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Rheumatology Advance Access published online on November 16, 2008
Rheumatology, doi:10.1093/rheumatology/ken398
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717

P. F. Sumariwalla1, C. D. Palmer1, L. B. Pickford2, M. Feldmann1, B. M. J. Foxwell1 and F. M. Brennan1

1Kennedy Institute of Rheumatology, Imperial College London, London, UK and 2Theracos Inc., Sunnyvale, CA, USA.

Correspondence to: P. F. Sumariwalla, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, Charing Cross Campus, Arthritis Research Campaign Building, 65 Aspenlea Road, Hammersmith, London W6 8LH, UK. E-mail: p.sumariwalla{at}imperial.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To evaluate the clinical efficacy of a novel synthetic peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) agonist, CLX-090717, in several in vitro cell culture systems and murine CIA, an experimental model of RA.

Methods. Peripheral blood monocytes purified by elutriation, and rheumatoid synovial cells isolated from clinical tissue were cultured with CLX-090717 and TNF-{alpha} release was measured. Molecular mechanism of action was analysed by western blotting and electrophoretic mobility shift assay. Thioglycollate-elicited murine peritoneal macrophages were cultured with CLX-090717 and lipopolysaccharide (LPS)-induced TNF-{alpha} release was assayed. Therapeutic studies were done in mice with established arthritis by evaluating clinical parameters and histology. In addition, type II collagen response of lymphocytes from mice with CIA was examined.

Results. CLX-090717 significantly inhibited spontaneous TNF-{alpha} release by RA synovial membrane cells, as well as LPS-induced TNF-{alpha} release from human and murine monocytic cells. Inhibition of TNF-{alpha} in monocytes was mediated partially through a nuclear factor-{kappa}B (NF-{kappa}B)-dependent pathway, as judged by sustained levels of I{kappa}B{alpha} in cytosolic extracts and a reduced level of LPS-induced NF-{kappa}B activity in nuclear extracts. CLX-090717 reduced clinical signs of arthritis and damage to joint architecture when administered therapeutically to arthritic mice. Mechanisms of action in CIA involved the reduction in proliferation of arthritic lymphocytes to antigen in vitro as well as reduced TNF-{alpha} release.

Conclusions. Our data suggest that the synthetic compound CLX-090717 has potential as a small molecular weight anti-inflammatory therapeutic for chronic inflammatory conditions.

KEY WORDS: Arthritis, Collagen, Cytokines, Monocytes, Synovium, Inflammation


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
RA is a common autoimmune chronic inflammatory disease in humans. Progress has been made in understanding its pathogenesis and its treatment using molecular targeted therapy [1]. We found that TNF-{alpha} blockade has profound effects on all aspects of the disease, including dramatic protection of joints [2]. Currently, two anti-TNF-{alpha} monoclonal antibodies, infliximab (Remicade®) and adalimumab (Humira®) and a TNF receptor Fc fusion protein etanercept (Enbrel®) are routinely used as treatment modalities in RA [3]. Blocking antigen presentation with CTLA4-human Fc Ig fusion protein abatacept (Orencia®) and targeted killing of B cells with anti-CD20 monoclonal antibody rituximab (Rituxan®, MabThera®) is also effective [4, 5], thus highlighting the contribution of both T- and B lymphocytes and antigen-presenting cells in the development and progression of RA [6].

Regrettably, all these effective therapeutics have drawbacks, as they are monoclonal antibodies or antibody receptor fusion proteins. They are injectables (either subcutaneously or intravenously), very costly, with consequent rationing and have a long half-life in circulation, so thus cannot be eliminated if there is toxicity. There is therefore a widespread search for possible other therapies, without these drawbacks; orally deliverable, easily cleared out and cheap.

Peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) agonists have been described to regulate macrophage and adipocyte function [7], and a novel agonist has been reported to reduce severity of CIA [8]. Calyx Therapeutics, Inc. (Hayward, CA, USA) had produced CLX-090717, a PPAR-{gamma} agonist that had properties in a variety of assays, which suggested that it may be a potential therapeutic in chronic inflammatory autoimmune disorders.

The aim of the present study was to test the anti-inflammatory/immunomodulatory efficacy of CLX-090717 using several well-described human and mouse in vitro and in vivo model systems, as used previously for defining TNF-{alpha} as a therapeutic target [9, 10]. Clinical efficacy of the compound in vivo was tested therapeutically in an extensively studied animal model of RA, type II CIA in the DBA/1 mouse [11].

Our findings indicated that CLX-090717 significantly inhibited release of the pro-inflammatory cytokine TNF-{alpha} from both human and mouse mononuclear cells, and that this effect in human monocytes was in part modulated through nuclear factor-{kappa}B (NF-{kappa}B). Importantly, CLX-090717 markedly inhibited TNF-{alpha} release from human rheumatoid synovial membrane cells in culture, ameliorated arthritis in mice and conferred joint protection when used therapeutically. These findings, taken together, suggest that the novel synthetic compound CLX-090717 may be useful in the management of chronic inflammatory disorders such as RA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Preparation of CLX-090717 solution for in vitro studies
The compound CLX-090717 (MW 517.57) from Calyx Therapeutics, Inc. was dissolved in DMSO (200 mM, Sigma Biosciences, Poole, UK). For use in in vitro culture systems (0.01–20 µM) serial dilutions were made in DMEM (PAA Laboratories, Paisley, UK). The lipopolysaccharide (LPS) preparation used in this study was Escherichia coli 026:B6 TCA extract (Sigma).

Culturing of human peripheral blood monocytes and rheumatoid tissue cells
Single donor plateletpheresis blood packs were obtained (North London Blood Transfusion service, Colindale, UK). Peripheral blood mononuclear cells were separated from them on a density gradient material (Lymphoprep, specific density 1.077 g/ml, Nycomed Pharma A.S., Oslo, Norway). Monocytes were obtained by elutriation of the isolated buffy coats, as described [12]. Cells were plated (2 x 105/100 µl/well), stimulated with LPS (10 ng/ml) in presence of CLX-090717 (0.01–10 µM) and 24 h culture supernatants harvested were assayed for TNF-{alpha} Cells from surgical samples of synovium were obtained, isolated and cultured as per established protocol [9]. CLX-090717 was added (0.01–10 µM) and 48-h culture supernatants harvested were assayed for spontaneous TNF-{alpha} release. An ELISA kit was used for detection of TNF-{alpha} as per the manufacturer's instructions (R&D Systems, Oxon, UK).

Culturing of murine peritoneal macrophages
Thioglycollate-elicited peritoneal macrophages were prepared and cultured (2 x 105/100 µl/well) as described elsewhere [13]. Briefly, a 3% sodium thioglycollate solution was injected intraperitoneally into mice and on day 4 post-elicitations peritoneal exudate cells were harvested as a lavage in chilled medium. Cells were treated with CLX-090717 (0.01–20 µM), stimulated with LPS (10 ng/ml), 24-h culture supernatants harvested, and assayed for TNF-{alpha}. An ELISA kit was used for detection of TNF-{alpha} as per the manufacturer's instructions (R&D Systems). At the end of all in vitro cell culture treatment experiments, cytotoxic effect of compound was assayed using either the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT assay, Sigma) [14] or the Alamar Blue (Serotec Ltd, Oxford, UK) dye reduction test [15].

Western blotting and electrophoretic mobility shift assay
Elutriated monocytes (5 x 106/ml) were pre-incubated with CLX-090717 (10 µM) or medium at 37°C/30 min, then either stimulated with LPS (10 ng/ml) or left unstimulated as controls. Cytosolic and nuclear extracts were prepared; I{kappa}B{alpha} in cytosolic extracts was detected by western blotting, while 32P-NF-{kappa}B oligonucleotide RNA probe (Promega, Southampton, UK) was added to nuclear extracts to detect NF-{kappa}B using established protocols [16].

Therapeutic treatment of CIA with CLX-090717 and culturing of lymph node cells for proliferation assays
Arthritis induced in DBA/1 mice (Harlan Laboratories UK Limited, Bicester, Oxon, UK) was monitored daily by recording clinical scores and hind paw thickness according to our previously established protocol [17]. Treatments were given daily i.p., and commenced on the first day of detectable limb inflammation. CLX-090717 was administered at 10 mg/kg, while a vehicle mixture [DMSO: 40% polyethylene glycol (PEG-300) in PBS, pH 7.2, BDH Laboratory Supplies, Poole, UK, 1 : 1 v/v] was injected in a separate group. A group of arthritic mice were left as untreated controls. Local ethical committee approval was obtained for all treatment studies; these followed the Helsinki Declaration principles. Histology was done on hind feet specimens fixed, sectioned and stained with haematoxylin and eosin (H&E) dye. Images were captured by a camera loaded on Leitz Dialux 22 EB microscope using the Windows Spot Advanced software (version 4.5 Sterling Heights, USA.).

Pooled draining lymph node cultures from arthritic mice were set up on day 5, in the presence of CLX-090717 (0.02–20 µM), stimulated with antigen. Alternatively, lymph node cultures from vehicle or CLX-090717-treated mice were separately set up with antigen for 72 h. Tritiated thymidine (Amersham Biosciences, Buckinghamshire, UK) added and incorporated during turnover was measured as described earlier [18].

Data analysis
Data were analysed by GraphPad Prism Software (CA, USA). Clinical data were analysed by one-way analysis of variance with Tukey's post test. Data on TNF-{alpha} levels were analysed by the Student's t-test, unpaired, two-tailed test. P-values of ≤0.05 were considered statistically significant. Bars and symbols represent arithmetic mean values ± S.E.M.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Inhibition of TNF-{alpha} release from human monocytes and rheumatoid mononuclear cells
Treatment of elutriated monocytes in vitro with CLX-090717 (0.01–10 µM) inhibited LPS-induced release of TNF-{alpha} in a concentration-dependent manner (Fig. 1A). At 1 µM a 66% and at 10 µM an 83% inhibition was seen (P < 0.001). Monocytes were viable at all concentrations of CLX-090717 assayed, by MTT assay (Fig. 1B). Efficacy of the compound in inhibiting release of spontaneous TNF-{alpha} from freshly isolated rheumatoid synoviocytes was tested in this previously established in vitro cell culture system bearing the most direct clinical relevance. CLX-090717 down-modulated TNF-{alpha} release in a concentration (0.1–10 µM)-dependent manner (Fig. 1C). Spontaneous TNF-{alpha} release from different RA membrane cell cultures is highly variable. Addition of CLX-090717 to all five membrane cell culture preparations significantly diminished release of TNF-{alpha} in a concentration-dependent manner, by up to 76% in membrane 5 (Fig. 1C). Viability of cells isolated from all these membranes was maintained upon treatment with CLX-090717 (Fig. 1D).


Figure 1
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  		FIG. 1. Concentration-dependent inhibition of TNF-{alpha} release (Figure 1) from monocytes. (A) CLX-090717 (0.01–10 µM) was added to cell cultures in vitro. TNF-{alpha} (pg/ml) was assayed from 24-h culture supernatants by a sandwich ELISA. Cells were incubated with diluted DMSO (10 µM) as the vehicle control. LPS was used as a stimulus at 10 ng/ml. Data were presented as mean ± S.E.M. of three experiments (n = 3). ***P < 0.0001 (Student's t-test, unpaired, two-tailed test, with 95% CIs applied). (B) MTT assay for CLX-090717 (0.01–10 µM)-treated monocytes. MTT reagent was added to cell cultures and incubated overnight. On the following day, indigo-coloured needle-shaped crystals were dissolved in 10% SDS 0.01 N HCl solution. The plates were read on a spectrophotometer at {lambda} = 574 nm. MTT readings (-•-) were represented on the ordinate axis as percentage value of control (LPS-stimulated cells with DMSO, 10 µM). (C) Ex vivo RA synovial membrane cell cultures (1 x 105 cells/100 µl) were set up in the absence or presence of CLX-090717 at the concentrations of 0.1 ({square}), 1.0 ({triangleup}) and 10 (x) µM as indicated in the figure. A sandwich ELISA measured TNF-{alpha} levels from 48-h culture supernatants. Results for five individual membranes were shown as the percent value of spontaneous TNF-{alpha} release from RA cells of the same membrane used as an intra 100% membrane control (baseline level). (D) MTT assay for CLX-090717 (0.01–10 µM)-treated rheumatoid synovial membrane cells. MTT reagent was added to cell cultures and incubated overnight. On the following day indigo coloured needle shaped crystals were dissolved in 10% SDS 0.01 N HCl solution. The plates were read on a spectrophotometer at {lambda} = 574 nm. MTT readings (-•-) were represented on the ordinate axis as percentage value of control (cells stimulated with DMSO, 10 µM). Data depicted in this graph represent three synovial membrane cell cultures ({square}, {triangleup} and x).

 
Analysis of mechanism of TNF-{alpha} inhibition
Treatment of monocytes with CLX-090717 (10 µM) prior to LPS stimulation partially prevented I{kappa}B{alpha} degradation (Fig. 2A). Comparable I{kappa}B{alpha} band densities were recorded for 30–60 min treatment with CLX-090717 when compared with PSi, an NF-{kappa}B inhibitor that prevents proteosomal degradation of ubiquinated I{kappa}B{alpha} thereby blocking NF-{kappa}B release and its translocation from cytoplasm into the nucleus (Fig. 2A). Electrophoretic mobility shift assay (EMSA) on nuclear extracts from monocytes treated with CLX-090717 prior to LPS stimulation showed a reduction in translocation of NF-{kappa}B to the nucleus, with weaker NF-{kappa}B band intensities at both 30- and 60-min treatments compared with LPS controls (Fig. 2B). PSi showed comparable reduction in NF-{kappa}B band intensity to the CLX-090717-treated group when compared with cells treated with LPS alone for 60 min (Fig. 2B). The effect of CLX-090717 on preventing I{kappa}B{alpha} degradation and reduction of NF-{kappa}B in the nuclear compartment suggests that NF-{kappa}B inhibition contributes towards the reduction of TNF-{alpha} release from CLX-090717-treated monocytes.


Figure 2
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  		FIG. 2. CLX-090717 inhibits TNF-{alpha} production from monocytes partially through the involvement of NF-{kappa}B pathway. (A) Western blot for I{kappa}B{alpha} and (B) EMSA for detection of NF-{kappa}B from CLX-090717-treated elutriated monocytes. Cells were treated with CLX-090717 at 10 µM for 30 min. LPS (10 ng/ml) was used as the stimulus for a time interval as shown in the figure. Cells were lysed and fractionated into cytosolic and nuclear extracts and western blotting and EMSA were performed on them, respectively. I{kappa}B{alpha} bands were detected by two-step antibody detection followed by an ECL visualization system. Uniform quantitative loading of protein was achieved by using β-actin as the loading control. Nuclear extracts were mixed with either 32P-labelled or unlabelled oligonucleotide sequence binding to NF-{kappa}B as shown in the figure. PSi (10 µM) was used as an inhibitor of NF-{kappa}B in both the assays. Representative gel blots from three experiments each have been shown in this figure. The band intensities from all three experiments were measured using a densitometer apparatus and analysed using Aida software systems (version 2.31, Raytest Isotopen Messgeraete GmbH, Strauberhardt, Germany). Densitometry data were normalized against unstimulated cell controls and represented as mean percent value of densitometry units of three experiments.

 
Inhibition of TNF-{alpha} release from murine peritoneal macrophages
We investigated the effect of CLX-090717 on primary murine thioglycollate-elicited peritoneal macrophages, to verify that the anti-inflammatory effects of CLX-090717 were not species specific. A statistically significant dose-dependent inhibition of TNF-{alpha} release was observed. We observed a 50% inhibition at 2 µM (P = 0.0008), which increased to a 70% inhibition at 20 µM (P = 0.0002, Fig. 3A). At none of these concentrations was CLX-090717 toxic to cells, as assayed by Alamar Blue dye reduction test (Fig. 3B).


Figure 3
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  		FIG. 3. Concentration-dependent inhibition of TNF-{alpha} release (Figure 3) from murine primary macrophages. (A) CLX-090717 (0.02–20 µM) was added to thioglycollate elicited peritoneal macrophage cultures in vitro. TNF-{alpha} (pg/ml) was assayed from 24-h culture supernatants by a sandwich ELISA. Cells were incubated with diluted DMSO (20 µM) as the vehicle control. LPS was used as a stimulus at 10 ng/ml. Data were presented as mean ± S.E.M. of triplicates. **P = 0.0008 at 2 µM and ***P = 0.0002 at 20 µM (Student's t-test, unpaired, two-tailed test, with 95% CIs applied). (B) Alamar Blue dye reduction assay for CLX-090717 (0.02–20 µM)-treated thioglycollate-elicited peritoneal macrophages. The redox dye was added at 10% volume of cell cultures and incubated overnight. On the following day, plates were read on a spectrophotometer at {lambda} = 574 nm. Absorbance readings were represented as percentage value of control (LPS-stimulated cells with DMSO, 20 µM).

 
Treatment of established CIA with CLX-090717 arrests clinical progression of disease and protects joints
The anti-inflammatory/immunomodulatory efficacy of CLX-090717 was investigated in a well-established animal model of RA, the DBA/1 mouse CIA model [11]. Therapeutic treatment with CLX-090717 (10 mg/kg body weight, intraperitoneally) reduced the clinical severity of established disease, compared with vehicle-treated and -untreated controls (Fig. 4A). On day 7 of established disease, the mean change in clinical scores ± S.E.M. from day 1 of arthritis in untreated, vehicle-treated and CLX-090717-treated mice were 2.317 ± 0.387, 2.005 ± 0.377 and 0.474 ± 0.102, respectively (Fig. 4A); a statistically significant reduction of 79.5% and 76.4% over the control groups (P < 0.001 and P < 0.05, respectively). Reduction of clinical scores closely mirrored the reduction achieved in hind paw swelling in CLX-090717-treated mice (Fig. 4B). On day 7 of disease, the mean paw swelling ({triangleup}mm) ± S.E.M. for untreated, vehicle-treated and CLX-090717-treated mice were 0.350 ± 0.050, 0.318 ± 0.021 and 0.160 ± 0.022, respectively, a statistically significant reduction (54.3%, P < 0.001 and 49.7%, P < 0.05, respectively) (Fig. 4B). The vehicle used in our experiments had a partial effect on reduction of both clinical score and paw swelling, which was not statistically significant when compared with the untreated group of mice. There was also a good degree of joint protection of the hind feet. Proximal phalange and medial cuneiform joints are most representative for arthritic changes [17]. In untreated- and vehicle-treated mice extensive hyperplasia and infiltration of the synovium was seen, marked with an erosive pannus causing cartilage, subchondral and trabecular bone destruction; this was considerably reduced in CLX-090717-treated mice as seen by H&E-stained histology of hind feet sections (Fig. 5).


Figure 4
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  		FIG. 4. Treatment of established CIA with CLX-090717 reduced clinical signs of disease. Mice were treated daily intraperitoneally with either vehicle (DMSO: 40% polyethylene glycol-300 in PBS, 1 : 1 v/v, n = 20, -{circ}-) or CLX-090717 (at 10 mg/kg body weight, n = 21, -•-). A group of arthritic mice were left as untreated controls (n = 20, -{blacksquare}-). The clinical signs of disease were monitored and represented as (A) clinical arthritis score, change from day 1 readings and (B) paw swelling measurements as change from day 1 of arthritis ({triangleup}mm) over the treatment period. Data from three experiments with comparable results were pooled and each point on the graph represents the mean value ± S.E.M. of (n) number of mice. Statistical significance was calculated by applying one-way analysis of variance test with Tukey's post test for multiple comparisons. *P < 0.05, **P < 0.001 when compared with CLX-090717-treated group of mice.

 

Figure 5
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  		FIG. 5. Histology of H&E-stained proximal phalange and medial cuneiform joint sections of mouse hind feet from untreated arthritic controls (A and B), vehicle-treated controls (C and D) and CLX-090717 (10 mg/kg)-treated mice (E and F). Hind feet were fixed in formalin, sectioned (6 µm thickness) and stained with H&E by Mayer's protocol. Representative sections were shown at a magnification of 125x.

 
Suppression of antigen-specific murine lymph node T-cell responses
Addition of CLX-090717 (0.02–2 µM) to pooled lymph node cultures from arthritic mice suppressed the antigen-specific (bovine CII) response, as measured by incorporation of tritiated thymidine (Fig. 6A). An 86% inhibition of the bCII proliferation response was seen at 2 µM of CLX-090717 (P < 0.0001 vs vehicle control). Cell viability of cultures was unaffected in both groups, as judged by Alamar Blue dye (Fig. 6B). Lymph node cells were also prepared as ex vivo cultures from mice treated with CLX-090717 (10 mg/kg body weight, i.p., daily for 3 days), or vehicle post-onset of arthritis. Treatment with CLX-090717 suppressed proliferative response of lymph node cells to bCII by 71%, when compared with vehicle control cultures (Fig. 6C). Cell viability remained unaffected in both the groups (Fig. 6D).


Figure 6
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  		FIG. 6. Suppression of antigen-specific murine lymph node cell responses. (A) CLX-090717 (-•-) was added at concentrations ranging from 0.02 to 2 µM to pooled arthritic lymph node cultures. Vehicle (diluted DMSO, -{circ}-) controls were set up alongside. Cells were stimulated for 72 h with bovine type II collagen (50 µg/ml) and incorporation of tritium (0.5 µCi) was assayed after an additional incubation period of 24 h. Each point on the graph represents the mean value ± S.D. of quadruplicate cultures. ***P < 0.0001 (Student's t-test, unpaired, two-tailed test with 95% CIs applied). (B) Alamar Blue dye reduction assay for in vitro CLX-090717 (0.02–2 µM)-treated (-•-) and vehicle treated (-{circ}-) pooled arthritic lymph node cultures. The redox dye was added at 10% volume of cell cultures and incubated overnight. On the following day, plates were read on a spectrophotometer at {lambda} = 574 nm. Absorbance readings were represented as percentage value of control (antigen-stimulated cells with DMSO, 2 µM). (C) Pooled ex vivo lymph node cell cultures were set up from day 3 arthritic mice treated with vehicle (DMSO: 40% PEG-300 in PBS, 1: 1 v/v) or CLX-090717 (10 mg/kg body weight) daily intraperitoneally. Cell cultures were stimulated in the presence of bovine type II collagen (50 µg/ml, -{blacksquare}-) or left unstimulated (-{square}-). Cells were pulsed with radioactive thymidine (0.5 µCi/culture/well) 72 h after stimulation and incorporation of activity was measured after an overnight period of incubation. Vertical bars represent mean value of pooled data from two experiments whereas hollow bars lines represent S.E.M. (D) Alamar Blue dye reduction assay for ex vivo lymph node cultures from vehicle- or CLX-090717-treated mice. Cell cultures were stimulated in the presence of bovine type II collagen (50 µg/ml, -{blacksquare}-) or left unstimulated (-{square}-). The redox dye was added at 10% volume of cell cultures and incubated overnight. On the following day, plates were read on a spectrophotometer at {lambda} = 574 nm. Absorbance readings were represented as percentage value of medium control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
There is an unmet medical need for novel oral therapeutics with low toxicity and high disease-modulating efficacy for use in the management of chronic inflammatory conditions such as RA, psoriasis, inflammatory bowel disease and Crohn's disease [19]. PPARs are a group of ligand-activated transcriptional factors belonging to the nuclear hormone receptor superfamily [20]. They are up-regulated in several inflammatory/immunomodulatory conditions [7, 21]. Here, we report the in vitro and in vivo anti-inflammatory/immunomodulatory properties of a novel synthetic compound, CLX-090717 (generated by Calyx Therapeutics Inc.).

CLX-090717 significantly suppressed LPS-mediated TNF-{alpha} release from human peripheral blood monocytes and was not toxic at concentrations tested. However, LPS-mediated IL-6 release from monocytes was unaffected (data not shown). PPAR-{gamma} agonists like 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2, a prostaglandin D2 metabolite of the arachidonic acid pathway) and troglitazone have been shown to inhibit production of monocyte inflammatory cytokines induced by phorbol esters, but not LPS [22], negatively regulate macrophage activation [23], and suppress monosodium urate monohydrate crystal-induced production of TNF-{alpha} and IL-1β from monocytes [24]. Differential effects of PPAR-{gamma} ligands on pro-inflammatory cytokine release from monocytes have also been reported in gouty arthritis [25, 26], just as seen by us in our study. In addition, we did not observe any modulation of IL-10 levels from human peripheral blood monocyte cultures upon in vitro treatment with CLX-090717.

We explored the possible molecular mechanism of action of the compound CLX-090717. There was no effect on the activation of any MAPK (p38, JNK, ERK) family members. However, CLX-090717 inhibited the degradation of I{kappa}B{alpha} in the cytosol. This in turn was reflected by partial inhibition of nuclear NF-{kappa}B activity as measured by EMSA. PPAR-{gamma} agonists, whose exact molecular mechanisms of inhibition of cytokine synthesis are as yet unknown, are believed to mediate their effects either transcriptionally or post-transcriptionally [20]. We have previously reported that inhibiting NF-{kappa}B by the use of adenoviruses overexpressing I{kappa}B{alpha} inhibits both TNF-{alpha} and IL-6 to the same degree [27]. The differential suppression of these two pro-inflammatory cytokines in our study thus indicates that there must be some other aspects of the mechanism of action apart from blockade of NF-{kappa}B, not known at present.

CLX-090717 significantly inhibited TNF-{alpha} release from primary murine peritoneal macrophages, while a modest inhibition was noted from RAW 264.7 cells (data not shown). We thus believe that the compound CLX-090717 has differential effects on suppression of pro-inflammatory cytokine production from different types of mononuclear cells, in both mouse and humans adding to the growing body of evidence that PPARs may be involved in regulating cytokine signals leading to monocyte/macrophage differentiation.

The significant down-modulation of LPS-driven TNF-{alpha} levels from monocytes and murine monocytic cells prompted us to further explore a therapeutic role of CLX-090717 in two well-established systems reflecting benefit in RA, namely spontaneous release of TNF-{alpha} from cultured human RA synoviocytes [9], and the DBA/1 mouse CIA model [11].

We found that CLX-090717 significantly inhibits spontaneous TNF-{alpha} production (by up to 76%) from cultured synoviocytes obtained from five RA synovial membranes. At present, despite the lack of knowledge regarding specific cellular/molecular target for CLX-090717, this profound inhibition noted in the spontaneous TNF-{alpha} production in the synovium suggests therapeutic potential in rheumatoid patients.

Recently, a novel PPAR-{gamma} agonist, THR0921, was reported to reduce clinical severity of CIA and protect joints [8]. We too found a similar amelioration of arthritis severity and joint protection with the use of CLX-090717. THR0921 acted in CIA through several mechanisms, such as reduced mRNA and protein levels of pro-inflammatory cytokines, proliferation of splenocytes and reduced circulating levels of antibodies to collagen [8]. We too in our CIA study have seen a significant reduction in anti-type II collagen immune responses. It is known that PPAR-{gamma} ligands possess anti-inflammatory activity in adjuvant-induced arthritis (AIA) mediated through the inhibition of NF-{kappa}B pathway, reduced nitrotyrosine formation in ankle joints and inhibition of protein expression of iNOS, COX-2 and ICAM-1 [28]. It has also been reported that PPAR-{gamma} ligands troglitazone and 15d-PGJ2 induce RA synoviocyte apopotosis, and reduce severity of AIA in Lewis rats [29]. In our CIA model, the observed reduction in paw swelling in combination with histological benefit indicated effective control of joint inflammation. However, we have not monitored any long-term disease-modulating effects of treatment with CLX-090717 in our studies. We thus believe that CLX-090717 may be a useful therapeutic in RA.

Lymphocytes obtained from arthritic mice showed a significant reduction of their proliferative responses to the eliciting antigen (bovine type II collagen) in the presence of CLX-090717. In addition, proliferative responses of draining lymph node lymphocyte cultures to specific antigen (bovine type II collagen) from arthritic mice treated with CLX-090717 were inhibited by 71%. PPAR-{gamma} ligands have been shown to control chronic inflammation by down-modulating vascular cell adhesion molecule-1 (VCAM-1) expression on endothelial cells [30], thus suggesting that perhaps they could control lymphocyte trafficking to inflammatory sites.

We believe that the observed disease-ameliorating action of CLX-090717 in our CIA model could be mediated through a combination of its ability to suppress bovine type II collagen proliferative responses and partial inhibition of NF-{kappa}B-mediated inflammatory cytokine gene transcription from mononuclear cells.

Several results presented in this article suggest that CLX-090717 may be of benefit in human RA: it inhibited human peripheral blood monocyte and mouse macrophage TNF-{alpha} release as well as inhibiting spontaneous TNF-{alpha} production from human RA synovial membrane cell cultures and significantly ameliorated CIA with joint protection against arthritic changes when used after disease onset. However, clinical trials are needed to establish CLX-090717 as a useful therapeutic in chronic inflammatory disorders.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors are grateful to the Orthopaedics Department and Rheumatology Clinic of Charing Cross Hospital and also to the Orthopaedics Department of Ravenscourt Park Hospital for providing clinical rheumatoid biopsy tissue. We also acknowledge the contribution of Ms Amy Peters and Ms Sarah Field for help provided in the preparation of cell suspensions from synovial membranes of rheumatoid surgical specimens. All staff members of the Biological Services Unit of the Kennedy Institute of Rheumatology Division, Imperial College London are duly acknowledged for their help provided in the care and maintenance of laboratory mice used in our studies. We also thank Ms Patricia Green and Mr Emmanouil Vernadakis for the help and assistance provided by them for experiments during the course of this study.

Funding: This project was funded by an industrial grant from Calyx Therapeutics, Inc. U.S.A., as well as a core grant to the Kennedy Institute of Rheumatology Division of Imperial College London by Arthritis Research Campaign (ARC).

Disclosure statement: F.M.B. and B.M.J.F. have served as consultants for Calyx Therapeutics, Inc. L.B.P. was employed by Calyx Therapeutics, Inc. All other authors have declared no conflicts of interest.


   Notes
 
Present address: C. D. Palmer, University College London, Rayne Building, 5 University Street, London WC1E 6JF, UK.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 2 June 2008; revised version accepted 15 September 2008.
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