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Effects of diclofenac, aceclofenac and meloxicam on the metabolism of proteoglycans and hyaluronan in osteoarthritic human cartilage

British Journal of Pharmacology (2000) 131, 1413 ± 1421 ã 2000 Macmil an Publishers Ltd Al rights reserved 0007 ± 1188/00 $15.00 E€ects of diclofenac, aceclofenac and meloxicam on the metabolism of proteoglycans and hyaluronan in osteoarthritic 1Laurent Blot, 1Annette Marcelis, 2Jean-Pierre Devogelaer & *,1,2Daniel-Henri Manicourt 1ICP Christian de Duve Institute of Cellular Pathology, Saint-Luc University Hospital, Catholic University of Louvain in Brussels, Brussels, Belgium and 2Department of Rheumatology, Saint-Luc University Hospital, Catholic University of Louvain in Brussels, Brussels, Belgium 1 Since nonsteroidal anti-in¯ammatory drugs (NSAIDs) may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan and hyaluronan (HA) molecules produced by aceclofenac, diclofenac and meloxicam in human osteoarthritic (OA) cartilage.
2 Explants were sampled from the medial femoral condyle and were classi®ed by use of the Mankin's histological-histochemical grading system. Cartilage specimens exhibited moderate (M) OA in 20 subjects and had severe (S) OA in 20.
3 Cartilage explants were pulsed with [-3H]-glucosamine and chased in the absence or in the presence of 0.3 ± 3 mg ml71 of either aceclofenac, diclofenac or meloxicam. After papain digestion, the labelled chondroitin sulphate ([-3H]-proteoglycans) and [-3H]-HA molecules present in the tissue and media were puri®ed by anion-exchange chromatography.
4 In cartilage with MOA and SOA, the metabolic balance of proteoglycan and HA was una€ected by diclofenac. In contrast, and in a dose-dependent manner, aceclofenac and meloxicam both increased the synthesis of proteoglycans and HA in explants with MOA and SOA; these two NSAIDs also reduced signi®cantly the net loss of [-3H]-proteoglycans and [-3H]-HA molecules from cartilage explants.
5 The data obtained in short-term in vitro cultures indicate that, at the concentrations found in synovial ¯uid, aceclofenac and meloxicam may exert a favourable e€ect on the overall metabolism of proteoglycans and HA in cartilage with MOA and SOA.
British Journal of Pharmacology (2000) 131, 1413 ± 1421 Keywords: Nonsteroidal anti-in¯ammatory drugs; diclofenac; aceclofenac; meloxicam; proteoglycan; hyaluronan; osteoarthritic cartilage Abbreviations: COX, cyclo-oxygenase; DMEM, Dulbecco's modi®ed Eagle's medium; DMSO, dimethylsulphoxide; HA, hyaluronan; HAS, hyaluronan synthase; OH-pro, hydroxyproline; M, moderate; NSAID, nonsteroidal anti- in¯ammatory drug; OA, osteoarthritis; Mol. wt, molecular weight; ODFR, oxygen-derived free radicals; PBS, phosphate-bu€ered saline; PG, prostaglandin; S, severe In the abundant extracellular matrix of articular cartilage, Nonsteroidal anti-in¯ammatory drugs (NSAIDs) are many polyanionic proteoglycan molecules bind non-cova- widely prescribed in patients su€ering from arthritides and lently and with high anity to a single ®lamentous molecule it is the inhibition of cyclo-oxygenase (COX), and hence the of hyaluronan (HA) to form huge multimolecular aggregates inhibition of prostaglandin (PG) production, that accounts at which then become unable to di€use out of the collagenous least in part for the anti-in¯ammatory properties of these meshwork (Hardingham, 1999). It is the presence of the large drugs (Vane, 1971). Two isoforms of COX have been proteoglycan aggregates ®rmly entrapped within the collage- identi®ed thus far: COX-1 which is constitutively expressed nous meshwork that creates a high ®xed charge density and in most tissues and COX-2 which is highly inducible in gives articular cartilage its load bearing properties. Proteo- response to proin¯ammatory cytokines and mitogens (as glycan aggregation also increases dramatically the rheological reviewed in Smith et al., 1996). It is generally believed that properties of proteoglycan molecules and, in so doing, a€ects the bene®cial e€ects of NSAIDs are related to their ability to the dynamic behaviour of cartilage in compression (Hard- inhibit COX-2 whereas the gastrointestinal and renal toxicity ingham et al., 1987). Therefore, any decrease in the cartilage of these drugs results from their inhibition of COX-1 (Vane, concentration of proteoglycans and HA, as occurs in 1994; Warner et al., 1999), a contention that has provided the osteoarthritis (OA), compromises the functional properties basis for the development of highly selective COX-2 of cartilage.
inhibitors. It should be however pointed out that COX-1- derived PGs can contribute to the in¯ammatory response (Gilroy et al., 1998; Wallace et al., 1998) and that COX-2- derived PGs perform physiologically important roles such as *Author for correspondence at: ICP, Tower 75, Floor 4, 75 Avenue Hippocrate, 1200 Brussels, Belgium.
the maintenance of normal renal function (Dinchuk et al., 1995; Morham et al., 1995) and the regulation of the female Effects of NSAIDs on cartilage metabolism reproductive system (Lim et al., 1997). Furthermore, COX-2- several weeks as well as known hereditary or congenital derived PGs have been implicated in the protection of the gastrointestinal tract from injury (Schmassmann et al., 1998; Joint pieces were immediately soaked into sterile phos- Gretzer et al., 1998) and also might have anti-in¯ammatory phate-bu€ered saline (PBS) solution and transported to properties (Gilroy et al., 1999).
culture facilities. Full thickness cartilage was sampled from Although NSAIDs undeniably produce relief of pain and the medial femoral condyle and the cartilage from osteo- improvement of joint mobility in patients su€ering from phytes was avoided. For each individual, three cartilage slices arthritides, the use of these drugs might be detrimental to the were taken at random, ®xed (PBS containing 10% formalin joints since e€ective pain relief could lead to overuse of a and 5% cetylpyridinium chloride) and embedded in paran disabled joint. Further, ex vivo and in vivo studies have prior to slicing. After staining with either Safranin-O, shown that some NSAIDs inhibit the synthesis of cartilage Toluidine blue or hematoxylin and eosin, cartilage slices proteoglycans whereas other do not (Brandt, 1987; Howell et were examined for the severity of the OA disease process al., 1991; Rainsford et al., 1997; Dingle, 1999). This according to Mankin's grading system (Mankin et al., 1971) di€erential e€ect of NSAIDs on cartilage metabolism is most which explores the structure (grade 0 for normality; 1 for relevant to clinical practice since any drug, that suppresses surface irregularities; 2 for pannus and surface irregularities; proteoglycan synthesis and impairs the chondrocyte to repair 3 for clefts to transitional zone; 4 for clefts to radial zone; 5 its damaged extracellular matrix, could potentially accelerate for deeper clefts; and 6 for complete disorganization), the the breakdown of the cartilage tissue. On the other hand, cellularity (grade 0 for normality; 1 for di€use hypercellular- although HA plays a central structural role in the ity; 2 for cloning; and 3 for hypo-cellularity) as well as the supramolecular organization of proteoglycan and, hence on intensity of safranin-O-staining (grade 0 for normality; 1 for the biomechanical properties of articular cartilage, the slight reduction; 2 for moderate reduction; and 3 for severe possible e€ects of NSAIDs on the metabolism of this reduction). Since the subchondral bone was not harvested, glycosaminoglycan has so far focused little investigative the integrity of the tide-mark (grade 0 for normality and 1 for attention (Manicourt et al., 1994).
vascular invasion) was not taken into account.
Aceclofenac is a phenylacetic acid derivative and melox- Mankin's grading system ranged from 2 ± 5 in 20 individuals icam is an acidic enolic derivative that is moderately selective (age range: 51 ± 70 years) who were classi®ed as having moderate for COX-2 (Warner et al., 1999). Although these two recently (M) OA whereas the grading system ranged from 6 ± 9 in the 20 marketed NSAIDs display good ecacy and tolerability in other patients (age range: 52 ± 72) who were classi®ed as having therapy for rheumatic disorders (Hunter et al., 1996; Distel et severe (S) OA. The median age was similar in both groups (63 al., 1996), knowledge of their possible e€ects on the versus 62 years). Since the total amount of cartilage obtained metabolism of articular cartilage is still fragmentary (Rains- from each patient was insucient to conduct pulse and chase ford et al., 1997; Dingle, 1999). We therefore investigated the studies in the presence of di€erent concentrations of NSAIDs, action of the two drugs on the metabolism of newly patients with MOA as well as patients with SOA were randomly synthesized HA and proteoglycan molecules in explant distributed into two subgroups (n=10, each), the pulse studies cultures from human OA cartilage. Results were compared being carried out in one subgroup and the chases studies in the with those obtained with diclofenac, a non-selective COX other subgroup.
inhibitor (Warner et al., 1999).
General culture procedures Tissue specimens obtained from each donor were cut into pieces of 3 ± 6 mg in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with penicillin (5000 i.u. ml71) and streptomycin (5000 mg ml71). The tissue was washed several [-3H]-glucosamine was from Amersham Pharmacia Biotech times with this medium and aspirated free from liquid.
(Roosendaal, The Netherlands). Culture plates and media Cartilage pieces were taken at random, weighted and were from Gibco BRL (Merelbeke, Belgium). Dialysis distributed into the di€erent wells of multiwell culture plates membranes (mol. wt. cut-o€: 3500) were from Spectrum (typically between 30 ± 60 mg tissue - well). Extra pieces were (Los Angeles, CA, U.S.A.). Streptomyces hyaluronidase, not cultured but lyophilized in order to assess the initial chondroitinase ABC, hyaluronan, twice crystallized papain content in collagen, proteoglycans and HA of the cartilage.
and diclofenac were from Sigma-Aldrich (Bornem, Belgium).
Culture medium supplemented with 20% v v71 foetal calf Econo-Pac Q cartridges were from Bio-Rad (Nazareth, serum (culture medium A) was then added to each well and Belgium). Aceclofenac was kindly provided by UCB Pharma the culture plates were incubated for 48 h at 378C.
(Brussels, Belgium) whereas meloxicam (4-hydroxy-2-methyl- For each experiment the cartilage from one individual was N - (5 - methyl - 2 - thiazolyl) - 2H - 1, 2 - benzthiazine- 3- carbox- used and tissue cultures were conducted in triplicate: that is, amide1, 1 dioxide) was a gift from Boehringer Ingelheim for the control culture as well for each NSAID concentration, (Brussels, Belgium). All other reagents were from Merck three cartilage explants were cultured separately. Reported values are the mean of the triplicate cultures.
Sampling and evaluation of cartilage tissue Cartilage was obtained from knee joints of 40 patients After two days of culture, the culture medium was aspirated undergoing arthroplasty for OA. Three weeks before surgery, and the explants were washed three times with 1 ml of NSAIDs were stopped, the patients being allowed to take DMEM. Explants were resuspended in culture medium A paracetamol and/or dextropropoxyphen HCl as needed.
(1 ml ± 50 mg tissue) supplemented with [-3H]-glucosamine Exclusion criteria were infections, articular injection of (50 mCi ml71) (culture medium B). To each well, a solution of steroids within 2 months before surgery, immobilization for NSAID dissolved in dimethylsulphoxide (DMSO) was added British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism (10 ml ml71 culture medium) to achieve the concentrations of the percentage of total incorporated d.p.m. found in the 0.3, 1, and 3 mg ml71. Control cultures received DMSO that medium samples of the 24-h period.
did not contain any NSAID (10 ml ml71). Culture wells were then incubated for 12 h.
Analytical methods Hydroxyproline (OH-pro) was determined by the method of Woessner (1961) and hexuronate by the method of Bitter & After 2 days of culture in medium A, the cartilage pieces were Muir (1962). HA was quanti®ed by a speci®c enzyme-linked aspirated free of medium, washed three times with 1 ml of immunosorbent assay (Li et al., 1989).
DMEM, resuspended in culture medium B (1 ml ± 50 mg tissue) and cultured for 12 h. After pulse labelling, the cartilage pieces were washed with DMEM and resuspended in culture medium A. DMSO alone or the appropriate The statistical signi®cance of the di€erences observed between NSAID dissolved in DMSO was added to each well the group of MOA and the group of SOA was evaluated by (10 ml ml71) as stated above and a non-radioactive chase the Mann ± Whitney U-test whereas, in each group, the period was conducted for 24 h.
signi®cance of the di€erences in HA and proteoglycan metabolism in the presence of di€erent NSAID concentra- Isolation and purification of proteoglycans and tions were evaluated by the Wilcoxon signed rank test. P values 50.05 were considered as statistically signi®cant.
At the end of the pulse labelling and non-radioactive chase periods, the culture media were removed and the cartilage pieces were washed with 0.15 M sodium chloride, 0.05 M sodium acetate, pH 6.0 (bu€er A). Media and corresponding Biochemical characterization of cartilage explants washes were combined. Bovine nasal proteoglycan monomers (500 mg ml71) and HA (10 mg ml71) were added to the Values found for the content of OH-pro, hexuronate and HA mixtures which were then dialysed against bu€er A before in cartilage explants from the two groups are given in being incubated with papain (10 mg ml71) for 24 h at 608C.
In close agreement with previous studies (Mankin et al., Cartilage specimens were lyophilized to obtain their dry 1971; Muir, 1986), the groups with MOA and SOA had a weight and then resuspended in bu€er A. Papain was added similar median content of OH-pro (70.5 versus 66.5 mg mg71 to each vial (0.1 mg ml71) and the tissues were digested for tissue dry weight, respectively; P=0.1718). On the other 24 h at 608C. Digests of cartilage and media were aliquoted.
hand, as reported previously (Mankin et al., 1971) the Samples were subjected either to biochemical determinations median content of hexuronate, and thus of proteoglycan, was or to ion-exchange chromatography on Econo-Pac Q higher in the group with MOA than in the group with SOA cartridge as previously reported (Manicourt et al., 1994).
(41.5 versus 31.5 mg mg71 tissue dry weight, respectively, Typically, two [-3H]-radiolabelled peaks were eluted from P50.0001). The group with MOA also had a higher content the Econo-Pac column: peak A at 0.23 M NaCl and peak B of HA (0.47 versus 0.83 mg mg71 tissue dry weight, at about 1 M NaCl. Fifty to 70 per cent of the material respectively; P50.0001) and this in close agreement with a present in peak A was sensitive to streptomyces hyaluroni- previous report (Manicourt et al., 1994). This overall dase and thus identi®ed to [-3H]-HA whereas the radiola- cartilage chemistry was not signi®cantly a€ected by standard belled material present in peak B was identi®ed as culture conditions and treatment with the NSAIDs examined chondroitin sulphate, and thus as [-3H]-proteoglycan, since over the 72-h period of culture (results not shown).
it was resistant to digestion with streptomyces hyaluronidase and completely digested by chondroitinase ABC.
Metabolic characterization of cartilage explants in the The rates of biosynthesis of HA and proteoglycans were absence of NSAIDs determined by the summation of [-3H]-HA and [-3H]- proteoglycan disintegrations per min. (d.p.m.) found in Since cartilage specimens with MOA and SOA had a similar papain-digested tissues and media at the end of the 12-h OH-pro content and since the loss of OH-pro from tissue pulse period.
specimens into the medium over a 72-h culture period was At the end of the 24-h non-radioactive chase period, the less than 5% of the amount present in cartilage pieces before total incorporation of [-3H]-glucosamine into HA and culture, the rates of proteoglycan and HA biosynthesis were proteoglycans was determined by the summation of [-3H]- expressed as d.p.m. [-3H]-HA and [-3H]-proteoglycan per h HA and [-3H]-proteoglycans d.p.m. found in the media and and per mg OH-pro. Values were distributed over a wide corresponding papain-digested tissue specimens. The radi- range in the two groups upper panels). The group olabelled material that accumulated in the medium during with MOA and the group with SOA had a similar median this non-radioactive chase period represents not only rate of proteoglycan synthesis (P=1). There was also no degraded HA and proteoglycan molecules but also intact statistically signi®cant di€erence in the median rate of HA HA and proteoglycan molecules that were being synthesized synthesis between the two groups (P=0.395).
at the end of the pulse period and that were not incorporated The net loss of [-3H]-proteoglycan and [-3H]-HA molecules into the matrix and lost in the medium during the subsequent from radiolabelled cartilage explants lower panels) chase period. Since the material present in the chase medium was also distributed over a wide range of values in the two was not characterized in the present study, it is dicult to groups. The median loss of [-3H]-proteoglycans was sig- assess the respective proportion of these two processes and, ni®cantly higher in the group with SOA than in the group accordingly, the radiolabelled material recovered in the with MOA (P50.0001). Likewise, the group with SOA had a medium during the 24-h chase period was described as net higher median loss of [-3H]-HA than the group with MOA loss rather than catabolism. This net loss was expressed as British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism Figure 2 Cartilage explants with moderate (M) and severe (S) osteoarthritis (OA) were cultured in the absence of nonsteroidal anti- in¯ammatory drugs. The distribution of values found for the rates of proteoglycan and hyaluronan (HA) synthesis is illustrated in the upper panels whereas the distribution of values found for the net loss of newly synthesized proteoglycan and HA molecules over a 24-h non-radioactive chase period is given in the lower panels. The horizontal line present in each column scatter corresponds to the median value. The Mann ± Whitney U-test disclosed that the groups with MOA and SOA had a similar median rate of proteoglycan synthesis (56 versus 58, respectively; U=43, P=1) as well as a similar median rate of HA synthesis (5.8 versus 5.5, respectively; U=44.5, P=0.395). On the other hand, during the 24-h non-radioactive chase period, the median net loss of labelled proteoglycans from cartilage tissue was higher in the group with SOA (26.5 versus 20.5, Figure 1 Distribution of values found for the hydroxyproline respectively; U=23, P50.0001); the group with SOA also exhibited content, the total hexuronate (and thus proteoglycan) content and a higher median net loss of labelled HA molecules (48.5 versus 38; the total hyaluronan content of cartilage explants with moderate and U=8, P50.0001).
severe osteoarthritis that were cultured in the absence of nonsteroidal anti-in¯ammatory drugs. The horizonal line present in each column scatter corresponds to the median value. The Mann ± Whitney U-test disclosed that the two groups had a similar median content of hydroxyproline (U=149; P=0.1718) but di€ered in their median content of hexuronate (U=34; P50.0001) and in their median content of hyaluronan (U=0; P50.0001).
the concentrations of 1 and 3 mg ml71 or with meloxicam at a concentration of 3 mg ml71 (P=0.002). In each group, the increase in proteoglycan synthesis was always stronger when explants were incubated with aceclofenac than with melox- These wide variations in metabolism that, in each group, icam (P=0.002). Further comparison between explants with were exhibited by the cartilage explants cultured in the MOA and SOA disclosed that the increase in proteoglycan absence of drug were likely to hamper the assessment of the synthesis was signi®cantly higher in explants with less e€ect of NSAIDs on proteoglycan and HA metabolism.
advanced OA lesions at a meloxicam concentration of Therefore, for each cartilage specimen of the two groups, the 3 mg ml71 (P50.001) and at an aceclofenac concentration rates of proteoglycan and HA metabolism that were obtained of 1 mg ml71 (P50.001) and 3 mg ml71 (P50.001).
in the presence of di€erent concentrations of NSAID were In explants with MOA and SOA, the total amounts of divided by the values observed in the absence of drug to yield newly synthesized HA molecules were una€ected by percentage changes.
diclofenac at a concentration of 0.3, 1 and 3 mg ml71 upper right panel) as well as by 0.3 mg ml71 of either Effects of NSAIDs on the total synthesis of proteoglycan aceclofenac middle right panel) or meloxicam lower right panel). On the other hand, at the concentrations of 1 and 3 mg ml71, both aceclofenac and In both groups, diclofenac at the three concentrations tested meloxicam enhanced HA synthesis in a relatively dose- upper left panel) as well as aceclofenac at a dependent manner in the two groups (P=0.002). The concentration of 0.3 mg ml71 middle left panel) and increase in HA synthesis observed at these two concentra- meloxicam at the concentrations of 0.3 and 1 mg ml71 tions was however stronger with aceclofenac than with lower left panel) did not change signi®cantly the total meloxicam (P=0.002) and this in the two groups. No amounts (tissue and medium) of newly synthesized proteo- statistically signi®cant di€erence in the increase of HA glycan molecules. In contrast, a signi®cant increase in synthesis could be disclosed between the two groups at the proteoglycan synthesis was observed when cartilage explants concentrations of 1 and 3 mg ml71 of either aceclofenac or from two groups were incubated with either aceclofenac at British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism Figure 3 E€ects of di€erent concentrations (mg ml71 of diclofenac, aceclofenac and meloxicam on the total amounts (tissue+medium) of newly synthesized proteoglycan and hyaluronan molecules in cartilage explants with moderate (open circles) and severe (closed circles) osteoarthritis. Results are expressed as the negative percentage (%) of values observed in corresponding explants cultured in the absence of drug. The horizontal line present in each column scatter corresponds to the median value. *P: comparison by the Wilcoxon signed-rank test.
Effect of NSAIDs on the relative amounts of newly In the two groups, the relative amounts [-3H]- synthesized proteoglycan and HA molecules incorporated proteoglycan molecules remaining in cartilage matrix were within cartilage matrix una€ected by diclofenac over the range of concentrations studied whereas a signi®cant increase was already observed During the pulse studies conducted in the absence of drug, a with both aceclofenac and meloxicam at a concentration of relative proportion of newly synthesized [-3H]-proteoglycan 1 mg ml71. The increase induced by these two NSAIDs and [-3H]-HA molecules were not incorporated into the matrix became even stronger at a concentration of 3 mg ml71.
and were lost into the culture medium. Further, in both In explants with MOA, the tissue levels of [-3H]- groups with MOA and SOA, this relative loss of radiolabelled proteoglycans were higher in the presence of aceclofenac molecules varied from one donor to another. Therefore, in than in the presence of meloxicam at the concentrations of each experiment, the amount of [-3H]-proteoglycans and 1 mg ml71 (P=0.0195) and 3 mg ml71 (P=0.0137). At the [-3H]-HA (as expressed in d.p.m. h ± 1 mg71 of OH-pro) end of the pulse labelling period of explants with SOA, the found in the explants at the end of the pulse-labelling period specimens bathed with aceclofenac had also a higher tissue conducted in the presence of di€erent concentrations of content in [-3H]-proteoglycans than the specimens exposed to NSAIDs were divided by the amounts observed in the control meloxicam at a concentration of either 1 mg ml71 (P=0.0137) explants pulse-labelled in the absence of drug to yield percent or 3 mg ml71 (P=0.0078).
The changes in the tissue content of [-3H]-HA observed at The changes in the tissue content of [-3H]-proteoglycans each NSAID concentration are shown in In the two obtained at each NSAID concentration are illustrated in groups, 1 mg ml71 of either aceclofenac or meloxicam had British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism Figure 4 E€ects of di€erent concentrations (mg ml71) of diclofenac, aceclofenac and meloxicam on the net loss of labelled proteoglycans from the tissue during the 24-h non-radioactive chase period (closed circles) and on the relative amounts of newly synthesized proteoglycan (open circles) molecules remaining within the matrix at the end of the pulse labelling period in cartilage explants with moderate (left panels) and severe (right panels) osteoarthritis. Results are expressed as the relative percentage (%) of values observed in corresponding explants cultured in the absence of drug. The horizontal line present in each column scatter corresponds to the median value. P: comparison by the Wilcoxon signed-rank test.
already produced a signi®cant increase in the tissue content can molecules and 3 mg ml71 of the two NSAIDs produced a of [-3H]-HA and this increase became even stronger at a further decrease in the net loss of labelled proteoglycans. At concentration of 3 mg ml71. Further, in explants with SOA, each concentration examined, there was no statistically the tissue content in [-3H]-HA observed at the end of the signi®cant di€erence between the e€ect of aceclofenac and pulse labelling period was signi®cantly higher in specimens that of meloxicam; there was also no statistically signi®cant incubated with aceclofenac than in specimens bathed with di€erence between explants with MOA and explants with meloxicam at a concentration of 3 mg ml71 (P=0.0156). On the other hand, over the range of concentrations tested, At the three concentrations tested, diclofenac did not diclofenac did not change signi®cantly the amount of [-3H]- change signi®cantly the net loss of [-3H]-HA molecules from HA molecules remaining within the matrix of cartilage explants of both groups In contrast, 1 mg ml71 of specimens with MOA and SOA.
either aceclofenac or meloxicam inhibited signi®cantly the net Comparison between the group of MOA and the group of loss of labelled HA from explants with MOA and SOA. At a SOA showed that, at the end of the pulse labelling period, the concentration of 3 mg ml71, the two NSAIDs reduced further tissue content in [-3H]-HA molecules was higher in explants the net loss of [-3H]-HA molecules from explants with MOA, with SOA cultured in the presence of aceclofenac and but not from cartilage specimens with more advanced OA meloxicam at the concentrations of 1 and 3 mg ml71 At a concentration of 1 mg ml71, aceclofenac was stronger than meloxicam in inhibiting the net loss of [-3H]-HA from Effects of NSAIDs on the net loss of newly synthesized explants with MOA (P=0.0039) and SOA as well proteoglycan and HA (P=0.0156). Three mg ml71 of aceclofenac exhibited also a stronger inhibition than 3 mg ml71 of meloxicam in the group In both groups, diclofenac did not change signi®cantly the with MOA (P=0.0488) and in the group with SOA net loss of [-3H]-proteoglycan molecules at the three concentrations tested On the other hand, in the Comparison between the two groups disclosed that the two groups, 1 mg ml71 of aceclofenac and meloxicam had e€ect of aceclofenac was stronger in explants with MOA at already signi®cantly reduced the net loss of [-3H]-proteogly- 1 mg ml71 (P50.0001) and 3 mg ml71 (P50.0001); the e€ect British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism Figure 5 E€ects of di€erent concentrations (mg ml71) of diclofenac, aceclofenac and meloxicam on the net loss of labelled hyaluronan from the tissue during the 24-h non-radioactive chase period (closed circles) and on the relative amounts of newly synthesized hyaluronan (open circles) molecules remaining within the matrix at the end of the pulse labelling period in cartilage explants with moderate (left panels) and severe (right panels) osteoarthritis. Results are expressed as the relative percentage (%) of values observed in corresponding explants cultured in the absence of drug. The horizontal line present in each column scatter corresponds to the median value. P: comparison by the Wilcoxon signed-rank test.
of meloxicam was also stronger in explants with MOA, but free) concentrations of these drugs that has been observed in only at the concentration of 3 mg ml71 (P50.0001).
human synovial ¯uid (Bort et al., 1996; Turck et al., 1996). It is however dicult to assess the actual e€ective concentration of drug acting on the tissue since this depends upon various factors such as the pH of the synovial ¯uid, the integrity of the surface of the cartilage, the partition coecient of the The data presented herein are the ®rst to describe the e€ects drug in the articular tissue as well as protein binding. Most of aceclofenac, diclofenac and meloxicam on the metabolism NSAIDs are extensively bound to plasma proteins and our of HA in explants of human OA cartilage.
culture media contained a relatively small proportion (20%) Cartilage composition and metabolism vary widely in the of foetal calf serum. Therefore, it is likely that, at the total di€erent topographical areas of the same joint and between concentration of 3 mg ml71, the concentration of the free the di€erent joints of the same individual (Muir, 1986; drug in our culture media was higher than that present in Holmes et al., 1988). Therefore, the articular tissue was synovial ¯uid bathing cartilage in vivo.
sampled from the same region of the knee joint to restrict Diclofenac una€ected the HA metabolism of OA cartilage, variations in the concentration and metabolism of both whereas, in a dose dependent manner, both aceclofenac and proteoglycan and HA. Further, as cartilage composition and meloxicam were able concomitantly to increase HA synthesis metabolism also change with age, the donors had a very and reduce the loss of newly synthesized HA molecules from similar range of age distribution in both groups, so that the articular tissue. Further, the action of aceclofenac was di€erences observed in response to NSAIDs could be more stronger than that of meloxicam. That these two NSAIDs closely related to the OA disease process rather than ageing.
had a positive e€ect on the metabolic balance of HA is worth The concentrations of the three NSAIDs used in our stressing since the progressive reduction in the HA content of culture system are similar to the range of total (bound and OA cartilage (Manicourt et al., 1988; Rizkalla et al., 1992) is British Journal of Pharmacology vol 131 (7) Effects of NSAIDs on cartilage metabolism likely to contribute, at least in part, to the apparent susceptibility of pathological articular cartilage has already irreversibility of the OA disease process (Pita et al., 1992) been observed in vivo and in vitro with several NSAIDs and further contrasts with the age-related increase in the HA (Brandt, 1987). Although its exact mechanism is unknown at content of normal articular cartilage (Holmes et al., 1988).
present, data in the literature suggest that the uptake of Our knowledge of the synthesis and degradation of HA in NSAID by cartilage is inversely related to the proteoglycan articular cartilage is still fragmentary. Chondrocytes do content of the matrix (Brandt, 1987). Accordingly, any express two di€erent HA synthases (HAS) which are decrease in the concentration of the negatively charged localized to the cell surface, but the enzymatic characteristics proteoglycans, which is proportional to the gravity of the of the two HAS isoforms as well as their mechanisms of OA process, would increase the permeability of the matrix to regulation, including the possible e€ects of NSAIDs, remain the acidically charged NSAID.
to be clari®ed (Hiscock et al., 2000). On the other hand, as The reduction in the net loss of proteoglycans produced by no hyaluronidase has been identi®ed thus far in the cartilage aceclofenac and meloxicam might be, at least in part, related matrix, it has been suggested that the degradation and loss of to the positive e€ect of these drugs on the overall metabolism HA molecules may result from the action of oxygen-derived of HA since any decrease in the HA content of cartilage is free radicals (ODFR) (Ng et al., 1995). Although NSAIDs likely to limit the aggregation of proteoglycans and, in so may block the production of ODFR (Minta & Williams, doing, favours the loss of newly synthesized proteoglycan 1985), it is likely that no single factor accounts for the molecules by di€usion or by proeolytic degradation (Heine- favourable e€ect of aceclofenac and meloxicam on the overall gard & Hascall, 1974). On the other hand, reports have metabolism of HA in the OA cartilage. Therefore, the exact shown that, at concentrations within the therapeutic range, mechanisms of action of the two drugs should be elucidated several NSAIDs inhibit the proteoglycanase and collagenase in further studies as they might be of great biological and activities present in OA cartilage (Vignon et al., 1992; therapeutic signi®cance in OA.
Barracchini et al., 1998). It remains to determine whether Aceclofenac and meloxicam also reduced the loss labelled this suppressive e€ect of NSAIDs on the loss of proteogly- proteoglycan molecules from the articular tissue and both cans is due either to an inhibition of the production of drugs concomitantly enhanced proteoglycan synthesis ODFR (Halliwell, 1995), or to a reduction in the synthesis of whereas diclofenac una€ected the overall metabolism of metalloproteinases (MMPs) and other proteolytic enzymes sulphated glycosaminoglycans. Previous in vitro studies have and/or to a stimulation of the synthesis and secretion of indeed shown that, at concentrations within therapeutic tissue inhibitors of proteolytic enzymes (Poole et al., 1995).
range, the e€ect of NSAIDs on the ability of chondrocytes Recent studies also suggest that NSAIDs could act either as to synthesize proteoglycans may be either stimulatory, reversible enzymatic inhibitors (Barracchini et al., 1998) or by inhibitory or neutral (Brandt, 1987; Dingle, 1999). Therefore, inhibiting COXs which mediate the induction of membrane- it is likely that a mechanism other than COX inhibition type metalloproteinase-1 (MMP-14), an enzyme able to accounts for these marked di€erences in the e€ects of these activate gelatinase A (MMP-2) and collagenase-3 (MMP-13) drugs on proteoglycan synthesis, a contention further (Takahashi et al., 1999).
strengthened by the report that the PG E1 analogue, In conclusion, although it remains to be established misoprostol, does not protect against the suppression of whether changes observed in cartilage metabolism over proteoglycan synthesis caused by NSAIDs (Brandt et al., short-term in vitro cultures would also occur in vivo as a 1991). Inhibition of IL-1 production and consequent result of long-term administration, the results presented expression of growth factor activity have been recently herein show that, in contrast to diclofenac, aceclofenac and, proposed as possible stimulatory mechanisms (Dingle, to a lesser extent, meloxicam at the concentrations found in 1999). On the other hand, some NSAIDs do have toxic synovial ¯uid both exert a favourable e€ect on the overall e€ects on chondrocyte metabolism such as inhibition of metabolism of proteoglycans and HA in OA cartilage.
glucuronyltransferase, an enzyme responsible for the elonga- Accordingly, the two drugs should not hamper the tion of chondroitin sulphate chains on the nascent biomechanical properties of the articular tissue and might proteoglycan molecules (Hugenberg et al., 1993).
delay joint failure in OA. Aceclofenac and meloxicam, That meloxicam enhanced the rate of proteoglycan however, did not normalize the changes in cartilage synthesis contrasts with the report of Rainsford et al.
metabolism seen in the OA tissue.
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