http://informahealthcare.com/ddi ISSN: 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 for intra-articular drug delivery system Laurent Be´douet1, Florentina Pascale2, Michel Bonneau2, and Alexandre Laurent1,2,3 Drug Dev Ind Pharm Downloaded from informahealthcare.com by North West University on 12/21/14 For personal use only.

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Occlugel SAS, Jouy-en-Josas, France, 2AP-HP, INRA, Center for Research of Interventional Imaging (CR2i), Jouy-en-Josas, France, and Department of Interventional Neuroradiology, AP-HP, Lariboisie`re Hospital, Paris, France

3

Abstract

Keywords

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the efficacy of S-(þ)-ibuprofen on cartilage degradation as drug candidate for DDS loading. Humeral cartilage and joint capsule explants were collected from healthy sheep shoulder joints and they were cultured in mono- or in co-culture for 13 days with LPS in combination with S-(þ)-ibuprofen at 50 mM and 1 mM. S-(þ)-ibuprofen (50 mM) blocked prostaglandins production in LPS-activated explants but did not reduce cartilage degradation. By contrast, 1 mM S-(þ)-ibuprofen treatment of cartilage explants reduced nitric oxide synthesis by 51% (p ¼ 0.0072), proteoglycans degradation by 35% (p ¼ 0.0114) and expression of serum amyloid protein – the main protein induced upon LPS challenge – by 44% (p50.0001). On contrary, in presence of synovial membrane, the protective 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 capsule either in mono- or in co-culture. This study performed on sheep explants shows that 1 mM S-(þ)-ibuprofen inhibited cartilage degradation via a mechanism independent of cyclooxygenase inhibition. Reduction of prostaglandins synthesis at 50 mM in all treatment groups and reduction of cartilage degradation observed at 1 mM suggest that S-(þ)-ibuprofen could be considered as a promising drug candidate for the loading of intra-articular DDS.

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

Introduction The non-steroidal anti-inflammatory drugs (NSAIDs) are effective in controlling pain in patients with mild to moderate osteoarthritis (OA)1. NSAIDs inhibit the cyclooxygenase (COX) pathway that accounts in part for their anti-inflammatory activity through the inhibition of the prostaglandins production from arachidonic acid2. During OA the expression of inducible COX-2 is elevated in synovial tissue and in cartilage with 50-fold more prostaglandin E2 (PGE2) levels than in healthy cartilage3. Catabolic effects of PGE2 on articular cartilage include the increase of matrix metalloproteinase (MMP) production in chondrocytes, synovial fibroblasts, and OA cartilage explants and the synergistic action with nitric oxide (NO) on chondrocytes apoptosis3,4. Reduction of PGE2 synthesis represents a therapeutic objective in OA therapy. The treatment of patients with severe knee OA during 3 months with aceclofenac and celecoxib reduced PGE2 concentration in synovial fluid and improved the patient mobility5. Among NSAIDs, ibuprofen is one of the most popular, is availabe in OTC6. In 1990, 3000 t of ibuprofen were consumed on Address for correspondence: Laurent Be´douet, Occlugel SAS, 12 rue Charles de Gaulle, Jouy-en-Josas 78350, France. E-mail: l.bedouet@ hotmail.fr

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

the US market7. Ibuprofen can inhibit PGE2 expression from IL-1 activated chondrocytes8 and protect in a concentration-dependent manner chondrocytes from apoptosis, suggesting a protective role of chondrocytes during OA9. In vitro, ibuprofen did not reduce the GAG loss and proteolytic activity of cartilage explants treated with IL-1 and TNF-a10. In patients with OA, ibuprofen was inefficient on the reduction of MMP-3 level in serum11 while it reduced significantly the release of cartilage and synovial degradation markers in patients with a flare of knee arthritis12. Clinical studies indicate that ibuprofen is effective and relatively safe in management of the mild-to-moderate OA of the knee and hip13. Recently, ibuprofen treatment in rat was effective in attenuating inflammation and early articular cartilage degeneration induced on wrist joints under mechanical stress for 12 weeks14. The systemic administration of NSAIDs is limited by dosedependent side effects such as gastric erosion, cardiovascular problems, liver and kidney damages that can appear for long-term treatment at high dosages15. NSAIDs can even have deleterious effects on hip and knee16. A novel therapeutic approach of OA consists of using intra-articular drug delivery systems (DDSs) with the ability to locally release NSAIDs17. Various formulations of DDS are currently investigated to achieve a sustained release of NSAIDs18–20. Intra-articular DDS should provide long-term sustained release of NSAIDs and diminish the amount of

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systemic drug exposition, which in turn reduces the relatedadverse events. Ibuprofen exists in two enantiomers, the isomer active on the cyclooxygenases activity is the S-(þ)-enantiomer21. For a local delivery of ibuprofen in joint cavity, loading of DDS with the active isomer rather than with the racemate mixture should in theory reduce the amount of biomaterial to be injected. S-(þ)-ibuprofen has proved to be an effective drug for patients suffering of OA22. Clinically, use of single active isomer of the profens class of NSAIDs instead of the racemate mixture may represent a reduction of dose during treatment leading to a less exposition to xenobiotics and a reduction of renal load23. In vitro, biological activities of several NSAIDs isomers on joint cells were analyzed: S-(þ)-ketoprofen and S-(þ)-flurbiprofen on human chondrocytes treated with interleukin-124 and carprofen isomers on equine chondrocytes and synoviocytes after lipopolysaccharides (LPS) treatment25,26. No such study was undertaken for S-(þ)-ibuprofen. In a previous work, we have tested the toxicity of S-(þ)-ibuprofen on chondrocytes and synovial fibroblasts collected from sheep shoulder joint27. We have observed that S-(þ)-ibuprofen at 50 mm and 1 mM did not induce toxicity on chondrocytes and type B synoviocytes as well as on cartilage and synovial membrane explants. Absence of cytotoxicity of S-(þ)-ibuprofen on joint cells authorizes the setting of an efficacy study on activated joint explants. In the present study we have explored whether S-(þ)-ibuprofen (50 mM and 1 mM) could inhibit experimentally induced degradation of sheep articular cartilage explants induced with LPS. This efficacy study performed on joint explants is a prerequisite prior to the preparation of intraarticular DDS containing S-(þ)-ibuprofen. The low concentration (50 mM) is in the range of IC50 values of the racemate ibuprofen to inhibit the COX-2 activity28 and corresponds to the higher concentration of racemic ibuprofen measured in synovial fluid of OA patients after oral treatment29. The millimolar dose of S-(þ)-ibuprofen was chosen to determine the activity of the NSAID regardless its inhibition of COX activity, since NSAIDs at high concentrations exert pharmacological activity different from inhibition of COX enzymes30,31. Consequently in an assessment of efficacy of a NSAID on cartilage degradation, low and high concentrations of drug must to be analyzed in regard to its different modes of action. We introduced a co-culture model of cartilage explant with synovial tissue to measure whether and to what extent capsular tissue changes the outcomes of S-(þ)-ibuprofen treatment on activated cartilage since previous studies had demonstrated the degradative capacity of synovium which promotes the catabolic pathways of chondrocytes during co-culture experiments32–34. We supposed that during in vitro efficacy studies of a drug, the procatabolic environment created during co-culture may have effects on activities of a drug on cartilage degradation. The effects of S-(þ)-ibuprofen at (50 mM and 1 mM) on cartilage degradation induced with LPS were studied and compared in cartilage co-cultured with explants of synovial membrane.

Materials and methods Materials Lipopolysaccharides, S-(þ)-ibuprofen, common culture medium supplements, trypsin, electrophoresis reagents, common chemicals and ‘‘In vitro toxcicology assay kit Lactate dehydrogenase based’’ were obtained from Sigma (Saint-Quantin Fallavier, France). The nitrite assay kit was purchased from Promega (Charbonnie`res, France) and PGE2 kit was from R&D Sytems (Lille, France).

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Animals and explants harvesting Pre´alpes sheep (n ¼ 7, 6–4 years old) were sacrificed by electronarcosis at the central slaughterhouse of INRA (Jouy en Josas, France) according to veterinary regulatory rules. Full-thickness cartilage slices from humerus surface were obtained after scalpel shaving. The capsular tissue including the intima, the sub-intima layer and the fibrous capsule was removed from each joint. Explants were placed in sterile medium (DMEM high-glucose, 100 U/mL penicillin, 100 mg/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 coculture, autologous fragments of capsule and cartilage were mixed. Wells were then filled with 2 mL of culture medium (10% FBS, 2 mM L-glutamine, penicillin (50 U/mL), streptomycin (50 mg/mL), 10 mM HEPES, DMEM-high glucose) and incubated at 37  C with 5% CO2. The explants (cartilage, capsule and coculture) were equilibrated for 5 days before the treatments. Cultures were performed with triplicate wells using tissue from one animal donor. Experiments were repeated 3 times, each time using tissue from a different animal. Preparation of S-(þ)-ibuprofen solutions and treatment of joint explants Explants were treated with 1 mL of culture medium alone (control explants), 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 solution of S-(þ)-ibuprofen (1 M) was prepared in absolute ethanol, aliquots were stored at 20 C. Addition of S-(þ)-ibuprofen in culture medium was performed before each medium change. For medium containing 1 mM of drug (206 mg/mL), S-(þ)-ibuprofen stock solution was progressively added under mild shaking. At end of drug addition to cell culture medium no precipitate was visible in accordance with the solubility of racemate ibuprofen in phosphate buffer saline measured at 6 mg/mL35. After 48 h of culture, the supernatants were harvested before storage at 20  C, and new media were added. Then, culture supernatants were collected after 6, 9 and 13 days. At the end of the culture, wet explants were collected from the culture wells and they were drained on filter paper before to be transferred in pre-weighed tubes. Drying of explants (n ¼ 144) occurred in an oven (90  C for 60 h) until a constant weight was obtained. The ratio of cartilage explants and synovium explants in the co-culture group was 1/3 (dry weight). For each explant group, the amounts of tissue involved in the four treatment groups were equivalent (Table 1). Cytotoxicity assay by lactate dehydrogenase release from explants Lactate dehydrogenase (LDH) release from explants to the medium 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 media according to the manufacturer’s instructions. The absorbance was measured (450–630 nm) and values were normalized to the explants 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 by ELISA at day 2 using a commercial kit. The cartilage, capsule and co-culture supernatants were diluted 10, 100 and 200 times in the calibrator diluent, respectively, before addition of 150 mL of

S-(þ)-ibuprofen efficacy on joint explants

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

Control LPS (10 mg/mL) LPS þ 50 mM S-(þ)-ibuprofen LPS þ 1 mM S-(þ)-ibuprofen KW

Co-culture

Cartilage (mg)

Capsule (mg)

Cartilage (mg)

Capsule (mg)

4.33  1.90 3.26  0.93 3.06  0.88 3.11  0.81 0.2412

10.41  4.96 12.15  5.34 8.76  4.14 11.73  6.10 0.5702

2.66  1.07 2.61  0.55 3.22  1.43 3.68  1.17 0.2200

10.38  2.88 9.06  4.25 7.83  2.35 10.12  10.84 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).

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dilutions to the microplate. The PGE2 expression (ng) was normalized with the explants dry weight (mg). Nitrite assay The assay of nitrite (stable metabolite of NO) in cartilage and coculture medium (25 mL) was performed in duplicate using the spectrophotometric Greiss assay according to the manufacturer’s protocol. Sample concentration in nitrite was determined by measuring the absorbance at 540 nm. A linear calibration curve was done with sodium nitrite (12.5–100 mM). The cumulative amount of NO synthesis was measured from day 2 to day 13 of culture. Nitrite concentration (mM) was normalized with the cartilage explants dry weight (mg). GAG assay The release of soluble glycosaminoglycans (GAG) in the cartilage and co-culture medium was used as an indicator of cartilage aggrecan degradation. The release of GAG was measured in duplicate using 10 mL of conditioned medium according to the 1,9-dimethylmethylene blue assay (DMMB) assay36 with shark chondroitin sulfate (Sigma) as standard. Absorbance of the soluble GAG-DMMB complex was measured at 515 nm. The cumulative amount of GAG release was measured from day 2 to day 13 of culture. The GAG amount (mg) was normalized with the cartilage explants dry weight (mg). Recovery of proteins secreted by explants and proteins electrophoresis Explants from cartilage and capsule were cultured alone or in co-culture in serum-free media and treated in triplicate with LPS (10 mg/mL) in presence of S-(þ)-ibuprofen (50 mM or 1 mM). Experiments were repeated twice, each time using tissues from two animal donors. Culture supernatants were collected 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 were lyophilized before proteins assay using the bicinchoninic acid method. Lyophilized proteins (50 mg) were separated on 15% SDS–polyacrylamide gel (SDS–PAGE)37 and stained with Coomassie blue R-250. Pieces of gel corresponding to selected proteins were sliced, washed (5% acetic acid) and dehydrated in acetonitrile before to in-gel reduction (10 mM dithiothreitol in 100 mM ammonium bicarbonate, 45 min, 56  C), alkylation (55 mM iodoacetamide, 30 min) and dehydration in acetonitrile. Trypsin was added (1 mg in 40 mL of 50 mM ammonium bicarbonate) per slice and incubated for 45 min on ice. Excess of enzyme was removed and digestion occurred overnight at 37  C. Tryptic peptides were extracted twice with 1% formic acid, and 100% acetonitrile. Supernatants were pooled and lyophilized.

Liquid chromatography and-mass spectrometry analysis of trypsin digests Separation of the tryptic peptides was done on a C18 column (150  1 mm, 4 mm, Modulo-cart UPISPHERE, Interchim, France) at a flow rate of 50 mL/min with 0.1% formic acid in water (eluent A), and acetonitrile (eluent B) according to a linear gradient from 5% to 50% eluent B in 45 min. Separated peptides were analyzed on line with an ESI-QqTOF hybrid mass spectrometer (pulsar I, Applied Biosystems) using information dependant acquisition (IDA) which allows to switch between MS and MS/MS experiments. The data were acquired and analyzed with the Analyst QS software. The mass spectra data were searched against NCBI non-redundant database using an in house MASCOT search engine (http://www.matrixscience.com). Proteins identified were validated based on the MASCOT Mowse score. Quantification of expression of serum amyloid protein Polyacrylamide gels after Coomassie blue staining were scanned and surface of serum amyloid protein (SAA) band was measured using ImageJ software. Level of expression of SAA protein in presence of S-(þ)-ibuprofen was normalized to SAA level measured in LPS-activated explants. All quantifications were performed 3 times on gels obtained from the two independent experiments composed of explants collected from two animal donors. Statistical analysis Statistical analyses were performed on StatView SAS 2000 (SAS institute, Cary, NC). The data are presented in box-and-whisker plots and continuous variables were expressed as median  median absolute deviation. For comparison of two groups, nonparametric Mann–Whitney (MW) test was used. The Kruskal– Wallis (KW) test was used to compare three or more independent groups. Significance was set at p50.05.

Results Sheep cartilage explants were induced to degrade alone or in coculture with synovial tissue by using LPS (10 mg/mL) and the chondroprotective effects of S-(þ)-ibuprofen at 50 mM and 1 mM were studied by co-treating the activated explants with drug for 13 days. We have cultured cartilage explants with fragments of joint capsule in order to mimic more closely the interactions which exist between the different joint tissues and analyze the effect of its association on the efficacy of S-(þ)-ibuprofen treatment of LPS-activated tissues. The conditioned media were collected and analyzed for their content of different markers of inflammation [PGE2, nitric oxide (NO)], and degradation of extracellular matrix molecules in cartilage explants (glycosaminoglycans (GAG)). The LDH leakage from explants to the

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medium was measured to quantify the cytotoxicity effects of drug treatments on explants during the culture period. Furthermore, protein expression changes of explants according to culture conditions were investigated using gel electrophoresis and liquid chromatography-mass spectrometry experiments. The aim was to identify proteins down-regulated or up-regulated during treatment of joint explants with LPS and S-(þ)-ibuprofen.

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Cytotoxicity of S-(þ)-ibuprofen during tissue cultures in presence of LPS Compared to control explants, no significant increase of LDH activity 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 the 13 days (Figure 1). On contrary, culture of explants in presence of 1 mM S-(þ)-ibuprofen reduced significantly (p50.05) the amount of LDH released into supernatants for each group of explants. For each group of explants, a better reduction of LHD leakage was achieved during culture with 1 mM of drug compared to 50 mM of drug (Table 2). Thus, treatment of explants with S(þ)-ibuprofen did not induce cell death and should have no cytotoxic or apoptotic effects on chondral and synovial tissues during the period of culture. Chondroprotective effects of S-(þ)-ibuprofen in sheep cartilage explants Expression of each marker of inflammation and cartilage degradation was determined before LPS challenge and after culture with endotoxins and S-(þ)-ibuprofen. Effects of synovial tissue on activities of S-(þ)-ibuprofen on cartilage degradation were examined. Results are summarized in Table 2. Effects of S-(þ)-ibuprofen on expression of PGE2

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co-culture was higher than during mono-culture of capsule (p ¼ 0.0209) and cartilage (p ¼ 0.0008) explants. Effect of S-(þ)-ibuprofen treatments on PGE2 production For each explant group, 50 mM and 1 mM of S-(þ)-ibuprofen reduced significantly the PGE2 synthesis (p ¼ 0.0008) compared to the LPS-activated explants. In presence of 50 mM ibuprofen, expression of PGE2 remained higher in the co-culture group compared to cartilage monoculture (p ¼ 0.0008) and equivalent to capsule group (p ¼ 0.0929). At 1 mM of drug, the residual PGE2 synthesis activity in the co-culture group was higher than in cartilage (p ¼ 0.0016) and capsule (p ¼ 0.0033) groups. Dose effect of S-(þ)-ibuprofen A better inhibition of PGE2 synthesis was observed with 1 mM S-(þ)-ibuprofen during mono-culture of capsule (p ¼ 0.0008) and co-culture of explants (p ¼ 0.0008) while for cartilage explant in mono-culture, 1 mM S-(þ)-ibuprofen did not enhance inhibition of PGE2 synthesis compared to drug at 50 mM (p ¼ 0.3446). Effects of S-(þ)-ibuprofen on synthesis of NO Basal NO synthesis The amounts of NO detected in cartilage and in co-culture supernatants were low and identical (p ¼ 0.2821), indicating that co-culture condition did not enhance NO synthesis (Figure 3). NO synthesis after LPS challenge LPS induced synthesis of NO during cartilage mono-culture and co-culture (p50.0001). In the synovial explants group, LPS challenge did not induce expression of NO compared to untreated explants (p ¼ 0.8466).

Basal PGE2 expression

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

Content of PGE2 in supernatant of control cartilage explants was lower than for capsule (p ¼ 0.0033). Co-culture of cartilage explant with joint capsule caused a 10-fold increase of PGE2 synthesis compared to cartilage (p ¼ 0.0008).

Compared to activated explants, S-(þ)-ibuprofen (50 mM) did not inhibit NO production during cartilage mono-culture (p ¼ 0.5907) and co-culture (p ¼ 0.8743). On the contrary, 1 mM S-(þ)-ibuprofen reduced by 51% the NO production in cartilage mono-culture (p ¼ 0.0072). In co-culture, a non-significant inhibition of NO synthesis (23%, p ¼ 0.0619) was measured with 1 mM S-(þ)-ibuprofen. At 1 mM of S-(þ)-ibuprofen, the cumulative synthesis of NO produced from cartilage explant in co-culture was higher than the expression achieved during cartilage mono-culture (p ¼ 0.0162). A more efficient inhibition

PGE2 production after LPS challenge Compared to the controls, LPS treatment stimulated by 53-, 45and 23-fold the expression of PGE2 for cartilage, capsule and co-culture, respectively (Figure 2). PGE2 produced during Figure 1. The cytotoxic effects of S-(þ)-ibuprofen during the culture of cartilage and joint capsule in mono- or in co-culture for 13 days. Explants were collected from three animal donors and culture was performed in triplicates. Data are presented in box-andwhisker plots with values corresponding to the median  median absolute deviation. Absorbance values (650–450 nm) were normalized to the explant dry weight (mg) and specific activities determined at days 2, 6, 9 and 13 were added leading to cumulative values for LDH leakage from joint explants. Comparisons between control, LPS and LPS þ 50 mM S-(þ)-ibuprofen groups were done using the Kruskal–Wallis (KW) nonparametric test. Comparisons between two groups were done using Mann–Whitney (MW) non-parametric test. *: significant reduction (MW) of LDH leakage compared to LPS treated explants.

S-(þ)-ibuprofen efficacy on joint explants

Comparison between culture conditions was done using Mann–Whitney non-parametric test. The values correspond to the median  median absolute deviation. *Comparison between control explants and LPS-treated tissues. yComparison between the S-(þ)-ibuprofen groups (dose effect).

LDH 97.66  69.53 78.47  44.74 107.61  88.33 17.51  17.51 188.32  29.52 244.59  99.19 239.56  29.26 133.63  40.68 316.66  69.73 289.09  19.63 312.99  148.15 187.17  48.17 (arbitrary unit) p ¼ 0.3589* p ¼ 0.0192y p ¼ 0.0498* p ¼ 0.0004y p40.9999* p ¼ 0.0114y PGE2 0.35  0.11 18.6  1.9 1.22  0.57 0.28  0.08 0.12  0.03 5.48  3.42 0.19  0.05 0.15  0.08 1.21  0.43 28.44  7.47 3.66  085 0.73  0.11 (ng/mg dry explant) p ¼ 0.0001* p ¼ 0.0008y p ¼ 0.0001* p ¼ 0.3446y p ¼ 0.0001* p ¼ 0.0008y Nitrite 0.162  0.162 0.87  0.87 0.189  0.189 0.428  0.428 2.18  1.68 15.77  9.03 17.13  8.46 7.74  4.08 1.38  2.33 24.45  13.66 19.34  5.36 18.98  7.88 (mM/mg dry explant) p ¼ 0.8466* p ¼ 0.8974y p50.0001* p ¼ 0.0011y p50.0001* p ¼ 0.3425y GAG 66.82  14.40 112.36  21.40 107.52  26.18 73.50  17.46 94.71  20.75 102.79  24.91 96.98  34.11 83.78  11.54 (mg/mg dry explant) p ¼ 0.0021* p ¼ 0.0577y p ¼ 0.4668* p ¼ 0.3113y

50 mM S-Ibu 50 mM S-Ibu 50 mM S-Ibu 0 Control

LPS (10 mg/mL)

1 mM S-Ibu

Control

0

LPS (10 mg/mL)

1 mM S-Ibu

Control

0

LPS (10 mg/mL)

Co-culture Cartilage Capsule

Table 2. Summary of activities of S-(þ)-ibuprofen (50 mM or 1 mM) on expression of inflammatory markers and degradation of cartilage proteoglycans induced with LPS.

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1 mM S-Ibu

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Figure 2. Activity of S-(þ)-ibuprofen on PGE2 expression (ng/mg dry weight) from cartilage and capsule explants cultured in mono- or in coculture. Explants were cultured as described in Figure 1. After 2 days of culture, PGE2 in medium of different groups (control, 10 mg/mL LPS, LPS þ 1 mM S-(þ)-ibuprofen and LPS þ 50 mM S-(þ)-ibuprofen) was assayed in duplicate. Comparison between culture conditions was done using Mann–Whitney non-parametric test. (¤p ¼ 0.0008 for comparison between controls and LPS, *p ¼ 0.0008 for comparison between S-(þ)ibuprofen at 50 mM or 1 mM to LPS groups).

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

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 only during mono-culture of cartilage explants (p ¼ 0.0011) compared to co-culture (p ¼ 0.3425). Effects of S-(þ)-ibuprofen on cartilage proteoglycans degradation Basal GAG loss from cartilage explant Degradation of cartilage matrix increased during co-culture of explant, compared to monoculture. The differences were

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significant at day 2 (p ¼ 0.004), day 6 (p50.0001) and day 9 (p ¼ 0.0014) and not at day 13 (p ¼ 0.0763) of culture (Figure 4). GAG degradation after LPS challenge The level of proteoglycans degradation of cartilage explants was increased in presence of LPS during cartilage monoculture. On contrary LPS did not enhance the GAG loss from cartilage in coculture compared to the control explants at each time of analysis (Figure 4).

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Effect of S-(þ)-ibuprofen treatments on cartilage degradation S-(þ)-ibuprofen (50 mM) did not inhibit the proteoglycans degradation of cartilage during mono- and co-culture of joint explants in presence of LPS. A significant reduction by 35% (p ¼ 0.0114) of the 13 days cumulative GAG loss from LPSactivated cartilage was measured after culture with 1 mM S-(þ)-ibuprofen (Figure 5). In presence of capsular tissue, a non-significant reduction of GAG release from cartilage was measured at 1 mM S-(þ)-ibuprofen (19%, p ¼ 0.0536). At 1 mM of S-(þ)-ibuprofen, the cumulative amount of soluble GAG released from cartilage explant was not different between the two groups of cartilage explants (mono- and co-culture) (p ¼ 0.8993).

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In this aim, cultures were performed without serum in order to observe the secreted proteins, which were present in lower quantities than proteins from serum. When exposed to LPS, the joint explants, cartilage, capsule and co-culture of both, secreted a low molecular weight protein (10 kDa) (Figure 6) which was also observed in presence of S-(þ)-ibuprofen (Figure 7). Identification of serum amyloid protein as inflammatory protein 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 analysis identified this protein as serum amyloid A3.2 protein (SAA) (Table 3). Low molecular weight protein at 10 kDa observed after S-(þ)-ibuprofen treatment of activated explants was also identified as serum amyloid protein. Some proteins whose expression did not appear to be modified by the culture conditions

Dose effect of S-(þ)-ibuprofen on reduction of GAG release At the end of the culture, a dose dependant inhibition of cartilage matrix degradation was not observed during mono-culture of cartilage explants (p ¼ 0.0557) and co-culture with synovial tissue (p ¼ 0.3113). Effects of S-(þ)-ibuprofen on protein expression in LPS-activated explants In order to investigate the effects of LPS challenge and the simultaneous treatment with S-(þ)-ibuprofen on protein expression of joint explants, proteins released in culture medium after 2 days of culture were recovered and separated by SDS–PAGE.

Figure 4. Effect of co-culture of cartilage with synovial tissue on degradation of cartilage proteoglycans. The accumulated GAG release to the medium (mg/mg dry weight cartilage) is indicated for cartilage monoculture and co-culture in absence (basal degradation) or in presence of LPS (10 mg/mL). Explants were cultured as described in Figure 1, and GAG content in supernatants was normalized to the cartilage dry weight (mg). Values determined at days 2, 6, 9 and 13 were added leading to cumulative GAG release from cartilage matrix. Comparison between culture conditions was done using Mann–Whitney non-parametric test. Comparison between co-culture and monoculture conditions for basal GAG release was performed at each day of analysis: a (p ¼ 0.004), b (p50.0001), c (p ¼ 0.0014) and d (p ¼ 0.07).

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

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

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Figure 7. Polyacrylamide gel electrophoresis analysis of effects of S-(þ)-ibuprofen (50 mM or 1 mM) on protein expression of LPSactivated explants. About 50 mg of desalted proteins recovered after 2 days of culture in serum-free medium were analyzed by SDS– PAGE 15% before staining with Coomassie Brilliant Blue R-250. Gel slices corresponding to 10-kDa protein were cut as indicated (7–14), digested with trypsin and the peptides were identified using LC-mass spectrometry (Table 3). Soluble proteins were collected from explants obtained from two animal donors. The experiment was repeated with explants obtained from two other animal leading to similar patterns.

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

Cartilage

Band n

Protein name

MW (Da)

Number of identified peptides

Control

1

Cartilage oligomeric protein (Rattus norvegicus) gi j 6978679 Chondroadherin precursor (Bos taurus) gi j 27806697 Chondroadherin (Bos taurus) gi j 241177896 Serum amyloid A3.2 protein (Capra hircus) gi j 147744634 Serum amyloid A3.2 protein (Ovis aries) gi j 165940902 Serum amyloid A3.2 protein (Ovis aries) gi j 165940902 Serum albumin (Ovis aries) gi j 57164373 Serum amyloid A3.2 protein (Capra hircus) gi j 147744634 Serum amyloid A3.2 protein (Ovis aries) gi j 165940902

85 234

3

4–71

41 373

10

29–429

40 306

8

25–355

14 638

3

22–88

11 307

4

37–139

11 307

5

41–131

71 139

13

23–356

14 638

3

22–82

11 307

6 6 6 5 4 4

44–196 44–230 44–242 40–138 31–106 37–137

2 LPS (10 mg/mL)

3 4 7

Capsule

Co-culture

Sequence coverage (%) and MASCOT protein score

Conditions of culture

LPS þ 50 mM S-ibu

8

Control

5

LPS (10 mg/mL)

6

LPS þ 50 mM S-ibu LPS þ 1 mM S-ibu LPS (10 mg/mL) LPS þ 50 mM S-ibu LPS þ 1 mM S-ibu

9 10 11 12 13 14

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

were identified in supernatants. In cartilage medium (Figure 6), the high molecular weight protein at 97 kDa was identified as cartilage oligomeric protein (COMP) and the polypeptide at 28 kDa probably correspond to fragment of chondroadherin (Table 3). In culture supernatant of capsule explant, the secreted protein with an apparent molecular weight of 70 kDa (Figure 6) was identified as serum albumin (Table 3).

observed for capsule (p ¼ 0.0031) and cartilage (p ¼ 0.0002) groups. On contrary, incubation of activated cartilage explant with 50 mM S-(þ)-ibuprofen increased faintly the expression of SAA2 protein (1.6-fold, p50.0001). Compared to the activated controls, S-(þ)-ibuprofen (50 mM) had no effect on the release of SAA protein from joint capsule and from explants maintained in coculture.

Effects of S-(þ)-ibuprofen on serum amyloid protein expression

Discussion

After 2 days of culture in serum-free medium, SDS–PAGE analysis indicated that treatment with 1 mM S-(þ)-ibuprofen reduced the release of SAA protein in medium from cartilage explant (Figure 7). Measures of SAA protein band surface stained with Coomassie blue following SDS–PAGE confirmed that S-(þ)-ibuprofen (1 mM) reduced by 44% the release of SAA protein from cartilage explants (p50.0001) and by 32% from joint capsule (p ¼ 0.0001), but was without effect on SAA expression during co-culture (Figure 8). Expression of SAA during co-culture with 1 mM of S-(þ)-ibuprofen was higher than

This work is a preliminary research aimed to the development of novel intra-articular DDS for local treatment of inflammation and pain during OA, the most common form of arthritis affecting millions of people worldwide. NSAIDs provide symptomatic relief but their systemic administration is limited by side effects. Intra-articular DDSs loaded with NSAIDs appears as appropriate tool to long-term treatment of joint inflammation during OA by reducing the systemic toxicity. Several natural and synthetic intraarticular DDS loaded with NSAIDs have been investigated for intra-articular injections, microspheres loaded with diclofenac38, naproxen39, flurbiprofen40, celecoxib41 and ibuprofen42. Among

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L. Be´douet et al.

Figure 8. Effects of S-(þ)-ibuprofen on expression level of serum amyloid protein in LPS-activated explants. After SDS–PAGE fractionation of proteins secreted during explants culture, the surface of serum amyloid protein in each group [LPS, LPS þ 50 mM S-(þ)-ibuprofen and LPS þ 1 mM S-(þ)-ibuprofen] was measured, and level of protein expression in the S-(þ)-ibuprofen-treated explants was normalized to that of LPS group. Measures were done using explants collected from 4 animals during two independent experiments. Comparison between treatment groups to LPS was carried out with the Mann–Whitney test.

the NSAIDs, 2-arylpropionic acids (ketoprofen, flurbiprofen, ibuprofen) exist as a racemic mixture of two enantiomers in which S-enantiomers are considered to be responsible for the inhibition of prostaglandins synthesis by inhibition of COX activity2. The active isomers of NSAIDs of 2-arylpropionic acids family are candidate for loading of intra-articular DDS instead of the racemate mixture. Before to undertake the preparation of such intra-articular-DDS loaded with S-(þ)-ibuprofen, exploration of biological activities of the purified isomer on activated cartilage is necessary. Our in vitro findings provide evidences that S-(þ)-ibuprofen at 50 mM only reduces the PGE2 synthesis in LPS-activated cartilage and synovial explants cultured in mono- or in co-culture. This effect is in accordance with the expected function of active isomer of profens on COX enzymes2,21,24. No effect of S-(þ)-ibuprofen at 50 mM on reduction of NO synthesis and proteoglycans degradation was found in contradiction with results obtained for active isomer of ketoprofen and S-(þ)-flurbiprofen which reduced at 10 mM the synthesis of NO from human chondrocytes treated with IL-124. These differences could be explained by the nature of the drugs, the origin of cells (sheep in our study versus human) and the culture conditions (explants in our study versus chondrocyte monolayers). We have noticed that S-(þ)-ibuprofen when used at millimolar level reduced cartilage degradation showing activities independent of the COX inhibition pathways achieved at micromolar level. On cartilage cultivated in mono-culture, 1 mM S-(þ)-ibuprofen reduced significantly the formation of NO from LPS-activated cartilage explants in agreement with observations made by43 who have shown on glial cells treated with LPS and interferon-g, that millimolar dose of racemic ibuprofen reduced the expressions of iNOS mRNA (IC50 ¼ 2 mM) and protein (IC50 ¼ 0.89 mM). During arthritic disorders, expression of inducible isoform of Nitric Oxide Synthase (iNOS) is induced within superficial chondrocytes and synovial fluid of arthritic patients is enriched in NO44. NO is involved in chondrocyte apoptosis4 and chondrocyte dedifferentiation31, and its reduction represent a therapeutic objective. Concomitantly, we observed that S-(þ)-ibuprofen at 1 mM reduces the GAG loss from the LPS-activated cartilage explants during monoculture. Such findings suggest that the reduction of NO expression by S-(þ)-ibuprofen may, at least partly, underlie the reduced degradation of cartilage in

Drug Dev Ind Pharm, 2015; 41(1): 85–94

monoculture. This hypothesis is supported by observations of45 who demonstrated on TNF-a-activated cartilage explants, that the inhibition of iNOS activity by N-methyl-arginine reduced the release of GAG from cartilage in medium, whereas the mRNA levels of aggrecanases were unchanged. Thus, it may be that NO can regulate aggrecanases activation at a post-transcriptional level. Taken those findings together, we hypothesize that the reduction of NO synthesis by 1 mM S-(þ)-ibuprofen in LPSchallenged cartilage gradually reduce the amount of activated aggrecanases, and consequently the degradation of the cartilage matrix. As additional chondroprotective activity of 1 mM S-(þ)-ibuprofen, we measured in each group of explants a reduction of cell lysis during the culture. This result is in accordance with former studies which had measured a protective activity of ibuprofen, but some differences exist about the active concentration. Racemic ibuprofen inhibits at high concentration (0.2–1 mM) apoptosis of human chondrocyte induced with NO31, while9 had measured a reduction of chondrocyte apoptosis induced with staurosporine in presence of low dose of racemic ibuprofen (106–1012 M). Again, differences between culture conditions may explain the contradictions about the concentration of ibuprofen efficient to reduce cell lysis. During this study we have examined the effects of S(þ)-ibuprofen treatment on protein expression of the LPSchallenged explants. LPS treatment of cartilage explants or chondrocytes increases expression of inflammatory mediators such prostaglandins and nitrite oxide46 but also modified protein expression by increasing the expression of proteins involved in innate immune response, such the Chitinase-3 like protein 1 and complement C3 and C1r proteins47. In our study, we found that the LPS challenge of cartilage and synovial membrane explants induced expression of one main protein identified as serum amyloid A3.2 protein. Our finding is in agreement with in vivo observations, as LPS injection into the radiocarpal joint of horse induced a local inflammatory response accompanied with expression of serum amyloid isoforms48. The functions of serum amyloid proteins in joint diseases are not elucitaded but it is supposed that they contribute to inflammation and cartilage destruction by chemoattracting properties for leukocytes and by activating angiogenesis49. In our experimental conditions, the effect of S-(þ)-ibuprofen on expression of serum amyloid protein was dose-dependent as an inhibition of the protein expression was seen only for cartilage and capsule explants cultures with 1 mM S(þ)-ibuprofen as observed previously for inhibition of NO expression and proteoglycans degradation. We did not analyze the mechanism by which 1 mM S(þ)-ibuprofen diminished on activated explants the extent of cell lysis, synthesis of NO, cartilage degradation and expression of serum amyloid protein, but we supposed that intracellular pathways are intercepted30. Indeed, when used at millimolar concentration, ibuprofen displays several cyclooxygenase-independent functions. Thus during experimental inflammation process induces by LPS on human monocytes,50 had shown that ibuprofen (1.2 to 3 mM) reduces the synthesis of IL-1 and TNF-a by interfering with the nuclear translocation of the pro-inflammatory NF-B transcription factor, and ibuprofen (0.25–1 mM) reduced apoptosis and dedifferentiation on bovine articular chondrocyte by blocking nitric oxide-induced activation of p38 kinase31. In conclusion, the mechanisms by which S-(þ)-ibuprofen protect cartilage from degradation in monoculture remain to be elucidated but probably involved several intracellular pathways controlling cell death and expression of inflammatory molecules. To better mimic natural situation where cartilage is surrounded by synovial tissue, we have added fragments of synovial tissue to cartilage explants in the aim to explore whether synovial

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DOI: 10.3109/03639045.2013.850704

secretions change the effects of S-(þ)-ibuprofen on activated cartilage. Few efficacy studies of chondroprotective drugs used co-culture model of cartilage and synovial tissue51,52 although it is well established that during in vitro culture, co-incubation of synovial membrane with cartilage generates soluble factors which accelerate matrix degradation32–34. To explain the degradative activities of synovium on cartilage it was proposed that tissuebound IL-1 increased the basal proteoglycans degradation on human OA cartilage during co-culture with synovial membrane53. Patwari et al.34 suggest that the catabolic pathways in chondrocytes induced during co-culture with cut synovium may be independent of the pro-inflammatory cytokines (IL-I and TNF-a), the inflammatory mediators secreted by synovium probably correspond to heat labile factors with a molecular weight of 20 kDa. In our study, we observed in the sheep model that without LPS, synovial explants added to cartilage explants increased basal expression of PGE2 and cartilage GAG loss, in agreement with observations made by others using explants isolated from bovine33 or human53 joints. In the pro-catabolic environment of co-culture, we found that the synovial tissue alter the efficacy of S-(þ)-ibuprofen on reduction of cartilage damages induced with LPS, ie NO synthesis, GAG degradation and secretion of serum amyloid protein. These findings are in accordance with the enhancement of basal cartilage degradation measured during co-culture of un-activated explants. We assume that inflammatory mediators secreted by capsular tissue could trigger specific catabolic pathways in chondrocytes which are resistant to the 1mM S-(þ)-ibuprofen treatment.

Conclusions In the aim to load novel intra-articular DDS with S-(þ)-ibuprofen instead of racemic ibuprofen, our in vitro study indicated that low dose of drug (50mM) reduces the synthesis of prostaglandins without obvious chondroprotective effects. Reduction of cartilage degradation achieved with 1mM S-(þ)-ibuprofen was abolished in presence of synovial membrane suggesting, at least in the sheep model, that the catabolic activity of synovial tissue could change the effects of a drug on cartilage metabolism. Taking together our results show that the delivery of S-(þ)-ibuprofen in a joint cavity using an intra-articular DDS could inhibit the synthesis of prostaglandins, while its delivery at higher dose (millimolar range) could exert chondroprotective effects.

Acknowledgements The authors would like to thank Julie Massonneau and Didier Mauchand for their help at the slaughterhouse (INRA, Domaine de Vilvert, Jouy en Josas, F-78352).

Declaration of interest The authors report no declarations of interest

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