K.A. Merritt

Study of intragastric administration of doxycycline: pharmacokinetics including body fluid, endometrial and minimum inhibitory concentrations

The objectives of this study were to determine the pharmacokinetics and tissue concentrations of doxycycline after repeated intragastric administration, and to determine the minimum inhibitory concentrations (MIC) for equine pathogenic bacteria. In experiment 1, 2 mares received a single intragastric dose of doxycycline hyclate (3 mg/kg bwt). Mean peak serum concentration was 0.22 microg/ml 1 h postadministration. In experiment 2, 5 doses of doxycycline hyclate (10 mg/kg bwt), dissolved in water, were administered to each of 6 mares via nasogastric tube at 12 h intervals. The mean +/- s.e. peak serum doxycycline concentration was 0.32+/-0.16 microg/ml 1 h after the first dose and 0.42+/-0.05 microg/ml 2 h after the fifth dose. The mean trough serum concentrations were > 0.16 microg/ml. Highest mean synovial concentration was 0.46+/-0.13 microg/ml and highest mean peritoneal concentration was 0.43+/-0.07 microg/ml, both 2 h after the fifth dose. Highest urine concentration was mean +/- s.e. 145+/-25.4 microg/ml 2 h after the last dose. Highest endometrial concentration was mean +/- s.e. 1.30+/-0.36 microg/ml 3 h after the fifth dose. Doxycycline was not detected in any of the CSF samples. Mean +/- s.e. Vd(area) was 25.3+/-5.0 l/kg and mean t1/2 was 8.7+/-1.6 h. In experiment 3, minimum inhibitory concentrations of doxycycline were determined for 168 equine bacterial culture specimens. The MIC90 was < or = 1.0 microg/ml for Streptococcus zooepidemicus and 0.25 microg/ml for Staphylococcus aureus. Based on drug concentrations achieved in the serum, synovial and peritoneal fluids and endometrial tissues and MIC values determined in the present study, doxycycline at a dose of 10 mg/kg bwt per os every 12 h may be appropriate for the treatment of infections caused by susceptible (MIC < 0.25 microg/ml) gram-positive organisms in horses.

Associations of horse age, joint type, and osteochondral injury with serum and synovial fluid concentrations of type II collagen biomarkers in Thoroughbreds.

OBJECTIVE To determine the effects of horse age, osteochondral injury, and joint type on a synthesis biomarker and 3 degradative biomarkers of type II collagen in Thoroughbreds. ANIMALS Healthy rested adult (3- to 12-year-old) Thoroughbreds (n = 19), yearling (1- to 2-year-old) Thoroughbreds (40), and Thoroughbred racehorses (2 to 7 years old) undergoing arthroscopic surgery for removal of osteochondral fragments that resulted from training or racing (41). PROCEDURES Samples of blood and metacarpophalangeal, metatarsophalangeal, or carpal joint synovial fluid (SF) were collected from all horses. Commercially available assays were used to analyze SF and serum concentrations of type II collagen biomarkers of synthesis (carboxy propeptide of type II collagen [CPII]) and degradation (cross-linked C-telopeptide fragments of type II collagen [CTX II], neoepitope generated by collagenase cleavage of type I and II collagen [C1,2C], and neoepitope generated by collagenase cleavage of type II collagen [C2C]). RESULTS Osteochondral injury affected concentrations of CPII, CTX II, C1,2C, and C2C in SF, serum, or both, compared with concentrations in healthy adult horses. Compared with adult horses, yearling horses had increased SF or serum concentrations of degradative biomarkers (CTX II, C1,2C, and C2C). Concentrations were higher in carpal than metacarpophalangeal or metatarsophalangeal joints for all biomarkers in osteochondral-injured horses. Variable differences in SF concentrations between joint types were detected in healthy adult and yearling horses. CONCLUSIONS AND CLINICAL RELEVANCE Horse age, osteochondral injury, and joint type all significantly affected type II collagen biomarker concentrations in SF and serum of Thoroughbreds.

High-mobility group box chromosomal protein 1 as a potential inflammatory biomarker of joint injury in Thoroughbreds

OBJECTIVE To investigate effects of osteochondral injury on high-mobility group box chromosomal protein 1 (HMGB-1) concentrations in synovial fluid (SF) from Thoroughbreds and to compare these results with radiographic and arthroscopic scores of severity of joint injury. ANIMALS 40 clinically normal rested Thoroughbreds (group 1) and 45 Thoroughbreds with osteochondral injury as a result of racing. PROCEDURES SF was obtained from the metacarpophalangeal (MCP) joints, metatarsophalangeal (MTP) joints, middle carpal joints, and radiocarpal joints. For group 2, radiographic and arthroscopic scores were determined. Concentrations of SF HMGB-1 were determined by use of an ELISA. RESULTS SF HMGB-1 concentrations in osteochondral-injured MCP-MTP joints were significantly higher than in normal MCP-MTP joints. Similarly, SF HMGB-1 concentrations in osteochondral-injured carpal joints were significantly higher than in normal carpal joints. Radiographic and arthroscopic scores were not correlated with SF HMGB-1 concentrations. Synovial fluid HMGB-1 concentrations > or = 11 ng/mL for MCP-MTP joints and > or = 9 ng/mL for carpal joints discriminated osteochondral-injured joints from normal joints. Horses with HMGB-1 concentrations > or = 11 ng/mL for MCP-MTP joints were twice as likely to have an osteochondral injury, and horses with HMGB-1 concentrations > or = 9 ng/mL for carpal joints were 4 times as likely to have an osteochondral injury. CONCLUSIONS AND CLINICAL RELEVANCE Osteochondral injury was associated with a significant increase in SF HMGB-1 concentrations in MCP-MTP and carpal joints, compared with results for clinically normal Thoroughbreds. Analysis of SF HMGB-1 concentrations may be useful for evaluation of joint injury in horses.

Regional and zonal variations in the sulfation patterns of chondroitin sulfate in normal equine corneal stroma.

OBJECTIVE To determine regional and zonal variation in sulfation patterns of chondroitin sulfate in normal equine corneal stroma. SAMPLE POPULATION 22 normal eyes from 11 horses. PROCEDURE Corneas were collected within 24 hours of death from equine necropsy specimens. After papain-chondroitinase digestion of corneal tissue, disaccharides deltaDi4S and deltaDi6S were quantified by use of capillary zone electrophoresis in the superficial, middle, and deep zones of central and peripheral regions of the cornea. RESULTS For the 2 regions combined, deltaDi6S/deltaDi4S values were significantly lower in the deep and middle zones, compared with that of the superficial zone. In the central region, deep and middle zones had significantly lower deltaDi6S/deltaDi4S values than the superficial zone did. In the peripheral region, the deep zone had significantly lower deltaDi6S/deltaDi4S values, compared with superficial and middle zones. In the deep zone, the peripheral region had significantly lower deltaDi6S/deltaDi4S values than the central region did. CONCLUSIONS AND CLINICAL RELEVANCE Distribution of deltaDi6S/deltaDi4S values follows a gradient across the healthy equine cornea, being smallest in the deep and middle zones of the central region and the deep zone of the peripheral region. Regional and zonal differences in the distribution of stromal deltaDi6S and deltaDi4S may influence the role of glycosaminoglycans in health, disease, and wound repair of the equine cornea.

Capillary zone electrophoresis for the determination of atovaquone in serum.

A rapid and simple capillary zone electrophoresis (CZE) method has been developed for the determination of atovaquone in serum. The drug was extracted from equine serum-chloroform (1:3, v/v) at greater than 80% recovery and assayed in buffer containing 25 mM sodium borate (pH 9.1) and 25% acetonitrile. A 100 microm I.D. fused-silica capillary was used and the detection was by UV-diode array at 254 nm; the migration time was approximately 8 min. Intra- and inter-assay variabilities were less than 7.8% and 5.8%, respectively, and the accuracy of the assay (expressed as % bias) ranged from 4.5 to -5.2%. The working assay range was from 2 to 100 microg/ml. This sensitivity could be increased by concentrating during the extraction procedure. Replacement of acetonitrile with 75 mM surfactant 3-(dimethyldodecylammonio)propanesulfonate gave similar sensitivity and provided an additional option to facilitate the separation of atovaquone on multiple-drug samples.

Concentrations of stromal cell-derived factor-1 in serum, plasma, and synovial fluid of horses with osteochondral injury

OBJECTIVE To determine whether stromal cell-derived factor-1 (SDF-1) concentrations in serum, plasma, and synovial fluid differed among untrained, race-trained, and osteochondral-injured Thoroughbred racehorses. ANIMALS 22 racehorses without osteochondral injury and 37 racehorses with osteochondral injury. PROCEDURES Horses without osteochondral injury were examined before and after 5 to 6 months of race training. Horses with osteochondral injury were undergoing arthroscopic surgery for removal of osteochondral fragments from carpal or metacarpophalangeal or metatarsophalangeal joints (fetlock joints). Serum, plasma, and fetlock or carpal synovial fluid samples were obtained and analyzed for SDF-1 concentration by use of an ELISA. RESULTS In horses with fetlock or carpal joint injury, mean synovial fluid SDF-1 concentrations were significantly higher, serum SDF-1 concentrations were significantly lower, and synovial fluid-to-serum SDF-1 ratios were significantly higher than in untrained and trained horses. Synovial fluid SDF-1 concentrations were not significantly different between trained and untrained horses. Plasma SDF-1 concentrations were not different among the 3 groups. Results obtained with serum, compared with synovial fluid and plasma, had better sensitivity for differentiating between osteochondral-injured horses and uninjured horses. In horses with fetlock joint osteochondral injury, serum SDF-1 concentrations were correlated with radiographic and arthroscopic inflammation scores, but not arthroscopic cartilage scores. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that serum SDF-1 concentrations were more sensitive than plasma and synovial fluid concentrations for detection of osteochondral injury in the fetlock or carpal joint of racehorses. Analysis of serum and synovial SDF-1 concentrations in horses with experimentally induced joint injury may help define the onset and progression of post-traumatic osteoarthritis and aid in the evaluation of anti-inflammatory treatments.

Vascular perfusion of the dorsal and palmar condyles of the equine third metacarpal bone

REASONS FOR PERFORMING STUDY Palmar osteochondral disease (POD) is an overload arthrosis that commonly affects fetlock joints of racing Thoroughbreds (TB) but the aetiopathogenesis of the disease has not been well defined. OBJECTIVES The aim of this study was to compare India ink perfusion in the dorsal and palmar condyles of the third metacarpal bone (McIII) in both passively flexed and maximally extended fetlock joints from paired equine cadaver limbs. STUDY DESIGN Descriptive cadaver study comparing perfusion of condyles of McIII in paired cadaver limbs in flexion (control group) and maximal extension (intervention group). METHODS Pairs of forelimbs were acquired from 5 TB horses subjected to euthanasia for reasons unrelated to lameness. Limb pairs were perfused intra-arterially with India ink and then randomly assigned to passive flexion or maximal extension of the fetlock joint. Limbs were sectioned sagittally in 3 mm sections through the fetlock and 12 sections per limb processed using a modified tissue-clearing technique. Sections were subsequently digitally imaged and bone perfusion evaluated with image analysis software. RESULTS Greater perfusion of the dorsal condyle than of palmar condyle was observed in 78% of sections from limbs in passive flexion and 92% of maximally extended sections. Perfusion to the palmar aspect of the condyle was significantly decreased (P < 0.0001) when the limbs were placed in maximal extension compared to passive flexion. CONCLUSIONS The palmar condyle of McIII had less perfusion than the dorsal condyle when the fetlock joint was in passive flexion and this difference was exacerbated by maximal extension. Based on the anatomical location of POD lesions, perfusion differences between the dorsal and palmar condyles of McIII may be associated with development of these lesions.

115 DEGRADATION TO SYNTHESIS RATIOS OF TYPE II COLLAGEN BIOMARKERS IN SYNOVIAL FLUID AND SERUM IN THOROUGHBRED RACEHORSES

115 – Table 1. Mean (± SD) concentrations of serum and SF type II collagen biomarkers and degradation to synthesis ratios for normal and osteochondral (OC) injured carpal joints Mean biomarker concentrations (± SD) Degradation:Synthesis C2C (pmol/mL) C1,2C (pmol/mL) CTX II (pg/mL) CPII (ng/mL) C2C:CP II C1,2C:CP II CTX II:CP II

118 TYPE II COLLAGEN SYNTHESIS TO DEGRADATION IMBALANCE IN SYNOVIAL FLUID AFTER OSTEOCHONDRAL INJURY IN THOROUGHBRED RACEHORSES

Purpose: High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that functions both as a regulator of gene transcription and as a proinflammatory cytokine. HMGB1 mediates many inflammatory diseases, including many forms of arthritis. The purposes of this study were to investigate the effects of osteochondral (OC) injury on HMGB1 concentrations in synovial fluid (SF) from Thoroughbred (TB) racehorses. Methods: SF was taken from 2 groups of TB racehorses: (1) rested horses (n = 40) and (2) OC injured horses: racehorses that had arthroscopic surgery for removal of OC fragments resulting from racing injury (n = 44). From group 1 horses, SF was obtained from 20 metacarpophalangeal joints (MCP), 10 middle carpal joints (MCJ), and 10 radiocarpal joints (RCJ) (n = 40). SF samples from group 2 horses were from 16 MCP, 6 metatarsophalangeal joints (MTP), 12 MCJ, and 10 RCJ (n = 44). SF samples were assayed using a commercially available ELISA (HMGB1, Shino-Test Corp.). Differences between groups were determined by an unpaired t-test for the metacarpo/metarsophalangeal (MP) and carpal joints. Positive and negative predictive value of SF HMGB1 for identifying OC injury was determined by Fisher’s exact test. P< 0.05 was considered significant. Results: SF HMGB1 concentrations in OC injured MP and carpal joints were significantly higher than in normal joints (P< 0.0001; Figure 1). SF HMGB1 concentrations 11 ng/mL for MP joints and 10 ng/mL for carpal joints were arbitrarily chosen to determine predictive value for discriminating OC injured horses from normal horses. This yielded a positive predictive value of 89% and a negative predictive value of 68% for MP joints, and positive predictive value of 90% and negative predictive value of 81% for the carpus. Conclusions: OC injury caused a significant increase in SF HMGB1 concentrations in MP and carpal joints compared to normal joints. The assay yielded good positive and negative predictive values. Based on these findings, SF HMGB1 analysis may be useful for evaluation of joint injury.

117 OSTEOCHONDRAL INJURY INCREASES HIGH MOBILITY GROUP BOX CHROMOSOMAL PROTEIN 1 (HMGB1) IN SYNOVIAL FLUID OF THOROUGHBRED RACEHORSES

Purpose: High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that functions both as a regulator of gene transcription and as a proinflammatory cytokine. HMGB1 mediates many inflammatory diseases, including many forms of arthritis. The purposes of this study were to investigate the effects of osteochondral (OC) injury on HMGB1 concentrations in synovial fluid (SF) from Thoroughbred (TB) racehorses. Methods: SF was taken from 2 groups of TB racehorses: (1) rested horses (n = 40) and (2) OC injured horses: racehorses that had arthroscopic surgery for removal of OC fragments resulting from racing injury (n = 44). From group 1 horses, SF was obtained from 20 metacarpophalangeal joints (MCP), 10 middle carpal joints (MCJ), and 10 radiocarpal joints (RCJ) (n = 40). SF samples from group 2 horses were from 16 MCP, 6 metatarsophalangeal joints (MTP), 12 MCJ, and 10 RCJ (n = 44). SF samples were assayed using a commercially available ELISA (HMGB1, Shino-Test Corp.). Differences between groups were determined by an unpaired t-test for the metacarpo/metarsophalangeal (MP) and carpal joints. Positive and negative predictive value of SF HMGB1 for identifying OC injury was determined by Fisher’s exact test. P< 0.05 was considered significant. Results: SF HMGB1 concentrations in OC injured MP and carpal joints were significantly higher than in normal joints (P< 0.0001; Figure 1). SF HMGB1 concentrations 11 ng/mL for MP joints and 10 ng/mL for carpal joints were arbitrarily chosen to determine predictive value for discriminating OC injured horses from normal horses. This yielded a positive predictive value of 89% and a negative predictive value of 68% for MP joints, and positive predictive value of 90% and negative predictive value of 81% for the carpus. Conclusions: OC injury caused a significant increase in SF HMGB1 concentrations in MP and carpal joints compared to normal joints. The assay yielded good positive and negative predictive values. Based on these findings, SF HMGB1 analysis may be useful for evaluation of joint injury.