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Background: Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids., Objectives: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS., Study Design: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation., Methods: Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation., Results: Estrone sulphate concentrations peaked by week 26 at almost 60 μg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed., Main Limitations: Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards., Conclusions: Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation., (© 2019 EVJ Ltd.)

Reason for Performing Study: The use of firocoxib in horses and its ability to affect performance and potential to allow a horse to compete when it otherwise should not, necessitates establishing appropriate withdrawal time guidelines prior to performance., Objectives: To describe plasma concentrations and characterise the pharmacokinetics of 3 firocoxib formulations following multiple administrations of the label dose, with respect to recommended plasma thresholds for performance horses., Study Design: Balanced 3-way crossover prospective study., Methods: Nine healthy mature horses were administered firocoxib injectable solution (0.09 mg/kg bwt i.v. s.i.d. for 5 days), firocoxib paste (0.1 mg/kg bwt per os s.i.d. for 14 days) and firocoxib tablets (57 mg s.i.d. for 14 days). Blood samples were collected at Time 0 and at various times post drug administration until plasma concentrations were below the limit of detection of the assay. Plasma samples were analysed using liquid chromatography-mass spectrometry and data analysed using noncompartmental analysis., Results: The mean plasma half-life was 1.64 ± 0.737, 1.70 ± 0.800 and 1.73 ± 0.767 days for injectable, paste and tablet formulations, respectively. Plasma concentrations fell below the Racing Medication and Testing Consortium's recommended threshold for racehorses (20 ng/ml) by 7 days post administration of the final dose for all formulations. Plasma concentrations never exceeded the threshold concentration (240 ng/ml) for horse competing in US Equestrian Federation events for any of the formulations., Conclusions: This study extends current knowledge regarding the pharmacokinetics of firocoxib and provides information that can be used to establish appropriate withdrawal time guidelines following multiple administrations, with respect to already established plasma regulatory threshold concentrations., (© 2013 EVJ Ltd.)

Reasons for Performing Study: Detomidine is commonly used i.v. for sedation and analgesia in horses, but the pharmacokinetics and metabolism of this drug have not been well described., Objectives: To describe the pharmacokinetics of detomidine and its metabolites, 3-hydroxy-detomidine (OH-detomidine) and detomidine 3-carboxylic acid (COOH-detomidine), after i.v. and i.m. administration of a single dose to horses., Methods: Eight horses were used in a balanced crossover design study. In Phase 1, 4 horses received a single dose of i.v. detomidine, administered 30 microg/kg bwt and 4 a single dose i.m. 30 microg/kg bwt. In Phase 2, treatments were reversed. Plasma detomidine, OH-detomidine and COOH-detomidine were measured at predetermined time points using liquid chromatography-mass spectrometry., Results: Following i.v. administration, detomidine was distributed rapidly and eliminated with a half-life (t1/2(el)) of approximately 30 min. Following i.m. administration, detomidine was distributed and eliminated with t1/2(el) of approximately one hour. Following, i.v. administration, detomidine clearance had a mean, median and range of 12.41, 11.66 and 10.10-18.37 ml/min/kg bwt, respectively. Detomidine had a volume of distribution with the mean, median and range for i.v. administration of 470, 478 and 215-687 ml/kg bwt, respectively. OH-detomidine was detected sooner than COOH-detomidine; however, COOH-detomidine had a much greater area under the curve., Conclusions and Potential Relevance: These pharmacokinetic parameters provide information necessary for determination of peak plasma concentrations and clearance of detomidine in mature horses. The results suggest that, when a longer duration of plasma concentration is warranted, the i.m. route should be considered.

Pirfenidone, a promising antifibrotic agent, was administered intravenously to six female sheep at 30 mg/kg. Four sheep received 14C-pirfenidone simultaneously. Plasma and urine were obtained for assay of pirfenidone and its metabolites over two days, and tissues were obtained via necropsy. Samples were analysed for pirfenidone and metabolites using HPLC-MS and flow scintillation spectrometry. Plasma pirfenidone disappeared with first order kinetics with a clearance of 1.2 l/kg/h, half-life of 24 min, and distribution volume of 0.71 l/kg. After 48 h, the organs containing the largest quantity of 14C were lungs, liver and intestinal wall. Tissues with the highest concentration of 14C were lung, kidney, brain, liver, lymph node and adipose. Metabolites found in plasma and urine were hydroxypirfenidone (half-life of 44 min) and carboxypirfenidone. Additional metabolites found in urine were hydroxypirfenidone glucuronide and acetoxypirfenidone. Approximately, 80% of the tracer eventually appeared in the urine, and approximately 50% of it was in the form of identifiable metabolites. Less than 1% of the dose appeared in the urine in the form of the parent drug. Quantitatively, most of the metabolites appeared in the urine within 2 h. Thus, the drug is rapidly and completely metabolized., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Reasons for Performing Study: In order to evaluate its potential as an adjunct to inhalant anaesthesia in horses, the pharmacokinetics of fentanyl must first be determined., Objectives: To describe the pharmacokinetics of fentanyl and its metabolite, N-[1-(2-phenethyl-4-piperidinyl)maloanilinic acid (PMA), after i.v. administration of a single dose to horses that were awake in Treatment 1 and anaesthetised with isoflurane in Treatment 2., Methods: A balanced crossover design was used (n = 4/group). During Treatment 1, horses received a single dose of fentanyl (4 microg/kg bwt, i.v.) and during Treatment 2, they were anaesthetised with isoflurane and maintained at 1.2 x minimum alveolar anaesthetic concentration. After a 30 min equilibration period, a single dose of fentanyl (4 microg/kg bwt, i.v.) was administered to each horse. Plasma fentanyl and PMA concentrations were measured at various time points using liquid chromatography-mass spectrometry., Results: Anaesthesia with isoflurane significantly decreased mean fentanyl clearance (P < 0.05). The fentanyl elimination half-life, in awake and anaesthetised horses, was 1 h and volume of distribution at steady state was 0.37 and 0.26 l/kg bwt, respectively. Anaesthesia with isoflurane also significantly decreased PMA apparent clearance and volume of distribution. The elimination half-life of PMA was 2 and 1.5 h in awake and anaesthetised horses, respectively., Conclusions and Potential Relevance: Pharmacokinetics of fentanyl and PMA in horses were substantially altered in horses anaesthetised with isoflurane. These pharmacokinetic parameters provide information necessary for determination of suitable fentanyl loading and infusion doses in awake and isoflurane-anaesthetised horses.

Reasons for Performing Study: Many racing jurisdictions monitor pre-race serum concentration of total carbon dioxide (TCO2) among racing horses. To our knowledge, factors influencing concentration of TCO2 among horses participating in racing have not been systematically evaluated and reported., Objectives: To determine if characteristics of horses and racing conditions routinely recorded were significantly associated with pre-race concentration of TCO2, while accounting for and estimating effects of trainer and horse., Methods: Pre-race serum TCO2 concentrations from 5028 starts made by 2,349 horses trained by 287 trainers at 2 racetracks in California during 2005 were examined. Data regarding characteristics of starters and race conditions obtained from a commercial database were recorded for each start. Data were analysed using mixed-effects, with TCO2 concentration as the dependent variable, and trainer and horse nested within trainer as random effects., Results: Sex, class and distance of race, frusemide administration and cloudy weather conditions were significantly (P<0.001) associated with pre-race TCO2 concentration. Horses that finished in the top 3 positions had values that were slightly (0.2 mmol/) but significantly (P<0.001) greater than horses not finishing in the top 3. There were significant effects of trainer on pre-race TCO2 concentration., Conclusions: A variety of factors may influence pre-race TCO2 concentration in horses. Horses with better performance tend to have higher pre-race TCO2 concentrations., Potential Relevance: TCO2 concentration is associated with improved performance although the magnitude of effect was quite small. Regulatory programmes based on monitoring should consider the influence of other factors on TCO2 concentration.

Reason for Performing Study: Human athletes run faster and experience fewer injuries when running on surfaces with a stiffness 'tuned' to their bodies. We questioned if the same might be true for horses, and if so, would running on surfaces of different stiffness cause a measurable change in the amount of energy required to move at a given speed?, Hypothesis: Different brands of commercial treadmills have pans of unequal stiffness, and this difference would result in different metabolic power requirements to locomote at a given speed., Methods: We tested for differences in stiffness between a Mustang 2200 and a Säto I commercial treadmill by incrementally loading each treadmill near the centre of the pan with fixed weights and measuring the displacement of the pan as weights were added or removed from the pan. We trained six 3-year-old Thoroughbreds to run on the 2 treadmills. After 4 months the horses ran with reproducible specific maximum rates of O2 consumption (VO2max/kg bwt, 2.62 +/- 0.23 (s.d.) mlO2 STPD/sec/kg) at 14.2 +/- 0.7 (s.d.) m/sec. They were alternately run on the 2 treadmills at identical grade (0.40 +/- 0.02%) and speeds (1.83 (walk), 4.0 (trot) and 8.0 (canter) m/sec, all +/- 0.03 m/sec) while wearing an open-flow mask for measurement of VO2., Results: The Mustang treadmill was over 6 times stiffer than the Säto. The VO2/kg bwt increased by approximately 4-fold over the range of speeds studied on both treadmills. Oxygen consumption was significantly lower at all speeds for the Mustang treadmill compared to the Säto. The fractional difference in energy cost decreased by a factor of 6 with increasing speed, although absolute difference in cost was relatively constant., Conclusions: We suggest it costs less energy for horses to walk, trot or canter on a stiffer treadmill than on a more compliant treadmill, at least within the ranges of stiffness evaluated., Potential Relevance: It may be possible to define a substrate stiffness 'tuned' to a horse's body enabling maximal energetic economy when running. The differences between treadmills allows more accurate comparisons between physiological studies conducted on treadmills of different stiffness, and might help to identify an ideal track stiffness to reduce locomotor injuries in equine athletes.