JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 JPET ThisFast article Forward. has not been Published copyedited andon formatted.June 11, The 2008 final versionas DOI:10.1124/jpet.108.139444 may differ from this version. JPET #139444

Licofelone suppresses prostaglandin E2 formation by interference with the inducible

microsomal prostaglandin E2 synthase-1*

Andreas Koeberle, Ulf Siemoneit, Ulrike Bühring, Hinnak Northoff, Stefan Laufer, Wolfgang

Albrecht and Oliver Werz

Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8, D-72076 Downloaded from Tuebingen, Germany (A.K., U.S., U.B., S.L., W.A., O.W.)

Institute for Clinical and Experimental Transfusion Medicine, University Medical Center jpet.aspetjournals.org Tuebingen, Hoppe-Seyler-Straße 3, 72076 Tuebingen, Germany (H.N.)

at ASPET Journals on September 27, 2021

1

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Running title: Licofelone inhibits mPGES-1

Correspondence to: Dr. Oliver Werz, Department of Pharmaceutical Analytics,

Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8, D-72076

Tuebingen, Germany. Phone: +4970712972470; Fax: +497071294565; e-mail: [email protected]

Downloaded from Number of text pages: 31

Tables: 0

Figures: 4 jpet.aspetjournals.org

References: 40

Words Abstract: 207

Introduction: 554 at ASPET Journals on September 27, 2021

Discussion: 1318

Abbreviations used: cPLA2, cytosolic phospholipase A2; cPGES, cytosolic prostaglandin E2 synthase; ELISA, enzyme-linked immunosorbent assay; FLAP, 5-lipoxygenase-activating protein; 12-HHT,

12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; mPGES, microsomal prostaglandin E2 synthase; NSAIDs, non-steroidal anti-inflammatory drugs; PBS, phosphate buffered saline; SDS; sodium dodecylsulphate.

Section: Cellular and molecular

2 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Abstract

The anti-inflammatory drug licofelone (= ML3000, 2-[6-(4-chlorophenyl)-2,2-dimethyl-7- phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid), currently undergoing phase III trials for osteoarthritis, inhibits the prostaglandin (PG) and leukotriene biosynthetic pathway.

Licofelone was reported to suppress the formation of PGE2 in various cell-based test systems, but the underlying molecular mechanisms are not entirely clear. Here, we examined the direct interference of licofelone with enzymes participating in PGE2 biosynthesis, that is, COX-1 Downloaded from and COX-2 as well as microsomal PGE2 synthase (mPGES)-1. Licofelone concentration- dependently inhibited isolated COX-1 (IC50 = 0.8 µM), whereas isolated COX-2 was less

affected (IC50 > 30 µM). However, licofelone efficiently blocked the conversion of PGH2 to jpet.aspetjournals.org

PGE2 mediated by mPGES-1 (IC50 = 6 µM) derived from microsomes of interleukin-1β- treated A549 cells, being about equipotent to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-

5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), a well-recognized mPGES-1 inhibitor. In at ASPET Journals on September 27, 2021 intact interleukin-1β-treated A549 cells, licofelone potently (IC50 < 1 µM) blocked formation of PGE2 in response to ionophore A23187 plus exogenous arachidonic acid, but the concomitant generation of 6-keto PGF1α, used as a biomarker for COX-2 activity, was not inhibited. We conclude that licofelone suppresses inflammatory PGE2 formation preferentially by inhibiting mPGES-1 at concentrations that do not affect COX-2, implying an attractive and thus far unique molecular pharmacological dynamics as inhibitor of COX-1, the

5-lipoxygenase pathway, and of mPGES-1.

3 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Introduction

Prostaglandins (PGs) and leukotrienes are powerful bioactive lipid mediators that are involved in numerous homeostatic biological functions but also in inflammation (Funk, 2001). The biosynthesis of PGs is initialized by COX isoenzymes, namely COX-1, a constitutively expressed enzyme in numerous cell types thought to provide PGs mainly for physiological functions, and COX-2, an inducible isoform in inflammatory cells, primarily producing PGs relevant for inflammation, fever and pain (Hawkey, 1999). Following conversion of Downloaded from arachidonic acid to PGH2 by COX enzymes, PGH2 is subsequently isomerized by three different PGE2 synthases to PGE2. Whereas the cytosolic PGE2 synthase (cPGES) and the

membrane-bound PGE2 synthase (mPGES)-2 are constitutive enzymes, the mPGES-1 is an jpet.aspetjournals.org inducible isoform (Samuelsson et al., 2007). Co-transfection experiments of COX-1/2 with

PGES isoenzymes imply that select molecular interactions between COX and PGES

isoenzymes cause preferential functional coupling (Murakami et al., 2000; Samuelsson et al., at ASPET Journals on September 27, 2021

2007). Thus, cPGES uses PGH2 produced by COX-1, whereas mPGES-1 receives PGH2 from

COX-2.

PGE2 plays a major role in the pathophysiology of inflammation, pain and pyresis, but also regulates physiological functions in the gastrointestinal tract, the kidney, and in the immune and nervous system (Smith, 1989). The classical non-steroidal anti-inflammatory drugs

(NSAIDs) reduce PGE2 biosynthesis by inhibiting both COX isoenzymes, and are potent suppressors of inflammation, fever and pain (Funk, 2001). Chronic use of these drugs is associated with severe side-effects mainly gastrointestinal injury and renal irritations, apparently due to suppression of COX-1-derived PGE2 (Rainsford, 2007). COX-2 selective inhibitors were designed to minimize gastrointestinal complications of traditional NSAIDs, but recent clinical studies indicated small but significantly increased risks for cardiovascular events (McGettigan and Henry, 2006).

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Licofelone (= ML3000, 2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H- pyrrolizin-5-yl] acetic acid, Fig. 1a) is an anti-inflammatory drug that inhibits the COX and 5- lipoxygenase pathway and is currently undergoing phase III trials for osteoarthritis (for review see Celotti and Laufer, 2001; Kulkarni and Singh, 2007). The effectiveness of licofelone has been demonstrated in animal models as well as in studies on humans, and is attributed mainly to the efficient suppression of PGE2 formation (Kulkarni and Singh, 2007).

Interestingly, in contrast to NSAIDs and selective COX-2 inhibitors, licofelone shows an Downloaded from improved gastrointestinal and potentially cardiovascular safety (Bias et al., 2004; Rotondo et al., 2006; Vidal et al., 2007). This effect of licofelone might be attributable to the

accompanied suppression of leukotrienes (Celotti and Laufer, 2001), which significantly jpet.aspetjournals.org contribute to gastric epithelial injury as well as to atherogenesis (Peters-Golden and

Henderson, 2007).

Although the effectiveness and tolerability of licofelone as an anti-inflammatory drug is well at ASPET Journals on September 27, 2021 documented, sparse data on its molecular mode(s) of action are available. We recently analyzed the molecular mechanism of the inhibition of the biosynthesis of 5-lipoxygenase products by licofelone and we found that licofelone-mediated suppression of leukotriene synthesis (IC50 = 1.7 µM) is mainly related to its interference with 5-lipoxygenase-activating protein (FLAP) rather than with 5-lipoxygenase (Fischer et al., 2007). MK-886 (3-[1-(4- chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), a well- known FLAP inhibitor, was shown to inhibit mPGES-1-activity (Claveau et al., 2003). As we hypothesized similarities between licofelone and MK-886, both in terms of structural conformation and mechanism of leukotriene synthesis inhibition (Fischer et al., 2007), we addressed the mode of action of licofelone in the suppression of PGE2 formation. Our data indicate that the potent inhibition of COX-2-mediated PGE2 formation by licofelone is seemingly a cause of interference with mPGES-1, rather than with COX-2.

5 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Methods

Materials

Licofelone, bovine insulin, and anti-6-keto PGF1α antibody were generous gifts by Merckle

GmbH (Ulm, Germany), Sanofi-Aventis (Frankfurt, Germany), and Dr. T. Dingermann

(University of Frankfurt, Germany), respectively. The thromboxane synthase inhibitor

CV4151 was synthesized according to Kato et al., 1985. DMEM/High glucose (4.5 g/l) medium, penicillin, streptomycin, trypsin/EDTA solution, PAA (Coelbe, Germany); PGH2, Downloaded from

Larodan (Malmö, Sweden); 11β-PGE2, PGB1, MK-886, 6-keto PGF1α, human recombinant

COX-2, ovine isolated COX-1, Cayman Chemical (Ann Arbor, MI); [5, 6, 8, 9, 11, 12, 14,

3 3

15- H] arachidonic acid ([ H]arachidonic acid), BioTrend Chemicals GmbH (Cologne, jpet.aspetjournals.org

Germany); Ultima GoldTM XR, Perkin Elmer (Boston, MA). All other chemicals were obtained from Sigma-Aldrich (Deisenhofen, Germany) unless stated otherwise.

at ASPET Journals on September 27, 2021

Cells and cell viability assay

Platelets were freshly isolated from leukocyte concentrates obtained at the Blood Center of the University Hospital Tuebingen (Germany) as described (Albert et al., 2002). In brief, venous blood was taken from healthy adult donors that did not take any medication for at least

7 days and leukocyte concentrates were prepared by centrifugation (4000×g, 20 min, 20 °C).

Platelets were immediately isolated by dextran sedimentation and centrifugation on Nycoprep cushions (PAA, Coelbe, Germany). The supernatants were mixed with PBS, pH 5.9 (3:2 v/v), centrifuged (2100×g, 15 min, RT) and the pelleted platelets were resuspended in PBS, pH

5.9/0.9% NaCl (1:1, v/v). Washed platelets were finally resuspended in PBS pH 7.4 and 1 mM CaCl2. For incubations with solubilized compounds, methanol or DMSO was used as vehicle, never exceeding 1% (v/v).

A549 cells were cultured in DMEM/High glucose (4.5 g/l) medium supplemented with heat- inactivated fetal calf serum (10%, v/v), penicillin (100 U/ml) and streptomycin (100 µg/ml) at

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37 °C and 5% CO2. After 3 days, confluent cells were detached using 1 × trypsin/EDTA solution and reseeded at 2 × 106 cells in 20 ml medium. Cell viability was measured using the colorimetric MTT dye reduction assay. A549 cells (4 × 104 cells/100 µl medium) were plated into a 96-well microplate and incubated at 37 °C and 5% CO2 for 16 h. Then, 10 µM of licofelone or solvent (DMSO) was added and the samples were incubated for another 5 h.

MTT (20 µl, 5 mg/ml) was added and the incubations were continued for 4 h. The formazan product was solubilized with SDS (10%, w/v in 20 mM HCl) and the absorbance of each Downloaded from sample was measured at 595 nm relative to that of vehicle (DMSO)-treated control cells using a multiwell scanning spectrophotometer (Victor3 plate reader, PerkinElmer, Rodgau-

Juegesheim, Germany). Licofelone did not significantly reduce cell viability within 5 h (data jpet.aspetjournals.org not shown), excluding possible acute cytotoxic effects of the compound in the cellular assays.

Induction of mPGES-1 in A549 cells and isolation of microsomes at ASPET Journals on September 27, 2021

Preparation of A549 cells was performed as described (Jakobsson et al., 1999). In brief, cells

(2 × 106 cells in 20 ml medium) were plated in 175 cm2 flasks and incubated for 16 h at 37 °C and 5% CO2. Subsequently, the culture medium was replaced by fresh DMEM/High glucose

(4.5 g/l) medium containing fetal calf serum (2%, v/v). In order to induce mPGES-1 expression, interleukin-1β (1 ng/ml) was added, and the cells were incubated for another 72 h.

Thereafter, cells were detached with 1 × trypsin/EDTA, washed with PBS and frozen in liquid nitrogen. Ice-cold homogenization buffer (0.1 M potassium phosphate buffer pH 7.4, 1 mM

PMSF, 60 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 2.5 mM glutathione and 250 mM sucrose) was added and after 15 min, cells were resuspended and sonicated on ice (3 × 20 sec). The homogenate was subjected to differential centrifugation at 10,000×g for 10 min and at 174,000×g for 1 h at 4 °C. The pellet (microsomal fraction) was resuspended in 1 ml

7 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444 homogenization buffer and the protein concentration was determined by the Coomassie protein assay.

Determination of PGE2 synthase activity in microsomes of A549 cells

Microsomal membranes of A549 cells were diluted in potassium phosphate buffer (0.1 M, pH

7.4) containing 2.5 mM glutathione (100 µl total volume), and test compounds or vehicle

(DMSO) was added. After 15 min, PGE2 formation was initiated by addition of PGH2 (20 Downloaded from µM, final concentration). After 1 min at 4 °C, the reaction was terminated with 100 µl of stop solution (40 mM FeCl2, 80 mM citric acid and 10 µM of 11β-PGE2), PGE2 was separated by

solid phase extraction on reversed phase (RP)-C18 material using acetonitrile (200 µl) as jpet.aspetjournals.org eluent, and analyzed by RP-HPLC (30% acetonitrile aq. + 0.007% TFA (v/v), Nova-Pak®

C18 column, 5 × 100 mm, 4 µm particle size, flow rate 1 ml/min) with UV detection at 195

β nm. 11 -PGE2 was used as internal standard to quantify PGE2 product formation by at ASPET Journals on September 27, 2021 integration of the area under the peaks.

Determination of PGE2 and 6-keto PGF1α formation in intact A549 cells

A549 cells (2 × 106 cells) were plated in a 175 cm2 flask and incubated for 16 h at 37 °C and

5% CO2. Thereafter, medium was replaced by fresh DMEM/High glucose (4.5 g/l) medium containing fetal calf serum (2%, v/v) and the cells were stimulated with interleukin-1β (1 ng/ml) for 72 h. After trypsination, the cells were washed twice with PBS. For determination

6 of PGE2, 2 × 10 cells resuspended in PBS (0.5 ml) containing CaCl2 (1 mM) were pre- incubated with the indicated compounds at 37 °C for 10 min and PGE2 formation was started by the addition of ionophore A23187 (2.5 µM), arachidonic acid (1 µM) and [3H]arachidonic acid (18.4 kBq). The reaction was stopped after 15 min at 37 °C and the samples were put on ice. After centrifugation (800×g, 5 min, 4 °C), the supernatant was acidified to pH 3 by addition of citric acid (20 µl, 2 M), and the internal standard 11β-PGE2 (2 nmol) was added. 8 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

PGE2 was separated by RP-18 solid phase extraction and eluted with acetonitrile (200 µl).

Solid phase extraction and HPLC analysis were performed as described above. The amount of

11β-PGE2 was quantified by integration of the area under the eluted peaks. For quantification of radiolabeled PGE2, fractions (0.5 ml) were collected and mixed with Ultima GoldTM XR

(2 ml) for liquid scintillation counting in a LKB Wallac 1209 Rackbeta Liquid Scintillation

Counter.

6 For determination of 6-keto PGF1α, 10 cells in 1 ml PBS containing CaCl2 (1 mM) were pre- Downloaded from incubated with the indicated compounds for 15 min at 37 °C, and 6-keto PGF1α formation was initiated by addition of arachidonic acid (30 µM). After 15 min at 37 °C, the reaction was

× stopped by cooling on ice. Cells were centrifuged (300 g, 5 min, 4 °C) and the amount of jpet.aspetjournals.org released 6-keto PGF1α was assessed by ELISA using a monoclonal antibody against 6-keto

PGF1α according to the protocol described by Yamamoto et al., 1987. For the ELISA, the

µ µ monoclonal antibody (0.2 g/200 l) was coated to microtiter plates via a goat anti-mouse at ASPET Journals on September 27, 2021 immunoglobulin G antibody. 6-Keto PGF1α (15 µg) was linked to bacterial β-galactosidase

(0.5 mg) and the enzyme activity bound to the antibody was determined in an ELISA reader at

550 nm (reference wavelength: 630 nm) using chlorophenol-red-β-D-galactopyranoside

(CPRG, Roche Diagnostic GmbH) as substrate.

Determination of COX-1 product formation in washed platelets

8 Freshly isolated platelets (10 /ml PBS containing 1 mM CaCl2) were pre-incubated with the indicated agents for 5 min at RT. After addition of 5 µM arachidonic acid and further incubation for 5 min at 37 °C, the COX-1 product 12(S)-hydroxy-5-cis-8,10-trans- heptadecatrienoic acid (12-HHT) was extracted and then analyzed by HPLC as described

(Albert et al., 2002).

Determination of PGE2 in whole blood

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Peripheral blood from healthy adult volunteers, who had not received any medication for at least two weeks under informed consent, was obtained by venipuncture and collected in syringes containing heparin (20 U/ml). For determination of PGE2, aliquots of whole blood

(0.8 ml) were mixed with the thromboxane synthetase inhibitor CV4151 (1 µM) and aspirin

(50 µM). A total volume of 1 ml was adjusted with sample buffer (10 mM potassium phosphate buffer pH 7.4, 3 mM KCl, 140 mM NaCl and 6 mM D-glucose). After pre- incubation with the indicated compounds for 10 min at RT, the samples were stimulated with Downloaded from lipopolysaccharide (10 µg/ml) for 5 h at 37 °C. PGE2 formation was stopped on ice, the samples were centrifuged (2300×g, 10 min, 4 °C) and citric acid (30 µl, 2 M) was added to

the supernatant. After another centrifugation step (2300×g, 10 min, 4 °C), solid phase jpet.aspetjournals.org extraction and HPLC analysis of PGE2 were performed as described above. The PGE2 peak (3 ml), identified by co-elution with authentic standard, was collected and acetonitrile was

removed under a nitrogen stream. The pH was adjusted to 7.2 by addition of 10 × PBS buffer at ASPET Journals on September 27, 2021 pH 7.2 (230 µl), before PGE2 was quantified using a PGE2 High Sensitivity EIA Kit (Assay

Designs, Ann Arbor, MI) according to the manufacturer's protocol.

Activity assays of isolated COX-1 and -2

Inhibition of the activities of isolated ovine COX-1 and human COX-2 was performed as described (Capdevila et al., 1995; Mitchell et al., 1993). Though the purified COX-1 is not of human origin, ovine COX-1 is generally used for inhibitor studies when examining the effectiveness of compounds on the activity of isolated COX-1 enzyme (Mitchell et al., 1993).

Briefly, purified COX-1 (ovine, 50 units) or COX-2 (human recombinant, 20 units) were diluted in 1 ml reaction mixture containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5

µM haemoglobin, 100 µM EDTA at 4 °C and pre-incubated with the test compounds for 5 min. Samples were pre-warmed for 60 sec at 37 °C and arachidonic acid (5 µM for COX-1, 2

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µM for COX-2) was added to start the reaction. After 5 min at 37 °C, the COX product 12-

HHT was extracted and then analyzed by HPLC as described (Albert et al., 2002).

Statistics

Data are expressed as mean ± SE. The program Graphpad Instat (Graphpad Software Inc., San

Diego, CA) was used for statistical comparisons. Statistical evaluation of the data was performed by one-way ANOVAs for independent or correlated samples followed by Tukey Downloaded from HSD post-hoc tests. Where appropriate, Student´s t test for paired and correlated samples was applied. A p value of <0.05 (*) was considered significant.

jpet.aspetjournals.org at ASPET Journals on September 27, 2021

11 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Results

Effects of licofelone on the activity of isolated COX-1/2

Previously, it was found that licofelone suppresses cellular COX-1 in bovine platelets (IC50 =

0.21 µM) (Laufer et al., 1994b) and thromboxane xB2 synthesis in ionophore-stimulated whole blood (IC50 = 3.9 µM) (Tries et al., 2002b) or COX-2-mediated PGE2 synthesis in ionomycin-stimulated human subchondral osteoblasts (IC50 < 0.8 µM) (Paredes et al., 2002).

In order to investigate the direct interference of licofelone with isolated COX enzymes, Downloaded from purified ovine COX-1 and purified human recombinant COX-2 were pre-incubated with licofelone (or vehicle, DMSO) for 5 min, arachidonic acid (5 µM for COX-1, 2 µM for COX-

2) was added and after 5 min, the formation of 12-HHT as the major COX-1/2-derived jpet.aspetjournals.org product under these experimental conditions (Capdevila et al., 1995) was determined.

Licofelone potently and concentration-dependently suppressed 12-HHT formation by COX-1

with an IC50 of 0.8 µM (Fig. 1b). In contrast, licofelone was less efficient in suppressing the at ASPET Journals on September 27, 2021 activity of COX-2 under comparable assay conditions with an IC50 value > 30 µM (Fig. 1c), excluding a strong direct interaction of licofelone with COX-2. In control experiments, the

COX-2-selective celecoxib (5 µM), strongly suppressed the formation of 12-HHT by COX-2 as expected (Fig. 1c).

Effects of licofelone on the activity of mPGES-1

Induction of COX-2 by pro-inflammatory stimuli coincides with the expression of mPGES-1 resulting in substantial PGE2 formation (Murakami et al., 2000; Thoren and Jakobsson, 2000).

Since licofelone potently reduced COX-2-coupled PGE2 formation in intact cells (Paredes et al., 2002), but caused no significant inhibition of isolated COX-2, we investigated whether licofelone could interfere with mPGES-1 activity. Expression of COX-2 and mPGES-1 in

A549 cells was induced by treatment with 1 ng/ml interleukin-1β for 72 h. Microsomes were isolated, pre-incubated with or without licofelone and PGE2 formation was induced by

12 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

addition of 20 µM PGH2. After 1 min incubation, PGE2 formation was terminated and PGE2 was determined by RP-HPLC. The mPGES-1 inhibitor MK-886 was used as reference drug, and in agreement with the literature (IC50 = 2.4 µM; (Claveau et al., 2003; Riendeau et al.,

2005)), concentration-dependently blocked PGE2 formation with an IC50 = 2 µM (Fig. 2a).

Licofelone potently and concentration-dependently suppressed PGE2 formation with an IC50 =

6 µM, being almost equipotent to MK-886 (Fig. 2a). In contrast, neither the COX-1/2 inhibitor indomethacin nor the COX-2 selective celecoxib (up to 10 µM, each) significantly Downloaded from reduced the enzymatic conversion of PGH2 to PGE2 in this assay (data not shown).

To investigate if licofelone blocks mPGES-1 in a reversible manner, microsomal preparations

of A549 cells were pre-incubated with licofelone at 1 and 10 µM for 15 min, and for jpet.aspetjournals.org comparison with MK-886 at 0.3 and 3 µM, each. The samples containing 3 µM MK-886 or

10 µM licofelone were divided: one part was diluted 10-fold with reaction buffer to obtain a

final concentration of 0.3 and 1 µM inhibitor, respectively. Control samples without inhibitor at ASPET Journals on September 27, 2021 received DMSO. Then, PGH2 was added to each sample to start the reaction. As shown in

Fig. 2b, both MK-886 as well as licofelone slightly reduced PGE2 formation at 0.3 µM or 1

µM, respectively, whereas at 3 µM or 10 µM, PGE2 synthesis was potently inhibited. Dilution of these incubations prior addition of substrate to a final concentration of 0.3 µM or 1 µM, respectively, reversed the inhibitory effect of both MK-886 and licofelone, implying that licofelone as MK-886 inhibit mPGES-1 in a reversible fashion.

It appeared reasonable that the potency of licofelone to inhibit mPGES-1 may depend on the substrate concentration. However, decreasing the PGH2 concentrations from 20 µM to 1 µM did not significantly affect the potency of licofelone, rather excluding a competitive inhibitory mechanism (Fig. 2c). The potency of the reference inhibitor MK-886 (10 µM) varied at 1 µM

PGH2 in individual microsomal preparations (data not shown), therefore we normalized the activity data in Fig. 2c to vehicle control (100%) and 10 µM MK-886 (0%). Similarly, the

13 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

effectiveness of MK-886 was essentially the same at 1 or 20 µM PGH2 (Koeberle, A.,

Pollastro, F., Northoff, H., and Werz, O., 2008, submitted).

Effects of licofelone on prostanoid formation in intact cells.

Interleukin-1β-stimulated A549 cells express mPGES-1 and COX-2, but not COX-1 (Asano et al., 1996; Thoren and Jakobsson, 2000). Since COX-2 preferably couples with mPGES-1

(Murakami et al., 2000; Thoren and Jakobsson, 2000), it is reasonable that PGE2 production Downloaded from in interleukin-1β-stimulated A549 cells is mainly dependent on COX-2-derived PGH2 and subsequent cleavage to PGE2 by mPGES-1. However, cPGES and mPGES-2 are still likely to

contribute to PGE2 formation, though to a lesser extent (Park et al., 2006). A549 cells (pre- jpet.aspetjournals.org treated with interleukin-1β for 72 h) were pre-incubated with licofelone, MK-886 or vehicle

(DMSO) for 10 min prior to stimulation of the cells with 2.5 µM A23187 plus 1 µM

3

arachidonic acid and [ H]arachidonic acid (18.4 kBq). Induction of PGE2 synthesis by at ASPET Journals on September 27, 2021

A23187 and exogenous arachidonic acid is thought to exclude possible effects of licofelone on the level of receptor-coupled signal transduction and/or on cytosolic phospholipase A2

(cPLA)2-mediated arachidonic acid release. Thus, cell stimulation by A23187 circumvents receptor-coupled signal transduction by activating the cell via massive elevation of intracellular Ca2+, and co-addition of exogenous arachidonic acid circumvents the requirement for the supply of endogenous substrate for PGE2 formation provided by cPLA2. Formed PGE2 was separated by RP-HPLC and quantified by liquid scintillation counting. Celecoxib (5 µM) almost completely abolished PGE2 production (Fig. 3a) and PGE2 was not detectable in the presence of 10 µM indomethacin (not shown). MK-886 was hardly active at 10 µM, and at 33

µM reduced PGE2 formation by 63%. The formation of PGE2 was potently and concentration- dependently reduced by licofelone with an IC50 = 0.1 µM (Fig. 3a). In so far, licofelone is about 60-fold more potent in the cellular assay than in cell-free systems, and more than 100- fold as potent as MK-886. In order to exclude the possibility that the potent suppression of

14 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

PGE2 is due to COX-2 inhibition by licofelone, we assessed the formation of the stable PGI2 degradation product 6-keto PGF1α in parallel as a parameter of cellular COX-2 activity in these A549 cells. However, neither licofelone nor MK-886 caused a significant reduction of

6-keto PGF1α formation in interleukin-1β-stimulated A549 cells, up to the highest concentrations (33 µM for licofelone and 30 µM for MK-886) tested (Fig. 3b). Control experiments using indomethacin (10 µM) or celecoxib (5 µM) led to an efficient decrease of the 6-keto-PGF1α levels under these conditions. On the other hand, for inhibition of cellular Downloaded from COX-1 activity we could confirm the efficacy of licofelone in intact human platelets reported previously (Laufer et al., 1994b), where an IC50 for 12-HHT formation of 0.24 µM was

apparent in our assay (Fig. 3c). jpet.aspetjournals.org

Effects of licofelone on the formation of PGE2 in human whole blood.

Although the effects of licofelone on general PGE2 formation using a simple ELISA at ASPET Journals on September 27, 2021 technique as read-out system in human osteoarthritis subchondral osteoblasts was shown by others before (Paredes et al., 2002), we established a novel assay that allowed us to specifically assess COX-2-mediated PGE2 synthesis in human whole blood (Koeberle, A.,

Pollastro, F., Northoff, H., and Werz, O., 2008, submitted). Heparinized human whole blood was pre-incubated with licofelone for 10 min, prior stimulation with lipopolysaccharide (10

µg/ml) for 5 h. In order to minimize the contribution of COX-1 in PGH2 synthesis, and thus, to establish experimental settings where PGE2 is primarily produced via the COX-2/mPGES-1 pathway, COX-1 was inactivated by the use of 50 µM aspirin and thromboxane formation by platelets was blocked using the thromboxane synthase inhibitor CV4151 (1 µM). For measuring PGE2, we first separated PGE2 from other arachidonic acid metabolites that may possibly interfere with commercially available PGE2 ELISA detection systems. Thus, PGE2 was isolated from plasma by solid phase extraction, separated by RP-HPLC and then quantified by ELISA. As shown in Fig. 4, licofelone concentration-dependently reduced

15 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

PGE2 synthesis with an IC50 of approx. 5 µM, but failed to completely decrease the PGE2 level and about 30% PGE2 still remained. Similarly, MK-886 only partially reduced PGE2 formation at 30 µM (44±8% versus vehicle control), and higher concentrations (up to 100

µM) caused no further decrease of PGE2 levels (not shown). Celecoxib (20 µM) and indomethacin (50 µM) efficiently reduced PGE2 formation to 24±13% and 16±4%, respectively (Fig. 4), implying that COX inhibition (by indomethacin or celcoxib) might be a more efficient mean to reduce PGE2 formation in whole blood as compared to mPGES-1 Downloaded from inhibition (by licofelone or MK-886). jpet.aspetjournals.org at ASPET Journals on September 27, 2021

16 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Discussion

Licofelone is an advanced dual inhibitor of the COX and 5-lipoxygenase pathway, exhibiting not only a remarkable efficacy as anti-inflammatory drug, but also exerts significantly less adverse effects under chronic use that are usually associated with NSAIDs (i.e. gastrointestinal injury and renal irritations) or selective COX-2 inhibitors (cardiovascular risks) (Kulkarni and Singh, 2007). Our data show that licofelone is a direct and potent inhibitor of COX-1 in cell-free assays, confirming previous conclusions drawn from analysis Downloaded from of the compound in cell-based test systems (Laufer et al., 1994b; Tries et al., 2002b). On the other hand, licofelone hardly inhibits isolated COX-2, and fails to reduce the formation of the

α COX-2 product 6-keto PGF1 in intact cells, despite potent suppression of COX-2-mediated jpet.aspetjournals.org

PGE2 formation. Since licofelone blocked the conversion of PGH2 to PGE2 catalyzed by mPGES-1 in a cell-free assay, we conclude that suppression of PGE2 synthesis in the cell is

due to interference with mPGES-1, rather than with COX-2. Taken together, the primary at ASPET Journals on September 27, 2021 mechanism of action of licofelone is as a COX-1 inhibitor but the additional suppression of mPGES-1 and of leukotriene biosynthesis may contribute to the overall efficacy and tolerability of this drug.

Initially, licofelone was proposed as a potent dual COX/5-lipoxygenase inhibitor based on its suppressive effect on COX and 5-lipoxygenase activity in intact bovine and human platelets and granulocytes (Laufer et al., 1994a; Laufer et al., 1994b). Several subsequent investigations confirmed inhibition of the 5-lipoxygenase pathway and of PGE2 or thromboxane B2 formation by licofelone in cell-based models (Fischer et al., 2007; Jovanovic et al., 2001; Paredes et al., 2002; Tries et al., 2002b; Vidal et al., 2007). Moreover, reduction of prostanoids (i.e. PGE2 and thromboxane B2) and leukotriene B4 by licofelone in vivo was shown in animal models such as experimental dog osteoarthritis (Jovanovic et al., 2001;

Lajeunesse et al., 2004) or fMLP-challenged rabbits (Rotondo et al., 2006). However, the experimental settings in all these studies did not allow the elucidation of the molecular

17 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444 mechanisms resulting in suppressed eicosanoid formation. In fact, an efficient suppression of leukotriene biosynthesis by licofelone was observed only in models with intact cells, where

FLAP represents a pivotal co-factor for leukotriene B4 formation. In leukocyte homogenates or in test systems with purified recombinant 5-lipoxygenase, the inhibitiory activity of licofelone was negligible (Fischer et al., 2007). FLAP inhibitors (e.g. MK-886 or BAY

X1005) are well-appreciated pharmacological tools for intervention with leukotriene synthesis

(Gillard et al., 1989), and licofelone shares structural similarities with MK-886 and exhibits Downloaded from similar pharmacological properties (Fischer et al., 2007).

In analogy to cellular leukotriene biosynthesis, several different enzymes are required for the

consecutive transformation of arachidonic acid to distinct PGs in the cell upon activation, jpet.aspetjournals.org including phospholipases A2, COX-1/2 and select PG synthases as well as the receptor for the external stimulus and the distal signaling molecules that eventually lead to release of

arachidonic acid and its metabolism (Funk, 2001). Consequently, any test compound resulting at ASPET Journals on September 27, 2021 in reduced biosynthesis of a certain PG in stimulated cells does not have to unequivocally act

(solely) on COX, but also may interfere with other components. Our data clearly show that licofelone is a potent inhibitor of isolated COX-1 (IC50 = 0.8 µM) but not of isolated human

COX-2 (IC50 > 30 µM). These data are in line with the observation that in stimulated A549 cells, PGE2 formation was potently inhibited (IC50 = 0.1 µM), whereas the generation of 6- keto PGF1α (the stable metabolite of PGI2) remained unaffected by licofelone. Since the COX-

1 route for supply of PGH2 can be ignored in A549 cells (Warner et al., 2006), the COX-1 inhibitory effect of licofelone might be negligible and we conclude that interference with mPGES-1 is mainly responsible for suppression of PGE2 by licofelone. Note that due to supply of exogenous arachidonic acid in the A549 cellular assay, suppression of phospholipase A2 enzymes (required for arachidonic acid liberation) by licofelone as possible point of attack can be ruled out, supported also by the lack of licofelone to reduce the

18 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444 formation of the 12-lipoxygenase product 12-hydro(pero)xyeicosatetraenoic acid from endogenously released arachidonic acid in thrombin-activated platelets (data not shown).

The effectiveness of licofelone as inhibitor of mPGES-1 was demonstrated in this study by (I) a cell-free assay measuring the direct conversion of PGH2 to PGE2 by mPGES-1, and (II) by a cellular assay determining PGE2 from exogenously added arachidonic acid. Interestingly, licofelone was about 60-fold more potent in intact A549 cells as compared to the cell-free assay which is not readily understood. It is puzzling that MK-886 showed no such different Downloaded from potencies, and in the whole blood assay the efficacy of licofelone was comparable as in the cell-free test system. Possibly, additional A549-specific (intra-)cellular

components/mechanisms are operative, or licofelone interferes with other steps in the jpet.aspetjournals.org formation of PGE2 from endogenous arachidonic acid such as the transfer of PGH2 from

COX-2 to mPGES-1. The loss of licofelone´s efficacy in whole blood versus intact A549 cells

could be due to binding to plasma albumin. at ASPET Journals on September 27, 2021

Pharmacological intervention with mPGES-1 in the therapy of PGE2-mediated disorders including rheumatoid arthritis, fever and pain is proposed to have advantages over general suppression of all PGs by applying COX inhibitors (Jachak, 2007; Samuelsson et al., 2007).

Drugs selective for COX-2 have originally been developed in order to reduce the gastrointestinal risk of unselective COX inhibitors, but the association with an increased incidence of major adverse cardiovascular events raises the need for alternative therapeutic strategies and targets. In particular, select mPGES-1 inhibition is proposed to avoid the increased incidence of major adverse cardiovascular events of COX-2 selective drugs due to the lack of impaired PGI2 levels (Wang et al., 2006). Thus, deletion of mPGES-1 in mice impaired inflammatory reactions and pain responses (Trebino et al., 2003), but neither affected thrombogenesis nor blood pressure, and caused augmented PGI2 expression (Cheng et al., 2006), implying mPGES-1 as a target for therapeutic intervention in patients with cardiovascular risk. To date, MK-886 that was originally developed as inhibitor of leukotriene

19 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444 biosynthesis (Gillard et al., 1989), and structural derivatives of MK-886 have been reported as potent and specific inhibitors of mPGES-1 (Claveau et al., 2003; Riendeau et al., 2005).

Recently, synthetic phenanthrene imidazoles were presented as selective, and orally active mPGES-1 inhibitors (IC50 = 0.42 to 1.3 µM in A549 cells and human whole blood, respectively) with significant analgesic effect in a guinea pig hyperalgesia model (Cote et al.,

2007). Taken together, preferential inhibition of mPGES-1 over COX-2 by licofelone might be advantageous in clinical use. Downloaded from There is accumulating evidence that inhibition of both the leukotriene and the PG biosynthetic pathway is superior over single interference, not only in terms of anti-inflammatory

effectiveness, but also due to a lower incidence of gastrointestinal toxicity, typically related to jpet.aspetjournals.org

COX inhibition (Celotti and Laufer, 2001; Kulkarni and Singh, 2007). In fact, licofelone markedly improved gastrointestinal tolerability in animal models and offered gastroprotection

against NSAIDs-induced gastropathy (Kulkarni and Singh, 2007; Wallace et al., 1994). In at ASPET Journals on September 27, 2021 healthy volunteers, licofelone showed significantly superior gastric tolerability and a lower incidence of ulcers compared with naproxen (Bias et al., 2004). Moreover, licofelone in contrast to COX-2-selective inhibitors may have a favorable cardiovascular profile because of the presumed lack of impaired PGI2 levels, and because of the ability to suppress the formation of leukotrienes which are mediators of atherogenesis and cardiovascular disease

(Peters-Golden and Henderson, 2007). In fact, licofelone reduced neointimal formation and inflammation in an atherosclerotic rabbit model (Vidal et al., 2007), protected rabbits from the cardiovascular derangement triggered by fMLP (Rotondo et al., 2006), and had a significant antithrombotic activity and a marked platelet aggregation inhibiting effect in rats (Tries et al.,

2002a). Though licofelone, based on its unique and favorable molecular pharmacological profile regarding intervention with eicosanoid biosynthesis, may represent a suitable drug for the therapy of chronic inflammatory diseases with low risks of adverse effects, long-term

20 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444 studies evaluating licofelone´s cardiovascular toxicity have to be awaited in order to judge its cardiovascular safety in chronic use.

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

21 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

Acknowledgments

We thank Gertrud Kleefeld for expert technical assistance. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

22 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

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*Footnotes:

Reprint requests should be send to: Dr. Oliver Werz, Department for Pharmaceutical

Analytics, Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8, D-

72076 Tuebingen, Germany. Phone: +49-7071-2972470; Fax: +49-7071-294565; e-mail: [email protected] Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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Legends for figures

Fig. 1 Effects of licofelone on the activity of isolated COX enzymes. (a) Chemical structure of licofelone. Effects of licofelone on the activity of isolated COX-1 (b) and COX-2 (c).

Purified ovine COX-1 (50 units) or human recombinant COX-2 (20 units) were added to a

COX reaction mix, containing 5 mM glutathione. The COX enzymes were pre-incubated with the test compounds for 5 min and then the reaction was started with 5 µM (COX-1) or 2 µM

(COX-2) arachidonic acid. After 5 min at 37 °C, the formation of 12-HHT was determined by Downloaded from RP-HPLC as described. Indomethacin (Indo, 10 µM) and celecoxib (Cele, 5 µM) were used as controls. Data are given as mean +/- S.E., n = 3-4, **p < 0.01, ***p < 0.001 vs vehicle

(0.1% DMSO) control, ANOVA + Tukey HSD post-hoc tests. jpet.aspetjournals.org

Fig. 2 Effects of licofelone and MK-886 on the activity of mPGES-1. (a) Concentration-

β

response curves for licofelone and MK-886. Microsomal preparations of interleukin-1 - at ASPET Journals on September 27, 2021 stimulated A549 cells were pre-incubated with vehicle (DMSO) or the test compounds at the indicated concentrations for 15 min at 4 °C and the reaction was started with 20 µM PGH2.

After 1 min at 4 °C the reaction was terminated using a stop solution, containing FeCl2 and

11β-PGE2 (1 nmol) as internal standard. (b) Reversibility of mPGES-1 inhibition by licofelone and MK-886. Microsomal preparations of interleukin-1β-stimulated A549 cells were pre-incubated with 3 µM MK-886 or 10 µM licofelone for 15 min at 4 °C. An aliquot was diluted 10-fold to obtain an inhibitor concentration of 0.3 µM and 1 µM, respectively.

For comparison, microsomal preparations were pre-incubated for 15 min with 0.3 µM MK-

886 or 1 µM licofelone, or with vehicle (DMSO) and then 20 µM PGH2 was added (no dilution). Then, all samples were incubated for 1 min on ice and PGE2 formation was analyzed as described by RP-HPLC in the Methods section. Data are given as mean + S.E., n

= 3-4, *p < 0.05 vs vehicle (0.1% DMSO) control, ANOVA + Tukey HSD post-hoc tests. (c)

The potency of licofelone for mPGES-1 inhibition was compared at 1 and 20 µM PGH2 as

29 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

substrate. The amount of PGE2 was quantified for 1 µM PGH2 by use of a PGE2 High

Sensitivity EIA Kit according to the manufacturer's protocol. PGE2 production at 10 µM MK-

886 was set 0% of vehicle control (100 %) in order to compare both data sets. Data are given as mean +/- S.E., n = 2-4.

Fig. 3. Effects of licofelone on prostanoid formation in intact cells. (a) PGE2 formation in

A549 cells. Interleukin-1β-stimulated A549 cells (4 × 106/ml) were pre-incubated with the Downloaded from indicated compounds or vehicle (DMSO) for 10 min and then 2.5 µM A23187 plus 1 µM arachidonic acid and [3H]arachidonic acid (18.4 kBq) were added. After 15 min at 37 °C,

3 formed [ H]PGE2 was analysed by RP-HPLC and liquid scintillation counting as described in jpet.aspetjournals.org the experimental section. (b) 6-keto PGF1α formation in A549 cells. Interleukin-1β-stimulated

A549 cells (106/ml) were pre-incubated with the indicated compounds (or vehicle (DMSO))

for 15 min, 30 µM arachidonic acid was added and after 15 min at 37 °C the amount of at ASPET Journals on September 27, 2021 released 6-keto PGF1α was assessed by ELISA as described in the Methods section. MK-886

(30 µM), indomethacin (Indo, 10 µM), and celecoxib (Cele, 5 µM) were used as controls. (c)

12-HHT formation in platelets. Washed human platelets (108/ml PBS containing 1 mM

CaCl2) were incubated with the indicated concentrations of licofelone. After 5 min at RT, 5

µM arachidonic acid was added to induce COX-1 product formation and after 5 min at 37 °C,

12-HHT was determined by RP-HPLC. Indomethacin (10 µM) was used as control. Data are given as mean +/- S.E., n = 3-4, **p < 0.01, ***p < 0.001 vs vehicle (0.1% DMSO) control,

ANOVA + Tukey HSD post-hoc tests.

Fig. 4. Effects of licofelone on PGE2 biosynthesis in human whole blood. Heparinized human whole blood, treated with 1 µM thromboxane synthase inhibitor and 50 µM aspirin, was pre-incubated with the indicated concentrations of licofelone for 10 min at RT and then

PGE2 formation was induced by addition of 10 µg/ml lipopolysaccharide. After 5 h at 37 °C,

30 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. JPET #139444

PGE2 was extracted from plasma by RP-18 solid phase extraction, separated by RP-HPLC and quantified by ELISA as described. MK-886 (30 µM), indomethacin (Indo, 50 µM), celecoxib (Cele, 20 µM) or vehicle (DMSO) were used as controls. Data are given as mean

+/- S.E., n = 3, *p < 0.05, **p < 0.01 or ***p < 0.001 vs vehicle (0.1% DMSO) control,

ANOVA + Tukey HSD post-hoc tests. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

31 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 11, 2008 as DOI: 10.1124/jpet.108.139444 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021