SCIENCE-BASED REGULATION OF PHARMACOLOGICAL SUBSTANCES IN COMPETITION HORSES

Current testing methodologies within equine forensic toxicology focus on arbitrary thresholds and zero-tolerance policy. Modern analytical chemistry’s limits of detection are low enough that oftentimes femtogram-per-milliliter amounts of a substance can readily be identified in both blood and urine of a horse. For most pharmacologically relevant compounds, these concentrations have no relevance to pharmacological effect. It is therefore crucial that testing methodologies to determine appropriate thresholds and cut-offs be developed that are driven by biological activity rather than arbitrary limits of detection. This dissertation looks to address this by suggesting a system of calculated Effective Plasma Concentrations by which a safety factor may be determined to calculate an Irrelevant Plasma Concentration that may act as a regulatory threshold. In support of such pharmacokinetic studies, novel synthetic pathways have been developed to produce reference standards and stably isotopically labelled deuterated internal standards. The research undertaken in this thesis is comprised of three primary areas of focus: (1) the development of novel synthetic pathways for certified reference standards and internal reference standards, (2) the pharmacokinetic description of biologically relevant compounds using modern techniques so as to guide both horsepersons and regulators in the horse industry, and (3) the description of environmental contamination risk for compounds and their ability to be taken up by the animals and later detected by forensic investigators from these residual exposures. Of the first category, three papers are presented, each representing the novel synthesis of a different compound of biological importance. The first compound synthesized was deuterated (d6) xylazine as use as an internal standard for quantitation of the therapeutic compound xylazine. The second compound was the novel synthesis and purification of barbarin, a naturally occurring glucosinolate which is believed to be responsible for the appearance of a DEA Schedule I compound in horse urine. The synthesis of barbarin as described is the first known chemical synthesis of the compound, which until this point had only been previously extracted from biological samples. The third paper covers the synthesis of a deuterated (d5) barbarin for use as an internal standard in its quantitation. The second category covers the pharmacokinetic studies, the first of which is a pilot study which looked at the terminal half-life of mepivacaine in thoroughbred horses. This study offers guidance for horsepersons and veterinarians on proper withdrawal times for the given regulatory thresholds of the compound when using therapeutic doses up to four-hundred milligrams as well as describing the pharmacokinetic model developed in determining these withdrawal times. The next study looks at methylprednisolone administration across multiple joints and the effects of specific joint injections and co-injections on the detection of the compound out to six days post-administration. This paper also offers guidance to withdrawal of animals after therapy and highlights many of the problems in current regulatory guidance. The final paper looks at betamethasone in harness racing and suggests testing thresholds based upon pharmacokinetic and statistical models at a six-day withdrawal timeline. The final paper discusses the likelihood of the commonly used non-steroidal anti-inflammatory drug naproxen to contaminate the environment of the horse. It also looks establishes a suggested screening limit of detection for the compound based upon the concentrations of a series of innocuous positives that appeared at a racetrack upon changing testing facilities. This paper highlights the importance of consistency between testing facilities as well as the need for expanded established limits instead of the zero-tolerance regulation of most compounds. Finally, the dissertation concludes with a synopsis of the research presented along with suggestions on how the racing industry may move forward in its testing of the animals in both a way that will be less burdensome in the future as well as more appropriate to protecting the health and livelihood of the animals on which it relies