Table Of ContentPHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF
TILMICOSIN IN SHEEP, CATTLE, AND RATS
BY
SANJA MODRlt
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
1997
To my dearest husband Tomislav, for his love, support and friendship
ACKNOWLEDGEMENTS
My warmest thanks go to Drs. Alistair Webb and Hartmut Derendorf, who served
as my mentors and guided me through my doctoral studies at the University ofFlorida. I
am thankful to Dr. Webb for his guidance, ideas, and advices since he assumed the
mentorship. Dr. Derendorfshowed a lot ofpatience, understanding and support for me,
and together with his students and postdoctoral fellows, helped solve many problems,
My
seemingly "dead ends", and other frustrations ofan inexperienced graduate student.
deep appreciation goes to Dr. Stephen Sundlof, who was my initial mentor, for his
enthusiasm, continuous support and involvement in the project. I would also like to thank
my committee members, Drs. Thomas Vickroy and Ronald Gronwall, for their many
suggestions and help in pursuing my doctoral degree. Dr. Maureen Davidson, who
supervised the rodent study, was very enthusiastic and supportive, for which I am very
thankful. It was both a pleasure and great experience to work with her and her group.
My very special thanks are extended to Ms. Kandi Crosier, who was always there
when I needed help, advice, orjust friendship in the seclusion ofour old lab. Her
understanding and help in many ways meant a whole lot to me. Mr. Jay King helped save
my days on numerous occasions, when I got lost in computer problems, which he was
always able and willing to solve. I am taking this opportunity to express my sincere
in
appreciation to Mr. Clifford Hall, for all his time and effort, both in the laboratory and in
working with animals. Cliffvolunteered many times for the middle-of-the-night samplings,
for which I was always very grateful. My very special thanks go to Ms. Sara Maria Becht,
my
who helped me learn HPLC, and whose friendship, ideas, and help really influenced
graduate studies at UF. I want to express my gratitude to Mr. Brian Lapham, for his
enthusiasm and help. We shared many laughs and discussions, but he will be mostly
remembered for his earnest, round-the-clock involvement in the sheep study.
Ms. Sally O'Connell, Dr. Philip C. Kosch and Dr. Charles Courtney deserve warm
thanks for their kindnes and concern for graduate students. My special thanks are
extended to Dr. Michael Fields for organization ofthe cattle experiments. His involvement
and support in providing help was greatly appreciated.
I wish to thank people in "Pliva", a pharmaceutical company fromZagreb, Croatia,
where I hold a position in their research center. They have been supportive and
understanding ever since I started receiving their scholarship as a veterinary student.
My sincere appreciation and warmest thanks go to my parents-in-law, and
especially to my dear parents, Durdica and Janko Moric, fortheir help and support.
Without my parents' endless love, understanding, and encouragement, this would have
been impossible. And, as they have always been very good and loving parents to me and
my brother, they are now as loving and caring grandparents, which helped me
my
tremendously in finishing studies.
Lastly, I would like to express my deepest thanks to my husband Tomislav, for
everything that he has done for me. This dissertation is his success as much as it is mine,
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for all his help, support, optimism, and most ofall, for his believing in me. Our sons,
Marko and Lovro, gave me the strength and persistence to finish my studies, through their
sweet laughs, hugs and kisses, for which I am endlessly grateful and happy.
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS
in
LIST OF TABLES ix
LIST OF FIGURES x
ABSTRACT
xi
CHAPTERS
INTRODUCTION
1. 1
2. REVIEW OF LITERATURE 7
2.1. Introduction 7
2.2. Tilmicosin 8
2.2.1. Physicochemical Properties 8
2.2.2. Pharmacology 9
2.2.2.1. Mechanism ofaction 11
2.2.2.2. Antibacterial activity 12
2.2.3. Pharmacokinetics 12
2.2.3.1. Absorption 13
2.2.3.2. Distribution 14
2.2.3.3. Biotransformation 16
2.2.3.4. Excretion 17
2.2.4. Therapeutic Uses 18
2.2.5. Dosage and Administration 19
2.3. Toxicity 19
2.3.1. Cardiovascular Toxicity ofTilmicosin 21
A
2.3.2. Cardiovascular Toxicity ofOther Antibiotics-- Review 23
2.3.2.1. Macrolides 25
2.3.2.2. Other antibiotics 28
2.3.2.3. Adverse effects in concurrent drug therapy 30
2.4. Factors Affecting Tissue Distribution ofDrugs 31
2.4.1. Physicochemical Properties ofDrugs 35
VI
2.4.2. pH as a Factor in Drug Distribution 36
2.4.3. Effects ofDisease on Drug Distribution 37
2.4.3.1. Effect ofdisease on the pharmacokinetics ofmacrolides 39
2.4.3.2. Effect ofdisease on the pharmacokinetics ofother antibiotics 41
2.5. Mycoplasmosis 45
2.5.1. Introduction 45
2.5.2. Experimental Respiratory Mycoplasmosis in Rodents 46
2.5.3. Clinical Signs and Virulence 46
2.5.4. Pathogenic Mechanisms 48
2.5.5. Antimicrobial Susceptibility ofMycoplasma 48
MATERIALS AND METHODS 50
3.
3.1. Determination ofTilmicosin Concentrations 50
3.1.1. Chemicals and Reagents 50
3.1.2. Tissue Preparation 51
3.1.2.1 Extraction oftilmicosin from serum 51
3.1.2.2. Extraction oftilmicosin from lung tissue 52
3.1.3. High Pressure Liquid Chromatography 53
3.1.3.1. Chromatographic conditions 53
3.1.3.2. Calculation ofHPLC results 56
3.1.3.3. HPLC method validation study 56
3.1.3.4. Quality control 63
3.1.3.5. Estimation ofpharmacokinetic parameters 63
3.2. Cardiopulmonary Monitoring in Sheep 68
3.3. Animal Handling 69
3.3.1 Experimental Animals 69
3.3.1.1. Sheep 69
3.3.1.2. Cattle 69
3.3.1.3. Rats 70
3.3.2. ExperimentalMycoplasmapulmonis Infection in the Rodent Study 70
3.3.3. Drug Administration 71
3.3.3.1. Tilmicosin administration in sheep and cattle 71
3.3.3.2. Tilmicosin administration in rats 71
EXPERIMENTAL DESIGN 72
4.
4.1. Introduction 72
4.1.1. Comparative Pharmacokinetics ofTilmicosin in Sheep and Cattle 73
4.1.1.1. Sample collection 76
4.1.1.2. Statistical analysis 78
4.1.2. Effect ofRespiratory Disease on Tilmicosin Pharmacokinetic in Rats 78
4.1.2.1. Sample collection 79
4.1.2.2. Statistical analysis 80
5. RESULTS 81
5.1. Serum Pharmacokinetics ofTilmicosin in Sheep and Cattle 81
5.1.1. Results ofthe Non-Compartmental Pharmacokinetic Analysis 81
vii
5.1.2. Results ofthe compartmental pharmacokinetic analysis and modeling 89
5.2. Cardiopulmonary Effects ofTilmicosin in Sheep 92
5.2.1. Blood Pressure 98
5.2.2. Heart Rate 100
5.2.3. Respiratory Rate 100
5.3. Effects ofTilmicosin on Blood Chemistry and Hematology in Sheep 103
5.4. Lung Tissue Distribution ofTilmicosin in Infected and Non-Infected Rats 104
5.4.1. pH Measurements ofthe Lung and Muscle Tissue 104
5.4.2. Tilmicosin Concentration in Serum and Lung Tissue 106
6. DISCUSSION 112
6.1. Pharmacokinetics ofTilmicosin in Sheep and Cattle 112
6.2. Cardiovascular Effects ofTilmicosin in Sheep 120
6.3. Lung Tissue Distribution ofTilmicosin in Rats 125
7. SUMMARY AND CONCLUSIONS 136
LIST OF REFERENCES 140
APPENDICES
A LIST OF SUPPLIERS FROM CHAPTER 3 155
B PHARMACOKINETIC EQUATIONS AS WRITTEN FOR "EXCEL" 156
C ANOVA TABLES FROM THE STATISTICAL ANALYSES OF THE
CARDIOVASCULAR DATA ON THE EFFECT OF TILMICOSIN IN
SHEEP 157
D BLOOD CHEMISTRY RESULTS IN SHEEP AFTER TILMICOSIN (OR
PLACEBO) TREATMENT 160
E HEMATOLOGY RESULTS IN SHEEP AFTER TILMICOSIN (OR
PLACEBO) TREATMENT 162
F ANOVA TABLES FROM THE STATISTICAL ANALYSES OF THE
EFFECT OF MYCOPLASMA INFECTION ON THE LUNG AND
MUSCLE TISSUE PH IN RATS 164
G INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)
APPROVAL
165
BIOGRAPHICAL SKETCH 167
vui
LIST OF TABLES
Table page
3-1: Validation results I 60
3-2: Validation results II 62
3-3: Summary ofthe quality control results 64
3-4: Equations used to calculate noncompartmental pharmacokinetic parameters 66
4-1: Schedule for data collection during the sheep experiments 74
5-1: Tilmicosin concentration over time inthe sheep serum 82
5-2: Tilmicosin concentration overtime in the cattle serum 83
5-3: Calculated pharmacokinetic parameters for tilmicosin in sheep (n = 10) 84
5-4: Calculated pharmacokinetic parameters for tilmicosin in cattle (n = 10) 85
5-5: Comparison ofthe half-life data (arithmetic vs. harmonic mean) for tilmicosin in
sheep and cattle 87
5-6: Comparison ofthe pharmacokinetic parameters for tilmicosin in sheep and cattle...88
5-7: The results ofthe non-compartmental pharmacokinetic analysis on tilmicosin serum
concentrations in rats 109
IX
1
LIST OF FIGURES
Figure page
2.1. Structural formula oftilmicosin with its two isomers 10
3-1: Examples ofthe UPLC chromatograms 55
3-2: Example ofan HPLC calibration curve 58
5-1: Tilmicosin concentrations over time in the sheep serum 90
5-2: Tilmicosin concentrations over time in the cattle serum 91
5-3a: Least squares fitting for serum tilmicosin concentrations in 5 sheep 93
5-3b: Least squares fitting for serum tilmicosin concentrations in 5 sheep 94
5-4a: Least squares fitting for serum tilmicosin concentrations in 5 cattle 95
5-4b: Least squares fitting for serum tilmicosin concentrations in 5 cattle 96
5-5: Summarized least squares fitting for serum tilmicosin concentrations in sheep and
97
cattle
5-6: The effect oftilmicosin on the systolic, diastolic and mean blood pressure in sheep.99
5-7: The effect oftilmicosin on the heart rate in sheep 101
5-8: The effect oftilmicosin on the respiratory rate in sheep 102
5-9: pH measurements in the lung and muscle tissue ofrats 105
5-10: Concentrations oftilmicosin in the serum and lung tissue ofrats 107
5-11: Comparison ofserum and lung tissue concentrations oftilmicosin for the non-
infected (A) and infected (B) rats 108
5-12: Lung:serum ratio over time for the infected and non-infected rats 11
