Summary The running abilities of 25 Thoroughbred racehorses were measured at distances of 1200, 1600 and 20000 m. Various physiological variables were measured subsequently on the treadmill and correlated with running speed. There was a negative correlation for running speed with the velocity (VLa4) and work rate (WLa4) at which blood lactate reaches a steady‐state concentration of 4 mmol/litre and a positive correlation with peak plasma lactate, suggesting that plasma lactate concentrations of faster horses rise more rapidly and to higher levels than do those of slower horses. The correlation between running speeds and heart rates (HR) was stronger for unfit than fit horses, suggesting that cardiovascular effects of training are more beneficial to slower horses. The significant correlation between running speeds and V200 suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and O2max suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and VO2max suggests that faster horses utilise more oxygen during maximal intensity exercise. The correlation of running speeds with minimum pH and minimum HCO3− suggests that faster horses maintain speed at higher hydrogen ion (H+) concentrations. Correlations between running speeds and the measured variables were consistently stronger for the longer distance runs. Because VLa4 and WLa4 were obtained during sub‐maximal exercise, these variables were determined to be the best correlates of running performance.

Lidocaine is a local anaesthetic agent that is widely used in equine medicine. It is also an Association of Racing Commissioners International (ARCI) Class 2 foreign substance that may cause regulators to impose substantial penalties if residues are identified in post race urine samples. Therefore, an analytical/pharmacological database was developed for this drug. Using our abaxial sesamoid local anaesthetic model, the highest no-effect dose (HNED) for the local anaesthetic effect of lidocaine was determined to be 4 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, administration of the HNED of lidocaine to eight horses yielded peak serum and urine concentrations of apparent lidocaine of 0.84 ng/mL at 30 min and 72.8 ng/mL at 60 min after injection, respectively. These concentrations of apparent lidocaine are readily detectable by routine ELISA screening tests (LIDOCAINE ELISA, Neogen, Lexington, KY). ELISA screening does not specifically identify lidocaine or its metabolites, which include 3-hydroxylidocaine, dimethylaniline, 4-hydroxydimethylaniline, monoethylglycinexylidine, 3-hydroxymonoethylglycinexylidine, and glycinexylidine. As 3-hydroxylidocaine is the major metabolite recovered from equine urine, it was synthesized, purified and characterized, and a quantitative mass spectrometric method was developed for 3-hydroxylidocaine as recovered from horse urine. Following subcutaneous (s.c.) injection of the HNED of lidocaine, the concentration of 3-hydroxylidocaine recovered from urine reached a peak of about 315 ng/mL at 1 h after administration. The mean pH of the 1 h post dosing urine samples was 7. 7, and there was no apparent effect of pH on the amount of 3-hydroxylidocaine recovered. Within the context of these experiments, the data suggests that recovery of less than 315 ng/mL of 3-hydroxylidocaine from a post race urine sample is unlikely to be associated with a recent local anaesthetic effect of lidocaine. Therefore these data may be of assistance to industry professionals in evaluating the significance of small concentrations of lidocaine or its metabolites in postrace urine samples. It should be noted that the quantitative data are based on analytical methods developed specifically for this study, and that methods used by other laboratories may yield different recoveries of urine 3-hydroxylidocaine.

Diclazuril (4-chlorophenyl [2,6-dichloro-4-(4,5-dihydro-3H-3,5-dioxo-1,2,4-triazin-2-yl)pheny l] acetonitrile), is a benzeneacetonitrile antiprotozoal agent (Janssen Research Compound R 64433) marketed as Clinacox . Diclazuril may have clinical application in the treatment of Equine Protozoal Myeloencephalitis (EPM). To evaluate its bioavailability and preliminary pharmacokinetics in the horse we developed a sensitive quantitative high-pressure liquid chromatography (HPLC) method for diclazuril in equine biological fluids. MS/MS analysis of diclazuril in our HPLC solvent yielded mass spectral data consistent with the presence of diclazuril. After a single oral dose of diclazuril at 2.5 g/450 kg (as 500 g Clinacox), plasma samples from four horses showed good plasma concentrations of diclazuril which peaked at 1.077 +/- 0.174 microg/mL (mean +/- SEM) with an apparent plasma half-life of about 43 h. When this dose of Clinacox was administered daily for 21 days to two horses, mean steady state plasma concentrations of 7-9 microg/mL were attained. Steady-state levels in the CSF ranged between 100 and 250 ng/mL. There was no detectable parent diclazuril in the urine samples of dosed horses by HPLC or by routine postrace thin layer chromatography (TLC). These results show that diclazuril is absorbed after oral administration and attains steady-state concentrations in plasma and CSF. The steady state concentrations attained in CSF are more than sufficient to interfere with Sarcocystis neurona, whose proliferation is reportedly 95% inhibited by concentrations of diclazuril as low as 1 ng/mL. These results are therefore entirely consistent with and support the reported clinical efficacy of diclazuril in the treatment of clinical cases of EPM.

No abstract

Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.

Summary The highest no effect doses (HNEDs) for the local anaesthetic (LA) effects of procaine, cocaine, bupivacaine and benzocaine were determined using the heat lamp/hoof withdrawal model of Kamerling et al. (1985b) and the abaxial sesamoid block model of local anaesthesia. The heat lamp rapidly (4 or 5 s) increased the temperature of the superficial skin layers of the pastern to about 90°C, at which point the animal sharply withdrew its hoof. Effective LA blockade precluded this response and superficial skin temperatures exceeded 120°C. Thermal stimulus experiments were routinely terminated after 10 s of exposure to prevent undue tissue damage. Following abaxial sesamoid block with bupivacaine, the HNED for that drug was about 0.25 mg/site. Increasing the dose to 2 mg/site apparently produced complete and prolonged LA blockade. Analogous work showed that the HNED for procaine was about 2.5 mg/site. Similarly, the dose response curve for procaine was parallel with that of bupivacaine but was shifted 10‐fold to the right The duration of the LA response following procaine injection was less than for bupivacaine with the statistically significant response following 40 mg/site injection lasting less than 45 min. Cocaine was less potent than procaine, showing a shallower dose response curve. The HNED for cocaine was less than 5 mg/site, although at this dose the duration of action was extremely short (<7.5 min). Benzocaine had no significant LA action when a dose of 800 mg was applied topically as a 5% preparation. These results show that the HNEDs for bupivacaine and procaine are remarkably low, that cocaine is somewhat less potent as a LA than might be expected, and that 5% topical benzocaine has no significant pharmacology. The small doses of bupivacaine and procaine producing effective local anaesthesia suggests that developing plasma thresholds for these agents is likely to be very challenging.

Summary Isoxsuprine is a therapeutic medication used to treat navicular disease and other lower limb problems in horses and is one of the more frequently detected therapeutic agents in racing horses. In a crossover study, horses were administered isoxsuprine i.v. to determine the character and duration of its pharmacological effects. Isoxsuprine significantly increased heart rate 5–150 min following injection. Unrestrained activity following isoxsuprine treatment was significantly greater than control activity for 105 min after treatment. There was an apparent, although statistically nonsignificant, increased cutaneous blood flow resulting in visible water vapour and sweat production 5–60 min after administration. Initially, there was no difference in skin temperature between control and isoxsuprine treatment values; however, skin temperature decreased below control values 45–120 min after injection. Concurrently, there was a significant decrease in rectal temperature reflecting a decrease in body core temperature. Using infrared thermography, a significant decrease in superficial skin temperature of the front legs occurred 30–240 min after treatment. Isoxsuprine also reduced smooth muscle tone, which was apparent by decreased tone of the internal anal sphincter 10–180 min after treatment. It was concluded that the measurable pharmacological effects of i.v. isoxsuprine are short lived, since none of the above responses were apparent 4 h or more after i.v. administration.

Amitraz, an acaricide used to control ectoparasites in animals has a complex pharmacological activity, including alpha2-adrenergic agonist action. The purpose of this research was to investigate the possible antinociceptive and/or sedative effect of amitraz in horses. The sedative effect of the intravenous (i.v.) injection of dimethylformamide (DMF, 5 mL, control) or amitraz (0.05, 0.10, 0.15 mg/kg), was investigated on the head ptosis test. The participation of alpha2-adrenergic receptors in the sedative effect provoked by amitraz was studied by dosing yohimbine (0.12 mg/kg, i.v.). To measure the antinociception, xylazine hydrochloride (1 mg/kg, i.v., positive control) and the same doses of amitraz and DMF were used. A focused radiant light/heat directed onto the fetlock and withers of a horse were used as a noxious stimulus to measure the hoof withdrawal reflex latency (HWRL) and the skin twitch reflex latency (STRL). The three doses of amitraz used (0.05, 0.10 and 0.15 mg/kg) provoked a dose-dependent relaxation of the cervical muscles. The experiments with amitraz and xylazine on the HWRL showed that after i.v. administration of all doses of amitraz there was a significant increase of HWRL up to 150 min after the injections. Additionally, there was a significant difference between control (DMF) and positive control (xylazine) values up to 30 min after drug injection. On the other hand, the experiments on the STRL show that after administration of amitraz at the dose of 0.15 mg/kg, a significant increase in STRL was observed when compared with the control group. This effect lasted up to 120 min after injection. However, no significant antinociceptive effect was observed with the 0.05 and 0.10 mg/kg doses of amitraz or at the 1.0 mg/kg dose of xylazine.