Pharmacology of procaine in the horse: procaine esterase properties of equine plasma and synovial fluid.

Procaine added to whole equine blood or diluted plasma was hydrolyzed with half times of approximately 9 and 12 minutes, respectively, at 37 C. This hydrolytic activity was sensitive to heating and physostigmine, but did not affect procainamide. At pharmacologic concentrations of procaine, the rate of the hydrolytic reaction depended directly on the concentrations of plasma or procaine in the system and was less in whole blood than in plasma. These properties are consistent with hydrolysis being due to plasma esterases operating at less than saturating procaine concentrations. These esterases were also inhibited cooling, sodium fluoride, or arsenite. Synovial fluid had approximately 20% of the procaine esterase activity of plasma. Comparison of hydrolytic activities of plasmas from Thoroughbred, Standardbred, and other breeds of horses showed statistically significant differences in the rates at which individual plasmas hydrolyzed procaine. A frequency distribution of these rates showed unimodal distribution, indicating that all horses tested may be regarded as members of a single population.

Pharmacology of procaine in the horse: evidence against the existence of a "procaine - penicillin" complex.

It has recently been suggested that procaine penicillin existed in solution in vitro and in vivo as a "procaine - penicillin" complex rather than as dissociated ions. In vivo, this complexed procaine was considered unavailable for hydrolysis by plasma esterases or for interaction with pharmacologic receptors for procaine. When procaine penicillin was intramuscularly given to horses, about 90% of the procaine in blood drawn from these horses was split at the same rate as authentic procaine or procaine penicillin added to equine blood in vitro. In vitro, procaine and procaine penicillin partitioned similarly from aqueous medium at physiologic pH into several organic solvents and were split at the same rate by blood or plasma esterases. Experiments on the time course of the partitioning of procaine from procaine penicillin into benzene showed no evidence for the existence of a "procaine - penicillin" complex within seconds after procaine penicillin was added to aqueous medium. Thin layer chromatography in 2 dimensions also yielded no evidence for the existence of this postulated complex. These results show no evidence in support of the "procaine - penicillin" hypothesis and argue against the physical and pharmacologic and forensic implications of this hypothesis.

Immunoassay detection of drugs in racing horses. VII. Detection of acepromazine in equine urine and blood by ELISA and PCFIA.

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.

Immunoassay detection of drugs in racing horses. IX. Detection of detomidine in equine blood and urine by radioimmunoassay.

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.