REFERENCE STANDARDS, INTERNAL STANDARDS AND CRITICAL REAGENTS/REGULATORY ANALYTES FOR ANALYTICAL/TOXICOLOGICAL APPROACHES TO NEEDS IN EQUINE MEDICINE AND RACING REGULATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010609
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Veterinary Science

Non Technical Summary
Proper medical care of competition horses requires treatment with approved therapeutic medications, some of which have potential to directly influence performance. About one hundred such approved and appropriate therapeutic medications are recognized worldwide, fifty have been recognized by the American Association of Equine Practitioners (AAEP) and US Racing Commissions and approaching thirty are currently recognized and approved by the Racing Medication and Testing Consortium (RTMC) and the Association of Racing Commissioners International (ARCI). As such, approved Therapeutic medications and Endogenous, Dietary and Environmental substances (EDEs) are now regulated by use of quantitative regulatory thresholds in serum/plasma, urine or, recently increasingly, in hair; such regulation requires accurate quantification of the medication or defined metabolites or metabolite fragments recovered from the test matrix, the substance quantified being the defined "Regulatory Analyte". This approach by definition creates a need for Certified Reference Standards and stable isotope Internal Standards for such "Regulatory Analytes."Many of these Certified Reference Standards and stable isotope Internal Standards are not readily available, and we have been supplying this worldwide regulatory requirement and niche market since 1995. This proposal outlines how we will continue our synthesis purification and characterization of the required Certified Reference Standards and stable isotope Internal Standards for this forensic area and, in cooperation with Frontier BioPharm LLC of Richmond, Kentucky under a UK Intellectual Property (IP) license, certification to ISO-17025 standards of any required Certified Reference Standard or Stable Isotope Internal Standard for an Equine Therapeutic Medication or an Endogenous, Dietary or Environmental (EDE) substance and making these standards available to the worlds equine forensic chemists. We have also responded to requests for synthesis of Certified Reference Standards and stable isotope Internal Standards for substances of specific research and regulatory interest such as ITPP, these requests generally coming from research colleagues in support of racing regulation related projects.Current needs include a deuterated internal standard for the tranquilizer Xylazine, which has been identified as the Regulatory Analyte for Xylazine by ARCI. A similar need exists for a deuterated internal standard for Albuterol, a recently ARCI approved bronchodilator. A pro-drug for which deuterated internal standards are required is Cortisol/Hydrocortisone, now being regulated in plasma via the Hydrocortisone Sodium Succinate pro-drug and for which a Certified Reference Standard and a Deuterated Internal Standard are required. Work is also in progress on a deuterated internal standard for 3-OH-Ropivacaine and also for Capsaicin, for which a number of requests have been received for a Certified Reference Standards and a Stable Isotope Internal Standard.We also note that our first Certified Reference Standards and stable isotope Internal Standards were research standards synthesized in small amounts and not rigorously certified. Following the essentially worldwide adoption of quantitative approaches to therapeutic medication regulation, we are resynthesizing these research standards in larger quantities and by improved methods for ISO-17025 equivalent certification. Standards requiring re-synthesis include deuterated Flunixin, deuterated Furosemide and also the Detomidine metabolite reference standards, carboxy-Detomidine and hydroxy-Detomidine. We also need to rework our synthesis of deuterated Flunixin because of the occurrence of a difficult to remove isotopic impurity associated with our original Flunixin synthesis. Other substances for which deuterated internal standards are required include the corticosteroid medications Flumethasone and Isoflupredone, as set forth in related proposals. Using these approaches we are also addressing an industry need for toxicologically useful quantities of Ergovaline and other difficult to obtain ergot alkaloids; we are also creating certified reference standards and deuterated internal standards for these ergot alkaloids with the goal of using these materials to create highly ergot alkaloid specific tests, including ELISA tests, in which area we have made considerable progress. Finally, we are working with VC Research Inc., on commercialization of a patented UK IP licensed to VC Research concerning new therapeutic approaches to fetal Toxoplasmosis.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3810	1180	100%

Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1180 - Pharmacology;

Keywords

equine forensic chemistry,

medication regulation

certified reference standards

stable isotope internal standards

chemical synthesis

purification, certification

worldwide distribution

therapeutic medication regulation

ergot alkaloids

fetal toxoplasmosis

, prevention and therapy of fetal toxoplasmosis

Goals / Objectives
1/ To identify, synthesize, purify and certify to ISO-17025 standards and make available to equine forensic chemists and veterinary researchers worldwide certified reference standards for the required regulatory analytes for unique equine therapeutic medications and Endogenous ,Dietary and Environmental substances [EDEs] including, where required urinary drug metabolite fragments for equine therapeutic medication regulation. 2/ To synthesize and make available to equine forensic chemists worldwide deuterated or other stable isotope internal reference standards for the unique equine urinary drug metabolite fragments of the classes of substancers set forth above, to allow scientifically and forensically accurate quantification of same. 3/ To identify and synthesize reference standards and stable isotope internal reference standards for glucuronide metabolites of high potency therapeutic medications with a view to their application in plasma-based quantitative medication regulation. 4/ To provide to equine researches and forensic chemists specific certified reference standards for substances of research and regulatory interest as we have on several occasions during our previous Hatch project. 5/ Following industry requests, we are working to develop cost effective methods for synthesizing toxicologically significant amounts of ergovaline and other ergot alkaloids and certified referemnce standards and stable isotope internal standards for same to allow characterization of the specific toxicological responses to individual ergot alkaloids and where appropriate to develop highly sensitive and specific ergot alkaloid analytical procedures including where appropriate ELISA tests. 6/ To pursue ongoing work related to a licensed UK IP with the goal of commercializing our patent protected efficacy of diclazuril and related benzeneacetonitrile substances alone and in combination with other substances in the prevention/treatment of fetal toxoplasmosis.

Project Methods
Albuterol-d9 will be synthesized as follows; salicaldehyde or its acetylated analog will be acylated with chloroacetylchloride and aluminium chloride to provide the chlorinated acetophenone derivative. This material will then be reacted with tert-butylamine-d9 and a non-nucleophilic base provides the labeled precursor which can be reduced with sodium borohydride to provide deuterium labeled albuterol. With regard to Flunixin, our first research synthesis of Flunixin-d3 produced a product containing small amounts of a difficult to remove isomer with similar MS characteristics but not identical to the desired structure. We now propose an unambiguous synthesis with the goal of providing the unambiguously pure correct isomer, as set forth in our research proposal avoiding our previous approach using a lithiation scheme which led to two possible isomers, with potentially troublesome amounts of the incorrect isomer. With regard to xylazine, regulatory control of xylazine will in future depend on quantifying parent Xylazine in plasma or, less likely a urinary metabolite fragment, 4-hydroxyxylazine. However it now appears that the preferred regulatory analyte in the US will be Xylazine itself in plasma\serum. We therefore propose to synthesize the required stable isotope internal standard of Xylazine, Xylazine-d6, as we now set forth. Labeled 2, 6-dimethylaniline will be converted to the isothiocyanate with thiophosgene and then reacted with 3-amino-1-propanol. After refluxing with conc. HCl the desired product (as the HCl salt) will be isolated and purified. We plan to synthesize deuterated Capsaicin, Capsaicin-d7 which will be prepared as follows: Ethyl-5-bromovalerate will be reacted with ethynyl magnesium bromide to provide the terminal alkyne which will be further alkylated with labeled isopropyl iodide to provide the substituted alkyne which will be selectively reduced to the E-alkene using Birch reduction techniques. Reaction of this ester with the benzylamine derived from vanillin will provides the desired labeled capsaicin-d7. With regard to Hydrocortisone, there is a long established international threshold/regulatory limit for Hydrocortisone of 1,000 ng/ml from/in urine, but this threshold is not considered satisfactory. Recent research has shown that detection of the Hydrocortisone sodium succinate pro-drug form in which Hydrocortisone is often administered to horses allows better regulation of this medication. We will synthesize deuterated Hydrocortisone sodium succinate to provide the required internal standards for optimal regulation of the therapeutic use of this substance. Cortisol-d4 will be prepared from cortisone by protecting it with paraformaldehyde and acid followed by labeling with deuterium at the 10 and 12 positions by exchange with labeled water under basic conditions. Following these steps, reduction of the 11-ketone to the alcohol with sodium borodeuteride provides the labeled and protected Cortisol with now non-exchangeable deuterium positions at C-10, 11 and 12. Any extra deuterium labeling at the 2 or 7 positions will be exchanged out in the deprotection step. The succinate half ester will be formed by reaction of the labeled Cortisol with succinic anhydride. The final product will be either the carboxylic acid, or it can be converted to the sodium salt. For the synthesis and certification of 3-carboxydetomidine-D4 and 3-Hydroxy-Detomidine-D4 HCl, the protected and d4 labeled 2-methyl-3-iodobenzoic acid derivative, supplied by Frontier BioPharm, LLC, will be converted to the Magnesium Iodide with magnesium and reacted with the protected imidazole aldehyde. The protected intermediate will then be simultaneously N-benzyl deprotected and hydrogenolyzed with palladium catalyzed hydrogenation, followed by deprotection of the carboxylic acid with aqueous acid to produce 3-carboxyDetomidine-d4 isolated as the HCl salt. This compound will then be converted to the similarly labeled 3-hydroxyDetomidine-d4 by reduction of the carboxylic acid of 3-carboxyDetomidine. The unlabeled analogs will be prepared by the same route using unlabeled starting material. Deuterated Dantrolene will be prepared by the following six step reaction sequence beginning with deuterated aniline. The first step will be protection of the deuterated aniline with acetic anhydride, followed by Friedel-Crafts nitration. Next, removal of the acetate protection group will be performed with deuterated solvents as there may be exchange with the aromatic deuteriums in the acidic conditions required for hydrolysis of the acetate protection group. These reactions will be followed by formation of the diazonium salt by well-established chemistry followed by coupling with 2-furaldehyde can be accomplished using catalytic copper (II) chloride as described in the literature. The 2-aminohydantoin will then be condensed with the aldehyde to provide d4 labeled Dantrolene as shown above. We will synthesize 3-HydroxyRopivacaine-d7 as follows: 3-HydroxyRopivacaine-d7 will be prepared by Condensation of 2, 6-dimethylaniline with the protected pipecholenic acid by standard peptide techniques provides the carbon skeleton of the pivacaine series. Reaction of the aliphatic amine with bromopropane provides Ropivacaine. This can also be prepared with bromopropane-d7 to provide the -d7 analog, Ropivacaine-d7. Nitration, reduction to the amine followed by conversion to the phenol provides the desired 3-HydroxyRopivacaine. We will resynthesize Furosemide-d5 by flowing cyanation of labeled furan, followed by reduction with NaBD4 and iodine to produce the d5 labeled furfurylamine. This will then be reacted directly with 2, 4-dichloro-5-sulfamoylbenzoic acid in the presence of a non-nucleophilic base to provide Furosemide-d5.The proposed process to synthesize ergovaline is as follows. The first peptide bond will be achieved by using commercially available starting materials Boc-Pro-OH and L-Valinemethylester HCl. The optimal peptide coupling reagent will likely be EDC. Other coupling agents have been evaluated, such as DCC, but so far they have provided insufficient product. The next step will be hydrolysis of intermediate number 3 using 2N KOH to form the carboxylic acid, to form the fourth step product. Then the Boc protecting group will be cleaved using TFA to form the fifth step product. The dipeptide intermediate required will then be achieved using EDC under the same conditions as above to obtain the desired dipeptide. For synthesis of ergovaline the synthetic process will be as follows. The first peptide bond will be achieved by using commercially available starting materials Boc-Pro-OH and L-Valinemethylester HCl. The optimal peptide coupling reagent will likely be EDC. Other coupling agents have been used, such as DCC, but so far they have provided insufficient yield of product. The next step will be hydrolysis of intermediate number 3 using 2N KOH to form the carboxylic acid product four. Then, the Boc protecting group will be cleaved using TFA to form product five. The dipeptide intermediate number six will then be achieved using EDC under the same conditions as above to obtain product number six, the required product. Projected work on the ergovaline tripeptide includes the following two routes to ergovaline. The primary approach will be synthesis of the tripeptide, followed by coupling with lysergic acid to produce ergovaline. The tripeptide will be prepared from the pyruvyl imide by reaction with sodium amide. The aminol base is expected to cyclize to the natural product stereochemistry, as this is the naturally derived stereochemistry. Reaction of the amine with lysergic acid, derived from the hydrolysis of the available ergopeptine via DCC coupling will then provide the requisite ergovaline. The requisite deuterium labeled standard can be provided by the usage of a CD3 labeled lactate derivative when preparing the tripeptide.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:The most directly and immediately influenced target audience is the world's racing chemists, the equine analytical chemists/ forensic scientists that perform that actual substance detection procedures that regulate drug and medication use in performance horses including racing horses worldwide. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The program directly enhances the professional development of each of its current major participants and coworkers and researchers. The major participants include the PI, Thomas Tobin, his research associate Dr. Sucheta Kudrimoti, his doctoral student, Mr. Jacob Joseph Machin and his close professional colleagues and collaborators, Dr. Rodney Eisenberg, and Dr. Clara Fenger. Additionally, the program has a long running professional interaction with the National Horsemen's Benevolent and Protective Association, the largest Horsemen's organization in the US, representing 30,000 or so Thoroughbred owners and trainers and which organization has keen professional interest in the area of Equine Forensic Science and most especially in ensuring accurate and reproducible quantitative testing of racing and sport horse samples for therapeutic medications and endogenous, dietary, and environmental substances and testing and fully scientifically validated regulatory practices. The primary non-academic scientific collaborator is Dr. Rodney Eisenberg, PhD, an experienced synthetic chemist who works for Frontier BioPharm LLC, London, Kentucky, the company which licenses the synthesized reference standards from the University of Kentucky. Frontier BioPharm then performs a rigorous chemical purification, characterization, and formulation stability tests on the provided reference standards to generate the final product that is marketed worldwide as an ISO-17025 equivalent Certified Reference Standard or a Stable Isotope Internal Standard. The chemical synthesis portion of this project is carried out in the Maxwell H. Gluck Equine Research Center and directly provides training and professional development experience for Mr. Jake Machin, a PhD candidate in the University of Kentucky Department of Toxicology and Cancer Biology, in which Department Dr. Tobin is also a Professor, and which PhD degree Mr. Machin has just completed, as set forth in this report. The program also provides training and professional development opportunities for Dr. Sucheta Kudrimoti, a Research Associate in the Department of Veterinary Science who works directly on the primary synthesis of these reference standards. The second major scientific collaborator is Dr. Clara Fenger, DVM, PhD, DACVIM, Equine Integrated Medicine, PLC, Georgetown, KY 40324, and Executive Director of the North American Association of Racetrack Veterinarians. Dr. Fenger is a veterinarian, Board Certified in Internal Medicine and with a PhD in Veterinary Science. With Dr. Fenger the program works on the closely related matter of defining regulatory thresholds for therapeutic medications, dietary and environmental substances in racing horse and competition horses worldwide, and Dr. Fenger is a frequent co-author with Dr. Tobin of both trade journal and scientific publications on matters related to the regulation of therapeutic medications and endogenous dietary and environmental substances in racing and sport horses. This work is supported by horsemen and other interested racing and sport horse groups, most particularly the National Horsemen's Benevolent and Protective Association and the Equine Health and Welfare Alliance Inc., whose members are among the primary beneficiaries of the research and reference standard and defined screening limits of detection output of the program. Additionally, the program has also contributed to the professional development of numerous academic veterinarians and related individuals and the ongoing process of defining regulatory "cut-offs" or Screening Limits of Detection (SLODs) for Therapeutic Medications and Endogenous, Dietary and Environmental substances (EDEs) in racing and sport horses worldwide. Because of the central role of these reference standards in racing and sport hors medication / foreign substance regulation these research products and research outcomes impact the training and professional development of regulatory professionals the entire racing and sport horse industry worldwide. On measure of this impact is the wide range of co-authors on the scientific publications from the program. These authors include Professor George Maylin of the New York Drug testing and Research program, whom we work closely with on the development and utilization of these reference standards. Similarly, Ms. Petra Hartmann of Industrial Laboratories, Denver, Colorado is an analytical chemist who performs analytical testing for a large number of racing jurisdictions and with whom we also work closely on matters involving reference standards. Similarly, we also work closely with Mr. Theodore Shults, J.D., M.S. Chairman of the American Association of Medical Review Officers (AAMRO), a parallel organization in humans forensic science. With respect to the application of these standards and technologies in equine medicine we work closely with a number of academic and practicing veterinarians including Dr Scott McClure, DVM, PhD, Dipl. ACVS, Dipl. ACVSMR, Dr. Kevin Kersh, DVM, Diplomate ACVS, of Iowa State University, Dr. Bradley A Brown DVM of Shelbyville, Ky, Dr. Wayne Duer, PhD of Duer Forensic Toxicology and Dr. Levent Dirikolu, DVM, PhD, Professor of Pharmacology, Director, Equine MedicationSurveillance Laboratory, Louisiana State University and Dr. Andreas Lehner PhD of Michigan State University. The program also has impacted training and professional development in the Spanish speaking veterinary communities, specifically South America and Spain and also in the middle east through a longtime professional development relationship with Dr. Abelardo Morales Briceno, DVM, and in the Caribbean through ongoing educational and professional development interactions with Dr. Pedro de Pedro DVM, MS. Dipl. ACVIM, of Ross University St Kitts and his student, Ms. Rojas Rivers and his colleagues, Dr. L. Dzikiti, PhD, and Dr. Greg Fox, DVM. How have the results been disseminated to communities of interest?These results have been disseminated to the communities of interest first and foremost by 1/ publication in the refereed scientific literature as detailed under products, by presentations at 2/ scientific and 3/ veterinary and 4/ professional horsemen's meetings and 5/ meetings of interest groups and also by use of the internet, via 6/ the University of Kentucky Maxwell H Gluck Equine Research Center web site , http://gluck.ca.uky.edu/ 7/ the Frontier BioPharm LLC web site https://frontierbiopharm.com/ and also 8/ via the PI's personal professional web site, www.thomastobin.com. Additionally, to the extent possible appropriate, the program is using 9/ the University of Kentucky Maxwell H. Gluck Equine Research Center Facebook page to communicate research findings and availability of certified reference standards to the equine forensic science and regulatory and scientific research communities and the equine sport community at large. Our research reports and communications are widely read, as per ResearchGate, where our publications are frequently noted as the most read contributions from our department. Regarding communication, our priority is always publication of our research findings and conclusions in the refereed scientific literature. Such publication immediately creates a record that is highly relevant in the scientific process, but also equally impactful in a regulatory / forensic field such as equine medication regulation. Analytical chemistry is basically directed towards identification of substances and with the great increase in the sensitivity of analytical chemistry, now about 1 billionfold more sensitive than 45 years ago, the pharmacological relevance and or forensic significance of low concentration identifications of pharmacologically active substances needs to be carefully evaluated. A classic example is the Barbarin / Aminorex project, where what may well appear on first review to be a blatant attempt to affect performance, i.e., an Aminorex identification in a post-race urine sample is actually evidence of the resourcefulness of life on this planet, with the matter of Aminorex identifications being the result of an overlap between an aggressive and highly successful plant family and racing chemistry activities. Publication of these findings in the refereed scientific literature, as we have been for many years, creates a published record that allows scientifically correct evaluation of the forensic significance of the continually increasing record of unexpected trace level identifications of pharmacologically active substances in post-race blood, urine, hair, and other tracing and sport horse regulatory and forensic samples. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The most significant scientific accomplishment of this most recent project research period has been the synthesis, certification and publication of certified reference standards and stable isotope internal standards for the plant substance barbarin, the completely unexpected most likely source of Aminorex identifications in post-race urines of sport and racing horses. Aminorex, (RS)-5- Phenyl-4,5-dihydro-1,3-oxazol-2-amine, is an amphetamine related substance at one time marketed as a prescription anorectic in human medicine and aminorex is currently a United States a Drug Enforcement Administration [DEA] Schedule 1 controlled substance. As such, aminorex identifications in horse urine can result in significant penalties for trainers and the connections of the horse, namely 1 year suspensions and $10,000.00 fines. Given this circumstance, the existence of largely unavoidable natural sources of Aminorex precursors in equine feedstuffs is of importance to both individual horsepersons and the racing and sport horse industries worldwide. Of particular importance in Kentucky is the fact the feeding of the Kentucky pasture weed Barbarea vulgaris, colloquially "yellow rocket", can give rise to such Aminorex identifications. While the barbarin found in Barbarea vulgaris and related plants worldwide is the most likely source of these identifications, we have synthesized and certified both reference standard barbarin and very recently d5 barbarin as an internal to allow racing chemists and equine researchers to unequivocally establish barbarin as the source of these equine urinary aminorex identifications. A further significant and highly important practical outcome of this research has been the publication in the Horsemen's Journal trade literature of a proposed interim Screening Limit Of Detection (SLOD) / regulatory threshold of 30 ng/ml of aminorex in equine urine as an interim regulatory "cut-off" for Aminorex in racing and sport horse urines. These barbarin related researches are in progress as we speak, and a number of research papers have been communicated and the paper on synthesis of deuterated barbarin is in final draft for publication. A closely related sequence of events has recently occurred with the human medication gabapentin, now the twelfth most commonly prescribed human medication. In humans the dose of gabapentin is large, up to 3 grams per day in some individuals, and gabapentin is, and this is very unusual, is excreted unchanged in human urine, which means that each medicated human contributes 3 grams/ day or so of Gabapentin to his/her environment. The result, not unexpectedly, is that humans prescribed gabapentin readily transfer trace levels amounts to horses, and we were requested by Ms. Renee Mancino, Executive Director of the Ohio Harness Horsemen's Association to investigate the source / origins of a large sequence of gabapentin identifications in Ohio Harness Racing. Approaching this matter, we first researched the likely pharmacological significance or lack thereof of the trace level concentrations of gabapentin being identified in these Ohio samples. Based on these researches and the ongoing 1 ng/ml or so trace level concentrations claimed identified in these samples by the Ohio laboratory, we suggested 5 ng/ml in plasma as a Screening Limit of Detection (SLOD) "cut-off" for gabapentin in equine plasma / serum, a serum/ plasma concentration that turned out to be in close agreement with the 8 ng/ml figure presented by an independent consultant retained by the Ohio Horse Racing Commission with respect to these matters. These events have been communicated in preliminary form in our Horsemen's Journal trade journal article and form the basis of a full case report scientific paper currently in final draft. In another somewhat similar matter, in the fall of 2020 we were contacted by Glanbia, a global nutrition company with a significant presence in the equine feed market concerning apparent contamination of some of their equine feed products by Zilpaterol, a cattle feed additive. On an emergency basis, i, e., 24 hours' notice, Glanbia requested an educational presentation on equine feed contamination by Zilpaterol for their English and French equine feed markets, the presentation to address the regulatory and equine health implications of these Zilpaterol identifications. The PI immediately reviewed the available scientific and regulatory literature, drafted, and shipped a three page summary scientific review to guide the video presentation, which video presentation was recorded the following day. This presentation was presented first in English, https://youtu.be/Vc5-fcg1uPQ, and in a later French subtitled version https://youtu.be/Vc5-fcg1uPQ. for the French speaking equine feedstuff market. We also drafted and published a Horsemen's Journal trade journal article on these and related Zilpaterol identifications where we noted that the common source of these Zilpaterol identifications was molasses to which Zilpaterol had been added for inclusion in cattle feed, which molasses was then inadvertently contaminating / entering horse feed. We also noted, as we had in the video presentations, that the concentrations of Zilpaterol identified in the equine samples were both pharmacologically insignificant and well below any concentration that would have an adverse health effect on the involved horses. As we were drafting this article for publication the Federation Equestre Internationale (FEI) announced a new Atypical Findings (ATF) policy designed to allow identification of atypical analytical findings such as these Zilpaterol findings as being of innocent environmental origins, and of no regulatory significance. Other accomplishments include the synthesis of a complete set of Certified Reference Standards for the 3 hydroxylated metabolites of the mepivacaine group of local anesthetics, namely 3-hydroxyMepivacaine, 3-hydroxyLidocaine, 3-hydroxy-Bupivacaine and 3-hydroxyRopivacaine, which standards we are purifying preparing for transfer to Frontier BioPharm for certification as we speak. Additionally, we are currently in the process of synthesizing the corresponding sequence of deuterated internal standards, which syntheses are proceeding more slowly as may be expected with the synthesis of deuterated molecules.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Jacob Machin3 George Maylin1, Clara Fenger2, and Thomas Tobin3* Plasma Pharmacokinetics of Betamethasone Following Intra-Articular Administration of 15mg of BetaVet� into Each Stifle Joint: A Review and Analysis, Submitted for publication, J vet Pharm Ther. in progress.
• Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Machin, JJ. Science-Based Regulation of Pharmacological Substances in Competition Horses: PhD Thesis, successfully defended August 11th, 2021. Final format thesis submitted for University of Kentucky Graduate School approval, September 22nd, 2021, Mentor: Dr. Thomas Tobin, Department of Veterinary Science, the Maxwell H. Gluck Equine Research Center, and Department of Toxicology and Cancer Biology, University of Kentucky.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Morales-Brice�o A1, M�ndez-S�nchez A1,P�rez-Ar�valo J1, Guil-Alcala P1, Brewer K2, Fenger C3, Tobin T4.(2021) Multicenter Case Study of Sudden Death associated with Exercise-Induced Pulmonary Hemorrhage in Endurance Racing Horses (Period 2006-2019). Poster presentation, XXXII Virtual Annual Meeting Spanish Society of Anatomy Veterinary Pathology, October 1st, 2021, Facultad Veterinaria, Universidad De Cordoba Av. de Medina Azahara, 3, 14005 C�rdoba, Spain
• Type: Journal Articles Status: Published Year Published: 2021 Citation: S. McClure, C. Fenger, K. Kersh ,B. Brown, G. Maylin, W. Duer, K. Brewer, J. Machin and T. Tobin; (2021) Dexamethasone serum concentrations after intravenous administration in horses during race training; Comparative Exercise Physiology:17 (3)- Pages: 263 272.
• Type: Other Status: Published Year Published: 2021 Citation: Kimberly Brewer, DVM MSc, Clara Fenger, DVM PhD DACVIM, Andreas Lehner PhD, Theodore F Shults JD Ms, Thomas Tobin MRCVS, PhD, DABT. Testing Times Following a sample from collection to a positive, how drug testing is done in 2021 and what it means The Horsemens Journal, Summer 2021 Pages 40- 45
• Type: Other Status: Published Year Published: 2021 Citation: Jacob Machin, MS, Kimberly Brewer, DVM, MSc, Clara K. Fenger, DVM, PhD, DACVIM, Sahar White Springer, PhD , Abelardo Morales-Briceno, DVM and Thomas Tobin, PhD, MRCVS, DABT, Zilpaterol and Ractopamine: Reaching Consensus on Trace-Level Transfers to Racehorses Worldwide clusters show trace-level positives typically result from contaminated feed. The Horsemens Journal, Fall 2021 Pages 38-40-42.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The most directly and immediately influenced target audience is the world's racing chemists, the equine analytical chemists/ forensic scientists that perform that actual substance detection procedures that regulate drug and medication use in performance horses including racing horses worldwide. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student was trained during the course of this work and multiple educational seminars were provided to equine practitioners. How have the results been disseminated to communities of interest?Peer reviewed publications and seminars geared toward both other sceintists and equine practitioners and other industry members. What do you plan to do during the next reporting period to accomplish the goals?We intend to work toward developing Certified Reference and Deuterated Internal Standards for 3-hydroxyMepivacaine, 3-hydroxyLidocaine, 3-hydroxyBupivacaine and 3-hydroxyRopivacaine, which have misuse potential and are of high interest to animal health regulators.

Impacts
What was accomplished under these goals? The primary goal of this project is to provide the racing and sport horse industries, specifically racing chemists and regulators, with certified reference standards and stable isotope internal standards for substances of regulatory concern detected and regulated in racing and sport horses. A closely related goal made possible by the availability of these standards is to specifically define the plasma or urinary concentrations of such substances which are, or are not, of regulatory concern in racing and sport horses. During this past year a number such certified reference standards and stable isotope internal standards have been synthesized and turned over to Frontier BioPharm for certification to ISO-17025 standards and subsequent worldwide distribution. Certified reference standards and stable isotope internal standards synthesized during the last 12 months include the following molecules/substances: 1. Barbarin: As presented in the "publications in the refereed scientific literature" section a Certified Reference Standard for barbarin has been synthesized and transferred to Frontier BioPharm for worldwide distribution, as set forth in the Frontier BioPharm website, and the synthesis and chemical characterization of this now certified reference standard has been reported in the scientific literature. 2. d5 Barbarin: We have also completed the synthesis and preliminary "on campus" purification and characterization of the corresponding required Stable Isotope Internal Standard d5-barbarin for use in barbarin related research and a publication on the synthesis and characterization of d5-barbarin is in final draft for publication. Additionally, the deuterated product has been transferred to Frontier BioPharm for final purification and certification and worldwide distribution as a d5 internal standard of barbarin for use in barbarin related research.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Machin, J. Childers, T, Kudrimoti, S. Eisenberg, R., Fenger, C., Hartmann, P., Maylin, G., Tobin, T., and Shults, T. (2020). Synthesis, Characterization and Certification of Barbarin, a Possible Source of Unexplained Aminorex Identifications in Forensic Science. Drug Testing and Analysis: October Oct;12(10):1477-1482. https://doi.org/10.1002/dta.2883

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audiences for this project are 1/ equine forensic chemistry scientists and laboratories worldwide and 2/ to the extent possible, human forensic science laboratories worldwide.T Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The chemical synthesis portion of this project is carried out in the Maxwell H Gluck Equine Research Center and directly provides training and professional development experience for Mr. Jake Machin, a PhD candidate in the University of Kentucky Department of Toxicology and Cancer Biology, in which Department Dr. Tobin is also a Professor. The program also provides training and professional development opportunities for Dr. Sucheta Kudrimoti, a Research Associate in the Department of Veterinary Science who works directly on the primary synthesis of these reference standards. A recent third addition to the Veterinary Science program is Ms. Taylor Childers, a Medical Sciences MSc program candidate who is writing her MSc thesis relevant to the synthesis of Barbarin and deuterated Barbarin and the Aminorex project. How have the results been disseminated to communities of interest?These results have been disseminated to the communities of interest first and foremost by 1/ publication in the refereed scientific literature as detailed under products, by presentations at 2/ scientific and 3/ veterinary and 4/ professional horsemen's meetings and 5/ meetings of interest groups and also by use of the internet, via 6/ the University of Kentucky Maxwell H Gluck Equine Research Center web site , http://gluck.ca.uky.edu/ 7/ the Frontier BioPharm LLC web site https://frontierbiopharm.com/ and also 8/ via the PI's personal professional web site, www.thomastobin.com. Additionally, to the extent possible appropriate, the program is using 9/ the University of Kentucky Maxwell H. Gluck Equine Research Center Facebook page to communicate research findings and availability of certified reference standards to the equine forensic science and regulatory and scientific research communities and the equine sport community at large. What do you plan to do during the next reporting period to accomplish the goals?The plan for the next reporting period is to continue the Barbarin/Aminorex related research to definitively establish the precise chemical source of the Aminorex identified in equine urine post Brassica vulgaris administration. We are working on publishing the Barbarin synthesis in the refereed scientific literature, including the purification and chemical characterization of our synthetic product, which paper is currently in close to final draft. The second project is to complete the synthesis of deuterated Barbarin for use as an internal standard in Barbarin related research and also, possibly, for equine administration to definitively identify Barbarin as the source of urinary Aminorex following consumption of Barbarea vulgaris by horses. As we speak these projects are at various stages of completion and these projects are of high priority for completion. The next highest priority is the creation of a deuterated internal standard for Tiludronic acid, an FDA approved Bisphosphonate medication, medications whose use in racing horse has come under increased scrutiny and whose use in racing horses is now closely regulated. Bisphosphonates are chemically unusual in that they are chemically closely related to pyrophosphate. As such, their charged nature means that during chemical analysis they are readily attracted to / bound to surface charges. Similarly, when administered to horses they bind tightly to bone, and they therefore have a long terminal plasma half live and remain detectable at trace concentrations in equine plasma and urine for 40 days or longer after the last administration. Given these chemical and pharmacokinetic characteristics, a suitable deuterated internal standard would greatly facilitate the development of high quality quantitative analytical tests and regulatory procedures for Bisphosphonate medications in equine sports. We will continue the purification, characterization and certification of d4-Tiludronic acid internal standard. Chemical purification and characterization of the synthesized intermediates (P1-P4) and final product d4-Tiludronic acid (FP) will be done by column chromatography and recrystallization and confirm the structures by NMR, C13, mass, and CHN analyses. We will also perform phosphorous NMR for the (FP) for confirmation of the d4-Tiludronic acid. Once synthesis and preliminary purification are complete the d4-Toulidine will be characterized in house for chemical identity and nominal purity using Nuclear Magnetic Resonance [NMR], Infrared [IR], Ultraviolet [UV], Mass Spectrometry [MS] and elemental analysis.

Impacts
What was accomplished under these goals? Mepivacaine is a generally approved therapeutic medication in performance horses. To guide the racing community with respect to its use close to regulated events, dosing regimens and withdrawal time guidelines with defined regulatory thresholds are required. The Association of Racing Commissioners International [ARCI] schedule is a 72-hour withdrawal after 0.07mg/kg, about 35.0 mg, subcutaneously [SQ] to comply with the regulatory threshold of 10 ng/mL of total 3-hydroxymepivacaine in urine. This pilot study was performed to provide guidance for a 400 mg SQ mepivacaine dose, which dose was administered to 6 horses, and urinary total 3-hydroxymepivacaine determined by liquid chromatography/tandem mass spectrometry for 5 days. The urinary concentrations of total 3-hydroxymepivacaine exceeded the ARCI 10 ng/mL urinary threshold in all samples collected through day 5. We created a three-compartment pharmacokinetic model of the urinary concentrations of total 3-hydroxymepivacaine and calculated withdrawal time estimates following doses of between 35 and 400 mg of mepivacaine. These pilot data provide estimates of withdrawal time guidelines for veterinarians treating Thoroughbreds horses with 400 mg SQ doses of Mepivacaine. Betamethasone is an anti-inflammatory corticosteroid used in equine medicine often as "BetaVet®" an approved and marketed combination of Betamethasone sodium phosphate 3.15 mg/ml and Betamethasone acetate 2.85 mg/ml injectable suspension, administered at up to 30 mg/horse. To determine detection times for Betamethasone following "BTM combination" which we administered as one 15 mg dose into each two medial femorotibial joints of 8 Standardbred horses. Blood samples were collected pre-administration and at ¼, ½, 1, 2, 4, 8, 12 and 24 hours post administration and then daily for ten days. Betamethasone concentrations were determined using Tandem Liquid Chromatography Mass Spectrometry and the data analyzed using compartmental pharmacokinetic modeling. Plasma Betamethasone peaked at 30 minutes at 22ng/ml and thereafter declined, with a second phase half-life of about 6.5 hours to around 55 pg./ml at 72 hours post administration. From 72 hours the plasma half-life was 126 hours, with a mean plasma concentration of 16.3pg/ml at 240 hours. These data were well fitted by a three-compartment model with absorption. Based on these data we calculated 95/95 and Gauss (Camp-Miedell) regulatory thresholds at each time point from 72 hours post administration on. Based on these data we suggest an interim Betamethasone regulatory threshold of 200 pg./ml with a six-day withdrawal guideline or 100 pg./ml with a ten-day withdrawal guideline following intraarticular administration of 30 mg of "BTM combination" for Standardbred racing. 5/ Dexamethasone (DXM) sodium phosphate is a corticosteroid used for inflammatory conditions in horses, regulated in equine sports by serum/plasma DXM concentrations. This study describes the variability of serum concentrations of DXM at 48 and 72 hrs after IV administration of 20 mg DXM sodium phosphate for 1-5 days. 74 actively racing horses, 39 Thoroughbreds, 13 Standardbreds, 22 Quarter Horses) met inclusion criteria for the study. Administration of 20 mg DXM sodium phosphate was performed for 1 to 5 days as part of clinical practice. Serum was collected pre-injection and at 48- and 72-hours post-injection. The effect of number of days and the effect of breed on serum concentrations of DXM determined by ANOVA. No differences were identified for the serum concentrations results for racing breeds, age, gender or the number of days of DXM sodium phosphate administration, so all-time point data were pooled. The DXM serum concentration at 48 hours (mean ± standard deviation (SD), pg./mL) was 2.18 ± 1.56. There was an extreme outlier at 48 hours, and two extreme outliers at 72 hours. The major conclusion is that Practitioners should be aware that regulatory thresholds for DXM may be exceeded without re-administration of DXM. The significance of a scientific product and or scientific contribution is how it changes the way people do things. By generating certified reference standards and deuterated internal standard products focused primarily on the equine regulatory chemistry field and equine related drug and medication detection, this program has contributed very substantially, in some cases made possible the ability of equine regulators and equine regulatory veterinarians and forensic scientists to scientifically regulate correct and appropriate therapeutic medication use in racing and sport horses. The requirement for reference standards used in this area is ongoing and during this last year the program renewed supplies of several reference standard materials for certification and distribution by our commercial licensee, Frontier BioPharm. A second and scientifically very interesting product has been the identification of plant Barbarin as the most likely potential source of urinary Aminorex identifications in racing horses and our synthesis and now availability of both Barbarin and deuterated Barbarin for research on its potential role in Aminorex identifications in racing and sport horses, as set forth in our just (December 23rd 2019) published communication and our proposed 30 ng/ml Screening Limit of Detection (SLOD) for Aminorex in post-race / event urine samples. A third area in which to program is contributing is in the area of regulatory threshold determination and evaluation. In this area the program has cooperated with Horsemen's groups, and also with equine practitioners with respect to unexpected identifications of therapeutic substances in post-race samples. For example, in the area of unexplained trace level detections of Naproxen the program worked with the Charles Town Horsemen's Benevolent and Protective Association to review of the circumstances of these trace level Naproxen identifications. This review focused on the dosage, usage patterns and chemical and pharmacokinetic characteristics of Naproxen, and which review led to a proposed Screening Limit of Detection (SLOD) of 250 ng/ml of Naproxen in equine plasma, a Screening Limit of Detection less that the Screening Limit in use in neighboring Mid-Atlantic states but sufficient to handle the problem of these irrelevant, i.e., less that 250ng/ml trace level detections of Naproxen that had been a considerable problem in Charles Town racing. Similar studies have been carried out on Methylprednisolone and Dexamethasone as reported earlier, and the role of these studies is to provide guidance to professional veterinarians administering these medications to performance horses close to a regulated event.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: George Maylin, Clara Fenger, Jacob Machin, Sucheta Kudrimoti, Rod Eisenberg, J D Green and Thomas Tobin 2019 Aminorex identified in Horse Urine following consumption of Barbarea vulgaris; a preliminary report Irish Veterinary Journal. 72:15.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Jacob Machina, Kimberly Brewer, Maria Catignanic, Theodore F. Shultsd, Clara Fengere, G.A. Maylinf, and Thomas Tobin, * 2019. Review and Analysis of an Interim Screening Limit of Detection for Naproxen in Post-race Samples. Comparative Exercise Physiology: 16 (2)- Pages: 153 - 160.
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: S. McClure, K. Kersh, B. Brown, C. Fenger, G. Maylin, W. Duer, L Dirikolu, J. Machin, & T. Tobin. Variability of dexamethasone serum concentrations after intravenous administration in racing horses: Submitted for publication, Equine Veterinary Journal.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jake Machin, Kimberly Brewer, Clara Fenger and Ted Shults. TRACE LEVELS OF ENVIRONMENTAL SUBSTANCES IN RACING SAMPLES: WHAT SHOWS UP AND WHY 5th Annual Meeting and Symposium of the North American Association of Racetrack Veterinarians [NAARV], Lexington, Kentucky, November 5-6th, 2018.
• Type: Other Status: Published Year Published: 2018 Citation: Fenger, C., Thomas Tobin., Catignani, M. and Shults, T. 2018 Hitting the Threshold of Common Sense: The time for screening limits to guard against environmental transfer is now Winter 2018 Issue of The Horsemens Journal.
• Type: Other Status: Published Year Published: 2019 Citation: Fenger, C., Brewer, K. Duer, W., and Thomas Tobin. Feed forage and bedding what is being called positive? p39-42 Spring 2019 Issue of The Horsemens Journal.
• Type: Other Status: Published Year Published: 2019 Citation: Brewer, K., Fenger, C., Thomas Tobin., The benefits of Lasix for exercise induced pulmonary hemorrhage; Fifty years of science and clinical experience; p43-46 Summer 2019 Issue of The Horsemens Journal.
• Type: Other Status: Published Year Published: 2019 Citation: Kimberly Brewer, Clara Fenger, and Thomas Tobin The Truth about Medications, Veterinary Care, and Breakdowns. Fall 2019 Issue of The Horsemens Journal, pages 36-41.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audiences for this project are 1/ equine forensic chemistry scientists and laboratories worldwide and 2/ to the extent possible, human forensic science laboratories worldwide. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The chemical synthesis portion of this project is carried out in the Maxwell H Gluck Equine Research Center and directly provides training and professional development experience for Mr. Jake Machin, a PhD candidate in the University of Kentucky Department of Toxicology and Cancer Biology, in which Department Dr. Tobin is also a Professor. The program also provides training and professional development opportunities for Dr. Sucheta Kudrimoti, a Research Associate in the Department of Veterinary Science who works directly on the primary synthesis of these reference standards. Additionally, the program has also contributed to the professional development of numerous academic veterinarians and related individuals. How have the results been disseminated to communities of interest?These results have been disseminated to the communities of interest first and foremost by 1/ publication in the refereed scientific literature as detailed under products, by presentations at 2/ scientific and 3/ veterinary and 4/ professional horsemen's meetings and 5/ meetings of interest groups and also by use of the internet, via 6/ the University of Kentucky Maxwell H Gluck Equine Research Center web site , http://gluck.ca.uky.edu/ 7/ the Frontier BioPharm LLC web site https://frontierbiopharm.com/ and also 8/ via the PI's personal professional web site, www.thomastobin.com. Additionally, to the extent possible appropriate, the program is using 9/ the University of Kentucky Maxwell H. Gluck Equine Research Center Facebook page to communicate research findings and availability of certified reference standards to the equine forensic science and regulatory and scientific research communities and the equine sport community at large. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aminorex is an amphetamine related central stimulant substance and a US DEA Schedule 1 controlled substance and an Association of Racing Commissioners International [ARCI] Class 1, Penalty class A foreign substance. Findings of Aminorex at any level in equine samples can give rise to significant penalties for horsemen. Such penalties include up to a one-yearsuspension and a US$10,000 fine for persons judged responsible. As such, identification of Aminorex in a post-event urine sample can be a significant concern for the connections of the horse. A previously identified unexpected source of Aminorex identification in equine urine samples was administration of the immune stimulant Levamisole to horses. Following elucidation of this unexpected source of Aminorex in horse urine the number of Aminorex identifications decreased significantly but did not entirely cease. Specifically, Aminorex was identified in a number of English sport horses with no history or evidence of either Levamisole or Aminorex exposure / administrations. Reviewing the analytical data from these English sport horse urine samples, the plant molecule Barbarin was suggested as a potential source of urinary Aminorex. Plants of the genus Barbarea, Brassicaceae family, contain GlucoBarbarin, a Barbarin precursor. In these plants GlucoBarbarin is hydrolyzed by myrosinase to an intermediate which spontaneously cyclizes to Barbarin, an insect repellant or attractant released when the plant structure is damaged. Barbarin is structurally related to Aminorex, and so it is possible that either GlucoBarbarin or Barbarin or a related substance in Brassicaceae plant fragments in equine feedstuffs may be the source of these unexpected equine urine Aminorex identifications. Pursuing this hypothesis, we synthesized, Barbarin in gram quantities. Availability of this standard allowed us to test the hypothesis that Brassicaceae plants could give rise to Aminorex identifications in equine urine, and we next botanically identified and harvested Kentucky Barbarea vulgaris, ("Yellow Rocket") and administered the plant orally to horses. Analysis of post-administration urine samples showed the presence of Aminorex, showing that consumption of Kentucky Barbarea vulgaris can give rise to Aminorex identifications in equine urine. These identifications are consistent with occasional low concentration identifications of Aminorex in equine samples submitted for drug testing. The source of these Aminorex identifications is therefore now believed to be the chemically related Barbarin, found as its precursor GlucoBarbarin in Kentucky Barbarea vulgaris and related Brassicaceae plants worldwide. Naproxen is a non-steroidal anti-inflammatory medication used in competition and racing horses. Given orally to horses, equine treatment stalls inevitably become contaminated with Naproxen. Naproxen is also both stable in the stall environment and well absorbed orally, so a horse remaining in a treatment stall is at risk of Naproxen exposure for an undefined period. Starting in 2015 Thoroughbred racing in Charles Town, West Virginia experienced a sequence of intermittent low concentration Naproxen identifications, 6.3 to 161ng/mL of plasma. These identifications were ongoing, so horsemen were unaware of their origins. These identifications are most likely associated with exposure of these horses to environmental traces of Naproxen. We were asked toidentify a Screening Limit of Detection [SLOD] below which these identifications would not be reported. Statistical analysis of the data suggested an SLOD of 200ng/mL, outlier analysis suggested an "extreme" outlier level at 247ng/mL, which was rounded up to 250ng/mL Naproxen. This SLOD is in good agreement with other US regulatory thresholds for therapeutic medications and this Interim SLOD was presented for review.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jacob Machin, MS1; Wayne Duer, PhD2; George Maylin, DVM, PhD3; Clara Fenger, DVM, PhD, DACVIM4*; Daniel Wilson, DVM5; Martin Ivey, DVM6; Brett Berthold, DVM5; Sean Allison, DVM5; Thomas Tobin, MRCVS, DABT, PhD1. A field investigation of methylprednisolone plasma concentrations six days after synovial injections in racing horses: Equine Vet J. doi: 10.1111/evj.13003. Epub 2018 Sep 5
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: W.C. Duer, J. Machin, G. Maylin, C. Fenger, T. Tobin. APPLICATION OF ROBUST REGRESSION ON ORDER STATISTICS AND DISTRIBUTION FREE STATISTICS IN THRESHOLD DETERMINATIONS FOR THERAPEUTIC MEDICATIONS, USING METHYLPREDNISOLONE AS AN EXAMPLE. 22nd International Conference of Racing Analysts and Veterinarians, Dubai, United Arab Emirates March 3-10th 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: J. Machin, G.A. Maylin, C.K. Fenger, R. Hunt, K.E. Bladon, O. Sacopulos, & T. Tobin. PILOT PHARMACOKINETIC STUDIES USING A HIGHEST NO EFFECT DOSE (HNED) AND A FULL CLINICAL DOSE OF MEPIVACAINE IN THOROUGHBRED HORSES. 22nd International Conference of Racing Analysts and Veterinarians, Dubai, United Arab Emirates March 3-10th 2018.
• Type: Other Status: Published Year Published: 2018 Citation: Fenger, C., P. Sacopulos and Thomas Tobin (2018). "Trace-level identifications are not positives: the scientifically correct and public-relations appropriate approach to medication regulation." Horsemen's Journal 65(2): 43-47.
• Type: Other Status: Published Year Published: 2018 Citation: Fenger, C., S. Selway and Thomas Tobin (2018). "Lasix lessons learned: science confirms what American horsemen knew 50 years ago." Horsemen's Journal 65(3): 48-50.
• Type: Other Status: Published Year Published: 2018 Citation: Shults, T. F., K. Brewer, J. Machin, E. Hamelback and Thomas Tobin (2018). "Rational reasoning on ractopamine." Horsemen's Journal 65(1): 44-45.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:TARGET AUDIENCES: The primary target audiences of this research project include the worldwide horse, camel and greyhound racing industries, with a particular emphasis on targeting racing chemists and regulators and an additional emphasis on the overall racing industries worldwide, with a secondary emphasis on Equine Forensic Science and Veterinary Diagnostic practice and Veterinary practice worldwide. Inthe third week of October 2016 the PI participated in and made four platform presentations on aspects of racingchemistry research at the Twenty First International Conference of Racing Analysts and Veterinarians held in Montevideo, Uruguay, from October 15th to 22nd. Presentationswere made onthe synthesis, certification and purification of a deuterated Certified ReferenceInternal Standardfor Xylazine, a widely used equine tranquilizer/therapeutic medication, a presentation was made on theanalytical and statistical determination of asuitable regulatorythreshold in plasmafor dexamethasoneand, in cooperationwith a Venezuelanveterinary colleague,Dr. AbelardoMorales Briceno, we presented an analysis of equineforensic science/equine drug testing in Venezuela, which drug testing is currently based largely the Enzyme Linked ImmunoSorbent Assay [ELISA] testingtechnology developed in our UK research program and licensed for worldwide distribution to Neogen Inc., which tests are now manufactured in and distributed worldwide from Lexington, Kentucky and which tests are now beginning to be widely used in human Forensic Science in the field of hair testing. Also with Dr. Abelardo Morales Briceno in Montevideo, we presented a unique review of the incidence of epistaxis in a traditional Spanish Romera, the first ever such report in the world veterinary literature. Other target audience activitiesincludeda presentation on November 20th, 2016 to a working group of the Louisiana State Racing Commission on therapeutic medication regulation, the Director of which regulatory laboratory at Louisiana State University, Dr. Levent Dirikolu, performed his doctoral research in Equine Forensic Science in association with this research program in the Maxwell H. Gluck Equine Research Center here at UK. In January 2107 the PI, along with a group of Veterinary and Farrier colleagues, organized and presented the first Tex Cauthen Memorial Seminar, an International Workshopfor Veterinarians and Farriers held on January 22nd in the Gluck Equine Research Center, attended by over 100 industry participants. The next target audience event was a presentation to a February 3rd, workshop on hair testing in Oklahoma, organized by the Oklahoma Horsemen's Benevolent and Protective Association [HBPA], followed on March 10th by a closely relayed presentation on "Hair testing as a regulatory technique in medication control" presented at theNational Horsemen's Benevolent and Protective Associationconference inLas Vegas. In late March and early April further target audience presentations to researchers and forensic scientists in Dubai were made following an invitationfrom Sheikh Mohammed bin Rashid Al Maktoum,Vice President and Prime Minister of the United Arab Emirates (UAE) and ruler of the Emirate of Dubaito present at the BuhootEquine Research Center in Dubai on March24th. Further target audience presentations on aspects of Equine Forensic Science included a presentation to the National Horsemen's Benevolent and Protective Association Executive Committee on June 30th, 2017 in Louisville, Kentucky, followed by related target audience presentations made on July 18th,-19th, in Bogota, Colombia,presentations concerning medication regulation to and onbehalf of Asoarabes Unaga, Colombia, with an emphasis on matters involving caffeine regulation. On September 27th at the invitation of the Ohio Horsemen's Benevolent and Protective Association the PI made an invited presentation on inconsequential trace levels ofsubstances identified in equine drug testing to the Ohio State Racing Commission in Columbus Ohio. Then on November 10th, the PI made a presentation beforetheCouncilof Arbitration for Sport, Lausanne, Switzerlandon the matter of "Trace level identifications of urinary ractopamine in show horses" on behalf of Cargill Inc. The PI was also author/co-author of a number of lay press presentations setting forth aspects of equine medication regulation of interest to horsemen, including Horsemen's Journal v.64, no.3 (Fall) 2017, p 32-35, Fenger, C.; Tobin, T. Medication migration: the Charles Town naproxen experience and why it matters to all racing jurisdictions., Horsemen's Journal v.64, no. 2(Summer) 2017, p. 27-30. Fenger, C.; Tobin, T.; Ecabert, J.O. No Lasix racing: good idea or failed experiment? Horsemen's Journal v. 64, no.1 (Spring) 2017, p. 40-42. Fenger, C.; Boulmetis, T.; Tobin, T. Out in the cold: ingredients in over-the-counter cold medicines can wreak havoc on a trainer's livelihood and an owner's prized racehorse. Horsemen's Journal v. 63, no. 4 (Winter) 2016, p. 43-47. Fenger, C.; Boulmetis, T.; Brewer, K.; Tobin, T. The Lowdown on out-of-competition testing. Horsemen's Journal v. 63, no. 3(Fall) 2016, p. 39-43. Fenger, C.; Boulmetis, T.; Brewer, K.; Stirling, K.; Tobin, T. Methamphetamine: a substance of emerging concern for horse racing. Changes/Problems:The principal major change in the project has been the acceleration of the shift from urine testing to blood/plasma/serum testing, which shift has been most marked in North American equine drug testing. This shift has expanded the need for reference standards and deuterated internal standards to include parent drugs as detected and quantified in plasma, as set forth in our recent synthesis, certification and release of certified reference deuterated internals standards for Detomidine and Xylazine. The second change has been the broadening of the requests for certified reference standard and deuterated internal standards away from regulated therapeutic medications and in the direction of unusual or atypical substances being identified in postrace testing worldwide. Emblematic of this change has been the request for a deuterated internal standard for capsaicin, a structurally complicated natural product without an associated well defined chemical synthesis literature, making the synthesis of a stable isotope internal standard for this substance challenging. Additionally, the shift in emphasis away urine testing and towards plasm testing has led us to re-evaluate our previous intent to synthesize reference standards for equine glucuronidated drug metabolites but the increasing shift in North American equine drug testing away from urine testing and in the direction of blood/plasma/serum testing has led us to reevaluate our planned synthesis of a number of glucuronidated medication reference standards and internal standards. What opportunities for training and professional development has the project provided?The program directly enhances the professional development of each of its current major participants. The Major participants include the PI, Thomas Tobin and two of his close professional colleagues/collaborators, Dr. Rodney Eisenberg and Dr. Clara Fenger. Additionally, the program has a long running professional interaction with the National Horsemen's Benevolent and Protective Association, the largest Horsemen's organization in the US and which organization has keen professional interest in the areas of Equine Forensic Science and drug and medication testing and regulation. The primary non-academic scientific collaborator is Dr. Rodney Eisenberg, PhD, an experienced synthetic chemist whose company, Frontier BioPharm Inc., London, Kentucky, licenses the synthesized reference standards from the University of Kentucky. Frontier Biopharm then performs a rigorous chemical purification, characterization and formulation stability tests on the provided reference standard to generate the final product that is marketed worldwide as a certified reference standard or a certified stable isotope internal standard. The synthetic portion of this project in the Maxwell H Gluck Equine Research Center directly provided training and professional development experience for Mr. Jake Machin, a PhD candidate in the University of Kentucky Department of Toxicology and Cancer Biology, in which Department Dr. Tobin is also a Professor. The program also provides training and professional development opportunities for Dr. Sucheta Kudrimoti, a Research Associate in the Department of Veterinary Science who works directly on the primary synthesis of these reference standards. The second major scientific collaborator is Dr. Clara Fenger, DVM, PhD, DACVIM, Equine Integrated Medicine, PLC, Georgetown,KY40324, and Executive Director of the North American Association of Racetrack Veterinarians. Dr. Fenger is a veterinarian, Board Certified in Internal Medicine and with a PhD in Veterinary Science. With Dr. Fenger the program works on the closely related matter of defining regulatory thresholds for therapeutic medications, dietary and environmental substances in racing horse and competition horses worldwide. This work is supported by Horsemen's and other racing groups, most particularly the National Horsemen's Benevolent and Protective Association, whose members are primary beneficiaries of the research and reference standard output of the program. How have the results been disseminated to communities of interest?Dissemination of the results of the research program to communities of interest takes place in a number of ways. First and most importantly and somewhat unusually, the results of the research program, in the form of certified reference standards and stable isotope internal standards are made directly available to the primary audience, equine forensic scientists, through the University of Kentucky licensing arrangement with Frontier Biopharm Inc., which means that these certified reference standards and stable isotope internal standards are available worldwide the equine forensic science and regulatory communities. Second, through the Frontier Biopharm web site the availability of these products is communicated on a 24/7 basis and requests for these standards are promptly attended to. Secondly, the PI published papers and reviews related to this research project as set forth earlier in this report and thirdly, the PI attends and presents at the ongoing bi-annual International Conferences of Racing Analysts and Veterinarians, which conference the PI has twice hosted in Kentucky and at which conferences he presents the most recent products from the conference and receives requests for s new standards. Four presentations were made at the 21st International Conference of Racing Analysts and Veterinarians, Montevideo, Uruguay, during the week of October 15-22nd, 2016, in November an invited presentation was made to the Louisiana State Racing Commission, "Optimal approaches to Regulation of Therapeutic Medications, Endogenous, Dietary and Environmental Substances" Sunday November 20th, 2016, Fairgrounds Race Track, New Orleans, Louisiana, on January 22nd, 2017 the PI and colleagues presented the first Tex Cauthen Memorial Seminar, initiating what appears to be becoming an annual seminar on the professional cooperation and interactions between veterinarians and farriers, on Feb 1st the PI made an educational presentation on behalf of Mr. Mark Ford and the New Jersey Harness Horsemen's Association on the matter of blood testing sampling procedures for the regulatory control of furosemide, on February 3rd the Pi participated in a round table discussion on "Hair testing as a regulatory procedure in racing horses", at the invitation of the Oklahoma HBPA, on March 10th the PI presented on hair testing to the NationalHBPA Annual Convention, Las Vegas, Nevada, on March 25th in association with an invitation to attend the 2017 Dubai World Cup the Pi made presentations to Mr. Azam Syed, Pharmacy in Charge, Zabeel Palace, Dubai and visits and related presentations to the Buhoot Equine Research Center, Marmoon, Dubai, UAE, July 19th and 20th, presentations on caffeine regulation at AGROEXPO 2017 and also top Fedequinas at their headquarters in Bogota, Colombia. September 26th presentation to the Ohio State Racing Commission, Columbus, Ohio on "Inconsequential Trace Level Identifications of "Environmental" Substances". Finally as set forth above in the target audience section the Pi collaborateswith professional colleagues and Horsemen's organizations and published outreach articles in the Horsemen's Journal as follows: , including Horsemen's Journal v.64, no.3 (Fall) 2017, p 32-35, Fenger, C.; Tobin, T. Medication migration: the Charles Town naproxen experience and why it matters to all racing jurisdictions., Horsemen's Journal v.64, no. 2(Summer) 2017, p. 27-30. Fenger, C.; Tobin, T.; Ecabert, J.O. No Lasix racing: good idea or failed experiment? Horsemen's Journal v. 64, no.1 (Spring) 2017, p. 40-42. Fenger, C.; Boulmetis, T.; Tobin, T. Out in the cold: ingredients in over-the-counter cold medicines can wreak havoc on a trainer's livelihood and an owner's prized racehorse. Horsemen's Journal v. 63, no. 4 (Winter) 2016, p. 43-47. Fenger, C.; Boulmetis, T.; Brewer, K.; Tobin, T. The Lowdown on out-of-competition testing. Horsemen's Journal v. 63, no. 3(Fall) 2016, p. 39-43. Fenger, C.; Boulmetis, T.; Brewer, K.; Stirling, K.; Tobin, T. Methamphetamine: a substance of emerging concern for horse racing. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue the project as previously to the best of our ability. First we will continue the reference standard synthesis, characterization, certification as ISO-17025 equivalent Certified Reference Standards and Stable Isotope Internal Standards and ensuring industry availability of these products. We review each substance request carefully and prioritize each synthesis based on the likelihood of reasonably rapid completion of the individual synthesis. In this area one requested stable isotope internal standard, has been very challenging, namely the request for a stable isotope internal standard for capsaicin and work on this very challenging synthesis has been assigned a lower priority. Additionally, work is ongoing with respect to the University of Kentucky Intellectual Property involving novel therapeutic approaches to parasitic disease licensed for patenting and commercialization to VC Equine Research, Inc., now Southern Research Inc. on September 24th, 2012. A provisional patent was filed by VC equine, now Southern Research Inc., and the utility patent made possible by this Intellectual Property disclosure was filed on August 4th, 2014 as follows; US Utility Patent E-filing, Filing, Oz, H and Tobin T "Novel Compositions & Methods for Preventing & Treating Apicomplexan Related Disease" utility patent application E-filed August 4th, 2014, EFS ID: 19771622, Application Number: 14451337 International Application Number: n/a Confirmation Number: 496. Details of this utility patent application are still being negotiated between Southern Research Inc., and the US Patent Office, and it is our understanding that awarding of this utility patent is expected shortly.

Impacts
What was accomplished under these goals? Impact of this project: This project is central the scientifically and forensically correct regulation of horse, camel and dog racing. Without the Certified Reference Standards and Stable Isotope Internal Standards produced by this program it would not be possible to scientifically and forensically correctly regulate many of the therapeutic substances currently approved for therapeutic use in horses, dogs and camel racing. Additionally the availability of these standards for a significant number of therapeutic medications in many cases underlies the ability to perform the basic research on which the regulatory thresholds for these substances are based. Additionally, a second and indirectly related area of research is the further development and application to human medicine of the highly specific anti-apicomplexan action identified in our earlier Equine Protozoal Myeloencephalitis research project , the application of which to human medical research we are pursuing with the Oz and Tobin "Novel Compositions & Methods for Preventing & Treating Apicomplexan Related Disease" utility patent application E-filed August 4th, 2014, which patent has been licensed out by UK and with reference to which patent application we are assisting the patent licensee. More broadly, the ongoing increases in the sensitivity of analytical techniques are driving a shift in equine forensic science from urine testing to blood/plasma/serum testing, a shift that is particularly marked in North American testing. This shift is redirecting equine forensic science towards testing for the parent medications in blood /plasma/serum rather than testing for urinary metabolites or metabolite fragments recovered from urine. One direct result of this shift has been an increased need for certified reference standards and deuterated internal standards forspecific high potency equine medications as the primary medication itself rather than forspecific drug metabolites or metabolite fragments recovered from urine samples, as had previously been the regulatory analytes of choice. In this areathe program has previously synthesized and certified reference standards for the regulatory analyte/metabolite fragment of Xylazine, namely 4-OH-Xylazine and also for the regulatory analyte/metabolite fragments of Detomidine namely carboxyDetomidine. However, given this recent regulatory shift in North American testing to blood/plasma serum testing for xylazine and detomidine the program has synthesized, certified and made available the industry certified reference internal standards for Xylazine, as reported at the Montevideo ICRAV and has also just reported the synthesis and certification of a deuterated internal standard for Detomidine, Other work approaching completion includes synthesis of a reference standard and a deuterated internal standard for OH-dantrolene and purification and certification of a reference standard for Guanabenz. We also had an industry request for a deuterated internal standard for capsaicin, an extremely challenging synthesis and considerable progress has been made in this area. A further challenging area has been the development of a useful synthetic pathway for toxicologically useful amounts of ergot alkaloids and considerable progress has been made in this area. With regard to the proposed synthesis of glucuronide metabolites of certain substances, this approach has been overtaken by the increases sensitivity of plasma testing as set forth above. In other work on the matter of commercializing our commercializing our patent applications on the efficacy of diclazuril and related benzeneacetonitrile substances alone and in combination with other substances in the prevention/treatment of fetal toxoplasmosis we are working with Southern Pharmaceutical Research of Frankfort, Kentucky on the commercialization of this technology. Additionally, in an unanticipated outreach area, in January of 2017 we launched a one-day educational seminar for professional farriers entitled the Tex Cauthen Memorial Seminar held in the Maxwell H. Gluck Equine Research Center on Sunday, January 22nd with well over 100 attendees and we currently organizing the second such seminar, now focusing on veterinarian/farrier interactions to be held also in the Gluck Equine Research Center on January 21st, 2018.

Publications

• Type: Other Status: Published Year Published: 2017 Citation: Machin J, Shults, T Fenger C and Tobin T, 2017: Inadvertent Environmental Transfer of Dextromethorphan from Groom to Racehorse results in Establishment of a Pharmacologically relevant Cutoff; ToxTalk, Vol 41 Issue #2 p 20 -23
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Nicola Pusterla, DVM, PhD,a,* Thomas Tobin, MVB, PhDb Update on EPM Therapeutics; Veterinary Clinics of North America: Equine Practice Equine Pharmacology, Vet Clin Equine 33 (2017) 87-97.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: J. Machin, S. Kudrimoti, K. Brewer, R. Eisenberg & T. Tobin SYNTHESIS, PURIFICATION AND CERTIFICATION OF XYLAZINE-d6 FOR EQUINE MEDICATION REGULATION " Platform presentation and in press, 21st International Conference of Racing Analysts and Veterinarians, Montevideo, Uruguay, pages 102-104, October 15-22nd, 2016.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Abelardo Morales Brice�o, Aniceto Mendez Sanchez, Kimberly Brewer, Thomas Tobin FOREIGN SUBSTANCES IDENTIFIED POST RACE IN THOROUGHBREDS AT LA RINCONADA RACETRACK, CARACAS VENEZUELA, A CLINICAL PATHOLOGICAL AND TOXICOLOGICAL STUDY " Platform presentation and in press, 21st International Conference of Racing Analysts and Veterinarians, Montevideo, Uruguay, pages 102-104, October 15-22nd, 2016. KAES Manuscript Number 17-14-089
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Jacob Machin, MS; Wayne Duer, PhD; George Maylin, DVM, PhD; Clara Fenger, DVM, PhD, DACVIM*; Daniel Wilson, DVM; Martin Ivey, DVM; Brett Berthold, DVM; Sean Allison, DVM; Thomas Tobin, MRCVS, DABT, PhD. A field investigation of methylprednisolone plasma concentrations six days after synovial injections in racing horses: Submitted for publication, Equine Vet J, September 2017 KAES Manuscript Number 17-14-088.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2018 Citation: Selway, S, Meittinis, N, Prendergast, J, Northrup, F, Carabajal, A, Duer, WC, Fenger, C, Bladon, KE, Tobin, T. A LONGITUDINAL EVALUATION OF THE EFFECT OF FUROSEMIDE ON PERFORMANCE IN A COHORT OF TWO-YEAR-OLD RACING THOROUGHBREDS Submitted and accepted for presentation and publication at the 22nd International Conference of Racing Analysts and Veterinarians, Dubai United Arab Emirates, March 3-10th, 2018.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2108 Citation: Machin, J, Maylin, GA, Fenger, CK, Hunt, R, Bladon, KE, Sacopulos, O, Tobin, T. Pilot Pharmacokinetic Studies using a Highest No Effect Dose (HNED) and a High Clinical Dose of Mepivacaine in Thoroughbred Horses THOROUGHBREDS Submitted and accepted for presentation and publication at the 22nd International Conference of Racing Analysts and Veterinarians, Dubai United Arab Emirates, March 3-10th, 2018
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Jake Machin, Sucheta Kudrimoti, Rodney Eisenberg and Thomas Tobin, SYNTHESIS AND CERTIFICATION OF A DEUTERATED INTERNAL STANDARD FOR DETOMIDINE: Submitted and accepted for presentation and publication at the 22nd International Conference of Racing Analysts and Veterinarians, Dubai United Arab Emiraets, March 3-10th, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Morales Brice�o A, M�ndez-S�nchez A, Brewer K, Tobin T. A�o: 2016 Pharmacoepidemiological study of foreign substances identified in urine samples post race associated mortality at a Racetrack La Rinconada, Caracas Venezuela, 2008-2012: A preliminary report. Boletim Informativo Geum, v. 7, n. 1, 2016 Disponible en: http://www.ojs.ufpi.br/index.php/geum/article/view/5059 Primera y �ltima p�gina: 1-6
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Morales Brice�o A, Brewer K, Tobin T, M�ndez S�nchez A, P�rez Ar�valo J. A�o: 2017.Hemorragia pulmonar padecimiento usual en caballos deportivos Volumen: Revista Mundo Equino Marzo-Abril 2017, Primera y �ltima p�gina: 58-59.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Morales Brice�o A, M�ndez-S�nchez A, Brewer K, Tobin T. A�o: 2016 Pharmacoepidemiological study of foreign substances identified in urine samples post race associated mortality at a Racetrack La Rinconada, Caracas Venezuela, 2008-2012: A preliminary report. Boletim Informativo Geum, v. 7, n. 1, 2016 Disponible en: http://www.ojs.ufpi.br/index.php/geum/article/view/5059 Primera y �ltima p�gina: 1-6.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Duer, WC, Machin, J, Maylin, GA, Fenger, CK, Tobin, T. Application of Robust Regression on Order Statistics and Distribution Free Statistics in Threshold Determinations for Therapeutic Medications, using Methylprednisolone as an Example: Submitted and accepted for presentation and publication at the 22nd International Conference of Racing Analysts and Veterinarians, Dubai United Arab Emirates, March 3-10th, 2018
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abelardo Morales Brice�o1,2, Antonio Lamprea Garrido2, Alicia Garcia Hermoso2, Aniceto Mendez Sanchez1, Kimberly Brewer3,Thomas Tobin5 EPISTAXIS IN UNMEDICATED EQUINE DURING INTENSE EXERCISE IN ALMONTE-HUELVA-SPAIN. A PRELIMINARY REPORT: Presented at the 21st International Conference of Racing Analysts and Veterinarians, Montevideo, Uruguay, October 15-22nd, 2016. KAES Manuscript Number 17-14-086. Also published as Morales A. M�ndez-S�nchez A, Brewer K, Thomas Tobin. Octubre, 2017. Estudio retrospectivo de la casu�stica de muerte s�bita en Almonte-Huelva, Andaluc�a, Espa�a. Proceeding XL Jornadas Cient�ficas Dra. Rosa Alba Salas Mora, a celebrarse en las conmemoraciones del LXXIX Aniversario del INHRR y el XLI Aniversario de la Sociedad Cient�fica del INHRR.Fecha: Ciudad y Pa�s: Caracas, Venezuela.