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A simple LC/MSMS method has been developed and fully validated to determine concentrations and characterize the concentration vs. time course of methocarbamol (MCBL) and guaifenesin (GGE) in plasma after a single intravenous dose and multiple oral dose administrations of MCBL to conditioned Thoroughbred horses. The plasma concentration-time profiles for MCBL after a single intravenous dose of 15 mg/kg of MCBL were best described by a three-compartment model. Mean extrapolated peak (C0 ) plasma concentrations were 23.2 (± 5.93) µg/mL. Terminal half-life, volume of distribution at steady-state, mean residence time, and systemic clearance were characterized by a median (range) of 2.96 (2.46-4.71) h, 1.05 (0.943-1.21) L/kg, 1.98 (1.45-2.51) h, and 8.99 (6.68-10.8) mL/min/kg, respectively. Oral dose of MCBL was characterized by a median (range) terminal half-life, mean transit time, mean absorption time, and apparent oral clearance of 2.89 (2.21-4.88) h, 2.67 (1.80-2.87) h, 0.410 (0.350-0.770) h, and 16.5 (13.0-20) mL/min/kg. Bioavailability of orally administered MCBL was characterized by a median (range) of 54.4 (43.2-72.8)%. Guaifenesin plasma concentrations were below the limit of detection in all samples collected after the single intravenous dose of MCBL whereas they were detected for up to 24 h after the last dose of the multiple-dose oral regimen. This difference may be attributed to first-pass metabolism of MCBL to GGE after oral administration and may provide a means of differentiating the two routes of administration.

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The disposition of plasma glycopyrrolate (GLY) is characterized by a three-compartment pharmacokinetic model after a 1-mg bolus intravenous dose to Standardbred horses. The median (range) plasma clearance (Clp), volume of distribution of the central compartment (V1 ), volume of distribution at steady-state (Vss), and area under the plasma concentration-time curve (AUC0-inf ) were 16.7 (13.6-21.7) mL/min/kg, 0.167 (0.103-0.215) L/kg, 3.69 (0.640-38.73) L/kg, and 2.58 (2.28-2.88) ng*h/mL, respectively. Renal clearance of GLY was characterized by a median (range) of 2.65 (1.92-3.59) mL/min/kg and represented approximately 11.3-24.7% of the total plasma clearance. As a result of these studies, we conclude that the majority of GLY is cleared through hepatic mechanisms because of the limited extent of renal clearance of GLY and absence of plasma esterase activity on GLY metabolism. Although the disposition of GLY after intravenous administration to Standardbred horses was similar to that in Thoroughbred horses, differences in some pharmacokinetic parameter estimates were evident. Such differences could be attributed to breed differences or study conditions. The research could provide valuable data to support regulatory guidelines for GLY in Standardbred horses.

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With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 10(11) vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

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This study investigated and compared the pharmacokinetics of intra-articular (IA) administration of dexamethasone sodium phosphate (DSP) into three equine joints, femoropatellar (IAS), radiocarpal (IAC), and metacarpophalangeal (IAF), and the intramuscular (IM), oral (PO) and intravenous (IV) administrations. No significant differences in the pharmacokinetic estimates between the three joints were observed with the exception of maximum concentration (Cmax ) and time to maximum concentration (Tmax ). Median (range) Cmax for the IAC, IAF, and IAS were 16.9 (14.6-35.4), 23.4 (13.5-73.0), and 46.9 (24.0-72.1) ng/mL, respectively. The Tmax for IAC, IAF, and IAS were 1.0 (0.75-4.0), 0.62 (0.5-1.0), and 0.25 (0.08-0.25) h, respectively. Median (range) elimination half-lives for IA and IM administrations were 3.6 (3.0-4.6) h and 3.4 (2.9-3.7) h, respectively. A 3-compartment model was fitted to the plasma dexamethasone concentration-time curve following the IV administration of DSP; alpha, beta, and gamma half-lives were 0.03 (0.01-0.05), 1.8 (0.34-2.3), and 5.1 (3.3-5.6) h, respectively. Following the PO administration, the median absorption and elimination half-lives were 0.34 (0.29-1.6) and 3.4 (3.1-4.7) h, respectively. Endogenous hydrocortisone plasma concentrations declined from a baseline of 103.8 ± 29.1-3.1 ± 1.3 ng/mL at 20.0 ± 2.7 h following the administration of DSP and recovered to baseline values between 96 and 120 h for IV, IA, and IM administrations and at 72 h for the PO.

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We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The intra and inter-batch imprecisions were <10% RSD and accuracy of the method ranged between 94 and 104%. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24h after oral administration. Analytical method validation requirements for linearity, specificity, precision, accuracy, stability, dilution integrity, matrix effect, and ruggedness have been fulfilled. The urine method described in this report is simple and efficient and is the first reported method with sufficient sensitivity, accuracy, and precision to regulate the use of glycopyrrolate in urine samples collected more than one day after dosing of horses. Urine to plasma glycopyrrolate concentration ratios were calculated and were approximately 100:1 in samples collected from 24h through the end of sample collection.

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A rapid, sensitive, and specific ultra-high-performance liquid chromatography with heated electrospray ionization-tandem mass spectrometry (UHPLC-HESI-MS-MS) method to detect and quantify glycopyrrolate in horse plasma has been developed and validated. We also determined glycopyrrolate in plasma after oral and intravenous administration of clinically relevant doses to Thoroughbred horses. Calibration was accomplished by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). Glycopyrrolate (GLY) and the IS (GLY-d(3)) were isolated from plasma matrices via weak cation exchange using a simple solid-phase extraction technique. Chromatographic analysis was achieved by reversed-phase UHPLC on a C(18) Acquity™ column. Extracts were analyzed in positive electrospray ionization mode and precursor and product ions were detected and quantified by MS-MS using a triple-stage quadrupole (TSQ) instrument. The method was characterized by a linear range of 0.125-25 pg/mL (R(2) > 0.998), a lower limit of quantification of 0.125 pg/mL and a lower limit of detection of 0.025 pg/mL. Recovery of GLY ranged from 78% to 96%, and intra- and interbatch precision were 3.3-14.4%CV and 3.4-14.4%CV, respectively. Glycopyrrolate was stable in plasma for up to 170 days at -80°C, through three freeze/thaw cycles, and for up to 48 h after extraction under 20°C autosampler conditions.

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Testosterone is an anabolic androgenic steroid (AAS) that is endogenously produced by both male and female horses that also has the potential for abuse when administered exogenously to race horses. To recommend appropriate withdrawal guidelines so that veterinarians can discontinue therapeutic use prior to competition, the pharmacokinetics and elimination of testosterone were investigated. An aqueous testosterone suspension was administered intramuscularly in the neck of Thoroughbred horses (n = 20). The disposition of testosterone from this formulation was characterized by an initial, rapid absorption phase followed by a much more variable secondary absorption phase. The median terminal half-life was 39 h. A second focus of this study was to compare the testosterone concentrations determined by two different laboratories using a percentage similarity model with a coefficient of variation of 16.5% showing good agreement between the two laboratories results. Based on the results of this study, a withdrawal period of 30 days for aqueous testosterone administered IM is recommended.

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REASONS FOR PERFORMING STUDY: Sildenafil, a phosphodiesterase-5 inhibitor vasodilator, increases cGMP concentrations by inhibiting enzymatic degradation. Marketed to treat erectile dysfunction in men, it also reduces pulmonary arterial pressure (PAP). Because it reduces PAP, sildenafil may enhance performance and/or prevent exercise induced-pulmonary haemorrhage (EIPH). OBJECTIVE: To determine if sildenafil citrate administration altered commonly measured indices of performance or reduced EIPH in exercised horses. METHODS: Thirteen athletically conditioned Thoroughbred horses (2 mares and 11 geldings, age 3-12 years) were administered sildenafil citrate or placebo in 2 crossover design exercise testing studies. In a step-wise test to exhaustion, inspired/expired gas analysis, blood lactate, heart rate, runtime and bronchoalveolar lavage (BAL) cytology were measured. In a 13 m/s test to exhaustion, blood lactate, heart rate, runtime, BAL cytology and pulmonary arterial pressure were measured. Data were analysed with paired and unpaired t tests, one-way ANOVA and Tukey's pair-wise multiple comparison and Friedman repeated measure analysis of variance on ranks. RESULTS: The administration of sildenafil did not alter mean inspired/expired gas measurements, plasma lactate concentrations or acute pulmonary haemorrhage in either exercise test or pulmonary arterial pressure measurement in the 13 m/s trial. Heart rates in both stress tests were significantly different at submaximal speeds and during the early recovery period. Run time was not affected by sildenafil administration in the step-wise trial (P = 0.622) or in the 13 m/s trial (P = 0.059). CONCLUSIONS: Sildenafil did not alleviate pulmonary haemorrhage or enhance performance-related indices in these trials. Sildenafil administration altered cardiovascular adaptation to intense exercise as evidenced by altered heart rates at submaximal speeds and post exercise. The effect of these alterations on other performance perimeters was not evident.

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REASONS FOR PERFORMING STUDY: Adrenergic activity accompanies intense exercise and mediates physiological and metabolic responses to exercise. Guanabenz, an antihypertensive drug marketed for human usage, depresses brain vasomotor and cardioaccelerator centres, blocks peripherally adrenergic neurons and is reportedly used as a calming agent in horses but little is known of its effects in the species. OBJECTIVES: To determine if guanabenz induces measurable signs of adrenergic suppression on fit Thoroughbred horses undergoing intense exercise. METHODS: In a random crossover design, 12 exercise conditioned Thoroughbred horses each received guanabenz (0.08 mg/kg bwt i.v.) and placebo at 3-week intervals. An incremental exercise test to exhaustion on a treadmill followed treatment by 1 h. Heart rate, oxygen consumption, carbon dioxide production, plasma lactate, catecholamines, adrenocorticotropic hormone (ACTH) and cortisol, and time to fatigue were monitored. Statistical analysis was performed using mixed-effects linear modelling. RESULTS: Mean heart rate during the exercise period was lower in guanabenz-treated horses (P = 0.04). Mean concentrations of plasma cortisol (P = 0.02) and adrenaline (P = 0.03) were lower for guanabenz-treated horses during the exercise period. Mean run time was slightly but not significantly longer for guanabenz-treated horses, (P = 0.053). No significant effects of guanabenz administration were found for oxygen consumption, carbon dioxide production nor for plasma lactate, noradrenaline and ACTH concentrations. CONCLUSION: Guanabenz administration induced signs of adrenergic suppression including plasma cortisol and adrenaline concentrations and heart rate and may enhance endurance, but did not eliminate increases in hormone concentrations induced by exercise. Clear determination of a positive performance effect of adrenaline, but not noradrenaline, suppression is needed before clinical significance can be determined.

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The objective of this study was to investigate the effects of ribose supplementation on blood ammonia-N, plasma lactic acid, plasma glucose, volume of oxygen consumption (VO2), heart rate, and performance in Thoroughbred geldings performing a maximal treadmill standardized exercise test (SET). The hypothesis tested was that ribose supplementation would decrease ammonia-N and lactic acid accumulation during exercise, and improve performance. Eight Thoroughbred geldings were assigned randomly to one of two groups: glucose or ribose. The glucose group received 0.15 g glucose/kg of BW, and the ribose group received 0.15 g of ribose/kg BW top-dressed on the feed twice daily. After 2 wk of glucose or ribose supplementation, a SET was performed. Blood was analyzed for blood ammonia-N, plasma lactic acid, and plasma glucose before exercise (0 min), every minute during SET, and at 15 and 30 min after exercise. Heart rate and VO2 were recorded for the duration of SET. After a 10-d washout period, geldings switched groups. Following another 2 wk of supplementation, a second SET was performed, and same data recorded. Blood ammonia-N and plasma lactic acid increased as duration of SET increased and reached a peak at 15 min after exercise. Peak plasma glucose was observed at 15 min after exercise, and peak heart rate and VO2 were recorded at highest speed during SET. Geldings supplemented with ribose had blood ammonia-N, plasma lactic acid, plasma glucose, VO2, heart rate, and performance similar to those of geldings supplemented with glucose. Results from this study show that supplementation with 0.15 g ribose/kg BW twice daily in the diet of conditioned Thoroughbred geldings for 2 wk does not influence blood ammonia-N, plasma lactic acid, plasma glucose, VO2, heart rate, or performance during SET or the first 30 min of recovery.

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Physical exertion is a stimulus for the upregulation of cytokine production including IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-10 and TNF-alpha in horses. To investigate that hypothesis, we initiated training of 5 stall-rested Thoroughbreds. Blood samples were drawn before and weekly during training. The relative transcription of mRNA within the leucocytes was measured using real time TaqMan quantitative PCR. The training protocol was walking (3 min), trotting (3 min) and cantering/galloping (6 min) increasing in intensity weekly (6 to 12 m/s) and culminating in an intense exercise period. Comparisons of mRNA concentrations were made using a repeated measures ANOVA on ranks and a Student-Newman-Keuls pair-wise multiple comparison (P<0.05). The training programme or intense exercise bout did not affect IL-2, IL-4, IL-6 and IL-10. IL1-beta and TNF-alpha transcription increased on Day 23. TNF-alpha peaked on Day 23 and IL1-beta on Day 30. Neither demonstrated a response to intense exercise. IL-1ra decreased significantly on Day 9; rose significantly from Day 9 to Days 16 and 23; remained significantly elevated through Days 30 and 37 over Day 9, and rose very slightly after intense exercise on Day 56. Alterations in leukocyte cytokine responses may influence susceptibility to infectious disease, metabolic responses to exercise or exercised induced syndromes.

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A diverse group of studies, which are equine exclusive, indicate that ribose administered to myocardial and skeletal muscle tissue stimulates ATP production and recovery. This study investigated the effects of ribose supplementation on blood and muscle metabolites and performance in Thoroughbred geldings performing a maximal treadmill standardised exercise test (SET). In Experiment 1, 6 conditioned Thoroughbred geldings performed a baseline SET and horses were assigned to one of 2 experimental treatment groups, placebo or ribose, based on VO2max. The placebo treatment group received 0.07 g glucose/kg bodyweight (bwt) and ribose treatment group received 0.07 g ribose/kg bwt top dressed on the feed twice daily. Following a 2 week treatment period, a second SET was performed. After a one-week washout period, the horses switched treatment groups. Following another 2 week treatment period, a third SET was performed. Blood ammonia-N was lower in the ribose treatment group at 15 min (P = 0.06) and 30 min (P = 0.02) postexercise. Plasma lactic acid was lower in the ribose treatment group at 30 min postexercise (P = 0.07). In Experiment 2, 1 h before a SET, 2 horses received 3 l water (control) and 3 horses 250 g of ribose dissolved in 3 l water (single ribose dose) via a nasogastric tube. Following a 2 week washout period, the horses switched treatment groups and another SET was performed. There were no differences in blood ammonia-N, plasma lactic acid or glucose between treatment groups. No differences in performance were detected between treatment groups in either experiment. In conclusion, the results from Experiment 1 show a trend that daily ribose supplementation may be beneficial during recovery from exercise. However, a single dose of ribose 1 h before exercise revealed no effect on the variables measured. Because moderate to intense daily exercise can cause a decrease in total adenine nucleotide (TAN) pool with no meaningful recovery even after 72 h rest, future experiments should be designed to futher elucidate the effects of ribose supplementation on TAN metabolism in horses exercising at high intensity.

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This study was designed to test the hypothesis that a specific beta 2 adrenergic receptor agonist, albuterol sulphate, when delivered by metered-dose inhaler (MDI) would affect the performance of Thoroughbred horses during incremental exercise testing. Six conditioned Thoroughbred horses were randomly assigned to one of 2 treatment groups: Group 1 horses received placebo and Group 2 received albuterol. Each horse received both treatments in a crossover design with a 3 week interval between trials. Horses inspired albuterol (900 micrograms) or placebo over a 5 min period followed by an incremental, peak oxygen consumption (VO2peak) exercise test. Facial arterial blood gases (PaO2, PaCO2), pHa, temperature, haemoximetry (O2Hb, COHb, MetHb, RHb) and electrolytes (Na+, K+, iCa2+, Cl-) were measured. Heart rate (HR), VO2peak, CO2 production (VCO2), plasma lactate, speed at failure, exercise duration, and urine levels of albuterol and metabolites (Alburine) were monitored. The Paratrend7 intravascular sensor provided continuous arterial blood gas data. Mixed effects linear modelling using generalised least-squares estimation was used to assess treatment and speed main effects interaction. Significant differences (P < 0.01) were found between placebo and albuterol-treated horses for total run time (mean +/- s.e. 405.8 +/- 8.8 vs. 430.5 +/- 6.7 s) and run time from completion of 8 m/s until fatigue (169.3 +/- 8.6 vs. 187.3 +/- 6.7 s), as well as VO2peak (121.7 +/- 3.7 vs. 130.3 +/- 3.8 ml/kg/min), respectively. In conclusion, inhaled albuterol sulphate has a small but significant effect on the performance of fit Thoroughbred horses during incremental, VO2peak exercise testing.

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Right third metacarpal bones (n = 24) from Thoroughbreds, 24 to 48 months old and in race training, were tested to failure in 3 point bending. The neutral load axis was estimated and the distance from the axis to the outer dorsal cortical surface measured. Mid-diaphyseal dorsopalmar and lateromedial outer cortical and medullary diameters were measured. Breaking strength, cortical area and area moment of inertia were also calculated. Significant correlations were demonstrated between months in training and dorsopalmar bone diameter, cortical area and area moment of inertia. Significant linear models were illustrated between the same 3 variables and number of months in training. It was concluded that increased duration of training significantly enlarges dorsopalmar bone diameter, cortical area and area moment of inertia. Training did not affect metacarpal bone breaking strength as determined by 3 point bending.

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In each of 4 horses, sterile synovitis was induced by intra-articular injection of 3 micrograms of Escherichia coli endotoxin (lipopolysaccharide, LPS) into one antebrachiocarpal joint; an equal volume (2 ml) of phosphate-buffered saline solution (PBSS) was injected into the opposite, control carpus. Blood and 1.5 ml of synovial fluid were obtained at postinjection hours (PIH) 0, 2, 4, 8, 12, 18, 42, 66, and 144. Synovial fluid sample collection was accomplished by use of an indwelling, intra-articular catheter through PIH 12, and by arthrocentesis subsequently. Joint fluid samples were analyzed for cell counts, protein concentration, cytologic variables, and tumor necrosis factor (TNF), interleukin 6 (IL-6), and prostaglandin E2 (PGE2) values. Tumor necrosis factor and IL-6 activities and WBC count were also measured in blood. To monitor local inflammation, skin temperature of each carpus was imaged, using a thermographic scanner prior to each sample collection time. Horses had minimal systemic effects. Mean (+/- SEM) rectal temperature increased significantly to 39.02 +/- 0.15 C only at PIH 18 after intra-articular injection of LPS. One horse had signs of mild depression from PIH 7 to 18, but its vital signs did not change appreciably. Each horse had mild signs of discomfort in the LPS-injected limb from PIH 1 to 3 until PIH 8 to 10. Mean peak surface temperature of the LPS-injected carpi was significantly higher than that of control carpi from PIH 8 to 144 (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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The pressure exerted on a flat level surface by recently trimmed, unshod hoofs of the front limbs of 23 sound, adult horses was measured using pressure-sensitive film and a specially built cassette. The horses were tranquilized and stood with one foot on the 2.9-cm-thick cassette and the other on a block of equal height. The hoofs were observed for motion during the measurement, and the developed film was examined for improper alignment of the film or slipping of the hoof. The center of pressure was located using the method of weighted proportions of Barrey. This static measurement system with a long measurement time and the number of measurements reduced the influence of variables inherent in the horses' behavior and the measuring system. The calculated point was recorded as falling medial to, lateral to or on a line bisecting the central sulcus of the frog. In the dorsal to palmar orientation the point was classified with reference to a line drawn halfway between the most dorsal and the most palmar mark on the film. Forty-six percent of the calculated centers of pressure were located in the medial heel area. Binomial analysis for large samples indicates that this was a significant variation from a random distribution. Seventy-six percent of the centers were located in or on the borders of the medial heel.

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Subchondral cyst-like lesions of the cubital joint were diagnosed in 7 horses at the teaching hospital between 1983 and 1987. Diagnosis of the lesions was made by administration of intra-articular local anesthesia and/or radiographically. Initial treatment for all horses consisted of stall rest for 60 to 90 days. In addition, 2 horses were administered sodium hyaluronate intra-articularly, 1 horse was given injections of polysulfated glycosaminoglycans IM, and 1 horse was given phenylbutazone orally. Follow-up information was compiled 6 weeks to 4 years after initial examination. At the time of follow-up inquiry, 6 horses were sound for intended use and only 1 horse became lame when exercised. A logical approach to choice of surgical or nonsurgical treatment is proposed on the basis of these findings and those reported in the literature.

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