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). 145
4.6b. 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). 146 -14- Page
4.7. Total ion chromatogram of isolated metabolic
fraction following methylation - gems analysis. 149
4.8. Gems analysis of a mixture of ds-acetophenone
and protonated acetophenone, isolated from
urine (upper spectra), and an authentic sample
of acetophenone (lower spectrum). 151
4.9. Gems analysis of a mixture of dg-cinnamic acid
methyl ester and protonated cinnamic acid
methyl ester isolated from urine (upper spectra)
and authentic cinnamic acid methyl ester
(lower spectrum). 152
4.10. Gems analysis of a mixture of dg-S-hydroxy
3-phenyl propionic acid methyl ester and
protonated 3-hydroxy 3-phenyl propionic
acid methyl ester, isolated from urine
(upper panel) and synthetic 3-hydroxy
3-phenyl propionic acid methyl ester
(lower panel). 153
4.11. Major ions in the fragmentation pathway
of ds and protonated 3-hydroxy 3-phenyl-
propionic acid methyl ester. 154
4.12. G.c. traces from single ion monitoring :
upper panel metabolic sample, lower panel
synthetic sample, 3-hydroxy 3-phenyl-
propionic acid. 157
13 5.1. C-NMR shifts for the carbonyl carbon in 166 potential metabolites of phenylacetic acid. -15-
Page
5.2. Cumulative excretion of 14 C following the oral . . 14 administration of C-phenylacetic acid. 175
5.3. Mass spectrum obtained by gems of the sole
metabolite isolated from Floral Song's urine,
following methylation. The m.s. corresponds
to a mixture of dg, d7 and protonated
phenylacetic acid. 176
5.4. Proposed fragmentation for the major ions
seen in the mass spectrum (fig. 5.3.),
Containing dg, d7 and protonated phenyl-
acetylglycine. 177
5.5. Gems of the major metabolite isolated from
Caspar's urine following methylation. 179
5.6. Gems of the methyl ester of phenylacetic
acid, upper panel material isolated from
Caspar's urine, and lower, synthetic sample. 180
5.7. The N-methyl ester of phenylacetylglycine methyl ester, from Caspar administration. 183 13 5.8. C-NMR spectrum of urine extract in D2O. 18 5 13 5.9. C-NMR spectrum of urine extract in D2O : (CD3)2 CO 85 : 15. 189 13 5.10 Proton decoupled C-NMR spectrum of urine
extract in D20 : (CD3)2CO 85 : 15, 24-50ppm. 192 13 13 5.11. C-NMR spectrum of C-phenylacetylglycine
isolated from urine in D20 : (CD3)2CO 85 : 15. 194 -16- Page
6.1. Routes of metabolism of 2-naphthylacetic acid. 200
6.2. Reaction scheme followed in the synthesis of 14 [ C-carboxyl]-2-naphthylacetic acid. 202
6.3. Direct insertion (E.I., 70 eV) mass spectrum 14 of [ C] 2-naphthylacetonitrile. 204
6.4. Direct insertion (E.I., 70eV) mass spectrum 14 of [ C] 2-naphthylacetic acid. 205
6.5. Major fragments in the mass spectra of
2-naphthylacetonitrile and 2-naphthylacetic
acid. 206
6.6 ^H-NMR spectrum of 2-naphthylacetylglycine. 209
6.7. "''H-NMR spectrum of a mixture of 2-naphthyl-
acetyltaurine and taurine in a ratio of 3:2. 211
6.8. "^H-NMR spectrum of 2-naphthylacetyltaurine. 212
6.9. Chromatograms obtained in A : isocratic system
and B : solvent programmed system. 217 14 6.10. Cumulative recovery of C following oral 14 dosing with C-2-naphthylacetic acid. 220
6.11. Radiochromatogram scan showing the major 14
C bands in urine, 2 naphthylacetic acid
administration. 221
6.12. Mass spectrum of 2-naphthylacetylglycine
methyl ester, upper panel:authentic sample,
lower:sample isolated from urine. 224
6.13. Assignments of major ions in the mass spectrum
of the methyl ester of 2-naphthylacetylglycine. 225
6.14. Quantitative pattern of metabolism for days
0-4 following an oral dose of 2-naphthylacetic
acid. 226 -13- Page
7.1. Isoxepac, (6,11-dihydrodibenz [b,e] oxepin-
2-acetic acid). 232
7.2. Routes of metabolism of isoxepac. 233
7.3. Cumulative excretion of radioactivity in urine 14 after oral administration of C -isoxepac :
Andrew. 242
7.4. Cumulative excretion of radioactivity following 14 oral administration of C -isoxepac :
Shepherd Boy. 243
7.5. Effect of light on the stability of isoxepac
in solutions buffered to different pH's
(2 hours exposure). 251
7.6. Radiochromatogram scan of Andrew's urine.
Isoxepac administration. 253
7.7. Radiochromatogram- scan of Shepherd Boy's
urine. Isoxepac administration. 254
7.8. Major decomposition product of isoxepac. 255
8.1. Routes of metabolism of fenclofenac. 259
8.2. Direct insertion mass spectrum of
fenclofenac (E.I., 70eV). 261
8.3. Direct insertion mass spectrum of 5-hydroxy-
fenclofenac (.E.I., 70eV). 262 14 8.4. Cumulative excretion of C following oral
administration of 14C -fenclofenac. 267
8.5. Plasma levels of radioactivity (dpm/ml), against time following the administration 14 of C-fenclofenac p.o. 269 -18- Page
8.6. Hplc separation of an ethyl acetate extract
of urine, fenclofenac administration. 274
8.7. Extract, shown in fig. 8.6., following
treatment with alkali. 275
8.8. The urinary metabolites of fenclofenac, the
cumulative totals expressed as percentage
of dose, Andrew. 278
8.9. The urinary metabolites of fenclofenac,
the cumulative totals expressed as
percentage of dose, Caspar. 279
9.1. Metabolism of 5-(41-chloro-n-butyl)
picolinic acid in the rat. 291
Al.l. Plot of loge % dose excreted per hour
versus time midpoint (hour) for Ginger,
salicylic acid administration. 302
A1.2. Plot of loge % dose excreted per hour
versus time midpoint (hour), for
Shepherd Boy, salicylic acid administration. 303 13 A5.1; C-NMR spectrum of synthetic phenylacetic
acid, in D20 : (CD3)2CO 85 : 15. 316 13 A5 . 2. C-NMR spectrum of synthetic phenylacetyl-
glycine in D20 : (CD3)2CO 85 : 15. 317 13 A5.3. C-NMR spectrum of synthetic hippuric acid,
in D20 : (CD3)2CO 85 : 15. 318 13
A5.4. C-NMR spectrum of synthetic phenylacetyl 319
taurine in D20 : (CD3)2CO 85 : 15. -15-
Page
14 A5.5. C-NMR spectrum of phenylacetone in D2O. 320 13 A5.6. C-NMR spectrum of phenylacetone in D2O ^H-noise decoupled. 321 13 A5.7. C-NMR spectrum of synthetic styrylacetic
acid in DgO : (CD3>2CO 65 : 35. 322 13 A5.8. C-NMR spectrum of 4-phenylbutyric acid
in D20 : (CD3)2CO 65 : 35. 323 13 A5.9. C-NMR spectrum of synthetic sodium
3-hydroxy propionate in D2O : (CD3)2CO
85 : 15. 324 13 A5.10. C-NMR spectrum of synthetic 3-hydroxy
propionic acid in D20 : (CD3)2CO 85 : 15. 325
A6.1. Loge % dose per hour eliminated against
time midpoint for each sample, Andrew, 337
Isoxepac administration. 337
A6.2. Loge % dose per hour eliminated against
time midpoint for each sample, Shepherd Boy,
Isoxepac administration. 338 14 A7.1. Loge [ C] plasma following fenclofenac administration. 346 -20-
Chapter One : Introduction
Page
The horse and horse racing - a brief history 21
Doping 25
Drug Metabolism and Disposition in the Horse 40
1. Anaesthetics 41
2. Methylxanthines 42
3. Amphetamine and other sympathomimetic
drugs. 45
4. Non-steroidal anti-inflammatory drugs 49
5. Steroids 54
6. Opiates 59
7. Barbiturates 51
8. Phenothiazines 61
9. Miscellaneous drugs 66
Conjugation Reactions 73
Group la Conjugation by formation of a
peptide bond 75
b Glutathione conjugation (mercapturic
acid synthesis) 76
Group 2a Acetylation 79
b Glucoside conjugation 81
c Glucuronic acid conjugation 82
d Methylation 82
e Sulphation 83
Summary 85 -21-
INTRODUCTIQN
The horse and horse racing - a brief history
Since ancient times the horse has served man, as a beast of burden, for transportation, in recreation and companionship.
Despite the ascendance of machines to displace the horse from some of these roles, the contribution of this species to leisure activities today is a valuable one, being central to many sports such as polo, show jumping and horse racing.
Although there are over 200 recognized breeds, the
Shire, Shetland and thoroughbred, for instance, all belong to the same species, Equus callabus.
The domestic horse has evolved over a period of 55 million years from a 12 inch high, four toed ancestor, called Eohippus, to the sometimes feet tall, single toed creatures known today.
The Equidae belong to the order Perissodactyla (which -22-
includes rhinoceroses), and within recent times
7 distinct species of wild equids have been recognised. These are Przewalskis horse
(E. przewalskii) which is native to Eastern Asia but only found now in parks and zoos; the onager and kiang (E. hemionus) of Mongolia and Tibet respectively, the 'zebras1; E. zebra of S.W. Africa E. burchelli
(or common zebra) of South African plains, and
E. grevyi of Somaliland and Northern Kenya, and the now extinct quagga, (E. quagga).
Of the hybrids man has produced, only one is fertile, the cross between Przewalski's horse and the domestic horse, these equids are nearest in diploid number.
(E. przewalskii 2n = 66, E. callabus 2n = 64,
E. zebra 2n = 32). From genetic and phenotypic similarities it would appear Przewalski's horse is the forefather of the domestic horse.
Domestication of E. Callabus occurred about 2500 -
3000 B.C. in the region of Turkestan. From there it spread into Macedonia, reaching Egypt by 1500 B.C. and later in to Western Europe. -23-
Racing developed into the sport recognisable today during the reigns of the Tudor and Stuart Kings.
Henry VIII (1509 - 1547) forbade the grazing of stallions on public land, to ensure the improvement of the horse population through the controlled use of well-bred stallions. Under James I (1603 - 1625) and especially Charles II (1660 - 1685) the sport thrived.
The racing stock in England then had great speed but lacked stamina and this was improved in the late
17th and early 18th centuries, by the importation of oriental breeds, Barbs, Turks and Arabians to cross with the English racing horses. Although many
Oriental stallions were used, all thoroughbreds can trace their lineage in the male line to one of three horses. These were the Byerley Turk (the Herod line), the Godolphin Barb (the Matchem line) and the Darley
Arabian (the Eclipse line).
It was at this time that the Jockey Club of Newmarket was founded (circa 1750) and the rules of racing established. Racing was initially run over any open space available, until a course was laid out at -24-
Newmarket in the mid seventeenth century and stewards
appointed to supervise and conduct meetings. -25-
Doping
The doping of horses is probably as old as racing itself, and has now come to mean any drug used to affect the performance or demeanour of a
Horse, but most especially with reference to stimulant effects.
The word 'dope' is derived from a Dutch word 'doop' which literally means to dip. With the movement of
Dutch settlers into S.E. Africa, it was incorporated into a dialect where ' dop' meant an alcoholic stimulant or spirit. It became an American slang expression to describe the use of drugging additives in tobacco, used by gypsies, as a preliminary to robbing travellers.
From this 'dope' has come to mean the illicit medication of racing animals to affect their performance.
Doping is not confined to horses, it can be applied to greyhounds, whippets and athletes.
The first reference to doping in England, was in 1666 when a regulation was drawn up to ban the use of -26-
'exciting1 substances at Worksop races. There follows a later series of attempts to poison horses, mainly with arsenic, resulting in the trial of Daniel Dawson at Cambridge Assizes in 1812 and the hanging of 2 or 3 men at the end of the eighteenth century following incidents of poisoning at Newmarket.
Doping itself did not become widespread until the turn of the 20th Century and is thought to have spread to Europe from America. In 1910, doping had become such a problem, that the Austrian Racing Authorities enlisted the services of a Racing Chemist, who claimed to be able to detect alkaloids in horse saliva.
He did not disclose his methods and his findings were not confirmed (whereupon he wisely retired to Russia) but it did have a controlling effect on the doping of racehorses.
In the 1930's doping became a problem in America, following the introduction of on-course betting, and an increase in the number of race meetings. The situation was so bad that racing stables were reputedly equipped like pharmacies, with prescriptions of elixirs of heroin, strychnine, nitroglycerine and -27-
digitalis recommended for pre- and post-race dosage.
The extent of the problem is illustrated by the fact that the Federal Bureau of Narcotics made over 100 arrests for horse-race connected violations.
The American authorities called on help from Europe and a French chemist introduced saliva testing, which screened mainly for alkaloids. The various analysts employed to carry out saliva tests started the
Association of Official Racing Chemists. Membership was limited to official racecourse analysts and members were barred from disclosing their methods.
New discoveries, rather than being published in scientific journals, were circulated in confidential documents. In England, doping increased after the second world war, with the widespread use of amphetamine and related drugs, which were not, initially easy to detect. At this time the punishments for offenders were very severe, meaning the withdrawal of training licence and a life-time ban for the horse
involved in the incident. There was no right of
appeal by the trainer (who was in danger of losing his livelihood), and the analyst's findings could not -28-
be questioned. When disqualifications were challenged,
the High Court action brought much publicity and
criticism of the analysts, but their findings were upheld.
Problems stemmed from the secrecy of the Association
of Official Racing Chemists (AORC), and the understandable difficulty of the Jockey Club in
interpreting results.
A turn of events came in 1960, when a committee,
appointed by the Jockey Club Stewards, investigated
the rules related to doping. The setup was radically
changed, with the appointment of an advisory scientific
board able to consult on analyst's reports, augmented
by the setting up of its own laboratory to analyse
racecourse samples and carry out basic research
into action of drugs in horses and methods for
their detection.
These laboratories were originally under the Animal
Health Trust, but now are called Racecourse Security
Services Limited. Routine dope testing was introduced
into Britain in 1963, initially this did not discourage
doping and there were several now notorious conspiracies
involving the administration of caffeine, phenobarbitone -29-
and acetylpromazine.
Today, most drug-positive cases, involve the administration of therapeutic substances, notably non-steroidal anti-inflammatory drugs, steroids or local anaesthetics too close to the time of racing, or accidental food contamination.
Doping can be subdivided into: a) doping to win b) doping to lose c) doping due to therapeutic use of medication
and d) doping accidentally or inadvertantly
a) Doping to win
This can be further subdivided into long term and short term medication. Long term doping involves giving a daily dose over a period of weeks or months, as in the use of anabolic steroids. It is a problem of recent years that these drugs can be used in yearlings to increase vigour and muscle mass, and when the horses are raced as two and three year olds, they have the advantage of apparently -30-
increased performance which will be virtually undetectable.
The problem is not so much in this performance advantage over a single race; but in that a horse, due to success, is used to continue the blood line through breeding. Vast amounts of money in the horse race industry are locked in stud value, and if one takes a Darwinian view of evolution, an acquired character- istic is not heritable. This type of interference will inevitably, in the long term, lead to a weakening in the quality of the thoroughbred blood line. Long term 'doping to win' can involve Vitamin E and Vitamin
Ifa diet supplements, or drugs which improve efficiency of glycogen utilization.
Short term medication is the administration of a drug immediately before the race, to influence the horse's performance. Drugs in this category are the stimulants such as caffeine, amphetamine and adrenaline.
b) Doping to lose
Doping to lose, is an attempt to diminish a horses performance. This could be achieved by the administration of a large dose of a sedative such as chloral hydrate, acetylpromazine or one of the -31-
barbiturates. This type of interference is always thought to take place without the knowledge of the racing stable, because there are less innocuous ways of stopping a horse without a trainer risking using drugs with unpleasant side-effects.
c) Therapeutic doping
This is the use of medication to treat a condition which would otherwise impede performance, e.g. using a local anaesthetic to remove discomfort from slight lameness, or calming an excitable horse with a minute dose of a tranquilliser.
Arthritic conditions may be treated with a drug such as phenylbutazone. The use of this drug has been the centre of some controversy in recent years, with reference to its use in show jumping and 3-day eventing horses. Due to financial pressures, show- jumping has become somewhat "circus-like" in that a small group of horses and riders travel the world to compete against each other at different venues.
This means the horse will have very little time between competitions to recover from joint strains etc. and is also competing more often. Many animals are kept in the competition arena with the use of phenyl- -32-
butazone. At present, show jumpers can use up to a maximum dose daily, which is an arbitrary figure not taking into account the inter-animal differences in pharmacokinetics, metabolism and elimination of the drug. In England, no pre-race medication is permitted in horse racing.
d) Inadvertant or accidental doping
This involves the ingestion of substances accidentally, usually through the materials in feeds. For instance Hayward (1978) found lupanine in the urine of South African racehorses. This plant product is closely related to the drug sparteine.
Another occurrence of this type was that reported by
Smyth and Pugh (1966) of perloline in horse urine, this is an alkaloid derived from rye grass.
The presence of nicotine and theobromine has been accounted for by the accidental contamination of food, water or bedding with tobacco or cacao husk.
Periodically, the Association of Official Racing
Chemists publish a list of drugs reported. Table 1.1 compiles those drugs reported between 1947-1973. -33-
A trend in the use of drugs in horses can be observed when one compares the data on Table 1.1. to a more recent list, issued by the AORC for the period of May 21st 1978 - December 31st 1978, given on Table 1.2.
The average incidence of the finding of a positive for the period, 1947 until 1973 was 136 per annum, and for the 9 month period examined in 1978, the total would come to 153 per annum. If drugs are grouped in families, on the basis of their pharma- cological action, trends in the use of particular drugs can be seen, in comparing the incidences for
1947 - 1973, and 1978. Table 1.3 lists the 8 different groups and their percentage contribution to the total number of drug positive samples observed.
Although the two groups of statistics are not directly comparable as there is a 30-fold difference
in sample size, two important trends can be observed.
Firstly, there is a decrease in the use of CNS stimulants (e.g. amphetamine) and the barbiturates.
Secondly, there is a marked increase in the non- Table 1.1 Drugs reported to the Associatl
Times Times Times Drug reported Drug reported Drug reported
Procaine 661 Amylocaine 8 Pangamic acid 2 Caffeine 482 Brucine 7 Sulphonamide 2 Amphetamine 452 Quinine 7 Acetophenazine 1 Phenylbutazone 267 Thozalinone 7 Amydricaine 1 Methylphenidate 193 Apomorphine 7 Berberine 1 Theobromine 127 Alcohol 6 Bromide 1 Methamphetamine 121 Mephenesin 6 Camphor 1 Dipyrone 116 Pemoline 6 Capsaicine 1 Polyethylene Glycol 111 Pyrilamine 6 Chlorobutanol 1 Phenobarbitone 86 Acetylsalicylic Acid 5 Cinchonine l Oxyphenbutazone 66 Butacaine 5 Cincophen 1 Morphine 62 Imipramine 5 Danthron l Ephedrine 59 Methoxamine 5 Dapsone 1 Strychnine 50 Propiomaxine 5 Thiamine 50 Tetracaine 5 Pentazocine 49 Chloroquine 4 Nikethamide 44 Hydrocortisone 4 Barbiturates 43 Levorphanol 4 Promazine 38 Prednisone 4 Methapyrilene 35 Mefenamic acid 4 Nicotine 33 Meperidine 4 Indomethacin 26 Cinchonidine 3 Ethylaminobenzoate 23 Propoxyphene 3
continued. Table 1.1 (continued)
Times Times Drug reported Drug reported
Atropine 22 Sulphanilamide Pipradrol 21 Sulphaphenazole Phenothiazine 18 Thiabendazole Lignocaine 16 Acepromazine Chlorpromazine 15 Antipyrine Prednisolone 13 Barbitone Theophylline 12 Codeine Mephentermine 11 Chloral Hydrate Leptazole 10 Dibucaine Acetophenetidin 9 Doxapram Cocaine 9 Guaiacol Methocarbamol 9 Phemitone Salicylic Acid 8 Meprobamate Hyoscine 8 Naphazoline -36-
Table 1.2 Drugs reported to the Association of Official Racing Chemists May 21st 1978 - December 31st 1978.
No. of times Drug reported
Procaine 17 Ethylaminobenzoate 16 Flunixin 14 Theophylline 10 Phenylbutazone 7 Caffeine 6 Theobromine 5 Furosemide 5 Dipyrone 5 Hydroxyphenylbutazone 4 Methylphenidate 4 Methampyrone 3 Pemoline 3 Phenylpropanolamine 3 Amphetamine 2 Oestran- 3, 17-diol 2 Phenothiazine 2 Salicylic Acid , 2 Apomorphine 1 Lignocaine 1 -37-
Table 1.3 Comparison of the type of drug positive samples found in 1947-1973 and 1978
% incidence
1947-1973 1978
Anaesthetics Procaine, lignocaine 19.7% 15.7% cocaine, butocaine, tetracaine,dibucaine, amydricaine
Methylxanthines Caffeine, theobromine 17.5% 18.3% theophylline
CNS Stimulants Amphetamine, methamphetamine, 23.3% 7. ephedrine, methylphenidate
Nonsteroidal anti- inflammatory drugs
a) Phenylbutazone, 9.4% 9.6% oxyphenbutazone, hydroxyphenylbutazone b) Indomethacin, salicylic 1.2% 13.9% acid, acetylsalicylic acid, mefenamic acid, flunixin c) Dipyrone (used in the 3.3% 4.3% treatment of colic)
Steroidal Drugs Oestran- 3, 17-diol 1.7% Prednisolone, hydrocortisone, 0.6% prednisone
continued. -38-
Table 1.3 (continued)
% incidence
1947-1973 1978
6. Narcotic Analgesics Morphine, apomorphine, 3.5% 3.5% pentazocine, pemoline
7. Barbiturates Phenobarbitone, barbitone 3.7%
8. Phenothiazines Promazine, acetyl- 1.6% 1.7% promazine, phenothiazine -39-
steroidal anti-inflammatory drugs (other than phenylbutazone-related drugs). -40-
Drug Metabolism and Disposition in the Horse
The amount of published material on drug metabolism in the horse is very small and there are several reasons for this. Firstly,the previously mentioned secrecy surrounding the work of the
Association of Official Racing Chemists; this restricted the circulation and publication of reports of any unusual finding of routes of metabolism and disposition of drugs.
Secondly, the horse had become less central to industry and agriculture (in comparison with pre- industrial revolution days), when there was the up- surge in pharmaceutical development in the 1940's onwards.
Horses, as experimental animals, are expensive to buy and maintain (requiring a high staff density and generous diet) and their size and temperament need special facilities for dosing and collection of biological samples.
The research into the actions and metabolism of drugs in the horse has been largely directed at particular -41-
drugs of abuse, mainly those found as drug
positive samples (see Table 1.1). The result is
a somewhat fragmented view of metabolic pathways
in the horse, some studies are incomplete, with the
main emphasis on detection, rather than looking at
a metabolic profile and disposition in general.
As the data available is fragmented, the groupings
of drugs given in Table 1.3 are maintained for the
discussion of the actions and metabolism of these
drugs in the horse. These are listed approximately
in the order of their frequency as drug positive
samples, with at the end, a miscellany of drugs,
which do not fit into the 8 major drug groups.
1. Anaesthetics
Cocaine is an alkaloid obtained from
the leaves of Erythroxylon coca, a plant indigenous
to Peru and Bolivia. Its toxic and addictive properties have led it to become replaced by
related drugs such as procaine and dibucaine, which
have more specific local anaesthetic properties.
Only the latter has been investigated in terms
of metabolism. -42-
Cocaine has an effect on the locomotor activity in the horse, due to its central effects (Tobin, 1982) but the pattern of response is erratic. After
1.v. administration, it is eliminated with a plasma half life of 45 minutes, with very little of the drug appearing in the urine unchanged.
Procaine produced signs of CNS excitement in thoroughbred mares for 4 min. following lg i.v., and was rapidly cleared, with a plasma half life of
25 minutes (Tobin, 1976). The drug is rapidly broken down by the action of plasma esterases.
Its metabolism has not been investigated.
Dibucaine has been administered subcutaneously to horses and urinary metabolites examined in a
0-6hr urine sample (Shinohara and Momose, 1982).
Two metabolites found in the horse were 2-butoxy- cinchonic acid, which has arisen by the hydrolysis of the amide bond, and 2-oxo-N-(2-diethylaminoethyl) cinchonamide formed by cleavage of the imino ether bond.
2. Methylxanthines
Caffeine
Caffeine is a stimulatory drug, and experiments -43-
on performance (Sanford 1971) have found doses of 2,4 and 9mg/kg significantly increased performance in speed tests.
Moss e^t al. (1979) have found that after a dose of caffeine, 7mg/kg p.o. the plasma half life of the - 14
C-labelled compound in one animal was 12 hours.
A total of 60% of the dose was recovered in the urine which could be divided into 10 metabolite fractions. The unchanged drug accounted for only 1% of dose, major metabolites were the dimethylxanthines, theophylline, theobromine and paraxanthine (totalling
15% of dose) and the methyl uric acids, 1,3- and
1,7-dimethyluric acids and 1,3,7-trimethyluric acid
(total 30% of dose). There was no evidence of conjugate formation. The metabolic routes are shown on Figure 1.1.
Kamei, Matsuda and Momose (1975) have found in the horse and other species a new metabolite of caffeine a-7-
(1,3-dimethylxanthinyl) methyl methylsulphoxide.
Theophylline 14 After an oral dose of C -theophylline 80% of the administered radioactivity was found in the -44-
V II 0 CHD CH
O' N N i H H
III VI H 0 H 0 CH CH i 'N NN I // C=0 O^N 0 N N' i I H CH3 CH3
DIMETHYLXANTHINES METHYL URIC ACIDS
I theobromine IV 1,3,7-trimethyluric acid II paraxanthine V 1,7-timethyluric acid III theophylline VI 1,3-timethyluric acid
Figure 1.1. The major metabolites of caffeine -45-
urine; excretion of radiolabel was complete in 4
days. 26% of this was unchanged and 65% as 1,3-
dimethyluric acid (Moss, 1977).
Theobromine 14 As in the above study, an oral C- labelled
dose of theobromine was excreted 4 days after dose, with a recovery of radiolabel of 75%. 56% of this was unchanged and 28% as 3,7-dimethyluric acid
(Moss, 1977).
3. Amphetamine and other sympathomimetic drugs
Amphetamine
Amphetamine is extensively metabolised by
the horse, this was first indicated after a study by
Baggot et al. (1972), where only 2% of a dose of (dl)-
amphetamine sulphate was excreted unchanged; urinary
pH was shown not to affect the biological half-life
of the drug (about 2 hours).
14 The study of the metabolism of C-(dl)-amphetamine
in two horses, has provided a quantitative assessment
of the different metabolic routes (Chapman and
Marcroft 1973a). 84% of the radiolabel was recovered, -46-
and the major metabolite (29% of dose) found to be a precursor of l-phenylpropan-2-one, which was not investigated further. Other metabolites found were conjugated and unconjugated, amphetamine (5%) and 4-hydroxyamphetamine (7%) hippuric acid (19%), l-phenylpropan-2-ol (12%) and benzoyl glucuronide (12%). These pathways are summarized on Fig. 1.2.
Ephedrine
Ephedrine is derived from Ephedra equistina, and related plants. It is a sympathomimetic drug different from adrenaline in that it acts for a longer period (7-10 times longer) and has a more marked effect on the central nervous system; both, however, have a lesser effect than amphetamine.
L-ephedrine (200mg p.o. or i.v.) was administered to one horse (Nicholson, 1970) and the urine voided examined by chromatography for l-ephedrine, nor- ephedrine, 4-hydroxyephedrine, 4-hydroxynorephedrine and mandelic acid. Only norephedrine
(7% of dose in alkaline urine, and 15% in acid urine), -47-
CH2CHNH2
CH3 amphetamine
/^yCH2chohch3 CHOCCHD
zll J I y^y 1-phenyl propan- ^^ 2-ol 0 1-phenyl propan- 2-one
/WCOOH r^rCH2CHNH2
Benzoic acid CH3 4-hydroxyamphetamine
r^VCOOC6H906 CONHCH2COOH
Hippuric acid Benzoyl glucuronide
Figure 1.2. The metabolism of amphetamine in the horse -48-
and conjugated hydroxyephedrines (3%) were detected.
In a similar experiment, Nicholson, (1972) analysed specifically for l-hydroxy-l-phenylpropan-2-one and 1,2-dihydroxy-l-phenylpropane. The latter was found conjugated with glucuronic acid (19% of dose); and when it was administered i.v., 68.5% of the dose was recovered in the urine. When 1-hydroxy- l-phenylpropan-2-one was administered i.v. , the dose was eliminated unchanged (1%), as a conjugate
(10-20%) and 1,2-dihydroxy-l-phenylpropane
(30-40%).
Adrenaline and Noradrenaline 14 14 (±)- C-Adrenaline and (+)- C-Noradrenaline metabolism has been studied and again the horse metabolises these sympathomimetic amines extensively
(Chapman and Marcroft, 1973b,c). The urinary metabolites include conjugated and unconjugated drug and the
3-methoxy-4-hydroxyamine derivatives and a variety of deaminated products. -49-
4. Non-steroidal anti-inflammatory drugs
Acetylsalicylic and salicylic acid
The metabolism of the salicylates is discussed
in more detail at the beginning of Chapter 3. The pathways observed in the horse are oxidation (to gentisic acid), glucuronidation (salicyl acyl and phenylglucuronides) and glycine conjugation (salicyluric
acid).
Phenylbutazone
Phenylbutazone is probably the most widely used anti-inflammatory drug in equine medicine, but
despite this a full study on the pharmacokinetics,
disposition and metabolism of this drug in the horse
has not yet been undertaken. The findings of the
five different investigations are summarized in
Table 1.5.
The drug was found not to change respiratory rate,
behaviour or haemocrit (Gabriel and Martin,1962).
However, a toxic side-effect of necrotizing phlebitis
of the portal veins was noted at a chronic dose of
2.8g/day. -50-
When lOg p.o. and 6g i.m. was administered to
the same horse on separate occasions (Schubert 1967),
analysis of urine showed the presence of parent
drug, oxyphenbutazone (ring hydroxylated metabolite)
and 1,2-diphenyl-4 (y-hydroxy-n-butyl) pyrazolidine
3,5-dione (aliphatic chain hydroxylation to the
'alcohol' metabolite). These pathways are shown
in Figure 1.3.
When the more usual route of administration for
this drug was used (intravenous injection), it
had a half-life of 3.5 hours (4.4mg/kg) and
3.7% of parent drug was recovered in 24 hours
(Piperno et al., 1968). In contrast, at 6mg/kg
i.v., the half life was found to be 7.0 hours
in plasma (Gandal 'et 'al., 1969), which was
similar to that found for oxyphenbutazone.
In the only detailed paper on the metabolism
of this drug, 4.4mg/kg i.v. was administered
daily for 4 days (Maylin, 1974); the results
are summarized on Table 1.4. This indicated
that there is a low urinary recovery of the
drug and the two hydroxylated metabolites are produced in equal proportions. O^N. N ; Q Phenylbutazone CH3CH2CH2CH2
OH
OH O^N N 0. I 4 4 0 0 CH3CHCH2CH2 CH3CH2CH2CH2 Oxyphenbut 'Alcohol' metabolite
Figure 1.3. Metabolism of phenylbutazone in the horse -52-
Table 1.4 The recovery of dose (percentage of total)
for every 24 hour period following a daily
dose of phenylbutazone (4.4mg/kg).
'Alcohol' Dose Phenylbutazone Oxyphenbutazone metabolite
1st 0.9 9.6 14.2
2nd 1.7 14.0 10.1
3rd 2.1 14.0 8.2
4th 1.6 10.5 6.4
(after Maylin, 1974) -53-
Table 1.5 Metabolism and elimination of phenyl- butazone in the horse
Dose Route Finding Reference
10g p.o. Phenylbutazone, Schubert, oxyphenbutazone (1967) 6g i .m. and alcohol metabolite found in urine
4.4mg/kg l. v. tA 3.5hrs,3.7% Piperno et al ricovery of (1968) parent drug in 24h
6mg/kg 1 .v t, 7.0 hours Gandal et^ al (1969)
2g 1 . v Oxyphenbut azone Finocchio et_ a^ detected as (1970) urinary metabolite
4.4mg/kg 1 . v Phenylbutazone.* Maylin (1974) daily oxyphenbut azone and alcohol metabolite found in urine -54-
Other non-steroidal anti-inflammatory
drugs (NSAID's)
Various newly developed NSAID's are used instead of salicylic acid and phenylbutazone, as they generally have less side-effects. The structures of some of these are given on Figure 1.4. The primary aim of the investigations has been the detection of the parent drug in the urine (Hunt et al.
1979). The findings give some idea of the rate of clearance of drug, after lg oral dose, as the time of the last urine sample in which the drug was detectable is quoted. Examples of the results; were mefenamic acid 15 and 29hr, flufenamic acid
10 and 11 hr, meclofenamic acid 7hr, ibuprofen 12 and lOhr, indomethacin 13 and 16hr, naproxen 30 and 25 hr, ketoprofen 17 and 12 hr and flunixin
21hr. The metabolism was not investigated.
5. Steroids
Anti-inflammatOry
Corticosteroids such as Cortisol and corticosterone and some synthetic steroids, prednisolone, dexamethasone, flumethasone are used in the treatment of inflammation. -55-
.COOH^.
1. Flufenamic acid N cf3 I H
COOH^
2. Flunixin N CF
H CH3
CHCOOH
CH Q |-| 3. Ibuprofen
3>CH2- CH
4. Indomethacin
CHOO CH2COOH
Figure 1.4. Some non-steroidal anti-inflammatory drugs used in equine medicine -56-
CHCOOH i CH3 5. Ketoprofen
0, PH CH. Nr 1 J
6. Meclofenamic acid
H CI
00H ^
7. Mefenamic acid
H CH3
CHCOOH 1 CH3 8. Naproxen CH
Figure 1.4. continued. Some non-steroidal anti-inflammatory drugs used in equine medicine -57-
Prednisolone
A study using tic examined the metabolism
of prednisolone in the horse (Moss and Rylance 1967).
Various doses were given to three animals, orally
and intramuscularly, and urinary steroids, not
detectable in control urine, found were prednisolone,
prednisone, 20-B-dihydroprednisolone and
20 3-dihydroprednisone (in roughly equal amounts),
and Cortisol, 17 deoxy-cortisol and the possible
finding of tetrahydrocortisol.
Dexamethasone
The metabolism of dexamethasone (9 a-fluoro-
16 ot—methyl —11 $ , 17ot —21—trihydroxypregna—1,
4-diene-3, 20-dione) has been studied in the horse i by Dumasia e£ al.(1979). The metabolites identified
in urine were dexamethasone, 11-oxodexamethasone,
20-dihydrodexamethasone, 17-oxo-dexamethasone,
6-hydroxydexamethasone, and 6-hydroxy-17-oxodexamethasone.
The recovery of radiolabel ( H) was 40-50% in 0-24hr.
A previous study by Moss (19 78) found after an
i.v. dose, the recovery in urine in 0-24hr was
44%. Of this 1% was present as dexamethasone,
and 4% as a dexamethasone -58-
conjugate (either the glucuronide or sulphate).
I.m. administration resulted in a similar recovery with dexamethasone and 11-dehydrodexamethasone as metabolites.
Anabolic Steroids
19-Nortestosterone
19-Nortestosterone, estrane-3,17,-diol, a metabolite of 19-nortestosterone was used to detect the parent steroid in horse urine
(Houghton e£ a£. 1978). Another urinary metabolite was 3-hydroestran-17-one.
Testosterone 14 After i.m. administration of [4- C]-testosterone,
50% of the administered radioactivity was excreted within 200h, with approximately 35% of drug related material in the urine in the form of conjugates.
Following acid hydrolysis, 2 neutral metabolites were identified as isomers of androstan-3,17-diol, i.e.
5 a-androstrane-3B, 173-diol, and 5 a -androstrane
3 3, 17a-diol. Also identified were testosterone and
3 3-hydroxy 5a~androstan-17-one (Houghton and Dumasia,
1979). -59-
The same authors (Dumasia and Houghton,1981) investigated the phase II metabolism of testosterone in more detail. They found isomers of 3,17-dihydroxy androstan-16-one, 3,16-dihydroxy-androstan-17-one and androstane, 3, 16, 17-triol as urinary metabolites.
These and other previously found metabolites, were found to be excreted conjugated with sulphate and glucuronic acid, usually one particular metabolite predominating in a particular conjugate fraction.
6. Opiates
This group includes, morphine, apomorphine, pethidine, fentanyl, methadone and pentazocine.
Most of these drugs have been tested for their effect on locomotor activity (Tobin,1981) but relatively little is known about their elimination and metabolism.
The glucuronides of apomorphine and pentazocine were analysed using different sources of B-glucuronidase to cause parent drug release, but the extent of glucuronidation was not quantified (Combie eit al, 1982).
Previously, 30% of an i.v. dose of pentazocine (lmg/kg) -60-
was eliminated in the urine as the glucuronic acid conjugate and was detectable in the urine (as the glucuronide) for up to 5 days post dosing (Tobin et al, 1979).
Morphine, similarly, is detectable for a number of days post dosing (up to six) of 0.lmg/kg (following
3-glucuronidase incubation) though the total recovery of drug was under 20% (Combie et^ al, 1981).
Pentazocine was administered to ponies (3mg/kg i.m.) and peak plasma concentration reached at 30 min. with the disappearance of drug from plasma following first order kinetics. The tx was 97 min, but metabolism was 2 not investigated (Davis and Sturm,1970).
Fentanyl was administered to a horse (70mg/500kg by venous infusion) and the urine examined for metabolites
(Frincke and Henderson, 1980). They identified the major metabolite as N-[l-(2-phenethyl-4-piperidinyl)] malonilinic acid. This comprised of 64% of dose
(total recovery was 80%); its formation involved the oxidation of the propionyl side chain, and is a metabolite of fentanyl not previously observed. -61-
7. Barbiturates
Only the metabolism of phenobarbitone and pentobarbitone has been investigated, (Alexander and
Nicholson, 1968) and the former would appear to be less extensively metabolised than the latter.
After an oral dose, of 5mg/kg sodium pentobarbitone
40% was recovered, 33% as the mixed alcohols, 5-ethyl
5- (3-hydroxy-l-methylbutyl) barbituric acid and
5-ethyl-5- (l-methyl-3-carboxypropyl)barbituric acid
and no parent drug was detected.
A lower recovery of dose was obtained following
sodium phenobarbitone administration, 4.4mg/kg
supplemented by further doses of 2mg/kg/hour, to
a total of lOmg/kg, only 11% of dose excreted
within 72 hours of dosing in the form of p-
hydroxyphenobarbitone glucuronide and unchanged
drug.
8. Phenothiazines
Phenothiazine is used as an anthelmintic, and
interest has centred on its effect upon physiological
and haematological parameters (Swales et al, 1942), of a
60g dose,-18.7% was excreted in the faeces unchanged with -62-
5.5% as oxidation products, and 23.3% as oxidation products in the urine.
In a larger study (Collier et al, 1943) where the recovery of drug related material was never in excess of 50%, the major metabolite was identified
as leucophenothiazine sulphate.
Phenothiazine 'tranquillisers'
Phenothiazine 'tranquillisers' are derivatives of phenothiazine possessing an amino-alkyl side chain attached to the ring nitrogen atom. Used as tranquillising
® V drugs, chlorpromazme was the prototype drug (Largactil ), but due to unpleasant side effects, acetylpromazine is now' the most commonly used drug in this class and has a marked effect in depressing the activity of the horse, as measured by blockade in a trotting response
(Tobin, 1979).
Carey and Sanford (1963) detected at least seven metabolites of promazine in horse urine, with a predominance of polar metabolites. Schubert (1967) found a slow excretion of chlorpromazine following an i.v. dose and a low recovery. Four metabolites were detected, the major one being chlorpromazine sulphoxide. -63-
Weir and Sanford (1969) measured the urinary excretion and metabolism of promazine in 5 horses.
There was a great inter-individual variation, with a predominance of glucuronic acid conjugates, with only a trace of sulphate conjugates and unchanged drug. Nine metabolites of promazine were detected, the major one was 3-hydroxypromazine glucuronide.
In a more recent study Dewey et al, (1980) looked at both the metabolism of promazine and acetyl- promazine, using more sensitive analytical techniques.
With promazine (50-500 mg dose, i.v.) the parent drug and four metabolites were detected in the urine. The two major metabolites were detected in the urine. The
two major metabolites were conjugated, and following
3-glucuronidase/aryl sulphatase incubation identified
as 3-hydroxypromazine and 3-hydroxydesmonomethy1- promazine. Two minor metabolites were promazine-N-
oxide and promazine-N-oxide sulphoxide.
In the horses, given acetylpromazine maleate
(5-50mg dose i.v.) three metabolites were found,
conjugated 7-hydroxyacetylpromazine, 2-(1-hydroxy-
ethyl) 7-hydroxypromazine and 2-(1-hydroxy-
ethyl)-promazine sulphoxide. The routes of (CH2)3N(CH3)2 N
(9) \(CHO ) DN (CHO)O (CHO) ON (CH 0)0 N N
Promazine-N-oxide 3-Hydroxy promazine I (0) I
(CH2)3N(CH3)2 (CH2)3NHCH
Promazine-N-oxide 3-Hydroxydesmonomethylpromazine Sulphoxide
The metabolism of promazine in the horse (CH2)3N.
R i N CHOHCH
hct^^S
7-hydroxyacetylpromazine 2-(1-hydroxyethyl)7-hydroxypromazine
CHOHCH
2-(1-hydroxyethyl)promazine sulphoxide
Figure 1.6. The metabolism of acetyl promazine in the horse -66-
metabolism of promazine and acetyl promazine are shown in Fig. 1.5 and 1.6 respectively.
9. Miscellaneous drugs
Chloral hydrate
Chloral hydrate, used as an anaesthetic, is metabolised to the active form, trichloroethanol.
Alexander e£ al, (1967) dosed two ponies with 25g of chloral hydrate and one with 20g of trichloroethanol.
Following rapid absorption, 60% of the dose of chloral hydrate was excreted in the urine as trichloroethanol glucuronide (urochloralic acid) with a trace of trichloroethanol and trichloroacetic acid. Following the trichloroethanol administration, the recovery was less than 50%,the majority in the form of the glucuronic acid conjugate.
Glyceryl guaiacolate
Glyceryl guaiacolate [3-(©-methoxyphenoxy)-
1,2-propanediol] is a centrally-acting skeletal muscle relaxant, one dose having an effect for 15-30min.
Using 10 ponies, the metabolism was studied by tic (one system), O-dealkylation to catechol -67-
followed by conjugation (with sulphuric or glucuronic acids) was the major route of metabolism, this was not quantitated (Davis and Wolff 1970).
Histamine
14 The metabolism of C-histamine has been studied in the horse (Eliassen 1971). Recovery of radiolabel in the urine following i.v. dosing, was
64-76% in 0-24hr. Methylation of histamine to 1,4, methyl histamine and further oxidation to 1,4, methyl— imidazoleacetic acid were quantitatively the most 14 important pathways of metabolism (63% of C). Minor products were unchanged drug and 1,5-methylimidazoleacetic acid.
Trimethoprim
Trimethoprim, a folic acid inhibitor, was excreted in the urine unchanged and as 3'- and 4'-0- demethylated trimethoprim (Alexander and Collett 1974), after a dose of 55mg/kg (i.m. and i.v.). -68-
Other compounds and drugs have been investigated in the horse measuring pharmacokinetic parameters, such as pethidine, chloramphenicol, oxytetracyline, quinidine
and reserpine but metabolism has not yet been
elucidated.
From the 9 groups of drugs certain metabolic pathways
which occur in the horse can be defined. Drug metabolism
pathways have been split into two groups, Phase I,
including oxidations, reductions and hydrolyses,and
Phase II, the conjugation reactions.
The major Phase I pathways in the horse are summarised
on Table 1.6 and those of Phase II on Table 1.7.
From a brief survey of the data for Phase I and Phase
II reactions in the horse, by comparing Table 1.6 and
1.7, it is apparent that the emphasis has been on
Phase I pathways, and relatively little is known about
Phase II reactions. In many studies, the description
of Phase II metabolism is restricted, mentioning
conjugation of drug or major metabolites without any
further investigation of the type of conjugate, or
the extent of its formation. Table 1.7 Metabolic Reactions of Drugs in the Horse. Phase II Reactions
Pathway Drug Metabolite Reference
1. Oxidation Aromatic Amphetamine 4-hydroxyamphetamine Chapman and Marcroft (1973(a)) hydroxylation Ephedrine 4-hydroxyephedrine Nicholson (1970) Salicylic acid Gentisic acid Schubert (1967) Phenylbutazone Oxyphenbutazone Maylin (1974)
Aliphatic Phenylbutazone 1,2-diphenyl-4 (y-hydroxy Maylin (1974) hydroxylation n-butyl) pyrazolidine Pentobarbitone 3-hydroxy- Nicholson (1968) pentobarbitone Fentanyl N- [l-(2-phenethyl-4- Frincke and Henderson (1980) piperidinyl] malonilinic acid
N-dealkylation Ephedrine Norephedrine Nicholson (1970) O-dealkylation Trimethoprim 3'- and 4' O-demethyl- Alexander and Collett (1974) trimethoprim Glyceryl Catechol Davis and Wolff (1970) guiaicolate
continued. Table 1.6 (continued)
Pathway Drug Metabolite Reference
1. Oxidation Oxidative Amphetamine l-phenylpropan-2-one Chapman and Marcroft (1973(a)) deamination
N-oxidation Promazine Promazine-N-oxide Dewey et. al. (1981) S-oxidation Chlorpromazine Chlorpromazine Schubert (1967) sulphoxide Acetyl promazine 2R( 1-hydroxyethyl )- Dewey et al. (1981) promazine sulphoxide
2. Reduction reduction of Chloral hydrate Trichloroethanol Alexander et al. (1967) aldehyde
3. Hydrolysis Splitting of Acetyl salicylic Salicylic acid Schubert (1967) an ester acid Splitting of 2-butoxycinchonic acid Shinohara and Momose (1982) an amide Dibucaine Splitting of 2-oxo-N-(2-diethyl- Shinohara and Momose (1982) an iminoether Dibucaine aminoethyl) bond cinchonamide Table 1.7 Metabolic Reactions of Drugs in the Horse. Phase II Reactions
Conjugated with Drug Administered Drug or Metabolite conjugate Reference
1. Glucuronic acid Amphetamine Benzoyl glucuronide Chapman and Marcroft (1973a) Apomorphine Apomorphine glucuronide Combie et al. (1982) Chloral hydrate Trichloroethanol glucuronide Alexander et al. (1967) Morphine Morphine glucuronide Combie et al. (1981) Pentazocine Pentazocine glucuronide Combie et_ al. (1982) Phenobarbitone p-Hydroxyphenobarbitone Nicholson (1968) glucuronide Promazine 31-Hydroxypromazine Weir and Sanford (1969) i glucuronide
2. Sulphate Phenothiazine Leucophenothiazine sulphate Collier et al. (1943) Amphetamine Hippuric acid Chapman and Marcroft (1973a) 3. Glycine Salicylic acid Salicyluric acid Schubert (1967) Caffeine a- 7-( 1, 3-Dimethylxanthinyl )pkOuiI Kamei, Matsuda and Momose (1975) 4. Thiomethyl methyl sulphoxide Histamine 1,4-Methyl histamine Eliassen (1971) 5. Methyl group -72-
If a drug is excreted largely in a conjugated form, a knowledge of the nature of the conjugate allows the elaboration of detection methods to confirm its presence or absence in a particular urine sample.
Conjugates pose specific analytical problems because they generally make the drug far more water soluble; and may need special conditions (e.g. use of ion pair reagents) or the least polar solvents for extraction.
The drug derived material may not be extractable from urine until hydrolysed (by acid, alkali or enzyme).
Also a specific problem can be rearrangements of structure (as described for acyl glucuronides in
Chapter 7), which may make the conjugate resistant to hydrolysis by 3-glucuronidase. This specific hydrolysis is usually used as conclusive evidence for glucuronic acid conjugation.
In conclusion, the study of conjugation reactions is of particular importance to the analytical chemist running a screening service for exogenous substances in urine, for the improvement of detection methods,and this is an area of drug metabolism so far poorly investigated in the horse. -73-
Conjugation Reactions
A conjugation reaction is one where a foreign compound or metabolite is combined through the mediation of an enzyme with a substance of endogenous origin to form a product (conjugate) which is usually polar and readily excreted. For a compound to be conjugated, it must possess a suitable functional group, to which the endogenous conjugating molecule can link.
These functional groups, for the majority of conjugation reactions are hydroxyl (phenolic, carboxyl or alcoholic), amino, heterocyclic nitrogen or thiol.
The products of conjugation reactions are generally inactive or at least less active than the parent compound, and the conjugation usually result in an increase in the water solubility of the xenobiotic, thus favouring renal elimination.
There are two mechanisms for the formation of the conjugate. The first (Group 1 on Table 1.8) involves the activation of the foreign compound prior to the addition of the conjugating agent, and the second (Group 2 on
Table 1.8) is where the conjugating agent is the Table 1.8 The major conjugation reactions
Reaction Conjugating entity Intermediate Functional group combined with
Group 1. a. Amino acid Glycine Coenzyme A -COOH conjugation Glutamine thioesters Taurine Ornithine b. Glutathione Glutathione Arene oxides, Aryl chloride, arene conjugation epoxides oxide, epoxide, carbonium ion
Group 2. a. Acetylation Acetyl CoA Acetyl coenzyme A OH, -NH2 £
b. Glucoside Glucose Uridine diphosphate -OH, -COOH, -SH 1 conjugation glucose (UDP glucose)
c. Glucuron idat ion Glucuronic acid Uridine diphosphate -OH, -COOH, -NH2 glucuronic acid (UDP glucuronic acid) -NR2, -SH, -C-H
d. Methylation Methyl group from S-adenosylmethionine -OH, -NH2 S-adenosylmethio- nine or 5-methyl- 5-methyltetrahydrofolate -OH, -NH2 tetrahydrofolic acid 1 e. Sulphation Sulphate Adenosine-3 -phosphate- -OH, -NH2 51-phosphosulphate (PAPS) -75-
activated entity (usually in combination with a nucleotide). The final step, in both situations, is the combination of the xenobiotic (or metabolite) and conjugating agent is catalysed by a specific transferase enzyme.
Group la Conjugation by formation of a peptide bond
The formation of a peptide bond is energy requiring, provided by the activation of the acid to a thio-ester derivative of coenzyme A, at the expense of
ATP, which is converted to AMP and pyrophosphate.
In common with many biochemical reactions, this reversible reaction is driven forward by the rapid removal of pyrophosphate, by the action of phosphodiesterase, the equilibrium thus favours thioester formation. The enzyme catalysing this reaction is one of a group of
ATP-dependent acid: CoA ligases (AMP) also termed acyl
CoA synthetases or acid activating enzymes.
In a separate reaction the coenzyme A thioester transfers its acyl group to the amino group of the acceptor amino -76-
acid, with the regeneration of the cofactor, coenzyme
A. The second reaction is irreversible and is catalysed by acyl CoA. amino acid N-acyl transferases, or amino acid acylases, the net reaction is given in
Fig. 1.7.
The amino acid used for the conjugation of a carboxylic acid depends on the substrate and the species, and the range of amino acids utilized in conjugation reactions seen in vertebrates is given in Table 1.9.
Conjugation involving the addition of a dipeptide have been found in isolated instances, such as the formation of the glycylvaline conjugate of 3-phenoxybenzoic acid in the mallard duck (Huckle et_ a£. 1981).
(b) Glutathione conjugation (mercapturic acid synthesis
Glutathione is a tripeptide, y-glutamyl cysteinylglycine and following conjugation it may be further metabolised to mercapturic acids (S-substituted
N-acetyl cysteines). Glutathione is a nucleophile, and the xenobiotic with which it is conjugated may have to be 1. RCOCT + ATPHCOASH RCOSCoA+AMP = +PPi3
2. RCOSCoA + R NH CoASH + RC0NHR'
I Net Reaction: ^
RC00"+ ATP4" RCONHR' + AMP= + PPi3"
Figure 1.7. Reactions involved in the formation of a peptide bond -78-
Table 1.9 Amino acid conjugates found in vertebrate animals
Amino acid Acid conjugated Species
Alanine p,p1-DDA Mouse, hamster
Aspartic acid o,p1-DDA Rat
Glutamine Arylacetic acids Primates 2-Naphthylacetic acid
Rat, rabbit, ferret Glutamic acid Benzoic acid
Fruit bats Glycine Aromatic, heterocyclic and acylic acids Mammals
Histidine Benzoic acid African bats
Ornithine Aromatic and arylacetic Galliform acids birds
After Caldwell, Idle and Smith (1980) -79-
activated to an electrophilic metabolite, e.g. arene oxides, epoxides, or not, e.g. halogenated alkanes and nitrobenzenes, and nitroalkanes.
The enzymes involved in the conjugation step are the glutathione S-transferases. Six forms have been delineated, which occur in the soluble fraction of the cell, of different tissues, notably the liver.
Intact glutathione conjugates are eliminated in the bile and mercapturic acids are excreted in the urine.
The steps involved in mercapturic acid formation are given in Fig. 1.8.
Glutathione conjugation is known to occur in cat, dog, hamster, man, mouse, rabbit and rat.
Group 2a Acetylation
Acetylation is an important route of metabolism for compounds which contain a primary amino group, most importantly endogenous molecules, e.g. aliphatic and aromatic amines, sulphonamides and amino acids. GLUTATHIONE CONJ. MERCAPTURIC ACID
GLY GLY I I I II III R-S-CYS R-S-CYS R-S CYS ^ R-S-CYS I I I i GLU NH2 NH2 NH COCH
I Transpeptidase I I Peptidase III N-Acetylase
Figure 1.8. Synthesis of mercapturic acid -81-
The formation of an amide bond, between the acetyl CoA
(from intermediary metabolism) and the -NIfa group is catalysed by an N-acetyltransferase. This enzyme exists in two forms. In the human population N-acetylation has been shown to exhibit genetic polymorphism and the population can be phenotypically divided into fast acetylators and slow acetylators, the frequency of each depending on ethnic group.
Acetylation makes xenobiotics less water soluble, in contrast to most other conjugation reactions, by decreasing the ionisation of the nitrogen atom at physiological pH.
b. Glucoside conjugation
Glucoside conjugation is common in insects and other invertebrates and has occasionally been observed in mammals. The glucose molecule is activated
in the form of UDP glucose, the C-l of the sugar combining with the functional group on the xenobiotic or metabolite, to form ester or ether $-glucosides. The UDP is released
and recycled in the cell. -82-
c). Glucuronic acid conjugation
Glucuronidation is the most versatile of the conjugation reactions, in terms of the variety of functional groups involved (see Table 1.7), and its widespread occurrence.
All glucuronic acid conjugates are 8-glycosides of D- glucuronic acid in the pyranose form. UDP glucuronic acid is the activated form and the transfer of the sugar is catalysed by UDP glucuronyl transferase, a membrane bound enzyme, found in the microsomal fraction of the liver and other tissues.
Other sugar conjugates
Other sugars conjugated in similar fashion have been found in isolated examples which are species and substrate dependent. Most commonly reported are xylose
(e.g. conjugated with bilirubin) and ribose (e.g. conjugated with 2-hydroxynicotinic acid).
d). Methylation
Methylation is quantitatively of minor importance in the conjugation of xenobiotics, (although of -83-
significance with endogenous substrates). The source of the methyl group is S-adenosylmethione, but the methylation of primary and secondary amines may involve
5-methyltetrahydrofolic acid. As seen with acetylation, methylation generally results in a decrease in polarity and water solubility of the foreign compound.
e). Sulphation
Sulphation involves the combination of sulphate with the accepting group. This is most frequently a reaction of phenols, but is also seen with alcohols, hydroxylamines and amino groups. The active form of sulphate is 3'-phosphoadenosine-5'-phosphosulphate
(PAPS), and after its formation, as detailed in Fig. 1.9, the sulphate is transferred to the xenobiotic or metabolite catalysed by a sulphotransferase.
The enzymes involved (see Fig. 1.9) are found in the cytosol of many tissues. Sulphate conjugation is ubiquitous throughout the animal kingdom, but quantitatively its importance is restricted, because the formation pathway is saturable, due to the low availability of inorganic sulphate and thus PAPS in the body. ATP-sulphurylase ATP + SO I ^ ADENOSINE -5 PHOSPHOSULPHATE (APST+ PPI=
ADP -phosphokinase ATP 4- APS"Q=± 3 PHOSPHOADENOSINE-5 PHOSPHOSULPHATE
(PAPS) + ADP + H+ oo
sulphotransferase PAPS+ R-OH • R-OSO3-3-PHOSPHOADENOSINE 5- PHOSPHATE
+ H +
Figure 1.9. Sequence of reactions in the formation of a sulphate conjugate -85-
Summary
This brief account of the major conjugations give an idea of the range and diversity of these reactions in many species and it is apparent that information about the metabolic pathways in the horse is quite limited.
The analysis and screening for doping substances would appear to be handicapped by a lack of knowledge about the distribution of drug and metabolites in the body fluids used for dope testing (urine, blood, saliva, sweat), and of the metabolic pathways by which drugs are detoxified and excreted.
The range of drugs covered by analytical procedures in dope testing is quite wide (Groups 1-8, Table 1.3), but the accumulation of information on how these drugs are handled and excreted by the horse has been disorganised and incomplete.
The study described here has concentrated on a limited range of compounds and drugs, (NSAID's) which all possess the carboxylic acid functional group, particularly to examine some of the conjugation reactions. -86-
The aim is to reveal some of the processes involved in the distribution and excretion of foreign compounds in the horse. -87-
Chapter Two : General Methods
Page
Dosing of animals and urine collection 88
Collection of saliva 90
Collection of blood 91
Radiochemical techniques 91
Chromatography 93
Thin layer chromatography 9 3
Paper chromatography 9 3
Detection methods 94
High pressure liquid chromatography 9 5
Treatment of urine 96
A. Alkaline hydrolysis 96
B. B-glucuronidase 96
C. Ammonolysis 98
D. Acid hydrolysis 98
XAD-2 column chromatography 98
Instrumentation 99 -88-
GENERAL METHODS
Dosing of Animals and Urine Collection
Two horses were used for each administration, either a thoroughbred and a pony, or two ponies. The weights of the animals were recorded a few days prior to dosing and a blank urine sample collected.
The dose solution was prepared on the morning of the administration. The acid, in each instance, was dissolved in 100ml of 5% sodium bicarbonate solution, which aided dissolution of the compound and minimized possible stomach irritation. Each compound was administered orally using a naso-gastric tube, the dose being followed by a further
200-300ml of water. The horses were then removed from the dosing stable to a stable designed for the convenient collection of samples, in which lateral movement of the horse was restricted. Hay and water were fed ad libitum for the duration of the collection period.
Each morning the animals were removed and exercised.
Samples are not voided during exercise, but the horses usually urinate soon afterwards. The collection of urine is achieved using the equipment shown in Fig. 2.1.
The harness over the horses back holds in place a fabric reinforced latex funnel, and its height adjusted so it -89-
Figure 2.1. Equipment used to collect urine samples from a horse -90-
surrounds the penis. The urine runs from the funnel, via plastic tubing into collection bottles placed in a pit below the level of the animal. Urine samples were collected individually, and overnight this was achieved using a large fraction collector. Working with a large animal such as the horse presents greater problems of sample collection and on isolated occasions some sample losses were unavoidable. These are discussed with the relevant compounds including methods by which losses could be estimated.
Urine was collected, the pH and volume measured, and duplicate aliquots of lOOjil removed for scintillation counting (see radiochemical techniques). The excretion of radiolabel was followed and the collection of samples terminated when the level of radioactivity in the urine had fallen to background. The urine samples were immediately frozen and transported, in this state, from
Newmarket to London and stored at -20°C until analysed.
Collection of Saliva
Saliva samples were collected in some experiments immediately following blood collection.
The ponies have previously established explanted parotid -91-
papillae, parotid saliva being produced in copious amounts when attractive food is fed to the animal. 1ml samples, 14
in duplicate, were counted for C.
Collection of Blood
Thoroughbred horses are too highly strung
to permit serial blood sampling under experimental
conditions. Where blood samples were collected from
ponies, an indwelling cannula was inserted into a jugular vein before drug administration, a 10ml blank sample
collected, and at suitable intervals during the course
of the experiment further 10ml samples collected into
lithium heparin tubes. The blood was centrifuged at
2500 rpm for 20 minutes, the plasma removed and duplicate 14 lml samples counted for C.
Radiochemical Techniques
14 Each compound administered was C-labelled,
and the means of detection and quantitation of excretion products was by using this radiolabelled marker. Urine, urine extracts, XAD-2 column eluates, plasma, saliva,
fractions from thin layer plates or HPLC eluates following 14 chromatography were assayed for C using liquid -92-
scintillation spectrometry. The scintillant was
Triton X-100-toluene based, and composition was
3.331 of Triton X-100, 6.661 of toluene, lg POPOP
(1,4-di-2-(5-phenyloxazoyl)-benzene) and 55g PPO (2,5-
diphenyloxazole). Initial counting at Newmarket was
carried out with a Packard Tricarb liquid scintillation
spectrometer model 3320. Correction of efficiency
in counting due to sample quenching was made using
the channels ratio method. All subsequent counting was
done with a Packard Tricarb liquid scintillation
spectrometer model 3385 with quench correction made with reference to an external standard. Quench correction 14
curves using C toluene (Packard) were prepared at regular intervals (3-6 monthly), using chloroform for
chemical quenching and urine for colour quenching.
For radiochromatogram scanning, plates and papers were cut to a width of 5cm, and scanned using a Packard radio- chromatogram scanner model 7201. Quantitation of the
14
C in the bands was then achieved by sectioning the chromatograms into 0.5cm or 1.0cm bands, and counting these directly in vials following the addition of scintillant. -93-
Chromatography
Thin layer chromatography (tic)
Tic was carried out using aluminium backed thin layer sheets, coated with silica gel 88p254» 8 • ^ mm thick (Cat. No. 5554, A.G. Merck, Darmstadt, F.R.
Germany) and silica gel 60, 0.2mm thick (Cat. No. 5553,
A.G. Merck, Darmstadt, F.R. Germany). The former contain a fluorescent indicator, so examination of a developed plate under u.v. light was a means of locating u.v. quenching bands (see below). Preparative work was carried out on glass backed plates with a concentration zone, coated with silica gel 60-^254' layer thickness of
0.25mm. (Cat. No. 11789, A.G. Merck, Darmstadt, F.R.
Germany). Plates were developed to a height of 15cm from the origin, the Rp values did not vary between the different types of plate used. Compounds were located following examination of fluorescent indicator- containing plates under u.v. light and by the use of spray reagents.
Paper Chromatography
Paper chromatography was performed on Whatman
No. 1 or 3MM papers developed using the ascending technique, to a height of 15cm from origin, and the descending technique to 35cm from the origin. -94-
Detection Methods
U.V. Lamp
Aromatic compounds were seen as purple spots under u.v. light (Ultra Violet Products Inc.
Chromato-vue Model CC-20), when developed on fluorescent indicator-containing plates.
Spray reagents
The following spray reagents were used in the identification of metabolites on thin layer and paper chromatograms.
1. Naphthoresorcinol reagent for glucuronic acid (and other uronic acids). 0.2% w/v Naphthoresorcinol
(Sigma) in ethanol and 85% orthophosphoric acid were mixed in 5:1 proportions (v:v), immediately before spraying onto a tic plate. The colour was developed by heating the plate at 100°C for 10 min. Glucuronic acid derivatives give a blue colour on a pink background.
2. p-Dimethylaminobenzaldehyde, (p-DMAB) for glycine conjugates of carboxylic acids. p-DMAB (2g,
Hopkin and Williams) was dissolved in acetic anhydride
(50ml), and a few crystals of sodium acetate added -95-
immediately before spraying. Glycine conjugates
of carboxylic acids given an orange colour on a
pale yellow background following heating at 100°C
for 5 minutes.
There are subtle differences in the colour developed
dependent on the carboxylic acid structure and these
are discussed with the relevant compounds.
3. Ninhydrin (1,2,3-Triketo hydrindene hydrate
1,2,3, Indantrione hydrate) for amino acids.
0.1% w/v Ninhydrin (Sigma) in acetone was sprayed onto plates, and amino acids visualised as blue-purple spots,
after heating at 100°C for 5 min.
High Pressure Liquid Chromatography (Hplc)
Two different assemblies of hplc equipment were used, designated A and B.
A comprised of Injector: Waters Associates U.6K valve loop injector
Pump: Waters Model 6000 A pump
Detector: Cecil 2012 u.v. variable wave- length detector -96-
B comprised of: Injector: Rheodyne valve loop injector 7125
Pump: HPLC Technology pump RR/015
Detector: Altex u.v. detector with 254nm or 280nm filter
Fractions of hplc column eluate were collected using a LKB
RediRac fraction collector.
Treatment of Urine
Urine was subject to different treatments to assist in metabolite identification. The methods for hydrolysis of ester glucuronides (A-C) are adapted from
Heirwegh and Compernolle (19 79).
A . Alkaline hydrolysis
Urine was adjusted to pH 9 with M NaOH and left at room temperature for 12 hours. The sample was adjusted to pH 7 with M HCI, before chromatographic analysis.
B . 3-glucuronidase incubation
Urine (2ml) was adjusted to pH 5 with glacial acetic acid, 0.2 M pH 5 acetate buffer (1ml) and 5000 -97-
Units of bovine liver B-glucuronidase (1ml Ketodase,
General Diagnostics, Eastleigh, Hampshire U.K.) were added, and after mixing, the mixture was incubated at 37°C for 20 hours. Controls to ensure enzyme activity and its specificity were made as outlined below:
(i) Positive control for enzyme activity.
Phenophthalein diglucuronide (1ml) was dissolved in a blank urine sample (2ml), and the sample treated as above. Following incubation, the appearance of a pink colour upon adjusting the sample to pH 11 with M NaOH confirmed enzyme activity.
(ii) Urine (2ml) was adjusted to pH 5 and incubated in 0.2 M pH 5 acetate buffer (2ml), in the same way as the samples containing enzyme. This gave a measure of non-specific hydrolysis.
(iii) Urine was treated in an identical manner to the test sample, with the exception that before incubation,
D-saccharic acid 1,4 lactone (Sigma),(final concentration of 1 x 10~4M),a specific inhibitor of the enzyme, was added. -98-
C . Ammonolysis
Urine (0.01 - 0.2ml) was streaked onto tic plates, which were then placed in an atmosphere of' ammonia overnight, by placing them in a sealed tank containing a small open beaker of ammonia solution
(sp. gravity 0.88). After the plates were removed, the ammonia was dispelled in a fume cupboard, using a cold air blower for up to 1 hour and then developed in an appropriate solvent.
D . Acid Hydrolysis
Urine (2ml) was adjusted to pH 1 with 6M HCI in a screw capped vial. After mixing, the vial was sealed, and heated at 130°C, in a heating block for
12 hours. After neutralization back to pH 7 it was examined by chromatography.
XAD-2 column chromatography
Amberlite XAD-2 resin (BDH) is an inert polymer which was employed in particular instances as a method of "cleaning up" and concentrating urine samples. -99-
Column Preparation
Mini columns containing the resin were prepared in Pasteur pipettes. XAD-2 resin, which is
supplied in a moist state, was initially washed with
successive volumes of acetone (2 x volume of acetone), until the cloudiness in the acetone on settling did
not persist. The resin was dried by Buchner
filtration and easily poured directly into pasteur
pipettes with a plug of glass wool in the base (0.4g
of resin/column), and after filling, another glass wool plug placed on top. The columns were washed with methanol (3.0ml) and distilled water (3.0ml). Urine
acidified to pH 1 with 6M HC1 was applied to the top
of the column (3-5ml), washed with acidified water
(0.05 M HC1, 3ml) and the retained material eluted with methanol (3ml). Each eluate was measured for radio-
activity, to ensure the recovery of radiolabel was
above 95%.
Instrumentation
Gas Chromatography Mass Spectrometry (Gems)
Gems in the EI mode was performed at RSS
Laboratories, Newmarket, with a Finnigan 4000 Instrument -100-
under the control of a Finnigan 6110 data system. The g.c. column was glass 1.8m x 3.2mm internal diameter, packed with 2Q 3E 30 (Pye Unicam, Cambridge, U.K.).
The carrier gas was helium, flow rate 20ml/min. The oven temperature was initially set at 200°C, programmed to rise at 10°C per minute to 280°C. The injection port and jet separator temperatures were both 240°C. The mass spectrometer source was held at
200°C, with an ionizing voltage of 70eV.
Direct Insertion Mass Spectrometry (MS)
MS was performed in the EI mode by direct insertion with a VG Micromass ZAB IF Instrument, with an ionizing voltage of 70eV. (University of London
Intercollegiate Mass Spectrometry Service).
Proton NMR ( "^H-NMR)
*H-NMR spectra were recorded at 250 MHz with a Bruker WM 250 spectrometer, with TMS as an internal standard. (University of London Intercollegiate NMR
Service, Kings College, London). -101-
Carbon-13 NMR (13C-NMR)
13 C-NMR spectra were recorded at 400 MHz with a Bruker WM 400 Spectrometer, with TMS as an internal standard. (University of London Intercollegiate NMR
Service, Queen Mary College, London).
Infra Red (IR) Spectrometry
IR spectra were recorded in Nujol mulls using
KBr discs with a Perkin-Elmer grating Spectrophotometer
(model 157G).
Melting Point Determination
Melting points were determined using an
Electrothermal apparatus, in open glass capillary tubes. Uncorrected values are quoted.
Colorimetry
Absorbance of various coloured products in assays, were measured, using a Pye-Unicam SP 30 UV spectrophotometer, in 1 cm quartz or glass cuvettes. -102-
Chapter Three : The metabolism and disposition
of salicylic acid
Page
Introduction 103
Materials and animals 107
Chromatography 108
Reverse isotope dilution for the determination of gentisic acid 108
Colorimetric estimation of gentisic acid 110
Determination of the acyl glucuronide 111
Solvent extraction 112
Results 114
Discussion 126 -103-
INTRODUCTION
Salicylic acid was first prepared from salicin in 1833, although its medicinal properties had been noted before this. Salicin is a bitter tasting glycoside present in the bark of willow trees
(Genus Salix), comprising of glucose and salicyl alcohol. In 1860, salicylic acid was prepared from phenol, and the first reported therapeutic use of sodium salicylate was in 1875. It is used today, after more than a century of therapeutic use, for the relief of pain, fever and inflammation, mainly in the form of the ester, aspirin. This was first synthesized by Hoffman in 1897 (see Florey, 1979) of the Bayer
Company and introduced under the name of aspirin soon after.
Salicylic acid is an endogenous component of horse urine, and it is assumed to be dietary in origin.
Normal values in concentration in horse urines quoted range from 3.97mg% +3.06 total salicylate (Hucklebridge,
1979) to 30mg% (Murdick, Ray and Noonan, 1968).
Salicylic acid and aspirin are commonly used in race- horses due to their analgesic and anti-inflammatory -104-
action and ready availability. Between 1947 and 1973
it was reported by the members of the Association of
Official Racing Chemists (AORC) as being illegally present in the urine of racehorses 13 times, 8 times
as salicylic acid and 5 times as acetylsalicylic
acid. As there is an endogenous level of salicylic
acid it is of importance to gain information on the pharmacokinetics of drug elimination and also identify
any metabolites which may indicate whether the drug has been administered pre-race.
The metabolism of salicylic and acetylsalicylic acids
has been extensively studied, particularly in man.
The metabolic pathways observed in man are given in
Fig. 3.1. Acetylsalicylic acid is rapidly hydrolysed
to salicylic acid and then metabolised predominantly
by conjugation pathways. The most important pathways
in many species are conjugation with glycine to form
salicyluric acid and with glucuronic acid to form the
acyl and phenyl glucuronides. Minor conjugation routes
are the formation of the glycine conjugate of gentisic
acid, gentisuric acid, this pathway has been observed in
patients chronically receiving aspirin (Wilson et al ,
1978), but not after a single dose (Hutt et al, 1982).
Another minor pathway is the formation of a diconjugate, -105-
mmCOOH .. .. Acetyl salicylic acid H3 (aspirin)
^faCOOH
'^Vj^^OH Salicylic acid
HO^^-COOH mmtOOtt
0C6H906 Salicylphenyl Gentisic acid glucuronide C00H CONHCH
Q Salicyluric ac 1 d
cooc6h9O6
Salicyl acyl glucuronide C00H 1 Homm conhch2cooh CONHCH
OH n^-0C6H906 Gentisuric acid Salicyluryl glucuronide
Figure 3.1. Routes of metabolism of Aspirin in man -106-
the glucuronic acid conjugate of salicyluric acid.
This has been observed in uremic patients (Zimmerman
et al, 1981) and following a dose of aspirin (Putter and
Daneels, 1974, Hutt et al, 1982).
Gentisic acid (2,5-dihydroxybenzoic acid) is formed
by oxidation at the 5-position of the aromatic ring
and is a metabolite in most species. In the rabbit,
oxidation can occur at other positions of the ring,
to form other dihydroxybenzoic acids (Bray et al, 1950).
A full study in man shows that gentisic acid is the
only dihydroxy metabolite present (Hutt et al, 1982).
The metabolism of salicylate has been previously
examined in the horse. The plasma half life in ponies
was approximately 1 hour following i.v. injection of
sodium salicylate, 44mg/kg (Davis and Westfall, 1972).
The majority of drug related material in the 0-24hr
urine was in the form of salicylic acid (67% of total),
salicyluric acid (22%), salicylglucuronides (10%) and
gentisic acid as a trace metabolite, the analyses being
by spectrophotometric techniques. Davis, Westfall and -107-
Short (1973) administered sodium salicylate i.v. to one foal at 1 day, 14 days and 30 days of age, finding salicylate as the major drug related urinary constituent, with the contribution of total conjugates
(glycine and glucuronic acid) increasing with age, to a total of 40%. Schubert (1967) found 99% of a dose of salicylic acid was excreted unchanged in three horses, again the analyses were by spectrophotometric techniques.
Materials
[Carboxvl-14C] Salicylic acid, specific activity 59mCi/mMol radiochemical purity > 99% was purchased from Amersham International, Amersham, Bucks,
U.K. Salicylic acid was supplied by St. Mary's Hospital
Pharmacy. Gentisic acid (2,5-dihydroxybenzoic acid) was obtained from Sigma Chemical Company, Poole, Dorset and salicyluric acid (2-hydroxyhippuric acid) from
Aldrich Chemical Company, Wembley, Middlesex.
Animals
Shepherd Boy, a thoroughbred gelding, weighing
450kg and Ginger, a pony gelding weighing 296kg were -108-
14 used. Each received C salicylic acid (35mg/kg, 0.254 mMoles/kg, lOOpCi/animal), p.o.
Chromatography
Paper Chromatography
Paper chromatography was carried out using the descending technique, developed using Solvent
System A. Isopropanol, ammonia solution (sp. gr. 0.88), water 8:1:1.
Thin Layer Chromatography (tic)
Tic was carried out using 3 solvent systems
B: Benzene, dioxan, glacial acetic acid 90:25:8
C: Ether, butanoic acid 30:1
D: Ether, butanoic acid 10:1
Rp values of reference compounds are given in Table 3.1.
Reverse Isotope Dilution for Gentisic Acid
For reverse isotope dilution, successive recrystallizations of material from urine are made Table 3.1 R^ Values of Salicylic Acid and Metabolites
System A B C D
Salicylic acid 0.59 0.59 0.65 0.94
Gentisic acid 0.47 0.34 0.36 0.73
Salicyluric acid 0.29 0.22 0.07 0.45 -110-
following the addition of a known weight of the metabolite of interest. It was found impossible to recrystallize gentisic acid directly, as a soluble quinone formed upon gentle heating. Therefore, a derivative, 5-acetyl-gentisic acid, was prepared, which had greater stability and more suitable crystallization properties.
Gentisic acid (1.54g) was dissolved in urine (10ml), and the whole reduced to dryness by rotary evaporation.
Sodium acetate (0.38g) and acetic anhydride (6ml) were added and refluxed for 1 hour, using a water cooled condenser. The reaction mixture was poured into iced water (25ml) and left to stand overnight. The crystals of 5-acetylgentisic acid which formed were separated, filtered and recrystallized from hot water to constant specific activity (mp 131-132°C,literature value 131-132°C, Beilstein).
Colorimetric estimation of Gentisic Acid
Gentisic acid in urine samples was estimated using a colorimetric assay, modified from that of Roseman and Dorfman (1951). Standards of gentisic acid were freshly prepared over the concentration range of 0-500pg/ml -111-
in blank horse urine. All samples and standards were treated in an identical manner. Urine (1ml), 7N HgSO^
(0.5ml) and distilled water (5ml) were mixed, and extracted with ether (10ml). Half of the ether layer was decanted off and extracted with 5% NaHCOg.
After this back extraction, an aliquot (2ml) of the bicarbonate layer was removed and conc. HCI added
(0.5ml) followed by Folin-Ciocalteu phenol reagent
(0.5ml) and water (2.0ml). After mixing the solution was left for the colour to develop and the absorbance read at 660nm after 20 minutes.
Determination of the Acyl Glucuronide
2M Hydroxylamine hydrochloride (4ml),adjusted to pH7 with M NaOH, was added to urine (2ml) and distilled water (7ml) and after mixing was left at room temperature for 2 hours. Any ester glucuronide in the sample is thereby converted to the hydroxyamate of the aglycone. The mixture was then extracted with ether (2 x 15ml), the extracts combined, washed with
2M phosphate buffer pH 7.0 (4ml followed by 2ml), and then dried with anhydrous sodium sulphate. The ether was removed by rotary evaporation and the residue taken 14 up in methanol (1ml) and counted for C. -112-
Solvent Extraction
After examining neat urine, the samples were extracted with organic solvents following pH adjustment. This served to clean up and concentrate 14 the urinary C and provide an alternative method of quantifying salicylic and salicyluric acids.
Carbon tetrachloride was shown to be specific in extracting only salicylic acid, all other metabolites remaining in the aqueous phase. Dichloroethane extracted both salicylic and salicyluric acids.
Extraction efficiencies for each were measured under the conditions employed.
For salicylic acid, the extraction efficiencies were 14 calculated by extracting known amounts of C salicylate in blank horse urine.
For salicyluric acid, the colorimetric method of Levy and Procknal (1968) was used after modification.
Standard salicyluric acid solutions were prepared over a concentration range of O-lOOmg/ml in the presence and absence of salicylic acid (lOmg/ml and 15mg/ml) in horse urine (2ml). To each, 6M HCI (0.5ml) was added, mixed and then extracted with dichloroethane -113-
(2 x 15ml). After pooling the organic layers, the dichloroethane (15ml) was transferred to a separate tube and Fe (NC>3)3 solution added (5ml). The tubes were shaken for 5 min. and centrifuged. The aqueous upper layer was decanted off, transferred to a glass cuvette (1ml) and absorbance read at 530nm. The standard curve was prepared by dissolving salicyluric acid in dichloroethane, and the steps following extraction (above) following in identical fashion.
The Fe (N03)3 solution was a 1:20 dilution with distilled water of a stock solution, 1% Fe (NOo)o in 0.07M HNOq.
Extraction with Carbon Tetrachloride and Dichloroethane
Urine (2ml) was acidified with 6M HCI (0.5ml) and extracted twice with the solvent (2 x 15ml). The extracts were combined, dried over anhydrous sodium sulphate and evaporated. Tic of the extracts confirmed the specificity of the extractions and the amount of each metabolite present.
The efficiency of extraction with dichloroethane and carbon tetrachloride for salicylic acid was 98.5% and 89% respectively, and for salicyluric acid 76% and 0%. -114-
RESULTS
Excretion of radiolabel
Urine, blood and saliva were collected from the pony (Ginger), and urine alone from the thoroughbred
(Shepherd Boy). In both horses, the radiolabel was rapidly excreted in the urine, as shown by the cumulative recovery of radiolabel plotted against time (Fig. 3.2).
The recovery of radioactivity in the urine is essentially complete in 24 hours, the total recovered was 96.4% of dose from Ginger (in 42 hours) and in Shepherd Boy 99.3%
(in 36 hours). The urinary elimination half-lives were
3.4 hours and 3.8 hours for Ginger and Shepherd Boy respectively, calculated from the log % dose excreted per hour"1 vs time midpoint plot given in Appendix One.
Plasma Pharmacokinetics: Ginger
14
The log C dpm/ml was plotted against time; the data is given in Appendix One and is shown in Fig. 3.3.
From the slope of the line (K) the plasma half-life obtained from the equation
tL = loge 2 2 K
Substituting in the values:-
tx = 0.693 0 Q , i = 2.8 hours 0.247 -115-
Cumulative excretion ( %)
14 Figure 3.2. Cumulative excretion of C in the urine 14
following [ C]-salicylic acid p.o.
Key ( A ) Ginger ( • ) Shepherd Boy 14 Figure 3.3. Ginger : plasma loge [ C] levels plotted against time
14 e[ C] Plasma
T 1 1 1 1 I 7 8 9 10 11 12 Time (Hrs)after dose -117-
Saliva Levels of Radioactivity
The level of radioactivity in saliva (in dpm/ml) is plotted on Fig. 3.4, for the first 7 hours 14 after drug administration. The level of C in plasma
(dpm/ml) is plotted for comparison on the same graph.
14 The rise in C level in saliva follows that seen in plasma, suggesting the drug moves passively from saliva to plasma and the amount in saliva is in proportion to plasma levels, at all time points.
Identification of Urinary Metabolites
Urine was examined by paper and thin layer chromatography, as described. In every sample, from both animals, the chromatogram scan indicated the presence of a single major peak, corresponding in Rp value to salicylic acid. The papers and plates were cut 14 into 0.5cm strips and C measured by liquid scintillation counting, which revealed the presence of several very minor bands in addition to that of salicylic acid. Two of these corresponded in Rj, value to salicyluric acid and gentisic acid. The presence of salicyluric acid was confirmed by tic following extraction with dichloroethane.
In addition, a minor band was present, which had low -118- Figure 3.4. Ginger:levels of 14.C in plasma and saliva plotted against time following oral dose of [14C]-salicylic acid
dpm/ml Key ( • ) Plasma 2000-1 ( A) Saliva
1000H
~r 0 2 5 6 7 Time (hr.) -119-
chromatographic mobility. The material was partially labile to hydrolysis by 6-glucuronidase, alkali and ammonia treatment, with a corresponding increase in each case in 14 the C salicylic acid band. On spraying plates with naphthoresorcinol reagent, a positive blue colour at the Rp value of this metabolite was observed, which is indicative of the presence of the glucuronic acid conjugates of salicylic acid. As alkali and ammonia partially hydrolysed the band, this provides evidence that it contains a mixture of phenolic glucuronide (stable to these conditions) and acyl glucuronide (labile). The amount of the acyl glucuronide was thus separately estimated by the formation of salicyl hydroxamate. The partial hydrolysis with B-glucuronidase is a phenomenon observed with some of the other carboxylic acids in the study, conjugated with glucuronic acid, and is discussed in detail in the Chapter on the metabolism and disposition of fenclofenac (Chapter 8).
The amounts of each metabolite and unchanged drug was quantified by the methods previously described and there was good agreement (+ 0.5%) in the percentage dose of each found between the different methods of analysis. The urinary metabolites of salicylic acid -120-
in Shepherd Boy and Ginger are listed in Table 3.2.
The amounts of these in the individual samples were assayed in Ginger (samples 1-20, 0-30hr) and Shepherd
Boy (samples 1-18, 0-24hr). These are plotted on Fig.
3.5 and 3.6 respectively,, with the data given in
Appendix One.
Over 90% of the dose in both animals is excreted in the urine unchanged, and as the major proportion of the dose in the urine is in the form of salicylic acid, the approximate amount of drug in mg/ml can be calculated for any time point. This will give an indication of the rapidity at which the single dose of salicylic acid is cleared and the rate at which it falls to endogenous levels. This is plotted on Figs. 3.7 and 3.8. The published 'normal' level of salicylic acid; 30mg%, is reached 17.5 hours after dosing (in the thoroughbred) and 24 hours (in the pony).
The major metabolite is gentisic acid (3.4, 2.9% of dose), which is formed by the hydroxylation of salicylic acid. Gentisuric acid, its glycine conjugate was not detected. Conjugation pathways were of minor importance, with phenolic glucuronidation predominating over acyl Figure 3.5. Urinary metabolites of salicylic acid 14 as percentage of total [ C] in sample in 0-30hr (Ginger)
Gentisic acid
Salicyluric acid
Salicyl glucuronides
r i 0 10 Hr. -122- Figure 3.6. Urinary metabolites of salicylic acid 100n 14 as percentage of total [ C] in sample in 0-24hr (Shepherd Boy) Salicylic acid
90-
OJ 82J CL E A to 101 Gentisic acid OJ A CZ ZD
JCZ LJ A CD
CZ 0-
i J * 6i Salicyluric acid
A -4— o
0J
6"! Salicyl glucuronides
0J
r o 10 20 ?4 Hr. -123-
Table 3.2 Urinary metabolites of salicylic acid
% dose
Shepherd boy Ginger (Thoroughbred) (Pony)
Salicylic acid 91.9 90.4
Salicyluric acid 0.4 0.4
Salicyl acyl glucuronide 0.1 0.3
Salicyl phenyl 1.9 2.0 glucuronide
Gentisic acid 3.4 2.9 -124-
Figure 3.7. Ginger : Salicylate levels in urine following salicylic acid p.o. estimated from the level of 14C.
Salicylate (mg/ml)
T I 1 1 1 1,1 1 1 0 10 20 30 AO 50 60 Time (h) -125-
Figure 3.8. Shepherd Boy : Salicylate levels in urine following Salicylic acid p.o. estimated f the level of [14C] -126-
glucuronidation. Formation of salicyluric acid, the glycine conjugate of salicylic acid, is a minor pathway of metabolism (0.4% of dose).
Analysis of the serial urine samples (Figs. 3.5 and
3.6), showed the extent of the conjugation pathways varied little over the recovery period. In the latter
samples, the fall in salicylic acid is compensated by an increased gentisic acid formation.
DISCUSSION
Following a single oral dose of 35mg/kg
salicylic acid, the drug was rapidly cleared with a plasma half life of 2.8hr, and urinary elimination half-
life of 3.6hr and 2.9 hr. This rapid clearance of
an acid is favoured by the high pH of horse urine
(pH 7-9 during this experiment). The drug appears to be exclusively eliminated via the kidneys, with the
level of salicylic acid in urine falling to endogenous
levels within 24 hours of dosing. This finding has
important implications within the Rules of Racing, where medication is not permitted within 8 days of
competing. This point is developed more fully
in the discussion. -127-
Salicylic acid appears to move passively from plasma to the saliva, but the level does not reach a concentration detectable by the analytical methods employed, if for instance, a post race mouth swab was taken for a sample. This body fluid would appear unsuitable for testing for illicit use of this drug.
The finding that salicylic acid is the major 14 urinary C component of urine, is consistent with the finding of other workers, not using radiolabelled techniques, and is in contrast to the metabolism seen in other species. The rabbit (Dutch), also a herbivore, and thought to be similar to the horse in terms of metabolism, excretes an identical dose of salicylic acid in conjugated form (personal observation, using the same analytical techniques), with 20% of dose as salicyluric acid, 18% as the phenyl glucuronide and
8% as the acyl glucuronide. It is possible that the horse has a low activity of salicylic acid-coenzyme
A transferase, which is required to activate the acid to salicyl CoA before amino acid conjugation. -128-
Chapter Four : The metabolism and disposition
of benzoic acid
Page
Introduction 129
Materials 132
Animals 136
Chromatography 136
Results 137
Summary 160 -129-
INTRODUCTION
Benzoic acid is the simplest aromatic carboxylic acid and its metabolism has been extensively studied in a wide variety of species, ranging from the usual laboratory animals such as mouse and rat to the more exotic, e.g. the gecko and hedgehog (Bridges et; al, 1970). Metabolism is exclusively by conjugation and the different options are illustrated in Fig. 4.1.
The urine of horses has long been known to contain high levels of hippuric acid. Hippuric acid was
first isolated from horse urine by Justus Liebig (1829).
Its name was coined by Liebig, from the Greek for
'horse' and 'urine'. The reason the horse is
exceptional in producing so much urinary hippuric acid
is unknown and it is thought to arise from material
in the diet.
The presence of such large amounts of an endogenous
component is a complicating factor in these metabolism
studies, particularly when examining the metabolism
of acids. Attempts to concentrate urine samples usually
result in the crystallization out of the hippuric acid -130-
Figure 4.1. Conjugation of benzoic acid
C00H
Benzoic acid
cooc6h9o6 conhch2cooh conhchcooh ch2
CH? Benzoylglucuronide Hippuric acid I 1 C00H
Benzoyl L(+)glutamic acid -131-
and with it is associated radioactivity from the administration, and also the volume of urine which can be examined by tic and hplc is limited not by the weight of drug related material present, but by the amount of hippurate.
These complications apart, hippuric acid is even more of a problem when looking at benzoic acid metabolism, where one would predict it to be present from both endogenous sources and the administration. The evidence for the formation of hippuric acid would be solely from co-chromatography of the radiolabel with the standard compound by tic and hplc.
Using benzoic acid labelled with the stable isotope deuterium, in all five positions of the aromatic ring, excellent evidence of the metabolic fate of exogenously administered benzoic acid can be obtained by mass spectrometry. All metabolites derived from (ring d^)- benzoic acid, will have fragmentation ions (with the ring intact) at 5 amu higher than the corresponding ions from any endogenous material.
The cost of the (ring d^)-labelled benzoic acid restricted -132-
its use solely to the pony, who received entirely 14 d^-labelled compound with the C-labelled benzoic acid.
MATERIALS
[Carboxyl-14C]-Benzoic acid, specific activity
56mCi/mMol, radiochemical purity >99% was purchased from
Amersham International, U.K. [ring-d^]-Benzoic acid, isotope abundance > 99%, and ethyl benzoylacetate were purchased from Aldrich Chemical Co. Wembley, Middlesex.
The [ring-d^] benzoic acid was analysed as its methyl ester by gems before use, see Fig 4.2.
trans-Cinnamic acid, benzoic acid, hippuric acid, aceto- phenone and sodium borohydride were purchased from Sigma
Chemical Co. Poole, Dorset, and benzoyl glucuronide was a sample prepared by Baldwin, Robinson and Williams,
(1959) .
3-Hydroxy 3-phenylpropionic acid
Ethyl benzoylacetate (5g) was dissolved in 90% aqueous ethanol (25ml) and sodium borohydride (lg) in
20% aqueous ethanol (10ml) was added dropwise, with stirring at room temperature. After 4hr, the reaction mixture was -133-
D 0
vFm c-och2d M+ 142
D-fa^D D D + D C=0 m/z 110
Dfa^D D D
m/z 82
D Relative abundance 100n 110
% 62
50 H 54
142
1 r h, t i—i—r 50 100 150 m/z
Figure 4.2. Mass spectrum of (ds) benzoic acid methyl ester (methylated in deuteromethanol) -134-
cooled in an icebath and 6M HCI added cautiously until the evolution of H2 ceased. M NaOH (100ml) was added and the mixture refluxed for 2 hours.
After cooling, the solution was extracted with 2 volumes of ether, which were discarded. The aqueous phase was adjusted to pH 1 (10M HCI) and extracted with 4x2 volumes of ether, the extracts were pooled and the ether removed on the rotary evaporator. The residue was dissolved in a small volume of methanol and stored for
7 days at -20°C; whereby white crystals of the title compound precipitated, which were harvested by filtration, m.p. 87-88°C, yield 31%.
Elemental analysis, CgH^gOg requires C, 65.1, H 6.0 found C65.6, H 6.4.
1H-NMR spectrum at 250MHz in CDClg (TMS as internal standard), 6 (ppm) 2.8 (M, 2, CH2), 5.15 (dd, 1, on SDC at 2.8 coalesced to a singlet C H), 6.2-7.0 (broad singlet, exchanges with D20-0H, -COOH), 7.3. (5 ArH). See Fig. 4.3.
MS (direct insertion E.I. at 70eV) m/z (relative abundance)
166 (25,M+), 148 (20), 131 (6), 120 (6), 107 (100), 106
(34), 105 (38), 103 (28), 79 (75), 78 (28), 77 (24),
60 (8), 52 (10), 51 (39), 50 (16), 44 (14), 39 (11). Figure 4.3. Proton-NMR of 3-hydroxy 3-phenylpropionic acid referenced to TMS
Decoupling at Decoupling at 2.8 ppm 5.15 ppm
chohch2cooh
250 MHz, Solvent cd3ci
CO cn I -136-
The 3-carbon (or 8-carbon) has a chiral centre, but the configuration of neither the synthetic nor metabolic acid was investigated. Kenyon et aJL. (1935) found the d-form has m.p. of 115°C, and the 1-form of 115-116°C.
Animals
Caspar, a gelded pony, weighing 374kg was dosed with 2g [ring-d^] benzoic acid (5.35mg/kg, 0.042 mMole/kg) and 14C-benzoic acid (150HCi, 0.33mg). Floral
Song, a thoroughbred gelding, weighing 560kg, was dosed 14 with 2.99g C benzoic acid (5.35mg/kg, 0.044mMole/kg,
150jiCi ) .
Chromatography
Paper chromatography
Paper chromatography was carried out using the descending technique, the solvent ran to approximately
35cm from the origin. The solvent used was J: butan-l-ol; ethanol: water: glacial acetic acid 3:1:1:0.1. Rp values are given in Table 4.1.
Thin Layer Chromatography (tic)
Four mobile phases were used, these were B: benzene :dioxan: glacial acetic acid 90:25:8, E :benzene -137-
acetone: glacial acetic acid 2:2:1, K:benzene*• acetone :
glacial acetic acid 6:2:1 , L: butan-l-ol: acetic acid: water 3:2:2. Rp values are given on Table 4.1.
High Pressure Liquid Chromatography (hplc)
Hplc was carried out using system A, equipped with 250 x 50mm stainless steel column packed with ODS-
Hypersil ( 5p particle size), with a mobile phase of 40% methanol, flow rate 2.0ml min"1. Retention times are
given on Table 4.2.
RESULTS
Excretion of radiolabel
The pattern of the excretion of radiolabel following the oral administration of benzoic acid is given
in Fig. 4.4, where cumulative urinary excretion expressed as percentage of dose is plotted against time. Excretion was extremely rapid and all the dose was recovered in the urine within 20 hours for Caspar and 33 hours for Floral
Song (in only 4 urine samples) of dosing,(data given in
Appendix Two). Table 4.1 R,-, values of benzoic acid and its metabolites
J B E K L Solvent System
Benzoic acid 0..9 2 0.,7 3 0.,8 9 0..7 2 0..7 5
Hippuric acid 0..8 3 0..1 6 0..6 3 0..2 6 0,.6 4
Acetophenone 0..8 1 0..9 1 0..8 2 0..7 1
trans-Cinnamic acid 0..7 3 0..9 1 0,.7 4 0,.7 5
3-Hydroxy-3-phenylpropionic acid 0,.4 5 0..8 0 0,.5 1 0,.6 7
3-Keto-3-phenylpropionic acid 0,.5 5
Benzoyl glucuronide o,.6 2 0,.0 0 0,.1 5 0,.0 1 -139-
Table 4.2 Hplc Retention times of benzoic acid and its metabolites
Rt (min)
Benzoic acid 7.2
Hippuric acid 2.4
Acetophenone 12.7 trans-Cinnamic acid 7.8
3-Hydroxy-3-phenylpropionic 3.5 acid -140-
Figure 4.4. Cumulative excretion of radio activity following oral administration of [-benzoic acid
Key ( A ) Caspar ( • ) Floral Song
Cumulative excretion (%)
Time (h) -141-
Identification of urinary metabolites
Urine from Caspar and Floral Song was examined by paper chromatography in System J, by tic in Systems B,E,K and L and by hplc. All these analyses revealed that the majority of the dose had been excreted in the form of hippuric acid i.e. identical Rp value and retention time to an authentic sample.
Three other radioactive bands were detected in Caspar's urine, and two of these were present in the urine of
Floral Song. The first of these (in order of polarity), was very polar, corresponding in Rp value to benzoyl glucuronide. It was completely cleaved by alkali and ammonia vapour, and partially hydrolysed by ^-glucuronidase.
The band gave a positive blue colour with naphthoresorcinol spray reagent, indicative of a glucuronic acid conjugate.
The second, which had polarity between that of hippuric acid and benzoic acid, was only found in the urine of
Caspar, and elucidation of its identity will be discussed fully in a later section.
The third peak was the most apolar and corresponded in Rp and hplc retention time to benzoic acid. -142-
Analysis of Hippuric Acid by Gas Chromatography Mass Spectrometry (gems)
Hippuric acid was isolated by hplc, from each of
the urine samples of Caspar, methylated with diazomethane
and subjected to gems. The metabolite isolated from urine
sample 1 gave the mass spectrum shown in Fig. 4.5 (upper
trace) with a mass spectrum of authentic hippuric acid methyl ester given for comparison (lower trace).
The metabolic sample yielded 2 spectra, both of hippuric
acid methyl ester, one derived from the d^-benzoic acid
administered and the other from endogenous hippuric acid methyl ester. The major ions containing the intact
ring differ by 5 amu i.e. m/z 193 and 198, 161 and 166,
134 and 139, and 105 and 110.
This demonstrates that hippuric acid is a metabolite
of exogenously administered benzoic acid, as well as being
a normal urinary constituent.
Single Ion Monitoring
Single Ion Monitoring (SIM) is a technique where
a single ion can be selected, and the g.c. trace from which the ion has been derived can be reconstructed. By -143-
100-1
conhch2cooch3 -0 50-
C>
lilil i m aj (}• i—i " r" i I—r i i 1 r LJ U £Z A "O c 3) JZ) <
£ 100
aj CC
50-
ji 1 r 0 T 1 —n 1 i 1— 200 50 100 m/z 150
Figure 4.5. Mass spectra of a mixture of d3 and protonated hippuric acid methyl ester, isolated from urine (upper spectra) and authentic hippuric acid methyl ester (lower spectrum); obtained by gems. -144-
integration, the amount of material under the curve can be calculated, and the mass of material from which the
ion has arisen be known. Selecting ions from the
spectrum which are indicative of the compound of interest
is a specific way of measuring the amount of material present
in the sample, particularly when the mass spectrometer
is analysing a mixture of compounds, i.e. d^-hippuric
acid and hippuric acid, these are separated on g.c. by only
fractions of a second, and would be impossible to
distinguish if specific ions to the two compounds were
not selected.
The ions monitored for were m/z 134 and 139, and m/z 193
and 198. The g.c. trace was recorded for each and the
area under the curve calculated from this the ratio of
d^-hippuric acid and protonated hippuric acid was
calculated for each sample. Knowledge of the percentage 14
of C dose, and thus the weight of d^-hippuric acid in
the urine sample, permits an estimate of the amount of
endogenous hippuric acid. These figures are presented on
Table 4.3 from the traces depicted in Fig. 4.6. The
calculated level of hippuric acid was found to be in the
range 36.5 - 54.6mg/100ml. -145- Figure 4.6. (a) 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). See text for details SAMPLE 1 _ SAMPLE 2 _ SAMPLE 3
L. M/Z19 8
M/Z193
M/Z 13 9
M/Z 134 iiiiiiiii|IIIIIIIII|II I'I • I 11 <111' I <|11 l|'HI"|flT| 0 100 0 100 100 Scan No. -146- Figure 4.6. (b) G.c. traces obtained by single ion monitoring for ions in the spectra of ds-hippuric acid (139,198) and protonated hippuric acid (134,193). See text for details SAMPLE 4 SAMPLE 5 SAMPLE 6
X 5 x 10 X 20
M/Z 198
M/Z 193
X 5 X10 X 20
M/Z 139
•I'i'i ii>|ii'i iuii|i|irn H'l'l'lu rnmrnro M/Z 134 I 0 100 0 100 Scan No. Table 4.3 Calculation of amount of hippuric acid normally present in urine
Ratio G.C. peak area d,- (g) of vol. of Hippuric acid Sample No. % Dose d^: H^ d§se sample mg/lOOml
69.1 0.605 1.383g 599 38
22.9 0.26 0.457g 376 46.7 -M I 3.7 0.085 0.073g 235 36.5
2.6 0.045 0.06g 291 39.5
1.8 0.02 0.035g 324 54.6
99.8 -148-
Identification of Unknown Metabolic Fraction
in Caspar's Urine
The unknown metabolite fraction was isolated by solvent extraction and preparative hplc. 25ml of the
0-24h urine of the horse were adjusted to pH 1 (6M-HC1), extracted with ether, and extracts were dried (anhydrous.
Na2 SO4) and the ether removed by rotary evaporation.
The residue was taken up in 2ml methanol and this
injected in lOOpl aliquots on to the hplc column.
Radioactivity eluting from the column between 3 and 4 minutes retention was collected and the pooled fractions concentrated iji vacuo. On tic in system A, scanning revealed a single radioactive band whose centre had
Rp 0.53. The residue was treated with ethereal diazomethane and examined by gems. The total ion chromatogram of this is given in Fig. 4.7, which shows the presence of one major and two minor peaks.
The MS of the first peak (A, Fig. 4.7) contains fragment ions diagnostic of acetophenone, and those ions containing the intact aromatic ring were accompanied by fragment ions
5amu higher in a ratio of 1.6:1. This shows that as well as being derived from the administered [ring-d5]- benzoic acid, acetophenone was also present as an endogenous component of horse urine. The MS of the metabolic sample, -149-
0 50 100
4.7. Total ion chromatogram of isolated metabolic fract ion following methylation — gems analysis -150-
together with that of an authentic sample of aceto- phenone which had the same GC retention time, is shown in Fig. 4.8.
The second minor peak (B, Fig. 4.7) in the GC trace gave the MS of the methyl ester of cinnamic acid, again with those ions containing the intact aromatic ring accompanied by ions 5 amu higher in a ratio of 1.6:1.
Fig 4.9 shows the MS of metabolic and synthetic samples of the methyl ester of cinnamic acid, this latter being produced by treatment of authentic trans-cinnamic acid with ethereal diazomethane and having the same GC retention time as the metabolic sample.
The MS of the major peak in the GC trace (C, Fig. 4.7) is shown in the upper panel of Fig. 4.10. The fragment- ation pattern shown in Fig. 4.11 proposes identities for the major ions in the mass spectrum. Diagnostic ions are seen at m/z 74, arising from the elimination of a neutral molecule of benzaldehyde from the molecular ion m/z 180 by a Y-hydrogen rearrangement (Williams and Fleming, 1966) and this ion fragments further by loss of a methoxy radical giving an ion at m/z 43. Fragment ions at m/z
107 (base peak) and 105 indicate the presence of an hydroxyl group on the benzylic carbon atom, m/z 105 arising by the loss of a hydrogen molecule to giving the benzoyl -151-
100-i
COCH- d5~ 50-
CD m •• i ..ill t r lj 0 i i t r c A TD d ZD jq <
% 100
CD cr
50-
ii .ill 0 t r t 1 1 1 1 50 100 150 200 m/e
Figure 4.8. Gems analysis of a mixture of d5~acetophenone and acetophenone isolated from urine (upper spectra), and an authentic sample acetophenone (lower spectrum) -152-
100-i
c A "o A ZD £ 100-1 cd a: 50- Q i—f V|L •III|I| '••I1- •j'1"1 , "'V !"'• n*—i—I—I—I 50 100 150 200 m/e Figure 4.9. Gems analysis of a mixture of d5~cinnamic acid methyl ester and cinnamic acid methyl ester isolated from urine (upper spectra) and authentic cinnamic acid methyl ester (lower spectrum) -153- 100-1 ch0hch2c00ch3 •O 50- ilIL ijj.lji JLJ 50- li I l.llll .T I I jl 0 l r U —r T r I 50 100 150 200 m/z Figure 4.10. Gems analysis of a mixture of ds~3 hydroxy 3 phenyl- propionic acid methyl ester and 3-hydroxy 3 phenyl- propionic acid methyl ester isolated from urine (upper panel) and synthetic 3-hydroxy 3-phenyl propionic acid methyl ester (lower panel) -154- 0^0 I' CH) (C + • v V \ 0CH3 OH A^ i H2 A M/Z 180/185 H2C OCH3 M/Z 74 AAn/CHOH AA M/Z 107/112 AAR-CH=CHC00CH3 H2C=C=0H A^ M/Z 162/167 M/Z 43 M/Z 105/110 M/Z 77/62 Figure 4.11. Major ions in the fragmentation pathway of dc- and protonated 3-hydroxy 3-phenylpropionic acid methyl ester - mass spectrum on figure 4.10 -155- cation, which further fragments by expulsion of CO giving an ion at m/z 77. A minor fragmentation route involves the elimination of water from the molecular ion, giving the ion of methyl cinnamate (m/z 162, ca 2% relative intensity) which further fragments to ions at m/z 131). All the ions and fragments with an intact aromatic ring system were paired with peaks of 5 amu greater mass derived from ring-d^ -benzoic acid, again in a ratio of 1.6:1. From this interpretation of the MS of the methylated sample, the major GC peak was identified as 3-hydroxy-3-phenylpropionic acid methyl ester. This acid was synthesized and characterized as described, and after methylation had identical GC characteristics with the metabolic sample. It gave the MS shown in the lower panel of Fig. 4.10. The GC/MS results would suggest that the unknown metabolite fraction isolated by HPLC contains 3-hydroxy-3-phenyl- propionic acid, acetophenone and cinnamic acid. However, comparison of the tic and hplc properties of these compounds (Tables 4.1 and 4.2) with those of the unknown metabolite suggests very strongly that both acetophenone and cinnamic acid are artefacts produced during the work- up of this fraction. -156- Acetophenone is proposed to arise by the decarboxylation of 3-keto-3-phenylpropionic acid. Such 6-keto acids are known to undergo facile decarboxylation to the corresponding ketones (March 1966) and this has been invoked to explain the formation of acetophenone during the metabolism of phenylpropionic acid (Dakin, 1909) and of p-methoxyacetophenone from the 3-keto acid metabolites of estragole and anethole (Solheim and Scheline 1973). Attempts to prepare 3-keto-3-phenylpropionic acid by the saponification of its ethyl ester, ethyl benzoylacetate, resulted in the formation of acetophenone in far greater quantities than the acid. On tic in system K, the unknown metabolite fraction had Rp 0.53 which corresponds to both 3-keto and 3-hydroxy-phenylpropionic acids (Table 4.1), indicating that the proposed precursor of acetophenone would have been isolated from urine during the procedure used. That cinnamic acid has been produced artefactually is suggested by Fig. 4.12, which shows the traces obtained by single ion monitoring (SIM) at m/z 180 and at m/z 162 of the unknown metabolite fraction and of authentic 3- hydroxy-3-phenyl-propionic acid methyl ester. m/z 162 m/z 180 100n 100-1 50 H 50 A 50 100 ol i 100-i 100-1 50 H 50 H T 0 50 100 Figure 4.12. Gc traces from single ion monitoringrupper panel metabolic sample lower panel:synthetic 3 hydroxy 3-phenyl propionic acid (see text for details) -158- SIM at m/z 180, the molecular ion of 3-hydroxy-3- phenylpropionic acid methyl ester, of the metabolic sample showed a single peak at scan number 71. Two peaks of m/z 162 are seen at scan numbers 63 and 71, that at scan number 71 being due to the formation of methyl cinnamate during the fragmentation of the 3- hydroxy-3-phenylpropionic acid methyl ester (see above) and that at scan number 63 being the molecular ion of the methyl ester of cinnamic acid (see above). The traces obtained with the methyl ester of an authentic sample of 3-hydroxy-3-phenylpropionic acid are identical with those given by the unknown metabolite. This shows that the cinnamic acid methyl ester present in the GC/MS of the unknown metabolite fraction is produced by the thermal dehydration of 3-hydroxy-3- phenylpropionic acid methyl ester in the GC prior to entry into the MS. The urinary metabolites of benzoic acid, expressed as the percentage dose for Caspar and Floral Song are given in Table 4.4, -159- Table 4.4 Urinary metabolites of benzoic acid % dose Caspar Floral Song Benzoic acid 0.8 0.8 Hippuric acid 94.5 97.7 Benzoyl glucuronide 2.9 1.5 3-Hydroxy-3-phenyl- 1.7 propionic acid Acetophenone* 0.1 *probably derived from 3-keto-3-phenylpropionic acid -160- SUMMARY Following the rapid urinary elimination of 14 C benzoic acid, the compound was extensively metabolized, with only 0.8% of the dose being recovered unchanged. The major metabolite was hippuric acid, the glycine conjugate of benzoic acid,and in the pony the use of deuterium labelling demonstrated that this was derived from the exogenously dosed material and from endogenous sources. Glucuronidation, again, was a minor pathway of metabolism (2.9% of dose in Caspar, 1.5% in Floral Song), as shown by comparison with a biosynthetic sample of benzoyl- glucuronide. The formation of the novel metabolites of benzoic acid in the horse suggests that xenobiotic metabolism can be linked with the normal endogenous processes of the body, specifically lipid metabolism. This is discussed in detail in the final chapter. -161- Chapter Five : The metabolism and disposition of phenylacetic acid Page Introduction 162 Materials 167 Animals 168 Chromatography 169 Solvent extraction 171 Isolation of metabolites by hplc 173 Results 174 Discussion 193 -162- INTRODUCTION Phenylacetic acid metabolism has been studied in a great number of species and has provided one of the landmarks in xenobiotic metabolism, as it was the first foreign compound found to be excreted conjugated with taurine (James et 'al, 1972). Before this observation, taurine conjugation was thought only to occur with bile acids. Phenacetyltaurine is formed by a variety of species, but is quantitatively important in the mouse, ferret and capuchin and rhesus monkeys. The metabolism of phenylacetic acid demonstrates interesting species variation. Glycine conjugation is extensive in rat, guinea pig, dog and prosimians; but in the New and Old World monkeys conjugation with glutamine is the predominant pathway. In addition, a variable proportion of the dose is excreted unchanged in most species (James et 'al, 1972). In the last century, phenylacetic acid was administered to horses (Salkowski, 1884) and the urinary excretion products investigated. It was found to be eliminated predominantly as the glycine conjugate, phenacetyl glycine (phenaceturic acid). The re-investigation of phenylacetic acid metabolism in the horse has particular pertinence, because among the -163- recently developed non-steroidal anti-inflammatory drugs, one group is substituted phenylacetic acids, e.g. alclofenac, diclofenac, and fenclofenac (see Chapter 8). Phenylacetic acid is the next aromatic carboxylic acid up in the series from benzoic acid, and from the metabolism studies in various species would appear to be activated to a similar extent. The activation to phenacetyl CoA is a prerequisite to amino acid conjugation. Formation of an acyl CoA was suggested to be a step before the addition of a 2-carbon fragment (Chapter 4, benzoyl CoA formation), and it is possible that analogous products may be seen with phenylacetic acid. With this possibility considered, the experiment was designed to increase the sensitivity of analysis, as, if present, they would represent only a small proportion of the dose. This was achieved by dosing 13 one horse with C-enriched phenylacetic acid, and analysing the dose related material in the urine by 13C-NMR. Carbon 13-NMR 13 Since the first C-NMR signals were recorded in 1957, the problems of using an isotope with such a low natural abundance have been overcome by improvements in techniques and instrumentation, so much so, that carbon -164- -13 NMR has developed into a very powerful technique in organic chemistry, and more recently in drug metabolism studies. Owing to the low sensitivity, 13 C-NMR spectra are recorded using broad band decoupling of the protons in the molecule. Under these conditions all protons are irradiated and hence 13 no coupling is seen between the C-atoms and the protons. Since (under normal circumstances) only 13 1% of the carbon atoms are C, the adjacent carbon atoms 12 will most likely be C (in 99.988% of cases) and no 13 13 coupling of C-nuclei to other C nuclei will occur. 13 Therefore C resonances appear as single peaks. Resonances for all carbon atoms can be observed, and unless they are equivalent, it is rare for two nuclei 13 to have the same chemical shifts. Thus C-NMR can be used to determine the different types of carbon atoms present in the molecule. The size of signals depends on whether or not a hydrogen atom is attached directly to the carbon atom. Therefore quaternary carbons will show the smallest signals in the spectrum. 13 The chemical shifts of C vary over 200 p.p.m. (much greater a range than "'"H). The shifts are basically determined by the electron density at the 13 carbon atom. It is therefore easier to predict C- chemical shifts, (as opposed to "'"H spectra, which are dependent on shielding) on the basis of the substituent present. -165- Using synthetic isotope labelling increases the sensitivity of the technique enormously. The spectra of such labelled compounds can be recorded under conditions where only the signals from isotopically enriched carbons are observed. Therefore it is possible to obtain spectra from enriched compounds in the presence of relatively large amounts of other compounds containing only the natural abundance 13 of C, such as the normal components of urine. 13 Fig. 5.1 shows the position of the - C label in the phenylacetic acid molecule, as well as the range of chemical shifts one might expect from the different potential metabolites of this compound. As can be seen, the chemical shifts are quite distinct and the presence of absence of a particular functional 13 group adjacent to the C-atom will be discerned from the spectrum of just a crude urine extract. Due to the cost of the stable label, only one animal was dosed with this compound, it was diluted from its 90% abundance, so the dose was equimolar with the benzoic acid administration, to 40% enrichment. The other horse was dosed with phenylacetic acid, labelled with deuterium in 7 positions. It had been observed that phenylacetic acid is a normal endogenous -166- Figure 5.1. 13 C-NMR shifts for the carbonyl carbon in potential metabolites of phenylacetic acid Sppm (TMS) CHoCOOH 162-183 fT'r1-^ 0 ch2cch2cooh 190-220 OH i CH2C-CH2C00H 60-80 H 0 II CH2C NHCHRCOOH -150 CH2CH=CHC00H 130-170 -167- component of horse urine. As in the benzoic acid study (chapter 4) the deuterium labelled metabolites can be identified by gems, distinguishing them from normal urinary constituents. MATERIALS 14 [carboxyl - C] Phenylacetic acid, specific activity 50mCi/mmole, radiochemical purity 13 >97% and [carboxyl— C] phenylacetic acid, 90% enriched, were purchased from CEA, Gif-Sur-Yvette, France. (d7)-Phenylacetic acid (98 at %D) was purchased from MSD Isotopes, (Merck, Sharpe and Dohme Canada Ltd), Montreal, Canada. Phenylacetic acid and phenylacetone were obtained from Sigma, Poole, Dorset and phenylacetyl-chloride, styrylacetic acid, 4-phenylbutyric acid, and B-hydroxypropionic acid were purchased from Aldrich Chemical Co. Gillingham, Dorset. Phenacetylglycine (phenaceturic acid) A solution of glycine (20.66g) in M NaOH (250ml) was stirred, during the dropwise addition of phenylacetylchloride (lOg) over a period of six hours. The acid chloride was sparingly soluble in alkali, and intervals of 15 min were required between each -168- addition of 0.1-0.5ml. The reaction mixture was left to stir overnight at room temperature (20-30°C), and phenacetylglycine precipitated upon the addition of conc. HCI. The precipitate was filtered, and washed thoroughly with distilled water to remove any excess hydrochloric acid. The product was recrystallised from hot water 3 times and dried in vacuo over silica gel. The yield was 1.85g (16.1%) 142-143°. Direct insertion MS (70ev E.I.) major ions m/z (abundance) 193 M+ (8.9%), 175 (8.9%) 149 (6.7%), 147 (3.9%), 119 (5.6%), 118 (17.8%), 120 (2.2%), 93 (6.7%), 92 (Base peak), 91 (89%), 90 (8.9%), 89 (6.7%), 74 (15.5%), 65 (14.4%), 64 (2.2%), 63 (5.5%), 56 (9.4%), 51 (4.4%). Phenacetyltaurine and phenacetylglutamine were available within the department. (James et al, 1972). Animals Caspar, a gelded pony, weighing 372kg, and Floral Song, a thoroughbred gelding, weighing 562kg were used. Both were dosed with phenylacetic acid at 6mg/kg (0.031mmole/kg). The dose for Caspar consisted of lg [l^C]-phenylacetic acid and 1.23g [l^C]- phenylacetic acid (100|jCi). Floral Song was -169- dosed with 3.37g (d7)—phenylacetic acid and phenylacetic acid (lOOpCi). Chromatography Thin Layer Chromatography (tic) Tic was carried out using one of the following solvent systems B: benzene: dioxan: glacial acetic acid 90:25:8, K: benzene: acetone: glacial acetic acid 6:2:1, R: butan-l-ol: glacial acetic acid: water 4:1:1. Rp values of compounds of interest are given in table 5.1. High pressure liquid chromatography (hplc) Hplc used assembly B, with the u.v. detector set at 254nm. The column was stainless steel 100mm x 5 mm i.d., packed with ODS-Hypersil (Shandon) of 5M particle size. The solvent system was [7] methanol:water:glacial acetic acid 75:25:2 with a flow rate of 1 ml min~l. Retention times are given in table 5.1. Table 5.1 Chromatographic properties of hippuric acid and phenylacetic acid and its conjugates Tic Rf Hplc (RT min) System B K R [7] Phenylacetic acid 0.83 0.88 0.89 15.2 0 1 Phenylacetylglycine 0.25 0.39 0.77 6.0 Phenylacetylglutamine 0.07 0.15 0.62 5.4 Phenylacetyltaurine 0.00 0.02 0.51 2.1 Hippuric acid 0.23 0.38 0.76 5.0 -171- Solvent Extraction 1) Caspar Urine The urine of Caspar was extracted, and this 13 crude extract used directly for C NMR analysis. An aliquot of Caspar urine (0.5-2.2hr) containing 55.9% of the dose (270ml) was adjusted to pHl with conc. HC1 and extracted with ether (3 x 270ml: 1st extract and 2 x 270ml : 2nd extract). These extracted 38.3% of 14C (21.4% of dose) and 18.2% of 14C (10.2% of dose) respectively. The urine was then extracted with ethyl acetate (2 x 270ml), 14 removing 30.4% of C (17% of dose), and further extracts 14 with ethyl acetate (1 x 2 70ml) removed 7.6% of C (4.2% of dose) and 2.7% of 14C (1.5% of dose). The total 14 C extracted was 97.2% representing 54.3% of the dose. With the material in the urine arising from the administration of phenylacetic acid, there are also endogenous substances. The acidic extract cleans the sample up a good deal, but organic acids, notably hippuric acid will be extracted with the compounds of interest. For NMR analysis, the sample has to be in solution, and the sensitivity of analysis is restricted by the amount of material which can be dissolved. For this reason, the ethyl acetate extract (17% of dose) was selected for the NMR analysis, as it had the highest 14 C content for the weight of material i.e. more of the -172- material in the sample was related to the dosed compound rather than being endogenous. This sample was dissolved in D20. The sample analysed was in a total volume of 0.6ml, and following the running of the spectrum, an aliquot (2 x 20pl) was 14 counted for C to provide an estimate of the amount of dose related material examined. This was calculated to be 7.4mmoles,. The nature of this aliquot was examined by tic and hplc, which confirmed it to have the same quantitative pattern as untreated urine. To assist in the interpretation of the spectrum 13 certain standard compounds were analysed by C-NMR. These were not all water soluble (D20), and so were dissolved in a mixture of acetone and D20 and a metabolic sample was re-examined using this solvent, as to compare shifts it is essential to keep the solvent composition consistent. The compounds analysed by NMR, with the solvents used in parenthesis were; phenylacetic acid (D20: (CD3>2CO 85:15), phenacetyl- glycine (85:15), hippuric acid (85:15), phenacetyl- taurine (85:15), phenacetylglutamine (85:15), phenyl- acetone (85:15 ), styrylacetic acid (65:35), 4-phenylbutyric acid (65:35) and B-hydroxypropionic acid (85:15). These spectra are given in Appendix 5. In addition, a sample of the isolated major metabolite (see later) was examined in (D20:(CD3)2CO 85:15 . -173- 2) Floral Song's Urine Floral Song's urine (0-llhr, 100ml) was adjusted to pHl with conc. HCI and extracted with ethyl acetate (3 x 60ml). This extracted 14 C with an efficiency of 80%, and the nature of both extractable and non-extractable material was examined by tic and hplc. Isolation of Metabolites by Hplc Using the extracts of urine, the metabolites were separated by hplc and the eluate corresponding to each collected. The injection volume was 5pl, (equivalent to 1ml Floral Song's urine and 5 ml of Caspar's urine). The fractions from each injection were pooled, extracted with ethyl acetate (2 vols) which was reduced to dryness by rotary evaporation. The residue was taken up in ether, and transferred to a screw capped vial, the solvent was removed under a stream of nitrogen, and the sample stored in a dessicator at 4°C before analysis. The major metabolite of phenylacetic acid in Caspar's 13 urine was divided, and half examined by C-NMR. The other half and rest of the samples were examined by gems following methylation with methyl iodide. -174- RESULTS Floral Song (thoroughbred) Following the oral administration of (d^)- 14 phenylacetic acid and C-phenylacetic acid, the radiolabel was rapidly eliminated in the urine, in a total of 4 samples, with a recovery of 84.9% of the dose in 0-46 hours. The cumulative excretion of 14 C plotted against time is presented in fig. 5.2, (data in Appendix 3). Analysis of the urine by tic and hplc revealed the 14 presence of only one C-component, which was identical in Rp and hplc retention time to synthetic phenacetyl- glycine. This was isolated by hplc, (and hence separated from hippuric acid), and after tic of the metabolite, a deep yellow colour developed with p-DMAB spray reagent, identical to the colour of the synthetic glycine conjugate (this colour is distinct from the orange colour found with hippuric acid). Following methylation and analysis by gems, the metabolite had an identical g.c. retention time (8.15min) to the methyl ester of phenacetylglycine, and yielded the mass spectra shown in fig. 5.3. This shows the sample yields spectra of a mixture of protonated phenacetylglycine (endogenous), and (dy)-phenacetylglycine and (dg)-phenacetylglycine (one proton has exchanged) which have arisen from the exogenous (d7)-phenylacetic acid. The fragmentation pattern proposed is given in fig. 5.4. Cumulative excretion i 1 1 1 10 20 30 40 Time after dose (hr) 14 Figure 5.2. Cumulative excretion of C following the oral administration of 14 C -phenylacetic acid 100 -1 M/Z 91(97,98 ) M/Z 88 ^3 c3 50- i M/Z M/Z M+207 (213,214) 116 118 llilil. ill il jo. I* . .IIII.1II1...I . II J dJL llLL J t—1 1 1 40 100 200 M/Z Figure 5.3. Mass spectrum obtained by gems of the sole metabolite isolated from Floral Song's urine, following methylation with methyl iodide. The m.s. corresponds to a mixture of dg, d7 and protonated phenylacetyl glycine. -177- m/z m /z m/z m/z 91 134 1148 176 It CH2!CONH!CH2 CO 0 CH M+ 207 Protonated phenylacetylglycine m/z m/z m/z 97 154 182 D D i it CH . CONH CH CO 0 CH M+ 213 D^F^ D D Dg-phenylacetylglycine m/z m/z m/z1 98 155 183! D Hi Dv^W CD? CONH CH C0:0 CH I J M + 214 D D7-phenylacetylglycine Figure 5.4. Proposed fragmentation for the major ions seen in the mass spectrum (Fig. 5.3.), containing (dg), (d7)- and protonated phenylacetyl glycine. -178- Caspar (pony) As was found with the thoroughbred, the 14 urinary elimination of C label in Caspar was extremely rapid, with 80% of the dose recovered within 5 hours. The total recovery was 87% (in 29 hours). The 14 cumulative excretion of C is plotted on fig. 5.2 with data in Appendix 3, Analysis of urine, directly by tic and hplc revealed the presence of three radiolabelled bands. The major metabolite (98.6% of urinary 14C) corresponded in Rp and retention time to phenacetylglycine. The most polar metabolite (0.8% of urinary 14C) was unchanged by alkali and B-glucuronidase hydrolyses, and ammonolysis, and corresponded in Rp value and hplc retention time to phenacetyltaurine. Due to the highly polar nature of this metabolite, it co-eluted with many urinary components and as it made up a small percentage of dose, isolation was not attempted. The smallest radioactive band (0.6% of urinary 14C) was least polar and was identical in chromatographical properties to phenylacetic acid. The bands corresponding to phenacetylglycine and phenylacetic acid were isolated by hplc, and following methylation were examined by gems. Fig 5.5 shows the spectra obtained from the fraction corresponding to phenacetylglycine. The isolated material 100-1 M/Z 91 UJ lj z M/Z92 < CD q 50- i oq < so M+ 207 M+208 lil I iL . .I, Jl .I L... II 100 200 M/Z Figure 5.5. Gems of the major metabolite isolated from Caspar's urine following 12 13 methylation. The spectrum corresponds to a mixture of C and C- phenylacetyl-glycine methyl ester -180- 100-1 91 lj z < a 50 H cq < 65 M + 150,1 u 1—R—R t—i—i—r 50 100 150 M/Z 100-1 91 •< 50 H ZD CD < no 65 M + 150 JJl "«—i—i—i—i—i—i—i—r •>—i 50 100 150 M/Z Figure 5.6 Gems of the methyl ester of phenylacetic acid, upper panel material isolated from Caspar's urine, showing a mixture of the 12c and 13c acid, and lower: synthetic sample -181- had an identical g.c. retention time to phenacetyl- glycine methyl ester. The spectra obtained corresponds 13 12 to C- and C-phenacetylglycine methyl ester. The 13 12 ratio of the % abundance of the C: C in the spectra for M/2 91/92 and M/2 207/208 is 1:1.6. This is higher than the dosed ratio of 1:1.3, suggesting that phenacetylglycine occurs normally in horse urine. 12 13 Fig. 5.6 shows the spectra of C- and C-phenylacetic acid methyl ester after isolation from urine and a spectrum of authentic phenylacetic acid methyl ester is given in the lower half of the figure for comparison. In addition to these, a third spectrum, of less polar material than phenacetylglycine, was obtained, shown 12 13 in fig. 5.7. This clearly contains C and C peaks, and is interpreted as being the N-methyl phenacetyl glycine methyl ester : i.e. phenacetylglycine has become methylated during work up, and a further methyl group added during the derivatisation. The quantitative pattern of metabolism of phenylacetic acid in Caspar and Floral Song is given in table 5.2. NMR Analysis The NMR obtained of reference compounds listed in materials are given in Appendix 5. Fig. 5.8 shows 13 the C-NMR spectrum obtained of the ethyl acetate extract -182- Table 5.2. Urinary metabolites of phenylacetic acid. % Dose Caspar (0-9hr) Floral Song (0-22hr) Phenacetylglycine 82.3% 84.4% Phenylacetyltaurine 0.8% Phenylacetic acid 1.7% 100 n M/Z 91,2 LU lj < o 50- M/Z 102,3 oq < M./Z 118,9 M+ 221,2 cr* M/Z 130,1 M/Z 176,7 yj J] 100 2J0 M/Z Figure 6.1.7 Routes. The of metabolismN-methyl este of 2-naphthylaceticr of phenylacety acidl glycin e methyl ester, from Caspar's administration. -184- in D20. There is a large peak at 175.1 ppm, which is indicative of a peptide bond. This has arisen from phenacetylglycine, which has previously been shown to be the major metabolite. A peak at 173.2 ppm, another peptide bond is probably arisen from the minor metabolite, phenacetyltaurine. One of the peaks in this region will have been derived from endogenous hippuric acid, from the natural carbon- 13 abundance (169.2 ppm for standard hippuric acid). The signal for the carboxyl carbon in phenylacetic acid is at 174.9 ppm, which probably corresponds to 175.1 ppm in the metabolic sample. The most interesting peaks in the spectrum are the four found at 71.2, 72.6, 74.5, 75.1 ppm, which are all of about equal intensity. These are in the region of the spectrum where signals arising from C-OH occur. The -OH group in 3-hydroxypropionic acid sodium salt gives a peak at 65.6 ppm, and of the free acid in a range of 64.5 - 69.6 ppm. At the far end of the spectrum, ppm 215.3, there is a small signal (intensity of 2.5), which is the region where a signal indicative of a carbonyl carbon occurs. This suggests that the sample contains metabolites 00 cn i 50 40 30 20 ppm 13 Figure 5.8a. C-NMR spectrum of urine extract in D2O 1 1—1 <3003 1 100 90 80 70 ppm 13 Figure 5.8b. C-NMR spectrum of urine extract in D2O 216 214 00 i w LjJL-J Vy 140 130 120 110 100 13 ppm Figure 5.8b. C-NMR spectrum of urine extract in D2O -188- where either a keto or hydroxyl group is attached to the carbon 13 atom. This gives the possibility that there are minor metabolites of phenylacetic similar to the unusual ones found previously with benzoic acid. The finding of 4 hydroxy-carbon signals was unexpected and it is possible that a family of similar metabolites have been formed. This spectrum was run in D20, and so difficult to interpret as the standard compounds were analysed in acetone:water. Fig. 5.9 shows the metabolic sample in this solvent, the 4 hydroxyl groups gave peaks at 69.8, 71.2, 73.2 and 73.7 ppm (change of solvent results in a slight shift downfield). The carbonyl probably gives the signal at 213.2 ppm, but the keto group from acetone, runs in this region. When this spectrum is re-run with decoupling the 4 hydroxyl signals move downfield, to 38.9, 39.3, 39.9 and 41.3 ppm (Fig. 5.10), which indicates they are all primary hydroxyls; i.e. only one hydroxyl group 13 is attached to the C-atom in each compound. Phenylacetone in D20 gives a carbonyl signal at 213.9 ppm, and in D20:acetone is 213.6 ppm which is slightly downfield from metabolic sample so absolute identification is not possible, (this will JLA_ 60 40 ppm 13 Figure 5.9(a). C-NMR spectrum of urine extract in D20 (CD3)2CO 85:15 JL JU il^jja xJ 140 120 13 ppm Figure 5.9(b) C-NMR spectrum of urine extract in D2O : (CD3)2CO 85:15 none decoupled i m co m i 220 180 160 13 ppm Figure 5.9(c) C-NMR spectrum of urine extract in D2O : (CD3)2CO 85:15 (none decoupled) i 1 1 i i 1 i 1 1 1 1 i i i i i i i ' » i i i i i i 50 40 30 ppm 13 Figure 5.10. Proton decoupled C-NMR spectrum of ethyl acetate extract of Caspar's urine in DoO (CD3)2 CO 85:15, 24-50ppm (non-decoupled in figure 5.9(a)) showing shift downfield of four signals (1-4), see text for explanation i -193- have been derived in an analogous way to acetophenone). Thus the NMR analysis demonstrates that unusual metabolites of phenylacetic acid have been formed. The sensitivity of the other techniques used in the study was not sufficient to allow further analysis or quantitation. As this is the only evidence for their formation one can assume that they are only very minor metabolites. The NMR spectrum of the isolated phenacetylglycine yielded the spectrum in fig. 5.11. The peptide bond peak is the very major band at 173.1. This spectrum also includes, one hydroxy-carbon signal suggesting one minor metabolite has been isolated with the glycine conjugate, but this was not seen when analysed by gems. DISCUSSION The metabolism of phenylacetic acid in the horse followed a pattern which one may have predicted from other species studied, i.e. extensive conjugation with glycine, and is in similarity to the finding with benzoic acid metabolism (see Chapter 4). Phenacetyl- glycine was the only metabolite seen in the thoroughbred, and using the stable isotope, deuterium, it was shown that this is a normal constituent of horse urine. CO i 70 40 20 13 13 ppm Figure 5.11a. C-NMR spectrum of C-phenylacetylglycine isolated from urine D20 : (CD3)2 CO 85:15 ^JJ) -xl^^vA^^^ —• 140 210 170 130 13 13 C0 Figure 5.11b. C-NMR spectrum of c-phenylacetylglycine isolated from urine in D20:(CD3)2 -196- In Caspar, the pony, in addition, a small percentage of the dose were excreted as the taurine conjugate and unchanged. These were measured at the limit 14 of detection (i.e. on 2 x background C on chromatogram). This may explain why they were not observed in the thoroughbred, where the dose was excreted in a larger urine volume, thus limiting the amount of dose related material examined, (the volume of urine which can be analysed is usually restricted by the amount of hippuric acid present.) 13 The use of C NMR indicated that unusual metabolites have been formed which arise from the addition of groups resulting in carbonyl-carbon hydroxyl, or keto bonds. These may have analogous structure to 3-hydroxy 3-phenyl- propionic acid, formed during benzoic acid metabolism 13 (see fig. 5.1). The finding of this by C-NMR demonstrates the sensitivity of the technique, as these were not detectable 13 by chromatography. The use of C in drug metabolism studies would have great advantages in speed and sensitivity of analysis. In the normal laboratory animals and man, when urine contains a small proportion of endogenous interfering material (in the horse, the sample size analysed is always restricted by the vast amounts of urinary components), the 14 use of C could be superseded. There are a few examples where this stable isotope has been used in drug metabolism studies and the subject has -197- been reviewed by Calder (1979), and Coutts and Jones (1980). Scott et al. (1973) studied the metabolism of the anticonvulsant/antiarrhythmic drug mexiletine 13 in humans, using C NMR, which elucidated the two ring-hydroxylated metabolites using both normal and decoupled spectra. Isopropylantipyrine metabolism investigated by Tateishi and Shimiza (1976) using 13 C NMR, found peaks indicative of enol glucuronide formation. Hawkins and Midgely (1978) examined the metabolism of amitriptyline in the rat, using 13 not only C-labelled amitriptyline but they also 13 prepared C-labelled putative metabolites. In this study the metabolic pattern was confirmed by gems, after the rapid initial analysis of a crude urine extract by NMR. -198- Chapter Six : The metabolism and disposition of 2-naphthylacetic acid Page Introduction 199 Materials 201 Animals 210 Chromatography 210 Isolation of the major metabolite by hplc 216 Reverse isotope dilution for 2-naphthylacetyl- taurine 218 Results 219 Discussion 228 -199- INTRODUCTION 2-Naphthylacetic acid has been used to elicit a variety of conjugation reactions, and has been found to be a useful probe compound for examining a species' ability to utilise different amino acids as conjugating agents. The metabolism of this compound has been investigated extensively in more usual laboratory species (Emudianughe 1980, Emudianughe et al 1980), and the findings of these studies are summarised in fig. 6.1. The major pathways observed, beside the excretion of the parent acid, are conjugation with glycine, glutamine, taurine and glucuronic acid. There are species differences in the routes of metabolism, and also dose dependent quantitative and qualitative differences, seen in some instances. 2-Naphthylacetic acid was the lead compound in a series of 2-naphthalene-acetic acids which have been found to possess anti-inflammatory, analgesic and antipyretic properties. The activity of 2-naphthylacetic acid is enhanced by the substitution of a lipophilic group on the 6 position, and also by addition of a methyl group on the a-carbon. Naproxen, one such non-steroidal anti-inflammatory drug, is (+) -a- (6-methoxy-2-naphthyl) propionic acid (Juby 19 74, Harrison et al 1970). 2-Naphthylacetic acid 0 0 0 II ,C I NH NH NH 0 I CH2 CH COOH CH2 I i l COOH CH? H9 l L CH 2 2-Naphthylacetylglycine CH S03H °6 /X H2N C00H 2-Naphthylacetyltaurine 2-Naphthyl- 2-Naphthylacetylglutamine acetylglucuronide Figure 6.1. Routes of metabolism of 2-naphthylacetic acid -201- Materials 14 Potassium [ C] cyanide, specific activity 60.2mCi/mMol, radiochemical purity > 99% was purchased from Amersham International U.K. 2-bromomethylnaphthalene was purchased from Aldrich Chemical Company, Wembley, Middlesex, 2-naphthylacetic acid from Koch-Light Laboratories, Colnbrook, Bucks. 14 [ C-carboxyl]-2-Naphthylacetic acid 14 [ C carboxyl]-2-Naphthylacetic acid was synthesized according to the scheme outlined in fig. 6.2. 14 Potassium [ C] cyanide (4mCi, 350mg), and 2-bromomethyl- naphthalene (1.19g) in 8 0% aqueous ethanol (25ml) were refluxed for 7 hours and left to stand overnight. The ethanol was removed by rotary evaporation, and further water added until a white precipitate was obtained. This was filtered through a sintered glass filter, porosity no. 3,under suction. A small portion was removed and examined by tic and mass spectrometry, which confirmed it to be 2-naphthylacetonitrile (see fig. 6.3). The crude nitrile was transferred to a 50 ml pear shaped flask, 30% NaOH was added (20ml) and heated under reflux with a silicone oil bath. The temperature of the bath was brought slowly to 128°C over an hour, and maintained at this temperature for a further 4 hours. The evolution of ammonia was observed, and the heating continued until -202- 2-Bromomethyl naphthalene 14 K CN 14 CHo'TN 2-Naphthylacetonit- L rile + H Br OH" [ C-carboxyl] 4 2-Naphthylacetic CH2 COOH acid HCN Figure 6.2. Reaction scheme followed in the synthesis 14 of [ C-carboxyl] 2-naphthylacetic acid -203- no more gas was produced (detected by turning moist red litmus paper blue). After cooling, the reaction mixture was adjusted to pH 1 with concentrated HCI, whereby a white precipitate formed. This was filtered and recrystallized from aqueous ethanol to yield crystals 14 of [ C-carboxyl]. 2-naphthylacetic acid, mp. 140-141°C. The yield by weight was 6 7%, with a specific activity of 5.5pCi/mg. This material gave a single radioactive spot by tic, radiochemical purity > 98%, corresponding in Rp value to authentic 2-naphthylacetic acid. Its structure was confirmed by mass spectrometry (fig. 6.4). Fig. 6.5 shows the major fragments in the spectra of 2-naphthylacetonitrile and 2-naphthylacetic acid. 2-Naphthylacetylglycine and 2-naphthylacetyl- taurine These two putative metabolites were synthesized using the Schotten-Baumann reaction. As aromatic acid chlorides are not as reactive as aliphatic acid chlorides the reaction can be carried out in an aqueous solution at room temperature. The acid chloride is added in small portions to a well stirred solution of the amino acid in base (NaOH or NaHC03), the base serving to neutralize the hydrogen chloride generated and also as a catalyst. Figure 6.3. Direct insertion (E.I. 70eV) mass spectrum of ( C]-2-naphthylacetonitrile % Relative abundance. 100-1 167 M + 140 I to ^0 50- 1 71 141 84 126 115 168 i—i—i—i—i—p—i—i—ItI i i'" i—i t i—|—i—i i "'i—i—i i i—r 50 100 150 200 m/z Figure 6.4. Direct insertion (E.I. 70eV) mass spectrum of [14C]-2-naphthylacetic acid 100 -i M/Z 141 M+186 M/Z 115 lj z < q 50 H ca < < i 50 100 150 200 M/Z -206- CH2CN CH2COOH M + 167 M+ 186 CO" M/Z 141 M/Z 142 M/Z 127 Figure 6.5. Major fragments in the mass spectra of 2-naphthylacetonitrile (left) and 2-naphthylacetic acid (right) -207- Th e acid chloride is synthesized by the substitution of a -CI for the -OH group of the carboxylic acid. Three reagents are commonly used, thionyl chloride (SO Cl2), phosphorus trichloride (P CI3) and phosphorus pentachloride (P CI5). Thionyl chloride was used in these reactions, because unreacted reagent can be conveniently removed by distillation (b.p. 79°C). 2-Naphthylacetyl chloride 2-Naphthylacetic acid (lOg) was dissolved in benzene (50-100ml), and thionyl chloride added (6.66g, 4.0ml), washed in with a further volume of benzene (ca. 10ml). The mixture was refluxed over a vigorously boiling water bath for 4 hours. Unreacted thionyl chloride was removed by distillation, and the last traces removed by the addition of 6 portions of benzene (20ml), each followed by distillation. The product was left to cool, solidifying to a light tan solid. This acid chloride was used without further purification, after storage overnight in a dessicator. 2-Naphthylacetylglycine Sodium bicarbonate (0.15g) and glycine (0.375g) were dissolved in distilled water at room temperature. 2-Naphthylacetyl chloride (2g) was added in 20 portions of 100 mg over 120 min. The mixture was left to stir at -208- room temperature (for 14hr.) and filtered. The filtrate was adjusted to pH with concentrated HCI, and a white precipitate appeared immediately. The product was filtered, washed with distilled water to remove any acid, recrystallized from aqueous ethanol and dried over P2O5 in vacuo, m.p. 145-146°C. The yield was 2.4g (83%) and the'product gave the expected mass spectrum and 4H-NMR spectrum in CD3OD, with TMS as the internal standard, 6 (ppm) 3.75 (2 CH2 CO), 3.93 (2 NH CH2), 7.44, 7.80 (7, Ar H), see fig. 6.6. 2-Naphthylacetyl-taurine Taurine (2.7g) was dissolved in M NaOH (25ml) and stirred over an ice bath (4°C) for five hours while 2-naphthylacetyl chloride (5g) was added in small portions Following the final addition, the ice bath was removed and the reaction mixture left to stir overnight at room temperature. The solution was adjusted to pH 2 with 2M HCI, and extracted with ether (3 x 50ml) to remove any free acid. The aqueous layer was reduced to dryness by rotary evaporation, the residue dissolved in hot methanol, and acetone added until a persistent cloudiness was observed. On cooling crystals formed, which were filtered and recrystallized from methanol: acetone (2:1) to give a white crystalline product. This material was examined by 4H-NMR and found to be a mixture 250MHz, Solvent CD3OD i to 0 CO 1 5 6 (PPM) H-NMR Spectrum of 2-naphthylacetylglyc -210- 2-naphthylacetyltaurine and taurine in an approximate ratio of 3:2 (fig. 6.7). It was then taken and purified by XAD-2 column chromatography (method in Chapter 2). Taurine is not retained on column, and is recovered in the aqueous eluate. The 2-naphthyl- acetyl taurine is eluted with methanol. "'"H-NMR analysis, in D2O using TMS as internal standard, 6 (ppm) 2.6 (t, CH2S03H), 3.4 (q, CH2 CH2 SO3H), 3.76 (Ar CH2 -CO), 7.41-7.94 (7, ArH), given in fig. 6.8, which is typical of taurine conjugates m.p. 293-294°C, yield 4.7g (54%). Animals Shepherd Boy a thoroughbred gelding, weighing 446kg and Ginger, a gelded pony weighing 289kg were 14 used. Each received C-2-naphthylacetic acid (2.24mg/kg, 0.012 mmoles/kg, HOpCi each); i.e. a total of Ig of material was administered to Shepherd Boy and 65 0mg to Ginger. The low dose was used due to the toxicity of 2-naphthylacetic acid noted in previous studies, particularly in the ferret (Idle, 1976). Chromatography Thin layer chromatography (tic) 2-Naphthylacetic acid and its amino acid conjugates were separated by tic in systems E - I. Their composition and Rp values are given in table 6.1. ch^onhch^sc^na: nh2ch2ch2s03na: 3: 250MHz, Solvent D20 5 6(Pf>M) Figure 6.7 H-NMR Spectrum of a mixture of 2-naphthylacetyl-taurine and taurine in a ratio of 3:2 Figure 6.8. 1H-NMR Spectrum of 2-naphthylacetyl-taurine referenced to TMS. ch^onhch^h^h 250 MHz, Solvent DMS0-d6 JUL'Taurin e •t^o D2O •a, 5 6 (PPM) Table 6.1. Chromatographic mobilities of 2-naphthylacetic acid and metabolites on tic. E F G H I 2-Naphthylacetic acid 0.72 0.78 0.92 0.64 0.77 2-Naphthylacetyl- • glycine 0.48 0.54 0.79 0.38 0.38 2-Naphthylacetyl- taurine 0.18 0.32 0.26 0.01 0.04 2-Naphthylacetyl- •glutamine 0.42 0.50 0.64 0.02 0.16 i to ft w Solvent : E Benzene:acetone:elaciaBenzene:acetone:glacial acetic aciacidd 22:2: :: 2 : 1 1 i system F Chloroform:methanol:glacial acetic acid 15 :: 4 : 1 G Hexane:acetone:glacial acetic acid 2 :: 2 : 1 H Benzene: ether:glacial acetic acid:methanol 60 :: 30 : 9 I Benzene: methanol:glacial acetic acid 90 ;: 16 : 8 -214- High pressure liquid chromatography (hplc) Hplc was carried out using system B, with a 100 x 8 mm i.d. stainless steel column packed with ODS-Hypersil (Shandon), uv detector set at 265 nm. Injection volumes of 50-500pl were used. Fractions collected directly from the column were counted for 14 C. The uv recording was then aligned with the radioactivity histograms and the radioactive peaks assigned. The mobile phases and retention times are given in table 6.2. Solvent programming As can be seen from the retention times given in table 6.2 improving the separation of the standards, resulted in a very long assay time (over 2 hours), increasing the methanol content resulted in the earlier elution of the acid. However separation of hippuric acid from dose-related material was only possible when the acid had a retention time of about one hour. A method of improving the assay so that analysis time is shortened, with the maintenance of resolution, is to change the composition of the solvent during the assay. Solvent programming has another advantage over an isocratic system, in that the shorter retention times give better peak shapes. Solvent programming was utilized in the analysis of 2-naphthylacetic acid and its amino Table 6.2. Hplc mobile phases, and retention times of 2-naphthylacetic acid and metabolites (1) (2) (3) 2-Naphthylacetic acid 71.6 52.5 153.5 2-Naphthylacetyl—glycine 25.0 17.9 57.3 2-Naphthylacetyl-taurine 3.2 1.8 7.5 2-Naphthylacetyl-glutamine 18.2 13.4 44.0 (1) 30% Methanol adjusted to pH3 with glacial acetic acid, flow rate 2.8 ml min"1. (2) 30% Methanol adjusted to pH3 with glacial acetic acid, flow rate 4.0 ml min"1. (3) 20% Methanol adjusted to pH3 with glacial acetic acid, flow rate 2.8 ml min"1. -216- acid conjugates. Using 2 pumps under the control of a microprocessor (Waters M720), a solvent gradient was produced which is shown in fig. 6.9. This gives a comparison of the chromatograms produced with the solvent programmed and isocratic systems. Peak shape is improved, the taurine conjugate is retained on column, away from the many u.v. absorbing endogenous components of horse urine which elute in or near the void volume, and the baseline separation of the glycine and glutamine conjugates is maintained. Isolation of the major metabolite by hplc The major radioactive component of horse urine was isolated by hplc by sucessive injections of XAD-2 eluate corresponding to 5 ml urine on column, and collection of the peak containing the highest level of radioactivity. The hplc solvent was evaporated, and the residue dissolved in methanol. The isolated material was divided and treated as follows. 1. The sample was streaked onto a tic plate, and developed with solvent systems E,F and I; and sprayed with p-dimethyl- aminobenzaldehyde (Chapter 2). 2. The sample was transferred to a screw capped tube, the methanol evaporated under a stream of nitrogen, and 6M HCI added (2ml). The tube was sealed and heated at 130°C for Figure 6.9. Chromatograms obtained in A : Isocratic system and B : Solvent programmed system A: Isocratic system (2) (3) (i) (4) o . • 1 -1 2-naphthyl 2-naphthylacetyl glycine 2-naphthyl- acet^_ 2-naphthylacetic taurine gluiamind acid J 0 10 20 30 40 50 60 70 Time (min) B :Solvent programmed system cd (2)1(3) (4) % PROGRAMME Methanol 50—1 I —1 10 20 30 40 0 10 20 30 40 Time(min) Time (min -218- 24 hours. After cooling, the acid hydrolysate was dried over NaOH pellets in vacuo, and the residue dissolved in water (1ml). This was then streaked on a tic plate without a fluorescent indicator (Cat. No. 5553) along with standards of amino acids, and developed in n-propanol : 34% ammonia solution 7:1. Following plate development, the positions of the reference amino acids and the amino acid in the metabolite hydrolysate were visualized using ninhydrin spray reagent (Chapter 2). 3. Following derivatisation with diazomethane, the nature of the metabolite was examined by gems. Reverse Isotope Dilution for 2-Naphthylacetyl— taurine 2-Naphthylacetyl-taurine (500mg) was dissolved in the 0-24 hour pooled urine (50ml). The urine was reduced to dryness on the rotary evaporator, the residue was dissolved in hot methanol (20ml) and 2-naphthylacetyl-taurine precipitated by the addition of acetone and cooling. It was recrystallised from methanol:acetone (2:1 v/v) until constant specific activity was attained. -219- Results Excretion of radioactivity Urine only was collected from both animals. The radiolabel was excreted very slowly in the urine, 24% of the dose from Ginger (pony) and 13% of the dose from Shepherd Boy (thoroughbred) being recovered in 24 hours. In six days, the total urinary recovery of 14 C was 77% and 53% respectively. The cumulative recovery of radiolabel in the urine is plotted against time for the first six day period for the two animals in fig 6.10. Sampling of urine continued for 1 month after dosing, a single sample per day, and the percentage dose excreted over this period was estimated at being 0.5% per day. Data is given in Appendix 4. Urinary Metabolites Aliquots of individual urine were combined into 4 pooled samples, 0-24hr, 24-48hr, 48-72hr, and 72-96hr, for analysis and also some individual samples which contained the highest levels of radiolabelled material were examined. Tic analysis showed the presence of 4 radioactive peaks, and a representative radiochromatogram scan is shown on fig. 6.11. The upper trace shows the separation of radioactive compounds in Ginger's urine developed in solvent system E, and the lower trace is the same sample following treatment with alkali, chromatographed in Cumulative excretion (%) Figure 6.10. Cumulative recovery of C following oral dosing with 14 C -2-naphthylacetic acid ( • ) Shepherd Boy ( a) Ginger -221- 14 Figure 6.11. Radiochromatogram scan showing the major C bands in urine. Solvent system E A: UNTREATED URINE Key: 1:2-naphthylacetyl- glucuronide 2-naphthylacetyl- taurine 2-naphthylacetyl- glycine 2-naphthylacetic acid origin t t 12. 3 Solvent front B: ALKALI TREATED URINE. Origin Solvent front -222- the same solvent system. The major metabolite corresponded in Rp to 2-naphthylacetyl-glycine. Two of the minor peaks corresponded in Rp to 2-naphthylacetyl-taurine and 2-naphthylacetic acid, and the third of low chromatographic mobility was labile to alkali treatment (absent from fig. 6.11 lower trace). It was also labile to ammonolysis, but was only partially hydrolysed with 3-glucuronidase. Each of these treatments caused a corresponding concomitant increase in the size of the 2-naphthylacetic acid peak. This metabolite gave a positive blue colour with the naphthoresorcinol spray. From this evidence the peak of low Rp was concluded to be the. glucuronic acid conjugate of 2-naphthylacetic acid (see later). The analysis of Shepherd Boy's urine showed only small quantitative differences in the pattern of metabolites from that described above. Hplc analysis confirmed the identity of the radioactive bands, by comparison of retention times with synthetic standards. The major metabolite, after isolation as described, was shown to yield glycine following acid hydrolysis. After tic, the radioactive band of the isolated material corresponded in Rp to synthetic -223- 2-naphthylacetyl-glycine, and gave an identical colour reaction with p-DMAB (pale orange, hippuric acid gives a deep orange). Following methylation of the metabolite with diazomethane, analysis by gems yielded a mass spectrum with major ions identical to an authentic sample of similarly derivatised 2-naphthylacetyl- glycine (see fig. 6.12). Fig. 6.13 presents assignments of the ions in the mass spectrum of the methyl ester of 2-naphthylacetylglycine. Fig. 6.14 presents a histogram of the amounts of each metabolite (as percentage of dose) for the 4 pooled urine samples from both animals. The recovery of dose from Ginger in 0-96 hours totalled 66.4%, and from Shepherd Boy 39.3%. The total metabolites over the entire time period are given in Table 6.3. -224- Fig. 6.12. Mass spectrum of 2-naphthylacetyl-glycine methyl ester. Upper : authentic sample, lower sample : isolated from urine : both derivatised with diazomethane 100-1 CH2CONHCH2COOCH3 50- ai 1 111 111iiiii .iii lJ -W £Z 0- -r r i—r A "o £Z z) -a < > 100- 0) CN 50- ILfJ ...111, , .lb h o- i—7 9—!'• "f "'i—rh—i—|— 50 100 150 200 250 m/e -225- Figure 6.13. Assignments of major ions in the mass spectrum of the methyl ester of 2-naphthylacetyl-glycine H i ch2c n ch2cooch3 0 M + 25 7 —I + CH=C=0 1 ' M/Z 142 M/Z 141 10 n SHEPHERD BOY 8 - 6 - 4 - 2 - illlllllls 0 o- 24 hr. 24 — 48 hr. 48- 72 hr. 72-96 hr. to to ACID co GINGER I I GLYCINE TAURINE HI GLUCURONIDE imniife^ 0-24 hr. 24-48 hr. 48-72 hr. 72-96hr. Figure 6.14. Quantitative pattern of metabolism for days 0-4 following an oral dose of 2-naphthylacetic acid -227- Table 6.3. Urinary metabolites of 2-naphthylacetic acid in 0-96 hours. Shepherd Boy Ginger 2-Naphthylacetic acid 5.7 15.8 2-Naphthylacetyl-glycine 26.0 37.9 2-Naphthylacetyl-taurine 4.3 5.8 2-Naphthylacetylglucuronide 3.9 6.9 -228- Discussion In contrast to all the other acids investigated, 2-naphthylacetic acid is eliminated very slowly in the urine of two horses following oral administration, and the total recovery is lower than seen with the other probe compounds. The study has yielded important information on the different conjugation pathways which the horse can utilise in foreign compound metabolism. The finding of 2-naphthylacetyl-glycine as a major metabolite (over 50% of 0-24hr urinary radioactivity) confirms that the horse can utilise glycine to a large extent, as seen with benzoic and phenylacetic acids, in the conjugation of carboxylic acids. More interestingly, this was the first occasion that conjugation of a xenobiotic with taurine had been observed in the horse (the metabolism of 2-naphthylacetic acid was studied chronologically before phenylacetic acid). Taurine conjugation had previously thought to predominate in the carnivorous species, particularly in the dog and ferret, so this finding was unexpected. Formation of taurine conjugates has important analytical implications, as the xenobiotic or drug molecule becomes very hydrophilic upon the addition of this sulphonic acid. -229- Th e chemical properties of taurine conjugates make the routine extraction of the compound very difficult; unless special conditions are employed, e.g. following the formation of an ion pair. Identification by methods such as gems and ms are more involved than routine analytical methods allow for, and a taurine conjugate usually requires derivatisation before analysis. A minor metabolite of 2-naphthylacetic acid was the glucuronic acid conjugate which, again ( as with salicylic acid and benzoic acid) was only partially hydrolysed by 3-glucuronidase. This observation is discussed further in Chapter 8. -230- Chapter Seven : The metabolism and disposition of isoxepac Page Introduction 231 Materials and Animals 234 Chromatography 237 Treatment of Urine 239 Results 240 Discussion 247 Appendix 249 -231- INTRODUCTION Isoxepac (6,11-dihydrodibenz [b,e] oxepin- 2-acetic acid, see fig. 7.1) is a non-steroidal anti- inflammatory drug (NSAID), which was under clinical trial in the U.K. until recently, when hepatotoxicity led to its withdrawal. The drug was developed by Hoechst Pharmaceuticals and its synthesis and properties have been described previously (Aultz et al 1977). The disposition and metabolism of isoxepac have been studied in the rat, rabbit, dog, rhesus monkey and man (Illing and Fromson 1978), and the routes of metabolism seen are shown on fig. 7.2. The major routes of metabolism are by conjugation of the carboxyl group, with glycine, taurine and glucuronic acid, and, as yet, an unidentified metabolite. There occurs a wide species variation quantitatively and qualitatively in the pathways favoured. Conjugation is also the major pathway of metabolism of a positional isomer of isoxepac, oxepinac, which has the acetic acid group attached to the 3-position of the aromatic ring. The conjugates formed are with taurine (rat, mouse), acyl glucuronide (man, dog) and glucose (mouse) (Hakusui et al 1978). * DENOTES THE POSITION OF 14C ATOM (CARBONYL CARBON) Figure 7.1. Isoxepac (6,11-dihydrodibenz [b,e] oxepin-2-acetic acid) Figure 7.2. Routes of metabolism of isoxepac ch2cooh isoxepac i to CO CO 0 ch2 CO NH CH? i COOH Glucuronic acid conjugate Taurine conjugate -234- Animals One gelded pony, Andrew, weighing 230kg and one thoroughbred gelding, Shepherd Boy, weighing 14 45 7kg were used. They were dosed with C-isoxepac (3mgkg-1, O.Ollmmoles kg-1, 125uCi) and urine only collected. Due to known photosensitivity of the drug, all samples were protected from light. In the case of one animal (Shepherd Boy), the routine was changed, as he developed flu-like symptoms, became lethargic and feverish (temperature 104°F) and had to be returned to his stable. The vet supervised the animal, confirming that this was unrelated to the dosing, and further urine samples collected using plastic bags. Losses were inevitably incurred, and because the collection was attended to by the head groom, protection of the samples from the light was no longer possible. Materials [carbonyl 14C]-Isoxepac and unlabelled isoxepac were gifts from Hoechst Pharmaceuticals Milton Keynes, Bucks., England. Following recrystallisation from acetonitrile, the radiolabelled compound had a radiochemical purity of > 98% by tic, specific activity of 0.29|jCi/mg. -235- Tetrabutyl-ammonium hydroxide (40%) was obtained from Aldrich Chemical Co. Ltd., Poole, Dorset, England. The taurine conjugate of isoxepac The taurine conjugate of isoxepac was synthesized by the Schotten-Baumann reaction. Isoxepac (4g) was dissolved in benzene (30ml) and thionyl chloride (2.2g) added, washed in with a further small volume of benzene (10ml). The flask and condensor were covered in aluminium foil to ensure the reaction was in darkness. The mixture was refluxed for 5 hours, and the benzene and unreacted thionyl chloride distilled off. The final traces of thionyl chloride were removed by addition of benzene and distillation (5 x 25ml). On cooling,the acid chloride, a brown oil, was stored dessicated in the dark overnight and used immediately without further purification. Taurine (3g) was dissolved in M NaOH (30ml) and stirred over an ice bath, while the acid chloride was added dropwise. After the final addition, the mixture was stirred for a further 2hr at 0-4°C and then left at room temperature (20°C) and stirred overnight. -236- The pH of the mixture was adjusted to pH 1 with 2M HCI, and extracted with 2x1 vol. of ether, which removed unreacted isoxepac. After neutralisation to pH 7 with 2M NaOH, the aqueous solution was reduced to dryness by rotary evaporation, and the residue taken up in hot methanol. Insoluble material was filtered and discarded, and crystals of the taurine conjugate of isoxepac appeared on cooling. This was recrystallised from hot methanol and the product, a white crystalline solid, was found to decompose at a temperature above 300°C (Yield 18%). Infra Red Spectrum The I.R. spectrum was measured in a Nujol mull using KBr discs. Absorptions observed included, 3420cm"1 (N-H stretching), 1640cm"1 (C=0 stretching), 1570cm"1 (Amide II band), 1240cm"1 and 1050cm"1 (S03 absorptions). These five bands are characteristic of taurine conjugates (Idle, Millburn and Williams 1978). 1H-NMR "''H-NMR spectrum was run in D20 at 250MHz, referenced to TMS. The following signals were observed 6(ppm) 3.07t (CH2-CH2-S03H), 3.56 complex multiplet (CONH CH2 CH2 -SO3H), 3.79m (-CH2 C0NHCH2CH2S03H) which are all characteristic of taurine conjugates, and 4.69, 5.03, 6.89q, 7.37 m, -237- 7.56m and 7.79 double doublet, characteristic of the isoxepac part of the molecule. Mass spectrometry of the taurine conjugate and its methyl ester following derivatisation yielded spectra related to isoxepac. Chromatography Thin layer chromatography (tic) Three mobile phases were employed K : benzene:acetone:glacial acetic acid 6:2:1 Q : chloroform: isopropanol: glacial acetic acid 90:5:2 R : butan-l-ol: glacial acetic acid: water 4:1:1 The Rp values are given on table 7.1. High pressure liquid chromatography (hplc) Hplc was performed using system A equipped with a lOOx 5 mm stainless steel column, packed with ODS-Hypersil (5p particle size). The mobile phase (6) was 40% aqueous methanol adjusted to pH3 with glacial acetic acid, with a flow rate of 2 ml min-1. The retention times are given on table 7.1. Table 7.1. Chromatographic properties of isoxepac and the taurine conjugate of isoxepac. Rp value in solvent system Isoxepac Taurine conjugate Q 0.60 0.01 R 0.81 0.45 K 0.70 0.03 Hplc retention time (min) System 6 14.8 2.0 -239- Treatment of Urine 1. Solvent extraction Urine (15ml) was acidified to pH 1 with M HCI, and extracted with ether (4 x 3 0ml). The extracts were pooled and reduced to dryness by rotary evaporation. The residue was taken up in methanol (5ml), and the efficiency of extraction calculated following liquid scintillation counting and the nature of the extracted material examined by chromatography. The extracted urine was retained, and residual ether was dispelled by bubbling air through,tetrabutyl- ammonium hydroxide (1ml) was added, and after shaking the urine was re-extracted with chloroform (3 x 30ml). The extracts were pooled, and solvent removed by rotary evaporation. The residue was taken up in ether, and transferred to a small vial where the solvent was removed under a stream of nitrogen. The extract was examined by tic and shown to contain a single radio- labelled band* Direct analysis of gems was attempted but only spectra related to isoxepac were obtained. Metabolite isolation by tic The radioactive band which had identical chromatographic properties as synthetic taurine conjugate of isoxepac was isolated from urine by preparative tic. The silica was scraped off, and eluted with hot methanol. -240- Examination of the isolated material by hplc showed it was free from any contaminating hippuric acid. The methanol was removed under a stream of nitrogen and the residue taken up in 6M HCI (2ml), and heated in a sealed tube at 130°C for 12 hr. The hydrolysate was dried over NaOH pellets in vacuo, and taken up in a small volume of water, and chromatographed with a series of authentic amino acids (in 10% propan-2-ol), in systems Q and R. In both systems a u.v. quenching spot corresponding in Rp value to isoxepac was visible, and radiochromatogram scanning confirmed this to be the only radioactive material on the plate. After spraying with ninhydrin (0.2% in acetone w/v), a single blue spot, indicative of an amino acid was observed in the metabolic sample which corresponded in colour reaction and Rp value to taurine. Results Excretion of radiolabel There was a rapid elimination of radioactivity in the urine of both horses following the oral administration 14 of C labelled isoxepac. In Andrew, 67% of the dose was recovered within 24 hours, and 98.4% in 72 hours, and the 14 urinary elimination half-life for [ C] was 6 hours. The cumulative excretion of radioactivity by Andrew plotted -241- against time is given in figure 7.3. With Shepherd Boy, there were a few problems associated with the collection of urine samples. Estimation was made for the losses by calculating the log percentage dose excreted per hour, and plotting this against the midpoint time for the sample. The data, calculations and graphs are given in Appendix 6. There were three known lost samples designated A, B and C; B was an overnight collection. Extrapolation back to the missing time points, gave these samples as A: 1.1% of dose, B: 7.1% of dose and C: 1.2%. There is a strong possibility that further samples were lost, which may account for the lower recovery of dose in Shepherd Boy. Figure 7.4 14 plots the cumulative recovery of [ C] in urine against time after dosing, showing the rapid elimination of radioactivity, with levels returning to background within 72 hours. The urinary elimination half-life in Shepherd Boy is approximately 10 hours (Data given in Appendix 6). To validate this plot, which accounted for losses (correlation coefficient was 0.899, for the line obtained by least squares regression),the data for Andrew has been treated in an identical way and is included in Appendix 6. -242- Figure 7.3. Cumulative excretion of radioactivity in urine after oral administration of 14, ^C -isoxepac (Andrew) ose Time(Hr) -243- Figure 7.4. Cumulative excretion of radioactivity following oral administration of l4C-isoxepac. Shepherd Boy Time (Hr) -244- Urinary metabolites Andrew's urine Analysis of the urine from Andrew by tic and hplc revealed that the majority of the dose had been excreted unchanged, i.e. the most abundant l4C band corresponded in Rp value and retention time to authentic isoxepac. The remainder of the urinary radioactivity, with the exception of ca. 2%, ran as a discrete band, which was far more polar than the parent drug. This was confirmed by solvent extraction, where isoxepac was exclusively and totally removed by ether extraction (demonstrated following analysis of the extractable and non-extractable components by tic). The metabolite remaining in the urine was extractable into chloroform following the addition of the ion pair reagent, tetrabutyl-ammonium hydroxide. This extract was examined by gems, but yielded only isoxepac-related spectra. This polar metabolite was stable to alkali, enzyme treatment and ammonolysis but labile to acid hydrolysis, which gave a corresponding increase in the amount of isoxepac present on the chromatogram. In tic and hplc systems the metabolite co-chromatographed with the synthetic taurine conjugate of isoxepac. Following isolation by preparative tic the metabolite was shown to yield taurine and isoxepac upon acid hydrolysis. -245- In addition, two very minor bands were seen. One was less polar than isoxepac (M4), and the other intermediate in polarity between isoxepac and the taurine conjugate (M2). The nature of these is discussed with the analysis of Shepherd Boy's urine (below). Shepherd Boy Analysis of urine from Shepherd Boy showed that the urine contained four radioactive components. The major radioactive band corresponded in tic RF value and hplc retention time to the taurine conjugate of isoxepac, and its identity was further confirmed by the methods described above for Andrew's urine. The second band, had identical chromatographical properties to the parent drug, and was thus identified as unchanged isoxepac. The other two radioactive bands (M2 and M4) are consistent in retention time and Ry value to those found in Shepherd Boy's urine. These are thought to have arisen from the light decomposition of isoxepac, and are thus artefacts arising in the urine subsequent to the voiding of the sample. -246- Table 7.2. Urinary metabolites of isoxepac in Andrew and Shepherd Boy (% dose) in 0-80hr. Andrew Shepherd Boy a Taurine conjugate 22.9 37.6 M2 1.9 10.6 Polarity Isoxepac 72.3 21.2 M4 1.2 8.4 -247- Table 7.2 presents the quantitative pattern of the metabolites in the urine of Andrew and Shepherd Boy. The two unknowns are designated M2 for the more polar and M4 for the less polar. Evidence that M2 and M4 are photodecomposition products of isoxepac is presented in an appendix to this chapter. DISCUSSION The administration of isoxepac and analysis of its urinary metabolites has shown that taurine conjugation is a major pathway of metabolism in the horse. Taurine conjugation has been seen previously in this species, with phenylacetic acid (Chapter 5) and 2-naphthylacetic acid (Chapter 6). In each case, this is quantitatively less important than glycine conjugation. Taurine conjugation of isoxepac has been found to occur in other species, notably the dog, where 30-50% of dose is excreted in this form (Illing and Fromson,1978). The dog is recognised as being able to utilise taurine in conjugation reactions, for example, with fenclofenac (80% of dose, Jordan and Ranee,1974). -248- The formation of a metabolite, such as a taurine conjugate raises problems for the detection of the drug in the urine. In the standard procedure, where the first step is solvent extraction of the urine, the taurine conjugate will remain in the aqueous phase. The taurine conjugate, in this instance, became extractable following the formation of an ion pair with tetrabutyl-ammonium hydroxide. Although detection of parent drugs can be straight- forward, evidence to support the finding of a drug administration to a horse is much enhanced by the detection of drug metabolites in horse body fluid. -249- Chapter Seven : Appendix. -250- Evidence that M2 and M4 have arisen as artefacts from the light decomposition of isoxepac. The precautionary measure of protecting the urine samples from light was taken, following the report of a g.c. assay for isoxepac in plasma where samples were- stored wrapped in foil to prevent photolytic breakdown (Bryce and Burrows,1978). This decomposition was originally examined by tic, using solutions of isoxepac, which were buffered to different pH's (0.067M phosphate buffer, pH 5.0-8.0). Following exposure to sunlight for 2hr, the samples were spotted onto a tic plate and developed in solvent system Q. The products were examined under u.v. light, and this is represented in fig. 7.5. Isoxepac decomposed while in solution to at least five different components separable by the system used. This decomposition was not seen in the sample kept frozen or in pH 5 solution. The extent of the photolytic decomposition is greater at higher pH. To obtain a quantitative assessment of this decomposition, -carbonyll-labelled isoxepac was left in buffered solution at pH 5 and pH 8, in the sunlight for 2 hr, 4 hr and 6 hr. These were compared by tic with a newly prepared solution, and one which was stored frozen. This demonstrated that the major decomposition product was -251- Solvent front Isoxepac Origin 1 1 1 1 1 1 1 frozen fresh pH5 pH6 pH7 pH7.4 pH8 solution solution SOLVENT SYSTEM Gl Figure 7.5. Effect of light on the stability of isoxepac in solutions buffered to different pH's. (2 hours exposure) -252- less polar than isoxepac, and breakdown occurred at a greater rate in more alkaline conditions. Finally, this photolytic decomposition was examined in the horse urine. A sample of Andrew's urine, was exposed to the light for 6 hr, and examined by tic before and after. Fig. 7.6 shows the radiochromatogram scan of the developed tic plates and fig. 7.7 shows a sample of Shepherd Boy's urine in the same solvent system. It is clearly shown that after exposure to light Andrew's urine takes on a similar appearance to Shepherd Boy's urine. The nature of these decomposition products has been investigated by Hoechst Pharmaceuticals (J. Fromson, personal communication). The sodium salt of isoxepac breaks down in alkaline solution at the rate of 6% per hour, the major product (>95% by glc) is shown on fig.7.8. The free acid decomposed, at a rate which was solvent dependent, such that less than 10% of the original acid remained after 65hr. Other decomposition products included a compound arising from the loss of CO2, to form a fluorene ring structure, and also the loss of H2CO from the methylene carbon from the central ring of isoxepac. origin c m solvent 5 ,u front 2. Andrew's urine following exposure to light for 6 hr i 1—; r- 1 1 1 1 1 1 1 1 1 1 1 1 t t- 1 origin solvent front -254- Figure 7.7. Radiochromatogram of urine : tic solvent system Q ISOXEPAC 1. Shepherd Boy's urine TAURINE CONJ.OF ISOXEPAC MARKER 1 — 1 * ' ' i l 1 1 1 1 1 1 1 1 1 1 on9in 5 10 solvent front -255- Figure 7.8. Major decomposition product of Isoxepac -256- Therefore it is suggested that M2 and M4 present in the urine of both horses have arisen from a light dependent breakdown of isoxepac. The two products can be attributed to have arisen from isoxepac, because the only other radioactive component in the urine, the taurine conjugate is not affected in chromatographic properties by exposure to the light. Although, there is probably photolytic breakdown of the isoxepac part of the molecule, the taurine conjugated to the drug has a greater influence on chromatographic behaviour i.e. it remains extremely polar. This light decomposition was more exaggerated in the urine of Shepherd Boy because light entered the samples during the collection period. The pH of the urine in this study ranged from pH 7.4-8.5, which favours breakdown of structure. Freezing the samples was shown to maintain the integrity of the molecule. -257- Chapter Eight : The metabolism and disposition of Fenclofenac Page Introduction 258 Materials 258 Animals 260 Chromatography 260 Treatment of plasma 263 Treatment of urine - solvent extraction 263 Naphthoresorcinol method for glucuronic acid determination 265 Results 266 Discussion 280 -258- INTRODUCTION Fenclofenac (2-(2,4-dichlorophenoxy) phenylacetic acid, Flenac ®) is a non-steroidal anti-inflammatory drug (NSAID), developed in the U.K., and marketed by Reckitt and Colman, and is one of a series of substituted phenylacetic acids used clinically in the treatment of inflammation. The major route of metabolism of fenclofenac, in other species, is by conjugation at the carboxyl group. These pathways are shown in figure 8.1. The taurine conjugate is formed exclusively by the dog among the species studied (Jordan and Ranee, 1974) and the glucuronic acid conjugate formed by man, guinea pig, rabbit, baboon and rat (Greenslade £t a_l, 1980). The hydroxylation products are found in the urine of man, guinea pig and baboon. In all these studies the dose was lOmg/kg, and this regime has been adopted for the administration to horses. Materials 14 [carboxy- C]-Fenclofenac (2-(2,4-dichloro- phenoxy) phenylacetic acid), specific activity 0.22pCi/mg (radiochemical purity by tic > 99%), unlabelled fenclofenac and 5-hydroxyfenclofenac were gifts from Reckitt and Colman, Pharmacetuical Division, Dansom Lane, Hull, U.K. The -259- Figure 8.1. Routes of metabolism of fenclofenac CH2COOH FENCLOFENAC CH2CONH CH< TAURINE GLUCURONIC CONJUGATE ACID CONJUGATE CH2SO2OH CI 0 CI CH2COOH 5-HYDROXY FENCLOFENAC + A DIHYDROXY- FENCLOFENAC t GLUCURONIC ACID CONJUGATES. -260- mass spectra (direct insertion, 70 eV E.I.) of these two compounds are given on figures 8.2 and 8.3. Animals Two gelded ponies were used, Andrew, weight 234kg and Caspar, weight 3 70kg. Both animals were dosed 14 with [ C]-fenclofenac (lOmg/kg, 0.034mMole/kg, 49nCi). In addition to urine collection, blood sampling was carried out at suitable intervals from both animals for up to 12 hours after dosing (details in Chapter 2). Chromatography Thin layer chromatography (tic) The following solvent systems were used: K: benzene: acetone: glacial acetic acid 6:2:1: M: butan-2-one: glacial acetic acid 75:10 N: chloroform: methanol: glacial acetic acid 8:5:1 O: cyclohexane: acetone: glacial acetic acid 2:2:1 P: chloroform: methanol: glacial acetic acid 90:10 Rp values are given in table 6.1. High pressure liquid chromatography (hplc) Hplc analysis of fenclofenac and its metabolites was carried out using System A, with a 100 x 5 mm stainless steel column packed with ODS-Hypersil, the mobile phase was (5) 60% aqueous methanol adjusted to pH3 with glacial -261- Figure 8.2. Direct insertion mass spectrum of fenclofenac (E.I., 70eV) 100-1 M/Z 152 M/Z77 M/Z 90 50- M/Z 104 M/Z 162 M/Z 131 i r —i " " l 1 r" 1 t— 1——i LU LJ 50 100 150 m/z ^00- M/Z 215 M+296 M/Z 181 M + 298 CQ < M/Z 251 M/Z 2 53 50- M/Z18 8 t ljt 1 200 250 300 m/z -262- Figure 8.3 Direct insertion mass spectrum of 5-hydroxy fenclofenac (E.I., 70eV) 100—1 M/Z 49 50 - M/Z 109 M/Z 123 M/Z 161 UJ LJ ui < r i i —i i—r—r—i i i—i i 50 100 150 m/z q ^00 M+313 cr* 50- M+ 315 M/Z 232 M/Z 234 M/Z 197 t 1 1 1 1 1——i 1 1 1 1 1 r t— 1 200 250 300 m/z -263- acetic acid, flow rate 2ml/min. The retention times in minutes are given in table 8.1. Treatment of plasma The nature of the radioactive components in the plasma were examined following pooling of the samples. The 0-2hr, 2.5-5hr and 6-12hr pooled samples from both animals were analysed. Each pooled sample was adjusted to pHl with M HCI, and extracted with 2 volumes of ethyl acetate. The ethyl acetate layer was separated by centrifugation (3,000 rpm for 10 min.), removed and reduced to dryness by rotary evaporation. The residue was dissolved in 1ml of methanol, 500|jl was counted for 14 C to measure extraction efficiency (>95%), and the remainder analysed by tic, solvent system K and assayed as previously described (Chapter 2). Treatment of urine-solvent extraction Urine was adjusted to pHl with the addition of 6 M HCI, and extracted with 2 volumes of ethyl acetate, the solvent layer was separated by centrifugation, removed, pooled and reduced to dryness by rotary evaporation. The residue was taken up in a small volume of methanol (l-2ml), 14 aliquots (2 x 50pl) were counted for C, and the efficency of extraction measured, (found to be greater than 95%). Table 8.1. Chromatographic properties of fenclofenac and 5-hydroxy fenclofenac tic Rp values Fenclofenac 5-Hydroxy fenclofenac System K 0.97 0.86 M 1.00 1.00 N 0.96 0.96- 0 0.96 0.96 P 0.92 0.92 hplc Retention Time (min) System 5 14.7 7.8 -265- Naphthoresorcinol method for glucuronic acid concentration A colorimetric assay, using naphthoresorcinol, measured the concentration of glucuronic acid in hplc eluate following chromatographic separation of the components of the ethyl acetate extract of urine. This method was adapted from Marsh (1966). Naphthoresorcinol (0.25g) was dissolved in distilled water (10 0ml), and kept in a shaking water bath for lhr at 37°C, to obtain maximum dissolution. The solution was filtered, the insoluble material discarded, and stored protected from the light, at 4°C. The hplc eluate (0.5 min fractions, 1 ml), naphthoresorcinol reagent (1ml) and concentrated HCI (1.5ml) were mixed well, the tubes capped with foil and heated in a water bath at 100°C for 2 hr. After cooling, ethyl acetate (5ml) was added, tubes mixed well, and the layers separated by centrifugation (3,000 rpm for 10 min). The coloured pigment (purple) produced moves into the ethyl acetate (upper layer). Standard D-glucuronic acid solutions (O-lOOjjg/ml) were treated in an identical way and absorbance of ethyl acetate layer from the standards and unknown fractions (1ml) read at 600nm. The standard curve was linear over this concentration range. Fig. 8.6 shows the hplc separation of a typical extract 14 of urine, with the C levels shown on the accompanying -266- histograms. Also indicated is the level of glucuronic acid in the eluate. Those containing detectable level of glucuronic acid, are marked by a (+), with the concentration being approximately represented by the number, i.e. (+) corresponds to 20-40|jg/ml, (++) 40-60pg/ml and (+++) above 60|jg/ml. Results Excretion of radioactivity 14 The excretion of C in the urine following the oral administration of fenclofenac is essentially complete within 20 hours after dosing. The total urinary recoveries were 86.4% (48hr) in Andrew, and 59.8% (49hr) in Caspar, by this time the level of radioactivity had fallen to background. The cumulative 14 excretion of C in urine is plotted on fig. 8.4, data in appendix 7. It is clear that there is a discrepancy between the total recoveries of dose in the two animals. The reason for the lower recovery in Caspar was due to an accident during the dosing procedure. During intubation and administration of radiolabelled dose, Caspar unfortunately coughed, and a portion of the dose spilt from the tubing and funnel. It was not possible to make a quantitative assessment of the Figure 8.4. Cumulative excretion of following the oral administration of enclofenac. Key ( • ) Andrew, ( a ) Caspar. -268- dose loss at the time, but from examination of plasma levels and the difference in urinary recoveries a retrospective estimate has been made. This adjustment to the true dose is required in order to examine the plasma level-time data pharmacokinetically. 14 Plasma levels of C The plasma levels ( in dpm/ml) are plotted against time for the two animals on figure 8.5. The peak plasma level in Caspar is 67% of that seen in Andrew, and the area under the curve is 79.3% of that obtained in 14 Caspar. The difference in urinary recovery of C between the two horses is 26.6%. Therefore, it would appear that some 20-30% of the dose was lost, and to account for this, it has been assumed that Caspar received 7mg/kg (33pCi) of fenclofenac i.e. 30% loss. This assumption is used for the pharmacokinetic analyses only. Plasma pharmacokinetics Analysis of plasma revealed the major radioact component to be unchanged fenclofenac (90-95%) and the remainder (5-10%) was a discrete band more polar than fenclofenac. The decline in plasma radioactivity with time was apparently mono-exponential following the absorption phase, in the 0-12hr period after dosing. 14 When loge plasma [ C] is plotted against time (0-12hr) the regression analysis gives a correlation coefficient -269- Figure 8.5. Plasma levels of radioactivity (dpm/ml), against 14 time following the administration of C - fenclofenac p.o. DPM/ML TIME(HR) DPM/ML TIME (HR) -270- of 0.99 for both animals (data in appendix 7) from this plot the elimination half-life of total radioactivity was calculated from the slope (K) of the least square regression line in each instance. The elimination half-life (t£) is calculated from equation 1. 2 ti = loge (1) K (loge dpm/ml/min) = 0.693 x 1/K dpm/ml min"1 The apparent volume of distribution of l4C (V^jss) was calculated using equation 2. V = Total dose (dpm) (2) DISS plasma radioactivity at t=o (dpm/ml) The apparent plasma clearance (CL) of radioactivity was calculated using equation 3. CL = VDISS X K (3) These calculated values are given on table 8.2, and where appropriate are corrected for the weight of the animal and for the loss of dose in Caspar. Table. 8.2. Plasma Disposition of Fenclofenac in the Horse Andrew Caspar Plasma pharmacokinetics Plasma elimination t\ (h) 2.2 2.4 1 VD (lKg" ) 0.1 0.056 Clearance (lKg "''hr 1) 0.033 0.016 -272- The time to peak plasma level in both animals is less than one hour indicating that fenclofenac is rapidly absorbed following oral administration (figure 8.5). Urinary Metabolites Analysis of urine by tic and hplc before and after solvent extraction, revealed that there were 4 discrete radioactive fractions, in both horses. Two were major and the two minor bands were only 14 discernible following concentration of urinary C by ethyl acetate extraction. The second largest radioactive component, in the urine of both Andrew and Caspar, was identical in Rp value and retention time by hplc to authentic fenclofenac. The major metabolite was extremely polar (R-p 0.16 on system M), was labile to acid and alkali treatment, and ammonolysis, all of which caused an accompanying increase in the amount of fenclofenac, but was only partially hydrolysed following 3-glucuronidase treatment. Following streaking and development of a plate with an ethyl acetate extract, spraying with naphthoresorcinol gave an intense blue colour at the Rp value of the major -273- metabolite. This is indicative of the presence of a glucuronic acid conjugate. The nature of this glycoside was examined further, and following ammonolysis, the only sugar liberated from the metabolic sample co-chromatographed with glucuronic acid and gave the identical colour reaction. Further evidence that the major metabolite was the glucuronic acid conjugate of fenclofenac was provided by hplc analysis. Fig. 8.6 presents a chromatogram of an ethyl acetate extract 14 of urine, showing both the u.v. absorption and the C content of 1ml (0.5 min) aliquots of the eluate. There are four major radioactive peaks present, one corresponding to fenclofenac (designated d on Fig. 8.6). The same sample was made alkaline by the addition of a few drops of M NaOH, shaken and left at room temperature for 3 0 minutes. The alkali was neutralized with M HCI, and then examined by hplc. Fig. 8.7 (left hand trace) shows the resulting u.v. absorbance trace, and the radioactivity level in the eluate fractions. The three major peaks (a,b,c in fig. 8.6 left hand trace) have disappeared, with a concomitant increase in the size of the fenclofenac peak (d). On a separate occasion, the original ethyl acetate extract was injected onto hplc, the eluate collected and tested with naphthoresorcinol reagent, as described previously. In fig. 8.6, those fractions giving a positive reaction dpm x 103 5-1 4 - 3 - i to i 2- 1- h+f|>rfl. i .lhp3-ca__cc -1* absorbance 5 10 15 20 Time(min) radioactivity in eluate Figure 8.6. Hplc separation of an ethyl acetate extract of urine dpm 900- 800- 700- 600- 500- i to cn i 400- 300- 200- 100- q-n-fT-rl h-[Th r-nirpTn-rT Thrrr^ 10 15 20 25 Time radioactivity in eluate Figure 8.7. Same extract of urine shown in fig. 8.6 following treatment with alkali -276- are denoted by a ( + ), the number of symbols giving an indication of amount of glucuronic acid present in the sample + = 20-40pg/ml, ++ = 40-60Mg/ml, +++ above 60Mg/ml. These values of glucuronic acid agree on a 1:1 molar basis with the amount of fenclofenac present in each fraction, as measured 14 by C level. This data suggests that the major metabolite consists of at least 3 different glucuronic acid esters (structural isomers) in horse urine. The two minor metabolites were both unchanged by enzyme, acid and alkali treatment, and ammonolysis. The larger of the two, had identical chromatographical properties to authentic 5-hydroxy fenclofenac and this metabolite, was not examined further. The other very minor metabolite which was less polar than 5-hydroxy- fenclofenac was not identified (Rp 0.52 in system K). Table 8.3 gives the metabolic profile of fenclofenac in the two horses, and these are represented on fig 8.8 and 8.9, as a cumulative plot of the amount of each metabolite over 0-12hr period for Andrew and 0-24 hr for Caspar. Table 8.3. Urinary metabolites of fenclofenac in the horse expressed as percentage of total dose Andrew (0-12hrs) Caspar (0-24hrs) Caspar (0-24hrs) Corrected for dose Fenclofenac 13.1 8.0 11.5 Fenclofenac 49.1 70.3 glucuronide 58.8 1.7 2.0 5-Hydroxy fenclofenac 8.1 1.0 1.5 Unidentified 3.2 -278- Figure 8.8. The urinary metabolites of fenclofenac, the cumulative totals expressed as percentage of dose ANDREW 70 i 60 - FENCLOFENAC QLUCURONIDE 50 - gj co o 40 - o ok 30 20 - FENCLOFENAC 10 - 5-HYDROXY FENCLOFENAC 0 0 8 10 12 Time(Hrs) - 2 t 9 - Figure 8.9. The urinary metabolites of fenclofenac, the cumulative totals CASPAR expressed as percentage of dose -280- Discussion 14 Following an oral dose of lOmg/kg of C- fenclofenac, the radioactivity was eliminated very rapidly by both ponies. The elimination half-life was found to be 2.2 and 2.4 hours, respectively for the two animals, which is shorter than in other species previously examined. Brewster et al. (1980) found elimination half-lives of 5.5hr (dog), 10.5hr (rat), 14.1hr (baboon) and 24hr (guinea pig). The low volume of distribution is consistent with the drug being confined to plasma (as calculated from the weight of the horse), and would indicate the drug is highly plasma protein bound. This is found in other species, where there is a variation in the degree of binding, e.g. 90% in the rat to 99.5% in man (Brewster et al,1980). Analysis of plasma by tic showed the major radioactive component was fenclofenac (90-95%), the remainder corresponding in Rp value to fenclofenac glucuronide (5-10%) The major urinary metabolite of fenclofenac was the ester glucuronide (58.7% and 49.1% of the dose). This was also the major metabolite in the rat, rabbit, guinea pig, baboon and man (Greenslade e£ al 1980). Glucuronic acid conjugation has been seen previously as a metabolic pathway in the horse, as a minor metabolite of salicylic acid (Chapter 3), 2-naphthylacetic acid (Chapter 4 -281- % and benzoic acid (Chapter 5). Extensive glucuronidation has been seen in the metabolism of chloral hydrate and trichloroethanol to urochloric acid (trichloroethyl- glucuronide), where over 60% of the dose was eliminated as this conjugate (Alexander et al., 1967). The hplc data suggests that several glucuronic acid esters occur in horse urine. It is known that bio- synthetic 1-acyl glucuronides can undergo intramolecular rearrangement at mild alkaline pH whereby the acyl function migrates to positions 2,3 and 4 of the glucuronic acid moiety (Blanckaert e^t £il. , 1978). It has been demonstrated that products resulting from this rearrangement are resistant to hydrolysis by B-glucuronidase (Illing and Wilson, 1981, Sinclair and Caldwell, 1981, 1982). Such a rearrangement would be favoured by the high pH (pH 8-9) of the horse urine and is consistent with the finding that the major excretion products are totally hydrolysed in alkaline conditions but only partially hydrolysed by B-glucuronidase. It appears that B-glucuronidase-resistant conjugates are formed from fenclofenac glucuronide in urine during collection and storage. This partial resistance had been noted in the glucuronides formed with salicylic, 2-naphthylacetic and benzoic acids, but due to their minor nature (in terms of percentage dose), they were not investigated further. ft -282- The instability of ester glucuronides in alkali has been demonstrated, which is of consequence in an animal like the horse, where the urine voided is in the pH range 7-9 under these experimental conditions. The amount of the ester glucuronide measured in the urine may be an underestimate of that produced biosynthetically as hydrolysis back to parent drug may occur in the bladder (some thoroughbreds only urinate twice a day), and in the period of time before the urine samples can be frozen, particularly overnight. One minor metabolite (3.2% and 1% of the dose) has not been identified, but it is likely to be the dihydroxylated metabolite of fenclofenac. Both mono-hydroxy (8.1%, 1.7% of dose in this experiment) and di-hydroxy fenclofenac have been found previously in the urine of guinea pig, baboon and man following an oral dose of fenclofenac (Greenslade et al., 1980). ft -283- Chapter Nine : Discussion ft -284- This thesis presents an investigation into the conjugation reactions in the horse. There is little systematic information concerning the metabolic fate of foreign compounds in this species. The reasons for this paucity of information are partly association with the problems of working with such a large animal, necessitating specialised and expensive facilities for housing and sample collection. The need for establishing a pattern of metabolism both quantitatively and qualitatively has recently become apparent with the growing use of drugs, which are also endogenous to horse urine, for example, cortisone and testosterone. A topical example of this is the use of anabolic steroids based upon 19-nortestosterone, which is now thought to be naturally occurring. This presents a new challenge in determining whether a particular substance in the urine is endogenous or exogenous in origin. There are two possible ways of solving this problem. Firstly, establishing the normal range of concentration of such substances in urine such that when concentrations are found in excess of this, ft -285- it can be confidently attributed to be from an external source. The second way is to measure both drug and metabolite(s) level in the urine and obtain a ratio. It is possible after drug administration that this ratio will change, if for example, the additional dose leads to saturation of a particular metabolic route. There is a need to expand research in this area, and so avoid misleading and inaccurate headlines, such as "Horse makes his own dope" which have become prevalent recently in the press. In view of the great sums of money invested in horse racing, in prize money and in blood stock, any contribution to the metabolic understanding of the horse will always have more than just academic interest. The drugs and compounds selected were designed to elicit a variety of conjugation reactions and establish the pathways of metabolism favoured in the horse. The following pathways have been observed and quantified, and serve to demonstrate the diversity of metabolic reactions found in the horse. Glycine conjugation Glycine conjugation has been established as a major route of metabolism, and is quantitatively important in the metabolism of phenylacetic, benzoic and 2-naphthyl- acetic acid. ft -286- It is interesting to note that salicylic acid is not conjugated with glycine to any great extent by the horse (>0.4% of the dose); salicyluric acid is a major metabolite in many other species. Although the glycine acyl transferase enzyme (from mammalian and bovine systems) is known to be twice as active . with benzoyl CoA than salicyl CoA (Tishler and Goldman, 1970), this difference would not account for the observed difference in glycine conjugation of these two acids, and it is more likely that the horse is defective in a form of the enzyme responsible for the activation of the acid, the ATP-dependent acid : CoA ligase. As the largest proportion of salicylic acid is excreted unchanged, a simpler explanation for this observation may be that excretion of this acid by the kidneys is very rapid, favoured by the alkaline pH of urine (pH 8-9). Glucuronic acid conjugation Although the conjugation of many drugs and foreign compounds with glucuronic acid has been observed previously, the evidence has been inconclusive relying on non-specific enzyme hydrolyses, and quantification may have been imprecise. In this study the versatility of this conjugation reaction was demonstrated, being seen with salicylic acid (acyl and ether glucuronides), benzoic acid, 2-naphthyl-acetic acid and the anti-inflammatory drug fenclofenac. ft -287- The analysis of the acyl glucuronide of each of these acids was complicated in that the sugar-aglycone bond was resistant to attack by the specific enzyme, 3-glucuronidase. This hydrolysis, together with specific inhibition and positive and negative controls (see Chapter 2), is accepted as being excellent evidence for the presence of a glucuronic acid conjugate. With fenclofenac, the chromatographic analysis separated 3 drug-related naphthoresorcinol positive peaks, which all yielded the parent acid following alkali hydrolysis. The exact structure of these metabolites could not be examined further, because they were present in low concentrations and the urine contains many interfering substances. More detailed studies of this phenomenon with other drugs explain the observations. Blanckaert et a!L (19 78) showed that 1-acyl glucuronides can undergo intramolecular rearrangement at mild alkaline pH, where the acyl function migrates to positions 2, 3 and 4 of the glucuronic acid group. This has now been shown to occur with drugs, for example, isoxepac (Illing and Wilson,1981) and clofibric acid (Sinclair and Caldwell,1982). The latter study has shown that biosynthetic 1-0-acyl glucuronides rearrange to form different isomers in a base-catalysed reaction, to yield enzyme resistant forms. This rearrangement is specific to acyl glucuronides. ft -288- Conjugation with Taurine Taurine conjugation has been established as an important route of metabolism for the first time in the horse. 2-Naphthylacetic acid and phenylacetic acid (in one animal) were conjugated with taurine to a small extent, and with isoxepac, the taurine conjugate was the major metabolite. The addition of a two carbon fragment A major discovery during the course of this study, was the finding of novel metabolites of benzoic acid. These are formed by the addition of a 2-carbon fragment to the carboxyl carbon to form 3-hydroxy 3-phenylpropionic acid, and also possibly 3-keto- 3-phenylpropionic acid, as its decarboxylation product, acetophenone was found in the urine. When the metabolism 13 of phenylacetic acid was investigated, C NMR analysis of a crude urine extract suggested that analogous products have been formed as minor metabolites, although this could not be confirmed by chromatography or mass spectrometry. There was a lower recovery of dose with phenylacetic acid, which may suggest that if such metabolites have been formed they are still in the body (e.g. in the fat depots) or excreted via the bile. Formation of these metabolites is of interest because it would appear that the metabolism of xenobiotics can be ft -289- associated with normal lipid metabolism in the body, examples of these types of reactions are listed on table 9.1. Four novel metabolites of 5(41-chloro-n-butyl) picolinic acid have been described (see fig. 9.1), all of which arise from the addition of a 2-carbon fragment. These are formed extensively, being 50% of the total 0-24hr urinary radioactivity in rats, and were also formed in the guinea pig, mice and man (Miyazaki e£ al, 1976). Addition of multiple 2-carbon units to a carboxylic acid group have been observed in a series of papers about the metabolism of cycloprate (Quistad et al, 1976, a,b,c). In the rat, two-thirds of residual tissue radiolabel was found as w-Cyclopropyl fatty acids, the major being 11-cyclopropylundecanoic acid, 13-cyclopropyl- tridecanoic acid and 15-cyclopropylpentadecanoic acid (18% of dose). In the cow, 14C residue in milk was found as triacylglycerols and 0-(cyclopropylcarbonyl) carnitine. Carnitine is the carrier molecule for long chain fatty acids across membranes and in the dog this carnitine conjugate was retained in the muscle (50% of dose). 4 Table 9.1. Reactions of xenobiotic metabolism and lipid biochemistry Functional Group Reaction Example Product found in Aldehyde Acrylic acid synthesis Furfural Urine (addition of a 2-carbon unit) Carboxylic acid Addition of a single 5(41-chloro-n-butyl) 2-carbon unit picolinic acid, benzoic acid Urine to Addition of multiple Cycloprate Tissue lipid g 2-carbon units Incorporation into 3-Phenoxybenzoic acid, triglycerides 4-benzyloxybenzoic acid Tissue lipid Chain elongation and Cycloprate incorporation into triglycerides Tissue lipid Carnitine conjugation Cycloprate Urine, muscle 1 6 Alcoholic hydroxyl Fatty acid esterification A and A -tetrahydro cannabinol (THC), pyridopyridines, dipyridamol, mopidamol etofenamate Urine and faeces (See Caldwell and Marsh, 1983) ft -291- ckch2)4^ N COOH COOH COOH COOH CHO CH? ^m^CCHoCOOH N z N L I L I II CH2 0 CH2 COOH COOH H00C(CH2) N CH=CHC00H Figure 9.1. Metabolism of 5-(41-chloro-n-butyl) picolinic acid in the rat -292- ft Implications of the formation of these metabolites to the analysis of urine for drugs and drug metabolites The conjugation pathways observed have important implications in the screening of urine samples from race horses for drugs, in that there is a variety of metabolites which can be formed, all leading to an increase in water solubility of the parent drug. This change in the properties of the exogenous material makes detection more difficult, as the primary analytical step is solvent extraction, to separate the acidic, basic and neutral components. Even under conditions favouring extractions with the commonly used solvents (e.g. ether), at best partial extraction of the conjugate will be achieved. In addition, the analysis of the extract is further complicated by the many endogenous urinary components which have similar chromatographic properties to the conjugates. It has been a problem throughout these analyses to separate endogenous hippuric acid from a drug-glycine conjugate, particularly as hippuric acid is present in much greater quantities than such metabolites. Taurine conjugates are particularly polar, demonstrated by the properties of the taurine conjugate of isoxepac, which was only extractable as its tetrabutyl ammonium ft -293- ion pair. Routine solvent extraction will not remove a taurine conjugate, and analysis is complicated in that they are unstable in the mass spectrometer and are difficult to derivatise. Although 4H-NMR and IR can give characteristic information for identification these techniques usually require > lmg of sample, for reliable results. The rearrangement of the glucuronic acid esters of carboxylic acids has been discussed, and this may have great relevance to dope detection in the horse, as the urinary pH generally favours such a rearrangement, and B-glucuronidase hydrolyses are often used in drug screening programmes (Combie et al, 1982). These enzyme resistant forms will remain unaltered following incubation, and remain in the aqueous phase when conditions are selected for extraction of the parent drug. The conjugation reactions in the horse are only a small part of the pharmacology of this species, and there are many other relevant areas un-investigated such as : enzyme induction, pharmacokinetics, influence of different routes of administration, biliary elimination of drugs, and inter-animal differences in drug effect and metabolism. ft -294- Any further understanding to the pharmacological responses of this animal, will assist veterinarians, and also contribute to the improvement of analytical techniques for the detection of drugs. ft -295- APPENDICES ft -296- APPENDIX ONE Urinary and plasma data following salicylic acid administration -297- 14 Table Al.l. Plasma data ( C levels) for Ginger (pony) following salicylic acid administration. 14 14 Sample number Time (hour) [ C]dpm/ml. loge[ C] 1 0.17 360 5.89 2 0.33 987 6.90 3 0.50 1398 7.24 4 0.61 1656 7.41 5 0.83 1822 7.51 6 1.00 1864 7.53 7 1.17 1870 7.54 8 1.33 1873 7.54 9 1.50 1825 7.51 10 1.67 1665 7.42 11 i . 83 1627 7.39 12 1.99 1530 7.33 13 2.27 1389 7.24 14 2.5 1263 7.14 15 2.76 1181 7.07 16 3.02 1104 7.00 17 3.27 912 6.82 18 3.53 935 6.84 19 3.75 874 6.77 20 4.00 803 6.69 21 4.50 720 6.58 22 5.01 614 6.42 23 5.53 566 6.34 24 6.02 509 6.23 25 7.01 400 5.99 26 8 .00 318 5.76 27 9.01 255 5.54 28 10.00 211 5.34 29 11.00 172 5.15 30 12.00 145 4.98 31 24.05 23 - 32 24.95 18 - 33 30.82 7 • % of total in each sample r I Sample Time %dose Cumulative Salicylic Gentisic Salicyluric Salicyl Number (hour) %dose acid acid acid glucuronides 1 1.5 7.3 7.3 . 95.5 2.7 0.4 1.6 2 2.5 24.5 31.8 96.3 2.2 0.1 1.3 3 3.5 19.7 50.9 96.0 2.2 0.1 1.7 4 5 9.8 60.7 95.5 2.5 0.3 1.7 5 6 8.3 69.0 93.9 2.6 1.5 1.9 6 7 5.6 74.6 94.8 2.8 0.3 2.1 7 8 3.7 78.3 94.4 3.0 0.2 2.4 8 9 3.9 82.2 93.3 2.9 1.4 2.4 9 10 2.6 84.8 93.5 3.0 1.0 2.4 10 11.5 2.2 87.0 92.5 3.9 1.0 2.6 11 13 2.0 89.0 93.0 3.6 1.4 2.1 12 14 1.5 90.5 94.7 2.5 0.5 1.9 13 15 0.9 91.4 92.3 1.0 1.5 2.1 14 16 1.0 92.4 92.4 3.9 0.2 2.2 15 17.5 0.9 93.3 93.6 3.0 0.5 2.1 16 19.5 0.8 94.1 91.7 4.0 0.3 3.0 17 20.5 0.6 94.7 93.2 3.0 0.8 2.3 18 24 0.7 95.4 90.1 4.2 1.1 2.9 19 26.5 0.4 95.8 87.6 4.5 2.5 3.1 20 29.5 0.2 96.0 87.0 5.6 1.6' 3.8 Table A1.2. Urinary data for Ginger (pony) following salicylic acid administration ft -299- Table A1.3. Urinary data for calculation of biological half-life for Ginger (pony)* following salicylic acid administration. Time of sample % dose excreted l°Se dose excreted midpoint, (hour) per hour per hour) 0.75 4.9 1.59 2.0 24.5 3.20 3.0 19.7 2.95 4.25 6.5 1.87 5.5 8.3 2.12 6.5 5.6 1.72 7.5 3.7 1.31 8.5 3.9 1.36 9.5 2.6 0.95 10.75 1.5 0.41 12.25 1.3 0.26 13.5 1.5 0.41 14.5 0.9 -0.11 15.5 1.0 0 16.75 0.6 -0.51 18.5 0.4 -0.92 20.0 0.6 -1.51 22.25 0.2 -1.61 25.25 0.2 -1.61 27.0 0.07 -2.66 This data relates to figure Al.l. # % of total in each sample Sample Time %dose Cumulative Salicylic Gentisic Salicyluric Salicyl lumber (hour) %dose acid acid acid glucuronides 1 1 9.5 9.5 94.6 2.2 0.3 1.3 2 2 29.7 39.2 94.2 2.8 0.1 1.5 3 3 17.0 56.2 94.4 3.1 0.1 1.9 4 5 8.1 64.3 92.9 3.7 0.4 2.2 5 5.5 5.3 69.6 92.6 4.4 0.2 2.6 6 6 4.6 74.2 93.4 4.0 0.2 2.4 7 7.5 6.9 81.1 92.2 4.4 0.5 2.6 8 9.5 4.6 85.7 90.2 5.1 0.9 2.9 i 91.8 4.5 0.6 2.6 CO 9 10.5 3.9 89.6 o 0 10 13 3.9 93.5 92.2 4.6 0.5 2.7 1 11 14 1.8 95.3 89.0 6.0 1.1 2.7 12 15 1.1 96.4 85.8 7.7 0.6 4.1 13 16 0.6 97.0 82.6 7.4 2.7 3.8 14 17.5 0.1 97.1 88.7 6.6 1.0 2.8 15 19.0 0.9 98.0 88.8 6.2 1.4 2.9 16 21.0 0.2 98.2 85.8 6.4 3.0 3.5 17 22.5 0.2 98.4 82.9 9.1 1.8 5.2 18 23.5 0.3 98.7 82.1 9.7 1.7 4.9 Table A1.4. Urinary data for Shepherd Boy (thoroughbred) following salicylic acid administration. ft -301- Table A1.5. Urinary data for calculation of biological half-life for Shepherd Boy (thoroughbred) following salicylic acid administration. Time of sample %dose excreted loge (%dose excreted midpoint,(hour) per hour per hour) 0.5 9.5 2.25 2.0 14.9 2.7 3.5 17.0 2.83 4.5 8.1 2.09 5.25 10.6 2.36 5.75 9.2 2.22 6.75 4.6 1.53 8.5 2.3 0.83 9.0 3.9 1.36 11.75 1.6 0.47 13.5 1.8 0.59 14.5 1.1 0.09 15.5 0.6 -0.51 16.75 0.06 -2.81 18.25 0.6 -0.51 20.0 0.1 -2.30 21.75 0.1 -2.30 23.0 0.3 -1.20 This data relates to figure Al.2. ft -302- Figure Al.l. Plot of loge % dose excreted per hour Figure A1.2. Plot of loge % dose excreted per hour ft -304- Kinetics of urinary drug elimination The urinary half-life of salicylic acid is obtained from the plot of loge (% dose excreted per hour) against the midpoint time of the sample, plotted on figure Al.l (Ginger) and figure A1.2 (Shepherd Boy). The best line for the data is derived from linear regression using the least squares method. From the slope (K), the biological half-life (t^) is calculated as : K = loge 2 . . t\ = 0.693 K Therefore for Ginger 0.693 ** = 07193 = 3'6hr and for Shepherd Boy t = = 2. 9hr 0.238 ft -305- APPENDIX TWO Urinary data following benzoic acid administration -306- 14 Table A2.1. Urinary elimination of C following benzoic acid administration to Caspar (pony) Sample number Time (hour) %dose Cumulative recovery of dose (%) 1 1.5 69.1 69.1 2 3.5 22.9 92.0 3 4.0 3.7 95.7 4 5.0 216 98.2 5 6.5 1.8 100.0 6 7.5 0.7 100.7 7 9.5 0.6 101.3 8 11.0 0.4 101.7 9 12.5 0.1 101.8 10 13.5 0.1 101.9 -307- 14 Table A2.2. Urinary elimination of C following benzoic acid administration to Floral Song (thoroughbred) Sample number Time(hour) % dose Cumulative recovery of dose (%) 1 11.0 87.7 87.7 2 21.0 10.0 97.7 3 33.0 5.6 103.3 4 45.0 0.7 104.0 ft -308- APPENDIX THREE Urinary data following phenylacetic acid administration -309- 14 Table A3.1. Urinary elimination of C following phenylacetic acid administration to Caspar (pony). Sample number Time % dose Cumulative recovery (hour) of dose (%) 1 0.5 0.1 0.1 2 2.2 66.3 66.4 3 4.2 12.8 79.2 4 5.2 3.3 82.5 5 6.2 1.2 83.7 6 9.0 1.1 84.8 7 10.0 0.5 85.3 8 11.0 0.3 85.6 9 13.0 0.2 85.8 10 15.0 0.2 86.0 11 17.0 0.04 86.0 12 19.0 0.2 86.2 13 21.0 0.3 86.5 14 22.5 0.2 86.7 15 23.8 0.1 86.8 16 24.7 0.03 86.9 17 27.0 0.06 86.9 18 28.0 0.03 87.0 19 29.0 0.03 87.0 -310- 14 Table A3.2. Urinary elimination of C following phenylacetic acid administration to Floral Song (thoroughbred). Sample number Time %dose Cumulative recovery of (hour) dose .(%) 7 11 65.3 65.3 2 22 19.1 84.4 3 34 0.4 84.8 4 46 0.1 84.9 ft -311- APPENDIX FOUR Urinary data following 2-naphthylacetic acid administration ft -312- Table A4.1. Urinary elimination of 14C following 2-naphthylacetic acid administration to Ginger (pony). Sample number Time (hour) %dose Cumulative recovery of dose (%) 1 10 0 2 3 2.4 2.4. 3 6 4.3 6.7 4 10 5.7 12.3 5 14 2.6 14.9 6 22 10.4 24.3 7 24 3.1 28.3 8 26 2.7 31.1 9 29 2.2 33.3 10 30 1.2 34.5 11 31 1.2 35.7 12 35 3.2 38.9 13 46 5.9 44.8 14 49 2.8 47.6 15 52 1.7 49.3 16 55 1.8 51.0 17 60 2.0 53.0 18 70 3.7 56.7 19 73 2.4 58.6 20 75 1.2 60.3 21 78 1.1 61.4 22 84 2.4 64.8 23 92 3.3 67.1 24 100 1.2 68.3 25 102 0.9 69.2 26 H8 3.5 72.7 27 130 0.8 73.5 28 142 2.4 75.1 29 146 0.9 75.8 30 149 0.5 76.3 31 166 2.5 78.8 32 170 0.3 79.1 33 171 0.6 79.7 34 193 0.4 82.1 "31.3" 14 Table A4.2. Urinary elimination of C following 2-naphthylacetic acid administration to Shepherd Boy (thoroughbred). Sample number Time (hour) %dose Cumulative recovery of dose (%) 1 2 0.7 0.7 2 4 0.7 1.4 3 6 1.1 2.6 4 8 1.3 3.9 5 10 0.7 4.7 6 12 1.2 5.8 7 14 1.0 6.8 8 15 1.9 8.7 9 17 0.4 9.1 10 19 1.2 10.3 11 20 0.9 11.2 12 21 0.5 11.7 13 24 1.2 12.9 14 25 1.5 14.4 15 28 1.8 16.1 16 29 0.2 16.4 17 31 1.3 17.7 18 33 1.3 19.0 19 36 1.4 20.4 20 37 1.0 21.3 21 44 0.2 21.5 22 46 0.6 22.2 23 47 0.7 22.9 24 51 1.3 24.2 25 53 1.0 25.2 26 56 1.3 26.5 27- 63 2.7 29.2 28 65 0.9 30.1 29 70 1.0 31.1 30 73 1.2 32.2 31 76 1.3 33.6 32 79 0.9 34.5 continued over page ft -314- Table A4.2. continued Sample number Time (hour) %dose Cumulative recovery of dose (%) 33 82 1.4 - 35.9 34 94 2.8 38.7 35 98 0.6 39.3 36 101 0.7 40.0 37 103 0.5 40.6 38 105 0.7 41.3 39 118 2.0 43.2 40 130 1.2 44.3 41 140 3.7 48.2 42 142 0.2 48.4 43 144 0.4 48.8 44 148 0.5 49.3 45 150 0.5 49.8 46 166 3.0 52.8 47 170 1.0 53.8 48 173 0.5 54.3 49 8 days 4.1 58.4 5 0 9 days 2.1 60.5 51 10 days 2.9 63.4 52 11 days 2.5 65.9 53 12 days 1.8 67.7 54 13 days 1.5 69.2 ft -315- APPENDIX FIVE 13 C-NMR spectra of compounds related to phenylacetic acid 4 i CO rt ai i JJL _j I I 1 1 J 1 I I I | 210 180 150 120 50 30 10 ppm 13 Figure A5.1. C-NMR spectrum of synthetic phenylacetic acid; in D2O : (CD3>3CO 85:15 i 00 i —i 1 i 210 190 170 150 J 1 130 50 30 13 ppm Figure A5.2. C-NMR spectrum of synthetic phenylacetylglycine in D20:(CD3)2CO 85:15 i CO (—4 00 i 40W 210 180 140 100 40 20 ppm 13 Figure A5.3. C-NMR spectrum of synthetic hippuric acid, in D20 : (CD3)2CO 85:15 « i CO h-» CD i 120 50 210 140 130 ppm 13 Figure A.5.5. C-NMR Spectrum of phenylacetone in D20 i 00 CO ft i l i j L 215 210 135 130 125 55 50 35 30 25 13 ppm 1 Figure A.5.6. C-NMR spectrum of phenylacetone in D20, H noise decoupled « i CO to DO i j l 200 150 110 40 20 0 PPM 13 Figure A.5.7. C-NMR spectrum of synthetic styrylacetic acid in D20: (CD3)2CO 65:35 4 i 00 CO 00 1 • ••im^ii iiiJi hy^ fttii i ii -j i— 200 150 100 40 20 ppm 13, Figure A5.8. C-NMR spectrum of 4-phenyl-butyric acid, in D20 : (CD3>2CO 65:35 Inset: scale expanded between 120 and 130 ppm. « 210 180 Figure A5.9. 13 C-NMR Spectrum of synthetic sodium 6-hydroxy-propionate, in D20 ppm (CD3)2 CO, 85 : 15 4 i CO to Cn i J L 30 20 200 170 70 60 40 210 ppm 13 Figure A5.10. C-NMR spectrum of B-hydroxy propionic-acid in D20 : (CD3)2CO 85^15 -326- 14 C-NMR Analysis : assignment of signals 1. Phenylacetic acid r^^CH2C00H b a,b 127.4,127.9 ppm c 125.9 ppm d 132.6 ppm e 39.2 ppm f 174.9 ppm 2. Phenylacetyl glycine ^CH2CONHCH2COOH a,b 127.4, 127.7 ppm c 125.9 ppm d 133.2 ppm e 39.8 ppm f 171.7 ppm g 40.6 ppm h 173.5 ppm ft -327- 3. Hippuric acid aCONHCH2COOH A^ b a, b 125. 7, 127 .4 ppm c, d 131. 0, 131 .3 ppm e 169. 2 ppm f 40.2 ppm g 171. 9 ppm 4. Phenylacetyl taurine CH2C0NHCH2CH2S020H b a,b 127.4, 127.8 ppm c 125.8 ppm d 133.4 ppm e 33.9 ppm f 172.7 ppm g»h 23.6, 24.0 ppm ft -328- 5. Phenylacetone (in D20) 0 a b ^^CHoCCHo | Af g D b a,b : 128.9, 129.8 ppm c : 127.3 ppm d : 134.3 ppm e : 49.8 ppm f : 213.9 ppm g : 29.1 ppm 6. Styrylacetic acid /FAD. CH=CHCH2COOH b a,b : 124.7, 127.3 ppm c : 120.7 ppm d : 131.7 ppm e : 135.4 ppm f : 126.2 ppm g : 36.4 ppm h : 173.4 ppm ft -329- 7. 4-Phenyl butyric acid br-^d,CH2CH2CH2COOH b a,b : 127.0, 127 . 0 ppm c : 124.6 ppm d : 140.1 ppm e : 24.9 ppm f,g : 31.7, 33.0 ppm h : 175.6 ppm 8. Sodium B-hydroxypropionate CH3CH0HC00Na a : 21.9 ppm b : 65.7 ppm c : 18 0.2 ppm ft -330- APPENDIX SIX Urinary data following isoxepac administration ft -331- Table A6.1. Urinary elimination of 14C following isoxepac administration to Andrew (pony) Sample number Time (hour) %dose Cumulative recovery of dose (%) 1 1.0 5.3 5.3 2 2.0 16.8 22.1 3 4.0 9.5 31.6 4 4.5 8.0 39.6 5 5.0 5.7 45.3 6 6.5 7.3 52.6 7 7.5 2.3 54.9 8 9.0 6.1 61.0 9 10.0 4.4 65.4 10 11.0 1.6 67.0 11 12.5 1.5 68.5 12 14.5 2.4 70.9 13 15.5 2.2 73.1 14 17.5 1.8 74.9 15 18.5 3.0 77.9 16 19.5 2.0 79.9 17 22.5 3.3 83.2 18 25.0 2.0 85.2 19 26.5 1.4 86.6 20 28.5 1.1 87.7 21 29.5 0.7 88.4 22 30.0 1.1 89.5 23 33.0 0.6 90.1 24 34.0 0.4 90.5 25 35.0 0.4 90.9 26 36.0 0.2 91.1 27 37.5 0.3 91.4 28 38.5 0.2 91.6 29 39.5 0.3 91.9 30 40.5 0.2 92.1 continued over page -332- Table A6.1. continued Sample number Time (hour) %dose Cumulative recovery of dose (%) 31 41.5 0.2 92.3 32 43.5 0.2 92.5 33 45.0 0.2 92.7 34 46.5 0.04 92.7 35 48.0 0.2 92.9 36 50.5 0.3 93.2 37 52.0 0.1 93.3 38 54.5 0.2 93.5 39 70.5 5.2 98.7 40 72.0 0.04 98.8 ft -333- Table A6.2. Urinary data following isoxepac administration, to Andrew (pony) (see figure A6.1.) 14 Sample number Time loge (percentage C (midpoint,hour) excreted per hour) 1 0.5 16.46 2 1.5 17.62 3 3.0 16.35 4 4.25 17.57 5 4.75 17.22 6 5.75 16.13 7 7.0 15.63 8 8.25 16.20 9 9.5 16.27 10 10.5 15.27 11 11.75 14.77 12 13.5 14.97 13 15.0 15.58 14 16.5 14.71 15 18.0 15.90 16 19.0 15.48 17 21.0 15.61 18 23.75 14.59 19 25.75 14.70 20 29.0 14.22 21 29.75 14.42 22 29.0 15.55 23 31.5 13.26 24 33.5 13.88 25 34.5 13.90 26 35.5 13.19 27 36.75 13.23 28 38.0 13.01 29 39.0 13.55 30 40.0 13.30 continued over page ft -334- Table A6.2. continued 14 Sample number Time loge (percentage C (midpoint,hour) excreted per hour) 31 41.0 13.04 32 42.5 12.59 33 44.25 12.64 34 45.75 11.15 35 47.25 12.56 36 49.25 12.79 37 51.25 12.47 38 53.25 12.10 39 62.25 11.41 40 71.0 10.88 -335- 14 Table A6.3. Urinary elimination of C following isoxepac administration to Shepherd Boy (thoroughbred). Sample number Time (hour) %dose Cumulative recovery of dose (%) 1 2.0 7.6 7.6 2 4.0 9.0 16.6 3 4.5 6.1 22.7 4 6.5 9.2 31.9 5 9.0 10.7 42.6 6 10.0 4.5 47.1 7 11.0 0.4 47.5 8 11.5 5.5 53.0 9 16.5 1.3 54.3 10 18.5 2.1 56.4 11 22.5 4.6 61.0 12 24.0 1.3 62.3 Estimated 1.1 63.4 13 28.5 2.4 65.8 14 29.5 0.4 66.2 15 31.0 0.6 66.8 Estimated 7.1 73.9 16 48.0 0.4 74.3 17 50.0 0.3 74.6 18 52.5 0.3 74.9 19 53.5 0.4 75.3 20 55 0.2 75.5 Estimated 1.2 76.7 21 70 0.2 76.9 22 72 0.1 77.0 ft -336- Table A6.4. Urinary data following isoxepac administration, to Shepherd Boy (thoroughbred) see figure A6.2. lo Sample number Midpoint time &e (%dose excreted (hour) per hour) 1 1.0 16.16 2 3.0 16.32 3 4.25 16.31 4 5.5 16.35 5 7.75 16.27 6 9.5 16.32 7 10.5 8 11.25 10-75 16.17 9 14.0 13.50 10 17.5 14.85 11 20.5 14.95 12 23.25 14.68 13 A 26. 13 - 14 29. 0 13.94 15 30.25 13.94 16 B 39.6 - 17 49.0 12.76 18 51.25 12.59 19 53.0 13.98 20 54.25 12.92 21 C 61.4 - 22 71.0 11.93 ft -337- Figure A6.1. Log % Dose Hr 1 plotted against midpoint time Andrew : Isoxepac Administration %Dose.HH 18 40 50 60 70 80 Time(Hr) -338- Figure A6.2. Log % dose excreted per hour plotted 17 H against midpoint time. Shepherd Boy : Isoxepac Administration 16 4 154 or 14- ZD CD CC lu cl CD uj i— lu CC 134 lj X LU LU U1 CD CD < A> 124 id o 11 10 20 30 40 50 60 70 80 MIDPOINT TIME (HR). % -339- Calculations to account for sample losses : Shepherd Boy Figure A6.2., which is the plot of the natural logarithm of the percentage dose excreted per hour against the midpoint time for each sample, is used to account for the lost samples. The three samples known to be lost are designated A,B and C. The amount of each is calculated from the straight line obtained by regression analysis using the least squares method. For Sample A In time period : the loge (%dose excreted per hour) changes from 14.8 to 14.5 . . this represents a difference of 693,686 dpm per hour. The collection time was 4.5 hr. . . total lost = 3,121,587 dpm = 1.1% of the dose. Similarly Sample B In time period : the loge (%dose excreted per hour) changes from 14.33 to 13.19 The collection period was 17 hours, thus this corresponds to a total of 19,342, 532 dpm = 7.1% of dose % -340- Sample C The loge (%dose excreted per hour) changes from 12.73 to 11.72 representing 214,722 dpm hr"1. The time period of collection was 15hr. to give a total of 3,220,830 dpm = 1.2% of the dose. % -341- APPENDIX SEVEN Urinary and plasma data following fenclofenac administration 0 -342- 14 Table A7.1. Plasma data ( C levels) for Andrew (pony) following fenclofenac administration. 14 Sample number Time (hr) dpm/ml loge( C, dpm/ml) 1 0.27 2271 7.73 2 0.5 3107 8.04 3 0.75 2746 7.92 4 1.0 2497 7.82 5 1.27 2312 7.75 6 1.5 2158 7.68 7 1.75 2012 7.61 8 2.0 1686 7.43 9 2.5 1672 7.42 10 3.0 1514 7.32 11 3.5 1464 7.29 12 4.0 1341 7.20 13 5.0 993 6.90 14 6.0 695 6.54 15 7.0 506 6.23 16 8.0 320 5.77 17 9.0 201 5.30 18 10.0 139 4.93 19 11.0 108 4.68 20 12.0 89 4.49 21 24.0 26 3.26 22 32.0 29 3.37 % -343- Table A7.2. Urinary elimination of 14C following fenclofenac administration to Andrew (pony) Sample number Time(hr) %dose Cumulative recovery of dose (%) 1 1.5 15.0 15.0 2 2.5 10.8 25.8 3 3.5 12.6 38.4 4 5.0 19.5 57.9 5 6.5 10.9 68 .8 6 8.0 5.7 74.5 7 9.0 4.0 78.5 8 10.0 1.8 80.3 9 11.0 1.6 81.9 10 12.0 1.6 83.5 11 14.0 0.7 84.2 12 16.0 0.4 84.6 13 19.0 0.5 85.1 14 21.0 0.3 85.4 15 23.0 0.2 85.6 16 24.5 0.1 85.7 17 27.0 0.1 85.8 18 29.0 0.1 85.9 19 30.0 0.03 20 31.5 0.05 21 34.0 0.04 86.0 22 35.0 0.03 23 35.5 0.03 24 47.0 0.3 86.3 25 48.0 0.03 84.4 -344- 14 Table A7.3. Plasma data ( C levels) for Caspar (pony) following fenclofenac administration 14, Sample number Time(hr) dpm/ml loge ( C, dpm/ml) 1 0.25 10 2.30 2 0.5 863 6.7.6 3 0.75 2147 7.67 4 1.0 2191 7.69 5 1.25 2132 7.66 6 1.5 2102 7.65 7 1.75 1949 7.58 8 2.0 1962 7.58 9 2.5 1864 7.53 10 3.0 1673 7.42 11 3.5 1536 7.34 12 4.0 1315 7.18 13 5.0 967 6.87 14 6.0 694 6.54 15 7.0 488 6.19 16 8.0 338 5.82 17 9.0 222 5.40 18 10.0 186 5.23 19 11.0 138 4.93 20 12.0 98 4.58 21 24.0 29 3.34 22 32.0 21 3.04 0 -345- 14 Table A7.4. Urinary elimination of C following fenclofenac administration to Caspar (pony) Sample number Time(hr) %dose Cumulative recovery of dose (%) 1 2.75 19.1 19.1 2 4.0 9.7 28.8 3 5.5 11.2 40.0 4 8.5 1.2 41.2 5 10.0 4.5 45.7 6 11.5 1.4 47.1 7 13.5 0.2 47.3 8 16.0 0.3 47.6 9 17.5 0.7 48.3 10 21.5 0.5 48.8 11 22.5 0.04 12 25.0 0.2 49.0 13 27.0 0.2 49.2 14 28.0 0.07 49.3 15 30.0 0.1 49.4 16 32.5 0.07 17 40 0.1 49.5 18 48 0.2 49.7 19 49 0.04 49.8 % -346- _ 14 Figure A7.1. 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