Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates.

Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example from each sub-class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11-month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilised a two-step solid phase extraction (SPE) consisting of polymeric reversed-phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed-phase prior to liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition. The LC-MS/MS analysis showed ZA was undetectable after 8 h post-administration while TA was detected up to the final collection point of 28 days post-administration. The LC-HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma.

Pharmacokinetics of Two Formulations of Altrenogest Administered to Mares.

Altrenogest is a synthetic progestin that suppresses reproductive behaviours and assists pregnancy maintenance in female horses. Two formulations are available, a 'weekly' intramuscular injection and a daily oral formulation. Altrenogest administration has returned positive swabs for steroids; consequently, using injectable altrenogest in racing mares is prohibited. Oral administration may be permitted in race mares if there is one clear day between dosing and racing. The only pharmacokinetic data available were generated from geldings. Therefore, to assist veterinarians and analysts in determining accurate dosing and detection intervals, pharmacokinetic analysis using mares is required. Blood samples were taken from 10 mares pretreatment to obtain baseline concentrations. Mares were administered altrenogest, either oral (PO; 0.044 mg/kg; daily for 15 days) or intramuscular (IM; 0.3 mg/kg; twice; Days 0 and 7). On the first and last treatment day, blood samples were taken at designated times post dosing. After a 3-week washout, mares received the alternative treatment with sampling repeated. At the initial dose, for IM administration mean (± SD) plasma altrenogest Cmax was 18.0 ± 6.6 ng/mL at 7.9 ± 3.9 h compared with PO dosing 13.2 ± 5.8 ng/mL at 0.8 ± 0.8 h. Plasma Cmax on the final day was significantly higher (p = 0.002 [IM]; p = 0.006 [PO]). At 24 h post final oral treatment, mean (± SD) plasma altrenogest was 1.0 ± 0.8 ng/mL and at 48 h were 0.65 ± 0.5 ng/mL. Plasma concentrations well exceeding this may indicate that the one clear day rule or dosage recommendations have not been adhered to.

3-Methoxytyrosine as an indicator of dopaminergic manipulation in equine plasma.

The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a rapid protein precipitation method was developed and validated to isolate target compounds from 100 µL equine plasma. A liquid chromatography-high resolution accurate mass (LC-HRAM) method using an IMTAKT Intrada amino acid column provided quantitative analysis of 3-methoxytyrosine (3-MTyr) with lower limit of quantification of 5 ng/mL. Reference population profiling (n = 1129) investigated the expected basal concentrations for raceday samples from equine athletes and showed a right-skewed distribution (skewness = 2.39, kurtosis = 10.65) which resulted from large variation (RSD = 71%) within the data. Logarithmic transformation of the data provided a normal distribution (skewness = 0.26, kurtosis = 3.23) resulting in the proposal of a conservative threshold for plasma 3-MTyr of 1,000 ng/mL at a 99.995% confidence level. A 12-horse administration study of Stalevo® (800 mg L-DOPA, 200 mg carbidopa, 1600 mg entacapone) revealed elevated 3-MTyr concentrations for 24-hours post-administration.

Horse Husbandry-Nutrition, Management and Welfare.

Nutrition is the foundation of health and welfare, going hand in hand with horse husbandry [...].

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes.

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.

Randomised clinical trial on the effect of a single oral administration of l-tryptophan, at three dose rates, on reaction speed, plasma concentration and haemolysis in horses.

Tryptophan (TRP) is marketed as a calmative for horses despite reservations about its efficacy. The aim of this study was to measure the effect of oral TRP administration on the reaction speed of horses. Sixty mature horses were used in a two stage randomised, blind, cross-over study, receiving a placebo and an oral dose of TRP (30, 60 or 120 mg/kg body weight), before undergoing a reaction speed test. Blood samples were taken up to 96 h after TRP administration, to identify signs of acute haemolytic anaemia. Plasma TRP concentrations were increased (P <0.001) by the administration of TRP paste. However, TRP had no effect on the reaction speed of horses when startled. There was no evidence of alterations in clinical pathology parameters in 432 blood samples. While the safety of these doses of TRP can be confirmed, there was no evidence to suggest that a single dose of TRP is an effective calmative for horses.

Effects of meloxicam and phenylbutazone on renal responses to furosemide, dobutamine, and exercise in horses.

OBJECTIVE: To compare the effects of 2 NSAIDs (phenylbutazone and meloxicam) on renal function in horses. ANIMALS: 9 Thoroughbred or Standardbred mares (mean ± SD age, 5.22 ± 1.09 years [range, 2 to 12 years]; mean body weight, 470 ± 25 kg [range, 442 to 510 kg]). PROCEDURES: A randomized blinded placebo-controlled crossover study was conducted to examine the effects of treatment with phenylbutazone, meloxicam, or a placebo (control solution) on renal responses to the administration of furosemide, dobutamine, and exercise (15 minutes at 60% of maximum heart rate). Renal function was assessed by use of bilateral ureteral catheterization for simultaneous determination of creatinine clearance, sodium excretion, and urine flow rate. RESULTS: Both phenylbutazone and meloxicam attenuated diuresis and natriuresis and reduced glomerular filtration rate, compared with results for the control solution, when horses were treated with furosemide. Mean arterial blood pressure, urine flow rate, and glomerular filtration rate were increased during or after (or both) dobutamine infusion. Both NSAIDs reduced urine flow rate and sodium excretion associated with dobutamine infusion and exercise but had no effect on glomerular filtration rate. CONCLUSIONS AND CLINICAL RELEVANCE: Responses to meloxicam, a cyclooxygenase (COX)-2 preferential agent, appeared comparable to those detected after phenylbutazone treatment, which suggested that COX-2 was the mediator of prostanoid-induced changes to renal function in horses and indicated that COX-2-preferential agents would be likely to have adverse renal effects similar to those for nonselective COX inhibitors in volume-depleted horses.

Acepromazine pharmacokinetics: a forensic perspective.

Acepromazine (ACP) is a useful therapeutic drug, but is a prohibited substance in competition horses. The illicit use of ACP is difficult to detect due to its rapid metabolism, so this study investigated the ACP metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS) as a potential forensic marker. Acepromazine maleate, equivalent to 30mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7days post-administration and analysed for ACP and HEPS by liquid chromatography-mass spectrometry (LC-MS). Acepromazine was quantifiable in plasma for up to 3h with little reaching the urine unmodified. Similar to previous studies, there was wide variation in the distribution and metabolism of ACP. The metabolite HEPS was quantifiable for up to 24h in plasma and 144h in urine. The metabolism of ACP to HEPS was fast and erratic, so the early phase of the HEPS emergence could not be modelled directly, but was assumed to be similar to the rate of disappearance of ACP. However, the relationship between peak plasma HEPS and the y-intercept of the kinetic model was strong (P=0.001, r(2)=0.72), allowing accurate determination of the formation pharmacokinetics of HEPS. Due to its rapid metabolism, testing of forensic samples for the parent drug is redundant with IV administration. The relatively long half-life of HEPS and its stable behaviour beyond the initial phase make it a valuable indicator of ACP use, and by determining the urine-to-plasma concentration ratios for HEPS, the approximate dose of ACP administration may be estimated.

The effect of oral metformin on insulin sensitivity in insulin-resistant ponies.

Metformin may be an effective therapeutic option for insulin-resistant (I-R) horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral tissues without stimulating insulin secretion. To determine the effect of metformin on insulin and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT). Metformin was administered at 15 mg/kg bodyweight (BW), orally, twice-daily, for 21 days to the metformin-treated group. The control group received a placebo. A FSIGT was conducted before and after treatment. The Minimal Model of glucose and insulin dynamics rendered indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). The body condition score (BCS), BW and cresty neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI, BW, BCS or CNS in response to metformin, or over time in the control group. There were no measurable benefits of metformin on SI, consistent with recent work showing that the bioavailability of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood concentrations. Alternatively, metformin may only be effective in obese ponies losing weight or with hyperglycaemia.

Pharmacokinetics of metformin after enteral administration in insulin-resistant ponies.

OBJECTIVE: To determine pharmacokinetics and plasma steady-state kinetics of metformin after oral or nasogastric administration in insulin-resistant (IR) ponies. ANIMALS: 8 IR ponies. PROCEDURES: Metformin (30 mg/kg) was administered to 8 ponies via nasogastric tube Blood samples were collected at intervals for 24 hours. Plasma concentrations of metformin were measured via liquid chromatography-electrospray tandem mass spectroscopy Pharmacokinetic variables were determined via noncompartmental analysis. Metformin (15 mg/kg, PO, twice daily [8 am and 5 pm]) was administered to 4 ponies for an additional 20 days, and blood samples were obtained every 2 days. Plasma concentration at steady state (Css) was determined. RESULTS: Mean±SD elimination half-life (t1/2) of metformin was 11.7±5.2 hours, maxima plasma concentration was 748±269 ng/mL at 54±32 minutes, mean area under the curve was 355±92 microg.h/mL, and apparent clearance was 90.6±28.1 mL/min/kg. The Css was 122±22 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Metformin reportedly enhances insulin sensitivity of peripheral tissues without stimulating insulin secretion, but bioavailability in horses is low. The t1/2 of metformin in IR ponies was similar to that in humans. Actual clearance of metformin adjusted for bioavailability in IR ponies was similar to that in humans; however, during chronic oral administration at dosages reported in efficacy studies, the Css of metformin was less than values associated with therapeutic efficacy in humans The apparent lack of long-term efficacy of metformin in horses is likely attributable to low bioavailability, rather than to rapid clearance.

Potential treatments for insulin resistance in the horse: a comparative multi-species review.

Insulin resistance and hyperinsulinaemia increase the risk of laminitis and horse owners and veterinarians should attempt to enhance insulin sensitivity in at-risk groups. In obese animals this may be achieved, in part, by promoting weight loss and increasing exercise, but such intervention may not be appropriate in non-obese insulin-resistant animals, or where exercise is contra-indicated for clinical reasons. An alternative approach to controlling insulin sensitivity in obese and non-obese horses may be the use of certain herbal compounds that have shown promise in humans and laboratory animals, although little is known of the effects of these compounds in horses. The herbs can be grouped according to their primary mechanism of action, including activators of the peroxisome proliferator-activated receptors, anti-obesity compounds, anti-oxidants, compounds that slow carbohydrate absorption, insulin receptor activators and stimulators of glucose uptake, with some herbs active in more than one pathway. Certain herbs have been prioritised for this review according to the quality and quantity of published studies, the reported (or extrapolated) safety profile, as well as potential for efficacy, all of which will hopefully motivate further research in this field.