1 Pharmacokinetic and Duration Of

Home , Fenamic acid, Phenylbutazone

PHARMACOKINETIC AND DURATION OF CYCLOOXYGENASE INHIBITION STUDIES OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE IN ATHLETIC THOROUGHBRED HORSES

JENNIFER NOELLE HATZEL

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2011

To the horses, without which, none of this would be possible

ACKNOWLEDGMENTS

I thank my committee members: Dr. Patrick Colahan, for the initial concept and subsequent support through the Racing Medication Testing Consortium (RMTC) for completion of these studies, Dr. Alison Morton for her constant encouragement and Dr.

Tom Vickroy for his thoughts and ideas, always in the nick of time. I thank Dr. David

Hurley and Natalie Norton from the University of Georgia for their patient guidance in

assisting me with learning to perform the assays. I thank Dr. Richard Sams for

bestowing me with a very basic knowledge of pharmacokinetics and Marc Rumpler for

his unwavering support and mentorship through every aspect of this process. I thank

Dan Neal, from the department of biostatistics for not only teaching me the basics, but

also assisting me when I was way over my head. This project would have never been

possible without the help of the entire staff of the University of Florida Equine

Performance Laboratory: Brett Rice, Allie Hreha, Amber Davidson and Megan

Davidson, for whom I am very grateful. Finally, I owe a great deal of support and encouragement from Jeremiah, always.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF EQUATIONS ...... 10

LIST OF ABBREVIATIONS ...... 11

ABSTRACT ...... 12

CHAPTER

1 INTRODUCTION ...... 15

The Eicosanoids ...... 15 History ...... 15 The Inflammatory Cascade ...... 16 Pharmaceutical Intervention ...... 19 The History and Mechanism of NSAIDs ...... 19 Adverse Effects Affiliated with NSAID Administration ...... 22 Novel Classes of Selective NSAIDs ...... 23 NSAID Use in Veterinary Medicine ...... 26 Equine NSAID Administration ...... 26 Drug Doping in the Performance Horse ...... 29 Monitoring Drug Abuse ...... 31 Summary and Objectives ...... 37

2 THE PHARMACOKINETICS OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION ...... 41

Background ...... 41 Materials and Methods ...... 42 Animals ...... 42 Standard Training Regimen for the University of Florida Equine Performance Lab ...... 42 Incremental Exercise Test to Exhaustion ...... 43 Standard Condition Test to Verify the Ability to Gallop One Mile in Two Minutes...... 43 Drug Administration and Sample Collection ...... 44 Drug Analysis ...... 44 Chemicals and Reagents ...... 44 Phenylbutazone ...... 45

Sample preparation ...... 45 Instrumentation ...... 46 Data analysis ...... 47 Ketoprofen ...... 48 Sample preparation ...... 48 Instrumentation ...... 49 Data analysis ...... 50 Flunixin Meglumine ...... 50 Sample preparation ...... 50 Instrumentation ...... 51 Data analysis ...... 53 Pharmacokinetic Modeling ...... 53 Results ...... 54 Phenylbutazone ...... 54 Ketoprofen ...... 54 Flunixin Meglumine ...... 55 Discussion ...... 55

3 THE DURATION OF CYCLOOXYGENASE INHIBITION FOR PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION ...... 69

Background ...... 69 Materials and Methods ...... 70 Animals ...... 70 Standard Training Regimen for the University of Florida Equine Performance Lab ...... 71 Drug Administration and Sample Collection ...... 71 Ex vivo COX-1 Assay ...... 73 Ex vivo COX-2 Assay ...... 73 Statistical Analysis ...... 74 Results ...... 74 Ex vivo Inhibition of Phenylbutazone ...... 74 Ex vivo Inhibition of Ketoprofen ...... 75 Discussion ...... 75

4 UTILIZING A BIOSTATISTICAL APPROACH TO MODEL COMPARISONS OF PHENYLBUTAZONE, KETOPROFEN, AND FLUNIXIN MEGLUMINE WITH SUPRESSED CONCENTRATIONS OF PGE2 AND TXB2 ...... 92

Background ...... 92 Materials and Methods ...... 94 Animals ...... 94 Method of Analysis ...... 94 Results ...... 95 Relating Phenylbutazone Concentrations to Prostaglandin (PGE) Concentrations ...... 95

Relating Phenylbutazone Concentrations to Thromboxane (TXB) Concentrations ...... 95 Relating Ketoprofen Concentrations to Prostaglandin (PGE) Concentrations .. 96 Relating Ketoprofen Concentrations to Thromboxane (TXB) Concentrations .. 96 Relating Flunixin Meglumine Concentrations to Prostaglandin (PGE) Concentrations ...... 97 Relating Flunixin Meglumine Concentrations to Thromboxane (TXB) Concentrations ...... 97 Example of Model Application ...... 98 Discussion ...... 98

5 CONCLUSIONS ...... 108

LIST OF REFERENCES ...... 111

BIOGRAPHICAL SKETCH ...... 116

LIST OF TABLES

Table page

1-1 Non-steroidal anti-inflammatory drugs currently available for use in the horse. . 39

1-2 Maximum allowable concentrations of NSAIDs in plasma for several equestrian sports...... 40

2-1 Plasma concentrations of phenylbutazone as determined by LC/MS/MS analysis ...... 60

2-2 Relevant pharmacokinetic parameters for phenylbutazone following a 2- compartmental analysis...... 62

2-3 Plasma concentrations of ketoprofen as determined by LC/MS/MS analysis ..... 63

2-4 Relevant pharmacokinetic parameters for ketoprofen following a 2- compartmental analysis...... 65

2-5 Plasma concentrations of flunixin meglumine as determined by LC/MS/MS analysis ...... 66

2-6 Relevant pharmacokinetic parameters for flunixin meglumine following a 2- compartmental analysis...... 68

3-1 Ex vivo TXB2 concentrations for phenylbutazone in six horses ...... 80

3-2 Ex vivo PGE2 concentrations for phenylbutazone in six horses ...... 82

3-3 Ex vivo TXB2 concentrations for ketoprofen in six horses ...... 84

3-4 Ex vivo PGE2 concentrations for ketoprofen in six horses ...... 86

3-5 Ex vivo TXB2 concentrations for flunixin meglumine in 6 horses ...... 88

3-6 Ex vivo PGE2 concentrations for flunixin meglumine in 6 horses ...... 90

LIST OF FIGURES

Figure page

2-1 Plasma elimination of phenylbutazone represented by concentration versus time up to 4 h post drug administration...... 61

2-2 Plasma elimination of phenylbutazone represented by concentration versus time up to 48 h post drug administration...... 61

2-3 Plasma elimination of ketoprofen represented by concentration versus time up to 4 h post drug administration...... 64

2-4 Plasma elimination of ketoprofen represented by concentration versus time up to 24 h post drug administration...... 64

2-5 Plasma elimination of flunixin meglumine represented by concentration versus time up to 4 h post drug administration...... 67

2-6 Plasma elimination of flunixin meglumine represented by concentration versus time up to 72 h post drug administration...... 67

3-1 Mean ± SD of % inhibition of phenylbutazone on TXB2 concentrations ...... 81

3-2 Mean ± SD % inhibition of phenylbutazone on PGE2 concentrations...... 83

3-3 Mean ± SD % inhibition of ketoprofen on TXB2 concentrations ...... 85

3-4 Mean ± SD % inhibition of ketoprofen on PGE2 concentrations ...... 87

3-5 Mean ± SD % inhibition of flunixin meglumine on TXB2 concentrations ...... 89

3-6 Mean ± SD % inhibition of flunixin meglumine on PGE2 concentrations ...... 91

4-1 Raw and Estimated log(PBZ/PGE) versus time...... 101

4-2 Raw and Estimated log(PBZ/TXB) versus time...... 102

4-3 Raw and Estimated log(KETO/PGE) versus time...... 103

4-4 Raw and Estimated log(KETO/TXB) versus time...... 104

4-5 Raw and Estimated log(FL/PGE) versus time...... 105

4-6 Raw and Estimated log(FL/TXB) versus time...... 106

LIST OF EQUATIONS

Equation page

4-1 Estimate of the relationship between time and log(PBZ/PGE)...... 101

4-2 Estimated time since administration for log(PBZ/PGE)...... 101

4-3 Lower bound of confidence interval...... 101

4-4 Upper bound of confidence interval...... 101

4-5 Estimate of the relationship between time and log(PBZ/TXB)...... 102

4-6 Estimated time since administration...... 102

4-7 Estimate of the relationship between time and log(KETO/PGE)...... 103

4-8 Estimated time since administration...... 103

4-9 Estimate of the relationship between time and log(KETO/TXB)...... 104

4-10 Estimated time since administration...... 104

4-11 Estimate of the relationship between time and log(FL/PGE)...... 105

4-12 Estimated time since administration...... 105

4-13 Estimate of the relationship between time and log(FL/TXB)...... 106

4-14 Estimated time since administration...... 106

4-15 Example of est. time since administration using log(PBZ/PGE) = 3.5...... 107

4-16 Example of determining lower bound for the confidence interval...... 107

4-17 Example of determining upper bound for the confidence interval...... 107

LIST OF ABBREVIATIONS

AQHA American quarter horse association

ARCI Association of racing commissioners international

ASA Acetylsalicylic acid

AUC Area under plasma concentration

CINOD Cyclooxygenase inhibiting nitric oxide donors

COX 1 & 2 Cyclooxygenase 1 & 2

DTSP Drug testing standards and practices program

EIPH Exercise induced pulmonary hemorrhage

FEI Federation equestre internationale

FL Flunixin meglumine

KETO Ketoprofen

LC/MS/MS Liquid chromatography with tandem mass spectrometry

LLOQ Lower Limit of Quantitation

LOD Limit of Detection

LPS Lipopolysaccharide

NSAID Non-steroidal anti-inflammatory drug

PBZ Phenylbutazone

PGE2 Prostaglandin E2

QAP Drug testing and quality assurance program

TXB2 Thromboxane B2

WADA World anti-doping agency

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

PHARMACOKINETIC AND DURATION OF CYCLOOXYGENASE INHIBITION STUDIES OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE IN ATHLETIC THOROUGHBRED HORSES

Jennifer Noelle Hatzel

December 2011

Chair: Patrick T. Colahan Major: Veterinary Medical Sciences

The Non-steroidal anti-inflammatory drugs (NSAIDs) represent a family of therapeutic agents that are one of the most widely administered medications in both

human and veterinary medicine. Equine veterinarians have been particularly keen of

utilizing their anti-inflammatory actions on anything from colic, to endotoxemia or to

alleviate pain associated with musculoskeletal injury. The Thoroughbred racing industry

has historically represented equine athletes at the peak of their performance abilities and consequently at great risk for injuries associated with this type of athleticism. A variety of NSAIDs such as flunixin meglumine, phenylbutazone and ketoprofen, have been administered to Thoroughbred racehorses in an effort to alleviate pain and reduce inflammation, ultimately with anticipation for continued racing. While NSAID administration for relief of pain is humane and necessary, racing authorities around the world prohibit the unscrupulous use of these drugs to enable a compromised horse to race.

The first objective described in these experiments was to characterize the pharmacokinetics of flunixin meglumine, phenylbutazone and ketoprofen in conditioned

Thoroughbred horses utilizing state of the art LC/MS/MS and establish updated

pharmacokinetic parameters for each drug. The second objective described in these

experiments aimed to identify the extent and duration of cyclooxygenase inhibition, after

a single dose of each drug to evaluate the inhibitory capabilities for an active

inflammatory event. Finally, the data obtained from these experiments was utilized in

creating a novel method of determining an estimated time of administration and

potentially enhance our knowledge of withdrawal times for each of these drugs.

In order to obtain this data 6 Thoroughbred horses (3 mares and 3 geldings) in

athletic condition sufficient to work 1 mile in 2 minutes without undue stresses were

included. Each horse in the study was administered a single intravenous dose of one

NSAID at the beginning of the week, while remaining in training. Blood samples were

collected one hour and immediately prior to drug administration. Following

administration of the NSAID, collections took place at 15 and 30 minutes, 1, 2, 3, 4, 6, 8,

24, 48 and 72 hours. Plasma samples were obtained following centrifugation and

placed in -80°C storage until pharmacokinetic analysis by the Florida Racing

Laboratory. Plasma and serum was obtained following an appropriate incubation period

for each sample and stored at -80°C until further processing. All samples were

subjected to PGE2 concentration (plasma) or TXB2 concentration (serum) analysis

through the use of a commercially available ELISA kit.

The pharmacokinetic data obtained for each NSAID provided an updated

overview, along with the ability to detect increasingly minute concentrations of drug, not

previously determined. Challenged by noxious stimuli, phenylbutazone was significantly inhibitory (p<0.5) towards TXB2 for 48 h and PGE2 for up to 72 h; ketoprofen

demonstrated inhibitory actions on both mediators for up to 48 h; and flunixin

meglumine significantly inhibited both inflammatory mediators up to 8 h. By combining

the data obtained from these studies, a statistical model was created to use both the

concentration of drug in a plasma sample along with the inhibited concentrations of

either inflammatory mediatory (PGE2 or TXB2) to determine an estimated time of

administration.

The increasing sensitivity of analytical techniques along with the inability to

completely correlate a plasma concentration with clinical efficacy, suggests a need to

create novel analytical methods to aid in governing the anti-doping industry in

Thoroughbred racing. The current study suggests a statistical model utilizing both

pharmacokinetic and pharmacodynamic parameters to estimate an administration time.

Potentially, this method could be used to enhance our existing knowledge regarding

withdrawal times and in turn, better educate veterinarians and trainers towards the

consequences of administering a monitored NSAID within a more scientifically

established window of time prior to post.

CHAPTER 1 INTRODUCTION

The Eicosanoids

History

The eicosanoid family of metabolites trace their known roots back to the identification of prostaglandin by Swedish scientist, Von Euler, in the early 1930s. His observations of smooth-muscle contraction and vasodepressor activities in accessory sex glands led him to the discovery that the active ingredient was a lipid-soluble acid in seminal fluid. He aptly bestowed it the name, prostaglandin, to denote the hypothesized origin, the prostate gland1. Concomitantly, two American gynecologists, Kurzrok and

Lieb, observed fluctuations in contractility of strips of uterine myometrium which were exposed to semen 2. Additional investigations throughout the 1960s led to the

discovery that prostaglandins were simply part of a larger family of acidic lipids sharing

a basic 20-carbon unsaturated carboxylic acid structure 3. By 1971, Vane, Smith and

Willis had demonstrated that aspirin and additional non-steroidal anti-inflammatory

drugs (NSAIDs) exert their therapeutic actions by inhibiting prostaglandin biosynthesis,

culminating in a shared Nobel Prize in 1982 for their landmark discoveries 4.

The family name of the prostaglandins, leukotrienes and related acidic lipids is

derived from the Greek word eikosi, translated to “twenty” in a reference to the 20

carbons shared by their precursor essential fatty acids. Initially, the nomenclature of the

prostaglandins utilized their chemical structure and were designated the letters A-F.

More recent scientific advances have led to the discovery of newer compounds such as

the cyclic endoperoxides PGG2 and PGH2, prostacyclin, thromboxane A2, leukotrienes,

and other eicosanoids 1. Pharmaceutical modulation of these and other enzymes within

the arachidonic acid cascade via NSAID administration is one of the most clinically

important therapeutic modalities in veterinary and human medicine today.

The Inflammatory Cascade

As previously mentioned, the classic prostaglandins are organized A-F according

to their substituents on the cyclopentane ring of the 20-carbon carboxylic acid

(prostanoic acid) of which they all share. They are derived from three different 20-

carbon polyunsaturated fatty acids: 8, 11, 14-eicosatrienoic acid, containing 3 double

bonds (dihomo-γ-linolenic acid); 5, 8, 11, 14-eicosatetraenoic acid, containing 4 double

bonds (arachidonic acid); and 5, 8, 11, 14, 17-eicosapentaenoic acid, containing 5

double bonds (EPA). These essential fatty acids yield prostaglandins with one, two or

three double bonds remaining on the side chains, respectively and are referred to as

mono-, di-, or triunsaturated. This categorization is denoted by a subscript to the letter;

5 for example PGE1, PGE2, and PGE3 .

Unlike other autacoids, the eicosanoids are not located locally or stored in tissue pools, rather their synthesis depends upon the release of their fatty acid precursor, arachidonic acid. An essential fatty acid, arachidonic acid is incorporated into phospholipids of cell membranes through ester links and can be released by acyl hydrolases like phospholipase A2. Chemical, physical, hormonal stimuli or other

2+ autocoids activate cytosolic phospholipase A2 by the Ca -dependent translocation of

group IV cytosolic phospholipase A2. This occurs through the hydroxylation of the sn-2

bond releasing arachidonate which is then rapidly oxygenated through a variety of

enzymatic pathways including cytochrome P450, cyclooxygenases and lipoxygenases 1.

Unesterified arachidonic acid is metabolized through oxygenation and cyclization by the smooth endoplasmic reticulum enzyme, endoperoxide G/H synthase, more

commonly referred to as cyclooxygenase (COX), yielding the cyclic endoperoxide

PGG2. The COX enzyme is bifunctional having both COX activity as well as hydroperoxidase (HOX) activity. Unlike lipoxygenase (LOX) which has been mainly isolated from the lung, platelets and white blood cells, COX is metabolized in many

5 tissue types in a wide variety of mammals . The cyclic endoperoxide PGG2 is then

converted to the related cyclic endoperoxide PGH2 through the HOX activity of the COX

enzyme 6. Both of these intermediates are highly unstable with half-lives of less than 5 minutes and are quickly transformed by cell-specific isomerases and synthases into prostaglandins, thromboxane and prostacyclin. These prostanoid products are thought to be moved through the cell membrane for release via a prostaglandin transporter

system 7.

The COX enzyme is comprised of three different folding units: an epidermal

growth factor-like domain, a membrane-binding motif and an enzymatic domain. An

entrance channel leading to the active site is formed by three structural helices, through

which arachidonic acid may access the interior of the bilayer active site. It is this active

site, where NSAIDs inhibit the access of arachidonate to the upper portion of the long

hydrophobic channel8. In 1991, two major isoforms of cyclooxygenase encoded by

different genes: COX-1 and COX-2 were elucidated. The first isoform, COX-1, is

considered a “housekeeping” prostanoid and is constitutively expressed by cells during

basal conditions modulating physiologic functions like gastro-protection of the mucosal

epithelial cells and renal blood flow regulation. The second isoform, COX-2, however is

not always detectable during basal conditions and is upregulated by inflammatory stimuli released from bacteria, cytokines discharged from macrophages, shear stress and growth factors, assigning it as the source of prostanoids formed in inflammation and cancer 1,8. Separating these two isoforms into simplistic categories of “good” vs. “evil” is

actually a misinterpretation as they function coordinately in certain circumstances 9.

Although their responsibilities differ, both isoforms share a similar crystal structure as a dimer homotypically inserted into the endoplasmic reticular membrane, along with a

61% similar amino acid identity 10. The most important difference in their structure lies within the active sites. The structural alteration found in the COX-2 isoform is a larger

catalytic channel with a side pocket such that arachidonic acid can “squeeze past”. This

difference has prompted the pharmaceutical industry to synthesize COX-2 specific

inhibitors that reportedly block anti-inflammatory actions but spare the gastric and anti-

platelet side-effects, which will be discussed in more detail 8.

In 2002, a splice variant of COX-1 was recognized and designated COX-3

retaining the intron-1 gene sequence which encodes a 30 amino acid sequence inserted

into the N-terminal hydrophobic signal peptide of the enzyme protein. Originally

discovered in dog tissues as a centrally acting cyclooxygenase inhibited by acetaminophen, the highest concentrations of this isoform have been isolated to the brain and nervous tissue. A mechanism for its conversion to an active enzyme has yet to be determined. It was suggested by Chandrasekharan et al. that this isoenzyme, may be a target of NSAID action by some therapeutic agents like acetaminophen, dipyrone 11 and paracetamol in mice 12 in the central nervous system that produces

analgesia and hypothermic properties. Such actions are not affected by COX-2.

The additional prostanoid products of arachidonic acid metabolism, thromboxane and prostacyclin, are also derived from the actions of enzymes on the intermediate endoperoxide PGH2. Initially isolated from thrombocytes, thromboxane synthase was

found to be the enzyme responsible for converting PGH2 into a substance referred to as

thromboxane A2 (TxA2) that contains an oxane ring in contrast to the cyclopentane ring found on prostaglandins. Thromboxane A2 plays a vital role in vasoconstriction and is a

proaggregate in thrombus formation. It has a half-life of around 30 seconds, and is

quickly degraded into the more stable form, thromboxane B2. Prostacyclin or PGI2 is a product of action of the enzyme, prostacyclin synthase, upon PGH2 within vascular

tissue. It is structurally different from the other two prostanoids by displaying a double-

ring, as well as demonstrating a short half-life of approximately 2-3 minutes before

conversion to a relatively inactive but stable 6-keto-PGF1α. Prostacyclin exerts its effects as a potent vasodilator as well as antiaggregatory action on blood platelets 5.

Pharmaceutical Intervention

The History and Mechanism of NSAIDs

The global therapeutic success, derived from the use of nonsteroidal anti- inflammatory drugs (NSAIDs), owes its existence to the early herbal folklore of its foundation. An excerpt from the first known pharmacopoeia, in reference to the willow tree reads: ‘The leaves of the willow being beaten small and drank with a little pepper and wine doe help such as are troubled with the Iliaca Passio (colic). The decoction of ye leaves and barck is an excellent foementation for ye Gout’ 13. With this in mind and

in an effort to create a more palatable formula with fewer gastrointestinal side effects for

his arthritic father, a chemist at Bayer Laboratories began tinkering with acetylsalicylic

acid (ASA) in 1899 and followed soon after with the marketing of aspirin14. From

inception, NSAIDs have become one of the most widely used therapeutics for anti- inflammatory disorders in both human and veterinary medicine.

Historically described, the classical signs of inflammation include: calor (warmth),

dolor (pain), rubor (redness), tumor (swelling) and functio laesa (loss of function)

invoked by a wide variety of noxious stimuli including infections, antibodies and physical

trauma. Upon induction of inflammation, prostanoid biosynthesis is upregulated leading to prostaglandin E2 and prostacyclin production, leading to the subsequent physiological

responses such as increased blood flow, vascular permeability and leukocyte infiltration.

It is through the inhibition of cyclooxygenase, consequently halting the biosynthesis of prostanoid products, which many NSAIDs exert their therapeutic action leading to a decline in cytokine production, leukocyte recruitment and ultimately inflammation.

NSAID administration obtunds pain by subduing the cytokines responsible for increasing the sensitivity of nocioceptive pain perception on nerve endings and thereby, reducing hypersensitivity caused by an inflammatory response. Similarly, fever is reduced as NSAIDs inhibit PGE2 synthesis, the prostanoid primarily responsible for

crossing the blood-brain barrier and triggering the hypothalamus to elevate body temperature and decrease heat loss14.

The NSAIDs available for clinical use are diverse and abundant. They share many

common therapeutic goals along with numerous undesirable side effects. This is much

in part due to their common hydrophobic structure, which allows access to the

hydrophobic arachidonate binding channel. The traditional NSAIDs (non COX-2

selective) are generally organic acids with relatively low pKa values and high uptake

following oral administration. Once absorbed, they are highly bound to plasma proteins

(95-99%), implying that the free active fraction of the drug excreted via glomerular filtration or tubular secretion is very small. Additionally, at sites of inflammation, the pH

is lower which encourages many NSAIDs to accumulate in these regions 14.

The propensity of NSAIDs to be highly protein bound, causes displacement of

other acidic drugs from their binding sites on plasma proteins if administered

concurrently. Dicumarol (Warfarin), as an example, is highly bound to albumin and is

displaced in the presence of NSAIDs in a dose-dependent manner. This potentially

leads to increased anticoagulant effects as a result of elevated concentrations of free

dicumarol in the blood. Similarly, aminoglycoside antibiotic administration in

simultaneous administration with an NSAID can potentially elevate the risk for

nephrotoxicity 15.

As previously described, activated arachidonic acid enters the active site of both

COX-1 and COX-2 enzymes through a hydrophobic channel to initiate the cascade of

events observed in inflammation. The mechanism of action for NSAIDs is thought to be

similar to that of acetylsalicylic acid to effectively inhibit this action, by irreversibly

blocking access to the channel through acetylation of serine 530 (COX-1) or serine 516

(COX-2) 10. Once bound, the NSAID COX inhibitors prevent arachidonic acid from entering the active site of the cyclooxygenase enzyme, thus preventing the formation of the characteristic prostanoid aromatic ring.

The therapeutic administration of NSAIDs has often been limited by their multisystemic adverse effects, particularly concerning the gastrointestinal system. Upon discovery that COX-2 represented the dominant form of inflammatory and cancerous prostanoids, many pharmaceutical laboratories pursued identification of NSAIDs that

selectively inhibited this specific isoenzyme. The amino acid conformation along with several subtle differences between the two isoforms accounts for the selectivity of COX-

1 versus COX-2 inhibitors. The smaller valine residues at sites 434 and 523 allows for a side-pocket to form in COX-2 isoforms, whereas COX-1 demonstrates larger isoleucine residues that effectively close down the entrance to a side-pocket formation.

Optimal orientation with this side-pocket allows for COX-2 selective diaryl heterocyclic compounds, to maintain their own bulky side group and form a bond with COX-2 and not COX-1 14. Additionally, COX-2 maintains a charged arginine residue at amino acid

513, whereas COX-1 has an aromatic histidine residue in this location. The charged

arginine residue interacts with sulphonamide groups often present on COX-2 selective

inhibitors, while the imidazole ring of the histidine residue is unable to interact,

demonstrating additional COX selectivity. Finally, certain residues present in COX-2 but

not COX-1 are able to form a hydrogen bond with COX-2 inhibitors, promoting further

selectivity 16.

Adverse Effects Affiliated with NSAID Administration

Although all NSAIDs provide antipyretic, analgesic and anti-inflammatory

properties (with the exception of acetaminophen which lacks anti-inflammatory activity),

relief is not afforded without a cost. Adverse side-effects resulting from the use of these

drugs have been well documented since the introduction of industrially produced

salicylic acid in 1874 and affect both human and veterinary patients. Body systems

most often affected by NSAID administration include the gastrointestinal tract, platelets, the renal system, the cardiovascular system, the central nervous system, the female reproductive tract and variable hypersensitivities. The constitutive isoform, COX-1, found in blood platelets yields thromboxane A2, of which inhibition leads to a loss of

normal platelet aggregation. The activation of COX-1 also produces antithrombogenic prostacyclin, which is released by the endothelium along with cytoprotective prostanoids in the gastric mucosa. Importantly, these eicosanoids inhibit acid secretion by the stomach, enhance mucosal blood flow and promote secretion of protective mucus in the lumen of the intestine 14. When these cytoprotective actions of prostanoids are

inhibited, gastric damage like mucosal ulceration leading to hemorrhage and potential

perforation can occur. This complication represents the major concern with NSAID administration in human medicine, especially regarding the geriatric population of patients. An additional mechanism for NSAID induced ulceration has been proposed to occur due to local irritation through direct contact with an orally administered formulation, however this risk is perceived minor when compared with inhibition of the cytoprotectivity actions of COX-1 induced prostanoids 14. COX-1 dependent

prostaglandin biosynthesis in the kidney primarily occurs in the renal medulla, the

ascending loop of Henle and the cortex. Prostaglandin synthesis controls several

aspects of renal physiology including total renal blood flow, distribution of renal blood

flow, sodium and water reabsorption and renin release. When these physiologic

functions are disrupted by NSAID administration, leading to a decrease in glomerular

filtration rate or sodium and water excretion, a variety of clinically relevant effects may

ensue including acute renal failure, and hypertension.

Novel Classes of Selective NSAIDs

Although our understanding has grown extensively since the discovery of COX-2

in 1991, it is still incomplete. Novel side effects of selective COX-2 NSAIDs, sprung

from the selective inhibition, became publically evident between 2001 and 2003. Prior

to this public controversy involving the ‘coxib’ drugs, Mukherjee et al. had raised a

cautionary flag based on a variety of studies indicating an increased risk of cardiovascular events associated with COX-2 inhibitors 17. Subsequent studies

determined that the adverse cardiovascular complications are associated with

prolonged administration of COX-2 inhibitors including increased risks of myocardial

infarction, destabization of controlled congestive heart failure and exacerbation of high

blood pressure. These complications ultimately lead to the withdrawal of some of the

COX-2 inhibitors from the market by the federal Food and Drug Administration18.

Ongoing pharmacological studies aimed at creating a more gastrotolerant and effective NSAIDs have followed several strategies. The concept that gastrointestinal safety can be improved by forming nitroso derivatives of the conventional nonselective

COX inhibitors, has led to the introduction of cyclooxygenase inhibiting nitric oxide donors (CINODs). The in vivo induction of nitric oxide release appears to be gastroprotective, and possibly demonstrates increased anti-inflammatory and analgesic potency. Although promising, to date no drugs in this class have been introduced into the veterinary market. A novel NSAID class, demonstrating dual inhibition has been introduced to both the human and veterinary industries recently. These drugs have been designed to inhibit both COX and LOX (specifically 5-LO) that produces a leukotriene from another pathway of the arachidonic acid cascade 13. It is believed that

inhibition of this secondary pathway could potentially increase gastrointestinal safety

and create greater analgesic effects. An example of this in the veterinary

pharmaceutical markets of both Europe and the United States is Tepoxalin (Zubrin®),

which is currently advertised for the use in dogs with osteoarthritis. Data has indicated

that this drug is more COX-1 inhibitory, but also inhibits LOX activity and has proven

increased gastrointestinal safety 11. Giorgi et al. has recently conducted several studies

investigating this drug administered orally to horses, but have found that it is rapidly

converted to its acidic metabolite, RWJ-20142. This metabolite has diminished COX-2

and 5-LO, inhibitory activities and thus ultimately raising concerns about efficacy as a

long-term therapeutic option for use in equine patients 19-21.

Conventionally, NSAIDs have been organized according to their chemical

structure dividing them into several classes: derivatives of salicylic acid, propionic acid,

acetic acid, enolic acid, fenamic acid, alkanones and diaryl heterocyclic compounds

(which represent the COX-2 selective agents or ‘coxibs’). With the introduction of

selective COX-2 inhibitors, a new classification system was created in an effort to

distinguish agents according to their specific selectivity. The selective COX-1 inhibitors,

including aspirin, are used at low dosages for their inhibition of platelet aggregation, and

are determined to be more damaging to the gastrointestinal tract at higher dose rates.

The nonselective COX inhibitors have been determined to inhibit both COX-1 and COX-

2, effectively inhibiting platelet aggregation as well as causing significant gastrointestinal

and renal disturbances. The selective COX-2 inhibitors have been expected to provide

decreased gastrointestinal and renal side-effects, permitting administration to patients in

whom platelet aggregation must be intact. Finally, the highly selective COX-2 inhibitors

represent a group of agents currently being tested, but not yet clinically relevant or

available. One further method of classifying these drugs is though a system based on

t1/2, allowing for visual interpretation of the persistence of plasma concentrations.

NSAID Use in Veterinary Medicine

Equine NSAID Administration

The NSAID pharmaceutical agents are amongst the most commonly prescribed therapeutics in equine medicine today. Discomfort associated with musculoskeletal pain and inflammation represents the most common indication for administration.

Phenylbutazone is commonly the drug of choice. Another commonly administered

NSAID in equine medicine, flunixin meglumine, is most often used to relieve pain associated with colic, fever, and soft-tissue inflammatory disorders. Furthermore,

flunixin meglumine, has been the primary NSAID used for the treatment of clinical signs

associated with endotoxemia, although other drugs such as ketoprofen and

phenylbutazone have been explored to determine their anti-endotoxic properties 22.

As science and technology have advanced, the number of NSAIDs available for

equine practitioners has grown substantially. Route, frequency, and ease of

administration are all considerations in NSAID selection. The majority of NSAIDs

available for use in the horse are administered either intravenously (i.v.) or orally (p.o.).

Intramuscular administration is not recommended due to the potential necrotizing

myositis at the injection site associated with this route of administration by most NSAID

preparations 23. Plasma concentration following oral administration of NSAIDs in equine

patients depend upon the drug’s tendency to bind to hay and digesta, leading to

incomplete immediate absorption followed by a prolonged absorption peak associated with hind gut fermentation 24. This affects the plasma concentration as well as the onset of action for these drugs. There is some clinical evidence regarding the superior

efficacy of individual NSAIDs in treating inflammatory conditions involving specific tissue

types. Although unsubstantiated, this has led to the practice of administering two

different NSAIDs in patients suffering from two different diseases simultaneously.

Although there is little proof that efficacy is improved utilizing this method. Evidence from human medicine that suggests the occurrences of side-effects are increased by simultaneous NSAID administration are increased, but minimal studies have been conducted in equine medicine 14. Furthermore, there is not an overwhelming amount of

evidence for the selection of one NSAID over another and that the decision should be

based upon the situation and owner (Table 1-1).

As in human medicine, the primary side effect of NSAID administration,

gastrointestinal ulceration, is the most prevalent side effect in horses. The

cytoprotective action of prostaglandins in the gastrointestinal tract of horses preserves

the integrity of the gastric mucosa through a variety of different mechanisms that are inhibited with administration of NSAIDs. Additionally, the acidity of the drug is hypothesized to directly irritate the gastric mucosa following oral administration, adding to an untoward effect 25. Horses also demonstrate a unique sensitivity of the right

dorsal colon to the ulcerogenic effects of NSAIDs even in the face of moderate dose rates 22. The pathophysiology of this sensitivity is currently unknown but has been

hypothesized to be due to the greater prostaglandin dependency of the blood flow and

subsequent vasodilatation in this anatomic region compared to other regions of the

colon 26.

Although not as notable as the gastrointestinal effects, renal side effects resulting

from NSAID administration to horses is documented in veterinary literature. Renal

papillary necrosis most commonly occurs as a sequela to routine doses of NSAID

therapy 27 often in dehydrated animals or those suffering preexisting impaired renal

perfusion, but does not appear to affect normal horses. In the kidney, prostaglandins induce afferent vasodilatation to maintain renal blood flow and glomerular filtration rate as well as maintain potassium homeostasis and water and sodium balance. Renal blood flow to the renal pelvis in horses has been hypothesized to be minimal such that even mild dehydration or hypotension increases the sensitivity to ischemic necrosis induced by NSAID administration 27.

As previously described, NSAIDs represent a family of drugs that are highly

bound to plasma proteins. Caution is indicated when administering these drugs

concurrently with other compounds that are also highly protein bound such as additional

NSAIDs, sulfonamides, warfarin and gentamicin. Careful monitoring of relevant pharmacokinetic and pharmacodynamic parameters is recommended to ensure that the

NSAID being used is not competitively inhibiting the binding ability of the other drug and consequently increasing the unbound fraction. Adjustments to dosage should be made in light of this interaction 22.

Hepatotoxicity induced by NSAID administration is a potential side effect,

reported in a variety of species, but not documented in the horse. The hepatotoxic

effects of NSAIDs in other species have been reported to be either idiosyncratic,

unpredictable/non-dose related or intrinsic, predictable and dose-related 11. Although

the horse does not appear particularly prone to suffering hepatotoxic effects induced by

NSAID administration, the potential for this to occur in conjunction with concomitant

herbal preparations administered by the owner may exist and veterinary consultation

should be performed 22.

The effect of NSAID administration on coagulatory function in the horse has been minimally investigated but historically, COX inhibition alters platelet aggregation.

Aspirin, despite a short half-life, has been demonstrated to be antithrombotic in horses irreversibly inactivating the aggregatory abilities of COX-1 28. Bleeding time is unaltered by the administration of flunixin meglumine or phenylbutazone 28, but the effect of any

other of the NSAIDs commonly utilized in equine practice is unknown in this regard.

With the recent availability of the more COX-2 selective products, an intense

interest in the effects of NSAIDs on cartilage as chondroprotective or

chondrodestructive, has developed. Studies have provided evidence supporting both

the protective and destructive properties 29 but more research is necessary to define the

effects of NSAIDs in both normal and arthritic joints 22.

Drug Doping in the Performance Horse

Athletic breakdown on racetracks is unfortunately a relatively common occurrence

in equine competition, causing injury to both horses and riders, tainting the image of

horse racing in the public’s eye. Many studies have evaluated different variables

potentially leading to a breakdown on the track but none have drawn specific

conclusions. A study was published by Dirikolu et al. in 2008 to evaluate the role of

nonsteroidal anti-inflammatory agents in musculoskeletal injuries of racing

Thoroughbred horses. The group examined 210 injuries occurring on Kentucky

racetracks between January 1, 1995 and December 31st, 1996 and included 161 in the

study based on injuries related to the musculoskeletal system. Plasma samples from

the injured horses were assayed for phenylbutazone, flunixin meglumine and naproxen

and were compared with drug concentrations in samples procured from the winner and

another horse in the race picked randomly by the stewards of the track. The average

apparent plasma concentration of phenylbutazone in samples from injured horses (5.84

μg/mL ± 0.563), was significantly greater than those from the race winner (4.337μg/mL

± 0.458) or the randomly selected horse (4.337 μg/mL ± 0.454). None of the concentrations were above an assumed pharmacologically effective level for this study,

7 μg/mL. In the same study, plasma concentrations of flunixin meglumine were significantly higher in plasma from the injured horses (1.632 μg/mL ± 0.158) and in samples from the winning horses (1.067μg/mL ± 0.078) compared with the samples from the randomly selected horses (0.695 μg/mL ± 0.069). Most injured horses (81%) had plasma concentrations higher than the assumed pharmacologically effective level of

0.1 μg/mL. Although no definitive cause and effect relationship may be drawn regarding the effects of NSAIDs on racetrack injuries, it remains a possibility that the injured horses had preexisting pathology, being masked by the administration of NSAIDs that was accentuated during the race. However, the role of additional risk factors including age, racetrack surface, length of race, gender, training program, and preexisting pathologic conditions must be examined further 30.

Many states allow the controlled use of NSAIDs during training to alleviate “sore”

horses. The question of NSAID administration effects on performance and the ethics

behind permitting such treatments may certainly be raised. Several studies have been

conducted examining the effects of NSAIDs on exercising horses and potential effects

on pharmacokinetics. Soma et al., in concurrence with other studies 31, concluded that

age but not training status influenced the disposition of the drug as defined by

depression of serum thromboxane levels 32. However, as pain is an indication for the

body to spare the use of afflicted tissues, competing under the influence of analgesic

drugs may cause further damage and possibly increase the incidence of breakdowns on the track.

Although performance enhancement by drug administration contaminates all aspects of equine sports, the racing industry has historically suffered the greatest abuse. The sport of horse racing is an ancient past-time with the first ever recorded ridden race occurring at the Greek Olympiad in 624 BC 33. With the cultivation of the

Thoroughbred in England and the Standardbred in France and the United States

throughout the 17th, 18th, and 19th centuries, the foundation for modern horseracing was

set and became a globally popular amusement. Alongside this entertainment,

undoubtedly ignited through human competitiveness, came the desire to utilize chemical

substances to improve the prospect of winning. However, a stimulatory effect was not

always the goal and in fact, the British practice of ‘nobbling’ was one in which competing

horses were subdued through chemical manipulation with the purpose of deceiving the

betting public 34. With the establishment of the English Jockey Club in 1752, came the

‘hay, oats and water’ rule, which has served as the guiding principle throughout the racing world over the past two centuries. With the invention of the hypodermic needle in

the 19th century and the development of pure injectable forms of stimulant drugs, arrived a new era of doping.

Monitoring Drug Abuse

As advancements within the pharmaceutical industry have substantially increased the number of available doping agents, the forensic laboratories strive to maintain the number and sensitivity of analytical techniques. During the 1940s, very few drugs were detectable and confirmation was difficult using melting point analytic technique, the primary technique available at the time. Chromatography was rediscovered in the

1950s and led to both gas chromatography and quickly thereafter, by the thin layer

chromatography in the 1960s, allowing for the identification of much lower concentration

of illegal drugs. The pharmaceutical industry continually evolved through the design of

products boasting higher affinities and greater specificity leading to increased potency

and reduced side effects at lower dosages. Throughout the 1980s many of these potent

substances began surfacing in test samples from the racetracks and the forensic

laboratories were challenged. With the use of chromatography/mass spectrometry, the

ability to detect minute concentrations of prohibited substances became a reality and

has become the cornerstone in drug rule enforcement 33. Ironically, the sensitivity of

current analytical techniques have become such that the detection of illegal substances

is possible for such a prolonged period following administration that there may be no

significant pharmacologic effect on performance from the drug. The debate involving

the establishment of threshold limits for certain substances and how those limits should

be set continues to be a ‘hot topic’ in the racing industry.

As the need for regulation on racetracks became more and more evident, the

Association of Racing Commissioners International (ARCI) was created in 1988 to

provide uniformity to the rules of racing. Pari-mutuel betting and racing are regulated at

the state rather than the national level in the United States, and currently there are 44

states regulating their own Rules of Racing. The members of the ARCI are politically

appointed and function to make recommendations on rules but do not serve as a

regulatory body. The Drug Testing and Quality Assurance Program (QAP) was

established in 1988 by the ARCI in an effort to monitor the testing laboratories involved

in processing racetrack samples and maintain a level of performance standard. In 1995

the QAP was replaced by another program called the Drug Testing Standards and

Practices Program (DTSP) that currently accredits 5 out of 18 laboratories throughout the U.S. receiving test samples from racetracks. Stewards and commissioners at the state level are responsible for determining the severity of disciplinary action enacted in the face of a post-race positive drug sample. Typically the trainer, who is considered the primary insurer of the horse’s fitness to race, receives the brunt end of a punishment, usually in the form of a fine and/or license suspension 33.

Although regulation on racetracks is still maintained at the state level, the ARCI

has developed a set of guidelines in which commissioners use as a template for their

own rules of racing in addition to maintaining legislation. These procedures have been

collected and published in the ‘Model Rules of Flat Racing’ which is a 25 chapter

publication containing a wide range of subjects, updated periodically with the most

current version published in October of 2010. The eleventh chapter of this publication

addresses equine health and medication and covers topics such as: the Uniform

Classification Guidelines, penalty recommendations, medication restrictions as well as

the permitted usage of phenylbutazone and furosemide. The Uniform Classification

Guidelines provides a tiered schematic, organizing foreign substances according to their

pharmacological capabilities of affecting a race along with their appropriateness for use

on racehorses in general. The QAP along with an additional ARCI subcommittee, the

Drug Penalty and Sample Selection, considered input from the Horsemen’s Benevolent

and Protective Association, the American Association of Equine Practitioners, the

American Horse Council and the Thoroughbred Racing Protective Bureau along with

others in order to draft the first edition of these guidelines along with their recommended

disciplinary actions in 1992. The criteria taken into consideration in formatting these guidelines included the overall rules of racing, the ability of a substance to alter

performance, and the pattern of use along with a drug’s acceptability to be used as an

equine veterinary therapeutic agent. Not only do the guidelines support uniformity

involving the pharmacological agents, but they also provide assistance to a

commissioner (who may or may not be savvy to this industry) in understanding the

significance surrounding a positive finding. Additionally, positive results may possibly

be the result of inattention or variability of a drug administration, and penalization should

be applied accordingly 33 .

As of December 2010, there are over 860 foreign materials listed within the

ARCI’s Uniform Classification of Foreign Substance Guidelines. Class one represents

the drugs that are among the greatest concern due to their pharmacological effect and

their potential outcome on a race. These drugs generally do not have acceptable

medicinal use on racehorses and include those in the family of opiates, amphetamines,

and pemoline. Class two drugs have a high potential to affect performance and are not

generally utilized in equine medicine consisting of psychotropic drugs used by humans, mood-elevating drugs and those used to reduce anxiety. The drugs found in the third class include those that may or may not be generally acceptable for use in the racehorse but demonstrate less potential to alter a race. Included in this class are bronchodilators, which may be used during training, procaine, which often accompanies administration of procaine penicillin G administration and antihistamines. The fourth class of substances includes a varied list of drugs more commonly used during training such as: the NSAIDs, steroids, diuretics (not including furosemide) and other

miscellaneous drugs. Finally, the fifth class of foreign substances includes those agents

that are commonly used in horses and are of little interest in affecting performance

including anti-ulcer drugs, certain antiallergic medications and anticoagulant drugs. The

ARCI has also developed a penalty recommendation scheme located at the end of the

Guidelines with advice for NSAID overages as well as for furosemide, which is

permitted for the prevention of exercise-induced pulmonary hemorrhage (EIPH).

Finally, there are several drug classes which are considered of no interest in equine

regulatory actions and include: antibiotics, sulfonamides, anthelmintics and vitamins

33,35. Every state implements the guidelines to suit its own needs in ways varying from

the amount of NSAID administration permitted and the limits of drugs to the time prior to post at which furosemide administration is allowed.

Several other countries have established unique methods for organizing veterinary medications used on equine racetracks. In Canada, all Pari-Mutuel Wagering is under the jurisdiction of Agriculture Canada, which maintains a published list of agents entitled

‘Schedule of Drugs’. The most recent edition, published in 2006, presents data from research conducted on Standardbred mares at the Agriculture Canada Equine Drug

Evaluation Centre displayed online as a graph of concentration versus time following administration until the concentration reaches the detection limit below the analytical

method. Trainers and veterinarians can use this chart as a reference but there are no

official recommendations or penalties available. Several of the Provinces will refer to

the ARCI Uniform Classification Guidelines for recommendations in penalties when

necessary 33,36. The International Agreement on Breeding, Racing and Wagering is a collection of articles, appendices and guidelines published by the International

Federation of Horseracing Authorities to recommend the best practices in significant areas of racing. Approximately fifty countries, including the United States, are in agreement upon the most recently updated version, published in April 2009. However,

not all countries adopt every article and for example, the United States excludes

sections of Article 6 – Prohibited Substances, due to the lack of specificity as well as

penalty recommendations within this publication. Owing to the international nature of

this document and in consideration of the diversity of cultures, a classification scheme

and/or penalty system would be virtually impossible to implement 33,37.

In examining other equestrian disciplines, recently the Federation Equestre

Internationale (FEI) has adopted the “Clean Sport” program, which prohibits the use of

any NSAIDs (amongst all other foreign prohibited substances) immediately prior to and during competition. These new guidelines, adopted in April 2010, mirror the World Anti-

Doping Agency (WADA) protocols used in human athletic events and were encouraged to be implemented prior to the 2010 World Equestrian Games in Lexington, Kentucky 38.

In the US, the United States Equestrian Federation (formerly the American Horse

Shows Association) specifically prohibits the use of more than one NSAID administered

at a time and has threshold limits for individual medications listed in chapter 4 of their

rulebook. Additionally, when an NSAID is administered within five days of a

competition, a disclosure form is required upon entry to the competition 39. The

American Quarter Horse Association (AQHA) refers trainers to the Uniform

Classification Guidelines recommended by the ARCI regarding racing Quarterhorses.

For showing purposes, the Uniform Classification Guidelines are again referenced, but

varying threshold limits are listed for individual drugs in addition to the specific

requirement of only one NSAID administered within 24 hours of performance time with

the proper disclosure form (Table 1-2).

Summary and Objectives

Upon initiation of the inflammatory cascade, a series of enzymatic events converts arachidonic acid release from the cell membrane into several endoperoxides, including

PGE2 and thromboxane B2. One of these essential enzymes is cyclooxygenase (COX),

whereupon the COX-1 isoform is known to be constitutively expressed and vital to

tissue homeostasis, but COX-2 is considered inducible and proinflammatory. Both the

human and veterinary medical fields have long utilized non-steroidal anti-inflammatory

drugs (NSAIDs) in order to inhibit COX, thereby restricting or reducing the production of prostanoids. While NSAID use is necessary for the relief of pain, authorities around the

world prohibit the unscrupulous use of these drugs to enable a compromised horse to

race or perform in any athletic event. This has led to the formation of a science

specifically responsible for monitoring the doping situation in equestrian sports, and

subsequently leading to tough decisions regarding topics such as threshold limits and

appropriate punishments.

The main focus of this investigation was to examine the inflammatory inhibition capabilities along with the decreasing concentrations of three non-steroidal anti- inflammatory drugs, commonly used in equine medicine, following a one-time administration. Through several experiments, we aimed to supplement the current knowledge of the effects of NSAIDs on athletic horses and potentially propose a novel method of determining their efficacy for commercial use. Specific objectives of the study were as follows:

1. Characterize the pharmacokinetics of phenylbutazone, ketoprofen and flunixin meglumine after intravenous administration to Thoroughbred horses in athletic condition

2. Determine the duration of cyclooxygenase (COX) inhibition of each drug

Table 1-1. Non-steroidal anti-inflammatory drugs currently available for use in the horse. Drug Formula Notes Acetylsalicylic acid Tablet, powder, paste & Not currently U.S. FDA (Aspirin) gel registered for veterinary use but some forms are marketed as if approved. A form in combination with methylpredenisolone is available for use in dogs. Diclofenac (Surpass®) Cream Dipyrone Injectable Only available through compounding in the U.S. but registered in Canada Eltenac (Telzenac®) Injectable Not currently available through the U.S. FDA Firocoxib (Equioxx®) Paste Previcoxx® also available for use in dogs in the U.S. Flunixin meglumine Injectable, paste & (Banamine®) granules Ketoprofen (Ketofen™) 10% Injectable solution Available in tablet form & as a 1% injectable soln. for use in dogs & cats in Europe & Canada Meclofenamic acid Granules Available in tablet form (Arquel) for dogs Naproxen (Equiproxen) Granules & injectable Phenylbutazone “bute” Granules, paste & (Butazolidine®) injectable Vedaprofen (Quadrisol) Gel or injectable Only available in Europe & Canada

Table 1-2. Maximum allowable concentrations of NSAIDs in plasma for several equestrian sports. NSAID ARCI: Uniform FEI USEF AQHA Classification (Jan. 2011) (April 2010) (Dec. 2010) Guidelines (Dec. 2010) Diclofenac Prohibited Prohibited 0.005 μg/mL 0.005 μg/mL (Surpass®) Firocoxib Prohibited Prohibited 0.240 μg/mL 0.240 μg/mL (Equioxx®) Phenylbutazone 2 μg/mL Prohibited 15.0 μg/mL 15.0 μg/mL (Butazolidin®) Flunixin 20 ng/mL Prohibited 1.0 μg/mL 1.0 μg/mL meglumine (Banamine®) Ketoprofen 10 ng/mL Prohibited 0.250 μg/mL 40.0 ng/mL (Ketofen®) Meclofenamic Prohibited Prohibited 2.5 μg/mL 2.5 μg/mL acid (Arquel®) Naproxen Prohibited Prohibited 40.0 μg/mL 40.0 μg/mL (Naprosyn®) Eltenac Prohibited Prohibited 0.1 μg/mL 0.1 μg/mL (Telzenac®/not yet approved) Salicylic acid Prohibited Prohibited Prohibited Prohibited

CHAPTER 2 THE PHARMACOKINETICS OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION

Background

From the inception of equine athletic competitive events, mankind has attempted

to create a “special advantage” in an effort to capture victory through the administration

of illegal substances. As the drugs became more complex, the analytical techniques

necessary to identify them in blood and urine samples strived to maintain the ability to

detect decreasing concentrations. With the advent of complimentary and tandem

chromatographic and mass spectrometric techniques, beginning in the 1980’s, scientists

were soon able to detect and quantify physiologically irrelevant concentrations of

prohibited substances in performance horses33. In conjunction with superior analytical

methodologies came the responsibility for understanding how certain concentrations of

such drugs may cause physiologic changes, which consequently alter the performance

ability of horses.

Pharmacokinetic and pharmacodynamic (PK-PD) modeling has been utilized in human medicine as a useful technique for over 20 years, and its introduction in veterinary medicine has also proven to be a valuable tool 40. Many of the

pharmacodynamic studies performed of NSAIDs on horses have utilized an in vitro

technique. However valuable, this method does not take into account the complexity of

drug disposition within the whole animal. It was the purpose of this study to evaluate

pharmacokinetic data extrapolated from an ex vivo scenario involving three commonly

administered NSAIDs to athletic horses, attempting to mimic the typical time frame

surrounding Thoroughbred racing situations.

Materials and Methods

Animals

Six adult Thoroughbred horses (3 mares and 3 geldings) between the ages of 3 to

10 years, weighing between 495-563 kg, in athletic condition sufficient to work 1 mile in

2 minutes without undue stress were used in this study. General examination of soundness was evaluated prior to inclusion. Routine farriery is performed monthly and dental care (regular floating) is performed annually. Vaccination is administered for tetanus, Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), West

Nile Virus (WNV), Venezuelan Equine Encephalitis (VEE), influenza and rabies.

Treatment with a rotating schedule of deworming agents is conducted every 6-8 weeks.

The horses were maintained in their typical paddocks prior to drug administration and for the duration of sample collection. They were supplemented with their normal daily ration of sweet feed (Seminole Feed™) and coastal Bermuda hay. This study was approved by and performed in facilities inspected by the University of Florida

Institutional Animal Care and Use Committee (IACUC).

Standard Training Regimen for the University of Florida Equine Performance Lab

In an effort to mimic a stringent exercise program for professional Thoroughbred racehorses, subjects utilized for these studies were trained three days per week on a high-speed Treadmill (Mustang 2200™) before and throughout the duration of the study period. The horses were trained for at least 2 months prior to the initiation of these studies and were administered one Standard Exercise Test to verify the level of fitness for inclusion in the study. The standard training regimen consists of trotting for

0.6 km at 4.0 m/s, galloping for 2 km at 8 m/s, and trotting for 0.6 km at 4 m/s. On

Monday of each week, the training was conducted on a horizontal treadmill inclined at

6° from horizontal. Individual records were kept on each animal indicating date of

training, speed, duration of training, and whether samples were collected.

Incremental Exercise Test to Exhaustion

Following their initial two months of training, the horses were subjected to an

Incremental Exercise Test to Exhaustion. The horses were warmed up on the treadmill at 4 m/s for 5 minutes before the start of the test. The horses were then exercised for 1 minute each at 9,10, 11, 12, 13 and 14 m/s until they are unable to maintain the speed of the treadmill with humane encouragement including vocal commands and limited use

(3-5 strikes) of a driving whip. Blood samples were collected via needle puncture of the jugular vein for testing of pH and blood lactate, determining whether the horses have met the criteria required for demonstrating an adequate level of exercise stress. Horses demonstrating a blood pH <6.95 and a lactate concentration >20 mM procured at the time of failure to maintain speed on the treadmill, were included in this study.

Standard Condition Test to Verify the Ability to Gallop One Mile in Two Minutes

The standard training regimen is designed to condition the horses so that they can gallop 1 mile in 2 minutes and recover promptly as indicated by the rate of decrease in their heart rate after the 1-mile gallop. To be included in this study, a horse must periodically demonstrate its ability to meet this ‘mile in 2 minutes’ goal by performing an exercise test at a distance of 1 mile at 13 m/s on the high-speed treadmill. The exercise test was conducted after a 4 minute warm up period conducted at 4 m/s. Following completion of the aforementioned Standard Condition Test, the horse was allowed to recover at 4 m/s for 4 minutes and the heart rate was monitored every 5 minutes. If the heart rate drops below 50 beats/m within 40 minutes of completing the Standard

Condition Evaluation, the horse was considered to have met the fitness goal and was

declared fit to participate in the drug studies. This challenge was repeated annually and after resumption of training for any horse that may have experienced a significant interruption in its training program.

Drug Administration and Sample Collection

Phenylbute Injection solution™ was calculated at the recommended dose of 4.4 mg/kg of body weight for each horse. Ketofen™ was calculated at the recommended dose of 2.2 mg/kg body weight for each horse and FluMeglumine™ calculated at the recommended dose of 1.1 mg/kg body weight for each horse. Each individual drug was administered intravenously as a single bolus using a 20-gauge hypodermic needle and syringe via the left jugular vein of each horse on Monday of the collection week for three weeks. Whole blood samples (7 mL) were collected utilizing venipuncture with a 20- gauge vacutainer needle of the jugular vein of each horse immediately prior to drug administration, 15 and 30 min and 1, 2, 3, 4, 6,8, 24, 48 and 72 h following drug administration. Samples destined for the pharmacokinetic analysis were directly collected into lithium heparinized, vacuumed blood collection tubes and maintained on ice until further processing. The blood samples harvested for pharmacokinetic analysis were immediately centrifuged at 1318 x g (3000 rpm) for 10 min at 4°C. Following

centrifugation, the plasma was separated and aliquoted into Cryovial storage tubes at -

80°C until drug analysis could be performed.

Drug Analysis

Chemicals and Reagents

Analytical grade drug standards including phenylbutazone (phenylbutazone-d9)™,

ketoprofen (d3-ketoprofen)™ and flunixin meglumine (d3-flunixin)™ were used for this

analysis. Reagent grade formic acid was obtained from ACROS Organics™. All

solvents including acetonitrile, methanol and dichloromethane were High Pressure

Liquid Chromatography (HPLC) grade and obtained from Thermo Fisher™. All of the

methods described in further detail below, were validated according to

recommendations made by the Food and Drug Administration (FDA).

Phenylbutazone

Sample preparation

All stock standard solutions were prepared from solid form and dissolved in

methanol. All working standard solutions were diluted to the appropriate concentrations

in methanol to prepare calibrators in plasma from 0.005-2.5 ng/mL. Calibrators and

positive control samples were prepared from independently prepared stock solutions.

Each calibrator and positive control sample was prepared from 1 mL of 0.2 M

phosphate buffer (pH 3.0) and 0.1 mL of drug-free control horse plasma, and fortified

with the appropriate volume of phenylbutazone working standard solution and 25 μL of

phenylbutazone-d9 working standard solution. The deuterated phenylbutazone

analogue was prepared in a working internal standard solution at a concentration of

1.25 ng/μL. The final internal standard concentration was 10 ng/mL of plasma.

In duplicate, a 0.1 mL aliquot of each plasma sample was added to 0.1 mL of ultra pure deonized water (with a resistivity greater than or equal to 18 mega ohms and organic content less than 10 ppb) and 20 μL of 1.25 ng/μL internal standard working solution in 15-mL screw cap disposable, centrifuge tubes. To adjust the pH to 3.5, 75

μL of 1 M phosphoric acid (H3PO4) was added followed by 3 mL of methyl tert butyl

ether. The tubes were roto-racked for 10-15 minutes to allow for thorough mixing and

then centrifuged for 15-20 minutes at 1508 x g (2800 rpm) or until adequate phase

separation was achieved. The top aqueous layer was aspirated into waste and a

constant volume (2 mL) of the organic layer was transferred into a clean 5 mL conical tube. The contents were evaporated under nitrogen on a TurboVap® LV evaporator .

Sample extracts were then dissolved in 100-150 μL of acetonitrile:water (30:70) containing 0.1% (v/v) formic acid and transferred to glass autosampler vials.

Instrumentation

LC/MS/MS analysis was performed on a Triple Stage Quadrupole (TSQ) Quantum

Ultra Mass spectrometer™ equipped with a heated electrospray ionization (HESI) source interfaced with a HTC PAL autosampler™ and Accela LC pump™. Xcaliber™ software version 2.0.7 and LCquan version 2.5.6 were used for data acquisition and analysis. Chromatographic separations were achieved with an Acquity™ UPLC HSS T3

(2.1 mm x 50 mm x 1.8 μm) column and a 2.1 mm x 5 mm, identically packed, guard column™. Gradient elution was begun with a mobile phase of 0.1% (v/v) formic acid in water (70%) (Solvent A) and 0.1% (v/v) formic acid in acetonitrile (30%) (Solvent B).The initial mixture, kept constant at a 400 μL/min flow rate, was held for 0.59 min, then

Solvent A was decreased linearly to 0% and Solvent B increased to 100 % over 5.40 minutes. The mobile phase was then returned to the initial conditions for the remaining

0.1 min for a total run time of 6.0 minutes. A divert valve was employed from 0-1.0 min and 2.5-4.0 minutes. The column temperature was isothermal at 45°C and a total of 20

μL of the sample extract dissolved in 100 μL of acetonitrile:water (20:80) containing

0.1% formic acid was injected. Mass spectral data were acquired in positive ion mode using the HESI and the following MS parameters: ESI spray voltage-3000, vaporizer

temperature-198°C, sheath gas pressure- 40, ion sweep gas-0, auxiliary gas pressure-

15, capillary temperature- 248°C, tube lens offset- 89, and skimmer offset- 10.

Identification and quantification of the analyte were based on selected reaction monitoring (SRM). Compound specific optimization (tuning) of MS/MS parameters was performed before sample via direct infusion of 10 ng/µL each of the analyte and internal standard dissolved in mobile phase. Tuning for phenylbutazone yielded collision energies of 32, 19, and 16 for transitions 309→92, 309→120, and 309→188, respectively. Tuning for phenylbutazone-d9 yielded a collision energy of 19 and tube

lens offset of 105 for transition 309→120.

Data analysis

The most abundant ion transmission (188→120) for the analyte was used for

quantification. The second and third most abundant transitions were used as qualifier

transitions. All standards, controls, calibrators, and samples were prepared in duplicate

and peak ion area ratios of the target analyte and internal standard were calculated for

each. Individual values of the duplicate concentrations were averaged. Calibration was

performed using a simple least squares regression analysis with a 1/Cp weighting factor, where Cp was the nominal plasma concentration. Quality control and sample

acceptance criteria have been outlined according to the following guidelines and

standard operating procedures of the University of Florida Racing Laboratory, Research

Division. The requirement is that the %CV for all calibrators, positive controls, and

samples must not exceed 10% (15% at the LLOQ). In addition, for calibrators the

difference between the back-calculated concentration and the nominal concentration

must not exceed 10% (15% at the LLOQ). All samples that did not meet such criteria

were re-analyzed.

Ketoprofen

Sample preparation

All stock standard solutions were prepared from solid form and dissolved in

methanol. All working standard solutions were diluted to the appropriate concentrations

in methanol to prepare calibrators in plasma from 0.1-100 ng/mL. Calibrators and

positive control samples were prepared from independently prepared stock solutions.

Each calibrator and positive control sample were prepared from 1 mL of drug-free control horse plasma, and fortified with the appropriate volume of ketoprofen working standard solution and 20 µL of d3-ketoprofen working standard solution. The deuterated ketoprofen analogue was prepared in a working standard solution at a concentration of

0.5 ng/μL. The final deuterated internal standard concentration was 10 ng/mL of plasma.

In duplicate, a 1.0 mL aliquot of each plasma sample was added to 1.0 mL of ultra pure de-ionized water (with a resistivity greater than or equal to 18 mega ohms and organic content less than 10 ppb) and 20 μL of 0.5 ng/μL internal standard working

solution in 15-mL screw cap disposable, centrifuge tubes. To adjust the pH to 3.5, 100

µL of 1M phosphoric acid (H3PO4) was added followed by 5 mL of dichloromethane.

The tubes were roto-racked for 10 min to allow for thorough mixing and then centrifuged

for 16 min at 1508 x g (2800 rpm) or until adequate phase separation was achieved.

The top aqueous layer was aspirated into waste and a constant volume (3-4 mL) of the organic layer was transferred into a clean 5 mL conical tube. The contents were evaporated under nitrogen on a TurboVap® LV evaporator™. Sample extracts were then dissolved in 100μL of acetonitrile:methanol:water (10:30:60) containing 0.1% formic acid and transferred to glass autosampler vials.

Instrumentation

LC/MS/MS analysis was performed on a Triple Stage Quadrupole (TSQ) Quantum

Ultra mass spectrometer™ equipped with a heated electrospray ionization (HESI) source interfaced with a HTC PAL autosampler™ and Accela LC pump™. Xcaliber™ software version 2.0.7 and LCquan version 2.5.6 were used for data acquisition and analysis.

Chromatographic separations were achieved with an Agilent Eclipse XDB-C18 (1 mm x 50 mm x 3.5 μm) column™ and a generic pre-column filter. A ternary gradient elution was begun with a mobile phase of 0.1% (v/v) formic acid in water (60%) (Solvent

A), 0.1% (v/v) formic acid in methanol (30%) (Solvent B) and 0.1% (v/v) formic acid in acetonitrile (10%) (Solvent C). The initial mixture, kept constant at a 300 μL/min flow rate, was held for 0.75 min, then Solvent A was decreased linearly to 0% and Solvent B increased to 100 % over 4.0 min and held for 1.0 min. The mobile phase was then returned to the initial conditions for the remaining 1.0 min for a total run time of 6.0 min.

A divert valve was employed from 0-3.50 min and 5.50-6.0 min. The column temperature was isothermal at 45°C and a total of 20 μL of the sample extract dissolved

in 100 μL of water:methanol:acetonitrile (60:30:10) containing 0.1% formic acid was

injected. Mass spectral data were acquired in positive ion mode using the HESI and the

following MS parameters: ESI spray voltage-4500, vaporizer temperature-200°C, sheath

gas pressure- 60, ion sweep gas-8, auxiliary gas pressure- 15, capillary temperature-

398°C, tube lens offset- 89, and skimmer offset- 10.

Identification and quantification of the analyte were based on selected reaction

monitoring (SRM). Compound specific optimization (tuning) of MS/MS parameters was

performed before analyses via direct infusion of 10 ng/µL each of the analyte and internal standard dissolved in mobile phase. Tuning for ketoprofen yielded collision energies of 37, 21, and 10 for transitions 255→76, 255→104, and 255→208, respectively. Tuning for d3-ketoprofen yielded a collision energy of 12 and tube lens

offset of 98 for transition 258→212.

Data analysis

The most abundant ion transmission (255→208) for the analyte was used for

quantification. The second and third most abundant transitions were used as qualifier

transitions. All standards, controls, calibrators, and samples were prepared in duplicate

and peak ion area ratios of the target analyte and internal standard were calculated for

each. Individual values of the duplicate concentrations were averaged. Calibration was

performed using a simple least squares regression analysis with a 1/Cp weighting factor, where Cp was the nominal plasma concentration. Quality control and sample

acceptance criteria have been outlined according to the following guidelines and

standard operating procedures of the UF Racing Laboratory, Research Division. The

requirement is that the %CV for all calibrators, positive controls, and samples must not

exceed 10% (15% at the LLOQ). In addition, for calibrators the difference between the

back-calculated concentration and the nominal concentration must not exceed 10%

(15% at the LLOQ). All samples that did not meet such criteria were re-analyzed.

Flunixin Meglumine

Sample preparation

All stock standard solutions were prepared from solid form and dissolved in

methanol. All working standard solutions were diluted to the appropriate concentrations

in methanol to prepare calibrators in plasma from 0.005-50 ng/mL. Calibrators and

positive control samples were prepared from independently prepared stock solutions.

Each calibrator and positive control sample were prepared from 1 mL of 0.2 M

phosphate buffer (pH 3.0) and 1.0 mL of drug-free control horse plasma, and fortified

with the appropriate volume of flunixin working standard solution and 20 µL of flunixin-d3 working standard solution. The deuterated flunixin analogue was prepared in a working standard solution at a concentration of 0.5 ng/μL. The final deuterated internal standard concentration was 10 ng/mL of plasma.

In duplicate, a 1.0 mL aliquot of each plasma sample was added to 1 mL of phosphate buffer (0.2 M, pH 3.0) and 20 μL of 0.5 ng/μL internal standard working solution in 5-mL disposable, centrifuge tubes. The tubes were centrifuged at 1508 x g

(2800 rpm) for 12 min and the buffered plasma samples were subjected to solid phase extraction. Oasis HLB 3-mL columns™ were sequentially conditioned with 2 mL each of methanol, water, and phosphate buffer. Buffered plasma specimens were loaded onto the columns and a positive pressure sufficient to achieve a flow rate of no more than 2 mL per minute was applied. The columns were sequentially washed with 2 mL each of water and methanol:water (10:90). The analyte was eluted with two 1-mL aliquots of hexanes:ethyl acetate (50:50). The elute was evaporated under nitrogen on a

TurboVap® LV evaporator™. Sample extracts were then dissolved in 100μL of

acetonitrile:water (10:90) containing 0.1% (v/v) formic acid and transferred to glass

autosampler vials.

Instrumentation

LC/MS/MS analysis was performed on a Triple Stage Quadrupole (TSQ) Quantum

Ultra mass spectrometer™ equipped with a heated electrospray ionization (HESI)

source interfaced with a HTC PAL autosampler™ and Accela LC pump™. Xcaliber™ software version 2.0.7 and LCquan version 2.5.6 were used for data acquisition and analysis.

Chromatographic separations were achieved with an Acquity™ UPLC HSS T3 (5 mm x 2 mm x 1.8 μm) column and a 2.1 mm x 5 mm, identically packed, guard column™.

Gradient elution was begun with a mobile phase of 0.1% (v/v) formic acid in water

(70%) (Solvent A) and 0.1% (v/v) formic acid in acetonitrile (30%) (Solvent B).The initial mixture, kept constant at a 300 μL/min flow rate, was held for 0.5 min, then Solvent A was decreased linearly to 0% and Solvent B increased to 100 % over 2.75 min and held for 0.5 min. The mobile phase was then returned to the initial conditions for the remaining 0.75 min for a total run time of 4.0 min. A divert valve was employed from 0-

1.0 min and 2.5-4.0 min. The column temperature was isothermal at 45°C and a total of

20 μL of the sample extract dissolved in 100 μL of acetonitrile:water (20:80) containing

0.1% (v/v) formic acid was injected. Mass spectral data were acquired in positive ion mode using the HESI and the following MS parameters: ESI spray voltage-3000, vaporizer temperature-198°C, sheath gas pressure- 40, ion sweep gas-0, auxiliary gas pressure- 15, capillary temperature- 248°C, tube lens offset- 89, and skimmer offset- 10.

Identification and quantification of the analyte were based on selected reaction monitoring (SRM). Compound specific optimization (tuning) of MS/MS parameters was performed before analyses via direct infusion of 10 ng/µL each of the analyte and internal standard dissolved in mobile phase. Tuning for flunixin yielded collision energies of 48, 38, and 23 for transitions 297→108, 297→264, and 297→279,

respectively. Tuning for d3-flunixin yielded a collision energy of 23 and tube lens offset of 133 for transition 300→281.

Data analysis

The most abundant ion transmission (297→279) for the analyte was used for quantification. The second and third most abundant transitions were used as qualifier transitions. All standards, controls, calibrators, and samples were prepared in duplicate and peak ion area ratios of the target analyte and internal standard were calculated for each. Individual values of the duplicate concentrations were averaged. Calibration was performed using a simple least squares regression analysis with a 1/Cp weighting factor, where Cp was the nominal plasma concentration. Quality control and sample

acceptance criteria have been outlined according to the following guidelines and

standard operating procedures of the UF Racing Laboratory, Research Division. The

requirement is that the %CV for all calibrators, positive controls, and samples must not

exceed 10% (15% at the LLOQ). In addition, for calibrators the difference between the

back-calculated concentration and the nominal concentration must not exceed 10%

(15% at the LLOQ). All samples that did not meet such criteria were re-analyzed.

Pharmacokinetic Modeling

Pharmacokinetic analysis was carried out by non-linear least squares regression

analysis determined through the use of a commercially available software package

(Phoenix WinNonlin 6.1 NLME 1.0 BUILD 6.1.0.173™). The calculations presented

were based on equations of pharmacokinetics described by Gabrielsson and Weiner41.

A two-compartmental model was determined the best fit for the data including all three

drugs in all six horses determined upon visual inspection of the data on a

semilogarthmic graph. The compartmental analysis was utilized to determine the area

under the plasma concentration versus time curve (AUC), calculated using the

trapezoidal rule and extrapolated to infinity. The values for maximum plasma

concentration (Cmax) are reported directly from the data. All values are reported as

mean ± SD, unless otherwise noted.

Results

Phenylbutazone

No adverse effects were observed throughout the course of this study. Plasma concentrations of phenylbutazone determined by LC/MS/MS, along with typical statistical values for all six horses in the study were determined (Table 2-1). The plasma concentrations of phenylbutazone versus time for all six horses were plotted for up to 4 hours (Figure 2-1) and 48 hours (Figure 2-2). The plasma concentration for all six horses at the 72 hour time point was below the limit of quantitation (< 10 ng/mL) and not included in the graphical interpretation. Phenylbutazone exhibited a long distribution half-life of 2.66 ± 3.40 h, as well as elimination half life of 117.7 ± 280 h. The median

(with range) Cmax determined was 29.8 (16.7 – 43.2) μg/mL which occurred at 0.29 ± 0.1

h following administration. Additional relevant pharmacokinetic parameters are

summarized in Table 2-2.

Ketoprofen

No adverse effects were observed throughout the course of this study. Plasma

concentrations of ketoprofen determined by LC/MS/MS, along with typical statistical

values for all six horses in the study were determined (Table 2-3). The plasma

concentrations of ketoprofen versus time disposition for all six horses were plotted for

up to 4 hours (Figure 2-3) and 24 hours (Figure 2-4). The plasma concentration for all

six horses at the 48 and 72 hour time points were below the limit of quantitation (< 0.25

ng/mL) and not included in the graphical interpretation. Ketoprofen exhibited a shorter

distribution half-life of 0.461 ± 0.082 h and a longer elimination half life of 3.05 ± 0.12 h.

The median (with range) Cmax reached was 6.86 (5.41 – 8.13) μg/mL, which occurred at

0.25 h following administration. Additional relevant pharmacokinetic parameters are

summarized in Table 2-4.

Flunixin Meglumine

No adverse effects were observed throughout the course of this study. Plasma

concentrations of flunixin meglumine determined by LC/MS/MS, along with typical

statistical values for all six horses in the study were determined (Table 2-5). The

plasma concentrations of flunixin meglumine versus time disposition for all six horses

were plotted for up to 4 hours (Figure 2-5) and 72 hours (Figure 2-6). The mean plasma

concentration at the 48 and 72 hour time points (0.001 μg/mL for each) were still above

both the LLOQ at 0.05 ng/mL (0.00005 μg/mL) and the LOD 0.01 ng/mL (0.00001

μg/mL). Flunixin meglumine exhibited a short distributional half life at 2.05 ± 0.2 h and

an elimination half life at 13.1 ± 3.21 h. The median (with range) Cmax reached was 6.51

(4.84-8.06) μg/mL, which occurred at 0.25 h following administration. Additional relevant pharmacokinetic parameters are summarized in Table 2-5.

Discussion

Although a large and diverse family, the pharmacology of most NSAIDs consists of

shared characteristics including a common mechanism of action leading to their

beneficial therapeutic actions. Additionally, many of the commonly administered

NSAIDs used on horses share similar adverse effects including gastrointestinal and

renal toxicity. Due to their commonality in administration, especially in racehorses, they

represent a group of drugs with extensive metabolic examination 42. Generally, the

volume of the central compartment along with the volume of distribution are low for

NSAIDs, most likely explained by their high propensity for protein binding 13. The half- life of most NSAIDs is relatively short which is typical of acidic drugs, once again, extensively bound to plasma proteins.

The pharmacokinetic parameters of phenylbutazone have been particularly scrutinized due to its long-time history as a common analgesic potentially abused in racehorses, although the majority of reports are 20-30 years old at this point in time. A more recent study, published in 2010, looked at the differences in pharmacokinetic parameters of phenylbutazone and dexamethasone in both resting and exercising horses to determine if exercise was a factor affecting the pharmacokinetic parameters43.

Although similar to the current study, their exercise protocol aimed to mimic a more rigorous endurance-type training regime which lasted for 3 hours and was hypothesized that a shorter amount of exercise (lasting a few minutes) would likely not alter drug concentrations in plasma or urine. The half-life determined from the horses in this study

(3.44 ± 2.42 h) was shorter than the values reported from the aforementioned study for both resting and exercising horses (7.42 ± 1.61 and 7.43 ± 0.99 h respectively). A similar half life was reported in a study conducted by Soma et al. with a half life reported at 6.16 h44. It is possible that the larger dose administered in both studies (8 mg/kg)

versus the recommended dose used in this study (4.4 mg/kg) created a prolonged half-

life. The current recommendations published by the RMTC establish a threshold limit of

2 μg/mL for racehorses. All six horses in this study demonstrated plasma

concentrations of phenylbutazone below this threshold limit at 24 h although one horse demonstrated a plasma concentration at 24 h close to the threshold limit (1.71 μg/mL).

The data reported from this study represents the most current pharmacokinetic parameters based on a typical one-time i.v. bolus administration of phenylbutazone.

Ketoprofen is a propionic acid based NSAID approved for the use in horses for alleviation of pain induced by inflammation and musculoskeletal disorders and by definition, contains a chiral center with two enantiomers. Although marketed and administered as a racemic mixture, it has previously been determined that each enantiomer must be regarded as two distinct drugs13 as stereoselective pharmacokinetics have been reported. One study in particular has already addressed the differences of pharmacokinetics involving the two enantiomers, S (+) and R (-), with regards to Ketoprofen45. Since the purpose of this study focused on the clinical effects of each drug, the pharmacokinetics included the total plasma concentration following the administration of the racemate. The published recommended threshold limits for ketoprofen through the RMTC are 0.01 μg/mL. All of the horses in this study had plasma concentrations below the allowable threshold at 24 h, but not at 8 h. The lower limits of quantitation (LLOQ) revealed in this study was 0.25 ng/mL, which is significantly lower than previously reported limits 10 ng/mL, 25 ng/mL and 100 ng/mL45-

47 respectively. The mean unconjugated plasma ketoprofen concentrations 15 minutes

after one administration were 6.94 ± 1.08 μg/mL, which is similar to previously reported

levels of 8.90 ± 1.90 μg/mL46. Sams et al. also reported plasma concentrations of 0.031

± 0.024 μg/mL at the 8 hour time point which is similar to the concentrations reported in

this study 0.014 ± 0.00 μg/mL. However, the ability to have a lower limit of quantitation

allowed for detection of plasma concentrations of ketoprofen for up to 24 hours following a one-dose administration. The terminal elimination half-life reported in this study (3.05

± 0.12 h) was longer than previously reported parameters (1.60 h and 1.09 h) 45,46, respectively. This could potentially be due to the lower limit of detection in the current study compared with the previous studies allowing for measurement of ketoprofen levels at a slower elimination phase.

Flunixin meglumine resides amongst the carboxylic acid nonsteroidal anti- inflammatory drugs. This NSAID has been classically used in treating horses for musculoskeletal pain and very commonly administered to treat pain associated with a

colic incident. Several racing jurisdictions allow this NSAID to be used up to 24 h prior

to a race and control potential abuse cases by establishing threshold limits. The current

threshold limit recommended by the RMTC is 20 ng/mL when collected following a race.

The concentrations determined in this study indicated that all but one horse (1/6) had

plasma concentrations of flunixin meglumine below the threshold limit established which

demonstrates the variability potential between horses. Previous studies have

demonstrated a half life of 2.46 ± 0.97 h31 which differs greatly from what this study revealed with a half life of 13.1± 3.21 h. This is most likely due to the differences in the

lower limit of quantification (LLOQ) detectable between each study. With the advanced

analytical techniques utilized in this study through LC/MS/MS, the LLOQ established

was 0.05 ng/mL versus previously reported limits of 0.1 μg/mL. The LLOQ established

for this study enabled the detection of flunixin meglumine up to 72 h, whereas the

previously established LLOQ were based upon concentrations only up to 24 h. This led

to an estimated half life much greater than previously reported which is an interesting

finding for this drug and a good example of the huge advancements made through the use of sophisticated analytical techniques.

Table 2-1. Plasma concentrations of phenylbutazone as determined by LC/MS/MS analysis. Concentrations are reported as μg/mL. Time(h) Annie Elle Ava Des Slip True Mean Median Min Max SD %CV Geomean 0 Below the limit of detection 0.25 43.2 20.1 36.4 26.3 33.3 16.7 29.3 29.8 16.7 43.2 10.1 8.09 27.8 0.5 39.4 15.9 31.4 22.2 26.6 10.3 24.3 24.4 10.3 39.4 10.5 8.43 22.2 1 27.6 11.0 27.5 18.2 17.5 8.83 18.4 17.9 8.83 27.6 7.92 6.34 16.9 2 27.1 10.8 23.2 17.4 16.1 6.47 16.8 16.7 6.47 27.1 7.61 6.09 15.2 3 22.4 9.91 17.5 17.7 10.7 5.61 14.0 14.1 5.61 22.4 6.25 5.00 12.7 4 18.7 8.32 6.29 15.2 8.2 6.32 10.5 8.25 6.29 18.7 5.20 4.16 9.58 6 14.3 5.94 3.94 12.1 7.02 4.41 7.95 6.48 3.94 14.3 4.27 3.42 7.07 8 13.2 3.26 3.54 6.76 6.16 4.32 6.20 5.24 3.26 13.2 3.69 2.95 5.49 24 0.83 0.47 0.75 1.71 0.70 0.49 0.83 0.73 0.47 1.71 0.45 0.36 0.75 48 0.07 0.03 0.06 0.10 0.04 0.02 0.05 0.05 0.02 0.10 0.03 0.02 0.05 72 Below 10.0 ng/mL (Lower limit of Quantitation)

Figure 2-1. Plasma elimination of phenylbutazone represented by concentration versus time up to 4 h post drug administration.

Figure 2-2. Plasma elimination of phenylbutazone represented by concentration versus time up to 48 h post drug administration.

Table 2-2. Relevant pharmacokinetic parameters for phenylbutazone following a 2- compartmental analysis. Pharmacokinetic Variable Mean ± SD T1/2alpha (h) 2.35 ± 3.71 T1/2beta (h) 5.73 ± 0.99 Tmax (h) 0.30 ± 0.11 Cmax (μg/mL) 34.3 ± 15.6 -1 k10 (h ) 0.30 ± 0.20 t1/2k10 (h) 3.44 ± 2.42 -1 k12 (h ) 1.14 ± 1.98 -1 k21 (h ) 0.47 ± 0.59 V1 (L/kg) 0.17 ± 0.11 V2 (L/kg) 3.53 ± 7.57 Cl (mL/h/kg) 37.9 ± 14.2 AUC (h*μg/mL) 132.5 ± 58.2 T1/2alpha, half-life of distribution; T1/2beta, half-life of elimination; Tmax,, time of maximum concentration; Cmax, maximum concentration; k10, first-order elimination rate constant; t1/2k10, half-life of elimination; k12, rate of transfer from central to peripheral compartment; k21, rate of transfer from peripheral to central compartment; V1, volume of distribution for the central compartment; V2, volume of distribution for peripheral compartment; Cl, systemic clearance; AUC, area under plasma concentration-time curve.

Table 2-3. Plasma concentrations of ketoprofen as determined by LC/MS/MS analysis. All concentrations are reported as μg/mL. Time (h) Annie Tia Ava Des Slip True Mean MedianMin MaxSD %CV Geomean 0 Below the limit of detection 0.25 8.07 8.13 6.45 7.26 5.41 6.31 6.94 6.86 5.4 8.1 1.08 15.5 6.86 0.5 3.82 4.14 3.06 4.02 3.01 3.38 3.57 3.60 3.0 4.1 0.49 13.8 3.54 1 1.50 1.91 1.24 1.66 1.62 1.17 1.52 1.56 1.2 1.9 0.28 18.3 1.49 2 0.32 0.45 0.27 0.48 0.59 0.32 0.40 0.39 0.3 0.6 0.12 30.3 0.39 3 0.13 0.18 0.11 0.17 0.24 0.12 0.16 0.15 0.1 0.2 0.05 29.5 0.15 4 0.07 0.09 0.07 0.08 0.11 0.07 0.08 0.07 0.1 0.1 0.02 20.9 0.08 6 0.03 0.04 0.03 0.03 0.03 0.02 0.03 0.03 0.0 0.0 0.00 13.2 0.03 8 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.0 0.0 0.00 23.4 0.01 24 0.00061 0.00069 0.00036 0.00033 0.00037 0.00036 0.000 0.000 0.0 0.0 0.00 34.8 0.00 48 Below 0.25 ng/mL (Lower limit of Quantitation) 72 Below 0.25 ng/mL (Lower limit of Quantitation)

Figure 2-3. Plasma elimination of ketoprofen represented by concentration versus time up to 4 h post drug administration.

Figure 2-4. Plasma elimination of ketoprofen represented by concentration versus time up to 24 h post drug administration.

Table 2-4. Relevant pharmacokinetic parameters for ketoprofen following a 2- compartmental analysis. Pharmacokinetic Variable Mean ± SD T1/2alpha (h) 0.46 ± 0.08 T1/2beta (h) 3.05 ± 0.12 Tmax (h) 0.25 ± 0.00 Cmax (μg/mL) 7.42 ± 1.50 -1 k10 (h ) 1.43 ± 0.20 t1/2k10 (h) 0.50 ± 0.09 -1 k12 (h ) 0.02 ± 0.01 -1 k21 (h ) 0.25 ± 0.01 V1 (L/kg) 307.1 ± 63.3 V2 (L/kg) 112.5± 15.9 Cl (mL/h/kg) 430.0 ± 59.7 AUC (h*μg/mL) 5.21 ± 0.79 T1/2alpha, half-life of distribution; T1/2beta, half-life of elimination; Tmax,, time of maximum concentration; Cmax, maximum concentration; k10, first-order elimination rate constant; t1/2k10, half-life of elimination; k12, rate of transfer from central to peripheral compartment; k21, rate of transfer from peripheral to central compartment; V1, volume of distribution for central compartment; V2, volume of distribution for peripheral compartment; Cl, systemic clearance; AUC, area under plasma concentration-time curve.

Table 2-5. Plasma concentrations of flunixin meglumine as determined by LC/MS/MS analysis. All values are reported as μg/mL. Time Annie Tia Ava Des Slip True Mean MedianMin Max SD %CV Geomean (h) 0 Below the limit of detection 0.25 8.06 6.77 4.84 6.45 6.57 5.66 6.39 6.51 4.84 8.06 1.086 17.0 6.31 0.5 5.71 6.09 3.25 5.62 5.06 4.86 5.10 5.34 3.26 6.09 1.011 19.8 5.0 1 4.00 4.37 3.02 4.29 3.63 3.98 3.88 3.99 3.02 4.36 0.496 12.8 3.85 2 2.29 2.55 2.58 3.08 2.47 2.74 2.62 2.56 2.29 3.08 0.271 10.4 2.61 3 1.53 1.86 1.96 2.07 1.59 1.60 1.77 1.73 1.53 2.07 0.226 12.8 1.76 4 1.07 1.18 1.28 1.48 1.05 1.17 1.21 1.17 1.07 1.48 0.159 13.1 1.20 6 0.57 0.63 0.80 0.82 0.54 0.61 0.66 0.62 0.54 0.82 0.119 17.9 0.65 8 0.29 0.29 0.39 0.42 0.31 0.33 0.34 0.32 0.29 0.42 0.054 15.9 0.33 24 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.004 29.5 0.01 48 0.00129 0.00103 0.001150.00168 0.00095 0.00074 0.001 0.001 0.0 0.00 0.000 28.1 0.001 72 0.00070 0.00049 0.000440.00073 0.00053 0.00033 0.001 0.001 0.0 0.00 0.000 28.3 0.0005

Figure 2-5. Plasma elimination of flunixin meglumine represented by concentration versus time up to 4 h post drug administration.

Figure 2-6. Plasma elimination of flunixin meglumine represented by concentration versus time up to 72 h post drug administration.

Table 2-6. Relevant pharmacokinetic parameters for flunixin meglumine following a 2- compartmental analysis. Pharmacokinetic Variable Mean ± SD T1/2alpha (h) 2.05 ± 0.20 T1/2beta (h) 13.1± 3.21 Tmax (h) 0.25 ± 0.00 Cmax (μg/mL) 5.04 ± 0.72 -1 k10 (h ) 0.33 ± 0.03 t1/2k10 (h) 2.08 ± 0.03 -1 k12 (h ) 0.004 ± 0.00 -1 k21 (h ) 0.05 ± 0.01 V1 (L/kg) 0.218 ± 0.03 V2 (L/kg) 0.014 ± 0.003 Cl (mL/h/kg) 72.7 ± 6.33 AUC (h*μg/mL) 15.1 ± 1.32 T1/2alpha, half-life of distribution; T1/2beta, half-life of elimination; Tmax,, time of maximum concentration; Cmax, maximum concentration; k10, first-order elimination rate constant; t1/2k10, half-life of elimination; k12, rate of transfer from central to peripheral compartment; k21, rate of transfer from peripheral to central compartment; VI, volume of distribution for central compartment; V2, volume of distribution for peripheral compartment; Cl, systemic clearance; AUC, area under plasma concentration-time curve.

CHAPTER 3 THE DURATION OF CYCLOOXYGENASE INHIBITION FOR PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION

Background

Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most

commonly administered pharmaceutical therapies in veterinary medicine. In modern

veterinary medicine the uses of NSAIDs are varied, from lowering body temperature in

animals with fever, to relieving respiratory distress in calves and piglets along with

controlling postoperative pain in both small and large animals13. Equine veterinarians,

in particular, have historically reached into their pharmacy cabinets for an NSAID to

treat pain associated with a colic event or musculoskeletal ailment. The mechanism of

action with which NSAIDs exert their therapeutic effect, involves inhibition of the

inflammatory mediator, cyclooxygenase (COX) which catalyses the conversion of

membrane bound arachidonic acid into prostanoids and thromboxanes22. In 1990, it was discovered that two isoforms of COX existed13. The isoform commonly referred to

as COX-1 has been considered the “housekeeping” isoform, constitutively expressed,

and mediates basic functions such as platelet aggregation, renal blood flow regulation

and gastric cytoprotection. The other isoform, COX-2 is considered to be a pro-

inflammation mediator and induced by noxious stimuli such as endotoxins, cytokines,

stress and injury48.

A variety of data has been accumulated regarding the efficacy in veterinary

species of both classic and newly synthesized NSAIDs that are COX isoform-selective

and sparing. Researchers have used an assortment of mechanisms to compare the effects of different NSAIDs but the comparisons made by different studies have been

difficult to correlate due to variations in study methods. Whole blood assays are well recognized as a convenient way to study the in vitro biochemical efficacy and selectivity of NSAIDs, and are the preferred method48. The advantages of utilizing a whole blood

assay are that: (1) they can be used in vitro in the pre-clinical assessment of COX

inhibitors as well as ex vivo; (2) they are sufficiently accurate to estimate potency and

selectivity for time-dependent COX inhibitors; (3) they compare clinically relevant target

cells (platelets and monocytes); and (4) they accommodate the drug to plasma protein

binding that occurs in vivo49.

It was one of the aims of this study to evaluate the percent inhibition induced by a one-time administration of three commonly utilized NSAIDs (phenylbutazone, ketoprofen, and flunixin meglumine) in athletic Thoroughbred horses, using a whole

blood assay. The goal of this study is to define the timeline for an NSAID’s ability to

provide inflammatory inhibition.

Materials and Methods

Animals

Six adult Thoroughbred horses (3 mares and 3 geldings) between the ages of 3 to

10 years, weighing between 495-563 kg, in athletic condition sufficient to work 1 mile in

2 minutes without undue stress were used in this study. Routine farriery was performed

monthly and dental care (regular floating) was performed annually. Vaccination for

tetanus, Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), West

Nile Virus (WNV), Venezuelan Equine Encephalitis (VEE), influenza and rabies was

conducted annually. Treatment with a rotating schedule of deworming agents was

conducted every 6-8 weeks. The horses were maintained in their typical paddocks for

the duration of this study.

The horses were supplemented with daily ration of sweet feed (Seminole Feed™)

twice daily and coastal Bermuda hay ad libidium. The study protocol was approved by

and performed in facilities inspected by the University of Florida Institutional Animal

Care and Use Committee (IACUC).

Standard Training Regimen for the University of Florida Equine Performance Lab

Standard training regimen, incremental test to exhaustion, and standard condition

test to verify the ability to gallop one mile in two minutes was previously described in

detail previously (Chapter 2).

Drug Administration and Sample Collection

Each NSAID was administered to each horse at the manufacture’s recommended dose rate, Phenylbute Injection solution™ at 4.4 mg/kg of body weight, Ketofen™ was

calculated at 2.2 mg/kg body weight, and FluMeglumine™ calculated at the recommended dose of 1.1 mg/kg body weight. Each drug was administered intravenously as a single dose through a 20-gauge hypodermic needle placed in the

right jugular vein. A one week washout period was imposed between administrations.

Whole blood samples (7 mL) were collected via venipuncture with a 20-gauge

vacutainer needle of the left jugular vein immediately prior to drug administration, and at

15 and 30 minutes and 1, 2, 3, 4, 6, 8, 24, 48 and 72 h following drug administration.

Blood samples destined for the measurement of prostaglandin E2 (PGE2) were

collected into partially evacuated blood collection tubes containing lithium heparin, and maintained on ice immediately following collection until processed. Samples to be assayed for thromboxane B2 (TXB) concentrations, were collected into partially

evacuated sterile blood collection tubes containing no anti-coagulant and maintained on ice until processed.

The samples obtained for COX-2 inhibition assay were stimulated for prostaglandin E2 (PGE2) production by incubating with 1 μg/mL lipopolysaccharide

(LPS, E. coli, serotype 0111:B4™) within 0.1% bovine serum albumin (Albumin, bovine fraction V™) in phosphate buffered saline (PBS) for 24 hours, as previously described48,50-52. One sample from each horse, was not incubated with LPS, to serve as

a negative control. An additional sample, obtained prior to drug administration, was

incubated with LPS to serve as a stimulated positive control. Following incubation,

meclofenamate sodium salt (100 μL mMol solution) was added to each sample tube and

53 gently inverted 3-5 times to mix thoroughly and halt the production of PGE2 . The

samples were then centrifuged at 20,000 x rpm for 10 minutes at 4°C and the plasma

separated and pipetted into 1 mL aliquots into cryovials to be preserved at -80°C until

the PGE2 assay was performed.

The blood serum samples procured for the COX-1 inhibition assay were allowed

to clot for 1 hour at 37°C to allow for maximal production of thromboxane (TXB2) as

previously described 48,50-52 and then centrifuged at 20,000 x rpm for 10 minutes at 4°C.

The serum was harvested and dispensed into 1 mL aliquots in cryovials for storage at -

80°C until assayed for thromboxane (TXB2). A sample obtained prior to drug

administration was immediately placed on ice and centrifuged at 20,000 x rpm for 10

minutes at 4°C and the serum dispensed into 1 mL aliquots for storage at -80°C to

serve as a negative control. Another sample obtained prior to drug administration,

incubated for 1 h at 37°C, served as a stimulated positive control.

Ex vivo COX-1 Assay

The aliquots of serum previously frozen for storage were allowed to thaw at room

temperature. Immediately after thawing, 50 μL of serum was added to 450 μL of methanol and centrifuged at 13,000 rpm for 5 minutes at 4°C. Following centrifugation,

400 μL of the methanol phase was extracted and added to a microfuge tube to be dried down. A CentriVap machine was utilized to dry down the samples overnight at 35°C in order to precipitate the protein component. The resulting precipitant was then

reconstituted with 400 μL TXB2 enzyme immunoassay (EIA) kit buffer, to yield a final 10- fold dilution. The TXB2 levels were then determined according to manufacturer’s

instructions for the commercially available TXB2 kit (Thromboxane B2 Express EIA

Kit™). The baseline and coagulation stimulated samples were determined and expressed as a mean ± standard error (SE).

Ex vivo COX-2 Assay

The aliquots of plasma previously prepared and frozen were allowed to thaw at room temperature. A 100 μL sample of plasma was added to 900 μL methanol maintaining a 1:10 dilution, vortexed to mix, and then centrifuged at 13,000 rpm for 5 minutes. Following centrifugation, 850 μL of the supernatant was extracted and placed into a microfuge tube. All samples were positioned within the CentriVap and allowed to dry down overnight at 35°C to precipitate the protein. The next day, the resulting precipitant was reconstituted with 170 μL PGE2 enzyme immunoassay (EIA) kit

buffer to achieve a 1:2 dilution for accurate results. The PGE2 levels were then determined according to manufacturer’s directions for the commercially available PGE2

kit (Prostaglandin E2 Express EIA Kit™). The baseline and LPS stimulated samples

were determined and expressed as a mean ± standard error (SE).

Statistical Analysis

The effect of coagulation and LPS stimulation on TXB2 and PGE2 maximally

stimulated samples respectively and was compared to inhibition through treatment with

phenylbutazone, ketoprofen and flunixin meglumine utilizing a two-way ANOVA with a

Dunnett’s post hoc test. All calculations were performed through a commercially available software program (SPSS™). Statistical significance was established at p<0.05.

Results

Ex vivo Inhibition of Phenylbutazone

The mean basal values of TXB2 and PGE2 in the six horses was found to be 326.5

± 232.3 pg/mL and 120.14 ± 67.5 pg/mL respectively. The mean stimulated values of

TXB2 and PGE2 were determined to be 40,785.8 ± 10,816.6 pg/mL and 568.7

± 231.4 pg/mL, respectively. These and all other time points are reported in Tables 3-1

and 3-2.

The effects of a clinically relevant one-time dose of phenylbutazone upon the

systemic concentrations of TXB2 (COX-1) and PGE2 (COX-2) are depicted in Figures 3-

1 and 3-2, respectively. Statistical analysis was performed utilizing a commercially

available software package to compare each time point to the maximally stimulated time

point. This was undertaken to evaluate the inhibition capabilities of phenylbutazone on

both inflammatory mediators and determine when a stimulatory effect would reach

maximum levels. Lack of inhibition reached levels similar to those determined with the maximally stimulated samples at 48 and 72 hours for TXB2 and PGE2 respectively.

Ex vivo Inhibition of Ketoprofen

The mean basal concentrations of TXB2 and PGE2 in the six horses were

determined to be 785.3 ± 1,304.1 pg/mL and 41.5 ± 37.0 pg/mL, respectively. The

mean stimulated values of TXB2 and PGE2 were found to be 45,713.4 ± 28,826 pg/mL

and 593.2 ± 179.7 pg/mL, respectively. These and all other time points are reported in

Tables 3-3 and 3-4.

The effects of a clinically relevant one-time dose of ketoprofen upon the systemic

concentrations of TXB2 (COX-1) and PGE2 (COX-2) are illustrated in Figures 3-3 and 3-

4, respectively. Following statistical analysis, the lack of inhibition as concentrations

approached those determined with the maximally stimulated sample, occurred at 48

hours for both TXB2 and PGE2.

Discussion

The present study utilized a whole blood assay, previously validated in the horse48

and humans 51, to determine the duration of inhibition of two inflammatory mediators

and ultimately cyclooxygenases after a one time administration of three commonly utilized NSAIDs in athletic Thoroughbred horses. Many other studies have previously

used a protocol involving a whole blood assay, primarily to determine selectivity of COX inhibition in the evaluation of novel selective pharmaceuticals 20,48,50. Those studies

have determined that TXB2 and PGE2 assayed via this method can be used as markers for COX-1 and COX-2 activity, respectively. Utilizing the same methodology, the present study focused on the duration of NSAID induced suppression for activity of two

inflammatory mediators, TXB2 and PGE2, expressed as a percent of the maximally

stimulated positive control sample.

The natural clotting process occurring in sterile anti-coagulant free blood tubes is a process known to stimulate platelets to aggregate and activate COX-1. This activation induces the synthesis of the unstable metabolite TXA2 metabolite of

5 arachidonic acid through the COX-1 pathway which is quickly hydrolyzed to TXB2 .

Although a rich source of COX-1, platelets do not normally produce COX-2, except in clinical conditions associated with a high platelet regeneration rate. In these situations, the newly released thrombocytes express COX-254. Brideau et al. had previously observed that the levels of TXB2 reached a plateau after the blood was allowed to clot

for 60 min and that period has since been used as the standard clotting period in

52 determining TXB2 levels in several studies .

The maximally stimulated concentrations for TXB2 were similar throughout all

three assays for phenylbutazone, ketoprofen, and flunixin meglumine (40785.8 ±

10816.6 pg/mL, 45713.4 ± 28826.3 pg/mL, and 53489.4 ± 24672.8 pg/mL respectively).

The slight differences observed reflect the variation of COX activity among individuals and their response to stimulation. The values obtained in this study are similar to previously reported values for stimulated levels for TXB2 using a similar assay (40.6 ±

8.5 and 51.6 ± 17.0 ng/mL)20. Both phenylbutazone and ketoprofen suppressed

induced levels of TXB2 for about 48 h. After 48 h TXB2 concentrations were statistically

indistinguishable from the maximally stimulated control values. This was identified as

the end of inhibitory efficacy. Twenty-four hours following the administration of flunixin

meglumine, the concentration of TXB2 was comparable to maximally stimulated levels, indicating the end of the inhibitory efficacy.

It has been well documented that the presence of lipopolysaccharide (LPS) stimulates cells, especially blood monocytes, to induce COX-2 to convert arachidonic

48 acid to PGH2 and subsequently PGE2 via PGE synthase . It has also been determined

that human monocytes responded to LPS in a time dependent manner that was slightly

enhanced at 4 h post exposure but markedly enhanced at 24 h. Isolated lymphocytes

and poly-morphologic neutrophils incubated for 24 hours with LPS produced

undetectable concentrations of PGE2, indicating that blood monocytes are the primary

circulating producers of prostaglandins51. Brideau et al. compared concentrations of

PGE2 production in response to plasma incubation with either LPS or PBS and found

that PBS failed to cause any increase in PGE2 activity. In contrast, PGE2

concentrations were comparable to other studies following incubation with LPS52.

Earlier research following similar protocols promoted the addition of aspirin to the blood

tubes to suppress any undesired COX-1 expression in COX-2 assays. Patrignani et al.

addressed this issue by incubating human plasma both in the presence and absence of

aspirin following exposure to LPS and found no significant difference in the PGE2

production51. Therefore, aspirin use was not included in the COX-2 assay used in the

current study. Several different concentrations of LPS have been used in the incubation

20,48 step of PGE2 stimulation, 10 μg/mL and 100 μg/mL , respectively. The Brideau study

determined from a preliminary LPS-dose response experiment that 100 μg/mL LPS/mL

was required for sustained and consistent PGE2 production in equine blood, but several

other studies have used a lower concentration and obtained similar results.

The PGE2 concentration obtained with maximal stimulation appeared comparable in all three studies (phenylbutazone: 568.7 ± 231.4 pg/mL, ketoprofen: 593.2 ± 179.7

pg/mL and flunixin meglumine: 466.6 ± 111.3 pg/mL respectively). The leukocytes of

individual horses responded differently to stimulation through LPS, but on average,

demonstrated similar production of PGE2. Concentrations of PGE2 in maximally

stimulated control samples were comparable to those measured in other studies in

which a similar method was carried out (316.6 ± 56.2 pg/mL)20. In this study,

phenylbutazone suppressed induced concentrations of PGE2 up to 72 h. Thereupon

PGE2 production was statistically indistinguishable from the maximally stimulated

positive control, indicating an end of inhibitory efficacy at this time point and beyond.

One dose of ketoprofen suppressed induced PGE2 concentrations up to 48 h at which time the inhibition efficacy ended and PGE2 concentrations were statistically

indistinguishable from those of the maximally stimulated positive control samples.

Finally, flunixin meglumine suppressed induced PGE2 concentrations until 24 h after

administration, whereupon induced PGE2 concentrations were statistically

indistinguishable from those in the maximally stimulated positive control samples.

This study used an ex vivo whole blood assay to determine the inhibitory efficacy

and duration following administration of each of three NSAIDs commonly used in equine

medicine (phenylbutazone, ketoprofen, and flunixin meglumine). From this data,

phenylbutazone suppresses COX-1 activity until 48 hours and COX-2 activity through

72 hours following a single administration at a 4.4 mg/kg dose rate. Ketoprofen

suppresses an induced inflammatory response from both COX-1 and COX-2 mediators

for up to 48 hours following a single administration at a 2.2 mg/kg dose rate. Finally,

flunixin meglumine suppressed COX-1 and COX-2 activity through 24 h following a single administration at 1.1 mg/kg dose rate.

Table 3-1. Ex vivo TXB2 concentrations for phenylbutazone in six horses, reported as pg/mL. Time (h) Ava Annie Elle True Slip Des Mean SD Base 695.4 499.1 47.8 195.4 247.7 273.8 326.5 232.3 Stim mean 30966.1 32763.3 39193.9 60999.0 37866.5 42925.9 40785.8 10816.6 0.25 5319.8 3657.1 631.2 5460.3 496.6 1360.4 2820.9 2290.9 0.5 544.7 652.8 1124.8 489.9 297.0 693.2 633.7 278.2 1 982.7 722.6 2292.5 612.7 508.9 721.7 973.5 665.2 2 1752.5 557.9 2822.4 2354.7 689.0 1350.7 1587.9 901.6 3 2104.9 1940.0 3177.2 4929.3 1638.7 2820.8 2768.5 1203.5 4 2201.2 2719.1 2370.8 3917.5 2786.2 4116.0 3018.5 805.5 6 2764.0 3662.0 8895.5 8256.9 3020.4 6397.0 5499.3 2718.4 8 2383.5 2603.0 24422.5 4395.6 825.5 2875.5 6250.9 8974.9 24 6951.7 7780.7 37318.922662.1 14917.3 10793.8 16737.411604.5 48 31994.2 35951.9 1845.3 57482.0 38976.9 47540.6 35631.8 18896.1 72 33047.3 50039.2 283.9 86497.5 51782.9 60292.5 46990.5 28795.6

Figure 3-1. Mean ± SD of % inhibition of phenylbutazone on TXB2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

Table 3-2. Ex vivo PGE2 concentrations for phenylbutazone in six horses, reported as pg/mL.

Time (h) Ava Annie Elle True Slip Des Mean SD Base 170.1 143.2 217.2 42.2 70.5 77.6 120.1 67.5 Stim mean 934.7 740.7 581.8 422.9 383.0 349.0 568.7 231.4 0.25 195.9 77.4 239.7 34.9 131.6 74.2 125.6 79.0 0.5 154.8 86.7 209.0 50.6 118.9 81.2 116.9 57.5 1 224.3 119.0 148.9 100.3 71.0 88.3 125.3 55.4 2 177.1 104.1 430.1 126.4 128.1 90.8 176.1 127.9 3 180.5 124.9 436.5 81.4 108.4 93.9 170.9 134.6 4 163.9 103.4 471.9 97.2 130.6 63.7 171.8 150.8 6 35.17 150.6 457.2 131.2 89.9 106.0 161.7 150.1 8 109.9 145.9 194.5 134.1 85.8 75.6 124.3 43.7 24 216.5 448.0 237.8 289.9 301.5 175.5 278.2 95.4 48 426.4 320.4 260.9 456.8 417.9 288.6 361.8 82.0 72 500.6 391.2 410.9 523.4 324.1 460.2 435.1 74.3

Figure 3-2. Mean ± SD % inhibition of phenylbutazone on PGE2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

Table 3-3. Ex vivo TXB2 concentrations for ketoprofen in six horses, reported as pg/mL. Time Ava Annie Tia True Slip Des Mean SD Base 3444.2 332.7 324.4 194.2 231.8 184.8 785.3 1304.1 Stim mean 30206.9 29149.3 40204.1 103818.6 32078.7 38822.6 45713.4 28826.3 0.25 182.3 228.3 384.3 300.3 338.6 263.2 282.8 73.7 0.5 320.6 100.2 232.1 217.9 205.6 281.1 226.2 75.3 1 327.7 79.4 160.6 299.5 224.7 402.8 249.1 118.0 2 349.1 206.1 288.9 305.3 240.7 481.0 311.9 96.8 3 344.6 156.7 377.5 236.2 301.7 476.5 315.5 111.6 4 413.0 255.5 533.6 473.0 883.9 772.6 555.3 233.5 6 614.6 400.4 514.1 1356.4 358.5 1394.1 773.0 475.2 8 951.7 643.9 465.8 1200.0 579.6 1278.9 853.3 340.6 24 10394.4 13071.6 14392.6 54501.8 10360.5 22763.3 20914.0 17074.1 48 12413.6 21340.0 40743.6 63643.9 28862.4 36333.6 33889.5 17789.1 72 30628.1 32131.9 50877.1 79246.1 42606.4 38409.2 45649.8 18033.9

Figure 3-3. Mean ± SD % inhibition of ketoprofen on TXB2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

Table 3-4. Ex vivo PGE2 concentrations for ketoprofen in six horses, reported as pg/mL. Time (h) Ava Annie Tia True Slip Des Mean SD Base 50.8 85.0 19.0 60.0 83.7 3.14 41.5 37.0 Stim mean 768.1 785.5 526.4 706.2 371.9 768.2 593.2 179.7 0.25 79.7 114.3 44.5 136.6 117.3 47.0 86.4 47.6 0.5 33.8 176.0 76.1 97.6 157.3 12.7 85.9 59.7 1 103.7 80.7 79.1 170.5 98.6 56.6 101.2 49.3 2 177.8 80.4 61.1 214.5 97.2 60.9 108.4 72.7 3 166.7 121.5 108.6 168.2 152.29 99.6 132.2 33.3 4 153.2 180.6 162.6 283.6 173.9 53.8 168.5 93.9 6 431.5 396.8 271.3 382.4 216.0 125.3 248.8 107.4 8 112.4 674.4 374.9 351.7 308.6 517.1 388.1 90.3 24 472.7 349.7 591.1 450.5 422.2 478.8 485.7 74.0 48 570.4 595.3 561.2 547.5 506.6 654.5 567.4 62.5 72 494.8 595.0 671.9 481.8 409.7 430.7 498.6 119.5

Figure 3-4. Mean ± SD % inhibition of ketoprofen on PGE2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

Table 3-5. Ex vivo TXB2 concentrations for flunixin meglumine in 6 horses, reported as pg/mL. Time Ava Annie Elle True Slip Des Mean SD Base 256.5 244.5 452.6 67.1 115.5 505.9 273.7 176.0 Stim 35732.2 47409.4 39558.4 100324.3 60418.2 37493.8 53489.4 24672.8 0.25 294.2 387.1 489.1 324.6 198.8 685.2 396.5 171.3 0.5 397.5 247.9 479.2 246.5 181.3 708.6 376.8 196.4 1 362.4 246.1 387.8 284.4 310.1 908.2 416.5 246.3 2 406.2 488.7 547.4 743.9 527.7 1169.3 647.2 279.1 3 660.0 3746.3 2641.0 1471.4 866.5 1797.3 1863.7 1161.9 4 575.8 728.5 918.8 710.8 628.1 1510.5 845.4 346.3 6 1328.9 971.5 804.6 3179.3 1450.2 2748.2 1747.1 980.5 8 4966.2 7693.3 3318.4 7012.2 2881.5 3434.3 4884.3 2048.9 24 14824.7 28087.7 18908.193986.2 44167.5 24832.5 37467.8 29475.5 48 38802.1 56210.5 44318.1109127.9 72826.1 39709.6 60165.7 27200.1 72 27029.9 52232.0 40742.5 185738.6 69186.6 62071.4 72833.5 57321.7

Figure 3-5. Mean ± SD % inhibition of flunixin meglumine on TXB2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

Table 3-6. Ex vivo PGE2 concentrations for flunixin meglumine in 6 horses, reported as pg/mL. Time Ava Annie Elle True Slip Des Mean SD Base 392.87 43.90 69.07 19.82 31.11 39.51 99.38 144.71 Stim mean 558.16 558.66 475.96 418.79 267.26 520.95 466.63 111.28 0.25 79.60 118.56 83.04 54.04 29.50 14.14 63.15 38.35 0.5 86.52 103.96 87.22 34.92 37.70 68.59 69.82 28.28 1 89.63 103.08 76.05 41.73 59.33 34.56 67.40 27.00 2 109.53 68.35 5.02 64.24 47.51 48.66 57.22 34.07 3 83.54 59.30 15.22 60.51 51.71 43.40 52.28 22.57 4 56.44 83.93 26.92 45.40 58.75 95.27 61.12 25.02 6 112.84 98.86 72.73 54.06 103.71 79.00 86.87 22.09 8 123.56 68.72 16.98 80.53 71.05 80.90 73.62 34.16 24 450.63 590.34 221.23322.56 345.88 300.05 371.78 130.30 48 508.26 705.72 297.87342.17 340.76 306.18 416.83 160.98 72 400.40 568.73 255.01 303.44 377.57 359.40 377.42 107.62

Figure 3-6. Mean ± SD % inhibition of flunixin meglumine on PGE2 concentrations. *Indicates a significant difference between that time point and the maximally stimulated control sample identifying an inhibitory effect.

CHAPTER 4 UTILIZING A BIOSTATISTICAL APPROACH TO MODEL COMPARISONS OF PHENYLBUTAZONE, KETOPROFEN, AND FLUNIXIN MEGLUMINE WITH SUPRESSED CONCENTRATIONS OF PGE2 AND TXB2

Background

In addition to the anabolic-androgenic steroids and sedatives/tranquilizers, the non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most extensively studied class of drugs in horses42. As previously discussed, the Association of Racing

Commissioners International place most NSAIDs within the 4th tier of drug classification,

however, some (novel COX-2 selective drugs for example) may be promoted to the 2nd

or 3rd tiers if they are not licensed for use in horses55. The decisions surrounding

withdrawal times and subsequent penalties regarding the use of NSAIDs on

performance horses remains very controversial today. Often control is based upon

which drug has been applied, whether it was administered prior to or during the

competition, and whether or not the drug has been declared. Most of these decisions

are based upon pharmacokinetic data that has been accumulated through numerous

studies to evaluate the characteristic pattern of a drug’s absorption, distribution,

metabolism, and excretion as a function of time. The scientific methodology utilized has

improved significantly and currently liquid-chromatography linked to mass spectrometry

is the method of choice for detection of drug concentrations. Historically, equine urine

had been the biological sample of choice for detection purposes, because it is easily

obtained under post-racing situations and because urine contains a higher

concentration of drug metabolites for a longer time than plasma. However, plasma has

gained popularity, especially in the United States, because it is easily and quickly

collected during training or pre-race time points, less sample preparation is required

compared to urine and blood drug concentrations correspond more closely to pharmacological effects42. Additionally, the once elusive minute amounts of drug within

plasma are now measurable using advanced screening techniques.

More recently, researchers interested in better understanding the pharmacologic

effects a drug has on a horse have employed the technique of

pharmacokinetic/pharmacodynamic (PK/PD) modeling. These models have proven to

be particularly useful in establishing competition drug thresholds, particularly because

drug effects do not always correlate with plasma concentrations56. Pharmacodynamics

describe the changes of a measurable response of a drug over time. Several different

models have been used with equine subjects including heart rate monitoring, spontaneous locomotor activity, head ptosis, and tissue inflammation. The last example, tissue inflammation, has been used in several experiments to examine the pathophysiolgical variable to correlate with drug concentration in PK/PD studies of

NSAIDs24. The model in Maihto’s study, used subcutaneously implanted tissue cages

and carrageenan-soaked sponges to monitor the penetration of phenylbutazone and eicosanoid concentrations in inflamed tissue. Similarly, our study used an ex vivo approach to stimulate inflammatory mediators and examined the inhibitory effect of three commonly administered NSAIDs in equine medicine (phenylbutazone, ketoprofen and flunixin meglumine). Finally, employment of a modern biostatistical analysis to correlate the concentration of each NSAID with its inhibitory effect on both prostaglandin

E2 and thromboxane B2, in the stimulated ex vivo inflammatory event represented the

final step. The ultimate use of our drug concentration to response model is to enable

extrapolation of an estimated administration time by determining both the inhibitory

effects and drug concentrations.

Materials and Methods

Animals

Plasma drug concentration and COX-1 and -2 inhibition data was obtained from

the six adult Thoroughbred horses (3 mares and 3 geldings) between the ages of 3 to

10 years, weighing between 495-563 kg, in athletic condition sufficient to work 1 mile in

2 minutes without undue stress previously described in this study. The pharmacokinetic

and pharmacodynamic data obtained and described previously was used in the

statistical analysis and development of a biostatistical model.

Method of Analysis

For each horse in the data set, we used either the prostaglandin (PGE) or

thromboxane (TXB) and phenylbutazone (PBZ), ketoprofen (KETO) and flunixin

meglumine (FL) concentrations from 0.25 hours to 72 hours. As our outcome variable,

we modeled the natural log of the ratio of each drug concentration to PGE or TXB

concentration. To determine the relationship between time and log(drug/PGE or TXB),

we used general estimating equations (SAS PROC GENMOD) employing a

commercially available statistical software package (SAS™), and treating each horse as

a random effect and modeling the within-horse variance between time points with an

autoregressive variance-covariance matrix structure. Since the relationship between

time and log(drug/PGE or TXB) was non-linear, we included a quadratic term in time to

improve model fit.

As the number of subjects was few and the object was to determine a relationship

that could be used to estimate the time since drug administration from observed

concentrations for all horses, we did not try to find the “best” model fit in order to avoid over fitting the data (creating a model that was a very good estimator for horses in the data set, but poor for other horses). Hence, we added only a quadratic modeling term even though in this analysis and most of those following, a cubic term or a more complicated non-linear model would have improved the fit of the model to the data. The relationships presented here are approximate, and valid confidence intervals that can easily be calculated for any given observation of the two compounds in question. The

University of Florida Biostatistical Consulting Lab, College of Public Health and Health

Professions, performed all statistical analyses.

Results

Relating Phenylbutazone Concentrations to Prostaglandin (PGE) Concentrations

The model created provided an estimate of the relationship between time and log(PBZ/PGE) illustrated in Equation 4-1. The P-values for all coefficients in this model were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients were as follows: Intercept: (5.023, 5.490), Time: (-0.230, -0.171), Time*time: (0.0006,

0.0014). In order to use this model, given an observed value of log(PBZ/PGE), one may solve the quadratic formula for an estimated time (Equation 4-2). The smaller of the two solutions is the model estimate of the time since administration. To get confidence limits for this estimate, the same formula can be used, but by substituting the lower and upper limits for the coefficients (Equations 4-3 & 4-4). Alternatively, an estimation of time can be extrapolated from the plot illustrated (Figure 4-1).

Relating Phenylbutazone Concentrations to Thromboxane (TXB) Concentrations

The model created here provided an estimate of the relationship between time and log(PBZ/TXB) illustrated in Equation 4-5. The P-values for all coefficients in the model

were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients were as follows: Intercept: (2.448, 3.208), Time: (-0.341, -0.245), Time*time: (0.0013,

0.0027). In order to use this model, given an observed value of log(PBZ/TXB), one may solve the quadratic formula for an estimated time (Equation 4-6). To get confidence limits for this estimate, the same formula can be used, however, appropriate coefficients would need to be used. Alternatively, an estimation of time can be extrapolated from the plot illustrated (Figure 4-2).

Relating Ketoprofen Concentrations to Prostaglandin (PGE) Concentrations

The model created here provided an estimate of the relationship between time and log(KETO/PGE) illustrated in Equation 4-7. The P-values for all coefficients in the model were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients were as follows: Intercept: (-3.191, -2.167), Time: (-1.327, -1.115),

Time*time: (0.0272, 0.0351). In order to use this model, given an observed value of log(KETO/PGE), one may solve the quadratic formula for an estimated time (Equation

4-8). To calculate confidence limits for this estimate, the same formula can be used however, appropriate coefficients would need to be used. Alternatively, an estimation of

time can be extrapolated from the plot illustrated (Figure 4-3).

Relating Ketoprofen Concentrations to Thromboxane (TXB) Concentrations

The model created here provided an estimate of the relationship between time and

log(KETO/TXB) illustrated in Equation 4-9. The P-values for all coefficients in the model

were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients

were as follows: Intercept: (-4.186, -3.568), Time: (-1.279, -1.077), Time*time: (0.0215,

0.0295). In order to use this model, given an observed value of log(KETO/TXB), one

may solve the quadratic formula for an estimated time (Equation 4-10). To calculate

confidence limits for this estimate, the same formula can be used however, appropriate

coefficients would need to be used. Alternatively, an estimation of time can be

extrapolated from the plot illustrated (Figure 4-4).

Relating Flunixin Meglumine Concentrations to Prostaglandin (PGE) Concentrations

The model created here provided an estimate of the relationship between time and

log(FL/PGE) illustrated in Equation 4-11. The P-values for all coefficients in the model

were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients

were as follows: Intercept: (-2.630, -2.006), Time: (-0.391, -0.372), Time*time: (0.0031,

0.0033). In order to use this model, given an observed value of log(FL/PGE), one may

solve the quadratic formula for an estimated time (Equation 4-12). To calculate

confidence limits for this estimate, the same formula can be used however, appropriate

coefficients would need to be used. Alternatively, an estimation of time can be

extrapolated from the plot illustrated (Figure 4-5).

Relating Flunixin Meglumine Concentrations to Thromboxane (TXB) Concentrations

The model created here provided an estimate of the relationship between time and

log(FL/TXB) illustrated in Equation 4-13. The P-values for all coefficients in the model

were highly significant (p<0.0001 for all). A 95% confidence interval for the coefficients

were as follows: Intercept: (-4.844, -4.287), Time: (-0.550, -0.479), Time*time: (0.0040,

0.0050). In order to use this model, given an observed value of log(FL/TXB), one may

solve the quadratic formula for an estimated time (Equation 4-14). To calculate

confidence limits for this estimate, the same formula can be used however, appropriate

coefficients would need to be used. Alternatively, an estimation of time can be

extrapolated from the plot illustrated (Figure 4-6).

Example of Model Application

Suppose you observe log(PBZ/PGE) is equivalent to 3.5. Then the estimated time since administration could be solved by application of Equation 4-15, obtaining the answer of 9.2 hours. Then, maintaining a starting point of 3.5 for the log(PBZ/PGE) value and the lower and upper bounds were applied to determine the confidence intervals (Equations 4-16 & 4-17), an estimation of time could be 95% confident that the drug administration occurred between 6.7 and 13 hours ago.

Discussion

Through the use of an ex vivo model of inflammation that employed a whole blood assay and advanced liquid chromatography with tandem mass spectrometry analytical techniques, this study examined both the pharmacokinetic and pharmacodynamic parameters of three NSAIDs (phenylbutazone, ketoprofen, and flunixin meglumine) commonly administered to athletic Thoroughbred horses. Previous studies have used similar methodologies to determine potency ratio for COX inhibition 48,50. Most of those studies initially focused on assay technique validation and then evaluated the COX selectivity of classic and novel NSAIDs. The current study developed biostatistical formulas to interpret the data and provide enhanced estimations associated with the clinical dose of an NSAID using both pharmacokinetic and pharmacodynamic data.

Studies measuring the level of inhibition of a single mediator to predict dosage are of

value from a mechanistic perspective but do not necessarily provide information on the clinical response. The degree of prostaglandin inhibition is assumed to correspond to the intensity of clinical responses but exact correlations are not known. Also, various clinical responses like analgesia, antipyresis and/or resolution of swelling may be induced at different levels of prostaglandin suppression13. The science of relating

plasma concentrations and inflammatory mediator inhibition to specific effects is

incompletely described and large deficiencies in our understanding of the clinical

efficacy of NSAIDs remain.

Recently, regulatory authorities have been faced with the challenge of determining the concentration of NSAIDs in post race samples that constitute a violation of racing regulations. The approach of reporting all drugs detected by very sensitive

analytical techniques can be problematic because very low but detectable

concentrations likely have no effect on racing performance. To simplify enforcement

and provide scientifically based regulatory decisions, selected cut-off values have been established through pharmacokinetic and pharmacodynamic data in conjunction with understanding what is acceptable or unacceptable to regulatory authorities57. The

definition of a “limit”, “threshold” or “cutoff” is based on any drug or metabolite

concentration in a biological fluid from a participant in a regulated event. When referring

to horse racing, concentrations greater than the stipulated limit induce regulatory action,

while those below, do not. In 1999, Tobin et al. proposed the formation of a database

containing limitations and withdrawal time data for drugs commonly used in equine

medicine, but restricted it to include those medications listed by the AAEP as

therapeutic agents. The purpose of this database would be to determine the “highest

no effect dose” of a particular drug and ultimately the relevant “no effect point” in plasma

or urine concentration. Difficulties in establishing this database include dose and route

of administration, along with the sensitivity of the analytical methodology used58. The lack of a database has long been deemed a worldwide problem in the horse racing industry and several different countries have taken a variety of approaches to alleviate

the ambiguity associated with drug testing. This study excluded pre-established

“threshold limits” but sought to describe plasma concentrations and inhibitory efficacy through measurement of two inflammatory mediators (PGE and TXB) following administration. This approach focused on the determination of an appropriate pre-race administration time to meet regulatory needs and was supported by drug effect data through inhibition of inflammatory mediators. The inhibition of inflammatory mediators could be used in conjunction with the routine screening process to retrospectively determine time of administration. This information enables humane considerations by providing the time required following NSAID administered in the course of proper veterinary care, to horses in training before participation in regulatory agency sanctioned events. In addition, regulatory agencies gain additional information about the duration and magnitude of the effects of NSAID administration and their correlation with plasma drug concentration. Potentially, these estimated formulas of administration time could be established for a variety of commonly administered foreign substances and provide accentuated information employed by regulatory agencies.

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Equation 4-1. Estimate of the relationship between time and log(PBZ/PGE).

Log(PBZ/PGE)=5.257 – 0.201*T + 0.001*T^2, where T=time (in hours)

Equation 4-2. Estimated time since administration for log(PBZ/PGE).

T = [0.201 – sqrt(0.201^2 – 4*0.001*(5.257 – observed log(PBZ/PGE)))]/(2*.001)

Equation 4-3. Lower bound of confidence interval.

CI = [0.230 – sqrt(0.230^2 – 4*0.0006*(5.023 - observed log(PBZ/PGE)))]/()]/(2*.0006)

Equation 4-4. Upper bound of confidence interval.

CI = [0.171 – sqrt(0.171^2 – 4*.0014*(5.490 - observed log(PBZ/PGE)))]/()]/(2*.0014)

Figure 4-1. Raw and Estimated log(PBZ/PGE) versus time.

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Equation 4-5. Estimate of the relationship between time and log(PBZ/TXB).

Log(PBZ/TBX)=2.828 – 0.293*T + 0.0020*T^2, where T=time (in hours)

Equation 4-6. Estimated time since administration.

T = [0.293 – sqrt(0.293^2 – 4*0.002*(2.828 - observed log(PBZ/TBX)))]/(2*.002)

Figure 4-2. Raw and Estimated log(PBZ/TXB) versus time.

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Equation 4-7. Estimate of the relationship between time and log(KETO/PGE).

Log(KETO/PGE)=-2.679 – 1.221*T + 0.0312*T^2, where T=time (in hours)

Equation 4-8. Estimated time since administration.

T = [1.221– sqrt(1.221^2 – 4*0.0312*(-2.679 - observed log(KETO/PGE)))]/(2*.0312)

Figure 4-3. Raw and Estimated log(KETO/PGE) versus time.

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Equation 4-9. Estimate of the relationship between time and log(KETO/TXB).

Log(KETO/TXB)=-3.877 – 1.178*T + 0.0255*T^2, where T=time (in hours)

Equation 4-10. Estimated time since administration.

T = [0.1.178 – sqrt(1.178^2 – 4*0.0255*(-3.877 - observed log(KETO/TXB)))]/(2*.0255)

Figure 4-4. Raw and Estimated log(KETO/TXB) versus time.

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Equation 4-11. Estimate of the relationship between time and log(FL/PGE).

Log(FL/PGE)=-2.318 – 0.381*T + 0.0032*T^2, where T=time (in hours)

Equation 4-12. Estimated time since administration.

T = [0.381 – sqrt(0.381^2 – 4*0.0032*(-2.318 - observed log(FL/PGE)))]/(2*.0032)

Figure 4-5. Raw and Estimated log(FL/PGE) versus time.

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Equation 4-13. Estimate of the relationship between time and log(FL/TXB).

Log(FL/TXB)=-4.565 – 0.515*T + 0.0045*T^2, where T=time (in hours)

Equation 4-14. Estimated time since administration.

T = [0.515 – sqrt(0.515^2 – 4*0.0045*(-4.565 - observed log(FL/TXB)))]/(2*.0045)

Figure 4-6. Raw and Estimated log(FL/TXB) versus time.

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Equation 4-15. Example of est. time since administration using log(PBZ/PGE) = 3.5.

T = [0.201 – sqrt(0.201^2 – 4*0.001*(5.257 - observed log(PBZ/PGE)))]/(2*.001) T = [0.201 – sqrt(0.201^2 – 4*0.001*(5.257-3.5))]/(2*.001) = 9.2 hours

Equation 4-16. Example of determining lower bound for the confidence interval.

CI = [0.230 – sqrt(0.230^2 – 4*0.0006*(5.023 - observed log(PBZ/PGE)))]/()]/(2*.0006) CI = [0.230 – sqrt(0.230^2 – 4*0.0006*(5.023 – 3.5))]/()]/(2*.0006) = 6.7 hours

Equation 4-17. Example of determining upper bound for the confidence interval.

CI = [0.171 – sqrt(0.171^2 – 4*.0014*(5.490 - observed log(PBZ/PGE)))]/()]/(2*.0014) CI = [0.171 – sqrt(0.171^2 – 4*.0014*(5.490 – 3.5))]/()]/(2*.0014) = 13.0 hours

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CHAPTER 5 CONCLUSIONS

The overall aim of this study was to enhance our knowledge of the pharmacological effects of three NSAIDs commonly administered to athletic

Thoroughbred horses. The pharmacokinetics of phenylbutazone, ketoprofen and flunixin meglumine were established following a single intravenous administration. A variety of NSAIDs have historically been administered to Thoroughbred racehorses in an effort to alleviate pain and reduce inflammation, ultimately with anticipation for continued racing. As mentioned, NSAID administration for relief of pain is humane and necessary, however racing authorities around the world prohibit the unscrupulous use of these drugs to enable a compromised horse to race. The drug testing industry has grown substantially for racing Thoroughbreds and the sophisticated analytical methods currently in use are capable of detecting minute amounts of drug concentrations in plasma for a long time after administration. This creates a challenging situation for not only track stewards faced with a positive sample, but for the veterinarians maintaining the health of these animals making the decisions regarding drug administration. The withdrawal times currently available are simple recommendations for veterinarians based upon published detection times but more dependent upon clinical judgment.

The study described provided an up to date analysis of the pharmacokinetic parameters of three commonly administered NSAIDs (phenylbutazone, ketoprofen and flunixin meglumine) through the use of advanced LC/MS/MS detection methodology.

Furthermore, this study aimed to determine the duration of cyclooxygenase (COX) inhibition of each drug and determine a comparable relationship (if any) between the two. Ultimately, the knowledge obtained was used to create a statistical model

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representing the relationship between plasma concentration and inhibitory efficacy of each NSAID. This model provided a novel method for determining an estimated time of administration for each drug.

The information identified through this study paves the way for a variety of future directions. The effects on inhibition of inflammation were determined through measurement of the concentrations of two inflammatory mediators (PGE2 and TXB) but the effects on pain through the use of these NSAIDs is still undetermined. A similar study utilizing a pain model would be beneficial in determining analgesic effects of these

NSAIDs. Additionally, this study used a one-time bolus approach to dose administration and it is well known that many of these drugs are administered over a longer period of time prior to racing. Therefore, extending this study to include a multi-dose administration protocol could enhance the current knowledge regarding the pharmacokinetics involving a more “real-life” method of administration.

Importantly, the knowledge gained through this study could potentially lead to more accurate withdrawal times based upon plasma concentrations and inhibitory effects derived from administered NSAIDs. In the future, veterinarians faced with the responsibility for educating their clients would be able to access information regarding the use of a certain drug in a racing Thoroughbred and determine the length of efficacy.

This method of providing a more customized withdrawal time for a drug could potentially discourage the use of drugs in a horse too compromised to race. The analytical methodology for detecting doping agents in Thoroughbred race horses, for the purpose of monitoring and control, will undoubtedly continue to increase in their level of sophistication. A novel technique for addressing this issue necessitates evaluation to

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protect veterinarians, trainers and most importantly, the welfare of Thoroughbreds and the sport of horseracing.

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BIOGRAPHICAL SKETCH

Jennifer Noelle Hatzel was born and raised in Katy, Texas and graduated from

High School in the same small town. She spent three years working towards a

Bachelor of Science degree in animal science from Texas Tech University in Lubbock,

Texas, but was accepted early to begin veterinary school in 2003. She successfully obtained a Doctor of Veterinary Medicine with the charter class at Western University of

Health Sciences in Pomona, California in May, 2007. Upon graduation, from 2007 to

2008, she completed a rotating hospital internship at Peterson and Smith Equine

Hospital in Ocala, Florida. In the spring of 2009, she completed a specialty fellowship in the area of Neonatal Intensive Care at Hagyard Equine Hospital in Lexington, Kentucky.

Upon completion of the fellowship, she began pursuing the Master of Science degree through the College of Veterinary Medicine at the University of Florida in January, 2010.

During her time at the University of Florida, she became increasingly interested in the field of equine theriogenology and proceeded by pursuing a residency training program.

She obtained and subsequently began this residency training in June of 2011, where she currently maintains a position at the Equine Reproductive Laboratory at Colorado

State University. She anticipates completing her residency training and sitting boards to become a Diplomate of the American College of Theriogenologists in August 2013. She currently resides in Fort Collins, Colorado with her husband Jeremiah, two dogs

(Sedona and Satchel), kitty (Sphinx) and longtime equine companion, Winchester.

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