Efficacy of intramuscular polysulfated glycosaminoglycan in a controlled study of equine carpitis

Verde, C., Ferrante, M., Simpson, M. I., Babusci, M., Broglia, G., Landoni I, M. F. Efficacy of intramuscular polysulfated glycosaminoglycan in a controlled study of equine carpitis. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01154.x. Twelve healthy horses were subject to the monoioidoacetate (MIA) carpitis model, which was allowed to develop for 7 days. The horses were then randomly divided into two groups. Group A (control) received an intramuscular injection of normal saline every 4 days for a total of seven injections while group B received 500 mg of a PSGAG (SYNTEX CSY36) intramuscularly every 4 days for seven treatments. Efficacy of the PSGAG was evaluated by three clinical outcomes: lameness score, carpal circumference and maximum carpal flexion. Clinical outcomes were measured on days ‐8 (previous to carpitis induction), 0 (previous to drug treatment), 7, 14, 21, 28 and 35. Areas under the curve clinical outcome as function of time were built and used as variables for the statistical analysis. There was less joint circumference enlargement and lameness and greater carpal flexion in PSGAG‐treated horses compared with that in controls. The studied compound has demonstrated to be efficacious on the treatment of a chemically induced carpitis in horses.     

Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.

The effect of intramuscular polysulfated glycosaminoglycan (PSG) on repair of cartilage injury was evaluated in eight horses. In each horse, one middle carpal joint had both a partial-thickness and a full-thickness articular cartilage defect created. In the contralateral middle carpal joint, chemical articular cartilage injury was created by intra-articular injection of 50 mg sodium monoiodoacetate (MIA). Horses were divided into two groups for treatment. Group 1 horses (control) received an intramuscular injection of normal saline every four days for a total of seven injections starting seven days after cartilage injury. Group 2 horses received 500 mg of PSG intramuscularly every four days for seven treatments starting seven days after cartilage injury. Horses were maintained for 12 weeks. Horses were evaluated clinically, and their middle carpal joints were evaluated radiographically and arthroscopically at the end of the study. Joint tissues were also collected and examined microscopically. The only significant difference between groups was slightly greater matrix staining intensity for glycosaminoglycans in the radiate articular cartilage layer in MIA injected and PSG treated joints. Partial-thickness defects had not healed and the predominant repair tissue in full-thickness defects was fibrous tissue. It was concluded that using this joint injury model, 500 mg PSG administered intramuscularly had no effect on the healing of articular cartilage lesions, and minimal chondroprotective effect from chemically induced articular cartilage degeneration.     

Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 micrograms of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.     

Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage

The effect of intra-articular polysulfated glycosaminoglycan (PSG) on repair of chemical and physical articular cartilage injuries was evaluated in 8 horses. In each horse, a partial- and a full-thickness articular cartilage defect was made on the distal articular surface of the radial carpal bone. In the contralateral middle carpal joint, a chemical articular cartilage injury was induced by injecting 50 mg of Na monoiodoacetate (MIA). Four of the 8 horses were not treated (controls), and 4 horses were treated by intra-articular injection of 250 mg of PSG into both middle carpal joints once a week for 5 treatments starting 1 week after cartilage injury. Horses were maintained for 8 weeks. There was less joint circumference enlargement in PSG-treated horses in MIA-injected and physical defect carpi, compared with that in controls. In MIA-injected joints, there was less articular cartilage fibrillation and erosion, less chondrocyte death, and greater safranin-O staining for glycosaminoglycans in PSG-treated horses. Evaluation of joints in which physical defects were made revealed no differences between control and PSG-injected joints. None of the partial-thickness defects had healed. Full-thickness defects were repaired with fibrous tissue (which was more vascular and cellular in PSG-injected joints) and occasionally small amounts of fibrocartilage. Seemingly, PSG had chondroprotective properties in a model of chemically induced articular cartilage damage, whereas PSG had no obvious effect in a physical articular cartilage-defect model.     

Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes

OBJECTIVE: To investigate effects of the anti-arthritic agents hyaluronan and polysulfated glycosaminoglycan (PSGAG) on inflammatory metabolism in cultured equine synoviocytes. SAMPLE POPULATION: Synoviocytes cultured from samples obtained from the metacarpophalangeal joints of 4 horses. PROCEDURE: Equine synoviocytes were grown in monolayer culture. Synoviocytes were stimulated with lipopolysaccharide (LPS) and simultaneously treated with various concentrations of hyaluronan or PSGAG for 48 hours. Three hyaluronan preparations were compared. Prostaglandin E2 (PGE2) concentrations in culture medium were measured, using radioimmunoassay. RESULTS: The highest concentrations of hyaluronan and PSGAG tested inhibited PGE2 production. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically achievable concentrations of hyaluronan and PSGAG inhibited PGE2 synthesis by cultured equine synoviocytes. This anti-inflammatory action may be a mechanism through which these agents exert anti-arthritic effects. The effect was obtained at concentrations that can be achieved by use of intra-articular, but not systemic, administration of hyaluronan or PSGAG.     

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

The uptake and distribution of intramuscularly (IM) administered tritium-labeled polysulfated glycosaminoglycan (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 mean ³H-PSGAG concentrations peaked at 2 hours post-injection. 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 (right 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.     

Uptake and distribution of tritium-labeled polysulfated glycosaminoglycan in serum, urine, and superficial digital flexor tendon of rabbits after intramuscular administration

OBJECTIVE: To determine concentrations of tritiated polysulfated glycosaminoglycans (3H-PSGAG) in serum, urine, and the superficial digital flexor tendon (SDFT) of rabbits after IM administration and molecular weight of 3H-PSGAG recovered from the SDFT. ANIMALS: Twenty-five 12-week-old New Zealand White rabbits. PROCEDURE: Rabbits were given a single dose of 3H-PSGAG (1.1 mg/kg [70 mCi of specific activity/kg] of body weight) IM. At each of 5 sample times (2, 24, 48, 96, and 192 hours), 5 rabbits were randomly selected and sedated, and blood and urine samples were collected. Rabbits were then euthanatized, and the SDFT were immediately harvested from the hind limbs. Scintillation spectrometry was used to detect concentration of 3H-PSGAG in fluid and tissue samples. Gel-filtration chromatography was used to determine molecular weight of recovered 3H-PSGAG. RESULTS: Mean concentrations of 3H-PSGAG in SDFT, serum, and urine were greatest 2 hours after administration. Tritiated PSGAG could be detected in all samples collected 192 hours after administration. Gel-filtration chromatography confirmed that 3H-PSGAG detected in SDFT samples was high molecular weight PSGAG. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that PSGAG is distributed to the SDFT, serum, and urine after IM administration in rabbits. Further study is needed to determine whether the same is true in horses and to determine what effect, if any, PSGAG has on inflammation of the SDFT.     

Povidone-iodine lavage treatment of experimentally induced equine infectious arthritis

Both tarsocrural joints of 4 horses were inoculated with 1.5 X 10(5) colony-forming units of Staphylococcus aureus. On days 1, 3, and 6, each horse had one tarsocrural joint lavaged with a balanced electrolyte solution and had the contralateral tarsocrural joint lavaged with 0.1% povidone-iodine solution. All horses were orally administered trimethoprim (5 mg/kg)/sufadiazine (25 mg/kg) combination twice daily and phenylbutazone (2 g) once daily for the duration of the study (21 days). On days 0, 1, 3, 6, 9, 14, and 21, synovial fluid specimens were collected and analyzed for color, clarity, total protein concentration, WBC count and differential, and mucin clot-forming ability. Synovial fluid specimens collected on days 1, 3, 6, 9, 14, and 21 were bacteriologically cultured. On day 21, all horses were euthanatized, the tarsocrural joints were opened and examined, synovial membrane specimens were collected, bacteriologically cultured, and histologically evaluated, and articular cartilage specimens were histochemically evaluated. Repeated measures analysis of variance were used to evaluate differences between lavage solutions and among days for objective measurements. A paired t test was used to evaluate differences between solutions for the indices of synovial membrane inflammation and articular cartilage staining intensity with safranin-O-fast green. To be considered significant, the probability of a type-I error was less than 0.05. Significant differences were not found between joints lavaged with electrolyte solution vs povidone-iodine solution for synovial total protein concentration, WBC count, results of synovial fluid and membrane bacteriologic culture, synovial membrane inflammation, or articular cartilage glycosaminoglycan concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of polysulfated glycosaminoglycan on DNA content and proteoglycan metabolism in normal and osteoarthritic canine articular cartilage explants

OBJECTIVE: To study the effect of polysulfated glycosaminoglycan (PSGAG) on proteoglycan metabolism and DNA content of control and osteoarthritic (OA) cartilage. STUDY DESIGN: An in vitro study comparing the effects of PSGAG on articular cartilage explants from canine stifle joints with and without chronic OA after transection of the left cranial cruciate ligament. SAMPLE POPULATION: Five large cross-breed dogs. METHODS: Cartilage explants (6 to 13 per treatment group) from the medial side of the femoral trochlea and medial femoral condyle from both stifles of each dog were incubated in a defined medium containing 0, 0.05, 0.5, or 5 mg/mL of PSGAG. After 72 hours in culture, explants were pulsed for 6 hours with sodium [35S]sulfate. Aminophenylmercuric acetate (APMA) was used to activate endogenous neutral matrix metalloproteinases (MMPs) and induce proteoglycan degradation in the radiolabeled explants. DNA content and radioactivity were measured in papain-digested explants, and radioactivity was measured in the medium by liquid scintillation counting. Proteoglycan synthesis and degradation were calculated. Cartilage was examined histologically for signs of OA. A mixed model analysis of variance and linear contrasts were used to test for significant (P < .05) effects of OA and treatment with PSGAG. RESULTS: Transection of the cranial cruciate ligament produced OA in operated joints. DNA content and proteoglycan synthesis of OA cartilage were significantly lower than in cartilage from control joints. For both DNA content and proteoglycan synthesis, significant interactions occurred between the concentration of PSGAG and whether the articular cartilage was from OA or control joints. The two lower concentrations of PSGAG (0.05 and 0.5 mg/mL) predominantly increased DNA content in OA cartilage (7 and 18%, respectively, compared with 0 mg/mL PSGAG) while the highest concentration (5 mg/mL) predominantly increased DNA content in control cartilage (30% compared with 0 mg/mL PSGAG). PSGAG at .05 mg/mL predominantly decreased proteoglycan synthesis in OA cartilage (19% reduction compared with 0 mg/mL PSGAG) while PSGAG at .5 and 5 mg/mL predominantly decreased proteoglycan synthesis in control cartilage (17 and 55% reduction, respectively, compared with 0 mg/mL PSGAG). Following activation of MMPs, PSGAG caused a dose-dependent decrease in degradation of radiolabeled proteoglycan in both OA and control cartilage. CONCLUSIONS: OA cartilage was responsive to treatment with PSGAG at 100-fold lower concentration than control cartilage. When treated with PSGAG, articular cartilage explants maintained or increased DNA content at the expense of proteoglycan synthesis. Following MMP activation, proteoglycan degradation was inhibited in OA and control explants in a dose-dependent manner. CLINICAL RELEVANCE: If the results of this study extend to in vivo use, treatment with PSGAG may modify the progression of OA in articular cartilage by maintaining chondrocyte viability or stimulating chondrocyte division as well as protecting against extracellular matrix degradation.     

Effects of treatment with polysulfated glycosaminoglycan on serum cartilage oligomeric matrix protein and C-reactive protein concentrations, serum matrix metalloproteinase-2 and -9 activities, and lameness in dogs with osteoarthritis

OBJECTIVE: To investigate the effects of polysulfated glycosaminoglycan (PSGAG) treatment on serum cartilage oligomeric matrix protein (COMP) concentration, matrix metal-loproteinase-2 (MMP-2) and -9 (MMP-9) activities, C-reactive protein (CRP) concentration, and lameness scores in dogs with osteoarthritis. ANIMALS: 16 dogs with osteoarthritis and 5 clinically normal dogs. PROCEDURES: Dogs with osteoarthritis had a history of chronic lameness, and osteophytes were observed on radiographic evaluation of the affected joint. Polysulfated glycosaminoglycan was administered IM twice a week for a total of 8 treatments to all dogs with osteoarthritis and to clinically normal control dogs. RESULTS: Lameness scores after PSGAG treatment in osteoarthritic dogs improved in 12 of the 16 dogs. Serum COMP concentrations in osteoarthritic dogs were significantly higher than in control dogs before treatment. Lameness scores in osteoarthritic dogs decreased significantly after treatment, compared with before treatment. Lameness scores of 9 dogs with hind limb lameness improved significantly after treatment; these dogs had corresponding decreases in serum COMP concentrations. After treatment, serum COMP concentrations and lameness scores of 7 dogs with forelimb lameness remained high and were significantly higher than those of dogs with hind limb lameness. Serum MMP-9 activities of dogs with forelimb lameness were significantly higher than in dogs with hind limb lameness after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: IM administration of PSGAG inhibited COMP degradation in dogs with osteoarthritis. Results indicate that decreases in serum COMP concentrations might be related to improvement in lameness after PSGAG treatment.     

Ceratohyoidectomy for treatment of equine temporohyoid osteoarthopathy (15 cases)

This study investigated 15 horses diagnosed with temporohyoid osteoarthopathy (THO) and treated by ceratohyoidectomy between 2004 and 2012. The presenting complaint, duration and nature of the clinical signs, additional diagnostic procedures, and complications were reviewed retrospectively. Long-term follow-up on horses was used to determine prognosis. All horses were diagnosed by guttural pouch endoscopy. Follow-up was available for 14 horses that survived to discharge. Eight of 10 horses that were used athletically prior to surgery returned to previous levels of use. Persisting clinical signs included mild facial nerve paralysis (3/14; 21.4%) or head tilt (6/14; 42.8%) but these were not functionally limiting. It was concluded that equine THO affects a wide range of breeds, disciplines, and ages of horses, and has a variety of presenting clinical signs most commonly associated with vestibular and facial nerves. Prognosis following ceratohyoidectomy is good for resolution of ataxia but some cranial nerve deficits may persist.     

Immunologic relationships between equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus)

The specificity of selected immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) was examined in 3 colostrum-deprived specific-pathogen-free foals. Single foals were vaccinated with inactivated EHV-1, inactivated EHV-4, or control cell lysate plus adjuvant followed by successive intranasal challenge exposures with EHV-1 and EHV-4 or with EHV-4 and EHV-1. Vaccination with inactivated virus preparations elicited cellular immune responses and antibody which were augmented by subsequent challenge exposures. Cellular immune responses, as measured by in vitro lymphocyte blastogenesis, were cross-reactive after foals were given either EHV-1 or EHV-4. Serum virus-neutralizing antibody responses were type-specific for foals given EHV-1, but were cross-reactive after EHV-4 administrations. It was concluded that diseases caused by EHV-1 and EHV-4 may be more effectively controlled with a bivalent vaccine containing both EHV-1 and EHV-4 than with the presently used monovalent vaccines based on EHV-1 alone.     

Theiler's disease associated with administration of tetanus antitoxin contaminated with nonprimate (equine) hepacivirus and equine parvovirus-hepatitis virus

An 11-year-old Trakehner gelding was presented for evaluation of lethargy, decreased appetite, mild icterus, and elevated hepatic enzyme activities. Physical examination, serum chemistry results, and liver biopsy histopathologic findings were supportive of Theiler's disease. Polymerase chain reaction (PCR) testing results of serum and liver tissue were positive for nonprimate (equine) hepacivirus (NPHV) and a novel equine parvovirus-hepatitis virus (EqPV-H). PCR testing of the lot of tetanus antitoxin administered to the gelding 3 months previously also yielded positive results for NPHV and EqPV-H. Treatment included supportive care and clinical signs resolved within 1 week, although hepatic enzyme activities remained elevated for several months. The horse successfully returned to work as a hunter/jumper for about 1 year until it developed a forelimb lameness and progressive atrophy of shoulder musculature (sweeney), prompting a decision for euthanasia 20 months after initial evaluation. Serial PCR testing of serum revealed persistent infection with both NPHV and EqPV-H and necropsy examination revealed chronic active hepatitis, mild liver atrophy, and positive PCR results for NPHV and EqPV-H in liver tissue. This case highlights the possible risk of administering potentially contaminated biologics of equine origin and the importance of screening for recently identified hepatic viruses in donors from which blood products are prepared.