In vivo kinetic study on uptake and distribution of intramuscular tritium-labeled polysulfated glycosaminoglycan in equine body fluid compartments and articular cartilage in an osteochondrial defect model

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IN VIVO KINETIC STUDY ON UPTAKEAND DISTRIBUTIONOF INTRAMUSCULAR TRITIUM-LABELEDPOLYSULFATED GLYCOSAMINOGLYCANIN EQUINE BODY FLUID COMPARTMENTSAND ARTICULAR CARTILAGEIN AN OSTEOCHONDRALDEFECT MODEL Daniel J. Burba, DVM1; Michael A. Collier, DVM Lawrence E. Default, PhD2; Olivia Hanson-Painton, PhD 2 Harold C. Thompson, Jr.3, Claude L. Holder3

SUMMARY The uptake and distribution of intramuscularly (IM) administered tritium-labeled polysulfated glyeosaminoglycan (3H-PSGAG) in serum, synovial fluid, and articular cartilage of eight horses was quantitated, and hyaluronic acid (HA) concentration of the middle carpal joint was evaluated in a pharmacokinetic study. A full-thickness articular cartilage defect, created on the distal articular surface of the left radial carpal bone of each horse served as an osteochondral defect model. 3H-PSGAG (500 mg) was injected IM, between 14 and 35 days after creation of the defects. Scintillation analysis of serum and synovial fluid, collected from both middle carpal joints at specific predetermined times up to 96 hours post-injection, revealed Authors' addresses: Department of Equine Surgery and Medicine, Boren Veterinary Medical Teaching Hospital, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma.1 Present Address: School of Veterinary Medicine, Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA.2Department of Pathology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK.3Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas. Acknowledgements: The authors graciously thank Martha Newman for her outstanding technical assistance and Brian Benjamin for his assistance in surgery and care of the animals. Recognition also goes to Dr. R.C. Wails for the statisticalanalysis and Dr. P. Pansefor his laboratoryanalysis. Supported by a grant from Luitpold Pharmaceuticals, Inc., Shidey, NY

696

mean 3H-PSGAG concentrations peaked at 2 hours postinjection. 3H-PSGAG was detected in cartilage and subchondral bone 96 hours post-injection in samples from all eight horses. There were no statistically significant differences in 3H-PSGAG concentration of synovial fluid or cartilage between cartilage defect and control (fight middle carpal) joints. HA assay of synovial fluid revealed concentrations significantly increased at 24, 48, and 96 hours post-injection in both joints. The concentration nearly doubled 48 hours post-injection. However, no statistically significant differences were found between synovial concentrations of HA in cartilage defect and control joints. 3H-PSGAG administered IM to horses, was distributed in the blood, synovial fluid, and articular cartilage. HA concentrations in synovial fluid increased after IM administration of polysulfated glycosaminoglycan.

INTRODUCTION Polysulfated glycosaminoglycan a (PSGAG) is used in the treatment of osteoarthritis in horses. 1 PSGAG consists of a polymeric chain of alternating units of hexuronic acid and hexosamine with a molecular weight of approximately aAdequan, Luitpold Pharmaceuticals, Inc., Shirley, NY.

J O U R N A L OF E Q U I N E V E T E R I N A R Y SCIENCE

10,000 daltons.1,2 It is chemically related to bepafin and the glycosaminoglycans (GAG's) in articular cartilage. 1 PSGAG a is extracted and purified from bovine tracheal tissue and approved for both intra-articular (IA) or intramuscular (IM) administration in horses.1 PSGAGis considered to have a chondroprotective effect, because of its ability to inhibit proteoglycan and collagen degrading enzymes. In vivo and in vitro studies indicate PSGAG inhibits lysosomal enzymes, neutral protease, stromelysin (a neutral metalloproteinase), and neutral serine proteinase (elastase). a9 Other reports indicate the presence of superoxide radical and interleukin-1 inhibitory properties. 1°,tl Less articular cartilage fibrillation, erosion, chondroeyte death, and greater safranin-O staining for GAG's occurred in horses with chemically induced cartilage damage when PSGAG a was administered IA, compared to control joints not injected with PSGAG. 12 PSGAG is described as having anti-inflammatory and possibly analgesic benefits attributable to decreased prostaglandin F_~ release as an indirect result of inhibition of lysosomal enzyme liberation, s'la A decrease in polymorphonuclear leukoeytes and an increase in lymphocytes in synovial fluid have been observed after IA injection of PSGAG in humans.9'14In vitro studies describe PSGAG as having a stimulatory effect on collagen, proteoglycan, and hyaluronie acid (HA) synthesis, as well. t5-18 Originally, PSGAGa was designed as an IA medication for horses. Transient joint inflammation and associated lameness has been occasionally observed after IA administration. Recent reports have indicated that IA use of PSGAG increased the incidence of sepsis in equine carpal joints injected with subinfective doses of Staphylococcus aureus, z°,21 Subsequently, PSGAG a has been found to be effective when administered IM. TM IA and IM use in human and equine clinical studies have resulted in no significant differences in response, tg'z2 Improvement in range of joint motion and decreasedjoint pain have been reported in humans and animals after IM administration of PSGAG. tf,la,z2'ia There have been anecdotal reports of improved clinical response in horses with non-infectious degenerative joint disease (DJD) following oral or IM administration of various dosages of PSGAG. m Numerous studies have reported the ability of PSGAG achieving therapeutic concentrations in synovial fluid and articular cartilage in humans and laboratory animals after IM administration,is,asa2 However, the uptake and distribution of tritium-labeled PSGAG (aH-PSGAG) administered IM in horses has not been reported previously. The purposes of this study were to determine the systemic uptake and distribution of 3H-PSGAG into equine body fluid compartments and articular cartilage after IM administration and to evaluate the effect of IM PSGAG on the HA concentration in synovial fluid of normal joints and joints with osteochondral defects.

Volume 13, Number 12, 1993

MATERIALS AND METHODS

Horses Eight mature horses (5 mares, 3 geldings, ages 2 to 14 years) with clinically and radiographically normal carpi were used in the study. Creation of Osteochondral Defect Model The horses were sedated with xylazineb (0.22 mg/kg, IV), and anesthesia induced with 5% guaifenesinc (30 to 35 gm) in combination with thiamylal sodiumd (6.6 mg/kg, IV). The horses were intubated, maintained on halothanee- oxygen anesthesia, and placed in dorsal recumbency. The left carpus was aseptically prepared, draped and suspended for arthroscopic surgery. A full thickness cartilage defect (approximately 5 mm x 10 ram) was created using arthroseopic techniqueaa with a 4 mm mortorized arthroburr, f on the distal dorsomedial articular surface of the left radial carpal bone of each horse. No osteochondral defect was made on the fight radial carpal bone, which served as the nonosteochondral defect control (referred to as control throughout the text). Records of articular cartilage surfaces in the fight and left middle carpal joints, both prior to and after creation of defect, were compiled onvideotapeg and computer video discsh via a 4 ram, 25* arthroscopeI and eamera.i Tritium-labeling of the PSGAG PSGAG was tritiated unspecifically in aqueous solution with tritium gas using palladium oxide as catalyst. The 3HPSGAG was purified by chromatography on BiG Gel P30 k with 2 N sodium chloride as eluent. The product was subsequently desalted by chromatography on BiG Gel P2 k with water. The molecular weight (MW) of the 3H-PSGAG was determined chromatographicallywith 2 N sodium chloride of a sample on BiG Gel P30 column, comparing with the elution pattern of MW standard substances. Dimethylene blue chemical determination of position of the peak yielded a MW of 5300 daltons, while radiometric determination yielded 4300 daltons. Electrophoresis of PSGAG and 3H-PSGAG on cellulose acetate foils using 35 mM sodium barbital as buffer showed homogenous spots with the same mobility. 3H-PSGAG (63.5 mg) was dissolved, together with bRompun, Bayvet Division, Cutter Laboratories, Shawnee Mission, KS.

CGecolate,Summit Hill Laboratories, Navesink, NJ. dBio-Tal, Bio-ceutin Laboratories, St. Joseph, MO. eHalothane, Fort Dodge Laboratories, Fort Dodge, ID. fWolf PAC-3000, Richard Wolf Medical Instruments Corp., Rosemont, IL. gMinolta 0. 5' SVHS System, Minoita Corp., Denver, CO. hvideo Roppy System, Hitachi, Elk Grove Village, IL. Ipanoview +, Richard Wolf Medical Instruments Corp. lArthroscopic Video Camera, Dyonics Video Division, Oklahoma City, OK. kBio Red Co., Rockville Centre, NY.

697

Table

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unlabeled PSGAG (4436.5 mg), in physiological saline to a total volume of 9.0 ml. The solution was subsequently sterilized by allowing it to pass through a 0.25 lxm Millex GV filter to yield 8 ampoules of 1.0 mI each, containing 500.0 mg 3H-PSGAG with a specific radioactivity of 1.739 mCi (64.3 MBq) and 3.806 x 10° disintegrations per minute (dpm).

middle carpal joints were disarticulated and the entire distal articular cartilage surface, with 3 to 4 mm of attached subchondral bone, of the left and right radial carpal bones were collected. These samples were sectioned sagittally through the 2nd carpal facet of the articular surface and fixed in 70% ethanol for at least 24 hours.

Drug Injection and Sample Collection The horses were paired and an identical sample collection protocol was followed for each of the 4 pairs (Table 1). Each horse was injected with a single 500 mg dose of 3HPSGAG into the serratus ventralis cervicis muscle (left lateral cervical region), 4 cm cranial to the cranial border of the scapula. The 4 pairs were injected at different times after creation of cartilage defects: pair 4 was injected at 14 days, pair 3 at 21 days, pair 2 at 28 days, and pair 1 at 35 days post defect creation. This detailed documentation of the injection times was made for each pair to allow observation for any possible influence age of cartilage lesion may have on 3HPSGAG concentration in the synovial fluid or articular cartilage. Urine, blood, and synovial fluid (left and right middle carpal joints) were collected into plain glass collection tubes prior to injection (time = 0 hour), and at 2, 4, 8,12, 24, 48, and 96 hours post-injection (Table 1). Synovial fluid (2 ml) was obtained with the animal standing, using the lateral approach. 34The blood samples were allowed to clot at room temperature, and serum was separated by centrifugation. Serum and urine were stored at 4"C and synovial fluid samples at -20"C until analyzed. The horses were monitored daily and handwalked to assess for evidence of joint pain related to the sample collections. Phenylbutazone (4.4 mg/kg PO, q 24 h) was administered if lameness was noted. The horses were sacrificed 96 hours post-injection. The

Scintillation Counting of Tritium in Body Fluids A 200 pl sample of urine, serum, or synovial fluid was added to 8 ml of scintillation fluidI and counted in a liquid scintillation spectrometer, m 3H-m-hexadecanen was used as the counting standard in the calculations of the internal counting efficiency for each fluid type. All data were corrected for background and counting efficiency and depicted as dpm of radioactivity per 200 ~ sample. The levels of radioactivity were converted to concentration of 3HPSGAG per sample and expressed as lxg/ml, using the following formula: (500 mg 3H-PSGAG/3.806xl09 dpm) x (1000 lxg/mg) x (RBF/200 ~tl) x (1000 lxl/ml) = lxg/ml ( R ~ = dpm of radioactivity in body fluid sample)

698

Scintillation Counting of Tritium in the Cartilage and Subchondral Bone Harvested at Necropsy The cartilage from the distal articular surface of the radial carpal bones harvested at necropsy (96 hours post-injection), was separated from the subchondral bone. Three samples of both the cartilage and subchondral bone from each radial carpal bone were then taken and each sample weighed in a gelatin capsule (lock caps size 3). Three capsules each with 100 mg samples were prepared. Each capsule was enveloped in a 2 cm x 2 cm slow match with an overlapping end of 2 cm IScintiverse II, Fisher, Plane, "IX. mTracor Analytic Delta 300, Elk Grove Village, IL. nAmershamTRR.6, Arlington Heights, IL.

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x 0.5 cm, cut out of residue and lignin free filter paper. The capsule was subsequently positioned into a platinum basket fixed to a 29 mm glass stopper of a 500 ml Erlenmeyer flask. The stopper was greased with non-burning silicone. After filling the flask with oxygen, the slow match was lit and the stopper was fixed to the flask by a spring clamp. After the completed combustion, the closed flask was stored for 20 minutes at -20"C. Scintillation cocktail° (18 ml) was added, and the closed flask was shaken vigorously for 2 minutes. After standing and warming to room temperature, the flask was shaken again for 10 seconds. A 15 ml aliquot was transferred to a scintillation counting vial and stored for a minimum of 16 hours at room temperature to allow chemiluminescence to decay. After counting for 5 minutes, the measurement was repeated after adding an internal standard of tritium-water. Background levels of radioactivity were determined by oxidation and counting of nonradioactive control samples. Data was expressed as lxg/gm using the same formula as for the body fluid samples with the exceptions being R representing the dpm of radioactivity per mg of cartilage (Re) or subchondral bone (Rb) and dry measures (mg) used instead of fluid.

Hyaluronic Acid Assay Synovial fluid was assayed for HA by a modiflcationP of a previously described dye-binding method,as A standard curve was prepared as follows: from a stock solution of 2 mg/ ml HA in 0.9% saline, dilutions were made to give 2, 10, 20, and 30 ~tg/tube. One ml of Alcian blue reagent (20 mg/ml in 45 mM sodium acetate, pH 5.8, 50 mM MgCI2) was added, followed by incubation at room temperature for 5 to 30 minutes.Absorbance of transmitted light at the wavelength of 620 nm was measured after dilution with 3 ml of 0.015 M phosphate buffer (pH 7.5), containing 0.15 M NaCI and 0.003 M KCI. Triplicate standards were analyzed and the correlation coefficient for each curve (total of 4 runs) was calculated to greater than 0.999. Internal reference standards of 1 mg/ml HA, 10 mg/ml bovine serum albumin were run in duplicate with each assay and varied no more than 6% from the standard value. Synovial fluid samples were analyzed in duplicate. If duplicates varied by more than 6% from their average, the sample was reanalyzed. Statistical Analysis Concentrations of 3H-PSGAG in the urine, serum, and in the synovial fluid from both the cartilage defect (left) joints and control (right) joints were described by a onecompartment open pharmacokinetic model for IM injections: Y=BokA[exp (kEt) -exp (-kAt)]/(kA-kE), where Y is the given response, t the time (hours) following injection, ka the absorption rate constant, kE the elimination rate constant, and BOa scale parameter. The model parameters were estimated by non-linear, least-squares, regression analysis for each Opicoflour 30, Packard Instruments PPersonal communication, Dr. P. Taw.

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horse and for the mean over all the horses using the NONLIN module of SYSTAT.q However, the area under the concentration-time curve (AUC) in each of these cases was estimated by the non-parametric trapezoidal method,s6 For each of the above responses, on each horse, the observed maximum concentration (CMAX) and the sampling time at which the maximum occurred (TMAX) were recorded as variables characterizing the response. The information provided by each set of 7 concentrations (observed at 2 through 96 hours) was transformed into estimates of 6 parameters: Bo, kA, kE, AUC, CMAX and TMAX. Hyaluronic acid in the synovial fluid was not modeled by the above equation. The AUC was estimated and the maximum concentration and time of maximum concentration were recorded for control and cartilage-defect joints on each horse and the mean over all horses. For any of the measures mentioned, theAUC is taken to be proportional to the average "exposure' of the joint to the substance being measured. The hyaluronic acid AUC was plotted versus each of the six parameter estimates (described above) for urine and for the synovial fluid in each joint to see if any correlations between these parameters and 'average' hyaluronic acid levels might be suggested. Synovial fluid concentrations of 3H-PSGAG and hyaluronic acid over hours 0 through 96 were analyzed by repeated measures of analysis of variance. Tests were made for differences between control and cartilage defect joints at each sampling time. For hyaluronic acid concentrations, the significance of the change from baseline (0 hour sample) was assessed at each sampling time. A separate repeated measures analysis of variance was done for the 96 hour levels of 3H-PSGAG in subchondral bone and cartilage samples. Differences between control and cartilage-defect joints were tested.

RESULTS

Effects of the Study on the Animals None of the horses exhibited any adverse reaction systemicallyor locally as a result of the 3H-PSGAG injection. Mild lameness was noted after performing repeated arthrocenteses. Scintillation of the Body Fluids, Cartilage Samples, and Subchondral Bone Scintillation analysis revealed 3H-PSGAG in the urine, in the serum, and in the synovial fluid of both the cartilage defect and control joints. Of the times urine was collected, the mean concentration of 3H-PSGAG was highest 2 hours postinjection. By 24 hours, most of the drug elimination via the urine occurred (Fig. 1). Mean concentrations of 3H-PSGAG in the serum and synovial fluid peaked 2 hours post drug injection, then rapidly qSYSTAT Inc., 1800 Sherman Blvd., Evanston, IL.

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declined over 24 hours post-injection (Fig. 2). The mean concentrations thereafter remained fairly constant for the remainder of the 96 hour test period. The peak mean concentration in the serum was 1.958 + 0.197 Ixg/ml.The peak mean in the synovial fluid of the cartilage defect joints was 0.331 ± 0.049 Ixg/ml, and 0.248 ± 0.043 lxg/ml in the synovial fluid of the control joints. The difference in the peak mean 3HPSGAG concentrations between cartilage defect and control 700

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Figure 4. Mean hyaluronic acid concentrations in the (right=control; left=cartilage defect) middle carpal joints of eight horses over a 96 hour period after IM administration of 500 mg of 3H-PSGAG.

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joints was not statistically significant. No significant differences in the synovial fluid 3H-PSGAG concentration were found between the cartilage defect and control joints at any sample time. Concentrations of 3H-PSGAG were detected in the cartilage of all eight horses 96 hours post drug injection. The concentration in the cartilage defect joints ranged from 0.204 to 0.456 ttg/gm with a mean of 0.290 ± 0.029 ~g/gm, and ranged from 0.149 to 0.628 I.tg/gm with a mean of 0.303 ± 0.051 ~g/gm in the control joints (Fig. 3). There were no statistically significant differences in the cartilage aHPSGAG concentration between the cartilage defect and control joints.Detectable levels of 3H-PSGAG were also found in the subehon&al bone 96 hours post drug injection (Fig. 3). The mean concentrations in subehondral bone of the cartilage defect and control joints were 0.161 ± 0.018 and 0.144 ± 0.019 ~g/gm, respectively. There were no significant differences in subchondral bone 3H-PSGAG concentration between the cartilage defect and control joints.

Hyaluronic Acid Assay The HA concentration in the cartilage defect and control joints showed an increase 24 hours after administration of3H PSGAG (Fig. 4 ). Hyaluronic acid levels in both the cartilage defect and control joints were significantly (P~).001 and 0.041) different from the pre-injection values (0-hour) at 24, 48, and 96 hours post injection. The mean pre-injeetion HA concentration for the cartilage defect joints was 0.438 + 0.062 mg/ml and 0.476 ± 0.087 mg/ml for the control joints. The mean HA concentrations of the cartilage defect and control joints respectively, 24 hours post-injection were 0.653 ± 0.027 and 0.660 ± 0.072 mg/ml; at 48 hours post-injection were 0.933 ± 0.097 and 0.921 ± 0.113 mg/ml; and at 96 hours postinjection were 0.775 ± 0.111 and 0. 865 ± 0.118 mg/ml. However, there were no significant differences in the HA concentrations between cartilage defect and control joints at any sample time.

DISCUSSION

Scintillation analysis was used to measure the amount of tritium activity in the samples. It was presumed that the tritium was still attached to the PSGAGin this study; work conducted by others, suggests the plausibility of this presumption,ar Concentrations of PSGAG sufficient to inhibit cartilage degrading enzymes in equine DJD have not been established. Concentrations of PSGAG necessary to inhibit certain degrading enzymes (ie, neutral protease, B-glucuronidaso, elastase) and to stimulate proteoglycan biosynthesis have been established in vitro;4,e.ae,aa inhibition of elastase and neutral pretense has been noted with PSGAG concentrations as low as 0.1 ~g/ml,4'e and B-glucuronidase may be inhibited with concentrations of approximately 1 ~tg/ml.39 In vitro studies have shown the stimulatory effects of

Volume 13, Number 12, 1993

PSGAG on matrix synthesis are dose-dependent) 7,4° PSGAG concentrations of 0.3 to 1 Ixg/ml are adequate to stimulate proteoglycan synthesis in diseased articular cartilage, aa Osteoarthritic cartilage appeared more sensitive than normal cartilage to stimulation ofprotcoglycan synthesis by exogenous PSGAG in in vitro studies.ts,iTArecent report however showed that PSGAG concentrations of 50 and 200 I~g/mlexerted no significant effect on proteoglycan synthesis in equine articular explants: 1 3H-PSGAG concentrations in the articular cartilage samples obtained in this study were of the level reported by others (0.1 I~g/ml) to inhibit certain cartilage degrading enzymes. The 3H-PSGAG concentrations in some cartilage samples was also of the levels reported (0.3 lxg/ml) to stimulate proteoglycan synthesis. However, the therapeutic usefulness of PSGAG depends on the progress and severity of cartilage degeneration. Considerable increase in the HA concentration of the synovial fluid occurred during this experiment. This was also observed during treatment of human osteoarthritis patients with PSGAG administered IM. 16 An increase in HA concentration in synovial fluid of cubital joints in lame boars was also observed after administration of PSGAG IM.23This may have been caused by competitive inhibition by PSGAG of the catabolism of HA, and a stimulation of compensatory biosynthesis of high polymeric HA from UDP-Nacetylglucosamine and UDP-glucuronie acid.5, 9 Chondrocytic HA synthesis has been stimulated in vitro by addition of PSGAG to either culture medium16.1a,42 or synovial fluid from osteoarthritis patients.4a It has been suggested that PSGAG amay link molecules of HA, forming complexes of high MW HA.'¢ Concentrations of H-PSGAG in the urine, serum and synovial fluids were adequately described by the proposed pharmacokinetic model. However, no obvious correlations were found between hyaluronic acid levels in the synovial fluid and either the estimated model parameters or any other transformation of the raw 3H-PSGAG concentrations in the urine, serum or synovial fluids. Consequently, no mechanism was suggested for a causal relationship between drug concentrations in body fluids and hyaluronic acid concentrations in the synovial fluids. Nevertheless, this study showed that 3H-PSGAG administered IM to horses diffused to the circulation, was transported into serum, synovial fluid, and was adsorbed by articular cartilage. However, there was no predilection of PSGAG for the carpal joints with articular damage. The data indicated that the dose given in this study does have the effect of increasing hyaluronic acid levels in the synovial fluid.

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glycosaminoglycan polysulfate (GAGPS) in articular cartilage and meniscus of rabbit knee joint. In: Proc 9th Europ Cong Rheumatol 68-80, 1979. 3. May, S.A., Hooke, R.E., and Lees, P. The effect of drugs used in the treatment of osteoarthritis on stromelysin (proteoglycanase) of equine synovial cell origin. Equine Vet J Supp 6 28-31, 1988. 4. Baici, A., Prathima, S., Fehr, K., and Boni, A. Inhibition of human lysosomal elastase from polymorphonuclear leucocytes by a glycosaminoglycan polysulfate (Arteparon). Biochem Pharmacol 29:1723-1727, 1979. 5. Greiling, H. Biochemical investigations of the mode of action of Artaparon. In: Proc 9th Europ Cong Rheumato/11-18, 1979. 6. Hamm, D., Goldman, L., and Jones, E.W. Polysulfated glycosaminoglycan: A new intra-articular treatment for equine lameness. Vet Med/Comp Anim 811-816, 1984. 7. Howell, D.S., Carreno, M.R., Pelletier, J.P., and Muniz, O.E. Articular cartilage breakdown in a lapine model of osteoarthritis. Action of glycosaminoglycan polysulfate ester (GAGPS) on proteoglycan degrading enzyme activity, hexuronate, and cell counts. C/in Orth Re/Res 213:69-76, 1986. 8. Kruze, D., Fehr, K., Menninger, H., et al. Effect of antirheumatic drugs on neutral protease from human leucocyte granules. Z Rheumato135:337-346, 1976. 9. Momburg, M., Stuhlsatz, H.W., Vogeli, H., et al. Changes in the clinical chemistry of synovial fluid after intra-articular injection of glycosaminoglycan polysulfate. Verh Dtsch Ges Rheumato14:383390, 1976. 10. Mcllwraith, C.W. and Vachon, J.A. Review of pathogenesis and treatment of degenerative joint disease. Equine Vet J Supp6 311, 1988. 11. Mcllwraith, C.W. Traumatic arthritis and its treatment in the athletic horse. EquineAth12:1,12-16,18, 1989. 12. Yovich, J.V., Trotter, G.W., Mcllwraith, C.W., and Nordin, R.W. Effects of polysulfated glycosaminoglyoan on chemical and physical defects in equine articular cartilage. Am J Vet Res48:14071414, 1987. 13. Stern, P., Nikulin, A., and Zeger-Vidovic, Z. Pharmacological analysis of a mucopolysaccharide polysulfate. Z Rheumaforsch 27:254-260, 1968. 14. Greiling, H., Gressner, A.M., K]eesiek, K., and Stuhlsatz, H.W. Biochemical studies on the therapy of osteoarthritis. In: Gastpar H, ed. Biology of the Articular Cartilage In Health and Disease; Pro 2nd Munich Syrup Biology of Connective Tissue. New York: Verlag. 459-465, 1980. 15. Adam, M., Krabcova, M., and Musilova, J., et al. Influence of Arteparon on disturbances of collagen and proteoglycan metabolism in osteoarthrotic cartilage. In: Pro 9th Europ Cong Rheumatol 30-38, 1979. 16. Verbruggen, G. and Veys, E.M. Treatment of chronic degenerative joint diseases with a glycosaminoglycan polysulfate. In: Pro 9th Europ Cong Rheumatot 50-67, 1979. 17. Glade, M.J. Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes. Am J Vet Res 51:779785, 1990. 18. Nishikawa, H., Mori, I., and Umemoto, J. Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane. Arch of Biochem & Biophys 240:146-153, 1985. 19. Hamm, D. and Jones, E.W. Intra-articular (IA) and intramuscular (IM) treatment of noninfectious equine arthritis (DJD) with polysulfated glycosaminoglycan (PSGAG). J Equine Vet Sci 8:456-459, 1988. 20. Gustafson, S.8., Mcllwreith, C.W., and Jones, R.L. Comparison of the effect of polysulfated glycosaminoglycan, corticosteroids, and sodium hyaluronate in the potentiation of a subinfective dose of Staphylococcus aureus in the midcarpal joint of horses. Am J Vet Res 50:2014-2017, 1989. 702

21. Gustafson, S.8., Mcllwraith, C.W., Jones, R.L., and DixonWhite, H.E. Further investigations in the potentiation of infection by intraarticular injection of polysulfated glycosaminoglycan and the effect of filtration and intra-articular injection of amikacin. Am J Vet Res 50:2018-2022, 1989. 22. Dettmer, N. The therapeutic affect of glycosaminoglycan polysulfate (Arteparon) in arthrosis with respect to its mode of application (intra-articular or intramuscular). In: Pro 9th Europ Cong Rheumato1132-135, 1979. 23. 8rennan, J.J., Aherne, F.X., and Nakano, T. Effects of glycosaminoglycan polysulfate treatment on soundness, hyaluronic acid content of synovialfluid and proteoglycan aggregate in articular cartilage in boars. Can J Vet Res 51:394-398, 1987. 24. White, G.W. Is oral supplementation of PSGAG a viable method of therapy for equine joint disease? J Equine Vet Sci9:232233, 1989. 25. Gallacr.hi, G., Gachter, A., Dick, W., and Muller, W. Accumulation <~f intramuscularly applied gtycosaminoglycan polysulfate in human articular cartilage. Akt Rheumatol 4:145-151, 1979. 26. Gallacchi, G., and Muller, W. Incorporation of intramuscularly injected glycosaminoglycan polysulfate in human joint cartilage. In: Pro 9th Europ Cong Rheumato199-102, 1979. 27. Hannan, N., Gosh, P., Bellenger, C., and Taylor, T. Systemic administration of glycosaminoglycan polysulphate (Arteparon) provides partial protection of articular cartilage from damage produced by meniscectomy in the canine. J Orthop Res 5:47-59, 1967. 28. Iwata, H. and Kaneko, M. Autoradiographic evidence of glycosaminoglycan polysulfate in articular cartilage in rat. In: Pro 9th Europ Cong Rheumato181-88, 1979. 29. Mueller, W., Panse, P., Brand, S., and Staubi, A. In vivo study of the distribution, affinity to cartilage and metabolism of glycosaminoglycan polysulfate (GAGPS, Arteparon). Zeitschr Rheumato142:355-361, 1983. 30. Mueller, W., Dick, J.W., and Panse, P. Concentrations of glycosaminoglycan polysulfate in serum, in the synovia and in the cartilage of humans after intramuscular injection. Therapiewoche 31:5902-5914, 1981. 31. Panse, P., Meske-Brand, S., and Mueller, W. Distribution, metabolism and analysis of glycosaminoglycan polysulfate (GAGPS, Arteparon). Symp Europ Leag Against Rheum 1984. 32. Panse, P., Zeiller, P., and Sensch, K.H. Distribution and excretion of a glycosaminoglycan polysulfate in rabbits after parental administration. AFzneim-Forsch(DrugRes) 26:2024-2029, 1976. :33. Mcllwraith CW: Diagnostic and SurgicalArthroscopy in the Horse. (2 ed), Lea & Febiger:Philadelphia, pp 22-25, 1990. 34. Kiely, R.G. and McMullan W. Lateral arthrocentesis of the equine carpus. Equine Pract 9:22,24, 1987. 35. Smith, R.E., Gilkerson, E., and Kohatsu, N., et aL Quantitative microanalysis of synovial fluid and articular cartilage glycosaminoglycan Anal Bloc 103:191-200, 1980. 36. Gibaldi, M. and Perrier, D. Pharmacokinetics. (2nd ed), Marcel Dekker:New York, pp 444-449, 1982. 37. Panse, P., Jakab, G., Barabas, K., et al. Pharmacokinetic studies on intra-articularly injected glycosaminoglycan polysulfate (GAGPS, Arteparon ) in rabbits and in man. In: Proceedings 12~ Europ Cong Rheumatol 1991. 38. Kleesiek, K., Olschewski, G., Schafer, N., and Greiling, H. Influence of antiphlogistic and antidegenerative compounds on the biosynthesis of hyaluronate and proteoglycan. Verh Dtsch Ges Rheumatol 7:527-532, 1981. 39. Voss, H., Sensch, K.H., and Panse, P. The effect of sulphated glycosaminoglycans in different virus-host systems as demonstrated by the plaque inhibition test. Zbl Bald Hyg IAbt Orig 229:1-36, 1974. 40. vonder Mark, K. Collagen synthesis in chondrocyte cultures under the influence of Arteparon. In: Pro 9th Europ Cong

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Rheumato139-49, 1979. 41. Caron, J.P., Eberhart, S.W., and Nachreiner, R. Influence of polysulphated glycosaminoglycan on equine articular cartilage in explant culture. Am J Vet Res 52:1622-1625, 1991. 42. Verbruggen, G. and Veys, E.M. Proteoglycan metabolism of connective tissue Cells: An in-vitro technique and its relevance to in-vivo conditions. In: Verbruggen G, Veys EM, eds. Degenerative Joints, Test Tubes, Tissues, Mode~s, Man. Amsterdam Excerpta Med Inter Cong Series 573.11-126, 1980. 43. Rainer, F. and Ribitsch, V. The influence of GAGPS on the viscosity of synovial fluid of activated osteoarthritis. Z Rheumatol 42:229-231, 1983.

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