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High Sensitivity Analytical/Toxicological Approaches to Problems in Equine Medicine
Department of Veterinary Sciences
The project addresses the problem of two classes of naturally occuring environmental toxins one of which causes substantial economic damage and the second of which is unique model of laminitis a common equine disease. The purpose of this project is to identify the specific toxins and the specific mechanisms of these toxicities; understanding of the mechanism of the toxicity will allow development of appropriate preventative, prophylactic and therapeutic approaches to these problems.
2009 Project Description
The program has 3 focus areas:
Project 1, REFERENCE STANDARDS, involves the synthesis and certification of reference standards for equine therapeutic medications, metabolites and metabolite fragments and the corresponding stable isotope internal standards.
Project 2 focuses on novel therapeutic approaches to infectious diseases caused by Toxoplasma gondii, while
Project 3, focuses on development of improved diagnostic tests for ergot alkaloid toxins.
During 2009, we worked on the synthesis and characterization of certified reference standards and deuterated internal standards for the equine urinary drug metabolite fragments hydroxyethylpromazine [HEP] and hydroxyethylpromazine sulfoxide [HEPS], a D6 internal standard for butorphanol, on synthesis of 4-hydroxyxylazine, the recovered equine drug metabolite fragment of the tranquilizer xylazine and its corresponding deuterated internal standard, and also on synthesis of deuterated standards for the equine drug metabolite fragments of mepivacaine and lidocaine, namely deuterated 3 and deuterated 4 hydroxymepivacaine, and deuterated 3 hydroxylidocaine.
Other projects commenced include synthesis of deuterated internal standards for two corticosteroids use routinely as therapeutic medications in equine medicine, namely, flumethasone and Isoflupredone.
These certified reference standards and internal standards are designed for use by racing chemists worldwide. They were initially created as part of a University of Kentucky research project focused on establishing "proof of principle" for the concept of equine therapeutic medication regulation by use of quantitative regulatory thresholds. This concept is now well-established and there is now significantly increased demand for these standards.
A new company, Frontier Biopharm, of Richmond Kentucky http://www.frontierbiopharm.com has been created to, in cooperation with the University of Kentucky and the Neogen Corp, assist with the commercial synthesis certification and marketing of these standards worldwide. Appropriate intellectual property disclosures have been made to the University of Kentucky and the Neogen Corp. The current projected line of reference standards includes standards for hydroxyethylpromazine [HEP], hydroxyethylpromazine sulfoxide [HEPS], D6 butorphanol, 4-hydroxyxylazine,3-and 4-hydroxy mepivacaine, 3-hydroxy lidocaine and their deuterated equivalents, deuterated flumethasone and Isoflupredone, deuterated clenbuterol, hydroxy and carboxy detomidine and their deuterated equivalents, deuterated furosemide, guafenesin, ketoprofen, methocarbamol, phenylbutazone, procaine, and 1-(4-Amino-3,5-Dichlorophenyl)ethane-1,2-diol, and deuterated pyrilamine.
Additionally we have made available standards for 7-OH fluphenazine and desmethyltramadol and this venture has gained significant research and commercial support.
Project number 2 has potentially the greatest impact of all three projects. The medication being evaluated is highly specific for apicomplexans and has an excellent record of success in veterinary medicine, including our highly successful patented application in the treatment of Equine Protozoal Myeloencephalitis [EPM]. This therapeutic efficacy is due to its unique biological mechanism of action and very low mammalian toxicity, in marked contrast with currently available anti-apicomplexan therapies.
Preliminary evaluations of our most recent mouse fetal experiments are very promising, and based on these data we propose to revisit our ocular toxoplasmosis experiments.
The third project addresses the problem of ergot alkaloid toxicosis, a major economic problem in the American South East, and also closely related alkaloid toxicoses occur throughout the world. We have shown the ergot alkaloids to be exceptionally potent toxins, and until recently it has been technically difficult to identify specific ergot alkaloid toxins in blood samples.
To solve this problem, inexpensive, sensitive and relatively specific tests for ergot alkaloids are required, and we are applying the ELISA technology successfully used in equine drug testing to the ergot alkaloid toxicoses. To this end, we have synthesized two highly specific ergot alkaloid haptens which should form the basis of two highly specific and sensitive and alkaloid ELISA immunoassays.
Dirikolu, L., Lehner, A.F., Hughes, C., Karpiesiuk, W., and Tobin, T. (2009) The effect of dimethyl sulfoxide on oral bioavailability of Triazine-based anti-protozoal agent Toltrazuril sulfone in horses. Proc. 16th International Conference of Racing Analysts and Veterinarians, Antalya, Turkey.
Dirikolu, L., Woods, W.E., Boyles, J., Lehner, A.F., Harkins, J.D., Fisher, M., Schaeffer, D.J., and Tobin, T. (2009) Musculoskeletal injuries and NSAIDS in Thoroughbred racehorses. Proc. 16th International Conference of Racing Analysts and Veterinarians, Antalya, Turkey, in press.
Tobin, T., and Stirling, K. (2009) National Horsemen's Benevolent and Protective Association Inc., Proposed National Policy on Drug Testing and Therapeutic Medication Regulation. Hershey, Pennsylvania Wind Publications Lexington, KY.
Dirikolu, L., Lehner, A.F., Harkins, J.D., Woods, W.E., Karpiesiuk, W., Gates, R.S., Fisher, M., and Tobin, T. (2009) Pyrilamine in the horse: Detection and pharmacokinetics of pyrilamine and its major urinary metabolite "O-desmethylpyrilamine". J Vet Pharmacol Ther. 32(1):66-78
Lehner, A.F., Petzinger, E., Stewart, J., Lang, D.G., Johnson, M.B., Harrison, L., Seanor, J.W., and Tobin, T. (2009) ESI+MS/MS confirmation of canine ivermectin toxicity J Mass Spectrom. 44(1):111-119.
Dhanjal, J.K. , Wilson, D.V., Robinson, N.E., and Tobin. T. (2009) Effects of intravenous tramadol in horses. Veterinary Anaesthesia and Analgesia, 1467-2995.2009.00492.x
Lehner, A.F., Hiltron, J.A., May, J., Hughes, C.G., Eisenberg, R., Timoney, P., and Tobin, T. (2009) Imidocarb Detection by ESI-MS/MS Mass Spectrometric Methods. Journal of Mass Spectrometry (pending).
Tobin, T., Dhanjal, J.K., Wilson, D.V., Hughes, C.G., Karpiesiuk, W., Spencer, W., Tharappel, J., Dirikolu, L., Lehner, A., and Robinson, N.E. (2009) Tramadol in the horse: A preliminary report on its detection, pharmacokinetics and pharmacodynamic responses. Proceedings of the 17th International Conference of Racing Analysts and Veterinarians, Antalya, Turkey.