Cartilage degradative enzymes in human osteoarthritis: Effect of a nonsteroidal antiinflammatory drug administered orally

Cartilage Degradative Enzymes in Human Osteoarthritis: Effect of a Nonsteroidal Antiinflammatory Drug Administered Orally ByEric INDEX WORDS: enzyme.

Vignon,

Pierre Mathieu,

NSAIDs: osteoarthritis;

Pierre Broquet,

cartilage;

N

ONSTEROIDAL antiinflammatory drugs (NSAIDs) are frequently administered to treat the pain and synovial inflammation of patients with osteoarthritis (OA). There has been some debate concerning the use of NSAIDs in OA because rapidly destructive arthropathy has been reported in patients taking high doses of indomethacin, and some NSAIDs have been shown to suppress proteoglycan synthesis by chondrocytes in vitro.lm4However, the suggested cartilage damage by NSAIDs has not been confirmed by a recent clinical study. By contrast some NSAIDs that are able to inhibit the enzymatic degradation of proteoglycans in vitro and have no adverse effect on matrix biosynthesis are now suggested to have a chondroprotective effect.6‘8 Proteoglycan and collagen degradation in cartilage by stromelysin and collagenase respectively, is now considered a plausible mechanism for OA cartilage destruction.9-‘3 The chondroprotective potential of tiaprofenic acid (TA) has been suggested in some animal experiments and could well be related with the dose dependent suppression of cartilage stromelysin activity observed in vitro.738,14As the in vivo effects of NSAIDs on human OA cartilage remains unknown, the cartilage degradative enzyme activity from patients with hip OA and treated with TA was determined before total joint replacement.

From the Laboratoire d’Exploration Articulaire. Pavilion F, Hbpital Edouard Herriot. Lyon, France. Eric Vignon, MD: Professor, Department of Rheumatology, HGpital Edouard Herriot, Lyon; Pierre Mathieu, MD: Department of Rheumatology, Hbpital Edouard Herriot, Lyon; Pierre Broquet, PhD: lnserm U 189, Lyon; Pierre Louisot, MD: Professor, Department of Biochemistry, LyonSud, Medical School, Oullins; Michel Richard, MD: Professor, Department of Biochemistry, Hapita Edouard Herriot, Lyon. Address reprint requests to Michel Richard, MD, Laboratoire de Biochimie. Hbpital Edouard Herriot, Place d’Arsonvaf. 69437 Lyon Cedex 03, France. 0 1990 by W.B. Saunders Company. 0049-0172/90/I 904/I 006$5.00/O 26

Pierre Louisot,

Paracetamol trols.

Michel Richard

treated patients were used as con-

MATERIALS In

AND

METHODS

Vivo Study

Eighteen patients referred for consideration of hip surgery were selected. The patients had radiographic evidence of hip OA. The severity of pain and disability justified total hip arthroplasty. Each patient gave a medical history and received radiographs of the pelvis, wrists, and knees, erythrocyte sedimentation rate, complete blood count, and an analysis of serum urea, urate, ferritin, calcium, and rheumatoid factor. The selected patients had no evidence of spondyloarthropathy, rheumatoid arthritis, chondrocalcinosis, or rapidly destructive arthropathy. They had no associated serious disease, did not take medication other than NSAIDs or analgesics and had not been treated previously with intraarticular injections of steroids. Patients were treated during 8 weeks with either TA, 600 mg daily or paracetamol, 3 g daily. Consecutive patients were allocated to two treatment groups according to a schedule derived from a table of random numbers. The treatment was discontinued 24 hours before surgery. Characteristics of patients from the two treatment groups are given in Table 1. At the time of surgery, blood samples were collected, centrifuged, and stored at -30°C for TA assay. Femoral heads were immediately collected and rinsed with physiological saline. Specimens of fibrillated or thinned cartilage found close to the region of bone exposure were excised and frozen at -30°C. Care was taken to avoid osteophytic tissues. Specimens of synovium were also taken from the lower pole of the femoral heads.

In Vitro Study The effect of TA and paracetamol on enzyme activities from OA fibrillated cartilage were also studied in vitro using 10 additional human OA femoral heads. The patients (four men, six women, average age 68.2, range 56 to 76 years) did not receive any NSAIDs for at least 8 days before surgery. Cartilage specimens were collected and stored as described above. The TA concentration used, 5 pg/mL, corresponded to a TA synovial fluid level found in vivo two hours after an concentration, 15 oral dose of 300 mg. I5 The paracetamol bg/mL, corresponded to the peak serum level observed after an oral dose of 1 g.16

Assays of TA Assays of TA were performed using high performance liquid chromatography, according to the instructions of Roussel Swindon.”

Assay of Stromelysin

and Collagenuse

Activity.

The assay was based on the digestion of exogenous (3H) labeled human proteoglycan and (3H) bovine type II colla-

Seminars in Arthritis end Rheumatism, Vol 19, No 4, Suppl 1 (February), 1990: pp 26-29

27

NSAID AND OA HUMAN CARTILAGE

Table 1. Characteristics

Symptoms(years) Onset tclsurgery Mean (Ranga)

Age IYears) Mean IRange) Tiaprofenic acid

of Patients Hip DA sup pole

Central

Concentric

61.7

In = 9)

(49-75)

Paracetamol

3/6

7.2 (2-15)

4

3

2

3/6

6 (2-16)

5

3

1

61.2

(n = 9)

(48-87)

gen. Enzyme activity was assayed using P-aminophenylmercurie acetate to activate the latent enzymes.’ Results are expressed as disintegrations per minute per hour of incubation per milligram of cartilage (wet weight).

serum TA was undetectable in 3 cases and low in the remainder (average: 0.53, range: 2.1 to 0.06

RESULTS

DISCUSSION

results of the in vitro study are given in Table 2. The statistical method used was the Student’s paired t-test. The activity of both stromelysin and collagenase was clearly unaffected by paracetamol when compared with untreated control. Tiaprofenic acid did not modify collagenase activity but induced a significant reduction of 37% in stromelysin activity (P < .OOl), when compared with control or paracetamol treated specimens. Table 3 shows the results of the in vivo study. The stromelysin activity of fibrillated cartilage from TA treated patients was significantly reduced by 43% when compared with specimens from paracetamol treated patients (P < .OOl). Cartilage collagenase was similar in both treatment groups. The stromelysin activity of synovium was extremely low, so that the results are expressed as disintegrations per minute for 48 hours of incubation per milligram of tissue. Stromelysin activity was 40% lower in TA treated patients than in the paracetamol group (P < .OO1). Total collagenase activity from synovium was lo-fold lower than in cartilage specimens and was similar in synovial specimens from both treatment groups. Tiaprofenic acid was not detected in the serum of the paracetamol group. In TA treated patients

Osteophyte growth and cartilage destruction, leading to bone exposure and erosion, are the characteristic lesions of OA. Osteophytes are clearly a repair process while cartilage destruction is responsible for the progressive deterioration of OA joints. Cartilage damage is probably the result of an imbalance between the destructive and reparative processes of the tissue. As chondrocytes have been shown to be actively engaged in the synthesis of proteoglycans and collagen at least in the early stages of OA, progressive cartilage destruction probably arises from an excessive catabolism of the matrix.‘7”8 Several biochemical and enzymatic studies indicate that OA tissue destruction is related to the action of catabolic enzymes such as metalloproteases.9-‘3 Nonsteroidal antiinflammatory drugs are widely used in patients with OA for treatment of pain and inflammation, but their effects on the degenerative cartilage process is poorly understood. Nonsteroidal antiinflammatory drugs were unable to block the degradation of proteoglycans by PMN elastase and cathepsin-G in vitro.” However, interleukin-1 stimulation of proteoglycanase in chondrocyte culture was found to be suppressed by diclofenac, piroxicam, and TA, and TA inhibition of proteoglycan degradation in human OA cartilage explants has been reported.6,7 Using in vitro studies, we found that TA, as well as several other NSAIDs, suppress, in a dose dependent fashion, the proteoglycanase activity in human OA cartilage, but do not affect the collagenase activity in the same tissue.E*20 The effect of TA on metalloprotease has been attributed to the inhibition of prostaglandins or cyclic AMP synthesis, but could also be related to other

The

Table 2. Metalloprotease

Activity in Human OA Cartilage.

In Vitro Effect of Tiaprofenic Paracetamol

Stromelysin Control TA Paracetamol lP < 0.001

Acid (6pg/mLl

and

f15pglmL) C0llsgWlsS.S

9,003

* 2.174

26,230

+ 13,862

“5,649

k 1,579

26,341

+ 14,082

9,021

-t 1,858

26,066

+ 12,965

Student’s t test.

Total enzyme activity expressed as dpm/h/mg of cartilage.

PgImL).

VIGNON ET AL

28

Table 3. Metalloprotease Activity in Human OA Cartilage and Synovium: Comparison of Specimens From Patients Treated Orally With Tiaprofenic Acid or Paracetamol Stromelysin Cartilags Tiaprofenic acid (600 mg/d) Paracetamol 13,000 mg/d)

COllagenase Synovium

Cartilage

Synovium

c3.101

+ 404

l8321

+ 1,093

44,903

+ 12,116

3,441

+ 457

5,462

f 703

13,880

+ 1,822

42,495

f 10,404

3,076

+ 450

Total activity expressed as dpm/h/mg of tissue. Stromelysin activity from synovium is expressed after 48 h of incubation. *Student’s t test P < 0.001

reported interactions of NSAIDs with the lipid bilayer of plasma membranes.7,8*21 The present work supports TA in vitro as an effective inhibitor of stromelysin at a concentration readily achieved in the synovial fluid of patients receiving the recommended dose, and shows that paracetamol has no effect on cartilage degradative enzymes. I5 As the synovial fluid concentration of paracetamol was unknown, the drug concentration was interpreted to correspond to the maximum serum level observed.16 The lack of effect of paracetamol on degradative enzymes could possibly be caused by its minimal effect on prostaglandins. To our knowledge this is the first reported study of the in vivo effect of NSAIDs on human OA cartilage catabolism. This in vivo study shows that stromelysin activity is very significantly lower in cartilage from TA treated patients than in the paracetamol group. The low or undetectable TA serum levels observed in TA treated patients is compatible with the short serum half-life and discontinuation of TA 24 hours before blood collection. The nearly 40% reduction in stromelysin activity between TA and paracetamol groups in vivo is similar to the in vitro results, and is observed in cartilage and synovium as well. The very low metalloprotease activity of synovium, when compared with cartilage specimens, is striking but in agreement with the mild OA synovitis. The TA induced suppression of OA synovium stromelysin was not observed previously in patients with rheumatoid

arthritis. ” The discrep ancy can probably be explained by a more complex and important stimulation of degradative enzymes formation and activation in rheumatoid arthritis than in OA. Thus the present findings suggest that TA as well as other NSAIDs shown to have no toxic effect on cartilage matrix biosynthesis probably possess a potential chondroprotective effect in OA.4*22 Assessment of NSAID suppression of progressive cartilage breakdown in human OA has been a difficult task. However, in vivo animal experiments, using the spontaneous model in C57 black mice or surgically induced joint instability, also suggest that some NSAIDs are able to reduce the progression of articular cartilage destruction.23324 SUMMARY

The activity of stromelysin and collagenase was determined in fibrillated human OA cartilage using labeled proteoglycans and type II collagen as substrates. In vitro paracetamol had no effect on metalloprotease whereas TA induced a significant inhibition of stromelysin. In cartilage and synovium from nine patients treated with TA and nine patients treated with paracetamol during 8 weeks before surgery for hip OA, stromelysin activity was significantly lower in the TA than in the paracetamol group. The results suggest that TA has a potential chondroprotective effect in OA.

REFERENCES 1. Mimer JC: Osteoarthritis of the hip and indomethacin. J Bone Joint Surg 54B:752, 1973 2. Solomon L: Drug-induced arthropathy and necrosis of the femoral head. J Bone Joint Surg .55B:246-261,1973 3. McKenzie LS, Horsburgh BA, Ghosh P, et al: Effect of anti-inflammatory drugs on sulphated glycosaminoglycan synthesis in aged human articular cartilage. Ann Rheum Dis 36~369-373, 1977 4. Palsmoski MJ, Brandt KD: Effects of some nonsteroidal anti-inflammatory drugs on proteoglycan metabolism and

organization in canine 23:1010-1020, 1980

articular

cartilage.

Arthritis

Rheum

5. Doherty M, Holt M, Mac Millan P, et al: A reappraisal of analgesic hip. Ann Rheum Dis 45:272-276, 1986 6. Shinmei M, Kiruchi T, Masuma K, et al: Effect of interleukin 1 and anti-inflammatory drugs on the degradation of human articular cartilage. Drugs 35:33-41, 1988 (suppl) 7. Martel-Pelletier action of tiaprofenic

J, Pelletier JP: Molecular basis for the acid on human osteoarthritic cartilage

NSAID AND OA HUMAN CARTILAGE

degradation.

Semin Arthritis

Rheum

29

18:19-26,

1989 (suppl

1) 8. Vignon E, Mathieu P, Louisot P, et al: Phospholipase A, activity in human osteoarthritic cartilage. J Rheumatol 16:35-38, 1989 (suppl 18) 9. Dean DD, Azzo W, Martel-Pelletier J, et al: Levels of metalloproteases and tissue inhibitor of metalloproteases in human osteoarthritic cartilage. J Rheumatol 14:43-44, 1987 (SUPPI) 10. Dodge CR, Poole AR: Immunochemical analysis of type II collagen degradation in human normal, rheumatoid and osteoarthritic cartilages and in explants of bovine articular cartilage cultured with interleukin 1. J Clin Invest 83:647-661, 1989 11. Martel-Pelletier J, Pelletier JP, Cloutier JM, et al: Neutral proteases capable of proteoglycan digesting activity in osteoarthritic and normal human articular cartilage. Arthritis Rheum 27:305-312, 1984 12. Pelletier JP, Martel-Pelletier J, Howell DS, et al: Collagenase and collagenolytic activity in human osteoarthritic cartilage. Arthritis Rheum 26:63-68, 1983 13. Ratcliffe A, Doherty M, Maini RN, et al: Increased concentrations of proteoglycans components in the synovial fluid of patient with acute but not chronic joint disease. Ann Rheum Dis 47:826-832, 1988 14. Burkhardt P, Ghosh I? Laboratory evaluation of antiarthritic drugs as potential chondroprotective agents. Semin Arthritis Rheum 17,2,3-34, 1987 (suppl) 15. Vignon E, Mathieu P, Couprie N, et al: Effects of tiaprofenid acid on interleukin 1, phospholipase A2 activity, prostaglandins, neutral protease and collagenase activity in Rheumatoid synovial fluid. Semin Athritis Rheum 18: 1I- 15, 1989 (suppl 1)

16. Gwilt JR, Robertson A, Goldman L, et al: The absorption characteristics of paracetamol tablets in man. J Pharm Pharmacol15:445-453,1963 17. Makin HJ, Dorfman H, Lippiello L, et al: Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. II Correlation of morphology with biochemical data. J Bone Joint Surg 53A:523-537, 1971 18. Eyre DR, MC Devitt CA, Billingham MEJ, et al: Biosynthesis of collagen and other matrix proteins by articular cartilage in experimental osteoarthritis. Biochem J 183: 823-837, 1980 19. Ghosh P: Therapeutic modulation of cartilage catabolism by nonsteroidal antiinflammatory drugs in arthritis. Semin Arthritis Rheum 18:2-6, 1989 (suppl 1) 20. Richard M, Vignon E, Mathieu P, et al: Effets in vitro des anti-inflammatoires non stbroidiens sur les enzymes de degradation du cartilage humain athrosique. Rev Rheum S&273,1989 21. Abramson SB, Weissmann G: The mechanism of action of non steroidal anti-inflammatory drugs. Arthritis Rheum 32: I-9, 1989 22. Muir H, Carney SL, Hall LG: Effects of tiaprofenic acid and other NSAIDs on proteoglycan metabolism in articular cartilage explants. Drugs 35:15-23, 1988 (suppl 1) 23. Maier R, Wilhelmi G: Preclinical investigations of drug effects in models of osteoarthritis, in Munthe E, Bjelle A (eds): Effects of Drugs on Osteoarthritis. Bern Stuttgart Vienna, Austria, Hans Huber, 1984, pp 69-72 24. Colombo C, Butler M, Hickman L, et al: A new model of osteoarthritis in rabbits. II Evaluation of anti-osteoarthritic effects of selected antirheumatic drugs administered systemically. Arthritis Rheum 26:1132-l 139, 1983