1332
BRIEF REPORT
IN VITRO EFFECT O F NONSTEROIDAL ANTIINFLAMMATORY DRUGS ON PROTEOGLYCANASE AND COLLAGENASE ACTIVITY IN HUMAN OSTEOARTHRITIC CARTILAGE ERIC VIGNON, PIERRE MATHIEU, PIERRE LOUISOT, and MICHEL RICHARD
The effects of several nonsteroidal antiinflammatory drugs, used at concentrationsachievable in synovial fluid, on human osteoarthritic (OA) cartilage metalloprotease activity in vitro was studied. Acetaminophen and ketoprofen had no effect; sodium salicylate, indomethacin, and diclofenac slightly decreased proteoglycanase activity. Piroxicam and tenoxicam suppressed proteoglycanase activity by 48.2% and 68.3%, respectively, and suppressed collagenase activity by 19.1 % and 36.8%, respectively. Use of these NSAIDs may help to decrease cartilage catabolism in patients with OA. Nonsteroidal antiinflammatory drugs (NSAIDs) are commonly used in the treatment of patients with osteoarthritis (OA), but their effects on articular cartilage metabolism and the course of human OA remains a subject of debate. It is believed that some NSAIDs, principally, salicylates and indomethacin, accelerate OA cartilage destruction by impairing proteoglycan synthesis by chondrocytes, while others have a chondroprotective effect (1-5). Theoretically, NSAIDs could help slow down the enhanced cataboFrom the Department of Rheumatology, and the Department of Biochemistry, Edouard Herriot Hospital, and the Department of Biochemistry, Jules Courmont Hospital, Claude Bernard University, Lyon, France. Eric Vignon, MD: Professor, Department of Rheumatology, Edouard Herriot Hospital; Pierre Mathieu, MD: Department of Rheumatology, Edouard Herriot Hospital; Pierre Louisot, MD: Professor, Department of Biochemistry, Claude Bernard University; Michel Richard, MD: Professor of Biochemistry, Edouard Herriot Hospital. Address reprint requests to Michel Richard, MD, Service de Biochimie B, HBpital Edouard Herriot, 69437 Lyon Cedex 03 France. Submitted for publication July 31, 1990; accepted in revised form May 9, 1991. Arthritis and Rheumatism, Vol. 34, No. 10 (October 1991)
lism of the OA cartilage matrix; however, such studies have yielded conflicting data, perhaps reflecting differences in NSAID classes, methodology, or source of cartilage used (3,6-9). The aim of the present study, therefore, was to evaluate in vitro the effects of several classes of NSAIDs on the proteoglycanase and collagenase activity of human OA cartilage. Patients and methods. Ten patients with hip OA were selected for study before undergoing total hip replacement surgery. There were 6 men and 4 women, ranging in age from 54 to 72. All had evidence of primary OA. We carefully evaluated each patient to assure the absence of articular disease such as rheumatoid arthritis, spondylarthropathy, osteonecrosis, Paget’s disease, and pyrophosphate arthropathy. All NSAIDs were stopped 48 hours before surgery. At the time of surgery, the femoral head was obtained and rinsed with physiologic saline. Affected cartilage from the anterior and posterior surfaces of the femoral head, which appeared as a sloping or shelving region adjacent to the eburnated bone, was excised and kept frozen at -80°C until used. Care was taken in cartilage sample selection to avoid including osteophytes, bone, and synovium with the cartilage sample. Cartilage from each femoral head was separately homogenized, using methods reported previously (9). Several aliquots were made from each homogenate obtained from each femoral head, and these were incubated with radiolabeled substrate, with or without (control) either acetaminophen (7.5 pg/ml), indomethacin (0.3 pglml), sodium salicylate (70 pg/ ml), ketoprofen (1.4 pg/ml), diclofenac (0.5 pg/ml), piroxicam (2 pg/ml), or tenoxicam (4 pglml). The NSAID concentration used was derived from levels
BRIEF REPORTS
found in the synovial fluid of patients receiving a normal therapeutic dosage. Metalloprotease activity was determined according to previously reported methods (9,lO).Proteoglycans extracted from normal human cartilage obtained at autopsy from grossly normal femoral heads were 3H-acetylated and incubated with OA cartilage homogenate. After centrifugation, the activity of degraded molecules from the supernatant was determined, and the results were expressed in disintegrations per minute per hour of incubation per milligram of cartilage wet weight. Labeled proteoglycans were chromatographed on Ultrogel Ac A22 and eluted in O.1M phosphate buffer, pH 7, containing 4M guanidine hydrochloride. Fractions were collected and assayed for radioactivity, protein content, and uronic acid content. Chondroitinase ABC treatment (37°C for 90 minutes in phosphate buffered saline, pH 7) of labeled proteoglycans used as substrate induced an almost complete loss of uronic acid in collected fractions, but had no1 effect on radioactivity, which remained closely correlated with protein content. Chromatography of labeled proteoglycans obtained after incubation with cartilage homogenate revealed degradation of the core protein by the presence of 3 new components of lower molec-. ular weight. Collagenase activity was similarly determinedl using 3H-acetylated soluble type I1 collagen as a1 substrate. Using polyacrylamide gel electrophoresis, the labeled collagen molecules degraded by OA cartilage were found to be similar to a pure collagenase obtained commercially (Sigma type VII, obtained from Clostridiurn histolyticurn and substantially free of n o n specific protease and clostripain; Sigma, St. Louis, MO). Pronase digestion demonstrated different products. Spontaneous collagenase activity could be seen on assay, but to identify significant proteoglycanase activity, APMA (17.5 mM) was needed. All measurements were made in duplicate or triplicate and in a blinded manner. Analysis of variance and the Neumann-Keuls test were used for the statistical analyses (11). Results. Table 1 shows the levels of metalloprotease activity in OA cartilage samples treated with the NSAIDs. These activities were clearly unaffected by acetaminophen and ketoprofen treatment compared with control values. Proteoglycanase activity was significantly reduced by all other NSAIDs ( P < 0.05 versus controls). The decrease reached 19.7% with salicylate, 21.2% with diclofenac, 23.8% with indo-
1333 Table 1. Effects of some NSAIDs on metalloprotease activity of human osteoarthritic cartilage in vitro* Proteoglycanase Concentration (dpm/hour/mg cartilage) (dmU Control Acetaminophen Ketoprofen Salicylate Diclofenac lndomethacin Piroxicam Tenoxicam
-
7.5 1.4 70 0.5 0.3 2 4
9,003 f 2,174 9,021 f 1,858 8,984 f 1,877 7,223 f 1,806 7,088 f 1,864 6,856 f 1,582 4,655 f 1,404 2,850 2 1,244
Collagenase (dpm/hour/mg cartilage) 26,230 f 13,662 26,066 f 12,965 26,613 2 13,407 24,717 f 13,086 26,078 2 13,418 25,843 f 13,196 21,205 f 11,099 16,566 f 9,393
* Values are the mean f SD dpm per hour of incubation per mg of cartilage wet weight, cartilage from 10 femoral heads. NSAIDs = nonsteroidal antiinflammatory drugs.
methacin, 48.2% with piroxicam, and 68.3% with tenoxicam. The decrease in proteoglycanase activity was significantly greater with tenoxicam treatment than with piroxicam, and with piroxicam than with the other NSAIDs (P < 0.05). Collagenase activity was found to be unaffected by NSAIDs, oxicams excepted. Piroxicam and tenoxicam significantly suppressed collagenase activity by 19.1%'and 36.8%, respectively (P < 0.05). The difference between tenoxicam and piroxicam was also significant (P < 0.05). The influence of the drug concentration on metalloprotease activity was similarly studied for 3 different NSAIDs using the cartilage from 6 additional OA femoral heads. The results, given in Table 2, confirm that ketoprofen did not affect metalloprotease activity even at a much higher concentration than is Table 2. Influence of NSAID concentration on metalloprotease activity of human osteoarthritic cartilage in vitro*
Concentration (PgW Control Ketoprofen
0.7 1.4 2.8
Indomethacin
0.1
Tenoxicam
0.3 0.6 2 4 8
Proteoglycanase (dpmlhourlmg cartilage)
Collagenase (dpm/hour/mg cartilage)
10,720 f 2,684 10,950 f 3,042 11,423 2 2,522 8,744 2,080 9,774 5 2,921 8,426 +- 1,951 4,895 f 1,643t 5,520 t 2,080t 3,248 f 1,512t 1,742 f 980t
32,140 f 15,722 33,086 f 16,750 31,065 ? 14,880 26,504 f 12,794 31,926 f 16,051 30,315 15,493 25,245 f 12,512 27,440 f 13,224 20,087 f 11,224 10,815 5,008t
*
*
*
* Values are the mean t SD dpm per hour of incubation per mg of cartilage wet weight, cartilage from 6 femoral heads. NSAID = nonsteroidal antiinflammatory drug. t P < 0.05 versus controls, by r-test.
1334 likely to be achieved in vivo (2.8 pg/ml). In contrast, the suppression of metalloprotease activity by indomethacin and by tenoxicam was clearly concentrationdependent. Tenoxicam was much more potent in any case. Discussion. Cartilage catabolism can be evaluated in vitro either by determining glycosaminoglycan release into the culture medium or by measuring metalloprotease activity. The use of a specific substrate and the characterization of the products of degradation allow the determination of specific enzyme activity. The spontaneously active form, rather than the total form, of the enzyme is probably of biological importance. The methods we used allowed the detection of the spontaneously active form of both collagenase and proteoglycanase. However, APMA activation of proteoglycanase was necessary in order to detect significant radioactivity (expressed per hour of incubation). Our findings show that NSAIDs differ with respect to OA cartilage degradative activity. Proteoglycanase activity was unaffected by ketoprofen but was slightly decreased by salicylate, diclofenac, and indomethacin, and markedly suppressed by oxicams, tenoxicam in particular. Oxicams alone had a significant suppressive effect on collagenase activity. The results are consistent with those of other studies using either OA cartilage or interleukin-lstimulated chondrocytes from normal cartilage, in which it was shown that salicylate, tiaprofenic acid, indomethacin, and naproxen decrease proteoglycan catabolism by 20-30% (7,9). In contrast, in other studies using unstimulated chondrocytes from normal cartilage, NSAIDs did not show any effect on proteoglycan catabolism (2,3,6). It is thus possible that some NSAIDs are active on proteoglycan catabolism only when stimulated. Metalloprotease activity is the final result of enzyme synthesis, activation, and inhibition, and the observed suppressive effects of NSAIDs on enzyme activity cannot yet be explained. Inhibition of collagenase activity, only induced by oxicams, could be related to a major suppression of proteoglycanase activity, since the latter is known as a potent activator of the former (12). The role of prostaglandins in cartilage catabolism has been assumed, but the observed lack of effect of ketoprofen, which is a potent inhibitor of prostaglandin formation, seems to exclude such a hypothesis, at least according to our findings (7). Inhibition of superoxide anion generation, which is induced to various degrees by different classes of
BRIEF REPORTS NSAIDs, could explain the present results since superoxide anions may activate latent proteoglycanase and inactivate the tissue inhibitor of metalloprotease (13). NSAIDs have, however, been shown to be active on a variety of enzymes, and many other explanations could be given (14). The in vivo extrapolation of the present in vitro findings may be questioned. In our experiments, NSAIDs were used at concentrations found in the synovial fluid of patients receiving long-term therapy. The synovial, rather than the serum, concentration of NSAID is found in articular cartilage, as previously reported with tenoxicam (15). A study with tiaprofenic acid indicates that inhibition of proteoglycanase activity in OA cartilage was similarly obtained in vitro and in the cartilage of patients treated 8 weeks before surgery (10). It is thus possible that the use of some NSAIDs demonstrated to be potent inhibitors of metalloproteases may help decrease the augmented cartilage catabolism in OA patients. The effect of such a decrease in enzyme activity on cartilage destruction and the course of OA, however, remains to be studied. REFERENCES 1. Rashad S, Revell P, Hemingway A, Low F, Rainsford K, Walker F: Effect of nonsteroidal anti-inflammatory drugs on the course of osteoarthritis. Lancet II:51!%522, 1989 2. Brandt KD: Effects of nonsteroidal anti-inflammatory drugs on chondrocyte metabolism in vitro and in vivo. Am J Med 83 (suppl 5A):29-34, 1987 3. Palmoski MJ, Brandt KD: Effects of some nonsteroidal antiinflammatory drugs on proteoglycan metabolism and organization in canine articular cartilage. Arthritis Rheum 23:lOlO-1020, 1980 4. McKenzie LS, Horsburgh BA, Ghosh P, Taylor TKF: Effect of anti-inflammatory drugs on sulphated glycosaminoglycan synthesis in aged human articular cartilage. Ann Rheum Dis 35:487-497, 1976 5. Burkhardt D, Ghosh P: Laboratory evaluation of antiarthritic drugs as potential chondroprotective agents. Semin Arthritis Rheum 17 (suppl 1):30-34, 1987 6. Herman JH, Appel AM, Khosla RC, Hess EV: The in vitro effect of select classes of nonsteroidal antiinflammatory drugs on normal cartilage metabolism. J Rheumatol 13:1014-1017, 1986 7. Pelletier JP, Cloutier JM, Martel-Pelletier JM: In vitro effect of tiaprofenic acid, sodium salicylate and hydrocortisone on the proteoglycan metabolism of human osteoarthritic cartilage. J Rheumatol 16:64&655, 1989 8. Shinmei M, Kiruchi T, Masuma K: Effect of interleukin
BRIEF REPORTS
9.
10.
11. 12.
1 and anti-inflammatory drugs on the degradation of human articular cartilage. Drugs 35:33-41, 1988 Vignon E, Mathieu P, Louisot P, Vilamitjana J , Harmand MF, Richard M: Phospholipase A, activity in human osteoarthritic cartilage. J Rheumatol 16 (suppl 18):35-38, 1989 Vignon E, Broquet P, Mathieu P, Louisot P, Richard M: Cartilage degradative enzymes in human osteoarthritis: effect of a nonsteroidal anti-inflammatory drug administered orally. Semin Arthritis Rheum 19 (suppl 1):1-4, 1990 Zar JH: Multiple comparisons, Biostatistical Analysis. Englewood Cliffs, NJ, Prentice-Hall, 1974 Murphy G, Cockett MJ, Stephens PE, Smith BJ,
1335
Doherty AJP: Stromelysin is an activator of procollagenase. Biochem J 248:265-268, 1987 13. Minta JO, Williams MD: Some nonsteroidal antiinflammatory drugs inhibit the generation of superoxide anions by activated polymorphs by blocking ligandreceptor interactions. J Rheumatol 12:75 1-757, 1985 14. Abramson SB, Weissmann G: The mechanisms of action of nonsteroidal antiinflammatory drugs. Arthritis Rheilm 32:1-9, 1989 15. Bannwarth B, Netter P, Lapicque F, Gilet B, Eener P, Delagoutte JP, Gaucher A: Tenoxicam distribution in articular tissues, XVIIth International League Against Rheumatism Congress of Rheumatology. Rio de Janeiro, September 17-23, 1989
BRIEF REPORT
IN VITRO EFFECT O F NONSTEROIDAL ANTIINFLAMMATORY DRUGS ON PROTEOGLYCANASE AND COLLAGENASE ACTIVITY IN HUMAN OSTEOARTHRITIC CARTILAGE ERIC VIGNON, PIERRE MATHIEU, PIERRE LOUISOT, and MICHEL RICHARD
The effects of several nonsteroidal antiinflammatory drugs, used at concentrationsachievable in synovial fluid, on human osteoarthritic (OA) cartilage metalloprotease activity in vitro was studied. Acetaminophen and ketoprofen had no effect; sodium salicylate, indomethacin, and diclofenac slightly decreased proteoglycanase activity. Piroxicam and tenoxicam suppressed proteoglycanase activity by 48.2% and 68.3%, respectively, and suppressed collagenase activity by 19.1 % and 36.8%, respectively. Use of these NSAIDs may help to decrease cartilage catabolism in patients with OA. Nonsteroidal antiinflammatory drugs (NSAIDs) are commonly used in the treatment of patients with osteoarthritis (OA), but their effects on articular cartilage metabolism and the course of human OA remains a subject of debate. It is believed that some NSAIDs, principally, salicylates and indomethacin, accelerate OA cartilage destruction by impairing proteoglycan synthesis by chondrocytes, while others have a chondroprotective effect (1-5). Theoretically, NSAIDs could help slow down the enhanced cataboFrom the Department of Rheumatology, and the Department of Biochemistry, Edouard Herriot Hospital, and the Department of Biochemistry, Jules Courmont Hospital, Claude Bernard University, Lyon, France. Eric Vignon, MD: Professor, Department of Rheumatology, Edouard Herriot Hospital; Pierre Mathieu, MD: Department of Rheumatology, Edouard Herriot Hospital; Pierre Louisot, MD: Professor, Department of Biochemistry, Claude Bernard University; Michel Richard, MD: Professor of Biochemistry, Edouard Herriot Hospital. Address reprint requests to Michel Richard, MD, Service de Biochimie B, HBpital Edouard Herriot, 69437 Lyon Cedex 03 France. Submitted for publication July 31, 1990; accepted in revised form May 9, 1991. Arthritis and Rheumatism, Vol. 34, No. 10 (October 1991)
lism of the OA cartilage matrix; however, such studies have yielded conflicting data, perhaps reflecting differences in NSAID classes, methodology, or source of cartilage used (3,6-9). The aim of the present study, therefore, was to evaluate in vitro the effects of several classes of NSAIDs on the proteoglycanase and collagenase activity of human OA cartilage. Patients and methods. Ten patients with hip OA were selected for study before undergoing total hip replacement surgery. There were 6 men and 4 women, ranging in age from 54 to 72. All had evidence of primary OA. We carefully evaluated each patient to assure the absence of articular disease such as rheumatoid arthritis, spondylarthropathy, osteonecrosis, Paget’s disease, and pyrophosphate arthropathy. All NSAIDs were stopped 48 hours before surgery. At the time of surgery, the femoral head was obtained and rinsed with physiologic saline. Affected cartilage from the anterior and posterior surfaces of the femoral head, which appeared as a sloping or shelving region adjacent to the eburnated bone, was excised and kept frozen at -80°C until used. Care was taken in cartilage sample selection to avoid including osteophytes, bone, and synovium with the cartilage sample. Cartilage from each femoral head was separately homogenized, using methods reported previously (9). Several aliquots were made from each homogenate obtained from each femoral head, and these were incubated with radiolabeled substrate, with or without (control) either acetaminophen (7.5 pg/ml), indomethacin (0.3 pglml), sodium salicylate (70 pg/ ml), ketoprofen (1.4 pg/ml), diclofenac (0.5 pg/ml), piroxicam (2 pg/ml), or tenoxicam (4 pglml). The NSAID concentration used was derived from levels
BRIEF REPORTS
found in the synovial fluid of patients receiving a normal therapeutic dosage. Metalloprotease activity was determined according to previously reported methods (9,lO).Proteoglycans extracted from normal human cartilage obtained at autopsy from grossly normal femoral heads were 3H-acetylated and incubated with OA cartilage homogenate. After centrifugation, the activity of degraded molecules from the supernatant was determined, and the results were expressed in disintegrations per minute per hour of incubation per milligram of cartilage wet weight. Labeled proteoglycans were chromatographed on Ultrogel Ac A22 and eluted in O.1M phosphate buffer, pH 7, containing 4M guanidine hydrochloride. Fractions were collected and assayed for radioactivity, protein content, and uronic acid content. Chondroitinase ABC treatment (37°C for 90 minutes in phosphate buffered saline, pH 7) of labeled proteoglycans used as substrate induced an almost complete loss of uronic acid in collected fractions, but had no1 effect on radioactivity, which remained closely correlated with protein content. Chromatography of labeled proteoglycans obtained after incubation with cartilage homogenate revealed degradation of the core protein by the presence of 3 new components of lower molec-. ular weight. Collagenase activity was similarly determinedl using 3H-acetylated soluble type I1 collagen as a1 substrate. Using polyacrylamide gel electrophoresis, the labeled collagen molecules degraded by OA cartilage were found to be similar to a pure collagenase obtained commercially (Sigma type VII, obtained from Clostridiurn histolyticurn and substantially free of n o n specific protease and clostripain; Sigma, St. Louis, MO). Pronase digestion demonstrated different products. Spontaneous collagenase activity could be seen on assay, but to identify significant proteoglycanase activity, APMA (17.5 mM) was needed. All measurements were made in duplicate or triplicate and in a blinded manner. Analysis of variance and the Neumann-Keuls test were used for the statistical analyses (11). Results. Table 1 shows the levels of metalloprotease activity in OA cartilage samples treated with the NSAIDs. These activities were clearly unaffected by acetaminophen and ketoprofen treatment compared with control values. Proteoglycanase activity was significantly reduced by all other NSAIDs ( P < 0.05 versus controls). The decrease reached 19.7% with salicylate, 21.2% with diclofenac, 23.8% with indo-
1333 Table 1. Effects of some NSAIDs on metalloprotease activity of human osteoarthritic cartilage in vitro* Proteoglycanase Concentration (dpm/hour/mg cartilage) (dmU Control Acetaminophen Ketoprofen Salicylate Diclofenac lndomethacin Piroxicam Tenoxicam
-
7.5 1.4 70 0.5 0.3 2 4
9,003 f 2,174 9,021 f 1,858 8,984 f 1,877 7,223 f 1,806 7,088 f 1,864 6,856 f 1,582 4,655 f 1,404 2,850 2 1,244
Collagenase (dpm/hour/mg cartilage) 26,230 f 13,662 26,066 f 12,965 26,613 2 13,407 24,717 f 13,086 26,078 2 13,418 25,843 f 13,196 21,205 f 11,099 16,566 f 9,393
* Values are the mean f SD dpm per hour of incubation per mg of cartilage wet weight, cartilage from 10 femoral heads. NSAIDs = nonsteroidal antiinflammatory drugs.
methacin, 48.2% with piroxicam, and 68.3% with tenoxicam. The decrease in proteoglycanase activity was significantly greater with tenoxicam treatment than with piroxicam, and with piroxicam than with the other NSAIDs (P < 0.05). Collagenase activity was found to be unaffected by NSAIDs, oxicams excepted. Piroxicam and tenoxicam significantly suppressed collagenase activity by 19.1%'and 36.8%, respectively (P < 0.05). The difference between tenoxicam and piroxicam was also significant (P < 0.05). The influence of the drug concentration on metalloprotease activity was similarly studied for 3 different NSAIDs using the cartilage from 6 additional OA femoral heads. The results, given in Table 2, confirm that ketoprofen did not affect metalloprotease activity even at a much higher concentration than is Table 2. Influence of NSAID concentration on metalloprotease activity of human osteoarthritic cartilage in vitro*
Concentration (PgW Control Ketoprofen
0.7 1.4 2.8
Indomethacin
0.1
Tenoxicam
0.3 0.6 2 4 8
Proteoglycanase (dpmlhourlmg cartilage)
Collagenase (dpm/hour/mg cartilage)
10,720 f 2,684 10,950 f 3,042 11,423 2 2,522 8,744 2,080 9,774 5 2,921 8,426 +- 1,951 4,895 f 1,643t 5,520 t 2,080t 3,248 f 1,512t 1,742 f 980t
32,140 f 15,722 33,086 f 16,750 31,065 ? 14,880 26,504 f 12,794 31,926 f 16,051 30,315 15,493 25,245 f 12,512 27,440 f 13,224 20,087 f 11,224 10,815 5,008t
*
*
*
* Values are the mean t SD dpm per hour of incubation per mg of cartilage wet weight, cartilage from 6 femoral heads. NSAID = nonsteroidal antiinflammatory drug. t P < 0.05 versus controls, by r-test.
1334 likely to be achieved in vivo (2.8 pg/ml). In contrast, the suppression of metalloprotease activity by indomethacin and by tenoxicam was clearly concentrationdependent. Tenoxicam was much more potent in any case. Discussion. Cartilage catabolism can be evaluated in vitro either by determining glycosaminoglycan release into the culture medium or by measuring metalloprotease activity. The use of a specific substrate and the characterization of the products of degradation allow the determination of specific enzyme activity. The spontaneously active form, rather than the total form, of the enzyme is probably of biological importance. The methods we used allowed the detection of the spontaneously active form of both collagenase and proteoglycanase. However, APMA activation of proteoglycanase was necessary in order to detect significant radioactivity (expressed per hour of incubation). Our findings show that NSAIDs differ with respect to OA cartilage degradative activity. Proteoglycanase activity was unaffected by ketoprofen but was slightly decreased by salicylate, diclofenac, and indomethacin, and markedly suppressed by oxicams, tenoxicam in particular. Oxicams alone had a significant suppressive effect on collagenase activity. The results are consistent with those of other studies using either OA cartilage or interleukin-lstimulated chondrocytes from normal cartilage, in which it was shown that salicylate, tiaprofenic acid, indomethacin, and naproxen decrease proteoglycan catabolism by 20-30% (7,9). In contrast, in other studies using unstimulated chondrocytes from normal cartilage, NSAIDs did not show any effect on proteoglycan catabolism (2,3,6). It is thus possible that some NSAIDs are active on proteoglycan catabolism only when stimulated. Metalloprotease activity is the final result of enzyme synthesis, activation, and inhibition, and the observed suppressive effects of NSAIDs on enzyme activity cannot yet be explained. Inhibition of collagenase activity, only induced by oxicams, could be related to a major suppression of proteoglycanase activity, since the latter is known as a potent activator of the former (12). The role of prostaglandins in cartilage catabolism has been assumed, but the observed lack of effect of ketoprofen, which is a potent inhibitor of prostaglandin formation, seems to exclude such a hypothesis, at least according to our findings (7). Inhibition of superoxide anion generation, which is induced to various degrees by different classes of
BRIEF REPORTS NSAIDs, could explain the present results since superoxide anions may activate latent proteoglycanase and inactivate the tissue inhibitor of metalloprotease (13). NSAIDs have, however, been shown to be active on a variety of enzymes, and many other explanations could be given (14). The in vivo extrapolation of the present in vitro findings may be questioned. In our experiments, NSAIDs were used at concentrations found in the synovial fluid of patients receiving long-term therapy. The synovial, rather than the serum, concentration of NSAID is found in articular cartilage, as previously reported with tenoxicam (15). A study with tiaprofenic acid indicates that inhibition of proteoglycanase activity in OA cartilage was similarly obtained in vitro and in the cartilage of patients treated 8 weeks before surgery (10). It is thus possible that the use of some NSAIDs demonstrated to be potent inhibitors of metalloproteases may help decrease the augmented cartilage catabolism in OA patients. The effect of such a decrease in enzyme activity on cartilage destruction and the course of OA, however, remains to be studied. REFERENCES 1. Rashad S, Revell P, Hemingway A, Low F, Rainsford K, Walker F: Effect of nonsteroidal anti-inflammatory drugs on the course of osteoarthritis. Lancet II:51!%522, 1989 2. Brandt KD: Effects of nonsteroidal anti-inflammatory drugs on chondrocyte metabolism in vitro and in vivo. Am J Med 83 (suppl 5A):29-34, 1987 3. Palmoski MJ, Brandt KD: Effects of some nonsteroidal antiinflammatory drugs on proteoglycan metabolism and organization in canine articular cartilage. Arthritis Rheum 23:lOlO-1020, 1980 4. McKenzie LS, Horsburgh BA, Ghosh P, Taylor TKF: Effect of anti-inflammatory drugs on sulphated glycosaminoglycan synthesis in aged human articular cartilage. Ann Rheum Dis 35:487-497, 1976 5. Burkhardt D, Ghosh P: Laboratory evaluation of antiarthritic drugs as potential chondroprotective agents. Semin Arthritis Rheum 17 (suppl 1):30-34, 1987 6. Herman JH, Appel AM, Khosla RC, Hess EV: The in vitro effect of select classes of nonsteroidal antiinflammatory drugs on normal cartilage metabolism. J Rheumatol 13:1014-1017, 1986 7. Pelletier JP, Cloutier JM, Martel-Pelletier JM: In vitro effect of tiaprofenic acid, sodium salicylate and hydrocortisone on the proteoglycan metabolism of human osteoarthritic cartilage. J Rheumatol 16:64&655, 1989 8. Shinmei M, Kiruchi T, Masuma K: Effect of interleukin
BRIEF REPORTS
9.
10.
11. 12.
1 and anti-inflammatory drugs on the degradation of human articular cartilage. Drugs 35:33-41, 1988 Vignon E, Mathieu P, Louisot P, Vilamitjana J , Harmand MF, Richard M: Phospholipase A, activity in human osteoarthritic cartilage. J Rheumatol 16 (suppl 18):35-38, 1989 Vignon E, Broquet P, Mathieu P, Louisot P, Richard M: Cartilage degradative enzymes in human osteoarthritis: effect of a nonsteroidal anti-inflammatory drug administered orally. Semin Arthritis Rheum 19 (suppl 1):1-4, 1990 Zar JH: Multiple comparisons, Biostatistical Analysis. Englewood Cliffs, NJ, Prentice-Hall, 1974 Murphy G, Cockett MJ, Stephens PE, Smith BJ,
1335
Doherty AJP: Stromelysin is an activator of procollagenase. Biochem J 248:265-268, 1987 13. Minta JO, Williams MD: Some nonsteroidal antiinflammatory drugs inhibit the generation of superoxide anions by activated polymorphs by blocking ligandreceptor interactions. J Rheumatol 12:75 1-757, 1985 14. Abramson SB, Weissmann G: The mechanisms of action of nonsteroidal antiinflammatory drugs. Arthritis Rheilm 32:1-9, 1989 15. Bannwarth B, Netter P, Lapicque F, Gilet B, Eener P, Delagoutte JP, Gaucher A: Tenoxicam distribution in articular tissues, XVIIth International League Against Rheumatism Congress of Rheumatology. Rio de Janeiro, September 17-23, 1989
