in vitro evaluation of s-(+)-ibuprofen as drug candidate for intra-articular drug...

http://informahealthcare.com/ddiISSN: 0363-9045 (print), 1520-5762 (electronic)

Drug Dev Ind Pharm, 2015; 41(1): 85–94! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.850704

RESEARCH ARTICLE

In vitro evaluation of S-(þ)-ibuprofen as drug candidate forintra-articular drug delivery system

Laurent Bedouet1, Florentina Pascale2, Michel Bonneau2, and Alexandre Laurent1,2,3

1Occlugel SAS, Jouy-en-Josas, France, 2AP-HP, INRA, Center for Research of Interventional Imaging (CR2i), Jouy-en-Josas, France, and3Department of Interventional Neuroradiology, AP-HP, Lariboisiere Hospital, Paris, France

Abstract

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locallyrelease non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen to reduce pain inpatients with osteoarthritis. The present study examines the efficacy of S-(þ)-ibuprofen oncartilage degradation as drug candidate for DDS loading. Humeral cartilage and jointcapsule explants were collected from healthy sheep shoulder joints and they were cultured inmono- or in co-culture for 13 days with LPS in combination with S-(þ)-ibuprofen at 50mM and1 mM. S-(þ)-ibuprofen (50mM) blocked prostaglandins production in LPS-activated explants butdid not reduce cartilage degradation. By contrast, 1 mM S-(þ)-ibuprofen treatment of cartilageexplants reduced nitric oxide synthesis by 51% (p¼ 0.0072), proteoglycans degradation by 35%(p¼ 0.0114) and expression of serum amyloid protein – the main protein induced uponLPS challenge – by 44% (p50.0001). On contrary, in presence of synovial membrane, theprotective effects of S-(þ)-ibuprofen on cartilage damages were significantly diminished.At 1mM, S-(þ)-ibuprofen reduced the cell lysis during culture of cartilage and joint capsuleeither in mono- or in co-culture. This study performed on sheep explants shows that 1 mMS-(þ)-ibuprofen inhibited cartilage degradation via a mechanism independent of cycloox-ygenase inhibition. Reduction of prostaglandins synthesis at 50mM in all treatment groups andreduction of cartilage degradation observed at 1 mM suggest that S-(þ)-ibuprofen could beconsidered as a promising drug candidate for the loading of intra-articular DDS.

Keywords

Cartilage, co-culture, drug delivery systems,osteoarthritis, S-(þ)-ibuprofen

History

Received 29 March 2013Revised 9 September 2013Accepted 19 September 2013Published online 30 October 2013

Introduction

The non-steroidal anti-inflammatory drugs (NSAIDs) are effect-ive in controlling pain in patients with mild to moderateosteoarthritis (OA)1. NSAIDs inhibit the cyclooxygenase (COX)pathway that accounts in part for their anti-inflammatory activitythrough the inhibition of the prostaglandins production fromarachidonic acid2. During OA the expression of inducible COX-2is elevated in synovial tissue and in cartilage with 50-fold moreprostaglandin E2 (PGE2) levels than in healthy cartilage3.Catabolic effects of PGE2 on articular cartilage include theincrease of matrix metalloproteinase (MMP) production inchondrocytes, synovial fibroblasts, and OA cartilage explantsand the synergistic action with nitric oxide (NO) on chondrocytesapoptosis3,4. Reduction of PGE2 synthesis represents a therapeuticobjective in OA therapy. The treatment of patients with severeknee OA during 3 months with aceclofenac and celecoxib reducedPGE2 concentration in synovial fluid and improved the patientmobility5.

Among NSAIDs, ibuprofen is one of the most popular, isavailabe in OTC6. In 1990, 3000 t of ibuprofen were consumed on

the US market7. Ibuprofen can inhibit PGE2 expression from IL-1activated chondrocytes8 and protect in a concentration-dependentmanner chondrocytes from apoptosis, suggesting a protective roleof chondrocytes during OA9. In vitro, ibuprofen did not reduce theGAG loss and proteolytic activity of cartilage explants treatedwith IL-1 and TNF-a10. In patients with OA, ibuprofen wasinefficient on the reduction of MMP-3 level in serum11 while itreduced significantly the release of cartilage and synovialdegradation markers in patients with a flare of knee arthritis12.Clinical studies indicate that ibuprofen is effective and relativelysafe in management of the mild-to-moderate OA of the knee andhip13. Recently, ibuprofen treatment in rat was effective inattenuating inflammation and early articular cartilage degener-ation induced on wrist joints under mechanical stress for12 weeks14.

The systemic administration of NSAIDs is limited by dose-dependent side effects such as gastric erosion, cardiovascularproblems, liver and kidney damages that can appear for long-termtreatment at high dosages15. NSAIDs can even have deleteriouseffects on hip and knee16. A novel therapeutic approach of OAconsists of using intra-articular drug delivery systems (DDSs)with the ability to locally release NSAIDs17. Various formulationsof DDS are currently investigated to achieve a sustained release ofNSAIDs18–20. Intra-articular DDS should provide long-termsustained release of NSAIDs and diminish the amount of

Address for correspondence: Laurent Bedouet, Occlugel SAS, 12 rueCharles de Gaulle, Jouy-en-Josas 78350, France. E-mail: [email protected]

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systemic drug exposition, which in turn reduces the related-adverse events.

Ibuprofen exists in two enantiomers, the isomer active on thecyclooxygenases activity is the S-(þ)-enantiomer21. For a localdelivery of ibuprofen in joint cavity, loading of DDS with theactive isomer rather than with the racemate mixture should intheory reduce the amount of biomaterial to be injected.S-(þ)-ibuprofen has proved to be an effective drug for patientssuffering of OA22. Clinically, use of single active isomer of theprofens class of NSAIDs instead of the racemate mixture mayrepresent a reduction of dose during treatment leading to a lessexposition to xenobiotics and a reduction of renal load23. In vitro,biological activities of several NSAIDs isomers on joint cellswere analyzed: S-(þ)-ketoprofen and S-(þ)-flurbiprofen onhuman chondrocytes treated with interleukin-124 and carprofenisomers on equine chondrocytes and synoviocytes after lipopoly-saccharides (LPS) treatment25,26. No such study was undertakenfor S-(þ)-ibuprofen.

In a previous work, we have tested the toxicity ofS-(þ)-ibuprofen on chondrocytes and synovial fibroblasts col-lected from sheep shoulder joint27. We have observed thatS-(þ)-ibuprofen at 50mm and 1 mM did not induce toxicity onchondrocytes and type B synoviocytes as well as on cartilage andsynovial membrane explants. Absence of cytotoxicity ofS-(þ)-ibuprofen on joint cells authorizes the setting of an efficacystudy on activated joint explants.

In the present study we have explored whetherS-(þ)-ibuprofen (50mM and 1 mM) could inhibit experimen-tally induced degradation of sheep articular cartilage explantsinduced with LPS. This efficacy study performed on jointexplants is a prerequisite prior to the preparation of intra-articular DDS containing S-(þ)-ibuprofen. The low concentra-tion (50mM) is in the range of IC50 values of the racemateibuprofen to inhibit the COX-2 activity28 and corresponds tothe higher concentration of racemic ibuprofen measured insynovial fluid of OA patients after oral treatment29. Themillimolar dose of S-(þ)-ibuprofen was chosen to determinethe activity of the NSAID regardless its inhibition of COXactivity, since NSAIDs at high concentrations exert pharmaco-logical activity different from inhibition of COX enzymes30,31.Consequently in an assessment of efficacy of a NSAID oncartilage degradation, low and high concentrations of drug mustto be analyzed in regard to its different modes of action.

We introduced a co-culture model of cartilage explant withsynovial tissue to measure whether and to what extent capsulartissue changes the outcomes of S-(þ)-ibuprofen treatment onactivated cartilage since previous studies had demonstrated thedegradative capacity of synovium which promotes the catabolicpathways of chondrocytes during co-culture experiments32–34. Wesupposed that during in vitro efficacy studies of a drug, the pro-catabolic environment created during co-culture may haveeffects on activities of a drug on cartilage degradation. Theeffects of S-(þ)-ibuprofen at (50mM and 1 mM) on cartilagedegradation induced with LPS were studied and compared incartilage co-cultured with explants of synovial membrane.

Materials and methods

Materials

Lipopolysaccharides, S-(þ)-ibuprofen, common culture mediumsupplements, trypsin, electrophoresis reagents, common chem-icals and ‘‘In vitro toxcicology assay kit Lactate dehydrogenasebased’’ were obtained from Sigma (Saint-Quantin Fallavier,France). The nitrite assay kit was purchased from Promega(Charbonnieres, France) and PGE2 kit was from R&D Sytems(Lille, France).

Animals and explants harvesting

Prealpes sheep (n¼ 7, 6–4 years old) were sacrificed by electro-narcosis at the central slaughterhouse of INRA (Jouy en Josas,France) according to veterinary regulatory rules. Full-thicknesscartilage slices from humerus surface were obtained after scalpelshaving. The capsular tissue including the intima, the sub-intimalayer and the fibrous capsule was removed from each joint.Explants were placed in sterile medium (DMEM high-glucose,100 U/mL penicillin, 100mg/mL streptomycin).

Joint explants culture

Cartilage and capsule fragments were aseptically cut in pieces(&5 mm in diameter) and placed into 24 well-plates. For co-culture, autologous fragments of capsule and cartilage weremixed. Wells were then filled with 2 mL of culture medium (10%FBS, 2 mM L-glutamine, penicillin (50 U/mL), streptomycin(50mg/mL), 10 mM HEPES, DMEM-high glucose) and incubatedat 37 �C with 5% CO2. The explants (cartilage, capsule and co-culture) were equilibrated for 5 days before the treatments.Cultures were performed with triplicate wells using tissue fromone animal donor. Experiments were repeated 3 times, each timeusing tissue from a different animal.

Preparation of S-(þ)-ibuprofen solutions and treatment ofjoint explants

Explants were treated with 1 mL of culture medium alone (controlexplants), or with LPS alone (10 mg/mL) or with LPS (10 mg/mL)in presence of 50 mM or 1 mM of S-(þ)-ibuprofen. Stock solutionof S-(þ)-ibuprofen (1 M) was prepared in absolute ethanol,aliquots were stored at �20�C. Addition of S-(þ)-ibuprofen inculture medium was performed before each medium change. Formedium containing 1 mM of drug (206 mg/mL), S-(þ)-ibuprofenstock solution was progressively added under mild shaking.At end of drug addition to cell culture medium no precipitate wasvisible in accordance with the solubility of racemate ibuprofen inphosphate buffer saline measured at 6 mg/mL35. After 48 h ofculture, the supernatants were harvested before storage at �20 �C,and new media were added. Then, culture supernatants werecollected after 6, 9 and 13 days. At the end of the culture, wetexplants were collected from the culture wells and they weredrained on filter paper before to be transferred in pre-weighedtubes. Drying of explants (n¼ 144) occurred in an oven (90 �C for60 h) until a constant weight was obtained. The ratio of cartilageexplants and synovium explants in the co-culture group was 1/3(dry weight). For each explant group, the amounts of tissueinvolved in the four treatment groups were equivalent (Table 1).

Cytotoxicity assay by lactate dehydrogenase release fromexplants

Lactate dehydrogenase (LDH) release from explants to themedium was measured to quantify the cell death by using the‘‘In vitro toxicology assay kit Lactate dehydrogenase based’’(TOX-07, Sigma). Measures were done on 25 mL of mediaaccording to the manufacturer’s instructions. The absorbance wasmeasured (450–630 nm) and values were normalized to theexplants dry weight (mg) to obtain an arbitrary unit of toxicity.Cumulative values were measured from day 2 to day 13.

Assay of PGE2

PGE2 in the explant supernatants was measured in duplicate byELISA at day 2 using a commercial kit. The cartilage, capsule andco-culture supernatants were diluted 10, 100 and 200 times in thecalibrator diluent, respectively, before addition of 150mL of

86 L. Bedouet et al. Drug Dev Ind Pharm, 2015; 41(1): 85–94

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dilutions to the microplate. The PGE2 expression (ng) wasnormalized with the explants dry weight (mg).

Nitrite assay

The assay of nitrite (stable metabolite of NO) in cartilage and co-culture medium (25mL) was performed in duplicate using thespectrophotometric Greiss assay according to the manufacturer’sprotocol. Sample concentration in nitrite was determined bymeasuring the absorbance at 540 nm. A linear calibration curvewas done with sodium nitrite (12.5–100 mM). The cumulativeamount of NO synthesis was measured from day 2 to day 13 ofculture. Nitrite concentration (mM) was normalized with thecartilage explants dry weight (mg).

GAG assay

The release of soluble glycosaminoglycans (GAG) in the cartilageand co-culture medium was used as an indicator of cartilageaggrecan degradation. The release of GAG was measured induplicate using 10 mL of conditioned medium according to the1,9-dimethylmethylene blue assay (DMMB) assay36 with sharkchondroitin sulfate (Sigma) as standard. Absorbance of thesoluble GAG-DMMB complex was measured at 515 nm. Thecumulative amount of GAG release was measured from day 2 today 13 of culture. The GAG amount (mg) was normalized with thecartilage explants dry weight (mg).

Recovery of proteins secreted by explants and proteinselectrophoresis

Explants from cartilage and capsule were cultured alone or inco-culture in serum-free media and treated in triplicate withLPS (10mg/mL) in presence of S-(þ)-ibuprofen (50 mM or1 mM). Experiments were repeated twice, each time usingtissues from two animal donors. Culture supernatants werecollected after 2 days of culture before dialysis against water(3 days, 4 �C) through a molecular weight cut-off of 6–8 kDa(Spectra/Por, Spectrum Laboratories). Desalted fractions werelyophilized before proteins assay using the bicinchoninic acidmethod. Lyophilized proteins (50 mg) were separated on 15%SDS–polyacrylamide gel (SDS–PAGE)37 and stained withCoomassie blue R-250. Pieces of gel corresponding to selectedproteins were sliced, washed (5% acetic acid) and dehydrated inacetonitrile before to in-gel reduction (10 mM dithiothreitol in100 mM ammonium bicarbonate, 45 min, 56 �C), alkylation(55 mM iodoacetamide, 30 min) and dehydration in acetonitrile.Trypsin was added (1 mg in 40mL of 50 mM ammoniumbicarbonate) per slice and incubated for 45 min on ice. Excessof enzyme was removed and digestion occurred overnight at37 �C. Tryptic peptides were extracted twice with 1% formicacid, and 100% acetonitrile. Supernatants were pooled andlyophilized.

Liquid chromatography and-mass spectrometry analysisof trypsin digests

Separation of the tryptic peptides was done on a C18 column(150� 1 mm, 4mm, Modulo-cart UPISPHERE, Interchim,France) at a flow rate of 50 mL/min with 0.1% formic acid inwater (eluent A), and acetonitrile (eluent B) according to a lineargradient from 5% to 50% eluent B in 45 min. Separated peptideswere analyzed on line with an ESI-QqTOF hybrid massspectrometer (pulsar I, Applied Biosystems) using informationdependant acquisition (IDA) which allows to switch between MSand MS/MS experiments. The data were acquired and analyzedwith the Analyst QS software. The mass spectra data weresearched against NCBI non-redundant database using an in houseMASCOT search engine (http://www.matrixscience.com).Proteins identified were validated based on the MASCOTMowse score.

Quantification of expression of serum amyloid protein

Polyacrylamide gels after Coomassie blue staining were scannedand surface of serum amyloid protein (SAA) band was measuredusing ImageJ software. Level of expression of SAA protein inpresence of S-(þ)-ibuprofen was normalized to SAA levelmeasured in LPS-activated explants. All quantifications wereperformed 3 times on gels obtained from the two independentexperiments composed of explants collected from two animaldonors.

Statistical analysis

Statistical analyses were performed on StatView SAS 2000 (SASinstitute, Cary, NC). The data are presented in box-and-whiskerplots and continuous variables were expressed as median�median absolute deviation. For comparison of two groups, non-parametric Mann–Whitney (MW) test was used. The Kruskal–Wallis (KW) test was used to compare three or more independentgroups. Significance was set at p50.05.

Results

Sheep cartilage explants were induced to degrade alone or in co-culture with synovial tissue by using LPS (10mg/mL) and thechondroprotective effects of S-(þ)-ibuprofen at 50 mM and 1 mMwere studied by co-treating the activated explants with drug for13 days. We have cultured cartilage explants with fragments ofjoint capsule in order to mimic more closely the interactionswhich exist between the different joint tissues and analyze theeffect of its association on the efficacy of S-(þ)-ibuprofentreatment of LPS-activated tissues. The conditioned media werecollected and analyzed for their content of different markers ofinflammation [PGE2, nitric oxide (NO)], and degradation ofextracellular matrix molecules in cartilage explants (glycosami-noglycans (GAG)). The LDH leakage from explants to the

Table 1. Details on joint explants collected from healthy sheep shoulder.

Monoculture Co-culture

Cartilage (mg) Capsule (mg) Cartilage (mg) Capsule (mg)

Control 4.33� 1.90 10.41� 4.96 2.66� 1.07 10.38� 2.88LPS (10 mg/mL) 3.26� 0.93 12.15� 5.34 2.61� 0.55 9.06� 4.25LPSþ 50mM S-(þ)-ibuprofen 3.06� 0.88 8.76� 4.14 3.22� 1.43 7.83� 2.35LPSþ 1 mM S-(þ)-ibuprofen 3.11� 0.81 11.73� 6.10 3.68� 1.17 10.12� 10.84KW 0.2412 0.5702 0.2200 0.3313

Data are expressed as dry weight (in mg� SD). The homogeneity between treatment groups is estimated by a non-parametric Kruskall–Wallis (KW)test. Nine explants were involved in each experimental group (culture in triplicate from three animal donors) leading to a total of 144 explants(cartilage and capsule).

DOI: 10.3109/03639045.2013.850704 S-(þ)-ibuprofen efficacy on joint explants 87

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medium was measured to quantify the cytotoxicity effects of drugtreatments on explants during the culture period. Furthermore,protein expression changes of explants according to cultureconditions were investigated using gel electrophoresis and liquidchromatography-mass spectrometry experiments. The aim was toidentify proteins down-regulated or up-regulated during treatmentof joint explants with LPS and S-(þ)-ibuprofen.

Cytotoxicity of S-(þ)-ibuprofen during tissue cultures inpresence of LPS

Compared to control explants, no significant increase of LDHactivity was detected in the conditioned medium of tissue cultures(capsule, cartilage and co-culture of both) treated with LPS and S-(þ)-ibuprofen, even in a concentration as high as 1 mM during the13 days (Figure 1). On contrary, culture of explants in presence of1 mM S-(þ)-ibuprofen reduced significantly (p50.05) theamount of LDH released into supernatants for each group ofexplants. For each group of explants, a better reduction of LHDleakage was achieved during culture with 1 mM of drug comparedto 50mM of drug (Table 2). Thus, treatment of explants with S-(þ)-ibuprofen did not induce cell death and should have nocytotoxic or apoptotic effects on chondral and synovial tissuesduring the period of culture.

Chondroprotective effects of S-(þ)-ibuprofen in sheepcartilage explants

Expression of each marker of inflammation and cartilagedegradation was determined before LPS challenge and afterculture with endotoxins and S-(þ)-ibuprofen. Effects of synovialtissue on activities of S-(þ)-ibuprofen on cartilage degradationwere examined. Results are summarized in Table 2.

Effects of S-(þ)-ibuprofen on expression of PGE2

Basal PGE2 expression

Content of PGE2 in supernatant of control cartilage explants waslower than for capsule (p¼ 0.0033). Co-culture of cartilageexplant with joint capsule caused a 10-fold increase of PGE2

synthesis compared to cartilage (p¼ 0.0008).

PGE2 production after LPS challenge

Compared to the controls, LPS treatment stimulated by 53-, 45-and 23-fold the expression of PGE2 for cartilage, capsule andco-culture, respectively (Figure 2). PGE2 produced during

co-culture was higher than during mono-culture of capsule(p¼ 0.0209) and cartilage (p¼ 0.0008) explants.

Effect of S-(þ)-ibuprofen treatments on PGE2 production

For each explant group, 50mM and 1 mM of S-(þ)-ibuprofenreduced significantly the PGE2 synthesis (p¼ 0.0008) comparedto the LPS-activated explants. In presence of 50 mM ibuprofen,expression of PGE2 remained higher in the co-culture groupcompared to cartilage monoculture (p¼ 0.0008) and equivalent tocapsule group (p¼ 0.0929). At 1 mM of drug, the residual PGE2

synthesis activity in the co-culture group was higher than incartilage (p¼ 0.0016) and capsule (p¼ 0.0033) groups.

Dose effect of S-(þ)-ibuprofen

A better inhibition of PGE2 synthesis was observed with 1 mMS-(þ)-ibuprofen during mono-culture of capsule (p¼ 0.0008) andco-culture of explants (p¼ 0.0008) while for cartilage explant inmono-culture, 1 mM S-(þ)-ibuprofen did not enhance inhibitionof PGE2 synthesis compared to drug at 50mM (p¼ 0.3446).

Effects of S-(þ)-ibuprofen on synthesis of NO

Basal NO synthesis

The amounts of NO detected in cartilage and in co-culturesupernatants were low and identical (p¼ 0.2821), indicating thatco-culture condition did not enhance NO synthesis (Figure 3).

NO synthesis after LPS challenge

LPS induced synthesis of NO during cartilage mono-culture andco-culture (p50.0001). In the synovial explants group, LPSchallenge did not induce expression of NO compared to untreatedexplants (p¼ 0.8466).

Effect of S-(þ)-ibuprofen treatments on NO synthesis

Compared to activated explants, S-(þ)-ibuprofen (50 mM) did notinhibit NO production during cartilage mono-culture (p¼ 0.5907)and co-culture (p¼ 0.8743). On the contrary, 1 mMS-(þ)-ibuprofen reduced by 51% the NO production in cartilagemono-culture (p¼ 0.0072). In co-culture, a non-significantinhibition of NO synthesis (23%, p¼ 0.0619) was measuredwith 1 mM S-(þ)-ibuprofen. At 1 mM of S-(þ)-ibuprofen, thecumulative synthesis of NO produced from cartilage explant inco-culture was higher than the expression achieved duringcartilage mono-culture (p¼ 0.0162). A more efficient inhibition

Figure 1. The cytotoxic effects of S-(þ)-ibu-profen during the culture of cartilage andjoint capsule in mono- or in co-culture for13 days. Explants were collected from threeanimal donors and culture was performed intriplicates. Data are presented in box-and-whisker plots with values corresponding tothe median�median absolute deviation.Absorbance values (650–450 nm) were nor-malized to the explant dry weight (mg) andspecific activities determined at days 2, 6, 9and 13 were added leading to cumulativevalues for LDH leakage from joint explants.Comparisons between control, LPS andLPSþ 50mM S-(þ)-ibuprofen groups weredone using the Kruskal–Wallis (KW) non-parametric test. Comparisons between twogroups were done using Mann–Whitney(MW) non-parametric test. *: significantreduction (MW) of LDH leakage comparedto LPS treated explants.


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of NO synthesis occurred during cartilage monoculture with S-(þ)-ibuprofen at 1 mM.

Dose effect of S-(þ)-ibuprofen

A dose dependant inhibition of NO synthesis was achieved onlyduring mono-culture of cartilage explants (p¼ 0.0011) comparedto co-culture (p¼ 0.3425).

Effects of S-(þ)-ibuprofen on cartilage proteoglycansdegradation

Basal GAG loss from cartilage explant

Degradation of cartilage matrix increased during co-cultureof explant, compared to monoculture. The differences wereT

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Figure 2. Activity of S-(þ)-ibuprofen on PGE2 expression (ng/mg dryweight) from cartilage and capsule explants cultured in mono- or in co-culture. Explants were cultured as described in Figure 1. After 2 days ofculture, PGE2 in medium of different groups (control, 10mg/mL LPS,LPSþ 1 mM S-(þ)-ibuprofen and LPSþ 50mM S-(þ)-ibuprofen) wasassayed in duplicate. Comparison between culture conditions was doneusing Mann–Whitney non-parametric test. (¤p¼ 0.0008 for comparisonbetween controls and LPS, *p¼ 0.0008 for comparison between S-(þ)-ibuprofen at 50mM or 1 mM to LPS groups).

Figure 3. Effect of S-(þ)-ibuprofen on 13-day accumulated nitrite releaseto the medium (mM/mg dry weight cartilage) as a measure of NOproduction of LPS-activated explants. Explants were cultured asdescribed in Figure 1. NO concentrations were normalized to thecartilage dry weight (mg) and values obtained at days 2, 6, 9 and 13 wereadded leading to cumulative values of NO synthesis. Comparisonbetween culture conditions was done using Mann–Whitney non-paramet-ric test (¤ for comparison between controls and LPS, * for comparisonbetween S-(þ)-ibuprofen groups to LPS groups).


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significant at day 2 (p¼ 0.004), day 6 (p50.0001) and day 9(p¼ 0.0014) and not at day 13 (p¼ 0.0763) of culture (Figure 4).

GAG degradation after LPS challenge

The level of proteoglycans degradation of cartilage explants wasincreased in presence of LPS during cartilage monoculture. Oncontrary LPS did not enhance the GAG loss from cartilage in co-culture compared to the control explants at each time of analysis(Figure 4).

Effect of S-(þ)-ibuprofen treatments on cartilage degradation

S-(þ)-ibuprofen (50mM) did not inhibit the proteoglycansdegradation of cartilage during mono- and co-culture of jointexplants in presence of LPS. A significant reduction by 35%(p¼ 0.0114) of the 13 days cumulative GAG loss from LPS-activated cartilage was measured after culture with 1 mMS-(þ)-ibuprofen (Figure 5). In presence of capsular tissue, anon-significant reduction of GAG release from cartilage wasmeasured at 1 mM S-(þ)-ibuprofen (19%, p¼ 0.0536). At 1 mMof S-(þ)-ibuprofen, the cumulative amount of soluble GAGreleased from cartilage explant was not different between the twogroups of cartilage explants (mono- and co-culture) (p¼ 0.8993).

Dose effect of S-(þ)-ibuprofen on reduction of GAG release

At the end of the culture, a dose dependant inhibition of cartilagematrix degradation was not observed during mono-culture ofcartilage explants (p¼ 0.0557) and co-culture with synovial tissue(p¼ 0.3113).

Effects of S-(þ)-ibuprofen on protein expression inLPS-activated explants

In order to investigate the effects of LPS challenge and thesimultaneous treatment with S-(þ)-ibuprofen on protein expres-sion of joint explants, proteins released in culture medium after2 days of culture were recovered and separated by SDS–PAGE.

In this aim, cultures were performed without serum in order toobserve the secreted proteins, which were present in lowerquantities than proteins from serum. When exposed to LPS, thejoint explants, cartilage, capsule and co-culture of both, secreted alow molecular weight protein (�10 kDa) (Figure 6) which wasalso observed in presence of S-(þ)-ibuprofen (Figure 7).

Identification of serum amyloid protein as inflammatoryprotein induced with LPS during explants culture

After trypsin digestion of the low molecular weight protein(10 kDa) induced with LPS (Figures 6 and 7), proteomic analysisidentified this protein as serum amyloid A3.2 protein (SAA)(Table 3). Low molecular weight protein at 10 kDa observed afterS-(þ)-ibuprofen treatment of activated explants was alsoidentified as serum amyloid protein. Some proteins whoseexpression did not appear to be modified by the culture conditions

Figure 4. Effect of co-culture of cartilage with synovial tissue ondegradation of cartilage proteoglycans. The accumulated GAG release tothe medium (mg/mg dry weight cartilage) is indicated for cartilage mono-culture and co-culture in absence (basal degradation) or in presence ofLPS (10 mg/mL). Explants were cultured as described in Figure 1, andGAG content in supernatants was normalized to the cartilage dry weight(mg). Values determined at days 2, 6, 9 and 13 were added leading tocumulative GAG release from cartilage matrix. Comparison betweenculture conditions was done using Mann–Whitney non-parametric test.Comparison between co-culture and monoculture conditions for basalGAG release was performed at each day of analysis: a (p¼ 0.004), b(p50.0001), c (p¼ 0.0014) and d (p¼ 0.07).

Figure 5. Effect of S-(þ)-ibuprofen on 13-day accumulated GAG releaseto medium (mg/mg dry weight cartilage). Explants were cultured asdescribed in Figure 1, and GAG content in supernatants was normalizedto the cartilage dry weight (mg). Values determined at each day ofsampling (days 2, 6, 9 and 13) were added leading to cumulative valuesof GAG release from cartilage matrix. Comparison between cultureconditions was done using Mann–Whitney non-parametric test. (¤ forcomparison between controls and LPS, * for comparison between S-(þ)-ibuprofen groups to LPS groups).

Figure 6. Polyacrylamide gel electrophoresis of proteins induced inshoulder joint explants after 2 days of culture with LPS (10 mg/mL) in aserum-free medium. Gels were cut into slices, as indicated (1–6), digestedwith trypsin and the peptides were identified using LC-mass spectrometry(Table 3). A second experiment performed on explants collected from twoother animals gave similar results with induction of a 10-kDa band in thepresence of LPS activation. MW: molecular weight standards inKilodalton (kDa). Ctrl: untreated explants.


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were identified in supernatants. In cartilage medium (Figure 6),the high molecular weight protein at 97 kDa was identified ascartilage oligomeric protein (COMP) and the polypeptide at28 kDa probably correspond to fragment of chondroadherin(Table 3). In culture supernatant of capsule explant, the secretedprotein with an apparent molecular weight of 70 kDa (Figure 6)was identified as serum albumin (Table 3).

Effects of S-(þ)-ibuprofen on serum amyloid proteinexpression

After 2 days of culture in serum-free medium, SDS–PAGEanalysis indicated that treatment with 1 mM S-(þ)-ibuprofenreduced the release of SAA protein in medium from cartilageexplant (Figure 7). Measures of SAA protein band surface stainedwith Coomassie blue following SDS–PAGE confirmed thatS-(þ)-ibuprofen (1 mM) reduced by 44% the release of SAAprotein from cartilage explants (p50.0001) and by 32% fromjoint capsule (p¼ 0.0001), but was without effect on SAAexpression during co-culture (Figure 8). Expression of SAAduring co-culture with 1 mM of S-(þ)-ibuprofen was higher than

observed for capsule (p¼ 0.0031) and cartilage (p¼ 0.0002)groups. On contrary, incubation of activated cartilage explant with50 mM S-(þ)-ibuprofen increased faintly the expression of SAA2protein (1.6-fold, p50.0001). Compared to the activated controls,S-(þ)-ibuprofen (50 mM) had no effect on the release of SAAprotein from joint capsule and from explants maintained in co-culture.

Discussion

This work is a preliminary research aimed to the development ofnovel intra-articular DDS for local treatment of inflammation andpain during OA, the most common form of arthritis affectingmillions of people worldwide. NSAIDs provide symptomaticrelief but their systemic administration is limited by side effects.Intra-articular DDSs loaded with NSAIDs appears as appropriatetool to long-term treatment of joint inflammation during OA byreducing the systemic toxicity. Several natural and synthetic intra-articular DDS loaded with NSAIDs have been investigated forintra-articular injections, microspheres loaded with diclofenac38,naproxen39, flurbiprofen40, celecoxib41 and ibuprofen42. Among

Figure 7. Polyacrylamide gel electrophoresisanalysis of effects of S-(þ)-ibuprofen (50 mMor 1 mM) on protein expression of LPS-activated explants. About 50mg of desaltedproteins recovered after 2 days of culture inserum-free medium were analyzed by SDS–PAGE 15% before staining with CoomassieBrilliant Blue R-250. Gel slices correspond-ing to 10-kDa protein were cut as indicated(7–14), digested with trypsin and the peptideswere identified using LC-mass spectrometry(Table 3). Soluble proteins were collectedfrom explants obtained from two animaldonors. The experiment was repeated withexplants obtained from two other animalleading to similar patterns.

Table 3. Identification by mass spectrometry of proteins secreted during culture of cartilage and capsule explants in serum-free medium.

Conditions ofculture Band n� Protein name MW (Da)

Number ofidentified peptides

Sequence coverage (%)and MASCOTprotein score

Cartilage Control 1 Cartilage oligomeric protein(Rattus norvegicus) gi j 6978679

85 234 3 4–71

2 Chondroadherin precursor (Bostaurus) gi j 27806697

41 373 10 29–429

LPS (10 mg/mL) 3 Chondroadherin (Bos taurus) gi j241177896

40 306 8 25–355

4 Serum amyloid A3.2 protein(Capra hircus) gi j 147744634

14 638 3 22–88

7 Serum amyloid A3.2 protein (Ovisaries) gi j 165940902

11 307 4 37–139

LPSþ 50mM S-ibu 8 Serum amyloid A3.2 protein (Ovisaries) gi j 165940902

11 307 5 41–131

Capsule Control 5 Serum albumin (Ovis aries) gi j57164373

71 139 13 23–356

LPS (10 mg/mL) 6 Serum amyloid A3.2 protein(Capra hircus) gi j 147744634

14 638 3 22–82

9 Serum amyloid A3.2 protein (Ovisaries) gi j 165940902

11 307 6 44–196LPSþ 50mM S-ibu 10 6 44–230LPSþ 1 mM S-ibu 11 6 44–242

Co-culture LPS (10 mg/mL) 12 5 40–138LPSþ 50mM S-ibu 13 4 31–106LPSþ 1 mM S-ibu 14 4 37–137

Proteins in media from control, LPS-stimulated explants in combination with S-(þ)-ibuprofen (50mM or 1mM) were separated by SDS–PAGE, andtryptic digests of gel slices were subjected to LC-MS/MS experiments. The proteins identified with a 95% confidence interval using MASCOT searchengine (http://matrixscience.com) are listed (S-ibu: S-(þ)-ibuprofen).


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the NSAIDs, 2-arylpropionic acids (ketoprofen, flurbiprofen,ibuprofen) exist as a racemic mixture of two enantiomers inwhich S-enantiomers are considered to be responsible for theinhibition of prostaglandins synthesis by inhibition of COXactivity2. The active isomers of NSAIDs of 2-arylpropionic acidsfamily are candidate for loading of intra-articular DDS instead ofthe racemate mixture. Before to undertake the preparation of suchintra-articular-DDS loaded with S-(þ)-ibuprofen, exploration ofbiological activities of the purified isomer on activated cartilage isnecessary.

Our in vitro findings provide evidences that S-(þ)-ibuprofen at50mM only reduces the PGE2 synthesis in LPS-activated cartilageand synovial explants cultured in mono- or in co-culture. Thiseffect is in accordance with the expected function of active isomerof profens on COX enzymes2,21,24. No effect of S-(þ)-ibuprofenat 50 mM on reduction of NO synthesis and proteoglycansdegradation was found in contradiction with results obtained foractive isomer of ketoprofen and S-(þ)-flurbiprofen which reducedat 10 mM the synthesis of NO from human chondrocytes treatedwith IL-124. These differences could be explained by the nature ofthe drugs, the origin of cells (sheep in our study versus human)and the culture conditions (explants in our study versus chondro-cyte monolayers).

We have noticed that S-(þ)-ibuprofen when used at millimolarlevel reduced cartilage degradation showing activities independ-ent of the COX inhibition pathways achieved at micromolar level.On cartilage cultivated in mono-culture, 1 mM S-(þ)-ibuprofenreduced significantly the formation of NO from LPS-activatedcartilage explants in agreement with observations made by43 whohave shown on glial cells treated with LPS and interferon-g, thatmillimolar dose of racemic ibuprofen reduced the expressions ofiNOS mRNA (IC50¼ 2 mM) and protein (IC50¼ 0.89 mM).During arthritic disorders, expression of inducible isoform ofNitric Oxide Synthase (iNOS) is induced within superficialchondrocytes and synovial fluid of arthritic patients is enriched inNO44. NO is involved in chondrocyte apoptosis4 and chondrocytededifferentiation31, and its reduction represent a therapeuticobjective. Concomitantly, we observed that S-(þ)-ibuprofen at1 mM reduces the GAG loss from the LPS-activated cartilageexplants during monoculture. Such findings suggest that thereduction of NO expression by S-(þ)-ibuprofen may, at leastpartly, underlie the reduced degradation of cartilage in

monoculture. This hypothesis is supported by observations of45

who demonstrated on TNF-a-activated cartilage explants, that theinhibition of iNOS activity by N-methyl-arginine reduced therelease of GAG from cartilage in medium, whereas the mRNAlevels of aggrecanases were unchanged. Thus, it may be that NOcan regulate aggrecanases activation at a post-transcriptionallevel. Taken those findings together, we hypothesize that thereduction of NO synthesis by 1 mM S-(þ)-ibuprofen in LPS-challenged cartilage gradually reduce the amount of activatedaggrecanases, and consequently the degradation of the cartilagematrix.

As additional chondroprotective activity of 1 mM S-(þ)-ibu-profen, we measured in each group of explants a reduction of celllysis during the culture. This result is in accordance with formerstudies which had measured a protective activity of ibuprofen, butsome differences exist about the active concentration. Racemicibuprofen inhibits at high concentration (0.2–1 mM) apoptosis ofhuman chondrocyte induced with NO31, while9 had measured areduction of chondrocyte apoptosis induced with staurosporine inpresence of low dose of racemic ibuprofen (10�6–10�12 M).Again, differences between culture conditions may explain thecontradictions about the concentration of ibuprofen efficient toreduce cell lysis.

During this study we have examined the effects of S-(þ)-ibuprofen treatment on protein expression of the LPS-challenged explants. LPS treatment of cartilage explants orchondrocytes increases expression of inflammatory mediatorssuch prostaglandins and nitrite oxide46 but also modified proteinexpression by increasing the expression of proteins involved ininnate immune response, such the Chitinase-3 like protein 1 andcomplement C3 and C1r proteins47. In our study, we found thatthe LPS challenge of cartilage and synovial membrane explantsinduced expression of one main protein identified as serumamyloid A3.2 protein. Our finding is in agreement with in vivoobservations, as LPS injection into the radiocarpal joint of horseinduced a local inflammatory response accompanied withexpression of serum amyloid isoforms48. The functions ofserum amyloid proteins in joint diseases are not elucitaded butit is supposed that they contribute to inflammation and cartilagedestruction by chemoattracting properties for leukocytes and byactivating angiogenesis49. In our experimental conditions, theeffect of S-(þ)-ibuprofen on expression of serum amyloid proteinwas dose-dependent as an inhibition of the protein expression wasseen only for cartilage and capsule explants cultures with 1 mM S-(þ)-ibuprofen as observed previously for inhibition of NOexpression and proteoglycans degradation.

We did not analyze the mechanism by which 1 mM S-(þ)-ibuprofen diminished on activated explants the extent of celllysis, synthesis of NO, cartilage degradation and expression ofserum amyloid protein, but we supposed that intracellular pathwaysare intercepted30. Indeed, when used at millimolar concentration,ibuprofen displays several cyclooxygenase-independent functions.Thus during experimental inflammation process induces by LPS onhuman monocytes,50 had shown that ibuprofen (1.2 to 3 mM)reduces the synthesis of IL-1 and TNF-a by interfering with thenuclear translocation of the pro-inflammatory NF-�B transcriptionfactor, and ibuprofen (0.25–1 mM) reduced apoptosis and dedif-ferentiation on bovine articular chondrocyte by blocking nitricoxide-induced activation of p38 kinase31. In conclusion, themechanisms by which S-(þ)-ibuprofen protect cartilage fromdegradation in monoculture remain to be elucidated but probablyinvolved several intracellular pathways controlling cell death andexpression of inflammatory molecules.

To better mimic natural situation where cartilage is surroundedby synovial tissue, we have added fragments of synovial tissue tocartilage explants in the aim to explore whether synovial

Figure 8. Effects of S-(þ)-ibuprofen on expression level of serumamyloid protein in LPS-activated explants. After SDS–PAGE fraction-ation of proteins secreted during explants culture, the surface of serumamyloid protein in each group [LPS, LPSþ 50mM S-(þ)-ibuprofen andLPSþ 1 mM S-(þ)-ibuprofen] was measured, and level of proteinexpression in the S-(þ)-ibuprofen-treated explants was normalized tothat of LPS group. Measures were done using explants collected from4 animals during two independent experiments. Comparison betweentreatment groups to LPS was carried out with the Mann–Whitney test.


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secretions change the effects of S-(þ)-ibuprofen on activatedcartilage. Few efficacy studies of chondroprotective drugs usedco-culture model of cartilage and synovial tissue51,52 although itis well established that during in vitro culture, co-incubation ofsynovial membrane with cartilage generates soluble factors whichaccelerate matrix degradation32–34. To explain the degradativeactivities of synovium on cartilage it was proposed that tissue-bound IL-1 increased the basal proteoglycans degradation onhuman OA cartilage during co-culture with synovial membrane53.Patwari et al.34 suggest that the catabolic pathways in chondro-cytes induced during co-culture with cut synovium may beindependent of the pro-inflammatory cytokines (IL-I and TNF-a),the inflammatory mediators secreted by synovium probablycorrespond to heat labile factors with a molecular weight of�20 kDa. In our study, we observed in the sheep model thatwithout LPS, synovial explants added to cartilage explantsincreased basal expression of PGE2 and cartilage GAG loss, inagreement with observations made by others using explantsisolated from bovine33 or human53 joints. In the pro-catabolicenvironment of co-culture, we found that the synovial tissue alterthe efficacy of S-(þ)-ibuprofen on reduction of cartilage damagesinduced with LPS, ie NO synthesis, GAG degradation andsecretion of serum amyloid protein. These findings are inaccordance with the enhancement of basal cartilage degradationmeasured during co-culture of un-activated explants. We assumethat inflammatory mediators secreted by capsular tissue couldtrigger specific catabolic pathways in chondrocytes which areresistant to the 1mM S-(þ)-ibuprofen treatment.

Conclusions

In the aim to load novel intra-articular DDS with S-(þ)-ibuprofeninstead of racemic ibuprofen, our in vitro study indicated that lowdose of drug (50mM) reduces the synthesis of prostaglandinswithout obvious chondroprotective effects. Reduction of cartilagedegradation achieved with 1mM S-(þ)-ibuprofen was abolished inpresence of synovial membrane suggesting, at least in the sheepmodel, that the catabolic activity of synovial tissue could changethe effects of a drug on cartilage metabolism. Taking together ourresults show that the delivery of S-(þ)-ibuprofen in a joint cavityusing an intra-articular DDS could inhibit the synthesis ofprostaglandins, while its delivery at higher dose (millimolarrange) could exert chondroprotective effects.

Acknowledgements

The authors would like to thank Julie Massonneau and Didier Mauchandfor their help at the slaughterhouse (INRA, Domaine de Vilvert, Jouy enJosas, F-78352).

Declaration of interest

The authors report no declarations of interest

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in vitro evaluation of s-(+)-ibuprofen as drug candidate for intra-articular drug...

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