Adequan: A review for the practicing veterinarian

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A D E Q U A N : A REVIEW FOR T H E P R A C T I C I N G V E T E R I N A R I A N .

Gary W. White, DVM

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INTRODUCTION It has been estimated that 1/3 of all equine lameness is due to non-septic equine joint diseas# t, making osteoarthritis (OA) and degenerative joint disease (DJD) a major cause of monetary loss in the equine IndusUT. Considering that 175,000 horses competed in at least one officially sanctioned race in the United States In 1987" the scope of the problem among racehorses alone is certainly significant in terms of lost time in training, cost of therapy and impaired performance. Much has been added to our knowledge of the etiology of equine DJD and the concept of"use-trauma" has become important In that understanding. Articular cartilage may suffer either direct or indirect damage~. Direct damage (traumatic arthritis) due to mechanical forces which exceed the capacity for cartilage compression resultsin injuryor death of the chondrocytes and directlossof the matrix components. Indircctdamage (synovitis) is due to the effects of enzymes and inflammatory mediators generated in response to softtissueinjury. These mediators gain cntreace intothe synovial fluidand subsequently to the articular cartilageand are a very important part of the etiologyof equine DJD. The equilibrium between synthesis, maintenance and breakdown of matrix components thatexistsin the normal joint= isdisturbedtoward catabolism by theseenzymes and mediators. This "cataboliccrisis''39can resultin a continuous, progressive dcst~ction of the articularcartilagematrix. The articularcartilagematrix is composed ofpmteoglycan (PG) complexes, collagen and water. P G are the basic"ouflding blocks" of articularcartilageand are composed of a proteincore to which many side chains of keratin and chondroitin sulfate (glycosaminoglyceas---GAGs) are attached. These complexes are attached by a link protein to a strand of hyalumnate. The polyeaionic nature of the GAGs resultsin repulsion of adjacent side chains resultingin a %ottle brush" configuration(Figure 1). Water is trapped within these complexes allowing for compressive resiliencyand the cxcheage of products of catabolism from the chondrocytes and substratesneeded forchondrocyte metabolism from the synovial fluid. This exchange is carriedout by a "pumping" action In response to loading and unloading of the cartilagewhich occurs in normal movement. ~ (Figure2). CoUaAuthor's address: LuitpoldPharmaceuticals,Inc. Shirley,N.Y. =Figuresfrom the Jockey Club,AQHA, and U.S. Trotting Club

Volume 8, Number 6, 1988

Figure 1. Articular cartilage matrix components. gcn fibers arc interspersed within the matrix in a pattern which enhances the structure and function of the PG-water complexes, optimiTes tensile strength, and acts to more evenly distribute mechanical forces on the cartilage into the surrounding subchondralbone. Loss ofPG frombe~nning DJD exposes the collagen fibers and chondrocytes to increased mechanical stress. Due to an increased water content in damaged matrix, the normal nutrient

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463

RAC N IG STRESS

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Figure 3. Suggested pathways for cartilage degeneration as it occurs in DJD in t~e high motion joints of the racehorse. ~

exchange capacity and resilience to compression is diminished resulting in a fua_her decrease in the chondrocyte's inherently limited capacity to repair the damage ~. All these mechanisms, if not arrested, resultin acontinuous loss of matrix and a decreased capacity of the articular cartilage to withstand stress. Eventually the collagen network undergoes an irreversible collapse leading to loss of the articular cartilage layer and loss of useful function of the joint (Figure 3). Pathologically these lesions begin as softening and thickening of the cartilage, and progress to partial thickness loss of articular cartilage, fragmenting and full thickness loss of cartilage, and marginal osteophyte formation,u Eventually, sclerosis of the subchondralbone, bony lysis andloss of joint space can be seen. Many types of therapy are employed in the treatment of DJD. Physical, medical and surgical treatments are available. Physical treatments such as rest and surgical treatments such as arthroscopy are of obvious benefit but are not always adequate alone or an acceptable economic alternative. Many systemic and intra-articularmedical therapies are available including steroids, nonsteroidal anti4nflammatory drugs, DMSO, sodium hyaluronateand polysulfated glycosaminoglycan. All these therapies produce beneficial effects when used according to indications. This discussionis limited to polysulfated glycosaminoglycan and its use:in DJD.

PHARMACOLOGY OF POLYSU LFATED GLYCOSAMINOGLYCAN (PSGAG) PSGAG is apolymeric chain of repeating units ofhexosamine and hexuronic acid andis extracted and purified from bovine tracheal tissue. |9 The molecular weight of PSGAG is 10,00012,000 daltons. Its chemical structure is closely related to the GAGs found in articular cartilage matrix and is also related to heparin. PSGAG's affinity for cartilage tissue and its deposition into cartilage, especially damaged cartilage, has been demonstrated

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tritium labeled PSGAG in OA patients the drug was dislributed evenly throughout the entire articular cartilage thickness and detectable levels persisted for up to 12 days after injection.|° It was also noted that the radio-labeleddrug could be detected in the contralateral untreated joint 9 days after injection; indicating that some of the drug passes into the circulation and reaches other joints. Afterparenteral admini.~tration PSGAG is excreted primarfly by the kidneys with only a partial breakdown of the active drug.'° Following IM injection in rabbits the drug is excreted in the urine at arate of 55% in 241iours and60% in48 hours. At48 hours post injection about 1.8% of the drug was excreted in the feces and 1% remained in the blood stream. When injected intraarticularly, laitiated PSGAG that did not attach to the cartilage passed into the bloodstream and was excreted within 48 hours by the kidneys. |° Large scale subacute and chronic toxicity studies have determined the drug to be safe at therapeutic levels. In rats a dose of 10 mg/kg/day injected subcutaneously for 6 months caused no evidence of intoxication.~ The intravenous L.D.50 for dogs was determined to be over 1000 mg/kg.~ Safety of PSGAG given intra-articularly to horses was demonstrated by the lack of adverse effects from weekly injection of 1250 mg into the intercarpal joint for 18 consecutive weeks./7 CLINICAL ACTIVITY

In 1981, Nizotek and White proposed that the ideal antiarthritic drug for the treatment of DJD in the horse would be one that would (1) promote synthesis of cartilage matrix components,(2) retard the catabolic process (3) decrease the inflammation of the synovia (4) restore synovial fluid to normal and (5) relieve pain29 These criteria will be used to examine the pharmacological effects of PSGAG and to determine if the drug does indeed fit these criteria.

Promote Synthesis of Cartilage Matritrlx Components The living component of articular cartilage, the chondmcyte, is responsible for the synthesis, maintenance and degradation of collagen and PG in the normal joinL Thus, the healthy condition of the chondrocytes is a critical factor in normal articular cartilage function. A study of osteoarthritic cartilage demonstrated that PSGAG effected an increased incorporation o f tritium labeled amino acids into PG and collagen./ (Figure 4.) This increase in chondrocyte synthesis in cell cultures was confirmed by other investigators.~.'m A study of the ultrastructare of chondrocytes in rats demonstrated a notable decrease in steroid-induced chondrocyte death rates after PSGAG administration.2 These studies demonstrating a positive contribution by PSGAG to chondroeyte metabolism have been supported by recent studies employing cell cultures of ¢ahritic equine cartilageJ 2

EQUINE VETERINARY SCIENCE

VETERINARY REVIEW Neutral Metalloprotease Activity dpmlmg

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Retard The Catabolic Process A number of catabolic enzymes released by leukocytes, synoviocytes and chondrocytes in responseto injury have been reported to contribute to the destruction of the components of the articular cartilage matrix. These enzymes include lysosomal elastase, cathepsin B1, cathepsin G, hyaluronidase, serine protease and neutral metalloproteinase (stromolysin, proteoglycanase).23~ Inhibition of many o f these enzymes by PSGAG has been demonstrated both in vilro and in vivo. PSGAG has been shown to inhibit the dissociation of PG and hyaluronate by lysosomal enzymes ~+and in vivo studies ofrshbits demonstrated that PSGAG resulted in a decrease loss ofPGs fromosteoarthritic cartilage,z; Other studies have shown PSGAG to be a potent inhibitor of the collagenase activity of cathepsin B P .4~ the proteoglycanase activity of the neutral proteases,~ and the local activity of lysosomal elastase2.s-n The serine proteases and neutral metalloproteinases have been shown to cause PG destructions at very low concentrations. Recent studies confirm PSGAG's ability to inhibit the activity of these enzymes,u (Figure 5.) PSGAGs collagenase inhibition activity was defined recently as an inhibition of activation of the enzyme from its inactive state, t6 The inhibition of neutral metalloproteinase (stromolysin) activity of the various anti-arthritic drugs was recently examined in an equine ca~lage cell culture test system. Only PSGAG showed signifcant inhibition of this enzyme at concentrations readily achievable in the equine joint? 1 (Figure 6.)

Volume 8, Number 6,1988

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Figure 5. PSGAG inhibitionof neutral proteaees. These enzymes have been found to be able to degrade cartilage proteoglycans at neutral pH and very low concentrations. The graphs above show net neutral metalloprotease and serine protease activity of rabbit articular femoral condylar cartilage from animals sacrificed at the end of the 12th and 20th weeks postmeniscectomy. Treatment with PSGAG: 1 mg/kg twice weekly by the intraarticular route. Values are x=SD and n=6 animals for each bar."

Decrease Inflammation of the Synovla Restores Synovlal Fluid to Normal The hyaluronate content of the synovial f u i d is important for normal joint function both as the boundary lubricantofthe soft tissue and as a barrier between the synovial membrane vasculalure and the synovial cavity. In inflammatory conditions the quality and quantity o f synovial fluid hyaluronate is diminished.4 This lowers syaovial fluid viscosity, increases soft tissue friction, and allows leukocytes and their enzymes to enter the synovial fluid and reach the articular caxtilage. An increase in the concentration of hyaluronate and relative viscosity was noted by Momberg et al and postulated to be due to inhibition of hyaluronidase and other enzymes. ~ These findings have been

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IETERINARY REVIE~h reaction. PSGAG has been shown to have anti-coagulanteffects? The sum of these effects combined with the positive effect on hyaluronate metabolism probably accounts for the anti-inflammatory and pain reducing properties of PSGAG.

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conrLrmed by further in vitro studies on synoviocyte cell cultures46 and in vivo studies of OA patients.47,s°(Table 1.) Improvement in synovial fluid viscosity and a decrease in synovial fluid protein (frequently elevated after equine joint injuries)was noted in a clinical study of PSGAG in equine DJDff Decrease I n f l a m m a t i o n of Synolva R e d u c e Pain Concurrent with a decrease in synovial inflammationand a restoration of synovial fluid to a more normal state, one would expect a reduction in pain; especially consideringthat pain due to DJD is highly dependent on soft tissue inflammation.~ One of the most important pain-producingmediators in inflammationis prostaglandinF_,2(PGE2). This mediator contributes to prolonged vasodilation, increased vascular permeability, and a reduction of the threshold of pain receptors.= In addition PGF_,2 also appears to exert a negative effect on PG metabolism.~ PSGAG has been shown to inhibit the synthesis of PGF_~in vitro9and this probably is an important contribution to the drug's ability to reduce pain. The complement and clotting systems are also an integral part of the inflammatory response." In viUo studies have shown that PSGAG has a distinct inhibitory effect on the beginning of the complement reaction.3° Blood coagulation can lead to fibrinolysis and cell lysis and thus contributes to local inflammatory

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PSGAG has been shown in laboratory and clinical studies described here to have a positive effect on the anabolic activity of chondrocytes and an inhibitoryeffect on the catabolic activity and tissue degradative effects of enzymes and inflammatory mediators. Thus the drug should decrease the "catabolic crisis" in an injured joint and aid in restoring the normal anabolic-catabolic equilibriuminthe articular cartilage. Inhibitionof the inflammatory pathways and restoration ofnormalhyaluronatemetabolism contributes to a decrease in soft rissue infl am marion which further decreases the release of catabolic enzymes and results in a decrease in the clinical manifestations of DJD (heat, pain, swelling and lameness). Thus an earlier restoration of function (and range of morion) and its inherent benefits would be expected. These activities are best described by the term chondroprotection. To the equine clinician, chondroprotecrion can be best summarized as protection of the cartilage from the deleterious effects of soft tissue and cartilage injury and the abatement of destruction in a joint that is already becoming degenerative.

A number of animal models of OA have been employed to demonstrate the chondroprotective benefits of PSGAG. In dogs resection of the lateral meniscus and the lateraltibialjointsurface in the stifle was performed to induce OA. 4~The use of PSGAG in this model demonstrated an increased preservation of cartilage and a more normal cartilage structure as compared to the untreated joints. Another study employed the use of a medial meniscectomy to produce OA in the canine stifle.2~ In this model PSGAG was shown to reduce histologic evidence of fibrillation arid to inogase the l ~ concentration in the affected articular cartilage as compared to the untreated dogs. These results indicate a chondmprotective benefit from PSGAG therapy. The chondroprotective effects of PSGAG was also demonstrated in models in which immobilizationwas used to create OA. DJminishmentofthedeleterious effects ofimmobifizationon the rabbitlmeeby controllingPG loss from the articularcartilagewas TABLE1

The effect of PSGAG on hy.aluronate concentration, mean molecular weight, specific viscosity in synovial fluid* increase 2-day increase 4-day Parameter post-injection posfinjection hyaluronate concentration 17°1o 27°1o mean molecular weight . ._ 2% 21% spocmc wscosny

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VETERINARY REVIEW demonstrated biochemically.13 This same animal model was used to demonstrate a marked reduction in the progression of OA by PSGAG as determined physically, histologically and radiographically.49 The induction of OA by means of chemically induced synovitis has also been employed as a model to demonstrate chondroprotection. Intra-articularinjectionof monoiodoacetate (MIA) was used to induce OA inlaboratory animals. This model was used to demonstrate chondroprotection by PSGAG based upon histologic, biochemical and radiographic findings.~ An MIA model was employed recently to induce carpal OA in horses and demonstrate chondroprotection based on gross, histologic, and biochemical l'mdings,s An experimental model of equine OA was created by the injection of Freund's complete adjuvant in the carpal joint. A dose-related response was established for equine carpitis based upon clinical, synovial fluid and radiographic findings.TM Chondroprotection by PSGAG was again demonstrated based on the above parameters. C L I N I C A L U S E O F P S G A G IN H O R S E S

The clinical use of PSGAG in horses was fu'st described in 1966.29 Adequanb was approved for use in the U.S. for the equine carpus in 1984. The dose was established at 250 mg by weekly intra-articular injection for up to 5 weeks. The clinical trials inwhich Adequanwas In'stused on actual clinical cases of DJD in the equine carpus further supported the use of the drug for this indication.~s Cases were selected on the basis of clinical findings and synovial fluid analysis. A total of 109 horses received up to 5 weekly intra-articular injections of 250 nag of Adequan. Heat, pain, swelling, maximumpermissible carpal flexion and lameness were determined at the initiation of the therapy and weekly thereafter. Synovial fluid analysis was performed prior to and at the termination of the treatment. At the initial clinical examination 93% of the horses were lame, 73% had pain on palpation, 87% had carpal swelling, and 92% hadhcatdetectable at the site of the lesion. About 25% were reported to have carpal chip fractures. Seventy horses received all 5 treatments. A significant response in 6 clinical parameters is summarized in Table 2. Seventy-two percent of the horses showed a decrease in synovial fluid protein verses their initial values. Viscosity was increased by an average of 24% in horses receiving all 5 treatments. When the 8 participating clinicians were asked to rate the clinical response in the 109 horses treated, 90.5% (99 horses) were judged to have a good or excellentresponse, 7.3% (8 horses) a fair response and 1.8% (2 horses) a poor response. The only observed side effectwas a transient lameness of less than4 hours in 2 horses. The widespread use of Adequan since its approval (appx. 300,000 doses) has further supported the chondroprotective benefits of the drug. The safety of the drug has also been clearly established; the reported rate of adverse reactions (<0.02%) has been much lower than the published expected reaction rate bLuitpold Pharmaceuticals, Inc. Shirley, N.Y.

Volume 8, Number 6, 1988

TABLE 2

Changes in clinical variables related to treatment with intraarticular PSGAG. 18 Variable improved* not improved* insufficient data* Lameness 90 2 17 Pain on palpation 71 0 38 Pain with flexion 74 0 35 Degree of flexion 54 3 52 Swelling 77 0 32 Heat 75 0 34 * Number of horses

(1.8%). This expected reaction rate (1.8%) is much lower than that of other specific equine intra-articular drugs?3 In addition to its approvedindicationsfor equine carpal joint arthritis, the successful use of Adequan has been reported in numerous otherjoints and for other joint related disorders as well (e.g. OCD, post-surgical, post septic arthritis). These reports point to the need for controlled studies of the dosage and efficacy of Adequan for these indications.19 A recent report describes a dose response study on the intramuscularuse of Adequan in horses and a study in which the effects of a series of intramuscular versus intra-articular injections were compared using both the adjuvant induced carpitis model and clinical cases./9 However, this route of administration has not yet been evaluated and approved by the FDA and further studies on the uptake, distributionand half-life of the drug in vivo in the equine would be helpful in supporting the use of the drug by the intramuscular route. In addition to its use in horses, there have been recent anecdotal reports of the use of Adequan in degenerative joint conditionsin dogs. Although chondroprotectionhas been clearly established in experimental models in the dog,2~,4sno controlled studies to determine the efficacy and dosage of Adequan in dogs have been completed. REFERENCES

1. Adam,M; Krabikova,M; Musilova,J; Pesakova, V: Arzneimittel-Forsch 30:H, 1730, 1980 2. Annefeld, M: Therapiewoche 34, 3476, 1984 3. Argirov,V: Unpublished 1976 4. Balasz,E: Univ. of Mich. Medical Center Journal 255, t968 5. Biachi,A, Fehr, K: IXEurop. Cong. RheumatolEular-Basil 1979 19-26 6. Biachi, A, Salgam, P; Fehr, K; Boni, A: Biochem. Pharmacol 29:12, 1723-1727, 1980 7. Dettmer,N: Z. Rheumaforsch, 25, 122-130, 1966 8. Dustmann,H: Z. Orthop. 112, 1188, 1974 9. Egg, D: PharmacologicalResearch Comm. 15:8 709-717, 1983 10. Ensalides,A; Die Med Welt 23, 733-735, 1972 11. Gallachi,G; Muller, W:/X Europ Cong Rheumatol Eular-Basil 99-102, 1979 12. Glade, M: Personal Comm 1988 13.Golding,J; Ghosh,P: Current Therapeutic Research 34:1,67-80, 1983 14. Greiling,H; Kaneko,M: Arzneimittel-Forsch 23,593-597,1973 15. Greiling,H; Kleesiek,K; Reinards,R: Articular Cartilage Biochem/stryRaven Press, NY, 1986p 195-209 16.Halverson,P; Cheung,H; McCartney,D: Proc49thArthritisand

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Rheumatism 1985 17. Harem,D; Goldman,L; Jones,W: Unpublished 1983 18. Harem,D; Goldman,L; Jones, W: VetMed6, 811-816, 1984 19. H a m , D; Jones, W: XXIII World Vet Cong. Montreal 1987 20. Handley, C; McQuillan, D; Campbell,M; Bolis, S: Articular Cartilage Biochem. Raven Press, NY, 1986p 163-176 21. Hannah,N; Ghosh,P; Bellinger,C; Taylor,T: J. OrthopRes. 5:1, 47-59, 1987 22. Higgins,A; Lees, P: Eq. Vet. J. 16:3, 163-174, 1984 23. Howell, D; Muniz, 0; Carreno, M: Advances in Inflammatory Research 35:6, 994-999, 1984 24. Iwata, H; Kaneko, M:/X Europ. Cong.Rheumatol Eular-Basil 68-80, 1979 25. Kahlben,D: Z. RheumatoL 42:4, 178-184, 1985 26. Kamata,T; Kosa, A; Nobusada,M; Yoshida,T: Kiso to Rinsho 9:2, 241-245, 1975 27. Kruze,K; Feb_r,K; Menninger,H; Boni,A:Z.Rheumato135,337, 1976 28. Kruze,K; Salgam,P; Fehr, K; Boni, A: Prospectives in lnflammat/on MTP Press 1977 361-370 29. Kubitza,G: Tierarztliche Umschau 8, 402, 1966 30. I.x~s, M; Heinz, H: Z. Rheumatol41, 155, 1982 31. May, S; Hooke, R; Lees, P: 8tit. J. Pharmaco193, 281, 1988 32. Menninger,H; Burkhardt,H; Roske, W; Ehlebracht,W; Hering, B; Gurr, E; Mohr, W; Mierau, H: Rheumatol. Int. 1, 73-81 1981 33. Menninger,H; Mohr, W: Therapiewoche 31 2134-2148, 1981 34. Momborg, M; Shuhlsatz, H; Vogeli, H; Voitisek, 0; Eylau, 0;

.,.

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,

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