Detection and pharmacokinetics of salmeterol in thoroughbred horses following inhaled administration.

Salmeterol is a man-made beta-2-adrenergic receptor agonist used to relieve bronchospasm associated with inflammatory airway disease in horses. Whilst judicious use is appropriate in horses in training, they cannot race with clinically effective concentrations of medications under the British Horseracing Authority's Rules of Racing. Salmeterol must therefore be withdrawn prior to race day and pharmacokinetic (PK) studies used to establish formal detection time advice. Salmeterol xinafoate (Serevent Evohaler® ) was administered (0.1 mg twice daily for 4.5 days) via inhalation to six horses. Urine and blood samples were taken up to 103 h postadministration. Hydrolysed samples were extracted using solid phase extraction. A sensitive Ultra high performance tandem mass spectrometry (UPLC-MS/MS) method was developed, with a Lower limit of quantification (LLOQ) for salmeterol of 10 pg/mL in both matrices. The majority of salmeterol plasma concentrations, postlast administration, were below the method LLOQ and so unusable for PK analysis. Urine PK analysis suggested a half-life consistent with duration of pharmacological effect. Average estimated urine concentration at steady-state was obtained via PK modelling and used to estimate a urine concentration of 59 ± 34 pg/mL as a marker of effective lung concentration. From this, potential detection times were calculated using a range of safety factors.

Pharmacokinetics of inorganic cobalt and a vitamin B12 supplement in the Thoroughbred horse: Differentiating cobalt abuse from supplementation.

BACKGROUND: While cobalt is an essential micronutrient for vitamin B12 synthesis in the horse, at supraphysiological concentrations, it has been shown to enhance performance in human subjects and rats, and there is evidence that its administration in high doses to horses poses a welfare threat. Animal sport regulators currently control cobalt abuse via international race day thresholds, but this work was initiated to explore means of potentially adding to application of those thresholds since cobalt may be present in physiological concentrations. OBJECTIVES: To devise a scientific basis for differentiation between presence of cobalt from bona fide supplementation and cobalt doping through the use of ratios. STUDY DESIGN: Six Thoroughbred horses were given 10 mL vitamin B12 /cobalt supplement (Hemo-15® ; Vetoquinol, Buckingham, Buckinghamshire, UK., 1.5 mg B12 , 7 mg cobalt gluconate = 983 µg total Co) as an i.v. bolus then an i.v. infusion (15 min) of 100 mg cobalt chloride (45.39 mg Co) 6 weeks later. Pre-and post-administration plasma and urine samples were analysed for cobalt and vitamin B12 . METHODS: Urine and plasma samples were analysed for vitamin B12 using an immunoassay and cobalt concentrations were measured via ICP-MS. Baseline concentrations of cobalt in urine and plasma for each horse were subtracted from their cobalt concentrations post-administration for the PK analysis. Compartmental analysis was used for the determination of plasma PK parameters for cobalt using commercially available software. RESULTS: On administration of a vitamin B12 /cobalt supplement, the ratio of cobalt to vitamin B12 in plasma rapidly increased to approximately 3 and then rapidly declined below a ratio of 1 and then back to near baseline over the next week. On administration of 100 mg cobalt chloride, the ratio initially exceeded 10 in plasma and then declined with the lower 95% confidence interval remaining above a ratio of 1 for 7 days. For two horses with extended sampling, the plasma ratio remained above one for approximately 28 days after cobalt chloride administration. The effect of the administration of the vitamin B12 /cobalt supplement on the urine ratio was transient and reached a peak value of 10 which then rapidly declined. However, a urine ratio of 10 was exceeded, with the lower 95% confidence interval remaining above a ratio of 10 for 7 days after cobalt chloride administration. For the two horses with extended sampling, the urine ratio remained above 10 for about 18 days (442 h) after cobalt chloride administration even though the absolute cobalt urine concentration had dropped below the international threshold of 100 ng/mL after 96 h. MAIN LIMITATIONS: Only one vitamin B12 /cobalt product was evaluated, a limited number of horses were included, the horses were not in full race training and the results may be specific to this population of horses. CONCLUSIONS: The results provide the basis for a potential strategy for allowing supplementation with vitamin B12 products, while controlling the misuse of high doses of cobalt, through a combination of international thresholds and ratios of cobalt to vitamin B12 , in plasma and urine.

Development and application of a scoring system for prognostic evaluation of equine liver biopsies.

REASONS FOR PERFORMING STUDY: The study was designed to investigate the prognostic value of liver biopsy during investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: That liver biopsy is the most prognostically useful technique in common usage in the investigation of suspected liver disease. METHODS: This study examined the prognostic value of liver biopsy during the investigation of suspected liver disease in 73 mature horses. Histopathological variables comprising fibrosis, irreversible cytopathology, inflammatory infiltration, haemosiderin accumulation and biliary hyperplasia were found to be significant predictors of nonsurvival and were used to formulate a weighted biopsy score representing a prognostically useful broad comparative index of histopathological severity. Minimum and maximum possible scores were 0 and 14 points, respectively. RESULTS: Retrospective application of the biopsy scoring system to the study population indicated that horses with scores of 0 or 1 were equally likely to survive to 6 months with a combined mortality of 4%. Horses with biopsy scores between 2 and 6 had a combined mortality of 33% and were at a 12-fold increased risk of nonsurvival within 6 months (hazard ratio = 12.04, 95% CI 134-107.81, P = 0.026) compared to horses with a biopsy score of 0. Horses with biopsy scores between 7 and 14 had a combined mortality of 86% and were at a 46-fold increased risk of nonsurvival (hazard ratio = 46.01, 95% CI 5.92-357.5, P < 0.001) compared to horses with biopsy score 0. CONCLUSIONS: Application of the biopsy scoring system to histopathological findings in liver biopsy specimens was very useful in predicting survival of cases of suspected liver disease. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, liver biopsy appears to be the most valuable prognostic technique employed during the investigation of suspected cases of liver disease in mature horses.

Retrospective analysis of historical, clinical, ultrasonographic, serum biochemical and haematological data in prognostic evaluation of equine liver disease.

REASONS FOR PERFORMING STUDY: Results of noninvasive tests of liver disease do not always correlate with the degree of hepatic disease nor outcome of the case. OBJECTIVE: To investigate the prognostic value of data collected using noninvasive tests during the investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: Much of the data gathered during the investigation of suspected hepatopathy cases offers little prognostic guidance and interpretation of such data can be misleading. METHODS: The results from a range of common and noninvasive diagnostic techniques applied in 116 mature horses with suspected liver disease, were assessed for their ability to predict survival within a 6 month period. RESULTS: A significantly poorer prognosis was found in association with clinical signs suggestive of liver disease, presence of hepatic encephalopathy, ultrasonographic abnormalities, increased serum globulins, increased total bile acids (TBA), increased alkaline phosphatase (AP), increased gamma-glutamyl transferase (gammaGT), erythrocytosis, leucocytosis, low serum albumin and low serum urea. Additional significant novel findings of interest included an association between increased plasma fibrinogen and low serum creatinine concentrations with nonsurvival in cases of liver disease, an association between raised serum concentrations of AP and gammaGT with biliary hyperplasia and also an association between hepatic fibrosis, haemosiderosis and biliary hyperplasia with ultrasonographically detected hepatic abnormalities. CONCLUSIONS: The most useful noninvasive prognostic test in cases of suspected liver disease in mature horses is the severity of clinical signs. Other data may be of some limited prognostic value. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, the findings should at least be considered when prognosis is based on similar criteria.