasts Suppresses Neuroblastoma Tumor Growth. EBioMedicine.
doi:10.1016/j.ebiom.2018.05.008
Kojima, F., Kapoor, M., Yang, L., Fleishaker, E. L., Ward, M. R., Monrad, S. U., et al.
(2008). Defective generation of a humoral immune response is associated with a
reduced incidence and severity of collagen-induced arthritis in microsomal
prostaglandin E synthase-1 null mice. J Immunol, 180(12), 8361-8368.
Korotkova, M., & Jakobsson, P. J. (2010). Microsomal prostaglandin e synthase-1 in
rheumatic diseases. Front Pharmacol, 1, 146. doi:10.3389/fphar.2010.00146
Leclerc, P., Idborg, H., Spahiu, L., Larsson, C., Nekhotiaeva, N., Wannberg, J., et al. (2013a).
Characterization of a human and murine mPGES-1 inhibitor and comparison to
mPGES-1 genetic deletion in mouse models of inflammation. Prostaglandins Other
Lipid Mediat, 107, 26-34. doi:10.1016/j.prostaglandins.2013.09.001
Leclerc, P., Pawelzik, S. C., Idborg, H., Spahiu, L., Larsson, C., Stenberg, P., et al. (2013b).
Characterization of a new mPGES-1 inhibitor in rat models of inflammation.
Prostaglandins Other Lipid Mediat, 102-103, 1-12.
doi:10.1016/j.prostaglandins.2013.03.005
Morimoto, K., Shirata, N., Taketomi, Y., Tsuchiya, S., Segi-Nishida, E., Inazumi, T., et al.
(2014). Prostaglandin E-2-EP3 Signaling Induces Inflammatory Swelling by Mast
Cell Activation. J Immunol, 192(3), 1130-1137. doi:10.4049/jimmunol.1300290
Morris, C. J. (2003). Carrageenan-induced paw edema in the rat and mouse. Methods Mol
Biol, 225, 115-121. doi:10.1385/1-59259-374-7:115
This article is protected by copyright. All rights reserved.

Murakami, M., Nakashima, K., Kamei, D., Masuda, S., Ishikawa, Y., Ishii, T., et al. (2003).
Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2
via both cyclooxygenases-1 and -2. J Biol Chem, 278(39), 37937-37947.
doi:10.1074/jbc.M305108200
Nakanishi, M., Menoret, A., Tanaka, T., Miyamoto, S., Montrose, D. C., Vella, A. T., et al.
(2011). Selective PGE(2) suppression inhibits colon carcinogenesis and modifies local
mucosal immunity. Cancer Prev Res (Phila), 4(8), 1198-1208. doi:10.1158/1940-
6207.CAPR-11-0188
Nakanishi, M., Montrose, D. C., Clark, P., Nambiar, P. R., Belinsky, G. S., Claffey, K. P., et
al. (2008). Genetic deletion of mPGES-1 suppresses intestinal tumorigenesis. Cancer
Res, 68(9), 3251-3259. doi:10.1158/0008-5472.CAN-07-6100
Nakanishi, M., & Rosenberg, D. W. (2013). Multifaceted roles of PGE2 in inflammation and
cancer. Semin Immunopathol, 35(2), 123-137. doi:10.1007/s00281-012-0342-8
Nissen, S. E., Yeomans, N. D., Solomon, D. H., Luscher, T. F., Libby, P., Husni, M. E., et al.
(2016). Cardiovascular Safety of Celecoxib, Naproxen, or Ibuprofen for Arthritis. N
Engl J Med, 375(26), 2519-2529. doi:10.1056/NEJMoa1611593
Norman, B. H., Fisher, M. J., Schiffler, M. A., Kuklish, S. L., Hughes, N. E., Czeskis, B. A.,
et al. (2018). Identification and Mitigation of Reactive Metabolites of 2-
Aminoimidazole-Containing Microsomal Prostaglandin E Synthase-1 Inhibitors
Terminated Due to Clinical Drug-Induced Liver Injury. Journal of Medicinal
Chemistry, 61(5), 2041-2051. doi:10.1021/acs.jmedchem.7b01806
Olesch, C., Sha, W., Angioni, C., Sha, L. K., Acaf, E., Patrignani, P., et al. (2015). MPGES-
1-derived PGE2 suppresses CD80 expression on tumor-associated phagocytes to
inhibit anti-tumor immune responses in breast cancer. Oncotarget.
This article is protected by copyright. All rights reserved.

Ozen, G., Gomez, I., Daci, A., Deschildre, C., Boubaya, L., Teskin, O., et al. (2017).
Inhibition of microsomal PGE synthase-1 reduces human vascular tone by increasing
PGI2 : a safer alternative to COX-2 inhibition. Br J Pharmacol, 174(22), 4087-4098.
doi:10.1111/bph.13939
Pai, R., Szabo, I. L., Soreghan, B. A., Atay, S., Kawanaka, H., & Tarnawski, A. S. (2001).
PGE(2) stimulates VEGF expression in endothelial cells via ERK2/JNK1 signaling
pathways. Biochem Biophys Res Commun, 286(5), 923-928.
doi:10.1006/bbrc.2001.5494
Pawelzik, S. C., Uda, N. R., Spahiu, L., Jegerschöld, C., Stenberg, P., Hebert, H., et al.
(2010). Identification of key residues determining species differences in inhibitor
binding of microsomal prostaglandin E synthase-1. J Biol Chem, 285(38), 29254-
29261. doi:10.1074/jbc.M110.114454
Raouf, J., Kirkby, N., Ahmetaj-Shala, B., Liu, B., Mazi, S., Korotkova, M., et al. (2016a).
MPGES-1 deletion increases prostacyclin and evades the elevated systemic ADMA
associated with COX-2 inhibitors: relevance to cardiovascular safety of Mpges-1
inhibitors. . Ann. Rheum. Dis., 75, A11-A12
Raouf, J., Mobarrez, F., Larsson, K., Jakobsson, P. J., & Korotkova, M. (2016b). mPGES-1
deletion affects platelet functions in mice. Clin Sci (Lond). doi:10.1042/CS20160463
Ren, J., Karpinski, E., & Benishin, C. G. (1995). Prostaglandin E2 contracts vascular smooth
muscle and inhibits potassium currents in vascular smooth muscle cells of rat tail
artery. J Pharmacol Exp Ther, 275(2), 710-719.
Robinson, D. R., Tashjian, A. H., & Levine, L. (1975). Prostaglandin-Stimulated Bone-
Resorption by Rheumatoid Synovia - Possible Mechanism for Bone Destruction in
Rheumatoid-Arthritis. Journal of Clinical Investigation, 56(5), 1181-1188. doi:Doi
10.1172/Jci108195
This article is protected by copyright. All rights reserved.

Samuelsson, B., Morgenstern, R., & Jakobsson, P. J. (2007). Membrane prostaglandin E
synthase-1: a novel therapeutic target. Pharmacol Rev, 59(3), 207-224.
doi:10.1124/pr.59.3.1
Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., & DuBois, R. N. (1998). Modulation
of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells.
Cancer Res, 58(2), 362-366.
Sheng, H., Shao, J., Washington, M. K., & DuBois, R. N. (2001). Prostaglandin E2 increases
growth and motility of colorectal carcinoma cells. J Biol Chem, 276(21), 18075-
18081. doi:10.1074/jbc.M009689200
Sjögren, T., Nord, J., Ek, M., Johansson, P., Liu, G., & Geschwindner, S. (2013). Crystal
structure of microsomal prostaglandin E2 synthase provides insight into diversity in
the MAPEG superfamily. Proc Natl Acad Sci U S A, 110(10), 3806-3811.
doi:10.1073/pnas.1218504110
Sondergaard, K. B., Weeke, P., Wissenberg, M., Schjerning Olsen, A. M., Fosbol, E. L.,
Lippert, F. K., et al. (2017). Non-steroidal anti-inflammatory drug use is associated
with increased risk of out-of-hospital cardiac arrest: a nationwide case-time-control
study. Eur Heart J Cardiovasc Pharmacother, 3(2), 100-107.
doi:10.1093/ehjcvp/pvw041
Stolina, M., Sharma, S., Lin, Y., Dohadwala, M., Gardner, B., Luo, J., et al. (2000). Specific
inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of
IL-10 and IL-12 synthesis. J Immunol, 164(1), 361-370.
Takeuchi, K. (2012). Pathogenesis of NSAID-induced gastric damage: importance of
cyclooxygenase inhibition and gastric hypermotility. World J Gastroenterol, 18(18),
2147-2160. doi:10.3748/wjg.v18.i18.2147
This article is protected by copyright. All rights reserved.

Tang, S. Y., Monslow, J., G, R. G., Todd, L., Pawelzik, S. C., Chen, L., et al. (2016).
Cardiovascular Consequences of Prostanoid I Receptor Deletion in Microsomal
Prostaglandin E Synthase-1-Deficient Hyperlipidemic Mice. Circulation, 134(4), 328-
338. doi:10.1161/CIRCULATIONAHA.116.022308
Tanioka, T., Nakatani, Y., Semmyo, N., Murakami, M., & Kudo, I. (2000). Molecular
identification of cytosolic prostaglandin E2 synthase that is functionally coupled with
cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem, 275(42),
32775-32782. doi:10.1074/jbc.M003504200
Trebino, C. E., Stock, J. L., Gibbons, C. P., Naiman, B. M., Wachtmann, T. S., Umland, J. P.,
et al. (2003). Impaired inflammatory and pain responses in mice lacking an inducible
prostaglandin E synthase. Proc Natl Acad Sci U S A, 100(15), 9044-9049.
doi:10.1073/pnas.1332766100
Wang, D., & Dubois, R. N. (2010). Eicosanoids and cancer. Nat Rev Cancer, 10(3), 181-193.
doi:10.1038/nrc2809
Wang, M., Ihida-Stansbury, K., Kothapalli, D., Tamby, M. C., Yu, Z., Chen, L., et al. (2011).
Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.
Circulation, 123(6), 631-639. doi:10.1161/CIRCULATIONAHA.110.973685
Wang, M., Zukas, A. M., Hui, Y., Ricciotti, E., Pure, E., & FitzGerald, G. A. (2006).
Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards
atherogenesis. Proc Natl Acad Sci U S A, 103(39), 14507-14512.
doi:10.1073/pnas.0606586103
This article is protected by copyright. All rights reserved.

Table 1. Selectivity of mPGES-1 inhibitors
Biochemical in vitro inhibition of enzymes involved in prostaglandin biosynthesis by
mPGES-1 inhibitors: 934, 117, 118, 322, and 323. No inhibition (No inh.) signifies less than
10% inhibition at 10 M compound concentration.
Table 2. In vitro performance of mPGES-1 inhibitors
Biochemical and cellular in vitro performance of mPGES-1 inhibitors 934, 117, 118, 322,
and 323 presented as mean IC50 value±SD.
This article is protected by copyright. All rights reserved.

Figure 1. Chemical structures of small molecule mPGES-1 inhibitors.
This article is protected by copyright. All rights reserved.

Figure 2. Inhibition of recombinant human mPGES-1 by 934, 117, 118, 322, and 323.
Potency was determined by MDA-TBA assay. Data are presented as mean ± SD of technical
duplicates from one representative experiment. The experiment was performed three times
for 118 and four times for 934, 117, 322, and 323.
This article is protected by copyright. All rights reserved.

Figure 3. Inhibition of PGE2 synthesis in intact A549 cells. A549 cells cultured in 2% FBS
were treated with IL-1 (10 ng/ml) for 24 h in the presence of 934, 117, 118, 322, and 323 at
indicated concentrations or vehicle control (1% DMSO). NS-398 at 10 µM was used as
positive control. PGE2 in supernatants was measured by EIA. Data are presented as mean ±
SD of technical duplicates. The absolute PGE2 concentration in the vehicle control was 10.8
±0.4 ng/ml for 934, 5.0 ± 0.5 ng/ml for 117 and 118, and 19.7 ± 1.8 ng/ml for 322 and 323.
The experiment was performed once.
This article is protected by copyright. All rights reserved.

Figure 4. Inhibition of PGE2 synthesis in human whole blood. Freshly drawn blood was
incubated with compounds at various concentrations or vehicle control (DMSO) for 30 min
and then treated with LPS (10 g/ml) for 24 h, when plasma was recovered. Diclofenac at 10
µM and NS-398 at 0.1 µM were used as reference compounds. PGE2 concentration was
measured by LC-MS/MS. Data are presented as mean ± SD of technical duplicates from one
representative experiment. The absolute PGE2 production in the vehicle control was 45.7 ±
3.7 ng/ml. The compounds were tested in two experiments.
This article is protected by copyright. All rights reserved.