Characterisation of some metabolic conjugation pathways in the horse by Mary Varwell Marsh a thesis submitted for the degree of Doctor of Philosophy in the University of London. March 1983 Department of Pharmacology, St. Mary's Hospital Medical School, London W2 IPG -2- ABSTRACT This thesis presents a study of the metabolic conjugation or Phase II reactions in the horse. There has been a paucity of systematic research on the metabolism of drugs in the horse, which is surprising in view of their widespread use in equine veterinary medicine and also their occasional use as doping substances. The approach adopted has been to investigate the in vivo metabolism of six carboxylic acids 14 ( C-labelled); the nature of the urinary metabolites was established by radiochemical and chromatographic techniques (paper and thin layer chromatography and hplc), and by NMR and mass spectrometry. The compounds administered were three carboxylic acids used as 'probes' for eliciting conjugation reactions, namely benzoic acid, phenylacetic acid and 2-naphthylacetic acid, and three non-steroidal anti-inflammatory drugs, salicylic acid, fenclofenac and isoxepac, all of which are metabolised to conjugates in other species. The study has established the horse can utilise glycine, glucuronic acid and taurine for the -3- metabolic conjugation of these acids. This is the first time that taurine conjugation has been observed to occur in the horse. A novel pathway of metabolism was uncovered which involves the addition of a 2-carbon fragment to benzoic acid, to form 3-hydroxy 3-phenylpropionic acid, this reaction is analogous to that which occurs in the natural elongation of fatty acids. The metabolic conjugation pathways which occur in the horse, and the disposition and renal elimination of some drugs representative of the widely used anti-inflammatories have been examined. The findings are of relevance in that they provide basic information on metabolic options for carboxylic acids in the horse. Also the findings contribute to the elaboration of sound means of detecting such drugs in the context of their illicit use. -4- CONTENTS Page Abstract 2 Acknowledgements 6 List of Tables 8 List of Figures 13 Chapter One : Introduction 20 Chapter Two : General Methods 87 Chapter Three : The metabolism and disposition of salicylic acid 102 Chapter Four : The metabolism and disposition of benzoic acid 128 Chapter Five : The metabolism and disposition of phenylacetic acid 161 Chapter Six : The metabolism and disposition of 2-naphthylacetic acid 198 -4- CONTENTS, continued Page Chapter Seven : The metabolism and disposition of isoxepac 230 Chapter Eight : The metabolism and disposition of fenclofenac 257 Chapter Nine : Discussion 283 Appendices 295 References 347 -2- ACKNOWLEDGEMENTS I would like to thank Professor R.L. Smith for giving me the opportunity to work in his department and for his constant enthusiastic guidance and encouragement. I am grateful to Dr John Caldwell for all his advice over the past three years, and also to my other colleagues at St Mary's especially Andrew Hutt, Tim Sloan, Lawrence Wakile and Nick Oates for their help in various ways. It would have been impossible for this work to be carried out without the co-operation of the employees of Racecourse Security Services Ltd., and I am grateful to my many friends at Newmarket, particularly to Dr Michael Moss and Marian Horner, Ed Houghton, Philip Teale, Pearl Blay and John, Brian and Paul in the stables. I am indebted to the expertise of Geoff Hawkes 13 of Queen Mary College who recorded the C-NMR spectra and helped with their interpretation. My special thanks are due to Philippa Chilvers and Jill Rogers for their expertise and patience in typing this thesis. -2- I am grateful to the Science and Engineering Research Council and R.S.S. Ltd. for financial support. Finally, I would like to thank my family for all their encouragement during my education. -8- List of Tables Page 1.1. Drugs reported to the Association of Official Racing Chemists 1947-1973. 34 1.2. Drugs reported to the Association of Official Racing Chemists May 21st 1978- December 31st 1978. 36 1.3. Comparison of the type of drug positive samples found in 1947-1973 and 1978. 37 1.4. The recovery of dose (percentage of total) for every 24 hour period following a daily dose of phenylbutazone. 52 1.5. Metabolism and elimination of phenylbutazone in the horse. 53 1.6. Metabolic reactions of drugs in the horse, Phase I reactions. 69 1.7. Metabolic reactions of drugs in the horse, Phase II reactions. 71 1.8. The major conjugation reactions. 74 1.9. Amino acid conjugates found in vertebrate animals. 78 3.1. Rji values of salicylic acid and metabolites. 109 3.2. Urinary metabolites of salicylic acid. 123 4.1. RF values of benzoic acid and its metabolites 138 4.2. Hplc retention time of benzoic acid and its metabolites. 139 -9- Page 4.3. Calculation of amount of hippuric acid normally present in urine. 147 4.4. Urinary metabolites of benzoic acid. 159 5.1. Chromatographic properties of hippuric acid and phenylacetic acid and its conjugates. 170 5.2. Urinary metabolites of phenylacetic acid. 182 6.1. Chromatographic mobilities of 2-naphthyl- acetic acid and metabolites on tic. 213 6.2. Hplc mobile phases, and retention times of 2-naphthylacetic acid and metabolites. 215 6.3. Urinary metabolites of 2-naphthylacetic acid in 0-96 hours. 227 7.1. Chromatographic properties of isoxepac and the taurine conjugate of isoxepac. 238 7.2. Urinary metabolites of isoxepac in Andrew and Shepherd Boy. 246 8.1. Chromatographic properties of fenclofenac and 5-hydroxyfenclofenac. 264 8.2. Plasma disposition of fenclofenac in the horse. 271 8.3. Urinary metabolites of fenclofenac in the horse expressed as percentage of total dose. 27 7 -10- Page 9.1. Reactions of xenobiotic metabolism and lipid biochemistry. 290 14 Al.l. Plasma data ( C levels) for Ginger (pony) following salicylic acid administration. 297 A1.2. Urinary data for Ginger (pony) following salicylic acid administration. 298 A1.3. Urinary data for calculation of biological half-life for Ginger (pony) following salicylic acid administration. 299 A1.4. Urinary data for Shepherd Boy (thoroughbred) following salicylic acid administration. 300 A1.5. Urinary data for calculation of biological half-life for Shepherd Boy (thoroughbred) following salicylic acid administration. 301 14 A2.1. Urinary elimination of C following benzoic acid administration to Caspar (pony). 306 14 A2.2. Urinary elimination of C following benzoic acid administration to Floral Song (thoroughbred). 307 14 A3.1. Urinary elimination of C following phenylacetic acid administration to Caspar (pony). 309 14 A3.2. Urinary elimination of C following phenylacetic acid administration to Floral Song (thoroughbred). 310 -11- Page 14 A4.1. Urinary elimination of C following 2-naphthylacetic acid administration to Ginger (pony). 312 14 A4.2. Urinary elimination of C following 2-naphthylacetic acid administration to Shepherd Boy (thoroughbred). 313 14 A6.1. Urinary elimination of C following isoxepac administration to Andrew (pony) 331 A6.2. Urinary data following isoxepac administration to Andrew (pony). 333 14 A6.3. Urinary elimination of C following isoxepac administration to Shepherd Boy (thoroughbred). 335 A6.4. Urinary data following isoxepac administration to Shepherd Boy (thoroughbred). 336 14 A7.1. Plasma data ( C levels) for Andrew (pony) following fenclofenac administration. 342 14 A7.2. Urinary elimination of C following fenclofenac administration to Andrew (pony). 343 14 A7.3. Plasma data ( C levels) for Caspar (pony) following fenclofenac administration. 344 14 A7.4. Urinary elimination of C following fenclofenac administration to Caspar (pony). 345 -12- List of Figures Page 1.1. The major metabolites of caffeine. 44 1.2. The metabolism of amphetamine in the horse. 47 1.3. The metabolism of phenylbutazone in the horse. 51 1.4. Some non-steroidal anti-inflammatory drugs used in equine medicine. 55 1.5. The metabolism of promazine in the horse. 64 1.6. The metabolism of acetylpromazine in the horse. 65 1.7. Reactions involved in the formation of a peptide bond. 77 1.8. Synthesis of mercapturic acid. 80 1.9. Sequence of reactions in the formation of a sulphate conjugate. 84 2.1. Equipment used to collect urine samples from a horse. 89 3.1. Routes of metabolism of aspirin in man. 105 14 3.2. Cumulative excretion of C in the urine following -salicylic acid p.o. 115 14 3.3. Ginger : plasma loge [ C] levels plotted against time. 116 14 3.4. Ginger : levels of C in plasma and saliva, plotted against time following an oral dose of 14C -salicylic acid. 118 3.5. Urinary metabolites of salicylic acid as 14 percentage of total C in sample in 0-30hr (Ginger). 121 -13- Page 3.6. Urinary metabolites of salicylic acid as 14 percentage of total C in sample in 0-24hr (Shepherd Boy). 122 3.7. Ginger : salicylate levels in urine following salicylic acid p.o., estimated from the level of 14C. 124 3.8. Shepherd Boy : salicylate levels in urine following salicylic acid p.o., estimated from the level of 14C. 125 4.1. Conjugation of benzoic acid. 130 4.2. Mass spectrum of (ds)-benzoic acid methyl ester (methylated in deuteromethanol). 133 4.3. ^H-NMR spectrum of 3-hydroxy 3-phenyl propionic acid in CD3CI. 135 4.4. Cumulative excretion of radioactivity following 14 oral administration of C -benzoic acid. 140 4.5. Mass spectra of a mixture of and protonated hippuric acid methyl ester, isolated from urine (upper spectra) and authentic hippuric acid methyl ester (lower spectrum), obtained by gems.143 4.6a. G.c. traces obtained by single ion monitoring for ions in the spectra of d5-hippuric acid (139, 198) and protonated hippuric acid (134, 193).