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Reference Standards, Internal Standards and Critical Reagents/Regulatory Analytes for Analytical/Toxicological Approaches to Problems in Equine Medicine
Department of Veterinary Sciences
1/ To identify, synthesize and make available to equine forensic chemists worldwide certified reference standards for unique urinary drug/metabolite fragments for therapeutic medication regulation.
2/ To synthesize and make available to equine forensic chemists worldwide deuterated or other stable isotope internal reference standards for the above unique urinary drug/metabolite fragment standards to allow accurate forensic quantification.
3/ To identify and synthesize certified reference standards and stable isotope internal standards for glucuronide metabolites of high potency therapeutic medications.
4/ To develop cost-effective methods of synthesizing ergovaline and related ergot alkaloids and haptens of same with the goal of creating highly sensitive and specific ELISA tests for these substances.
5/ To complete ongoing work on establishing the therapeutic efficacy of diclazuril in the therapy and prophylaxis of certain Toxoplasma related syndromes.
2011 Project Description
Three projects are presented in this report.
The first involves creation of certified reference standards and deuterated internal standards for equine therapeutic medication metabolite fragments. During this calendar year a number of reference standards were synthesized, purified, and certified to the point that they are appropriate for use as forensic science standards. These standards are for use in equine forensic chemistry, but have other uses, including human forensic science.
Additionally, there is also a parallel requirement for deuterated internal standards of the same equine medication reference standards. These standards have been created in cooperation with Dr. Rodney Eisenberg of Frontier BioPharm LLC, of Richmond, Kentucky; however, the intellectual property and the reference standards themselves are the intellectual property of the University of Kentucky [UK]. In March 2011 the index reference standard, 4-OH-xylazine, was licensed by UK to Frontier BioPharm LLC, of Richmond, Kentucky; overall, 12 more or less standards have been synthesized and are undergoing certification and marketing. These include standards of the equine tranquilizer acepromazine, namely HydroxyEthylPromazine Sulfoxide maleate, [HEPS], and also D4 HydroxyEthylPromazine Sulfoxide, the corresponding deuterated internal standard.
Additionally, reference standards for the two major urinary metabolite fragments of the equine tranquilizer detomidine, 3-Hydroxydetomidine and carboxydetomidine and their corresponding internal standards, deuterated 3-Hydroxydetomidine and deuterated carboxydetomidine have been synthesized.
Other standards synthesized include Inositol-Tris-Pyro-Phosphate sodium salt, [ITPP], reputed to be a major performance enhancing drug in racehorses. ITPP was synthesized on an emergency basis to enable development of suitable equine forensic tests for this substance. Other substances synthesized include deuterated methocarbamol, deuterated guaifenesin and the two equine hydroxylated metabolite fragments of the local anesthetic mepivacaine, namely 3-and 4-hydroxymepivacaine and their parallell deuterated internal standards, deuterated 3- and 4-hydroxymepivacaine. Other standards for which synthesis is underway include deuterated furosemide, and considerable work has been carried out on the synthesis of deuterated butorphanol and deuterated pyrilamine.
A second project has approached extending the demonstrated efficacy of diclazuril agents in the treatment of equine protozoal myelitis [EPM] and related conditions. In January of this year two intellectual property [IP] disclosures in this area were made to the UK Intellectual Property office and work is proceeding on commercialization of these innovations.
A third project is involved with development of new and improved the ELISA tests for ergovaline and related ergot alkaloids is in its early stages, with a number of unique haptens synthesized and considerable progress being made towards developing cost-effective syntheses for pharmacologically significant quantities of ergovaline and stable isotope/deuterated ergovaline and corresponding haptens for ELISA development
Appropriate care of performance horses means that they must have access to full veterinary care, include use of therapeutic medications with the potential to affect performance. Use of potentially performance enhancing medications is currently regulated by the use of quantitative regulatory thresholds in either plasma/serum or urine. Such regulation requires that forensic chemists have access to appropriate certified reference standards and stable isotope internal standards for these medications and/or their metabolites. We have therefore been identifying, synthesizing and certifying reference standards for these equine therapeutic medications, including medication metabolites and or metabolite fragments recovered from equine samples.
Furthermore, since therapeutic medication regulation is an ongoing process, we elected to commercialize these standards by licensing them to Frontier BioPharm of Richmond Kentucky through the University of Kentucky Technology Transfer program. Frontier BioPharm then makes these certified standards available to racing chemists/forensic scientists worldwide. In this way we have helped create a high-tech industry in Richmond, Kentucky, Frontier BioPharm LLC, which company is certifying and distributing these reference standards worldwide.
A further benefit of this approach is that we were in a position to synthesize an urgently required substance, Inositol-Tris-Pyro-Phosphate, [ITPP], reputed to be a major performance enhancing drug in racehorses. On short notice we synthesized this substance and provided it to researchers in Pennsylvania and Hong Kong seeking to evaluate the threat to the integrity of racing posed by ITPP.
In another project we are evaluating the therapeutic potential of diclazuril agents in selected medical conditions with a view to extend the therapeutic use of these substances to areas outside of infectious intestinal diseases. In this regard, a project supported by the Kentucky Science and Engineering Foundation has been completed and two related intellectual property disclosures made to the University of Kentucky. At this point the immediate goal of this project is to identify commercial support for filing for patent protection for these findings and the pursuit of further application of this technology in both human and veterinary medicine.
The third project involves improved diagnostic approaches to the problem of fescue toxicosis in domestic herbivores, particularly cattle and horses. Research in this area is hindered by the difficulty and expense of synthesis of significant quantities of ergovaline and the lack of highly sensitive and specific ergovaline directed ELISA tests.
Directly approaching this problem, we have been working on identifying more cost-effective routes of ergovaline synthesis, as well as the synthesis of unique ergovaline/ergovaline peptide haptens to allow the creation of highly sensitive and specific anti-ergovaline antibodies for ELISA test development. Another expected outcome of this work is the likely creation of stable isotope [deuterated] internal standards of ergovaline and related ergot alkaloids for use in analytical toxicology research
Thomas Tobin, Kimberly Brewer, and Kent H. Stirling (2011) Equine Drug Testing & Therapeutic Medication Regulation; 2011 Policy of the National Horsemen's Benevolent and Protective Association, Inc. Wind Publications, 600 Overbrook Dr, Nicholasville, KY 40356, USA pp 370 KAES Article # 11-014-096 UK # 397.
Lehner Andreas F., Jennifer M. Duringerb, Charles T. Estillc, Thomas Tobind, A. Morrie Craige ESI-Mass spectrometric and HPLC elucidation of a new ergot alkaloid from perennial ryegrass hay silage associated with bovine reproductive problems, In press, Toxicology Mechanisms and Methods, 2011 KAES Article # 11-14-031 UK # 396.
Gutierrez Julio a, Rodney Eisenbergb, Gabrielle Herrensmitha, Thomas Tobina, Tonglei Lic, Sihui Longc Solvatomorphism in (E)-2-(2,6-dichloro-4-hydroxybenzylidene)hydrazinecarboximidamide, Submitted for publication February 2011, Acta Crystallographica Reference: sq3285 UK# 394, PMID: 21817799,[PubMed]
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. In press, Proceedings of the 18th International Conference of Racing Analysts and Veterinarians,Queens Town, New Zealand.
Tobin, T., Gutierrez, J., and Eisenberg, R., (2010) Experiences as an Expert: Educating People While Under Oath, 25 Years since Hong Kong. In press, Proceedings of the 18th International Conference of Racing Analysts and Veterinarians,QueensTown, New Zealand
Dirikolu L, Karpiesiuk, W, Lehner F and Tobin T., Toltrazuril Sulfone Sodium Salt: Synthesis, Analytical Detection, and Pharmacokinetics in the Horse J Vet Pharmacol Ther 2011 Kentucky Ag Expt Station Number 11-14-046 16 JUN 2011