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Hordenine is an alkaloid occurring naturally in grains, sprouting barley, and certain grasses. It is occasionally found in post race urine samples, and therefore we investigated its pharmacological actions in the horse. Hordenine (2.0 mg/kg bodyweight [bwt]) was administered by rapid intravenous (iv) injection to 10 horses. Typically, dosed horses showed a flehmen response and defecated within 60 secs. All horses showed substantial respiratory distress. Respiratory rates increased about 250 per cent and heart rates were approximately double that of resting values. All animals broke out in a sweat shortly after iv injection, but basal body temperature was not affected. These effects were transient, and the animals appeared normal within 30 mins of dosing. Treated horses were tested in a variable interval responding apparatus 30 mins after dosing and no residual stimulation or depressant effects of hordenine were apparent. Animals dosed orally with 2.0 mg/kg bwt of hordenine showed no changes in heart rate, respiratory rate, basal body temperature or behaviour. After iv injection of hordenine, (2.0 mg/kg bwt) plasma reached a maximum value of about 1.0 micrograms/ml, and declined thereafter in a biexponential fashion. Kinetics of plasma concentration satisfied the concept of a two compartment open system, with an alpha-phase half-life of about 3 mins, and a beta-phase half-life of about 35 mins. Total urinary concentrations of hordenine (free and conjugated) peaked at about 400 micrograms/ml, and then declined exponentially to background levels by 24 h after dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Our investigation of the urine of grazing horses at the University of Kentucky shows that the mean pH level is about 7.9, and if their diet is supplemented with grain, it is about 7.4. There appears to be no significant effect of time of day or year on urine pH levels in horses. However, horses taken from pasture and supplemented with grain in a stalled environment show a slight decrease in urine pH. Additionally, we investigated the effects of storage on pH levels. Equine urine samples appear to be quite stable with regard to pH for 48h, but then show a marked increase. Urine pH can have a great effect on the urine concentration of some drugs and therefore, uncertainties can arise when data generated in grazing horses are compared or extrapolated to racing horses whose urine pH can be quite low. In an effort to simulate the drop in urine pH seen in some racing horses, we examined the effects of ammonium chloride, ascorbic acid, lactic acid and methionine on urine pH in research horses. Both oral and intravenous routes of administration were used. Although all agents tested showed varying degrees of efficacy, oral administration of ascorbic acid proved to be the safest and most effective agent to model the rapid acidification of urine seen in post race samples.

The effects of procaine, mepivacaine and phenylbutazone on pain perception in the equine were studied using two behavioral assays of nociception; the thermal evoked hoof withdrawal reflex and skin twitch reflex. Pain perception threshold was measured as the latency from onset of thermal stimuli to reflex withdrawal of the forelimb or contraction of the cutaneous musculature. Procaine 2% and mepivacaine 2% prolonged the hoof withdrawal reflex latency when administered locally by producing a block of the palmar and metacarpal nerves. Significant analgesia lasted 90 min and 210 min for procaine and mepivacaine, respectively. Phenylbutazone (7.3 mg/kg) failed to alter pain thresholds measured over a 36 h post-treatment period. However, pain thresholds rose over time with successive trials. These data suggest that in the equine (1) phenylbutazone does not alter normal cutaneous pain perception, and (2) successive presentation of painful stimuli increases nociceptive thresholds.

Sensitive methods for measuring the analgesic, physiological and behavioural effects of opioids in the horse have recently been developed. Fentanyl, a prototypic mu-opiate receptor agonist, has been previously shown to produce a syndrome characterized by marked analgesia and locomotor stimulation as well as tachycardia, tachypnoea and behavioural arousal. To determine whether other opiate receptors mediate some of the actions of the narcotic analgesics in the horse, an agent with activity at kappa- and to lesser extent mu-receptors was studied using a vigorous experimental protocol. Like fentanyl, ethylketazocine (EKC) (0.0025-0.012 mg kg-1 i.v.) produced marked dose-related analgesia to noxious thermal stimuli. Modest dose-related increases in locomotor activity, pupil diameter and rectal temperature were also observed. However, in contrast to fentanyl, EKC failed to produce any change in cardiac or respiratory rates and produced behavioural sedation rather than arousal. These data suggest that mu- and possibly kappa-receptors can mediate the actions of narcotics in the horse.

The dose-related effects of intravenously administered fentanyl (0.010, 0.005, 0.0025 mg/kg) and saline were studied in mature performance horses using a rigorous experimental protocol. Fentanyl produced a dose-related prolongation of the skin twitch reflex latency but did not increase the hoof withdrawal reflex latency. Dose related increases in stepping frequency, cardiac and respiratory rats were observed following fentanyl, while changes in rectal temperature and pupil area were not. These data indicate that fentanyl, a prototypic mu-agonist, produces a syndrome characterized by analgesia, locomotor and sympathetic stimulation in the horse.

Pain perception and its alteration by analgesic drugs is difficult to measure in the horse. The latency to onset of flexion of a limb in response to a noxious thermal stimulus has been used as a nociceptive end point for analgesic studies in many species. While this method has been employed in the horse, it may be confounded by the spontaneous locomotor activity observed after administration of narcotic analgesics. Consequently, an alternative method of assaying narcotic analgesia that did not involve the equine locomotor apparatus was developed. This report describes the use of the heat-evoked skin-twitch reflex as a reproducible measure of pain threshold and its alteration by the narcotic analgesic fentanyl. This method is compared with the heat-evoked hoof-withdrawal reflex, and the apparatus necessary to elicit both reflexes in the horse is described. Fentanyl, administered at intravenous doses of 0.010, 0.005, and 0.0025 mg/kg, produced a dose-related prolongation of the skin-twitch reflex but failed to alter the latency to hoof withdrawal following noxious thermal stimulation. The skin-twitch reflex is therefore a more sensitive assay of narcotic analgesia in the horse than is the hoof-withdrawal reflex.

We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test for sufentanil and a 125I radioimmunoassay test for alfentanil as part of a panel of pre- and post-race tests for narcotic analgesics in racing horses. Our sufentanil ELISA test detects sufentanil with an I-50 of about 0.5 ng/ml. The test is rapid and economical in that it can be read with an inexpensive spectrophotometer, or even by eye. The test readily detects the presence of sufentanil or its metabolites in equine blood and urine from 1 to 24 hours respectively after administration of therapeutic or sub-therapeutic doses of this drug. Our sufentanil assay also cross-reacts with fentanyl, the methylated analogs of fentanyl (designer fentanyls), and carfentanil and detected these drugs in urine for several hours after their administration to horses. It does not, however, cross-react significantly with alfentanil. We have also developed an 125I radioimmunoassay for alfentanil. This test allows detection of alfentanil in blood and urine of horses for up to 4 hours after administration of this drug. As such, these tests are capable of improving the quality and reducing the cost of pre-race and post-race testing for fentanyl, sufentanil, carfentanil and alfentanil and a number of their congeners in racing horses. Similarly, these tests are capable of screening for these drugs in human drug abuse monitoring.

Pharmacologic effects of alpha-methylfentanyl and 3-methylfentanyl, analogs of fentanyl, were investigated in mares. The ability of an 125I-labeled fentanyl radioimmunoassay (125I-RIA) to detect these methylated fentanyl analogs in individual and pooled urine samples from horses was evaluated. Also, the ability of 7 fentanyl antibodies to react with fentanyl and fentanyl derivatives (sufentanil, alfentanil, and carfentanil) was investigated. Mares were studied in a locomotor test to determine the amount of stimulation methylated fentanyl analogs might induce. Two mares each were given alpha-methylfentanyl at 1, 2, 4, 8, or 13 micrograms/kg of body weight, IV, or 3-methylfentanyl at 0.4, 0.7, or 1 microgram/kg IV. The cross-reactivity of sufentanil, alfentanil, carfentanil, alpha-methylfentanyl, and 3-methylfentanyl with 7 fentanyl antibodies was studied, using the 125I-RIA. All fentanyl analogs, with the exception of alfentanil, cross-reacted well with a C1 antibody raised to fentanyl. Less satisfactory cross-reactivity was determined with 6 other antibodies raised to fentanyl derivatives. When the C1 antibody was combined with an iodinated analog to fentanyl, good detectability of alpha-methylfentanyl and 3-methylfentanyl, in terms of fentanyl equivalents, was obtained from urine samples of dosed mares. The ability of the 125I-RIA to detect methylated fentanyl analogs in forensic urine samples pooled in groups of up to 20 samples was evaluated. When these methylated analogs were administered to mares in doses that induced measurable locomotor stimulation, the analog's presence was readily detected in individual or pooled samples.

We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test and a particle concentration fluorescence immunoassay (PCFIA) test for acepromazine as part of a panel of pre- and post-race tests for illegal medications in racing horses. These tests are rapid, sensitive and economical and development of the tests occurred in less than seven months. The ELISA test detects acepromazine with an I-50 of about 150 pg/ml. In vivo, it readily detects the presence of acepromazine or its metabolites in equine blood and urine from 8 to 72 hours or longer, respectively, after administration of sub-therapeutic doses. In vitro, the ELISA test cross-reacts with analogs of acepromazine, suggesting that it will also detect the use of other phenothiazine tranquilizers. The PCFIA test detects acepromazine with an I-50 of about 10 ng/ml. When applied to pre-race screening of serum samples as part of the pre-race testing program at a midwestern racetrack, the PCFIA test detected a number of cases of acepromazine abuse. Screening of stored post-race urine samples from associated horses by the ELISA test 'flagged' numerous samples for acepromazine, suggesting a pattern of acepromazine abuse. To date about twenty of these acepromazine flagged samples have been confirmed positive on mass spectrometry. As such the ELISA and PCFIA tests described in this communication are capable of substantially improving the quality of pre- and post-race testing programs for phenothiazine tranquilizers in racing horses.

1. Drug administration studies using diisopropylamine dichloroacetate (DADA) and diisopropylamine (DIPA) were conducted in Thoroughbred and Standardbred horses to assess physiological effects and develop detection methods. 2. Four horses received 0.08 mg DADA/kg body wt and showed no changes in heart and respiratory rates or body temperature as measured over a 1-hr period after administration. A transient diuretic effect was found to occur in 2 mares dosed with 0.80 mg DADA/kg body wt. 3. A qualitative detection method using thin-layer chromatography was developed to detect DIPA, the major metabolite of DADA in equine urine. A quantitative detection method (lower limit of detection 0.5 micrograms/ml urine) for this metabolite was also developed using gas chromatography. 4. Neither DADA or the free base, DIPA, were detectable in equine blood samples using the above-mentioned methodologies.

A one step enzyme-linked immunosorbent assay (ELISA) and a particle concentration fluorescent immunoassay (PCFIA) test for furosemide were evaluated as part of a panel of pre- and post-race tests for illegal medication of racing horses. These tests are very sensitive to furosemide with an I-50 for furosemide of about 20 ng/ml. The test is also rapid; an average pre-race complement of 10 samples can be analyzed in 90 minutes or less. The ELISA test results can be read with an inexpensive spectrophotometer, or even by eye. Both the PCFIA test and the ELISA test readily detect the presence of furosemide in equine blood for up to five hours after administration of the recommended therapeutic dose of this agent. The principal utility of these tests lies in rapid screening of samples for compliance with regulations governing the use of furosemide. Thus these tests can be used pre-race to determine whether horsemen have treated their horses with furosemide, and post-race to perform an initial evaluation of whether certain blood concentrations of furosemide have been exceeded. Pilot trials with these systems in Kentucky and Illinois suggest that these tests are economical and effective, and can form part of an analytical approach to substitute for the detention barn system of monitoring furosemide administration.

Narcotic analgesics produce pharmacological effects by interacting with specific opiate receptors. At least five major types of opiate receptors have been recognised. These include mu (morphine) and kappa (ethylketazocine) receptor types. Narcotic analgesics which interact with mu receptors produce locomotor and autonomic stimulation at doses that produce little or no analgesia. Therefore, use of these drugs as analgesics in equine medicine has not been very satisfactory. Theoretical considerations suggested that the role of kappa agonists in equine analgesia be investigated. Using a pure kappa agonist, U-50, 488H, good analgesia was produced in the horse with little or no locomotor stimulation or autonomic effects. These data suggest that kappa agonists may be superior analgesics for clinical use in the horse. On the other hand, the locomotor stimulant effects of mu agonist analgesics enable their use as illegal medications. Specifically, these agents produce a good running response, signs of central nervous stimulation and analgesia, all potentially useful effects in a racehorse. Regulatory control of most narcotic analgesics can be obtained by high performance thin layer chromatographic screening. However, effective screening for the fentanyls and small doses of etorphine can only be achieved by use of immunoassay.

Phenylbutazone is an acidic, lipophilic, non-steroidal anti-inflammatory drug (NSAID). It is extensively metabolized in the horse. The metabolites so far identified, oxyphenbutazone, gamma-hydroxyoxyphenbutazone, account for some 25-30% of administered dose over 24 h. The plasma half-life of phenylbutazone and termination of its pharmacological action are determined primarily by its rate of hepatic metabolism. Phenylbutazone acts by inhibiting the cyclooxygenase enzyme system, which is responsible for synthesis of prostanoids such as PGE2. It appears to act on prostaglandin-H synthase and prostacyclin synthase, after conversion by prostaglandin-H synthase to reactive intermediates. It markedly reduces prostanoid-dependent swelling, edema, erythema, and hypersensitivity to pain in inflamed tissues. Its principal use in the horse is for treatment of soft tissue inflammation. Phenylbutazone is highly bound (greater than 98%) to plasma protein. After i.v. injection, blood levels decline with an elimination half-life of 3-10 h. The plasma kinetics of phenylbutazone may be dose dependent, with the plasma half-life increasing as the drug dosage level increases. Plasma residues of the drug at 24 h after a single i.v. dose of 2 g/450 kg average about 0.9 microgram/ml, but considerable variation occurs. If dosing is repeated, the plasma residue accumulates to give mean residual blood levels of approximately 4.5 microgram/ml on Day 5 after 4 days of dosing. Approximately similar blood levels are found after a combination of oral and i.v. dosing. Experiments on large numbers of horses in training have been undertaken to ascertain the population distributions of residual blood levels after such dosing schedules. Absorption of phenylbutazone from the gastrointestinal tract is influenced by the dose administered and the relationship of dosing to feeding. Access to hay can delay the time of peak plasma concentration to 18 h or longer. Under optimal conditions, the bioavailability of oral phenylbutazone is probably in the region of 70%. Paste preparations may be more slowly absorbed than other preparations and yield higher residual plasma levels at 24 h after dosing, but further controlled studies are required. Phenylbutazone is easily detected in the plasma and urine of horses but concentrations in saliva are low. It is quantitated for forensic purposes by HPLC. The variability of this method between laboratories is about +/- 25%. Increasing urinary pH increases the urinary concentration of phenylbutazone and its metabolites up to 200-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Detomidine is a potent non-narcotic sedative agent which is currently in the process of being approved for veterinary clinical use in the United States. Since no effective screening method in horses is available for detomidine, we have developed an 125I radioimmunoassay for detomidine in equine blood and urine as part of a panel of tests for illegal drugs in performance horses. Our 125I radioimmunoassay has an I-50 for detomidine of approximately 2 ng/ml. Our assay shows limited cross-reactivity with the pharmacodynamically similar xylazine, but does not cross-react with acepromazine, epinephrine, haloperidol or promazine. The plasma kinetic data from clinical (greater than or equal to 5 mg/horse) as well as sub-clinical doses indicate first-order elimination in a dose-dependent manner. Within the first 30 minutes after intravenous (IV) administration of 30 mg/horse, plasma levels peak at approximately 20 ng/ml and then decline with an apparent plasma half-life of 25 minutes. Diuresis can occur with administration of clinical doses of detomidine and this effect was accounted for in the analysis of urine samples. Using this method, administration of 30 mg/horse can be readily detected in equine urine for up to 8 hours after IV injection. Additionally, doses as low as 0.5 mg/horse can be detected for short periods of time in blood and urine with use of this assay. Utilization of this assay by research scientists and forensic analysts will allow for the establishment of proper guidelines and controls regarding detomidine administration to performance horses and assurance of compliance with these guidelines.

To improve the sensitivity and specificity of screening for etorphine in horses, an 125I-labeled etorphine analog was synthesized and an antibody to etorphine was raised in rabbits. A radioimmunoassay (RIA) for etorphine was developed, using these reagents. Bound and free 125I-labeled etorphine was separated by a double-antibody method that reduced interference from materials associated with equine urine. The 125I-labeled etorphine binding was rarely greater than 250 pg of background etorphine equivalents/ml in raw urine and was 100 pg/ml in hydrolyzed urine. The 125I-RIA was capable of detecting etorphine equivalents in urine above these background values. Etorphine equivalents were detected in equine urine samples for about 7 days after 4 mares were dosed with 0.22 microgram of etorphine/kg of body weight, IV. The stability of etorphine in urine from these mares was evaluated. Urine from these dosed mares was held in constant -20 C storage, and aliquots were repeatedly frozen and thawed. When analyzed for etorphine equivalents using an 125I-RIA, etorphine and its metabolites in urine samples were stable for less than or equal to 38 days if continuously frozen and also were resistant to repeated freezing and thawing.

We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test for fentanyl as part of a panel of pre- and post-race tests for narcotic analgesics in racing horses. This ELISA test detects fentanyl with an I-50 of about 100 pg/ml. The test is economical in that it can be read with an inexpensive spectrophotometer, or even by eye. The test is rapid, and ten samples, a normal pre-race complement, can be analyzed in about twenty minutes. The test readily detects the presence of fentanyl or its metabolites in equine blood and urine from two and twenty-four hours respectively after administration of sub-therapeutic doses. The two antibodies evaluated also cross-react with the methylated analogs of fentanyl, sufetanil and carfentanil and the test detected these drugs shortly after their administration to horses. When introduced into routine screening, this test, in combination with another immunoassay test previously described, yielded 10 sufentanil positives. As such this test is capable of both improving the quality and reducing the cost of pre-race and post-race testing for fentanyl and a number of its congeners in racing horses.