PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF
TILMICOSIN IN SHEEP, CATTLE, AND RATS
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
To my dearest husband Tomislav, for his love, support and friendship
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
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
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
184.108.40.206. Mechanism of action....................................................... 11
220.127.116.11. Antibacterial activity ................. .......................................... 12
2.2.3. Pharm acokinetics.............................................................. .................. 12
2.2.3. 1. A bsorption.............................................................. ................... 13
2.2 .3.2 D distribution ............................................................... ................ 14
18.104.22.168. Biotransform ation.................................................... ..................... 16
22.214.171.124 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
126.96.36.199. O their antibiotics ....................................... .... ........ .................. 28
188.8.131.52. 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
184.108.40.206. Effect of disease on the pharmacokinetics of macrolides................ 39
220.127.116.11. 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
18.104.22.168 Extraction of tilmicosin from serum ............................................ 51
22.214.171.124. Extraction of tilmicosin from lung tissue .................................... 52
3.1.3. High Pressure Liquid Chromatography ............................................... 53
126.96.36.199. Chromatographic conditions ...................................................... 53
188.8.131.52. Calculation of HPLC results....................................................... 56
184.108.40.206. HPLC method validation study .................................................. 56
220.127.116.11. Q quality control......................................................... .................. 63
18.104.22.168. 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
22.214.171.124. 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
126.96.36.199. Tilmicosin administration in sheep and cattle................................. 71
188.8.131.52. 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
184.108.40.206. Statistical analysis.................................................. .................... 78
4.1.2. Effect of Respiratory Disease on Tilmicosin Pharmacokinetic in Rats........ 78
220.127.116.11. Sample collection ....................................................................... 79
18.104.22.168. 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
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
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
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
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.
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
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
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
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
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.
REVIEW OF LITERATURE
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.
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.
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 HO N(CH3)2
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).
22.214.171.124. 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).
126.96.36.199. 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.
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).
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).
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,
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.
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.
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.,
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
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
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
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.
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
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
188.8.131.52. 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
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).
184.108.40.206. 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
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.
220.127.116.11. 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
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
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).
18.104.22.168. 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 22.214.171.124.), 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
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.
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,
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.,
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).
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.
126.96.36.199 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.
188.8.131.52. 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.
184.108.40.206. 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
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).
220.127.116.11. 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
(18.104.22.168. and 22.214.171.124.). 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
126.96.36.199. 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.
188.8.131.52.1. 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
-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
0.053 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.
184.108.40.206.2. 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).
220.127.116.11.3. 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
18.104.22.168.4. 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
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
22.214.171.124.5. 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).
126.96.36.199.6. 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).
188.8.131.52.7. 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
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
Mean 0.062 106.9
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
Mean 0.099 99.3
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
Mean 1.025 102.5
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
Mean 1.931 96.55
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.
184.108.40.206. 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.
220.127.116.11. 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.).
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
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);
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.
ke [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
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
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
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:
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
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
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.
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).
18.104.22.168. 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
22.214.171.124. 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.
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
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.
126.96.36.199. 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.
188.8.131.52. 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.
184.108.40.206. 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.
Serum and lung samples were subsequently used for HPLC analysis as described in
220.127.116.11. 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.
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,
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
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
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
AUMC 885 690 672 729 1041 768 539 665 955 1168 811 195
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
AUMC 432 580 720 416 562 522 451 694 980 1137 649 241
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  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
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
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.
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  10906 1602 1651 318.8 <0.001