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Pharmacokinetic and pharmacodynamic properties of tilmicosin in sheep, cattle, and rats

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Title:
Pharmacokinetic and pharmacodynamic properties of tilmicosin in sheep, cattle, and rats
Creator:
Modric, Sanja, 1966-
Publication Date:
Language:
English
Physical Description:
xii, 166 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibiotics ( jstor )
Cattle ( jstor )
Dosage ( jstor )
Infections ( jstor )
Lungs ( jstor )
Macrolides ( jstor )
Pharmacokinetics ( jstor )
Rats ( jstor )
Sheep ( jstor )
Subcutaneous injections ( jstor )
Anti-Bacterial Agents -- pharmacokinetics ( mesh )
Anti-Bacterial Agents -- pharmacology ( mesh )
Anti-Bacterial Agents -- toxicity ( mesh )
Cardiovascular System -- drug effects ( mesh )
Cattle ( mesh )
Department of Physiological Sciences thesis Ph.D ( mesh )
Dissertations, Academic -- College of Veterinary Medicine -- Department of Physiological Sciences -- UF ( mesh )
Lung -- drug effects ( mesh )
Macrolides -- pharmacokinetics ( mesh )
Macrolides -- pharmacology ( mesh )
Macrolides -- toxicity ( mesh )
Mycoplasma Infections -- drug therapy ( mesh )
Rats ( mesh )
Research ( mesh )
Sheep ( mesh )
Tylosin -- analogs & derivatives ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Bibliography: leaves 139-153.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Sanja Modric.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
002287023 ( ALEPH )
48663387 ( OCLC )
ALP0174 ( NOTIS )

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PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF
TILMICOSIN IN SHEEP, CATTLE, AND RATS
















BY


SANJA MODRIC


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 of Florida. I

am thankful to Dr. Webb for his guidance, ideas, and advices since he assumed the

mentorship. Dr. Derendorf showed a lot of patience, understanding and support for me,

and together with his students and postdoctoral fellows, helped solve many problems,

seemingly "dead ends", and other frustrations of an inexperienced graduate student. My

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, or just friendship in the seclusion of our 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








appreciation to Mr. Clifford Hall, for all his time and effort, both in the laboratory and in

working with animals. Cliff volunteered 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,

who helped me learn HPLC, and whose friendship, ideas, and help really influenced my

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 of the cattle experiments. His involvement

and support in providing help was greatly appreciated.

I wish to thank people in "Pliva", a pharmaceutical company from Zagreb, 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 Morid, for their 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

tremendously in finishing my 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,








for all his help, support, optimism, and most of all, 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 ............................................................................................. iii

L IST O F T A B L E S ................................................... ................................................ ix

LIST O F FIG U R E S.................................................................................. ..................x...

A B STR A C T .......... ..... .................... ................................ .............................. xi

CHAPTERS

1. IN TR O D U C T IO N.................................................................................. ................ 1

2. REVIEW OF LITERATURE ................................... 7
2.1. Introduction ................................................................... ........... ......... ................ 7
2 .2 T ilm ico sin ...................................................................................... ......... ............ 8
2.2.1. Physicochemical Properties................................................................... 8
2.2.2. Pharm acology..................................................................... .................. 9
2.2.2.1. Mechanism of action....................................................... 11
2.2.2.2. Antibacterial activity ................. .......................................... 12
2.2.3. Pharm acokinetics.............................................................. .................. 12
2.2.3. 1. A bsorption.............................................................. ................... 13
2.2 .3.2 D distribution ............................................................... ................ 14
2.2.3.3. Biotransform ation.................................................... ..................... 16
2.2.3.4 E xcretion ....................................... ........................ ................ 17
2.2.4. Therapeutic U ses.............................................................. .................. 18
2.2.5. Dosage and Administration........................................................ 19
2.3. Toxicity ....................................... .... .................................. 19
2.3.1. Cardiovascular Toxicity of Tilmicosin................................................. 21
2.3.2. Cardiovascular Toxicity of Other Antibiotics--A Review ....................... 23
2.3.2 .1. M acrolides.............................................................. .................. 25
2.3.2.2. O their antibiotics ....................................... .... ........ .................. 28
2.3.2.3. Adverse effects in concurrent drug therapy .................. ................. 30
2.4. Factors Affecting Tissue Distribution of Drugs............................................ 31
2.4.1. Physicochemical Properties of Drugs................................................... 35








2.4.2. pH as a Factor in Drug Distribution.................................................... 36
2.4.3. Effects of Disease on Drug Distribution.............................................. 37
2.4.3.1. Effect of disease on the pharmacokinetics of macrolides................ 39
2.4.3.2. Effect of disease on the pharmacokinetics of other antibiotics........... 41
2.5. M ycoplasm osis............................................ .......................... ................... 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 of Mycoplasma ........................................ 48

3. MATERIALS AND METHODS......................................................................... 50
3.1. Determination of Tilmicosin Concentrations................................................ 50
3.1.1. Chemicals and Reagents ........ ............. .............................................. 50
3.1.2. Tissue Preparation....... ................................ ................................ 51
3.1.2.1 Extraction of tilmicosin from serum ............................................ 51
3.1.2.2. Extraction of tilmicosin from lung tissue .................................... 52
3.1.3. High Pressure Liquid Chromatography ............................................... 53
3.1.3.1. Chromatographic conditions ...................................................... 53
3.1.3.2. Calculation of HPLC results....................................................... 56
3.1.3.3. HPLC method validation study .................................................. 56
3.1.3.4. Q quality control......................................................... .................. 63
3.1.3.5. Estimation of pharmacokinetic parameters ...................................... 63
3.2. Cardiopulmonary Monitoring in Sheep ........................................................ 68
3.3. A nim al H handling ............................................................................................... 69
3.3.1 Experimental Animals .......................................................................... 69
3.3.1.1. Sheep ...... .................. ................................. 69
3.3 .1.2 C attle...................................................................... ................... 69
3 .3 .1.3 R ats ........................................................................... .................... 7 0
3.3.2. Experimental Mycoplasma pulmonis Infection in the Rodent Study .......... 70
3.3.3. D rug A dm inistration........................................ ............... ................. 71
3.3.3.1. Tilmicosin administration in sheep and cattle................................. 71
3.3.3.2. Tilmicosin administration in rats................................................. 71

4. EXPERIMENTAL DESIGN............................................................................... 72
4 .1. Introduction ......................................................................... ...................... 72
4.1.1. Comparative Pharmacokinetics of Tilmicosin in Sheep and Cattle............. 73
4. 1.1. 1. Sam ple collection..................................................... .................. 76
4.1.1.2. Statistical analysis.................................................. .................... 78
4.1.2. Effect of Respiratory Disease on Tilmicosin Pharmacokinetic in Rats........ 78
4.1.2.1. Sample collection ....................................................................... 79
4.1.2.2. Statistical analysis....................................... 80

5 R E SU L T S .......................... ................ .. .......... ....... ............... ........ ........................ 8 1
5.1. Serum Pharmacokinetics of Tilmicosin in Sheep and Cattle............................. 81
5.1.1. Results of the Non-Compartmental Pharmacokinetic Analysis................ 81








5.1.2. Results of the compartmental pharmacokinetic analysis and modeling ....... 89
5.2. Cardiopulmonary Effects of Tilmicosin in Sheep ................................................ 92
5.2.1. B lood P pressure ................................................................. .................. 98
5.2.2. H eart R ate.................................. ................................................ 100
5.2.3. R respiratory R ate.................................................................................. 100
5.3. Effects of Tilmicosin on Blood Chemistry and Hematology in Sheep ............ 103
5.4. Lung Tissue Distribution of Tilmicosin in Infected and Non-Infected Rats....... 104
5.4.1. pH Measurements of the Lung and Muscle Tissue ............................... 104
5.4.2. Tilmicosin Concentration in Serum and Lung Tissue............................ 106

6. D ISC U SSIO N .................................................... .................. ........... .................. 112
6.1. Pharmacokinetics of Tilmicosin in Sheep and Cattle .................................. 112
6.2. Cardiovascular Effects of Tilmicosin in Sheep ........................................... 120
6.3. Lung Tissue Distribution of Tilmicosin 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
SH E E P .................... ................................................. 157

D BLOOD CHEMISTRY RESULTS IN SHEEP AFTER TILMICOSIN (OR
PLACEBO) TREATM ENT....... ........... ........... ...................... 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)
A P P R O V A L ................................................... ................................................. 165

BIOGRAPHICAL SKETCH .................................. ......................................... 167














LIST OF TABLES


Table page

3-1: V alidation results I ......................................................................... ................. 60
3-2: Validation results II ........................ .................... 62
3-3: Summary of the 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 in the sheep serum...................................... 82
5-2: Tilmicosin concentration over time 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 of the half-life data (arithmetic vs. harmonic mean) for tilmicosin in
sheep and cattle............................................................................... ................. 87
5-6: Comparison of the pharmacokinetic parameters for tilmicosin in sheep and cattle... 88
5-7: The results of the non-compartmental pharmacokinetic analysis on tilmicosin serum
concentrations in rats ............................. .................................................. 109














LIST OF FIGURES


Figure page

2.1. Structural formula of tilmicosin with its two isomers......................... 10
3-1: Examples of the HPLC chromatograms ........................................................... 55
3-2: Example of an 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
cattle ..................... ......... ........ ..... ............ ..... ..... ............... .................. 9 7
5-6: The effect of tilmicosin on the systolic, diastolic and mean blood pressure in sheep.99
5-7: The effect of tilmicosin on the heart rate in sheep........................................... 101
5-8: The effect of tilmicosin on the respiratory rate in sheep................................... 102
5-9: pH measurements in the lung and muscle tissue of rats......................................... 105
5-10: Concentrations of tilmicosin in the serum and lung tissue of rats ..................... 107
5-11: Comparison of serum and lung tissue concentrations of tilmicosin for the non-
infected (A) and infected (B) rats ................................................... ................ 108
5-12: Lung:serum ratio over time for the infected and non-infected rats ................... 111














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


PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF
TILMICOSIN IN SHEEP, CATTLE, AND RATS

By

Sanja Modrid

May 1997

Chairman: Dr. Alistair I. Webb
Major Department: Veterinary Medical Sciences

Tilmicosin is a relatively new long-acting macrolide antibiotic approved for the

treatment of bovine respiratory disease in cattle in the USA. A high degree of similarity is

expected among domestic ruminants in the distribution and elimination of drugs, such as

tilmicosin, that are not highly metabolized. The pharmacokinetic properties of tilmicosin in

serum after subcutaneous injection were compared between cattle and sheep. For both

species, tilmicosin concentration in serum followed a pharmacokinetic two-compartment

body model. There were no significant differences in the elimination rates, maximum

serum concentrations, half-lives, areas under the curve, areas under the first--moment

curve, and mean residence times. The volume of distribution and clearance, when

normalized by body weight, were also similar. The only significantly different non-








compartmental parameter was the time when the maximum serum concentration was

reached (t"), with sheep having the t,,m of 3.9 hours, compared to 0.5 hours in cattle.

Although macrolides in general are considered to be one of the safest anti-infective drugs,

adverse cardiovascular effects of several macrolides have been reported. Tilmicosin has

been found to have a potential for causing cardiopulmonary toxicity, with a manifestation of

positive chronotropic and negative inotropic effects. The cardiopulmonary effects of

tilmicosin (heart rate, ECG, blood pressure, and respiratory rate) were monitored in

healthy adult sheep after receiving a single subcutaneous injection of tilmicosin at the dose

of 10 mg/kg. No significant changes were found in the cardiopulmonary parameters

monitored for six hours after tilmicosin administration in sheep. In a study on tilmicosin

tissue distribution, rats were used as a model for studying the effects of a chronic

respiratory disease on tilmicosin pharmacokinetics. It was found that tilmicosin

consistently had higher lung tissue concentrations in the infected than in the non-infected

animals. There was no correlation between the local pH of the lung tissue and

inflammation resulting from the infection with Mycoplasma pulmonis. In summary, the

present study provides evidence that tilmicosin can be safely used in sheep, with no

adverse cardiopulmonary effects. Good penetration of the drug into infected pulmonary

tissue implies a possible therapeutic advantage of tilmicosin in treating lung infections.














CHAPTER 1
INTRODUCTION

Respiratory diseases in cattle, and especially the bovine respiratory disease (BRD)

complex, cause important economic losses in the beef industry. Pasteurella haemolytica is

recognized as the major pathogen in the etiology of the BRD, although a combination of

environmental and husbandry factors are believed to play a role in the full development of

the disease (Jordan et al., 1993). Although not as prevalent as in cattle, respiratory

diseases still have a marked influence on the sheep industry. Similar pathogens to those in

BRD have been recognized in chronic respiratory disease in sheep, the most important of

these being P. haemolytica and Mycoplasma spp. (Fraser, 1986).

Tilmicosin, a relatively new antibiotic from the macrolide class, has been found to

have good in vitro activity against many gram positive bacteria, as well as some gram-

negative organisms. It has excellent activity against Pasteurella, Mycoplasma and

Actinobacillus spp. (Barragry, 1994; Moore et al., 1996a; Musser et al., 1996; Ose, 1987)

all of which have been recognized as the pathogens involved in the BRD complex. Besides

its wide spectrum of antibacterial activity and proven efficacy against the BRD, tilmicosin

has other features which justify its popularity in comparison with other veterinary

antibacterial products of similar activity. It has a prolonged duration of antibacterial

activity in vivo, which allows for effective treatment of most animals with a single low-








volume injection of the drug, thereby greatly reducing handling risks to already stressed

animals (Jordan et al., 1993).

Tilmicosin has been available in the United States since 1992, and is approved for

treatment of the BRD in beef cattle and non-lactating dairy cattle, associated with

Pasteurella and Mycoplasma species (Crosier, 1996). It has also recently been approved

for use in swine as a feed additive for the control of respiratory disease associated with

Actinobacillus pleuropneumoniae and P. multocida (Federal Register, 1996). Tilmicosin

has not been approved for use in sheep. Sheep are expected to demonstrate similar

pharmacokinetic properties for tilmicosin as have already been shown for cattle.

Therefore, because sheep are considered a minor species, the approval for tilmicosin will

be based on demonstrated pharmacokinetic similarities of the drug between the two

species.

Drug metabolism can vary markedly even between related species, but a high

degree of similarity is expected among domestic ruminants in the distribution and

elimination of drugs that are not highly metabolized (Short, 1994). Tilmicosin is eliminated

from the body for the most part unchanged, with approximately three quarters of total

excretion consisting of parent compound (Donoho et al., 1988; Giera and Peloso, 1988).

The first aim of this study was to compare the pharmacokinetic properties of tilmicosin in

cattle and sheep serum, with the hypothesis that there would be no major differences in

tilmicosin pharmacokinetics between the two species.

Macrolide antibiotics are considered to be among the safest anti-infective drugs in

clinical use, with severe adverse reactions being rare (Bryskier and Labro, 1994; Periti et








al., 1993). Jordan et al. (1993) reported on the clinical evidence of tilmicosin toxicity as

primarily a manifestation of the positive chronotropic and negative inotropic

cardiovascular effects. Although the adverse effects of several macrolides on the

cardiovascular system have been reported in literature (Freedman et al., 1987; Tamargo et

al., 1982; Wakabayashi and Yamada, 1972), they have always included doses much

greater than therapeutic, and/or the effects were seen in the subjects with already

compromised cardiac status or impaired renal function. Similarly, cardiovascular toxicity

of tilmicosin, as observed in cattle and dogs, as well as in in vitro studies on the cardiac

muscle tissue (Jordan et al., 1993), was a result of large doses of tilmicosin, given by

routes other than the labeled subcutaneous injection (Elanco Animal Health, 1994).

The second aim of the tilmicosin project was to study the cardiopulmonary effects

oftilmicosin in healthy adult sheep, after receiving a single subcutaneous injection of

tilmicosin at the labeled dose of 10 mg/kg body weight. Therefore, concurrently with the

pharmacokinetic study in sheep, the heart rate, ECG, blood pressure, and respiratory rate

were monitored, and compared between the sheep that received either tilmicosin treatment

or a placebo injection of the saline solution. It was hypothesized that there would be no

effect of a therapeutic dose of tilmicosin on any of the cardiopulmonary parameters tested.

The sheep were monitored for the first six hours after administration oftilmicosin or

saline, after which no adverse cardiopulmonary effects were considered likely to occur,

because of the decline in the serum drug concentrations.

In an extension of the pharmacokinetic study of tilmicosin, the reported

accumulation of tilmicosin in the lung tissue was investigated using the rat as a model. As








with other macrolide antibiotics, such as azithromycin (Bergogne-Berezin, 1995b) and

erythromycin (Foumet et al., 1989), tilmicosin exhibits good tissue penetration, reaching

much higher concentrations in the lung than in the serum (Brown et al., 1995; Ziv et al.,

1995). However, various drugs have been reported to respond differently to infection and

inflammation. Some drugs, such as azithromycin (Bergogne-Berezin, 1995b; Veber et al.,

1993), show better tissue penetration in infected than non-infected animals, while others,

such as erythromycin (Burrows, 1985) and ceftazidime (McColm et al., 1986), have

impaired penetration as a result of infection. Since drugs are intended for use in subjects

that are ill, it is necessary to know how the infection and/or inflammation might affect the

pharmacokinetics of a drug being used for treatment. Increasingly, clinical studies have

directed attention to this often neglected aspect of clinical pharmacokinetics (Gibaldi,

1991). In parallel with this current trend, regulatory agencies now require the

pharmacokinetics of a new drug to be studied in patient populations. Therefore, the aim of

the rodent study was to determine the effect of disease on tilmicosin pharmacokinetics by

comparing its tissue distribution between healthy rats and rats infected with Mycoplasma

pulmonis.

The pharmacokinetic profile of tilmicosin in rats was expected to be similar to the

one previously described for cattle and sheep (Patel et al., 1992; Thomson and Peloso,

1989). In mice, tilmicosin concentrations in lung homogenates were ten-fold higher than

concurrent plasma concentrations two hours after drug injection (Brown et al., 1995),

which is in agreement with findings from cattle and sheep. Based on these data, it was

hypothesized that the pharmacokinetics in rats would be similar to that found in ruminants.








Mycoplasma pulmonis was chosen as an infectious agent in the rodent study because (a) it

can cause a chronic respiratory disease in rats (somewhat similar to the BRD), and (b)

tilmicosin is effective against various Mycoplasma spp (Ose, 1987). The recognized

morphologic similarities and similar natural histories of chronic bronchitis, bronchiectasis,

and emphysema and of M. pulmonis respiratory disease in rats and mice make the latter a

particularly useful model for study of the pathogenesis of chronic pulmonary inflammation

(Cassell, 1982).

While testing the susceptibility of M pulmonis to tilmicosin was not a part of this

project, some preliminary results indicate that tilmicosin may be effective against M

pulmonis in rats (Davidson, Personal Communication). The aim of the rodent study was to

compare the lung tissue distribution of tilmicosin between the infected and non-infected

rats. It is commonly believed that inflammation has profound effects on the tissue

distribution of antibiotics, in some cases resulting in raising, and in other cases, lowering

of the drug concentrations (Schentag and Gengo, 1982; Wise, 1986).

Inflammation can increase capillary permeability or the rate of blood flow thereby

permitting some antibiotics to enter the sites that are usually impenetrable. However,

blood flow to a local area of infection may be decreased, or energy-dependent transport

processes can be destroyed or altered by inflammation. The hypothesis of the rodent study

was that the infected rats would have higher tilmicosin levels in the lung when compared

to the non-infected rats.

To further characterize lung tissue accumulation of tilmicosin, the pH of the lung

tissue was measured and compared between the infected and non-infected animals. The








hypothesis was that there would be a decrease in the pH of the infected lung due to the

inflammatory processes resulting from the infection. Furthermore, because of the pKa of

tilmicosin, its lipophilicity and slightly basic nature, it was hypothesized that it would be

trapped within the acidified lung tissue. Concurrently with the lung pH, the pH of the

muscle tissue was measured in order to determine whether the hypothesized change in the

pH as a result of infection would be reflected systemically. It is reasonable to expect some

decrease in systemic pH in chronic lung disease because of the hypoventilation and

accumulation of CO2.

In summary, the aims of the presented project were to determine:



1) the serum pharmacokinetic profile of tilmicosin in sheep and cattle, and to

compare the two species with regard to drug absorption, distribution and elimination

processes;



2) the potential of tilmicosin to cause cardiopulmonary toxicity in sheep when

given in the therapeutic doses, and;.



3) the effects of an experimentally induced respiratory disease on tissue

distribution of tilmicosin, in order to understand tilmicosin accumulation in the lung tissue.














CHAPTER 2
REVIEW OF LITERATURE


2.1. Introduction


Respiratory tract diseases cause important economic losses in feeder calves

(Picavet et al., 1991). Church and Radostits (1981) and Thomson (1980) reported that the

bovine respiratory disease complex (BRD) is the most economically important infectious

disease of calves in North American feedlots. Moreover, it is known to be the second most

common cause of death in female dairy calves from birth to weaning (Musser et al., 1996).

The etiology of the BRD is complex, but is considered to be the result of a combination of

environmental factors, husbandry factors, and infectious agents (Laven and Andrews,

1991). Of the infectious agents, Pasteurella haemolytica is recognized as the key

pathogen in the BRD complex (Jordan et al., 1993). Tilmicosin is a relatively new

antibiotic from the macrolide class, with excellent in vitro and in vivo activity against the

various microorganisms involved in the pathology of BRD (Debono et al., 1989; Jordan,

1993; Ose, 1987).

Although not as prevalent as in cattle, respiratory diseases still have a marked

influence on the sheep industry. The importance of sheep respiratory diseases depends on

their prevalence, which fluctuates seasonally. Other factors include the effect of

respiratory diseases on the sheep productivity, and their world-wide spread (Fraser, 1986).








Many bacteria, viruses, and mycoplasmas have now been recovered from the respiratory

tract of sheep, but not all have been shown to cause disease. The importance of some

bacteria and mycoplasmas has been established in pneumonia of sheep, most notable of

which are Pasteurella and Mycoplasma spp.


2.2. Tilmicosin


The first macrolide antibiotics were marketed over 40 years ago. These drugs

constitute a homogeneous class with very similar mechanisms of action and patterns of

bacterial resistance (Aubert, 1988). Interest in the macrolide antibiotics has recently

increased because of their specific antibacterial spectrum, activity against intracellular

bacteria, and their mild side-effects. The macrolides are considered the most broad

spectrum class of oral antibacterial agents available for the treatment of respiratory

infections (Butts, 1994).

Tilmicosin is a long-acting, semisynthetic macrolide antibiotic derived from tylosin

(Jordan et al, 1993), which is a product of the controlled fermentation of Streptomyces

fradiae (Picavet et al., 1991). Tylosin, although an effective macrolide for treatment of

BRD, is primarily effective against mycoplasmas (Barragry, 1994). Tilmicosin not only

retains this potent antimycoplasmal activity, but the efficacy against other microorganisms

has been improved through chemical modification of tylosin and desmycosin.


2.2.1. Physicochemical Properties

Tilmicosin shares a common structure with all antibiotics from the macrolide

family (of which the most widely used and clinically important one is erythromycin) a








many-membered macrocyclic lactone ring to which one or more deoxy sugars are attached

(Sande & Mandell, 1985).

Tilmicosin, 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (Debono et al.,

1989; Ose, 1987), has been prepared by chemical modification ofdesmycosin, a

microbiologically active degradation product of tylosin (Hamill et al., 1961). Desmycosin

is readily produced from tylosin by mild acid hydrolysis to remove the terminal sugar

mycarose (Ose, 1987). Tilmicosin is then synthesized from desmycosin by reductive

amination of the C-20 aldehyde group with a mixture of cis and trans-3,5-

dimethylpiperidin (Figure 2-1.; Ose, 1987). The commercially available tilmicosin is a

mixture of 85% cis and 15% trans isomers (Debono et al., 1989).

Tilmicosin is a white crystalline powder with the molecular formula C4HsoN2013. It

has a molecular weight of 869.15 and pKa of 7.4, and 8.5 (Debono et al., 1989). Water

solubility of tilmicosin is very dependent on temperature and pH; at pH 9, the solubilities

are 7.7 mg/ml at 250C, and 72.5 mg/ml at 50C. At pH 7.0 and 250C, the solubility is 566

mg/ml (Walker, 1993). Its solubility in alcohol and most organic solvents is >5000 mg/1.


2.2.2. Pharmacology

Since 1952, when the first macrolide antibiotic, erythromycin, was developed,

there has been a search for structurally related compounds with a wider spectrum of

activity and better pharmacokinetic and safety profiles (Nahata, 1996). Modifications of

the macrocyclic lactone ring structure and of the substituent groups have resulted in

compounds with various antibacterial activities and pharmacokinetic properties. Macrolide

























CH3


OCH3


OCH3


CH3


R=-N

CIS cH3


0
CH3

R

CH3 HO N(CH3)2

0 H


OH CH3






R=- N

TRANS CH3


Figure 2-1. Structural formula of tilmicosin with its two isomers








antibiotics inhibit bacterial protein synthesis both in vivo and in vitro with different

potencies. As a group, they are generally bacteriostatic, although some of them may be

bactericidal at high concentrations (Brisson-Noel et al., 1988). Their main use has been as

second-line antibiotics against gram-positive bacteria and mycoplasmas. They may be of

particular value in conditions such as pneumonia and mastitis because of their tendency to

achieve high tissue concentrations (Barragry, 1994).


2.2.2.1. Mechanism of action

Macrolides inhibit protein synthesis by binding to the 50S ribosomal subunit of

sensitive microorganisms (Sande and Mandell, 1985), where they stimulate dissociation of

peptidyl-tRNA from ribosomes, probably during translocation (Brisson-Noel et al., 1988;

Menninger and Otto, 1982; Vannuffel and Cocito, 1996). The peptidyl transferase center

has been identified at the 50S surface, and the binding sites of inhibitors have been mapped

within this domain. Macrolides interfere with the formation of long polypeptides and cause

a premature detachment of incomplete peptide chains.

Microbial resistance to antibiotics develops mainly by inactivation of inhibitors and

modification of targets (mutations of ribosomal proteins or rRNA genes) (Vannuffel and

Cocito, 1996). Alterations of rRNA bases can induce resistance to a single inhibitor or to a

group of antibiotics. It is proposed that mutations and modifications of rRNA bases induce

conformational ribosomal changes that prevent antibiotics binding to the target (Vannuffel

and Cocito, 1996).








2.2.2.2. Antibacterial activity

Similar to other macrolides, the antibacterial activity of tilmicosin is principally

directed against gram-positive bacteria, as well as some gram-negative organisms, such as

Pasteurella (Moore et al., 1996a; Musser et al., 1996; Ose, 1987), Mycoplasma

(Barragry, 1994; Musser et al., 1996; Ose, 1987) and Actinobacillus species (Moore et

al., 1996a). It exhibits good in vitro antimicrobial activity against P. haemolytica and P.

multocida (MIC range of 0.78 to approximately 6.25 jig/ml) as well as Mycoplasma

species (MIC range of 0.39 to approximately 6.25 pg /ml) (Debono et al., 1989).

Ose (1987) reported that in vitro, 95 % of the P. haemolytica isolates were

inhibited by the tilmicosin concentration of 3.12 jg /ml. Growth of A. pleuropneumoniae,

Streptococcus suis, Actinomyces pyogenes and certain other bacteria was inhibited at

levels of 6.25 pg/ml or less. Although tilmicosin is mainly bacteriostatic, concentrations

equivalent to 4 times the MIC value were bactericidal for Pasteurella spp. (Ose, 1987).

Debono et al. (1989) studied the in vivo activity of subcutaneously administered

tilmicosin against experimental Streptococcus pyogenes infections in mice and reported the

effective dose (EDso) of 2.7 mg/kg.


2.2.3. Pharmacokinetics

Most of the data regarding tilmicosin pharmacokinetics are unpublished

proprietary material held by Eli Lilly. Only three reports dealing with the pharmacokinetic

patterns of tilmicosin in the mouse, rabbit, and dairy cattle have been published (Brown et

al., 1995; McKay et al, 1996; Ziv et al., 1995).








A prominent feature of tilmicosin is its prolonged duration of action, which allows,

in most cases, for an effective single-dose treatment. This single-dose, low-volume

treatment greatly reduces handling risks to already stressed animals and also reduces labor

costs (Jordan et al., 1993). This makes tilmicosin a well-liked antibiotic among

veterinarians for treatment of the common respiratory diseases in cattle. The popularity of

tilmicosin is supported by the fact that it is highly lipid-soluble and is found in lung

homogenate at concentrations several fold higher than concurrent plasma concentrations

(Brown et al., 1995).


2.2.3.1. Absorption

A single subcutaneous injection oftilmicosin at the dose of 10 mg/kg to neonatal

calves produces a peak mean tilmicosin level in plasma (Cmax) of 1.55 utg/ml following

administration in the dorsolateral chest region, and 1.6 ug/ml with the lateral neck location

(Thomson, 1989a). For both sites, the maximum concentration was reached at the one

hour sample (tmax = 1 h). When the same dose was given to feedlot Hereford steers and

heifers, the Cmna was 0.97 pg/ml following subcutaneous administration in the dorsolateral

chest region, and 0.71 jig/ml for the subcutaneous administration in the lateral neck

region. In both cases, the tmax was reached at 1 hour (Thomson, 1989b). In a third

pharmacokinetic study with cattle, the same dose was given to Holstein dairy cows, and

the average Cax for 5 cows was 0.13 pg/ml and tnmx 1.84 hours (Ziv et al., 1995). The

differences between those studies appear to be more related to the value of Cax, while the

trx value was more constant across the studies. Those differences may be attributed to








different sites of tilmicosin administration, as well as to different breeds and age groups

neonatess, subadult, and adult cattle) included in the studies.

When tilmicosin was given to adult sheep subcutaneously at a dosage of 10 mg/kg,

the Cmax was 0.44 ug/ml, which was reached 1 hour after administration of the drug

(Cochrane and Thomson, 1990). In another study, young sheep weighing 20 kg were

given a single subcutaneous dose oftilmicosin at the 20 mg/kg level, and Cm,a of 1.42

ug/ml and tm, of 3.8 hours were reported (Elsom et al., 1993). In either case, the site of

injection has not been reported.

Among other species in which tilmicosin was investigated, rabbits treated with a

subcutaneous dose of 25 mg/kg tilmicosin had a Cma, of 1.91 pg/ml and tm~, of 2 hours

(McKay et al., 1996). In mice receiving the dose of 10 mg/kg subcutaneously, the average

plasma concentration, measured at 2 hours after tilmicosin injection, was 0.51 ug/ml

(Brown et al., 1995).

Ziv et al. (1995) reported that binding of tilmicosin to serum proteins was in

correlation with drug concentration (Ziv et al., 1995). Higher concentrations resulted in

lower percentage of protein binding and vice versa (25% binding at concentration of 7.8

Ig/ml as opposed to 64% at 0.15 Ig/ml).


2.2.3.2. Distribution

After a single injection, tilmicosin is well distributed throughout the body, but

especially high levels are found in the lung, liver, and kidney. This has been reported for

various species: cattle (Giera et al., 1986; Giera and Peloso, 1988), sheep (Elsom et al.,

1993), mice (Brown et al., 1995), and rabbits (McKay et al., 1996).








In cattle administered 20 mg/kg tilmicosin subcutaneously, the concentration of

tilmicosin in liver 3 days after the dose, as detected from the mean 14C residue, was 36

pg/g, in kidney 39.2 ug/g, and in lung 14.3 pg/g (Giera et al., 1986). At the same time, the

concentrations in muscle and subcutaneous fat were only 1.96 and 2.03 pg/g, respectively.

When feedlot cattle were administered tilmicosin at a dose rate of 10 mg/kg (Thomson

and Peloso, 1989), the serum and lung tilmicosin concentrations 8 hours following

injection were 0.35 pg/ml and 5.50 pg/g, respectively, which represented a lung-to-serum

ratio of 16:1.

Following a single 20 mg/kg subcutaneous injection of tilmicosin in sheep, the

concentrations 3 days post administration, as detected from the mean 14C residues, were

21.09 pg/g in the kidney, 9.98 pg/g in the liver, and 5.11 pg/g in the lung (Elsom et al.,

1993). Muscle and fat concentrations were 1.26 and 1.24 pg/g, respectively. In a study

using 6 month-old sheep, which were administered tilmicosin subcutaneously at a dose

rate of 10 mg/kg, the serum and lung tilmicosin concentrations 8 hours following injection

were 1.18 ug/ml and 14.8 pg/g, respectively, which represented a lung-to-serum ratio of

13:1 (Patel et al., 1992). This ratio kept increasing until 72 hours after tilmicosin

administration, when it reached the maximum of 106:1, after which it started to decline.

The pharmacokinetic profile of tilmicosin in rats would be expected to be similar to

that described for cattle and sheep. A similar pharmacokinetic pattern has been observed in

mice (Brown et al., 1995), where tilmicosin concentrations in lung homogenates were ten-

fold higher than concurrent plasma concentrations two hours after drug injection. The

mice were injected subcutaneously with tilmicosin at a dose rate of 10 mg/kg, and two








hours after the dosing, the lung and serum concentrations were 5.5 pLg/g and 0.51 Ag/ml,

respectively.

In a rabbit study, animals were given a dose of 25 mg/kg tilmicosin

subcutaneously, and the serum Ca was 1.91 .tg/ml, which was reached 2 hours after the

dosing (McKay et al., 1996). Concurrent lung concentration reached a maximum of 14.43

tg/ml at the same time point, resulting in the lung: serum ratio of 8:1. At eight hours, the

ratio was 10:1.


2.2.3.3. Biotransformation

In the study on cattle injected with 10 mg/kg tilmicosin, urinary radioactivity was

approximately three quarters parent tilmicosin, while the fecal radioactivity was

approximately 22% parent tilmicosin, 22% metabolite T-1, and the remainder comprised

minor metabolites or non-extractable residue (Donoho et al., 1988). However, in vitro

degradation was demonstrated in control fortified feces, and so the true portions of fecal

tilmicosin are suggested to be higher than reported 22%. Parent tilmicosin was the

predominant radioactive component in liver, kidney, and the injection site.

The tilmicosin metabolites were further identified (Donoho, 1988). Metabolite T-1

was characterized as N-desmethyl tilmicosin (i.e. tilmicosin minus CH3), and is the only

major tilmicosin metabolite. Metabolite T-2 was later called an impurity from tilmicosin

technical material; and T-3 was determined to be a minor degradation product resulting

from the replacement of-N(CH3)2 on the mycaminose sugar with -OH.

Tilmicosin metabolites were also investigated in sheep (Elsom et al., 1993). Similar

to the cattle, the major component in urine was the unchanged tilmicosin, accounting for








approximately 75% of the total urinary radioactivity, and the majority of the rest of

radioactivity was identified as metabolite T-1. The major component of the radioactivity

detected in the liver, kidney, and urine was parent tilmicosin.


2.2.3.4. Excretion

Elimination pattern of 14C tilmicosin was studied in Hereford steers injected

subcutaneously with a dose of 30 mg/kg (Giera et al., 1987). Urine and fecal samples were

obtained daily for 15 days, and a total recovery of the dose was 72% in the feces and 19%

in urine. Radioactivity in both excreta decreased progressively between days I and 15.

When the therapeutic dose of 10 mg/kg was administered to steers, the total recovery of

the dose from excreta over 14 days was lower than when compared to the higher dose

from the previously mentioned study, with a mean of 73% (Donoho et al., 1988).

In sheep, the proportions of tilmicosin excreted in feces and urine after

subcutaneous injection are similar to those determined for cattle. In the lamb metabolism

study by Elsom et al. (1993) using the dose of 20 mg/kg of radioactive-labeled tilmicosin,

the mean total of radioactivity recovered within 7 days of dosing was about 85% (from

which 72% was from feces, and 13% from urine).

Parker and Walker (1993) analyzed serum samples of lambs weighing 16-18 kg,

that received a dose of 20 mg/kg tilmicosin. The authors reported a plasma half-life of

approximately 7 hours, for the initial elimination phase (between 6 and 24 hours after

dosing); and the terminal half-life (between 48 and 96 hours after dosing) of 41 hours.

Based on a semilogarithmic plot of serum depletion data, a two-compartment body model

was indicated for distribution and elimination of tilmicosin. In a study of six-month old








sheep injected with 10 mg/kg of tilmicosin, the serum half-lives of approximately 8 and 30

hours, for the initial and later elimination phases, respectively, were reported (Patel et al.,

1992).


2.2.4. Therapeutic Uses

Tilmicosin is active against many of the infectious pathogens commonly associated

with respiratory tract infections, including Pasteurella and Mycoplasma spp. (Musser,

1996). It is currently labeled for the treatment of BRD associated with P. haemolytica in

the beef cattle and non-lactating dairy cattle (Crosier, 1996; Darling, 1993). It was initially

evaluated for treatment of the BRD (shipping fever) of feedlot cattle (Schumann et al.,

1991), but was later found also to be effective as a treatment for naturally occurring

pneumonia (enzootic calf pneumonia) in calves during their first weeks of life (Laven and

Andrews, 1991; Ose and Tonkinson, 1988; Picavet et al., 1991). The prophylactic

administration of injectable tilmicosin for pneumonia in weaned beef calves was also

investigated, and it was suggested that tilmicosin given prophylactically had a beneficial

effect on the incidence and severity of pneumonia (Morck et al., 1993). Ziv et al. (1995)

determined that the MIC of tilmicosin for S. aureus isolates from the bovine udder was

0.78 tg/ml and they suggested its possible use as a systemic therapy for udder infections

in dry cows.

Tilmicosin has recently been widely investigated for its use in species other than

cattle. Moore et al. (1996a and 1996b) determined the effective dosage of tilmicosin

phosphate when fed to pigs for the control of pneumonia attributable to A.

pleuropneumoniae. The drug has recently been approved in the US for use in swine for








treatment of the respiratory disease associated with A. pleuropneumoniae and P.

multocida (Federal Register, December 1996; 21 CFR Parts 556 and 558). McKay et al.

(1996) investigated the efficacy of tilmicosin for treatment of pasteurellosis in rabbits and

reported a treatment success rate of 93%. Jordan and Horrocks (1996) determined the

minimum inhibitory concentration of tilmicosin and tylosin for Mycoplasma gallisepticum

and Mycoplasma synoviae and compared their efficacy in the control ofM gallisepticum

infection in broiler chicks.


2.2.5. Dosage and Administration

A single dose of 10 mg of tilmicosin per kilogram body weight for use in cattle is

listed in the label (Elanco Animal Health: Micotil 300 Injection, 1994). The FDA approved

route of administration is subcutaneous. It is recommended in the label that no more than

15 ml of MICOTIL be administered per injection site, due to the possibility of tissue

irritation and damage.

In view of tilmicosin's prolonged kinetic excretory pattern, it is contraindicated for

use in lactating animals. A withholding time of 56 days is recommended for meat

(Barragry, 1994).


2.3. Toxicity


Toxicity of tilmicosin has been studied after administration by a variety of routes to

various laboratory animals (Jordan et al., 1993). A median lethal dose (MLD) for fasted

Sprague-Dawley rats given by gavage was 825 mg/kg, but if the rats were fed before

tilmicosin administration, then the MLD increased to 2,250 mg/kg. The MLD for mice








was 100 mg/kg, and in both species signs of toxicity were non-specific, and no systemic

lesions were found at necropsy. Clinical signs in surviving animals were also non-specific,

and animals usually appeared normal by 48 hr after dosing.

Rabbits were used to test the potential effects of accidental dermal or ocular

exposure to MICOTIL and it was reported that MICOTIL produced very slight irritation

of the skin and a slight to moderate conjunctivitis (Jordan et al., 1993). A negative

response in a guinea pig sensitization study indicated that aqueous tilmicosin was not an

allergen, neither did it have an effect on the primary antibody response during an immune

testing in mice (Jordan et al., 1993).

Although tilmicosin is toxic for pigs when given intramuscularly or intravenously

(Barragry, 1994; Jordan, 1993), it has been proven to be safe when formulated as a feed

additive (Jordan et al., 1993; Moore et al., 1996a and b). Acute studies in horses and

goats indicate high risk of toxicity, including death, at intravenous doses of 10 mg/kg or

less, and at subcutaneously or intramuscularly administered doses above 10 mg/kg (Jordan

et al., 1993).

The results of tilmicosin administration to laboratory animals and domestic

livestock suggest that the cardiovascular system is the target of acute tilmicosin toxicity

(Jordan et al., 1993; McGuigan, 1994), with the primary effects of tachycardia and

decreased inotropy. However, the labeled dose of 10 mg/kg was well tolerated in both

cattle and sheep, the only adverse effect being a transient swelling at the site of injection.

Not only did the labeled dose never result in overt cardiovascular toxicity, but tilmicosin

had an adequately wide margin of safety for injection in both species; five-fold multiple








doses (50 mg/kg) repeated 3 times produced only mild toxicity, while 30 mg/kg/day

represented a no-adverse-effect dose.

McGuigan (1994) reported on human exposures to tilmicosin that had occurred

since the drug had been on the market. Thirty six cases of accidental human exposure to

tilmicosin were collected and analyzed. About 75% of the patients included in the study

were exposed to probably less than 1 ml of tilmicosin (less than 300 mg), with 72% of

exposures resulting from needle punctures. While local symptoms predominated, there

appeared to be no unexpected local tissue reaction, and there was no clinical evidence of

systemic toxicity in any of the reported cases.


2.3.1. Cardiovascular Toxicity of Tilmicosin

The toxicity dose response varies among the laboratory animal and domestic

livestock species, but in general, large doses of tilmicosin will manifest positive

chronotropic and negative inotropic effects (Jordan et al., 1993).

An intravenous administration of sublethal doses of tilmicosin (0.25 mg/kg) to

conscious dogs resulted in a pronounced sinus tachycardia, myocardial depression

(negative inotropy), and a reduction in arterial pulse pressure (Jordan et al., 1993). The

authors concluded that partial blockade of the tilmicosin-induced tachycardia by

propranolol was in part mediated thorough the stimulation of cardiac 3-receptors. In

anesthetized dogs, tilmicosin had no remarkable effect upon cardiovascular or

electrocardiographic parameters at the dose of 0.5 mg/kg (Jordan et al., 1993). However,

higher doses (1.0 and 5.0 mg/kg) produced prominent tachycardia, peripheral

vasoconstriction, increased pulmonary artery pressure, increased pulmonary artery wedge








pressure, and increased pulmonary vascular resistance, as well as decreased cardiac

output, stroke volume, stroke work index, femoral artery flow, and marked hypotension.

In in vitro smooth muscle tissue studies, tilmicosin did not elicit contractile activity

in the guinea pig ileum, rat uterus, or rat vas deferens, but there was a significant dose-

dependent decrease in the force of contractions of the spontaneously beating guinea pig

atria, as well as a significant increase in the rate of contractions (Jordan et al., 1993).

Tilmicosin markedly antagonized the contractile force response of the atria to

isoproterenol, as well as the contractile rate response to two other positive inotropic and

chronotropic agents (norepinephrine force/rate; calcium agonist BAY K 8644 -

force/rate), and the antagonism was not readily reversible.

Main et al. (1996) studied the cardiovascular effects of sublethal doses (0.25 to 5.0

mg/kg) of tilmicosin administered intravenously to conscious mixed-breed dogs. Left

ventricular function, systemic arterial blood pressure, and heart rate responses to

tilmicosin alone and in combination with propranolol or dobutamine were evaluated.

Cardiovascular variables were recorded, and the peak value of the first derivative of left

ventricular pressure (dp/dt(max)) was used as an index of left ventricular inotropic state.

Tilmicosin caused dose dependent decreases in (dp/dt(max)) and aortic pulse pressure.

Heart rate increased dose-dependently. Left ventricular end-diastolic pressure increased at

the 2.5 and 5.0 mg/kg dosages. Left ventricular systolic pressure was reduced

dose-dependently at the 2.5 and 5.0 mg/kg dosages. Treatment with propranolol

exacerbated the negative inotropic effect and the decrease in left ventricular systolic

pressure, but did not attenuate the tachycardia associated with tilmicosin treatment.








Dobutamine attenuated the changes in ventricular inotropic state in a dose-dependent

manner. Dobutamine infusion also restored left ventricular systolic pressure at dosages of

3 or 10 itg/min/kg. The authors concluded that toxic doses of tilmicosin may have a

negative inotropic effect in conscious dogs. Heart rate increased in a dose-dependent

manner and was not the result of beta 1-receptor stimulation, which is to the contrary of

findings from Jordan et al. (1993). Dobutamine reversed some, but not all, of the effects

caused by tilmicosin administration.

Main et al. (1996) suggested that the mechanism of tilmicosin cardiovascular

toxicity might be mediated through intracellular calcium. A rapid depletion of intracellular

calcium through interference with sarcolemmal calcium channels or some other mechanism

could result in negative inotropic effect of the drug. It has been reported for other

macrolides, such as josamycin and erythromycin, that they were capable of inhibiting

transmembrane calcium flux (Tamargo et al., 1982), a similar mechanism as involved in

the effect of calcium channel blockers on the heart (Boddeke et al., 1988).

Safety of tilmicosin was tested in feeder cattle administered subcutaneous doses of

10, 30, and 50 mg/kg of tilmicosin on three consecutive days (Jordan et al., 1993).

Clinically, no overt evidence of toxicity was observed. The only side-effect in the treated

animals was the presence of small foci of necrosis in the papillary muscle of the left

ventricle of the heart, observed in 2 of 8 cattle treated with the 50 mg/kg dose.


2.3.2. Cardiovascular Toxicity of Other Antibiotics--A Review

Clinical reports on the treatment of infection and efficacy of antibiotics

predominate in the literature, while studies on the pharmacological actions, and especially








adverse effects, have not been as numerous (Wakabayashi and Yamada, 1972). The main

reason for this may be that the toxicity of antibiotics is milder than that of most other

synthetic drugs, and there is seldom any recorded cardiovascular action at the usual

therapeutic dosages.

New drug candidates are frequently identified in highly specific assays designed to

target certain receptors or disease states. It therefore becomes increasingly important to

verify the selectivity of new compounds in broad pharmacological profiling, which also

identifies potential secondary activities that could result in functional adverse effects or

toxicity (Colbert et al., 1991). Antibiotics, in particular, are targeted to affect

microorganisms and are therefore expected to be relatively inert from a pharmacological

perspective. They are routinely used as prophylactic antiinfectious agents in surgery

(DiPiro et al., 1981; Stinner et al., 1995), but several hazards may arise by the wide-spread

use of these drugs, including neglect of their physiological effects on the patient and their

potential for modulating cardiovascular stability following complicated surgery (Stinner et

al., 1995). Although cardiovascular activity of antibiotics is not potent, it may play an

important role in patients with already compromised cardiac status or with impaired renal

function (Adams, 1975; Cohen et al., 1970; Freedman et al., 1987). The importance of

dosage considerations in assessment of drug toxicity was stressed by Adams (1975) in his

review of acute adverse effects of antibiotics. It was noted there that in most experimental

studies, doses of antibiotics much greater than the therapeutic doses have been used to

demonstrate the potential for adverse effects, while the vast majority of antibiotics never

showed any serious adverse effects at therapeutic concentrations.








Toxic injury is one of the many ways by which the functional integrity of the heart

may become compromised (Combs and Acosta, 1990). Any of the subcellular elements

may be the target of toxic injury, including various membranes and organelles.

Understanding the mechanisms underlying cardiotoxicity may lead to treatment of the

toxicity or to its prevention by designing new drugs that will not have secondary

cardiovascular affinity.

A review of selected clinical reports in humans and experimental studies in various

animal species suggests that, under certain circumstances, several commonly used

antibiotics may cause cardiovascular depression, respiratory difficulties, or alter the

metabolic breakdown of other drugs (Adams, 1975; Kuenneke et al., 1996). With regard

to the cardiovascular system, some groups of antibiotics (aminoglycosides, tetracyclines,

macrolides) have been shown to cause different adverse effects, most common of which is

cardiovascular depression, including hypotension bradycardia, myocardial depression, and

decreased cardiac output (Adams and Parker, 1982). These untoward responses are

believed to be due to the direct effects of antibiotics on specific physiologic functions,

rather than related to allergic reactions or cytotoxic lesions. Severe pathologic conditions,

over-dosage, or concomitant exposure to other potent drugs may predispose a patient to

these acute adverse effects.


2.3.2.1. Macrolides

Macrolides are an old and well established class of antimicrobial agents that

account for 10-15% of the worldwide oral antibiotic market (Periti et al., 1993) They are

considered to be one of the safest anti-infective drugs in clinical use, with severe adverse








reactions being rare (Bryskier and Labro, 1994; Periti et al., 1993). Newer products with

improved features have recently been discovered and developed, thereby maintaining or

significantly expanding the role of macrolides in the management of infection. In their

review of the adverse effects of macrolide antibacterials, Periti et al. (1993) divided the

macrolides' adverse effects into two groups: (1) expected and established effects; and (2)

unusual and rare or questionable effects. However, cardiovascular toxicity was not

described as either one of those, suggesting a very low occurrence of cardiovascular

toxicity associated with the macrolide therapy. In general, gastrointestinal reactions

represent the most frequent disturbance among macrolides, occurring in 15 to 20% of

patients on erythromycin and in 5% or fewer patients treated with some recently

developed macrolide derivatives. The hepatotoxic potential of macrolides, which rarely or

never form nitrosoalkanes, is low or negligible, depending on the antibiotic used. Transient

deafness and allergic reactions to macrolide antibacterials are highly unusual and have

definitely been shown to be more common following treatment with erythromycin than

with the recently developed 14-, 15- and 16-membered macrolides. In contrast to Periti et

al. (1993), other authors, including Adams (1975), Tamargo et al. (1982), and

Wakabayashi and Yamada (1972), have reported on cardiovascular toxicity of macrolides.

Tamargo et al. (1982) examined and compared the cardiovascular effects of

macrolides (josamycin, erythromycin, spiramycin and oleandomycin) and related

antibiotics (clindamycin and lincomycin) in spontaneously beating right atrial preparations

and in electrically driven left atrial preparations of rats. Josamycin and erythromycin

produced a dose-dependent decrease in heart rate and contractile force, while spiramycin,








oleandomycin, clindamycin, and lincomycin all produced notable changes only at the

highest concentrations tested. The negative inotropic effect ofjosamycin was not modified

by pretreating the atria with atropine or with a mixture of antagonists containing

phentolamine, practolol, diphenhydramine, cimetidine, methysergide and indomethacin. In

isolated right atria, josamycin did not block the positive inotropic and chronotropic

responses to isoprenaline but shifted the dose-response curve to Ca+ to the right.

Josamycin and erythromycin reduced in a dose-dependent manner the slow responses

induced in K-depolarized right atria by isoprenaline, but this effect was reversed by

increasing the Ca' concentration in the bathing media. Those findings demonstrated a

direct negative inotropic effect ofjosamycin and suggested that this effect could be

explained by inhibiting transmembrane Ca"+ influx into atrial cells. A similar mechanism of

cardiovascular toxicity has been suggested for tilmicosin by Main et al. (1996), as

described in chapter 2.3.1.

Another mechanism of cardiovascular toxicity of macrolides has been suggested by

Wakabayashi and Yamada (1972). Their study on the mechanism of the cardiovascular

depressor effect of several macrolide antibiotics revealed the absence of any influence

from bilateral cervical sympathectomy, bilateral cervical vagotomy, transsection of the

cervical spinal cord, or pretreatment with atropine. It was observed in the same study that

the histamine concentration in the blood following administration of the macrolide

antibiotics rose up to 30 times its pretreatment level, suggesting that macrolides might be

histamine releasers, and therefore, that histamine might induce depressor effect on the

blood pressure.








Treatment with erythromycin has been associated with ventricular

tachyarrhythmias and QT prolongation of the electrocardiogram (ECG) (Gueugniaud et

al., 1985; McComb et al., 1984), as well as ventricular repolarization (Freedman et al.,

1987). It had been suggested earlier that the mechanism for erythromycin-induced

arrhythmias might be myocardial potassium efflux, which is a predisposing factor for

ventricular tachycardia (Regan et al., 1969). However, Freedman et al. (1987) suggested

that the erythromycin effect was mediated by the sympathetic nervous system, because of

the abolition of the erythromycin effect by propranolol therapy and left cervicothoracic

sympathetic ganglionectomy.


2.3.2.2. Other antibiotics

Toxicity is a major limitation to the therapeutic usefulness of the aminoglycoside

group of antibiotics, most notable of which is ototoxicity and nephrotoxicity (Sande and

Mandel, 1985), but other adverse effects of the aminoglycosides, including depression of

cardiac function, have been also reported. Intravenous administration of streptomycin has

been found to cause a dose-dependent depression of cardiovascular functions, including a

decrease in cardiac output, mean arterial pressure, and contractile force (Cohen et al.,

1970). Similar depression was demonstrated after administration of kanamycin, as well as

some non-aminoglycoside antibiotics, such as tetracycline, vancomycin, erythromycin, and

colymycin (Cohen et al., 1970).

Adams et al. (1979) examined the acute cardiovascular activities of gentamicin,

tobramycin, sodium penicillin-G, and sodium cephalothin on dogs during experimental

circulatory shock induced by Escherichia coli endotoxin. Intravenous administration of








gentamicin or tobramycin resulted in transient cardiovascular depression, as reflected by

dose-related decreases of systemic blood pressure, cardiac output, and contractile force,

while the heart rate was affected little. Equally large doses of penicillin or cephalothin,

however, had no discernible circulatory effects in either control dogs or dogs subjected to

endotoxin shock.

Doxorubicin and its analogs are very important cancer chemotherapeutic agents

that can cause cardiotoxicity. The most important cardiotoxic mechanisms proposed for

doxorubicin include oxidative stress with its resultant damage to myocardial elements,

changes in calcium homeostasis, decreased ability to produce ATP, and systemic release of

cardiotoxic humoral mediators from tissue mast cells (Ringenberg et al., 1990). It is

suggested that doxorubicinol, one of the metabolites of doxorubicin, may be responsible

for the cardiotoxicity.

Pirlimycin adenylate is a clindamycin analog possessing antiarrhythmic activity

(Kopia et al., 1983). In the anesthetized dog, the sustained ventricular tachycardia

produced by ouabain intoxication was converted to a normal sinus rhythm with pirlimycin

adenylate. The drug failed, however, to decrease arrhythmia. It was concluded by the

authors that pirlimycin adenylate might be an interesting prototype antiarrhythmic agent

and further chemical modification of the drug molecule might increase the spectrum of

antiarrhythmic activity without altering the drug's toxicity.

Loracarbef is a carbacephem antibiotic targeted for use in the treatment of

infectious disease. A safety study was performed using high oral or intravenous doses, and

loracarbef was found to cause changes in cardiovascular system, including increase in








mean pressure, cardiac output, heart rate, and femoral flow. However, the doses used in

the safety study represented significant multiples of the therapeutic dose and therefore, it

was concluded that loracarbef has a very low potential to produce adverse effects at

therapeutic doses (Shetler et al., 1993).

The anthracycline antibiotics, adriamycin and daunomycin, are potent antitumor

agents, but their clinical use is limited by pronounced acute and chronic cardiotoxicity

(Lefrak et al., 1973; Singer et al., 1978). Pirarubicin, a newer anthracycline antibiotic, was

found to have a similar antitumor effect, but much lower cardiotoxicity (Matshushita et al.,

1985). Anthracyclines depress the blood pressure, acting directly on blood vessels, and

also have a positive inotropic effect, that may be mediated through the release of histamine

(Hirano et al., 1991).


2.3.2.3. Adverse effects in concurrent drug therapy

It has become evident that the effects of many drugs, when given concurrently, are

not predictable on the basis of knowledge of their individual effects. The pharmacological

responses from drug interactions may result from enhancement of the effects of one or the

other drug, the development of totally new effects that are not seen when either drug is

used alone, the inhibition of the effect of one drug by another, or no change whatever in

the net effect despite the fact that the kinetics and metabolism of one or both of the drugs

may be altered substantially (Murad and Gilman, 1995).

Macrolide antibiotics interact with many commonly used drugs by altering

metabolism due to complex induction and inhibition of cytochrome P-450 IIIA4

(CYP3A4) in the liver and enterocytes (Nahata, 1996; von Rosensteil and Adam, 1995).








2.4. Factors Affecting Tissue Distribution of Drugs


Although information concerning the concentration of drugs in the blood is of

great importance in studying their absorption and excretion, it may be of little or no value

in ascertaining the actual quantity of antibacterial substance at the site of infection

(Weinstein et al., 1951). Serum pharmacokinetics constitutes the first step in determining

the potential efficacy of a drug against pathogens, but most drugs are unevenly distributed

in tissues and concentrations achieved in a given tissue cannot be accurately predicted

from serum pharmacokinetics (Bergogne-Berezin, 1996). Therefore, tissue penetration

studies have become an important aspect of the assessment of antimicrobials.

Effective antimicrobial therapy requires adequate penetration of the agent from the

intravascular phase into the focus of infection. This entails passage of drug across the

capillary walls to the interstitial space, across barriers surrounding abscesses, and past the

lining of body cavities (Bergan, 1981). The kinetics of drug distribution can have profound

effects on the time course of drug action. Drugs that move relatively slowly in and out of

body "compartments", thereby confer pharmacokinetic characteristics of a

multicompartment system and elicit a considerably different time course of

pharmacological effect than do drugs which are distributed extremely rapidly in the body

(Gibaldi et al., 1971).

The general ability and velocity with which molecules pass through body systems is

regulated by various factors. With regard to the characteristics of a drug molecule,

important factors include molecular weight, molecular size, electrostatic charge, pKa,

protein binding, and lipid solubility. Factors affected by the target tissue include local pH,








pH gradient, concentration gradient, vascularity, and membrane permeability (Bergan,

1981; Mazzei et al., 1991). For the portion of drug not bound to protein, passage follows

Fick's law of diffusion:

dc/dt = Dq d2c/dx2

where dc/dt designates change in concentration over time; dc/dx is concentration gradient

over the distance x; D is diffusion coefficient expressed in m2/sec; and q is the area

expressed in metric units across which diffusion may occur.

Following injection, an antibiotic is absorbed and distributed by the blood and

undergoes one of two fates: (1) limited tissue distribution and rapid elimination from the

body; or (2) extensive tissue distribution and slow elimination from the body (Young et

al., 1995). The rate at which an antibiotic is eliminated and the characteristics by which it

accumulates in various body tissues have a significant impact on determining an

antibiotic's therapeutic utility. Although rapid absorption from the injection site is

necessary for effective treatment, the relationship of blood antibiotic levels and overall

therapeutic utility is highly variable. The antibiotic has to be present in target tissue in

sufficient quantities to inhibit microorganisms responsible for the infection.

Respiratory infections and other soft tissue infections are difficult to treat

effectively, partly because many antibacterial agents have poor tissue penetration (Butts,

1994). Furthermore, these respiratory infections are caused by a diverse variety of

organisms, many with differing mechanisms of resistance. One of the more difficult genre

of organisms to effectively treat is the facultative intracellular organism, such as

Mycoplasma spp.








The ability of antibiotics to penetrate tissues is best evaluated by use of the ratio of

the area under the concentration-time curve (AUC) for an antibiotic in the peripheral locus

to the AUC for serum (Bergan, 1981). There have been several principle approaches in

determining tissue concentrations of drugs, of which the most common one is still to use

whole tissue homogenates (Bergan, 1981; Brown et al., 1996; Ryan and Cars, 1980 and

1983). Data obtained using whole tissue or tissue homogenates infer a uniform distribution

of drug throughout the tissue mass, i.e. from the average of at least three distinct tissue

compartments: interstitial fluid, vascular system, and cellular mass. Because of that, the

tissue homogenate method, although widely used, is highly criticized for often resulting in

under- or over- estimations of the true levels of drugs. This is especially true for the drugs

that have relatively poor intracellular penetration, such as O3-lactam antibiotics (Brown et

al., 1995; Ryan and Cars, 1980), where the whole tissue levels are sometimes 5-10 times

lower than the drug concentration in the extracellular fluid. However, for antibiotics with a

good cellular penetration, drug levels can be accurately measured in the whole tissue

(Brown et al., 1995; Ryan and Cars, 1983).

Despite methodological and interpretive problems associated with studies of

antibiotic concentrations in tissues, it is important to confirm the presence of a drug in

significant concentrations in tissues and fluids at a desired site (Bergogne-Berezin, 1995a).

For antibiotics used in the treatment of respiratory infections, tissue distribution at sites of

potential infection in the respiratory tract has been related to clinical outcome.

Measurement of antibiotic concentrations achieved in lung parenchyma epithelial lining

fluid, bronchial mucosa or bronchial secretions has indicated significant concentrations for








beta-lactams and macrolides. Many respiratory infections are caused by obligate or

facultative intracellular pathogens, which may be eradicated as a result of intracellular

penetration and accumulation of macrolides. This has been shown in several models of

phagocytic cells, and of intracellular antibacterial activities. For bacteria multiplying in

alveolar macrophages, the high concentrations of the new macrolides that can be achieved

in extravascular and intracellular fluids should have clinical relevance.

Macrolide antibiotics in general are known to achieve very high intracellular

concentrations within phagocytic cells (Butts, 1994). These high intracellular

concentrations are important for the treatment of infections caused by intracellular

pathogens. In contrast, streptomycin, tetracyclines, and chloramphenicol are relatively

ineffective against intracellular microorganisms, which is believed to be due to their

inability to penetrate the phagocytic cell membrane (Butts, 1994; Shaffer et al, 1953). A

mechanism for the accumulation of macrolides in phagocytic cells has been proposed by

Renard et al. (1987). Macrolides are considered to be weak organic bases and, therefore,

are unprotonated to a certain degree in the extracellular fluids. After intracellular ingestion

into a more acidic environment, the macrolides become protonated, and thus concentrate

in the phagosome.

Although the specific intra-tissue location of tilmicosin has not been determined

explicitly, Brown et al. (1995) suggested that accumulation within pneumocytes or binding

to membranes and/or organelles must occur for concentrations to exceed concurrent

plasma concentrations. When lungs were perfused with drug-free fluid, measured

tilmicosin concentrations were similar to concentrations in the lungs that were not








perfused. That suggested that tilmicosin was present in lung in locations within the tissue

that are not in rapid equilibrium with plasma. It was, therefore, concluded by the authors

of that study that the whole tissue concentrations would represent true drug levels in the

tilmicosin case and that the whole tissue homogenization is a valid method for studying

tilmicosin lung distribution.


2.4.1. Physicochemical Properties of Drugs

The chemical and physical properties of a drug are of primary concern to the

formulator, because these characteristics can affect drug stability, absorption and

distribution characteristics, and ease of formulation (Young, 1995). Important

characteristics of a drug with regard to its absorption and distribution are molecular size,

solubility at the site of absorption, degree of ionization, and relative solubility of its

ionized and nonionized forms (Benet and Sheiner, 1985).

Drugs cross membranes either by passive processes or by mechanisms involving

the active participation of components of the membrane (Benet and Sheiner, 1985). In the

former, which is the dominant mechanism of drug passage across membranes, the drug

molecule usually penetrates by passive diffusion along a concentration gradient by virtue

of its solubility in the lipid bilayer. For nonelectrolytes, the concentration of the free drug

is the same on both sides of the membrane, after a steady-state is attained. For ionic

compounds, however, the steady-state concentrations will depend on differences in pH

across the membrane, which may influence the state of ionization of the molecule on each

side of the membrane, and on the electrochemical gradient for the ion.








Most drugs are weak acids or bases that are present in solution as both the

unionized and ionized species (Benet and Sheiner, 1985). The nonionized molecules are

usually lipid soluble and can diffuse across the cell membrane, while the ionized fraction is

usually unable to penetrate the lipid membrane because of its low lipid solubility. The

distribution of a weak electrolyte is usually determined by its pKa and the pH gradient

across the membrane.

Another factor that influences drug distribution into tissues is drug binding to

plasma and tissue proteins. In general, acidic drugs bind to albumin, while basic drugs tend

to bind to ai-acid glycoprotein. An agent that is totally or strongly bound has no access to

cellular sites of action, nor can it be metabolized and eliminated. Bound drugs may

accumulate in tissues in higher concentrations than would be expected from diffusion

equilibrium as a result of pH gradients, because of binding to intracellular constituents, or

partitioning into lipid (Benet and Sheiner, 1985). Drug that has accumulated in a given

tissue may serve as a reservoir that prolongs drug action in the same tissue (as is the case

with tilmicosin), or at a distant site reached through recirculation.


2.4.2. pH as a Factor in Drug Distribution

The pH difference between intracellular and extracellular fluids is small (7.0 vs.

7.4) (Benet and Sheiner, 1985), but for drugs that have pKa values close to the

physiological pH in the body, transfer across membrane will be greatly influenced by even

slight changes in pH. In general, weak bases are concentrated slightly inside of cells, while

the concentration of weak acids is slightly lower in the cells than in extracellular fluids.

Lowering the pH of extracellular fluid increases intracellular concentration of weak acids








and decreases that of weak bases, provided that the intracellular pH does not also change.

These predictions are based upon the assumption that the pH change does not

simultaneously affect the binding, biotransformation, or excretion of the drug. Elevating

the pH produces the opposite effects.

Based on the pH of tissues and the pKa of tilmicosin, there will be an equilibrium

of its unionized and ionized forms at the physiological pH (Young et al., 1995). The

pharmacokinetics of tilmicosin, and macrolides in general, indicate that as weak organic

bases, they are highly lipid soluble and partially protein bound, the latter being dependent

on tilmicosin concentration (Ziv et al, 1995). These characteristics allow tilmicosin to pass

through cell membranes freely and distribute quickly throughout the body.

Similar to tilmicosin, tetracycline and erythromycin concentrate highly in human

and rat pulmonary tissues (Fournet et al., 1989). High intrapulmonary concentrations of

tetracycline and erythromycin could be explained by a passive diffusion, dependent on the

pH variation between the intra- and extra- tissue compartments, the percentage of

un-ionized form present, and their lipid solubility.


2.4.3. Effects of Disease on Drug Distribution

It is believed that inflammation has profound effects on the tissue distribution of

antibiotics, in some cases raising, and in other cases lowering the drug levels (Schentag

and Gengo, 1982; Wise, 1986). Inflammation increases capillary permeability or the rate

of flow, thus permitting antibiotics to enter sites usually impenetrable. This is the case, for

example, with 13-lactam antibiotics in meningitis, where inflammation allows penetration of

the drugs into the central nervous system (Mazzei et al., 1991). On the contrary, blood








flow to a local area of infection may be decreased, or energy-dependent transport

processes can be destroyed or altered by inflammation

In mammals, tissue damage and/or invasion of pathogenic microorganisms induce

systemic changes, based on the inflammation process (van Miert, 1990). These systemic

changes are collectively known as the "acute phase immune response" (Bauman et al.,

1992; van Miert, 1990), and are mediated by proteins capable of binding the bacterial cell

wall product endotoxin (Bauman et al., 1992). Among the alterations are fever and

changes in blood flow to various organs (van Miert, 1990), which can influence drug

absorption and distribution. The intensity of these different reactions may vary depending

upon the type of invading microorganism or bacterial toxin present.

The passage of drugs across biological barriers may be as simple as movement

across capillary endothelium (as discussed in chapter 4.1.), but can also be as complex as

penetration of bronchial epithelium, intrabronchial mucus, bacterial and cellular debris, and

possibly chronic fibrotic scar tissue (Pennington, 1981). The latter series of organic

barriers is representative of the blood-bronchus barrier present in many patients with

chronic bronchitis. Physicochemical characteristics of antibiotics influence their

penetration into sputum (Saggers and Lawson, 1966). Benzene rings, as found in

erythromycin, and lipid solubility, appear to offer an advantage in terms of penetration.

High molecular weight antibiotics seem to have better penetration rates than smaller

molecules, possibly because of some form of gel filtration that takes place in mucus,

therefore trapping small molecules in pores in the mucin. In addition, the integrity of the

blood-bronchus barrier may be damaged by factors such as bronchial inflammation or








bronchial injury, the factors that cause anatomical alterations in tissue barriers. In

bronchitis and bronchopneumonia, increased local inflammation may enhance permeability

to antibiotic molecules, which may therefore gain access to bronchial secretions by leakage

across inflamed tissues (Pennington, 1981). From the disease-induced changes in

pharmacokinetics, it follows that more attention should be paid to drug disposition in

actual patients, in whom the drugs are meant to be used.


2.4.3.1. Effect of disease on the pharmacokinetics of macrolides

Since tilmicosin is a weak organic base, it would tend to concentrate in acidic

environments, such as pneumonia-affected lungs, resulting in unequal distribution across

membranes. The non-ionized fraction is greater in serum than in a more acidic

environment, because tilmicosin, as a base, has a tendency to move into the more acidic

lung tissue, where it becomes ionized and therefore trapped. A similar mechanism can be

used to explain the penetration and accumulation of tylosin in milk, which also has lower

pH than plasma (Ziv and Sulman, 1973).

Burrows (1985) investigated the effects of experimentally induced P. haemolytica

pneumonia on the pharmacokinetics of erythromycin in the calf. The distribution and

elimination rates of erythromycin were significantly increased, and half-life decreased in

pneumonia when compared to healthy animals. There also was a decrease in apparent

volume of distribution with pneumonia, while the lung tissue concentrations in the

pneumonic lung areas were as high or higher than those in non-affected lung tissues in the

same animals.








Bergogne-Berezin (1995b) studied tissue pharmacology of azithromycin, which,

when compared to reference compounds such as erythromycin or roxithromycin, is

characterized by (1) much lower serum concentrations; (2) a much longer elimination

half-life (48-96 h); (3) high and persistent tissue concentrations. It was found that in lung

parenchyma, azithromycin concentrations were higher and more persistent in infected mice

as compared to controls, possibly suggesting high intracellular concentrations in

polymorphonuclear leukocytes and release of the drug at pulmonary sites of infection.

Veber et al. (1993) investigated the correlation between the pharmacokinetics and

efficacy of erythromycin, roxithromycin, clarithromycin, spiramycin and azithromycin in

pneumococcal pneumonia. No differences were found between infected and control mice

in terms of the serum pharmacokinetic profiles, while the lung pharmacokinetic parameters

showed more pronounced differences between the two groups, with improved tissue

penetration of azithromycin and spyramycin in the infected animals. When azithromycin

was administered to infected mice with severe leukopenia, the elimination half life in serum

was shorter, and the serum AUC was five-fold lower than in normal mice, suggesting that

leukocytes or leukocyte products may facilitate transport of macrolides to sites of

infection.

In order to further characterize the role of phagocytes in azithromycin tissue

distribution, Girard et al. (1996) investigated the correlation of increased azithromycin

concentrations with phagocyte infiltration into sites of localized infection. Since

azithromycin reaches high concentrations in phagocytic and other host cells, this suggests

that it may be transported to specific sites of infection. When azithromycin was given








during a period of little or no inflammation, there was marginal difference between

concentrations found in infected or non-infected sites. However, when the compound was

given during a period of profound inflammation (at 5-24 h after dosing), considerably

higher drug concentrations were found in infected sites than in non-infected sites. The data

indicated that increased azithromycin concentrations occurred at sites of localized

infection, which is in correlation with the presence of inflammation and is associated with

the cellular components of the inflammatory response. It was therefore suggested that

phagocytes might be important vehicles for delivering azithromycin to and sustaining

azithromycin concentrations at sites of infection.

Phagocytic cells in general (in the lung these are primarily polymorphonuclear

leukocytes and alveolar macrophages) are responsible for the non-specific defense

mechanisms of the host to various microorganisms. They are activated in response to a

chemotactic factor elicited by bacteria undergoing their normal metabolic processes

(Butts, 1994). Upon contact with any foreign substance (e.g. bacteria), the phagocytes

typically extend their cell membrane around the bacteria until they are completely

enclosed. Then, the cell membrane containing the bacteria breaks away from the main cell

membrane and forms a phagosome within the cytoplasm of the cell. The phagosome

eventually merges with a larger intracellular vacuole, lysosome, filled with oxidative

enzymes that usually destroy the bacteria (Steinberg et al., 1988).


2.4.3.2. Effect of disease on the pharmacokinetics of other antibiotics

Unlike the macrolide antibiotics, which always showed improved tissue penetration

as a result of infection/inflammation (reviewed above in 2.4.3.1.), various other antibiotic








drugs have shown less uniform response to experimental infections, some showing

increased tissue penetration, others no change, yet others impaired tissue distribution, as a

result of disease.

The intrapulmonary concentration of tetracycline was found to be significantly

increased in rat lungs infected by Legionella pneumophila when compared to non-infected

lung (Fournet et al., 1989). In contrast, erythromycin was shown to posses the same intra-

tissue penetration in healthy and infected rat lungs.

The activity of ceftazidime was examined in a murine model of Klebsiella

pneumoniae pneumonia (McColm et al., 1986). There was no difference in respiratory

tract penetration between uninfected mice and mice infected with K. pneumoniae with

regard to the peak concentrations, half-lives, AUCs, and percentage penetration. It was

suggested, however, that perhaps animals with more advanced or chronic infections might

show differences in antibiotic kinetics compared to uninfected animals.

Pharmacokinetics of cefodizime, a newer cephem antibiotic, was studied in mice

with systemic infection by E. coli and those with respiratory infection by S. pneumoniae

(Arai et al., 1989). It was found that in mice with systemic infection, disappearance of the

drug from plasma and tissues was obviously delayed, and there was a decrease in

elimination constant and increase in the apparent volume of distribution as compared with

the control group. In the group with respiratory infection, terminal half-life and AUC for

the hepatic drug level and terminal half-life for the renal drug level increased but in the

other organs, including lungs, there was no great difference from the control group.








The intestinal wall in Crohn's disease represents a good model for studying the

effect of inflammation on the tissue penetration of drugs. Mazzei et al. (1991) studied the

effect of inflammation on cefotetan tissue distribution in the intestinal wall of patients with

Crohn's disease. It was found that the mean tissue levels of cefotetan in inflamed intestinal

wall were constantly higher than in normal wall. Both the MRT and AUC were

significantly higher in inflamed wall than in normal, suggesting facilitated penetration of

cefotetan into the inflamed intestinal wall.

Agapitova and Bobrov (1984) and Agapitova and Iakovlev (1987) studied the

effect of protein binding on penetration of antibiotics into infected inflammation foci. It

was found that the penetration depended on the level of antibiotic binding to serum

proteins. Low binding antibiotics provided the highest levels of the free antibiotic in both

serum and the inflammation foci, while the highly bound drugs were not available to

penetrate inflamed tissues.

Influence ofPneumocystis carinii pneumonia on serum and tissue concentrations

of pentamidine was studied in rats (Mordelet-Dambrine et al., 1992). It was found that the

serum concentration of pentamidine base administered by the tracheal route was higher in

the infected rats than in the control animals, while the lung concentration was lower.

Respiratory clearance, an index of the permeability of the respiratory epithelium, was also

higher in infected animals, suggesting a more rapid diffusion of pentamidine from the

alveolar lumen to the pulmonary circulation. The morphologic data suggest that the

increase in the permeability of the respiratory epithelium may be due to structural

modifications and/or inflammation as a result of pneumocystosis.








Vallee et al. (1991) studied the pharmacokinetic parameters of fluoroquinolones in

a mouse model ofS. pneumoniae-infected lung. Fluoroquinolones in general exhibit good

activity at the site of infection. When the pharmacokinetics for each drug was compared

between the infected and non-infected animals, it was found that all four drugs reached

higher lung concentrations and more persistent activity in the infected lungs, suggesting

probable trapping of the drugs at the site of infection.

Hansen et al. (1973) compared trimethoprim concentration in normal and

pathological human lung tissue and found significantly higher concentration of

trimethoprim in infected than non-infected lung tissue. Similar to tilmicosin, trimethoprim

has the pKa value of 7.6, and its lung concentrations are also significantly higher than

serum.

Infection does not only have an effect on drug pharmacokinetics, as discussed

above, but it can also affect drugs' adverse behavior. Adams et al. (1979) examined the

effect of the experimental circulatory shock induced by E. coli endotoxin on the acute

cardiovascular activities of gentamicin, tobramycin, sodium penicillin-G, and sodium

cephalothin. It was found that the cardiovascular effects of gentamicin and tobramycin

were relatively more pronounced during the endotoxin shock than during the control state,

while penicillin and cephalothin had no discernible circulatory effects in either control dogs

or dogs subjected to endotoxin shock.








2.5. Mycoplasmosis


2.5.1. Introduction

The first Mycoplasma species was described by Nocard and Roux (1898) as a

causative agent of contagious bovine pleuropneumonia. Mollicutes (wall-free prokaryotes,

such as Mycoplasma and Ureaplasma) have subsequently been isolated from humans and

many species of animals, including all the common domestic and laboratory animals.

Mycoplasmal diseases are economically important in agriculture as well as in biomedical

research (Simecka et al., 1992), and are primarily associated with diseases of the lung,

genitourinary tract and joints.

Chronic respiratory disease primarily due to Mycoplasma pulmonis remains the

major intercurrent disease problem encountered in laboratory rodents (Cassell and Hill,

1979; Lindsey, 1986). Recent surveys indicate that M. pulmonis infection remains a

common problem, not only in conventionally maintained colonies, but also in

cesarean-derived, barrier-maintained animals (Cassell et al., 1981). Klieneberger and

Steabben (1937) and Nelson (1937) first reported the occurrence of mycoplasma-like

organisms in association with the disease. The organism was later designated Mycoplasma

pulmonis (Edward and Freundt, 1956). Besides causing the respiratory disease (Cassell et

al., 1986; Lindsey and Cassell, 1973), M. pulmonis causes genital disease in both rats and

mice (Brown and Reyes, 1991; Brown and Steiner, 1996; Simecka et al., 1992).

Few diseases of laboratory animals have been as troublesome to research workers

or as enigmatic to microbiologists as murine respiratory mycoplasmosis (MRM). This

disease of laboratory rats and mice is caused by M pulmonis, but its expression is








markedly influenced by a variety of environmental, host, and organismal factors (Cassell,

1982; Lindsey, 1986; Lindsey et al., 1985; Simecka et al., 1992). The presence of

mycoplasmas in animal facilities is practically synonymous with the presence of rats and

mice (Cassell, 1982; Lindsey, 1986). More insidious than the direct loss resulting from the

respiratory mycoplasmosis (due to morbidity and mortality) is the undermining of the

validity of scientific experiments that utilize these animal species (Lindsey et al., 1971).

Mycoplasmas are difficult to diagnose and, except for the terminal stage of the

disease (when weight loss, roughened hair coat, serosanguinous nasal and ocular

discharges, and dyspnea are seen), the natural form of MRM is usually a clinically silent

disease (Cassell and Hill, 1979; Simecka et al., 1992). However, the infection induces

changes in physiology and behavior, and even subtle changes can compromise research

utilizing rats and mice (Simecka et al., 1991).


2.5.2. Experimental Respiratory Mycoplasmosis in Rodents

Experimental respiratory mycoplasmosis resulting from M pulmonis infection in

rats provides a useful model for the study of immunological and inflammatory mechanisms

operative in the respiratory tract (Simecka et al., 1991). The experimental diseases

represent useful models for the study of various human and animal diseases, particularly

mechanisms involved in chronic pulmonary inflammation and reproductive failure (Cassell,

1982; Simecka et al., 1991).


2.5.3. Clinical Signs and Virulence

Mycoplasma disease can vary from subtle, low-level disease that is subclinical to

overt severe disease that can result in death of the host (Simecka et al., 1992). By varying








the dose ofM. pulmonis in mice, it is possible to produce three reasonably distinct

clinicopathological syndromes: (1) minimal lesions that regress spontaneously; (2) an

acute disease with edematous fluid and large number of neutrophils in alveolar spaces,

pulmonary congestion, and hemorrhage with occasional pleuritis; (3) chronic

bronchopneumonia (Lindsey and Cassell, 1973). In general, the most consistent features of

respiratory mycoplasmosis are lymphoid hyperplasia and chronic inflammation (Simecka et

al., 1992). The experimental disease in rats is also dose-dependent, but rats do not develop

the acute disease (Cassell and Hill, 1979). In the rat, the full spectrum of respiratory

mycoplasmosis develops more slowly; if unaltered by other factors, it can take as long as

265 days (Whittlestone et al., 1972). It has been shown that strains of rats differ in

susceptibility to M. pulmonis respiratory disease (Davis et al., 1982; Davis and Cassell,

1982). Davis et al. (1982) found that differences in lesion severity and progression were

associated with changes in lung lymphocyte populations. Lung lesions in LEW rats, when

compared to F344 rats, developed earlier after infection, became more severe, and were

characterized by continued proliferation of all classes of lymphoid cells (Davis and Cassell,

1982; Davis et al., 1982; Simecka et al., 1991).

Virulence of mycoplasmas appears to be related to the ability of the organisms to

evade nonspecific defense mechanisms (Davidson et al., 1988). In the lungs, mucociliary

clearance and intrapulmonary killing mediated by alveolar macrophages are the major

processes responsible for nonspecific resistance (Green and Goldstein, 1966).








2.5.4. Pathogenic Mechanisms

The pathogenesis of mycoplasma disease is a complex process influenced by the

genetic background of both the host and the organism, environmental factors, and the

presence of other infectious agents (Simecka et al., 1992). Although many virulence

factors have been suggested for various mycoplasmas, there is no clear case of cause and

effect between these factors and pathogenicity. There are a number of attributes of

mycoplasmas that are likely to affect disease pathogenicity, including the ability to attach

to mucosal surfaces, to cause cell injury, to vary phenotype at a high frequency, and to

modulate and resist the host immune response (Simecka et al., 1982).

The interaction of mycoplasmas with eukaryotic membranes is likely the initial

event in most infections. However, it is quite unlikely that mere attachment to host cell

surfaces could produce the wide variety of cellular changes associated with M. pulmonis

infections, i.e. loss of cilia, cytoplasmic vacuolization, disruption of mitochondria,

epithelial hyperplasia and metaplasia, and giant cell formation (Cassell and Hill, 1979).

Mycoplasmas establish intimate contact with host cells, which may lead to cell injury

through production of toxic substances or deprivation of nutrients (Simecka et al., 1992).


2.5.5. Antimicrobial Susceptibility of Mycoplasma

It is commonly accepted that the first choice in treatment ofMycoplasma

infections involves therapy with tetracyclines or erythromycin (Gray, 1984; Sande and

Mandell, 1985; Vogel, 1995). Treatment of pneumonia with either tetracycline or

macrolides results in a shorter duration of fever, cough, malaise, fatigue, pulmonary rales,








and roentgenographic changes in the lungs (Levieil et al., 1989; Sande and Mandell,

1985).

Among macrolides, azithromycin was found to be significantly more effective

against M. pneumoniae than erythromycin or clarithromycin in the same regimens (Ishida

et al., 1994). In domestic animals, tylosin has been found to have a potent

antimycoplasmal activity, which has been retained and even improved in its newer

derivative, tilmicosin (Barragry, 1994). Ose (1987) reported that in vitro antibacterial

activity of tilmicosin includes M hyopneumoniae, M. hyorhinis, M. gallispeticum, M

dispar, M alkalescens, M bovirhinis, and M bovoculi. The in vivo effectiveness of

tilmicosin against mycoplasma infections in cattle has been mostly reported in association

with bovine respiratory disease (Barragry, 1994; Gourlay et al., 1989; Musser et al.,

1996).

In rodents, a few investigators have claimed success in eliminating M pulmonis

infection through programs involving rigid selection, administration of antibacterial drugs,

vaccination or principles of cesarean derivation combined with strict isolation procedures

(Cassell and Hill, 1979). With regard to antibacterial treatment, tylosin administration in

drinking water was found effective in the treatment of M. pulmonis pneumonia in rats

(Carter et al., 1987). The slow release form of oxytetracycline in rats was able to maintain

serum levels greater than the minimum inhibitory concentration of M. pulmonis (Curl et

al., 1988). However, despite different approaches in prevention and treatment,

mycoplasmosis continues to be one of the major intercurrent disease problem encountered

in laboratory rodents (Cassell et al., 1981).














CHAPTER 3
MATERIALS AND METHODS


3.1. Determination of Tilmicosin Concentrations


Quantitative analysis of tilmicosin concentrations in serum and lung tissue was

done using high pressure liquid chromatography (HPLC) with ultraviolet spectroscopy.

An analytical method for determination of tilmicosin concentrations in various tissues was

initially developed by the Eli Lilly Research group (Peloso and Thomson, 1988), but has

been modified for this study.


3.1.1. Chemicals and Reagents

Dried tilmicosin reference standard (technical grade) containing tilmicosin base

was provided by Eli Lilly1 (Appdx A), and was kept refrigerated at 40C (Lot # RS0164). The

defined potency of the standard was: 756.7 mg cis and 130.4 mg trans isomers of

tilmicosin per gram, when dried for three hours at 600C under a vacuum. The reference

standard was used in method validation study, as well as for preparation of working

tilmicosin standard solutions for calibration and quality control samples.

The commercial preparation of tilmicosin, MICOTIL 300 (Elanco2) was used in

all animal studies. This was kept at room temperature with minimal exposure to light due

to known photosensitivity of tilmicosin (Eli Lilly, Pers. Comm.). The active ingredient in

MICOTIL 300is tilmicosin phosphate, and each milliliter contains: 300 mg tilmicosin








base, 250 mg propylene glycol, phosphoric acid to adjust the pH to 5.8-5.9, and water for

injection to 1 ml.

All chemicals used for preparation of mobile phases for HPLC, with exception of

dibutylamine (methanol, water, acetonitrile, phosphoric acid), were HPLC-grade and were

purchased from Baxter3. Dibutylamine and most of the chemicals used for both solid phase

and liquid-liquid extraction procedures (chloroform, sodium chloride, sodium phosphate

dibasic, sodium hydroxide, potassium phosphate monobasic) were of analytical grade and

were purchased from Fisher4. Potassium phosphate dibasic and carbon tetrachloride were

also of analytical grade, and were purchased from Aldrich5.


3.1.2. Tissue Preparation

Samples were prepared for HPLC analysis by means of either solid phase or liquid-

liquid extraction, for serum and lung tissue, respectively.


3.1.2.1 Extraction of tilmicosin from serum

For solid phase extraction (SPE), Bond Elut Varian6 cartridges were used.

Tilmicosin was extracted from serum using cartridges with 500 mg C18 packing and

eluted in the Varian Vac-Elut vacuum manifold. The cartridges were activated with

methanol and water prior to addition of serum. In the sheep and cattle experiments, 2 ml

of serum were used, whereas in the rat study the amount of serum was decreased to 1 ml.

After the serum was drained through the cartridge by applying vacuum, the cartridge was

washed with water, then with 5% ammonium hydroxide in water. The water wash was

then repeated. Tilmicosin was finally eluted from the cartridge with 2 ml of a mixture of

5% methanol in 100% acetic acid, glacial. The collected eluent was evaporated to dryness








under a nitrogen stream, and reconstituted in sample diluent. The sample diluent consisted

of 475 ml water, 500 ml methanol and 25 ml of 1 M dibutylammonium phosphate

(DBAP). The sheep and cattle samples were reconstituted to 1 ml, whereas rat samples

were reconstituted in 0.5 ml of sample diluent. After the extraction, the reconstituted

samples were injected into the HPLC system.


3.1.2.2. Extraction of tilmicosin from lung tissue

Liquid-liquid extraction of tilmicosin from lung tissue samples was done from the

whole lung specimens, which had the approximate average weight of 1 g. After

determining the weight of sample, lung tissue was homogenized in the Polytron7 tissue

homogenizer with 4 ml of methanol. Homogenate was centrifuged at 485 g for 10

minutes, and the resulting supernatant was collected. The pellet was reconstituted with 4

ml of methanol, rehomogenized and centrifuged at 485 g for 10 minutes. This new

supernatant was added to the previously collected methanol fraction, to which 5 ml of

10% sodium chloride was added. The pH of the confluated methanol fraction was adjusted

to 2.5 with IM hydrochloric acid. The solution was then transferred to a 30-ml separatory

funnel, and 3 ml of carbon tetrachloride was added. The solution was shaken for 1 minute

and the bottom layer discarded. Another 3 ml of carbon tetrachloride was added, the

solution was mixed for 1 minute, and the bottom layer discarded. The collected

supernatant was then alkalized to pH 9.0 with IM sodium hydroxide, and tilmicosin in the

solution was extracted into chloroform. This was done by mixing the solution in the

separatory funnel with 3 ml of chloroform for 1 minute, and collecting the bottom layer.

Then, another 3 ml of chloroform was added and mixed for 1 minute. Again the bottom








chloroform layer was collected. The total collected chloroform was evaporated to dryness

under nitrogen stream, and reconstituted in 0.7 ml sample diluent. Extracted samples were

then ready for injection into the HPLC system.


3.1.3. High Pressure Liquid Chromatography

Reversed phase chromatographic analysis was performed using a Beckman8

System Gold apparatus, which consisted of three separate modules: a high pressure binary

pump (model 126), a spectrophotometric UV detector (model 166) and an autosampler

(model 502) equipped with a 50 pl injection loop. A Regis9 Hi-Chrom Reversible HPLC

column (25 cm x 4.6 mm) with 5 p phenyl particles was used for tilmicosin analysis. To

prolong the life of the column, a guard column (Regis phenyl guard column, 4.6 mm x 5

cm) had been placed in front of the analytical column and was replaced when a loss of

peak resolution on the chromatogram became visible. Beckman System Gold software

(version 6.01) was used for chromatogram and data collection and storage.


3.1.3.1. Chromatographic conditions

A modified Peloso and Thomson method was used for this analysis, with a binary

instead of tertiary pump, and with slightly altered mobile phase compositions to obtain

improved chromatography. The mobile phases were prepared as follows:

mobile phase A: a mixture of acetonitrile/water (50/50; v/v) adjusted to pH 2.5

with orthophosphoric acid;

mobile phase B: water adjusted to pH 2.5 with orthophosphoric acid;

mobile phase C: a mixture of 80 ml IM DBAP in water, with volume brought to

1000 ml. DBAP was prepared by adding 168 ml of dibutylamine to 700 ml of water, and








by adjusting pH to 2.5 with orthophosphoric acid. After the solution was cooled to room

temperature, the volume was brought to 1000 ml.

Solvent Mix II was then prepared as a mixture of 55% mobile phase A, 30% of

mobile phase B, and 15% of mobile phase C. Mobile phase A and solvent mix II were the

two final mobile phase solutions used for chromatography in a gradient manner. All mobile

phases were filtered through a 0.45 p.m pore filters and degassed in an ultrasonic bath

before use in the HPLC apparatus. The time-table for the gradient conditions used for

tilmicosin detection, with the pump A pumping mobile phase A, and pump B pumping

solvent mix II is as follows:


Time (min.) Pump A% Pump B%
(mobile phase A) (solvent mix II)

0 100 0
4 0 100 (ramping for 1 min.)
12 100 0 (ramping for 1 min.)
20 End of run

The flow rate was 1.5 ml/min., and the run time 20 minutes. The detector

wavelength was set at 280 nm. The average retention time for tilmicosin from both serum

and lung tissue samples using this method was 11 minutes.

Examples of chromatograms of a tilmicosin-free sheep serum sample, and a sheep

serum sample fortified with tilmicosin at the concentration of 1.0 .g/ml are shown in

Figure 3.1.







Absoroance
0.0000


-0.0100


10.00










19.99
0.00


10.00


0.0100


10.20 tilaicosin


19.99 iE t' ,ho o r
Figure 3-1: Examples of the HPLC chromatograms


Chromatograms of a tilmicosin-free sheep serum sample (A), and a sheep serum sample
containing tilmicosin at the concentration of 1.0 pg/ml (B).








3.1.3.2. Calculation of HPLC results

With each analytical run, a calibration curve with at least 4 and up to 6

concentration levels was established. The standards were prepared from aqueous

tilmicosin solutions ranging in concentrations from 2 to 100 jig/ml. For the serum sample

calibration curve, tilmicosin-free serum samples were fortified with tilmicosin standard

solutions to achieve final calibrator concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0

pg/ml. Final concentrations for the lung tissue calibration curve were 0.1, 0.5, 1.0, 5.0,

and 10.0 pg/g. Both lung and serum samples for calibration curve were fortified at the

time of HPLC analysis and carried through the extraction procedure as described earlier

(3.1.2.1. and 3.1.2.2.). Calculations in the HPLC analysis were conducted blindly.

Since the calibration curve was found to be linear in the concentration range of

interest, a least-square straight line was drawn through all data points of the calibration

curve to determine the detector response factor (y = ax + b; where y represents

concentration, x is analytical response, a is slope, b is intercept). The analytical response

was defined as the chromatographic area of a peak of interest (i.e. peak area), and

calculations were always based on that parameter, rather than peak height.

Chromatographic quantitation was done by comparing the peak areas of the standards

with unknowns.


3.1.3.3. HPLC method validation study

A validated assay method is pivotal to the acceptability of any pharmacokinetic

study. The Food and Drug Administration recommends that the following parameters

should be assessed in the process of method validation (in: Recommendations for








evaluating analytical methods, Center for Veterinary Medicine, US FDA, 1994):

concentration range and linearity, limit of detection (LOD), limit of quantitation (LOQ),

specificity, accuracy (recovery), precision reproducibilityy), and analyte stability.

Concentration range and linearity test should be performed to determine that the new

matrix does not contain elements that would interfere with the accuracy or sensitivity of

the method. No data points are allowed to be extrapolated later in the analysis if they fall

below or above the calibration curve points. LOD is the lowest concentration that can be

determined to be statistically different from background. LOQ is the lowest concentration

that the method can measure reliably. Specificity determines whether the matrix of interest

contains any elements that could interfere with elution of the peak of interest. Accuracy is

a measure of the exactness of measured value to a known or actual value. Precision is a

measure of the degree to which several analytical runs are reproducible and it determines

the magnitude of random errors in the method. Stability of the analyte in the biological

matrix (serum or tissue) should be determined under the conditions of the experiment and

would include any period for which samples were stored before analysis.

3.1.3.3.1. Linearity and range. For the linearity and range, seven replicates of the

calibration curve were analyzed and the ability to obtain linear curves over the entire range

of expected concentrations for each calibration curve was demonstrated. An example of a

typical calibration curve is shown in Figure 3-2.















Utilities Calibrations SOLMt6I

CILIBRATION


-Uieti orI -


Coponentf # 1: tilaicosin Metho d:
Y = 8.714394 X # 8.828489
Coefficient of Detemination = 8.99909


2.20 Calibtion Cve linear L R Conc. Area

ii 1 1 8.0858 8 e.85839M
a2 1 8.1888I 8.124428
x3 1 8.29881 8.277112
S4 1 8.58814 0 8.632843
/ |5 1 1.8888M 1.329594
t n6 1 2.888888 2.796189
0
U







0.053 X -- _reX 3.07A r









Figure 3-2: Example of an HPLC calibration curve

The calibration curve is derived from the chromatograms of the HPLC analysis of
tilmicosin concentrations in the sheep serum fortified at 0.1, 0.2, 0.5, 1.0, and 2.0 pg/ml.


m m








3.1.3.3.2. Limit of detection. Limit of detection was determined based on seven

replicates of the calibration curve, with the means and standard deviations for each level,

including the zero control. The LOD of a method is defined as that point which is 3

standard deviations above the analytical response (peak area, in this case) in the zero drug

sample. It was calculated using the following equation: LOD = 3 o / m, where ao is the

standard deviation of the analytical response at zero concentration, and m is the slope of

the analytical curve. Calculated LOD for the method was 0.0026 pg/ml (Table 3-1).

3.1.3.3.3. Limit of quantitation. Limit of quantitation of a method is defined as that

point which is 10 standard deviations above the analytical response in the zero drug

sample. It was calculated using the following equation: LOQ = 10 Go/ m. Calculated

LOQ for the method was 0.0087 pg/ml (Table 3-1), but based on the expected blood

concentrations of tilmicosin in sheep from literature and our pilot study, an LOQ of 0.05

pg/ml was adopted for further validation parameters, because lower concentrations were

not expected.

3.1.3.3.4. Specificity. Specificity was determined by analyzing 6 independent

sources of control matrix (as described below) and demonstrating that the calibration

curve was comparable to the one produced under chemically defined conditions (i.e. that

there was nothing in the matrix that would interfere with the elution of the peak of

interest). A diverse source of sheep blood was used in the validation study, including the

Health Science Center Animal Resources Department (HSCARD), Animal Sciences

Department, Veterinary Medicine








Table 3-1: Validation results I

Determination of the limit of detection (LOD) and limit of quantitation (LOQ) of the
HPLC method for analysis of tilmicosin in serum. The LOD and LOQ were calculated
from the values of mean and standard deviation (St. Deviation), based on 6 replicates of
tilmicosin-free sheep serum samples.


Sample # Peak Area Calibration Curve
at Zero Conc. Slope

1 0.00049 0.774
2 0.00216 0.951
3 0.00032 0.804
4 0.00145 0.791
5 0.00076 0.858
6 0.00037 0.854

Std. Deviation 0.000734
Mean 0.000925 0.839
LOD 0.002624
LOQ 0.008745








Teaching Hospital (VMTH), and Department of Physiology. There was never any

interference found in any chromatograms around the time of tilmicosin elution in any of

the samples.

3.1.3.3.5. Accuracy. Accuracy was evaluated using three concentrations of

analyte, one being the LOQ (0.1 V.g/ml for the sheep samples' analysis; 0.05 p.g/ml for the

cattle samples), one in the middle of the range of the standard curve (1.0 p.g/ml), and one

at the high end of the standard curve (2.0 p.g/ml). The accuracy of the method, based upon

the mean value of six replicate injections, was within 80-120% of the nominal

concentration at each level. The accuracy was calculated as the ratio of the calculated

concentration of the sample analyzed as "unknown" and actual concentration at which the

sample was fortified (Table 3-2).

3.1.3.3.6. Precision. Precision was determined by analyzing 6 replicates of samples

of known concentration at three different concentration levels (as described for accuracy),

and expressing them as the coefficient of variation (COV = standard deviation / mean).

The COV of six replicate injections was within +/- 10% for all three levels of

concentrations. (Table 3-2).

3.1.3.3.7. Analyte stability. To evaluate stability, serum samples were fortified at 3

concentration levels (high, medium, and low; as described for accuracy) on the day that

experimental samples were collected. The stability samples were then stored at -200C, and

run together with unknowns at the time of the actual sample analysis.












Table 3-2: Validation results II


Accuracy and precision data for the HPLC method for analysis of tilmicosin in serum. The
results are presented as the arithmetic means of 6 replicate samples for each, low (0.05
gg/ml for the cattle experiment, and 0.1 p.g/ml for the sheep experiment), medium, and
high level of concentration. Accuracy is expressed as percentage, and the coefficient of
variation (COV) is a measure of precision, based on standard deviation (St.Dev.).


Concentration Sample # Precision Accuracy
g/ml %
1 0.058 99.5
2 0.057 98.0
LOW I 3 0.066 116.2
(0.05 gg/ml) 4 0.061 105.4
5 0.060 102.3
6 0.068 119.8
St.Dev. 0.004
Mean 0.062 106.9
COV 7.244
1 0.106 105.7
2 0.109 109
LOW II 3 0.092 91.7
(0.1 gg/ml) 4 0.097 96.9
5 0.097 96.8
6 0.096 95.7
St.Dev. 0.007
Mean 0.099 99.3
COV 6.646
1 0.984 98.4
2 0.977 97.7
MEDIUM 3 1.079 107.9
(1.0 pg/ml) 4 1.115 111.5
5 0.979 97.9
6 1.017 101.7
St.Dev. 0.059
Mean 1.025 102.5
COV 5.720
1 2.061 103.1
2 1.858 92.9
HIGH 3 1.889 94.4
(2.0 g/ml) 4 1.847 92.4
5 2.054 102.7
6 1.876 93.8
St.Dev. 0.099
Mean 1.931 96.55
COV 5.145








The longest time period for which samples were tested for stability was 36 days. There

was no decrease in the detector response when those stability samples were compared to

freshly fortified and analyzed samples; their concentrations were calculated as 120, 107,

and 109% (as calculated in accuracy) of the nominal values for each concentration.


3.1.3.4. Quality control

Besides the method validation study, which was performed before the actual

animal experiments, to be in compliance with the Good Laboratory Practices (GLP)

procedures (Federal Register, 1987), quality control (QC) samples were analyzed

contemporaneously with test samples, evenly dispersed throughout each analytical run.

For the QC samples, tilmicosin-free serum samples were fortified with tilmicosin at three

different levels of concentrations (6 replicates per level). The mean and standard deviation

for each level was calculated and compared to the standard values of the nominal standard

material. The mean for each concentration level agreed to within +/- 20%, and the

standard deviation to within the established precision (COV of 10% for concentrations at

or above 1.0 upg/ml, and 20% for concentrations below 1.0 gg/ml). A summary of quality

control results is shown in Table 3-3.


3.1.3.5. Estimation of pharmacokinetic parameters

The following pharmacokinetic parameters for tilmicosin were determined for each

animal in both cattle and sheep groups using standard non-compartmental data analysis

techniques (Gibaldi and Perrier, 1982):

elimination rate constant (k.);








Table 3-3: Summary of the quality control results.


Results are presented for each analytical run separately (3 analyses in the sheep and 3 in
the cattle group). The results are presented as the arithmetic means of 6 replicate samples
for each, low (0.05 pg/ml for the cattle experiment, and 0.1 ag/ml for the sheep
experiment), medium, and high level of concentration. Accuracy is expressed as
percentage, and the coefficient of variation (COV) is a measure of precision, based on
standard deviation (St.Dev.).



Cattle Sheep
FDA Limits Analysis Low Medium High Low Medium High

Mean 0.047 0.915 2.041 0.102 0.918 1.926
Accuracy % 80-120% I 93.1 91.5 102.1 102.4 91.8 96.3
St. Dev. 0.005 0.041 0.082 0.009 0.043 0.200
COV % 10 (20) % 10.4 4.5 4.0 8.5 4.6 10.3

Mean 0.049 1.080 2.017 0.118 1.024 2.106
Accuracy % 80-120% II 98.4 108.0 100.9 117.7 102.4 105.3
St. Dev. 0.001 0.017 0.038 0.005 0.047 0.207
COV% 10(20)% 1.8 1.5 1.9 4.3 4.6 9.8

Mean 0.055 1.017 2.224 0.114 1.010 2.099
Accuracy % 80-120% III 110.6 101.7 111.2 114.4 101.0 104.9
St. Dev. 0.001 0.022 0.046 0.003 0.041 0.051
COV % 10(20)% 1.5 2.2 2.1 2.7 4.0 2.4








half-life (tl/2);

area under the serum concentration versus time curve (AUC);

area under the first-moment curve (AUMC);

mean residence time (MRT);

maximum drug concentration in serum (Cmax)

time at which Cmax was reached (tmax);

clearance (Cl);

volume of distribution (Vd).

Calculation of pharmacokinetic parameters was performed using a commercial

spreadsheet program (Excel by Microsoft10, version 5.0c). The equations used in the

pharmacokinetic analysis, as written for Excel, are listed in Appendix B. The overall

elimination rate constant was calculated from the terminal slope of a natural log-linear plot

of the individual serum concentration vs. time curve. Half-life, the time necessary for the

concentration of drug in the plasma to decrease by one-half, was determined from the

value of ke. Area under the serum concentration vs. time curve, and area under the first

moment curve, were calculated by the trapezoidal rule. Mean residence time, the average

time a drug spends in the body, was calculated from the values of AUC and AUMC.

Clearance describes the removal of drug from a volume of plasma in a given unit of time,

and was calculated based on the dose and AUC. The apparent volume of distribution is the

hypothetical volume of serum in which the drug distributes, and was determined from the

clearance and elimination rate constant. Table 3-4 shows the equations used to calculate

all noncompartmental parameters described above.











Table 3-4: Equations used to calculate noncompartmental pharmacokinetic parameters

Abbreviations used in the table: ke = elimination rate constant; t,2 = half-life; AUC = area under the
serum concentration versus time curve; AUMC = area under the first-moment curve; MRT = mean
residence time; Cnax = maximum drug concentration in serum; tmx = time at which Cmax was reached; Cl
= clearance; Vd = volume of distribution; f = bioavailability.


PK Parameter


Equation


slope
ke [h -'
t1/2 [h]
AUC [tg/ml*h]

AUMC [tg/mi*h2]
MRT [h]
CL/f [1/h]


m =(In C2 In C) / t2- t
ke = slope
tl/2 = 0.693 / ke
AUC = J C(t) dt
AUMC = f C(t)*t dt
AUMC / AUC
Dose / AUC


Cl / f ke


Vd/f [I]








The same pharmacokinetic parameters for tilmicosin were calculated in the rodent

study, using the mean serum concentration data for each time point for both infected and

non-infected animals.

In the pharmacokinetic modeling of the sheep and cattle data, serum concentration

vs. time profiles for each individual animal were fitted to a three-exponential equation

corresponding to a two-compartment model with first-order input, as proposed for

tilmicosin:

C = A e'at + B e -pt (A + B) e -kat

where C is the serum drug concentration at time t, coefficients A and B are intercept

terms; exponents a and 13 are the hybrid rate constants which are functions of the

microconstants for distribution and elimination; and the ka is the absorption rate constant.

The equation was fitted to the experimental data by use of a curve stripping and

fitting program (Scientist, version 2.0 by Micromath"), and was applied separately for

each individual animal. The program applies least squares fitting using a modified Powell

algorithm to find a local minimum, possibly the global minimum, of the sum of squared

deviations between observed data and model calculations. The dependent variable

(concentration) was weighed, if needed, to get a better fit, and a weight factor of 1 or 2

was applied. In general, data weighting is applied to transform the statistical error term

into a fractional error rather than an absolute error. If weighting was employed, it

transformed data so that each point was assigned a weight inversely proportional to the

absolute value of the data, raised to some power given by the weighting factor. The same

principle was applied in the non-compartmental pharmacokinetic analysis, to assign more








weight to lower concentration points, in order to have a better fit for the low range, where

most of the data were placed, rather than for the high range.


3.2. Cardiopulmonary Monitoring in Sheep


The following cardiopulmonary parameters were monitored in sheep to study

possible adverse effects of tilmicosin administration:

heart rate;

electrocardiogram (ECG)

respiratory rate;

systolic blood pressure;

mean blood pressure

diastolic blood pressure.

Heart rate was determined from the ECG by counting QRS complexes, and was

automatically recorded by the Datascope Passport12 multichannel oscilloscope, together

with the blood pressure data. ECG tracings were collected using modified Lead II

electrodes, which were placed at the sternal notch on the ventral neck, on the sternum at

the level of the elbow, and on the back between the shoulder blades, for the left-arm,

right-arm, and left-leg electrode, respectively. Blood pressure measurements were

obtained using the indirect oscillometric method, with a child-size pressure cuff (18-27

cm) placed on a front leg, in the antebrachial region. The osilloscopic method utilizes

microprocessor-controlled electro-pneumatic acquisition system which senses

displacements of the artery wall (Weiss et al., 1995). Amplified, digitally converted,

filtered and transformed data were displayed on the screen as digital pressure values for








the systolic (SBP), diastolic (DBP) and mean blood pressure (MBP). Mean blood pressure

was calculated automatically as: MBP = DBP + 1/3 pulse pressure. Respiratory rate was

obtained by observing the excursions over a 1-min. time period.


3.3. Animal Handling



3.3.1 Experimental Animals


3.3.1.1. Sheep

Ten cross-bred non-pregnant adult female sheep were used for the study. The

original source for the animals was a livestock market in St. Angelo, TX, and the animals

had been at the UF Animal Sciences Department farm for at least 6 months before the

study was commenced. The age range of the animals was 2-6 years, and the body weights

ranged between 120 and 170 lbs (54 to 77 kg). The sheep were individually identified by

ear tags.


3.3.1.2. Cattle

Ten young Angus cows, recently weaned of their calves, were used for the cattle

experiment. The source of animals was the UF Animal Sciences Department, Beef

Research Unit. The experiments were conducted at the Animal Sciences Department's

Physiology Unit, Colson Tract, where the animals had been maintained for 2 months

before the study. The cows were aged between 2-3 years, with body weights ranging

between 865 and 1065 lbs (392 483 kg). They were individually identified by ear tags.








3.3.1.3. Rats

A total of 72 LEW pathogen-free rats was used in the rodent study. Animals were

purchased from Harlan Sprague Dawley"1 (Indianapolis, IN), and were allowed to

acclimate for at least I week before study initiation. All animals were young adult females,

approximately weighing 100-120 g.

Rats were housed in a barrier facility with limited access to protect their pathogen-

free status. They were housed in the sterile microisolator cages (Lab Products'4), supplied

with sterile hardwood chip bedding "Beta-Chip""15 and were provided with sterile

autoclaved food (PMI rodent diet #501016). and water ad libitum. The rats were kept on a

12:12 light: dark cycle (6 am: 6 pm) and their cages were changed twice per week.


3.3.2. Experimental Mycoplasma pulmonis Infection in the Rodent Study

Experimental infection with Mycoplasma pulmonis was used to induce a chronic

respiratory infection in rats. The inoculation dose of 106 colony forming units (CFUs) of

M pulmonis (strain UAB XI 048) was used so that all rats would acquire an infection

resulting in grossly visible lung lesions, but without making them too ill (showing sniffing,

roughness of fur, hunched posture, lethargy or inappetence; Davidson, Pers. Comm.). The

animals were lightly anesthetized with 0. 1 ml of ketamine/xylazine mixture per animal

given intramuscularly (0.15 ml of 100 mg/ml xylazine and 10 ml of 100 mg/ml ketamine).

The animals were inoculated intranasally by placing 25 pl of a broth culture into each

nostril. Controls were inoculated with sterile medium.








3.3.3. Drug Administration

MICOTIL 300 is a 30% solution of tilmicosin in propylene glycol, and the label

indicates a single subcutaneous injection of 10 mg/kg (1.5 ml MICOTIL 300 per 100

lbs.) (Elanco Animal Health, 1994).


3.3.3.1. Tilmicosin administration in sheep and cattle

Both sheep and cattle were administered a single dose of tilmicosin at the rate of

10 mg tilmicosin free base equivalents per kg body weight. The drug was injected

subcutaneously between the scapulae or on both sides of the neck for the sheep and cattle

groups, respectively. Because of the larger volume of drug administered to cattle, the dose

was divided between the two sides of the neck, to avoid any possible tilmicosin-induced

tissue irritation.


3.3.3.2. Tilmicosin administration in rats

On Day 31 after the initial treatment (Mycoplasma or control), both infected and

non-infected rats were administered a single subcutaneous dose of tilmicosin at a level of

20 mg/kg body weight. MICOTIL 300 was diluted with propylene glycol to achieve a

dosing solution of 10 mg/ml.














CHAPTER 4
EXPERIMENTAL DESIGN


4.1. Introduction


The aim of this study of pharmacokinetic and pharmacodynamic properties of

tilmicosin consisted of three parts:

1. To compare the pharmacokinetics of tilmicosin in two ruminants, cattle and

sheep, following subcutaneous injection;

2. To investigate cardiopulmonary effects of tilmicosin in the sheep. These data

were collected simultaneously with (1), so the relationship of any change with

tilmicosin concentration could be assessed; and

3. To assess the prolonged retention time of tilmicosin in treating respiratory

infections, the lung tissue distribution of tilmicosin was studied in the rat. The rat

was used as a model because controlled experimental respiratory infection could be

established using Mycoplasma pulmonis.

The sheep and cattle study was performed in compliance with the GLP

recommendations (Federal Register, Sept. 4, 1987; 21 CFR Part 58). The clinical part of

the sheep study was performed in the HSCARD, and the cattle study at the Animal

Sciences Department's Beef Research Unit farm. The HPLC analysis for determination of

tilmicosin in sheep and cattle serum was performed in the Department of Physiological








Sciences, College of Veterinary Medicine, University of Florida. The project was

approved by the University of Florida (UF) Institutional Animal Care and Use Committee

(IACUC) prior to study initiation, with a protocol approval number 4102 (Appendix G)

The in vivo part of the rodent study was performed in the HSCARD's Infectious

Diseases Suite, and the tissue tilmicosin assay in the Department of Physiological

Sciences, College of Veterinary Medicine, University of Florida. This project was

approved by the UF IACUC prior to study initiation, with a protocol approval number

8072 (Appendix G).


4.1.1. Comparative Pharmacokinetics of Tilmicosin in Sheep and Cattle

The sheep experiment was of a cross-over design with the animals randomized into

initial treatment and placebo groups, and a two-week washout period between the

treatments. There was a single study factor (drug treatment) with two levels (tilmicosin

and placebo, i.e. saline administration). Tilmicosin was administered as described in

paragraph 3.3.3., while the control animals received an equal volume of saline

subcutaneously. The group that started the experiment with the administration of saline

received tilmicosin two weeks later and vice versa. Blood samples were collected for

analysis of tilmicosin concentrations in serum and cardiopulmonary parameters were

monitored concurrently, as detailed in Table 4.1. For both serum drug concentration

determinations and pharmacodynamic monitoring, the response variable was measured on

a continuous scale.









Table 4-1: Schedule for data collection during the sheep experiments.
Hem. / Chem. represents the blood sample collected for hematology and chemistry
analyses; RR denotes respiratory rate measurement; HR is heart rate measurement; BP is
blood pressure measurement; Temp. is body temperature measurement; Attit. is the
description of attitude; Appet. is the description of appetite; Elimin. is the description of
elimination patterns.; Behav. is the description of behavior; Abnor. is a list of any
abnormalities observed.


Time HPLC Hem. / RR HR BP Temp. Dep./ Appet. Elimin. Behav. Abnor.
Point sample Chem. Attit.
0 min + + + + + + + + + + +
5 min + + + + + + + + + +
15 min + + + + + + + + + +
30 min + + + + + + + + + +
I hr + + + + + + + + + +
1.5 hr + + + + + + + + + +
2 hr + + + + + + + + + +
3hr + + + + + + + + + +
4hr + + + + + + + + + +
5 hr + + + + + + + + + +
6 hr + + + + + + + + + +
8 hr +
10 hr +
12 hr +
18 hr +
24 hr + + + + + + + + +
30 hr +
36 hr +
48 hr + + + + + + + + +
60 hr +
72 hr + + + + + + + + +
96 hr + + + + + + + +








In the cattle study, all cows received a single subcutaneous dose of tilmicosin at

the beginning of the experiment, with blood sampling being performed at the same time

points as described for sheep (Table 4.1). Experimental design, therefore, was without

factors or blocking, and the response variable (tilmicosin blood concentration) was

measured on a continuous scale.

Physical examinations were performed on all study animals prior to entrance to the

study. The animals had to be in normal health in order to be accepted for the study.

Samples for blood analyses were collected and analyzed by the University of Florida

VMTH Clinical Pathology Service. The analysis included a complete blood count (CBC)

and blood chemistry panel. The value for each parameter was required to fall within the

mean 95% confidence limit for the VMTH Clinical Pathology Service, for an animal to be

accepted for the study. All parameters for both blood analyses are listed in Appendices D

and E for the blood chemistry and hematology, respectively.

During the study, the sheep were provided with food (2,000 g alfalfa-based pellets

daily) and water ad libitum. They were housed individually on a 12:12 light:dark cycle (6

am:6 pm) under controlled environmental conditions, at approximately 16-210C and 40-

60% humidity. Prior to the study and during the cross-over period, sheep were maintained

at the Animal Sciences Department Farm, where they were provided with food (corn-soy-

based sheep diet, plus bermuda hay) and water ad libitum.

The cows were kept on pasture at the Animal Science's Physiology Unit, where

they had free access to water. During the first 4 hours of the experiment the cows were

kept in the chutes, with their heads restrained in head-catch devices only for the time of








blood collection. After four hours, they were allowed to move within a pen, and after 8

hours were released onto the pasture. For each subsequent sample collection they were

brought back into the chutes and restrained in the head-catch devices.


4.1.1.1. Sample collection

The tilmicosin or saline placebo treatments were always administered between

0700 and 1000 hours. For the sheep experiment, animals were held in transporting carts

for the first six hours of testing, and after that period returned to their regular pens until

the end of experiment, 96 hours after drug administration. Before drug administration,

indwelling 16 g x 5 1/2 in. over-the-needle catheters (Becton Dickinson"7) were placed in

each jugular vein and connected to an intravenous (IV) fluid extension tube. An initial

(pre-drug) blood sample was collected at that time. Fluid balance was maintained by

continuous intravenous administration of 0.9% saline solution (at the rate of 2 ml/min) for

the first six hours of experiment, when the sampling was most frequent. Sheep were

connected to the multichannel oscilloscope (Datascope) for continuous monitoring of the

cardiopulmonary parameters (ECG; heart rate; systolic, diastolic, and mean blood

pressure), until 6 hours after tilmicosin administration, when the monitoring setup was

detached. Data for the cardiopulmonary monitoring were collected as described in chapter

3.2, according to the same schedule as blood sampling (Table 4.1).

An abbreviated physical examination was performed daily on each sheep during the

actual experimental period. This included measuring respiratory rate, heart rate, and body

temperature, as well as the evaluation of animal's attitude, appetite, elimination, behavior,

and any abnormalities.








Blood samples for the sheep study were collected using an indwelling venous

catheters (inserted before zero sample was collected) for the first 36 hours after tilmicosin

administration. After that time, to minimize risk of infection at the site of catheter

insertion, the catheters were removed and subsequent samples were collected directly by

venipuncture using the Vacutainer brand (Becton Dickinson) blood collection system (15

cc serum collection tubes and 20 g x 1 1/2 in. needles with holder).

In the cattle study, blood samples were all collected by venipuncture, using the

same type of evacuated tubes and needles as described above for sheep. In both sheep and

cattle groups, blood was collected at the time points as described in Table 4-1. Care was

taken to avoid exposing blood samples to light by keeping the blood tubes in aluminum

foil and additionally protecting them by storage in cardboard mailer boxes both before and

after refrigeration. Blood samples were left at room temperature for at least 2 hours, and

up to 24 hours, after collection, to allow clotting to take place. They were then

centrifuged at 756 g for 20 minutes. The harvested serum was stored at -200C until

assayed for tilmicosin.

Blood was collected for hematology and blood chemistry analyses in the sheep

experiment prior to drug administration, and again at 24 and 72 hours post tilmicosin

injection (Table 4-1). Samples in EDTA (hematology) and clotted blood samples

(chemistry) were submitted to the VMTH Clinical Pathology Service immediately after

collection for analysis. For the cattle study, samples for hematology and blood chemistry

analyses were collected only prior to drug administration, as a part of determination of the

animals' health status.








4.1.1.2. Statistical analysis

A commercial microcomputer program was used for the statistical analysis (Sigma

Stat19, Version 2.0). All pharmacokinetic parameters were compared between the cattle

and sheep groups using a two sample t-test for two independent samples. When the t-

test assumptions for normal distribution and equal variance were not met, the Mann-

Whitney Rank Sum test was used instead. As in all statistical analyses throughout this

project, a p-value less than 0.05 was considered significant.

For the statistical analysis of the cardiopulmonary effects of tilmicosin in sheep, a

two-way analysis of variance (ANOVA) was performed. Statistical analysis of the blood

chemistry and hematology data was performed using a two sample t-test for two

independent samples, where each parameter of the hematology and chemistry analyses was

compared between the two treatment groups.


4.1.2. Effect of Respiratory Disease on Tilmicosin Pharmacokinetic in Rats

Seventy two rats were randomly assigned to two equally sized experimental

groups. One group was infected with M pulmonis (hereafter termed "infected" or "inf")

and the other group received only sterile broth and served as negative controls (hereafter

termed "non-infected" or "n-inf"). All animals in both groups received a single dose of

tilmicosin 1 month after inoculation, and were killed at defined time points after tilmicosin

administration, as described below.

After inoculation, the rats were observed daily for any signs of illness, including

sniffing, roughness of fur, hunched posture, lethargy or inappetence. No animals were

observed to develop any of the aforementioned signs.








4.1.2.1. Sample collection

Six rats per group (infected and non-infected) were killed according to the

following schedule: 0 (before drug treatment), 1, 3, 7, 24, and 72 hours after tilmicosin

administration. The rats were euthanized with the intramuscular injection of 0.4 ml of

ketamine/xylazine mixture per animal (0.15 ml of 100 mg/ml xylazine and 10 ml of 100

mg/ml ketamine), and exsanguinated. This allowed the collection of 3-4 ml of blood from

each animal. The blood samples were allowed to clot and were stored at +40C for 24

hours. At this time, the serum was harvested after 10 minutes of centrifugation at 485 g,

and stored at -200C. Whole lungs and approximately 2 g of quadriceps muscle from the

hind leg were collected from each rat concurrently with blood samples. All samples were

placed in test tubes and stored at -200C until analysis.

Lung and muscle samples were used for determination of tissue pH. Within 30

days of sample collection, samples were first thoroughly thawed and tissue pH was

measured at room temperature. The pH measurements were made using a portable pH

meter (Extech"') with a specifically designed combination electrode for tissue penetration.

This allowed for the measurement of tissue pH without homogenization or mixing of

sample with solutions (Bager and Petersen, 1983; Korkeala et al., 1986). To obtain

reliable measurements, the tapered tip of the electrode was completely immersed into a

tissue sample. The electrode was cleaned with distilled water after each sample, and after

every six samples it was wiped with an alcohol swab. At that time, the pH meter electrode

was recalibrated according to the manufacturer's instructions, using buffer solutions of pH

4.0 and 7.0.





80

Serum and lung samples were subsequently used for HPLC analysis as described in

Chapter 3.


4.1.2.2. Statistical analysis

In the statistical analysis, a two-way ANOVA was performed to determine possible

interactions between the type of treatment (infected and non-infected), pH measurements,

and tissue and blood tilmicosin concentrations.















CHAPTER 5
RESULTS


5.1. Serum Pharmacokinetics of Tilmicosin in Sheep and Cattle


The concentrations of tilmicosin in sheep and cattle serum were determined for up

to 96 hours after drug administration. Since the concentrations oftilmicosin for the last

time point (96 hours) fell below the LOQ, the pharmacokinetic parameters were calculated

based on the end-point of 72 hours (or less; depending on the concentration) after

tilmicosin injection. However, the 96-hour time point was included in the graphical and

tabular presentations of the raw data.


5.1.1. Results of the Non-Compartmental Pharmacokinetic Analysis

The HPLC analysis was performed, as described in chapter 3.1., to determine

concentrations of tilmicosin in the cattle and sheep serum. Tilmicosin concentrations for

each individual animal at each time point are presented in Tables 5-1 and 5-2, for the

sheep and cattle groups, respectively. The non-compartmental pharmacokinetic analysis

was performed on these data. The computed pharmacokinetic parameters for each

individual animal are displayed in Tables 5-3 and 5-4, for the sheep and cattle group,

respectively.

The mean elimination rate constant for 10 sheep was 0.021 h-' ( 0.005 standard

deviation, St.D.), resulting in the mean terminal half-life of 34.6 hours ( 8.1), or 32.8








Table 5-1: Tilmicosin concentration over time in the sheep serum.

Serum (12 ml) was collected at 22 time points (up to 96 hours) after subcutaneous
administration of 10 mg/kg of Micotil to 10 sheep (S-145 to S-205). The arithmetic mean
and standard deviation (St.D.) were calculated for each time point. A hyphen indicates a
missing sample.

Time Concentration (tg/ml) in sheep serum Mean St.D.
(h) S-145 S-192 S-193 S-194 S-196 S-197 S-198 S-199 S-201 S-205
0 0 0 0 0 0 0 0 0 0 0 0 0
0.08 0.770 0.224 0.212 0.120 0.116 0.195 0.134 0.171 0.456 0.203 0.260 0.204
0.25 0.908 0.626 0.449 0.185 0.184 0.338 0.306 0.335 0.663 0.435 0.443 0.229
0.5 0.783 0.691 0.549 0.214 0.193 0.468 0.401 0.445 0.926 0.569 0.524 0.233
1 0.959 0.824 0.550 0.261 0.245 0.438 0.618 0.657 0.718 0.891 0.616 0.247
1.5 0.992 0.901 0.518 0.271 0.423 0.409 0.864 0.664 0.895 0.879 0.682 0.258
2 0.969 0.762 0.624 0.328 0.468 0.700 0.567 0.700 1.127 0.885 0.713 0.237
3 1.047 0.868 0.439 0.341 0.612 0.718 0.588 0.603 1.154 0.981 0.735 0.268
4 0.729 0.639 0.574 0.336 0.581 0.629 0.565 0.286 1.156 0.694 0.619 0.237
5 0.642 0.551 0.415 0.391 0.625 0.622 0.312 0.409 1.090 0.767 0.582 0.228
6 0.422 0.581 0.692 0.312 0.559 0.617 0.446 0.321 0.919 0.797 0.567 0.199
8 0.447 0.522 0.828 0.398 0.547 0.587 0.457 0.506 0.825 0.593 0.571 0.148
10 0.433 0.524 0.686 0.398 0.566 0.428 0.473 0.407 0.628 0.589 0.513 0.101
12 0.313 0.327 0.381 0.374 0.333 0.457 0.252 0.323 0.546 0.515 0.382 0.095
18 0.277 0.230 0.432 0.333 0.305 0.327 0.238 0.192 0.334 0.260 0.293 0.069
24 0.166 0.172 0.302 0.363 0.272 0.139 0.232 0.151 0.259 0.231 0.229 0.073
30 0.151 0.110 0.299 0.273 0.211 0.162 0.092 0.187 0.188 0.186 0.068
36 0.105 0.106 0.147 0.189 0.151 0.145 0.189 0.104 0.156 0.148 0.144 0.031
48 0.104 0.074 0.106 0.136 0.111 0.127 0.081 0.081 0.142 0.101 0.106 0.023
60 0.095 0.069 0.097 0.107 0.109 0.094 0.071 0.070 0.114 0.090 0.092 0.017
72 0.066 0.063 0.071 0.078 0.091 0.073 0.065 0.086 0.085 0.075 0.010
96 0.062 0.044 0.061 0.039 0.074 0.064 0.038 0.063 0.040 0.054 0.014









Table 5-2: Tilmicosin concentration over time in the cattle serum.

Serum (12 ml) was collected at 22 time points (up to 96 hours) after subcutaneous
administration of 10 mg/kg of Micotil to 10 cows (C-1 to C-10). The arithmetic mean and
standard deviation (St.D.) were calculated for each time point. A hyphen indicates a
missing sample.


Time J Concentration (pg/ml) in cattle serum Mean St.D.
(h) C-I C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10
0 0 0 0 0 0 0 0 0 0 0 0 0
0.08 0.680 0.865 0.316 0.748 0.058 0.159 0.493 0.283 0.991 1.421 0.601 0.422
0.25 0.545 1.015 0.446 0.552 0.778 0.429 0.749 0.713 0.944 1.941 0.811 0.443
0.5 0.537 0.971 0.507 0.606 0.646 0.416 0.700 0.684 0.685 1.334 0.709 0.265
1 0.667 0.973 0.437 0.287 0.685 0.325 0.638 0.575 0.566 0.891 0.604 0.221
1.5 0.364 1.055 0.595 0.548 0.514 0.288 0.548 0.701 0.501 0.811 0.592 0.220
2 0.649 1.112 0.462 0.571 0.494 0.320 0.540 0.610 0.507 0.786 0.605 0.216
3 0.526 0.958 0.525 0.482 0.461 0.383 0.416 0.592 0.526 0.769 0.564 0.175
4 0.515 0.955 0.517 0.417 0.515 0.362 0.466 0.630 0.628 0.716 0.572 0.171
5 0.516 0.873 0.482 0.426 0.528 0.425 0.464 0.649 0.638 0.723 0.572 0.146
6 0.609 0.660 0.434 0.374 0.394 0.345 0.431 0.543 0.520 0.668 0.498 0.119
8 0.386 0.556 0.429 0.305 0.505 0.368 0.351 0.549 0.544 0.572 0.456 0.100
10 0.442 0.517 0.415 0.349 0.395 0.315 0.457 0.453 0.464 0.423 0.062
12 0.226 0.403 0.337 0.267 0.341 0.265 0.267 0.455 0.439 0.443 0.344 0.086
18 0.146 0.279 0.243 0.213 0.230 0.213 0.219 0.266 0.267 0.231 0.040
24 0.122 0.186 0.248 0.159 0.169 0.154 0.178 0.242 0.245 0.218 0.192 0.044
30 0.171 0.169 0.213 0.154 0.174 0.178 0.141 0.217 0.208 0.167 0.179 0.025
36 0.114 0.145 0.166 0.120 0.142 0.131 0.097 0.153 0.171 0.148 0.139 0.023
48 0.087 0.093 0.130 0.069 0.109 0.112 0.089 0.121 0.139 0.137 0.108 0.024
60 0.060 0.075 0.105 0.064 0.066 0.066 0.060 0.095 0.101 0.096 0.079 0.019
72 0.048 0.058 0.071 0.044 0.059 0.054 0.045 0.073 0.093 0.088 0.063 0.017
96 0.043 0.040 0.060 0.028 0.044 0.028 0.025 0.066 0.066 0.078 0.048 0.019









Table 5-3: Calculated pharmacokinetic parameters for tilmicosin in sheep (n = 10).

The arithmetic mean and standard deviation (St.D.) were calculated for each time point.

Abbreviations used in the table: ke = elimination rate constant; tl/2 = half-life; AUC = area under the
serum concentration versus time curve; AUMC = area under the first-moment curve; MRT = mean
residence time; C. = maximum drug concentration in serum; tmx = time at which Cn.x was reached; Cl
= clearance; Vd = volume of distribution; f = bioavailability.


PK Parameter S-145 S-192 S-193 S-194 S-196 S-197 S-198 S-199 S-201 S-205 Mean SLD.
k [h-1] 0.016 0.019 0.029 0.028 0.018 0.021 0.028 0.018 0.020 0.015 0.021 0.005
tvz [h] 43.9 36.7 23.8 24.8 38.8 32.6 24.9 39.1 34.4 46.8 34.6 8.1
AUC 19.8 17.6 21.2 19.0 21.4 19.6 15.5 15.2 26.0 23.9 19.9 3.4
[mg/ml*hl
AUMC 885 690 672 729 1041 768 539 665 955 1168 811 195
[mg/ml*hz]
MRT[h] 44.6 39.1 31.7 38.4 48.7 39.2 34.8 43.7 36.7 48.9 40.6 5.7
C. [mg/ml] 1.05 0.90 0.83 0.40 0.63 0.72 0.86 0.70 1.16 0.98 0.82 0.22
t. [h] 3 1.5 8 8 5 3 1.5 2 4 3 3.9 2.4
CL/f [/h] 34.1 37.0 36.1 40.3 32.6 32.2 44.2 35.5 25.1 22.6 34.0 6.4
Vd/f[l] 2158 1961 1243 1444 1825 1516 1591 1999 1247 1525 1651 319









Table 5-4: Calculated pharmacokinetic parameters for tilmicosin in cattle (n = 10).
The arithmetic mean and standard deviation (St.D.) were calculated for each time point.

Abbreviations used in the table: ke = elimination rate constant; t1/2 = half-life; AUC = area under the
serum concentration versus time curve; AUMC = area under the first-moment curve; MRT = mean
residence time; Cmx = maximum drug concentration in serum; tx = time at which Ca, was reached; Cl
= clearance; Vd = volume of distribution; f = bioavailability.


PK Parameter C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 Mean St.D.
lk [h1] 0.029 0.025 0.024 0.029 0.026 0.025 0.025 0.024 0.020 0.016 0.024 0.004
tI/2 h] 23.9 27.3 29.4 24.0 27.1 27.5 27.2 28.9 35.3 43.9 29.4 6.0
AUC 13.8 19.8 18.3 13.2 16.1 13.3 13.8 18.3 21.8 23.7 17.2 3.8
[mg/ml*h]
AUMC 432 580 720 416 562 522 451 694 980 1137 649 241
[mg/ml*h2]
MRT [h] 31.3 29.2 39.2 31.5 34.9 39.1 32.7 38.0 45.0 47.9 36.9 6.1
C. [mg/ml] 0.68 1.11 0.60 0.75 0.78 0.43 0.75 0.71 0.99 1.94 0.87 0.42
t. [h] 0.08 2.00 1.50 0.08 0.25 0.25 0.25 0.25 0.08 0.25 0.50 0.67
CUf [/h] 335.7 196.4 224.4 327.9 251.5 318.7 332.8 262.4 204.3 189.6 264.4 59.9
Vd/f [1] 11598 7723 9507 11348 9830 12635 13075 10937 10398 12006 10906 1602








hours, when the harmonic mean was calculated (Table 5-5). The AUC of 19.9 mg/ml h

( 3.4) and the AUMC of 811.2 mg/ml h2 ( 195) was calculated, resulting in the MRT

of 40.6 h ( 5.7). The maximum concentration reached in serum of sheep was 0.82 uig/ml

( 0.2) and it was achieved at 3.9 h ( 2.4) after tilmicosin injection (tmax = 3.9 h 2.4).

Tilmicosin in sheep had the clearance of 34.0 1/h ( 14.3), and volume of distribution of

1,651 1 ( 798).

The cattle group had the mean elimination rate constant of 0.024 h-1 ( 0.004), and

the mean terminal half-life of 29.4 hours ( 6.0), or 28.6 hours, when the harmonic mean

was calculated (Table 5-5). The AUC was 17.2 mg/ml h ( 3.8) and the AUMC 649.3

mg/ml h2( 241), resulting in the MRT of 36.9 h ( 6.1). The Cmax for the cattle was

0.873 utg/ml ( 0.4) and tmax was 0.5 h ( 0.7). In the cattle group, tilmicosin had the

clearance of 264.4 1/h ( 113), and volume of distribution of 10,906 1 ( 3049).

The results of the non-compartmental analysis were compared between the two

species in order to test the hypothesis that there would be no difference in tilmicosin

pharmacokinetics between sheep and cattle. The summary of comparative results, as well

as the summary of results of the statistical analysis, are presented in Table 5-6. The results

for each pharmacokinetic parameter are based on the arithmetic mean value of 10

sheep/cattle per group. A comparison between the arithmetic and harmonic mean in

calculation of half-life is presented in Table 5-5. Harmonic mean was used because in the

cattle group, the half-life data did not have a normal distribution.








Table 5-5: Comparison of the half-life data (arithmetic vs. harmonic mean) for tilmicosin
in sheep and cattle

Arithmetic and harmonic mean values ( standard deviation, St. D.) are displayed for the
sheep and cattle groups (n = 10 animals/group), together with their respective p-values
resulting from the statistical analysis (t-test for two independent samples).


SHEEP CATTLE p-value
MEAN Mean St.Dev. Mean St.Dev.


Arithmetic 34.6 +/- 8.1 29.4 +/- 6.0 0.122


Harmonic 32.8 -10.9 to 6.5 28.6 -5.6 to 4.0 0.151











Table 5-6: Comparison of the pharmacokinetic parameters for tilmicosin in sheep and
cattle

Arithmetic mean values ( standard deviation, St. D.) are displayed for the sheep and
cattle groups (n = 10 animals/group), together with their respective p-values resulting
from the statistical analysis (t-test for two independent samples or Mann-Whitney rank
sum test).

Abbreviations used in the table: ke = elimination rate constant; tI/2 = half-life; AUC = area under the
serum concentration versus time curve; AUMC = area under the first-moment curve; MRT = mean
residence time: Cmax = maximum drug concentration in serum; tax = time at which Cmax was reached; Cl
= clearance; Vd = volume of distribution; f = bioavailability.


Cattle Sheep
PK Parameter Mean St.D. Mean St.D. p-value


k. (h) 0.024 0.004 0.021 0.005 0.15
ti2 [h] 29.4 6.0 34.6 8.1 0.122
AUC [pg/ml*h] 17.2 3.8 19.9 3.4 0.111
AUMC [pg/ml*h2] 649.3 241.4 811.2 195.5 0.117
MRT [h] 36.9 6.1 40.6 5.7 0.178

Cmax [Ig/ml] 0.873 0.420 0.822 0.221 0.734
tmax [h] 0.50 0.67 3.90 2.41 <0.001
CL/f [1/h] 264.4 59.9 34.0 6.4 <0.001
Vd/f [1] 10906 1602 1651 318.8 <0.001




Full Text

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PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF TILMICOSIN IN SHEEP CATTLE AND RATS BY SANJA MODRIC 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

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To my dearest husband Tomislav for his love support and friendship

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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 of Florida I am thankful to Dr. Webb for his guidance ideas and advices since he assumed the mentorship Dr. Derendorf showed a lot of patience understanding and support for me and together with his students and postdoctoral fellows helped solve many problems seemingly dead ends ", and other frustrations of an inexperienced graduate studen t. M y 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 alwa y s there when I needed help advice or just friendship in the seclusion of our old lab. Her understanding and help in many ways meant a whole lot to me Mr. Jay King helped sav e my days on numerous occasions when I got lost in computer problems w hich he w as always able and willing to solve I am taking this opportunity to express my sincere lll

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appreciation to Mr. Clifford Hall, for all his time and effort both in the laboratory and in working with animals Cliff volunteered 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, who helped me learn HPLC and whose friendship ideas, and help really influenced my 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 of the cattle experiments His involvement and support in providing help was greatly appreciated I wish to thank people in 'Pliva", a pharmaceutical company from Zagreb 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 Morie for their 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 tremendously in finishing my studies Lastly I would like to express my deepest thanks to m y husband Tomisla v, for everything that he has done for me This dissertation is his success as much as it is mine IV

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for all his help support optimism and most of all for his believing in me Our sons Marko and Lovro gave me the strength and persistence to finish my stud i es through their sweet laughs hugs and kisses for which I am endlessly grateful and happy. V

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TABLE OF CONTENTS ACKNOWLEDGMENTS .... ..... ..... ......... .... . ..... . .... ... .......................... .............. ...... .. iii LIST OF TABLES ... ....... ........ .... ..... . ..... ... ... ...... ...... ... ..... ..... ..... ......... ..... . .... ....... . . ix LIST OF FIGURES .... ........... ..... ..... ............. ......... ..... ................ . .......... . . ...... .... ...... X ABSTRACT .............. .... . ..................... ..... .............. ............ ..... . ......... ....... ... ... ... . ... .... xi CHAPTERS 1 INTRO DUCTION ... . ..... .... .............. ... ......... .......................... ... ..... . ... ... .......... . .... 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 of action ...... ... ... ... ................ ... ... ......... ................ .... 1 1 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 of Tilmicosin ...................... ............................... . 21 2 3.2 Cardiovascular Toxicity of Other Antibiotics--A Review ... ........... ............ 23 2.3 2 1 Macrolides ......... ............................................................................ . 2 5 2.3 2 2. Other antibiotics ....................... .... .... ....... ... . . ....... ....... .... ............... 28 2.3 2 .3. Adverse effects in concurrent drug therap y ........... ... .... .... .... ... ......... 30 2.4 Factors Affecting Tissue Distribution ofDrugs ........ ................ .... .... ..... ...... . ... 31 2 4 1 Physicochemical Properties of Drugs ... ............. ......... ............. ........... ..... 35 VI

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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 of disease on the pharmacokinetics of macrolides ............... ... 39 2.4 3 2 Effect of disease on the pharmacokinetics of other 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 of Mycoplasma .......... .......... .... . ....... ....... .... 48 3 MATERIALS AND METHODS .... ..... ............ .......... . ............ .............................. 50 3 1 Determination of Tilmicosin Concentrations ............... . ... . ... ............... .......... .... 50 3 1 1 Chemicals and Reagents . .... ....... .... ........... ..... . ....... ... ... ...... . ... .... .... ... 50 3 1.2 Tissue Preparation ... ... ............... ... .................. . .... .... . ........... ................. 51 3 1.2 1 Extraction of tilmicosin from serum ... . . .... ........ ............ .... .. .... ..... 5 I 3 1. 2 2 Extraction of tilmicosin from lung tissue . .... .... .... ..... .... ..... ....... ... 5 2 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 of pharmacokinetic 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. Experimental Mycoplasma pulmonis 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 4 EXPERIMENTAL DESIGN ......... ... ......... . .... ... . ... ....... .......... .. ..... . ... ... .... .......... 72 4 1 Introduction ... . ... . .... ..... . . . ... ... ... . . .............. . ..... ..... ..... .... .... ..... . ...... ...... 72 4 1 .1. Comparative Pharmacokinetics of Tilmicosin in Sheep and Cattle ............. 73 4 1.1.1. Sample collection .... . ... . ........... ...... ............... .... ........ .... ........ ... . ... 76 4 1 1 2. Statistical analysis ... ....... ... ... ... ...... ...... .... .... . ....... ..... ... ..... . . ... ... 78 4 1 2 Effect of Respiratory Disease on Tilmicosin Pharmacokinetic in Rats . ..... 7 8 4.1 2 1 Sample collection ... ... ... ..... ...... . ........................... . ... . . . ........ ...... 79 4 1 2 2 Statistical analysis .... ...... . ..... . . ..... ... ... ... ....... . ...... ... .................... 8 0 5 RESlJLTS ......... . ... .... ............... .... .... ... ........ .... ... .... . ........ ... ...... .... .. . ....... .... ... 81 5 1. Serum Pharmacokinetics of Tilmicosin in Sheep and Cattle ... ........... ....... . ..... ... 81 5 1 1 Results of the Non-Compartmental Pharmacokinetic Analysis ........ ......... . 8 1 Vll

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5 1 2 Results of the 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 of the Lung and Muscle Tissue ....... .... ... ... ............... 104 5.4 2 Tilmicosin Concentration in Serum and Lung Tissue ............................... 106 6 DISCUSSION ................................................................... ... ... ................. ....... ...... 112 6 .1. Pharmacokinetics of Tilmicosin in Sheep and Cattle ... .... ..... .............. ..... .... . 112 6 2 Cardiovascular Effects of Tilmicosin in Sheep . . ..... . ....................... ..... ........ 120 6 3 Lung Tissue Distribution of Tilmicosin in Rats ... . ..... .. .. ... .... .......... ....... ..... ... 125 7 SUMMARY AND CONCLUSIONS ........... .......... ...... .............. ..... ..... .... ... .......... 136 LIST OF REFERE N CES ... ........ .... ...... ... ............................... ......... ....... ........... . ...... 140 APPENDICES A LIST OF SUPPLIERS FROM CHAPTER 3 ... ................. ..... ............. .......... . ... .... 155 B PHARMACOKINETIC EQUATIONS AS WRITTEN FOR ''EXCEL .... ... ....... ... 156 C ANOV AT ABLES 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) TREATME N T ..... ... . ......... . .... ............................. ... ... ........... ... . ...... 162 F ANOV AT ABLES FROM THE STATISTICAL ANALYSES OF THE EFFECT OF MYCOPLASMA INFECTION ON THE LUNG AND MUSCL E TISSUE PH IN RA TS .... ........... .......... . .... .... .............. ....... ........ .... . 164 G INS T ITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) APPROVAL ... ... ... ................... .......... ... .... .... ...... . .... .......... ......... .... .... ............. 165 BIOGRAPHICAL SKETCH ....................................... ....... ... ... ........... .... ..... ..... .... ...... 167 Vlll

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LIST OF TABLES 3-1 : Validation results I ........ . ............. . ............................................. ... .... ............ .... 60 3-2 : Validation results II ..... ... .... ........ .... . . ......... . ... ...... ....... ........ ............ ........ .... 62 3-3 : Summary of the 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 in the sheep serum .. ... .......................... ..... .... 82 5-2 : Tilmicosin concentration over time 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 of the half-life data (arithmetic vs harmonic mean) for tilmicosin in sheep and cattle ........... ......... ...... ................... ......... ..... ................ ... ... ..... . . .... ... 87 5-6 : Comparison of the pharmacokinetic parameters for tilmicosin in sheep and cattle . 88 57 : The results of the non-compartmental pharmacokinetic analysis on tilmicosin serum concentrations in rats ........... ............. ............. .... ...... ... ...... ... .... ... .... ......... . . ... 109 lX

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LIST OF FIGURES Figure 2 1. Structural formula of tilmicosin with its two isomers ........... ..... . ........ .... . ........... . 10 3-1: Examples of the HPLC chromatograms ... . ...... . .... . ... ....... . . ...................... ..... 55 3-2: Example of an 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 cattle ............................ . .......................... .... ....... . . ...... ... .... ..... . ... ............. ....... 97 5-6 : The effect of tilmicosin on the systolic, diastolic and mean blood pressure in sheep 99 5-7 : The effect oftilmicosin on the heart rate in sheep .. ............. .... .... . ........ .... .... ... 10 1 5-8 : The effect oftilmicosin on the respiratory rate in sheep .............. ............ ......... . ... 102 5-9 : pH measurements in the lung and muscle tissue of rats . .... .... .......... ........... . . ... 105 5-10 : Concentrations oftilmicosin in the serum and lung tissue ofrats ..... .... . . ..... ... 107 5-11 : Comparison of serum and lung tissue concentrations of tilmicosin for the noninfected (A) and infected (B) rats ........... ... .......... ........... . ........ ....... ... . .......... 108 5-12 : Lung : serum ratio over time for the infected and non-infected rats ...... ................ 111 X

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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 PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF TILMICOSIN IN SHEEP CATTLE AND RATS By Sanja Modric May 1997 Chairman : Dr. Alistair I. Webb Major Department: Veterinary Medical Sciences Tilmicosin is a relatively new long-acting macrolide antibiotic approved for the treatment of bovine respiratory disease in cattle in the USA. A high degree of similarit y is expected among domestic ruminants in the distribution and elimination of drugs such as tilmicosin that are not highly metabolized. The pharmacolcinetic properties of tilmicosin in serum after subcutaneous injection were compared between cattle and sheep For both species tilmicosin concentration in serum followed a pharmacokinetic two-compartment body model. There were no significant differences in the elimination rates maximum serum concentrations half-lives areas under the curve areas under the first--moment curve and mean residence times The volume of distribution and clearance when normalized by body weight were also similar The onl y significantly d i fferent non-X I

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compartmental parameter was the time when the maximum serum concentration was reached (tmax), with sheep having the tmax of 3 9 hours compared to 0 5 hours in cattle Although macrolides in general are considered to be one of the safest anti-infective drugs adverse cardiovascular effects of several macrolides have been reported Tilrnicosin has been found to have a potential for causing cardiopulmonar toxicity with a manifestation of positive chronotropic and negative inotropic effects The cardiopulmonary effec t s of tilmicosin (heart rate ECG, blood pressure, and respiratory rate) were monitored in healthy adult sheep after receiving a single subcutaneous injection of tilmicosin at the dose of 10 mg/kg. No significant changes were found in the cardiopulmonary parameters monitored for six hours after tilrnicosin administration in sheep. In a study on tilmicosin tissue distribution, rats were used as a model for studying the effects of a chronic respiratory disease on tilmicosin pharmacokinetics. It was found that tilrnicosin consistently had higher lung tissue concentrations in the infected than in the non-infec t ed animals There was no correlation between the local pH of the lung tissue and inflammation resulting from the infection with Mycoplasma pulmon is In summary the present study provides evidence that tilrnicosin can be safely used in sheep with no adverse cardiopulmonary effects Good penetration of the drug into infected pu l monary tissue implies a possible therapeutic advantage of tilmicosin in treating lung infections XII

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CHAPTER l INTRODUCTION Respiratory diseases in cattle and especially the bovine respiratory disease (BRD) complex, cause important economic losses in the beef industry Pasteur ella haemolytica is recognized as the major pathogen in the etiology of the BRD, although a combination of environmental and husbandry factors are believed to play a role in the full development of the disease (Jordan et al 1993) Although not as prevalent as in cattle respiratory diseases still have a marked influence on the sheep industry Similar pathogens to those in BRD have been recognized in chronic respiratory disease in sheep the most important of these being P. haemolytica and Mycoplasma spp (Fraser, 1986) Tilrnicosin a relatively new antibiotic from the macrolide class, has been found to have good in vitro activity against many gram positive bacteria, as well as some gram negative organisms It has excellent activity against Pasteurella Mycoplasma and Actinobacillus spp (Barragry 1994 ; Moore et al., 1996a ; Musser et al ., 1996 ; Ose 1987) all of which have been recognized as the pathogens involved in the BRD complex Besides its wide spectrum of antibacterial activity and proven efficacy against the BRD tilrnicosin has other features which justify its popularity in comparison with other veterinary antibacterial products of similar activity It has a prolonged duration of antibacterial activity in vivo which allows for effective treatment of most animals with a single low-l

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2 volume injection of the drug, thereby greatly reducing handling risks to already stressed animals (Jordan et al ., 1993) Tilmicosin has been available in the United States since 1992 and is approved for treatment of the BRD in beef cattle and non-lactating dairy cattle, associated with Pasteurella and Mycoplasma species (Crosier 1996) It has also recently been approved for use in swine as a feed additive for the control of respiratory disease associated with Achnobacillus pleuropneumoniae and P. multocida (Federal Register 1996). Tilmicosin has not been approved for use in sheep. Sheep are expected to demonstrate similar pharmacokinetic properties for tilmicosin as have already been shown for cattle Therefore because sheep are considered a minor species the approval for tilmicosin will be based on demonstrated pharmacokinetic similarities of the drug between the two species Drug metabolism can vary markedly even between related species but a high degree of similarity is expected among domestic ruminants in the distribution and elimination of drugs that are not highly metabolized (Short, 1994 ) Tilmicosin is eliminated from the body for the most part unchanged, with approximately three quarters of total excretion consisting of parent compound (Donoho et al ., 1988 ; Giera and Peloso 1988) The first aim of this study was to compare the pharmacokinetic properties of tilmicosin in cattle and sheep serum with the hypothesis that there would be no major differences in tilmicosin pharmacokinetics between the two species Macrolide antibiotics are considered to be among the safest anti-infective drugs in clinical use with severe adverse reactions being rare (Bryskier and Labro, 1994 ; Periti et

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3 al., 1993) Jordan et al. (1993) reported on the clinical evidence oftilmicosin toxicity as primarily a manifestation of the positive chronotropic and negative inotropic cardiovascular effects Although the adverse effects of several macrolides on the cardiovascular system have been reported in literature (Freedman et al ., 1987 ; Tamargo et al., 1982 ; Wakabayashi and Yamada 1972) they have always included doses much greater than therapeutic and /or the effects were seen in the subjects with already compromised cardiac status or impaired renal function Similarly cardiovascular toxicity of tilmicosin as observed in cattle and dogs as well as in in vitro studies on the cardiac muscle tissue (Jordan et al ., 1993) was a result oflarge doses oftilmicosin given by routes other than the labeled subcutaneous injection (Elanco Animal Health, 1994) The second aim of the tilmicosin project was to study the cardiopulmonary effects oftilmicosin in healthy adult sheep after receiving a single subcutaneous injection of tilmicosin at the labeled dose of IO mg/kg body weight. Therefore concurrently with the pharmacokinetic study in sheep the heart rate ECG, blood pressure and respiratory rate were monitored and compared between the sheep that received either tilrnicosin treatment or a placebo injection of the saline solution It was hypothesized that there would be no effect of a therapeutic dose of tilmicosin on any of the cardiopulmonary parameters tested The sheep were monitored for the first six hours after administration of tilmicosin or saline after which no adverse cardiopulmonary effects were considered likely to occur because of the decline in the serum drug concentrations In an extension of the pharmacokinetic study of tilmicosin, the reported accumulation of tilrnicosin in the lung tissue was investigated using the rat as a model. As

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4 with other macrolide antibiotics such as azithromycin (Bergogne-Berezin, 1995b) and erythromycin (Fournet et al., 1989) tilmicosin exhibits good tissue penetration, reaching much higher concentrations in the lung than in the serum (Brown et al 1995 ; Ziv et al., 1995) However various drugs have been reported to respond differently to infection and inflammation Some drugs such as azithromycin (Bergogne-Berezin, 1995b ; Veber et al., 1993) show better tissue penetration in infected than non-infected animals while others such as erythromycin (Burrows 1985) and ceftazidime (McColm et al ., 1986) have impaired penetration as a result of infection Since drugs are intended for use in subjects that are ill, it is necessary to know how the infection and/or inflammation might affect the pharmacokinetics of a drug being used for treatment. Increasingly clinical studies have directed attention to this often neglected aspect of clinical pharmacokinetics (Gibaldi 1991 ) In parallel with this current trend regulatory agencies now require t he pharmacokinetics of a new drug to be studied in patient populations. Therefore the aim of the rodent study was to determine the effect of disease on tilmicosin pharmacokinetics by comparing its tissue distribution between healthy rats and rats infected with M y coplasma pulmonis. The pharmacokinetic profile of tilmicosin in rats was expected to be similar to the one previously described for cattle and sheep (Patel et al ., 1992 ; Thomson and Peloso 1989) In mice tilmicosin concentrations in lung homogenates were ten-fold higher than concurrent plasma concentrations two hours after drug injection (Brown et al ., 1995) which is in agreement with findings from cattle and sheep Based on these data it was hypothesized that the pharmacokinetics in rats would be similar to that found in ruminants

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5 Mycoplasma pulmonis was chosen as an infectious agent in the rodent study because (a) it can cause a chronic respiratory disease in rats (somewhat similar to the BRD), and (b) tilrnicosin is effective against various Mycoplasma spp (Ose 1987) The recognized morphologic similarities and similar natural histories of chronic bronchitis bronchiectasis and emphysema and of M pulmonis respiratory disease in rats and mice make the latter a particularly useful model for study of the pathogenesis of chronic pulmonary inflammation (Cassell 1982) While testing the susceptibility of M pulmonis to tilrnicosin was not a part of this project some preliminary results indicate that tilrnicosin may be effective against M. pulmonis in rats (Davidson Personal Communication) The aim of the rodent study was to compare the lung tissue distribution of tilrnicosin between the infected and non-infected rats It is commonly believed that inflammation has profound effects on the tissue distribution of antibiotics in some cases resulting in raising and in other cases lowering of the drug concentrations (Schentag and Gengo, 1982 ; Wise 1986) Inflammation can increase capillary permeability or the rate of blood flow thereby permitting some antibiotics to enter the sites that are usually impenetrable However, blood flow to a local area of infection may be decreased or energy-dependent transport processes can be destroyed or altered by inflammation The hypothesis of the rodent study was that the infected rats would have higher tilmicosin levels in the lung when compared to the non-infected rats To further characterize lung tissue accumulation of tilmicosin t he pH of the lung tissue was measured and compared between the infected and non-infected animals The

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6 hypothesis was that there would be a decrease in the pH of the infected lung due to the inflammatory processes resulting from the infection Furthermore because of the pKa of tilmicosin its lipophilicity and slightly basic nature it was hypothesized that it would be trapped within the acidified lung tissue Concurrently with the lung pH, the pH of the muscle tissue was measured in order to determine whether the hypothesized change in the pH as a result of infection would be reflected systemically It is reasonable to expect some decrease in systemic pH in chronic lung disease because of the hypoventilation and accumulation of CO2 In summary the aims of the presented project were to determine : 1) the serum pharmacokinetic profile of tilmicosin in sheep and cattle and to compare the two species with regard to dru g absorption distribution and elimination processes ; 2) the potential of tilmicosin to cause cardiopulmonary toxicity in sheep when given in the therapeutic doses and ; 3) the effects of an experimentally induced respiratory disease on tissue distribution of tilmicosin, in order to understand tilmicosin accumulation in the lung tissue

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CHAPTER2 REVIEW OF LITERATURE 2 1. Introduction Respiratory tract diseases cause important economic losses in feeder calves (Picavet et al., 1991) Church and Radostits (1981) and Thomson (1980) reported that the bovine respiratory disease complex (BRD) is the most economically important infectious disease of calves in North American feedlots Moreover it is known to be the second most common cause of death in female dairy calves from birth to weaning (Musser et al ., 1996) The etiology of the BRD is complex, but is considered to be the result of a combination of environmental factors husbandry factors and infectious agents (Laven and Andrews 1991 ) Of the infectious agents Pasteurella haemolytica is recognized as the key pathogen in the BRD complex (Jordan et al. 1993) Tilmicosin is a relatively new antibiotic from the macrolide class with excellent in vitro and in vivo activity against the various microorganisms involved in the pathology ofBRD (Debono et al., 1989 ; Jordan, 1993 ; Ose 1987) Although not as prevalent as in cattle respiratory d i seases still ha v e a marked influence on the sheep industry The importance of sheep respiratory diseases depends on their prevalence which fluctuates seasonally Other factors include the effect of respiratory diseases on the sheep productivity and their world-wide spread (Fraser 1986). 7

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8 Many bacteria viruses, and mycoplasmas have now been recovered from the respiratory tract of sheep, but not all have been shown to cause disease The importance of some bacteria and mycoplasmas has been established in pneumonia of sheep most notable of which are Pasteurella and Mycoplasma spp 2 2. Tilmicosin The first macrolide antibiotics were marketed over 40 years ago These drugs constitute a homogeneous class with very similar mechanisms of action and patterns of bacterial resistance (Aubert 1988) Interest in the macrolide antibiotics has recently increased because of their specific antibacterial spectrum activity against intracellular bacteria and their mild side-effects The macrolides are considered the most broad spectrum class of oral antibacterial agents available for the treatment of respiratory infections (Butts, 1994) Tilmicosin is a long-acting semisynthetic macrolide antibiotic derived from tylosin (Jordan et al 1993) which is a product of the controlled fermentation of Streptomyces fradiae (Picavet et al., 1991) Tylosin although an effective macrolide for treatment of BRD is primarily effective against mycoplasmas (Barragry 1994) Tilmicosin not only retains this potent antimycoplasmal activity, but the efficacy against other microorganisms has been improved through chemical modification of tylosin and desmycosin 2.2 .1. Physicochemical Properties Tilmicosin shares a common structure with all antibiotics from the macrolide family (of which the most widely used and clinically important one is erythromycin) -a

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9 many-membered macrocyclic lactone ring to whi ch one or more deoxy sugars are attached (Sande & Mandell 1985) Tilmicosin 20-deoxo-20-(3,5-dimethylpiperidin-l-yl)desmycosin (Debono et al., 1989 ; Ose, 1987), has been prepared by chemical modification of desmycosin a microbiologically active degradation product of tylosin (Hamill et al., 196 1 ) Desmycosin is readily produced from tylosin by mild acid hydrolysis to remove the terminal sugar mycarose (Ose 1987). Tilmicosin is then synthesized from desmycosin by reductive amination of the C-20 aldehyde group with a mixture of cis and trans-3 5-dimethylpiperidin (Figure 2-1. ; Ose, 1987) The commercially available tilmicosin is a mixture of85% cis and 15% trans isomers (Debono et al., 1989) Tilmicosin is a white crystalline powder with the molecular formula C46Hs0N2013. It has a molecular weight of869. 15 and pKa of7.4, and 8.5 (Debono et al., 1989) Water solubility of tilmicosin is very dependent on temperature and pH; at pH 9 the solubilities are 7 7 mg/ml at 25 C and 72 5 mg/ml at 5 C. At pH 7 0 and 25 C the solubility is 566 mg/ml (Walker 1993) Its solubility in alcohol and most organic solvents is > 5000 mg/I. 2 2 2 Pharmacology Since 1952 when the first macrolide antibiotic erythromycin was developed there has been a search for structurally related compounds with a wider spectrum of activity and better pharmacokinetic and safety profiles (Nahata 1996) Modifications of the macrocyclic lactone ring structure and of the substituent groups have resulted in compounds with various antibacterial activities and pharmacokinetic properties Macrolide

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10 0 0 OCH3 CH3 CIS CH3 Figure 2-1 Structural formula of tilmicosin with its two isomers

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11 antibiotics inhibit bacterial protein synthesis both in vivo and in vitro with different potencies As a group they are generally bacteriostatic although some of them may be bactericidal at high concentrations (Brisson-Noel et al., 1988). Their main use has been as second-line antibiotics against gram-positive bacteria and mycoplasmas They may be of particular value in conditions such as pneumonia and mastitis because of their tendency to achieve high tissue concentrations (Barragry, 1994) 2.2 2 .1. Mechanism of action Macrolides inhibit protein synthesis b y binding to the SOS ribosomal subunit of sensitive microorganisms (Sande and Mandell 1985) where they stimulate dis sociation of peptidyl-tRNA from ribosomes probably during translocation (Brisson-Noel et al. 1988 ; Menninger and Otto 1982 ; Vannuffel and Cocito 1996) The peptidyl transferase center has been identified at the SOS surface and the binding sites of inhibitors ha v e been mapped within this domain Macrolides interfere with the formation of long polypeptides and cause a premature detachment of incomplete peptide chains Microbial resistance to antibiotics develops mainly by inactivation of inhibitors and modification of targets ( mutations of ribosomal proteins or rRNA g enes) (Vannuffel and Cocito 1996) Alterations ofrRNA bases can induce resistance to a single inhibitor or to a group of antibiotics It is proposed that mutations and modifications ofrRNA bases induce conformational ribosomal changes that prevent antibiotics binding to the target (V annuffel and Cocito 1996)

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12 2 2 2.2 Antibacterial activity Similar to other macro lid es, the antibacterial activity of tilmicosin is principally directed against gram-positive bacteria as well as some gram-negative organisms, such as Pasteurella (Moore et al., 1996a ; Musser et al., 1996 ; Ose 1987) Mycoplasma (Barragry, 1994 ; Musser et al. 1996; Ose 1987) and Actinobacillus species (Moore et al., 1996a) It exhibits good in vitro antimicrobial activity against P. haemolytica and P. multocida (MIC range of 0 78 to approximately 6 25 g/ml) as well as Mycoplasma species (MIC range of0. 39 to approximately 6 .25 g / ml) (Debono et al., 1989). Ose ( 1987) reported that in vitro 95 % of the P. haemolytica isolates were inhibited by the tilmicosin concentration of 3 .12 ~Lg /ml. Growth of A pleuropneumoniae Streptococcus suis, Actinomyces pyogenes and certain other bacteria was inhibited at levels of 6 .25 g/ml or less Although tilmicosin is mainly bacteriostatic concentrations equivalent to 4 times the MIC value were bactericidal for Pasteurella spp ( Ose 1987) Debono et al. (1989) studied the in vivo activity of subcutaneously administered tilmicosin against experimental Streptococcus pyogenes infections in mice and reported the effective dose (ED5 0 ) of 2 7 mg/kg 2 2 3 Pharmacokinetics Most of the data regarding tilmicosin pharmacokinetics are unpubl ished proprietary material held by Eli LiJly. Only three reports dealin g with the pharmacokinetic patterns of tilmicosin in the mouse rabbit and dairy cattle have been published (Brown et al., 1995 ; McKay et al, 1996 ; Ziv et al., 1995)

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13 A prominent feature of tilmicosin is its prolonged duration of action, which allows in most cases, for an effective single-dose treatment. This single-dose low-volume treatment greatly reduces handling risks to already stressed animals and also reduces labor costs (Jordan et al., 1993) This makes tilmicosin a well-liked antibiotic among veterinarians for treatment of the common respiratory diseases in cattle The popularity of tilmicosin is supported by the fact that it is highly lipid-soluble and is found in lung homogenate at concentrations several fold higher than concurrent plasma concentrations (Brown et al., 1995) 2 2 3 .1. Absorption A single subcutaneous injection of tilmicosin at the dose of 10 mg/kg to neonatal calves produces a peak mean tilrnicosin level in plasma (Cmax) of 1 .55 g/ml following administration in the dorsolateral chest region and 1 6 g/rnl with the lateral neck l ocation (Thomson 1989a) For both sites, the maximum concentration was reached a t the one hour sample (tmax = 1 h). When the same dose was given to feedlot Hereford steers and heifers the Cmax was 0 97 g/ml following subcutaneous administration in the dorsolateral chest region, and 0 .71 g/rnl for the subcutaneous administration in the lateral neck region In both cases the tmax was reached at 1 hour (Thomson 1989b ) In a third pharmacokinetic study with cattle the same dose was given to Holstein dairy cows and the average Cma. ... for 5 cows was 0 .13 g/rnl and tma.x 1 84 hours (Ziv et al., 1995) The differences between those studies appear to be more related to the value of Cma,c, while the tmax value was more constant across the studies. Those differences may be attributed to

PAGE 26

14 different sites of tilmicosin administration as well as to different breeds and age groups (neonates subadult and adult cattle) included in the studies When tilmicosin was given to adult sheep subcutaneously at a dosage of 10 mg/kg the Cmax was 0.44 g/ml which was reached 1 hour after administration of the drug (Cochrane and Thomson, 1990) In another study young sheep weighing 20 kg were given a single subcutaneous dose of tilmicosin at the 20 mg/kg level and Cmax of 1.42 g/rnl and tmax of 3 8 hours were reported (Elsom et al., 1993) In either case the site of injection has not been reported Among other species in which tilmicosin was investigated rabbits treated with a subcutaneous dose of 25 mg/kg tilmicosin had a Cmax of 1 91 g/rnl and tmax of 2 hours (McKay et al., 1996) In mice receiving the dose of 10 mg/kg subcutaneously the average plasma concentration, measured at 2 hours after tilmicosin injection was 0 .51 g/rnl (Brown et al., 1995) Ziv et al. ( 1995) reported that binding of tilmicosin to serum proteins was in correlation with drug concentration (Ziv et al. 1995) Higher concentrations resulted in lower percentage of protein binding and vice versa (25% binding at concentration of7. 8 g/rnl as opposed to 64% at 0 .15 g/rnl) 2 2 3 2 Distribution After a single injection, tilmicosin is well distributed throu g hout the body but especially high levels are found in the lung liver and kidney This has been reported for various species : cattle (Giera et al. 1986 ; Giera and Peloso 1988) sheep (Elsom et al., 1993) mice (Brown et al., 1995) and rabbits (McKay et al. 1996)

PAGE 27

15 In cattle administered 20 mg/kg tilmicosin subcutaneously the concentration o f tilmicosin in liver 3 days after the dose as detected from the mean 14C residue was 36 gig, in kidney 39.2 gig and in lung 14 3 ~Lglg (Giera et al. 1986) At the same time the concentrations in muscle and subcutaneous fat were only 1.96 and 2 03 gig respectively When feedlot cattle were administered tilmicosin at a dose rate of 10 mg/kg (Thomson and Peloso 1989) the serum and lung tilmicosin concentrations 8 hours following injection were 0 35 glml and 5 50 gig, respectively which represented a lung-to-serum ratio of 16 : 1 Following a single 20 mg/kg subcutaneous injection of tilmicosin in sheep the concentrations 3 days post administration as detected from the mean 14C residues were 21.09 gig in the kidney 9 98 gig in the liver and 5 .11 gig i n the lung (Elsom et al ., 1993) Muscle and fat concentrations were 1 26 and 1 24 gig respectively. In a stud y using 6 month-old sheep which were administered tilmicos i n subcutaneously a t a dose rate of 10 mg/kg the serum and lung tilmicosin concentrations 8 hours following inject ion were 1.18 glml and 14 8 gig respectively which represented a lung-to-serum ratio of 13: 1 (Patel et al 1992) This ratio kept increasing until 72 hours after tilmicosin administration when it reached the maximum of 106 : 1 after which it started to decline The pharmacokinetic profile of tilmicosin in rats would be expected to be similar to that described for cattle and sheep A similar pharmacokinetic pattern has been observed in mice (Brown et al 1995) where tilmicosin concentrations in lung homogenates were ten fold higher than concurrent plasma concentrations two hours after drug injection The mice were injected subcutaneously with tilrnicosin at a dose rate of 10 mg/kg and two

PAGE 28

16 hours after the dosing the lung and serum concentrations were 5.5 gig and 0 .51 g/ml respectively In a rabbit study animals were given a dose of25 mg/kg tilmicosin subcutaneously and the serum Cmax was 1 .91 g/ml which was reached 2 hours after the dosing (McKay et al 1996) Concurrent lung concentration reached a maximum of 14.43 g/ml at the same time point resulting in the lung : serum ratio of 8 : 1. At eight hours the ratio was 10 : 1. 2 2.3.3. Biotransformation In the study on cattle injected with IO mg/kg tilmicosin, urinary radioactivity was approximately three quarters parent tilmicosin, while the fecal radioactivity was approximately 22% parent tilmicosin 22% metabolite T-1 and the remainder comprised minor metabolites or non-extractable residue (Donoho et al ., 1988) However, in vitro degradation was demonstrated in control fortified feces and so the true portions of fecal tilmicosin are suggested to be higher than reported 22%. Parent tilmicosin was the predominant radioactive component in liver, kidney and the injection site The tilmicosin metabolites were further identified (Donoho 1988) Metabolite T-1 was characterized as N-desmethyl tilmicosin (i e tilmicosin minus CH3 ) and is the only major tilmicosin metabolite Metabolite T-2 was later called an impurity from tilmicosin technical material ; and T-3 was determined to be a minor degradation product resulting from the replacement of -N(CH3)i on the mycaminose sugar with -OH. Tilmicosin metabolites were also investigated in sheep (Elsom et al. 1993) Similar to the cattle the major component in urine was the unchanged tilmicosin, accounting for

PAGE 29

17 approximately 75% of the total urinary radioactivity, and the majority of the rest of radioactivity was identified as metabolite T-1 The major component of the radioactivity detected in the liver, kidney, and urine was parent tilmicosin 2 2 3.4 Excretion Elimination pattern of 14C tilmicosin was studied in Hereford steers injected subcutaneously with a dose of 30 mg/kg (Giera et al., 1987) Urine and fecal samples were obtained daily for 15 days and a total recovery of the dose was 72% in the feces and 19% in urine Radioactivity in both excreta decreased progressively between days 1 and 15. When the therapeutic dose of 10 mg/kg was administered to steers the total recovery of the dose from excreta over 14 days was lower than when compared to the higher dose from the previously mentioned study with a mean of 73% (Donoho et al., 1988) In sheep the proportions of tilmicosin excreted in feces and urine after subcutaneous injection are similar to those determined for cattle In the lamb metabolism study by Elsom et al. ( 1993) using the dose of 20 mg/kg of radioactive-labeled tilmicosin the mean total of radioactivity recovered within 7 days of dosing was about 85% (from which 72% was from feces and 13% from urine) Parker and Walker (1993) analyzed serum samples of lambs weighing 16-18 kg that received a dose of 20 mg/kg tilrnicosin The authors reported a plasma half-life of approximately 7 hours for the initial elimination phase (between 6 and 24 hours after dosing) ; and the terminal half-life (between 48 and 96 hours after dosing) of 41 hours Based on a semilogarithmic plot of serum depletion data, a two-compartment body model was indicated for distribution and elimination of tilmicosin In a study of six-month old

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18 sheep injected with 10 mg/kg of tilmicosin the serum halfl ives of approximate l y 8 and 30 hours for the initial and later elimination phases respectively were reported (Pa t el e t al., 1992) 2 2.4 Therapeutic Uses Tilmicosin is active against man y of the infectious pathogens commonl y associated with respiratory tract infections including Past e urella and Myc oplasma s pp. (Musser 1996) It is currently labeled for the treatment of BRD associated w ith P. haemolytica in the beef cattle and non-lactating dairy cattle (Crosier 1996 ; Darling 1993) It was init i ally evaluated for treatment of the BRD (shipping fever) of feedlot catt l e (Schumann et al. 1991 ) but was later found also to be effective as a treatment for naturally occurring pneumonia ( enzootic calf pneumonia) in calves during their first weeks of life (L a v en and Andrews 1991 ; Ose and Tonkinson 1988 ; Picavet et al., 1991). The prophylactic administration of injectable tilmicosin for pneumonia in weaned beef calves was also investigated and it was suggested that tilmicosin given proph y lacticall y had a benefic i a l effect on the incidence and severit y of pneumonia (Morck et al., 1 99 3) Ziv e t al. ( 199 5) determined that the MIC oftilmicosin for S. aure u s isolates from the bovine udder was 0 78 g/rnl and they suggested its possible use as a systemic therapy for udder infections in dry cows Tilmicosin has recently been widel y investigated for its use in spec i es o t her than cattle Moore et al ( 1996a and 1996b) determined the effective dosage of tilmicosin phosphate when fed to pigs for the control of pneumonia attributable to A. pleuropneumoniae. The drug has recently been approved in the US for use in swine for

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19 treatment of the respiratory disease associated with A pleuropneumon i a e and P. multocida (Federal Register December 1996 ; 21 CFR Parts 556 and 558) McKa y et al. ( 1996) investigated the efficacy of tilmicosin for treatment of pasteurellosis in rabbits and reported a treatment success rate of93%. Jordan and Horrocks (1996) determined the minimum inhibitory concentration of tilmicosin and tylosin for Mycoplasma gallis epticu m and Mycoplasma synoviae and compared their efficacy in the control of M galli septicu m infection in broiler chicks 2 2 5 Dosage and Administration A single dose of 10 mg of tilmicosin per kilogram body weight for use in cattle is listed in the label (Blanco Animal Health : Micotil 300 Injection 1994) The FDA approved route of administration is subcutaneous It is recommended in t he label that no more than 15 ml of MICOTIL be administered per injection site due to the possibilit y of tissue irritation and damage In view of tilmicosin s prolonged kinetic excretory pattern i t is contraindicated for use in lactating animals A withholding time of 56 days is recommended for meat (Barragry 1994) 2 3 Toxicity Toxicity oftilmicosin has been studied after administration by a variety of routes to various laboratory animals (Jordan et al., 1993) A median lethal dose (MLD) for fasted Sprague-Dawley rats given by gavage was 825 mg/kg but if the rats were fed before tilmicosin administration, then the MLD increased to 2 250 m g/kg The MLD for mice

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20 was 100 mg/kg and in both species signs of toxicity were non-specific and no systemic lesions were found at necropsy Clinical signs in surviving animals were also non-specific and animals usually appeared normal by 48 hr after dosing Rabbits were used to test the potential effects of accidental dermal or ocular exposure to MICOTIL and it was reported that MICOTIL produced very slight irritation of the skin and a slight to moderate conjunctivitis ( Jordan et al., 1993) A negative response in a guinea pig sensitization study indicated that aqueous tilmicosin was not an allergen neither did it have an effect on the primary antibody response during an immune testing in mice (Jordan et al., 1993) Although tilmicosin is toxic for pigs when given intramuscularl y or intravenously (Barragry 1994 ; Jordan, 1993) it has been proven to be safe when formulated as a feed additive (Jordan et al ., 1993 ; Moore et al. 1996a and b ) Acute studies in horses and goats indicate high risk of toxicity including death at intravenous doses of 10 mg/kg or less and at subcutaneously or intramuscularly administered doses above 1 0 mg/kg ( Jordan et al., 1993) The results of tilmicosin administration to laboratory animals and domestic livestock suggest that the cardiovascular system is the target of acute tilmicosin toxicit y (Jordan et al ., 1993 ; McGuigan, 1994) with the primary effects of tachycardia and decreased inotropy However the labeled dose of 10 mg/kg was well tolerated in both cattle and sheep, the only adverse effect being a transient swelling at the site of injection. Not only did the labeled dose never result in overt cardiovascular toxicity but tilmicosin had an adequately wide margin of safety for injection in both species ; five-fold multiple

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21 doses (50 mg/kg) repeated 3 times produced only mild toxicity while 30 mg/kg/day represented a no-adverse-effect dose McGuigan (1994) reported on human exposures to tilmicosin that had occurred since the drug had been on the market. Thirty six cases of accidental human exposure to tilmicosin were collected and analyzed. About 75% of the patients included in the study were exposed to probably less than I ml of tilmicosin (less than 300 mg), with 72% of exposures resulting from needle punctures While local symptoms predominated there appeared to be no unexpected local tissue reaction and there was no clinical evidence of systemic toxicity in any of the reported cases 2 3 1 Cardiovascular Toxicity ofTilmicosin The toxicity dose response varies among the laboratory animal and domestic livestock species but in general large doses of tilmicosin will manifest positive chronotropic and negative inotropic effects (Jordan et al ., 1993) An intravenous administration of sublethal doses of tilmicosin (0. 25 mg/kg) to conscious dogs resulted in a pronounced sinus tachycardia myocardjal depression (negative inotropy) and a reduction in arterial pulse pressure (Jordan et al ., 1993) The authors concluded that partial blockade of the tilmicosin-induced tachycardia by propranolol was in part mediated thorough the stimulation of cardiac f3-receptors In anesthetized dogs tilmicosin had no remarkable effect upon cardiovascular or electrocardiographic parameters at the dose of0. 5 mg/kg (Jordan et al ., 1993) However higher doses ( I 0 and 5 0 mg/kg) produced prominent tachycardia, peripheral vasoconstriction, increased pulmonary artery pressure increased pulmonary artery wedge

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22 pressure and increased pulmonary vascular resistance as well as decreased cardiac output stroke volume stroke work index femoral artery flow and marked hypotension In in vitro smooth muscle tissue studies tilmicosin did not elicit contractile activity in the guinea pig ileum rat uterus, or rat vas deferens but there was a significant dose dependent decrease in the force of contractions of the spontaneously beating guinea pig atria as well as a significant increase in the rate of contractions (Jordan et al., 1993). Tilmicosin markedly antagonized the contractile force response of the atria to isoproterenol, as well as the contractile rate response to two other positive inotropic and chronotropic agents (norepinephrine force/rate ; calcium agonist BAY K 8644 force / rate) and the antagonism was not readily reversible Main et al. (1996) studied the cardiovascular effects of sublethal doses (0 25 to 5 0 mg/kg) of tilmicosin administered intravenously to conscious mixed-breed dogs Left ventricular function, systemic arterial blood pressure and heart rate responses to tilmicosin alone and in combination with propranolol or dobutamine were evaluated Cardiovascular variables were recorded and the peak value of the first derivative of left ventricular pressure (dp / dt(max)) was used as an index ofleft ventricular inotropic state Tilmicosin caused dose dependent decreases in (dp / dt(max)) and aortic pulse pressure Heart rate increased dose-dependently Left ventricular end-diastolic pressure increased at the 2.5 and 5.0 mg/kg dosages. Left ventricular systolic pressure was reduced dose-dependently at the 2 5 and 5 0 mg/kg dosages Treatment with propranolol exacerbated the negative inotropic effect and the decrease in left ventricular systolic pressure but did not attenuate the tachycardia associated with tilmicosin treatment.

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23 Dobutamine attenuated the changes in ventricular inotropic state in a dose-dependent manner. Dobutamine infusion also restored left ventricular systolic pressure at dosages of 3 or 10 g/min/kg The authors concluded that toxic doses of tilmicosin may have a negative inotropic effect in conscious dogs Heart rate increased in a dose-dependent manner and was not the result of beta I-receptor stimulation which is to the contrary of findings from Jordan et al. (1993) Dobutamine reversed some but not all, of the effec t s caused by tilmicosin administration Main et al. ( 1996) suggested that the mechanism of tilmicosin cardiovascular toxicity might be mediated through intracellular calcium A rapid deple t ion of i ntracellular calcium through interference with sarcolemmal calcium channels o r some othe r mechanism could result in negative inotropic effect of the drug It has been reported for other macrolides such as josam y cin and erythromycin that they were capable of inhibiting transmembrane calcium flux (Tamargo et al., 1982) a similar mechanism as in v ol v ed i n the effect of calcium channel blockers on the heart ( Boddeke et al., 1988) Safety of tilmicosin was tested in feeder cattle administered subcutaneous doses of 10, 30 and 50 mg/kg of tilmicosin on three consecutive da y s ( Jordan et al. 1993) Clinically no overt evidence of toxicity was observed The onl y side-effect in the treated animals was the presence of small foci of necrosis in the papillary muscle of the left ventricle of the heart observed in 2 of 8 cattle treated with the 50 mg/kg dose 2 3 2 Cardiovascular Toxicity of Other Antibiotics--A Review Clinical reports on the treatment of infection and efficacy of ant i biotics predominate in the literature while studies on the pharmacological actions and especiall y

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24 adverse effects have not been as numerous (Yvakabayashi and Yamada 1972) The main reason for this may be that the toxicity of antibiotics is milder than that of most other synthetic drugs and there is seldom any recorded cardiovascular action at the usual therapeutic dosages New drug candidates are frequently identified in highly specific assays designed to target certain receptors or disease states It therefore becomes increasingly important to verify the selectivity of new compounds in broad pharmacological profiling which also identifies potential secondary activities that could result in functional adverse effects or toxicity (Colbert et al., 1991) Antibiotics in particular are targeted to affect microorganisms and are therefore expected to be relatively inert from a pharmacological perspective They are routinely used as prophylactic antiinfectious agents in surgery (DiPiro et al., 1981 ; Stinner et al., 1995) but several hazards may arise by the wide-spread use of these drugs including neglect of their physiological effects on the patient and their potential for modulating cardiovascular stability following complicated surgery (Stinner et al., 1995) Although cardiovascular activity of antibiotics is not potent it may play an important role in patients with already compromised cardiac status or with impaired renal function (Adams 1975 ; Cohen et al., 1970 ; Freedman et al., 1987) The importance of dosage considerations in assessment of drug toxicity was stressed by Adams (1975) in his review of acute adverse effects of antibiotics It was noted there that in most experimental studies doses of antibiotics much greater than the therapeutic doses have been used to demonstrate the potential for adverse effects while the vast majority of antibiotics never showed any serious adverse effects at therapeutic concentrations

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25 Toxic injury is one of the many ways by which the functional integrity of the heart may become compromised (Combs and Acosta 1990). Any of the subcellular elements may be the target of toxic injury including various membranes and organelles Understanding the mechanisms underlying cardiotoxicity may lead to treatment of the toxicity or to its prevention by designing new drugs that will not have secondary cardiovascular affinity A review of selected clinical reports in humans and experimental studies in various animal species suggests that under certain circumstances several commonly used antibiotics may cause cardiovascular depression respiratory difficulties or alter the metabolic breakdown of other drugs (Adams 1975 ; Kuenneke et al., 1996) With regard to the cardiovascular system some groups of antibiotics (aminoglycosides tetracyclines, macrolides) have been shown to cause different adverse effects most common of which is cardiovascular depression including hypotension bradycardia, myocardial depression and decreased cardiac output (Adams and Parker 1982) These untoward responses are believed to be due to the direct effects of antibiotics on specific physiologic functions rather than related to allergic reactions or cytotoxic lesions Severe pathologic conditions over-dosage or concomitant exposure to other potent drugs may predispose a patient to these acute adverse effects 2 3 2 .1. Macrolides Macrolides are an old and well established class of antimicrobial agents that account for 10-15% of the worldwide oral antibiotic market (Periti et al 1993) They are considered to be one of the safest anti-infective drugs in clinical use with severe adverse

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26 reactions being rare (Bryskier and Labro 1994 ; Periti et al., 1993) Newer products with improved features have recently been discovered and developed thereby maintaining or significantly expanding the role of macrolides in the management of infection In their review of the adverse effects of macrolide antibacterials Peri ti et al. (1993) divided the macrolides adverse effects into two groups : (1) expected and established effects ; and (2) unusual and rare or questionable effects. However cardiovascular toxicity was not described as either one of those suggesting a very low occurrence of cardiovascular toxicity associated with the macrolide therapy In general gastrointestinal reactions represent the most frequent disturbance among macrolides occurring in 15 to 20% of patients on erythromycin and in 5% or fewer patients treated with some recently developed macrolide derivatives The hepatotoxic potential of macrolides which rarely or never form nitrosoalkanes is low or negligible depending on the antibiotic used Transient deafness and allergic reactions to macrolide antibacterials are highly unusual and have definitely been shown to be more common following treatment with erythromycin than with the recently developed 14, 15and 16-membered macrolides In contrast to Periti et al. (1993) other authors including Adams (1975) Tamargo et al. (1982) and Wakabayashi and Yamada (1972) have reported on cardiovascular toxicity of macrolides Tamargo et al. (1982) e x amined and compared the cardiovascular effects of macrolides Gosamycin erythromycin spiramycin and oleandomycin) and related antibiotics ( clindamycin and lincomycin) in spontaneously beating right atrial preparations and in electrically driven left atrial preparations of rats Josamycin and erythromycin produced a dose-dependent decrease in heart rate and contractile force while spiramycin

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27 oleandomycin, clindamycin, and lincomycin all produced notable changes only at the highest concentrations tested The negative inotropic effect of josamycin was not modified by pretreating the atria with atropine or with a mixture of antagonists containing phentolamine practolol diphenhydramine cirnetidine methysergide and indomethacin In isolated right atria josamycin did not block the positive inotropic and chronotropic responses to isoprenaline but shifted the dose-response curve to Ca++ to the right Josamycin and erythromycin reduced in a dose-dependent manner the slow responses induced in K depolarized right atria by isoprenaline but this effect was reversed by increasing the Ca++ concentration in the bathing media Those findings demonstrated a direct negative inotropic effect of josamycin and suggested that this effect could be explained by inhibiting transmembrane Ca++ influx into atrial cells A similar mechanism of cardiovascular toxicity has been suggested for ti1micosin by Main et al. ( 1996) as described in chapter 2 3 1 Another mechanism of cardiovascular tox icity of macrolides has been suggested by Wakabayashi and Yamada (1972) Their study on the mechanism of the cardiovascular depressor effect of several macrolide antibiotics revealed the absence of any influence from bilateral cervical sympathectomy bilateral cervical vagotomy transsection of the cervical spinal cord or pretreatment with atropine It was observed in the same study that the histamine concentration in the blood following administration of the macrolide antibiotics rose up to 30 times its pretreatment l evel suggesting that macrolides might be histamine releasers and therefore that histamine might induce depressor effect on the blood pressure

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28 Treatment with erythromycin has been associated with ventricular tachyarrhythmias and QT prolongation of the electrocardiogram (ECG) (Gueugniaud et al., 1985 ; McComb et al., 1984) as well as ventricular repolarization (Freedman et al. 1987) It had been suggested earlier that the mechanism for erythromycin-induced arrhythmias might be myocardial potassium efflux which is a predisposing factor for ventricular tachycardia (Regan et al., 1969) However Freedman et al. (1987) suggested that the erythromycin effect was mediated by the sympathetic nervous system because of the abolition of the erythromycin effect by propranolol therapy and left cervicothoracic sympathetic ganglionectomy 2 3 2 2 Other antibiotics Toxicity is a major limitation to the therapeutic usefulness of the aminoglycoside group of antibiotics most notable of which is ototoxicity and nephrotoxicity (Sande and Mandel 1985) but other adverse effects of the aminoglycosides including depression of cardiac function, have been also reported Intravenous administration of streptomycin has been found to cause a dose-dependent depression of cardiovascular functions including a decrease in cardiac output mean arterial pressure and contractile force (Cohen et al., 1970) Similar depression was demonstrated after administration ofkanamycin as well as some non-arninoglycoside antibiotics such as tetracycline vancomycin, erythromycin, and colymycin (Cohen et al., 1 9 70) Adams et al. (1979) examined the acute cardiovascular activities of gentarnicin tobramycin sodium penicillin-G and sodium cephalothin on dogs during experimental circulatory shock induced b y E s c h e richia coli endotoxin Intravenous administration of

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29 gentamicin or tobramycin resulted in transient cardiovascular depression, as reflected by dose-related decreases of systemic blood pressure cardiac output and contractile force while the heart rate was affected little Equally large doses of penicillin or cephalothin however had no discernible circulatory effects in either control dogs or dogs subjected to endotoxin shock. Doxorubicin and its analogs are very important cancer chemotherapeutic agents that can cause cardiotoxicity The most important cardiotoxic mechanisms proposed for doxorubicin include oxidative stress with its resultant damage to myocardial elements changes in calcium homeostasis decreased ability to produce ATP and systemic release of cardiotoxic humoral mediators from tissue mast cells (Ringenberg et al 1990) It is suggested that doxorubicinol one of the metabolites of doxorubicin, may be responsible for the cardiotoxicity Pirlimycin adenylate is a clindamycin analog possessing antiarrhythmic activity (Kopia et al., 1983) In the anesthetized dog the sustained ventricular tachycardia produced by ouabain intoxication was converted to a normal sinus rhythm with pirlimycin adenylate The drug failed however to decrease arrhythmia It was concluded by the authors that pirlimycin adenylate might be an interesting prototype antiarrhythmic agent and further chemical modification of the drug molecule might increase the spectrum of antiarrhythmic activity without altering the drug's toxicity Loracarbef is a carbacephem antibiotic targeted for use in the treatment of infectious disease A safety study was performed usin g high oral or intravenous doses and loracarbef was found to cause changes in cardiovascular system including increase in

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30 mean pressure, cardiac output heart rate, and femoral flow. However the doses used in the safety study represented significant multiples of the therapeutic dose and therefore it was concluded that loracarbef has a very low potential to produce adverse effects at therapeutic doses (Shetler et al. 1993). The anthracycline antibiotics adriamycin and daunomyc~ are potent antitumor agents but their clinical use is limited by pronounced acute and chronic cardiotoxicity (Lefrak et al., 1973 ; Singer et al., 1978) Pirarubicin a newer anthracycline antibiotic, was found to have a similar antitumor effect but much lower cardiotoxicity (Matshushita et al., 1985) Anthracyclines depress the blood pressure acting directly on blood vessels and also have a positive inotropic effect that may be mediated through the release of histamine (Hirano et al., 1991) 2 3 2 3 Adverse effects in concurrent drug therapy It has become evident that the effects of many drugs when given concurrently are not predictable on the basis of knowledge of their individual effects. The pharmacological responses from drug interactions may result from enhancement of the effects of one or the other drug the development of totally new effects that are not seen when either drug is used alone the inhibition of the effect of one drug by another or no change whatever in the net effect despite the fact that the kinetics and metabolism of one or both of the drugs may be altered substantially (Murad and Gilman 1995) Macrolide antibiotics interact with many commonly used drugs by altering metabolism due to complex induction and inhibition of cytochrome P-450 IIIA4 (CYP3A4) in the liver and enterocytes (Nahata 1996 ; von Rosensteil and Adam, 1995 )

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31 2.4. Factors Affecting Tissue Distribution of Drugs Although information concerning the concentration of drugs in the blood is of great importance in studying their absorption and excretion, it may be of little or no value in ascertaining the actual quantity of antibacterial substance at the site of infection (Weinstein et al. 1951) Serum pharmacokinetics constitutes the first step in determining the potential efficacy of a drug against pathogens but most drugs are unevenly distributed in tissues and concentrations achieved in a given tissue cannot be accurately predicted from serum pharmacokinetics (Bergogne-Berezin 1996). Therefore tissue penetration studies have become an important aspect of the assessment of antimicrobials Effective antimicrobial therapy requires adequate penetration of the agent from the intravascular phase into the focus of infection This entails passage of drug across the capillary walls to the interstitial space across barriers surrounding abscesses and past the lining of body cavities (Bergan 1981 ) The kinetics of drug distribution can have profound effects on the time course of drug action Drugs that move relatively slowly in and out of body "compartments", thereby confer pharmacokinetic characteristics of a multicompartment system and elicit a considerably different time course of pharmacological effect than do drugs which are distributed extremely rapidly in the body (Gibaldi et al., 1971) The g eneral ability and velocity with which molecules pass through body systems is regulated by various factors With regard to the characteristics of a drug molecule important factors include molecular weight molecular size electrostatic charge pKa protein binding and lipid solubility Factors affected by the target tissue include local pH,

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32 pH gradient concentration gradient vascularity and membrane permeability (Bergan 1981 ; Mazzei et al 1991 ) For the portion of drug not bound to protein, passage follows Fick's law of diffusion : dc / dt = -Dq d2c/dx2 where dc/dt designates change in concentration over time ; dc / dx is concentration gradient over the distance x ; Dis diffusion coefficient expressed in m2/ sec ; and q is the area expressed in metric units across which diffusion may occur Following injection an antibiotic is absorbed and distributed by the blood and undergoes one of two fates : (1) limited tissue distribution and rapid elimination from the body ; or (2) extensive tissue distribution and slow elimination from the body (Young et al., 1995) The rate at which an antibiotic is eliminated and the characteristics by which it accumulates in various body tissues have a significant impact on determining an antibiotic s therapeutic utility Although rapid absorption from the injection site is necessary for effective treatment the relationship of blood antibiotic levels and overall therapeutic utility is highly variable The antibiotic has to be present in target tissue in sufficient quantities to inhibit microorganisms responsible for the infection Respiratory infections and other soft tissue infections are difficult to treat effectively partly because many antibacterial agents have poor tissue penetration (Butts 1994) Furthermore these respiratory infections are caused by a diverse variety of organisms many with differing mechanisms of resistance One of the more difficult genre of organisms to effectively treat is the facultative intracellular org anism, such as Mycoplasma spp

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33 The ability of antibiotics to penetrate tissues is best e v aluated b y use of the ratio of the area under the concentration-time curve (AUC) for an antibiotic in the peripheral locus to the AUC for serum (Bergan, 1981 ) There have been several principle approaches in determining tissue concentrations of drugs, of which the most common one is still to use whole tissue homogenates (Bergan 1981 ; Brown et al., 1996 ; Ryan and Cars 1980 and 1983) Data obtained using whole tissue or tissue homogenates infer a uniform distribution of drug throughout the tissue mass i e from the average of at least three distinct tissue compartments : interstitial fluid vascular system and cellular mass Because of that the tissue homogenate method, although widely used is highly criticized for often resulting in under-or o v erestimations of the true levels of drugs This is especially true for the drugs that have relatively poor intracellular penetration such as ~-lactam antibiotics (Brown et al., 1995 ; Ryan and Cars 1980) where the whole tissue le v els are sometimes 5-10 times lower than the drug concentration in the extracellular fluid However for antibiotics with a good cellular penetratio n, drug le v els can be accuratel y measured in the whole tissue (Brown et al., 1995 ; Ryan and Cars 1983) Despite methodological and interpretive problems associated with studies of antibiotic concentrations in tissues it is important to confirm the presence of a drug in significant concentrations in tissues and fluids at a desired site (Ber g ogne-Berezin 1995a) For antibiotics used in the treatment of respiratory infections tissue distribution at sites of potential infection in the respiratory tract has been related to clinical outcome Measurement of antibiotic concentrations achieved in lung parenchyma epithelial linin g fluid bronchial mucosa or bronchial secretions has indicated significant concentrations for

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34 beta-lactams and macrolides. Many respiratory infections are caused by obligate or facultative intracellular pathogens which may be eradicated as a result of intracellular penetration and accumulation of macrolides This has been shown in several models of phagocytic cells and of intracellular antibacterial activities For bacteria multiplying in alveolar macrophages the high concentrations of the new macrolides that can be achieved in extravascular and intracellular fluids should have clinical relevance Macrolide antibiotics in general are known to achieve very high intracellular concentrations within phagocytic cells (Butts 1994) These high intracellular concentrations are important for the treatment of infections caused by intracellular pathogens In contrast streptomycin tetracyclines and chloramphenicol are relatively ineffective against intracellular microorganisms which is believed to be due to their inability to penetrate the phagocytic cell membrane (Butts 1994 ; Shaffer et al, 1953) A mechanism for the accumulation of macrolides in phagocytic cells has been proposed by Renard et al. (1987) Macrolides are considered to be weak organic bases and therefore are unprotonated to a certain degree in the extracellular fluids After intracellular ingestion into a more acidic environment the macrolides become protonated and thus concentrate in the phagosome. Although the specific intra-tissue location of tilmicosin has not been determined explicitly Brown et al (1995) suggested that accumulation within pneumocytes or binding to membranes and /or organelles must occur for concentrations to exceed concurrent plasma concentrations When lungs were perfused with drug-free fluid measured tilmicosin concentrations were similar to concentrations in the lungs that were not

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35 perfused That suggested that tilmicosin was present in lung in locations within the tissue that are not in rapid equilibrium with plasma It was therefore concluded by the authors of that study that the whole tissue concentrations would represent true drug levels in the tilmicosin case and that the whole tissue homogenization is a valid method for studying tilmicosin lung distribution 2.4 1 Physicochemical Properties ofDrugs The chemical and physical properties of a drug are of primary concern to the formulator because these characteristics can affect drug stability absorption and distribution characteristics and ease of formulation (Young 1995) Important characteristics of a drug with regard to its absorption and distribution are molecular size solubility at the site of absorption degree of ionization and relative solubility of its ionized and nonionized forms (Benet and Sheiner 1985) Drugs cross membranes either by passive processes or by mechanisms involving the active participation of components of the membrane (Benet and Sheiner 1985). In the former which is the dominant mechanism of drug passage across membranes the drug molecule usually penetrates by passive diffusion along a concentration gradient by virtue of its solubility in the lipid bilayer For nonelectrolytes the concentration of the free drug is the same on both sides of the membrane after a steady-state is attained For ionic compounds however the steady-state concentrations will depend on differences in pH across the membrane which may influence the state of ionization of the molecule on each side of the membrane and on the electrochemical gradient for the ion

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36 Most drugs are weak acids or bases that are present in solution as both the unionized and ionized species (Benet and Sheiner 1985) The nonionized molecules are usually lipid soluble and can diffuse across the cell membrane while the ionized fraction is usually unable to penetrate the lipid membrane because of its low lipid solubility The distribution of a weak electrolyte is usually determined by its pKa and the pH gradient across the membrane Another factor that influences drug distribution into tissues is drug binding to plasma and tissue proteins In general acidic drugs bind to albumin, while basic drugs tend to bind to a1-acid glycoprotein An agent that is totally or strongly bound has no access to cellular sites of action nor can it be metabolized and eliminated. Bound drugs may accumulate in tissues in higher concentrations than would be expected from diffusion equilibrium as a result of pH gradients because of binding to intracellular constituents or partitioning into lipid (Benet and Sheiner 1985) Drug that has accumulated in a given tissue may serve as a reservoir that prolongs drug action in the same tissue ( as is the case with tilmicosin) or at a distant site reached through recirculation 2.4 2 pH as a Factor in Drug Distribution The pH difference between intracellular and extracellular fluids is small (7 0 vs 7.4) (Benet and Sheiner 1985) but for drugs that have pKa values close to the physiological pH in the body transfer across membrane will be greatly influenced by even slight changes in pH. In general weak bases are concentrated slightly inside of cells while the concentration of weak acids is slightly lower in the cells than in extracellular fluids Lowering the pH of extracellular fluid increases intracellular concentration of weak acids

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37 and decreases that of weak bases provided that the intracellular pH does not also change. These predictions are based upon the assumption that the pH change does not simultaneously affect the binding biotransformation, or excretion of the drug Elevating the pH produces the opposite effects Based on the pH of tissues and the pKa of tilmicosin there will be an equilibrium of its unionized and ionized forms at the physiological pH (Young et al. 1995) The pharmacokinetics of tilmicosin and macrolides in general indicate that as weak organic bases they are highly lipid soluble and partially protein bound the latter being dependent on tilmicosin concentration (Ziv et al, 1995) These characteristics allow tilmicosin to pass through cell membranes freely and distribute quickly throughout the body Similar to tilmicosin tetracycline and erythromycin concentrate highly in human and rat pulmonary tissues (Fournet et al. 1989) High intrapulmonary concentrations of tetracycline and erythromycin could be explained by a passive diffusion dependent on the pH variation between the intraand extratissue compartments the percentage of un-ionized form present and their lipid solubility 2.4 3 Effects of Disease on Drug Distribution It is believed that inflammation has profound effects on the tissue distribution of antibiotics in some cases raising and in other cases lowering the drug levels (Schentag and Gengo 1982 ; Wise 1986). Inflammation increases capillary permeability or the rate of flow thus permitting antibiotics to enter sites usually impenetrable This is the case for example with f3-lactam antibiotics in meningitis where inflammation allows penetration of the drugs into the central nervous system (Mazzei et al. 1991) On the contrary blood

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38 flow to a local area of infection may be decreased, or energy-dependent transport processes can be destroyed or altered by inflammation In mammals tissue damage and/or invasion of pathogenic microorganisms induce systemic changes based on the inflammation process (van Miert 1990) These systemic changes are collectively known as the acute phase immune response (Bauman et al ., 1992 ; van Miert 1990) and are mediated by proteins capable of binding the bacterial cell wall product endotoxin (Bauman et al., 1992) Among the alterations are fever and changes in blood flow to various organs (van Miert 1990) which can influence drug absorption and distribution The intensity of these different reactions may vary depending upon the type of invading microorganism or bacterial toxin present. The passage of drugs across biological barriers may be as simple as movement across capillary endothelium (as discussed in chapter 4 1.) but can also be as complex as penetration of bronchial epithelium intrabronchial mucus bacterial and cellular debris and possibly chronic fibrotic scar tissue (Pennington, 1981 ) The latter series of organic barriers is representative of the blood-bronchus barrier present in many patients with chronic bronchitis Physicochemical characteristics of antibiotics influence their penetration into sputum (Saggers and Lawson, 1966) Benzene rings as found in erythromycin and lipid solubility appear to offer an advantage in terms of penetration. High molecular weight antibiotics seem to have better penetration rates than smaller molecules possibly because of some form of gel filtration that takes place in mucus therefore trapping small molecules in pores in the mucin. In addition the integrity of the blood-bronchus barrier may be damaged by factors such as bronchial inflammation or

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39 bronchial injury the factors that cause anatomical alterations in tissue barriers In bronchitis and bronchopneumonia increased local inflammation may enhance permeability to antibiotic molecules which may therefore gain access to bronchial secretions by leakage across inflamed tissues (Pennington 1981 ) From the disease-induced changes in pharmacokinetics it follows that more attention should be paid to drug disposition in actual patients in whom the drugs are meant to be used 2 4 3 1 Effect of disease on the pharmacokinetics of macrolides Since tilmicosin is a weak organic base it would tend to concentrate in acidic environments such as pneumonia-affected lungs resulting in unequal distribution across membranes The non-ionized fraction is greater in serum than in a more acidic environment because tilrnicosin, as a base has a tendency to move into the more acidic lung tissue where it becomes ionized and therefore trapped A similar mechanism can be used to explain the penetration and accumulation of tylosin in milk, which also has lower pH than plasma (Ziv and Sulman 1973) Burrows (1985) investigated the effects of experimentally induced P haemolyt ica pneumonia on the pharmacokinetics of erythromycin in the calf The distribution and elimination rates of erythromycin were significantly increased and half-life decreased in pneumonia when compared to healthy animals There also was a decrease in apparent volume of distribution with pneumonia while the lung tissue concentrations in the pneumonic lung areas were as high or higher than those in non-affected lung tissues in the same animals

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40 Bergogne-Berezin (1995b) studied tissue pharmacology of azithromycin, which when compared to reference compounds such as erythromycin or roxithromycin is characterized by (1) much lower serum concentrations ; (2) a much longer elimination half-life (48-96 h) ; (3) high and persistent tissue concentrations It was found that in lung parenchyma, azithromycin concentrations were higher and more persistent in infected mice as compared to controls possibly suggesting high intracellular concentrations in polymorphonuclear leukocytes and release of the drug at pulmonary sites of infection Veber et al. ( 1993) investigated the correlation between the pharmacokinetics and efficacy of erythromycin, roxithromycin clarithromycin spiramycin and azithromycin in pneumococcal pneumonia No differences were found between infected and control mice in terms of the serum pharmacokinetic profiles while the lung pharmacokinetic parameters showed more pronounced differences between the two groups with improved tissue penetration of azithromycin and spyramycin in the infected animals When azithromycin was administered to infected mice with severe leukopenia the elimination half life in serum was shorter and the serum AUC was five-fold lower than in normal mice suggesting that leukocytes or leukocyte products may facilitate transport of macrolides to sites of infection In order to further characterize the role of phagocytes in azithromycin tissue distribution, Girard et al. (1996) investigated the correlation of increased azithromycin concentrations with pha g ocyte infiltration into sit e s of localized infection Since azithromycin reaches high concentrations in phagocytic and other host cells this suggests that it may be transported to specific sites of infection When azithromycin was given

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41 during a period of little or no inflammation, there was marginal difference between concentrations found in infected or non-infected sites However when the compound was given during a period of profound inflammation (at 5-24 h after dosing) considerably higher drug concentrations were found in infected sites than in non-infected sites. The data indicated that increased azithromycin concentrations occurred at sites of localized infection which is in correlation with the presence of inflammation and is associated with the cellular components of the inflammatory response It was therefore suggested that phagocytes might be important vehicles for delivering azithromycin to and sustaining azithromycin concentrations at sites of infection Phagocytic cells in general (in the lung these are primarily polymorphonuclear leukocytes and alveolar macrophages) are responsible for the non-specific defense mechanisms of the host to various microorganisms They are activated in response to a chemotactic factor elicited by bacteria undergoing their normal metabolic processes (Butts 1994) Upon contact with any foreign substance ( e g bacteria) the phagocytes typically extend their cell membrane around the bacteria until they are completely enclosed Then the cell membrane containing the bacteria breaks away from the main cell membrane and forms a phagosome within the cytoplasm of the cell The phagosome eventually merges with a larger intracellular vacuole lysosome filled with oxidative enzymes that usually destroy the bacteria (Steinberg et al., 1988) 2.4 3 2 Effect of disease on the pharmacokinetics of other antibiotics Unlike the macrolide antibiotics which always showed improved tissue penetration as a result of infection/inflammation (reviewed above in 2.4 3 1.) various other antibiotic

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42 drugs have shown less uniform response to experimental infections some showing increased tissue penetration, others no change, yet others impaired tissue distribution as a result of disease. The intrapulmonary concentration of tetracycline was found to be significantly increased in rat lungs infected by Legionella pneumophila when compared to non-infected lung (Fournet et al. 1989). In contrast erythromycin was shown to posses the same intra tissue penetration in healthy and infected rat lungs. The activity of ceftazidirne was examined in a murine model of Klebsiella pneumoniae pneumonia (McColm et al. 1986) There was no difference in respiratory tract penetration between uninfected mice and mice infected with K. pneumoniae with regard to the peak concentrations half-lives AUCs, and percentage penetration It was suggested however that perhaps animals with more advanced or chronic infections might show differences in antibiotic kinetics compared to uninfected animals. Pharmacokinetics of cefodizime a newer cephem antibiotic was studied in mice with systemic infection by E. coli and those with respiratory infection by S. pneumoniae (Arai et al., 1989) It was found that in mice with systemic infection disappearance of the drug from plasma and tissues was obviously delayed and there was a decrease in elimination constant and increase in the apparent volume of distribution as compared with the control group In the group with respiratory infection terminal half-life and AUC for the hepatic drug level and terminal half-life for the renal drug level increased but in the other organs including lungs there was no great difference from the control group.

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43 The intestinal wall in Crohn s disease represents a good model for studying the effect of inflammation on the tissue penetration of drugs. Mazzei et al. ( 1991) studied the effect of inflammation on cefotetan tissue distribution in the intestinal wall of patients with Crohn's disease It was found that the mean tissue levels of cefotetan in inflamed intestinal wall were constantly higher than in normal wall Both the MRT and AUC were significantly higher in inflamed wall than in normal, suggesting facilitated penetration of cefotetan into the inflamed intestinal wall. Agapitova and Bobrov (1984) and Agapitova and Iakovlev (1987) studied the effect of protein binding on penetration of antibiotics into infected inflammation foci It was found that the penetration depended on the level of antibiotic binding to serum proteins Low binding antibiotics provided the highest levels of the free antibiotic in both serum and the inflammation foci while the highly bound drugs were not available to penetrate inflamed tissues Influence of Pneumocystis carinii pneumonia on serum and tissue concentrations ofpentamidine was studied in rats (Mordelet-Dambrine et al., 1992) It was found that the serum concentration of pentamidine base administered by the tracheal route was higher in the infected rats than in the control animals while the lung concentration was lower. Respiratory clearance an index of the permeability of the respiratory epithelium, was also higher in infected animals suggesting a more rapid diffusion of pentamidine from the alveolar lumen to the pulmonary circulation The morphologic data suggest that the increase in the permeability of the respiratory epithelium may be due to structural modifications and/or inflammation as a result of pneumocystosis

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44 Vallee et al. (1991) studied the pharmacokinetic parameters offluoroquinolones in a mouse model of S pneumoniae-infected lung Fluoroquinolones in general exhibit good activity at the site of infection When the pharmacokinetics for each drug was compared between the infected and non-infected animals, it was found that all four drugs reached higher lung concentrations and more persistent activity in the infected lungs, suggesting probable trapping of the drugs at the site of infection. Hansen et al (1973) compared trimethoprim concentration in normal and pathological human lung tissue and found significantly higher concentration of trimethoprim in infected than non-infected lung tissue. Similar to tilmicosin trimethoprim has the pKa value of 7 6 and its lung concentrations are also significantly higher than serum Infection does not only have an effect on drug pharmacokinetics as discussed above but it can also affect drugs' adverse behavior Adams et al (1979) examined the effect of the experimental circulatory shock induced by E. coli endotoxin on the acute cardiovascular activities of gentarnicin tobramycin sodium penicillin-G and sodium cephalothin. It was found that the cardiovascular effects of gentamicin and tobramycin were relatively more pronounced during the endotoxin shock than during the control state, while penicillin and cephalothin had no discernible circulatory effects in either control dogs or dogs subjected to endotoxin shock.

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45 2 5 Mycoplasmosis 2.5.1. Introduction The first Mycoplasma species was described by Nocard and Roux (1898) as a causative agent of contagious bovine pleuropneumonia. Mollicutes (wall-free prokaryotes, such as Mycoplasma and Ureaplasma) have subsequently been isolated from humans and many species of animals, including all the common domestic and laboratory animals Mycoplasmal diseases are economically important in agriculture as well as in biomedical research (Simecka et al., 1992), and are primarily associated with diseases of the lung genitourinary tract and joints Chronic respiratory disease primarily due to Mycoplasma pulmonis remains the major intercurrent disease problem encountered in laboratory rodents (Cassell and Hill 1979 ; Lindsey 1986) Recent surveys indicate that M pulmonis infection remains a common problem, not only in conventionally maintained colonies, but also in cesarean-derived barrier-maintained animals (Cassell et al. 1981) Klieneberger and Steabben (1937) and Nelson (1937) first reported the occurrence ofmycoplasma-like organisms in association with the disease The organism was later designated Mycoplasma pulmonis (Edward and Freundt 1956) Besides causing the respiratory disease (Cassell et al. 1986 ; Lindsey and Cassell 1973 ) M pulmonis causes genital disease in both rats and mice (Brown and Reyes, 1991 ; Brown and Steiner 1996 ; Simecka et al. 1992) Few diseases oflaboratory animals have been as troublesome to research workers or as enigmatic to microbiologists as murine respiratory mycoplasmosis (MRM) This disease of laboratory rats and mice is caused by M pulmonis but its expression is

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46 markedly influenced by a variety of environmental host and organismal factors (Cassell 1982 ; Lindsey 1986 ; Lindsey et al ., 1985 ; Simecka et al 1992) The presence of mycoplasmas in animal facilities is practically synonymous with the presence of rats and mice (Cassell 1982 ; Lindsey 1986) More insidious than the direct loss resulting from the respiratory mycoplasmosis (due to morbidity and mortality) is the undermining of the validity of scientific experiments that utilize these animal species (Lindsey et al. 1971) Mycoplasmas are difficult to diagnose and except for the terminal stage of the disease (when weight loss roughened hair coat serosanguinous nasal and ocular discharges and dyspnea are seen) the natural form ofMRM is usually a clinically silent disease (Cassell and Hill 1979 ; Simecka et al. 1992) However the infection induces changes in physiology and behavior and even subtle changes can compromise research utilizing rats and mice (Simecka et al. 1991) 2 5 2. Experimental Respiratory Mycoplasmosis in Rodents E x perimental respiratory mycoplasmosis resulting from M pulmoni s infection in rats provides a useful model for the study of immunological and inflammatory mechanisms operative in the respiratory tract (Simecka et al., 1991 ) The experimental diseases represent useful models for the study of various human and animal diseases particularly mechanisms involved in chronic pulmonary inflammation and reproductive failure (Cassell 1982 ; Simecka et al. 1991) 2 5 3 Clinical Signs and Virulence Mycoplasma disease can vary from subtle low-level disease that is subclinical to overt severe disease that can result in death of the host (Simecka et al., 1992) By varying

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47 the dose of M pulmoni s in mice it is possible to produce three reasonably distinct clinicopathological syndromes : (1) minimal lesions that regress spontaneously ; (2) an acute disease with edematous fluid and large number of neutrophils in alveolar spaces pulmonary congestion and hemorrhage with occasional pleuritis ; (3) chronic bronchopneumonia (Lindsey and Cassell 1973) In general the most consistent features of respiratory mycoplasmosis are lymphoid hyperplasia and chronic inflammation (Simecka et al. 1992) The experimental disease in rats is also dose-dependent but rats do not develop the acute disease (Cassell and Hill 1979) In the rat the full spectrum of respiratory mycoplasmosis develops more slowly ; if unaltered by other factors, it can take as long as 265 days (Whittlestone et al., 1972) It has been shown that strains ofrats differ in susceptibility to M pulmonis respiratory disease (Davis et al ., 1982 ; Davis and Cassell 1982) Davis et al. ( 1982) found that differences in lesion severity and progression were associated with changes in lung lymphocyte populations. Lung lesions in LEW rats when compared to F344 rats developed earlier after infection became more severe and were characterized by continued proliferation of all classes of lymphoid cells (Davis and Cassell 1982 ; Davis et al., 198 2; Simecka et al., 1991). Virulence of mycoplasmas appears to be related to the ability of the organisms to evade nonspecific defense mechanisms (Davidson et al. 1988) In the lungs mucociliary clearance and intrapulmonary killing mediated by alveolar macrophages are the major processes responsible for nonspecific resistance (Green and Goldstein 1966)

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48 2 5.4 Pathogenic Mechanisms The pathogenesis of mycoplasma disease is a complex process influenced by the genetic background of both the host and the organism environmental factors and the presence of other infectious agents (Simecka et al. 1992) Although many virulence factors have been suggested for various mycoplasmas, there is no clear case of cause and effect between these factors and pathogenicity There are a number of attributes of mycoplasmas that are likely to affect disease pathogenicity, including the ability to attach to mucosa! surfaces to cause cell injury to vary phenotype at a high frequency and to modulate and resist the host immune response (Simecka et al. 1982) The interaction of mycoplasmas with eukaryotic membranes is likely the initial event in most infections However it is quite unlikely that mere attachment to host cell surfaces could produce the wide variety of cellular changes associated with M pulmonis infections i e loss of cilia cytoplasmic vacuolization disruption of mitochondria epithelial hyperplasia and metaplasia and giant cell formation (Cassell and Hill, 1979) Mycoplasmas establish intimate contact with host cells which may lead to cell injury through production of toxic substances or deprivation of nutrients (Simecka et al. 1992) 2. 5 5 Antimicrobial Susceptibility of Mycoplasma It is commonly accepted that the first choice in treatment of Mycoplasma infections involves therapy with tetracyclines or erythromycin (Gray 1984 ; Sande and Mandell 1985 ; Vogel 1995) Treatment of pneumonia with either tetracycline or macrolides results in a shorter duration of fever cough, malaise fatigue pulmonary rales

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49 and roentgenographic changes in the lungs (Levieil et al., 1989 ; Sande and Mandell 1985) Among macrolides azithromycin was found to be significantly more effective against M. pneumoniae than erythromycin or clarithromycin in the same regimens (Ishida et al 1994). In domestic animals tylosin has been found to have a potent antimycoplasmal activity which has been retained and even improved in its newer derivative tilmicosin (Barragry 1994). Ose (1987) reported that in vitro antibacterial activity of tilmicosin includes M hyopneumoniae M hyorhinis, M gallisp e ticum M dispar M alkal e sc e ns, M bovirhinis, and M bovoculi The in vi v o effectiveness of tilmicosin against mycoplasma infections in cattle has been mostly reported in association with bovine respiratory disease (Barragry 1994 ; Gourlay et al ., 1989 ; Musser et al ., 1996) In rodents a few investigators have claimed success in eliminating M pulmonis infection through programs involving rigid selection administration of antibacterial drugs vaccination or principles of cesarean derivation combined with strict isolation procedures (Cassell and Hill 1979). With regard to antibacterial treatment tylosin administration in drinking water was found effective in the treatment of M pulmoni s pneumonia in rats (Carter et al 1987) The slow release form of oxytetracycline in rats was able to maintain serum levels greater than the minimum inhibitory concentration of M pulmonis (Curl et al 1988) However despite different approaches in prevention and treatment mycoplasmosis continues to be one of the major intercurrent disease problem encountered in laboratory rodents ( Cassell et al., 1981)

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CHAPTER3 MATERIALS AND METHODS 3 1 Determination of Tilmicosin Concentrations Quantitative analysis of tilmicosin concentrations in serum and lung tissue was done using high pressure liquid chromatography (HPLC) with ultraviolet spectroscopy An analytical method for determination of tilmicosin concentrations in various tissues was initially developed by the Eli Lilly Research group (Peloso and Thomson, 1988) but has been modified for this study. 3 1 1 Chemicals and Reagents Dried tilmicosin reference standard (technical grade) containing tilmicosin base was provided by Eli Lilly1 < Appendi xA>, and was kept refrigerated at 4 C (Lot# RS0164) The defined potency of the standard was: 756.7 mg cis and 130.4 mg trans isomers of tilmicosin per gram when dried for three hours at 60 C under a vacuum The reference standard was used in method validation study as well as for preparation of working tilmicosin standard solutions for calibration and quality control samples The commercial preparation of tilrnicosin MICOTIL 300 (Elanco2 ) was used in all animal studies This was kept at room temperature with minimal exposure to light due to known photosensitivity of tilmicosin (Eli Lilly Pers Comm ) The active ingredient in MICOTIL 300 is tilmicosin phosphate and each milliliter contains : 300 mg tilmicosin 50

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51 base 250 mg propylene glycol, phosphoric acid to adjust the pH to 5 8-5.9 and water for injection to 1 ml All chemicals used for preparation of mobile phases for HPLC, with exception of dibutylamine (methanol water acetonitrile phosphoric acid), were HPLC-grade and were purchased from Baxter3 Dibutylamine and most of the chemicals used for both solid phase and liquid-liquid extraction procedures ( chloroform, sodium chloride sodium phosphate di basic, sodium hydroxide potassium phosphate monobasic) were of analytical grade and were purchased from Fisher4 Potassium phosphate dibasic and carbon tetrachloride were also of analytical grade and were purchased from Aldrich5 3 1 2. Tissue Preparation Samples were prepared for HPLC analysis by means of either solid phase or liquid liquid extraction for serum and lung tissue, respectively 3 1.2.1 Extraction oftilmicosin from serum For solid phase extraction (SPE) Bond Elut Varian6 cartridges were used Tilmicosin was extracted from serum using cartridges with 500 mg C 18 packing and eluted in the Varian Vac-Elut vacuum manifold The cartridges were activated with methanol and water prior to addition of serum In the sheep and cattle experiments 2 ml of serum were used whereas in the rat study the amount of serum was decreased to 1 ml After the serum was drained through the cartridge by applying vacuum, the cartridge was washed with water then with 5% ammonium hydroxide in water The water wash was then repeated Tilmicosin was finally eluted from the cartridge with 2 ml of a mixture of 5% methanol in I 00% acetic acid glacial. The collected eluent was evaporated to dryness

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52 under a nitrogen stream and reconstituted in sample diluent The sample diluent consisted of 475 ml water 500 ml methanol and 25 ml of 1 M dibutylammonium phosphate (DBAP) The sheep and cattle samples were reconstituted to 1 ml, whereas rat samples were reconstituted in 0 5 ml of sample diluent. After the extraction the reconstituted samples were injected into the HPLC system. 3 1 2 2 Extraction of tilmicosin from lung tissue Liquid-Liquid extraction of tilmicosin from lung tissue samples was done from the whole lung specimens, which had the approximate average weight of 1 g After determining the weight of sample lung tissue was homogenized in the Polytron 7 tissue homogenizer with 4 ml of methanol. Homogenate was centrifuged at 485 g for 10 minutes and the resulting supernatant was collected The pellet was reconstituted with 4 ml of methanol rehomogenized and centrifuged at 485 g for 10 minutes This new supernatant was added to the previously collected methanol fraction to which 5 ml of I 0% sodium chloride was added The pH of the confluated methanol fraction was adjusted to 2 5 with IM hydrochloric acid. The solution was then transferred to a 30-ml separatory funnel and 3 ml of carbon tetrachloride was added The solution was shaken for 1 minute and the bottom layer discarded Another 3 ml of carbon tetrachloride was added the solution was mixed for 1 minute and the bottom layer discarded The collected supernatant was then alkalized to pH 9 0 with IM sodium hydroxide and tilrnicosin in the solution was extracted into chloroform. This was done by mixing the solution in the separatory funnel with 3 ml of chloroform for 1 minute and collecting the bottom Layer. Then another 3 ml of chloroform was added and mixed for 1 minute Again the bottom

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53 chloroform layer was collected. The total collected chloroform was evaporated to dryness under nitrogen stream, and reconstituted in 0 7 ml sample diluent. Extracted samples were then ready for injection into the HPLC system 3 1 3 High Pressure Liquid Chromatography Reversed phase chromatographic analysis was performed using a Beckrnan8 System Gold apparatus which consisted of three separate modules : a high pressure binary pump (model 126) a spectrophotometric UV detector (model 166) and an autosampler (model 502) equipped with a 50 l injection loop A Regis9 Hi-Chrom Reversible HPLC column (25 cm x 4 6 mm) with 5 ~L phenyl particles was used for tilrnicosin analysis To prolong the life of the column a guard column (Regis phenyl guard column 4 6 mm x 5 cm) had been placed in front of the analytical column and was replaced when a loss of peak resolution on the chromatogram became visible Beckman System Gold software (version 6.01) was used for chromatogram and data collection and storage. 3 1 3 1 Chromatographic conditions A modified Peloso and Thomson method was used for this analysis with a binary instead of tertiary pump and with slightly altered mobile phase compositions to obtain improved chromatography The mobile phases were prepared as follows : mobile phase A : a mixture of acetonitrile/water (50/50 ; v / v) adjusted to pH 2 5 with orthophosphoric acid ; mobile phase B : water adjusted to pH 2 5 with orthophosphoric acid ; mobile phase C: a mixture of 80 ml IM DBAP in water with volume brought to 1000 ml DBAP was prepared by adding 168 ml of dibutylarnine to 700 ml of water and

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54 by adjusting pH to 2 5 with orthophosphoric acid After the solution was cooled to room temperature the volume was brought to 1000 ml. Solvent Mix II was then prepared as a mixture of 55% mobile phase A, 30% of mobile phase B and 15% of mobile phase C. Mobile phase A and solvent mix II were the two final mobile phase solutions used for chromatography in a gradient manner. All mobile phases were filtered through a 0.45 m pore filters and degassed in an ultrasonic bath before use in the HPLC apparatus The time-table for the gradient conditions used for tilmicosin detection, with the pump A pumping mobile phase A, and pump B pumping solvent mix II is as follows : Time (min ) Pump A% PumpB% (mobile phase A) (solvent mix II) 0 100 0 4 0 100 (ramping for 1 min ) 12 100 0 ( ramping for 1 min ) 20 End of run The flow rate was 1 5 ml/min. and the run time 20 minutes The detector wavelength was set at 280 nm The average retention time for tilmicosin from both serum and lung tissue samples using this method was 11 minutes E x amples of chromatograms of a tilmicosin-free sheep serum sample and a sheep serum sample fortified with tilmicosin at the concentration of 1 0 g/rnl are shown in Figure 3 1

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A B -0.0100 0.00 i 10.00 l 19,99 0.00 10.00 19.99 I 7 I i I 1 I l I 55 Absorbance 0.0000 Figure 3-1: Examples of the HPLC chromatograms 0.0100 10.20 til1icosin Chromatograms of a tilmicosin-free sheep serum sample (A) and a sheep serum sample containing tilrnicosin at the concentration of 1.0 g/ml (B)

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56 3.1.3.2 Calculation ofHPLC results With each analytical run a calibration curve with at least 4 and up to 6 concentration levels was established The standards were prepared from aqueous tilmicosin solutions ranging in concentrations from 2 to 100 g/ml For the serum sample calibration curve tilmicosin-free serum samples were fortified with tilmicosin standard solutions to achieve final calibrator concentrations of 0 05 0 1 0.2 0 5 1.0 and 2 0 g/ml. Final concentrations for the lung tissue calibration curve were 0 1 0 5 1.0 5 0 and 10 0 gig Both lung and serum samples for calibration curve were fortified at the time ofHPLC analysis and carried through the extraction procedure as described earlier (3. 1 2 1 and 3 1 2.2 ) Calculations in the HPLC analysis were conducted blindly Since the calibration curve was found to be linear in the concentration range of interest, a least-square straight line was drawn through all data points of the calibration curve to determine the detector response factor (y = ax + b ; where y represents concentration, xis analytical response a is slope bis intercept). The analytical response was defined as the chromatographic area of a peak of interest (i e peak area) and calculations were always based on that parameter, rather than peak height. Chromatographic quantitation was done by comparing the peak areas of the standards with unknowns 3 1 3 3 HPLC method validation study A validated assay method is pivotal to the acceptability of any pharmacokinetic study The Food and Drug Administration recommends that the following parameters should be assessed in the process of method validation (in : Recommendations for

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57 evaluating analytical methods Center for Veterinary Medicine US FDA, 1994) : concentration range and linearity limit of detection (LOD) limit of quantitation (LOQ) specificity accuracy (recovery) precision (reproducibility) and analyte stability Concentration range and linearity test should be performed to determine that the new matrix does not contain elements that would interfere with the accuracy or sensitivity of the method No data points are allowed to be extrapolated later in the analysis if they fall below or above the calibration curve points LOD is the lowest concentration that can be determined to be statistically different from background LOQ is the lowest concentration that the method can measure reliably Specificity determines whether the matrix of interest contains any elements that could interfere with elution of the peak of interest. Accuracy is a measure of the exactness of measured value to a known or actual value Precision is a measure of the degree to which several analytical runs are reproducible and it determines the magnitude of random errors in the method Stability of the analyte in the biological matrix (serum or tissue) should be determined under the conditions of the experiment and would include any period for which samples were stored before analysis 3 1.3 3 .1. Linearit y and range For the linearity and range seven replicates of the calibration curve were analyzed and the ability to obtain linear curves over the entire range of expected concentrations for each calibration curve was demonstrated An e x ample of a typical calibration curve is shown in Figure 3-2.

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Utilities Calibrations Uie11 or nodify-calibration results: --. r.o.pooent 1: tilaicosin Y = 0.714394 X + 0.828489 58 llBRATHlt Coefficient of Deterllinatioo = 0.99')09 2.200 V C 0 u > 0 000 ~;------::;---.--------D .053 X -~reo 3 DU Figure 3-2 : Example of an HPLC calibration curve Hetood: I CRTTLE3 I Dian: LR fm:. ha I 1 1 0 .09IOOO 8 .058399 I 2 1 0.10fBI 0.124428 I 3 1 0.2'BBJ 8.277112 I 4 1 0.5lDm 8.632843 I 5 1 1.tlDBl 1.329594 I 6 1 2.~ 2.796189 The calibration curve is derived from the chromatograms of the HP L C anal y sis of tilmicosin concentrations i n the sheep serum fortified at 0 1 0 2 0 5 1.0 and 2 0 g/ml.

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59 3 1.3 3 2 Limit of detection Limit of detection was determined based on seven replicates of the calibration curve with the means and standard deviations for each level including the zero control. The LOD of a method is defined as that point which is 3 standard deviations above the analytical response (peak area in this case) in the zero drug sample It was calculated using the following equation : LOD = 3 cr0 / m where cr0 is the standard deviation of the analytical response at zero concentration and m is the slope of the analytical curve Calculated LOD for the method was 0 0026 ~Lg/ml (Table 3-1) 3 1 3 3 3 Limit of quantitation Limit of quantitation of a method is defined as that point which is 10 standard deviations above the analytical response in the zero drug sample It was calculated using the following equation : LOQ = 10 cr0 / m Calculated LOQ for the method was 0 0087 g/ml (Table 3-1) but based on the expected blood concentrations oftilmicosin in sheep from literature and our pilot study an LOQ of 0 05 g/ml was adopted for further validation parameters because lower concentrations were not expected 3 1.3 3.4 Specificity Specificity was determined by analyzing 6 independent sources of control matrix (as described below) and demonstrating that the calibration curve was comparable to the one produced under chemically defined conditions (i e that there was nothing in the matrix that would interfere with the elution of the peak of interest) A diverse source of sheep blood was used in the validation study including the Health Science Center Animal Resources Department (HSCARD) Animal Sciences Department Veterinary Medicine

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60 Table 3-1: Validation results I Determination of the limit of detection (LOD) and limit of quantitation (LOQ) of the HPLC method for analysis of tilmicosin in serum The LOD and LOQ were calculated from the values of mean and standard deviation (St. Deviation) based on 6 replicates of tilmicosin-free sheep serum samples Sample# Peak Area Calibration Curve at Zero Cone. Slope 1 0 00049 0.774 2 0 00216 0.951 3 0 00032 0.804 4 0.00145 0.791 5 0 00076 0 858 6 0 00037 0 854 Std. Deviation 0.000734 Mean 0 000925 0 839 LOD 0.002624 LOQ 0.008745

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61 Teaching Hospital (VMTH) and Department of Physiology There was never any interference found in any chromatograms around the time of tilmicosin elution in any of the samples 3 1 3 3 5 Accuracy Accuracy was evaluated using three concentrations of analyte one being the LOQ (0 1 g/ml for the sheep samples analysis ; 0 05 g/ml for the cattle samples) one in the middle of the range of the standard curve ( 1 0 g/ml) and one at the high end of the standard curve (2 0 g/ml) The accuracy of the method based upon the mean value of si x replicate injections was within 80-120% of the nominal concentration at each level. The accuracy was calculated as the ratio of the calculated concentration of the sample analyzed as 'unknown and actual concentration at which the sample was fortified (Table 3-2) 3.1.3 3.6 Precision Precision was determined by analyzin g 6 replicates of samples of known concentration at three different concentration levels (as described for accuracy) and expressing them as the coefficient of variation (COY= standard deviation / mean) The COY of six replicate injections was within +/-10% for all three levels of concentrations (Table 3 2) 3.1.3 3 7 Analyte stability. To evaluate stability serum samples were fortified at 3 concentration levels (high, medium and low ; as described for accuracy) on the day that experimental samples were collected The stability samples were then stored at -20 C and run together with unknowns at the time of the actual sample anal y sis

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62 Table 3-2 : Validation results II Accuracy and precision data for the HPLC method for analysis of tilmicosin in serum The results are presented as the arithmetic means of 6 replicate samples for each, low (0 05 g/ml for the cattle experiment and O 1 g/ml for the sheep experiment) medium, and high level of concentration Accuracy is expressed as percentage and the coefficient of variation (COV) is a measure of precision based on standard deviation (St.Dev. ) Concentration Sample# Precision Accuracy ua/ml % 1 0 058 99 5 2 0 057 98 0 LOWI 3 0 066 116 2 (0.05 g/ml) 4 0 .061 105.4 5 0 060 102 3 6 0 068 119 8 St.Dev. 0 004 Mean 0 062 106 9 cov 7 244 1 0 106 105 7 2 0 109 109 LOW II 3 0 092 91. 7 (0.1 g/ml) 4 0 097 96. 9 5 0.097 96.8 6 0 096 95 7 St.Dev. 0 007 Mean 0 099 99 3 cov 6 646 1 0 984 98 4 2 0.977 97 7 MEDIUM 3 1 079 107 9 (1.0 g/ml) 4 1.115 111.5 5 0 979 97.9 6 1 017 101. 7 St.Dev. 0 059 Mean 1 025 102.5 cov 5 720 1 2 .061 103.1 2 1 858 92. 9 HIGH 3 1 889 94.4 (2 0 g/ml) 4 1 847 92 4 5 2.054 102 7 6 1.876 93 8 St.Dev. 0 099 Mean 1.931 96 55 cov 5 145

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63 The longest time period for which samples were tested for stability was 36 days There was no decrease in the detector response when those stability samples were compared to freshly fortified and analyzed samples ; their concentrations were calculated as 120 107 and 109% (as calculated in accuracy) of the nominal values for each concentration 3.1.3.4 Quality control Besides the method validation study which was performed before the actual animal experiments to be in compliance with the Good Laboratory Practices (GLP) procedures (Federal Register 1987) quality control (QC) samples were analyzed contemporaneously with test samples evenly dispersed throughout each analytical run For the QC samples tilmicosin-free serum samples were fortified with tilmicosin at three different levels of concentrations (6 replicates per level) The mean and standard deviation for each level was calculated and compared to the standard values of the nominal standard material The mean for each concentration level agreed to within+/20% and the standard deviation to within the established precision (COY of 10% for concentrations at or above 1.0 g/ml, and 20% for concentrations below 1.0 g/ml) A summary of quality control results is shown in Table 3-3 3 .1.3. 5 Estimation of pharmacokinetic parameters The following pharmacokinetic parameters for tilmicosin were determined for each animal in both cattle and sheep groups using standard non-compartmental data analysis techniques (Gibaldi and Perrier 1982) : elimination rate constant (ke);

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64 Table 3-3 : Summary of the quality control results Results are presented for each analytical run separately (3 analyses in the sheep and 3 in the cattle group) The results are presented as the arithmetic means of 6 replicate samples for each low (0 05 g/ml for the cattle experiment and 0.1 g/ml for the sheep experiment) medium and high level of concentration Accuracy is expressed as percentage and the coefficient of variation (COV) is a measure of precision based on standard deviation (St.Dev .) Cattle Sheep FDA Limits Analysis Low Medium High Low Medium High Mean 0 047 0 915 2.041 0 102 0.918 1.926 Accuracy% 80 -120 % I 93 1 91. 5 102.1 102.4 91. 8 96.3 St. Dev. 0 005 0 .041 0 082 0 009 0 043 0 200 COV% 10 (20) % 10 4 4 5 4 0 8.5 4.6 10.3 Mean 0 049 1 080 2 017 0 118 1 024 2 106 Accuracy% 80 -120 % II 98.4 108 0 100 9 117.7 102 4 105 3 St. Dev. 0 .001 0 017 0 038 0 005 0 047 0.207 COV% 10 (20) % 1.8 1.5 1 9 4 3 4 6 9 8 Mean 0 055 1 017 2 224 0 114 1.010 2.099 Accuracy% 80 -120 % 111 110 6 101. 7 111 2 114 4 101.0 104.9 St. Dev. 0 .001 0.022 0 046 0 003 0 .041 0.051 COV% 10 (20) % 1 5 2 2 2 1 2 7 4 0 2 4

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65 half-life (t112 ) ; area under the serum concentration versus time curve (AUC) ; area under the first-moment curve (AUMC) ; mean residence time (MRT) ; maximum drug concentration in serum (Cmax) time at which Cmax was reached (tmax) ; clearance (Cl) ; volume of distribution (V d) Calculation of pharmacokinetic parameters was performed using a commercial spreadsheet program (Excel by Microsofl:10 version 5 0c) The equations used in the pharmacokinetic analysis as written for Excel are listed in Appendix B. The overall elimination rate constant was calculated from the terminal slope of a natural log-linear plot of the individual serum concentration vs time curve Half-life the time necessary for the concentration of drug in the plasma to decrease by one-half was determined from the value of kc. Area under the serum concentration vs. time curve and area under the first moment curve were calculated by the trapezoidal rule Mean residence time the average time a drug spends in the body was calculated from the values of AUC and AUMC Clearance describes the removal of drug from a volume of plasma in a given unit of time and was calculated based on the dose and AUC. The apparent volume of distribution is the hypothetical volume of serum in which the drug distributes and was determined from the clearance and elimination rate constant. Table 3-4 shows the equations used to calculate all noncompartmental parameters described above

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66 Table 3-4 : Equations used to calculate noncompartmental pharmacokinetic parameters Abbreviations used in th e table: k., = elimination rat e constant ; t112 = half-life ; A U C = area under the serum concentration v ersus time curve ; AUMC = area under the first-moment curve ; MRT = mean residence time Cmax = maximun1 drug concentration in serum ; tmax = time at w hich Cru..~ w as reach e d ; Cl = clearance ; Vd = v olume of distribution ; f = bioa v ailabili ty slope ke [h-1 ) t11 2 [h] PK Parameter AUC [g/ml"'h] AUMC [g/ml"'h2 ] MRT [h] CL/f [1/h] Vd/f [I] Equation m = (ln C 2 -ln C 1) / h -t 1 ke = -slope tl/2 = 0 693 I ke A U C = J C(t) dt AUMC = J C(t) t dt AUMC / AUC Dose / AUC Cl/ f ke

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67 The same pharmacokinetic parameters for tilmicosin were calculated in the rodent study using the mean serum concentration data for each time point for both infected and non-infected animals In the pharmacokinetic modeling of the sheep and cattle data serum concentration vs time profiles for each individual animal were fitted to a three-exponential equation corresponding to a two-compartment model with first-order input as proposed for tilmicosin : C =A* e -a t + B e P t -(A + B) e -k a t where C is the serum drug concentration at time t coefficients A and B are intercept terms ; exponents a. and are the hybrid rate constants which are functions of the microconstants for distribution and elimination ; and the ka is the absorption rate constant. The equation was fitted to the experimental data by use of a curve stripping and fitting program (Scientist version 2 0 by Micromath11) and was applied separately for each individual animal. The program applies least squares fitting using a modified Powell algorithm to find a local minimum possibly the global minimum, of the sum of squared deviations between observed data and model calculations The dependent variable ( concentration) was weighed if needed to get a better fit and a weight factor of 1 or 2 was applied In general data weighting is applied to transform the statistical error term into a fractional error rather than an absolute error If weighting was employed it transformed data so that each point was assigned a weight inversely proportional to the absolute value of the data raised to some power given by the weighting factor. The same principle was applied in the non-compartmental pharmacokinetic analysis to assign more

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68 weight to lower concentration points in order to have a better fit for the low range where most of the data were placed rather than for the high range. 3 .2 Cardiopulmonary Monitoring in Sheep The following cardiopulmonary parameters were monitored in sheep to study possible adverse effects of tilmicosin administration : heart rate ; electrocardiogram (ECG) respiratory rate ; systolic blood pressure ; mean blood pressure diastolic blood pressure Heart rate was determined from the ECG by counting QRS complexes and was automatically recorded by the Datascope Passport 1 2 multichannel oscilloscope together with the blood pressure data ECG tracings were collected using modified Lead II electrodes which were placed at the sternal notch on the ventral neck, on the sternum at the level of the elbow and on the back between the shoulder blades for the left-a~ right-a~ and left-leg electrode respectively Blood pressure measurements were obtained using the indirect oscillometric method with a child-size pressure cuff ( 18-27 cm) placed on a front leg in the antebrachial region The osilloscopic method utilizes microprocessor-controlJed electro-pneumatic acquisition system which senses displacements of the artery wall (Weiss et al., 1995) Amplified digitally converted filtered and transformed data were displayed on the screen as digital pressure values for

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69 the systolic (SBP) diastolic (DBP) and mean blood pressure (MBP) Mean blood pressure was calculated automatically as : MBP = DBP + 1 / 3 pulse pressure Respiratory rate was obtained by observing the excursions over a 1-min time period 3 3 Animal Handling 3 3 1 Experimental Animals 3 3 1.1. Sheep Ten cross-bred non-pregnant adult female sheep were used for the study The original source for the animals was a livestock market in St Angelo TX, and the animals had been at the UF Animal Sciences Department farm for at least 6 months before the study was commenced The age range of the animals was 2-6 years and the body weights ran g ed between 120 and 170 lbs (54 to 77 k g ) The sheep were individuall y identified b y ear tags 3 3 1.2 Cattle Ten young Angus cows recently weaned of their calves were used for the cattle experiment. The source of animals was the UF Animal Sciences Department Beef Research Unit. The e x periments were conducted at the Animal Sciences Department s Physiology Unit Colson Tract where the animals had been maintained for 2 months before the study The cows were aged between 2-3 years with body weights ranging between 865 and 1065 lbs (392 483 kg) They were individuall y identified by ear tag s

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70 3 3 .1. 3 Rats A total of 72 LEW pathogen-free rats was used in the rodent study Animals were purchased from Harlan Sprague Dawley13 (Indianapolis IN), and were allowed to acclimate for at least 1 week before study initiation All animals were young adult females approximately weighing 100-120 g Rats were housed in a barrier facility with limited access to protect their pathogen free status They were housed in the sterile rnicroisolator cages (Lab Products14 ) supplied with sterile hardwood chip bedding 'Beta-Chip"15 and were provided with sterile autoclaved food (PMI rodent diet #50101 6 ) and water ad libitum The rats were kept on a 12: 12 light:dark cycle (6 am : 6 pm) and their cages were changed twice per week. 3 3.2 ExperimentalMycoplasmapulmonis Infection in the Rodent Study Experimental infection with Mycoplasma pulmonis was used to induce a chronic respiratory infection in rats The inoculation dose of 106 colony forming units ( CFU s) of M pulmonis (strain UAB X1048) was used so that all rats would acquire an infection resulting in grossly visible lung lesions but without making them too ill (showing sniffing roughness of fur hunched posture lethargy or inappetence ; Davidson, Pers Comm ) The animals were lightly anesthetized with 0 1 ml of ketarnine /xy lazine mixture per animal given intramuscularly (0 .15 ml of 100 mg/ml xylazine and 10 ml oflO0 mg/ml ketarnine) The animals were inoculated intranasally by placing 25 l of a broth culture into each nostril Controls were inoculated with sterile medium

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71 3 3 3. Drug Administration MICOTIL 300 is a 30% solution oftilmicosin in propylene glycol and the label indicates a single subcutaneous injection of 10 mg/kg ( 1 5 ml MICOTIL 300 per 100 lbs ) (Blanco Animal Health 1994). 3.3.3.1. Tilmicosin administration in sheep and cattle Both sheep and cattle were administered a single dose of tilrnicosin at the rate of 10 mg tilmicosin free base equivalents per kg body weight. The drug was injected subcutaneously between the scapulae or on both sides of the neck for the sheep and cattle groups respectively Because of the larger volume of drug administered to cattle the dose was divided between the two sides of the neck to avoid any possible tilmicosin-induced tissue irritation 3 3 3 .2 Tilmicosin administration in rats On Day 31 after the initial treatment (Mycoplasma or control) both infected and non-infected rats were administered a single subcutaneous dose of tilmicosin at a level of 20 mg/kg body weight. MICOTIL 300 was diluted with propylene glycol to achieve a dosing solution of 10 mg/ml

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CHAPTER4 EXPERIMENT AL DESIGN 4 1 Introduction The aim of this study of pharmacokinetic and pharmacodynamic properties of tilmicosin consisted of three parts : 1 To compare the pharmacokinetics of tilmicosin in two ruminants, cattle and sheep, following subcutaneous injection ; 2 To investigate cardiopulmonary effects of tilmicosin in the sheep These data were collected simultaneously with ( 1 ) so the relationship of any change with tilmicosin concentration could be assessed ; and 3 To assess the prolonged retention time of tilmicosin in treating respiratory infections the lung tissue distribution of tilmicosin was studied in the rat. The rat was used as a model because controlled experimental respiratory infection could be established using Mycoplasma pulmonis The sheep and cattle study was performed in compliance with the GLP recommendations (Federal Register Sept. 4 1987 ; 21 CFR Part 58). The clinical part of the sheep study was performed in the HSCARD and the cattle study at the Animal Sciences Department s Beef Research Unit farm The HPLC analysis for determination of tilmicosin in sheep and cattle serum was performed in the Department of Physiological 72

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73 Sciences College of Veterinary Medicine, University of Florida. The project was approved by the University of Florida (UF) Institutional Animal Care and Use Committee (IACUC) prior to study initiation with a protocol approval number 4102 (Appendix G) The in vivo part of the rodent study was performed in the HSCARD s Infectious Diseases Suite and the tissue tilmicosin assay in the Department of Physiological Sciences College of Veterinary Medicine, University of Florida This project was approved by the UF IACUC prior to study initiation with a protocol approval number 8072 (Appendix G). 4 1 1 Comparative Pharmacokinetics of Tilmicosin in Sheep and Cattle The sheep experiment was of a cross-over design with the animals randomized into initial treatment and placebo groups and a two-week washout period between the treatments There was a single study factor (drug treatment) with two levels (tilmicosin and placebo i e saline administration). Tilmicosin was administered as described in paragraph 3. 3 3 ., while the control animals received an equal volume of saline subcutaneously The group that started the experiment with the administration of saline received tilmicosin two weeks later and vice versa Blood samples were collected for analysis of tilmicosin concentrations in serum and cardiopulmonary parameters were monitored concurrently as detailed in Table 4 1 For both serum drug concentration determinations and pharmacodynamic monitoring the response variable was measured on a continuous scale.

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74 Table 4-1: Schedule for data collection during the sheep experiments Hem I Chem represents the blood sample collected for hematology and chemistry analyses ; RR denotes respiratory rate measurement ; HR is heart rate measurement ; BP is blood pressure measurement; Temp is body temperature measurement; Attit. is the description of attitude ; Appet. is the description of appetite ; Elimin. is the description of elimination patterns .; Behav is the description of behavior ; Abnor. is a list of any abnormalities observed Time HPLC Hem I RR HR BP Temp. Dep./ Appet. Elimin. Behav Abnor. Point sample Chem Attit. 0 min + + + + + + + + + + + 5 min + + + + + + + + + + 15 min + + + + + + + + + + 30 min + + + + + + + + + + 1 hr + + + + + + + + + + 1.5 hr + + + + + + + + + + 2 hr + + + + + + + + + + 3 hr + + + + + + + + + + 4 hr + + + + + + + + + + 5 hr + + + + + + + + + + 6 hr + + + + + + + + + + 8 hr + 10 hr + 12 hr + 18 hr + 24 hr + + + + + + + + + + 30 hr + 36 hr + 48 hr + + + + + + + + + 60 hr + 72 hr + + + + + + + + + + 96 hr + + + + + + + + +

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75 In the cattle study all cows received a single subcutaneous dose of tilmicosin at the beginning of the experiment with blood sampling being performed at the same time points as described for sheep (Table 4 1) Experimental design, therefore was without factors or blocking and the response variable (tilmicosin blood concentration) was measured on a continuous scale Physical examinations were performed on all study animals prior to entrance to the study The animals had to be in normal health in order to be accepted for the study Samples for blood analyses were collected and analyzed by the University of Florida VMTH Clinical Pathology Service The analysis included a complete blood count (CBC) and blood chemistry panel. The value for each parameter was required to fall within the mean 95% confidence limit for the VMTH Clinical Pathology Service for an animal to be accepted for the study All parameters for both blood analyses are listed in Appendices D and E for the blood chemistry and hematology respectively During the study the sheep were provided with food (2 ,000 g alfalfa-based pellets daily) and water ad libitum. They were housed individually on a 12 : 12 light:dark cycle (6 am : 6 pm) under controlled environmental conditions at approximately 16-21 c and 4060% humidity Prior to the study and during the cross-over period sheep were maintained at the Animal Sciences Department Farm, where they were provided with food (com-soy based sheep diet plus bermuda hay) and water ad libitum The cows were kept on pasture at the Animal Science s Physiology Unit where they had free access to water. Durin g the first 4 hours of the experiment the cows were kept in the chutes with their heads restrained in head-catch devices only for the time of

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76 blood collection. After four hours they were allowed to move within a pen and after 8 hours were released onto the pasture. For each subsequent sample collection they were brought back into the chutes and restrained in the head-catch devices 4 .1.1.1. Sample collection The tilmicosin or saline placebo treatments were always administered between 0700 and 1000 hours For the sheep experiment, animals were held in transporting carts for the first six hours of testing, and after that period returned to their regular pens until the end of experiment, 96 hours after drug administration Before drug administration, indwelling 16 g x 5 1/2 in. over-the-needle catheters (Becton Dickinson17 ) were placed in each jugular vein and connected to an intravenous (IV) fluid extension tube An initial (pre-drug) blood sample was collected at that time. Fluid balance was maintained by continuous intravenous administration of 0.9% saline solution (at the rate of2 ml/min) for the first si x hours of experiment when the sampling was most frequent. Sheep were connected to the multichannel oscilloscope (Datascope) for continuous monitoring of the cardiopulmonary parameters (ECG ; heart rate ; systolic diastolic and mean blood pressure) until 6 hours after tilmicosin administration when the monitoring setup was detached Data for the cardiopulmonary monitoring were collected as described in chapter 3 2 according to the same schedule as blood sampling (Table 4 1 ) An abbreviated physical examination was performed daily on each sheep during the actual experimental period This included measuring respiratory rate heart rate and body temperature as well as the evaluation of animal s attitude appetite elimination, behavior and any abnormalities

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77 Blood samples for the sheep study were collected using an indwelling venous catheters (inserted before zero sample was collected) for the first 36 hours after tilmicosin administration After that time to minimize risk of infection at the site of catheter insertion the catheters were removed and subsequent samples were collected directly by venipuncture using the Vacutainer brand (Becton Dickinson) blood collection system (15 cc serum collection tubes and 20 g x 1 1/2 in. needles with holder) In the cattle study blood samples were all collected by venipuncture using the same type of evacuated tubes and needles as described above for sheep In both sheep and cattle groups blood was collected at the time points as described in Table 4-1 Care was taken to avoid exposing blood samples to light by keeping the blood tubes in aluminum foil and additionally protecting them by storage in cardboard mailer boxes both before and after refrigeration. Blood samples were left at room temperature for at least 2 hours and up to 24 hours after collection to allow clotting to take place They were then centrifuged at 756 g for 20 minutes The harvested serum was stored at -20 C until assayed for tilmicosin. Blood was collected for hematology and blood chemistry analyses in the sheep experiment prior to drug administration, and again at 24 and 72 hours post tilmicosin injection (Table 4-1 ) Samples in EDTA (hematology) and clotted blood samples (chemistry) were submitted to the VMTH Clinical Pathology Service immediately after collection for analysis For the cattle study samples for hematology and blood chemistry analyses were collected only prior to drug administration as a part of determination of the animals health status

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78 4 .1.1. 2 Statistical analysis A commercial microcomputer program was used for the statistical analysis (Sigma Stat19 Version 2.0) All pharmacokinetic parameters were compared between the cattle and sheep groups using a two sample t-test for two independent samples When the ttest assumptions for normal distribution and equal variance were not met the Mann Whitney Rank Sum test was used instead As in all statistical analyses throughout this project a p-value less than 0 05 was considered significant. For the statist i cal analysis of the cardiopulmonary effects of tilmicosin in sheep a two-way analysis of variance (ANOVA) was performed. Statistical analysis of the blood chemistry and hematology data was performed using a two sample t-test for two independent samples where each parameter of the hematology and chemistry analyses was compared between the two treatment groups 4 1 2 Effect of Respiratory Disease on Tilmicosin Pharmacokinetic in Rats Seventy two rat s were randomly assi g ned to two equally s iz ed e x perimental groups One group was infected with M pulmoni s (hereafter termed infected or "inf' ) and the other group received only sterile broth and served as negative controls (hereafter termed non-infected or "n-inf' ) All animals in both groups recei ved a sin g le dose of tilmicosin 1 month after inoculation and were killed at defined time points after tilmicosin administration, as described below After inoculation the rats were observed daily for an y signs of illness including sniffin g rou g hnes s of fur hunched postur e lethar gy or inappeten c e No animals were observed to develop an y of the aforementioned sig n s

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79 4 1 2 1 Sample collection Si x rats per group (infected and non-infected) were killed according to the following schedule : 0 (before drug treatment) 1 3 7 24 and 72 hours after tilmicosin administration The rats were euthanized with the intramuscular injection of 0 4 ml of ketamine/xylazine mixture per animal (0 .15 ml of 100 mg/ml xylazine and 10 ml of 100 mg/ml ketarnine) and exsanguinated This allowed the collection of 3-4 ml of blood from each animal. The blood samples were allowed to clot and were stored at + 4 C for 24 hours At this time the serum was harvested after 10 minutes of centrifugation at 485 g and stored at -20 C. Whole lungs and approximately 2 g of quadriceps muscle from the hind leg were collected from each rat concurrently with blood samples All samples were placed in test tubes and stored at -20 C until analysis Lung and muscle samples were used for determination of tissue pH. Within 30 days of sample collection samples were first thoroughly thawed and tissue pH was measured at room temperature The pH measurements were made using a portable pH meter (Extech1 8 ) with a specifically designed combination electrode for tissue penetration This allowed for the measurement of tissue pH without homogenization or mixing of sample with solutions (Bager and Petersen 1983 ; Korkeala et al., 1986) To obtain reliable measurements the tapered tip of the electrode was completely immersed into a tissue sample The electrode was cleaned with distilled water after each sample, and after eve ry six sample s it w as wiped with an alcohol swab At that time the pH meter electrode was recalibrated according to the manufacturer s instructions using buffer solutions of pH 4 0 and 7 0

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80 Serum and lung samples were subsequently used for HPLC analysis as described in Chapter 3 4 1.2.2. Statistical analysis In the statistical analysis a two-way ANOVA was performed to determine possible interactions between the type of treatment (infected and non-infected) pH measurements and tissue and blood tilmicosin concentrations.

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CHAPTERS RESULTS 5 1 Serum Pharmacokinetics of Tilmicosin in Sheep and Cattle The concentrations of tilmicosin in sheep and cattle serum were determined for up to 96 hours after drug administration Since the concentrations of tilmicosin for the last time point (96 hours) fell below the LOQ, the pharmacokinetic parameters were calculated based on the end-point of 72 hours (or less ; depending on the concentration) after tilmicosin injection However, the 96-hour time point was included in the graphical and tabular presentations of the raw data 5 1 1 Results of the Non-Compartmental Pharmacokinetic Analysis The HPLC analysis was performed as described in chapter 3 1 ., to determine concentrations of tilmicosin in the cattle and sheep serum Tilmicosin concentrations for each individual animal at each time point are presented in Tables 5-1 and 5-2 for the sheep and cattle groups respectively The non-compartmental pharmacokinetic analysis was performed on these data The computed pharmacokinetic parameters for each individual animal are displayed in Tables 5-3 and 5-4 for the sheep and cattle group respectively The mean elimination rate constant for 10 sheep was 0 021 h -1 ( 0 005 standard deviation St.D ) resultin g in the mean terminal half-life of 34. 6 hours( 8 1) or 3 2 8 81

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82 Table 5-1: Tilmicosin concentration over time in the sheep serum Serum (12 ml) was collected at 22 time points ( up to 96 hours) after subcutaneous administration of 10 mg/kg of Micotil to 10 sheep (S-145 to S-205) The arithmetic mean and standard deviation (St. D ) were calculated for each time point. A hyphen indicates a missing sample Time Concentration (g/ml) in sheep serum Mean St.D. (h) S-145 S-192 S-193 S-194 S-196 S-197 S-198 S-199 S-201 S-205 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .08 0.770 0 224 0 212 0 120 0 116 0 195 0.134 0 .171 0.456 0.203 0.260 0 204 0 .25 0 908 0 626 0.449 0 .185 0 184 0 338 0 306 0 335 0 663 0.435 0.443 0 229 0.5 0 .783 0 .691 0 549 0 214 0 .193 0 468 0 401 0.445 0 926 0 569 0.524 0 233 1 0.959 0 824 0 550 0 .261 0 245 0.438 0.618 0.657 0.718 0.891 0.616 0 247 1.5 0 992 0 .901 0.518 0 .271 0.423 0.409 0 864 0 664 0 .895 0 .879 0 682 0 258 2 0 969 0 762 0 624 0 328 0.468 0 700 0 567 0.700 1.127 0.885 0.713 0 237 3 1 047 0 868 0.439 0 .341 0.612 0 718 0 588 0 603 1 154 0.981 0.735 0.268 4 0 729 0.639 0 574 0 336 0 .581 0 629 0.565 0 286 1.156 0 694 0 619 0 237 5 0 642 0 .551 0.415 0 .391 0 625 0 622 0.312 0.409 1.090 0.767 0 582 0 228 6 0.422 0 581 0 692 0 312 0 559 0 617 0.446 0 .321 0.919 0.797 0.567 0 199 8 0.447 0 522 0 828 0 398 0 547 0 587 0.457 0 506 0 825 0 593 0 .571 0 148 10 0.433 0 524 0 686 0 398 0.566 0.428 0.473 0.407 0 628 0.589 0.513 0.101 12 0 313 0 327 0 .381 0 374 0.333 0.457 0 252 0 323 0.546 0.515 0.382 0 095 18 0 277 0 230 0.432 0 333 0 305 0 327 0 238 0 192 0 334 0 260 0.293 0 069 24 0 166 0 172 0 302 0 363 0 272 0.139 0 232 0.151 0 259 0 231 0 229 0 073 30 0 .151 0 110 0 299 0 273 0 .211 0 162 -0 092 0.187 0 188 0.186 0 068 36 0 105 0 106 0 147 0 189 0 .151 0 145 0 189 0 104 0.156 0.148 0.144 0 .031 48 0 104 0 074 0 106 0 136 0 .111 0 127 0 .081 0 .081 0 142 0.101 0 106 0 023 60 0.095 0 069 0.097 0 107 0 109 0 094 0 .071 0.070 0.114 0 090 0 092 0 017 72 0.066 0 063 0 .071 0 078 0 .091 0.073 0 065 -0.086 0 085 0.075 0 010 96 0 062 0 044 0 .061 0 039 0 074 0 064 0 038 -0 063 0 040 0 054 0 014

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83 Table 5-2 : Tilmicosin concentration over time in the cattle serum Serum (12 ml) was collected at 22 time points (up to 96 hours) after subcutaneous administration of 10 mg/kg ofMicotil to 10 cows (C-1 to C-10) The arithmetic mean and standard deviation (St.D ) were calculated for each time point. A hyphen indicates a missing sample Time Concentration (g/ml) in cattle serum Mean St.D. (h) C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 08 0 680 0 .865 0 316 0.748 0 058 0 159 0.493 0.283 0 991 1.421 0 601 0.422 0 .25 0 545 1.015 0.446 0 552 0 778 0.429 0 749 0 .713 0 944 1 941 0 811 0.443 0 5 0 537 0 .971 0 507 0 606 0 646 0.416 0 700 0 684 0 685 1 334 0 709 0 265 1 0 667 0 973 0.437 0 287 0 685 0 325 0 638 0 575 0 566 0 .891 0 604 0 .221 1.5 0 364 1.055 0 595 0 548 0 514 0 288 0 548 0 .701 0 .501 0 .811 0.592 0 220 2 0 649 1.112 0.462 0 .571 0.494 0 320 0 540 0 610 0 507 0 786 0 605 0 216 3 0 526 0 958 0 525 0.482 0 .461 0 383 0.416 0 592 0 526 0 769 0 564 0 175 4 0 515 0 955 0 517 0.417 0 515 0 362 0.466 0 630 0 628 0 716 0 572 0 .171 5 0.516 0 .873 0.482 0.426 0.528 0.425 0.464 0.649 0.638 0.723 0.572 0.146 6 0 609 0 660 0.434 0.374 0 394 0 345 0.431 0 543 0 520 0 668 0.498 0 119 8 0 386 0 556 0.429 0 305 0 505 0 368 0 .351 0 549 0 544 0 572 0.456 0 100 10 0.442 0 517 0.415 0 349 0 395 -0 315 0.457 0.453 0.464 0.423 0 062 12 0 226 0.403 0.337 0 267 0 .341 0 265 0 267 0.455 0.439 0.443 0 344 0 086 18 0 146 0 279 0 243 0 213 0 230 0.213 0 219 -0 266 0 267 0.231 0 040 24 0 122 0 186 0 248 0 159 0 169 0 154 0 178 0 242 0 245 0 218 0 192 0 044 30 0 .171 0 169 0 213 0 154 0 174 0 178 0 .141 0.217 0 208 0 167 0 179 0 025 36 0 114 0 145 0 166 0 120 0 142 0.131 0 097 0.153 0 .171 0 148 0 139 0 023 48 0 087 0 09 3 0 130 0 069 0 109 0 112 0 089 0 .121 0 139 0 137 0 108 0 024 60 0 060 0 075 0 .105 0 064 0 066 0 066 0 060 0 095 0 .101 0 096 0 079 0 019 72 0 048 0 058 0 .071 0 044 0.059 0 054 0 045 0 073 0 093 0 088 0 063 0 017 96 0 043 0 040 0 060 0 028 0 044 0 028 0 025 0 066 0 066 0 078 0 048 0 019

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84 Table 5-3 : Calculated pharmacokinetic parameters for tilmicosin in sheep (n = 10) The arithmetic mean and standard deviation (St.D.) were calculated for each time point. Abbreviations used in the table: k., = elimination rate constant ; t112 = half-life ; AUC = area under the serum concentration vers us time curve ; AUMC = area under the first-moment curve ; MRT = mean residence time ; Cmax = maximum drug concentration in serum ; t.nax = time at which Cmax was reached ; Cl = clearance ; V d = volume of distribution ; f = bioa vaila bility PK Parameter S-145 S-192 S-193 S-194 S-196 S-197 S-198 S-199 S-201 S-205 Mean St.D. 1..,w 0.016 0.019 0 029 0 028 0 018 0 021 0.028 0.018 0.020 0.015 0.021 0.005 tin [h] 43. 9 36 7 23 8 24 8 38 8 32.6 24 9 39.1 34.4 46 8 34.6 8 1 AUC 19 8 17. 6 21.2 19 .0 21.4 19 6 15.5 15. 2 26.0 23 9 19 .9 3.4 [mg/ml*h) AUMC 885 690 672 729 1041 768 539 665 955 1168 811 195 [mg/ml*h2 ] MRT [h] 44 6 39.l 31.7 38 4 48 7 39 2 34 8 43 7 36 7 48 9 40 6 5 7 Cmax [ mg/ml] 1.05 0 90 0 .83 0 40 0 63 0 72 0.86 0 70 1.16 0.98 0.82 0.22 tmax [h] 3 1.5 8 8 5 3 1.5 2 4 3 3 9 2.4 CUf(l/h] 3 4.1 37.0 36. 1 40 3 32.6 32.2 44.2 35 5 25.1 22.6 34 0 6 4 Vd/f [I] 2158 1961 1243 1444 1825 1516 1591 1999 1247 1525 1651 319

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85 Table 5-4 : Calculated pharmacokinetic parameters for tilmicosin in cattle (n = 10) The arithmetic mean and standard deviation (St.D .) were calculated for each time point. Abbreviations used in the table : k., = elimination rat e constant ; t1 2 = half-Life; AUC = area under the serum concentration versus time curve ; AUMC = area under the first-moment curve ; MRT = mean residence time ; Cru.x = maximum drug concentration in serum ; t.n..x = time at which Cma.x was reached ; CL = clearance ; Vd = volume of distribution ; f = bioavailability PK Parameter C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 Mean St.D. ke[h")] 0 029 0 025 0 024 0 029 0 026 0 025 0 025 0 024 0 020 0 016 0.024 0 004 tin1h] 23.9 27 3 29 4 24 0 27.l 27 5 27 2 28 9 35 3 43 9 29.4 6 0 AUC 13. 8 19. 8 18. 3 13.2 16 l 13.3 13. 8 18. 3 21.8 23. 7 17 2 3 8 [mg/mJ h] AUMC 432 580 720 416 562 522 451 694 980 1137 649 241 [mg/ml"h2 ] MRT fh] 31.3 29. 2 39.2 31.5 34 9 39 l 32. 7 38 0 45.0 47. 9 36.9 6 1 Cmax (mg/ml] 0 68 1.11 0 60 0 75 0 78 0 43 0 75 0 .71 0 99 1.94 0 87 0 42 tmax fh] 0 08 2.00 1.50 0 08 0 25 0 25 0 25 0 25 0 08 0 25 0 .50 0 67 Cllf fl/h] 335.7 196.4 224.4 3 27 9 251.5 318 7 332 8 262 4 204. 3 189.6 264.4 59 9 Vd/f fl] 11598 7723 9507 11348 9830 12635 13075 10937 10398 12006 10906 1602

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86 hours when the harmonic mean was calculated (Table 5-5). The AUC of 19.9 mg/ml h ( 3.4) and the AUMC of811.2 mg/ml* h2 ( 195) was calculated resulting in the MRT of 40. 6 h ( 5 7). The maximum concentration reached in serum of sheep was 0 82 g/ml ( 0 .2) and it was achieved at 3 9 h ( 2.4) after tilmicosin injection (tmax = 3 9 h 2.4) Tilmicosin in sheep had the clearance of34.01/h ( 14 .3), and volume of distribution of 1 651 I ( 798) The cattle group had the mean elimination rate constant of 0 024 h-1 ( 0 004), and the mean terminal half-life of 29.4 hours( 6 .0) or 28. 6 hours when the harmonic mean was calculated (Table 5-5) The AUC was 17.2 mg/ml h ( 3 .8) and the AUMC 649 3 mg/ml* h2 ( 241) resulting in the MRT of 36. 9 h ( 6 1) The Cmax for the cattle was 0 873 g/ml ( 0.4) and tma1< was 0 5 h ( 0.7) In the cattle group, tilmicosin had the clearance of 264.4 1/h ( 113) and volume of distribution of 10 ,906 1 ( 3049). The results of the non-compartmental analysis were compared between the two species in order to test the hypothesis that there would be no difference in tilmicosin pharmacokinetics between sheep and cattle The summary of comparative results as well as the summary of results of the statistical analysis are presented in Table 5-6 The results for each pharmacokinetic parameter are based on the arithmetic mean value of 10 sheep/cattle per group. A comparison between the arithmetic and harmonic mean in calculation of half-life is presented in Table 5-5 Harmonic mean was used because in the cattle group, the half-life data did not have a normal distribution.

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87 Table 5-5 : Comparison of the half-life data (arithmetic vs harmonic mean) for tilmicosin in sheep and cattle Arithmetic and harmonic mean values( standard deviation St. D ) are displayed for the sheep and cattle groups (n = 10 animals/group), together with their respective p-values resulting from the statistical analysis (t-test for two independent samples) SHEEP CATTLE p-value MEAN Mean St.Dev. Mean St.Dev. Arithmetic 34 6 +/8 1 29.4 +/-6 0 0.122 Harmonic 32.8 -10 9 to 6.5 28 6 -5 6 to 4 0 0 151

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88 Table 5-6 : Comparison of the pharmacokinetic parameters for tilmicosin in sheep and cattle Arithmetic mean values( standard deviation St. D ) are displayed for the sheep and cattle groups (n = 10 animals/group) together with their respective p-values resulting from the statistical analysis (t-test for two independent samples or Mann-Whitney rank sum test) Abbreviations used in the table : k., = elimination rate constant ; t112 = half-life ; AUC = area under the serum concentration versus time curve ; AUMC = area under the first-moment curve ; MRT = mean residence time: Cmax = maximum drug concentration in serum ; tmax = time at which Cmax was reached ; CJ = clearance ; V d = volume of distribution ; f = bioavailability Cattle Sheep PK Parameter Mean St.D. Mean St.D. p-value ke (h -1 ) 0 024 0 004 0 021 0 005 0 .15 t112 [h] 29.4 6 0 34 6 8 1 0 122 AUC [g/ml*h] 17 2 3 8 19 9 3.4 0 .111 AUMC [g/ml h2 ] 649 3 241.4 811.2 195 5 0 117 MRT [h] 36 9 6.1 40 6 5 7 0 178 Cmax [g/ml] 0 873 0.420 0 822 0 221 0 734 tmax [h] 0 50 0 67 3 90 2.41 < 0 001 CL/f [1/h] 264 4 59.9 34.0 6.4 < 0 001 Vd/f [I] 10906 1602 1651 318 8 < 0 001

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89 Statistical analysis of the data was done using the unpaired t-test or, when the normality and/or equal variance test failed, the Mann-Whitney rank sum test. The statistical analysis revealed no significant difference between the cattle and sheep in the following pharmacokinetic parameters : elimination rate half-life AUC AUMC MRT and CmaxParameters that showed a significant difference between the two animal groups are : tmax, clearance and volume of distribution. However, when the clearance was expressed per kilogram of body weight the results between the cattle and sheep were not significantly different (0 60 as opposed to 0 52 1/h/kg for the cattle and sheep respectively) Similarly there was no significant difference in the volume of distribution when it was normalized for body weight (25 0 1/kg in both groups) leaving the tmax as the only significantly different pharmacokinetic parameter between the sheep and cattle While the tmax in sheep was 3 9 h the maximum tilmicosin concentration in cattle was reached at 0 5 hours after injection of tilmicosin although the Cmax in both groups was almost identical. 5.1.2. Results of the compartmental pharmacokinetic analysis and modeling When the logarithmic concentrations of tilmicosin were plotted against time the resulting curves for the sheep and cattle groups (Figs 5-1 and 5-2 respectively) consisted of two distinct parts indicating a two-compartment body model. The curved portion of the curvilinear profile in both groups (approximately the first 36 hours after tilmicosin administration) reflects the absorption and rapid distribution processes (into and out of the central compartment) while the terminal linear portion of the curve reflects the post distribution equilibrium between the peripheral and central compartments together with

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e Cl :::l C .Q '! 0 100 c GI u C 0 u g, -I 90 0 010 +---~-+---+-------+-----1-----+-----+----+-----i 0 12 24 36 48 Time (h) 60 72 Figure 5-1 : Tilmicosin concentrations over time in the sheep serum 84 Tilmicosin was injected subcutaneously at the dose of 10 mg/kg and the time concentration curve represents the arithmetic mean for 10 sheep ( arithmetic mean standard error) 96

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C !! 0 100 c GI u C 0 0 CJI 0 ..J 91 0 010 +-----+---+-----+----+-----+------;~----r---0 12 24 36 48 Time (h) 60 72 Figure 5-2 : Tilmicosin concentrations over time in the cattle serum 84 96 Tilmicosin was injected subcutaneously at the dose of 10 mg/kg and the time concentration curve represents the mean for 10 cattle (arithmetic mean standard error)

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92 the elimination from the central compartment. Based on the figures of the serum concentration versus time curves for sheep and cattle (Figs 5-1 and 5-2), it can be observed that the disposition profiles of tilmicosin did not differ substantially between the two species Particularly the later portions of the curves had a very similar shape while the initial portion (representing a relatively short time period after administration) showed different profiles for sheep and cattle which in the non-compartmental analysis is reflected as the significantly different tmax-The experimental data were then fitted to the bi-exponential equation corresponding to a two-compartment body model with first-order input (as described in chapter 3 .1.3. 5 ) The curve fitting was not used to compute the pharmacokinetic parameters and the results are presented here just to demonstrate a goodness of fit to a theoretical body model. The results of the curve stripping and fitting for each individual animal are presented in Figure 5 3 (a and b) for the sheep and in Figure 5-4 (a and b) for the cattle The average concentrations for each animal group were plotted and fitted using the same equation and the combined results per group are shown in Figure 5-5 5.2 Cardiopulmonary Effects of Tilmicosin in Sheep Because of the reported cardiotoxicity of tilmicosin, the effects of the therapeutic dose oftilmicosin on the cardiopulmonary system were studied in sheep The animals were monitored for signs of cardiopulmonary toxicity after receiving either tilmicosin ( at a dose of 10 mg/kg) or the same volume of saline subcutaneously as described in chapter 4 1 The following cardiopulmonary parameters were monitored for the first six hours after tilmicosin administration ( as described in chapters 3 2 and 4 1 1 ) : systolic diastolic and

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I C: .Q c 8 C: 0 (.) i C: 0 c 8 C: 0 (.) C: 0 10 10' 102 100 10-' 101 C: 8 C: 0 (.) 0 0 0 18 18 18 S-145 36 Timeh S-193 36 Timeh S-196 36 Timeh 54 54 54 93 S-192 100 e C: 0 10 -1 c 8 C: 0 (.) 1 0 -2 72 0 18 36 54 72 Timeh S-194 10 i C: 0 10 -1 c 8 C: 0 (.) 72 0 14 29 43 58 72 Timeh 72 Figure 5-3a : Least squares fitting for serum tilmicosin concentrations in 5 sheep Tilmicosin was injected subcutaneously at a dose of 10 mg/kg. Diamonds ( +) represent the experimental data points and curves represent the lea st squares fitting for a twocompartment body model.

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S-197 10" i C: 2 101 c 8 C: 0 0 1 0-2 0 18 36 72 Timeh 5-199 1 0" I C: 0 10 1 c 8 C: 0 0 10 2 ...J.-~~~-.--.--,--,--,--,--,--,-...-...-...-..,......., I C: 0 1 0 c 8 C: 0 0 0 18 0 18 36 Timeh S-205 36 Time h 54 72 54 72 94 10" I C: 0 10 1 c G) 0 C: 0 0 1 0 2 0 101 I 10" C: 0 c 8 C: 1 0 1 0 0 0 18 18 S-198 36 Timeh S-201 36 Timeh 54 72 54 72 Figure 5-3b : Least squares fitting for serum tilmicosin concentrations in 5 sheep Tilmicosin was injected subcutaneously at a do se of IO mg/kg Diamonds ( +) represent the e x perimental data points and curves represent the lea st squares fitting for a twocompartment body model.

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95 C-1 C-2 100 10 1 i I 10" C: C: 0 0 10-1 c c B B C: C: 10 1 0 0 C) C) 10-0-1----,...-----.---r--.---.----.--,-------,--~ 0 24 48 72 0 24 48 72 Timeh Timeh C-3 C-4 100...---.----.---.--.--.--.-----,---,-----.--,------,--,-----.----,----,---, i E C: C: 0 0 10-1 10 1 C: c B 8 C: C: 0 0 C) C) 10-2 10-2 0 24 48 72 0 18 36 72 Timeh Timeh C-5 102 101 i C: 0 100 I C: 0 C) 10-1 10-0 0 24 48 72 Timeh Figure 5-4a : Least squares fitting for serum tilmicosin concentrations in 5 cattle. Tilmicosin was injected subcutaneously at a dose of 10 mg/kg Diamonds ( +) represent the experimental data points, and curves represent the least squares fitting for a two compartment body model.

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i C 0 c B C 0 (.) I C .Q C B C 0 (.) I C 0 :;:; !! c B C 0 (.) 102 101 100 10' 10-2 0 24 100 10 1 0 18 1 0 1 C-6 Timeh C-8 36 Timeh C-10 48 72 54 72 102 +-~...-..--r-................ ---. ...................... """'T"....,... ................ --1 0 18 36 Time h 72 96 100 I C 0 10' C B C 0 (.) 10-2 0 100 e g C 0 10 1 C B C 0 (.) 0 18 18 C-7 36 Timeh C-9 36 Timeh 54 72 54 72 Figure 5-4b : Least squares fitting for serum tilmicosin concentrations in 5 cattle. Tilmicosin was injected subcutaneously at a dose of 10 mg/kg Diamonds ( +) represent the experimental data points and curves represent the least squares fitting for a two compartment body model.

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C: 0 :;:; 10-1 ..... C: C: 0 (.) C: 0 :;:; 10 -1 ..... C: (I) u C: 0 (.) 0 0 18 18 97 Sheep 36 Timeh Cattle 36 Timeh 54 72 54 72 Figure 5-5 : Summarized least squares fitting for serum tilrnicosin concentrations in sheep and cattle Tilrnicosin was injected subcutaneously at the dose of 10 mg/kg Diamonds ( +) represent the average for the experimental data points of 10 sheep triangles (.A) represent the arithmetic mean for the experimental data points of 10 cattle and the curves represent the least squares fitting for the two-compartment body model. Data are presented as arithmetic mean standard error.

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98 mean blood pressure ; ECG; heart rate ; and respiratory rate Blood samples for the pharmacokinetic study were collected concurrently with the pharmacodynamic monitoring. The scheme for the sampling schedule was presented in chapter 4 (Table 4-1 ) A two-way repeated measures ANOV A was performed to compare each cardiopulmonary parameter between the tilmicosinand saline-treated sheep The drug and saline treatment were repeated for each sheep The ANOVA table for each cardiopulmonary parameter is presented in Appendix C 5 2 .1. Blood Pressure The effect of tilmicosin on the systolic diastolic and mean blood pressure in sheep is shown in Figure 5-6 When the data for all three blood pressure parameters are summarized tilmicosin-treated animals had lower blood pressures than the control animals in all but 3 (out of 33) data collection points In the statistical analysis the effect of treatment was found to be significant for the systolic and mean blood pressure while it was not significant for the diastolic blood pressure However when the data on systolic and mean blood pressure were further analyzed using the Dunnett s multiple comparison procedure there was no significant difference between the treated and non-treated animals The effect of time was found to be significant for both animal groups in the analysis of the systemic blood pressure There was no significant interaction between the treatment and time in the analysis of the effect of tilmicosin on either one of the blood pressure parameters

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99 Systolic Blood Pressure (SBP) 200 --------------------------, 160 120 E o. 80 = "' l I r I rL I ;r:, 1 I 40 a Tilmico 0 +-"-Y_ -'-+-__ -L+-' ~ +-"'L...L.t--'-'-t~ -'--+' ~ ~+-~--Y 0 0.08 0 25 0 5 1.5 2 6 a P l acebo llme / h Diastolic Blood Pressure (DBP) JOO r-------r-------------1 I I "I I I L I 80 'ol, :: 60 E ;j 40 Q 20 I T ilm i cos 0 +-'-'-+--'-+-~Y---~+-'-'-+~-'-+~.._._.~......-~....----+~---< O P l acebo 140 120 100 'ol, :: 80 E 60 .. = 40 20 0 0 0 .08 0.25 0 5 1.5 2 llme / h Mean Blood Pressure (MBP) I I [ L I L I 0 0 .08 0 25 0 5 1.5 2 3 llme / h 6 L 1 : 6 i i Tilmicosin O P l acebo Figure 5-6 : The effect of tilmicosin on t he systolic diastolic and mean blood pressure in sheep Using the repeated-measures design, ten sheep received tilmicosin (10 mg/kg) or the same volume of saline and their systolic diastolic and mean blood pressure was measured for the first six hours after treatment. Bars represent standard error for the group

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100 5 2 2 Heart Rate The effect of tilmicosin on the heart rate of sheep is shown in Figure 5-7 The data show that the heart rate in sheep was slightly increased in tilmicosin-treated animals, except for the first two time points (before treatment and first point after the treatment) However in the statistical analysis there was no significant difference in heart rate between the treated and non-treated sheep The effect oftime was significant for both treatment groups There was no significant difference in the interaction of treatment and time in the analysis of the effect of tilmicosin on the heart rate. 5 2.3 Respiratory Rate Figure 5-8 depicts the graphical presentation of the effect of tilmicosin on the respiratory rate in sheep In both tilmicosin and saline-treated animals the respiratory rate steadily decreased over time of the experiment However there was not a uniform trend when comparing the two treatment groups : while in the earlier and later time points after treatment sheep receiving tilmicosin seemed to have slightly higher respiratory rate than controls ; the more pronounced difference but in the opposite direction, was observed in the middle of the data-collection period Statistical analysis revealed no significant effect of tilmicosin on the sheep respiratory rate The effect of time was found to be significant in both treatment groups so that there was a notable decrease in respiratory rate over time There was no significant interaction between the treatment and time in the analysis of the effects of tilmicosin on heart rate in sheep

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101 120 100 I. II. ;c, [ lI. rI. 1 80 :r: a: II. :r: - T ilmi cosin :i 60 "' O P l ac e bo .. = t: "' 40 .. I = 2 0 1 0 0 0 .08 0 .2 5 0 5 1.5 2 3 4 5 6 lime/ h Figure 5 7 : The effect of tilmicosin on the heart rate in sheep Usin g the repeated-measures design ten sheep received tilmicosin (10 mg/k g ) or the same volume of saline and their heart rate was measured over a 1-minute period for the first six hours after treatment. Bars represent standard error of the arithmetic mean for the group

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102 140 120 100 [ [ = a 80 "' = 0 [ J [ "' !: -a 60 .5 40 'It , 20 0 -4 + + + + 4 4 0 0 08 0.25 0 5 1 1.5 2 3 4 5 llme/b Figure 5-8 : The effect of tilmicosin on the respiratory rate in sheep 6 T ilm i cosin O P l acebo Using the repeated-measures de s ign ten sheep received tilmicosin (10 mg/k g ) or the same volume of saline and their respiratory rate was measured over a I-minute period for the first s ix hours after treatment. Bars r epresent standard error of the arithmetic mean for the group.

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103 5 3 Effects of Tilmicosin on Blood Chemistry and Hematology in Sheep Before any experiments were started on sheep and cattle the initial samples were collected for the hematology and blood chemistry analyses as a part of the initial animal health status determination. The results for all animals fell within the normal ranges established by the VMTH Clinical Pathology Service for each species involved. Complete blood count (CBC) for hematology (including 16 parameters) and 19 blood chemistry parameters were monitored in sheep after administration of 10 mg/kg of tilmicosin or saline (as described in chapter 4 1.) Samples for the CBC and chemistry analyses ( blood with and without anticoagulant respectively) were collected before tilmicosin/saline injection and at 24, and 72 hours after the treatment. The results of the CBC/ Chemistry analysis followed in each animal for three days are shown in Appendi x D Statistical analysis using the two-way repeated measures ANOVA revealed that there was no significant effect of treatment on any parameters of the CBC/Chemistry analysis However there was a significant effect oftime for several parameters tested In the chemistry analysis the amount of potassium and alkaline phosphatase increased while there was a decrease in the levels of glucose and albumin in the blood over a three-day period in both treatment groups Of the CBC parameters the numbers of segmented WBCs and lymphocytes decreased over time while the total number ofWBCs and RBCs increased as well as the hemoglobin level hematocrit number of platelets and spun hematocrit.

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104 5.4 Lung Tissue Distribution ofTilmicosin in Infected and Non-Infected Rats Lung tissue distribution of tilmicosin was examined in rats infected with M pulmonis and compared to non infected animals It was hypothesized that in the infected lungs there would be a decrease in the pH as a result of the inflammatory processes and that the more acidic environment might be responsible for the higher tilmicosin concentrations in the lung when compared to the healthy animals. Therefore the lung tissue pH was measured, and serum and lung tissue samples were collected concurrently for the HPLC determination of tilmicosin concentration 5. 4 1 pH Measurements of the Lung and Muscle Tissue Lung tissue pH was measured at various time points after tilmicosin administration (as described in chapter 4 2 1 ). The pH of the leg muscle was also measured in order to determine whether the hypothesized changes in lung pH would be reflected systemically Summary of the results of the pH measurements for both tissues is shown in Fig 5-9. When the muscle pH was compared to the lung pH using the Mann-Whitney rank sum test it was found that the mean values in the muscle pH group were significantly lower than the pH of the lung tissue A two-way ANOVA was performed to determine the effect of infection on the lung tissue pH. The ANOVA tables for analyses of both lung and muscle tissue are shown in Appendix F. It was found that there was no significant difference in the lung pH between infected and non-infected rats The time effect for the lung pH was found to be significant in both treatment groups and the interaction between time and treatment was also significant.

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.. ::, 7.40 ] 7 20 z 0. 7 .00 6 .80 0 105 Lung Tissue pH Measurements 3 7 2 4 72 Time (h) Muscle Tissue pH Measurements 6 .60 ~------------------, .. ::, 6.40 ] 6 .20 z 0. 6 .00 5 .80 0 3 7 2 4 72 Time (h) Figure 5-9 : pH measurements in the lung and muscle tissue of rats Treated rats (n = 36 ; "inf' ) were inoculated intranasally withM pulmonis and control rats (n = 36 ; "n-inf ) were administered the same volume of sterile broth intranasally Both groups received a single subcutaneous dose of 20 mg/kg of tilmicosin 31 days after the initial treatment, and were sacrificed at 0 1 3 7 24 and 72 hours after tilmicosin administration Number of rats per group per collection point was 6 Bars represent standard error of the arithmetic mean for the group

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106 In the analysis of the muscle pH, there was no significant effect of treatment but the time effect was found to be significant as well as the interaction between treatment and time 5.4 2 Tilmicosin Concentration in Serum and Lung Tissue Tilmicosin concentration in lung and serum was determined using the HPLC method described in chapter 3 1 Tilmicosin was extracted from serum by solid phase extraction with C18 cartridges as described in chapter 3 1.2.1. and from the lung using liquid-liquid extraction with methanol and chloroform ( chapter 3 1 2 2 ) The concentrations of tilmicosin in lung samples were consistently higher than the serum concentrations (Figure 5-10) That was true for all time points and for infected and non-infected animals alike (Figure 5-11 ) Statistical analysis using the paired t-test confirmed that the difference between the lung and serum concentrations was statistically significant. The results of the non-compartmental analysis for the infected and non-infected rats are shown in Table 5-7. There appeared to be no major differences for any pharmacokinetic parameters between the infected and non-infected animals Statistical analysis of the lung tissue concentrations of tilmicosin in the infected and non-infected rats was done using a two-way ANOV A. It was found that the effect of treatment (Mycoplasma or plain broth) was significant so that the infected animals had significantly higher lung tissue concentrations than the non-infected animals The effect of time was also found to be significant

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107 Tilrria)gn 0n:s11ratia1 in Senm 04~----------~0. 3 C: _g 0.2 c fl 0 1 C: 0 0 0 3 7 Tme(h) Tilnicmn Onl!rntioo in urg Ti91.e 3 7 lirre(h) 24 24 72 I 11o~n1 1 n Figure 5-10 : Concentrations of tilmicosin in the serum and lung tissue of rats Treated rats (n = 36) were inoculated intranasally with M pulmonis and control rats (n = 36) were administered the same volume of sterile broth intranasally. Both groups received a single subcutaneous dose of 20 mg/kg of tilmicosin 31 days after the initial treatment and were sacrificed at 0 1 3 7 24 and 72 hours after tilmicosin administration Number of rats per group per collection point was 6. Bars represent standard error of the arithmetic mean for the group

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108 A -NorHnfected Rats 7 ;zs g> 5 .2 Cl4 :1. 5 3 vi I l ~ j2 ~1 0 1tT 31T 71T 241T Time 8-ll"R!dedRJls 9 ;zs 0,7 C: ~6 ~5 54 (L) : 3 e2 ~1 0 1 IT 31T 71T 241T Time Figure 5-11 : Comparison of serum and lung tissue concentrations of tilmicosin for the non-infected and infected rats Treated rats (n = 36) were inoculated intranasally with M pulmonis and control rats (n = 36) were administered the same volume of sterile broth intranasally Both groups received a single subcutaneous dose of 20 mg/kg of tilmicosin 31 days after the initial treatment and were sacrificed at 0 1, 3 7 24, and 72 hours after tilmicosin administration Number ofrats per group per collection point was 6 Bars represent standard error of the arithmetic mean for the group

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109 Table 5-7 : The results of the non-compartmental pharmacokinetic analysis on tilmicosin serum concentrations in rats Treated rats (n = 36) were inoculated intranasally withM pulmonis and control rats (n = 36) were administered the same volume of sterile broth intranasally Both groups received a single subcutaneous dose of 20 mg/kg of tilmicosin 31 days after the initial treatment and were sacrificed at 0 1 3, 7 24 and 72 hours after tilmicosin administration. Number of rats per group per collection point was 6, and results are expressed as the arithmetic mean for the group, except for the half-life (expressed as the harmonic mean). Abbre vi ations used in the table : k., = elimination rate constant ; t112 = half-life ; AUC = area under the serum concentration versus time curve ; AUMC = area under the first-moment curve ; MRT = mean residence time ; C.U..X = maximum drug concentration in serum ; tmax = time at which C.U..X was reached ; Cl = clearance ; Vd = volume of distribution ; f= bioavailability PK Parameter Non-infected Infected Mean Mean ke [h-1 ] 0 093 0 092 t1,2 [h] (harmonic) 5 .7 6 2 AUC [mg/ml"'h] 2 75 2.46 AUMC[mg/ml"'h2 ] 27 23 24.42 MRT [h] 9.90 9.94 Cmax [mg/ml] 0 294 0.309 tmax [h] 1 1 CL/f [1/h] 0.800 0.895 Vd/f [I] 8.61 9 76

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110 The changes in the lung : serum ratio over time for the infected and non-infected rats are shown in Figure 5-12 The lung : serum ratio always seemed to be higher in Mycoplasma-infected animals than in the controls but the difference was most remarkable at 24 hours after tilmicosin administration The ratio in both groups increased over time and at 24 hours it was 86 : 1 for the non-infected and 178 : 1 for the infected rats

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111 Ll.llJ: Senm Rlto 2fD :a:D E 1!i> Cl> Ill g> 100 ...J !i) 0 1 IT 31T ?IT 241T lime Figure 5-12: Lung : serum ratio over time for the infected and non-infected rats Treated rats (n = 36) were inoculated intranasally withM. pulmonis and control rats (n = 36) were administered the same volume of sterile broth intranasally Both groups received a single subcutaneous dose of 20 mg/kg of tilmicosin 31 days after the initial treatment and were sacrificed at 0 1 3 7 24, and 72 hours after tilmicosin administration Number of rats per group per collection point was 6 Bars represent standard error of the arithmetic mean for the group

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CHAPTER6 DISCUSSION 6 1. Pharmacokinetics of Tilmicosin in Sheep and Cattle In both sheep and cattle data on serum concentrations of tilmicosin showed an agreement with the proposed two-compartment body model for tilmicosin In both species the serum-concentration versus time curves had a similar shape with the terminal portion of the curve (36 hours post tilmicosin administration) being almost identical There were no significant differences between the sheep and cattle in the elimination rate constants Cmax, half-lives AUCs, AUMCs and MR Ts. As expected in animals of grossly different sizes clearance and volume of distribution were significantly different between the two groups However when they were normalized for body weight there were no significant differences in either parameter between cattle and sheep Therefore the only non-compartmental parameter that was truly different between the two species was tmax. The initial portion of the serum-concentration versus time curve in sheep showed a gradual increase in tilmicosin concentration until the Cmax was reached at almost 4 hours The rate of absorption following subcutaneous injection of a drug is often sufficiently constant and slow to provide a sustained effect (Benet and Sheiner 1985) Therefore it is expected to observe a lag from the time of drug administration until the maximum concentration is reached due to the process of drug absorption from the site of injection The data in sheep are in agreement with the Eli Lilly study reports where the tmax in sheep 112

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113 depending on the study was from 3 8 hours (Elsom et al., 1993) to 8 hours (Cochrane and Thomson 1990) However when the results of this study were compared to that of Cochrane and Thomson (1990) using the same subcutaneous dose of 10 mg/kg it was found that the Cmax was much higher in this study (0 9 g/ml as opposed to 0.44 g/ml) In the report by Parker and Walker (1993), sheep had a similar Cmax as in this study (0 .96 g/ml) but the dose used in that study was twice as much as used in this study The half life of tilmicosin in the sheep from this study was 3 5 hours, which is in good agreement with the Eli Lilly reports : a half-life of 31 hours was reported in adult sheep by Patel et al. (1992), and in a study with lambs using a dose of20 mg/kg a half-life of 41 hours was reported (Parker and Walker 1993) While sheep showed a gradual increase in tilmicosin serum concentration until the Cmax was reached at 3 9 hours the cattle data, with a tmax of O. 5 hours imply a fast absorption from the site of injection The tilmicosin pharmacokinetics in neonatal cattle (Thomson, 1989a) showed that the absorption from the injection site was faster in cattle than in sheep averaging approximately 1 hour (as opposed to 4-8 hours in sheep). That study used a 10 mg/kg dose of tilmicosin administered subcutaneously in the lateral neck region (the same dose route and site as used in the cattle study presented here) In Thomson s study (1989a) three injection sites were compared for the speed of absorption and significant differences in the tmax values were found Subcutaneous injection into the lateral neck region resulted in the fastest absorption (tmax = 1 h) followed by intramuscular injection into the semitendinosus muscle (tmax = 4 h) followed by subcutaneous injection into dorsolateral chest region (tmax = 6 hours) The Cmax for the three different sites was 0 97 g/ml 0.71 g/ml and 0 .81 g/ml for subcutaneous (neck) intramuscular and

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114 subcutaneous (chest) administrations respectively Those results are similar to findings from this study in cattle (Cmax = 0 87 g/ml) In another study of tilmicosin pharmacokinetics a single subcutaneous dose of 10 mg/kg tilmicosin was administered in the lateral neck region of dairy cattle and it produced a Cmax of O .13 g/ml and tmax of 1. 8 hours (Ziv et al ., 1995) The results of the study comparing injection sites (Thomson, 1989a) suggest that different tmax values in sheep and cattle may be attributed to more than one factor First the difference in injection sites may have contributed to the difference in initial absorption In sheep the drug was injected subcutaneously between the scapulae where it could have been retained in the fat tissue resulting in the slower absorption from the injection site. In contrast the lateral neck region was used for injection of tilmicosin in the cattle where the drug might have entered the bloodstream faster supposedly because of the presence of less subcutaneous fat when compared to the interscapular region of sheep Another difference in the experimental conditions in the two studies was that the sheep study was performed under controlled climate conditions (approximate temperature of 16-21 C and humidity of 40-60 % ) whereas the cattle study was conducted outside where the animals were exposed to warm summer temperatures Environmental temperature has been recognized as an important factor in the pharmacokinetics of drugs for cold-blooded animals (Dorrestein, 1993) but little has been reported on the effect of the environmental temperature on drug disposition in warm-blooded animals. Osborne and MacKillop ( 198 7) studied the effects of e x posure of ovary cells to elevated temperatures on the plasma membrane permeability to adriamycin in vitro. They found an increase in permeability and drug uptake with increased temperatures. The effect of environmental

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115 factors on the percutaneous absorption of parathion was studied in excised porcine skin in a flow-through diffusion cell system (Chang and Riviere 1991) It was found that high relative humidity and elevated temperature conditions significantly increased parathion penetration Nawaz and Nawaz (1983) compared the pharmacokinetics of sulphadimidine after a single intravenous injection in sheep during summer and winter seasons It was found that zero time plasma concentration of the drug was higher during summer than in winter while the elimination half-life of the drug was not influenced by changes in the environmental temperature These observations can be related to the results of this study of tilmicosin pharmacokinetics in sheep and cattle where the animals were maintained under different environmental conditions and the difference was found in the tmax value between the two groups but not in the elimination half-life Kolendorf et al ( 1979) studied the effect of changes in blood flow on insulin disappearance from subcutaneous tissue It was reported that both adipose tissue blood flow and insulin disappearance were significantly increased after local heating of the skin surface (to 45 C) in the area of injection Similarly local cooling of the skin surface (to I0C) resulted in a significant decrease in the adipose tissue blood flow and insulin disappearance from the subcutaneous tissue These findings support the hypothesis that even non-extreme changes in temperature can affect d i sposition of drugs after subcutaneous injection On the other hand the sheep although not exposed to the heat stress were e x posed to stressful environmental conditions ( due t o the catheter placement frequent monitoring and blood collections) This was especially important in the first two hours of the e x periment when most of the sheep were restless and obviously agitated In addition they were enclosed and basically immobile in small cages throughout the first 6 hours of

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116 the experiment. It has been known that exposure of animals to noxious or stressful stimuli elicits the release of catecholamines (Danner et al., 1981 ; Funk and Stewart, 1996; Kaji et al., 1989). Although it was not determined in this study, one may expect to have seen a rise of catecholamines as a result of animal handling and stressful experimental conditions In his review on the subcutaneous absorption of drugs, Schou ( 1961) reported that epinephrine has been long recognized as an important factor able to influence the rate of absorption of drugs given subcutaneously Epinephrine constricts the terminal vascular bed in the zone of absorption constricting arteries, arterioles, capillaries and venules, and therefore, significantly depressing the blood flow through the absorbing area resulting in the reduced absorption Therefore the absorption of tilmicosin in sheep might have been slower than otherwise expected due to the effect of catecholamines on tilmicosin absorption Finally there may be a true species difference in the absorption phase of tilmicosin pharmacokinetics, for which there is some evidence Similar interspecies differences for the same pharmacokinetic parameters were found between the sheep (Cochrane and Thomson 1990 ; Elsom et al ., 1993) and cattle (Thomson 1989a and b). Clearance of tilmicosin in the sheep and cattle differed significantly when expressed in liters per hour When the data were normalized for animal weight the clearance values (0 6 l/h/kg in cattle, compared to 0 5 l/h/kg in sheep) were not statistically different. However when the allometric weight (weight to the power of 0 75) was used in calculations of clearance per body weight there was again a significant difference between the species with cattle showing higher clearance rates than sheep (2 78 and 1.47 l/h/kg0 7 5 ) This result is in contrast to findings oflngs (1990) who compared different

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117 pharmacokinetic parameters across species and their relationship with body weight. It has been observed for clearance and half-life that if they are plotted against body weight a distinct non-linear curve will be obtained showing a disproportional increase in those parameters with increased body weight. Other parameters such as volume of distribution, produce a linear curve when plotted against body weight (Ings 1990 ; Jezequel 1994 ; Khor et al., 1997) It has been therefore suggested for extrapolation of clearance values across species to employ allometric scaling while it was not necessary for the volume of distribution Allometric scaling relies on established extrapolation relationships using power functions of body weight and is often used for the extrapolation of physiological pharmacokinetic parameter distributions across species (Hayton, 1989 ; Hill and Wands 1989 ; Ings 1990 ; Watanabe & Bois 1996) The power function of0. 75 has been used most commonly for the allometric calculation of clearance and it has been found to be reasonably constant for a range of different drugs (lngs 1990) On the other hand the power function for volume of distribution approximates 1 suggesting a direct proportionality between volume of distribution and body weight In his review of pharmacokinetics in sheep as compared to other ruminants Short (1994) concluded that in estimating pharmacokinetic parameters between species the best comparison would be found between species of like size and that kinetic parameters would scale more closely between sheep and goats than between sheep and cattle. However he found that for drugs that do not undergo extensive metabolism but are eliminated by passive processes (such as renal g lomerular filtration) there is a high degree of similarity among domestic ruminants in the distribution and elimination of such drugs

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118 The similarity in pharmacokinetics of tilmicosin between sheep and cattle is in accordance with Short s observations since tilmicosin is an example of a drug that is eliminated mostly unchanged, and is not highly metabolized. In both sheep and cattle tilmicosin had a large apparent volume of distribution, 25 0 I/kg when normalized for animal body weight. In general if the V d calculated from plasma ( or serum) concentrations is much higher than the physiologic volume in which a drug distributes it indicates an extensive tissue distribution of the drug (DiPiro et al 1988) This was shown to be true for tilmicosin in the rat experiment where in both rats infected with M pulmonis and non-infected rats there was a significant difference (p<0 01) between the serum and lung concentrations oftilmicosin, with lung concentrations always being much higher. The half-life of tilmicosin in sheep and cattle based on the calculated arithmetic means per group did not differ significantly (35 versus 29 hours). It has been argued that elimination rate constant (ke), rather than elimination t l/2,, is the primary experimentally derived variable (Greenblatt et al., 1989) Therefore mean population values should focus on mean values of ke rather than mean values of t112, which is analogous to calculating the harmonic mean half-life (because t112 is derived from the reciprocal of ke). When the harmonic means were used for calculating the tl/2 in sheep and cattle the difference between the two groups was even smaller (33 hours in sheep versus 29 hours in cattle) but still very similar to the arithmetic means-based calculations These results are in agreement with Greenblatt et al (1989) who found no marked difference in the value of half-life based on arithmetic versus harmonic mean calculations

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119 Tilmicosin has been found to have much higher peak concentrations and AUC in the lung tissue than in serum in cattle (Thomson and Peloso 1989) sheep (Patel et al., 1992) mouse (Brown et al. 1995) rabbit (McKay et al., 1996) and rat (the results ofthis study). In all species the ratios between concentrations in the lung and serum ranged from 11: 1 (mouse) to 106 : 1 (sheep) and 178 : 1 (rats infected with M pulmonis) with the rat i o increasing with time Similar patterns have been observed for other macrolide antibiotics which have good tissue penetration (Aubert 1988 ; Bergogne-Berezin 1996 ; Nilsen 1995) In fact impro v ed tissue penetration was one of the main goals in developin g newer macrolides While erythromycin the first antibiotic from the macrolide class showed a relatively poor penetration into tissues (Fournet 1989) azithromycin reaches very high concentrations in the pulmonary tissues (Azoulay-Dupuis 1991 ; Leophonte 1995 ; Veber and Pocidalo 1995 ) Similar patterns of tissue distribution were also reported for other newer macrolides such as roxithromycin and clarithromycin (Nilsen, 1995 ; Veber et al., 1993) In this stud y of tilmicosin pharmacokinetics the relati v e activities of the c i s and tran s -stereo isomers of tilmicosin were not determined Therefore in the interspecies compari s on o f tilmico sin it w a s assumed that if the AU Co-inf v alues are equivalent the serum contained similar concentrations of the two isomers This assumption was based on the e x perience that interspecies differences in stereoselective absorption are not likely to occur with a parenteral dosa g e form or for drugs in which biotransformation does not play an important role in the dru g elimination (Landoni and Lees 1 9 96) T herefore since tilmicosin was administered parenterall y and does not under g o extensi v e biotransformation

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120 in the process of the drug elimination determination of relative activities of the two stereoisomers was not deemed necessary. 6 2 Cardiopulmonary Effects ofTilmicosin in Sheep Tilmicosin has been reported to cause cardiovascular toxicity in various species if given in high doses (Jordan et al., 1993) The aim of this set of experiments was to determine whether there would be any changes in cardiopulmonary physiology in 10 healthy sheep after receiving a single therapeutic dose of 10 mg/kg tilmicosin subcutaneously It was found that the systolic and mean blood pressures were lower in the tilmicosin-treated sheep than in the controls suggesting that tilmicosin, when given at a therapeutic dose might have a negative effect on blood pressure In the statistical analysis of the effect oftilmicosin on blood pressure the two-way repeated measures ANOVA was used to compare the blood pressure parameters between treatment groups The statistical analysis showed a significant difference between the two treatments for the systolic and mean blood pressure while there was no significant difference in the diastolic blood pressure. However, when multiple comparisons were done for the systolic and mean blood pressure analyses using the Dunnett s method (which compares numbers against zero or control) no significant differences were found between the treatments and the starting or control pressure Moreover when all systolic blood pressure measurements were normalized against zero time there was no difference between the treatments This normalization step was done because at zero time (before tilmicosin treatment) all three

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121 blood pressure measurements were lower in the tilmicosin-treated group of sheep than in the controls Based on the findings from the blood pressure monitoring experiments it is concluded that tilmicosin did not have a significant effect on any blood pressure parameters in sheep when given at the therapeutic dose of 10 mg/kg However the results suggest that there is a potential for seeing a decrease in blood pressure, if tilmicosin is given in doses that are higher than therapeutic. This hypothesis is based on the observed although non-significant decrease in the systolic and mean blood pressures Decreases in blood pressure after tilmicosin administration have been reported in dogs (Jordan et al., 1993 ; Main et al ., 1996) and cattle (Jordan et al., 1993) but in both cases only very high doses (several-fold multiples of the labeled 10 mg/kg) were able to produce a notable decrease in blood pressure. Similar to tilmicosin, several other macrolides such as erythromycin, oleandomycin spiramycin and leucomycin have been reported to cause a marked decrease in blood pressure (Wakabayashi and Yamada 1972) It was concluded in the study by Wakabayashi and Yamada that this depressor effect is correlated to an increase of histamine in the blood suggesting that macrolides might be histamine releasers In the same study the authors reported the absence of any sympathetic influence on the observed decrease in blood pressure for all four macrolides investigated Although tilmicosin seemed to slightly increase the heart rate in sheep the difference in the heart rate between the treated and control animals was not found to be statistically significant. The greatest difference between the treatment groups was observed at 1 hour after the treatment and the second greatest at 4 hours which was

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122 around the time when the maximum tilmicosin concentration in serum was reached (!max in sheep = 3 9 h) These results, if extrapolated to higher tilmicosin concentrations might support the hypothesis of tilmicosin cardiotoxicity in sheep This potential for tilmicosin induced adverse effects on the heart has been previously shown in dogs (Jordan et al 1993 ; Main et al. 1996) and cattle (Jordan et al. 1993) but has not yet been studied in sheep Jordan et al. (1993) concluded from their studies in dogs that the mechanism for cardiovascular toxicity of tilmicosin was at least partially mediated through the stimulation of cardiac f3-receptors In contrast to Jordan et al. (1993) Main et al. (1996) found that in dogs administered tilmicosin, treatment with propranolol did not attenuate tilmicosin induced tachycardia It was, therefore concluded in that study that the tilmicosin effect on the heart rate was not the result of f3-receptor stimulation. The authors suggested instead that the mechanism of tilmicosin cardiotoxicity might be mediated through intracellular calcium A rapid depletion of intracellular calcium through interference with sarcolemmal calcium channels or some other mechanism could result in negative inotropic effect of tilmicosin A similar mechanism has been suggested for josamycin and erythromycin, which were found to be capable of inhibiting transmembrane calcium flux (Tamargo et al 1982) The effect of tilmicosin on the respiratory rate in sheep was not significantly different from the effect in the control group. It appears that from about 0.5 hours until 4 hours after treatment there was a slight decrease in the respiratory rate in the sheep receiving tilmicosin when compared to the controls but at other times saline-treated sheep had lower respiratory rate than the tilrnicosin-treated ones In both treatment groups the respiratory rate steadily decreased with time and this effect of time was found

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123 to be statistically significant. The most likely explanation for that, as already discussed earlier in this chapter is that the sheep were more agitated and restless at the beginning of the experiments, which was most notable during the first 1 or 2 hours. It was seen less often as time passed and most of the animals became visibly calm over time, some of them even laying down to rest. The hypothesis that tilmicosin would not cause any changes in the hematology and blood chemistry panel in sheep was supported by the results ofthis study No treatment effect was found for any of the 16 parameters in the complete blood count analysis nor in any of the 19 blood chemistry parameters. However, there was a significant effect of time for several parameters tested in both tilmicosin-treated and control animals Changes over time were seen more frequently in the CBC than in the chemistry tests, with 50% of the CBC parameters versus 21 % of the chemistry parameters being changed over time These changes over a three-day testing period may possibly be attributed to the concurrent collection ofblood samples (there was a total of 21 12-ml samples collected) and resulting change of blood composition The non-significant results of the hematology and chemistry analyses are in accordance with the results of the toxicity studies on tilmicosin in cattle (Jordan et al. 1993) In that study after multiple subcutaneous doses of 150 mg/kg (IS-fold higher than the labeled IO mg/kg dose), the cattle showed only mild haemoconcentration and neutrophilia In the chemistry analysis there was an increase in the activities of lactate dehydrogenase and creatine phosphokinase and mild decreases in serum levels of alkaline phosphatase proteins and electrolytes Neither change was considered of toxicological importance and both were attributed to tissue damage and edema at the injection site All

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124 other parameters of the CBC and chemistry analyses were either within established normal ranges or were not significantly different from those of concurrent controls Ziv et al (1995) reported that the only abnormal blood enzyme activity in cattle after receiving 10 mg/kg of tilmicosin subcutaneously was a transient significant rise in serum creatine phosphokinase activity In this study the activity of serum creatine phosphokinase was not determined but all enzyme activity tests performed (results are shown in Appendix D) showed that there were no significant differences between the tilmicosin-treated and control sheep 6 3 Lung Tissue Distribution ofTilmicosin in Rats The study in rats compared serum and tissue pharmacokinetics of tilmicosin between the animals infected with Mycoplasma pulmoni s and healthy animals The rats were first treated with Mycoplasma or plain broth and were administered a single subcutaneous injection of 20 mg/kg tilmicosin 31 days after the initial treatment. Lung blood and muscle samples were collected 31 to 34 days after the microorganism or broth inoculation when the chronic respiratory disease was established in the infected group There were no outward clinical signs of the disease at the time of sampling but at necropsy the infected animals had visible gross pathologic changes in the lung including scattered lesions atelectasis and tissue consolidation It was found that the infected lungs were edematous and increased in wei g ht with the avera g e weight of the infected lung being 1.01 g compared to 0 94 gin the non-infected group These findings are in agreement with typical pathologic changes resulting from the respiratory M y copla s ma infection (Cassell and Hill 1979 ; lntraraksa et al 1984 ; Lindsey and Cassell 1973).

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125 Although testing of the susceptibility of M pulmonis to tilmicosin was not a part of this project some preliminary results indicate that tilmicosin may be effective against M pulmonis in rats (Davidson Pers. Comm.). This speculation is based on the observation that Mycoplasma-infected rats that were subsequently treated with tilmicosin had fewer lung lesions and other pathologic changes in the lungs at the time of sacrifice than what would be expected 40 days post infection Because of the important economic and research losses due to the mycoplasma infection in laboratory rodents future studies should determine the efficacy of tilmicosin against M pulmonis in vitro and in vivo for both rats and mice In the HPLC analysis of the levels of tilmicosin in the lung tissue the whole lung of an animal was used as a specimen, and therefore the concentrations were expressed per lung rather than per gram of tissue Previously it had been decided to use that approach for quantification of tilmicosin in lung tissue because an increase in the weight of lung was expected as a result of infection / inflammation. Since tilmicosin is hypothesized to accumulate intracellularly or to be bound to cell membranes and/or organelles (Brown et al., 1995) the inflammatory processes and edema should not greatly influence the results of the drug analysis if calculated per lung In contrast if the results were calculated per gram of tissue the true tilrnicosin concentrations in the lung could be underestimated The results of the pharmacokinetic study showed that similar to other macrolides (Bergogne-Berezin 1995b ; Veber et al., 1993) tilmicosin had a good tissue penetration, consistently exhibiting higher lung tissue concentrations than serum concentrations This was true for both infected and non-infected animals but the lung : serum ratio was always higher in the infected than non-infected group

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126 In the non-compartmental pharmacokinetic analysis of data from the rat study there seemed to be no major differences between the infected and non-infected rats for any parameters calculated suggesting that infection I inflammation does not have an effect on the serum pharmacokinetics of tilmicosin However statistical analyses were not performed on those data, since the average results for each time point were pooled from 6 different rats per group (for which the rats were sacrificed at that time) so the same animals could not have been monitored over time for their tilmicosin concentration in the lung and serum Veber et al. ( 1993) compared the lung tissue and serum pharmacokinetics of erythromycin roxithromycin clarithromycin spiramycin and azithromycin in mice with and without pneumonia They found no major differences in the serum pharmacokinetics of the macro Ii des tested while the lung tissue concentrations were significantly higher in the infected mice than in the controls Azithromycin was found to have the highest lung tissue concentrations when compared to serum and erythromycin the lowest. These results agree with the data collected from this study where tilmicosin serum pharmacokinetics seemed to be unchanged by infection while the distribution into lung tissue was significantly increased in the infected animals when compared to controls The results of the non-compartmental pharmacokinetic analysis in rats were compared to those from the sheep and cattle. It was presumed based on the literature that tilmicosin pharmacokinetics in rats would show enough similarities with cattle and sheep and therefore the rat was used as a model for studying the tissue distribution of tilmicosin When the results of the non-compartmental pharmacokinetic analyses were compared across the three species it was found that the half-life was much shorter in rats

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127 than ruminants (8 hours as opposed to 30-35 hours respectively) Rats had very low serum concentrations of tilmicosin already at 24 hours after administration of the drug and by 72 hours, it was impossible not only to quantify but in most cases even to detect tilmicosin in serum at all. Therefore the values for AUC AUMC and MRT were much lower in the rat group when compared to the cattle and sheep Both infected and non infected rats had tmax value of 1 hour but there were no samples taken earlier than 1 hour after subcutaneous tilmicosin administration, nor between 1 and 3 hours so the actual 1:max could have occurred anywhere within that window However it still seems that the absorption of tilmicosin from the injection site in the rats resembles more the cattle model (1:max = 0 5 h) rather than sheep (tmax = 3.9 h) Both clearance and volume of distribution seemed to be increased in the infected rats when compared to controls and in both groups when the parameters were expressed per kilogram body weight they were higher in rats than in ruminants The difference in V d between the species does not seem to be striking (25 I/kg for the sheep and cattle versus 29, and 33 I/kg for the non-infected and infected rats respectively) This is in agreement with findings that the relationship between volume of distribution and body weight is linear and does not require allometric scaling (Ings 1990) Unlike the volume of distribution, clearance was found to be approximately five-fold higher in rats than in the ruminants (0 5-0 61/h/kg for the sheep and cattle versus 2 7 and 3 1/h/kg for the non-infected and infected rats respectively) The variation in clearance between the ruminants and rats is not unexpected because in small laboratory animals in general drug elimination is more rapid due to the higher rate of basal metabolism (Baggot 1992) Therefore the allometric scaling was employed for the interspecies comparison of clearance It was found that the

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128 difference in clearance between the healthy rats and the ruminants diminished considerably when the results were normalized for body weight (1.95 l/kg0 75 in the rats, compared to 2.7 l/kg0 75 in the sheep and 1.5 l/kg0 75 in the cattle) This finding is again in agreement with Ings (1990) showing that clearance variable correlates with body weight in a non linear way and indicating that the allometric scaling should be used for interspecies compansons The two hypotheses of the rat study were that: (1) Tilrnicosin concentration in the lung tissue would be higher in the infected than in the non-infected rats ; and (2) the pH of the lung tissue would be lower in the infected than in the non-infected animals. While the first hypothesis was found to be supported by the data from this study, the second hypothesis was not. Rats infected with M pulmonis had significantly higher concentrations of tilrnicosin in the lung tissue than non-infected rats This was observed already at 1 hour after tilrnicosin administration (first sample collection) and was persistent until 24 hours after tilrnicosin treatment. The only time point where there was no difference in tilmicosin lung concentration between the infected and non-infected rats was at 72 hours However it may be that the true average lung concentration for the non-infected group at 72 hours is considerably lower than presented here ( and therefore considerably lower compared to the infected rats) because one of the rats had the unusually high tilmicosin concentration of 5.6 g/lung (while all the others in the group ranged between 0 .6 8 and 1.55 g/lung and moreover none of the animals had a concentration higher than 3 5 g/lung at 24 hours after tilmicosin administration) If this one sample is considered an outlier then

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129 tilmicosin concentration in the infected rats was significantly higher than in the controls for all time points included in the study These findings of increased lung concentrations of tilmicosin in the Mycoplasmainfected rats suggest that infection / inflammation improves tissue penetration of tilmicosin which is a typical feature of antibiotics from the macrolide class (Bergogne Berezin, 1995b ; Veber et al., 1993) The only exception to that is erythromycin, which not only lacked a better tissue penetration in Pasteurella-infected calves (Burrows 1985) but also had significantly different serum pharmacokinetic parameters (increased distribution and elimination rates decreased half-life and V d) in the infected animals when compared to the controls (which are both in contrast to the findings of this study) Antibiotics from other classes such as ampicillin (Agapitova and lakovlev 1987) ceftazidime (McColm et al., 1986) and pentamidine (Mordelet-Dambrine et al ., 1992) have also been shown to have impaired tissue penetration during experimental infections While the tmax for tilmicosin in serum was 1 hour in lung tissue the maximum concentration was reached at 3 hours after tilmicosin administration for the infected and at 7 hours for the non-infected rats Again, because of the sampling schedule it was not possible to determine the exact time oftmax, but some conclusions can be drawn : (1) the penetration into lung tissue appeared faster in the infected animals than in the non infected ; and (2) in both groups as expected there was a time lag between the tmax reached in serum and lung tissue Mycoplasma-infected rats exhibited higher lung : serum ratios than the controls at each time point of sample collection, but the difference became most marked at 24 hours (last quantitation point for serum samples) after tilmicosin administration when the ratio

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130 in the infected group was 178 : 1 as opposed to 86 : 1 in the non-infected If these results are extrapolated one could expect to see an even bigger difference in the lung: serum ratio after 24 hours This presumption is supported by the findings from the sheep study of Patel et al. (1992) where the highest lung :plasma ratio (106 : 1) was observed at 72 hours after tilmicosin administration This suggests that tilmicosin s tendency to accumulate more in the infected tissue than in the non-infected tissue could result in an increased efficacy of the drug in patient populations as compared to antibiotics with poorer tissue penetration such as erythromycin (Azoulay-Dupuis, 1991) A similar finding of improved efficacy was reported for azithromycin, which also tends to have a better tissue penetration in the infected than non-infected tissues ( Azoulay-Dupuis 1991 ; Bergogne-Berezin, 1995 ; Girard et al ., 1996 ; Leophonte 1995) Girard et al. (1996) reported that azithromycin was extensively associated with the cellular component of the inflamed middle ear bulla wash as opposed to the fluid component as was reported for some other antibiotics Furthermore azithromycin was found to have a reduced tissue AUC in leukopenic mice indicating that leukocytes may help transport macrolides to the sites of infection (Leophonte 1995 ; Veber et al., 1993) Therefore the suggested explanation for the improved tissue penetration of azithromycin in infection was that the leukocytes release azithromycin after having migrated into the infectious site thus allowing to increase the antibiotic concentration at the infection site (Leophonte 1995) Although the exact site oftilmicosin action is not known Brown e t al. (1995) hypothesized that it accumulates within pneumocytes or binds to membranes and/or organelles However the comparison oflung and serum concentrations oftilmicosin in

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131 that study was done in healthy animals and their hypothesis of pneumocyte accumulation is not supported by the results of this study where the lung accumulation was increased in infected animals when compared to controls In future studies on tilmicosin pharmacokinetics it would be interesting to investigate whether a similar mechanism of accumulation in the infected tissues and particularly into leukocytes as proposed for azithromycin applies to tilrnicosin No correlation was found between the lung tissue pH and tilmicosin concentration in the lung with regard to the Mycoplasma infection nor did the treatment (Mycoplasma or plain broth administration) have any effect on the muscle pH. Therefore it seems that some other mechanisms are involved in the observed increase in tilmicosin tissue penetration during infection It is also possible that there was indeed a shift in the lung pH, but that it was not detectable with the method used in which the average pH of all components of the lung tissue was measured. The hypothesis of the tissue distribution study was that animals infected with M pulmonis would have higher lung tissue concentrations of tilrnicosin than non-infected animals and that it would be explained by trapping of tilrnicosin within an acidic environment of the lung tissue resulting from infection Tilrnicosin is a highly lipophilic weak base with pKa values of 7 4 and 8 5 so it easily crosses cell membranes and distributes quickly until equilibrium is reached However, if the pH across the membrane becomes even slightly acidic tilrnicosin will become ionized and therefore trapped within the acidic tissue Evidence does exist that the pH decreases locally in respiratory diseases as indicated by lowering of the pH of the pleural fluid (Chavalittamrong et al. 1979 ; Hoff et

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132 al. 1989) respiratory mucus (Karnad et al., 1990) and in the bronchi (Bodem et al., 1983) Moreover the degree of acidification was found to be proportional to and predictive of the severity ofrespiratory disease (Chavalittamrong et al. 1979 ; Hoff et al., 1989) Karnad et al. (1990) suggested that a decrease in pH of the respiratory mucus could predict the presence of pneumonia with a positive predictive value of90%. They found that similar to cerebrospinal, ascitic and synovial fluids respiratory mucus became acidic in acute bacterial infections due to lactate production by bacteria and leukocytes An analogous type of prediction with a similar predictive value was suggested by Payne et al. ( 1988) but in that case a decrease in blood pH to less than 7 25 was recommended to be used tog ether with some other criteria to evaluate and predict the outcome of the Group B streptococcal infection. lntraraksa et al. (1984) reported that in swine e x perimentally infected w ith Mycoplasma hyopne um o nia e there was a decrease in arterial blood pH, arterial partial pressures of 0 2 and CO 2 and arterial concentration ofHCO3 The suggested e x planation for the observed changes in the blood gas parameters is that as a result of poor diffusion of o xy gen at the alveoli there was a marked decrease in partial pressure of o xy gen in arterial blood resultin g in metabolic acidosis and a slight decrease in the pH of arterial blood However the blood pH values in most infected animals remained near control v alues because of the compensatory mechanisms produced by respiratory alkalosis In this stud y both mu s cle and lun g pH were measu r ed usin g a direct method with the penetration probe inserted into the muscle and lun g tissue (as e x plained in chapter 4 2 1.) Different electrometric method s (as opposed to colorimetric and indicator methods) for measurin g t issue pH have been suggested for use by researchers in the meat

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133 science Some workers prefer the tissue homogenate method, others favor the direct method still others use a water-muscle mixture or meat extracts for measuring the pH (Bager and Petersen 1983 ; Korkeala et al 1986). In this study the direct measurement method was used because of all the methods available that involved the least manipulation of samples This was critical since lung tissue samples were to be subsequently used in the HPLC analysis and were therefore to be subjected to the liquid liquid extraction procedure so nothing could have been added to samples before extraction. Korkeala et al. ( 1986) compared various methods for determination of the pH in meat and found that although there were some differences between the methods (homogenate, direct meat-water mixture) none of the methods currently used could be considered better than the others. On the other hand Bager and Petersen (1983) found that the probe method did not differ significantly from the tissue homogenate method To summarize this part of the tilmicosin study although the correlation was not found between tilmicosin lung concentration and pH of the lung tissue the possibility that it does exist cannot be ruled out. Knowing that alterations from the physiological pH are usually not of great magnitude, and being aware of the limitations of the method used for determination of pH, the possible significance of local tissue pH on tilmicosin distribution might have been underestimated by the results of this study Indeed it can be speculated that the hypothesized local decrease in the lung pH as a result of infection was not detected because of the method used If the tissue homogenate method was used, the process would break cells and cellular elements including lysosomes therefore releasing oxidative enzymes and decreasing the pH. About 40 hydrolytic enzymes are known to be contained in lysosomes and all are acid hydrolases optimally active near the pH 5

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134 maintained within lysosomes (Alberts et al., 19890. The number ofphagocytic cells containing lysosomes increases during infection and inflammation (Butts 1994 ; Leophonte 1995; Veber et al. 1993) so there would be a decrease oflung pH as a result of the migration of phagocytic cells into infectious site This new hypothesis further justifies the importance of studying within-tissue distribution of tilmicosin in both infected and healthy hosts However even if the hypothesis of decreased lung pH in infection is supported by findings from future studies there must be yet another mechanism responsible for tilmicosin partitioning in the lung tissue It has been found in this and previous studies that tilmicosin concentrations in the lung tissue of healthy animals were also higher than the serum concentrations (although lung : serum ratios were significantly higher in the infected group) so the mechanism of the lung tissue partitioning of tilmicosin (in healthy and infected tissue alike) remains unclear

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CHAPTER 7 SUMMARY AND CONCLUSIONS The present study was undertaken to compare the pharmacokinetic properties of tilmicosin between the sheep and cattle, to learn about the adverse effects of tilmicosin on the cardiopulmonary system in sheep and to determine the effect of disease on tilmicosin tissue distribution Based on the studies of other drugs a high degree of similarity was expected between ruminants with regard to distribution and elimination of tilmicosin The aim of the first portion of the study was to quantify and compare serum pharmacokinetic parameters between the two ruminants There were no significant differences between the two species in the elimination rate half-life AUC AUMC MRT Cl (per kg body weight), and Vd (per kg body weight) The tmax was the only significantly different non-compartmental pharmacokinetic parameter with cattle having a tmax of 0.5 hours versus 3 9 hours in sheep This finding suggests that tilmicosin was absorbed faster from the site of injection in cattle than it was in sheep Several approaches can be used to explain this difference First although in both species the drug was administered subcutaneously in sheep it was injected between the scapulae whereas in cattle the dose was given in the lateral neck region. There is evidence in the literature that different injection sites can result in different absorption rates Secondly there were some differences in the environmental conditions between the two experimental set-ups Sheep were maintained inside in a temperatureand 135

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136 humiditycontrolled environment while the cattle experiment was performed outside during summer when both temperature and humidity were high Findings from several studies have indicated that high environmental temperatures can influence pharmacokinetic properties of drugs both in vivo and in vitro. Those results support the hypothesis that high environmental temperatures to which the cattle were exposed during the tilmicosin experiment might have affected the rate of absorption of the drug from the site of injection On the other hand the levels of catecholamines might have been increased in the sheep as a result of stressful experimental conditions This could have caused depression of the blood flow through the absorbing area and therefore slower absorption of tilmicosin from the site of injection in sheep And finally a genuine species difference might exist between the two ruminants in the rate of absorption of tilmicosin from the site of injection In conclusion the comparative study of tilmicosin pharmacokinetics in sheep and cattle showed that with the exception of the tmax difference all other non-compartmental pharmacokinetic parameters were not significantly different. This suggests a fair degree of similarity between the two species In the compartmental analysis it was determined that tilmicosin was best described by a two-compartment body model in both species, and except for the initial uptake portion of the serum concentration versus time curve the curves proved to be very similar The aim of the pharmacodynarnic study was to determine the adverse effects of a therapeutic dose of tilmicosin on the cardiopulmonary system in healthy sheep The potential for causing cardiovascular toxicity has been reported for tilmicosin in various species but all studies included either high doses of the drug and/or the unlabeled routes

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137 of administration of tilmicosin In this study using the labeled dose of IO mg/kg body weight and the labeled subcutaneous route of injection no adverse effects of tilmicosin were found on the blood pressure heart rate nor respiratory rate These results suggest that tilmicosin can be safely used in sheep for effective treatment of respiratory infections without an increased risk of cardiovascular toxicity In the study of the tissue distribution of tilmicosin the effects of a chronic respiratory disease on the pharmacokinetics of the drug were determined Tilmicosin was found to have not only good tissue penetration ( when serum concentrations are compared to lung tissue concentrations) but this finding was shown to be more pronounced in the animals with chronic respiratory disease than in the healthy animals That effect of good penetration of tilmicosin in infected tissues and the demonstrated ability to retain those elevated concentrations for longer periods of time promises an effective single-dose treatment of respiratory disease with this antibiotic In conclusion, it has been proven by the results of this study that tilmicosin can be used safely and effectively in sheep The fate of tilmicosin in sheep closely resembles its pharmacokinetic properties in cattle Therefore the same dose of 10 mg/kg for both sheep and cattle should result in concentrations high enough and retained for long enough time above the MIC of the microorganisms involved in respiratory infections This dose of tilmicosin has been found to cause no adverse effects on the cardiopulmonary system, suggesting that tilmicosin in sheep can be used as safely as in cattle Finally this study has proven the ability oftilmicosin to reach high lung tissue concentrations that are necessary to inhibit or kill the microorganisms Moreover it has been found that the lung tissue

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138 distribution of tilmicosin improves as a result of respiratory disease implying a possible therapeutic advantage of tilmicosin in treating lung infections

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LIST OF REFERENCES Adams HR (1975) Acute adverse effects of antibiotics. J. Am. Vet .Med. Assoc. 166 : 983-987 Adams HR & Parker, JL (1982) Cardiovascular depressant effects of antibiotics IN: CARDIOVASCULAR TOXICOLOGY. Ed. VanStee, EW (Raven Press, New York). pp 327-351. Adams HR; Parker, JL; Mathew, BP (1979) Cardiovascular manifestations of acute antibiotic toxicity during E coli endotoxin shock in anesthetized dogs. Circ Shock, 6 : 391-404 Agapitova IV & Bobrov, VI (1984) Cephalosporin and carbenicillin penetration into the tissues of rats with aseptic inflammation Antibiotiki, 29:427-430 Agapitova, IV & lakovlev VP (1987) Antibiotic pharmacokinetics in rats with an infected inflammation Antibiot. Med. Biotekhnol. 32: 508-511 Alberts B ; Bray, D ; Lewis J ; Raff, M ; Roberts, K ; Watson, JD (Eds. ) (1989) Intracellular sorting and the maintenance of cellular compartments IN: MOLECULAR BIOLOGY OF THE CELL, 2nd edition (Garland Publishing Inc New York, NY). pp. 405-480. Arai S ; Tabata, S ; Kobayashi S ; Inazu M ; Hayashi S (1989) Pharmacokinetic study of cefodizime in experimentally infected animals Arzneimittelforschung, 39: 877-882 Aubert G (1988) Macrolides Rev. Pneumo!. Clin 44 122-127 Azoulay-Dupuis E ; Vallee E ; Bedos, JP; Muffat-Joly M ; Pocidalo JJ (1991) Prophylactic and therapeutic activities of azithromycin in a mouse model of pneumococcal pneumonia Antimicrob Agents Chemother. 35: 1024-1028 Bag er, F. & Petersen, GV ( 1983) The relative precision of different methods of measuring pH in carcass muscles Nord. Vet. Med., 35 : 86-90. Baggot, JD (1992) Clinical pharmacokinetics in veterinary medicine Clin Pharmacokinet. 22: 254-273 139

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140 Barragry TB (1994) General Clinical Pharmacology : Macrolides and Lincosamides IN: VETERINARY DRUG THERAPY. (Lea & Febiger Malvern PA). pp. 251-262 Baumann H ; Marinkovic-Pajovic S ; Won, KA;. Jones VE; Campos SP ; Jahreis GP; Morella KK (1992) The action of interleukin 6 and leukaemia inhibitory factor on liver cells. Ciba Found. Symp ., 167 : 100-114 Benet LZ & Sheiner LB (1985) Pharmacokinetics : The dynamics of drug absorption, distribution and elimination. IN: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS. Eds. Goodman Gilman A; Goodman, LS ; Rall TW; Murad, F (Macmillan Publishing Company New York) pp. 3-34. Bergan, T ( 1981) Pharmacokinetics of tissue penetration of antibiotics Reviews of Infectious Diseases 3 :45-66. Bergogne-Berezin, E (1995a) Predicting the efficacy of antimicrobial agents in respiratory infections--is tissue concentration a valid measure? J. Antirnicrob. Chemother. 35 : 363-371 Bergogne-Berezin E ( 1995b) Azithromycin : tissue pharmacology Pathol. Biol. (Paris) 43 : 598-504 Bergogne-Berezin E (1996) Tissue pharmacokinetics of antibiotics Theoretical bases and new pharmacological approaches Presse Med., 25 : 399-406 Boddeke HW; Wilffert B ; Heynis JB; van Zwieten, PA (1988) Investigation of the mechanism of negative inotropic activity of some calcium antagonists L. Cardiovasc Pharmacol. 11:3213 25 Bodem, CR; Lampton, LM; Miller DP; Tarka, EF; Everett ED (1983) Endobronchial pH. Relevance of aminoglycoside activity in gram-negative bacillary pneumonia Am. Rev. Respir Dis ., 127:39-41. Brisson-Noel A ; Trieu-Cuot P ; Courvalin P (1988) Mechanism of action of spiramycin and other macrolides J. Antimicrob Chemother. 22 (Suppl. B): 13-23 Brown, MB & Reyes L (1991) Immunoglobulin classand subclass-specific responses to Mycoplasma pulmonis in sera and secretions of naturally infected Sprague-Dawley female rats. Infect. Immun ., 59 : 2181-2185 Brown, MB & Steiner DA (1996) Experimental genital mycoplasmosis : time of infection influences pregnancy outcome. Infect. Immun 64 : 2 3 15 2321.

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141 Brown, SA; Deleeuw NR; Stahl GL; Roof, RD (1995) Characterization of plasma and lung concentrations after ceftiofur sodium and tilmicosin phosphate administered subcutaneously to mice J. Vet. Pharmacol. Therap ., 18 : 385 -387. Bryskier A & Labro, MT (1994) Macrolides. New therapeutic prospects Presse Med., 23 : 1762-1766 Burrows, GE ( 1985) Effects of e x perimentally induced Pasteurella haemolytica pneumonia on the pharmacokinetics of erythromycin in the calf Am. J. Vet. Res., 46: 798-803 Butts, JD (1994) Intracellular concentrations of antibacterial agents and related clinical implications. Clio. Pharmacokinet. 27: 63-84 Carter KK; Hietala S ; Brooks, DL; Baggot, JD (1987) Tylosin concentrations in rat serum and lung tissue after administration in drinking water. Lab. Anim Sci ., 37:468-470 Cassell GH (1982) Derrick Edward Award Lecture The pathogenic potential of mycoplasmas : Mycoplasma pulmonis as a model. Rev. Infect. Dis. 4 :Sl8-S34. Cassell GH; Davis JK ; Lindsey JR (1981) Control ofMycoplasma pulmonis infection in rats and mice : detection and elimination vs vaccination Isr. J. Med. Sci 17 : 674-677 Cassell GH; Davis JK ; Simecka JW; Lindsey JR; Cox, NR; Ross, S ; Fallon, M (1981) Mycoplasma infections : Disease pathogenesis implications for biomedical research and control. IN: VIRAL AND MYCOPLASMAL INFECTIONS OF LABORATORY RODENTS. EFFECTS O N BIOMEDICAL RESEARCH. Eds. Bhatt PN; Jacoby RO; Morse, HC; New, AE (Academic Press Inc., Orlando FL) pp 87-130 Cassell GH & Hill A. (1979) Murine and other small-animal Mycoplasmas IN: THE MYCOPLASMAS, VOL. II, HUMAN AND ANIMAL MYCOPLASMAS. Eds. Tully and Whitcomb (Academic Press) pp 235-273 Chang SK & Riviere JE ( 1991) Percutaneous absorption of parathion in vitro in porcine skin : effects of dose temperature humidity and perfusate composition on absorptive flux Fundam Appl. Toxicol. 17 : 494-504 Chavalittamrong B ; Angsusingha K ; Tuchinda M ; Habanananda S ; Pidatcha P ; Tuchinda, C (1979) Diagnostic significance of pH, lactic acid dehydrogenase lactate and glucose in pleural fluid Respiration 38 : 112-120 Church TL & Radostits OM ( 1981) A retrospective survey of diseases of feedlot cattle in Alberta Can Vet. J., 22: 27-30

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142 Cochrane RL & Thomson, TD (1990) Toxicology and pharmacology oftilmicosin following administration of subcutaneous and intravenous injections to sheep Lilly Research Study Report T5C768908 (Eli Lilly Pers. Comm. : Tilmicosin Injection Application to Extend License to Include Sheep) Cohen, LS; Wechsler AS ; Mitchell JH; Glick, G (1970) Depression of cardiac function by streptomycin and other antimicrobial agents Am. J. Cardiol. 26 : 505-511. Colbert WE; Turk, JA; Williams PD; Buening MK (1991) Cardiovascular and autonomic pharmacology of the macrolide antibiotic L Y2813 89 in anesthetized beagles and in isolated smooth and cardiac muscles Antimicrob Agents Chemother. 35 : 1365-1369 Combs, AB & Acosta, D (1990) Toxic mechanisms of the heart : a review Toxicol. Pathol., 18 : 583-596 Crosier KK; Riviere JE; Craigrnill AL (Eds. ) (1996) Tilmicosin phosphate. IN: THE FOOD ANIMAL RESIDUE AVOIDANCE DATABANK. A COMPREHENSIVE COMPENDIUM OF FOOD ANIMAL DRUGS. 10th Ed. (Publications and Distribution Center, University of Florida Gainesville, FL). p 386. Curl JL; Curl JS ; Harrison JK (1988) Pharmacokinetics oflong acting oxytetracycline in the laboratory rat. Lab. Anim Sci ., 38:430-434 Danner SA; Endert, E ; Koster, RW; Dunning AJ (1981) Biochemical and circulatory parameters during purely mental stress Acta Med. Scand., 209: 305-308. Darling L. (1993) Micotil 300 Injection. Tilmicosin Phosphate. IN: VETERINARY PHARMACEUTICALS AND BIOLOGICALS, 8th e Ed. Darling, L. (Vet. Med. Publishing Company Madison WI). pp. 573-574. Davidson MK; Davis JK; Lindsey JR; Cassell GH (1988) Clearance of different strains ofMycoplasma pulmonis from the respiratory tract of C3H/HeN mice. Infect. Immun ., 56 : 2163-2168 Davis JK & Cassell GH (1982) Murine respiratory mycoplasmosis in LEW and F344 rats : strain differences in lesion severity. Vet. Pathol. 19 :280293 Davis JK; Thorp, RB; Maddox, PA; Brown, MB; Cassell, GH (1982) Murine respiratory mycoplasmosis in F344 and LEW rats: evolution of lesions and lung lymphoid cell populations Infect. lmmun. 36: 720-729

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1 4 3 Debono M ; Willard, KE; Kirst HA; Wind JA; Crouse GD; Tao, EV; Vicenzi JT; Counter FT; Ott, JL; Ose EE; Omura, S (1989) S y nthesis and antimicrobial evaluation of 20-deoxo-20-(3 5-dimethylpiperidin-l-yl)desmycosin (tilmicosin, EL-870) and related cyclic amino derivatives The Journal of Antibiotics 4 : 12531267 DiPiro JT; Blouin, RA; Pruemer JM; Spruill WJ ( 1988) Volume of distribution and body fluids IN: CONCEPTS IN CLINICAL PHARMACOKINETICS. A SELFINSTRUCTIONAL COURSE. (American Society of Hospital Pharmacists Bethesda, MD) pp. 19-20 DiPiro JT; Record KE; Schanzenbach KS ; Bivins BA (1981)Antimicrobial prophylaxis in surgery: Part 1. Am. J. Hosp. Pharm., 38: 320-334 Donoho, AL ( 1988) comparative metabolism of 1 4C tilmicosin in cattle and rats Lilly Research Study Report ABC-0395 (Eli Lilly Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Donoho, AL; Peloso JS ; Thomson TD ( 1988) 14C tilmicosin tissue residue study in cattle Lilly Research Study Report ABC-0383 (Eli Lilly Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Dorrestein GM (1993) Problems and recommendations concerning treatment of e x otic pets. Tijdschr Diergeneeskd 118 : 47 Edward, DG & Freundt EA (1956) The classification and nomenclature of organisms of the pleuropneumonia group. J. Gen. Microbiol. 14 : 197-207 Elanco Animal Health (1994) Micotil 300 Injection Tilmicosin Phosphate Manufacturer s drug insert (revised April 8 1994) Indianapolis IN. Elsom LF; Hawkins DR; Dighton MH; Kaur, A ; Cameron DM (1993) The metabolism and residues of 1 4C tilmicosin following subcutaneous administration to sheep Huntingdon Research Centre Study Report HRC/LLY36 / 930477 (Eli Lill y, Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Federal Register Rules and Regulations (Sept. 4 1987) : 21 CFR Part 58 -Good Laboratory Practice Regulations Final Rule. IN: FEDERAL REGISTER, 52(172): 33768-33782 Department of Health and Human Services Food and Drug Administration Federal Register Rules and Regulations (Dec. 27, 1996) : 21 CFR Parts 556 and 558 Animal Drugs, Feeds and Related Products; Tilmicosin Phosphate Type A Medicated Article IN: FEDERAL REGISTER, 61(250) : 68147-68148. Department of Health and Human Services Food and Drug Administration

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144 Fournet MP; Zini R; DeForges, L ; Lange, F ; Lange, J ; Tillement JP (1989) Tetracycline and erythromycin distribution in pathological lungs of humans and rat. J. Pharm. Sci. 78: 1015-1019 Fraser CM; Mays, A ; Amstutz HE ; Archibald J ; Armour, J ; Blood, JC; Newberne, PM; Snoeyenbos, GH; Huebner, RA (Eds.) (1986) Respiratory diseases of sheep and goats IN: MERCK VETERINARY MANUAL (Merck & Co. Inc ., Rahway NJ). pp. 722728 Freedman, RA; Anderson KP; Green LS; Mason, JW (1987) Effect of erythromycin on ventricular arrhythmias and ventricular repolarization in idiopathic long QT syndrome Am. J. Cardiol. 59 : 168-169 Funlc, D & Stewart J ( 1996) Role of catecholamines in the frontal cortex in the modulation of basal and stress-induced autonomic output in rats. Brain Res. 741:220-229 Gibaldi M (1991) Pharmacokinetic variability Disease IN: BIOPHARMACEUTICS AND CLINICAL PHARMACOKINETICS. Lea & Febiger (Malvern, PA) pp. 272-304. Gibaldi M ; Levy G ; Weintraub H (1971) Drug distribution and pharmacologic effects Clin Pharmacol. Ther. 12 : 734-742. Gibaldi M & Perrier D (1982) IN: PHARMACOKINETICS. Marcel Dekker, New York. Giera DD; Herberg JR; Klink PR; Thomson, TD (1986) 1 4C EL-870 tissue residue decline study and balance-e x cretion study in cattle Lilly Research Study Report ABC-0340 (Eli Lilly Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Giera DD; Herberg JR; Thomson, TD (1987) 14C EL-870 balance excretion and tissue residue stud y in a steer. Lill y Research Study Report ABC-0299 (Eli Lilly Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Giera DD & Peloso, JS (1988) Characterization of radioactive residues in cattle tissues following therapeutic dose of 14C EL-870. Lilly Research Study Report ABC-0353 (Eli Lilly Pers. Comm.: Tilmicosin Injection. Application to Extend License to Include Sheep) Girard AE; Cimochowski CR; Faiella J A ( 1996) Correlation of increased azithromycin concentrations with phagocyte infiltration into sites of localized infection L Antimicrob Chemother ., 37 (Suppl. C):9-19.

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145 Gourlay RN ; Thomas LH; Wyld SG; Smith, CJ (1989) Effect of a new macrolide antibiotic ( tilmicosin) on pneumonia experimentally induced in calves by Mycoplasma bovis and Pasteurella haemolytica Res. Vet. Sci., 47 : 84-89 Gray JA (1984) Antimicrobial drugs. IN: CLINICAL PHARMACOLOGY. Ed. Girdwood RH (Bailliere Tindall University press Cambridge UK) pp 1-104 Green, GM & Goldstein E (1966) A method for quantitating intrapulmonary bacterial inactivation by individual animas. J. Lab Clin Med ., 68 : 669-677 Greenblatt, DJ; Harmatz JS; Friedman H (1989) Arithmetic versus harmonic mean values of elimination half-life : A study of triazolam. J. Clin Pharmacol. 29 : 655-656 Gueugniaud PY; Guerin C ; Mahul P ; Due, C ; Robert D (1985) Torsades de pointe induites par I association lidocaine-erythromycine et insuffisance hepatique Presse Med. 14 : 896 Hamill RL ; Haney ME; Stamper M ; Wiley P (1961) Tylosin a new antibiotic : II Isolation, properties and preparation of desmycosin, a microbiologically active degradation product. Antibiotics and Chemotherapy 11, 328-334 Hansen, I ; Lykkegaard Nielsen, M ; Heerfordt L ; Henriksen, B ; Bertelsen S (1973) Trimethoprim in normal and pathological human lung tissue Chemotherapy 19:221-234 Hayton WL (1989) Pharmacokinetic parameters for interspecies scaling using allometric techniques Health Phys ., 57 : 159-164 Hill TA; Wands RC (1989) Serial allometric factor extrapolation: compartmental and physiological pharmacokinetic approaches Health Phys. 57 : 395-401 Hirano S ; Agata N ; Hara Y ; Iguchi H ; Shirai M ; Tone H ; Urakawa, N (1991) Effects of pirarubicin, an antitumor antibiotic on the cardiovascular system Cancer Chemother Pharmacol. 28 : 266-272. Hoff, SJ ; Neblett WW 3d ; Heller RM; Pietsch JB ; Holcomb GW Jr; Sheller, JR; Harmon TW (1990) Postpneumonic empyema in childhood : selecting appropriate therapy J. Pediatr. Surg 24 : 659-663 Ings RM (1990) Interspecies scaling and comparisons in drug development and toxicokinetics Xenobiotica 20 : 1201-1231

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146 Ishida K ; Kaku M ; Irifune K ; Mizukane R ; Takemura H ; Yoshida R; Tanaka H ; Usui T ; Suyama N ; Tomono K ; Koga H ; Kohno, S ; Izumikawa K ; Hara, K. (1994) In vitro and in vivo activities of macrolides against Mycoplasma pneumoniae Antimicrob. Agents Chemother. 38: 790-798 Intraraksa Y; Engen, RL; Switzer WP (1984) Pulmonary and hematologic changes in swine with Mycoplasma hyopneumoniae pneumonia Am. J. Vet. Res 45:474-477 Jezequel SG (1994) Fluconazole : interspecies scaling and allometric relationships of pharmacokinetic properties. J. Pharm Pharmacol. 46: 196-199 Jordan FT & Horrocks BK (1996) The minimum inhibitory concentration oftilmicosin and tylosin for mycoplasma gallisepticum and Mycoplasma synoviae and a comparison of their efficacy in the control ofMycoplasma gallisepticum infection in broiler chicks. Avian Dis ., 40: 326-334 Jordan WH; Byrd RA; Cochrane RL; Hanasono GK; Hoyt JA; Main, BW; Meyerhoff, RD; Sarazan, RD (1993) A review of the toxicology of the antibiotic MICOTIL 300 Vet Hum Toxicol. 35 : 151-158. Kaji, Y ; Ariyoshi K ; Tsuda Y ; Kanaya, S ; Fujino T ; Kuwahara, H (1989) Quantitative correlation between cardiovascular and plasma epinephrine response to mental stress Eur. J. Appl. Physiol. 5 9 : 221-226 Karnad DR; Mhaisekar DG; Moralwar KV (1990) Respiratory mucus pH in tracheostomized intensive care unit patients: Effects of colonization and pneumonia. Crit. Care Med, 18 : 699-701. Khor SP ; Amyx, H ; Davis ST ; Nelson D ; Baccanari DP; Spector T (1997) Dihydropyrimidine dehydrogenase inactivation and 5-fluorouracil pharmacokinetics: allometric scaling of animal data pharmacokinetics and toxicodynamics of 5-fluorouracil in humans Cancer Chemother. Pharmacol. 39: 233-238 Klieneberger E & Steabben, DB (1937) On a pleuropneumonia-like organism in lung lesions of rats with notes on the clinical and pathological features of the underlying condition. J. Hyg. 37: 143-152 Kolendorf, K ; Bojsen J ; Nielsen, SL (1979) Adipose tissue blood flow and insulin disappearance from subcutaneous tissue Clin Pharmacol. Ther. 25 : 598-604 Kopia GA; Driscoll EM; Yeung KF ; Lucchesi BR (1983) Antiarrhythmic and cardiovascular actions of the new antibiotic agent pirlimycin adenylate Pharmacology. 27 : 255-266

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147 Korkeala, H; Maki-Petays, O ; Alanko T; Sorvettulla 0 (1986) Determination of pH in meat. Meat Science 18 : 121-132 Kuenneke, M ; Stinner, B ; Celik, I ; Lorenz, W (1996) Cardiovascular adverse effects of antimicrobials in complex surgical cases Eur. J. Surg Suppl., 576:24-28 Landoni MF & Lees P (1996) Chirality: a major issue in veterinary pharmacology J. Vet. Pharmacol. Ther. 19 : 82-84 Laven, R & Andrews AH (1991) Long-acting antibiotic formulations in the treatment of calf pneumonia : a comparative study of tilmicosin and oxytetracycline Vet. Rec., 129 : 109-111. Lefrak EA; Pitha J ; Rosenheim S ; Gottlieb JA (1973) A clinicopathologic analysis of adriamycin cardiotoxicity Cancer 32 : 302-314 Leophonte P (1995) Azithromycin and bronchopulmonary infections Pathol. Biol. 43 : 534-541. Levieil ML; Valeyre D ; Tandjaoui H (1989) Mycoplasma pneumoniae infections Rev. Pneumol. Clin 45: 5-13 Lindsey JR (1986) Prevalence of viral and mycoplasmal infections in laboratory rodents IN: VIRAL AND MYCOPLASMAL INFECTIONS OF LABORATORY RODENTS. EFFECTS ON BIOMEDICAL RESEARCH. Eds. Bhatt, PN; Jacoby RO; Morse, HC; New, AE (Academic Press Inc. Orlando, FL). pp 801-808 Lindsey JR; Baker, HJ; Overcash RG; Cassell GH; Hunt, CE (1971) Murine chronic respiratory disease Significance as a research complication and experimental production with Mycoplasma pulmonis. Am J. Pathol. 64: 675-716 Lindsey JR & Cassell GH (1973) Experimental Mycoplasma pulmonis infection in pathogen-free mice Models for studying mycoplasmosis of the respiratory tract.,_ Amer. J. Pathol. 72 : 63-90 Lindsey JR; Davidson MK; Schoeb TR; Cassell GH (1985) Mycoplasma pulmonis-host relationships in a breeding colony of Sprague-Dawley rats with enzootic murine respiratory mycoplasmosis Lab Anim Sci. 35 : 597-608 Main, BW; Means JR; Rinkema LE; Smith WC; Sarazan RD (1996) Cardiovascular effects of the macrolide antibiotic tilmicosin administered alone and in combination with propranolol or dobutarnine in conscious unrestrained dogs. J. Vet. Pharmacol. Therap 19 : 225-232

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148 Matsushita Y ; Kumagai H ; Yoshimoto A ; Tone, H ; Ishikura T ; Takeuchi T ; Umezawa H (1985) Antitumor activities of (2"R)-4'-O-tetrahydropyranyl-adriamycin (THP) and its combination with other antitumor agents on murine tumors J. Antibiot. 38 : 1408-1419 Mazzei T ; Tonelli F ; Anastasi A ; Ficari F ; Novelli A ; Periti P (1991) Tissue distribution of cefotetan in patients with Crohn's disease Chemotherapy. 37: 297-302 McColm, AA; Shelley E ; Ryan, DM; Acred P ( 1986) Evaluation of ceftazidime in experimental Klebsiella pneumoniae pneumonia : comparison with other antibiotics and measurement of its penetration into respiratory tissues and secretions J. Antimicrob Chemother. 18:599-608 McComb JM; Campbell NPS; Cleland J (1984) Recurrent ventricular tachycardia associated with QT prolongation after mitral valve replacement and its association with intravenous administration of erythromycin Am. J. Cardiol. 54: 922-923 McGuigan, MA (1994) Human exposures to tilmicosin (MICOTIL) Vet. Human Toxicol. 36 : 306-308 McKay SG; Morck, DW; Merrill JK; Olson, ME; Chan SC; Pap, KM (1996) Use of tilmicosin for treatment of pasteurellosis in rabbits. AJVR 57 : 1180-1184 Menninger JR & Otto, DP (1982) Erythromycin carbamycin and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes Antimicrob Agents Chemother ., 21: 811-818 Moore, GM; Mowrey, DH; Tonkinson, L V ; Lechtenberg KF; Schneider JH (1996a) Efficacy dose determination study of tilmicosin phosphate in feed for control of pneumonia caused by Actinobacillus pleuropneumoniae in swine AJVR 57 : 220223 Moore, GM; Mowrey DH; Tonkinson LV; Lechtenberg KF; Schneider JH (1996b) Clinical field trials with tilmicosin phosphate in feed for the control of naturally acquired pneumonia caused by A c tinobacillus ple uropneumoniae and Pasteurella multocida in swine AJVR 57 : 224-228 Morck, DW; Merrill, JK; Thorlakson BE; Olson ME; Tonkinson, LV; Costerton, JW (1993) Prophylactic efficacy oftilmicosin for bovine respiratory tract disease J. Am. Vet. Med. Assoc 202 : 273-277

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149 Mordelet-Dambrine M ; Danel C; Farinotti R ; Urzua, G ; Barritault, L ; Huchon GJ ( 1992) Influence of Pneumocystis carinii pneumonia on serum and tissue concentrations of pentamidine administered to rats by tracheal injections Am. Rev. Respir. Dis. 146 : 735-739 Murad F & Gilman, AG (1995) Drug Interactions IN: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS Eds. Goodman Gilman A; Goodman, LS; Rall TW; Murad F (Macmillan Publishing Company New York) pp 1734-11736 Musser JD ; Mechor GD; Grohn YT; Dubovi EJ; Shin, S (1996) Comparison of tilmicosin with long-acting oxytetracycline for treatment of respiratory tract disease in calves JAYMA, 208 : 102-106 Nahata, M (1996) Drug interactions with azithromycin and the macrolides: an overview L Antimicrob Chemother ., 37 (Suppl. C): 133142. Nawaz, M & Nawaz, R (1983) Pharmacokinetics and urinary excretion of sulphadimidine in sheep during summer and winter Vet. Rec., 112 : 379-381. Nelson, JB (1937) Infectious catarrh of mice I A natural outbreak of the disease J. Exp Med., 65 : 833-860 Nilsen OG (1995) Pharmacokinetics ofmacrolides. Comparison of plasma t i ssue and free concentrations with special reference to roxithromycin Infection, 23 : S5-S9 Nocard MM & Roux, ER (1898) Le microbe de la peripneumonie Ann. Inst. Pasteur (Paris) 12:240-262 Osborne EJ & MacKillop WJ (1987) The effect of e x posure to elevated temperatures on membrane permeability to adriamycin in Chinese hamster ovary cells in vitro Cancer Lett., 37:213-224 Ose, EE (1987) In v itro antibacterial properties ofEL870 a new semi-synthetic macrolide antibiotic The Journal of Antibiotics 40: 190-194 Ose EE & Tonkinson LV (1988) Single-dose treatment of neonatal calf pneumonia with the new macrolide antibiotic tilmicosin Vet. Rec., 123 : 367-369 Parker RM & Walker AM (1993) Metabolism and residues of 1 4C-tilmicosin following subcutaneou$ administration to sheep : HPLC analysis of plasma and tissues for the parent compound Central Veterinary Laboratory Weybridge Study Report CVLS4 / 92 (Eli Lill y, Pers Comm. : Tilmicosin Injection Application to E xtend License to Include Sheep)

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150 Patel RKP; Parker, RM; Simmons HA (1992) Tilmicosin : Pharmacokinetics in Sheep Central Veterinary Laboratory Weybridge Study Report CVLSS / 91 (Eli Lilly Pers. Comm.: Tilmicosin Injection. Application to Extend License to Include Sheep). Payne NR; Burke, BA; Day, DL; Christenson, PD; Thompson, TR; Ferrieri P (1988) Correlation of clinical and pathologic findings in early onset neonatal group B streptococcal infection with disease severity and prediction of outcome. Pediatr Infect. Dis. J. 7 : 836-847 Peloso JS & Thomson, TD ( 1988) Tilmicosin tissue residue decline study in cattle. Lilly Research Study Report AAC8701 (Eli Lilly Pers. Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Pennington, JE (1981) Penetration of antibiotics in respiratory secretions Rev. Inf Dis., 3 : 67-73 Periti P ; Mazzei T ; Mini, E ; Novelli A (1993) Adverse effects ofmacrolide antibacterials Drug Saf. 9 : 346-364 Picavet T ; Muylle E ; Devriese LA; Geryl J (1991) Efficacy oftilmicosin in treatment of pulmonary infections in calves Vet. Rec., 129:400-403 Regan TJ; Khan, MI; Oldewurtel HA; Passannante AJ (1969) Antibiotic effect on myocardial K + transport and the production of ventricular tachycardia J. Clin Invest. 48 :68a. Renard C ; Vanderhaeghe HG; Claes PJ; Zenebergh A ; Tulkens PM (1987) Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages Antimicrob Agents Chemother ., 31 :410-416 Ringenberg QS; Propert KJ; Muss, HB; Weiss RB; Schilsky RL; Modeas, C ; Perry MC; Norton, L ; Green, M (1990) Clinical cardiotoxicity of esorubicin ( 4'-deoxydoxorubicin ,DxDx): prospective studies with serial gated heart scans and reports of selected cases A Cancer and Leukemia Group B report Invest. New Drugs 8 : 221-226 Ryan DM & Cars 0 (1980) Antibiotic assays in muscle : Are conventional tissue levels misleading as indicators of the antibacterial activity ? Scand J. Infect. Dis ., 12 :307-309 Ryan DM & Cars 0 (1983) A problem in the interpretation of P-lactam antibiotic le v els in tissues J. Antimicr Ther. 12 : 281-284

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151 Saggers BA & Lawson, D (1966) Some observations on the penetration of antibiotics through mucus in vitro J. Clin Pathol. 19 : 313-317. Sande MA & Mandell GL (1985) Antimicrobial agents Tetracyclines chloramphenico~ erythromycin, and miscellaneous antibacterial agents IN: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS. Eds. Goodman Gilman A, Goodman, LS; Rall TW; Murad, F (Macmillan Publishing Company New York) pp. 1170-1198 Schentag JJ & Gengo FM (1982) Principles of antibiotic tissue penetration and guidelines for pharmacokinetic analysis. Med. Clin North. Am. 6681:39-49 Schou, J ( 1961) Absorption of drugs from subcutaneous connective tissue Pharm Revs ., 13 :441-464 Schumann, FJ; Janzen ED; McKinnon JJ (1991) Prophylactic medication of feedlot calves with tilmicosin Vet. Rec., 128 : 278-280 Shaffer JM; Kucera CJ; Spink WW (1953) The protection of intracellular brucella against therapeutic agents and the bactericidal action of serum J. Exp. Med., 97: 77-90 Shetler T ; Bendele A, Buening M ; Clemens J ; Colbert W ; Deldar A ; Helton, D ; McGrath, J ; Shannon, H ; Turk, J ; Williams P (1993) General pharmacology of loracarbef in animals Arzneimittelforschung 43 : 60-70. Short CR (1994) Consideration of sheep as a minor species : Comparison of drug metabolism and disposition with other domestic ruminants Vet. Hum. Toxicol. 36:24-40 Simecka JW; Davis JK; Davidson, MK; Ross, SE; Stadtlander CT; Cassell K H (1992) Mycoplasma Diseases of animals IN: MYCOPLASMAS, MOLECULAR BIOLOGY AND PATHOGENESIS. Eds. Manilofl: McElhaney Finch and Baseman American Society for Microbiology Washington, D C. pp 391-415 Simecka JW; Patel P ; Davis JK; Ross, SE; Otwell P ; Cassell GH (1991) Specific and nonspecific antibody responses in different segments of the respiratory tract in rats infected with Mycoplasma pulmonis Infect. Immun 59 : 3715-3721. Singer JR; Narahara KA; Ritchie JL; Hamilton GW; Kennedey JW (1978) Timeand dose-dependent changes in ejection fraction determined by radionuclide angiography after anthracycline therapy. Cancer Treat. Rep., 62: 945-948 Steinberg TH; Swanson JA; Silverstein SC (1988) A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of 1774 macrophages J. Cell Biol. 107:887-896

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152 Stinner, B ; Kunneke M; Thiel T ; Hasse C ; Kapp B ; Lorenz, W (1995) Modification of cardiovascular response and histamine release by prophylactic antibiotic drugs in complicated surgery : a prospective randomized trial in a pig experimental model. lnflamm Res 44: S78-S79 Tamargo J; De Miguel, B; Tejerina, MT (1982) A comparison ofjosamycin with macrolides and related antibiotics on isolated rat atria. Eur. J. Pharmacol., 80: 285-293 Thomson RG (1980) A perspective on respiratory disease in feedlot cattle. Can Vet. J., 21:181-185 Thomson TD (1989a) Serum tilmicosin profiles following a single 10 mg/kg administration of the proposed tilmicosin bovine parenteral formulation in neonatal calves in several anatomical sites Lilly Research Study Report T5C768804 (Eli Lilly Pers Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Thomson TD ( 1989b) Serum tilmicosin profiles following a single 10 mg/kg administration of the proposed tilmicosin bovine parenteral formulation feedlot type cattle in several anatomical sites Lilly Research Study Report T5C768805 (Eli Lilly Pers Comm. : Tilmicosin Injection Application to Extend License to Include Sheep) Thomson, TD & Peloso JS (1989) Serum and lung tilmicosin levels in feedlot-type cattle follo:wing a single 10 mg/kg subcutaneous injection with the bovine parenteral formulation Lilly Research Study Report T5C768902 (Eli Lilly Pers Comm.: Tilmicosin Injection Application to Extend License to Include Sheep) Vallee E ; Azoulay-Dupuis E ; Pocidalo JJ ; Bergogne-Berezin, E (1991) Pharmacokinetics of four fluoroquinolones in an animal model of infected lung J. Antimicrob Chemother., 28 (Suppl. C): 39-44 van Miert A (1990) Influence of febrile disease on the pharmacokinetics of veterinary drugs Ann. Rech Vet. 21: 11 S-28S Vannuffel P & Cocito C (1996) Mechanism of action of streptogramins and macrolides Drugs, 51: 20-30. Veber B & Pocidalo JJ (1995) The particular case of azalides : antibiotic diapedesis Experimental data from a murine model of pneumococcal pneumonia Pathol. Biol. 43 : 524-528

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153 Veber B ; Vallee E ; Desmonts JM; Pocidalo JJ ; Azoulay-Dupuis E (1993) Correlation between macrolide lung pharmacokinetics and therapeutic efficacy in a mouse model of pneumococcal pneumonia J. Antimicrob Chemother. 32:473-482 Vogel F (1995) A guide to the treatment of lower respiratory tract infections Drugs, 50 : 62-72 von Rosensteil NA & Adam D (1995) Macrolide antibacterials Drug interactions of clinical significance Drug Saf, 13: 105-122 Wakabayashi K & Yamada S (1972) Effects of several macrolide antibiotics on blood pressure of dogs. Jap J. Pharmacol. 22: 799-807 Walker D (1993) Safety and Residues Documentation (Eli Lilly Pers. Comm.: Tilmicosin Injection. Application to Extend License to Include Sheep) Watanabe KR & Bois, FY ( 1996) Interspecies extrapolation of physiological pharmacokinetic parameter distributions Risk Anal. 16 : 741-754 Weinstein L ; Daikos GK; Perrin TS ( 1951) Studies on the relationship of tissue fluid and blood levels of penicillin J. Lab Clio Med., 38 : 712-718 Weiss BM; Spahn DR; Keller E ; Seifert B ; Pasch T (1995) Continuous non-invasive blood pressure monitoring by brachia! artery displacement method in high-risk surgical patients Eur. J. Anaesthesiol. 12 : 555-563 Whittlestone P ; Lemcke RM; Olds RJ (1972) Respiratory disease in the colony of rats II. Isolation of Mycoplasma pulmonis from the natural disease and the experimental disease induced with the cloned culture ofthis organism J. Hyg. 70 : 387-407 Wise R (1986) Methods for evaluating the penetrat ion ofbeta-lactam antibiotics into tissues Infect. Dis. 8 (Suppl. 3): S325-S332 Young C ; Laudert SB ; Thomson TD ( 1995) Accurate evaluation of pharmacokinetic action key to treatment success Tech Report Research information for the veterinarian from Blanco Animal Health. AI8124 (5/95) Ziv G & Sulman, FG (1973) Serum and milk concentrations of spectinomycin and tylosin in cows and ewes Am. J. Vet. Res., 34: 329-333 Ziv G ; Shem-Tov M ; Glickman A ; Winkler M ; Saran, A (1995) Tilmicosin antibacterial activity and pharmacokinetics in cows. J. Vet. Pharmacol. Ther. 18 :340-3 45

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APPENDIX A LIST OF SUPPLIERS FROM CHAPTER 3 1 Eli Lilly & Co. Greenfield IN 2 Elanco Animal Health Indianapolis IN 3 Baxter Diagnostics Inc McGaw Park IL 4 Fisher Scientific Co., Pittsburg, PA 5 Aldrich Chemical Company Inc ., Milwaukee WI 6 Varian Sample Preparation Products Harbor City CA 7 Polytron Brinkmann Instruments Rexdale ON 8 Beckman Instruments Inc. Fullerton CA 9 Regis Chemical Company Morton Grove IL 1 0 Microsoft Corporation Redmond WA 11 Micromath Scientific Salt Lake City UT 1 2 Datascope Corp ., Paramus NJ 13 Harlan Sprague Dawley Indianapolis IN 14 Lab Products Inc ., Maywood NJ 1 5 Northeastern Products Corp ., Warrensburg NY 1 6 PMI Feeds Inc St Louis MO 1 7Becton Dickinson and Co., Franklin Lakes NJ 18 Extech Instruments Waltham MA 1 9 Jandel Corporation Chicago IL 154

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APPENDIXB PHARMACOKINETIC EQUATIONS AS WRITTEN FOR 'EXCEL Example : Data set (concentrations) for which the pharmacokinetic parameters are to be calculated is located in rows 4-24 column B. Time points for each concentration are located in rows 4-24 column A The points included for calculation of k are the points that lie on the terminal straight-line portion of the curve : k [h-1] = -LINEST(LN(B20 : B24) $A20 : $A24) t112 [h] = LN(2)/k AUC [g/ml*h] =SUM((B5 : B24 + B4 : B23)/2*($A5:$A24-$A4 : $A23)) +B24/k AUMC [g/ml h2] = SUM((B5 : B24*$A5 : $A24+B4 : B23*$A4 : $A23) / 2 ($A5 : $A24$A4 : $A23) )+B24 *$A24/B27 +B24/k/\2 MRT [h] =AUMC /AUC Cmax [g/ml] = MAX(B5 : B23) tmax [h] = observed from data Dose [ mg] = total dose given in mg CL/f [l/h] = Dose/A UC Vd/f [l] =Cl/k 155

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APPENDIXC ANOV AT ABLES FROM THE STATISTICAL ANALYSES OF THE CARDIOVASCULAR DAT A ON THE EFFECT OF TILMICOSIN IN SHEEP A-A Two-Way Repeated Measures (RM) ANOVA Table of the Effect ofTilmicosin on the SYSTOLIC BLOOD PRESSURE Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep# 9 49320.7 5480 1 7.94 0 018 Treatment 1 16636 3 16636 3 18.61 0.002 Treat. x Sheep 9 8056 9 895 2 Time 10 10706 6 1070 7 2.44 0 012 Timex Sheep 90 39263 7 436 3 Treat. x Time 10 9558 3 955.8 1.49 0 158 Residual 88 56627 9 643 5 Total 217 189344 7 872.6 B -A Two-Way RM ANO VA Table of the Effect of Tilmicosin on the SYSTOLIC BLOOD PRESSURE (using the values normalized against zero time) Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep# 9 17672 7 1963 6 0 388 0 911 Treatment 1 251.6 251. 6 0 0488 0 830 Treat. x Sheep 9 46590 1 5176 7 Time 10 6206 2 620 6 2 319 0 018 Timex Sheep 90 23991.4 266 6 Treat. x Time 10 4674.4 467.4 1 205 0 299 Residual 88 34142 9 388 0 Total 217 134042 6 617 7 156

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157 C -A Two-Way RM ANO VA Table of the Effect of Tilmicosin on the DIASTOLIC BLOOD PRESSURE Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep# 9 15572 6 1730 3 0 902 0 560 Treatment 1 7399 9 7399.9 3 888 0.080 Treat. x Sheep 9 17187 5 1909 7 Time 10 2722 8 272 3 0 741 0 684 Timex Sheep 90 33083.4 367 6 Treat. x Time 10 4657 0 465 7 1.296 0 245 Residual 88 31629 2 359.4 Total 217 111370 2 513 2 D -A Two-Way RM ANO VA Table of the Effect of Tilmicosin on the MEAN BLOOD PRESSURE Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep # 9 17878 8 1986.5 1.783 0 197 Treatment 1 5844.4 5844.4 5 446 0 044 Treat. x Sheep 9 9682 9 1075.9 Time 10 5441.1 544 1 1.158 0 329 Timex Sheep 90 42308 0 470 1 Treat. x Time 10 4924 6 492 5 1.141 0 342 Residual 88 37990 7 431.7 Total 217 127490 0 587 5 E-A Two-Way RM ANOVA Table of the Effect ofTilmicosin on the HEART RATE Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep# 9 17468 6 1941.0 1.620 0 241 Treatment 1 621.9 621.9 0 524 0.488 Treat. x Sheep 9 10730 7 1192 3 Time 10 4819 0 481 9 3 044 0 002 Timex Sheep 90 14250 9 158 3 Treat. x Time 10 1747 9 174 8 1 149 0 336 Residual 88 13384.3 152 1 Total 217 64242 0 296 0

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158 F A Two-Way RM ANOVA Table of the Effect ofTilmicosin on the RESPIRATORY RATE Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Sheep # 9 98453 1 10939 2 5 628 0 008 Treatment 1 1132 8 1132 .8 0.589 0.463 Treat x Sheep 9 17380.9 193 1.2 Time 10 48734.0 4873.4 16. 626 < 0 001 Timex Sheep 90 26391.2 293 2 Treat. x Time 10 1533.4 153 3 0 546 0 853 Residual 88 24709 2 280 8 Total 217 219260 9 1010.4

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Ani. T 145 T 145 T 145 T 145 p 145 p 145 p 192 T 192 T 192 T 192 p 192 p 192 p 193 T 193 T 193 T 193 p 193 p 193 p 194 T 194 T 194 T 194 p 194 p 194 p 196 T 196 T 196 T 196 p 196 p 196 p APPENDIXD BLOO D CHEMISTRY RE SUL TS IN SHEE P AFTER TILMIC O SIN ( O R PLACEBO) TREATMENT Abbreviation s in the table s : Ani. = animal ID D = da y after treatmen t T = tilmico s in tr ea tmen t P = placebo ( saline ) tre a tment (Na = sodium; K = potassium ; Cl = cloride ; CO2 = carbon dio xide; An G = anion gap ; BUN = blood urea nitrog en; Crtn = creatinin; Ca = calcium ; Gluc = gluco se ; P = pho s phorus ; Bili = bilirubin ; Mg = magnesium ; Prot. = prot ein; Alb = albumin; Blob = globulin; A/G = albumin / globulin ratio ; AP = alkalin e phosp h atase ; SOO T = serum glutamate o x aloacetate transamina s e ; GGT = g amma glutam y ltran s ferase ) D Na K C l CO 2 An G B UN C rtn C a G luc p B ili. M g P r o t. A l b G l ob A/ G AP SG O GGT 0 140 8 4 5 113 22.9 9 4 14. 8 0 8 8 50.8 4 3 0 1 1 .68 5 7 2 2 3 5 0 6 56 61 76 1 144 4 4 1 114 28 6 5 14 7 0 7 8 3 61. 7 5 8 0 1 1 .62 5 5 2 1 3 3 0 6 57 59 70 3 142 6 4 6 114 24.2 9 13 0 6 8 9 57. 9 4.4 0.1 1 .58 5 6 2 2 3 4 0 6 91 65 85 0 142 8 4.1 111 26.1 9 8 1 5 0 6 8 7 57. 7 4 4 0.1 1 .62 5 9 2 2 3 6 0 6 73 79 141 1 141. 2 4 110 30 6 4 6 13 9 0 6 8 5 52 5 4 7 0 1 1 .59 6.1 2 1 4 0 5 82 78 143 3 140 7 4 7 111 25 6 8 8 13 8 0 6 8 4 35 4 4 0 2 1 .64 5 9 2 1 3 7 0 6 72 84 128 0 143.3 4 2 113 24 7 9 8 18 4 0 7 8 5 62. 3 5 9 0 1 1 7 6 6 2 5 4 1 0 6 91 68 66 1 144.6 4 4 115 25 8 8 2 1 8 0 7 8 5 59. 1 5 7 0 1 1 .87 6.6 2 3 4 3 0 5 119 64 60 3 142 4 6 114 25.5 7 1 17 0 7 9 2 61. 4 4 5 0 1 1 .82 6 9 2.5 4 5 0.6 132 69 64 0 145 1 4 4 1 13 24 1 2 6 1 5 2 0 8 8 9 81. 6 4 6 0 2 1.79 6 1 2 6 3 5 0.7 127 57 71 1 145 4 4 7 113 25 8 11. 2 16 5 0 8 8 4 47. 8 4.9 0 1 1 .68 6 3 2 4 3 9 0 6 135 56 68 3 143 2 5 113 25 8 9 4 16 1 0 7 9 3 60. 1 4 9 0 1 1 .67 6 9 2 5 4 5 0.5 133 58 69 0 146 2 4 2 108 23 6 18 8 17 3 1 8 1 92 7 11 0.1 1 .83 6 4 2 9 3 5 0 8 111 59 80 1 144 8 4 2 111 26 7 1 1 3 16. 6 0 9 8 78 9 8 1 0 1 2 .23 6 3 3 3 3 0.9 100 54 78 3 143 9 4 9 111 26. 8 11 15. 8 0 8 8 8 64.8 7 8 0 1 1 79 6.4 2 9 3.4 0 9 144 53 77 0 167 5 5 4 124 28 20. 9 20. 7 0 8 8 1 72 6 10 0 1 1 .66 5 7 2 7 3 1 0.9 159 58 73 1 142 6 4 3 107 32 7 9 19 0 9 8 6 71. 6 8 3 0 1 2 15 6 3 2 8 3 5 0 8 1 52 60 76 3 141. 5 4.8 109 27 7 9 7 16 8 0 9 8 6 59 5 6.2 0 1 2 04 6 2 3 3 2 0.9 135 58 74 0 171 7 4.7 128 27 4 21 17. 8 1 1 0 161 7 4 0 1 1 77 6 2 2 7 3.5 0 8 200 63 60 1 144 8 4 3 111 28.4 9 7 1 7 2 0.9 72 3 5 5 0 1 1 .88 6.4 2 7 3 7 0 7 210 65 62 3 146 8 4 5 112 29 5 9 8 20. 3 0 9 8 8 62. 7 5 9 0 1 1 .79 6 3 2.6 3 7 0 7 242 64 66 0 143 1 4.3 110 28 6 8 8 17 0 7 9 3 96. 9 6.1 0 1 1 .91 6 1 2 7 3 5 0 8 245 57 60 1 144 3 4 1 112 28 3 8 1 19. 7 0 9 9 1 77.2 6 0 1 2 15 6 3 2 7 3 6 0 8 266 55 61 3 144 7 4.4 110 21. 8 17. 3 25. 3 1 2 8 8 93 6 6 1 0 1 2 06 7 2 9 4 2 0 7 200 69 68 0 143 4 3 108 27 9 11. 4 21.8 1 5 10 111 7 3 0 2 1 .85 7 3 1 3 9 0.8 35 68 71 1 143 3 4 5 110 29 6 8 2 18 6 1 3 9 2 70 5 4 9 0 2 1 .63 6 2 2 7 3 5 0 8 33 60 62 3 146 2 4 8 111 28 9 11. 1 19. 4 1 1 8 6 61. 6 8.3 0.1 1 72 5 9 2 5 3 3 0 8 46 52 61 0 144.8 4 5 111 28. 3 10 21 1 8 8 77 8 0.1 1 8 5 9 2 8 3 1 0.9 36 53 68 1 145 6 4 7 1 1 2 27 9 10 4 2 1 1 1 1 8.4 68. 1 8 5 0 1 1 8 1 5 7 2 6 3 1 0.9 56 49 67 3 145 8 4 7 110 21.7 1 8 8 27. 1 1 5 8 9 85. 9 9 6 0 1 2 .34 6 2 7 3.4 0 8 48 6 1 72 159

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160 Ani. T D Na K C l CO2 A n G B UN C rt n Ca G l uc p B ili. Mg P r ot. A l b b l ob NG AP $GO GG T 197 T 0 144 4 4.8 109 24. 2 15. 9 19. 1 1 2 9 138 6 8 0.3 1 .82 6 1 2.8 3.4 0 8 47 56 68 197 T 1 144. 5 4 2 114 26.3 8 4 18 2 1 2 7 7 90. 6 4 6 0 2 2 .19 6 1 2 5 3 6 0 7 41 57 64 197 T 3 145. 6 4.7 111 30.2 9 1 14.4 1.1 9 2 71.5 6 3 0 2 1 .58 5 9 2 6 3 3 0 8 51 59 60 197 p 0 143. 8 4.1 112 21. 6 14. 3 21 1 3 8 6 119 7 7 0 3 1 .95 6 3 3 1 3 2 1 48 69 70 197 p 1 146. 3 4 4 115 26. 3 9.4 12. 8 1.1 8.4 77. 1 6 6 0 2 1 .77 6 2 8 3 2 0.9 48 59 66 197 p 3 144.7 4 6 111 32.6 5 7 18 1 9 6 68. 6 7 1 0 2 1 63 6 2 9 3.1 0.9 44 55 63 198 T 0 146. 1 4 1 109 24. 8 16. 4 19. 5 1 1 6 6 74. 9 8 0 3 2.23 7 3 1 3 8 0 8 54 11 74 198 T 1 146. 8 4 5 113 26. 9 11. 4 13 5 1 7 70. 2 8 0.2 2 03 6 7 3 3 8 0 8 63 99 69 198 T 3 147. 5 4 8 112 28 6 11. 7 16 0 8 11 57. 6 6.9 0 1 1 52 6.4 2 9 3 5 0 8 52 83 68 198 p 0 143 4 4 5 109 26. 1 12 8 21. 7 0 8 6 5 56. 5 7 8 0 1 1 68 6 2 2 6 3 6 0 7 87 70 70 198 p 1 145. 8 4 7 110 27. 8 12. 7 22 4 0 8 6.4 56. 8 7 5 0 1 2 .04 6 3 2 6 3 7 0 7 99 72 71 198 p 3 182. 7 5 6 140 32. 4 15. 9 23 3 0 8 7 5 57. 8 7 5 0 1 1 66 6 1 2 5 3 6 0 7 104 72 68 199 T 0 145.2 4 3 113 23 6 12 9 13. 9 1 1 8 7 108 8 1 0.3 1 92 6 2 9 3 1 0 9 60 54 75 199 T 1 144. 8 4 5 116 25. 9 7 4 10 0 9 8 7 67 5 6 7 0 3 1 .99 5 8 2 8 3 0 9 68 55 71 199 T 3 147 5 1 1 3 26.9 1 2 1 14 7 1 2 8 7 60 7 0 3 1 69 6 1 2 7 3 4 0 8 56 61 73 199 p 0 144 7 4 7 114 23. 2 12 2 15. 2 1 8 114 6 2 0 2 1 8 6 1 2 8 3 3 0 8 93 49 70 199 p 1 191. 5 5 5 147 36. 5 13. 5 17 2 1 1 9 5 81. 7 7 1 0 1 2 12 6 7 2 8 3 8 0.7 103 58 78 199 p 3 146. 9 5 6 112 3 1 9 5 16 1 9 2 72. 6 7 2 0 1 1 .82 6 6 2 9 3 7 0 8 128 59 76 201 T 0 143. 6 4 2 109 27 5 11. 3 13. 7 1 8 8 121 6 2 0 1 1 .43 6 2 7 3 2 0 8 36 48 58 201 T 1 142 9 4 6 1 1 2 29. 6 5 9 14 1 9 5 73. 5 5 3 0 1 1 .59 5 9 2 5 3 4 0 7 39 49 54 201 T 3 143 4 4 8 110 31.2 7 16 0 9 9 4 75. 4 5 5 0 2 1 .54 6 1 2 6 3 5 0 8 60 52 58 I 201 p 0 141. 7 4.4 108 27. 2 10 9 20. 7 1 1 8 6 163 4 4 0 2 1 .86 6 3 2 6 3 7 0.7 49 53 64 201 p 1 186 2 5 4 143 38. 7 9 9 22 4 1 1 9 84. 7 5 0 1 1 .87 6 2 5 3 4 0.7 48 49 64 201 p 3 143. 6 5 1 111 31.4 6 3 17. 9 0 9 9 1 62 8 6 8 0 1 1 7 3 6 2 2 6 3 6 0.7 55 44 61 205 T 0 145 4 4 1 112 23. 9 13 6 13. 6 1 3 9 3 133 3 4 0 6 1 .51 6 8 3 3 7 0 8 56 54 70 205 T 1 143.1 3 7 111 25. 7 10.1 10. 9 1 2 9 3 78. 9 8 2 0 4 1 .56 5 7 2.4 3 3 0 7 54 45 56 205 T 3 146. 4 4 3 114 27 9 6 11 0 9 9 1 74. 6 7 1 0 3 1 66 6 2 6 3 4 0 8 58 53 62 205 p 0 146.1 3 2 110 26.4 12 9 8 9 1 1 8.3 74. 7 5 3 0 8 1 .11 6 4 2 8 3 6 0.8 58 73 60 205 p 1 147 3 5 111 28. 5 10 9 8 1 9 1 67 2 5 3 0 5 1 2 6 6 2 7 3 9 0 7 60 74 60 205 p 3 171 5 4 129 35. 5 11. 9 12. 3 0 9 9 1 70. 8 5 3 0.3 1.25 5 7 2 3 3 4 0.7 71 69 54

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Ani. T 145 T 145 T 145 T 145 p 145 p 145 p 192 T 192 T 192 T 192 p 192 p 192 p 193 T 193 T 193 T 193 p 193 p 193 p 194 T 194 T 194 T 194 p 194 p 194 p 196 T 196 T 196 T 196 p 196 p 196 p APPENDIXE HEMATOLOGY RESULTS IN SHEEP AFTER TILMICOSIN (OR PLACEBO) TREATMENT (WBC = white blood cells ; Seg. = segmented WBC ; Lym = lymphocytes ; Mon = monocytes ; Eos. = eosinophil.s ; Bas. = basophil s; RBC = red blood cell s; HGB = heinoglobin ; HCT = hematocrit ; MCV = mean cell volume ; MCH = mean corpuscular cell volume ; MCHC = mean corpuscular cell volume concentration ; RDW = red cell distribution width; Pit= platelet estimate ; S HCT = s pun hematocrit ; Prot. = total protein ; Fibm. = fibrinogen ) D WBC Seg Lym Mon Eos Bas RBC HGB HCT MCV MCH MCHC ROW P L T ~ .HCl Prot. F i brn. 0 12 3 72 23 5 0 0 7 28 7 .68 22.4 30 9 10 6 34 2 22 356 24 6 7 200 1 10 9 67 28 5 1 0 6 37 6 .65 19. 6 30 7 10.4 34 20.4 438 22 6.1 300 3 12 2 77 18 5 0 1 6.2 6.4 18. 8 30 3 10.3 34 2 20 7 617 20 6.2 300 0 1 23.4 90 5 2 0 1 6 28 6 7 19 2 30 6 10. 7 34.8 21.4 116 21 7 1 300 3 20. 2 81 16 2 1 0 6 74 6 .88 20. 3 30. 1 10. 2 33 9 22. 8 619 22.8 6 9 700 0 8 18 68 24 5 4 0 9.49 9 8 27. 5 29 10. 3 35 7 20 2 132 30 7.5 300 1 8 .01 56 31 3 9 1 9 39 9.82 27. 4 29 1 10.5 35 9 19 1 144 30 7.3 200 3 6 .01 53 32 9 6 0 9 97 10 8 29.5 29.6 10 8 36.5 19. 8 227 32 7.7 300 0 6 78 30 66 3 1 0 9 57 9 .68 27. 1 28.3 1.01 35 8 19 143 30 7 4 200 1 7.27 56 36 5 3 1 9 24 9 16 26. 1 28 3 9 92 35 1 19 2 57 8 26 2 7.3 300 3 7 36 62 29 6 1 2 10.4 10 6 29.8 28.6 10. 2 35 7 20 303 32 7 6 300 0 6.13 70 26 4 0 0 8 8 9 .52 28. 7 32 4 10. 8 33.1 19 7 301 30 7 2 300 1 6 14 61 37 1 0 1 7 92 8 .54 25. 7 32 5 10. 8 33 2 18 3 203 28 7 300 3 5.92 29 66 5 0 0 8 89 9 56 28. 9 32.5 10 8 33 1 19. 4 581 30 7 300 0 6 38 47 48 3 1 1 9 78 11.2 32. 9 33.6 11. 4 34 19. 7 46.5 35 6 9 100 1 5 39 47 46 4 4 0 8 34 9 .32 27. 6 33 1 11. 2 33 8 184 145 29 6 9 200 3 6.41 28 67 1 3 1 8 88 10 29. 2 32 9 11. 3 34.4 19. 3 410 31. 5 6 6 200 0 2 36 71 27 1 1 0 9 .21 10 8 31.3 34 11. 7 34 3 18.3 24 33 6 8 100 1 4 09 57 35 6 1 1 8.72 10 3 29. 5 33 8 11. 8 34 9 18 35.4 31 7 300 3 4.44 36 59 2 3 0 9 52 11. 1 31. 9 33 5 11. 7 34 8 18.1 27. 3 34 6 9 300 0 2.85 59 35 3 3 0 8 12 9 8 28 34 5 12. 1 35 18 3 30 6 6 200 1 3.5 19 26 4 1 0 8 76 10 7 30. 3 34 6 12 2 35 4 19 3 17 33 4 6 8 200 3 3 2 29 65 1 5 0 11. 5 14 3 40. 5 35 2 12. 4 35. 3 21. 6 44 7 3 200 0 3 6 62 32 3 2 1 8.68 9 54 27.4 31. 6 11 34 8 17 49.8 29 6 7 300 1 5 2 70 22 3 4 1 7 72 8.44 24. 3 31. 5 10. 9 34 7 17.9 282 26 6 7 200 3 3 .88 39 56 1 3 1 8 18 9 15 25. 3 30. 9 11. 2 36 2 17 3 550 27 6 4 200 0 4 29 41 46 9 2 2 7 8 8 .55 24. 3 31.2 11 35.1 16.9 24 4 6 3 200 1 4 34 62 36 0 2 0 7 93 8 .87 25 31. 6 11. 2 35.4 17. 5 34. 5 28 6 2 200 3 3.46 37 59 2 2 0 11. 3 13 5 37 32 6 11. 9 36 4 18. 9 413 42 6.4 200 161

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162 Ani. T D WBC Seg Lym Mon Eos Bas RBC HGB HCT MCV MCH MCHC ROW P L T s .HCl P rot. Fi brn. 197 T 0 6 39 76 19 4 0 1 8 88 10 5 29. 5 33.3 11. 8 35.4 18 5 48 4 33 6 3 300 197 T 1 8 85 14 1 0 0 9 77 11. 5 32. 6 33.4 11. 8 35 2 18 4 63 7 35 6 6 400 197 T 3 3 73 50 35 4 11 0 10 1 12 2 33 9 33 7 12. 1 35 9 19 9 261 36 7 300 197 p 0 3.77 57 36 1 5 1 9 .62 11. 7 32 5 33 8 12. 2 36 2 19 3 375 34 2 7 300 197 p 1 4 85 77 18 1 4 0 10 9 13 5 36 6 33.7 12. 4 36 7 19.8 338 38 6 6 200 197 p 3 4 .71 50 41 3 5 1 11. 3 13 7 38. 1 33.8 12. 1 35.8 21. 1 39.9 6 6 300 198 T 0 7 12 85 12 3 1 0 9 76 11. 3 30 3 31 11. 6 37 3 21 524 33 7 7 300 198 T 1 6 39 63 31 3 3 0 10.5 12 5 32 7 31 11.9 38 3 20.1 537 34 4 7 5 300 198 T 3 3.69 41 51 4 4 0 1 1 .6 13 5 35. 9 31 11. 7 37 6 21.1 39.7 7 1 400 198 p 0 3 5 33 65 2 1 0 9 85 11. 1 30 30 5 11. 3 37 19.6 44 2 34 6 8 300 198 p 1 4 39 44 48 2 2 1 10. 7 12 32 4 30 4 11. 3 37.1 20 7 532 34 6 7 200 198 p 3 3 1 43 51 3 2 1 10. 4 12 33 2 31. 9 11. 5 36 2 20 9 368 35 7 2 300 199 T 0 3 79 56 43 1 1 0 8 4 9 .19 27. 6 32 8 10 9 33.4 19.4 399 28 8 6 7 300 199 T 1 3 83 52 45 1 1 1 9 78 11 32 4 33 1 11. 2 34 20 5 34 2 6 6 300 199 T 3 3 8 42 52 3 2 1 1 1 .4 12.8 37 8 33 2 11. 3 34 21.4 420 39 8 6 7 500 199 p 0 5 .81 72 23 3 1 1 7 13 9 09 29.4 12 7 43.4 25 7 455 29 6 6 100 199 p 1 4 .21 61 33 5 0 1 9 47 10 5 31. 1 32 9 11.1 33 6 20.5 121 32 7 4 100 199 p 3 4 25 48 46 4 2 0 10. 8 11. 9 35 4 32 8 11 33 6 20 6 353 38 6 8 200 201 T 0 4 26 55 38 6 1 0 9 .71 10.3 28. 1 29 10. 6 36.5 19.7 593 29 6 9 300 201 T 1 5 68 51 40 2 5 1 1 0 6 11. 5 31 29. 2 10 9 37 2 20 2 32 8 6 6 400 201 T 3 2 64 40 51 6 2 1 10. 3 10 9 30 1 29 3 10 6 36 1 19 5 723 31. 6 6 8 400 201 p 0 4 89 63 34 3 1 0 9.4 9 59 27. 1 28. 8 10.2 35.4 18 3 42 4 29 6 8 300 201 p 1 5 2 76 19 4 2 0 9 .41 9 .74 27. 6 29 3 10 3 35 3 18 7 41. 9 30 6 6 300 201 p 3 3 3 31 61 6 2 0 1 0 8 1 1 .2 30. 9 28 8 10 4 36.1 19.1 451 33 6.5 200 205 T 0 4 77 85 12 3 1 0 10. 9 12 2 34 9 32 1 11. 2 34 9 18 9 385 38 7 5 200 205 T 1 5 .21 68 29 2 2 1 8 98 9 .92 28. 7 32 11 34 5 1 8 31 30 8 6.4 200 205 T 3 4 73 58 35 3 4 0 9 3 10.4 29. 7 32 11. 2 35 1 18. 5 30 6 6 7 300 205 p 0 4 .64 77 16 5 1 1 8 89 9 88 27. 9 31. 4 11. 1 35 3 17 2 502 29 7 1 300 205 p 1 6 .64 78 18 3 2 0 9.27 10 2 29. 2 31. 5 11 34 8 17.4 492 32 7 1 300 205 p 3 4 05 60 32 6 2 0 9 35 10 3 29. 9 32 11 34 6 17.3 141 31 7 200

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APPENDIXF ANOVA TABLES FROM THE STATISTICAL ANALYSES OF THE EFFECT OF MYCOPLASMA INFECTION ON THE LUNG AND MUSCLE TISSUE PH IN RATS A-A Two-Way ANOVA Table of the Effect ofDisease on the Muscle Tissue pH Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Treatment 1 0.00390 0 00390 0.394 0 533 Hours 5 0 862 0 172 17.406 < 0.001 Treat. x Hours 5 0 125 0 0250 2 526 0 038 Residual 60 0.595 0.00991 Total 71 1.586 0 0223 B -A Two-Way ANOVA Table of the Effect of Disease on the Lung Tissue pH Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Treatment 1 0 000450 0.000450 0.0480 0 827 Hours 5 0.403 0 0806 8.596 < 0 .001 Treat. x Hours 5 0 140 0 0280 2.984 0 018 Residual 60 0 562 0 00937 Total 71 1 106 0 0156 C -A Two-Way ANOVA Table of the Effect of Disease on Tilmicosin Concentration in the Lung Tissue Source of Degrees of Sum of Mean F Test p value Variation Freedom Squares Squares Statistic Treatment 1 23.839 23.839 6.012 0 018 Time 5 436 27 87 254 22 005 <0 001 Residual 52 206 .19 3 965 Total 58 314.65 5.425 163

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APPENDIXG INSTITUTIONAL ANIMAL CARE AND USE COM1vfITTEE (IACUC) APPROVAL ........................................................................................................................ REAPPROVAL T 0 US E ANIMALS ....................................................................................................... ....................................................................................... PROJECT #4102 EXPIRES 06/23/97 Responsible Faculty: ALISTAIR I WEBB Address: PHYSIOLOGICAL SCIENCES BOX 100144 Project Title: A NATIONAL AGRICULTURAL PROGRAM TO APPROVE ANIMAL DRUGS FOR MINOR SPECIES AND USES -NATIONAL RESEARCH SUPPORT PROJECT .................................................................................. ............................................................................................................ .. Your project using animals has been approved for another year for the activity shown above. This approval has been granted by the Institutional Animal Care and Use Committee (IACUC). The original approval date of this project was 06/24/94. This approval expires on the date listed above. You'll be contacted next year about this time with instructions to extend, modify or withdraw this approval. This project was last approved on 05/31/96. Farol Tomson, DVM P.O. Box 100142 University of Florida Gainesville, FL 32610-0142 (904) 392-9917 163

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164 APPROVAL TO USE ANIMALS PROJECT #6072 EXPIRES 05/15/97-Responsible Faculty: ALISTAIR I WEBB Address: PHYSIOLOGICAL SCIENCES BOX 100144 Project Title: COMPARISON OF TILMICOSIN LEVELS IN NORMAL AND PATHOLOGIC LUNG TISSUE IN RODENTS ................................................................................................................ A project using animals has been approved for the researcl: or teaching activity shown above. This approval has been granted by the Institutional Animal Care and Use Committee (IACUC). Animals may be purchased and used for the above project and housed in any IACUC approved facility. This approval is for one year and expires on the above dat You will be contacted in ten months with instructions abou extending, modifying or withdrawing this approval. ................................................................................................... This project was approved on 05/15/96. Farol Tomson, DVM P.O. Box 100142 University of Florida Gainesville, FL 32610-0142 (904) 392-9917

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BIOGRAPHICAL SKETCH Sanja Modric maiden name Morie, was born in Zagreb Croatia on February 17, 1966 She graduated with honors from the College of Veterinary Medicine University of Zagreb in April 1990. In 1990 she started a graduate program at the Biology Department University of Texas at El Paso where she obtained a Master of Science degree in Biology in July 1992. She started a Ph. D. program in the Department of Physiological Sciences at the University of Florida in January 1993 She worked under the mentorship of Dr. Stephen Sundlofuntil he accepted the position at the Food and Drug Administration, when Dr. Alistair Webb assumed the mentorship In May 1997 she completed the requirements for the degree of Doctor of Philosophy She holds a research scientist position in the Research Center of" Pliva" a major pharmaceutical company in Zagreb Croatia. 166

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I certify that I have read this study and that in my op1ruon it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy ebb, Chair Pr fessor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Maureen K. Davidson, Associate Professor of Veterinary Medicine I certify that I have read this study and that in my op1ruon it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy A-<. VJ~V\ olQJ f Hartmut C. Derendorf, Professor of Pharmaceutics I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy onwall Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is full y adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy Stephen F Sundlof, Professor of Veterinary Medicine

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I certify that I have read this study and that in my op1IDon it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy ~W."J~ Thomas W Vickroy Associate Professor of Veterinary Medicine This dissertation was submitted to the Graduate Faculty of the College of Veterinary Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy May 1997 C. Dean, edicine Dean Graduate School

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