Research Accomplishment Reports 2010

High Sensitivity Analytical/Toxicological Approaches to Problems in Equine Medicine

T. Tobin
Department of Veterinary Sciences

 

Non-Technical Summary

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.

2010 Project Description

The project involves synthesis of standards for equine drugs/toxins and therapy of diseases caused by Toxoplasma gondii and has yielded 14 research publications, a commercialization license under negotiation and a UK patent disclosure. The standards project is synthesizing and making available to racing chemists standards certified to ISO-17025 standards for equine medications and metabolites. We have synthesized the urinary metabolite of xylazine, 4-OH xylazine, are completing synthesis of 4-Hydroxyxylazine-d6, hydroxyethylpromazine (HEP), hydroxyethyl-promazine sulfoxide [HEPS] and their internal standards, d4 hydroxyethyl promazine [HEP-d4] and [HEPS-d4], also carboxydetomidine, hydroxydetomidine and carboxydetomidine-d4 and hydroxydetomidine-d4. Others standards include 3-Hydroxylidocaine, 3-Hydroxylidocaine-d3, 4-Hydroxylidocaine, 3-Hydroxymepivacaine, 3-Hydroxymepivacaine-d3, 4-Hydroxymepivacaine-d3, 7-Hydroxyfluphenazine, 3-Hydroxypromazine, O-Desmethylpyrilamine, O-Desmethylpyrilamine-d3, O-Desmethyltramadol, O-Desmethyltramadol-d4. Standards being synthesized in larger quantities include Phenylbutazone-d9,Furosemide-d5, Clenbuterol-d9, Flunixin-d3, Ketoprofen-d4, Methocarbamol-d4, Guaifenesin-d4, Procaine-d10, Butorphanol-d6, Pyrilamine-d3 Tetrahydrogestrinone-d4 Tramadol-d3 and others. These outcomes have been communicated in the scientific literature and this technology is being licensed for commercialization. We are also exploring a novel total synthesis pathway for ergovaline and parallel development of highly ergovaline specific ELISA tests. Recent work has shown that serum ergovaline concentrations in horses on ergovaline positive [E+] pastures are very low, lower than concentrations usually associated with toxicological effects, in agreement with work by Strickland showing that the in vitro vasoconstrictive actions of ergovaline are long-lasting, consistent with ergovaline being the critical toxin in fescue toxicosis. We are focusing on the novel total synthesis of ergovaline to enable in vivo ergovaline toxicity experiments, on creating reference standards and internal standards for ergovaline, and also synthesizing unique haptens to allow creation of specific ergovaline ELISA tests. This project is well underway and we have made considerable progress with regard to synthesis of the unique ergovaline ergopeptide. The third project addresses wider application of the therapeutic efficacy of diclazuril in the treatment of Toxoplasmosis. In many species, man included, Toxoplasma infection produces fetal damage with significant lifelong adverse consequences for the fetus. Fetal treatment in utero is challenging because currently used therapeutics, pyrimethamine -sulfonamide combinations, interfere with nucleic acid metabolism and are fetoxic. Given the unique and highly specific anti-apicomplexan mechanism of diclazuril, entirely unrelated to nucleic acid metabolism, and its low mammalian toxicity, we have demonstrated the therapeutic efficacy diclazuril in a fetal toxoplasmosis model, which data formed the basis of a recent patent/intellectual property disclosure to the University of Kentucky.

2010 Impact

This project addresses three audiences.

Project 1 addresses racing regulation and horse racing worldwide and Kentucky's Thoroughbred industry. The increase in drug testing sensitivity has created a requirement for certified reference standards and internal standards for equine therapeutic medication regulation. About 20 of these reference standards have been synthesized and are in the process of being licensed to, Frontier Biopharm, a Kentucky company, which will market these standards worldwide.

This project underlies the regulation of medication in racing horses, and as such the image and integrity of racing worldwide. This project has been challenging. Most of the easy reference standards have been synthesized, so synthesis of the needed standards has been challenging, and the need to synthesize the matching stable isotope standard adds further complexity. Another consideration is the need to add the stable isotope material late in the synthesis process to minimize synthesis costs. Given these technical challenges, we are pleased to report considerable progress in this area, including the ongoing licensing of this product line to Frontier Biopharm.

The second project addresses a significant portion of the world population. This project builds on our highly successful technology patented in 1999, US Patent # 5, 883, 095". Formulations and methods to treat and prevent Equine Protozoal Myeloencephalitis [EPM]" based on diclazuril. Between 30 and 60% of humans worldwide are infected with Toxoplasma gondii, and susceptible to drugs we have demonstrated to be highly effective in equines. Three human toxoplasmosis syndromes are described, ocular, central nervous system, and fetal toxoplasmosis. With regard to fetal toxoplasmosis, we have just demonstrated the prophylactic/therapeutic efficacy of diclazuril in a mouse model of fetal toxoplasmosis, establishing proof of principle for the use of diclazuril in the treatment of human fetal toxoplasmosis. An intellectual property disclosure has just been presented to the University of Kentucky and the goal of this project is to produce a highly effective and unusually safe approach to human fetal toxoplasmosis, a serious medical condition affecting many thousands of humans worldwide every year.

The third project addresses development of improved diagnostic/detection methods for fescue toxicosis in domestic animals. Ergot alkaloid/fescue toxicosis causes > $1 billion/year in economic losses in the US. Research is hindered by the non-availability of sufficient quantities of chemically pure ergot alkaloids to study the clinical toxicosis in domestic animals and identify the specific causative alkaloid(s). We are developing a novel and inexpensive synthesis of pure ergovaline and the required stable isotope internal standard for toxicological research.

Additionally, we are synthesizing specific ergovaline peptide haptens with the goal of developing specific ELISA tests for individual ergot alkaloids, starting with ergovaline, the candidate alkaloid toxin. This work is ongoing but has not yet reached the stage of licensing or patent disclosure attained by projects one and two.

2010 Publications

Tobin T, Eisenberg, R., Gutierrez, J., Long, S., Li, T., Tharappel, J., Hughes, C., Armstrong, E., Mayer, B., Stanley, E., Lyons, M and Karpiesiuk, W. (2010) Certified Reference Standards and Stable Isotope Internal Standards for Equine Therapeutic Medication Regulation. Proceedings of the 18th International Conference of Racing Analysts and Veterinarians, Queenstown, New Zealand.

Tobin, T., Gutierrez, J., and Eisenberg, R. (2010) Experiences as an Expert: Educating People While Under Oath, 25 Years since Hong Kong. Proceedings of the 18th International Conference of Racing Analysts and Veterinarians, Queenstown, New Zealand.

Gutierrez, J., Eisenberg, R.L., Koval, N.J., Armstrong, E.R., Tharappel, J., Hughes, C.G., and Tobin, T. (2010) Pemoline and Tetramisole Positives In English Racehorses Following Levamisole Administration. Irish Vet J 63(8):498-500.

Lehner, A., Hitron, J., May, J., Hughes, and CG. Eisenberg, R., Schwint, N., Timoney, P. and Tobin, T. (2010) Evaluation of Mass Spectrometric Methods for Detection of the Anti-protozoal Drug Imidocarb. Accepted for publication, Journal of Analytical Toxicology.

Tobin T, Gutierrez, J, Schwartz, E, Camargo, F, Hughes,C G, Eisenberg, R, Lehner, A and Stirling, K, 2010; Equine Drugs, Medications, and Performance Altering Substances: Their Performance Effects, Detection, and Regulation: www.thomastobin.com presentation reworked for incorporation into EQS 214 Equine Healthcare Management, 3 credits, Midway College, Midway, Kentucky.