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Pharmacokinetics and Pharmacodynamics of Glycopyrrolate in the Horse

Permanent Link: http://ufdc.ufl.edu/UFE0044126/00001

Material Information

Title: Pharmacokinetics and Pharmacodynamics of Glycopyrrolate in the Horse
Physical Description: 1 online resource (218 p.)
Language: english
Creator: Rumpler, Marc J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: glycopyrrolate -- horse -- lcmsms -- pharmacodynamics -- pharmacokinetics -- thoroughbred
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Glycopyrrolate is a muscarinic receptor antagonist typically used in veterinary medicine to inhibit gastrointestinal motility and block the effects of vagal stimulation during anesthesia. It is reportedly used in horseracing to reduce bronchial secretions despite the fact that it is a prohibited substance. Regulating legitimate medications in horseracing relies on the development of industry threshold limits and recommended withholding times. Therefore pharmacokinetics studies are important for determining drug plasma and urine concentrations following a typical often clinically relevant dosage. The following research describes a rapid, sensitive, selective and fully validated liquid-chromatography mass spectrometry analytical method for the detection and quantification of glycopyrrolate in horse plasma and urine. The Pharmacokinetics of glycopyrrolate have been evaluated after a 1 mg intravenous dose in Thoroughbreds and Standardbreds. Additionally, the pharmacodynamics have been evaluated following a clinically relevant intravenous infusion (8 micrograms/kg) in Thoroughbreds. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Pharmacokinetic parameters were best estimated using a three-compartment model for plasma concentration versus time data from 0-24 h, although glycopyrrolate was detectable for 168 h after administration. Glycopyrrolate disposition in the horse was characterized by a steep decline in plasma concentration immediately after dosing, extensive distribution from the central compartment, rapid clearance, and a prolonged terminal elimination phase. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24 h after oral administration.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marc J Rumpler.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Vickroy, Thomas W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044126:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044126/00001

Material Information

Title: Pharmacokinetics and Pharmacodynamics of Glycopyrrolate in the Horse
Physical Description: 1 online resource (218 p.)
Language: english
Creator: Rumpler, Marc J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: glycopyrrolate -- horse -- lcmsms -- pharmacodynamics -- pharmacokinetics -- thoroughbred
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Glycopyrrolate is a muscarinic receptor antagonist typically used in veterinary medicine to inhibit gastrointestinal motility and block the effects of vagal stimulation during anesthesia. It is reportedly used in horseracing to reduce bronchial secretions despite the fact that it is a prohibited substance. Regulating legitimate medications in horseracing relies on the development of industry threshold limits and recommended withholding times. Therefore pharmacokinetics studies are important for determining drug plasma and urine concentrations following a typical often clinically relevant dosage. The following research describes a rapid, sensitive, selective and fully validated liquid-chromatography mass spectrometry analytical method for the detection and quantification of glycopyrrolate in horse plasma and urine. The Pharmacokinetics of glycopyrrolate have been evaluated after a 1 mg intravenous dose in Thoroughbreds and Standardbreds. Additionally, the pharmacodynamics have been evaluated following a clinically relevant intravenous infusion (8 micrograms/kg) in Thoroughbreds. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Pharmacokinetic parameters were best estimated using a three-compartment model for plasma concentration versus time data from 0-24 h, although glycopyrrolate was detectable for 168 h after administration. Glycopyrrolate disposition in the horse was characterized by a steep decline in plasma concentration immediately after dosing, extensive distribution from the central compartment, rapid clearance, and a prolonged terminal elimination phase. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24 h after oral administration.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marc J Rumpler.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Vickroy, Thomas W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044126:00001


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1 PHARMACOKI NETICS AND PHARMACODYNAMICS OF GLYCOPYRROLATE IN THE HORSE By MARC J RUMPLER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Marc J Rumpler

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3 To Tyler and Evelyn: Whatever it is you wish to accomplish in life, may you pursue it with passion, attack it with perseverance, triumph with success and reminisce with pride.

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4 ACKNOWLEDGMENTS original advisor, Dr. Richard Sams, who had given me the opportunity to study and become a University of Florida graduat e student and part of the Florida Racing Laboratory staff. I have been fortunate to gain from his knowledge and experience. I also appreciate our personal candid conversations. He was an avid listener and easy to talk to. I would like to thank Nancy Szabo for our short time together. With her patience, thoughtful insight, and positive attitude I felt at ease during my first year of the doctoral program. She was a renewable resource on my c ommittee even from a distance. Tebbet t for hi program and later as a doctorate committee advisor. Dr. Genther Hochhaus has provided constructive feedback for my research pertaining to pharmacokinetics pharmacodynamics Dr. Thomas Vickroy has guided me through particula rly challenging times such as the qualifying examinations and dissertation defense. I a m grateful that he accepted the role and responsibility I would also like to thank the Florida Racing Laboratory staff for their patience and assistance throughout the past several years. I have had consistent support and encouragement from Margare t Wilding throughout my tenure and Patrick Russell has given me technical advice on occasion. I would like to thank the staff at the Equine Performance Laboratory, including Brett Rice, Allison Hreha and Dr. Ted Broome for their generous assistance with the drug administration studies and animal care. I would like to thank certain faculty members of the College o f Veterinary Medicine (CVM) who have assisted me with my research including Drs. Chris Sanchez, Sheila Robertson and Patrick Colahan. I am grateful for the CVM graduate school advisors, such as Dr. Charles Courtney and

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5 discussions with Physiological Sciences departmental faculty including Drs. Roger Reep, Ri ck Johnson, and Paul Davenport. Finally, I would like to thank those agencies which have provided me with the financial support necessary to conduct this research and complete my program of study: The University of Florida Racing Laboratory a nd the Racing Med ication and Testing Consortium. On a personal note I could not have pursued my education without the love and support from all of my family, who has indirectly contributed to my success.

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6 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 9 LIST OF FIGURES ................................ ................................ ................................ ....................... 12 LIST OF ABBR EVIATIONS AND SYMBOLS ................................ ................................ .......... 17 ABSTRACT ................................ ................................ ................................ ................................ ... 22 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ................................ .............................. 24 Overview and History ................................ ................................ ................................ ............. 2 4 Basic Ph armacology of Glycopyrrolate ................................ ................................ .................. 25 Therapeutic and Clinical Applications of Glycopyrrolate ................................ ...................... 28 Analytical Methodology ................................ ................................ ................................ ......... 31 Glycopyrrolate Doping in Race Horses ................................ ................................ .................. 33 Hypothesis ................................ ................................ ................................ .............................. 35 2 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION ................................ .. 37 Methods ................................ ................................ ................................ ................................ .. 38 Chemicals and Reagents ................................ ................................ ................................ .. 38 Standard Preparation ................................ ................................ ................................ ....... 39 So lid Phase Extraction ................................ ................................ ................................ ..... 40 Liquid Chromatography ................................ ................................ ................................ .. 40 Data Analysis ................................ ................................ ................................ ................... 41 Method Validation ................................ ................................ ................................ ........... 42 System Suitability ................................ ................................ ................................ ............ 43 Sample Acceptance Criteria ................................ ................................ ............................ 45 Linearity and Range ................................ ................................ ................................ ........ 45 Sensitivity ................................ ................................ ................................ ........................ 46 Accuracy and Pre cision ................................ ................................ ................................ ... 46 Carryover ................................ ................................ ................................ ......................... 47 Matrix Effect, Extraction Efficiency, Process Efficiency ................................ ............... 47 Dilution Integrity ................................ ................................ ................................ ............. 48 Specificity ................................ ................................ ................................ ........................ 49 Stability ................................ ................................ ................................ ............................ 50 Ruggedness ................................ ................................ ................................ ...................... 50 Stock and Working Standard Solution Stability ................................ .............................. 50 Analyte Confirmation ................................ ................................ ................................ ...... 51 Statistical Analysis ................................ ................................ ................................ .......... 51

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7 Result s ................................ ................................ ................................ ................................ ..... 51 Method Validation ................................ ................................ ................................ .................. 52 Linearity ................................ ................................ ................................ .......................... 52 Sensitivity ................................ ................................ ................................ ........................ 53 Accuracy and Precision ................................ ................................ ................................ ... 53 Carryover ................................ ................................ ................................ ......................... 54 Matrix Effect, Extraction Efficiency, Process Efficiency ................................ ............... 54 Dilution Integrity ................................ ................................ ................................ ............. 55 Specificit y ................................ ................................ ................................ ........................ 56 Stability ................................ ................................ ................................ ............................ 56 Ruggedness ................................ ................................ ................................ ...................... 57 Analyte Confirmation ................................ ................................ ................................ ...... 58 Discussion ................................ ................................ ................................ ............................... 58 3 PHARMACOKINETICS OF GLYCOPYRROLATE IN THOROUGHBREDS ................. 90 Methods ................................ ................................ ................................ ................................ .. 91 Animals ................................ ................................ ................................ ............................ 91 Conditioning ................................ ................................ ................................ .................... 91 Dosing ................................ ................................ ................................ .............................. 92 Specimen Collection ................................ ................................ ................................ ........ 92 Pharmacokinetic Analysis ................................ ................................ ............................... 93 Statistical Analysis ................................ ................................ ................................ .......... 94 Results ................................ ................................ ................................ ................................ ..... 94 Intravenous Administration ................................ ................................ ............................. 94 Oral Administration ................................ ................................ ................................ ......... 96 Discussion ................................ ................................ ................................ ............................... 97 4 PHARMACOKINETICS OF GLYCOPYRROLATE IN STANDARDBREDS ................ 121 Methods ................................ ................................ ................................ ................................ 122 Animals ................................ ................................ ................................ .......................... 122 Dosing ................................ ................................ ................................ ............................ 122 Specimen Collection ................................ ................................ ................................ ...... 122 Plasma E sterase Stability ................................ ................................ ............................... 123 Pharmacokinetic Analysis ................................ ................................ ............................. 124 Statistical Analysis ................................ ................................ ................................ ........ 124 Results ................................ ................................ ................................ ................................ ... 125 Discussion ................................ ................................ ................................ ............................. 126 5 PHARMACODYNAMICS OF GLYCOPYRROLATE IN THOROUGHBREDS ............ 152 Methods ................................ ................................ ................................ ................................ 153 Animals ................................ ................................ ................................ .......................... 153 Dosing ................................ ................................ ................................ ............................ 154 Specimen Collection ................................ ................................ ................................ ...... 154 Determination of Plasma Protein Binding ................................ ................................ ..... 155

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8 Pharmacokinetic Analysis ................................ ................................ ............................. 157 Physiological Endpoints ................................ ................................ ................................ 157 PK P D Modeling ................................ ................................ ................................ ........... 158 Statistical Analysis ................................ ................................ ................................ ........ 158 Results ................................ ................................ ................................ ................................ ... 159 Protein Binding ................................ ................................ ................................ .............. 159 Pharmacokine tics ................................ ................................ ................................ ........... 159 Physiological Endpoints ................................ ................................ ................................ 160 PK PD Modeling ................................ ................................ ................................ ........... 161 Discussion ................................ ................................ ................................ ............................. 162 6 CONCLUSIONS AN D FINAL REMARKS ................................ ................................ ........ 189 Analytical Methodology ................................ ................................ ................................ ....... 189 Pharmacokinetics and Pharmacodynamics ................................ ................................ ........... 189 Regulatory Control ................................ ................................ ................................ ............... 191 APPENDIX DRUGS AND INSTRUMENT ................................ ................................ ................................ .... 193 HORSE PICTURES ................................ ................................ ................................ ..................... 195 LIST OF REFERENCES ................................ ................................ ................................ ............. 198 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 218

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9 LIST OF TABLES Table page 2 1 Preparation of working standard solutions for plasma analysis. ................................ ........ 61 2 2 Preparation of working standard solutions for urine analysis. ................................ ........... 62 2 3 Gradient table for GLY LC method. A ACN with 0.1% formic acid; B DI Water with 0.1% formic acid. ................................ ................................ ................................ ....... 63 2 4 Characteristics of plasma GLY calibration curves (n=5) used for linearity assessment. ................................ ................................ ................................ ......................... 64 2 5 Plasma GLY calibrator concentration taken from the accuracy and precision studies. ..... 65 2 6 Characteristics of urine GLY calibration curves (n=5) used for linearity assessment. ..... 67 2 7 Urine GLY calibrator concentration taken from the accuracy and precision studies. ....... 68 2 8 Accuracy and Precision (plasma) ................................ ................................ ....................... 75 2 9 Summary of Accuracy and Precision (plasma) ................................ ................................ .. 77 2 10 Accuracy and Precision (urine) ................................ ................................ .......................... 78 2 11 Summary of Accuracy and Precision (urine) ................................ ................................ ..... 80 2 12 Matrix effect, Extraction Efficiency, and Process Efficiency data for GLY in horse plasma. ................................ ................................ ................................ ............................... 81 2 13 Relative matrix effect in plasma. ................................ ................................ ....................... 82 2 14 Matrix effect, Extraction Efficiency, and Process Efficiency data for GLY in horse urine. ................................ ................................ ................................ ................................ .. 82 2 15 Relative mat rix effect in urine. ................................ ................................ .......................... 82 2 16 Plasma sample dilution integrity. ................................ ................................ ....................... 83 2 17 Urine sample dilution integrity. ................................ ................................ ......................... 83 2 18 Storage stability of GLY in plasma. ................................ ................................ .................. 84 2 19 Storage stability of GLY in urine. ................................ ................................ ...................... 85 2 20 Method ruggedness evaluation. ................................ ................................ ......................... 86

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10 2 21 Determination of the product ion ratio for confirmation of the presence of GLY in horse plasma. ................................ ................................ ................................ ...................... 88 2 22 Determin ation of the product ion ratio for confirmation of the presence of GLY in horse urine. ................................ ................................ ................................ ......................... 88 2 23 Comparison of evaluated method validation parameters between the current study and three previously published reports ................................ ................................ ............. 89 3 1 Demographics of study subjects. E ach number represents a horse. ................................ 102 3 2 Plasma GLY concentrations (pg/mL) following intravenous administration of 1 mg to each of 20 horses. ................................ ................................ ................................ ......... 103 3 3 Pharmacokinetic parameter estimates of GLY, determined using noncompartmental analysis, following intravenous administration of 1mg to eight (n=8) healthy adult Thoroughbred horses. ................................ ................................ ................................ ...... 105 3 4 Pharmacokinetic parameter estimates of GLY, determined using a three compartmental model, following intravenous administration of 1 mg to eight (n=8) healthy adult Thoroughbred horses. ................................ ................................ ................. 106 3 5 Model comparison using diagnostic criteria. ................................ ................................ ... 109 3 6 Urine GLY concentrations (pg/mL) following intravenous administration of 1 mg to each of 20 horses. ................................ ................................ ................................ ............. 113 3 7 Median (range) of estimated renal clearance (mL/min/kg) using the range of urinary flow rate estimates from various reports. ................................ ................................ ......... 120 4 1 Demographics of Standardbred study subjects. Each number represents a horse. .......... 131 4 2 Diagnostic values for a three compartment model fit to the concentration vs. time data for each horse. ................................ ................................ ................................ .......... 134 4 3 Plasma concentration of GLY after a single intravenous dose of 1 mg to each of 6 Standardbred horses. ................................ ................................ ................................ ........ 135 4 4 Pharmacokinetic parameter estimates of GLY, determined using a three compartmental model, following intravenous administration of 1 mg to six (n=6) healthy adult Standardbred horses. ................................ ................................ .................. 139 4 5 Comparison of pharmacokinetics parameters of Standardbreds and Thoroughbreds. .... 140 4 6 Calculated p values using the Mann Whitney U test for comparisons of pharmacokinetic parameter estimates between horse breeds. ................................ .......... 141 4 7 Urine GLY excretion data following intravenous administration of 1 mg to eight (n=6) Standardbred horses. ................................ ................................ .............................. 144

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11 4 8 Urine GLY renal clearance following intravenous administration of 1 mg to six (n=6) Standardbred horses. ................................ ................................ .............................. 150 5 1 Demographics of PK PD study subjects. ................................ ................................ ......... 168 5 2 Percent protein binding (mean SD) of GLY in the plasma of six ( n =6) healthy horses. ................................ ................................ ................................ .............................. 168 5 3 Plasma GLY co ncentrations (ng/mL) for six Thoroughbred horses following a two hour CRI of 8 g/kg. ................................ ................................ ................................ ........ 169 5 4 Pharmacokinetic parameter estimates of GLY, determined using a three compartmental model, following a two hour intravenous infusion of 4 g/kg/h of body weight to six (n=6) healthy adult Thoroughbred horses. ................................ ........ 173 5 5 Pharmacodynamic model parameters. ................................ ................................ ............. 185

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12 LIST OF FIGURES Figure page 1 1 Chemical structure of atropine. ................................ ................................ .......................... 36 1 2 Chemical structure of scopolamine. ................................ ................................ ................... 36 1 3 Chemical structure of GLY. ................................ ................................ ............................... 36 2 1 A weak cation exchange sorbent that consists of a carboxylic acid group bonded to ................................ ................................ ........... 62 2 2 sorbent is neutralized, and the elution is facilitated by the addition of a high ionic strength solvent. The analyte elutes from the sorbent because there are no forces to retain it. ................................ ................................ ................................ .............................. 63 2 3 Ion spectrum for GLY (m/z 318.1) ................................ ................................ .................... 64 2 4 GLY calibration curve weighted 1/x ( R 2 > 0.999) corresponding to Set 1. PAR Peak Ar ea Ratio. ................................ ................................ ................................ ................ 66 2 5 Plot of the response factor vs. the nominal calibrator concentrations. Slope = 12.86, y intercept = 152766. ................................ ................................ ................................ ......... 66 2 6 Plot of the response factor of the peak area ratio (PAR) vs. the nominal calibrator concentrations. Slope = 1.36 x 10 4 y intercept = 0.01680. ................................ ............ 67 2 7 GLY calibration curve weighted 1/x (R2 > 0.999) corresponding to Set 1. PAR Peak Area Ratio. ................................ ................................ ................................ ................ 69 2 8 Plot of the response factor vs. the nominal calibrator concentrations. Slope = 11.02, y intercept = 73825. ................................ ................................ ................................ ........... 69 2 9 Plot of the response factor of the peak area ratio (PAR) vs. the nominal calibrator concentrations. Slope = 3.51 x 10 8 y intercept = 0.0112. ................................ ............. 70 2 10 SRM chromatograms for GLY in horse plasma at the LLOQ (0.125 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram. ................................ ........... 71 2 11 SRM chromatograms for GLY in horse plasma at ULOQ (25 pg/mL) and the deuterated in ternal standard. TIC Total Ion Chromatogram. ................................ ........... 72 2 12 SRM chromatograms for GLY in horse urine at LLOQ (5 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram. ................................ ............................. 73

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13 2 13 SRM chromatograms for GLY in horse urine at ULOQ (2500 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram. ................................ ........... 74 2 14 GLY present as carryover in a mobile phase blank (2) following CAL 9 (1000 pg/mL) (1) and a second consecutive mobile phase blank (3) exhi biting no further GLY carryover. ................................ ................................ ................................ .................. 81 2 15 Comparison of the ion intensity ratios between a GLY reference standard (25 pg/mL) (A), a plasma (B) and urine (C) sample after GLY administration. ................................ .. 87 3 1 Illustration of a three compartment model. ................................ ................................ ..... 104 3 2 Observed (open circles) and the predicted concentrations (line) versus time when a two compartment model is applied. ................................ ................................ ................. 107 3 3 Observed (open circles) and the predicted concentrations (line) versus time when a three compartment model is applied. ................................ ................................ ............... 108 3 4 Plasma concentration (ng/mL) vs. time (h) data from 0 24 h and for GLY administered intravenously to eight healthy athletic adult Thoroughbreds. .................... 110 3 5 Plasma concentration (ng/mL) vs. time (h) data from 0 1 h and for GLY administered intravenously to eight healthy athletic adult Thoroughbreds. .................... 111 3 6 Plasma concentration (ng/mL) vs. time (h) data from 0 168 h and for GLY administered intravenously to eight healthy athletic adult Thoroughbreds. .................... 112 3 7 Plot of urine concentration (ng/mL) vs. time (h) after intravenous administration of GLY (1 mg) to 20 horses. ................................ ................................ ................................ 114 3 8 ) concentrations for 20 horses administered a single 1 mg intravenous dose of GLY. ................................ .................... 115 3 9 Plot of median (range) urine to plasma concentration ratios for 20 horses administered a single 1 mg intravenous dose of GLY. ................................ .................... 116 3 10 Plot of plasma concentration (pg/mL) vs. time (h) after oral administration of GLY (10 mg/mL) to six horses. ................................ ................................ ................................ 117 3 11 ) administered GLY in horse ( n =6) plasma. ................................ ................................ ....... 118 3 12 Plot of concentration (pg/mL) vs. time (h) of orally administered GLY (10 mg/mL) in horse (n=6) urine. ................................ ................................ ................................ ......... 119 4 1 Observed concentrations (open circles) and the predicted concentrations (line) versus time when a two compartment model i s applied to six Standardbred horses. ................. 132

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14 4 2 Observed (open circles) and the predicted concentrations (line) versus time when a three compartment model is applied to six Standardbred horses. ................................ ... 133 4 3 Plasma concentration (ng/mL) vs. time (h) data from 0 24 h and for GLY administered intravenously to six healthy athletic adult Standardbreds. ......................... 136 4 4 Plasma concentration (ng/mL) vs. time (h) data from 0 1 h and for GLY administered intravenously to six healthy athletic adult Standardbreds. ......................... 137 4 5 Plasma conc entration (ng/mL) vs. time (h) data from 0 168 h and for GLY administered intravenously to six healthy athletic adult Standardbreds. ......................... 138 4 6 Distribution of PK parameter estimates in Standardbreds ( n =6) and Thoroughbreds ( n =6). ................................ ................................ ................................ ................................ 142 4 7 Linear regression of concentration vs. incubation time demonstrating the degree of plasma esterase activity on GLY in horse plasma. ................................ .......................... 143 4 8 Cumulativ e GLY excretion in six horses administered 1 mg of GLY intravenously. ..... 146 4 9 Amount remaining to be excreted (ARE). ................................ ................................ ....... 147 4 10 Urinary excretion rate of GLY following a single 1 mg intravenous dose to six Standardbred horses. ................................ ................................ ................................ ........ 148 4 11 Mean (SD) plasma concentration (ng/mL) vs. time (h) data from 0 24 h and for GLY 149 5 1 Plasma concentration (ng/mL) vs. time (h) data from 0 26 h and for GLY administered by an intravenous infus ion in each of six healthy adult Thoroughbreds. ... 170 5 2 Plasma concentration (ng/mL) vs. time (h) data from 0 26 h and for GLY ad ministered by an intravenous infusion in six healthy adult Thoroughbreds. ............... 171 5 3 Plasma concentration (ng/mL) vs. time (h) data from 0 4 h and for GLY administered by an intravenous infusion in six healthy adult Thoroughbreds. ............... 171 5 4 Observed (circles) and predicted (line) plasma GLY concentrations after a two hour intravenous infusion of 8 g/kg of body weight to six (n=6) healthy adult Thoroughbred horses and pharmacokinetic analysis using a three compartment model. ................................ ................................ ................................ ............................... 172 5 5 Heart rate for each subject following a 2 h CRI of GLY. ................................ ................ 174 5 6 Mean (SD) heart rate (bpm) for six horses during the direct observation period. ........... 175 5 7 Respi ratory rate for each subject following a 2 h CRI of GLY. ................................ ...... 176

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15 5 8 Mean (SD) respiratory rate (breathes/min) for six horses during the direct observation period. ................................ ................................ ................................ ........... 177 5 9 Mean (SD) frequency of bowel movements for the entire direct observation period (t 0 = sta rt of the infusion) for six healthy horses administered GLY (8 g/kg of body weight) over a two hour intravenous infusion. ................................ ................................ 178 5 10 Mean (SD) frequency of bowel movements for the entire direct observation period (t 0 = start of the infusion) for six healthy horses administered GLY (8 g/kg of body weight) over a two hour intravenous infusion. ................................ ................................ 178 5 11 Mean (SD) time elapsed from the discontinuation of the infusion (t=0) until the first bowel movement for six healthy horses administered GLY (8 g/kg of body we ight) over a two hour intravenous infusion. ................................ ................................ ............. 179 5 12 Hysteresis (counterclockwise) plot demonstrating that the there is a temporal lag between the physiologic endpoint (heart rate) and plasma GLY concentration. Heart rate represents mean (SD) of all subjects. ................................ ................................ ........ 180 5 13 The effect compartment model. ................................ ................................ ....................... 181 5 14 Mean plasma GLY concentration and mean heart rate effects over time for each subject. Error bars are removed for clarity. ................................ ................................ ..... 182 5 15 Plot demonstrating that the there is a temporal lag between the mean p hysiologic endpoint (heart rate) and mean plasma GLY concentration. Error bars are removed for clarity. ................................ ................................ ................................ ......................... 183 5 16 PK PD linked model fit for heart rate for each of six horses. ................................ .......... 184 5 17 Mean plasma GLY concentration and mean respiratory rate effects over t ime for each subject. Error bars are removed for clarity. ................................ ............................. 186 5 18 Plot demonstrating that the mean maximum effect for the physio logic endpoint (respiratory rate) occurs before mean maximum plasma GLY concentration. Error bars are removed for clarity. ................................ ................................ ............................ 187 5 19 Mean (SD) respiratory rate as a function of concentration for six horses demonstrating proteresis (clockwise) plot. ................................ ................................ ...... 187 5 20 Individual hysteresis showing the plasma concentration and the predicted effect compartment concentration after the PK PD link model was applied. ............................ 188 A 1 GLY (Robinul V) injectable. ................................ ................................ ........................ 193 A 2 Generic GLY injectable used for intravenous injection. ................................ ................. 193 A 3 Robinul V tablets (1 mg each). ................................ ................................ ........................ 194

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16 A 4 Triple Stage Quadrupole (TSQ) Quantum Ultra mass spectrometer (ThermoFisher, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) source and interfaced with a HTC PAL autosampler (Leap Technologies, C arrboro, NC, USA) and Accela LC pump (ThermoFisher) ................................ ................................ ... 194 B 1 Study horse being conditioned on a treadmill. ................................ ................................ 195 B 2 Study horse receiving an intravenous infusion of GLY. ................................ .................. 196 B 3 During the PK PD study, horses were housed in pairs, reducing anxiety and normalizing behavior. ................................ ................................ ................................ ...... 197

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17 LIST OF ABBREVIATION S AND SYMBOLS z elimination rate constant A intercept at time=0 for the first phase ACh acetylcholine ACN acetonitrile AIC Akaike Information Criteria Alpha slope for the first phase of the model equation ANOVA analysis of variance AORC Association of Official Racing Chemists ARCI Association of Racing Commissioners International ARE amount remaining to be excreted AUC area under the curve AUMC area under the moment curve B intercept at time=0 for the second phase Beta slope for the second phase of the model equation BLQ below the limit of quantitation bpm beats per minute C concentration C b bound concentration C e effect site drug concentration C f free concentration C last observed plasma drug concentration at the last timepoint C max maximum plasma drug concentration C p plasma drug concentration C p(last) last measured plasma drug concentration

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18 C pss plasma drug concentration at steady state C t total concentration C u ur ine drug concentration CBA carboxy propyl phase CBC complete blood count CID collision induced dissociation Cl total clearance Cl P plasma clearance Cl R renal clearance Cl H hepatic clearance CRI continuous rate infusion CV coefficient of variation D dose DCM dichloromethane E Effect E H extraction ratio E 0 baseline effect E max maximum effect produced (efficacy) ECG electrocardiography EC 50 concentration that produces half of the maximal effect (potency) EE extraction efficiency ESI electrospray ionization F A formic acid FDA Food and Drug Administration fu fraction unbound or free drug fraction

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19 Gamma slope for the third phase of the model equation GC gas chromatography GLY glycopyrrolate h hour HESI heated electrospray ionization HR heart rate IS internal standard IV intravenous k 12 transfer rate constant from the first to the second compartment k 21 transfer rate constant from the second to the first compartment k 10 elimination rate constant from the first compartment k 1e transfer rate constant from the fir st to the effect compartment k a absorption rate constant k e0 elimination rate constant from the effect compartment LC liquid chromatography LLE liquid liquid extraction LLOD lower limit of detection LLOQ lower limit of quantitation ME matrix effect MeOH m ethanol mg milligrams min minutes mL milliliters MRM multiple reaction monitoring MRT mean residence time

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20 MS mass spectrometry MS/MS tandem mass spectrometry MW molecular weight m/z mass to charge ratio n sigmoidicity factor (hill coefficient) NA nonspeci fic adsorption ng nanograms PAR peak area ratio PB protein binding PD pharmacodynamics PE process efficiency pg picograms PK pharmacokinetics R 2 coefficient of determination R o rate of intravenous infusion RP reversed phase RMTC Racing Medication Testing Consortium RR respiratory rate RSD relative standard deviation SBC Schwarz Bayesian Criteria SD standard deviation S/N signal to noise ratio SOP standard operating procedure SPE solid phase extraction t time

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21 t 1/2 half life t 1/2 ke0 half life for equilibrat ion t max time to reach peak plasma concentration TI tolerance interval TIC total ion chromatogram TSQ triple stage quadrupole UF ultrafiltration g micrograms ULOQ upper limit of quantitation rate of urine formation V 2 volume of distribution of the second compartment V 3 volume of distribution of the third compartment Vd volume of distribution Vd ss volume of distribution at steady state Vc volume of central compartment Vz apparent volume of distribution during the terminal phase

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22 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE PHARMACOKINETICS AND PHARMCODYNAMICS OF GLYCOPYRROLATE IN THE HORSE By MARC J RUMPLER May 2012 Chair: Tom Vickroy Major: Veterinary Medical Sciences Glycopyrrolate (GLY) is a muscarinic receptor antagonist typically used in veterinary medicine to inhibit gastrointestinal motility and block the effects of vagal stimulation during anesthesia. It is reportedly used in horseracing to reduce bronchial secretions despite the fa ct that it is a prohibited substance Regulating legitimate medications in horseracing relies on the development of industry threshold limits and recommended withholding times. Therefore pharmacokinetics studies are important for determining drug plasma an d urine conc entrations following a typical often clinically relevant dosage. The following research describes a rapid, sensitive, selective and fully validated liquid chromatography mass spectro metry analytical method for the detection and quantification of GLY in horse plasma and urine. The Pharmacokinetics of GLY have been evaluated after a 1 mg intravenous dose in Thoroughbreds and Standardbreds. Additionally, the pharmacodynamics have been evaluated following a clinically relevant intravenous infusion (8 g/kg) in Thoroughbreds. Further, we investigated the elimination of GLY in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Pharmacokinetic parameters were best estimated using a three compartmen t model for plasma concentration versus time data from 0 24 h, although GLY was detectable for 168 h after administration. GLY

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23 disposition in the horse was characterized by a steep decline in plasma concentration immediately after dosing, extensive distrib ution from the central compartment, rapid clearance, and a prolonged terminal elimination phase. GLY remained detectable in urine samples collected through 168 h after intravenous administration and through 24 h after oral administration.

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24 CHAPTER 1 INTRODUCTION AND LIT ERATURE REVIEW Overview and History Glycopyrrolate (GLY) is a synthetic anticholinergic compound with a chemical name 3[(cyclopentylhydroxyphenylacetyl)oxy] 1, 1 dimethyl pyrrolidinium bromide that was derived from several Solanaceae ) plants. It was synthesized in the 1950s in an effort to d evelop safer anticholinergic compounds that could not easily penetrate the centr al nervous system (CNS) where they may impart adverse effects (Franko & Lunsford 1960) addition of a quaternary amm onium substituent group that is permanently ionized at physiological pH values. A nticholinergic agent s competitively inhibit the action s of acetylcholine (ACh) in tissues innervated by po stganglionic cholinergic nerve terminals and on smooth muscles that respond to ACh but lack cholinergic innervation (Grum & Osborne, 1991) The prototypical drug in this class is atropine which, along with its analog ue s, competes predominately at the muscarinic receptor with little action at th e nicotinic receptor (Doods et al 1987) Thus, these compounds are also known as muscarinic antagonists or antimuscarinic drugs. Cholinergic antagonists all contain similar structural elements such as a cationic head group with a tertiary or quaternary a mmonium group; alicyclic or aromatic rings, for hydrophobic interactions with the receptor; an interconnecting structural moiety such as an amide or ester; and one or more hydroxyl groups (Wess et al 1990) The two most frequently used anticholinergics d rugs are atropine and scopolamine, both isolate d from Atropa belladonna and having long medicinal histories. They are organic esters formed by the combination of an aromatic acid, tropic acid, and a comple x organic base, either

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25 tropine as in the case of at ropine ( Figure 1 1 ) or scopine in the case of scopolamine ( Figure 1 2 ). Scopine differs from tropine only in having a n epoxyl group in the tropine structure. The intact ester of tropine and tropic acid is essential for the antimuscarinic action of atropine, sin ce neither the free acid nor the b a s e exhibit significant anticholinergic activity. The presence of a free OH group in the acid portion of the ester also is important for activity. Substitution of other aromatic acids for tropic acid modifies but does not necessarily abolish the anticholinergic activity. Both atropine and scopolamine contain a te rtiary ammonium group ( pKa 7.5 10.2 ) which allows the drug to permeate biological membranes causing unwanted side effects as a result of interaction with central n ervous system receptors when the drugs were used to treat peripheral conditions. The introduction of GLY into human medicine provided a n antimuscarinic agent that was specifically designed to penetrate barriers less effectively and to act in the periphery without causing side effects that could outweigh the potential benefits. To date, GLY has been used as an alternat ive to these antimuscarinic agents in the treatment of several conditions of the cardiovascular, respiratory, glandular and gastrointestinal s ystems. Basic Pharmacology of Glycopyrrolate Glycopyrronium occurs as a white, odorless crystalline powder and is known as GLY when it is accompanied by a bromide counter ion in order to maintain electric neutrality. GLY consists of two components (mandelic acid and an organic base) bound together by an ester linkage. The quaternary ammonium substituent was formed with the addition of a second methyl group on the tertiary nitrogen atom (Franko & Lunsford 1960) The compo und is water soluble due to the quaternary amine structure ( Figure 1 3 ). The partition coefficient of GLY in an n octanol/water system is 0.304 (log10 P= 1.52) at roo m temperature (24C).

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26 Muscarinic receptor antagonists block the effects of ACh on muscarinic cholinergic receptors in the peripheral ganglia as well as in the CNS. GLY differs from other muscarinic antagonists because it has difficulty permeating biologic al membranes due its highly polar quaternary ammonium group and its permanent ionization at physiological pH (Franko 1962) In contrast to its naturally occurring analogues it is poorly absorbed or ally (Rautakorpi et al 1998) does not cross the placen tal barrier (Proakis & Harris 1979) and does not interfere with central nervous system (CNS) cholinergic function (Ali Melkkila et al ., 1990) Consequently, without such membrane permeating potential, GLY produces markedly reduced CNS effects compared to its more lipoph ilic congeners, atropine and scopolamine (Shereff, 1985) Peripheral cholinergic receptors are predominately present in the autonomic effector cells of smooth muscle, cardiac muscle, the sin oatrial node, the atrioventricular node, exocrine glands, and, to a lesser extent, in the autonomic ganglia (Brown & Taylor 2006) However, due to the nonselective blockade of muscarinic receptors in other organ systems, GLY also inhibits a number of para sympathetically mediated functions, causing decreased airway and gastrointestinal secretions, bronchodilation, and inhibition of gastrointes tinal motility (Barocelli et al 1993) GLY competitively antagonizes the effect of ACh on the muscarinic receptors by occupying post synaptic receptor sites (Lau & Szilagyi 1992) It has a high affinity for the muscarinic receptor family (with little effect at nicotinic sites) with little selectivity for any of the receptor subtypes (Haddad, et al ., 1999) At present there are five well defined subtypes of muscarinic cholinergic receptors in humans, denoted M1 to M5, each encoded by a unique gene and differing in their amino aci d sequence (Bonner et al 1987) Until the early 1980s, all muscarinic receptors were t hought to be alike given the lack of muscarinic agonists or antagonists presenting specific effects (Goyal, 1989) Since then, different

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27 muscarinic receptor subtypes have been shown to be responsible for mediating the effects of AC h on heart rate, smooth m uscle contractile activity, central nervous system activity and exocrine gland function. M1 and M4 receptors are predominately located in the brain and are involved with behavioral and cognitive functions (Hammer & Giachetti 1982) M2 receptors are the on ly subtype located in the cardiac smooth muscle (Caulfield, 1993) and the M3 subtype exists in the glandular tissue as well as in the smooth muscle of the airways (Doods et al 1987) gastrointestinal tract (Eglen R. 2001) and urinary bladder (Hedge, 2006) Little research has been conducted on M5 receptors but they are also believed to be physiologically relevant in the CNS (Yamada et al 2001) Functional studies in humans suggest that parasympathetically mediated intestinal smooth muscle contractio n occurs via M3 receptor stimulation (Eglen et al ., 1992) whereas M2 receptor stimulation may be involved in abrogating sympathetically mediated smooth muscle relaxation (Elgen et al ., 1996) Radioligand binding studies have shown the presence of receptor subtypes M2 and M3 in intestinal smooth muscle of guinea pigs and dogs (Giraldo et al ., 1987; Zhang et al ., 1991) In the horse species, the M3 receptors are responsible for mediating ACh induced tr acheal (van Nieuwastadt et al ., 1997) and jejunal (Teixeira Neto et al ., 2011) smooth muscle contraction. Even though relaxation of the airway of the horse by M3 receptor antagonists may have potential therapeutic implications in horses presented with heaves, M3 receptor antagonism may also have a major i nhibitory effect on bethanechol induced smooth muscle contraction in the horse (Marti et al ., 2005) Although GLY atropine and scopolamine are non specific antagonists at these receptors, atropine has a two fold preference f or M1 (CNS) receptors (Gomez et al ., 1995) M3 receptors (glandular secretion, blockade producing a reduction in sali vation) are much more sensitive to

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28 anticholinergics than M2 receptors (cardiac, blockade causing an increase in heart rate). Thus, for all three drugs, a greater dose is needed to prevent bradycardia than to reduce salivation (Jongerius et al ., 2003) Therapeutic and Clinical Applications of Glycopyrrolate GLY and other muscarinic cholinergic antagonists, such as atropine have been used clinically for several decades for a variety of conditions as a result of their receptor non selectivity GLY is administered almost exclusively by the parenteral route, due to its poor absorption from the gastrointestinal tract. GLY was introduced in 1961 as a long acting anticholinergic wi th initial reports confined to gastroenterology to reduce the volume and free acidity of gastric secretions (Sun, 1962; Moeller, 1962) until 1970 when Boatright et al ., reported on its preoperative uses to decrease complication of gastric juice aspiration during tonsillectomy (Boatright et al 1970) GLY protects against the peripheral muscarinic effects (e.g. bradycardia and excessive secretions) of cholinergic agents such as neostigmine and pyridostigmine which are given to reverse the neuromuscular blockade due to non depolariz ing muscle relaxants (Gye rmek, 1975; Ramamurthy, 1972) Atropine is often used in emergency situations but the lack of CNS side effects and better matched onset and offset time with neostigmine makes GLY the agent of choice in reversal of neuromuscular blockade, especially in neon ates and the elderly (Salem & Ahearn, 1986) Typical doses for adults are 0.2 mg intravenously per 1 mg of neostigmine or the equivalent dose o f pyridostigmine and a dose of 0.01 0.015 mg intravenously with 0.05 mg/kg neostigmine or equivalent dose of pyri dostigmine. In anesthesia GLY is indicated for use as a preoperative antimuscarinic agent to reduce salivary, tracheobron chial and pharyngeal secretions, to reduce the volume and free acidity of

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29 gastric secretions, and to block cardiac vagal inhibitory r eflexes during induction of anesthesia and intubation. GLY i njectable may be used intra operatively to counteract drug induced or vagal traction reflexes with the associated arrhythmias. In small animals GLY has been used with success to treat vagally medi ated bradycardia during anesthesia without causing major complications in other organ systems (Dyson et al ., 1999) In horses, however, GLY has been reported to cause prolonged intestinal hypomotility and colic when used to prevent introperative bradycardia (Singh et al ., 1997; Teixeira Neto et al 2004) Therefore clinicians have taken a conservative approach when using GLY during intra operative procedures. Typical does for adult humans are 0.2 mg to 0.4 mg intravenously or intramuscularly bef ore the induction of anesthesia. Alternatively, a dose of 4 5 g/kg of body weight up to a maximum of 0.4 mg may be used In dogs and horses typical pre anesthetic doses range from 5 10 g/mg of body weight. GLY antisialagogue properties have been known for several decades (Wyant & Kao, 1974; Mirakhur et al ., 1978) More recently, GLY has been investigated to treat sialorrhea ( excessive drooling ) in adolescents with neurological disorders (Tscheng, 2002) and adults with Parkinson disease (Arbouw et al ., 2 010) Despite some reports detailing only a modest improvement (Blasco & Stansbury, 1996) and adverse effects (Madan & Beck, 2006) the medication is well tolerated (Stern, 1997) and a liquid oral formulation (Cuvposa) has recently been FDA approved for this purpose based on encouraging clinical trial results (Zeller et al ., 2012; Zeller et al ., 2012) Similarly, GLY has been used topically for the treatment of hyperhidrosis (excessi ve sweating) (Seukeran & Highet, 1998; Luh & Blackwell, 2002; Shaw et al ., 1997; Atkin & Brown, 1996; Bajaj & Langtry, 2007) an embarrassing condition th at effects a small population.

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30 Chronic obstructive pulmonary disease (COPD) is characterized by progr essive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of t he human muscarinic (M3) receptor subtype (Brown & Taylor, 2006) Clinical studies have shown that inhaled GLY displays bronchodilator activity in COPD and asthmatic patients, an effect apparently lasting for 8 12 h (Walker et al ., 1987) (Tzelepis et al 1996) (Schroeckenstein et al ., 1988) A ddition ally nebulized GLY has been found to have a synergistic effect when combined with albuterol in treating acute exacerbations of asthma (Gilman et al ., 1990) (Cydulka & Emerman, 1994) and COPD (Cydulka & Emerman 1995) It has also been demonstrated to be a more effective bronchodilator over a longer duration when compared to atropine, for the treatment of exercise induced asthma (Johnson et al ., 1984) In normal human subjects, GLY has been shown to cause prolon ged bronchodilation after intravenous (10 g/kg) (Gal & Suratt, 1981) and nebulized (Gal et al ., 1984) (Alex et al ., 1999) administration without the systemic ant icholinergic cardiac effects of the shorter acting atropine. It was shown that GLY inhibited electrical field stimulation induced contractions of human and guinea pig isolated airways with a longer duration of action than ipratropium and that both agents exhibited no selectivity toward M1, M2, and M3 receptors (Giraldo et al ., 1987) Ot her human studies have demonstrated that intratracheally administered GLY provide s superior bronchoprotection and slower receptor dissociation over ipratropium bromide in patients with asthma (Hansel et al ., 2005) and COPD (Ogoda et al ., 2011) There is al so opt imism for product s under development following clinical trials for an inhaled therapeutic of GLY alone (Sechaud et

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31 al ., 2012) and a combinatio n of GLY with a long acting beta 2 agonist (formoterol) to treat COPD. GLY has n ot been reported to be used routinely to treat respiratory conditions in horses. Atropine was u sed to a limited extent before the development of more selective drugs such as clenbuterol with reduced side effects (Murphy et al ., 1980) Instead most research in horses is 2 selective agonists because of their potent and prolonged duration of action with limited adverse effects (Adams, 2001) GLY is reportedly used to reduce bronchial secretions in race horses, an effect that has benefite d human patients in surgery (Sengupta et al ., 1980) and in trauma with life threatening conditions (Bhalla et al ., 2011) GLY is also preferred in obstetrics due to its lack of propensity to reach neonates (Abboud et al ., 1981) GLY has been investigated for its effect on hypotension during a nesthesia conducted for caesarean section, but has been met with conflicting results. Previous studies found that GLY reduced (Ure et al ., 1999) increased (Quiney & Murphy, 1995) or had no effect (Yentis et al ., 2000) (Rucklidge et al ., 2002) on maternal hypotension after spinal anesthesia for caesarean section. Studies evaluating GLY as a prophylactic antiemetic for spinal anesthesia have shown that the drug is of little benefit for this purpose (Quiney & Murphy, 1995) (Thakur et al ., 2011) Analytical M ethodology Early investigations into human and horse GLY disposition use d a radioreceptor assay (Kaila et al 1990) or an enzyme linked immunoabsorbent assay (Leavitt et al ., 1991) P harmacokinetic studies could not be performed earlier due to the availability of methods that were too complex (Kaltiala et al 1974) or demonstrated inadequate sensitivity (Murray et al 1984) By their nature, radioreceptor and immuno assays are rapid, highly sensitive, reliable,

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32 precise, accurate and simp le to perform. Their primary disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand The use of such techniques has been eclipsed by hyphenated separatio n and mass spectrometric met hods due to their ability to identify and quantify a substance For many years gas chromatography (GC) has cemented its role as an analytical powerhouse in analytical chemistry/toxicology and is likely the most widely used tool in analytical laboratories. However, analysis of heat labile and polar compounds, long analysis time and tedious sample preparation procedures have left room for much innovation. Gas chromatography has been successfully used for GLY detection in horse uri ne (Matassa et al 1992) The method employs a selective solid phase extraction and can be sensitive down to 250 pg/mL. However, a large sample volume (50 mL) and an intensive deriva tization procedure put a strain on repeat/duplicat e analysis and the anal yst. Liquid chromatography (LC) is quickly winning favor among investigators as an alternative to GC methods This is evident by the number of published method validation studies performed using LC MS compared to GC MS. In recent years researchers have fou nd that (Alder et al ., 2006) capabilities (Lee & Kerns, 1999) analytical drug detection laboratories have been rapidly validating methods over the past several years to incorporate this technology into routine screening and confirmation procedures (Maurer 2005; Maurer H., 2005) Limitations for LC MS include ion s uppression (Cappiello et al ., 2008) and cost In the most recent work Storme et al (2008) use d liquid chromatography tandem mass spectrometry to quantify GLY using a method that employ ed volatile ion pairing reagents to extract GLY from human plasma (Sto rme et al

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33 2008) In this method time of flight (TOF) mass spectrometry was used. A structurally similar compound, mepenzolate was used as the internal standard. While sensitivity was substantially improved (100 pg/mL) compared to previous methods, there appeared to be unwanted matrix effects and recovery remained unacceptable for the objectives proposed in this research Mass spectrometry has become the analytical tool of choice for the confirmation of small molecules because it allows the unequivocal id entification and qu antification of substances at very low concentrations in complex mixtures such as plasma and urine Tandem mass spectrometry (MS n ), which incorporates consecutive stages of MS analysis can derive structural information (Chen et al ., 2007 ) and further improve compound selectivity by mapping fragmentation pathways (Watson & Sparkman, 2007) Glycopyrrolate Doping in Race Horses The detection and confirmation of identity of drugs and metabolites in bio logical specimens of racing animals is a challenging analytical undertaking Laboratories are tasked with the goal to detect and quantify substances that may affect performance. This includes compounds that may enhance the physical capabilities and or provide legitimate therapeutic value. For substances that provide no therapeutic benefits, their presence alone may result in regulatory sanctions. GLY is designated a class 3 substance by the Association of Racing Commissioners International (ARCI), Inc. (Association of Racing Commissioners Inte rnational, 2011) and regulated in racing horses due to its potential to affect performance and degree of therapeutic value. Although it has several veterinary clinical applications by inhibiting parasympathetic activity, its use near race day is prohibite d and positive reports from post horserace samples in the US are relatively common (reference RMTC). Accordingly, the American Association of

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34 Equine Practitioners (AAEP) has identified GLY as a therapeutic substance used by race track practitioners for leg itimate therapeutic purposes. Consequently, the Racing Medication and Testing Consortium (RMTC) has provided financial support for studies of the disposition of GLY as part of its efforts to acquire rel iable data upon which to establish thresholds and with drawal time recommendations for therapeutic substances used in racing horses. A threshold limit is a concentration, either in plasma, serum or urine that, if exceeded, will result in a positive finding. Whereas threshold limits cannot easily be interpreted by non scientists, a drug withdrawal time is also recommended by regulatory bodies. A withdrawal time is an estimated time period in which veterinarians or horseman should withhold a medication before the start of a race. While threshold limits and withdr awal times may be determined from properly designed and sophisticated pharmacokinetic studies, it is important to determine the duration of pharmacological actions relative to plasma drug concentrations for compounds that may alter performance. According to the ARCI guidelines a class 3 substance has potential medicinal value with the possibility to affect performance (Short et al ., 1998) In horse racing, GLY is potentially exploited for its bronchodilatory effects and favored for its lack of effects on t he central nervous system (CNS) compared to other muscarinic antagonists as described above. The pharmacokinetics and pharmacodynamics of GLY and other anticholinergics substances have been studied t horoughly in humans. Several accounts of the effects of G LY have been studied in horses (Dyson et al 1999 ; Singh et al ., 1997; Teixeira Neto et al ., 2004) but there are no reports that have combined pharmacokinetic and pharmacodynamic analysis and have performed predicative modeling. Such studies may benefit the developme nt of regulatory guidelines and clinical research.

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35 Hypothesis A sensitive and selective quantitative analytical method can be developed to enable accurate determination of GLY in plasma and urine in the horse after clinically relevant doses. Further the method will allow meaningful pharmacokinetic analysis and the development of an integrated pharmacokinetic pharmacodynamic (PK PD) model to describe the complete time course of common clinical effects. To test the hy pothesis of the study, the following specific aims were purposed: Specific Aim 1: To develop and fully val idate a quantitative liquid chromatography tandem mass spectrometric method for the confirmation of GLY in horse plasma and urine. Specific Aim 2 : T o investigate and characterize the pharmacokinetic profile after intravenous and oral administration to horses. Specific Aim 3 : To investigate the pharmacodynamic effects after a continuous rate intravenous infusion in horses. Specific Aim 4 : To apply a m athematical model to correlate GLY plasma concentrations with the pharmacological response.

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36 Figure 1 1 Chemical structure of atropine. Figure 1 2 Chemical structure of scopolamine. Figure 1 3 Chemical structure of GLY

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37 CHAPTER 2 ANALYTICAL METHOD DE VELOPMENT AND VALIDA TION The physiochemical properties of glycopyrrolate ( GLY ) present certain analytical challenges. Its quaternary ammonium structure can increase the difficulty of isolat ing and extract ing it from biological fluids Recovery of the compound could only be done with a selective sorbent and optimiz ed pH conditions, solvent wash steps and the selective elution of analytes While the linearity of previous methods was sufficient for routine detection, a larger dynamic range would likely be necessary for the proposed analysis of samples collected after in travenous and oral administration of GLY to the horse. Therefore we sought a stable isotope labeled analogue for use as an internal standard to appropriately track the analyte and possibly reduce matrix effects. Finally, the intention for this work was to produce reliable and meaningfu l pharmacokinetic analysis. Selection of an appropriate internal standard (IS) is important to the development of a quantitative assay as in the case of LC MS/MS (Avery, 2003) During method development, the selection of the IS should be such that it will adequately track the analy te throughout sample analysis. An IS is added to samples to compensate for unavoidable sample losses (e.g., extraction transfer losses, ionization effects and injection issues) and to account for th e presence of competing substances in the extracts and to minimize their influence Thus, a reliable IS should be structurally similar to the analyte of interest. Typically the best alternative, if available, is a stable isotopically labeled analogue of th e analyte (Jian et al 2010; Stokvis et al 2005) GLY may be chromatographed on a wide range of analytical LC columns. Ideally, the best match would offer superior polar retention, aqueous and acidified mobile phase compatibility, rapid analysis and the ability to sustain high pressures, although the latter in not a nece ssity. Column selection is based upon the likelihood of accommodating these features. Use of a guard

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38 column is necessary to prevent unwanted matrix components from entering the analytical column, thus extending its life. Ultra performance liquid chromatography (UPLC), a recent advancement (Sherma, 2005) i s appropriate for this method because the columns are packed with smaller diameter particles requiring a shorter column length and facili tating faster run times with comparable resolution to traditional LC columns (Fan et al 2007) (Wren & Tchelitcheff 2006) Analytical method validation is required across several disciplines and is essential in order to acquire reliable data for analyst interpretation (Stckl et al 2009) Without such guidelines for analytical integrity, assay determinations are capricious and could impose speculative and/or unsupported conclusions. Analysts, laboratories and even organizations may become accountable for misleading, inaccurate or erroneous information. From a medico legal perspective such misinformation may result in unsubstantiated legal ramifications. Several governing agencies have published requirements and recommendations for validation studies (CITAC/EURACHEM, 2002; Guidance for Industry, Bioana lytical Method Validation, 2001; Guideline on Validat ion of Analytical Methods, 2009; General Requirements for the Competence of Testing and Calibration Laboratories) Consequently, the validation of bioanalytical methods has become a rate limiting step for the reliable analysis of research samples. Methods Chemicals and Reagents Reagent g rade formic acid was obtained from ACROS Organics (Morris Plains, NJ, USA). All solvents including acetonitrile, methanol, and methylene chloride were HPLC grade and were obtained from Therm o Fisher (Pittsburg, PA, USA).

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39 Standard Preparation GLY ((United S tates Adopted Name (USAN)) is also known as glycopyrronium bromide (Recommended International Nonproprietary Name) (Organization, 1997) (Council) GLY has the elemental composition C 19 H 28 BrNO 3 and, as such, includes the bromide counter ion. Therefore, con centrations of GLY are reported herein without adjustment of the mass for the bromide ion component, consistent with the USAN definition for GLY. All stock solutions used to prepare calibrators and controls were prepared from certified reference standards. Drug standards including GLY and GLY iodide d 3 were obtained from United States Pharmacopeial Convention (Rockville, MD, USA) and Toronto Research Chemicals (North York, Ontario, Canada), respectively. All stock standard solutions were prepared from solid form and dissolved in acetonitrile. All working standard solutions were diluted to the appropriate concentrations in acetonitrile to prepare calibrators in urine and in plasma from 1 2500 pg/mL and 0.05 25 pg/mL, respectively. Working standard solutions u sed to prepare calibrators and positive control samples were prepared from independently prepared stock solutions. For plasma analysis each calibrator and positive control sample was prepared using 1 mL of phosphate buffer (50 mM, pH 7.0) and 1.0 mL of dru g free control horse plasma, and fortified with the appropriate volume of GLY working standard solution ( Table 2 1 ) and 25 L of GLY d 3 standard solution. The deuterated GLY analog was prepared in a working standard solution at a concentration of 0. internal standard concentration was 100 pg/mL of plasma. For urine analysis each calibrator and positive control sample w as prepared using 1 mL of phosphate buffer (50 mM, pH 7.0) and 0.5 mL of drug free control horse urine, and fortified with

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40 the appropriate volume of GLY working standard solution ( Table 2 2 ) and 25 L of GLY d 3 standard solution. The deuterated GLY analog was prepared in a working standard solution at a concentration of 0 internal standard concentration was 200 pg/mL of urine. Solid Phase Extraction The tubes were centrifuged at 1508 x g (2800 rpm) for 12 min and the buffered plasma and urine samples were subjected to solid phase extraction. Isolute CBA 3 mL columns (Biotage, Charlottesville, VA, USA) were sequentially conditioned with 2 mL each of m ethanol, water, and phosphate buffer (50 mM, pH 7.0). Samples 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, methanol, and dichloromethane. The analyte was eluted with two 0.5 mL aliquots of 1% formic acid in acetonitrile and the eluate was evaporated under nitrogen on a TurboVap LV evaporator (Zymark, Hopkington, MA, USA). Sample extracts were then dissolved in (10:90) and transferred to glass autosampler vials. Liquid Chromatography y packed, guard column (Waters, Taunton, MA, USA). Gradient elution was begun with a mobile phase of 0.1% (v/v) formic acid in water (80%) (Solvent A) and 0.1% (v/v) formic acid in acetonitrile (20%) (Solvent B). The initial mixture, kept constant at a 500 Solvent A was decreased linearly to 5% and Solvent B increased to 95 % over 2.25 min and held for 0.25 min. The mobile phase was then returned to the initial conditions for the remaining 0.5 min fo r a total run time of 3.5 min ( Table 2 3 ). The flow into the mass spectrometer was diverted

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41 from 0 0.75 min and 2.5 er (10:90) containing 0.1% formic acid was injected. All LC MS/MS analyses were performed on a Triple Stage Quadrupole (TSQ) Quantum Ultra mass spectrometer (ThermoFisher, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) source and interfaced with a HTC PAL autosampler (Leap version 2.0.7 and LCquan version 2.5.6 were used for data acquisition and analysis. The autosampler syringe was washed befor e and after injection five times each with 2% formic acid in acetonitrile (wash 1) followed by 10% methanol in water (wash 2). The post injection rinse was followed by an injection valve rinse using five repetitions each of wash 1 followed by wash 2. All r inse solvents were diverted directly in to the waste stream after use. Mass spectral data were acquired in positive ion mode using a heated electrospray ionization technique with the following MS parameters: ESI spray voltage; 4100, vaporizer temperature; 2 40 C, sheath gas pressure; 40 (arbitrary units), ion sweep gas; 0 (arbitrary units), auxiliary gas pressure; 6 (arbitrary units), capillary temperature; 300 C, tube lens offset; 89 V and skimmer offset; 0 V. Data Analysis Identification and quantificatio n 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/L each of the analyte and internal standard dissolved in mobile phase Tuning for GLY yielded collision energies of 39, 50, and 33 for transitions GLY d 3 yielded a collision energy of

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42 on (i.e., transitions were used as qualifier transitions Figure 2 3 repre sents the ion spectrum for GLY All standard solutions controls, calibrators, and test samples were prepared in duplicate and peak ion area ratios of the analyte and internal standard were calculated for each. Individual values of the duplicate concentrations were averaged. Calibration was performed using a simple least squares linear regre ssion analysis with a 1/C weighting factor, where C was the nominal plasma or urine concentration. Quality control and sample acceptance criteria have been specified according to the following requirements and standard operating procedures of the UF Racing Laboratory, Research Section. The requirement is that the % CV for all calibrators, positive controls, and test samples must not exceed 10% (15% at the L LOQ). In addition, for calibrators the difference between the back calculated concentration and the nom inal concentration must not exceed 10% (15% at the L LOQ). Al l samples that did not meet these criteria were re analyzed. Method Validation The method was validated in accordance with the U.S Food and Drug Administration recommended guidelines (Guidance for Industry, Bioanalytical Method Validation, 2001) for specificity, sensitivity, linearity, accuracy, precision, extraction efficiency and stability. Other parameters such as carryover, dilution integrity and matrix effect were assessed in accordance with t he European Medicines Agency recommended guidelines (EURACHEM, 1998) Also, I have consulted additional reference materials such as those referenced above for guidance with method validation procedures and specifications.

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43 System Suitability System suitability is a valuable component of any analytical procedure and ensures the performance of the analytical system(s) (Briscoe et al 2007) Each validation and test sample batch contained ten calibrators prepared in plasma or urine, three non fo rtified (analyte) control samples, and five analyte and internal standard fortified positive control samples spanning the range of the calibration line all prepared in duplicate. The three non fortified analyte control samples consisted of a reagent blank (diluent only), matrix blank (matrix + diluent) and negative control (internal standard + matrix + diluent). Run acceptability was determined by the accuracy, precision and minimum acceptance criteria (as described below) of the calibration standards and positive control samples, the coefficient of determination of the standard curve, and the absence or degree of GLY present in the negative control samples. If carryover or contamination exist ed in the negative control samples, attempts wer e made to minimi ze this effect before analyzing the calibration curve All calibration points included in the curve calculation must adhere to the following minimum acceptance criteria: 1. Each duplicate measurement from each calibration point to be included in the calibrati on curve must generate valid data (see conditions below). 2. For the average of the replicate determinations the absolute difference between the back calculated and nominal (intended) concentration must be <15% for the LOQ standard and <10% for all other stan dards. 3. The coefficient of variation between duplicate measurements of the same standard must be <10%. 4. For the entire batch to qualify, a minimum of 75% of the calibrator s or at least six points must be included and within the aforementioned limits. Additi onal measurements falling outside of these limits may be excluded from calculation provided this does not adversely affect the model.

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44 If one of the calibrator s does not meet any of the criteria outline in points 1 3, the calibrator should be rejected and the calibration curve without the failed calibration standard should be re evaluated, and regression analysis performed. Removal of additional calibrator s can be performed under the condition that the calibration curve adheres to point 4. If the criteria i n point 4 are not met, the analytical run should be rejected. The following conditions outline minimum acceptance criteria for positive control samples: 1. For the average of the replicate determinations the absolute difference between the back calculated and nominal (intended) concentration must be <10% for all positive control standards. 2. The coefficient of variance between duplicate measurements of the same standard must be <10%. 3. The concentrations of the positive control samples must be spaced across th e calibration curve and span the range of the unknown samples as per FDA Guidance (Guidance for Industry, Bioanalytical Method Validation, 2001). 4. A minimum of 3 out of 5 (60 % ) of positive control samples must meet the requirements in points 1 3 above. Fail ure to meet the criteria should result in the rejection of the analytical run. The following criteria describe conditio ns under which calibrator s or positive controls may generate no valid data: 1. Improper sample collection or preparation procedures. 2. A power failure occurs during injection, analysis, or data acquisition for the calibrators or positive controls in question. 3. A mechanical injection failure prevents t he necessary volume of sample from be ing introduced into the system. 4. A lack or loss of sample leads to an incomplete injection of a fraction of the required sample volume.

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45 Sample Acceptance Criteria During sample analysis all test samples were bracketed by positive control samples to ensure accuracy throughout the run. Test samples were a lways prepared in duplicate. Reanalysis was necessary if samples did not meet the acceptance criteria outlined below. Samples that did not generate valid data due to a mechanica l failure or power outage were re tested. Data that appeared anomalous in the c ontext of other measurements or variable compared to duplicate measurements may be accepted provided it meets all crit eria outlined in this document. Acceptance criteria guidelines: 1. The samples must be prepared with an established validated protocol. In th e case of method development, the sample preparation, including concentrations used, necessary laboratory notebook. 2. The sample(s) must be preceded with a successful and appropriate standard curve and a set of negative and positive controls. 3. Each sample duplicate must generate valid data (see below for conditions). 4. The coefficient of variation between duplicate measurements of the same sample must be <10%. Duplicate measur ements must occur consecutively with or without a blank in between. The following conditions describe when no v alid data could be generated: 1. The sample mixture was not fortified with the proper concentration of appropriate internal standard during the samp le preparation procedure. 2. The instrument experiences a mechanical failure or power outage during sample injection or data acquisition. 3. A lack or loss of sample leads to an incomplete injection of a fraction of the required sample volume. Linearity and Rang e The linearity defines the ability of the method to obtain test results proportional to the concentration of the analyte (Green, 1996) Linearity was assessed using a simple least squares

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46 regression with a 1/C p or 1/C u weighting factor to compensate for h eteroscedasticity (Almeida et al 2002) where C p and C u were the nominal concentration of GLY in plasma and urine calibrators respectively. Evidence of linearity was provided when calibrator quantification was within 15% and 10% of the nominal concentra tion at the lower limit of quantification ( LLOQ ) and all other concentrations, respectively. Linearity was also evaluated by plotting the response factor against the nominal concentration, visually inspecting residuals plots, and calculating the coefficie nt of determination ( R 2 ). Sensitivity Sensitivity was assessed by determining the limit of detection (LOD) and lower limit of quantification (LLOQ) for the analyte using the proposed method. The limit of detection was defined as the lowest concentration of analyte that could be detected with acceptable chromatography, the presence of quantifier and qualifier ions each with a signal to noise ratio of at least 3, and a retention time within 0.2 min of the average retention time. The lower limit of quantifica tion was the lowest concentration that met the LOD criteria but with a signal to noise ratio of 10 and acceptable accuracy and precision as defined below. Both LOD and LLOQ were determined with decreasing analyte concentrations in fortified plasma and urin e. The upper limit of quantification (ULOQ) was defined as the concentration of the highest calibration point. Accuracy and Precision Accuracy is defined as the degree of closeness of the measured concentration to the true concentration. Precision is the degree of scatter for repeated measured concentrations from one homogenous sample (Bansal et al 2007) Accuracy and precision were inv estigated at five positive control concentrations for plasma (0.125, 1.25, 5, 12.5, and 22.5 pg/mL) and urine (5, 125, 500, 750 and 1250 pg/mL). Intra and inter batch accuracy and precision were assessed in

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47 each matrix with five replicates per concentrati on over 1 (n=5) and 4 days (n=20), respectively. An estimate of precision, expressed as percentage relative standard deviation (%RSD), was obtained using a one way analysis of variance (ANOVA), using Microsoft Excel (Desilva et al 2003) Precision estima tes were required to be within 20% for the lowest positive control concentration and 15% for all other positive control concentrations Accuracy was determined by comparing the mean (n=20) measured concentration of the analyte to the target or nominal va lue. Accuracy was expressed as a percent of the target concentration with an acceptance criterion being 100% 20% of the nominal concentration. Carryover Carryover, a common issue encountered with LC MS/MS, is caused by residual analyte from a sample anal yzed earlier in the run sequence (Hughes et al 2007) Eliminating or minimizing carryover confirms th at a high concentration sample will not contribute to the quan tification of the next sample (Clouser Roche et al 2008) Carryover was evaluated by obse rving the ion intensities of the characteristic ions of GLY in a negative plasma and urine sample analyzed immediately after each of the four highest calibrators. Concentrations in the negative plasma and urine samples were calculated and carryover was det ermined to occur if the analyte concentrations exceeded the limit of detection. Matrix Effect, Extraction Efficiency, Process Efficiency Matrix effect, extraction efficiency (recovery), and process efficiency were evaluated using the three set method out lined by Matuszewski et al (Matuszewski et al 2003) The first set (A) solution. Set 2 (B) was negative control plasma or urine extracts that were fortifie d with analyte and internal standard solutions following solid phase extraction. The third set (C) was negative

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48 control plasma or urine fortified with analyte and internal standard solutions before solid phase extraction. Absolute matrix effect, extr action efficiency and process efficiency, all expressed as a percentage, were calculated using the following equations: Matrix Effect (%) = (B/A) x 100 (2 1) Extraction Efficiency (%) = (C/B) x 100 (2 2) Process Efficiency (%) = (C/A) x100 (2 3) where A, B and C are the mean absolute peak areas obtained with a neat preparation, with plasma or urine extracts fortified with analyte and internal standard solutions following extraction, and with plasma or urine fortified with analyte and internal standard soluti ons before solid phase extraction, respectively. The process efficiency incorporates matrix effect and provides a more accurate estimation of the analyte recovery than does extraction efficiency In addition, to evaluate the influence of different sources of matrices on analyte quantification, five different lots of negative control plasma or urine were compared (Matuszewski B. 2006) Dilution Integrity It was presumed that concentrations of GLY in plasma and in urine samples collected immediately after d rug administration would exceed the upper limit of the calibration range, for the respective methods, used for validation. Proving that dilution does not affect quantification is essential for obtaining accurate results for high concentration specimens Hen ce sample dilutions were likely required for both matrices and an appropriate validation procedure is recommended (Bansal et al 2007) Dilution integrity was assessed by supplementing negative control plasma with GLY at four concentrations (0.02, 1, 5 a nd 10 ng/mL) and diluting the samples over the range of dilution factors used for the study samples. Dilution factors evaluated were 1:2, 1:100,

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49 1:500 and 1:1000. Dilutional integrity was considered acceptable if replicate (n=5) values were within 20% of 10 pg/mL. Dilution integrity for urine samples was assessed by supplementing negative control urine with GLY at four concentrations (0.05, 2.5, 10.0 ng/mL) and diluting the samples over the range of dilution factors used for the study samples. Dilution fa ctors used and evaluated were 1:5, 1:250, and 1:1000. Dilutional integrity was considered acceptable if replicate (n=5) values were within 20% of 10 pg/mL. All dilutions, in both plasma and urine, were prepared using ultra pure (resistivity greater than or equal to 18 megaohms and organic content less than 10 ppb) de ionized water. Specificity Specificity is the ability of the method to accurately measure the analyte response in the presence of all potential sample components (Kushnir et al 2005) Spec ificity of the method was assessed by supplementing negative control horse plasma or urine with various therapeutic substances that are allowed in horse racing if present at a concentration less than the regulatory threshold The purpose of this study was to determine whether such compounds altered the response of the analyte or internal standard or both. For plasma three replicates each of five concentrations (0.125, 1.25, 5, 12.5 and 22.5 pg/mL) of positive controls samples were evaluated in the presence of 1 4 g/mL of phenylbutazone and furosemide, substances that are frequently present in post race horse specimens. For urine three replicates each of five GLY concentrations (25, 125, 500, 750 and 1250 pg/mL) of positive controls samples were evaluated in the presence of 500 ng/mL each of phenylbutazone and furosemide.

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50 Stability The evaluation of stability under the appropriate conditions is necessary in order to verify chemical integrity and assure reliable quantification results throughout the entire ana lytical process. The stability test conditions are determined by the method and length of storage of the biological samples. Stability of the analyte in both plasma and urine was evaluated over short term intervals at 0 C, 20 C and 80 C storage. Long term stability was evaluated over six, twelve and eighteen months at 80 C. Freeze thaw stability was evaluated following three freeze/thaw cycles. Extracted analyte stability was evaluated at 24, 48 and 72 h in 20C autosampler conditions. All GLY stab ility samples were assessed with three replicates at each of three concentrations for plasma (1, 5 and 25 pg/mL) and urine (5, 100, 2500 pg/mL). Ruggedness Method ruggedness was investigated to determine whether small variations in sample preparation affected analyte quanti fication Positive control samples at five concentrations (0.125, 1.25, 5, 12.5 and 22.5 pg/mL) were evaluated under various test conditions and compared to positive control samples prepared under the standard conditions Stock and Working Standard Solution Stability As noted above all stock and working standard solutions were prepared in methanol. These solutions are independent from the dry certified drug standard. Therefore prepared stock and working standard solutions cannot be a ssigned a similar expiration date as the dry standard. One stock solution and four working standard solutions that are routinely prepared from the stock solution were evaluated for stability over several intervals for up to 2 yr under 0C conditions. All s olutions were compared to one fresh stock solution prepared directly from the dry reference

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51 material and fresh working standards that had been prepared from the fresh stock solution (Nowatzke & Woolf 2007) Analyte C onfirmation In order to demonstrate tha t the GLY in a specimen obtained from a treated horse is chemically identical to the certified reference standard used for calibration the io n intensity ratios were compared The GLY product ions used for analyte confirmation were at m/z 58, 88, and 116. An ion intensity ratio was calculated according to peak height and relative abundance of the total ion chromatogram (TIC) as previously described (de Zeeuw, 2004) As such, the ion intensity ratio for the reference standard (calibr ators and positive contro ls) were compared to the ion intensity ratio for unknown substance in a plasma sample using the following equation: Ion intensity ratio similarity (%) = (R unknown /R standard ) x 100 (2 4) where R unknown represents the ion intensity ratio for the unknown su bstance present in a plasma specimen obtained from a horse treated with GLY and R standard represents the ratio for the GLY certified reference standard used to prepare the calibrators and positive control samples. Statistical Analysis All p values were de test and were computed using Microsoft Excel 2010. A p value of less than 0.05 was considered statistically significant. (Graph Pad Software, San Diego, CA, USA). Results GLY is a quaternary ammonium compound and as such contains a permanent positive charge. Thus, weak cationic exchange is the preferred method of extraction because the quaternary ammonium moiety makes the compound difficult to isolate by other means. The

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52 cation exchange sorbent (CBA carboxyl propyl phase) has a pKa of 4.8 ( Figure 2 1 ). Therefore the sorbent is negatively charged at pH 6.8 and greater and neutralized at pH 2.8 and lower. GLY is adsorbed onto the stationary phase due to the attraction between the negative charge on the sorbent and the positive charge of GLY ( Figure 2 2 (A)). Interfering matrix components can be eluted with methanol washes by interrupting hydrophobic interactions without concomitant loss of cationic analytes. Final elution of the drug can be achieved by disrupting the ionic interactions by suppressing ionization of the sorbent while ensuring th at hydrophobic interactions between the phase and the target analyte do not occur ( Figure 2 2 (B)). Method Validation Linearity Plasma: Method linearity for GLY in plasma was demonstrated with five calibration curves each spanning the range of 0.025 2 5 pg/mL. In all instances (n=5) a coefficient of determination (R2) of >0.998 was obtained ( Table 2 4) The back calculated concentrations of GLY in calibrators were within 85 1 15% and 90 1 10% of the target concentration for the LLOQ and all other concentr ations, respectively ( Table 2 5 ). Figure 2 4 illustrates a standard curve taken from the linearity experiments. Linear regression analysis of the response factors (based on the areas of the quantifier ion) vs. the nominal GLY calibrator concentrations demonstrated a slight decrease in response with increasing GLY concentrations and a slope of 12.86 ( Figure 2 5 ). Linear regression analysis of the response factor (based on the ion area ratios) vs. the nominal GLY calibrator concentrations demonstrated a response that was not statistically different from zero across the range of concentrations ( Figure 2 6 ). Urine: Method linearity was demonstrated with five calibration curves each spanning the range of 5 2500 pg/mL. In all instances (n=5) a coefficient of determination (R2) of >0.999 was

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53 obtained ( Table 2 6). The back calculated concentrations of G LY in calibrators were within 85 1 15% and 90 1 10% of the target concentration for the LLOQ and all other concentrations, respectively ( Table 2 7 ). Figure 2 7 illustrates a standard curve taken from the linearity experiments. Linear regression analysis of the response factors (based on the areas of th e quantifier ion) vs. the nominal GLY calibrator concentrations demonstrated a decreasing response with increasing GLY concentrations and a slope of 11.02 ( Figure 2 8 ). However, linear regression analysis of the response factor (based on the ion area ratios) vs. the nominal GLY calibrator concentrations demonstrated a response that was not statistically different from zero across the range of concentrat ions ( Figure 2 9 ). Sensitivity Plasma: The corresponding LOD, LOQ and ULOQ were 0.025 (CAL 1), 0.125 (CAL 2) and 25 pg/mL (CAL 10) of plasma, respectively. Chromatograms for the LLOQ and ULOQ are shown in Figure 2 10 and Figure 2 11 respectively. Urine: The corresponding LOD, LOQ and ULOQ were 1 (CAL 1), 5 (CAL 2) and 2500 pg/mL (CAL 10 ) of urine, respectively. Chromatograms for the LLOQ and the ULOQ are shown in Figure 2 12 and Figure 2 13 respectively. Accuracy and Precision Plasma: Precision and accuracy of the method were evaluated at five concentrations over the linear dynamic range (0.125, 1.25, 5, 12.5, 22.5 pg/mL) and res ults are provided i n Table 2 8 The intra batch (n=5) and inter batch (n=20) precisions, expressed as % RSD were < 10. Accuracy was calculated as the percentage difference from the mean measured values to the target value and was determined to have a range of 94 104% ( Table 2 9 ).

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54 Urine: Precision and accuracy of the method were evaluated at five concentrations over the linear dynamic range (25, 125, 500, 750, 1250 pg/mL) and results are provided in Table 2 10 The intra batch (n=5) and inter batch (n=20) precisions, expressed as % RSD were < 10. Accuracy was calculated as the percentage difference from the mean measured values to the target value and was determine d to have a range of 94 104% ( Table 2 11 ). Carryover Plasma: Carryover and possible contamination of GLY throughout the entire LC MS/MS system occurre d and was potentially detrimental to the determination of low concentrations when comparatively high concentrations were analyzed during method validation studies. GLY sequestration within the system had been determined to occur largely in the syringe, inj ection valve, and wash stations of the autosampler. We therefore incorporated the extensive syringe and injection valve washing steps outlined above. Under these conditions the extent of GLY carryover was <20% of the LOD. Urine: GLY carryover was observed in one blank injection each following the 1000 and 2500 pg/mL calibrators. However, this carryover was <1% of the total area response and was eliminated with the addition of a second consecutive mobile phase only injection. Hence, s tudy sample duplicates were separated by a minimum of two blank mobile phase injections. Matrix Effect, Extraction Efficiency, Process Efficiency Plasma: The matrix effect was evaluated using five different lots of matrix at five concentrations (0.125, 1.2 5, 5, 12.5, and 22.5 pg/mL) of GLY for five replicates each (n=5) using the three set experimental design described by Matuszewski et al Absolute matrix effect was observed in all five lots of plasma with a range of 85 99%. Extraction efficiency ranged fr om 79 96% for all concentrations. Overall process efficiency, taking into account the matrix

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55 effect, ranged from 67 95%). Relative matrix effect between five lots of plasma was expr essed as coefficient of variation of five slopes generated from five pr epared standard curves within a set. These values were less than 6% indicating minimal matrix interferences and increased reliability across different sources of plasma (Table 2 13). Urine: Extraction efficiency, taking into account the matrix effect, was determined at 5, 20, 50, 250, and 1000 pg/mL (n=5) for each concentration. It ranged from 91 108% for all concentrations, except for the low concentration (5.0 pg/mL), which was 120%. Overall, process efficiency, calculated from the ratio of the pre extr action over the neat preparations, ranged from 82 105%. Absolute matrix effect was observed in all five positive control concentrations with a range of 82 90% ( Table 2 14) Relative matrix effect between five lots of urine was expr essed as coefficient of v ariation of five slopes generated from five prepared standard curves within a set. These values were less than 4% indicating minimal matrix interferences and increased reliability across different sources of urine ( Table 2 15 ). Dilution Integrity Plasma: Dilutional Integrity was evaluated at four dilution factors (1:2, 1:100, 1:500, and 1:1000), with five determinations at each factor, encompassing the range of dilution s that were required for sample analysis. The average back calculated concentration did not differ from the target concentration more than 5%. Comparing the average of five replicates for each dilution factor with the nominal value produced p values > 0.05 ( Table 2 16 ). Urine: Dilutional Integrity was evaluated at three dilution factors 5 (low), 250 (medium) and 1000 (high), at five determinations for each factor, to encompass the range of dilutions that were required for sample analysis The average back calculated concentration did not differ from

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56 the target concentration more than 10%. Comparing the average of five replicates for each dilution factor with the nominal value produced p values > 0.05 ( Table 2 17 ). Specificity Plasma: No interferences with the determination of the target analyte GLY or the IS were detected in the analysis of plasma samples fortified with phenylbutazone or furosemide. High selectivity was assessed by the retention time of both GLY and its internal standard, and the accuracy to the target value for all three concentrations (96 101%) of the control samples. Urine: No interferences with the determination of the GLY or the IS wer e detected in the analysis of positive control urine samples fortified with phenylbutazone or furosemide. This high both GLY and the IS as well as sufficient accuracy (97 103 % ) compared to positive control samples that did not contain phenylbutazone or furosemide. The identity of GLY in plasma and urine was confirmed by comparing the product ion intensity ratio. The product ions used for compound identification were m/z 116, 88, and 58. The ion intensity ratio was obtained using the peak area and calculated relative to the total ion chromatograph (see Analyte Confirmation). Stability Plasma: The stability of GLY from extracted quality control samples over the range of the calibration curve was evaluated under 20 C autosampler conditions for up to 72 hours. The mean GLY concentration after storage for 48 h on the autosampler tray differed les s than a 10% compared to freshly prepared samples whereas those determined after storage for 72 hours were greater than 10%. Additionally, the stability of GLY through three freeze thaw cycles at 80 C was demonstrated as no appreciable degradation was fo und compared to freshly prepared

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5 7 positive control samples. Short term stability of GLY at three concentrations in plasma after storage at 0 C, 20 C and 80 C for 14, 60, and 60 days, was evaluated. Long term storage stability was evaluated at 80 C fo r 170 days at these concentrations. Other validation study results for stability are reported in Table 2 18 Urine: The stability of GLY from extracted quality control samples over the range of the calibration curve was evaluated under 20 C autosampler conditions for up to 72 hours. The mean GLY concentration after storage for 72 h on the autosampler tray differed less than 5 % compared to freshly prepared samples. Addi tionally, the stability of GLY through three freeze thaw cycles at 80 C was demonstrated as no appreciable degradation was found compared to freshly prepared positive control samples. Short term stability of GLY was assessed at three concentrations in ur ine after storage at 0 C, and 20 C for 30 and 60 days, respectively. Long term storage stability was evaluated at 80 C for 170 days at these concentrations. Other validation study results for stability are reported in Table 2 19 Ruggedness Plasma: Changes in the composition of the solution used to dissolve the extraction residue and the volume of the rinse phase of the solid phase extraction procedure had minimal effects on GLY response. We investigated the solid phase extraction elution step for ruggedness by removing the 1% formic acid from the elution solvent (acetonitrile). T h e results demonstrated no detectable response for GLY at the concentratio ns examined. However, when the elution volume (1 mL) was reduced to 0.5 mL, mean accuracy and precision ranged from 85.5 263.2% and 5.4 171.2%. All concentrations were out of specification for accuracy, precision, or both. Results indicate that the volume of the elution solvent and the presence of formic acid in the elution

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58 solvent are critical variables in the solid phase extraction process. For entire results refer to Table 2 20 Analyte C onfirmation Figure 2 15 illustrates a graphical comparison of the product ion intensity ratios between the GLY standard (A) the unknown test plasma (B) and urine (C) samples. The ion intensity ratio similarity for all three GLY product ions between the standard and plasma samples are presented in Table 2 21 and range from 82 106 % The ion intensity ratio similarity for all three GLY product ions between the standard and urine samples are presented i n Table 2 22 and range from 94 101 % Discussion GLY has been extracted from biological matrices using solid phase (SPE) (Matassa et al 1992) and liquid liquid extractions (LLE) (Storme et al 2008; Tang et al 2001) Liquid liquid extractions are difficult to automate and often require large volumes of hazardous solven t The liquid liquid extraction procedure is labor intensive, time consuming and often less reproducible than SPE procedures Additionally, the cationic properties of GLY are not ideal for partitioning into the liquid phase Tang et al present ed a liquid liquid extraction (LLE) for the screening and confirmation of eight quaternary ammonium compounds in horse urine, including GLY which was characterized b y a variable but modest recovery (74 % ) Solid phase extraction produces cleaner extracts greater recoveries and is available for automation (Zief & Kokodkar 1994) Such advantages and the wide availability of sorbent chemistries have made solid phase ext raction ideal for this analysis (Wynne 2000) The goal of the method development process was to make improvements where necessary to exis ting methodologies and to utilize newer technologies to accommodate the specific aims

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59 for this project Linea rity and range of the method have been improved and i ncreased by the addition of an internal standard The m ethod s use a stable isotope labeled analogue of the analyte of interest (GLY) to account for the presence of competing substances in the extracts and to min imize their influence. Our experience with GLY before the use of this internal standard was that the methods were not adequate in terms of accuracy and precision. A solid phase extraction for the isolation and purification of GLY has increased recovery and reduced unwanted matrix interferences. The sample extraction technique has also been simplified with the use of a commonly available sorbent type and minimal procedural steps. Method analysis time has been reduced with the incorporation of a short but ef ficient UPLC analytical column while maintaining excellent peak shape and resolution. Sensitivity and selectivity for the analyte have been improved by the choice of instrumentation, a TSQ mass spectrometer. In a previous report (Storme et al 2008) a c apillary electrophoresis tandem mass spectrometry (CE MS/MS) was compared to LC MS/MS for the detection of GLY in human plasma The authors reported numerous benefits using CE MS/MS over the conventional LC MS/MS methods including, but not limited to, impr oved sensitivity and separation with a limit of detection of 1 ng/mL. For comparison, the LLOQ for the horse plasma method described above is 0.125 pg/mL. The advantages of CE MS are well documented (Suntornsuk, 2007) but its use has been stifled because of a history of poor interfacing issues with an MS detector (Hommerson et al 2011) While these problems have been largely eliminated many laboratories are not equip ped and do not have trained operators for CE instrumentation. Additionally, CE MS method validations are uncommon, thus forensic laboratories are less likely to incorporate them into their standard oper ating procedures. The current methods also improve on previously published LC MS/MS methods for the quantification of GLY in urine and plasma by including a

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60 thorough method validation study and clearly describing identification criteria and procedures to quantify analytes of interest. Additionally, carryover, dilution integrity, and specificity with respect to potentially interfering exogenous analytes were included in the current validation but not in previously published reports (Table 2 23).

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61 Table 2 1 Preparation of working standard solutions for plasma analysis. Calibrators Concentration Working Standard Concentration Volume of working sta ndard (pg/mL) (ng/ L) (L) CAL 1 0.025 0.00000125 20 CAL 2 0.05 40 CAL 3 0.25 0.0000125 20 CAL 4 0.5 40 CAL 5 1.0 80 CAL 6 2.5 0.000125 20 CAL 7 5 .0 40 CAL 8 10 .0 80 CAL 9 17.5 140 CAL 10 25 .0 0.00125 20 Positive Controls PC A 0.125 0.000005 25 PC B 1.25 0.00005 PC C 5 .0 100 PC D 12.5 0.0005 25 PC E 22.5 45

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62 Table 2 2 Preparation of worki ng standard solutions for urine analysis Calibrators Concentration Working Standard Concentration Volume of working standard (pg/mL) (ng/ L) (L) CAL 1 5 0.00 0125 20 CAL 2 10 40 CAL 3 20 8 0 CAL 4 3 5 1 40 CAL 5 50 0. 0 0 125 2 0 CAL 6 100 4 0 CAL 7 250 10 0 CAL 8 500 0.0 125 2 0 CAL 9 1000 40 CAL 10 2500 10 0 Positive Controls PC A 0.125 0.000 5 5 PC B 1.25 0.00 5 12.5 PC C 5 .0 50 PC D 12.5 75 PC E 22.5 0.05 12.5 Figure 2 1 A weak cation exchange sorbent that consists of a carboxylic acid group bonded to

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63 Figure 2 2 Solid phase extraction of GLY. A) At ioniz sorbent is neutralized, and the elution is facilitated by the addition of a high ionic strength solvent. The analyte elutes from th e sorbent because there are no forces to retain it Table 2 3 Gradient table for GLY LC method. A ACN with 0.1% formic acid; B DI Water with 0.1% formic acid. Time (min) A (%) B (%) Flow Rate (L/min) 0.00 80.0 20.0 500 0.50 80.0 20.0 500 2.75 5.0 95.0 500 3.00 5.0 95.0 500 3.01 80.0 20.0 500 3.50 80.0 20.0 500

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64 Figure 2 3 Ion spectrum for GLY (m/z 318.1) Table 2 4 Characteristics of plasma GLY calibration curves (n=5) used for linearity assessment. Run Y intercept Slope R 2 Set 1 0.00115 0.00960 0.9997 Set 2 0.00220 0.00904 0.9994 Set 3 0.00003 0.00702 0.9984 Set 4 0.00182 0.00631 0.9997 Set 5 0.00005 0.00934 0.9994

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65 Table 2 5 Plasma GLY calibrator concentration taken from the accuracy and precision studies. Nominal Concentration (pg/mL) A&P Run 0.025 0.05 0.25 0.5 1 .0 2.5 5 .0 10 .0 17.5 25 .0 Set 1 0.025 0.049 0.244 0.506 1.02 2.54 5.01 9.93 17.6 25.0 Set 2 0.024 0.054 0.256 0.484 0.964 2.47 5.04 10.0 17.5 25.0 Set 3 0.026 0.054 0.237 0.492 0.954 2.40 5.01 10.0 17.7 25.0 Set 4 0.025 0.048 0.273 0.518 0.983 2.37 4.91 10.0 17.5 25.2 Set 5 0.028 0.053 0.237 0.453 0.907 2.60 5.00 10.0 17.6 24.9 Mean 0.026 0.052 0.249 0.500 0.965 2.44 4.99 10.0 17.6 25.0 SD 0.00 0.00 0.02 0.02 0.04 0.07 0.05 0.05 0.07 0.10 %RSD 7.10 6.17 6.07 3.03 4.28 3.00 0.94 0.48 0.38 0.41 %RE 2.57 3.40 0.21 0.04 3.45 2.27 0.15 0.04 0.40 0.01 n 5 .0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

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66 Figure 2 4 GLY calibration curve weighted 1/x ( R 2 > 0.999) corresponding to Set 1. PAR Peak Area Ratio. Figure 2 5 Plot of the response factor vs. the nominal calibrator concentrations. Slope = 12.86, y intercept = 152766.

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67 Figure 2 6 Plot of the response factor of the peak area ratio (PAR) vs. the nominal calibrator concentrations. Slope = 1.36 x 10 4 y intercept = 0.01680. Table 2 6 Characteristics of urine GLY calibration curves (n=5) used for linearity assessment. Run y intercept Slope R 2 Set 1 0.01002 0.00429 0.99990 Set 2 0.00393 0.00407 0.99995 Set 3 0.00265 0.00394 1.0000 Set 4 0.00354 0.00406 0.99995 Set 5 0.00144 0.00514 0.99970

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68 Table 2 7 Urine GLY calibrator concentration taken from the accuracy and precision studies. Nominal Concentration (pg/mL) A&P Run 5 10 20 35 50 100 250 500 1000 2500 Set 1 5.43 10.5 19.4 33.2 48.9 97.7 246.4 499.9 1004.9 2503.6 Set 2 5.42 10.0 19.4 34.6 50.7 98.5 237.9 500.0 998.8 2514.6 Set 3 5.19 10.1 19.5 33.3 50.1 101.2 249.3 507.0 1003.1 2491.6 Set 4 5.24 10.3 19.7 33.8 49.5 98.2 248.3 505.4 1004.5 2495.3 Set 5 5.04 9.56 19.2 33.3 49.4 98.2 254.2 499.2 1003.5 2498.5 Mean 5.26 10.1 19.4 33.6 49.7 98.8 247.2 502.3 1002.9 2500.7 SD 0.17 0.34 0.17 0.59 0.68 1.39 5.94 3.63 2.43 8.92 %RSD 3.14 3.38 0.85 1.76 1.37 1.41 2.40 0.72 0.24 0.36 %RE 5.27 0.79 2.76 3.88 0.59 1.22 1.12 0.46 0.29 0.03 n 5 5 5 5 5 5 5 5 5 5

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69 Figure 2 7 GLY calibration curve weighted 1/x (R2 > 0.999) corresponding to Set 1. PAR Peak Area Ratio. Figure 2 8 Plot of the response factor vs. the nominal calibrator concentrations. Slope = 11.02, y intercept = 73825.

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70 Figure 2 9 Plot of the response factor of the peak area ratio (PAR) vs. the nominal calibrator concentrations. Slope = 3.51 x 10 8, y intercept = 0.0112.

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71 Figure 2 10 SRM chromatograms for GLY in horse plasma at the LLOQ (0.125 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram.

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72 Figure 2 11 SRM chromatograms for GLY in horse plasma at ULOQ (25 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram

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73 Figure 2 12 SRM chromatograms for GLY in horse urine at LLOQ (5 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram.

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74 Figure 2 13 SRM chromatograms for GLY in horse urine at ULOQ (2500 pg/mL) and the deuterated internal standard. TIC Total Ion Chromatogram.

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75 Table 2 8 Accuracy and Precision (plasma) Intra batch Statistics Sample Batch n Mean SD % CV %RE Ancillary Statistics PC1 1 5 0.131 0.012 9.88 0.080 MS w = 0.000 0.125 2 5 0.119 0.014 10.9 0.975 MS b = 0.000 3 5 0.126 0.031 25.0 0.052 MS t = 0.000 4 5 0.127 0.012 9.40 1.037 s t = 0.018 s b = 0.000 Intra batch statistics (Pooled) 5 0.126 0.018 14.4 0.50 p = 4 Inter batch statistics (ANOVA): 20 0.126 0.018 14.4 0.50 PC2 1 5 1.27 0.069 5.55 1.55 MS w = 0.006 1.25 2 5 1.26 0.045 3.57 0.73 MS b = 0.014 3 5 1.21 0.126 10.1 3.09 MS t = 0.007 4 5 1.34 0.025 1.97 7.33 s t = 0.085 s b = 0.042 Intra batch statistics (Pooled): 5 1.27 0.076 6.10 1.63 p = 4 Inter batch statistics (ANOVA): 20 1.27 0.087 6.95 1.63 PC3 1 5 5.15 0.074 1.48 2.98 MS w = 0.045 5 2 5 4.98 0.187 3.74 0.47 MS b = 0.211 3 5 5.04 0.312 6.23 0.76 MS t = 0.071 4 5 5.44 0.203 4.06 8.80 s t = 0.266 s b = 0.182 Intra batch statistics (Pooled): 5 5.20 0.211 4.23 3.02 p = 4 Inter batch statistics (ANOVA): 20 5.20 0.279 5.58 3.02

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76 Table 2 8 : Continued Intra batch Statistics Sample Batch n Mean SD % CV %RE Ancillary Statistics PC4 1 5 12.9 0.345 2.76 2.91 MS w = 0.222 12.5 2 5 12.8 0.419 3.35 2.07 MS b = 0.747 3 5 13.1 0.538 4.30 4.98 MS t = 0.305 4 5 13.6 0.554 4.43 9.00 s t = 0.553 s b = 0.324 Intra batch statistics (Pooled): 5 13.1 0.472 3.77 4.74 p = 4 Inter batch statistics (ANOVA): 20 13.1 0.572 4.58 4.74 PC5 1 5 22.6 0.526 2.34 0.241 MS w = 0.542 22.5 2 5 22.7 0.694 3.09 0.872 MS b = 0.781 3 5 22.7 0.712 3.16 0.702 MS t = 0.580 4 5 23.4 0.951 4.23 4.08 s t = 0.762 s b = 0.219 Intra batch statistics (Pooled): 5 22.8 0.736 3.27 1.47 p = 4 Inter batch statistics (ANOVA): 20 22.8 0.768 3.41 1.47 Sample concentrations are in pg/mL. MS w ANOVA mean square for intra batch samples; MS b ANOVA mean square for inter batch samples; MS t ANOVA mean square for all samples; s t ANOVA variance component for all samples; s b ANOVA variance component for inter batch samples

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77 Table 2 9 Summary of Accuracy and Precision (plasma) Nominal Concentration (pg/mL) PC1 PC2 PC3 PC4 PC5 Characteristic Statistic 0.125 1.25 5 12.5 22.5 # Results N 20 20 20 20 20 Accuracy Mean Bias (%RE) 0.533 1.62 3.05 4.75 1.56 *LCL 5.62 5.22 3.52 1.82 1.32 **UCL 6.62 8.48 9.56 9.66 4.27 Precision Intra batch (%CV) 14.4 6.15 4.20 3.84 3.39 Inter batch (%CV) 14.4 7.00 5.60 4.60 3.45 Accuracy + Precision |Mean| + Inter batch 14.9 8.58 8.60 9.32 4.89 90% Expectation Lower Limit (%RE) 24.6 11.2 7.91 3.92 4.74 Tolerance Interval Upper Limit (%RE) 25.6 14.4 13.9 13.4 7.61 *Lower Confidence Limit for the Mean Bias; **Upper Confidence Limit for the Mean Bias

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78 Table 2 10 Accuracy and Precision (urine ) Intra batch Statistics Sample Batch n Mean SD % CV %RE Ancillary Statistics PC1 1 5 5.04 0.039 0.780 0.840 MSw = 0.004 5 2 5 5.02 0.075 1.50 0.400 MSb = 0.001 3 5 5.03 0.074 1.48 0.560 MSt = 0.004 4 5 5.05 0.063 1.25 0.960 st= 0.060 sb = 0.000 Intra batch statistics (Pooled) 5 5.04 0. 060 1.20 0.690 p = 4 Inter batch statistics (ANOVA): 20 5.04 0.060 1.20 0.690 PC2 1 5 125.1 1.5 3 1.221 0.037 MSw = 2.17 1 25 2 5 126.4 1.2 8 1.022 1.09 MSb = 1.45 3 5 125.7 1.1 4 0.910 0.538 MSt = 2.05 4 5 125.7 1.8 5 1.477 0.556 st= 1.43 sb = 0.00 Intra batch statistics (Pooled): 5 125.7 1.43 1.146 0.556 p = 4 Inter batch statistics (ANOVA): 20 125.7 1.43 1.146 0.556 PC3 1 5 507.6 18. 6 3.728 1.52 MSw = 125. 5 5 00 2 5 503.5 5.19 1.038 0.7 1 MSb = 29.3 3 5 508.3 9.59 1.918 1.6 7 MSt = 110. 3 4 5 504.1 5.96 1.193 0.8 3 st= 10.5 sb = 0.182 Intra batch statistics (Pooled): 5 505.9 10.5 2.10 1.18 p = 4 Inter batch statistics (ANOVA): 20 505.9 10.5 2.10 1.18

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79 Table 2 10 : Continued Intra batch Statistics Sample Batch n Mean SD % CV %RE Ancillary Statistics PC4 1 5 757.7 11.42 1.523 1.028 MSw = 82.42 750 2 5 753.1 8.916 1.189 0.416 MSb = 80.27 3 5 747.9 7.785 1.038 0.275 MSt = 82.08 4 5 752.1 7.685 1.025 0.294 st= 9.060 sb = 0.000 Intra batch statistics (Pooled): 5 752.7 9.060 1.208 0.366 p = 4 Inter batch statistics (ANOVA): 20 752.7 9.060 1.208 0.366 PC5 1 5 1253.6 33.59 2.687 0.291 MSw = 406.5 1250 2 5 1257.5 15.06 1.204 0.600 MSb = 51.36 3 5 1250.5 10.44 0.835 0.041 MSt = 350.5 4 5 1251.0 12.75 1.020 0.079 st= 18.72 sb = 0.000 Intra batch statistics (Pooled): 5 1253.2 18.72 1.498 0.253 p = 4 Inter batch statistics (ANOVA): 20 1253.2 18.72 1.498 0.253 Sample concentrations are in pg/mL. MS w ANOVA mean square for intra batch samples; MS b ANOVA mean square for inter batch samples; MS t ANOVA mean square for all samples; s t ANOVA variance component for all samples; s b ANOVA variance component for inter batch samples

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80 Table 2 11 Summary o f Accuracy and Precision (urine ) Nominal Concentration (pg/mL) PC1 PC2 PC3 PC4 PC5 Characteristic Statistic 5 125 500 750 1250 # Results N 20 20 20 20 20 Accuracy Mean Bias (%RE) 0.690 0.556 1.18 0.366 0.253 *LCL 2.83 0.131 0.410 0.484 0.155 **UCL 1.10 1.24 1.95 1.2 2 0.661 Precision Intra batch (%CV) 1.20 1.15 2.10 1.2 1 1.49 Inter batch (%CV) 1.20 1.15 2.10 1.2 1 1.49 Accuracy + Precision |Mean| + Inter batch 1.89 1.70 3.28 1.57 1.75 90% Expectation Lower Limit (%RE) 1.40 1.5 0 2.5 4 1. 78 2.4 1 Tolerance Interval Upper Limit (%RE) 2.78 2.6 0 4.8 2 2.47 2. 89 *Lower Confidence Limit for the Mean Bias; **Upper Confidence Limit for the Mean Bias

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81 Figure 2 14 GLY present as carryover in a mobile phase blank (2) following CAL 9 (1000 pg/mL) (1) and a second consecutive mobile phase blank (3) exhibiting no further GLY carryover. Table 2 12 Matrix effect, Extraction Efficiency, and Process Efficiency data for GLY in horse plasma. Positive Control concentration (pg/mL) Absolute Matrix Effect (%) Extraction Efficiency (%) Process Efficiency (%) 0.125 85.3 78.5 66.9 1.25 97.6 90.5 88.4 5 98.7 95.7 94.5 12.5 99.0 91.0 90.1 22.5 97.3 95.2 92.6

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82 Table 2 13 Relative matrix effect in plasma. Neat Standards (set 1) Plasma extracts fortified after extraction (set 2) Plasma extracts fortified before extraction (set 3) Slope a 0.0100 0.0098 0.0097 SD 0.00017 0.00055 0.00045 %CV 1.73 5.57 4.64 For each set, five different standard curves using five different plasma matrix lots were prepared. Each standard curve was constructed using five concentrations (positive controls). a Mean values of five slopes ( n =5), each obtained in a different plasma matrix lot. The slope of a standard curve was calculated using y=mx+b Table 2 14 Matrix effect, Extraction Efficiency, and Process Efficiency data for GLY in horse urine. Table 2 15 Relative matrix effect in urine. Neat Standards (set 1) Plasma extracts fortified after extraction (set 2) Plasma extracts fortified before extraction (set 3) Slope a 0.0052 0.0045 0.0044 SD 0.0001 0.0001 0.0001 %CV 2.09 1.79 3.06 For each set, five different standard curves using five different matrix lots of urine were prepared. Each standard curve was constructed using five concentrations (positive controls). a Mean values of five slopes ( n =5), each obtained in a different urine matrix lot. The slope of a standard curve was calculated using y=mx+b Positive Control concentration (pg/mL) Absolute Matrix Effect (%) Extraction Efficiency (%) Process Efficiency (%) 5 87.4 119.8 104.6 20 87.6 101.0 88.5 50 82.4 107.6 88.6 250 89.8 91.0 81.7 1000 90.4 90.8 82.0

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83 Table 2 16 Plasma sample dilution integrity. Dilution Factor* % CV (n=5) % difference from nom inal concentration p value 2 4.12 1.75 0.521 100 3.49 2.91 0.117 500 3.32 2.04 0.124 1000 2.41 4.24 0.181 *Diluted concentrations were multiplied by the appropriate dilution factor to obtain a mean (n=5) sample concentration. This value was compared to the nominal concentration of the positive control prior to dilution. Table 2 17 Urine sample dilution integrity. Dilution Factor* % CV (n=5) % difference from nominal concentration p value 5 4.35 8.31 0.09 250 2.89 2.75 0.445 1000 2.67 3.03 0.295 *Diluted concentrations were multiplied by the appropriate dilution factor to obtain a mean (n=5) sample concentration. This value was compared to the nominal concentration of the positive control prior to dilution.

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84 Table 2 18 Storage stability of GLY in plasma. Storage Conditions Positive Control Concentration (pg/mL) 1 5 25 Fresh Samples Mean Conc. (pg/mL) 1.00 5.05 24.47 Difference (%) 0.00 0.00 0.00 CV (%) 3.76 1.21 1.76 0C (14 days) Mean Conc. (pg/mL) 0.97 4.91 24.8 Difference (%) 2.67 2.86 1.36 CV (%) 4.89 1.84 3.92 p value 0.426 0.1 44 0.720 20C (60 days) Mean Conc. (pg/mL) 0.98 4.93 25.5 Difference (%) 2.03 2.34 4.03 CV (%) 7.27 1.71 5.04 p value 0.431 0.281 0.361 80C (60 days) Mean Conc. (pg/mL) 1.04 4.87 24.7 Difference (%) 4.68 3.61 0.85 CV (%) 3.43 0.89 1.94 p value 0.369 0.09 4 0.692 80C (170 days) Mean Conc. (pg/mL) 1.08 4.88 23.7 Difference (%) 8.64 3.35 3.22 CV (%) 1.49 3.92 5.0 p value 0.108 0.364 0.472 Extracts (24 h) Mean Conc. (pg/mL) 0.98 4.89 23.3 Difference (%) 1.60 3.30 4.64 CV (%) 5.76 8.38 4.46 p value 0.632 0.592 0.143 Extracts (48 h) Mean Conc. (pg/mL) 0.93 4.65 23.1 Difference (%) 6.21 8.01 5.66 CV (%) 5.49 3.24 9.57 p value 0.025 § 0.077 0.314 Extracts (72 h) Mean Conc. (pg/mL) 0.90 4.04* 22.1 Difference (%) 10.0 20.1 § 9.75 CV (%) 5.74 15.8 11.6 p value 0.169 n/a** 0.257 3 Freeze/thaw cycles ( 80C) Mean Conc. (pg/mL) 0.98 4.98 24.08 Difference (%) 1.46 1.48 1.58 CV (%) 7.81 2.29 2.50 p value 0.747 0.135 0.552 The % difference compares the mean concentration of replicates (n=3) under the test condition to the mean concentration of replicates prepared fresh. The P t test. §Values are out of specification. *Value was deter mined with two replicates instead of three due to a failed injection. **p value could not be generated with unequal number of replicates.

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85 Table 2 19 Storage stability of GLY in urine. Storage Conditions Positive Control Concentration (pg/mL) 5 100 2500 Fresh Samples Mean Conc. (pg/mL) 4.98 97.8 2559.2 Difference (%) 0.00 0.00 0.00 CV (%) 3.09 4.17 0.28 0C (30 days) Mean Conc. (pg/mL) 5.46 95.2 2553.9 Difference (%) 9.66 2.66 0.21 CV (%) 5.10 1.35 2.57 p value 0.125 0.326 0.911 20C (60 days) Mean Conc. (pg/mL) 4.81 91.5 2333.0 Difference (%) 3.44 6.49 8.84 CV (%) 9.47 1.88 4.55 p value 0.489 0.171 0.070 80C (147 days) Mean Conc. (pg/mL) 4.78 93.5 2514.7 Difference (%) 4.04 4.47 1.74 CV (%) 2.66 2.20 2.82 p value 0.219 0.068 0.360 Extracts (24 h) Mean Conc. (pg/mL) 4.92 98.4 2525.4 Difference (%) 1.12 0.60 1.32 CV (%) 5.21 2.84 0.71 p value 0.824 0.869 0.143 Extracts (48 h) Mean Conc. (pg/mL) 4.88 98.5 2474.8 Difference (%) 2.06 0.68 3.30 CV (%) 1.31 2.99 0.36 p value 0.330 0.884 0.002 § Extracts (72 h) Mean Conc. (pg/mL) 4.82 96.3 2506.4 Difference (%) 3.11 1.56 2.06 CV (%) 2.83 3.43 1.10 p value 0.006 § 0.686 0.051 3 Freeze/thaw cycles ( 80C) Mean Conc. (pg/mL) 4.82 98.8 2501.0 Difference (%) 3.27 1.00 2.27 CV (%) 7.77 0.84 2.54 p value 0.646 0.716 0.222 The % difference compares the mean concentration of replicates (n=3) under the test condition to the mean concentration of replicates prepared fresh. The P t test. §Values are out of specification.

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86 Table 2 20 Method ruggedness evaluation. Positive control Concentration (pg/mL) 0.125 1.25 5 12.5 22.5 Sample preparation condition tested Mean accuracy (%) with CV (%) Rinse with 1 mL of water, MeOH, DCM instead of 2 mL water, MeOH, DCM 104.0 (12.90) 109.1 (2.33) 93.0 (2.86) 95.9 (2.57) 89.5 (1.66) SPE rinse with water and MeOH (no DCM) 101.3 (10.40) 106.0 (8.72) 94.6 (8.20) 98.4 (2.42) 93.1 (1.33) Dissolved in 3 0 :7 0 instead of 80:20 ACN:Water (0.1% formic acid) 107.2 (8.60) 98.2 (8.32) 92.4 (3.23) 93.3 (3.47) 92.2 (1.46) Dissolved in 10:90 instead of 80:20 ACN:Water (0.1% formic acid) 102.4 (3.92) 108.1 (5.59) 94.6 (0.59) 98.5 (2.26) 93.9 (1.81) Eluted with 0.5 mL instead of 1.0 mL 240.9 (120.4) 263.2 (171.2) 85.5 (69.1) 101.9 (5.40) 16.9 (87.1) Eluted with ACN instead of ACN w/ 1% FA < LOD < LOD < LOD < LOD < LOD Mean accuracy (%) is obtained by comparing the mean (n=3) measured concentration under test conditions to the mean measured concentration of positive control samples prepared under standard conditions. Precision of the replicates is expressed as coefficient of variation (% CV).

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87 Figure 2 15 Comparison of the ion intensity ratios between a GLY reference standard (25 pg/mL) (A) a plasma (B) and urine (C) sample after GLY administration.

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88 Table 2 21 Determination of the product ion ratio for confirmation of the presence of GLY in horse plasma. Product ions Intensity Ratio standard ( n =10) Intensity Ratio unknown ( n =10) Ion Intensity Ratio Similarity (%) 58 9.34 0.67 9.85 0.45 105.5 88 5.38 0.96 4.42 0.63 82.2 116 85.3 0.34 85.7 0.21 100.2 TIC was used as the denominator in calculating the ion intensity ratio Table 2 22 Determination of the product ion ratio for confirmation of the presence of GLY in horse urine. Product ions Intensity Ratio standard ( n =10) Intensity Ratio unknown ( n =10) Ion Intensity Ratio Similarity (%) 58 9.34 0.67 8.82 94.4 88 5.38 0.96 5.31 98.7 116 85.3 0.34 85.8 100.6

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89 Table 2 23 Comparison of evaluated method validation parameters between the current study and three previously published reports 1 Current Study 2 Storme et al ., 2008 3 Tang et al ., 2001 3 Matassa et al ., 1992 Sensitivity X X X X Linearity X Carryover X Accuracy X X X X Imprecision X X X X Recovery X X X X Matrix Effect X X Process Efficiency X X Matrix Interference X Dilution Integrity X System Suitability X Specificity X Stability X Ruggedness X X Measurement Uncertainty X 1 Method validation study performed in horse plasma and urine 2 Method validation study performed in human plasma 3 Method validation study performed in horse urine

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90 CHAPTER 3 PHARMACOKINETICS OF GLYCOPYRROLATE IN TH OROUGHBREDS Until the early 19 9 0s, the pharmacokinetics of glycopyrrolate ( GLY ) were poorly understood primari ly due the lack of sufficiently sensitive analytical meth ods (Kaltiala et al ., 1974; Murray et al 1984) when Kaila et al introduced a radioreceptor assay for this purpose (Kaila et al 1990) Since then GLY pharmacokinetics have been reported mainly in adult humans through intramuscular (Ali Melkki la et al ., 1990; Ali Me lkkila et al ., 1993) and intravenous (Ali Melkkila et al 1989) administration and in children (Rautakorpi, et al ., 1994; Rautakorpi et al 1998) T following rumored useage at performance racetracks have not been reported due to limitations in sensitivity of the methods that are commonly used. Yet positiv e post race findings for GLY have surfaced at a steady rate in recent years. Therefore this study investigated the disposition of GLY following intravenous administration of a 1 mg dose in the horse a dose thought to be used during the prohibite d administ ration period before racetime In order to provide the appropriate regulatory control for therapeutic substances that may also have the ability to affect performance, threshold limits must be determined in blood (plasma), urine or both (Tobin et al ., 1999; Kollias Baker, 2001; Report, 1991) Furthermore, it is important to determine the relationship between urine and plasma concentrations after a single intravenous and clinically relevant dose of GLY. Such investigations could contribute to the RMTC effort to establish a plasma threshold and to recommend a withdrawal time for this drug in race horses. The urine to plasma concentration ratio is useful forensically since it permits analysts to predict the concentration in one matrix (e.g., urine) from a result in the other matrix. This is

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91 useful in such cases in which the drug is regulated with a plasma threshold but for which screening methods are based on analysis of urine samples. For example, a finding in urine can be dismissed or pursued based on a simple calculation of dividing the estimated urine concentration by the concentration ratio and comparing the predicted plasma concentration to the plasma threshold. Additionally, a laboratory finding in an official post race sample of comparatively higher urine to plasma ratio may indicate that the drug was administered close to race time. Methods Animals Twenty, adult, athletically conditioned Thoroughbreds (6 mares and 14 geldings) ranging in age from 4 10 years and weighing from 485 602 kg were used in these studies. Eight horses (1 mares and 7 geldings) ranging in age from 4 10 years and weighing from 502 580 kg were used in the pharmacokinetic studies. All horses were dosed intravenously and six of these horses (1 mare and 5 geldings) ranging in age from 8 1 0 years and weighing from 518 580 kg were dosed orally following a sufficient (30 day) washout period. All horses were housed in grass paddocks at the UF Veterinary Medical Center, kept on a diet of commercially available grain mixture, and had open access to water and hay at all times. Horses were regularly exercised (3 days/week) before and throughout the duration of the study. Conditioning All horses were conditioned using a high speed Sto treadmill (Equine Dynamics, Lexington, KY, USA) at the UF Equine Performance Laboratory. For two months before the study, horses were conditioned using a standard training regimen and were subjected to an exhaustion test. The conditioning regimen was designed to prepare the horses to complete a mile in 2 min at a steady gallop without undue stress. Horses were evaluated for this goal through a

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92 condition test before the start of the dose administration stud y. The standardized training regimen continued throughout the course of the study and consisted 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 4.0 m/s. The treadmill belt was horizontally orientated one day per w eek (Monday) and at a 6 inclination two days per week (Wednesday and Friday). Dosing For the intravenous study, horses were administered 1 mg (1.66 2.06 g/kg) of GLY (glycopyrronium bromide, American Regent, Inc., Shirley, NY, USA) into the right jugular vein. Oral administration was carried out using 50 mL of 0.2 mg/mL GLY solution for a total of 10 mg orally. Demographics for the current study are summarized in ( Table 3 1 ) Specimen Collection Plasma: Whole blood samples were collected from the left jugular vein via needle venipuncture into partially evacuated tubes containing lithium heparin. Blood samples were stored on ice until the plasma was conc entrated by centrifugation (2500 3000 rpm or 776 1318 x g) at 4 C for 15 min. Harvesting of plasma took place within 1 hour of sample collection and 2 4 mL aliquots of plasma were immediately frozen at 20 C and stored within 24 h at 80 C until analyz ed. Collection times were before drug administration and at 4, 8, 24, 48, 72, 96, and 168 h after intravenous administration. For horses included in the pharmacokinetic studies, colle ction times included a sample collection before drug administration and 5 10, 15, 20, 30 and 45 min and 1, 2, 3, 4, 6, 8, 24, 48, 72, 96, and 168 h after intravenous administration. Collections times for oral administration were before drug administration and at 15, 30, 45, 60 and 90 min and 2, 3, 4 6, 8 and 24 h after dosing

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93 Urine: All horses were trained to urinate on command and the urine from each horse was collected via the free catch method into separate clean 1 L containers. Urine specimens were divided into single use aliquots in 15 mL sterile, disposable, polypropyle ne centrifuge tubes and stored at 20C immediately and at 80C within 48 h. Collection times were before drug administration and at 4, 8, 24, 48, 72, 96, and 168 h after intravenous administration. For oral administration collection times were before dru g administration and at 2, 4, 6, 8, and 24 h after dosing. The experimental protocol, including animal conditioning and drug administration and collection, was approved and facilities were inspected by the University of Florida Institutional Animal Care an d Use Committee (IACUC). Pharmacokinetic Analysis Nonlinear least squares regression analysis was performed on plasma GLY concentration versus time data and pharmacokinetic parameters for all horses were estimated with both noncompartmental (NCA) and compartmental analysis using Phoenix WinNonlin 6.1 (Pharsight, St. Louis, MO, USA). For compartmental analysis, the Gauss Newton (Levenberg and Hartley) method was used and goodness of fit and the appropriate weighting factor were selected based on t (Yamaoka, 1978) and Schwar z's Bayesian Criterion (Schwarz, 1978) as well as visual analysis of the graphical output (including residual plots). Secondary parameters calculated include area under the curve (AUC), terminal half life (t ), apparent volumes of distribution, total plasma clearance (Cl p ), and micro distribution rate con stants. For the NCA analysis, the area under the plasma concentration vs. time curve (AUC 0 24 ) from time 0 to 24 h w as calculated using the log linear trapezoidal method with linear interpolation. The pharmacokinetic parameters calculated

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94 included the observed maximum plasma concentration (C max ), area under the plasma concentration vs. time curve to the last determined plasma concentration (AUC t ), terminal half life (t 1/2 ), total plasma clearance (Cl p ), mean residence time (MRT), and steady state volume of distribution (V ss ). All calculations for pharmacokinetic parameters were based on methods described by Gibaldi and P errier (Gibaldi & Perrier 1982) All pharmacokinetic parameter estimates were calculated for each horse and values are reported as median and range (minimum maximum). Statistical Analysis Plots of urine and plasma concentrations were performed using Gra 5.0 for Windows (GraphPad Software, San Diego, CA, USA) and p lasma and urine concentrations of GLY are expressed as mean and standard deviation. Tolerance intervals for urine and plasma concentrations at different collection times were computed to contain at least 99 % of the population with 95 % confidence and were calculated using the software program JMP 8.0 (SAS Institute, Inc., Cary, NC, USA). Results Intravenous Administration Plasma: After intravenous administration of 1 mg, plasm a GLY was detectable for up to 168 h in fourteen of the twenty horses used for the study. The mean plasma s.d. concentration at 24 h after dosing was 1.36 0.41 pg/mL. The upper limit of the tolerance interval was calculated (n=20) for each collection i nterval. Notably, the upper limit of the 99/95% tolerance interval (n=20) at 24 h in plasma was 3.02 pg/mL. Plasma concentrations and descriptive sta tistics at collection times for all horses (n=20) are summarized in Table 3 2

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95 For sub jects (n=8) with a more extensive blood collection s pharmacokinetic analysis was performed. After intravenous administration of 1 mg of GLY, the observed plasma concentration versus time profile could be best described by a three compartment model ( Figure 3 1 ). The derived equation (Jacobs, 1988) based on macr o constants for this model is: C t = A exp + B exp + C exp (3 1) where C t is the plasma concentration at time (t), A, B and C are the zero time intercepts for and exp is the base of the natural logarithm (Gabrielsson & Weiner 2007) The weighting factor chosen with this model was 1/ (Y 2 ) where Y was the observed plasma concentration. A three compartment model was chosen over a two compartment model based on visual observations of the observed and predicted concentrations versus time for a two ( Figure 3 2 ) and three ( Figure 3 3 ) compartment analysis and other diagnostic criteria ( Table 3 5 ). Estimates for a number of pharmacokinetic parameters following noncompartmental and compartmental model analysis are reported in Table 3 3 and Table 3 4 respectively. Plasma GLY concentration vs. time plots for all eight horses are depicted in Figure 3 4 and Figure 3 5 The drug concentrations remained ( Figure 3 6 ) Urine: Urine GLY concentrations, determined using the method described above, were above the lower limit of quantification (5 pg/mL) in urine samples collected through 96 h in all horses a nd in all but three samples through 168 h after IV administration. All urine concentrations are reported in Table 3 6 Peak urine concentrations of GLY were observed at the 4 h collection time for all horses. Urine concentrations (n=20) of GLY through 168 h are graphed in Figure 3 7 The upper limit of the tolerance interval was calculated for each collection time. Notably, the upper limit of the 99/95% tolerance interval at 24 h in urine was 325 pg/mL.

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96 The urine to plasma concentration ratio of any substance is determined by the renal clearance of t hat substance and the rate of urine formation: (3 2) where CL R is the renal clearance, is the rate of urine formation, C U is the urine concentration, and C p is the corresponding plasma concentration. The urine to plasma GLY concentration ratios for samples collected daily after intravenous administration are shown in Figure 3 9 The urine to plasma concentration ratio at 4 and 8 h after administration had a median (range) of 3453.3 (505.8 15969.3) and 232.6 (505.8 15969.3), respectively. From 24 168 h after administ ration the urine to plasma concentration ratio ranged from 90 150 with a mean of 131. The mean (n=20) at 24 h after administration was 103. Oral Administration Plasma: GLY was detected in plasma samples collected after oral administration of 10 mg of GLY in aqueous solution. The measured mean peak concentrations were 4.7 2.6 pg/mL and occurred at 15 min after dosing. At 1 h after dosing the plasma GLY concentrations in all horses were below 0.5 pg/mL and those at all other collection times up to 24 h wer e determined to be less than the LLOQ. Therefore, bioavailability could not be calculated using the conventional methods as described in Gibaldi and Perrier (Gibaldi & Perrier 1982) Figure 3 10 displays plasma concentrations versus time after oral administration of 10 mg to each horse. Figure 3 11 d isplays the plasma concentrations versus time following oral and intravenous administration. Urine: GLY urine concentrations were above the limit of detection in samples collected up to 24 h after oral administration in all horses. Peak urinary concentrati ons of GLY were observed within 2 4 h after oral administration. Median (range) concentrations at 2 were 26.8 (17.5 67.4) pg/mL. Figure 3 12 displays urine concentrations versus time after oral administration of 10 mg

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97 to each horse. Median (range) concentrations at 4 h after oral administration were 23.8 (16.3 83.2) pg/mL, not very different from the median value (22.4 pg/mL) when the highest value of 8 3.2 pg/mL was excluded. Discussion The data indicate that GLY d isposition in the horse exhibited tri exponential disappearance from plasma after intravenous administration. This is characterized by an early rapid decline in plasma concentrations followed b y a slow terminal phase with concentrations above the LLOQ of the method for up to 168 h. All horses exhibited plasma concentrations above 1 ng/mL 5 min after drug administration followed by a precipitous decline through 20 min. Although the three compartm ent model estimates for C max are higher than the noncompartmental estimates due to the extrapolation back to time 0 in the compartmental model, it is believed that the inclusion of these values in the model is necessary to describe the disposition of GLY (Beaufort et al 1999) Moreover, data in humans suggest a similar pharmacokinetic profile (Penttila et al 2001) Schenin et al ., have reported a rapid initial distribution/elimination phase with a two compartment mode l in humans following a single intr avenous 5 g/kg dose (Scheinin et al 1999) However, an insufficient analytical method may have prevented the discovery of an additional decay phase. Relevant factors in the rapid decline in the plasma GLY concentration were the large volume of distribut ion and the swift transfer into the tissue compartment and not due to renal clearance. The long terminal half life is the result of the distribution into a deeper third compartment. Noncompartmental analysis a model independent approach, was also performe d and provided physiologically reasonable parameter estimates. However, the median (range) volume of distribution based on the terminal phase (V z ) was unrealistically large (14.4 (7.56

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98 27.9) L/kg), likely accounted for by the rapid elimination during the i nitial phase and low plasma GLY concentrations during the terminal phase (Toutain & Bousquet Molou 2004) Moreover, NCA analysis typically provides limited information regarding the plasma drug concentration vs. time profile due to the numerous assumption s made (Rosenbaum, 2011) Total plasma clearance is attributed to hydrolysis of GLY and renal clearance. While this study has revealed that some GLY is eliminated unchanged in the urine, we did not perform volumetric urine collections in this study and the refore cannot estimate renal clearance of GLY. The median (range) of the total GLY plasma clearance reported from the pharmacokinetic study was 22.4 (14.2 31.2) mL/min/kg and is approximately equal to previous estimates of hepatic blood flow in the horse ( Dyke et al 1998) suggesting that GLY is appreciably cleared by metabolic transformation The observed plasma clearance cannot be attributed exclusively to renal clearance because it exceeds the effective renal plasma flow and therefore the maximum value for renal clearance in the horse (Kohn and Strasser 1986; Woods, 2000) Since the renal clearance estimates from these horses are approximately equal to the glomerular filtration rate and are substantially lower than estimates of total plasma clearance, it is evident that GLY is substantially cleared by non renal mechanisms in the horse. The metabolism of GLY in the horse and other species has not been extensively investigated. So me investigators have reported that most of the human dose is excreted unchanged in the urine (Kaltiala et al 1974) indicating that renal clearance is responsible for much of the plasma clearance in contrast to our finding in the horse. In contrast more recent studies in humans, following a single intravenous dose, estimate plasma clearance values to be 16.8 3.83 (mean s.d.) and 18.1 (10 23.8) (median and range) mL/min/kg (Penttil a et al 2001; Rautakorpi, et al ., 1994) closely approximating human hepatic blood flow (Davies & Morris 1993) Further, these high total clearance estimates exceed the

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99 effective renal blood flow rate in man and rule out the possibility that GLY clearance can be mostly attributed to renal mechanisms. The upper limits of the 99/95 tolerance intervals for GLY in horse plasma were calculated and reported Several plasma measurements for samples collected at 72, 96, and 168 h were orde r to calculate the tolerance interval (Hornung & Reed 1990) GLY urine concentrations were not detectable or were below the LOD in pre administration urine samples from nineteen out of twenty horses. An exception was one horse in which the urine sample co ncentration was 12.2 pg/mL. The presence of GLY in this sample was confirmed through re analysis and a review of the full scan product spectra. This finding can likely be attributed to an error in the sample collection process or post c ollection processing It is suspected that the pre administration sample was inadvertently contaminated with a post administration sample from a different c ollection time. A review of the sample collection and aliquot handling procedures has been performed but has not identified a definitive explanation for this finding. LLOQ at 168 h. These values were included in calculations of the 168 h upper limit of the 99/95 tolerance inter val in order to r educe the bias associated with replacing these values with zero, replacing them with the LLOQ or a fraction of the LLOQ, or omitting the values from the calculations (Beal, 2001; Duval & Karlsson, 2002; Ahn et al 2008) The urine to pla sma concentration ratio for any substance is a dimensionless value that is equal to the renal clearance of the substance divided by the volumetric urine flow rate. It is therefore possible to estimate the renal clearance of GLY by multiplying the ratio obt ained from

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100 the GLY concentrations in paired urine and plasma samples by the measured or estimated volumetric flow rate of urine. The urine flow rate was not measured in the present study because volumetric urine collections were not made. However, normal u rine flow rates of 0.52, 0.92, 1.12, and 1.24 mL/h/kg have been reported in healthy horses with no restrictions to feed and water (Tasker, 1967; Watson et al ., 2002; Thurmon et al ., 1984; Rumbaugh et al 1982) Using these estimates of the urinary flow ra te in horses, we estimated renal clearance of GLY by multiplying the urine to plasma concentration ratios at various times by th ese urine flow rates and report them in Table 3 7 The renal clearance estimates from 24 168 h ranged from 0.84 3.43 mL/min/kg and are similar to estimates of the glomerular filtration rate in horses suggesting that GLY is cleared renally primarily by filtration in th e horse. Si nce GLY is not expected to be reabsorbed in the distal tubules due to its polarity and because GLY is not appreciably bound to plasma proteins, tubular secretion must not account for much of the renal clearance due to the similarity between reported values of glomerular filtrati on and the estima tes of renal clearance of GLY. The estimated value of renal clearance from these studies is affected by the timing of sample collections particularly in the early period after drug administration when concentrations were changing rapidly. For this reason, renal clearance studies often use the plasma concentration at the midpoint of the urine collection interval rather than one at either end for renal clearance calculations. In the study reported here, urine samples were collected at the sam e times as blood samples so it was anticipated that the ratios in the early period after GLY administration would be affected by the timing of sample collections and it was predicted that the urine to plasma ratios would be higher than those obtained later because plasma concentrations declined very rapidly for the first 8 h after administration. In fact, urine to plasma

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101 GLY ratios were higher at 4 and 8 hours after administration than they were at any later time. From 24 h after administration, the ratio w as relatively constant and was approximately 100 to 1 with a median (range) of 97.3 (83.1 123.5). Samples used to regulate drugs in horse racing are typically collected in the period from 30 120 min after the end of the race during which a brief period of increased urine flow rate has been observed (Schott et al 1991) The increase in urine flow rate is likely due to increased renal blood flow in which case the renal clearance and urine flow rate would be expected to increase proportionately. If this is t he case, the urine to plasma concentration ratio would be exp ected to be unchanged during this period. Analysis of GLY in plasma samples collected after a 10 mg oral administration resulted in concentrations that would have been undetectable with methods d escribed in the literature, due to its extremely low oral bioavailability as predicted from its permanent ionization. In the only other account, Rautakorpi et al report a median (range) bioavailability of 3.3% (1.3 13.3) following a 50 g/kg dose in child ren (Rautakorpi et al 1998) Therefore, a method with greater sensitivity is necessary in order to detect GLY administration by this route for more than a few hours after dosing.

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102 Table 3 1 Demographics of study subjects. Each number represents a horse. Horse Gender Age (yr) Weight (kg) IV Administration Oral Administration PK Analysis 1 G 9 549 X X X 2 G 9 570 X X X 3 G 9 546 X X X 4 G 9 545 X X X 5 G 7 580 X X X 6 G 9 518 X X X 7 G 7 494 X 8 G 9 571 X X 9 G 7 502 X X 10 G 9 534 X 11 G 6 602 X 12 G 6 538 X 13 G 6 536 X 14 G 4 495 X 15 M 5 554 X 16 M 6 485 X 17 M 7 602 X 18 M 6 565 X 19 M 6 518 X 20 M 4 515 X M Mare, G Gelding

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103 Table 3 2 Plasma GLY concentrations (pg/mL) following intravenous administration of 1 mg to each of 20 horses. Time (h) Horse 4 8 24 48 72 96 168 1 8.27 3.88 1.54 0.510 0.102 0.029 0.093 2 9.36 4.45 1.17 0.850 0.209 0.110 0.069 3 11.9 3.91 1.92 0.970 0.479 0.163 0.119 4 16.6 6.49 2.25 1.21 0.313 0.209 0.097 5 9.40 3.82 0.95 0.376 0.283 0.146 0.080 6 9.69 4.86 0.860 0.294 0.138 0.025
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104 Figure 3 1 Illustration of a three compartment model.

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105 Table 3 3 Pharmacokinetic parameter estimates of GLY, determined using noncompartmental analysis, following intravenous administration of 1mg to eight (n=8) healthy adult Thoroughbred horses. Parameter Horse Median Min Max 1 2 3 4 5 6 7 8 z (h 1 ) 0.097 0.066 0.089 0.102 0.084 0.082 0.067 0.054 0.083 0.066 0.102 t 1/2 z (h) 7.14 10.5 7.79 6.78 8.28 8.48 10.4 12.9 8.38 6.78 12.9 C max (ng/mL) 5.48 4.72 4.21 8.27 5.14 4.07 2.43 4.55 4.64 2.43 8.27 C last (ng/mL) x 10 3 1.11 1.54 1.17 0.860 1.25 0.953 2.25 1.92 1. 21 0.860 2.25 AUC 0 24 (h*ng/mL) 1.67 1.40 1.38 2.49 1.54 1.43 0.953 1.50 1.46 0.953 2.49 AUC 0 (h*ng/mL) 1.68 1.42 1.40 2.50 1.55 1.44 0.987 1.53 1.49 0.987 2.50 V z (L/kg) 12.5 19.4 14.1 7.56 13.1 14.6 27.9 22.2 14.4 7.56 27.9 Cl P (mL/min/kg) 20.3 21.3 21.0 12.9 18.3 19.9 31.0 19.9 20.1 12.9 31.0 AUMC 0 24 (h*h*ng/mL) 1.06 0.963 1.04 0.952 0.990 0.783 1.40 1.20 1.01 0.783 1.40 MRT 0 24 (h) 0.636 0.688 0.750 0.383 0.644 0.548 1.47 0.798 0.666 0.383 1.47 V ss (L/kg) 1.05 1.68 1.35 0.383 1.08 1.00 5.13 2.10 1.22 0.383 5.12 z elimination rate constant; t 1/2 z terminal half life; C max observed maximum plasma GLY concentration; C last observed plasma GLY concentration at 24 h; AUC 0 24 area under the plasma concentration vs. time curve from time 0 to 24 h; V z volume of distribution based on the terminal phase; Cl P observed total plasma clearance; AUMC 0 24 area under the first moment curve from time 0 to 24 h; MRT 0 24 mean residence time from time 0 to 24 h; V ss volume of distribution at steady state;

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106 Table 3 4 Pharmacokinetic parameter estimates of GLY, determined using a three compartmental model, following intravenous administration of 1 mg to ei ght (n=8) healthy adult Thoroughbred horses. Parameter Horse Median Min Max 1 2 3 4 5 6 7 8 A (ng/mL) 9.72 5.96 7.49 27.3 9.24 22.8 4.51 7.32 8.37 4.51 27.3 B (ng/mL) 0.436 0.076 0.371 2.23 0.281 0.954 0.935 0.331 0.404 0.076 2.23 C (ng/mL) 0.012 0.069 0.011 0.014 0.011 0.098 0.015 0.071 0.011 0.069 0.015 Alpha (h 1 ) 9.16 6.77 7.30 17.3 8.73 23.5 10.8 8.86 9.01 6.77 23.5 Beta (h 1 ) 1.73 0.809 1.53 3.86 1.57 2.95 2.45 1.09 1.65 0.809 3.86 Gamma (h 1 ) 0.101 0.063 0.095 0.119 0.092 0.102 0.080 0.056 0.094 0.056 0.119 C max (ng/mL) 10.2 6.04 7.88 29.5 9.54 23.8 5.46 7.66 8.71 5.46 29.5 V 1 (L/kg) 0.201 0.302 0.223 0.065 0.179 0.073 0.336 0.239 0.212 0.065 0.336 K 21 (h 1 ) 2.05 0.884 1.80 4.88 1.79 3.78 3.89 1.43 1.93 0.884 4.88 K 31 (h 1 ) 0.110 0.070 0.104 0.125 0.101 0.110 0.098 0.062 0.102 0.062 0.125 K 10 (h 1 ) 7.08 5.58 5.69 13.0 7.03 17.1 5.58 6.10 6.56 5.58 17.1 K 12 (h 1 ) 1.12 0.505 0.838 2.60 0.830 4.32 2.58 1.76 1.44 0.505 4.32 K 13 (h 1 ) 0.628 0.603 0.494 0.668 0.646 1.23 1.19 0.656 0.651 0.494 1.23 K 10 _HL (h) 0.098 0.124 0.122 0.053 0.099 0.041 0.124 0.114 0.106 0.041 0.124 t (h) 0.076 0.102 0.095 0.040 0.079 0.030 0.064 0.078 0.077 0.030 0.102 t (h) 0.401 0.857 0.454 0.180 0.441 0.235 0.283 0.635 0.421 0.180 0.857 t (h) 6.89 11.0 7.28 5.82 7.52 6.77 8.61 12.5 7.40 5.82 12.5 AUC 0 24 (h*ng/mL) 1.43 1.08 1.39 2.27 1.36 1.39 0.979 1.26 1.37 0.979 2.27 Cl P (mL/min/kg) 23.8 28.0 21.1 14.2 20.9 20.7 31.2 24.3 22.4 14.2 31.2 AUMC 0 24 (h*h*ng/mL) 1.47 1.98 1.52 1.20 1.52 1.09 2.43 2.65 1.51 1.09 2.65 V ss (L/kg) 1.46 3.07 1.39 0.449 1.41 0.967 4.64 3.08 1.43 0.449 4.64 V 2 (L/kg) 0.110 0.172 0.104 0.035 0.083 0.083 0.222 0.295 0.107 0.035 0.295 V 3 (L/kg) 1.15 2.60 1.06 0.349 1.14 0.812 4.08 2.54 1.15 0.349 4.08 A,B and C, intercepts at t=0 for the model equation; alpha, beta and gamma, slopes for the model equation; C max extrapolated plasma GLY concentration at time 0; V 1 V 2 V 3 volumes of the central, second and third compartments, respectively; k 21 k 31 k 12 k 13 distribution rate constants; k 10 elimination rate constant; t phase 1 half life; t phase 2 half life; t phase 3 half life; AUC, area under the plasma concentration vs. time curve; Cl P total plasma clearance; AUMC, area under the f irst moment curve; V ss volume o f distribution at steady state.

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107 Figure 3 2 Observed (open circles) and the predicted concentrations (line) versus time when a two compartment model is applied

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108 Figure 3 3 Observed (open circles) and the predicted concentrations (line) versus time when a three compartment model is applied.

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109 Table 3 5 M odel comparison using diagnostic criteria. 2 compartment 3 compartment Subject # AIC SBC AIC SBC 1 138. 8 136.5 167. 9 164. 5 2 129. 9 127.6 143. 5 140. 1 3 137.1 134. 9 159.1 155.7 4 143. 4 141.1 152.1 148. 8 5 134. 9 132.6 155. 4 151.9 6 138.0 135. 8 142.4 139.0 7 120.3 118. 1 123.4 120. 1 8 118. 9 116.6 129.4 126.0 AIC riteria SBC Schwar z Bayesian C riteria

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110 Figure 3 4 Plasma concentration (ng/mL) vs. time (h) data from 0 24 h and for G LY administered intravenously to eight healthy athletic adult Thoroughbreds.

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111 Figure 3 5 Plasma concentration (ng/mL) vs. time (h) data from 0 1 h and for G LY administered intravenously to eight healthy athletic adult Thoroughbreds.

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112 Figure 3 6 Plasma concentration (ng/mL) vs. time (h) data from 0 168 h and for G LY administered intravenously to eight healthy athletic adult Thoroughbreds.

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113 Table 3 6 Urine GLY concentrations (pg/mL) following intravenous administration of 1 mg to each of 20 hor ses. Time (h) Horse 4 8 24 48 72 96 168 1 45820 1596 157 71.0 49.8 24.9 8.02 2 5082 794 123 58.1 40.1 23.0 7.03 3 10160 1366 188 101 58.9 34.9 11.3 4 8396 665 104 57.7 27.5 28.5 8.24 5 11790 413 138 71.7 36.4 24.1 6.20 6 30316 482 83.2 45.1 27.9 14.1 4.74 7 25040 1072 154 124 60.6 13.6 12.0 8 23939 1092 117 60.6 32.5 26.8 5.28 9 12932 1631 109 31.2 15.8 10.4 5.70 10 9415 188 79.7 34.1 14.8 13.0 6.25 11 209198 3675 262 93.8 41.7 35.7 12.8 12 68240 1135 80.6 34.0 14.5 10.4 4.86 13 64186 510 103 35.2 16.6 11.4 6.22 14 11939 423 61.5 17.3 10.4 7.06 3.22 15 11121 519 145 69.7 34.1 18.5 7.47 16 34568 2387 196 104 63.3 45.0 14.7 17 173489 9708 132 75.9 34.7 24.3 11.3 18 119981 2049 132 117 45.8 18.7 14.5 19 77124 944 144 80.2 39.6 23.7 10.8 20 43033 2378 113 52.1 25.6 18.8 5.81 Geomean 32276 1073 124 59.6 30.7 19.2 7.66 Median 32442 1082 127 65.1 34.4 20.9 7.25 Min 5082 188 61.5 17.3 10.4 7.06 3.22 Max 209198 9708 262 124 63.3 45.0 14.7 TI (urine) 441837 9232 282 203 108 60.7 20.3 values have been included in calculations to obtain measures of central tendency and dispersion and the tolerance interval. Geomean geometric mean TI tolerance interval (99 % /95 % ) *Values have been calculated from previously reported data.

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114 Figure 3 7 Plot of urine concentration (n g/mL) vs. time (h) after intravenous administration of GLY (1 mg) to 20 horses.

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115 Figure 3 8 Plot of median (range) urine and plasma ( ) concentrations for 20 horses administered a single 1 mg intravenous dose of GLY.

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116 Figure 3 9 Plot of median (range) urine to plasma concentration ratios for 20 horses administered a single 1 mg intravenous dose of GLY.

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117 Figure 3 10 Plot of plasma concentration (pg/mL) vs. time (h) after oral administration of GLY (10 mg/mL) to six horses.

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118 Figure 3 11 ) administered GLY in horse ( n =6) plasma.

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119 Figure 3 12 Plot of concentration (pg/mL) vs. time (h) of orally administered GLY (10 mg/mL) in horse (n=6) urine.

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120 Table 3 7 Median (range) of estimated renal clearance (mL/min/kg) using the range of urinary flow rate estimates from various reports. Urinary Flow Rate (mL/h/kg) Time (h) 0.52 0.92 1.12 1.24 4 29.9 (4.38 138.4) 53.0 (7.76 244.9) 64.5 (9.44 298.1) 71.4 (10.5 330.0) 8 2.02 (0.28 14.48) 3.57 (0.49 25.6) 4.34 (0.60 31.2) 4.81 (0.66 34.5) 24 0.84 (0.37 1.94) 1.49 (0.66 3.43) 1.82 (0.81 4.17) 2.01 (0.89 4.62) 48 1.11 (0.32 3.59) 1.96 (0.56 6.35) 2.39 (0.68 7.74) 2.65 (0.75 8.57) 72 1.13 (0.25 5.03) 3.33 (0.44 8.89) 2.43 (0.53 10.8) 2.69 (0.59 12.0) 96 1.44 (0.42 12.4) 2.54 (0.74 21.9) 3.10 (0.90 26.7) 3.43 (1.00 29.5) 168 1.06 (0.43 5.09) 1.87 (0.76 9.00) 2.27 (0.93 11.0) 2.52 (1.03 12.13)

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121 CHAPTER 4 PHARMACOKINETICS OF GLYCOPYRROLATE IN ST ANDARDBREDS We hypothesized that the urinary clearance of unchanged glycopyrrolate ( GLY ) accounts for a minor fraction of the total clearance because the estimated total plasma clearance is substantially greater than effective renal plasma flow and approaches estimates of hepatic blo od flow in the horse However, our previous studies did not employ an experimental design that would permit estimat ion of the renal clearance. This requires volumetric urine collections for a specified time period following drug administration. Once this i s done, renal clearance may be calculated using several methods, all of which are based upon the rate of unchanged GLY excretion and the plasma GLY concentration. T he presence and degree of plasma esterase activity in the blood may contribute to GLY elimi nation (Chen & Hsieh, 2005) Compounds containing ester groups such as GLY, may be particularly susceptible to plasma esterases, a heterogeneous family of enzymes that catalyze the hydrolysis of esters (Satoh et al ., 2002) Other studies have indicated a minimal inhibitory effect of GLY on plasma cholinesterases in humans, but d id not determine the presence or extent of GLY hydrolysis by these or other enzymes (Mirakhur, 1985; Zsigmond et al ., 1985) The following study wil l determine whether plasma hydrolysis occurs and the extent to which this pathway contributes to GLY clearance in the horse. Additionally, this study uses a different horse breed (Standardbred) from the previous (Thoroughbred) study This will allow a bre ed comparison of pharmacokinetic disposition and parameter estimates. We hypothesize that there are no major differences in pharmacokinetic disposition between the two horse breeds.

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122 Methods Animals Six, healthy, adult, and athletically conditioned Standar dbreds (1 mare and 5 geldings) ranging in age from 4 9 years and weighing from 445 510 kg were used in these studies All h orses were fed a diet of commercially available grain mixture and were housed indoors at the UF Veterinary Medical Center in climate controlled stalls for 1 h before and 24 h after drug administration. From 24 h after administration until the conclusion of the study (168 h) all horses were turned out to grass paddocks and had open access to water and hay at all times. Horses were regula rly exercised (3 days/week) before and throughout the duration of the study. The conditioning protocol is described in detail in Chapter 2 Dosing All horses were administered 1 mg (1.96 2.25 g/kg) of GLY (glycopyrronium bromide, American Regent, Inc., Sh irley, NY, USA) intravenously into the right jugular vein. Demographics for the study animals are presented in Table 3 1 Specimen Collection Plasma: Whole blood samples were collected using the procedures described in Chapter 3 Collection times were before drug administration and at 5, 10, 15, 20, 30 and 45 min and 1, 2, 3, 4, 6, 8, 24, 48, 72, 96, and 168 h af ter intravenous administration. Urine: A ll horses were trained to urinate on command and the urine from each horse was collected via the free catch method into separate clean 1 L containers. Total urine volume was collected through 24 h. Designated c ollection intervals were every hour. If a su bject produced no urine during a collection interval the next available specimen was taken. Additional urine collections occurred at 48, 72, 96 and 168 h following administration. Urine specimens were

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123 aliquoted into 15 mL sterile, disposable, polypropylen e centrifuge tubes and stored at 20C immedi ately and at 80C within 48 h. Plasma Esterase Stability One hundred milliliters of venous blood was collected from a 3 yr old gelding into tubes containing lithium heparin (BD Vacutainer, 10 mL, Becton Dickins on, Franklin Lakes, NJ, USA). The horse had been drug free for the previous 30 days and was considered healthy, based upon physical examination, complete blood count, serum chemistry profile, and pl asma fibrinogen concentration. Fresh harvested plasma was obtained within 1 h of blood collection through centrifugat ion of the blood samples for 15 min at 2,000 g All plasma was pooled and the pH was adjusted to 7.4, if necessary. Stock and working standard solutions of GLY and GLY d 3 were prepared according to th e procedures outlined above in C hapter 2. Calibrators and positive controls were prepared according to Table 2 1 using the fresh plasm a obtained for this experiment (Nunc, Roskilde, Denmark). The tubes were fortified with 32 L of 0.00125 ng/L GLY working standard solution for a final concentration of 10 pg/mL. The zero time sample was immediately capped and flash frozen using liquid nitrogen and stored at 20C until analysis. The remaining samples were capped and incubated in a water bath at 38C for 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min, flash frozen in liquid nitrogen upon removal and stored in 20C until analyzed. All samples were analyzed in duplicate according to the LC MS/MS me thod outlined in Chapter 2.

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124 Pharmacokinetic Analysis Nonlinear least squares regression analysis was performed on plasma GLY concentration versus time data and pharmacokinetic parameters for all horses were estimated with compartmental analysis using Phoenix WinNonlin 6.1 (Pharsight, St. Louis, MO, USA). The Gauss Newton (Levenberg and Hartley) method was used and goodness of fit and the Information Criterion (AIC) (Yamaoka et al ., 1978) and Schwar z's Bayesian Criterion (SBC) (Schwarz, 1978) as well as visual analysis of the graphica l output (including residual plots). Secondary parameters calculated include area under the curve (AUC), terminal half life (t ), apparent volumes of distribution, total plasma clearance (Cl p ), and micro distribution rate constants. All calculations for pharmacokinetic parameters were based on methods described by Gibaldi and Perrier (Gibaldi & Perrier, 1982) All pharmacokinetic parameter estimates were calculated for each horse and values are reported as media n and range (minimum maximum). Statistical Analysis Plots of urine and plasma concentrations 5.0 for Windows (GraphPad Software, San Diego, CA, USA) and p lasma and urine concentrations of GLY are expressed as mean and standard deviation. Mann Whitney U a nd Kruskall Wallis rank sum tests were used for statistical comparisons (nonparametric) of pharmacokinetic parameters between breeds (Powers, 1990; Hollander & Wolfe, 1973) Analysis of variance (ANOVA) was used for parametric analysis. A p value of less t han 0.05 was considered statistically significant. Statistical analysis was performed using JMP Version 8.0 (SAS Institute Inc., Cary, NC, USA)

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125 Results Plasma: After intravenous administration of 1 mg of GLY, the observed plasma concentration versus time profile could be best described by a three compartment model. The equation based on macro constants for this model is: C t = A exp + B exp + C exp (4 1) where C t is the plasma concentration at time (t), A, B and C are the zero time intercepts for and exp is the base of the natural logarithm (Gabrielsson & Wei ner 2007) The weighting factor chosen with this model was 1/ (Y 2 ) where Y was the observed plasma concentration. The model was chosen based on visual inspection of the observed and predicted concentration versus time graphs for a two ( Figure 4 1 ) and three ( Figure 4 2 ) compartment analysis and other diagnostic criteria ( Table 4 2 ). Plasma GLY concentrat ion vs. time plots for all six horses are depicted in Figure 4 3 and Figure 4 4 pg/mL) for all horses through 4 8 h and only two horses 168 h after dosing ( Figure 4 5 ). Mean (SD) plasma concentrations are presented in Table 4 3 The median (range) C max in six Standardbreds following a 1 mg i ntravenous dose of GLY wa s 12.4 (10.0 19.0) ng/mL. Median (range) estimates for plasma clearance (Cl p ) volume of distribution of the central compartment (V 1 ) and area under the plasma concentration time curve (AUC 0 24 ) were 16.7 (13.6 21.7) mL/min/kg 0. 167 ( 0.103 0.215) L/kg, and 2.13 (1.67 2.57) ng*h/mL, respectively Estimates for all of the pharmacokinetic parameters followin g compartmental model analysis are reported in Table 4 4 The median (range) values for pharmacokinetic parameter estimates for Standardbreds and Thor oughbreds are presented in Table 4 5 The AUC 0 24 MRT a nd terminal half life of the Standardbred horses were significant ly differe nt when compared to those of six Thoroughbreds

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126 ( Table 4 6 ) Figure 4 6 illustrates the distribution of twelve horses separated by breed for each of six PK parameter estimates Statistical comparisons were made using the M ann Whitney test (Powers, 1990) With regard to plasma hydrolysis, the measured concentrations of GLY in plasma incubated at 38 degrees for various times did not differ from the fortified concentrations by mo re than 10% and the GLY concentrations of all incubated samples differed from the zero time sample by < 5%. Average concentrations of GLY for duplicate analysis at the end of each incubation period through 360 min ranged from 9.05 10.3 pg/mL ( Figure 4 7 ) Urine: For five of six horses, urine GLY concentrations peaked at the first collection interval Complete urine results for each horse are provided in Table 4 7 The total GLY accumulated urinary excretion theough 24 h for six horses had a median (range) of 0.140 (0. 113 0.246) mg or 14 (11.3 24.6) % of the total administered dose of 1 mg. Cumulative urinary excretion of GLY for each horse is shown in Figure 4 8 Greater than 9 5% of GLY that was renal ly cleared was excreted with in 4 h after intravenous administration ( Figure 4 9 ) The urinary GLY excretion rate is plotted for each horse in Figure 4 10 Median (range) renal clearance for all six horses was 2.65 (1.92 3.59) mL/min/kg. Renal clearance represented approximately 11.3 24.7 % of the total plasma clearance Discussion The pharmacokinetic profile of a single intravenous administration of GLY in Standardbred horses followed a tri exponen tial decay, similar to its disposition i n the Thoroughbred horse ( Figure 4 11 ) A t hree compartment model compared to a two compartment model was a superior model fi t based on graphical inspection a nd low er diagnostic values for both AIC and SBC GLY disposition was characterized by a rapid drop in plasma concentrations

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127 beginning immediately after administration, a small volume of distribution and a slow terminal elimination attributed to the redistribution o f GLY from the third compartment to the central compartment Although the Thoroughbred and Standardbred horses show ed a similar GLY disposition following intravenous administration differences in some pharmacokinetic p arameter estimates were evident when compared using a nonparametric rank sum test. The difference in AUC 0 24 was attributed to the larger CL T exhibited in Standardbreds (16.7 (13.6 21.7) mL /min/kg) compared to Thoroughbreds (22.4 (14.2 31.2) mL /min/kg ). Wh ile an identical dose was used for both studies, a reason for the difference in peak plasma concentrations is unknown other than the wide degree of subject variability for parameter estimates in both studies Additionally, w hile differences in drug distrib ution between donkeys and horse s have been reported for phenylbutazone (Mealey et al ., 1997) guaifenesin (Matthews et al ., 1997) and sulphamethoxasole (Peck et al ., 2002) there is little data detailing pharmacological differences, if any, between Standard bred and Thoroughbred horse breeds. The cumulative excretion of unchanged GLY in horse urine indicates that less than 25% of the total dose is cleared through renal mechanisms. This contrasts with pharmacokinetic studies in humans that have reported over 8 0% (Kaltiala et al 1974) and nearly 50 % (Ali Melkkila et al ., 1990) of the intramuscularly administered dose was excreted unchanged in the urine. Consistent with the present research, however, the latter study in humans reported that unchanged GLY was el iminated within three hours of administration. The amount remaining to be excreted (ARE) plots ( Figure 4 9 ) demonstrate that, for this study, over 95% of the unchanged GLY wa s excreted within 4 h after drug administration. However, it is recognized that ARE calculations are subject to error if com plete urine collectio ns are not made Since this study relied

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128 use catheteriz ation collection volumes may not be completely accurate due to incomplete bladder emptying Yet the cumulative excretion remai ns unaffected by this limitation. Another method for analyzing urine data for half life (t 1/2 ) and elimination rate constant (k el ) is to plot the averag e rate of excretion against the time, which in most case s is best represented by the midpoint of the collection interval. The quality of the excretion rate plot has limitations, namely an uncertainty of complete bladder emptying and the need to collect accurately timed urine samples over short intervals, relativ e to the elimination half life of the drug (Gibaldi, 1986) However, a significant advantage of the rate of excretion plot is that each data point is essentially independent, especially if the bladder is fully voided for each sample. A missed sample or da t a points is not critical to this analysis. The GLY excretion rate plots ( Figure 4 10 ) showed a large degree of scatter when displayed on a semi log pl ot. Renal clearance of a particular drug can be determined by several methods, all of which are based on the relationship between the excretion rate and the plasma concentration as demonstrated in Equation 4 2. (4 2) where Au is the amount of drug in the urine t is the time interval, Cl R is the renal clearance and C p is the plasma concentration As shown in the equation above, renal clearance is the constant of proportionality between the rate of excretion and the plasma concentration. Thus, a plot of the rate of excretion against the corresponding plasma concentration yields a straight line with a slope equal to renal clearance. Renal clearance is calculated using the rate of excretion method for each time interval in Table 4 8

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129 The maximum rate at which GLY can be filtered by the glomerulus is the glomerular filtration rate (GFR). Estimates of the GFR can be obtained by measuring creatinine becaus e the substance is filtered but neither secreted nor reabsorbed. Creatinine clearance is calculated similar to renal clearance in the equation above (4 2) using plasma and urine creatinine measurements and the urine volume taken in the time interval ( Table 4 8 ) We have determined the creatinine clearance to range between 1.8 2.0 mL/min/kg. This indicates that s ecretion of GLY from the bloo d into the tubular lumen is l ikely because estimates for GLY renal clearance are higher than the GFR (creatinine clearance). Reabsorption of unchanged GLY from the tubular lumen back into the blood via passive diffusion polarity. Therefore, th is data suggests that GLY undergo es renal excre tion through glomerular filtration and tubular secretion Plasma esterase activity appeared to have little or no effect on the concentration of GLY, ruling out the possibility that a substantial proportion of t he drug was being hydrolyzed in the plasma (La Du, 1972) such as the case with other ester containing compounds (Lehner et al ., 2000; Jordan et al ., 1996; Egan, 1995) Knowing this and in light of the urinary GLY excretion data, it can be hypothesized tha t the majority of non renal clearance is hepatic clearance, and further, the majority of total clearance is attributed to hepatic clearance as demonstrated using the following equation (Wilkinson et al ., 1987) (4 3) where Cl is the total body clearance, Cl R is the renal clearance, Cl H is the hepatic clearance, and Cl other represents the sum of all other clearance processes Thus hepatic clearance may be estimated from this study by subtracting Cl R from Cl assuming that CL other is relatively small. Total plasma clearance in Standardbreds wa s characterized by a median (range) of 16.7

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130 (13.6 21.7) mL/min/kg. Therefore using the renal clearance estimated from this study of 2.65 (1.92 3.59) mL/min/kg, non renal clearance wa s approximately 14.1 (11.7 18.1) mL/min/kg. The median (range) estimates of total plasma clearance in both Thoroughbreds (22.7 (14.2 31.2) mL/mi n/kg) and Standardbreds (16.7 (13.6 21.7) mL/min/kg) closely approximates previous estimates of liver blood flow i n the horse (15 20 mL/min/kg) (Dyke et al ., 1998) Under such conditions whereby the plasma clearance is equivalent to the hepatic blood flow, the drug is said to be highly extracted by the liver (Toutain & Bousquest Molou, 2004) (4 4 ) where Cl H is the hepatic blood clearance, Q is the liver blood flow, and E H is the hepatic extraction ratio, a term representing the fraction of dose undergoing metabolism and thus irreversibly removed Additionally, for drugs with a high extraction ratio changes in liver blood flow are a major determinant of hepatic clearance. Also, the degree of, or changes in the protein binding of GLY have little influence on hepatic clearance of drugs with a high extraction ratio In the horse GLY is extensively metabolized i n the liver by hydrolysis of the ester moiety to 2 cyclopentyl 2 hydroxy 2 phenylacetic acid (cyclopentylmandelic acid) and 1,1 dimethyl 3 hydroxypyrrolidine 1 ium. Although possible that the decrease in total plasma cleara nce observed for Standardbreds compared to Thoroughbreds was due to decreased liver blood flow as a result of confinement (anxiety) in an indoor stall for 24 h during the study

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131 Table 4 1 Demographics of Standardbred study subjects. Each number represents a horse. Horse Gender Age (yr) Weight (kg) IV Administration Total Urine Collection PK Analysis 1 M 4 4 45 X X X 2 G 7 508 X X X 3 G 8 510 X X X 4 G 9 457 X X X 5 G 4 450 X X X 6 G 4 4 78 X X X M Mare, G Gelding

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132 Figure 4 1 Observed concentrations (open circles) and the predicted concentrations (line) versus time when a two compartment model is applied to six Standardbred horses.

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133 Figure 4 2 Observed (open circles) and the predicted concentrations (line) versus time when a three compartment model is applied to six Standardbred horses.

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134 Table 4 2 Diagnostic values for a three compartment model fit to the concentration vs. time data for each horse. 2 compartment 3 compartment Subject # AIC SBC AIC SBC 1 101. 7 99.4 129. 6 126.2 2 111. 8 109.5 132.6 129.2 3 124. 7 122.4 159.5 156.1 4 112. 3 109.9 152. 7 149.2 5 103.8 101.6 125.5 122.1 6 93.9 91.6 120.1 116.7 AIC riteria SBC Schwar z Bayesian C riteria

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135 Table 4 3 Plasma concentration of GLY after a s ingle intravenous dose of 1 mg to each of 6 Standardbred horses Time (h) Mean SD (ng/mL) Range (ng/mL) 0 < LOD
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136 Figure 4 3 Plasma concentration (ng/mL) vs. time (h) data from 0 24 h and for G LY administered intravenously to six healthy athletic adult Standardbreds.

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137 Figure 4 4 Plasma concentration (ng/mL) vs. time (h) data from 0 1 h and for G LY administered intravenously to six healthy athletic adult Standardbreds.

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138 Figure 4 5 Plasma concentration (ng/mL) vs. time (h) dat a from 0 1 68 h and for GLY administered intrav enously to six healthy athletic adult Standardbreds.

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139 Table 4 4 Pharmacokinetic parameter estimates o f GLY, determined using a three compartmental mode l, following intravenous administration of 1 mg to six (n=6) healthy adult Standardbred horses. Parameter Horse Median Min Max. 1 2 3 4 5 6 A (ng/mL) 13.9 12.3 18.5 9.70 11.7 9.78 12.0 9.70 18.5 B (ng/mL) 0.912 0.401 0.521 0.419 0.313 0.229 0.410 0.229 0.912 C (ng/mL) 0.012 0.007 0.009 0.007 0.009 0.012 0.009 0.007 0.012 Alpha (h 1 ) 8.61 7.06 9.88 8.431 8.39 8.75 8.52 7.06 9.88 Beta (h 1 ) 1.41 1.04 1.24 1.35 0.978 0.791 1.14 0.791 1.41 Gamma (h 1 ) 0.039 0.039 0.082 0.034 0.035 0.030 0.037 0.030 0.082 C max (ng/mL) 14.8 12.7 19.0 10.1 12.0 10.0 12.4 10.0 19.0 V 1 (L/kg) 0.152 0.154 0.103 0.216 0.185 0.209 0.1 67 0.103 0.21 5 K 21 (h 1 ) 1.85 1.23 1.48 1.64 1.17 0.975 1.36 0.975 1.85 K 31 (h 1 ) 0.044 0.042 0.086 0.039 0.040 0.038 0.041 0.038 0.086 K 10 (h 1 ) 5.76 5.509 7.92 6.05 6.11 5.58 5.91 5.51 7.92 K 12 (h 1 ) 1.63 0.90 1.35 1.22 1.21 1.48 1.29 0.904 1.63 K 13 (h 1 ) 0.776 0.456 0.368 0.859 0.873 1.50 0.817 0.368 1.50 K 10 _HL (h) 0.120 0.126 0.088 0.114 0.113 0.124 0.117 0.088 0.126 t (h) 0.080 0.098 0.070 0.082 0.083 0.079 0.081 0.070 0.098 t (h) 0.492 0.666 0.557 0.514 0.709 0.876 0.612 0.492 0.876 t (h) 17.9 17.7 8.46 20.2 19.8 23.1 18.9 8.46 23.1 AUC 0 24 (h*ng/mL) 2.57 2.31 2.40 1.67 1.96 1.80 2.13 1.67 2.57 Cl T (mL/min/kg) 14.5 14.1 13.6 21.7 18.9 19.4 16.7 13.6 21.7 AUMC 0 24 (h*h*ng/mL) 8.73 5.23 1.88 6.54 7.62 13.5 7.08 1.88 13.5 V ss (L/kg) 2.96 1.92 0.640 5.10 4.41 8.73 3.69 0.640 8.73 V 2 (L/kg) 0.133 0.113 0.094 0.160 0.191 0.317 0.146 0.094 0.317 V 3 (L/kg) 2.68 1.66 0.443 4.72 4.03 8.20 3.36 0.443 8.20 A,B and C, intercepts at t=0 for the model equation; alpha, beta and gamma, slopes for the model equation; C max extrapolated plasma GLY concentration at time 0; V 1 V 2 V 3 volumes of the central, second and third compartments, respectively; k 21 k 31 k 12 k 13 distribution rate constants; k 10 elimination rate constant; t phase 1 half life; t phase 2 half life; t phase 3 half life; AUC, area under the plasma concentration vs. time curve; Cl P total p lasma clearance; AUMC, area under the first moment curve; V ss volume o f distribution at steady state.

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140 Table 4 5 Comparison of pharmacokinetics parameters of Standardbreds and Thoroughbreds. Standardbreds Thoroughbreds Parameter Median Min Max Median Min Max A (ng/mL) 12.0 9.70 18.5 8.37 4.51 27.3 B (ng/mL) 0.410 0.229 0.912 0.404 0.076 2.23 C (ng/mL) 0.009 0.007 0.012 0.011 0.069 0.015 Alpha (h 1 ) 8.52 7.06 9.88 9.01 6.77 23.5 Beta (h 1 ) 1.14 0.791 1.41 1.65 0.809 3.86 Gamma (h 1 ) 0.037 0.030 0.082 0.094 0.056 0.119 C max (ng/mL) 12.4 10.0 19.0 8.71 5.46 29.5 V 1 (L/kg) 0.170 0.103 0.215 0.212 0.065 0.336 K 21 (h 1 ) 1.36 0.975 1.85 1.93 0.884 4.88 K 31 (h 1 ) 0.041 0.038 0.086 0.102 0.062 0.125 K 10 (h 1 ) 5.90 5.51 7.92 6.56 5.58 17.1 K 12 (h 1 ) 1.28 0.904 1.63 1.44 0.505 4.32 K 13 (h 1 ) 0.817 0.368 1.50 0.651 0.494 1.23 K 10 _HL (h) 0.117 0.088 0.126 0.106 0.041 0.124 t (h) 0.081 0.070 0.098 0.077 0.030 0.102 t (h) 0.612 0.492 0.876 0.421 0.180 0.857 t (h) 18.9 8.46 23.1 7.40 5.82 12.5 AUC 0 24 (h*ng/mL) 2.13 1.67 2.57 1.37 0.979 2.27 Cl p (mL/min/kg) 16.7 13.6 21.7 22.4 14.2 31.2 AUMC 0 24 (h*h*ng/mL) 7.08 1.88 13.5 1.51 1.09 2.65 MRT (h) 3.64 0.784 7.50 1.43 0.449 4.64 V ss (L/kg) 3.69 0.640 8.73 0.107 0.035 0.295 V 2 (L/kg) 0.146 0.094 0.317 1.15 0.349 4.08 CLD2 3.92 2.32 5.15 8.37 4.51 27.3 V 3 (L/kg) 3.36 0.443 8.20 0.404 0.076 2.23 CLD3 2.33 0.634 5.21 0.011 0.069 0.015 Min minimum ( n =6) Max maximum ( n =6)

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141 Table 4 6 Calculated p values using the Mann Whitney U test for comparisons of pharmacokinetic parameter estimates between horse breeds. PK Parameter Estimate p value Significance Cl p 0.065 No AUC 0 24 0.015 Yes V 1 0.394 No C max 0.392 No MRT 0.041 Yes t 0.015 Yes

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142 Figure 4 6 Distribution of PK parameter estimates in Standardbreds ( n =6 ) and Thoroughbreds ( n =6)

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143 Figure 4 7 Linear regression of concentration vs. incubation time demonstrating the degree of plasma esterase activity on GLY in horse plasma.

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144 Table 4 7 Urine GLY excretion data following intravenous administration of 1 mg to eight (n=6) Standardbred horses Time Interval (h) t (h) Urine Volume (mL) Urine Concentration (ng/mL) Amount ( g) Cumulative Amount Excreted ( g) Midpoint time (h) Rate of excretion ( ( g/h ) A.R.E. ( g) Subject 1 0.5 1 0.5 950 176.6 167.7 167.7 0.75 335.5 78.3 1 2 1.0 40 103.0 4.12 171.9 1.5 4.12 74.1 2 3 1.0 720 94.2 67.8 239.7 2.5 67.8 6.33 3 4 1.0 90 26.1 2.35 242.0 3.5 2.35 3.98 4 6 2.0 80 8.64 0.692 242.7 5 0.346 3.29 8 12 4.0 750 2.46 1.84 244.6 10 0.461 1.44 12 16 4.0 1270 0.797 1.01 245.6 14 0.253 0.430 16 20 4.0 480 0.687 0.330 245.9 18 0.082 0.100 20 24 4.0 180 0.557 0.100 246.0 22 0.025 0.000 Subject 2 0 0.5 0.5 440 189.5 83.4 83.4 0.25 166.8 113.5 0.5 1 0.5 460 86.1 39.6 123.0 0.75 79.3 73.9 1 2 1.0 1780 32.1 57.1 180.1 1.5 57.1 16.7 2 3 1.0 600 15.9 9.53 189.7 2.5 9.53 7.21 4 6 2.0 570 6.37 3.63 193.3 5 1.81 3.59 8 12 4.0 740 2.38 1.76 195.1 10 0.440 1.83 12 16 4.0 1270 0.916 1.16 196.2 14 0.291 0.662 16 20 4.0 800 0.565 0.452 196.7 18 0.113 0.210 20 24 4.0 470 0.447 0.210 196.9 22 0.053 0.000 Subject 3 0.5 1 0.5 1040 18.7 19.5 19.5 0.75 39.0 121.8 1 2 1.0 740 27.2 20.1 39.6 1.5 20.1 101.6 2 3 1.0 1370 29.5 40.4 80.0 2.5 40.4 61.3 3 4 1.0 2210 26.9 59.4 139.4 3.5 59.4 1.83 8 12 4.0 720 0.922 0.664 140.1 10 0.166 1.17 16 20 4.0 2750 0.339 0.931 141.0 18 0.233 0.237 20 24 4.0 1720 0.138 0.237 141.3 22 0.059 0.000

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145 Table 4 7 Continued Time Interval (h) (h) Urine Volume (mL) Urine Concentration (ng/mL) Amount ( g) Cumulative Amount Excreted ( g) Midpoint time (h) Rate of excretion ( ( g/h ) A.R.E. ( g) Subject 4 0.5 1 0.5 860 125.6 108.0 108.0 0.75 216.1 14.6 1 -2 1.0 240 36.2 8.70 116.7 1.5 8.70 5.88 4 -6 2.0 570 7.45 4.249 121.0 5 2.12 1.63 8 -12 4.0 950 0.781 0.742 121.7 10 0.185 0.892 12 -16 4.0 940 0.642 0.604 122.3 14 0.151 0.289 16 20 4.0 680 0.239 0.162 122.5 18 0.041 0.126 20 24 4.0 780 0.162 0.126 122.6 22 0.032 0.000 Subject 5 0 0.5 0.5 140 345.0 48.3 48.3 0.25 96.6 90.4 0.5 1 0.5 250 286.6 71.7 120.0 0.75 143.3 18.8 1 2 1.0 150 65.5 9.83 129.8 1.5 9.83 8.95 2 3 1.0 170 26.3 4.48 134.3 2.5 4.48 4.48 3 4 1.0 90 13.5 1.21 135.5 3.5 1.21 3.27 4 6 2.0 350 4.72 1.65 137.1 5 0.827 1.61 8 12 4.0 830 0.698 0.579 137.7 10 0.145 1.03 12 16 4.0 860 0.503 0.432 138.2 14 0.108 0.600 16 20 4.0 820 0.317 0.260 138.4 18 0.065 0.341 20 24 4.0 1240 0.275 0.341 138.8 22 0.085 0.000 Subject 6 0 0.5 0.5 1140 69.9 79.7 79.7 0.25 159.3 33.7 0.5 1 0.5 490 37.4 18.3 98.0 0.75 36.7 15.3 8 12 4.0 1870 7.89 14.8 112.8 10 3.69 0.564 12 16 4.0 650 0.412 0.268 113.0 14 0.067 0.297 16 20 4.0 620 0.242 0.150 113.2 18 0.038 0.146 20 24 4.0 760 0.193 0.146 113.3 22 0.037 0.000

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146 Figure 4 8 Cumulative GLY excretion in six horses administered 1 mg of GLY intravenously.

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147 Figure 4 9 Amount remaining to be excreted (ARE)

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148 Figure 4 10 Urinary excretion rate of GLY following a single 1 mg intravenous dose to six Standardbred horses.

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149 Figure 4 11 Mean (SD) p lasma concentration (ng/mL) vs. time (h) data from 0 24 h and for GLY administered intravenous ly to

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150 Table 4 8 Urine GLY renal clearance following intravenous administration of 1 mg to six (n=6) Standardbred horses Time Interval (h) (h) Cp at midpoint (ng/mL) Renal Clearance (mL/min/kg) Urine Creatinine (mg/dL) Plasma Creatinine (mg/dL) Creatinine C learance (mg/mL/min) Subject 1 0.5 1 0.5 0.350 35.89 1 2 1.0 0.121 1.270 2 3 1.0 0.038 67.139 3 4 1.0 0.017 5.1 4 6 2.0 0.011 1.207 8 12 4.0 0.008 2.1 239.9 1.2 6.2 12 16 4.0 0.007 1.4 16 20 4.0 0.006 0.5 20 24 4.0 0.005 0.2 318.1 1.3 1.8 Subject 2 0 0.5 0.5 2.420 2.261 0.5 1 0.5 0.252 10.3 1 2 1.0 0.091 20.6 2 3 1.0 0.036 8.7 4 6 2.0 0.008 7.4 8 12 4.0 0.005 3.01 197.2 1 6.08 12 16 4.0 0.004 2.33 16 20 4.0 0.003 1.06 20 24 4.0 0.003 0.58 265.3 1.1 4.723 Subject 3 0.5 1 0.5 0.225 5.670 1 2 1.0 0.089 7.4 2 3 1.0 0.031 43.150 3 4 1.0 0.013 144.0 8 12 4.0 0.004 1.36 144.5 1.2 3.61 16 20 4.0 0.002 3.657 20 24 4.0 0.001 1.294 115.9 1.2 6.922

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151 Table 4 8 Continued Time Interval (h) (h) Cp at midpoint (ng/mL) Renal Clearance (mL/min/kg) Urine Creatinine (mg/dL) Plasma Creatinine (mg/dL) Creatinine Clearance (mg/mL/min) Subject 4 0.5 1 0.5 0.176 44.6 1 2 1.0 0.062 5.10 4 6 2.0 0.007 11.7 8 12 4.0 0.005 1.31 294.1 1.3 8.95 12 16 4.0 0.004 1.226 16 20 4.0 0.004 0.38 20 24 4.0 0.003 0.34 227.3 1.3 5.683 Subject 5 0 0.5 0.5 1.680 2.130 0.5 1 0.5 0.180 29.448 1 2 1.0 0.081 4.5 2 3 1.0 0.035 4.717 3 4 1.0 0.018 2.5 4 6 2.0 0.010 3.2 8 12 4.0 0.006 0.9 157.1 1.1 4.94 12 16 4.0 0.005 0.746 16 20 4.0 0.005 0.516 20 24 4.0 0.004 0.779 215.9 1 11.155 Subject 6 0 0.5 0.5 1.295 4.3 0.5 1 0.5 0.152 8.429 8 12 4.0 0.009 14.7 192.6 1 15.0 12 16 4.0 0.008 0.3 16 20 4.0 0.007 0.2 20 24 4.0 0.006 0.21 197.5 1.2 5.21

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152 CHAPTER 5 PHARMACODYNAMICS OF GLYCOPYRROLATE IN TH OROUGHBREDS Muscarinic an tagonists, such as glycopyrrolate ( GLY ) cause positive chronotropism and dromotropism (improved atrioventricular conduction) by competitively blocking the effects of acetylcholine at muscarinic receptors in the heart (Ali Melkklia et al ., 1991) However, because of the nonselective blockade of muscarinic receptors in other organ systems, GLY also inhibit s a number of parasympathetically mediated functions, causing decreased airway and gastrointestinal secretions, bronchodilation, and inhibition of gastrointestinal motility (Fuder & Meincke, 199 3) During the pharmacokinetic studies outlined in Chapters 3 and 4, the administration of a single intravenous dose of 1 mg produced no apparent clinical or behavioral effects in horses. Although these studies did not specifical ly monitor clinical effects associated with the drug, it was assumed that the low dosag e and rapid metabolism had induced only transient effects and none that were observable without ded icated instrumentation for a measureable amount of time Pharmacokinetic parameters representing drug disposition may be substantially affected by the extent of plasma protein binding (Schmidt et al ., 2010) It has been mathematically demonstrated that the volume of distribution, clearance, half life and hepatic bioavailability have the potential to be influenced by the fract ion of the drug that is unbound (Rowland & Tozer, 1995) Unbound drugs are free and available for extensive distribution into the tissue and thus pharmacologically active. From a pharmacodynamic perspective, clinical effects of mo st drugs are often minimally affected due to changes in plasma protein binding. Benet and Hoener (2002) make evident that only drugs which are given intravenously and primarily eliminated through hepatic mechanisms or orally and eliminated through non hepa tic mechanism are subject to changes in clinical effects due to an increased unbound percentage (Benet & Hoener, 2002) In

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153 order to further characterize the pharmacology and disposition of GLY in the horse an in vitro experiment was conducted to determine the extent of plasma protein binding at various concentrations. No reports of the pharmacokinetics of GLY following a clinically relevant dosage in horses are found in the literature. Thus data correlating plasma concentrations of GLY with the pharmacody namics are not available, leaving a void of information for clinicians as well as agencies that regulate the drug in performance horse s The purpose of this study was to investigate the pharmacokinetics and pharmacodynamics (PK PD) following a continuous r ate infusion of GLY to the horse Additionally, we developed a model that offers a plasma concentration effect predictive relationship for regulators who encounter plasma or urine concentrations in post performance specimens. Methods Animals Six, adult, T horoughbred geldings ranging in age from 9 11 years and weighing from 540 595 kg were used in these studies ( Table 5 1 ) All horses were determined to be healthy prio r to the study based on physical examination, complete blood count (CBC), horse blood chemistry panel, urine analysis, indirect blood pressure, and electroca rdiography (ECG). One week before the start of the study two horses per day for three days underwen t a full 5 hour mock up of the infusion study in order to acquaint them with the equipment, procedures and handling, thereby reducing the potential behavioral effects or stress of the experiment. On the morning of the study, horses were weighed and allowed to feed on a commercially a vailable grain mixture 2 h before drug administration. Each horse was housed indoors at the University of Florid a (UF) Veterinary Medical Center, in an individual climate controlled (26C) stable throughout the

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154 dosing and direct observation period and had open access to water at all times. Following the direct observation period horses were released outdoors to grass pa ddocks. The experimental protocol in cluding drug administra tion and sample collection, was approved and facilit ies were inspected periodically by the UF Institutional A nimal Care and Use Committee. Dosing The study consisted of a 2 way crossover design where in the six participating horses each received a continuous rate infusion (CRI) of GLY and a saline (0.9 % NaCl ) control with a 10 day washout period between treatments. Thus each horse served as its own control. On the days during which drug administration took place, the study always began at 0800 in order to reduce variability associated with circadian rhythm c hanges. Baselin e observations took place for 1 h (0800 0900) before drug administration. Following the baseline observation period, horses were administered GLY (glycopyrronium bromide, American Regent, Inc., Shirley, NY) using local lidocaine anesthesia an d a 14 guage catheter aseptically placed into the right jugular vein, at an intravenous CRI (Medex 3010, Duluth, GA) of 4 g/kg per hour for 2 h for a total drug dose of 8 g/kg. The dose was based on previous pharmacokinetic analysis (Chapter 3) in a s imi lar group of horses and was intended to achieve steady state conditions. After the end of the infusion period a ll horses were directly observed in the stalls until the first bowl movement was passed. After a satisfactory general health assessment for abdom inal discomfort and colic the horses were re leased to outside paddocks Specimen Collection Plasma: Whole blood samples were collected from the contralateral left jugular vein via needle venipuncture into partially evacuated tubes containing lithium hepar in. Blood samples were stored on ice until the plasma was concentrated by centrifugation (2500 3000 rpm or 776

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155 1318 x g) at 4 C for 15 min. Harvesting of plasma took place within 1 hour of sample collection and 2 4 mL aliquots of plasma were immediately frozen at 20 C and stored within 24 h at 80 C until analyzed. Collection times were relative to the start of the infusion and included a sample collection before drug administration and at 5, 10, 15, 20, 30, 45, 60, 90, 120 (end of infusion), 122.5, 12 5, 130, 135, 140, 150, 165 min and 3, 3.5, 4, 5, 6, 8, 10, 14, and 26 h after the start of the infusion. Samples were stored for no longer than 6 weeks under 80 C (4 weeks) and 20 C (2 weeks) conditions which were well within the validated stability li mitations previously r eported in Chapter 2. Urine: All horses were trained to urinate on command and the urine from each horse was collected via the free catch method into separate clean 1 L containers. Urine specimens were aliquoted into 15 mL sterile, disposable, polypropylene centrifuge tubes and store d at 20C immediately and at 80C within 48 h. Collection times are relative to the start of the infusion and included a sample collection before drug administration and at 1, 2, 3, 4, 6, 8, 10, 14, and 26 h after the start of the infusion. Determination of Plasma Protein Binding One hundred milliliters of v enous blood was collected from each of six horses, ages 3 8 yrs old, into tubes containing lithium heparin V acutainer, 10 mL, Becton Dickinso n, Franklin Lakes, NJ, USA). Each horse had been drug free f or the previous 30 days and was considered healthy, based upon physical examination, complete blood count, serum chemistry analysis, and pl asma fibrinogen concentration. The storage of plasma has been known to affect the plasma protein binding of certain d rugs (Paxton, 1981) For this reason fresh harvested plasma was obtained within 1 h of blood collection through centrifugation of the blood samples for 15 min at 2,000 g All plasma was

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156 pooled and the pH was adjusted to 7.4. The extent of GLY plasma protei n binding was determined by UF methodology as described previously (Wright et al ., 1996 ; Sebille et al ., 1990) Stock and working standard solutions of GLY and GLY d 3 were prepared according to th e procedures outlined above in C hapter 2. Calibrators were prepared using the fresh plasma obtained for this experiment. A set of positive control samples was prepared in triplicate for each horse ( n =6) in fresh plasma and a set of protein free negative control samples was prepared in ph osphate buffered saline (pH 7.4), to achieve end concentratio ns of 25, 10, 5, 1, 0.5 and 0.1 ng/mL. A 1 mL aliquot of each plasma dilution and of the negative control solution was then d at 37C for 20 min, flash frozen in liquid nitrogen, C until analyzed. A second 1 mL aliquot of each sample was transferred into the sample reservoir of the Centrifree ultrafiltration device (Millipore Corp, Bedford, MA, USA ) and incubated at 37C for 10 min to allow drug plasma protein binding equilibrium. The samples were then centrifuged at 2,000 g and 37C for 15 min. After centrifugation, the filtrate cup was disconnected from the filtration device, sealed with a cap, fl ash frozen, C until analyzed. Concentrations of GLY in the unfiltered samples and the filtered samples were determined by use of high performance liquid chromatography tandem mass spectrometry as described above in cha pter 2. The concentration of protein bound drug (C b ) was calculated as: C b = C t C f ( 5 1) where C t is the total GLY concentration and C f is the concentration of the free fraction of GLY. The percent plasma protein binding (PB) was calculated as: PB (%) = C b /C t 100, (5 2)

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157 Similarly, the degree of nonspecific adsorption ( N A) of GLY to the filtration device was determined based on the phosphate buffered saline diluted, protein fr ee negative control samples as: N A = C b /C t 100. (5 3) Pharmacokinetic Analy sis Nonlinear least squares analysis was performed on plasma GLY concentration versus time data and pharmacokinetic parameters for all horses were estimated with compartmental analysis using Phoenix WinNonlin 6.1 (Pharsight, St. Louis, MO, USA). The Gauss Newton (Levenberg and Hartley) method was used and goodness of fit and the appropriate weighting factor were selected based on the coefficients (Yamaoka et al ., 1978) and Schwar z's Bayesian (Schwarz, 1978) C riteria (SBC) as well as visual analysis of the graphical output (including residual plots). Secondary parameters calculated include area under the curve (AUC), terminal half life (t ), mean residence time (MRT), apparent volumes of distribution, total plasma cleara nce ( Cl p ), and microdistribution rate constants. All calculations for pharmacokinetic parameters were based on methods described by Gibaldi and Perrier (1982). The plasma drug concentration at steady state (C p ss ) was calculated as: C p ss = R o /Cl p (5 4 ) wh ere R o is the drug infusion rate and Cl p is the systemic (total) drug clearance. All pharmacokinetic parameters were calculated separately for each horse and values are reported as media n and range (minimum maximum). Physiological Endpoints Horses were kept under constant direct observation from 0800 (1 h before administration ) until 1300 (2 h following the end of the infusion). Any cli nical signs of drug response were

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158 carefully monitored and recorded. Heart rate and respiratory rate were recorded for ba seli ne every 10 min for 1 h before drug administration and again every 10 min for 4 h after the start of the infusion. Heart rate was measured using a telemetric device fastened with a girth for continuous monitoring. Respiratory rate was measured by direc t counting. D efecation, urinary incidence and stool consistency were recorded throughout the observation period. Horses were retained in indoor stalls until the first bowel movement after the end of drug administration was r ecorded. Following a routine h ealth evaluation, horses were turned out to pasture In addition to direct observation all subjects were video recorded from 0800 (1 h before administration) until 1300 (2 h after administration) for a total of 5 h. Abnorm al behavior was evaluated based o n a detailed rubric (Price et al ., 2003) PK PD Modeling The relationship of the plasma GLY concentrations vs. pharmacodynamic effect was assessed for heart and respiratory rate each. Pharmacokinetic pharmacodynamic linked analysis was performed using Phoe nix WinNonlin 6.1 (Pharsight, St. Louis, MO, USA). The appropriate model was selected based on AIC and a visual inspection of the fitted graph. Minimum Akaike Information Criteria estimates were applied to discriminate the best fitting model and improved fit of data was achieved by re weighting. Statistical Analysis Plasma GLY concentrations and pharmacokinetic parameter estimates are reported as mean standard deviation and median and range (minimum maximum), respectively Differences in plasma PB between different nominal GLY concentrations were assessed using one way repeated measures ANOVA with Tukey's post hoc test. Differences between treatment and control groups for physiological endpoints were assessed using two way A NOVA with

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159 post hoc test. All analyses were performed using commercial statistical software (Microsoft Office Excel 2003, Microsoft Corporation, Redmond, WA, USA; and GraphPad A p value of less than 0.05 was considered statistically significant. Results Protein B inding Percent of GLY bound to plasma proteins under a range of plasma concentration s (0.1 25 ng/mL) were between 37 44% and are summarized in Table 5 2 The extent o f nonspecific adsorp tion was 0.9 2.9% suggesting that adsorption of GLY to the filt ration device was negligible Pharmacokinetics GLY plasma concentrations for all six horses are presented in Table 5 3 and plotted in Figure 5 1 A composite plasma GLY concentration vs. time plot for all six horses from 0 until 24 h after the discontinuation of the infusion ( Figure 5 2 ) and from 0 until 4 h after the discontinuation of the infusion ( Figure 5 3 ) is depicted and represents mean SEM. After the discontinuation of a two hour CRI administration of 4 g/kg /hr of GLY the observed plasma concentration versus time profile could be best described by a three compartment model. The equation based on macro constants for this model is: C t = Aexp + Bexp + Cexp (5 5) where C t is the plasma concentration at time (t), A, B and C are the zero time intercepts for and exp is the base of the natural log arithm (Gabrielsson & Weiner, 2007) The weighting factor chosen with this model was 1/ (Y 2 ) where Y was the observed plasma co ncentration P lasma

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160 GLY concentration versus time curves demonstrating observed and model predicted concentrations are illustrate d in Figure 5 4 Pharmacokinetic parameter estimates determined from compartmental model analysis are presented in Table 5 4 After the infusion was discontinued the plasma GLY concentration declined rapidly exhibiting a tri exponential decay, similar to the disposition following a bolus in travenous dose of 1 mg ( C hapter 3 & 4 ). The initial distribution phase was characterized by an estimated median half life (t ) of 0.12 h (7.2 min) and the rapid (t ) and slower (t ) elimination phases were 0.78 h and 13.2 h, respectively. The median (minimum maximum) plas ma GLY steady state concentration was 5.10 (3.90 6.18) ng/mL. Physiological Endpoints The heart rate s during the direct observation period for GLY and saline adm inistrations for each subject are plotted over time in Figure 5 5 The mean heart rate of the GLY treated group differed significantly from that of the control gr oup beginning approximately 50 min after the start of the drug infusion and ending 40 min after the discontinuation of the infusion. A plot of the mean heart rate (beats per minute) over time for the treated and control groups is displayed in Figure 5 6 In dividual plots demonstrating the respiratory rate response from the treated and control experiments are presented in Figure 5 7 The mean respiratory rate of the GLY t reated group differed significantly from that of the control group beginning approximately 20 min after the start of the drug infusion and ending 20 min before the discontinuation of the infusion. A plot of the mean (SD) respiratory rate (breathes per minu te) over time for t he treated and control groups are displayed in Figure 5 8

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161 All horses that received GLY exhibited slowed gastrointestinal motility an d reduced def ecation incidence compared to the control group. Figure 5 9 illustrates the frequency of bowel movements during the entire 5 h direct observation period and Figure 5 10 displays the same data in separate time bins. It is evident that bowel movements in the treatment group are occurring during the pre administration period and the first hour of the drug infusion, presumably before the drug exerts its effect s on the gastro intestinal tract The treated horses experienced extended periods until the first bowel movement since the first hour of the drug infusion. The mean SD time elapsed until the first bowel movement for the treated horse was 6 2.03 h ( Figure 5 11 ) with one horse experiencing a 9 h period without defecation. During the study it was observed that a ll horses had exhibited a loss of appetite and refused treats within sixt y minutes after the start of the infusion while control horses readily accepted them throughout. All horses showed mild behavioral changes in the form of shifting weight from one leg to the other and occasional muscle fas ciculatio ns Two horses showed sign s of colic and received flunixin. These horses were evaluated on the following morning and were found to be in normal condition and medically sound PK PD Modeling Examination of plots of GLY plasma concentrations against the corresponding heart rate effec ts revealed that these pharmacodynamic response s were lagging behind the plasma concentrations ( Figure 5 12 ) In order to accommodate this temporal disconnect, the model chosen included an effect compartment, and the relationship between the effect compartment concentration and response was assumed to be linear (Meibohm & Derendorf, 1997) Effect compartment mo deling with heart rate effects following GLY administration was performed using a sigmoidal E max model with baseline effec t ( Figure 5 13 ) in which the

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162 indiv idual pha rmacokinetic parameter estimates obtain ed were used as constants The equation for the effect site concentration (C e ) during and after the constant GLY infusion is, (5 6 ) where k 0 is the zero order infusion rate, k e0 is the elimination rate constant from the hypothetical effect compartment and V c k 21 k 31 1 2 3 are the modeled PK parameters, T is the elapsed time during the infusion (after the infusion T=2 h) and t is time af ter the infusion (Coburn, 1981) Equa tion 5 6 and Equation 5 7 (5 7 ) were modeled simultaneously which provided estimates of the baseline effect (E 0 ), maximal drug effect (E max ), concentration at 50% of Emax (EC 50 ), sigmoidicity factor (n hill coefficient) and k e0 for each horse (H olford & Sheiner, 1981) Hysteresis for the effect of heart rate can also be illustrated when concentration and effect are plotted versus time for each subject ( Figure 5 14 ) and for the entire group ( Figure 5 15 ) The observed and predicted pharmacological response during th e direct observation period from the proposed PK PD linked model for each subject is plotted in Figure 5 16 Linked model PD estimates are presented in Table 5 5 Discussion Ultrafiltration is a commonly used technique to determine the binding of a drug to plasma proteins because of its rapidity and ease of use. It utilizes a two chambered reservoir separated by a filter membrane that allows passage of the drug but not that of drug bound to plasma proteins Drug fortified plasma is pipetted into the upper reservoir and the sample is centrifuged.

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163 Free drug is filtered into the lowe r reservoir and its concentration subtracted from the total fortified concentration in order to calculate a percentage of the drug which is protein bound. The primary disadvantage includes the non specific binding of the free drug to the filter membrane (H oward et al ., 2010) Protein binding of GLY appeared to decrease with increasing concentrations indicating possible drug saturation at protein binding sites in horses. The degree of plasma protein binding of GLY determined in this study suggests that the potential for displacement interactions with other drugs is unlikely to be clinically significant. However, we did not specifically investigate the influence of GLY metabolites o r other drugs on the protein bindi ng of GLY Since GLY is approximately 40 % protein bound in the horse, previous estimates of glomerular filtration may be overstated. Only the free form of GLY may be filtered by the glomerulus Therefore in order to calculate the amount of GLY that underg oes filtration, the fraction unbound (fu) must be multiplied by the glomerular filtration rate (GFR). Therefore approximately 1.2 mL/min/kg is estimated to undergo filtration while the remainder is likely to be actively secreted in the tubular lumen. GLY disposition in the horse following a CRI resulted in a rapid decrease in plasma concentrations early and a prolonged terminal elimination, although this study did not look at GLY plasma or urine concentrations beyond 24 h following the end of the infusion. This profile was similar to that reported following a single rapid bolus administration in horses in Chapters 2 and 3. GLY distributed very rapidly from the central to the peripheral compartments as demonstrated by the initial distribution phase median ha lf life (t ) of 0.12 h (7.2 min). Evidence of wide distribution and prolonged elimination were noted with the rapid (t ) and slower (t ) elimination phases of 0.78 h and 13.2 h, respectively. Compared to single

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164 intravenous injections, GLY plasma c oncentration versus time curves demonstrated little variability between subjects after a continuous rate intravenous infusion. GLY g/kg intravenous dose. Mean heart ra te (n=5) after the 2.5 g/kg dose did not demonstrate a significant difference from the control group for measurements taken up to 120 min. However, the 5 and 10 g/kg doses both produced significantly elevated mean heart rates from the control group begin ning 5 min after and ending 60 min after treatment in awake horses (Singh et al ., 1997), in agreement with the current study. In other studies with horses GLY is used to attenuate the cardiovascular depressive effects of anesthetic agents, such as xylazine Singh et al (1995) report a 2.5 g/kg dose is effective at reducing atrioventricular bock, but doses such as 5 and 10 g/kg were associated with a profound loss of gastrointestinal motility and therefore determined unsafe. Teixeira et al (2003) noted a 53 % increase in cardiac output over the control group when horses (n=6) were intravenously administered 5 g/kg of GLY and anesthetized with xylazine, while low intestinal auscultation scores were evident. A third study in anesthetized horses recognized a significant increase in mean heart rate after 5 g/kg intravenously but also cautioned unwanted gastrointestinal effects (Dyson et al 1999). of GLY have not bee n reported in horses. However, in humans intravenous GLY has been shown to cause bronchodilatation (Gal and Suratt, 1981), while nebulized administration caused a longer duration of bonchodilatation without the systemic anticholinergic effects of inhaled a tropine (Gal et al ., 1984; Walker et al ., 1987). In the horse GLY would be expected to antagonize muscarinic receptors on the airway smooth muscle and submucosal glands, to cause bronchodilation and reduced mucus secretions (Coulson and Fryer, 2003). Pharm acokinetic

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165 pharmacodynamic modeling was not performed using respiratory rate as a physiological parameter in these studies. However, respiratory rate and GLY plasma concentrations were plotted against time for each ( Figure 5 17 ) and all ( Figure 5 18 ) subjects I n contrast to heart rate, a clockwise hysteresis (proteresis) was ob served between GLY concentrations and the physiological endpoint of respiratory rate ( Figure 5 19 ). Proteresis generally occurs when a subject develops tolerance for a given compound. Despite the limited value of respiratory rate as an indicator of airway function, changes were evident between the treatment and control groups. The depressant effects of muscarinic receptor antagonists on intestinal motility have been documented and t he horse is particularly sensitive to the gastrointestinal effects of these compounds. GLY has been used successfully to treat vagally mediated bradycardia during anesthesia in small animals (Dyson & James Davies, 1999) and in goats (Pablo et al ., 1995) without causing major complications in other organ systems Yet in horses, doses as low as 5 g/kg of GLY have demonstrated lower auscultation scores and in some case intestinal impaction and colic (Dyson et al., 1999; Singh et al ., 1997; Tei xeira Neto et al ., 2004; Singh et al ., 1995 ). This study demonstrates that GLY has the potential to slow defecation incidence and frequency in the period following drug administration compared to horses administered saline. Although in this study, motility was not directly quantified, it is assumed th at a reduced fecal frequency and extended periods of withheld defecation are the result of gastrointestinal hypomotility. As such, it has been demonstrated through this and other studies that GLY should be used conservatively at CRI doses below 10 g/kg, e specially in horses with pre existing gastrointestinal conditions or those undergoing surgery.

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166 This study attempted to evaluate behavioral effects associated with the administration of GLY to the horse and the subjects were video monitored for five hour s beginning from the time they were led into the stalls. There was no clear evidence of behavioral changes with any of the appeared more relaxed when a companion animal was housed in an adjacent stall for the duration of the observation period. Behavioral variation ranged from circling the stall continuously to standing still. There was obvious hysteresis observed when heart rate and plasma GLY concentrations wer e plotted against time and when the effect was plotted against plasma GLY concentrations that was minimized by the incorporation of an effect compartment model ( Figure 5 20 ). This finding is unusual mainly because cardiac receptors are located in the periphery and GLY concentrations at the effect site are expected to be similar to measured concentrations. While hysteresis appears to be a characteristic of PK PD modeling of several drugs active in the central nervous system (Mandema et al ., 1991; Danhof et al ., 1992), t he current study does not permit us to establish whether distribution is the major determinant of the observed hysteresis. However, the ra tionale for this delay is easily understood if one assumes that the resulting pharmacological effect or response is preceded by drug distribution to the site of action. Nonetheless, since GLY is unlikely to cross the central nervous system barrier due to i ts polarity a temporal delay due to drug distribution to these sites is unlikely. Also, there is no consistent report demonstrating the role of other factors in the biophase equilibration of GLY which are known to cause hysteresis, such as coupling mechan isms and effectuation process that follow the drug receptor interaction. The hysteresis has been successfully modeled by the effect compartment approach, which postulates the existence of a hypothetical effect compartment linked to the plasma site by a fi rst

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167 order process (k e0 ). This approach is based on the assumption that distribution kinetics between plasma and effect site are linear and that the same effect site concentration always evokes the same response, independent of time. This assumption may not hold when active metabolites are formed or when there is development of acute tolerance. However, there are no reports indicating that active metabolites of GLY are formed in humans (Kaila et al ., 1990) or horses (Matassa et al ., 1992). M oreover, there is no evidence of the development of tolerance toward the anticholinergic effects of GLY in these models. In the case of purely peripheral effects, a more direct link of the plasma pharmacokinetics to the corresponding pharmacodynamic effect would have been expected. However, the physiological situation may be more complex. A long equilibration half life can also ensue from a high affinity of the drug for the cardiac tissue leading to prolongation of the (peripheral) effects of GLY on the heart. Moreover, hea rt rate is determined by complex peripheral cardiovascular regulatory systems, and changes in one part of the system, such as muscarinic blockade, will likely cause compensatory changes in the components.

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168 Table 5 1 Demographics of PK PD study subjects Subject # Gender Age (yr) Weight (kg) CRI Administration PK Analysis PD Analysis 1 G 12 530 X X X 2 G 11 590 X X X 3 G 12 5 60 X X X 4 G 12 548 X X X 5 G 12 540 X X X 6 G 12 550 X X X Table 5 2 Percent protein binding (mean SD) of GLY in the plasma of six ( n =6) healthy horses. Nominal plasma GLY concentration (ng/mL) Analyte 0.1 0.5 1 5 10 25 Protein Binding (%) 42.9 8 .9 43.5 3 .4 44.1 2 .1 37.3 3.2 40.1 5 .8 37.5 7 5 Nonspecific Adsorption (%) 1.5 2 .2 2.3 1.9 0.9 1.1 3.3 3.6 2.9 1.1 1.8 1.4

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169 Table 5 3 Plasma GLY concentrations (ng/mL) for six Thoroughbred horses following a two hour CRI of 8 g/kg. Plasma GLY Concentration (ng/mL) Subject # Time (h) 1 2 3 4 5 6 Mean SD Median MIN MAX 0.08 1.29 1.45 1.30 1.06 1.56 1.63 1.38 0.209 1.38 1.06 1.63 0.17 2.77 3.02 3.08 2.96 2.78 3.02 2.94 0.131 2.99 2.77 3.08 0.33 3.47 3.59 3.92 3.90 4.05 4.20 3.85 0.278 3.91 3.47 4.20 0.50 3.67 3.60 4.22 4.38 4.54 4.73 4.19 0.461 4.30 3.60 4.73 0.67 3.92 3.98 4.49 4.78 4.87 5.14 4.53 0.496 4.64 3.92 5.14 1.00 4.39 4.28 5.09 5.31 5.15 5.49 4.95 0.498 5.12 4.28 5.49 1.50 4.77 4.66 5.31 6.06 5.90 5.62 5.39 0.581 5.47 4.66 6.06 2.00 4.86 4.72 5.35 5.99 6.04 5.88 5.47 0.584 5.62 4.72 6.04 2.04 2.72 2.91 3.83 4.75 4.93 5.45 4.10 1.12 4.29 2.72 5.45 2.08 2.17 3.20 2.95 4.91 3.31 4.15 3.45 0.957 3.26 2.17 4.91 2.17 1.26 1.47 1.77 2.74 2.17 2.98 2.06 0.693 1.97 1.26 2.98 2.25 0.793 0.755 1.29 1.78 1.49 2.11 1.37 0.538 1.39 0.755 2.11 2.33 0.531 0.533 0.933 1.13 1.05 1.29 0.911 0.316 0.991 0.531 1.29 2.50 0.340 0.276 0.541 0.713 0.649 0.896 0.569 0.234 0.595 0.276 0.896 2.75 0.220 0.165 0.414 0.393 0.351 0.616 0.360 0.160 0.372 0.165 0.616 3 0.133 0.112 0.265 0.253 0.254 0.463 0.247 0.125 0.253 0.112 0.463 3.5 0.080 0.053 0.174 0.148 0.134 0.214 0.134 0.059 0.141 0.053 0.214 4 0.055 0.041 0.124 0.096 0.086 0.147 0.092 0.040 0.091 0.041 0.147 5 0.027 0.021 0.049 0.067 0.043 0.082 0.048 0.023 0.046 0.021 0.082 6 0.020 0.017 0.034 0.040 0.030 0.051 0.032 0.013 0.032 0.017 0.051 8 0.014 0.012 0.013 0.023 0.021 0.030 0.019 0.007 0.018 0.012 0.030 10 0.010 0.008 0.009 0.015 0.015 0.021 0.013 0.005 0.012 0.008 0.021 14 0.006 0.004 0.006 0.008 0.011 0.010 0.007 0.003 0.007 0.004 0.011 26 0.003 0.002 0.003 0.006 0.007 0.008 0.005 0.002 0.005 0.002 0.008

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170 Figure 5 1 Plasma concentration (ng /mL) vs. time (h) data from 0 26 h and fo r GLY administered by an intravenous infusion in each of six healthy adult Thoroughbreds.

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171 Figure 5 2 Plasma concentration (ng /mL) vs. time (h) data from 0 26 h and fo r GLY administered by an intravenous infusion in six healthy adult Thoroughbreds. Figure 5 3 Plasm a concentration (ng /mL) vs. time (h) data from 0 4 h and fo r GLY administered by an intravenous infusion in six healthy adult Thoroughbreds

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172 Figure 5 4 Observed (circles) and predicted (line) plasma GLY concentrations after a two hour intravenous infusion of 8 g/kg of body weight to six (n=6) healthy adult Thoroughbred horses and pharmacokinetic analysis using a three compartment model

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173 Table 5 4 Pharmacokinetic parameter estimates o f GLY, determined using a three compartmental model, following a two hour intravenous infusion of 4 g/kg /h of body weight to six (n=6) healthy adult Thoroughbred horses. Para meter Subject # 1 2 3 4 5 6 Median Min Max. A (ng/mL) 47.7 47.7 41.6 40.8 51.0 47.3 47.5 40.8 51.0 B (ng/mL) 0.806 0.556 1.13 0.367 1.19 0.928 0.867 0.367 1.19 C (ng/mL) 0.018 0.018 0.013 0.010 0.023 0.019 0.018 0.010 0.023 Alpha (h 1 ) 7.03 6.59 5.73 4.22 5.50 4.56 5.61 4.22 7.03 Beta (h 1 ) 1.05 1.071 0.822 0.473 0.995 0.650 0.908 0.473 1.07 Gamma (h 1 ) 0.072 0.095 0.056 0.019 0.050 0.036 0.053 0.019 0.095 C max (ng/mL) 3.74 3.86 4.20 5.08 5.17 5.73 4.64 3.74 5.73 V 1 (L/kg) 0.165 0.166 0.187 0.194 0.153 0.166 0.166 0.153 0.194 K 21 (h 1 ) 1.150 1.135 0.952 0.507 1.10 0.725 1.02 0.507 1.15 K 31 (h 1 ) 0.075 0.097 0.057 0.020 0.052 0.038 0.055 0.020 0.097 K 10 (h 1 ) 6.22 6.07 4.82 3.76 4.77 3.91 4.80 3.76 6.22 K 12 (h 1 ) 0.511 0.308 0.650 0.246 0.413 0.399 0.406 0.246 0.650 K 13 (h 1 ) 0.203 0.147 0.127 0.175 0.210 0.172 0.173 0.127 0.210 K 10 _HL (h) 0.112 0.114 0.144 0.184 0.145 0.177 0.145 0.112 0.184 t (h) 0.099 0.105 0.121 0.164 0.126 0.152 0.124 0.099 0.164 t (h) 0.660 0.647 0.843 1.47 0.697 1.07 0.770 0.647 1.47 t (h) 9.59 7.32 12.4 35.8 14.0 19.1 13.2 7.32 35.8 AUC 0 24 (h*ng/mL) 7.80 7.94 8.88 10.9 10.9 12.3 9.91 7.80 12.3 Cl p (mL/min/kg) 17.1 14.1 15.0 12.2 12.2 10.8 13.6 10.8 17.1 AUMC 0 24 (h*h*ng/mL) 13.0 5.23 16.1 40.3 23.4 31.5 19.7 11.6 40.3 V ss (L/kg) 0.686 0.462 0.729 1.96 0.831 1.01 0.780 0.462 1.963 V 2 (L/kg) 0.073 0.045 0.128 0.094 0.058 0.091 0.082 0.045 0.128 V 3 (L/kg) 0.448 0.251 0.414 1.67 0.620 0.751 0.534 0.251 1.675 C pss (ng/mL) 3.90 4.73 4.45 5.48 5.48 6.18 5.10 3.90 6.18 A,B and C, intercepts at t=0 for the model equation; alpha, beta and gamma, slopes for the model equation; C max extrapolated plasma GLY concentration at time 0; V 1 V 2 V 3 volumes of the central, second and third compartments, respectively; k 21 k 31 k 12 k 13 distribution rate constants; k 10 elimination rate constant; t phase 1 half life; t phase 2 half life; t phase 3 half life; AUC, area under the plasma concentration vs. time curve; Cl P total p lasma clearance; AUMC, area under the first moment curve; V ss volume o f distribution at steady state; C pss plasma GLY concentration at steady state.

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174 Figure 5 5 Heart rate for each subject following a 2 h CRI of GLY.

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175 Figure 5 6 Mean (SD) heart rate (bpm) for six horses during the direct observation period

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176 Figure 5 7 Respiratory rate for each subject following a 2 h CRI of GLY.

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177 Figure 5 8 Mean (SD) respiratory rate (b reathes/ m in ) for six horses during the direct observation period

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178 Figure 5 9 Mean (SD) frequency of bowel movements for the entire direct observation period (t 0 = start of the infusion) for six healthy horses administered GLY (8 g/kg of body weight) over a two hour intravenous infusion. Figure 5 10 Mean (SD) frequency of bowel movements for the entire direct observation period (t 0 = start of the infusion) for six healthy horses administered GLY (8 g/kg of body weight) over a two hour intravenous infusion.

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179 Figure 5 11 Mean (SD) time elapsed from the discontinuation of the infusion (t=0) until the first bowel movement for six healthy horses administered GLY (8 g/kg of body weight) over a two hour intravenous infusion.

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180 Figure 5 12 Hysteresis (counterclockwise) plot demonstrating that the there is a temporal lag between the physiologic endpoint (heart rate) and plasma GLY concentration. Heart rate represents mean (SD) of all subjects.

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181 Figure 5 13 The effect compartment model.

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182 Figure 5 14 Mean plasma GLY concentration and mean heart rate effects over time for each subject Error bars are removed for clarity.

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183 Figure 5 15 Plot demonstrating that the there is a temporal lag between the mean physiologic endpoint (heart rate) and mean plasma GLY concentration. Error bars are removed for clarity.

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184 Figure 5 16 PK PD linked model fit for heart rate for each of six horses

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185 Table 5 5 Pharmacodynamic model parameters. Subject # Parameter (units) 1 2 3 4 5 6 Median Minimum Maximum E max (bpm) 87.2 73.5 49.9 41.9 61.9 46.0 55.9 41.9 87.2 EC 50 (ng/mL) 4.66 3.60 4.72 3.22 3.47 1.76 3.54 1.76 4.72 E0 (bpm) 44.5 38.6 33.9 37.9 40.3 41.3 39.5 33.9 44.5 n 2.81 3.99 1.87 3.70 1.96 2.01 2.41 1.87 3.99 k e0 (h 1 ) 1.61 0.697 1.63 1.58 0.811 1.41 1.49 0.697 1.63 t 1/2 ke0 (h) 0.430 0.994 0.425 0.439 0.855 0.491 0.465 0.425 0.994 E max maximal effect; EC 50 plasma drug concentration producing 50% of E max ; n Hill coefficient; k e 0 rate constant of eq uilibrium of drug compartment; t 1/2 k e 0 half life of equilibrium of drug in effect compartment.

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186 Figure 5 17 Mean plasma GLY concentration and mean resp iratory rate effects over time for each subject. Error bars are removed for clarity.

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187 Figure 5 18 Plot demonstrating that the mean maximum effect for the physiologic endpoint (respiratory rate) occurs before mean maximum plasma GLY concentration. Error bars are removed for clarity. Figure 5 19 Mean (SD) respiratory rate as a func tion of concentration for six horses demonstrating p roteresis (clockwise) plot.

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188 Figure 5 20 Individual hysteresis showing the plasma concentration and the predicted effect compartment concentratio n after the PK PD link model was applied

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189 CHAPTER 6 CONCLUSIONS AND FINA L REMARKS Analytical Methodology The results of m ethod development studies described herein have demonstrated that the identification of glycopyrrolate ( GLY ) in horse plasma and urine sa mples can be accomplished at sub picogram per milliliter concentrations using common analytical laboratory procedures and modern analytical instrumentation. Moreover the method s described have undergone the nec essary validation procedu res to demonstrate that they are acc eptable for their intended use for quantification of GLY in horse plasma and urine, and meet the minimum requirements set forth by United States Food and Drug Administration (USFDA) (Guidance for Industry, Bioanalytical Method Validation, 2001) The methods as described have been successfully applied to the analysis of plasma and urine specimens collected from research horses after administrations of GLY for elimination and pharmacokinetic pharmacodynamic studies. Currently, the reported methods for the determination of GLY in horse plasma and urine are believed to be the first reported with adequate sensitivity and selectivity for regu latory control of this drug in performance horses Such investigations could contribute to the RMTC effort to establish a plasma threshold and to recommend a withdrawal time for this drug in race horse s Further improvements to the describ ed methods can be accomplished with advanced sample preparation techniqu es and more sophisticated instrumental technologies. Yet, for the purposes of clinical evaluations and for the regulatory control of this drug as a performance enhancer in both humans and horses the method is adequate and robust Pharmacokinetics and Phar macodynamics Thoroughbred or Standardbred horse have not previously been investiga ted. These studies have suggested that the

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190 plasma pharmacokinetics of GLY in the horse following a single intrav enous and clinically relevant dose can be characterized by a three compartment mammillary model. Extensive distribution from the central compartment, rapid clearance and prolonged terminal half life were observed. Further, we have demonstrated that GLY was detectable in horse urine for at least 168 h after intravenous administration of a clinically relevant dose and 24 h after oral dosing. Post race plasma analysis could be complementary to urine analysis in order to provide adequate regulatory control of t he use of GLY. The relationship between plasma and urinary GLY concentrations following a single intravenous dose is a noteworthy observation that should be considered when evaluating the pharmacologic significance of the presence of GLY and regulatory con trol in official post race urine samples. The results of this research can be used to develop thresholds and withdrawal guidelines for regulating the use of GLY in the horseracing industry. The plasma pharmacokinetics of GLY have also been demonstrated in Standardbred horses and PK parameter estimates have been compared between t w o different breeds of horses. Obvious differences are apparent between breeds. However, differences in study environments may contribute to such differences in PK parameter estimat es. Further studies are needed to confirm differences in drug distribution among breeds. Clearance of GLY in the horse oc curs predominately in the liver and contrast s with previous data obtained from human studies. Renal clearance of unchanged GLY accounts for approximately 10 25% of the total clearance of GLY and plasma hydrolysis of the drug does not occur appreciably in horses. Active secretion of GLY into the renal tubular lumen is unlikely because the rate of excretion does not exceed rate of glomerular filtration and unchanged GLY is Further, the effect

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191 of protein binding on the clearance of GLY is thought to be minimal because of the limited degree to which the drug is renally cleared. The results show that the use of concomitant PK PD modeling provided independent, accurate information about the transport of GLY to the site of action, un derstanding about the nature of the observed effects and the underlying concentration effect relationship. Heart rate is significantly increased for at least 60 min following IV administration. Respiratory rate is also affected but the effect has a shorter duration of action. Defecation incidence and frequency are slowed following GLY administration and the drug must be used conservatively due to intestinal complications Regulatory Control GLY is a class 3 drug as categorized by the ARCI and therefore its identification in official post race track samples may lead to violations and sanctions for the trainer or owner or both. However, GLY is also well documented in the medical and scientific lit erature to be an effective human and veterinary therapeutic drug. In order to allow for the legitimate treatment of horses before racing or performance events, withdrawal periods or threshold limits (allowable drug concentrations) for each regulated drug m ay be set by the various associations Concentrations of GLY have been documented to be present in samples taken from post competition horses. Therefore, it is imperative to determine drug concentrations which no longer provide a performance altering effe ct. The capability to detect and quantify small amounts or concentrations of drug substances is critically important for research and regulatory purposes. However, for routine drug analysis in performance horses such sensitivity may reveal pharmacologicall y insignificant concentrations of drugs that w ere legitimately used for therapeutic purposes.

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192 One way in which analytical testing laboratories respond to industry concerns over drug use in performance horses is to develop and validate methods and utilize i nstruments that offer superior sensitivity. Although such increases in sensitivity are necessary for substance s that have no practical medicinal value, therapeutic medications should undergo a different level of scrutiny. In fact, it is not uncommon to det ect small concentrations of therapeutic medications and it can be argued that the presence of such substances at small concentrations will have no As part of this drug monitoring process, bioanalytical methods mus t be available to determine drug concentrations in plasma and urine. Since bioanalytical methods have become highly sensitive, physiologically relevant concentrations of drugs must be determined in order to regu late threshold drug concentrations in plasma and urine that are to be referenced for drug testing, instead of relying on the detection limits of the analytical methods. The investigations detailed above, describe GLY plasma and urine concentrations and disposition following different dosing regimens and can provide regulatory agencies with sufficient data to support certain threshold limits and withdrawal guidelines for the horserace industry

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193 APPENDIX A DRUGS AND INSTRUMENT Figure A 1 GLY (Robinul V) injectable. Figure A 2 Generic GLY injectable used for intravenous injection.

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194 Figure A 3 Robinul V tablets (1 mg each ). Figure A 4 Triple Stage Quadrupole (TSQ) Quantum Ultra mass spectrometer (ThermoFisher, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) source and interfaced with a HTC PAL autosampler (Leap Technologies, Carrboro, NC, USA) and Accela LC pump (ThermoFisher)

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195 APPENDIX B HORSE PICTURES Figure B 1 Study horse being conditioned on a treadmill.

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196 Figure B 2 Study horse receiving an intravenous infusion of GLY.

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197 Figure B 3 During the PK PD study, horses were housed in pairs, reducing anxiety and normalizing behavior.

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198 LIST OF REFERENCES Abboud T K., Read, J., Miller, F., Chen, T., Valle, R. & Henriksen, E. ( 1981 ) Use of glycopyrrolnium in the paturient: effect on the maternal and fetal heart and uterine activity Obstetrics and Gynecology 57 224 226. Adams H.R. ( 2001 ) Veterinary Pharmacology and Therapeutics Ames: Iowa State University Press. Ahn J.E., Karlsson, M.O., Dunne, A. & Ludden, T.M. ( 2008 ) Likelihood based approaches to handling data below the quantification limit using NONMEM VI. Journal of Pharmacokinetics and Pharmacodynamics 35 401 421. Akaike H. 1976. An information criterion (AIC ) Mathmatical Sciences 14 5 9. Alder L., Greulich, K., Kempe, G. & Vieth, B. ( 2006 ) Residue analysis of 500 high priority pesticides: better by GC MS or LC MS? Mass Spectrometry Reviews 25 (6), 838 865. Alex C.G., Sko rodin, M.S. & Shilstone, J. ( 1994 ) Glycopyrrolate inhalation aerosol in the treatment of chronic obstructive pulmonary disease. American Review of Respiratory Distress 149 p.309. Alex C.G., Skorodin, M.S. & Karafilidis, J. ( 1999 ) Nebulized glycopyrrolat e cause significant and long lasting bronchdilation in patients with chronic obstructive pulmonary disease. American Journal of Respiratory Critical Care Medicine 159 p.823. Ali melkkila T., M., Kaila, T., Kanto, J. & IIsalo, E. ( 1990 ) Pharmacokinetics of I.M. glycopyrronium. British Journal of Anaesthesia 64 667 669. Ali melkkila T., T. Kaila, & J. Kanto. ( 1989 ) Glycopyrrolate: pharmacokinetics and some pharmacodynamic findings. Acta Anaesthesiol Scandanavia 33 513 7. Ali melkkila T., T. Kaila, J. Kanto & E. Isalo. ( 1990 ) Pharmacokinetics of glycopyrronium in parturients. Anaesthesia 45 634 7. Ali melkkila T. M., T. Kaila, J. Kanto, & E. Iisalo. ( 1990 ) Pharmacokinetics of i.m. glycopyrronium. British Journal of Anesthesia 64 667 9. Ali melkki la, T., J. Kanto, & E. Iisalo. ( 1993 ) Pharmacokinetics and related pharmacodynamics of anticholinergic drugs. Acta Anaesthesiol Scandanavia 37 633 42. Ali melkklia T., Kai la, T., Antila, K., Halkola, L. & lisalo, E. ( 1991 ) Effects of glycopyrrolate and atropine on heart rate variability. Acta Anaesthesiology Scandanavia 35 436 441. Almeida, A.M., Castel Branco, M.M. & Falcao, A.C. ( 2002 ) Linear regression for calibration lines revisited: weighting schemes for bioanalytical methods. Journal of Chromatography B 215 222.

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218 BIOGRAPHICAL SKETCH Marc Ja son Rumpler was born in August 1978 in Troy, New York The younger of two sons, h e graduate d with his high school diploma from LaSalle Institute (grades 7 12), an all male catholic military school, receiving exclusively average grades. Rumpler was admitted to the Bouv College of Health Sciences at Northeastern Un iversity (Boston, Massachusetts) in August 1996, with an undetermined major and later went on to join the t oxicology program under the gu idance of Robert Schatz, PhD. From January of 1998 until May of 2001 Rumpler interned with several organizations including Biogen Idec, Inc. and the U niversity of Massachusetts (UMass) Medical School (Worcester MA) Rumpler graduated in 2001 with a Bachelor of Science degree in t oxicology and was chosen by his peers to represent his c lass with a speech at commencement After several years in private industry performing wet chemistry and routine analytical methods and procedures, Rumpler entered the University of Florida online program for forensic toxicology. In 2007, Rumpler earned his Certificate in Forensic Toxicology and o ne year later, a Master o f Science degree in Forensic Toxicology. Later in 2008, Marc was accepted into the College of Veterinary Medicine at the University of Florida as a Physiological Sciences doctoral student. Here he was introduced to the Florida Racing L aboratory under the m entoring of Drs. Ric hard Sams and Nancy Szabo. Over four years, Marc was fortunate to participate on several different projects between the Racing and Equine Performance Laboratories. In March 2012 he Exce by t he University Of Florida College Of Veterinary Medicine. I n May 2012, Marc received his Ph.D. and later assume d a post doctoral position with the National Institute on Drug Abuse (Baltimore, Maryland) under the guidance of Dr. Marilyn Huestis Marc was the first person in his family to obtain a four year degree, and the first t o obtain a graduate degree. Marc has a wife of 10 years and tw o children ages 3 and 14 at the time of this writing.