SUMMARY A radioiodinated analog of fentanyl was synthesized for use with a commercially available radioimmunoassay for fentanyl. The sensitivity of the modified assay was at least 100 times greater than that of the original assay. Using this modified assay, concentrations of fentanyl as low as 1 pg/ml of fentanyl or fentanyl equivalents in equine urine were detected. Doses of fentanyl 100 times smaller than the minimum dose for a pharmacologic effect were detectable and a pharmacologically effective dose of fentanyl was detectable for up to 96 hours or more. The high sensitivity of the assay indicated that large numbers of urine samples (ie, 10 to 20) probably could be pooled and screened simultaneously, which would result in an economical analysis for fentanyl in the urine of horses after a race. Sufentanil and its metabolites also were detectable, using this assay, but at only about 1% of the efficiency at which fentanyl was detectable.
Summary Narcotic analgesics produce pharmacological effects by interacting with specific opiate receptors. At least five major types of opiate receptors have been recognised. These include μ (morphine) and kappa (ethylketazocine) receptor types. Narcotic analgesics which interact with μ 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 μ 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.
Summary Current opiate receptor theory suggests that kappa agonists should provide good analgesia without producing marked central nervous system stimulation. U‐50,488H is an experimental narcotic analgesic that is a selective kappa agonist. In the present study, U‐50,488H produced good analgesia in horses using both the skin twitch and hoof withdrawal reflex assays. Further, the analgesia was relatively long lasting (120 mins) compared to other μ‐agonists tested in horses. The locomotor response to U‐50,488H was less than observed with ethylketazocine and butorphanol, and has yielded the smallest locomotor response of any of the narcotic analgesics tested to date. Other work showed that the autonomic responses to U‐50,488H were less than those of other narcotic analgesics, and that the analgesic response to this drug was blocked by naloxone. Based on its ability to produce analgesia with little other stimulatory action, U‐50,488H shows promise of becoming a useful narcotic analgesic in equine medicine.
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.
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.
SUMMARY Pharmacologic effects of α-methylfentanyl and 3-methylfentanyl, analogs of fentanyl, were investigated in mares. The ability of an 125 I-labeled fentanyl radioimmunoassay ( 125 I- 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 α-methylfentanyl at 1, 2, 4, 8, or 13 μg/kg of body weight, iv , or 3-methylfentanyl at 0.4, 0.7, or 1 μg/kg iv . The cross-reactivity of sufentanil, alfentanil, carfentanil, α-methylfentanyl, and 3-methylfentanyl with 7 fentanyl antibodies was studied, using the 125 I- ria . All fentanyl analogs, with the exception of alfentanil, cross-reacted well with a CI antibody raised to fentanyl. Less satisfactory cross-reactivity was determined with 6 other antibodies raised to fentanyl derivatives. When the CI antibody was combined with an iodinated analog to fentanyl, good detectability of α-methylfentanyl and 3-methylfentanyl, in terms of fentanyl equivalents, was obtained from urine samples of dosed mares. The ability of the 125 I- 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.
Experiments to determine the residual plasma concentrations of phenylbutazone and its metabolites found in horses racing on a 'no-race day medication' or 24-h rule were carried out. One dosing schedule (oral-i.v.) consisted of 8.8 mg/kg (4 g/1000 lbs) orally for 3 days, followed by 4.4 mg/kg (2 g/1000 lbs) intravenously on day 4. A second schedule consisted of 4.4 mg/kg i.v. for 4 days. The experiments were carried out in Thoroughbred and Standardbred horses at pasture, half-bred horses at pasture, and in Thoroughbred horses in training. After administering the i.v. schedule for 4 days to Thoroughbred and Standardbred horses at pasture, the mean plasma concentrations of phenylbutazone increased from 0.77 microgram/ml on day 2 to 2.5 micrograms/ml on day 5. The shape of the frequency distribution of these populations was log-normal. These data are consistent with one horse in 1,000 yielding a plasma level of 8.07 micrograms/ml on day 5. After administration of the oral-i.v. schedule to Thoroughbred and Standardbred horses at pasture, the mean plasma concentrations of phenylbutazone were 3.4 micrograms/ml on day 2 and 3.5 micrograms/ml on day 5. The range on day 5 was from 1.4 to 8.98 micrograms/ml and the frequency distribution was log-normal. These data are consistent with one horse in 1000 having a plasma level of 15.8 micrograms/ml on day 5. In a final experiment, the oral dosing schedule was administered to 62 Thoroughbred horses in training. Plasma concentrations on day 5 in these horses averaged 5.3 micrograms/ml. The range was from 1.3 to 13.6 micrograms/ml and the frequency distribution was log-normal. Statistical projection of these values suggests that following this oral dosing schedule in racing horses about one horse in 1000 will yield a plasma level of 23.5 micrograms/ml of phenylbutazone 24 h after the last dose.
